Sustained Aerobic Oxidation of Vinyl Chloride at Low Oxygen

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Environ. Sci. Technol. 2010, 44, 1405–1411

Sustained Aerobic Oxidation of Vinyl Chloride at Low Oxygen Concentrations JAMES M. GOSSETT* School of Civil and Environmental Engineering, 220 Hollister Hall, Cornell University, Ithaca, New York 14853

Received November 8, 2009. Revised manuscript received December 30, 2009. Accepted January 2, 2010.

One possible explanation for unexplained disappearance of vinyl chloride (VC) from what are thought to be anaerobic subsurface environments is that the environments are, in fact, not anaerobic. Rather, they might be subject to low, steady influx of oxygen, and aerobic oxidation could be occurring at extremely low oxygen concentrations. Studies were conducted with VC-oxidizing transfer cultures derived from two chloroethene-contaminated sites, as well as with microcosms constructed from sediment and groundwater from one of these sites. Oxygen was steadily delivered to the experimental systems using permeation tubes to maintain low dissolved oxygen throughout the time-course of investigation. VC oxidation was sustained at dissolved oxygen concentrations below 0.02 mg/L in the two transfer cultures, and below 0.1 mg/L in the microcosms. This supports the possibility thatsat least at some sitessapparent loss of VC from what are thought to be anaerobic zones might, in fact, be due to aerobic pathways occurring under conditions of low oxygen flux (e.g., via diffusion from surrounding aerobic regions and/or from recharge events).

Introduction Chlorinated ethenes such as tetrachloroethene (PCE) and trichloroethene (TCE) are well-known to undergo biological reductive dechlorination in subsurface anaerobic zones at contaminated sites, resulting in less-chlorinated daughter products such as dichloroethenes (DCEs), vinyl cloride (VC), ethene, and ethane (1-3). These daughter products can undergo mineralization through aerobic oxidation, if the contaminants migrate into oxygenated regions. Such aerobic oxidation can be either cometabolic (4-6) or growth-coupled (7-11), depending upon the particular microbial populations and upon presence or absence of possible cosubstrates (e.g., methane, toluene, ammonia). At some sites, however, the absence of a mass-balance in anaerobic zones between parent compounds and expected products of reductive dechlorination has led investigators to evaluate the possibility of oxidative mechanisms occurring under anaerobic conditions, with alternative electron acceptors such as nitrate, sulfate, Fe(III), Mn(IV), and/or humic substances (12-20). However, despite the many laboratory studies that have appeared to demonstrate links between disappearance of VC and/or DCEs and addition of such alternative electron acceptors, no anaerobic oxidizer of VC or DCEs has yet been isolated. An alternative explanation for unexplained disappearance of VC at some sites, in what are thought to be anaerobic * Tel: 607-255-4170; e-mail: [email protected]. 10.1021/es9033974

 2010 American Chemical Society

Published on Web 01/21/2010

subsurface environments, is that the environments are, in fact, not anaerobic. Rather, they might be subject to low, steady influx of oxygen, and aerobic oxidation could be occurring at extremely low oxygen concentrations. Several previous reports provide the genesis for this hypothesis. First, the half-velocity constants and oxygen thresholds measured for five different pure cultures of aerobic VC-degraders were all extremely low (8). Thresholds for oxygen use ranged from 0.02 to 0.1 mg/Lsconcentrations low enough that comparable subsurface environments would likely be classified as “anaerobic” in field assessments. Additionally, Schmidt and Tiehm (21) observed that aerobic VC-oxidizing bacteria survived anaerobic conditions for up to a year and maintained their ability for aerobic degradation, which they observed at dissolved oxygen concentrations between 0.1 and 0.3 mg/L. Finally, there was the isolation of an aerobic, VC-oxidizing Mycobacterium from what was thought to be an anaerobic microcosm (22). Recently, in studies conducted in our lab while unsuccessfully prospecting for anaerobic VC-oxidizers, several microcosms that had been incubated anaerobically for five months were subsequently amended with oxygen. VCoxidation commenced within 42 days in three of them, and a second addition of VC was degraded completely within an additional 8 days (23). This led to the studies reported in this papersinvestigation of the potential for aerobic oxidation of VC at extremely low oxygen concentrations (below 0.02 mg/L). Studies were conducted with VC-oxidizing transfer cultures derived from two chloroethene-contaminated sites, as well as with microcosms constructed from sediment and groundwater from one of these sites. Oxygen was steadily delivered to the experimental systems using permeation tubes to maintain low dissolved oxygen throughout the time-course of investigation.

