PHYTOREMEDIATION RHIZOREMEDIATION

FOCUS ON BIOTECHNOLOGY
Volume 9A

Series Editors
MARCEL HOFMAN
Centre for Veterinary and Agrochemical Research, Tervuren, Belgium

JOZEF ANNÉ
Rega Institute, Catholic University of Leuven, Belgium

Volume Editors
MARTINA MACKOVA
Institute of Chemical Technology,
Prague, Czech Republic

DAVID DOWLING
Institute of Technology,
Carlow, Ireland

THOMAS MACEK
Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic,
Prague, Czech Republic

COLOPHON

Focus on Biotechnology is an open-ended series of reference volumes produced by

Springer in co-operation with the Branche Belge de la Société de Chimie Industrielle
a.s.b.l.

The initiative has been taken in conjunction with the Ninth European Congress on
Biotechnology. ECB9 has been supported by the Commission of the European
Communities, the General Directorate for Technology, Research and Energy of the
Wallonia Region, Belgium and J. Chabert, Minister for Economy of the Brussels
Capital Region.
Phytoremediation
Rhizoremediation
Edited by

MARTINA MACKOVA
Institute of Chemical Technology,
Prague, Czech Republic

DAVID DOWLING
Institute of Technology,
Carlow, Ireland

and

THOMAS MACEK
Institute of Organic Chemistry and Biochemistry,
Academy of Sciences of the Czech Republic,
Prague, Czech Republic
A C.I.P. Catalogue record for this book is available from the Library of Congress.

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TABLE OF CONTENTS

1. Introduction .................................................................................................................. 1
John Fletcher

2. The Chemical Ecology of Pollutant Biodegradation: Bioremediation and

Phytoremediation from Mechanistic and Ecological Perspectives ........................ 5
Andrew C. Singer

3. Dendroremediation: The Use of Trees in Cleaning up Polluted Soils ............. 23

Tamas Komives and Gabor Gullner

4. Methods for Rhizoremediation Research: Approaches to Experimental

Design and Microbial Analysis ............................................................................. 33
Mary Beth Leigh

5. Constructed Wetlands for Phytoremediation: Rhizofiltration,

Phytostabilisation and Phytoextraction ................................................................... 57
Marinus L. Otte and Donna L. Jacob

6. Influence of Helophytes on Redox Reactions in their Rhizosphere ..................... 69

A. Wiessner, P. Kuschk, U. Kappelmeyer, O. Bederski, R.A. Müller
and M. Kästner

7. Exploitation of Fast Growing Trees in Metal Remediation ............................ 83

Pavel Tlustoš, Daniela Pavlíková, JiĜina Száková, Zuzana Fischerová
and JiĜí Balík

8. Using Hyperaccumulator Plants to Phytoextract Soil Cd ..................................103

Autumn S. Wang, Rufus L. Chaney, J. Scott Angle and Marla S. McIntosh

9. Enhanced Heavy Metal Phytoextraction.............................................................. 115

Domen Leštan

10. Enzymes Transferring Biomolecules to Organic Foreign Compounds:

A Role for Glucosyltransferase and Glutathione S-transferase in
Phytoremediation ..................................................................................................... 133
Peter Schröder

11. Phytoremediation of Polychlorinated Biphenyls ................................................. 143

Martina Mackova, Diane Barriault, Katerina Francova, Michel Sylvestre,
Monika Möder, Blanka Vrchotova, Petra Lovecka, Jitka Najmanová, Katerina
Demnerova, Martina Novakova, Jan Rezek and Tomas Macek
vi Table of Contents

12. Metabolism and Genetic Engineering Studies for Herbicide

Phytoremediation ..................................................................................................... 169
Melissa P. Mezzari and Jerald L. Schnoor

13. Pesticides Removal Using Plants: Phytodegradation Versus

Phytostimulation ...................................................................................................... 179
Jean-Paul Schwitzguébel, Joana Meyer and Petra Kidd

14. Phytoremediation of Volatile Organic Compounds ........................................... 199

Joel G. Burken and Xingmao Ma

15. In vitro Propagation of Wetland Monocots for Phytoremediation ............... 217

Mihály Czakó, Xianzhong Feng, Yuke He, Sharada Gollapudi and
László Márton

16. Modifying a Plant’s Response to Stress by Decreasing Ethylene

Production ................................................................................................................ 227
Bernard R. Glick

17. Mycorrhizal Fungi as Helping Agents in Phytoremediation

of Degraded and contaminated Soils.................................................................. 237
Miroslav Vosátka, Jana Rydlová, Radka Sudová and Martin Vohník

18. Assessing Risks and Containing or Mitigating Gene Flow

of Transgenic and Non-transgenic Phytoremediating Plants ........................... 259
Ton Rotteveel, Hani Al-Ahmad and Jonathan Gressel

19. Human Exposure Assessment for Food – One Equation for all
Crops is not Enough................................................................................................. 285
Stefan Trapp and Ales Kulhanek
INTRODUCTION

JOHN FLETCHER
University of Oklahoma, Department of Botany, Norman, USA

To appreciate the history of phytoremediation and its importance, it is helpful to start

with a brief review of common remediation strategies used around the world to treat soil
contaminated with toxic metals and/or organic chemicals. Three widely used strategies
are: 1) immobilization or retention of toxicants within a confined area (i.e. the soil at the
site of their release or in contaminated soil placed in a landfill, 2) removal of
contaminants from the soil, 3) destruction of organic pollutants by chemical, physical, or
biological means. These strategies either individually or in combination with each other
have been routinely implemented by the remediation industry to successfully treat
contaminated soil. Unfortunately, implementations have often required extensive earth
moving, expensive equipment, and costly construction; all features that have aroused
public resistance on occasion and have sometimes been tagged, rightly or wrongly, as
more threatening to the environment than the contaminants themselves. In any event,
public concern for the implementation features listed above have been instrumental in
keeping pressure on the remediation industry to develop more cost effective and friendly
methods, including bioremediation.
Bioremediation started over 50 years ago with research examining the fate of
pesticides in agricultural soils. In view of the wide range of catabolic reactions mediated
by bacterial enzymes, it is not surprising that from the beginning bioremediation
research focused on bacteria. The capacity for bacteria to degrade xenobiotics was so
impressive that other living organisms were virtually ignored for 30 years. As a result
bioremediation became thought of as degradation of organic contaminants by bacteria
even though the bio prefix suggested involvement of all life forms. Early investigators in
plant remediation work were confronted with an attitude held by some persons that if
remediation of a contaminant could not be achieved by bacteria with their diversified
array of catabolic enzymes it sure couldn’t be achieved with plants. This attitude is
perhaps why investigators striving to call attention to the unique remediation features of
plants felt obliged to establish a separate remediation field, phytoremediation, and
include several subdivisions (i.e. phytoextraction, rhizoremediation, etc.).
Unique remediation features possessed by plants are easily illustrated by returning to
the 3 common remediation strategies listed earlier to treat contaminated soil: 1)
immobilization, 2) removal, and 3) destruction. Partial immobilization of water soluble
contaminants is brought about by plant transpiration (soil water taken up, transported,
and evaporated from leaf surfaces) since the process removes soil water that would

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© 2006 Springer.
2 John Fletcher

otherwise cause contaminant leaching and movement. Removal of toxic metals from
contaminated soil occurs when inorganic ions are taken up by plant roots and
translocated through the stem to aboveground plant parts. Regarding contaminant
destruction, plants, because of their autotrophic nature, were rarely examined for
catabolic properties until phytoremediation emerged and studies conducted then with
nonphotosynthetic tissue culture cells and axenic roots clearly showed that plant
enzymes degrade some organic pollutants. The use of plants to foster the degradation or
organic soil contaminants has been further advanced by studies showing that soil
microflora under the chemical influence of plant roots (rhizosphere zone) can be
important in xenobiotic metabolism. The catabolic activity within the rhizosphere has
been attributed to both bacteria and fungi whose presence and enzymatic expression are
believed to be modulated by organic chemicals released from both living and dead roots.
Since both root physiology and biosynthetic pathways vary considerably among plant
species it is anticipated that rhizoremediation properties will also vary among plants.
The most useful species for rhizoremediation may be previously unexplored species with
no commercial importance prior to their use in rhizoremediation. Both the direct and
indirect degradation of soil contaminants can potentially occur at the lowest depth of
root penetration, a special feature of plant remediation. Thus, through the efforts of a
relatively small group of scientists working around the world over the last 20 years,
phytoremediation has become a well established, multifaceted technology capitalizing
on three plant properties: transpiration, ion uptake, and metabolism with the later having
both direct and indirect influences.
As phytoremediation technology has evolved it has become increasingly apparent
that no single plant species excels in all three plant remediation properties, nor does any
single species show maximum uptake of all toxic metals or foster degradation of all
organic contaminants. Therefore, successful treatment of soils with mixed waste requires
a combination of plant species with appropriate remediation properties, and also the
inclusion of plant species hosting rhizosphere communities (bacteria and fungi) active
against specific contaminants that are present. Thus, a major contribution that has
emerged from the field of phytoremediation is the biosystems approach to soil
remediation where the joint actions of several different organisms functioning in unison
or in sequence are used to treat contaminated soil. The concept of a plant driven
remediation system may be foreign in some remediation circles but it is certainly
consistent with the fundamental principals of terrestrial ecology where the central role of
plants as the primary producers and greatest users of water is well established.
Accepting the central role played by plants in biosystem remediation, raises many
unanswered questions on how to assemble and manage the most effective biosystem.
What are the best plant species to use since the physiology, biochemistry, and
rhizosphere of very few of the thousands of native species have been studied? How does
the microflora of a dead root and its degradative properties compare with that of a living
root? Are genetically altered organisms necessary to degrade some soil contaminants?
Do some plant roots release surfactants? Can plant species that move deep groundwater
Introduction 3

to surface root zones be capitalized on in rhizoremediation? Should plants be introduced

as single annual crops or as perennial communities? Should plant succession be
encouraged? As such questions are addressed and new remediation technology emerges
it is very likely that phytoremediation employing ecologically and physiologically sound
biosystems will be accepted as a necessary and first step in successful ecological
restoration of contaminated habitats.
THE CHEMICAL ECOLOGY OF POLLUTANT BIODEGRADATION

Bioremediation and phytoremediation from mechanistic and ecological perspectives

ANDREW C. SINGER
Centre for Ecology & Hydrology–Oxford, Mansfield Rd, Oxford OX1
3SR, United Kingdom, E-mail: [email protected]

1. Introduction

As the yachtswoman Dame Ellen MacArthur returned to the south coast of Britain in
early 2005 after a record 71-day solo circumnavigation of the globe on a trimaran, she
noted pointedly, “It’s funny when you smell the land and you have not smelled it for two
months”. MacArthur’s comment reflects the multitude of odours originating from terra
firma and highlights an important and underappreciated feature of our world – a
dizzying abundance and diversity of chemicals surround us and in some subtle, as well
as some very direct ways, dictate the actions and reactions of all life.
Among the numerous sources of chemicals in our environment, molecules of plant
origin are arguably the most abundant and best characterised. This chapter aims to
highlight the ecological functions of plant-derived chemicals and discuss their roles in
both multi-trophic interactions and (pollutant-degrading) enzyme evolution. Evidence to
support these positions has largely been generated in the past decade and will be
reviewed in the later part of the chapter.
Rhizodeposition, the release of carbon compounds from living plant roots into the
surrounding soil, is dominated by low molecular mass solutes such as sugars, amino
acids and organic acids. There are numerous studies which aim to understand the
regulation and ecological significance of rhizodeposition, for which the reader is
directed to three excellent reviews [1-3]. Although rhizodeposition plays a central role in
establishing and sustaining a soil system, this chapter will focus on a class of
compounds, secondary plant metabolites (SPMe), that are nearly four-orders of
magnitude more diverse than the typical rhizodeposits. Over 100 000 low-molecular-
mass SPMe have been described with an estimated 400 000 yet to be discovered [4].
Many of these SPMe contain one of the following chemical structural backbones:
isoprene, phenylpropene, alkaloid or fatty acid/polyketide (Figure 1) [5].

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© 2006 Springer.
6 Andrew C. Singer

O
N

N OH

Isoprene Phenylpropene Alkaloid Fatty acid/polyketide

Figure 1. Typical skeletal backbones for the majority of secondary plant metabolites.

Although referred to as “secondary metabolites”, implying a function of only secondary

importance to the plant, SPMe fulfill a range of vital functions: (1) antimicrobial
activity; (2) insect and microbial attraction; (3) insect and microbial deterrent; (4) plant-
plant signal; (5) stress response; and (6) germination and growth inhibition [6].
Volatile low-molecular mass SPMe, consisting of a range of functional groups
(hydrocarbons, alcohols, aldehydes, ketones, ethers and esters), are integral in how
plants interact with their environment. Volatile emissions from flowers and fruits, for
example, provide clues to animals, pollinators and seed disseminators, while those from
vegetative tissues contribute to plant defence systems by repelling microorganisms and
animals or attracting herbivore predators, thereby protecting the plant through tritrophic
interactions [7].

1.1. THE NATURE OF THE PROBLEM

The Twenty-Fourth Report by the Royal Commission on Environmental Pollution stated

that there are between 30 000 and 100 000 chemicals on the market [8]. Every year,
approaching 2000 novel xenobiotic chemicals are added to this list, the vast majority of
which have not been tested for even the most basic indications of environmental hazard.
It is now recognised that this policy has been responsible for a number of environmental
catastrophes such as: (1) reproduction failures in songbirds resulting from the
organochlorine pesticide 4,4’-(2,2,2-trichloroethane–1,1-diyl)bis(chlorobenzene) (DDT)
which was highlighted by Rachel Carson’s landmark book, Silent Spring in 1962 [9]; (2)
bioaccumulation of the organochlorine polychlorinated biphenyl (PCB) and
reproduction failures at all levels of the food web from fish to eagles and humans [10];
and (3) depletion of the ozone layer induced by the release of chlorofluorocarbons
(CFC) [11].
DDT, the chemical for which Carson is most noted for highlighting, was banned in
the United States at the end of 1972, eight years after her untimely death from cancer.
Although the DDT ban spread to many temperate countries, few tropical countries
acceded to the ban, largely due to the pesticide’s efficacy to control the spread of
malaria and other insect-borne diseases. DDT has been shown to dissipate much more
rapidly in tropical than temperate soils [12]. The mechanism for the latter is partly
attributed to increased temperature-mediated volatility, but more importantly increased
microbial biodegradation. The mechanisms underpinning chemical persistence in the
environment are complex but are thought to be heavily influenced by the rarity of the
chemical’s structure and substituents.
Chemical Ecology of Pollutant Biodegradation 7

My contention is that the chemical ecology of a site should also be considered as an

important variable in determining a chemical’s persistence. In this chapter, the term
chemical ecology is used as defined by the International Society of Chemical Ecology,
“the chemical mechanisms which help control intra- and interspecific interactions among
living beings”. Owing to key differences in the local chemical ecology, a recalcitrant
molecule in one locale might be readily biodegraded in another. In part, the local flora
provides the evolutionary mechanism for the development and modification of SPMe-
degrading enzymes in the metagenome, which, it is argued, is fortuitously responsible
for the presence of pollutant-degrading enzymes, a priori the chemicals synthesis by
chemists.

1.2. TRITROPHIC TRINITY

Although it has been widely proposed that pollutant-degrading enzymes evolved from
isozymes in response to industrial production and environmental release of xenobiotics,
the a priori existence of readily mutable pollutant-degrading isozymes remains largely
absent from the literature [13-16]. This chapter will contribute to the dialogue on the
source and developmental mechanism of these pollutant-degrading enzymes.
The evolution of plants and their natural enemies was, arguably, responsible for
generating much of the Earth’s biological diversity [17]. A corollary was proposed [18],
stating that the synergistic and antagonistic relationships between plants, micro-
organisms, and insects (P-M-I) are responsible for the diversity of SPMe. A second
corollary then suggested that the P-M-I tritrophic interactions serve as one of the main
driving forces of pollutant-degrading enzyme evolution [19].

1.2.1 Theories on the evolution of pollutant-degrading isozymes

In “The Fractal Geometry of Nature”, Mandelbrot highlights the fractal structure of
many natural systems [20]. In this chapter, catabolic enzymatic systems are proposed to
conform to a fractal architecture. Elucidation of the organisation and evolution of
catabolic systems will aid in the investigation into the origins of pollutant-degrading
enzymes.
The classic example of a fractal structure can be found in the form of a tree (Figure
2). The long tree trunk provides the foundation from which a repeating series of shorter
branches are serially connected. At the metaphorical “leaves” of the tree lie molecules
which necessitate an individualised enzymatic step before moving into more central
metabolic pathways located at the base of the tree (e.g. citrate cycle, glycolysis). Their
location in the periphery of the tree can be attributed to their relatively unusual chemical
structure, substituent, or both. Whereas chemicals metabolised in well connected, more
centralised locales of the tree (nearer the “trunk”), consist of relatively more common
chemical structures and substituents [21]. Elaborating upon Firn and Jones (2000), it is
proposed that substrate specificity of biodegradative enzymes is proportional to the
distance of the enzymatic reaction from the “leaves”, i.e. enzymes with a low substrate
specificity are located in the “leaves” while enzymes with a high substrate specificity are
typical of central metabolic enzymatic reactions (i.e. “trunk”). This metabolic
architecture can be helpful in developing a mechanistic understanding of the evolution of
8 Andrew C. Singer

pollutant-degrading enzymes. For example, in the event a microorganism encounters a

novel molecule, it might perish if the molecule is toxic, as are many SPMe, or a mutant
enzyme might emerge from the population, enabling the molecule’s detoxification or

Figure 2. Fractal structure of catabolic enzymatic system within microorganisms. Novel

xenobiotic and natural chemical structures are catabolised in the outer branches,
funnelling metabolites to more central, substrate specific enzymatic steps.

metabolism. The mutant enzyme would develop in the “leaves” as it is the location of
enzymes which are responsible for interacting with the environment (e.g. detoxification,
communication, assimilation). Moreover, as the novel chemical is metabolised it may
continue down the “tree” into central metabolic pathways, or it may persist without any
further metabolism [14, 22]. Persistent metabolites would be further evidence of
enzymatic activity in the “leaves”, as catabolism of molecules with relatively novel
structures or substituents: (1) might produce equally novel metabolites requiring
additional modification to indigenous enzymes for further metabolism; (2) require low
substrate specific enzymes for primary and secondary catabolism, which is a
characteristic of enzymes residing in the “leaves”.
Networked databases, such as MetaRouter and the University of Minnesota
Biocatalysis/Biodegradation Database, provide a useful framework to visualise pollutant
catabolic pathways (http://pdg.cnb.uam.es/MetaRouter/index.html) [23], UM-BBD;
http://umbbd.ahc.umn.edu/) [24] and discern their fractal, interwoven structure.
Integrating this information into a “suprametabolism” network (i.e. incorporating all
pathways), will enable predictions to be made on the fate of both current and future
environmental pollutants [22, 24].

2. Chemical ecology of pollutant degradation

In the early 1990’s, researchers began to theorise about the “natural substrate” of
pollutant-degrading enzymes. Among the first pollutants to be scrutinised were PCBs.
Higson [25] and Furukawa [26] postulated that lignin may be the natural substrate for
the PCB-degrading enzyme. Rhodococcus erythropolis TA421, a microorganism
isolated from a wood-feeding termite ecosystem, was shown to degrade the recalcitrant
Chemical Ecology of Pollutant Biodegradation 9

pollutant PCB [27, 28]. The association of TA421 with a wood-feeding termite provided
the researchers with an opportunity to link lignin-degrading ability with the capacity to
catabolise PCBs. Maeda et al., confirmed the presence of three PCB-degrading genes
(bphC) in TA421, each with a different, yet narrow, substrate specificity [27], which
might correlate with the three monomers of lignin (Figure 3). This, however, has yet to
be tested.

HO HO
O

O
Cl Cl

X Y
OH Cl Cl

A B C
Figure 3. Structure similarity between: (A) Lignin monomer structure, where (1) X = Y = H
(p-coumaryl alcohol), (2) X = OMe; Y = H (coniferyl alcohol), (3) X = Y = OMe (sinapyl
alcohol) [29]; (B) 2,4-dichlorobiphenyl, PCB congener; and (C) 2-4-
dichlorophenoxyacetate.

Among the first studies that specifically investigated the link between plant-derived
chemicals and pollutant remediation was that by Donnelly et al., The authors
demonstrated that a range of flavonoids could support the growth of PCB-degrading
microorganisms [30]. The best growth substrate and concentration were determined for
each of three PCB-degrading microorganisms: Ralstonia eutrophus strain H850;
Burkholderia cepacia LB400; and Corynebacterium sp. MB1. Each bacterium was
subjected to a congener depletion assay [31], which was designed to show in a 24-hour
period the extent of congener degradation after growth on a particular flavanoid.
Naringin proved the best growth substrate for H850 and supported its greatest metabolic
activity on PCBs. Myricetin induced the greatest PCB degradation by LB400, which
catabolised 16 of the 19 congeners tested. Strain MB1 degraded thirteen PCB congeners
in the presence of coumarin in excess of the biphenyl controls.
The researchers suggested that fine plant roots may ultimately serve as a “naturally
occurring injection system,” capable of dispensing phenolic plant-derived compounds
into the rhizosphere. They advocated the use of these exudates as a means to support the
growth of PCB-degrading microorganisms. A major step in the development of the
chemical ecology of pollution was provided by Focht (1995) who proposed that plant
terpenes, rather than biphenyl [32], might be the natural substrates for PCB catabolising
enzymes. Subsequent studies by Hernandez et al., showed that soils enriched with
10 Andrew C. Singer

orange peel, ivy leaves, pine needles or eucalyptus leaves resulted in 105 times more
biphenyl (unchlorinated PCB) utilizers (108 g–1) than their unsupplemented control (103
g–1), which suggested that terpenes found in these plants might be natural substrates for
biphenyl-utilizing bacteria. Notably, complete disappearance of Aroclor 1242 was
observed in soils amended with orange peel, ivy leaves, pine needles and eucalyptus
leaves [33]. The authors examined the efficacy of terpene-degrading isolates to
biotransform Aroclor 1242 in broth. Three bacteria isolated from the experimental soil
with the capacity to utilize cymene as a sole carbon source exhibited enhanced (20-80%)
transformation of Aroclor 1242, in comparison with glucose-grown cultures. Four of five
limonene-utilising isolates exhibited elevated (43-83%) Aroclor 1242 transformation
compared with the controls. In conclusion, the authors speculated that biphenyl may
provide soil microorganisms with a relatively labile source of carbon, which is rapidly
utilized by fast-growing soil microorganisms (copiotrophs). They argued that slow-
growing microorganisms (oligotrophs), which rely on low concentrations and slowly
delivered secondary plant metabolites, might be more effective in degrading PCBs [33].
Evidence for the interaction between pollutant degradation and the availability of labile
carbon sources is highlighted in several papers referenced in this chapter.
In a similar vein, Dzanto and Woolston, demonstrated removal of 10, 21 and 24%
PCB (Aroclor 1248) from soils supplemented with pine needles, biphenyl and orange
peel, respectively, compared with control soil, although the differences were not deemed
mathematically significant (P > 0.05) [34]. The authors suggested that further promotion
might be achieved by biostimulating the rhizosphere with specific inducing substrates
for the target pollutant. This approach has since been demonstrated successfully in two
classic studies by Narasimhan et al. [35] and Kupier et al. [36, 37], as discussed
elsewhere [19].
The first methodical screening and isolation of active inducing compounds in plants
was achieved by Gilbert and Crowley. The authors examined a number of plant extracts
(spearmint, pennyroyal, basil, barley, green bean, dill, avocado litter and garden
compost) to determine if any stimulated the degradation of 4-4’-dichlorobiphenyl by a
known PCB-degrading bacterium, Arthrobacter sp. strain B1B [38]. Spearmint extract
resulted in approximately 33% of the metabolites produced by the known PCB-inducing
compound, biphenyl. Subsequent analysis of this extract identified carvone as the
principal component responsible for catabolism induction. Ten terpenoids of similar
structure to carvone were assayed for their ability to induce PCB (50 mg l–1)
degradation: p-cymene, isoprene, (S)-(+)-carvone, (R)-(–)-carvone, (S)-(–)-limonene,
(R)-(+)-limonene, carvacrol, cumene, trans-cinnamic acid and thymol. These plant-
derived compounds are commonly found in dill and caraway seed, spearmint, pine
needles, citrus, juniper, oregano, thyme and numerous other aromatic plants. With the
exception of cumene, trans-cinnamic acid and thymol, all terpenes enhanced 4-4’-
dichlorobiphenyl metabolism, while p-cymene and isoprene accelerated catabolism in
comparison with biphenyl (P < 0.05). The workers highlighted that not only was PCB
degradation induced by nonaromatic compounds, but also among the most effective was
isoprene, which lacks a ring structure. It was proposed that the relatively high
antimicrobial activities of terpenes might induce a P450-like detoxification and
fortuitous degradation of the PCBs. Cytochrome P450 enzymes are a large family of
Chemical Ecology of Pollutant Biodegradation 11

enzymes that have been shown to oxidize terpenes, such as camphor (P450cam), as well
as pollutants, such as polycyclic aromatic hydrocarbons (PAHs; e.g. naphthalene and
pyrene [39]), chlorinated phenols [40], and biphenyls [41].
p-Cymene is among the more frequently investigated SPMe in pollutant-degradation
studies, and is arguably among the more effective. It is a natural aromatic hydrocarbon
that occurs in the oils of over 100 gymnospermic and angiospermic plants, including
eucalyptus, cumin, thymine, cypress, coriander, sage, star anise and cinnamon [42, 43].
Its efficacy might stem from: (1) its structural similarity to many pollutants (e.g. toluene,
xylene, ethylbenzene, biphenyl, chlorobenzene); and (2) a common evolutionary origin
of the genes encoding the catabolic pathways [43].
Encouraged by carvone induction of PCB degradation by Arthrobacter sp. strain
B1B, Park et al., demonstrated expression of the bphC gene (2,3-dihydroxybipheyl 1,2-
dioxygenase) in the PCB degrader Ralstonia eutrophus H850 following induction by
(R)-(–)-carvone (50 mg l–1) [44]. The researchers concluded that carvone might induce a
different degradative pathway, potentially generating different congener specificity to
that of biphenyl-induced cells. Jung et al., examined the efficacy of carvone or limonene
to induce the bphC gene of R. eutrophus H850 in soil. Although biphenyl was capable of
inducing the bphC gene up to 4 days after addition to the soil, neither carvone nor
limonene were able to maintain the induction [45]. The authors concluded that the
presence of potential inducing compounds in situ does not necessarily ensure that
induction will occur, and that a greater understanding of induction is needed before field
implementation [45].
Using a similar approach to Jung et al., Oh et al., examined the ability of terpene to
prolong the survival of a known PCB-degrading bacterium, Pseudomonas
pseudoalcaligenes KF707, in soil [46]. The addition of 50 mg l–1 p-cymene or 50 mg l–1
Į-terpinene increased KF707 survival by 10- to 100-fold compared with biphenyl-
supplemented and control mesocosms. Rhodococcus sp. strain T104, a PCB-degrading
bacterium, was shown to catabolise biphenyl as well as the SPMe limonene, cymene,
pinene and abietic acid as sole sources of carbon. Limonene was capable of inducing the
biphenyl degradation pathway. The bacterium contains three genes, T1, T3 and T5,
which potentially code for aromatic-degrading compounds. T1 was induced by limonene
and cymene, and to a much lower extent, biphenyl. Notably, glucose exerted a similar
degree of induction to that of limonene. Cymene was the strongest inducer of T3, while
limonene and cymene induced T5 more strongly than biphenyl and glucose [47-49]. Kim
et al., further demonstrated that T104 is responsible for three distinct catabolic pathways
for phenol, biphenyl and limonene the last of which can induce both the upper and lower
pathways for biphenyl degradation [49]. Therefore, the authors concluded that
microorganisms might harbour several mechanisms for degrading structurally similar
compounds [49].
Rhodococci play an important role in the carbon cycle due to their ability to degrade
many semi-recalcitrant organic compounds. One of the three linear plasmids from a
well-studied PCB-degrading bacterium, Rhodococcus sp. strain RHA1, was sequenced
to elucidate the number, structure and regulation of the open reading frames [50]. The
smallest of the linear plasmids is divided into three clusters, one of which contains
limonene degradation genes, which are potentially responsible for its ability to grow on
12 Andrew C. Singer

limonene, as well as carveol and carvone as sole sources of carbon. Interestingly, the
plasmid contains three cytochrome P450-encoding genes. Earlier, the bacterium had
been shown to possess multiple isozymes (three bph-type ring-hydroxylating
dioxygenases and seven bph-type ring cleavage enzymes) for PCB degradation. The
identification of aromatic- and terpene-degrading genes as well as cytochrome P450 on
the same plasmid suggests that the bacterium employs both broad and narrow substrate
range enzymes to: (a) make maximum utilization of available carbon sources,
particularly those deemed recalcitrant by specialised bacteria; and (b) detoxify
compounds that may associate with, otherwise, labile carbon sources. Thus, RHA1 is an
excellent model microorganism to examine the link between SPMe and pollutant
degradation.
Through the use of a chromosomally-encoded lacZ reporter, Master and Mohn
gained insight into the differential induction of bphA, the large subunit of the biphenyl
dioxygenase, in two PCB-degrading bacteria, Pseudomonas sp. strain Cam-1 and
Burkhoderia xenovorans LB400 [51]. The latter exhibited constitutive expression of bphA
in the presence of twelve different inducers, including many plant-derived compounds
such as pinene, limonene, cymene, cumene, carvone and salicylate. Due to its
constitutive PCB-degrading capacity, however, the authors suggested a cautious
interpretation of the efficacy of the inducing compounds. In contrast, the biphenyl-
induced strain Cam-1 demonstrated a bphA activity six times greater than the basal level
in cells at 30°C in the presence of pyruvate, indicating the need for induction prior to
bioaugmentation of PCB-contaminated soil. Of the twelve SPMe examined, only
salicylate induced Cam-1 bphA activity to levels greater than basal levels recorded for
pyruvate-exposed cells.
Tandlich et al., used carvone and limonene to stimulate biodegradation of Delor 103
(a commercial mixture of PCBs) by Pseudomonas stutzeri. An expansion of PCB
congener removal was achieved after supplementation with 10 mg l–1 carvone compared
to glucose-grown control cells [52]. It is interesting to note that the spectrum of
congeners degraded decreased with the addition of 20 mg l–1 carvone, which suggested
that terpene induction may be compound- and concentration-specific. Limonene and
glycerol-cultured cells increased the range of congeners degraded as well as the total
PCB catabolised compared to the controls. Increased congener depletions were recorded
with elevated limonene concentrations from 10 to 20 mg l–1, while a decline in PCB
degradation resulted with the co-addition of biphenyl and carvone or limonene.
Specifically, 90% of a tri-ortho-substituted PCB congener was removed by biphenyl-
induced cells while no removal was observed in the presence of carvone. Furthermore,
biodegradation in the presence of glycerol or xylose, with carvone or limonene addition,
increased the suite of congeners degraded.
Nishio et al., demonstrated the broad substrate specificity for p-cymene
monooxygenase (CMO) found in the soil microorganism Pseudomonas putida F1
(PpF1). The bacterium can grow on p-cymene as a sole carbon and energy source by
employing a different degradative pathway compared with cultivation on the structurally
similar pollutant, toluene. CMO was shown to actively biotransform 4-ethyltoluene,
styrene, m- and p-xylene, 4-chlorostyrene, 4-(methylthio)toluene, 3-chlorotoluene,
4-chlorotoluene, 4-fluorotoluene and 4-nitrotoluene [53]. Interestingly, the highest
Chemical Ecology of Pollutant Biodegradation 13

biotransformation rate was found not with cymene but with 4-chlorostyrene, which
shares the same chemical substructure with flavones such as anthocyanidin and
isoflavone as well as the lignin monomer p-coumaryl alcohol (Figure 4; [53]).

HO
O

Cl OH

A B C D E

Figure 4. Structural similarities are bolded between: (A) toluene (pollutant); (B) 4-
chlorostyrene (pollutant); (C) p-cymene (SPMe); (D) isoflavone (SPMe); and (E) p-
coumaryl alcohol – lignin monomer.

Qui et al., assessed the influence of the addition of two flavonoids, morin and flavone,
on benz[a]pyrene (B[a]P) degradation in rhizosphere soil [54]. The soils were exposed
to 0, 0.1, 1, 10, 100 µmoles of the flavonoids for 60 days. Both morin and flavone-
supplemented soils recorded decreased mineralization of 14C-B[a]P with flavonoid
concentrations as low as 10 µmoles. Flavone-supplemented soils lowered B[a]P
bioavailability as monitored by decreased recovery from serial extractions with hexane,
water and ethyl acetate. The researchers suggested that morin might have either inhibited
the enzyme system responsible for B[a]P degradation or was preferentially used as a
carbon source by the native B[a]P-degrading population. Due to a decrease in its
extractability, flavone might have stimulated B[a]P transformation only and not
mineralization, and so resulted in the sequestration, sorption or humification of the
metabolite. The authors supported the need for an understanding of the complicated and
potentially confounding effects of root exudation, secondary plant metabolite selection,
and the specific soil system on the rate and extent of pollutant degradation in soils
treated by phytoremediation [54].
Recent studies of the consumption of atmospheric methane in forest soils has
indicated that monoterpenes can inhibit methanotrophy (40-100%), with (–)-α-pinene
the most effective [55]. The degree of inhibition was found to be species and
monoterpene specific in mono-cultures (Methylosinus trichosporium OB3b), for
unsaturated, cyclic hydrocarbon forms such as (–)-α-pinene, (S)-(–)-limonene, (R)-(+)-
limonene and χ-terpinene) [56]. Amaral and Knowles applied an aqueous extract of two
depths of forest soil to examine if natural substrates inhibit methanotrophy [55]. They
observed a concentration-dependent and transient inhibition after the addition of 0-5 cm
14 Andrew C. Singer

depth soil extracts, whereas extracts from deeper soil (5-12 cm) proved non-inhibitory.
Consistent with the current literature, monoterpene depositions from plant leaves might
accumulate within the upper soil horizon and result in methanotrophy inhibition. Owing
to its global implications, this system provides an interesting and environmentally
important model to study the chemical ecology of terpene- and pollutant-degrading
genes.

2.1. STRUCTURAL- AND STEREO-ISOMERS

Many of the environmental pollutants controlled under international agreements, such as

the United Nations Economic Commission for Europe Persistent Organic Pollutants
Protocol (U.N.E.C.E. P.O.P.s Protocol) and the United Nations Environment
Programme PoPs Convention, are mixtures of structural- and stereoisomers (e.g. aldrin,
chlordane, dieldrin, DDT, heptachlor, hexabromobiphenyl, hexachlorocyclohexane,
PCBs, dioxin). Detailed investigation of differential biological activity on, and
biodegradation of, these complex isomeric mixtures of PoPs, universally demonstrates
highly variable activities and persistence [57]. Isomers are molecules with the same
chemical formula. Structural-isomers have different bonding patterns whereas stereo-
isomers have identical bonding patterns but differ only in the geometric position of the
bond. Hence, it is misleading to discuss the efficacy of a remediation approach when
addressing structural- or stereo-isomeric mixtures without acknowledging the potential
for differential isomeric activity. Similarly, when investigating the recalcitrance of
inducing pollutant degradation with SPMe, one must be cautious of the differential
effects of structural- and stereo-isomers. Two studies are presented here as evidence of
the differential effects of structural- and stereo-isomers in both the pollutant and the
secondary plant metabolite.
Strong evidence for the induction of alternative PCB catabolic pathways using SPMe
within many of the well-known PCB-degrading bacteria was demonstrated by Singer
et al. [58] through the use of stereoselective degradation. Five PCB-degrading bacteria,
Ralstonia eutrophus H850, Burkholderia xenovorans LB400 ([59]), Rhodococcus
globerulus P6, Rhodococcus sp. strain ACS and Arthrobacter sp. strain B1B were
assessed for their ability to differentially degrade four atropoisomeric PCBs (one
tetrachlorobiphenyl and three pentachlorobiphneyls). Catabolism was assessed for each
bacterium after growth on tryptic soy broth and in the presence of biphenyl, (S)-(+)-
carvone or p-cymene. Stereoselectivity varied with respect to strain, congener and co-
substrate. The authors concluded that the inducing compounds might facilitate
alternative PCB-degradation pathways within the bacterium, thereby accounting for the
observed stereoselective degradation pattern. The stereoselective degradative pattern for
each enzyme can exist owing to the enzyme and the chemicals’ chirality. Hence, changes
in metabolic pathway might be detectable through the use of chiral chemical substrates
as they might be differentially degraded by each enzymatic system.
Chemical Ecology of Pollutant Biodegradation 15

2.2. RHIZOSPHERE ECOLOGY

Yu et al., reported the recovery, by three to four orders of magnitude, of more resin acid
degraders (tricyclic terpenoids originating from softwood trees) in hydrocarbon-
contaminated soils than in pristine Arctic tundra soil [60]. Notably, the soil samples
were collected thousands of kilometres from the nearest source of resin acids (conifer
forest) and contained no native resin acids. The bacteria isolated in the study,
Pseudomonas and Sphingomonas, are hydrocarbons degraders, which suggested that
their ability to mineralise resin acid and xenobiotics may not be purely coincidental. The
results from Yu et al., were particularly interesting in light of a publication by Button
who discovered that over 10% of the bacteria in a litre of seawater near Seward, Alaska
(similar Arctic region to that studied by Yu et al.,), catabolised terpenes [61]. The author
postulated that very heavy precipitation on the conifer forest of the Pacific Northwest
carries the canopy drip and guttation fluid into the surface water and, ultimately, the sea.
However, due to the Alaska Coastal Current, the dissolved terpenes are carried into the
estuaries upstream, thereby sustaining a terpene-based food web [61]. The distribution
of large quantities of SPMe in the Arctic region may provide the elusive mechanism Yu
et al. [60] sought for the presence of resin acid (and hydrocarbon) degraders.

2.2.1 Induction by plant phenolics and root recycling

It has been proposed that fine plant root recycling can provide the stimulus needed to
sustain pollutant-degrading microorganisms in the rhizosphere [62, 63]. The researchers
demonstrated that a majority of fine roots (<1 mm diameter) from mulberry (Morus sp.)
die at the end of a 6-month growing season. Flavones, such as morusin, morusinol and
kuwanon C, contribute to approximately 4% of the fine root biomass (dry weight) after a
full growing season. The authors have demonstrated that a wide range of flavones
sustain the growth of the PCB-degrading bacterium Burkholderia xenovorans LB400
and concluded that a continual supply in the rhizosphere, through fine root recycling,
might facilitate the structure and function of the microbial populations facilitating
degradation of otherwise recalcitrant pollutants [30, 62]. The biphenyl dioxygenase of
Pseudomonas pseudoalcaligenes KF707, a well studied PCB-degrading microorganism,
has also been shown to catalyze both flavone and 5,7-dihydroxyflavone [64], and as
discussed earlier, was shown to exhibit protracted survival in soil supplemented with p-
cymene or Į-terpinene [46].
The fine root recycling hypothesis was evaluated by Parrish et al., who, following
application of an herbicide to kill the roots of fescue (Festuca arundinacea Schreb.) and
yellow sweet clover (Melilotus officinalis Lam.), assessed the rate and extent of PAH
degradation in the plant rhizospheres. Although they demonstrated differences in the
extent of PAH removal by the two species, there was no enhancement of PAH removal
due to “induced root death” [65].
Addressing a similar question, Shaw and Burns demonstrated that Trifolium pratense
exudates and a supplement of roots grown in non-sterile soil, increased the maximum
2,4-dichlorophenoxyacetic acid (2,4-D) degradation rate and decreased the lag time to
the maximal 2,4-D degradation rate [66]. Notably, both these promotions also resulted
16 Andrew C. Singer

with supplementation of autoclaved roots. Conversely, gnotobiotic hydroponic and sand-

grown roots did not increase the rate of 2,4-D degradation, which suggested that the
stimulatory component was both a function of the plant and cultivation medium. The
authors also found evidence that unfractionated legume rhizodeposits enhanced 2,4-D
mineralization. The implication was that flavonoids, as major signalling components of
the rhizobia-legume symbiosis [1], might select for microorganisms capable of
detoxifying and utilising the flavanoid signals or their metabolites [66]. For example,
cinnamic acid is one of the possible metabolites of flavanoid degradation and has been
shown to induce TfdA, the gene responsible for the first step of 2,4-D catabolism [66].
2,4-D is structurally analogous to p-coumaryl alcohol, a lignin monomer, which has been
proposed to be a natural inducer of PCB degradation (Figures 3 and 4) [26].

2.2.2 Salicylate
Akin to p-cymene, salicylate is another SPMe that has been studied extensively, not only
for its efficacy to stimulate pollutant degradation but also in relation to its role as a
plant-plant signalling compound [18]. Salicylate has been shown to induce biphenyl,
xylene and toluene degradation in Pseudomonas paucimobilis Q1 [67] and PAH
degradation in P. saccharophila P15 and P. putida 17484 and PpG1 [68-70]. Filonov
et al., demonstrated preferential expression of the ortho-pathway for catechol cleavage
(a metabolite of PAHs and salicylate), as well as the presence of silent genes for the
meta-catechol cleavage pathway. Recognition of this alternative pathway extends the
range of substrates utilized although at a potential cost of cell death if the microorganism
is exposed to particular halogenated isomers [71].
A considerable body of results has amassed that demonstrates the funnelling of PAH
metabolites through one of two intermediate pathways, salicylate and phthalate [72].
However, the discovery of naphthalene-, phenanthrene-, anthracene-, chrysene-,
fluorine-, pyrene-degrading bacteria, which do not grow on salicylate or phthalic acid,
suggests that a variety of pathways and inducers exist for the degradation of PAHs
[72-74].

2.2.3 Pollutant-degrading pathway repression

Rentz et al., examined the effect of hybrid willow (Salix alba × matsudana) root
exudates on the phenanthrene-degrading activity of P. putida 17484. Although salicylate
was expected to increase phenanthrene degradation, it was repressed by approximately
21% of its maximum [70]. The researchers concluded that the prevalence of alternative
carbon sources in the rhizosphere exerted catabolite repression [3]. However, in this and
a previous study with Pseudomonas fluorescens HK44, it was suggested that increased
numbers of total heterotrophs and pollutant-degrading bacteria, as well as increased
metabolic activity, can, potentially, compensate for catabolite repression [70, 75].
Global carbon source regulation was implicated by a decline in phenanthrene
degradation in cells exposed to 2.0 mM acetate, lactate, pyruvate, glucose and
glutamate. The amino acids aspartic acid and glutamate, quantified as up to 3.9% of the
total organic carbon of willow root exudates, might have contributed to the repression.
In previous studies, it has been demonstrated that the availability of amino acids in the
Chemical Ecology of Pollutant Biodegradation 17

concentration range of 0.001 to 0.1% can suppress the Pseudomonas-derived DmpR-Po

ı54-dependent regulatory system, and so delay expression of the (methyl)phenol
catabolic enzyme. The authors emphasised that the appropriate transcriptional response
to specific signals in their environment are contingent on the physiological status of the
cell [76,77]. Notably, the ı54 promoter for the toluene/xylene catabolic TOL plasmid has
also been shown to be growth-phase regulated in rich media [78]. Hence, the efficacy of
SPMe induction will likely be dependent on the availability of carbon sources (e.g.
amino acids) and the growth stage of the catabolic microorganism (e.g. stationary
phase). Coordinated expression of pollutant-degrading genes upon entry into stationary
phase was also demonstrated by Denef et al., in the PCB-degrading bacterium B.
xenovorans LB400 [79]. Rentz et al., were careful to note that root-derived substrate
repression is likely to vary among different microbial strains and plant species [70].
Yoshitomi and Shann confirmed this in a study that involved continuous application of
corn (Zea mays L.) root exudates to pyrene contaminated soil for 90 days. The
researchers observed enhanced pyrene mineralization in root-exudate supplemented as
compared with controls [80].

3. The tortoise and the hare: Exponential silencing

Repression of a microbial catabolic gene in log-phase growth on nutrient rich medium is

termed exponential silencing [81]. This phenomenon has been studied in only a few
microorganisms (Pseudomonas putida pWWO [82], Acinetobacter sp. ADP1 [83] and
Burkholderia xenovorans [79]). The insights gained from understanding this process
have immediate implications towards rhizostimulation and the chemical ecology of
pollutant remediation.
Exponential silencing might suggest that copiotrophic rhizosphere-competent
bacteria will preferentially exploit labile carbon substrates (e.g. pyruvate, malate, citrate,
succinate) before degrading less labile molecules, such as pollutants (e.g. toluene,
xylene, biphenyl). However, on entering stationary phase, the copiotroph experiences a
general stress response, which up-regulates ı54-dependent promoters and activates
enzymes (e.g. monooxygenases, dioxygenases) with broad substrate specificity thus
enabling utilization of semi-recalcitrant, lower energy-yielding carbon sources, which in
some cases, might (fortuitously) be a pollutant. Conversely, oligotrophs, which arguably
rely more on lower energy-yielding carbon sources might thereby avoid exponential
silencing and thereby carry out a significant proportion of what is termed “natural
attenuation”. In this way, the tortoise (oligotroph) could provide more extensive pollutant
removal than the hare (copiotrophs). If validated, laboratory studies demonstrating the
efficacy of copiotroph-mediated pollutant attenuation might be in vitro anomalies, unre-
presentative of complex soil microbial systems.
This chapter has highlighted the value of consolidating interdisciplinary knowledge
to generate new hypotheses for pollutant degradation. Due to the increasing literature
base in all fields of science, it is now possible (and necessary) to initiate interdisciplinary
collaboration between microbiology, ecology, biochemistry, botany and entomology, to
resolve this complex problem.
18 Andrew C. Singer

References
[1] Dakora, FD and Phillips, DA (2002) Root exudates as mediators of mineral acquisition in low-nutrient
environments. Plant Soil 245: 35-47
[2] Jones, DL; Hodge, A and Kuzyakov, Y (2004) Plant and mycorrhizal regulation of rhizodeposition. New
Phytol 163: 459-480
[3] Hutsch, BW; Augustin, J and Merbach, W (2002) Plant rhizodeposition - an important source for carbon
turnover in soils. J Plant Nutr Soil Sci - Z Pflanzenernaehr Bodenkd 165: 397-407
[4] Hadacek, F (2002) Secondary metabolites as plant traits: Current assessment and future perspectives.
CRC Crit Rev Plant Sci 21: 273-322
[5] Dixon, R (2001) Natural products and plant disease resistance. Nature (London) 411: 843-847
[6] Farmer, EE (2001) Surface-to-air signals. Nature (London) 411: 854-856
[7] Dudareva, N and Negre, F (2005) Practical applications of research into the regulation of plant volatile
emission. Curr Opin Plant Biol 8: 113-118
[8] Anonymous (2003) Chemicals in Products: Safeguarding the Environment and Human Health. Cm
5827, in Royal Commission on Environmetal Pollution, Twenty-Fourth Report
[9] Carson, R (1962) Silent Spring. Houghton Mifflin Company. New York
[10] Jensen, S (1966) Report of a new chemical hazard. New Scientist 32: 612
[11] Farman, JC; Gardiner, BG and Shanklin, JD (1985) Large losses of total ozone in Antarctica reveal
seasonal ClO and NOx interaction. Nature (London) 315: 207-210
[12] Rasche, ME; Hicks, RE; Hyman, MR and Arp, DJ (1990) Oxidation of monohalogenated ethanes and n-
chlorinated alkanes by whole cells of Nitrosomonas europaea. J Bacteriol 172: 5368-5373
[13] Wackett, LP (2003) Evolution of new enzymes and pathways: Soil microbes adapt to s-triazine
herbicides. 37-50, in MA Boston, JJ Gan, Eds, Pesticide Decontamination and Detoxification; American
Chemical Society, Washington DC
[14] Vandermeer, JR; Devos, WM; Harayama, S and Zehnder, AJB (1992) Molecular mechanisms of genetic
adaptation to xenobiotic compounds. Microbiol Rev 56: 677-694
[15] Vandermeer, JR (1994) Genetic adaptation of bacteria to chlorinated aromatic-compounds. FEMS
Microbiol Rev 15: 239-249
[16] Liu, S and Suflita, JM (1993) Ecology and evolution of microbial populations for bioremediation.
Trends Biotechnol 11: 344-352
[17] Rausher, MD (2001) Co-evolution and plant resistance to natural enemies. Nature (London) 411:
857-864
[18] Singer, AC; Crowley, DE and Thompson, IP (2003) Secondary plant metabolites in phytoremediation
and biotransformation. Trends Biotechnol 21: 123-130
[19] Singer, AC; Thompson, IP and Bailey, MJ (2004) The tritrophic trinity: A source of pollutant-degrading
enzymes and its implications for phytoremediation. Curr Opin Microbiol 7: 1-6
[20] Mandelbrot, BB (1982) The Fractal Geometry of Nature. WH Freeman. San Francisco, CA, USA
[21] Firn, RD and Jones, CG (2000) The evolution of secondary metabolism - a unifying model. Mol
Microbiol 37: 989-994
[22] Pazos, F; Valencia, A and De Lorenzo, V (2003) The organization of the microbial biodegradation
network from a systems-biology perspective. EMBO Reports 4: 994-999
[23] Pazos, F; Guijas, D; Valencia, A and De Lorenzo, V (2005) MetaRouter: Bioinformatics for
bioremediation. Nucleic Acids Res 33: D588-D592
[24] Ellis, LBM; Hou, BK; Kang, WJ and Wackett, LP (2003) The University of Minnesota
Biocatalysis/Biodegradation Database: Post-genomic data mining. Nucleic Acids Res 31: 262-265
[25] Adams, R; Huang, C; Higson, F; Brenner, V and Focht, D (1992) Construction of a 3-chlorobiphenyl-
utilizing recombinant from an intergeneric mating. Appl Environ Microbiol 58: 647-654
[26] Furukawa, K (1994) Molecular genetics and evolutionary relationship of PCB-degrading bacteria.
Biodegradation 5: 289-300
[27] Maeda, M; Chung, S-Y; Song, E and Kudo, T (1995) Multiple genes encoding 2,3-dihydoroxybiphenyl
1,2-dioxygenase in the Gram-positive polychlorinated biphenyl-degrading bacterium Rhodococcus
erythropolis TA421, isolated from a termite ecosystem. Appl Environ Microbiol 61: 549-555
[28] Chung, S-Y; Maeda, M; Song, E; Horikoshi, K and Kudo, T (1994) Isolation and characterization of a
Gram-positive polychlorinated biphenyl-degrading bacterium, Rhodococcus erythropolis strain TA421,
from a termite ecosystem. Biosci Biotechnol Biochem 58: 2111-2113
Chemical Ecology of Pollutant Biodegradation 19

[29] Burdon, J (2001) Are the traditional concepts of the structures of humic substances realistic. Soil Sci
166: 752-769
[30] Donnelly, PK; Hegde, RS and Fletcher, JS (1994) Growth of PCB-degrading bacteria on compounds
from photosynthetic plants. Chemosphere 28: 981-988
[31] Bedard, DL; Unterman, R; Bopp, LH; Brennan, MJ; Haberl, ML and Johnson, C (1986) Rapid assay for
screening and characterizing microorganisms for the ability to degrade polychlorinated biphenyls. Appl
Environ Microbiol 51: 761-768
[32] Focht, DD (1995) Strategies for the improvement of aerobic metabolism of polychlorinated biphenyls.
Curr Opin Biotechnol 6: 341-346
[33] Hernandez, BS; Koh, SC; Chial, M and Focht, DD (1997) Terpene-utilizing isolates and their relevance
to enhanced biotransformation of polychlorinated biphenyls in soil. Biodegradation 8: 153-158
[34] Dzantor, EK and Woolston, JE (2001) Enhancing dissipation of Aroclor 1248 (PCB) using substrate
amendment in rhizosphere soil. J Environ Sci Health 36: 1861-1871
[35] Narasimhan, K; Basheer, C; Bajic, VB and Swarup, S (2003) Enhancement of plant-microbe
interactions using a rhizosphere metabolomics-driven approach and its application in the removal of
polychlorinated biphenyls. Plant Physiol 132: 146-153
[36] Kuiper, I; Bloemberg, GV and Lugtenberg, BJJ (2001) Selection of a plant-bacterium pair as a novel
tool for rhizostimulation of polycyclic aromatic hydrocarbon-degrading bacteria. Mol Plant Microbe
Interact 14: 1197-1205
[37] Kuiper, I; Kravchenko, LV; Bloemberg, GV and Lugtenberg, BJJ (2002) Pseudomonas putida strain
PCL 1444, selected for efficient root colonization and naphthalene degradation, effectively utilizes root
exudate components. Mol Plant Microbe Interact 15: 734-741
[38] Gilbert, ES and Crowley, DE (1997) Plant compounds that induce polychlorinated biphenyl
biodegradation by Arthrobacter sp. strain B1B. Appl Environ Microbiol. 63: 1933-1938
[39] England, PA; Harford-Cross, CF; Stevenson, JA; Rouch, DA and Wong, LL (1998) The oxidation of
naphthalene and pyrene by cytochrome P450(cam). FEBS Lett 424: 271-274
[40] Jones, JP; O’Hare, EJ and Wong, LL (2001) Oxidation of polychlorinated benzenes by genetically
engineered CYP101 (cytochrome P450(cam)). Eur J Biochem 268: 1460-1467
[41] Wolkers, J; Burkow, IC; Lydersen, C; Dahle, S; Monshouwer, M and Witkamp, RF (1998) Congener
specific PCB and polychlorinated camphene (toxaphene) levels in Svalbard ringed seals (Phoca hispida)
in relation to sex, age, condition and cytochrome P450 enzyme activity. Sci Total Environ 216: 1-11
[42] Harms, G; Rabus, R and Widdel, F (1999) Anaerobic oxidation of the aromatic plant hydrocarbon
p-cymene by newly isolated denitrifying bacteria. Arch Microbiol 172: 303-312
[43] Eaton, R (1996) p-Cumate catabolism pathway in Pseudomonas putida F1: Cloning and
characterization of DNA carrrying the cmt operon. J Bacteriol 178: 1341-1362
[44] Park, YI; So, JS and Koh, SC (1999) Induction by carvone of the polychlorinated biphenyl (PCB)-
degradative pathway in Alcaligenes eutrophus H850 and its molecular monitoring. J Microbiol
Biotechnol 9: 804-810
[45] Jung, KJ; Kim, BH; Kim, E; So, JS and Koh, SC (2002) Monitoring expression of bphC gene from
Ralstonia eutropha H850 induced by plant terpenes in soil. J Microbiol 40: 340-343
[46] Oh, ET; Koh, SC; Kim, E; Ahn, YH and So, JS (2003) Plant terpenes enhance survivability of
polychlorinated biphenyl (PCB) degrading Pseudomonas pseudoalcaligenes KF707 labeled with gfp in
microcosms contaminated with PCB. J Microbiol Biotechnol 13: 463-468
[47] Choi, KY; Kim, D; Koh, SC; So, JS; Kim, JS and Kim, E (2004) Molecular cloning and identification of
a novel oxygenase gene specifically induced during the growth of Rhodococcus sp. strain T104 on
limonene. J Microbiol 42: 160-162
[48] Kim, D; Park, MJ; Koh, SC; So, JS and Kim, E (2002) Three separate pathways for the initial oxidation
of limonene, biphenyl, and phenol by Rhodococcus sp. strain T104. J Microbiol 40: 86-89
[49] Kim, BH; Oh, ET; So, JS; Ahn, Y and Koh, SC (2003) Plant terpene-induced expression of multiple
aromatic ring hydroxylation oxygenase genes in Rhodococcus sp. strain T104. J Microbiol 41: 349-352
[50] Warren, R; Hsiao, WWL; Kudo, H; Myhre, M; Dosanjh, M; Petrescu, A; Kobayashi, H; Shimizu, S;
Miyauchi, K; Masai, E, et al. (2004) Functional characterization of a catabolic plasmid from
polychlorinated-biphenyl-degrading Rhodococcus sp. Strain RHA1. J Bacteriol 186: 7783-7795
[51] Master, ER and Mohn, WW (2001) Induction of bphA, encoding biphenyl dioxygenase, in two
polychlorinated biphenyl-degrading bacteria, psychrotolerant Pseudomonas strain Cam-1 and
mesophilic Burkholderia strain LB400. Appl Environ Microbiol 67: 2669-2676
20 Andrew C. Singer

[52] Tandlich, R; Brezna, B and Dercova, K (2001) The effect of terpenes on the biodegradation of
polychlorinated biphenyls by Pseudomonas stutzeri. Chemosphere 44: 1547-1555
[53] Nishio, T; Patel, A; Wang, Y and Lau, PCK (2001) Biotransformations catalyzed by cloned p-cymene
monooxygenase from Pseudomonas putida F1. Appl Microbiol Biotechnol 55: 321-325
[54] Qiu, XJ; Reed, BE and Viadero, RC (2004) Effects of flavonoids on C-14 7,10 -benzo a pyrene
degradation in root zone soil. Environ Eng Sci 21: 637-646
[55] Amaral, JA and Knowles, R (1997) Inhibition of methane consumption in forest soils and pure cultures
of methanotrophs by aqueous forest soil extracts. Soil Biol Biochem 29: 1713-1720
[56] Amaral, JA; Ekins, A; Richards, SR and Knowles, R (1998) Effect of selected monoterpenes on methane
oxidation, denitrification, and aerobic metabolism by bacteria in pure culture. Appl Environ Microbiol
64: 520-525
[57] Kohler, H-PE; Angst, W; Giger, W; Kanz, C; Muller, S and Suter, MJF (1997) Environmental fate of
chiral pollutants-the necessity of considering stereochemistry. Chimia 51: 947-951
[58] Singer, AC; Wong, CS and Crowley, DE (2002) Differential enantioselective transformation of
atropisomeric polychlorinated biphenyls by multiple bacterial strains with different inducing
compounds. Appl Environ Microbiol 68: 5756-5759
[59] Goris, J; De Vos, P; Caballero-Mellado, J; Park, J; Falsen, E; Quensen, JF; Tiedje, JM and Vandamme,
P (2004) Classification of the biphenyl- and polychlorinated biphenyl-degrading strain LB400(T) and
relatives as Burkholderia xenovorans sp nov. Int J Syst Evol Microbiol 54: 1677-1681
[60] Yu, Z; Stewart, GR and Mohn, WW (2000) Apparent contradiction: Psychrotolerant bacteria from
hydrocarbon-contaminated Arctic tundra soils that degrade diterpenoids synthesized by trees. Appl
Environ Microbiol 66: 5148-5154
[61] Button, DK (1984) Evidence for a terpene-based food chain in the Gulf of Alaska. Appl Environ
Microbiol 48: 1004-1011
[62] Leigh, MB; Fletcher, JS; Fu, XO and Schmitz, FJ (2002) Root turnover: An important source of
microbial substrates in rhizosphere remediation of recalcitrant contaminants. Environ Sci Technol 36:
1579-1583
[63] Olson, PE; Wong, T; Leigh, MB and Fletcher, JS (2003) Allometric modeling of plant root growth and
its application in rhizosphere remediation of soil contaminants. Environ Sci Technol 37:638-643
[64] Kim, SY; Jung, JY; Lim, YH; Ahn, JH; Kim, SI and Hur, HG (2003) Cis-2’, 3’-dihydrodiol production
on flavone B-ring by biphenyl dioxygenase from Pseudomonas pseudoalcaligenes KF707 expressed in
Escherichia coli. Antonie Van Leeuwenhoek 84: 261-268
[65] Parrish, ZD; Banks, MK and Schwab, AP (2005) Effect of root death and decay on dissipation of
polycyclic aromatic hydrocarbons in the rhizosphere of yellow sweet clover and tall Fescue. J Environ
Qual 34: 207-216
[66] Shaw, LJ and Burns, RG (2005) Rhizodeposition and the enhanced mineralization of 2,4-
dichlorophenoxyacetic acid in soil from the Trifolium pratense rhizosphere. Environ Microbiol 7:
191-202
[67] Furukawa, K; Simon, JR and Chakrabarty, AM (1983) Common Induction and regulation of biphenyl,
xylene toluene, and salicylate catabolism in Pseudomonas paucimobilis. J Bacteriol 154: 1356-1362
[68] Yen, K-M and Gunsalus, IC (1982) Plasmid gene organization: Naphthalene/salicylate oxidation. Proc
Natl Acad Sci USA 79: 874-878
[69] Chen, S-H and Aitken, MD (1999) Salicylate stimulates the degradation of high-molecular weight
polycyclic aromatic hydrocarbons by Pseudomonas saccharophilia P15. Environ Sci Technol 33:
435-439
[70] Rentz, JA; Alvarez, PJJ and Schnoor, JL (2004) Repression of Pseudomonas putida phenanthrene-
degrading activity by plant root extracts and exudates. Environ Microbiol 6: 574-583
[71] Filonov, AE; Karpov, AV; Kosheleva, IA; Puntus, IF; Balashova, NV and Boronin, AM (2000) The
efficiency of salicylate utilization by Pseudomonas putida strains catabolizing naphthalene via different
biochemical pathways. Process Biochem 35: 983-987
[72] Bogan, BW; Lahner, LM and Paterek, JR (2001) Limited roles of salicylate and phthalate in bacterial
PAH bioremediation. Bioremed J 5: 93-100
[73] Uz, I; Duan, YP and Ogram, A (2000) Characterization of the naphthalene-degrading bacterium,
Rhodococcus opacus M213. FEMS Microbiol Lett 185: 231-238
Chemical Ecology of Pollutant Biodegradation 21

[74] Aitken, MD; Stringfellow, WT; Nagel, RD; Kazunga, C and Chen, SH (1998) Characteristics of
phenanthrene-degrading bacteria isolated from soils contaminated with polycyclic aromatic
hydrocarbons. Can J Microbiol 44: 743-752
[75] Kamath, R; Schnoor, JL and Alvarez, PJJ (2004) Effect of root-derived substrates on the expression of
nah-lux genes in Pseudomonas fluorescens HK44: Implications for PAH biodegradation in the
rhizosphere. Environ Sci Technol 38: 1740-1745
[76] Sze, CC and Shingler, V (1999) The alarmone (p)ppGpp mediates physiological-responsive control at
the sigma54-dependent Po promoter. Mol Microbiol 31: 1217-1228
[77] Sze, CC; Moore, T and Shingler, V (1996) Growth phase-dependent transcription of the sigma(54)-
dependent Po promoter controlling the Pseudomonas-derived (methyl)phenol dmp operon of pVI150. J
Bacteriol 178: 3727-3735
[78] Hugouvieuxcottepattat, N; Kohler, T; Rekik, M and Harayama, S (1990) Growth-phase-dependent
expression of the Pseudomonas putida Tol plasmid Pww0 catabolic genes. J Bacteriol 172: 6651-6660
[79] Denef, VJ; Park, J; Tsoi, TV; Rouillard, J-M; Zhang, H; Wibbenmeyer, JA; Verstraete, W; Gulari, E;
Hashsham, SA and Tiedje, JM (2004) Biphenyl and benzoate metabolism in a genomic context:
outlining genome-wide metabolic networks in Burkholderia xenovorans LB400. Appl Environ
Microbiol 70: 4961-4970
[80] Yoshitomi, KJ and Shann, JR (2001) Corn (Zea mays L.) root exudates and their impact on C-14-
pyrene mineralization. Soil Biol Biochem 33: 1769-1776
[81] Cases, I and de Lorenzo, V (2005) Promoters in the environment: Transcriptional regulation in its
natural context. Nat Rev Microbiol 3: 105-118
[82] Cases, I; deLorenzo, V and PerezMartin, J (1996) Involvement of sigma(54) in exponential silencing of
the Pseudomonas putida TOL plasmid Pu promoter. Mol Microbiol 19: 7-17
[83] Huang, WE (Personal Communication). Oxford, UK; 2005
DENDROREMEDIATION: THE USE OF TREES IN CLEANING
UP POLLUTED SOILS

TAMAS KOMIVES AND GABOR GULLNER

Plant Protection Institute of the Hungarian Academy of Sciences,
Herman Otto ut 15, 1022 Budapest, Hungary, FAX +36-1-4877555,
E-mail: [email protected]

1. Introduction

Forests have provided shelter and habitat for our ancestors for many millennia. During
historical times, man grew trees for a number of uses, including energy, furniture,
building material, production of paper, fruit and rubber, etc. Recently, trees were
introduced for use in dendroremediation, i.e. to depollute contaminated soils. The word
dendroremediation comes from the Ancient Greek dendron meaning “tree” and Latin
remediare meaning “reuse” [1-3]. Dendroremediation is an emerging phytoremediation
[4-7] technology for cleaning up environment contaminated with organic or inorganic
pollutants by using living trees to remove, sequester, or chemically decompose the
pollutant [1, 2].
From the point of view of dendroremediation a tree may be considered as a solar
driven pump-and-treat system, which may contain a contaminant plume and prevent the
spread of contamination by reducing the movement of contaminated water and the
erosional transport of contaminated soil. The efficiency dendroremediation has been
proven in cleaning up soils polluted with crude oil, explosives, landfill leachates, metals,
pesticides, polycyclic aromatic hydrocarbons, and solvents [1-7].
Trees are woody plants characterized with a large biomass, a permanent central self-
supporting stem, a stable root system, and a long lifespan. Trees are highly efficient
competitors for light, nutrients, and water and tend dominate the vegetation wherever
conditions are favourable for plant growth. Since trees are exposed to highly variable
biotic and abiotic stresses during their long lifespan they had evolved mechanisms to
cope with them. For example, formation of wood can be viewed as an adaptive
mechanism that enables trees to secure a dominant position in ecosystems. Wood has
many functions that may be important for efficient dendroremediation, e.g. water and
nutrient transport and storage of organic compounds and gases [8].
Dendroremediation considers the tree with its physical and biological environment
including the soil and the associated microflora [2]. Tree roots are know to produce and
release organic chemicals and create a rhizosphere zone more amenable to the microbes
that degrade the contaminant. Root exudates such as organic acids and ketones may

23
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 23 –31.
© 2006 Springer.
24 Tamas Komives and Gabor Gullner

promote microbial growth, as may the increase in soil organic matter caused by the
roots. Microorganisms fostered by trees in their root zone may contribute significantly to
the success of dendroremediation by enhancing the availability of the pollutant for
uptake by the plant root system, as well as by degrading some organic pollutants [9, 10].
Much is expected from a plant to be successful in dendroremediation. For efficient
uptake of the pollutant a large and deep penetrating root system and a high transpiration
rate is important. Large biomass producing, fast-growing, stress-tolerant trees are
preferred that are characterised with low nutrient and soil-quality requirement and
are capable to survive in a hostile environment and tolerate the phytotoxic effects of
the pollutants. In addition, feasible reproduction, propagation and production of the trees
are also highly important. Recently, the genera Salix (willows and osiers) and Populus
(i.e. poplars, including aspens and cottonwoods) have emerged as the most efficient
systems for dendroremediation [1-7]. Very importantly, the power of poplar as a model
system among tree species has been dramatically enhanced by the recent sequencing of
P. trichocarpa (black cottonwood) [11].

2. Uptake and translocation of the pollutants in trees

Although the binding of pollutant molecules to soil particles can be irreversible, usually
desorption occurs: pollutants may move with the soil solution and ultimately reach the
groundwater. Efficacy of dendroremediation strongly depends on the bioavailability of
the pollutant. Bioavailability is determined by the physical and chemical properties of
the pollutant, as well as those of the soil. Uptake of aqueous solutions of inorganic and
organic pollutants and their translocation within tree tissues are usually passive
processes, regulated by the water transport into the cells. Alternatively, they may also be
mediated by membrane-bound transporter systems [6]. Thus, uptake and translocation of
a pollutant in trees depends on the pollutant’s concentration in the soil solution, its
efficiency to enter the root system, and the rate of transpiration in the tree. Trees are
known to take up large amounts of water lost from the leaf surface in the transpiration
stream. For example, mature poplar trees can transpire 200–1000 liters of water per day
[12].

2.1. UPTAKE AND TRANSLOCATION OF INORGANIC POLLUTANTS

In soils metal ions are usually strongly bound to soil particles. To improve the
bioavailability of metal micronutrients trees have evolved several strategies [6], e.g.
producing and secreting metal-chelating chemicals which, by chelation, mobilize iron,
copper and zinc, as well as exuding protons in order to change the pH of the soil in the
root zone, thereby solubilising the soil-bound metal ions [13]. The physiological and
biochemical mechanisms that explain differences in metal mobility in trees are not well
understood [8]. Since in trees metals are transported through the xylem their mobility
towards the shoots may be strongly retarded by the high cation exchange capacity of the
xylem cell walls. As a result, anionic metal-chelate complexes are more efficiently
transported in the transpiration stream. Thus, in the practice of dendroremediation,
uptake and accumulation of metals in aerial tissues of plants can be enhanced through
The Use of Trees in Cleaning up Polluted Soils 25

the application synthetic and/or natural chelating amendments, such as EDTA and citric
acid to the soil [14, 15].

2.2. UPTAKE AND TRANSLOCATION OF ORGANIC POLLUTANTS

Physicochemical and structural properties determine the uptake of organic chemicals by

plant roots from the soil [16]. Aqueous solutions of moderately hydrophobic organic
chemicals (characterized with an octanol-water partition coefficient [log Kow] of 1.0-
3.5), such as low molecular weight aliphatics and aromatics, and chlorinated solvents
dissolved in water are readily taken up by roots of trees and translocated to the aerial
parts of the plant. Uptake of hydrophilic (log Kow < 1.0) and strongly hydrophobic (log
Kow > 3.5) compounds is much slower and they may be practically unavailable for
uptake because of their strong bonding to soil particles or to the roots of the tree. It is
interesting to note that efficiencies of uptake of organic pesticides into the crop plant
barley [17] and organic pollutants into poplars [16] are closely correlated. Although
bioavailability of organic contaminants is typically low when compared to water-soluble
inorganics, much less is known about the roles of amendments in the dendroremediation
of soils polluted with organic compounds. Thus, contamination by benzene and its alkyl-
derivatives (toluene, ethylbenzene, and xylenes) seems to be ideally suited for
dendroremediation. However, removal of these aromatics from soil is possible only by
increasing their apparent water-solubility. A new approach takes advantage of the ability
of cyclodextrins to increase the elution of organic compounds from soils. Cyclodextrins
have dual solubilising potency: they may act as surfactants as well as complexing agents
that form inclusion complexes with hydrophobic compounds [18].

3. Biotransformation of pollutants in trees

Plant tissues are capable of transforming pollutants by a wide variety of chemical/bio-

chemical metabolic reactions. Rate of metabolism of a pollutant is the main factor in
determining sensitivity/tolerance between plant species and has been found to play an
important role in the development of stress-resistant plants. Biotransformation reac-
tions of xenobiotics are generally referred to as Phases I and II, where Phase I in-
cludes oxidation of xenobiotics and Phase II deals with the conjugation of Phase I
products.
In trees, the oxidative metabolism in the Phase I system is usually mediated by
cytochrome P-450-containing mixed function oxygenases (CYP, E.C.1.14.-.-) [19, 20].
These enzymes support the oxidative, peroxidative and reductive metabolism of both
endogenous and xenobiotic substrates. They comprise a superfamily of heme-thiolate
proteins present in every class of organism, including Archaea, and in humans they are
responsible for 70-80% of all Phase I dependent metabolism of clinically used drugs
[21]. In plants there are a surprisingly high number of CYP genes: 246 in Arabidopsis
(representing approximately 1% of the plant’s gene complement) compared with less
than a 100 in humans [22]. CYP enzymes are characterized by the high diversity of
reactions that they catalyze and the high range of their chemically divergent substrates.
26 Tamas Komives and Gabor Gullner

Increasing emphasis on functional genomic approaches to CYP research recently has

greatly advanced our understanding of CYP-mediated reactions in plants [22].
In the Phase II systems hydrophobic xenobiotics functionalized by the Phase I
system are converted to more hydrophilic forms via conjugation with sugars or
sulfhydryl (-SH) group-containing tripeptides, such as glutathione (γ-L-glutamyl-L-
cysteinyl-glycine, GSH) [23, 24]. Since endogenous sulphhydryl group-containing
chemicals give protection against toxic metal ions as well as against alkylating organic
compounds, it is not surprising that GSH-homeostasis in trees is powerfully regulated by
pollutants such as the heavy metal cadmium, or the -SH reactive chloroacetanilide
herbicides [23].

Xenobiotic (hydrophobic)

Phase I

Functionalized derivative (less hydrophobic)

Phase II

Conjugate (hydrophilic)

Phase III

Terminal metabolite (compartmentised)

Figure 1. Metabolic scheme of xenobiotics in plants.

3.1. TRANSFORMATION PRODUCTS

Detailed information on the chemistry of transformation products of inorganic and

organic pollutants and metabolic pathways in susceptible and tolerant trees are scarcely
available. In tolerant trees heavy-metal ions may be detoxified via chemical
transformation into insoluble forms or chelated with cellular thiols or carboxylic acids
and are eventually sequestered into the cell vacuole as described in herbaceous plants
[8]. Of the different Phase II reactions that are most commonly involved in pollutant me-
tabolism in trees, conjugation with GSH is one of the most important reactions and often
the rate limiting step in the detoxification of an organic compound [23]. GSH trans-
ferases (GST, EC. 2.5.1.18) mediate the GSH-conjugation of chloroacetanilide
herbicides in poplar trees according to the reaction:
The Use of Trees in Cleaning up Polluted Soils 27

GST
GSH + X-R GS-R + XH

X-R = pollutant
GS-R = glutathione conjugate of the pollutant

Figure 2. Glutathione-conjugation reaction of a pollutant containing an X- leaving group.

GSTs represent a family of enzymes with usually broad and overlapping substrate
specificities, which facilitate the above reactions of hydrophobic, electrophilic
substrates. Our knowledge on plant GSTs in trees has expanded greatly in recent years.
Evidence is accumulating on the regulation of gene expression, molecular characte-
ristics, and specific catalytic action of the multiple forms of these enzymes. The
majority of the information on plant GSTs concerns enzymes which are involved in the
detoxification of a number of herbicides [23, 24], but evidence is gathering that plant
GSTs have a much wider role, and may be involved in general plant stress phenomena
[25]. Although the Phase II conjugation system is regarded as a detoxification process of
xenobiotics, GSH conjugates are not devoid of biological activity. Therefore, processes
reducing the concentration of GSH conjugates in the cytosol are important detoxification
steps [23].

3.2. BIOTRANSFORMATION OF INORGANIC POLLUTANTS

Because of their active metal-uptake systems plants are vulnerable to toxic levels of
metals in their rhizosphere. Plants have evolved several mechanisms to reduce high
concentrations of free metal ions in their tissues. These involve metal-chelate forma-
tion with amino acids and carboxylic acids, as well as with thiol-rich proteins
(metallothioneins, MTs) [26] and oligopeptides (phytochelatins, PCs) [27] followed by a
transport of the metal-ligand complex to a subcellular location or to a specific tissue of
the plant. MTs are gene-encoded, low-molecular-weight proteins rich in cysteine. They
are induced by copper and have high affinity for this metal [26]. PCs, on the other hand,
are cysteine-rich oligopeptides that are synthesized from GSH in an enzyme
(phytochelatin synthase, EC. 1.2.3.4) catalyzed reaction which is powerfully upregulated
by traces of heavy metal ions in the cytosol [27]. Plants can detoxify such metals and
metalloids as arsenic, chromium, mercury, and selenium by chemically reducing the
element and/or incorporation it into organic compounds [4].

3.3. BIOTRANSFORMATION OF ORGANIC POLLUTANTS

Petroleum products (such as o-xylene, Figure 3) and chlorinated organic solvents, such
as carbon tetrachloride, chloroform, and trichloroethylene (TCE, Figure 3) are used in
large quantities and are among the most common of the toxic substances found at
hazardous waste sites. Leaking underground gasoline storage tanks are the most
probable sources of groundwater contamination at numerous polluted sites. One major
component of gasoline is methyl-t-butyl ether or MTBE (Figure 3), a volatile, toxic
28 Tamas Komives and Gabor Gullner

chemical. Poplars were successfully used in the dendroremediation of these low-

molecular weight compounds [28, 29].

H3C
O Cl
Cl Cl NH O
N CH3 N N
N
O CH3 H3C NH N NH CH3 H3C H3C CH3

acetochlor atrazin chlortoluron

H3 C

CH3
O 2N NO2 CH3
CH3
H3C CH3 Cl Cl CH3

O
CH3 H Cl NO2

MTBE pyrene TCE TNT o-xylene

Figure 3. Chemical structures of acetochlor, atrazin, chlortoluron, MTB, pyrene, TCE,

TNT, and o-xylene.

Soil and groundwater contamination due to explosives such as glycerol trinitrate,

hexahydro-1,3,5-trinitro-1,3,5-triazine, and 2,4,6-trinitrotoluene (TNT, Figure 3) is a
problem at many ammunition plants. The presence of trees did enhance removal of these
explosives from groundwater [25-27].
Contamination of some soils with herbicides has become a serious environmental
problem. Chloroacetanilides, especially acetochlor (Figure 3), alachlor and metolachlor,
the chlorotriazine herbicide atrazin (Figure 3) and the urea-derivative chlortoluron
(Figure 3) are common contaminants in agricultural settings and at herbicide manu-
facturing sites [28-30]. Since poplar trees tolerate low concentrations of these herbi-
cides, dendroremediation is an attractive option to reduce their levels in the soil and in
the roundwater [28, 29].
Dendroremediation of pollutants that are highly phytotoxic (such as herbicides) or
highly persistent (such as polyaromatic hydrocarbons [PAHs], polychlorinated biphenyls
[PCBs], and dioxins) are most difficult. [31-34]. PAHs (for example pyrene [Figure 3]
and POPs are very hydrophobic. Therefore, their uptake by roots is very low. In
addition, chemically they are very stable: plant and microbial enzymes can degrade them
only very slowly. In spite of all the difficulties, introduction of xenobiotics-detoxifying
enzymes from other plants or from heterologous species (animals and bacteria) to trees
has been shown to enhance metabolism of organic pollutants. For example, poplar trees
expressing the mammalian cytochrome P450 2E1 also metabolized TCE at an elevated
rate [35]. Poplars, containing the bacterial γ-EC synthase had increased ability to
tolerate and detoxify chloroacetanilide herbicides [23].
The Use of Trees in Cleaning up Polluted Soils 29

Plants lack the excretion system of animals. In plant cells toxic metabolites and
pollutants are sequestered into the vacuole. This Phase III type process is an active one
and is catalyzed by membrane-bound ATP-driven pumps. A recent study indicated the
existence of a Phase III system in poplar [36].

4. Detoxification of the active oxygen species generated by the pollutant in trees

Activated oxygen species (hydrogen peroxide, superoxide anion, and hydroxyl radical)
are produced at various electron transfer sites or via different oxidation reactions in tree
tissues [37]. Under chemical stress conditions production of these radicals is powerfully
enhanced. For example, cadmium-induced inhibition of ascorbate peroxidase (E.C.
1.11.1.11) and catalase (E.C. 1.11.1.6) was also associated with H2O2 accumulation and
growth retardation in the poplar roots [38].
Trees contain a variety of defences to protect against the damaging effects of oxygen
radicals that are produced. It has been shown that a critical balance exists between
oxyradical-generating factors and the activity of the systems that protect the cell from
their harmful effects. Antioxidant defences belong to three general classes including:
• water-soluble reductants, e.g. compounds that contain thiol-groups
(cysteine, GSH, etc.), ascorbate and catechols;
• lipid-soluble compounds, e.g. Ȗ-tocopherol and ȕ-carotene; and
• enzymatic antioxidants, e.g. GSH peroxidase (GP, E.C. 6.4.11.6), ascor-
bate peroxidase, catalase, and superoxide dismutase (E.C. 1.15. 1.1) [39].
Microsomal and cytosolic GST enzymes in mammals may act as GP by catalyzing the
reaction between GSH and lipophilic hydroperoxides (Figure 4) thereby protecting cell
membrane polyunsaturated fatty acid moieties against lipid peroxidation. Poplar trees
were recently shown to contain a number of GST isozymes [36], including those capable
of detoxifying lipid hydroperoxides.

GST
GSH + ROOH GS-SG + ROH + H2O

ROOH = lipid hydroperoxide

ROH = lipid alcohol

Figure 4. Glutathione-peroxidase activity of plant GST enzymes.

4. Conclusions

Trees are well suited to be the key plants for phytoremediation – a vehicle with which
the growing needs to depollute agricultural, industrial, military and communal sites can
be fulfilled. Poplars seem to have many advantages as the first choice for the above
purpose but continued research is necessary to fully exploit their potential. This can be
achieved if simple, standardized methods for characterizing clone properties useful for
the dendroremediation of a range of inorganic and organic pollutants alone or in
30 Tamas Komives and Gabor Gullner

mixtures are developed and a functional analysis of the genes that are most important for
tree growth, development, and stress tolerance will be available. It is also critical to
identify and characterize the genes that are involved in the uptake, translocation and
detoxification (accumulation) of pollutants in plants to make dendroremediation
applicable in under various environmental conditions. Finally, further research is
necessary to clarify the roles woody tissues play in adaptation of trees to environmental
stress.

References
[1] Dickmann, DI; Nguyen, PV and Pregitzer, KS (1996) Effects of irrigation and coppicing on above-
ground growth, physiology, and fine-root dynamics of two field-grown hybrid poplar clones. J Forest
Ecology and Management 80: 163-174
[2] Schoenmuth, BW and Pestemer, W (2004) Dendroremediation of trinitrotoluene (TNT). Part 1:
Literature overview and research concept. Environ Sci Pollut Res 11: 273-278
[3] Schoenmuth, BW and Pestemer, W (2004) Dendroremediation of trinitrotoluene (TNT). Part 2: Fate of
TNT in morphological compartments of trees. Environ Sci Pollut Res 11: 331-339
[4] Komives, T and Gullner, G (2000) Phytoremediation, in: RE Wilkinson, Ed., Plant-Environment
Interactions, pp. 437-452. Marcel Dekker Inc., New York, U.S.A.
[5] Macek, T; Mackova, M and Kas, J (2000) Exploitation of plants for the removal of organics in
environmental remediation. Biotechnol Advances 18: 23-35
[6] Pilon-Smits, EAH (2005) Phytoremediation. Ann Rev Plant Biol 56: 15-39
[7] Dietz, AC and Schnoor, JL (2001) Advances in phytoremediation. Environmental Health Perspectives,
109: 163-168
[8] Pulford, ID and Watson, C (2003) Phytoremediation of heavy metal-contaminated land by trees - a
review. Environ Int 29: 529-540
[9] Tesar, M; Reichenbauer, TG and Sessitsch, A (2002) Bacterial rhizosphere populations of black poplar
and herbal plants to be used for phytoremediation of diesel fuel. Soil Biol Biochem 34: 1883-1892
[10] Jordahl, JL; Foster, L; Schnoor, JL and Alvarez, PJJ (1997) Effect of hybrid poplar trees on microbial
populations important to hazardous waste bioremediation. Environ Toxicol Chem16: 1318-1321
[11] Brunner, AM; Busov, VB and Strauss, SH (2004) Poplar genome sequence: functional genomics in an
ecologically dominant plant species. Trends in Plant Science 9: 49-55
[12] Wullschleger, S; Meinzer, F and Vertessy, RA (1998) A review of whole-plant water use studies in trees.
Tree Physiol 18: 499-512
[13] Robinson, BH; Mills, TM; Petit, D; Fung, LE; Green, SR and Clothier, BE (2000) Natural and induced
cadmium-accumulation in poplar and willow: Implications for phytoremediation, Plant and Soil 227:
301-306
[14] Fodor, F; Gaspar, L; Morales, F; Gogorcena, Y; Lucena, JJ; Cseh, E; Kropfl, K; Abadia, J and Sárvári, E
(2005) Effects of two iron sources on iron and cadmium allocation in poplar (Populus alba) plants
exposed to cadmium. Tree Physiology 25: 1173-1180
[15] Liphadzi, MS; Kirkham, MB; Mankin, KR and Paulsen, GM (2003) EDTA-assisted heavy-metal uptake
by poplar and sunflower grown at a long-term sewage-sludge farm. Plant and Soil 257: 171-182
[16] Burken, JG and Schnoor, JL (1998) Predictive relationships for uptake of organic contaminants by
hybrid poplar trees. Environ Sci Technol 32: 3379-3385
[17] Briggs, GG; Bromilow, RH and Evans, AA (1982) Relationship between lipophilicity and root uptake
and translocation of nonionized chemicals by barley. Pestic Sci 13: 495-504
[18] Tanada, S; Nakamura, T; Kawasaki, N; Torii, Y and Kitayama, S (1999) Removal of aromatic
hydrocarbon compounds by hydroxypropyl-cyclodextrin. J Colloid Interface Sci 217: 417-419
[19] Komives, T and Gullner, G (2005) Phase I xenobiotic metabolic systems in plants. Z Naturforsch C 60:
179-185
[20] Coleman, JOD; Frova, C; Schroder, P and Tissut, M (2002) Exploiting plant metabolism for the
phytoremediation of persistent herbicides. Environ Sci Pollut Res Int 9: 18-28
The Use of Trees in Cleaning up Polluted Soils 31

[21] Danielson, PB (2002) The cytochrome P450 superfamily: biochemistry, evolution and drug metabolism
in humans. Current Drug Metab 3: 561-97
[22] Nelson, DR; Schuler, MA; Paquette SM, Werck-Reichhart, D and Bak, S (2004), Comparative genomics
of rice and arabidopsis. Analysis of 727 cytochrome P450 genes and pseudogenes from a monocot and a
dicot. Plant Physiol 135: 756-772
[23] Gullner, G; Komives, T and Rennenberg, H (2001) Enhanced tolerance of transgenic poplar plants
overexpressing Ȗ-glutamylcysteine synthase towards chloroacetanilide herbicides. J Exp Bot 52: 971-979
[24] Schroeder, P and Collins, C (2002) Conjugating enzymes involved in xenobiotic metabolism of organic
xenobiotics in plants. Int J Phytoremediation 4: 247-265
[25] Tausz, M; Gullner, G; Komives, T and Gril, D (2003) The role of thiols in plant adaptation to
environmental stress, in Sulphur in Plants, Abrol,YP and Ahmad, A, Eds., Kluwer Academic Publishers,
Dordrecht, The Netherlands. pp. 221-244
[26] Kohler, A; Blaudez, D; Chalot, M and Martin, F (2004) Cloning and expression of multiple
metallothioneins from hybrid poplar. New Phytologist 164: 83-93
[27] Cobbett, CS (2000). Phytochelatins and their roles in heavy metal detoxification. Plant Physiol 123:
825-832
[28] Hong, MS; Farmayan, WF; Dortch, IJ; Chiang, CY; McMillan, S. and Schnoor, JL (2001)
Phytoremediation of MTBE from a groundwater plume. Environ Sci Technol 35: 1231-1239
[29] Gordon, M; Choe, N; Duffy, J; Ekuan, G; Heilman, P; Muiznieks, I; Ruszaj, M; Shurtleff, BB; Strand,
S; Wilmoth, J and Newman, LA (1998) Phytoremediation of trichloroethylene with hybrid poplars.
Environ Health Perspect 106: 1001-1004
[25] Thompson, PL; Ramer, LA and Schnoor, JL (1998) Uptake and transformation of TNT by hybrid poplar
trees. Environ Sci Technol 32: 975-980
[26] Thompson, PL; Ramer, LA and Schnoor, JL (1999) Hexahydro-1,3,5-trinitro-1,3,5-triazine translocation
in hybrid poplar trees. Environ Toxicol Chem 18: 279-284
[27] Yoon, JM; Oh, BT; Just, CL and Schnoor, JL (2002) Uptake and leaching of octahydro-1,3,5,7-
tetranitro-1,3,5,7-tetrazocine by hybrid poplar trees. Environ Sci Technol 36: 4649-55
[28] Komives, T; Gullner, G; Rennenberg, H and Casida, JEC (2003) Ability of poplar (Populus spp.) to
detoxify chloroacetanilide herbicides. Water, Air and Soil Pollution: Focus. 3: 277-283
[29] Burken, JG and Schnoor, JL (1997) Uptake and metabolism of atrazine by poplar trees. Environ Sci
Technol 31: 1399-1406
[30] Mezzari, MP; Walters, K; Jelinkova, M, Shih, M-C; Just, CL and Schnoor JL (2005) Gene expression
and microscopic analysis of Arabidopsis exposed to chloroacetanilide herbicides and explosive
compounds. A phytoremediation approach. Plant Physiol 138: 858-869
[31] Liste, HH and Alexander, M (2000) Plant-promoted pyrene degradation in soil. Chemosphere 40: 7-10
[32] Francova, K; Mackova, M; Macek, T and Sylvestre, M (2004) Ability of bacterial biphenyl
dioxygenases from Burkholderia sp. LB400 and Comamonas testosteroni B-356 to catalyse oxygenation
of ortho-hydroxychlorobiphenyls formed from PCBs by plants. Environmental Pollution, 127: 41-48
[33] Campanella, B; Bock, C and Schroder, P (2002) Phytoremediation to increase the degradation of PCBs
and PCDD/Fs. Environ Sci Pollut Res 9: 73-85
[34] Macek, T; Sura, M; Pavlikova, D; Francova, K; Scouten, WH; Szekeres, M; Sylvestre, M and Mackova,
M (2005) Can tobacco have a potentially beneficial effect to our health? Z Naturforsch C 60: 292-299
[35] Strand, SE; Dossett, M; Harris, C; Wang, X and Doty, SL (2005) Mass balance studies of volatile
chlorinated hydrocarbon phytoremediation Z Naturforsch C 60: 325-330
[36] Rishi, AS; Munir, S; Kapur, V; Nelson, ND and Goyal, A (2004) Identification and analysis of safener-
inducible expressed sequence tags in Populus using a cDNA microarray. Planta 220: 296-306
[37] Noctor, G; Arisi, A-C M., Jouanin, L; Kunert, KJ; Rennenberg, H and Foyer, CH (1998) Glutathione:
biosynthesis, metabolism and relationship to stress tolerance explored in transformed plants. J Exp
Botany 49: 623-47
[38] Schutzendubel. A; Nikolova, P; Rudolf, C and Polle, A (2002) Cadmium and H2O2-induced oxidative
stress in Populus x canescens roots. Plant Physiol Biochem 40: 577-584
[39] Komives, T; Gullner, G and Kiraly, Z (1997) The ascorbate-glutathione cycle and oxidative stresses in
plants, in KK Hatzios, Ed., Regulation of Enzymatic Systems Detoxifying Xenobiotics in Plants,
Dordrecht: Kluwer Academic Publishers, pp. 85-96
METHODS FOR RHIZOREMEDIATION RESEARCH

Approaches to experimental design and microbial analysis

MARY BETH LEIGH

Center for Microbial Ecology, Michigan State University, 540 Plant and
Soil Sciences Building, East Lansing, MI 48824-1325 USA,
Fax:(517)353-2917, E-mail: [email protected]

1. Introduction

Rhizoremediation is an elegant form of bioremediation that seeks to harness light energy

via plants to biostimulate pollutant degradation by the indigenous soil microbial
community. In pursuit of this goal, rhizoremediation draws upon the fields of
rhizosphere ecology and microbial biodegradation, and in doing so confronts many of
the same questions that have challenged these fields for decades, including: What
mechanisms dictate microbial community structure and activity in the rhizosphere?
Which microorganisms and degradative genes are actively involved in biodegradation,
and how does their activity respond to different treatments? One major obstacle to
answering these fundamental mechanistic questions over the years, and subsequently
developing successful rhizoremediation technology, has been our limited methodo-
logical capabilities to quantify the degradative potential of microbial communities and to
demonstrate and understand their response to the unique and multi-faceted environment
of the plant-soil interface.
Microbiological research methods are the windows through we view the hidden
world of microorganisms, with each being of different size and dimension, yet none
affording a complete view of the diversity, function and abundance of microbial
populations within a community. Cultivation methods are notoriously limited in their
ability to detect the uncultivable majority of organisms in the environment, yet remain
informative and indispensable for studying the metabolic capabilities of particular
strains. Molecular biological methods like functional gene detection and stable isotope
probing open much larger windows by circumventing culture bias and allowing the
direct examination of microbial populations important to bioremediation, although they
are limited somewhat by available sequence data. One aim of this chapter is to provide
an overview of microbial methodologies, both cultivation-dependent and independent,
which have already or are likely to provide new insight into the multitude of mechanistic
and applied rhizoremediation questions to be answered.

33
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 33– 55.
© 2006 Springer.
34 Mary Beth Leigh

Microbial analyses are most valuable to rhizoremediation when conducted within the
context of comprehensive, carefully designed experiments to evaluate the effectiveness
of different plant species and to uncover the mechanisms that drive rhizodegradation.
Accomplishing conclusive, informative experiments can be a challenging task in light of
the extraordinary heterogeneity of soil and rhizosphere systems and the long-term nature
of rhizostimulation. This chapter considers a variety of different experimental app-
roaches, ranging from controlled greenhouse pot experiments to forensic field studies,
and discusses the relationship between the hypothesized mechanism of rhizostimula-
tion and selection of the appropriate experimental system.
While this overview is primarily drawn from rhizoremediation studies of organic
aromatic pollutants like polychlorinated biphenyls (PCBs) and polyaromatic hydro-
carbons (PAHs), the guidelines are easily adaptable to many other organic contami-
nants. Together, the experimental design strategies and microbial analytical methods
discussed are aimed at assisting researchers in the creation of studies that will success-
fully answer an array of rhizoremediation questions, both mechanistic and applied.

2. Experimental approaches and design

When planning a study to assess the influence of plants on pollutant degradation,

selecting the appropriate experimental approach is critical. Will conclusive results best
be achieved by pot studies, field plots or less conventional approaches like forensic field
studies of long-term sites? The answer lies in part in the mechanisms and time scale by
which detectable rhizostimulation and/or contaminant disappearance is hypothesized to
occur. In this section, the benefits and drawbacks of different experimental approaches
are discussed and guidelines are provided to assist in their successful implementation.

2.1. FORENSIC FIELD STUDIES

While conventional wisdom may dictate a progression from bench to greenhouse to

field-testing of remedial technologies, there are distinct advantages to starting directly in
the field to examine the rhizoremediation capabilities of natural vegetation. This
approach affords a view of the long-term impacts of a diverse array of plant species,
including mature trees, on microbial communities and/or contaminant disappearance
otherwise inaccessible due to the practical time limitations of most planted studies.
Forensic studies are also invaluable as a means of screening species for their ability to
prosper in contaminated soil and under environmental stresses (i.e. drought and nutrient
stress) without active cultivation. However, the numerous abiotic heterogeneities and
lack of controlled conditions at natural field sites can make determinations of true
rhizoremediation effects challenging.
The forensic approach was first applied by Olson et al. [1-4] at a naturally-vegetated
PAH-contaminated sludge disposal basin in Texas City, Texas. Historical aerial
photographs and tree ring analyses revealed that plants colonized the basin over the
course of 16 years following the drainage of standing water. Through plant invasion and
succession, a diverse plant community (51 species and 22 families) developed that was
Methods for Rhizoremediation Research 35

dominated by mulberry trees (Morus spp.), Bermuda grass (Cynodon dactylon) and
common sunflowers (Helianthus annuus). In the absence of baseline data regarding
original contaminant levels, PAH concentrations beneath the root zone were considered
representative of the parent sludge which averaged 16,854 mg/kg total PAHs. Coring,
excavation and chemical analyses throughout the site indicated that PAH concentrations
in the root zones of plants were reduced to 10-50% of that in the original sludge.
Microbial analyses indicated that cultivable PAH utilizing bacteria were five-fold more
numerous (p < 0.01) in the mulberry root zone than in non-rooted samples of the same
depth, and these results were confirmed with real-time PCR quantitation of degradative
genes [5-8]. The close correspondence between root zone depth, sludge PAH
concentration and abundance of PAH-degrading bacteria suggests that plants and
associated microflora facilitated pollutant degradation.
Areas with deep and relatively homogeneous contamination like the sludge basin in
Texas City are fortuitous opportunities for forensic rhizoremediation studies based on
chemical disappearance. However in many cases contaminated field sites are a result of
heterogeneous surface spillage of chemicals, making a forensic determination of
chemical disappearance nearly impossible. Even in the absence of chemical
disappearance data, naturally vegetated contaminated sites afford a good opportunity to
screen various plant species for their tolerance to contaminants and their long-term
influence on the degradative potential of the associated microbial community.
A forensic field study focusing on rhizosphere microbial populations was performed
in a PCB contaminated site near Uherské Hradiste in South Moravia, Czech Republic [9-
11]. Accidental spillage of PCBs onto the soil occurred from the mid 1950s to the mid
1980s, resulting in a heterogeneously contaminated area with soil PCB concentrations
ranging from 1-500 mg/kg. The site was naturally vegetated with 25 different plant
species, including 5 tree species, all rooted directly in contaminated soil of varying
concentrations. The abundance and identity of cultivable PCB-metabolising bacteria
were determined in soil and rhizosphere samples collected at various depths beneath the
trees, grasses and forbs on four different dates during one year. In order to distinguish
effects of plants from abiotic factors on microbial populations, PCB concentration and
soil moisture content were also analysed but were not found to significantly correlate
with numbers of PCB-degrading bacteria. Austrian pine (Pinus nigra) and goat willow
(Salix caprea) trees fostered significantly higher numbers of cultivable PCB-
metabolising bacteria in their root zones than other plant species or non-rooted soil of
equivalent depth (collected beneath shallow-rooted grasses). The results imply that long
term growth of certain plant species can increase numbers of contaminant degraders in
the bulk soil.
Forensic field studies, by virtue of their scale and scope, are large interdisciplinary
undertakings that incorporate plant taxonomy and ecology, soil biochemistry,
soil/rhizosphere microbiology and complex statistical analyses. If planning to embark on
a forensic field study, is it recommended that the assistance of collaborators with
expertise in these fields be sought early in the process. As much historical information
as possible should be collected regarding the time of contamination, previous
measurements of concentration, presence of co-contaminants and vegetation history. A
comprehensive sampling strategy should be planned (see Sampling Methods section)
36 Mary Beth Leigh

and discussed in depth with an ecological statistician. A particular challenge in forensic

studies is distinguishing true plant-mediated effects from those created by numerous
abiotic heterogeneities. Multiple measurements (i.e. contaminant concentration, soil
moisture, pH, nutrients, depth, etc.) should be made of each sample and subjected to
statistical analyses along with microbial population data. Multivariate statistical methods
are particularly well-suited to this purpose, and a number of useful books on ecological
multivariate statistics are available to provide guidance [12-15].

2.2. PLANTED STUDIES

Rhizoremediation experiments conducted by planting and monitoring microbial

populations and/or contaminant levels over time are powerful approaches since they can
be constructed and operated with replication, randomisation and controlled conditions.
Perhaps the major limitation of planted studies is time, since for reasons of practicality
planted studies are typically only run for 1-3 years. Because roots are estimated to
occupy less than 1% of soil [16], it is reasonable to anticipate that many years of root
exploration would be required to thoroughly treat a contaminated area. Rates of
contaminant disappearance also indicate that rhizoremediation should be regarded as a
long-term process [17]. Nonetheless, losses in soil pollutant concentration and/or
increased populations of degraders have been observed within several years or less
[17-21]. In the event that pollutant disappearance occurs too slowly to detect, a
comprehensive planted study still affords an excellent opportunity to investigate
mechanisms of remediation, such as microbial population shifts at the root-soil interface.
When designing planted studies, as with any controlled experiment, careful and
statistically conscious design is critical to ensure conclusive results. Replicates of each
treatment including unplanted soil as a control should be included. Numbers of
replicates should be as high as reasonably achievable without compromising the quality
of the data since higher numbers of replicates produce more accurate results and a lower
likelihood of producing false positive/negative results. Randomised arrangements of
treatments, whether in pots or field plots, should be employed to ensure that differences
observed are due to treatments rather than variations in light, soil composition, moisture,
etc. associated with different locations in a greenhouse or outdoor area. Additional
guidance may be found in books focusing on basic statistics [22] and experimental
design [23]. The following sections discuss additional design considerations specific to
pot or field plot studies.

2.2.1 Pot studies

Performing studies in containers is an immediately attractive option because it affords
greater homogeneity than can be achieved in an intact field site, keeps plant roots
contained and therefore maintains discrete species-specific influences on soil. However,
pot studies are subject to criticism regarding their ability to realistically replicate natural
conditions that may impact rhizoremediation processes, especially when conducted in a
greenhouse.
Pot experiments performed in a greenhouse are convenient in that they provide
independence from seasonal limitations on outdoor research. Unfortunately, plants are
Methods for Rhizoremediation Research 37

shielded from environmental cues that influence plant physiological responses important
to some rhizoremediation mechanisms. One issue is that greenhouse glass filters out a
portion of ultraviolet light from solar radiation. Ultraviolet light is reported to promote
the production of plant secondary compounds [24], which are thought to play a major
role in biostimulation and/or induction of aromatic pollutant degrading bacteria [25]
(detailed in the following chapter by Andrew C. Singer). If studies are conducted in the
greenhouse, supplementation with UV light is recommended since it has been
demonstrated to increase the concentrations of phenolic compounds produced by plants
to levels similar to those obtained under outdoor growth conditions [24].
Greenhouse studies also shield perennial plants from seasonal signals that dictate
cycles of senescence and dormancy, including tree root turnover events [26]. Root
turnover may be an important mechanism in the delivery of secondary plant metabolites
that support the growth of PCB-degrading bacteria [27]. If a concern, this can be
remedied by running the pot study outdoors where plants can be exposed to natural
environmental conditions. For additional realism, pots may be buried in the soil to help
maintain ambient soil temperature. Whether indoors or outdoors, root growth in pots is
often very different from bulk soil. Roots tend to grow along the pot edges, coil along
the bottom, and may be significantly less branched than field-grown roots, which could
deleteriously effect rhizoremediation results (John Fletcher, 2005, personal
communication).
Pot studies are advantageous over field studies since they afford the opportunity to
minimize heterogeneity of contaminant concentrations. To take advantage of this, care
should be taken to homogenize soil thoroughly in a large soil mixer or similar equipment
before filling pots, since soil either collected from a contaminated site or manually
spiked with pollutants tends to be heterogeneous. At or prior to the initiation of the
study, chemical analyses of replicate samples should be performed to assess the
variability of pollutant concentrations within and among pots and help determine
adequate sample replication. When natural environmental soils are used without
additives to provide loose texture (i.e. vermiculite), they can become very compacted
after continued watering. If considering the addition of organic material to the potting
mix, note that some materials (i.e. bark mulch) contain high concentrations of plant
aromatic compounds that can interfere with gas chromatographic analyses of
polyaromatic hydrocarbons in soil as well as impact microbial communities.
Another consideration with pot studies is that contaminants may leach out of
drainage holes posing not only a chemical hygiene problem but also creating a false
positive response when chemical disappearance data are analyzed. Care should be taken
to avoid excessive watering, and leachates should be collected for chemical analyses
especially if working with water-soluble contaminants. Unplanted controls introduce
unique complications with watering regime. If watered uniformly with planted
treatments, soil in unplanted pots may remain saturated, generating hypoxic conditions
distinctly different from soil conditions in planted pots. This compromises important
control data for background levels of microbial activity as well as for volatilization of
pollutants through soil pores. A solution to this problem is to maintain soil moisture at
similar levels rather than to use equal water volumes, and to monitor outflow for
escaping contaminants.
38 Mary Beth Leigh

2.2.2 Field plots

Planted field plots afford the opportunity to evaluate the rhizoremediation potential of a
variety of plant species under more realistic environmental conditions than pot studies.
While heterogeneity of soil contamination is often greater, it can generally be overcome
with a randomised layout such as Latin square or randomised block design and careful
statistical analyses [22, 23]. Many precautions mentioned in the previous section also
apply, including attention to the watering regime and chemical analysis of preliminary
samples to determine contaminant variability.
A unified experimental design strategy for field investigations of rhizoremediation
has been developed by the U.S. Environmental Protection Agency’s Remediation
Technologies Development Forum (RTDF) Phytoremediation Action Team. The
strategy was designed to evaluate the efficacy of various agricultural and non-crop
herbaceous plants for the rhizoremediation of weathered petroleum hydrocarbons. Use
of the unified protocol permits direct comparisons of data by researchers in a wide range
of geographic locations and climates. Having been carefully developed and debated by
numerous researchers, the RTDF experimental design provides a useful general
framework for field plot studies, and may be easily adapted to study other plants or
contaminants. The experimental method involves three different planted treatments in a
randomised block design with each block being a minimum of 6.1 m square in size. The
three treatments are 1) a mixture of species optimised for local conditions which may
include grasses and trees 2) an unplanted control and 3) a standard seed mixture,
including some flexibility in plant species to accommodate local conditions, of 10-15%
rye, 20-25% legume and 60-70% fescue. Additional blocks may be added with plants of
particular interest to the investigator. Plots are planted and monitored over the course of
3 growing seasons for chemical disappearance and microbial populations. Detailed
sampling methods, chemical, microbial and plant analyses are provided in the protocol,
which is available at http://www.engg.ksu.edu/HSRC/appa.html.

3. Sampling methods

Strategies for sampling forensic or planted rhizoremediation studies are an integral

component of experimental design, and ultimately will impact how accurately the data
reflect true conditions. The first step in developing a sampling strategy is to define the
compartments of interest within the complex root-soil interface (rhizosphere, rhizoplane,
bulk soil, etc.) and select a sampling method that is both appropriate and feasible.
Multiple samples must be collected from each replicate treatment in a representative
manner, with consideration of temporal and spatial issues. Lastly, samples must be
transported and stored for microbial or chemical analyses in ways that will minimize the
introduction of artefacts.

3.1. DEFINING AND SAMPLING THE ROOT-SOIL INTERFACE

Before sampling the root-soil interface, one has first to define it. When the term
“rhizosphere” was first coined by Lorenz Hiltner in 1904, it was defined simply as the
zone of soil in which the microflora is influenced by plant roots. The zone of influence
Methods for Rhizoremediation Research 39

has since been subdivided into several subgroups to distinguish microbial consortia
based on their physical location relative to the root. Although the terminologies and
definitions of each subgroup vary somewhat, in general the root-influenced microbial
community is divided into those residing in the interior of roots (endophytes or
endorhizosphere), on the root surface (rhizoplane), in the soil immediately surrounding
the root (rhizosphere), and beyond the root into the root zone or bulk soil. Dead roots
are rarely included in rhizosphere studies, however they may harbour distinct microbial
populations including bacteria relevant to rhizoremediation [27, 28].
In practice, the rhizosphere is defined operationally. The traditional method for
sampling the rhizosphere is to excavate roots carefully and then to shake off loose soil,
keeping roots with the small amount of soil that remains adhered for analysis [29].
Although this rhizosphere sampling method may be imprecise since the amount of soil
that adheres varies with soil moisture and composition, it remains the most practical,
widely used and accepted technique. Following transport to the laboratory, the
rhizosphere soil is separated from the root by washing with a diluent, and the resultant
suspension is subjected to microbial analyses. The remaining root may then be further
processed to recover rhizoplane and endorhizosphere organisms, as detailed later in the
microbial analysis section.
The soil beneath a plant that is not defined operationally as the rhizosphere is often
referred to as the bulk soil or the root zone. Plant species-specific effects on microbial
communities are frequently observed in this soil fraction [9, 30-32], including effects on
numbers of pollutant degraders in rhizoremediation studies [9, 30]. These findings
indicate that the plant’s influence extends beyond the immediate vicinity of roots, which
is not surprising considering that fine roots are continually exploring new regions of soil
and dying back, impacting the soil both with exudates from living roots and lysates from
detritus.
In the field, sampling the root zone of trees and shrubs is achieved by excavation in
the densely rooted region of soil, which generally occupies the area beneath the canopy.
Root zone soil samples can be accessed by excavation or coring, although the former is
often most practical, especially when root and rhizosphere samples are targeted as well.
It is recommended that sampling tools (shovels, augers) be disinfected with alcohol
between samples to minimize cross contamination.
For pot studies, soil and root sampling may easily be performed by removal of the
soil and root complex from the pot followed by dissection. If plants are to be repeatedly
sampled, coring may also be used to collect soil samples, although it is generally
difficult to obtain sufficient root material for analysis from small cores.

