*".
United States
Environmental Protection
Agency
Robert S. Kerr Environmental
Research Laboratory
Ada, OK 74820
Research and Development
EPA/600/S2-89/033 Aug. 1989
4>EPA Project Summary
In-Situ Aquifer Restoration of
Chlorinated Aliphatics by
Methanotrophic Bacteria
Paul V. Roberts, Lewis Semprini, Gary D. Hopkins, Dunja Qrbic-Qalic,
Perry L. McCarty, and Martin Reinhard
This protect evaluated the
potential of an innovative approach to
aquifer restoration: enhanced in-situ
biotransformation of chlorinated
aliphatic solvents by a bacterial
community grown on methane under
aerobic conditions. The target
chlorinated compounds were
trichloroethene (TCE), cis- and trans-
1,2-dlchloroethene (DCE), and vinyl
chloride (VC). Laboratory studies
were conducted to improve
understanding of the microbial
growth and transformation rates and
to characterize important transport
properties. In the field experiments,
biostimulation was accomplished by
introducing methane and oxygen into
a shallow, confined, sand and gravel
aquifer to encourage the growth of a
native bacterial community. Methane
utilization commenced rapidly, within
ten days in the first biostimulation
attempt, and within one day in
subsequent biostimulation episodes.
Biotransformation of the target
organic compounds ensued
immediately after commencement of
methane utilization, and reached
steady-state values within three
weeks. The approximate extents of
transformation were as follows: VC,
95%; trans-DCE, 85%; cis-DCE, 40%;
and TCE, 20%.These amounts of
biotransformation were achieved in a
relatively small biostlmulated zone,
with travel distances of 1 to 4 m and
travel times of 8 to 25 hrs.
Mathematical modeling of the
transport and transformation process
confirmed that the behavior observed
in the field demonstration was
consistent with the results of the
laboratory research and theoretical
expectations. This technology has
been demonstrated to be effective In
continuous operation under carefully
controlled condtttens in a real
subsurface environment at small
scale, and is a viable candidate for
consideration at real contamination
sites where conditions are favorable.
This Project Summary was
developed by EPA's Robert S. Kerr
Environmental Research Laboratory,
Ada, OK, to announce key findings of
the research project that Is fully
documented tn a separate report of
the same title (see Project Report
ordering Information at back).
Introduction
The in-situ remediation of aquifers
contaminated with halogenated aliphatic
contaminants is a promising alternative in
efforts to protect and restore groundwater
quality. Approaches based on extracting
the contaminated groundwater by
pumping and subsequently treating
above ground have proven effective for
the restoration of aquifers contaminated
by these compounds, but often entail
great expense as well as a risk of
transferring the contaminants to another
medium, e.g., the atmosphere. To
circumvent these difficulties, in-situ
treatment of the contaminants is being
considered as a potentially favorable
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alternative, with development efforts
centering on promoting biotransformation
of the contaminants.
This project has assessed under field
conditions the ability of native
microorganisms, i.e., bacteria indigenous
to groundwater zone, to degrade
halogenated organic contaminants when
proper conditions are provided to
enhance bacterial growth. Specifically,
the growth of methane-utilizing microbial
communities was stimulated in a field
situation by providing ample supplies of
dissolved methane and oxygen. Under
biostimulation conditions, the
transformation of representative
halogenated organic contaminants,
including trichloroethene (TCE), cis- and
trans-1,2-dichloroethene (cis- and trans-
DCE), and vinyl chloride (VC), was
assessed by means of controlled
addition, frequent sampling, quantitative
analysis, and mass balance comparisons.
To provide guidance for the field work as
well as a firm foundation for
interpretation, and to improve basic
understanding of key microbial and
physical processes, laboratory
experiments were also performed.