Materials and Methods Chemicals and Media. Vinyl chloride was from SigmaAldrich, 99.5+ % purity. It was added to serum bottles via a gastight syringe with locking valve. Amounts added were calculated from the ideal gas law, incorporating local temperature and barometric pressure. Transfer cultures were maintained in minimal salts medium (MSM) containing trace metals as described by Coleman et al. (7). MSM was buffered at pH 7.1-7.2 with 20 mM phosphate. Site Materials. Industrial Site 4 (“Site 4”). Subsurface sediment and groundwater were obtained from an electronics manufacturing site in Southern California. Shallow groundwater was primarily impacted by TCE and 1,1-DCE (a product of 1,1,1-trichloroethane’s abiotic transformation). cis-DCE and VC were also present, but VC attenuated as it left the site. Soil samples were collected (within 6 m of the surface) from the down-gradient area where anaerobic conditions transitioned to mildly aerobic conditions, and aerobic biodegradation of VC was potentially occurring. Groundwater was obtained from the nearest well. The soil from this site was sandy-silt, with small amount of clay. Cecil Field (“CF”). Streambed sediment and groundwater were obtained from the former NAS Cecil Field, Jacksonville, FL. The location was Rowell Creek, Site 3, where Bradley and Chapelle (13) had earlier obtained material used in their investigation of VC oxidation under Fe(III)-reducing conditions. Sediment was obtained from 5-25 cm below the sediment/water interface. Groundwater was obtained from a shallow monitoring well within 3 m of the sedimentVOL. 44, NO. 4, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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sampling location. Sediment from this site was essentially viscous, black muck. Microcosms and Transfer Cultures. Anaerobic Source Microcosms. From both Site-4 and CF materials, microcosms were constructed and monitored by Jingshuang Fang (23) for anaerobic studies in 160-mL glass serum bottles. Site-4 microcosms contained 10 g (wet) sediment and 90 mL of groundwater; for CF, the mix was 25 g (wet) sediment plus 90 mL of groundwater. Through 190 days of monitoring, Site-4 microcosms evidenced no significant loss of VC. Through 140 days of monitoring, CF microcosms evidenced substantial reductive dechlorination of VC to ethene. After the extended anaerobic incubation, 10 mL of oxygen was added to each. When re-examined after 42 days of aerobic incubation, VC was absent from both Site-4 and CF aerobic microcosms (the residual ethene that accumulated from previous anaerobic incubation in the CF microcosm was also gone). Second additions of 16 µmol VC to each of the Site-4 and CF aerobic microcosms were depleted within an additional 8 days. Transfer Cultures for Permeation Studies. Aerobic transfer cultures, 10% (v/v), were prepared from each of the two, now-aerobic Site-4 and CF microcosms by delivering 10 mL of the mixed contents of each to 90 mL of minimal salts medium (MSM) in 160-mL serum bottles with ambient-air headspace, sealed with Teflon-lined, butyl-rubber septum and aluminum crimp. Each was repetitively spiked with VC (initially16 µmol, but later 42 µmol/bottle) and O2 (10 mL) as necessary when either was depleted. Incubation occurred at 22 °C in the dark, on a rotary shaker (120 rpm) with bottles inverted. Five additions of VC were degraded over a 12-day period in the Site-4 transfer culture; and eight additions of VC were degraded over a 25-day period in the CF transfer culture. Then each of these first-generation cultures was used to prepare eight replicate second-generation 10% (v/v) aerobic transfer cultures in fresh MSM. These were initially given 42 µmol VC, and reamended (Day 4 for the Site-4 cultures; Day 6 for the CF cultures) when VC had been nearly depleted in all. After this second addition of VC had been approximately 50% degraded, the replicates were taken for use in oxygen-permeation studies. For both Site-4 and CF second-generation transfer cultures, their respective eight replicates tracked one another almost identically. Site-4 Microcosms for Permeation Studies. Six replicate microcosms were prepared, each with 25 g (wet wt.) soil and 75 mL of groundwater from Site 4. Each was also amended with 10 mL of MSM (to provide buffer and nutrients) and 1 mL of second-generation Site-4 transfer culture. The headspace of each was ambient air. Bottles were sealed with septa and crimps as usual, then provided with 10.4 µmol VC. Incubation occurred at 22 °C in the dark, on a rotary shaker (120 rpm) with bottles inverted. The intent of the microcosm study was to assess the potential for sustained aerobic oxidation of VC in a mixedculture environment that better approximated in situ conditions than did the enriched transfer cultures. However, the available Site-4 materials had been stored (4 °C) for over a year; therefore, aerobic VC oxidation activity was likely low. Since it is important that the oxygen-permeation rate not exceed the potential oxygen-demand rate, the modest inoculum was added for insurance, as was the MSM. The lower-than-usual VC addition was intended to minimize growth of VC-oxidizers, providing a more natural environment than in the transfer cultures. When VC-degradation activity was first detected (after 17 days), these microcosms were transitioned to an oxygen-permeation experiment. Oxygen Measurement. Oxygen was measured in transfer cultures, microcosms, and permeation studies by gas chromatography (GC) with a 3-ft × 1/8-in. column packed with 60/80 Molecular Sieve 5A (Supelco, Inc.), held isothermally at 30 °C. A thermal-conductivity detector was 1406

