Relative Contributions of Dehalobacter and Zerovalent Iron in the

Mar 12, 2015 - Relative Contributions of Dehalobacter and Zerovalent Iron in the. Degradation of Chlorinated Methanes. Matthew Lee,*. ,†. Eliza Well...
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Relative Contributions of Dehalobacter and Zerovalent Iron in the Degradation of Chlorinated Methanes Matthew Lee,*,† Eliza Wells,† Yie Kuan Wong,† Joanna Koenig,† Lorenz Adrian,‡ Hans H. Richnow,‡ and Mike Manefield† †

School of Biotechnology and Biomolecular Science, University of New South Wales, Kensington, Sydney 2052, Australia Department of Isotope Biogeochemistry, Helmholtz Centre for Environmental Research−UFZ, Permoser Strasse 15, Leipzig 04318, Germany

‡

S Supporting Information *

ABSTRACT: The role of bacteria and zerovalent iron (Fe0) in the degradation of chlorinated solvents in subsurface environments is of interest to researchers and remediation practitioners alike. Fe0 used in reactive iron barriers for groundwater remediation positively interacted with enrichment cultures containing Dehalobacter strains in the transformation of halogenated methanes. Chloroform transformation and dichloromethane formation was up to 8-fold faster and 14 times higher, respectively, when a Dehalobacter-containing enrichment culture was combined with Fe0 compared with Fe0 alone. The dichloromethanefermenting culture transformed dichloromethane up to three times faster with Fe0 compared to without. Compound-specific isotope analysis was employed to compare abiotic and biotic chloroform and dichloromethane degradation. The isotope enrichment factor for the abiotic chloroform/Fe0 reaction was large at −29.4 ± 2.1‰, while that for chloroform respiration by Dehalobacter was minimal at −4.3 ± 0.45‰. The combined abiotic/biotic dechlorination was −8.3 ± 0.7‰, confirming the predominance of biotic dechlorination. The enrichment factor for dichloromethane fermentation was −15.5 ± 1.5‰; however, in the presence of Fe0 the factor increased to −23.5 ± 2.1‰, suggesting multiple mechanisms were contributing to dichloromethane degradation. Together the results show that chlorinated methane-metabolizing organisms introduced into reactive iron barriers can have a significant impact on trichloromethane and dichloromethane degradation and that compound-specific isotope analysis can be employed to distinguish between the biotic and abiotic reactions involved.

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and ethenes yields ethane,9 while tetra- and trichloromethane yield dichloromethane, methane, formate, carbon monoxide, and carbon dioxide7,10,11 (Figure S1, Supporting Information). The reaction of Fe0 with water in reactive iron barriers also leads to the formation of hydrogen (eq 1), supporting the growth of hydrogenotrophic microorganisms that can further influence the degradation of the barriers target compound(s).12−18 For example, a methanogenic consortia stimulated by Fe0 was able to accelerate the removal of 1,1,1-trichloroethane; however, at the same time, it appeared to inhibit the abiotic degradation of perchloroethylene.13 In other studies, the application of hydrogenotrophic organohalide respiring bacteria (ORB) enabled accelerated and complete dechlorination of perchloroethylene15 and complete dechlorination of 1,2-dichloroethane a compound that does not react with Fe0.18

INTRODUCTION Carbon tetrachloride (IUPAC name: tetrachloromethane), chloroform (IUPAC name: trichloromethane), and dichloromethane are common groundwater pollutants because of their extensive industrial production and historical improper handling and disposal practices. The three compounds are present at 244 (18%), 474 (36%) and 389 (30%) of the 1319 priority polluted sites in the USA, respectively.1 Organochlorine solvents are inhibitory to most anaerobic microbial processes with inhibitory potency increasing with decreasing water solubility.2 Further, chlorinated methanes are inhibitory to microbial organohalide respiration of chlorinated ethanes and ethanes,3,4 which can confound attempts to bioremediate sites polluted with solvent mixtures. In such a scenario, pretreatment to remove chlorinated methanes maybe necessary or an abiotic strategy maybe employed. One abiotic approach for the treatment of organohalidecontaminated groundwater is by chemical reduction with Fe0. This can be achieved in situ by introducing a permeable reactive iron barrier into the flow path of contaminated groundwater.5 Fe0 facilitates the reductive transformation of chlorinated solvents to benign products.6−8 Reaction of Fe0 with chlorinated ethanes © 2015 American Chemical Society

