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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.

Electronic waste is termed as e-waste and on recycling it produces environmental pollution. Among these e-waste pollutants, polychlorinated biphenyls (PCBs) are significantly important due to ubiquitous, organic in nature and serious health and environmental hazards. PCBs are used in different electrical equipment such as in transformers and capacitors for the purposes of exchange of heat and hydraulic fluids. Bioremediation is a reassuring technology for the elimination of the PCBs from the environment. In spite of their chemical stability, there are several microbes which can bio-transform or mineralize the PCBs aerobically or anaerobically. In this review paper, our objective was to summarize the information regarding PCB-degrading enzymes and microbes. The review suggested that the most proficient PCB degraders during anaerobic condition are Dehalobacter, Dehalococcoides, and Desulfitobacterium and in aerobic condition are Burkholderia, Achromobacter, Comamonas, Ralstonia, Pseudomonas, Bacillus, and Alcaligenes etc., showing the broadest substrate among bacterial strains. Enzymes found in soil such as dehydrogenases and fluorescein diacetate (FDA) esterases have the capability to breakdown PCBs. Biphenyl upper pathway involves four enzymes: dehydrogenase (bphB), multicomponent dioxygenase (bphA, E, F, and G), second dioxygenase (bphC), hydrolase, and (bphD). Biphenyl dioxygenase is considered as the foremost enzyme used for aerobic degradation of PCBs in metabolic pathway. It has been proved that several micro-organisms are responsible for the PCB metabolization. The review provides novel strategies for e-waste-contaminated soil management.

Chloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. Complete dechlorination of CF has been reported under anaerobic conditions by microbes that respire CF to DCM and others that biodegrade DCM. The objectives of this study were to ascertain if a commercially available bioaugmentation enrichment culture (KB-1® Plus) uses an oxidative or fermentative pathway for biodegradation of DCM; and to determine if the products from DCM biodegradation can support organohalide respiration of CF to DCM in the absence of an exogenous electron donor. In various treatments with the KB-1® Plus culture to which 14C-CF was added, the predominant product was 14CO2, indicating that oxidation is the predominant pathway for DCM. Recovery of 14C-DCM when biodegradation was still in progress confirmed that CF first undergoes reductive dechlorination to DCM. 14C-labeled organic acids, including acetate and propionate, were also recovered, suggesting that synthesis of organic acids provides a sink for the electron equivalents from oxidation of DCM. When the culture was washed to remove organic acids from prior additions of exogenous electron donor and only CF and DCM were added, the culture completely dechlorinated CF. The total amount of DCM added was not sufficient to provide the electron equivalents needed to reduce CF to DCM. Thus, the additional reducing power came via the DCM generated from CF reduction. Nevertheless, the rate of CF consumption was considerably slower in comparison to treatments that received an exogenous electron donor.IMPORTANCEChloroform (CF) and dichloromethane (DCM) are among the more commonly identified chlorinated aliphatic compounds found in contaminated soil and groundwater. One way to address this problem is to add microbes to the subsurface that can biodegrade these compounds. While microbes are known that can accomplish this task, less is known about the pathways used under anaerobic conditions. Some use an oxidative pathway, resulting mainly in carbon dioxide. Others use a fermentative pathway, resulting in formation of organic acids. In this study, a commercially available bioaugmentation enrichment culture (KB-1® Plus) was evaluated using carbon-14 labelled chloroform. The main product formed was carbon dioxide, indicating the use of an oxidative pathway. The reducing power gained from oxidation was shown to support reductive dechlorination of CF to DCM. The results demonstrate the potential to achieve full dechlorination of CF and DCM to nonhazardous products that are difficult to identify in the field.