3.2. SPATIAL SAMPLING STRATEGY

Soil is notoriously heterogeneous at both the macro and the micro scale, and plant roots
introduce further variation. The main focus of this section is to provide guidelines on
sampling schemes that can provide an accurate and statistically valid representation of
the treatment in question. As discussed previously, the appropriate sample size can be
determined by careful preliminary studies to determine the variability within the
experimental system.
40 Mary Beth Leigh

When sampling a field plot study with a randomised block design, multiple replicate
samples should be collected within each block. The arrangement of the sampling
locations within the block may be either random or systematic. There are a number of
ways to design systematic sampling schemes, such as along a grid pattern or several
transects [33, 34]. Composing of samples from several different locations is not
recommended because it reduces sample size and precludes determinations of variability
that are important to determining statistical differences among treatments.
In pot studies, multiple samples should be collected from each pot. If the whole plant
root system is harvested, different zones of soil may be separated, such as into quadrants
and by depth. Analysing multiple separate samples from each pot helps provide the
statistical power needed to discern relatively small but significant effects with
confidence, as well as to reveal the heterogeneity of the system.
In forensic studies, the distribution of plants will be random and hence rhizosphere
sampling will be dictated primarily by the location of the plants. Multiple replicate
samples should be collected from each plant, such as in a circumference around a tree.
For purposes of site characterization and mapping of contaminant concentration and
other factors, it is recommended that a comprehensive set of samples also be collected
throughout the entire site on at least one time point using either a randomised or
systematic pattern.

3.3. SEASONAL SAMPLING CONSIDERATIONS

Seasonal fluxes in environmental conditions are unavoidable, and not necessarily

detrimental, in real-world applications of rhizoremediation technology. Since both plant
root physiology (i.e. root turnover) [26] and soil microbial communities [35, 36]
respond to seasonal changes, it is anticipated that degradative potential of the microbial
community will also fluctuate. For this reason, research efforts should seek whenever
possible to understand rhizoremediation process not just in the active summer growing
season but throughout the seasonal cycle and under varying moisture conditions.
Recognizing these influences on microbial populations important to rhizoremediation
may help to formulate site management practices and provide new insights into the
mechanisms of rhizoremediation.
At minimum, it is recommended that samples be collected from outdoor
rhizoremediation studies at 3-4 times per year to reflect early spring, summer and late
autumn conditions. Because major root turnover events typically occur in the autumn in
rough synchrony with leaf senescence [26], samples should be collected after occurrence
of complete leaf fall for deciduous plants or visible shoot dieback for annuals or
biennials. While more frequent sampling events are desirable, care should be taken to
ensure that analysing the large number of samples resulting from aggressive sampling is
feasible.
At a PCB-contaminated site in the Czech Republic, populations of PCB-degrading
bacteria were enumerated in June, August, November and the following May.
Significant seasonal differences in population size were detected beneath the Austrian
pine tree, in which numbers of degraders increased significantly between August and
November [9], coinciding with expected root turnover events. Although the cause of the
Methods for Rhizoremediation Research 41

increase remains unknown, it exemplifies the seasonal flux in degradative populations

that can occur in a rhizoremediation setting.

3.4. SAMPLE STORAGE

Root and soil samples collected in the field can be conveniently stored in self-sealing
thin-walled plastic bags, which permit slow gas exchange (O2 and CO2) while preventing
soil drying [33]. If samples are destined for chemical analyses of contaminants, exposure
to common flexible plastics should be avoided because they can introduce phthalate
esters into the sample that interfere with analyses of many pollutants including pesticides
and PCBs. Instead, inert sample vessels such as glass bottles should be used. For more
detailed recommendations regarding sample storage for chemical analyses for particular
pollutants, consult U.S. EPA Methods such as Method 8081.
Samples are commonly transported to the laboratory as quickly as possible on ice to
reduce microbial activity, and then they are stored at 4°C until analysis. Cultivable or
direct microbial analyses should be performed as soon as possible, since sample storage
results in changes in microbial properties [33]. For molecular-based analyses of
microbial communities, subsamples should be frozen at –20°C or –70°C for later
extraction and analyses of DNA or RNA, respectively. Commercially available RNA
protectants may be added before freezing to inhibit RNase activity when samples thaw.
However, nucleic acid extraction recovery in the presence of the protectant should be
tested in advance since these protectants can interfere with some protocols.
If microbial, molecular or pollutant analyses are to be reported per unit of dry weight
of soil or root material, then aliquots of each samples should be weighed and dried to
obtain ratios of fresh weight to dry weight.

4. Microbial analyses

Once samples have been collected, one turns to the task of investigating the microbial
populations contained therein. What is the best approach to identify, quantify or
characterize pollutant degraders and to compare them among samples? There are a host
of different microbiological methods for these purposes, each with its own set of
advantages and limitations. Cultivation-based methods present a notoriously incomplete
picture of the microbial community due to the “great plate count anomaly”, in which
only 0.1 - 10% of bacteria in the environment are cultivable in the laboratory. However,
cultivation methods are widely used for their simplicity and cost-effectiveness, and
because they remain the primary way to demonstrate metabolic capabilities of individual
strains. Molecular tools including stable isotope probing, quantitative real-time PCR,
high-throughput sequencing and microarrays provide unprecedented access to data not
subject to issues of culture bias, yet are somewhat constrained by our existing
knowledge of target genes and sequence diversity. Because of the unique insights
provided by both culture-based and direct molecular approaches, their combined
application can provide a complementary view of the microbial community and its
function. This section aspires to acquaint investigators embarking on rhizoremediation
42 Mary Beth Leigh

studies with an array of both common and innovative approaches to studying the
diversity, abundance and activity of microorganisms important to biodegradation.

4.1. CULTIVATION-BASED METHODS

4.1.1 Microbial recovery from the soil-root interface

In preparation for cultivation, it is often necessary to extract microorganisms from the
soil, rhizosphere, rhizoplane or endorhizosphere. Typically a bacterial suspension is
created that may then be serial diluted or used directly as an inoculum using the
following methods.
Soil bacteria are suspended by shaking or vortexing soil in liquid along with sterile
glass beads [37], sometimes followed by standing for 30 min or short low-speed
centrifugation (500-1000 × g) [33] to separate soil particles from suspended bacteria.
Sodium pyrophosphate solution is an effective soil aggregate dispersal agent [38] and so
is commonly used as a suspension medium [33, 37]. Alternatively, other buffers, saline
solution or simply sterile water may be used. Media containing carbon sources should be
avoided since growth may occur that would skew populations during processing. A side-
by-side study evaluating cell recovery from soil using water, sodium pyrophosphate and
several buffers showed no significant difference (Terence Marsh, 2003, personal
communication).
Rhizosphere soil bacteria can be suspended as described for soil, although glass
beads may be omitted. Some rhizoplane organisms will invariably be recovered along
with the rhizosphere, however gentle washing is thought to leave the rhizoplane largely
intact since it reportedly removes only 10% of the bacteria removed by vigorous shaking
with glass beads [39]. After shaking a defined quantity (by fresh weight or length) of
root with adhering rhizosphere soil in liquid to suspend cells, the roots may be
recovered, dried and weighed so that rhizosphere bacterial numbers can be based on root
dry weight.
Following removal of the rhizosphere soil, the rhizoplane and endorhizosphere
microflora may be recovered separately or together for analyses. Rhizoplane organisms
remaining on the surface of the root after rhizosphere washing may be extracted by
vigorous treatments such as shaking with glass beads, vortexing, or using a Stomacher
blender [40, 41]. For recovery of endorhizosphere organisms, roots may be ground with
a small amount of water or buffer in a Warring blender [42] or mortar and pestle [43] to
create a suspension. To ensure that truly endophytic organisms are recovered in the
absence of rhizoplane contaminants, the roots may be surface disinfected prior to
grinding [43].

4.1.2 Liquid enrichment cultures

Because pollutant-degrading bacteria typically comprise a small proportion of the total
microbial community, enrichment methods are useful to generate a mixed culture in
which degradative bacteria are predominant. Many of the earliest isolates of bacteria
capable of degrading pesticides and pollutants were isolated using enrichment methods
[44, 45]. Constructing enrichment cultures is a simple matter of inoculating a flask, tube
or bottle containing sterile minimal salts medium with an environmental sample, taking
Methods for Rhizoremediation Research 43

care to minimize introduction of foreign substrates, providing the compound of interest

as the sole carbon source, and then incubating with shaking for a period of days to weeks
until turbid. Often the enrichment is subjected to several passages to achieve a stable
consortium.
Although enrichment cultures are valuable for producing isolates for study, they are
not quantitative reflections of the diversity or relative abundance of contaminant-
degrading populations within a sample. Studies of environmental samples have
demonstrated dramatically reduced diversity of 2,4-D and PAH degrading organisms as
well as degradative genes when cultivated using enrichment cultures in comparison to
direct agar plates or biofilm culture methods [46, 47]. The difference is explained by
competitive interactions in the enrichment culture in which slower-growing organisms
are outcompeted by others with higher maximum specific growth rates. Thus,
enrichment methods are not recommended for making comparisons of diversity or
relative abundance among different samples.

4.1.3 Direct agar plate methods

Direct agar plating methods are a useful means to both enumerate and investigate the
diversity of the cultivable fraction of bacteria capable of utilizing pollutants. As opposed
to enrichment cultures, direct plates afford the opportunity for organisms of the same
functional group but with different growth rates to form colonies and be detected [48].
Direct plates are also relatively simple to perform, require little specialized equipment
and can easily be appended to existing protocols for plate counts of total cultivable
bacteria. For these reasons, direct plate methods are the most widely used technique for
studying abundance and diversity of microbial populations important to pollutant
degradation.
Direct plating of soil or rhizosphere samples is performed by suspending bacterial
cells from a defined quantity of soil or roots, spread-plating a dilution series onto a
minimal medium with the contaminant (or an analogue) provided as a sole carbon
source, incubating and colony counting. A secondary screening procedure, such as a
clearing-zone test, may be employed to help verify that colonies are truly utilizing the
substrate rather than impurities in the medium or carryover from the soil suspension.
Details of these procedures are discussed below. For accurate plate counts, 2-3 replicate
plates should be inoculated from each dilution generated from a sample and counts
averaged. Likewise, multiple subsamples from each soil/root sample should be plated for
statistically valid enumeration.

Growth conditions In order to selectively cultivate organisms that utilize a sole carbon
source, care should be taken in the design and preparation of agar media to avoid
introduction of unwanted growth substrates. A variety of different defined minimal
media recipes may be used, taking care to avoid inclusion of any potential energy
sources. Addition of yeast extract or vitamins should be avoided unless absolutely
necessary for growth since they also function as carbon sources, even in low
concentrations. Although expensive, highly purified Noble Agar is preferable over
common agars, since most agars contain impurities that act as non-specific carbon
sources. However, secondary screening methods discussed later can be used to
44 Mary Beth Leigh

distinguish true contaminant utilisers from non-degradative colonies growing on agar

plates.
The contaminant or an analogue can be provided as a growth substrate in a number
of ways depending on the nature of the compound. Water-soluble compounds may be
added directly to the agar medium, preferably when relatively cool following
autoclaving to prevent thermal decomposition. Practically insoluble but highly volatile
compounds like biphenyl or naphthalene are easily provided in the vapour phase.
Biphenyl is commonly provided by sprinkling a few crystals in the lid of the Petri plate
[49]. For chemical hygiene, plates are then sealed with Parafilm or enclosing in a plastic
sleeve or chamber to minimize volatilization into the ambient air. Naphthalene can be
provided to plates all together in a sealed container [37]. Less volatile compounds may
be added to the surface of agar plates by dissolving in water, ethanol or acetone and
spreading, followed by removal of organic solvents by evaporation from open plates in a
laminar flow hood [37]. Alternatively, compounds may be suspended in a small amount
of agar or agarose and poured over the basal mineral agar layer [50].
Plates are commonly incubated or below 25°C, which presumably simulates
environmental conditions better than the higher temperatures commonly used in
microbiological research. The length of incubation required for colony formation of
pollutant degraders varies depending on the nature of the population and the compound.
It is recommended that when embarking on a new study that a pilot test be performed in
which colony forming units are counted repeatedly over a period of up to 1 month in
order to identify appropriate incubation times. Brief incubation periods can result in
erroneously low counts and bias data toward the faster growing fraction of the
population. A biphenyl-utilizing bacterial population comprised predominantly of
relatively slow-growing Gram-positive rhodococci required 3 weeks for new colony
formation to cease [9].

Colony screening methods Screening colonies pre-grown on agar plates for degradative
abilities is often desirable since non-degradative colonies can grow on substrates other
than the target compound such as impurities in agar, carryover from the soil suspension,
or when cofactors in media are required. Several clearing-zone and colorimetric
techniques for secondary screening are described below that that can help confirm that
colonies are truly degrading the target substrate. Some of these methods may also be
applied to colonies grown intentionally on a rich medium to detect degraders among the
total cultivable community.
Following colony formation, clearing zone assays can be performed by depositing a
thin cloudy layer of the target compound over the agar plate surface and then incubating
until zones of clearing form. A 5 - 10% w/v solution of the target compound prepared in
ether or acetone solution may be sprayed onto the agar surface using thin layer
chromatography plate spraying apparatus in a fume hood. Alternatively, compounds may
be overlayed by sublimation [51]. Spray methods have been used for a variety of
aromatic compounds including biphenyl and chlorobiphenyl [52, 53], as well as
phenanthrene, fluoranthene, pyrene, naphthalene and anthracene [53, 54]. Agar plates
are then sealed in plastic sleeves to minimize volatilization of the compound and
Methods for Rhizoremediation Research 45

incubated for days to several weeks and checked periodically for the formation of zones
of clearing around colonies.
Clearing zone assays may also be performed simultaneously with initial colony
formation for compounds such as phenanthrene. The inoculum is mixed in an agarose
suspension containing phenanthrene crystals and then spread onto a minimal medium
agar plate. As phenanthrene-utilizing colonies form they create clearing zones [50]. This
approach is advantageous over spraying methods by reducing the contamination of
laboratory fume hoods with chemicals and preventing exposure of organisms to organic
solvents, and also provides an effective means for efficient delivery of non-volatile
substrates to organisms.
Colonies with aromatic ring dioxygenase activity can be screened using a simple
colour indication assay that capitalizes on the ability of naphthalene enzyme to produce
indigo from indole [54, 55]. Crystals of indole placed in the lid of the Petri dish will
result in rapid (1 day or less) appearance of the blue indigo pigment in colonies in which
aromatic dioxygenases are active. However, the assay only detects activity of
dioxygenases enzymes that form cis-dihydrodiols from aromatic hydrocarbons [55].

4.1.4 Most probable number (MPN) method

Another useful approach for enumeration of pollutant degraders is the most probable
number (MPN) method, which has been adapted for efficient use in 96-well microtiter
plates to quantify bacteria that degrade polyaromatic hydrocarbon and alkanes [56] and
crude oil [57]. In essence, a suspension of bacterial cells is diluted to extinction in a
microtiter plate containing specific substrates as carbon sources. For PAH-degrading
organisms, growth is detected in the wells by the presence of a yellow to green-brown
colour generated by PAH oxidation products. For substrates without pigmented products
such as alkanes, detection using the colour change of an iodonitrotetrazolium violet dye
can be used. Empirical testing demonstrated that microtiter MPN produced the same
result as enumeration by direct plate count for PAH-degraders [56]. The MPN method
may be easily adapted for use with other compounds. When degrader population sizes
are low and growth is only detected in wells with very low dilution factors, care should
be taken to ensure that the signal is due to utilization of the substrate and not compounds
carried over from the soil solution. To minimize carryover, cell suspensions may be
washed by repeated centrifugation and resuspension to remove unwanted substrates prior
to inoculation.

4.2. MOLECULAR ECOLOGICAL METHODS

Molecular microbial ecology methods bypass culture-bias to provide direct measures of

a variety of microbial community parameters. This section highlights some methods that
are of particular value to bioremediation studies because of their ability to directly detect
the presence, activity, diversity and abundance of degradative organisms.

4.2.1 DNA and RNA extraction methods

Many molecular ecological analyses require bacterial DNA and/or RNA to be extracted
from soil. Depending on both the soil itself and the protocol applied, results vary widely
46 Mary Beth Leigh

in terms of the quantity, size and purity of nucleic acids yielded. Numerous extraction
methods exist in the published literature and in the form of commercial kits. Since the
effectiveness of extraction protocols varies among soil types, it is recommended that
several methods be tested, compared or sometimes adapted to identify a successful
approach for a particular soil.
For the simultaneous extraction of DNA and RNA, a crude nucleic acid extract is
first obtained using methods such as the rapid bead-beating method developed by
Griffiths et al. [58] or a freeze grinding approach by Hurt et al. [59]. Bead beating tends
to shear nucleic acids, so if high molecular weight extracts are needed (i.e. for
metagenomics) then freeze grinding may be preferable. Precipitation of nucleic acids
with polyethylene glycol (PEG) [58] is particularly effective in removal of humic
materials that interfere with PCR, and may be used with other protocols when needed.
RNA and DNA can then be separated from each other by gravity column systems such
as the Qiagen DNA/RNA extraction kit (Qiagen Inc., Carlsbad, CA), followed by RNase
or DNase treatment to remove residual amounts of undesirable nucleic acids.
Alternatively, the crude extract may be split and then each fraction directly subjected to
RNase or DNase treatment.
To extract DNA only, simple and effective commercial bead beating kits are
commonly used such as those available from QBiogene, Inc. (Carlsbad, CA) or MoBio
(Carlsbad, CA) or using a freeze-grinding method [60]. RNA extraction techniques are
similar to DNA methods, however since RNA is very labile, extracting it from soil is
much more challenging and requires conditions that inhibit RNase activity. Soil RNA
extraction protocols are available in the literature [61], and commercial kits have
recently arrived on the market (MoBio, Carlsbad, CA and QBiogene Inc., Carlsbad,
CA).
To simultaneously extract endophytes, rhizoplane and rhizosphere soil, methods
above may be adapted for use with whole roots and adhering soil. For extraction
of endophytic root organisms only, other methods in the literature can be consulted
[62-64].

4.2.2 Functional gene detection methods

Direct detection of genes that function in degradative processes may be performed on
bacterial community DNA extracts either by PCR amplification or using microarrays.
The primary limitations of these approaches lie in effective primer or probe design and
detection limits. Primers and probes can be designed using bioinformatic methods to
either broadly target groups of similar sequences or to be highly sequence specific.
Inclusive primers or probes are designed by aligning multiple sequences and identifying
conserved regions. Thus, they are limited by current knowledge of sequence diversity in
the environment, which may prevent detection of unknown sequences. Detection limits
for both PCR and microarray methods can be an obstacle since pollutant degraders often
comprise a small proportion of the total community. Nonetheless, direct functional gene
detection is a very powerful tool to investigate the presence, diversity and in some cases
quantity of certain degradative genes in the microbial community.
Methods for Rhizoremediation Research 47

PCR and quantitative real-time PCR PCR methods for detecting genes important to
aromatic pollutant degradation have frequently targeted initial aromatic dioxygenase
genes, which encode enzymes catalyzing the first, rate-limiting hydroxylation step of
degradation. The aromatic dioxygenase gene family spans a wide substrate range,
including PCBs, mono- and polyaromatic hydrocarbons, and are multimeric enzymes
including a reductase subunit, a ferredoxin subunit, a large (alpha) and a small (beta)
iron sulphur protein. Sequence variations in the large subunit are associated with
substrate specificity [65]. For this reason, as well as the presence of a conserved region
encoding the Rieske centre, the large subunit is an attractive target for PCR primer
design. PCR primer sets have been successfully developed that can amplify groups of
initial dioxygenase genes specific to certain substrates [66], while other primer sets are
more organism-specific [66, 67]. Baldwin et al., [66] developed multiple primer systems
to differentially detect and enumerate subfamilies of aromatic oxygenase genes for
biphenyl, naphthalene, toluene dioxygenases as well as monooxygenases involved in
toluene/xylene and phenol degradation. Primers have been effectively designed targeting
dioxygenases catalysing later steps in aromatic degradation as well [67, 68] For
example, Erb and Wagner-Dobler successfully amplified bphC encoding the di-
oxygenase enzyme catalysing the meta-cleavage step in biphenyl degradation from the
environment and achieved a detection limit of five copies per reaction mixture or 100
cells per g wet weight of sediment.
The genetic diversity of catabolic genes can be investigated when PCR products are
subjected to sequence analysis. Following PCR amplification of degradative genes,
clone libraries may be constructed for sequencing, or alternatively amplicons may be
separated by methods such as denaturing gradient gel electrophoresis (DGGE) and
excised bands sequenced. The relationship of sequences to previously known genes can
be established by searching for similar sequences in public databases such as BLASTn
searches of GenBank [69] and conducting phylogenetic analyses [70].
The degradative potential of a microbial community can be quantified using real-
time PCR (RTm-PCR) with primers targeting functional genes. In RTm-PCR, a PCR
reaction is performed in the presence of either fluorescent probes that hybridize to the
specific target sequence (i.e. TaqMan ® probes, Applied Biosciences, Foster City, CA)
or alternatively a non-specific fluorescent dye that binds to double stranded DNA (i.e.
SYBR green). RTm-PCR thermocyclers are specially designed to measure fluorescence
in the PCR reaction at each amplification cycle, permitting quantitation relative to a
standard curve containing known copy numbers of the target sequence. When amplifying
variable regions of functional genes, use of SYBR green is preferable over highly
specific probes, however extra care must be taken to ensure that non-specific products
are not being produced and measured. Running a melting curve analysis at the end of the
reaction is an important quality control step for this purpose. For additional information
regarding RTm-PCR methods, the reader is referred to books dedicated to the subject
[71, 72]. RTm-PCR methods to enumerate aromatic dioxygenases have been developed,
which used SYBR green and produced detection limits down to 2 × 102 copies per
reaction using pure culture DNA [66].
48 Mary Beth Leigh

Functional gene microarrays Using microarray techniques, it is now possible to probe

community DNA samples for thousands of target sequences simultaneously, opening an
enormous window into the functional gene diversity of microbial communities.
Microarrays have already been designed that successfully detect a broad range of genes
involved in the degradation of pollutants, including monoaromatic and polyaromatic
hydrocarbons, PCBs and aliphatics [73-75]. Careful probe design, including the design
of several probes per target when possible, followed by comprehensive testing is critical
to help ensure the specificity of hybridisation and to prevent false positives or negatives.
Unfortunately, as in the design of PCR primers, probe design is limited by current
knowledge of the diversity of functional gene sequences so some important genes may
not be detected.
Although not yet applied for quantitative purposes, strong linear relationships have
been reported between signal intensity of hybridised probes and the number of gene
copies in a sample with some functional gene arrays [73-75]. Thus, arrays may
eventually be used to evaluate both the diversity and abundance of target genes in a
sample.
A major challenge in applying arrays to bioremediation studies is their detection
limits. As reported by Denef et al. [73], single-copy genes can only be detected from
organisms that comprise 1% or more of the community with current technology. This is
an issue for many environmental samples where the degradative population often
comprises a small proportion of the total community. The detection limits of arrays
could likely be overcome, however, if combined with stable isotope probing methods
detailed in the following section.

4.2.3 Stable isotope probing

Stable isotope probing (SIP) methods now allow researchers to directly answer the
previously evasive question: Which microorganisms are truly active in pollutant
degradation? In addition to linking phylogeny with function, SIP also provides the
opportunity to thoroughly investigate the genomic content of active degradative
organisms. Although it does not provide quantitative measures of degrader abundance,
SIP can provide insights into the identity and genetic diversity of organisms that are
truly active in rhizoremediation.

SIP methodologies SIP involves provision of a substrate that is labelled with a heavy
stable isotope, typically 13C, to a microbial community. Organisms that actively
incorporate the substrate are detected by virtue of the presence of 13C in biomarkers such
as phospholipid fatty acids (PLFAs), DNA or RNA. SIP based on nucleic acids provides
higher resolution phylogenetic data than PLFAs [76], as well as access to functional
genes of active species [77]. For DNA or RNA-SIP, nucleic acid extracts are subjected
to density gradient centrifugation to separate heavier, 13C-labelled nucleic acids from
those of the inactive community. Heavy fractions may then be subject to PCR
amplification, community profiling and/or sequence analyses. RNA-SIP is more
sensitive than DNA-SIP, and is also advantageous in that growth (and potential
community change) during the incubation is not necessary [78-80]. However, if
functional genes are to be targeted, DNA-SIP is more attractive because of the difficulty
Methods for Rhizoremediation Research 49

of extracting mRNA from environmental samples. Recently, SIP methods have been
developed to capitalize on the stable isotope, 15N [81], which opens a window into the
microorganisms that biodegrade N-containing contaminants.
To perform SIP of soil samples, microcosms are typically constructed in the
laboratory, or using an in situ method that has also been developed [82] which may
prove valuable in studying intact rhizospheres. Closed systems are desirable because
they permit the sampling and monitoring of 13CO2 evolution in the headspace as an
indicator of substrate utilization. Substrates may be provided to soil in aqueous solution
or, if volatile, as crystals enclosed in the microcosm. For SIP with 13C-biphenyl, crystals
on the interior wall of a sealed serum bottle were effective for enriching bacteria in soil
spread loosely on the bottom [83, 84].
Care should be taken to avoid lengthy incubation times to minimize flow of 13C to
non-degraders via degradative intermediates or the biomass of primary degraders, and to
avoid community change during the incubation. Thus, sampling over a time-course is
recommended to ensure that primary utilisers are identified. In a timecourse SIP
experiment with 13C-biphenyl, significant 13CO2 evolution into the headspace was
detected within 24 hours and continued to be produced over the course of the 14-day
incubation, indicating that biphenyl was being metabolised [83, 84].
Separation of 13C-labeled DNA from unlabeled DNA can be performed by density
gradient centrifugation in caesium chloride (CsCl) [79, 85] or in caesium trifluoroacetate
(CsTFA) solutions [83, 84], while RNA is separated in CsTFA with the addition of
formamide to reduce secondary structure [86]. Following ultracentrifugation, it is
strongly recommended that density gradients be fractionated rather than extracting bands
using a syringe needle since fractionation permits the detection of partially labelled
nucleic acids. Fractionating samples along with appropriate controls is important to
ensuring that organisms detected are truly labelled, since low levels of unlabeled nucleic
acids are present throughout the density gradient. Control gradients should be
constructed with unlabeled nucleic acids from the same community, such as a sample
collected at the initiation of the experiment (t = 0). Control fractions should be subjected
to the same fractionation and downstream molecular analyses. Then, any sequences
detected in the background heavy control fractions can be subtracted from those
identified in the 13C-treated sample.
Following fractionation and nucleic acid recovery, PCR amplification of 16S rRNA
genes (or RT-PCR of 16S rRNA) and community analyses are helpful to locate the 13C-
labeled fractions and to track changes in the labelled populations with increased
incubation time. Denaturing gradient gel electrophoresis (DGGE) [78, 86] or terminal
restriction fragment length polymorphism (T-RFLP) [79, 80] are common community
profiling methods used in SIP.
Following the identification of the fractions containing 13C-labeled nucleic acids,
sequence analysis may be performed to phylogenetically identify organisms that derived
carbon from the substrate. Clone libraries may be constructed from PCR amplicons, or if
DGGE is used, bands of interest may be excised, reamplified and sequenced. Sequence
analyses of 13C-labeled DNA and RNA have focused primarily on the SSU rRNA for the
purposes of obtaining phylogenetic information. However, a wealth of functional gene
information is available in the labelled fraction that can prove informative in
50 Mary Beth Leigh

biodegradation studies. Functional gene analyses of SIP-derived 13C-DNA have been

reported for methylotrophs [77], but not yet for environmental pollutants.

SIP in bioremediation and the rhizosphere To date, SIP studies targeting aromatic
pollutant-degrading bacteria have been achieved in a variety of settings, including
phenol in activate sludge [78, 87] naphthalene and phenanthrene in a bioreactor treating
soil [88], 2,2’-dichlorobiphenyl in a biofilm growing on PCB droplets [89], naphthalene
in sediment [90], toluene in soil [91, 92], phenol, caffeine and naphthalene in soil [82].
Although as yet unexplored, SIP also provides an excellent means for identifying fungi
that are significant in bioremediation by simply amplifying 18S rRNA genes in heavy
nucleic acid fractions using universal fungal rRNA primers [93].
In the field of rhizosphere ecology, SIP has been applied in efforts to identify
microbial communities that derive carbon from root exudates following provision of
13
CO2 to aboveground plant parts [94, 95]. A major challenge of this approach is the
dilution of 13C with the predominantly unlabeled carbon in the plant which can result in
isotopic enrichment of the microbial biomarkers below levels needed for SIP [94]. If
combined with functional gene detection, SIP of root exudates may prove valuable to
demonstrate that pollutant-degrading bacteria are deriving carbon from the plant in the
rhizosphere. Unfortunately this approach simultaneously probes for microbes using any
of the plethoras of compounds exuded by plant roots, and thus does not permit
differentiation of specific plant compound-utilizing bacteria. In order to determine which
components of root exudates or lysates specifically feed pollutant degraders, SIP may be
performed in a rhizosphere setting, or in root zone samples, with 13C-labelled plant
compounds provided individually.

5. Conclusions and research needs

Through the array of windows opened by powerful molecular ecological tools in

combination with traditional microbial methods and carefully constructed experiments,
new insights can be gained into bioremediation processes in the rhizosphere. What are
the mechanisms for rhizostimulation of contaminant degraders? Which bacteria, fungi
and degradative genes are most important? Which plant species, communities, or
biosystems are most effective at rhizoremediation of various contaminants? Methods
now available invite us to answer these questions and assemble the fundamental
understanding of rhizoremediation ecology necessary for its successful implementation.

References
[1] Olson, PE; Wong, T; Leigh, MB and Fletcher, JS (2003) Allometric modeling of plant root growth
andits application in rhizosphere remediation of soil contaminants. Environ Sci Technol 37(3): 638-643
[2] Olson, PE; Fletcher, JS and Philp, PR (2001) Natural attenuation/phytoremediation in the vadose zone
of a former industrial sludge basin. Environmental Science and Pollution Research 8(4): 243-249
[3] Olson, PE and Fletcher, JS (1999) Field evaluation of mulberry root structure with regard to
phytoremediation. Bioremediat J 3(1): 27-33
[4] Olson, PE and Fletcher, JS (2000) Ecological recovery of vegetation at a former industrial sludge basin
and its implications to phytoremediation. Environ Sci Pollut R 7(4): 195-204
Methods for Rhizoremediation Research 51

[5] Kyle, MD (2002) Rhizoremediation: Statistical and molecular analysis of PAH-utilizing bacteria
enriched in the rhizozone of mulberry growing in a contaminated site, in American Society for
Microbiology Missouri Valley Branch Joint Annual Meeting. 2002. Kansas City, MO, USA
[6] Kyle, MD; Fletcher, JS and Nagle, DP (2001) Apparent enrichment for PAH-degrading bacteria in the
mulberry rhizozone of contaminated soil, in 8th International Petroleum Environmental Conference:
Issues and Solutions in Exploration, Production and Refining. 2001. Houston, TX, USA
[7] Nagle, DP; Kyle, MD; Olson, PE and Fletcher, JS (1998) Bacterial microflora from a vegetated, PAH-
contaminated industrial site, in IBC Third International Conference on Phytoremediation: Strategies and
evaluation of phytoremediation's performance in the field. 1998. Houston, TX: International Business
Communications, Inc.
[8] Nagle, DP; Kyle, MD; Olson, PE and Fletcher, JS (1998) Polyaromatic hydrocarbon-degrading
microflora associated with vegetation at a phytoremediated site, in Fifth International Petroleum
Environmental Conference. 1998. Albuquerque, NM.
[9] Leigh, MB (2003) Rhizosphere ecology of PCB-degrading bacteria at a contaminated site and
implications for bioremediation, PhD Dissertation, Department of Botany and Microbiology, University
of Oklahoma: pp. 88.
[10] Leigh, MB; Fletcher, JS; Nagle, DP; Mackova, M and Macek, T (2001) Vegetation and fungi at Czech
PCB-contaminated sites as bioremediation candidates. International In Situ and On-Site Bioremediation
Symposium, 6th, San Diego, CA, United States, June 4-7, 2001, 5: 61-68
[11] Demnerova, K; Stiborova, H; Leigh, MB; Pieper, D; Pazlarova, J; Brenner, V; Mackova, M and Macek,
T (2001) Degradation of PCBs and CBs by Indigenous Bacteria Isolated from Contaminated Soil, in N.
Kalogerakis, Ed., Proceedings of the First European Bioremediation Conference. 2001. Chania, Crete,
Greece.
[12] Legendre, P and Legendre, L (1998) Numerical ecology. Amsterdam: Elsevier.
[13] Braak, CJF; Jongman, RHG; Ter-Braak, CJF and Van-Tongerer, OFR (1995) Data analysis in
community and landscape ecology. Cambridge: Cambridge University. 299
[14] Ludwig, JA and Reynolds, JF (1988) Statistical ecology: a primer on methods and computing. Wiley:
New York.
[15] Manly, BFJ (1994) Multivariate statistical methods: a primer. 2nd ed. London: Chapman & Hall.
[16] Jungk, AO (1991) Dynamics of nutrient movement at the soil-root interface, in Y Waisel, A Eshel and U
Kafkafi, Eds., Plant Roots the Hidden Half, Marcel Dekker, New York. 455-481
[17] Banks, MK; Schwab, AP; Liu, B; Kulakow, PA; Smith, JS and Kim, R (2003) The effect of plants on
the degradation and toxicity of petroleum contaminants in soil: a field assessment, in Phytoremediation,
DT Tsao, Ed., Springer, Berlin. 206
[18] Demnerova, K; Mackova, M; Kucerova, P; Leigh, MB; Novakova, H; Burkhard, J and Macek, T (2000)
Practical use of bioremediation for PCB removal from contaminated soil, in M. Hofman and J. Anné,
Series Eds., Focus on Biotechnology, Vol. 3, Kluwer, Dordrecht
[19] Chekol, T; Vough, LR and Chaney, RL (2004) Phytoremediation of polychlorinated biphenyl-
contaminated soils: the rhizosphere effect. Environ Int 30(6): 799-804
[20] Siciliano, SD and Germida, JJ (1998) Mechanisms of phytoremediation: biochemical and ecological
interactions between plants and bacteria. Environ Rev 6: 65-79
[21] Miya, RK and Firestone, MK (2000) Phenanthrene-degrader community dynamics in rhizosphere soil
from a common annual grass. Journal of Environmental Quality 29(2): 584-592
[22] Dytham, C (1999) Choosing and Using Statistics: A Biologists Guide. Oxford: Blackwell Science Ltd.
218
[23] Montgomery, DC (2005) Design and Analysis of Experiments. 6th ed. Hoboken, NJ: John Wiley &
Sons. 643
[24] Rice, EL (1974) Allelopathy, Vol. 352. Academic Press: New York
[25] Singer, AC; Crowley, DE and Thompson, IP (2003) Secondary plant metabolites in phytoremediation
and biotransformation. TRENDS in Biotechnology 21(3): 123-130
[26] Vogt, KA and Bloomfield, J (1991) Tree root turnover and senescence, in Y Waisel and U Kafkafi, Eds.,
Plant Roots the Hidden Half, Marcel Dekker: New York, 297-306
[27] Leigh, MB; Fletcher, JS; Fu, XO and Schmitz, FJ (2002) Root turnover: An important source of
microbial substrates in rhizosphere remediation of recalcitrant contaminants. Environ Sci Technol 36(7):
1579-1583
52 Mary Beth Leigh

[28] Miya, RK and Firestone, MK (2001) Enhanced phenanthrene biodegradation in soil by slender oat root
exudates and root debris. J Environ Quality 30: 1911-1918
[29] Rovira, AD (1965) Interactions between plant roots and soil microorganisms. Annu Rev Microbiol 19:
241-266
[30] Siciliano, SD; Germida, JJ; Banks, K and Greer, CW (2003) Changes in microbial community
composition and function during a polyaromatic hydrocarbon phytoremediation field trial. Appl Environ
Microbiol 69(1): 483-489
[31] Nusslein, K and Tiedje, JM (1999) Soil bacterial community shift correlated with change from forest to
pasture vegetation in a tropical soil. Appl Environ Microbiol 65(8): 3622-3626
[32] Saetre, P and Baath, E (2000) Spatial variation and patterns of soil microbial community structure in a
mixed spruce-birch stand. Soil Biol Biochem 32(7): 909-917
[33] van Elsas, JD and Smalla, K (1997) Methods for sampling soil microbes, in CJ Hurst, GR Knudsen, MJ
McInerney, LD Stetzenbach and MV Walter, Eds., Manual of Environmental Microbiology, ASM Press:
Washington, DC, 893
[34] Tan, KH (1996) Soil Sampling, Preparation and Analysis. Marcel Dekker: New York, 408
[35] Smalla, K; Wieland, G; Buchner, A; Zock, A; Parzy, J; Kaiser, S; Roskot, N; Heuer, H and Berg, G
(2001) Bulk and rhizosphere soil bacterial communities studied by denaturing gradient gel
electrophoresis: Plant-dependent enrichment and seasonal shifts revealed. Appl Environ Microbiol
67(10): 4742-4751
[36] Buckley, DH and Schmidt, T (2003) Diversity and dynamics of microbial communities in soils from
agro-ecosystems. Environ Microbiol 5(6): 441
[37] Kastner, M; Jammali, MB and Mahro, B (1994) Enumeration and characterization of the soil microflora
from hydrocarbon-contaminated soil sites able to mineralize polycyclic aromatic hydrocarbons (PAH).
Appl Microbiol Biotechnology 41: 267-273
[38] Strickland, TC; Sollins, P; Schimel, DS and Kerle, EA (1988) Aggregation and aggregate stability in
forest and range soils. Soil Sci Soc Am J 52: 829-833
[39] Rovira, AD; Newman, EI; Bowen, HJ and Campbell, R (1974) Quantitative assessment of the rhizoplane
microflora by direct microscopy. Soil Biol Biochem 6: 211-216
[40] Dandurand, L-MC and Knudsen, GR (1997) Sampling microbes from the rhizosphere and phyllosphere,
in CJ Hurst, GR Knudsen, MJ McInerney, LD Stetzenbach and MV Walter, Eds., Manual of
Environmental Microbiology, ASM Press, Washington, DC, 893
[41] Wieland, G; Neumann, R and Backhaus, H (2001) Variation of microbial communities in soil,
rhizosphere, and rhizoplane in response to crop species, soil type and crop development. Appl Environ
Microbiol 67(12): 5849-5854
[42] Rouatt, JW and Katznelson, H (1961) A study of the bacteria on the root surface and in the rhizosphere
soil of crop plants. J Appl Bacteriol 24(2): 164-171
[43] Siciliano, SD and Germida, JJ (1999) Taxonomic diversity of bacteria associated with the roots of field-
grown transgenic Brassia napus cv. Quest, compared to the non-transgenic B. napus cv. Excel and B.
rapa cv. Parkland. FEMS Microbiol Ecol 29: 263-272
[44] Furukawa, K (1982) Microbial degradation of polychlorinated biphenyls (PCBs), in AM Chakrabarty,
Ed., Biodegradation and detoxification of environmental pollutants, CRC Press: Boca Raton, FL. 147
[45] Bartha, R (1990) Isolation of microbes that metabolize xenobiotic compounds, in Isolation of
Biotechnological Organisms from Nature, DP Labeda, Ed., McGraw Hill: New York. 283-305
[46] Dunbar, J; White, S and Forney, LJ (1997) Genetic diversity through the looking glass: effect of
enrichment bias. Appl Environ Microbiol 63: 1326-1331
[47] Stach, JE and Burns, RG (2002) Enrichment versus biofilm culture: a functional and phylogenetic
comparison of polycyclic aromatic hydrocarbon-degrading microbial communities. Environ Microbiol
4(3): 169-182
[48] Dunbar, J; Wong, DCL; Yarus, DC and Forney, LJ (1996) Autoradiography method for isolation of
diverse bacterial species with unique catabolic traits. Appl Environ Microbiol 62: 4180-4185
[49] Gilbert, ES and Crowley, DE (1997) Plant compounds that induce polychlorinated biphenyl
biodegradation by Arthrobacter sp. strain B1B. Appl Environ Microbiol 63(5): 1933-1938
[50] Bogardt, AH and Hemmingsen, BB (1992) Enumeration of phenanthrene-degrading bacteria by an
overlayer technique and Its use in evaluation of petroleum-contaminated sites. Appl Environ Microbiol
58(8): 2579-2582
Methods for Rhizoremediation Research 53

[51] Alley, JF and Brown, LR (2000) Use of sublimation to prepare solid microbial media with water-
insoluble substrates. Appl Environ Microbiol 66(1): 439-442
[52] Sylvestre, M (1980) Isolation method for bacterial isolates capable of growth on p-chlorobiphenyl. Appl
Environ Microbiol 39(6): 1223-1224
[53] Kiyohara, H; Nagao, K and Yana, K (1982) Rapid screen for bacteria degrading water-insoluble, solid
hydrocarbons on agar plates. Appl Environ Microbiol 43(2): 454-457
[54] Ahn, Y; Sanseverino, J and Sayler, G (1999) Analyses of polycyclic aromatic hydrocarbon-degrading
bacteria isolated from contaminated soils. Biodegradation 10(2): 149-157
[55] Ensley, BD; Ratzkin, BJ; Osslund., TD and Simon, MJ (1983) Expression of naphthalene oxidation
genes in Escherichia coli results in the biosynthesis of indigo. Science 222: 167-169
[56] Wrenn, BA and Venosa, AD (1996) Selective enumeration of aromatic and aliphatic hydrocarbon
degrading bacteia by a most probable number procedure. Can J Microbiol 42: 252-258
[57] Haines, JR; Wrenn, BA; Holder, EL; Strohmeier, KL; Herrington, RT and Venosa, AD (1996)
Measurement of hydrocarbon-degrading microbial populations by a 96-well plate most-probable-number
procedure. J Ind Microbiol 16: 36-41
[58] Griffiths, RI; Whiteley, AS; O’Donnell, AG and Bailey, MJ (2000) Rapid method for coextraction of
DNA and RNA from natural environments for analysis of ribosomal DNA- and RNA-based microbial
community composition. Appl Environ Microbiol 66(12): 5488-5491
[59] Hurt, RA; Qiu, X; Wu, L; Roh, Y; Palumbo, AV; Tiedje, JM and Zhou, JZ (2001) Simultaneous
recovery of RNA and DNA from soils and sediments. Appl Environ Microbiol 67(10): 4495-4503
[60] Zhou, JZ; Bruns, MA and Tiedje, JM (1996) DNA recovery from soils of diverse composition. Appl
Environ Microbiol 62(2): 316-322
[61] Sessitsch, A; Gyamfi, S; Stralis-Pavese, N; Weilharter, A and Pfeifer, U (2002) RNA isolation from soil
for bacterial community and functional analysis:evaluation of different extraction and soil conservation
protocols. J Microbiol Methods 51(2): 171-179
[62] Garbeva, P; Van Overbeek, LS; Van Vuurde, JWL and Van Elsas, JD (2001) Analysis of endophytic
bacterial communities of potato by plating and denaturing gradient gel electrophoresis (DGGE) of 16S
rDNA based PCR fragments. Microbial Ecology 41(4): 369-383
[63] Mullin, BC; Joshi, PA and An, CS (1983) The isolation and purification of endophyte DNA from Alnus
glutinosa nodules. Can J Bot 61(11): 2855-2858
[64] Conn, V and Franco, CMM (2004) Analysis of the endophytic actinobacterial population in the roots of
wheat (Triticum aestivum L.) by terminal restriction fragment length polymorphism and sequencing of
16S rRNA clones. Appl Environ Microbiol 70(3): 1787-1794
[65] Parales, RE; Lee, K; Resnick, SM; Jiang, H; Lessner, DJ and Gibson, DT (2000) Substrate specificity of
naphthalene dioxygenase: effect of specific amino acids at the active site of the enzyme. Curr Opin
Biotechnol 182(6): 1641-1649
[66] Baldwin, BR; Nakatsu, CH and Nies, L (2003) Detection and enumeration of aromatic oxygenase genes
by multiplex and real-time PCR. Appl Environ Microbiol 69(6): 3350-3358
[67] Ringelberg, DB; Talley, JW; Perkins, EJ; Tucker, SG; Luthy, RG; Bouwer, EJ and Frederickson, HL
(2001) Successon of phenotypic, genotypic and metabolic community characteristics during in vitro
bioslurry treatment of polycyclic aromatic hydrocarbon-contaminated sediments. Appl Environ
Microbiol 67(1542-1550)
[68] Erb, RW and Wagner-Dobler, I (1993) Detection of polychlorinated biphenyl degradation genes in
polluted sediments by direct DNA extraction and polymerase chain reaction. Appl Environ Microbiol
59(12): 4065-4073
[69] Altschul, SF; Madden, TL; Schäffer, AA; Zhang, J; Zhang, Z; Miller, W and Lipman, DJ (1997) Gapped
BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:
3389-3402
[70] Hall, BG (2004) Phylogenetic Trees Made Easy, A How-To Manual. 2nd ed. Sunderland, Mass.: Sinauer
Associates.
[71] Edwards, K; Logan J and Saunders, N Ed. (2004) Real-time PCR: an essential guide. Wymondham,
Norfolk, UK: Horizon Bioscience.
[72] Meuer, S; Wittwer, C and Nakagawara, K Ed. (2001) Rapid cycle real-time PCR: methods and
applications. Berlin: Springer.
54 Mary Beth Leigh

[73] Denef, VJ; Park, J; Rodrigues, JLM; Tsoi, TV and Hashsham, S (2003) Validation of a more sensitive
method for using spotted oligonucleotide DNA microarrays for functional genomics studies on bacterial
communities. Environ Microbiol 5(10): 933-943
[74] Rhee, SK; Liu, XD; Wu, LY; Chong, SC; Wan, XF and Zhou, JZ (2004) Detection of genes involved in
biodegradation and biotransformation in microbial communities by using 50-mer oligonucleotide
microarrays. Appl Environ Microbiol 70(7): 4303-4317
[75] Wu, LY; Liu, XD; Schadt, CW; Tiedje, JM and Zhou, JZ (2005) Microarray-based detection of fento-to-
nanogram levels of genomic DNA from microbial genomes and microbial communities using whole
community genome amplification, in Proc. of American Society for Microbiology 105th General
Meeting, Atlanta, GA.
[76] Manefield, M; Whiteley, AS and Bailey, MJ (2004) What can stable isotope probing do for
bioremediation? International Biodeterioration and Biodegradation 54(2-3): 163-166
[77] McDonald, IR; Radajewski, S and Murrell, JC (2005) Stable isotope probing of nucleic acids in
methanotrophs and methylotrophs: A review. Org Geochem 36(5): 779-787
[78] Manefield, M; Whiteley, AS; Griffiths, RI and Bailey, MJ (2002) RNA stable isotope probing, a novel
means of linking microbial community function to phylogeny. Appl Environ Microbiol 68(11): 5367-
5373
[79] Lueders, T; Manefield, M and Friedrich, MW (2004) Enhanced sensitivity of DNA- and rRNA-based
stable isotope probing by fractionation and quantitative analysis of isopycnic centrifugation gradients.
Environ Microbiol 6(1): 73-78
[80] Lueders, T; Wagner, B; Claus, P and Friedrich, MW (2004) Stable isotope probing of rRNA and DNA
reveals a dynamic methylotroph community and trophic interactions with fungi and protozoa in oxic rice
field soil. Environ Microbiol 6(1): 60-72
[81] Cadisch, G; Espana, M; Causey, R; Richter, M; Shaw, E; Morgan, JAW; Rahn, C and Bending, GD
(2005) Technical considerations for the use of 15N-DNA stable-isotope probing for functional microbial
activity in soils. Rapid Commun Mass Spectrom 19(11): 1424-1428
[82] Padmanabhan, P; Padmanabhan, S; DeRito, C; Gray, A; Gannon, D; Snape, JR; Tsai, CS; Park, W;
Jeon, C and Madsen, EL (2003) Respiration of 13C-labeled substrates added to soil in the field and
subsequent 16S rRNA gene analysis of 13C-labeled soil DNA. Appl Environ Microbiol 69(3): 1614-
1622
[83] Leigh, MB; Park, J; Tiedje, JM and Ostrom, N (2004) Stable isotope probing for (polychlorinated)-
biphenyl degrading bacteria in soil, in Proc. of 10th International Symposium on Microbial Ecology
(ISME), Cancun, Mexico
[84] Leigh, MB; Park, J; Bailey, MJ and Tiedje, JM (2005) Identification of (polychlorinated) biphenyl-
degrading bacteria in contaminated soils, in Proc. of American Society for Microbiology (ASM) 105th
General Meeting, Atlanta, GA, USA
[85] Radajewski, S; McDonald, IR and Murrell, JC (2003) Stable-isotope probing of nucleic acids: a window
to the function of uncultured microorganisms. Curr Opin Biotechnol 14(3): 296-302
[86] Manefield, M; Whiteley, AS; Ostle, N; Ineson, P and Bailey, MJ (2002) Technical considerations for
RNA-based stable isotope probing: An approach to associating microbial diversity with microbial
community function. Rapid Commun Mass Spectrom 16(23): 2179-2183
[87] Manefield, M; Griffiths, RI; Leigh, MB; Fisher, R and Whiteley, AS (2005) Functional and
compositional comparison of two activated sludge communities remediating coking effluent. Environ
Microbiol 7(5): 715-722
[88] Singleton, DR; Powell, SN; Sangaiah, R; Gold, A; Ball, LM and Aitken, MD (2005) Stable-isotope
probing of bacteria capable of degrading salicylate, naphthalene, or phenanthrene in a bioreactor treating
contaminated soil. Appl Environ Microbiol 71(3): 1202-1209
[89] Tillmann, S; Strompl, C; Timmis, KN and Abraham, WR (2005) Stable isotope probing reveals the
dominant role of Burkholderia species in aerobic degradation of PCBs. FEMS Microbiol Ecol 52(2):
207-217
[90] Jeon, CO; Park, W; Padmanabhan, P; DeRito, C; Snape, JR and Madsen, EL (2003) Discovery of a
bacterium, with distinctive dioxygenase, that is responsible for in situ biodegradation in contaminated
sediment. Proc Natl Acad Sci 100(23): 13591-13596
[91] Hanson, JR; Macalady, JL; Harris, D and Scow, KM (1999) Linking toluene degradation with specific
microbial populations in soil. Appl Environ Microbiol 65(12): 5403-5408
Methods for Rhizoremediation Research 55

[92] Pelz, O; Chatzinotas, A; Zarda-Hess, A; Abraham, W-R and Zeyer, J (2001) Tracing toluene-
assimilating sulfate-reducing bacteria using 13C-incorporation in fatty acids and whole-cell
hybridization. FEMS Microbiol Ecol 38: 123-131
[93] Smit, E; Leeflang, P; Glandorf, B; van Elsas, JD and Wernars, K (1999) Analysis of fungal diversity in
the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and
temperature gradient gel electrophoresis. Appl Environ Microbiol 65(6): 2614-2621
[94] Griffiths, RI; Manefield, M; Ostle, N; McNamara, N; O'Donnell, AG; Bailey, MJ and Whiteley, AS
(2004) 13CO2 pulse labelling of plants in tandem with stable isotope probing: methodological
considerations for examining microbial function in the rhizosphere. J Microbiol Methods 58(1): 119-129
[95] Rangel-Castro, JI; Killham, K; Ostle, N; Nicol, GW; Anderson, IC; Scrimgeour, CM; Ineson, P; Meharg,
A and Prosser, JI (2005) Stable isotope probing analysis of the influence of liming on root exudate
utilization by soil microorganisms. Environ Microbiol 7(6): 828-838
CONSTRUCTED WETLANDS FOR PHYTOREMEDIATION

Rhizofiltration, phytostabilisation and phytoextraction

MARINUS L. OTTE AND DONNA L. JACOB

Wetland Ecology Research Group, School of Biological and
Environmental Science, College of Life Sciences, University College
Dublin, Belfield, Dublin 4, Ireland Tel:+353-17162019, Fax:+353-
17161153, E-mail: [email protected]

1. Introduction

The utilisation of wetlands for remediation of polluted soils and waters via
rhizofiltration, phytostabilisation and phytoextraction has been increasing steadily over
the past decades. The use of wetlands for quality improvement of wastewater, referred to
as rhizofiltration, is the best known and most researched application of constructed
wetlands. Wetlands as a cover over polluted soils and sediments, such as mine tailings,
are referred to as phytostabilisation. It is a less common utilisation of wetlands, no doubt
because of its more limited range of applications compared to rhizofiltration. The least
applied and studied utilization of wetlands is that of phytoextraction, in which removal
of substances is carried out through uptake by plants.
This paper aims to give an overview of the utilization of wetlands for the three
approaches in phytoremediation described above. It is not intended to be an exhaustive
review, but to give an impression of the versatility of wetlands and the wide range of
potential applications.

2. The importance of biogeochemistry to wetlands for phytoremediation

2.1. GENERAL BIOGEOCHEMISTRY OF WETLAND SOILS AND SEDIMENTS

Of paramount importance to using wetlands for phytoremediation purposes, particularly

rhizofiltration and phytostabilisation are the biogeochemical characteristics of wetlands.
Wetland substrates are typically waterlogged, at least during part of the annual cycle [1].
Because oxygen diffuses through water 10,000 times slower than through air, the supply
of oxygen to wetland substrates is much slower than to dryland substrates. Upon
flooding, the oxygen levels in well aerated dryland soils rapidly decrease, and generally

57
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 57 – 67.
© 2006 Springer.
58 Marinus L. Otte and Donna L. Jacob

wetland soils are low in or devoid of oxygen. These anaerobic conditions do not just
require special adaptations in plants and sediment infauna, but also in the microbial
community. Depending on the chemical composition of the substrate, a wide range of
terminal electron acceptors other than oxygen are used for respiration by these
organisms [2, 3]. Which terminal electron acceptor is used depends on their availability
and that of other substances. This is best explained by considering what happens upon
flooding of a well-aerated, dry soil, because under those conditions the redox reactions
that occur due to the respiration activity of micro-organisms follow a thermodynamic
sequence, from more oxidised to more reduced [2, 3]. After the disappearance of
oxygen, nitrate is reduced to N2 or N2O. Because this process leads to the formation of
gases and the subsequent removal of nitrogen from the substrate, this process is referred
to as denitrification. After all nitrate has been removed, ferric iron, Fe(III), may be
–
reduced to ferrous iron, Fe(II). Upon removal of most ferric iron, sulphate (SO42 ) may
2–
be reduced to sulphide (S ), and when most sulphate is removed carbon dioxide (CO2)
may be reduced to methane (CH4). This is a highly simplified explanation of what is, in
fact, a highly complex process, but these are the reactions that are most important for the
purpose of this paper. The reactions are more or less exclusive, in that denitrification
will not take place in the presence of oxygen, iron reduction will not happen in the
presence of nitrate, and so forth. This is because the reduction of, for example, ferric to
ferrous iron, is thermodynamically unfavourable in the presence of nitrate. Like all soils
and sediments, wetland substrates contain a wide range of inorganic and organic redox-
sensitive compounds, and which reactions take place not only depends on the
thermodynamic characteristics, but also on the different micro-organisms competing for
oxidisable substrates [4]. Therefore, while the redox potential of a wetland sediment
gives some indication of the chemical status, it cannot be used to assess the actual
chemical composition. As a general rule however, the following observations apply:
• flooding of any soil or sediment leads to rapid denitrification, and wetland
sediments typically contain low levels of nitrate,
• most wetlands contain relatively high concentrations of ferrous iron, and
• because of the continuous influx of sulphate from flooding with seawater
in coastal and estuarine wetlands (salt marshes, coastal lagoons, mud flats),
their sediments are typically rich in sulphide, but rarely produce methane.
The pH of soils and sediments is also affected by waterlogging [3]. Upon water-
logging of acid soils, the pH increases towards neutral, mainly due to the reduction of
Fe(III) to Fe(II), while in alkaline soils the pH decreases towards neutral due to the
build-up of CO2 and subsequent formation of carbonic acid. As a result, wetland
substrates tend to be circum-neutral. Exceptions are wetlands that have organic-matter
rich, ferric-iron poor soils, such as peat bogs, which tend to be more acidic, and
wetlands with substrates that are very poor in organic matter, such as wetlands formed
on metal mine tailings, which may be alkaline.
Constructed Wetlands for Phytoremediation 59

2.2. PLANTS AND HETEROGENEITY OF WETLAND SOILS

AND SEDIMENTS

Wetland substrates are normally highly heterogeneous. While the bulk soil may be
chemically reduced, the surface layer of the substrate is typically oxidised, particularly if
the substrate is regularly exposed to the air, for example due to fluctuating water levels.
This layer can be very thin (a few millimetres), as is for example typically found in
coastal mudflats, but its importance in element cycling must not be underestimated.
More important to substrate heterogeneity, however, is the presence of plants. Roots
penetrating the soil often form a very dense network and take up a large volume. They
affect the soil immediately surrounding them (the rhizosphere) due to organic root
exudates and organic matter derived from die-back [e.g. 5, 6, 7] and due to radial
oxygen loss [e.g. 8, 9]. The above-ground biomass as well contributes significantly to
input of organic matter into wetland substrates [10]. The result is a three-dimensional
patchwork of areas in the substrate greatly varying in organic matter content, redox
status and elemental content. While organic compounds are supplied by the plants in one
area, they are consumed by oxidation/reduction reactions in other areas, perhaps only
millimetres away. Similarly, while ferrous iron is being oxidised very near the root
surface, ferric iron is being reduced by anaerobic bacterial activity at the interface
between oxidised rhizosphere and chemically reduced bulk substrate. The substrate
heterogeneity makes it difficult to predict the mobility and bioavailability of nutrients
and pollutants in wetlands, at least at the scale of the rhizosphere [11]. Despite this
however, wetlands have proven to be very effective in retaining pollutants from water
and in stabilising contaminated sediments.

3. Rhizofiltration

3.1. RANGE OF POLLUTANTS AND EFFICIENCY

Rhizofiltration refers to the use of wetlands for retention of pollutants from water. While
the prefix “rhizo” suggests that the activity of the plants in these systems is limited to the
roots, it is in fact the interaction of the whole plants with the substrates that is the key to
the success of wetlands for this application. Natural and constructed wetlands have been
successfully utilised for the removal of a wide range of pollutants, including plant
macronutrients, metals, organic compounds and biological contaminants, such as
coliform bacteria and parasites, and they also improve pH, Biological Oxygen Demand
(BOD) and Chemical Oxygen Demand, (COD), for examples see Table 1.
While an overwhelming amount of reports exists to show that wetlands can
successfully remove a wide range of pollutants, not all pollutants are equally removed by
all types of wetlands. In fact, a wetland may retain one set of pollutants while acting as a
source of another. Birch and co-workers [18] for example, reported efficient removal of
Cr, Cu, Pb, Ni and Zn, but also negative removal efficiencies of -84% for Fe and -294%
for Mn. Therefore, wetlands generally act as sinks of pollutants, but can also act as
sources. Whether or not a wetland acts as a sink will depend on its size and structure
60 Marinus L. Otte and Donna L. Jacob

(e.g. availability of binding sites for the pollutant, presence of appropriate micro-flora
and plant species). Samecka-Symerman et al. [17] and Best et al. [21], for example,
showed that the removal efficiency of wetlands strongly depended on the type of plants
present in the systems. The removal efficiency will also depend on the season, as has
been shown for both natural and constructed wetlands [13, 24, 25, 26]. As a general rule
though, wetlands show net removal of pollutants over longer periods of time, as long as
they are large enough to provide enough treatment capacity relative to the pollutant
loading rate.
Table 1. Examples of applications and efficiency of wetlands for quality improvement of water

Type of pollutant Efficiency (% removal) References

Macronutrients
Ammonium-N 16-67 12, 14
Nitrate/nitrite 40 13
Total Kjeldahl nitrogen (TKN) 49-81 14, 20
Organic nitrogen 82 14
Soluble Reactive Phosphate (SRP) 56 13
Total-P (TP) 44-68 12, 13
Metals and metalloids
Al 81-97 17
As 65 15
Ba 70-95 17
Cd 58-71 17
Co 39-98 16
Cu 49-65 17, 18
Fe 91-97 16, 17
Mn 91-99 16, 17
Ni 22-67 16, 17, 18
Se 69 22
Sr 24-51 17
Zn 52-95 15, 17, 18
V 100 17
Organic compounds, including 63-100 19, 20, 21
explosives
Coliform bacteria 26-98 18
Eggs of human parasites 94-100 23
COD 81 20
BOD5 72-89 12, 14, 20
Suspended solids (SS) 43-94 12, 14

3.2. PROCESSES INVOLVED IN REMOVAL OF POLLUTANTS

Why wetlands are so successful in removing a wide range of pollutants form wastewater
becomes clear when the underlying processes are considered. Key to these processes is
the heterogeneity of the substrate. The removal of nitrogen is a good example to
illustrate this. Mineralisation of nitrogen from plant-derived organic compounds leads
initially to the formation of ammonium. In the aerobic environment of the surface layer
Constructed Wetlands for Phytoremediation 61

of the sediments and in the rhizosphere of plants, ammonium may be further oxidised to
nitrate. The latter process (nitrification) does not occur under the anaerobic conditions of
the bulk wetland substrate and so inverse gradients in the nitrate and ammonium
concentrations are formed between the aerobic (high in nitrate, low in ammonium) and
anaerobic areas (low in nitrate, high in ammonium) in the substrate. As a consequence,
ammonium diffuses from anaerobic to aerobic areas, where it is converted to nitrate.
Nitrate, in turn, diffuses from aerobic into anaerobic areas, where it is subsequently
converted to nitrous oxide (N2O) or gaseous nitrogen (N2) by denitrifying bacteria.
Because both N2O and N2 are gases, denitrification effectively leads to the removal of
nitrogen from the system. In a similar fashion, and equally dependent on alternation of
aerobic and anaerobic areas in the substrate, sulphur forms volatile compounds (e.g. H2S
and dimethyl sulphide), which leads to an efflux of sulphur from wetland substrates (for
detailed diagrams of the cycles of N and S associated with wetlands, see [1]). The
interactions between plants, bacteria [27] and the wetland substrate, particularly by the
formation of an oxidised rhizosphere in an otherwise chemically reduced bulk substrate;
also favour the volatilisation via the lacunal spaces of macrophytes of elements that can
be methylated, such as Se [22] and Hg [28]. The efficacy of wetlands for the removal of
a wide range of organic compounds is also due to the heterogeneity of wetlands. Both
anaerobic and aerobic degradation of organic compounds occurs [e.g. 29, 30, 31], while
removal of the lighter compounds can at least in part be explained by volatilisation [30,
32].
The plant-induced heterogeneity in redox status of wetland substrates is also the
main driver for the immobilisation and retention of metals such as Fe, Cd, Cu and Zn. In
the anaerobic, chemically reduced bulk soil, many metals form insoluble, and therefore
immobile, sulphides, provided of course that sulphides are formed in sufficient supply
[33]. In the rhizosphere, the roots affect the redox status by the supply of oxygen
through radial oxygen loss [8,9] and by supplying habitat for iron-oxidizing bacteria
[34], both of which in turn lead to the oxidation and precipitation of iron and manganese
oxides in the rhizosphere and on the roots (iron plaque) [35, 36]. Many metals and
metalloids have high binding affinities for iron and manganese oxides, and iron and
manganese deposits on the roots and in the rhizospheres of wetland plants therefore
contain relatively high concentrations (compared to the bulk substrate) of those elements
[e.g. 37, 38, 39].
The slow flow rates through wetlands are the main reason why suspended solids are
removed – they simply precipitate out of suspension. The micro-pores of the substrate
act as filters and this too contributes to the retention of suspended solids and eggs of
parasites from the water. Removal of coliform bacteria is attributed to some extent to
filtration as well [40], but the incidence of sunlight and UV-radiation is thought to be a
more important factor [41].
62 Marinus L. Otte and Donna L. Jacob

4. Phytostabilisation

4.1. WHY COVER POLLUTED SOILS AND SEDIMENTS UNDER

WETLANDS?

Wetlands for phytostabilisation of polluted soils and sediments have so far been limited
to mining activities, for example for coal [42, 43], lignite [44, 45] and uranium [46].
One reason to cover soils and sediments under water may be the prevention of erosion
and dispersal of pollutants due to wind and dust blows. Even in humid climates
occasional droughts may cause dust blows, as happened at the abandoned lead-zinc
mines of Silvermines, Ireland [47]. Another reason is that, as described for
rhizofiltration above, the biogeochemistry of wetlands favours the chemical
immobilisation of pollutants, particularly metals. The anaerobic, reduced conditions of
the bulk substrate may lead to the formation of insoluble metal sulphides, while the
aerobic, oxidised conditions of the plant rhizospheres and the surface layer favour
immobilisation due to adsorption on and co-precipitation with iron and manganese
oxides and phosphates. The plants also contribute by supplying organic matter.
Pollutants are taken up by the roots and returned to the substrate bound to organic matter
after the plants die off, or they may bind to plant-derived organic matter (through die-
back or exudation) in the substrates [48]. While all these processes favour
immobilisation, there is of course the problem that plants, and other organisms, may also
mobilise pollutants.

4.2. MOBILISATION OF POLLUTANTS BY WETLAND ORGANISMS

While most studies agree that wetlands lead to overall net immobilisation, organisms
may mobilise pollutants on a smaller scale, both in time and in space. The burrowing
activity of sediment infauna, for example, may lead to re-suspension of sediments into
the water column and oxidation of reduced compounds and subsequent release of metals
into the water column [49]. Similarly, the growth of plant roots into the anaerobic,
reduced bulk soil will, at least temporarily, mobilise metals from sulphides in the
rhizosphere due to the oxidising activity of the roots and associated micro-organisms
[11, 50, 51, 52]. While this is a matter of concern, there are no reports of mass
mobilisation of pollutants from wetland covered soils or sediments.

4.3. METAL TOLERANCE IN WETLAND PLANTS

Particularly in the case of pollutants that are potentially toxic to the plants, such as
metals, the question arises whether or not the wetland plants selected for the purpose of
phytostabilisation should be tolerant. This problem is particularly well described for
remediation of dry mine tailings, where only metal-tolerant populations of certain
species, mostly grasses, can be used for revegetation of mine wastes. Classic studies on
this topic were for example carried out by Bradshaw and co-workers [53]. However, it
has only relatively recently been established that wetland plants typically show innate
tolerance to metals.
Constructed Wetlands for Phytoremediation 63

McNaughton and co-workers [54] in 1974 were the first to report on tolerance to zinc,
cadmium and lead in Typha latifolia and observed that this trait existed in this species
without the development of specific metal-tolerant populations. Tolerance to copper and
nickel in the same species was confirmed ten years later by Taylor and Crowder [55].
More recently, Ye and co-workers [56] confirmed the findings for T. latifolia and the
same research team made similar observations in Phragmites australis [57, 58]. Then
Otte and co-workers reported that populations of floating sweetgrass, Glyceria fluitans,
which had not previously been exposed to zinc, were capable of growing well in zinc-
lead mine tailings containing highly elevated levels of metals [59]. Research by the same
group has now established the existence of innate metal tolerance in many wetland
plants [60, 61, 62, 63, 64]. This has been confirmed for several other species by Wong
and co-workers [65]. What this means is that wetland plants for applications in both
rhizofiltration or phytostabilisation of metal-contaminated water or soils need not be
sourced from specific metal-tolerant populations, as is the case for dryland conditions
[53], but that they can be obtained from any population, anywhere. This is an important
consideration, as it reduces the costs of supply of suitable plants and it avoids depletion
of specific plant populations.

5. Phytoextraction

5.1. REMOVAL OF POLLUTANTS VIA PLANTS

Phytoextraction refers to the removal of pollutants from soils or water through plant
uptake [66]. In this approach, the pollutants are not simply recycled back into the
wetland, as is the case in phytostabilisation, but they are removed from the system by
harvesting the plants or by volatilisation through the plants. The latter only applies to
substances that can form gaseous compounds, such as Se [22, 67] and Hg [28]. For
phytoextraction through uptake and harvest to be effective, uptake by the plants must be
high relative to the amount of pollutant present. This is the case when the amount of
pollutant is clearly defined, for example when relatively small volumes of water are
contaminated. While the concentrations of pollutants resulting from uptake in aquatic
plants may not be very high, the sheer volumes of the plants may be enough to
successfully remove the pollutants from the water within a foreseeable period of time
[68, 69, 70]. This is different, however, when attempts are made to use emergent plants
for removal of pollutants from soils or sediments. In that case, the amount of pollutants
present in the soils is often not clearly defined and much larger than that taken up by the
plants annually. Some plants are much better at translocating pollutants to the
harvestable parts (typically the shoots) than others [e.g. 71, 72, 73], but the amounts
removed are typically not enough to reduce the levels of pollution in the soils within
reasonable time [74].
One particular approach to phytoextraction, that of phytomining, has not had much
attention in general, let alone in relation to wetlands, yet may have great potential.
Phytomining uses the uptake and accumulation in plants to extract valuable metals from
diffuse sources for commercial exploitation. The plants are harvested after growing in
64 Marinus L. Otte and Donna L. Jacob

moderately metal-rich soils for time and the metals extracted and concentrated from the
plant biomass. While the effort is not worth the gain for readily available metals like
zinc and lead, Brooks and co-workers [75] show that even at current prices, it may in
fact be worthwhile for precious metals, like gold, under certain circumstances.
According to the Science Citation Index, the search for “wetland and phytomining”
returns no matching reports. However, recent work by Otte and Jacob [76] suggests that
plants from mineral rich wetlands, such as those associated with hot springs, show
exceptional accumulation of less studied, yet valuable metals, such as Th, U and W,
suggesting that there is potential for the use of plants wetland plants for phytomining.

6. Conclusions

The efficacy of wetlands for applications in phytoremediation, particularly for

rhizofiltration, is a proven fact. Not only can they be applied to a wide range of
pollutants, but they also provide suitable habitat for wildlife [77]. At the same time,
while other reviews agree with this conclusion [78, 79, 80], care must be taken in the
design, and failures have occurred [81]. Design mistakes may include building the
system too small (rhizofiltration), with too little organic matter (rhizofiltration,
phytostabilisation), and/or creating a system that is too homogeneous.
There is much scope for further development of wetlands for phytoremediation,
particularly in the areas of phytostabilisation and phytomining. Where the latter is
concerned, bioengineering (wetland) plants to improve uptake of valuable metals is
likely to be worthwhile undertaking [82].

References
[1] Mitsch, WJ and Gosselink JG (2000) Wetlands. 3 rd edition, John Wiley & Sons, New York
[2] Gambrell RP and Patrick WH, Jr (1978) Chemical and microbiological properties of anaerobic soils and
sediments, in DD Hook and RMM Crawford, Eds., Plant life in anaerobic environments. Ann Arbor
Publishers, Michigan, USA, Chapter 13, pp. 375-424
[3] Ponnamperuma, FN (1984) Effects of flooding on soils, in TT Kozlowski, Ed., Flooding and plant
growth. Academic Press, Orlando, Fl, USA, pp. 9-45
[4] Van Bodegom, PM and Scholten, JCM (2001) Microbial processes of CH4 production in a rice paddy
soil: Model and experimental validation. Geochim Cosmochim Ac 65: 2055-2066
[5] Bertin, C; Yang, XH and Weston, LA (2003) The role of root exudates and allelochemicals in the
rhizosphere. Plant Soil 256: 67083
[6] Lu, YH; Watanabe, A and Kimura, M (2004) Contribution of plant photosynthates to dissolved organic
carbon in a flooded rice soil. Biogeochemistry 71: 1-15
[7] Saarnio, S; Wittenmayer, L and Merbach, W (2004) Rhizosphere exudation of Eriophorum vaginatum
L. – Potential link to methanogenesis. Plant Soil 267: 343-355
[8] Purnobasuki, H and Suzuki, M (2004) Aerenchyma formation and porosity of a mangrove plant,
Sonneratia alba (Lytharaceae). J Plant Res 117: 465-472
[9] Bezbaruah, AN and Zhang, TC (2005) Quantification of oxygen release by bulrush (Scirpus validus)
roots in a constructed treatment wetland. Biotechnol Bioeng 89: 308-318
[10] Anderson, CJ and Cowell, BC (2004) Mulching effects on the seasonally flooded zone of west-central
Florida, USA, wetlands. Wetlands 24: 811-819
[11] Jacob, DL and Otte, ML (2003) Conflicting processes in the wetland plant rhizosphere: metal retention
or mobilization? Water Air Soil Poll Focus 3: 91-104
Constructed Wetlands for Phytoremediation 65

[12] Klomjek, P and Nitisoravut, S (2005) Constructed treatment wetland: a study of eight plant species
under saline conditions. Chemosphere 58: 585-593
[13] Fink DF and Mitsch WJ (2004) Seasonal and storm event nutrient removal by a created wetland in an
agricultural watershed. Ecol Eng 23: 313-325
[14] Yirong, C and Puetpaiboon, U (2004) Performance of constructed wetland treating wastewater from
seafood industry. Water Sci Technol 49: 289-294
[15] Beining, BA and Otte, ML (1997) Retention of metals and longevity of a wetland receiving mine
leachate, in JE Brandt; JR Galevotic; L Kost and J Trouart, Eds., Proc. 14th Annual National Meeting -
Vision 2000: An Environmental Commitment. American Society for Surface Mining and Reclamation,
Austin, Texas, May 10 -16, 1997, pp. 43-46
[16] Ye, ZH; Whiting, SN; Lin, ZQ; Qian, JH and Terry, N (2001) Removal and distribution of iron,
manganese, cobalt, and nickel within a Pennsylvania constructed wetland treating coal combustion by-
product leachate. J Environ Qual 30: 1464-1473
[17] Samecka-Symerman, A; Stepien, D and Kempers, AJ (2004) Efficiency in removing pollutants by
constructed wetland purification systems in Poland. J Toxicol Env Heal A 67: 265-275
[18] Birch, GF; Matthai, C; Fazeli, MS and Suh, J (2004) Efficiency of a constructed wetland in removing
contaminants from stormwater. Wetlands 24: 459-466
[19] Keefe, SH; Barber, LB; Runkel, RL and Ryan, JN (2004) Fate of volatile organic compounds in
constructed treatment wetlands. Environ Sci Technol 38: 2209-2216
[20] Ji, GD; Sun, T; Zhou, QX; Sui, X; Chang, SJ and Li, PJ (2002) Constructed subsurface flow wetland for
treating heavy oil-produced water of the Liaohe oilfield in China. Ecol Eng 18: 459-465
[21] Best, EPH; Sprecher, SL; Larson, SL; Fredrickson, HL and Bader FD (1999) Environmental behavior of
explosives in groundwater in groundwater from the Milan army ammunition plant in aquatic and
wetland plant treatments. Removal, mass balances and fate in groundwater of TNT and RDX.
Chemosphere 38: 3383-3396
[22] Lin, ZQ and Terry, N (2003) Selenium removal by constructed wetlands: quantitative importance of
biological volatilization in the treatment of selenium-laden agricultural drainage water. Environ Sci
Technol 37: 606-615
[23] Stott, R; May, E and Mara, DD (2003) Parasite removal by natural wastewater treatment systems:
performance of waste stabilisation ponds and constructed wetlands. Water Sci. Technol. 48: 97-104
[24] Gauci, V; Fowler, D; Chapman, SJ and Dise, NB (2004) Sulphate deposition and temperature controls
on methane emission and sulfur forms in peat. Biogeochemistry 71: 141-162
[25] Qiu, S; McComb, AJ; Bell, RW and Davis, JA (2004) Phosphorus dynamics from vegetated catchment
to lakebed during seasonal refilling. Wetlands 24: 828-836
[26] Wiessner, A; Kappelmeyer, U; Kuschk, P and Kastner, M (2005) Influence of the redox condition
dynamics on the removal efficiency of a laboratory-scale constructed wetland. Water Res 39: 248-256
[27] Benoit, JM; Gilmour, CC and Mason, RP (2001) The influence of sulfide on solid-phase mercury
bioavailability for methylation by pure cultures of Desulfobulbus propionicus (1pr3). Environ Sci
Technol 35: 127-132
[28] Lindberg, SE; Dong, WJ; Chanton, J; Qualls, RG and Meyers, T (2005) A mechanism for bimodal
emission of gaseous mercury from aquatic macrophytes. Atmos Environ 39: 1289-1301
[29] Guerin, TF (1999) The anaerobic degradation of endosulfan by indigenous microorganisms from low
oxygen soils and sediments. Environ Pollut 106: 13-21
[30] Lahvis, MA; Baehr, AL and Baker, RJ (1999) Quantification of aerobic biodegradation and
volatilization rates of gasoline hydrocarbons near the water table under natural attenuation conditions.
Water Resour Res 35: 753-765
[31] Armenante, PM; Kafkewitz, D; Lewandowski, GA and Jou, CJ (1999) Anaerobic-aerobic treatment of
halogenated phenolic compounds. Water Res 33: 681-692
[32] Keffe, SH; Barber, LB; Runkel, RL and Ryan, JN (2004) Fate of volatile organic compounds in
constructed wastewater treatment wetlands. Environ Sci Technol 38: 2209-2216
[33] Allen, HE; Fu, G and Deng, B (2003) Analysis of acid-volatile sulfide (AVS) and simultaneously
extracted metals (SEM) for the estimation of potential toxicity in aquatic sediments. Environ Toxicol
Chem 12:1441-53
[34] Emerson, D; Weiss, JV and Megonical, JP (1999) Iron-oxidizing bacteria are associated with ferric
hydroxide precipitates (Fe-plaque) on the roots of wetland plants. Appl Environ Microb 65: 2758-2761
66 Marinus L. Otte and Donna L. Jacob

[35] Mendelssohn, IA; Kleiss, BA and Wakeley, JS (1995) Factors controlling the formation of oxidized root
channels: a review. Wetlands 15: 37-46
[36] Wang, T and Peverly, JH (1996) Oxidation states and fractionation of plaque iron on roots of common
reeds. Soil Sci Soc Am J 60: 323-329
[37] Otte, ML; Rozema, J; Koster, L; Haarsma, MS and Broekman, RA (1989) Iron plaque on roots of Aster
tripolium L.: interaction with zinc uptake. New Phytol 111: 309-317
[38] St.-Cyr L and Campbell PGC (1996) Metals (Fe, Mn, Zn) in the root plaque of submerged aquatic plants
collected in situ: relations with metal concentrations in the adjacent sediments and in the root tissue.
Biogeochemistry 33: 45-76
[39] Doyle, MO and Otte, ML (1997) Organism-induced accumulation of iron, zinc and arsenic in wetland
soils. Environ Pollut 96: 1-11
[40] Arias, CA; Cabello, A; Brix, H and Johansen, NH (2003) Removal of indicator bacteria from municipal
wastewater in an experimental two-stage vertical flow constructed wetland system. Water Sci Technol
48: 35-41
[41] Mayo, AW (2004) Kinetics of bacterial mortality in granular bed wetlands. Phys Chem Earth 29: 1259-
1264
[42] Cole, CA and Lefebvre, EA (1991) Soil and water characteristics of a young surface mine wetland.
Environ Manage 15: 403-410
[43] Sistani, KR; Mays, DA and Taylor, RW (1995) Biogeochemical characteristics of wetlands developed
after strip mining for coal. Commun Soil Sci Plan 26: 3221-3229
[44] King, SL (1996) The effects of flooding on bottomland hardwood seedlings planted on lignite mine spoil
in east Texas. Tex J Sci 75-84
[45] Johns, D; Williams, H; Farrish, K and Wagner, S (2004) Denitrification and soil characteristics of
wetlands created on two mine soils in east Texas, USA. Wetlands 24: 57-67
[46] Pappin Willianen, S; Beckett, P and Courtin, G (1998) Progress in establishing wetland plants in
permanently-flooded uranium tailings. Min Pro Ext Met Rev 19: 47-60
[47] Farrell, L (2002) Rehabilitation of Silvermines. Extractive Industry of Ireland Annual Review 3: 41-43
[48] Weis, JS and Weis, P (2004) Metal uptake, transport and release by wetland plants: implications for
phytoremediation and restoration. Environ Int 30: 685-700
[49] Banta, GT and Andersen, O (2003) Bioturbation and the fate of sediment pollutants - Experimental case
studies of selected infauna species. Vie Milieu 53: 233-248
[50] Stolz, E and Greger, M (2002) Cottongrass effects on trace elements in submerged mine tailings.
J Environ Qual 31: 1477-1483
[51] Jacob, DL and Otte, ML (2004) Influence of Typha latifolia and fertilization on metal mobility in two
different Pb-Zn mine tailings types. Sci Total Environ 333: 9-24
[52] Jacob DL and Otte ML (2004) Long-term effects of submergence and wetland vegetation on metals in a
90-year old abandoned Pb-Zn mine tailings pond. Environ Pollut 130: 337-345
[53] Smith, RA and Bradshaw, AD (1979) The use of metal tolerant plant populations for the reclamation of
metalliferous wastes. J Appl Ecol 16: 595-612
[54] McNaughton, SJ; Folsom, TC; Lee, T; Park, F; Price, C; Roeder, D; Schmitz, J and Stockwell, C (1974)
Heavy metal tolerance in Typha latifolia without the evolution of tolerant races. Ecology 55: 1163-1165
[55] Taylor, GJ and Crowder, AA (1984) Copper and nickel tolerance in Typha latifolia clones from
contaminated and uncontaminated environments. Can J Bot 62: 1304-1308
[56] Ye, ZH; Baker, AJM; Wong, MH and Willis, AJ (1997) Zinc, lead and cadmium tolerance, uptake and
accumulation by Typha latifolia. New Phytol 136: 469-480
[57] Ye, ZH; Baker, AJM; Wong, MH and Willis, AJ (1997) Zinc, lead and cadmium tolerance, uptake and
accumulation by common reed, Phragmites australis Cav Trin ec Steudel. Ann Bot 80: 363-370
[58] Ye, ZH; Wong, MH; Baker, AJM and Willis, AJ (1998) Comparison of biomass and metal uptake
between two populations of Phragmites australis grown in flooded and dry conditions. Ann Bot 82:
83- 87
[59] McCabe OM and Otte ML (2000) The wetland grass Glyceria fluitans for revegetation of mine tailings.
Wetlands 20: 548-559
[60] Moran BM and Otte ML (2004) Innate zinc tolerance in the wetland grass Glyceria fluitans. Phyton 44:
95-108
[61] Matthews DJ; Moran BM and Otte ML (2004) Zinc tolerance, uptake and accumulation in the wetland
plants Eriophorum angustifolium, Juncus effusus and Juncus articulatus. Wetlands 24: 859-869
Constructed Wetlands for Phytoremediation 67

[62] Matthews DJ; Moran BM; McCabe PF and Otte ML (2004) Zinc tolerance, uptake, accumulation and
distribution in plants and protoplasts of five European populations of the wetland grass Glyceria
fluitans. Aquat Bot 80: 39-52
[63] Matthews DJ; Moran BM and Otte ML (2005) Screening the wetland plant species Alisma plantago-
aquatica, Carex rostrata and Phalaris arundinacea for innate tolerance to zinc and comparison with
Eriophorum angustifolium and Festuca rubra Merlin. Environ Pollut 134: 343-351
[64] Matthews DJ; Gallagher TF and Otte ML (2005) An analysis of genetic variation in zinc-tolerant
European populations of the wetland grass Glyceria fluitans using amplified fragment length
polymorphism (AFLP) analysis. Aquat Bot In press
[65] Deng, H; Ye, ZH and Wong, MH (2004) Accumulation of lead, zinc and copper and cadmium by
twelve wetland plant species thriving in metal contaminated sites in China. Environ Pollut 132: 29-40
[66] Salt, DE; Blaylock, M; Kumar, NPBA; Dushenkov, V; Ensley, BD; Chet, I and Raskin, I (1995)
Phytoremediation: a novel strategy for the removal of toxic metals from the environment using plants.
Biotechnology 13: 468-474
[67] Lin, ZQ; de Souza, M; Pickering, IJ and Terry, N (2002) Evaluation of the macroalga, muskgrass, for
the phytoremediation of selenium-contaminated agricultural drainage water by microcosms. J Environ
Qual 31: 2104-2110
[68] Kamal, M; Ghaly, AE; Mahmoud, N and Cote, R (2004) Phytoaccumulation of heavy metals by aquatic
plants. Environ Int 29: 1029-1039
[69] Mkandawire, M; Tauert, B and Dudel, EG (2004) Capacity of Lemna gibba L. (Duckweed) for uranium
and arsenic phytoremediation in mine tailing waters. Int J Phytoremediat 6: 347-362
[70] Al Rmalli, SW; Harrington, CF; Ayub, M and Haris, PI (2005) A biomaterial based approach for arsenic
removal from water. J Environ Monitor 7: 279-282
[71] Stolz, E and Greger, M (2002) Accumulation properties of As, Cd, Cu, Pb and Zn by four wetland plant
species growing in submerged mine tailings. Environ Exp Bot 47: 271-280
[72] Fritioff, A and Greger, M (2003) Aquatic and terrestrial plant species with potential to remove heavy
metals from stormwater. Int J Phytoremediat 5: 211-224
[73] Gothberg, A; Greger, M; Holm, K and Bengtsson, BE (2004) Influence of nutrient levels on uptake and
effects of mercury, cadmium, and lead in water spinach. J Environ Qual 33: 1247-1255
[74] Hinton, TG; Knox, AS; Kaplan, DI and Sharitz, R (2005) Phytoextraction of uranium by native trees in a
contaminated wetland. J Radioanal Nucl Ch 264: 417-422
[75] Brooks, RR; Chambers, MF; Nicks, LJ and Robinson, BH (1998) Phytomining. Trends Plant Sci 3:
359-362
[76] Otte, ML and Jacob, DL (2005) Chemical fingerprinting of plants from contrasting wetlands – salt
marsh, geothermal and mining-impacted. Phyton, in press
[77] Horstman, AJ; Nawrot, JR and Woolf, A (1998) Mine-associated wetlands as avian habitat. Wetlands
18: 298-304
[78] Williams, JB (2002) Phytoremediation in wetland ecosystems: progress, problems and potential. Crit
Rev Plant Sci 21: 607-635
[79] Kennedy, G and Mayer, T (2002) Natural and constructed wetlands in Canada: an overview. Water
Qual. Res J Can 37: 295-325
[80] Li, Q-F; Li, Z-A; Ren, H; Du, W-B; Tian, S-N and Peng S-L (2004) The role of wetland plants and soil
in decontamination of heavy metals. J Trop Subtrop Bot 12: 273-279
[81] Barton, CD and Karathanasis, AD (1999) Renovation of a failed constructed wetland treating acid mine
drainage. Environ Geol 39: 39-50
[82] Dushenkov, S; Skarzhinskaya, M; Glimelius, K; Gleba, D and Raskin, I (2002) Bioengineering of a
phytoremediation plant by means of somatic hybridization. Int J Phytoremediat 4: 117-126
INFLUENCE OF HELOPHYTES ON REDOX REACTIONS
IN THEIR RHIZOSPHERE

ARNDT WIESSNER, PETER KUSCHK, UWE

KAPPELMEYER, OLIVER BEDERSKI, ROLAND-ARNO
MÜLLER AND MATTHIAS KÄSTNER
Department of Remediation Research, UFZ Centre for Environmental
Research Leipzig-Halle, Permoserstraße 15, 04318 Leipzig, Germany
Tel.: +49-341-235-2821; Fax: +49-341-235-2492.
E-mail:[email protected]

1. Introduction

In recent decades, wetland sites have become increasingly important for wastewater
treatment [1, 2]. Treating wastewater in semi-natural plant systems is a technique which
can in principle be applied in natural wetlands such as marshes, moors and wet fields, in
artificial ponds and lagoons, and in constructed wetlands. Constructed wetlands exist in
a number of different basic designs featuring different flow characteristics [3, 4]. Today
the use of constructed wetlands, particularly to treat wastewater from decentralized
sources making for cheap and simple treatments (including in combination with other
technologies) has become the state of the art, and general design and operation guide-
lines have been published [5, 6].
The processes inside the rhizosphere in principle and the role of the plants within the
complex system of plants, microorganisms, solids and pollutants are crucial for most
natural treatment wetlands and for relatively simple constructed wetlands with horizontal
subsurface slow flow characteristics [7]. Judged by experiments and practical experience,
species of helophytes (marsh plants) work best of all in semi-natural wastewater
treatment systems. This is because helophytes have certain characteristics of growth
physiology that guarantee their survival even under waterlogged rhizosphere conditions.
Although all the plant species listed in Table 1 are suitable, reed along with types of
rushes and cattails are the ones most frequently used. Recently, the suitability of fast-
growing trees such as willows has also been examined [8].

69
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 69– 82.
© 2006 Springer.
70 Arndt Wiessner et al.

Table 1. Plant species frequently used in constructed wetlands (adapted from [7])

Scientific name English name

Acorus calamus L. sweet flag
Carex spp. sedges
Glyceria maxima (Hartm.) Holmb reed grass
Iris pseudacorus L. yellow flag
Juncus spp. rushes
Phragmites australis (Cav.) Trin. ex Steud. common reed
Scirpus spp. bulrushes
Typha angustifolia L. narrow-leaved cattail
Typha latifolia L. broad-leaved cattail

One of the decisive advantages of wetlands for wastewater treatment is the simultaneous
effective use of very different turnover processes. The wetland rhizosphere allows
fundamentally different oxidation–reduction conditions to exist in the same ‘reactor’ at
the same time due to aerobic and anaerobic microbiological processes and varied
physical and chemical processes. The reasons for these multireactive conditions are the
characteristics of the waterlogged soil, the enormous biodiversity of the micro-organisms
established under these conditions, and the plants’ specific adaptational responses to the
flooding of their rhizosphere.
The objectives of this paper are in particular to shed light on the influence of the
helophytes on the redox reactions in their rhizosphere and to present the latest results of
investigations into the redox dynamics in the rhizosphere of treatment wetlands.

2. Characterisation of the rhizosphere of helophytes

The conditions in the waterlogged soils of treatment wetlands can be defined as extreme
for the survival of macrophytes. When soil is flooded, a chain of physical, chemical and
biological processes initiate reducing soil conditions [9-13]. The exclusion of
atmospheric gases results in oxygen depletion in the soil following chemical changes,
including the accumulation of CO2, N2, H2 and methane, and the microbial reduction of
nitrate, nitrite, manganese, iron and sulphate, as well as the microbial accumulation of
acetic and other small-chain fatty acids. The redox state becomes more negative [10, 11,
14], reaching an Eh lower than –300 mV [15]. The wastewater may also cause extreme
conditions in the rhizosphere of the plants depending on its pH (acidic or alkaline), the
presence of toxic compounds such as phenols, tensides, biocides and heavy metals, etc.,
and its salinity [7].
These extreme reducing conditions result in particular in various stress symptoms for
the wetland plants owing to the restricted oxygen supply of plant tissues [16-20],
changes in the availability and/or concentrations of essential nutrients [13], the
formation of phytotoxic compounds [9, 11, 21] causing injury to the plants [10, 22], the
inhibited growth of the roots [23, 24] or even the entire plants [18-20, 25-27,], and other
adverse effects on plants [18-20, 28-30]. Furthermore, reduced conditions have been
found to initiate internal water stress [31, 32], decreases in photosynthetic rates [16, 33,
34], and reduced biomass growth [35-37].
Influence of Helophytes on Redox Reactions 71

Although many findings have been published on plant responses under flooded soil
conditions [17, 36, 38-44], our understanding of the relationship between the redox state
of the soil and the physiology of wetland plants is far from complete [13].

3. Responses of helophytes to waterlogged soil conditions

3.1. INTERNAL GAS TRANSPORT MECHANISM

Despite the extreme conditions in waterlogged soils, most wetland plants are able to
survive, grow and reproduce there. In contrast to typical land plants, wetland plants have
anatomical and physiological attributes necessary for their long-term survival under
waterlogged soil conditions [17-20, 44]. The degree of adaptation is specific to
individual species and their survival varies from a few hours to several months [45].
This adaptation is based on an extensive gas (oxygen) transport mechanism that enables
the plants to provide their roots with atmospheric or internal photosynthetic oxygen for
respiration and to oxidize reduced compounds in the rhizosphere [17]. This process has
been considered as a major mechanism for coping with soil anaerobiosis [17-20, 22, 36,
38-43, 46-48]. Gas transport of air (oxygen) from aboveground parts of the plants to the
underground parts for respiration and release into the flooded rhizosphere occurs by
special internal tissues forming open channels with low flow resistance, known as
aerenchyma [17]. Depending on the degree of adaptation, aerenchyma can account for as
much as 60% of the total tissue volume [49]. In addition, this tissue enhances the
potential for oxygen to be transported to the remote underground parts of the plant by
diminishing the internal volume of respiring tissues and oxygen consumption [17-20,
50]. The genesis of aerenchyma structures by cell lysis or cell formation as well as their
anatomical peculiarities has been thoroughly studied physiologically and anatomically
[17, 42, 51-53].
Diffusion is one mechanism for moving the oxygen within the plants [54]. In
addition, intensive convective gas flow induced by pressure gradients and known as
“ventilation” or “through flow” exists in many wetland plants [18-20, 55-60]. The
pressure gradients are formed by low pressure in oxygen-consuming tissues of the plants
caused by different solubilities of the oxygen consumed and the carbon dioxide formed
in this process, and by high pressure in the plant’s leaves resulting from the inflow of
atmospheric gases. The higher pressure in the leaves causes air to flow throughout the
entire body of the plant, including the whole root system, by entering the aerenchyma
tissues and leaving the plant through older leaves with lower stomatal conductance [49,
61]. The types and combination of the transport mechanisms (diffusion and/or
convection) involved are specific to each plant species. Intensive through flow has been
observed in Typha latifolia (cattail) and Phragmites australis (reed) for example [57,
62]. The inflow of air into the leaves to produce high internal pressure is mainly induced
by temperature and humidity gradients between the inside of the leaves and the ambient
air [18, 20, 49, 52, 58, 61-64].
Different mechanisms of oxygen transport to and through the plants, including the
processes of the temperature and humidity induced turnover of air and its effects, are
72 Arndt Wiessner et al.

described in detail in [50] and [51]. The processes involved in ventilation in plants have
been studied since the mid-19th century [65]. This interest has been increasingly revived
since the 1980s, at least in connection with the growing attention paid to the
biotechnological usage of helophytes to clean wastewater in constructed wetlands [65].

3.2. OXYGEN RELEASE BY ROOTS INTO RHIZOSPHERE OF HELOPHYTES

Oxygen reaching the root aerenchyma is respired by the root tissues and diffuses towards
to the root apex and radially to the rhizosphere, to be consumed in the surrounding soil
[54]. The oxygen flux from the root aerenchyma to the rhizosphere, known as “radial
oxygen loss” (ROL), is determined by the concentration gradient, the consumption of
oxygen by the cells along the radial path, and the physical resistance to oxygen diffusion
[54, 56].
As a result, 30–40% of the oxygen supplied via the aerenchyma to the root apex is
lost to the rhizosphere [54]. The radial oxygen loss causes oxygenation and therefore
significant chemical and biological changes within the rhizosphere relevant to microbial
populations [66], nutrient availability [67-70], and concentrations of potentially toxic
substances [71-74]. However, the oxygen release from the root system to the rhizosphere
mainly occurs at the apex, as well as at lateral parts which tend to be shorter, thinner and
of lower porosity than the main axes [75, 76]. The lateral parts were described as the
major source of oxygen loss to the rhizosphere in some species [77]. The oxygen
released into the rhizosphere protects the young, sensitive parts of the root system
against reduced soil compounds and helps the plants penetrate the reduced soil [19, 54,
78-81].
The oxygen released may result in a protective “layer” on the root surface [82]. This
“layer” may have a thickness of between 1 and 4 mm depending on the reduced state of
the root environment and it is characterized by a redox gradient ranging from about –250
mV as frequently measured in reduced rhizospheres to about +500 mV directly at the
root surface [82]. Oxygen is more or less continuously released from the root system,
counterbalancing chemical and biological oxygen consumption. This ability of
helophytes to oxygenate their rhizosphere is of particular interest for biotechnological
application [83, 84].
Different methods to estimate oxygen flow rates have been used, mainly in plant-
physiological investigations [50]. Rates of 126 µmol O2/h g root dry mass for Juncus
ingens (giant rush) and 120–200 µmol O2/h g root dry mass for Typha latifolia (cattail)
determined by the titanium-citrate method are of technological relevance [85, 86].
Furthermore, model calculations for Phragmites australis (reed) resulting in oxygen
input rates of 5–12 g O2/m2 patch area per day [86] and investigations with individual
plantlets of different species in hydroponic cultures resulting in the highest oxygen
release rates of 1.4 mg O2/h plantlet for T. latifolia [84] highlight from a more
biotechnological view the considerable potential of helophytes to release oxygen. Some
studies have revealed that the redox state of the rhizosphere has a significant effect on
the intensity of oxygen release of various helophytes, with oxygen release rates
increasing as the soil Eh becomes more reduced [85, 87, 88, 84]. In hydroponics model
investigations, plantlets of various species showed the highest intensity of oxygen
Influence of Helophytes on Redox Reactions 73

release at –250 mV < Eh < –150 mV and for extremely reduced conditions of Eh < –250
mV the release intensities were found to be lower [84].
Additional investigations emphasize the importance of the above and underground
portions of the plants for the intensity of oxygen release [89]. The release rates by the
roots of T. latifolia and J. effusus were found to be determined by the growth state of the
aboveground part of the plants and relatively independent from the size of the root
systems [89]. To clarify the influence of ambient conditions (air temperature and air
humidity) determining the gas input at the leaves on the intensity of oxygen release into
the rhizosphere, additional investigations should be performed.
The ventilation inside the helophytes causes air flow through the plants, i.e. the
transport of mainly O2 and also N2. The behaviour of the atmospheric nitrogen (given
radial losses from the roots) and its importance inside the rhizosphere has still not been
investigated.
Gas exchange at the appropriate parts of the root system not only causes the
oxygenation of the rhizosphere but also enables the flux of gases from the rhizosphere
together with gases generated inside the plant tissues to the atmosphere by internal
diffusion and/or ventilation. Although this exchange has been investigated for ethylene,
carbon dioxide and methane, for example [90-92], information about nitrogen generated
by denitrification inside the rhizosphere is still lacking. The processes involved depend
on the species [83, 93-100]. In connection with the gas exchange, determining the
phytohormone ethylene as a marker for plant-physiology studies is of particular interest
[52, 92, 101-108]. Carbon dioxide may be generated by microbial mineralization in the
soil or be derived from respiration in underground tissues of the plants [95, 109] and can
also be fixed in photosynthesis by the plants [110]. Because of the role of methane as a
“greenhouse gas” – after all, up to 90% of methane emissions from flooded soils may be
transferred by the plants to the atmosphere [98] – interest in the process of methane
release by plants has grown in recent years [111].

4. Release of organic compounds from plants

Knowledge about the input of carbon from plants into the rhizosphere (rhizodeposition)
mainly comes from agricultural research. The quantity of C-compounds released by
agricultural crops has been estimated at 10–40% of net photosynthetic production [112].
The composition of the exudates is highly diverse and species-specific. For example,
sugars and vitamins such as thiamine, riboflavine and pyridoxine etc., organic acids such
as malate, citrate, amino acids, benzoic acids, and phenolic compounds have all been
identified [113]. Rhizodeposition may initiate the mobilization of nutrients [114],
allelopathic effects [113], and the stimulation of microbial growth and activity inside the
rhizosphere [112, 115-117].
Generally speaking, our understanding of the composition of root exudates of
helophytes is very limited. Various substituted aromatic derivatives with hydroxyl,
methoxyl, aldehyde and carboxyl groups were found in rhizome extracts of Scirpus
lacustris (bulrush) [118]. These compounds can be used as a carbon source by micro-
organisms in the rhizosphere, causing oxygen depletion and decreasing the redox state.
If not enough oxygen is available, other electron acceptor such as nitrate, nitrite, Fe3+,
74 Arndt Wiessner et al.

sulphate or carbonate (methanogenesis) are used. The resulting metabolic products,

especially H2S, influence the redox conditions of the rhizosphere.

5. Dynamics of redox conditions and the removal of contaminants inside

the rhizosphere of helophytes – results of model investigations

Because of the usually low hydrodynamics, rapid microbial consumption by more or less
adsorbed micro-organisms and fast chemical reactions (fast oxidation in the strongly
reduced surroundings) changes in redox reactions inside the rhizosphere due to gas
exchange and the release and uptake of compounds mainly affect the area close to the
root surface. Evaluating such micro-gradient processes near the root surface in
constructed wetlands is very difficult due to the diurnal dynamics of different interacting
redox reactions, such as nitrification, denitrification, the mineralisation of carbon
substances, methanogenesis, sulphate reduction, sulphur/sulphide oxidation etc. [2, 7,
13].
Despite the overall reduced redox conditions detectable in the rhizosphere of
constructed wetlands, highly effective oxidation processes can also be determined by
evaluating the inflow and outflow concentrations of oxidizable contaminants. In addition
to oxidation processes enhanced by the input of atmospheric oxygen at the surface of the
wetland [119], diurnal micro-gradient oxidation processes near the rhizoplane clearly
also play an important role. Furthermore, temporal gradients overlapping the diurnal
gradients corresponding to changes in the oxygen release at the rhizoplane have to be
considered, too, due to daily and annual variations of plant-physiological and/or ambient
gas exchange conditions (temperature, humidity, illumination etc).
To shed more light on the dynamics of the redox conditions enhanced by the plants
and the changes in micro-gradient redox reactions, investigations in a specially designed
laboratory-scale reactor [120] were carried out [121]. This enabled the treatment of an
artificial wastewater in a planted (J. effusus) gravel bed to be evaluated, and daily
variations in the redox state depending on the intensity of illumination by daylight were
observed, as shown in Figure 1.
Particularly in summer, these variations ranging from Eh § –150 to –230 mV at night
to Eh § +50 to +350 mV at midday and in the early afternoon could only have been
caused by changes in the light-enhanced ventilation inside the plants, resulting in
changes in the oxygen release by the roots. This effect was interpreted as fundamental
proof of the daily temporal variation of the redox state in the areas of the rhizosphere
near the roots. Further investigations are needed to evaluate the correlations between
these short-term redox changes and certain removal processes. The pH was also found to
change daily to a small but still significant extent in response to the intensity of the
daylight (Figure 2).
This pH variability has to be interpreted as a reaction to changes in the removal
processes, including the consumption of nitrate, as well as changes to carbon dioxide
transport out of the rhizosphere that are enhanced by the plants.
Influence of Helophytes on Redox Reactions 75

350 100
Eh Light
250
80

Intensity of Daylight [kLx]

150
60
Eh [mV]

50

40
−50

20
−150

−250 0
0:00

0:00

0:00

0:00

0:00

0:00

0:00

0:00

0:00
Daytime

Figure 1. Changes of the redox state in the rhizosphere related to the intensity of daylight shown for several
days in mid-summer (adapted from [121]).

The correlation between the gas exchange by the plants and carbon removal inside the
rhizosphere also can be derived from a long-term seasonal evaluation, as shown in
Figure 3. The increase in global radiation during the summer causes more intensive
internal ventilation inside the plants. The resulting higher intensities of oxygen release
into the rhizosphere on the other hand cause higher intensities of carbon removal and
minimum outflow concentrations of COD, respectively. Contrasting behaviour is
observed during darker seasons.

6. Outlook

Especially in natural and in simple horizontal subsurface flow constructed treatment

wetlands; the interactions of the helophytes and their rhizosphere are of importance for
the efficiency of the removal processes. The gas exchange by the plants is mainly
responsible for influencing the redox conditions and the removal processes inside the
rhizosphere. Above all, oxygenation initiates diurnal and temporal gradients of redox
states and oxygen availability in the area around the roots and enables different chemical
and biological oxidation and anoxic processes to take place simultaneously close
together.
76 Arndt Wiessner et al.

Our knowledge of these correlations in the rhizoplane and the roots’ environment is
by no means complete. However, they must be understood if treatment wetlands are to
be ideally designed and operated.

6.20 80
pH
Light

Intensity of Daylight [kLx]

6.15 60

6.10 40
pH

6.05 20

6.00 0
10:19

19:55

5:33

15:07

0:43

10:19

19:55

5:31

15:07
Daytime

Figure 2. Changes of the pH in the rhizosphere correlated to the redox state shown for several days in mid-
summer (adapted from [121]).

The problem is that because technologists mainly evaluate overall removal efficiencies
by “black box” investigations of treatment wetlands, too little is known about the main
processes and their phytobiological, microbiological and chemical interactions in the
rhizosphere. Moreover, plant biologists extensively investigate the fundamental
anatomical and physiological aspects of plants’ gas exchange independently of technical
application. Future biotechnological investigations should be based on current biological
and technological expertise and should favour a combination of methods. Such
investigations should be focused on: i) quantifying the amounts of gas transported into
and out of the rhizosphere; ii) evaluating the variability of oxygen release by the plants
into their rhizosphere; iii) investigating micro-gradient processes inside the rhizosphere
by using suitable laboratory-scale techniques; iv) evaluating the microbial diversity
inside the rhizosphere.
Influence of Helophytes on Redox Reactions 77

40 250
COD
MSSP
200

30
COD [mg L−1]

150

100
20

50

10 0
28-Jul

5-Nov

13-Feb

24-May

1-Sep

10-Dec

20-Mar

28-Jun

Figure 3. Mean COD outflow concentrations and the monthly sum of sunshine periods (MSSP) for a two
years operation period (adapted from [121]).

References
[1] White, KD and Burken, JG (1999) Natural treatment and on-site processes. Water Environment
Research 71(5): 676-685
[2] Williams, JB (2002) Phytoremediation in wetland ecosystems: Progress, problems, and potential.
Critical Reviews in Plant Sciences 21(6): 607-635
[3] Kadlec, R (1987) Nothern natural wetland water treatment systems, in KR Reddy and WH Smith, Eds.,
Aquatic plants for water treatment and resource recovery. Magnolia Publ., Orlando, FL, USA, 83-98
[4] Wissing, F (1995) Wasserreinigung mit Pflanzen. Verlag Eugen Ulmer, Stuttgart, Germany
[5] Anonymous (1998) ATV instructions. Principles for the Dimensioning, Construction and Operation of
Plant Beds for Communal Wastewater with Capacities up to 1000 Total Number of Inhabitants and
Population Equivalents. Society for Promotion of Wastewater Treatment Technology e.V. (GFA),
Hennef, Germany
[6] Kadlec, R; Knight, R; Vymazal, J; Brix, H; Cooper, P and Haberl, R (2000) Constructed Wetlands for
Pollution Control. IWA Publishing, London, UK
[7] Stottmeister, U; Wiessner, A; Kuschk, P; Kappelmeyer, U; Kästner, M; Bederski, O; Müller, RA and
Moormann, H (2004) Effects of plants and microorganisms in constructed wetlands for wastewater
treatment. Biotechnology Advances 22(1-2): 93-117
[8] Greenway, M and Bolton, KGE (1996) From wastes to resources - turning over a new leaf: Melaleuca
trees for wastewater treatment. Environmental Res. Forum 5-6: 363-366
[9] Ponnamperuma, FN (1972) The chemistry of submerged soil. Advances in Agronomy 24: 29-96
78 Arndt Wiessner et al.

