~-Potential for anaerobic
contaminated
aquifers
bioremediation
of BTEX in petroleum-
OR Lovley
Department
of Microbiotogy,
University of Massachusetts,
Amherst, Massachusetts
01003, USA
Laboratory incubations of aquifer material or enrichments derived from aquifer material as well as geochemical
data have suggested that, under the appropriate conditions, BTEX components of petroleum (benzene, toluene,
ethylbenzene and xylene) can be degraded in the absence of molecular oxygen with either Fe(III), sulfate, or nitrate
serving as the electron acceptor. BTEX degradation under methanogenic conditions has also been observed. How.
ever, especially for benzene, the BTEX contaminant of greatest concern, anaerobic degradation is often difficult to
establish and maintain in laboratory incubations. Although studies to date have suggested that naturally occurring
anaerobic BTEX degradation has the potential to remove significant quantities of BTEX from petroleum-contaminated aquifers, and mechanisms for stimulating anaerobic BTEX degradation in laboratory incubations have been
developed, further study of the organisms involved in this metabolism and the factors controlling their distribution
and activity are required before it will be possible to design rational strategies for accelerating anaerobic BTEX
degradation in contaminated aquifers.
Keywords:
benzene; toluene; groundwater;
Fe(lll) reduction; sulfate reduction
Introduction
Contamination of shallow aquifers as the result of terrestrial
oil spills and leaking underground storage tanks is a serious
environmental problem. The benzene. toluene. ethyl benrene, and xylene (BTEX) components of petroleum products are of particular concern becauseof their toxicity and
because these compounds are relatively soluble and thus
migrate with the groundwater. Therefore. there is substantial interest in mechanisms which can remove BTEX from
contaminated groundwater. either naturally or under engineered conditions. All BTEX components can be degraded
by aerobic microorganisms and aerobic degradation can
play an impol1ant role in removing BTEX from
groundwater naturally [54]. Ful1hermore. addition of oxygen to contaminated aquifers in order to stimulate aerobic
degradation of BTEX is a common bioremediation practice [32].
Significant portions of petroleum-contaminated aquifers
are anaerobic as the result of microbial respiration consuming the low concentrations of oxygen that can dissolve in
ground water and because rates of reoxygenation as a result
of oxygen diffusion from the atmosphere are relatively
slow. As detailed below. within the last decade it has
become apparent that BTEX can be degraded in these
anaerobic 7.oncsand that such anaerobic BTEX degradation
may be responsible for significant removal of at least some
BTEX components from contaminated groundwater. However. until recently anaerobic BTEX degradation was considered to have only limited usefulnessas a bioremediation
components. was not degraded under anaerobic conditions.
The recent finding that benzene can be rapidly degraded
under
Fe(III)and
sulfate-reducing
conditions
[3.17.39.43,44,45] has enhanced the potential benefit of
anaerobic metabolism to BTEX remediation as it demonstrates that if the appropriate conditions are supplied. becnzene might be removed from anoxic aquifers without the
requirement of adding oxygen. which can be technically
difficult and expensive [32,54]. The purpose of this review
is to summarize recent advances in the understanding of
anaerobic BTEX degradation in petroleum-contaminated
aquifers.
Potential electron acceptors for anaerobic BTEX
oxidation
Potential el~tron acceptors for anaerobic oxidation of
organic matter in subsurface environments include: nitrate,
Mn(IV), Fe(III), and sulfate [36]. With the exception of
Mn(IV), one or more BTEX components can be oxidized
with the reduction of each of these el~tron acceptors, as
detailed below. Although it does not appear to have been
investigated, it is likely that BTEX oxidation coupled to
Mn(IV) reduction is also possible as at least one Fe(lll)
reducer that can oxidize aromatic compounds is known to
use Mn(IV) as an el~tron acceptor for the oxidation of
other el~tron donors [40]. Of these electron acceptors,
Fe(lll) typically provides the greatest potential el~tronaccepting capacity in shallow aquifers [14,33.34]. However, as discussed below, it is possible that nitrate and sulfstrategy because numerous studies (1.4.8.9.22,29.31.32. ate reduction can be important in some instances,especially
50.56J indi.:alcd that benzene. the most toxic of the BTI,:X
if they are added to groundwater
ColTespondc:ncc:: Dr DR Lovlc:y, Dc:par1mc:nlof Microbiology,
of Massachusells. AmhC:~I. Massachu5c:lIs01003, USA
Rc:cc:ived 21 November 1995: accc:plc:d20 Fc:bruary 1996
Univc:rsilY
in order to stimulate
BTEX oxidation coupled to nitrate or sulfate reduction.