Reductive dehalogenases (RDases) are a family of redox enzymes that are required for anaerobic organohalide respiration, a microbial process that is useful in bioremediation. Structural and mechanistic studies of these enzymes have been greatly impeded due to challenges in RDase heterologous expression, potentially because of their cobamide-dependence. There have been a few successful attempts at RDase production in unconventional heterologous hosts, but a robust method has yet to be developed. Here we outline a novel respiratory RDase expression system using Escherichia coli. The overexpression of E. coli's cobamide transport system, btu, and anaerobic expression conditions were found to be essential for production of active RDases from Dehalobacter - an obligate organohalide respiring bacterium. The expression system was validated on six enzymes with amino acid sequence identities as low as 28%. Dehalogenation activity was verified for each RDase by assaying cell-free extracts of small-scale expression cultures on various chlorinated substrates including chloroalkanes, chloroethenes, and hexachlorocyclohexanes. Two RDases, TmrA from Dehalobacter sp. UNSWDHB and HchA from Dehalobacter sp. HCH1, were purified by nickel affinity chromatography. Incorporation of the cobamide and iron-sulfur cluster cofactors was verified; though, the precise cobalamin incorporation could not be determined due to variance between methodologies, and the specific activity of TmrA was consistent with that of the native enzyme. The heterologous expression of respiratory RDases, particularly from obligate organohalide respiring bacteria, has been extremely challenging and unreliable. Here we present a relatively straightforward E. coli expression system that has performed well for a variety of Dehalobacter spp. RDases. IMPORTANCE Understanding microbial reductive dehalogenation is important to refine the global halogen cycle and to improve bioremediation of halogenated contaminants; however, studies of the family of enzymes responsible are limited. Characterization of reductive dehalogenase enzymes has largely eluded researchers due to the lack of a reliable and high-yielding production method. We are presenting an approach to express reductive dehalogenase enzymes from Dehalobacter, a key group of organisms used in bioremediation, in E. coli. This expression system will propel the study of reductive dehalogenases by facilitating their production and isolation, allowing researchers to pursue more in-depth questions about the activity and structure of these enzymes. This platform will also provide a starting point to improve the expression of reductive dehalogenases from many other organisms.

This study is aimed at investigating the performance of the continuous-flow, two-stage anaerobic column for the reductive dechlorination of perchloroethene (PCE) in a trichloroethene (TCE)-contaminated aquifer without bioaugmentation. Results indicated that complete dechlorination of PCE to cis-dichloroethene (DCE) was observed within the first 12 days after the addition of lactate and PCE (620:1 mg mg−1 mass ratio). The PCE conversion to ethene was initially observed in the 2nd column after 70 days (during phase II) upon the increase of lactate to PCE mass ratio to 5000:1 (mg mg−1). However, the degradation of PCE to ethene occurred in the 1st column after 30 days operation of phase IV (217–310 d) under the high PCE feed conditions (33.65 ± 2.86 μM). 16S rDNA clone library sequence analysis confirmed that Dehalobacter sp., Desulfuromonas sp., Desulfovibrio sp. Geobacter sp., Methanosaeta sp., Methanobacterium paludism, and M. spelaei were the dominant species in the original aquifer, and their relative abundance were further changed following lactate addition. Dehalococcoides mccartyi sequences were retrieved after lactate stimulation. Overall, the continuous-flow, two-stage anaerobic column process exhibited the maximum dechlorination (6.23 μmol L−1 hr−1) of PCE without bioaugmentation, indicating its potential up-scaling application in PCE dechlorination.

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.

Trichloroethylene (TCE) is one of the most common groundwater pollutants. It is carcinogenic, teratogenic, mutagenic and poses a serious threat to human health and the environment. Therefore, reducing the environmental toxicity of TCE is of great significance. Anaerobic sludge was cultured and acclimated in an upflow anaerobic sludge blanket (UASB) reactor in this study. The Chemical Oxygen Demand (COD) concentration of the influent was approximately 2500 mg L−1, and the TCE concentration of the influent ranged from 1.46 mg L−1 to 73 mg L−1. After biodegradation of the anaerobic microflora, the COD removal rate was approximately 85%, and the TCE removal rate was over 85%. The microbial community of anaerobic sludge was analysed by 16 S rDNA clone libray and 454 high-throughput sequencing. Through analysis of the sequencing results, we found that there were a variety of acid-forming bacteria, anaerobic dechlorinating bacteria, and methanogenic bacteria. Based on the analysis of microflora function, it was speculated that the TCE metabolic pathway took place in UASB reactors. Desulfovibrio and Syntrophobacter provided an anaerobic environment, and acid-forming bacteria metabolise organic compounds into hydrogen. With Dehalobacter and Geobacter, TCE as an electron acceptor is dechlorinated and reduced under the anaerobic environment, in which hydrogen acts as an electron donor. By this, we clarified the metabolic pathway for improving TCE bioremediation.

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 non-desirable and toxic lower chlorinated compounds such as monochlorobenzene (MCB) and benzene, among others, has been broadly documented. Here, we demonstrate for the first time that complete biotransformation of lindane to non-toxic 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 spp. as chlorobenzene- and HCH-organohalide-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.

