Environ. Sci. Technol. 1997, 31, 786-791
Long-Term Biodegradation of Trichloroethylene Influenced by Bioaugmentation and Dissolved Oxygen in Aquifer Microcosms J U N K O M U N A K A T A - M A R R , * ,† V. GRACE MATHESON,‡ LARRY J. FORNEY,‡ JAMES M. TIEDJE,‡ AND PERRY L. MCCARTY† Department of Civil Engineering, Stanford University, Stanford, California 94305-4020, and Center for Microbial Ecology, Michigan State University, East Lansing, Michigan 48824-1325
Aerobic trichloroethylene (TCE) biodegradation was investigated in small-column aquifer microcosms repeatedly fed solutions containing 6.5 mg/L phenol or 15 mg/L lactate and 250 µg/L TCE every two to three days. The effectiveness of TCE cometabolism by an indigenous phenolfed microbial population declined significantly during a 280day experiment. This behavior, possibly due to the negative selective pressure of TCE cometabolism, which leads to the formation of toxic products, has not been observed previously in shorter term TCE transformation experiments. The addition of microorganisms Burkholderia cepacia G4 or PR1301 to microcosms along with phenol or lactate initially allowed for substantial TCE degradation but led to the eventual depletion of dissolved oxygen and a decline in TCE transformation. After termination of bioaugmentation, dissolved oxygen levels recovered in all microcosms and those microcosms that continued to receive phenol returned to or surpassed previous TCE transformation levels, while unfed and lactate-fed microcosms lost degradative activity. The introduced organisms, however, did not appear to be responsible for the recovered TCE degradation in the phenol-fed, formerly bioaugmented microcosms. The source of activity in these microcosms was not identified but is likely to have been efficient TCE-transforming indigenous organisms selected by the operating conditions within the microcosms.
Introduction Bacterial cultures grown aerobically on primary substrates such as methane (1-11), aromatics (12-17), ammonia (18), isoprene (19), and propylene (20) have been observed to produce oxygenases that fortuitously degrade or cometabolize trichloroethylene (TCE), a common groundwater contaminant. Stimulation of indigenous organisms with a specific primary substrate for in situ bioremediation of TCE may enrich for a population that is unable to cometabolize the target compound or else does so slowly. Bioaugmentation of * Author to whom correspondence should be addressed: Environmental Science & Engineering Division, Colorado School of Mines, Golden, CO 80401-1887. Telephone: (303) 273-3421; fax: (303) 2733413; e-mail:
[email protected]. † Stanford University. ‡ Michigan State University.
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contaminated groundwater systems through addition of bacterial cultures known to transform TCE rapidly may enhance in situ 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 (2128) , but successful demonstrations of bioaugmentation have tended to be associated with readily metabolizable target compounds and with added organisms distributed throughout readily aerated topsoils. Bioaugmentation for the degradation of cometabolic substrates has shown limited success (29-32). The ability to cometabolize TCE varies considerably between pure cultures given primary substrates such as phenol (13, 16, 17, 33). In addition, the transformation of TCE has been observed to generate products that are toxic to the organisms carrying out the transformation (1, 2, 20, 34-38). Nonspecific covalent binding of TCE degradation products to cellular proteins in a number of pure cultures (34, 36-38) has been attributed to cometabolic intermediates such as hydrolysis products of TCE epoxide or chloral. Such transformation product toxicity to organisms that readily transform TCE leads to the concern that long-term bioremediation efforts may be hindered by a negative selective pressure against the better TCE-degrading microorganisms. However, in situ testing of TCE cometabolism over extended time periods has been limited (8, 15, 29, 39-44). Bioaugmentation may provide a means to circumvent such toxicity-related selection pressure. A comparative laboratory-scale aquifer microcosm study was conducted to evaluate the potential of bioaugmentation with organisms known to cometabolize TCE. The early portion of this study (first 44 days) was presented previously (45). The long-term study, continued from the previous study, is described here.