Furthermore, some BTEX components may also be
degraded under mcthanogenic conditions. The reduction of
nitrate, Mn(IV). Fe(III), or sulfate as well as methano-
'"
'""
:~
'"
.,
~
~
16
gel1c~i~are referred 10 a~ lerminal eleclron-accepling procc~~c~(TEAPs) [36,371.
In aquifers conlaminated v<ith pctroleum or other organic
p<)llutants such as landfill leachate, the anaerohic TEAPs
arc generally segregated into dislinct zone~ (Figure I)
15.6,11.37,47). Such zonalion is most likely Ihe resull of
Ihe availabilily of electron acceplors and the competition
of microorganisms for electron acceptors [36). In aquifers
that have been contaminated for some time, melhanogenesis typically predominates upgradient. closest to the source
of contamination. This is because near the source of contamination, potential alternative electron acceptors (Fe(III),
sulfate. etc) have already been depleted by BTEX degradation. If Fe(III) or sulfate are available. methanogenesis
is generally excluded because Fe(III) reducers and sulfate
reducersare able to outcompetethe methanogeniccommunity for electron donors [36]. This competition was evident from geochemical data on a petroleum-contaminated
aquifer in Bemidji. Minnesota [35]. It appeared that shortly
after contamination and the development of anaerobic conditions immediately downgradient from the contaminant
source. Fe(III) reduction was the predominant TEAP in the
anaerobic zone. However. as Fe(III) was depleted from the
upgradient sediments closest to the source of the contamination. Fe(III) reduction moved further downgradient and
methanogenesisbecamethe pr~~minant TEAP upgradient.
It seems likely that this progression (Figure 2) is a common
phenomenon in petroleum-contaminated aquifers.
As discussed below. sulfate concentrations in some
groundwaters are sufficiently high that sulfate reduction can
be an important mAP. When Fe(III) is available. Fe(III)
reducers can often outcompete sulfate reducers for electron
donors or sulfate reducersmay preferentially reduce Fe(III)
[15.36]. Thus. when there is significant sulfate in the
groundwater. a sulfate reduction zone can be expected
downgrCldicnl from Ihe sulfale-deplclcd melhanogenic lone
bUI upgradicnl from Ihe zone in which Fe(lll) is slill available for FeCIII) reduClion (Figure I). When presenl. MnCIV)
and/or nilrale-reducing zones are expected to be downgradienl from the Fe(III) reduction zone. This is because FeCIII)
reduction is generally excluded in the presence of MnCIV)
and nilrate due to: I) preferenlial utilization of nitrate and
MnCIV) by Fe(lll) reducers; 2) the ability of nitrate reducers
10 oulcompete Fe(III) reducers for electron donors; and
3) rapid oxidation of Fe(ll) by Mn(IV) prevenling nel
Fe(lll) reduction Unlil Mn(IV) is depleted [36].
The TEAP zones in anaerobic aquifers can sometimes
be roughly discerned through standard geochemical
measuremenlsof loss of electron acceptors (sulfate. nitrate)
from groundwater or accumulation of dissolved endproducts (Fe(II). Mn(II). sulfide. methane). However. reduced
endproducts produced in upgradienl zones tend to persisl
in anaerobic groundwater as it moves further downgradienl
and this can obscure the true TEAP distribut;ions [14.37].
Measuremenls or calculations of redox potential are also
not effective in delineating the TEAP distribution because
there is no consistent relationship between redox potentials
aDd the predominating TEAPs in sedimentary environmeniS [41).
Thus. in the past it has generally been necessaryto obtain
cores from aquifers to accurately defennine the TEAP. One
way in which this can be done is to perfonn incubations
of sediments in which rates of all of the various anaerobic
processes are measured. However. this is labor-intensive
and time-consuming. A simpler method is to add [2-ICC]
acetate to the sediments and monitor the production of
ICCO2and 'cCHc [37]. From the production of radiolabelled
end products and simple geochemical measurements the
predominant TEAP in the sediments can be rapidly and
simply determined (Figure 3).
~~~~'!!~~~~~A~
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:::::-::::-:.:::.~::::::::::
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--,. -~':'-:-- ..
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source
of
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..:'
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.,.,:':,.,:':'.':':'.':.;'.1...:'.'...
