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Organohalide respiring bacteria (ORB) are capable of utilising organohalides as electron acceptors for the generation of cellular energy and consequently play an important role in the turnover of natural and anthropogenically-derived organohalides. In this study, the response of a Dehalobacter sp. strain UNSWDHB to the addition of trichloromethane (TCM) after a 50 h period of its absence (suffocation) was evaluated from a transcriptomic and proteomic perspective. The up-regulation of TCM reductive dehalogenase genes (tmrABC) and their gene products (TmrABC) was confirmed at both transcriptional and proteomic levels. Other findings include the upregulation of various hydrogenases (membrane-associated Ni-Fe hydrogenase complexes and soluble Fe-Fe hydrogenases), formate dehydrogenases, complex I and a pyrophosphate-energized proton pump. The elevated expression of enzymes associated with carbon metabolism, including complete Wood Ljungdahl pathway, during TCM respiration raises interesting questions on possible fates of intracellular formate and its potential role in the physiology of this bacterium. Overall, the findings presented here provide a broader view on the bioenergetics and general physiology of Dehalobacter UNSWDHB cells actively respiring with TCM.

Dehalobacter sp. strain UNSWDHB can dechlorinate up to 4 mM trichloromethane at a rate of 0.1 mM per day to dichloromethane and 1,1,2-trichloroethane (1 mM, 0.1 mM per day) with the unprecedented product profile of 1,2-dichloroethane and vinyl chloride. 1,1,1-trichloroethane and 1,1-dichloroethane were slowly utilized by strain UNSWDHB and were not completely removed, with minimum threshold concentrations of 0.12 mM and 0.07 mM respectively under growth conditions. Enzyme kinetic experiments confirmed strong substrate affinity for trichloromethane and 1,1,2-trichloroethane (Km = 30 and 62 µM respectively) and poor substrate affinity for 1,1,1-trichloroethane and 1,1-dichloroethane (Km = 238 and 837 µM respectively). Comparison of enzyme kinetic and growth data with other trichloromethane respiring organisms (Dehalobacter sp. strain CF and Desulfitobacterium sp. strain PR) suggests an adaptation of strain UNSWDHB to trichloromethane. The trichloromethane RDase (TmrA) expressed by strain UNSWDHB was identified by BN-PAGE and functionally characterized. Amino acid comparison of homologous RDases from all three organisms revealed only six significant amino acid substitutions/deletions, which are likely to be crucial for substrate specificity. Furthermore, strain UNSWDHB was shown to grow without exogenous supply of cobalamin confirming genomic-based predictions of a fully functional cobalamin synthetic pathway.

Summary 1,1,1-Trichloroethane (TCA) and chloroform are two notorious groundwater pollutants. Here we report the isolation and characterization of Desulfitobacterium sp. strain PR that rapidly dechlorinates both compounds. In pyruvate-amended medium, strain PR reductively dechlorinates ∼ 1.0 mM TCA completely to monochloroethane within 15 days. Under the same conditions, strain PR dechlorinates ∼ 1.2 mM chloroform to predominantly dichloromethane (∼ 1.14 mM) and trace amount of monochloromethane (∼ 0.06 mM) within 10 days. Strain PR shares 96.7% 16S rRNA gene sequence similarity with its closest relative – Desulfitobacterium metallireducens strain 853-15; however, it distinguishes itself from known Desulfitobacterium strains by its inability of utilizing several of their commonly shared substrates such as lactate, thiosulfate and sulfite. A reductive dehalogenase gene (ctrA) in strain PR was identified to be responsible for dechlorination of both TCA and chloroform, showing a maximum expression level of 5.95 ∼ 6.25 copies of transcripts cell-1. CtrA shares 94% amino acid sequence identity with CfrA in Dehalobacter sp. strain CF50 and DcrA in Dehalobacter sp. strain DCA. Interestingly, strain PR could tolerate high aqueous concentrations (up to 0.45 mM) of trichloroethene, another groundwater pollutant that often coexists with TCA/chloroform. As the first chloroform-respiring and the second TCA-respiring isolate that has been identified, Desulfitobacterium sp. strain PR may prove useful in remediation of halogenated alkanes with trihalomethyl (-CX3) groups.

This article presents a case study of the source-area treatment of tetrachloroethene (PCE) in a low-permeability formation using zero-valent iron (ZVI). Evidence of the stimulation of biological reduction processes within the treatment zone occurred. Pneumatic fracturing and injection of microscale ZVI slurry in the overburden and weathered bedrock zones was performed at a commercial brownfields redevelopment site in Maryland. A 20,000-square-foot source area impacted with PCE at concentrations greater than 15,000 µg/L was treated at depths ranging from 10 to 70 feet bgs. An average ZVI dosage of 0.0024 iron-to-soil mass ratio within the overburden zone led to a 75 percent decrease in PCE mass in less than one year. For the weathered bedrock zone, an average 0.0045 iron-to-soil mass ratio resulted in a 92 percent decrease in PCE mass during the same period. The reducing environment and hydrogen generated by the ZVI may have stimulated Dehalobacter populations, as evidenced by concentrations up to 104 cells per milliliter measured within the treatment area despite a groundwater pH as high as 9. The biological reductive dechlorination of the chlorinated ethenes explains the temporary increase in trichloroethene and cis-1,2-dichloroethene concentrations. © 2013 Wiley Periodicals, Inc.

