Aerobic bacteria that grow on vinyl chloride (VC) have been isolated previously, but their diversity and distribution are largely unknown. It is also unclear whether such bacteria contribute to the natural attenuation of VC at chlorinated-ethene-contaminated sites. We detected aerobic VC biodegradation in 23 of 37 microcosms and enrichments inoculated with samples from various sites. Twelve different bacteria (11 Mycobacterium strains and 1 Nocardioides strain) capable of growth on VC as the sole carbon source were isolated, and 5 representative strains were examined further. All the isolates grew on ethene in addition to VC and contained VC-inducible ethene monooxygenase activity. The Mycobacterium strains (JS60, JS61, JS616, and JS617) all had similar growth yields (5.4 to 6.6 g of protein/mol), maximum specific growth rates (0.17 to 0.23 day ؊1 ), and maximum specific substrate utilization rates (9 to 16 nmol/min/mg of protein) with VC. The Nocardioides strain (JS614) had a higher growth yield (10.3 g of protein/mol), growth rate (0.71 day ؊1 ), and substrate utilization rate (43 nmol/min/mg of protein) with VC but was much more sensitive to VC starvation. Half-velocity constant (K s ) values for VC were between 0.5 and 3.2 M, while K s values for oxygen ranged from 0.03 to 0.3 mg/liter. Our results indicate that aerobic VC-degrading microorganisms (predominantly Mycobacterium strains) are widely distributed at sites contaminated with chlorinated solvents and are likely to be responsible for the natural attenuation of VC.Vinyl chloride (VC) is a common groundwater contaminant (49) which is of concern due to its carcinogenicity (7). Although VC can be produced naturally at very low levels in some soils (32), the industrial synthesis of polyvinyl chloride plastics (27 million tons per year globally [33]) and the bacterial metabolism of chlorinated solvents (36, 41) are the most problematic sources of VC contamination. Many anaerobic bacteria can reductively dechlorinate the widely used solvents tetrachloroethene (PCE) and trichloroethene (TCE), producing cis-dichloroethene (cDCE), VC, or ethene (ETH) (19,30,35,40,59). However, microbes capable of reducing VC to ETH are often absent or inactive in subsurface ecosystems, and thus, VC commonly accumulates as an end product of anaerobic dechlorination (17,37,39).VC can be oxidized to CO 2 under anaerobic conditions in the presence of Fe(III) or humic acids (4, 5), but the microbiology and biochemistry of such anaerobic oxidations have not been investigated. Aerobic bacteria can catalyze the cometabolic oxidation of VC in the presence of monooxygenase inducers such as methane (18), ethane (20), ETH (34), propane (38), propene (15), isoprene (16), toluene (48), and ammonia (56). Bioremediation strategies based on aerobic cometabolism have been examined for VC and other chlorinated ethenes (36, 47), but there are several problems with cometabolic systems-electron donors are required (2), the growth-supporting substrate and the pollutant compete for the same enzymes (1...

Extensive use and inadequate disposal of chloroethenes have led to prevalent groundwater contamination worldwide. The occurrence of the lesser chlorinated ethenes [i.e. vinyl chloride (VC) and cis-1,2-dichloroethene (cDCE)] in groundwater is primarily a consequence of incomplete anaerobic reductive dechlorination of the more highly chlorinated ethenes (tetrachloroethene and trichloroethene). VC and cDCE are toxic and VC is a known human carcinogen. Therefore, their presence in groundwater is undesirable. In situ cleanup of VC- and cDCE-contaminated groundwater via oxidation by aerobic microorganisms is an attractive and potentially cost-effective alternative to physical and chemical approaches. Of particular interest are aerobic bacteria that use VC or cDCE as growth substrates (known as the VC- and cDCE-assimilating bacteria). Bacteria that grow on VC are readily isolated from contaminated and uncontaminated environments, suggesting that they are widespread and influential in aerobic natural attenuation of VC. In contrast, only one cDCE-assimilating strain has been isolated, suggesting that their environmental occurrence is rare. In this review, we will summarize the current knowledge of the physiology, biodegradation pathways, genetics, ecology, and evolution of VC- and cDCE-assimilating bacteria. Techniques (e.g. PCR, proteomics, and compound-specific isotope analysis) that aim to determine the presence, numbers, and activity of these bacteria in the environment will also be discussed.

