xvEPA
United States
Environmental Protection
Agency
Robert S. Kerr Environmental
Research Laboratory
Ada, OK 74820
Research and Development EPA/600/M-90/024 February 1991
ENVIRONMENTAL
RESEARCH BRIEF
Anaerobic Biotransformation of Contaminants
in the Subsurface
Joseph M. Suflita* and Guy W. Sewell"
Abstract
Anaerobic conditions predominate in contaminated aquifers and
are not uncommon in noncontaminated areas. Comparatively
little is known about degradative processes and nutrient cycling
under anaerobic conditions. However, it is apparent these
processes are fundamentally different and more complex than
comparable aerobic processes. Research in this area is critical
to our understanding of the fate of contam inants in the subsurface
environment and for the design and operation of efficient and
effective treatment technologies. The objective of this research
brief is to report the current status of research directed toward
defining anaerobic microbial metabolic processes which occur in
the subsurface environment.
Introduction
Bioremediation technologies hold great promise as economical
and permanent solutions for contaminants in the environment.
This is particularly true for the terrestrial subsurface where
technical or economic constraints may preclude physical removal
of a contaminant and thus its treatment by sorption, incineration
or containment methods. Bioremediation technologies have
several other inherent advantages. Instead of transferring
• Department of Botany and Microbiology, University of Oklahoma,
Norman, OK.
" U.S. EPA, Robert S. Kerr Environmental Research Laboratory,
Ada, OK.
contaminants from one environmental medium to another,
complete breakdown or mineralization of contaminants is often
possible. Further, the partial transformation of a contaminant
can sometimes make it more environmentally acceptable.
Bioremediation is also usually cheaperthan conventional physical
or chemical treatment schemes.
To date, most subsurface biotrealment processes have relied on
aerobic microbial metabolism. In these cases oxygen serves as
a terminal electron acceptor for the microorganisms and may be
supplied in variousforms (compressed air, liquid oxygen, hydrogen
peroxide or ozone). Such systems have shown great success in
the cleanup of a wide variety of contaminants, but they can also
suffer from several drawbacks. For example, some compounds,
particularly halogenated solvents, are resistant to biodegradation
under aerobic conditions. Also, most of the costs associated with
aerobic bioremediation are due to the low solubility of molecular
oxygen and the difficulties associated with the introduction and
transport of this electron acceptor in the subsurface.
However, microbial metabolism also occurs in the absence of
molecular oxygen, but there is a general lack of appreciation for
the metabolic potential of anaerobic microorganisms in the
subsurface. Historically, this may be attributed to the difficulties
associated with research on anaerobic microorganisms
(specialized equipment, slow growth rates, poorly defined growth
requirements), the misconceptions about the numbers and
activities of microorganisms in the subsurface, and the expense
and technical limitations of sampling the subsurface environment.
uuD Printed on Recycled Paper
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Toagreater or lesser extentthese problems have been addressed
by the advancement of technology and the results of investigations
into the subsurface environment. New methods and equipment
for the isolation, cultivation, and manipulation of anaerobic
microorganisms have made their study less of an art and more
a set of widely used and accepted techniques. In recent years
microbiological studies of the subsurface have demonstrated the
abundance, diversity and important activities of bacteria in this
environment. Technical developments have allowed the recovery
of high quality microbiological samples from both the near and
deep subsurface. Together these advances and discoveries
have allowed us access to the subsurface and techniques for the
characterization of the indigenous microorganisms. However,
many questions surrounding anaerobic metabolic abilities remain,
including: 1) What types of contaminants are susceptible to
anaerobic decay and which are not? 2) What structural features
of the contaminants favor its bioconversion under anaerobic
conditions? 3) Are pollutants mineralized or only partially
transformed? 4) What rates of transformation can be expected?
5) How do such transformations impact predictionsof the transport
and fate characteristics of contaminants?