In an aquifer-aquitard system in the subsoil of the city of Ferrara (Emilia-Romagna region, northern Italy) highly contaminated with chlorinated aliphatic toxic organics such as trichloroethylene (TCE) and tetrachloroethylene (PCE), a strong microbial-dependent dechlorination activity takes place during migration of contaminants through shallow organic-rich layers with peat intercalations. The in situ microbial degradation of chlorinated ethenes, formerly inferredby the utilization of contaminant concentration profiles and Compound-Specific Isotope Analysis (CSIA), was here assessed using Illumina sequencing of V4 hypervariable region of 16S rRNA gene and by clone library analysis of dehalogenase metabolic genes. Taxon-specific investigation of the microbial communities catalyzing the chlorination process revealed the presence of not only dehalogenating genera such as Dehalococcoides and Dehalobacter but also of numerous other groups of non-dehalogenating bacteria and archaea thriving on diverse metabolisms such as hydrolysis and fermentation of complex organic matter, acidogenesis, acetogenesis, and methanogenesis, which can indirectly support the reductive dechlorination process. Besides, the diversity of genes encoding some reductive dehalogenases was also analyzed. Geochemical and 16S rRNA and RDH gene analyses, as a whole, provided insights into the microbial community complexity and the distribution of potential dechlorinators. Based on the data obtained, a possible network of metabolic interactions has been hypothesized to obtain an effective reductive dechlorination process.

In our previous research, two-stage anaerobic co-fermentation (TSAC) treating lipid-extracted microalgae waste (LEMW) mixed with food waste leachate (FWL) was successfully designed to recover the additional energy (H2 and CH4) while mitigating the inhibition of leftover chloroform (CF). This study aimed to elucidate the change in the microbial community and their association with the CF degradation in the TSAC. 90% of CF was dechlorinated in the acidogenic reactor of TSAC when LEMW was fed with FWL at a ratio of 40:60. The results of microbial analysis clearly showed that Dehalobacter (5.3%) played the role in dehalorespiration while Clostridium (8.9%) and Enterobacter (3.6%) promoted cometabolic dechlorination. In contrast, the microbial consortia were completely inhibited by CF when LEMW was fed without FWL. In the methanogenic reactor of TSAC, increased Methanosarcina (57.8%), Methanobacterium (10.4%), and Methanosaeta (8.6%) were observed. Those were involved in successful conversion of acetate and H2 into CH4.

Trichloromethane (TCM) is a pollutant frequently detected in contaminated aquifers, and only four bacterial strains are known to respire it. Here, we obtained a novel Dehalobacter strain capable of transforming TCM to dichloromethane, which was denominated Dehalobacter sp. strain 8M. Besides TCM, strain 8M also completely transformed 1,1,2-trichloroethane to vinyl chloride and 1,2-dichloroethane. Quantitative PCR analysis for the 16S rRNA genes confirmed growth of Dehalobacter with TCM and 1,1,2-trichloroethane as electron acceptors. Carbon and chlorine isotope fractionation during TCM transformation was studied in cultured cells and in enzymatic assays with cell suspensions and crude protein extracts. TCM transformation in the three studied systems resulted in small but significant carbon (εC = −2.7 ± 0.1‰ for respiring cells, −3.1 ± 0.1‰ for cell suspensions, and − 4.1 ± 0.5‰ for crude protein extracts) and chlorine (εCl = −0.9 ± 0.1‰, −1.1 ± 0.1‰, and − 1.2 ± 0.2‰, respectively) isotope fractionation. A characteristic and consistent dual Csingle bondCl isotope fractionation pattern was observed for the three systems (combined ΛC/Cl = 2.8 ± 0.3). This ΛC/Cl differed significantly from previously reported values for anaerobic dechlorination of TCM by the corrinoid cofactor vitamin B12 and other Dehalobacter strains. These findings widen our knowledge on the existence of different enzyme binding mechanisms underlying TCM-dechlorination within the genus Dehalobacter and demonstrates that dual isotope analysis could be a feasible tool to differentiate TCM degraders at field studies.

