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Cyanobacteria as bioindicators and
bioreporters of environmental analysis in
aquatic ecosystems

Pilar Mateo, Francisco Leganés, Elvira

Perona, Virginia Loza & Francisca
Fernández-Piñas

Biodiversity and Conservation

ISSN 0960-3115
Volume 24
Number 4

Biodivers Conserv (2015) 24:909-948

DOI 10.1007/s10531-015-0903-y

1 23
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 Author's personal copy
Biodivers Conserv (2015) 24:909–948
DOI 10.1007/s10531-015-0903-y

REVIEW PAPER

Cyanobacteria as bioindicators and bioreporters

of environmental analysis in aquatic ecosystems

Pilar Mateo1 • Francisco Leganés1 • Elvira Perona1 •

Virginia Loza1 • Francisca Fernández-Piñas1

Received: 22 December 2014 / Revised: 24 February 2015 / Accepted: 4 March 2015 /

Published online: 17 March 2015
Ó Springer Science+Business Media Dordrecht 2015

Abstract Knowledge of the incidence of anthropogenic pressure on water ecosystems is

one of the main focus of integrated water resource management. The use of biological
methods to assess water quality is of particular importance since organisms show an
integrating response to their environment. Tolerances or ecological ranges of individual
species can differ depending on the taxon, which leads to distinct bioindicator values of
cyanobacterial taxa. In addition, a number of morphological and physiological features are
known to relate with the environment in which they occur, which makes them excellent
environmental indicators. Therefore, we review literature data of the main cyanobacterial
methods used to obtain information about changes in running water quality, mainly related
to eutrophication processes, which are found as the main cause of disturbance in rivers,
with the focus on benthic cyanobacteria, as habitat recommended for monitoring studies.
Further, their trophic independence and ease of cultivation make them very useful in the
field of bioreporters of environmental monitoring and ecotoxicology. In fact, several
cyanobacterial strains have been already genetically engineered to construct bioreporters
which respond to different types of pollutants as well as limiting nutrients. The potential of
cyanobacteria both as in situ bioindicators as well as bioreporters of environmental ana-
lysis in aquatic ecosystems will be discussed.

Keywords Environmental monitoring
Eutrophication
Nutrients
Toxicology

Transgenic cyanobacteria

Abbreviations
BMWPc Biological Monitoring Working Party adapted to Catalonian streams
IBD Biological Diatom Index (Indice Biologique Diatomées)

Communicated by Anurag chaurasia.

& Francisca Fernández-Piñas

[email protected]
1
Departamento de Biologı́a, Universidad Autónoma de Madrid, Cantoblanco, 28049 Madrid, Spain

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Introduction

Anthropogenic pressure on water bodies which receive diverse pollutants, leading to

multiple negative ecological impacts on these habitats, is a serious problem, whereby
knowledge of the state of water quality is essential for water management systems. In the
last decades, eutrophication problems have replaced organic pollution as the dominant
chemical pressure in large river systems being caused by high nutrient (nitrogen, N, and
phosphorus, P), fluxes from population pressure and intensive agriculture (Jarvie et al.
2013; Mischke et al. 2011). For watershed management, P has been regarded as the
primary limiting nutrient for algal growth as nuisance in freshwaters. Accordingly, con-
trolling P inputs has been the primary goal for resource managers, reducing freshwater P
loads but this is not the case for N, which is more mobile throughout the environment,
being therefore both N and P important in eutrophication processes (Paerl et al. 2011).
Traditionally the assessment of river water quality had been based on the measurement
of physico-chemical characteristics, but although these measurements were efficient for
regulating effluent discharges and protecting humans, they were not very useful for large-
scale management of catchments or for assessing whether river ecosystems are being
protected. Therefore, different biological methods were developed to assess river condition
since effects on biota are usually the final point of environmental degradation and pollution
of these systems (Norris and Thoms 1999). Two important arguments in favour of bio-
logical methods are (a) due to organisms having an integrating response to their envi-
ronment, fluctuations in water quality, which may be missed by intermittent physico-
chemical analysis, are recorded, and (b) if we wish to maintain healthy, diverse biological
communities, it is more appropriate to monitor the aquatic community rather than physico-
chemical variables only (Cox 1991). The advantage of monitoring with the use of
bioindicators is that biological communities reflect overall ecological quality by integrating
the effects of different stressors providing a broad measure of their impact and an eco-
logical measurement of fluctuating environmental conditions. Overall routine monitoring
of biological communities is reliable and relatively inexpensive compared to the cost of
assessing toxicant pollutants (Iliopoulou-Georgudaki et al. 2003). Other arguments about
the use of biological methods versus physico-chemical analysis are summarized in Fig. 1.
The forerunner works of bioindicator systems for surface water quality assessment
started more than a century and a half ago by Kolenati (1848) and Cohn (1853), both
quoted in Liebmann (1962), De Pauw and Vanhooren (1983) and Iliopoulou-Georgudaki
et al. (2003). This ancient literature described that organisms occurring in organically
polluted water were different from those in clean waters. Since then, the bioindicator
concept has evolved substantially and is now widely applied in situations ranging from the
verification of the compliance of industries to integrated water resource management, with
a shift towards using environmental indicators of anthropogenic impact within a regulatory
framework ((EC) 2000; Carignan and Villard 2002; USEPA 2000).
A range of biological assemblages has been used to monitor and assess environmental
contamination or long-term changes. Although the methods used for a long time were
based mostly on heterotrophic bacteria or macroinvertebrates, algae have long been used to
assess environmental conditions in aquatic habitats (Stevenson and Smol 2003; Whitton
1999), being routinely used for monitoring rivers (Whitton 2012). Cyanobacteria, as pri-
mary producers with a key role in the N and C cycles, are useful bioindicators, given that
any detrimental effect on this phototrophic community may have a negative effect on
nutrient availability to organisms at higher trophic levels.

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Fig. 1 Characteristics of methods for assessment of aquatic contamination: abiotic vs biotic

The present review gives a short summary of the main cyanobacterial methods that are
used to obtain information about changes in running water quality, and mainly related to
eutrophication processes, which are found as the main cause of disturbance in rivers. The
focus will be on benthic cyanobacteria, as habitat recommended for monitoring studies
(Kelly et al. 1998; Round 1991), given that rivers are characterized by their unidirectional
flow along a continuum from source to entry to the sea or any other large water body, and
benthic organisms, as relatively sessile, are directly in contact with the flowing water
detecting the early effects of disturbances in the ecosystem that may arise. A second part of
the review focuses on the use of cyanobacterial bioreporters able to sense and detect
different types of pollution from general toxicity to specific pollutants, such as heavy
metals or Fe, P and N chemical species that may control eutrophication processes.

Cyanobacteria as bioindicators

Although cyanobacteria have generally been regarded as a problematic symptom of eu-

trophic conditions, it must be emphasized that their occurrence should not be always linked
to an ecological decline. On the contrary, some species are characteristics of clean waters,
emerging their use as bioindicators for monitoring running waters. Besides, there are
specific responses to nitrogen and phosphorus enrichment depending on the cyanobacteria,
which are linked to changes in species dominance along eutrophic gradients in running
waters, as will be described below.
On the other hand, cyanobacteria are also been regarded as a widely and ubiquitously
distributed group. However, it must be emphasized that the occurrence and predominance
of cyanobacteria in a vast array of habitats is a result of several general characteristics and
of some features characterizing certain cyanobacterial species. Many species are gener-
alists and will tolerate a great range of environmental conditions including extreme en-
vironments that usually exclude eukaryotic algae (Castenholz 2001). Thus, not all

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cyanobacterial taxa are ubiquitous but have ecological niches that are constrained by their
ecological properties (Komárek 1994). Tolerances or ecological ranges of individual
species can differ depending on the taxon. Some broad-niched species are recognized by
many authors, while others are found to be confined to a more restricted range of eco-
logical conditions (Komárek and Anagnostidis 1999). For instance, some species are
characteristic of unpolluted waters whereas other pollution-tolerant species can survive in
waters so heavily contaminated with organic matter that they become deoxygenated
(Fjerdingstad 1964; Fogg et al. 1973; Palmer 1969).
The first serious attempt to use cyanobacteria as bioindicators of water quality was the
saprobic system (Kolkwitz and Marsson 1908), which showed that water conditions de-
termined the composition of the algal flora. A range of methods were subsequently de-
veloped although the principal tools for monitoring rivers, which are extensively
employed, are indices based on diatom communities (Stevenson et al. 2010). However, a
number of researchers have suggested that the floristic composition of other groups in the
benthos besides diatoms could be useful for monitoring rivers (Kelly and Whitton 1998).
Methods based solely on diatoms, with no parallel assessment of non-diatom phytobenthos
are potentially worrying, as it indicates that monitoring will fail to detect situations where
pressures have resulted in shifts in the balance between different groups of phytobenthos
(Kelly 2013). In addition, it has been pointed out that the diatom-based methodology,
which has been widely developed for monitoring lowland rivers is less suited for upland
rivers, partly because the individual cells are too short-lived, but also because little is
known about the response of different species to nutrient types and ratios and/or other
factors (Whitton 2013). However, many upland rivers have slow-growing cyanobacterial
species which are colonial, so particular species may eventually prove to be good indi-
cators of certain combinations of nutrient conditions (Kelly and Whitton 1998). Besides, in
some fluvial systems lower values of biotic indices such as the BMWPc (based on
macroinvertebrates) and IBD (based on diatoms) than expected have found in Spanish
rivers without anthropogenic influences (Aboal et al. 2002). These results suggest a
cyanotoxic effect on the communities studied and cyanobacterial dominance in such fluvial
systems, and highlight the necessity to consider the cyanobacterial communities.
Some countries use the full or a wide range of periphyton taxa, including cyanobacteria,
for routine monitoring programs, such as several countries in central Europe, including
Austria, Germany, Czech Republic and Poland (Kelly 2013; Whitton 2013). In Austria, the
use of diatoms combined with other benthic algae, including cyanobacteria, is mandatory
for ecological quality impairment analyses, and the use of diatoms alone is only permitted
for regional case studies or when the abundance of non-diatom benthic algae is low (Rott
and Schneider 2014). In Norway and other regions of northern Europe, the Periphyton
Index of Trophic status (PIT), based on presence/absence of the full range of cyanobacteria
and eukaryotic algae other than diatoms, has been adopted for phytobenthos assessment
(Schneider and Lindstrøm 2011). An algae Index of Biotic Integrity (IBI) using also non-
diatom algae, including cyanobacteria, for bioassessment of southern California streams,
was recently developed by (Fetscher et al. 2014), and species optima calculations com-
bined with indicator species analysis identified more than 40 algal species as potential
indicators of nutrient conditions in these streams (Stancheva et al. 2012, 2013). Barinova
and coworkers analysed algal communities, including the cyanobacteria, in running waters
of Israel, Russia and Georgia in order to select bioindicators species (Barinova et al. 2011,
2006, 2008; Barinova and Tavassi 2009). Studies of water quality in rivers of Iran con-
firmed the use of cyanobacterial species as bioindicators for monitoring eutrophication
(Soltani et al. 2012).