Fe 0 + 2H 2O → Fe 2 + + 2OH− + H 2 Received: Revised: Accepted: Published: 4481

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October 27, 2014 February 28, 2015 March 12, 2015 March 12, 2015 DOI: 10.1021/es5052364 Environ. Sci. Technol. 2015, 49, 4481−4489

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Environmental Science & Technology For chlorinated methanes, methanogens stimulated by Fe0 have been shown to cometabolize tetrachloromethane and trichloromethane at low concentrations (∼1 mg/L).13,14,16 However, bioremediation of chlorinated methanes by methanogenic cometabolism is not an effective strategy, as these compounds are inhibitory to methanogens,19−21 with the inhibitory potency decreasing with increasing water solubility (e.g., tetrachloromethane 1.4 μM, trichloromethane, 7.8 μM, dichloromethane, 2.4 mM).19 Microorganisms that can use chlorinated methanes as primary grown substrates are the preferred strategy for bioremediation as they proliferate as the pollutant is removed, which leads to faster rates of removal. Importantly, these organisms have higher tolerance toward these compounds. For example a trichloromethane-respiring Dehalobacter strain can transform 250 μM trichloromethane to dichloromethane per day, which can then be fermented to acetate and hydrogen at 380 μM per day.22 Compound specific isotope analysis (CSIA) is an analytical technique that can measure changes in stable isotope ratios during compound degradation. This technique can therefore be used to differentiate between degradation mechanisms if isotope fractionation signatures are significantly different. CSIA has been used to differentiate between biotic and abiotic degradation of chlorinated ethanes in groundwater sediments23 and biotic and abiotic contribution of trichloroethylene degradation in a reactive iron barrier.12 There have been several studies documenting carbon isotope fractionation in chlorinated ethanes and ethenes via reaction with Fe0.24−28 However, there is no analogous data published for chlorinated methanes, and in general, there is a lack of fractionation data for these compounds. Current reported data include: large carbon fractionation in tetrachloromethane reacting with iron oxides and sulfides (−29.3‰ and −15‰),29 trichloromethane respiration by Dehalobacter strain CF (−27.5 ± 0.9‰),30 and dichloromethane oxidation by methylotrophic organisms (− 41 to −61‰).31 Thus, the aim of this study is to investigate how two recently described Dehalobacter strains capable of growth-linked metabolism of trichloromethane and dichloromethane might impact upon chlorinated methane depletion in a reactive iron barrier. Additionally, we employ CSIA to distinguish between biotic and abiotic transformations of trichloromethane and dichloromethane and reveal previously unreported isotope enrichment factors for trichloromethane reacting with Fe0 and dichloromethane while being consumed by dichloromethanefermenting organisms.

