United States
Environmental Protection
Agency
Robert S. Kerr Environmental
Research Laboratory
Ada, OK 74820
Research and Development
EPA/600/S2-90/060 Mar. 1991
v/EPA Project Summary
In-Situ Biotransformation of
Carbon Tetrachloride Under
Anoxic Conditions
Lewis Semprini, Gary D. Hopkins, Dick B. Janssen, Margaret Lang,
Paul V. Roberts, and Perry L. McCarty
This project evaluated the potential
for enhanced in-situ biotransformation
of chlorinated aliphatic solvents under
anoxic conditions. The target test
compound was carbon tetrachloride
(CT). The transformation of 1,1,1-
trichloroethane (TCA) and two chlo-
rofluorocarbons (Freon-11 and
Freon-113) present as background
contaminants in the test zone ground-
water was also evaluated. Laboratory
column studies were performed initially
and confirmed that transformation of
CT was likely under the conditions of
the proposed field tests, and indicated
that chloroform was a product likely to
result from the transformation. In the
field experiments, biostimulation of a
native microbial population in a shallow
confined aquifer was accomplished
through the introduction of acetate as
the electron donor and substrate for
growth, in the absence of oxygen and
the presence of nitrate, which was used
as the electron acceptor. Acetate and
nitrate utilization commenced within a
few days upon the addition of acetate.
The disappearance of CT commenced
2 weeks after active denitrification be-
gan, and the rate accelerated following
nitrate depletion. The appearance of
chloroform as an Intermediate product
coincided with the disappearance of
CT in the 10-week test and represented
approximately 30% of the CT trans-
formed. The laboratory studies sug-
gested that the other major product of
CT transformation by an alternate
pathway was CO}. The other haloge-
nated solvents were also significantly
transformed, but at slower rates than
CT. The percent transformation within
2 meters of travel in the test zone was
as follows: TCA, 15%; Freon-113, 20%;
Freon-11, 68%; and CT, 95%. With all
the halogenated aliphatics observed,
the disappearance commenced some
time after the beginning of active deni-
trification, and the rate appeared to ac-
celerate after the nitrate was depleted,
suggesting that the transformation may
have been mediated by a microbial
subpopulation other than the active
denitriflers. A mathematical model
which included the transport and
transformation processes thought to be
important successfully mimicked the
behavior observed in the field study.
The model results supported the hy-
pothesis that the growth of a secondary
population was responsible for the bio-
transformation, and that different com-
pounds were transformed by the same
process, but at different rates. This
research demonstrates that it is pos-
sible to promote in-sftu biotransforma-
tion of halogenated aliphatics in the
subsurface under anoxic conditions. A
problem confronting the use of anoxic
bioremediation processes is the for-
mation of halogenated intermediate
products.
This Project Summary was developed
by EPA's Robert S. Kerr Environmental
Research Laboratory, Ada, OK, to an-
nounce key findings of the research
project that Is fully documented In a
separate report of the same title (see
^yV) Printed on Recycled Paper
-------
Project Report ordering Information at
back).
Introduction
Chlorinated aliphatic compounds with
one or two carbon atoms are widely used
as solvents, degreasing agents, and inter-
mediates in chemical synthesis. Their
widespread use and uncontrolled disposal
has resulted in the contamination of
groundwater supplies. There is an urgent
need to better understand the behavior of
the contaminants in the subsurface, to
develop methods for monitoring the distri-
bution and movement of the chemicals,
and to clean up contamination once its
extent is delineated. In-situ bioremediation
of contamination by halogenated aliphatics
is a promising alternative for aquifer resto-
ration, since the process may lead to
complete mineralization to non-toxic end
products and/or may create intermediate
products that are less harmful, are more
easily removed from the aquifer, and are
more readily treated by other processes.