Objectives
The specific objectives of this project
were the following: 1) to demonstrate
whether the proposed method is
effective, by conducting controlled
experiments in a regulated natural
groundwater setting; 2) to quantify the
rate of decomposition and to identify
intermediate transformation products, if
any; 3) to determine the factors that
govern biodegradation rates; 4) to bracket
the range of conditions under which the
method is effective; 5) to quantify the
sorption of the chlorinated compounds on
the aquifer solids, and its effect on
transport and exchange between
porewater and solids; and 6) to simulate
the in-situ biodegradation process using
a mathematical model that incorporates
the principal biological and transport
processes, and to develop suitable
models for that purpose.
Field Demonstration
Methodology
An effective methodology was
developed to evaluate objectively and
quantitatively the effectiveness of the
biorestoration approach for stimulating
the growth of the desired bacterial
populations and transforming the target
organic compounds under natural
conditions at a field site. The
methodology entails creating a flow field
dominated by pumping from an
extraction well, while introducing solutes
in known amounts at a nearby injection
well and measuring concentrations
regularly at the injection, extraction, and
intermediate observation points.
Evidence of biotransformation can
then be assessed by qualitative
examination of the concentration histories
of the various solutes at the several
monitoring points, comparing results
under biostimulation .conditions with
results obtained under similar conditions
in the absence of biostimulation
measures. A specially designed,
automated data acquisition and control
system proved capable of providing
continuous records of high-accuracy data
over sustained periods that enabled us to
compute mass balances with relative
errors of only a few percent.
Site Characterization
The site chosen for the field
demonstration, at Moffett Naval Air
Station, offered a near-ideal combination
of characteristics. The site was
representative of a typical situation of
groundwater contamination, in which a
shallow sand-and-gravel aquifer is
contaminated by chlorinated aliphatic
compounds widely used as solvents.
Drilling logs revealed that the shallow
aquifer at the test site consisted of a
layer of sand and gravel, approximately 5
m below the ground surface and 1.2 m
thick, well confined above and below by a
silty clay layer of low permeability. The
solids exhibited a wide size range, with
approximately 70 wt% > 2 mm and 10
wt% < 0.1 mm. The average organic
carbon content of the aquifer solids was
0.11% and the specific surface area was
5 m2/g.
The formation groundwater was also of
appropriate composition for the field
experiments. The water was moderately
saline and was substantially
contaminated by chlorinated organic
compounds, mainly 1,1,1-trichloroethane,
but was devoid of the chlorinated
alkenes--TCE, 1,2-DCE isomers, and
VC--chosen as target compounds for this
study. There were no appreciable
amounts of toxic metals. Both nitrate and
phosphorus were naturally present in the
subsurface in amounts adequate to
support the anticipated biological growth.
Sustained pump tests showed that the
transmissivity was sufficiently high
(approximately 100 m2/day) to permit
extracting water at the design rate
(approximately 10 l/min) without
excessive drawdown at the extraction
well. Extensive tracer tests, conducted
while extracting at 10 l/min, '
undertaken to quantify transport veto'
and residence times in the test
(Table 1). These tracer tests confi
that the aquifer was virtually compl
permeated by the injected fluid ir
observation zone, as evidences
complete breakthrough of bromide t
at the observation wells-Si, 82,
under the chosen experime
conditions. Further, the overall i
balances, comparing the amount
tracer injected and extrac
demonstrated that the tracer recove
the extracted water was essen
complete: after raising the injectior
extraction rates in the second and
years of field work, the amoui
bromide extracted agreed withir
percent with the amount injected (
1). This was necessary to assure
validity of the experimental approac
quantify the extent of biotransformat
the organic solutes by comp.
instantaneous concentrations al
injection and monitoring points, d
steady-state periods after
advection/sorption transients.
The hydraulic residence times (
1) between the injection well and th
nearest observation wells (S1 and
quantified by the tracer tests i
forced-gradient conditions, were fot
be in the range of 8 to 23 hrs.
residence time between the injectio
extraction well was 30 to 40 hrs. "
residence times were later found
suitable for quantifying the transforn
rates of interest in this work.
retardation factors for the organic sc
evaluated from relative mobility
obtained in the field, were in the rar
two to twelve (Table 2).
