ARTICLE pubs.acs.org/est
Pathway-Dependent Isotope Fractionation during Aerobic and Anaerobic Degradation of Monochlorobenzene and 1,2, 4-Trichlorobenzene Xiaoming Liang,† Michael R. Howlett,† Jennifer L. Nelson,‡ Gavin Grant,§ Sandra Dworatzek,|| Georges Lacrampe-Couloume,† Stephen H. Zinder,‡ Elizabeth A. Edwards,^ and Barbara Sherwood Lollar*,† †
Department of Geology, University of Toronto, Toronto, Ontario M5S 3B1, Canada Department of Microbiology, Cornell University, Ithaca, New York 14853, United States § Geosyntec Consultants Inc., Guelph, Ontario N1G 5G3, Canada SiREM, Guelph, Ontario N1G 5G3, Canada ^ Department of Chemical Engineering and Applied Chemistry, University of Toronto, Toronto, Ontario M5S 3E5, Canada
)
‡
ABSTRACT: Stable carbon isotope fractionation is a valuable tool for monitoring natural attenuation and to establish the fate of groundwater contaminants. In this study, we measured carbon isotope fractionation during aerobic and anaerobic degradation of two chlorinated benzenes: monochlorobenzene (MCB) and 1,2,4-trichlorobenzene (1,2,4-TCB). MCB isotope fractionation was measured in anaerobic methanogenic microcosms, while 1,2,4-TCB isotope experiments were carried out in both aerobic and anaerobic microcosms. Large isotope fractionation was observed in both the anaerobic microcosm experiments. Enrichment factors (ε) for anaerobic reductive dechlorination of MCB and 1,2,4-TCB were 5.0% ( 0.2% and 3.0% ( 0.4%, respectively. In contrast, no significant isotope fractionation was found during aerobic microbial degradation of 1,2,4-TCB. The cleavage of a CCl σ bond occurs during anaerobic reductive dechlorination of MCB and 1,2,4-TCB, while no σ bond cleavage is involved during aerobic degradation via dioxygenase. The difference in isotope fractionation for aerobic versus anaerobic biodegradation of MCB and 1,2,4-TCB can be explained by the difference in the initial step of aerobic versus anaerobic biodegradation pathways.
’ INTRODUCTION Chlorinated benzenes (CBs) are frequently detected groundwater contaminants due to their widespread use as solvents, pesticides, and industrial intermediates.13 CBs have become an environmental issue because of their toxicity and persistence in aquifers.3,4 Under aerobic condition, CBs with four or fewer chlorine substituents can serve as growth substrates for a variety of bacteria and are oxidized to chlorocatechol intermediates.1,57 On the other hand, anaerobic reductive dechlorination of CBs [e.g., trichlorobenzenes (TCBs),8,9 dichlorobenzenes (DCBs),10,11 and monochlorobenzene (MCB)10] removes chlorine atoms sequentially and preferentially from more highly chlorinated CBs and leads to the formation of less chlorinated daughter products under both methanogenic and sulfate-reducing conditions. Some anaerobic bacteria have been shown to use CBs as terminal electron acceptors for growth. For instance, Dehalococcoides strain CBDB1 can reductively dechlorinate CBs with three or more chlorines,12,13 whereas Dehalococcoides strain 195,14 Dehalococcoides strain DCMB5,15 and bacterium DF-116 dechlorinate a smaller spectrum of CB substrates. Transformation of MCB to benzene is less than common, but it has been observed r 2011 American Chemical Society
in a methanogenic enrichment culture.17 Based on the extremely low yield of benzene, this is likely a cometabolic process. Fung et al.10 recently described complete reductive dechlorination of DCBs and MCB to benzene in anaerobic microcosms. However, the microbial community in the microcosms has not yet been described. Compound-specific isotope analysis (CSIA) has been recently used to identify1821 and quantify2224 in situ biodegradation of various contaminants. The preferential rate of cleavage of chemical bonds containing lighter isotopes (e.g., 12C and 1H) relative to those containing heavier isotopes (e.g., 13C and 2H) leads to enrichment of heavier isotopes in the remaining substrate during the biodegradation process.3134 Isotope fractionation typically can be described by the Rayleigh model:25 R ¼ R0 f ðεbulk =1000Þ
