United States
                     Environmental Protection
                     Agency
Robert S. Kerr Environmental
Research Laboratory
Ada OK 74820
*
                     Research and Development
                                                        EPA/600/S2-87/096 Jan. 1 988
v°/EPA          Project  Summary
                    A Field  Evaluation of  In-Situ
                     Biodegradation  for  Aquifer
                     Restoration

                    Lewis Semprini, Paul V. Roberts, Gary D. Hopkins, and Douglas M. Mackay
                      The in-situ remediation  of aquifers
                     contaminated with  halogenated ali-
                     phatic compounds, commonly known
                     in water supply as chlorinated solvents,
                     is a promising alternative in efforts to
                     protect ground water.
                      The full report presents the experi-
                     mental methodology and the initial
                     results of a field experiment evaluating
                     the feasibility of in-situ biotransforma-
                     tion  of trichloroethylene  (TCE)  and
                     related compounds. The method being
                     tested relies on the ability of methane-
                     oxidizing  bacteria to degrade these
                     contaminants to stable, non-toxic, end
                     products.
                      The field site is located at the Moffett
                     Naval Air Station, Mountain View, CA.
                     The test zone is a shallow, confined
                     aquifer composed of coarse grained
                     alluvial sediments.
                      This Project Summary  was devel-
                     oped by EPA's Robert S.  Kerr Envir-
                     onmental  Research Laboratory,  Ada,
                     OK. to announce key findings of the
                     research project that is fully  docu-
                     mented in 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, commonly known in
                     water supply as chlorinated solvents, is
                     a promising alternative  in efforts to
                     protect ground water quality.
                      This project aims to assess under field
                     conditions the capacity of native microor-
                     ganisms, i.e., bacteria indigenous to the
                     subsurface environment, to metabolize
                     halogenated  synthetic organic contam-
inants, when proper  conditions  are
provided to enhance microbial growth.
Specifically,   the    growth    of
methanotrophic bacteria is being stim-
ulated in a field situation by providing
ample supplies of dissolved methane and
oxygen. Under biostimulation conditions,
the transformation of representative
halogenated organic contaminants, such
as trichloroethylene (TCE), is assessed by
means of controlled addition, frequent
sampling, quantitative analysis, and
mass balance comparisons.
  The field demonstration study is being
conducted at  Moffett Naval Air Station,
Mountain View, CA, with the support of
the Robert  S. Kerr  Environmental
Research laboratory of the U.S. Environ-
mental Protection Agency (EPA), and
with the cooperation of the U.S. Navy.
To provide guidance for and confirmation
of the field work, laboratory experiments
and analyses are also being conducted,
both at Stanford University's Water
Quality Control Research Laboratory and
at the  Kerr Laboratory.
  The full report summarizes the exper-
imental approach taken  in the field study
and the characterization of the test zone
before the  initiation of the evaluation
experiments.  The  results  of the first
phase  of the  field  evaluation  are
presented.

Research Objectives
  The overall  objective of this work is to
assess the efficacy of a proposed method
for enhancing the in-situ degradation of
the  halogenated aliphatic compounds.
The specific objectives of the field study
are:

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  1.  To demonstrate whether the pro-
     posed  method of promoting the
     enzymatic  decomposition  of TCE
     and related compounds is effective
     under controlled experiments per-
     formed  in-situ,  in an aquifer
     representing conditions typical  of
     ground water environments;
  2.  To quantify the rate of decompo-
     sition and to identify intermediate
     transformation products,  if  any;
     and