In situ bioremediation (ISB) using reductive dechlorination is a widely accepted but relatively slow approach compared to other technologies for the treatment of groundwater contaminated by chlorinated ethenes (CVOCs). Due to the known positive kinetic effect on microbial metabolism, thermal enhancement may be a viable means of accelerating ISB. We tested thermally enhanced ISB in aquifers situated in sandy saprolite and underlying fractured granite. The system comprised pumping, heating and subsequent injection of contaminated groundwater aiming at an aquifer temperature of 20-30°C. A fermentable substrate (whey) was injected in separate batches. The test was monitored using hydrochemical and molecular tools (qPCR and NGS). The addition of the substrate and increase in temperature resulted in a rapid increase in the abundance of reductive dechlorinators (e.g., Dehalococcoides mccartyi, Dehalobacter sp. and functional genes vcrA and bvcA) and a strong increase in CVOC degradation. On day 34, the CVOC concentrations decreased by 87% to 96% in groundwater from the wells most affected by the heating and substrate. On day 103, the CVOC concentrations were below the LOQ resulting in degradation half-lives of 5 to 6days. Neither an increase in biomarkers nor a distinct decrease in the CVOC concentrations was observed in a deep well affected by the heating but not by the substrate. NGS analysis detected Chloroflexi dechlorinating genera (Dehalogenimonas and GIF9 and MSBL5 clades) and other genera capable of anaerobic metabolic degradation of CVOCs. Of these, bacteria of the genera Acetobacterium, Desulfomonile, Geobacter, Sulfurospirillum, Methanosarcina and Methanobacterium were stimulated by the substrate and heating. In contrast, groundwater from the deep well (affected by heating only) hosted representatives of aerobic metabolic and aerobic cometabolic CVOC degraders. The test results document that heating of the treated aquifer significantly accelerated the treatment process but only in the case of an abundant substrate.

It is acknowledged that organohalide-respiring bacteria (OHRB) can degrade polybrominated diphenyl ethers (PBDEs); however, very little is known about the distribution of OHRB or their response to PBDE contamination in natural sediments. We collected sediments from 28 sampling sites in Taihu Lake, China, and investigated the spatial distribution and diversity of OHRB, and the relationships between the PBDE contamination levels and the PBDE removal potential. The abundances of five typical OHRB genera, namely Dehalobacter, Dehalococcoides, Dehalogenimonas, Desulfitobacterium, and Geobacter, ranged from 0.34 × 104 to 19.4 × 107 gene copies g-1 dry sediment, and varied significantly among different areas of Taihu Lake. OHRB were more abundant in sediments from Meiliang and Zhushan Bay, where the PBDE concentrations were higher, and the phylotype diversity of the OHRB belonging to the family Dehalococcoidaceae was lower, than reported for other areas. While the sulfate concentrations explained much of the spatial distribution of OHRB, PBDE concentrations were also a strong influence on the abundance and diversity of OHRB in the sediments. For Dehalococcoides, Dehalogenimonas and Geobacter, the abundance of each genus was positively related to its own potential to remove PBDEs. The dominant OHRB genus, Dehalogenimonas, may contribute most to in situ bioremediation of PBDEs in Taihu Lake.

Bioremediation of polychlorinated biphenyls (PCBs) is impeded by difficulties in massively cultivating bioinoculant. Meanwhile, sewage sludge is rich in pollutant-degrading microorganisms and nutrients, drawing our attention to investigate their potential to be used as a supplement for bioremediation of PCBs. Here we reported extensive microbial reductive dechlorination of PCBs by waste activated sludge (WAS) and digestion sludge (DS), which were identified to harbor multiple putative organohalide-respiring bacteria (i.e., Dehalococcoides, Dehalogenimonas, Dehalobacter, and uncultivated Dehalococcoidia) and PCB reductive dehalogenase genes (i.e., pcbA4 and pcbA5). Consequently, amendment of 1-20% (w/w) fresh WAS/DS enhanced the attenuation of PCBs by 126-544% in a soil microcosm compared with the control soil, with the fastest dechlorination of PCBs being achieved when spiked with 20% fresh WAS. Notably, dechlorination pathways of PCBs were also changed by sludge amendment. Microbial and physicochemical analyses revealed that the enhanced dechlorination of PCBs by sludge amendment was largely attributed to the synergistic effects of sludge-derived nutrients, PCB-dechlorinating bacteria, and stimulated growth of beneficial microorganisms (e.g., fermenters). Finally, risk assessment of heavy metals suggests low potential ecological risks of sludge amendment in soil. Collectively, our study demonstrates that sewage sludge amendment could be an efficient, cost-effective and environment-friendly approach for in situ bioremediation of PCBs.