ð1Þ
Received: April 11, 2011 Accepted: August 18, 2011 Revised: August 18, 2011 Published: August 18, 2011 8321
dx.doi.org/10.1021/es201224x | Environ. Sci. Technol. 2011, 45, 8321–8327
Environmental Science & Technology where R is the isotope ratio of the remaining substrate at any time, R0 is its isotopic ratio at time zero, f is the fraction of the remaining substrate at a given time (i.e., C/C0), and εbulk is the bulk enrichment factor. εbulk is often used to describe the magnitude of isotope fractionation, and the extent of in situ biodegradation can be estimated by use of the Rayleigh model combined with isotope data from field samples.22,23,26 Carbon isotope fractionation has been investigated systematically for degradation of chlorinated ethenes1821,2729 and aromatic hydrocarbons.20,3036 Carbon isotope fractionation during biodegradation of chlorinated benzenes has received much less attention to date. Only two studies have tried to determine the specific carbon isotope fractionation factors associated with biodegradation of chlorinated aromatics. No carbon isotope fractionation was found during aerobic degradation of 1,2,4-TCB by the strain Pseudomonas sp. P51.37 Similarly, negligible isotope fractionation with εbulk values from 0.1% ( 0.1% to 0.4% ( 0.1% was reported during the mineralization of MCB by aerobic MCBdegrading strains, including Ralstonia sp. DSM 8910, Pseudomonas veronii B547, Acidovorax facilis B517, and Rhodococcus erythropolis B528.38 Even less information is available regarding carbon isotope fractionation of chlorinated aromatics under anaerobic conditions. Significant fractionation was reported during anaerobic reductive dechlorination of 1,2,3-TCB (εbulk = 3.4% ( 0.3%) and 1,2,4-TCB (εbulk = 3.2% ( 0.5%) by the pure strain Dehalococcoides sp. strain CBDB1 in one study.37 To our knowledge, no other laboratory microcosm studies have been conducted to investigate the fractionation factors associated with biodegradation of CBs under anaerobic conditions, and no studies have yet reported the specific fractionation factors associated with biodegradation by mixed microbial communities in sediments taken from contaminated field sites. Field studies have simply documented the observations that MCB was more 13C-enriched at the fringes of a MCB-contaminated plume relative to its source zone,38 or at a second site, that there was an enrichment of the cumulative isotope composition of all CBs in field samples,39 suggesting that anaerobic reductive dechlorination of chlorinated aromatics may occur in the field. In recent studies, characterization of carbon isotope fractionation factors during biodegradation investigations of chlorinated ethenes and chlorinated ethanes by pure strains and mixed microbial communities and during laboratory and field studies have all demonstrated that a range of fractionation factors may be observed, even if compounds are degraded by the same pathway.1821,2729 The overall goal of this study was to move beyond studies of pure strains alone to determine carbon isotope fractionation factors for MCB, and for 1,2,4-TCB, under a range of conditions (aerobic and anaerobic), and using mixed microbial populations in microcosms created from materials taken directly from contaminated field sites. MCB and 1,2,4-TCB were chosen as model CBs compounds in this study because (1) MCB is the most persistent chlorobenzene to reductive dechlorination and (2) 1,2,4-TCB is one of the most widely used chlorobenzenes and is subject to both aerobic and anaerobic degradation. The first objective of this study was to characterize carbon isotope fractionation during anaerobic reductive degradation of MCB in microcosms prepared from sediments from the DuPont Chambers Works site (Salem County, New Jersey). The second objective was to measure εbulk for both aerobic and anaerobic degradation of 1,2,4-TCB in microcosms prepared from sediments from a historically CBscontaminated site (Sunnyvale, CA) and to compare these results to previous work on 1,2,4-TCB done with pure cultures.37