[10] Gambrell, RP and Patrick, WH (1978) Chemical and microbiological properties of anaerobic soils and
sediments, in DD Hook and RMM Crawford, Eds., Plant life in anaerobic environments. Ann Arbor
Science, Michigan, USA, 375-423
[11] Ponnamperuma, FN (1984) Effects of flooding on soils, in TT Kozlowski, Ed., Flooding and Plant
Growth. Academic Press, Orlando, FL, USA, 1-44
[12] Gambrell, RP; DeLaune, RD and Patrick, WH (1991) Redox processes in soil following oxygen
depletion, in MB Jackson, DD Davies and H Lambers, Eds, Plant Life Under Oxygen Deprivation:
Ecology, Physiology, and Biochemistry. SPB Academic Publisher, The Hague, Netherlands, 101-117
[13] Pezeshki, SR (2001) Wetland plant responses to soil flooding. Environmental and Experimental
Botany 46(3): 299-312
[14] Patrick, WH and DeLaune, RD (1977) Chemical and biological redox systems affecting nutrient
availability in the coastal wetlands. Geoscience and Man 17: 131-137
[15] Turner, FT and Patrick, WH (1968) Chemical changes in waterlogged soils as a result of oxygen
depletion, in Transactions of the 9th congress of the International Society of Soil Science 4: 53-56
[16] DeLaune, RD; Pezeshki, SR and Pardue, JH (1990) An oxidation-reduction buffer for evaluating
physiological response of plants to root oxygen stress. Environ Exp Bot 30: 243-247
[17] Armstrong, W; Braendle, R and Jackson, MB (1994) Mechanisms of flood tolerance in plants. Acta
Botanica Neerlandica 43: 307-358
[18] Armstrong, J; Armstrong, W; Beckett, PM; Halder, JE; Lythe, S; Holt, R and Sinclair, A (1996)
Pathways of aeration and the mechanisms and beneficial effects of humidity- and Venturi-induced
convections in Phragmites australis (Cav) Trin ex Steud. Aquatic Botany 54(2-3): 177-197
[19] Armstrong, J; Armstrong, W and VanderPutten, WH (1996) Phragmites die-back: Bud and root death,
blockages within the aeration and vascular systems and the possible role of phytotoxins. New
Phytologist 133(3): 399-414
[20] Armstrong, W; Armstrong, J and Beckett, PM (1996) Pressurised aeration in wetland macrophytes:
Some theoretical aspects of humidity-induced convection and thermal transpiration. Folia Geobotanica
and Phytotaxonomica 31(1): 25-36
[21] DeLaune, RD; Patrick, WH and Buresh, RJ (1978) Sedimentation rates determined by Cs-137 dating
in a rapidly accreting salt marsh. Nature 275: 532-533
[22] Drew, MC and Lynch, JM (1980) Soil Anaerobiosis, Microorganisms, and Root Function. Annual
Review of Phytopathology 18(1): 37-66
[23] Tanaka, A; Mulleriyawa, RP and Yasu, T (1968) Possibility of hydrogen sulphide induced iron
toxicity of the rice plant. Soil Science and Plant Nutrition 4: 1-6
[24] Allam, AI and Hollis, JP (1972) Sulphide inhibition of oxidases in rice roots. Phytopathology 62:
634-639
[25] King, GM; Klug, MJ; Wiegert, RG and Chalmers, AG (1982) Relation of soil water movement and
sulfide concentration to Spartina alterniflora production in a Georgia saltmarsh. Science 218: 61-63
[26] Ingold, A and Havill, DC (1984) The influence of sulphide on the distribution of higher plants in salt
marshes. Journal of Ecology 72: 1043-4054
[27] Havill, DC; Ingold, A and Pearson, J (1985) Sulphide tolerance in coastal halophytes. Vegetatio 62:
279-285
[28] Rao, DN and Mikkelsen, DS (1977) Effects of acidic, propionic, and butyric acids on rice seedling
growth and nutrition. Plant and Soil 47: 323-334
[29] Armstrong, J; Afreen Zobayed, F and Armstrong, W (1996) Phragmites die-back: Sulphide- and acetic
acid-induced bud and root death, lignifications, and blockages within aeration and vascular systems.
New Phytologist 134(4): 601-614
[30] Armstrong, J and Armstrong, W (1999) Phragmites die-back: toxic effects of propionic, butyric and
caproic acids in relation to pH. New Phytologist 142(2): 201-217
[31] Kramer, PJ (1940) Causes of decreased absorption of water by plants in poorly aerated media. Am J
Bot 27: 216-220
[32] Hiron, RWP and Wright, STC (1973) The role of endogenous abscisic acid in the response of plants to
stress. Journal of Experimental Botany 24: 769-781
[33] Pezeshki, SR (1993) Differences in patterns of photosynthetic responses to hypoxia in flood-tolerant
and flood-sensitive tree species. Photosynthetica 28: 423-430
[34] Pezeshki, SR and Anderson, PA (1997) Response of three bottomland woody species with different
flood-tolerance capabilities to various flooding regimes. Wetland Ecol Manag 4: 245-256
Influence of Helophytes on Redox Reactions 79

[35] Kludze, HK and DeLaune, RD (1994) Methane emission and growth of Spartina patens in response to
soil redox intensity. Soil Science Society of America Journal 58: 1838-1845
[36] Pezeshki, SR (1994) Plant responses to flooding, in RE Wilkinson, Ed, Plant-Environment
Interactions. Marcel Dekker, New York, NY, USA, 289-321
[37] Kludze, HK and DeLaune, RD (1995) Straw application effects on Methane and oxygen exchange and
growth in rice. Soil Science Society of America Journal 59: 824-830
[38] Hook, DD and Crawford, RMM (1978) Plant life in anaerobic environments. Ann Arbor Science
Publishers, Michigan, MA, USA
[39] Kozlowski, TT (1984) Flooding and Plant Growth. Academic Press, New York, NY, USA
[40] Kozlowski, TT (1984) Plant responses to flooding of soil. Bioscience 34: 1626-167
[41] Drew, MC (1990) Sensing soil oxygen. Plant Cell and Environment 13: 681-693
[42] Drew, MC (1997) Oxygen deficiency and root metabolism: Injury and acclimation under hypoxia and
anoxia. Annual Review of Plant Physiology and Plant Molecular Biology 48: 223-250
[43] Kozlowski, TT (1997) Response of woody plants to flooding and salinity. Tree Physiology Monograph
1: 1-29
[44] Vartapetian, BB and Jackson, MB (1997) Plant adaptations to anaerobic stress. Annals of Botany 79:
3-20
[45] Crawford, RMM and Braendle, R (1996) Oxygen deprivation stress in a changing environment.
Journal of Experimental Botany 47(295): 145-159
[46] Teal, JM and Kanwisher, JW (1966) Gas transport in the marsh grass Spartina alterniflora. Journal of
Experimental Botany 17: 355-361
[47] Armstrong, W; Justin, SHFW; Beckett, PM and Lythe, S (1991) Root adaptation to soil waterlogging.
Aquatic Botany 39(1-2): 57-73
[48] Perata, P and Alpi, A (1993) Plant responses to anaerobiosis. Plant Science 93(1-2): 1-17
[49] Grosse, W and Schröder, P (1986) Pflanzenleben unter anaeroben Umweltbedingungen, die
physikalischen Grundlagen und anatomischen Voraussetzungen. Ber Dtsch Bot Ges 99: 367-81
[50] Colmer, TD (2003) Long-distance transport of gases in plants: a perspective on internal aeration and
radial oxygen loss from roots. Plant Cell and Environment 26(1): 17-36
[51] Allen, LH (1997) Mechanisms and rates of O2 transfer to and through submerged rhizomes and roots
via aerenchyma. Soil and Crop Science Society of Florida Proceedings 56: 41-54
[52] Jackson, MB and Armstrong, W (1999) Formation of aerenchyma and the processes of plant
ventilation in relation to soil flooding and submergence. Plant Biology 1(3): 274-287
[53] Soukup, A; Votrubova, O and Cizkova, H (2000) Internal segmentation of rhizomes of Phragmites
australis: protection of the internal aeration system against being flooded. New Phytologist 145(1):
71-75
[54] Armstrong, W (1979) Aeration in higher Plants. Advances in Botanical Research 7: 225-332
[55] Dacey, JWH (1981) Pressurized Ventilation in the Yellow Waterlily. Ecology 62(5): 1137-1147
[56] Armstrong, W and Beckett PM (1987) Internal aeration and the development of stelar anoxia in
submerged roots: A multishelled mathematical model combining axial diffusion of oxygen in a cortex
with radial losses to the stele, the wall layers and the rhizosphere. New Phytologist 105: 221-245
[57] Armstrong, J and Armstrong, W (1991) A convective through-flow of gases in Phragmites australis
(Cav.) Trin. ex Steud. Aquatic Botany 39(1-2): 75-88
[58] Armstrong, J; Armstrong, W and Beckett, PM (1992) Phragmites australis: venturi- and humidity-
induced pressure flows enhance rhizome aeration and rhizosphere oxidation. New Phytologist 120:
197-207
[59] Sorrell, BK; Brix, H and Orr, PT (1997) Eleocharis sphacelata: Internal gas transport pathways and
modelling of aeration by pressurized flow and diffusion. New Phytologist 136(3): 433-442
[60] Vretare, V and Weisner, SEB (2000) Influence of pressurized ventilation on performance of an
emergent macrophyte (Phragmites australis). Journal of Ecology 88(6): 978-987
[61] Grosse, W (1989) Thermoosmotic air transport in aquatic plants affecting growth activities and oxygen
diffusion to wetland soils, in DA Hammer, Ed, Constructed Wetlands for Wastewater Treatment:
Municipal, Industrial and Agricultural. Lewis Publisher, Chelsea, UK, 416-469
[62] Bendix, M; Tornbjerg, T and Brix, H (1994) Internal gas transport in Typha latifolia L. and Typha
angustifolia L. 1. Humidity-induced pressurization and convective throughflow. Aquatic Botany
49(2-3): 75-89
80 Arndt Wiessner et al.

[63] Grosse, W (1996) The mechanism of thermal transpiration (equals thermal osmosis). Aquatic Botany
54(2-3): 101-110
[64] Woermann, D; Metzner, H and Grosse, W (2002) Humidity-induced convection of air across porous
membranes. Journal of Membrane Science 206(1-2): 69-85
[65] Grosse, W; Armstrong, J and Armstrong, W (1996) A history of pressurised gas-flow studies in plants.
Aquatic Botany 54(2-3): 87-100
[66] Gilbert, B and Frenzel, P (1998) Rice roots and CH4 oxidation: The activity of bacteria, their
distribution and the microenvironment. Soil Biology and Biochemistry 30(14): 1903-1916
[67] Reddy, KR; Patrick, WH and Lindau, CW (1989) Nitrification-denitrification at the plant root-
sediment interface in wetlands. Limnology and Oceanography 34: 1004-1013
[68] Kirk, GJD and Bajita, JB (1995) Root-induced iron oxidation, pH changes and zinc solubilisation in
the rhizosphere of lowland rice. New Phytologist 131: 129-137
[69] Saleque, MA and Kirk, GJD (1995) Root-induced solubilisation of phosphate in the rhizosphere of
lowland rice. New Phytologist 129: 325-336
[70] Christensen, KK and Wigand, C (1998) Formation of root plaques and their influence on tissue
phosphorus content in Lobelia dortmanna. Aquatic Botany 61(2): 111-122
[71] Laanbroek, HJ (1990) Bacterial cycling of minerals that affect plant growth in waterlogged soils: a
review. Aquatic Botany 38(1): 109-125
[72] Begg, CBM; Kirk, GJD; MacKenzie, AF and Neue, HU (1994) Root-induced iron oxidation and pH
change in the lowland rice rhizosphere. New Phytologist 128: 469-477
[73] Mendelssohn, IA; Keiss, BA and Wakeley, JS (1995) Factors controlling the formation of oxidised
root channels: a review. Wetlands 15: 37-46
[74] St-Cyr, L and Campbell, PGC (1996) Metals (Fe, Mn, Zn) in the root plaque of submerged aquatic
plants collected in situ: relations with metal concentrations in the adjacent sediments and in the root
tissue. Biogeochemistry 33: 45-76
[75] Sorrell, BK; Mendelssohn, IA; McKee, KL and Woods, RA (2000) Ecophysiology of wetland plant
roots: A modelling comparison of aeration in relation to species distribution. Annals of Botany 86(3):
675-685
[76] Bouma, TJ; Koutstaal, BP; van Dongen, M and Nielsen, KL (2001) Coping with low nutrient
availability and inundation: root growth responses of three halophytic grass species from different
elevations along a flooding gradient. Oecologia 126(4): 472-481
[77] Armstrong, W; Armstrong, J and Beckett, PM (1990) Measurment and modelling of oxygen release
from roots of Phragmites australis, in P Cooper and BC Findler, Eds, The Use of Constructed
Wetlands in Water Pollution Control. Pergamon Press, Oxford, UK, 41-54
[78] Green, MS and Etherington, JR (1977) Oxidation of ferrous iron by rice (Oriza sativa) roots: a
mechanism for waterlogging tolerance? Journal of Experimental Botany 28: 678-690
[79] Jaynes, ML and Carpenter, SR (1986) Effects of Vascular and Nonvascular Macrophytes on Sediment
Redox and Solute Dynamics. Ecology 67(4): 875-882
[80] Koncalova, H (1990) Anatomical adaptations to waterlogging in roots of wetland graminoids:
limitations and drawbacks. Aquatic Botany 38(1): 127-134
[81] Chabbi, A; McKee, KL and Mendelssohn, IA (2000) Fate of oxygen losses from Typha domingensis
(Typhaceae) and Cladium jamaicense (Cyperaceae) and consequences for root metabolism. American
Journal of Botany 87(8): 1081-1090
[82] Flessa, H (1991) Redoxprozesse in Böden in der Nähe von wachsenden und absterbenden
Pflanzenwurzeln. Verlag Marie L. Leidorf, Buch am Erlbach, Germany
[83] Brix, H; Sorrell, BK and Schierup, HH (1996) Gas fluxes achieved by in situ convective flow in
Phragmites australis. Aquatic Botany 54(2-3): 151-163
[84] Wiessner, A; Kuschk, P; Kästner, M and Stottmeister, U (2002) Abilities of helophyte species to
release oxygen into rhizospheres with varying redox conditions in laboratory-scale hydroponic
systems. International Journal of Phytoremediation 4(1): 1-15
[85] Sorrell, BK and Armstrong, W (1994) On the difficulties of measuring oxygen release by root systems
of wetland plants. Journal of Ecology 82: 177-183
[86] Jespersen, DN; Sorrell, BK and Brix, H (1998) Growth and root oxygen release by Typha latifolia and
its effects on sediment methanogenesis. Aquatic Botany 61(3): 165-180
[87] Kludze, HK and DeLaune, RD (1996) Soil redox intensity effects on oxygen exchange and growth of
Cattail and Sawgrass. Soil Science Society of America Journal 60(2): 616-621
Influence of Helophytes on Redox Reactions 81

[88] Sorrell, BK (1999) Effect of external oxygen demand on radial oxygen loss by Juncus roots in titanium
citrate solutions. Plant Cell and Environment 22(12): 1587-1593
[89] Wiessner, A; Kuschk, P and Stottmeister, U (2002) Oxygen release by roots of Typha latifolia and
Juncus effusus in laboratory hydroponic systems. Acta Biotechnologica 22(1-2): 209-216
[90] Smith, KA and Russell, RS (1969) Occurrence of ethylene, and its significance in anaerobic soil.
Nature 222: 769-771
[91] Smith, KA and Restall, SWF (1971) The occurrence of ethylene in anaerobic soil. Journal of Soil
Science 22: 430-443
[92] Visser, EJW; Bogemann, GM; Blom, C and Voesenek, L (1996) Ethylene accumulation in
waterlogged Rumex plants promotes formation of adventitious roots. Journal of Experimental Botany
47(296): 403-410
[93] Dacey, JWH (1979) Methane efflux from lake sediments through water lilies. Science 203: 1253-1255
[94] Dacey, JWH and Klug, MJ (1982) Tracer transport in Nuphar: 18O2 and 14CO2 transport. Physiologia
Plantarum 56: 361-366
[95] Higuchi, T; Yoda, K and Tensho, K (1984) Further evidence for gaseous CO2 transport in relation to
root uptake of CO2 in rice plant. Soil Science and Plant Nutrition 30: 125-136
[96] Sebacher, DI; Harriss, RC and Bartlett, KB (1985) Methane emissions to the atmosphere through
aquatic plants. Journal of Environmental Quality 14: 40-46
[97] Sorrell, BK and Boon, PI (1994) Convective gas flow in Eleocharis sphacelata R. Br.: methane
transport and release from wetlands. Aquatic Botany 47(3-4): 197-212
[98] Shannon, RD; White, JR; Lawson, JE and Gilmour, BS (1996) Methane efflux from emergent
vegetation in peatlands. Journal of Ecology 84(2): 239-246
[99] Butterbach-Bahl, K; Papen, H and Rennenberg, H (1997) Impact of gas transport through rice cultivars
on methane emission from rice paddy fields. Plant Cell and Environment 20(9): 1175-1183
[100] Yavitt, JB and Knapp, AK (1998) Aspects of methane flow from sediment through emergent cattail
(Typha latifolia) plants. New Phytologist 139(3): 495-503
[101] Smith, KA and Robertson, PD (1971) Effect of ethylene on root extension of cereals. Nature
234(5325): 148-9
[102] Jackson, MB and Campbell, DJ (1975) Movement of ethylene from roots to shoots, a factor in the
response of tomato plants to waterlogged soil conditions. New Phytologist 74: 397-406
[103] Drew, MC; Jackson, MB and Giffard, S (1979) Ethylene-promoted adventitious rooting and
development of cortical air spaces (aerenchyma) in roots may be adaptive responses to flooding Zea
mays L. Planta 147: 83-88
[104] Konings, H and Jackson, MB (1979) A relationship between rates of ethylene production by roots and
the promoting or inhibiting effects of exogenous ethylene and water on root elongation. Zeitschrift für
Pflanzenphysiologie 92: 385-397
[105] Jackson, MB (1989) Regulation of aerenchyma formation in roots and shoots by oxygen and ethylene,
in DJ Osborne and MB Jackson, Eds, Cell Separation in Plants: Physiology, Biochemistry and
Molecular Biology. H35. Springer-Verlag, Berlin, 263-274
[106] Liu, J; Mukherjee, I and Reid, DM (1990) Adventitious rooting in hypocotyls of sunflower
(Helianthus annuus) seedlings. III. The role of ethylene. Physiol Plant 78(2): 268-276
[107] Jackson, MB (1991) Ethylene in root growth and development, in AK Mattoo and JC Shuttle, Eds.,
The Plant Hormone Ethylene. CRC Press, Boca Raton, FL, USA, 259-278
[108] Visser, EJW; Nabben, RHM; Blom, C and Voesenek, L (1997) Elongation by primary lateral roots and
adventitious roots during conditions of hypoxia and high ethylene concentrations. Plant Cell and
Environment 20(5): 647-653
[109] Brix, H (1990) Uptake and photosynthetic utilization of sediment-derived carbon by Phragmites
australis (Cav.) Trin. ex Steudel. Aquatic Botany 38(4): 377-389
[110] Constable, J and Longstreth, DJ (1994) Aerenchyma Carbon Dioxide can be assimilated in Typha
Iatifolia L. Leaves. Plant Physiol. 106(3): 1065-1072
[111] Lelieveld, J; Crutzen, PJ and Bruhl, C (1993) Climate effects of atmospheric methane. Chemosphere
26(1-4): 739-768
[112] Helal, HM and Sauerbeck, D (1989) Carbon turnover in the rhizosphere. Zeitschrift für
Pflanzenernährung und Bodenkunde 152: 211-216
[113] Miersch, J; Krauss, G-J and Sclee, D (1989) Allelochemische Wechselbeziehungen zwischen Pflanzen -
eine kritische Wertung. Wiss Ztg Univ Halle 38: 59-74
82 Arndt Wiessner et al.

[114] Hoffland, E; van de Boogaard, R; Nelemans, J and Findenegg, G (1992) Biosynthesis and root
exudation of citric and malic acids in phosphate-starved rape plants. New Phytologist 122: 675-680
[115] Donnelly, PK; Hegde, RS and Fletcher, JS (1994) Growth of PCB-degrading bacteria on compounds
from photosynthetic plants. Chemosphere 28(5): 981-988
[116] Horswell, J; Hodge, A and Killham, K (1997) Influence of plant carbon on the mineralisation of
atrazine residues in soils. Chemosphere 34(8): 1739-1751
[117] Moormann, H; Kuschk, P and Stottmeister, U (2002) The effect of rhizodeposition from helophytes on
bacterial degradation of phenolic compounds. Acta Biotechnologica 22(1-2): 107-112
[118] Kaitzis, G (1970) Mikrobiozide Verbindungen aus Scirpus lacustris L. (Ein Beitrag zur Ökochemie
des Wurzelraumes). Universität Göttingen, Göttingen, Germany.
[119] Wu, MY; Franz, EH and Chen, SL (2001) Oxygen fluxes and ammonia removal efficiencies in
constructed treatment wetlands. Water Environment Research 73(6): 661-666
[120] Kappelmeyer, U; Wiessner, A; Kuschk, P and Kästner, M (2002) Operation of a Universal Test Unit
for Planted Soil Filters - Planted Fixed Bed Reactor. Engineering in Life Sciences 2(10): 311-315
[121] Wiessner, A; Kappelmeyer U; Kuschk, P and Kästner M (2005) Influence of the redox condition
dynamics on the removal efficiency of a laboratory-scale constructed wetland. Water Research 39:
248-256
EXPLOITATION OF FAST GROWING TREES IN METAL REMEDIATION

PAVEL TLUSTOŠ, DANIELA PAVLÍKOVÁ, JIěINA

SZÁKOVÁ, ZUZANA FISCHEROVÁ AND JIěÍ BALÍK
Czech University of Agriculture, Department of Agrochemistry
and Plant Nutrition, 165 21 Prague 6, Czech Republic, E-mail:
[email protected]

1. Advantages of using fast growing trees for phytoextraction

The cultivation of fast-growing tree species for remediation purpose and the production
of renewable energy from contaminated biomass is an approach to the use of post-
mining polluted areas which offers an alternative to more traditional types of land use
[1-4]. Compared to herbaceous species, fast-growing trees have several advantageous
characteristics, such as a deeper root system, a high productivity and transpiration
activity.
Bungart and Hüttl [1, 2] investigated growth dynamics and biomass production of 8-
yeard-old hybrid poplar clones (Populus spp., section Tacamahaca). Aboveground
biomass production ranged from 24 to 49 t dry matter per hectare at age 8. The high-
yield clones were Rap (46.7 t per ha) and Beaupré (48.6 t per ha). Differences in mean
aboveground biomass production between the hybrid poplar clones were largely the
result of genetic and ecophysiological causes. Investigation of plant nutrition and the
water budget indicated that these two factors might have an influence on the productivity
of the clones, measured in terms of the accumulated aboveground biomass. Armstrong
et al. [5] found the yield of poplar dry biomass from 3.6 to 13.6 t per ha per year. The
highest yields were achieved at the site with the highest average annual rainfall. Moffat
et al. [6] confirmed significant effect of irrigation for poplar biomass production.
Application of sewage sludge was not effective in increasing biomass yield in this
experiment. According to Sebastiani et al. [3] poplar clones responded to waste
treatment through increasing plant growth, although the investigated clones differed in
their response.
Field experiments with four willow species (Salix amygdalina, S. viminalis, S.
americana, S. purpurea) irrigated by municipal wastewater were performed by Kowalik
and Randerson [7]. Annual yields for 2000 mm annual irrigation in two-year plant
rotation were 5 – 14.4 t dry matter per ha. According to Cannell et al. [8] the potential
production of stem dry biomass for a 1- or 2-year-old uncoppiced stand of Salix
viminalis could be more than 12 t.ha–1 per year. Yield of willow dry matter in one-year
cutting cycle (14.1 t.ha–1 per year) was significantly increased to 16.1 and 21.6 t.ha–1 per

83
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 83 – 102.
© 2006 Springer.
84 Pavel Tlustoš et al.

year when harvest was performed in two and three years cycle, respectively. The highest
yield was found for Salix viminalis 082 clone cut in three years cycle, amounted up to
–
26.4 t.ha 1 per year [9]. Hytönen and Issakainen [10] investigated biomass production of
birches (Betula pubescens) and according to their results birch is not suitable for
biomass production when using annual harvest.
The concentration of elements for optimum nutrition presented in Table 1 describes
the range values refer to tree populations less than 9 years old. Clonal differences in
biomass production seemed to be related to differences in the use-efficiency of nutrients
for the purposes of internal N and P translocation. Bungart and Hüttl [2] recommended
fertilization of poplar clones in the form of the application of an extended NPK
fertilization regime after the culmination of height and diameter stem growth in the
seventh year in order to achieve a higher and more stable biomass production. Given the
local conditions, Bungart and Hüttl [2] suggest focusing on cultivation of a polyclonal
mixture of high-yielding hybrid poplar clones for biomass production.

Table 1. Range of values (mg g-1 DM) for optimum nutrition of Populus spp. [2]

N P K Ca Mg
mg.g–1 DM
17 – 30 1.0 – 4.4 7 – 20 3.0 – 17 1.4 – 4.0

Planting density according to Adegbidi et al. [11], and Bergkvist and Ledin [12] did not
significantly affect annual biomass production, nutrient removal and nutrient use
efficiency. The most important factors taken into consideration for yield optimisation of
stem wood are the number of plants per area and the length of the cutting cycle. The
results of Bergkvist and Ledin [12] show during the first cutting cycle of willow, that the
yield was positively correlated with the number of plants per ha up to 20000, but not
with higher numbers. Tharakan et al. [4] investigated variation among 3-year-old 30
willow clones and recommended to grow willow shrubs in densities of 10000 – 20000
plants per ha.
Fast-growing species used in short-rotation forestry are dependent on a substantial
and continuous availability of nutrients and water to give high yield. Increased biomass
yields in willow and poplar stands have been observed as a result of fertilization with N,
P and K, soil amendment with lime and irrigation. Rytter [13] published that the annual
willow uptake of N during the last 2 years of the study was nearly 200 kg.ha–1 per year
and the N concentration in fully developed leaves near the shoot tip varied between 2.9 –
4.4% N. The results of Adegbidi et al. [11] showed that annual biomass production of
willow clones of 15 – 22 t.ha–1 removed 75 – 86 kg N, 10 – 11 kg P, 27 – 32 kg K, 52 –
79 kg Ca and 4 – 5 kg Mg.ha–1 per year.
Growth and associated nutrient uptake during the first rotation of a willow plantation
is summarized in Table 2. The estimation of the nitrogen amounts required for leaf
growth in well established willow plantations is in the rate of 150 to 200 kg.ha–1 per year
[14]. Between one and two thirds of the leaf N and P contents can be transported back
from senescing leaves to woody tissues and stored for use in coming season. The genus
Alnus differs from Populus and Salix and translocation of N from senescing leaves is
Exploitation of Fast Growing Trees 85

small. In poplar plantations more than 50% of the aboveground N requirement and 30%
of P can come from retranslocation the previous year [14]. Stems constitute the major
storage organ for resorbed nutrients in willows when the process of nutrient withdrawal
is allowed to be completed, but hybrid poplars has been associated the major storage
pool of retranslocated leaf-N to large structural roots. Leaf litter from species belonging
to the genera Alnus, Populus and Salix may be regarded as easily decomposed. A release
of N in the order of 30% of the initial N-content from 1-year leaf litter is reported for
alders and willows after one year of decomposition [15]. A nitrogen budget during the
first rotation of a willow plantation on good agricultural soil is presented in Table 3.
Table 2. Calculated growth and nutrient accumulation in roots, stems and leaves during
the first rotation of a Salix viminalis plantation [14]

Planting year
Year 1 Year 2 Year 3
Biomass (t.ha–1)
Leaves 3.5 5.0 5.2
Stems 8.0 11.0 13.0
Roots 1.8 2.4 2.6
Nitrogen uptake (kg.ha –1)
Leaves 123 175 182
Stems 40 44 46
Roots 22 29 31
Total uptake 185 248 259
Potassium and phosphorus uptake (kg.ha –1)
K - total uptake 133 186 200
P – total uptake 28 39 42

Sander and Ericsson [16] studied the vertical distribution of elements in the woody
biomass of two willows. In the 5-year-old shoots, the concentration of all elements
increased significantly from shoot base to shoot top (Table 4). The general increase in
plant nutrient and heavy metal concentrations from the shoot base towards the top can be
explained by distribution patterns of wood and bark. The concentration of plant nutrients
is generally higher in bark than wood.
Nielsen [17] published optimal proportions by weight of the essential mineral
nutrient components in a fertilizer for achieving maximum willow production (Table 5).
Biomass production, nutrient removal and nutrient use efficiency in fast-growing tree
plantations are strongly affected by clonal selection. Fertilization, irrigation and a long
harvest cycle significantly increased annual biomass production. Nutrient use efficiency
is increased by extension of harvest cycle and fertilizer application.
86 Pavel Tlustoš et al.

Table 3. Estimated nitrogen uptake, nitrogen recycling and nitrogen fertilizer requirements
of a high yielding willow plantation on good agricultural soil during the first rotation

Planting year
Year 1 Year 2 Year 3
Nitrogen (kg.ha–1)
Total N uptake* 185 248 259
Soil** 50 50 50
Retranslocation - 41 58
Leaf litter - 27 66
Root litter 11 14 15
Atmosphere 10 10 10
N-leakage ? ? ?
Fertilizer needs 114 106 60
Recommended 80 - 110 60 - 80 60 - 80
Outtake at harvest 128
* from Table 2; ** mineral soil, N-content 5000 kg .ha–1;

Table 4. Plant nutrient and heavy metal concentrations at different shoot levels in 5-year-
old shoots of Salix viminalis on a clay soil. Shoots were sampled in late March to early
April 1995 [16]

element Level above ground (m)

0.5 1.5 2.5 3.5 4.5 5.5
mg.g–1
P 0.53 0.48 0.53 0.70 1.11 2.38
K 1.5 1.7 2.1 2.8 3.7 5.1
Ca 3.4 3.3 3.7 4.3 5.5 7.8
Mg 0.47 0.44 0.47 0.56 0.79 1.35
S 0.31 0.31 0.36 0.46 0.65 1.13
mg.kg–1
Mn 18.2 17.2 18.4 21.8 29.6 46.3
Zn 45.3 48.6 51.0 54.5 67.3 87.3
Cu 2.3 2.2 2.4 2.9 3.8 10.0
Ni 0.98 0.86 1.01 1.25 1.86 6.04
Cd 0.78 0.78 0.81 0.85 0.91 0.92

2. Metal uptake by fast growing trees and metal distribution in plants

The application of fast growing trees, especially willows and poplars (Salicaceae), for
phytoremediation use was discussed at the soils contaminated to deeper horizons [18].
The uptake and distribution of potentially risk elements were investigated predominantly
in genes Salix spp. and Populus spp., and also Betula spp., Alnus spp. (Betulaceae), and
Acer spp. (Aceraceae), but possible remediation use was checked only in the case of
Salix spp. The remediation potential of trees including their metal tolerance, metal
uptake by trees grown on contaminated substrates, metal distribution within trees, and
phytoremediation using trees was deeply reviewed by Pulford and Watson [19].
Analyses of leaves and twigs of Salix sp. and Populus sp. showed relatively high
contents of elements in the first part of vegetation period because of higher nutrient
uptake compared to the growth rate. During the vigorous growth dilution effect resulting
Exploitation of Fast Growing Trees 87

in lower element contents was observed until the flowering stage where the minimum
values for almost all elements were observed. Senescence of trees usually results in
increase of metal levels due to concentration caused by loss of fluids [20]. The
variations in element contents in leaves of trees are demonstrated in Figure 1. Ali et al.
[21] demonstrated that Salix acmophylla was able to accumulate considerable amounts
of Cu, Ni and Pb in different plant parts and exhibited high tolerance to these metals.
The plants showed ability for their detoxification by antioxidant enzymes and cellular
antioxidants like cysteine and thiols.

Table 5. Optimal proportions by weight of the essential mineral nutrient components in a

fertilizer for achieving maximum willow production (the amount of nitrogen is set at 100 %
and the other are relative to that) [17]

Macro nutrients Micro nutrients

element proportion element proportion
Nitrogen 100 Iron 0.6
Potassium 72 Manganese 0.4
Phosphorus 14 Boron 0.1
Magnesium 8.5 Copper 0.03
Calcium 7 Zinc 0.06
Sulphur 9 Chlorine 0.003
Molybdenum 0.007
Sodium 0.003

Individual tree species differ significantly in ability to transport metals from roots to
shoots and to accumulate them in individual plant parts. Pulford and Watson [19]
concluded that within the tree Pb, Cr, and Cu are usually immobilized in roots while Cd,
Ni, and Zn are more easily transported to shoots. Figure 2 indicates opposite pattern for
Cr and Ni but different soil properties of different sites and/or different sampling time
and climate conditions must be taken into account in this case.
Nissen and Lepp [22] found that the concentrations of Cu and Zn varied between
plant parts and between species. There was no consistent pattern of metal concentration.
Copper concentrations decreased in the order twigs > leaves > wood > bark, and those of
Zn in the order leaves > bark > twigs > wood indicating the low retention of Zn in the
xylem tissues. Salix x sericans and Salix cinerea had high concentrations of both metals;
S. purpurea had high concentrations of Zn but low concentrations of Cu, whilst Salix
fragilis and Salix viminalis had low concentrations of both metals. Concentration factors
for both elements in shoot tissues were consistent, despite the variations in associated
metal concentrations. Concerning cadmium the willow leaves contain approximately 1.6-
fold higher level compared to wood representing 49% of total shoot cadmium content.
Roots contained 90% of cadmium contents in shoots [23]. Distribution of arsenic within
tree was leaves > branch bark > stem bark > wood, where stem position on the tree was
significant for leaf arsenic content [24].
88 Pavel Tlustoš et al.

Figure 1. Seasonal element variations in (a) Salix spp. and (b) Populus nigra leaves
according to Dinelli and Lombini [20].

Willows are generally characterized by high production of aboveground biomass and

intensive uptake of cadmium [25] where close relationships of these parameters are
evident. This dependence can be significantly affected by water uptake and transpiration
of plants differing among individual clones [26]. High amount of risk elements in
cultivation medium reduces yield of willow shoots (S. viminalis) compared to plants
grown in uncontaminated environment [27]. The total cadmium uptake by plants is
affected by both yield of aboveground biomass and element content in leaves and twigs,
and these parameters must be taken into account for an evaluation of phytoremediation
potential [25]. Willows characterized by higher production of twigs usually contain
lower cadmium levels and simultaneously grow at less acidic soils with lower cadmium
mobility compared to willows with lower production of twigs. The portion of leaves
represent in the case of willows 17 - 37% of total shoot yield whereas cadmium uptake
by willow leaves represents 21 - 48% of total cadmium uptake by aboveground biomass
[25]. LuĖáþková et al. [28] investigated the cadmium impact on roots of Salix alba and
Populus × euroamericana cv. Robusta cuttings grown in 10 µmol.l–1 solution of
Cd(NO3)2 (direct treatment) or in Knop solution and afterwards in 10 µmol.l–1 solution
of Cd(NO3)2 (indirect treatment). The cumulative length, number and biomass of willow
roots, pigment and starch contents, leaf net photosynthetic rate and dry mass/leaf area
ratio of willow leaves were positively influenced by indirect treatment. However,
indirectly treated poplars were more sensitive to Cd than directly treated ones. Indirect
treatment lowered root Cd uptake in willow, Cd accumulation in cuttings of both species
and Cd accumulation in poplar shoots. Structural changes caused by Cd were similar in
both species and in both treatments. Root apices, rhizodermis and cortex were the most
seriously damaged root parts. Pulford and Watson [19] summarized that survival of fast
growing trees at contaminated site seems to be due to facultative tolerance (avoidance of
Exploitation of Fast Growing Trees 89

highly contaminated substrate by roots and/or immobilization of elements in the root)

and genetically transmitted tolerance systems were not evolved. However, tolerance of
plants can be increased by acclimation of trees to elevated concentrations of risk
elements.

100%

80%

60%

40%

20%

0%
As Cd Cr Cu Ni Pb Zn Hg

leaves twigs roots

Figure 2. Distribution of selected elements into individual parts of Salix fragilis.

Perttu et al. [29] focused on some important processes that are likely to be driving
variables for Cd fluxes in the soil – willow system. These included plant growth and
processes that are linked with biomass production. In the prevailing northern European
climate, where precipitation normally exceeds evapotranspiration, soil fluxes of elements
are seldom directed towards the soil surface. Such fluxes therefore depend on living
plants and their root uptake. The principal flows of Cd are: i) root uptake of subsoil Cd,
transported to the foliage which eventually is shed as litter fall, ii) Cd leakage from deep
penetrating roots passing the topsoil, iii) decomposition of roots that pass the topsoil
with organically bound Cd, and iv) fertilisation with wood ashes, containing Cd. The
first one is the most likely process for transporting Cd from the subsoil to the topsoil.
The results indicated that the efficiency of Cd uptake is about the same, regardless of
where the roots are situated. Most of the Cd taken up by the deep penetrating roots is
attached and tightly bound to these; only about 5 - 11% is transported upwards to the
shoots and 3 - 7% to the foliage. The latter will end up in the topsoil after litter fall. A
very small part, probably less than 1%, of the Cd in the shoot ends up in the topsoil as
the stool remains after final harvest. About 40% of the Cd taken up by the deep
penetrating roots passes into the shoot part. Consequently, the root turn over rate is an
important factor to consider when discussing Cd translocation. The Cd that is adhered to
organic material (stems and roots) will remain there and eventually be released into the
soil when the material is broken down and humified. No Cd is leaking out from the
living roots penetrating into deeper soil layers. The result was expected as mineral and
90 Pavel Tlustoš et al.

element uptake only occurs close to the root tips and older parts of the suberised root
functions as a transport link to the above ground parts of the plant. Relocation of Cd
within the root system, i.e. from deep penetrating roots into the topsoil roots is negligible
as the element-uptake processes is from the root tip towards the foliage.

3. Metal accumulation capacity of fast growing trees

A large number of species and hybrids of Salix spp. suggests wide genetic variability
within the genus and some species are known to colonise contaminated soil. For
example S. alba, S. dasyclados, S. viminalis (containing up to 4.1 mg Cd.kg–1 in stems
and 7.3 mg Cd.kg–1 in leaves), S. cinerea, and S. caprea naturally invade polluted
dredged sediment disposal sites [19, 25, 30]. Some willow or poplar varieties do not
retain metals in roots but transfer them to aboveground plant tissues [19, 23, 31]. The
advantage of these species is their greater harvestable biomass in contrast to
hyperaccumulators mostly with only a small aboveground part [32]. Poplar trees grown
in mine spill affected soil were able to take up the Cd and Zn contents exceeding normal
values in plants (up to 15 mg Cd.kg–1 and 1312 mg Zn.kg–1) [33]. These levels can be
usually considered as phytotoxic for plants. Greger and Landberg [34] determined
cadmium contents in shoots of 104 clones of S. viminalis grown in medium enriched by
1, 3, and 10 µmol.l–1 solution of Cd in range between 0.2 and 8.5 mg.kg–1. In the case of
zinc the element contents in shoots varied between 14 and 1813 mg.kg–1 confirming also
the ability of willows and poplars to take up higher Cd and Zn contents compared to
other plant species [35].
Plant accumulation capacity and remediation capability were tested in two years pot
experiment [36]. Anthropogenically contaminated (by industrial activity) Cambisol
containing 28 mg As.kg–1, 5.46 mg Cd.kg–1, 956 mg Pb.kg–1, and 279 mg Zn.kg–1 was
applied in this case. Detailed characteristics of the site were described in Šichorová et al.
[37]. Plants were selected from two groups: hyperaccumulators – Arabidopsis halleri,
and Thlaspi caerulescens; and trees with high element accumulation capacity and a great
biomass production – Salix smithiana, Salix dasyclados, Salix caprea, Populus
trichocarpa, and Populus nigra. Figure 3 confirms good willingness of all tested species
to transport Cd and Zn from roots to aboveground biomass. All species deposit As and
Pb in roots preferentially. The highest shoot/root Cd ratio was recognised for A. halleri,
S. dasyclados, P. trichocarpa and P. nigra. Significantly higher transport of Zn was
progressed in both poplars compare to other species. This is in concordance with results
of many authors they had described the highest concentration of Cd and Zn in poplars or
willows leaves compare to other plant parts [19]. We are able to resume that poplar trees
are really disposed to transport and deposit Cd and Zn in shoots, but their remediation
factor (calculated as percentage of element removed from total amount of element in pot
soil) is lower in contrast to both hyperaccumulators and S. dasyclados. The
hyperaccumulation possibility of A. halleri and T. caerulescens for Cd and great
accumulation ability for other studied elements was confirmed but very good
accumulation was determined in tested trees, too. Especially willow S. dasyclados
compensated lower metal content in shoots by higher biomass production compare to
hyperaccumulators which have higher element content but lower aboveground biomass
Exploitation of Fast Growing Trees 91

resulting in similar remediation capability. Although poplar trees showed the best
willingness to transport Cd and Zn from roots to shoots, their remediation potential do
not achieve S. dasyclados or hyperaccumulators level.
Madejon et al. [33] surveyed the content of eight trace elements (As, Cd, Cu, Fe,
Mn, Ni, Pb and Zn) in leaves and stems of 25 white poplar (Populus alba) trees in the
riparian forest of the Guadiamar River (S. Spain), one year after this area was
contaminated by a mine spill, and 10 trees in non-affected sites. The spill-affected soils
had significantly higher levels of available cadmium (mean of 1.25 mg.kg–1), zinc (117
mg.kg–1), lead (63.3 mg.kg–1), copper (58.0 mg.kg–1) and arsenic (1.70 mg.kg–1), than
non-affected sites. The concentration of trace element in poplar leaves was positively
and significantly correlated with the soil availability for cadmium and zinc, and to a
lesser extent for arsenic. Thus, these results confirmed that poplar leaves could be used
as biomonitors for soil pollution of Cd and Zn, and moderately for As.

0
As Cd Pb Zn

A. halleri T. caerulescens S. smithiana S. dasyclados

S. caprea P. trichocarpa P. nigra

Figure 3. Average metal accumulation shoot/root ratio of different plant species [36].

Similarly Roselli et al. [38] compared phytoextraction capacity of five tree species
grown at metal contaminated site (total element content in the soil was 557 ± 51 mg.kg–1
of Cu, 620 ± 41mg.kg–1 of Zn, and 1.8 ± 0.2 mg.kg–1 of Cd), and determined fairly good
bioconcentration factors (calculated as ratio of total element content in plant tissue and
in soil) of Salix viminalis and Betula pendula for cadmium and zinc in contrast to other
species as summarized in Table 6. In the case of copper, however, the bioconcentration
factors were negligible regardless of tree species.
92 Pavel Tlustoš et al.

Table 6. Bioconcentration factors to the aboveground plant tissues for copper, zinc and
cadmium for the trees growing on the contaminated site [33]

Plant species Bioconcentration factor Bioconcentration factor Bioconcentration factor

for Cu for Zn for Cd
leaves twigs leaves twigs leaves Twigs
Alnus incana 0.05 0.03 0.07 0.06 a A
Betula pendula 0.02 0.03 0.37 0.32 0.06 0.11
Fraxinus excelsior 0.01 0.04 0.02 0.05 a a
Salix viminalis 0.01 0.03 0.37 0.28 0.83 0.72
Sorbus mougeotti 0.01 0.02 0.03 0.05 a a
a…data under detection limit

For assessment of clonal variability of willow plants seven clones of high biomass
production willows (S. x smithiana S-218, S. x smithiana S-150, S. viminalis S-519, S.
alba S-464, S. alba “Pyramidalis” S-141, S. dasyclados S-406, S. x rubens S-391) were
planted in a pot experiment for three vegetation periods in two soils differing in total
content of risk elements [39] as characterized in Table 7. Because of serious symptoms
of Zn phytotoxicity at extremely contaminated Fluvisol the experiment was terminated
after second vegetation period at this soil. Comparing the remediation factors,
reasonable phytoextraction potential of willows was obtained for cadmium and zinc at
moderately contaminated Cambisol where aboveground biomass removed about 30% Cd
and 5% Zn of total element content, respectively. Clones showed different ability to
remove Cd and Zn, depending on soil type and contamination level: S. x smithiana
(S-150) and S.x.rubens (S-391) demonstrated the highest phytoextraction effect for Cd
and Zn. The differences in accumulation between the clones are due to the property of
clone and not to the soil element concentration or properties [24, 35, 40]. The
mechanisms behind the accumulation, transport, and tolerance are specific for each of
the different metals and the uptake property of the clone is stable [34]. In extremely
contaminated Fluvisol production of willow biomass was limited by phytotoxicity of
zinc resulting in phytoextraction efficiency not exceeding 1% for both Cd and Zn
(Figure 4). Concerning arsenic and lead, poor ability of willows to translocate these
elements from roots to aboveground biomass led to low removal of these elements from
soil (less than 1%) and application of these plants for cleaning of As and Pb
contaminated soil is not reasonable. Similarly Nissen and Lepp [22] found the evidence
for the exclusion of Cu from shoot tissue indicating low potential for depletion of the
plant available soil Cu pool as a consequence of repeated cropping and for concentration
in combustion residues.
The cultivation of willows can decrease bioavailable portion of soil cadmium even in
deeper horizons. The annual removal of 3 – 4% of plant-available portion of Cd was
demonstrated in dependence on mobilization/immobilization processes of cadmium
bound in less mobile soil fractions [19, 25, 41-42]. Moreover, potential of willow trees
to reduce element leaching to groundwater as well as to stabilize soil characteristics was
already described [41-42]. Difference between cadmium uptakes by roots within soil
profile was not observed [25]. Perttu et al. [29] also described the uptake of Cd in Salix
from deeper soil layers. The actual decrease in the topsoil after harvesting is therefore
less than what may be theoretically calculated. However, the risk of increasing Cd
Exploitation of Fast Growing Trees 93

concentrations, because of the relocation from deeper soil layers, is small as long as the
percentage is less than about 60% of total uptake. Normally, more than 70% of the
active roots are situated in the topsoil and there is no indication that the roots penetrating
into the subsoil are more effective than roots in the topsoil.
Table 7. Basic characteristics and total element contents in experimental soils [39]

Corg. As Cd Pb Zn
Soil pH
(%) mg.kg–1 mg.kg–1 mg.kg–1 mg.kg–1
Cambisol 6.1 2.1 37.5 4.73 1158 180
Fluvisol 5.3 1.8 64.1 30.5 2297 3718

Shann [43] investigated the “target-neighbour” cocropping approach to determine the

effect of planting density on the uptake of metal (in this case, selenium) by known
accumulating (mustard) and nonaccumulating, sensitive species (tomato). If the resource
for which plants are competing is a toxin or a contaminant, density should determine the
amount that each individual acquires from the substrate. The concentration of metal in
plant tissue may be highest for the individual at low density, but total metal found in a
stand of vegetation should occur at the density where biomass is maximal. If
accumulating plants are to be managed in a manner that optimises the amount of metal
removed from the substrate, then this maximal biomass density needs to be determined.
In addition, the cocropping of an accumulator with a sensitive species will provide
information on the possibility of growing crop species with accumulators, thus
minimizing the amount of metal that is taken up by an individual. This could decrease
metal phytotoxicity or could limit the tissue concentration of metals to a level considered
safe for consumption. Liu et al. [44] investigated a cocropping technique using a known
zinc hyperaccumulator Sedum alfredii with a grain crop, Zea mays. After a 3-month
growth trial, the results indicated that when Z. mays is cocropped with S. alfredii, heavy
metals accumulated in the grains were significantly reduced when compared to
monoculture cropping. Cocropping improved the growth of both plant species and seems
to be an effective approach to reduce the risk of contaminant uptake in edible crops.
Hyperaccumulator Thlaspi caerulescens is able to change conditions in rhizosphere
shared with other plant species and subsequently affect the bioavailability of selected
elements for adjacent plants. Hyperaccumulator cocropped with other plant species can
either mobilize soil elements such as cadmium or on the contrary immobilize other
elements [45]. The effect of cocropping of willow and hyperaccumulator Thlaspi
caerulescens was investigated in pot experiment where the plants were planted either
separately or in cocropping version. Moderately contaminated Cambisol and extremely
contaminated Fluvisol (Table 7) were used as cultivation medium.
94 Pavel Tlustoš et al.

Figure 4. Total remediation factors (%) of cadmium (a) and zinc (b) obtained for
aboveground biomass of willow plants during three years of cultivation in pot experiment
at moderately contaminated Cambisol and extremely contaminated Fluvisol [39].

Concerning cadmium contents in plants the most significant differences were observed
in the leaves of T. caerulescens grown at the soils with different level of contamination.
Cocropping of T. caerulescens and willow did not result in change of cadmium content
in aboveground biomass of T. caerulescens at moderately contaminated Cambisol
whereas Cd content in penny-cress plants grown together with willow at extremely
contaminated Fluvisol significantly decreased. The contents of lead and arsenic in
penny-cress biomass dropped down significantly in cocropping variant at both
investigated soils while zinc contents remained unchanged.
Exploitation of Fast Growing Trees 95

In the case of aboveground biomass of Salix sp. the cadmium contents in leaves
significantly decreased in plants grown together with penny-cress plants regardless of
experimental soil. For As, Pb, and Zn this effect was observed only at the extremely
contaminated Fluvisol. Moreover, the growth of individually cultivated willows at the
extremely contaminated soil was significantly limited due to phytotoxic effect of this soil
whereas the willow trees cultivated together with T. caerulescens were able to survive.
Phytoremediation factors of both plants (Figure 5) also demonstrate the beneficial effect
of cocropping of willow and penny-cress plants at extremely contaminated site.
Evidently, penny-cress plants accumulated sufficiently high amount of mobile cadmium
and zinc to improve conditions of cultivation of willows especially at extremely
contaminated soil. For arsenic and lead the removal of these elements from both soils
remains very low in both individual and cocropping variants of planting.
15 4

(a) (b)
3
10

% %2

5
1

0
0
Salix

Salix
Salix+Thlaspi

Salix+Thlaspi
Thlaspi

Thlaspi
Salix+Thlaspi

Salix+Thlaspi
Salix

Thlaspi

Salix

Thlaspi

Cambisol Fluvisol Cambisol Fluvisol

Figure 5. The effect of cocropping of Salix sp. and hyperaccumulator Thlaspi caerulescens
on phytoremediation factors of cadmium (a) and zinc (b) at moderately contaminated
Cambisol and extremely contaminated Fluvisol.

4. Modification of soil properties for improvement of tree phytoextraction

potential

The present results suggest that fast-growing trees and especially willows have very
promising potential for phytoremediation use because of their large biomass and good
ability to accumulate risk elements [19]. However, some limitations reduce significantly
these perspectives. In extremely contaminated soil the production of willow biomass is
limited by phytotoxicity of elements resulting in phytoextraction efficiency not
96 Pavel Tlustoš et al.

exceeding 1% for both Cd and Zn. For these soils, stabilization and immobilization of
toxic elements is required before application of phytoremediation technologies [46]. The
enhancement of soil organic carbon in soil belongs to the possible approaches tested for
decreasing of soil element mobility [47]. Selected clone of high biomass production
willow (S. dasyclados S-406) was planted in a pot experiment in two highly
contaminated soils (Table 8) for one vegetation period [48]. The soils were supplied by
nutrients and i) sewage sludge, ii) farmyard manure, and iii) wood chips in the rate
representing 20 t.ha–1. The addition of organic matter improved the soil properties of soil
A resulting in significantly higher yield of aboveground biomass of willow plants
whereas at soil B no changes and even decrease of biomass yield at wood chips
treatment was observed. The total uptake of individual elements from experimental soils
by aboveground biomass of willows taking into account the changes in plant yield in
treated pots is summarized in Table 9. The highest effect of organic matter addition was
reported for As at soil A where total uptake represented from 280 to 530% of control
value. At the soil B the increasing uptake of arsenic was observed only at manure treated
pots (180 %). The enhancement of total uptake of Cd and Zn in treated pots was evident
at both soils except the wood chips treatment at the soil B (due to lower plant yield at
this treatment) and represented 120 – 180% of control value. In the case of Pb the soil
treatment by organic matter did not lead to significant change in its uptake by willow
plants at soil A and even decreased the uptake of this element at soil B. The mobility of
elements in soil determined by single (0.01 mol.l–1 CaCl2) and sequential extraction
procedure was not significantly affected by the addition of different forms of organic
matter after one vegetation period and long-term investigation of these effects will be
necessary in further research.
Table 8. Basic characteristics and total element contents in experimental soils [48]

Soil Soil type pH Corg. (%) As (mg.kg–1) Cd (mg.kg–1) Pb (mg.kg–1) Zn (mg.kg–1)

A Fluvisol 5.3 1.8 64.1 30.5 2297 3718
B Chernozem 6.8 1.8 118 40.4 29.3 2087

Hasselgren [49] also found that the stem biomass production was enhanced by sludge
application rate and it also led to more uniform growth and a greater shoot number than
in control plots. Rockwood et al. [24] also reported improved willow yields after
application of sewage effluent where the wastewater application did not enrich the heavy
metal (Cd, Cu, Pb, Zn) content in the wood. Tsakou et al. [50] observed no changes in
both growth parameters and element content in poplar cultures grown on sewage sludge
treated soil, as well.
Present results also indicated the specificity of phytoremediation potential of willows
for cadmium and zinc whereas other elements in soil such as lead are not removed by
these plants due to low mobility of lead in soil. Therefore, the possibilities to enhance
lead mobility in soil and subsequently the uptake by plants are intensively investigated.
Plant roots are able to release specific chelating agents to rhizosphere characterized by
binding ability for metals [51, 52]. The effect of soil application of synthetic chelating
agents on increasing uptake of Pb, Cd, and Zn by plants was confirmed by many authors
Exploitation of Fast Growing Trees 97

[53-61]. Following chemicals were tested for this purpose: EDTA (ethylendiamino-
tetraacetic acid), CDTA (trans-1,2-diaminocyclohexane-N,N,N´,N´-tetraacetic acid),
DTPA (diethylentriamino-pentaacetic acid), EGTA (ethylenglycol-bis(β-amino-
ethylether)), HEDTA (N-(2-hydroxyethyl)-ethylen-diamino-tetraacetic acid), NTA
(nitrilotriacetic acid) [57, 61-63]. The effectivity of these agents depends on soil
properties, experimental plant, and investigated element but EDTA is considered as the
most effective one [61, 62]. The EDTA – metal complexes are taken up by plant roots
and transported to aboveground biomass via xylem [61]. The EDTA application can
increase the effectivity of phytoextraction even at the soils multicontaminated by more
elements [64].
Table 9. Total uptake of elements by aboveground biomass of willows according to the
individual treatments (mg) [48]

Treatment As (mg) Cd (mg) Pb (mg) Zn (mg)

soil A soil B soil A soil B soil A soil B soil A soil B
Control 32.8 76.0 1382 1183 120 28.4 39981 17444
Sludge 117 70.2 2480 1407 174 12.6 71456 22803
Manure 174 134 2163 2141 90.3 19.4 49558 31650
Wood chips 94.0 65.3 2031 801 148 16.2 66005 11777

Robinson et al. [23] investigated the effect of chelating agents on cadmium uptake by
two clones of poplar and one clone of willow. Although two weeks after application of
chelating agents cadmium content significantly increased in leaves, in the end of
experiment no significant differences were found between treated and untreated variants.
These results indicate that if chelating agents are used to enhance plant Cd uptake in a
phytoremediation operation, then the plants should be harvested shortly after their
addition to take immediate advantage of the surge in uptake. Moreover, two of the
chelating agents (2 g.kg–1 EDTA and 0.5 g.kg–1 NTA of soil) also resulted in a
significant reduction in growth, as well as abscission of leaves. Plants treated with
0.5 g.kg–1 EDTA showed reduced growth and leaf discoloration. Schmidt [61] also
observed reduced plant growth after application of chelates during vigorous growth of
plants. Thus, the application of chelates is recommended after achievement of required
size of plants. The results of Wenzel et al. [65] indicate that metal uptake and
translocation from roots to shoots is limited in the absence of EDTA. Split chelate agent
applications are generally more effective to enhance Pb and Zn shoot concentrations in
canola (Brassica napus) than the same amounts of EDTA added at once (Figure 6). The
plateau or maximum is reached at about 0.4 g EDTA kg–1 soil for Zn. Lead accu-
mulation in shoots further increases at larger EDTA application rates. It is less effective
to apply the same amount of EDTA at once 12 days before harvest, and a plateau is
obtained even at about 0.25 g EDTA kg–1 soil [65].
On a soil containing 5 mg Cd.kg–1, trees harvested shortly after a pulse of EDTA
would have a Cd concentration in the dry aboveground biomass of 53 mg.kg–1. The
biomass production of poplars and willows under optimal conditions is 30 tones per
hectare per annum. Thus, a single crop would remove 1.06 kg of Cd per hectare. This
equates to 106 years of regular P fertiliser addition. A soil containing 5 mg.kg–1 Cd in
98 Pavel Tlustoš et al.

the top 100 mm of soil contains 6 kg of Cd per hectare, assuming a bulk density of 1.2.
Four crop rotations of willows or treated poplars would reduce the soil Cd burden to
2.35 mg.kg–1 [23].

Figure 6. Lead and zinc contents in shoots of Brassica napus in the pot experiment
as a function of EDTA applied and mode of application (single versus split) [65].

5. Future prospective for technological application of phytoextraction

at contaminated sites

Summarizing the representative set of papers dealing with evaluation of phytoextraction

capacity of different plant species there is very well described a potential of tested plant
groups and their capacity to remediate contaminated areas. Unfortunately majority was
done in laboratory or in a small-scale experiments and there is very difficult to estimate
their responses in real conditions up to date. Rockwood et al. [24] summarized the
possible beneficial phytoremediation use of fast-growing trees for following purposes: i)
where former industrial land has little potential for development, fast-growing trees have
an opportunity to introduce the land into economic use; ii) fast-growing trees can be
grown at agricultural land contaminated by atmospheric deposition or waste disposal
where food crops are not permitted, and iii) fast-growing trees can be grown at the
agricultural land contaminated from rock phosphate fertilizers where the soil quality can
be improved within 10 - 15 years.
To extend the environmental friendly and socio-economically highly accepted
technology into regular use there is necessary to meet several criteria for successful
phytoextraction of metals from contaminated sites as follows: i) The recognition, extend
and distribution of contaminants: Fast growing trees show promising results only for
several elements mainly for mobile ones like cadmium and zinc presented in medium
level of contamination so far. Salix spp. as well as Populus spp. showed high
phytotoxicity at soils showing high content of total Zn (more than 2000 mg.kg–1) with
Exploitation of Fast Growing Trees 99

relation to soil properties. There is necessary to provide immobilization treatment at first

or exclude phytoextraction as suitable method. Sensitivity to other elements is not seen
in such extent [46]. There is also necessary to recognize distribution of contaminants
within profile, because phytoextraction is limited by the length of roots and their
penetration through the profile. Contamination of deep subsoil less than 80 cm as well as
topsoil from 0 to 10 cm is not suitable for phytoextraction. ii) Suitable conditions for
plant growth: The yield of aboveground biomass belongs among crucial parameters
affecting the remediation potential. Agricultural soils usually have relatively suitable
conditions for plant growth, but there are many other contaminated areas without
suitable soil fertility caused by soil properties or lack of nutrients. In such conditions it is
required to improve soil properties first. The application of fertilizers in relation to soil
is able to maximize yield and substantially increase removal of metals. iii) Planting of
trees and their harvest: Plant development and maximization of yield can be discussed
from the point of view of wood harvest. The use of poplar or willow for
phytoremediation would require annual harvesting to avoid the recycling of leaf-bound
Cd in autumn [23]. The annual wood biomass yield showed the highest if cutting is done
once in two or three year interval unfortunately leaves containing high amount of metals
are lost, therefore annual harvest of wood with leaves show higher total removal of
metals without any risk of contaminant penetration into surrounding environment.
Annual harvest increases the need of nutrients applied for maximum growth. iv) Post
harvest manipulation: Burning of harvested biomass can reduce the volume of biomass
and only 1 - 3% of total dry matter mass is left as the ash. The risk of possible metal
losses during burning is not satisfactory solved. The great advantage of burning is the
production of renewable energy. Gasification (i.e. pyrolysis), which occurs under reducing
conditions, seems to be a better method than incineration under oxidizing conditions to
increase volatilization and, hence subsequently recovery, of Cd and Zn from plants. It
would also allow the recycling of the bottom ash as fertilizer [66].

Acknowledgements

Financial support for these investigations was provided by MSM project No.
6046070901.

References
[1] Bungart, R and Hüttl, RF (2001) Production of biomass for energy in post-mining landscapes in Lusatia
and nutritient dynamics. Biomass Bioenergy 20: 181-187
[2] Bungart, R and Hüttl, RF (2004) Growth dynamics and biomass accumulation of 8-yeard-old hybrid
poplar clones in a short-rotation on a clayey-sandy mining substrate with respect to plant nutrition and
water budget. Eur J Forest Res 123: 105-115
[3] Sebastiani, L; Scebba, F and Tognetti, R (2004) Heavy metal accumulation and growth responses in
poplar clones Eridano (Populus deltoids x maximowiczii) and I-214 (P. x euramericana) exposed to
industrial waste. Environ Exp Bot 52: 79-88
[4] Tharakan, PJ; Volk, TA; Nowak, CA and Abrahamson, LP (2005) Morphological traits of 30 willow
clones and their relationship to biomass production. Can J For Res 35: 421-431
[5] Armstrong, A; Johns, C and Tubby, I (1999) Effects of spacing and cutting cycle on the yield of poplar
grown as an energy crop. Biomass Bioenergy 17: 305-314
100 Pavel Tlustoš et al.

[6] Moffat, AJ; Armstrong, AT and Ockleston, J (2001) The optimization of sewage sludge and effluent
disposal on energy crops of short rotation hybrid poplar. Biomass Bioenergy 20: 161-169
[7] Kowalik, PJ and Randerson, PF (1994) Nitrogen and phosphorus removal by willow stands irrigated
with municipal waste water – a review of the polish experience. Biomass Bioenergy 6: 133-139
[8] Cannell, MGR; Milne, R; Sheppard, LJ and Unsworth, MH (1987) Radiation interception and
productivity of willow. J Appl Ecol 24: 261-278
[9] Szczukowski, S; Tworkowski, J; Klasa, A and Stolarski, M (2002) Productivity and chemical
composition of wood tissues of short rotation willow coppice cultivated on arable land. Rostl Výr 48:
413-417
[10] Hytönen, J and Issakainen, J (2001) Effect of repeated harvesting on biomass production and sprouting
of Betula pubescens. Biomass Bioenergy 20: 237-245
[11] Adegbidi, HG; Volk, TA; White, EH; Abrahamson, LP; Briggs, RD and Bickelhaupt, DH (2001)
Biomass and nutrient removal by willow clones in experimental bioenergy plantations in New York
State. Biomass Bioenergy 20: 399-411
[12] Bergkvist, P and Ledin, S (1998) Stem biomass yields at different planting designs and spacings in
willow coppice systems. Biomass Bioenergy 14: 149-156
[13] Rytter, R-M (2001) Biomass production and allocarion, including fine-root turnover, and annual N
uptake in lysimeter-grown basket willows. For Ecol Manage 140: 177-192
[14] Ericsson, T (1994) Nutrient cycling in energy forest plantations. Biomass Bioenergy 6: 115-121
[15] Slapokas, T. and Granhall, U. (1991). Decomposition of litter in fertilized short-rotation forests on a
low-humified peat bog. For Ecol Manage 41: 143-165
[16] Sander, M-L and Ericsson, T (1996) Vertical distribution of plant nutrients and heavy metals in Salix
viminalis stems and their implications for sampling. Biomass Bioenergy 14: 57-66
[17] Nielsen, KH (1994) Environmental aspects of using waste waters and sludges in energy forest
cultivation. Biomass Bioenergy 6: 123-132
[18] Saxena, PK; KrishnaRaj, S; Dan, T; Perras, MR and Vettakkorumakankav, NN (1999)
Phytoremediation of heavy metal contaminated and polluted soils, in Prasad, MNV and Hagemeyer, J
(Eds.), Heavy Metal Stress in Plants - From Molecules to Ecosystems, Springer-Verlag Berlin
Heidelberg, pp. 305-329
[19] Pulford, ID and Watson, C (2002) Phytoremediation of heavy metal-contaminated land by trees - A
review. Environ Intern 29: 529-540
[20] Dinelli, E and Lombini, A (1996) Metal distributions in plants growing on copper mine spoils in
Northern Apennines, Italy: the evaluation of seasonal variations. Appl Geochem 11: 375-385
[21] Ali, MB; Vajpayee, P; Tripathi, RD; Rai, UN; Singh, SN and Singh, SP (2003) Phytoremediation of
lead, nickel, and copper by Salix acmophylla Boiss.: Role of antioxidant enzymes and antioxidant
substances. Bull Environ Contam Toxicol 70: 462-469
[22] Nissen, LP and Lepp, NW (1997) Baseline concentrations of copper and zinc in shoot tissues of a range
of Salix species. Biomass Bioenergy 12: 115-120
[23] Robinson, BH; Mills, TM; Petit, D; Fung, LE; Green, SR and Clothier, BE (2000) Natural and induced
cadmium-accumulation in poplar and willow: Implications for phytoremediation. Plant Soil 227:
301-306
[24] Rockwood, DL; Naidu, CV; Carter, DR; Rahmani, M; Spriggs, TA; Lin, C; Alker, GR; Isebrands, JG
and Segrest, SA (2004) Short-rotation woody crops and phytoremediation: Opportunities for
agroforestry? Agroforest Syst 61: 51-63
[25] Klang-Westin, E and Eriksson, J (2003) Potential of Salix as phytoextractor for Cd on moderately
contaminated soils. Plant Soil 249: 127-137
[26] Greger, M and Landberg, T (2001) Investigations on the relation between biomass production and
uptake of Cd, Cu, and Zn, in Greger, M; Landberg, T and Berg, B (Eds.): Salix clones with different
properties to accumulate heavy metals for production of biomass. Akademitryck AB, Edsbruk, Sweden,
ISBN 91-631-1493-3, 19-27
[27] Greger, M. and Landberg, T. (2001). Tolerance to and uptake of metals in different clones of Salix
viminalis grown in wastewater, in Greger, M; Landberg, T and Berg, B (Eds.): Salix clones with
different properties to accumulate heavy metals for production of biomass. Akademitryck AB, Edsbruk,
Sweden, ISBN 91-631-1493-3, 28-37
[28] LuĖáþková, L; Šottníková, A; Masaroviþová, E; Lux, A and Streško, V (2003) Comparison of cadmium
effect on willow and poplar in response to different cultivation conditions. Biol. Plant. 47, 403-411
Exploitation of Fast Growing Trees 101

[29] Perttu, K; Eriksson, J; Greger, M; Göransson, A; Blombäck, K; Klang-Westin, E and Landberg, T

(2003) Förråd och flöden av kadmium i systemet mark-Salix. (Content and fluxes of cadmium in the soil
– willow – system). Rep. ER 19:2003, Energimyndigheten, Box 310, SE-63104 Eskilstuna, Sweden
[30] Vandecasteele, B; De Vos, B and Tack, FMG (2002) Cadmium and zinc uptake by volunteer willow
species and elder rooting in polluted dredged sediment disposal sites. Sci Tot Environ 299; 191-205
[31] Vysloužilová, M; Tlustoš, P and Száková, J (2003) Cadmium and zinc phytoextraction potential of
seven clones of Salix spp. planted on heavy metal contaminated soils. Plant Soil Environ 49: 542-547
[32] Cobbett, CS and Meagher, RB (2002) Arabidopsis and the genetic potential for the phytoremediation of
toxic elements and organic pollutants, in: Somerville, C and Meyerowitz, E (Eds.): The Arabidopsis
Book, American Society of Plant Biologists, USA, 1543-8120
[33] Madejón, P; Maratón, T; Murilloa, JM and Robinson, B (2004) White poplar (Populus alba) as a
biomonitor of trace elements in contaminated riparian forests. Environ Pollut 132: 145-155
[34] Greger, M and Landberg, T (1999) Use of willow in phytoextraction. Int J Phytoremed 1: 115-123
[35] Riddell-Black D (1994) Willow vegetation filters for municipal wastewaters and sludges. A biological
purification system. Proc Study Tour, Conference and Workshop in Sweden, Department of Ecology and
Environmental Research, Swedish University of Agricultural Sciences, Uppsala, 145-151
[36] Fischerová, Z; Tlustoš, P; Száková, J and Šichorová, K (2005) A comparison of phytoremediation
capability of selected plant species for cadmium and lead. Proc 8th ICOBTE, Adelaide, Australia,
274-275
[37] Šichorová, K., Tlustoš, P., Száková, J., KoĜínek, K., and Balík, J. (2004). Horizontal and vertical
variability of heavy metals in the soil of a polluted area. Plant Soil Environ 50: 525-534
[38] Roselli, W; Keller, C and Boschi, K (2003) Phytoextraction capacity of trees growing on a metal
contaminated soil. Plant Soil 256: 265-272
[39] Tlustoš, P; Száková, J; Vysloužilová, M and KoláĜ, J (2005) The phytoremediation potential of fast
growing trees on anthropogenically contaminated soil. Proc 8th ICOBTE, Adelaide, Australia, 816-817
[40] Greger, M and Landberg, T (1995) Use of willow clones with high Cd accumulating properties in
phytoremediation of agricultural soils with elevated Cd levels. Proc 3rd ICOBTE, Paris, France, 505-511
[41] Eriksson, J and Ledin, S (1998) Changes in phytoavailability and concentration of cadmium in soil
following long term Salix cropping. Water Air Soil Pollut 114: 171-184
[42] King, R; Royle, A; Putwain, P and Dickinson, N (2005) The potential of phytoremediation: Monitoring
changes in contaminant bioavailability of a sediment in a 3-year field trial. Proc 8th ICOBTE, Adelaide,
Australia, 260-261
[43] Shann, JR (1995) The role of plants and plant/microbial systems in the reduction of exposure. Environ
Health Perspect 103: 13-15
[44] Liu, XM; Wu, QT and Banks, MK (2005) Effect of simultaneous establishment of Sedum alfredii and
Zea mays on heavy metal accumulation in plants. Int J Phytoremed 7: 43-53
[45] Gove, B; Hutchinson, JJ; Young, SD; Craigon, J and McGrath, SP (2002) Uptake of metals by plants
sharing a rhizosphere with the hyperaccumulator Thlaspi caerulescens. Int J Phytoremed 4: 267-281
[46] Vysloužilová, M; Tlustoš, P; Száková, J and Pavlíková, D (2003) As, Cd, Pb and Zn uptake by different
Salix spp. grown at soils enriched by high loads of these elements. Plant Soil Environ 49: 191-196
[47] Halim, M; Conte, P and Piccolo, A (2003) Potential availability of heavy metals to phytoextraction from
contaminated soils induced by exogenous humic substances. Chemosphere 52: 265-275
[48] Száková, J; Tlustoš, P; Pavlíková, D; Najmanová, J and Balík, J (2005) The response of extractable
fractions of potentially toxic elements on alteration of organic matter content in contaminated soil. Proc
8th ICOBTE, Adelaide, Australia, 106-107
[49] Hasselgren, K (1999) Utilization of sewage sludge in short-rotation energy forestry: A pilot study. Waste
Manage Res 17: 251-262
[50] Tsakou, A; Roulia, M and Christodoulakis, NS (2003) Growth parameters and heavy metal
accumulation in poplar tree cultures (Populus euroamericana) utilizing water and sludge from a sewage
treatment plant. Bull Environ Contam Toxicol 71: 330-337
[51] Kinnersely AM (1993). The role of phytochelates in plant growth and productivity. Plant Growth Regul
12: 207-217
[52] Marschner, H (1995) Mineral Nutrition of Higher Plants. 2nd Edition, Academic Press, London, ISBN
0-12-473543-6, 889
[53] Huang, JW; Chen, J; Berti, WR and Cunningham, SD (1997) Phytoremediation of lead-contaminated
soils: Role of synthetic chelates in lead phytoextraction. Environ Sci Technol 31: 800-805
102 Pavel Tlustoš et al.

[54] Huang, JW and Cunningham, SD (1996) Lead phytoextraction: species variation in lead uptake and
translocation. New Phytol 134: 75-84
[55] Vassil, AD; Kapulnik, Y; Raskin, I and Salt, DE (1998) The role of EDTA in lead transport and
accumulation by Indian mustard. Plant Physiol 117: 447-453
[56] Wenzel, WW; Adriano, DC; Salt, D and Smith, R (1999) Phytoremediation: A plant-microbe-based
remediation system, in: Adriano, DC; Bollag, J-M; Frankenberger, Jr, WT and Sims RC (Eds.):
Bioremediation of Contaminated Soils, Madison, Wisconsin USA, 457-508
[57] Bricker, TJ; Pichtel, J; Brown, HJ and Simmons, M (2001) Phytoextraction of Pb and Cd from a
superfund soil: Effects of amendments and croppings. J Environ Sci Health 36; 1597-1610
[58] McGrath, SP; Zhao, F-J and Lombi, E (2001) Plant and rhizosphere processes involved in
phytoremediation of metal-contaminated soils. Plant Soil 232: 207-214
[59] Pereira, BFF; Abreu, CA and Berton, RS (2005) EDTA effect on soil solution ionic speciation and lead
availability to jack bean and sunflower in an oxisol. Proc. 8th. ICOBTE, Adelaide, Australia, 98-99
[60] Piechalak, A; Tomaszewska, B and Barałkiewicz, D (2003) Enhancing phytoremediative ability of
Pisum sativum by EDTA application. Phytochemistry 64: 1239-1251
[61] Schmidt, U (2003) Enhancing phytoextraction: The effect of chemical soil manipulation on mobility,
plant accumulation, and leaching of heavy metals. J Environ Qual 32: 1939-1954
[62] Chen, H and Cutright, T (2001) EDTA and HEDTA effects on Cd, Cr, and Ni uptake by Helianthus
annuus. Chemosphere 45: 21-28
[63] Kos, B and Leštan, D (2003) Induced phytoextraction/in situ soil washing of lead using biodegradable
chelate and permeable barriers. Environ Sci Technol 37: 624-629
[64] Lai, H-Y and Chen, Z-S (2005) The interaction of Cd, Zn, and Pb on the phytoextraction of Rainbow
Pink growing in combined metals-contaminated soils. Proc. 8th. ICOBTE, Adelaide, Australia, 286-287
[65] Wenzel, WW; Unterbrunner, R; Sommer, P and Sacco, P (2003) Chelate-assisted phytoextraction using
canola (Brassica napus L.) in outdoors pot and lysimeter experiments. Plant Soil 249: 83-96
[66] Keller, C; Ludwig, C; Davoli, F and Wochele J (2005) Thermal treatment of metal-enriched biomass
produced from heavy metal phytoextraction. Environ Sci Technol 39: 3359-3367
USING HYPERACCUMULATOR PLANTS TO PHYTOEXTRACT SOIL Cd

AUTUMN S. WANG1, RUFUS L. CHANEY2, J. SCOTT ANGLE1

AND MARLA S. MCINTOSH1
1
University of Maryland at College Park, Maryland, USA, 20742,
E-mail: [email protected], 2USDA-ARS, Animal Manure and
By-Products Lab, Beltsville, Maryland, 20705 USA

1. Why is Cd phytoextraction being developed?

Adverse effects of soil Cd on humans were observed in Toyoma, Japan in 1969, and
subsequently found in many locations in Japan and China. In these cases, mining or
smelting of non-ferrous metals (Zn, Pb, Cu, Ag) caused dispersal of mine tailings which
contaminated rice paddies where farmers grew their own food for many years. Such
contamination localized to the rice irrigation network is one example of how rice uptake
to grain of soil Cd harmed humans [1]. Similar contamination has now been found in
other countries where rice is produced by subsistence farm families and where it is likely
that human disease is occurring or will occur in time.
Much has been learned about transfer of Cd from paddy soils to rice grain which
helps explain why high incidence of Cd disease (renal proximal tubular dysfunction) has
been seen among subsistence rice farm families, but not in Europeans or North
Americans with even higher soil Cd concentrations. Chaney et al. summarized data from
rice Cd research, and bioavailability of food-borne Cd to humans [2]. Further analysis of
the Japanese epidemiologic data showed that urinary Cd had to exceed a threshold
before any adverse effects on tubular function occurred, and these preceded any effect
on bone [3]. These re-interpretations of the susceptibility of humans to dietary Cd
illustrated the importance of malnutrition of Fe and Zn in subsistence rice farm families
which causes them to absorb a much higher fraction of diet Cd than persons with more
nutritious diets. Rice is also remarkable because grain Cd is greatly increased with no
increase in grain Zn even though the soils contain 100-times more Zn than Cd [4, 5]. No
other crop grown on geogenic Cd rich soils causes such high transfer of soil Cd to
human diets in bioavailable forms. Tobacco is the other crop that can comprise risk to
humans when the crop is grown on geogenic Cd contaminated soils. Because part of the
Cd enters smoke and is inhaled and absorbed very efficiently in the lung, factors which
would reduce bioavailability of ingested Cd cannot protect against absorption of tobacco
smoke Cd. Tobacco with minimal yield reduction can reach as high as 25 mg Cd kg–1
dry weight in leaves, high enough to cause adverse effects in smokers after many years.

103
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 103 – 114.
© 2006 Springer.
104 Autumn S. Wang et al.

The bioavailability of Cd in specific foods has been studied for many years, and it is
now evident that foods rich in supply of Fe, Zn, Ca, or phytate can inhibit absorption of
Cd. Cadmium rich shellfish also carry high levels of Fe and Zn which prevent high
absorption of shellfish Cd ingested by humans. High phytate and fibre in whole grains
reduce Cd bioavailability. Because polished rice has such low levels of bioavailable Fe
and Zn, intestinal processes to absorb Cd are increased substantially compared to
persons with higher bioavailable Fe and Zn in their diets [6]. Thus, although many
locations in Europe and North America have become contaminated by mining and
smelting of Zn ores, none are now recognized to have caused adverse effects on humans
through food-chain transfer of Cd from contaminated soils. This change in
understanding of soil Cd risk thus focuses attention on rice soils, and soils which are
contaminated by Cd sources with high Cd:Zn ratio such as Cd pigments, Cd plating, high
Cd biosolids, phosphate mine wastes, and Cd-Ni battery wastes.

2. Where are those possible commercial applications for Cd phytoextraction?

Thus paddy rice land in Japan, China, Korea, Vietnam and Thailand which has been
shown to be contaminated by geogenic Zn + Cd sources is the largest area of soils
requiring Cd remediation. No method for inactivation of soil Cd has been identified that
can persistently reduce Cd phytoavailability. Recent evaluation of the contamination of
rice fields in Japan suggests that 500,000 ha would need Cd remediation if the Cd limit
for marketed rice were set at 0.2 mg kg-1, with about 100,000 ha needing remediation if
the rice Cd limit is 0.4 mg kg-1. The improved understanding of Cd effects on kidney
function reported by Ikeda et al. [3] and new population studies reported by Horiguchi et
al. [7] indicate that with present nutritional status of Japanese farm families, setting the
rice limit at 0.4 mg Cd kg-1 would provide the needed protection for the most sensitive
and exposed individuals.
One can estimate the cost of soil Cd phytoextraction remediation by removal and
replacement of contaminated soil depth needed for rice soils which produce brown rice
with Cd greater than 0.4 mg kg-1 in Japan by multiplying the area times the cost per ha
[100,000 ha -times- $2,500,000 ha-1] = $250 billion if the traditional soil removal and
replacement approach were used as shown in the remediation of about 646 ha in Japan in
the 1980s [8]. The extreme cost of remediation of this area by soil removal and
replacement has delayed government decisions on soil Cd remediation. Fortunately,
phytoextraction of this Cd would cost < 1% of soil removal. Rice farmers could be paid
to produce high Cd T. caerulescens biomass during a short clean up period, and be paid
based on the amount of Cd in delivered biomass (mass times concentration) to
encourage best phytoextraction practice for production of the phytoextraction crop. Soils
contaminated by sources with high Cd:Zn ratio comprise the remaining soils where
remediation will be required to protect humans from accumulation of Cd in soils. In the
case of geogenic Cd+Zn contamination, a crop such as lettuce will attain only about 4-5
mg Cd kg-1 DW when it reaches 400-500 mg Zn kg-1 DW and suffers Zn phytotoxicity.
When Zn phytotoxicity is observed, growers can add limestone and prevent economic
loss due to Zn and further reduce Cd concentration in the plant tissues. But when the Cd
source has high Cd:Zn ratio, leafy crops can reach over 100 mg Cd kg-1 DW with no
Using Hyperaccumulator Plants 105

yield reduction and no visible symptoms of the crop contamination. Thus soils with high
Cd:Zn ratio also require remediation and Cd phytoextraction appears to be the only
economic method which is available.

3. Phytoextraction concept

The concept of using hyperaccumulator plants to accumulate high quantities of metals in

plant biomass to remove heavy metals from contaminated soils was first suggested by
Chaney [9]. In addition to the low cost, phytoextraction has several other important
advantages over the traditional soil removal/replacement remediation methods. For
example, it is in situ, preserves top soil, reduces the secondary waste stream, is
environmental sustainable and the plant ash may also have economic value [10, 11]. It is
important to note that only hyperaccumulator plants can make it practical. Non-
hyperaccumulator plants have no practical value in phytoextraction [12].
Hyperaccumulator plants typically contain >100 mg Cd kg–1, >1000 mg Ni kg–1, or
>10,000 mg Zn kg–1 in their leaf tissue (dry weight). Most plants suffer toxicity and
experience yield reduction when leaves contain about 400-500 mg Zn kg–1 or 50-100 mg
Ni kg–1.

4. Thlaspi caerulescens

To date, about 400 taxa have been identified as hyperaccumulators [13]. The majority of
them are Ni hyperaccumulators. Thlaspi caerulescens is primarily a Zn and Cd
hyperaccumulator and is the most extensively studied. It actually requires abnormal
amounts of Zn to be able to grow normally [14]. Concentrations can exceed 3% and
0.1% of Zn and Cd, respectively, in shoot dry matter without yield reduction. Thlaspi
caerulescens is not uniform in metal accumulation capability [15]. The accumulation
rates vary among populations [16, 17], and are influenced by the physical and chemical
characteristics of soils. Some populations of T. caerulescens from the south of France
exhibited extraordinary Cd hyperaccumulating ability where foliar Cd concentrations
could reach 3000 mg kg–1 [18-20]. These Southern France populations appear to have
the great potential to make Cd phytoextraction a reality.

5. Metal phytoavailability and T. caerulescens hyperaccumulation

Chemical fractionation procedures have been proposed as a means to identify plant

available forms of heavy metals in soil. Different sequential extraction procedures (SEP)
have been used to partition metals into fractions as soluble, exchangeable, absorbed,
organically bound, oxide-bound, precipitated, occluded and residual [21, 22].
Researchers have for many years tried to correlate metals in these fractions with plant
concentrations. Although SEP is useful as an indicator of metal bioavailability,
correlation studies are of less value. Metal bioavailability only correlated with plant
tissue concentration when it is a limiting factor for plant uptake due to low soil buffering
capacity or low plant solubilization. In many cases, especially for T. caerulescens,
106 Autumn S. Wang et al.

metals released from formerly non-available forms comprised more than 50% of the
metals accumulated in plants [23-25]. Thus, the dynamic cyclic process: depletion due to
plant uptake and replenishment due to solubilization and desorption are generally not at
equilibrium. Measured metal concentrations can only capture a “moment in time” while
plant metal concentration is an accumulation of uptake over time. In mathematical terms,
it is an integration of numerous “moments” of metal concentrations. This may explain
similar studies gave varied results.
Progress in making phytoextraction a practical commercial technology is hindered by
a lack of strategies to optimize plant uptake of metals. Although T. caerulescens has
extraordinary ability to transfer high amount of Zn and Cd from soil into the shoot, its
use for commercial remediation of contaminated soils is limited by the restricted
phytoavailability of metals in a contaminated soil. Although the mechanisms of hyper-
accumulation remain unclear, it is generally agreed that hyperaccumulation process
involves three major steps: rapid uptake of metals by roots, high rates of translocation
from roots to shoots, and huge storage capacity by vacuolar compartmentalization [15,
26, 27]. The first step is usually rate-limiting. Plant uptake of metals is confined by
metal availability. Numerous observations have suggested that metal supply rate in soil
is more limiting than plant uptake of the metals.

6. Mechanisms by which T. caerulescens scavenges metals

Instead of avoiding metal polluted spots, Thlaspi caerulescens roots preferentially

colonize Zn and Cd-polluted areas [28]. The allocation and morphology of roots are
strongly influenced by Zn and Cd content, form, and location in soil. When all roots
were in homogeneous soil polluted with a soluble Zn salt (ZnSO4), root growth was
severely inhibited. The positive response of roots to metals is specific, only to Zn and
Cd. There was no response to Pb localization [29]. This specificity and precision of
distribution of the root system is considered an important factor in determining the
efficient removal of metals.
Once roots have proliferated in metal rich soil, there is still a problem that T.
caerulescens has to overcome: how to make the metals available? Rhizosphere
acidification and release of root exudates are two common mechanisms by which plants
modify the rhizosphere to acquire nutrients. A study by Luo et al. [30] investigated soil
solution Zn and pH dynamics during phytoextraction by T. caerulescens. Soil solution
pH decreased initially and then increased slightly in both planted and unplanted soil
zones. From 60 to 84 days after transplanting, the pH of the rhizosphere soil solution
was higher than that of non-rhizosphere soil solution. This indicated that rhizosphere
acidification was not the primary mechanism for mobilization of Zn in soil for T.
caerulescens. Similar result was found in a pot study [31]. Root exudates do not appear
to play a role in metal mobilization by T. caerulescens hyperaccumulation, either [32].
On the contrary, it was repeatedly found that T. caerulescens was able to access less
soluble Zn fractions in soil. In McGrath’s study, decreases in the mobile fraction of Zn
accounted for less than 10% of the total uptake of T. caerulescens; that is, more than
90% of the Zn must have come from the non-mobile fractions [31]. These authors also
found that rhizosphere soils tended to have higher concentrations of mobile Zn than the
Using Hyperacumulator Plants 107

non-rhizosphere soils. Similarly, in a study, the decrease of Zn in soil solution after

growth accounted for only 1% of the total Zn uptake by T. caerulescens. The authors
suggested that either T. caerulescens was highly efficient at mobilizing Zn which was
not initially soluble, or the soil could replenish solution Zn rapidly due to high buffering
capacity [23].
To test which one of the above two possible mechanisms was more important,
Whiting et al. used co-cultivated plants to see if mobilization of Zn by T. caerulescens
affected Zn concentrations of a co-cultivated indicator plants (Thlaspi arvense or
Festuca rubra) provided that they shared the same rhizosphere. Thlaspi caerulescens did
not increase Zn concentrations in either of the indicator plants, suggesting that T.
caerulescens does not “strongly” mobilize Zn in its rhizosphere [25]. In another
experiment, Whiting et al. used five Zn compounds of different solubility (ZnS,
Zn3(PO4)2, ZnO, ZnCO3, and ZnSO4•7H2O) to test how Zn hyperaccumulation was
influenced by Zn bioavailability. In a Clough Wood soil, the use of Zn-sulphate resulted
in the greatest total Zn in plant biomass, while in a Prayon soil, highest uptake was from
the Zn-oxide fraction. In the unenriched and ZnS enriched treatments, about 70% and
50% of T. caerulescens biomass Zn came from previously non-soluble forms. But Zn
hyperaccumulation in these two treatments was less than that from the other four
treatments. This may indicate that the solubilization effect of Zn by T. caerulescens was
not strong [24]. But comparing the nitrate-extractable Zn in day 0 and day 90, there was
a significant increase in all of the five Zn-enriched treatments. In the Zn-sulphide
treatment, there was an almost 10-fold increase in both soils. If this was caused by T.
caerulescens, it was obviously a very strong solubilization effect. Unfortunately they did
not study an un-planted treatment to exclude the possible effect of soil microbial
oxidation of ZnS during incubation. The paper by Gérard et al. showed that Thlaspi
absorbed Cd from the same labile pool as other plant species, only much faster [18]. It
seems likely to us that the dense, fine root system of Thlaspi caerulescens gives the plant
access to more soil volume within the rhizocylinder (the soil within the root hair distance
from root surfaces). High root surface area coupled with rapid uptake of Cd and Zn lets
this species achieve hyperaccumulation needed for phytoextraction.

7. Localization of Cd in leaves of T. caerulescens

In a recent approach, Cosio et al. reported that in both Ganges and Prayon ecotypes, Cd
was found primarily at leaf edges. Scanning electron microscopy coupled with energy
dispersive X-ray microanalysis (cryo-SEM-EDXMA) and tissue fractionation revealed
similar distribution patterns of Cd in leaf cells of both ecotypes. Cadmium was in both
inside the cells and in the cell walls of large epidermal cells as well as small epidermal
cells [33].

8. Development of Cd phytoextraction technology

Chaney et al. identified the systematic approaches to develop commercial phytoex-

traction technique:
108 Autumn S. Wang et al.

• collection of plant genetic diversity so that improved phytoremediation

cultivars can be selected and/or bred;
• valid comparison of genotypic differences in yield and hyperaccumulation;
• breeding improved plant cultivars which are effective in metal-rich fields;
• identification of soil and plant management practices needed to attain high
yields and high metal concentrations in the biomass including tillage,
fertilization, soil conditioners, pH adjustment, herbicides, etc.;
• identification of methods to plant, grow, harvest and market the biomass;
• selection of methods to economically recover the metals from the biomass
(e.g. a method to burn the biomass which retains the metals in a form that
they can be sold as a high grade metal ore), and the biomass energy may be
used for power generation;
• identification of methods to recover the metals from the ash;
• identification of farming systems which allow use of this technology to
produce jobs and profits for growers as well as smelters [11]
Chaney et al. summarised the philosophy and progress of our team in developing Cd
phytoextraction technology: “The approach…is based on our understanding of the
science and the potential market for Cd and Zn phytoextraction. Because the Zn and Cd
accumulated in biomass have much lower value than Ni, the value of metal in the
biomass will not drive development and use of this technology. Biomass energy can
reduce the cost of soil Cd cleanup, but is unlikely to make Zn+Cd phytoextraction
profitable by phytomining. Rather, the value of the cleanup will drive the market. Risk
from Zn can be controlled by soil pH management, but for Cd, risk from rice soils,
tobacco soils, and soils with high Cd:Zn ratio must be remediated to be able to produce
crops safe for consumption. Most of the Cd phytoextraction market will be in paddy rice
land contaminated by emissions of Zn-Pb-Cu-Ag mining and smelting. It is now clear
that paddy rice lands in Japan, China, Korea, Thailand and Vietnam have been
contaminated enough to require remediation or change in land use.” [12].
“We adopted the agricultural paradigm to develop a Cd phytoextraction technology.
Comparing different models for Zn and Cd phytoextraction by crop and
hyperaccumulator species, the value of southern France genotypes of Thlaspi is evident.
We selected Thlaspi caerulescens for development because it was the largest Zn
hyperaccumulator which also accumulated Cd. The “Prayon” population was initially
studied by researchers because Alan Baker shared collected seed. Some other species
which can accumulate Cd exist, but were even smaller than Thlaspi (e.g., Arabidopsis
halleri, Arabis gemmifera), or did not accumulate favourable levels of Cd
(Dichapetalum gelonioides).” [12].
“After our initial studies to understand the potential of Thlaspi to accumulate soil and
solution Cd and Zn [34, 35], we evaluated 20 different Thlaspi genotypes in nutrient
solutions and a smaller subset in contaminated soils. The genetic screening in nutrient
solutions demonstrated high accumulation of Cd by southern France populations
compared to other populations [36]. A similar outcome was observed when we grew
Thlaspi genotypes in smelter contaminated field plots at Palmerton, PA [37] (Fig. 1).
We concluded that natural variation in Cd accumulation by Thlaspi populations would
support breeding of improved cultivars useful for practical Cd phytoextraction. Strong
Using Hyperacumulator Plants 109

promise was also reported by Keller and Hammer [38] who grew T. caerulescens in
several contaminated fields” [12].
“Phytoextraction Associates LLC, a new company created to commercialize Cd
phytoextraction, is working with USDA-ARS to develop and commercialize improved T.
caerulescens cultivars with higher yields and the high Cd:Zn accumulation of southern
France genotypes. Based on the findings of Reeves et al. who analyzed T. caerulescens
collected across Europe, the southern France genotypes are of special importance [20].
In the study of Perner et al. [16], eight plants were grown from seeds of each of 25
mother plants collected at these southern France Zn-Cd-contaminated sites. The wide
variation among siblings (due to mixed inbreeding and out-crossing) suggests that Cd
hyperaccumulation is a quantitative trait [39], while the southern France genotypes
appear to have a single gene which causes 10-fold higher Cd accumulation than
“Prayon” genotypes. Although more research is being conducted to understand these
genetic differences, it is clear that normal plant breeding can be used to develop
cultivars with higher yield and high annual Cd removal needed to achieve remediation of
soils which require Cd remediation to protect human health” [12].
As indicated previously in this paper, restricted bioavailability of metals in
contaminated soils is a limitation for phytoextraction efficiency. Researchers have tried
different methods to increase metal solubility. Apparently, EDTA or other chelating
Shoot Cd concentration, mg/kg

1500 1500

1000 1000

500 500
Prayon

0 0
1 6 8 11 12 14 15 16 17 18 19 20 21
Genotype Number

Figure 1. Cd accumulation into shoots of Thlaspi caerulescens ecotypes grown in the field
at Palmerton, PA, where soil contains 15,000 mg Zn and 150 mg Cd kg–1 dry weight [37].
The “Prayon” ecotype is marked. All of the much higher Cd ecotypes came from the
southern French populations. (Figure source: Chaney et al., 2004)
110 Autumn S. Wang et al.

induced treatments is not a solution due to high cost and the concern of ground-water
contamination. Instead, using S addition to reduce soil pH to enhance metal availability
could be a good strategy. Elemental S will be oxidized into sulphate salts (CaSO4, etc),
excessive salts will be drained out of the soil by natural rainfall. We conducted a
greenhouse study by using two contaminated soils differing in levels of Cd and Zn (5 mg
Cd kg–1 and 450 mg Zn kg–1 in the lower metal soil and 25 mg Cd kg–1 and 1500 mg Zn
kg–1 in the higher metal soil) and which were adjusted to 5 or 6 different pH levels (from
7.27 to 4.74). Reducing pH significantly increased 0.1 M Sr(NO3)2 extractable Cd and
Zn concentrations in both soils. Further, pH adjustment had significant effect on both
plant yield and metal concentrations in plant biomass. However, pH adjustment caused
different responses in these two soils. For the higher metal soil, yield continued to
increase with the decrease of pH, and was the highest at the lowest pH treatment.
Cadmium and Zn concentration in plant tissue as well as the total metal in plant shoots
were increased with the reduction of pH. The highest shoot metal concentration was
observed at pH 5.28 (Fig. 2). For the lower metal soil, plant yield, metal concentration in
plant tissue and total metal extracted by plant shoot all increased with decreasing pH,
reached a peak at pH treatment of 6.07 and rapidly decreased at further reduced pH
treatments. Soluble Al and Mn concentrations reached toxic levels in lower pH
treatments in the lower metal soil. For the higher metal soil, from the highest pH (6.88)
to the lowest pH (4.74), Al concentration only increased by about 30%. However, there
was 8 to 11 fold increase in soluble Al concentration for the lower metal soil. Al toxicity
in the lower metal soil restricted root development and hindered the root ability to
accumulate metals [40].
The extraordinary Cd phytoextraction ability of T. caerulescens was further
demonstrated in this experiment. In the optimum pH treatments, Thlaspi caerulescens
extracted 45% and 37% of total Cd in the lower and higher metal soils, respectively,
with just one 6-month planting. One of the major criticisms of phytoextraction is the
long time requirement. In order to remove all contaminants or reduce their level to
legislatively allowed limits, phytoremediation often requires decades to be effective. If
constant uptake by T. caerulescens in subsequent croppings can be achieved-where
Keller and Hammer’s study suggested that it is possible [38]- reducing pH would
shorten the required remediation time from 5 yr (the original soil pH) to 3 yr (the
optimum soil pH 5.28 in the higher metal soil and 6.07 in the lower metal soil) to
remove all the Cd from both soils. For Zn, reducing pH would shorten the remediation
time from 60 yr to 46 yr for the higher metal soil and 38 yr to 14 yr for the lower metal
soil. We recommend focus on Cd phytoextraction because risks from soil Zn can be
alleviated by liming, but soil Cd risks are not so easily remediated.
We concluded that reducing pH is an effective method to enhance Cd
phytoavailability and T. caerulescens uptake for both Cd and Zn. It also can greatly
shorten the time span for phytoremediation to be complete and has the potential to
overcome the problems associated with the long time requirement of phytoremediation.
Using S to reduce soil pH is advantageous over chelating-induced methods because it is
cost-effective and with few toxic effects on plant growth. The proper and effective pH
range for maximum metal uptake may differ for individual soils and therefore must be
identified to ensure successful phytoremediation. For Cd phytoextraction, the higher
Using Hyperacumulator Plants 111

Cd:Zn accumulation in plants allows Cd removed at high rates without Zn phytotoxicity

reducing yields (which limits annual Cd removed by “Prayon” types).

400
Low metal soil shoot
350
Cd conc. (mg kg−1)

root
300
250
200
150
100
50
0
4.74 5.28 6.07 6.37 6.88 7.27
pH

1800 High metal soil shoot

Cd conc. (mg kg−1)

1500 root

1200

900

600

300

0
4.74 5.28 6.07 6.37 6.88
pH

Figure 2. Means and standard errors of T. caerulescens tissue Cd concentration in higher

metal and lower metal soils with different pH treatments.

9. Ecological considerations of Cd phytoextraction

Recently, there is increasing awareness of how to define a truly successful

phytoextraction. The ultimate goal of soil remediation is to achieve a healthy soil
ecosystem and crops safe for lifetime consumption. Metal removal is one of the methods
to achieve this, not the remediation purpose itself. Therefore a successful
112 Autumn S. Wang et al.

phytoextraction include not only metal removal from soil, but also return of a healthy
soil ecosystem. One of the major misunderstandings during phytoextraction is the sole
focus on a simple calculation of how much pollutants were removed. The subject of
whether the soil health is eventually recovered has been largely ignored. Therefore, it is
possible that the process of remediation itself brings a more severe environmental
problem (leaching of metal-EDTA to groundwater; reduction of soil microbial function,
etc). It is important to realize that monitoring soil health throughout the remediation
process is vital in remediation.
When investigating the effect of reducing soil pH to enhance Cd phytoextraction, we
selected numerous soil quality indicators to monitor soil ecosystem health conditions
responding to reducing pH treatment both before and after phytoextraction. Soil enzyme
activities, nitrification, respiration, number of bacteria, actinomycetes, fungi, and
rhizobia were tested both under low pH treatments and after pH neutralization. This
research is the first attempt to examine whether and to what degree the soil biological
activities and soil microbial populations return to the background levels after soil pH is
re-adjusted to normal conditions after phytoextraction. Except for acid phosphatase
activity and the fungal population, reducing pH significantly reduced all tested activities
and microbial populations. However, soil retained the capacity to recover toward the
condition before acidification treatment even at the lowest pH treatment. But full
recovery may take a much longer time and may be very difficult to achieve original
levels seen in the lowest pH treatment. It is recommendable to keep the pH values above
6.1 and 5.3 for lower and higher metal soils tested in this experiment, respectively.
Above this value, most of the soil biological activities and all tested microbial
populations can return to background levels within a short period. Moreover, metal
leaching was negligible if soil pH were maintained at above these values. Based on our
results, soil biological activities and microbial populations can be severely damaged
when soil pH is reduced below a certain threshold. Precaution must be exercised when
we try to enhance phytoextraction by reducing soil pH. Fortunately, our research
revealed that it is not necessary to reduce pH too much. Optimum plant growth and
phytoextraction can be achieved before pH being lowered to a value where possibly
permanent soil ecologic damage would occur. The pH threshold can vary according to
the soil type, mineral composition and other properties. There is no unique solution to
all different kinds of soils [41].
More ecological considerations should be integrated into Cd phytoextraction
process. Representative and sensitive soil health indicators may be used to monitor the
soil ecosystem health and function changes accompanying phytoextraction processes
after returning the soil to a pH required by most crops, and cropping to feed the soil
microbes and aid population growth.

References
[1] Kobayashi, J (1978) Pollution by cadmium and the itai-itai disease in Japan. pp. 199-260, in FW
Oehme, Ed., Toxicity of Heavy Metals in the Environment. Marcel Dekker, Inc., New York
[2] Chaney, RL and Ryan, JA (1994) Risk Based Standards for Arsenic, Lead and Cadmium in Urban Soils,
Dechema, Frankfurt. ISBN 3-926959-63-0
Using Hyperacumulator Plants 113

[3] Ikeda, M; Ezaki, T; Tsukahara, T; Moriguchi, J; Furuki, K; Fukui, Y; Ukai, H; Okamoto, S and Sakura,
H (2003) Threshold levels of urinary cadmium in relation to increases in urinary ȕ2-microglobulin
among general Japanese populations. Toxicol Lett 137: 135-141
[4] Chaney, RL; Reeves, PG; Ryan, JA; Simmons, RW; Welch, RM and Angle, JS (2004) An improved
understanding of soil Cd risk to humans and low cost methods to remediate soil Cd risks. BioMetals 17:
549-553
[5] Simmons, RW; Pongsakul, P; Chaney, RL; Saiyasitpanich, D; Klinphoklap, S and Nobuntou, W (2003)
The relative exclusion of zinc and iron from rice grain in relation to rice grain cadmium as compared to
soybean: Implications for human health. Plant Soil 257: 163-170
[6] Reeves, PG and Chaney, RL (2002) Nutritional status affects the absorption and whole-body and organ
retention of cadmium in rats fed rice-based diets. Environ Sci Technol 36, 2684-2692
[7] Horiguchi, H; Oguma, E; Sasaki, S; Miyamoto, K; Ikeda, Y; Machida, M and Kayama, F (2004).
Dietary exposure to cadmium at close to the current provisional tolerable weekly intake does not affect
renal function among female Japanese farmers. Environ Res 95: 20-31
[8] Iwamoto, A (1999) Restoration of Cd-polluted paddy fields in the Jinzu River basin - Progress and
prospects of the restoration project. pp. 179-183, in K Nogawa; M Kurachi and M Kasuya, Eds.,
Advances in the Prevention of Environmental Cadmium and Countermeasures. Eiko Laboratory,
Kanazawa, Japan
[9] Chaney, RL (1983) Plant uptake of inorganic waste constituents. pp. 50-77, in JF Parr; PB Marsh and
JM Kla, Eds, Land Treatment of Hazardous Wastes. Noyes Data Corp., Park Ridge, NJ
[10] Baker, AJM; McGrath, SP; Reeves, RD and Smith, JAC (2000) Metal hyperaccumulator plants: a
review of the ecology and physiology of a biological source for phytoremediation of metal-polluted soil.
pp. 85-107, in N Terry and GS Bañuelos, Eds, Phytoremediation of Contaminated Soil and Water. Lewis
Publishers, Boca Raton
[11] Chaney, RL; Li, YM; Angle, JS; Baker, AJM; Reeves, RD; Brown, SL; Homer, FA; Malik, M and Chin,
M (2000) Improving metal hyperaccumulator wild plants to develop commercial phytoextraction
systems: approaches and progress. pp. 131-160, in N Terry and GS Bañuelos, Eds, Phytoremediation of
Contaminated Soil and Water. CRC Press, Boca Raton, FL
[12] Chaney, RL; Angle, JS; McIntosh, MS; Reeves, RD; Li ,YM; Brever, EP; Chen, KY; Roseberg, RJ;
Perner, H; Synkowski, EC; Broadhurst, CL; Wang, AS and Baker, AJM (2004) Using hyperaccumulator
plants to phytoextract soil Ni and Cd. Z Naturforsch C 60: 190-190
[13] Baker, AJM; Reeves, RD and Hajar, ASM (1994) Heavy metal accumulation and tolerance in British
populations of the metallophyte Thlaspi caerulescens J and C Presl. (Brassicaceae). New Phytol 127:
61-68
[14] Shen, ZG; Zhao, FJ and McGrath, SP (1997) Uptake and transport of zinc in the hyperaccumulator
Thlaspi caerulescens and the non-hyperaccumulator Thlaspi ochroleucum. Plant Cell Environ 20:
898-906
[15] Pollard, AJ; Harper, KD and Smith, JAC (2002) The genetic bases of metal hyperaccumulation in plants.
Crit Rev Plant Sci 21, 539-566
[16] Perner H; Chaney, RL; Reeves, RD; Römheld, V; McIntosh, MS; Angle, JS and Baker, AJM (2004)
Variation in Cd- and Zn-accumulation by French genotypes of Thlaspi caerulescens J and C Presl.
grown on a Cd and Zn contaminated soil. Submitted to: Plant Soil
[17] Molitor, M; Dechamps, C; Gruber, W and Meerts, P (2005) Thlaspi caerulescens on nonmetalliferous
soil in Luxembourg: ecological niche and genetic variation in mineral element composition. New Phytol
165: 503-512
[18] Gérard, E; Echevarria, G; Sterckeman, T and Morel, JL (2000) Cadmium availability to three plant
species varying in Cd accumulation pattern. J Environ Qual 29: 1117-1123
[19] Lombi, E; Zhao, FJ; Dunham, SJ and McGrath, SP (2000) Cd uptake in populations of Thlaspi
caerulescens and Thlaspi goesingense. New Phytol 145: 11-20
[20] Reeves, RD; Schwartz, C; Morel, JL and Edmondson, J (2001) Distribution and metal-accumulating
behaviour of Thlaspi caerulescens and associated metallophytes in France. Int J Phytorem 3: 145-172
[21] Davidson, CM; Thomas, RP; McVey, SE; Perala, R; Littlejohn, D and Ure, AM (1994) Evaluation of a
sequential extraction procedure for the speciation of heavy metals in sediments. Analytica Chimica Acta
291: 277-286
114 Autumn S. Wang et al.