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employed with helium carrier. Headspace samples (0.25mL) were acquired from experimental serum bottles using a locking, gastight syringe. Because very low oxygen levels were to be assayed, the syringe and needle were flushed with anoxic N2 prior to acquisition of each sample. A “zero oxygen” serum bottle containing a N2 headspace above a saturated solution of Na2SO3 (s) with 100 mg/L CoCl2 catalyst was kept near the GC for this purpose. Oxygen calibration was performed by delivering measured volumes of pure oxygen to serum bottles (without liquid) of known volume that had been previously purged for 20 min with oxygen-free N2 (scrubbed through titanium(III)-citrate solution (24)). The ideal gas law was invoked (with local measurement of temperature and barometric pressure) to calculate moles of O2 added to these standards, and thus to calculate resulting volumetric gaseous concentrations (Cg, mg/L). A locking, gastight syringe was used to sample the standards at Cg concentration, and GC peak units from 0.25-mL injection were related to Cg. For convenience, calibration curves were expressed in terms of the aqueous dissolved oxygen concentrations (Cw, mg/L) corresponding to the Cg standards. Cg and Cw are related by pseudodimensionless Henry’s constant, (Hc), where Cg ) HcCw. At 22 °C, Hc ) 31.676 [mg/L gas per mg/L water], based on an oxygen solubility in pure water (1 atm) of 8.743 mg/L (25). The detection limit for dissolved oxygen (DO) was ( 0.01 mg/L of true zero. This was determined by noting the standard deviation of DO values obtained from injecting samples of headspaces from “zero oxygen” bottles described earlier. There was always a small oxygen peak noted by the GC, even from these “zero oxygen” bottles. The source appears to be some combination of oxygen that desorbed from the syringe and oxygen entrained in the act of sample-injection to the GC. The mean of these peaks represented a small Y-intercept on the calibration regression relating GC peak-units to corresponding DO. Variance in underlying mechanisms responsible for the intercept explains why negative values of DO are reported in some instances. To put the issue in context, consider that a 0.25-mL sample of truly oxygen-free gas, subsequently contaminated with only 1.5 µL of air (i.e., 0.6% of sample volume), would give a GC peak the size of the mean intercept. Vinyl Chloride Measurement. VC was measured in transfer cultures, microcosms, and permeation studies by GC with a 16-ft × 1/8-in. column packed with 1% SP1000 on 60/80 Carbopack B (Supelco, Inc.), held isothermally at 150 °C. A flame-ionization detector was employed with nitrogen carrier. Headspace samples (0.25-mL) were acquired from experimental serum bottles using a locking, gastight syringe. VC calibration was performed by delivering measured volumes of VC gas to sterile microcosms and serum bottles prepared exactly as experimental bottles (i.e., with or without permeation tubes, as appropriate). The ideal gas law was invoked (with local measurement of temperature and barometric pressure) to calculate moles of VC added to these standards, and calibration regressions were prepared that related GC peak units (PU) from headspace injection to total µmoles of VC in bottles. Detection limit was 0.03 µmol/bottle. Permeation Tubes. Permeation tubes were sections of low-density polyethylene (LDPE) tubing (Freelin-Wade 1J074), 3/8-in. OD × 1/4-in. ID × 0.062-in. wall. The length of each tube was 6.3 cm, and the ends were sealed with custom, shop-made, tapered PVC plugs. With plugs in place, the internal length was 4.5 cm, giving a volume of 1.43 mL, corresponding to 59.5 µmol of gas at 1 atm, 20 °C (the usual conditions at which the tubes were filled). Permeation tubes were filled with either N2 or O2 by the following procedure: A PVC plug was fully inserted into one end of a tube, while a plug rested loosely in the other

