Nitrous oxide inhibition of methanogenesis represents an underappreciated greenhouse gas emission feedback.

Methane (CH4) and nitrous oxide (N2O) are major greenhouse gases that are predominantly generated by microbial activities in anoxic environments. N2O inhibition of methanogenesis has been reported, but comprehensive efforts to obtain kinetic information are lacking. Using the model methanogen Methanosarcina barkeri strain Fusaro and digester sludge-derived methanogenic enrichment cultures, we conducted growth yield and kinetic measurements and showed that micromolar concentrations of N2O suppress the growth of methanogens and CH4 production from major methanogenic substrate classes. Acetoclastic methanogenesis, estimated to account for two-thirds of the annual 1 billion metric tons of biogenic CH4, was most sensitive to N2O, with inhibitory constants (KI) in the range of 18-25 µM, followed by hydrogenotrophic (KI, 60-90 µM) and methylotrophic (KI, 110-130 µM) methanogenesis. Dissolved N2O concentrations exceeding these KI values are not uncommon in managed (i.e. fertilized soils and wastewater treatment plants) and unmanaged ecosystems. Future greenhouse gas emissions remain uncertain, particularly from critical zone environments (e.g. thawing permafrost) with large amounts of stored nitrogenous and carbonaceous materials that are experiencing unprecedented warming. Incorporating relevant feedback effects, such as the significant N2O inhibition on methanogenesis, can refine climate models and improve predictive capabilities.

Integrated Advanced Molecular Tools Predict In Situ cVOC Degradation Rates: Field Demonstration.

Chlorinated volatile organic compound (cVOC) degradation rate constants are crucial information for site management. Conventional approaches generate rate estimates from the monitoring and modeling of cVOC concentrations. This requires time series data collected along the flow path of the plume. The estimates of rate constants are often plagued by confounding issues, making predictions cumbersome and unreliable. Laboratory data suggest that targeted quantitative analysis of Dehalococcoides mccartyi (Dhc) biomarker genes (qPCR) and proteins (qProt) can be directly correlated with reductive dechlorination activity. To assess the potential of qPCR and qProt measurements to predict rates, we collected data from cVOC-contaminated aquifers. At the benchmark study site, the rate constant for degradation of cis-dichloroethene (cDCE) extracted from monitoring data was 11.0 ± 3.4 yr-1, and the rate constant predicted from the abundance of TceA peptides was 6.9 yr-1. The rate constant for degradation of vinyl chloride (VC) from monitoring data was 8.4 ± 5.7 yr-1, and the rate constant predicted from the abundance of TceA peptides was 5.2 yr-1. At the other study sites, the rate constants for cDCE degradation predicted from qPCR and qProt measurements agreed within a factor of 4. Under the right circumstances, qPCR and qProt measurements can be useful to rapidly predict rates of cDCE and VC biodegradation, providing a major advance in effective site management.

Dehalogenimonas etheniformans sp. nov., a formate-oxidizing, organohalide-respiring bacterium isolated from grape pomace.

A strictly anaerobic, organohalide-respiring bacterium, designated strain GPT, was characterized using a polyphasic approach. GPT is Gram-stain-negative, non-spore-forming and non-motile. Cells are irregular cocci ranging between 0.6 and 0.9 µm in diameter. GPT couples growth with the reductive dechlorination of 1,2-dichloroethane, vinyl chloride and all polychlorinated ethenes, except tetrachloroethene, yielding ethene and inorganic chloride as dechlorination end products. H2 and formate serve as electron donors for organohalide respiration in the presence of acetate as carbon source. Major cellular fatty acids include C16 : 0, C18 : 1ω9c, C16 : 1, C14 : 0 and C18 : 0. On the basis of 16S rRNA gene phylogeny, GPT is most closely related to Dehalogenimonas formicexedens NSZ-14T and Dehalogenimonas alkenigignens IP3-3T with 99.8 and 97.4 % sequence identities, respectively. Genome-wide pairwise comparisons based on average nucleotide identity, average amino acid identity and digital DNA-DNA hybridization do not support the inclusion of GPT in previously described species of the genus Dehalogenimonas with validly published names. On the basis of phylogenetic, physiological and phenotypic traits, GPT represents a novel species within the genus Dehalogenimonas, for which the name Dehalogenimonas etheniformans sp. nov. is proposed. The type strain is GPT (= JCM 39172T = CGMCC 1.17861T).

Cometabolic Vinyl Chloride Degradation at Acidic pH Catalyzed by Acidophilic Methanotrophs Isolated from Alpine Peat Bogs.

