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: ------- 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 ------- 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) ------- 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. ------- 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. ------- 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. ------- <§ 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 ------- United States Environmental Protection Agency Center for Environmental Research Information Cincinnati OH 45268 BULK RATE POSTAGE & FEES EPA PERMIT No. G-: Official Business Penalty for Private Use $300 EPA/600/S2-87/096 0000329 PS CHICAGO ------- |