trichloromethane to dichloromethane using hydrogen as the electron donor. The second inoculum, known as DCMD, is a methanogenic mixed culture and contains a member of the Dehalobacter genus capable of using dichloromethane as the sole source of organic carbon and energy producing acetate and hydrogen.22 Degradation of Chlorinated Methanes with Fe0 and Enrichment Cultures CFH2 and DCMD. Experiments were conducted in 60 mL glass flasks. Bicarbonate-buffered anaerobic mineral media (50 mL) as described previously22 was used; however, cysteine and sulfide were excluded where Fe0 was present. Unless otherwise stated, 500 mg of Fe0 was supplied (17.9 mmol, 0.14 m2). Flasks were sealed with Teflon-lined butyl septa. The gas phase was then exchanged to N2/CO2 (4:1). Chlorinated methanes were added through the septa from concentrated aqueous stock solutions using a sterile glass syringe. The inoculation ratio was 10% (v/v). For negative controls the inoculum was injected though a 0.22 μm filter with a syringedriven filter unit (Millex). Cultures were incubated at 30 °C with agitation at 100 rpm on a horizontal shaker. Experiments were performed at least in triplicate. Inhibition of Trichloromethane Respiration by Tetrachloromethane. Four sets of triplicated trichloromethane respiring cultures were set up in 120 mL flasks containing 90 mL of anaerobic media inoculated with CFH2 (10 mL, 10% v/v). The chemical reducing agents sulfide and cysteine were supplied at 0.2 mM each, and hydrogen (0.2 bar overpressure) was supplied as the electron donor. Each flask was supplied with trichloromethane (100 μM), and then the triplicates were supplied with tetrachloromethane at 0, 13, 32, and 65 μM, respectively. Controls included autoclaved cultures to observe abiotic losses of tetrachloromethane due to reaction with sulfide and cysteine. Hydrogen and Carbon Monoxide Analysis. Hydrogen and carbon monoxide were analyzed by gas chromatography (GC) using a molecular sieve column (30 m × 0.32 mm × 25 μm). The carrier gas was nitrogen (2 mL min−1), inlet temperature was 250 °C, split ratio 10:1, and the oven was kept isothermal (40 °C). Headspace samples of 100 μL were withdrawn from culture flasks with a gastight glass syringe. Concentrations were quantified by comparison of peak areas with a three-point calibration curve (0−2000 μmol flask−1). Hydrogen was measured with a thermal conductivity detector, whereas CO was measured with a mass-selective detector (Agilent 5973 MSD) in selective ion-monitoring mode (masses 16 and 28). Chlorinated Methanes and Hydrocarbon Analysis. All four chlorinated methanes and hydrocarbon gases were analyzed by GC−FID using a GASPRO GS column (60 m × 0.32 mm, Agilent Technologies). The carrier gas was helium (3 mL min−1), inlet temperature was 250 °C, split ratio 10:1. Oven temperature program: isothermal 100 °C (1 min) and then 25 °C min−1 to 250 °C and held for 1 min. Headspace samples of a 100 μL volume were withdrawn from culture flasks with a pressure lockable gastight glass syringe. Concentrations were quantified by comparison of the peak area with a four-point calibration curve (0−20 μmol flask−1). Chlorinated methane standards were made in flasks with the same headspace to liquid ratio to account for partitioning of the compound between the gas and liquid phase. Formic Acid Analysis. Formic acid was analyzed using an enzyme-based assay kit (Megazyme catalog no. K-FORM). Briefly, 200 μL of filtered sample or standard was transferred to a 96 well plate, along with buffer and NADH. The absorbance was

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MATERIALS AND METHODS Chemicals. Chloromethanes and Fe0 (catalog no. 44890) were obtained from Sigma-Aldrich Pty Ltd. (Australia). The Fe0 particle size was 6−9 μm, and its surface area was determined to be 0.28 ± 0.002 m2/g by Brunauer−Emmett−Teller (BET) surface area analysis. High purity ethane, ethene, methane, carbon monoxide, and hydrogen were obtained from BOC gases (Australia). A mixed hydrocarbon gas standard containing 15 ppmv butane, propane, propene, propyne, ethane, ethene, ethyne, and methane in nitrogen was obtained from SigmaAldrich (USA). Inocula. The two inocula used in this study were enriched from an organochlorine-contaminated aquifer in Sydney, Australia.22 The first inoculum, the trichloromethane-respiring culture CFH2, is a nonmethanogenic enrichment that has been maintained on trichloromethane and hydrogen for three years. It contains the Dehalobacter strain UNSWDHB respiring 4482