Remediation of toxic chlorinated ethenes via microbial reductive dechlorination can lead to ethene formation; however, the process stalls in acidic groundwater, leading to the accumulation of carcinogenic vinyl chloride (VC). This study explored the feasibility of cometabolic VC degradation by moderately acidophilic methanotrophs. Two novel isolates, Methylomonas sp. strain JS1 and Methylocystis sp. strain MJC1, were obtained from distinct alpine peat bogs located in South Korea. Both isolates cometabolized VC with CH4 as the primary substrate under oxic conditions at pH at or below 5.5. VC cometabolism in axenic cultures occurred in the presence (10 µM) or absence (<0.01 µM) of copper, suggesting that VC removal had little dependence on copper availability, which regulates expression and activity of soluble and particulate methane monooxygenases in methanotrophs. The model neutrophilic methanotroph Methylosinus trichosporium strain OB3b also grew and cometabolized VC at pH 5.0 regardless of copper availability. Bioaugmentation of acidic peat soil slurries with methanotroph isolates demonstrated enhanced VC degradation and VC consumption below the maximum concentration level of 2 µg L-1. Community profiling of the microcosms suggested species-specific differences, indicating that robust bioaugmentation with methanotroph cultures requires further research.

Anaerobic Microbial Metabolism of Dichloroacetate.

Dichloroacetate (DCA) commonly occurs in the environment due to natural production and anthropogenic releases, but its fate under anoxic conditions is uncertain. Mixed culture RM comprising "Candidatus Dichloromethanomonas elyunquensis" strain RM utilizes DCA as an energy source, and the transient formation of formate, H2, and carbon monoxide (CO) was observed during growth. Only about half of the DCA was recovered as acetate, suggesting a fermentative catabolic route rather than a reductive dechlorination pathway. Sequencing of 16S rRNA gene amplicons and 16S rRNA gene-targeted quantitative real-time PCR (qPCR) implicated "Candidatus Dichloromethanomonas elyunquensis" strain RM in DCA degradation. An (S)-2-haloacid dehalogenase (HAD) encoded on the genome of strain RM was heterologously expressed, and the purified HAD demonstrated the cofactor-independent stoichiometric conversion of DCA to glyoxylate at a rate of 90 ± 4.6 nkat mg-1 protein. Differential protein expression analysis identified enzymes catalyzing the conversion of DCA to acetyl coenzyme A (acetyl-CoA) via glyoxylate as well as enzymes of the Wood-Ljungdahl pathway. Glyoxylate carboligase, which catalyzes the condensation of two molecules of glyoxylate to form tartronate semialdehyde, was highly abundant in DCA-grown cells. The physiological, biochemical, and proteogenomic data demonstrate the involvement of an HAD and the Wood-Ljungdahl pathway in the anaerobic fermentation of DCA, which has implications for DCA turnover in natural and engineered environments, as well as the metabolism of the cancer drug DCA by gut microbiota.IMPORTANCE Dichloroacetate (DCA) is ubiquitous in the environment due to natural formation via biological and abiotic chlorination processes and the turnover of chlorinated organic materials (e.g., humic substances). Additional sources include DCA usage as a chemical feedstock and cancer drug and its unintentional formation during drinking water disinfection by chlorination. Despite the ubiquitous presence of DCA, its fate under anoxic conditions has remained obscure. We discovered an anaerobic bacterium capable of metabolizing DCA, identified the enzyme responsible for DCA dehalogenation, and elucidated a novel DCA fermentation pathway. The findings have implications for the turnover of DCA and the carbon and electron flow in electron acceptor-depleted environments and the human gastrointestinal tract.

Impact of microbial iron oxide reduction on the transport of diffusible tracers and non-diffusible nanoparticles in soils.

In subsurface bioremediation, electron donor addition promotes microbial Fe(III)-oxide mineral reduction that could change soil pore structure, release colloids, and alter soil surface properties. These processes in turn may impact bioremediation rates and the ultimate fate of contaminants. Columns packed with water-stable, Fe-oxide-rich soil aggregates were infused with acetate-containing artificial groundwater and operated for 20 d or 60 d inside an anoxic chamber. Soluble Fe(II) and soil colloids were detected in the effluent within one week after initiation of the acetate addition, demonstrating Fe(III)-bioreduction and colloid formation. Diffusible Br-, less diffusible 2,6-difluorobenzoate (DFBA), and non-diffusible silica-shelled silver nanoparticles (SSSNP) were used as tracers in transport experiments before and after the bioreduction. The transport of Br- was not influenced by the bioreduction. DFBA showed earlier breakthrough and less tailing after the bioreduction, suggesting alterations in flow paths and soil surface chemistry during the 20-d bioreduction treatment. Similarly, the bioreduction increased the transport of SSSNP very significantly, with mass recovery increasing from 1.7% to 25.1%. Unexpectedly, the SSSNP was completely retained in the columns when the acetate injection was extended from 20 to 60 d, while the mass recovery of DFBA decreased from 89.1% to 84.1% and Br- showed no change. The large change in the transport of SSSNP was attributed to soil aggregate breakdown and colloid release (causing mechanical straining of SSSNP) and the exposure of iron oxide surfaces previously unavailable within aggregate interiors (facilitating attachment of SSSNP). These results suggest a time-dependent fashion of microbial effect on the transport of diffusivity-varying tracers.

