Enhancement of Trichloroethylene Degradation in Aquifer Microcosms

Environ. Sci. Technol. 1996, 30, 2045-2052

Enhancement of Trichloroethylene Degradation in Aquifer Microcosms Bioaugmented with Wild Type and Genetically Altered Burkholderia (Pseudomonas) cepacia G4 and PR1 J U N K O M U N A K A T A - M A R R , * ,† PERRY L. MCCARTY,† MALCOLM S. SHIELDS,‡ MICHAEL REAGIN,‡ AND STEPHEN C. FRANCESCONI§ Department of Civil Engineering, Stanford University, Stanford, California 94305-4020, Center for Environmental Diagnostics and Bioremediation, University of West Florida, Pensacola, Florida 32514, and National Research Council, U.S. EPA National Health and Environmental Effects Laboratory, Gulf Breeze, Florida 32561

The effects of bioaugmentation on the aerobic cometabolism of trichloroethylene (TCE) in groundwater were investigated using small-column aquifer microcosms. Nonsterile nonbioaugmented microcosms fed phenol as a primary substrate mimicked observed in-situ behavior at the Moffett Field site (1), cometabolizing approximately 60 µg/L TCE while fed 6.5 mg/L phenol. High-density single bioaugmentation with Burkholderia (Pseudomonas) cepacia G4 increased TCE removal in sterile aquifer material, while producing mixed results in nonsterile material. Low-density semicontinuous bioaugmentation enhanced TCE transformation in nonsterile microcosms. A nonrecombinant NTG-induced mutant of G4 (PR1301) capable of uninduced constitutive degradation of TCE in the absence of phenol or toluene was developed for environmental release. Phenol-fed microcosms augmented with either B. cepacia strain G4 or PR1301 transformed twice as much TCE as the nonaugmented phenol-fed microcosm. In addition, should primary substrate addition be a regulatory concern, TCE degradation was observed without primary substrate addition through bioaugmentation using organisms expressing the TCE-transforming enzyme.

* Author to whom correspondence should be addressed telephone: (415) 723-0315; fax: (415) 725-3162; e-mail address: [email protected]. † Stanford University. ‡ University of West Florida. § National Research Council.

S0013-936X(95)00834-0 CCC: $12.00

 1996 American Chemical Society

Introduction Trichloroethylene (TCE), a common groundwater contaminant, has been found to be fortuitously degraded (cometabolized) by organisms grown on a variety of substrates such as methane (2-12), aromatics (13-18), ammonia (19), isoprene (20), and propylene (21). However, stimulation of native organisms with a specific substrate for in-situ bioremediation of TCE may enrich for a population either unable to cometabolize the target compound or else does so slowly. Bioaugmentation of contaminated groundwater systems through the addition of bacterial cultures known to transform TCE rapidly may enhance native biodegradation or even provide the sole means of degradation in systems without indigenous TCE-degrading organisms. Such biological enhancement has been investigated for degradation of many different compounds (e.g., refs 22-29). Daughton et al. (22) provided a good summary of early work. Successful demonstrations of bioaugmentation have tended to be associated with readily metabolizable target compounds and with added organisms distributed throughout soils. A difficulty posed by aerobic in-situ TCE bioremediation schemes is the requirement of a primary growth substrate to induce TCE-degrading enzymes. Many of the compounds shown to effectively support TCE-transforming cultures, however, pose mass transfer and/or potential regulatory problems with their addition to groundwater systems. An alternative to circumvent these problems is the use of strains of bacteria selected not only for their ability to degrade TCE but also to do so in the absence of inducing compounds. Such bacteria may grow on harmless water-soluble substrates and still maintain their ability to transform TCE. However, such substrates may be less selective, and the introduced organisms may encounter strong competition from indigenous organisms, resulting in little TCE degradation. Tn5 mutagenesis was previously demonstrated to result in the production of a constitutive TCE-degrading strain (30), but the insertion of additional genetic information, particularly antibiotic resistance, in this recombinant strain may subject its release to strict regulatory and public approval. As an alternative, a nonrevertible regulatory mutant selected for spontaneous constitutive TCE transformation through N-methyl-N′nitro-N-nitrosoguanidine (NTG) mutagenesis was produced and tested. To evaluate the TCE degradation potential of bioaugmentation with both wild type and genetically altered organisms, small-column microcosms were biologically augmented and monitored for TCE degradation under conditions resembling an in-situ treatment scheme.

