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In Situ Soil Remediation: Bacteria or Fungi?

a a
TERESA J. CUTRIGHT & SUNGGYU LEE
a
Process Research Center Department of Chemical Engineering , University of Akron , Akron,
Ohio, USA
Published online: 27 Apr 2007.

To cite this article: TERESA J. CUTRIGHT & SUNGGYU LEE (1995) In Situ Soil Remediation: Bacteria or Fungi?, Energy Sources,
17:4, 413-419, DOI: 10.1080/00908319508946090

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In Situ Soil Remediation: Bacteria or Fungi?

TERESA J. CUTRIGHT
SUNGGYULEE
Process Research Center
Department of Chemical Engineering
University of Akron
Akron, Ohio, USA
Downloaded by [McGill University Library] at 16:50 19 February 2015

Contamination of the environment is not a new problem. For most of recorded

history, the unwanted by-products of industrial and residential processes have been
dumped into unlined pits or nearby streams. Althrougb disposal techniques have
greatly improved, significant quantities of hazardous materials are still being released
to the environment via accidental spills and leaking underground storage tanks. One
particular group of contaminants of critical environmental concern is polycyclic
aromatic hydrocarbons (PAHs). Pnli-contaminated sites typically cover large areas;
therefore, the development of in situ remediation techniques such as bioremediation
is strongly emphasized. In situations when inherent microorganisms are not capable
of degrading the contaminants, foreign strains must be used. Bioremediation experi-
ments were conducted to compare the remediation efficiencies of a bacteria and a
fungus for an industrially PAH contaminated soil. Specifically, the use of three
supplemental nutrient solutions were investigated in conjunction with the bacteria
Achromobacter sp. and fungus Cunninghamella echinulata var. elegans.
Keywords Achromobacter sp., biorernediation, Cunninghamella echinulata var.
elegans, polycyclic aromatic hydrocarbons

Polycyclic aromatic hydrocarbons (PARs) are a group of organic priority pollutants

of growing environmental concern. This concern stems from the fact that PARs are
a known class of potent carcinogens. For instance, benzoialpyrene, the most
extensively studied PAR, has been proven to form cancer-producing metabolites
when acted upon by enzymes within the body (Manahan, 1991). Another growing
concern is that PAR contamination in soil and groundwater is associated with a
wide range of hazardous waste sites.
The first documented source of PAR contamination occurred in Germany
during the early 1800s. At that time, increased production of dyes and organic
chemicals developed from the coal tar industry led to "PAH poisoning" (Manahan,
1991). Approximately 50 years later, the primary source of PAR contamination was
from manufactured gas plant (MGP) sites (Cutright et al., 1991). At that time,
heating and lighting of both the industrial and residential communities came from
coal gasification. To increase the heating value, light hydrocarbons were cracked
with the coal, producing a liquid by-product rich in PAHs. As time and technology
progressed, electricity replaced coal gasification, and the MGPs were abandoned.
Althrough MGPs are no longer in use, the sites are still heavily contaminated and
awaiting remediation.

Received 26 June 1994: accepted 17 July 1994.

Address correspondence to Dr. Sunggyu Lee, Department of Chemical Engineering, Uni-
versity of Akron, Akron, OH 44325-3906, USA.

413
 414 T. J. Cutright and S. Lee

PAHs are still being produced today by simple processes such as smoking and
industrial processes such as petroleum refining, coke production, and wastewater
facilities. The increasing occurrence of leaking underground storage tanks (Song &
Bartha, 1990) and accidential spills such as the Exxon Valdez in Alaska (Atlas &
Atlas, 1991; Mihe\cic & Luthy, 1988) also contributes to the amount of PAHs being
released to the environment. To data, there are an estimated 3,000-4,000 sites
contaminated with PAHs from MGP sites and accidential spills in North America
alone. In addition, it is estimated that over 30% of the 7 million underground
storage tanks in the United States are leaking (Kovalick, 1991).
Perhaps the simplest method for the treatment of large contaminated PAH
sites is bioremediation (Hicks & Caplan, 1993; Levin & Gealt, 1993; Thayer, 1991;
Vervalin, 1989). In bioremediation, the microorganisms use the contaminants as
the primary substrate (food) for their growth. If the indigenous microorganisms are
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not sufficient to degrade the contaminants, foreign strains can be introduced at the
site. Supplemental nutrients may be added to enhance the remediation process,
regardless of whether indigenous or foreign strains are used (Aggarwal et a\., 1991).
Under aerobic conditions, the contaminants are completely mineralized to carbon
dioxide, water, biomass, and salts, thus making bioremediation environmentally
friendly. Bioremediation has the added advantages that it is inherently versatile,
keeps site disruption to a minimum, able to be coupled with other technologies,
eliminates long-term liability, and less expensive than other technologies.