ARTICLE
’ EXPERIMENTAL SECTION Chemicals. The following chemicals were purchased from SigmaAldrich (St. Louis, MO): 1,2,4-TCB (99%), 1,2-DCB (99%), 1,3-DCB (99%), 1,4-DCB (99%), MCB (99.5%), benzene (99.8%), hexane (99%), bicarbonate (99.5%), and yeast extract. Microcosm Setup. Triplicate bottles for MCB microcosm experiments were prepared in an anaerobic glovebox (Labconco Co., Kansas City, MO) in 120-mL glass bottles capped with Mininert valves. Anaerobic deionized water (50 mL) and 20 g of wet sediment collected from the DuPont Chambers Works site were added to each bottle. In order to complete experiments within a reasonable period of time, we added 2 mL of slurry from an actively reductively dechlorinating microcosm to each fresh microcosm. The sediment in the active microcosm was obtained from the same site as that in fresh microcosms. The microcosms were then amended with 1 g/L sodium bicarbonate as a buffer and 0.2 g/L yeast extract as an electron donor. The headspace of the bottles consisted of 80% N2 and 20% CO2. The bottles were spiked with neat MCB to produce an initial aqueous MCB concentration (C0) of approximately 700 μM. Experimental bottles were manually shaken about 30 min in the anaerobic chamber in order to ensure equilibrium partitioning between headspace and liquid phase. The microcosms were incubated in the dark at room temperature in the anaerobic chamber. Abiotic controls containing only deionized water and MCB were also prepared. Triplicate bottles of for 1,2,4-TCB microcosm experiments were conducted in 250-mL Mininert sealed glass bottles. Each bottle contained 200 mL of groundwater and 50 mL of wet sediment (bulk volume) obtained from a site in Sunnyvale, CA. The headspace in each bottle was approximately 20 mL. Groundwater and sediments for aerobic and anaerobic microcosms came from an aerobic zone and an anaerobic zone of the site, respectively. Neat 1,2,4-TCB was added to aerobic and anaerobic microcosms separately to produce initial aqueous concentrations (C0) of approximately 23 μM and 60 μM, respectively. Experimental bottles were shaken manually for approximately 30 min to ensure equilibrium partitioning between headspace and liquid phase. Typically, 24 h was allowed for the pure phase to dissolve before sampling. Sterile controls were also prepared by following the same procedures but adding 2.8 mL of 2.7% HgCl2 and 0.5 mL of 5% NaN3 in order to prevent or reduce any intrinsic microbial activity. Analytical Methods. For MCB experiments, concentrations of MCB and benzene were determined by manually injecting 300 μL of headspace sample from each experimental bottle into a Varian 3400 gas chromatograph (GC) equipped with a flame ionization detector (FID) and an Agilent J&W GS-Q capillary column (30 m 0.53 mm). The injector temperature was 200 C and the detector temperature was 210 C. The oven temperature was initially 100 C, immediately ramped to 210 at 30 C/min, and held isothermally at 210 C for 7 min. Threepoint external calibration curves were prepared daily. Relative standard deviations for samples and standards by this method were typically (5%. For analysis of 1,2,4-TCB and degradation products (DCBs and MCB), 9.5 mL of microcosm liquid was withdrawn by syringe, and 9 mL was stored in 9-mL glass vials without headspace and stored at 4 C for less than 5 h. CBs in the 9 mL of liquid were extracted by adding 180 μL of hexane after the method of Dempster et al.,40 8322