FIGURE 1. Permeation of O2 from LDPE tubes (22 °C). Dashed curve is simulation with permeation coefficient, Kp ) 0.21 cm/d. end; a needle was used to deliver flowing gas to the tube by insertion alongside the loose PVC plug; and after several seconds, the needle was extracted while simultaneously forcing the tapered plug tightly into the tube. In parallel with the several permeation studies with live cultures and microcosms, abiotic controls were run with permeation tubes in bottles with MSM, to allow estimation of the permeation coefficient for O2 with these tubes. Results from all controls (six bottles from three separate runs) are plotted together in Figure 1; experimental conditions were identical to those in all other permeation experiments (22 °C, with bottles mounted in an inverted position on an orbital shaker agitated at 120 rpm). A permeation coefficient (Kp) of 0.21 cm/dsmore fully, (mg transferred d-1 cm-2 surface) /(mg/cm3 differential driving force in liquid-concentration units)swas obtained by fitting these data using Stella (ISEE Systems) dynamic modeling. The model simulated the rise in dissolved oxygen over time, and included three phases (water, headspace, and permeation tube). Equilibrium was assumed between headspace and aqueous phases. From previous studies, it was known that mass transfer between these two phases was sufficiently rapid to justify this assumption (8, 26). For perspective, consider that under the experimental conditions employed (temperature, bottle volume, gas/liquid ratio, inverted position on the orbitalshaker, and rpm), the applicable overall mass-transfer coefficient (Kla) for gas/solution exchange of oxygen is 43.8 h-1, estimated using the method of ref 26. At this Kla, an equilibrium deficit is 95% removed within 4 min. The permeation-model simulation of DO vs time, with Kp ) 0.21 cm/d, is shown as dashed curve in Figure 1. Literature values for O2 permeability through LDPE are generally reported in units that are not specific to a particular thickness and geometryse.g., cm3 mm m-2 d-1 atm-1. On that basis, my measured Kp converts to 104 cm3 mm m-2 d-1 atm-1, which is within the range (102-188 cm3 mm m-2 d-1 atm-1) that Massey (27) gives of permeation coefficients for O2 through LDPE at 23 °C. Note that environmental conditions matter, as they can affect the properties of the permeation material. For example, I measured a consistent 20% increase in Kp when the LDPE tubes were placed in a phosphoric acid solution (0.2%(v/v) of 85% phosphoric acid in distilled water). For that reason, Kp was measured with tubes in MSM, simulating conditions of the permeation experiments with live cultures. Permeation Studies With Live Cultures. Three studies were conducted to investigate whether VC oxidation could be sustained at low DO supplied via permeation tubes. Two

studies used second-generation aerobic transfer cultures derived from Site-4 and CF formerly anaerobic microcosms; and one used newly constructed microcosms from Site-4 materials (but inoculated with 1 mL of Site-4 transfer culture). In all studies, eight bottles were used: three live controls contained N2-filled permeation tubes; and three live bottles contained O2-filled tubes. The remaining two live bottles were used in VC-O2 stoichiometry studies without permeation tubes, with O2 added directly to headspaces. Duplicate, sterile, MSM-only controls accompanied the permeation studies. Bottles were purged with N2 for 20 min, recapped with Teflon-lined, butyl-rubber septa and aluminum crimps, and injected with 5 mL of N2 to provide excess pressure, ensuring that a vacuum would not develop in bottles from repeated sampling over the subsequent monitoring period. Each was then injected with the desired, initial VC levels20.8 µmol (13 mg/L nominal concentration, if partitioning to headspace, sediment, and permeation tubing is ignored) for the two studies with transfer cultures; and 10.4 µmol for the Site-4 microcosm study. The rationale for use of lower VC level in the microcosm study is that a lower VC-oxidation rate (and more competition for oxygen) was expected within the microcosms than in the transfer cultures; given the limited oxygen available within permeation tubes, I wanted to ensure that a significant fraction of added VC could be degraded over the monitoring period. Bottles were incubated in the dark at 22 °C, in an inverted position on an orbital shaker at 120 rpm.

