Biodegradation of Lindane (γ-Hexachlorocyclohexane) To Nontoxic End Products by Sequential Treatment with Three Mixed Anaerobic Microbial Cultures.

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.

Nanoscale zero-valent iron reduction coupled with anaerobic dechlorination to degrade hexachlorocyclohexane isomers in historically contaminated soil.

Hexachlorocyclohexane (HCH) isomers pose potential threats to the environment and to public health due to their persistence and high toxicity. In this study, nanoscale zero-valent iron (nZVI) coupled with microbial degradation by indigenous microorganisms with and without biostimulation was employed to remediate soils highly polluted with HCH. The degradation efficiency of total HCHs in both the "nZVI-only" and "Non-amendment" treatments was approximately 50 %, while in the treatment amended with nZVI and acetate, 85 % of total HCHs was removed. Addition of nZVI and acetate resulted in enrichment of anaerobic microorganisms. The results of quantitative PCR (qPCR) and 16S rRNA gene amplicon sequencing revealed that Desulfotomaculum, Dehalobacter, Geobacter, and Desulfuromonas likely contributed to the depletion of HCH isomers. Moreover, some abiotic factors also favored this removal process, including pH, and the generation of iron sulfides as revealed by the result of Mössbauer spectrometer analysis. Our research provides an improved remediation strategy for soils polluted with HCH isomers and an understanding of the synergistic effect of nZVI and indigenous microorganisms.

Insights into origins and function of the unexplored majority of the reductive dehalogenase gene family as a result of genome assembly and ortholog group classification.

Organohalide respiring bacteria (OHRB) express reductive dehalogenases for energy conservation and growth. Some of these enzymes catalyze the reductive dehalogenation of chlorinated and brominated pollutants in anaerobic subsurface environments, providing a valuable ecosystem service. Dehalococcoides mccartyi strains have been most extensively studied owing to their ability to dechlorinate all chlorinated ethenes - most notably carcinogenic vinyl chloride - to ethene. The genomes of OHRB, particularly obligate OHRB, often harbour multiple putative reductive dehalogenase genes (rdhA), most of which have yet to be characterized. We recently sequenced and closed the genomes of eight new strains, increasing the number of available D. mccartyi genomes in NCBI from 16 to 24. From all available OHRB genomes, we classified predicted translations of reductive dehalogenase genes using a previously established 90% amino acid pairwise identity cut-off to identify Ortholog Groups (OGs). Interestingly, the majority of D. mccartyi dehalogenase gene sequences, once classified into OGs, exhibited a remarkable degree of synteny (gene order) in all genomes sequenced to date. This organization was not apparent without the classification. A high degree of synteny indicates that differences arose from rdhA gene loss rather than recombination. Phylogenetic analysis suggests that most rdhA genes have a long evolutionary history in the Dehalococcoidia with origin prior to speciation of Dehalococcoides and Dehalogenimonas. We also looked for evidence of synteny in the genomes of other species of OHRB. Unfortunately, there are too few closed Dehalogenimonas genomes to compare at this time. There is some partial evidence for synteny in the Dehalobacter restrictus genomes, but here too more closed genomes are needed for confirmation. Interestingly, we found that the rdhA genes that encode enzymes that catalyze dehalogenation of industrial pollutants are the only rdhA genes with strong evidence of recent lateral transfer - at least in the genomes examined herein. Given the utility of the RdhA sequence classification to comparative analyses, we are building a public web server () for the community to use, which allows users to add and classify new sequences, and download the entire curated database of reductive dehalogenases.

Microbial Communities Associated with Sustained Anaerobic Reductive Dechlorination of α-, ß-, γ-, and δ-Hexachlorocyclohexane Isomers to Monochlorobenzene and Benzene.