  3.  To bracket  the range of conditions
     under which the  method is effec-
     tive, and to establish  criteria for
     dependable treatment  of  a real
     contamination incident.
Field Experiment Methodology
  The basic approach of the evaluation
experiments is to create a test zone  in
the subsurface.  An array of injection,
extraction,  and  monitoring wells  is
installed within a confined aquifer. An
induced  flow  field is created  by the
injection and  extraction  of fluid.  The
chemicals  of  interest for  a  specific
experiment are metered  into a stream
comprising  10 to 15 percent of the
extracted ground water and then rein-
jected. The concentrations of the specific
chemicals  are  monitored  at several
locations, including the injected fluid, the
three monitoring wells, and the extracted
fluid. The spatial and temporal response
of the  chemicals in  the  test zone  is
determined by  frequent  monitoring,
using an automated data acquisition and
control system located at the site.
  The  sequence of  field experiments
using this approach is outlined in Table
1. The  initial experiments study the
transport of bromide ion as a conserva-
tive  tracer. The  experiments determine
fluid residence times in the system, the
degree  of dispersion, and the  recovery
of the injected fluid at the extraction well.
In later  experiments, bromide, dissolved
oxygen   and the chlorinated  aliphatic
compounds of  interest  are  injected
simultaneously.  The retardation factors
of the different chemicals with respect
to bromide, owing  to  sorption, are
determined. The transformation of the
chlorinated aliphatic compounds in these
experiments  is  evaluated based  on
comparisons with the bromide  tracer.
Two criteria are used; (1)  the degree of
steady-state  fractional  breakthrough
achieved at the monitoring wells, and  (2)
mass balances on the amounts injected
Table  1.    Sequence of Experiments and Processes Studied During the First Phase of th
          Field Evaluation
     Injected Chemicals
                                                            Process Studied
(21 Br~+ O2


(3) Br'+TCE + O2
(5)
       02+(nutrients)

       Oi+( nutrients)* TCE
A dvection/Dispersion

Retardation/Dispersio
  fTCA - Elution)

Retardation
  (Transformation)

Biostimulation

Biotransformation
and extracted. These tracer experiments
therefore,  serve as pseudo-controls,
permitting a comparison of the observed
responses before and after the test zone
is biostimulated.
  The   biostimulation  experiments
involve  adding  methane, oxygen,  and
nutrients (if required),  to stimulate the
growth of methane-consuming bacteria
in the test zone. The transient response
of the different  chemical components is
monitored,  as previously discussed. This
experiment determines: (1) how easily
the  methane-oxidizing  bacteria  are
stimulated  and  whether  nutrients are
required, (2) stoichiometric requirements
of oxygen to methane, (3) information on
the kinetics and the rate of growth, and
(4) the areal extent  over which biostim-
ulation is achieved.
  The degree of biotransformation of the
chlorinated aliphatic compound (TCE) is
evaluated in the final stage (Stage 5) of
the program. Known quantities of  TCE
are  introduced  into the biostimulated
zone along with methane,  oxygen  and
bromide. The extent of transformation of
TCE is determined based on both mass
balances and steady-state breakthrough
concentrations  at  monitoring points,
compared  to those of bromide as a
conservative tracer. The results are also
compared with those obtained during the
earlier transport and retardation exper-
iments (Stage 3) before the test zone was
biostimulated.

Field Site Description
  After a  reconnaissance study of  sev-
eral sites, a location at the Moffett Naval
Air Station, Mountain View,  CA,  was
chosen. The experimental site is located
in a region where the ground water is
contaminated   with  several  organic
solutes for which this  biorestoration
method might be applied. Thus, if effec-
tive, the treatment method may have
direct  use in  the  area  where  it  was
evaluated.
                                          Well  logs indicate the  aquifer  i
                                        approximately 1.2 meters thick with a to
                                        4.4 to 4.6  meters below  the groum
                                        surface; the bottom ranges from 5.3  t
                                        5.7 meters  below the  surface. Thi
                                        aquifer  is confined between silty cla
                                        layers. The aquifer consists of fine-  t
                                        coarse-grained sand and appears wel
                                        bedded in most cores. Gravel lenses witl
                                        pebbles up to 2.5 cm in diameter occu
                                        in some cores within the sand layers.
                                          Pump test results indicate  an esti
                                        mated hydraulic conductivity of 100 m/i
                                        (based on  an aquifer thickness  of 1.'
                                        meters). The hydraulic conductivity is i
                                        the range  of values typical for  coars
                                        sand(20-100 m/d), gravel (100-1000 m,
                                        d) and sand-gravel mixes (20-100 m/d
                                        and is consistent with the  particle siz
                                        distributions  measured for  cores  mate
                                        rials taken from the aquifer. The pum
                                        tests  indicated that the site had severe
                                        favorable  hydraulic features:  (1)  hig
                                        transmissivity should permit the require
                                        pumping and injection of fluids into th
                                        test  zone;  (2) loss of permeability  b
                                        clogging due to biological growth  c
                                        chemical precipitation, would be limitec
                                        due to the original high permeability; (;.
                                        the aquifer is semi-confined,  thus th
                                        test zone is fairly well bounded in verticc
                                        direction; and (4) the aquifer was capabl
                                        of supplying ground  water  at  rate
                                        required for the experiments with les
                                        than  one  meter of drawdown  at th
                                        extraction well.