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Morphological and physiological indicator characteristics

Morphological and/or physiological features not restricted to a particular species, but

occurring in a number of genera or species, may provide useful insights to the nutrient
status of running waters. Some filamentous species form specialized cells (heterocysts or
heterocytes) (Fig. 2a), which differentiate from an ordinary vegetative cell, and are the site
of nitrogen fixation. The fixed nitrogen moves from heterocysts to vegetative cells. Ex-
perimental studies have shown that heterocyst formation can be prevented if there are high
enough concentrations of nitrate and/or ammonium in the growth medium, so the presence
of many heterocysts in a filament is an indication that the organism is growing in an
environment relatively deficient in combined nitrogen compared to other nutrients
(Whitton 2002; Whitton and Mateo 2012). Analyses of nitrogenase gene expression to
assess N2 fixation along a NO3-N gradient showed that nitrogenase activities specific to
Nostoc and Calothrix were only detected in streams with low values of nitrates (Stancheva
et al. 2013). Similarly, Horne and Carnmiggelt (1975), as well as Grimm and Petrone
(1997) documented high N2 fixation rates in northern California and Arizona desert
streams, respectively, in relation to low levels of NO3-N found in waters. Therefore,
nitrogenase activity can also be used as an indication of low levels of combined N (NO3-N,
NO2--N, NH4?-N).

Fig. 2 Morphological indicator characteristics of cyanobacteria. a Rivularia filaments showing heterocysts

(h). b Rivularia filaments showing multicellular hairs (ha). c Phormidium filament showing stained
polyphosphate granules (pp). d Phormidium filaments showing calyptras (arrows). Solid scale
bar = 10 lm. Dotted scale bar = 40 lm

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Another feature of many filamentous forms is that part of the filament tapers, forming
multicellular hairs (Fig. 2b), in which the cells are much narrower, elongated, highly
vacuolated and usually colorless (Whitton and Mateo 2012). Phosphatase activity, which
hydrolyzes organic phosphorus esters into an organic moiety and orthophosphate, being
associated with P-limitation, is mainly located at hairs, when present, whereby hair fre-
quency increases with increasing P limitation. The hairs not only enhance the surface area
for phosphatase activities, but also aid in P acquisition from environments where the
ambient P concentration is mostly low, but with occasional much higher pulses. A large
number of hairs has been observed associated to phosphorus limitation in streams (Ber-
rendero et al. 2008; Mateo et al. 2010; Muñoz-Martı́n et al. 2014a; Stancheva et al. 2013).
In the same way, increased values of phosphatase activity have also been reported as good
indicator of P limitation (Mateo et al. 2010; Muñoz-Martı́n et al. 2014a; Sabater et al.
2000; Whitton et al. 1998). Therefore, those species which have both heterocysts and well
developed hairs can be used as bioindicators of environments which are deficient in
combined nitrogen during part of the time, and of phosphorus for the rest of time (Whitton
2002; Whitton and Potts 2000).
On the other hand, if phosphate is added to a culture with hairs, polyphosphate (polyP)
granules are formed rapidly in cells (Fig. 2c). Therefore cyanobacteria accumulate polyP
granules when the cells are P-rich, being the relative abundance of these granules an
indication of P-limitation or P-enrichment (Muñoz-Martı́n et al. 2014a; Whitton and Mateo
2012).
Furthermore, it has been suggested that the development of a thickened cap, the ca-
lyptra, which forms on the outer wall of apical cells in some cyanobacteria, (e.g. Phor-
midium) (Fig. 2d), could play a role in detecting features of their environment such as
phosphate or other nutrient gradients (Whitton and Neal 2011; Whitton and Potts 2012).
Muñoz-Martı́n et al. (2014a) found fewer filaments of Phormidium developing a calyptra
in mats from streams sites with higher P concentrations than those of upstream oligotrophic
waters.
Therefore, a programme combining a survey of macroscopically obvious phototrophs
with physiological assays should provide sufficient information to assess whether condi-
tions at the site are undergoing long-term changes regarding eutrophication.

Shifts in cyanobacterial community structure

Changes in cyanobacterial species richness, abundance, and diversity have been observed
in several Spanish rivers in relation to eutrophic gradients (Douterelo et al. 2004; Loza
et al. 2013a, b, c, 2014; Perona et al. 1998; Rodriguez et al. 2007). The cyanobacterial
community of the upstream sites was different from that of the downstream communities,
where anthropogenic influences caused an increase in nutrients. The community compo-
sition also changed with the water quality, whereby certain species were absent from more
perturbed locations, while those remaining proliferated (Douterelo et al. 2004; Loza et al.
2013a, b, c, 2014; Perona and Mateo 2006). Particularly, there were greater Oscillatoriales
species numbers at downstream sites with high trophic levels, while species belonging to
the Nostocales were less abundant and fewer under these conditions. Heterocystous species
were basically observed mainly at upstream sites where the nutrient loads were low
(Douterelo et al. 2004; Perona et al. 1998; Perona and Mateo 2006; Rodriguez et al. 2007).
Similar results were obtained in studies in Brazilian Rivers (Branco and Pereira 2002)
where the proportion of non-heterocystous relative to heterocystous species was proposed
as a good tool for environmental evaluation in tropical regions. The great majority of the

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cyanobacteria present in running waters of a National Park in Northern Italy with very low
nitrate values were heterocystous species (Cantonati 2008). However, the excess of N
(especially nitrates) could explain the clear predominance of non-heterocystous forms in
the Southern Alps (Cantonati et al. 1996, 2006). In North American flowing waters
heterocystous cyanobacteria have been proposed as indicators of nutrient status, since the
relative abundance of nitrogen-fixing species was negatively correlated with nitrogen
concentrations (Porter et al. 2008; Stancheva et al. 2012, 2013). In addition, as stated
above, nitrogen fixation could be used as an indication of low levels of combined N. These
results enhance the suitability of using heterocystous cyanobacteria as bioindicators for
N-limiting conditions.

Species specific responses to water quality: bioindicator values

of cyanobacterial taxa

Table 1 shows a compilation of literature data for bioindicator species, in which only
species with at least two references found were included. The table also lists literature
values for saprobic and/or trophic status. As previously stated, the first descriptions of
differential occurrence of distinct cyanobacteria depending on the water quality of running
waters were those of saprobic system (Kolkwitz and Marsson 1908), in which, as well as in
subsequent reviews (Fjerdingstad 1964; Rott et al. 1997; Sládecek 1973) several species,
such as Oscillatoria putrida, O. chlorina, O. splendida, and O. ornata were described as
bioindicators of organically polluted waters. On the other hand, others, such as Calothrix
parietina and C. fusca, and Tolypotrix distorta, were commonly associated with clean
water.
As long as organic pollution was reduced in running waters, but eutrophication prob-
lems became increasing, indices to assess trophic status were developed and applied. A
number of species were recorded earlier from oligotrophic conditions: the genus Rivularia
and other members of the Rivulariaceae, such as genera Dichothrix and Calothrix are
useful as indicators of environments with waters with low levels of nutrients (Aboal 1988;
Charlton and Hickman 1984; Lindstrøm and Traaen 1984). Since several species of the
genus Tolypothrix had similar ecological optima with respect to nutrient levels (Dell’Uomo
1991; Lindstrøm and Traaen 1984), this genus has been catalogued as oligotrophic taxon
(Schneider and Lindstrøm 2011). Other studies described several species characteristic of
low nutrient conditions, such as species of the genera: Nostoc (N. parmeloides, N. ver-
rucosum) (Aboal 1988; Dell’Uomo 1991; Sabater 1983), Chamaesiphon (C. polonicus, C.
fuscus, C. geitleri, C.investiens, C. minutus, C. starmachii) (Aboal 1988; Rott and Pfister
1988; Sabater 1989), Schizothrix (S. latierita, S. penicillata, S. tinctoria) (Gutowski and
Foerster 2009; Rott et al. 1999; Sabater 1989), and Gloeocapsa (G. alpina, G. sanguinea)
(Rott et al. 1999, 2006), being associated all these genera with oligotrophic conditions
(Schneider and Lindstrøm 2011). Homoeothrix juliana was recorded as occurring in
pristine waters with low level of nutrients (Garcı́a and Aboal 2014; Rott et al. 1999;
Sabater 1989; Soltani et al. 2012), and Phormidium corium has also been found in un-
polluted and slightly influenced environments (Gutowski and Foerster 2009; Gutowski
et al. 2004; Loza et al. 2013b; Rott et al. 1999). Lindstrøm (1999) suggested that, in
Norwegian streams, Stigonema mamillosum is especially sensitive to eutrophication and
should be considered as a ‘red-list’ organism.
However, many data in the literature showed certain cyanobacterial taxa to be indicators
of enriched waters: Oscillatoria species, such as O. limosa, O. princeps, and O. tenuis were
associated to highly eutrophic waters (Gutowski et al. 2004; Palmer 1969; Sierra and

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Table 1 Bioindicator cyanobacteria from literature data

Species Bioindicator value/characteristic Reference

Ammatoidea simplex S: 1.4 Rott et al. (1997)

TW: 1.2 Rott et al. (2006)
Aphanocapsa fonticola TW: 0.6 Rott et al. (1999)
A/B Gutowski and Foerster (2009)
Calothrix fusca Oligosaprobic Dell’Uomo (1991)
Pristine nature Sabater (1989)
S: 1.4 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)
TW: 1.2 Rott et al. (2006)
Calothrix parietina Pristine nature Sabater (1989)
S: 1.4 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)
Oligotrophic water, low DIN Garcı́a and Aboal (2014)
Calothrix gypsophila Oligotrophic waters Lindstrøm and Traaen (1984)
Low nutrient Lindstrøm et al. (2004)
Calothrix sp. In saprobic katharobic zone Persoone and De Pauw (1979)
Decreasing with eutrophication Kann (1982)
IV: 5.21 Schneider and Lindstrøm (2011)
Chamaesiphon Oligotrophic waters Lindstrøm and Traaen (1984)
confervicola S: 1.3 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)
B Gutowski et al. (2004)
TW: 1.2 Rott et al. (2006)
IV: 6.61 Schneider and Lindstrøm (2011)
Chamaesiphon fuscus Unpolluted streams Kann (1978)
S: 1.6 Rott et al. (1997)
TW: 0.7 Rott et al. (1999)
Low nutrient Lindstrøm et al. (2004)
Low NO3-N and conductivity Schaumburg et al. (2004)
A Gutowski et al. (2004)
TW: 0.7 Rott et al. (2006)
IV: 5.09 Schneider and Lindstrøm (2011)
Chamaesiphon geitleri Unpolluted streams Kann (1978)
TW: 0.6 Rott et al. (2006)
S: 1.4 Rott et al. (1997)
TW: 0.6 Rott et al. (1999)
Chamaesiphon S: 2.0 Rott et al. (1997)
incrustans TW: 1.7 Rott et al. (1999)
B Gutowski et al. (2004)
IV: 20.38 Schneider and Lindstrøm (2011)

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Table 1 continued

Species Bioindicator value/characteristic Reference

Chamaesiphon investiens Low nutrient Mulholland and Rosemond

(1992)
S: 1.4 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)
Low nutrient Loza et al. (2013a)
Chamaesiphon minutus Oligotrophic waters Lindstrøm and Traaen (1984)
S: 1.2 Rott et al. (1997)
TW: 0.6 Rott et al. (1999)
TW: 0.6 Rott et al. (2006)
IV: 3.47 Schneider and Lindstrøm (2011)
Chamaesiphon polonicus In saprobic katharobic zone Persoone and De Pauw (1979)
Clean waters Aboal (1988)
Poor in nutrients Sabater (1989)
S: 1.5 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)
A Gutowski et al. (2004)
TW: 1.2 Rott et al. (2006)
Chamaesiphon S: 2.4 Rott et al. (1997)
polymorphus TW: 2.3 Rott et al. (1999)
B Gutowski et al. (2004)
IV: 16.11 Schneider and Lindstrøm (2011)
Low TN Stancheva et al. (2012)
High nutrient Loza et al. (2013a)
Chamaesiphon S: 1.1 Rott et al. (1997)
rostafinskii TW: 0.3 Rott et al. (1999)
IV: 4.37 Schneider and Lindstrøm (2011)
Chamaesiphon Nutrient-poor Rott and Pfister (1988)
starmachii S: 1.7 Rott et al. (1997)
TW: 1.7 Rott et al. (1999)
A Gutowski et al. (2004)
Chamaesiphon S: 1.7 Rott et al. (1997)
subglobosus TW: 1.4 Rott et al. (1999)
Nutrient poor water Lindstrøm et al. (2004)
B Gutowski et al. (2004)
Chlorogloea S: 1.9 Rott et al. (1997)
microcystoides TW: 1.3 Rott et al. (1999)
TW: 1.3 Rott et al. (2006)
Chroococcus turgidus Pristine nature Sabater (1989)
Oligotrophic waters Komárek and
Anagnostidis(1999)
Chroococopsis gigantea TW: 3.0 Rott et al. (1999)
S: 1.7 Rott et al. (1997)
B Gutowski et al. (2004)