[22] Welter, E; Calmano, W; Mangold, S and Tröger, L (1999) Chemical speciation of heavy metals in soils
by use of XAFS spectroscopy and electron microscopical techniques. Fresenius J Anal Chem 364:
238-244
[23] Knight, BP; Zhao, FJ; McGrath, SP and Shen, ZG (1997) Zinc and cadmium uptake by the
hyperaccumulator Thlaspi caerulescens in contaminated soils and its effects on the concentration and
chemical speciation of metals in soil solution. Plant Soil 197: 71-78
[24] Whiting, SN; Leake, JR; McGrath, SP and Baker, AJM (2001) Zinc accumulation by Thlaspi
caerulescens from soils with different Zn availability: a pot study. Plant Soil 236: 11-18
[25] Whiting, SN; Leake, JR; McGrath, SP and Baker, AJM (2001) Assessment of Zn mobilization in the
rhizosphere of Thlaspi caerulescens by bioassay with non-accumulator plants and soil extraction. Plant
Soil 237:147-156
[26] Chaney, RL; Malik, M; Li, YM; Brown, SL; Brewer, EP; Angle, JS and Baker, AJM (1997)
Phytoremediation of soil metals. Curr Opin Biotech 8: 279-284
[27] McGrath, SP; Zhao, FJ and Lombi, E (2002). Phytoremediation of metals, metalloids, and radionuclides.
Adv Agron 75, 1
[28] Whiting, SN; Leake, JR; McGrath, SP and Baker, AJM (2000) Positive responses to Zn and Cd by roots
of the Zn and Cd hyperaccumulator Thlaspi caerulescens. New Phytol 145: 199-210
[29] Schwartz, C; Morel, JL; Saumier, S; Whiting, SN and Baker, AJM (1999) Root development of the zinc-
hyperaccumulator plant Thlaspi caerulescens as affected by metal origin, content and localization in
soil. Plant Soil 208: 103-115
[30] Luo, YM; Christie, P and Baker, AJM (2000) Soil solution Zn and pH dynamics in non-rhizosphere soil
and in the rhizosphere of Thlaspi caerulescens grown in a Zn/Cd contaminated soil. Chemosphere 41:
161-164
[31] McGrath, SP; Shen, ZG and Zhao, FJ (1997) Heavy metal uptake and chemical changes in the
rhizosphere of Thlaspi caerulescens and Thlaspi ochroleucum grown in contaminated soils. Plant Soil
188: 153-159
[32] Zhao, FJ; Hamon, RE and McLaughlin, MJ (2001) Root exudates of the hyperaccumulator Thlaspi
caerulescens do not enhance metal mobilization. New Phytol 151: 613-620
[33] Cosio, C; DeSantis, L; Frey, B; Diallo, S and Keller, C (2005) Distribution of cadmium in leaves of
Thlaspi caerulescens. J Exp Bot 56: 765-775
[34] Brown, SL; Chaney, RL; Angle, JS and Baker, AJM (1994) Phytoextraction potential of Thlaspi
caerulescens and Bladder Campion for zinc and cadmium-contaminated soil. J Environ Qual 23:
1151-1157
[35] Brown, SL; Chaney, RL; Angle, JS and Baker, AJM (1995) Zinc and cadmium uptake by
hyperaccumulator Thlaspi caerulescens and metal tolerant Silene vulgaris grown on sludge-amended
soils. Environ Sci Technol 29: 1581-1585
[36] Li, YM; Chaney, RL; Angle, JS; Chen, KY; Kerschner, BA and Baker, AJM (1996) Genotypical
differences in zinc and cadmium hyperaccumulation in Thlaspi caerulescens. Agron Abstr 1996, 27
[37] Li, YM; Chaney, RL; Chen, KY; Kerschner, BA; Angle, JS and Baker, AJM (1997) Zinc and cadmium
uptake of hyperaccumulator Thlaspi caerulescens and four turf grass species. Agron Abstr 1997, 38
[38] Keller, C and Hammer, D (2004) Metal availability and soil toxicity after repeated croppings of Thlaspi
caerulescens in metal contaminated soils. Environ Pollut 131: 243-254
[39] Synkowski, EC; McIntosh, MS; Chaney, RL; Angle, JS and Reeves, RD (2004) RAPD variation in two
Thlaspi caerulescens populations: Implications for breeding. Proc. Fourth International Serpentine
Ecology Conference, Havana, Cuba
[40] Wang, AS; Angle, JS; Chaney, RL; Delorme, TA and Reeves, RD (2005) Soil pH effects on uptake of
Cd and Zn by Thlaspi caerulescens. Plant Soil (In press)
[41] Wang, AS; Angle, JS; Chaney, RL; Delorme, TA and McIntosh, MS (2005) Ecological Risks of
Reducing Soil pH during Zn and Cd Phytoextraction. Submitted to: J Environ Qual
ENHANCED HEAVY METAL PHYTOEXTRACTION

DOMEN LEŠTAN
Agronomy Department, Biotechnical Faculty, University of Ljubljana,
Jamnikarjeva 101, 1000 Ljubljana, Slovenia – Fax 386 1 423 1161
Email: [email protected]

1. Introduction

Phytoextraction refers to the use of metal-accumulating plants, which are able to

transport and concentrate inorganic contaminants, most importantly heavy metals but
also metalloids and radionuclides, from the soil into their harvestable, above-ground
parts. Metal-enriched plant biomass can be safely disposed of as a hazardous material or,
if economically feasible, used for metal recovery. For phytoextraction to be possible, the
metals must be in soil horizons within a plant’s root zone, be bioavailable for plants and
plants must have a genetic predisposition for compartmentalisation of extracted metals.
Some metals are readily bioavailable for plants: Cd, Ni, Zn, As, Se, Cu, and some have a
low bioavailability: Pb, Cr, U, Hg [1].
There are two general approaches to phytoextraction: continuous and chemically
enhanced phytoextraction [2]. The first approach uses naturally hyperaccumulating
plants with the ability to accumulate an exceptionally high metal content in the shoots.
Hyperaccumulating plants usually hyperaccumulate only a specific metal and metals that
are primarily accumulated (Ni, Zn and Cu) are not among the most important
environmental pollutants. No plant species has yet been found that demonstrates a wide
spectrum of hyperaccumulation [3]. Hyperaccumulators are also mostly slow growing,
low biomass-producing species, lacking in good agronomic characteristics [4]. There is
no evidence that natural hyperaccumulator plants can access a less soluble and bio-
available pool of metals in soil.
No effective hyperaccumulating plant, with high Pb uptake and high biomass
essential for efficient phytoextraction, has been reported so far for Pb, one of the most
widespread and important metal contaminants. Vegetation growing in heavily
contaminated areas often has less than 50 mg g–1 Pb in shoots [4] and Pb is mostly
confined to the roots, with minimal transport to the green parts of plants. In fact, there is
only one reliable report of Pb hyperaccumulation. Puschenreiter et al. [5] reported that
the small alpine plant Thlaspi goesingense accumulated 2840 mg Pb kg–1 in its shoots.
However, when we tested the same reported hyperaccumulator for phytoextraction of
soil contaminated with 1170 Pb and 750 mg kg–1 Zn from Mežica Valley in Slovenia,
contaminated by mining and the smelting industry, the concentration of Pb in leaves of

115
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 115–132.
© 2006 Springer.
116 Domen Leštan

T. goesingense did not exceed 20 mg kg–1 and was statistically comparable to the Pb
concentration in leaves of lettuce (Lactuca sativa) [6]. These results might indicate that
the Pb hyperaccumulating capacity of T. goesingense is limited only to certain soil and
contamination types.

Harvesting

Metal
removal

Metal
accumulation Mobilising
agent

Metal Metal
translocation mobilisation

Metal Metal
plant uptake leaching

Figure 1. Conceptual representation of heavy metal phytoextraction enhanced by the

addition of mobilizing (i.e. chelating) agent.

In non-hyperaccumulating plants, factors limiting their potential for phytoextraction

include small root uptake and little root-to-shoot translocation of heavy metals.
Chemically enhanced phytoextraction has been shown to overcome the above problems
[7-9]. Common crop plants with high biomass can be triggered to accumulate high
amounts of low bioavailable metals, when their mobility in the soil and translocation
from the roots to the green part of plants was enhanced by the addition of mobilizing
agents when the crop had reached its maximum biomass. The feasibility of chemically
enhanced phytoextraction has been primarily studied for Pb and chelating agents as soil
additives, less attention has been given to other metals and radionuclides or their
mixtures.

2. Fractionation of metals in soil

The geochemical forms of metals in contaminated soils affect their solubility, which
directly influences their availability to plants. Plant uptake of metals therefore shows
marked dependence on the chemical speciation and soil fractionation of the metals. For
example, Pichtel et al. [10] reported that a significant correlation exists between Pb
uptake by dandelion (Taraxacum officinale) and bioavailable fractions of soil Pb soluble
in the soil solution and exchangeable from soil colloids to the soil solution. The fraction
of metals soluble in the soil solution consists of free hydrated ions, water-soluble organic
and inorganic complexes and metals sorbed on dissolved organic matter. They are the
Enhanced Heavy Metal Phytoextraction 117

most mobile forms of metals in soil and directly available to plants. A plausible
explanation for the lack of Pb hyperaccumulating plants is therefore that in most soils
capable of supporting plant growth, only a very small portion of the Pb in the soil is
present in soil solution or exchangeable from soil colloids and thus does not allow
substantial Pb mobility and plant uptake. Indeed, Pb from most contaminated sites tends
to persist in the soil surface layers, although some Pb bound to the soluble part of the
soil organic matter can be transported with percolates through the soil profile [11].
The fractionation of metals into soluble in soil solution and exchangeable from soil
colloids (two bioavailable fractions), and metals strongly bound into the soil solid phase
is probably controlled by many types of reactions, a) Adsorption/desorption reactions
due to chemical bonding, complex formation, and ion-exchange. Partly covalent bonds
of metals with mineral/humic colloids and hydrous oxides are formed. Coordinate bonds
and complexes of metals with soluble soil organic matter and polycarboxylic organic
acids of microbial origin are also formed. Reversible electrostatic exchange occurs
between metals and other cations from the negatively charged surface of soil colloids.
Adsorption/desorption reactions mostly depend on the pH of the soil solution. b)
Precipitation of metals, generally with anions such as phosphate, carbonate, sulphate and
as hydroxides. Precipitation is more unlikely in acidic soil. c) Penetration into the crystal
structure of minerals and isomorphic exchange with cations and d) Biological
mobilization and immobilization. Metals bioaccumulate and biomagnify in the food
chain [12]. These reactions are presumably determined and constrained by soil
properties: soil texture, content of organic matter, content and type of clay minerals and
Al, Fe and Mn oxides, and prevailing physicochemical conditions in the soil: soil
saturation, soil aeration, pH, and redox potential. A considerable number of studies have
tried to infer these relationships. Sauve et al. [13], for example, reported that the
concentration of dissolved Pb and activity of free Pb2+ ions were highly significantly
correlated to soil pH and total Pb content. Janssen et al. [14] reported that pH was in
fact the only soil parameter of significance for partitioning Pb, Zn and other metals
between the soil solution and soil solid phases. This association between metal
adsorption on exchangeable surfaces of soil colloids and pH is due partly to competition
of H+ for adsorption sites at low pH, resulting in decreased metal adsorption [11].
Sequential extraction analysis remains a widely used procedure for identifying the
metal fractionation, despite some difficulties: sensitivity to procedural variables, limited
selectivity of extractants, re-adsorption of metals at different phases during extraction,
and overload of the chemical system if the content of metals is too high [15, 16]. The
distribution pattern of Pb in different soil fractions from contaminated sites in Mežica
Valley and the Celje region in Slovenia is shown in Table 1 [17-18]. We found Pb
mostly bound to soil organic matter and carbonates or remaining in the residual form. A
strong association of Pb with organic matter has also been described by Kabata-Pendias
[19], based on the results of many investigators. Li and Thornton [20] reported a
significant association of Pb with the soil carbonate phase. As also observed before by
other authors [15, 16, 21], a very small fraction of the total Pb was determined in the soil
solution and exchangeable from the soil colloids to the soil solution.
118 Domen Leštan

Table 1. A modified analytical procedure according to Tessier et al. [22] was used to
determine fractionation of Pb in soils from Mežica Valley and the Celje region in Slovenia,
contaminated by the metal smelting industry.

Fractions Mežica Valley1 Celje region2

(range of 12 samples, %) (range of 30 samples, %)
soil solution 0.05-0.57 0.0-0.08
exchangeable 0.01-6.4 0.0-1.6
carbonate 4.9-67.1 2.04-43.5
Fe and Mn oxides 0.11-8.3 0.0-16.1
organic matter 31.5-74.7 35.8-71.1
residual 5.9-23.8 10.4-53.4
1
Soils pH range 3.7-7.3
2
Soils pH range 4.6-7.5

3. Enhanced phytoextraction

In addition to the inherent metal characteristics and soil factors, which determine
fractionation and, consequently, the bioavailability of metals, plants can themselves
change metal mobility. Plants can regulate metal solubility by acidification of the
rhizosphere due to the extrusion of H+ from roots and by exuding their own chelating
agents, phytosiderophores and organic acids; for example, malic and citric acids. A
chelating agent is a substance whose molecules can form several coordinative bonds to a
single metal ion (Figure 2). A chelating agent is therefore a multidentate metal ligand
and forms complexes with metals. The localized excretion of plant chelating agents
mobilizes nutrients such as Zn, Fe, Mn and other metals. Water-soluble chelating agents
increase and maintain metal concentration in the soil aqueous phase.

O 2-
C
O CH2 CH2
H2C
CH2 N C O
N
Pb O CH2
H2C O
C O C
O O

Figure 2. Pb complexes with EDTA. Dotted bonds to Pb are coordinate.

These plant mechanisms are sufficient to desorb readily bioavailable polluting metals,
such as Cd, which predominantly form easily hydrolysable, low-energy bonds to the soil
solid phase [23]. To make plants take up Pb and other low bioavailable metals, which
are much more strongly bound to the soil solid phase than Cd, strong synthetic chelating
agents have been used to bring metals into the soil solution. For example, the addition of
Na salt of ethylenediamine-tetracetic acid (EDTA) to soil shifted Pb mostly from the
carbonate fraction to the fraction soluble in the soil solution, in which the Pb con-
centration increased from 3 to 362 mg kg–1 (Figure 3).
Enhanced Heavy Metal Phytoextraction 119

Figure 3. Fractionation of Pb in soil sample from contaminated site in Mežica Valley,

Slovenia, before and after mobilization with 5 mmol kg–1 EDTA. Fractions: 1 soil solution,
2 exchangeable, 3 carbonate, 4 Fe and Mn oxides, 5 organic matter, 6 residual.

Theoretically, the metal-chelating efficiency of chelating agents depends on the stability

constant (logK) of the metal-complex formation. Martell and Smith [24] compiled an
extensive database of stability constants for different metals and chelating agents.
Synthetic chelating agent such as EDTA and its structural analogues have a strong
affinity for Pb and some other important metallic soil pollutants (Table 2). The resulting
complexes are very stable, prevent precipitation and sorption of metals, and do not
release their metal ions unless there is a significant drop in soil pH. However, the
chelating agents are not specific to a particular metal in the soil and are subject to
numerous interferences with other cations, most notably Fe and Ca, usually present in
soil at much higher concentrations than the polluting metal [25, 26]. Furthermore,
complex formation is controlled by the kinetic of all complexation (coordination)
reactions, adsorption in the soil solid phases, mineral dissolution and possible
degradation of the chelating agent or its metal complexes [27]. In practice, these
interactions often limit the mobilization efficiency of chelating agents for a specific
polluting metal in the soil.
Synthetic chelating agents, mostly EDTA, are widely used. In agriculture, for
example, as ingredients of mineral fertilizers to increase the phytoavailability of Fe
and other soil micronutrients, and to maintain the solubility of micronutrients in
hydroponic solutions. Chelating agents are also used for ex situ chemical extraction of
polluting metals from contaminated soils. Two technological approaches are in use;
extraction of metals from soil slurry in reactors and leaching of metals from soil heaps or
columns. The literature to date reports a number of chelating agents that have been
tested for enhanced heavy metal phytoextraction. The ability of a chelating agent to
facilitate phytoextraction does not necessarily always relate to its theoretical affinity
for metals (Table 2). Blaylock et al. [8] compared five chelating agents for their ability
to enhance Pb accumulation in the shoots of Indian mustard (Brassica juncea) and found
EDTA to be the most effective. For Cd uptake by B. juncea, Blaylock et al. [8] tested
120 Domen Leštan

CDTA, DTPA, EDTA and EGTA and found the last named the most effective. Of the
organic acids tested as chelating agents (acetic, citric and malic) Huang et al. [28] found
citric acid to be the most effective in enhancing the U concentration in shoots of the
same plant species. Zn binding by DTPA is so strong, that plants cannot use Zn from its
complex and potentially suffer from Zn deficiency. We compared four chelating agents
(citric acid, EDTA, DTPA and [S,S] isomer of EDDS) for enhanced phytoextraction of
Cu into Chinese cabbage (Brassica rapa) and found [S,S]-EDDS to be the most efficient
[29].
Table 2. Most important chelating agents tested for enhanced phytoextraction of metals
from the soil and their stability constant of complex formation (logK at T 20-25 oC and
ionic strength 0.1-1.0) for low bioavailable Pb, Cr and U in the soil, and readily available
Fe and Ca (source: Martel and Smith [24]).

Name Acronym LogK

Pb2+ Cr3+ U4+ Fe3+ Ca2+
ethylenediamine tetraacetic acid EDTA 18.0 23.4 25.8 25.1 10.6
trans-1,2-diaminocyclohexane-N,N,N′,N′- CDTA 20.2 23.0 27.6 30.1 13.1
tetraacetic acid
dietylenetriamine pentaacetic acid DTPA 18.8 / 28.8 27.8 10.7
ethylenediamine dissucinic acid EDDS 12.7 / / / 4.72
nitrilotriacetic acid NTA 11.5 6.2 9,61 15.9 6.3
N-hydroxyethylethylenedioamine triacetic HEDTA 15.6 6.1 / 19.7 8.1
acid
ethylenebis(oxyethylenetrinitrilo)-N,N,N',N' EGTA 14.6 / 9.41 20.5 10.8
tetraacetic acid
1
citric acid / 4.4 / 7.4 11.2 3.5
1
UO22+
Several plants have been used in combination with chelating agents for enhanced
phytoextraction. The ideal plant should be fast growing and produce a large biomass
while accumulating high concentrations of polluting metals. It should also be tolerant
enough to grow in contaminated soils and be resistant to the chelating agent. It should
have known agronomics practice and produce usable fruit or biomass to generate some
financial income after harvesting. B. juncea possesses several of these characteristics
and is the most commonly used plant species in this remedial approach.
Chelating agents are almost exclusively used as mobilizing agents for enhanced
phytoextraction. However, different mineral acids and salts have also been tested.
Screening of potential mobilizing agents for radionuclide 137Cs among chelating agents,
reducing agents, mineral acids and salts, showed ammonium and potassium salts to be
the most effective [30]. In a pot study, Lasat et al. [31] showed that the application of
NH4+ (40 – 80 mmol kg–1) increases the accumulation of 137Cs in different plants by 2-12
times. Hammer et al. [32] tested sulphur as a soil amendment. Elemental sulphur is
oxidized into sulphuric acid by lithoautotrophic soil microorganisms. This acidifies the
soil and possibly mobilizes toxic metals. They used high biomass crops such as willow
(Salix viminalis) to extract Cd and Zn from one calcareous and one acidic soil. However,
the addition of elemental sulphur to the soil did not yield any additional benefit.
Once a metal is chelated by a chelating agent, the complex has to move from the bulk
of the soil to the root’s xylem. A threshold concentration of chelating agent is usually
required to disrupt the physiological barriers that control metal root uptake under normal
Enhanced Heavy Metal Phytoextraction 121

conditions [33]. Two pathways are then possible: the solution with complexed metals
can enter the symplast by crossing the cell membranes, or move via the apoplast. Further
translocation of metal complexes from the roots to the green parts of the plants is driven
by plant transpiration. We reported that a single dose addition of 10 mmol EDTA kg–1 to
soil contaminated with Pb, Zn and Cd increased their concentration in the aboveground
biomass of B. rapa by 104.6, 3.2 and 2.3-times, respectively, while the concentration of
the same elements in the roots was 1.7, 3.5 and 3-times lower compared to the
corresponding plant tissues from control treatments [34]. These data indicate that metals
are probably translocated as a complex with the chelating agent. Indeed, an ultra-
structure study using a transmission electron microscope [35] located un-complexed Pb
predominantly in the root tissue of Chamaectisus palmensis plants, while HEDTA and
EDTA chelated Pb was mainly taken up by the shoots. In B. juncea, measurements of a
Pb-EDTA complex in xylem confirmed that the majority of Pb was transported as metal
complex in the transpiration stream [33, 36].
Soil contamination is seldom monometallic, and several polluting metals are usually
simultaneously present in elevated concentrations. It would therefore be of great
practical advantage if the use of a single chelating agent allowed phytoextraction of
multi-contaminated soils. Many studies support this. For example, we reported that the
addition of 10 mmol kg–1 [S,S]-EDDS to soil contaminated with 1100 mg Pb kg–1, 800
mg Zn kg–1, and 5.5 mg Cd kg–1, enabled the uptake of 1053 ± 125, 211 ± 16 and
5.4 ± 0.8 mg kg–1 of Pb, Zn and Cd, respectively, in the biomass of hemp (Cannabis
sativa). This was 105-, 2.3- and 31.7-times higher, respectively, than in the control
treatment [37]. However, other authors have reported that the use of chelating agents
such as EDTA and DTPA did not enhance, and in some cases even reduced, plant heavy
metal uptake [1].

4. Efficiency of chelating agent enhanced phytoextraction

The efficiency of phytoextraction is determined by two key factors: biomass production

and the metal bioconcentration factor. The bioconcentration factor is defined as the ratio
of metal concentration in the plant shoot to metal concentration in the soil. For
phytoextraction to be feasible, the bioconcentration factor of the plant must be greater
than 1, regardless of how large the achievable biomass is. Another way to measure the
efficiency of phytoextraction is by using the phytoextraction potential. This can be
calculated from soil and plant metal concentrations and dry biomass plant yield, as the
total amount of metal extracted per ha of soil, in a single phytoextraction cycle, and
expressed as kg ha–1. In order to be economically viable, plants for Pb phytoextraction
should be able to accumulate at least 10,000 mg Pb kg–1 in their green parts (harvesting
the roots is not practical) and achieve a dry biomass of 20 t ha–1 [7].
Shen et al. [38] used 3.0 mmol kg–1 EDTA to treat soil from a mining site in Hong
Kong, heavily contaminated with over 10,000 mg kg–1 of Pb. Application of EDTA in
three separate doses was the most effective and enabled the Pb concentration in the
shoots of B. rapa to exceed 5000 mg kg–1 of dry plant biomass. As explained above,
transpiration is believed to be a major force that drives heavy metal accumulation in
plant shoots. It has been shown that EDTA soil treatment is toxic for plants and could
122 Domen Leštan

decrease the plant transpiration rate [33]. Applying EDTA in three separate additions
Shen et al. [38], therefore, presumably minimized its adverse effect on the transpiration
rate. Barosci et al. [39] applied EDTA in multiple doses to provide time for plants to
initiate their adaptation mechanisms and raise their damage threshold against EDTA
phytotoxicity. In contrast to these results, we found application of EDTA in multiple
doses to be less effective for phytoextraction of Pb, Zn and Cd by B. rapa. [34].
Other authors have reported even higher plant metal concentrations induced by
chelating agent application. Blaylock et al. [8] used 3 week-old seedlings and measured
more than 15,000 mg Pb kg–1 in the dry weight of shoots of B. juncea after a 10 mmol
kg–1 EDTA addition. Huang et al. [36] determined 8960 mg kg–1 and 2410 mg Pb kg–1 in
two-week old pea and corn shoots transplanted into a soil substrate pre-treated with 1.5
mmol EDTA kg–1. However, experimental conditions were used in these studies that
would be unrealistic in field conditions and full-scale remediation. These include the use
of very young plants, a Pb soil fractionation that was favourable for phytoextraction and
achieved by “artificial” soil contamination with water-soluble metal salts, and an
experimental set up in which no losses of the Pb complex due to leaching occurred.
Since phytoextraction is a long-term technology, it is imperative to keep areas
undergoing phytoremediation productive to achieve economically viable and socially
acceptable decontamination. Industrial plants, i.e. energy crops or crops for bio-diesel
production, are therefore the prime candidates for phytoextraction plants. The use of
energy and/or bio-diesel crops for metal phytoextraction would give contaminated soil a
productive value and decrease remediation costs. Furthermore, if the phytoextraction
cost falls within the margin of interest, or even turns a profit, then the time needed for
the operation becomes less important. We evaluated the Pb, Zn and Cd phytoextraction
potential of a selection of potential energy plants (C. sativa, Sorghum vulgare, Arundo
donax), bio-diesel plants (Brassica napus, Raphanus sativus oleiformis, Sinapis alba)
and other plants (Amaranthus spp., Linum usitatissimum, Trifolium pratense, Trifolium
repens, Medicago sativa, Zea mays, B. rapa) [37]. Chelating agents EDTA and [S,S]-
EDDS were applied when some plants were in the juvenile vegetative and some in the
adult vegetative growth phase. The most effective was treatment with 10 mmol kg–1
[S,S]-EDDS and C. sativa, in which the Pb phytoextraction potential reached 26.3
kg ha–1. We used literature data for plant biomass yields in our calculation and
potentials were thus probably overestimated. Older, full-grown plants are likely to
concentrate less heavy metals than younger ones. With the obtained phytoextraction
potential, the percentage of Pb phytoextracted in a single cycle was only approx. 0.6%
of the total Pb present in the upper 30 cm of soil. The achieved Pb concentration in C.
sativa was clearly far from concentrations required for efficient soil remediation within a
reasonable time span. Pb concentrations 10-times higher than actually obtained
(exceeding 10,000 mg kg–1 dry biomass) would be required to reduce soil Pb
concentrations from an initial 1100 to 300 mg kg–1 Pb (the regulatory limit set by
European Union Council, directive 86/278/EEC) in approx. 10-15 years.
There have been very few field demonstrations of chelating agent enhanced
phytoextraction. Blaylock [40] showed a significant decrease in soil Pb concentration
over two years at two sites in the United States using B. juncea and EDTA. However,
the possible leaching of the Pb complex through the soil profile was not determined and
Enhanced Heavy Metal Phytoextraction 123

it therefore remains uncertain how much Pb the plants really removed. It is well known
that EDTA and other chelating agents act as chemical ploughs, redistributing surface
contamination from the surface to lower soil horizons, which gives rise to concerns
about the environmental safety of enhanced phytoextraction.

5. Concerns relating to the use of chelating agents for enhanced phytoextraction

The ideal chelating agent for enhanced phytoextraction should be specific for targeting
metal contaminants, water soluble in its free form to allow easy soil application, and be
able to form more lipophilic metal complexes, easily absorbed by plants, and thus
decrease the risk of leaching. It should also be non-toxic and inexpensive. EDTA, the
most widely tested chelating agent, is not ideal for a number of reasons. EDTA forms
highly soluble complexes with Pb and other polluting metals, which can therefore be
leached from the soil to the groundwater. EDTA is toxic, especially in its free form [41,
42], and poorly photo-, chemo- and biodegradable in soil [43]. A combined widespread
use in fertilizers and slow decomposition has already led to background concentrations
of EDTA in European surface waters in the range 10-50 mg L–1 [44].
The increase of soluble metal complexes in pore water following the application of
chelating agents raises health, safety and environmental concerns. In a soil column
study, we examined the effect of EDTA addition and different watering regimes on the
uptake of Pb, Zn and Cd by B. rapa, the leaching of polluting metals and the toxicity
effect of EDTA additions on plants and soil microfauna. The most effective was a single
dose of 10 mmol EDTA kg–1 soil, whereby 0.06% of soil Pb, 0.03% of Zn and 0.13% of
Cd were extracted into shoots, whereas up to 38% of initial total Pb in the soil, 10.5% of
Zn and 56% of Cd were leached down the soil profile. EDTA addition had a strong
phytotoxic effect on red clover (Trifolium pratense) and inhibited the development of
arbuscular mycorrhiza on plants. The results of phospholipid fatty acid analyses
indicated a toxic effect of EDTA addition on soil fungi and increased stress of soil
microfauna in general, indicated by increased fatty acids trans/cis ratio [34]. Soil
microorganisms depend directly on the soil solution for uptake of food, and elevated
heavy metal concentrations might be responsible for the toxic effects. Soil
microorganisms are largely responsible for important soil processes, such as
mineralization and synthesis of soil organic matter, soil micro-aggregate formation,
nitrification and denitrification etc., and disturbed microbial activity could thus effect
soil functioning. Soil microorganisms are also at the base of the soil food chain.
Römkens et al. [45] reported that the number of microbivorous nematodes was greatly
reduced during EGTA-enhanced phytoextraction, presumably due to a smaller
availability of food. Another hazard is the potential contamination of the food chain if
animals graze on the metal contaminated phytoextracting vegetation.
Several other studies investigating phytoextraction enhanced by chelating agents
have highlighted the risk of possible mobilization of Pb and other toxic metals from soil
to groundwater [9, 46]. Wenzel et al. [47] used canola (B. napus) in a pot and out-door
lysimeter experiment and also reported that leaching losses of Cu, Pb and Zn far
exceeded the amounts of metal taken up by plants after EDTA was applied. These
results indicate that the application of EDTA and other biologically persistent chelating
124 Domen Leštan

agents may be limited to field conditions in which soil containment and hydrological
control of the metal–enriched leachates can be safely achieved, for example to sites
where the connection to receiving water has been “broken”. In soils that are not
hydrologically isolated, control over metal leaching could be possible by maintaining a
neutral or negative soil water balance. If a chelating agent is applied and soil irrigation
adjusted to natural precipitation in such a way that losses of soil water due to plant
transpiration and evaporation are higher or equal to soil water gains, then leaching of
metal complexes should in principle not occur. In practice, however, the control of
natural factors affecting the soil water balance (precipitation, evapotranspiration) would
be very difficult to achieve. To maximize Pb accumulation by plants and reduce the
environmental risk of leaching, Epstein et al. [48] proposed that the chelating agent
application rate should be selected that maximizes the concentration of complexed Pb,
based on the extractability of Pb by the chelating agent. For example; Shen et al. [38]
reported that application of EDTA in three separate doses was not just more effective in
enhancing the accumulation of Pb in B. rapa, but also reduced mobility and the potential
risk of soluble Pb movement into the groundwater.
To extend the time available for plants to accumulate heavy metals and in this way to
reduce the leaching of heavy metals from the soil, we tested controlled release as an
alternative method of chelating agent application [49]. Controlled release pesticide
formulation was first proposed in agriculture to reduce pesticide leaching while
maintaining control of pests in soil. We entrapped EDTA in different hydrogel carriers
and used it in a column experiment with B. rapa. EDTA in acrylamide, starch, and
carrageenan hydrogel granules increased Pb accumulation in a test plant by up to 9.4-
times compared to the control. However, the addition of the corresponding amount of
EDTA in water solution (5 mmol kg–1 in four separate doses) was more effective and
increased the Pb plant concentration by 26.7-times. Controlled release of EDTA was not
particularly successful in reducing the Pb leaching, either; 5.4 to 23.6% of initial total Pb
was leached through the soil profile. EDTA applied in water solution leached 49.6 % of
the total initial Pb.

6. Use of the biodegradable chelating agents for enhanced phytoextraction

The use of biodegradable instead of biologically persistent chelating agents for enhanced
phytoextraction could curb off-site migration of their heavy metal complexes. For
example, NTA is biodegradable in both aerobic (where it degrades as fast as glucose and
citric acid) and anaerobic soil conditions [50]. Kulli et al. [46] used NTA for enhanced
phytoextraction of soil contaminated with Cd (2 mg kg–1), Cu (530 mg kg–1) and Zn (700
mg kg–1), with lettuce and Italian ryegrass as test plants. At the highest NTA dose (5.3
mol m–2 soil), the metal concentration in the aboveground plant biomass was 4 to 24
times greater than in the control plants. At this NTA dose, plant growth was almost
completely inhibited. Severe visual symptoms indicated metal toxicity as the likely
cause. Citric acid used for enhanced U phytoextraction could also represent a useful
alternative to more persistent chelating agents. It induces very rapid accumulation of U
in plants. Huang et al. [28] reported that shoot U concentrations of B. juncea and
Brassica chinensis grown in a U-contaminated soil (750 mg kg–1) increased from less
Enhanced Heavy Metal Phytoextraction 125

than 5 mg kg–1 to more than 5000 mg kg–1 in citric acid-treated soils. Citric acid
complex with U is degraded in soil within a few days. The resulting readsorption of
residual U, not extracted by the plants, in the soil reduces the environmental hazard
related to potential leaching of U to groundwater.
The [S,S]-isomer of EDDS is a particularly interesting chelating agent because it
combines high biodegradability with high chelating strength. [S,S]-EDDS was first
isolated as a metabolite of the soil actinomycete Amycolatopsis orientalis [51] and is
naturally present in the soil. In addition, the environmental risk of its use in detergent
application has already been assessed [52]. The toxicity to fish and daphnia was low
(EC50 > 1000 mg/L). The [S,S]-EDDS is readily biodegradable using the criteria
stipulated by the Organization for Economic Co-operation and Development (OECD).
The OECD criteria state that 60% of the compound must biodegrade within 28 days. For
[S,S]-EDDS, the final CO2 yield exceeded 80% after 20 days, assessed by the modified
Sturm test [53, 54]. [S,S]-EDDS biodegradation in a concentration of 0.0034 mmol kg–1
occurred rapidly in various environmental compartments. In unacclimated sludge
amended soil, the half-life of [S,S]-EDDS was ca. 2.5 days and mineralization was
completed in 28 days. [S,S]-EDDS was transformed into benign degradation products,
first into N-(2-aminoethyl) aspartic acid, which significantly decreased the chelating
capacity [54, 55].
To test the feasibility of using [S,S]-EDDS in enhanced phytoextraction, we
compared its efficiency against a benchmark chelating agent EDTA [56]. Applied in a
single 10 mmol kg–1 dose, both EDTA and [S,S]-EDDS were almost equally effective in
increasing the concentrations of Pb (94.2 and 102.3-fold) and, to a lesser extent, also of
Zn and Cd in the leaves of the test plant B. rapa. In separate doses, EDTA was more
effective than [S,S]-EDDS, but caused leaching of approx. 22% of Pb, while [S,S]-
EDDS leached only 0.8% of initial total Pb concentrations. A biotest with red clover
(Trifolium pratense) indicated a greater phytotoxic effect of EDTA than [S,S]-EDDS
addition. EDDS was also less toxic to soil fungi, as determined by PLFA analysis, and
caused less stress to soil microorganisms, as indicated by the trans/cis PLFA ratio. [S,S]-
EDDS and EDTA (5 mmol kg–1, single dose) equally effectively promoted Zn and Cd
uptake by oilseed rape (B. napus), amaranth (Amaranthus spp.), Chinese cabbage (B.
rapa) and hemp (C. sativa) [37]. Generally, [S,S]-EDDS was also less efficient for Pb
plant uptake, except for C. sativa, where the Pb biomass concentration was higher than
in the EDTA treatment by 42%. The stability constant for complexes with Pb is
substantially higher for EDTA than for [S,S]-EDDS (Table 2). These data therefore
illustrate that data on logK does not provide sufficient information on the potency of
specific chelating agents for enhanced phytoextraction. The efficiency of chelating
agents for phytoextraction seems to be plant-specific, as well as being controlled by the
stability constant and soil conditions, as already described above.
To retain the chelating agent solution in the topsoil and thus (i) improve chelating
agent availability for Pb mobilisation and plant uptake, and (ii) reduce leaching of Pb-
chelate complexes by prolonging the retention time available for [S,S]-EDDS complexes
biodegradation in soil, we modified the soil water holding capacity by using synthetic
acrylamide hydrogel [57]. Gel-forming soil conditioners are known from agronomy and
forestry practice to be effective in increasing the soil water holding capacity, decreasing
126 Domen Leštan

deep percolation, and minimising losses of water solution through leaching. The addition
of 0.2% (w/w) of hydrogel amendment increased soil field water capacity from an initial
24.6% to 31.3%. The use of 5 mmol kg–1 [S,S]-EDDS in hydrogel amended soil
increased Pb uptake by B. rapa by 18 times while only 0.2% of total initial Pb was
leached through the soil profile. In the control, soil without hydrogel [S,S]-EDDS
leached 1.2% of Pb. EDTA was more effective for phytoextraction but caused much
higher Pb leaching in all treatments, under any of the soil water sorption condition
tested. Using a higher 10 mmol kg–1 [S,S]-EDDS dose in hydrogel amended soil
significantly reduced plant Pb uptake and increased Pb leaching to up to 44.2% of initial
soil content. This was presumably caused by the toxic effect of high concentrations of
[S,S]-EDDS on soil microorganisms (slower biodegradation) and plants (lower Pb
uptake). In all treatments in which 10 mmol kg–1 EDTA or [S,S]-EDDS was applied,
visual symptoms (necrotic lesions on the leaves of B. rapa) of toxicity were observed.
The effect was much less pronounced in treatments with lower amounts of chelating
agents.
A serious limitation of [S,S]-EDDS based enhanced phytoextraction is the high price
of the chelating agent. The current price for 1 ton of [S,S]-EDDS is approx. 5000 GBP.
As [S,S]-EDDS has been substituted for traditional chelating agents in a number of
commercial products, e.g. industrial detergents, the price is expected to decrease. In
future, the biosynthesis of EDDS by A. orientalis, which produces a chelating agent
exclusively in the biodegradable S,S-configuration, instead of the current chemical
synthesis, could significantly reduce the production costs [58].

7. Horizontal permeable reactive barriers in enhanced phytoextraction

To further reduce the hazard of heavy metal leaching and off-site migration from treated
soil to the environment, we proposed the use of biodegradable chelating agents together
with horizontal permeable reactive barriers placed below the contaminated topsoil [59].
A flowsheet of the method is shown in Figure 4. The construction and reactive materials
in the barriers are a layer of nutrient enriched substrate with a high water sorption
capacity and with extensive surfaces on which microbial films could form, and a layer of
absorbent (apatite) for precipitation of metals. The main purpose of the substrate is to
enhance the microbial degradation of the metal complexes. Once metal ions are released
from the complex they are bound by absorbent, become immobilized and are no longer
subject to leaching. After completion of the soil remediation process, the barrier can be
excavated and removed.
We borrowed the concept of horizontal permeable reactive barriers from polluted
groundwater remediation. Here, a vertical permeable reactive barrier is constructed
below ground as a vertical underground wall, filled with reactive materials. The barrier
is built by digging a long, narrow trench in the path of the polluted groundwater. Clean
groundwater flows out of the other side of the wall. Reactive materials in the barrier trap
harmful chemicals or change the chemicals into harmless ones. For example, zero-valent
iron can be used for the reduction of toxic Cr6+ to harmless Cr3+, and limestone for Pb
precipitation.
Enhanced Heavy Metal Phytoextraction 127

[S,S]-EDDS
solution
Soil: heavy metal mobilisation,
phytoextraction, leaching

Reactive barrier: heavy

metal accumulation
“Clean” solution
Groundwater

Figure 4. Conceptual representation of combined enhanced phytoextraction of heavy

metals with biodegradable chelating agent and in situ soil washing using horizontal
permeable reactive barriers.

In our first study, barriers were placed 20 cm deep in soil columns and tested for their
ability to prevent leaching of Pb during enhanced phytoextraction using [S,S]-EDDS,
EDTA and B. rapa [59]. The reactive materials in the barriers were nutrient enriched
vermiculite, peat or agricultural hydrogel, and apatite as absorbent. EDTA and [S,S]-
EDDS addition (10 mmol kg–1) increased Pb concentrations in the test plant by 158 and
89 times compared to the control. In EDTA treatments, approx. 25% of total initial soil
Pb was leached in a single cycle of chelating agent addition. This was expected, since
EDTA is nonbiodegradable, so none of the reactive materials in the barriers were
effective. The biodegradability of the chelating agent and metal complexes and binding
capacity of adsorptive materials in the barrier for released heavy metals are essential for
this type of barrier to function. In [S,S]-EDDS treatments, 20% of the initial Pb was
leached from columns with no barrier, while barriers with vermiculite or hydrogel and
apatite decreased leaching by more than 60 times, to 0.35%. In total 11.6% of initial Pb
was removed from the soil above the barrier with vermiculite and apatite. However,
almost all removed Pb was simply washed from the soil and accumulated in the barrier,
only a fraction (0.03%) of Pb was phytoextracted. These results pointed toward the
possibility of enhanced phytoextraction combined with in situ soil washing. With current
plants, however, phytoextraction can be expected to make only a minor contribution.
Most soils are multi-contaminated, so we tested the feasibility of using reactive
barriers for remediation of soil contaminated simultaneously with Pb, Zn and Cd. Hemp
(C. sativa) was used as the phytoextracting plant [60]. The addition of [S,S]-EDDS (10
mmol kg–1 dry soil) yielded concentrations of 1026 ± 442, 330 ± 114 and 3.8 ± 1.5 mg
kg–1 of Pb, Zn and Cd in the dry above-ground plant biomass, respectively. These
concentrations were 1926-, 7.5-, and 11- times higher, respectively, compared to
treatments with no chelating agent addition. Horizontal permeable barriers, composed of
128 Domen Leštan

a 3 cm high layer of nutrient enriched sawdust and vermiculite and a 3 cm layer of soil
mixed with vermiculite and apatite, were positioned at different soil depths. The barrier
placed 30 cm deep reduced leaching of Pb, Zn and Cd by 435-, 4- and 53-times,
respectively, compared to columns with no barrier. The lower positioned barrier did not
prevent leaching of Zn. In total, 2.5% of initial Pb, 7.3% of Zn and 2.8% of Cd was
removed from the contaminated soil in a single [S,S]-EDDS treatment and mostly
accumulated in the barrier. The contribution of in situ soil washing prevailed over
enhanced phytoextraction, despite a marked increase in Pb plant uptake induced by
[S,S]-EDDS. The relative inefficiency of the barrier to prevent leaching of Zn indicates
either a lower biodegradability of the Zn-[S,S]-EDDS complex or poor adsorption of
released Zn ions. Indeed, Van Devivere et al. (2001) reported that the microbial
degradation of Pb-[S,S]-EDDS was much faster than that of Zn-[S,S]-EDDS, and while
apatite is an effective absorption material for Pb by conversion into pyromorphite, a
poorly soluble Pb phosphate mineral [61], the sorption mechanisms of apatite for metals
other than Pb are less clear.
In the follow-up study, the treatment of a vineyard soil with 5 mmol kg–1 [S,S]-
EDDS increased the accumulation of Cu in B. rapa by 3.3-times over the control. The
reactive barriers composed of nutrient enriched sawdust and apatite were effective and
only 0.53±0.32% of the initial Cu was leached, while 36.7% of Cu was washed from the
layer of contaminated soil and accumulated in the barriers [29]. Again, these results
indicate that with current plants, the use of reactive barriers could be more justified for
controlled and environmentally safe in situ soil washing of metals rather than for
enhanced phytoextraction.

8. Conclusions

The use of hyperaccumulating plants and continuous phytoextraction is limited to

situations in which all of the pollutants present are bioavailable and can be tolerated by
plants. At present, it is difficult to judge whether chelating agent assisted phytoextraction
can be economically feasible and environmentally sustainable. On the other hand, it is
the only phytoextraction option for soils contaminated with highly insoluble metals such
as Pb and U. The use of both phytoextraction methods is limited by the climatic and
geologic conditions of the site to be cleaned.
The risk of metal leaching is one of the most important limitations of enhanced
phytoextraction. We showed that it could be efficiently prevented by the use of a
biodegradable chelating agent and reactive barriers. Further research might provide a
new environmentally safe mobilizing agent, more effective in enhancing metal
accumulation in plants than currently known agents. New methods of chelating agent
application, such as alternative formulations allowing the controlled release, could be
developed. Another possible option is the use of plants genetically modified to exude
strong, metal-selective mobilizing compounds in their rhizosphere. Indeed,
environmentally safe methods of enhanced heavy metal phytoextraction must be fully
developed and tested before steps towards further commercialization of this remediation
technology are attempted.
Enhanced Heavy Metal Phytoextraction 129

Since it is the bioavailable fraction and not the total concentration of metals in soil
that interact with biological targets and pose an environmental and health threat,
phytoextraction research should focus on stripping the bioavailable metal fractions,
instead of trying to reduce the concentration of metals in soil below limits set by
legislation. Nevertheless, to allow remediation within a reasonable period, the plant
heavy metal uptake must be enhanced dramatically. This may be achieved by
engineering high-biomass producing plants with as yet unidentified hyper-accumulating
genes. One possible strategy to increase heavy metal plant uptake is to increase
translocation of chelate complexes through the plant transpiration stream. This could be
achieved using high-transpiration plants. For example, Gleba et al. [62] reported that a
high-transpiration species of B. juncea phytoextracted 104 % more Pb than the wild-type
plant.
Plants exhibiting a high capacity for heavy metal uptake when assisted by chelating
agents, i.e. B. juncea, are also generally not very resistant to high levels of Pb and other
heavy metals in their foliage. This requires harvesting soon after chelating agent
application. There are some indications that the ability to hyperaccumulate heavy metals
is the result of high resistance to the metals rather than greater rates of metal uptake [63].
Vacuolar sequestration is likely to be a key component of metal tolerance and
hyperaccumulation. To increase resistance to metal by genetic manipulation is therefore
another potential approach to improve the efficiency of phytoextraction. Freeman et al.
[64] reported that glutathione biosynthesis might play a role in Ni tolerance in
hyperaccumulator Thlaspi goesingense. Meagher [65] reported that bacterial genes
encoding Hg reductase and organomercurial lyase were successfully transferred into the
genome of several plants, including poplar, and that transformed plants showed
increased tolerance to Hg.
Although fast progress is being made and studies have demonstrated the power of
genetic engineering, more knowledge of the molecular mechanisms responsible for plant
metal accumulation, root to shoot transfer and vacuolar sequestration, is necessary
before the genetic traits can be transferred into high biomass plants. Practical aspects of
enhanced heavy metal phytoextraction also need further research. These might include
optimisation of agronomic practices (fertilization, crop protection, harvesting, irrigation)
and development of economically feasible techniques for the disposal of metal-enriched
plants or, when practical, for metal recovery. Finally, apart from deeper basic research,
more pilot-scale outdoor studies and field trials are needed objectively to evaluate the
real potential and feasibility of enhanced heavy metal phytoextraction.

References
[1] Alkorta, I; Hernandez-Allica, J; Becerril, JM; Amezaga, I; Albizu, I; Onaindia, M and Garbisu, C (2004)
Chelate-enhanced phytoremediation of soils polluted with heavy metals. Rev Environ Sci Biotech 3: 55-
70
[2] Salt, DE; Smith, RD and Raskin, I (1998) Phytoremediation. Annu Rev Plant Physiol Plant Mol Biol 49:
643-668
[3] Watanabe, ME (1997) Phytoremediation on the brink of commercialization. Environ Sci Technol 31:
182-186
[4] Cunningham, SC; Berti, WR and Huang, JW (1995) Phytoremediation of contaminated soils. TIBtech
13: 393-397
130 Domen Leštan

[5] Puschenreiter, M; Stoger, G; Lombi, E; Horak, O and Wenzel, WW (2001) Phytoextraction of heavy
metal contaminated soils with Thlaspi goesingense and Amaranthus hybridus: Rhizosphere
manuipulation using EDTA and ammonium sulfate. J Plant Nut Soil Sci 164: 615-621
[6] Finžgar, N; Kos, B and Leštan, D (2005) Heap leaching of lead contaminated soil using biodegradable
chelator [S,S]-ethylenediamine disuccinate. Environ Technol 26: 553-560
[7] Huang, JW and Cunningham, SD (1996) Lead phytoextraction: species variation in lead uptake and
translocation. New Phytol 134: 75-84
[8] Blaylock, MJ; Salt, DE; Dushenkov, S; Zakharova, O; Gussman, C; Kapulnik, Y; Ensley, BD and
Raskin, I (1997) Enhanced accumulation of Pb in Indian mustard by soil-applied chelating agents.
Environ Sci Technol 31: 860-865
[9] Cooper, EM; Sims, JT; Cunningham, SD; Huang, JW and Berti, WR (1999) Chelate-assisted
phytoextraction of lead from contaminated soils. J Environ Qual 28: 1709-1719
[10] Pichtel, J; Kuroiwa, K and Sawyer, HT (2000) Distribution of Pb, Cd and Ba in soils and plants of two
contaminated sites. Environ Pollut 110: 171-178
[11] Rieuwerts, JS; Thornton, I; Farago, ME and Ashmore, MR (1998) Factors influencing metal
bioavailability in soils: preliminary investigations for the development of a critical loads approach for
metals. Chem Spec Bioavail 10: 61-75
[12] Levy, DB; Barbarick, KA; Siemer, EG and Sommers LE (1992) Distribution and partitioning of trace
metals in contaminated soil near Leadville, Colorado. J Environ Qual 21: 185-195
[13] Sauve, S; McBride, MB and Hedershot, WH (1997) Speciation of lead in contaminated soils. Environ
Pollut 98: 149-155
[14] Janssen, RPT; Peijnenburg, WJGM; Posthuma, L and Van Den Hoop, MAGT (1997) Equilibrium
partitioning of heavy metals in Dutch field soils. 1. Relationship between metal partitioning coefficient
and soil characteristics. Environ Toxicol Chem 16: 2470-2478
[15] Ramos, L; Hernandez, LM and Gonzales, MJ (1994) Sequential fractionation of copper, lead, cadmium
and zinc in soil from or near Donana national park. J Environ Qual 23; 50-57
[16] Chlopecka, A; Bacon, JR; Wilson, MJ and Kay, J (1996) Heavy metals in the environment. J Environ
Qual 25: 69-79
[17] Leštan, D and Grþman, H (2001) Speciation of lead, zinc and cadmium in contaminated soils from
Mežica Valley. Zb Bioteh Fak 77: 205-214
[18] Leštan, D; Grþman, H; Zupan, M and Baþac, N (2003) Relationsip of soil properties to fractionation of
Pb and Zn in soil and their uptake into Plantago lanceolata. Soil Sediment Contam 12: 507-522
[19] Kabata-Pendias, A (2000) Trace Elements in Soils and Plants. CRC Press: Boca Raton. 432
[20] Li, X and Thornton, I (2001) Chemical partitioning of trace and major elements in soils contaminated by
mining and smelting activities. Appl Geochem 16: 1693-1706
[21] Maiz, I; Arambarri, I; Garcia, R and Millan, E (2000) Evaluation of heavy metal availability in polluted
soils by two sequential extraction procedures using factor analysis. Environ Pollut 110: 3-9
[22] Tessier, A; Campbell, PGC and Bisson, M (1979) Sequential extraction procedure for the speciation of
particulate trace metals. Anal Chem 51: 844-851
[23] Alloway, BJ (1995) Cadmium, in Heavy Metals in Soils, BJ Alloway, Ed., Blackie Academic and
Professional: Glasgow. 368
[24] Martell, AE and Smith, RM (2003) NIST critically selected stability constants of metal complexes;
Version 7.0. NIST: Gaithersburg.
[25] Sun, B; Zhao, FJ; Lombi, E and McGrath, SP (2001) Leaching of heavy metals from contaminated soils
using EDTA. Environ Pollut 113: 111-120
[26] Theodoratos, P; Papassiopi, N; Georgoudis, T and Kontopoulos, A (2000) Selective removal of lead
from calcareous polluted soils using the Ca-EDTA salt. Water Air Soil Poll 122: 351-368
[27] Nowack, B (2002) Environmental chemistry of aminopolycarboxylate chelating agents. Environ Sci
Technol 36: 4009-4016
[28] Huang, JW; Blaylock, MJ; Kapulnik, Y and Ensley, BD (1998) Phytoremediation of uranium-
contaminated soils: role of organic acids in triggering uranium hyperaccumulation in plants. Environ Sci
Technol 32: 2004-2008
[29] Kos, B and Leštan, D (2003) Chelator induced phytoextraction and in situ soil washing of Cu. Environ
Pollut 132: 333-339
[30] McGrath, SP; Zhao, FJ and Lombi, E (2002) Phytoremediation of metals, metalloids and radionuclides.
Advances in Agronomy 75: 1-56
Enhanced Heavy Metal Phytoextraction 131

[31] Lasat, MM; Norvell, WA and Kochian, LV (1997) Potential of phytoextraction of 137Cs from a
contaminated soil. Plant Soil 195: 99-106
[32] Hammer, D; Kayser, A and Keller, C (2003) Phytoextraction of Cd and Zn with Salix viminalis in field
trials. Soil Use Manag 19: 187-192
[33] Vasssil, AD; Kapulnik, Y; Raskin, I and Salt, DE (1998) The role of EDTA in lead transport and
accumulation by Indian mustard. Plant Physiol 117: 447-453
[34] Grþman, H; Velikonja-Bolta, Š; Vodnik, D; Kos, B and Leštan, D (2001) EDTA enhanced heavy metal
phytoextraction: metal accumulation, leaching and toxicity. Plant Soil 235: 105-114
[35] Jarvis, MD and Leung, DWM (2001) Chelated lead transport in Chamaecytisus proliferus (L. f.) ling
ssp. proliferus var. palmensis (H. Chris): an ultrastructural study. Plant Sci 161: 433-441
[36] Huang, JW; Chen, J; Berti, WR and Cunningham, SD (1997) Phytoremediation of lead contaminated
soils: Role of synthetic chelates in lead phytoextraction. Environ Sci Technol 31: 800-805
[37] Kos, B; Grþman, H and Leštan, D (2003) Phytoextraction of lead zinc and cadmium from soil by
selected plants. Plant Soil Environ 49: 548-553
[38] Shen, Z-G; Li, X-D; Wang, C-C; Chen, H-M and Chua, H (2002) Lead phytoextraction from
contaminated soil with high-biomass plant species. J Environ Qual 31: 1893-1900
[39] Barocsi, A; Csintalan, Z; Kocsanyi, L; Duschenkov, S; Kuperberg, JM; Kucharski, R and Richter, PI
(2003) Optimizing phytoremediation of heavy-metal contaminated soil by exploring plants stress
adaptation. Int J Phytoremediation 5: 13-23
[40] Blaylock, MJ (2000) Field demonstration of phytoremediation of lead contaminated soils, in
Phytoremediation of Contaminated Soil and Water, N Terry and TN Banuelos, Eds., Lewis Publishers,
Boca Raton. 389
[41] Dirilgen, N (1998) Effects of pH and chelator EDTA on Cr toxicity and accumulation in Lemma minor.
Chemosphere 37: 771-783
[42] Sillanpaa, M and Oikari, A (1996) Assessing the impact of complexation by EDTA and DTPA on heavy
metal toxicity using Microtox bioassay. Chemosphere 32: 1485-1497
[43] Nörtemann, B (1999) Biodegradation of EDTA. Appl Microbiol Biotechnol 51: 751-759
[44] Kari, FG; Hilger, S and Canonica, S (1995) Determination of the reaction quantum yield for the
photochemical degradation of Fe(III)-EDTA: Implications for the environmental fate of EDTA in surface
waters. Environ Sci Technol 29: 1008-1017
[45] Römkens, P; Bouwman, L; Japenga, J and Draisma, C (2002) Potentials and drawbacks of chelate-
enhanced phytoremediation of soils. Environ Pollut 116: 109-121
[46] Kulli, B; Balmer, M; Krebs, R; Lothenbach, B; Geiger, G and Schulin, R (1999) The influence of
nitrilotriacetate on heavy metal uptake of lettuce and ryegrass. J Environ Qual 28: 1699-1705
[47] Wenzel, WW; Unterbrunner, R; Sommer, P and Sacco, P (2003) Chelate-assisted phytoextraction using
canola (Brassica napus L) in outdoors pot and lysismeter experiments. Plant Soil 249: 83-96
[48] Epstein, AL; Gussman, CD; Blaylock, MJ; Yermiyahu, U; Huang, JW; Kapulnik, Y and Orser, CS
(1999) EDTA and Pb-EDTA accumulation in Brassica juncea grown on Pb-amended soil. Plant Physiol
208: 87-94
[49] Grþman, H and Leštan, D (2003) Use of hydrogels in EDTA induced Pb phytoextraction. Fresen
Environ Bull 12, 1044-1049
[50] Tiedje, JM and Mason, BB (1974) Biodegradation of nitrilotriacetate (NTA) in soil. Soil Sci Soc Am
Proc 38: 278-283
[51] Nishikiori, T; Okuyama, A; Naganawa, T; Takita, T; Hamida, M; Takeuchi, T; Aoyagi, T and
Umezawa, H (1984) Production of actinomycetes (S,S)-N,N’-ethylenediamine-dissuccinic acid, an
inhibitor of phospholipase. C J Antibiot 37: 426-427
[52] Jaworska, JS; Schowanek, D and Feijtel, TCJ (1999) Environmental risk assessment for trisodium [S,S]-
ethylene diamine disuccinate, a biodegradable chelator used in detergent applications. Chemosphere 38:
3597-3625
[53] Jones, PW and Williams, DR (2001) Chemical speciation used to assess [S,S]-ethylendiamine-
disuccinic acid (EDDS) as a readily-biodegradable replacement for EDTA in radiochemical
decontamination formulations. Appl Radiat Isotopes 54: 587-593
[54] Schowanek, D; Feijtel, TCJ; Perkins, CM; Hartman, FA; Federlen, TW and Larson, RJ (1997)
Biodegradation of [S,S], [R,R] and mixed stereoisomers of ethylene diamine disuccinic acid (EDDS), a
transition metal chelator. Chemosphere 34: 2375-2391
132 Domen Leštan

[55] Bucheli-Witschel, M and Egli, T (2001) Environmental fate and microbial degradation of
aminopolycarboxylic acids. FEMS Microb Reviews 25: 69-106
[56] Grþman, H; Vodnik, D; Velikonja-Bolta, Š and Leštan D (2003) Ethylenediamine-disuccinate as a new
chelate for environmentally safe enhanced lead phytoextraction. J Environ Qual 32: 500-506
[57] Kos, B and Leštan, D (2003) Influence of a biodegradable ([S,S]-EEDS) and nondegradable (EDTA)
chelate and hydrogel modified soil water sorption capacity on Pb phytoextraction and leaching. Plant
Soil 253: 403-411
[58] Zwicker, N; Theobald, U; Zähner, H and Fiedler, HP (1997) Optimization of fermentation conditions for
the production of ethylene-diamine-disuccinic acid by Amycolatopsis arientalis. J Ind Microbiol
Biotechnol 19:280-285
[59] Kos, B and Leštan D (2003) Induced phytoextraction/soil washing of lead using biodegradable chelate
and permeable barriers. Environ Sci Technol 37: 624-629
[60] Kos, B and Leštan D (2004) Soil washing of Pb, Zn and Cd using biodegradable chelator and permeable
barriers and induced phytoextraction by Cannabis sativa. Plant Soil 263: 43-51
[61] Laperche, V; Traina, SJ; Gaddam, P and Logan TJ (1996) Chemical and mineralogical characterization
of Pb in a contaminated soil: reactions with synthetic apatite. Environ Sci Technol 30: 3321-3326
[62] Gleba, D; Borisjuk, NV; Borisjuk, LG; Kneer, R; Poulev, A; Skarzhinskaja, M; Dushenkov, S;
Logendra, S; Gleba, YY and Raskin, I (1999) Use of plant roots for phytoremediation and molecular
framing. Proc. Natl Acad Sci USA 96: 5973-5977
[63] Kramer, U; Smith, RD; Wenzel, WW; Raskin, I and Salt, DE (1997) The role of metal transport and
tolerance in nickel hyperaccumulation by Thlaspi goesingense Halacsy. Plant Physiol 115: 1641-1650
[64] Freeman, JL; Persans, MW; Nieman K; Albrecht, C; Peer, W; Pickering, IJ and Salt, DE (2004)
Increased glutathione biosynthesis plays a role in nickel tolerance in Thlaspi nickel hyperaccumulators.
Plant Cell 16: 2176-2191
[65] Meagher, RB (2000) Phytoremediation of toxic elemental and organic pollutants. Curr Opin Plant Biol
3: 153-162
ENZYMES TRANSFERRING BIOMOLECULES TO ORGANIC FOREIGN
COMPOUNDS: A ROLE FOR GLUCOSYLTRANSFERASE AND
GLUTATHIONE S-TRANSFERASE IN PHYTOREMEDIATION

PETER SCHRÖDER
Institute of Soil Ecology, Department Rhizosphere Biology, GSF National
Research Center for Environment and Health, D-85758 Neuherberg,
FRG. E-mail: [email protected]

1. Introduction

In the general enzyme list of the Nomenclature Committee of the International Union
of Biochemistry and Molecular Biology (NC-IUBMB, first published in 1961 and with
the last printed edition in 1992) EC 2 is reserved for the enzyme family of transfe-
rases. Generally, transferases are enzymes transferring a functional group, for example,
methyl- or glycosyl-groups, from one substrate (regarded as donor) to another sub-
strate (regarded as acceptor). Hence, the classification is based on the scheme
“donor:acceptor-group transferase”. The common names of the enzymes belonging to
this group are normally derived from acceptor group-transferase or donor group-
transferase. In many cases the donor is a cofactor (coenzyme) carrying the group to be
finally transferred.
Whereas most members and subclasses of EC2 are confined to the metabolism of
biogenic and natural compounds two subgroups, the glycosyltransferases of EC 2.4 and
the aryl/alkyl transferases of EC 2.5, have been recognized as having crucial functions in
the metabolism of foreign compounds, xenobiotics, in both animals and plants.
This role is very important, as all organisms are frequently exposed to an array of
potentially toxic substances. Organic chemicals are particularly threatening. They may
have natural sources e.g. fires, volcano eruptions or processes of biodegradation. They
may also be the products of microbial or animal metabolism, or from the secondary
metabolism of plants [1]. These organic substances may play a role in defence or in
allelopathic reactions. Furthermore, increasing industrialization has provided two novel
sources of foreign compounds: (1) through the invention and use of agrichemicals for
the protection of crops from pests and weeds, and (2) through the emission of organic
xenobiotics in chemical manufacturing processes or the use of synthetic chemicals. The
latter compounds of solely anthropogenic origin represent a threat to our environment as
these synthetic chemicals are emitted without any control. For plants, the situation is
especially difficult as they are rooted in the ground and are dependent on that site for
survival. Plants therefore, have to rely on effective detoxification mechanisms.

133
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 133 – 142.
© 2006 Springer.
134 Peter Schröder

The uptake of xenobiotics from polluted media, i.e. air, water or soil, follows the laws
of phase distribution and diffusion. Plants therefore, have only limited possibilities to
avoid accumulation of xenobiotics in their tissue and the associated detrimental conse-
quences. In recent years, some plant species have been recognized as potent accumu-
lators or detoxifiers of such compounds. These plants are capable of removing these
dangerous chemicals from the environment. Hence, they are to be utilized in the green
technology of phytoremediation, helping to solve some of our environmental problems
in aninexpensive, reliable and natural manner. However, information on the under-
lying biochemical principles involved in these processes is generally scarce.

2. EC 2.4 Glycosyltransferases

All enzymes transferring glycosyl groups to acceptor molecules belong to this class.
Some of these enzymes also catalyse hydrolysis, which can be regarded as transfer of a
glycosyl group from a donor molecule to water. Also, inorganic phosphate can act as an
acceptor in the case of phosphorylases; phosphorolysis of glycogen is regarded as
transfer of one sugar residue from glycogen to phosphate. However, the more usual
scenario is the transfer of a sugar from an oligosaccharide or a high-energy compound to
another carbohydrate molecule as acceptor. This subclass, EC2.4 is further subdivided,
according to the nature of the sugar residue being transferred, into hexosyltransferases
(EC 2.4.1), pentosyltransferases (EC 2.4.2) and those transferring other glycosyl groups
(EC 2.4.99). This mechanism is widespread in the plant kingdom, and the resulting
glycosides represent the largest group of natural substances in plants, contributing
factors to whether plants are colourful, tasty, or poisonous. The mechanism of sugar
transfer in plants was discovered early in plant biochemistry [2]. The earliest reports of
plant glucosides were associated with the metabolism of secondary compounds, such as
flavonoids, anthocyanins and phenylpropanoids. Intermediates as well as storage forms
of these compounds are frequently glucosylated. Glucosides possess lower reactivity
than aglyca [3], they have a high hydrophilicity [4], they are used in detoxification of
endogenous products and xenobiotics [5], and they may be compartmentalized in the
plant [3]. A physiological role for glucosides is seen in pathogen defence, allelopathy
and plant inherent signals [6, 7].
The transfer of glucose to a xenobiotic molecule requires the presence of an acceptor
group on the target. Such an acceptor functional group might be an –OH, -NH or –SH
function, and correspondingly, the plant glucosyltransferases are named O-glucosyl-, N-
glucosyl-, and S-glucosyltransferases.
Molecules that do not bear these functional groups may be conjugated with sugars
after chemical activation, i.e. hydroxylation by one of the plant P450 monooxygenases.
For a significant number of herbicides, activation by P450 prior to detoxification by O-
glycosyltransferases has been reported. Recently the formation of plant cyanoglucosides
via stepwise activation by P450 and final glucosylation (e.g. dhurrin or triglochin) has
attracted considerable interest [8, 9].
The second requirement for glucosyltransfer reactions is the availability of activated
sugars, such as UDP-glucose (uridine[5´]diphospho-[1]-α-D-glucose, Fig. 1A). This
metabolite is formed via phosphorylation of glucose in an ATP-driven reaction, to yield
Enzymes Transferring Biomolecules 135

glucose-6-phosphate, followed by conversion to glucose-1-phosphate. Glucose-1-

phosphate reacts with UTP (uridine triphosphate), yielding UDP-glucose plus
pyrophosphate. The UDP-glucose is the final activated intermediate that donates its
glucose residue to the xenobiotic in an energetically favourable reaction. It has to be
noted that the reaction of UDP-glucose on hydroxylated ring systems is the most
frequently described reaction of sugar transfer in plants. Usually ß-1-D bonds are formed
between the sugar moiety and the second substrate, but ß-5 and ß-3-conjugates have also
been reported [3].

Figure 1. A) UDP-glucose, B) glucosyl transfer to a target molecule; C) conjugation of the

herbicide, Bentazon, after activation by P450.

Numerous herbicides are conjugated to sugars via O-glucosyl-transfer or N-glucosyl-

transfer in tolerant plants. The non-identity of the responsible enzymes has been
demonstrated several times, although overlapping activities have been found in some
cases. Conjugation may occur either at OH-groups of the molecule to form O-glucosides
or at carboxy-groups to form acylglucosides. For N-glucosyltransfer, coupling to NH2-
groups of the molecule is crucial. From a practical point of view, predominantly
phenolic pollutants, as well as components of ammunition (TNT and metabolites) or
136 Peter Schröder

pesticide spills, might be candidates for detoxification via glucose transfer in plants. A
possible exploitation of these enzymatic mechanisms has recently been reviewed [10].
It has been shown that glucosyl-transferase activities are hardly inducible and might
thus represent a class of housekeeping enzymes. Attempts to increase their activity using
herbicide safeners are rare. Recently Brazier and co-workers [11] demonstrated the
increase of O-GT in black grass after dichlormid or cloquintocet mexyl treatment. In
each of the treatments increased activity was found for the conjugation of quercetin but
not for xenobiotic compounds. On the other hand, evidence has been found to suggest
that the individual enzymes responsible for these reactions might well be under develop-
mental control and that the conjugation of single xenobiotics can not be expected to
proceed throughout the plant’s life and in every plant part [12, 13]. Of course, this fact
has consequences for the practical use of plants in the detoxification of foreign
compounds, and it is especially important when considering plants for use in
phytoremediation, because it has to be ensured that the detoxification capacity meets
with the xenobiotic burden of the system.
Table 1: Examples for xenobiotic substrates of glucosyl transferases in plants (adapted
from [14])

O-glucosyl transfer N-glucosyl transfer

Direct conjugation after activation direct conjugation after activation
1,2,5-Trichlorophenol 2,4-D Chloramben Dinoben
2,4-dichloroanilin Chlorpropham Metribuzin Propanil
4-nitrophenol Cisanilide Pyridate
4-nitrophenol DDT / DDE
Chloramben Dicamba
Clopyralid Bentazon
Dimethenamid Diclofop
Fenoxaprop ethyl Diphenamid
Maleic hydrazide Methylphenylureas
MCPA Perfluridone
Pentachlorophenol Sulfonylureas
Picloram Terbacil
Quinclorac

It is important to note, that glucosyl conjugates may be cleaved by glucosidases and, in

the case of acylglucosides (-COOH substitution) by esterases. Both enzyme activities are
abundant in plant cells. However, these activities might be compartmentalized or under
developmental control. The action of these enzymes will yield the respective aglyca that
are spontaneously reprotonated under the conditions of the cytosol. Thus, the original
xenobiotic substrate may be regenerated. This reversibility represents a great dis-
advantage of glucosylation for its practical consideration in phytoremediation, because
previously detoxified compounds may regain their toxicity under certain conditions. In
oats, the formation of an acylglucoside from Diclofop in the presence of an esterase
explains this plant’s susceptibility to this herbicide. In wheat, an O-glucoside is formed
from Diclofop that is not readily cleaved [15].
This reaction chain of activating, conjugating and releasing a certain compound
makes sense in the course of natural compound formation. It has also been shown that
Enzymes Transferring Biomolecules 137

homeostasis of salicylic acid and indole acetic acid (auxin) is maintained in plant cells
by this mechanism. For practical application, it has to be ensured that the cleavage of the
glycosyl-conjugate happens only under conditions where the aglycon can be inserted
into the cell wall and covalently bound to lignin or other polymerous structures.

3. EC 2.8 Glutathione S-transferases

Various xenobiotics possess electrophilic centres, i.e. centres of low electron density that
can accept an electron pair to form a covalent bond. This feature makes them dangerous
because they can to react spontaneously with corresponding nucleophilic sites of
proteins and genetic material, i.e. DNA and RNA and thereby disturb metabolic
networks.
The action of such electrophilic xenobiotics appears to be dependent on particular
cellular enzymes called glutathione S-transferases (GSTs) [16, 14]. Electrophilic centres
necessary for GSH conjugation are found in arene-oxides, aliphatic and arylic halides, in
α−β-unsaturated carbonyls, organonitro-esters and organic thiocyanates. Industrial
substrates for GSTs are haloalkanes, chlorobenzenes, thiocarbamates, diphenylethers,
triazines, chloracetanilides [see 17, 18]. In animals the oxidants acrolein, propenals,
lipid hydroperoxides, chlorambucil and fosfomycin are additional substrates [19].
Such compounds will not be conjugated by glucosyl transferases. Instead, the
reactions are performed by a somewhat heterogeneous class of enzymes, GSTs, which
catalyze the transfer of aliphatic, aromatic, or heterocyclic radicals as well as epoxides
and arene oxides to glutathione. The transfer reaction takes place at the sulphur atom
and has been annotated as the enzyme class coding EC 2.5.1.18. GST enzymes occur
ubiquitously [20]. The binding of the foreign compound and the transfer of glutathione
follows two mechanisms catalyzed by glutathione S-transferases [21, 22]:
• (a) Nucleophilic displacement of an alkyl or aryl halogen or a nitro group
is the most frequently observed step. Conjugation of many pesticides like
atrazine, propachlor or pentachloronitrobenzene (PCNB) are examples of
this type of reaction. Halogens or nitrogroups of these molecules are soft
electrophiles and react readily with GSH. Further substitution reactions are
found in the detoxification of diphenylether herbicides (e.g. fluorodifen,
fenoxaprop-ethyl). Here, an ether bond is cleaved and substituted by the
thiolate. Moreover, the standard enzyme assays for GST activity use
1-chloro-2,4-dinitrobenzene (CDNB) or 1,2-dinitro-4-chlorobenzene
(DCNB) as substrates (see Fig. 2, B).
• (b) Nucleophilic addition (Michael reaction): Addition of the thiolate to
carbon-carbon-double bonds is a special type of reactions on compounds
with reactive carbon-carbon double bonds neighboured by an electron
withdrawing group [23]. Conjugation of tridiphane or cinnamic acid may
be examples for this type of reaction [24, 25]. The conjugation on these
bonds leads to a labile conjugate that may be sensitive to pH changes.
138 Peter Schröder

Figure 2. (A) reduced glutathione (γ-glutamyl-cysteinyl-glycin), (B) S-dinitrobenzyl-

glutathione; (C) four typical plant GST substrates: chlorodinitrobenzene,
dichloronitrobenzene, nitrobenzylchloride, and fluorodifen.

In contrast to the metabolism of glucosyl conjugates, and with the exception of the
cleavage of “Michael-reaction”-type conjugates formed at double bonds of molecules
(see above), the dissolution of glutathione conjugates does not lead to the liberation of
the original toxic or lipophilic foreign compound. This is due to the fact that
nucleophilic substitution or displacement removes the significant electrophilic centre
from the target molecule to introduce the glutathione thio-function. When cleaved, the
electrophilic moiety is lost, and the toxicity of any released parent residue is
significantly lowered.
The unravelling of the Arabidopsis genome has confirmed the observation that the
multiple reactions of GST enzyme activity are attributes to a large number of
isoenzymes encoded for by more than 50 GST genes. These genes, many of them
formed by gene reduplication in the simple Arabidopsis genome, can be clustered in four
distinct groups. According to the mammalian GST system, which names the GSTs with
Greek letters, a comprehensive nomenclature of plant GSTs has been proposed by
Edwards and coworkers [26].
To date, five distinct classes of GSTs, Lambda (and DHAR, dehydroascorbate
reductases), Phi, Tau, Theta and Zeta have been identified in plants. According to
Edwards & Dixon [27], class Tau, the predominant class, catalyzes the detoxification of
xenobiotics by nucleophilic substitution reactions. Class Phi and Tau GSTs, the next
most abundant, appear to catalyze reactions with endogenous toxic metabolites or are
involved in the metabolism of reactive oxygen species. The two remaining, classes
Lambda and Zeta, are unusual, Lambda GSTs occur as monomeric enzymes or enzymes
with transmembrane regions, functioning as redox mediators. Zeta GSTs are isomerases.
Enzymes Transferring Biomolecules 139

The multitude of GSTs in plants reflects their ability to conjugate to GSH a large
array of different substrates, many of them of anthropogeneous origin. Furthermore,
GSTs are inducible by different forms of stress, including xenobiotics [28]. This
property also confers herbicide tolerance or resistance in many plant species. Whereas
herbicides are designed to kill weeds and leave crops unaffected, organic pollutants
might have properties that stress plants in a similar manner. GST activities have been
shown to be crucial for the detoxification of a number of these compounds.

4. Physiological roles of conjugates

Summarizing the above results, we must conclude that glutathione and sugar conjugation
lead to the formation of detoxified and more or less stable products in the plant cytosol.
Upon chemical contact or under conditions of pesticide application, plants may
encounter relatively high concentrations of the respective foreign compounds, and,
provided the defence enzymes work properly, they will form significant amounts of
conjugates from them. Besides eventual chemical lability this accumulation of con-
jugates will lead to an unfavourable situation as conjugates might inhibit enzymes by
feedback mechanisms or may affect specific binding.
Glucosyl- and glutathione-conjugates do not accumulate in the cytosol of plant cell,
but are translocated into the vacuole through ABC-transporters [29, 30, 15]. The
presence of a conjugate is mandatory for this translocation. An interesting observation is
that glucosylconjugates build up in pools inside the plant, whereas glutathione
conjugates do not. They undergo rapid and complete metabolism [31]. As well as the
ABC-transporters on the tonoplast, ABC-transporters have recently been found in the
plasmalemma. They accept the same conjugates and allow for long range transport of
these metabolites. Some hints exist that a physiological role for conjugates might be the
signalling of pollutant stress, and induction of detoxification reactions has been
described after application of xenobiotics to plant cells [32]. In the context of
phytoremediation, such conjugates might also play a role in enhancing the plants
capacity for detoxification.

5. Conjugation reactions and options for phytoremediation

The use of plants in phytoremediation of organic pollutants has been reviewed

thoroughly in the frame of the COST action 837 [33, 34, 10, 14). Literature on the
degradation of herbicides in crops demonstrates clearly that xenobiotic conjugates are
usually further processed to more complex conjugates [21, 31], or cleaved to form
reactive molecules that are excreted from the living cell and reside in the cell wall or the
apoplast [35]. In our context of phytoremediation it is, of course, crucial to know which
type of primary conjugation occurred, because this determines the final fate of the
compound [36, 37].
Further breakdown steps include incorporation of metabolites into the cell wall in the
pectin, lignin, hemicellulose and cellulose fraction [35, 38, 39]. This has been
demonstrated with numerous cereals, soybean and the respective cell cultures. Few
140 Peter Schröder

metabolites have also been found in the rhizosphere, where they might disappear in
microbial and mycorrhizal metabolism, and single findings point to the volatilization of
metabolites after the action of methyl transferases [31]. One crucial question remaining
is how and why the conjugating enzymes detect and meet the foreign compounds
attacking the cells. Given the large number of isoforms of detoxification enzymes on the
one hand, and the diverse group of xenobiotics on the other, it is hard to imagine how
the different reactions occur in the cytosol in an orderly manner. Even if the respective
enzymes would occupy distinct positions in a metabolic network, the question would
remain how the detoxification is channelled in a cell or a tissue. Our present knowledge
is that the conjugating enzymes have low specificity, i.e. that they are able to accept a
larger number of endogenous and xenobiotic substrates. Only very specifically designed
poisons at higher concentrations will represent a severe threat.
Historically, our present state of knowledge on foreign compound metabolism is
focused on crops and a small number of ornamental plants. Only a few reports exist on
plants that might be interesting with respect to their potential in phytoremediation.
Conjugative metabolism in the outstanding candidates Arundo donax, Brassica juncea,
Phragmites sp.., Typha sp.., Plantago majus, Populus sp.., Salix sp., to name but a few,
has not been investigated in any depth. This situation is awkward as there are already
numerous existing field sites that seem to be very successful in the removal of
xenobiotics from soil and water. Knowing about the mechanisms involved, the efficiency
of these systems could probably be improved when methods to increase metabolism
rates would be applied. One option could be to utilize gene manipulation methods to
over express the desired enzymes. Another option could be to add inducers of herbicide
resistance to the plants. Finally it is possible that xenobiotic uptake and transport to
tissues with high degradative activity would be enhanced. Each of these attempts would
improve commercial and public acceptance of the use of plants to improve the
environment with biological methods. Plants have a large potential to cope with their
environment [40], and they will also be able to help us solve some of our pollution
problems in an environmentally friendly way.

References
[1] Naumann, K (1993) Chlorchemie der Natur. Chemie in unserer Zeit 27(1): 33-41
[2] Ciamician, GL and Ravenna, C (1916), without title, RC Acad Linei 18: 419
[3] Hösel, W (1981) Glycosylation and glycosidases, in The Biochemistry of Plants, pp. 725-753, Conn, EE,
Ed., New York, Academic Press
[4] Bartz, W; Köster, J; Weltring, KM and Strack, D (1985) Recent advances in the metabolism of phenolic
compounds in plants and animals, in The Biochemistry of Plant Phenolics. Ann Proc Phytochem Soc
Europe, 25, pp. 307-347, VanSumere, CF and Lea, PL, Eds., Oxford, Clarendon
[5] Gallant, ER and Balke, NE (1995) Xenobiotic glucosyltransferase activity from suspension cultured
Glycine max cells. Pestic Sci 43: 31-40
[6] Matile, P (1976) Biochemistry and function of vacuoles. Ann Rev Plant Phys 29: 193-213
[7] Balke, NE and Schulz, M (1987) Potential impact of enzyme glucosylation of allelopathic phenolic
compounds, in Invited Lectures, Section 4: Industrial Chemistry, 31st International Congress on Pure
and Applied Chemistry, Sophia, Bulgarian Academy of Sciences, pp. 17-23
Enzymes Transferring Biomolecules 141

[8] Bak, S; Olsen, CE; Halkier, BA and Moller, BL (2000) Transgenic tobacco and Arabidopsis plants
expressing the two multifunctional Sorghum cytochrome P450 enzymes, CYP79A1 and CYP71E1, are
cyanogenic and accumulate metabolites derived from intermediates in dhurrin biosynthesis. Plant
Physiol 123: 1437-1448
[9] Nielsen, JS and Moller, BL (1999) Biosynthesis of cyanogenic glucosides in Triglochina maritima and
the involvement of cytochrome P450 enzymes. Arch Biochem Biophys 368: 121-130
[10] Harvey, P; Campanella, B; Castro, PML; Harms, H; Lichtfouse, E; Schäffner, A; Smrzek, S and Werck-
Reichhart, D (2001) Phytoremediation of Polyaromatic Hydrocarbons, Anilines and Phenols. ESPR
Environmental Sciences and Pollution Research 9(1): 29-41
[11] Brazier, M; Cole, JD and Edwards, R (2002) O-Glucosyltransferase activities toward phenolic natural
products and xenobiotics in wheat and herbicide-resistant and herbicide-susceptible black-grass
(Alopecurus myosuroides). Phytochem 59: 149-156
[12] Haas, M (1997) Metabolisierung von Xenobiotika durch pflanzliche Zellkulturen und Enzyme.
Dissertation, University Weihenstephan
[13] Schröder, P (2001) The role of glutathione and glutathione S-transferases in the adaptations of plants to
xenobiotics, in Significance of Glutathione in Plant Adaptation to the Environment. Handbook Series of
Plant Ecophysiology, pp. 157-182, Grill, D, Tausz, M and DeKok, LJ, Eds., Boston, Dordrecht, London,
Kluwer Acad Publishers
[14] Schröder, P and Collins, CJ (2002) Conjugating enzymes involved in xenobiotic metabolism of organic
xenobiotics in plants, Int J Phytorem 4(4): 247-265
[15] Shimabukuro, RH; Walsh, WC and Hoerauf, RA (1979) Metabolism and selectivity of Diclofop-methyl
in wild oat and wheat. J Agric Food Chem 27: 615-623
[16] Coleman JOD; Randall RA and Blake-Kalff, MMA (1997) Detoxification of xenobiotics by plants:
chemical modification and vacuolar compartimentation. TIPS 2: 144-151
[17] Schröder, P (1997) Fate of glutathione S-conjugates in plants: Cleavage of the glutathione moiety, in
Hatzios, KK, Ed., Regulation of enzymatic systems detoxifying xenobiotics in plants. NATO ASI Series
Vol. 37, pp. 233-244, Kluwer, NL
[18] Sheehan, D; Meade, G; Foley, VM and Dowd, CA (2001) Structure, function and evolution of
glutathione transferases: implications for classification of non-mammalian members of an ancient
enzyme superfamily. Biochem J 360: 1-16
[19] Hayes, JD and Pulford, DJ (1995) The glutathione S-transferase supergene family: Regulation of GST
and the contribution of the isoenzymes to cancer chemoprotection and drug resistance. Crit Rev
Biochem Mol Biol 30: 445-600
[20] Pflugmacher, S; Sandermann, H and Schröder, P (2000) Taxonomic distribution of plant glutathione
S-transferases acting on xenobiotics. Phytochemistry 54: 267-273
[21] Lamoureux, GL and Rusness, DG (1989) The role of glutathione and glutathione S-transferases in
pesticide metabolism, selectivity and mode of action in plants and insects, in Dolphin, D, Poulson, R
and Avramovic, O, Eds., Glutathione: Chemical Biochemical and Medical Aspects, Vol IIIB, Ser:
Enzyme and Cofactors, pp. 153-196, New York, Wiley & Sons
[22] Lamoureux, GL; Rusness, DG and Schröder, P (1993) Metabolism of a diphenylether herbicide to a
volatile thioanisole and a polar sulphonic acid metabolite in spruce (Picea). Pestic Biochem Physiol 47:
8-20
[23] Talalay, P; de Long, MJ and Prochaska, HJ (1988) Identification of a common chemical signal
regulating the induction of enzymes that protect against chemical carcinogenesis. PNAS 85: 8261-8265
[24] Lamoureux, GL and Rusness, DG (1986) Xenobiotic conjugation in higher plants, in Paulson, GD,
Caldwell, J, Hutson, DH, and Menn, JJ, Eds., Xenobiotic Conjugation Chemistry, pp. 62-105,
Washington, Am Chem Soc
[25] Dean, JV; Devarenne, TP; Lee, IS and Orlofsky, LE (1995) Properties of a maize glutathione
S-transferase that conjugates coumaric acid and other phenylpropanoids. Plant Physiol 108: 985-994
[26] Dixon, DP; Cole, JD and Edwards, R (2001) Cloning and characterization of plant theta and zeta class
GSTs: implications for plant GST classification. Chem Biol Interact 133: 33-36
[27] Edwards, R and Dixon, DP (2004) Metabolism of natural and xenobiotic substrates by the plant
glutathione S-transferase superfamily, in Sandermann H, Ed., Molecular ecotoxicology of plants, Ecol
Studies Vol. 170, Springer, Berlin, Heidelberg, pp. 17-52
[28] Dean, JV; Gronwald, JW and Eberlein, CV (1991) Induction of glutathione S-transferase isozymes in
sorghum by herbicide antidotes. Plant Physiol 92: 467-473
142 Peter Schröder

[29] Marrs, KA (1996) The functions and regulation of glutathione S-transferases in plants. Annu. Rev. Plant
Physiol. 47: 127-158
[30] Wolf, A.E; Dietz, K.J and Schröder, P (1996) A carboxypeptidase degrades glutathione conjugates in the
vacuoles of higher plants. FEBS Lett 384: 31-34
[31] Lamoureux, GL and Rusness, DG (1993) Glutathione in the metabolism and detoxification of the xeno-
biotics in plants, in De Kok, LJ, Stulen, I, Rennenberg, H, Brunold, C and Rauser, W, Eds., Sulphur
Nutrition and Assimilation in Higher Plants, pp. 221-239. The Hague, SPB Acad. Press
[32] Diekmann, F; Nepovim, A and Schröder, P (2004) Influence of Serratia liquefaciens and a xenobiotic
glutathione conjugate on the detoxification enzymes in a hairy root culture of horse radish (Armoratia
rusticana). J Appl Bot 78: 64-67
[33] Chaudhry, Q; Schröder, P; Werck-Reichhart, D; Grajek, W and Marecik R (2001) Prospects and
Limitations of Phytoremediation for the Removal of Persistent Pesticides in the Environment. ESPR -
Environmental Sciences & Pollution Research 9(1): 4-17
[34] Coleman, JOD; Frova, C; Schröder, P and Tissut, M (2002) Exploiting plant metabolism for the
phytoremediation of persistent herbicides. ESPR, Environmental Sciences and Pollution Research 9(1):
18-28
[35] Schmidt, B; Ebing, W and Schupahn, I (1988) Einsatz eines Pflanzenzellkultur-Tests zur Ermittlung der
Metabolisierbarkeit von Pflanzenschutzmitteln. Gesunde Pflanzen 40: 245-249
[36] Frear, DS (1976) Pesticide conjugates – glycosides, in Kaufman, DD, Still, GG, Paulson, GD, and
Bandal, SK, Eds., Bound and conjugated pesticide residues, ACS Symposium 29, pp. 35-54.
Washington DC, American Chemical Society.
[37] Kreuz, K; Tommasini, R and Martinoia, E (1996) Old enzymes for a new job. Herbicide detoxification
in plants. Plant Physiol 111: 349-353
[38] Langebartels, C and Harms, H (1986) Plant cell suspension cultures as test systems for an ecotoxi-
cological evaluation of chemicals. Angew Bot 60: 113-123
[39] Sandermann, H; Haas, M; Meßner, B; Pflugmacher, S; Schröder, P and Wetzel, A (1997) The role of
glucosyl and malonyl conjugation in herbicide selectivity, in Hatzios, KK, Ed., Regulation of Enzymatic
Systems Detoxifying Xenobiotics in Plants. NATO ASI Series Vol. 37 pp. 211-232 Kluwer, Dordrecht
[40] Schnoor, JL; Licht, LA; McCutcheon, SC; Wolfe, NL and Carreira, LH (1995) Phytoremediation of
organic and nutrient contaminants, Env Sci Tech 29: 318-323
PHYTOREMEDIATION OF POLYCHLORINATED BIPHENYLS

MARTINA MACKOVA1,2, DIANE BARRIAULT3, KATERINA

FRANCOVA1, MICHEL SYLVESTRE3, MONIKA MÖDER4,
BLANKA VRCHOTOVA1,2, PETRA LOVECKA1, JITKA
NAJMANOVA1, KATERINA DEMNEROVA1, MARTINA
NOVAKOVA1,2, JAN REZEK1,2 AND TOMAS MACEK2
1
Dept. Biochemistry and Microbiology., Faculty of Food and
Biochemical Technology, ICT Prague, Technicka 3, Prague, 166 28
Czech Republic, 2 Dept. of Natural Products, Institute of Organic
Chemistry and Biochemistry, Academy of Science of the Czech
Republic, Flemingovo n. 2, 166 10 Prague, Czech Republic, E-mail:
[email protected] 3Institut National de la Recherche
Scientifique, INRS-IAF, 245 Boul. Hymus, Pointe-Claire, Québec,
Canada, H9R 1G6, 4 Laboratory of Analytical Chemistry, UFZ
Leipzig-Halle, Permoserstrasse 17, Leipzig, Germany, 4

1. Introduction

Many hydrophobic organic compounds including polychlorinated biphenyls are

priority soil contaminants because of their toxicity and tendency to persist in soils,
sediments and to escape biological degradation. The fate of contaminants in the
environment is controlled by a combination of interacting processes. They can be
classified as physical, chemical or biological. Physical processes are responsible for
the movement of contaminants through the soil and subsurface away from their
source. Chemical and biological processes determine the extent to which compounds
will be transformed. In some cases the most important transformations occur
abiotically, while in other cases they are mediated by microorganisms with the help of
other living organisms. Knowing the physicochemical properties of environmental
contaminants is a prerequisite for predicting the fate of organic contaminants or new
products in the environment, predicting the efficiency of treatment systems, and
assessing ecotoxicological and human health risks. Unfortunately such data are
available only for a relatively small number of compounds. At this time, several
reviews described the pathway used by bacteria and fungi to transform PCBs in the
environment as well as the various alternate pathways created by engineered
organisms. However, the contribution of plants to the ecodeposition of PCBs has not

143
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 143 – 167.
© 2006 Springer.
144 Martina Mackova et al.

yet been well investigated. In this review, we will place emphasis on studies
regarding the pathways used by plants to transform PCB in the environment.

2. Chemistry and use of PCBs

PCBs were manufactured from 1929 until their use begins to be restricted in the
1970s and their production banned in most countries in 1979. Because of their
excellent chemical and thermal stabilities they were used on a large scale as dielectric
fluids in transformers and capacitors, hydraulic fluids and lubricants, organic diluents,
paints, carbon-less copy paper, etc but also in small household electrical appliance
and domestic products. The extensive application of the PCBs resulted in their wide
distribution in the environment. PCBs are actually a large family of 209 molecules
that all share the biphenyl backbone but differ by the number and the position of the
chlorine atoms on the biphenyl ring. We call isomer the compounds having the same
number of chlorine atoms and congeners those, which bear different number of
chlorine. The congeners are designated by describing the position of the chlorine
atoms on the biphenyl ring or, more simply, by the IUPAC numbering system. The
congeners differ in their physical properties according to the number and the position
of chlorine atoms. The high-chlorinated biphenyls are less water-soluble and less
volatile than the low-chlorinated ones. The degree of chlorine substitution influences
their biodegradability that decreases with increasing chlorination. The toxicity for the
biota is also related to the number of chlorines but their position on the biphenyl ring
is of prime importance. The congeners that take a co-planar configuration such as
3,4,3’,4’-TCB (IUPAC #77 ) are the more toxic ones.
The PCBs are prepared by direct chlorination of the biphenyl ring. The com-
mercial formulations display various overall percentages of chlorine and different
congener distribution. For example, Aroclor 1242 contains 42% of chlorine with a
predominance of congeners bearing three and four chlorine atoms; Aroclor 1260 has
60% chlorine content with a predominance of six- and seven-chlorinated congeners.
These mixtures typically contain more than 70 different congeners [1] and were sold
under different names (Aroclor, Phenoclor, Clophen, Delor and Kanechlor),
depending on the manufacturer.

3. PCBs in the environment

The PCBs have been detected in the environment for the first time in 1966 by Jensen
[2] along with DDT contamination. Since then, PCBs were found everywhere,
including polar regions, both Artic and Antarctic [3]. They are now considered as the
most widespread pollutant on the planet. In industrial countries, the contamination
originates from inadequate disposal and leaks from equipments. In remote areas
where PCBs were not used, the contamination resulted from atmospheric transport
[4]. The high chemical stability of PCBs explains their persistence in the environment.
Their low water solubility results in their accumulation in the biota (in fats) and their
Phytoremediation of Polychlorinated Biphenyls 145

concentration in the food chain [5]. The marine mammals at the top of the food chain
concentrate high level of PCBs [6, 7]. Human are contaminated as well and breast
milk contains measurable content of PCBs [8]. In the industrialized countries the
PCB blood level of an individual increases with age [9, 10].
The distribution of the PCBs in the different environmental compartments in the
United Kingdom has been extensively studied by Harrad et al. [11]. They found that
the PCB concentration peaked in the early to mid 1960s, declined rapidly following
restrictions of their use in the 1970s and continued to decrease since then, but at a
lesser rate. According to their estimates, most of the PCBs reside in soil (92.5%), sea
water (3.5%) and marine sediments (2%). Up to 0.2% might be in humans. The
amount in soil represents about 370 tons for UK only. The pattern for other
industrialized countries should be similar, adjusted upscale or downscale depending
of the amount of PCBs used and the duration of their use. Today, even if the PCB
production has ceased, there is still a flux of PCBs originating from leaks of
transformers and capacitors remaining in use (0.2-0.3 tons/year for UK) and from
volatilization from soil (8,1 to 40 tons/year for UK) [11].
The composition of PCB mixtures found in the environment differs from the
original commercial preparations. At or near contamination points there may be a
shift to the more highly chlorinated congeners because the lowest chlorinated
congeners being more volatile and more easily degradable have either migrated or
have been transformed by microorganisms. So the pattern observed for long term
contaminated soil displays a higher proportion of high chlorinated congeners than the
mixture at the origin of the contamination. In remote areas where PCBs were not
used, the shift is toward low-chlorinated congeners which are more volatile and more
easily translocated through the atmospheric currents. Contaminated sediments also
present a higher proportion of low-chlorinated congeners that is attributable to
reductive dechlorination resulting from microbiological activity.
Although toxicological studies of PCBs are complex, because they should take
into account numerous different molecules with possible synergic effects, studies
suggest toxic and carcinogenic properties, particularly for co-planar congeners [12,
13]. Extensive studies in vivo and in vitro have been conducted on the toxicological
effects of PCBs in different mammalian systems. One area that has received less
attention is the uptake, translocation and transformation of PCBs in terrestrial plants.
To investigate the mobility of PCBs in any phase of the environment, it is very
essential to understand their physical and chemical properties, transport, translocation
and accumulation in the food chain.
Proof of biotransformation is not itself sufficient to guarantee protection, the
biotransformation must not result in the production of harmful intermediates with
long lifetimes. For this reason, biotransformation pathways must also be understood.
Additionally, good mass balances are difficult to make. Alternative approaches are
often necessary to provide proof that the desired transformation process is occurring.
Evidence is provided by detection of intermediates and products or of the consump-
tion of other chemicals that are associated with biotransformation.
146 Martina Mackova et al.

4. Bacterial degradation of PCBs

Despite the high chemical stability of PCBs, the finding that microorganisms are able
to degrade some congeners opened the door to implement bioremediation
technologies. Essentially, bacteria can degrade PCBs by two processes: aerobic
degradation via the biphenyl pathway and anaerobic dechlorination. The former being
the most documented.

4.1. AEROBIC PATHWAY

The first report on the isolation of PCB degrading bacteria dates back to 1973 when
Ahmed and Focht found two species of Achromobacter capable of degrading PCBs
[14]. Rapidly, other strains were isolated from PCB contaminated sites, some with
better degradation efficiencies and broader spectrum of activity [15, 16, 17, 18, 19,
20]. These strains were extensively studied and the biphenyl degradation pathway
was elucidated. The genes encoding the different enzymes of the pathway were
sequenced and in some cases cloned in high expression vectors to obtain large amount
of enzymes. The enzymes produced and purified from clones or from native strains
were used to assess in vitro activity against specific PCB congeners or mixtures of
them [21, 22].
The biphenyl pathway (Fig. 1) is identical for all the aerobic bacterial strains.
Biphenyl dioxygenase is a multi-component enzyme constituted by an iron-sulfur
protein, (ISPbph), which interacts directly with the substrate to introduce two oxygen
atoms on adjacent positions on the biphenyl ring. Electrons are transferred from
NADH to ISPbph by the two other components of the dioxygenase, a ferredoxine and a
reductase. The biphenyl dioxygenase components are encoded by four genes
clustered in a single operon in most of the strains: bphA (Į-subunit of ISPbph), bphE
(ȕ-subunit of ISPbph), bphF (FERbph) and bphG (REDbph). The product of the first
enzymatic step is cis-(2R,3S)-dihydroxy-1-phenylcyclohexa-4,6-diene. A dehydrogenase
(encoded by bphB gene) further transforms the first metabolite to a dihydro-
xybiphenyl that is transformed by a second dioxygenase (encoded by bphC gene)
in an open-ring compound. Finally a hydrolase (encoded by bphD gene) cleaves
the molecule to form benzoic acid. This is the so-called higher pathway. The minera-
lization of benzoic acids is performed by another group of enzymes constituting
the lower pathway.
Chlorinated biphenyls are processed by the same pathway. However not all
congeners can be degraded and biphenyl dioxygenases isolated from different
bacterial strains have different specificity patterns. Biphenyl dioxygenases generally
have a good activity towards congeners containing three or less chlorine atoms, but
lower or no activity towards more substituted biphenyl. Burkholderia xenovorans
LB400 is the most potent natural occurring strain known to date. It can degrade most
of chlorinated biphenyls containing 3 chlorine atoms and some containing 4 and 5
chlorine atoms [23, 24]. Several evolved biphenyl dioxygenases have been reported
which exhibit extended catalytic activity toward PCBs [25, 26, 27, 28]. However, in
some case the metabolites produced by these modified enzymes or even by native
Phytoremediation of Polychlorinated Biphenyls 147

NAD+ NADH+H+

REDBPH REDBPH
(red) BphG (ox) COOH
FERBPH FERBPH H OH OH
(ox) BphF OH
(red)
H COOH
ISPBPH ISPBPH BphB
BphAE OH OH O
(red) (ox) BphC BphD benzoate
O2 NAD+ NADH+H+ O2
OH

biphenyl 2-hydroxy-6-oxo- CH2 COOH

cis-(2R,3S)-dihydroxy-1- 2,3-dihydroxybiphenyl
phenylcyclohexa-4,6-diene 6-phenyl-hexa- 2-hydroxypenta-
2,4-dienoate 2,4-dienoate

Figure 1. Aerobic bacterial biphenyl degradation pathway.

large spectrum dioxygenases cannot be further processed downstream, the first

bottleneck being at the third step of the pathway (2,3-dihydroxybiphenyl 1,2-
dioxygenase) [29]. Thus the development of more efficient PCB degrading strains
must address the improvement of every enzyme of the pathway.

4.2. ANAEROBIC DECHLORINATION

The observation that PCB contaminated sediments display a higher content of low
chlorinated congeners than the mixture at the origin of the contamination was the first
clue that anaerobic bacteria are able to degrade PCBs by a reductive dechlorination
process. This process has been well documented for several aquatic sediments [30,
31, 32]. Anaerobic enrichment cultures able to dechlorinate PCBs were maintained in
laboratory conditions, with and without sediment [33, 34, 35, 36, 37]. The
dechlorination release preferentially meta and para chlorines [35, 38] but there are
some reports of ortho chlorine release [39, 40, 41]. Brown et al described eight
different microbiological processes responsible for the different dechlorination
patterns and congener specificities [30, 42]. It is thought that these processes are
performed by different strains and different enzymes. Until recently, all attempts to
obtain a pure culture able to dechlorinate PCBs had failed. Now, one pure anaerobic
strain and one consortium have been reported [43, 44]. However, isolation of more
strains and studies of the enzymes responsible for the dechlorination are still to come.
The effect of anaerobic dechlorination is double: first the removal of meta and para
chlorines reduces the toxicity of the contaminated sediments by reducing the
occurrence of co-planar congeners [45]. Second, the reduction of the number of
chlorine atoms on the biphenyl ring potentially facilitates the aerobic degradation
through the biphenyl pathway.
148 Martina Mackova et al.

5. Degradation of PCBs in higher organisms

The first reports of PCBs degradation by white rot fungi date back to 1985 [46, 47].
The enzymes that are potential candidates for this activity are peroxidases and
laccase. These enzymes are involved in lignin degradation and are also active against
a wide range of aromatic compounds (PAH, substituted phenols, chlorinated
pesticides [48, 49] etc.). The mechanism of degradation by lignin-peroxidase,
manganese-peroxidases and laccases is described in degradation of many environ-
mental pollutants. The ligninolytic enzyme system is non-specific, extracellular
and free radical based that allows them to degrade structurally diverse range
of xenobiotic compounds. Lignin peroxidase and manganese peroxidase carry
out direct and indirect oxidation as well as reduction of xenobiotic compounds.
Indirect reactions involved redox mediators such as veratrylalcohol and Mn2+.
Reduction reactions are carried out by carboxyl, superoxide and semiquinone
radicals, etc. Methylation is used as detoxification mechanism by WRF (white rot
fungi). Highly oxidized chemicals are reduced by transmembrane redox potential.
However, the mechanism of PCB degradation has not yet been fully understood.
Several authors documented transformation of PCBs determined by fungi and some
studies concluded that neither lignin peroxidases nor Mn-dependent peroxidases are
involved in PCB degradation [50, 51]. A major problem is the adsorption of the
different PCB congeners to the biomass which introduces a bias when the degradation
is measured by substrate depletion. One alternative is to measure the complete
mineralisation by using radiolabeled substrates but it is likely that many congeners
cannot go through the entire process. The other alternative is the identification of the
metabolites resulting from PCB degradation. Dietrich et al. [52] reported the
presence of two metabolites produced from 4,4’-dichlorobiphenyl by P.
chrysosporum confirming that this fungi can actually degrade this congener. Schultz
et al. [53] described dehalogenation and metabolisation of chlorinated hydro-
xybiphenyls by laccase from white rot fungi Pycnoporus cinnabarinus, Romero
et al. [54] documented biotransformation of biphenyl by ascomyceteous fungi
Talaromyces helicus giving hydroxylated biphenyls and 4-phenyl-2-pyrone-
6-carboxylic acid. Several studies showed capacity of fungal cultures on di, tri, tetra-
and penta-chlorinated phenols when first step is characterized by oxidative
dehalogenation mediated by extracellular peroxidases to form benzoquinones. The
level of degradation of halogenated aromatics, reported in scientific literature, is very
different from one study to the other. More extensive investigation will be necessary
to elucidate the PCB degradation pathway in these eukaryotic microorganisms.

5.1. TRANSFORMATION OF PCBS BY PLANT CELLS

5.1.1 Metabolism of PCBs by plant cells and products formed

Plants have shown the capacity to withstand relatively high concentrations of metals
or organic chemicals without significant toxicity symptoms. Also in some cases they
can uptake and transform organic compounds to less phytotoxic metabolites. Several
Phytoremediation of Polychlorinated Biphenyls 149

investigations have shown that PCBs can be translocated from soil to various parts of
the plants and can accumulate in particular tissues in higher concentrations than in
others.
The metabolism of PCBs varies between the plant species and is affected by the
substitution pattern and the degree of chlorination [55]. Wilken et al. [55] studied
metabolism of 10 different congeners of PCBs in cell cultures of 12 plant species.
They stated observed that the metabolism of defined PCB congeners was dependent
of the plant species. They detected various monohydroxylated and dihydroxylated
compounds that were likely PCB metabolites. Macková et al. [56, 57, 58] studied the
ability of the callus, root and shoot cultures of various species cultivated in vitro to
degrade PCBs. Root cultures derived from single plant cells genetically transformed
by the Ri-plasmid of Agrobacterium rhizogenes, the so-called hairy roots, proved to
be a very useful tool to investigate the metabolic routes used by plants for PCB
conversion [57, 58, 59, 60]. About 40 axenic cell cultures of different plant species
[62, 63, 64, 65] were screened for the ability to transform PCBs. Delor 103 - a
mixture of PCBs commercially produced in the former Czechoslovakia until mid
eighties (similar to Aroclor 1242) [61, 62] was used in the screening as model
mixture of PCBs in these investigations. This PCB mixture contains about 59
congeners with an average of 3 chlorine substitution per biphenyl molecule.
Degradative abilities in relation to the origin and morphology of the cultures have
been evaluated. The PCB transformation capacities varied considerably within
different cultures of different but even the same species [58]. The growth of plant
biomass in the presence of individual congeners was lower compared to the controls
without PCBs and correlated with the metabolic activities toward PCBs. In general,
lesser-chlorinated congeners were metabolised more rapidly than those with higher
number of chlorine atoms. However, there are some exceptions, which indicate that
not only the number of chlorine atoms, but also the position of chlorine substitution
and molecular structure are also important factors controlling the PCB metabolism by
plants. Previous studies [65, 55] have documented that as the degree of chlorination
of the biphenyl ring increases, the rate of their conversion by plant cells of different
species decreases, mostly in agreement with the increasing phytotoxicity of the tested
compound. Comparison of different clones of one plant species proved, that clones
can significantly differ in their ability to metabolise PCBs within one species and thus
the ability to transform PCBs should always be checked and compared with
metabolism of the normal plants. Differentiated and transformed plant cell cultures
metabolised xenobiotics with higher efficiency than amorphous and non-transformed
ones of the same species. This ability was comparable with that of intact plants.
Differentiated cultures of horseradish and black nightshade cells exhibited the
best transformation abilities as well as growth characteristics [65, 66]. Transformed
roots are excellent model systems for screening higher plants that are tolerant to
various inorganic and organic pollutants, and for determining the role of the root
matrix in the uptake and further metabolism of contaminants [67].
The results of the removal of PCB congeners by plant cell cultures exposed to
Delor 103 are shown in Table 1 and Table 2. Both plant species were chosen for
further studies to investigate not only the capacity of cell cultures to remove PCBs
150 Martina Mackova et al.

but also the PCB degrading ability of the whole plants and those grown under natural
conditions in real contaminated soil. Plant tissue cultures of two other species, plant
tissue cultures of Nicotiana tabacum (tobacco) and Medicago sativa (alfalfa), grew
well in the presence of PCBs. Other reasons motivated the choice of these two
species: tobacco is a well studied general plant model, especially for molecular
biology and genetic studies; alfalfa is a plant species with high metabolic activities
within rhizosphere and thus could potentially serve as model for investigations
addressing the interaction between the plant of rhizosphere interactions of plants and
indigenous bacteria.
Table 1. Assignment of individual PCB congeners to peaks of Delor 103 analysed by
gas chromatography with electron capture detector and their conversion shown as the
residual amount of each congener after 14 days incubation, expressed as % of residual
PCB content in the dead cell control flasks (hairy root clone SNC-9O at initial PCB
(Delor 103) content 5 mg per flask and 2.5 mg per flask with 100 ml of media).
Modified according to [65].

Peak No. IUPAC No. Cl-substitution of congeners Residual content of PCB (%)
in the GC peak Initial Delor 103 conc.
*+D *+D
5mg/10ml 2.5mg/100ml
1 5+8 2,3 + 2,4 91 33
2 15 + 18 4,4’ + 2,2 ’,5 90 36
3 17 2,2’,4 90 29
4 16 + 32 2,2’,3 + 2,4’,6 80 35
5 26 2,3’,5 82 35
6 31 2,4’,5’ 84 36
7 28 2,4,4 ’ 80 34
8 20 + 33+53 2,3,3’ + 2’,3,4 + 2,2’,5,6 ’ 86 33
9 45 2,2’,3,6 80 28
10 52 2,2’,5,5’ 78 26
11 49 2,2’,4,5’ 77 26
12 47 + 75 2,2’,4,4’ + 2,4,4’,6 81 26
13 48 2,2’,4,5 80 29
14 44 2,2’,3,5’ 76 27
15 37 + 42+ 59 3,4,4’ + 2,2’,3,4’ + 2,3,3’,6 79 39
16 41 + 64 2,2’,3,4 + 2,3,4’,6 77 43
17 96 2,2’,3,6,6’ 79 35
18 74 2,4,4’,5 75 35
19 70 2,3 ’,4’,5 75 38
20 66 + 88+ 95 2,3,4,4’ + 2,2’3,4.6 + 75 40
2,2’,3,5’,6
21 101 2, 2’,4,5,5 ’ 75 37
22 77 + 110 3,3’,4,4’ + 2,3,3’,4’,6 69 45
+D = mixture of PCBs (Delor 103) was added in described amounts. The results were
calculated from 3 independent experiments, each GC value was measured in 2 parallels.

Studies were focused to investigate the fate of PCB congeners in plant cells, to
identify the enzymes involved in their metabolism and the products formed. These
experiments were performed at laboratory under aseptic conditions and in real soil
conditions. The study was performed first with PCB mixture – Delor 103 and then
Phytoremediation of Polychlorinated Biphenyls 151

with individual monochlorobiphenyls and dichlorobiphenyls in concentration of 0.3

mg /100ml [65, 66, 67].
The results showed slight transformation of all mono and dichlorobiphenyl
congeners except 4,4’-dichlorobiphenyl [67]. 3- and 4-Chlorobiphenyl, PCB 4 – 2,2’-
dichlorobiphenyl and PCB 10 – 2,6-dichlorobiphenyl were the most easily degraded
congeners [68]. All these three congeners are relatively well soluble in water their log
Kow is rather low. These features are likely to increase their bioavailability for plants.
Similar results were reported by the group of prof. Harms [68]. The authors showed
that 12 plant species were able to metabolise 10 different PCBs. Except 4,4’-
dichlorobiphenyl, all other PCBs congeners tested were at least slightly metabolised.
They hypothesised that 4,4’- dichlorobiphenyl is sterically protected from the enzyme
attack in spite of being more soluble in water and having lower Kow.
Table 2. Assignment of PCB congeners to peaks of Delor 103 analyzed by gas
chromatography and their conversion by horse radish cultured cells K54 after 2 weeks
of incubation with PCB content 2.5, 5mg/100 ml of medium, expressed as the residual
amount of each congener in % of the control. Modified according to [65].