Results Stoichiometry of VC Oxidation. For later analysis/interpretation of data from permeation studies with VC-oxidizers, it was useful to know the stoichiometric relationship between VC oxidation and oxygen consumption. If microbial synthesis is ignored, theoretically 2.5 mol of O2 would be consumed per mole of VC completely mineralized: H2C ) CHCl + 2.5 O2 f 2 CO2 + H2O + H+ + Cl(1) However, only the fraction of VC used in energyproduction (fe) involves O2; the fraction of VC used in synthesis (fs) does not. These fractions can be estimated from measurements of cellular yield. Coleman et al. (8) reported measurements of yield for four different strains of VCdegrading Mycobacterium, averaging about 6 g of protein/ mol VC. If protein is assumed 55% of cellular organics (28), then this yield converts to 10.9 g of organic solids per mol VC degraded. A commonly invoked empirical formula for the major elements of bacterial organic biomass is C5H7O2N, FW ) 113 g/mol (29), representing 20 electron equivalents (eeq) per empirical mole. This converts to 5.65 g of organic solids per eeq of cells. VC has 10 eeq/mol. Therefore, 10.9 g organic solids/mol VC (5.65 g organic solids/eeq cells) × (10 eeq VC/mol VC) ) 0.19 (2)

fs )

and fe ) (1 - fs) ) 0.81. The expected stoichiometry between O2 consumption and VC degradation would be 0.81 × (2.5) ) 2.025 mol O2/mol VC. During the O2 permeation studies with second-generation transfer cultures, duplicates without permeation tubes were run in which O2 was added directly to the headspaces. Figure 2 presents results for Site-4 and CF bottles, respectively. Each chart shows measured DO (mg/L) and VC (µmol), accompanied by dashed curves that show the expected remaining VC levels predicted from observed O2 depletions, based on the assumed fe ) 0.81. The good agreement between observations and predictions in all cases suggests two things: (i) that the predicted stoichiometry is reasonably close to VOL. 44, NO. 4, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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FIGURE 2. VC and O2 consumptions in (A) Site 4 and (B) Cecil Field transfer cultures. Dashed curves show predicted VC depletion curves based on observed O2 depletions and a stoichiometry of 2.025 mol O2 per mol VC. reality; and (ii) that whatever VC was not being converted to biomass was completely mineralized. On the basis of these findings, fe ) 0.81 was used in subsequent predictions of expected VC depletion from O2 permeation. Based on a stoichiometry of 2.025 mol O2 consumed per mol VC degraded, permeation tubes containing 1.43 mL (59.5 µmol) of O2 would be expected to support the degradation of up to 29.4 µmol VC. Permeation Studies: VC Oxidation at Low Oxygen Concentrations. Figure 3 presents results of the permeation study with second-generation, Site-4 transfer culture. The top chart shows VC and DO levels in triplicates with N2-filled permeation tubes, and the bottom, for triplicates with O2filled tubes. While there was perhaps a slow apparent loss from bottles with N2-filled tubes, the losses did not exceed those from abiotic MSM controls (not shown). On the other hand, VC was almost completely degraded from bottles with O2 permeation tubes by the end of 19-day study. Other than a couple of early DO levels of ca. 0.03 mg/L, the DO levels in these O2-permeation systems remained e0.02 mg/L throughout. Sustained degradation of VC occurred while DO remained below 0.02 mg/L. Also shown in the bottom chart of Figure 3 (dashed curve) is the remaining VC predicted from stoichiometry, based on the expected O2 flux (at Kp ) 0.21 cm/d with steady-state DO ) 0.01 mg/L) and an assumed stoichiometry of 2.025 mol O2/mol VC. This prediction assumes that all O2 delivered to the systems was used by aerobic VC-degraders. The good agreement between ob1408

