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Abstract Background Chlorophenoxy compounds represent a group of selective herbicides widely used around the world. Chlorophenoxy herbicides are toxic, chemically stable, and can migrate into groundwater through soil leaching, posing a significant threat to drinking water safety and human health. Chlorophenoxy herbicides in groundwater aquifers are subject to anaerobic processes; however, the pathway and microbiology involved in the attenuation of chlorophenoxy herbicides under anaerobic condition are largely unknown. Here, the anaerobic degradation process of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a typical chlorophenoxy herbicide, was investigated. Results The initial 52.5 ± 2.3 μM 2,4,5-T was completely degraded by a sediment-derived microbial consortium, with 3,4-dichlorophenol, 2,5-dichlorophenol, 3-chlorophenol (3-CP) and phenol being identified as the intermediate products. Reductive dechlorination of 3-CP to phenol and the subsequent elimination of phenol were the key transformation steps in the overall degradation process of 2,4,5-T. Amplicon sequencing suggested that Dehalobacter, Sulfuricurvum, Bacteroides, Acetobacterium, and Clostridium sensu stricto 7 might contribute to the transformation of 2,4,5-T to phenol, and Smithella, Syntrophorhabdus, Methanofollis and Methanosaeta likely cooperated to accomplish the complete mineralization of phenol. Conclusions This study reported the anaerobic degradation of 2,4,5-T via reductive dechlorination and the subsequent syntrophic metabolization of phenol, an intermediate product transformed from 2,4,5-T. Dehalobacter was identified as the organohalide-respiring population catalyzing the reductive dechlorination reaction. Syntrophorhabdus and methanogenic populations were likely involved in anaerobic phenol oxidation and facilitated the complete mineralization of 2,4,5-T.

Background: Chlorophenoxy compounds represent a group of selective herbicides widely used around the world. Chlorophenoxy herbicides are toxic, chemically stable, and can migrate into groundwater through soil leaching, posing a significant threat to drinking water safety and human health. Chlorophenoxy herbicides in groundwater aquifers are subject to anaerobic processes; however, the pathway and microbiology involved in the attenuation of chlorophenoxy herbicides under anaerobic condition are largely unknown. Here, the anaerobic degradation process of 2,4,5-trichlorophenoxyacetic acid (2,4,5-T), a typical chlorophenoxy herbicide, was investigated. Results: The initial 52.5 ± 2.3 μM 2,4,5-T was completely degraded by a sediment-derived microbial consortium, with 3,4-dichlorophenol, 2,5-dichlorophenol, 3-chlorophenol (3-CP) and phenol being identified as the intermediate products. Reductive dechlorination of 3-CP to phenol and the subsequent elimination of phenol were the key transformation steps in the overall degradation process of 2,4,5-T. Amplicon sequencing suggested that Dehalobacter, Sulfuricurvum, Bacteroides, Acetobacterium, and Clostridium sensu stricto 7 might contribute to the transformation of 2,4,5-T to phenol, and Smithella, Syntrophorhabdus, Methanofollis and Methanosaeta likely cooperated to accomplish the complete mineralization of phenol. Conclusions: This study reported the anaerobic degradation of 2,4,5-T via reductive dechlorination and the subsequent syntrophic metabolization of phenol, an intermediate product transformed from 2,4,5-T. Dehalobacter was identified as the organohalide-respiring population catalyzing the reductive dechlorination reaction. Syntrophorhabdus and methanogenic populations were likely involved in anaerobic phenol oxidation and facilitated the complete mineralization of 2,4,5-T. [ABSTRACT FROM AUTHOR]

Chloroform (CF) and dichloromethane (DCM) are groundwater contaminants of concern due to their high toxicity and inhibition of important biogeochemical processes such as methanogenesis. Anaerobic biotransformation of CF and DCM has been well documented but typically independently of one another. CF is the electron acceptor for certain organohalide-respiring bacteria that use reductive dehalogenases (RDases) to dechlorinate CF to DCM. In contrast, known DCM degraders use DCM as their electron donor, which is oxidized using a series of methyltransferases and associated proteins encoded by the mec cassette to facilitate the entry of DCM to the Wood-Ljungdahl pathway. The SC05 culture is an enrichment culture sold commercially for bioaugmentation, which transforms CF via DCM to CO2. This culture has the unique ability to dechlorinate CF to DCM using electron equivalents provided by the oxidation of DCM to CO2. Here, we use metagenomic and metaproteomic analyses to identify the functional genes involved in each of these transformations. Though 91 metagenomeassembled genomes were assembled, the genes for an RDase--named acdA--and a complete mec cassette were found to be encoded on a single contig belonging to Dehalobacter. AcdA and critical Mec proteins were also highly expressed by the culture. Heterologously expressed AcdA dechlorinated CF and other chloroalkanes but had 100-fold lower activity on DCM. Overall, the high expression of Mec proteins and the activity of AcdA suggest a Dehalobacter capable of dechlorination of CF to DCM and subsequent mineralization of DCM using the mec cassette. [ABSTRACT FROM AUTHOR]