Residual amount of PCBs

Peak No. IUPAC No. Cl-substitution of after transformation (%)
congeners in the GC peak
+D* +D*
5mg/100 ml 2.5mg/100 ml
1 5+8 2,3+2,4’ 54 7
2 15+18 4,4’+2,2’,5 55 10
3 17 2,2’,4 50 8
4 16+32 2,2’,3+2,4’,6 54 10
5 26 2,3’,5 46 8
6 31 2,4’,5’ 41 10
7 28 2,4,4’ 36 12
8 20+33+53 2,3,3’+2’,3,4 +2,2’,5,6’ 47 10
9 45 2,2’,3,6 50 8
10 52+69 2,2’,5,5’+2,4,6,3 ’ 43 12
11 49 2,2’,4,5’ 35 11
12 47+75 2,2’,4,4’+2,4,4’,6 38 9
13 48 2,2’,4,5 41 8
14 44 2,2’,3,5’ 42 10
15 37+42+59 3,4,4’+2,2’,3,4’+2,3,3’ 47 9
16 41+64 2,2’,3,4 + 2,3,4’,6 38 12
17 96 2,2’,3,6,6’ 42 8
18 74 2,4,4’,5 39 8
19 70 2,3’,4’,5 43 9
20 66+88+95 2,3,4,4’+2,2’3,4.6 31 9
+2,2’,3,5’,6
21 101 2, 2’,4,5,5’ 38 8
22 77+110 3,3’,4,4’+2,3,3’,4’,6 35 22
+D = mixture of PCBs (Delor 103) was added in described amounts. The results were
calculated from 3 independent experiments, each GC value was measured in 2 parallels.

In our studies we further paid attention to the metabolites generated by the plant cells.
In order to identify the structure of the metabolites and the position of attack to
152 Martina Mackova et al.

investigate number and structure of the products, using improved and more sensitive
analytical procedures and increasing the biomass used for the assays, we showed that
although 4,4’-dichlorobiphenyl was metabolised poorly, one hydroxylated product
generated from this congener (Table 3) was formed.
Analyses of the transformation products of plant cultures exposed to dichloro-,
trichloro- and tetrachlorobiphenyls showed that even the most persistent 3,3’,4,4’-
tetrachlorobiphenyl can be metabolised [69].
Table 3. Hydroxylated derivatives of monochlorobiphenyls formed from individual
monochlorobiphenyls during 14 days incubation with plant cells of tobacco, black-
nightshade horseradish and alfalfa.

PCB Plant species Number of Identified structure

hydoxy
products
PCB 1, Tobacco 4 No product was precisely
2-chlorobiphenyl identified
Black nightshade 5 One product identified as
2Cl-5OH biphenyl
Horseradish 5 Two products were
identified as 3Cl-2OH
biphenyl and
2Cl-5OH biphenyl
Alfalfa 0 No product was identified
PCB 2, Tobacco 3 No product was precisely
3-chlorobiphenyl identified
Black nightshade 4 Two products identified
3Cl4-OH biphenyl, 3Cl-
6OH biphenyl
Horseradish 4 One product identified
as 3Cl-4OH biphenyl
Alfalfa 0 No product was identified
PCB 3, Tobacco 3 4Cl-4’OH biphenyl
4-chlorobiphenyl Black nightshade 2 4Cl-4’OH biphenyl
Horseradish 4 4Cl-4’OH biphenyl
Alfalfa 1 4Cl-4’OH biphenyl
PCB 4 Tobacco 3 Dichlorohydroxybiphenyls
2,2’dichlorobiphenyl No product was precisely
identified
Black nightshade 3 Dichlorohydroxybiphenyls
No product was precisely
identified
PCB 9 Tobacco 2 2,5diChl-3OH biphenyl
2,5 -dichlorobiphenyl 2,5diChl-4OH biphenyl
Black nightshade 6 2,5diChl-2OH biphenyl
2,5diChl-3OH biphenyl
2,5diChl-4OH biphenyl

To identify chemical structure, position of chlorines and hydroxyl groups plant cells
of four different species were incubated with monochlorobiphenyls. GC-MS analysis
Phytoremediation of Polychlorinated Biphenyls 153

revealed that metabolites identified in plant tissue were mono- and dihydroxy-
compounds. Some of them could be identified by GC-MS by comparison with the
structures of the standards. The results are summarized in Table 3. Altogether, data
showed that although different plant species metabolise PCB congeners following a
similar pattern for any PCB congener, the different plant species form the same types
of hydroxychloroderivatives but number of products and positions of hydroxyl
group(s) differ with the plant species. Alfalfa has quite low potential to metabolise
PCBs, and its ability is limited only to transformation of 4-chlorobiphenyl, which is
metabolised to 4Cl-4’OH biphenyl, the same product found in cells of other plant.

5.1.2 Enzymes responsible for PCB transformation in plants

As was previously documented in case of many environmental xenobiotics,
phytotransformation refers a process by which organic contaminants are uptake by
plants of organic contaminants from soil and groundwater and subsequently the
xenobiotics are metabolised or transformed. Various plant xenobiotics-converting
enzymes, cytochrome P450, peroxidases, glutathione–S-transferases, carboxyesterases,
O-glucosyl transferases, O-malonyl transferases, N-glucosyl transferases and N-
malonyl transferases, were isolated and purified. They were proved to metabolise
efficiently various xenobiotics such as PCBs, organic solvents and chlorinated
pesticides [70, 71].
Organic pollutants undergo in plants a three -stage metabolic processes similar to
mammalian metabolism [72]. First phase which includes oxidation, reduction or
hydrolysis of xenobiotics often generates metabolites with increased polarity [67, 71,
72, 73]. These reactions are catalyzed by mixed functional oxidases (cytochrome
P450) and/or peroxidases to introduce an hydroxyl group or to substitute common
functional groups (nitro, carboxyl, alkyl or halogens) [68]. Plants hydroxylate PCBs
to form more soluble hydroxyderivatives [67, 74, 75, 76]. This reaction suggests that
polychlorinated biphenyls oxidases, cytochrome P450 and/or peroxidases present in
plant cells are involved in PCB conversion. The identity of the enzymes responsible
for plant transformation of xenobiotics was already a matter of discussion in the early
seventieth, when the first reports about PCB metabolisation were published [77]. In
1992 it was discussed by Lee and Fletcher [74] who studied involvement of
cytochrome P450 and peroxidase in PCB transformation in presence of specific
inhibitors for cytochrome P450 and peroxidases. Their study provided evidence that
cytochrome rather than peroxidases were the major contributors to plant metabolism
of PCB. More recently, we got evidence that peroxidases are also likely [62, 63]
involved in PCB transformation. It was shown that plant tissue cultures of various
species, exhibiting higher peroxidase activities in PCB presence, possess higher
capabilities to transform them. Chroma et al. [63] used different specific inhibitors
for both enzymic systems and measured not only PCB removal but also peroxidase
activities. Presence of all inhibitors resulted in a decrease of PCB degradation
efficiency compared to the controls without any inhibitor. Nevertheless, further
following analyses showed a decrease of peroxidase activity not only when
peroxidase’s but also cytochrome P450 inhibitors were added (see Table 4). This
154 Martina Mackova et al.

phenomenon supported idea that both these enzymic systems are involved in the
metabolism of PCBs in plant cells.
Tab. 4. The effect of inhibitors on peroxidase of SNC-9O and its PCB transformation.
Modified according [63].

Inhibitor PCB Removal POX activity

(Enzyme inhibited) of PCB [%] [RLUd × 107/mg
protein]
None – – 390
None + 45 318
Propylgallate (POX) + 10 48
Sodium benzoate (POX) + 15 84
Aminobenztriazol.(cyt P450) + 20 49
Metyrapone (cyt P450) + 12 37

Transformation and degradation of organic pollutants by peroxidases has been

already described in many reports [49, 50, 51, 52, 78, 79, 80, 81, 82, 83, 84]. Most
of these addressed the peroxidases of the so-called white rot fungi (WRF),
basidiomycetes strains involved in wood decay. Degradation of a number of
environmental pollutants by ligninolytic system of white rot fungi was described
many times [49, 84, 85].
Plant peroxidases, namely horseradish peroxidase [86] and their mechanism of
catalytic reaction on various xenobiotics [87] (including phenolic compounds [88,
89]), were described in detail. The classical reaction catalysed by heme-peroxidases
is oxidative dehydrogenation, although they also catalyze a variety of related
reactions, including oxygen transfer, hydrogen peroxide cleavage, and peroxidative
halogenations.
The plant enzyme-mediated decomposition and biodegradation of PCB were
recently investigated [76]. 5-dichlorobiphenyl (PCB 9) and 2,2’,5,5’- tetrachloro-
biphenyl (PCB 52) were used as model compounds to study efficiency and mech-
anism of the degradation processes. It was found that the application of commercial
horseradish peroxidase (HRP) together with defined amounts of hydrogen pero-
xide removed 90% of the initial concentration of PCB 9 and 55% of the initial
concentration of PCB 52 from an aqueous solution after a reaction period of
220 min. Dechlorination was observed as the initial step. Although the metabolites
identified were mainly chlorinated hydroxybiphenyls, benzoic acids and non-
substituted 1,10-biphenyl, some higher chlorinated biphenyl isomers were also
detected. As a comparison can be added that the biodegradation of PCB 9 using the
white rot fungus Trametes multicolor took about four weeks and reduction was about
80% of the initial concentration. The metabolites produced (dichlorobenzenes,
chlorophenols and alkylated benzenes) were not quite the same as those observed
upon incubation with HRP.
To investigate the capacity of peroxidases of different plant species to transform
PCBs, plant peroxidases from the cells of tobacco, black nightshade and alfalfa were
isolated, partially purified and concentrated by ammonium sulphate precipitation.
Phytoremediation of Polychlorinated Biphenyls 155

Then transformation experiments with various PCB congeners (mono and

dichlorobiphenyls) were performed. All cultures (hairy root of black nightshade,
callus of tobacco and callus of alfalfa) synthesized both intracellular and extracellular
peroxidases. Peroxidases of tobacco and black nightshade changed in the presence of
PCBs their total activities and some changes in isoenzyme patterns were also visible
after native electrophoresis. Total peroxidase activity of alfalfa exhibited the highest
values when measured in absence of PCB presence. Its activity decreased and
isoenzyme pattern was not influenced by the presence of PCBs and this plant was
generally less active in PCB transformation (data not shown) [63]. Peroxidases
isolated from tobacco and black nightshade transformed various PCBs with different
efficiency. Significant differences were detected when congeners with chlorines at
different positions were used for the reaction with peroxidases. In several cases
peroxidases from tobacco or nightshade exhibited different potential to transform the
same congeners. The highest yield was obtained after reaction with PCB 3 and PCB
9, the lowest transformation was detected in cases of PCB 13 and 15. The results are
summarized in Table 5. These experiments confirmed that peroxidases of different
plant species can actively metabolise polychlorinated biphenyls, but reaction
specificity is not clear yet. Efficiency of degradation of structurally different PCB is
related to their chemical structure, position and number of chlorines, physico-
chemical properties but also plant species, and PCB toxicity to plant cells of diverse
species.
Small amounts of numerous products formed from PCBs during reactions of
peroxidases, isolated from black nightshade, with were detected by GC-MS after
silylation. Reactions with tobacco peroxidase did not give any obvious products, only
traces of compounds that are possibly di-OH-dichlorobiphenyl occurred after reaction
of tobacco peroxidase with PCB 9. After derivatization the concentration of products
was under detection limit and original hypothesis could not be confirmed. In reaction
mixtures containing peroxidase from black nightshade, dechlorination was observed
as the initial step. As metabolites less chlorinated biphenyls than the original ones or
even the non-substituted biphenyl were detected. Reaction of horseradish peroxidase
with PCBs gave very low concentrations of hydroxychlorobiphenyls [76]. In our
study surprisingly no hydroxylated chlorobiphenyls were found. This is most likely
explained by further fast reactions following the first dechlorination step without any
accumulation of intermediates. Regarding oxidative degradation, the cleavage of the
ring system and subsequent reactions gave benzoic acid and hydroxybenzoic acids as
the products (see Figure 2). As a result of POX radical mechanism, higher chlorinated
isomers were formed. Traces of phenylacylchlorides were also found in reaction
mixtures [63].
156 Martina Mackova et al.

Table 5. Transformation of mono- and dichlorobiphenyls by intracellular plant

peroxidases isolated from cells of black nightshade and tobacco.

IUPAC No. PCB Degradation of PCB by Degradation of PCB by

POX from black POX from tobacco (%)
nightshade (%)
Delor 103 Mix 45 ± 5 20 ± 3
PCB 1 2-Cl 35 ± 5 –*
PCB 2 3-Cl 35 ± 7 –*
PCB 3 4-Cl 100 ± 10 79 ± 10
PCB 4 2,2’- diCl 56 ± 9 60 ± 8
PCB 5 2,3 - diCl 63 ± 8 17 ± 3
PCB 7 2,4 - diCl 59 ± 7 47 ± 6
PCB 8 2,4’- diCl 16 ± 2 44 ± 5
PCB 9 2,5 - diCl 66 ± 4 90 ± 8
PCB10 2,6 - diCl 14 ± 2 48 ± 5
PCB 11 3,3’- diCl 28 ± 4 66 ± 5
PCB 12 3,4 - diCl –* 59 ± 8
PCB 13 3,4’- diCl –* 0
PCB 14 3,5 - diCl 16 ± 2 12 ± 2
PCB15 4,4’- diCl 8±1 –*
* Transformation was not measured
Residual amount of individual congeners were analysed after 20 hours of the reaction of
peroxidases and 5-10µmol/l of PCB congeners. Reaction was started with 5mmol/l of
hydrogen peroxide
Our results have proved that an important role in PCB transformation can be attributed
also to plant peroxidases. In plant biochemistry peroxidases are mostly studied as
functional enzymes in plant cell growth and development, ethylene biosynthesis,
lignification, important also in protection of plants during stress as protective
enzymes against reactive oxygen species generated during biotic and abiotic stress
[90]. Our results confirmed that peroxidases from different plant sources (tobacco,
black-nightshade) than horseradish (the most studied model peroxidase) can be
involved not only in antioxidative defense mechanisms, but they even could play
important role in direct transformation and degradation of xenobiotics [91].

5.1.3 Cooperation and interactions between PCB metabolising bacteria and plants
can affect their removal from the environment
Microbial activity has been deemed the most influential and significant cause of PCB
removal. Numerous studies have been conducted on microbial consortia and
enrichment, and several diverse genera of bacteria have been isolated (for a review,
see reference [92]). Recent work has indicated that the stimulation of microbial
activity in the rhizosphere of plants can also stimulate biodegradation of various toxic
Phytoremediation of Polychlorinated Biphenyls 157

dichlorobiphenyl
Cl

Cl Cl
monochlorobiphenyl

OH

benzoic acid

OH

tri(di)hydroxybenzoic
(OH)n n=2-3 acid
Cl OH

hydroxychlorobenzoic
Cl 2 (OH)n n=2-3 acid

trichlorobiphenyl

Cl

Figure 2. Products identified after reaction of peroxidase isolated from black

nightshade with individual congeners of PCBs (modified according [91]).

organic compounds [93]. The rhizosphere soil has been described as the zone of soil
under the direct influence of plant roots and usually extends a few millimetres from
the root surface and is a dynamic environment for microorganisms. The rhizosphere
microbial community is comprised of microorganisms with different types of
metabolism and adaptive responses to variation in environmental conditions. The
production of mucilaginous material and the exudation of a variety of soluble organic
compounds by the plant root play an important part in root colonization and
maintenance of microbial growth in the rhizosphere. Microbial activity is thus
generally higher in the rhizosphere due to readily biodegradable substrates exuded
from the plants [94, 95, 96, 97, 98]
Recent studies indicate that degradation of PAHs and polychlorinated biphenyls
(PCBs) in soils can be enhanced by plants. Published results support the hypothesis
and provide mechanistic understanding of rhizosphere degradation {98, 99]: (1)
purified natural plant compounds (i.e. flavanoids) stimulate the growth and activity of
PCB degrading bacteria [99]; (2) Plant roots release phenolic compounds that support
the growth of PCB degrading bacteria, but all plant species are not effective [100];
(3) Flavanoid compounds that support the growth of PAH-degrading bacteria
158 Martina Mackova et al.

accumulate in aging/dying fine roots of mulberry [100]. The combined interpretation

of these data is that the roots of some plant species are capable of growing to
immobile soil contaminants (PCBs, PAHs) and deliver cometabolites (i.e. flavanoids)
upon fine root death. These natural cometabolites foster the growth and activity of
degradative microbes. The dead/decayed roots also create soil cavities that facilitate
soil aeration.
Plant-microbial interactions in contaminated soil taking place on the level of
growth and support of degradation abilities were already studied and a lot of
information about cooperative mechanism was described. Less information is
available about possible linking up the metabolism of bacteria and plants as a result
of followed metabolisation of metabolic products formed during primary degradation
of PCBs in both types of organisms. Particularly our interest was directed to ability of
plants originally transforming PCB, to metabolise also bacterial PCB degradation
products, i.e. chlorobenzoates. On the contrary if bacteria degrading PCBs are able to
transform plant primary metabolites – hydroxychlorobiphenyls [101]. Similarly
Sondossi et al. [102, 103] studied transformation of nonchlorinated hydroxybiphenyls.
Commercially available hydroxychlorobiphenyls were transformed in vitro by bacterial
enzymes isolated from two PCB degrading bacteria Pseudomonas xenovorans LB400
and Comamonas testosteroni B356 [101]. Products of each step of bacterial PCB
pathway were detected after derivatization by GC-MS. Data obtained after the first
reaction step of three hydroxychlorobiphenyls, 3Cl-2OH; 5Cl2OH and 3,5Cl2OH
with biphenyldioxygenase (BPDO) confirmed, that both B-356 BPDO and LB400
BPDO oxygenate mono-substituted-hydroxy- and hydroxychlorobiphenyls on the
non-substituted ring. A meta-para oxygenation of the ortho-hydroxylated ring would
have required a dehalogenation of 2-hydroxy-3,5-dichlorobiphenyl, but no
dehalogenated metabolites were detected when this compound was used as substrate.
This confirms that neither LB400 BPDO nor B-356 BPDO can oxygenate the
substituted ring of the ortho-hydroxylated biphenyl. Furthermore, the fact that the
metabolites generated by LB400 BPDO were identical to those generated by B-356
BPDO for all three hydroxychlorobiphenyls tested exclude any other mode of attack
of these compounds by LB400 BPDOs than the ortho-meta oxygenation. When the
reaction was done with 3Cl-2OH; 5Cl-2OH and 3,5Cl-2OH, and BphAEFGBC,
metacleavage compound was produced. Thus, these compounds could be converted,
probably efficiently to HOPDA. However, it seemed that BphD did not work on the
HOPDAs carrying an hydroxyl group on the phenyl ring. When 2Cl-4OH and 3Cl-
4OH were submitted to BphAEFGBC, a lots of HOPDA was produced, thus the
triOH was a good substrate for BphC. Similarly, when submitted to BphAEFGBCD,
hydroxybenzoic acid was detected. This observation indicated that with respect to the
catalytic activity of BPDO, the mode of oxygenation of substrate analogs as well as
the stability of the metabolites produced can greatly differ depending on the type of
substituents present on the biphenyl ring. Thus caution should be taken when
transposing data reported for the bacterial degradation of PCB congeners to the
bacterial degradation of hydroxylated PCB metabolites derived from plants. From
above mentioned results can be concluded that some intermediates of plant
transformation of polychlorinated biphenyls can be further degraded by the same
Phytoremediation of Polychlorinated Biphenyls 159

bacteria which degrade original PCBs. More soluble hydroxychlorobiphenyls are

more available than original PCBs and can be released during decaying of plants and
root turnover. On the other hand they are more toxic [104], especially for non-
degrading bacteria. In context of this observation we suggest that PCB degrading
bacteria possess unique ability and the action of microbial associations can be
responsible for generally wider degrading capacity of biological systems important
when evaluating final degradation potential in contaminated environment.
Chlorobenzoic acids (CBAs) are one of the end products of so-called upper
aerobic bacterial degradation pathway (see Figure 1) of PCBs and they can be
available to plants growing in consortia with PCB degrading microbes. To explain the
fate of xenobiotics and their intermediates we also intended to find the answer, if
plants were able to metabolise CBAs. As a model we chose four plant tissue cultures
used before to examine their PCB transformation potential and peroxidases activities,
namely black nightshade (Solanum nigrum), tobacco (Nicotiana tabacum), and
horseradish (Armoracia rusticana) alfalfa (Medicago sativa) [105]. Plant cells of
horseradish and black nightshade showed to be the most active cultures in respect of
CBAs metabolism. Both species metabolised about 70% of 2-chloro and 2,5-
dichlorobenzoic acids within one week (see Table 6). 2CBA is probably the most
suitable substrate due to its best solubility and bioavailability. Cells of alfalfa and
tobacco plants did not degrade significant amounts of CBAs and all chosen CBAs
proved much higher toxic effect on vitality and growth of these two species in
comparison with horseradish and black nightshade cultures. When evaluating the
results of CBAs transformation by plants it can be concluded that ortho-position in
molecule of chlorobenzoate supports the transformation potency. As well as in
previous cases [55, 63], chlorine in ortho-position is crucial for transformation not
only of PCBs [55, 75], but also in metabolism of chlorobenzoic acids.
Our study proved that certain plant species are able to degrade some
chlorobenzoic acids entering the environment due to microbial PCB degradation but
also industrial and agricultural activities. From the literature it is known, that plants
help the rhizospheric microflora to remove several organic and inorganic pollutants
by supporting its activities and living conditions. Our experiments showed that plant
cells could help to remove CBAs also by their direct metabolisation and removal
from the environment.
Direct and indirect interactions and mutual cooperation of plants and growth
promoting rhizosphere bacteria were already studied and the aspects, advantages and
pitfalls of the processes were described for many types of inorganic and organic
chemicals [106, 107, 108, 109]. Many laboratories are studying processes of
biological degradation by different systems to increase the existing knowledge about
fate, toxicity and effect of contaminants. Such approach needs not only detailed
information about uptake, transport, metabolism and toxicity of pollutants and their
products, but also physiology of individual organisms, enzymes involved and
complex interactions of organisms included within the environment [110, 111, 112,
113].
The storage of organic compounds, the presence of transformed and conjugated
derivatives are also causes for concern [114] to explain unknown metabolic and
160 Martina Mackova et al.

toxicological impacts. Our study shows further possibility of interactions between

bacteria and plants in contaminated environment and on example of model
contaminant – PCB, gives more detailed information about abilities of biological
systems to metabolised original xenobiotics and also their intermediates and products.
TABLE 6. Degradation of chlorobenzoic acids during 14-days cultivation with plant
tissue cultures of Armoracia rusticana K54. Solanum nigrum SNC 9O and Nicotiana
tabacum WSC 38.

Residual CBA [%]

CBA Armoracia rusticana Solanum nigrum Nicotiana tabacum

Day 14 Day 14 Day 14

2-CBA 5±3 13 ± 2 88 ± 1
3-CBA 90 ± 3 91 ± 4 95 ± 4
4-CBA 82 ± 5 99 ± 2 94 ± 3
2,3-di CBA 77 ± 5 58 ± 10 99 ± 6
2,4-di CBA 65 ± 2 61 ± 10 79 ± 9
2,5-di CBA 77 ± 5 65 ± 3 85 ± 4
2,6-di CBA 66 ± 10 80 ± 5 98 ± 2
3,4-di CBA 93 ± 3 89 ± 12 98 ± 6
3,5-di CBA 92 ± 6 95 ± 12 100 ± 5
2,3,5-tri CBA 95 ± 7 88 ± 11 96 ± 9
2,4,6-tri CBA 90 ± 2 95 ± 2 99 ± 3
Initial concentration of CBAs was 200 mg/l. As controls cells killed by boiling for 20
minutes were used.

6. Conclusions

During last 15 years many examples describing various plant species accumulating
and/or metabolising organic xenobiotics were described [55, 56, 57, 63, 75, 105, 107,
108, 109, 110, 111, 112, 113]. First interest was directed to uptake and translocation
of organics within plants [114], later the approach was shifted to experiments
investigating the metabolic routes leading to detoxification of xenobiotics in
individual organisms. Sandermann [115], Burken and Schnoor [114] described
general so-called “green liver model” showing common steps in plant and
mammalian metabolism, others studied particular metabolisation of xenobiotics.
Many laboratories have been studying plant metabolism on level of plant tissue
cultures (in vitro systems) or hydroponic systems that provide information on the
metabolism, phytotoxicity, products formed and enzymes involved. Many
investigations showed that qualitatively the metabolic pathways in plant tissue
cultures and intact plants are the same [75]. As a result of more comprehensive
information about natural systems, more sophisticated analytical methods and
development of molecular biology and genetic engineering, also metabolic pathways
and interactions between all involved natural organisms and complexity of their
action became important in phytoremediation studies [71, 74, 92, 94, 95, 96, 97, 98,
Phytoremediation of Polychlorinated Biphenyls 161

99, 100, 105, 106]. Nowadays, laboratories are focused on processes for the removal
of the contaminants based on cooperation of different systems, optimizing agronomic
and agricultural practises, post-harvesting processes, ecological risk assessments
or construction of genetically modified plants combining autotrophic energy
independence of plants with heterotrophic enzymatic processes needed for complete
mineralization [114, 116]. The metabolic degradation routes of microorganisms
(namely bacteria) and higher organisms are different [91, 117, 118, 119] and lead to
different products from the point of structure and toxicity [110, 111, 116]. From this
point of view transgenic plants have great potential for field applications.
It seems that in spite we have available a lot of information to use the plants for
phytoremediation purposes, still much work is necessary to forecast all aspects of the
beneficial application of plants in the field [107, 108, 112, 114]. In this context needs
for bench and field-testing are still actual to extend theoretical knowledge and
practical experience.

Acknowledgement

The authors thank to grant agencies of Ministry of Education of the Czech Republic
which sponsored the experiments and work described above by the grant No. MSM
6046137305, No. 1PO5ME 745 and Z40550506, Grant Agency of the Czech
Republic – grants No. GACR 521/05/0500, and 203/06/0563

References
[1] Schulz, DE; Petrick, G and Duinker, JC (1989) Complete characterization of polychlorinated
biphenyl congeners in commercial Aroclor and Clophen mixtures by multidimensional gas
chromatography –electron capture detection. Environ Sci Technol 23: 852-859
[2] Jensen, S (1966) Report of a new chemical hazard. New Science 32: 612
[3] Risebrough, RW; Walker, W; Schmidt, TT; deLappe, BW and Connors, CW (1976) Transfer of
chlorinated biphenyls to Antarctica. Nature 264(5588): 738-739
[4] Montone, RC; Taniguchi, S and Weber, RR (2001) Polychlorinated biphenyls in marine sediments
of Admiralty Bay, King George Island, Antarctica. Mar Pollut Bull 42(7): 611-614
[5] Safe, S (1980) Polyhalogenated aromatic compounds, in R Kimbrough, Ed., Halogenated
biphenyls, naphtalenes, dibenzodioxins and related products. Elsevier North Holland Pub,
Amsterdam
[6] Ross, PS; Jeffries, SJ; Yunker, MB; Addison, RF; Ikonomou, MG and Calambokidis, JC (2004)
Harbour seals (Phoca vitulina) in British Columbia, Canada, and Washington State, USA, reveal a
combination of local and global polychlorinated biphenyls, dioxin and furan signals. Environ
Toxicol Chem 23(1): 157-165
[7] Kannan, K; Kajiwara, N; Watanabe, M; Nakata, H; Thomas, NJ; Stephenson, M; Jessup, DA and
Tanabe, S (2004) Profiles of polychlorinated biphenyls congeners, organochlorine pesticides and
butyltins in southern sea otters and their prey. Environ Toxicol Chem 23(1): 49-56
[8] Solomon, GM and Weiss, PM (2002) Chemical contaminants in breast milk: time trends and
regional variability. Environ Health Perspect 110(6): A339-347
[9] Bates, MN; Buckland, SJ; Garrett, N; Ellis, H; Needham, LL; Patterson, DG Jr; Turner, WE and
Russell, DG (2004) Persistent organochlorines in the serum of non-occupationally exposed New
Zeland population. Chemosphere 54(10): 1431-1443
162 Martina Mackova et al.

[10] Orloff, KG; Dearwent, S; Metcalf, S; Kathman, S and Turner, W (2003) Human exposure to
polychlorinated biphenyls in a residential community. Arch Environ Contam Toxicol 44(1):
125-131
[11] Harrad, JS; Sewart, AP; Alcock, R; Boumphrey, R; Burnett, V; Duarte-Davidson, R; Halsall, C;
Sanders, G; Waterhouse, K; Wild, SR and Jones, KC (1994) Polychlorinated biphenyls (PCBs) in
the British environment: sink, sources and temporal trends. Environ Pollut 85(2): 131-146
[12] Golden, R; Doull, J; Waddell, W and Mandel, J (2003) Potential human cancer risks from exposure
to PCBs: a tale of two evaluations. Crit Rev Toxicol 33(5): 543-580
[13] Faroon, OM; Keith, S; Jones, D and De Rosa, C (2001) Carcinogenic effects of polychlorinated
biphenyls. Toxicol Ind Health Mar 17(2): 41-62
[14] Ahmed, M and Focht, DD (1973) Degradation of polychlorinated biphenyls by two species of
Achromobacter. Can J Microbiol 19: 47-52
[15] Furukawa, K; Matsumura, F and Tonomura, K (1978) Alcaligenes and Acinetobacter capable of
degrading polychlorinated biphenyls. Agr Biol Chem 42: 543-548
[16] Sylvestre, M and Fauteux, J (1982) A new facultative anaerobe capable of growth on
4-chlorobiphenyl. J Gen Appl Microbiol 28: 61-72
[17] Bopp, LH (1986) Degradation of highly chlorinated PCBs by Pseudomonas strain LB400. J Ind
Microbiol 1: 23-29
[18] Bedard, DL; Wagner, RE; Brennan, MJ; Haberl, ML and Brown, JF Jr (1987) Extensive
degradation of Aroclors and environmentally transformed polychlorinated biphenyls by
Alcaligenes eutrophus H850. Appl Environ Microbiol 53(5): 1094-1102
[19] Asturias, JA and Timmis KN (1993) Three different 2,3-dihydroxybiphenyl-1,2-dioxygenase genes
in the Gram-positive polychlorobiphenyl-degrading bacterium Rhodococcus globerulus P6.
J Bacteriol 175(15): 4631-4640
[20] Sierra, I; Valera, JL; Marina, ML and Laborda, F (2003) Study of the biodegradation process of
polychlorinated biphenyls in liquid medium and soil by a new isolated aerobic bacterium
(Janibacter sp). Chemosphere 53(6): 609-618
[21] Haddock, JD and Gibson, DT (1995) Purification and characterization of the oxygenase
component of biphenyl 2,3-dioxygenase from Pseudomonas strain LB400. J Bacteriol 177:
5834-5839
[22] Hurtubise, Y; Barriault, D; Powlowski, J and Sylvestre, M (1995) Purification and characterization
of the Comamonas testosteroni B-356 biphenyl dioxygenase components. J Bacteriol 177:
6610-6618
[23] Haddock, JD; Horton, JR and Gibson, DT (1995) Dihydroxylation and dechlorination of
chlorinated biphenyls by purified biphenyl 2,3-dioxygenase from Pseudomonas sp. Strain LB400.
J Bacteriol 177: 20-26
[24] Seeger, M; Zielinski, M; Timmis, KN and Hofer, B (1999) Regiospecificity of dioxygenation of di-
to penta-chlorobiphenyls and their degradation to chlorobenzoates by the bph-encoded catabolic
pathway of Burkholderia sp strain LB400. Appl Environ Microbiol 65: 3614-3621
[25] Kumamaru, T; Suenaga, H; Mitsuoka, M; Watanabe, T and Furukawa, K (1998) Enhanced
degradation of polychlorinated biphenyls by directed evolution of biphenyl dioxygenase. Nat
Biotechnol 16(7): 663-666
[26] Brühlmann, F and Chen, W (1999) Tuning biphenyl dioxygenase for extended substrate
specificity. Biotechnol Bioeng 63(5): 544-551
[27] Suenaga, H; Watanabe, T; Sato, M; Ngadiman, T and Furukawa, K (2002) Alteration of
regiospecificity in biphenyl dioxygenase by active-site engineering. J Bacteriol 184(13):
3682-3688
[28] Barriault, D and Sylvestre, M (2004) Evolution of the biphenyl dioxygenase from Burkholderia
xenovorans LB400 by random mutagenesis of multiple sites in region III. J Biol Chem 279 (46):
47480-47488
[29] Dai, S; Vaillancourt, FH; Maaroufi, H; Drouin, NM; Neau, DB; Snieckus, V; Bolin, JT and Eltis,
LD (2002) Identification and analysis of a bottleneck in PCB biodegradation. Nat Struct Biol
9(12): 934-939
[30] Brown, JF and Wagner, RE (1990) PCB movement, dechlorination and detoxification in the
Acushnet estuary. Environ Toxicol Chem 9: 1215-1233
Phytoremediation of Polychlorinated Biphenyls 163

[31] Lake, JL; Pruell, RJ and Osterman, FA (1992) An examination of dechlorination processes and
pathways in New Bedford harbor sediments. Marine Environ Res 33: 1
[32] Abramowicz, DA; Brennan, MJ; Van Dort, HM and Gallagher, EL (1993) Factors influencing the
rate of PCB dechlorination in Hudson River sediments. Environ Microbiol 58: 1057
[33] Hartcamp-Commandeur, LCM (1996) Reductive dehalogenation of polychlorinated biphenyls by
anaerobic microorganisms enriched from Dutch sediments. Chemosphere 32: 1275-1286
[34] Williams, WA (1997) Stimulation and enrichment of two microbial polychlorinated biphenyl
reductive dechlorination activities. Chemosphere 34: 665-669
[35] Bedard, DL; Van Dort, HM; May, RJ and Smullen, LA (1997) Enrichment of microorganisms that
sequentially meta, para-dechlorinate the residue of Aroclor 1260 in Housatonic River sediments.
Environ Sci Technol 31: 3300-3307
[36] Kuipers, B; Cullen, WR and Mohn, WW (1999) Reductive dechlorination of nonachlorobiphenyls
and selected octachlorobiphenyls by microbial enrichment cultures. Environ Sci Technol 33: 3577-
3583
[37] Cuttel, L; Sowers, KR and May, HD (1998) Microbial dechlorination of 2,3,5,6,- tetrachlo-
robiphenyl undr anaerobic conditiond in the absence of soil or sediment. Appl Environ Microbiol
64(8): 2966-2969
[38] Quensen, JF; Tiedje, JM and Boyd, SA (1988) Reductive dechlorination of PCBs by anaerobic
microorganisms from sediments. Science 242: 752-754
[39] Berkaw, M; Sowers, KR and May, HD (1996) Anaerobic ortho dechlorination of polychlorinated
biphenyls by estuarine sediments from Baltimore Harbor. Appl Environ Microbiol 62: 2534-2539
[40] Van Dort, HM and Bedard, DL (1991) Reductive ortho and meta dechlorination of a
polychlorinated biphenyl congener by anaerobic microorganisms. Appl Environ Microbiol 57:
1576-1578
[41] Wu, Q; Bedard, DL and Weigel, J (1997) Effect of incubation temperature on the route of
microbial reductive dechlorination of 2,3,4,6-tetrachlorobiphenyl in polychlorinated biphenyl
(PCB)-contaminated and PCB-free freshwater sediments. Appl Environ Microbiol 63: 2836-2843
[42] Brown, JF; Wagner, RE; Feng, H; Bedard, DL; Brennan, MJ; Carnahan, JC and May, RJ (1987)
Environmental dechlorination of PCBs. Environ Toxicol Chem 6: 579-593
[43] Cutter, LA; Watts, JE; Sower, KR and May, HD (2001) Identification of a microorganism that
links its growth to the reductive dechlorination of 2,3,5,6-chlorobiphenyl. Environ Microbiol
3(11): 699-709
[44] Wu, Q; Watts, JE; Sower, KR and May, HD (2002) Identification of a bacterium that specifically
catalyzes the reductive dechlorination of polychlorinated biphenyls with doubly flanked chlorines.
Appl Environ Microbiol 68(2): 807-812
[45] Tiedje, JM; Quensen, JF; Chee-Sanford, J; Schimel, JP and Boyd, SA (1993-1994) Microbial
reductive dechlorination of PCBs. Biodegradation 4(4): 231-240
[46] Bumpus, JA; Tien, M; Wright, D and Aust, SD (1985) Oxidation of persistent environmental
pollutants by a white rot fungus. Science 228: 1434-1436
[47] Eaton, D (1985) Mineralization of polychlorinated biphenyls by Phanerochaete chrysosporium: a
lignolytic fungus. Enzyme Microb Technol 7: 194-196
[48] Hundt, K; Jonas, U; Hammer, E and Schauer, F (1999), Transformation of diphenyl ethers by
Trametes versicolor and characterization of ring cleavage products. Biodegradation 10: 279-286
[49] Davila-Vazquez, G; Tinoco, R; Pickard, MA and Vazquez-Duhalt, R (2005) Transformation of
halogenated pesticides by versatile peroxidase from Bjerkandera adusta. Enzyme Microb Technol
36: 223-231
[50] Thomas, DR; Carswell,, KS and Georgiou, G (1992) Mineralization of biphenyl and PCBs by the
white rot fungus Phanerochaete chrysosporiun. Biotechnol Bioeng 40: 1395-1402
[51] Beaudette, LA; Davies, S; Fedorak, PM; Ward, OP and Pickard, MA (1998) Comparison of gas
chromatography and mineralization experiments for measuring loss of selected polychlorinated
biphenyl congeners in cultures of white rot fungi. Appl Environ Microbiol 64(6): 2020-2025
[52] Dietrich, D; Hickey, WJ and Lanar, R (1995) Degradation of 4,4’-dichlorobiphenyl, 3,3’,4,4’-
tetrachlorobiphenyl and 2,2’,4,4’,5,5’-hexachlorobiphenyl by white rot fungus Phanerochaete
chrysosporium Appl Environ Microbiol 61: 3904-3909
164 Martina Mackova et al.

[53] Schultz, A; Jonas, U; Hammer, E and Schauer F (2001) Dehalogenation of chlorinated

hydroxybiphenyls by fungal laccase. Appl Environ Microbiol 67: 4377-4381
[54] Romero, MC; Hammer, E; Hanschke, R; Arambarri, AM and Schauer, F (2005) Biotransformation
of biphenyl by the filamentous fungus Talaromyces helicus. World J Microbiol Biotechnol 21:
101-106
[55] Wilken, A; Bock, C; Bokern, M and Harms, H (1995) Metabolism of different PCB congeners in
plant cell cultures. Environ Chem Toxicol 14: 2017-2022
[56] Mackova, M; Macek, T; Kucerova, P; Burkhard, J; Pazlarova, J and Demnerova, K (1997)
Degradation of polychlorinated biphenyls by hairy root culture of Solanum nigrum. Biotechnol Let
19: 787-790
[57] Mackova, M; Macek, T; Burkhard, J; Ocenaskova, J; Demnerova, K and Pazlarova, J (1997)
Biodegradation of polychlorinated biphenyls by plant cells. Int Biodeterior Biodegrad 39: 317-325
[58] Macek, T; Kotrba, P; Suchová, M; Skácel, F; Demnerová, K and Ruml, T (1994) Accumulation of
cadmium by hairy-root cultures of Solanum nigrum. Biotechnol Let 16 (6): 621-624
[59] Macek, T; Kotrba, P; Ruml, T; Skácel, F and Macková, M (1997) Accumulation of cadmium by
hairy root cultures, in PM Doran, Ed., Hairy Roots, pp. 133-138, Gordon and Breach/Harwood
Academic, London, Sydney, UK
[60] Demnerová, K; Burkhard, J; Kostál, J; Macek, T; Macková, M; Pazlarová, J, Kastánek, F and
Kuncová, G (1997) Biodegradation of alkanes and PCBs. Experience in the Czech Republic, in
FW Holm, Ed., Mobile Alternative Technologies, 53-70, Kluwer Academic Publishers, Dordrecht,
Boston, London
[61] Kas, J; Burkhard, J; Demnerová, K; Kostál J; Macek, T; Macková, M and Pazlarová, J (1997)
Perspectives in biodegradation of alkanes and PCBs. Pure Appl. Chem. 69, (11): 2257-2369
[62] Kucerová, P; Macková, M; Poláchová, L; Burkhard, J; Demnerová, K; Pazlarová, J and Macek, T
(1999) Correlation of PCB transformation by plant tissue cultures with their morphology and
peroxidase activity changes. Coll Czech Chem Commun 64 (9): 1497-1509
[63] Chromá, L; Macková, M; Kucerova, P; in der Wiesche, C; Burkhard, J and Macek, T (2002)
Enzymes in plant metabolism of PCBs and PAHs. Acta Biotechnol 22: 35-41
[64] Groeger, AW and Fletcher, JS (1988) The influence of increasing chlorine content on the
accumulation and metabolism of polychlorinated biphenyls by Paul’s Scarlet rose cells. Plant Cell
Rep 7: 329-332
[65] Macková, M; Chromá, L; Kucerová, P; Burkhard, J; Demnerová, K and Macek, T (2001) Some
aspects of PCBs metabolism by horseradish cells. Int J Phytorem 7 (7): 401-414
[66] Pletsch, M; de Araujo, BS and Charlwood, BV (1999) Novel biotechnological approaches in
environmental remediation. Biotechnol Advances 17: 679-687
[67] Kucerová, P; Macková, M; Chromá, L; Burkhard, J; Tríska, J; Demnerová, K and Macek, T (2000)
Metabolism of polychlorinated biphenyls by Solanum nigrum hairy root clone SNC-9O and
analysis of transformation products. Plant Soil 225: 109-115
[68] Bock, C; Kolb, M; Bokern, M; Harms, H; Mackova M; Chroma, L; Macek, T; Hughes, J; Just, C
and Schnoor, J (2002) PCB – approaches to possible removal from the environment, in D Reible
and K Demnerova, Eds., Advances in phytoremediation, phytotransformation, NATO ASI Series,
pp. 115-140. Kluwer Academic Publishers, Dordrecht
[69] Rezek, J; Macek, T and Macková, M (2004) The effect of vegetation on decrease of PAH and PCB
content in long-term contaminated soil, in Proceedings ESEB 2004, (W Verstraete, Ed.), pp.
833-837, Taylor and Francis Group, London
[70] Suresh, B and Ravishankar, GA (2004) Phytoremediation – A novel and promising approach for
environmental clean-up. Crit Rev Biotechnik 24: 97-124
[71] Macek, T; Macková, M and Kás, J (2000) Exploitation of plants for the removal of organics in
environmental remediation. Biotechnol Adv 18 (1), 23-35
[72] Schnoor, JL; Licht, A; McCutcheon, SC; Wolfe, NL and Carreira, LH (1995) Phytoremediation of
organic and nutrient contaminants. Environ Sci and Technol 29, 318-323
[73] Susarla, S; Medina, VF and McCutcheon, SC (2002) Phytoremediation: An ecological solution to
organic chemical contamination. Ecol Engineer 18, 647-658
[74] Lee, I and Fletcher, JS (1992) Involvement of mixed function oxidase systems in PCB metabolism
by plant cells. Plant Cell Rep 11: 97-100
Phytoremediation of Polychlorinated Biphenyls 165

[75] Harms, H; Bokern, M; Kolb, M and Bock, C (2003) Transformation of organic contaminants by
different plant systems, in SC McCutcheon and JL Schnoor, Eds., Phytoremediation.
Transformation and control of contaminants: 285-316, Wiley Interscience, John Wiley and Sons,
Inc., Hoboken, New Jersey
[76] Koeller, G; Moeder, M and Czihal, K (2000) Peroxidative degradation of selected PCB: a
mechanistic study. Chemosphere 41: 1827-1834
[77] Moza, P; Weisberger, I; Klein, W and Korte, F (1973) Distribution and metabolism of carbon-
labelled 2,2’- dichlorobiphenyl in the higher marsh plants Veronica beccabunga. Chemosphere 5:
217-222
[78] Van Aken, B; Hofrichter, M; Scheibner, K; Hatakka, A; Naveau, H and Agathos, S (1999)
Transformation and mineralization of 2,4,6-trinitrotoluene (TNT) by manganese peroxidase from
the white-rot basidiomycete Phlebia radiata. Biodegradation 10: 83-91
[79] Ikehata, K; Buchanan, I and Smith, D (2004) Recent developments in the production of
extracellular fungal peroxidases and laccases for waste treatment. J Environ Engin Science 3: 1-19
[80] Wesenberg, D; Kyriakides, I and Agathos, SN (2003) White-rot fungi and thein enzymes for the
treatment of industrial dye effluents. Biotechnol Adv 22: 161-187
[81] Cameron, S; Timofeevski, G and Aust, SD (2000) Mini-review: Enzymology of Phanerochaete
chrysosporium with respect to the degradation of recalcitrant compounds and xenobiotics. Appl
Microbiol Biotechnol 54: 751-758
[82] Novotny, C; Erbanova, P; Cajthaml, T; Rothschild, N.; Dosoretz, C and Sasek, V (2000) Irpex
lacteus, a white rot fungus applicable to water and soil bioremediation. Appl Microbiol Biotechnol
54: 850-853
[83] Reddy GVB and Gold MH (2000) Degradation of pentachlorophenol by Phanerochaete
chrysosporium: intermediates and reactions involved, Microbiology 146: 405-413
[84] Cajthaml, T; Moeder, M; Kacer, P; Sasek, V and Popp, P (2002) Study of fungal degradation
products of polycyclic aromatic hydrocarbons using gas chromatography with ion trap mass
spectrometry detection. J Chromatog A 974: 213-22
[85] Shrivastava, CHV; Shukla, R; Modi, HA and Vyas, BR (2005) Degradation of xenobiotic
compounds by lignin-degrading white-rot fungi: enzymology and mechanisms involved. Indian J
Exp Biol 43: 301-12
[86] Veitch, NC (2004) Horseradish peroxidases: A modern view of a classic enzyme, Phytochemistry
65: 249-259
[87] Moeder, M; Martin, CM and Koeller, G (2004) Degradation of hydroxylated compounds using
laccase and horseradish peroxidases immobilized on microporous propylene hollow fiber
membranes, J Membrane Sci 245: 183-190
[88] Klibanov, AM; Tu, TM and Scott, KL (1983) Peroxidase catalyzed removal from coal-conversion
waste waters. Science 221: 259-260
[89] Cooper, VA and Nicell, JA (1996) Removal of phenols from foundry wastewater using HRP. Wat
Res 30, 954-959
[90] Inze, D and Van Montagu, M, Eds., (2002) Oxidative Stress in Plants. Taylor & Francis, London.
[91] Chromá, L; Moeder, M; Kucerova, P; Macek, T; and Mackova, M (2003) Plant enzymes in
metabolism of PCBs. Fres Environ Bull 12: 291-295
[92] Pieper, DH (2005) Aerobic degradation of polychlorinated biphenyls. Appl Microbiol Biotechnol
67: 170-191
[93] Gilbert, ES and Crowley, DE (1997) Plant compounds that induce polychlorinated biphenyl
biodegradation by Arthrobacter sp. strain B1B. Appl Environ Microbiol 63: 1933-1938
[94] Gilbert, ES and Crowley, DE (1998) Repeated application of carvone-induced bacteria to enhance
biodegradation of polychlorinated biphenyls in soil. Appl Microbiol Biotechnol 50: 489-494
[95] Singer, AC; Smith, D; Jury, WA; Hathuc, K and Crowley, DE (2003) Impact of the plant
rhizosphere and augmentation on remediation of polychlorinated biphenyl contaminated soil.
Environ Toxicol Chem 22(9): 1998-2004
[96] Singer, AC; Crowley, DE and Thompson, IP (2003) Secondary plant metabolites in
phytoremediation and biotransformation. Review. Trends Biotechnol 21(3): 123-30
166 Martina Mackova et al.

[97] Leigh, MB; Fletcher, JS; Nagle, DP; Mackova, M and Macek, T (2001) Vegetation and fungi at
Czech PCB-contaminated sites as bioremediation candidates, in A Leeson, EA Foote, MK Banks,
V Magar, Eds., Phytoremediation, Wetlands and Sediments, the Sixth International In Situ and On-
Site Bioremediation Symposium, San Diego, 61-68, Battelle Press, Columbus, OH.
[98] Donnelly, PK; Hedge, RS and Fletcher, JS (1994) Growth of PCB-degrading bacteria on
compounds from photosynthetic plants. Chemosphere 28: 981-988
[99] Fletcher, JS and Hegde, RS (1995) Release of phenols by perennial plant roots and their potential
importance in bioremediation. Chemosphere 31: 3009-16
[100] Leigh, MB; Fletcher, JS; Fu, X and Schmitz, FJ (2002) Root turnover: an important source of
microbial substances in rhizosphere remediation of recalcitrant contaminants. Environ Sci Technol
36: 1579-83
[101] Francova, K; Mackova, M; Macek, T and Sylvestre, M (2004) Ability of bacterial biphenyl
dioxygenases from Burkholderia sp. LB400 and Comamonas testosteroni B-356 to catalyse
oxygenation of ortho-hydroxybiphenyls formed from PCBs by plants. Environ Pollution 127/1:
41-48
[102] Sondossi, M; Sylvestre, M; Ahmad, D and Massé, R (1991) Metabolism of hydroxybiphenyl and
chlorohydroxybiphenyl by biphenyl/chlorobiphenyl degrading Pseudomonas testosteroni, strain B-
356. J Ind Microbiol 7: 77-88
[103] Sondossi, MD; Barriault, D and Sylvestre, M (2004) Metabolism of 2,2’- and 3,3’-
Dihydroxybiphenyl by the Biphenyl Catabolic Pathway of Comamonas testosteroni B-356. Appl
Environ Microbiol 70: 174-181
[104] Lovecká, P; Macková, M; Zlamaliková, J; Kochanková, L; Ryslavá, E and Demnerová, K (2005)
Use of biological systems for measurement of ecotoxicity during biological degradation of PCBs,
in N. Kalogerakis, Ed., Proceedings of 3rd European Bioremediation Conference, P139, Chania,
Greece, CD-ROM, Tech University of Crete
[105] Mackova, M; Francova, K; Vrchotová, B; Najmanová, J; Kochánková, L; Sylvestre M; Zídková, J;
Demnerova, K and Macek, T (2005) Biotransformation of PCBs by plants and bacteria –
consequences of plant microbe interactions, in N Kalogerakis, Ed., Proceedings of 3rd European
Bioremediation Conference, P63, Chania, Greece, CD-ROM, Tech University of Crete
[106] Vilacieros, M; Whelan, CM; Mackova, M; Molgaard, J; Sanchez-Contreras, M; Lloret, J; Aguirre
de Carcer, D; Bolanos, L; Oruezabal, RI; Macek, T; Karlson, U; Dowling, DN; Martin, M and
Rivilla, R (2005) PCB rhizoremediation by Pseudomonas fluorescens F113 derivatives using a
Sinorhizobium meliloti nod system to drive bph gene expression. Appl Environ Microbiol 71 (4):
2687-2694
[107] McCutcheon, SC and Schnoor, JL (2003) Phytoremediation. Transformation and control of
contaminants. Wiley Interscience, John Wiley and Sons, Inc., Hoboken, New Jersey
[108] Macek, T; Francova, K; Kochankova, L; Lovecka, P; Ryslava, E; Rezek, J; Sura, M; Triska, J;
Demnerova, K and Mackova, M (2004) Phytoremediation: Biological cleaning of a polluted
environment. Rev Environ Health 19 (1): 63-82
[109] Trapp, S and Karlson, U (2001) Aspects of phytoremediation of organic pollutants. J Soil
Sediments 1(1): 37-43
[110] Meagher, RB (2000) Phytoremediation of toxic elemental and organic pollutants. Cur Opin Plant
Biol 3: 153-162
[111] Newman, LA and Reynolds, CM (2004) Phytodegradation of organic compounds. Curr Opin
Biotechnik 14: 225-230
[112] Glick, BR (2003) Phytoremediation: synergistic use of plants and bacteria to clean up the
environment. Biotech Advances 21: 383-393
[113] Winter, AC; Crowley, DE and Thompson, IP (2003) Secondary plant metabolites in
phytoremediation and biotransformation. Trends Biotechnol 21 (3): 123-130
[114] Burken, JG (2003) Uptake and metabolism of organic compounds: Green liver model, in SC
McCutcheon and JL Schnoor, Eds., Phytoremediation. Transformation and control of
contaminants. Wiley Interscience, John Wiley Sons, Inc., Hoboken, New Jersey
[115] Sandermann, H Jr (1994) Higher plant metabolism of xenobiotics: the “green liver concept”
Pharmacogenetics 4: 225-241
Phytoremediation of Polychlorinated Biphenyls 167

[116] Macek, T; Surá, M; Pavlíková, D; Francová, K; Scouten, WH; Szekeres, M; Sylvestre, M and
Macková, M (2005) Can Tobacco Have Potentially Beneficial Effect to our Health? Zeitschrift fuer
Naturforschung C, 60: 292-299
[117] Furukawa, K; Suenaga, H and Goto, M (2004) Biphenyl dioxygenases: functional versatilities and
directed evolution. J Bacteriol 186: 5189-5196
[118] Sylvestre, M (2004) Genetically Modified Organisms to Remediate Polychlorinated Biphenyls.
Where Do We Stand? Intl Biodeterioration Biodegradat 54: 153-162
[119] Unterman, R (1996) A history of PCB degradation, in Biotechnology Research Series:
Bioremediation Principles and Applications. RL Crawford and DL Crawford, Eds., Cambridge,
U.K.: Cambridge University Press, 209-253
METABOLISM AND GENETIC ENGINEERING STUDIES
FOR HERBICIDE PHYTOREMEDIATION

MELISSA P. MEZZARI1 AND JERALD L. SCHNOOR2

1
Biochemistry and Cell Biology, Rice University, Houston, Texas, USA
2
Civil and Environmental Engineering, University of Iowa, Iowa City,
IA, 52242, USA – Fax 01 319 3355660
E-mail: [email protected]

1. Introduction

Herbicides are extensively used for the purpose of crop cultivation and still remain as an
important strategy for the commercial agriculture. However, the widespread use of
herbicides has resulted in both point source and non-point source contamination of
shallow groundwater and surface waters, which became a major issue and a serious
environmental problem [1]. Phytoremediation is an alternative technique to effectively
cleanup herbicides from soil and groundwater, since plants possess highly efficient
systems for the removal and transformation of these compounds. Plant-based
remediation can accelerate natural attenuation processes from contaminated sites by
taking up significant quantities of water, and herbicides may be transformed into less
toxic forms.
Some of the most commonly used herbicides in the world can be removed from the
environment using phytoremediation techniques with several plant species. The structure
of the major herbicides are shown in Figure 1.
The use of non-target and tolerant plants, such as trees, shrubs and grasses, have been
proposed for phytoremediation of pesticide-contaminated soil and water [2]. Poplar trees
are able to uptake, hydrolyze, and dealkylate atrazine to less toxic metabolites [3, 4].
The retention ability of pesticides, such as simazine, atrazine (triazines), isoproturon,
linuron (phenylureas), carbaryl (carbamates), fenamiphos (organophosphorus) and
permethrin (pyrethroids) was also evaluated in Lupinus angustifolius seeds, and the
apparent degradation of these pesticides in seeds showed to be promissing in water
contaminated sites [5]. In addition, mixed prairie grasses (big bluestem, yellow
indiangrass, and switchgrass) have been considered as potential tools for
phytoremediation of atrazine, alachlor, metolachlor, and pendimethalin [2, 6]. These
studies have indicated three successful types of plant-based remediation techniques: the
containment of contaminated groundwater plumes by trees; sequestration of pesticides
from water and sediments using aquatic plants; and uptake of pesticides from
contaminated soils by terrestrial plants.

169
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 169 – 178.
© 2006 Springer.
170 Melissa P. Mezzari and Jerald L. Schnoor

Figure 1. Structures of chemicals that are commonly used worldwide as herbicides.

The elucidation and understanding of degradation pathways in plants used for

phytoremediation is of great importance to discern the end point transformation of
herbicides. Mineralization of these chemicals by selected plants and the rhizosphere in
degraded environments would be the final goal for successful phytoremediation
processes.

2. Herbicide transformation pathways in plants

Plant herbicide metabolism is mediated by three groups of enzymes involved in phase I

(conversion), phase II (conjugation) and phase III (compartmentation). Conversion and
transformation reactions are often catalyzed by cytochrome P450 monooxygenases,
where herbicides are converted to more hydrophilic metabolites [7]. Phase II of
xenobiotic metabolism is usually mediated by a sugar, an amino acid, glutathione S-
transferases (GST), or a cellular macromolecule, which generally form polar and water
soluble products. The formation of insoluble, non-extractable bound components of a
variety of toxic exogenous compounds, such as herbicides, is often performed by GSTs
[8]. In Phase III, ATP-dependent membrane pumps recognize the conjugates and
transfer them across membranes, where GST conjugates are either sequestered in the
vacuole or transferred to the apoplast [9].
Numerous studies have contributed to the understanding of enzymes in plants and the
overall plant metabolism towards herbicides. Evolution of GSTs, P450s, and other
enzymes towards herbicide tolerance is mostly related to the complex and versatile
chemistry developed by higher plants to synthesise a large number of natural products. A
more complete picture of the main enzymes and pathways that can be explored for
enhanced transformation processes of herbicides in planta are represented in Table 1.
Metabolism and Genetic Engineering Studies 171

Table 1. Plant enzymes involved on transformation processes of some herbicides commonly present in
surface and ground waters of Europe and North America

Herbicide (Chemical Mode of Action Metabolism in Resistant References

Class) Plants
Atrazine, Simazine Photosynthetic Dechlorination followed [10-13]
(triazines) inhibitors by hydroxylation; N-
dealkylation; GST
conjugation
Acetochlor, Protein and lipid GST conjugation followed [14-16]
Metolachlor synthesis inhibitors by vacuolar
(chloroacetanilides) compartmentation; O-
demethylation by
cytochrome P450s
2,4-Dichlorophenoxyacetic Systemic hormone- Hydroxylation; [17, 18]
acid (phenoxyalkanoic type that causes conjugation with sugars or
acid) abnormal growth and amino acids
development
Glyphosate (unique Inhibition of the Compartmentation to [19-22]
chemical class) biosynthesis of vacuole and organelles or
aromatic compounds; insensitivity of the EPSPS
inhibition of 5- enzyme
enolpyruvylshikimate-
3-phosphate synthase
enzyme (EPSPS)
Chlorotoluron, Photosynthetic N-demethylation by [23-25]
chlorosulfuron, inhibitors P450s and C-
diuron, isoproturon, hydroxylation followed by
linuron (ureas) glucose conjugation

3. Exploitation of plant genes

Herbicides have important characteristics for their removal from the environment or
detoxification during phytoremediation, since they are designed to kill unwanted plants
by blocking vital biochemical and physiological processes [26]. However, weed and
crop resistance to various herbicides have been reported, and the resistance could result
from modification of the target site or by induced expression of genes that can lead to
enhanced metabolism, and alteration in uptake, translocation, and compartmentation of
the herbicide [26]. Plant herbicide tolerance can be achieved or improved by using
metabolic engineering of enzymes. However, genetic engineering has been mostly
focused and applied into crop plants in order to confer herbicide selectivity and enhance
crop safety and production.
Genomic sequencing information from poplar (Populus sp.) has been progressively
developing for gene discovery, expression profiling, genetic mapping, and exploitation
in forest biotechnology and phytoremediation [27]. In addition, genomics of the model
plant Arabidopsis and major agronomic crops, such as corn (Zea mays L.) and rice
(Oryza sativa L.) have provided useful information about the genetic, physiological, and
biochemical mechanisms involved in xenobiotic transformations in plants. The genomic
sequence of Arabidopsis, with an estimate of 25,500 genes in 11,000 gene families, is
widely accepted as a model for detailed functional characterization of plant genes, which
172 Melissa P. Mezzari and Jerald L. Schnoor

comprises the gene families that could be used in phytoremediation applications,

including regulatory networks (e.g. transcription factors) and tissue-specific transporters
[28]. The functional definition of each gene of a gene family involved in xenobiotic
metabolism is now the challenge for phytoremediation applications.
Studies on herbicide biochemical pathways now comprise modern biochemistry with
metabolic processes and other mechanisms present at the cellular and molecular level.
Plant molecular biology has rapidly evolved and several advanced methods are available
for the isolation of a large number of genes that are thought to be involved in xenobiotic
transformation. Genes that express the various enzymes involved in the metabolism of
herbicides can be identified by several available methods such as screening of
complementary DNA (cDNA) using protein-specific antibodies or a polymerase chain
reaction (PCR) based approach [29]. The accelerating determination of genome
sequences and their interpretation (genomics) in the last few years have provided new
methods for phytoremediation approaches such as serial analysis of gene expression
(SAGE), development of genetically modified organisms, and DNA microarrays. These
efficient methods are available to isolate desired genes and to express them in relevant
plant species or lines for phytoremediation, and to improve catalysts for specific
functions. A combination of these tools and approaches provides an enormous resource
for the development of new and more efficient detoxification and/or remediation by
plant enzymes. Thus, the biggest challenge in modern bioscience is to determine the
structure, function and expression of all the corresponding proteins that are encoded in
these genes of interest.

3.1. C-DNA LIBRARIES

Numerous enzyme sequences have been identified in Arabidopsis, but little headway has
been made in matching specific enzymes with their function on herbicide substrates.
Molecular information on enzymes and function identification towards herbicides has
been studied based on cDNA libraries. cDNA libraries are prepared from the mRNA of
interest that is synthesized to DNA (cDNA) strand, which contains a specific coding
region of a genome that is inserted into vectors and cloned [30].
Studies on P450 metabolism of herbicides have reported the use of two yeast-
expressed plant enzymes, CYP73A1 [31] and CYP81B1 [32], in which ring-methyl
hydroxylation of chlortoluron was observed. In both cases, however, the reaction was
extremely slow if compared to a previous investigation, where a very fast N-
demethylation catalysis was observed with CYP76B1 [33]. It was found that CYP76B1
catalyzes the mono- and di-N-demethylation of both chlortoluron and isoproturon in
higher plants, and plays a significant role in the detoxification of most phenylureas,
including the methoxylated forms [33]. Other studies showed that 2,4-D treated S401
tobacco cells could metabolize chlorotoluron using four different P450 enzymes
identified as CYP71A11, CYP81B2, CYP81C1 and CYP81C2 [34]. Because only
CYP71A11 and CYP81B2 were highly induced, both cDNA clones were expressed in
yeast S. cerevisiae, which exhibited enhanced chlorotoluron metabolism [34].
Recent studies have investigated specific GST isoform function in response to
herbicides (2,4-dichlorophenoxyacetic acid and metolachlor) and other chemicals by
overexpressing specific GST cDNAs from plants in E. coli [35]. Results from this
Metabolism and Genetic Engineering Studies 173

research showed that three GST isoform genes (AtGSTF2, AtGSTF6 and AtGSTF8) are
increased in the presence of metolachlor or 2,4-D [35]. Further literature studies with
Arabidopsis plants exposed to chloroacetanilide herbicides have supported the
information about AtGSTF2 induction not only for metolachlor but also for acetochlor
[36]. However, it was also observed that both herbicides induced other GST isoform
genes, such as AtGSTU24 and AtGSTU1 [36]. The expression of Arabidopsis GSTs has
also been studied in response to herbicide safeners – chemicals used to enhance
herbicide tolerance of crop plants by a number of different mechanisms, specially by the
induction of GST activity [37]. Studies from the proteomic analysis of Arabidopsis
GSTs reported an effective strategy for examining the Arabidopsis GST family in
response to the benoxacor safener, where it was observed a 6-fold induction of the gene
AtGSTF9 [38].
Phase III transformation pathway in plants rely on the transport of xenobiotics to the
vacuole and storage in this compartment. The transporters involved in this process are
mainly the ATP-binding cassete (ABC), which contain a cytoplasmic domain (the ABC
protein) that binds and hydrolyzes ATP to energize solute translocation accross the
cytoplasmic membrane [39, 40]. Induction of transport activity in Arabidopsis treated
with herbicides and herbicide safeners has been observed previously, indicating that
several glutathione-conjugate transporters exist [40]. Recent studies have functionally
analyzed a full length cDNA of Arabidopsis coding for a protein (AtMRP3) that exhibits
high homologies to MPR1 and YCF1, two gluthathione-conjugate transporters of
humans and yeast, respectively [40]. It was observed that AtMRP3 was not only a
glutathione-conjugate but also a chlorophyll catabolite transporter [40]. This investi-
gation confirmed the multifunctionality between ABC-transporters in plants and opens
the need for further identification and functional analysis of other members of the MRP
family. Additional studies have provided evidence that the oligopeptide transporter
(OPT) family in Arabidopsis mediate the transport of glutathione derivatives and metal
complexes, thus participation in stress resistance [41]. RNA blots on yeast cell
suspensions and real-time reverse transcription-PCR on Arabidopsis plants indicated that
AtOPT6 expression is strongly induced by the herbicide primisulfuron [41].

3.2. SAGE

Serial analysis of gene expression (SAGE) is a method that allows the quantitative and
simultaneous analysis of a large number of transcripts [42]. The first application of this
technique in higher plants was used for profiling expressed genes in rice (Oryza sativa
L.) seedlings [43]. Recent applications of SAGE technique in plants consist of wood
formation in loblolly pine [44]; gene expression pattern in the crop plant cassava
(Manihot esculenta) from viral disease resistance and susceptible genotypes; and a few
studies of gene expression patterns in Arabidopsis plants [45-49]. Only one study was
directly related to phytoremediation applications, in which genes involved in the
metabolism of TNT were identified in Arabidopsis plants [46]. This study provided
important information for our understanding of the mechanisms that might be involved in
plant root TNT degradation. Therefore, the SAGE technique could strengthen some
existing theories of plant tolerance and metabolism of xenobiotic compounds, including
herbicides.
174 Melissa P. Mezzari and Jerald L. Schnoor

3.3. MICROARRAY

The cDNA microarray has been successfully applied to the simultaneous expression of
many thousands of known and unknown genes, and to large-scale gene discovery, as
well as polymorphism screening and mapping of genomic DNA clones. cDNA
microarrays were first developed for Arabidopsis, in which differential gene expression
was simultaneously assayed in roots and shoots using a microarray of 48 duplicate
cDNA elements [50]. Microarray experiments have been analyzed for plant response to
drought and cold stresses, mechanical wounding and insect feeding, herbivory, nitrate
treatments, and others [51].
Phytoremediation studies using cDNA microarrays are very few and some interesting
articles report on metals [52-54]. A few studies have provided further information on the
expressed genes in plants towards herbicides. GST sequences from soybean (Glycine
max) and maize (Zea mays) were identified, expressed in cDNA libraries and studied in
microarray analysis [55]. These studies have discussed the diversity of GSTs in plants
which has enabled herbicide tolerance in plants. Other studies using a DNA array
(MetArray) investigated stress-induced Arabidopsis in response to herbicides, UV-B
radiation, endogenous stress hormones, and pathogen infection [56]. Arabidopsis
transcriptional reaction was investigated towards sulfonylurea herbicides and
bromoxynil, and results from this study supported the induction of specific cytochrome
P450s, glycosyltransferases, glutathione S-transferases and ABC transporters [56]. The
MetArray is a tool to detect interactions and important overlaps between abiotic and
biotic stress responses, as indicated by the potential mutual interactions of pathogen
defense and response to herbicides.
Populus is an ideal model system among tree species and microarray studies have
revealed the putative functions of several genes involved in wood formation, root
development, and gene expression patterns under different environmental conditions
[57]. A novel investigation has reported the use of Populus as a model tree species to
examine the genes up-regulated by safeners and their role in herbicide metabolism and
the molecular mechanisms of herbicide detoxification [58]. These studies also showed
induction of the enzymes involved in phase I (oxidation), phase II (conjugation)
and phase III (sequestration). A few safener-induced genes that were not previously
reported to be induced by safeners, such as 12-Oxophytodienoate reductase, MtN19
and thioredoxin h, were significantly expressed [58]. The newly identified genes could
have potential for application in genetic engineering of plants for herbicide detoxifi-
cation and tolerance.

3.4. GENETICALLY MODIFIED ORGANISMS

Changes in gene expression can be informative and useful in developing transgenic

plants that respond to contaminants for phytoremediation enhancement. Plant
metabolism rarely mineralizes xenobiotic contaminants and genetic engineering is a
necessary approach to achieve sustainable in situ recycling of contaminants [59].
Genetically modified crops which can tolerate or metabolise herbicides are well known
[7, 60-62]. Engineering of herbicide tolerance in higher plants can result from the
overexpression of an endogenous enzyme, modification of the metabolic activity through
Metabolism and Genetic Engineering Studies 175

introduction and expression of novel genes, or improved physiological capabilities, such

as alterations in uptake and translocation, using classical breeding [7, 59, 63].
A few recent investigations have been developed for the tolerance of herbicides
using transgenic crops [7, 60, 64]. Thus, direct herbicide phytoremediation approaches
using transgenic plants have been reported by overexpressing a bacterial gamma-
glutamylcysteine synthetase in Populus [65]. This study showed that the transgenic
poplar was more tolerant to chloroacetanilide herbicides than the wild-type.
Phytoremediation and genetic engineering are a recent combination of technologies
that have shown, in laboratory experiments, a great potential for the decontamination of
areas with little risk of biomagnification of xenobiotics in the food chain. At this point, a
potential solution for the phytoremediation of contaminated areas could be achieved,
however, engineering plants for herbicide resistance raises an environmental concern
regarding the transfer of foreign genes via pollen dispersion, consequently resulting in
resistant weeds or genetic pollution among other crops [61]. Agrobiotechnology
companies such as Monsanto and Novartis have recognized the risk of horizontal gene
spreading and new technologies are being developed to avoid major environment
impact. Genetic engineering of the chloroplast genome offers a novel way to obtain high
expression without the risk of spreading the transgene via pollen [61, 66, 67]. This
technique relies on the engineering of the maternal chloroplast DNA, which is lost
during pollen maturation and hence is not transmitted to the next generation [68].

4. Conclusions

Understanding transformation pathways and determining the enzymes involved in plant

tolerance to xenobiotics is vital for feasible phytoremediation application. Herbicides are
of major concern due to the worldwide contamination of soil and associated
groundwater. There is a complex array of plant enzymes that are involved in the
detoxification of these compounds, however, the exact number of plant enzymes
potentially involved in the metabolism of herbicides remains unknown. New
biotechnological approaches are now tools for the identification of missing xenobiotic
pathways. The large amount of genomic sequence information presents a challenge not
only to the plant biology community but also to environmental engineers that are
exploring new phytoremediation approaches to reduce human exposure to toxic
pollutants.

References
[1] Suri CR; Raje M and Varshney GC (2002) Immunosensors for pesticide analysis: Antibody production
and sensor development. Crit Rev Biotechnol 22: 15-32
[2] Karthikeyan R; Davis LC; Erickson LE; Al-Khatib K; Kulakow PA; Barnes PL; Hutchinson SL and
Nurzhanova AA (2004) Potential for plant-based remediation of pesticide-contaminated soil and water
using nontarget plants such as trees, shrubs, and grasses. Crit Rev Plant Sci 23: 91-101
[3] Burken JG and Schnoor JL (1997) Uptake and metabolism of atrazine by poplar trees. Environ Sci
Technol 31: 1399-1406
[4] Schnoor JL; Licht LA; McCutcheon SC; Wolfe NL and Carreira LH (1995) Phytoremediation of
Organic and Nutrient Contaminants. Environmental Science and Technology 29: A318-A323
176 Melissa P. Mezzari and Jerald L. Schnoor

[5] Garcinuno RM; Fernandez-Hernando P and Camara C (2003) Evaluation of pesticide uptake by Lupinus
seeds. Water Res 37: 3481-3489
[6] Belden JB; Phillips TA and Coats JR (2004) Effect of prairie grass on the dissipation, movement, and
bioavailability of selected herbicides in prepared soil columns. Environ Toxicol Chem 23: 125-132
[7] Kawahigashi H; Hirose S; Inui H; Ohkawa H and Ohkawa Y (2005) Enhanced herbicide cross-tolerance
in transgenic rice plants co-expressing human CYP1A1, CYP2B6, and CYP2C19. Plant Science 168:
773-781
[8] Dixon DP; Cummins I; Cole DJ and Edwards R (1998) Glutathione-mediated detoxification systems in
plants. Current Opinion in Plant Biology 1: 258-266
[9] Marrs KA (1996) The functions and regulation of glutathione S-transferases in plants. Annual Review of
Plant Physiology and Plant Molecular Biology 47: 127-158
[10] Beynon KI; Stoydin G and Wright AN (1972) A comparison of the breakdown of the triazine herbicides
cyanazine, atrazine and simazine in soils and in maize. Pestic Biochem Physiol 2: 153-161
[11] Deprado R; Romera E and Menendez J (1995) Atrazine Detoxification in Panicum dichotomiflorum and
Target Site Polygonum lapathifolium. Pestic Biochem Physiol 52: 1-11
[12] Hatton PJ; Dixon D; Cole DJ and Edwards R (1996) Glutathione transferase activities and herbicide
selectivity in maize and associated weed species. Pestic Sci 46: 267-275
[13] Schmidt B; Siever M; Thiede B; Breuer J; Malcherek K and Schuphan I (1997) Biotransformation of
ring-U-C-14 atrazine to dealkylated and hydroxylated metabolites in cell-suspension cultures. Weed
Research 37: 401-410
[14] Scarponi L; Perucci P and Martinetti L (1991) Conjugation of 2-Chloroacetanilide Herbicides With
Glutathione - Role of Molecular-Structures and of Glutathione-S-Transferase Enzymes. J Agric Food
Chem 39: 2010-2013
[15] Jablonkai I and Hatzios KK (1993) In-Vitro Conjugation of Chloroacetanilide Herbicides and Atrazine
with Thiols and Contribution of Nonenzymatic Conjugation to Their Glutathione-Mediated Metabolism
in Corn. J Agric Food Chem 41: 1736-1742
[16] Moreland DE; Corbin FT; Fleischmann TJ and McFarland JE (1995) Partial Characterization of
Microsomes Isolated from Mung Bean Cotyledons. Pestic Biochem Physiol 52: 98-108
[17] Scheel D and Sandermann H (1981) Metabolism of 2,4-Dichlorophenoxyacetic Acid in Cell-Suspension
Cultures of Soybean (Glycine max L.) and Wheat (Triticum aestivum L.) .1. General Results. Planta 152:
248-252
[18] Chkanikov DI; Makeyev AM; Pavlova NN; Grygoryeva LV; Dubovoi VP and Klimov OV (1976)
Variety of 2,4-D Metabolic Pathways in Plants - Its Significance in Developing Analytical Methods for
Herbicides Residues. Arch Environ Contam Toxicol 5: 97-103
[19] Hetherington PR; Marshall G; Kirkwood RC and Warner JM (1998) Absorption and efflux of
glyphosate by cell suspensions. J Exp Bot 49: 527-533
[20] Simarmata M; Kaufmann JE and Penner D (2003) Potential basis of glyphosate resistance in California
rigid ryegrass (Lolium rigidum). Weed Sci 51: 678-682
[21] Feng PCC; Tran M; Chiu T; Sammons RD; Heck GR and Jacob CA (2004) Investigations into
glyphosate-resistant horseweed (Conyza canadensis): retention, uptake, translocation, and metabolism.
Weed Sci 52: 498-505
[22] Koger CH and Reddy KN (2005) Role of absorption and translocation in the mechanism of glyphosate
resistance in horseweed (Conyza canadensis). Weed Sci 53: 84-89
[23] Mougin C; Cabanne F and Scalla R (1992) Additional Observations on the Chlorotoluron Hydroxylase
and N-Demethylase Activities in Wheat Microsomes. Plant Physiology and Biochemistry 30: 769-778
[24] Hall LM; Moss SR and Powles SB (1995) Mechanism of resistance to chlorotoluron in two biotypes of
the grass weed Alopecurus myosuroides. Pestic Biochem Physiol 53: 180-192
[25] Glassgen WE; Komossa D; Bohnenkamper O; Haas M; Hertkorn N; May RG; Szymczak W and
Sandermann H (1999) Metabolism of the herbicide isoproturon in wheat and soybean cell suspension
cultures. Pestic Biochem Physiol 63: 97-113
[26] Coleman JOD; Frova C; Schroder P and Tissut M (2002) Exploiting plant metabolism for the
phytoremediation of persistent herbicides. Environmental Science and Pollution Research 9: 18-28
[27] Bhalerao R; Nilsson O and Sandberg G (2003) Out of the woods: forest biotechnology enters the
genomic era. Curr Opin Biotechnol 14: 206-213
Metabolism and Genetic Engineering Studies 177

[28] Kolukisaoglu HU; Bovet L; Klein M; Eggmann T; Geisler M; Wanke D; Martinoia E and Schulz B
(2002) Family business: the multidrug-resistance related protein (MRP) ABC transporter genes in
Arabidopsis thaliana. Planta 216: 107-119
[29] Zhen RG and Singh BK (2001) From inhibitors to target site genes and beyond-herbicidal inhibitors as
powerful tools for functional genomics. Weed Sci 49: 266-272
[30] Lodish H; Berk A; Zipursky LS; Matsudaira P; Baltimore D and Darnell J (2000) Molecular Cell
Biology, vol 2, New York: WH Freeman & Company
[31] Pierrel MA; Batard Y; Kazmaier M; Mignottevieux C; Durst F and Werck-Reichhart D (1994) Catalytic
Properties of the Plant Cytochrome-P450 Cyp73 Expressed in Yeast - Substrate-Specificity of a
Cinnamate Hydroxylase. Eur J Biochem 224: 835-844
[32] Cabello-Hurtado F; Batard Y; Salaun JP; Durst F; Pinot F and Werck-Reichhart D (1998) Cloning,
expression in yeast, and functional characterization of CYP81B1, a plant cytochrome P450 that
catalyzes in-chain hydroxylation of fatty acids. J Biol Chem 273: 7260-7267
[33] Robineau T; Batard Y; Nedelkina S; Cabello-Hurtado F; LeRet M; Sorokine O; Didierjean L and
Werck-Reichhart D (1998) The chemically inducible plant cytochrome P450 CYP76B1 actively
metabolizes phenylureas and other xenobiotics. Plant Physiology 118: 1049-1056
[34] Yamada T; Kambara Y; Imaishi H and Ohkawa H (2000) Molecular cloning of novel cytochrome P450
species induced by chemical treatments in cultured tobacco cells. Pestic Biochem Physiol 68: 11-25
[35] Wagner U; Edwards R; Dixon DP and Mauch F (2002) Probing the diversity of the arabidopsis
glutathione S- transferase gene family. Plant Mol Biol 49: 515-532
[36] Mezzari MP; Walters K; Jelinkova M; Shih MC; Just CL and Schnoor JL (2005) Gene expression and
microscopic analysis of Arabidopsis thaliana exposed to chloroacetanilide herbicides and explosive
compounds: a phytoremediation approach. Plant Physiology. In Press
[37] Davies J and Caseley JC (1999) Herbicide safeners: a review. Pestic Sci 55: 1043-1058
[38] Smith AP; DeRidder BP; Guo WJ; Seeley EH; Regnier FE and Goldsbrough PB (2004) Proteomic
analysis of Arabidopsis glutathione S-transferases from benoxacor- and copper-treated seedlings. J Biol
Chem 279: 26098-26104
[39] Davies TGE and Coleman JOD (2000) The Arabidopsis thaliana ATP-binding cassette proteins: an
emerging superfamily. Plant Cell Environ 23: 431-443
[40] Tommasini R; Vogt E; Schmid J; Fromentau M; Amrhein N and Martinoia E (1997) Differential
expression of genes coding for ABC transporters after treatment of Arabidopsis thaliana with
xenobiotics. FEBS Lett 411: 206-210
[41] Cagnac O; Bourbouloux A; Chakrabarty D; Zhang MY and Delrot S (2004) AtOPT6 transports
glutathione derivatives and is induced by primisulfuron. Plant Physiology 135: 1378-1387
[42] Velculescu VE; Zhang L; Vogelstein B and Kinzler KW (1995) Serial Analysis of Gene-Expression.
Science 270: 484-487
[43] Matsumura H; Nirasawa S and Terauchi R (1999) Transcript profiling in rice (Oryza sativa L.) seedlings
using serial analysis of gene expression (SAGE). Plant J 20: 719-72
[44] Lorenz WW and Dean JFD (2002) SAGE Profiling and demonstration of differential gene expression
along the axial developmental gradient of lignifying xylem in loblolly pine (Pinus taeda). Tree Physiol
22: 301-310
[45] Jung SH; Lee JY and Lee DH (2003). Use of SAGE technology to reveal changes in gene expression in
Arabidopsis leaves undergoing cold stress. Plant Mol Biol 52: 553-567
[46] Ekman DR; Lorenz WW; Przybyla AE; Wolfe NL and Dean JFD (2003) SAGE analysis of
transcriptome responses in Arabidopsis roots exposed to 2,4,6-trinitrotoluene. Plant Physiology 133:
1397-1406
,
[47] Chakravarthy S; Tuori RP; D Ascenzo MD; Fobert PR; Despres C and Martin GB (2003) The tomato
transcription factor Pti4 regulates defense-related gene expression via GCC box and non-GCC box cis
elements. Plant Cell 15: 3033-3050
[48] Fizames C; Munos S; Cazettes C; Nacry P; Boucherez J; Gaymard F; Piquemal D; Delorme V; Commes
TS; Doumas P; Cooke R; Marti J; Sentenac H and Gojon A (2004) The Arabidopsis root transcriptome
by serial analysis of gene expression. Gene identification using the genome sequence. Plant Physiology
134: 67-80
[49] Munos S; Cazettes C; Fizames C; Gaymard F; Tillard P; Lepetit M; Lejay L and Gojon A (2004)
Transcript profiling in the chl1-5 mutant of Arabidopsis reveals a role of the nitrate transporter NRT1.1
in the regulation of another nitrate transporter, NRT2.1. Plant Cell 16: 2433-2447
178 Melissa P. Mezzari and Jerald L. Schnoor

[50] Schena M; Shalon D; Davis RW and Brown PO (1995) Quantitative Monitoring of Gene-Expression
Patterns with a Complementary-DNA Microarray. Science 270: 467-470
[51] Aharoni A and Vorst O (2002) DNA microarrays for functional plant genomics. Plant Mol Biol 48:
99-118
[52] Weber M; Harada E; Vess C; von Roepenack-Lahaye E and Clemens S (2004). Comparative microarray
analysis of Arabidopsis thaliana and Arabidopsis halleri roots identifies nicotianamine synthase, a ZIP
transporter and other genes as potential metal hyperaccumulation factors. Plant J 37: 269-281
[53] Bovet L; Feller U and Martinoia E (2005) Possible involvement of plant ABC transporters in cadmium
detoxification: a cDNA sub-microarray approach. Environ Int 31: 263-267
[54] Kovalchuk I; Titov V; Hohn B and Kovalchuka O (2005) Transcriptome profiling reveals similarities
and differences in plant responses to cadmium and lead. Mutation Research-Fundamental and Molecular
Mechanisms of Mutagenesis 570: 149-161
[55] McGonigle B; Keeler SJ; Lan SMC; Koeppe MK and O’Keefe DP (2000) A genomics approach to the
comprehensive analysis of the glutathione S-transferase gene family in soybean and maize. Plant
Physiology 124: 1105-1120
[56] Glombitza S; Dubuis PH; Thulke O; Welzl G; Bovet L; Gotz M; Affenzeller M; Geist B; Hehn A;
Asnaghi C; Ernst D; Seidlitz HK; Gundlach H; Mayer KF; Martinoia E; Werck-Reichhart D; Mauch F
and Schaffner AR (2004) Crosstalk and differential response to abiotic and biotic stressors reflected at
the transcriptional level of effector genes from secondary metabolism. Plant Mol Biol 54: 817-835
[57] Sterky F; Bhalerao RR; Unneberg P; Segerman B; Nilsson P; Brunner AM; Charbonnel-Campaa L;
Lindvall JJ; Tandre K; Strauss SH; Sundberg B; Gustafsson P; Uhlen M; Bhalerao RP; Nilsson O;
Sandberg G; Karlsson J; Lundeberg J and Jansson S (2004) A Populus EST resource for plant functional
genomics. Proc Natl Acad Sci USA 101: 13951-13956
[58] Rishi AS; Munir S; Kapur V; Nelson ND and Goyal A (2004) Identification and analysis of safener-
inducible expressed sequence tags in Populus using a cDNA microarray. Planta 220: 296-306
[59] McCutcheon SC; Medina VF and Larson SL (2003) Proof of phytoremediation for explosives in water
and soil, in SC McCutcheon and JL Schnoor, Eds., Phytoremediation: transformation and control of
contaminants. Hoboken, New Jersey, John Wiley & Sons, Inc, 429-480
[60] Inui H; Shiota N; Motoi Y; Ido Y; Inoue T; Kodama T; Ohkawa Y and Ohkawa H (2001) Metabolism of
herbicides and other chemicals in human cytochrome P450 species and in transgenic potato plants co-
expressing human CYP1A1, CYP2B6 and CYP2C19. J Pestic Sci 26: 28-40
[61] Daniell H; Khan MS and Allison L (2002) Milestones in chloroplast genetic engineering: an
environmentally friendly era in biotechnology. Trends Plant Sci 7: 84-91
[62] Morant M; Bak S; Moller BL and Werck-Reichhart D (2003) Plant cytochromes P450: tools for
pharmacology, plant protection and phytoremediation. Curr Opin Biotechnol 14: 151-162
[63] Didierjean L; Gondet L; Perkins R; Lau SMC; Schaller H; O’Keefe DP and Werck-Reichhart D (2002)
Engineering herbicide metabolism in tobacco and Arabidopsis with CYP76B1, a cytochrome P450
enzyme from Jerusalem artichoke. Plant Physiology 130: 179-189
[64] Inui H and Ohkawa H (2005) Herbicide resistance in transgenic plants with mammalian P450
monooxygenase genes. Pest Manag Sci 61: 286-291
[65] Gullner G; Komives T and Rennenberg H (2001) Enhanced tolerance of transgenic poplar plants
overexpressing gamma-glutamylcysteine synthetase towards chloroacetanilide herbicides. J Exp Bot 52:
971-979
[66] Daniell H and Dhingra A (2002) Multigene engineering: dawn of an exciting new era in biotechnology.
Curr Opin Biotechnol 13: 136-141
[67] Daniell H (2002) Molecular strategies for gene containment in transgenic crops. Nat Biotechnol 20:
581-586
[68] Daniell H; Wiebe PO and Millan AFS (2001) Antibiotic-free chloroplast genetic engineering - an
environmentally friendly approach. Trends Plant Sci 6: 237-239
PESTICIDES REMOVAL USING PLANTS: PHYTODEGRADATION
VERSUS PHYTOSTIMULATION

JEAN-PAUL SCHWITZGUÉBEL1, JOANA MEYER1

AND PETRA KIDD2
1
Laboratory for Environmental Biotechnology, Station 6, EPFL,
CH-1015 Lausanne, Switzerland, Fax+41216934722, E-mail: jean-
[email protected] 2Soil Biochemistry, IIAG, Consejo
Superior de Investigaciones Científicas (CSIC), Aptdo. 122, E-15780
Santiago de Compostela, Spain, E-mail: [email protected]

1. Introduction

Pesticides are chemicals used for crop protection and pest control, and are probably the
most widely distributed contaminants in the environment over the last century. Although
it is extremely difficult to obtain precise figures concerning their production and use per
country [1], millions of tons of pesticides are produced and spread annually all over the
world. Thousands of different synthetic molecules are used as pesticides: carbamates,
thiocarbamates, dipyridyls, triazines, phenoxyacetates, coumarins, nitrophenols, pyrazoles,
pyrethroids, etc. Most of these chemicals contain chlorine, phosphorus, tin, mercury,
arsenic or copper atoms. Pesticides are divided into different groups according to their
target, e.g. herbicides, against weeds and toxic vegetation; insecticides, against harmful
insects; fungicides, against blights, mildews, mould and rusts; algaecides, for the
sanitary control of lakes, channels, water pools, reservoirs; bactericides, against some
pathogenic microbes; etc.
To be efficient, a pesticide must remain in the appropriate environmental
compartment and be stable enough to act against the target pest for a certain period of
time. However, less than 5% of these products are estimated to reach the target
organism, the remainder being deposited on the soil and nontarget organisms, as well as
moving into the atmosphere and water [2].
Once in the environment, the persistence of a pesticide depends on its chemical
stability, degradability by microorganisms, climatic conditions (influencing pesticide
degradation through soil genesis), soil physicochemical properties (especially amount
and nature of organic matter) and uptake by terrestrial and aquatic species including
plants. The degree of environmental contamination is thus dependent on many factors
and on physicochemical properties of the pesticide: volatility, reactivity, absorption and
adsorption, solubility in water, partition between polar and non-polar phases (log Kow)

179
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 179 – 198.
© 2006 Springer.
180 Jean-Paul Schwitzguébel et al.

and between soil and water (Kd). Depending upon their properties, many pesticides used
in the field end up in surface and groundwater (Figure 1).

Figure 1. Fate of pesticides in the environment.

Figure 2. Factors affecting the formation of metabolites and mineralization of pesticides in

soil, plant and rhizosphere.

Widespread and large-scale use of pesticides during the last decades has led to a global
problem of pollution of soils and water resources. Heavy environmental contamination
by pesticides may arise from industrial point sources such as accidental spillage during
Pesticides Removal Using Plants 181

production, wastewater from pesticide production plants, leakage from old stockpiles,
storage and transport of pesticides, or as leachate from former dumping sites and
municipal waste. The disposal of unwanted or obsolete pesticide stocks has also resulted
in many long-term contaminated sites with very high levels of pesticides. In contrast,
sources of pollution arising from agricultural use are considered to be diffuse as the
compounds are distributed over large areas and at rather low concentrations [3].
Pesticides, like other organic pollutants, are more easily bioavailable in freshly
spiked soils, as compared to aged or long-term contaminated soils [4-6]. The stability
and toxicity of many pesticides make them hazardous when incorporated into the food
chain. There is also increasing concern about their transformation products because
these can be present at higher levels in soil than the parent pesticide itself. In some cases
these products are even more toxic and more mobile, representing a greater risk to the
environment than parent molecules.
A persistent organic pollutant does not undergo biodegradation in certain
environments, whereas a recalcitrant compound resists biodegradation in most
environments studied so far [7]. While partial biodegradation can be achieved by only
one or a few biochemical reactions, total biodegradation involves more extensive
metabolism leading to mineralization. Slow degradation rates often limit the practical
use of microorganisms in remediating contaminated sites. Therefore, pesticides removal
using plants and their interactions with rhizospheric microorganisms appears to be a
promising approach, based on three basic principles (Figure 2):
• the accessibility or bioavailability of the contaminants to the biological
system (plant roots, microorganisms);
• the biochemistry of plants or microbes, which transform the contaminant to
a less toxic product; and
• the possibility of optimising the biological activity to efficiently remove
the organic pollutant [8-12].
As examples of the potential of plants and rhizospheric microorganisms to remove
pesticides from contaminated environment, we shall consider here only two pesticides
with different properties: atrazine (ATR, 2-chloro-4-(ethyl-amino)-6-isopropylamino-s-
triazine) and lindane (1, 2, 3, 4, 5, 6 hexachlorocyclohexane), amenable to different
phytoremediation strategies, phytodegradation and phytostimulation, respectively.

2. Atrazine

Triazines are a large family of herbicides, widely used over the last decades, especially
atrazine, simazine, propazine, prometryn and a few others (Figure 3). Even if the exact
and present figures are not freely available, the annual use of atrazine (ATR) alone has
been estimated to be around 40,000 tons in the USA [13], 5,000 tons in China [14] and
more than 2,000 tons in Europe [15]. Triazines are pre-emergence, selective systemic
herbicides used mainly for the control of annual grasses and broad-leaf weeds in a
variety of cultivated crops, such as maize, sorghum, fruit orchards, sugar cane or cotton.
Once taken up by the roots, triazines are evenly distributed throughout the plant via the
xylem, and act in leaves by inhibiting photosynthesis.
182 Jean-Paul Schwitzguébel et al.

Figure 3. Chemical structure of some triazines (log Kow between 2 and 3).

To be effective, pre-emergence herbicides once applied to the soil, must have an

agronomical remanence of several weeks or months, since they need time to exert their
phytotoxicity and to kill any weeds germinating after their application. Herbicides are
generally retained in the soil by adsorption in the superficial soil horizons, but washing
can occur during the first rain or irrigation event following application. Additional losses
occur through leaching to deeper soil horizons, and washing of soil particles from
unvegetated surfaces. The potential danger of groundwater contamination has been
recognized and it appears that atrazine and simazine application should be avoided in
sandy soils, and should only be used in non-irrigated crops.
Leaching and agricultural runoff are therefore the primary mechanisms by which
herbicides such as atrazine, and some of their degradation products, reach surface water
and groundwater. Once atrazine reaches the surface water system, it is transported
without substantial loss, and ends in lakes, reservoirs and alluvial aquifers, where it
shows minimal loss by volatilization, sorption or transformation. Atrazine, other
triazines and their metabolites are thus frequently detected in groundwater, lakes,
streams and rivers of many countries [16].
Due to its persistence, atrazine is the second most common ground and surface water
contaminant in the USA, and its metabolite deethylatrazine has also been found in
groundwater and soils, sometimes at concentrations greater than that of the parent
compound [17]. Moreover, atrazine is often used in combination with many other
herbicides, such as alachlor, metolachlor, cyanazine, simazine, amitrole + simazine, or
diuron + simazine. In most pesticide-contaminated agrochemical facilities, atrazine is
thus found in combination with other widely used agricultural chemicals. Therefore,
remediation strategies must often cope with a multiple-contaminant situation.

2.1. BIODEGRADATION, BIOREMEDIATION

In alkaline soils, a biological transformation of atrazine takes place, whereas in acidic

soils both chemical and microbial transformations occur. By the action of bacteria,
atrazine is mainly degraded to deethylatrazine (DEA, 6-Chloro-N4-isopropyl-1,3,5-
triazine-2,4-diamine) and deisopropylatrazine (DIA, 6-Chloro-N2-ethyl-1,3,5-triazine-
2,4-diamine) and to a lower extent to didealkylatrazine (DDA). In contrast, a chemical
Pesticides Removal Using Plants 183

dechlorination into hydroxyatrazine occurs in anaerobic environments and acidic soils.

Dealkylates are thus the main metabolites of atrazine found in soils, and since they still
have some herbicidal activity and are mobile compounds, they can also contaminate
aquifers [17, 18].
Predominance of degradation by dealkylation has been shown by Shapir and
Mandelbaum [19]: significant atrazine disappearance is observed in subsurface soil due
to the activity of indigenous microorganisms in the upper part of soil, but only 1%
mineralization occurs. Panshin et al. [20] observed that after atrazine application, DEA
is the dominant degradation product detected the following year. It was shown that
cultivated common soil microorganisms such as Pseudomonas, Nocardia, and
Rhodococcus sp. are able to degrade atrazine, predominantly by dealkylation of the side
chains on the triazine ring.
The limiting factor of atrazine degradation seems to be the absence of efficient
atrazine-mineralizing microorganisms in soils, whereas atrazine-dealkylating bacteria are
active. Although a few bacterial strains (such as the Pseudomonas sp. strain ADP) able
to cleave the triazine ring and mineralize atrazine completely have been isolated and
characterized, and their genetic potential estimated, the majority of bacteria in atrazine-
contaminated fields cannot further metabolize the dealkylated products [21-25]. These
bacteria initiate atrazine degradation with hydrolytic dechlorination, catalysed by ATZA
(atrazine chlorohydrolase), followed by two amidohydrolytic reactions catalysed by
ATZB and ATZC, which altogether transform atrazine to cyanuric acid, which can be
mineralized to CO2 and NH3 by three other hydrolases, ATZD (cyanuric acid
amidohydrolase), ATZE (biuret hydrolase) and ATZF (allophanate hydrolase).
However, if atzA, B and C genes are widely distributed in different bacterial species, the
other genes are less common. Other known genes involved in triazine degradation
include trzD and trzN; trzN encodes the triazine chlorohydrolase, whereas trzD encodes
the cyanuric acid amidohydrolase that catalyses the ring cleavage of cyanuric acid.
Although atrazine has been applied in the field for decades, its persistence in the
environment and the absence of large scale demonstrations of microbial mineralization
indicate the difficulty of rapid microbial breakdown in the field and the rarity of atrazine
mineralizers. Many studies investigating the efficacy of bioremediation have been
carried out on a bench scale and under ideal laboratory conditions. However,
environmental conditions such as soil pH, temperature, nutrient availability and
contaminant bioavailability vary from site to site and greatly affect the bioremediation
process and growth of atrazine-mineralizers. Therefore, the removal of atrazine in
contaminated soils by bioremediation is at present only a dream, and there is still a long
way to go before a bioaugmentation approach can be successfully applied. Other
biological tools should thus be explored, and phytoremediation appears to be the most
promising [26-29].

2.2. PHYTOREMEDIATION

Phytoremediation or the use of vegetation at waste sites or contaminated soils can

overcome limitations of microbial cleanup such as low bacterial population or
inadequate microbial activity. Improving the quality of surface water and reducing
184 Jean-Paul Schwitzguébel et al.

nonpoint source pollution can be achieved using wetland vegetation or vegetative filter
strips to reduce herbicide runoff.
Plants can remediate environments contaminated with organic compounds directly
via root uptake, detoxification by phytotransformation and conjugation with glutathione
or sugars, and subsequent storage of nonphytotoxic metabolites in plant tissues; and/or
indirectly by the release of exudates or enzymes which can enhance degradation by
rhizosphere microorganisms [30, 31].
To be useful for the removal of herbicides from contaminated soil or preventing their
runoff, a plant must first be able to grow in the presence of the target compounds without
being harmed. The plant must not only be resistant to the pollutant, but also be able to
remove it from the environment and to transform it into non-toxic metabolites or end-
products. Differences in the ability of various plant species to accumulate and
metabolize a particular pollutant have been shown, indicating that natural biodiversity
should be better explored and exploited in order to choose the most appropriate plant
species or variety in the development of any phytoremediation process [32]. Plant
taxonomy and phytochemistry can help to use biochemical specificities of plants
producing natural chemicals, whose structures are similar to xenobiotic compounds.
Publications on plant metabolism of herbicides in species useful for phytoremediation
are scarce compared to publications about plant metabolism for agronomic purposes. In
the case of atrazine however, many results obtained in agronomy studies are useful when
choosing the most appropriate families or genera for phytoremediation.
The use of vegetative filter strips is a low cost and practical option for improving the
quality of runoff water from intensively farmed agricultural production areas [33].
Hybrid-poplar buffer strips were first initiated and planted in rows along a portion of a
stream at the end of the 1980’s [34]. Poplar is able to take up atrazine with transpiration
stream, showing that such buffer strips are also effective in removing atrazine from
agricultural percolation and runoff water. The only extensive study of plant metabolism
of atrazine for a phytoremediation purpose is precisely in poplar (Populus deltoides x
nigra), able to transform the herbicide mainly into dealkylates and to a lesser extend,
into polar ammeline [26, 27].
Grasses and semiaquatic plants can also remove nutrients and chemicals; reduce
transport of contaminants like atrazine from runoff water by reducing flow which
promotes deposition of sediment-adsorbed herbicides; and thus allow time for plant
uptake and metabolism or infiltration of pollutants into soils and subsequent degradation
before entering water systems. The use of common cattails (Typha latifolia) to remove
simazine from contaminated water has also been sucessfully tested [31], whereas the
atrazine mineralization potential of wetlands has been shown [35]; and a constructed
wetland is able to treat efficiently atrazine present in nursery irrigation runoff [36].
Anderson and Coats [37] have evaluated the degradation of atrazine in rhizosphere
soil of 15 plant species used for vegetative filter strips. Enhanced mineralization was
found in rhizopshere soil collected from kochia (Kochia acoparia), common
lambsquarters (Chenopodium album), foxtail barley (Hordeum jubatum), witchgrass
(Panicum capillare), catnip (Nepeta cataria) and musk thistle (Carduus nutans). On the
other hand, the efficiency of a natural filter of bluegrass (Poa annua) and fescue
(Festuca sp.) strips located immediately down slope of a standard erosion plot of 9%
Pesticides Removal Using Plants 185

slope has been investigated [28]. Trapping efficiency of atrazine by a 4.5 m wide strip
was 93%, in the same magnitude as dissolved phosphorus, nitrate, ammonium and
sediments. This study emphasises the relevance of grass filters as buffer strips, but the
mechanism underlying atrazine disappearance was not studied.
Decontamination of water polluted with 6 ppm atrazine by several marsh plants,
common club-rush (Schoeplectus lacustris), bulrush (Typha latifolia), yellow iris (Iris
pseudacorus) and common reed (Phragmites australis) was observed and the
disappearance of atrazine was suggested to be due to the action of rhizosphere
microorganisms [38]. The action of plants themselves was not explored, but was not
excluded. Fernandez et al. [39] have also evaluated semiaquatic herbaceous perennial
plants for their use in herbicide phytoremediation, such as canna (Canna generalis),
pickerel (Pontaderia cordata), and iris (Iris x Charjoys Jan), and concluded that these
taxa were not optimal for phytoremedation, since the plants exposed to herbicides
showed significantly reduced biomass.
Phytoremediation can also prevent leaching of contaminants to groundwater from
unplanted fields, after crop harvest [16, 31, 35]. On the other hand, higher rates of
atrazine and simazine removal have been found in soil planted with Pennisetum
clandestinum than in unplanted soil [29]. This could be due to plant uptake, degradation
by enzymes secreted by plant roots, or increased microbial activity in the rhizosphere.

2.3. VETIVER AS A CANDIDATE FOR ATRAZINE PHYTOREMEDIATION

Vetiver (Chrysopogon zizanioides Nash) is a perennial tropical grass, native to India.

Vetiver is by nature a hydrophyte, but often thrives under xerophytic conditions: it
grows particularly well on river-banks and in rich marshy soil. It can withstand periods
of flood, as well as extreme drought, survives temperatures of between –9°C and 45°C,
is fire resistant, and is able to grow in any type of soil regardless of fertility, salinity, or
pH. Vetiver is a tall (up to 3 meters high), fast growing perennial grass with stiff and
tough stems which form a dense hedge with compact rhizome clumps (crown) when
planted closely in rows [40].
The distribution of vetiver is pantropical, and some boundary strips are found in
vetiver’s native region of India. It was introduced recently in Southern regions of
Europe, such as Italy, Portugal and Spain. Non-seeding vetiver plants are used in many
countries for soil erosion control and many other applications: vetiver grass was first
introduced for soil conservation and land stabilization in Fiji in the early 1950s.
Recognizing the potential in combating land degradation, the World Bank has promoted
the vetiver grass system since the mid 1980s, which is now used worldwide as a low-
cost, low-technology and effective means of soil and water conservation and land
stabilization in developing countries. The US Board of Science and Technology for
International Development has reported successful vetiver applications for stabilization
of slopes, terraces and channel banks in numerous tropical and subtropical countries.
Vetiver plantation for soil erosion control is mainly performed linearly, along fields,
terraces, canals, streams, or rivers, where the erosive force of water is at its greatest,
lakeshores, artificial embankments, and little canals for irrigation or water drainage. It
can even be planted across the river itself to slow down the flow of water [41].
186 Jean-Paul Schwitzguébel et al.

As a result of the available literature it was deemed relevant to study vetiver uptake
potential to intercept and remove not only atrazine, but also dealkylates from soil. The
uptake of DEA and DIA was also tested because of their toxicity; relatively high
bioavailability due to low sorption on soil matrix (log Kow 1.38 for DEA, and 1.7 for
DIA); and their frequent occurence in groundwater, suggesting possible penetration and
translocation in plants [42].
The ability of vetiver to take up ATR, DEA and DIA was thus investigated in a
model system: 8-month old vetiver plants cultivated in hydroponics and under sterile and
moderate transpiring conditions [43]. The disappearance of atrazine and dealkylates
from the hydroponic system was dependent on transpiring flux of the plant, showing that
the influence of microorganisms was negligible. This relationship was not linear because
the concentration of the tested compounds was not constant: water was refilled to the
initial level before each sampling. When the herbicide concentration was decreasing in
the medium, a progressively lower quantity of herbicide was absorbed. After 20 days,
uptake of DEA and DIA per liter of transpired water was not significantly different from
that of ATR (Table 1).
Table 1. Herbicide taken up by Vetiver plant per volume of transpired water. Values were
calculated as cumulated quantity of herbicide taken up by the plant per total transpired
water after 20 days exposure to 10 uM atrazine or dealkylates

Compound Herbicide uptake per transpired water

(mmol l–1)
ATR 5.20 ± 0.40
DEA 4.90 ± 0.05
DIA 4.80 ± 0.88

Vetiver could thus take up DEA and DIA in the same range as atrazine. This observation
indicates that DEA and DIA uptake appeared to be largely a passive process closely
associated with the movement of water, as reported by Wilson et al. [44] in Canna
hybrida, by Raveton et al. [45] in Zea mays and Acer platanus protoplasts, and in P.
deltoides x nigra by Burken and Schnoor [26, 27].
Since the uptake of DEA and DIA was passive and the transpiration stream
constantly lowered their concentration in the roots, vetiver caused a global loss of
dealkylates from the medium. The potential of vetiver for control of dealkylates
produced by microorganisms in soil and atrazine runoff is believed to be high, since the
plant was observed to absorb and tolerate atrazine, the first requirement of
phytoremediation. The deep and dense root system of vetiver will physically retard the
runoff of water loaded with atrazine and its primary metabolites, and retain soil and
sediments on which atrazine and metabolites are adsorbed, allowing plant uptake and
phytotransformation to occur, resulting in the removal of atrazine from the environment.
Since its leaf surface area is small compared to phreatophytes, and the highest uptake
of atrazine and dealkylates is dependent on the volume of water transpired, vetiver is not
thought to be very efficient for phytoremediation of highly contaminated soil or water.
However, due to its highly dense root system, vetiver should be an ideal system against
non-point pollution by atrazine and dealkylates. As vetiver is a huge grass, it is expected
that this plant could remove pesticides in the same way as the smaller grasses, festuca
Pesticides Removal Using Plants 187

and poa, in temperate climates. Vetiver is not a crop plant, but has already been shown
to have a wide range of applications; an understanding of the effect of vetiver on
atrazine and dealkylates should open a new application window. In the near future,
vetiver could play an important ecological role for water protection, especially in
developing tropical countries. To our knowledge, most of the plants studied until now
for phytoremediation of atrazine are adapted to temperate climates of developed
countries.
The resistance mechanism of vetiver to atrazine was investigated to further the
assessment of its potential for phytoremediation of atrazine-contaminated environments.
Plants known to metabolise atrazine rely on hydroxylation mediated by benzoxazinones,
conjugation catalyzed by glutathione-S-transferases and dealkylation probably mediated
by cytochromes P450. All three possibilities were thus explored in mature vetiver grown
in hydroponics. Whereas the role of benzoxazinones in the chemical hydroxylation of
atrazine is only marginal [46], conjugation to glutathione was found to play a major role
in the detoxification of atrazine by vetiver [47]. Dealkylates are further conjugated with
glutathione in sorghum. Vetiver, close to sorghum, could also detoxify DEA and DIA by
conjugation. This would be highly beneficial for the environment.
The use of a hydroponic system is the first step towards a comprehensive knowledge
of the fate of pesticides in plants, but it is also a useful tool for the assessment of
phytotreatments of industrial wastewater, agricultural runoff, surface and groundwater
contaminated with pesticides. On the other hand, over-concentration of atrazine was
observed in oil from roots grown in soil, suggesting that during plant ageing, partition
might play a non-negligible role in retaining atrazine from agricultural runoff. Studies
with other pesticides are required to see if vetiver as a tool against pesticide runoff could
be extended to include other contaminants.

