Oxygen and chlorine isotopic fractionation during perchlorate biodegradation: laboratory results and implications for forensics and natural attenuation studies

Perchlorate is a widespread environmental contaminant having both anthropogenic and natural sources. Stable isotope ratios of O and Cl in a given sample of perchlorate may be used to distinguish its source(s). Isotopic ratios may also be useful for identifying the extent of biodegradation of perchlorate, which is critical for assessing natural attenuation of this contaminant in groundwater. For this approach to be useful, however, the kinetic isotopic fractionations of O and Cl during perchlorate biodegradation must first be determined as a function of environmental variables such as temperature and bacterial species. A laboratory study was performed in which the O and Cl isotope ratios of perchlorate were monitored as a function of degradation by two separate bacterial strains (Azospira suillum JPLRND and Dechlorospirillum sp. FBR2) at both 10 degrees C and 22 degrees C with acetate as the electron donor. Perchlorate was completely reduced by both strains within 280 h at 22 degrees C and 615 h at 10 degrees C. Measured values of isotopic fractionation factors were epsilon(18)O = -36.6 to -29.0% per hundred and epsilon(37)Cl = -14.5 to -11.5% per hundred, and these showed no apparent systematic variation with either temperature or bacterial strain. An experiment using (18)O-enriched water (delta(18)O = +198% per hundred) gave results indistinguishable from those observed in the isotopically normal water (delta(18)O = -8.1% per hundred) used in the other experiments, indicating negligible isotope exchange between perchlorate and water during biodegradation. The fractionation factor ratio epsilon(18)O/epsilon(37)Cl was nearly invariant in all experiments at 2.50 +/- 0.04. These data indicate that isotope ratio analysis will be useful for documenting perchlorate biodegradation in soils and groundwater. The establishment of a microbial fractionation factor ratio (epsilon(18)O/ epsilon(37)Cl) also has significant implications for forensic studies.     

Combined application of stable carbon isotope analysis and specific metabolites determination for assessing in situ degradation of aromatic hydrocarbons in a tar oil-contaminated aquifer

To evaluate the intrinsic bioremediation potential in an anoxic tar oil-contaminated aquifer at a former gasworks site, groundwater samples were qualitatively and quantitatively analyzed by compound-specific isotope analysis (CSIA) and signature metabolites analysis (SMA). 13C/12C fractionation data revealed conclusive evidence for in situ biodegradation of benzene, toluene, o-xylene, m/p-xylene, naphthalene, and 1-methylnaphthalene. In laboratory growth studies, 13C/12C isotope enrichment factors for anaerobic degradation of naphthalene (epsilon = -1.1 +/- 0.4) and 2-methylnaphthalene (epsilon = -0.9 +/- 0.1) were determined with the sulfate-reducing enrichment culture N47, which was isolated from the investigated test site. On the basis of these and other laboratory-derived enrichment factors from the literature, in situ biodegradation could be quantified for toluene, o-xylene, m/p-xylene, and naphthalene. Stable carbon isotope fractionation in the field was also observed for ethylbenzene, 2-methylnaphthalene, and benzothiophene but without providing conclusive results. Further evidence for the in situ turnover of individual BTEX compounds was provided by the presence of acetophenone, o-toluic acid, and p-toluic acid, three intermediates in the anaerobic degradation of ethylbenzene, o-xylene, and p-xylene, respectively. A number of groundwater samples also contained naphthyl-2-methylsuccinic acid, a metabolite that is highly specific for the anaerobic degradation of 2-methylnaphthalene. Additional metabolites that provided evidence on the anaerobic in situ degradation of naphthalenes were 1-naphthoic acid, 2-naphthoic acid, 1,2,3,4-tetrahydronaphthoic acid, and 5,6,7,8-tetrahydronaphthoic acid. 2-Carboxybenzothiophene, 5-carboxybenzothiophene, a putative further carboxybenzothiophene isomer, and the reduced derivative dihydrocarboxybenzothiophene indicated the anaerobic conversion of the heterocyclic aromatic hydrocarbon benzothiophene. The combined application of CSIA and SMA, as two reliable and independent tools to collect direct evidence on intrinsic bioremediation, leads to a substantially improved evaluation of natural attenuation in situ.     

