|
Kevin Finneran, Derek Lovley, and
Ellen Moyer
INTRODUCTION
Many laboratories have been
researching methods to accelerate in situ bioremediation of
methyl tert butyl ether (MTBE) and tert butyl alcohol (TBA).
Most of the focus has been on aerobic bioremediation because
there are several aerobes known to utilize MTBE as a sole
carbon and energy source. However, past research with the
benzene, toluene, ethylbenzene, and xylenes (BTEX) compounds
indicates that anaerobic bioremediation can be just as
effective as aerobic bioremediation (1). The following reviews
anaerobic bioremediation research to date for MTBE and TBA,
with an emphasis on novel strategies for enhanced in situ
remediation.
ANAEROBIC
MTBE BIOREMEDIATION
The source zone of
petroleum-affected subsurface environments is typically
anaerobic. Aerobic microbial respiration rapidly depletes the
oxygen with the influx of large concentrations of electron
donor (1). The anaerobic zone can become quite large in a
short period of time, and depending on the factors controlling
natural attenuation, chemicals can spread downgradient from
the source even after it is removed. The anaerobic
portion of the aquifer develops distinct areas dominated by
specific terminal electron accepting processes (TEAPs). The
competing electron acceptors most prevalent in anaerobic
aquifers are nitrate, Fe (III), and sulfate (1). Oxidation of
BTEX compounds and MTBE is thermodynamically favorable with
all of these electron acceptors. Therefore, free energy values
by themselves cannot explain why these distinct zones arise.
Competition amongst the microorganisms for the electron donors
leads to this TEAP distribution. Nitrate yields the most
energy of the anaerobic electron acceptors, and the
denitrifying organisms can metabolize the electron donors at
concentrations that are too low to sustain the Fe (III)
reducers. When nitrate is depleted then the next most
favorable acceptor is Fe (III). The Fe (III)-reducing microbes
metabolize electron donors at concentrations too low to
sustain the sulfate-reducers. In turn, sulfate-reducers can
out-compete the methanogenic microorganisms. This is how the
TEAP zones arise, and it creates a hierarchy of processes that
can be monitored with existing monitoring wells using
dissolved hydrogen, a key electron donor, as an indicator of
anaerobic respiration (2). Methanogenesis generally dominates
in the area immediately adjacent to the source of petroleum
contamination. Methanogenesis is the least energetically
favorable anaerobic process, and dominates only after nitrate,
Fe (III), and sulfate are depleted. These electron acceptors
are depleted almost immediately within close proximity to the
source because of the high concentration of electron donors
localized in the area.
Adding oxygen to the subsurface
to stimulate the aerobic microbial community is a typical
strategy for accelerating contaminant degradation. However,
this is complicated in the source zone because of the highly
reduced geochemical conditions and heavy biochemical oxygen
demand (3). Oxygen is not very soluble, and adding it to
groundwater is an inefficient process in source zones. In
addition, oxygen that enters the source area can be depleted
by chemically reacting with reduced compounds such as Fe (II).
The newly formed Fe (III) oxides can plug oxygen injection
wells or monitoring wells, further complicating the process.
The anaerobic electron acceptors are more soluble and less
technically difficult to introduce into the subsurface.
Previous results with benzene
indicate that anaerobic bioremediation may be just as
effective as aerobic bioremediation. Benzene was previously
thought to be recalcitrant under all but aerobic conditions,
but it is now generally regarded as susceptible to anaerobic
degradation. Different TEAPs support benzene degradation
including Fe (III) reduction, sulfate reduction, and
methanogenesis (4-6). The successes with benzene provide a
foundation on which to develop MTBE-degradation strategies. In
the case of Fe (III)- and sulfate-reduction the processes can
be stimulated by adding the appropriate electron acceptor.
Several studies indicate that
benzene is degraded with the concomitant reduction of Fe (III)
in contaminated environments. At one petroleum-contaminated
site in Minnesota benzene was degraded under in situ
conditions within the Fe (III)-reducing zone. Upon further
analysis it was found that the bacteria responsible were
members of the Geobacteraceae, known Fe (III) reducers (7).
Several methods for stimulating Fe (III) reduction were
successful in also stimulating benzene degradation.