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.

Grape pomace compost harbors organohalide-respiring Dehalogenimonas species with novel reductive dehalogenase genes.

Organohalide-respiring bacteria have key roles in the natural chlorine cycle; however, most of the current knowledge is based on cultures from contaminated environments. We demonstrate that grape pomace compost without prior exposure to chlorinated solvents harbors a Dehalogenimonas (Dhgm) species capable of using chlorinated ethenes, including the human carcinogen and common groundwater pollutant vinyl chloride (VC) as electron acceptors. Grape pomace microcosms and derived solid-free enrichment cultures were able to dechlorinate trichloroethene (TCE) to less chlorinated daughter products including ethene. 16S rRNA gene amplicon and qPCR analyses revealed a predominance of Dhgm sequences, but Dehalococcoides mccartyi (Dhc) biomarker genes were not detected. The enumeration of Dhgm 16S rRNA genes demonstrated VC-dependent growth, and 6.55±0.64 × 108 cells were measured per µmole of chloride released. Metagenome sequencing enabled the assembly of a Dhgm draft genome, and 52 putative reductive dehalogenase (RDase) genes were identified. Proteomic workflows identified a putative VC RDase with 49 and 56.1% amino acid similarity to the known VC RDases VcrA and BvcA, respectively. A survey of 1,173 groundwater samples collected from 111 chlorinated solvent-contaminated sites in the United States and Australia revealed that Dhgm 16S rRNA genes were frequently detected and outnumbered Dhc in 65% of the samples. Dhgm are likely greater contributors to reductive dechlorination of chlorinated solvents in contaminated aquifers than is currently recognized, and non-polluted environments represent sources of organohalide-respiring bacteria with novel RDase genes.

The corrinoid cofactor of reductive dehalogenases affects dechlorination rates and extents in organohalide-respiring Dehalococcoides mccartyi.

Corrinoid auxotrophic organohalide-respiring Dehalococcoides mccartyi (Dhc) strains are keystone bacteria for reductive dechlorination of toxic and carcinogenic chloroorganic contaminants. We demonstrate that the lower base attached to the essential corrinoid cofactor of reductive dehalogenase (RDase) enzyme systems modulates dechlorination activity and affects the vinyl chloride (VC) RDases BvcA and VcrA differently. Amendment of 5,6-dimethylbenzimidazolyl-cobamide (DMB-Cba) to Dhc strain BAV1 and strain GT cultures supported cis-1,2-dichloroethene-to-ethene reductive dechlorination at rates of 107.0 (±12.0) µM and 67.4 (±1.4) µM Cl(-) released per day, respectively. Strain BAV1, expressing the BvcA RDase, reductively dechlorinated VC to ethene, although at up to fivefold lower rates in cultures amended with cobamides carrying 5-methylbenzimidazole (5-MeBza), 5-methoxybenzimidazole (5-OMeBza) or benzimidazole (Bza) as the lower base. In contrast, strain GT harboring the VcrA RDase failed to grow and dechlorinate VC to ethene in medium amended with 5-OMeBza-Cba or Bza-Cba. The amendment with DMB to inactive strain GT cultures restored the VC-to-ethene-dechlorinating phenotype and intracellular DMB-Cba was produced, demonstrating cobamide uptake and remodeling. The distinct responses of Dhc strains with BvcA versus VcrA RDases to different cobamides implicate that the lower base exerts control over Dhc reductive dechlorination rates and extents (that is, detoxification), and therefore the dynamics of Dhc strains with discrete reductive dechlorination capabilities. These findings emphasize that the role of the corrinoid/lower base synthesizing community must be understood to predict strain-specific Dhc activity and achieve efficacious contaminated site cleanup.

Interference of ferric ions with ferrous iron quantification using the ferrozine assay.

The ferrozine assay is a widely used colorimetric method for determining soluble iron concentrations. We provide evidence for a heretofore unrecognized interference of ferric ions (Fe(3+)) on ferrous iron (Fe(2+)) measurements performed in the dark. Fe(3+) concentrations affected the absorbance measurements, which linearly increased with incubation time.

Shewanella spp. Use acetate as an electron donor for denitrification but not ferric iron or fumarate reduction.