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FIGURE 3. Site-4 transfer cultures. VC degradation and dissolved oxygen (DO) concentrations in cultures with (A) N2-filled or (B) O2-filled permeation tubes. Dashed curve in (B) is predicted VC depletion if all O2 delivered (Kp ) 0.21 cm/d) was used by VC oxidizers with a stoichiometry of 2.025 mol O2 per mol VC. served VC and predicted VC suggests that there were no other significant O2 sinks. Figure 4 presents results of the O2 permeation study with second-generation, CF transfer culture. Unfortunately, one of the triplicate bottles with O2-permeation tube experienced an obvious leak of O2 from its tube, with sudden rise in DO and a concomitant, rapid VC-depletion. It is not shown. The remaining two evidenced a significant VC degradation that can be attributed to O2 permeation, when compared to results from bottles with N2-filled tubes. The major difference between this study and the previous one with Site-4 transfer culture is that apparently only about one-half the oxygen delivered via permeation to CF culture was used in VCoxidation. This is evident from the large difference between predicted VC depletion (based on expected oxygen permeation) and actual VC depletion. The experiment was terminated after 26 days because it was estimated that oxygen had been excessively depleted from permeation tubes. The CF material from which the original microcosms were constructed consisted of stream-sediment mucksvery black and organic-rich. Even these second-generation transfers (containing only 1% of original microcosm content) had observable black particles in them. It is therefore not surprising that there would be other sinks for O2 besides VC-oxidation. Indeed, the greater background O2 consumption in CF vs Site-4 transfer cultures is evident in Figure 2; the rate of O2 depletion in the first 4 d after VC had been completely consumed was 60-80% higher in CF culture than in Site-4 culture. Because of greater competition for O2 in CF

FIGURE 4. Cecil-Field transfer cultures. VC degradation and dissolved oxygen (DO) concentrations in cultures with (A) N2-filled or (B) O2-filled permeation tubes. Dashed curve in (B) is predicted VC depletion if all O2 delivered (Kp ) 0.21 cm/d) was used by VC oxidizers with a stoichiometry of 2.025 mol O2 per mol VC. culture, VC degradation was slower and O2 became depleted in permeation tubes before VC was completely consumed. However, sustained VC degradation did occur while DO was maintained below 0.02 mg/L, as was true with the Site-4 studies above. Figure 5 presents results of the permeation study with newly constructed, Site-4 microcosms. It is again apparent that O2-permeation supported VC oxidation. As with the CFtransfers, only about one-half the O2 delivered via permeation to Site-4 microcosms was used for VC oxidation; some other sink(s) existed. The most significant difference between these microcosm results and those from transfer cultures is that the aerobic activity was initially too low at very low DO to keep pace with O2 permeation; DO rose to about 0.1 mg/L, and only came down after VC-degradation activity increased. Note, therefore, that the “predicted” VC degradation based on O2 permeationsdashed curve in the bottom chart of Figure 6swas in this case based upon a steady-state DO of 0.1 mg/L. Use of a tube with slower permeation characteristics would possibly have prevented this. Still, VC degradation was sustained at DO concentrations at or below 0.1 mg/L, with DO concentrations considerably lower in latter stages of the experimental run.

Discussion With VC-oxidizing transfer cultures and microcosms derived from authentic site materials, VC oxidation was sustained at

FIGURE 5. Site-4 microcosms. VC degradation and dissolved oxygen (DO) concentrations in microcosms with (A) N2-filled or (B) O2-filled permeation tubes. Dashed curve in (B) is predicted VC depletion if all O2 delivered (Kp ) 0.21 cm/d) was used by VC oxidizers with a stoichiometry of 2.025 mol O2 per mol VC. DO concentrations below 0.02 and 0.1 mg/L, respectively. This supports the possibility thatsat least at some sitessapparent loss of VC from what are thought to be anaerobic zones might, in fact, be due to aerobic pathways occurring under conditions of low oxygen flux (e.g., via diffusion from surrounding aerobic regions and/or from recharge events). Groundwaters with DO concentrations of 0.1 mg/L would likely be labeled “anaerobic”; and those with DO of 0.02 mg/L, most assuredly would be. Field measurement of dissolved oxygen is generally accomplished with membrane electrodes, and low DO concentrations are typically considered to indicate “anaerobic conditions,” since the time-constant of the membrane electrode is rather long (i.e., the approach of the reading to the true value is increasingly slow as the true value approaches zero). Furthermore, DO instrumentation is typically calibrated at high DO, not at low DO concentrations. If geochemical data show typical end-products of anaerobic processes (e.g., methane, reduced iron, sulfide), that usually solidifies a conclusion of anaerobic conditions. However, monitoring wells are often screened over >1 m depth intervals; and the depth interval of an aquifer contributing water to a pumped sample can exceed the screened interval considerably, resulting in a sample that represents a mix of different in situ strata (some anaerobic and some aerobic), complicating assessment of biogeochemical conditions. Very little oxygen would be required for aerobic degradation to have significant impact. The stoichiometry measured in this study (2.025 mol O2/mol VC) converts to almost exactly 1:1 on a mass basis. Thus, a VC concentration of 100 ppb would require VOL. 44, NO. 4, 2010 / ENVIRONMENTAL SCIENCE & TECHNOLOGY