One of these techniques is the
addition of humic acid substances (HS) to accelerate Fe (III)
reduction. HS are naturally occurring compounds that result
from the breakdown of complex organic matter. Fe
(III)-reducing bacteria can directly reduce HS, and Fe(III),
which is oxidized, can abiotically accept electrons from
reduced HS. The HS become re-oxidized and are again free to
accept electrons in microbial metabolism. As such the HS act
as an electron shuttle between the microorganism and the Fe
(III). This is significant because the prevalent form of iron
in subsurface environments is Fe (III) oxides. It is generally
accepted that bacterial cells must physically contact the
insoluble Fe (III) oxides in order to reduce them. However,
because they are not freely soluble the Fe (III) oxides are
occluded from rapid reduction. Soluble HS stimulates Fe (III)
reduction because of this electron shuttling phenomenon (8),
and increased Fe (III) reduction hastens the degradation of
organic electron donors.
Synthetic electron shuttles
have also been tested because of the recent discovery that
quinone moieties on the HS are responsible for the electron
accepting, and consequently the electron shuttling capacity
(9). One such synthetic HS analog is
anthraquinone-2,6-disulfonate (AQDS). In laboratory
incubations it was shown that AQDS stimulates Fe (III)
reduction to the same extent as naturally occurring HS (10).
Because of its catalytic nature very little AQDS (or HS) is
needed to accelerate Fe (III) reduction. It is constantly
recycled – alternating between variant oxidized and reduced
states.
Because Fe (III) is often the most prevalent electron acceptor
in anaerobic environments, large zones of Fe (III) reduction
develop in gasoline and petroleum-contaminated aquifers. Fe
(III) reduction has the potential to yield large amounts of
free energy, and many microorganisms within the Bacteria and
Archaea are known that reduce Fe (III). Stimulating Fe (III)
reduction with electron shuttling compounds was applied to
MTBE remediation.
One gasoline-contaminated site
in coastal South Carolina was tested for its capacity to
degrade MTBE. This site was characterized by the United States
Geological Survey (USGS), and the MTBE plume was found to be
more extensive than the BTEX plume. Landmeyer et al. set up
laboratory incubations with sediment from the Fe
(III)-reducing zone of this aquifer. No exogenous Fe (III) or
electron shuttling compounds were added. 14C-MTBE was added to
determine if indigenous microorganisms could degrade MTBE.
After seven months 3% of the 14C-MTBE was mineralized to
14CO2. This loss was attributed to Fe (III) reduction, because
oxygen and nitrate were depleted, and there was no significant
Mn(IV) or sulfate (11). This was the first demonstration of
the possibility of anaerobic mineralization of MTBE.
Further studies with the same
sediment in our laboratory demonstrated that MTBE could be
degraded to a much greater extent under Fe (III)-reducing
conditions if HS were added as an electron shuttle (12).
Although Fe (III) chelators have previously been shown to
stimulate anaerobic benzene degradation in similar sediments
(13) the addition of chelators did not stimulate MTBE
degradation. When HS and additional Fe (III) oxide were added
to the sediments, MTBE was degraded rapidly and repeatedly
each time MTBE was introduced. When Fe (III) was depleted MTBE
degradation ceased, but degradation ensued upon adding more Fe
(III) oxide. HS were never re-added to the sediment because
they are constantly recycled. MTBE was also degraded when AQDS
was added along with Fe (III) oxide (12).
Freshwater aquatic sediment
from the Potomac River that had previously been used to
develop a benzene-degrading enrichment (14) was screened for
its capacity to degrade MTBE (10). Uniformly labeled
[14C]-MTBE added to this sediment was oxidized to 14CO2 and
14CH4 if no exogenous electron acceptors were added. Three
times as much 14CO2 was produced as 14CH4 (12). This ratio of
carbon dioxide to methane production suggested that there were
probably several different microbial populations involved in
MTBE degradation in this sediment. When Fe (III) alone, or Fe
(III) plus electron shuttling compounds, was added to the
sediment no 14CH4 was produced, only 14CO2. Mineralization was
rapid under these conditions with almost one-third of the
initial MTBE added converted to carbon dioxide in as little as
60 days (12).
Recently researchers with the
U.S. Environmental Protection Agency have provided evidence
for the degradation of MTBE under methanogenic conditions in
the Elizabeth City aquifer in North Carolina (15). MTBE
concentrations at selected monitoring wells were tested over
the length of the methanogenic portion of a contaminant plume.
Over time the concentration of MTBE decreased, and TBA was
detected in some wells as a metabolite of MTBE. Laboratory
incubations support the possibility of MTBE degradation within
the methanogenic portion of the aquifer (15). Experiments with
14C-MTBE would provide more evidence that this is a viable
process at this site.