Intensive historical and worldwide use of pesticide formulations containing hexachlorocyclohexane (HCH) has led to widespread contamination. We derived four anaerobic enrichment cultures from HCH-contaminated soil capable of sustainably dechlorinating each of α-, ß-, γ-, and δ-HCH isomers stoichiometrically to benzene and monochlorobenzene (MCB). For each isomer, the dechlorination rates, inferred from production rates of the dechlorinated products, MCB and benzene, increased progressively from <3 to ∼12 µM/day over 2 years. The molar ratio of benzene to MCB produced was a function of the substrate isomer and ranged from ß (0.77 ± 0.15), α (0.55 ± 0.09), γ (0.13 ± 0.02), to δ (0.06 ± 0.02) in accordance with pathway predictions based on prevalence of antiperiplanar geometry. Data from 16S rRNA gene amplicon sequencing and quantitative PCR revealed significant increases in the absolute abundances of Pelobacter and Dehalobacter, most notably in the α-HCH and δ-HCH cultures. Cultivation with a different HCH isomer resulted in distinct bacterial communities, but similar archaeal communities. This study provides the first direct comparison of shifts in anaerobic microbial communities induced by the dechlorination of distinct HCH isomers. It also uncovers candidate microorganisms responsible for the dechlorination of α-, ß-, γ-, and δ-HCH, a key step toward better understanding and monitoring of natural attenuation processes and improving bioremediation technologies for HCH-contaminated sites.

Sustained Dechlorination of Vinyl Chloride to Ethene in Dehalococcoides-Enriched Cultures Grown without Addition of Exogenous Vitamins and at Low pH.

Trichloroethene (TCE) bioremediation has been demonstrated at field sites using microbial cultures harboring TCE-respiring Dehalococcoides whose growth is cobalamin (vitamin B12)-dependent. Bioaugmentation cultures grown ex situ with ample exogenous vitamins and at neutral pH may become vitamin-limited or inhibited by acidic pH once injected into field sites, resulting in incomplete TCE dechlorination and accumulation of vinyl chloride (VC). Here, we report growth of the Dehalococcoides-containing bioaugmentation culture KB-1 in a TCE-amended mineral medium devoid of vitamins and in a VC-amended mineral medium at low pH (6.0 and 5.5). In these cultures, Acetobacterium, which can synthesize 5,6-dimethylbenzimidazole (DMB), the lower ligand of cobalamin, and Sporomusa are dominant acetogens. At neutral pH, Acetobacterium supports complete TCE dechlorination by Dehalococcoides at millimolar levels with a substantial increase in cobalamin (∼20-fold). Sustained dechlorination of VC to ethene was achieved at pH as low as 5.5. Below pH 5.0, dechlorination was not stimulated by DMB supplementation but was restored by raising pH to neutral. Cell-extract assays revealed that vinyl chloride reductase activity declines significantly below pH 6.0 and is undetectable below pH 5.0. This study highlights the importance of cobamide-producing populations and pH in microbial dechlorinating communities for successful bioremediation at field sites.

A switch of chlorinated substrate causes emergence of a previously undetected native Dehalobacter population in an established Dehalococcoides-dominated chloroethene-dechlorinating enrichment culture.

Chlorobenzenes are soil and groundwater pollutants of concern that can be reductively dehalogenated by organohalide-respiring bacteria from the genera Dehalococcoides and Dehalobacter. The bioaugmentation culture KB-1® harbours Dehalococcoides mccartyi spp. that reductively dehalogenate trichloroethene to ethene. It contains more than 30 reductive dehalogenase genes; some of them are highly similar to genes found in the chlorobenzene-respiring Dehalococcoides mccartyi strain CBDB1. We explored the chlorobenzene dehalogenation capability of the KB-1 enrichment culture using 1,2,4-trichlorobenzene (1,2,4-TCB). We achieved adaptation of KB-1 to 1,2,4-TCB that is dehalogenated to a mixture of dichlorobenzenes, and subsequently to monochlorobenzene and benzene. Surprisingly, a native Dehalobacter population, and not a Dehalococcoides population, couples the dechlorination of 1,2,4-TCB to growth achieving an average yield of 1.1 ± 0.6 × 1013 cells per mole of Cl- released. Interestingly, the dechlorination of 1,2,4-TCB occurs alongside the complete dechlorination of trichloroethene to ethene in cultures fed both electron acceptors. Dehalobacter was not previously identified as a major player in KB-1, but its ecological niche was favoured by the introduction of 1,2,4-TCB. Based on 16S rRNA phylogeny, Dehalobacter populations seem to cluster into specialised clades, and are likely undergoing substrate specialisation as a strategy to reduce competition for electron acceptors.