Laboratory Studies
Sorption
The retardation factors quantifiec
the field data were consistent wil
results of laboratory studies of soi
The sorption of the organic solut
aquifer core samples from the ft
site confirmed that sorption equili
was approximately linear, justifyin
use of a distribution coefficiei
interpreting and reporting the so
equilibrium data. Sorption was str<
for TCE and weakest for VC. amoi
compounds studied. The retan
factors calculated from the labo
sorption data agreed closely with
estimated from the transport experi
conducted in the field. The exti
sorption was approximately equal
grain size fractions, but equilibriur
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Table 1. Comparison of Bromide Tracer Tests Under Induced Gradient Conditions
Injection Rate (I/mm)
Extraction Rate (l/min)
Percent Steady-State Breakthrough
Time to 50% Breakthrough (hrs)
Percentage Recovered at the
Extraction Well
Monitoring
Point1"
S1
S2
S3
Ext
S1
S2
S3
Ext
TR8"
1.36
10.0
100
98
84
13
8
16
20
30
105
TR11
1.5
10.0
102
100
96
14
9
23
27
40
94
TR12
1.5
10.0
100
99
95
15
8
21
27
42
ND
aTR8 = Tracer experiment, etc.
bDistances from injection well to monitoring wells: S1,1.0 m; S2, 2.2 m; S3, 4 m; and Extraction well (Ext), 6 m.
Table 2. Residence Times and Retardation Factors for the Chlorinated Organic Compounds Based on the Time
Required to Achieve 50% Fractional Breakthrough
Experiment
Tracers
Tracerl 1
Tracerl 2
Compound
TCE
trans-DCE
cis-DCE
TCE
trans-DCE
cis-DCE
Vinyl chloride
Well S1
^50%
(hrs)
60
50
30
50
120
45
13
Well S2
^50%
(hrs)
150
115
70
175
280
90
42
R
7
6
3
6
13
5
1.6
R
(S2)
8
7
4
8
12
4
2.0
reached much more slowly in large
grains than in small ones. This finding
Doints out that deviations from sorption
equilibrium owing to rate limitations may
3e an important factor influencing
ransport and biotransformation behavior.
3rowth and Transformation
=?ates
Biotransformation studies of several
[inds were conducted in the laboratory to
:haracterize the populations of
nethanotrophic bacteria at the field site.
"hese included studies with enriched
nixed cultures and isolated pure cultures
irown on nutrient media, as well as
ixperiments with the natural population
irown on aquifer solids under conditions
imulating the field experiments, in batch
xchange soil columns and a
ontinuously fed column.
The experiments with mixed cultures
nriched from Moffett samples evaluated
te ability of populations grown on
several substrates-methane, propane,
and ethylene-to transform TCE as the
target compound. Methane oxidizers
transformed TCE about one hundred
times faster than ethylene oxidizers;
propane oxidizers showed no ability to
transform TCE. Pure cultures of both
methane- and ethylene-oxidizing
organisms were isolated from the
corresponding mixed cultures, and were
shown to be capable of transforming
TCE. Acetylene inhibited both methane
oxidation and TCE transformation,
implying that the methane
monooxygenase (MMO) enzyme was
responsible for both processes.
Experiments with varying methane
concentration revealed that high methane
concentration slpws or stops the
transformation of TCE, presumably
through the competition between
methane and TCE for the MMO enzyme.
The properties of the various cultures
enriched from the Moffett aquifer material
differed somewhat with respect to
transformation rates and the effects of
environmental variables on rates. In
some, but not ail, cultures, TCE
concentrations above 10 mg/l were found
to inhibit the rates of both methane
oxidation and TCE transformation.
Extremely high concentrations of oxygen
(i.e. > 30 mg/l) also exercised a slight
inhibitory effect, Cultures containing
storage compounds (PHB granules) were
able to transform TCE as rapidly in the
absence of methane as in the presence
of low methane concentrations; this
finding illustrates the importance of the
availability of reducing power in
sustaining the normal function of MMO.