The potential for anaerobic
MTBE oxidation coupled to sulfate reduction has not been
studied extensively. Mormile and Suflita published reports of
MTBE degradation to TBA in a single bottle of a triplicate
series in sediment (16). The TBA was not further degraded. The
authors attributed the degradation to sulfate reduction or
methanogenesis. If MTBE could be oxidized with sulfate this
would be an ideal electron acceptor because sulfate has a
greater electron accepting capacity than oxygen, it is freely
soluble, and it will not react with any reduced compounds in
the source zone. Again drawing on benzene bioremediation as an
example, sulfate amendment was tested as a stimulant in
laboratory batch and column benzene degradation studies. It
was so effective at stimulating benzene degradation that it
was eventually used in situ to remediate a
petroleum-contaminated aquifer (5). Future studies should test
MTBE degradation in sulfate-reducing sediment, or in sediment
to which sulfate has been added.
Dissimilatory nitrate reduction
and denitrification are two other anaerobic processes that
have been tested in the past. Yeh and Novak published reports
of anaerobic MTBE degradation under denitrifying conditions in
soil that had been amended with starch and nutrients (17).
However it is uncertain if the conditions were strictly
anaerobic. This study differentiated between “anoxic” and
“anaerobic,” leading to the conclusion that oxygen may have
been present in the denitrifying incubations. Many of the
nitrate-utilizing pathways are carried out by facultative
anaerobes. Borden et al. attribute loss of MTBE at a field
test site to anaerobic degradation, but the laboratory
incubations did not confirm the results (18). Denitrification
was the dominant anaerobic process in the laboratory
incubations, and the only anaerobic process in the aquifer.
However, no direct evidence was presented confirming anaerobic
MTBE degradation.
ANAEROBIC TBA
BIOREMEDIATION
A major concern
about MTBE biodegradation is the possible accumulation of the
intermediate TBA. Several studies have suggested that, under
anaerobic conditions, MTBE is degraded to TBA without further
degradation. Previous research by Yeh and Novak indicated that
TBA was degraded in soil under denitrifying conditions (17).
However, as noted above, conditions may not have been strictly
anaerobic. Studies with aquatic sediment (Potomac River) found
that [14C]-TBA was readily mineralized to both 14CO2 and 14CH4
without any lag. As much as 30% of the [14C]-TBA was recovered
as 14CO2 and 9% as 14CH4. When 900 µmol/kg of unlabelled TBA
was added to sediments it was degraded in the sediment in less
than 50 days (12). The rate of TBA consumption was much
greater than the rate of MTBE degradation. These results
suggest that TBA is unlikely to accumulate under anaerobic
conditions.
IMPLICATIONS
FOR MTBE AND TBA REMEDIATION
Anaerobic strategies are
potential alternatives for source zone remediation when
pump-and-treat technologies are too expensive and aerobic
bioremediation may not be feasible or practical. If successful
these anaerobic strategies could remove MTBE before it
spreads. In contrast, current aerobic bioremediation
strategies are most effective when they are employed far from
the source after the MTBE has migrated.
All of the anaerobic strategies
benefit from being relatively inexpensive and easy to
implement. They require delivering the appropriate electron
acceptor (and in the case of electron shuttling the
appropriate shuttling compound) to the subsurface, and
monitoring groundwater to document the loss of MTBE
downgradient from the injection gallery. All of the
electron acceptors that have been discussed above can be
easily delivered to the subsurface via injection wells. Once
the injection gallery is in place and operational it would
require infrequent monitoring. In some instances,
bioaugmentation with microorganisms capable of anaerobically
degrading MTBE might accelerate the degradative process. For
example, laboratory studies of anaerobic benzene oxidation
coupled to sulfate reduction demonstrated that, in some
instances, the addition of a benzene-oxidizing,
sulfate-reducing consortia enhanced anaerobic benzene
degradation better than the addition of sulfate alone (14).
Bioaugmentation might “prime the pump” to help anaerobic, MTBE-degrading
microbes gain a foothold in the source zone, and increase
initial degradation rates.
More research is necessary to
optimize these anaerobic strategies. Compared to the work that
has been done for anaerobic BTEX degradation very little is
actually known about anaerobic MTBE remediation. However, the
results to date are promising. ENSR International and Lyondell
Chemical Company are currently initiating a project to test
enhanced anaerobic bioremediation using electron shuttling
compounds and Fe (III) at several gas station sites.
When the chemicals are not
immediately impinging on a drinking water source these
enhanced anaerobic techniques may be the best option. They may
allow source zone remediation with minimal oversight. Few
sites have been explored for potential anaerobic communities,
which may degrade MTBE with little environmental manipulation.
Preliminary studies suggest that microorganisms eventually
adapt to anaerobically degrade MTBE. Perhaps techniques for
stimulating this metabolism can be applied to hasten the
process and add another remedial option to a growing list of
solutions.