3. Lindane

The organochlorine 1, 2, 3, 4, 5, 6 hexachlorocyclohexane (HCH) is an efficient insec-

ticide, available in two formulations: technical-grade HCH (a mixture of different iso-
mers, mainly Į, ȕ, į, and Ȗ-HCH) and lindane (almost pure Ȗ-HCH). The eight possible
isomers differ in the axial or equatorial orientations of the different chlorine atoms
(Figure 4). Typically, the technical mixture consists of 60-70% Į-HCH, 5-12% ȕ-HCH,
10-15% Ȗ-HCH, 6-10% į-HCH and smaller amounts of other isomers. Lindane, the
only isomer with insecticidal properties, is isolated from technical-grade HCH by
crystallization. It is a rather hydrophobic compound, with a log Kow of 3.72.
HCH is toxic and considered as a potential carcinogen, but because of its low-cost
production and its effective pesticide properties, it is ubiquitously used in tropical
countries to reduce vector-transmitted diseases, to protect livestock and to increase
agricultural yields. Global use of lindane and technical HCH are estimated to be as high
as 6 and 11 millions of tons, respectively. Its simple application, efficacy, and economic
return may explain the popularity of this broad-spectrum insecticide, and why it was
produced in large quantities worldwide until the discovery of its toxicity.
Being a persistent organic pollutant, technical-grade HCH can stay as long as 15
years after the last application in the field; HCH is nowadays found all over the world in
188 Jean-Paul Schwitzguébel et al.

air, water and soil samples [1, 48-50]. In addition to agricultural soils, contaminated
sites are found where isomers were disposed of in areas surrounding manufacturing
centres. Remediation strategies are thus urgently needed to remove HCH isomers from
environmental compartments so that they do not end up in food samples through growth
of crop plants.

3.1. BIODEGRADATION, BIOREMEDIATION

The chemical structure and polarity of pesticides affect the solubility, sorption and
volatility properties, and thus influence their transport, persistence and biodegradability.
The ring structure of HCH prevents rotation around the C-C bonds, making vicinal
elimination of chlorines dependent on the presence of axial chlorines oriented opposite
to each other (anti-parallel positions). For example, β-HCH has no such chlorine pairs,
since all chlorine atoms are equatorially oriented, which stabilize the molecule, and it is
thus the most persistent isomer (Figure 4). In contrast, γ-HCH has 3 axial chlorines and
2 chlorine pairs, and α-HCH 4 axial chlorines and 1 chlorine pair, giving these isomers
available sites for enzymatic attack (dehydrohalogenase). The physical properties and
persistence in the environment of the different HCH isomers thus differ because of the
different, axial or equatorial, chlorine orientations. Nevertheless, the four major isomers,
having log Kow between 3.7 and 4.1, are considered as toxic and recalcitrant [50-52].
Even trickier, isomerisation of HCH can occur under both biotic and abiotic conditions.
Bioisomerisation has been observed for bacterial cultures, as well as for bench-scale
studies of sediment and soil slurries. This phenomenon should thus be taken into account
in any bioremediation process, especially if the formation of the more stable β-HCH is
significant.
HCH seems to be biodegradable under both oxic and anoxic conditions, but
mineralization occurs only under oxic conditions. Biodegradation of HCH has been
widely studied at laboratory scale, but information on full scale in situ bioremediation of
industrial sites contaminated by HCH isomers, including lindane, is still very scarce
[53]. Furthermore, the effect of HCH concentration on the biodegradation process is not
yet known, especially in soils, but it has been recently reported that bacterial growth in
liquid culture is decreased above 1 mM lindane and totally inhibited at 2.4 mM [54].

Lindane-degrading microorganisms have been isolated from different contaminated

soils: among others, several Clostridium sp., Pseudomonas and especially
Sphingomonas paucimobilis B90A (or Sphingobium indicum) and UT26 (or
Sphingobium japonicum) [55]. Some of these strains are able to grow on γ-HCH as the
only carbon source [52]. The aerobic degradation of lindane by S. paucimobilis is the
best described and involves several novel enzymes encoded by linA, linB, linC, linD,
linE, linF, and linX genes, leading to a possible mineralization. The LinA enzyme, a γ-
HCH dehydrochlorinase, catalyses the dehydrochlorination of HCH to pentachloro-
cyclohexene, then to 1,3,4,6-tetrachloro-1,4-cyclohexadiene. This metabolite is converted
to 2,4,5-trichlorocyclohexenol, then to 2,5-dichloro-2,5-cyclohexadiene-1,4-diol (2,5-DDOL)
by the LinB protein, a halidohydrolase. LinC, as well as LinX, catalyse the oxidation
Pesticides Removal Using Plants 189

Figure 4. Chemical structures of HCH isomers, showing the equatorial and axial positions
of the chlorine atoms: Alpha = aaaaee; Beta = eeeeee; Gamma = aaaeee; Delta = aeeeee;
Epsilon = aeeaee; Eta = aaeaee; Theta = aeaeee.

of 2,5-DDOL to 2,5-dichlorohydroquinone (2,5-DCHQ) by a NAD+-dependent

dehydrogenase reaction. The linD gene encodes a reductive dehalogenase, catalysing the
rapid conversion of 2,5-DCHQ to chlorohydroquinone, then very slowly to
hydroquinone. The linE gene encodes a dioxygenase able to cleave the aromatic ring of
190 Jean-Paul Schwitzguébel et al.

both chlorohydroquinone and hydroquinone, forming 2-chloromaleylacetate or

maleylacetate (MA), respectively. MA is then reduced to β-ketoadipate by an MA
reductase, the product of gene linF [56]. LinA, linB and linC are genes expressed
constitutively, existing separately from one another on the genome, and belong to the so-
called “upstream pathway”. LinD, linE and linF are inducible genes forming the
“downstream pathway”; linD and linE are organised within an operon with a
transcriptional regulator called LinR, and are induced at 7 mg l–1 γ-HCH, but not at 0.7
mg l–1 [52, 57, 58].
S. paucimobilis is not the only aerobic HCH-degrading bacterium. Escherichia coli
is also able to degrade lindane but only up to 10% level. Another bacterium,
Rhodanobacter lindaniclasticus, degrades lindane with higher removal capacity [52].
Recently, two bacteria able to remove from 45.5% to 90% of lindane within 2 to 8
weeks of incubation were identified as Pandorea sp. [59]. These bacteria can use
lindane as a sole source of carbon and energy. Some cyanobacteria, e.g. Anabaena sp.
strain PCC7120, Nostoc ellipsosporum also possess lindane-degradation capacity [60]. It
should be mentioned however that mineralization of lindane by such microorganisms is
rarely encountered, with the exception of S. paucimobilis strains.
Several anaerobic bacteria, such as Clostridium sphenoides, C. rectum, Citrobacter
freundii and Desulfovibrio sp. have been shown to degrade lindane by reductive
dechlorination, but no mineralization seems to occur [52].
Finally, lignin-degrading fungi like Phanerochaete chrysosporium, Trametes
hirsutus, Cyathus bulleri and several Pleurotus species have been shown to degrade
lindane by extracellular lignin-degrading non-specific peroxidases [52].
Until now however, bioaugmentation with indigenous microorganisms failed to
enhance degradation of either isomer [52]. Survival and activity of the inoculum are not
always guaranteed, but the cultivation of appropriate plant species should increase the
success of such a strategy. Bioremediation of HCH-contaminated soil is probably not
realistic for crop fields where application rates were low, since HCH is expected to be
removed by natural attenuation. In contrast, bioremediation should be appropriate and
useful on industrial post-production or waste dumping sites, and storage sites.

3.2. PHYTOREMEDIATION

The uptake of pesticides by plants depends on the physicochemical properties of the

compound, mode of application, soil type, climatic factors and plant species. Once in the
roots, the chemical may be translocated to shoot via xylem. The permeation from plant
roots to xylem is optimal for moderately hydrophobic molecules, with a log Kow
between 0.5 and 3.5. More hydrophobic chemicals tend to bind with lipid membranes or
oil possibly present in plant roots. Translocation of non-ionic pesticides thus varies
greatly between plant species and depends on the properties of the chemical. Therefore,
the uptake and translocation of hydrophobic compounds (log Kow > 4) is limited, and
consequently so is their phytodegradation. On the other hand, the transformation of
pesticides by rhizosphere microorganisms could result in metabolites more efficiently
absorbed and translocated by plants. Thus any factor enhancing rhizospheric microbial
activity should also increase the overall efficiency of pesticide phytoremediation.
Pesticides Removal Using Plants 191

With regards to HCH isomers, phytoextraction should not be favoured, since they are
hydrophobic chemicals, theoretically bound to soil particles and also to plant roots, thus
preventing plant uptake [61-64]. However, it has been recently shown that the predicted
partition of lindane with lipids using the log Kow is notably lower than the measured
sorption in roots and shoots of ryegrass and wheat seedlings, due to underestimation of
the plant lipid contents and to the fact that octanol is less effective than plant lipids as a
partition medium [65].
Nevertheless, the persistence of all isomers was found to be lower in cropped plots
of maize (Zea mays), wheat (Triticum sp.) or pigeon pea (Cajananus cajan) than in
uncropped plots, therefore sustaining the idea of remediation of polluted sites with plants
[66]. Revision of available literature concerning contamination of the aerial part of
plants reveals that the α, β and γ isomers have been detected in many plants, including
Lactuca sativa (lettuce), Sesamum indicum (sesame), Hydrilla verticillata (hydra),
Lagernia siceraira (bottle gourd), Memordica charantia (bitter gourd), Luffa cylindrical
(sponge gourd), Citrullus varifistulosus (tinda punjab), Spinacia oleracea (spinach) and
Brassica campestris (rape). These species were not selected for testing in hydroponics
however, since the detected residues were due to a direct contact with lindane, and not as
a result of translocation from roots to shoots. Barriada-Pereira et al. [67] also attributed
the presence of lindane in the shoots of Rubus ulmifolius and Paranthropophytia to
atmospheric deposition and not translocation. However, chilli (Capsicum annuum) and
coriander (Coriander sativum) cultivated in lindane-contaminated soil were reported to
contain Ȗ-HCH residues in their aerial parts [43]. This fact was considered as a clue for
the selection of plants able to take up lindane from a hydroponic experimental system.
Within 9 days, lindane concentration in the medium decreased by 70% with chilli and
86% with coriander (Figure 5). Adsorption on roots of chilli and coriander accounted for
29% and 40% of γ-HCH disappearance, respectively. The 23% and 30% remaining loss
was called “plant effect”, which could include the increasing of pH from 5.0 to 6.8,
leading to enhanced hydrolysis, and a possible uptake of γ-HCH in unknown quantities
with the transpiration flux [43, 68]. However, the translocation of lindane from roots to
shoots should be low, due to lindane hydrophobicity, unless special molecules are
produced by plants, able to increase the apparent aqueous solubility of hydrophobic
pollutants, such as those shown by Campanella and Paul [69] for dioxin absorption by
zucchini (Cucurbita pepo L.).
In further experiments, vetiver plants were grown in Hoagland solution with 14C
lindane (2 mg/l). At the end of 30 days, it was observed that 12% of lindane disappeared
from the solution. Since lindane was found to accumulate in vetiver roots where essential
oil is produced, an attempt was made to use a plant, which has oil in the shoot as well.
Lemon grass (Cymbopogan citrates) was thus grown in Hoagland solution for 19 days in
the presence of 14C lindane, but again more 14C residues were found in roots (11.4%)
than in shoots (4.2%). All the plants tested for uptake of 14C lindane, Iris sp., Sesuvium
portulacastrum, Zea mays and Cymbopogan sp. showed that 14C residues were retained
in roots and there was no significant translocation to shoots.
Another study has shown that the concentration of lindane in ryegrass, cultivated in
hydroponics, slowly increases with uptake time to reach a plateau after a few days,
indicating that plant metabolism and formation of bound residues are minimal in such a
192 Jean-Paul Schwitzguébel et al.

system [70]. Other authors [71, 72] aimed to evaluate the bioaccumulation of HCH
isomers in plants growing on areas surrounding a production centre and to investigate

3.0

2.5
γ-HC H [mg/L ]

2.0

1.5

1.0

0.5

0.0
0 3 6 9
Days

Figure 5. Time course experiment of lindane disappearance in solution with chilli and
coriander. Controls (white columns), chilli (black columns), coriander (grey columns).
Data points represent the means of 5 replicates determinations. Bar = SD.

the effect of the rhizosphere on these compounds. Five plant species were harvested and
separated into roots, stems and leaves: Chenopodium vulgare, Solanum nigrum, Cytisus
striatus, Vicia sativa and Avena sativa. Concentrations of total HCH in plant organs
ranged between 1.7 and 62.5 mg kg–1, depending on plant species and organs. Leaves
systematically contained the highest amount of HCH, probably due to the volatilization
of HCH isomers from the soil surface and subsequent sorption by leaves. In one species,
C. striatus, the metabolites pentachlorocyclohexene, cyclopentiltrichloroethene and 6
trichloro, 4-en-hexanoic derivates were also detected in the all plant tissues analysed
[71]. Data obtained from the bulk and rhizosphere soils from C. striatus and A. sativa
suggest that both plant species tend to reduce the levels of all HCH isomers in the
rhizosphere. This could be due to the enhanced biodegradation in the rhizosphere, root
exudation of enzymes able to dechlorinate HCH isomers and/or sequestration by
partitioning into the lipophilic plant tissues or uptake by the roots.
In the case of HCH, phytoremediation has thus shown its own limitations, even in the
favourable case of hydroponic systems, where the pollutant is made highly bioavailable.
Since rhizosphere microbial activity is known to aid the release of bound pesticide
residues in soil, which can in turn enhance uptake and transformation, by plants, a
Pesticides Removal Using Plants 193

combination of bioremediation and phytoremediation, or the phytostimulation of

rhizosphere microorganisms, is likely to be more successful.
Phytostimulation of bacteria present or added in soil seems the most promising
approach to remove lindane from contaminated sites. It has long been known that plants
release a vast range of organic materials through roots into the rhizosphere. These
exudates contain water soluble, insoluble and volatile compounds including sugars,
amino acids, organic acids, flavonones, phenolic compounds and even enzymes [12, 73-
77]. The root exudates can enhance the acquisition of nutrients by plants; stimulate
microbial growth in the rhizosphere; and change pH, water flux and availability of
oxygen. Microorganisms able to use phenolic compounds as a carbon source often have
enzymes that can co-metabolise pollutants with similar structures. Plants have also a
great capacity to release secondary metabolites having a surfactant activity, which is
favourable for phytoremediation purposes. Thus the degradation of several chlorinated
pesticides has been reported to be higher in a vegetated soil than a non-vegetated soil.
For example, the biodegradation of HCH isomers is enhanced in rhizosphere soils of
Kochia sp., as compared to bulk soil, even if the mechanism by which this occurs is not
yet known [78].
A dynamic synergy does exist between plant roots and soil microorganisms. The
microbial activity in the immediate vicinity of the root (rhizosphere) seems to offer a
favourable environment for co-metabolism of soil-bound and recalcitrant chemicals. The
microbial transformations of organic compounds are not always driven by energy needs,
but also by the necessity to reduce toxicity for which microbes may have to suffer an
energy deficit. Thus, the processes may be helped and driven by the abundant energy
provided by root exudates. Certain soil microorganisms can also produce biosurfactant
compounds that may facilitate the removal and degradation of organic chemicals by
increasing their availability to plants. Plants can thus take advantage of an increased
bioavailability of nutrients and degradation of phytotoxic soil contaminants.
A successful example of exploring rhizosphere microorganisms for the
decontamination of pesticide-contaminated soils has been highlighted by the recent
findings of a coordinated Indo-Swiss project [77]. The project was aimed at
investigating possibilities to remediate agricultural soils contaminated with lindane,
using a plant-rhizosphere system. Mineralisation of 14C lindane by rhizospheric soils of
plants growing in lindane-contaminated fields indicated that microorganisms capable of
degrading the insecticide were present. Enrichment culture techniques resulted in the
isolation of bacteria growing in the presence of lindane. Klebsiella sp., Pseudomonas
sp., and Pseudoarthrobacter sp. degraded up to 50% of lindane with the formation of
2,3,4-trichlorobenzene. Root exudates of some plants stimulated the growth of lindane
degrading bacterium Pseudomonas sp., indicating the need for an approach, which
includes both plants and interacting microorganisms for an efficient degradation of
pesticides. To treat lindane-contaminated soils, it thus appears that phytostimulation
would be the most appropriate technique.
Soil composition influences sorption, soil pH, bulk density and water retention, all of
which affecting aeration, nutrient availability and thus bioavailability and
biodegradability of contaminants [61]. A high density of indigenous S. paucimobilis was
found in the plant debris fraction of soil and it was postulated that plant organic matter
194 Jean-Paul Schwitzguébel et al.

integrated into the soil aggregates served as a microhabitat rich in growth substrates.
This can be the basis for the application of plant-derived organic amendments to soil as
a phytostimulation strategy, like rice straw or other cellulosic material, because of their
efficiency, availability in large quantities and low cost [52]. For example, the
Daramend® technology for bioremediation of HCH-contaminated soils is based on the
application of solid plant-derived organic matter providing nutrients and a non-toxic
habitat for indigenous microorganisms; it creates a concentration gradient that facilitates
diffusion of organic contaminants from pockets of higher to lower concentrations on the
amendment surface, where they are more bioavailable [79]. Since the texture, nutrient
requirements and microbial populations of each soil are specific, the usefulness and
composition of amendments should be assessed on a case-by-case basis.
Detailed strategies for optimising treatments on sites contaminated with HCHs
remain to be established and could involve enhanced natural attenuation and opti-
misation of environmental conditions, to stimulate growth and biodegradation by
indigenous microorganisms. Supplemental nutrients and/or organic amendments could
be added to enrich the soil and stimulate the bacteria degrading HCH. Bioaugmentation
with phytostimulation or a vegetative cover should also be tested, either by increasing
the population of microorganisms able to degrade HCH isomers, or by increasing the
bioavailability of the insecticide.

4. Conclusions and perspectives

Both examples developed here show that plants and soil microorganisms have certain
limitations with respect to their individual abilities to remove and degrade organic
pollutants like pesticides, and other molecules containing chlorines and/or aromatic ring
structures. However, plants and bacteria have very specific and complementary
metabolic pathways, and their combined appropriate use can breakdown many man-
made chemicals. Therefore, a synergy between rhizospheric microorganisms leading to
increased availability of hydrophobic compounds and plants leading to their removal
and/or degradation, may overcome many of the limitations, thus providing a sound basis
for enhancing biological remediation of contaminated environments.
Phytostimulation or rhizoremediation is of particular importance because it refers to
an important contribution that microorganisms in the root-zone (rhizosphere) make to
the overall breakdown and removal of organic pollutants by plants. Plant-microbial
interactions in the rhizosphere are thus of utmost importance for the degradation of
recalcitrant chemicals in the environment [12, 30, 80].
However, further research into the mechanisms by which plants can stimulate
biodegradation and the complexity of the soil-plant-microbe system due to its
interwoven nature is thus required to better explore and exploit their huge potential.
Such studies must be done not only at laboratory scale, but also under real conditions, as
demonstration projects, to optimise the phytoremediation process and convince
regulators and the general public of the technique’s feasibility [81, 82]. To increase its
acceptance as a remediation concept, phytoremediation must also become an eco-
nomically interesting approach and biomass disposal or use after the treatment is thus
an important issue to consider. For example, the biomass of fibres, oil or fragrance
Pesticides Removal Using Plants 195

producing plants like vetiver, could be used to recover these added-value products, if
however their level of contamination is nil or low enough. Alternatively, contaminated
biomass could be used for renewable energy generation, either by direct combustion,
gasification or pyrolysis, or indirectly via biogas or biofuel production [83, 84].

References
[1] Courdouan, A; Marcacci, S; Gupta, S and Schwitzguébel, JP (2004) Lindane and technical HCH
residues in Indian soils and sediments – A critical appraisal. Journal of Soils and Sediments 4: 192-196
[2] Pimental, D and Levitan, L (1986) Pesticides: amounts applied and amounts reaching pests. Bioscience
36: 86-91
[3] Chaudhry, Q; Schröder, P; Werck-Reichhart, D; Grajek, W and Marecik, R (2002) Prospects and
limitations of phytoremediation for the removal of persistent pesticides in the environment.
Environmental Science and Pollution Research 9: 4-17
[4] Alexander, M (2000) Aging, bioavailability and overestimation of risk from environmental pollutants.
Environmental Science and Technology 34: 4259-4265
[5] Anhalt, JC; Arthur, EL; Anderson, TA and Coats JR (2000) Degradation of atrazine, metolachlor and
pendimethalin in pesticides-contaminated soils: Effects of aged residues on soil respiration and plant
survival. Journal of Environmental Science and Health B 35: 417-438
[6] Hatzinger, PB and Alexander, M (1995) Effect of aging chemicals in soil on their biodegradability and
extractability. Environmental Science and Technology 29: 537-545
[7] Dua, M; Singh, A; Sethunathan, N and Johri, AK (2002) Biotechnology and bioremediation: successes
and limitations. Applied Microbiology and Biotechnology 59: 143-152
[8] Atterby, H; Smith, N; Chaudhry, Q and Stead, D (2002) Exploiting microbes and plants to clean up
pesticide contaminated environments. Pesticide Outlook 13: 9-13
[9] Davis, LC; Castro-Diaz, S; Zhang, QZ and Erickson, LE (2002) Benefits of vegetation for soils with
organic contaminants. Critical Reviews in Plant Sciences 21: 457-491
[10] Belden, JB; Clark, BW; Phillips, TA; Henderson, KL; Arthur, EL and Coats, JR (2004) Detoxification
of pesticides residues in soil using phytoremediation. Pesticide Decontamination and Detoxification
ACS Symposium Series 863: 155-167
[11] Karthikeyan, R; Davis, LC; Erickson, LE; Al-Khatib, K; Kulakow, PA; Barnes, PL: Hutchinson, SL and
Nurzhanova, AA (2004) Potential for plant-based remediation of pesticides-contaminated soil and water
using nontarget plants such as trees, shrubs and grasses. Critical Reviews in Plant Sciences 23: 91-101
[12] Kuiper, I; Lagendijk, EL; Bloemberg, GV and Lugtenberg, BJJ (2004) Rhizoremediation: a beneficial
plant-microbe interaction. Molecular Plant-Microbe Interactions 17: 6-15
[13] Radosevich, M; Traina, SJ; Hao, YL and Tuovinen, OH (1994) Degradation and mineralization of
atrazine by a soil bacterial isolate. Applied and Environmental Microbiology 61: 297-302
[14] Jin, R and Ke, J (2002) Impact of atrazine disposal on the water resources of the Yang river in
Zhangjiakou area in China. Bulletin of Environmental Contamination and Toxicology 68: 893-900
[15] Vighi, M and Funari, E (1995) Pesticide Risk in Groundwater. Lewis Publishers, Boca Raton, FL, USA,
ISBN 0-87371-439-3, 275 p
[16] Coleman, JOD; Frova, C; Schröder, P and Tissut, M (2002) Exploiting plant metabolism for the
phytoremediation of persistent herbicides. Environmental Science and Pollution Research 9: 18-28
[17] Mersie, W and Seybold, C (1996) Adsorption and desorption of atrazine, deethylatrazine,
deisopropylatrazine and hydroxyatrazine on levy wetland soil. Journal of Agricultural and Food
Chemistry 44: 1925-1929
[18] Qiao, X; Ma, L and Hummel, HE (1996) Persistence of atrazine and occurrence of its primary
metabolites in three soils. Journal of Agricultural and Food Chemistry 44: 2846-2848
[19] Shapir, N and Mandelbaum, RT (1997) Atrazine degradation in subsurface soil by indigenous and
introduced microorganisms. Journal of Agricultural and Food Chemistry 45: 4481-4486
[20] Panshin, SY; Carter, DS and Bayless, RE (2000) Analysis of atrazine and four degradation products in
the pore water of the vadose zone, Central Indiana. Environmental Science and Technology 34: 2131-
2137
196 Jean-Paul Schwitzguébel et al.

[21] Singh, BK; Kuhad, RC; Singh, A; Lal, R and Tripathi, KK (1999) Biochemical and molecular basis of
pesticide degradation by microorganisms. Critical Reviews in Biotechnology 19: 197-225
[22] Piutti, S; Hallet, S; Rousseaux, S; Philippot, L; Soulas, G and Martin-Laurent, F (2002) Accelerated
mineralisation of atrazine in maize rhizosphere soil. Biology and Fertility of Soils 36: 434-441
[23] Rhine, ED; Fuhrmann, JJ and Radosevich, M (2003) Microbial community responses to atrazine
exposure and nutrient availability: linking degradation capacity to community structure. Microbial
Ecology 46: 145-160
[24] Martin-Laurent, F; Cornet, L; Ranjard, L; Lopez-Gutiérrez, JC; Philippot, L; Schwartz, C; Chaussod, R;
Catroux, G and Soulas, G (2004) Estimation of atrazine-degrading genetic potential and activity in three
French agricultural soils. FEMS Microbiology Ecology 48: 425-435
[25] Smith, D; Alvey, S and Crowley, DE (2005) Cooperative catabolic pathways within an atrazine-
degrading enrichment culture isolated from soil. FEMS Microbiology Ecology 53: 265-273
[26] Burken, JG and Schnoor, JL (1996) Phytoremediation: plant uptake of atrazine and role of root exudates.
Journal of Environmental Engineering 122: 958-963
[27] Burken, JG and Schnoor, JL (1997) Uptake and metabolism of atrazine by poplar trees. Environmental
Science and Technology 31: 1399-1406
[28] Barfield, B; Blevins, R; Fogle, A; Madison, C; Inamdar, S; Carey, D and Evangelou, V (1998) Water
quality impacts of natural filter strips. American Society of Agricultural Engineering 41: 371-381
[29] Singh, N; Megharaj, M; Kookana, RS; Naidu, R and Sethunathan, N (2004) Atrazine and simazine
degradation in Pennisetum rhizosphere. Chemosphere 56: 257-263
[30] Van Eerd, LL; Hoagland, RE and Hall, JC (2003) Pesticide metabolism in plants and microorganisms.
Weed Science 51: 472-495
[31] Wilson, PC; Whitwell, T and Klaine, SJ (2000) Metalaxyl and simazine toxicity to and uptake by Typha
latifolia. Archives of Environmental Contamination and Toxicology 39: 282-288
[32] Marcacci, S and Schwitzguébel, JP (2005) Using plant phylogeny to predict detoxification of triazine
herbicides, in Willey, N Ed., Phytoremediation: Methods and Reviews. Humana Press, NJ, USA,
Chapter 20, (in press)
[33] Krutz, LJ; Senseman, A; Zablotowicz, RM and Matocha, MA (2005) Reducing herbicide runoff from
agricultural fields with vegetative filter strips: a review. Weed Science 53: 353-367
[34] Nair, DR; Burken, JG; Licht, LA and Schnoor, JL (1993) Mineralization and uptake of triazine
pesticides in soil-plant systems. Journal of Environmental Engineering 119: 842-854
[35] Anderson, KL; Wheeler, KA; Robinson, JB and Tuovinen, OH (2002) Atrazine mineralization potential
in two wetlands. Water Research 36: 4785-4794
[36] Runes, HB; Jenkins, JJ; Moore, JA; Bottomley, PJ and Wilson, BD (2003) Treatment of atrazine in
nursery irrigation runoff by a constructed wetland. Water Research 37: 539-550
[37] Anderson, TA and Coats, JR (1995) Screening rhizosphere soil samples for the ability to mineralize
elevated concentrations of atrazine and metolachlor. Journal of Environmental Science and Health B 30:
473-484
[38] McKinlay, R and Kasperek, K (1998) Observations on decontamination of herbicide-polluted water by
marsh plant systems. Water Research 33: 505-511
[39] Fernandez, TR; Whitwell, T; Riley, MB and Bernard, CR (1999) Evaluating semi-aquatic herbaceous
perennials for use in herbicide phytoremediation. Journal of American Society of Horticultural Science
124: 539
[40] Bertea, CM and Camusso, W (2002) Vetiveria: anatomy, biochemistry and physiology, in Maffei, M
Ed., Vetiveria. Taylor and Francis, London and New York, ISBN, 0-415-27586-5, pp. 19-43
[41] Truong, P (2002) Vetiver grass technology, in Maffei, M Ed., Vetiveria, Taylor and Francis, London and
New York, ISBN, 0-415-27586-5, pp. 114-132.
[42] Briggs, GC; Bromilow, RH and Evans, AA (1982) Relationships between lipophilicity and root uptake
and translocation of non-ionized chemicals by barley. Pesticide Science 13: 495-504
[43] Marcacci, S (2004) A phytoremediation approach to remove pesticides (atrazine and lindane) from
contaminated environment, PhD thesis Nr 2950, EPFL, Lausanne, Switzerland.
[44] Wilson, PC; Whitwell, T and Klaine, SJ (1999) Phytotoxicity, uptake and distribution of 14C-simazine
in Canna hybrida “Yellow King Humbert”. Environmental Toxicology and Chemistry 18: 1462-1468
[45] Raveton, M; Ravanel, P; Serre, AM; Nurit, F and Tissut, M (1997) Kinetics of uptake and metabolism
of atrazine in model plant system. Pesticide Science 49: 157-163
Pesticides Removal Using Plants 197

[46] Marcacci, S; Raveton, M; Ravanel, P and Schwitzguébel, JP (2005) The possible role of hydroxylation
in the detoxification of atrazine in mature vetiver (Chrysopogon zizanioides Nash) grown in
hydroponics. Zeitschrift für Naturforschung 60c: 427-434
[47] Marcacci, S; Raveton, M; Ravanel, P and Schwitzguébel, JP (2006) Conjugation of atrazine in vetiver
(Chrysopogon zizanioides Nash) grown in hydroponics. Environmental and Experimental Botany (in
press)
[48] Walker, K; Vallero, DA and Lewis, RG (1999) Factors influencing the distribution of lindane and other
hexachlorocyclohexanes in the environment. Environmental Science and Technology 33: 4373-4378
[49] Wania, F; Mackay, D; Li, YF; Bidleman, TF and Strand, A (1999) Global chemical fate of α-
hexachlorocyclohexane. 1. Evaluation of a global distribution model. Environmental Toxicology and
Chemistry 18: 1390-1399
[50] Willett, KL; Ulrich, EM and Hites, RA (1998) Differential toxicity and environmental fates of
hexachlorocyclohexane isomers. Environmental Science and Technology 32: 2197-2207
[51] Deo, PG; Karanth, NG and Karanth, NGK (1994) Biodegradation of hexachlorocyclohexane isomers in
soil and food environment. Critical Reviews in Microbiology 20: 57-78
[52] Phillips, TM; Seech, AG; Lee, H and Trevors, JT (2005) Biodegradation of hexachlorocyclohexane
(HCH) by microorganisms. Biodegradation 16: 363-392
[53] Van Liere, H; Staps, S; Pijls, C., Zwiep, G., Lassche, R. and Langenhoff, A. (2003) Full scale case:
successful in situ bioremediation of a HCH contaminated industrial site in Central Europe (The
Netherlands). Proceedings of the 7th International HCH and Pesticides Forum, Kyiv, Ukraine, 5-7 June
2003, ISBN 966-8187-31-8, pp. 128-132
[54] Pesce, SF and Wunderlin, DA (2004) Biodegradation of lindane by a native bacterial consortium
isolated from contaminated river sediment. International Biodeterioration and Biodegradation 54:
255-260
[55] Pal, R; Bala, S; Dadwahl, M; Kumar, M; Dhingra, G; Prekash, O; Prabagaran, SR; Shivaji, S; Cullum, J;
Holliger, C and Lal, R (2005) The hexachlorocyclohexane-degrading bacterial strains Sphingomonas
paucimobilis B90A, UT26 and Sp. having similar lin genes are three distinct species, Sphingobium
indicum sp. nov.; Sphingobium japonicum sp. nov.; and Sphingobium francense sp. nov. and
reclassification of Sphingomonas chungburkensis as Sphingobium chungbukense comb. nov.
International Journal of Systematic and Evolutionary Microbiology (in press)
[56] Endo, R; Kamakura, M; Miyauchi, K; Fukuda, M; Ohtsubo, Y; Tsuda, M and Nagata, Y (2005)
Identification and characterization of genes involved in the downstream degradation pathway of
γ-hexachlorocyclohexane in Sphingomonas paucimobilis UT26. Journal of Bacteriology 187: 847-853
[57] Miyauchi, K; Lee, HS; Fukuda, M; Takagi, M and Nagata, Y (2002) Cloning and characterization of
linR, involved in regulation of the downstream pathway for γ-hexachlorocyclohexane degradation in
Sphingomonas paucimobilis UT26. Applied and Environmental Microbiology 68: 1803-1807
[58] Suar, M; van der Meer, JR; Lawlor, K; Holliger, C and Lal, R (2004) Dynamics of multiple lin gene
expression in Sphingomonas paucimobilis B90A in response to different hexachlorocyclohexane
isomers. Applied and Environmental Microbiology 70: 6650-6656
[59] Okeke, BC; Siddique, T; Arbestain, MC and Frankenberger, WT (2002) Biodegradation of
γ-hexachlorocyclohexane (Lindane) and α-hexachlorocyclohexane in water and soil slurry by a
Pandoraea species. Journal of Agricultural and Food Chemistry 50: 2548-2555
[60] Kuritz, T (1999) Cyanobacteria as agents for the control of pollution by pesticides and chlorinated
organic compounds. Journal of Applied Microbiology 85: 1865-1925
[61] Agnihotri, NP and Barooah, AK (1994) Bound residues of pesticides in soil and plant – A review.
Journal of Scientific and Industrial Research 53: 850-861
[62] Bromilow, RH and Chamberlain, K (1995) Principles governing uptake and transport of chemicals, in
Trapp, S and McFarlane, JC Eds., Plant Contamination – Modeling and Simulation of Organic Chemical
Processes. Lewis Publishers, Boca Raton, FL, USA, ISBN 1-56670-078-7, pp. 37-68
[63] Sicbaldi, F; Sacchi, GA; Trevisan, M and Del Re, AAM (1997) Root uptake and xylem translocation of
pesticides from different chemical classes. Pesticide Science 50: 111-119
[64] Burken, JG (2003) Uptake and metabolism of organic compounds: green-liver model, in McCutcheon,
SC and Schnoor, JL Eds., Phytoremediation: Transformation and Control of Contaminants. Wiley
Interscience, Hoboken, NJ, USA, ISBN 0-471-39435-1, pp. 59-84
[65] Li, H; Sheng, G; Chiou, CT and Xu, O (2005) Relation of organic contaminant equilibrium sorption and
kinetic uptake in plants. Environmental Science and Technology 39: 4864-4870
198 Jean-Paul Schwitzguébel et al.

[66] Singh, G; Kathpal, T; Spencer, W and Dhankar, J (1991) Dissipation of some organochlorine
insecticides in cropped and uncropped soil. Environmental Pollution 70: 219-239
[67] Barriada-Pereira, M; Concha-Grana, E; González-Castro, MJ; Muniategui-Lorenzo, S; López-Mahía, P;
Prada-Rodríguez, D and Fernández-Fernández, E (2003) Microwave-assisted extraction versus Soxhlet
extraction in the analysis of 21 organochlorine pesticides in plants. Journal of Chromatography A 1008:
115-122
[68] Marcacci, S; Paratte, S and Schwitzguébel, JP (2002) Phytoextraction of lindane by chilli and coriander
in hydroponic system, in T Macek, M Mackova and K Demnerova, Eds, Proceedings of the 12th
International Biodeterioration and Biodegradation Symposium, Prague, Czech Republic, CSBMB
Prague, JPM Tisk, p. 193, ISBN 80-86313-08-5
[69] Campanella, B and Paul, R (2000) Presence, in the rhizosphere and leaf extracts of zucchini (Cucurbita
pepo L.) and melon (Cucumis melo L.), of molecules capable of increasing the apparent aqueous
solubility of hydrophobic pollutants. International Journal of Phytoremediation 2: 145-158
[70] Li, H; Sheng, G; Sheng, W and Xu, O (2002) Uptake of trifluralin and lindane from water by ryegrass.
Chemosphere 48: 335-341
[71] Barriada-Pereira, M; González-Castro, MJ; Muniategui-Lorenzo, S; López-Mahía, P; Prada-Rodríguez,
D and Fernández-Fernández, E (2005) Organochlorine pesticides accumulation and degradation
products in vegetation samples of a contaminated area in Galicia (NW Spain). Chemosphere 58: 1571-
1578
[72] Monterroso, MC; Camps Arbestain, M; Calvelo Pereira, R; Gomez Garrido, B; Lorenzo, SM; Lopez-
Mahia, P; Prada, D and Macias, F (2002) Environmental fate and behavior of HCH isomers in a soil-
plant system in a contaminated site. Organohalogen Compounds 59: 307-310
[73] Fletcher, JS and Hedge, RS (1995) Release of phenols by perennial plant roots and their potential
importance in bioremediation. Chemosphere 31: 3009-3016
[74] Yoshitomi, KJ and Shann, JR (2001) Corn (Zea mays L.): root exudates and their impact on 14C-pyrene
mineralization. Soil Biology and Biochemistry 33: 1769-1776
[75] Singer, AC; Crowley, DE and Thompson, IP (2003) Secondary plant metabolites in phytoremediation
and biotransformation. Trends in Biotechnology 21: 123-130
[76] Valant-Vetschera, KM; Roitman, JN and Wollenweber, E (2003) Chemodiversity of exudates
flavonoids in some members of the Lamiaceae. Biochemical Systematics and Ecology 31: 1279-1289
[77] Chaudhry, Q; Blom-Zandstra, M; Gupta, S and Joner, EJ (2005) Utilising the synergy between plants
and rhizosphere microorganisms to enhance breakdown of organic pollutants in the environment.
Environmental Science and Pollution Research 12: 34-48
[78] Singh, N (2003) Enhanced degradation of hexachlorocyclohexane isomers in rhizosphere soil of Kochia
sp. Bulletin of Environmental Contamination and Toxicology 70: 775-782
[79] Phillips, TM; Seech, AG; Trevors, JT and Piazza, M (2000) Bioremediation of soils containing
hexachlorocyclohexane, in Wickramanayake, GB; Gavaskar, AR; Gibbs, JT and Means JL Eds., Case
Studies in the Remediation of Chlorinated and Recalcitrant Compounds. Battelle Press, Columbus, OH,
USA, pp. 285-292
[80] Schwitzguébel, JP (2001) Hype of hope: the potential of phytoremediation as an emerging green
technology. Remediation 11(4): 63-78
[81] Van der Lelie, D; Schwitzguébel, JP; Glass, DJ; Vangronsveld, J and Baker, A (2001) Assessing
phytoremediation’s progress in the United States and Europe. Environmental Science and Technology
35: 446A-452A
[82] Schwitzguébel, JP; Van der Lelie, D; Baker, A; Glass, DJ and Vangronsveld, J (2002) Phytoremediation:
European and American trends, success, obstacles and needs. Journal of Soils and Sediments 2: 91-99
[83] Singhal, V and Rai, JPN (2003) Biogas production from water hyacinth and channel grass used for
phytoremediation of industrial effluents. Bioresource Technology 86: 221-225
[84] Schwitzguébel, JP (2004) Potential of phytoremediation, an emerging green technology: European
trends and outlook. Proceedings of the Indian National Science Academy B70: 131-152
PHYTOREMEDIATION OF VOLATILE ORGANIC COMPOUNDS

JOEL G. BURKEN AND XINGMAO MA

Department of Civil, Architectural & Environmental Engineering; Butler
Carlton Civil Engineering Hall; University of Missouri-Rolla; Rolla,
Missouri; 65409-0030 USA
Phone 573-341-6547; FAX 573-341-4729; E-mail: [email protected]

1. Introduction

Many volatile organic compounds pose a unique type of threat to our environment,
existing over vast areas in multiple media: water, soil and vapour. Phytoremediation
applications are also unique as they are both highly visible and publicly acceptable and
are also both long-term and low-energy to apply. These attribute pairings are quite
unusual, being mutually exclusive for all other remedial approaches. The other attribute
that is unmatched by any other technology is the ability to concurrently remediate and
ecologically restore the environment. The listed phytoremediation traits do not yet
include: rapid, highly effective, or well understood. Research and engineering findings
covered herein address these concerns and reveal how and why phytoremediation can
offer an unmatched possibilities to treat volatile organic compounds in particular.
Numerous contaminants are classified as ‘volatile organic compounds’ that are
commonly referred to as VOCs. The definition of a VOC is generally a compound that
has a vapour pressure of greater than 0.1 mm of mercury. As this definition is broad and
only considers one chemical property, the term VOC in this work specifically refers to
research performed on chlorinated solvents and a number of petroleum hydrocarbons.
The extensive use as solvents and fuels lead to widespread releases through spills and
leaks in the handling and transport due to the immense volumes. Additionally, in
previous decades these compounds were disposed via methods that are quite improper
in light of today’s knowledge, but at the time simple dumping on unused land or even
dumping out of the back door of a dry cleaner or automechanic shop were common
disposal practices and accepted. Release to the environment occurred in almost all
places where these compounds were used. The inherent physico-chemical properties that
made these compounds so desirable are the same properties that make these compounds
a bane to current efforts to address the earlier releases. The chlorinated solvents are
particularly problematic because these compounds with low reactivity, relatively low
water solubility, dense liquid phases, and high volatility now pose large dissolved
groundwater plumes with overlying vapour contamination of the vadose (unsaturated)
zones, and pools of pure compounds that are difficult to access. Large enduring plumes
of these compounds are difficult to address, even though decades of effort were spent to
remediate contaminated environments.

199
M. Mackova et al. (ed.), Phytoremediation Rhizoremediation, 199– 216.
© 2006 Springer.
200 Joel G. Burken and Xingmao Ma

Current remedial approaches for VOC-contaminated groundwater and unsaturated

zones include: air sparging, vacuum-enhanced recovery, aqueous pump and treat, in situ
chemical reactions (reduction or oxidation), in-situ bioremediation (microbial-based),
permeable reactive barriers, and almost all conceivable combinations of these and even
a few other technologies. Many of these technologies are certainly effective at removing
and/or destroying these compounds, but become cost prohibitive and energy intensive
when considering the scope of many VOC plumes or do not address the long-term and
multimedia problem that the VOCs pose. In addition, many of the existing sites have a
relatively low risk associated, as the most atrocious sites have been addressed through
existing technologies once sites were identified. The resulting legacy is many large,
relatively low risk sites that include multiple contaminated media, but still a treatment is
desired. Phytoremediation can fit this niche, offering a long-term, relatively low cost
treatment option.
Increased understanding of phytoremediation applied to volatile organic compounds
(VOCs) has been developed, showing volatilisation to the atmosphere as a dominant
fate, occurring via diffusion from the trunk/stem/roots and leaves. Other findings
include uptake from the vadose zone profile. These findings generate a new view of
VOC phytoremediation. This new view includes fate considerations, new sampling and
monitoring potential, and evidence that all three media (water, soil, vapour) can be
addressed via phytoremediation. This treatment is unique in that it offers a highly visible
and yet publicly acceptable treatment option and, as shown in this body of work, can
address the multiple-media nature of VOC contaminants. Phytoremediation for VOCs
can not be considered as a quick option, with clearly identified endpoints. Time and
careful consideration of the impacts expected are requirements of phytoremediation.

2. Background

The recent advancements in the field of VOC phytoremediation has come not in
developing the concept. VOC phytoremediation is not conceptually new, as full-scale
applications and pilot-scale testing have been ongoing for roughly a decade. One of the
first large scale applications was planted in 1996 at the J-Field Site at Aberdeen Proving
Ground (APG). The site, planted by Applied Natural Systems®, is also the field site
discussed later in this chapter. A pilot-scale test system initiated at the same time also
revealed tremendous potential, as the control-dosed system showed greater than 90%
sustained reduction in mass [1]. Recent discovery in VOC phytoremediation has
occurred in understanding the fate and unravelling the specific plant-contaminant
interactions that are unique to volatile compounds, many of these interactions were not
considered and certainly not fully understood at the time the application concept was
developed.
Phytoremediation for organic compounds focuses on two main process: uptake and
rhizodegradation. For many organic compounds, the rhizodegradation is a dominant
fate, and overall the in-situ destruction is a desired fate. The specific research findings
and discussion in this chapter focus on the uptake and relate briefly to rhizosphere
degradation, but detailed specifics rhizodegradation are covered in great detail in
separate sections of the compilation.
Phytoremediation of Volatile Organic Compounds 201

When considering vegetative uptake in remediation, there are a number of

subsequent fates, which will be discussed herein; but the uptake and entry into plants is
a universal step to all plant-mediated removal or metabolic degradation. Plant uptake of
organics was one of the initial research topics attracting interested in the
phytoremediation of organics.
Table 1. The Mass Balance of TCE in Phytoremediation Experiments. Quantitative values
presented from published laboratory studies.

Note Rhizodegradation Uptake & Metabolism Residues* Others

Transpiration
1 23% 56%# NDo ND 20%
2 N/A N/A >1.5% (CO2) 3.7% 88.1%
3 N/A N/A >1.5% (CO2) 3.9% 94.4%
4 1.06% 0.083% ND 0.55% 91.6%
5 0.84% 0.084% ND 0.37% 92.9%
6 1.06% 0.027% ND 0.237% 93.0%
7 1.00% ND ND 0.17% 95.4%
8 0.71% ND ND 0.143% 96.57%
9 N/A 63.1+7.4% ND 0.6+0.4% 9.1+2.1%
10 N/A 44.7+10.0% ND 0.7+0.4% 4.4+1.6%
11 N/A 21.4+5.3% N/A 3.4+0.9% 10.8+3.6%
1. Laboratory study with alfalfa [10].
2-3. Data from batch axenic cell culture experiment [6].
4-8. Data from whole plant study with hybrid poplars. [7].
9-10. Data from tomato study with two different doses, 0.58 µCi and 0.15µCi [11].
11. Data from whole plant study with hybrid poplars. [3]
*Residues include both extractable and non-extractable 14C.
#
The percentage was calculated from evapotranspiration (evaporation + transpiration).
N/A = Not Available, ND = Not Detectable.

From the initial research and applications there were inherent disconnects in the research
approach for VOCs in lab studies conducted and in the field scale evaluation. This
disconnect was partly due to the development paths for phytoremediation concepts.
Initial phytoremediation studies were targeting non-volatile compounds, and much of
the research was adapted and developed from existing agrochemical research on the fate
and activity of herbicides and insecticides, inherently non-volatile compounds. The
transport of these compounds followed the transpiration stream from the subsurface and
the resulting fate was largely in the leaf tissues, where the compounds would reside
either intact or as a metabolite that was stored in the leaf tissues. From the earliest
information on plant uptake and volatilisation of VOCs, notation regarding the specific
transport pathway referred to the leaves as the potential source of the VOCs emanating
from the plant tissues [2].
Initial research on phytoremediation of specific VOCs such as trichloroethylene
(TCE) targeted uptake and two specific fates: persistence/degradation in plant tissues
and volatilisation from the leaves [3-7]. These studies all lead to valuable findings
including; overall mass balances showing that the compounds were indeed translocated,
TCE levels in plant tissues, metabolic products and pathways, presence of bound
residues in tissues, and volatilisation to the atmosphere. The relative weighting of each
fate varied considerably in these independent studies, Table 1, which is not surprising as
202 Joel G. Burken and Xingmao Ma

each effort focused upon different aspects of experiment design in complex, whole-plant
systems. To further complicate the dissimilarity, sampling of the field-scale systems that
were already in place revealed an even greater disconnect, showing little agreement with
the laboratory studies [1, 8]. In full-scale and pilot-scale systems TCE was not detected
in high quantities in evapotranspiration from leaves nor was TCE or metabolites found
at levels to explain removal rates anticipated. Metabolism of TCE and other compounds
does occur in plants and the reactions have been outlined. Trichloroacetic acid (TCAA)
has been identified repeatedly as a primary metabolite. Metabolites have been shown to
reach levels in tissues up to 30 mg per kg [1, 8, 9]. Subsequent studies revealed VOC
transport mechanisms and pathways unique to volatile compounds that had not been
considered or evaluated previously. These transport mechanisms and pathways are
discussed in detail herein.

3. VOC: laboratory studies

3.1. UPTAKE AND DIFFUSION FROM PLANT TISSUES

Contaminant presence in plant tissues when dosed in laboratory studies or when

growing over VOC-contaminated groundwater clearly indicated the uptake occurs for a
variety of organic compounds and specifically VOCs [3, 4]. In this line of research a
number of compounds were identified in plant tissues and the volatilisation of benzene,
TCE, nitrobenzene, toluene, ethylbenzene and m-xylene were shown. Later research
revealed that the concentrations in plant tissues were linearly related to the aqueous
exposure concentration in laboratory experiments, Figure 1. Vroblesky et al. [12] had
used this direct concept in field investigation and discovered the presence of a
decreasing concentration gradient with elevation. Volatile compounds were proposed to
escape to the atmosphere directly from the transpiration stream in tree trunks [12]. This
discovery helped to increase attention on vapour phase mechanisms, including diffusion.
Earlier efforts have been presumptuous in that volatile compounds emanated from
leaves, along with the transpired water [2-4, 6, 13]. Once the VOC-specific diffusion
mechanisms were considered and targeted in research, a more thorough understanding
developed.
Detailed research to elucidate VOC transport sought to isolate and quantify the
vapour phase mechanisms, which potentially lead to the observed gradient. An initial
step was to directly isolate the diffusion and volatilisation from the stem of poplar
cuttings growing in contaminated media. The experimental arrangement was developed
to directly capture and measure the flux of chlorinated VOCs in laboratory studies. The
experimental apparatus employed to directly measure the efflux from tissues was termed
a “diffusion trap” and was installed on hybrid poplar cuttings. The cuttings were planted
in 1 litre glass reactors, filled with soil to promote natural rooting condition. The
reactors were watered with a TCE-spiked feed solution. Prior to dosing, the upper
portions of the cuttings were each fitted with 2 of the diffusion traps, which consisted of
a 2.5 cm quartz glass tube around the cutting and sealed with two Teflon lined septa to
seal the trap to the cutting. Gas flow was controlled through the trap via two hypodermic
needles in the septa, and the purged gas phase was passed through an activated carbon
trap to accumulate any TCE diffused through the stem. The diffusion traps were placed
Phytoremediation of Volatile Organic Compounds 203

at two different heights to evaluate the height specific diffusion expected if the gradient
observed was indeed due to diffusion along the transpiration stream. A detailed
schematic of the experimental arrangement has been previously published [15]. The
reactors were dosed in pairs at a variety of concentrations to further evaluate the impact
of concentration. After 28 days of dosing the feeding of TCE was curtailed and clean
water was dosed to the plants to follow the diffusive flux with time. Plants were grown
under controlled conditions in a walk-in fume hood, to eliminate any potential leaks
from the soil compartments, which were sealed, but minimal loss of the highly volatile
TCE was expected and placement in the hood would ensure that any leaked TCE was
rapidly swept away.

18
concentration, Cx (mg/l)
Calculated xylem flow

Cx = 0.26 Ca + 0.7262
15 2
R = 0.9757
12
9
6
3
0
0 10 20 30 40 50 60
Aqueous Feedwater TCE Concentration, Ca (mg/l)

Figure 1. Concentration in xylem flow calculated from headspace sampling plotted versus
the aqueous concentration of TCE in controlled laboratory study. Figure adapted from Ma
and Burken [14].

Findings from the uptake and diffusion experiments confirmed that TCE did directly
volatilise from the cutting, and that the diffusive flux was related to height. Results
shown in Figure 2 reveal a slow increase in diffusive flux over the first 7 to 10 days.
After this period the diffusion from the cuttings remained constant while the TCE feed
was constant. After the TCE was removed from the feedwater, the flux quickly
decreased. In most cases the TCE diffusion was non-detectable after 10 days of feeding
with clean water. The rapid flushing of the TCE from the system indicates that there is
not a high transient storage in the plant tissues and that the transport and residence of the
TCE is minimal in this system. Longer residence would be expected in a full-scale tree,
as will be discussed in later sections on modelling and field scale sampling.
The uptake and diffusion experiment also showed two other relationships. Diffusive
flux was clearly related to height, with the upper trap accumulating less TCE than the
lower trap in all sampling events for all plants. This trend is exhibited clearly in figure 2,
and in all reactor sets [15]. The gradient with height supports that the diffusive loss is a
major fate of TCE translocated from the subsurface, in this experiment and in field
settings. Another clear relationship was observed as the linear correlation of diffusion to
exposure concentration. Linear relationship was noted between the feed concentration
and the mass diffused. In associated hydroponic experiments however, decreased
transpiration due to apparent toxicity at higher exposure concentrations indicated that
204 Joel G. Burken and Xingmao Ma

transpiration rates were also linked, which agrees with accepted theory regarding uptake
and chemical translocation. The apparent toxicity in the hydroponic study occurred at
concentrations of 0.4 mM, which is in the range of observed impacts but below the zero-
growth concentration of 0.9 mM noted by Dietz and Schnoor [16], so decreased
transpiration is not unexpected.

9
Upper Trap, 550 ppm
Diffused TCE mass, mg

8
Lower Trap, 550 ppm
7
Upper Trap, 50 ppm
6
Lower Trap, 50 ppm
5
4
3
2
1
0
0 10 20 30 40 50 60
Time, days

Figure 2. TCE volatilisation from hybrid poplar stems in a controlled laboratory. Diffusion
and volatilisation reached consistent levels within 8 days of dose initiation and levels off
within 10 days following the removal of TCE from the feedwater on day 28. Details of
experimental arrangement appear in Ma and Burken [15]

Related research was also performed for methyl-t-butyl ether (MTBE), presented in
detail elsewhere [17]. MTBE was observed to be taken up and to diffuse from the
transpiration stream of the xylem tissue. Diffusion was also related to height of the dif-
fusion trap on the stem and the dose concentration, Figure 3. The transpiration rate was
also a clear impact on the diffusive flux, as the diffusion normalized with the rate of
water use was shown to be a linear relationship, Figure 3b.
Observing MTBE diffusion and volatilisation processes confirms that the vapour
pressure of the VOCs in question is of significance. Earlier research evaluated vapour
pressure and the Henry’s law constant as the primary properties relating to volatilisation
extent in phytoremediation experiments [4]. MTBE volatilisation suggests that the
vapour pressure correlates better with diffusion and volatilisation from the transpiration
stream than does the Henry’s constant. In previous work the degree of volatilisation was
Phytoremediation of Volatile Organic Compounds 205

20
Upper trap
Total diffused MTBE
16
mass (mg/inch)
Lower trap
12

0
148 296 593 1186
Feedwater concentrations (ppm)
Transpiration Normalized

14
diffusion (ug/d.inch.mL)

12 Upper trap 2
R = 0.97
10 Lower trap
8
2
6 R = 0.97
4
2
0
0 500 1000 1500
Feedwater concentrations (mg/l)

Figure 3a. Total MTBE collection from the vacuum diffusion traps over the 7-day
experiment. The error bars represent the maximum and minimum values of two replicates;
3b. MTBE accumulation in plants normalized for the volume of water transpired. Figure
adapted from Ma and Burken, 2004 [17].

noted to be related to the vapour pressure, although the impact of a compound’s Henry’s
constant, relating activity in air and in water, was not discounted. When considering
MTBE’s vapour pressure of 0.33 atm relative to the empirical relationship previously
proposed, volatilized of MTBE from plant tissues is expected, but it should also be
noted that the Vp for MTBE is higher than the compounds previously evaluated.
Conversely, the low Henry’s law constant for MTBE, KH = 0.02 dimensionless at 25° C,
206 Joel G. Burken and Xingmao Ma

would indicate very limited volatilization could occur if the relationship of volatilization
to Henry’s law constant was valid. In the more recent MTBE research, volatilization
was dominant when compared to the concentration in plant tissues [17]. Therefore,
when considering the potential diffusion and volatilization of organic compounds from
plant tissues, compounds with a higher vapour pressure pose the highest potential for
volatilization.

3.2. CHLORINATED VOC DEGRADATION IN PLANT TISSUES

Plant tissues are also capable of degrading chlorinated solvents. Degradation products of
TCE detected in plant tissues include trichloroethanol, trichloroacetic acid,
dichloroacetic acid and trichloroethanol glycoside [1, 5, 7, 8, 18-21]. These oxidative
metabolites have been identified in controlled cell culture experiments, whole plant
laboratory experiments and full-scale field settings. When evaluating oxidative
metabolite levels in plants, caution and background sampling should take place as
TCAA is also produced in the atmosphere and is a common atmospheric pollutant, and
has been detected in plant tissues world-wide at concentrations up to 300 µg per kg [22,
23].
Degradation pathways for TCE in particular have been evaluated; however no
established pathway is presented to date. Common metabolites listed above strongly
suggest an oxidative pathway. Mammalian and bacterial metabolic pathways have also
shown the metabolites as intermediates. Shang et al. [9] outlined these pathways in
detail, including a number of the enzymes involved. The plant metabolism model posed
for anthropogenic compounds could also explain the metabolite signature that is
common to many experiments and field sites. The metabolic process termed the “Green
liver model” has been supported in numerous studies focusing on contaminant transport.
The main metabolites observed and the production of bound residues does coincide with
the three phase metabolic processes, Table 2.
In this process xenobiotics are initially transformed to intermediates that can be
readily conjugated. The resulting conjugates undergo two potential fates. Some
conjugates are covalently bound into the cell wall via lignification and some are targeted
by transporter enzymes in the vacuole membrane, resulting in removal from the cell
cytosol and any interference with metabolic activities. While not directly identified as
the reactions taking place, the comparison of the commonly identified metabolites of
TCE and the established reactions clearly shows that plant metabolism of TCE can be
explained by the green-liver phases.

4. Diffusion model considerations

Whole-plant and tissue-specific models have been generated and experimentally veri-
fied. A fundamental model was developed, and while the trends in model output
Phytoremediation of Volatile Organic Compounds 207

Table 2. Green-liver model phases with comparison of established reactions to identified

metabolites in TCE phytoremediation studies.

Green Liver Phase Established Reactions (Enzymes) Metabolites noted for TCE
I: Activation or Oxidation or hydroxylation Trichloroacetic acid,
transformation (Cytochrome P-450) or reduction trichloroethanol, dichloroacetic
(dehalogenase) acid
II: Conjugation Glucoside (O- or N- Trichloroethanol glycoside
glucosyltransferase) or Glutathione
(glutathione S-transferase)
III: Sequestration or Glucoside- or Glutathione-conjugate Vacuole-bound xenobiotic-
compartmentalisation transporter enzymes or lignification conjugates and Bound residues

would result in an exponential decrease in VOC concentrations with elevation along the
tree trunk, an explicit solution for the model was not attained. A simplified model was
based on assumptions that the radial diffusion could be estimate as planar diffusion with
some geometric simplifications [24]. Model input values included the concentration in
the xylem flow at ground level, dimensions of the tree stem, the transpiration rate for the
cutting and the height of the diffusion trap used in analysis and data collection. The lone
fitting parameter in the model was D, termed effective diffusivity. The resulting model
generated a plant tissue profile that very closely mimicked the concentration profiles for
TCE measured in the tree tissues for hybrid poplars grown in the laboratory and dosed
with aqueous TCE at various concentrations. The model also generated a profile for
diffusive flux from the tree. The profile closely followed the trend of the data but under-
predicted the flux, by approximately 10-40% [24]. The underestimation is hypothesized
to result three primary causes. One cause is that both dispersion and diffusion result in
VOC transport in the xylem flow As the two processes can not be isolated in live plant
experiments, the term “effective diffusivity” was adopted, as noted above. Another
potential cause has to do with the continuous nature of the model and the discrete nature
of the diffusion traps, which covered a 2.5 cm length of the stem. The measured value
was plotted at the median height of the trap. A third potential cause is the tissue
differentiation of xylem – vascular cambium-phloem. The tissue boundaries that exist in
this region and possible gas phase in the outer periderm can pose a considerable
diffusive resistance, causing lower than predicted diffusive efflux from this simpler
model, which considers only one diffusivity value for all tissues. The deviation in the
data and model is likely a combination of the posed causes. To evaluate the diffusivity
independently, separate experiments are being conducted on individual tissues rather
than actively transpiring, whole plants. Diffusivity values for common contaminants
have been calculated in a number of studies, ranging from tests with dried sections of
xylem tissue to small scale, active plant systems [24-27]. Reported diffusivities range
widely in these studies, due to experimental arrangements and approaches, so care
should be taken in interpreting these results. Tr a pp et al. [28] and Mackay and
Gschwend [26] present predictive relationships. These relationships should be thought
of as “order of magnitude” estimates from chemical and tissue properties, measured
values could stray greatly from these predictions. The complex hetrogeneous nature
of plant tissues likely prohibits one or two variant models from being truely represen-
tative.
208 Joel G. Burken and Xingmao Ma

Overall these modelling approaches applied to whole plant tests reveal that chemical
concentrations in the stems and flux to the atmosphere for trees taking up VOCs can be
modelled considering the processes of translocation in and diffusion through the xylem
tissues. The processes of metabolism, phloem transport and sorption were also
incorporated into the model development discussed above [24]. In solving the model for
this arrangement however, these processes were considered to be in equilibrium
(sorption), at magnitudes that were not significant (metabolism and phloem transport).
These processes are discussed in greater detail later in this chapter.

5. Vapour phase uptake

Uptake of VOCs into plant tissues was clearly displayed in the work outlined above,
however comprehensive understanding of how VOCs enter plant tissues in the
subsurface has had considerable recent discovery and is still not completely understood.
Early work on the uptake of organics targeted herbicide uptake and activity [29], and the
transpiration stream concentration factor (TSCF) concept was developed. The TSCF is
calculated as the aqueous transpiration stream concentration divided by the bulk
aqueous concentration in the root zone. The TSCF therefore range from 0 for a
compound that is not taken up and translocated to 1.0 for a compound that transports
without impediment. The TSCF values for compounds were compared to many
chemical properties, with a bell-shaped curve as a function of log Kow being the most
commonly accepted, and inherent relation to Kow is expected as uptake includes
movement through and past organic phases as part of an aqueous stream. The evaluation
of TSCF values and relationships for VOCs led to widely different values. TSCF values
range from 0.02 to 0.75 for TCE in laboratory studies [3, 6, 7, 9, 19, 30]. The wide
range of values determined for TCE uptake casts doubt on the methods utilized. Recent
research findings suggest that vapour phase transport is a major transport process that
was not fully considered and could explain some of the variation noted.
Vapour phase uptake was identified during tree coring investigation at the New
Haven Missouri Riverfront site. Results showed a disconnect between the tree coring
analysis for tetrachloroethylene (PCE) and the underlying aqueous concentrations [31].
Further investigation of the contaminant distribution at the site revealed that the tree
cores collected at the site were in better agreement with the vadose zone PCE
concentrations. Uptake and transport of contaminants from the vadose zone was
hypothesized to occur and be a dominant process at the field site. Laboratory studies to
investigate the process in controlled setting, clearly showed that PCE is taken up from
the vadose zone [32]. In this study identical PCE mass was introduced either to the
saturated zone or the vadose zone of soil filled reactors planted with hybrid poplar
cuttings. Diffusion from the cuttings was monitored and the tissue concentrations were
determined at the termination of the experiment. The study also revealed that increasing
gas exchange rate in the vadose zone decreased the translocation and volatilization of
PCE. Recent research has shown the processes also apply to a wide variety of VOCs;
including benzene, toluene, xylene, naphthalene TCE and MTBE [33]. These results
suggest that VOCs can not only be taken up from the vadose zone, but also released to
the vadose zone from roots following uptake from a deeper soil profile. This concept,
termed hydraulic lift, is well studied for water as plants can transport water from the
Phytoremediation of Volatile Organic Compounds 209

saturated zone and release to the upper vadose zone when transpiration rates decrease in
dark periods. The same concept can be considered for VOCs. A potential transport can
be explained considering hydraulic transport and the chemical activity of the com-
pounds. VOCs transport into root tissues via two pathways, an advective transport
with the water flow into roots systems and a diffusive transport as chemical activity
in the root reaches equilibrium between the surrounding contaminated soil. Com-
pounds are then transported in the roots, primarily via advection, through a relatively
clean soil profile and the VOCs diffuse from the roots to approach chemical activity
equilibrium.
The proposed vapour phase transport explains data observed in the laboratory
studies conducted and the disparity observed in the early uptake studies to determine the
TSCF values. Diffusion of vapour phase compounds was not considered in the initial
studies conducted regarding TSCF values for volatile compounds. These studies varied
greatly in how the root zone was treated, some being sealed air tight and some being
actively aerated. The definition of TSCF inherently excludes any vapour phase
transport, either to or from root tissues. Therefore, the “TSCF-concept” of should not be
applied for volatile compounds.

6. Field scale: contaminant fate and hydraulic control

Full-scale phytoremediation of VOCs has been active at contaminated sites since 1995
and 1996. In the decade since then, findings at the field sites have varied. In part the
results are sketchy as some processes that are important were not previously considered
and data collection is limited and largely unpublished. The full-scale phytoremediation
site at the APG J-field site in Maryland is among the best characterized and analysed.
Sampling at the site provided some of the first clear delineation of the vapour phase
mechanisms taking place, summarised in Table 2. Sampling of the trees on-site have
shown VOC efflux from the stems and leaves of trees, a decreasing trend in VOC
concentrations in the transpiration stream with height, and a gradient in the radial
direction in the trunks of tree. The gradient of contaminants in the tissue going from the
centre of the trunks to the outer bark, as shown in Figure 4, reveals a clear driving force
for diffusion, and allows estimation of diffusivities in modelling efforts. Using tedlar
bags, VOCs were detected in leaf bags in the canopy, at low levels [8], and diffusion
traps placed on trees verified both TCE and TeCA volatilisation from the trunk via three
different analytic methods; solid phase microextraction (SPME), 2-liter summa canister,
and gas phase purge with collection onto activated carbon or Tenax [34]. The flow-
through purge and trap captured over 100 µg/hour while sampling only a 0.3 m section
of tree trunk [35]. A rapid decrease of VOC concentrations in root tissues was also
shown in the subsurface when root tissues were sampled for TCE and TeCA. Overall
the contaminant fate at the APG site has shown VOCs are largely volatilized to the
atmosphere from plant tissues, but are also subject to degradation in plant tissues and
potentially redistribution into the vadose zone. Hydraulic control of the plume at the
APG J-Field site has also been noted, as discussed below.
While APG is a well-studied site, numerous other sites also have measurements and
estimates for contaminant removal rates and contaminant fate. Volatilisation and
phytodegradation were also shown at Hill Air Force base. The main VOC removal
210 Joel G. Burken and Xingmao Ma

mechanisms was noted to be phytovolatilisation with rate estimates ranging from 2 to 50

grams per tree per year, while metabolism in the plants was also a noted fate with
metabolites measured in some but not all trees sampled [19]. In other full-scale research,
performed at Carswell Airfield, uptake and volatilisation were again noted, but not
quantified. Degradation in the root zone was cited as a primary fate and is discussed
below in the rhizoremediation section [36].
Even at these well-studied sites, quantifiable estimates of remediation rates,
contaminant removal rates and periods for site remediation remain unclear. The the
complexity of water transport between the soil zones (vadose and saturated) and into
plant roots from these zones makes quantification quite difficult water transport alone,
much less the compounding complexity of VOCs being present in three phases. In field
testing, the importance of rainfall or other water sources has also been noted, as
infiltration of rainwater is used preferentially to groundwater, [19, 37]. The source of
transpired water should be carefully considered in field-scale sampling of any potential
phytoremediation mechanism.
At numerous sites, phytoremediation impacts on groundwater levels and control are
undeniable. In pilot scale work, Newman and colleagues revealed the hydraulic impacts
of hybrid poplars when planted cells the pilot scale system were dewatered at the peak
of the growing season, making sampling of groundwater impossible [1]. This work
clearly shows the potential of phytoremediation by presenting a comprehensive water
budget for the pilot scale system. Use and management of contaminated plumes is the
goal of many remediation efforts. At one chlorinated solvents site, Phytokinetics®
designed a phytoremediation application to work in tandem with a pump and treat
system. Trees planted at the site removed an estimated 6.5 x 106 litres over the 2004
growing season, allowing the pumps to run at a much lower capacity [38]. VOC fate
was not analysed.
Plume capture was most clearly shown at the APG J-Field site as work by Schneider
et al. [39] clearly revealed a complete hydraulic gradient reversal through the
progression of the growing season. Hydraulic gradients were not only inward
horizontally from the plume boundaries, but also upward from strata below the
contaminated zone. The hydraulic profile data was clearly response to the phyto-
remediation design on the site as a diurnal fluctuation was observed, with the lowest
hydraulic potential occurring in late afternoon (average at 17:30) and the greatest
potential occurring at daybreak (average at 04:00).
When the hydraulic findings from the APG site are paired with the chemical analysis
performed on site, the results undeniably support VOC phytoremediation with respect to
control of large plumes, Table 3. Methods to evaluate transpiration from phytome-
diation applications and the hydraulic impacts are now becoming established [39, 40]
and further comprehension and proof of hydraulic control will follow.
Phytoremediation of Volatile Organic Compounds 211

1.00
TCE
TeCA
0.75
C/Ccenter

0.50

0.25

0.00
Center Mid Outer

Figure 4. Gradient of 1,1,2,2-tetrachloroethane (TeCA) and trichloroethylene (TCE) in

xylem tissue at the Aberdeen Proving Ground J-Field site, supporting the mechanistic
understanding of the volatilization measured from trees on site.

Table 3. Delineation of processes and hydraulic impacts at the Aberdeen Proving Ground J-
Field site, Edgewood Maryland.

Process Evidence Citation

Uptake and translocation VOCs and metabolites in plant tissues [8, 15, 34]
Plant Metabolism Metabolites (TCAA) in plant tissues [8]
Diffusion to Atmosphere Decreasing VOC profile with height, [15, 34]
measured VOCs in diffusion traps
Subsurface Vapour Exchange Decreasing root VOC concentrations [15, 34]
Hydraulic plume control Reversing hydraulic gradient, diurnal [39, 41]
fluctuations in water levels.

7. VOC rhizodegradation

While major processes are discussed in detail in this work with specific data presented
from the authors’ research, there are other aspects that should be considered in
approaching research or implementation. Degradation of contaminants prior to
contacting the plants is a desirable fate, limiting transfer into above ground tissues and
to the atmosphere. The rhizosphere effect is covered in detail elsewhere in this volume
(Leigh MB). If rhizodegradation is complete, all plant – VOC interactions noted above
are insignificant. In research at Carswell Air Force Base, reductive dechlorination of
chlorinated solvents was noted to occur, preceding plant mediated removal [42]. Under
the phytoremediation plot, ground water oxidation reduction potential was sharply
212 Joel G. Burken and Xingmao Ma

reduced, background electron acceptor pools were depleted and products of reductive
dechlorination (dichloroethylene, chloride ions) were clearly detected. The potential for
plant systems to lead to reducing conditions in the subsurface is established, as plants
can contribute a tremendous flux of readily degradable organic carbon. While the
reductive dechlorination is desirable, the predominant trend is for aerobic conditions to
be enhanced in the rhizosphere through soil dewatering and diurnal fluctuations of the
water table drawing air and oxygen into the subsurface. This trend towards aerobic
conditions is a general statement regarding terrestrial, not wetland, applications of
phytoremediation.
In a number of cases the aerobic conditions can lead to enhanced hydrocarbon
degradation. Degradation of hydrocarbon fuel spills has been noted [43, 44] and can
decrease the role of processes that are directly plant mediated, like uptake and
translocation. While numerous applications of phytoremediation for petroleum are in
place and VOC fractions are likely present, little published work exists on the VOCs in
particular. Increased benzene degradation in planted reactors was clearly noted in a
laboratory study, although the artefacts associated with the laboratory study likely
increase the extent of degradation enhancement [45]. To evaluate the impacts of
terrestrial phytoremediation on hydrocarbon VOCs, more research is needed.

8. Engineering improvements

While understanding the plant-VOC interactions for existing systems is important,

applying this understanding for engineering improvements to phytoremediation and
generating new approaches hold the greatest potential. Engineering can specifically
target the plant or microbial populations involved through genetic engineering or can
target the engineering the plant introduction to specifically target the contaminated
media, improving the plant-contaminant interactions.

8.1. ADVANCING PLANT-CONTAMINANT INTERACTIONS

The upward transport of contaminated groundwater noted at APG is a substantial

finding as phytoremediation is commonly cited to be limited to shallow contamination.
Accessing deep groundwater contamination is the goal of engineered phytoremediation
systems. At the Portsmouth Gas Diffusion Plant, Piketon, Ohio, a phytoremedia-
tion system was designed to target contaminated strata roughly 10 meters below ground
surface. The targeted TCE plume was primarily in the sandy-gravel Gallia aquifer,
overlain with a clay aquitard. Augured holes were placed to connect the contaminated
aquifer and trenches at the ground surface, and the holes were backfilled with sand to
allow a low resistance transport path between the contaminated zone and the trenches,
which were planted with hybrid poplars. Hydraulic impact on the contaminated aquifer
was recorded with pressure transducers. The system was shown to significantly impact
the deep, contaminated Gallia aquifer, revealing a daily piezometric drop of up to 0.4m,
and the daily piezometric drop was correlated to the solar radiation recorded on the site.
Phytoremediation of Volatile Organic Compounds 213

Over the course of one growing season, over 2.1 x 106 litres were extracted as recorded
with sap flow measurements [46]. Results from this site show that once the specific
mechanisms are known, the mechanisms and site specific conditions can be exploited
through engineering to increase the effectiveness and potential for phytoremediation to
treat VOC plumes. Other improvements could be as simple as decreasing infiltration in
contaminated areas, to promote evapotranspiration of contaminated groundwater rather
than clean, infiltrating rainfall.

8.2. MOLECULAR ENGINEERING ADVANCEMENTS.

Concurrently as the understanding of VOC phytoremediation have advanced, molecular

engineering approaches to enhance processes are occurring and innovative applications
to exploit specific plant systems have been designed. One line of molecular engineering
has been to take advantage of the plant-microbe symbiosis. By selecting root-colonizing
bacteria as recipients for enhanced degradation genes, the improved degradation of TCE
has been observed in sealed reactors containing plants inoculated with recombinant
strains as compared to plants alone or plants with the wild type microbes [47-50]. In
these studies, toluene-o-monooxygenase (TOM) was incorporated into the chromosomal
DNA of the wild type bacteria to provide an aerobic degradation pathway for
degradation. The plant-bacterial symbiosis was also observed to depend on the host
plant selection, as wheat and poplar colonizers were not able to thrive in the root zone of
other plant species [48]. While degradation potential was clear, the survival rate of the
inoculants and the residence time of the VOC in the root zone were a concern for the
systems tested.
Another recombinant bacterial approach tested recently targeted endophytic bacteria,
organisms colonizing the interior plant cells, rather than root-colonizers. In work by
Barac and colleagues endophytic bacteria were isolated from different tissues of hybrid
poplars and engineered with a TOM gene [51]. In the inoculated whole plant studies,
toluene degradation was proven to be enhanced and the presence of the recombinant
organisms was show using a fluorescent tag. The engineering of endophytic bacteria has
a clear advantage in terms of survival as competition in the plant interior is greatly
reduced. However the compounds must be able to pass into the plant tissues where the
bacteria colonize. For VOC applications, the xylem tissues are ideal, as compounds
have the longest residence times in the xylem.
Another molecular engineering approach is to target the plant metabolic pathways.
In doing so, the competition of the microbial populations is avoided, and survival and
thriving of the plant is much easier to evaluate and promote. Work on TCE degradation
includes GM plants to express a mammalian cytochrome P450 gene with high activity
for TCE [52]. Enhanced TCE degradation and maintenance of the gene was shown to
occur. Although not targeting VOC in the particular tests, rice has been successfully
modified to express a human P450 CYP2B6 [53]. The degradation potential for GM
plants is literally bounded only by the known, identified degradation genes and the ability
to integrate these genes into plants. However the compounds of interest, in the same
aspect of the endophytic approach, must pass into the tissues where the genes are
expressed and have a residence time that allows for degradation.
214 Joel G. Burken and Xingmao Ma

9. Summary

Expansive plumes associated with many volatile contaminants make phytoremediation

an attractive approach. The current state of understanding shows that certain compounds
can be removed from the subsurface through plant uptake, which includes uptake from
both the saturated zone and the unsaturated vadose zone. Vadose zone uptake may be
useful in efforts to address vapour transport and vapour intrusion concerns. In targeting
dissolved contaminants, understanding hydraulic aspects of phytoremediation reveal
that engineering can target groundwater and can clearly increase phytoremediation
impacts on groundwater plumes, including accessing deeper groundwater profiles.
Following uptake, volatilization to the atmosphere is a major fate and occurs from the
transpiration stream directly as well as from leaf tissues. Phytodegradation within the
leaf tissues is significant for the contaminants reaching the leaves, with trichloroacetic
acid being a most common metabolite identified in TCE applications.
The complexity of site hydrogeologic conditions, contaminant distribution between
media (vapor, aqueous or sorbed) and in different zones (vadose or saturated), along with
variable rooting patterns of plants makes quantification of remediation at a larger site
very difficult. Improved quantifiable estimates are being developed using recent VOC
fate measurements and advancements in hydraulic understanding of phytoremediation
applications.

References
[1] Newman, LA; Wang, XP; Muiznieks, IA; Ekuan, G; Ruszaj, M; Cortellucci, R; Domroes, D; Karscig,
G; Newman, T; Crampton, RS; Hashmonay, RA and Yost, MG (1999) Remediation of
trichloroethylene in an artificial aquifer with trees: A controlled field study. Environ Sci Technol
33(13): 2257-2265
[2] McFarlane, JC; Pfleeger, T and Fletcher, J (1987) Transpiration Effect on the Uptake and Distribution
of Bromacil, Nitrobenzene, and Phenol in Soybean Plants. J Environ Qual 16(4): 372-376
[3] Burken, JG and Schnoor, JL (1998) Predictive Relationships for Uptake of Organic Contaminants by
Hybrid Poplar Trees. Environ Sci Technol 32(21): 3379-3385
[4] Burken, JG and Schnoor, JL (1999) Distribution and volatilization of organic contaminants following
uptake by hybrid poplar trees. Int J Phytoremediation 1(2): 139-152
[5] Gordon, M; Choe, N; Duffy, J; Ekuan, G; Heilman, P; Muiznieks, I; Ruszaj, M; Shurtleff, BB; Strand,
S; Wilmoth, J and Newman, L (1998) Phytoremediation of Trichloroethylene with Hybrid Poplars.
Environ Health Perspect 106(Aug): 1001-1004
[6] Newman, LA; Strand, SE; Choe, N; Duffy, J; Ekuan, G; Ruszaj, M; Shurtleff, BB; Wilmoth, J;
Heilman, PE and Gordon, MP (1997) Uptake and Biotransformation of Trichloroethylene by Hybrid
Poplars. Environ Sci Technol 31(4): 1062-1067
[7] Orchard, BJ; Doucette, WJ; Chard, JK and Bugbee, B (2000) Uptake of trichloroethylene by hybrid
poplar trees grown hydroponically in flow-through plant growth chambers. Environ Toxicol Chem
19(4): 895-903
[8] Compton, HR; Haroski, DM; Hirsch, SR and Wrobel, JG (1998) Pilot-Scale Use of Trees to Address
VOC Contamination, in GB Wickramanayake and RE Hinchee, Eds., International Conference on
Remediation of Chlorinated and Recalcitrant Compounds, Battelle Press: Monterey, CA. p. 245-250
ISBN 1-57477-059-4
[9] Shang, TQ; Newman, LA and Gordon, MP (2003) Fate of Trichloroethylene in Terrestrial Plants, in
SC McCutcheon and JL Schnoor, Eds., Phytoremediation: Transformation and Control of Contami-
nants, John Wiley and Sons Inc.: Hoboken, New Jersey. p. 529-561. ISBN 0-471-39435-1
[10] Narayanan, M; Davis, LC and Erickson, LE (1995) Fate of Volatile Chlorinated Organic Compounds
in a Laboratory Chamber with Alfalfa Plants. Environ Sci Technol 29(9): 2437-2444
Phytoremediation of Volatile Organic Compounds 215

[11] Schnabel, WE; Dietz, AC; Burken, JG; Schnoor, JL and Alvarez, PJ (1997) Uptake and
Transformation of Trichloroethylene by Edible Garden Plants. Water Res 31(4): 816-824
[12] Vroblesky, DA; Neitch, CT and Morris, JT (1999) Chlorinated Ethenes from Groundwater in Tree
Trunks. Environ Sci Technol 33(1): 510-515
[13] McFarlane, JC; Pfleeger, T and Fletcher, JS (1990) Effect, Uptake and Disposition of Nitrobenzene in
Several Terrestrial Plants. Environ Toxicol Chem 9: 513-520
[14] Ma, X and Burken, JG (2002) VOCs Fate and Partitioning in Vegetation: Use of Tree Cores in
Groundwater Analysis. Environ Sci Technol 36(21): 4663-4668
[15] Ma, X and Burken, JG (2003) Diffusion of TCE to the Atmosphere in Phytoremediation Applications.
Environ Sci Technol 37(11): 2534-2539
[16] Dietz, AC and Schnoor, JL (2001) Phytotoxicity of Chlorinated Aliphatics to Hybrid Poplar.
Environmental Toxicology and Chemistry 20(2): 389-393
[17] Ma, X; Richter, A; Burken, JG and Albers, S (2004) Phytoremediation of MTBE with Hybrid Poplar
Trees. Int J Phytoremediation 6(2): 157-167
[18] Doucette, W; Bugbee, B; Hayhurst, S; Plaehn, W; Downey, D; Taffinder, S and Edwards, R (1998)
Phytoremediation of Dissolved-Phase Trichloroethylene Using Mature Vegatation, in GB
Wickramanayake and RE Hinchee, Eds., The First International Conference on Remediation of
Chlorinated and Recalcitrant Compounds, Battelle Press: Columbus Ohio. p. 251-256. ISBN 1-57477-
059-4
[19] Doucette, WJ; Bugbee, BG; Smith, SC; Pajak, CJ and Ginn, JS (2003) Uptake, Metabolism, and
Phytovolatilization of Trichloroethylene by Indigenous Vegetation: Impact of Precipitation, in SC
McCutcheon and JL Schnoor, Eds., Phytoremediation: Transformation and Control of Contaminants,
John Wiley and Sons Inc.: Hoboken, New Jersey. p. 561-588. ISBN 0-471-39435-1
[20] Nzengung, VA and Jeffers, P (2001) Sequestration, Phytoreduction, and Phytooxidation of
Halogenated Organic Chemicals by Aquatic and Terrestrial Plants. Int J Phytoremediation 3(1): 13-40
[21] Shang, TQ and Gordon, MP (2002) Transformation of [C-14] Trichloroethylene by Poplar Suspension
Cells. Chemosphere 47(9): 957-962
[22] Forczek, ST; UhlíĜová, H; Gryndler, M; Albrechtová, J; Fuksová, K; Vágner, M; Schröder, P and
Matucha, M (2004) Trichloroacetic Acid In Norway Spruce/Soil-System. II. Degradation in the Plant.
Chemosphere 56: 327-333
[23] Chlor, E (2000) Trichloroacetic Acid in the Environment. http://www.eurochlor.org/chlorine/publica
tions/publications.htm: Brussels, Belgium. p. 46
[24] Ma, X and Burken, JG (2004) Modeling of TCE Diffusion to the Atmosphere and Distribution in Plant
Stems. Environ Sci Technol 38(17): 4580-4586
[25] Trapp, S; Miglioranza, K and Mosebek, H (2001) Sorption of Lipophilic Organic Compounds to Wood
and Implications for Their Environmental Fate. Environ Sci Technol 35: 1561-1566
[26] Mackay, AA and Gschwend, PM (2000) Sorption of Monoaromatic Hydrocarbons to Wood. Environ
Sci Technol 34(5): 839-845
[27] Davis, LC; Zhang, Q and Erickson, LE (2002) Benefits of Vegetation for Soils with Organic
Contaminants. Critical Reviews in Plant Sciences 21(5): 457-491
[28] Trapp, S and Karlson, U (2001) Aspects of Phytoremediation of Organic Pollutants. J Soil and
Sediments 1: 37-43.
[29] Shone, MGT and Wood, AV (1972) Factors Affecting Absorbtion and Translocation of Simazine by
Barley. J Exp Bot 23(74): 141-51
[30] Davis, LC; Vanderhoff, S; Dana, J; Selk, K; Smith, K; Golpen, B and Erickson, LE (1998) Movement
of Chlorinated Solvents and Other Volatile Organics Through Plants Monitored by Fourier Transform
Infrared (FT-IR) Spectrometry. J Hazardous Waste 1-4: 1-19
[31] Schumacher, JG; Struckhoff, GC and Burken, JG (2004) Assessment of Subsurface Chlorinated
Solvent Contamination Using Tree Cores at the Front Street Site and Former Dry Cleaning Facility at
the Riverfront Superfund Site, New Haven Missouri, 1999-2003. US Geological Survey. p. 35
[32] Struckhoff, G; Burken, JG and Schumacher, J (2005) Phytoremediation of Vadose Zone VOCs.
Environ Sci Technol 39(6): 1563-1568
[33] Breite, SR (2005) Uptake and Transport of Vapor Phase VOCs in Phytoremediation, in Civil
Architectural and Environmental Engineering. University of Missouri Rolla: Rolla, MO
[34] Burken, JG and Ma, X (2002) Chlorinated Solvents Phytoremediation: Uptake and Diffusion, in AR
Gavakar and ASC Chen, Eds., Remediation of Chlorinated and Recalcitrant Compounds, Battelle
Press: Columbus, OH, p 2B-24 ISBN 1-57477-132-9
216 Joel G. Burken and Xingmao Ma

[35] Burken, J (2005) Phytoremediation of VOC’s: Understanding Fate and Innovative Applications, in
Third International Phytotechnologies Conference. 2005. Atlanta, GA, USA: http://www.cluin.org/phyt
oconf/
[36] Eberts, SM; Harvey, GJ; Jones, SA and Beckman, SW (2003) Multiple Process Assessment for a
Chlorinated-Solvent Plume, in SC McCutcheon and JL Schnoor, Eds., Phytoremediation:
Transformation and Control of Contaminants, John Wiley and Sons Inc.: Hoboken, New Jersey, p.
589-633 ISBN 0-471-39435-1
[37] Vroblesky, DA; Clinton, BD; Vose, JM; Casey, CC; Harvey, GJ and Bradley, PM (2004) Groundwater
Chlorinated Ethenes, In Tree Trunks: Case Studies, Influence of Recharge and Potential Degradation
Mechanism. Ground Water Monit Remediation 24(3): 124-139
[38] Ferro, AM; Zollinger, N and Thompson, B (2005) Groundwater Phytoremediation System
Performance at the SRSNE Superfund Site, in Third International Phytotechnologies Conference.
2005. Atlanta, GA, USA: http://www.cluin.org/phytoconf/
[39] Schneider, WH; Hirsh, SR; Compton, HR; Burgess, AE and Wrobel, JG (2002) Analysis of Hydrologic
Data to Evaluate Phytoremediation System Performance, in AR Gavakar and ASC Chen, Eds.,
Remediation of Chlorinated and Recalcitrant Compounds, Battelle Press: Columbus, OH.
[40] Vose, JM; Harvey, GJ; Elliot, KJ and Clinton, BD (2003) Measuring and Modeling Tree and Stand
Level Transpiration, in SC McCutcheon and JL Schnoor, Eds., Phytoremediation: Transformation and
Control of Contaminants, John Wiley and Sons Inc.: Hoboken, New Jersey. p. 263-282. 0-471-39435-1
[41] Schneider, WH; Wrobel, JG; Hirsh, SR; Compton, HR and Haroski, DM (2000) The influence of an
integrated remedial system on groundwater hydrology, in GEA Wickramanayake, Ed., Battelle
International Conference on Remediation of Chlorinated and Recalcitrant Compounds, Battelle Press:
Columbus, OH, p. 477-484 ISBN 1-57477-094-2
[42] Godsy, E; Warren, E and Paganelli, V (2003) The role of microbial reductive dechlorination of TCE at
a phytoremediation site. Int J Phytoremediation 5(1): 73-87
[43] Carman, E; Crossman, T and Gatliff, E (1998) Phytoremediation of No. 2 Fuel-Oil Contaminated Soil.
J Soil Contam 7(4): 455-466
[44] Wiltse, C; Rooney, W; Schwab, A and Banks, M (1998) Greenhouse Evaluation of Agronomic and
Crude Oil-Phytoremediation Potential among Alfalfa Genotypes. J Environ Qual 27:169-173
[45] Burken, JG; Ross, C; Harrison, LM; Marsh, A; Zetterstrom, L and Gibbons, JS (2001) Benzene
Toxicity and Removal, in Laboratory Phytoremediation Studies, ASCE Practice Periodical of
Hazardous, Toxic, and Radioactive Waste Management 5(3): 161-171
[46] Sokol, J; Rieske, D; Ritchey, J; Childers, S and Galanti, M (2005) Measuring Effectiveness of
Phytoremediation For a TCE-Contaminated Groundwater Plume Using Sap Flow Instrumentation, in
2005 Third International Phytotechnologies Conference. 2005. Atlanta, GA, USA: http://www.cluin.org/p
hytoconf/
[47] Wood, TK; Shim, H; Ryoo, D; Burken, JG and Gibbons, JS (200) Root colonizing genetically
engineered bacteria for trichloroethylene phytoremediation, in Battelle International Conference on
Remediation of Chlorinated and Recalcitrant Compounds. 2000. Monterey, CA
[48] Shim, H; Chauhan, S; Ryoo, D; Bowers, K; Thomas, S; Canada, K; Burken, J and Wood, T (2000)
Rhizoshpere Competitiveness of Trichloroethylene-Degrading, Poplar-Colonizing Recombinant
Bacteria. Appl Environ Microbiol. 66(11): 4673-4678
[49] Yee, DC; Maynard, JA and Wood, TK (1998) Rhizoremediation of Trichloroethylene by a
Recombinant, Root-Colonizing Pseudomonas fluorescens Strain Expressing Toluene ortho-
Monooxygenase Constitutively. Appl Environ Microbiol 64(1): 112-118
[50] Gilbertson, AW; Burken, JG; Fitch, MW and Wood, TK (2002) Fluorescent, Root-Colonizing
Recombinant Bacteria to Enhance the Rhizosphere Degradation of TCE, in AR Gavakar and ASC
Chen, Eds., Remediation of Chlorinated and Recalcitrant Compounds, Battelle Press: Columbus, OH.
1-57477-132-9
[51] Barac, T; Taghavi, S; Borremans, B; Provoost, A; Oeyen, L; Colpaert, J; Vangronsveld, J and van der
Lelie, D (2004) Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile,
organic pollutants. Nature Biotechnology 22(5): 583-588
[52] Doty, SL; Shang, TQ; Wilson, AM; Tangen, J; Westergreen, AD; Newman, LA; Strand, SE and
Gordon, MP (2000) Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing
mammalian cytochrome P450 2E1. P Natl Acad Sci USA 97(12): 6287-6291
[53] Hirose, S; Kawahigashi, H; Ozawa, K; Shiota, N; Inui, H; Ohkawa, H and Ohkawa, Y (2005)
Transgenic Rice Containing Human CYP2B6 Detoxifies Various Classes of Herbicides. Agr Food
Chem 53: 3461-3467
IN VITRO PROPAGATION OF WETLAND MONOCOTS
FOR PHYTOREMEDIATION

MIHÁLY CZAKÓ1, XIANZHONG FENG2, YUKE HE2,

SHARADA GOLLAPUDI1, AND LÁSZLÓ MÁRTON1
1
Department of Biological Sciences, University of South Carolina, 700
Sumter St, Columbia, SC 29208, USA. Fax: 1-803-777-4002; E-mail:
[email protected] 2National Laboratory of Plant Molecular Genetics,
Shanghai Institute of Plant Physiology, Chinese Academy of Sciences,
300 Fenglin Road, Shanghai 200032, People’s Republic of China

1. Introduction

Wetlands are home to very important natural remediators of pollutants. Plants in

wetlands are often exposed to pollutants because of their very location in an ecosystem
that often occupies the boundaries of land and water. Monocot plants that can form
monocultures are important components of wetland plant communities. Genetic
improvement of wetland monocots by introduction of key transgenes can significantly
improve their remediation abilities. Tissue culture is prerequisite for genetic
manipulation, and advances are reported here in culture and propagation in vitro of
wetland monocots of various ecological tolerances regarding water depth, flow, seasonal
fluctuations and salinity. The monocots represent numerous genera in various families
such as Cyperaceae, Juncaceae, Poaceae, and Typhaceae. The reported species are in
various stages of in vitro propagation including embryogenic tissue, multishoot culture,
micropropagation, and genetic engineering.
Phytoremediation is the use of plants to remediate pollutants in contaminated soil,
water and air, encompassing a number of methods for the degradation (phyto- and
rhizodegradation), removal (phytoextraction, rhizofiltration and phytovolatilisation), or
immobilization (hydraulic control and phytostabilization) of contaminants [1].
Phytoremediation of natural ecosystems like wetlands is a not straightforward
because the ecological balance and species diversity is supposed to be restored to
original or at least retained at the pre-remediation state. Remediation of diverse wetlands
is not likely to be achievable by a single all-purpose wetland species. Instead, several
species from each habitat concerned should be considered.
Grass like plants are important as ornamentals, sources of energy and biomass, raw
materials, animal and potentially to carry out industrial processes such as phytoremediation

217
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 217 – 225.
© 2006 Springer.
218 Mihály Czakó et al.

[2]. Environmentally important wetland monocots have been neglected by plant

breeders; however, their remediation potential could certainly be expanded by
somaclonal breeding [3-7] and introduction of key genes to increase the remediation
ability [8].
Therefore, a method is needed for propagation that is independent of fertility and
seasons, and is more rapid and more efficient than conventional methods and is
sustainable at a high rate of multiplication. In vitro culture, if properly optimized, can
satisfy all these requirements and it also facilitates genetic manipulation, e.g. for
enhanced remediation ability. Tissue culture and transgenic technology needs to be
developed for multiple species.
In vitro propagation of the great majority of monocot (mostly Poaceae) species that
have been reported, was based on callus initiated on high concentrations of a strong
auxin, such as 2,4-dichlorophenoxyacetic acid (2,4-D) [9]. Induction of the regenerable
callus cultures from monocot species is conventionally attempted from germinating
seeds, immature embryos or immature inflorescences. These explants have been used
successfully in a number of wetland monocots (Table 1).
Our laboratory has a program for developing cell culture, propagation and gene
transfer technologies for numerous species from various wetlands, including salt
marshes, brackish water, rivers, riverbanks, lakes, ponds, and bogs. The monocots
represent multiple genera in several families including Cyperaceae, Juncaceae, Poaceae,
and Typhaceae. Here we report progress on in vitro culture methods for 41 wetland
monocot species.

1.2. MATERIALS AND METHODS

Immature inflorescences of Poaceae and Typhaceae were harvested while unemerged

and still covered by leaf sheaths. For Cyperaceae and Juncaceae, inflorescences were
gathered while the flowers were small early in their development. The explants were
surface sterilized by dilute bleach solution then cultured on primary explant medium
DM-8 in the dark at 26-28oC. DM-8 medium [10] contained (in mg l–1, unless indicated
otherwise): Murashige and Skoog (MS) salts [11], Sigma Fine Chemicals) 4,300;
Miller’s salt solution [6% (w/v) KH2PO4], 3 ml; myo-inositol, 100; Vitamix [12], 2 ml;
sucrose, 30,000; supplemented with the plant growth regulators adenine hemisulfate,
400 µM; picloram, 0.12; indole-3-butyric acid, 1; 2,4-dichlorophenoxyacetic acid, 0.5;
isopentenyladenine, 0.5; trans-zeatin, 0.5; thidiazuron (TDZ) 3.0; and solidified with
Phytagel (Sigma Fine Chemicals), 2000. The secondary medium (0201) contained fewer
plant growth regulators at reduced levels: 0.2 2,4-D and 0.1 µM TDZ [8]. The tertiary,
shoot induction/multiplication medium contained no vitamins or myo-inositol and only
0.1 µM TDZ as plant growth regulator [8]. The pH of all tissue culture media was
adjusted to 5.8 before sterilization in a pressure cooker (109oC, 35 kPa, 25 min). Callus
was subcultured every four weeks for maintenance. Regenerated and rooted plants were
separated, potted in the greenhouse, and initially kept under plastic wrap cover to help
acclimatization.
In vitro Propagation of Wetland Monocots 219

Table 1. In vitro culture of wetland monocots reported previously.

Species Family References Species Family References

Cyperaceae Lemnaceae
Carex acutiformis Ehrh. [8] Lemna spp. [13]
Carex divisa Huds. [8] Poaceae
Carex gracilis Curt. [8] Arundo donax L. [8,14,15]
Carex hyalinolepis Steud. [8] Distichlis spicata Greene [6,16]
Carex lurida Wahlenberg [17] Erianthus giganteus P. Beauv. [8]
Carex melanostachya Willd. [8] Erianthus strictus Baldwin [8]
[8,14,18-
Carex nigra Reichard [8] Miscanthus sinensis Andersson
21]
Carex pendula Huds. [8] Oryza sativa L. [22]
Carex riparia Curtis [8] Panicum dichotomum L. [8]
Carex spicata Huds. [8] Paspalum urvillei Steud. [8]
Carex vulpine L. [8] Phragmites australis Steud. [8,23-27]
Cladium jamaicense Crantz [8] Saccharum officinarum L. [28,29]
Cyperus giganteus Vahl. [8] Setaria gigantea Makino [8]
Cyperus haspan L. [8] Sorghum halepense Pers. [8]
Cyperus iria L. [8] Spartina alterniflora Loisel [7,8,30,31]
Cyperus pseudovegetus Steud. [8] Spartina argentinensis Parodi [32-36]
Cyperus retrorsus Chapm. [8] Spartina cynosuroides Roth. [8,37]
Eleocharis palustris Roem. & Schult. [8] Spartina patens Muhl. [8,38-40]
Eleocharis vivipara Link [8] Spartina pectinata Link [8,41]
Lepironia articulate Domin. [8] Spartina spartinae Hitchc. [8]
Schoenoplectus californicus Soják [8] Sporobolus virginicus Kunth [5,42]
Schoenoplectus tabernaemontani
[8,43] Vetiveria zizanioides Nash [8,44]
Palla
Scirpus americanus Pers. [8] Typhaceae
Scirpus polyphyllus Vahl. [17] Typha angustifolia L. [8,17]
Scirpus robustus Pursh. [45] Typha dominguensis Pers. [8]
Juncaceae Typha x glauca Godr. [8]
Juncus acuminatus Michaux [8,46] Typha latifolia L. [2,8,17]
Juncus articulatus L. [8]
Juncus compressus Jacq. [8]
Juncus dichotomus Elliott [8]
Juncus effusus L. [8,47]
Juncus roemerianus Scheele [8]
Juncus tenuis Willd. [8]
220 Mihály Czakó et al.

2. In vitro culture and micropropagation

Developing cell culture and micropropagation protocols for a diverse group of wetland
monocot species was facilitated by the availability of a universal cell culture initiation
medium, which had successfully been used for a great number of species [8, 10, 15]. The
method comprised using immature inflorescences as explants and cultivating the tissue
on a primary medium to produce totipotent embryogenic tissue culture, cultivating the
embryogenic tissue on a secondary medium to produce regenerating cultures, then
sustaining shoot multiplication and production of complete plantlets having roots and
shoots of tertiary medium before acclimating the plantlets in soil.
Establishment of primary callus from the 41 species listed in Table 2 table was
straightforward. It was preferred that the explant be obtained from an immature
inflorescence. The tips of field-grown or greenhouse-grown pre-flowering shoots of
Poaceae and Typhaceae with leaf sheaths completely enclosing a developing but yet
unemerged immature inflorescence, whose surface has been sterilized, were stripped of
the leaves and the inflorescences were cut into cross-sectional pieces, which were then
cultivated on a solid-type primary medium containing plant hormones. Primary callus
and, in certain species, shoot formation, but not elongation, occur on the primary
medium, and so the method is therefore suitable for sustained maintenance and
propagation of the totipotent tissue culture.

3. Phytoremediation

Wetland plants in general are considered to be useful for phytoremediation [2]. Spartina
alterniflora in particular has received more attention [7] and was subject to biochemical
studies. S. alterniflora’s natural capacities for pollutant remediation have been evaluated
before. The chemical similarity of selenium to sulfur allows Se to enter the DMSP
pathway and this is the basis of S. alterniflora’s potential use for abatement of selenium
pollution by phytovolatilisation [48, 49]. S. alterniflora also has tolerance to certain
man-made organic pollutants. It is uniquely adapted to the salt marsh which is impacted
both by halocompounds elaborated by marine organisms and also man-made
halogenated-organics [50]. Spartina’s natural biodegradation activities towards 2,4,6-
trichlorophenol (TCP) [8, 51] and trichloroethene (TCE) [52], respectively, have been
assessed in hydroponic experiments. With the introduction of organomercurial lyase and
mercuric reductase genes, Spartina alterniflora has become a potentially multipurpose
plant for phytoremediation [8], not only for salt marshes, but for non-saline constructed
wetlands as well as it grows best under freshwater conditions when competition is
absent. Spartina alterniflora has successfully been introduced into temperate and
In vitro Propagation of Wetland Monocots 221

subtropical regions of all continents [53]. Therefore, the potential areas of Spartina’s
application include nearly all wetlands globally.

4. Gene transfer

Spartina alterniflora was an attractive target species because it is a globally widespread,

large, perennial grass, which forms monocultural stands in salt marshes [54]. Transgenic
S. alterniflora lines have been generated [8]. The bacterial organomercurial lyase
(merB) [55] and mercuric reductase (merA) [56] genes were introduced by
Agrobacterium-mediated gene transfer. Co-introduction and co-expression of two genes
from separate Agrobacterium strains was possible and the transgenic lines were resistant
to both organomercurials and ionic mercury salts. Although the overall frequency of
transformation was low, the co-transformation is of significance because essentially the
complete mercury detoxification pathway has been incorporated into an ecologically
important species in one step. Highly poisonous organic mercury compounds form
naturally via biological processes in inorganic mercury contaminated sites. Therefore a
transgenic plant that can mineralise organomercurials to much less toxic inorganic
mercury to be removed by volatilisation after reduction is very advantageous [48].
The generation of other transgenic wetland monocots from the species listed in the
Tables 1 and 2 will be certainly facilitated by the availability of embryogenic cell
cultures and plant regeneration systems.
Table 2. Wetland monocots newly introduced into in vitro culture and progress in previously reported
species.

Primary Embryogeni Multishoot Acclimatised

Species Family
callus c callus culture plants
Cyperaceae
Baumea articulata S. T. Blake +
Bolboschoenus maritimus Palla +
Bolboschoenus robustus Soják +
Carex divisa Huds. + + + +
Carex elata All. + +
Carex gracilis Curt. + + +
Carex hirta L. +
Carex lacustris Willd. +
Carex leporina L. +
Carex lurida Wahlenberg +
222 Mihály Czakó et al.

Carex melanostachya Willd. + + + +

Carex nigra Reichard + + + +
Carex riparia Curtis + + + +
Carex spicata Huds. + + + +
Carex spissa L. H. Bailey +
Carex stenophylla Wahlenberg +
Carex vesicaria L. + +
Carex vulpine L. + + + +
Cyperus longus L. + + +
Cyperus pseudovegetus Steud. + + +
Eleocharis palustris Roem. & Schult. + + +
Lepironia articulate Domin. + + + +
Kyllinga brevifolia Rottb. + +
Schoenoplectus acutus Muhl. +
Schoenoplectus lacustris Palla +
Scirpus atrovirens Willd. + +
Scirpus cyperinus Kunth. + + +
Scirpus polyphyllus Vahl. + +
Scirpus silvaticus L. + +
Juncaceae
Juncus acuminatus Michaux + +
Juncus glaucus Wahlenberg +
Juncus gymnocarpus Coville +
Juncus inflexus L. +
Poaceae
Miscanthus sinensis Anderss. subsp.
+ + + +
Condensatus T. Koyama “Cosmopolitan”
Miscanthus sacchariflorus Hack. +
Phragmites communis Trin. var. variegata
+
Hitchc. ex L.H. Bailey
Vetiveria zizanioides Nash + +
Zizaniopsis miliacea Doell & Ascherson +
Typhaceae
Typha minima Funck ex Hoppe +

Acknowledgements

This research was supported by grants from the South Carolina Sea Grant Consortium.
In vitro Propagation of Wetland Monocots 223

References
[1] Cunningham, SD; Shann, JR; Crowley, DE and Anderson, TA (1997) Phytoremediation of contaminated
water and soil, in Kruger, EL Anderson, TA and Coats, JR, Ser. Eds., Vol. 664 from ACS Symposium
Series, Phytoremediation of Soil and Water Contaminants, pp. 2-17, Oxford University Press,
Cambridge, MA, ISBN 0-8412-3503-1
[2] Rogers, SD; Beech, J and Sarma, KS (1998) Shoot regeneration and plant acclimatization of the wetland
monocot cattail (Typha latifolia). Plant Cell Rep 18: 71-75
[3] Larkin, PJ and Scowcroft, WR (1981) Somaclonal variation - a novel source of variability from cell
culture for plant improvement. Theor Appl Genet 60: 197-214
[4] McClintock, B (1984) The significances of responses of the genome to challenge. Science 226: 792-801
[5] Seliskar, DM (1998) Natural and tissue culture-generated variation in the salt marsh grass Sporobolus
virginicus: Potential selections for marsh creation and restoration. Hortscience 33: 622-625
[6] Seliskar, DM and Gallagher, JL (2000) Exploiting wild population diversity and somaclonal variation in
the salt marsh grass Distichlis spicata (Poaceae) for marsh creation and restoration. Am J Bot 87:
141-146
[7] Wang, JB; Seliskar, DM and Gallagher, JL (2003) Tissue culture and plant regeneration of Spartina
alterniflora: Implications for wetland restoration. Wetlands 23: 386-393
[8] Czakó, M; Feng, X; He, Y; Liang, D and Márton, L (2005) Genetic modification of wetland grasses for
phytoremediation. Z Naturforsch C 60c: 285-291
[9] Conger, BV and Kuklin, AI (1995) In vitro culture and plant regeneration in gramineous crops, in Terzi,
M; Cella, R and Falavigna, A, Eds, Current Issues in Plant Molecular and Cellular Biology: Proceedings
of the VIIIth International Congress on Plant Tissue and Cell Culture, Florence, I, pp. 59-68, Kluwer
Academic Publishers BV, Dordrecht, The Netherlands, ISBN 0-7923-332-25
[10] Czakó, M and Márton, L (2001) A heartwood pigment in Dalbergia cell cultures. Phytochemistry 57:
1013-1022
[11] Murashige, T and Skoog, F (1962) A revised medium for rapid growth and bio-assays with tobacco
tissue cultures. Physiol Plant 15: 473-497
[12] Márton, L and Browse, J (1991) Facile Transformation of Arabidopsis. Plant Cell Rep 10: 235-239
[13] Márton, L and Czakó, M (2004) Sustained totipotent culture of selected monocot genera. U.S. Patent
No. 6,821,782 B2.
[14] Meagher, RB (2000) Phytoremediation of toxic elemental and organic pollutants. Curr Opin Plant Biol
3: 153-162
[15] Ansede, JH; Pellechia, PJ and Yoch, DC (1999) Selenium biotransformation by the salt marsh cordgrass
Spartina alterniflora: Evidence for dimethylselenopropionate formation. Environ Sci Technol 33:
2064-2069
[16] Sanger, DM; Holland, AE and Scott, GI (1999) Tidal creek and salt marsh sediments in South Carolina
coastal estuaries: I. Distribution of trace metals. Arch Environ Contam Tox 37: 445-457
[17] Márton, L; Chen, YP and Czakó, M (2000) Method for decomposing toxic organic pollutants U.S.
Patent No 6,087,547.
[18] Macek, T; Francova, K; Kochankova, L; Lovecka, P; Ryslava, E; Rezek, J; Sura, M; Triska, J;
Demnerova, K and Mackova, M (2004) Phytoremediation: Biological cleaning of a polluted
environment. Reviews on Environmental Health 19: 63-82
[19] Chung, CH (1989) Ecological engineering of coastlines with saltmarsh plantations, in Mitsch, WJ and
Jorgensen, SE, Eds, Ecological Engineering: an Introduction to Ecotechnology, pp. 255-289, John Wiley
and Sons, New York, ISBN 0-4716-255-90
[20] Mobberley, D (1956) Taxonomy and distribution of the genus Spartina. Iowa State Coll J Sci 30:
471-574
[21] Bizily, SP; Rugh, CL; Summers, AO and Meagher, RB (1999) Phytoremediation of methylmercury
pollution: merB expression in Arabidopsis thaliana confers resistance to organomercurials. Proc Natl
Acad Sci USA 96: 6808-6813
[22] Rugh, CL; Wilde, HD; Stack, NM; Thompson, DM; Summers, AO and Meagher, RB (1996) Mercuric
ion reduction and resistance in transgenic Arabidopsis thaliana plants expressing a modified bacterial
merA gene. Proc Natl Acad Sci USA 93: 3182-3187
[23] Stomp, A-M and Rajbhandari, N (2000) Genetically Engineered Duckweed. U.S. Patent No. 6,040,498.
224 Mihály Czakó et al.