Batch soil column experiments with
cultures grown on Moffett solids largely
confirmed the results of the experiments
with cultures grown on nutrient media,
and served to demonstrate the
applicability of the results to the aquifer
at the Moffett site. The experiments
showed conclusively that a native
methanotrophic community could be
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stimulated in a porous medium consisting
of Moffett aquifer material, without the
addition of microbes or nutrients. The
natural system contained sufficient nitrate
and phosphate as nutrient sources; the
column experiments showed that
transformation rates were not enhanced
by supplying additional nitrogen and
phosphorus.
Columns fed methane and oxygen
began to utilize the methane within 7
days, and partial TCE transformation
ensued within 80 days, reaching
approximately 20% after a year. No
significant amounts of intermediate
transformation products of TCE were
found. Mass balances on columns
previously saturated with sorbed TCE
and then purged with water for prolonged
periods, with and without biostimulation,
showed that the TCE was removed from
the solids twice as fast by the
combination of biodegradation and
desorption as by desorption alone. Vinyl
chloride (VC) degraded much more
rapidly than TCE, being removed about
one-half as fast as methane itself. Within
two days, VC degradation was essentially
complete.
The concentration observations from
the column experiments generally
supported the hypothesis of enzyme
competition, and showed tftat methane
should not be present at too high a
concentration. It was further
demonstrated that methane does not
have to be added continuously for TCE
degradation to proceed; TCE
transformation persisted for several days
after methane depletion, and indeed
seemed to be more rapid at very low
methane concentrations.
The caMiton.uo.us JJow column
experiments tctasely simulated the
conditions of the field experiment. Tte
experiments were conducted with
continuous feed of methane and oxygen,
with a hydraulic residence time of one
day, corresponding approximately to the
travel times between the injection well
and the observation wells at the field site.
In the initial biostimulation with methane
and oxygen, substantial methane
utilization commenced 20 days after
beginning the methane feed, increasing
rapidly over the next 5 days to the point
where methane was completely utilized.
Following attainment of complete
methane utilization, transformation of
TCE began, ultimately reaching
approximately 20%. The transformation
of TCE was not improved by raising the
influent methane concentration from 4.5
to 6.5 mg/l. On the contrary, TCE
transformation was improved
substantially (from 22% to 29%) by
temporarily ceasing the methane input for
a period of up to 20 days. The
transformation of trans-DCE under similar
conditions was much greater than that of
TCE (85% vs 22%). Transformation of
trans-DCE in the continuous column
persisted unabated for more than 40
days after the methane input was ceased.
Field Demonstration of
Biostimulation and
Biotransformation
The biostimulation and
biotransformation evaluations conducted
in the field were consistent in most major
respects with expectations based on the
laboratory results and theory.
It was confirmed that a native
community of methane-oxidizing bacteria
could be stimulated by introducing
dissolved methane and oxygen into the
aquifer In proper amounts, without any
other supplementary nutrients. In the
first year's biostimulation experiment, the
population of methane utilizers had grown
to the point of utilizing substantial
amounts of methane within ten days, and
within another five days methane
utilization was complete (Figure 1).
Clogging of the injection well and
borehole could be controlled effectively
by alternately pulsing methane and
oxygen, 0.9. for time periods of 4 and 8
hrs, a strategy which also served to
spread the microbial growth more
uniformly over a larger domain around
the injection point. The ratio of oxygen
consumption to methane consumption
was 2.5 g/g, consistent with literature
data and laboratory results on
methanotrophic metabolism.
In order to ewatate transformations of
the target chlorinated organics, Shey m&ie
added to the injection water (at
concentrations in the range of 50 to 100
ng/l), in the absence of methane, until the
soil was saturated as evidenced by
complete breakthrough at the monitoring
wells. The feed was then supplemented
with dissolved oxygen and methane.
Transformation of the organic target
compounds ensued immediately
following the beginning of methane
utilization, increasing with time as the
bacterial population grew, and ultimately
reaching a steady-state value that
differed among the compounds as shown
in Figure 2 for the third year's
experimental results.