Chloroform and trichloroethanes are pervasive groundwater contaminants for which bioremediation has been an effective treatment strategy. Reductive dehalogenase (RDase) enzymes from organohalide-respiring bacteria are essential for their remediation under anaerobic conditions. RDases are responsible for dehalogenating these chlorinated solvents, leading to their removal. This work explores the kinetic characteristics of three closely related Dehalobacter chloroalkane-reductases─TmrA, CfrA, and AcdA─and identifies differences between their activity on chloroform (CF), 1,1,1-trichloroethane (TCA), and 1,1,2-TCA. The side-by-side comparison of these enzymes has emphasized that TmrA and AcdA are specialized toward CF with both having a 4-fold higher maximum specific activity ( V max ) on CF than 1,1,1-TCA, whereas CfrA has very similar rates on both CF and 1,1,1-TCA. AcdA is the most sensitive to substrate inhibition by CF and 1,1,2-TCA and inhibition by a common cocontaminant trichloroethene. Finally, the reduction of 1,1,2-TCA, which can produce both 1,2-dichloroethane and vinyl chloride, was assessed for each enzyme. Interestingly, each enzyme has a distinct preference for the major product it produces, indicating a favored reaction pathway. Despite over 95% sequence identity, TmrA, CfrA, and AcdA exhibit substantial differences in kinetic behavior, highlighting the importance of understanding such nuances for informed bioremediation strategies.

Here we present two metagenomes and two Dehalobacter metagenome-assembled genomes from subcultures of an anaerobic chloroform and dichloromethane degrading microbial community used for bioremediation. Our objective was to assemble and curate the genome(s) of Dehalobacter , key biodegraders in the culture, through repeated sequencing and joint assembly with previous datasets., Competing Interests: The authors declare no conflict of interest.

Dehalobacter is a genus of organohalide-respiring bacteria that is recognized for its fastidious growth using reductive dehalogenases (RDases). In the SC05 culture, however, a Dehalobacter population also mineralizes dichloromethane (DCM) produced by chloroform dechlorination using the mec cassette, just downstream of its active RDase. A closed genome of this DCM-mineralizing lineage has previously evaded assembly. Here, we present the genomes of two novel Dehalobacter strains, each of which was assembled from the metagenome of a distinct subculture from SC05. A pangenomic analysis of the Dehalobacter genus, including RDase synteny and phylogenomics, reveals at least five species of Dehalobacter based on average nucleotide identity, RDase and core gene synteny, as well as differential functional genes. An integration hotspot is also pinpointed in the Dehalobacter genome, in which many recombinase islands have accumulated. This nested recombinase island encodes the active RDase and mec cassette in both SC05 Dehalobacter genomes, indicating the transfer of key functional genes between species of Dehalobacter . Horizontal gene transfer between these two novel Dehalobacter strains has implications for the evolutionary history within the SC05 subcultures and of the Dehalobacter genus as a whole, especially regarding adaptation to anthropogenic chemicals.

Using bioelectrochemical systems (BESs) to provide electrochemically generated hydrogen is a promising technology to provide electron donors for reductive dechlorination by organohalide-respiring bacteria. In this study, we inoculated two syntrophic dechlorinating cultures containing Dehalobacter and Dehalobacterium to sequentially transform chloroform (CF) to acetate in a BES using a graphite fiber brush as the electrode. In this co-culture, Dehalobacter transformed CF to stoichiometric amounts of dichloromethane (DCM) via organohalide respiration, whereas the Dehalobacterium-containing culture converted DCM to acetate via fermentation. BES were initially inoculated with Dehalobacter, and sequential cathodic potentials of −0.6, −0.7, and −0.8 V were poised after consuming three CF doses (500 μM) per each potential during a time-span of 83 days. At the end of this period, the accumulated DCM was degraded in the following seven days after the inoculation of Dehalobacterium. At this point, four consecutive amendments of CF at increasing concentrations of 200, 400, 600, and 800 μM were sequentially transformed by the combined degradation activity of Dehalobacter and Dehalobacterium. The Dehalobacter 16S rRNA gene copies increased four orders of magnitude during the whole period. The coulombic efficiencies associated with the degradation of CF reached values > 60% at a cathodic potential of −0.8 V when the degradation rate of CF achieved the highest values. This study shows the advantages of combining syntrophic bacteria to fully detoxify chlorinated compounds in BESs and further expands the use of this technology for treating water bodies impacted with pollutants.