[24] Tóth, S and Mix-Wagner, G (1998) Embryogenic callus induction of different explants of Miscanthus
sinensis, Miscanthus x giganteus, and Arundo donax genotypes. Sust Agric Food, Energy, and Ind
249-253
[25] Straub, PF; Decker, DM and Gallagher, JL (1989) Tissue culture and regeneration of Distichlis spicata
(Gramineae). Am J Bot 76: 1448-1451
[26] Rogers, SMD (2003) Tissue culture and wetland establishment of the freshwater monocots Carex,
Juncus, Scirpus, and Typha. In Vitro Cell Dev Biol Plant 39: 1-5
[27] Lewandowski, I (1997) Micropropagation of Miscanthus x giganteus, in Bajaj, YPS, Ser. Ed., Vol. 39,
Biotechnology in Agriculture and Forestry, High-Tech and Micropropagation V, pp. 239-255, Springer,
New York, ISBN 3-540-61606-3
[28] Gawel, NJ; Robacker, CD and Corley, WL (1987) Propagation of Miscanthus sinensis through tissue
culture. Hortscience 22: 1137-1137
[29] Gawel, NJ; Robacker, CD and Corley, WL (1990) In vitro propagation of Miscanthus sinensis.
Hortscience 25: 1291-1293
[30] Holme, IB and Petersen, KK (1996) Callus induction and plant regeneration from different explant types
of Miscanthus x ogiformis Honda “Giganteus”. Plant Cell Tissue Organ Cult 45: 43-52
[31] Heaton, ACP; Rugh, CL; Kim, T; Wang, NJ and Meagher, RB (2003) Toward detoxifying mercury-
polluted aquatic sediments with rice genetically engineered for mercury resistance. Environ Toxicol
Chem 22: 2940-2947
[32] Lauzer, D; Dallaire, S and Vincent, G (2000) In vitro propagation of reed grass by somatic
embryogenesis. Plant Cell Tissue Organ Cult 60: 229-234
[33] Mathe, C; Hamvas, MM; Grigorszky, I; Vasas, G; Molnar, E; Power, JB; Davey, MR and Borbely, G
(2000) Plant regeneration from embryogenic cultures of Phragmites australis (Cav.) Trin. ex Steud.
Plant Cell Tissue Organ Cult 63: 81-84
[34] Poonawala, IS; Jana, MM and Nadgauda, RS (1999) Factors influencing bud break and rooting and
mass-scale micropropagation of three Phragmites species: P. karka, P. communis and P. australis. Plant
Cell Rep 18: 696-700
[35] Sangwan, R and Gorenflot, R (1975) In vitro culture of Phragmites tissues: callus formation, organ
differentiation and cell suspension culture. Z Pflanzenphysiol 75s: 256-269
[36] Straub, PF; Decker, DM and Gallagher, JL (1988) Tissue culture and long-term regeneration of
Phragmites australis (Cav.) Trin. ex Steud. Plant Cell Tissue Organ Cult 15: 73-78
[37] Arencibia, AD; Carmona, ER; Tellez, P; Chan, MT; Yu, SM; Trujillo, LE and Oramas, P (1998) An
efficient protocol for sugarcane (Saccharum spp. L.) transformation mediated by Agrobacterium
tumefaciens. Transgenic Res 7: 213-222
[38] Chengalrayan, K; Gallo-Meagher, M and English, RG (2001) Novel selection agents for sugarcane
transformation. Soil and Crop Science Society of Florida Proceedings 60: 81-87
[39] Utomo, HS; Wenefrida, I and Croughan, TP (2001) Smooth cordgrass synthetic seeds: Production,
storage and potential use for coastal erosion controls. TurfGrass Trends 10: 12-15
[40] Croughan, TP; Cao, HX; Regan, RP; Meche, MM; Wang, XH; Xie, QJ; Trumps, DB and Materne, MD
(1993) Biotechnological applications to coastal erosion control. Ann Res Rep LA State Univ Baton
Rouge 85: 157-160
[41] Bueno, MS; Petenello, C; Feldman, SR and Ortiz, JP (2005) Obtención de variantes de Spartina
argentinensis Parodi mediante el cultivo in vitro de inflorescencias inmaduras, in BairesBiotec 2005,
Congreso internacional, VI Simposio Nacional de Biotecnología- REDBIO Argentina 2005. Encuentro
Trinacional REDBIO Argentina-Chile-Uruguay. Buenos Aires
[42] Bueno, MS; Chincuini, DAE; Soumoulou, M; Feldman, SR and Ortiz, JP (2002) Obtención de callos a
partir de hojas de Spartina argentinensis Parodi, in XXII Reunión anual de la Sociedad de Biología de
Rosario. Diciembre 2002
[43] Bueno, MS; Chincuini, DAE; Soumoulou, M; Feldman, SR and Ortiz, JP (2003) Estudio del
comportamiento in vitro de diferentes explantos de Spartina argentinensis Parodi, in II Congreso
Nacional de Pastizales Naturales. VI Jornada Regional. IV Reunión de la Asociación Argentina de
Prosopis
[44] Bueno, MS; Chincuini, DAE; Soumoulou, M; Feldman, SR and Ortiz, JP (2003) Citodiferenciación de
callos de Spartina argentinensis Parodi, in Proc. of XXIII Reunión anual de la Sociedad de Biología de
Rosario
In vitro Propagation of Wetland Monocots 225

[45] Bueno, MS; Feldman, SR and Ortiz, JP (2004) Utilización de cariopses como explantos para el cultivo
de tejidos de Spartina argentinensis Parodi, in Proc. of Simposio Internacional de Biotecnología. II
Simposio Argentino-Italiano de Bacterias Lácticas, Tucumán
[46] Li, XG and Gallagher, JL (1996) Tissue culture and plant regeneration of big cordgrass, Spartina
cynosuroides: Implications for wetland restoration. Wetlands 16: 410-415
[47] Li, XG; Seliskar, DM; Moga, JA and Gallagher, JL (1995) Plant regeneration from callus cultures of
salt-marsh hay, Spartina patens, and its cellular-based salt tolerance. Aquat Bot 51: 103-113
[48] Rao, JD; Seliskar, DM and Gallagher, JL (1995) The effect of phytohormones, NaCl, and carbon source
on plant shoot regeneration from callus cultures of Spartina patens. Plant Physiol 108: 48-48
[49] Rao, JD; Seliskar, DM and Gallagher, JL (1996) In vitro shoot regeneration from callus cultures of
Spartina patens, a halophytic C4 grass. Plant Physiol 111: 486-486
[50] Hogan, CJ (1988) Preprophase bands in a suspension culture of the monocot Spartina pectinata. Exp
Cell Res 175: 216-222
[51] Straub, PF; Decker, DM and Gallagher, JL (1992) Characterization of tissue-culture initiation and plant
regeneration in Sporobolus virginicus (Gramineae). Am J Bot 79: 1119-1125
[52] Gollapudi, S (2005) Development of a tissue culture system in great bulrush for biotechnological
applications. M.Sc. Thesis, The University of South Carolina, Columbia, SC, USA
[53] Maffei, M (2002) Vetiveria: The Genus Vetiveria, Taylor & Francis, New York, ISBN 0-415-27586-5
[54] Wang, JB; Seliskar, DM and Gallagher, JL (2004) Plant regeneration via somatic embryogenesis in the
brackish wetland monocot Scirpus robustus. Aquat Bot 79: 163-174
[55] Sarma, KS and Rogers, SMD (1998) Plant regeneration and multiplication of the emergent wetland
monocot Juncus accuminatus. Plant Cell Rep 17: 656-660
[56] Sarma, KS and Rogers, SMD (2000) Plant regeneration from seedling explants of Juncus effusus. Aquat
Bot 68: 239-247
MODIFYING A PLANT’S RESPONSE TO STRESS BY DECREASING
ETHYLENE PRODUCTION

BERNARD R. GLICK
Department of Biology, University of Waterloo
200 University Avenue West, Waterloo, ON, Canada N2L 3G1
Fax: +1-519-746-0614, E-mail: [email protected]

1. Introduction

Plants that are relatively tolerant of various environmental contaminants are often used
as components of phytoremediation strategies. Nevertheless, as a consequence of the
stress that the environmental contaminant(s) imposes on plants, they are often unable to
proliferate to any great extent and remain small in the presence of high levels of the con-
taminant. To remedy this situation, plant growth-promoting bacteria that facilitate the
proliferation of plants under environmentally stressful conditions may be added to the
system. These bacteria have been selected or engineered to lower the level of growth-
inhibiting stress ethylene within the plant and also to directly promote plant growth,
usually by either providing the plant hormone indoleacetic acid or facilitating the
acquisition of iron from the soil. The net result of adding these bacteria to plants is a
significant increase in both the number of seeds that germinate and the amount of
biomass that the plants are able to attain in contaminated soil, making phytoremediation
in the presence of plant growth-promoting bacteria a much faster and more efficient
process. In addition, some of the benefits conferred upon plants by plant growth-
promoting bacteria may be provided by genetically modifying plants to have lower
ethylene levels.

1.1. PLANT GROWTH-PROMOTING BACTERIA

Beneficial free-living soil bacteria are often referred to as plant growth-promoting

rhizobacteria, or PGPR, and are commonly found in association with the roots of plants
[1, 2]. There is usually a high concentration of bacteria in the plant rhizosphere because
of the presence of high levels of nutrients that are exuded from the roots of most plants,
and these nutrients can be used to support bacterial growth and metabolism [3-5].
Plant growth-promoting bacteria can positively influence plant growth and develop-
ment in two different ways: indirectly or directly [1, 2]. The indirect promotion of plant
growth occurs when these bacteria decrease or prevent some of the deleterious effects of
phytopathogenic organisms. For example, many biocontrol bacteria indirectly promote

227
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 227– 236.
© 2006 Springer.
228 Bernard R. Glick

plant growth by synthesizing pathogen-inhibiting antibiotics. Bacteria can directly

promote plant growth by providing the plant with a compound synthesized by the
bacterium or by facilitating the uptake of nutrients from the environment by the plant.
Thus, plant growth-promoting bacteria may fix atmospheric nitrogen; synthesize
siderophores which can solubilize and sequester iron from the soil; synthesize
phytohormones including auxins and cytokinins which can enhance plant growth;
solubilize phosphorus from the soil; and contain enzymes that can modulate plant
growth and development [1, 2, 6-11]. A particular bacterium may affect plant growth
and development using any one, or more, of these mechanisms and may utilize different
mechanisms under different conditions. For example, bacterial siderophore synthesis is
induced only in soils that do not contain sufficient levels of iron. Similarly, bacteria do
not fix nitrogen when sufficient fixed nitrogen is available.

1.2. ACC DEAMINASE AND THE REDUCTION OF PLANT ETHYLENE

In higher plants, ethylene is produced from L-methionine via the intermediates,

S-adenosyl-L-methionine (SAM) and 1-aminocyclopropane-1-carboxylic acid (ACC)
[12]. The enzymes involved in this metabolic sequence are SAM synthetase, which
catalyzes the conversion of methionine to SAM [13]; ACC synthase, which is
responsible for the hydrolysis of SAM to ACC and 5-methylthioadenosine [14]; and
ACC oxidase, which metabolizes ACC to ethylene, carbon dioxide and cyanide [15].
In 1978, an enzyme capable of degrading ACC was isolated from Pseudomonas sp.
strain ACP [16]. Since then, ACC deaminase has been detected in several fungi and
yeasts [17, 18] as well as in bacterial strains [1, 2, 19-26] This enzyme cleaves the plant
ethylene precursor, ACC, to produce ammonia and α-ketobutyrate. It has been proposed
[11] that microorganisms that contain the enzyme ACC deaminase can all promote plant
growth since they act as a sink for ACC and thereby lower ethylene levels in a
developing or stressed plant.
Ethylene is important for normal development in plants as well as for their response
to stress [27]. Ethylene is important during the early phase of plant growth; it is required
by many plant species for seed germination, and the rate of ethylene production
increases during germination and seedling growth [28]. Ethylene also induces some
plant defences including induced systemic resistance [29]. However, high levels of
ethylene can lead to inhibition of root elongation and the onset of senescence.
Strains of ACC deaminase-containing plant growth-promoting bacteria can reduce
the amount of ACC within plant tissues that is detectable by HPLC, and hence the
ethylene levels in plants are also lowered [5, 30]. As a consequence of this activity, ACC
deaminase-containing plant growth-promoting bacteria promote root elongation in a
variety of (ethylene sensitive) plants [31]. In addition to lowering ethylene levels during
plant development, ACC deaminase-containing plant growth-promoting bacteria
decrease the levels of “stress ethylene” — the accelerated biosynthesis of ethylene
associated with biological and environmental stresses and pathogen attack [32]. Thus,
the deleterious effects of flooding, high salt or drought on tomato plants [26, 33, 34]
were decreased and the shelf life of the petals of ethylene sensitive cut flowers was
prolonged, following treatment with ACC deaminase-containing plant growth promoting
Modifying a Plant’s Response to Stress 229

bacteria [35]. Moreover, biocontrol strains of bacteria carrying ACC deaminase genes
were better able to protect plants against various phytopathogens [36]. In addition,
canola seedlings grown in the presence of high levels of nickel, produced much less
ethylene when the seeds were inoculated with an ACC deaminase-containing nickel-
resistant plant growth-promoting strain that also produced indoleacetic acid and high
levels of siderophores [22]. In each of these situations, the ‘‘stress ethylene’’ produced
and the damage caused by it, was reduced by the activity of ACC deaminase.

2. Plant growth-promoting bacteria and phytoremediation

While plants grown on metal contaminated soils might be able to withstand some of the
inhibitory effects of high concentrations of metals within a plant, two features of most
plants could result in a decrease in plant growth and viability. In the presence of plant
inhibitory levels of metals, most plants (i) synthesize stress ethylene and (ii) become
severely depleted in the amount of iron that they contain. Fortunately, ACC deaminase-
containing plant growth-promoting bacteria may be used to relieve some of the toxicity
of metals to plants. This can occur in two different ways (i) a decrease in the level of
stress ethylene in plants growing in metal-contaminated soil and (ii) utilization by plants
of complexes between bacterial siderophores and iron. Plant siderophores bind to iron
with a much lower affinity than bacterial siderophores so that in metal-contaminated
soils a plant is generally unable to accumulate a sufficient amount of iron (and often
becomes chlorotic) unless bacterial siderophores are present.
In one study, in an effort to overcome the inhibition of plant growth by nickel, a
bacterium was isolated from a nickel contaminated soil sample; the bacterium was (i)
nickel-resistant, (ii) capable of synthesizing the auxin indoleacetic acid, (iii) able to
grow at the cold temperatures (i.e., 5–10°C) that one expects to find in nickel
contaminated soil environments in northern climes such as Canada, and (iv) an active
producer of ACC deaminase [22]. In order to isolate plant growth-promoting bacteria,
all of the nickel-resistant bacterial isolates from a nickel-contaminated rhizosphere soil
sample were tested for the ability to grow on minimal medium with ACC as the sole
source of nitrogen [37]. Nickel-resistant bacterial strains that were also able to grow on
ACC were tested for the ability to produce siderophores and grow in cold temperatures.
It was ascertained in laboratory tests, that the selected bacterium could promote plant
growth (both roots and shoots) in the presence of high levels (1–6 mM) of nickel
[22, 38].
Subsequently, a spontaneous siderophore overproducing mutant of this bacterium
was selected. When the wild-type bacterium and the siderophore overproducing mutant
were tested in the laboratory, both of them were observed to promote the growth of
tomato, canola and Indian mustard plants in soil that contained otherwise inhibitory
levels of nickel, lead or zinc. In addition, the siderophore overproducing mutant
decreased the inhibitory effect of the added metal on plant growth significantly more
than the wild-type bacterium. Metal contamination of soils is often associated with iron-
deficiency of the plants grown in these soils [39]. The low iron content of plants that are
grown in the presence of high levels of metals generally results in these plants becoming
230 Bernard R. Glick

chlorotic, since iron deficiency inhibits both chloroplast development and chlorophyll
biosynthesis [40]. Moreover, iron deficiency is a stress that causes the plant to synthesize
stress ethylene. However, once they have bound iron, bacterial iron-siderophore complexes
can be taken up by plants and thereby serve as an iron source for plants [41].
Thus, there is (at least) a dual role for bacteria that facilitate plant growth in metal-
contaminated soils. On the one hand, the bacteria lower the level of stress ethylene in the
plant thereby allowing it to develop longer roots and thus better establish itself during
early stages of growth [11, 22]. On the other hand, the bacterium helps the plant to
acquire sufficient iron for optimal plant growth, in the presence of levels of metals that
might otherwise make the acquisition of iron difficult [42]. When the siderophore
overproducing mutant was tested in the field with nickel-contaminated soil, it was
observed that both the number of seeds that germinated, and the size that the plants were
able to attain was increased by 50–100% by the presence of the bacterium.
In another study, the common reed, Phragmites australis, a plant that has often been
suggested for use in the phytoremediation of wetlands, was grown from seed in the
laboratory in copper-contaminated soil. It was observed that the addition of a copper-
resistant strain of Pseudomonas asplenii that had been genetically transformed to
express a bacterial ACC deaminase gene significantly stimulated seed germination in the
presence of high levels of copper where the native form of this bacterium had no
stimulatory effect on seed germination (M.L.E Reed, B. Warner and B.R. Glick,
submitted for publication). This is consistent with the notion that one reason that plant
germination is often inhibited by the presence of high levels of soil contaminants is that
a high level of ethylene is produced in seeds as a response to the contaminant. In this
case, lowering seed ethylene levels so that they are no longer inhibitory should promote
seed germination in the presence of a range of contaminants. Moreover, in these
experiments, both the native and the transformed strain of Pseudomonas asplenii had a
small but reproducible stimulatory effect on Phragmites australis root and shoot growth.
This indicates that, at least for Phragmites australis, growth inhibition by copper is not
solely a consequence of stress ethylene synthesis but rather likely mainly reflects copper
inhibition of plant metabolic processes.
In a separate but similar study, the growth of canola roots and shoots in copper-
contaminated soil was stimulated (significantly) but to the same extent by both native
and ACC deaminase-transformed Pseudomonas asplenii (M.L.E. Reed and B.R. Glick,
submitted for publication). In this case, the promotion of plant growth by both native
and transformed Pseudomonas asplenii was attributed to the production of indoleactic
acid by the added bacteria since this strain does not produce siderophores (and therefore
could not be involved in providing iron to the plant). Only the transformed strain has
ACC deaminase activity so that, as with Phragmites australis, decreasing ethylene levels
is not a factor in growth promotion, and bacterial indoleacetic acid has previously been
shown to be capable of directly promoting plant growth [43].
Given the extreme toxicity of arsenate to most plants and bacteria, the development
of a phytoremediation scheme for the detoxification of arsenate-contaminated soils is
not a simple matter. For example, unlike what has been observed with nickel, lead,
copper and zinc, arsenate-resistant plant growth-promoting bacteria do not significantly
protect plants from arsenate inhibition.
Modifying a Plant’s Response to Stress 231

Polycyclic aromatic hydrocarbons (PAHs) are a particularly recalcitrant group of

contaminants and are known to be highly persistent in the environment. In situ microbial
remediation (i.e., bioremediation) has been attempted, but it is difficult to generate
sufficient biomass in natural soils to achieve an acceptable rate of movement of
hydrophobic PAHs (which are often tightly bound to soil particles) to the microbes
where they can be degraded. In addition, relatively few microorganisms can use high
molecular weight PAHs as a sole carbon source. More recently, there have been some
improvements in the strategies for bacterial remediation of contaminated soil, including
inoculation with bacteria that were selected from PAH contaminated sites, or
supplementing contaminated soils with nutrients [44]. However, there has only been
limited success with these techniques. For bioremediation to be effective, the overall rate
of PAH removal and degradation must be accelerated above current levels. One way to
achieve this is to increase the amount of biomass in the contaminated soil. For this
reason, the use of phytoremediation has received considerable attention [45-47].
Although using plants for remediation of persistent contaminants may have
advantages over other methods, many limitations exist for the large-scale application of
this technology. For example, many plant species are sensitive to contaminants including
PAHs so that they grow slowly, and it is time consuming to establish sufficient biomass
for meaningful soil remediation. In addition, in most contaminated soils, the number of
microorganisms is depressed so that there are not enough bacteria either to facilitate
contaminant degradation or to support plant growth. To remedy this situation, both
degradative and plant growth-promoting bacteria may be added to the plant rhizosphere.
Phytoremediation (where contaminant degradation is dependent solely on plants) is not
significantly faster than bioremediation (where biodegradation of the organics is by
microorganisms independent of plants) for removal of PAHs or TPHs (Total Petroleum
Hydrocarbons) [48-50]. However, cultivating plants together with plant growth-
promoting bacteria allows the plants to germinate in the presence of soil contaminants to
a much greater extent than they would otherwise, and then to grow well under stressful
conditions and accumulate a larger amount of biomass than plants grown in the absence
of plant growth-promoting bacteria. In addition, the plant growth-promoting bacteria in
these experiments significantly increase the amount of PAH or TPH that is removed
from the soil. In heavily contaminated soils, plant growth-promoting bacteria increased
seed germination and plant survival, increased the plant water content, helped plants to
maintain their chlorophyll contents and chlorophyll a/b ratio, and promoted plant root
and shoot growth. In the case of PAHs, this is most likely due to a combination of the
direct promotion of plant growth by bacterial indoleacetic acid and a lowering of the
concentration of stress ethylene by bacterial ACC deaminase (MLE Reed and BR Glick,
submitted for publication). As a consequence of the treatment of plants with plant
growth-promoting bacteria, the plants provide a greater sink for the contaminants since
they are better able to survive and proliferate.
232 Bernard R. Glick

3. Phytoremediation with plants engineered to produce less ethylene

If ACC deaminase-containing plant growth-promoting bacteria, bound to plant roots, can

act as a sink for some of the excess ACC produced as a consequence of environmental
stress, then transgenic plants expressing a bacterial ACC deaminase gene should behave
similarly and have a level of stress ethylene lower than non-transformed plants and
consequently be less susceptible to the deleterious effects of the stress. In fact, in two
separate studies transgenic plants expressing ACC deaminase were shown to proliferate
to a much greater extent than the comparable non-transformed plants in the presence of
metals [51, 52]. In one study, transgenic tomato plants expressing a bacterial ACC
deaminase gene under the transcriptional control of two tandem 35S cauliflower mosaic
virus promoters (constitutive expression), the rolD promoter from Agrobacterium
rhizogenes (root specific expression) or the pathogenesis related PRB-1b promoter from
tobacco, were compared to non-transgenic tomato plants in their ability to grow in the
presence of cadmium, cobalt, copper, magnesium, nickel, lead or zinc and to accumulate
these metals [51]. These transgenic tomato plants acquired a greater amount of metal
within the plant tissues, and were less subject to the inhibitory effects of the metals on
plant growth than were non-transformed plants. Moreover, plants in which the ACC
deaminase gene was under the transcriptional control of the rolD promoter were more
resistant to the various metals than were the other transgenic plants.
Of course, there is no expectation that transgenic tomato plants will ever become part
of a phytoremediation strategy. Nevertheless, the results that were obtained with tomato
plants were intriguing and served as a starting point for the development of other
transgenic plants with lowered ethylene concentrations that could be used as a
component of a phytoremediation scheme. Thus, both transgenic tobacco (because of its
potentially large leaf biomass) and canola (because of its previously demonstrated ability
to be a moderate accumulator of numerous metals) were transformed with bacterial ACC
deaminase genes under the transcriptional control of either the 35S or rolD promoters
(Li, Q, Shah, S, Saleh-Lakh, S and GLick, BR, submitted for publication; Stearns, JC,
Shah, S, Dixon, DG, Greenberg BM and Glick, BR, submitted for publication). When
they were tested, in laboratory and greenhouse experiments, the transgenic tobacco and
canola plants responded similarly to the presence of nickel in the soil to the previously
constructed transgenic tomatoes. In all instances, transgenic plants in which the
exogenous ACC deaminase gene was controlled by the rolD promoter demonstrated the
highest level of resistance to growth inhibition by nickel. Moreover, rolD canola plants
were also resistant to growth inhibition by high levels of salt in the soil (Sergeeva, E,
Shah, S and Glick, BR, submitted for publication). Reminiscent of the protection from
salt stress that is afforded by a salt-resistant plant growth-promoting bacterium [34].
From these and other data, it appears that the behaviour of plants to a variety of stresses
(metals, salt, flooding and pathogens), transformed with an exogenous ACC deaminase
gene controlled by the rolD promoter, is similar to the way in which these plants respond
when ACC deaminase-containing plant growth-promoting bacteria have colonized the
plant roots. In both cases, root-associated ACC deaminase acts as a sink for ACC and
thereby prevents the formation of growth inhibitory levels of stress ethylene. The major
difference between these two scenarios is that, in addition to lowering ethylene levels,
Modifying a Plant’s Response to Stress 233

the bacteria can directly promote plant growth by providing the hormone indoleacetic
acid or siderophores that help the plant to obtain a sufficient amount of iron. In fact,
in laboratory and greenhouse experiments, ACC deaminase-containing plant growth-
promoting bacteria generally are a greater stimulus to plant growth under a range of
stressful and potentially inhibitory conditions than are ACC deaminase transgenes
expressed exclusively in the roots. Unfortunately, as a consequence of a number of
environmental factors (such as weather and the presence of predators in the soil) plant
growth-promoting bacteria may not always be as persistent in field conditions as they are
in the greenhouse. One way around this problem may be to select or engineer endophytic
bacterial strains that promote plant growth by employing some of the above mentioned
bacterial mechanisms [53-55]. Finally, it should also be noted that plant ethylene levels
may be decreased through a variety of genetic manipulations (e.g., the use of antisense
versions of ACC oxidase) other than ACC deaminase. [29].
In another study, the growth of canola plants expressing ACC deaminase under the
control of two tandem 35S cauliflower mosaic virus promoters in the presence of
arsenate was monitored [52]. About 70-80% of the transgenic plants germinated while a
maximum of 25-30% of the non-transformed plants germinated. Although a small
ethylene pulse is important in breaking seed dormancy in many plants, too much
ethylene can inhibit plant seed germination [56]. In the presence of arsenate, ACC
deaminase may enhance the process of germination by hydrolyzing any excess ACC that
forms as a consequence of the stress, hence lowering the inhibitory level of ethylene in
seeds. Transgenic canola also had much higher fresh and dry weights of roots and
shoots, and higher leaf chlorophyll contents, than non-transformed canola grown in the
presence of arsenate. Moreover, the addition of plant growth-promoting bacteria to the
roots of transgenic canola plants grown in arsenate-contaminated soils helped the plants
to grow to a slightly larger size. In this case, growth promotion is probably attributable
to the bacterial indoleacetic acid. When biomass and rate of seed germination are
considered in calculating arsenate accumulation, for each seed planted, transgenic canola
expressing ACC deaminase takes up approximately eight times as much arsenate as non-
transformed canola. This notwithstanding, considerable work remains to be done before
a practical system for the phytoremediation of arsenic can be implemented.

4. Summary and conclusions

Microbial activities exerted in the rhizosphere can influence plant growth, development
and metabolism at both the root and the shoot levels, and can reduce the effects of
various stresses. More specifically, traits that directly contribute to the promotion of
plant growth and stress reduction include the synthesis of indoleacetic acid, siderophores
and the enzyme ACC deaminase. Several strains of plant growth-promoting bacteria
with different properties are already commercially available and are being used to
increase crop yields.
Given the current reluctance on the part of many consumers worldwide to embrace
the use of foods derived from genetically modified plants, it may be advantageous to use
either natural or genetically engineered plant growth-promoting bacteria as a means to
234 Bernard R. Glick

promote growth or reduce disease through induction of resistance, rather than genetically
modifying the plant itself to the same end. Moreover, given the large number of different
plants, the various cultivars of those plants and the multiplicity of genes that would need
to be engineered into plants, it is not feasible to genetically engineer all plants to be
resistant to all pathogens and environmental stresses. Rather, it seems more logical to
engineer plant growth-promoting bacteria to do this job; the first step in this direction
could well be the introduction of appropriately regulated ACC deaminase genes. While
ethylene signalling is required for the induction of systemic resistance elicited by
rhizobacteria, a significant increase in the level of ethylene is not. Hence, lowering of
ethylene levels by bacterial ACC deaminase is not incompatible with the induction of
systemic resistance. Indeed, some bacterial strains possessing ACC deaminase also
induce systemic resistance.

Acknowledgements

Work from the author’s laboratory was supported by grants from the Natural Science
and Engineering Research Council, CRESTech (a province of Ontario Centre of
Excellence), Ontario Hydro, and Inco. The following individuals contributed to the work
reviewed here: Genrich Burd, George Dixon, XiaoDong Huang, Sibdas Ghosh, Bruce
Greenberg, Varvara Grichko, Jiping Li, Qiaosi Li, Wenbo Ma, Shimon Mayak, Barbara
Moffatt, Lin Nie, Cheryl Patten, Donna Penrose, Lucy Reed, Saleema Saleh-Lakha,
Elena Sergeeva, Saleh Shah, Jennifer Stearns, Tsipi Tirosh, Chunxia Wang and Barry
Warner.

References
[1] Glick, BR (1995) The enhancement of plant growth by free-living bacteria. Can J Microbiol 41:
109-117.
[2] Glick, BR; Patten, CL; Holguin, G and Penrose, DM (1999) Biochemical and genetic mechanisms used
by plant growth promoting bacteria. Imperial College Press, London, UK, ISBN 1-86094-152-4.
[3] Whipp, JM (1990) Carbon utilization, in Lynch JM, Ed. The rhizosphere. pp 59-97, John Wiley,
Chichester, UK, ISBN 0471925489.
[4] Bayliss, C; Bent, E; Culham, DE; MacLellan, S; Clarke, AJ; Brown, GL and Wood, J (1997) Bacterial
genetic loci implicated in the Pseudomonas putida GR12-2R3-canola mutualism: identification of an
exudate-inducible sugar transporter. Can J Microbiol 43: 809-18.
[5] Penrose, DM and Glick, BR (2001) Levels of 1-aminocyclopropane-1-carboxylic acid (ACC) in
exudates and extracts of canola seeds treated with plant growth-promoting bacteria. Can J Microbiol 47:
368-72.
[6] Brown, ME (1974) Seed and root bacterization. Annu Rev Phytopathol 12: 181-97.
[7] Davison, J (1988) Plant beneficial bacteria. Bio/technology 6: 282-286.
[8] Kloepper, JW; Lifshitz, R and Zablotowicz, RM (1989) Free-living bacterial inocula for enhancing crop
productivity. Trends Biotechnol 7: 39-43.
[9] Lambert, B and Joos, H (1989) Fundamental aspects of rhizobacterial plant growth promotion research.
Trends Biotechnol 7: 215-9.
[10] Patten, CL and Glick, BR (1996) Bacterial biosynthesis of indole-3-acetic acid. Can J Microbiol 42:
207-20.
[11] Glick, BR; Penrose, DM and Li, J (1998) A model for the lowering of plant ethylene concentrations by
plant growth promoting bacteria. J Theor Biol 190: 63-8.
Modifying a Plant’s Response to Stress 235

[12] Yang, SF and Hoffman, NE (1984) Ethylene biosynthesis and its regulation in higher plants. Annu Rev
Plant Physiol 35: 155-89.
[13] Giovanelli, J; Mudd, SH and Datko, AH (1980) Sulphur amino acids in plants, in: Miflin, BJ, Ed,
Amino acids and derivatives. The biochemistry of plants: a comprehensive treatise, Vol. 5, pp 435-505,
Academic Press, New York, USA, ISBN 0-12-675416-0.
[14] Kende, H (1989) Enzymes of ethylene biosynthesis. Plant Physiol 91: 1-4.
[15] John, P (1991) How plant molecular biologists revealed a surprising relationship between two enzymes,
which took an enzyme out of a membrane where it was not located, and put it into the soluble phase
where it could be studied. Plant Mol Biol Rep 9: 192-4.
[16] Honma, M and Shimomura, T (1978) Metabolism of 1-aminocyclopropane-1-carboxylic acid. Agric
Biol Chem 42: 1825-31.
[17] Honma, M (1993) Stereospecific reaction of 1-aminocyclopropane-1-carboxylate deaminase, in: Pech,
JC; Latché, A and Balagué, C, Eds. Cellular and molecular aspects of the plant hormone ethylene. pp
111-6 Kluwer Academic Publishers, Dordrecht, The Netherlands, ISBN 0-7923-2169-3.
[18] Minami, R; Uchiyama, K; Murakami, T; Kawai, J; Mikami, K; Yamada, T; Yokoi, D; Ito, H; Matsui,
H and Honma, M (1998) Properties, sequence, and synthesis in Escherichia coli of
1-aminocyclopropane-1-carboxylate deaminase from Hansenula saturnus. J Biochem 123: 1112-8.
[19] Klee, HJ and Kishore, GM (1992) Control of Fruit Ripening and Senescence in Plants. United States
Patent Number: 5,702,933.
[20] Jacobson, CB; Pasternak, JJ and Glick, BR (1994) Partial purification and characterization of
1-aminocyclopropane-1-carboxylate deaminase from the plant growth promoting rhizobacterium
Pseudomonas putida GR12-2. Can J Microbiol 40: 1019-25.
[21] Campbell, BG and Thomson, JA (1996) 1-Aminocyclopropane-1-carboxylate deaminase genes from
Pseudomonas strains. FEMS Microbiol Lett 138: 207-10.
[22] Burd, GI; Dixon, DG and Glick, BR (1998) A plant growth promoting bacterium that decreases nickel
toxicity in plant seedlings. Appl Environ Microbiol 64: 3663-8.
[23] Belimov, AA; Safronova, VI; Sergeyeva, TA; Egorova, TN; Matveyeva, VA; Tsyganov VE, Borisov,
AY; Tikhonovich, IA; Kluge, C; Preisfeld, A; Dietz, KJ and Stepanok VV (2001) Characterization of
plant growth promoting rhizobacteria isolated from polluted soils and containing 1-aminocyclopropane-
1-carboxylate deaminase. Can J Microbiol 47: 642-52.
[24] Ghosh, S; Penterman, JN; Little, RD; Chavez, R and Glick, BR (2003) Three newly isolated plant
growth-promoting bacilli facilitate the growth of canola seedlings. Plant Physiol Biochem 41: 277-81.
[25] Ma, W; Sebestianova, S; Sebestian, J; Burd, GI; Guinel, F and Glick, BR (2003) Prevalence of
1-aminocyclopropane-1-carboxylate deaminase in Rhizobia spp. Antoine van Leeuwenhoek 83: 285-91.
[26] Mayak, S; Tirosh, T and Glick, BR (2004) Plant growth-promoting bacteria that confer resistance to
water stress in tomato and pepper. Plant Sci 166: 525-530.
[27] Deikman, J (1997) Molecular mechanisms of ethylene regulation of gene transcription. Physiol Plant
100: 561-6.
[28] Abeles, FB; Morgan, PW and Saltveit, Jr, ME (1992) Ethylene in plant biology. 2nd ed.: Academic
Press New York, USA, ISBN 0-12-041451-1.
[29] Stearns, J and Glick, BR (2003) Transgenic plants with altered ethylene biosynthesis or perception.
Biotechnol Adv 21: 193-210.
[30] Penrose, DM; Moffatt, BA and Glick BR (2001) Determination of 1-aminocyclopropane-1-carboxylic
acid (ACC) to assess the effects of ACC deaminase-containing bacteria on roots of canola seedlings.
Can J Microbiol 47: 77-80.
[31] Hall, JA; Peirson, D; Ghosh, S and Glick BR (1996) Root elongation in various agronomic crops by the
plant growth promoting rhizobacterium Pseudomonas putida GR12-2. Isr J Plant Sci 44: 37-42.
[32] Morgan, PW and Drew, CD (1997) Ethylene and plant responses to stress. Physiol Plant 100: 620-30.
[33] Grichko, VP and Glick, BR (2001) Amelioration of flooding stress by ACC deaminase-containing plant
growth-promoting bacteria. Plant Physiol Biochem 39: 11-7.
[34] Mayak, S; Tirosh, T and Glick, BR (2004) Plant growth-promoting bacteria that confer resistance in
tomato to salt stress. Plant Physiol Biochem. 42: 565-572.
[35] Nayani, S; Mayak, S and Glick, BR (1998) The effect of plant growth promoting rhizobacteria on the
senescence of flower petals. Ind J Exp Biol 36: 836-9.
236 Bernard R. Glick

[36] Wang, C; Knill, E; Glick, BR and Défago, G (2000) Effect of transferring 1-aminocyclopropane-1-
carboxylic acid (ACC) deaminase genes into Pseudomonas fluorescens strain CHA0 and its gacA
derivative CHA96 on their growth promoting and disease-suppressive capacities. Can J Microbiol 46:
898-907.
[37] Penrose, DM and Glick, BR (2003) Methods for isolating and characterizing ACC deaminase-
containing plant growth-promoting rhizobacteria. Physiol Plant 118: 10-15.
[38] Ma, W; Zalec, K and Glick, BR (2001) Effects of the bioluminescence-labelling of the soil bacterium
Kluyvera ascorbata SUD165/26. FEMS Microbiol Ecol 35: 137-44.
[39] Mishra, D and Kar, M (1974) Nickel in plant growth and metabolism. Bot Rev 40: 395-452.
[40] Imsande, J (1998) Iron, sulfur, and chlorophyll deficiencies: a need for an integrative approach in plant
physiology. Physiol Plant 103: 139-44.
[41] Bar-Ness, E; Chen, Y; Hadar, Y; Marschner, H and Romheld V (1991) Siderophores of Pseudomonas
putida as an iron source for dicot and monocot plants. Plant Soil 130: 231-41.
[42] Burd, GI; Dixon, DG and Glick, BR (2000) Plant growth-promoting bacteria that decrease heavy metal
toxicity in plants. Can J Microbiol 46: 237-45.
[43] Patten, CL and Glick BR (2002) The role of bacterial indoleacetic acid in the development of the host
plant root system. Appl Environ Microbiol 68: 3795-801.
[44] Suthersan, SS (2002) Natural and enhanced remediation systems. pp 239-267, CRC Press, Boca Raton,
USA, ISBN 1566702828.
[45] Cunningham, SD and Berti, WR (1993) Remediation of contaminated soils with green plants: an
overview. In Vitro Cell Dev Biol 29P: 207-12.
[46] Cunningham, SD; Berti, WR and Huang, JW (1995) Phytoremediation of contaminated soils. Trends
Biotechnol 13: 393-7.
[47] Cunningham, SD and Ow, DW (1996) Promises and prospects of phytoremediation. Plant Physiol 110:
715-9.
[48] Huang, X-D; El-Alawi, Y; Penrose, DM; Glick, BR and Greenberg, BM (2004) Responses of plants to
creosote during phytoremediation and their significance for remediation processes. Environ Pollut 130:
453-463.
[49] Huang, X-D; El-Alawi, Y; Penrose, DM; Glick, BR and Greenberg, BM (2004) Multi-process
phytoremediation system for removal of polycyclic aromatic hydrocarbons from contaminated soils.
Environ Pollut 130: 465-476.
[50] Huang, X-D; El-Alawai, Y; Gurska, J; Glick, BR and Greenberg, BM (2004) A multi-process
phytoremediation system for decontamination of Persistent Total Petroleum Hydrocarbons (TPHs) from
soils. Microchem J, in press
[51] Grichko, VP; Filby, B and Glick, BR (2000) Increased ability of transgenic plants expressing the
bacterial enzyme ACC deaminase to accumulate Cd, Co, Cu, Ni, Pb, and Zn. J Biotechnol 81: 45-53.
[52] Nie, L; Shah, S; Burd, GI; Dixon, DG and Glick, BR (2002) Phytoremediation of arsenate contaminated
soil by transgenic canola and the plant growth-promoting bacterium Enterobacter cloacae CAL2. Plant
Physiol Biochem 40: 355-61.
[53] Glick, BR (2004) Teamwork in phytoremediation. Nature Biotechnol 22: 526-527.
[54] Barac, T; Taghavi, S; Borremans, B; Provoost, A; Oeyen, L; Colpaert, JV; Vangronsveld, J and van der
Lelie, D (2004) Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile,
organic pollutants. Nature Biotechnol 22: 583-588.
[55] Sessitsch, A; Coenye, T; Sturz, AV; Vandamme, P; Ait Barka, E; Wang-Pruski, G; Faure, D; Reiter, B;
Glick, BR and Nowak, J (2005) Burkholderia phytofirmins sp. Nov., a novel plant-associated bacterium
with plant beneficial properties. Int J Syst Evol Microbiol, in press.
[56] Bewley, JD and Black, M (1985) Dormancy and the control of germination, in Seeds: physiology of
development and germination, pp. 175-235, Plenum Press, New York, USA, ISBN 0-30-641687-5.
MYCORRHIZAL FUNGI AS HELPING AGENTS IN PHYTOREMEDIATION
OF DEGRADED AND CONTAMINATED SOILS

MIROSLAV VOSÁTKA, JANA RYDLOVÁ, RADKA SUDOVÁ

AND MARTIN VOHNÍK
Institute of Botany, Academy of Sciences of the Czech Republic, 252 43
PrĤhonice, Czech Republic, E-mail: [email protected]

1. Introduction

Plant roots were defined from phytoremediation point of view as “exploratory, liquid-
phase extractors that can find, alter and/or translocate elements and compounds against
large chemical gradients” [1]. Since the roots of majority of higher plants live naturally
in symbiosis with different types of mycorrhizal fungi [2], this association should be
regarded as an organic component of the phytoremediation systems [3, 4, 5]. However,
relatively few studies have focused on the effects of mycorrhiza on phytoremediation
and vice versa, despite the widely acknowledged importance of mycorrhizal symbionts
for plant growth and fitness particularly in harsh environments.
By various mechanisms mycorrhizal fungi are able to take either direct or indirect
part in different processes of phytoremediation of contaminated soils including
phytostabilisation, phytoextraction or phytodegradation. Indirectly, mycorrhiza can
increase plant ability to withstand soil phytotoxicity due to improved nutrition,
particularly in the soils with relatively immobile phosphorus, protect plants against root
pathogens and drought stress and enhance soil aggregation and consequently increase
retention of xenobiotics. These functions are of particular importance mainly in
degraded and contaminated soils that are often poor in nutrients, with low water holding
capacity and adverse physical conditions. Through altered root exudation, mycorrhiza
may also affect composition and activity of microbial communities in the rhizosphere
towards the microflora more effective in xenobiotics degradation or microflora, which
stimulates plant growth (plant growth promoting rhizobacteria such as nitrogen-fixing
bacteria). Furthermore, mycorrhizal fungi also directly help the plant to escape from the
build-up of phytotoxic concentrations of certain pollutants by secreting specific
detoxifying compounds (e.g. organic acids) or by binding the pollutants into fungal
tissues associated with the roots and thus creating a physical barrier against their
translocation to the shoots of the host plant. Important role in direct interactions of all
types of mycorrhiza with soil contamination plays so called extraradical fungal
mycelium (ERM) radiating from the colonised root cortex far into the surrounding soil.
This mycelial network represents extensive interface between roots and pollutants

237
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 237 – 257.
© 2006 Springer.
238 Miroslav Vosátka et al.

dispersed in soil. Until now, some experimental data were published on possible role of
different types of mycorrhiza in phytoremediation of xenobiotics, however, majority of
those represents results of microcosm experiments and is focused mainly on heavy
metals. This chapter is not a detailed review of the literature which is rather extensive in
particular for ectomycorrhizal symbiosis, but it focuses on basic features of the role of
mycorrhizas in phytoremediation, shifts in occurrence of mycorrhizal fungi in degraded
and contaminated soils, the effects of xenobiotics (heavy metals – HMs, polyaromatic
hydrocarbons – PAHs and polychlorinated biphenyls – PCBs) on development of
symbioses and the effects of symbioses on plant tolerance and fitness in contaminated
environment.
The role of mycorrhiza in phytoremediation should be viewed from the perspective
that there are different types of mycorrhizal symbiosis varying in host plant species and
each mycorrhizal type can also exhibit different mechanisms of interaction with
xenobiotics. Two basic types of mycorrhizal symbiosis are endomycorrhiza and
ectomycorrhiza. The endomycorrhiza is further divided into several subtypes among
them arbuscular mycorrhizal and ericoid mycorrhizal being the most important and
having a relevant role in phytoremediation, other subtypes such as orchideoid or
arbutoid mycorrhizas are rather marginal from this point of view.

2. Potential of mycorrhizal fungi in phytoremediation of xenobiotics

2.1. ARBUSCULAR MYCORRHIZAL (AM) FUNGI

The arbuscular mycorrhiza is the most ubiquitous as the arbuscular mycorrhizal fungi
belonging to the phylum Glomeromycota [6] form symbiosis with more than 80% of
vascular plant species [2]. This type of mycorrhiza occurs in majority of the terrestrial
ecosystems, however, it is difficult to be observed by naked eye in nature as the AM
fungi form only spores in the soil and not the fruiting bodies and symbiosis does not
change morphology of the colonised roots.
The AM fungi have not been considered as an important component of HM
phytoremediation protocols in early studies because many metal hyperaccumulators, often
small and slowly growing, belong to plant families Brassicaceae or Caryophyllaceae that
predominantly do not form mycorrhizal symbiosis [7]. However, a range of plant species
intended for phytoremediation practices has been extended also to the plants capable of
forming AM symbiosis and readily producing large biomass (e.g. maize, tobacco, hemp,
sunflower or some hardwood trees), partly along with the progress in genetic
engineering and possibility to introduce genes inducing hyperaccumulation of
xenobiotics [8]. Regarding different phytoremediation strategies feasible for HM
contaminated soils, AM fungi are of interest especially for phytoextraction and
phytostabilisation, whereas their potential for rhizofiltration technique is negligible due
to a limited ability of AM fungi to survive under long-term flooding [4].
Mycorrhizal Fungi as Helping Agents 239

2.2. ERICOID MYCORRHIZAL (ERM) FUNGI

Ericoid mycorrhiza is a distinctive subtype of endomycorrhiza formed mostly by

ascomycetous fungi inhabiting fine roots of the members of Ericaceae [9]. Ericaceous
species have worldwide distribution occurring and sometimes dominating over large
areas in both Northern and Southern Hemisphere. Their habitats are characterized by
challenging environmental conditions including acidic, nutrient poor substrates with high
carbon/nitrogen ratio, low decomposition rate, poor drainage and not least by elevated
level of toxic compounds including heavy metals [2, 9].
The key role of the ErM fungi for the establishment of ericaceous vegetation at HM
polluted habitats is widely accepted [10, 11, 12], as ericoid endophytes show high
resistance to metal toxicity as compared to other mycorrhizal fungi [13]. Since
ericaceous species occurring at HM contaminated sites are relatively slowly growing
dwarf shrubs, their role in phytoextraction is unlikely to reach significant levels on
industrial scale. However, the ErM fungi are suggested to be the key factors enabling
ericaceous dwarf shrubs to dominate at HM polluted sites [11]. The ericaceous plants
are often the first colonisers of such habitats [12], which together with their ability to
grow at nutrient poor substrates underline their phytostabilisation effect in natural
succession on contaminated sites.

2.3. ECTOMYCORRHIZAL (ECM) FUNGI

Ectomycorrhiza occurs in majority of coniferous trees and also in numerous broadleaved

trees mainly from the families Fagaceae, Betulaceae and Ulmaceae. The EcM
associations are formed mainly by basidiomycetous or ascomycetous fungi, most of them
produce fruiting bodies known as mushrooms. Unfortunately, the diversity of fruiting
bodies aboveground is not necessarily connected with the diversity of fungi forming
ectomycorrhizas underground. In contrast to other types of mycorrhiza, these
associations change distinctly morphology of the root system by forming typically
thickened ectomycorrhizal root tips, from which dense fungal mycelium spreads into
surrounding soil exploiting its nutrient sources. Extraradical ectomycorrhizal mycelium
often forms thick and dense hyphal cords called rhizomorphs serving for water and
nutrient transport between distant parts of mycelium.
Plants growing on contaminated substrates benefit from the associations with the
EcM fungi, which can support their growth by improving their nutrient and water uptake
and also alleviate HM toxicity. Areas polluted by HM are often re-vegetated with trees
and EcM fungi are regarded as positive factors influencing survival of tree species at
such areas [14] thus significantly affecting site phytostabilisation.
240 Miroslav Vosátka et al.

3. Effect of xenobiotics on occurrence of mycorrhizal fungi and development

of mycorrhizal symbioses

3.1. ARBUSCULAR MYCORRHIZAL FUNGI

There are relatively scarce data on the effects of soil contamination on field abundance
and diversity of populations of AM fungi. It was found that contamination of soils with
heavy metals decreased numbers of AM propagules and reduced mycorrhizal infectivity
of soils as compared to uncontaminated sites [15, 16, 17]. Nevertheless, spores of the
AM fungi and relatively high levels of root colonisation were reported even from highly
contaminated mine spoils as well as polluted agricultural soils [18, 19, 20, 21, 22]. A
great variability in HM tolerance of different AM fungi isolates have been shown [23,
24, 25], with higher HM tolerance often reported for indigenous isolates obtained from
polluted soils as compared with non-indigenous ones [26, 27, 28]. Especially the isolates
capable to tolerate extreme soil contamination represent a valuable material for potential
inoculation of plants within phytoremediation programmes. More attention should be,
however, paid to the stability of high HM tolerance of these isolates during sub-culturing
process since significant shifts in tolerance to contamination have been recently reported
under keeping in conditions without selection pressure. For example, a decline in Mn
tolerance of a Glomus sp. isolate from a Mn contaminated substrate was shown after its
2-year maintenance in metal-free substrate, compared with the lineage grown constantly
under HM stress in the original substrate [28]. A similar decrease in Al tolerance of
Glomus clarum isolates was reported, with the tolerance decreasing with increasing time
under conditions without metal exposure [25].
Negative effects of heavy metals were observed at any ontogenetic stage of AM
development, from spore germination, germ tube growth, root penetration and
colonisation to proliferation of the extraradical mycelium into surrounding soil and
formation of new spores. Considerable sensitivity of spore germination to HMs was
firstly described almost thirty years ago [29, 30] and this stage was repeatedly shown as
more sensitive than subsequent germ tube growth [23, 31]. A majority of results on HM
effect on AM development refers, however, to the level of root colonisation in HM
contaminated substrate. Generally lower percentage of root colonisation in contaminated
soil is a complex phenomenon caused by inhibition of spore germination, lower
extension of hyphae, unsuccessful root penetration and disruption of the internal hyphae
[11]. In comparison to intraradical phase of colonisation, only little attention has been
paid to the effect of HMs on the development of extraradical mycelium. Both higher [17,
32] and lower [33] sensitivity of ERM to HM exposure has been reported in comparison
to root colonisation.
As regards the interaction of AM fungi with organic pollutants, in one of the few
studies [34] there was found that mycorrhizal colonisation of several plant species by
indigenous AM population was not significantly affected by addition of a single PAH
(anthracene) in concentrations up to 10 g.kg–1. However, colonisation of clover and leek
decreased when industrial PAH polluted soil was added into unpolluted soil, while
maize and ryegrass colonisation was not affected. Spiking of soil with a mixture of three
PAHs reduced colonisation of clover by a non-adapted AM fungus Glomus mosseae
Mycorrhizal Fungi as Helping Agents 241

BEG69 to a half of that in non-spiked soil [35]. Uptake of phosphorus was maintained in
mycorrhizal clover when PAHs were added, but was reduced in non-inoculated clover
and in inoculated clover plants that received surfactant to increase PAH availability.
This may indicate higher PAH sensitivity of clover as compared to the AM fungus. No
such effects were observed for ryegrass. By contrast to the previous results, the
colonisation of alfalfa by G. caledonium was not significantly affected in soil artificially
contaminated with a single PAH (benzopyrene) up to 10 mg.kg–1 but significantly
decreased at 100 mg.kg–1 [36]. In two pot experiments with PCB contaminated soils
(mixture of Delor 103 and Delor 106, total PCB concentrations 25 and 94 g.kg–1,
respectively), relatively high colonisation of tobacco (up to 98%) and alfalfa (up to
52%) roots by two non-adapted AM isolates was observed, depending on plant species,
cultivar and AM fungal isolate [37].

3.2. ERICOID MYCORRHIZAL FUNGI

The data on the effects of HMs on occurrence of ericoid mycorrhizal fungi in the soil are
very limited due to rather difficult quantification of ericoid mycorrhiza in the field
samples – the ErM fungi generally do not form spores or fruiting bodies. However,
occurrence of ericoid mycorrhizal fungi can be linked together with occurrence of their
ericaceous hosts, which are usually highly mycorrhizal at both disturbed and undisturbed
sites.
For the ErM fungi, there are only few reports on how heavy metals influence
development of symbiosis and growth of the host plants. Screening the effects of lead on
the growth of ericoid mycorrhizal mycobiont Hymenoscyphus ericae cultivated in vitro
showed that this fungus was able to grow on media containing up to 400 µg.ml–1 Pb
[38]. The same fungus was able to grow over all iron concentrations tested (0-144
µg.ml–1), exhibiting greater resistance than its host plants [39]. The growth of another
ErM fungus Oidiodendron maius isolated from mycorrhizal roots of Vaccinium
myrtillus growing in heavily contaminated soil was investigated in the presence of zinc
ions [40] and the authors found strong specificity of HM tolerance for each fungal strain.
In the presence of increasing concentrations of Zn salts (especially at higher ion
concentrations), better performance of the mentioned isolate was observed in
comparison with isolates from unpolluted soils. Differential resistance was also observed
among populations of H. ericae isolated from Calluna vulgaris from natural heathland
soils and mine-site soils contaminated with AsO43– and Cu2+ [41]. H. ericae populations
from the mine sites demonstrated resistance to AsO43– compared with the heathland
population; the mine-site populations produced significant growth at the highest AsO43–
concentrations (4.7 mol.m–3), whereas growth of the heathland population was almost
completely inhibited. All isolates produced identical responses to increasing copper
concentrations with no differences observed between mine-site and heathland fungal
populations. Thus, H. ericae on the contaminated sites has developed an adaptive
resistance to arsenate whereas resistance to copper appears to be constitutive.
242 Miroslav Vosátka et al.

3.3. ECTOMYCORRHIZAL FUNGI

Negative effects of heavy metals on the development of EcM symbiosis have been
repeatedly reported, both in vitro as well as in association with host plants. Decreasing
abundance and diversity of Ectomycorrhiza morphotypes with increasing concentration
of heavy metals was observed in a vicinity of the fertiliser factory [42]. Similarly, lower
average number of EcM root tips in Pinus sylvestris was reported from site polluted with
acid rain and with high aluminium availability than from a reference unpolluted site [43].
On the contrary, no evidence for a reduction of the genetic variation of subpopulations
of the EcM fungus Suillus luteus caused by HM contamination was revealed in another
field study [44]. High chromium and nickel concentrations were reported to decrease
EcM colonisation by Pisolithus tinctorius in Eucalyptus urophylla [45]. Five different
heavy metals were shown to negatively influence nitrogen acquisition efficacy of
ectomycorrhizal birch seedlings [46]. On the contrary, no adverse effects of lead
exposure on development of association of two EcM fungi with Norway spruce were
found in another study [47]. Increased concentrations of cadmium and zinc reduced
EcM colonisation of Scots pine with Paxillus involutus and EcM colonisation was found
to be more sensitive to elevated HM levels than host Pinus sylvestris itself [48]. The
authors also showed that both metals negatively influenced not only root colonisation
but also dispersal of EcM fungi from colonised roots towards non-mycorrhizal roots.
This was prominent mainly in the case of cadmium, which suppressed cross-colonisation
of non-mycorrhizal roots with mycelium from roots already colonised by EcM fungi to a
greater extent than the colonisation of already EcM roots. However, it should be noted
that HM tolerance of ectomycorrhizal fungus in pure culture and symbiotic state does
not always correlate. For example, strains of Paxillus involutus and Laccaria bicolor
exhibited similar HM tolerance in pure culture, but in association with Norway spruce
roots only the colonisation by L. bicolor but not by P. involutus was decreased by
cadmium [49].
Similarly as for the other mycorrhizal types, a strong inter- and intraspecific variation
in HM tolerance has been reported for the EcM fungi [46, 50, 51]. The isolates of EcM
fungi originating from polluted soils with high selection pressure were repeatedly shown
to perform better in the presence of increasing concentrations of heavy metals when
compared to isolates from unpolluted soils [50, 52, 53, 54]. For example, experiments
screening HM tolerance of ectomycorrhizal strains of Amanita muscaria, Paxillus
involutus, Pisolithus tinctorius, Suillus bovinus, S. luteus and Thelephora terrestris
isolated from polluted or unpolluted soils revealed that the strains originating from
unpolluted soils were strongly inhibited by HM amendment into growth media, whereas
most of the strains isolated from sporocarps growing in polluted soils exhibited tolerance
to HMs. Some of these strains were able to grow at zinc concentrations up to 1 mg.g–1 in
medium [55]. The authors supposed EcM strains to be naturally selected for HM
tolerance in polluted soil, but at the same time noted the occurrence of HM sensitive
EcM strains at polluted sites, which was attributed to unequal distribution of HMs in
polluted soils. However, HM tolerance of different geographical strains of the EcM
fungi from polluted and unpolluted sites is not always predetermined by their origin, as it
Mycorrhizal Fungi as Helping Agents 243

was the case of different Paxillus involutus strains screened for alleviation of aluminium
stress [56].

4. Effect of mycorrhizal fungi on xenobiotics uptake and tolerance of the host

plants

4.1. ARBUSCULAR MYCORRHIZAL PLANTS

Inoculation with AM fungi has been repeatedly reported to modify HM uptake by host
plants, however, the results are contradictory [4, 11]. In general, AM plants are more
efficient than non-mycorrhizal in the acquisition of micronutrients such as copper, iron,
manganese and zinc when available at low concentrations [57, 58, 59, 60, 61]. However,
when grown in excess of micronutrients or in soils contaminated by HMs with unknown
biological function such as cadmium, chromium or lead, both AM-mediated increases
[62, 63, 64, 65, 66] and decreases [57, 60, 67, 68] of HM concentrations in plant tissues
were observed, depending strongly on plant-fungus combination and cultivation
conditions.
Protective role of AM fungi for plants growing in HM contaminated soils together
with lower HM concentrations in plant tissues were frequently reported for inoculated
plants [59, 69, 70], although the opposite observations, i.e. growth inhibition connected
with higher HM concentrations were also shown [62, 66, 71]. Growth stimulation
induced by inoculation with AM fungi is not always connected with lower HM
concentrations in plant tissues, for example alleviation of zinc phytotoxicity in
mycorrhizal plants without any effect of inoculation on Zn concentrations was observed
[72]. In some cases, AM can be of fundamental importance for plant survival in heavily
contaminated substrate: non-inoculated plants of several species (maize, barley, alfalfa)
were not able to survive long-term without inoculation with an isolate from the
rhizosphere of a metallophyte species Viola calaminaria [21]. When inoculated with
reference fungal isolate of the same species from unpolluted soil, the positive effect on
plant growth and HM uptake was less pronounced [73]. Differences between AM fungal
isolates of different origin were reported also in other study [74] where AM fungi from a
zinc contaminated site were more effective in increasing Adropogon gerardii biomass at
higher levels of Zn in the soil whereas plant growth at lower levels of soil Zn was better
with mycorrhizal fungi from an uncontaminated site. In some cases, positive effect of
AM inoculation on host plant growth is more pronounced under conditions of HM
contamination than in control conditions or under slight contamination levels [75, 76]. It
was observed that only an obligately mycotrophic species Andropodon gerardii but not
facultatively mycotrophic Festuca arundinacea benefited from AM in uncontaminated
soil whereas both species benefited from inoculation under HM contamination [77].
Protective effect of AM can vary not only among plant species but even varieties [69].
Recently, also indigenous soil bacteria were shown to significantly modify the effect of
AM on HM uptake by host plants [78, 79]. Therefore, fungal isolates should be
thoroughly screened prior to the final inoculation of a particular host plant species not
244 Miroslav Vosátka et al.

only in greenhouse experiments, but also directly on contaminated sites where AM fungi
inocula interact with the whole microbial community.
The data on the uptake of organic xenobiotics by AM plants are very scarce.
Amendment of soil with up to 5% of a heavily PAH polluted soil (8.1 g PAHs kg–1)
reduced growth of ryegrass but AM colonisation with a non-adapted fungus (G. mosseae
BEG69) was not affected [34]. At 5 g of PAHs kg–1 only mycorrhizal plants survived.
The fate of PAHs in the rhizosphere and mycorrhizosphere of ryegrass inoculated with
G. mosseae BEG69 was investigated in soil spiked with 5 g.kg–1 of anthracene or with 1
g.kg–1 of a mixture of eight PAHs [80]. Proportion of the total PAH amount that was
taken up to plant tissues or adsorbed to roots was negligible and major part of PAH
dissipation in the rhizosphere was due to biodegradation or biotransformation. The
authors found no difference between inoculated and non-inoculated ryegrass in PAH
dissipation, however, shoot concentrations of PAHs and their adsorption to roots were
lower in mycorrhizal than in non-mycorrhizal plants.

4.2. ERICOID MYCORRHIZAL PLANTS

Results of the studies confirmed mycorrhizal colonisation with the ErM fungi as an
important factor positively influencing the resistance to heavy metals. Ericaceous plant
species themselves display HM resistance [81], but it is supposed that successful
colonisation of certain HM polluted sites requires adaptations from both host plant and
ericoid mycorrhizal fungus [12]. In general all reports on experiments with the ErM
fungi showed lower content of heavy metals in shoots of mycorrhizal plants as compared
to non-mycorrhizal plants but often increased HM accumulation in the roots. Seedlings
of Vaccinium macrocarpon colonised by Hymenoscyphus ericae showed increased
tolerance to lead expressed in reduced translocation of Pb to the shoots when compared
to non-mycorrhizal seedlings [38]. In another study, two races of Calluna, one from a
HM polluted site, the other from an unpolluted natural heathland, were compared under
mycorrhizal and non-mycorrhizal conditions in sand cultures with different levels of
copper and zinc [82]. The colonisation by ErM fungi lead to significant reduction of the
HM content in shoots of experimental plants. The ErM fungi from the study were Cu
resistant regardless of the site of their origin. Mycorrhizal endophytes isolated from
Calluna vulgaris, Vaccinium macrocarpon and Rhododendron ponticum were reported
to protect their host plants against metal toxicity when grown together in sand with
addition of copper (concentrations from 0 to 75 mg.l –1) and zinc (concentrations from
0-150 mg.l–1) [83]. All ErM fungal endophytes were able to grow even at the highest
concentrations of both elements in pure cultures. Mycorrhizal plants also showed at least
some growth in all treatments, whereas non-mycorrhizal plants failed to grow at all but
the lowest Cu and Zn concentrations. Mycorrhizal plants had lower concentrations of
metals in their shoots than non-mycorrhizal but higher metal concentrations in roots. The
role of the ErM colonisation in the regulation of iron uptake was investigated in a study
where ericaceous plants were exposed to various Fe concentrations corresponding to
those occurring in the extracts from heathland soil (0-144 µg.ml–1) [39]. The authors
observed very high affinity of mycorrhizal roots of V. macrocarpon and C. vulgaris for
absorption of Fe at low concentrations, which was not observed for non-mycorrhizal
roots. They suggested the involvement of a hydroxamate siderophore in the absorption
Mycorrhizal Fungi as Helping Agents 245

of Fe by mycorrhizal plants at low external concentrations. At higher concentrations, the

presence of fungal endophyte decreased Fe uptake to shoots resulting in lower
concentrations of Fe in shoots comparing to non-mycorrhizal plants. A strain of H.
ericae decreased Fe content in shoots of Vaccinium macrocarpon and reduced HM
toxicity symptoms of the host plant [84].

4.3. ECTOMYCORRHIZAL PLANTS

Regarding heavy metal uptake by host plants, EcM fungi can decrease the uptake of
range of elements, e.g. aluminium [85], cadmium [50], copper [86], lead [87] and zinc
[50] and thus alleviate HM toxicity to the host plants. For example, the seedlings of
birch, pine and spruce were reported to be less susceptible to toxic concentrations of
zinc, copper, nickel and aluminium when formed associations with several EcM fungi
[88]. In another study, Paxillus involutus ameliorated the toxicity of cadmium and zinc
to Scots pine seedlings in terms of root length [89]. Even though cadmium inhibited
ectomycorrhiza formation in seedlings, colonisation with P. involutus decreased
cadmium and zinc transport to the plant shoots and also altered the ratio of zinc
transported to the roots and shoots, with higher amount of cadmium retained in the roots
of the seedlings. Investigations on the effect of Paxillus involutus, Suillus luteus and
Thelephora terrestris on the copper resistance of Pinus sylvestris revealed that although
Suillus luteus was more sensitive to increased copper concentrations than the other two
fungi, it prevented HM accumulation in the needles of Pinus sylvestris; this ability was
absent in T. terrestris [86]. Alleviating effect of the EcM fungi on HM toxicity was
observed across wide range of fungal species, therefore, it seems to be universal feature:
protective effects of Thelephora terrestris, Laccaria laccata, Scleroderma citrinum,
Paxillus involutus, Suillus luteus and S. bovinus against cadmium in Pinus sylvestris
seedlings were demonstrated [50].
On the other hand, some studies report that EcM fungi do not limit uptake of heavy
metals by their host plants, moreover, they can increase such an uptake, which was the
case of zinc in Pinus sylvestris inoculated with Thelephora terrestris [54]. Increased
uptake of aluminium by host plants mediated by EcM fungi was show as well [90]. EcM
fungi can affect HM stress in host plants differently according to the metal screened:
Laccaria proxima, Lactarius hibbardae, L. rufus and Scleroderma flavidum increased
tolerance of Betula papyrifera to different concentrations of nickel but negative growth
effect of ectomycorrhizal fungi were observed at elevated levels of copper [91]. Also the
concentration of the metal in the medium plays a significant role: regulation of zinc
uptake by EcM fungi in Pinus sylvestris was dependent on the concentration of metal in
the mycorrhizosphere [92]. At low Zn concentrations, the EcM fungi increased its
uptake, whereas at high external Zn concentrations, the EcM fungi were able to maintain
shoot Zn concentrations at relatively low levels. This effect of EcM fungi was
hypothesised to be involved also in uptake of other heavy metals and it might explain
also contradictory data reported on HM uptake [93].
Little information is available on EcM effects on host tolerance to organic pollutants.
Tolerance of pine seedlings to m-toluate was unaltered regardless of the presence or
absence of the EcM fungus Suillus bovinus or biodegradative bacteria in microcosms
containing expanded clay and growth media [94]. On the contrary, fungus tolerance was
246 Miroslav Vosátka et al.

significantly increased when grown in symbiosis with pine: it was able to withstand two-
order higher concentration of m-toluate as compared to the pure culture on agar plates
(0.02 vs. 2.0 mg.kg–1). Fungal survival on agar was increased in a co-culture with the
degradative bacterial strain.

5. Possible mechanisms of the interaction of mycorrhizal fungi and their host plants
with xenobiotics

5.1. HEAVY METALS

Most of the references on potential mechanisms of plants-mycorrhizae-xenobiotics

interaction are again related to heavy metals. It is generally acknowledged that
mycorrhizal amelioration of metal toxicity is achieved via several physiological
processes rather than through a single mechanism and that participation of single
mechanisms in metal detoxification is metal- and species-specific [4, 95, 96].
Firstly, extraradical fungal mycelium radiating from the roots and exploiting non-
rhizosphere soil enables the mycorrhizal plants to capture nutrients, mainly immobile
ones such as phosphorus, zinc, copper and ammonium, also from sources non-available
to non-mycorrhizal plants. Due to better nutrition, growth stimulation is often observed
in mycorrhizal plants, sometimes connected with lower concentrations of HMs in plant
biomass [e.g. 68, 97]. However, this “growth dilution effect” cannot explain all the
experimental results and also another, direct mechanisms must be involved.
As the main mechanism, immobilisation of HMs in both intra- and extraradical
fungal structures and resulting restriction of metal transfer into plant tissues has been
suggested [4, 98]. In accordance with this theory, it was shown that inoculation with
different EcM strains decreased zinc and cadmium uptake into pine shoots and the
fungus producing more extensive extraradical mycelium showed greater effect on
overcoming metal toxicity and lowering shoot metal concentrations [50, 54]. Non-
mycorrhizal pine seedlings were reported to transport more copper to above-ground
parts, however, roots and extraradical mycelium of inoculated plants accumulated up to
two times more metal than non-mycorrhizal roots [99]. Similarly, EcM colonisation
decreased Zn and Cd transport to the plant shoots and influenced the proportion of Zn
transported to the roots and shoots, with a higher proportion retained in the roots of the
seedlings [100]. Recently, accumulation of large amounts of aluminium at the fungal
mantle and in areas with the Hartig net was described in the pine roots colonised by an
EcM fungus [101]. Increased root but decreased shoot HM concentrations were also
reported for three ericoid mycorrhizal plant species [83] and the authors hypothesised
that fungus provided adsorptive surfaces for binding of metals and thus facilitated
exclusion of metals from the shoots. Concerning AM fungi, metal immobilisation in
fungal tissues and lower metal partitioning to the shoots of inoculated plants was also
repeatedly demonstrated [57, 102]. For example, root-to-shoot ratio of Cd
concentrations of 3.15 was reported in mycorrhizal clover plants compared to only 1.66
in non-mycorrhizal plants [67]. However, retention of the HMs in fungal structures does
not seem to be a universal phenomenon as inoculation was shown to lead to the
Mycorrhizal Fungi as Helping Agents 247

completely opposite results in some cases and significantly enhanced translocation of

HMs from roots to shoots of mycorrhizal plants was observed [68, 103]. Retention of
HMs in fungal structures inside colonised roots was supported also by several
microanalytical studies. Accumulation of metals within intracellular AM hyphae, mainly
in phosphate-rich materials in the vacuoles, was reported in the mycorrhizal roots of the
fern Pteridium aquilinum from a contaminated soil [104]. A massive accumulation of
heavy metals in inner cortical cells of AM colonised maize roots where arbuscules and
intraradical hyphae were located was also confirmed using different microbeam
techniques [73]. Further, a high cadmium and zinc sorption capacity of the ERM of AM
fungi in comparison with other microorganisms was shown [105] and a metal tolerant
Glomus mosseae isolate showed significantly higher binding capacity than non-tolerant
isolate of the same species. Similarly, a very high cation exchange capacity for lead
absorption was reported for the fungal mycelium of two EcM fungi [106]. Different
HMs can be detoxified via different mechanisms, as shown by a study where Cd was
complexed predominantly extracellularly in the Hartig net hyphae, whereas zinc was
sequestered also in the cytosol of mantle hyphae [107]. Binding of cadmium onto fungal
cell walls as well as accumulation of Cd in the vacuolar compartments was described for
in vitro grown EcM fungus Paxillus involutus [108]. Cytoplasmatic sequestration of
metals may be realised via different thiol compounds, including reduced glutathione,
phytochelatins and metallothioneins that are essential components of HM detoxification
pathways in various organisms. Using a specific histochemical staining, induction of
protein-bound thiolate clusters in EcM hyphae in response to cadmium was detected
[109] and the sulphur-rich clusters were hypothesised to be derived from metallothionein
or metallothionein-like proteins. Later, a metallothionein-like protein was detected in two
tolerant EcM fungi in response to copper exposure and this compound associated with as
much as 90% of the total copper [110]. Recently, a drastically increased production of
the Cd-modulated compound most probably related to a metallothionein was detected in
Cd-exposed EcM mycelium of Paxillus involutus [111]. In contrast, neither
phytochelatins nor metallothioneins were detected in EcM fungus exposed to Cd, in
spite of increased glutathione levels [112] and the authors concluded that rather
glutathione and glutamylcystein were involved in metal detoxification. Similarly,
increased contents of glutathione and lack of phytochelatin induction in two different EcM
fungi exposed to cadmium were reported recently [111, 113]. As far as AM fungi,
increased synthesis of metallothioneins by mycorrhizal plants was also proposed [114,
115], however, higher synthesis of peptides containing thiol groups was not revealed in
roots of AM-inoculated plants exposed to high Cd and Cu concentrations, in spite of
increased concentrations of cysteine, glutathione and glutamycysteine. Recently, a novel
metallothionein-like gene that was selectively up-regulated by Cu exposure was
identified in the ERM of AM fungi [116].
Apart from metal binding onto cell walls, accumulation of copper in the
mucilaginous outer hyphal wall zone of the ERM of AM fungi was reported [117] and
the authors suggested an employment of extracellular glycoprotein glomalin into HM
detoxification. Similarly in EcM fungi, zinc was shown to be bound not only to
extraradical hyphal walls, but also to extrahyphal slime polymers [53]. In accordance
with this finding, a frequent excretion of extracellular mucilaginous substances by EcM
248 Miroslav Vosátka et al.

fungal cultures exposed to higher metal concentrations was observed [118]. The
correlation between excretion of loosely adhering extrahyphal slime and zinc tolerance
of fungal strains and the degree of amelioration of zinc toxicity to mycorrhizal host
plants was reported also for ErM fungi [119]. A significantly higher production of oxalic
acid by EcM pine seedlings than non-mycorrhizal controls was reported in response to
aluminum exposure [120] and it was assumed that ectomycorrhizae produce or stimulate
the roots to secrete organic acids that bind Al and prevent its absorption. Study on a Zn
and Cd tolerant strain of ErM fungus Oidiodendron maius showed that the increase in
concentration of Zn ions in cultivation medium induced a shift in the spectrum of
proteins secreted by the fungus towards more basic, low molecular weight polypeptides
[121]. Among other products, two superoxide dismutase isoforms were found as well.
These are known to act in HM response in plants, animals and microorganisms and the
authors hypothesised that they could play similar role also in ErM fungi. As another
option how mycorrhizal plants may influence metal uptake, modification of metal
solubility mediated by changes in pH of soil solution was suggested [122] as higher pH
values and lower zinc concentrations in soil solution were observed in mycorrhizal
treatments.
Interesting mechanism of eliminating arsenate toxicity was reported for the ErM
fungi growing in arsenate-polluted soils. Arsenate is analogical to phosphate and can be
transported across the plasma membrane via the phosphate co-transporter [123]. The
ErM accumulating arsenate in their hyphae would thus transport phosphorus together
with arsenate into plant tissues resulting in negative consequences to host plants. But
Hymenoscyphus ericae strains from arsenic-polluted sites had the ability to reduce
arsenate to arsenite and excrete the latter from their hyphae [124, 125]. By this way, H.
ericae actively accumulated phosphate while eliminating arsenate and transported
phosphate to the host plant. Ericaceous host plants appear to lack altered phosphate-
arsenate uptake system and seem to be dependent on their mycobionts regarding
alleviation of arsenate toxicity [125].
Also other mechanisms how mycorrhizal fungi cope with elevated concentrations
heavy metals in their environment have been proposed (e.g. chelating by pigments with
metal binding ability such as melanin, enzymes resistant to inactivation by metal
binding, modified uptake systems at the plasmalemma, ability of membranes to
withstand attack from oxygen radicals), their role, however, remains unravelled [12, 96,
126].

5.2. ORGANIC COMPOUNDS

It was shown that dissipation of condensed PAHs might be enhanced in the presence of
AM fungi in the soil spiked with a mixture of different PAHs [127]. These results were
consecutively verified in pot experiments conducted in two different industrially
polluted soils [128]. In general, following explanations were suggested by the authors to
elucidate the contribution of AM to PAH degradation: i) mycorrhiza modifies root
enzyme activity, exudation and architecture in a manner that stimulates PAH
degradation, either by root-derived enzymes or by rhizosphere microorganisms, ii)
mycorrhizal colonisation affects root surface or rhizosphere soil properties that act on
PAH availability through adsorption. Several examples of above mentioned capabilities
Mycorrhizal Fungi as Helping Agents 249

of AM fungi can be found in the literature. Mycorrhiza was reported to enhance amount
of hydrogen peroxide in the roots [129] and to stimulate oxidoreductase activities in the
roots and the rhizosphere [130]. These mechanisms may contribute to increase of PAH
dissipation associated with mycorrhizal roots. Mycorrhizal colonisation also modifies
root exudation both quantitatively and qualitatively [131, 132], which could have further
effects on the composition and activity of microbial communities in the rhizosphere
[133, 134]. This mycorrhiza-associated microflora may be more effective in organics
degradation in comparison with that related to non-mycorrhizal roots. Accumulation of
phenolics in the roots or rhizosphere soil of mycorrhizal plants could induce degradation
of more complex aromatic compounds [135].
Effects of AM on root longevity and proportion of higher order lateral roots with
short life span was documented for several plant species [136]. Accumulation of root
debris together with the ability of mycorrhizal plants to enrich the soil in organic matter
[137] may contribute to enhanced PAH adsorption in plant rhizosphere. Finally, AM
colonisation results in formation of an extensive network of extraradical mycelium,
which can modify surrounding environment by extrusion of glycoproteins [138] and
extracellular enzymes [139]. Hyphal biomass together with the two latter substances can
serve as substrates for microbial growth. Nutrients derived from extraradical hyphae of
AM fungi were hypothesised to drive co-metabolic degradation of PAHs within small soil
pores where PAHs are spatially unavailable to roots [127].
Most of the ectomycorrhizal fungi have some limited ability to use polymers such as
lignin and cellulose as substrates for their growth [2], however, their ability to degrade
polymers of this kind was supposed to be much lower as compared to wood
decomposers or even of some ericoid mycorrhizal fungi [140, 141, 142]. As it was
recently reviewed, most of the EcM fungi screened for degradation of persistent organic
pollutants are able to transform these compounds [143]. On the other hand, most reports
concern degradation in pure cultures, rather than in symbiosis with plants. It has been
demonstrated that EcM fungi can degrade e.g. trinitrotoluene [144], dichlorphenol [145],
atrazine [146] and several 3-5 ring PAHs [147]. Only limited PAH degrading abilities of
EcM fungi and decreased mineralisation of some PAHs in microcosms with EcM pine
seedlings vs. unplanted microcosms inoculated with ectomycorrhizosphere soil were
documented [148]. On the contrary, mineralisation of dichlorphenol was stimulated when
EcM fungi were cultivated in symbiosis with pine than when grown in absence of the
host [145].
Also degradation of lower chlorinated PCBs by eight of 13 studied species of EcM
fungi was reported, however, only two species were able to degrade 4 and 5 chlorinated
biphenyls [149]. While EcM fungi could sequentially hydroxylate a halogenated
biphenyl ring (parent compound is then more polar and bioavailable), they were unable
to cleave the ring [150]. However, hydroxylation of the biphenyl ring can be an
important initial metabolic step facilitating degradation of PCBs by other rhizosphere
organisms that have the capacity to degrade compounds further [143]. In this context it
can be hypothesised that the presence of EcM fungi may negate the need for the
presence of co-substrates. Although the tolerance of EcM fungi to different aromatic
compounds is lower as compared to white rot fungi and depends on the compound type,
its external concentration and the fungal species, the degradative capabilities of different
250 Miroslav Vosátka et al.

fungi varied between species but not generally between the biotrophic and saprotrophic
fungi [151].

6. Conclusion

Application of mycorrhizal fungi as supportive agents for phytoremediation can

generally eliminate or reduce some known limitation of phytoremediation processes.
Mycorrhizal fungi can help plants to acquire nutrients more effectively, increase their
tolerance to edaphic stress and change accumulation of pollutants in plant tissues.
Reduced HM concentrations in plant tissues of mycorrhizal plants, together with an
amplified barrier against metal translocation from plant roots to shoots, are favourable
for phytostabilisation that is aimed at prevention of contamination spreading into the
surroundings. The presence of AM fungi leading to decreased HM concentrations in
plant shoots can be also an important factor for quality and safety of plants which enter
food chains such as forage crops, medicinal herbs and vegetables. For example, tobacco
was found to accumulate significantly less cadmium in the leaves when inoculated with
selected AM fungi [8].
On the other hand, enhanced HM concentrations in the shoots of mycorrhizal plants
induced by some mycorrhizal fungal isolates represent optimal conditions for
phytoextraction technique. In some cases, elimination of present AM fungi populations (in
particular if they involve strains decreasing HM translocation to the shoots) might be
recommended before phytoextraction beginning. For example, application of the
fungicide benomyl detrimental to mycorrhiza was shown to significantly decrease root
colonisation and simultaneously to increase Pb concentrations in plant shoots [153].
Mycorrhizal fungi may be crucial also for re-vegetation efforts after heavy metal
removal as the rate of site re-vegetation may be accelerated when AM fungi are present
in soil. However, little is known about mycorrhiza functioning under conditions imposed
by particular metal remediation protocols. First investigations have appeared showing
that the quantity and species composition of glomalean propagules and the functioning
of AM symbiosis could be significantly influenced by phytoextraction treatments (the
choice of plant species, i.e. non-mycotrophic vs. mycotrophic, soil supplements etc.) [7].
Recently, negative effects of synthetic chelates used for chelate-induced HM
phytoextraction such as EDTA on AM development were also described [102, 152].
Based on the intended phytoremediation strategy, the appropriate management of
native fungi and/or application of artificial inocula should be chosen. For the
introduction of artificial inocula, it seems to be essential to formulate specific products
(mixtures of strains compatible with target plants and environment) rather than to use
generic products all over the scale of edaphic conditions. However, it should considered
that the effect of mycorrhizal inoculation may interact e.g. with fertilisation regime as
demonstrated for the grasses grown in mine tailings containing high levels of zinc: plant
growth was best after inoculation combined with nitrogen and phosphorus fertilisation,
whereas neither mycorrhiza nor fertilisation alone had any effect on plant biomass [77].
Therefore, mycorrhizal inoculation cannot be considered as panacea and should be
combined with other practices such as appropriate fertilisation or soil amendments to
maximise re-vegetation success.
Mycorrhizal Fungi as Helping Agents 251

To conclude, an extension of knowledge on the involvement of mycorrhizal fungi in

phytoremediation should still be achieved. Great attempt should be also undertaken to
increase awareness of potential users of mycorrhizal inoculants regarding all possible
functions and impacts of mycorrhiza applications in phytoremediation processes.

Acknowledgement

Presentation of this contribution was supported by the grant 526/04/0996 of the Grant
Agency of the Czech Republic and by the Research Centre for Bioindication and
Revitalisation funded by the Ministry of Education, Youth and Sports of the Czech
Republic (grant 1M67985939).

References
[1] Cunningham, SD; Berti, WR and Huang, JW (1995) Phytoremediation of contaminated soils. Trends
Biotechnol 12: 393-397
[2] Smith, SE and Read, DJ (1997) Mycorrhizal symbiosis. Academic Press, London.
[3] Donnelly, PK and Fletcher, JS (1994) Potential use of mycorrhizal fungi as bioremediation agents. In:
Anderson T.A. (ed.) Bioremediation through Rhizosphere Technology. ACS Symposium series No.
563, American Chemical Society, pp. 93-99
[4] Leyval, C; Turnau, K and Haselwandter, K (1997) Effect of heavy metal pollution on mycorrhizal
colonization and function: physiological, ecological and applied aspects. Mycorrhiza 7: 139-153
[5] Gaur, A and Adholeya, A (2004) Prospects of arbuscular mycorrhizal fungi in phytoremediation of
heavy metal contaminated soils. Curr Sci 86: 528-534
[6] Schüssler, A; Schwarzott, D and Walker, C (2001) A new fungal phylum, the Glomeromycota:
phylogeny and evolution. Mycol Res 105: 1413-1421
[7] Pawlowska, TE; Chaney, RL; Chin, M and Charvat, I (2000) Effects of metal phytoextraction practices
on the indigenous community of arbuscular mycorrhizal fungi at a metal-contaminated landfill. Appl
Env Microbiol 66: 2526-2530
[8] Janoušková, M; Pavlíková, D; Macek, T and Vosátka, M (2005) Arbuscular mycorrhiza decreases
cadmium phytoextraction by transgenic tobacco with inserted metallothionein. Plant Soil 272: 29-40
[9] Read, DJ (1996) The structure and function of the ericoid mycorrhizal root. Ann Bot 77: 365-374
[10] Cairney, JWG (2000) Evolution of mycorrhiza systems. Naturwissenschaften 87: 467-475
[11] Meharg, AA and Cairney, JWG (2000) Co-evolution of mycorrhizal symbionts and their hosts to
metal-contaminated environments. Adv Ecol Res 30: 69-112
[12] Cairney, JWG and Meharg, AA (2003) Ericoid mycorrhiza: a partnership that exploits harsh edaphic
conditions. Eur J Soil Sci 54: 735-740
[13] Shaw, G and Read, DJ (1989) The biology of mycorrhiza in the Ericaceae. XIV. Effects of iron and
aluminium on the activity of acid phosphatase in the ericoid endophyte Hymenoscyphus ericae (Read)
Korf and Kernan. New Phytol 113: 529-533
[14] Wilkinson, DM and Dickinson, NM (1995) Metal resistance in trees: the role of mycorrhizae. Oikos
72: 298-300
[15] Boyle, M and Paul, EA (1988) Vesicular-arbuscular mycorrhizal associations with barley on sewage-
amended plots. Soil Biol Biochem 20: 945-948
[16] Leyval, C; Singh, BR and Joner, EJ (1995) Occurrence and infectivity of arbuscular mycorrhizal fungi
in some Norwegian soils influenced by heavy metals and soil properties. Water Air Soil Poll 84:
203-216
[17] del Val, C; Barea, JM and Azcón-Aguilar, C (1999) Assessing the tolerance to heavy metals of
arbuscular mycorrhizal fungi isolated from sewage sludge-contaminated soils. Appl Soil Ecol 11:
261-269
252 Miroslav Vosátka et al.

[18] Ietswaart, JH; Griffioen, WAJ and Ernst, WHO (1992) Seasonality of VAM infection in three
populations of Agrostis capillaris (Graminae) on soil with or without heavy metal enrichment. Plant
Soil 139: 67-73
[19] Weissenhorn, I; Mench, M and Leyval, C (1995) Bioavailability of heavy metals and arbuscular
mycorrhizas in a sewage sludge amended sandy soil. Soil Biol Biochem 27: 287-296
[20] Pawlowska, TE; Blaszkowski, J and Rühling, A (1996) The mycorrhizal status of plants colonizing a
calamine soil mound in southern Poland. Mycorrhiza 6: 499-505
[21] Hildebrandt, U; Kaldorf, M and Bothe, H (1999) The zinc violet and its colonization by arbuscular
mycorrhizal fungi. J Plant Physiol 154: 709-717
[22] Turnau, K; Ryszka, P; Gianinazzi-Pearson, V and van Tuinen, D (2001) Identification of arbuscular
mycorrhizal fungi in soils and roots of plants colonizing zinc wastes in southern Poland. Mycorrhiza
10: 169-174
[23] Weissenhorn, I; Leyval, C and Berthelin, J (1993) Cd-tolerant arbuscular mycorrhizal (AM) fungi from
heavy-metal polluted soils. Plant Soil 157: 247-256
[24] Bartolome-Esteban, H and Schenck, NC (1994) Spore germination and hyphal growth of arbuscular
mycorrhizal fungi in relation to soil aluminium saturation. Mycologia 86: 217-226
[25] Kelly, CN; Morton, JB and Cumming, JR (2005) Variation in aluminium resistance among arbuscular
mycorrhizal fungi. Mycorrhiza 15: 193-201
[26] Gildon, A and Tinker, PB (1983) Interactions of vesicular-arbuscular mycorrhizal infection and heavy
metals in plants. I. The effects of heavy metals on the development of vesicular-arbuscular
mycorrhizas. New Phytol 95: 247-261
[27] Weissenhorn, I and Leyval, C (1995) Root colonization of maize by a sensitive and a Cd-tolerant
Glomus mosseae and cadmium uptake in sand culture. Plant Soil 175: 233-238
[28] Malcová, R; Rydlová, J and Vosátka, M (2003) Metal-free cultivation of Glomus sp. BEG 140 isolated
from Mn-contaminated soil reduces tolerance to Mn. Mycorrhiza 13: 151-157
[29] Hepper, CM and Smith, GA (1976) Observations on the germination of Endogone spores. Trans Br
Mycol Soc 66: 189-194
[30] Hepper, CM (1979) Germination and growth of Glomus caledonius spores: the effects of inhibitors
and nutrients. Soil Biol Biochem 11: 269-277
[31] McIlveen, WD and Cole, H (1978) Influence of zinc on development of the ectomycorrhizal fungus
Glomus mosseae and its mediation of phosphorus uptake by Glycine max “Amsoy 71”. Agric
Environm 4: 245-256
[32] Batkhuugyin, E; Rydlová, J and Vosátka, M (2000) Effectiveness of indigenous and non-indigenous
isolates of arbuscular mycorrhizal fungi in soils from degraded ecosystems and man-made habitats.
Appl Soil Ecol 14: 201-211
[33] Arines, J; Porto, ME and Vilariño, A (1992) Effect of manganese on vesicular-arbuscular mycorrhizal
development in red clover plants and on soil Mn-oxidizing bacteria. Mycorrhiza 1: 127-131
[34] Leyval, C and Binet, P (1998) Effect of polyaromatic hydrocarbons in soil on arbuscular mycorrhizal
plants. J Environ Qual 27: 402-407
[35] Joner, EJ and Leyval, C (2001) Influence of arbuscular mycorrhiza on clover and ryegrass grown
together in a soil spiked with polycyclic aromatic hydrocarbons. Mycorrhiza 10: 155-159
[36] Liu, SL; Luo, YM; Cao, ZH; Wu, LH; Ding, KQ and Christie, P (2004) Degradation of benzo[a]pyrene
in soil with arbuscular mycorrhizal alfalfa. Environ Geochem Hlth 26: 285-293
[37] Rydlová, J; Stárek, L; Macková, M; Vosátka, M and Macek, T (2005) Interactions of arbuscular
mycorrhizal fungi and PCB degrading bacteria in two soils contaminated with PCB. Biodegradation
(submitted)
[38] Hashem, AR (1990) Hymenoscyphus ericae and the resistance of Vaccinium macrocarpon to lead.
Trans Myc Soc Jap 31: 345-353
[39] Shaw, G; Leake, JR; Baker, AJM and Read, DJ (1990) The biology of mycorrhiza in the Ericaceae.
XVII. The role of mycorrhizal infection in the regulation of iron uptake by ericaceous plants. New
Phytol 115: 251-258
[40] Martino, E; Turnau, K; Girlanda, M; Bonfante, P and Perotto, S (2000) Ericoid mycorrhizal fungi
from heavy metal polluted soils: their identification and growth in the presence of zinc ions. Mycol
Res 104: 338-344
[41] Sharples, JM; Meharg, AA; Chambers, SM and Cairney, WG (2001) Arsenate resistance in the ericoid
mycorrhizal fungus Hymenoscyphus ericae. New Phytol 151: 265-270
Mycorrhizal Fungi as Helping Agents 253

[44] Stankenkeviþiene, D and Peþiulyte, D (2004) Functioning of ectomycorrhizas and soil microfungi in
deciduous forests situated along a pollution gradient next to a fertilizer factory. Pol J Environ Stud 13:
715-721
[45] Rudawska, M; Kieliszewska-Rokicka, B and Leski, T (1996) Effect of acid rain and aluminium on the
mycorrhizas of Pinus sylvestris, in Azcón-Aguillar C and Barea JM (Eds.), Mycorrhizas in integrated
systems. From genes to plant development. European Commission, Luxembourg, pp. 452-454
[46] Muller, LAH; Lambaerts, M; Vangronsveld, J and Colpaert, JV (2004) AFLP-based assessment of the
effects of environmental heavy metal pollution on the genetic structure of pioneer populations of
Suillus luteus. New Phytol 164: 297-303
[47] Aggangan, NS; Dell, B and Malajczuk, N (1989) Effects of chromium and nickel on growth of the
ectomycorrhizal fungus Pisolithus tinctorius and formation of ectomycorrhizas on Eucalyptus
urophylla Blake. S.T. Geoderma 84: 15-27
[48] Blaudez, D; Jacob, C; Turnau, K; Colpaert, JV; Ahonen-Jonnarth, U; Finlay, R; Botton, B and Chalot,
M (2000) Differential reponses of ectomycorrhizal fungi to heavy metals in vitro. Mycol Res 104:
1366-1371
[49] Jentschke, G; Fritz, E and Godbold, DL (1991) Distribution of lead in mycorrhizal and non-
mycorrhizal Norway spruce seedlings. Physiol Plantarum 81: 417-422
[50] Hartley, J; Cairney, JWG and Meharg, A (1999) Cross-colonization of Scots pine (Pinus sylvestris)
seedlings by the ectomycorrhizal fungus Paxillus involutus in the presence of inhibitory levels of Cd
and Zn. New Phytol 142: 141-149
[51] Jentschke, G; Winter, S and Godbold, DL (1999) Ectomycorrhizas and cadmium toxicity in Norway
spruce seedlings. Tree Physiol 19: 23-30
[52] Colpaert, JV and van Assche, JA (1993) The effects of cadmium on ectomycorrhizal Pinus sylvestris
L. New Phytol 123: 325-333
[53] Vodnik, D; Byrne, AR and Gogala, N (1998) The uptake and transport of lead in some
ectomycorrhizal fungi in culture. Mycol Res 102: 953-958
[54] Egerton-Warburton, LM and Griffin, BJ (1995) Differential responses of Pisolithus tinctorius isolates
to aluminium in vitro. Can J Bot 73: 1229-1233
[55] Denny, HJ and Wilkins, DA (1987) Zinc tolerance in Betula spp. 4. The mechanism of
ectomycorrhizal amelioration of zinc toxicity. New Phytol 106: 545-553
[56] Colpaert, JV and van Assche, JA (1992) Zinc toxicity in ectomycorrhizal Pinus sylvestris. Plant Soil
143: 201-211
[57] Colpaert, JV and van Assche, JA (1987) Heavy metal resistance in some ectomycorrhizal fungi. Funct
Ecol 1: 415-421
[58] Rudawska, M and Leski, T (1998) Aluminium tolerance of different Paxillus involutus Fr. strains
originating from polluted and non-polluted sites. Acta Soc Bot Pol 67: 115-122
[59] Dehn, B and Schüepp, H (1989) Influence of VA mycorrhizae on the uptake and distribution of heavy
metals in plants. Agr Ecosyst Environ 29: 79-83
[60] El-Kherbawy, M; Angle, JS; Heggo, A and Chaney, RL (1989) Soil pH, rhizobia, and vesicular-
arbuscular mycorrhizae inoculation effects on growth and heavy metal uptake of alfalfa (Medicago
sativa L.). Biol Fertil Soils 8: 61-65
[61] Heggo, A; Angle, JS and Chaney, RL (1990) Effects of vesicular-arbuscular mycorrhizal fungi on
heavy metal uptake by soybeans. Soil Biol Biochem 22: 865-869
[62] Loth, FG and Höfner, W (1995) Einfluss der VA-Mykorrhiza auf die Schwermetallaufnahme von
Hafer (Avena sativa L.) in Abhängigkeit vom Kontaminationsgrad der Böden. Z Pflanzenernaehr
Bodenk 158: 339-345
[63] Liu, A; Hamel, C; Hamilton, RI; Ma, BL and Smith, DL (2000) Acquisition of Cu, Zn, Mn and Fe by
mycorrhizal maize (Zea mays L.) grown in soil at different P and micronutrient levels. Mycorrhiza 9:
331-336
[64] Killham, K and Firestone, MK (1983) Vesicular arbuscular mycorrhizal mediation of grass response to
acidic and heavy metal depositions. Plant Soil 72: 39-48
[65] Carneiro, MAC; Siqueira, JO and Moreira, FMD (2001) Establishment of herbaceous plants in heavy
metal contaminated soils inoculated with arbuscular mycorrhizal fungi. Pesqui Agropecu Bras 36:
1443-1452
[66] Jamal, A; Ayub, N; Usman, M and Khan, AG (2002) Arbuscular mycorrhizal fungi enhace zinc and
nickel uptake from contaminated soil by soybean and lentil. Int J Phytorem 4: 205-221
254 Miroslav Vosátka et al.

[67] Liao, JP; Lin, XG; Cao, ZH; Shi, YQ and Wong, MH (2003) Interactions between arbuscular
mycorrhizae and heavy metals under sand culture experiment. Chemosphere 50: 847-853
[68] Yu, X; Cheng, J and Wong, MH (2005) Earthworm-mycorrhiza interaction on Cd uptake and growth
of ryegrass. Soil Biol Biochem 37: 195-201
[69] Joner, EJ and Leyval, C (1997) Uptake of 109Cd by roots and hyphae of a Glomus mosseae/Trifolium
subterraneum mycorrhiza from soil amended with high and low concentrations of cadmium. New
Phytol 135: 353-360
[70] Weissenhorn, I; Leyval, C; Belgy, G and Berthelin, J (1995) Arbuscular mycorrhizal contribution to
heavy metal uptake by maize (Zea mays L.) in pot culture with contaminated soil. Mycorrhiza 5:
245-251
[71] Rivera-Becerril, F; Calantzis, C; Turnau, K; Caussanel, JP; Belimov, AA; Gianinazzi, S; Strasser, RJ
and Gianinazzi-Pearson, V (2002) Cadmium accumulation and buffering of cadmium-induced stress
by arbuscular mycorrhiza in three Pisum sativum L. genotypes. J Exp Bot 53: 1177-1185
[72] Chen, BD; Li, XL; Tao, HQ; Christie, P and Wong, MH (2003) The role of arbuscular mycorrhiza in
zinc uptake by red clover growing in a calcareous soil spiked with various quantities of zinc.
Chemosphere 50: 839-846
[73] Dosskey, MG and Adriano, DC (1993) Trace element toxicity in VA mycorrhizal cucumber grown on
weathered coal fly ash. Soil Biol Biochem 25: 1547-1552
[74] Dueck, TA; Visser, P; Ernst, WHO and Schat, H (1986) Vesicular-arbuscular mycorrhizae decrease
zinc-toxicity to grasses growing in zinc-polluted soil. Soil Biol Biochem 18: 331-333
[75] Kaldorf, M; Kuhn, AJ; Schröder, WH; Hildebrandt, U and Bothe, H (1999) Selective element deposits
in maize colonized by a heavy metal tolerance conferring arbuscular mycorrhizal fungus.
J Plant Physiol 154: 718-728
[76] Shetty, KG; Hetrick, BAD and Schwab, AP (1995) Effects of mycorrhizae and fertilizer amendments
on zinc tolerance of plants. Environ Pollut 88: 307-314
[77] Davies, FT; Puryear, JD; Newton, RJ; Egilla, JN and Grossi, JAS (2001) Mycorrhizal fungi enhance
accumulation and tolerance of chromium in sunflower (Helianthus annuus). J Plant Physiol 158:
777-786
[78] Chen, BD; Liu, Y; Shen, H; Li, XL and Christie, P (2004a) Uptake of cadmium from an
experimentally contaminated calcareous soil by arbuscular mycorrhizal maize (Zea mays L.).
Mycorrhiza 14: 347-354
[79] Hetrick, BAD; Wilson, GWT and Figge, DAH (1994) The influence of mycorrhizal symbiosis and
fertilizer amendments on establishment of vegetation in heavy metal mine soil. Environ Pollut 86:
171-179
[80] Vivas, A; Barea, JM and Azcon, R (2005) Interactive effect of Brevibacillus brevis and Glomus
mosseae, both isolated from Cd contaminated soil, on plant growth, physiological mycorrhizal fungal
characteristics and soil enzymatic activities in Cd polluted soil. Environ Pollut 134: 257-266
[81] Vivas, A; Vörös, A; Biró, B; Barea, JM; Ruiz-Lozano, JM and Azcón, R (2003) Beneficial effects of
indigenous Cd-tolerant and Cd-sensitive Glomus mosseae associated with a Cd-adapted strain of
Brevibacillus sp. in improving plant tolerance to Cd contamination. Appl Soil Ecol 24: 177-186
[82] Binet, P; Portal, JM and Leyval, C (2000) Fate of polycyclic aromatic hydrocarbons (PAH) in
rhizosphere and mycorrhizosphere of ryegrass. Plant Soil 227: 207-213
[83] Marrs, RH and Bannister, P (1978) The adaptation of Calluna vulgaris (L.) Hull to contrasting soil
types. New Phytol 81: 753-761
[84] Bradley, R; Burt, AJ and Read, DJ (1981) Mycorrhizal infection and resistance to heavy metal toxicity
in Calluna vulgaris. Nature 292: 335-337
[85] Bradley, R; Burt, AJ and Read, DJ (1982) The biology of mycorrhiza in the Ericaceae. VIII. The role
of mycorrhizal infection in heavy metal resistance. New Phytol 91: 197-209
[86] Hashem, AR (1995) The role of mycorrhizal infection in the tolerance of Vaccinium macrocarpon to
iron. Mycorrhiza 5: 451-454
[87] Hentschel, E; Godbold, DL; Marschner, P; Schlegel, H and Jentschke, G (1993) The effect of Paxillus
involutus Fr. on aluminium sensitivity of Norway spruce seedlings. Tree Physiol 12: 379-390
[88] van Tichelen, KK; van Straelen, T and Colpaert, JV (1999) Nutrient uptake by intact mycorrhizal
Pinus sylvestris seedlings: a diagnostic tool to detect copper toxicity. Tree Physiol 19: 189-196
[89] Dixon, RK (1988) Response of ectomycorrhizal Quercus rubra to soil cadmium, nickel and lead. Soil
Biol Biochem 20: 555-559
Mycorrhizal Fungi as Helping Agents 255

[90] Cumming, JR and Weinstein, LH (1990) Aluminum-mycorrhizal interactions in the physiology of

pitch pine seedlings. Plant Soil 125: 7-18
[91] Hartley-Whitaker, J; Cairney, JWG and Meharg, AA (2000) Sensitivity to Cd or Zn of host and
symbiont of ectomycorrhizal Pinus sylvestris L. (Scots pine) seedlings. Plant Soil 218: 31-42
[92] Schier, G and McQuattie, C (1995) Effect of aluminium on the growth, anatomy, and nutrient content
of ectomycorrhizal and nonmycorrhizal eastern white pine seedlings. Can J For Res 25: 1252-1262
[93] Jones, MD and Hutchinson, TC (1986) The effect of mycorrhizal infection on the response of Betula
papyrifera to nickel and copper. New Phytol 102: 429-442
[94] Bucking, H and Heyser, W (1994) The effect of ectomycorrhizal fungi on Zn uptake and distribution
in seedlings of Pinus sylvestris L. Plant Soil 167: 203-212
[95] Khan, AG; Kuek, C; Chaudhry, TM; Khoo, CS and Hayes, WJ (2000) Role of plants, mycorrhizae and
phytochelators in heavy metal contaminated land reclamation. Chemosphere 41: 197-207
[96] Sarand, I; Timonen, S; Koivula, T; Peltola, R; Haahtela, K; Sen, R and Romantchuk, M (1999)
Tolerance and biodegradation of m-toluate by Scots pine, a mycorrhizal fungus and fluorescent
pseudomonads individually and under associative conditions. J Appl Microbiol 86: 817-826
[97] Hartley, J; Cairney, JWG and Meharg, AA (1997) Do ectomycorrhizal fungi exhibit adaptive tolerance
to potentially toxic metals in the environment? Plant Soil 189: 303-319
[98] Jentschke, G and Godbold, DL (2000) Metal toxicity and ectomycorrhizas. Physiol Plantarum 109:
107-116
[99] Kucey, RMN and Janzen, HH (1987) Effects of VAM and reduced nutrient availability on growth and
phosphorus and micronutrient uptake of wheat and field beans under greenhouse conditions. Plant Soil
104: 71-78
[100] Godbold, DL; Jentschke, G; Winter, S and Marschner, P (1998) Ectomycorrhizas and amelioration of
metals stress in forest trees. Chemosphere 36: 757-762
[101] van Tichelen, KK; Colpaert, JV and Vangronsveld J (2001) Ectomycorrhizal protection of Pinus
sylvestris against copper toxicity. New Phytol 150: 203-213
[102] Hartley-Whitaker, J; Cairney, JWG and Meharg, AA (2000) Toxic effects of cadmium and zinc on
ectomycorrhizal colonization of Scots pine (Pinus sylvestris L.) from soil inoculum. Environ Toxicol
Chem 19: 694-699
[103] Moyer-Henry, K; Silva, I; Macfall, J; Johannes, E; Allen, N; Goldfarb, B and Rufty, T (2005)
Accumulation and localization of aluminium in root tips of loblolly pine seedlings and the associated
ectomycorrhiza Pisolithus tinctorius. Plant Cell Environ 28: 111-120
[104] Chen, B; Shen, H; Li, X; Feng, G and Christie, P (2004) Effects of EDTA application and arbuscular
mycorrhizal colonization on growth and zinc uptake by maize (Zea mays L.) in soil experimentally
contaminated with zinc. Plant Soil 261: 219-229
[105] Citterio, S; Prato, N; Fumagalli, P; Aina, R; Massa, N; Santagostino, A; Sgorbati, S and Berta, G
(2005) The arbuscular mycorrhizal fungus Glomus mosseae induces growth and metal accumulation
changes in Cannabis sativa L. Chemosphere 59: 21-29
[106] Turnau, K; Kottke, I and Oberwinkler, F (1993) Element localization in mycorrhizal roots of Pteridium
aquilinum (L.) Kuhn collected from experimental plots treated with cadmium dust. New Phytol 123:
313-324
[107] Joner, EJ; Briones, R and Leyval, C (2000) Metal-binding capacity of arbuscular mycorrhizal
mycelium. Plant Soil 226: 227-234
[108] Marschner, P; Jentschke, G and Godbold, DL (1998) Cation exchange capacity and lead sorption in
ectomycorrhizal fungi. Plant Soil 205: 93-98
[109] Frey, B; Zierold, K and Brunner, I (2000) Extracellular complexation of Cd in the Hartig net and
cytosolic Zn sequestration in the fungal mantle of Picea abies – Hebeloma crustaliniforme
ectomycorrhizas. Plant Cell Environ 23: 1257-1265
[110] Blaudez, D; Botton, B and Chalot, M (2000) Cadmium uptake and subcellular compartmentation in
the ectomycorrhizal fungus Paxillus involutus. Microbiology 146: 1109-1117
[111] Morselt, AFW; Smits, WTM and Limonard, T (1986) Histochemical demonstration of heavy metal
tolerance in ectomycorrhizal fungi. Plant Soil 96: 417-420
[112] Howe, R; Evans, RL and Ketteridge, SW (1997) Copper-binding proteins in ectomycorrhizal fungi.
New Phytol 135: 123-131
[113] Courbot, M; Diez, L; Ruotolo, R; Chalot, M and Leroy, P (2004) Cadmium-responsive thiols in the
ectomycorrhizal fungus Paxillus involutus. Appl Environ Microb 70: 7413-7417
256 Miroslav Vosátka et al.