Materials and Methods Microcosm Preparation and Operation. Small-column microcosms (17 mL total volume, 5.0-6.5-mL pore volume) containing aquifer material from Moffett Federal Air Station, Mountain View, CA (Moffett Field) were prepared and operated in a batch-fed mode as described previously (45). The microorganisms used for bioaugmentation were Burkholderia (Pseudomonas) cepacia G4 (G4), a strain isolated from a holding pond at an industrial waste treatment facility in Pensacola FL (14), and B. cepacia PR1301 (PR1), a chemically induced mutant strain of G4 (45). G4 cometabolizes TCE with the enzyme toluene ortho-monooxygenase (Tom), which is normally induced by phenol or toluene (46), while PR1 constitutively expresses Tom while grown on substrates such as lactate (45). The microcosms, the microorganisms added, and the substrates on which the cultures were grown are listed in Table 1. Fluid in each nonbioaugmented column was exchanged by passing 10 mL of feed solution through the column every 2-3 days. At each exchange, each bioaugmented microcosm received 1.0 mL of culture (∼70 µg dry weight of cells (45)) followed by 9 mL of feed solution. Bioaugmentation was performed until day 83, at which time organisms were no longer added to the microcosms but fluid and substrate exchange continued, with 10 mL of feed solution flushed through each column at each exchange. Feed solutions consisted of oxygenated Moffett groundwater supplemented with ∼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
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1997 American Chemical Society
TABLE 1. Bacterial Strains, Their Growth Substrates Prior to Bioaugmentation, and the Microcosm Feed Substrates for Exchanges during the First 83 Days of the Long-Term Studya microcosm
C1 C2 C3 C4 C5
C6
C7
C8 C9 C10
culture added - - G4 G4 G4 PR1 PR1 G4 G4 G4 growth substrate - - P L L L L P P P feed substrate - P - P P P P/L/Pb L L -/Pc a P, phenol; L, lactate. b Feed usually contained phenol, but between day 26 and day 61, feed was switched to lactate. c Feed initially contained no primary substrate; after day 26, feed contained phenol.
oxidation to CO2 and water. The feed substrates for each microcosm are listed in Table 1. During each exchange, the first 0.5 mL of effluent from each microcosm was discarded. The subsequent 2.0 mL of effluent was collected to measure the post-incubation dissolved oxygen (DO) and TCE concentrations in the microcosm. For the DO and TCE samples, a gas-tight syringe was used to subsample the 2.0-mL effluent collected in a glass barrel syringe. After collecting the sample, 5.5 mL of effluent was discarded, then a 1.0-mL effluent sample was taken for microbial analysis, and the final 1.0 mL of effluent was collected for additional TCE analysis. The final effluent TCE concentration, typically very similar to the measured influent concentration, was averaged with the influent concentration to estimate the initial microcosm TCE concentration. Analytical Methods. Phenol was measured using a modification of the direct photometric method (47) with visual estimation of concentration by comparison with a standard color calibration chart. DO was measured with a DO microelectrode (Hansatech Instruments Ltd., Kings Lynn, U.K.) using a saturated potassium chloride solution as the electrolyte and 0.5-mL samples. TCE was measured by pentane extraction and gas chromatography as previously described (45). Microbial Detection. The ninth milliliter of effluent from various column fluid exchanges was tested for the presence of G4 or PR1 using repetitive extragenic palindromic (REP)PCR amplification of DNA in the effluent followed by hybridization to strain-specific probes (48). The probes were made by cloning DNA fragments amplified from genomic DNA of G4 using primers specific for REP sequences (49). Cloned fragments were gel-purified using 1% SeaPlaque (FMC Bioproducts) low-melting temperature agarose, extracted with the Geneclean II Kit (Bio 101), and labeled with digoxigenindUTP using the DIG DNA Labeling and Detection Kit (Boehringer-Mannheim). These probes were shown to be specific to REP-PCR amplification products of G4 and PR1 and could detect as few as 10 cfu of G4 in a mixture with 105 cfu nontarget strains (48). To detect G4 or PR1 in microcosm effluents, 1 µL of each effluent was amplified by REP-PCR using the conditions described by de Bruijn (50). After PCR, 12 µL of each reaction was electrophoresed in 1.5% agarose with 0.5× TAE buffer (51). The DNA was transferred to a Hybond-N nylon membrane (Amersham) by capillary blotting (52) and crosslinked to the membrane with a UV Stratalinker 1800 (Stratagene). Prehybridization and high stringency hybridization to the probes were performed as described previously (45). Filters were developed for chemiluminescent detection with Lumigen PPD (Boehringer-Mannheim) according to the manufacturer’s instructions. Filters were exposed to Kodak X-OMAT film for up to 3 h during maximum luminescence for autoradiographic detection of hybridization signals. Samples were stored at -80 °C until the analyses were performed.
Results The long-term TCE degradative activity of phenol-fed indigenous organisms in microcosm C2 is displayed in Figure
FIGURE 1. Effluent TCE concentration in phenol-fed microcosm: (0) C1, control; (b) C2, phenol.