This project assessed under field condi-
tions the capacity of native organisms,
i.e., bacteria indigenous to the subsurface
environment, to metabolize halogenated
synthetic organics when the proper condi-
tions were provided to enhance microbial
growth. Reducing conditions were pro-
moted in the field by simulating a consor-
tium of denitrifying bacteria, and perhaps
sulfate-reducing bacteria, through the ad-
dition of acetate as a primary substrate
for growth to the aquifer that contained
both nitrate and sulfate. Under biostimu-
lated conditions the transformation of tar-
get compounds, including CT, TCA,
Freon-11, and Freon-113, was assessed
by controlled addition, frequent sampling,
quantitative analysis, and mass-balance
comparisons. To provide guidance for
the field work, laboratory studies were also
performed to obtain a more basic under-
standing of key microbial and physical
processes involved.
Objectives
The specific objectives of this project
were the following: 1) to demonstrate in a
controlled field experiment the ability to
biostimulate an indigenous population of
denitrifying bacteria under conditions rep-
resentative of groundwater environments;
2) to quantify the extent of enhanced bio-
degradation of CT, 1,1,1-TCA, Freon-11,
and Freon-113 in the biostimulated zone,
and the formation of intermediate prod-
ucts; 3) to determine how to modify
biostimulation conditions to achieve more
complete mineralization of the halogenated
aliphatics; 4) to evaluate laboratory proce-
dures for simulating field results; and 5) to
use mathematical models that incorporate
key microbial and transport processes for
interpreting the results of laboratory and
field experiments.
Field Demonstration
Methodology
A methodology was developed to
evaluate objectively and quantitatively the
effectiveness of the approach for stimulat-
ing anoxic microbial growth in order to
transform the target organic compounds
under natural conditions at the field site.
The methodology entails creating a flow
field dominated by pumping from an ex-
traction well, while introducing solutes in
known amounts at a nearby injection well
and by measuring concentrations regu-
larly at the injection, extraction, and inter-
mediate observation points. Evidence of
transformation was then assessed by
quantitative examination of the concen-
tration histories of the various solutes at
the several monitoring points, and com-
paring results under biostimulation condi-
tions with results obtained under similar
conditions in the absence of biostimulation
measures. A specially designed auto-
mated data acquisition and control sys-
tem provided continuous records of high-
accuracy data over sustained periods,
which enabled mass balances to be made
with relative errors of only a few percent.
Site Characterization
The Moffett Field Naval Air Station,
Mountain View, CA, site chosen for this
demonstration was used earlier to study
in-situ restoration of chlorinated aliphatics
by methanotrophic bacteria (EPA/600/S2-
89/033), and has been well characterized.
The site is characteristic of typical ground-
water contamination, where a shallow
sand-and-gravel aquifer is contaminated
by chlorinated compounds widely used as
solvents. Drilling logs revealed that the
shallow aquifer of the test site consisted
of a layer of sand and gravel, approxi-
mately 5 m below the surface and 1.2 m
thick, well confined above and below by a
silty clay layer of low permeability. The
transmissivity of the test zone is high (ap-
proximately 100m2/day), which permits
extraction of water at the design rate (ap-
proximately 101/min) without excessive
drawdown at the extraction well.
The formation groundwater was also of
appropriate composition for the field ex-
periments. The dissolved oxygen con-
centration was below detection. Nitrate
and sulfate, two potential electron accep-
tors, were present at concentrations of
25 mg/l (as nitrate) and 700 mg/l (as sul-
fate). The groundwater was contaminated
with TCA (50 jig/1), Freon-113 (6 u.g/1), and
Freon-11 (3 u,g/l). The target compound,
CT, was not present and therefore was
continuously added in a controlled man-
ner to the injected water. The other halo-
genated aliphatics that were present in
the extracted groundwater were reinjected
along with CT into the test zone. There
were no appreciable amounts of toxic
metals. Both nitrate and phosphorus,
naturally present in the subsurface, served
as sources of N and P so that their addi-
tion was not required during biostimulation
of the test zone.
The schematic of the test zone, includ-
ing the injection, extraction, and monitor-
ing wells, is shown in Figure 1. Tracer
experiments were performed along the two
legs to determine whether the north leg
(Nl, N1, N2, N3, P) or the south leg (SI,
S1, S2, S3, P) was best suited for the
biostimulation-biotransformation experi-
ments. Under the induced gradient condi-
tions of injection and extraction, only 80%
of the bromide was recovered at the ex-
traction well when injected into the Nl
well, while over 90% was recovered when
it was injected into the SI well. A strong
regional flow from north to south caused
the lower recovery with the north leg, and
so the south leg was used for the
biostimulation experiments.