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 CF) 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 CF culture to which 14C-CF was added, the predominant product was 14CO2, indicating that oxidation is the predominant pathway for DCM. Recovery of 14CDCM 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 biomass 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 lower compared to that of treatments that received an exogenous electron donor. [ABSTRACT FROM AUTHOR]

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 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; however, 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 Escherichia 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. [ABSTRACT FROM AUTHOR]

Tetrabromobisphenol A (TBBPA), a widely used brominated flame retardant in electronics manufacturing, has caused global contamination due to improper e-waste disposal. Its persistence, bioaccumulation, and potential carcinogenicity drive studies of its transformation and underlying (a)biotic interactions. This study achieved an anaerobic enrichment culture capable of reductively dehalogenating TBBPA to the more bioavailable bisphenol A. 16S rRNA gene amplicon sequencing and quantitative PCR confirmed that successive dehalogenation of four bromide ions from TBBPA was coupled with the growth of both Dehalobacter sp. and Dehalococcoides sp. with growth yields of 5.0 ± 0.4 × 10 8 and 8.6 ± 4.6 × 10 8 cells per μmol Br - released ( N = 3), respectively. TBBPA dehalogenation was facilitated by solid humin and reduced humin, which possessed the highest organic radical signal intensity and reducing groups -NH 2 , and maintained the highest dehalogenation rate and dehalogenator copies. Genome-centric metatranscriptomic analyses revealed upregulated putative TBBPA-dehalogenating rdhA (reductive dehalogenase) genes with humin amendment, cprA -like Dhb_rdhA1 gene in Dehalobacter species, and Dhc_rdhA1/Dhc_rdhA2 genes in Dehalococcoides species. The upregulated genes of lactate fermentation, de novo corrinoid biosynthesis, and extracellular electron transport in the humin amended treatment also stimulated TBBPA dehalogenation. This study provided a comprehensive understanding of humin-facilitated organohalide respiration.

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 x 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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Dehalobacter sp. strain TeCB1 was isolated from groundwater near Sydney, Australia, that is polluted with a range of organochlorines. The isolated strain is able to grow by reductive dechlorination of 1,2,4,5-tetrachlorobenzene to 1,3- and 1,4-dichlorobenzene with 1,2,4-trichlorobenzene being the intermediate daughter product. Transient production of 1,2-dichlorobenzene was detected with subsequent conversion to monochlorobenzene. The dehalogenation capability of strain TeCB1 to respire 23 alternative organochlorines was examined and shown to be limited to the use of 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene. Growth on 1,2,4-trichlorobenzene resulted in the production of predominantly 1,3- and 1,4-dichlorobenzene. The inability of strain TeCB1 to grow on 1,2-dichlorobenzene indicated that the production of monochlorobenzene during growth on 1,2,4,5-tetarchlorobezene was cometabolic. The annotated genome of strain TeCB1 contained only one detectable 16S rRNA gene copy and genes for 23 full-length and one truncated Reductive Dehalogenase (RDase) homologs, five unique to strain TeCB1. Identification and functional characterization of the 1,2,4,5-tetrachlorobenzene and 1,2,4-trichlorobenzene RDase (TcbA) was achieved using native-PAGE coupled with liquid chromatography tandem mass spectrometry. Interestingly, TcbA showed higher amino acid identity with tetrachloroethene reductases PceA (95% identity) from Dehalobacter restrictus PER-K23 and Desulfitobacterium hafniense Y51 than with the only other chlorinated benzene reductase [i.e., CbrA (30% identity)] functionally characterized to date. [ABSTRACT FROM AUTHOR]

Dechlorination patterns of three tetrachlorobenzene isomers, 1,2,3,4-, 1,2,3,5-, and 1,2,4,5-TeCB, were studied in anoxic microcosms derived from contaminated harbor sludge. The removal of doubly, singly, and un-flanked chlorine atoms was noted in 1,2,3,4- and 1,2,3,5-TeCB fed microcosms, whereas only singly flanked chlorine was removed in 1,2,4,5-TeCB microcosms. The thermodynamically more favorable reactions were selectively followed by the enriched cultures with di- and/or mono-chlorobenzene as the main end products of the reductive dechlorination of all three isomers. Based on quantitative PCR analysis targeting 16S rRNA genes of known organohalide-respiring bacteria, the growth of Dehalococcoides was found to be associated with the reductive dechlorination of all three isomers, while growth of Dehalobacter, another known TeCB dechlorinator, was only observed in one 1,2,3,5-TeCB enriched microcosm among biological triplicates. Numbers of Desulfitobacterium and Geobacter as facultative dechlorinators were rather stable suggesting that they were not (directly) involved in the observed TeCB dechlorination. Bacterial community profiling suggested bacteria belonging to the phylum Bacteroidetes and the order Clostridiales as well as sulfate-reducing members of the class Deltaproteobacteria as putative stimulating guilds that provide electron donor and/or organic cofactors to fastidious dechlorinators. Our results provide a better understanding of thermodynamically preferred TeCB dechlorinating pathways in harbor environments and microbial guilds enriched and active in anoxic TeCB dechlorinating microcosms. [ABSTRACT FROM AUTHOR]

