Biodegradation of cis-Dichloroethene as the Sole Carbon Source by a β-Proteobacterium

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 β-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 μM, 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 Genome of Polaromonas sp. Strain JS666: Insights into the Evolution of a Hydrocarbon- and Xenobiotic-Degrading Bacterium, and Features of Relevance to Biotechnology

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.     

Characterization of a highly enriched dehalococcoides-containing culture that grows on vinyl chloride and trichloroethene

A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described. The Dehalococcoides strain in this enrichment culture had a yield of (5.6 +/- 1.4) x 10(8) 16S rRNA gene copies/micromol of Cl(-) when grown on VC and hydrogen. Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 +/- 1.3) x 10(8) 16S rRNA gene copies/micromol of Cl(-). The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1. A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H(2) enrichment culture. This second Dehalococcoides sequence was identical to that of BAV1. As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group.     

Phylogenetic and kinetic diversity of aerobic vinyl chloride-assimilating bacteria from contaminated sites

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 micro 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.     

Genome Sequence of the ethene- and vinyl chloride-oxidizing actinomycete Nocardioides sp. strain JS614

Nocardioides sp. strain JS614 grows on ethene and vinyl chloride (VC) as sole carbon and energy sources and is of interest for bioremediation and biocatalysis. Sequencing of the complete genome of JS614 provides insight into the genetic basis of alkene oxidation, supports ongoing research into the physiology and biochemistry of growth on ethene and VC, and provides biomarkers to facilitate detection of VC/ethene oxidizers in the environment. This is the first genome sequence from the genus Nocardioides and the first genome of a VC/ethene-oxidizing bacterium.     

Influence of Hydraulic Retention Time on Extent of PCE Dechlorination and Preliminary Characterization of the Enrichment Culture

The extent of tetrachloroethene (PCE) dechlorination in two chemostats was evaluated as a function of hydraulic retention time (HRT). The inoculum of these chemostats was from an upflow anaerobic sludge blanket (UASB) reactor that rapidly converts PCE to vinyl chloride (VC) and ethene. When the HRT was 2.9 days, PCE was converted only to cis-dichloroethene (cDCE). When the HRT was 11 days, the end products were VC and ethene. Further studies showed that the dechlorinating microbial community in the UASB reactor contained two distinct populations, one of which converted PCE to cDCE and the other cDCE to VC and ethene. Methanogenic activity was very low in these cultures. The cDCE dechlorinating culture apparently has a lower growth rate than the PCE dechlorinating culture, and as a result, at a shorter HRT, the cDCE dechlorinating culture was washed out from the system leading to incomplete dechlorination of PCE. Both enrichment cultures used pyruvate or hydrogen as electron donors for dechlorination. Acetate was the carbon source (but not energy source) when hydrogen was used. Both cultures had undefined nutrient requirements and needed supplements of cell extract obtained from the mixed culture in the UASB reactor. However, the two cultures were different in their response to the addition of an inhibitor of methanogenesis (2-bromoethanesulfonate [BES]). BES inhibited the dechlorinating activity of the enriched cDCE dechlorinating culture, but had no influence on the PCE dechlorinating culture. Preliminary studies on BES inhibition are presented.     

Biodegradation of vinyl chloride, <Emphasis Type="Italic">cis</Emphasis>-dichloroethene and 1,2-dichloroethane in the alkene/alkane-oxidising <Emphasis Type="Italic">Mycobacterium</Emphasis> strain NBB4

Mycobacterium chubuense strain NBB4 can grow on both alkanes and alkenes as carbon sources, and was hypothesised to be an effective bioremediation agent for chlorinated aliphatic pollutants. In this study, the ability of NBB4 to biodegrade vinyl chloride (VC), cis-dichloroethene (cDCE) and 1,2-dichloroethane (DCA) was investigated under pure-culture conditions and in microcosms. Ethene-grown NBB4 cells were capable of biodegrading VC and cDCE, while ethane-grown cells could biodegrade cDCE and DCA. The stoichiometry of inorganic chloride release (1 mol/mol in each case) indicated that VC was completely dechlorinated, while cDCE and DCA were only partially dechlorinated, yielding chloroacetate in the case of DCA, and unknown metabolites in the case of cDCE. The apparent maximum specific activities (k) of whole cells against ethene, cDCE, ethane and DCA were 93 ± 4.6, 89 ± 18, 39 ± 5.5, and 4.8 ± 0.9 nmol/min/mg protein, respectively, while the substrate affinities (K_S) of whole cells with the same substrates were 2.0 ± 0.15, 46 ± 11, 11 ± 0.33 and 4.0 ± 3.2 μM, respectively. In microcosms containing contaminated aquifer sediments and groundwater, NBB4 cells removed 85-95% of the pollutants (cDCE or DCA at 2 mM) within 24 h, and the cells remained viable for >1 month. Due to its favourable kinetic parameters, and robust survival and biodegradation activities, strain NBB4 is a promising candidate for bioremediation of chlorinated aliphatic pollutants.     