Trihalomethanes such as chloroform and bromoform, although well-known as a prominent class of disinfection by-products, are ubiquitously distributed in the environment due to widespread industrial usage in the past decades. Chloroform and bromoform are particularly concerning, of high concentrations detected and with long half-lives up to several hundred days in soils and groundwater. In this study, we report a Dehalobacter- and Desulfovibrio-containing co-culture that exhibits dehalogenation of chloroform (~0.61 mM) to dichloromethane and bromoform (~0.67 mM) to dibromomethane within 10-15 days. This co-culture was further found to dechlorinate 1,1,1-trichloroethane (1,1,1-TCA) (~0.65 mM) to 1,1-dichloroethane within 12 days. The Dehalobacter species present in this co-culture, designated Dehalobacter sp. THM1, was found to couple growth with dehalogenation of chloroform, bromoform, and 1,1,1-TCA. Strain THM1 harbors a newly identified reductive dehalogenase (RDase), ThmA, which catalyzes chloroform, bromoform, and 1,1,1-TCA dehalogenation. Additionally, based on the sequences of thmA and other identified chloroform RDase genes, ctrA, cfrA, and tmrA, a pair of chloroform RDase gene-specific primers were designed and successfully applied to investigate the chloroform dechlorinating potential of microbial communities. The comparative analysis of chloroform RDases with tetrachloroethene RDases suggests a possible approach in predicting the substrate specificity of uncharacterized RDases in the future.

Taxonomic assignments of anaerobic dichloromethane (DCM)-degrading bacteria remain poorly constrained but are important for understanding the microbial diversity of organisms contributing to DCM turnover in environmental systems. We describe the taxonomic classification of a novel DCM degrader in consortium RM obtained from pristine Rio Mameyes sediment. Phylogenetic analysis of full-length 16S rRNA gene sequences demonstrated that the DCM degrader was most closely related to members of the genera Dehalobacter and Syntrophobotulus, but sequence similarities did not exceed 94% and 93%, respectively. Genome-aggregate average amino acid identities against Peptococcaceae members did not exceed 66%, suggesting that the DCM degrader does not affiliate with any described genus. Phylogenetic analysis of conserved single-copy functional genes supported that the DCM degrader represents a novel clade. Growth strictly depended on the presence of DCM, which was consumed at a rate of 160±3μmolL-1 d-1. The DCM degrader attained 5.25×107±1.0×107 cells per μmol DCM consumed. Fluorescence in situ hybridization revealed rod-shaped cells 4±0.8μm long and 0.4±0.1μm wide. Based on the unique phylogenetic, genomic, and physiological characteristics, we propose that the DCM degrader represents a new genus and species, 'Candidatus Dichloromethanomonas elyunquensis'.

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.

Halogenated compounds such as polychlorinated biphenyl (PCBs) and polybrominated diphenyl ethers (PBDEs) enter wastewater treatment plants (WWTPs) via the sewage system. These organic contaminants partition between the aqueous and the biosolid phase, where the former is discharged as wastewater effluent. Biosolids from a WWTP provide a hydrophobic surface for adsorption and thus the presence and potential growth of organohalide respiring (OHR) bacteria. In this study, the aim was to assess the potential organohalide respiration capacity in wastewater biosolids by investigating actively organohalide respiring bacteria with a focus on organohalide respiration of PCBs and PCE. The results of the biosolids analysis showed increased amounts of products from PCB respiration. Simultaneously, experiments with organohalide respiration of PCE in biosolids samples showed significant decreases PCE concentration after 46 days (28–92%). Subsequently, it was evaluated if the OHR microbial populations in biosolids were similar to those present in intestinal human biofilms by applying a bioinformatic approach. The OHR populations of the communities were analyzed from existing American and Chinese human intestinal microbiomes. The overall groups Proteobacteria, Bacteroides, Actinobacteria, and Firmicutes phyla dominated the microbiomes in all datasets. The OHR groups in biosolids and intestinal biofilms included Dehalogenimonas, Dehalobacter, Desulfitibacter, Desulfovibrio, Sulfurospirillum, Clostridium, and Comamonas. The results of this study showed that several OHR phyla were present in all samples independent of origin. Wastewater and intestinal microbiomes also contained OHR phyla. Overall, the results points towards using bacterial communities in biosolids as indicators of organohalide respiration in wastewater and intestinal microbiomes, which is related to ingestion or halogenated compounds.