Methods and Materials

Soil and Fungi Origin

An industrially contaminated MGP soil sample labeled FC2-2 was used for all of
the bioremediation experiments. The sample originated from the Alberta Research
Council (ARC) Primary clean-up facility in Alberta, Canada. The initial PAH
contamination level was determined to vary between 5% and 20% by mass. To
eliminate any error due to this variation, the soil was completely homogenized. The
soil was then analyzed to determine the extent of contamination before conducting
the individual experimental sets.
Both the bacteria and fungi were purchased from the American Type Culture
Collection (ATCC) in Rockville, Maryland. The first set of experiments used the
bacteria Achromobacter sp. (ATCC 21910). The second set of experiments used the
fungi Cunninghamella echinulata var. elegans (26269). Each of the experiments
were conducted batchwise over an 8-week period. Hydrogen peroxide was used as
the oxygen source at a concentration of 0.1 g HzO z per liter -of nutrient solution.
The exact methodology used in the experiments was presented in a previous paper
(Cutright & Lee, 1994).

Supplemental Mineral Salt Solutions

Three supplemental mineral salt (MS) solutions were selected for use in the
experiments. The first solution, MS1, was developed by Travis (1990) and consists
of 0.25 g NH 4N0 3 , 0.1 g K zHP0 4 , and 1.0 L distilled water.
The other two mediums, MS2 and MS3, were developed by Bailey and Coffey
(1986) and Gauger et a\. (l990);respectively. MS2 consists of 0.3 g NH 4N0 3 , 0.2 g
K zHP04 , 0.2 g MgS04 • 7H zO, 0.2 g CaS0 4 , 2.0 g malt extract, 2.0 g glucose, 2.0 g
 In Situ Soil Remediation: Bacteria or Fungi? 415

casamino acid, and 1 L distilled water. MS3 contains 4.0 g K 2HP04 , 4.0 g
Na 2HP04 , 0.2 g MgCI 2 , 2.0 g NH 4Cl, 0.001 g CaCI 2 , 1.42 g Na 2S04 , 0.001 g FeCI),
and 1 L distilled water.

Extent of Contamination and Molecular Identification

Initial contaminant concentration of the original sample was determined by Soxhlet
extraction to be 10.216 mgy kg and 24.496 mgy kg for the first and second
experimental sets, respectively. The Soxhlet extraction methodology has been
described in a previous paper (Cutright & Lee, 1994).
The molecular identification of the contaminants was determined using a
Hewlett-Packard high-performance liquid chromatography (HPLC) model 1050.
The HPLC was operated with a gradient method consisting of acetonitrile and
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water at 1.5 ml.yrnin and 35°C. A 50:50 acetonitrile:water mixture was used for the
first 3 min, after which the solvent ratio was rampled to 100% acetonitrile over a
12-min time span. The final 7 min of the analysis used 100% acetonitrile. Quantita-
tive results (relative weight of contaminant present) were obtained by the use of
reference factors for each compound in conjunction with the area output from the
HPLC analysis.

Results
The first set of experiments showed that the bacteria Achromobacter sp., in
conjunction with the three supplemental nutrient solutions (MS), was capable of
degrading the majority of the PAHs present. Table I contains the relative weights
of each contaminant that was present after 8 weeks of treatment. As shown,
Achromobacter sp. was successful at remediating over 95% of the PAHs for all
three of the bacteria-nutrient solution combinations. The first combination, MSI,
had the best results, obtaining a 99% remediation efficiency of the contaminants.
MS2 achieved 96% remediation, this slight decrease in efficiency due to the
inability to degrade f1uoranthene, benztaranthracene, and benzofbjfluoranthcne.
However, MS2 was the only combination in this experimental set to completely
degrade indeno(l23cd)pyrene.
MS3, unlike MS2, was able to degrade f1uoranthene, benzfajanthracene, and
benzofbjfluoranthcne. None of the bacteria-nutrient solution combinations were
capable of remediating pyrene in its entirety.
Table 2 contains the apparent mass of each contaminant after 8 weeks of
remediation for all three mineral solutions with Cunninghamella echinulata var.
elegans. As indicated, the soil sample used for these experiments had a much
higher initial contaminant concentration than that used for the Achromobacter sp.
experiments; however, this did not hinder the bioremediation treatment. The best
remediation (97%) was achieved using MS1. The PAHs that were degraded by the
fungi can be divided into three general classification types. Type I consists of
anthracene, 4-terphenyl-d I4 , chrysene-d.y, and pyrene. These compounds have the
same general shape for their degradation trend. Type II consists of benzotghilpery-
lene, anthracene, and dibenztahlanthracene. These compounds were completely
degraded within one week. The final classification, Type III, consists of the
remaining PAHs. For these contaminants, the majority of the remediation was
completed within the first 4 weeks of treatment.
 416 T. 1. Cutright and S. Lee

Table 1
Contaminant weight in soil after 8 weeks of remediation with Achromobacter sp.