Monitoring biodegradation of methyl tert-butyl ether (MTBE) using compound-specific carbon isotope analysis

Methyl tert-butyl ether (MTBE), the most common gasoline oxygenate, is frequently detected in surface water and groundwater. The aim of this study was to evaluate the potential of compound-specific isotope analysis to assess in situ biodegradation of MTBE in groundwater. For that purpose, the effect of relevant physical and biological processes on carbon isotope ratios of MTBE was evaluated in laboratory studies. Carbon isotope fractionation during organic phase/gas-phase partitioning (0.50 +/- 0.15@1000), aqueous phase/gas-phase partitioning (0.17 +/- 0.05@1000), and organic phase/aqueous-phase partitioning (0.18 +/- 0.24@1000) was small in comparison to carbon isotope fractionation measured during biodegradation of MTBE in microcosms based on aquifer sediments of the Borden site. In experiments with MTBE as the only substrate and a cometabolic experiment with 3-methypentane as primary substrate, MTBE became enriched in 13C by 5.1 to 6.9@1000 after 95 to 97% degradation. For both experiments, similar isotopic enrichment factors were obtained (-1.52 +/- 0.06 to -1.97 +/- 0.05@1000). Biodegradation of TBA, which accumulated transiently in the cometabolic microcosms, was also accompanied by carbon isotope fractionation, with an isotopic enrichment factor of -4.21 +/- 0.07@1000. This study suggests that carbon isotope analysis is a potential tool to trace in situ biodegradation of MTBE and TBA and thus to better understand the fate of these contaminants in the environment.     

Variations in 13C/12C and D/H enrichment factors of aerobic bacterial fuel oxygenate degradation

Reliable compound-specific isotope enrichment factors are needed for a quantitative assessment of in situ biodegradation in contaminated groundwater. To obtain information on the variability on carbon and hydrogen enrichment factors (epsilonC, epsilonH) the isotope fractionation of methyl tertiary (tert-) butyl ether (MTBE) and ethyl tert-butyl ether (ETBE) upon aerobic degradation was studied with different bacterial isolates. Methylibium sp. R8 showed a carbon and hydrogen isotope enrichment upon MTBE degradation of -2.4 +/- 0.1 and -42 +/- 4 per thousand, respectively, which is in the range of previous studies with pure cultures (Methylibium petroleiphilum PM1) as well as mixed consortia. In contrast, epsilonC of the beta/-proteobacterium L108 (-0.48 +/- 0.05 per thousand) and Rhodococcus ruber IFP 2001 (-0.28 +/- 0.06 per thousand) was much lower and hydrogen isotope fractionation was negligible (epsilonH < or = -0.2 per thousand). The varying isotope fractionation pattern indicates that MTBE is degraded by different mechanisms by the strains R8 and PM1 compared to L108 and IFP 2001. The carbon and hydrogen isotope fractionation of ETBE by L108 (epsilonC = -0.68 +/- 0.06 per thousand and epsilonH = -14 +/- 2 per thousand) and IFP 2001 (epsilonC = -0.8 +/- 0.1 per thousand and epsilonH = -11 +/- 4 per thousand) was very similar and seemed slightly higher than the fractionation observed upon MTBE degradation by the same strains. The low carbon and hydrogen enrichment factors observed during MTBE and ETBE degradation by L108 and IFP 2001 suggest a hydrolysis-like reaction type of the ether bond cleavage compared to oxidation of the alkyl group as suggested for the strains PM1 and R8. The variability of carbon and hydrogen enrichment factors should be taken into account when interpreting isotope pattern of fuel oxygenates with respect to biodegradation in contamination plumes.     