The south experimental leg had been
used previously for bioremediation stud-
ies using methanotrophic bacteria, an
aerobic treatment process. Thus, in using
the same experimental leg, a determination
was possible of whether both aerobic and
anoxic transformation processes could be
enhanced in the same test zone.
A tracer test was performed along the
south experimental leg to study the rela-
tive rate of transport of CT and a bromide
tracer under the induced gradient condi-
tions created by continuous injection and
extraction. The test determined the ex-
tent to which CT was retarded in its
transport, and also served to indicate
whether substantial losses of CT occurred
in the test zone before it was biostimulated.
This was necessary to assure the validity
of the experimental approach and to
quantify the extent of biotransformation
before and after the test zone was
biostimulated. The hydraulic residence
times (Table 1) between the injection and
the three observation wells, S1, S2, and
S3, were found to be in the range of 8 to
28 hrs. CT residence times were longer
due to sorption onto the aquifer solids and
ranged from 12 to 57 hrs. The resulting
retardation factors ranged from 1.5 to 2.0.
CT was much less strongly sorbed than
cis- and trans-dichloroethylene (DCE) and
trichloroethylene (TCE), whose retardation
-------
Sampling
Injection Wells
Well •
Extraction
Well
Sampling
Wells Injection
Well
2-
4-
Cl
San
\
A
C
ay
d and
\ Gravel
'SS
1
'////>
/xxx
'/S/S/S
l/////////////'//////'/r/r//r///rs///////////s/s/s/
ay
SI S1 S2 S3 P N3 N2 N1 Nl
9 I I 1 I 1 1 1 1 1 1 1 1 1
4 6
Distance from Well SI, m
10
12
;igure 1. Sc/7emaf/c of the injection system.
fable 1. Results of Tracer 14 Test
Well S1
Well 52
Well S3
formalized
3r Breakthrough (C/C0)
formalized
DT Breakthrough (C/C0)
Fime to 50% Br
Breakthrough (hr)
fime to 50% CT
Breakthrough (hr)
1.00
0.98
8
12
0.98
0.99
24
44
0.94
0.98
28
57
Estimated Retardation
Factor T/T
1.5
1.8
2.0
factors in previous studies ranged from 6
to 12.
The tracer test also confirmed that the
injected fluid completely permeated the
test zone around the S1 and S2 wells, as
indicated by the normalized breakthroughs
of near unity (Table 1). CT also reached
a normalized concentration near unity, in-
dicating minimal transformation and sorp-
tion losses with prolonged injection. A
minor amount of chloroform (CF) produc-
tion was observed early upon CT addi-
tion, with the maximum CF concentration
representing 3 to 4% of the CT added.
Thus, minor CT transformation was ob-
served before biostimulation of the test
zone through acetate addition.
Laboratory Studies
Sorptlon
Batch sorption studies were performed
on pulverized aquifer solid samples. A
linear sorptbn isotherm was measured that
yielded a KD estimate of 1.0 I/kg. The es-
timated retardation factor based on the
laboratory measured KD value was 6.0, a
factor of 3 greater than that estimated in
the field test. There are several possible
reasons for the higher laboratory estimate.
Pulverization limited diffusional processes
that were likely occurring in the field. Dif-
fusional limited sorption would have re-
sulted in low estimated values of retarda-
tion based on the time to 50% break-
through of CT and bromide used in the
field retardation estimates. The samples
used in the laboratory tests may not have
been representative of those of the test
zone, due to aquifer heterogeneities and
the inability to obtain intact aquifer cores
from the test zone's highly permeable
zone.
Laboratory Column Studies
Batch exchange soil column experi-
ments were performed to determine the
applicability of laboratory results to field
studies. The experiments showed con-
clusively that CT could be transformed to
a significant extent under anoxic condi-
tions, biostimulated through the addition
of a primary substrate for growth. The
columns were batch-fed a range of pri-
mary substrates for growth (ethanol, ac-
etate, methanol, and glucose) that were
added to groundwater from the field site
along with unlabeled and 14C-labeled CT.
