Search

The γ isomer of hexachlorocyclohexane (HCH), also known as lindane, is a carcinogenic persistent organic pollutant. Lindane was used worldwide as an agricultural insecticide. Legacy soil and groundwater contamination with lindane and other HCH isomers is still a big concern. The biotic reductive dechlorination of HCH to nondesirable and toxic lower chlorinated compounds such as monochlorobenzene (MCB) and benzene, among others, has been broadly documented. Here, we demonstrate that complete biodegradation of lindane to nontoxic end products is attainable using a sequential treatment approach with three mixed anaerobic microbial cultures referred to as culture I, II, and III. Biaugmentation with culture I achieved dechlorination of lindane to MCB and benzene. Culture II was able to dechlorinate MCB to benzene, and finally, culture III carried out methanogenic benzene degradation. Distinct Dehalobacter populations, corresponding to different 16S rRNA amplicon sequence variants in culture I and culture II, were responsible for lindane and MCB dechlorination, respectively. This study continues to highlight key roles of Dehalobacter as chlorobenzene- and HCH -respiring bacteria and demonstrates that sequential treatment with specialized anaerobic cultures may be explored at field sites in order to address legacy soil and groundwater contamination with HCH.

Trichloromethane (TCM) is a frequently detected and persistent groundwater contaminant. Recent studies have reported that two closely related Dehalobacter strains (UNSWDHB and CF) transform TCM to dichloromethane, with inconsistent carbon isotope effects (e(13)C(UNSWDHB) = -4.3 +/- 0.45 parts per thousand; e13C(CF) = -27.5 +/- 0.9 parts per thousand). This study uses dual element compound specific isotope analysis (C; Cl) to explore the underlying differences. TCM transformation experiments using strain CF revealed pronounced normal carbon and chlorine isotope effects (e13C(CF) = -27.9 +/- 1.7 parts per thousand; e37Cl(CF) = -4.2 +/- 0.2 parts per thousand). In contrast, small carbon and unprecedented inverse chlorine isotope effects were observed for strain UNSWDHB (e13C(UNSWDHB) = -3.1 +/- 0.5 parts per thousand; e37Cl(UNSWDHB) = 2.5 +/- 0.3 parts per thousand) leading to opposing dual element isotope slopes (?CF = 6.64 +/- 0.14 vs lambda(UNSWDHB) = -1.20 +/- 0.18). Isotope effects of strain CF were identical to experiments with TCM and Vitamin B12 (e13C(Vitamin B12) = -26.0 +/- 0.9 parts per thousand, e37Cl(Vitamin B12) = -4.0 +/- 0.2 parts per thousand, lambda(Vitamin) (B12) = 6.46 +/- 0.20). Comparison to previously reported isotope effects suggests outer-sphere-single-electron transfer or S(N)2 as possible underlying mechanisms. Cell suspension and cell free extract experiments with strain UNSWDHB were both unable to unmask the intrinsic KIE of the reductive dehalogenase (TmrA) suggesting that enzyme binding and/or mass-transfer into the periplasm were rate-limiting. Nondirected intermolecular interactions of TCM with cellular material were ruled out as reason for the inverse isotope effect by gas/water and gas/hexadecane partitioning experiments indicating specific, yet uncharacterized interactions must be operating prior to catalysis.

Dehalococcoides mccartyi strain CBDB1 and Dehalobacter strain 14DCB1 are organohalide-respiring microbes of the phyla Chloroflexi and Firmicutes, respectively. Here, we report the transformation of chloroanilines by these two bacterial strains via dissimilar dehalogenation pathways and discuss the underlying mechanism with quantum chemically calculated net atomic charges of the substrate Cl, H, and C atoms. Strain CBDB1 preferentially removed Cl doubly flanked by two Cl or by one Cl and NH2, whereas strain 14DCB1 preferentially dechlorinated Cl that has an ortho H. For the CBDB1-mediated dechlorination, comparative analysis with Hirshfeld charges shows that the least-negative Cl discriminates active from nonactive substrates in 14 out of 15 cases and may represent the preferred site of primary attack through cob(I)alamin. For the latter trend, three of seven active substrates provide strong evidence, with partial support from three of the remaining four substrates. Regarding strain 14DCB1, the most positive carbon-attached H atom discriminates active from nonactive chloroanilines in again 14 out of 15 cases. Here, regioselectivity is governed for 10 of the 11 active substrates by the most positive H attached to the highest-charge (most positive or least negative) aromatic C carrying the Cl to be removed. These findings suggest the aromatic ring H as primary site of attack through the supernucleophile Co(I), converting an initial H bond to a full electron transfer as start of the reductive dehalogenation. For both mechanisms, one- and two-electron transfer to Cl (strain CBDB1) or H (strain 14DCB1) are compatible with the presently available data. Computational chemistry research into reaction intermediates and pathways may further aid in understanding the bacterial reductive dehalogenation at the molecular level.

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.