                                        Chemical Characteristics
                                          Analyses of the ground water provide
                                        information on the quality of the  groun
                                        water in the area of the test  zone an
                                        determined  whether  the  aquifer wa
                                        contaminated with chlorinated aliphatic
                                        of interest. Four  volatile organic corr
                                        pounds  were detected. The highe;
                                        concentrations  in the  native groun
                                        water   were   found    for    1,1,1
                                        trichloroethane  (TCA) which is preset

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at a  concentration on the order of 100
//g/l, varying over a range of 56-131 fjg/
I  for analyses conducted over several
months. Trichloroethylene (TCE) was not
detected in these samples.
  The results of the initial inorganic and
organic analyses indicated that the
ground water was of a suitable chemical
composition for  performing the experi-
ments. The chemical composition would
not inhibit the stimulation of the metha-
notrophic bacteria, and it appears feas-
ible  to  inject and transport dissolved
oxygen in the test zone without  undue
consumptive losses.

Aquifer Solids Characterization
  Core samples  of the aquifer material
were obtained to characterize the aquifer
material's physical, chemical, and micro-
biological properties. Some of the core
material was to be used for microbiolog-
ical studies in  the laboratory. Aseptic
procedures were used for obtaining the
cores samples  and  transferring the
materials to storage containers.
  The  acridine  orange-epifluorescence
procedure was used to enumerate the
active bacteria attached to solid samples
from the test zone. The analysis indicated
that  the microorganisms were typically
attached to particles of organic matter.
The  bacterial numbers per gram of dry
solids varied  from 2  x 106 to 39 x 106,
within the range obtained in previous
subsurface investigations. No apparent
trend with  depth was  indicated. The
highest value, however, was observed in
the sand and gravel zone, 17 -  17.5 ft
below the surface, and is believed to be
associated with the high permeability of
this  zone  and a corresponding greater
flux of nutrients.
  The presence  of methanotrophic bac-
teria  was  not  established  using this
enumeration procedure,  since the
method is not type specific. The presence
of methane-consuming bacteria  on
aquifer solids was,  however, demon-
stratred  in  column  experiments con-
ducted at  the Kerr Laboratory. In these
studies, columns were packed with core
solids  obtained from  well  SI.  After
exchanging the  pore water with water
containing methane and oxygen, oxygen
and   methane   consumption  was
observed.  This  study and the bacteria
enumeration study indicated that the test
zone had an  indigenous  microbial pop-
ulation that could  be successfully
biostimulated.
  The  degree of  sorption  of  several
chlorinated  aliphatic  compounds onto
aquifer solid samples was determined in
batch sorption experiments. It was found
that TCA sorbs less than TCE, while PCE
sorbs more strongly. Linear fits resulted
in Kd values of 0.42, 1.4 and 4.0 cm3/
g for TCA, TCE, and PCE respectively.
  The  Kd  values were also  predicted
based on established partitioning rela-
tionships,  according to which Kd  is
dependent on the organic carbon fraction
of the aquifer solids, measured as 0.001
at the Moffett site.  The  predicted Kd
values were 0.266,0.318, and 1.06 cm3/
g for TCA, TCE and PCE, respectively.
  Estimates of the degree of retardation
of the sorbing  solutes  relative to a
nonsorbing solutes were made based on
the retardation factor given by R = 1 +
pt>Kd/n, where pb is the bulk density of
the aquifer  material (g/cm3); n  is the
porosity (cmVcm3); and Kd is the equil-
ibrium distribution coefficient.  The
estimated retardation factors  are pres-
ented in Table 2. Based on these esti-
mates,the movement of TCE through the
test zone  would be  expected to be
retarded  by a factor  of 6.5 to 8.5. This
has important implications for the time
required  to establish steady-state  con-
centrations during  the tests, and  the
effect the sorption process may have on
the biotransformation of the TCE.