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0 0
Typical distribution or anaerobic terminal electron-accepting processes(TEAPs) in a pelroleum-contaminated aQuifer-
~
werobic
bioremediation of BT£X
00 lo..iey
Pristine
71
aerobic
Figure 2
Evolution or TEAP distribution at a pctroleum-<:ontaminated aquirer rollowing the introduction or crude oil.
Is I.CH. the
Methanogenesis
is the TEAP
predominant product?
~
NO
Is 14CO2 production
inhibited by molybdate?
-<9>-
Sulfate reduction
is the TEA?
Y
NO
Are sediments
nitrate-depleted?
Is Fe{lI) present
in the sample?
-e-
Nitrate and/or
Mn(lV) reduction
is theTEAP
--e-
Mn(lV) reduction
is the TEAP
y
Fe(lll) reduction
is the TEAP
Figure 3
Decision trtt
-
~
for detennining the TEAP in sediments with
[2-"CJ-acetate.
A simpler technique for determining the TEAP taking
place in a particular anaerobic zone of an aquifer that does
not require sampling of the sediments has recently been
developed (37). This approach relies on the fact that. under
quasi steady-state conditions in anaerobic sedimentary
environments. there are specific concentrations of dissolved
H2 that are characteristic of methanogenic. sulfate-reducing.
Fe(III)-reducing. and Mn(IV)- and/or nitrate-reducing conditions (37.41). Comparisons of predictions of TEAPs
derived from
measurements of dissolved H..- and direct
,
measurements of TEAPs in laboratory incubations of
aquifer sediments demonstrated that dissolved H2 measurements could accurately predict TEAPs in petroleum-contaminated aquifers in which it was impossible to detennine
the TEAP by measuring concentrations of microbial end
products or by estimates of redox potential [37].
BTEX oxidation coupled to Fe(lIl) reduction
As discussed above, with the development of anaerobic
conditions in petroleum-contaminated aquifers, Fe(Ill) is
generally the most abundant potential electron acceptor for
organic matter oxidation. A pure culture of a toluene-oxidiring, Fe(III)-reducing microorganism known as Geobacrer
metallireducens has been described [35,40,42]. G. metallireducenswas the first organism found to degrade aromatic
hydrocarbons under strict anaerobic conditions. l1~xidi7P_~
toluene directly to carbon dioxide with Fe(lli) as the sole
electron acceptor without the production of detectable
extracellular intennediates. Pure cultures of Fe(III) reducers
capable of oxidizing other BlEX components have not
been described. However, in sediment enrichments of benzene-oxidizing Fe(Ill) reducers, the stoichiometry of benzene oxidation and Fe(lli) reduction was consiStent with
complete oxidation of benzene to carbon dioxide with
Fe(III) serving as the sole electron acceptor [44].
One of the first suggestions that Fe(lli) reduction could
be important for BlEX degradation in petroleum-contarninated aquifers carne from geochemical data collected by
Baedecker and colleagues at the US-Geological Survey's
Groundwater Toxics Site in Bemidji, Minnesota [6,7,35].
These studies demonstrated loss of BTEX components in
the anaerobic portion of the aquifer that could not be attributed to sorption or volatilization. The initial accumulation
of isotopically light carbon dioxide in the anaerobic ground
water suggested that the early anaerobic loss of BTEX
components was the result of their oxidation to carbon
dioxide. Preliminary evidence that at least some of the
BTEX degradation was coupled to Fe(III) reduction was
the finding that Fe(Il) accumulated in the water over time
and that the sediments in the contaminated portion of the
aquifer contained much less Fe(III) than sedimenls from a
nearby uncontaminaled portion of the aquifer. Subsequenl
geochemical modeling suggesled that even in the most contaminated portion of the aquifer, some of which was
already depleted of Fe(III), approximately half of the BTEX
oxidation could be attributed to Fe(IlI) reduction [6].
Anierobic bioremediitionof BTEX
~ l o.tey
Rcccnlly. direct evidence r()r BTEX degradalion in Ihe
FeCIII) reduclion zone or the Bemidji aquifer wa~ oblained
by moniloring Ihe producl!on of "CO~ rrom Iring-'.CIloluene and 1"CI-benzene injecled inlo sedimenl.. incu.
baled under anaerobic condilion~. Bolh aromalic hydrocarbons were rapidly oxidized 10 "CO2 withoul a lag 131.