Lactate but not acetate oxidation was reported to support electron acceptor reduction by Shewanella spp. under anoxic conditions. We demonstrate that the denitrifiers Shewanella loihica strain PV-4 and Shewanella denitrificans OS217 utilize acetate as an electron donor for denitrification but not for fumarate or ferric iron reduction.

Localized plasticity in the streamlined genomes of vinyl chloride respiring Dehalococcoides.

Vinyl chloride (VC) is a human carcinogen and widespread priority pollutant. Here we report the first, to our knowledge, complete genome sequences of microorganisms able to respire VC, Dehalococcoides sp. strains VS and BAV1. Notably, the respective VC reductase encoding genes, vcrAB and bvcAB, were found embedded in distinct genomic islands (GEIs) with different predicted integration sites, suggesting that these genes were acquired horizontally and independently by distinct mechanisms. A comparative analysis that included two previously sequenced Dehalococcoides genomes revealed a contextually conserved core that is interrupted by two high plasticity regions (HPRs) near the Ori. These HPRs contain the majority of GEIs and strain-specific genes identified in the four Dehalococcoides genomes, an elevated number of repeated elements including insertion sequences (IS), as well as 91 of 96 rdhAB, genes that putatively encode terminal reductases in organohalide respiration. Only three core rdhA orthologous groups were identified, and only one of these groups is supported by synteny. The low number of core rdhAB, contrasted with the high rdhAB numbers per genome (up to 36 in strain VS), as well as their colocalization with GEIs and other signatures for horizontal transfer, suggests that niche adaptation via organohalide respiration is a fundamental ecological strategy in Dehalococccoides. This adaptation has been exacted through multiple mechanisms of recombination that are mainly confined within HPRs of an otherwise remarkably stable, syntenic, streamlined genome among the smallest of any free-living microorganism.

Detoxification of vinyl chloride to ethene coupled to growth of an anaerobic bacterium.

Tetrachloroethene (PCE) and trichloroethene (TCE) are ideal solvents for numerous applications, and their widespread use makes them prominent groundwater pollutants. Even more troubling, natural biotic and abiotic processes acting on these solvents lead to the accumulation of toxic intermediates (such as dichloroethenes) and carcinogenic intermediates (such as vinyl chloride). Vinyl chloride was found in at least 496 of the 1,430 National Priorities List sites identified by the US Environmental Protection Agency, and its precursors PCE and TCE are present in at least 771 and 852 of these sites, respectively. Here we describe an unusual, strictly anaerobic bacterium that destroys dichloroethenes and vinyl chloride as part of its energy metabolism, generating environmentally benign products (biomass, ethene and inorganic chloride). This organism might be useful for cleaning contaminated subsurface environments and restoring drinking-water reservoirs.

Complete detoxification of vinyl chloride by an anaerobic enrichment culture and identification of the reductively dechlorinating population as a Dehalococcoides species.

A major obstacle in the implementation of the reductive dechlorination process at chloroethene-contaminated sites is the accumulation of the intermediate vinyl chloride (VC), a proven human carcinogen. To shed light on the microbiology involved in the final critical dechlorination step, a sediment-free, nonmethanogenic, VC-dechlorinating enrichment culture was derived from tetrachloroethene (PCE)-to-ethene-dechlorinating microcosms established with material from the chloroethene-contaminated Bachman Road site aquifer in Oscoda, Mich. After 40 consecutive transfers in defined, reduced mineral salts medium amended with VC, the culture lost the ability to use PCE and trichloroethene (TCE) as metabolic electron acceptors. PCE and TCE dechlorination occurred in the presence of VC, presumably in a cometabolic process. Enrichment cultures supplied with lactate or pyruvate as electron donor dechlorinated VC to ethene at rates up to 54 micromol liter(-1)day(-1), and dichloroethenes (DCEs) were dechlorinated at about 50% of this rate. The half-saturation constant (K(S)) for VC was 5.8 microM, which was about one-third lower than the concentrations determined for cis-DCE and trans-DCE. Similar VC dechlorination rates were observed at temperatures between 22 and 30 degrees C, and negligible dechlorination occurred at 4 and 35 degrees C. Reductive dechlorination in medium amended with ampicillin was strictly dependent on H(2) as electron donor. VC-dechlorinating cultures consumed H(2) to threshold concentrations of 0.12 ppm by volume. 16S rRNA gene-based tools identified a Dehalococcoides population, and Dehalococcoides-targeted quantitative real-time PCR confirmed VC-dependent growth of this population. These findings demonstrate that Dehalococcoides populations exist that use DCEs and VC but not PCE or TCE as metabolic electron acceptors.