DOI: 10.1021/es5052364 Environ. Sci. Technol. 2015, 49, 4481−4489

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the electron donor instead of Fe0, trichloromethane depletion took 17 days (Figure 1A), while the abiotic reaction of trichloromethane with Fe0 took 39 days (Figure 1C). There was no difference in the rate of depletion of trichloromethane in the abiotic control supplied filter sterilized inocula, thus demonstrating that microbially produced electron shuttles had no involvement in trichloromethane transformations as has been witnessed with supernatants from methanogenic cultures.14,34 In the abiotic reaction, dichloromethane accounted for approximately 6% of the supplied trichloromethane, whereas in the biological treatments CFH2/ H2 and CFH2/Fe0 dichloromethane accounted for 95% and 85% of the trichloromethane supplied, suggesting a biological transformation was taking place where Fe0 was the electron donor. The 3.5 fold increase in the rate of biological trichloromethane removal where Fe0 was the electron donor maybe due to increased hydrogen partial pressure in the Fe0 supplied flasks. In a parallel experiment the hydrogen partial pressure reached approximately 4 bar in biotic and Fe0 amended flasks after 7 days (Figure 2), which is 20-fold higher than when hydrogen is supplied externally (0.2 bar). The large increased hydrogen partial pressure would lead to faster mass transfer of hydrogen to Dehalobacter and therefore faster rates of dechlorination if hydrogen mass transfer was the rate-limiting step. Additionally, the ORB may also benefit from the lower redox conditions and extra surface area provided by the Fe0. Also note worthy is hydrogen partial pressures in biotic/Fe0 flasks were 2 fold higher than in abiotic flasks, indicating accelerated corrosion of Fe0 in the presence of microorganisms according with previous reports.13,35 However, given the accumulation of hydrogen it would seem that the acceleration is not due to consumption of hydrogen by hydrogenotrophic organisms as has been speculated previously.13 In addition to dichloromethane, 14 other hydrocarbons ranging from methane to hexane were also detected in the different experiments (Table 1). Hydrocarbons are produced by the reaction of carbon monoxide with Fe0 in a process known as the Fischer−Tropsch synthesis.36 This process involves the absorption of carbon monoxide to Fe0 where it is hydrogenated to −CH2−. Iron surface absorbed −CH2− moieties then polymerize to form longer chain hydrocarbons, and the chain polymerization terminates when the hydrocarbon desorbs from the iron surface (Figure S1, Supporting Information). As the hydrocarbon chain length increases so too does the compound hydrophobicity; this has been reported to lead to a substantial proportion of hydrocarbons remaining sorbed to the iron surface.37 This phenomenon could in part explain the low mass balance in abiotic reactions observed in the present study (Table 1). Additionally, carbon dioxide, which can be an abiotic degradation product,38 could not be measured in the current study, as all treatments are bicarbonate buffered, with 20% carbon dioxide in the headspace. In the abiotic reaction of trichloromethane with Fe 0 hydrocarbons accounted for ∼15% of the total trichloromethane supplied compared with ∼4% when CFH2 and Fe0 were combined. Formate amounts detected in the abiotic experiments were not different from those in organochlorine-free controls. Interestingly, in CFH2/Fe0 flasks 39 μmol of formate was produced, exceeding the amount that could be produced from trichloromethane (10 μmol), suggesting biological formate production (Table 1). Formate was not detected in the CFH2/H2 control. Formate production by a range of anaerobic bacteria and archaea has been observed when hydrogen and carbon dioxide were supplied.39,40 Both studies showed formate production was positively correlated to increasing hydrogen partial pressure.

measured at 340 nm before adding formate dehydrogenase. The plate was incubated at 21 °C for 20 min before remeasuring. Compound Specific Isotope Ratio Analysis (CSIA). Triplicate batch treatments were prepared in 250 mL culture flasks containing 200 mL of anaerobic media. Fe0 was supplied at 2 g (36 mmol) per flask, and flasks were inoculated with cultures CFH2 or DCMD at a volume ratio of 10% (v/v) where required. At regular intervals, 2 mL of liquid was withdrawn from the flasks and transferred to 10 mL headspace vials containing Na2SO4 (500 mg) and H2SO4 (1 M, 500 μL). Prior to CSIA, each vial was analyzed by headspace GC−FID to determine the aqueous concentration of chlorinated compounds. Aqueous phase concentrations were calculated using dimensionless Henry’s law constants (Hcc, 30 °C) for trichloromethane (0.18) and dichloromethane (0.12) where Hcc = Cw/Cg. Henry’s law constants were obtained from the US-EPA Web site (http://www.epa.gov/ athens/learn2model/part-two/onsite/esthenry.html). The 13C/12C isotopic ratio composition of the analytes was determined by a GC-combustion isotope ratio mass spectrometry system (GC−C-IRMS). The system was composed of an Agilent 7890 GC, Thermo Scientific GC IsoLink combustion interface, and a Finnigan MAT 253 mass spectrometer (Thermo Finnigan, Germany). The combustion temperature was 1000 °C. Headspace gas (1 mL) was injected into a split/splitless inlet at 250 °C with a split ratio of 1:10. Chloromethanes were separated on a CP-PoraBOND Q column (50 m × 0.32 mm × 5 μm, Varian). The GC oven was held at 100 °C for 1 min and then ramped at 20 °C min−1 to 250 °C and held for 1 min. Samples were analyzed in at least duplicate, and the instrument precision for replicate analyses was in general less than ±0.3‰. The carbon isotope composition is reported in δ-notation (per mil, ‰) relative to the Vienna Pee Dee Belemite standard (V-PDB, IAEA-Vienna). The isotopic enrichment factors (ε) were calculated applying the Rayleigh equation (eq 2)32 ln((δ13Ct + 1000)/(δ13C0 + 1000)) = ε /1000· ln f = (α − 1) ·ln f