Carbon isotopic fractionation during anaerobic biotransformation of methyl tert-butyl ether and tert-amyl methyl ether

The fuel oxygenate methyl tert-butyl ether (MTBE) has been frequently detected in groundwater and surface water. Since contaminated sites are often subsurface, anaerobic degradation of MTBE will likely be significant for remediation. As traditional approaches to evaluate biodegradation generally involve laboratory microcosm studies which require time and resources, innovative approaches are needed to demonstrate active in situ biodegradation of MTBE. This study was conducted to gather information at the laboratory level to evaluate the potential of applying carbon isotope fractionation as an indicator for in situ biodegradation of the fuel oxygenates MTBE and tert-amyl methyl ether (TAME). In this study, MTBE utilization was observed in a methanogenic sediment microcosm after a lengthy lag period of about 400 days. MTBE utilization was sustained upon refeeding and subculturing. tert-Butyl alcohol (TBA) was found to accumulate after propagation of cultures. The MTBE-grown cultures also utilized TAME and produced tert-amyl alcohol (TAA). The detection of TBA and TAA indicated that ether bond cleavage was the initial step in degradation for both compounds. Carbon isotope fractionation during anaerobic MTBE and TAME degradation was studied, and isotopic enrichment factors (epsilon) with 95% confidence intervals of -15.6 +/-4.1% and -13.7+/-4.5% were estimated for anaerobic MTBE and TAME degradation, respectively. Addition of 2-bromoethanesulfonic acid, an inhibitor of methanogenesis, substantially prolonged the lag period before transformation, but did not influence carbon isotope fractionation. Our experiment provided strong evidence of significant carbon isotope fractionation during anaerobic MTBE and TAME degradation, demonstrating that this technique can be used as an indicator for in situ MTBE and TAME degradation.     

Treatment of perchlorate-contaminated groundwater using highly selective, regenerable ion-exchange technologies

Treatment of perchlorate-contaminated water using highly selective, regenerable ion-exchange and perchlorate-destruction technologies was demonstrated at a field site in California. Four treatment and four regeneration cycles were carried out, and no significant deterioration of resin performance was noted in 2 years. The bifunctional resin (Purolite A-530E) treated about 37,000 empty bed volumes (BVs) of groundwater before a significant breakthrough of perchlorate occurred at an average flow rate of 150 gpm (or 1 BV/min) and a feed perchlorate concentration of about 860 microg/L. Sorbed perchlorate (approximately 20 kg) was quantitatively recovered by eluting with as little as 1 BV of the FeCl3-HCl regenerant solution. The eluted ClO4- was highly concentrated in the third quarter of the first BV of the regenerant solution with a concentration up to 100,000 mg/L. This concentrated effluent greatly facilitated subsequent perchlorate destruction or recovery by precipitation as KClO4 salts. High perchlorate destruction efficiency (92-97%) was observed by reduction with FeCl2 in a thermoreactor, which enabled recycling of the FeCl3-HCl regenerant solution, thereby minimizing the need to dispose of secondary wastes containing ClO4-. This study demonstrates that a combination of novel selective, regenerable ion-exchange and perchlorate-destruction and/or recovery technologies could potentially lead to enhanced treatment efficiency and minimized secondary waste production.     

Stable carbon isotope fractionation during aerobic biodegradation of chlorinated ethenes

Stable isotope analysis is recognized as a powerful tool for monitoring, assessing, and validating in-situ bioremediation processes. In this study, kinetic carbon isotope fractionation factors (epsilon) associated with the aerobic biodegradation of vinyl chloride (VC), cis-1,2-dichloroethylene (cDCE), and trichloroethylene (TCE) were examined. Of the three solvents, the largest fractionation effects were observed for biodegradation of VC. Both metabolic and cometabolic VC degradation were studied using Mycobacterium aurum L1 (grown on VC), Methylosinus trichosporium OB3b (grown on methane), Mycobacterium vaccae JOB5 (grown on propane), and two VC enrichment cultures seeded from contaminated soils of Alameda Point and Travis Air Force Base, CA. M. aurum L1 caused the greatest fractionation (epsilon = -5.7) while for the cometabolic cultures, epsilon values ranged from -3.2 to -4.8. VC fractionation patterns for the enrichment cultures were within the range of those observed for the metabolic and cometabolic cultures (epsilon = -4.5 to -5.5). The fractionation for cometabolic degradation of TCE by Me. trichosporium OB3b was low (epsilon = -1.1), while no quantifiable carbon isotopic fractionation was observed during the cometabolic degradation of cDCE. For all three of the tested chlorinated ethenes, isotopic fractionation measured during aerobic degradation was significantly smaller than that reported for anaerobic reductive dechlorination. This study suggests that analysis of compound-specific isotopic fractionation could assist in determining whether aerobic or anaerobic degradation of VC and cDCE predominates in field applications of in-situ bioremediation. In contrast, isotopic fractionation effects associated with metabolic and cometabolic reactions are not sufficiently dissimilar to distinguish these processes in the field.     