Biodegradation of dichloromethane (DCM) under reducing conditions is of major concern due to its widespread detection in contaminated groundwaters. Here, we report an anaerobic enrichment culture derived from a membrane bioreactor operating in an industrial wastewater treatment plant, capable of fermenting DCM and the brominated analogue dibromomethane (DBM). Comparative analysis of bacterial 16S rDNA-DGGE profiles from fresh liquid medium inoculated with single colonies picked from serial dilution-to-extinction agar vials showed that cultures degrading DCM contained a predominant band belonging to Dehalobacterium , however this band was absent in cultures unable to degrade DCM. Analysis of the microbial composition of the enrichment by bacterial 16S rRNA gene amplicon paired-end sequencing confirmed the presence of Dehalobacterium together with three additional phylotypes belonging to Acetobacterium , Desulfovibrio , and Wolinella , representing all four operational taxonomic units > 99.9% of the retrieved sequences. The carbon isotopic fractionation (ε) determined for DCM degradation in this culture was − 27 ± 2‰. This value differs from the ε previously reported for the DCM-fermentative bacteria Dehalobacter (− 15.5 ± 1.5‰) but they are both significantly different from those reported for facultative methylotrophic organisms (ranging from − 45 to − 61‰). This significant difference in the ε allows differentiating between hydrolytic transformation of DCM via glutathione-dependent dehalogenases and fermentation pathway. Capsule The carbon isotopic fractionation of dichloromethane by an enriched Dehalobacterium -containing culture has significant potential to monitor biodegradation of DCM in groundwaters. [ABSTRACT FROM AUTHOR]

Organohalides are recalcitrant pollutants that have been responsible for substantial contamination of soils and groundwater. Organohalide-respiring bacteria (ORB) provide a potential solution to remediate contaminated sites, through their ability to use organohalides as terminal electron acceptors to yield energy for growth (i.e. organohalide respiration). Ideally, this process results in non- or lesser-halogenated compounds that are mostly less toxic to the environment or more easily degraded. At the heart of these processes are reductive dehalogenases (RDase), which are membrane bound enzymes coupled with other components that facilitate dehalogenation of organohalides to generate cellular energy. This review focuses RDases, concentrating on those which have been purified (partially or wholly) and functionally characterized. Further, the paper reviews the major bacteria involved in organohalide breakdown and the evidence for microbial evolution of RDases. Finally, the capacity for using ORB in a bioremediation and bioaugmentation capacity are discussed.

The genomes of two closely related Dehalobacter strains (strain CF and strain DCA) were assembled from the metagenome of an anaerobic enrichment culture that reductively dechlorinates chloroform (CF), 1,1,1-trichloroethane (1,1,1-TCA) and 1,1-dichloroethane (1,1-DCA). The 3.1 Mbp genomes of strain CF (that dechlorinates CF and 1,1,1-TCA) and strain DCA (that dechlorinates 1,1-DCA) each contain 17 putative reductive dehalogenase homologous (rdh) genes. These two genomes were systematically compared to three other available organohalide-respiring Dehalobacter genomes (Dehalobacter restrictus strain PER-K23, Dehalobacter sp. strain E1 and Dehalobacter sp. strain UNSWDHB), and to the genomes of Dehalococcoides mccartyi strain 195 and Desulfitobacterium hafniense strain Y51. This analysis compared 42 different metabolic and physiological categories. The genomes of strains CF and DCA share 90% overall average nucleotide identity and greater than 99.8% identity over a 2.9 Mbp alignment that excludes large insertions, indicating that these genomes differentiated from a close common ancestor. This differentiation was likely driven by selection pressures around two orthologous reductive dehalogenase genes, cfrA and dcrA, that code for the enzymes that reduce CF or 1,1,1-TCA and 1,1-DCA. The many reductive dehalogenase genes found in the five Dehalobacter genomes cluster into two small conserved regions and were often associated with Crp/Fnr transcriptional regulators. Specialization is on-going on a strain-specific basis, as some strains but not others have lost essential genes in the Wood-Ljungdahl (strain E1) and corrinoid biosynthesis pathways (strains E1 and PER-K23). The gene encoding phosphoserine phosphatase, which catalyzes the last step of serine biosynthesis, is missing from all five Dehalobacter genomes, yet D. restrictus can grow without serine, suggesting an alternative or unrecognized biosynthesis route exists. In contrast to Dehalococcoides mccartyi, a complete heme biosynthesis pathway is present in the five Dehalobacter genomes. This pathway corresponds to a newly described alternative heme biosynthesis route first identified in Archaea. This analysis of organohalide-respiring Firmicutes and Chloroflexi reveals profound evolutionary differences despite very similar niche-specific metabolism and function.