Rapid biostimulation of the columns was
observed upon addition of the growth
substrates, with complete nitrate removal
occurring within 10 days. The decreases
in aqueous CT concentrations were more
gradual and occurred over a period of 60
days. CT concentrations were most re-
duced in columns fed acetate or ethanol,
with 80 to 90% removal observed com-
pared to a non-sterile control column to
which no growth substrate was added.
The 14C-labeled CT studies confirmed
the transformation of the CT; 35 to 50% of
the CT added was completely mineralized
to CO2, while 30 to 40% was transformed
to CF. Denitrifying strains from the col-
umn effluent strains did degrade CT.
These pure culture studies suggested that
denitrifiers were not the microbes respon-
sible for the transformations in the labora-
tory columns.
The column studies proved useful as a
means of assessing the effect of
biostimulation as a means of facilitating
transformation of CT under controlled
laboratory conditions. The tests indicated
that acetate would be an appropriate, non-
toxic growth substrate for the field test;
the test zone should be rapidly
biostimulated, but CT transformation was
expected to significantly lag behind the
uptake of nitrate and acetate. Partial min-
eralization of CT to CO2 might be realized
in the field; however, the formation of CF
-------
as an intermediate product was also pre-
dicted. The lag in time before transfor-
mation of CT was observed, combined
with the lack of CT transformation by
denitrifying cultures, indicated that the main
population of denitrifiers was not likely to
be responsible for CT transformation.
Field Demonstration of
Biostimulation and
Biotransformation
The biostimulation and biotransforma-
tion evaluations conducted in the field were
consistent in most major respects with
expectations from laboratory results and
theory. It was confirmed that a native
bacterial community could be rapidly
stimulated by introducing acetate as a
growth substrate into an aquifer that con-
tained nitrate and sulfate as potential
electron acceptors, without any supple-
mental nutrients. In the initial biostimulation
experiment, the utilization of acetate and
nitrate rapidly commenced, with virtually
complete nitrate utilization occurring after
100 hrs of acetate addition. A transitory
buildup of nitrite concentration was ob-
served within the first 60 hrs of addition,
in response to the establishment of deni-
trifying conditions. Clogging of the injec-
tion well and borehole was effectively
controlled by adding the acetate in a high
concentration pulse for a period of one
hour in a 13-hr pulse cycle, while continu-
ously recycling nitrate in the native
groundwater. More than 80 to 90% of the
acetate was consumed within the first
meter of transport. The stoichiometric ra-
tios of nitrate to acetate consumption were
approximately 1 mg NO3 per milligram
acetate, which is lower than the ratio cal-
culated for complete respiration of nitrate
to nitrogen gas, due to the incorporation
of an estimated 40% of the acetate into
cell biomass during biostimulation, consis-
tent with literature reports.
In order to evaluate transformation of
CT, the target organic compound, CT was
continuously injected at a concentration of
40 ug/l until the soil was saturated, as
evidenced by the complete breakthrough
at the monitoring wells (Table 1), in the
absence of acetate addition. CT injection
into the test zone was continued upon the
addition of acetate. CT transformation, as
indicated by a decrease in its concentra-
tion at monitoring locations, and the for-
mation of CF as an intermediate product
significantly lagged behind the uptake of
acetate and nitrate in the test zone (Figure
2). Decreases in CT concentration and
increases in CF concentration were ob-
served after approximately 400 hrs, with
gradual decreases over the 1250-hr pe-
riod that acetate and nitrate were injected
into the test zone. Transformation of CT
and the formation of CF as an intermedi-
ate product were more rapid and more
complete at the S2 observation well, 2
meters from the injection well (Figure 2),
compared to the S1 well, 1 meter from the
injection well. The response indicated
that the most rapid rates of transformation
did not occur in the first meter of transport,
where most of the acetate and nitrate
were consumed, but in the zones further
removed, where significantly less acetate
and nitrate were consumed.