Materials and Methods Microcosm Preparation. Column microcosms were prepared using cores of aquifer material from a slightly contaminated groundwater site at Moffett Federal Air Station, Mountain View, CA (Moffett Field). The aquifer materials were sieved under a laminar flow hood through an ethanol-flamed size 8 Taylor sieve (2.36 mm mesh opening) into an autoclaved Mason jar. Sieved aquifer material for sterile column studies was autoclaved for 30 min (120 °C) on each of three consecutive days. All columns were constructed as described by Dolan and McCarty (31),

VOL. 30, NO. 6, 1996 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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TABLE 1

Bacterial Strains Used in This Study strain

B. cepacia G4 B. cepacia PR123 B. cepacia G4301 B. cepacia PR1301 B. cepacia 17762 a

phenotypea

treatment reference none, wild type isolate spontaneous revertant of Tn5 mutant NTG mutant spontaneous revertant of G4301 none, wild type isolate

Tol+,

Phe+,

TCEi

Tol+, Phe+, TCEc Tol-, Phe-, TCETol+, Phe+, TCEc Phe-

ref 15 30 this paper this paper 34, 37

Tol, toluene metabolism; Phe, phenol metabolism; TCE, TCE cometabolism; (+) positive, (-) negative, (i) induced, (c) constitutive.

except that 17-mL Pyrex test tubes containing approximately 27 g of sieved material and closed with screw-caps and Teflon-lined rubber septa were used. Bromide tracer tests indicated pore volumes of 5-6.5 mL. Columns were stored at room temperature (20-23 °C) during experiments. Bacterial Cultures for Bioaugmentation. The bacterial strains used for bioaugmentation in this study are summarized in Table 1. The wild-type strain evaluated was Burkholderia (Pseudomonas) cepacia G4 (G4), a strain isolated from a holding pond at an industrial waste treatment facility in Pensacola, FL (15). This organism cometabolizes TCE with the enzyme toluene ortho-monooxygenase (Tom), which is normally induced by phenol or toluene (32). Two mutants of G4 were used: B. cepacia PR123 (PR123), described previously (33) and developed by Tn5 insertion, and PR1301 (PR1301), developed through chemical mutation as described in this paper. Both mutants constitutively express Tom while grown on substrates such as lactate. In addition, B. cepacia strain no. 17762 (ATCC 17762) was obtained from the American Type Culture Collection for comparison. Frozen stock cultures of the above were prepared in sterile 1.5-mL Eppendorf tubes by vortexing 1.0-mL aliquots of the cultures briefly together with 35 µL of dimethyl sulfoxide (DMSO) (99+%, Aldrich Chemical Co., Milwaukee), storing on wet ice for 15 min, briefly vortexing again with an additional 35 µL of DMSO, flash freezing in an ethanol/dry ice bath, and storing at -80 °C. Cultures to be used were withdrawn from the frozen aliquots with autoclaved toothpicks and streak-plated onto agar containing a standard mineral base (SMB) (34) and 20 mM D-glucose. To prepare suspended cultures for each exchange in the low-density bioaugmentation study, colonies from these plates were used to seed tubes containing SMB and 190 mg/L sodium lactate. The following day, the resulting cultures were used to inoculate new SMB + 190 mg/L lactate tubes with G4 or PR1301 and an SMB + 170 mg/L phenol tube with G4. These cultures were then incubated for 2 days to allow maximum growth and complete primary substrate utilization before each culture was used for bioaugmentation. Development of PR1301. Because of occasional concern regarding the introduction of recombinant strains such as PR123 into the environment, a nonrecombinant strain (PR1301) capable of constitutive TCE degradation was developed. Bacterial cultures used for PR1301 development were grown on two formulations of media based on a basal salts minimal medium (BSM) (35), BSM-lactate (BSM, 20 mM lactate) and BSM-phenol-TTC [BSM, 0.025 mg/mL triphenyl tetrazolium chloride (TTC) (Sigma Chemical Co., St. Louis), 0.2 mg/mL proteose peptone (Fisher Scientific), 2 mM phenol (Sigma Chemical Co., St. Louis)]. NTG mutagenesis and enrichment for Tol- and Phe- mutants