An aerobic bacterium capable of growth on cis-dichloroethene (cDCE) as a sole carbon and energy source was isolated by enrichment culture. The 16S ribosomal DNA sequence of the isolate (strain JS666) had 97.9% identity to the sequence from Polaromonas vacuolata, indicating that the isolate was a ␤-proteobacterium. At 20°C, strain JS666 grew on cDCE with a minimum doubling time of 73 ؎ 7 h and a growth yield of 6.1 g of protein/mol of cDCE. Chloride analysis indicated that complete dechlorination of cDCE occurred during growth. The half-velocity constant for cDCE transformation was 1.6 ؎ 0.2 M, and the maximum specific substrate utilization rate ranged from 12.6 to 16.8 nmol/min/mg of protein. Resting cells grown on cDCE could transform cDCE, ethene, vinyl chloride, trans-dichloroethene, trichloroethene, and 1,2-dichloroethane. Epoxyethane was produced from ethene by cDCE-grown cells, suggesting that an epoxidation reaction is the first step in cDCE degradation.The extensive use of chloroethenes as solvents and synthetic feedstocks has resulted in widespread environmental contamination (22), which is of concern due to the toxicity and carcinogenicity of such compounds. Microbial metabolism is an important factor in determining the fate of chloroethenes in the biosphere (14). Several anaerobic bacteria use tetrachloroethene (perchloroethene) and trichloroethene (TCE) as electron acceptors, producing cis-1,2-dichloroethene (cDCE), vinyl chloride (VC), and ethene as end products (10,15,16,17). Under aerobic conditions, ethene and VC can serve as carbon and energy sources for bacterial growth (3, 8), but thus far, no conclusive evidence exists for aerobic growth on any of the dichloroethenes (cis-dichloroethene, trans-dichloroetheneBecause cDCE accumulation is often a limiting factor in the biodegradation of chloroethenes in subsurface ecosystems, aerobic bacteria capable of growth on cDCE would provide a crucial missing link in the chain of microbial metabolism for this class of compounds. Thermodynamic calculations suggest that cDCE contains sufficient energy to support aerobic growth (4), and enzymes active on cDCE are known in hydrocarbonoxidizing bacteria (5,6,13,20,24,25). In addition, aerobic oxidation of cDCE to CO 2 has been observed in microcosm and enrichment culture studies (2, 12). Encouraged by the facts described above, we hypothesized that aerobic growth on cDCE was possible and searched at a variety of contaminated sites for microorganisms able to use this compound as a sole source of carbon and energy. MATERIALS AND METHODSChemicals and media. cis-Dichloroethene (97%), tDCE (98%), TCE (99.5%), and 1,2-dichloroethane (1,2-DCA) (99.8%) were obtained from Sigma-Aldrich. VC (99.5%) was obtained from Fluka, and ethene (99.5%) was obtained from Scott. All other chemicals were reagent grade. A minimal salts medium (MSM) based on that of Hartmans et al. (9) was used for enrichment cultures, with the following modifications: the phosphate concentration was reduced to 20 mM, the ammonium concentration was red...

Nocardioides sp. strain JS614 utilizes vinyl chloride and ethene as carbon and energy sources. JS614 could be influential in natural attenuation and biogeochemical ethene cycling, and useful for bioremediation, biocatalysis and metabolic engineering, but a fundamental understanding of the physiological and genetic basis of vinyl chloride and ethene assimilation in strain JS614 is required. Alkene monooxygenase (AkMO) activity was demonstrated in whole-cell assays and epoxyalkane:coenzyme M transferase (EaCoMT) activity was detected in JS614 cell-free extracts. Pulsed-field gel electrophoresis revealed a 290-kb plasmid (pNoc614) in JS614. Curing experiments and PCR indicated that pNoc614 encodes vinyl chloride/ethene-degradation genes. JS614 vinyl chloride/ethene catabolic genes and flanking DNA (34.8 kb) were retrieved from a fosmid clone. AkMO and EaCoMT genes were found in a putative operon that included CoA transferase, acyl-CoA synthetase, dehydrogenase, and reductase genes. Adjacent to this gene cluster was a divergently transcribed gene cluster that encoded possible coenzyme M biosynthesis enzymes. Reverse transcription-PCR demonstrated the vinyl chloride- and ethene-inducible nature of several genes. Genes encoding possible plasmid conjugation, integration, and partitioning functions were also discovered on the fosmid clone.