Pathway dependent isotopic fractionation during aerobic biodegradation of 1,2-dichloroethane

1,2-Dichloroethane (1,2-OCA) is a widespread groundwater contaminant known to be biodegradable under aerobic conditions via enzymatic oxidation or hydrolytic dehalogenation reactions. Current literature reports that stable carbon isotope fractionation of 1,2-DCA during aerobic biodegradation is large and reproducible (-27 to -33/1000). In this study, a significant variation in the magnitude of stable carbon isotope fractionation during aerobic biodegradation was observed. Biodegradation in experiments involving microcosms, enrichment cultures, and pure microbial cultures produced a consistent bimodal distribution of enrichment factors (epsilon) with one mean epsilon centered on -3.9 +/- 0.6/1000 and the other on -29.2 +/- 1.9/1000. Reevaluation of epsilon in terms of kinetic isotope effects 12k/13k gave values of 12k/13k = 1.01 and 1.06, which are typical of oxidation and hydrolytic dehalogenation (S(N)2) reactions, respectively. The bimodal distribution is therefore consistent with the microbial degradation of 1,2-DCA by two separate enzymatic pathways. This interpretation is further supported in this study by experiments with pure strains of Xanthobacter autotrophicus GJ10, Ancylobacter aquaticus AD20, and Pseudomonas sp. Strain DCA1 for which the enzymatic degradation pathways are well-known. A small fractionation of -3.0/1000 was measured for 1,2-DCA degradation by Pseudomonas sp. Strain DCA1 (monooxygenase enzyme), while degradation by the hydrolytic dehalogenase enzyme by the other two pure strains was characterized by fractionation of -32.3/1000.     

Large Carbon Isotope Fractionation during Biodegradation of Chloroform by Dehalobacter Cultures

Compound specific isotope analysis (CSIA) has been applied to monitor bioremediation of groundwater contaminants and provide insight into mechanisms of transformation of chlorinated ethanes. To date there is little information on its applicability for chlorinated methanes. Moreover, published enrichment factors (ε) observed during the biotic and abiotic degradation of chlorinated alkanes, such as carbon tetrachloride (CT); 1,1,1-trichloroethane (1,1,1-TCA); and 1,1-dichloroethane (1,1-DCA), range from -26.5‰ to -1.8‰ and illustrate a system where similar C-Cl bonds are cleaved but significantly different isotope enrichment factors are observed. In the current study, biotic degradation of chloroform (CF) to dichloromethane (DCM) was carried out by the Dehalobacter containing culture DHB-CF/MEL also shown to degrade 1,1,1-TCA and 1,1-DCA. The carbon isotope enrichment factor (ε) measured during biodegradation of CF was -27.5‰ ± 0.9‰, consistent with the theoretical maximum kinetic isotope effect for C-Cl bond cleavage. Unlike 1,1,1-TCA and 1,1-DCA, reductive dechlorination of CF by the Dehalobacter-containing culture shows no evidence of suppression of the intrinsic maximum kinetic isotope effect. Such a large fractionation effect, comparable to those published for cis-1,2-dichloroethene (cDCE) and vinyl chloride (VC) suggests CSIA has significant potential to identify and monitor biodegradation of CF, as well as important implications for recent efforts to fingerprint natural versus anthropogenic sources of CF in soils and groundwater.     

