United States
Environmental Protection
Agency
National Risk Management
Research Laboratory
Ada, OK 74820
Research and Development
EPA/600/SR-99/012
February 1999
&EPA Project Summary
Biotransformation of Gasoline-
Contaminated Groundwater Under
Mixed Electron-Acceptor Conditions
Jeffrey R. Barbara, Barbara J. Butler, and James F. Barker
Introduction
The main objective of the research
was to evaluate nitrate-based
bioremediation as an enhanced
bioremediation technology in a gasoline
source area under controlled,
experimental conditions. Because the
quantity of mass in a source is typically
very large in relation to the g rou ndwater
plume, the source area exerts an
influence on plume longevity, and
therefore on plume remediation
strategies, including natural attenuation.
Consequently, hydrocarbon source
remediation remains an important
groundwaterquality issue. In this study
the soluble, plume-forming aromatic
hydrocarbons, benzene, toluene,
ethylbenzene, xylene isomers,
trimethylbenzene isomers, and
naphthalene (referred to as BTEXTMB)
were designated as the target
compounds.
Electron-acceptor mixtures were
evaluated in an attempt to maximize the
biotransformation of these target
compounds. In addition to NO.,-,
microaerophilic dissolved O2 (2 mg/L or
less) was present in most experimental
treatments to potentially enhance mass
losses of the soluble compounds such
as benzene that are recalcitrant under
denitrifying conditions. Multiple lines
of evidence were gathered to evaluate
this mixed electron-acceptor approach
in the Borden aquifer. These included a
series of laboratory microcosm
experiments, microbial characterization
studies, and afield demonstration. The
in situ demonstration under highly-
controlled, dynamic conditions
provided the most realistic and useful
assessment of this technology.
ThisProjectSummarywasdeveloped
by EPA's National Risk Management
Research Laboratory's Subsurface
Protection and Remediation Division,
Ada, OK, to announce key findings of
the research project that is fully
documented in a separate report of the
same title (see Project Report ordering
information at back).
Experimental Approach
The enhanced bioremediation of
aromatic hydrocarbons in the presence of
NO3~ and mixtures of NO3~ and O2 was
investigated at both the laboratory and
field scales. Prior to the field
demonstration, a series of preliminary
laboratory microcosm experiments were
performed to determine the effects of O2,
NO3~ concentration, and inorganic nutrients
on the biotransformation of neat BTEX in
pristine Borden sand. Following these
experiments, another series of microcosm
experiments was performed with gasoline-
contacted groundwater to determine
whether BTEX biotransformation would be
enhanced under mixed electron-acceptor
conditions (microaerophilic/denitrifying ).
This was done by comparing mixed-
electron-acceptor microcosms with
microaerophilic only and anaerobic,
denitrifying microcosms. These initial
laboratory experiments were performed
with substrate concentrations that
corresponded to a 10x dilution of gasoline-
saturated water (10-15 mg/L total
aromatics). However, after the gasoline
was spilled in the field it became apparent
that aromatic-hydrocarbon concentrations
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would greatly exceed these levels
throughout the treatment cells. An
additional series of microcosm experiments
was therefore performed to evaluate
aromatic-hydrocarbon biotransformation
under conditions more similar to the field.
These experiments included treatments
with gasoline-saturated water(ca. 100mg/L
total aromatics), and were performed with
the American Petroleum Institute gasoline
(API 91-01) used in the field.
The field experiment was performed in
the Borden aquifer. Seventy liters of
API 91-01 gasoline were first released into
two sealed, sheet-piling treatment cells
(2m by 2m) to create gasoline-
contaminated source areas below the
ambient water table. A schematic of the
treatment cells is shown in Figure 1. Six
months after the spills, clean groundwater
amended with different combinations of
electron acceptors was flushed vertically
through the cells to stimulate microbial
activity. Water was flushed continuously
through the cells under steady flow
conditions. The "Nitrate Cell" received a
mixture of microaerophilic O2 and NO3',
and the "Control Cell" received
microaerophilic O2 only. Groundwater
samples were then analyzed for aromatic-
hydrocarbons, added electron-acceptors,
metabolites, and other geochemical
indicators of biotransformation during both
flushing and static periods over a 13 month
period. After this experiment was
completed, cores were collected from the
treatment cells to perform a follow-up
microcosm experiment to confirm the
results obtained in the Nitrate Cell, and to
investigate changes in microbial biomass
and dehydrogenase activity in response to
nearly two years of gasoline exposure.