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Table 1 continued

Species Bioindicator value/characteristic Reference

Clastidium setigerum Oligotrophic waters Lindstrøm and Traaen (1984)

S: 1.2 Rott et al. (1997)
TW: 0.4 Rott et al. (1999)
TW: 0.4 Rott et al. (2006)
Low nutrient Lindstrøm et al. (2004)
IV: 4.76 Schneider and Lindstrøm (2011)
Clastidium rivularis S: 1.2 Rott et al. (1997)
TW: 0.8 Rott et al. (1999)
TW: 0.8 Rott et al. (2006)
Cyanophanon mirabile Oligotrophic waters Lindstrøm and Traaen (1984)
S: 1.1 Rott et al. (1997)
TW: 0.3 Rott et al. (1999)
TW: 0.3 Rott et al. 2006)
IV: 4.39 Schneider and Lindstrøm (2011)
Dichothrix gypsophila S: 1.6 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)
IV: 4.20 Schneider and Lindstrøm (2011)
Unpolluted waters Komárek (2013)
Dichothrix orsiniana S: 1.1 Rott et al. (1997)
TW: 0.6 Rott et al. (1999)
TW: 0.6 Rott et al. (2006)
IV: 4.42 Schneider and Lindstrøm (2011)
Geitlerinema TW: 3.0 Rott et al. (1999)
acutissimum IV: 24.22 Schneider and Lindstrøm (2011)
Geitlerinema splendidum TW: 3.5 Rott et al. (1999)
IV: 43.42 Schneider and Lindstrøm (2011)
Gloeocapsopsis magma TW: 0.6 Rott et al. (1999)
IV: 2.74 Schneider and Lindstrøm (2011)
Gloeocapsa sanguinea S: 1.1 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)
TW: 1.2 Rott et al. (2006)
IV: 2.74 Schneider and Lindstrøm (2011)
Gloeocapsa alpina S: 1.1 Rott et al. (1997)
TW: 0.6 Rott et al. (2006)
Heteroleibleinia S: 1.4 Rott et al. (1997)
kuetzingii TW: 0.8 Rott et al. (1999)
B Gutowski and Foerster (2009)
IV: 5.32 Schneider and Lindstrøm (2011)
Homoeothrix crustacea Pristine nature Sabater (1989)
S: 1.7 Rott et al. (1997)
TW: 2.2 Rott et al. (1999)
A Gutowski and Foerster (2009)
Oligotrophic water Garcı́a and Aboal (2014)

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Table 1 continued

Species Bioindicator value/characteristic Reference

Homoeothrix gracilis S: 1.4 Rott et al. (1997)

TW: 0.8 Rott et al. (1999)
TW: 0.8 Rott et al. (2006)
Homoeothrix janthina Poor in nutrients Sabater (1989)
S: 1.8 Rott et al. (1997)
TW: 1.5 Rott et al. (1999)
B Gutowski et al. (2004)
TW: 1.5 Rott et al. (2006)
A/C Gutowski and Foerster (2009)
IV: 12.53 Schneider and Lindstrøm (2011)
Homoeothrix juliana Pristine nature Sabater (1989)
S: 1.9 Rott et al. (1997)
TW: 1.3 Rott et al. (1999)
Low nutrient Soltani et al. (2012)
Oligotrophic water Garcı́a and Aboal (2014)
Homoeothrix varians Unpolluted streams Kann (1978)
S: 1.8 Rott et al. (1997)
TW: 1.4 Rott et al. (1999)
B Gutowski et al. (2004)
TW: 1.4 Rott et al. (2006)
B Gutowski and Foerster (2009)
IV: 6.14 Schneider and Lindstrøm (2011)
Hydrococcus rivularis S: 1.5 Rott et al. (1997)
TW: 1.7 Rott et al. (1999)
A Gutowski et al. (2004)
IV: 8.5 Schneider and Lindstrøm (2011)
Hydrococcus cesatii S: 2.2 Rott et al. (1997)
TW: 1.8 Rott et al. (1999)
A Gutowski et al. (2004)
Leptolyngbya foveolarum TW: 2.2 Rott et al. (1999)
C Gutowski et al. (2004)
D Gutowski and Foerster (2009)
High TP Stancheva et al. (2012)
Leptolyngbya frigida S: 1.3 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)
Leptolyngbya S: 1.5 Rott et al. (1997)
nostocorum Low nutrient Loza et al. (2013a)
Leptolyngbya perforans S: 1.4 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)
Leptolyngbya tenuis S: 2.2 Rott et al. (1997)
TW: 3.5 Rott et al. (1999)
Low nutrient Loza et al. (2013a)

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Table 1 continued

Species Bioindicator value/characteristic Reference

Merismopedia glauca s’j: 2.04 Walley et al. (2001)

A Gutowski and Foerster (2009)
IV: 5.33 Schneider and Lindstrøm (2011)
Merismopedia punctata s’j: 2.22 Walley et al. (2001)
IV: 3.77 Schneider and Lindstrøm (2011)
Nostoc parmeloides S: 1.3 Rott et al. (1997)
TW: 0.6 Rott et al. (1999)
Very low nutrient Mollenhauer et al. (1999)
A Gutowski and Foerster (2009)
IV: 7.14 Schneider and Lindstrøm (2011)
Nostoc verrucosum Oligosaprobic Dell’Uomo (1991)
Clean waters Aboal (1988)
S: 1.4 Rott et al. (1997)
Low nutrient Mollenhauer et al. (1999)
Low nutrient Mateo et al. (2010)
IV: 7.34 Schneider and Lindstrøm (2011)
Low TN Stancheva et al. (2012)
Nostoc sp. s’j: 1.16 Walley et al. (2001)
TW: 0.6 Rott et al. (2006)
IV: 7.02 Schneider and Lindstrøm (2011)
Low nutrient Loza et al. (2013b)
Oscillatoria chlorina Pollution-tolerant species Palmer 1969)
Polysaprobic Persoone and De Pauw (1979)
S: 3.6 Rott et al. (1997)
TW: 3.5 Rott et al. (2006)
High nutrient Soltani et al. (2012)
Oscillatoria limosa Pollution-tolerant species Palmer (1969)
Waste water pollution Rott and Pfister (1988)
High nutrient Sabater (1989)
S: 2.6 Rott et al. (1997)
TW: 3.5 Rott et al. (1999)
s’j: 2.29 Walley et al. (2001)
High P content Sabater et al. (2003)
B Gutowski et al. (2004)
High organic matter Sierra and Gómez (2007)
C Gutowski and Foerster (2009)
IV: 39.10 Schneider and Lindstrøm (2011)
Oscillatoria ornata Organic pollution species Tseng and Wang (1982)
Organically polluted waters Branco and Pereira (2002)

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Table 1 continued

Species Bioindicator value/characteristic Reference

Oscillatoria princeps Pollution-tolerant species Palmer (1969)

S: 2.8 Rott et al. (1997)
TW: 3.9 Rott et al. (1999)
TW: 3.9 Rott et al. (2006)
C Gutowski and Foerster (2009)
Oscillatoria putrida Pollution-tolerant species Palmer (1969)
Polysaprobic Persoone and De Pauw (1979)
Oscillatoria splendida Pollution-tolerant species Palmer (1969)
IV: 40.99 Schneider and Lindstrøm (2011)
Oscillatoria tenuis Pollution-tolerant species Palmer (1969)
High P content Sabater et al. (2003)
High organic matter Sierra and Gómez (2007)
IV: 44.24 Schneider and Lindstrøm (2011)
Eutrophic conditions Loza et al. (2013b)
Phormidium aerugineo- Oligotrophic conditions Charlton and Hickman (1984),
caeruleum Dell’Uomo (1991), Branco and
Pereira (2002), Serrano et al.
(2004), Perona and Mateo
(2006) and Loza et al. (2013b)
S: 2.6 Rott et al. (1997)
TW: 3.5 Rott et al. (1999)
Phormidium ambiguum S: 2.1 Rott et al. (1997)
TW: 3.0 Rott et al. (1999)
C Gutowski and Foerster (2009)
Phormidium atumnale Pollution-tolerant species Palmer (1969)
Unpolluted streams Kann (1978)
Poor in nutrients Sabater (1989)
S: 2.7 Rott et al. (1997)
TW: 1.7 Rott et al. (1999)
s’j: 2.35 Walley et al. (2001)
B Gutowski et al. (2004)
Nutrient poor water Lindstrøm et al. (2004)
Low NO3-N and conductivity Schaumburg et al. (2004)
TW: 1.7 Rott et al. (2006)
High nutrient Loza et al. (2013b)
Eutrophic saltwaters Garcı́a and Aboal (2014)
Phormidium corium S: 1.3 Rott et al. (1997)
TW: 1.6 Rott et al. (1999)
A Gutowski et al. (2004)
A/B Gutowski and Foerster (2009)
Oligotrophic conditions Loza et al. (2013b)
Phormidium foveolarum S: 3.1 Rott et al. (1997)
s’j: 2.64 Walley et al. (2001)

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Table 1 continued

Species Bioindicator value/characteristic Reference

Phormidium hetropolare Low nutrient Lindstrøm et al. (2004)

IV: 3.40 Schneider and Lindstrøm (2011)
Phormidium incrustatum Pristine nature Sabater (1989)
S: 1.7 Rott et al. (1997)
TW: 2.4 Rott et al. (1999)
C Gutowski et al. (2004)
A/C Gutowski and Foerster (2009)
Phormidium inundatum Oligosaprobic Persoone and De Pauw (1979)
IV: 35.81 Schneider and Lindstrøm (2011)
Phormidium retzii Poor in nutrients Sabater (1989)
S: 1.7 Rott et al. (1997)
TW: 2.6 Rott et al. (1999)
Low conductance (nutrient) Branco et al. (2001)
C Gutowski et al. (2004)
C Gutowski and Foerster (2009)
IV: 32.02 Schneider and Lindstrøm (2011)
Phormidium subfuscum Oligo-b-mesosaprobic Dell’Uomo (1991)
Slightly eutrophic Sabater (1989)
S: 2.2 Rott et al. (1997)
TW: 1.6 Rott et al. (1999)
High NO3-N and conductivity Schaumburg et al. (2004)
C Gutowski et al. (2004)
TW: 1.6 Rott et al. (2006)
C Gutowski and Foerster (2009)
Phormidium tenue Enriched waters Branco and Pereira (2002)
High nutrient Soltani et al. (2012)
Phormidium terebriforme High nutrient Kolkwitz and Marsson (1908),
Fjerdingstad (1964), Sládecek
(1973) and
Loza et al. (2013b)
S: 3.4 Rott et al. (1997)
TW: 3.5 Rott et al. (1999)
Phormidium tinctorum S: 1.6 Rott et al. (1997)
IV: 52.77 Schneider and Lindstrøm (2011)
Phormidium uncinatum Pollution-tolerant species Palmer (1969)
Oligo-b-mesosaprobic Dell’Uomo (1991)
Plectonema S: 1.5 Rott et al. (1997)
tomasinianum TW: 1.7 Rott et al. (1999)
B Gutowski and Foerster (2009)
IV: 17.60 Schneider and Lindstrøm (2011)
Pleurocapsa aurantiaca Poor in nutrients Sabater (1989)
S: 1.1 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)