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where δ Ct and δ C0 are the δ C values of the analyte at time t and time zero respectively, f is the fraction of analyte remaining, and α is the isotope fractionation factor. The slope of ln f versus ln ((δ13Ct + 1000)/(δ13C0 + 1000)) plot equates to α − 1, and ε = 1000·(α − 1). In these plots uncertainty is expressed as 95% confidence intervals. For comparison of experimentally determined enrichment factors (ε) between different compounds it is useful to express this value in terms of the apparent kinetic isotope effect (AKIE) (eq 3).30,33 The AKIE takes into account the number of carbon atoms in the molecule (n), the number of carbon atoms in reacting positions (x) and the number of carbons in chemically equivalent positions (z). Compounds with one carbon atom n, x, and z equal one. 13

AKIE =

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13

1 1 + (nxεz /1000)

13

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RESULTS AND DISCUSSION Transformations of Trichloromethane by Fe0 in the Presence or Absence of Trichloromethane Respiring Culture. Analysis of trichloromethane transformation rates by Fe0 and/or the nonmethanogenic enrichment culture CFH2 showed the fastest trichloromethane removal and dichloromethane/methane production rates occurred when both Fe0 and the enrichment culture were combined (5 days) (Figure 1B). In cultures with hydrogen as 4483

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Figure 1. Dechlorination of chlorinated methanes with trichloromethane (TCM) respring and dichloromethane (DCM) fermenting enrichment cultures CFH2 and DCMD. (A) Trichloromethane with CFH2 and H2. (B) TCM with CFH2 and Fe0. (C) TCM with filter sterilized CFH2 and Fe0. (D) TCM with Fe0. (E) Tetrachloromethane (TeCM) with CFH2 and Fe0. (F) TeCM with Fe0. (G) DCM depletion by fermentation by DCMD in the presence or absence of Fe0. (H) Methane production by dichloromethane fermenting cultures in the presence and absence of Fe0. Error bars represent one standard deviation (n = 3).

Transformation of Tetrachloromethane by Fe0 in the Presence and Absence of Trichloromethane Respiring Enrichment Culture CFH2. Inhibition experiments to determine the effect of tetrachloromethane on trichloromethane

Therefore, the relatively low hydrogen partial pressure in Fe0-free CFH2/H2 treatments (0.2 bar) compared with Fe0-supplied flasks (up to 4 bar, Figure 2) may explain why formate is not detected in the Fe0-free controls. 4484

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(8.5 μmol per flask) was completely removed within 2 days (Figure 1E,F). At the time of complete tetrachloromethane depletion trichloromethane and dichloromethane accounted for 90% and 8% of the supplied tetrachloromethane in both treatments. It was anticipated that in inoculated flasks the rate of subsequent trichloromethane removal would be accelerated by biological activity. However, biological activity was not evident, and the subsequent rate of trichloromethane depletion was similar in both inoculated and uninoculated flasks taking 22 days. The depletion of trichloromethane was again accompanied by the production of dichloromethane (30% of trichloromethane) and hydrocarbons (20% of trichloromethane) in both inoculated and uninoculated flasks. To further investigate the lack of biological activity, treatments were prepared where CFH2 was inoculated before and after tetrachloromethane had been consumed by reaction with Fe0 (Figure S3 A,B, Supporting Information). Hydrogen partial pressure was measured at the time of inoculation and was found to be 1.8 bar demonstrating that Dehalobacter was not electron donor limited. Inoculation after tetrachloromethane depletion made little difference with a lack of biological activity in both treatments. Trichloromethane depletion was slow and ∼50% complete after 20 days. This finding suggests that a product from the abiotic dechlorination of tetrachloromethane has and inhibitory effect on trichloromethane respiring Dehalobacter. Potential candidates include hexachloroethane and perchloroethene that can form by the dimerization of trichloromethyl radicals.42 However, these compounds were not present when reactions products were analyzed by GC/MS, therefore more work is required to elucidate the reason behind observed inhibition. In the context of reactive iron barrier function it can only be concluded that tetrachloromethane will prevent biological trichloromethane transformation by Dehalobacter species.