This i~ thc first documenled in~lance of rapid and exlensive
anaerobic benzene oxidalion in laboratory incubalion\ wilhOUIlong lag periods or exlensive manipulation of Ihc sedimenl. These dala strongly suggesl thaI benzene can be rapidly oxidized to carbon dioxide in the anaerobic ponions
of some aquifers.
Funher geochemical evidence for involvemenl of Fe(lll)
reducers in BTEX degradalion was the disappearance of
BTEX components and the accumulation of Fe(ll) in
ground water at a North Carolina site [12J. In IwO aquifers
contaminated with a variety of organics, including BTEX.
there was significant loss of BTEX components, including
benzene, as the ground waler moved through the zone in
which Fe(lll) reduction was considered to be the TEAP
[46,53J. There was little or no loss of BTEX in the upgradient methanogeniclsulfate-reducing zones.
At a petroleum-contaminated aquifer located in South
Carolina, toluene was degraded in laboratory incubations
of sediments from the Fe(III)-reducing zone, but there was
no degradation of benzene,even after extended incubations
[44J. However, benzene was rapidly oxidized to carbon
dioxide with the reduction of Fe(III) if synthetic Fe(lll)
chelators were added to the sediments [44,45J. The chelators stimulate benzene and toluene oxidation coupled to
Fe(III) reduction by solubilizing Fe(III) [43J. The soluble
Fe(III) is much more accessible to the Fe(III) reducers than
the insoluble Fe(III) oxides that are the natural source of
Fe(III) in aquifer material. Addition of chelated Fe(lII) as
an electron donor in order to stimulate anaerobic degradation of BTEX has been proposed as an alternative to oxygen addition for remediation of petroleum-contaminated
aquifers [44].
Addition of humic acids also stimulates benzene degradation under anaerobic conditions [45]. Although humic
acids can chelate Fe(llI), the primary mechanism by which
they stimulate Fe(ill) reduction and, thus presumably benzene oxidation coupled to Fe(lII) reduction, is nor by solubilizing Fe(III). Rather, the humics act as an electron shuttle
between the Fe(ill)-reducing microorganisms and the
Fe(III) oxides [38J. Fe(III) reducers are able to donate electrons to humics which then can nonenzymatically reduce
Fe(III), regenerating an oxidized form of the humic. Fe(lII)
reducers can conserve energy to support growth from the
electron transport to the humics. Thus, this represents a
novel form of microbial respiralion. It may also have praclical significance as the addition of humics to petroleumcontaminated aquifers in order to stimulate BTEX degradation coupled to Fe(III) reduclion may be environmentally
more acceptable than addition of synthetic Fe(lII) chelalors.
BTEX oxidation coupled to sulfate reduction
Although ground waters in freshwater aquifers are often low
in sulfate, sulfate concentrations may be increased due to
high sulfate concentrations in the rainwater providing
recharge. fertilizer amendmenl\ to \oil\. conlaminatilll\ willi
septage or landfill leachate. or proximity to \alll.\'alt:r
sources 110.19.56.581. Furthermorc. bccause of thc good
solubility of \ulfate. high concentrations of sulfatc f.:lluld
readily bc addcd to an aquifer. if it was found that provi,jon
of this electron acceptor could stimulate BTEX degrada
tion.
A pure culture of a toluene-oxidizing sulfate reducer ha,
been isolated from marine sedimenls (51I. This organism
which has requirements for high concentrations of sodium
and magnesium is unlikely to be important in toluenc oxi.
dation in freshwater aquifers.
However. degradation of toluene and xylenes has been
documented in laboratory incubations of aquifer material
under sulfate-reducing conditions (10,13,19,22.26.501. In
some instances [10,19] oxidation of aromatics was confirmed with radiolabelled tracers, and stoichiometries of
loss of the aromatic and reduction of sulfate were consistent
with sulfate serving as the sole electron acceptor for oxi.
dation. Rates of toluene loss along a groundwater flow path
in the sulfate reduction zone of a petroleum-contaminated
aquifer were consistent with measured rates of toluene oxi.
dation estimated by the injection of (U-14C]-toluene inlo
laboratory incubations of aquifer sediments [13].