FIGURE 2. Effluent TCE and DO concentrations in unfed microcosm augmented with phenol-grown G4. Arrows indicate when bioaugmentation was stopped. (0) C1, control; (b) C3, G4 + no substrate. 1. Within the first 100 days, the microcosm consumed as much as 100 µg/L TCE during each exchange period relative to the control. This is consistent with observations made for short-term field studies at the Moffett Field site (42, 43). However, this activity declined as the experiment progressed, so that after 200 days only 30 µg/L TCE was lost relative to the control between each exchange. Dissolved oxygen within the microcosms remained above 20 mg/L throughout the experiment and should not have adversely affected TCE degradation activity. Microcosm C3, augmented with phenol-grown G4 but no primary substrate (Figure 2), initially degraded a similar amount of TCE as microcosm C2 (Figure 1). However, the DO level began to decrease around day 40, becoming completely depleted by day 70, at which point the effluent TCE concentration began to increase. After bioaugmentation was stopped on day 83, more than 20 days were required for the DO to return to significant levels. The TCE concentration continued to increase steadily and quickly became indistinguishable from the control microcosm C1. Similarly, microcosms C8 and C9 were amended with phenol-grown G4. However, these microcosms also received lactate as a primary substrate, as previous studies showed enhanced TCE degradation in its presence (42). The averaged
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FIGURE 3. Average effluent TCE and DO concentrations in microcosms augmented with phenol-grown G4 and lactate. Error bars display 95% confidence intervals: (0) C1, control; (b) average of C8 and C9, G4 + lactate.
FIGURE 4. Effluent TCE and DO concentrations in microcosm augmented with phenol-grown G4. Microcosm fed phenol starting day 26: (0) C1, control; (b) C10, G4 + phenol. results are shown in Figure 3. The general trends were similar to those observed in C3, except that in C8 and C9 TCE removal was better, especially during the early period, and TCE levels took longer to reach the control concentration after bioaugmentation was stopped. The DO concentrations, however, remained much lower. Figure 4 displays the behavior of microcosm C10, augmented with phenol-grown G4 but no primary substrate, initially. Then, starting on day 26, phenol was added in addition to G4. Dissolved oxygen dropped when phenol was added and was depleted by day 50. Up to this point, ∼80
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FIGURE 5. Average effluent TCE and DO concentrations in microcosms augmented with G4 or PR1 and phenol. Error bars display 95% confidence intervals: (0) control; (b) average of C4, C5, C6, and C7 (only with phenol feed). µg/L TCE was degraded in C10, 30 µg/L more than in C3 without phenol feed. When dissolved oxygen became limiting, activity toward TCE decreased, resuming after bioaugmentation was stopped on day 83 and sufficient DO eventually returned. Effluent TCE concentrations stabilized at ∼135 µg/L TCE less than the control. The averaged results from the remaining microcosms C4, C5, C6, and C7 are shown in Figure 5. These microcosms, all fed phenol and bioaugmented with lactate-grown G4 or PR1, behaved similarly and degraded the most TCE of the microcosms tested. The results for C7 during the brief period that it was fed lactate (days 26-61) were discussed previously (45) and are omitted from the averages. Before complete oxygen depletion around day 80, ∼120 µg/L TCE was removed compared to the control. Again, as DO levels decreased, TCE removal dropped. After bioaugmentation was stopped and DO once again became available, steady TCE removal of ∼140 µg/L TCE was achieved, indistinguishable from the other phenol-fed, initially bioaugmented microcosm C10, the primary difference being that phenol addition began later in microcosm C10. G4 and PR1 were never observed in the effluents from nonbioaugmented microcosms C1 and C2. In contrast, G4 and PR1 were regularly detected in the bioaugmented column effluents throughout the bioaugmentation period (days 0-83) and, indeed, for several fluid exchanges beyond that (Table 2). However, by the day 93 exchange, G4 or PR1 was found in only one column effluent and was not detected after that in any of the column effluents (days 95, 97, 105, 113, 137, 191, 236, and 237).