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only 0.1 mg/L DO for its complete degradation by aerobes. Furthermore, reports suggest that aerobic VC-oxidizers are common at chloroethene-contaminated sites (8), and can survive prolonged exposure to truly anaerobic conditions (21). One of the aerobic, VC-oxidizing cultures in this present study (Cecil Field) was derived from a microcosm that had exhibited reductive dechlorination and methane production over 140 days of anaerobic incubation, before it was administered oxygen to commence aerobic oxidation of VC. What of the potential for aerobic oxidation in studies intended to be anaerobic? Bradley et al. recognized the potential problem with oxygen contamination of experiments designed to investigate “anaerobic oxidation” (30). To avoid such artifacts, they suggested that many investigators had gone to the alternate extreme of establishing excessively high reducing conditions, incompatible with processes such as iron or manganese reduction. And therein lies the dilemma: experiments designed to study the role of iron- or manganesereducing bacteria in chloroethene transformation must operate under conditions only slightly more reducing than microaerophilic regimes. The potential for artifact resulting from small amounts of oxygen introduction is therefore significant. In the context of a typical experimental microcosm with 100 mL of liquid, a nominal concentration of 100 ppb VC (0.16 µmol VC) would require only 0.32 µmol O2 for its aerobic degradation. That requirement would be met by the cumulative introduction of only 40 µL of air over the course of microcosm operation. Given multiple sampling events and the possibility of developing a vacuum in bottles from repeated sampling, introduction of such quantities of air is not unreasonable. Obviously, the potential for significant aerobic contribution to observed VC disappearance increases at lower VC levels. These studies underscore both the need to assess the potential for aerobic VC oxidation at sites, as well as the difficulty in doing so. Given that sustained VC oxidation was observed at DO concentrations below 0.02 mg/L, it is not practical to exclude its possibility through measurement of DO. Assessment of aerobic VC oxidation activity can be done with microcosms, of course. However, aerobic VC-degraders are likely to be limited in distribution to a relatively small zone at the interface between truly anaerobic and aerobic portions of a contaminant plumesi.e., degradation is so rapid that VC might not persist far into the aerobic zone. Microcosm studies are relatively expensive, a factor tending to restrict the number of sampling locations investigated. Given the number of sampling points required to detect an activity likely to be narrowly distributed at an ill-defined anaerobic/ aerobic interface, the most cost-effective way would seem to be through application of molecular biological tools targeting aerobic processes. DNA-based tools would enable investigators to determine the current (or at least recent) presence of aerobic VC-oxidizers. Better still, tools based on transcription (mRNA) of VC-degradative genes would allow detection of ongoing activity in otherwise mischaracterized “anaerobic” environments. One apparent impediment to development of such tools is the lack of distinguishing genotypical features between aerobic ethene-degraders capable of VC-oxidation and those that are not (31). However, a recent study (32) found that extended exposure of ethene-oxidizers to VC caused their adaptation to use of VC as growth substrate, perhaps making it less important to distinguish among phenotypes of etheneoxidizers. Detection of generic, aerobic, “ethenotrophic” bacteria would likely be sufficient to distinguish low-oxygen aerobic plumes from truly anaerobic ones.

Acknowledgments This study was supported by the Strategic Environmental Research and Development Program (SERDP, ER-1557) through Geosyntec Consultants (Evan Cox, Principal 1410

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Project Manager). Site-4 samples were generously provided by David Freedman (Clemson University) and Leo Lehmicke (Hargis+Associates); Cecil Field samples were provided by Mike Singletary (NAVFAC) through logistical coordination with Carey Austrins (Geosyntec Consultants). Jingshuang Fang (Cornell University) prepared and monitored the original anaerobic microcosms from which the aerobic transfer cultures were derived.

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