To address someofthesequest ions, researchers at the University
of Oklahoma, through the National Center for Ground Water
Research and the Robert S. Kerr Environmental Research
Laboratory (RSKERL) of the U.S. EPA have been investigating
anaerobic biotransformations of pollutant chemicals in material
collected from the terrestrial subsurface. They have found an
abundant and diverse microflora existing in anoxic aquifers.
Further, the microorganisms within these aquifers are more
metabolically diverse than originally believed. It appears that
these organisms can catalyze biotransformation reactions which
will be useful for refining the predictions of the transport and fate
of contaminants and possibly form the basis for novel
bioremediation strategies.
Metabolic Principles
Heterotrophic organisms (like humans and most bacteria) oxidize
organic compounds to obtain energy. In this process, electrons
or reducing equivalents from the oxidizable organic compound
(substrate) are transferred to and ultimately reduce an electron
acceptor. The electron acceptor may be an organic or inorganic
compound. During this electron transfer process, usable energy
is recovered through a complex series of oxidation-reduction
(redox) reactions by the formation of energy storage compounds
or electrochemical gradients. The oxidation of organic compounds
coupled lo the reduction of molecular oxygen is termed aerobic
heterotrophic respiration (Table 1).
When oxygen is unavailable, redox reactions can still occur. In
anaerobic respiration, the oxidation of organic matter can be
coupled with a number of other organic or inorganic electron
acceptors. Some microorganisms carry out a process known as
fermentation. Fermenting organisms utilize their substrate as
both an electron donor and acceptor. In this process an organic
compound is metabolized with a portion of that compound
becoming a reduced end product and another becoming an
oxidized product. A common example of this process is the
alcoholic fermentation of starch to CO2 (oxidized product) and
ethanol (reduced product). Fermentative organisms play a
critical role in anaerobic consortia by transforming organic
substrates into simple products which can then be used by other
members of the community. Still other organisms can utilize
Table 1. Selected types of aerobic and anaerobic respiration
Involved In the microbial metabolism of organic matter.
Process Electron Metabolic Relative
Acceptor Products Potential
Energy
Aerobic O2 CO2, H2O ,
Heterotrophic
Respiration
Denitrification NOj" CO2, N2
Iron Reduction Fe3* CO2, Fe2*
Sulfate Reduction SO42' CO2, H2S
Methanogenesis CO2 CO2, CH4
i
HIGH
LOW
alternate electron acceptors (Table 1). The potential energy
available from the oxidation of a particular substrate coupled with
the reduction of different electron acceptors varies considerably.
A higher energy yielding process will tend to predominate if the
required electron acceptor is available. Under anaerobic
conditions, microorganisms may enter into very tightly linked
metabolic consortia. That is, the catalytic entity responsible for
the destruction of a contaminant is often not a single type of
microorganism. Such consortia can develop regardless of the
nature of the terminal electron acceptor (see below).
Impact of Contaminants on Subsurface Ecology
When readily degradable organic matter enters the subsurface
in sufficient quantities, it can produce a variety of chronologically
and spatially defined metabolic zones (Figure 1). These zones
are not necessarily mutually exclusive and are dependent on the
availability of electron acceptors. As organic matter enters an
oxygenated aquifer, whether it is a human produced
(anthropogenic) contaminant or an influx of "natural" material,
indigenous aerobic heterotrophic microorganisms metabolize
the organic material and consume the available oxygen in the
process. When this occurs, aerobic respiration slows and
eventually stops. This allows for the development of anaerobic
metabolic communities. If nitrate is available, dissimilatory
nitrate-reduction (denitrification) tends to become the dominant
metabolic process linked to the degradation of organic
contaminants. Sulfate tends to serve as the electron acceptor
(sulfate reduction) when nitrate is depleted. Sulfate reduction
may lead to the formation of sulfides. Organic matter can still be
consumed when sulfate is depleted by coupling its metabolism
with the reduction of CO2 in the process of methanogenesis
(Figure 1). The metabolic processes for the consumption of
organic matter illustrated in Table 1 and Figure 1 are far from
complete. Other electron acceptors such as iron or manganese
may also participate in this respect. Due to the abundance of
oxidized iron (Fe3*) in the subsurface and the apparent catabolic
diversity of the organisms responsible for iron reduction (Lovely
and Lonergan, 1990), the environmental importance of such
reactions may be quite considerable. There is no reason to
suspect that the biodegradation potential in different metabolic
zones will necessarily be similar. This potential will be based on
the energetics associated with the dominant redox processes,
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AEROBIC
HETEROTROPHIC
RESPIRATION
SULFATE-REDUCTION
METHANOGENESIS
DENITRIFICATION
Figure 1. Changes in Chemical Species and Microbial
Processes in Contaminated Subsurface Material.