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Table 1 continued

Species Bioindicator value/characteristic Reference

Pleurocapsa minor Waste water pollution Rott and Pfister (1988)

S: 2.6 Rott et al. (1997)
TW: 2.3 Rott et al. (1999)
C Gutowski et al. (2004)
C Gutowski and Foerster (2009)
Porphyrosiphon S: 1.5 Rott et al. (1997)
martensianus TW: 1.9 Rott et al. (1999)
(Syn.: Lyngbya
martensiana) s’j: 1.48 Walley et al. (2001)
Low nutrient Perona and Mateo (2006)
B Gutowski and Foerster (2009)
Pseudanabaena catenata IV: 35.91 Schneider and Lindstrøm (2011)
D Gutowski and Foerster (2009)
S: 3.3 Rott et al. (1997)
TW: 1.8 Rott et al. (1999)
Pseudanabaena frigida Oligotrophic waters Komárek and Anagnostidis
(2005)
IV: 3.63 Schneider and Lindstrøm (2011)
Rivularia biasolettiana Pristine nature Sabater (1989)
Low nutrient Mateo et al. (2010)
IV: 4.55 Schneider and Lindstrøm (2011)
Rivularia haematites S: 1.4 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)
Low nutrient Mateo et al. (2010)
IV: 8.75 Schneider and Lindstrøm (2011)
Rivularia sp. Decreasing with eutrophication Kann (1982)
s’j: 1.25 Walley et al. (2001)
IV: 4.99 Schneider and Lindstrøm (2011)
Schizothrix lacustris S: 1.6 Rott et al. (1997)
TW: 0.6 Rott et al. (1999)
IV: 4.35 Schneider and Lindstrøm (2011)
Schizothrix latierita S: 1.5 Rott et al. (1997)
TW: 0.3 Rott et al. (1999)
IV: 4.29 Schneider and Lindstrøm (2011)
Schizothrix penicillata Pristine nature Sabater (1989)
S: 1.3 Rott et al. (1997)
TW: 0.6 Rott et al. (1999)
Schizothrix tinctoria S: 1.7 Rott et al. (1997)
TW: 0.8 Rott et al. (1999)
B Gutowski and Foerster (2009)
Scytonematopsis S: 1.0 Rott et al. (1997)
starmachii TW: 0.3 Lindstrøm et al. (2004)
Nutrient poor water Rott et al. (2006)
IV: 3.08 Schneider and Lindstrøm (2011)

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Table 1 continued

Species Bioindicator value/characteristic Reference

Scytonema crispum S: 1.4 Rott et al. (1997)

TW: 2.4 Rott et al. (1999)
Scytonema crustaceum S: 1.1 Rott et al. (1997)
TW: 0.6 Rott et al. (1999)
Scytonema myochrous S: 1.0 Rott et al. (1997)
TW: 0.6 Rott et al. (1999)
Stigonema hormoides TW: 0.6 Rott et al. (1999)
S: 1.1 Rott et al. (1997)
IV: 1.87 Schneider and Lindstrøm (2011)
Stigonema mamillosum TW: 0.3 Rott et al. (1999)
Low nutrient Lindstrøm et al. (2004)
IV: 3.88 Schneider and Lindstrøm (2011)
Stigonema ocellatum IV: 3.34 Schneider and Lindstrøm (2011)
TW: 0.6 Rott et al. (1999)
S: 1.1 Rott et al. (1997)
Tolypothrix distorta S: 1.4 Rott et al. (1997)
TW: 0.5 Rott et al. (1999)
Low nutrient Lindstrøm et al. (2004)
IV: 7.71 Schneider and Lindstrøm (2011)
Tolypothrix distorta var. Oligotrophic waters Lindstrøm and Traaen (1984)
penicillata S: 1.2 Rott et al. (1997)
TW: 0.6 Rott et al. (1999)
TW: 0.6 Rott et al. (2006)
Low nutrient Mateo et al. (2010)
IV: 5.20 Schneider and Lindstrøm (2011)
Tolypothrix tenuis S: 1.5 Rott et al. (1997)
TW: 1.2 Rott et al. (1999)
Low nitrate Cantonati (2008)
IV: 6.45 Schneider and Lindstrøm (2011)
Low nutrient Loza et al. (2013a)
Only species with at least two references found were included (see descriptions of bioindicator value
categories below)
S Saprobic range: oligosaprobic: 1.0–1.5, oligosaprobic to b-mesosaprobic [1.5 to B1.8, b –mesosaprobic
[1.8 to B2.3, b-mesosaprobic to a-mesosaprobic [2.3 to B2.7, a-mesosaprobic [2.7 to B3.2, a-mesos-
aprobic to polysaprobic [3.2 to B3.5, polysaprobic [3.5 to B4.0. The katharobic zone comprises the very
pure waters, i.e. those waters without any trace of pollution. Oligosaprobic organisms are those indicators
for scarcely polluted waters. b-mesosaprobic organisms are those indicators for moderately polluted waters
a-mesosaprobic organisms are those indicators for highly polluted waters. Polysaprobic organisms are those
indicators for extremely polluted waters. s’j revised saprobic values. TW trophic value: ultra-oligotraphentic
all values \0.5, oligotraphentic: 0.6–1.0, oligomesotraphentic: 1.1–1.5, mesotraphentic: 1.6–2.0, meso-
eutraphentic 2.1–2.5, eutraphentic: 2.6–3.0, eu-polytraphentic: 3.1–3.5. Category description of A, B, C,
D—A: sensitive species, characteristic of certain types of water bodies (found at the most pristine sites), B:
less sensitive species, more widely distributed, indicate good conditions, C: tolerant species, indicate eu-
trophication when highly abundant, D: species prefer strongly eutrophic conditions. IV Indicator value. IV
has been calculated as the average log (TP) to the power of 10 at the sites where the taxon occurs. Indicator
values range from 1.87 to 68.91. DIN dissolved inorganic nitrogen. TP total phosphorus. TN total nitrogen

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Gómez 2007; Walley et al. 2001). Leptolyngbya foveolarum (Gutowski and Foerster 2009;
Gutowski et al. 2004; Rott et al. 1999; Stancheva et al. 2012), Phormidium terebriforme
(Fjerdingstad 1964; Kolkwitz and Marsson 1908; Loza et al. 2013b; Rott et al. 1997, 1999;
Sládecek 1973), and P. ambiguum (Gutowski and Foerster 2009; Rott et al. 1999) were
also associated to nutrient-rich conditions.
In general, a greater number of unicellular and heterocystous genera/species were as-
sociated with low trophic levels, while the number of non-heterocystous filamentous
genera/species was greater in high nutrient conditions.

Ecophysiological strategies

Variations in benthic cyanobacterial species distribution could be explained on the basis of

ecophysiological differences between cyanobacteria in which nutrient loading can be a strong
selective force shaping cyanobacterial communities. Recent studies showed distinct patterns
in growth and competitive response of benthic cyanobacteria under N and P regimes (Loza
et al. 2014). Nutrient gradient bioassays in monoculture as well as in competition experiments
with a mixture of species indicated predominance of certain cyanobacteria over others,
depending on the concentration of phosphorus and nitrogen growth was either stimulated or
inhibited. Some species grew better at high nutrient concentrations, while higher yields were
recorded for others under low nutrient regimes, according to the differential distribution of
these cyanobacteria in the river from which they were isolated in which a group of species
occurred mostly in downstream nutrient-rich locations, while others were typical of upstream
oligotrophic conditions (Loza et al. 2013a, b, c). Therefore, ecological ranges of individual
cyanobacterial populations can differ with nutrient concentration, and the persistence of
several species under low or high nutrient concentrations in running waters can be explained
on the basis of their different ecophysiological properties.
A key ecological feature at upstream sites with typically low nutrients may be the ability
to have nitrogenase activity. The property of field populations to show marked phosphatase
activity allows their occurrence in environments where organic phosphate is an important
source of phosphate, even when inorganic phosphate is below detectable levels (Whitton
1987). In addition, a possible environmental explanation for the shifts in cyanobacterial
distribution could be related to differential growth and/or utilization of P and N reserves.
Since P supply tends to come in pulses in running water (Bhaya et al. 2000; Turner et al.
2003) with subsequent falling in water concentration, the ability of taking up P in excess of
immediate requirement and store it intracellularly in a luxury consumption (Dodds and
Welch 2000), can be advantageous, allowing for minimum growth, survival, till a new P
pulse arrives. Similar behaviour could be extrapolated for N in non-fixing species.
Therefore, the ability to scavenge nutrients during rare periods of high concentrations,
rather than a general tolerance of low-nutrient conditions, has been proposed as an adaptive
strategy at upstream sites with typically low nutrients (Kelly and Whitton 1998). A greater
ability to store P and slower utilization of intracellular polyP granules has been found in
Calothrix cultures with respect to Nostoc, isolated from a Spanish river with eutrophic
gradients (Mateo et al. 2006). Rott et al. (2000) suggested two strategies used for species to
overcome resource limitations, such as rapidly growing r-strategists (e.g. Phormidium)
with increased phosphatase synthesis during the peak growing periods, and the persistent
k-strategists (e.g. Rivularia) showing seasonal variations in light adaptation and moderate
variations in the nutrient pool/phosphatase synthesis. Other strategies exploit the presence
of mucilage, which helps these organisms to survive in nutrient poor media, where they
play an important role in nutrient uptake and assimilation, or the possibility of increasing

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bioavailability of P at the thin layer of fine sediment, commonly observed under well-
developed cyanobacterial mats (Loza et al. 2014, and references therein).

Molecular approaches

Studies on cyanobacterial diversity and community structure based mainly on the accurate
taxonomic identification of the individuals found is often time-consuming due to diffi-
culties identifying species that exhibit extreme morphological variability and whose tax-
onomy is controversial (Komárek and Anagnostidis 1999). Moreover, in many cases, the
only means of identification is through the comparison of live material with cultured field
samples, which introduces more difficulties due to culture selectivity and the fact that
many species frequently develop anomalous morphological states in culture (Komárek and
Anagnostidis 1999; Perona et al. 2003). Molecular techniques can obviate many of these
difficulties and are especially well suited to differentiate closely related organisms, al-
lowing morphological comparisons in parallel, and the consequent identification using both
molecular and morphological data. Therefore, sequencing of ribosomal 16S rRNA genes
has become a widespread practice in the detection and characterization of cyanobacteria in
the environment (Komárek 2013). In addition, nucleic acid extraction from polluted lo-
cations and their subsequent amplification by polymerase chain reaction (PCR) has proved
extremely useful in assessing the changes in microbial community structure by several
profiling techniques (Malik et al. 2008). Among these PCR-based tools, molecular fin-
gerprinting methods, such as Denaturing/Temperature Gradient Gel Electrophoresis
(DGGE/TGGE) have been used to analyze the diversity of bacterial assemblages in dif-
ferent environments (Fromin et al. 2002). In particular, TGGE has been used to measure
changes in cyanobacterial diversity along a pollution gradient in a Spanish river and
compared it with that of using microscopic observations of field-fixed and cultured samples
(Loza et al. 2013c; Rodriguez et al. 2007). These studies were based on comparisons of
cyanobacterial communities in biofilms in order to evaluate and characterize their eco-
logical responses depending on the stream water quality. The different 16S rDNA genes
present in the community of each sampling point of the river were separated by TGGE,
giving a characteristic pattern of bands for each site. This pattern represents a ‘‘fingerprint’’
of the community, allowing direct comparisons of the different samples. Band profiles
differed among sampling sites depending on differences in water quality, whereby TGGE
band richness decreased in a downstream direction, and there was a clear clustering of
phylotypes on the basis of their origins from different locations according to their eco-
logical requirements. Some phylotypes were associated with low nutrient concentrations
and high levels of dissolved oxygen, while other phylotypes were associated with eu-
trophic-hypertrophic conditions. Results were consistent with those obtained from mi-
croscopic observations of field-fixed samples, and it allowed the identification of the
occupants of particular ecological niches, so that the particular ecologically relevant taxa,
different ecotypes, could be selected as bioindicators of water quality. Therefore, an
ecotype-based model of the structure of cyanobacterial populations has been proposed in
which all related inhabitants of a unique ecological niche are thought to belong to a single,
stable ecotype. Results of the TGGE studies supported the view that once a community has
been characterized and its fingerprint obtained, this band pattern could be used as a bar
code that, like a detector or sentinel, acts as an ‘early warning’ device alerting us of the
presence of pollutants in the environment (Loza et al. 2013c; Rodriguez et al. 2007).
In 2005, a new technology referred to as ‘Next Generation Sequencing (NGS)’ was
introduced that leads to the determination of millions of DNA sequences in a single