Weight (mg /kg)

Untreated
Compound soil MSI MS2 MS3

Acenaphthylene 1.853
Phenanthrene 0.138
Anthracene 0.205
Fluoranthene 0.207 0.120
Pyrene 0.282 0.054 0.093 0.056
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4-Terphenyl-d 14 1.222
Chrysene-d 12 0.570
Benztajanthracene 1.194 0.073
Chrysene 0.305
Benzofbjfluoranthene 0.766 0.124
Benzo(k)fluoranthene 0.064
Dibenz(ah)anthracene 1.622
Benzo(ghi)perylene 1.622
Indeno(I23cd)pyrene 0.166 0.031 0.318
Total 10.216 0.085 0.410 0.374
Remediation efficiency (%) 99 96 96
Dash indicates no containment detected.

Treatment with MS2 was only able to achieve 73% remediation. As with
Cunninghamella echinulata vaT. elegans and nutrient solution 1 (CE1), the contami-
nants can be divided into the same three types. The final result with the third
nutrient solution was 91% remediation of the PAHs. Unlike the previous two
experiments with the fungi, the contaminants could not be divided into three
classification types.

Discussion
The susceptibility of individual PAHs to microbial degradation has been well
documented. The most widely studied PAH is benzotajpyrene (Manahan, 1991;
Park et aI., 1990). The studies on individual contaminants have generated several
general rules-of-thumb that can be applied to sites containing multiple PAHs. For
instance, the ability of PAHs to undergo microbial degradation is inversely propor-
tional with the number of benzene rings incorporated in the compound (Baker &
Herson, 1994; Kostecki & Calabrese, 1992). In other words,

where Co represents one benzene ring; C I' two benzene rings "fused" together;
etc.
 In Situ Soil Remediation: Bacteria or Fungi? 417

Table 2
Contaminant weight in soil after 8 weeks of remediation with Cunninghamella
echinulata var. elegans

Weight (mg /kg)

Untreated
Compound soil MSI MS2 MS3

Naphthalene 11.117
Acenaphthylene 1.101
Acenaphthene 3.992 3.042
Phenanthrene 0.254
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Anthracene 0.052
Fluoranthene 0.703 0.114 0.263 0.148
Pyrene 0.485 0.103 0.237 0.115
4-Terphenyl-d 14 2.033 0.822 0.386
Chrysene-d 12 2.066 1.174 0.554
Bcnzfalanthraccnc 0.685 0.137 0.415 0.201
Chrysene 0.435 0.098 0.257 0.134
Bcnzotkjfluoranthcnc 0.058 0.062
Benzo(a)pyrene 0.224 0.101
Dibenztahjanthracene 0.309
Bcnzotghijperylcne 0.079 0.207 0.165
Indeno(123cd)pyrene 0.029
Total 24.496 0.787 6.434 2.035
Remediation efficiency (%) 97 73 91
Dash indicates no containment detected.

The experimental results using Achromobacter sp. did in fact follow this
general rule. The lower ringed structures (napthalene -> anthracene) had been
completely degraded within the first week of treatment. The larger structures, such
as benzofbjfluoranthene, did not undergo degradation until after the lower ringed
structures had been completely remediated. Even then, the largest contaminants
degraded at a much slower rate.
Fungal degradation of PAHs ranging from 2 to 5 rings has been demonstrated,
but does not follow the rules of thumb associated with microbial degradation
(McFarland et aI., 1990). In controlled environments made up of only one PAH,
the degradation occurs by the reaction sequence called the "NIH shift" (Baker &
Herson, 1994). However, the exact methodology that occurs at an actual site is still
unclear. This is due primarily to the fact that the contaminated sites contain
multiple contaminants with varying concentrations. More research will have to be
conducted on multiple PAH contaminants in a controlled environment before the
rules of thumb could be developed and validated.

Conclusions
Laboratory-scale remediation with both the bacteria and fungi has very promising
results with all of the supplemental nutrient solutions investigated. Achromobacter
 418 T. J. Cutright and S. Lee

sp. achieved 99%, 96%, and 96% remediation with MS1, MS2, and MS3, respec-
tively. Cunninghamella echinulata var. elegans had 97% remediation with MS1 and
achieved 73% and 91% with MS2 and MS3, respectively. As indicated in Tables 1
and 2, the initial contaminant concentration for the fungi experiments was approxi-
mately 3 times higher than that of the experiments with the bacteria. However, this
did not deter the success of the remediation. This indicates that the process would
be successful at an actual site, where the levels of contamination vary.
The primary difference between the bacteria and fungi was with the individual
contaminants that could not be degraded within the 8 weeks of treatment. Achro-
mobacter sp. was not able to degrade indenot l Bcdlpyrenc. Cunninghamella echinu-
lata var. elegans could degrade indeno(l23cd)pyrene, but not benzotghilperylene.
Therefore, the choice between bacteria and fungi would depend on the contami-
nants present.
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However, there are two areas that may pose a problem for using the fungi over
large contaminated areas. The first is associated with the long-term storage of the
starter culture. Fungal strains are more temperature sensitive than conventional
bacterial strains. Second, for subsurface applications, mass transfer and pumping
limitations may occur. For these reasons, fungi treatment should probably be
limited to either small contaminated areas or ex situ batch treatments. The
efficiency of using both Achromobacter sp. and Cunninghamella echinulata var.
elegans at the same site should be investigated.

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