The Well Field
   Figure 1  shows the locations of the
wells installed at the test site. The well
field was designed to permit simultane-
ous experiments by creating two test
zones through the injection of fluids at
both the south (SI) and north (Nl) injection
wells, and  extraction at the  central
extraction well (P). The operation of the
extraction well is intended to dominate
the regional flow field in the study area
in an approximation  of radial  flow. The
injection wells are located 6 meters from
the extraction well. The monitoring wells
are located 1.0, 2.2, and 4.0 meters from
the injection wells. This spacing should
result in roughly equivalent fluid resi-
dence times between monitoring  wells
if radial flow conditions exist.
  An automated data acquisition system
has been devised at the site to implement
the field experiments. The system per-
mits the continuous measurement of the
experiment's principal parameters: the
concentrations of the bromide ion tracer,
methane, halogenated  aliphatic  com-
pounds of interest, and dissolved oxygen,
as well as pH. A schematic of the system
is shown in Figure 2. The system is driven
by a microcomputer.  A data acquisition
and  control program (DAC) has  been
designed and  programmed that can be
operated in either manual or automated
mode. Manual mode permits selection of
samples, creation of a sample sequence
for automated operation, calibration of
    13
                                   O
         Scale, meters
 72
                               11
Figure  1.   Layout of the well field at
           the Moffett site.
Table 2.    Measured and Predicted Ka Values for PCE, TCE, and 1,1,1-TCA. and Estimated
           Retardation Factors
Compound
TCA
TCE
PCE
Measured
Sorption^
Coefficient
Ks
(cm/g)
0.42
1.4
4.0
Predicted
Sorption2
Coefficient
Kd
(cm/g)
0.27
0.32
1.06
Retardation3
Factor
R
2.5-3
65-8.5
17-22
 'Based on measured linear sorption isotherm
 2Based on the empirical correlation with water solubility of Kanckhoff et a/. (1979) and the
 measured foc = 0.001
 3Based on Eq 1 .range p*=f1.6-1 9 g/cm3), range n=(0.3-Q 4)

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                             Automated Data Acquisition
                                 and Control System
Figure 2.   Schematic of the automated data acquisition and control system.
various instruments, and  graphing  the
results as the sampling proceeds.
  In order to realize real-time control and
interpretation, measurements are made
continually for several weeks or months
at a frequency of approximately two per
hour. The sampling points are typically
six  in  number, the  mj jcted  fluid,
extracted fluid, three intermediate mon-
itoring points, and the effluent from the
air stripper  (for  monitoring the ground
water discharged to a storm sewer). In
order to obtain precise and reproducible
measurements  during an experiment,
the instruments are calibrated daily.
  A series  of experiments  were per-
formed using the DAC system to study
the transport characteristics of the test
zone under  a variety of flow conditions.
Natural gradient tests were performed in
order  to estimate the ground  water
velocity and direction at the site. Induced
flow  tests  were performed,  in  which
ground water was injected  and extracted,
to  study transport under  conditions
similar to those  used in the biostimula-
tion and biodegradation  stages of  the
experiment. The DAC system was found
to work reliably and generated sufficient
data to observe the transient responses
at observation locations.
   The natural  gradient  tracer tests
indicated that the ground water flow was
primarily in  a northerly direction, with an
average velocity of 2.6 m/d Two induced
flow tracer experiments were performed
under the  conditions used in the later
evaluation experiments, quantifying the
transport  of bromide  ion,  dissolved
oxygen,  and TCE through  the test zone.
Figure  3 shows the response of both
bromide and TCE at the S1 observation
well, during the early stages of the Tracer
5 experiment.  The movement of TCE is
shown to be  retarded with respect  to
bromide, with  a more gradual approach
to the injected  concentration.
  The average  fluid reside nee times from
the injection to the observation wells and
correpsonding fluid velocities  were
estimated based  on the results of the
tracer experiments and  the initial bios-
                             timulation experiment. The average flui
                             residence times  based on the bromid
                             tracer are 7.3 hrs and 16.0 hrs from th
                             injection  well to the S1 and S2 obser
                             vation wells, respectively. This corres
                             ponds to an average fluid velocity of 0.1 •
                             m/hr in  both cases. Methane and D(
                             analyses  were  found  to yield simila
                             residence time  estimates as  obtaine
                             using the bromide. This result  indicate
                             that  these dissolved  gases  are easil
                             transported through the test zone and ar
                             not retarded.
                               The data for  TCA and TCE indicat
                             retardation factors of 1.4 for TCA an
                             5.75 for TCE. Estimates based on the S
                             well  data yield retardation values of 1.
                             and 9.8 for TCA and TCE, respectively
                             The values are in good agreement wit
                             those predicted  from the batch exper
                             ments performed in the laboratory (Tabl
                             2).
                               The results of the tracer experiment
                             demonstrate that reproducible transpoi
                             experiments can be performed in the tes
                             zone. The fluid residence times in the tes
                             zone are  fairly short, about 8 hrs to th
                             first  observation  well to 30  hrs at th
                             extraction well. Owing to the high groun
                             water velocity under natural  gradier
                             conditions, longer transport times are nc
                             possible,  since an  extraction rate of  £
                             least 8  l/min  is  required  to ensur
                              Br and TCE Response—Tracer 5