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process. Through the use of the massive amounts of sequence data produced by NGS
platforms, researchers have been able to observe the slight changes in community structure
that may occur following anthropogenic or natural environmental fluctuations (Shokralla
et al. 2012). Therefore, the use of high-throughput NGS approaches in biodiversity science
has the potential to further extend the application of DNA information for routine
biomonitoring applications to an unprecedented scale (Hajibabaei et al. 2011). However, in
the available literature, there are few studies of microbial community composition and
diversity in rivers based on NGS, and consequently, more studies are needed to better
determine the cyanobacterial diversity and community structure contained within rivers
(Schultz et al. 2013; Tytgat et al. 2014).

Cyanobacteria as bioreporters

The term bioreporter refers to transgenic organisms (usually microorganisms) which

harbor, either in the chromosome or in a replicative plasmid, a fusion between a sensing
element [a gene(s) promoter responsive to the stimulus/compound to be detected] and a
promoterless reporter element encoding easily detectable output signals (i.e. light,
fluorescence, color development). These recombinant organisms are designed to produce a

Fig. 3 Bioreporters turn-off and turn-on concepts. a Turn-off bioreporters detect general toxicity as a
decrease in the reporter signal; in these bioreporters the reporter system is fused to a constitutive promoter
and toxicity leads to inhibition of the reporter quantifiable product. A second class of turn-off bioreporters
are some used to detect nutrient bioavailability which are induced in response to nutrient deficiency and
gradually turned-off when the nutrient is added. b In turn-on bioreporters, the reporter system is fused to
gene promoters responsive to pollutants/groups of pollutants with similar chemical structure which are
turned on (induced) in response to these specific chemicals. A second class of turn-on bioreporters are those
denoted as semi-specific where the promoter system is fused to gene promoters responsive to cellular
stresses such as oxidative stress, DNA damage or membrane damage

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measureable signal in response to an analyte (i.e. toxin or pollutants) or a stress situation.

As depicted in Fig. 3, regarding specificity of detection, bioreporters are usually divided
into three categories (Belkin 2003): non-specific, semi-specific and specific. In the field of
toxicology, non-specific or ecotoxicity bioreporters, also known as general toxicity
bioreporters, provide data on the toxicity of a sample but do not detect specific pollutants;
they usually consist of a reporter element fused to a constitutive promoter so that the
reporter product is continuously produced but in the presence of a toxin that impairs
metabolism, the bioreporter response is reduced or even fully inhibited; in other words, the
reporter response is turned off, so these strains may be denoted as turn-off bioreporters
(Fig. 3a). The semi-specific bioreporters report on pollutants/stressors that cause cellular
responses to stresses such as oxidative stress, genotoxicity (DNA damage) or membrane
damage; the promoters are activated in response to global changes in the cell; these
bioreporters harbor a fusion between the reporter element and stress responsive gene
promoters; as in this case, the bioreporter response is induced (turned on), these strains
may be denoted as turn-on bioreporters (Fig. 3b). The specific bioreporters are able to
detect a specific pollutant or group of related pollutants as they have a fusion between a
pollutant-responsive gene(s) regulatory element and the promoterless reporter system; they
are usually turn-on strains but in some cases, such as some promoters responsive to
nutrient deficiency used to develop bioreporters of nutrient bioavailability (see below),
turn-off strains are constructed. In toxicology, microbial bioreporters are an interesting
alternative/complement to classic toxicity bioassays as they are usually less laborious,
quicker in their response and more cost-effective with no animal sacrifice (Belkin 2003;
Sorensen et al. 2006; van der Meer and Belkin 2010).
There are several reporter systems that are usually used: lacZ encoding the b-galac-
tosidase of Escherichia coli has been largely used as there are a wide range of detection
methods; i.e. chemiluminescence, colorimetry, electrochemistry and fluorescence; how-
ever, it needs exogenous substrate addition which may require cell membrane permeabi-
lization and does not allow real-time in vivo expression studies; it also may involve time
and labor-consuming operations. Fluorescent proteins such as GFP encoded by genes
found in the jellyfish Aequorea victoria and other marine invertebrate do not require the
addition of exogenous substrate but lower sensitivity, autofluorescence of the organisms/
samples, the need of O2 for maturations of the fluor and lag time before expression limit
their use to measure quick cellular responses although a clear advantage is that a whole
myriad of mutants with different color varieties is available ((Heim et al. 1995; Heim and
Tsien 1996). Finally, the luciferase reporter systems are widely used due to their high
sensitivity and ease of measurement. The luciferase systems product is bioluminescence
and may be based either in bacterial luxCDABE genes or in eukaryotic luc genes. Both
luciferases catalyze the oxidation of a substrate, long chain aldehydes in the case of
bacterial luciferases and luciferin/pholasin/coelentarazine in the case of eukaryotic lu-
ciferases, both reactions require O2. Bacterial bioluminescence lux genes are present in
bacteria found in marine, freshwater and terrestrial ecosystems. They are all gram negative
belonging to the genera: Vibrio, Aliivibrio, Photobacterium, Shewanella and Photorhabdus
(previously Xenorhabdus). The bacterial luciferase encoded by luxAB catalyzes the
oxidation of FMNH2 and a long chain fatty acid to produce the oxidized flavin (FMN) and
a long chain fatty acid with the emission of blue-light (490 nm). The fatty aldehyde is
produced by a fatty acid reductase complex that include a reductase, transferase and a
synthetase encoded by luxC, D and E genes, respectively. A luxCDABE bioreporter is self-
luminescent and allows for real time in vivo measurements while luxAB-based bioreporters
need the addition of exogenous aldehyde (usually n-decanal or nonanal) to luminesce

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allowing single time point measurement rather than continuous monitoring. The firefly
luminescent systems is the best characterized of the eukaryotic systems (also including
click beetles, railroad worms or the sea pansy); it catalyzes the O2 and ATP dependent
decarboxylation of the luciferin, emitting light in the green-yellow region of the spectrum
(maximum around 560 nm). As in the case of luxAB-based bioreporters, this system needs
the exogenous addition of the luciferin precluding in vivo continuous light monitoring;
however, the quantum yield of the firefly luciferase is the highest of any of the so far
characterized bioluminescence systems resulting in the highest sensitivity; besides, the
different luc genes and mutant variants allow multicolor luciferase assays which might be
interesting for developing bioreporters useful in environmental monitoring; (Branchini
et al. 2005; Nakajima and Ohmiya 2010; Roda and Guardigli 2012). As the biolumines-
cence reactions depend on cell metabolism due to the requirement of ATP or reducing
power, any pollutant affecting metabolism, may decrease light emission in a dose–response
manner, being denoted as lights-off (from turn-off bioreporters, see Fig. 3a). These lights-
off bioreporters are able to detect the general toxicity of a sample but cannot identify the
pollutant(s); another kind of lights-off bioreporters are some that are used to detect nutrient
bioavailability; while, by analogy, the lights-on bioreporters (from turn-on, see Fig. 3b) are
induced by a specific stress, pollutant or group of related pollutants (Belkin 2003; Sorensen
et al. 2006).
An important feature of bioreporters is that they provide complementary data of en-
vironmental samples to those obtained by analytical chemistry methods; while these give a
sensitive and accurate measurements of environmental pollutants, bioreporters provide
data on the bioavailability of the pollutants (the biologically relevant fraction of the
chemical compound that is actually available to the reporter cell, capable of interact-
ing/passing through cellular membranes) and/or the global toxicity of a sample.
In the field of environmental microbial bioreporters, cyanobacteria are of utmost rele-
vance due to their photosynthetic nature, being ubiquitous primary producers in both
aquatic and terrestrial ecosystems (Bachmann 2003). Cyanobacteria are at the base of
trophic webs and anything having a detrimental effect on cyanobacteria may seriously
affect the functioning of the whole ecosystem; in fact, reports show that marine
cyanobacteria Prochlorococcus sp. and Synechococcus sp. account for up to 80 % of
primary production (Goericke and Welschmeyer 1993; Liu et al. 1997). Besides, more than
100 cyanobacterial genomes have been sequenced (http://genome.microbebd.jp/
cyanobase) (Shih et al. 2013) and many are genetically amenable.
To date, all reported cyanobacterial environmental bioreporters are based on lumines-
cence systems, so that lights-off and lights-on cyanobacterial bioreporters will be described
in this review; also, so far, and to our knowledge, only cyanobacterial bioreporters to detect
general toxicity, nutrient bioavailability and heavy metals have been constructed; there is a
lack of semi-specific bioreporters able to detect oxidative stress or genotoxicity although
efforts to construct such reporters are under way (F. Fernández-Piñas; personal
communication).