Nucleic acid amplification of biomarkers is increasingly used to monitor microbial activity and assess remedial performance in contaminated aquifers. Previous studies described the use of filtration, elution, and direct isothermal amplification (i.e. no DNA extraction and purification) as a field-able means to quantify Dehalococcoides spp. in groundwater. This study expands previous work with direct loop mediated isothermal amplification (LAMP) for the detection and quantification of Dehalobacter spp. in groundwater. Experiments tested amplification of DNA with and without crude lysis and varying concentrations of humic acid. Three separate field-able methods of biomass concentration with eight aquifer samples were also tested, comparing direct LAMP with traditional DNA extraction and quantitative PCR (qPCR). A new technique was developed where filters were amplified directly within disposable Gene-Z chips. The direct filter amplification (DFA) method eliminated an elution step and provided a detection limit of 10 2 Dehalobacter cells per 100 mL. LAMP with crudely lysed Dehalobacter had a negligible effect on threshold time and sensitivity compared to lysed samples. The LAMP assay was more resilient than traditional qPCR to humic acid in sample, amplifying with up to 100 mg per L of humic acid per reaction compared to 1 mg per L for qPCR. Of the tested field-able concentrations methods, DFA had the lowest coefficient of variation among Dehalobacter spiked groundwater samples and lowest threshold time indicating high capture efficiency and low inhibition. While demonstrated with Dehalobacter , the DFA method can potentially be used for a number of applications requiring field-able, rapid (< 60 min) and highly sensitive quantification of microorganisms in environmental water samples. [ABSTRACT FROM AUTHOR]

Bioremediation strategies, including bioaugmentation with chlorinated ethene-degrading enrichment cultures, have been successfully applied in the cleanup of subsurface environments contaminated with tetrachloroethene (PCE) and/or trichloroethene (TCE). However, these compounds are frequently found in the environment as components of mixtures that may also contain chlorinated ethanes and methanes. Under these conditions, the implementation of bioremediation may be complicated by inhibition effects, particularly when multiple dehalorespirers are present. We investigated the ability of the 1,1,2,2-tetrachloroethane (TeCA)-dechlorinating culture WBC-2 to biotransform TeCA alone, or a mixture of TeCA plus PCE and carbon tetrachloride (CT), in microcosms. The microcosms contained electron donors provided to biostimulate the added culture and sediment collected from a wetland where numerous “hotspots” of contamination with chlorinated solvent mixtures exist. The dominant TeCA biodegradation mechanism mediated by the WBC-2 culture in the microcosms was different in the presence of these wetland sediments than in the sediment-free enrichment culture or in previous WBC-2 bioaugmented microcosms and column tests conducted with wetland sediment collected at nearby sites. The co-contaminants and their daughter products also inhibited TeCA biodegradation by WBC-2. These results highlight the need to conduct biodegradability assays at new sites, particularly when multiple contaminants and dehalorespiring populations are present. [ABSTRACT FROM AUTHOR]

Organohalides are recalcitrant pollutants that have been responsible for substantial contamination of soils and groundwater. Organohalide-respiring bacteria (ORB) provide a potential solution to remediate contaminated sites, through their ability to use organohalides as terminal electron acceptors to yield energy for growth (i.e., organohalide respiration). Ideally, this process results in non- or lesser-halogenated compounds that are mostly less toxic to the environment or more easily degraded. At the heart of these processes are reductive dehalogenases (RDases), which are membrane bound enzymes coupled with other components that facilitate dehalogenation of organohalides to generate cellular energy. This review focuses on RDases, concentrating on those which have been purified (partially or wholly) and functionally characterized. Further, the paper reviews the major bacteria involved in organohalide breakdown and the evidence for microbial evolution of RDases. Finally, the capacity for using ORB in a bioremediation and bioaugmentation capacity are discussed. [ABSTRACT FROM AUTHOR]

De novo corrinoid biosynthesis represents one of the most complicated metabolic pathways in nature. Organohalide-respiring bacteria (OHRB) have developed different strategies to deal with their need of corrinoid, as it is an essential cofactor of reductive dehalogenases, the key enzymes in OHR metabolism.In contrast to Dehalococcoides mccartyi, the genome of Dehalobacter restrictus strain PER-K23 contains a complete set of corrinoid biosynthetic genes, of which cbiH appears to be truncated and therefore non-functional, possibly explaining the corrinoid auxotrophy of this obligate OHRB. Comparative genomics within Dehalobacter spp. revealed that one (operon-2) of the five distinct corrinoid biosynthesis associated operons present in the genome of D. restrictus appeared to be present only in that particular strain, which encodes multiple members of corrinoid transporters and salvaging enzymes. Operon-2 was highly up-regulated upon corrinoid starvation both at the transcriptional (346-fold) and proteomic level (46-fold on average), in line with the presence of an upstream cobalamin riboswitch. Together, these data highlight the importance of this operon in corrinoid homeostasis in D. restrictus and the augmented salvaging strategy this bacterium adopted to cope with the need for this essential cofactor.

