Mineralization versus fermentation: evidence for two distinct anaerobic bacterial degradation pathways for dichloromethane.

Dichloromethane (DCM) is an anthropogenic pollutant with ozone destruction potential that is also formed naturally. Under anoxic conditions, fermentation of DCM to acetate and formate has been reported in axenic culture Dehalobacterium formicoaceticum, and to acetate, H2 and CO2 in mixed culture RM, which harbors the DCM degrader 'Candidatus Dichloromethanomonas elyunquensis'. RM cultures produced 28.1 ± 2.3 µmol of acetate from 155.6 ± 9.3 µmol DCM, far less than the one third (i.e., about 51.9 µmol) predicted based on the assumed fermentation model, and observed in cultures of Dehalobacterium formicoaceticum. Temporal metabolite analyses using gas chromatography-mass spectrometry (GC-MS) and nuclear magnetic resonance (NMR) spectroscopy revealed that no 13C-labeled acetate was formed in 13C-DCM-grown RM cultures, indicating acetate was not a direct product of DCM metabolism. The data were reconciled with DCM mineralization and H2 consumption via CO2 reduction to acetate and methane by homoacetogenic and methanogenic partner populations, respectively. In contrast, Dehalobacterium formicoaceticum produced 13C-labeled acetate and formate from 13C-DCM, consistent with a fermentation pathway. Free energy change calculations predicted that organisms with the mineralization pathway are the dominant DCM consumers in environments with H2 <100 ppmv. These findings have implications for carbon and electron flow in environments where DCM is introduced through natural production processes or anthropogenic activities.

Biotic and Abiotic Dehalogenation of 1,1,2-Trichloro-1,2,2-trifluoroethane (CFC-113): Implications for Bacterial Detoxification of Chlorinated Ethenes.

Chlorofluorocarbons including 1,1,2-trichloro-1,2,2-trifluoroethane (CFC-113) often occur in groundwater plumes comingled with chlorinated solvents such as trichloroethene (TCE). We show that CFC-113 inhibits reductive dechlorination by Dehalococcoides mccartyi (Dhc) in a concentration-dependent manner, causing cis-1,2-dichloroethene (cis-DCE) stalls. Following a 17-day exposure of Dhc-containing consortium SDC-9 to 76 µM CFC-113, cis-DCE dechlorination activity did not recover after CFC-113 removal. River sediment microcosms demonstrated that CFC-113 was subject to microbial degradation under anoxic conditions, and chlorotrifluoroethene (CTFE) was observed as a transformation product. No degradation of CFC-113 was observed in killed controls and in incubations with reactive minerals including mackinawite, green rust, magnetite, and manganese dioxide. In vitro experiments with reduced corrinoid (i.e., vitamin B12) mediated reductive dechlorination of CFC-113 to CTFE and trifluoroethene (TFE) followed by reductive defluorination of TFE to cis-1,2-difluoroethene (cis-DFE) as an end product. This biomimetic degradation of CFC-113 to cis-DFE was also demonstrated in vivo using the corrinoid-producing homoacetogen Sporomusa ovata, suggesting the cometabolic microbial reductive dechlorination and reductive defluorination of CFC-113 to cis-DFE is feasible under anoxic in situ conditions. The CFC-113 degradation intermediates CTFE, TFE, and cis-DFE did not inhibit TCE dechlorination by Dhc, indicating that the initial reductive transformation step can overcome cis-DCE stalls.

Proteogenomics Reveals Novel Reductive Dehalogenases and Methyltransferases Expressed during Anaerobic Dichloromethane Metabolism.

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.

Dual Carbon-Chlorine Isotope Analysis Indicates Distinct Anaerobic Dichloromethane Degradation Pathways in Two Members of Peptococcaceae.

Dichloromethane (DCM) is a probable human carcinogen and frequent groundwater contaminant and contributes to stratospheric ozone layer depletion. DCM is degraded by aerobes harboring glutathione-dependent DCM dehalogenases; however, DCM contamination occurs in oxygen-deprived environments, and much less is known about anaerobic DCM metabolism. Some members of the Peptococcaceae family convert DCM to environmentally benign products including acetate, formate, hydrogen (H2), and inorganic chloride under strictly anoxic conditions. The current study applied stable carbon and chlorine isotope fractionation measurements to the axenic culture Dehalobacterium formicoaceticum and to the consortium RM comprising DCM degrader Candidatus Dichloromethanomonas elyunquensis. Degradation-associated carbon and chlorine isotope enrichment factors (εC and εCl) of -42.4 ± 0.7‰ and -5.3 ± 0.1‰, respectively, were measured in D. formicoaceticum cultures. A similar εCl of -5.2 ± 0.1‰, but a substantially lower εC of -18.3 ± 0.2‰, were determined for Ca. Dichloromethanomonas elyunquensis. The εC and εCl values resulted in distinctly different dual element C-Cl isotope correlations (ΛC/Cl = Δδ13C/Δδ37Cl) of 7.89 ± 0.12 and 3.40 ± 0.03 for D. formicoaceticum and Ca. Dichloromethanomonas elyunquensis, respectively. The distinct ΛC/Cl values obtained for the two cultures imply mechanistically distinct C-Cl bond cleavage reactions, suggesting that members of Peptococcaceae employ different pathways to metabolize DCM. These findings emphasize the utility of dual carbon-chlorine isotope analysis to pinpoint DCM degradation mechanisms and to provide an additional line of evidence that detoxification is occurring at DCM-contaminated sites.

Mutualistic interaction between dichloromethane- and chloromethane-degrading bacteria in an anaerobic mixed culture.

The microbial mixed culture RM grows with dichloromethane (DCM) as the sole energy source generating acetate, methane, chloride and biomass as products. Chloromethane (CM) was not an intermediate during DCM utilization consistent with the observation that CM could not replace DCM as a growth substrate. Interestingly, cultures that received DCM and CM together degraded both compounds concomitantly. Transient hydrogen (H2 ) formation reaching a maximum concentration of 205 ± 13 ppmv was observed in cultures growing with DCM, and the addition of exogenous H2 at concentrations exceeding 3000 ppmv impeded DCM degradation. In contrast, CM degradation in culture RM had a strict requirement for H2 . Following five consecutive transfers on CM and H2 , Acetobacterium 16S rRNA gene sequences dominated the culture and the DCM-degrader Candidatus Dichloromethanomonas elyunquensis was eliminated, consistent with the observation that the culture lost the ability to degrade DCM. These findings demonstrate that culture RM harbours different populations responsible for anaerobic DCM and CM metabolism, and further imply that the DCM and CM degradation pathways are mechanistically distinct. H2 generated during DCM degradation is consumed by the hydrogenotrophic CM degrader, or may fuel other hydrogenotrophic processes, including organohalide respiration, methanogenesis and H2 /CO2 reductive acetogenesis.

Complete Genome Sequence of Dehalobacterium formicoaceticum Strain DMC, a Strictly Anaerobic Dichloromethane-Degrading Bacterium.

Dehalobacterium formicoaceticum utilizes dichloromethane as the sole energy source in defined anoxic bicarbonate-buffered mineral salt medium. The products are formate, acetate, inorganic chloride, and biomass. The bacterium's genome was sequenced using PacBio, assembled, and annotated. The complete genome consists of one 3.77-Mb circular chromosome harboring 3,935 predicted protein-encoding genes.