Experimental Results
Laboratory Experiments
In the preliminary experiments with low
concentrations of benzene, toluene,
ethylbenzene, and the xylene isomers,
mass losses under aerobic and denitrifying
conditions were generally consistent with
results from previous investigations of
Borden aquifer material. All of these
compounds were degradable underaerobic
conditions, but microbial activity was limited
by inorganic nutrients. Under anaerobic,
denitrifying conditions, both toluene and
ethylbenzene biotransformed most
consistently, while the other aromatic
compounds appeared to be recalcitrant.
We did not observe nutrient limitations
under denitrifying conditions; mass losses
were generally small and the minor
assimilatory requirement for N may have
been satisfied by NO3', eliminating the need
for an additional source of supplied N.
In the laboratory, the effect of
microaerophilic O2 was found to depend on
the concentrations of aromatic
hydrocarbons and other carbon
compounds present in the microcosm.
When aqueous concentrations of the
aromatics were low (10x dilution of
gasoline-saturated water), and there were
no other sources of labile carbon, the
mass of O2 in a microcosm was fairly large
relative to the mass of carbon, and aerobic
mass losses were observed. Notably,
however, benzene mass losses were
typically minimal under these conditions.
On the other hand, in gasoline-
contaminated microcosms less extensive
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Figure 1. Injection-system schematic for the Nitrate Cell with selected instrumentation to illustrate positions of injection/extraction wells
and multilevel piezometer ports. The system for the Control Cell was identical except for NCy addition equipment.
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mass losses were observed, presumably
as a result of O2 consumption by other
gasoline hydrocarbons. When aqueous
concentrations of the aromatic
hydrocarbons were increased to gasoline-
saturated levels to better reflect field
conditions, negligible losses of the
aromatics were observed despite rapid
consumption of the microaerophilic O2.
Under these conditions, the mass of O2
was probably too lowto observe any losses,
even if the aromatics were utilized in
preference to othergasoline hydrocarbons,
and abiotic demands were minimal.
In pristine aquifer material, NO3'
utilization was frequently observed under
anaerobic conditions, but consumption was
slow relative to O2, occurring over time
periods on the order of months to years,
and losses were limited to toluene,
ethylbenzene, and less consistently,
/7>xylene. When substrate concentrations
were increased to gasoline-saturated
levels, negligible NO3" utilization was
observed in pristine aquifer material. This
suggested that the indigenous denitrifying
population was inhibited by high aqueous
substrate concentrations. In contrast, after
in situ exposure, denitrifying activity was
apparently not inhibited by gasoline
constituents; NO3'utilization was observed
in microcosms prepared with gasoline-
contaminated aquifer material and
gasoline-saturated groundwater, but
consumption of the labile aromatic
hydrocarbons was not evident. These
compounds did not appear to be the
preferred substrates in this carbon-rich
environment. For example, in microcosms
amended initially with microaerophilic O2,
NO3~, and gasoline-saturated water, the
conditions most similarto those established
in situ, there were negligible aromatic-
hydrocarbon losses under anaerobic,
denitrifying conditions until pure O2 was
added to microcosm headspaces on
incubation day 154 (Figure 2). Overall,
laboratory results with gasoline-
contaminated material showed that
benzene, toluene, ethylbenzene, /7>xylene,
/j-xylene, 1,2,4-trimethylbenzene, and
naphthalene would biotransform readily at
the expense of O2.
In general, under mixed electron-
acceptor conditions the patterns of O2, and
NO3', and aromatic-hydrocarbon
concentrations suggested that O2 and NO3'
were used sequentially; most aromatic-
hydrocarbon biotransformation occurred
within the first few days of incubation, likely
at the expense of microaerophilic O2, with
additional losses of toluene and
ethylbenzene occurring under denitrifying
conditions over longertime periods. When
the initial concentrations of the aromatic
Microaerophilic/ NO3
Gasoline-Saturated
0.9
8
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0.6
0.3
0 30 60 90 120 150 180
Time (days)
- x -Ben --O--TO!