(adapted from Bouwer and McCarty, 1984)
the metabolicdiversity of the microbial communities, the immediate
geochemical conditions and the chemical nature of the
contaminant of concern.
In the last 20 years, fundamental developments in the theory,
microbiological techniques, and understanding of anaerobic
processes have occurred. The impact of these devebpments
has been for researchers to reevaluate the types of chemicals
that are subjecttobiodegradationunderanaerobicconditions. In
only the last 10 years, several new anaerobic processes such as
reductive dehalogenation and anaerobic alky) benzene
degradation have been identified. The future will undoubtedly
harbor even more exciting and significant developments.
However, a glimpse of future progress must be firmly rooted in an
assessment of the current state of the art. The following
subsections will hopefully serve to help provide such an
assessment.
Anaerobic Biotransformations
The metabolic pathways utilized by indigenous subsurface
microorganisms for the degradation of environmental
contaminants and natural analogs is an area of intense ecological
research. As noted above, under anaerobic conditions most
organic compounds are degraded by groups of interacting
microorganisms referred to as a consortium. In the consortium,
individual types of organisms carry out different specialized
reactions which, when combined, can lead to the complete
mineralizationofaparticularcompound. The metabolic interaction
between organisms can be complex and may be so tightly linked
under a given set of conditions that stable consortia have been
mistakenly identified as a single species. Without all the individual
members of the consortium, the degradation of the initial substrate
tends to be inhibited. In a hypothetical sutfate-reducing consortium
growing on toluene, the sulf ate-reducing bacteria would probably
not be oxidizing the toluene directly, but rather a product of a
primary or secondary transformation. Together, the reactions
tend to be pulled in the forward direction. An example of the
complexity of a methanogenic consortium is shown in Figure 2.
COMPLEX
ORGANIC
MATTER
T
FERMENTATIVE
BACTERIA
LOW MOLECULAR
WEIGHT ALCOHOLS
AND CARBOXYLIC
ACIDS
ACETOGENIC
BACTERIA
HOMOACETOGENIC
BACTERIA
METHANOGENIC
BACTERIA
ACETATE
Figure 2. Degradation of Organic Material by Methanogenic
Consortia. (adapted from Schink, 1988)
There seems to be several advantages to the evolution of
microbial consortia: 1) This arrangement allows for the creation
of microenvironments where certain types of organisms can
survive in otherwise hostile conditions. 2) Reactions that are
thermodynamically unfavorable can be driven by favorable
reactions when they are metabolically linked within the consortium.
3) Toxic or inhibitory compounds may be removed by resistant
members. 4) This system takes advantage of the diverse
metabolic capabilities of microorganisms by allowing for the
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formation and enrichment of associations that can utilize an
introduced nutrient much faster than a given species could
evolve a novel complex degradation pathway.
Aromatic and Oxygen-Substituted Aromatic
Compounds
Aromatic hydrocarbons are among the most common ground
water contaminants. Although the aerobic biodegradation of
alkylbenzenes has been extensively studied, until recently
anaerobic degradation of these compounds was considered
unlikely (Wilson and McNabb, 1983). New research has shown
that alkylbenzenes such as toluene are degraded under nitrate-
reducing (Kuhn et. al. 1988), methanogenic (Wilson et.al. 1986)
and iron-reducing conditions (Lovely and Lonergan, 1990).