Discussion The importance of having dissolved oxygen present for TCE biodegradation is reflected in the decline in TCE transformation that occurred in all microcosms when oxygen was completely depleted. Regular injection of the small mass of organisms used for bioaugmentation generated a significant and extended oxygen demand, likely due to the slow decay of the added biomass. However, with the addition of a smaller mass of microorganisms, less enhancement of TCE degrada-
TABLE 2. Detection of G4 or PR1 in Microcosm Effluents of Bioaugmented Microcosms on Different Sampling Daysa microcosm time, days
C3
C4
C5
C6
C7
C8
C9
C10
0b 0c 2 5 14 20 31 40 52 58 66 73 79
+ + + + +
+ + + + + + + + + + +
+ + + + + + + + + + +
+ + + + + + + + + -
+ + + + + + + +
+ + + + + + +
+ + + + + + -
+ + + + + + +
+ -
+ -
+ + + + -
85 87 89 93 95 97 105 113 137 191 236 237
+ + + -
Bioaugmentation Stopped + + + + + + + -
a (+) detectable levels of G4 or PR1; (-) G4 or PR1, not detected. First milliliter of effluent during first exchange. c Ninth milliliter of effluent during first exchange. b
tion is likely to have occurred. This emphasizes the conflicting need for sufficient microbial populations as well as DO levels to maintain good activity for aerobic TCE degradation. The mass of organisms added during each exchange would have exerted an oxygen demand of 15-20 mg/L O2 upon mineralization, assuming full retention of the organisms. The maximum DO required by both organism and substrate mineralization would then have been 31-36 mg/L DO, greater than the 30 mg/L DO provided in the feed solution. Assuming complete substrate mineralization, the additional oxygen required for mineralization of completely retained microorganisms would have been 1-6 mg/L DO per fluid exchange. The cumulative excess demand after 32 microbial additions would total 32-192 mg/L O2. Because substrate addition, accompanied by 16 mg/L oxygen demand, continued after bioaugmentation, 3-14 exchanges (8-30 days) would then be required for sufficient oxygen supply following the final addition of organisms. In addition, microorganisms and organic matter contained in the aquifer material exerted some oxygen demand as observed in the control microcosm C1, which consumed over 70 mg/L DO during the first 20 days of operation (data not shown). While these estimates represent a maximum DO demand, they demonstrate the possibility that cellular decay consumed a significant portion of available oxygen. The age of cultures may have affected TCE transformation activity as well. The same plates were used to seed culture growth tubes for the duration of the bioaugmentation, and although the tube cultures initially consumed all phenol (170 mg/L) within 2 days, by day 80 ∼20 mg/L phenol remained in the tubes after 2 days of incubation. This apparent decline in activity could be due to a decreased rate of phenol metabolism or differences in culture growth (e.g., prolonged lag phase) and may have affected TCE transformation in the microcosms. Any residual substrates in the culture medium also may have affected DO levels in the microcosms.
Lactate and phenol feed concentrations were calculated to generate equivalent oxygen demands based on complete mineralization. However, the oxygen demand in lactate-fed microcosms was consistently greater than that in phenol-fed microcosms. This could be due to different proportions of substrate oxidized for energy and synthesized into new cells. Incomplete utilization of phenol compared to lactate might also create such a discrepancy, although this seems an unlikely cause as phenol was not detected in microcosm effluents. The gradual decline in TCE degradation observed in the phenol-fed microcosm C2 is interesting and is cause for concern. Extended competition among phenol-metabolizing indigenous species may have naturally led to a shift in population away from TCE-transforming to nontransforming organisms. However, TCE transformation has been directly observed to produce products that are toxic toward the transforming organisms (1, 2, 20, 34-38). Exposure to TCE over a long period of time most likely would put TCEtransforming organisms at a competitive disadvantage due to TCE transformation product toxicity, leading to their eventual displacement. This possibility has been of concern, but the results with microcosm C2 are the first direct evidence justifying the concern. In situ remediation efforts may have time frames of many months, if not years, and this declining activity within microcosm C2 over a nine-month period has important implications for in situ schemes. Bioaugmentation may provide a technique for maintaining activity when indigenous TCE degradation declines. In microcosms C3, C8, and C9, augmented with preinduced G4 and no additional oxygenase inducer, TCE transformation was relatively good during bioaugmentation. Dissolved oxygen was limiting, though, and once bioaugmentation was halted, activity was lost quickly in the absence of an enzyme inducer. The TCE degradation observed in these microcosms during bioaugmentation demonstrates that the addition of induced TCE-transforming organisms may, in some cases, provide sufficient degradation for in situ treatment in the absence of indigenous TCE-degrading activity or oxygenaseinducing substrate. The lactate-fed microcosms with G4 (C8, C9) appeared to degrade TCE better than the unfed microcosm with G4 (C3) after termination of bioaugmentation. Lactate may have supplied needed reducing power and helped G4 maintain its activity. However, as lactate does not induce Tom, it is unlikely to have supported Tom activity for the duration of the postbioaugmentation period. When bioaugmentation was stopped, aqueous effluent TCE concentrations were ∼150 µg/L in C3 and 110 µg/L in C8 and C9. The rise in concentration in these three microcosms after halting bioaugmentation was comparable to the initial rise observed in the control microcosm C1, within the scatter of the data. This comparison suggests that the apparent removal of TCE in these microcosms during the period following bioaugmentation was in fact due to the influence of sorption. This aquifer material is known to have slow kinetics and high capacity for sorption (8, 45). In contrast, the phenol-fed bioaugmented microcosms continued to transform TCE after bioaugmentation was stopped. Microcosm C10, after dissolved oxygen returned to sufficient levels, removed over 100 µg/L TCE relative to the control for over 90 days (settling at ∼135 µg/L TCE transformed) and showed no signs of losing activity during that time. Similarly, after ceasing bioaugmentation and relieving DO absence, microcosms C4-C7 consumed over 100 µg/L TCE for 140 days (stabilizing at ∼140 µg/L TCE removed), again showing no signs of loss of activity. In comparison, phenol-fed nonbioaugmented microcosm C2 achieved a maximum degradation of ∼100 µg/L relative to the control within 60 days of exposure to TCE, with declining degradation after that.