Natural perchlorate has a unique oxygen isotope signature

Perchlorate is known to be a minor component of the hyperarid Atacama Desert salts, and its origin has long been a subject of speculation. Here we report the first measurement of the triple-oxygen isotope ratios (18O/16O and 17O/16O) for both man-made perchlorate from commercial sources and natural perchlorate extracted from Atacama soils. We found that the delta 18O values (i.e., normalized 18O/ 16O ratios) of man-made perchlorate were at -18.4+/-1.2%, whereas natural perchlorate has a variable delta 18O value, ranging from -4.5% to -24.8%. The delta 18O and delta 17O values followed the bulk Earth's oxygen isotope fractionation line for man-made perchlorate, but all Atacama perchlorates deviated from this line, with a distinctly large and positive 170 anomaly ranging from +4.2% to +9.6%. These findings provide a tool for the identification and forensics of perchlorate contamination in the environment. Additionally, they confirm an early speculation that the oxidation of volatile chlorine by 03 and the formation of HClO4 can be a sink (albeit a minor one) for atmospheric chlorine.     

Impact of bioavailability restrictions on microbially induced stable isotope fractionation. 2. Experimental evidence

Stable isotope fractionation analysis (SIFA) of contaminants is an emerging technique to characterize in situ microbial activity. The kinetic isotope effect in microbial degradation reactions, or enzyme catalysis, is caused by the preferential cleavage of bonds containing light rather than heavy isotopes. This leads to a relative enrichment of the heavier isotopes in the residual substrate pool. However, a number of nonisotopically sensitive steps preceding the isotopically sensitive bond cleavage may affect the reaction kinetics of a degradation process, thus reducing the observed (i.e., the macroscopically detectable) isotope fractionation. Low bioavailability of contaminants poses kinetic limitations on the biodegradation process and can significantly reduce the observed kinetic isotope fractionation. Here we present experimental evidence for the influence of bioavailability-limited pollutant biodegradation on observed stable isotope fractionation. Batch laboratory experiments were performed to quantify the toluene hydrogen isotope fractionation of Pseudomonas putida mt-2 (pWWO) subjected to different small concentrations of toluene with and without deuterium label, which corresponded to realistic environmental mass transfer scenarios. Detected isotope fractionations depended significantly on the toluene concentration, hence confirming the influence of substrate mass transfer limitation on observed isotope fractionation, hypothesized by Thullner et al. (Environ. Sci. Technol. 2008, 42,6544-6551). Our results indicate that the bioavailability of a substrate should be considered during quantitative analysis of microbial degradation based on SIFA.     

Hydrogen isotopic enrichment: an indicator of biodegradation at a petroleum hydrocarbon contaminated field site

Compound-specific carbon and hydrogen isotope analysis was used to investigate biodegradation of benzene and ethylbenzene in contaminated groundwater at Dow Benelux BV industrial site. delta13C values for dissolved benzene and ethylbenzene in downgradient samples were enriched by up to 2+/-0.5 per thousand, in 13C, compared to the delta13C value of the source area samples. delta2H values for dissolved benzene and ethylbenzene in downgradient samples exhibited larger isotopic enrichments of up to 27+/-5 per thousand for benzene and up to 50+/-5 per thousand for ethylbenzene relative to the source area. The observed carbon and hydrogen isotopic fractionation in downgradient samples provides evidence of biodegradation of both benzene and ethylbenzene within the study area at Dow Benelux BV. The estimated extents of biodegradation of benzene derived from carbon and hydrogen isotopic compositions for each sample are in agreement, supporting the conclusion that biodegradation is the primary control on the observed differences in carbon and hydrogen isotope values. Combined carbon and hydrogen isotope analyses provides the ability to compare biodegradation in the field based on two different parameters, and hence provides a stronger basis for assessment of biodegradation of petroleum hydrocarbon contaminants.     

Insight into methyl tert-butyl ether (MTBE) stable isotope fractionation from abiotic reference experiments