Anaerobic metabolism of toluene appears to require the oxidation
of the methyl group or ring with water serving as the source of
oxygen atoms (Grbic-Galic and Vogel, 1986; Vogel and Grbic-
Galic, 1986). Oxygen-substituted aromatic compounds are
important ground water contaminants in their own right as well as
intermediates of anaerobic alkylbenzene degradation. Phenol,
cresol and benzoic acid appear to degrade under several
anaerobic systems. Table 2 summarizes reports of degradation
of aromatics and oxygen-substituted aromatics in soils and
subsurface material under different anaerobic conditions. Under
aerobic conditions, aromatic degradative pathways tend to feed
into a few common intermediates such as catechol and
protocatechuic acid. From the information available at this time,
it appears that a similar situation may also occur under anaerobic
conditions. Benzoic acid appears as an intermediate in numerous
anaerobic aromatic degradation pathways.
Oxygen, Nitrogen, and Sulfur Heterocycllc
Compounds
Two thirds of the approximately 4 million known organic
compounds have a chemical structure made up of carbon, and
either oxygen, nitrogen or sulfur atoms arranged in a ring. These
compounds are referred to as heterocycles and are widely used
components of, or precursors for, the synthesis of
Pharmaceuticals, pesticides, explosives, dyes and food additives.
These compounds are also found naturally in fossil fuel deposits
and serve in important metabolic roles (such as vitamins, nucleic
acids, proteins and carbohydrates) in living systems.
Many of the heterocyclic compounds which are found as ground
water contaminants are derivatives of pyridines, furans and
thiophenes, but a few saturated heterocycles such as dioxane
are also of concern. Encouragingly, most of these derivatives
were anaerobically biotransformed under methanogenic or sulfate-
reducing conditions (Table 3). Substitution of a carboxyl group
increased the biodegradability of the O, N, and S heterocycles.
These results are similar to those reported for non-heterocyclic
aromatic compounds. In general it appears that oxygen and
nitrogen heterocyclic compounds are more susceptible to
anaerobicdegradationthanthose containing a sulfur heteroatom.
Nitrogen-Substituted and Sulfonated Benzenes
Substituted anilines, benzenesulfonamides and benzamides are
frequent aquifer contaminants. The methylated or higher alky lated
derivatives of these compounds are of particular importance. In
1987, the annual production of aniline alone exceeded 900
million pounds. Recent research has shown that the amino
Table 2. Reported anaerobic biotransformations of aromatic
compound* by subsurface microorganisms or with
pure cultures.
Compound Inoculum
Source*
Toluene
Toluene,
Xylenes
Toluene
Toluene,
Xylenes
Cresols
Phenol,
Benzoale,
Hydroxybenzoate
Phenol,
Cresol,
Benzoate,
Hydroxybenzoate
Phenoxy-
acetate
Methoxy-
benzoate
Hydroxy-
biphenyl
AqM
AqM
PC
AqM
AqM
AqM
PC
AqM
PC
AqM
Incubation
Conditions"
M
NR
NR.IR
NR
M.SR
M.SR.NR
IR.NR
M
An
SR
Reference
Wilson etal.,
1986.
Kuhn etal.,
1988.
Lovely and
Lonergan, 1990.
Hutchins et al.,
1990.
Smolensk! and
Suflita, 1987.
Kuhn et al.,
1989.
Lovely and
Lonergan, 1990.
Gibson and
Suflita, 1986.
DeWeerd et al.,
1988.
Suflita et al.,
1990.