Halogenated organic compounds (organohalides) are globally prevalent, recalcitrant toxic, and carcinogenic environmental pollutants. Select microorganisms encode enzymes known as reductive dehalogenases (EC 1.97.1.8) that catalyze reductive dehalogenation reactions resulting in the generation of lesser-halogenated compounds that may be less toxic and more biodegradable. Recent breakthroughs in enzyme structure determination, elucidation of the mechanisms of reductive dehalogenation, and in heterologous expression of functional reductive dehalogenase enzymes have substantially increased our understanding of this fascinating class of enzymes. This knowledge has created opportunities for more versatile ( in situ and ex situ ) biologically-mediated organohalide destruction strategies. [ABSTRACT FROM AUTHOR]

Organohalide contaminants such as triclosan and triclocarban have been well documented in municipal wastewater treatment plants (WWTPs), but the degradation of these contaminants is not well understood. One possible removal mechanism is organohalide respiration by which bacteria reduce the halogenated compound. The purpose of this study was to determine the abundance of organohalide-respiring bacteria in eight WWTP anaerobic digesters. The obligate organohalide respiring Dehalococcoides mccartyi was the most abundant and averaged 3.3 × 107 copies of 16S rRNA genes per gram, while the Dehalobacter was much lower at 2.6 × 104 copies of 16S rRNA genes per gram. The genus Sulfurospirillum spp. was also detected at 1.0 × 107 copies of 16S rRNA genes per gram. No other known or putatively organohalide-respiring strains in the Dehalococcoidaceae family were found to be present nor were the genera Desulfitobacterium or Desulfomonile. [ABSTRACT FROM AUTHOR]

De novo corrinoid biosynthesis represents one of the most complicated metabolic pathways in nature. Organohalide-respiring bacteria (OHRB) have developed different strategies to deal with their need of corrinoid, as it is an essential cofactor of reductive dehalogenases, the key enzymes in OHR metabolism. In contrast to Dehalococcoides mccartyi, the genome of Dehalobacter restrictus strain PER-K23 contains a complete set of corrinoid biosynthetic genes, of which cbiH appears to be truncated and therefore non-functional, possibly explaining the corrinoid auxotrophy of this obligate OHRB. Comparative genomics within Dehalobacter spp. revealed that one (operon-2) of the five distinct corrinoid biosynthesis associated operons present in the genome of D. restrictus appeared to be present only in that particular strain, which encodes multiple members of corrinoid transporters and salvaging enzymes. Operon-2 was highly up-regulated upon corrinoid starvation both at the transcriptional (346-fold) and proteomic level (46-fold on average), in line with the presence of an upstream cobalamin riboswitch. Together, these data highlight the importance of this operon in corrinoid homeostasis in D. restrictus and the augmented salvaging strategy this bacterium adopted to cope with the need for this essential cofactor. [ABSTRACT FROM AUTHOR]

Microcosm studies investigated the effects of bioaugmentation with a mixed Dehalococcoides ( Dhc)/ Dehalobacter ( Dhb) culture on biological enhanced reductive dechlorination for treatment of 1,1,1-trichloroethane (TCA) and chloroethenes in groundwater at three Danish sites. Microcosms were amended with lactate as electron donor and monitored over 600 days. Experimental variables included bioaugmentation, TCA concentration, and presence/absence of chloroethenes. Bioaugmented microcosms received a mixture of the Dhc culture KB-1 and Dhb culture ACT-3. To investigate effects of substrate concentration, microcosms were amended with various concentrations of chloroethanes (TCA or monochloroethane [CA]) and/or chloroethenes (tetrachloroethene [PCE], trichloroethene [TCE], or 1,1-dichloroethene [1,1-DCE]). Results showed that combined electron donor addition and bioaugmentation stimulated dechlorination of TCA and 1,1-dichloroethane (1,1-DCA) to CA, and dechlorination of PCE, TCE, 1,1-DCE and cDCE to ethane. Dechlorination of CA was not observed. Bioaugmentation improved the rate and extent of TCA and 1,1-DCA dechlorination at two sites, but did not accelerate dechlorination at a third site where geochemical conditions were reducing and Dhc and Dhb were indigenous. TCA at initial concentrations of 5 mg/L inhibited (i.e., slowed the rate of) TCA dechlorination, TCE dechlorination, donor fermentation, and methanogenesis. 1 mg/L TCA did not inhibit dechlorination of TCA, TCE or cDCE. Moreover, complete dechlorination of PCE to ethene was observed in the presence of 3.2 mg/L TCA. In contrast to some prior reports, these studies indicate that low part-per million levels of TCA (<3 mg/L) in aquifer systems do not inhibit dechlorination of PCE or TCE to ethene. In addition, the results show that co-bioaugmentation with Dhc and Dhb cultures can be an effective strategy for accelerating treatment of chloroethane/chloroethene mixtures in groundwater, with the exception that all currently known Dhc and Dhb cultures cannot treat CA. [ABSTRACT FROM AUTHOR]