Methyl group oxidation, SN2-type hydrolysis, and SN1-type hydrolysis are suggested as natural transformation mechanisms of MTBE. This study reports for the first time MTBE isotopic fractionation during acid hydrolysis and for oxidation by permanganate. In acid hydrolysis, MTBE isotopic enrichment factors were epsilon(C) = -4.9 per thousand +/- 0.6 per thousand for carbon and epsilon(H) = -55 per thousand +/- 7 per thousand for hydrogen. Position-specific values were epsilon(C), reactive position = -24.3 per thousand +/- 2.3 oer thousand and epsilon(H,reactive position) = -73 per thousand +/- 9 per thousand, giving kinetic isotope effects KIE(C) = 1.025 +/- 0.003 and KIE(H) = 1.08 +/- 0.01 consistent with an SN1-type hydrolysis involving the tert-butyl group. The characteristic slope of deltadelta2H(bulk)/deltadelta13C(bulk) approximately epsilon(bulk,H)/ epsilon(bulk,C) = 11.1 +/- 1.3 suggests it may identify SN1-type hydrolysis also in settings where the pathway is not well constrained. Oxidation by permanganate was found to involve specifically the methyl group of MTBE, similar to aerobic biodegradation. Large hydrogen enrichment factors of epsilon(H) = -109 per thousand +/- 9 per thousand and epsilon(H,reactive position) = -342 per thousand +/- 16 per thousand indicate both large primary and large secondary hydrogen isotope effects. Significantly smaller values reported previously for aerobic biodegradation suggest that intrinsic fractionation is often masked by additional non-fractionating steps. For conservative estimates of biodegradation at field sites, the largest epsilon values reported should, therefore, be used.     

Stable carbon isotope fractionation during enhanced in situ bioremediation of trichloroethene

Time-series stable carbon isotope monitoring of volatile organic compounds (VOCs) atthe Idaho National Engineering and Environmental Laboratory's (INEEL) field site Test Area North (TAN) was conducted during a pilot study to investigate the treatment potential of using lactate to stimulate in situ biologic reductive dechlorination of trichloroethene (TCE). The isotope ratios of TCE and its biodegradation byproducts, cis-dichloroethene (c-DCE), trans-dichloroethene (t-DCE), vinyl chloride (VC), and ethene, in groundwater samples collected during the pilot studywere preconcentrated with a combination of purge-and-trap and cryogenic techniques in order to allow for reproducible isotopic measurements of the low concentrations of these compounds in the samples (down to 0.04 microM, or 5 ppb, of TCE). Compound-specific stable isotope monitoring of chlorinated solvents clearly differentiated between the effects of groundwater transport, dissolution of DNAPL at the source, and enhanced bioremediation. Isotope data from all wells within the zone of lactate influence exhibited large kinetic isotope effects during the reduction of c-DCE to VC and VC to ethene. Despite these large effects, the carbon isotope ratio of ethene in all these wells reached the carbon isotope ratios of the initial dissolved TCE, confirming the complete conversion of dissolved TCEto ethene. Conversely, the carbon isotope ratios of t-DCE were only marginally affected during the study, indicating that minimal biologic degradation of t-DCE was occurring.     

Stimulation and molecular characterization of bacterial perchlorate degradation by plant-produced electron donors

Root homogenate from poplar trees (Populus deltoides x nigra DN34, Imperial Carolina) stimulated perchlorate degradation in microcosms of soil and water samples collected at a perchlorate contaminated site, the Longhorn Army Ammunition Plant (LHAAP), located outside Karnack, Texas. Direct use of root products by perchlorate-degrading bacteria was shown for the first time as six pureculture bacteria isolated from LHAAP perchlorate-degrading microcosms degraded perchlorate when given root products as the sole exogenous source of carbon and electron donor. Nonenriched environmental consortia were able to utilize root products for perchlorate degradation, regardless of prior exposure to perchlorate. Microcosms that contained perchlorate-contaminated groundwater (MW-3) or uncontaminated surface water (Harrison Bayou) as inoculum degraded approximately 240 and 160 mg L(-1) perchlorate, respectively, using root products (approximately 440 mg L(-1) as COD) over 38 days. The predominant bacterial species in these aqueous microcosms, identified by DGGE, depended only upon the source inoculum as similar sequences were obtained whether root products or lactate was the electron donor. Sequences from DGGE bands that matched species within Dechloromonas, a genus consisting of many perchlorate degraders, were identified in all perchlorate-degrading microcosms. This study demonstrates the ability of root products to drive perchlorate respiration by bacteria and the potential for successful achievement of perchlorate rhizodegradation using in situ phytoremediation.     