dx.doi.org/10.1021/es201224x |Environ. Sci. Technol. 2011, 45, 8321–8327
Environmental Science & Technology followed by the injection of 2 μL of the solvent extract into a Varian 3380 GC with a FID and a Restek VOCOL capillary column (30 m 0.25 mm 1.5 μm). In future studies, addition of an internal standard into the hexane before extraction of the 1,2,4TCB might improve reproducibility. The oven temperature was isothermal at 120 C for 15 min, ramped at 15 C/min to 210 C, and held isothermally at 210 C for 2 min. Each extracted sample was analyzed in duplicate. External calibration standards for GC/ FID analysis were prepared in hexane. Relative standard deviations for the liquidliquid extraction method were typically (15%. Stable carbon isotope analysis for MCB experiments was conducted by direct injection of headspace samples into a Varian 3400 GC coupled with a Finnigan Mat 252 isotope ratio mass spectrometry via a combustion interface (GC-C-IRMS) after the method of Slater et al.41 The combustion oven temperature was maintained at 980 C. GC separation was done on a VOCOL capillary column (60 m 0.25 mm 1.5 μm). The GC temperature was initially 40 C, immediately ramped to 210 at 25 C/min, and held at 210 C for 10 min. The injector temperature was 180 C and the split ratio was 6:1. Samples for stable carbon isotope analysis for 1,2,4-TCB and less chlorinated transformation products were prepared by hexane solvent extraction as described above. Isotope analysis was done by injecting 10 μL extracts into the GC-C-IRMS with the split ratio 6:1 on the GC column as described above. The GC temperature program was 1 min isothermal at 40 C, ramped at 3 C/min to 100 C, increased at 1 C/min to 133 C, ramped at 10 C/min to 200 C, and isothermal at 200 C for 15 min. Isotope standards were run daily. The total analytical uncertainty (reproducibility and accuracy) for each sample was (0.5%.42 Carbon isotope ratios are reported as δ13C with respect to the international standard V-PDB (in units of %). ! ! 13 13 C C 12 12 C C sample std ! δ13 C ¼ 1000 ð2Þ 13 C 12 C std
’ RESULTS Abiotic and Sterile Controls. During the course of degradation experiments, no significant variation in the concentration of MCB (or 1,2,4-TCB) was observed in abiotic or sterile controls, and the δ13C values in control bottles were within (0.5% of initial δ13C0 for all experiments. Reductive Dechlorination of Monochlorobenzene in Anaerobic Microcosms. Figure 1A shows that MCB was almost completely transformed to benzene within 140 h. Dechlorination rates in three replicate bottles were in good agreement with each other. Significant carbon isotope fractionation was observed during the reductive dechlorination of MCB (Figure 1B). Values of δ13C for MCB became more enriched from 25.6% initially to 12.4% when approximately 90% of MCB was degraded (f = 0.1) (Figure 1B). Experimentally measured δ13C values of parent compound MCB fit a Rayleigh isotopic model (eq 1) (Figure 1B). The εbulk value is 5.0% ( 0.2%, which was calculated on the basis of combined data from three replicate bottles (Table 1). Carbon isotope values of the degradation product benzene were always more depleted than that of the parent compound MCB and also became more enriched during the degradation
ARTICLE
Figure 1. Reductive dechlorination of MCB in anaerobic microcosms. (A) Concentrations of MCB and benzene versus time. Error bars represent (5% error as described in text. (B) δ13C of remaining MCB and benzene versus fraction (f) of MCB remaining. Error bars on C/C0 represent (7% total uncertainty, calculated by propagation of error in concentration (see text), and error bars on δ13C represent (0.5% (reproducibility and accuracy). The solid line represents a Rayleigh model fit as described by eq 1, and the dashed line represents the calculated isotope ratios of benzene according to eq 3.