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using a toluene vapor feeder was performed as previously described (32). Tol- and Phe- mutants were detected using the TTC dye-reduction assay described by Shields et al. (32) with the following changes: phenol (2 mM) was used as the primary carbon source instead of toluene vapor, and 0.2 mg/mL proteose peptone was added to the BSMphenol-TTC purified agar plates. Mutagenized cells were diluted to give approximately 150 colonies/100 µL plated. Assays for Tom through the oxidation of trifluoromethyl phenol (TFMP) to the yellow trifluoromethylheptadienoic acid (TFHA) were performed as previously described (32). Stock Solutions for Bioaugmentation. An aqueous solution saturated with TCE was prepared by adding approximately 10 mL of TCE (99+%, Aldrich Chemical Co., Milwaukee) to an autoclaved 65-mL amber glass bottle containing 50 mL of deionized water, 2 glass beads, and capped with a Mininert valve. A stock concentrated phenol solution was made by diluting 1.32 g of liquid phenol (90%, Fluka, Ronkonkoma, NY) to 10 mL with autoclaved deionized water and storing in an autoclaved 20-mL amber glass bottle capped with an open-hole screw cap and Teflonlined silicone septum. Ten milliliters of a concentrated lactate solution was prepared by diluting 6.64 g of sodium lactate syrup (60% w/w, racemic mixture, Sigma Cell Culture, St. Louis) in deionized water and autoclaving in a 20-mL amber glass bottle capped with an open-hole screw cap and Teflon-lined silicone septum. Microcosm Operation. Fluid in each column was exchanged periodically by pumping 10 mL of feed solution (described below) held in a gas-tight glass-barrel syringe (Spectrum, Houston, TX) into the column influent port at a rate of 2 mL/min with a Cole-Parmer 74900 Series syringe pump (Cole-Parmer, Niles, IL). The first 0.5 mL of the resulting effluent was wasted, and the subsequent 2.0 mL of effluent was collected for dissolved oxygen (DO) and TCE measurements. In the high-density bioaugmentation study, after collecting the DO and TCE sample, the microcosm was flushed with an additional 7.5 mL of feed solution, wasting 6.5 mL of the effluent and collecting the final 1.0 mL of effluent for TCE analysis. The low-density study was similar, but 5.5 mL of effluent was wasted after collecting the DO and TCE sample, then a 1.0-mL effluent microbial analysis sample was taken, and finally a 1.0-mL TCE sample was collected. All effluent samples were collected in glass-barrel Luer-lok syringes with glass plungers. (i) High-Density Single Bioaugmentation. In the highdensity study, both sterile and nonsterile aquifer columns were used. Pure cultures were added through the column influent port only once and prior to any column fluid exchanges. The different B. cepacia strains added to separate columns are listed in Table 2. Column fluid was exchanged every 2-5 days using feed solutions consisting

TABLE 2

Bacterial Strains and Substrates Added to High-Density Single Bioaugmentation Microcosmsa sterile microcosm

H1

culture added mg cells added feed substrate a

nonsterile

H2

H3

H4

G4 11 P

ATCC 17762 1 P

PR123 7 L

H5

H6

P

H7

H8

H9

H10

H11

L

G4 11 P

PR123 7 L

G4 7 P

PR1301 5 L

P, phenol; L, lactate.

TABLE 3

Bacterial Strains and Their Growth and Microcosm Feed Substrates for Low-Density Semicontinuous Bioaugmentation Studya microcosm

C1

culture added growth substrate feed substrate a

P, phenol; L, lactate.

C2

P b

C3

C4

C5

C6

C7

C8

C9

G4 P

G4 L P

G4 L P

PR1301 L P

PR1301 L P/Lb

G4 P L

G4 P L

Feed initially contained phenol; from day 26 on, feed was switched to lactate.