General toxicity or ecotoxicity bioreporters

Only two cyanobacterial general toxicity bioreporters have been described up to date; both
are lights-off; one is based on the unicellular Synechocystis strain PCC 6803 (Shao et al.
2002) and one based on the filamentous heterocystous cyanobacterium Anabaena sp. PCC
7120 (now renamed by many authors as Nostoc sp. PCC 7120; both terms will appear in
this review indistinctly) denoted as Anabaena CPB4337 (Fernandez-Piñas and Wolk 1994;

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Garcia et al. 2013; González-Pleiter et al. 2013; Rodea-Palomares et al. 2009, 2010a, b,
2011).
The freshwater Synechocystis chromosome was double tagged with both luc (from firely
Photinus pyralis) and luxAB genes (from marine bacterium Aliivibrio fischeri, formerly
Vibrio fischeri) under the control of the tac promoter (Shao et al. 2002). They characterized
the bioluminescence of the reporter strain with respect to batch growth and pH. As
aldehyde (substrate for bacterial luciferase) addition was toxic to the culture, Shao and
coworkers measured the luminescence of the luc gene; in this way, they tested the toxicity
of four herbicides with different modes of action, two heavy metals (copper and zinc) and
the organic 3,5 dichlorophenol (DCP) and compared the sensitivity of the cyanobacterial
bioreporter with other heterotrophic bacterial bioreporters (based on E. coli; Pseudomonas
putida or P. fluorescens). As expected, the cyanobacterial bioreporter proved to be more
sensitive to the herbicides but in the same range of sensitivity to the heavy metals or 3,5
DCP. The Synechocystis bioreporter was not self-luminescent and the luciferin substrate
had to be added at each time point for measurement precluding its use for continuous
monitoring; besides, it does not appear to have been tested with environmental samples.
By contrast, the filamentous Anabaena (Nostoc) CPB4337 (Fernández-Piñas and Wolk
1994) is a self-luminescent bioreporter which bears in its chromosome a tn5 derivative
with luxCDABE from the luminescent terrestrial bacterium Photorhabdus luminescens.
This strain shows a high constitutive luminescence (the gene promoter has not been
identified yet) and does not need the addition of exogenous aldehyde. Luminescence has
been shown to be high even at increasing temperatures (up to 30 °C) in contrast with
luciferases from marine organisms like Vibrio and Aliivibrio which is in agreement with
Photorhabdus luminescence having the greatest thermal stability (Fernandez-Piñas et al.
2000; Meighen 1991). The strain has been successfully used in combination with a battery
of standardized toxicity bioassays based on organisms form different trophic levels (Lu-
minescent bacterium A. fischeri; the crustacean Daphnia magna and the green alga
Pseudokirchneriella subcapitata) to study the toxicity of different priority (heavy metals,
organic solvents) and emerging pollutants (pharmaceuticals, perfluorinated surfactants and
nanomaterials) (González-Pleiter et al. 2013; Rodea-Palomares et al. 2009, 2011, 2012;
Rosal et al. 2010a, b); it has also been tested in environmental matrices of different
complexity (Garcia et al. 2013; Rodea-Palomares et al. 2010; Rosal et al. 2010a) and for
the first time in the case of a cyanobacterial bioreporter, it has been used to study the
toxicology of pollutants’ mixtures by the Combination Index-Isobologram Equation
method (Rodea-Palomares et al. 2010, 2012). The authors applied the Combination Index
method to study the toxicological interactions of priority and emerging pollutants in the
above mentioned aquatic organisms including Anabaena CPB4337 finding that the nature
of the interaction (either synergism or antagonism) strongly depended on the test species
and also in the effect level exerted by the pollutant mixture in the organism (Rodea-
Palomares et al. 2010, 2012; Rosal et al. 2010b). In a study with mixtures of antibiotics, by
using bioluminescent Anabaena CPB4337, they were able to demonstrate that certain
mixtures of antibiotics could pose an ecological risk towards aquatic environments
(González-Pleiter et al. 2013).

Cyanobacterial bioreporters to detect nutrient bioavailability

The environmental relevance of cyanobacteria as primary producers both in freshwater and

marine environments make cyanobacterial bioreporters a useful tool to assess nutrient
bioavailability in water bodies. Iron, phosphate and nitrogen are essential for primary

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production and usually limiting in certain aquatic environments such as the ocean or large
lakes; on the other hand, an excess of P and/or N may lead to eutrophication of water
bodies and developments of algal blooms which may lead to toxin production by
cyanobacteria (Dodds 2006). Bullerjahn and coworkers already published an extensive and
comprehensive review on the use of cyanobacterial bioreporters as sensors of nutrient
availability, namely Fe, P and N (Bullerjahn et al. 2010); in this review we shall revise all
what has been done in this field highlighting and discussing the most recent reports.

Fe bioavailability

Most cyanobacterial bioreporters constructed to detect Fe have been based on the Fe-
responsive isiAB promoter which is in part regulated by Fur (ferric uptake regulator). When
cells are depleted of iron, the isiAB promoter is induced; when increasing concentrations of
iron are supplied to the cells, the isiAB promoter is down-regulated in a dose–response
manner; so that these bioreporters may be considered as lights-off strains.
The isiAB promoter has been fused to luxAB from Vibrio harveyi and transformed in the
unicellular freshwater Synechococcus sp. PCC 7942 yielding strain S. KAS101 (Durham
et al. 2002); in the unicellular marine Synechococcus sp. strain PCC 7002 yielding strain
S. BM004, deposited as Synechococcus sp. CCMP 2669 (Boyanapalli et al. 2007) and in
the model unicellular Synechocystis sp. PCC 6803 yielding strain S. MpILisi (Kunert et al.
2000). For strains KAS101 and BM004, bioluminescence was found to be a function of the
free ferric ion concentration in metal-buffered media so that the dynamic range of Fe
concentrations over which the bioreporters may be used as quantitative tools for
bioavailable Fe was obtained; in the case of KAS101, the dynamic range was between free
Fe3? 10-21.1 to Fe3? 10-20.6 M and in the case of BM004 a three parameter-sigmoidal
curve was generated in a range of free Fe3? between 10-22.4 and 10-19.4 M. Both reporter
strains have been used in environmental systems such as the Laurentian great lakes
(Hassler et al. 2008, 2009; McKay et al. 2005; Porta et al. 2003, 2005) and marine
environments such as the Baltic sea or subarctic Pacific (Boyanapalli et al. 2007). These
strains are not self-luminescent and aldehyde had to be added for each measurement.
Recently Zha et al. (2012) have reported a novel Fe-bioreporter based in the filamentous
Nostoc (Anabaena) PCC 7120 which harbors the iron-regulated schizokinen transporter
alr0397 gene promoter fused to V. fischeri luxAB (also non self-luminescent); The alr0397
gene is also highly inducible under iron deficiency (Dong and Xu 2009); so, it is also a
lights-off strain. Dose–response relationships between luciferase activity and free Fe3?
were obtained and the dynamic range of performance of the bioreporter was found to be
free Fe3? 10-21.5–10-19.6 M; this range makes the bioreporter useful in environmental
samples with high bioavailable iron; the bioreporter has been tested successfully with
water samples from three eutrophic Chinese lakes.

P bioavailability

In most aquatic ecosystems P is usually a limiting nutrient, barely detectable in olig-

otrophic systems (Whitton 2008). On the other hand, an excess of P may lead to eu-
trophication of water bodies and algal blooms (Conley et al. 2009). Assessing the
bioavailability of P in environmental samples is not an easy issue as this element may
appear in various organic and inorganic species both dissolved and particulate (Neal et al.
2010). Dissolved Inorganic Phosphorus (DIP) is a measure of dissolved orthophosphate
which is probably the most readily available species to photosynthetic organisms; the

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Dissolved Organic Fraction of Phosphorus (DOP) may also be available to algae and
aquatic plants (Bostrom et al. 1988; Neal et al. 2010). Cyanobacteria respond to P defi-
ciency by increasing surface phosphatase activities such as alkaline phosphatases which
scavenge P from dissolved organic sources and have broad substrate specificity (Hoppe
2003); cyanobacteria may, then, be useful to determine which fraction of total P is actually
detected by the phytoplancktonic cell.
At a genetic level, microorganisms sense P bioavailability and respond regulating the
expression of a series of genes that constitute the Pho regulon, such as pho encoding the
alkaline phosphatases or pst encoding phosphate transporters (Su et al. 2007; Van Bogelen
et al. 1996). In cyanobacteria this system has been well characterized in Synechococcus sp.
PCC 7942 and in Synechocystis sp. PCC 6803 (Su et al. 2007); a strain of Synechococcus
sp. PCC 7942 in which the phoA promoter has been fused to the reporter genes luxAB from
A. fischeri was the first P bioavailability cyanobacterial bioreporter reported (Gillor et al.
2002; Schreiter et al. 2001); the strain has been named as S. APL, it is not self-luminescent
and needs the addition of exogenous aldehyde. The phoA gene is induced under P-defi-
ciency and luminescence decreases with the addition of increasing inorganic P (DIP) in a
dose–response manner so it is a lights-off strain. The authors tested also organic P species
finding that ADP and D-fructose-6-phosphate almost completely inhibited bioluminescence
and that D-glucose 6 phosphate, q-nitrophenol-phosphate and b-glycerol phosphate only
partially inhibited luminescence; so the bioreporter is also responsive to DOP. Cells needed
to be pre-starved for P during 30 h before being transferred to media with different PO43-
concentrations. The authors tested it with water samples from Lake Kinneret (Israel)
finding a good correlation between luminescence measurements and total dissolved P
concentrations as measured chemically; they also immobilized P-starved cells in agar in
microtiter plates which could be stored fully active for three weeks at 4 °C and denoted the
immobilized strain as CyanoSensor. The CyanoSensor showed a dynamic detection range
of 0.3–8 lM with an incubation time of 8 h (Schreiter et al. 2001). This bioreporter has
been recently used in combination with a glnA based N- bioreporter (see below) to measure
nutrient bioavailability for over two years in water samples from the same lake collected
monthly, finding that the bioavailable P fraction estimated with the bioreporter was very
low indicating that in this lake P rather than N is the limiting nutrient for primary pro-
duction by cyanobacteria (Gillor et al. 2010).
Synechococcus sp. PCC 7942 APL strain may be considered as representative of the
picophytoplankton but not of the whole phytoplankton community; in this regard, Muñoz-
Martı́n et al. (2011) have constructed novel self-luminescent cyanobacterial bioreporters
based on the filamentous Anabaena (Nostoc) sp. PCC 7120. Three bioreporters were
constructed, one denoted Anabaena AP harboring the Anabaena phoA (all2843 gene)
promoter fused to the thermostable luxCDABE operon from P. luminescens; Anabaena AP-
L with a luxCDABE fusion to a phoA-like gene (alr5291) promoter and Anabaena PST
with a luxCDABE fusion to the pst promoter from the phosphate transport cluster
pstS1C1A1B1 (all4575 to all4572 genes). Strains A. AP-L and A. PST responded to P
starvation by induction of luminescence while A. AP did not show any induction and was
discarded for further studies; when increasing quantities of PO43- were added to the
P-starved cultures, luminescence decreased in a dose response manner both in A. AP-L and
A. PST which means that both are lights-off bioreporters. After 24 h of incubation with
PO43-, the dynamic range of performance of both bioreporters could be calculated and was
between 1 and 57 lM for the A. AP-L strain and between 0.5 and 57 lM for the A. PST
strain. Re-feeding experiments with organic P-sources indicated that both strains re-
sponded to the addition of D-glucose 6-phosphate and ADP by lowering their

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bioluminescence, ADP decreased bioluminescence to a higher extent than did D-glucose

6-phosphate. Both bioreporters were tested to sense P bioavailability in environmental
samples from upstream and downstream course of a large river in the Madrid area (central
Spain) and from the influent and effluent of an urban sewage treatment plant (STP) finding
a very good correlation between the estimates of bioavailable P as measured by the two
bioreporters and the chemically determined P concentrations indicating that most of the P
in the samples was present as chemical species bioavailable to the cyanobacterium. The A.
AP-L bioreporter has also been recently used in a polyphasic approach to monitor P
bioavailability in five rivers of central Spain in combination with cyanobacterial mor-
phological features related to P bioavailability (the presence of hairs, polyP granules and
calyptras) and the determination in situ of alkaline phosphatases activities of cyanobacteria
found at the sampling sites. As expected, there was an inverse correlation between
phosphatase activity and P-bioavailability as measured by the bioreporter. The bioavailable
P detected by the bioreporter was in general low and correlated with a high measurement of
phosphatase activity, lower abundance of polyP granules and higher abundance of hairs
and calyptra in those cyanobacteria present in the sampling sites able to develop such
structures, indicating low bioavailable P concentrations in the sampling sites (Muñoz-
Martı́n et al. 2014a). Strains S. APL and A. AP-L and A. PST may complement each other
and provide a global picture of P bioavailability for phytoplankton in aquatic
environments.