[114] Galli, U; Meier, M and Brunold, C (1993) Effects of cadmium on non-mycorrhizal and mycorrhizal
Norway spruce seedlings [Picea abies (L.) Karst.] and its ectomycorrhizal fungus Laccaria laccata
(Scop. Ex Fr.) Bk. & Br.: Sulphate reduction, thiols and distribution of the heavy metal. New Phytol
125: 837-843
[115] Ott, T; Fritz, E; Polle, A and Schützendübel, A (2002) Characterisation of antioxidative systems in the
ectomycorrhiza-building basidiomycete Paxillus involutus (Bartsch) Fr. and its reaction to cadmium.
FEMS Microbiol Ecol 42: 359-366
[116] Galli, U; Schüepp, H and Brunold, C (1994) Heavy metal binding by mycorrhizal fungi. Physiol
Plantarum 92: 364-368
[117] Galli, U; Schüepp, H and Brunold, C (1995) Thiols of Cu-treated maize plants inoculated with the
arbuscular-mycorrhizal fungus Glomus intraradices. Physiol Plantarum 94: 247-253
[118] Lafranco, L; Bolchi, A; Ros, EC; Ottonello, S and Bonfante, P (2002) Differential expression of a
metallothionein gene during the presymbiotic versus the symbiotic phase of an arbuscular mycorrhizal
fungus. Plant Physiol 130: 58-67
[119] Gonzalez-Chavez, C; D’Haen, J; Vangronsveld, J and Dodd, JC (2002) Copper sorption and
accumulation by the external mycelium of different Glomus ssp. (arbuscular mycorrhizal fungi)
isolated from the same polluted soil. Plant Soil 240: 287-297
[120] Tam, PCF (1995) Heavy metal tolerance by ectomycorrhizal fungi and metal amelioration by
Pisolithus tinctorius. Mycorrhiza 5: 181-187
[121] Denny, HJ and Ridge, I (1995) Fungal slime and its role in the mycorrhizal amelioration of zinc
toxicity to higher plants. New Phytol 130: 251-257
[122] Ahonen-Jonnarth, U; van Hess, PAW; Lundström, US and Finlay, RD (2000) Organic acids produce
by mycorrhizal Pinus sylvestris exposed to elevated aluminium and heavy metal concentrations. New
Phytol 146: 557-567
[123] Martino, E; Franco, B; Piccoli, G; Stocchi, V and Perotto, S (2002) Influence of zinc ions on protein
secretion in a heavy metal tolerant strain of the ericoid mycorrhizal fungus Oidiodendron maius. Mol
Cell Biochem 231: 179-185
[124] Christie, P; Li, X and Chen, B (2004) Arbuscular mycorrhiza can depress translocation of zinc to
shoots of host plants in soils moderately polluted with zinc. Plant Soil 261: 209-217
[125] Meharg, AA and Macnair, MR (1992) Suppression of the high affinity phosphate uptake system: a
mechanism of arsenate tolerance in Holcus lanatus L. J Exp Bot 43: 519-524
[126] Sharples, JM; Meharg, AA; Chambers, SM and Cairney, JWG (2000) Evolution: Symbiotic solution
to arsenic contamination. Nature 404: 951-952
[127] Sharples, JM; Meharg, AA; Chambers, SM and Cairney, JWG (2000) Mechanism of arsenate
resistance in the ericoid mycorrhizal fungus Hymenoscyphus ericae. Plant Physiol 124: 1327-1334
[128] Perotto, S and Martino, E (2001) Molecular and cellular mechanisms of heavy metal tolerance in
mycorrhizal fungi: what perspectives for bioremediation? Minerva Biotecnol 13: 55-63
[129] Joner, EJ; Johansen, A; Loibner, AP; de la Cruz, MA; Szolar, OHJ; Portal, JM and Leyval, C (2001)
Rhizosphere effects on microbial community structure and dissipation and toxicity of polycyclic
aromatic hydrocarbons (PAHs) in spiked soil. Environ Sci Technol 35: 2773-2777
[130] Joner, EJ and Leyval, C (2003) Rhizosphere gradients of polycyclic aromatic hydrocarbon dissipation
in two industrial soils and the impact of arbuscular mycorrhiza. Environ Sci Technol 37: 2371-2375
[131] Salzer, P; Corbiere, H and Boller, T (1999) Hydrogene peroxide accumulation in Medicago truncatula
roots colonized by the arbuscular mycorrhiza-forming fungus Glomus intraradices. Planta 208:
319-325
[132] Criquet, S; Joner, EJ; Léglize, P and Leyval, C (2000) Anthracene and mycorrhiza affect the activity of
oxidoreductases in the roots and the rhizosphere of lucerne (Medicago sativa L.). Biotechnol Lett 22:
1733-1737
[133] Mada, RJ and Bagyaraj, DJ (1993) Root exudation from Leucaena leucocephala in relation to
mycorrhizal colonization. World J Microb Biot 9: 342-344
[134] Jones, DL; Hodge, A and Kuzyakov, Y (2004) Plant and mycorrhizal regulation of rhizodeposition.
New Phytol 163: 459-480
[135] Secilia, J and Bagyaraj, DJ (1987) Bacteria and actinomycetes associated with pot cultures of
vesicular-arbuscular mycorrhizas. Can J Microbiol 33: 1069-1073
[136] Kandeler, E; Marschner, P; Tscherko, D; Gahoonia, TS and Nieksen, NE (2002) Microbial community
composition and functional diversity in the rhizosphere of maize. Plant Soil 238: 301-312
Mycorrhizal Fungi as Helping Agents 257

[137] Vierheilig, H; Gagnon, H; Strack, D and Maier, W (2000) Accumulation of cyclohexanone derivates in
barley, wheat and maize roots in response to inoculation with different arbuscular mycorrhizal fungi.
Mycorrhiza 9: 291-293
[138] Hooker, JE and Atkinson, D (1996) Arbuscular mycorrhizal fungi-induced alteration to tree-root
architecture and longevity. Z Pflanzenernaehr Bodenk 159: 229-234
[139] Quintero-Ramos, M; Espinoza-Victoria, D; Ferrera-Cerrato, R and Bethlenfalvay, GJ (1993) Fitting
plants to soil through mycorrhizal fungi – mycorrhiza effects on plant growth and soil organic matter.
Biol Fert Soils 15: 103-106
[140] Wright, SF and Upadhyaya, A (1998) A survey of soils for aggregate stability and glomalin, a
glycoprotein produced by hyphae of arbuscular mycorrhizal fungi. Plant Soil 198: 97-107
[141] Joner, EJ; van Aarle, IM and Vosátka, M (2000) Phosphatase activity of extraradical arbuscular
mycorrhizal hyphae: A review. Plant Soil 226: 199-210
[142] Trojanowski, J; Haider, K and Hüttermann, A (1984) Decomposition of 14C-labelled lignin,
holocellulose amd lignocellulose by mycorrhizal fungi. Arch Microbiol 139: 202-206
[143] Haselwandter, K; Bobleter, O and Read, DJ (1990) Degradation of 14C-labelled lignin and
dehydroplymer of coniferyl alcohol by ericoid and ectomycorrhizal fungi. Arch Microbiol 153:
352-354
[144] Cairney, JWG and Burke, RM (1994) Fungal enzymes degrading plant cell walls: their possible
significance in ectomycorrhizal symbiosis. Mycol Res 98: 1345-1356
[145] Meharg, AA and Cairney, JWG (2000) Ectomycorrhizas – extending the capabilities of rhizosphere
remediation? Soil Biol Biochem 32: 1475-1484
[146] Meharg, AA; Cairney, JWG and Maguire, N (1997) Mineralization of 2,4-dichlorphenol by
ectomycorrhizal fungi in axenic culture and in symbiosis with pine. Chemosphere 34: 2495-2504
[147] Meharg, AA; Dennis, GR and Cairney, JWG (1997) Biotransformation of 2,4,6-trinitrotoluen (TNT)
by ectomycorrhizal basidiomycetes. Chemosphere 35: 513-521
[148] Donnelly, PK; Entry, JA and Craftword, DL (1993) Degradation of atrazine and 2,4-
dichlorphenoxyacetic acid by mycorrhiozal fungi at three nitrogen concentrations in vitro. Appl
Environ Microbiol 59: 2642-2647
[149] Braun-Lüllemann, A; Hüttermann, A and Majcherczyk, A (1999) Screening of ectomycorrhizal fungi
for degradation of polycyclic aromatic hydrocarbons. Appl Microbiol Biot 53: 127-132
[150] Genney, DR; Alexander, IJ; Killham, K and Meharg, AA (2004) Degradation of the polycyclic
aromatic hydrocarbon (PAH) fluorene is retarded in a Scots pine ectomycorrhizosphere. New Phytol
163: 641-649
[151] Donnelly, PK and Fletcher, JS (1995) PCB metabolism by ectomycorrhizal fungi. B Environ Contam
Tox 54: 507-513
[152] Green, NA; Meharg; AA; Till, C; Troke, J and Nicholson, JK (1999) Degradation of 4-fluorbiphenyl
by mycorrhizal fungi as determined by F-19 nuclear magnetic resonance spectroscopy and C-14
radiolabelling analysis. Appl Environ Microbiol 65: 4021-4027
[153] Dittmann, J; Heyser, W and Bucking, H (2002) Biodegradation of aromatic compounds by white rot
and ectomycorrhizal fungal species and the accumulation of chlorinated benzoic acid in
ectomycorrhizal pine seedlings. Chemosphere 49: 297-306
[154] Jurkiewicz, A; Orłowska, E; Anielska, T; Godzik, B and Turnau, K (2004) The influence of
mycorrhiza and EDTA application on heavy metal uptake by different maize varieties. Acta Biol
Cracov Bot 46: 7-18
[155] Hovsepyan, A and Greipsson, S (2004) Effect of arbuscular mycorrhizal fungi on phytoextraction by
corn (Zea mays) of lead-contaminated soil. Int J Phytorem 6: 305-321
ASSESSING RISKS AND CONTAINING OR MITIGATING GENE FLOW
OF TRANSGENIC AND NON-TRANSGENIC PHYTOREMEDIATING
PLANTS

TON ROTTEVEEL1, HANI AL-AHMAD2 AND JONATHAN

GRESSEL2
1
Plant Protection Service, NL-6700 HC Wageningen, The Netherlands
and 2Plant Sciences, Weizmann Institute of Science, Rehovot, Israel
Fax +972-8-934-4181, E-mail: [email protected]

1. Introduction - needs for preventing gene flow in phytoremediating species

Plants have been used to correct human error over the ages. The few species capable of
revegetating Roman lead and zinc mine tailings in Wales [2] taught us that there are a
limited number of species that can withstand toxicants: some by exclusion, and others
that can withstand toxic wastes after they have been taken up. Plants with the latter type
mechanism are of interest for phytoremediation. Ideally, one might consider that it is
best to use the species that naturally take up particular toxic wastes, but these are often
slow growing (e.g. mosses, lichens, or the Thlaspi species that take up heavy metals) [3]
or may have a potential to be weedy. If the desired wild species do not exist locally,
there may be a reticence or legal issues about introducing them into the ecosystem, toxic
as it may be, due to fear that the plants or their genes may spread to other areas.
Two types of multi-cut species are used, with the cut material burnt to extract the
heavy metals or to oxidize the organic wastes: herbaceous species such as Brassica
juncea and Spartina spp. (cord grasses), which are most efficient at dealing with surface
wastes, and trees such as Populus spp., for dealing with deeper wastes [4]. Brassica
juncea (Indian mustard) wild type had been used commercially, because it grows
rapidly, and is easy to cultivate as a crop, but especially because of its inherent ability to
take up some heavy metals. This ability has been enhanced by mutant selection (in tissue
culture) for heavy metal resistance [5], from Thlaspi by protoplast fusion (along with
many other genes) [6], but it was better yet to transgenically transfer genes leading to
enhanced glutathione content [7, 8] to make the necessary phytochelatins.
A single cropping of B. juncea does not clean up a toxic site; many growth cycles are
required, with multiple harvests and natural reseeding. B. juncea, even more than its
close relative B. napus (oilseed rape) is not fully domesticated, and the multiple cycles
of cropping would allow the possibility of selecting for feral forms that may persist or
crossing the genes into related Brassica species, or cultivated varieties of Indian
mustard. Thus, gene containment and/or mitigation seem necessary to prevent volunteers

259
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 259 – 284.
© 2006 Springer.
260 Ton Rotteveel et al.

from becoming feral and to prevent crossing into related species. Similarly, many
oppose introducing transgenic or non-transgenic phytoremediating tree species such as
poplars unless they can be prevented from establishing outside of the contaminated area
or from hybridising with related native or introduced species.
The herbaceous plants, shrubs, and trees used for phytoremediation pose certain
biological risks, whether transgenic or not. Many of the species are semi-domesticated
and introduced from habitats far removed from the site requiring phytoremediation.
Such species pose a risk of becoming established in the contaminated site after the
contaminant is remediated, and also pose a risk of spread to adjacent areas, displacing
native or other desirable species, or hybridising with other varieties of the same species
or even other varieties of the same species. Spartina [9, 10] and Populus [11, 12] are
often proposed for phytoremediation, yet they commonly form hybrids with other
species in their genera. In the case of Spartina, the results were devastating when the
new world Spartina alterniflora crossed with the European S. maritime around 1870, the
hybrids massively displaced all other native species from the ecosystem [13]. Populus
species easily form hybrids [14], and native species could easily be displaced by
hybrids. An added concern is that transgenes in the phytoremediation species may
introgress into related species. If a non-transgenic species poses a risk, the addition of
specific transgenes can actually reduce the risk.
We describe below the molecular tools that can be used to contain gene flow within
the bioremediation site, and separately, molecular mitigation tools that can prevent
establishment of such transgenes should they leak out of the phytoremediation site,
which are appropriate for non-transgenic and transgenic bioremediating species alike.
Molecular solutions to gene flow problems for non-transgenic phytoremediation species
may sound oxymoronic in the present climate surrounding transgenics. Still, if the
scientifically determined risk of spread of a phytoremediating species outweighs the
utility of the species for phytoremediation, such molecular solutions should be sought to
allow effective phytoremediation while preventing gene flow.
Genes can flow from bioremediation sites in three forms – seeds carried by various
vectors, vegetative propagules, and pollen. Typically pollen is thought of as the source
of gene movement, but even without human intervention seeds carrying an undesirable
trait can move large distances; e.g. maternally inherited triazine resistance in Solanum
nigrum has moved 20 km per year from a single site – the distance a bird flies from
eating berries to defecating [15]. Some species can move long distances as vegetative
propagules, e.g. feral forms of asexually propagated Jerusalem artichoke (Helianthus
tuberosus) have become widely spread in Europe along riverbanks [16]. The number 1
and 16th Worst Weeds of the World, Cyperus spp., are primarily spread asexually [17].
This review will not cover the toxicological risks of the pollutants sequestered in or
vaporized from plants used for phytoremediation, or the toxicological risks of not
phytoremediating a contaminated site.

2. Assessing the likelihood of risks

Species used for phytoremediation are often no ordinary agricultural species, nor are
they used in agricultural contexts. In their natural surroundings any species occupies a
Risk assessment and gene flow 261

specific niche that determines its occurrence in space, time and function of the
ecosystem concerned. Genetic modification might alter a niche, and does certainly so
when phytoremediation traits are attached to species formerly sensitive to the chemicals
involved, and equally certain if genetic modification changes fitness. However, the basic
biological traits will be the same as for any plant species.
In this section we examine the interaction between different factors that might
produce an ecological risk situation. First the factors are indicated and briefly described
as we did for the biotechnology derived herbicide resistant plants [18]. However, the
situation is clearly more complicated since phytoremediation traits always operate under
natural conditions compared to herbicide resistance, which requires that the herbicide be
applied to be operational.
For contaminated sites it might be thought that there is no risk as the purpose of the
use of phytoremediation plants is cleaning of the site by being grown there. However,
there are many rare, sometimes endemic species that are specialised on growing on
(heavy metal) containing soils. An example is Viola calaminaria, which is endemic in
the Netherlands, Belgium and Western Germany, and completely restricted to zinc, lead,
and/or cadmium containing soils [19]. Such a rare and endangered species may easily be
out competed by an engineered if phytoremediating species are used on, or invade the
Viola habitats, and in this way a loss of biodiversity may occur. Conversely, this species
may be used to mine the genes for zinc phytoremediation, to be transformed into faster
growing species, for use elsewhere.
The theoretical case of a remediation species becoming invasive on uncontaminated
sites is currently a remote possibility but cannot completely ruled out, especially as they
can flow via pollen to related species, forming hybrid swarms.

2.1. DECISION TREES FOR ASSESSING THE LEVEL OF RISK

Here we combine the different factors through the use of decision trees. We want to state
clearly that a decision tree is not a quantitative tool producing a quantified risk. It is an
aid in risk evaluation providing unbiased guidance in indicating hazards attached to a
certain species. When a hazard is indicated, more detailed and quantified data
acquisition will often be necessary.
Different levels of certainty apply to the various factors used. For instance:
invasiveness is highly unpredictable whereas the presence or absence of vegetative
propagation is not.
The keys are layered; first assessing the “biological hazard” (Key 1), which is equal
for engineered and non-engineered species, and then we examine the extent to which
measures aimed at containment and mitigation affect the total risk (Key 2). Finally, the
keys are designed to assist in determining whether the growing conditions might trigger
identified hazards into real risks, both on contaminated sites (Key 3) and in an
uncontaminated environment (Key 4).

2.1.1 Key 1 Assessing basic hazards imposed by biology.

1. Invasiveness
1a. The plant has no known invasive characters:
262 Ton Rotteveel et al.

Basic Biological Hazard low, go to Key 2A

1b. The plant is a known invasive, go to 2
2. The plant is a known invasive
2a. The plant has no sexual propagation (Most species have a sexual propagation
pathway. However, some species may almost never propagate sexually, or when
cultivated, propagation might be entirely vegetative), go to 3
2b. The plant has sexual propagation, go to 4
3. Known invasive, without sexual propagation
3a. The plant has a proven capacity for efficient natural long range dissemination:
Basic Biological Hazard high: go to Key 2C
3b. The plant has little capacity for efficient natural long range dissemination;
dissemination takes place with the aid of people:
Basic Biological Hazard medium: go to Key 2B
4. Known invasive with sexual propagation
4a. The species is cross-pollinating - go to 5
4b. The species is self- pollinating - go to 6
5. Cross pollinating invasive species
5a. The plant has a proven capacity for efficient natural long range dissemination:
Basic Biological Hazard very high: go to Key 2D
5b. The plant has little capacity for efficient natural long range dissemination;
dissemination takes place with the aid of people:
Basic Biological Hazard high: go to Key 2C
6. Self pollinating invasive species
6a. The plant has a proven capacity for efficient natural long range dissemination
Basic Biological Hazard high: go to Key 2C
6b. The plant has little capacity for efficient natural long range dissemination;
dissemination takes place with the aid of people:
Basic Biological Hazard medium: go to Key 2B

2.1.2 Key 2A Assessing hazards imposed by containment and mitigation measures -

Basic Biological hazard low.
1. Presence of added genetic containment and mitigation measures
1a. No measures new to the species added:
Biological hazard low, go to Key 3B and Key 4
1b. New measures have been added aimed at containment and/or mitigation:
Biological Hazard very low; go to Key 3A and Key 4

2.1.3 Key 2B Assessing hazards imposed by containment and mitigation measures -

Basic Biological hazard medium.
1. Presence of added genetic containment and mitigation measures
1a. No measures new to the species added. (If containment or mitigation genes present
in the species gene pool are used in cultivar breeding, no new possibilities are
introduced in the species, and hence the hazard to the environment is estimated as
equal to the basic biological hazard): Biological Hazard medium, go to Key 3C and
Key 4
Risk assessment and gene flow 263

1b. New measures have been added aimed at containment and/or mitigation, go to 2
2. Decreasing gene flow
2a. The plant has added genes, not in the parent, enhancing containment, go to 3
2b. The plant has added genes, not present in the parent, enhancing mitigation, go to 5
3. New containment genes present
3a. Containment genes added at random, go to 4
3b. Containment genes present as tandem constructs:
Hazard very low, go to Key 3A and Key 4
4. Random containment genes present, presence of mitigation genes
4a. The plant has added genes engineered in tandem, enhancing mitigation:
Hazard very low, go to Key 3A and Key 4
4b. The plant has added genes incorporated at random, enhancing mitigation:
Hazard very low, go to Key 3A and Key 4
4c. The plant has no mitigation genes added: Hazard low, go to Key 3b and Key 4
5. No containment genes present, presence of mitigation genes
5a. The plant has added genes engineered in tandem, enhancing mitigation:
Hazard very low: go to Key 3A and Key 4
5b. The plant has added genes incorporated at random, enhancing mitigation:
Hazard low, go to Key 3B and Key 4

2.1.4 Key 2C Assessing hazards imposed by containment and mitigation measures -

Basic Biological hazard high.
1. Presence of added genetic containment and mitigation measures
1a. No measures new to the species added:
Biological hazard high: go to Key 3D and Key 4
1b. New measures have been added aimed at containment and/or mitigation, go to 2
2. Decreasing gene flow
2a. The plant has added genes, not in the parent, enhancing containment, go to 3
2b. The plant has added genes, not in the parent species, enhancing mitigation, go to 6
3. New containment genes present
3a. Containment genes added at random, go to 4
3b. Containment genes present as tandem constructs, go to 5
4. Random containment genes present, presence of mitigation genes
4a. The plant has added genes engineered in tandem, enhancing mitigation:
Hazard very low, go to Key 3A and Key 4
4b. The plant has added genes incorporated at random, enhancing mitigation:
Hazard very low, go to Key 3A and Key 4
4c. The plant has no mitigation genes added. Hazard medium, go to Key 3C and Key 4
5. Tandem containment genes and mitigation genes present
5a. The plant has added genes engineered in tandem, enhancing mitigation:
Hazard very low, go to Key 3A and Key 4
5b. The plant has added genes incorporated at random, enhancing mitigation:
Hazard very low, go to Key 3A and Key 4
5c. No mitigation genes present: Hazard low, go to Key 3B and Key 4
6. No containment genes present, presence of mitigation genes
264 Ton Rotteveel et al.

6a. The plant has added genes engineered in tandem, enhancing mitigation:
Hazard very low: go to Key 3A and Key 4
6b. The plant has added genes incorporated at random, enhancing mitigation:
Hazard medium, go to Key 3C and Key 4

2.1.5 Key 2D Assessing hazards imposed by containment and mitigation measures -

Basic Biological hazard very high.
1. Presence of added genetic containment and mitigation measures
1a. No novel measures species added:
Biological hazard very high: go to Key 3E and Key 4
1b. New measures have been added aimed at containment and/or mitigation, go to 2
2. Decreasing gene flow
2a. The plant has added genes, not in the parent, enhancing containment, go to 3
2b. The plant has added genes, not in the parent species, enhancing mitigation, go to 6
3. New containment genes present
3a. Containment genes added at random, go to 4
3b. Containment genes present as tandem constructs, go to 5
4. Random containment genes present, presence of mitigation genes
4a. The plant has added genes engineered in tandem, enhancing mitigation:
Hazard very low, go to Key 3a and 4
4b. The plant has added genes incorporated at random, enhancing mitigation:
Hazard low, go to Key 3B and 4
4c. The plant has no mitigation genes added: Hazard high, go to Key 3D and Key 4
5. Tandem containment genes present, presence of mitigation genes
5a. The plant has added genes engineered in tandem, enhancing mitigation:
Hazard very low, go to Key 3A and Key 4
5b. The plant has added genes incorporated at random, enhancing mitigation:
Hazard very low, go to Key 3A and Key 4
6. No containment genes present, presence of mitigation genes
6a. The plant has added genes engineered in tandem, enhancing mitigation:
Hazard low: go to Key 3A and Key 4
6b. The plant has added genes incorporated at random, enhancing mitigation:
Hazard high, go to Key 3D and Key 4

2.1.6 Key 3A Assessing risks to contaminated sites - Biological hazard very low.
Contaminated site refers to all sites contaminated with the compound(s) for which the
plant may be used in cleaning up. There still may be unwanted side-effects to some
naturally occurring contaminated sites because they are inhabited by rare and protected
wild species that evolved to withstand the contamination. These species may be out-
competed by the “bioremediating” species.
1a. The species (transgenic or not) needs management to survive at the site, go to 2
1b. The species (transgenic or not) survives at a site without management, go to 4
2. Species only surviving under management
2a. The species poses a risk to higher trophic levels on the site, go to 3
2b. The species poses no risk to higher trophic levels on the site:
Risk assessment and gene flow 265

Risk very low for contaminated sites, go to Key 4

3. Species survives under management, posing risk to higher trophic levels on the site
3a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
3b. To species not confined to this specific contaminated environment:
Risk low for contaminated sites, go to Key 4
4. Species survives without management
4a. Species unable to spontaneously invade a contaminated site, go to 5
4b. Species able to invade and dominate the contaminated site, go to 7
5. Species survives but unable to invade contaminated sites
5a. The species poses a risk to higher trophic levels on the site, go to 6
5b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
6. Species survives, but unable to spontaneously invade, yet poses a risk to higher
trophic levels on the site
6a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
6b. To species not confined to this specific contaminated environment.
Risk low for contaminated sites, go to Key 4
7. Species survives and able to spontaneously invade contaminated sites
7a. The species poses a risk to higher trophic levels on the site, go to 8
7b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
8. Species survives, can spontaneously invade, and poses a risk to higher trophic levels
on the site
8a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
8b. To species not confined to this specific contaminated environment:
Risk low for contaminated sites, go to Key 4

2.1.7 Key 3B Assessing risks to contaminated sites - Biological hazard low.

1a. The species (transgenic or not) needs management to survive at a site, go to 2
1b. The species (transgenic or not) survives at a site without management, go to 4
2. Species only survives under management
2a. The species poses a risk to higher trophic levels on the site, go to 3
2b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
3. Species survives under management posing risk to higher trophic levels on the site
3a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
3b. To species not confined to this specific contaminated environment:
Risk low for contaminated sites, go to Key 4
4. Species survives without management
4a. Species unable to spontaneously invade a contaminated site, go to 5
4b. Species can invade and dominate a contaminated site, go to 7
266 Ton Rotteveel et al.

5. Species survives but unable to spontaneously invade contaminated sites

5a. The species poses a risk to higher trophic levels on the site, go to 6
5b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
6. Species survives, but unable to spontaneously invade and poses risk to higher
trophic levels on the site
6a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
6b. To species not confined to this specific contaminated environment:
Risk low for contaminated sites, go to Key 4
7. Species survives and able to spontaneously invade contaminated sites
7a. The species poses a risk to higher trophic levels on the site, go to 8
7b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
8. Species survives and can spontaneously invade, and poses a risk to higher trophic
levels on the site
8a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
8b. To species not confined to this specific contaminated environment.
Risk low for contaminated sites, go to Key 4

2.1.8 Key 3C Assessing risks to contaminated sites - Biological hazard medium.

1a. The species (transgenic or not) needs management to survive at a site, go to 2
1b. The species (transgenic or not) survives at a site without management, go to 4
2. Species only survives under management
2a. The species poses a risk to higher trophic levels on the site, go to 3
2b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
3. Species survives only under management, but poses a risk to higher trophic levels
on the site
3a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
3b. To species not confined to this specific contaminated environment.
Risk low for contaminated sites, go to Key 4
4. Species survives without management
4a. Species unable to spontaneously invade contaminated site, go to 5
4b. Species can spontaneously invade and dominate a contaminated site, go to 7
5. Species survives but unable to spontaneously invade contaminated sites
5a. The species poses a risk to higher trophic levels on the site, go to 6
5b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
6. Species survives, yet unable to spontaneously invade, but poses risk to higher
trophic levels on the site
6a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
Risk assessment and gene flow 267

6b. To species not confined to this specific contaminated environment:

Risk low for contaminated sites, go to Key 4
7. Species survives and is able to spontaneously invade contaminated sites
7a. The species poses a risk to higher trophic levels on the site, go to 8
7b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
8. Species survives, can spontaneously invade and poses risk to higher trophic levels
on the site
8a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
8b. To species not confined to this specific contaminated environment:
Risk low for contaminated sites, go to Key 4

2.1.9 Key 3D Assessing risks to contaminated sites - Biological hazard high.

1a. The species (transgenic or not) needs management to survive at a site, go to 2
1b. The species (transgenic or not) survives at a site without management, go to 4
2. Species only survives under management
2a. The species poses a risk to higher trophic levels on the site, go to 3
2b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
3. Species survives only under management yet poses a risk to higher trophic levels on
the site
3a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
3b. To species not confined to this specific contaminated environment:
Risk low for contaminated sites, go to Key 4
4. Species survives without management
4a. Species unable to spontaneously invade a contaminated site, go to 5
4b. Species spontaneously invades and dominates a contaminated site, go to 7
5. Species survives but unable to spontaneously invade contaminated sites
5a. The species poses a risk to higher trophic levels on the site, go to 6
5b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
6. Species survives, is unable to spontaneously invade, yet poses risk to higher trophic
levels on the site
6a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
6b. To species not confined to this specific contaminated environment:
Risk low for contaminated sites, go to Key 4
7. Species survives and can spontaneously invade contaminated sites
7a. The species poses a risk to higher trophic levels on the site, go to 8
7b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
8. Species survives, can spontaneously invade and poses a risk to higher trophic levels
on the site
268 Ton Rotteveel et al.

8a. To species confined to this specific contaminated environment:

Risk medium for contaminated sites, go to Key 4
8b. To species not confined to this specific contaminated environment:
Risk low for contaminated sites, go to Key 4

2.1.10 Key 3E Assessing risks to contaminated sites - Biological hazard very high.
1a. The species (transgenic or not) needs management to survive at a site, go to 2
1b. The species (engineered or not) survives at a site without management, go to 4
2. Species only survives under management
2a. The species poses a risk to higher trophic levels on the site, go to 3
2b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
3. Species survives under management posing risk to higher trophic levels on the site
3a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
3b. To species not confined to this specific contaminated environment:
Risk low for contaminated sites, go to Key 4
4. Species survives without management
4a. Species unable to spontaneously invade contaminated site, go to 5
4b. Species can spontaneously invade and dominate a contaminated site, go to 7
5. Species survives but unable to spontaneously invade contaminated sites
5a. The species poses a risk to higher trophic levels on the site, go to 6
5b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
6. Species survives, is unable to spontaneously invade, yet poses a risk to higher
trophic levels on the site
6a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
6b. To species not confined to this specific contaminated environment:
Risk low for contaminated sites, go to Key 4
7. Species survives and can spontaneously invade contaminated sites
7a. The species poses a risk to higher trophic levels on the site, go to 8
7b. The species poses no risk to higher trophic levels on the site:
Risk very low for contaminated sites, go to Key 4
8. Species survives, can spontaneously invade, and poses a risk to higher trophic levels
on the site
8a. To species confined to this specific contaminated environment:
Risk medium for contaminated sites, go to Key 4
8b. To species not confined to this specific contaminated environment:
Risk low for contaminated sites, go to Key 4

2.1.11 Key 4 Assessing risks to the natural, uncontaminated environment.

1. The basic biological hazard has been estimated in Key 2 as:
1a. biological hazard very low: non-invasive species, new containment and/or
mitigation genes added: Risk very low
Risk assessment and gene flow 269

1b. Biological hazard low, go to 2

1c. Biological hazard medium, go to 3
1d. Biological hazard high, go to 5
1e. Biological hazard very high, go to 7
2. Biological hazard low – species can invade uncontaminated environment
2a. Species unable to spontaneously invade and dominate an uncontaminated site:
Risk to uncontaminated environment very low
2b. Species able to spontaneously invade and dominate an uncontaminated site:
Risk to uncontaminated environment low
3. Biological hazard medium - species can invade uncontaminated environment
3a. Species unable to spontaneously invade and dominate an uncontaminated site:
Risk to uncontaminated environment very low
3b. Species able to invade and dominate an uncontaminated site, go to 4
4. Invasive on uncontaminated sites: risk to trophic levels
4a. The species poses a risk to higher trophic levels
Risk to uncontaminated environment high
4b. The species poses no risk to higher trophic levels:
Risk to uncontaminated environment medium
5. Biological hazard high - ability to invade uncontaminated environment
5a. Species unable to spontaneously invade and dominate an uncontaminated site:
Risk to uncontaminated environment very low
5b. Species able to invade and dominate an uncontaminated site, go to 6
6. Invasive on uncontaminated sites: risk to trophic levels
6a. The species poses a risk to higher trophic levels:
Risk to uncontaminated environment very high
6b. The species poses no risk to higher trophic levels:
Risk to uncontaminated environment high
7. Biological hazard very high - ability to invade uncontaminated environment
7a. Species unable to spontaneously invade and dominate a uncontaminated site:
Risk to uncontaminated environment very low
7b. Species able to invade and dominate a uncontaminated site:
Risk to uncontaminated environment very high

3. Dealing with the risks

3.1. GENE FLOW

Genes do flow in nature, not only within species, but also among related species that do
not readily cross, in a process coined “diagonal” gene transfer [20] to readily distinguish
between vertical gene transfer in readily crossing species and horizontal gene transfer
between totally unrelated species. For example, a DNA sequence typical of hexaploid
wheat, found in modified form in some progenitors of wheat, was not found in
>90 accessions of Aegilops peregrina (syn. Ae. variabilis) but was found in two
270 Ton Rotteveel et al.

geographically distinct populations of that species with >99% sequence identity to wheat
[21]. In agroecosystems, such inadvertent gene flow may be undesirable.
There are two general approaches to dealing with gene flow: (1) “contain” the
transgenes in the novel variety so that gene inflow, gene outflow or both are precluded
depending on the mechanism; (2) “mitigate” gene flow effects if there are inevitable
“leaks” in the containment system, which should also prevent volunteer populations of
the phytoremediation species from establishing and/or reaching maturity so that they
cannot evolve into problems. Most discussions so far have dealt with “containing” gene
flow from managed ecosystems to “natural” ecosystems with less on “mitigation” of the
effects of gene flow after it has occurred [22-27]. Only recently has discussion begun
dealing with gene flow within the agroecosystems, both on preventing and mitigating
endo-feral (evolution within the biotype) and exo-feral (evolution of less domesticated
forms by crossing with wild or weedy forms) dedomestication of species as volunteer
weeds [28]. Containment and mitigation are discussed below in the general context of
bi-directional containment as well as mitigation.

3.2. CONTAINING GENE FLOW

Several molecular mechanisms have been suggested for containing gene flow (i.e., to
prevent gene flow between the phytoremediating species and relatives), especially by
pollen, ignoring the other routes of sporophyte propagule (seeds and asexual parts)
movement, especially transgenes within the phytoremediating species (i.e., to prevent
outflow to related species), or to mitigate the effects of transgene flow once it has
occurred [20, 22, 26, 29]. It is more important to prevent gene flow from the phyto-
remediating species to outside the contaminated site than to prevent influx into the
phytoremediation site, as the phytoremediating species should be most fit to live the
contaminated site. Even though the hybrids may be the same in either direction,
the likelihood of such a hybrid establishing on a phytoremediation site is minimal.

3.2.1 Containment by targeting genes to a cytoplasmic genome

The most widely discussed containment possibility is to integrate the transgene of choice
in the plastid or mitochondrial genomes [30-32]. There are good reasons to engineer
phytoremediating genes into chloroplasts besides the presumed biosafety. The
chloroplasts are often the targets of environmental contaminants and need protection.
Additionally, many genes of value come from bacteria with similar codon usage as
chloroplasts. Such genes often need to be re-engineered to plant codon usage before
inserting into the nuclear genome [33]. Indeed the bacterial genes merA/merB that
convert organomercurials into elemental mercury (which is later volatilised) were
successfully introduced into chloroplasts of tobacco [34]. Still, the same genes were
active in Arabidopsis when the merB was augmented with a peptide that targeted the
gene product into the endoplasmic reticulum, despite the bacterial codon usage dif-
ferences [35].
The opportunity of gene outflow is limited due to the predominantly maternal
inheritance of these genomes in many, but far from all species. This is presently an
arduous technology, which so far is limited to a few species. It does not preclude the
Risk assessment and gene flow 271

outside species from pollinating the bioremediating species, and then acting as the
recurrent pollen parent, but this is less of a problem on a bioremediation site than off
site.
The claim of strict maternal inheritance of plastome-encoded traits [32, 36, 37] was
not substantiated. Tobacco [38] and other species [39] often have between a 10–3–10–4
frequency of pollen transfer of plastid inherited traits. Pollen transmission of plastome
traits can only be easily detected using both large samples and selectable genetic
markers. A large-scale field experiment utilized a Setaria italica (foxtail or birdseed
millet) with chloroplast-inherited atrazine resistance (bearing a nuclear dominant leaf
marker) crossed with five different male sterile herbicide susceptible lines. Chloroplast-
inherited resistance was pollen transmitted at a frequency of 3 × 10 – 4 in >780,000 hybrid
offspring [40]. At this transmission frequency, the probability of transgene movement
via plastomic gene flow is orders of magnitude greater than by spontaneous nuclear
genome mutations. Thus, chloroplast transformation is probably unacceptable for
preventing transgene outflow, unless stacked with additional mechanisms, and as noted
above, will not at all impede gene inflow. Maliga [32] discounts the relevance of the
findings with tobacco and Setaria as being due to an origin of the plastids from
interspecific (closely related) cytoplasmic substitution, where pollen transmission
barriers can break down [41]. Setaria viridis, the wild progenitor of Setaria italica is
biologically con-specific with it [42]. There are two problems with this denigration of
the relevance of pollen movement of plastome encoded genes: 1) it is just such
interspecific movement that could be a problem between phytoremediating species and
related species; 2) he [32] ignores the discussion in Darmency et al. [39] of cases of
intraspecific transmission of plastomic traits by pollen at about the same frequency,
within the same species, as reported above between species.

3.2.2 Male sterility coupled with transplastomic traits

A novel additional combination that considerably lowers the risk of plastome gene
outflow within a field (but not gene influx from related strains or species) can come
from utilizing male sterility with transplastomic traits [40]. Introducing plastome-
inherited traits into varieties with complete male sterility would vastly reduce the risk of
transgene flow, except in the small isolated areas required for line maintenance. Such a
double failsafe containment method might be considered sufficient where there are
highly stringent requirements for preventing gene outflow to interbreeding species
adjacent to the phytoremediation sites. Plastome-encoded transgenes for non-selectable
traits (e.g. for phytoremediation) could be transformed into the chloroplasts together
with a trait such as tentoxin or atrazine resistance as a selectable plastome marker. With
such mechanisms to further reduce out-crossing risk, plastome transformation can
possibly meet the initial expectations.

3.2.3 Genetic use restriction technologies and recoverable block of function

Other molecular approaches suggested for transgene containment include: seed sterility,
utilizing the genetic use restriction technologies (GURT) (‘terminator gene’) [43, 44],
272 Ton Rotteveel et al.

and recoverable block of function (RBF) [45] to prevent transgene flow. Such proposed
technologies control both the gene influx of exo-ferality and endo-feral volunteer seed
dispersal, but theoretically if the controlling element of the transgene is silenced,
expression would occur, rendering a critical defect in principle and practice. The
frequency of loss of such controlling elements is yet unclear, as there have been no
large-scale field trials to test this.

3.2.4 Repressible seed lethal technologies

An impractical technology has been proposed to use a “repressible seed-lethal system”
[46]. The seed-lethal trait and its repressor must be simultaneously inserted at the same
locus on homologous chromosomes in the hybrid used for phytoremediation (in our
specific case), to prevent recombination (crossing over), a technology that is not yet
workable in plants. The hemizygote transgenic seed lethal parent of the hybrid cannot
reproduce by itself, as its seeds are not viable. If the hybrid could be made, half the
progeny would not carry the seed lethal trait (or the trait of interest linked to it) and they
would have to be culled, which would not be easy without a marker gene. A containment
technology should leave no viable volunteers with the transgene, but this complex
technology would kill only 25% of the progeny and 50% would be like the hybrid
parents and 25% would contain just the repressor. Thus, the repressor can cross from the
volunteers to related weeds, and so can the trait of choice linked with the lethal, and
viable hybrid plants could form. The death of a quarter of the seeds in all future
generations is inconsequential to plants that copiously produce seed, as long as the
transgenic trait provides some selective advantage.
In summary, none of the above containment mechanisms is absolute, but the risk
could be reduced by stacking a combination of containment mechanisms, compounding
the infrequency of gene introgression. Still, even at very low frequencies of gene
transfer, once gene transfer occurs, the new bearer of the transgene could disperse
throughout the population if it has just a small fitness advantage.

3.2.5 Transient transgenics

It is possible to insert certain phytoremediation traits encoding transgenes on RNA
viruses or in endomycorrhizae that are expressed in the plant, but are not carried through
meiosis into reproductive cells, and thus there will be no gene flow via seeds or pollen.
Attempts had been made to use endophytes to carry useful genes into plants by pressure-
infiltrating the endophytes into seeds [47, 48]. The advantage of the technology was that
it was not variety specific, such that indigenous species or varieties can be used. There
are endophytes that naturally participate in phytoremediation processes, e.g. the
Methylobacterium sp. that inhabits poplars and degrades explosives [49]. Genes from
this or similar species can and have been engineered into other endophytic bacteria, with
quite promising results [50, 51].
The same or other infection procedures could be used to introduce phytoremediation
traits by disarmed plant disease viruses as the vector. The possibility that such a
procedure might work was borne out in many cases with dicots showing that they
express virus-encoded genes, e.g. [52]. It was possible to infect Arabidopsis with
tobacco etch virus carrying the bar gene; the gene was fully expressed in the plants [53].
Risk assessment and gene flow 273

Cucurbits artificially infected with an attenuated zucchini yellow mosaic potyvirus

containing the same transgene s were resistant in the field [54]. An NTPII carrying
wheat streak mosaic virus was used to infect various grains, and the gene was expressed
(immunologically) [55]. The virus carrying the genes was expressed in the roots
following leaf infection, though not in all tissues.
Considerable technological obstacles of infection of the phytoremediating species
will have to be worked out. While no gene flow from the plants is expected, endophytic
bacteria are prone to horizontal gene transfer among themselves, an issue which
bacterial biosafety experts will have to consider. There are biosafety issues relating to
the mode of disarming to be considered, and it must be demonstrated that there is no
gene introgression from the virus to the plant chromosomes, as well as no-extra-nuclear
transmission of the virus through ovules or pollen in very large numbers of individuals.
It is necessary to transfect the phytoremediating species every generation, which may be
easier with perennials, such as poplar, and it may be more cumbersome with annual
species.

3.3. PREVENTING ESTABLISHMENT BY TRANSGENIC MITIGATION

If a transgene confers even a small fitness disadvantage, the less fit transgenic volunteers
and their own or hybrid progeny should only be able to exist as a very small proportion
of the population. Therefore, it should be possible to mitigate volunteer establishment
and gene flow by lowering the fitness of transgene recipients below the fitness of
competitors, so that the volunteer or hybrid offspring will reproduce with considerably
less success than its non-transgenic competitors. A concept of “transgenic mitigation”
(TM) was proposed [22], in which mitigator genes are linked or fused to the desired
primary transgene. Thus, a transgene with a desired trait is directly linked to a transgene
that decreases fitness in volunteers (Fig. 1). TM could also be used as a stand-alone
procedure with non-transgenic phytoremediating species to reduce the fitness advantage
of hybrids and their rare progeny, and thus substantially reduce the risk of exo-feral
hybrid volunteer persistence.
This TM approach is based on the premises that: 1) tandem constructs act as tightly
linked genes, and their segregation from each other is exceedingly rare; 2) the gain of
function dominant or semi-dominant TM traits chosen are neutral or favourable to
phytoremediating species, but deleterious to volunteer progeny and their hybrids due to
a negative selection pressure; and 3) individuals bearing even mildly harmful TM traits
will be kept at very low frequencies in volunteer/hybrid populations because strong
competition with their own wild type or with other species should eliminate even
marginally unfit individuals, and prevent them from persisting in the field population
[22].
Thus, it was predicted that if the primary gene(s) for phytoremediation advantage
being engineered into a phytoremediating species or a crop will not persist in future
generations if it is flanked by TM gene(s), such as genes (for crops) encoding dwarfing,
strong apical dominance to prevent tillering (in grains) or multi-heading (in crops like
sunflowers), determinate growth, non-bolting genes, uniform seed ripening, non-
shattering, anti-secondary dormancy. When they are in such a tandem construct, the
overall effect would be deleterious to the volunteer progeny and to hybrids. Indeed a
274 Ton Rotteveel et al.

TM gene such as anti-shattering should decrease re-seeding, and thus the number of
initial volunteers. With crops or phytoremediating species there is typically a small
amount of shattering due to imperfect harvesting equipment, which may leave a few
seeds behind. Because the TM genes will reduce the competitive ability of the rare
hybrids, they should not be able to compete and persist in easily measurable or
biologically significant frequencies in agroecosystems [20, 22].
Once TM genes are isolated, the actual cost of cloning them into TM constructs is
minimal, compared to the total time and effort in producing a transgenic phyto-
remediating species. The cost is even inconsequential in systems where biolistic co-
transformation allows introducing genes into the same site such that the tandem
construct is made by the plant.

3.3.1 Demonstration of Transgenic Mitigation in tobacco and oilseed rape

We used tobacco (Nicotiana tabacum) as a model plant to test the TM concept: a
tandem construct was made containing an ahasR (acetohydroxy acid synthase) gene for
herbicide resistance as the primary desirable gene of choice, and the dwarfing ∆gai
(gibberellic acid-insensitive) truncated gene as a mitigator [23].
Dwarfing would be disadvantageous to the rare weeds introgressing the TM
construct, as they could no longer compete, but is desirable in many crops, preventing
lodging and producing less stem with more leaves. The dwarf and herbicide resistant
TM transgenic hybrid tobacco plants (simulating a TM introgressed hybrid) were more
reproductive than the wild type when cultivated alone (without herbicide). They formed
many more flowers than the wild type when cultivated by themselves, which is
indicative of a higher harvest index. Conversely, the TM transgenics were weak
competitors and highly unfit when co-cultivated with the wild type in ecological
simulation of competition. The inability to achieve flowering on the TM plants in the
competitive situation resulted in zero reproductive fitness of the TM plants grown in an
equal mixture with the wild type at typical field spacing of plants resulting from seed
rain of volunteer weeds [23].
From the data above it is clear that transgenic mitigation should be advantageous to
a phytoremediation species growing alone, while disadvantageous to a hybrid with it
living in the competitive environment of the phytoremediation site, or off site. If a rare
pollen grain bearing tandem transgenic traits bypasses containment, it must compete
with multitudes of wild type pollen to produce a hybrid. Its rare progeny must then
compete with more fit wild type cohorts during self-thinning and establishment. Even a
small degree of unfitness encoded in the TM construct would bring about the elimination
of the vast majority of progeny in all future generations, as long as the primary gene
provides no selective advantage that counterbalances the unfitness of the linked TM
gene. Most phytoremediating genes have a drag, not an increased fitness off the
phytoremediation site. We have inserted the same construct into oilseed rape and
have tested the selfed progeny, as well as hybrids with the weed Brassica campestris
Risk assessment and gene flow 275

Fig. 1. Transgenic Mitigation to prevent establishment of (A) volunteers and (B) hybrids
between phytoremediation species and relatives. The phytoremediation species bears
desirable transgenes coupled in tandem with transgenes encoding traits that are neutral or
positive for the phytoremediating species, but render volunteers or hybrids unfit to compete
outside of cultivation. Source: From ref. [1], with permission of Springer Verlag.

x B. rapa. When cultivated alone, the dwarf transgenic oilseed rape grew at almost the
same rate as the transgenic (Fig. 2A), but produced twice as much seed as the non-
transgenic isoline (Fig. 2C). When the TM transgenic oilseed rape plants were co-
cultivated in competition with the wild type, they were unable to grow normally (Fig.
2B), and hardly set seed (Fig. 2C) because they were so unfit to reproduce.
276 Ton Rotteveel et al.

Fig. 2. Suppression of B. growth and C. seed yield of TM (transgenic mitigator) bearing

oilseed rape plants carrying a dwarfing gene in tandem with a herbicide resistance gene
(closed symbols and bars) when in competition with non-transgenic plants (open symbols
and bars)), and A. near-normal growth of the transgenics and C. much higher seed yield of
the transgenics when cultivated separately without herbicide at 3 cm spacing in a
biocontainment screenhouse. (Unpublished data: Al-Ahmad and Gressel, 2005).

The rare hybrid offspring from escaped pollen bearing transgenic mitigator genes would
not pose a dire threat, especially to wild species outside fields, as the amount of pollen
reaching the pristine wild environment would only be at a minuscule fraction of the
pollen from the wild type. This is dependent on the distance, source size, and on fertility
barriers. Large-scale cultivation creates large pollen sources, and in theory a wild
population having its niche on “the edge of agriculture” with coincident pollen shed
could be swamped. There has been pollen flow, but no swamping with native DNA of
wheat sporadically appearing in a ruderal Aegilops sp. [21]. Presently, there are no well
documented cases where fertility barriers do not prevent more than the formation of a
few infertile hybrids near the borders, as well as the rare introgressions, as have been
happening for time immemorial. Any unfit hybrids and their rare backcross offspring
containing transgenes linked to TM genes should still be eliminated. Further large-scale
field studies will be needed with crop/weed pairs to continue to evaluate the positive
implications of risk mitigation.

3.3.2 Risk that introgression of TM traits will affect relatives of the phytoremediating
species
A model by Haygood et al. [56] claims to “prove” the premise that “demographic
swamping” by transgenes would cause “migrational meltdown” of wild species related
to the crop or phytoremediating species, especially if the introgressed genes confer
unfitness. This proposition that recurrent gene flow from crops or phytoremediating
species, even TM gene flow, could affect wild relatives deserves some discussion, as it
negates the concept of transgenic mitigation.
They claim that their model demonstrates that recurrent gene flow from transgenic
crops or phytoremediating species with less fit genes will cause wild populations to
Risk assessment and gene flow 277

shrink. Firstly, conventional crops already belie this possibility. There are few if any
major domesticated crops that are fit to live in a wild ecosystem, so their normal genes
should confer a modicum of unfitness. Such crop x wild hybrids continually form, yet no
evidence is presented that demographic swamping has occurred due to recurrent gene
flow from the crops or phytoremediating species, nor could we locate any published data
to that effect. Indeed, considerable evidence has been presented that many crops exist
near their wild or weedy progenitors, without causing the extinction of the progenitors,
despite gene flow.
There are other mundane yet fatal flaws in their model based on shaky premises and
assumptions not borne out by plant biology. Three problematic issues that seem to
invalidate the relevance of their model for the vast majority of conceivable crop or
phytoremediating species/wild species systems, are discussed below:
• to get the level of swamping that they [56] discuss, the wild relative and
the phytoremediating species would have to live in the same ecosystem.
There are typically geographic separations between phytoremediation
ecosystems and wild ecosystems, with the extent of pollen flow decreasing
exponentially with distance between them – usually to a low asymptote due
to wind currents or insects not fully following simple physics. There
should always be far more wild pollen in the wild ecosystems, so
hybridisation events in the wild from crop pollen will be rare, even with
masses of pollen occurring within the agroecosystem. Thus their basic
assumption of transgenic pollen swamping wild type pollen in the wild is
invalid. Indeed, even when they assume an enormous 10% of
hybridisations in the wild each generation coming from transgenic pollen,
according to their model it will take about 20 generations of recurrent
pollination for the unfit allele to become fixed in half the population, and
50 generations for an unfit gene to asymptotically reach 80% of the
population. As discussed below, their other assumptions leading to these
numbers are also off target, so it should actually take much longer;
• they assume synchronous flowering, no self-fertilization, and no genetic or
other barriers to cross-fertilization; indeed, this negates the definition of
speciation. It is exceedingly rare for pollen from one species to fertilize
another species without any genetic barrier in the wild relative. Of the
species mentioned in [57], this might only occur with con-specific wild
sunflowers, which might fit this criterion, but even in this case there are
genomic deterrents to introgression (as reviewed in [26]. The flow of
genes between con-specific rice and red (weedy)-rice does not fit their
assumptions because they are cleistogamous, predominantly self-fertilizing
before the flowers open, and the amount of outcrossing possible is very
low. Of course weedy rice is not a wild species (by definition), so it too is
not really relevant to their case. There are fertilization barriers of different
chromosome numbers, non-homology etc, which limit fertilization of wild
relatives of oilseed rape and wheat, so they are outside the models;
• their models assume animal-type replacement rates – a few progeny per
mating, where lower fitness can indeed become fixed. Most wild relatives
278 Ton Rotteveel et al.

of phytoremediating herbaceous or tree species produce copious amounts

of seed to replace parents. Hundreds to thousands typically germinate in
the area occupied by a parent and the process of self-thinning is
ferociously competitive, eliminating less fit individuals. Our experimental
data show that at realistic seed output and seeding rates, unfit individuals
are eliminated or remain at a low frequency, just as unfit mutations are
maintained in populations at some low frequency (the relative fitness
multiplied by the mutation frequency).

Their conclusion that “the most striking implication of this model is the possibility of
thresholds and hysteresis, such that a small increase in (unfit gene) immigration can lead
to fixation of a disfavoured crop allele…..” [56] flies in the face of evolutionary
evidence, and decades of classic and contemporary field data showing that only near-
neutral genes exist in pockets of the evolutionary landscape of plants, and blatantly unfit
plant genes are not known to exist in such pockets unless all the fit genes are somehow
removed. Just as endogenous unfavored gene mutations exist in the wild at a frequency
lower than the mutation rate, transgenes from phytoremediating species that have a
fitness penalty will exist in the wild at a rate lower than the immigration rate. As
discussed above, the immigration rate to the wild is perforce very low. Unfit genes are
eliminated from populations of plants that produce large numbers of seeds, whereas the
genes could be fixed in populations of animals with few progeny. When a model
contradicts reams of data, it is more likely than not that the model is invalid.
Haygood et al. [56] further contend that their model would work if the
phytoremediating species were heterozygous for the unfit gene (and many transgenic
hybrids have the transgene in a single parent and are thus hemizygous). The data in Fig.
2 clearly demonstrate that when even half of the backcross progeny contain a TM
construct, they cannot compete with their non-transgenic sibs, let alone the wild type.
Part of the problem may be that Haygood et al. [56] (p. 1880 column 2) “assume (that)
the number of plants surviving to maturity does not vary from one generation to the
next”, a questionable assumption for unfit phenotypes when they must compete with fit
cohorts and other species.
In summary, where might their model have some validity? Even though, despite their
claims, the model has limited validity for the “wild” ecosystems, the model might be
valid for a few weeds (not wild species) related to phytoremediating species. Weeds are
man-made domesticated species (of a sort), and they are dependent on human controlled
agro-ecosystems. These systems change continuously, which leads to continual shifting
weed populations with an ever-changing composition. Over time new species invade,
and old species go extinct, adapt, or are once more confined to their original natural
environments. This is the nature of agriculture itself. It is likely that weeds that are
evolutionarily threatened by the flow of unfit genes would evolve exclusionary
mechanisms that block extinction;, e.g., they could evolve a shift to predominant self-
fertilization that would protect them from transgenic pollen bearing unfit genes. The
model of Haygood et al. [56] may be right for certain animal systems but irrelevant for
the vast majority of plant systems. They fail to mention specific plant systems where
their model might be valid. Indeed, the species that naturally phytoremediate mine sites
Risk assessment and gene flow 279

(for the last 2000 years in the case of Roman sites) are so unfit to compete off of mine
sites that the heavy metal resistant genes are not found in the same species of wind-
pollinated grasses a few cm from the edge of mine tailings [58]. Some pollen flowed, but
the hybrid offspring cannot compete with wild-type offspring.

3.3.3 Following transgene flow to volunteers and feral forms

Using the various containment and mitigation strategies it should be possible to keep
transgene “leaks” below risk thresholds, which have to be specified by science-based
regulators on a case-to-case basis. As the numbers of transgenic species being released is
increasing, and the problems of monitoring for such genes increases geometrically, we
suggested that a uniform biobarcodeTM system be used, where a small piece of non-
coding DNA having uniform recognition sites are at the ends (for single PCR primer
pair amplification) with an assigned variable region in between. Thus, PCR-automated
sequencing could be used to determine the origin of “leaks”, contamination, liability, as
well as intellectual property violations [59].

4. Special transgenic mitigation genes for phytoremediation

As more genes become isolated and their properties elucidated, it appears that many
might be specifically utilizable to contain and mitigate gene flow in plants used for
phytoremediation. Some genes that can be used for containment might be better used for
mitigation. For example, various Populus species have been genetically engineered and
field-tested out of doors for heavy metal tolerance or for metabolising halogenated
hydrocarbons, as well as male sterility, and lack of fertility [60], but necessarily linked
in tandem, so the traits can segregate. Male sterility and lack of fertility can prevent gene
outflow, albeit typically leaky. Thus, some pollen bearing the phytoremediation traits
can escape to the wild, and some pollen from the wild can fertilize the few flowers
appearing on a tree. In the case of vegetatively propagated species such as poplars, male
sterility can be coupled with female sterility, which will prevent pollen from nearby
related species from effectively pollinating the phytoremediating poplar. Additionally,
floral ablation can be used (no pollination in either direction) can be used, as described
in a review of the earlier literature [61]. A presently used cytotoxin gene under the
control, of a PTD flower promoter imparts “high levels” of floral ablation in poplar, a
species commonly used for phytoremediation [62], with complete loss of flower buds in
some lines tested in the greenhouse, in plants also engineered for early flowering.
Whether they are leaky and allow some flowering as plants mature is being tested in
field trials now in progress (S.H. Strauss, Oregon State Univ., pers. comm. 2004). If the
infertility is not 100% and the genes are just used for containment, i.e., not engineered in
a tandem construct with the phytoremediation genes, the infertility genes can segregate
from the phytoremediation genes in further generations, giving fertile plants with the
phytoremediation traits. If the same infertility genes are engineered in a tandem
construct or in such a way that they will be linked in planta (as happens with most
biolistic co-transformants), the two sets of traits will remain linked, and the rare escapee
280 Ton Rotteveel et al.

bearing infertility and phytoremediation will remain “mitigated”, i.e., in a perennially

low proportion of the population.
Some traits are appropriate containing/mitigating both tree, shrub, and herbaceous
phytoremediating plants, for example: the overexpression of a cytokinin oxidase [63],
which reduces the levels of isopentenyl and zeatin type cytokinins. This in turn leads to
phenotypes with far reduced shoot systems (unfitness to compete) but with faster
growing more extensive root systems [64], all the better for extracting toxic wastes.
Irreversible sterility is best for trees and shrubs that can be vegetatively propagated,
reversible male sterility is better for herbaceous species, as it allows seed production, as
described below.

4.1. CONTAINMENT/MITIGATION FOR HERBACEOUS

PHYTOREMEDIATION AGENTS

Mitigating genes should easily prevent or delay flowering in rosette type herbaceous
species such as the Brassica spp. that are two phase species, where the vegetative
material is harvested, and flowering (bolting) is detrimental. This could easily be
effected by preventing gibberellic acid biosynthesis [65], either in a TM construct and/or
by permanent mutation of the kaurene oxidase gene using a chimeraplastic gene
conversion system [66], a system that as yet is hard to use in plants. Kaurene oxidase
suppression would require the use of gibberellic acid to ‘force’ flowering for seed
production. There should be a concomitant biosafety requirement that seed production
areas be far removed from areas where weedy or other feral or wild relatives grow to
prevent pollen transfer.
Delaying of bolting and flowering by using a different transgene has recently been
demonstrated. Curtis et al. [67] engineered a fragment of the GIGANTEA gene, the gene
encoding a protein that is part of the photoperiod recognition system, into radish using
an antisense approach. Bolting was considerably delayed, and thus seed production
could come about without reversal mechanisms if seed producers waited long enough. If
despite all isolation distances, a TM construct or a mutant in a seed production area
introgresses with a wild species, the progeny will also be delayed, i.e., the transgenic
hybrid would be non-competitive with cohorts.

4.2. SPECIAL CONTAINMENT/MITIGATION GENES FOR PHYTORE-

MEDIATING TREES

In forestry, the possibility of gene flow is especially problematic as the duration until
long-term implications of gene movement become apparent can be longer than human
lifetimes. The introgression of traits from these species to wild populations has been
extensively discussed by [20, 68] and thus containment/mitigation requirements should
be stringent. Some phytoremediating species such as the poplars are vegetatively
propagated and thus flowers and seeds are not important – indeed may provide a
metabolic/genetic drag. Such phytoremediating trees can be vegetatively propagated,
and if sterile, besides possibly higher yield and biosafety, allergy-causing pollen clouds
and messy fruits would be prevented. An ideal gene for doing this is barnase under the
Risk assessment and gene flow 281

T29 tapetum-specific promoter [69]. The ribonuclease is only produced in the tapetum
and prevents pollen formation with no other ill effects.
If one has an important phytoremediating species in which transgenics are
exceedingly worthwhile, yet the risks of cultivation too great, one could envisage using a
pollen sterility system coupled with flower drop, as described above and the crop could
be propagated by artificial seed, e.g., artificially encased somatic embryos produced in
mechanized tissue culture systems. As noted above, such genes are being tested [60], but
whether in tandem with phytoremediation traits, or separate is not clear.
Poplar height is under control of gibberellic acid, just as it is with herbaceous species
[70]. The GAI and related dwarfism genes are thus being tested in poplar to ascertain
whether the shorter, fatter trees concept cited will grow any faster and be less
competitive under competition. So far a field trial has been growing for one year and the
researches at Oregon State University have many short, fattish trees (size varies from 1/3
to 2m)...but it will take several more years to ascertain the capacity to mitigate (Steven
Strauss, personal communication, 2004). They believe that better genes or more specific
promoters may be needed to really make the concept work. The professional foresters
are quite sceptical, given that tall and straight trees is what they have been taught to seek
all their careers (Steven Strauss, personal communication, 2004).
Another approach by scientists at Oji Paper Company in Japan for an analogous
situation has been announced (in a news release) [71]. They engineered Eucalyptus to
withstand very acid soils, and graft non-transgenic rapidly growing Eucalyptus on the
transgenic acid-tolerant rootstock. There can be no transgene flow from these plants,
unless suckers or shoots form on the rootstocks. Similar grafting approaches could be
used with many bioremediating tree species.

5. Concluding remarks

Systems exist that can theoretically preclude a phytoremediating species from becoming
established outside the contaminated area being treated, whether by containing gene
flow or by preventing the establishment of hybrids by mitigation. There is evidence that
some of these systems are efficient in crops, and there is no reason they could not be
used in phytoremediating species, where a risk of transgene flow is perceived. Thus, if a
risk of establishment is discerned using the enabling decision tree proved above, such a
risk should not preclude developing transgenic phytoremediation species – it should
stimulate the imagination to devise and test systems to deal with the potential problems.

Acknowledgements

The second and last authors’ research on transgenic mitigation was supported by the
Levin Foundation, by INCO–DC contract no. ERB IC18 CT 98 0391, and by a bequest
from Israel and Diana Safer. This chapter is a heavily augmented and updated version of
Gressel and Al-Ahmad [1], and updated portions from that review are included with the
permission of the Springer Verlag.
282 Ton Rotteveel et al.

References
[1] Gressel, J and Al-Ahmad, H (2005) Assessing and managing biological risks of plants used for
bioremediation, including risks of transgene flow. Z Naturforsch C, 60: 154-165
[2] Smith, RAH and Bradshaw, AD (1979) Use of metal tolerant plant populations for the reclamation of
metalliferous wastes. J Appl Ecol 16: 595-603
[3] Kramer, U; Smith, RD; Wenzel, WW; Raskin, I and De, S (1997) The role of metal transport and
tolerance in nickel hyperaccumulation by Thlaspi goesingense Halacsy. Plant Physiol 115: 1641-1650
[4] Pilon-Smits, E and Pilon, M (2002) Phytoremediation of metals using transgenic plants. Crit Rev Plant
Sci 21: 439-456
[5] Schulman, RN; Salt, DE and Raskin, I (1999) Isolation and partial characterization of a lead-
accumulating Brassica juncea mutant. Theor Appl Genet 99: 398-404
[6] Dushenkov, S; Skarzhinskaya, M; Glimelius, K; Gleba, D and Raskin, I (2002) Bioengineering of a
phytoremediation plant by means of somatic hybridization. Intl J Phytoremed 4: 117-126
[7] Zhu, YL; Pilon-Smits, EAH; Tarun, AS; Weber, SU; Jouanin, L and Terry, N (1999) Cadmium
tolerance and accumulation in Indian mustard is enhanced by overexpressing gamma-glutamylcysteine
synthetase. Plant Physiol 121: 1169-1177
[8] Bennett, LE; Burkhead, JL; Hale, KL; Terry, N; Pilon, M and Pilon-Smits, EAH (2003) Analysis of
transgenic Indian mustard plants for phytoremediation of metal-contaminated mine tailings. J Envir
Qual 32: 432-440
[9] Weis, JS and Weis, P (2004) Metal uptake, transport and release by wetland plants: implications for
phytoremediation and restoration. Envir Intl 30: 685-700
[10] Rugh, CL; Gragson, GM; Meagher, RB and Merkle, SA (1998) Toxic mercury reduction and
remediation using transgenic plants with a modified bacterial gene. Hortscience 33: 618-621
[11] Bittsanszky, A; Komives, T; Gullner, G; Gyulai, G; Kiss, J; Heszky, L; Radimszky, L and Rennenberg,
H (2005) Ability of transgenic poplars with elevated glutathione content to tolerate zinc(2+) stress.
Envir Intl 31: 251-254
[12] Schoenmuth, BW and Pestemer, W (2004) Dendroremediation of trinitrotoluene (TNT) - Part 2: Fate of
radio-labelled TNT in trees. Envir Sci Pollution Res 11: 331-339
[13] Gray, AJ; Marshall, DF and Raybould, AF (1991) A century of evolution in Spartina angelica. Adv
Ecol Res 21: 1-62
[14] Kowarik, I (2005) Urban Ornamentals Escaped from Cultivation, in J Gressel, Ed., Crop Ferality and
Volunteerism, pp. 97-121, CRC Press, Boca-Raton
[15] Stankiewicz, M; Gadamski, G and Gawronski, SW (2001) Genetic variation and phylogenetic
relationships of triazine-resistant and triazine-susceptible biotypes of Solanum nigrum - analysis using
RAPD markers. Weed Res 41: 287-300
[16] Bervillé, A; Muller, M-H; Poinso, B and Serieys, H (2005) Ferality : risks of gene flow between
sunflower and other Helianthus species, in J Gressel, Ed., Ferality and volunteerism in plants, pp. 209-
230, CRC Press, Boca-Raton
[17] Holm, LG; Plucknett, JD; Pancho, LV and Herberger, JP (1977) The World’s Worst Weeds, Distribution
and Biology. University Press of Hawaii (Vol. pp. 609) Honolulu
[18] Gressel, J and Rotteveel, T (2000) Genetic and ecological risks from biotechnologically-derived
herbicide resistant crops: decision trees for risk assessment. Plant Breeding Rev 18: 251-303
[19] Bizoux, JR; Brevers, F; Meerts, P; Graitson, E and Mahy, G (2004) Ecology and conservation of
Belgian populations of Viola calaminaria, a metallophyte with a restricted geographic distribution.
Belgian J Bot 137: 91-104
[20] Gressel, J (2002) Molecular biology of weed control, Taylor and Francis, London
[21] Weissmann, S; Feldman, M and Gressel, J (2003) Evidence for sporadic introgression of a DNA
sequence from polyploid wheat into Aegilops peregrina (Ae. variabilis), in Proceedings - 10th
International Wheat Genetics Symposium, Paestum (Italy), 539-542
[22] Gressel, J (1999) Tandem constructs: preventing the rise of superweeds. Trends Biotech 17: 361-366
[23] Al-Ahmad, H; Galili, S and Gressel, J (2004) Tandem constructs to mitigate transgene persistence:
tobacco as a model. Mol Ecol 13: 697-710
[24] Al-Ahmad, H; Galili, S and Gressel, J (2005) Poor competitive fitness of transgenically mitigated
tobacco in competition with the wild type in a replacement series. Planta (in press)
Risk assessment and gene flow 283

[25] Ellstrand, NC (2003) Dangerous liaisons – when cultivated plants mate with their wild relatives, Johns
Hopkins University Press, Baltimore MD
[26] Stewart, CN; Halfhill, MD and Warwick, SI (2003) Transgene introgression from genetically modified
crops to their wild relatives. Nature Rev Genet 4: 806-817
[27] Jenszewski, E; Ronfort, J and Chevre, AM (2003) Crop-to-wild gene flow, introgression, and possible
fitness effects of transgenes. Envir Biosafety Res 2: 9-24
[28] Gressel, J and Al-Ahmad, H (2005) Molecular mitigation of ferality, in J Gressel, Ed., Crop Ferality and
Volunteerism, pp. 371-388, CRC Press, Boca-Raton
[29] Daniell, H (2002) Molecular strategies for gene containment in transgenic crops. Nat Biotechnol 20:
581-586
[30] Khan, MS and Maliga, P (1999) Fluorescent antibiotic resistance marker for tracking plastid
transformation in higher plants. Nat Biotechnol 17: 910-915
[31] Maliga, P (2002) Engineering the plastid genome of higher plants. Curr Opin Plant Biol 5: 164-172
[32] Maliga, P (2004) Plastid transformation in higher plants. Annu Rev Plant Biol 55: 289-313
[33] Tian, JL; Shen, RJ and He, YK (2002) Sequence modification of merB gene and high organomercurial
resistance of transgenic tobacco plants. Chinese Sci Bull 47: 2084-2088
[34] Ruiz, ON; Hussein, HS; Terry, N and Daniell, H (2003) Phytoremediation of organomercurial
compounds via chloroplast genetic engineering. Plant Physiol 132: 1344-1352
[35] Bizily, SP; Kim, T; Kandasamy, MK and Meagher, RB (2003) Subcellular targeting of methylmercury
lyase enhances its specific activity for organic mercury detoxification in plants. Plant Physiol 131:
463-471
[36] Daniell, H; Datta, R; Varma, S; Gray, S and Lee, SB (1998) Containment of herbicide resistance
through genetic engineering of the chloroplast genome. Nat Biotechnol 16: 345-348
[37] Bock, R (2001) Transgenic plastids in basic research and plant biotechnology. J Mol Biol 312: 425-438
[38] Avni, A and Edelman, M (1991) Direct selection for paternal inheritance of chloroplasts in sexual
progeny of Nicotiana. Mol Gen Genet 225: 273-277
[39] Darmency, H (1994) Genetics of herbicide resistance in weeds and crops, in SB Powles and JAM
Holtum, Eds., Herbicide Resistance in Plants: Biology and Biochemistry, pp. 263-298, Lewis, Boca-
Raton
[40] Wang, T; Li, Y; Shi, Y; Reboud, X; Darmency, H and Gressel, J (2004) Low frequency transmission of
a plastid encoded trait in Setaria italica. Theor Appl Genet 108: 315-320
[41] Kiang, A-S; Connolly, V; McConnell, DJ and Kavanagh, TA (1994) Paternal inheritance of
mitochondria and chloroplasts in Festuca pratensis-Lolium perenne intergeneric hybrids. Theor Appl
Genet 87: 681-688
[42] Darmency, H (2005) Incestuous relations of foxtail millet (Setaria italica) with its parents and cousins,
in J Gressel, Ed., Crop Ferality and Volunteerism, pp. 81-96, CRC Press, Boca-Raton
[43] Oliver, MJ; Quisenberry, JE; Trolinder, NLG and Keim, DL (1998) Control of plant gene expression.
US Patent 5,723,765
[44] Crouch, ML (1998) How the terminator terminates: An explanation for the non-scientist of a remarkable
patent for killing second generation seeds of crop plants http://www.bio.indiana.edu/people/
terminator/html, The Edmonds Institute, Edmond WA, USA
[45] Kuvshinov, V; Koivu, K; Kanerva, A and Pehu, E (2001) Molecular control of transgene escape from
genetically modified plants. Plant Sci 160: 517-522
[46] Schernthaner, JP; Fabijanski, SF; Arnison, PG; Racicot, M and Robert, LS (2003) Control of seed
germination in transgenic plants based on the segregation of a two-component genetic system. Proc Natl
Acad Sci USA 100: 6855-6859
[47] Fahey, JW and Anders, J (1995) Delivery of beneficial clavibacter microorganisms to seeds and plants.
US Patent 5,415,672
[48] Tomasino, SF; Leister, RT; Dimock, MB; Beach, RM and Kelly, JL (1995) Field performance of
Clavibacter xyli subsp. cynodontis expressing the insecticidal protein gene cryIA(c) of Bacillus
thuringiensis against european corn borer in field corn. Biol Control 5: 442-448
[49] Van Aken, B; Yoon, JM and Schnoor, JL (2004) Biodegradation of nitro-substituted explosives 2,4,
6-trinitrotoluene, hexahydro-1,3,5-trinitro-1,3,5-triazine, an octahydro-1,3,5,7-tetranitro-1,3,
5-tetrazocine by a phytosymbiotic Methylobacterium sp. associated with poplar tissues (Populus
deltoides x nigra DN34). Appl Environ Microbiol 70: 508-517
284 Ton Rotteveel et al.

[50] Barac, T; Taghavi, S; Borremans, B; Provoost, A; Oeyen, L; Colpaert, JV; Vangronsveld, J and van der
Lelie, D (2004) Engineered endophytic bacteria improve phytoremediation of water-soluble, volatile,
organic pollutants. Nat Biotechnol 22: 583-588
[51] Newman, LA and Reynolds, CM (2005) Bacteria and phytoremediation: new uses for endophytic
bacteria in plants. Trends Biotechnol 23: 6-8
[52] Koo, M; Bendahmane, M; Lettieri, GA; Paoletti, AD; Lane, TE; Fitchen, JH; Buchmeier, MJ and
Beachy, RH (1999) Protective immunity against murine hepatitis virus (MHV) induced by intranasal or
subcutaneous administration of hybrids of tobacco mosaic virus that carries an MHV epitope. Proc Natl
Acad Sci USA 96: 7774-7779
[53] Whitham, SA; Yamamoto, ML and Carrington, JC (1999) Selectable viruses and altered susceptibility
mutants in Arabidopsis thaliana. Proc Natl Acad Sci USA 96: 772-777
[54] Shiboleth, YM; Arazi, T; Wang, Y and Gal-On, A (2001) A new approach for weed control in a cucurbit
field employing an attenuated potyvirus-vector for herbicide resistance. J Biotech 92: 37-46
[55] Choi, I-R; Stenger, DC; Morris, TJ and French, R (2000) A plant virus vector for systemic expression of
foreign genes in cereals. Plant J 23: 547-555
[56] Haygood, R; Ives, AR and Andow, DA (2003) Consequences of recurrent gene flow from crops to wild
relatives. Proc Roy Soc London B 270: 1879-1886
[57] Gressel, J, Ed. (2005) Crop Ferality and Volunteerism, CRC Press
[58] Bradshaw, AD (1982) Evolution of heavy metal resistance - an analogy for herbicide resistance? In HM
LeBaron and J Gressel, Eds., Herbicide Resistance in Plants, pp. 293-307, Wiley, New York
[59] Gressel, J and Ehrlich, G (2002) Universal inheritable barcodes for identifying organisms. Trends Plant
Sci 7: 542-544
[60] USDA-APHIS (2004) Search Results of the Field Test Release Permits Database for the U.S.,
http://www.nbiap.vt.edu/cfdocs/fieldtests3.cfm
[61] Meilan, R; Brunner, AM; Skinner, JS and Strauss, SH (2001) Modification of flowering in transgenic
trees, in N Morohoshi and A Komamine, Eds., Molecular breeding of woody plants, pp. 247-256,
Elsevier, Amsterdam
[62] Skinner, JS; Meilan, R; Ma, CP and Strauss, SH (2003) The Populus PTD promoter imparts floral-
predominant expression and enables high levels of floral-organ ablation in Populus, Nicotiana and
Arabidopsis. Molec Breeding 12: 119-132
[63] Bilyeu, KD; Cole, JL; Laskey, JG; Riekhof, WR; Esparza, TJ; Kramer, MD and Morris, RO (2001)
Molecular and biochemical characterization of a cytokinin oxidase from maize. Plant Physiol 125:
378-386
[64] Werner, T; Motyka, V; Laucou, V; Smets, R; Van Onckelen, H and Schmulling, T (2003) Cytokinin-
deficient transgenic Arabidopsis plants show multiple developmental alterations indicating opposite
functions of cytokinins in the regulation of shoot and root meristem activity. Plant Cell 15: 2532-2550
[65] Hedden, P and Kamiya, Y (1997) Gibberellin biosynthesis: Enzymes, genes and their regulation. Annu
Rev Plant Physiol Plant Molec Biol 48: 431-460
[66] Zhu, T; Mettenburg, K; Peterson, DJ; Tagliani, L and Baszczynski, CL (2000) Engineering herbicide-
resistant maize using chimeric RNA/DNA oligonucleotides. Nat Biotechnol 18: 555-558
[67] Curtis, IS; Nam, HG; Yun, JY and Seo, K-H (2002) Expression of an antisense GIGANTEA (GI) gene
fragment in transgenic radish causes delayed bolting and flowering. Transgenic Res 11: 249-256
[68] Llewellyn, DJ (2000) Herbicide tolerant forest trees, in SM Jain and SC Minocha, Eds., Molecular
Biology of Woody Plants (Vol. 2), pp. 439-466, Kluwer, Dordrecht
[69] Mariani, C; Debeuckeleer, M; Truettner, J; Leemans, J and Goldberg, RB (1990) Induction of male
sterility in plants by a chimeric ribonuclease gene Nature 347: 737-741
[70] Busov, VB; Meilan, R; Pearce, DW; Ma, C; Rood, SB and Strauss, SH (2003) Activation tagging of a
dominant gibberellin catabolism gene (GA 2-oxidase) from poplar that regulates tree stature. Plant
Physiol 132: 1283-1291
[71] Anonymous (2004) Oji grafts natural eucalyptus onto gene-altered eucalyptus. Nikkei Report, Aug 6,
see: http://www.agbioworld.org/newsletter_wm/index.php?caseid=archive&newsid=2209
HUMAN EXPOSURE ASSESSMENT FOR FOOD – ONE EQUATION
FOR ALL CROPS IS NOT ENOUGH

STEFAN TRAPP1 AND ALES KULHANEK2

1
Environment & Resources DTU, Technical University of Denmark,
DK-2800 Kongens Lyngby, E-mail: [email protected]
2
Department of Environmental Chemistry, Institute of Chemical
Technology, Technická 5, Prague 160 00, Czech Republic

Keywords: Benzo(a)pyrene, bioconcentration, health risk, models, plant uptake, soil

1. Introduction

Several risk assessment tools for contaminated soils have been developed, i.e. the
Contaminated Land Exposure Assessment Model – CLEA (UK), CETOX (Denmark),
CSOIL (The Netherlands), the Soil Screening Guidance – SSG (USA) or the (more
general) European Union System for the Evaluation of Substances – EUSES (EU). Each
of these tools uses a different approach for the calculation of the transfer into food [1].
The methods differ, and the estimation of transfer into food has a very high uncertainty
when calculating human exposure. Another shortcoming is that only one type of crop,
usually green vegetables, is considered. However, green vegetables represent only a
small fraction of vegetable food, compared to bread, potatoes, juice and other beverages.
Besides this, the uptake into leaves does not necessarily correlate with uptake into other
plant parts. The uptake and transport behavior of neutral organic compounds can be
adequately described with the available theory [2]. Plant specific models (leafy
vegetables, root vegetables, fruits from trees) have been developed [3, 4, 5]. The results
from crop-specific exposure assessment can not only provide more detailed information
for risk management, they may also lead to different conclusions [6].
The objective of this chapter is to gain an insight into the underlyimg equations of
the models, to show the similarities, but also the differences, and to compare the
outcome to the empirical equation of Travis & Arms [7]. Crop-specific models were
developed by describing the basic processes of convective or diffusive uptake, chemical
equilibrium between plant tissue (roots, wood, leaves, tubers) and surrounding soil or
air, as well as fluxes inside the plants. This mechanistic principle of building is similar
for all crop-specific models. However, processes and the parameterization depend on the
type of crop (Figure 1). The models are briefly described below but for more detailed
descriptions and the limits of applicability we refer the reader to the recent report of
Samsøe-Petersen et al. [8] or published original work [3, 4, 5].

285
M. Mackova et al. (eds.), Phytoremediation Rhizoremediation, 285 – 300.
© 2006 Springer.
286 Stefan Trapp and Ales Kulhanek

Figure 1. Principles of crop-specific plant uptake models.

Example compound: Benzo(a)pyrene (BaP) is used as example for the calculations. BaP
is a polycyclic aromatic hydrocarbon (PAH). It is one of the strongest carcinogens
registered. Its sources are incomplete burning processes from heating, traffic, industry,
and smoking. It occurs practically everywhere in the environment. BaP is very lipophilic
(characterized by the partition coefficient between n-octanol and water KOW, log KOW =
6.13) and semivolatile (characterized by the partition coefficient between air and water,
KAW, also known as dimensionless Henry’s Law Constant, KAW = 1.39 x 10–5) [9].

1. Chemical equilibrium

1.1. SORPTION TO SOIL MATRIX

The natural bulk soil consists of soil matrix, soil solution and soil gas. The distribution
coefficient between soil matrix and water Kd (L water/kg soil) of organic chemicals is
related to the fraction of organic carbon in soil OC (kg/kg):

CM / CW = Kd = OC x KOC

CW is the equilibrium concentration in the aqueous solution (mg/L), CM is the

concentration sorbed to the soil matrix (mg/kg). It follows the expression to calculate the
dissolved concentration of a chemical in soil (mg/L) from the total concentration in soil
CSoil (mg/kg, wet weight):
Human Exposure Assessment for Food 287

CW ȡ wet 1 (1)
= = KWS =
C Soil OC × K OC × ȡ dry + PW + K AW × PA K SW

ȡwet is the density of the wet soil (kg/L), ȡdry is the density of the dry soil, and PW and PA
are the volume fractions of water and air in the soil (L/L) (data see Table 1). KAW is the
partition coefficient between air and water (also named dimensionless Henry's Law
constant) and KOC is the partition coefficient between organic carbon and water and can
be estimated from [10]:

log KOC = 0.81 log KOW + 0.1

Example calculation. The concentration of BaP in wet soil is 1 mg/kg. What is the
concentration in soil solution?

KOC = 100.81 log KOW + 0.1 = 100.81 x 6.13 + 0.1 = 116 225

ȡ wet
CW = × C soil
OC × K OC × ȡ dry + PW + K AW × PA

1.95kg/L
= × 1mg/kg
0.02g/g × 116225L / kg × 1.6kg / L + 0.35L / L + 0.1L / L × 1.39 × 10−5

= 0.0005 mg/L

Table 1. Typical soil data.

Parameter Symbol Value Unit

Soil wet density ȡwet 1.95 kg/L
Organic carbon content OC 0.02 kg/kg
Soil pore water PW 0.35 L/L
Soil gas pores PA 0.1 L/L
Soil dry density ȡdry = ȡwet - PW kg/L

1.2. ROOT CONCENTRATION FACTOR RCF

Briggs et al. [11] macerated barley roots and made shaking experiments in water with
chemicals of different KOW. They expressed the result as “root concentration factor”
RCF (L/kg):
concentration in roots (mg/kg)
RCF = ņņņņņņņņņņņņņņņņņņ
concentration in water (mg/L)
288 Stefan Trapp and Ales Kulhanek

The RCF increased with KOW. The fit curve between RCF and KOW was

log (RCF – 0.82) = 0.77 log KOW – 1.52 or RCF = 0.82 + 0.03 KOW0.77

The RCF can be rewritten as KRW (L/kg), which describes the equilibrium partitioning
between root concentration CR (mg/kg fresh weight) and water CW (mg/L). The
partitioning occurs into the water, the lipid and the gas phase of the root:

KRW = WR + LR a KOW b + PA(root) KAW (2)

KOW is the equilibrium partition coefficient between n-octanol and water, W and L are
water and lipid content of the plant root, ‘b’ for roots is 0.77, ‘a’ = 1/ȡoctanol = 1.22.
Partitioning into the gas phase of the root, PA(root), is usually negligible.
Table 2. Water and lipid content of carrots.

Parameter Symbol Value Unit

Carrot water content WR 0.89 L/kg
Carrot lipid content (*) LR 0.025 kg/kg
Carrot air pores PA(carrot) 0.05 L/kg
(*) includes all lipid-like compounds, also waxes like suberin and cutin.

Example Root concentration factor of BaP:

BaP by Briggs’ equation

log (RCF – 0.82) = 0.77 log KOW – 1.52 = 0.77 x 6.13 – 1.52 = 3.20

RCF – 0.82 = 103.20 = 1584.8

RCF = 0.82 + 1584.8 = 1586 L kg–1

or

RCF = 0.82 + 0.03 KOW0.77 = 0.82 + 0.03 x 1 348 9630.77 = 1576 L kg–1

By equation 2
Human Exposure Assessment for Food 289

KRW = 0.89 L kg–1 + 0.025 kg kg–1 x 1.22 L kg–1 x 1 348 9630.77+ 0.05 L kg–1

x 1.39 x10 – 5 = 1602 L kg–1

2. Dynamic root uptake model for neutral lipophilic organics

The “carrot model” calculates uptake into root with the transpiration water [4]. The
change of chemical mass in roots = + flux in with water – flux out with water

dm R
= CW × Q − C Xy × Q
dt

mR is the mass of chemical in roots, Q is the transpiration stream (L/d), CXY is the
concentration in the xylem (mg/L) at the outflow of the root. Diffusive uptake is not
considered (the carrot is peeled!). From mass, we get the concentration by dividing
through the mass of the root M:

d (C R × M ) dm R
= = CW × Q − C Xy × Q
dt dt

If growth is exponential, and the ratio Q/M (transpiration to plant mass) is constant, the
growth by exponential dilution can be considered by a first-order growth rate k (d–1):

dC R
= CW × Q / M − C Xy × Q / M − k × C R
dt

If the xylem sap is in equilibrium with the root, the concentration CXy = CR/KRW. Then,

dC R
= CW × Q / M − C R / K RW × Q / M − k × C R
dt

Setting this to steady-state (dCR/dt = 0) gives us

Q
CW × Q / M = C R × ( − k)
K RW × M

And we solve for the concentration in the root CR:

Q
CR = CW
Q
+ kM
K RW
290 Stefan Trapp and Ales Kulhanek

The ratio of the concentration in soil water CW to that in bulk soil CSoil is KWS, and for
the bioconcentration factor BCF (ratio of concentrations in plant and soil) between
carrot and bulk soil follows:

CR C Q (3)
BCF = = R × K WS = × K WS
C Soil CW Q
+ kM
K RW

The model approach is strictly limited to non-ionising compounds, log KOW >1. The
parameterization of the model is for 1 m2 soil, with 1 kg roots, a transpiration of 1 L d–1
and a root growth rate of 0.1 d–1. This parameterization is not idealized, the values are
realistic (Table 3).
Table 3. More data for carrots.

Parameter Symbol Value Unit

Transpiration stream Q 1 L/d
Root mass M 1 Kg
1st order growth rate k 0.1 d-1

Example calculation for BaP.

From before: KWS = 0.0005 kg/L, KRW = 1602

Q 1 L/ d
BCF = × KWS = × 0.0005 kg / L = 0.005 kg / kg
Q 1 L/ d + 0.1 d −1 ×1 kg
+ kM 1602 L / kg
KRW

2.1. TRANSLOCATION UPWARDS

The water, which is taken up by the roots, does not stay there but is translocated in the
xylem to the leaves and evaporates from the stomata. Only 1-2% is taken up into the
plant cells. Chemicals, which are dissolved in the “transpiration stream” (= the xylem
sap), can be moved upwards, too. The ‘Transpiration Stream Concentration Factor’
TSCF is defined as the concentration ratio between xylem sap and external solution
(water).

TSCF = CXy/CW
The TSCF can be calculated from the root model. The xylem sap consists of water, and
with CXy = CR / KRW follows

C Xy Q / K RW
=
CW Q
+ kM
K RW (4a)
Human Exposure Assessment for Food 291

Alternatively, empirical relations exist. The TSCF is related to the KOW [11] by a bell-
shaped (Gaussian) curve:

­– (log K OW –1.78) 2 ½
TSCF = 0.784 × exp® ¾
¯ 2.44 ¿ (4b)

For poplar trees, a similar relation was found by [12] (corrected by authors):

­– (log K OW – 2.50) 2 ½
TSCF = 0.756 × exp ® ¾
¯ 2.58 ¿ (4c)

It is recommended to use Briggs’ equation (4b) [11] for herbal plants and the green
vegetables model (experiments were done with the grass barley), and Burken &
Schnoors equation (4c) [12] for woody plants and the fruit tree model (experiments were
done on poplars).
The calculated TSCF (4a) is equivalent to the empirically derived fit curves of
Briggs et al. (4b) and Burken and Schnoor (4c), but only as long as log KOW is not <1.5
(Figure 2). At lower values, reduced uptake was found in the experimentally determined
TSCF-curves, possibly due to decreasing membrane permeability for polar compounds.
These experiments were done in hydroponic solution. The TSCF depends on diffusion
processes, and the root surface area is a limiting factor for exchange [13]. In soil, roots
will - differently from hydroponic solution - form root hairs, which increase largely the
surface area. Therefore, the uptake limitation for polar compounds does probably not
exist. Experiments to clarify this are lacking so far.

1.2

0.8
TSCF

0.4

0
−1 1 3 5

log Kow

Briggs fit Briggs B+S

CXy B+S fit

Figure 2. Comparison of empirical and calculated TSCF; ‘Briggs fit’ is eq. 4b; ‘ǻ Briggs’ is original data
from Briggs et al. [11]. ‘B+S’ is original data from Burken and Schnoor [12]; ‘CXY’ is calculated with eq.
4a; ‘B+S fit’ is eq. 4c.
292 Stefan Trapp and Ales Kulhanek

Example calculation TSCF.

1L / d
C Xy Q / K RW 1602 L / kg
= = = 0.0062 L / L
CW Q
+ kM
1L / d + 0.1d −1 × 1kg
1602 L / kg
K RW
­– (6.13 - 1.78)2 ½
TSCF = 0.784 × exp® ¾ = 0.00034 L / L
¯ 2.44 ¿

­– (6.13 –2.50)2 ½
TSCF = 0.756 × exp ® ¾ = 0.0046 L / L
¯ 2.58 ¿

All three models predict low translocation of BaP, the root model's theoretical curve is
in between the empirical predictions.

3. Fruit tree model

Based on Trapp et al. [14]. Trees differ from herbs by their woody stem. Wood is
composed of cellulose, hemicellulose and lignin. Lignin is a giant macromolecule with
lipophilic sorption properties. The ratio between a chemical’s concentration in wood and
its concentration in the water is named “KWood” (mg chemical per g dry wood to mg
chemical per mL water). The log KWood is significantly correlated to the log KOW of the
chemical and the following regression was established [15]:

Oak: log KWood = – 0.27 + 0.632 log KOW

The tree model considers chemical flux into stem via xylem, flux out of the stem after
equilibration, dilution by growth and first order metabolism. Exchange with air and bark
and phloem flow are (in this version) neglected. The transport of the compound is
considered as a passive transport, taking place with the transpired water from the xylem
of the roots:

influx per time = Q × CXy = Q x CW x TSCF

where Q is the transpiration stream (m3/year) and CXy is the concentration of the
compound in the xylem sap, which is TSCF x CW. The chemical is transported out of the
stem with the water transpired from the xylem of the stem into leaves and fruits:

loss per time = Q × CStem /KWood

Human Exposure Assessment for Food 293

where CStem is the concentration of the chemical in the stem (mg/kg). Then, the mass
balance of the stem is

dm Stem
= + Q × C W × TSCF − Q × C Stem /K Wood
dt

and by replacing mass with concentration, and with exponential growth rate kG:

dCStem Q × CW × TSCF Q × CStem

= − − kG × C Stem
dt M KWood × M

The steady-state solution (dC/dt = 0) for the concentration in the stem is

Q
C Xy ×
C Stem (t = ∞ ) = M
Q
+ kG
M × K Wood (5)

Uptake into fruits. Transport from stem into fruits can occur both with the phloem and
xylem sap. It may be assumed that the water flow into fruits is about 20 times the dry
matter content. The calculated water flow per kg fruit (fresh weight) is:

QF = dw × 20

where QF is the water flow (L) into 1 kg of fruit, and dw is the dry weight fraction of the
fruit. The amount of chemical m (mg) transported into the fruit can then be calculated
from the concentration in the stem by assuming chemical equilibrium between wood,
phloem and xylem:

m = QF × CStem /KWood

The chemical concentration in the fruit CF (mg/kg fresh weight) is obtained by dividing
the chemical mass m by the weight of the fruit MF (1 kg):

CF = m / MF or in one step:

CF QF C
= × Stem
C Soil K Wood × M F C Soil (6)
294 Stefan Trapp and Ales Kulhanek

Example calculation for an apple tree stand with 100 tons/ha dry wood and 300 mm/year
(3000 m3 ha–1 year–1) transpiration, a growth rate of 0.01 year–1, and the water flux for
apples (3.12 L water per kg fruit). Results we have from before: Dissolved concentration
in soil CW = 0.0005 mg L–1; TSCF [12] = 0.0046 L L–1.

log KWood = –0.266 + 0.632 log KOW = 3.61; KWood = 4057.

Concentration in stem (Equation 5)

3000
0.0005 × 4.6 × 10 −3 ×
C Stem 100 = 0.004 mg/kg fresh plant
=
C Soil 3000 mg/kg wet soil
+ 0.01
100 × 4057

Concentration in apples (Equation 6); Water flux into apples:

QF = dw × 20 = 0.156 × 20 = 3.12 L/kg

CF QF C 3.12 L
= × Stem = × 0.004 kg / kg = 3.1 × 10 − 6 kg/kg
C Soil KWood × M F C Soil 4057 L / kg × 1kg

4. TGD-model for leafy vegetables

Based on Trapp and Matthies [3]. This model was developed for the uptake of neutral
organic substances into leafy vegetable or green fodder. The main objective of the model
was the integration into multi-media fate models, which are used in risk assessment of
new and existing chemicals. The model was adopted in the Technical Guidance
Documents (TGD) of the European Union System for the Evaluation of Substances
(EUSES) [10]. For leafy vegetables, there is uptake from soil via the xylem and
exchange with air. The change of mass in leaves = + translocation from stem + uptake
from air - loss to air

dm L C × g × A× ρ
= + Q × TSCF × CW + C Air × g × A − L
dt K LA

The differential equation for the concentration in leaves considers again additionally the
dilution by exponential growth:

dC L Q C ×g×A C × g × A× ρ
=+ × TSCF × CW + Air − k Growth × C L − L
dt ML ML K LA × M L
Human Exposure Assessment for Food 295

where L is the index for leaves. The equation can be rewritten and gives the standard
linear differential equation

dC L b
= b − aC L C L (t )= C L (0) × e − at + (1 − e − at )
dt with the solution a (7)

where
A× g × ρ A
a= + k Growth b = CW × TSCF × Q / M L + C Air × g ×
K LA × M L and ML

The steady-state solution is

b
C L (t = ∞) =
a
Table 4. Parameterization of the leafy vegetables model, normalized to 1 m2 (data taken from the original
publication [3]).

Parameter Symbol Value Unit

Shoot mass ML 1 kg
Leaf area A 5 m2
Shoot density ȇ 500 kg/m3
Transpiration Q 1 L/d
Lipid content L 0.02 kg/kg
Water content W 0.8 L/kg
Conductance G 10–3 m s–1
Growth rate kL 0.035 d –1
Time to harvest T 60 d

Example calculation for benzo(a)pyrene. CSoil is 1 mg/kg, CAir is 1 ng m–3. What is the
concentration in leaves? Is the uptake from soil or from air? Data we know: CW is 0.5 x
10–3 mg/L; TSCF (Briggs) = 0.00036

A× g × ρ
Loss term: a= + k Growth
K LA × M L

= 5 m2 x 10–3 x 86400 m d–1 x 500 kg m–3/(1.2 x 109 m3 : m3 x 1 kg) + 0.035 d–1

= 0.035 d–1
296 Stefan Trapp and Ales Kulhanek

A
Input term: b = CW × TSCF × Q / M L + C A × g ×
ML
= 0.5 x 10–3 mg/L x 0.00036 x 1 L/d / 1kg + 10–6 mg m–3 x 10–3 x 86400 m d–1 x 5 m2 / 1kg

= 0.18 x 10–6 mg kg–1 d–1 + 0.00043 mg kg–1 d–1 = 0.00043 mg kg–1 d–1

From an inspection of this last line it can be seen that the uptake into leaves from air is
240 times faster than from soil. The concentration in leaves at t = 60 days is:

b
C L (t ) = C L (0) × e −at + (1 − e − at )
a
0.00043 mg kg −1 d −1
=0+ −1
× (1 − e −0.0035 × 60 ) = 0.011 mgkg −1
0.0035 d

And steady-state:

b/a = 0.00043 mg kg–1 d–1 / 0.035 d–1 = 0.012 mg/kg.

If the uptake is only from soil:

b/a = 0.18 x 10–6 mg kg–1 d–1/0.035 d–1 = 5.1 x 10–6 mg/kg.

Note: This model is used in the EU risk assessment for new and existing chemicals
(EUSES), but it lacks one important pathway: deposition of soil on leaves. Many green
vegetables are contaminated by attached soil (e.g., lettuce). To consider this, there is
always a default soil-to-plant transfer with particles of 1% attached soil assumed, which
means a minimum BCF plant/soil of 0.01 (wet weight based):

BCF with soil = BCF model + 0.01 = 5.1 x 10–6 + 0.01 § 0.01mg/kg
Human Exposure Assessment for Food 297

5. The regression of Travis and Arms (T&A)

Travis and Arms [7] established a regression that estimates uptake of neutral organic
chemicals into above-ground plants. The regression has been obtained from a range of
empirical data acquired by the authors from literature. Mainly data from uptake of
pesticides with a log KOW ranging from 1.15 to 9.35 were used but no particular type
of vegetation was targeted in the original study. The form of the regression is:

log BV (dry) = 1.588 – 0.578 log KOW (8)

where BV is the bioconcentration factor vegetation and is the concentration ratio

between plants (dry weight) and soil (dry weight). The BV was converted into wet
weight bioconcentration factors (BCF) using

BCF (wet) = BV (dry) x (1 - W) x ȡwet / ȡdry

where W is the water content of the plants, and ȡ is the density of the soil.

In practice, the regression of Travis and Arms needs lesser efforts to calculate BCFs.
Therefore, it might serve as an “early warning”. The advantage of the crop-specific
models is the possibility to adapt them to specific situations. So they might be used when
the regression signals a critical situation.

BCF of BaP in carrot with the Travis and Arms regression:

log BV (dry) = 1.588 – 0.578 x 6.13 = –1.955 ĺ BV (dry) = 0.01

Carrot water content; W = 0.89

BCF (wet) = BV (dry) x (1 – 0.89) x 1.95 / 1.6 = 0.134 x BV(dry) = 0.0013

6. Exposure and risk assessment

For the calculation of exposure, the concentrations in the different food crops need to be
multiplied with the consumption of these foodstuffs. The consumption, expressed as
daily dietary intake, may vary for different groups of society (e.g., children, vegetarians)
and different regions. To derive the risk associated with food consumption, toxicological
values, such as acceptable daily intake, have to be compared with the actual uptake.
298 Stefan Trapp and Ales Kulhanek

6.1. DAILY DIETARY INTAKE (DDI)

For the calculation of the daily dietary intake (DDI), the consumption of vegetables is
multiplied with the calculated concentration in the crop types, i.e. root vegetable, fruits,
potatoes and leafy vegetable. The daily dietary intake DDI is then

DDI [mg/d] =™i concentration in crop(i) [mg kg–1] x mass of crop(i) consumed [kg d–1]

Table 5. Average consumption [g person – 1 d – 1] in the Czech Republic [16].

Crop type Example Consumption

Leafy vegetable Spinach, lettuce 2.4
Tree fruit Apples, pears 62.7
Root vegetable Carrots 13.5
Sum 78.6

The DDI with the Travis & Arms regression (equation 8), neglecting the differences in
water content, is:

DDI = 0.0013 mg/kg x 0.079 kg/d = 102 ng/d

This is a quite similar result to the crop-specific calculation (see example below), giving
confidence in both methods. However, the crop-specific approach allows a much more
situation-specific estimation. E.g., the use of a BaP-contaminated garden for growing
vegetables might yield a quite considerable additional exposure to BaP, whereas the use
for a grass lawn plus some apple trees would lead to a very small additional exposure of
humans to BaP. “Crop-taylored management” of polluted soils is a chance to reduce the
risk associated with soil pollution with very low costs.
Example calculation: Daily Dietary Intake DDI of BaP in the Czech Republic by consumption of various
crops

Crop Concentration Consumption DDI DDI

(mg/kg) (kg/d) (mg/d) (ng/d)
Root vegetable 0.005 13.5 x 10–3 6.75 x 10–5 67.5
Tree fruit 3.1 x 10–6 62.7 x 10–3 1.94 x 10–7 0.19
Leafy vegetable 5.1 x 10–6 2.4 x 10–3 1.22x 10–8 0.01
Leafy veg. + attached soil 0.01 2.4 x 10–3 2.4 x 10–5 24
Sum 0.079 9.7 x10–6 91.7

6.2. ACCEPTABLE DAILY INTAKE (ADI)

“Sola dosis facit venenum” (Paracelsus, 16th century) – everything is toxic, it is only the
dosis that makes a thing a poison or a remedy. An acceptable daily intake of the
chemical under investigation has to be defined by human toxicologists.
Human Exposure Assessment for Food 299

A “virtually safe dose of BaP as a marker of the mixture of carcinogenic PAH in

food would be in the range 0.06 to 0.5 ng BaP kg–1 bw d–1” [17]. If we chose the lowest
value of this virtually safe dose as acceptable, the acceptable daily intake (ADI) of BaP
via food for an adult weighing 70 kg would be 4.2 ng d–1. If the upper limit of the
virtually safe dose is used, the ADI of BaP would be 35 ng d–1 (the unit “ng BaP kg–1 bw
d–1”means nanogram benzo(a)pyrene per kilogram bodyweight and day).

6.3. ACCEPTABLE SOIL CONCENTRATION (ASC)

To derive an acceptable soil concentration, we expect that the life-long consumption of

food produced from this soil does not lead to oral intake above the virtually safe dose.
Using the linearity of BCF to concentration in soil, the acceptable soil concentration
(ASC) can be estimated from DDI (calculated for a concentration in soil of 1 mg/kg):

ASC [mg kg–1 wet wt] = {ADI [mg d –1]/ DDI [mg d –1]} x 1 mg kg–1 wet wt

What would be a “virtually safe” or acceptable concentration of BaP in soil?

with ADI = 4.2 ng/d ASC = 4.2 / 91.7 = 0.046 mg BaP/kg soil

with ADI = 35 ng/d ASC = 35 / 91.7 = 0.38 mg BaP/kg soil

Note: This is only for uptake with root vegetables, tree fruits and leafy vegetables. The
legal standards for BaP in soil are: Czech Republic: 0.1 mg/kg (dry weight), US-EPA
0.087 mg/kg (dry weight); Denmark: 3 mg/kg (since 2005).

7. Conclusions

The concept of crop-specific human exposure assessment for soil pollutants allows
adapting to different food habits, different life-styles, different regions and different
accepted risks. Models for transfer into major food crops are combined with regional
food baskets to give the exposure of the population, which can be compared to tolerable
risks based on health considerations. Backwards, tolerable concentrations of pollutants
in soil for the agricultural production and gardening can be derived. The approach might
therefore be used to derive rational soil quality standards for a large variety of chemicals
with reasonable effort. It could also be the basis for an internationally harmonized
procedure in soil risk assessment [18].

Acknowledgements

We are grateful for the international students funding Erasmus/Socrates awarded to Ales
Kulhanek and for the founding MSM 223200003 from the government of the Czech
Republic. We furthermore thank J.-P. Schwitzguébel for organising the COST 859 (and
300 Stefan Trapp and Ales Kulhanek

other actions). In particular, we thank Lise Samsøe-Petersen and Dorte Rasmussen, DHI
Hørsholm, and the Danish EPA for support.

References
[1] Versluijs, CW; Koops, R; Kreule, P and Waitz, MFW (1998) The accumulation of soil contaminants in
crop, location-specific calculation based on the CSOIL module. Part 1: Evaluation and suggestion for
model development. RIVM Report No 711 701 008, Bilthoven, The Netherlands
[2] Trapp, S and Mc Farlane, JC (1995) Plant Contamination. Modeling and Simulation of Organic
Chemical Processes. Boca Raton, Florida: Lewis Pub
[3] Trapp, S and Matthies, M (1995) Generic One-Compartment Model for Uptake of Organic Chemicals
by Foliar Vegetation. Environ Sci Technol 29, 2333-2338; Erratum 30: 360
[4] Trapp, S (2002) Dynamic root uptake model for neutral lipophilic organics. Environ Toxicol Chem 21:
203-206
[5] Trapp, S; Rasmussen, D and Samsøe-Petersen, L (2003) Fruit Tree Model for Uptake of Organic
Compounds from Soil. SAR - QSAR Environ Res 14: 17-26
[6] Kulhanek, A; Trapp, S; Sismilich, M; Janku, J and Zimova, M (2005) Cropp-Specific Human Exposure
Assessment for Polycyclic Aromatic Hydrocarbons in Czech Soils. Sci Tot Environ (in press)
[7] Travis, C and Arms, A (1988) Bioconcentration in beef, milk and vegetation. Environ Sci Technol 22:
271-274
[8] Samsøe-Petersen, L; Rasmussen, D and Trapp, S (2003) Modellering af optagelse af organiske stoffer i
grøntsager og frugt. Rapport til Miljøstyrelsen, Miljøprojekt Nr 765 2003
[9] Rippen, G (2002) Handbuch Umweltchemikalien. ecomed, Landsberg a.L, D
[10] EC European Commission (1996) Technical Guidance Document in Support of Commission Directive
93/67/EEC on Risk Assessment for New Notified Substances and Commission Regulation (EC) No
1488/94 on Risk Assessment for existing substances. European Commission, Office for Official
Publications of the European Communities, Luxemburg, Luxemburg
[11] Briggs, GG; Bromilow, RH and Evans, AA (1982) Relationships between lipophilicity and root uptake
and translocation of non-ionised chemicals by barley. Pestic Sci 13: 495-504
[12] Burken, JG and Schnoor, JL (1998) Predictive relationships for uptake of organic contaminants by
hybrid poplar trees. Environ Sci Technol 32: 3379-3385
[13] Trapp, S (2000) Modeling uptake into roots and subsequent translocation of neutral and ionisable
organic compounds. Pest Manag Sci 56: 767-778
[14] Trapp, S; Rasmussen, D and Samsøe-Petersen, L (2003) Fruit Tree Model for Uptake of Organic
Compounds from Soil. SAR - QSAR Environ Res 14: 17-26
[15] Trapp, S; Miglioranza, KSB and Mosbæk, H (2001) Sorption of Lipophilic Organic Compounds to
Wood and Implications for their Environmental Fate. Environ Sci Technol 35: 1561-1566
[16] Ruprich, J (2000) Spotrebni kos potravin pro Ceskou republiku - 2000, Expozicni faktory CR 1997.
National Institute of Public Health, Prague, Czech Republic
[17] EC European Commission (2002) Health and Consumer Protection Directorate (ed.): Opinion of the
scientific committee on food on the risks to human health of polycyclic aromatic hydrocarbons in food.
Brussels, Belgium. http://europa.eu.int/comm/food/fs/sc/scf/index_en.html
[18] Trapp, S (2003) Harmonized European Assessment for the Transfer of Chemicals from Soil into Food.
Journal of Soils and Sediments 3: 6