The steady-state transformations
observed during the third year's field
work (Table 3), quantified by
normalization to the bromide fractional
breakthrough, were as follows: TCE,
29%; cis-DCE, 33 to 45%; trans-DCI
to 90%; and VC, 90 to 95%. Of
values cited, the lower end of the r
represents the nearest observation
(1 m distant, 8 hr residence ti
whereas the upper end of the r
represents more distant observ;
points with longer residence times (2
m; 16 to 27 hr). A chlorinated al
present as a background contami
1,1,1-trichloroethane (TCA), was
degraded to any appreciable exter
analysis of water samples during •<
biotransformation of trans-DCE pro
evidence of an intermed
transformation product identifie
laboratory studies to be the epoxii
trans-DCE, which was present in ami
equivalent to a few percent of the p
compound. No other intermei
products were detected.
Termination of the methane feec
followed by cessation of transform
activity on approximately the same
scale as that of organic trans
suggesting that the microbial popi
remained active in the absent
methane for only a short time t
ceasing to transform the target 01
compounds. These results differ
some of the laboratory evidence,
suggests continued activity for I
periods m the absence of methane.
The concentration osciMatioi
response to the alternate pulsii
methane and oxygen did mai
definite signs of methane inhibition:
examination of the concentr
variations showed that the or
compounds were transformed mon
when the methane concentrator
lower.
Employing peroxide as a mee
increasing the electron acceptor
permitted operating at a higher r
methane feed for increased biol
growth, but did not enhance the i
transformation of the target 01
compounds.
Transient experiments in
formate and methanol were subs
for methane, showed that me
inhibition effects could be overcon
higher transformation rates COL
achieved temporarily, i.e., for s
days.
Overall, the field results confirrr
existence of a natural commur
methane oxidizers that cou
stimulated by introducing methai
oxygen, demonstrated that quan
comparisons could confirm the e>
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o
3
s_x
z
O
Z
u
o
z
o
o
MODEL SIMULATION AND FIELD RESULTS
(METHANE AND DO B1OSTIM1 - WELL S2)
200
400
600
TIME (MRS)
Figure 1. Observed methane (+) and DO O responses at the S2 well due to biostimulation of methanotrophs in the first season of
field testing and corresponding model simulations (solid lines). Four-hour and eight-hour alternate pulses of metfjane and
DO were started at 454 hrs.
transformation within five percent, and
showed that substantial transformation of
TCE, cis- and trans-DCE, and VC
occurred within a distance of a few
meters and residence times on the order
of a few days.
Mathematical Modeling
A non-steady-state model developed
for simulating the results of the field
experiments proved extraordinarily useful
in interpreting the results and comparing
with the laboratory data. The model
incorporated advection, dispersion,
sorption with and without rate limitation,
and the microbial processes of substrate
utilization, growth, halogenated aliphatic
transformation, and competitive inhibition.
The transport was simplified by assuming
one-dimensional, uniform flow, as a
computational compromise to permit
more rigorous representation of the
biological processes. Input parameters
were estimated based on the results of
the laboratory research, or on values
from the literature. Only the initial
btomass of methane-utilizing bacteria was
allowed to vary as an unconstrained
fitting parameter.
The model was able to simulate the
dynamic behavior of the biostimulated
system very closely (Figure 1). The
observed transient responses of the
target organic compounds also were
closely matched by the model
simulations (Figure 2), using rate
parameters (Table 4) that were consistent
with the values inferred from rate
experiments conducted in the laboratory.
The transformation rate parameter values
suggest that vinyl chloride and trans-DCE
were transformed about as rapidly as
methane, whereas cis-DCE and TCE
were transformed one and two orders of
magnitude less rapidly, respectively.
Model simulations of the effects of
competitive inhibition and rate-limited
sorption-desorption also agreed well with
the observed dynamic behavior in
response to the pulsed injection of
methane and oxygen, showing substantial
attenuation of the organic solute
concentrations due to both these
processes.