of oxygenated Moffett Field groundwater (filter-sterilized for sterile columns) supplemented with about 31 mg/L O2, 250 µg/L TCE, and 13 mg/L PO43-. The microcosms containing G4 and ATCC strains initially received feed solutions containing 13 mg/L phenol while those containing PR123 received feed solutions containing 30 mg/L lactate, concentrations that were calculated to create equivalent oxygen demands of 31 mg/L, assuming complete conversion to CO2 and water. Because complete DO consumption was regularly observed in the columns at these substrate levels, feed solution concentrations were subsequently halved to 6.5 mg/L phenol and 15 mg/L lactate. (ii) Low-Density Semicontinuous Bioaugmentation. In the low-density bioaugmentation study, nonsterile microcosms were operated using microorganism addition together with column fluid exchanges every 2-3 days. Feed solutions consisted of oxygenated Moffett Field groundwater supplemented with about 250 µg/L TCE and a primary substrate. The primary substrate consisted of either phenol or lactate at concentrations of 6.5 and 15 mg/L, respectively, which created equivalent oxygen demands of 16 mg/L assuming complete oxidation to CO2 and water. The culture additions and fluid exchanges were performed by adding 1.0 mL of the appropriate tube culture through the influent port of each augmented column, followed by 9 mL of feed fluid. Cell mass added to each bioaugmented column during each exchange was approximately 70 µg dry weight, based on measured yields of 0.45 and 0.4 mg dry cell weight/mg of substrate for phenol and lactate, respectively. This is equivalent to cell concentrations of 7 mg/L dry weight if uniformly distributed throughout the feed solution, concentrations that are approximately double that which would grow on the 6.5 mg/L phenol added in the feed and about equal to that resulting from growth on the 15 mg/L lactate in the feed. Nonbioaugmented columns were exchanged with 10 mL of feed fluid at the same substrate concentrations. Duplicate columns were generally operated for all bioaugmented columns, with the exceptions noted in Table 3. Analytical Methods. DO was measured with a DO microelectrode (Hansatech Instruments Ltd., Kings Lynn, England) using a saturated potassium chloride solution as

the electrolyte. Samples of 0.5 mL were diluted in 0.5 mL of air-equilibrated deionized water. Oxygen- and nitrogenpurged deionized water samples were used for calibration. For TCE analysis from the microcosms, 1.0-mL samples were transferred to 2-mL glass vials capped with Teflonlined silicone septa and open-hole screw caps. Gravimetrically prepared TCE/methanol stock solutions were used to prepare standards. After the samples were collected, each vial was uncapped, 0.5 mL of pentane (99+%, Aldrich Chemical Co., Milwaukee) was added, and the vial was recapped, shaken vigorously by hand, and stored inverted. TCE measurements using this method were comparable to those with pentane injection through the septum and with addition of the sample to pentane in the vial (data not shown), so headspace losses were considered negligible. All vials were shaken on a table-top rotary shaker at 350 rpm for 30 min. A 6.0-µL sample from the pentane phase was injected into an Hewlett-Packard HP5890II gas chromatograph with a 30-m DB-1 column and electron-capture detector. Helium was the carrier gas at 23 mL/min, argon/ methane provided the makeup gas at 67 mL/min, and the septum purge was 4 mL/min. The temperature program typically used was 2 min at 60 °C, ramp to 80 °C at 70 °C/min, 0.2 min at 80 °C, and 2.8 min data acquisition time. TCE concentrations in batch cultures were monitored with headspace samples. Gas samples of 500 µL from cultures sealed in 165-mL serum bottles were analyzed with a gas chromatograph similar to that described above but equipped with a photoionization detector. Phenol was measured using a modification of the direct photometric method (36) with visual estimation of concentration by comparison with a standard color calibration chart. Cell density in the high-density single bioaugmentation study and growth yield calculations were based on volatile suspended solids measurements as previously reported (1).

Results Constitutive Strain Selection. In order to develop a nonrecombinant strain that constitutively degrades TCE, NTG-mutagenized cells were subjected to five enrichment cycles of toluene-cycloserine/pipericillin counterselection


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FIGURE 1. Effluent TCE concentration in phenol-fed microcosm: (0) H5, control; (b) H6, phenol.