* Source of microorganisms in laboratory biotransformation
experiments; AqM- Aquifer material, PC- Model Pure culture.
b Incubation conditions; M- Methanogenic, SR- Sulfate-reducing, NR-
Nitrate-reducing, IR- Iron-reducing, An- Anaerobic (Fermentation).
substituted benzenes were relatively easily anaerobically
biodegraded when the aromatic nucleus was substituted with a
carboxyl group (Table 4). This is in contrast to aniline and the
methylated anilines (toluidines) which proved recalcitrant under
methanogenic conditions. There were some indications of
anaerobic biodegradation of m-toluidine and perhaps aniline
itself under sulfate-reducing conditions, but the evidence is not
strong. In contrast, aminobenzoic acids can be readily degraded
under nitrate-reducing, sulfate-reducing and methanogenic
conditions.
Benzamides were biodegraded under sulfate-reducing and
methanogenic conditions (Table 5). When properly positioned,
methyl group substituents did not render these derivatives
resistant to biodegradation. However, multiple methyl
substitutionsor complicated alkylation patterns severely inhibited
anaerobic decay (Table 5). Similarly, as a class of compounds,
the aryl sulfonates tend to resist biodegradation under sulfate-
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Table 3. Anaerobic biotransformation of heterocyclic
compounds under methanogenlc conditions.
Table 5. Anaerobic biotransformations of alkylated benzamldes
In aquifer microcosms.
Compound
Chemical
structure
Furan
2-Methylfuran
2-Furoic acid
Thiophene
2-Methylthiophene
3-Methylthiophene
2-Thiophene
carboxylic acid
R
R
H
COOH
H
COOH
Biotrans-
formation*
Pyridine
4-Picoline
3-Picoline
2-Picoline
Nicotinic acid
^\ H
r/^YI CH3 +
K-7rR CH3
>w~^<^ CH
N COOH +
"Loss of test compound and production of methane relative to abiotic
controls during 8 month anaerobic incubation in aquifer derived
methanogenic microcosms. Adapted from Kuhn and Suflita, 1989c.
Compound
Benzamide
N-Methyl-
benzamide
N,N-Dimethyl-
benzamide
p-Toluamide
N,N-Diethyl-
m-toluamide
3 Substituents Biotransformation'
M"
SR<
H H
H H ChL
H CH3 CH3
CH3 H H
CH3 C2H5 C2H5
* Loss of target compound relative to abiotic controls during 11 month
anaerobic incubation.
b Methanogenic aquifer microcosms.
c Sulfate-reducing aquifer microcosms.
Adapted from Kuhn and Suflita, 1989a.
Table 4. Anaerobic biotransformation of amino-substituted
benzenes in aquifer microcosms.
R
group
Biotransformation'
Mb SRC
Compound
Aniline
o-Toluidine
m-Toluidine
p-Toluidine
o-Aminobenzoate
m-Aminobenzoate
p-Aminobenzoate
COOH
COOH
COOH
" Loss of target compound relative to abiotic controls during 10
month anaerobic incubation.
b Methanogenic aquifer microcosms.
0 Sulfate-reducing aquifer microcosms.
Adapted from Kuhn and Suflita, 1989a.
reducing or methanogenic conditions (Table 6). The only exception
was the carboxyl substituted compounds (p-benzosulfonic acid)
which was only partially transformed. Benzenesulfonamide and
the aryl methyl benzenesulfonamide (p-toluenesufonamide) were
degraded under methanogenic conditions. As in the case of
benzamides, complicated patterns of alky lation inhibited anaerobic
biotransformation.
