Insights into enzyme kinetics of chloroethane biodegradation using compound specific stable isotopes.

While compound specific isotope analysis (CSIA) has been used extensively to investigate remediation of chlorinated ethenes, to date considerably less information is available on its applicability to chlorinated ethanes. In this study, biodegradation of 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA) was carried out by a Dehalobacter-containing mixed culture. Carbon isotope fractionation factors (ε) measured during whole cell degradation demonstrated that values for 1,1,1-TCA and 1,1-DCA (-1.8‰ and -10.5‰, respectively) were significantly smaller than values reported for abiotic reductive dechlorination of these same compounds. Similar results were found in experiments degrading these two priority pollutants by cell free extracts (CFE) where values of -0.8‰ and -7.9‰, respectively, were observed. For 1,1,1-TCA in particular, the large kinetic isotope effect expected for cleavage of a C-Cl bond was almost completely masked during biodegradation by both whole cells and CFE. Comparison to previous studies demonstrates that these patterns of isotopic fractionation are not attributable to transport effects across the cell membrane, as had been seen for other compounds such as PCE. In contrast these results reflect significant differences in the kinetics of the enzymes catalyzing chlorinated ethane degradation.

Quantification of biotransformation of chlorinated hydrocarbons in a biostimulation study: added value via stable carbon isotope analysis.

Stable carbon isotope analysis of chlorinated aliphatic compounds was performed at an in situ biostimulation pilot test area (PTA) at a site where 1,2-dichloroethane (1,2-DCA) and trichloroethene (TCE) were present in groundwater. Chlorinated products of TCE reductive dechlorination (cis-dichloroethene (cDCE) and vinyl chloride (VC)) were present at concentrations of 17.5 to 126.4 micromol/L. Ethene, a potential degradation product of both 1,2-DCA dihaloelimination and TCE reductive dechlorination was also present in the PTA. Emulsified soybean oil and lactate were added as electron donors to stimulate anaerobic dechlorination in the PTA. Stable carbon isotope analysis provided evidence that dechlorination was occurring in the PTA during biostimulation, and a means of monitoring changes in dechlorination efficiency over the 183 day monitoring period. Stable carbon isotope analysis was also used to determine if ethene production in the PTA was due to dechlorination of TCE, 1,2-DCA, or both. Fractionation factors (alpha) were determined in the laboratory during anaerobic biotransformation of 1,2-DCA via a dihaloelimination reaction in four separate enrichment cultures. These alpha values (as well as the previously published ranges of alpha for the dechlorination of TCE, cDCE and 1,2-DCA) were used, along with isotopic values measured during the pilot test, to derive quantitative estimates of biotransformation during the pilot test. Dechlorination was found to account for 10.7 to 35.9%, 21.9 to 74.9%, and 54.4 to 67.8% of 1,2-DCA, TCE and cDCE concentration loss respectively in the PTA. Stable carbon isotope analysis indicates that dechlorination of 1,2-DCA, TCE and cDCE were all significant processes during the pilot test, while ethene production during the pilot test was dominated by 1,2-DCA dihaloelimination. This study demonstrates how stable carbon isotope analysis can provide more conservative estimates of the extent of biotransformation than do conventional protocols. In addition, in a complex mixed plume such as this, compound specific isotope analysis is shown to be one of the few methods available for clarifying dominant biotransformation pathways where breakdown products are non-exclusive (i.e. ethene).

Isotope analysis as a natural reaction probe to determine mechanisms of biodegradation of 1,2-dichloroethane.

1,2-Dichloroethane (1,2-DCA), a chlorinated aliphatic hydrocarbon, is a well-known groundwater contaminant. In this study, fractionation of stable carbon isotope values of 1,2-DCA during biodegradation was used as a novel reaction probe to provide information about the mechanism of 1,2-DCA biodegradation under both aerobic (O2-reducing) and anaerobic (NO3-reducing) conditions. Under O2-reducing conditions, an isotopic enrichment value (epsilon) of -25.8 +/- 1.1 per thousand (+/-95% confidence intervals) was measured for the enrichment culture. Under NO3-reducing conditions, an epsilon-value of -25.8 +/- 3.5 per thousand was measured. The microbial culture produced isotopic enrichment values (epsilon) that are not only large and reproducible, but also are the same whether O2 or NO3 was used as an electron acceptor. Combining data measured under both O2- and NO3-reducing conditions, an isotopic enrichment value (epsilon) of -25.8 +/- 1.6 per thousand is measured for the microbial culture during 1,2-DCA degradation. The epsilon-value can be converted into a kinetic isotope effect (KIE) value to relate the observed isotopic fractionation to the mechanism of degradation. This KIE value (1.05) is consistent with degradation via hydrolytic dehalogenation under both electron-accepting conditions. This study demonstrates the added value of compound-specific isotope analysis not only as a technique to verify the occurrence and extent of biodegradation in the field, but also as a natural reaction probe to provide insight into the enzymatic mechanism of contaminant degradation.

An approach for assessing total instrumental uncertainty in compound-specific carbon isotope analysis: implications for environmental remediation studies.

Determination of compound-specific carbon isotope values by continuous flow isotope ratio mass spectrometry is impacted by variation in several routine operating parameters of which one of the most important is signal size, or linearity. Experiments were carried out to evaluate the implications of these operating parameters on both reproducibility and accuracy of delta13C measurements. A new method is described for assessing total instrumental uncertainty of routine compound-specific delta13C analysis, incorporating both accuracy and reproducibility. These findings have important implications for application of compound-specific isotope analysis in environmental geochemistry and in particular for the rapidly developing field of isotopic investigation of biodegradation and remediation of organic chemicals in contaminant hydrogeology.

Effects of trace element concentration on enzyme controlled stable isotope fractionation during aerobic biodegradation of toluene.

The effects of iron concentration on carbon and hydrogen isotopic fractionation during aerobic biodegradation of toluene by Pseudomonas putida mt-2 were investigated using a low iron medium and two different high iron media. Mean carbon enrichment factors (epsilonc) determined using a Rayleigh isotopic model were smaller in culture grown under high iron conditions (epsilonc = -1.7+/-0.1%) compared to low iron conditions (epsilonc = -2.5+/-0.3%). Mean hydrogen enrichment factors (epsilonH) were also significantly smaller for culture grown under high iron conditions (epsilonH = -77 +/-4%) versus low iron conditions (EpsilonH = -159+/-11%). A mechanistic model for enzyme kinetics was used to relate differences in the magnitude of isotopic fractionation for low iron versus high iron cultures to the efficiency of the enzymatic transformation. The increase of carbon and hydrogen enrichment factors at low iron concentrations suggests a slower enzyme-catalyzed substrate conversion step (k2) relative to the enzyme-substrate binding step (k-l) at low iron concentration. While the observed differences were subtle and, hence, do not significantly impact the ability to use stable isotope analysis in the field, these results demonstrated that resolvable differences in carbon and hydrogen isotopic fractionation were related to low and high iron conditions. This novel result highlights the need to further investigate the effects of other trace elements known to be key components of biodegradative enzymes.

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.