followed by survivor recovery in BSM-lactate. The optical density of the culture was monitored and found to increase from A600 ) 0.12 following the first cycle to 3.85 following the fifth cycle. The total viable cell count recovered from this final cycle was 2.5 × 107/mL on Luria Bertani (LB) medium. Colorless colonies were picked from the BSMphenol-TTC purified agar plates as presumptive Phe- and checked for growth on BSM-phenol (2 mM) or BSMtoluene plates. A total of 291 presumptive Phe- mutants was picked as such colorless colonies. Seven were identified as presumptive tomB mutants as indicated by the dark brown discoloration of these colonies in the presence of toluene or phenol, previously shown to be due to catechol accumulation (32). Thirty of the Phe- mutants were confirmed as such by their inability to grow on 2 mM phenol-BSM plates. These strains were challenged to spontaneously revert to phenol utilization following inoculation of 5 mL of BSM-phenol and incubation for 5 days at 30 °C. Fifteen of these showed slight turbidity. Isolates from each were obtained on LB plates and grown in LB broth overnight. These cells were then assayed for constitutive expression of tomA and tomB by their ability to produce TFHA from TFMP without prior exposure to aromatic inducers (30). One strain (PR1301) was capable of TFHA production within the standard 20min assay period used for phenol-induced G4 or PR123. Thirteen of the remaining 14 were capable of doing so after 180 min, while one was not. Batch BSM-phthalate-grown PR1301 with and without phenol produced TFHA at rates similar to G4 with phenol and PR123 with and without phenol (data not shown). In addition, a batch-suspended culture study performed prior to the low-density bioaugmentation experiment indicated similar rate and extent of TCE degradation by phenol-grown G4 and PR1301 in groundwater, about half of the rate and extent demonstrated by lactate-grown PR1301 in groundwater (data not shown). High-Density Single Bioaugmentation. The effluent TCE concentrations in both sterile and nonsterile control microcosms H1 and H5 (Figures 1 and 2) increased rapidly during the first 30 days and then more gradually throughout the remainder of the experiment, reaching a level of approximately 70% of the influent concentration (250 µg/ L) within 120 days. This behavior is representative of initially fast and then slow sorption processes and confirms the high TCE sorption capacity of Moffett Field aquifer material reported previously (9). In the phenol-only microcosm H6, TCE breakthrough was observed initially (Figure 1), but after four exchanges

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FIGURE 2. Effluent TCE concentrations with sterile microcosm material: (0) H1, control; (2) H2, G4 + phenol; ([) H3, ATCC + phenol; (4) H4, PR123 + lactate.

FIGURE 3. Effluent TCE concentrations with nonsterile microcosm material. H7 behaved similarly to H5 and is not shown for clarity: (0) H5, control; (b) H6, phenol; (2) H8, G4 + phenol; (4) H9, PR23 + lactate; (1) H10, G4 + phenol, half substrate; (3) H11, PR1301 + lactate, half substrate.

(18 days) the effluent concentration began to decline, reaching levels below 6 µg/L after 7 exchanges. This behavior, believed to result from the gradual buildup of an indigenous TCE-degrading population, is similar to or somewhat better than that observed under similar conditions in the field (1), an indication that the column microcosm adequately mimicked field behavior. The results for the microcosms initially containing sterilized aquifer material (microcosms H1-H4) are depicted in Figure 2. In the G4-bioaugmented microcosm H2, no TCE breakthrough was observed through day 37, indicating that bioaugmentation was effective. During the same time period, microcosm H3, bioaugmented with ATCC 17762, degraded TCE to a concentration of 65 µg/L (55% degradation relative to control). This culture was intended to act as a negative control, as it was not known to metabolize phenol (34, 37) and did not show significant TCE degradation in a batch experiment (data not shown). The TCE degradation observed in microcosm H3 was thus not expected and perhaps resulted from incomplete sterilization of the aquifer material. The TCE level in microcosm H4 with PR123 and lactate did not vary significantly from that in the sterile control, suggesting that the constitutive expression of Tom was not effective in this system. Figure 3 displays the results obtained from the microcosms containing nonsterile aquifer material. During the first 65 days, the bioaugmented microcosms H8 and H9

FIGURE 5. Effluent TCE concentrations in microcosms bioaugmented with phenol-grown G4: (0) C1, control; (b) C2, phenol; (open plus sign) C3, G4 + no substrate; (4) C8, G4 + lactate; (3) C9, G4 + lactate.