Halogenated Compounds
Chlorinated aliphatic and aromatic compounds are undoubtedly
some of the most pervasive and troubling ground water
contaminants known. Such compounds may be toxic in bw
concentrations, carcinogenic and tend to resist aerobic
degradation. However, recent research has shown that anaerobic
microorganisms can transform many such compounds. They do
so by catalyzing a reductive dehalogenation process (Suflita et
al. 1982). During a reductive dehalogenation reaction, a
chlorinated compound (for instance) acts as an electron acceptor
in a novel type of anaerobic microbial respiration. The chloride
moiety is removed from the molecule and replaced by a hydrogen
(Vogel et al., 1987). Such a process usually renders the resulting
compound more susceptible to subsequent transformations. Of
course this reduction must be coupled to the oxidation of an
electron donor. Recent research suggests that the required
reducing equivalents may be supplied in a variety of forms
(Gibson and Suflita, 1990). With chlorinated aromatics, in some
cases, the compound can serve as both donor and acceptor, but
with other compounds such as chloroethenes another source of
reducing equivalents must be supplied. For their part, the
bacteria likely gain energy during the process of reductive
dehalogenation. Such reactions then form a promising scientific
foundation upon which to build novel bioremediation strategies.
Chlorinated aromatic compounds are widely used components
or synthetic precursors of solvents, pesticides, plastics and many
other products of modern society. To date, over 30 different
mono-, di-, tri-, and tetrahalogenated aromatic compounds have
been tested for their susceptibility for anaerobic decay in aquifer
microcosms. These compounds belonged to several chemical
classes including the benzoates, phenols, phenoxyacetates,
anilines, and a nitrogen heterocyclic compound. The list included
numerous priority pollutants, several pesticides (2,4-D; 2,4,5-T;
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Table 6. Anaerobic biotransformation of benzenesulfonic acids
and benzenesulfonamldes in aquifer microcosms.
n Substituents Biotransformation1
R R
Compound 1 2 Mb SRC
Benzenesulfonic acid H
Orthanilic acid NH2(o)
Metanilic acid NH2(m)
Sulfanilic acid NH2(p)
p-Toluenesulfonic
acid CH3
p-Phenolsulfonic
acid OH
p-Benzosulfonic
acid COOH
Benzene-
sulfonamide H
p-Toluene-
sulfonamide CH3
N-Methylbenzene-
sulfonamide H
N,N-Diethyl-p-toluene-
sulfonamide CH3
N-Ethyl-p/m-toluene-
sulfonamide CH3
OH
OH
OH
OH
OH
OH
OH + +
NH2 ±
NH2
NHCH3
N(C2H5)2 -
NH(C2H5) -
• Loss of target compound relative to abiotic controls during anaerobic
incubation (13 months for benzenesulfonic acids and 10 months for
benzenesulfonam ides).
b Methanogenic aquifer microcosms
c Sulfate-reducing aquifer microcosms.
Adapted from Kuhn and Suflita, 1989a.
bromacil) and chemicals known to be recalcitrant under aerobic
conditions. Of the haloaromatic test chemicals, only four proved
recalcitrant in anoxic aquifer microcosms. The others were
reductively dehalogenated. For most compounds, the
dehalogenation process continued in a sequential manner until
all halogens were removed. Others were converted to products
that were more susceptible to subsequent aerobic microbial
metabolism. However, it is important to note that reductive
dehalogenation reactions were confined to methanogenic
microcosms and did not occur in sulfate-reducing microcosms.
To illustrate with a model compound, when 2,4,5-
trichlorophenoxyacetate (2,4,5,-T) was incubated in aquifer
sample from a methanogenic site and from a nearby sutfate-
reducing site, the pesticide was dehalogenated only in the former
incubations (Gibson and Suflita, 1990). Additional studies
indicate that the requisite dehalogenating microorganisms were
present at the sulfate-reducing site, but their metabolic activity
was controlled, at least in part, by the high levels of sulfate
(Gibson and Suflita, 1986). In consistent fashion, the addition of
sulfate to microcosms made from the methanogenic aquifer
material severely inhibited but did not completely preclude the
dehalogenation of 2,4,5-T. The same types of findings were
obtained when other haloaromatic compounds were similarly
examined (Kuhn et al., 1990; Kuhn and Suflita, 1989b).