References
1) Lovley, D. R. Potential for
Anaerobic Bioremediation of BTEX in Petroleum-Contaminated
Aquifers. J. Industrial Microbiol. and Biotechnol.
1997, 18, 75-81.
2) Lovley, D.R.; Chapelle, F.H.;
Woodward, J.C. Use of Dissolved H2 Concentrations to Determine
Distribution of Microbially Catalyzed Redox Reactions in
Anoxic Groundwater. Environ. Sci. Technol. 1994,
28, 1205-1210.
3) Hutchins, S. R. Optimizing
BTEX Biodegradation Under Denitrifying Conditions. Environ.
Toxicol. Chem. 1991, 10, 1437-1448.
4) Lovley, D. R.; Woodward, J.
C.; Chapelle, F. H. Stimulated Anoxic Biodegradation of
Aromatic Hydrocarbons Using Fe(III) Ligands. Nature
1994, 370, 128-131.
5) Anderson, R. T.; Lovley, D.
R. Anaerobic Bioremediation of Benzene Under Sulfate-Reducing
Conditions in a Petroleum-Contaminated Aquifer. Environ.
Sci. Technol 2000, 34, 2261-2266.
6) Weiner, J. M.; Lovley, D. R.
Rapid Benzene Degradation in Methanogenic Sediments from a
Petroleum-Contaminated Aquifer. Appl. Environ.
Microbiol. 1998, 64, 1937-1939.
7) Anderson, R.; Rooney-Varga,
J.; Gaw, C.; Lovley, D. Anaerobic Benzene Oxidation in the
Fe(III) Reduction Zone of Petroleum-Contaminated Aquifers.
Environ. Sci. Technol. 1998, 32, 1222-1229.
8) Lovley, D. R.; Fraga, J. L.;
Blunt-Harris, E. L.; Hayes, L. A.; Phillips, E. J. P.; Coates,
J. D. Humic Substances as a Mediator for Microbially Catalyzed
Metal Reduction. Acta Hydrochim. Hydrobiol. 1998,
26, 152-157.
9) Scott, D. T.; McKnight, D.
M.; Blunt-Harris, E. L.; Kolesar, S. E.; Lovley, D. R. Quinone
Moieties Act as Electron Acceptors in the Reduction of Humic
Substances by Humics-Reducing Microorganisms. Environ. Sci.
Technol. 1998, 32, 2984-2989.
10) Lovley, D.R.; Coates, J.D.;
Blunt-Harris, E.L.; Phillips, E.J.P.; Woodward, J.C. Humic
Substances as Electron Acceptors for Microbial Respiration.
Nature. 1996, 382, 445-448.
11) Landmeyer, J. E.; Chapelle,
F. H.; Bradley, P. M.; Pankow, J. F.; Church, C. D.; Tratnek,
P. G. Fate of MTBE Relative to Benzene in a
Gasoline-Contaminated Aquifer (1993-1998). GWMR 1998,
Fall 1998, 93-102.
12) Finneran, K. T.; Lovley, D.
R. Anaerobic Degradation of Methy tert-Butyl Ether (MTBE) and
tert-Butyl Alcohol (TBA). Environ. Sci. Technol. 2001,
(accepted for publication).
13) Lovley, D.R.; Woodward,
J.C.; Chapelle, F.H. Rapid Anaerobic Benzene Oxidation with a
Variety of Chelated Fe(III) Forms. Appl. Environ. Microbiol.
1996, 62, 288-291
14) Weiner, J. M.; Lovley, D.
R. Anaerobic Benzene Degradation in Petroleum-Contaminated
Aquifer Sediments after Inoculation with a Benzene-Oxidizing
Enrichment. Appl. Environ. Microbiol. 1998, 64,
775-778.
15) Wilson, J. T.; Cho, J. S.;
Wilson, B. H.; Vardy, J. A. Natural Attenuation of MTBE in
the Subsurface Under Methanogenic Conditions. U.S. EPA,
2000.
16) Mormile, M. M.; Liu, S.;
Suflita, J. M. Anaerobic Biodegradation of Gasoline
Oxygenates: Extrapolation of Information to Multiple Sites and
Redox Conditions. Environ. Sci. Technol. 1994,
28, 1728-1732.
17) Yeh, C. K.; Novak, J. T.
Anaerobic Biodegradation of Gasoline Oxygenates in Soils.
Water Environ. Res. 1994, 66, 744-752.
18) Borden, R. C.; Daniel, R.
A.; LeBrun IV, L. E.; Davis, C. W. Intrinsic Biodegradation of
MTBE and BTEX in a Gasoline-Contaminated Aquifer. Water
Resour. Res. 1997, 33, 1105-1115.
Top |