FIGURE 4. (a) Dissolved oxygen levels in sterile microcosms. Break in data due to DO probe downtime: (0) H1, control; (2) H2, G4 + phenol; ([) H3, ATCC + phenol; (4) H4, PR123 + lactate. (b) Dissolved oxygen levels in nonsterile microcosms. On day 67, operation of H7, H8, and H9 was stopped and operation of H10 and H11 was started. Break in data occurred due to DO probe downtime: (0) H5, control; (b) H6, phenol; (2) H8, G4 + phenol; (1) H10, G4 + phenol, half substrate; (O) H7, lactate; (4) H9, PR123 + lactate; (3) H11, PR1301 + lactate, half substrate.

performed no better than the H5 control and considerably worse than the phenol-only microcosm H6. A major problem in these bioaugmented microcosms can be seen in Figures 4a and 4b. In all of the substrate-fed columns, DO levels were consistently less than 3 mg/L and may have inhibited TCE degradation which requires oxygen, thus raising uncertainties about factors causing the above TCE transformation results. The problem was most severe in the bioaugmented microcosms, except for H3 which had a smaller population addition. To address this concern, phenol and lactate concentrations were halved in all feeds after day 36. This change did not improve degradation in columns containing sterile aquifer material (Figure 2). TCE concentrations increased in microcosms H2 and H3 bioaugmented with G4 and ATCC 17762, respectively, while the DO levels increased to 20 mg/L. The previously observed poor results with microcosm H4 containing PR123 did not improve, perhaps because the DO concentration remained below 2 mg/L. Because no change in TCE degradation or DO concentration was observed in either of the bioaugmented nonsterile microcosms H8 and H9 (Figures 3 and 4b), two new microcosms H10 and H11 were started on day 67 (Figure 3). Microcosms H10 and H11, bioaugmented with 7 mg of G4 and 5 mg of the newly developed nonrecombinant PR1301, respectively, received feed solutions containing phenol and lactate, respectively, at the lower primary substrate concentrations. The PR1 microcosm H11 again

showed no difference from the control H5, while the G4 microcosm H10 behaved similarly to the phenol-only microcosm H6. The TCE concentration in microcosms H6 and H10 increased to about 95 µg/L. While the microcosm H6 DO increased to 20 mg/L, the DO in microcosms H10 and H11 remained below 2 mg/L. Hydrogen peroxide (up to 20 mg/L as O2) was added to the lactate-fed PR123 sterile microcosm H4 and PR1301 nonsterile microcosm H11 in an attempt to increase available oxygen, but DO was still consumed in both microcosms within 36 h (data not shown) and again, no improvement in effluent DO concentration or TCE degradation was observed. Low-Density Semicontinuous Bioaugmentation. The low-density bioaugmentation study was conducted to determine whether small continuous addition of microorganisms might be a better bioaugmentation strategy for maintaining culture activity while reducing the DO problem associated with large culture addition. During the 44 days of this study, dissolved oxygen was generally present at levels greater than 5 mg/L in all semicontinuous bioaugmentation microcosms (data not shown), thus DO should not have been a limiting factor in the results. The control microcosm C1 displayed sorption behavior as in the highdensity microcosm study (Figure 5). Compared to control C1, the phenol-only microcosm C2 degraded TCE to an extent (55 µg/L) similar to the previously run microcosm at a comparable feed concentration (H6, Figure 3). Bioaugmentation with phenol-grown G4 alone (microcosm C3) generated TCE removal very close to that of the phenol-only microcosm C2 (Figure 5). Because previous studies demonstrated enhanced TCE degradation with phenol-grown mixed cultures in the presence of lactate (1), microcosms C8 and C9 were operated with phenolgrown G4 and lactate feed. Interestingly, these microcosms displayed a 70% greater TCE degradation over G4 alone or phenol alone. Batch suspended culture studies showed that lactategrown G4 could be readily induced to degrade TCE with low concentrations of phenol (as low as 1 mg/L phenol, data not shown), so lactate-grown G4 was added to columns C4 and C5, which were also fed phenol. Lactate-grown PR1301 was similarly added to columns C6 and C7, to provide a direct comparison. The results are displayed in Figures 6 and 7. These microcosms consumed more than twice as much TCE as the phenol-only C2, about 130 µg/L, with this efficient degradation observed almost immediately. How-


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FIGURE 6. Effluent TCE concentrations in microcosms bioaugmented with lactate-grown G4: (0) C1, control; (b) C2, phenol; (open plus sign) C4, G4 + phenol; (cross) C5, G4 + phenol.