|—a—m+p-Xyl --»--o-Xyl
-Eben
-1,2,4-TMB
Figure 2. Normalized concentrations of
selected aromatic hydrocarbons
in gasoline-contaminated aquifer
material amended with
microaerophilic O2, NO3~, and
gasoline-saturated water. Lines
join means of replicate
microcosms.
hydrocarbons were low, there was a
beneficial effect of dual electron acceptors
in laboratory microcosms: mass losses in
microaerophilic / NO3" microcosms were
more extensive than in comparable
microaerophilic and anaerobic, denitrifying
microcosms. This showed that under
certain conditions the extent of mass loss
could be maximized by the presence of
these two electron acceptors. However,
as mentioned above, in experiments with
gasoline-contaminated aquifer material,
which were more representative of in situ
conditions, mass losses were either very
small or negligible under mixed
microaerophilic/NO3" conditions. These
microcosm results were consistent with
field observations.
Borden Field Experiment
Mean injection flow rates were
250 ml/min and 237 ml/min for the Nitrate
and Control Cells, respectively. The length
of the vertical flow path was about 2 m, and
these rates corresponded to cell residence
times of approximately 9 days. Roughly 20
cell pore volumes were flushed over six
months of continuous operation. The mean
electron-acceptor concentrations in
injected water were 2.3 mg/L O2 and
116mg/L NCy in the Nitrate Cell, and
2.3 mg/L O2 in the Control Cell.
Data from multilevel piezometers showed
that dissolved O2was consumed rapidly to
a non-zero threshold concentration in both
treatment cells. Because dissolved O2was
depleted at sampling ports located 60 cm
below ground surface (bgs), and water
was injected at about 50 cm bgs, O2 was
apparently consumed within the first 10 cm
of the vertical flowpath. On the basis of the
laboratory data, the O2 was utilized primarily
by microbial activity, but it could not be
determined whetherthe aqueous aromatic
hydrocarbons or other gasoline
constituents were serving as substrates.
Utilization in abiotic reactions may also
have occurred in the field. In contrast to
the rapid O2 consumption, NO3~ utilization
was low, but the production of NO2"
suggested that some biological NO3'-
reduction had been induced. Nitrate
concentrations in injection water and
piezometer ports at 60-cm and 180-cm
depths are shown in Figure 3. A mass
balance indicated that only 12% of added
NO3' was consumed over the six-month
flushing experiment. Nitrate concentrations
were also monitored during a period when
the cells were static and the residence time
was much larger. Under these conditions,
complete NO3" depletion was observed over
a time period on the order of 100 days, but
accompanying losses of labile compounds
such as toluene and ethylbenzene were
not evident.
Dissolved aromatic hydrocarbon
concentrations remained near gasoline-
x—Injection Port
.-n--. 60 cm Ports
90 120 150 180
...D... 60cm Ports
0 cm Ports
I
0 30 60 90 120 150 180
Time (days)
Figure 3. Nitrate concentrations in injection
water and 60- and 180-cm bgs
piezometer ports during six
months of continuous injection
into the Nitrate Cell. Plotted
values for60- and 180-cm depths
are means and standard
deviations from five piezometers.
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saturated levels in both cells throughout
the experiment. Concentration trends were
generally consistent with the dissolution of
a multicomponent liquid (i.e., relatively
rapid depletion of soluble compounds such
as benzene), and there was no clear
evidence of preferential removal of labile
compounds from denitrifying activity.
These trends can be seen in the extraction-
well breakthrough curves for the two
treatment cells (Figure 4). After the
experiments were completed cores were
collected from the cells to measure the
mass of BTEXTMB remaining in the
residual gasoline and mass balances were
completed. In terms of total BTEXTMB,
81% and 83% of the initial mass was
recovered in the Control and Nitrate Cells,
respectively, which correspond to roughly
2,500 g of unrecovered mass per cell.
These losses probably resulted from a
combination of physical losses and
systematic error associated with the mass
balance procedure.
Mass balance results for the added
electron acceptors were used to estimate
the amount of aromatic-hydrocarbon mass
loss that could reasonably be attributed to
biotransformation. The results suggested
thatthe mass of microaerophilic O2 injected
-BENZENE 0 TOLUENE D ETHYLBENZENE
-TOTAL XYLENES X — TOTAL TMB - + -NAPHTHALENE
Figure 4.Concentrations of dissolved
aromatic hydrocarbons in
samples collected from
extraction-well ports in the Nitrate
and Control Cells.
into the treatment cells was too low to
observe any losses even if all of the O2 was
consumed in mineralization reactions with
aromatic compounds. Similarly, given the
limited NCy utilization, the mass loss of
compoundsthatare labile underdenitrifying
conditions was likely very small relative to
the amount of mass in the Nitrate Cell.