Previously, we demonstrated the reductive dehalogenation of dichlorobenzene (DCB) isomers to monochlorobenzene (MCB), and MCB to benzene in sediment microcosms derived from a chlorobenzene-contaminated site. In this study, enrichment cultures were established for each DCB isomer and each produced MCB and trace amounts of benzene as end products. MCB dehalogenation activity could only be transferred in sediment microcosms. The 1,2-DCB-dehalogenating culture was studied the most intensively. Whereas Dehalococcoides spp. were not detected in any of the microcosms or cultures, Dehalobacter spp. were detected in 16S rRNA gene clone libraries from 1,2-DCB enrichment cultures, and by PCR using Dehalobacter-specific primers in 1,3-DCB and 1,4-DCB enrichments and MCB-dehalogenating microcosms. Quantitative PCR showed Dehalobacter 16S rRNA gene copies increased up to 3 orders of magnitude upon dehalogenation of DCBs or MCB, and that nearly all of bacterial 16S rRNA genes in a 1,2-DCB-dehalogenating culture belonged to Dehalobacter spp. Dehalobacter 16S rRNA genes from DCB enrichment cultures and MCB-dehalogenating microcosms showed considerable diversity, implying that 16S rRNA sequences do not predict dehalogenation-spectra of Dehalobacter spp. These studies support a role for Dehalobacter spp. in the reductive dehalogenation of DCBs and MCB, and this genus should be considered for its potential impact on chlorobenzene fate at contaminated sites.

Dehalococcoides ethenogenes strain 195 dechlorinates tetrachloroethene to vinyl chloride and ethene, and its genome has been found to contain up to 17 putative dehalogenase gene homologues, suggesting diverse dehalogenation ability. We amended pure or mixed cultures containing D. ethenogenes strain 195 with 1,2,3,4-tetrachlorodibenzo-p-dioxin, 2,3,7,8-tetrachlorodibenzo-p-dioxin, 2,3-dichlorodibenzo-p-dioxin, 1,2,3,4-tetrachlorodibenzofuran, 2,3,4,5,6-pentachlorobiphenyl, 1,2,3,4-tetrachloronaphthalene, various chlorobenzenes, or a mixture of 2-, 3-, and 4-chlorophenol to determine the dehalogenation ability. D. ethenogenes strain 195 dechlorinated 1,2,3,4-tetrachlorodibenzo-p-dioxin to a mixture of 1,2,4-trichlorodibenzo-p-dioxin and 1,3-dichlorodibenzo-p-dioxin. 2,3,4,5,6- Pentachlorobiphenyl was dechlorinated to 2,3,4,6- and/or 2,3,5,6-tetrachlorobiphenyl and 2,4,6-trichlorobiphenyl. 1,2,3,4-Tetrachloronaphthalene was dechlorinated primarily to an unidentified dichloronaphthalene congener. The predominant end products from hexachlorobenzene dechlorination were 1,2,3,5-tetrachlorobenzene and 1,3,5-trichlorobenzene. We did not detect dechlorination daughter products from monochlorophenols, 2,3-dichlorodibenzo-p-dioxin or 2,3,7,8- tetrachlorodibenzo-p-dioxin. D. ethenogenes strain 195 has the ability to dechlorinate many different types of chlorinated aromatic compounds, in addition to its known chloroethene respiratory electron acceptors. Remediation of sediments contaminated with aromatic halogenated organic pollutants such as polychlorinated biphenyls and polychlorinated dibenzo-p-dioxins could require billions of dollars in the coming years. Harnessing microorganisms such as Dehalococcoides spp. that detoxify these compounds via removal of halogens may lead to cost-effective biotechnological approaches for remediation.

cis-Dichloroethene (DCE) and vinyl chloride (VC) often accumulate in contaminated aquifers in which tetrachloroethene (PCE) or trichloroethene (TCE) undergo reductive dechlorination. "Dehalococcoides ethenogenes" strain 195 is the first isolate capable of dechlorinating chloroethenes past cis-DCE. Strain 195 could utilize commercially synthesized cis-DCE as an electron acceptor, but doses greater than 0.2 mmol/L were inhibitory, especially to PCE utilization. To test whether the cis-DCE itself was toxic, or whether the toxicity was due to impurities in the commercial preparation (97% nominal purity), we produced cis-DCE biologically from PCE using a Desulfitobacterium sp. culture. The biogenic cis-DCE was readily utilized at high concentrations by strain 195 indicating that cis-DCE was not intrinsically inhibitory. Analysis of the commercially synthesized cis-DCE by GC/mass spectrometry indicated the presence of approximately 0.4% mol/mol chloroform. Chloroform was found to be inhibitory to chloroethene utilization by strain 195 and at least partially accounts for the inhibitory activity of the synthetic cis-DCE. VC, a human carcinogen that accumulates to a large extent in cultures of strain 195, was not utilized as a growth substrate, and cultures inoculated into medium with VC required a growth substrate, such as PCE, for substantial VC dechlorination. However, high concentrations of PCE or TCE inhibited VC dechlorination. Use of a hexadecane phase to keep the aqueous PCE concentration low in cultures allowed simultaneous utilization of PCE and VC. At contaminated sites in which "D. ethenogenes" or similar organisms are present, biogenic cis-DCE should be readily dechlorinated, chloroform as a co-contaminant may be inhibitory, and concentrations of PCE and TCE, except perhaps those near the source zone, should allow substantial VC dechlorination.