FIGURE 7. Effluent TCE concentrations in microcosms bioaugmented with lactate-grown PR1: (0) C1, control; (b) C2, phenol; (×) C6, PR1 + phenol; (cross) C7, PR1 + variable.

ever, duplicate microcosms C4 and C5 displayed some divergence in results, the cause of which is unknown but may reflect some form of mixed culture dynamics. The results from the comparative study with PR1301 are shown in Figure 7. The effective degradation of TCE with phenol feed in duplicate columns was encouraging, as the high-density augmentation attempt with PR1301 and lactate feed had failed. The feed for one of the duplicates, C7, was then switched to lactate in order to determine the extent to which TCE degradation would occur under less selective conditions. The effect was dramatic. TCE removal decreased significantly after one exchange, and the TCE effluent concentration quickly rose to the levels of the phenol-grown G4 with no substrate in microcosm C3 and the phenol-only microcosm C2. Such behavior indicates that PR1 does not degrade TCE as well in aquifer materials when fed lactate as when fed phenol.

Discussion

The small-column microcosms that were operated under conditions similar to those in the field generated responses comparable to observed field data (1). Such results suggest that this simple microcosm technique may be used to predict field behavior. Because the available field data are limited, the true reliability of the small-column microcosm method has yet to be determined, but the method clearly shows good potential for rapidly assessing the effects of various operating parameters on cometabolic transformations in aquifer materials. The development of a method to distribute added organisms throughout the microcosms was initially a

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concern. However, as enhanced TCE degradation downstream of the influent port was immediately evident, the addition of organisms followed by feed fluid through the influent port seemed sufficient for microbial dispersion. Distribution of microorganisms will, of course, vary by type of aquifer material and microorganism and may have been nearly ideal in this uniform sandy gravel system. Clogging was generally not observed in the microcosms. When pressure did build up within the microcosms, it was typically due to fine sand or silt blocking the effluent tube. In the low-density semicontinuous bioaugmentation systems, microcosm C3 with added G4 but no substrate achieved the same degree of TCE degradation as microcosm C2 to which only phenol was added. This result indicates that simple bioaugmentation without primary substrate addition can allow for biodegradation of TCE. The efficiency of transformation as measured by transformation yield (Ty, g of TCE transformed/g of phenol used) is another method of comparing these results. Ty was higher when only phenol was added (microcosm C2) than when both bioaugmentation and phenol addition were used (microcosm C3). With phenol alone, Ty was 0.008 g of TCE/g of phenol consumed, while with bioaugmentation and phenol addition, Ty was only 0.002 g of TCE/g of phenol. The latter considers both the phenol used to grow the culture added to the microcosm and the phenol added to the microcosm in the feed solution. It also assumes complete retention of the added bacterial culture within the microcosm. If, for example, only half of the added culture remained in the microcosm at the end of a column fluid exchange, the calculated Ty would have a higher value of 0.004 g of TCE/g of phenol. Folsom and Chapman observed yields between 0.007 and 0.02 g of TCE/g of phenol using G4 in suspended bioreactor studies (38), while transformation yields between 0.002 and 0.062 g of TCE/g of phenol were measured in nonbioaugmented in-situ studies at Moffett Field (1, 39). The transformation yields observed in microcosms C2 and C3 are comparable to those reported in the above studies at similar concentrations. Because transformation yields are a function of the concentration of TCE present (1), higher transformation yields may be achieved at TCE concentrations higher than those used in this study. The addition of formate, acetate, or lactate has been shown to enhance TCE degradation in phenol-grown resting mixed culture cell suspensions (1). Similar behavior was observed during this experiment in microcosms C9 and C10, which were augmented regularly with G4 and lactate. In these microcosms, 45 µg/L more TCE was degraded than in microcosm C3, to which only G4 was added. The mechanism for such enhancement has not been elucidated, but it may be due to energetic benefits from the addition of lactate, similar to that obtained by adding formate with methanotrophic TCE biodegradation (2, 40). These results indicate that, in addition to the benefits from bioaugmentation with induced organisms alone for transforming TCE, the simultaneous addition of a harmless substance such as lactate can enhance the effects of bioaugmentation significantly. As an additional consideration, small quantities of trace nutrients from the growth media were undoubtedly added along with bacterial cultures during bioaugmentation. However, it is not apparent from these data that this preferentially benefited the bioaugmented columns. Dense cultures of G4 can be readily grown on lactate, a significant advantage over growing such cultures on phenol due to difficulties posed by phenol toxicity when