The results indicate that the main deni-
trifying population did not participate in
the transformation process to the same
extent as microbes stimulated further
away. The transformation of CT by
denitrifiers may have been strongly inhib-
ited by the presence of nitrate in the test
zone. Another possibility is that a sec-
ondary mierobial population, living on ac-
etate or decay products of the stimulated
denitrifiers were slowly growing and were
responsible for the transformation. The
growth of this population and/or its trans-
formation of CT may have been inhibited
by the presence of nitrate.
A transient experiment was performed
to study the effect that nitrate had on the
biotransformation, and to determine
whether more effective CT transformation
could be achieved in the first meter of
transport. Nitrate was completely removed
from the injected fluid through use of a
surface bioreactor fed acetate. The tran-
sient test was initiated at 1260 hrs (Figure
3). A significant decrease in CT was
observed over the 300-hr period of the
test. Chloroform concentration increased
to a lesser extent, indicating either that
less was being formed in a parallel trans-
formation pathway or CF was being de-
graded at higher transformation rates.
Before nitrate was completely eliminated
from the test zone, 55 to 67% of the
transformed CT appeared as CF, while
only 30 to 40% was observed after nitrate
addition was terminated. Chloroform was
the main chlorinated intermediate product
found. Dichlorornethane and chloro-
methane, possible intermediate products
of CT transformation, were not detected
at a detection limit of 1 u.g/1.
There was no direct evidence for the
stimulation of sulfate-reducing bacteria or
methanogenic bacteria when nitrate was
completely removed. Neither sulfide nor
methane were detected in groundwater
extracted from the test zone. If sulfate-
reducing conditions were established,
however, reactions with test zone miner-
als may have scavenged sulfide from the
groundwater.
The transformation of background con-
taminants, including Freon-11, Freon-113,
and TCA, was also observed in the
biostimulated zone. The responses of the
halogenated aliphatics were similar to that
of CT, but with slower rates of transforma-
tion (Figures 4 and 5). Rates of trans-
formation were also enhanced when ni-
trate was removed from the test zone.
The degrees of transformation (Table 2),
quantified by normalization to the bromide
breakthrough, were as follows: CT, 70-
97%; Freon-11, 42-75%; Freon-113, 0-
30%; TCA, 5-19%. Of the values cited,
the lower value represents the nearest
observation well and the lower of the 95%
confidence intervals, and the higher value
represents the farther observation wells
and the upper 95% confidence intervals.
As indicated in Figures 4 and 5, steady-
state transformation conditions had not
been achieved by the end of the experi-
ments. Thus these transformation extents
are considered as conservative estimates.
Overall the field results confirmed the
ability of indigenous bacteria to promote
the biotransformation of CT, Freon-11,
Freon-113, and TCA under anoxic condi-
tions. Denitrification was readily accom-
plished through the addition of acetate to
the test zone. The responses indicate,
however, that the main population of deni-
trifying bacteria was not responsible for
the CT transformation, but that a second-
ary population was responsible. CT trans-
formations of 95% or greater were
achieved in the test zone. Chloroform,
however, was produced as an intermedi-
ate transformation product, and accounted
for 30 to 40% of the CT transformed.
Mathematical Modeling
A non-steady-state model that was de-
veloped for simulating the biostimulation
and biotransformation tests proved useful
in interpreting the results of the field ex-
periments. The model accounts for the
basic phenomena of mierobial growth,
electron donor and electron acceptor utili-
zation, biotransformation of the chlorinated
compounds, and the formation of interme-
diate products. The model simulates the
growth and metabolism of two mierobial
populations: a denitrifying population and
a second assumed population that utilizes
the respiration products of the denitrifiers.
The approach adequately simulated the
transient decreases in CT concentration
due to its transformation and the increase
in CF concentration due to its formation
as an intermediate product. Some pa-
rameter adjustments were necessary to
achieve the model fits. The model also fit
well the observed field transformation of
other halogenated aliphatics (Figures 4
-------
Table 2. Estimates of the Degree of Transformation Based on Mean Calculated Values from 1450-
1550 Hrs
Percent Blotransformation
Chemical
Well
Average
95% Confidence
Interval
CT
Freon-1 1
Freon-11 3
TCA
S1
S2
S3
Extraction
S1
S2
S3
S1
S2
S3
S1
S2
S3
74
95
96
93
46
68
72
8
20
18
9
15
9
70-78
94-96
95-97
89-96
42-50
65-71
69-75
0-16
10-30
8-27
5-13
11-19
2-16
and 5), indicating that these transforma-
tions were mediated by the same pro-
cesses, but at different rates.