Chlorophenols are widely used as biocides, leading them to being prevalent environmental contaminants that pose toxic threats to ecosystems. In this study, a Dehalobacter species strain TCP1 was isolated from a digester sludge sample, which is able to dechlorinate 2,4,6-trichlorophenol (2,4,6-TCP) to 4-monochlorophenol (4-MCP) with H as the sole electron donor and acetate as the carbon source. Strain TCP1 also distinguishes itself from other Dehalobacter species with its capability to dechlorinate tetrachloroethene or trichloroethene (TCE) to both cis- and trans-dichloroethenes in a ratio of 5.6 (±0.2):1. The growth yields of strain TCP1 on TCE and 2,4,6-TCP were 4.14 × 10 and 5.77 × 10 cells mol of Cl released, respectively. Strain TCP1 contains five unusually long 16S rRNA gene copies per genome, and the extra length is due to the ~110 bp insertion sequences at their 5′-ends. This suggests that strain TCP1 may represent a novel Dehalobacter species. A putative chlorophenol reductive dehalogenase gene- debcprA-was identified to catalyze the ortho-chlorine removal from 2,4,6-TCP. Both the culture-dependent and housekeeping rpoB gene-based approaches indicate the purity of the culture. Strain TCP1 can serve as a promising candidate for the bioremediation of 2,4,6-TCP contaminated sites, and its discovery expands our understanding of metabolic capabilities of Dehalobacter species. [ABSTRACT FROM AUTHOR]

Highlights: [•] A TBBPA-debrominating microbial consortium was obtained. [•] The debrominating activity of the consortium was dependent on humin. [•] A Dehalobacter species was involved in the debromination of TBBPA. [•] The Dehalobacter species was reliant on humin for its debromination activity. [Copyright &y& Elsevier]

Across the U. S. Upper Midwest, a natural geographical sulfate gradient exists in lakes. Sediment grab samples and cores were taken to explore whether this sulfur gradient impacted organohalide-respiring Chloroflexi in lake sediments. Putative organohalide-respiring Chloroflexi were detected in 67 of 68 samples by quantitative polymerase chain reaction. Their quantities ranged from 3.5 × 104 to 8.4 × 1010 copies 16 S r RNA genes g−1 dry sediment and increased in number from west to east, whereas lake sulfate concentrations decreased along this west-to-east transect. A terminal restriction fragment length polymorphism ( TRFLP) method was used to corroborate this inverse relationship, with sediment samples from lower sulfate lakes containing both a higher number of terminal restriction fragments ( TRFs) belonging to the organohalide-respiring Dehalococcoidetes, and a greater percentage of the TRFLP amplification made up by Dehalococcoidetes members. Statistical analyses showed that dissolved sulfur in the porewater, measured as sulfate after oxidation, appeared to have a negative impact on the total number of putative organohalide-respiring Chloroflexi, the number of Dehalococcoidetes TRFs, and the percentage of the TRFLP amplification made up by Dehalococcoidetes. These findings point to dissolved sulfur, presumably present as reduced sulfur species, as a potentially controlling factor in the natural cycling of chlorine, and perhaps as a result, the natural cycling of some carbon as well. [ABSTRACT FROM AUTHOR]

Two novel reductive dehalogenases (RDases) that are highly similar to each other but catalyse distinct dechlorination reactions were identified from Dehalobacter-containing mixed cultures. These two RDases were partially purified from crude protein extracts of anaerobic dechlorinating enrichment cultures using blue native polyacrylamide gel electrophoresis. Gel slices were assayed for dechlorinating activity, and associated proteins were identified using liquid chromatography tandem mass spectrometry with the metagenome of the parent culture as the reference database. The two RDases identified, annotated as CfrA and DcrA, share an amino acid identity of 95.2 per cent, but use different substrates: CfrA dechlorinates chloroform (CF) and 1,1,1-trichloroethane (1,1,1-TCA), but not 1,1-dichloroethane; DcrA dechlorinates 1,1-dichloroethane, but not CF or 1,1,1-TCA. These two novel RDases share no more than 40 per cent amino acid identity to any other known or putative RDases, but both have a twin-arginine motif and two iron--sulfur binding motifs conserved in most RDases. Peptides specific to two putative membrane anchor proteins, annotated as CfrB and DcrB, were also detected in gel slices. [ABSTRACT FROM AUTHOR]