                                  Observation Well S1
Figure 3.
                                               i	1	1	r
                                              120     140     16(
                           Time Ihrs)
Normalized response of bromide and TCE at theSJ observation well in the Trac
5 experiment.

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effective recovery of the injected fluid at
the extraction well. The tracer experi-
ments indicated recovery of 60 to 75 of
the bromide injected. TCE was recovered
to the same degree as bromide, indicat-
ing negligible loss of TCE. There is some
dilution  of the injected ground water by
the native ground water with the degree
of dilution increasing with distance from
the injection well.
Pulsed Injection
  To enhance the effectiveness of bios-
timulation, it was decided to introduce
the methane  (primary substrate)  and
oxygen (electron acceptor) as alternating,
timed pulses. This decision was reached
based upon consideration of two crucial
requirements: (1)  the need to avoid
clogging of the injection well and bore-
hole i nterface, and (2) the need to achieve
as uniform  a distribution of microbial
growth  as possible throughout a sub-
stantial  portion of the aquifer. Failure to
fulfill the first requirement would cause
loss of hydraulic capacity and premature
termination of our experiments, as the
drastic  chemical  measures such as
chlorination or strong acid treatment that
are customarily employed to rejuvenate
clogged wells would interfere  with
biostimulation. Failure  to satisfy the
second  requirement would lead to con-
ditions  of extremely  high microbial
densities near the injection point and low
bacterial populations elsewhere, which
would not be conducive to secondary
substrate utilization  as  needed to
degrade  halogenated aliphatic  com-
pounds  by methanotrophs.
  It was thought that introducing the two
essential additives, methane and oxygen,
as alternating timed pulses would assure
their separation in the injection well and
borehole,  thus discouraging biological
growth in  that critical  region.  The
methane  and oxygen would then mix
gradually, owing to the action of hydro-
dynamic  dispersion and associated
mixing  processes,  during transport
through the aquifer, stimulating the
growth  of methanotrophic bacteria over
the  zone  in  which  mixing  occurs. In
designing the pulsed injection system,
two  important  variables  had to be
selected:  (1)  the ratio of the individual
pulses of  methane and oxygen, and (2)
the overall pulse length.