Carbon and hydrogen isotopic fractionation during biodegradation of methyl tert-butyl ether

Carbon and hydrogen isotopic fractionation during aerobic biodegradation of MTBE by a bacterial pure culture (PM1) and a mixed consortia from Vandenberg Air Force Base (VAFB) were studied in order to assess the relative merits of stable carbon versus hydrogen isotopic analysis as an indicator of biodegradation. Carbon isotopic enrichment in residual MTBE of up to 8.1/1000 was observed at 99.7% biodegradation. Carbon fractionation was reproducible in the PM1 and VAFB experiments, yielding similar enrichment factors (epsilon) of -2.0/1000 +/- 0.1/1000 to -2.4/1000 +/- 0.3/1000 for replicates in the PM1 experiment and -1.5/1000 +/- 0.1/1000 to -1.8/1000 +/- 0.1/1000 for replicates in the VAFB experiment. Hydrogen isotopic fractionation was highly reproducible for the PM1 pure cultures, with epsilon values of -33/1000 +/- 5/1000 to -37/1000 +/- 4/1000 for replicate samples. In the VAFB microcosms, there was considerably more variability in epsilon values, with values of -29/1000 +/- 4/1000 and -66/1000 +/- 3/1000 measured for duplicate sample bottles. Despite this variability, hydrogen isotopic fractionation always resulted in 2H enrichment of the residual MTBE of >80/1000 at 90% biodegradation. The reproducible carbon fractionation suggests that compound-specific carbon isotope analysis may be used to estimate the extent of biodegradation at contaminated sites. Conversely, the large hydrogen isotopic fractionation documented during biodegradation of MTBE suggests that compound-specific hydrogen isotope analysis offers the most conclusive means of identifying in-situ biodegradation at contaminated sites.     

Assessing iron-mediated oxidation of toluene and reduction of nitroaromatic contaminants in anoxic environments using compound-specific isotope analysis

We evaluated compound-specific isotope analysis (CSIA) as a tool to assess the coupling of microbial toluene oxidation by Fe(III)-reducing bacteria and abiotic reduction of nitroaromatic contaminants by biogenic mineral-bound Fe(II) species. Examination of the two processes in isolated systems revealed a reproducible carbon isotope fractionation for toluene oxidation by Geobacter metal-lireducens with a solid Fe(III) phase as terminal electron acceptor. We found a carbon isotope enrichment factor, epsilonC, of -1.0 +/- 0.1 per thousand, which corresponds to an apparent kinetic isotope effect (AKIE(C)) of 1.0073 +/- 0.0009 for the oxidative cleavage of a C-H bond. Nitrogen isotope fractionation of the reduction of nitroaromatic compounds (NAC) by mineral-bound Fe(ll) species yielded a nitrogen isotope enrichment factor, epsilonN, of -39.7 +/- 3.4 per thousand for the reduction of an aromatic NO2-group (AKIE(N) = 1.0413 +/- 0.0037) that was constant for variable experimental conditions. Finally, AKIE values for C and N observed in coupled experiments, where reactive Fe(II) was generated through microbial activity, were identical to those obtained in the isolated experiments. This study provides new evidence on isotope fractionation behavior during contaminant transformation and promotes the use of CSIA for the elucidation of complex contaminant transformation pathways in the environment.     

Identifying competing aerobic nitrobenzene biodegradation pathways by compound-specific isotope analysis

Nitroaromatic compounds that contaminate soil and groundwater can be biodegraded by different, sometimes competing reaction pathways. We evaluated the combined use of compound-specific stable C and N isotope analysis to distinguish between enzymatic nitrobenzene oxidation by Comamonas sp. strain JS765 and partial reduction by Pseudomonas pseudoalcaligenes strain JS45 under aerobic conditions. Bulk 13C and 15N enrichment factors for nitrobenzene dioxygenation with JS765 were -3.9 per thousand +/- 0.09 per thousand (+/- 1sigma) and -0.75 per thousand +/- 0.09 per thousand, respectively. The corresponding primary apparent kinetic isotope effects (AKIE) of 1.0241 +/- 0.0005 for 13C and a secondary 15N AKIE of 1.0008 +/- 0.0001 are in very good agreement with the proposed enzymatic addition of dioxygen to the aromatic ring to form a cis-dihydrodiol in the rate-limiting step of nitrobenzene degradation. For the partial reduction pathway with JS45, epsilonC and epsilonN values were -0.57 per thousand +/- 0.06 per thousand and -26.6 per thousand +/- 0.7 per thousand. The 13C and 15N AKIEs amount to 1.0034 +/- 0.0003 and 1.0273 +/- 0.0008, respectively, and are consistent with the two-electron reduction and dehydration of the aromatic NO2 group to nitrosobenzene. The combined evaluation of delta13C and delta15N changes in nitrobenzene, based on the isotope enrichment behavior found in this laboratory study, provide an excellent starting point for assessing of the extent of nitrobenzene biodegradation via competing pathways in contaminated environments.     