Dehalobacter restrictus strain PER-K23 is an obligate organohalide respiring bacterium, which displays extremely narrow metabolic capabilities. It grows only via coupling energy conservation to anaerobic respiration of tetra- and trichloroethene with hydrogen as sole electron donor. Dehalobacter restrictus represents the paradigmatic member of the genus Dehalobacter, which in recent years has turned out to be a major player in the bioremediation of an increasing number of organohalides, both in situ and in laboratory studies. The recent elucidation of the D. restrictus genome revealed a rather elaborate genome with predicted pathways that were not suspected from its restricted metabolism, such as a complete corrinoid biosynthetic pathway, the Wood--Ljungdahl (WL) pathway for CO2 fixation, abundant transcriptional regulators and several types of hydrogenases. However, one important feature of the genome is the presence of 25 reductive dehalogenase genes, from which so far only one, pceA, has been characterized on genetic and biochemical levels. This study describes a multi-level functional genomics approach on D. restrictus across three different growth phases. A global proteomic analysis allowed consideration of general metabolic pathways relevant to organohalide respiration, whereas the dedicated genomic and transcriptomic analysis focused on the diversity, composition and expression of genes associated with reductive dehalogenases. [ABSTRACT FROM AUTHOR]

Abstract: The degradation of chlorinated ethenes and ethanes in clay till was investigated at a contaminated site (Vadsby, Denmark) by high resolution sampling of intact cores combined with groundwater sampling. Over decades of contamination, bioactive zones with degradation of trichloroethene (TCE) and 1,1,1-trichloroethane (1,1,1-TCA) to 1,2-cis-dichloroethene (cis-DCE) and 1,1-dichloroethane, respectively, had developed in most of the clay till matrix. Dehalobacter dominated over Dehalococcoides (Dhc) in the clay till matrix corresponding with stagnation of sequential dechlorination at cis-DCE. Sporadically distributed bioactive zones with partial degradation to ethene were identified in the clay till matrix (thickness from 0.10 to 0.22m). In one sub-section profile the presence of Dhc with the vcrA gene supported the occurrence of degradation of cis-DCE and VC, and in another enriched δ13C for TCE, cis-DCE and VC documented degradation. Highly enriched δ13C for 1,1,1-TCA (25‰) and cis-DCE (−4‰) suggested the occurrence of abiotic degradation in a third sub-section profile. Due to fine scale heterogeneity the identification of active degradation zones in the clay till matrix depended on high resolution subsampling of the clay till cores. The study demonstrates that an integrated approach combining chemical analysis, molecular microbial tools and compound specific isotope analysis (CSIA) was required in order to document biotic and abiotic degradations in the clay till system. [Copyright &y& Elsevier]

Abstract: 1,1,1-Trichloroethane (TCA) in groundwater is susceptible to a variety of natural degradation mechanisms. Evidence of intrinsic decay of TCA in aquifers is commonly observed; however, TCA remains a persistent pollutant at many sites and some of the daughter products that accumulate from intrinsic decay of TCA have been determined to be more toxic than the parent compound. Research advances from the past decade indicate that in situ enhanced reductive dechlorination (ERD) offers promise as a cost-effective solution toward the cleanup of groundwater contaminated with TCA and its transformation daughter products. Laboratory studies have demonstrated that pure or mixed cultures containing certain Dehalobacter (Dhb) bacteria can catalyze respiratory dechlorination of TCA and 1,1-dichloroethane (1,1-DCA) to monochloroethane (CA) in groundwater systems. 16S rRNA Dhb gene probes have been used as biomarkers in groundwater samples to both assess ERD potential and quantify growth of Dhb in ERD applications at TCA sites. Laboratory findings suggest that iron-bearing minerals and methanogenic bacteria that co-occur in reduced aquifers may synergistically affect dechlorination of TCA. Despite these advances, a number of significant challenges remain, including an inability of any known cultures to completely dechlorinate TCA to ethane. CA is commonly observed as a terminal product of the biological reductive dechlorination of TCA and 1,1-DCA. Also important is the lack of rigorous field studies demonstrating the utility of bioaugmentation with Dhb cultures for remediation of TCA in the field. In this paper we review the state-of-the-science of TCA degradation in aquifers, examining results from both laboratory experiments and twenty-two field case studies, focusing on the capabilities and limits of ERD technology, and identifying aspects of the technology that warrant further development. [Copyright &y& Elsevier]