Slightly contrasting findings were obtained when the fate of
halogenated aliphatic compounds were examined in comparable
aquifer microcosms. Halogenated aliphatic compounds are
widely used as cleaning and degreasing agents. Chloroethenes
are among the most common and tightly regulated of ground
watercontaminants. Comparisonsofthefateoftetrachloroethene
(PCE) in methanogenic and sulfate-reducing aquifer systems
indicate that PCE and trichloroethene (TCE) undergo reductive
dechlorination under both conditions (Suflita et al., 1988). The
reductive dechlorination of TCE and PCE led to the formation of
intermediates that were more susceptible to aerobic microbial
metabolism. However, while PCE and TCE are suspected
carcinogens, the dehalogenated intermediate vinyl chloride
(monochloroethene) is a known carcinogen. This compound
also tends to be more mobile in the subsurface than the higher
chloroethenes. This fact serves to illustrate the importance of
transport and fate studies for accurate assessment of
environmental risk and damage. The additional dehalogenation
of vinyl chloride would eliminate this risk and has been shown
(Vogel and McCarty, 1985; Freedman and Gossett, 1989).
However, additional research on the environmental factors and
microorganisms which influence reductive dechlorination
processes is needed before anaerobic bioremediation of such
compounds is safe and feasible.
Table 7 is a selected list of halogenated compounds known to be
susceptible to anaerobic biotransformation reactions in soil,
subsurface material, or by pure cultures of microorganisms.
Extrapolation of Metabolic Information
Studies with subsurface microorganisms or microcosms are
extremely useful for exploring the limits of anaerobic
biodegradation potential. It is, of course, important to question
whether information generated in this fashion is environmentally
meaningful. Yet microcosms haveproventhemselvesrepeatedly
as tools for conducting scientifically rigorous experiments with
limited quantities of subsurface material. That is, they afford an
opportunity to conduct replicate trials employing appropriate
negative and positive controls, while avoiding field contamination.
Moreover, microcosms can help researchers and regulators
anticipate whether anaerobic biotransformation is likely, the
conditions under which biodegradation occurs, the metabolic
intermediates that may arise and the ultimate end products. As
such, microcosms can assist researchers in their understanding
of field contamination incidents. Laboratory studies are important
in identifying new or unproven degradative processes and in
defining the environmentalfactors that serve to retard or stimulate
such reactions. The ability to make such determinations is
independent of the inoculum source. Thus it matters little if
subsurface microorganisms prove to be novel or unusual.
Microbial metabolism tends to be a unifying feature of life and
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Table 7. Reported haloorganic anaerobic biotransformations in
subsurface- derived microcosms and pure culture.
Compound Inoculum Incubation Reference
Source8 Conditions'1
CI12-Phenols AqM M
Cl, 2-Benzoates
CI23-Phenoxy acetates
CI^-Anilines AqM M,SR
Bromacil AqM M
Tetrachloroethene, AqM, PC M,SR
Trichloroethene
(PCE, TCE)
PCE, TCE PC M
TCE AqM M
cis 1,2-Dichloroethene
trans 1,2-Dichloroethene
1,1-Dichloroethene
1,2-Dibromoe thane
Tetrachloromethane AqM An
1,1,1-Trichloroethane
(CT, TCA)
Trichloromethane PC An
(CF)
TCA, CT
1,2-Dichloroethane, PC M,SR
CT
Bromoform, CF, CT PC M
Vinyl Chloride AqM An
Suflita et al.,
1988.
Kuhn etal.,
1990.
Adrian and
Suflita, 1990.
Suflita et al.,
1988.
Fathepure et
al., 1987.
Wilson etal.,
1986.
Parsons etal.,
1985
Galli and
McCarty, 1989.
Eglietal., 1987.
Mikesell and
Boyd, 1990.
Barrio-Lage et
al., 1990.
* Source of microorganisms in laboratory biotransformation
experiments; AqM- Aquifer material, PC- Model Pure culture.
b Incubation conditions; M- Methanogenic, SR- Sulfate-reducing,
An- Anaerobic (Undefined).
is no guarantee that it will occur at a given subsurface location.