N bioavailability

As stated above, primary production, particularly in freshwater systems, is limited by P

bioavailability but recent studies emphasize that N can act also as a primary limiting
nutrient (Conley et al. 2009; Dolman et al. 2012; Lewis et al. 2011). As with Fe and P,
chemical determination of total N concentrations does not correlate with the bioavailability
of N species to photosynthetic organisms. Several cyanobacterial bioluminescent biore-
porters to detect N bioavailability have been constructed. Mbeunkui et al. (2002) were
probably the first to report on a cyanobacterial bioreporter to detect bioavailable nitrate, the
strain N1luxKm (Richaud et al. 2001). This strain bears a luxAB fusion to the nblA1
promoter in the chromosomal DNA of the unicellular Synechocystis sp. PCC 6803. The
nblA1 gene encodes the regulator NblA which is essential for phycobilisome degradation
(Collier and Grossman 1994). Upon N deprivation, the nblA1 gene is up-regulated; the
strain is a lights-off bioreporter as bioluminescence is inversely proportional to nitrate
concentration in the range 4–100 lM; it needs an incubation of 10 h; besides nitrate, it also
responds to ammonium, so it is not really specific for nitrate. The authors immobilized the
strain in a way similar to the S. APL strain (Schreiter et al. 2001) and denoted it as
CyanoSensor N1luxkm; apparently, it has not been used to detect N bioavailability in
environmental samples.
Gillor et al. (2003) reported the construction of a Synechococcus strain bearing a fusion
between the glnA promoter and luxAB which was denoted as strain S. GSL to detect N
bioavailability; in the unicellular non-diazotrophic Synechococcus sp. PCC 7942, the glnA
gene encodes a glutamine synthetase (GS) involved in the assimilation of ammonium by
amidation of glutamate to yield glutamine; the global regulator NtcA regulated glnA
expression which is severely repressed by ammonium and activated in the absence of a
combined nitrogen source. Strain S. GSL luminescence was indeed induced in response to
N deprivation and increasing ammonium concentrations progressively decreased biolu-
minescence (also a lights-off strain); the dynamic range of performance was between 1 lm

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and 1 mM of ammonium; it was also found that the reporter strain also responded to nitrate
and nitrite with a limit of detection (LOD) of 1 lM but also to organic compounds with
LODs 10–50-fold higher. The bioreporter strain was tested with samples of the upper and
lower layers of Lake Kinneret and the bioreporter response correlated well with the levels
of Total Dissolved Nitrogen (TDN); so it seems that this bioreporter is non-specific and
may be useful to assess total N bioavailability in aquatic environments (Gillor et al. 2003,
2010).
A different approach was used by Ivanikova et al. (2005) who constructed a luminescent
cyanobacterial bioreporter using the unicellular Synechocystis sp. PCC 6803 based on the
NtcA/B dependent nitrate/nitrite activated nirA promoter fused to V. harveyi luxAB. The
fusion was integrated in the chromosome and the strain was denoted as AND100. It is a
lights-on strain whose luminescence increases with increasing nitrate concentration and has
a dynamic range of performance between 10 and 50 lM of nitrate. Although the strain
might also respond to nitrite, as nitrate concentrations in most freshwater systems are
considerably higher than those of nitrite, the bioreporter may be considered specific to
detect nitrate bioavailability. The bioreporter has been tested in field samples from Lake
Superior where it underestimated the level of nitrate; the authors amended the environ-
mental samples with P and Fe that were apparently limiting in these samples and the
bioreporter was able to detect nitrate accurately (Ivanikova et al. 2005, 2007). The above
mentioned N-responsive bioreporters are all non-self-luminescent and based on unicellular
cyanobacteria; recently, Muñoz-Martı́n et al. (2014b) constructed a battery of self-lumi-
nescent bioreporters of N bioavailability based on the filamentous diazotrophic hetero-
cystous Nostoc (Anabaena) sp. PCC 7120 and also tested the usefulness for N detection of
an alternative GS gene (glnN) present in the unicellular Synechococcus sp. PCC 7942 that
encodes a class II GS strongly activated under nitrogen starvation (Aldehni and Forch-
hammer 2006). The three Nostoc (Anabaena) strains were named as N. GLA bearing the
promoter of the Nostoc glnA gene to P. luminescens luxCDABE; N. NIR which bears a
PnirA::luxCDABE fusion and N. GIF which bears the promoter of the gifA gene also fused
to luxCDABE, the gifA gene encodes IF7A, a post translational regulator of GS, it is
activated when there is an excess of ammonium, such that increased levels of IF7A binds
to GS and inactivate it (Galmozzi et al. 2010); the Synechococcus strain sp. FAM431
harbors the glnN gene fused to luxAB (Aldehni and Forchhammer 2006; Leganes et al.
2009) and has been named as S. GLN.
The N. GLA and S. GLN bioluminescence was significantly activated in N-deplete
medium and when cultures were re-supplemented with combined N sources (nitrate or
ammonium), bioluminescence decreased in a dose–response fashion (lights-off biore-
porters). Calibration curves (24 h incubation time) were calculated and the dynamic range
of performance of N. GLA was 50–500 lM of nitrate or ammonium and for S. GLN was
10–500 lM of nitrate or ammonium; S. GLN was more sensitive as its LOD was con-
siderably lower. N. NIR bioluminescence was induced in the nitrate replete medium and N.
GIF bioluminescence activated in ammonium replete medium (both lights-on biore-
porters). A calibration curve (6 h incubation time) was calculated for the N. NIR finding a
dynamic range for nitrate concentrations between 10 and 100 lM. As ammonium inhibits
nir operon expression (Cai and Wolk 1997), the bioreporter performance was studied in the
absence or presence of increasing concentrations of ammonium, finding that concentrations
of 125 lM and above completely inhibited bioluminescence. A calibration curve (24 h
incubation time) was also calculated for the N. GIF strain, the dynamic range was between
100 and 600 lM of ammonium, the presence of nitrate up to 150 lM did not affect N. GIF

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bioluminescence but the addition of 300 lM of nitrate enhanced bioluminescence so that

the dynamic range shifted to 50–1000 lM of ammonium.
The bioreporters were tested with environmental samples form upstream and down-
stream courses of two rivers in central Spain whose concentrations of ammonium, nitrate
and dissolved inorganic nitrogen (DIN; the sum of both) was determined chemically and
compared to those estimated by the bioreporters. S. GLN detected both nitrate and am-
monium (DIN) with the detected amounts very similar to the chemically determined
values. The N. GLA strain detected also both N species but only in two samples as the
others were below its LOD, The N. NIR strain which detects nitrate estimated quite well
the amount of this N species except in samples with high ammonium concentrations as
expected. The N. GIF strain only detected ammonium in two of the samples with the
highest ammonium concentrations as the other samples had ammonium levels outside its
dynamic range. When used in combination these bioreporters enabled the measurement of
specific N-species as well as total N bioavailability in the wide range of concentrations
present in the tested samples; besides, due to its detection range, the N. GIF bioreporter
may be particularly useful to predict blooms in eutrophic environments. As stated for P
bioreporters above, the use of both unicellular and filamentous cyanobacterial bioreporters
is a powerful tool to assess the capacity of phytoplankton to assimilate various N species in
aquatic ecosystems.

Cyanobacterial bioreporters responsive to heavy metals

Heavy metal responsive bioreporters are engineered to provide information on the

bioavailable fraction of the metal(s) which is the fraction that may exert toxicity to the
biota in the environment. The sensor element is usually the promoter of a metal(s) re-
sponsive gene fused to a promoterless reporter gene and a gene encoding a transcriptional
regulator that when a certain metal(s) is present in the cell activates the promoter (Hyn-
ninen et al. 2010; Osman and Cavet 2010). Based on this concept, all the heavy metal
bioluminescent bioreporters are lights-on as bioluminescence is induced in the presence of
the metal(s) but it should be taken into account that at a certain heavy metal concentration,
bioluminescence might decrease due to toxicity; it is, then, important to clearly define the
dynamic range of operation of these bioreporters.
To our knowledge, the first cyanobacterial bioreporter able to detect heavy metals was
constructed by Erbe et al. (1996). It was based on the smt locus of the unicellular Syne-
chococcus sp. PCC 7942, this locus consists of the cyanobacterial metallothionein gene
smtA and smtB, a divergently transcribed gene encoding the transcriptional repressor of
smtA; binding of metal ions by SmtB induces conformational changes in it that promote
RNA polymerase accessibility to smtA (Huckle et al. 1993; Morby et al. 1993). smtA
transcription is induced in the presence of several metals: Zn (as preferred metal), Cd, Cu,
Hg, Co and Ni (Huckle et al. 1993; Osman and Cavet 2010). The authors fused part of the
smt locus including smtB, the smt operator/promoter region and the first codon of smtA to
luxCDABE of A. fischeri. The strain, although self-luminescent, did not generate enough
endogenous aldehyde and dodecanal was added at each sample before measurement. They
tested the performance of the bioreporter in the presence of the metal salts ZnCl2, CuSO4
and CdCl2. ZnCl2 induced bioluminescence in the range 0.5–2, 4 lM ZnCl2 was already
toxic as luminescence began to decline; CdCl2 also induced luminescence in a shorter
concentration range: 0.5–1.5 lM; CuSO4 was less effective in inducing luminescence than
the other metal salts and toxicity was found also at higher concentrations, 15 lM. Ap-
parently, there are no further reports in the literature concerning this particular bioreporter.

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Peca et al. (2008) constructed self-luminescent novel cyanobacterial bioreporters able to

detect nickel, cobalt and zinc. They fused the coaT promoter which is inducible by Co and
Zn and the nrsBACD Ni responsive promoter to luxAB from V. harveyi; the constructions
were transformed into a Synechocystis sp. PCC 6803 strain harboring the fatty acid re-
ductase complex genes luxCD,E from V. harveyi. The constructs were integrated into the
chromosome; the regulatory genes coaR and nrsRS controlling the expression of the coaT
and nrsBACD genes were cloned in the same vectors. The resulting bioreporters were
denoted as coaLux and nrsLux strains.
The bioreporters were tested for specificity with a battery of metal salts with an incu-
bation time of 3 h. Form the tested metals, only Zn and Co significantly induced the
bioluminescence of the coaLux strain while the nrsLux strain was specific to Ni. The
dynamic detection range for Co of the coaLux was 0.3–6 and 1–3 lM for Zn; the nrsLux
strain detected Ni in the range 0.2–6 lM. When the performance of the bioreporters was
tested in darkness, the detection range of the coaLux strain for Co and Zn increased to 26 and
13 lM, respectively; the nrsLux strain showed no detectable luminescence. The coaLux
bioreporter was tested for quantification of bioavailable Zn in soil samples; the concentration
of Zn estimated by the bioreporter represented about 90 % of the atomic absorption spec-
troscopy measured Zn concentrations indicating a high bioavailability of the metal.

Aequorin-based cyanobacterial bioreporters

Cyanobacteria are ancient organisms that date from the Precambrian era and during the
course of evolution have developed signal transduction systems to sense and respond to
changes in their environment, including anthropogenic pollution. Free Ca2? is a common
intracellular messenger in eukaryotes (Berridge et al. 2000, 2003; Clapham 1995) and
reported evidences indicate that it also has this role in prokaryotes (Dominguez 2004).
Increases in intracellular free Ca2? have been shown to be induced in response to a wide
range of abiotic and biotic stimuli; in fact, there are solid evidences that indicate that the
specificity of the Ca2? response is encoded in the recorded Ca2? signal in response to the
stimulus, the so-called Ca2? signature, according to the amplitude, duration, frequency,
rise time, time to return to basal Ca2? levels, source and intracellular location of the Ca2?
signal (Kudla et al. 2010; Whalley and Knight 2013).
The photoprotein apoaequorin is a sensitive Ca2? indicator whose gene has been cloned
form the jellyfish Aequorea victoria which has been expressed in a number of cell systems, both
prokaryotic and eukaryotic, to measure Ca2? signals in response to a variety of environmental
stresses (Dominguez 2004; Torrecilla et al. 2000; Whalley and Knight 2013). Apoaequorin can
be fully reconstituted to aequorin by the addition of the hydrophobic luminophore coelenter-
azine. In Fig. 4a the reaction of this protein with Ca2? is depicted; as shown, once Ca2? ions are
bound to aequorin, the photoprotein catalyzes the oxidation of coelenterazine by oxygen
resulting in blue light luminescence; Fig. 4 also shows some relevant parameters taken into
account to define the specificity of the Ca2? signatures (Fig. 4b).
Torrecilla et al. (2000) were the first to clone and express apoaequorin in a cyanobac-
terium, the filamentous Anabaena (Nostoc) sp. PCC 7120, denoted as Anabaena pBG2001a,
which enables continuous and in vivo monitoring of intracellular free Ca2? concentrations.
The strain has been used to record and analyze Ca2? signatures in response to a wide spectrum
of environmental stimuli (Torrecilla et al. 2000, 2004a, b). The same group has also expressed
apoaequorin in the unicellular Synechococcus sp. PCC 7942 under the control of the promoter
of the petJ gene (encoding cytochrome c553) and so far, it has been used to record Ca2?
signatures in response to N deprivation (Leganes et al. 2009).