The bioremediation of chloroethene contaminants in groundwater polluted systems is still a serious environmental challenge. Many previous studies have shown that cooperation of several dechlorinators is crucial for complete dechlorination of trichloroethene to ethene. In the present study, we used an explorative functional DNA microarray (DechloArray) to examine the composition of specific functional genes in groundwater samples in which chloroethene bioremediation was enhanced by delivery of hydrogen-releasing compounds. Our results demonstrate for the first time that complete biodegradation occurs through spatial and temporal variations of a wide diversity of dehalorespiring populations involving both Sulfurospirillum, Dehalobacter, Desulfitobacterium, Geobacter and Dehalococcoides genera. Sulfurospirillum appears to be the most active in the highly contaminated source zone, while Geobacter was only detected in the slightly contaminated downstream zone. The concomitant detection of both bvcA and vcrA genes suggests that at least two different Dehalococcoides species are probably responsible for the dechlorination of dichloroethenes and vinyl chloride to ethene. These species were not detected on sites where cis-dichloroethene accumulation was observed. These results support the notion that monitoring dechlorinators by the presence of specific functional biomarkers using a powerful tool such as DechloArray will be useful for surveying the efficiency of bioremediation strategies.

Bioremediation of 1,1,1-trichloroethane (TCA) is more challenging than bioremediation of other chlorinated solvents, such as tetrachloroethene (PCE) and trichloroethene (TCE). TCA transformation often occurs under methanogenic and sulfate-reducing conditions and is mediated by Dehalobacter. The source area at the project site contains moderately permeable medium sand with a low hydraulic gradient and is approximately 0.5 acre. TCA contamination generally extended to 35 feet, with the highest concentrations at approximately 20 feet. The concentrations then decreased with depth; several wells contained 300 to 600 mg/L of TCA prior to bioremediation. The area of treatment also contained 2 to 30 mg/L of TCE from an upgradient source. Initial site groundwater conditions indicated minimal biotic dechlorination and the presence of up to 20 mg/L of nitrate and 90 mg/L of sulfate. Microcosm testing indicated that TCA dechlorination was inhibited by the site's relatively low pH (5 to 5.5) and high TCA concentration. After the pH was adjusted and TCA concentrations were reduced to less than 35 mg/L (by dilution with site water), dechlorination proceeded rapidly using whey (or slower with sodium lactate) as an electron donor. Throughout the remediation program, increased resistance to TCA inhibition (from 35 to 200 mg/L) was observed as the microbes adapted to the elevated TCA concentrations. The article presents the results of a full-scale enhanced anaerobic dechlorination recirculation system and the successful efforts to eliminate TCA- and pH-related inhibition. © 2012 Wiley Periodicals, Inc.

Chloroform (CF), or trichloromethane, is an ubiquitous environmental pollutant because of its widespread industrial use, historically poor disposal and recalcitrance to biodegradation. Chloroform is a potent inhibitor of metabolism and no known organism uses it as a growth substrate. We discovered that CF was rapidly and sustainably dechlorinated in the course of investigating anaerobic reductive dechlorination of 1,1,1-trichloroethane in a Dehalobacter-containing culture. Like 1,1,1-trichloroethane dechlorination in this culture, CF dechlorination was a growth-linked respiratory process, requiring H(2) as an electron donor and CF as an electron acceptor. Moreover, the same specific reductive dehalogenase likely catalyzed both reactions. This Dehalobacter population appears specialized for substrates with three halogen substituents on the same carbon atom, with widespread implications for bioremediation.

An anaerobic microcosm set up with aquifer material from a 1,1,2,2-tetrachloroethane (TeCA) contaminated site and amended with butyrate showed a complete TeCA dechlorination to ethene. A structure analysis of the microbial community was performed by fluorescence in situ hybridization (FISH) with already available and on purpose designed probes from sequences retrieved through 16S rDNA clone library construction. FISH was chosen as identification tool to evaluate in situ whether the retrieved sequences belong to primary bacteria responsible for the biodegradative reactions. FISH probes identified up to 80% of total bacteria and revealed the absence or the marginal presence of known TeCA degraders and the abundance of two well-known H(2)-utilizing halorespiring bacteria, Sulfurospirillum (32.4 +/- 8.6% of total bacteria) and Dehalococcoides spp. (14.8 +/- 2.8), thereby providing a strong indication of their involvement in the dechlorination processes. These results were supported by the kinetic and thermodynamic analysis which provided indications that hydrogen was the actual electron donor for TeCA dechlorination. The specific probes, developed in this study, for known dechlorinators (i.e., Geobacter, Dehalobacter, and Sulfurospirillum species) represent a valuable tool for any future in situ bioremediation study as well as a quick and specific investigation tool for tracking their distribution in the field.

Summary The importance of Dehalobacter species in bioremediation as dedicated degraders of chlorinated organics has been well recognized. However, still little is known about Dehalobacter's full genomic repertoires, including the genes involved in dehalogenation. Here we report the first insights into the genome sequence of Dehalobacter sp. E1 that grows in strict co-culture with Sedimentibacter sp. B4. Based on the co-culture metagenome and the genome of strain B4 (4.2 Mbp) we estimate the genome sequence of strain E1 to be 2.6 Mbp. Ten putative reductive dehalogenase homologue (Rdh)-encoding gene clusters were identified. One cluster has a putative tetrachloroethene Rdh-encoding gene cluster, similar to the pceABCT operon previously identified in Dehalobacter restrictus. Metagenome analysis indicated that the inability of strain E1 to synthesize cobalamin, an essential cofactor of reductive dehalogenases, is complemented by Sedimentibacter. The metagenomic exploration described here maps the extensive dechlorinating potential of Dehalobacter, and paves way for elucidation of the interactions with its co-cultured Sedimentibacter.