Figure 2. Hydrogen produced by the corrosion of Fe0 abiotically and in flasks inoculated with CFH2. Chlorinated compounds were not supplied in these treatments. Error bars represent one standard deviation (n = 3).

respiration revealed, first, that Dehalobacter UNSWDHB is not capable of tetrachloromethane respiration and, second, that tetrachloromethane concentration greater than 1 μM is inhibitory to trichloromethane respiration by Dehalobacter UNSWDHB (Figure S2A−D, Supporting Information). The level of inhibition imposed by tetrachloromethane on trichloromethane respiration reported here is in line with other reported inhibitory values for organohalide respiration (i.e., perchloroethene dechlorination (99% similarity in 16S rRNA sequence).44,45 The discovery of a low enrichment factor of −4.3 ± 0.45‰ (AKIE = 1.004) found here for Dehalobacter UNSWDHB was therefore surprising and shows activity of this strain in a reactive iron barrier could be detected using CSIA in contrast to strain CF. The opposite would the case outside a reactive iron barrier setting. The reason for the disparity in the enrichment factors is not clear as both organisms possess the gene encoding for the trichloromethane reductive dehalogenase (CfrA) with 95% similarity.45,46 CfrA has been functionally characterized for strain CF46 but not strain UNSWDHB. Strain UNSWDHB has three unique putative Rdh encoding genes; potentially one of these proteins is responsible for trichloromethane dechlorination. Regardless, this result shows that care should be taken when interpreting CSIA data, and detailed knowledge of participating organisms beyond 16S rRNA phylotyping maybe required. Primers targeting one of the unique Rdh genes in strain UNSWDHB would enable differentiation of the two trichloromethane-respiring Dehalobacter strains.

Because of the large difference in the isotope enrichment factors between biological dechlorination by strain UNSWDHB and the abiotic dechlorination by Fe0 it is possible to determine the extent of biotic versus abiotic dechlorination. The ratio of two competing degradation pathways (F) can be calculated from the observed isotope enrichment factor for the overall degradation process (εoverall) and from individual contributing processes (i.e., εCFH2 and εFe0) (eq 4).47 The overall enrichment factor for the two competing biotic abiotic reactions for the dechlorination of trichloromethane reported here (−8.3 ± 0.7‰) confirms that biological dechlorination predominated and accounted for 84% of the activity. ε − ε Fe0 Fabiotic/biotic = overall εCFH2 − ε Fe0 (4) This number is in general agreement with biotic versus abiotic degradation (75%) calculated using hydrocarbon production as a proxy for the abiotic activity (1.6 μmol abiotic and 0.39 μmol biotic/abiotic from Table 1). Dichloromethane Transformation with Fe0 in the Presence or Absence of a Dichloromethane Fermenting Methanogenic Culture DCMD. Because CFH2 produced dichloromethane from trichloromethane with or without Fe0 as electron donor, the microbial transformation of dichloromethane was studied also in the presence of Fe0. Microbial use of dichloromethane as a terminal electron acceptor is not known; however, there have been recent discoveries of dichloromethanefermenting Dehalobacter species.22,48 We have previously shown a syntrophic codependence of the dichloromethane fermenter in our DCMD culture on methanogens to remove hydrogen as hydrogen partial pressures as low as 0.2 bar were inhibitory to dichloromethane fermentation.22 In the presence of Fe0, DCMD completely removed 5 μmol of dichloromethane within 14 days, whereas DCMD without Fe0 (i.e., standard cultivation conditions) removed 5 μmol of dichloromethane in 22 days (Figure 1G). Dichloromethane was not depleted in uninoculated control flasks containing Fe0. Methane reached 58 μmol in DCMD/Fe0 flasks and 2 μmol per flask in DCMD flasks without Fe0 (Figure 1H). The increased rate of dichloromethane removal could be due to cometabolism by methanogens, which has been observed previously for the acetoclastic/methylotrophic methanogen (i.e., Methanosarcina thermophila), and the activity was attributed to the presence of hydrogen and unknown excreted biomolecule.14 Another possibility is the aforementioned benefit from the lower redox environment and extra surface area provided by Fe0. Stable Carbon Isotope Fractionation during Dichloromethane Fermentation in the Presence and Absence of Fe0. Dichloromethane was consumed in both Fe0-amended and Fe0-free treatments within 24 days (Figure 3D). Approximately 10-fold more methane was produced where Fe0 was present (36 ± 5 μmol), compared with 3.9 ± 0.4 μmol being produced in the Fe0-free set (data not shown). No dichloromethane disappearance was observed where Fe0 alone was supplied, and no change in the δ13C values was detected throughout the duration of the experiment (−33.3 ± 0.2‰ over 24 days, n = 5, data not shown). Enrichment of 13C was observed in both inoculated treatments with δ13C values as high as −8.0‰ in the DCMD culture and +3.0‰ in DCMD culture with Fe0 (Figure S5B,F, Supporting Information). The plot of ln f versus ln ((δ13Ct + 1000)/(δ13C0 + 1000)) showed a good fit of the data to the Rayleigh model, with linear regression R2 values of approximately 0.97 for both treatment types (Figure 3H). The ε values for the 4486