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Fig. 4 a Aequorin reaction showing the emission of bioluminescence. b Relevant parameters that encode
the specificity of the recorded Ca2? signatures in response to environmental stimuli or pollution. c Ca2?
signature induced in Anabaena pBG2001a in response to binary mixtures of Zn2? ? Pb2? at constant ratio
(1:1)

It is known that many pollutants interfere with Ca2? homeostasis in eukaryotic or-
ganisms (Kozlova et al. 2005; Ogunbayo et al. 2008; Ohta and Suzuki 2007; Wang et al.
2007); however such studies were lacking in prokaryotes. Barran-Berdon et al. (2011) used
the apoaequorin expressing Anabaena (Nostoc) pBG2001a strain to systematically record
and analyze the Ca2? signatures elicited by a variety of environmental pollutants: cationic
and anionic heavy metals, the metalloid As, naphthalene, organic solvents (acetone,
ethanol, toluene) and pharmaceuticals such as lipid regulators (fibrates) and antibiotics
(fluoroquinolones); also the Ca2? signatures induced by binary mixtures of some of these
pollutants and the signature induced by a real wastewater sample which could be mimicked
by mixing it main constituents at environmental concentrations were recorded. The results
indicated that all the tested pollutants elicited a fast and specific Ca2? signature which was
highly reproducible and dose-dependent; the Ca2? signatures were previous to toxicity as
measured by the lights-off bioreporter Anabaena CPB4337. The Ca2? signatures induced
by binary mixtures of pollutants could predict the nature of pollutants interactions
(Fig. 4c). It was concluded that intracellular free Ca2? signatures elicited by pollutants
could serve as an early biomarker of exposure to environmental pollution.
Table 2 provides a summary of main features of cyanobacterial bioreporters described
to date.

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Table 2 Main features of cyanobacterial bioreporters

Cyanobacterium Promoter- Type of Targets and dynamic range of References
(name of strain if reporter fusion bioreporter detection (if indicated)
indicated)

Synechocystis sp. Ptac-luc-luxAB Lights-off General toxicity bioreporter. Shao et al. (2002)
PCC 6803 Tested with herbicides, Cu,
Zn and 3,5DCP. Not tested
in environmental samples
Anabaena Non-identified. Lights-off General toxicity bioreporter. Fernández-Piñas
(Nostoc) sp. Constitutive Tested with heavy metals, and Wolk
PCC 7120 P-luxCDABE organic solvents, (1994), Rodea-
(Anabaena pharmaceuticals, Palomares et al.
CPB4337) perfluorinated surfactants (2009), Rosal
and nanomaterials. Tested et al. (2010a, b)
with pollutants mixtures Rodea-Palomares
and in environmental et al. (2010,
matrices of different 2011,
complexity 2012), Garcia
et al. (2013) and
González-Pleiter
et al. (2013)
Synechococcus PisiAB-luxAB Lights-off Fe3?; 10-21.1–10-20.6 M. Durham et al.
sp. PCC 7942 Tested with environmental (2002), McKay
(S. KAS101) samples et al. (2005),
Porta et al.
(2003, 2005)
and Hassler
et al. (2008,
2009)
Synechococcus PisiAB-luxAB Lights-off Fe3?; 10-22.4–10-19.4 M. Boyanapalli et al.
sp. PCC 7002 Tested with environmental (2007)
(S. BM004 or samples
S. CCMP
2669)
Synechocystis sp. PisiAB-luxAB Lights-off Fe3?. Not tested with Kunert et al.
PCC 6803 environmental samples (2000)
(S. MpILisi)
Anabaena Palr0397-luxAB Lights-off Fe3?; 10-21.5–10-19.6 M. Zha et al. (2012)
(Nostoc) sp. Tested with environmental
PCC 7120 samples
Synechococcus PphoA-luxAB Lights-off DIP (PO43-) and DOP (ADP; Schreiter et al.
sp. PCC 7942 D-fructose-6-phosphate; D- (2001) and
(S. APL) glucose-6-phosphate; q- Gillor et al.
nitrophenol-phosphate). (2002, 2010)
Tested with environmental
samples
Anabaena PphoAlike- Lights-off DIP (PO43-) and DOP (ADP; Muñoz-Martin
(Nostoc) sp. luxCDABE D-glucose-6-phosphate). et al. (2011,
PCC 7120 1–57 lM PO43-. Tested with 2014a)
(A. APL-L) environmental samples
Anabaena Ppst-luxCDABE Lights-off DIP (PO43-) and DOP (ADP; Muñoz-Martı́n
(Nostoc) sp. D-glucose-6-phosphate). et al. (2011)
PCC 7120 (A. 0.5–57 lM PO43-. Tested
PST) with environmental
samples

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Table 2 continued

Cyanobacterium Promoter- Type of Targets and dynamic range of References

(name of strain if reporter fusion bioreporter detection (if indicated)
indicated)

Synechocystis sp. PnblA1-luxAB Lights-off 4–100 lM nitrate; it also Mbeunkui et al.

PCC 6803 (S. responds to ammonium. (2002)
N1luxkm) Not tested with
environmental samples
Synechococcus PglnA-luxAB Lights-off Ammonium, nitrate and Gillor et al. (2003,
sp. PCC 7942 nitrite. 2010)
(S. GSL) 1 lM to 1 mM ammonium.
Nitrate and nitrite with
LOD of 1 lM. Organic N
compounds with LODs
10–50-fold higher. Tested
with environmental
samples
Synechocystis sp. PnirA-luxAB Lights-on 10–50 lM nitrate. Tested Ivanikova et al.
PCC 6803 (S. with environmental (2005, 2007)
AND100) samples
Nostoc PglnA-luxCDABE Lights-off 50–500 lM nitrate or Muñoz-Martı́n
(Anabaena) sp. ammonium. Tested with et al. (2014b)
PCC 7120 (N. environmental samples
GLA)
Nostoc Pnir-luxCDABE Lights-on 10–100 lM nitrate. Muñoz-Martı́n
(Anabaena) sp. Concentrations of 125 lM et al. (2014b)
PCC 7120 (N. ammonium and above
NIR) inhibit bioluminescence.
Tested with environmental
samples
Nostoc PgifA-luxCDABE Lights-on 100–600 lM ammonium. Muñoz-Martı́n
(Anabaena) sp. The addition of 300 lM et al. (2014b)
PCC 7120 (N. nitrate shifted the dynamic
GIF) range of detection of
ammonium to
50–1000 lM. Tested with
environmental samples
Synechococcus PglnN-luxAB Lights-off 10–500 lM nitrate or Muñoz-Martı́n
sp. PCC 7942 ammonium. Tested with et al. (2014b)
(S. GLN) environmental samples
Synechococcus smtB-PsmtBA- Lights-on 0.5–2 lM ZnCl2 Erbe et al. (1996)
sp. PCC 7942 luxCDABE 0.5–1.5 lM CdCl2
CuSO4 lees effective as
inducer. Not tested with
environmental samples
Synechocystis sp. coaR-PcoaT-luxAB Lights-on 0.3–6 lM Co Peca et al. (2008)
PCC 6803 (S. transformed in a 1–3 lM Zn. In darkness, the
coaLux) luxCD,E detection range for Zn
S. expressing strain increased to 13 lM and to
26 lM for Co. Tested with
environmental samples
Synechocystis sp. nrsRS-PnrsBACD- Lights-on 0.2–6 lM Ni. Not tested with Peca et al. (2008)
PCC 6803 (S. luxAB environmental samples
nrsLux) transformed in a
luxCD,E
S. expressing strain

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Table 2 continued

Cyanobacterium Promoter- Type of Targets and dynamic range of References

(name of strain if reporter fusion bioreporter detection (if indicated)
indicated)

Anabaena Apoaequorin gene Lights-on Specific Ca2? signatures in Torrecilla et al.

(Nostoc) sp. expressed in an response to a variety of (2000, 2004a, b)
PCC7120 Anabaena stimuli/pollutants. Tested and
(A. pBG2001a) (Nostoc) with environmental Barran-Berdon
replicative samples et al. (2011)
plasmid (pDU1
replicon)

Concluding remarks

Cyanobacteria are ecologically relevant organisms, ubiquitous in aquatic and terrestrial

environments; as primary producers they are a fundamental component of trophic webs
and have a crucial role in the N and C cycles. Biological methods based on benthic
cyanobacteria to assess environmental contamination in running waters include different
levels of organization, such as cellular, populations and communities, with the corre-
sponding use of the changes in morphological and/or physiological characteristics, use of
specific autecological data of cyanobacterial taxa, with a specific value of bioindicators
populations, and/or shifts in the community structure, as indicator of nutrient status.
Cyanobacterial identification performed by light microscopy, while remains commonplace,
requires trained taxonomists with the ability to distinguish differences between taxa or
morphological variations. Therefore, lately, molecular methods, which can obviate many
of these difficulties, are being developed and used to analyse changes in the communities
in relation to variations in water quality. New technology referred to as ‘NGS’, which leads
to the determination of millions of DNA sequences in a single process, has the potential to
further extend the application of DNA information for routine biomonitoring applications
to an unprecedented scale.
Regarding cyanobacteria as bioreporters, they offer a low cost, low maintenance al-
ternative to heterotrophic bacterial bioreporters. Due to their role in nutrient cycling and in
the formation of algal blooms under eutrophic conditions in water reservoirs, the majority
of cyanobacterial bioreporters constructed to date have focused on the detection of Fe, N
and P chemical species; many of these bioreporters have also been validated with actual
environmental samples; however, regarding other sources of pollution, cyanobacteria have
been very little exploited as bioreporters: only two cyanobacterial strains have been
constructed and used for general ecotoxicity testing but only one of them, Anabaena
CPB4337 has been tested in environmentally realistic scenarios with real samples and
complex mixtures of pollutants; also an Anabaena strain which allows in vivo monitoring
of intracellular calcium levels and which is responsive to a broad range of pollutants has
been constructed. With respect to specific pollutants, only two cyanobacterial bioreporters
have been constructed able to detect heavy metals and none to detect specific organic
pollutants. In this regard, there is much to be done in the field of cyanobacterial biore-
porters, they could serve as hosts for sensing elements from other bacteria, new reporter
systems should be evaluated (most of them are bioluminescent, with fluorescence/other
reporter elements seldom used) and also, given the fact that over 100 cyanobacterial

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genomes have been sequenced, novel genetic elements responsive to pollutants could be
identified and used to construct new and useful bioreporters.

Acknowledgements This study was funded by MINECO grants CTM2013-45775-C2-2-R and CGL2013-
44870-R.

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