Anoxic aquifers suffer from energy limitations due to the unavailability of organic substrates, as dictated by hydrogen (H2) for various electron-accepting processes. This deficiency often results in the accumulation of persistent organic pollutants, where bioremediation using organic compounds often leads to secondary contamination. This study involves the reductive dechlorination of pentachlorophenol (PCP) by dechlorinators that do not use H2 directly, but rather through a reduced state of humin&mdash, a solid-phase humic substance&mdash, as the extracellular electron donor, which requires an organic donor such as formate, lactate, etc. This shortcoming was addressed by the development of an anaerobic mixed culture that was capable of reductively dechlorinating PCP using humin under autotrophic conditions induced by homoacetogenesis. Here, H2 was used for carbon-dioxide fixation to acetate, the acetate produced was used for the reduction of humin, and consequently used for dechlorination through reduced humin. The 16SrRNA gene sequencing analysis showed Dehalobacter and Dehalobacterium as the possible dechlorinators, while Clostridium and Oxobacter were identified as the homoacetogens. Thus, this work contributes to the development of an anaerobic consortium that balanced H2 dependency, where efficiency of humin reduction extends the applicability of anaerobic microbial remediation in aquifers through autotrophy, syntrophy, and reductive dechlorination.

Chlorinated ethanes are environmental pollutants found frequently at many contaminated industrial sites. 1,1,1-Trichloroethane (1,1,1-TCA) can be dechlorinated and detoxified via abiotic transformation or biologically by the action of dechlorinating microorganisms such asDehalobacter(Dhb). At a field site, it is challenging to distinguish abiotic vs biotic mechanisms as both processes share common transformation products. In this study, we evaluated using theDhb16S rRNA gene and specific reductive dehalogenase genes as biomarkers for 1,1,1-TCA and 1,1-dichloroethane (1,1-DCA) dechlorination. We analyzed samples from laboratory groundwater microcosms and from an industrial site where a mixture of granular zero valent iron (ZVI) and guar gum was injected for 1,1,1-TCA remediation. Abiotic and biotic transformation products were monitored and the changes in dechlorinating organisms were tracked using quantitative PCR (qPCR) with primers targeting theDhb16S rRNA gene and two functional genescfrAanddcrAencoding enzymes that dechlorinate 1,1,1-TCA to 1,1-DCA and 1,1-DCA to chloroethane (CA), respectively. The abundance of thecfrA- anddcrA-like genes confirmed that the two dechlorination steps were carried out by two distinctDhbpopulations at the site. Using cell yields established in laboratory microcosms along with measured abundances of theDhb16S rRNA gene in site samples, biotic and abiotic transformation of 1,1,1-TCA at the site was estimated. The biomarkers used in this study proved useful for tracking biodechlorination of 1,1,1-TCA and 1,1-DCA where both abiotic (e.g. with ZVI) and biotic processes co-occur.

Dichloromethane (DCM) is susceptible to microbial degradation under anoxic conditions and is metabolized via the Wood-Ljungdahl pathway; however, mechanistic understanding of carbon-chlorine bond cleavage is lacking. The microbial consortium RM contains the DCM degrader “Candidatus Dichloromethanomonas elyunquensis” strain RM, which strictly requires DCM as a growth substrate. Proteomic workflows applied to DCM-grown consortium RM biomass revealed a total of 1,705 nonredundant proteins, 521 of which could be assigned to strain RM. In the presence of DCM, strain RM expressed a complete set of Wood-Ljungdahl pathway enzymes, as well as proteins implicated in chemotaxis, motility, sporulation, and vitamin/cofactor synthesis. Four corrinoid-dependent methyltransferases were among the most abundant proteins. Notably, two of three putative reductive dehalogenases (RDases) encoded within strain RM’s genome were also detected in high abundance. Expressed RDase 1 and RDase 2 shared 30% amino acid identity, and RDase 1 was most similar to an RDase of Dehalococcoides mccartyi strain WBC-2 (AOV99960, 52% amino acid identity), while RDase 2 was most similar to an RDase of Dehalobacter sp. strain UNSWDHB (EQB22800, 72% amino acid identity). Although the involvement of RDases in anaerobic DCM metabolism has yet to be experimentally verified, the proteome characterization results implicated the possible participation of one or more reductive dechlorination steps and methyl group transfer reactions, leading to a revised proposal for an anaerobic DCM degradation pathway. IMPORTANCE Naturally produced and anthropogenically released DCM can reside in anoxic environments, yet little is known about the diversity of organisms, enzymes, and mechanisms involved in carbon-chlorine bond cleavage in the absence of oxygen. A proteogenomic approach identified two RDases and four corrinoid-dependent methyltransferases expressed by the DCM degrader “Candidatus Dichloromethanomonas elyunquensis” strain RM, suggesting that reductive dechlorination and methyl group transfer play roles in anaerobic DCM degradation. These findings suggest that the characterized DCM-degrading bacterium Dehalobacterium formicoaceticum and “Candidatus Dichloromethanomonas elyunquensis” strain RM utilize distinct strategies for carbon-chlorine bond cleavage, indicating that multiple pathways evolved for anaerobic DCM metabolism. The specific proteins (e.g., RDases and methyltransferases) identified in strain RM may have value as biomarkers for monitoring anaerobic DCM degradation in natural and contaminated environments.