Conclusions and
Recommendations
This project demonstrated
conclusively the efficacy of enhanced in-
situ biotransformation of chlorinated
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0
O
O
O
O
U
O
z
BIOTRANSFORMATION OF VC, T-DCE, C-DCE
(MODEL AND FIELD RESULTS - WELL S2)
20
VINYL CHLORIDE
TIME (HRS)
O TRANS-DCE
Figure 2. The biotransformation response of vinyl chloride, trans-DCE, and cis-DCE to biostimulation in the third test year. Model
simulations include the processes of growth, competitive inhibition transformation of kinetics, and rate-limited sorption-
desorption of the chlorinated organics.
Table 3. Extent of Biotransformation—Third
Field Season
Percent Transformed8
Well
VC t-DCE c-DCE TCE
SI
S2
S3
Ext
85
96
95
87
85
90
90
80
31
41
43
47
10
17
19
10
aEstimated by adjusting for bromide fractional
breakthrough.
alkenes by microbial communities
comprising methanotrophic and
heterotrophic bacteria. It proved easy to
stimulate the growth of the native
population of methanotrophic bacteria by
providing oxygen and methane in the
proper amounts. Once stimulated, the
mixed methane-grown communities
metabolized the target chlorinated
compounds at rates that ranged from
moderately rapid (one to two orders of
magnitude less than the primary
substrate) to very rapid (same order as
the primary substrate). The
transformations appeared to progress
completely to stable, harmless end
products, for the most part, although in
one case a transitory intermediate
product was identified.
Incorporating experimental controls
and quantitative mass balances to the
extent possible is essential for
meaningful experimentation, in the field
as in the laboratory. Strong dyn
forcing is helpful in stimulating po
characteristic responses that ai
identifying mechanisms and in te
hypotheses and mathematical mo
Moreover, the laboratory research
field work reinforced one another ti
extent that the results and conclu
were consonant, and hence pernr
stronger statements regarding
governing mechanisms and rel«
processes than otherwise would
been possible. This kind of synei
expressed itself throughout the
reported here, as the overall picture
one of general agreement betweei
results of the field and the labor
work. The combination of f
laboratory, and modeling studies o
kind can provide a reliable engine
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Table 4. Model Parameters for Simulation of Chlorinated Organics in BiostimS (Figure 2)
Compound
Methane
VC
trans-DCE
cis-DCE
TCE
Kd
(l/mg)
0.0
0.40
1.60
1.90
2.25
o
(d-1)
0.00
0.33
0.33
0.33
0.33
wk',
2.0
2.0
2.0
0.10
0.025
Ks
(mg/l)
1.0
2.0
1.0
1.0
1.0
k/K
(l/mg-sd)
2.0
1.0
2.0
0.1
0.025
Kd = sorption distribution coefficient [l/mg].
a = rate coefficient for sorption [d-1 ].
k = maximum transformation rate [d-1]-
Ks = half-saturation coefficient [mg/l].
scientific basis for evaluating and cometabolize targeted chlorinated consists in large part of the compounds
designing in-situ biorestoration measures. compounds as secondary substrates, for which methanotrophic transformation
merits full consideration for application to has been shown effective in the
This innovative biorestoration rea| aquifer remediation cases. This demonstration phase of the present work:
technology, premised on the ability of technology should be considered as an namely, VC, trans- and cis-DCE, and
methane-oxidizing bacteria to alternative where the contamination TCE.
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Paul V. Roberts, Lewis Semprini, Gary D. Hopkins, Dunja Grbic-Galic, Perry L
McCarty, and Martin Reinhard are with Stanford University, Stanford, CA
94305.
Wayne C. Downs is the EPA Project Officer (see below).
The complete report, entitled "In-Situ Aquifer Restoration of Chlorinated
Aliphatics by Methanotrophic Bacteria," (Order No. PB 89-2T9 992; Cosf:
$28.95, 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:
Robert S. Kerr Environmental Research Laboratory
U.S. Environmental Protection Agency
Ada, OK 74820
United States
Environmental Protection
Agency
Center for Environmental Research
Information
Cincinnati OH 45268
BULK RATE
POSTAGE & FEES f
EPA
PERMIT No. G-3!
Official Business
Penalty for Private Use $300
EPA/600/S2-89/033
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