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Figure 3. CSIA experiments involving the degradation of trichloromethane (TCM) and dichloromethane (DCM). (A−C) Degradation of TCM (A) biologically with culture CFH2 and H2; (B) abiotically with Fe0; (C) combined biotic/abiotic degradation with culture CFH2 and Fe0. (E−G) Respective Rayleigh plots for δ13C in TCM. (D) Degradation of DCM by culture DCMD in the presence and absence of Fe0, and the respective Rayleigh plots are shown in the panels. Error bars in panels B−D represent one standard deviation (n = 3). Error bars for ln f in Rayleigh plots represent 5% variability in analyzing TCM and DCM concentration. In panel A, individual replicates are plotted due to the differences in lag phases in the TCM respiring culture. Uncertainties for isotope enrichment factors are 95% confidence intervals.

this difference will allow bioremediation practitioners to discriminate between the two processes. The difference in enrichment factors confirms the conclusion made from Figure 1G that a process other than dichloromethane fermentation is contributing to dichloromethane degradation in the presence of Fe0. One possible explanation could be related to the proliferation of methanogens evident by the increased methane production in the presence of Fe0 (Figure 1H). It has been shown previously that corrinoids and coenzyme F430

degradation of dichloromethane in the DCMD culture versus DCMD/Fe0 were significantly different, with values of −15.5 ± 1.5‰ and −23.5 ± 2.1‰ (±95% CI), respectively (Figure 3H). The first conclusion to be drawn here is that CSIA can be used to detect dichloromethane attenuation via fermentation in or outside a reactive iron barrier. While the enrichment factor for dichloromethane fermentation is large at −15.5‰ it is significantly smaller than that for methylotrophic dichloromethane oxidation, which ranges between −41 and −66‰.31,49 Therefore, 4487

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Environmental Science & Technology

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produced by methanogens can catalyze dichloromethane dechlorination with titanium citrate as the electron donor.50,51 Currently, there are no studies that have shown the extent of carbon isotope fractionation in chlorinated methanes during abiotic cobalamin-catalyzed reactions. However, studies have observed stable isotope fractionation in chlorinated ethenes and showed a larger fractionation in the cobalamin catalyzed reaction than that catalyzed by microorganisms.52,53 These findings are consistent with the shift in isotope fractionation in dichloromethane reported here; however, more work is required to fully understand the differences. Environmental Significance. The data presented here demonstrate that Dehalobacter-containing mixed cultures support trichloromethane transformation by Fe0, and bioaugmentation therefore has the potential to increase significantly trichloromethane removal rates in reactive iron barriers. However, the production of unreactive dichloromethane would be significantly increased, an effect that could also be induced by naturally populating endogenous trichloromethane respiring organisms (i.e., biostimulation). Large differences of the isotope-enrichment factors between abiotic trichloromethane transformation and reduction by strain UNSWDHB allow the quantitative differentiation of these two processes. Because such a strong difference was not found in Dehalobacter strain CF30 it might be important to select carefully the cultures used in bioaugmentation approaches to allow in situ monitoring of the bioaugmentation success via CSIA. Large carbon isotope fractionation observed in dichloromethane fermentation shows that CSIA could be a useful approach for monitoring microbial degradation of dichloromethane in the environment. It may also be possible to identify the mechanism of dichloromethane degradation due to differences in isotope enrichment for dichloromethane fermentation versus oxidation.

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ASSOCIATED CONTENT

S Supporting Information *

This material is available free of charge via the Internet at http:// pubs.acs.org/.

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AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Fax: +612 9385 1483. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We acknowledge the funding support of the Australian Research Council (LP110200610 and FT100100078), Orica Australia Pty Ltd., and Dow Chemical (Australia) Ltd. We also thank Benjamin Scheer, Ursula Günter, and Matthias Gehre for assistance with CSIA.

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REFERENCES

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Article

Environmental Science & Technology

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