ISCO using activated sodium persulphate is a widely used technology for treating chlorinated solvent source zones. In sensitive areas, however, high groundwater sulphate concentrations following treatment may be a drawback. In situ biogeochemical transformation, a technology that degrades contaminants via reduced iron minerals formed by microbial activity, offers a potential solution for such sites, the bioreduction of sulphate and production of iron sulphides that abiotically degrade chlorinated ethenes acting as a secondary technology following ISCO. This study assesses this approach in the field using hydrochemical and molecular tools, solid phase analysis and geochemical modelling. Following a neutralisation and bioaugmentation, favourable conditions for iron- and sulphate-reducers were created, resulting in a remarkable increase in their relative abundance. The abundance of dechlorinating bacteria (Dehalococcoides mccartyi, Dehalobacter sp. and Desulfitobacterium spp.) remained low throughout this process. The activity of iron- and sulphate-reducers was further stimulated through application of magnetite plus starch and microiron plus starch, resulting in an increase in ferrous iron concentration (fromLOQ to 337 mg/l), a decrease in sulphate concentration by 74-95% and production of hydrogen sulphide (fromLOQ to 25.9 mg/l). At the same time, a gradual revival of dechlorinators and an increase in ethene concentration was also observed. Tetrachloroethene and trichloroethene concentrations decreased by 98.5-99.98% and 75.4-98.5%, respectively. A decline in chlorine number indicated that biological dechlorination contributed to CVOC removal. This study brings new insights into biogeochemical processes that, when properly engineered, could provide a viable solution for secondary treatment.

Dicamba is an important herbicide that is widely used in agriculture. However, its residue pollutes the environment and thus its catabolism raises great concern. Anaerobic degradation is the main dicamba degradation pathway in the environment, however, the whole degradation pathway and the microorganisms involved remain unknown. In this study, an anaerobic sludge capable of efficiently degrading high concentration of dicamba was enriched from a river sediment. The degradation capacity reached 1.74 μmol dgs−1 days−1 which was 47-fold higher than that of the inoculated sediment. The acclimated sludge could degrade dicamba, 3,6-dichlorosalicylic acid, 6-chlorosalicylic acid, 3-chlorophenol and phenol but not salicylic acid, 3-chlorosalicylic acid, 4-chlorosalicylic acid, 2-chlorophenol or benzoic acid. Based on the identification of 3,6-dichlorosalicylic acid, 6-chlorosalicylic acid as well as four new dicamba metabolites, 3-chlorohydroquinone, 3-chlorophenol, phenol and butyric acid, a novel anaerobic degradation pathway was proposed for dicamba. Firstly, dicamba was O-demethylated to 3,6-dichlorosalicylic acid and then dechlorinated to 6-chlorosalicylic acid. Next, 6-chlorosalicylic acid was decarboxylated to 3-chlorohydroquinone, then dehydroxylated to 3-chlorophenol, and dechlorinated to phenol, which was further converted to butyric acid. The microbial community of the acclimated sludge was dominated by bacteria belonging to the genera Desulfovibrio, Exilispira, Caldisericum, Aminicenantales_unclassified, Syntrophus, Dehalobacter, Romboutsia, Syntrophorhabdus, and Saccharimonadales_unclassified, as well as archaea belonging to the genera Methanosaeta, Methanolinea, Bathyarchaeia_unclassified, and Methanomethylicus, all of which might be involved in the degradation of dicamba. This study provides a basis for further elucidation of the anaerobic catabolism of dicamba and development of anaerobic technology to treat high concentration dicamba wastewater.