Co-occurrence of genes for aerobic and anaerobic biodegradation of dichloroethane in organochlorine-contaminated groundwater.

1,2-Dichloroethane (DCA) is a problematic groundwater pollutant. Factors influencing the distribution and activities of DCA-degrading bacteria are not well understood, which has hampered their application for bioremediation. Here, we used quantitative PCR to investigate the distribution of putative DCA-dehalogenating bacteria at a DCA-impacted site in Sydney (Australia). The dehalogenase genes dhlA, tceA and bvcA were detected in all groundwater samples (n = 15), while vcrA was found in 11/15 samples. The 16S rRNA gene sequences specific to the dehalogenating genera Dehalobacter, Desulfitobacterium and Dehalogenimonas were detected in 15/15, 13/15 and 13/15 samples, respectively, while Dehalococcoides sequences were found in 9/15 samples. The tceA, bvcA and vcrA genes occurred in the same samples as Dehalococcoides and Dehalobacter. Microcosm experiments confirmed the presence of bacteria capable of dechlorination under anoxic conditions. The abundance of the dhlA gene, which is found in hydrolytic DCA degraders, was positively correlated to the DCA concentration, and was unexpectedly most abundant in samples with low oxygen conditions. A dhlA-containing bacterium isolated from the site (Xanthobacter EL8) was capable of anaerobic growth on DCA under denitrifying conditions. The presence of diverse DCA-dehalogenating bacteria at this site indicates that natural attenuation or biostimulation could be valid approaches for site cleanup.

Particles and enzymes: Combining nanoscale zero valent iron and organochlorine respiring bacteria for the detoxification of chloroethane mixtures.

Nanoscale zero valent iron (nZVI) and organochlorine respiring bacteria (ORB) are two technologies used to detoxify chlorinated aliphatic hydrocarbons (CAHs). nZVI can rapidly detoxify high CAH concentrations, but is quickly oxidised and unable to degrade certain CAHs (e.g., 1,2-dichlorothane). In contrast, ORB can dechlorinate CAHs resistant to nZVI (e.g., 1,2-dichlorothane) but are inhibited by other CAHs of concern degradable by nZVI (e.g., chloroform and carbon tetrachloride). Combining the two was proposed as a unique treatment train to overcome each technology's shortcomings. In this study, this combined remedy was investigated using a mixture of 1,2-dichloroethane, degradable by ORB but not nZVI, and 1,1,2-trichloroethane, susceptible to both. Results indicated that nZVI rapidly dechlorinated 1,1,2-trichloroethane when supplied above 0.5 g/L, however ORB were inhibited and unable to dechlorinate 1,2-dichloroethane. pH increase and ionic species associated with nZVI did not significantly impact ORB, pinpointing Fe(0) particles as responsible for ORB inhibition. Below 0.05 g/L nZVI, ORB activity was stimulated. Results suggest that combining ORB and nZVI at appropriate doses can potentially treat a wider range of CAHs than each individual remedy. At field sites where nZVI was applied, it is likely that in situ nZVI concentrations were below the threshold of negative consequences.

Relative contributions of Dehalobacter and zerovalent iron in the degradation of chlorinated methanes.

The role of bacteria and zerovalent iron (Fe(0)) in the degradation of chlorinated solvents in subsurface environments is of interest to researchers and remediation practitioners alike. Fe(0) 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 Fe(0) compared with Fe(0) alone. The dichloromethane-fermenting culture transformed dichloromethane up to three times faster with Fe(0) 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/Fe(0) 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 Fe(0) 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.

Tolerance of anaerobic bacteria to chlorinated solvents.

The aim of this research was to evaluate the effects of four chlorinated aliphatic hydrocarbons (CAHs), perchloroethene (PCE), carbon tetrachloride (CT), chloroform (CF) and 1,2-dichloroethane (1,2-DCA), on the growth of eight anaerobic bacteria: four fermentative species (Escherichia coli, Klebsiella sp., Clostridium sp. and Paenibacillus sp.) and four respiring species (Pseudomonas aeruginosa, Geobacter sulfurreducens, Shewanella oneidensis and Desulfovibrio vulgaris). Effective concentrations of solvents which inhibited growth rates by 50% (EC50) were determined. The octanol-water partition coefficient or log Po/w of a CAH proved a generally satisfactory measure of its toxicity. Most species tolerated approximately 3-fold and 10-fold higher concentrations of the two relatively more polar CAHs CF and 1,2-DCA, respectively, than the two relatively less polar compounds PCE and CT. EC50 values correlated well with growth rates observed in solvent-free cultures, with fast-growing organisms displaying higher tolerance levels. Overall, fermentative bacteria were more tolerant to CAHs than respiring species, with iron- and sulfate-reducing bacteria in particular appearing highly sensitive to CAHs. These data extend the current understanding of the impact of CAHs on a range of anaerobic bacteria, which will benefit the field of bioremediation.

Successful microcosm demonstration of a strategy for biodegradation of a mixture of carbon tetrachloride and perchloroethene harnessing sulfate reducing and dehalorespiring bacteria.

Carbon tetrachloride (CT) is known to inhibit the transformation of perchloroethene (PCE) to ethene by dehalorespiring bacteria, creating a challenge for the bioremediation of environments contaminated with both compounds. We report on the sequential use of sulfate reduction and dehalorespiration as a microbial strategy for the transformation of a mixture of CT (10 µM) and PCE (14 µM). Sulfide production in Desulfovibrio vulgaris cultures led to complete CT disappearance in as little as 12 days. The addition of amorphous ferric oxide decreased the proportion of chloroform (CF) produced from 65% to 30%. CT conversion rates were enhanced more than 13-fold where vitamin B(12) (5 µM) was added. In vitamin B(12)-containing D. vulgaris cultures, no chlorinated products were detected and carbon disulfide was the major product of CT transformation. PCE concentrations were not impacted upon by D. vulgaris activity. The subsequent inoculation of a PCE-respiring enrichment culture resulted in microbial PCE dechlorination to ethene.

Complete chloroform dechlorination by organochlorine respiration and fermentation.

Chloroform (CF, CHCl(3)) is a recalcitrant and toxic environmental pollutant. In this communication we report for the first time a microbial community capable of complete CF dechlorination by metabolic processes. Cultures derived from subsurface soil (3.5 m) could sustain complete dechlorination of CF at levels of least 360 µM at a rate of 40 µM per day. Scrutiny of CF dechlorination revealed two metabolic processes at work. First, CF was respired to dichloromethane (DCM, CH(2) Cl(2)), which was then fermented to acetate, hydrogen and carbon dioxide. Elevated hydrogen partial pressures were found to inhibit the fermentation process. Interspecies hydrogen transfer was observed in the form of methanogenesis and acetogenesis. This suggests that the dechlorination process required syntrophic partners to maintain low hydrogen partial pressures. (13)C-labelled DCM was employed to help elucidate the chemistry of the process and identify bacterial community members involved. CF respiring cultures, where emulsified vegetable oil was supplied as the electron donor and DCM fermenting cultures, where DCM was supplied as the sole organic carbon source were studied separately. Pyrosequencing of these cultures revealed Dehalobacter lineages as a predominant community member in both. Subsequent growth experiments confirmed that the proliferation of Dehalobacter was linked directly to both the dehalorespiration and dehalofermentation processes.