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The relatively efficient TCE degradation by microcosms C4-C7 and C10 after bioaugmentation was stopped is curious, as there is no evidence that bioaugmented microcosm activity was due to the persistence of the added organisms. In addition to their absence in effluent samples, G4 and PR1 were not found in the aquifer materials from microcosms C5 and C6 after they were dismantled at the end of the experiment (data not shown), at which time good TCE degradation was still being observed. No cause for the good performance of these bioaugmented microcosms in the latter period of this study could be identified. Transfer of the plasmid encoding for Tom to indigenous organisms either did not occur or the transconjugants were not selected, as no DNA samples extracted from previously bioaugmented microcosms hybridized to a Tomspecific catabolic gene probe (data not shown) which can detect 102-103 cells/g of soil within a background of 109 cells/g of soil (53). Selection of more effective TCE-degrading organisms from within the indigenous community in these microcosms seems the most plausible explanation, although this appears contrary to the argument for selection via transformation product toxicity. Numerous distinct indigenous microorganisms capable of TCE cometabolism have been isolated from the aquifer (54). Perhaps an organism less susceptible to TCE transformation product toxicity was favored. It may be that addition of G4 or PR1 altered the relative dominance of organisms, either through direct competition for substrates or through the development of anaerobic conditions. As it appears that G4 and PR1 did not persist in the microcosms, other organism(s) may have been given the opportunity to dominate. It is also possible that addition of trace nutrients in the culture media during bioaugmentation may also have improved the effectiveness of the indigenous community or shifted the population toward a more effective organism. No suitable controls were used that might confirm or refute this possibility. The loss of G4 and PR1 from these phenol-fed microcosms following the end of bioaugmentation is interesting because it demonstrates that addition of a large population of microorganisms for an extended period of time, along with growth-supporting primary substrates, is not necessarily sufficient for maintenance of that culture within a system. This phenomenon is unfavorable for in situ bioremediation, as extended survival of added organisms might eliminate the need for regular bioaugmentation. However, if instead long-term survival following introduction of a nonnative culture was not desired, the lack of establishment would be beneficial. The requirement of a primary growth substrate to induce TCE-degrading enzymes complicates in situ bioremediation, as many of the primary substrates shown to effectively support TCE-transforming cultures pose mass transfer and/or potential regulatory problems with their addition to groundwater systems. One possible way to circumvent this difficulty is to use bacterial strains that exhibit constitutive expression of TCE-degrading oxygenases and can grow on compounds that are easier to use and not of regulatory concern. However, as described previously (45), microcosm C7 bioaugmented with the constitutive strain PR1 degraded ∼60% less TCE when fed lactate than when fed phenol. One explanation for the reduced activity is that lactate-fed PR1 was washed out of the columns more readily than phenolfed PR1. As presented here, however, PR1 was detected equally in column effluents from phenol- and lactate-fed microcosms (C6 and C7, days 26-61). The lack of TCE degradation by PR1 previously observed under lactate feed was thus probably due to some other factor, as yet undetermined, such as lower enzyme expression, reduced viability, or competitive disadvantage.
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Acknowledgments This work was supported through cooperative agreement CR 822029 with the United States Environmental Protection Agency, Gulf Breeze Environmental Research Laboratory, and by a National Science Foundation graduate fellowship. As it has not been subjected to agency review, no official endorsement should be inferred.
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Received for review June 4, 1996. Revised manuscript received November 4, 1996. Accepted November 5, 1996.X ES960480N X
Abstract published in Advance ACS Abstracts, January 15, 1997.
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