However, the more environmentally realistic the microcosm, the
greater the ease in extrapolating the resulting information from
the laboratory to the field. Certainly, microcosms allow for the
identification of environmental compartments where conditions
limit or preclude degradative processes. Once the limiting
factors are identified, microcosms provide experimental
opportunities to try and overcome these limitations and to
effectively stimulate desirable metabolic transformations.
In situ Anaerobic Bioremediation
The public and scientific community's increasing awareness of
the impact of contaminants on the environment has been the
impetus for the development and usage of bioremediation type
technobgies for the treatment of contaminants. This awareness
notwithstanding, realization of the costs associated with alternate
remedial measures also stimulates these biotechnological
developments.
Nitrate-reducing based in s/fubioremediation of petroleum-based
fuels, which has been in limited use for a number of years, has
recently undergone a rigorous field evaluation by RSKERL
personnel (Hutchins et al. 1989). Early results suggest that this
approach appears to be a viable technology. A field evaluation
of the'natural"anaerobicbiodegradation processes in agasoline-
contaminated aquifer is currently being conducted under the
direction of Dr. John T. Wilson (RSKERL). It may be that the
methanogenic fermentation of such contaminants will ultimately
prove a useful treatment technology.
For chlorinated compounds anaerobic bacterial remediatbn
designs may prove to be the most cost effective measure
available. Such approaches are among the few to actually
reduce the mass of such contaminants in situ. In low-
permeability aquifers, the indigenous anaerobic population may
be amenable to stimulation by introduction of only small doses of
electron donors (or other required growth factors) and still produce
the desired effects. Anaerobic treatment processes may not
require the alteration of the in situ redox conditions in aquifers
contaminated with complex waste mixtures. Hence, the addition
of oxygen with its inherent problems, limitations and costs can be
avoided. If we can learn to control and harness the reductive
dechlorination process in an effective in situ technology, it will
likely prove to have numerous advantages over existing aerobic
bioremediation and physical-chemical treatment methods.
Conclusion
factors which influence the degradation of contaminants in one
ecosystem, whether on the surface or subsurface, will likely also
impact others. This is demonstrated in studies (Gibson and
Suflita, 1986) in which methanogenic and sulfate-reducing aquifer
systems are compared to sewage sludge and pond sediments for
the degradation of a number of aromatic and chloroaromatic
compounds. Forthe most part, general agreement was observed,
but sludge generally proved to be a poor surrogate inoculum for
aquifer sediments.
Lastly, microcosms inherently incorporate the important site-
specific variables that influence the success orfailure of treatment
schemes. To be sure, the mere demonstration that a particular
degradative process canoccur under certain ecological conditions
Research has shown that anaerobic microorganisms are much
more metabolically versatile than originally believed. Indeed the
major differences between anaerobic and aerobic systems may
not be in whether a particular compound will degrade but what
pathway is involved, which terminal electron acceptor is used,
and what rates of bioconversion can be expected. Results
summarized here are applicable to fate and transport models,
assimilative capacity studies and biotreatment design
considerations. Although the list of compounds that are known
to be degradable under anaerobic conditions has increased
greatly in recent years and steady progress has been made in
delineating the appropriate metabolic pathways, the study of the
microbial ecology of the subsurface, below the rhizosphere, is
still in its infancy. This information gap limits our ability to
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extrapolate these results without careful site-specific
investigations. These considerations serve to identify subsurface
ecology as a prime candidate for core research.
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Quality Assurance Statement
All research projects making conclusions or recommendations
based on environmentally related measurements and funded by
the Environmental Protection Agency are required to participate
in the Agency Quality Assurance Program. This project was
conducted under an approved Quality Assurance Program Plan.
The procedures specified in this plan were used without exception.
Information on the plan and documentation of the quality assurance
activities and results are available from the Principal Investigator.
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