Biostimulation and
Biotransformation Experiments
  The biostimulation and biotransforma-
tion experiments in the first (1986) field
season were conducted in two stages.
First, the test zone was biostimulated by
the pulse  injection  of methane and
oxygen into the test zone. After the zone
had been  successfully stimulated, TCE
injection was commenced.
  The injection system uses two counter-
current columns to sorb the methane and
oxygen  to approximately 80 percent
saturation, resulting  in  concentrations
that are approximately 20 mg/l  for CH4
and 32 mg/l for DO. These solutions are
alternately pulsed, with a pulse time ratio
of about 2:1 (methane:oxygen), based on
the stoichiometric requirements. A pulse
timer  permits the ratio  and the length
of the pulses to be  varied.  The other
components  of  the  injection  system
permit the  accurate and continuous
addition of the bromide  tracer and TCE
into the injection stream, the monitoring
of the injection rates, and the sampling
of the injection fluid,  while maintaining
a constant rate of injection.
  The  biostimulation experiment was
performed under same induced flow
conditions as the earlier tracer tests. The
pulse  cycle  for the  injection of either
methane or oxygen  containing ground
water was varied during the  course of
the experiment,  from  less than 1  hr
during start-up to ensure the  pulsing
would not interfere with growth, to a 12-
hr period during the later  stages to
                            distribute growth in  the test zone.  No
                            additional nutrients were added to the
                            ground water.
                              The first signs of consumption were
                            observed in the extraction well and the
                            S3 observation well after approximately
                            200 hrs of injection.  The concentration
                            at the extraction well decreased below
                            the detection  limit  after  300  hrs of
                            injection. Owing  to the  continuous
                            removal  by  microorganisms,   the
                            decrease in DO was  greater the longer
                            the travel paths through the aquifer. As
                            time proceeds, the increase in the growth
                            of microbial population throughout the
                            test zone results in an increase in the
                            DO consumption along the flow path. The
                            methane response was similar to that
                            observed for the DO, which is expected,
                            as methane is the electron donor and
                            oxygen the electron acceptor for microb-
                            ial growth. Figure 4 shows the response
                            of the  methane and  DO  at  the S2
                            observation  well.  The  fairly  rapid
                            decrease in the methane concentration
                            over the  period  of  200 to  400  hrs
                            indicates fairly rapid growth  kinetics
                            typical of aerobic  microorganisms. A
                            significant amount of methane substrate
                            is also incorporated into cells. Based on
                            the concentration values  during  the
                            period  of  350 -  375  hrs,  the ratio of
                            oxygen to methane consumed was 2.25
                            mg  O2/mg CH4, which is significantly
                                Biostimulation Experiment
                                Methane and DO Well S2
  I
                         100
                                         200
                                       Time (Hrs)
                                             300
                                                            400
Figure 4.
The response  of methane and DO at the S2 observation well  during the
biostimulation of the test zone.

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lower than the ratio of 4 required  for
complete  oxidation.  The  lower value
suggests incorporation of the methane
substrate into the cell mass, with a yield
coefficient of approximately 0.5 mg cells
per mg CH4.
  After 450 hrs of injection, the methane
concentration at the S2 observation well
decreased  below  the  detection  limit,
indicating that the microbial mass was
increasing near the injection well. The
pulse cycles were therefore lengthened
to 12 hrs in order to prevent biofouling
near the wellbore. Figure  5  shows the
response of the system to the pulsing at
the S2 observation well. Peak methane
values are shown to increase from below
detection to approximately 1  mg/l, as a
result of the longer pulse duration. Peak
methane concentrations are noted  to
occur when minimum DO concentrations
are observed, which is anticipated based
on transport theory.
  Long  pulse  cycles were  continued
throughout the biostimulation  and bio-
degradation experiments, with durations
ranging from 6 hrs to 12 hrs. Based on
continued methane breakthrough at the
observation wells, the pulsing is believed
to have helped to distribute the microbial
population in the test zone and prevented
biofouling of the  aquifer.
  The biostimulation experiment demon-
strated that methane-oxidizing bacteria
could be successfully established in the
test zone. No additional nutrients were
required to stimulate growth. The tran-
sient methane and DO responses indi-
cated that a population was stimulated
which grew closer to the injection well
with time. The response indicates that
microorganisms have fairly rapid growth
kinetics, typical  of aerobic  organisms.
Thus, pulsing was required to distribute
the growth in the test zone and to prevent
biofouling of the  aquifer.