used at high concentrations (41). This is an important consideration for field applications, as large amounts of organisms would be needed in a large-scale bioaugmentation project. The dense lactate-grown G4 culture can then be readily induced for Tom with as little as 1 mg/L of phenol (data not shown), lowering the amount of phenol needed to be introduced into an aquifer or into the culture for TCE transformation. This benefit was illustrated in phenol-fed microcosms C5 and C6 that were bioaugmented with lactate-grown G4 and degraded TCE very well, as did phenol-fed microcosms C7 and C8 bioaugmented with lactate-grown PR1301. Thus, bioaugmentation with cultures known to transform TCE more than doubled the extent of TCE degradation achieved in the microcosm to which the primary substrate alone was added. Though this technique still requires the addition of phenol, the quantity required for a specific degree of TCE degradation would be lowered significantly with bioaugmentation. Bioaugmentation also ensured that an active TCE-degrading population was present. The poorer performance of the lactate-fed, relative to the phenol-fed, PR1 microcosm raises many questions. The constitutive expression of Tom by PR1 was expected to eliminate the need for phenol addition. However, the lactate-fed system did not degrade TCE well under the conditions described here. The semicontinuous lowdensity bioaugmentation results do not suggest that PR1301 reverted to a nonconstitutive strain, as some TCE degradation was observed. However, the degree of degradation was similar to that by G4 addition alone, suggesting that PR1301 did not benefit measurably from the addition of lactate. In contrast, the addition of lactate enhanced TCE degradation in microcosms C8 and C9, bioaugmented with phenol-grown G4. One possible explanation for this puzzling behavior is that PR1301 may have had a disadvantage relative to both native organisms and G4 in competing for lactate, or it may simply have reduced viability compared with that of G4. It may also be that native organisms dominated over both G4 and PR1301 in the competition for phenol. Stimulation of indigenous TCE degradation using phenol but not lactate could account for the difference in cometabolism of TCE. However, this explanation does not account for the enhancement of TCE degradation with lactate addition to induced G4 but not to PR1301. Another possible explanation is a difference in enzyme levels. Lower expression of Tom by lactate-fed PR1301 compared with phenol-fed G4 and PR1301 may be the cause of the reduced TCE degradation in columns augmented with the former culture. These possibilities warrant further study. Dissolved oxygen, as expected, appears to be a significant limiting factor for bioremediation in bioaugmented systems. In the single high-density bioaugmentation experiment, all phenol- and lactate-fed microcosms consumed essentially all of the oxygen at the higher primary substrate concentrations. Even after the primary substrate concentrations were halved, the DO level remained low in the bioaugmented columns and may have limited the extent of TCE degradation observed in these microcosms. These results illustrate the need to keep the levels of bioaugmentation with substrate addition within the bounds dictated by the amount of oxygen that can be supplied. Finally, a major concern in bioaugmented systems is the survival of introduced organisms. Cometabolism may be diminished if the added organisms fail to compete with

native strains for primary substrate or if sufficient predation occurs. The fate of added organisms within the microbial community in the microcosms is thus of great interest and requires further study. In summary, a nonrecombinant constitutive TCEdegrading culture, PR1301, was developed for environmental release. From the microcosm studies, bioaugmentation appears to be a promising technique for in-situ bioremediation schemes. Immediate activity toward phenol and TCE can be attained, eliminating long startup periods accompanied by high phenol concentrations and ensuring the presence of an active, efficient TCE-degrading population. In addition, bioaugmentation can increase substantially the degree of TCE degradation achieved. By working within the bounds dictated by dissolved oxygen requirements, the addition of an active bacterial culture capable of degrading a target contaminant such as TCE can significantly enhance the effectiveness and efficiency of in-situ treatment.

Acknowledgments This work was supported through Cooperative Agreements CR 822029 and CR 820704 with the United States Environmental Protection Agency, Gulf Breeze Environmental Research Lab, and through National Science Foundation graduate and post-doctoral support. As the paper has not been subjected to agency review, no official endorsement should be inferred.

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Received for review November 7, 1995. Revised manuscript received February 12, 1996. Accepted February 13, 1996.X ES950834V X

Abstract published in Advance ACS Abstracts, April 1, 1996.

Enhancement of Trichloroethylene Degradation in Aquifer Microcosms

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