Chlorine and carbon isotopes fractionation during volatilization and diffusive transport of trichloroethene in the unsaturated zone

To apply compound-specific isotope methods to the evaluation of the origin and fate of organic contaminants in the unsaturated subsurface, the effect of physicochemical processes on isotope ratios needs to be known. The main objective of this study is to quantify chlorine and carbon isotope fractionation during NAPL-vapor equilibration, air-water partitioning, and diffusion of trichloroethene (TCE) and combinations of these effects during vaporization in porous media. Isotope fractionation is larger during NAPL-vapor equilibration than air-water partitioning. During NAPL-vapor equilibration, carbon, and chlorine isotope ratios evolve in opposite directions although both elements are present in the same bond, with a normal isotope effect for chlorine (ε(Cl) = -0.39 ± 0.03‰) and an inverse effect for carbon (ε(C) = +0.75 ± 0.04‰). During diffusion-controlled vaporization in a sand column, no significant carbon isotope fractionation is observed (ε(C) = +0.10 ± 0.05‰), whereas fairly strong chlorine isotope fractionation occurs (ε(Cl) = -1.39 ± 0.06‰) considering the molecular weight of TCE. In case of carbon, the inverse isotope fractionation associated with NAPL-vapor equilibration and normal diffusion isotope fractionation cancel, whereas for chlorine both processes are accompanied by normal isotope fractionation and hence they cumulate. A source of contamination that aged might thus show a shift toward heavier chlorine isotope ratios.     

New evaluation scheme for two-dimensional isotope analysis to decipher biodegradation processes: application to groundwater contamination by MTBE

Compound-specific analysis of stable carbon and hydrogen isotopes was used to assess the fate of the gasoline additive methyl tert-butyl ether (MTBE) and its major degradation product tert-butyl alcohol (TBA) in a groundwater plume at an industrial disposal site. We present a novel approach to evaluate two-dimensional compound-specific isotope data with the potential to identify reaction mechanisms and to quantify the extent of biodegradation at complex field sites. Due to the widespread contaminant plume, multiple MTBE sources, the presence of numerous other organic pollutants, and the complex biogeochemical and hydrological regime atthe site, a traditional mass balance approach was not applicable. The isotopic composition of MTBE steadily changed from the source regions along the major contaminant plume (-26.4% to +40.0% (carbon); -73.1% to +60.3% (hydrogen)) indicating substantial biodegradation. Constant carbon isotopic signatures of TBA suggest the absence of TBA degradation at the site. Published carbon and hydrogen isotope fractionation data for biodegradation of MTBE under oxic and anoxic conditions, respectively, were examined and used to determine both the nature and the extent of in-situ biodegradation along the plume(s). The coupled evaluation of two-dimensional compound-specific isotope data explained both carbon and hydrogen fractionation data in a consistent way and indicate anaerobic biodegradation of MTBE along the entire plume. A novel scheme to reevaluate empiric isotopic enrichment factors (epsilon) in terms of theoretically based intrinsic carbon (12k/13k) and hydrogen (1k/2k) kinetic isotope effects (KIE) is presented. Carbon and hydrogen KIE values, calculated for different potential reaction mechanisms, imply that anaerobic biodegradation of MTBE follows a SN2-type reaction mechanism. Furthermore, our data suggest that additional removal process(es) such as evaporation contributed to the overall MTBE removal along the plume, a phenomenon that might be significant also for other field sites at tropic or subtropic climates with elevated groundwater temperatures (25 degrees C).