Biotransformation Experiments

   Biotransformation  experiments  were
performed  after  the  test  zone was
biostimulated. TCE was  continuously
injected over a three-month  period.
During the initial stages, TCE was
injected at an  average concentration of
100 ug/\. During the later stages,  the
concentration  was lowered to 60 (JQ/\
Methane and oxygen (no nutrients) were
continuously pulse-injected during this
period to support the methane-oxidizing
microorganisms   that  had   been
biostimulated.
   During the initial phase of the exper-
iment, the TCE  concentrations slowly
   I
   I
   c
   u
   c
   o
  ID-



   S'

   7-

   6-

   5-

   4-

   3

   2-

   1-
                                  Pulsed Biostimulation
                                 Methane and DO Well S2
          0
                                                      DO
                                                        	
           400   420    440   460   480   500   520   540   560   580   60>

                                        Time (Hrs)

Figure 5.    The effect of long-term pulsing of DO  and methane on the response at the S
            observation well.
approached steady-state values.  The
early breakthrough results indicated that
degradation is on the order of 30 percent.
The degradation of TCE  is illustrated in
Figure 6, which shows the time series
observations of TCE concentrations  at
monitoring wells  S1 and S2  during
steady-state operation under biostimula-
tion conditions.  Comparisons of mass
balances of the amount  injected  and
extracted in the two experiments  also
                                 confirms that TCE was degraded durini
                                 the biotransformation experiment.
                                   Figure 7 represents a summary of th<
                                 biostimulation experiments, where th
                                 fractional breakthroughs of TCE relativ
                                 to bromide ion (TCE/Br) at the  observa
                                 tion wells are compared. The ratios rang
                                 from 70 percent to 80 percent, indicatin
                                 a maximum degree of degradation of 3<
                                 percent. Degradation is noted  to occu
                                 in the  area  of the test zone  in whic
   o
   O
   •o
   I
   "5

   I
  1 •

0.9

0.8

0.7

0.6

0.5

04

03

02

0.1

  0
                 Extract,
           0
                   40
                    80
                                                             240     280
Figure 6.
                        120      160     200
                           Time (Hrs)
Steady-state TCE concentrations during the biostimulation experiment.

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  <§
  o
  .o
  I
1.1

  1 -

0.9 -

0.8 -

0.7 -

0.6 -

0.5 -

0.4 -

0.3 -

0.2 -

0.1 -

  0
                             \\X\\X\l
                                  S2
                                            S3
                                                                   Extract
              \S/\  Average
                            l\.\l Standard Deviation
 Figure 7.
     Estimated TCE biotransformation based on comparisons  with bromide as  a
     conservative tracer.
methane is present  to  support the
methane-oxidizing bacteria.
  The different methods of assessing the
degree of degradation—including mass
balances, comparison of  TCE  break-
through concentrations with the pseudo
control  experiment, and  comparisons
with bromide concentrations at steady-
state within  an experiment— yield
similar estimates of the degree  of TCE
degradation  in the test zone. The  degree
of degradation is in the range of 20 to
30%. The results demonstrate  that, if
sufficient care is taken in obtaining the
experimental data, quantitative evidence
of degradation can be obtained in a real
aquifer situation.
  A mass balance for TCE over the course
of the TCE  biostimulation experiment
shows that of the total 10.1 g that wsre
injected during the course of this exper-
iment, 4.5 g  were recovered in the water
pumped from the extraction well, repres-
enting a  recovery of 45 percent. During
this same overall period,  65 percent  of
the bromide tracer was recovered. The
lower recovery  of  TCE  supports the
conclusion that 25 to 30 percent of the
injected TCE was degraded.
  This interim report was submitted  in
partial fulfillment  of Cooperative Agree-
ment No. R-812220  by Stanford Univer-
sity under the sponsorship of the U  S.
Environmental Protection Agency.
                                    Lewis Semprini, Pau! V. Roberts, Gary D. Hopkins, and Douglas M. Mackay
                                      are with Stanford University, Stanford, CA 94305.
                                    Jack W. Keeley is the EPA Project Officer (see below).
                                    The complete report,  entitled "A Field Evaluation of In-Situ Biodegradation for
                                      Aquifer Restoration," (Order No. PB 88-130 257/AS; Cost: $14.95) 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
                                            P.O. Box 1198
                                            Ada, OK 74820

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