Polaromonas sp. strain JS666 can grow on cis-1,2-dichloroethene (cDCE) as a sole carbon and energy source and may be useful for bioremediation of chlorinated solvent-contaminated sites. Analysis of the genome sequence of JS666 (5.9 Mb) shows a bacterium well adapted to pollution that carries many genes likely to be involved in hydrocarbon and xenobiotic catabolism and metal resistance. Clusters of genes coding for haloalkane, haloalkanoate, n-alkane, alicyclic acid, cyclic alcohol, and aromatic catabolism were analyzed in detail, and growth on acetate, catechol, chloroacetate, cyclohexane carboxylate, cyclohexanol, ferulate, heptane, 3-hydroxybenzoate, hydroxyquinol, gentisate, octane, protocatechuate, and salicylate was confirmed experimentally. Strain JS666 also harbors diverse putative mobile genetic elements, including retrons, inteins, a miniature inverted-repeat transposable element, insertion sequence transposases from 14 families, eight genomic islands, a Mu family bacteriophage, and two large (338-and 360-kb) plasmids. Both plasmids are likely to be self-transferable and carry genes for alkane, alcohol, aromatic, and haloacid metabolism. Overall, the JS666 genome sequence provides insights into the evolution of pollutant-degrading bacteria and provides a toolbox of catabolic genes with utility for biotechnology.

Contamination of drinking water source zones by vinyl chloride (VC), a known human carcinogen and common groundwater contaminant, poses a public health risk. Bioremediation applications involving aerobic, VC-assimilating bacteria could be useful in alleviating environmental VC cancer risk, but their evolution and activity in the environment are poorly understood. In this study, adaptation of ethene-assimilating Mycobacterium strains JS622, JS623, JS624, and JS625 to VC as a growth substrate was investigated to test the hypothesis that VC-assimilating bacteria arise from naturally occurring ethene-assimilating bacteria. VC consumption in the absence of microbial growth was initially observed in cultures grown in both ethene and 1/10-strength trypticase soy agar + 1% (w/v) glucose. After extended incubations (55-476 days), all strains commenced growth-coupled VC consumption patterns. VC-adapted cultures grown on 20 mM acetate subsequently retained their ability to assimilate VC. Three independent purity check methods (streak plates, 16S rRNA gene sequencing, and repetitive extragenic palindromic polymerase chain reaction) verified culture purity prior to and following VC adaptation. Overall, our results suggest that ethene-assimilating mycobacteria have a widespread ability to adapt to VC as a growth substrate.

We conducted experiments to determine whether bioaugmentation with aerobic, polychlorinated biphenyl (PCB)-degrading microorganisms can mitigate polychlorinated biphenyl (PCB) emissions from contaminated sediment to air. Paraburkholderia xenovorans strain LB400 was added to bioreactors containing PCB-contaminated site sediment. PCB mass in both the headspace and aqueous bioreactor compartments was measured using passive samplers over 35 days. Time-series measurements of all 209 PCB congeners revealed a 57% decrease in total PCB mass accumulated in the vapor phase of bioaugmented treatments relative to non-bioaugmented controls, on average. A comparative congener-specific analysis revealed preferential biodegradation of lower-chlorinated PCBs (LC-PCBs) by LB400. Release of the most abundant congener (PCB 4 [2,2′-dichlorobiphenyl]) decreased by over 90%. Simulations with a PCB reactive transport model closely aligned with experimental observations. We also evaluated the effect of the phytogenic biosurfactant, saponin, on PCB bioavailability and biodegradation by LB400. Time-series qPCR measurements of biphenyl dioxygenase (bphA) genes showed that saponin better maintained bphA abundance, compared to the saponin-free treatment. These findings indicate that an active population of bioaugmented, aerobic PCB-degrading microorganisms can effectively lower PCB emissions and may therefore contribute to minimizing PCB inhalation exposure in communities surrounding PCB-contaminated sites.

Vinyl chloride (VC) is a known human carcinogen that is primarily formed in groundwater via incomplete anaerobic dechlorination of chloroethenes. Aerobic, ethene-degrading bacteria (etheneotrophs), which are capable of both fortuitous and growth-linked VC oxidation, could be important in natural attenuation of VC plumes that escape anaerobic treatment. In this work, we developed a quantitative, real-time PCR (qPCR) assay for etheneotrophs in groundwater. We designed and tested degenerate qPCR primers for two functional genes involved in aerobic, growth-coupled VC- and ethene-oxidation (etnC and etnE). Primer specificity to these target genes was tested by comparison to nucleotide sequence databases, PCR analysis of template DNA extracted from isolates and environmental samples, and sequencing of qPCR products obtained from VC-contaminated groundwater. The assay was made quantitative by constructing standard curves (threshold cycle vs log gene copy number) with DNA amplified from Mycobacterium strain JS60, an etheneotrophic isolate. Analysis of groundwater samples from three different VC-contaminated sites revealed that etnC abundance ranged from 1.6 × 10(3) - 1.0 × 10(5) copies/L groundwater while etnE abundance ranged from 4.3 × 10(3) - 6.3 × 10(5) copies/L groundwater. Our data suggest this novel environmental measurement method will be useful for supporting VC bioremediation strategies, assisting in site closure, and conducting microbial ecology studies involving etheneotrophs.