The rate coefficients determined from
model fits to the field observations were in
the range of those reported in the litera-
ture for microbial transformation under
su If ate-reducing conditions, and for a pure
Clostridium culture. Rate coefficients for
the apparent specific first-order rate con-
stants (in units of literTng cells 1'day1) were
as follows: CT, 0.4; Freon-11, 0.16; CF,
0.08; Freon-113, 0.04; and TCA, 0.01.
There was a factor of 40 difference be-
tween the rate of CT (the most rapidly
transformed) and TCA (the least rapidly
transformed). CF was estimated to be
degraded at a rate five times slower than
CT. These differences in rates are con-
sistent with those reported in the literature.
0.3
o
O
0.2 -
0.1
00
Nifrate (x10-3)
CT
Chloroform
250
500 750
Time (Hours;)
1000
1250
Figure 2. Nitrate, CT, and CF concentration histories at the S2 well for the first 1250 hrs of
biostimulation with acetate.
o
o
200 400
600 800 1000
Time (Hours)
1200 1400 1600
Figure 3. Response of CT and CF at well 51 to nitrate removal from tho injected fluid after 1260 hrs.
The CF values represent net values after subtracting CF concentration present in the
recycled injection water.
Summary
The results of this project showed that
CT was transformed to a significant extent
and at a rapid rate under subsurface con-
ditions in the absence of dissolved oxy-
gen, when a native population was
biostimulated by the addition of acetate in
the presence of nitrate. Chloroform was
formed as an intermediate product. Labo-
ratory column studies, conducted under
similar conditions as the field tests, con-
firmed that a significant amount of CT was
completely mineralized to CO2. Labora-
tory soil column studies also predicted the
responses that were later observed in the
field experiments.
Freon-11, Freon-113, and TCA, back-
ground contaminants in the test zone, were
also transformed to significant extents in
the field. Transformation was more com-
plete after nitrate was completely removed
from the test zone and in zones that lacked
the main population of denitrifiers. The
response observed in the field and in the
laboratory columns indicated that a sec-
ondary microbial population, and not the
main denitrifying population, was respon-
sible for the transformation. Modeling
studies supported the hypothesis of a
secondary population being responsible
for the transformation. The modeling re-
sults were consistent with the hypothesis
that the halogenated aliphatics were
transformed by a similar biological pro-
cess as CT, but at slower rates. The
rates of transformation determined from
model fits to the field response were in
the range of those reported in the literature.
-------
1 2
1.1 -
1 -
0.9
0.8-
0.7-
0.6-
0.5-
0.4-
0.3-
0.2-
0.1 -
0
^* •£ ».^\ *V * A
D CT
A 1.1.1-TCA
0 02 0.4 0.6 06 1 1.2
(Thousands)
Time (Hours)
1 4
1.6
Figure 4. Model simulations and field concentration histories of TCA and CT
at the S2 observation well.
o
y
o
(Thousands)
Time (Hours)
Figure 5. Model simulations and concentration histories of Freon-11 and Freon-113
at the S2 observation well.
T^rU.S. GOVERNMENT PRINTING OFFICE: 19*1 - 548-028/40089
-------
-------
Lewis Semprini, Gary D. Hopkins, Dick B. Janssen, Margaret Lang, Paul V. Roberts
and Perry L. McCarty are with Stanford University, Stanford, CA.
Wayne C. Downs is the EPA Project Officer (see below).
The complete report, entitled "In-Situ Biotransformation of Carbon Tetrachloride Under
Anoxic Conditions," (Order No. PB91-148 346/AS; Cost: $23.00, 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:
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 PAID
EPA PERMIT NO. G-35
Official Business
Penalty for Private Use $300
EPA/600/S2-90/060
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