experiment (Figure 1B). The isotope composition of benzene was continually monitored even after the reaction proceeded beyond 140 h (data not shown). The final δ13C value of benzene at ∼350 h was 26.0%, which is within error ((0.5%) of the initial isotope ratio of the substrate MCB (25.6%), indicating quantitative conversion of MCB to benzene. For a product accumulating in a closed system, the cumulative δ13C value of the product can be calculated on the basis of the following equation from ref 25: RAP ¼
R0 fR 1f
ð3Þ
where RAP is the isotope ratio of the accumulated product and all other symbols are as previously defined in eq 1. From this equation, the dashed line in Figure 1B shows the expected δ13C values for benzene produced from biodegradation of MCB. Experimentally measured δ13C values of benzene were very close to those calculated values, and this isotope mass balance provides additional evidence that benzene was the only product from the transformation of MCB. Aerobic and Anaerobic Degradation of 1,2,4-Trichlorobenzene in Microcosms. Nearly complete degradation of 1,2,4TCB was observed within 80110 h in three active aerobic microcosms (Figure 2A). No other CBs were detected in active microcosms during the course of experiments, suggesting that 8323
dx.doi.org/10.1021/es201224x |Environ. Sci. Technol. 2011, 45, 8321–8327
Environmental Science & Technology
ARTICLE
Table 1. Measured Carbon Bulk Enrichment Factors (εbulk) and Apparent Kinetic Isotope Effects for Carbon (AKIEC) for Biodegradation of MCB and 1,2,4-TCB condition
εbulka (%)
R2
nb
xb
zb
AKIECc
ref
MCB Ralstonia sp. DSM 8910/aerobic
0.4 ( 0.1
NAd
6
4
4
1.0024 ( 0.0006
38
Pseudomonas veronii B547/aerobic Acidovorax facilis B517/aerobic
0.2 ( 0.2 0.1 ( 0.1
NA NA
6 6
4 4
4 4
1.001 ( 0.001 1.0006 ( 0.0006
38 38
Rhodococcus erythropolis B528/aerobic
0.30 ( 0.04
NA
6
4
4
1.0018 ( 0.0002
anaerobic microcosm
5.0 ( 0.2
0.98
6
1
1
1.031 ( 0.001
38 this study
1,2,4-TCB Dehalococcoides sp. strain CBDB1
3.2 ( 0.5
NA
6
1
1
1.020 ( 0.003
37
anaerobic microcosm
3.0 ( 0.4
0.93
6
1
1
1.018 ( 0.003
this study
Pseudomonas sp. strain P51/aerobic
NFe
37
aerobic microcosm
NFe
this study
a
Enrichment factors are calculated on the basis of combined data from all replicate bottles for each microcosm, and uncertainties are 95% confidence intervals determined from the slope of the linear regression of ln f versus ln (R/R0) through all replicates. b Regardless of pathway, n = 6 for both MCB and 1,2,4-TCB. For aerobic degradation of MCB, x = 4 and z = 4 because four C atoms at positions 2, 3, 5, and 6 (Figure 4) have equal potential for bond cleavage from the attack of reactive oxygen species. For anaerobic degradation of both MCB and 1,2,4-TCB, x = 1 and z = 1 because the C atom at position 1 in MCB and the C atom at position 2 in 1,2,4-TCB (Figure 4) are the only carbons with potential for CCl bond cleavage during the initial dechlorination process. c Uncertainties are 95% confidence intervals calculated from propagation of error. d NA indicates that an R2 value was not reported. e NF indicates no significant fractionation.
aerobic mineralization was the major degradation mechanism responsible for the disappearance of 1,2,4-TCB.5,6,37 There was no change in carbon isotope composition of 1,2,4-TCB during aerobic degradation (Figure 2B). The average stable carbon isotope signature of 1,2,4-TCB during the course of the experiment was 28.5% ( 0.3% for all active aerobic microcosms (Figure 2B), within error of stable carbon isotope value of 1,2,4TCB (28.8% ( 0.5%) for sterile control aerobic microcosms. Figure 3A shows significant removal of 1,2,4-TCB in anaerobic microcosms over 70150 days. The major product observed was 1,4-DCB, while a small amount of MCB (