Benzene degradation under sulfate-reducing conditions
has also been observed in some instances. An enrichmenl
established with aquifer sediments oxidized benzene to carbon dioxide after lag periods of 30-60 days [17]. Keys to
establishmenl of the benzene-oxidizing enrichmenl were
omitting the addition of toluene and xylenes to the sediments along with the benzene and maintaining benune
concentrations below 200 p.M. Although it was not demonstrated that benzene oxidation was coupled to sulfate
reduction, subsequentstudies with marine sediments demonstrated that be!:tzenecan be rapidly oxidized to carbon
dioxide with sulfate serving as the sole electron acceptor
[39]. A pure culture which can serve as a model for this
metabolism has yet to be isolated.
Very slow rates of 14C-benzeneoxidation to 14CO2were
observed in sediments from the sulfate-reduction zone.of a
petroleum-contaminated aquifer. These slow rates were in
accordance with the finding that there was little removal of
benzene from the groundwater as it moved through the sulfate reduction zone [13].
BTEX degradation under methanogenlc
conditions
Loss of each BTEX component has been observed under
methanogenic conditions in incubations of contaminated
aquifer material [1.18.59.60]. Mineralization of toluene and
o-xylene have been confirmed with radiolabel [18.59) and
stoichiometric production of methane from these substrates
has also been reported [18). Although benzene disappearance from aquifer material under methanogenic conditions
has been reponed [59.60]. the fate of [he benzene that disappeared was not determined and other studies have found
that benzene persists under methanogenic conditions
[1,18.44). Further evidence for the potential for benzene
degradation under methanogenic conditions includes the
finding of partial mineralization of benzene to carbon diox-
-
An~erobic bioremedi~tion 01 BTEX
DII LOvley
~
19
BTEX oxidation coupled to nitrate reduction
Although nilrale i!i typically nOI available in high concen.
trations in pelroleum-contaminated aquifer!i. it could read.
ily be added a~ an electron acceptor. Thu!i. there has been
substanlial investigation into BTEX degradation under
nitrate-reducing conditions in aquifers. Pure cultures of
denitrifying microorganisms capable of oxidizing toluene.
ethyl benzene. and m-xylene have been reponed
(2,16.20,23.52,55) and metabolism of 0- and p-xylenes
have been documented in enrichment cullures (21,27). No
well-substantiated benzene-degrading denitrifying enrichment or pure cultures have been identified.
The potential for degradation of toluene and m-xylene
under denilrifying conditions in aquifers was first demonstrated in laboratory columns of aquifer material (30.31,61 ).
Degradation of other xylenes and ethyl benzene. as well as
funher confirmation of toluene and m-xylene degradation.
have subsequently been observed in laboratory incubations
of aquifer sediments or ground water (4,8.22.28.29.31.
48-50J.
The potential for benzene oxidation under denitrifying
conditions is less clear. The vast majority of studies failed
to detect benzene oxidation under denitrifying conditions
[4.8.22.28.29.31). Although another study (24J has been
cited (49J as evidence for benzene oxidation coupled to
nitrate reduction. no data specifically demonstrating benzene degradation were presented.
To date. the best evidence for benzene oxidation with
the reduction of nitrate are two studies which demonstrated
nitrate-dependent loss of benzene in anaerobic incubations
of aquifer sediments or ground water (48,49J. However.
neither of these studies demonstrated oxidation of benzene
to carbon dioxide or established stoichiometries. of benzene
loss with the expected reduction of nitrate. Thus. anaerobic
benzene oxidation coupled to nitrate reduction is less welldocumented than benzene degradation with other TEAPs.
As an indication of the often fickle nature of benzenedegradation. at one of the sites where nitrate-dependent benzene
loss had been observed (48J. a subsequent study could nOI
repeat this observation (8J.
Conclusions
Perusal of the peer-reviewed literature demonstrates that
there is the potential for all of the BTEX components to
be degraded in petroleum-contaminated aquifers with either
Fe(III), nitrate, or sulfate as the electron acceptor and
degradation has even been observed under methanogenic
conditions. In some instances. especially for ~nzene
metabolism, the evaluation of this metabolism has been at
a relatively superficial level. This is due. in part, to the fact
(hat benzene oxidation is not consistenlly seen in samples
from anaerobic aquifers. Furthermore, in many cases where
benzenc uptake has been observed (here have been no follow-up studies to further investigate this metabolism. Isolation of an anaerobic benzene-oxidizing organism would
greatly benefit research in this area.
Acknowledgements
This research was supported by National Science Foundation grant DEB-9523932 and the American Petroleum
Institute.
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ICnc h~ ml'.:11
2)4.-2(~J
'""Ihan{)~cnic
cullurc'
Appl
of lulucnc and hcn
I:nviron
Microhi<11 ~,
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