Consequently, although there was
evidence from metabolite formation that
some aromatic-hydrocarbon biotrans-
formation had occurred, the bulk of the
experimental data indicated that mass
losses from biotransformation were quite
small in both cells. These observations
indicated, therefore, that abiotic gasoline
dissolution was the dominant mass removal
mechanism in both treatment cells.
Conclusions and Implications
Although there was evidence that
microbial activity had been stimulated, the
field and laboratory data indicated overall
that nitrate-based bioremediation was not
an effective source-area remedial
technology in this aquifer. The field
evidence for activity included 1) NO3'
consumption and NO2~ production, 2) O2
consumption, and 3) metabolite production.
However, NCy utilization was slow relative
to the residence time in the treatment cell,
and utilization of labile aromaticcompounds
was not apparent. It is not clear why a large
denitrifying population capable of rapid
aromatic-hydrocarbon biotransformation
did not develop in the treatment cell in
response to extended exposure to
abundant substrate and NO3'. One
possibility is that the denitrifying population
remained relatively sensitive to the
presence of a gasoline phase and
associated high aqueous concentrations.
Dissolved O2 was utilized rapidly in both
laboratory and field experiments,
demonstrating that aerobic activity was
not inhibited, but under microaerophilic
conditions mass losses were limited bythe
quantity of O2 available for reaction, and
possibly by abiotic demand in the field.
Based on the laboratory results, dissolved
O2 may have been used to oxidize
compounds that otherwise would have
been recalcitrant under anaerobic,
denitrifying conditions, but in situ losses
appeared small relative to the mass of
gasoline hydrocarbons in the cells. This
suggests that the partial oxidation of
recalcitrant parent compounds by
microaerophilic O2 was a relatively
unimportant process in this system. In
addition, there was no evidence that other
terminal electron acceptors were being
utilized in the treatment cells. As expected
fromthese electron-acceptortrends, mass
losses were not enhanced in the cell treated
with microaerophilic O2 and NCy relative to
the unremediated control, and effluent
breakthrough curves in both cells were
consistent with concentration trends
expected to result from abiotic gasoline
dissolution.
These conclusions pertain to the specific
experimental system evaluated in this study
(i.e., a recent gasoline spill flushed for a
relatively short period of time and monitored
over a short flow path). It is conceivable
that this approach, particularly with respect
to the effects of microaerophilic O2, would
be more effective during the latter stages
of an enhanced bioremediation project
when source-area concentrations were
lower, or for downgradient plume control
using a reactive wall or other semi-passive
remedial technology. Similarly, although
NO3' utilization was minor over the flow
path evaluated here, adaptation resulting
in the development of a substantial
population capable of degrading TEX may
have occurred with continued exposure.
Based on previous studies in this aquifer,
it is also possible that substantial NO3"
utilization would have occurred further
downgradient (beyond this experimental
system) in the anaerobic core of the plume,
providing a benefit to an enhanced or
intrinsic remediation strategy.
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Jeffrey R. Barbara, Barbara J. Butler, and James F. Barker are with University of
Waterloo, Waterloo, Ontario, Canada N2L3G1
Stephen R. Hutchins is the EPA Project Officer (see below).
The complete report, entitled "Biotransformation of Gasoline-Contaminated Groundwater
Under Mixed Electron-Acceptor Conditions, "(Order No. PB99-139677; Cost: $36.00,
subject to change) will be available only from:
National Technical Information Service
5285 Port Royal Road
Springfield, VA 22161
Telephone: 703-487-4650
The EPA Project Officer can be contacted at:
U. S. En vironmental Protection Agency
National Risk Management Research Laboratory
Subsurface Protection and Remediation Division
P.O. Box1198
Ada, OK 74820
United States
Environmental Protection Agency
Technology Transfer and Support Division (CERI)
Cincinnati, OH 45268
BULK RATE
POSTAGE & FEES PAID
EPA
PERMIT No. G-35
Official Business
Penalty for Private Use $300
EPA/600/SR-99/012
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