Distribution of the coenzyme M pathway of epoxide metabolism among ethene- and vinyl chloride-degrading Mycobacterium strains

An epoxyalkane:coenzyme M (CoM) transferase (EaCoMT) enzyme was recently found to be active in the aerobic vinyl chloride (VC) and ethene assimilation pathways of Mycobacterium strain JS60. In the present study, EaCoMT activity and genes were investigated in 10 different mycobacteria isolated on VC or ethene from diverse environmental samples. In all cases, epoxyethane metabolism in cell extracts was dependent on CoM, with average specific activities of EaCoMT between 380 and 2,910 nmol/min/mg of protein. PCR with primers based on conserved regions of EaCoMT genes from Mycobacterium strain JS60 and the propene oxidizers Xanthobacter strain Py2 and Rhodococcus strain B-276 yielded fragments (834 bp) of EaCoMT genes from all of the VC- and ethene-assimilating isolates. The Mycobacterium EaCoMT genes form a distinct cluster and are more closely related to the EaCoMT of Rhodococcus strain B-276 than that of Xanthobacter strain Py2. The incongruence of the EaCoMT and 16S rRNA gene trees and the fact that isolates from geographically distant locations possessed almost identical EaCoMT genes suggest that lateral transfer of EaCoMT among the Mycobacterium strains has occurred. Pulsed-field gel electrophoresis revealed large linear plasmids (110 to 330 kb) in all of the VC-degrading strains. In Southern blotting experiments, the strain JS60 EaCoMT gene hybridized to many of the plasmids. The CoM-mediated pathway of epoxide metabolism appears to be universal in alkene-assimilating mycobacteria, possibly because of plasmid-mediated lateral gene transfer.     

Characterization of a Highly Enriched Dehalococcoides-Containing Culture That Grows on Vinyl Chloride and Trichloroethene

A highly enriched culture that reductively dechlorinates trichloroethene (TCE), cis-1,2-dichloroethene (cDCE), and vinyl chloride (VC) to ethene without methanogenesis is described. The Dehalococcoides strain in this enrichment culture had a yield of (5.6 ± 1.4) × 10⁸ 16S rRNA gene copies/μmol of Cl⁻ when grown on VC and hydrogen. Unlike the other VC-degrading cultures described in the literature, strains VS and BAV1, this culture maintained the ability to grow on TCE with a yield of (3.6 ± 1.3) × 10⁸ 16S rRNA gene copies/μmol of Cl⁻. The yields on an electron-equivalent basis measured for the culture grown on TCE and on VC were not significantly different, indicating that both substrates supported growth equally well. PCR followed by denaturing gradient gel electrophoresis, cloning, and phylogenetic analyses revealed that this culture contained one Dehalococcoides 16S rRNA gene sequence, designated KB-1/VC, that was identical (over 1,386 bp) to the sequences of previously described organisms FL2 and CBDB1. A second Dehalococcoides sequence found in separate KB-1 enrichment cultures maintained on cDCE, TCE, and tetrachloroethene was no longer present in the VC-H₂ enrichment culture. This second Dehalococcoides sequence was identical to that of BAV1. As neither FL2 nor CBDB1 can dechlorinate VC to ethene in a growth-related fashion, it is clear that current 16S rRNA gene-based analyses do not provide sufficient information to distinguish between metabolically diverse members of the Dehalococcoides group.     

Distribution of the Coenzyme M Pathway of Epoxide Metabolism among Ethene- and Vinyl Chloride-Degrading Mycobacterium Strains

An epoxyalkane:coenzyme M (CoM) transferase (EaCoMT) enzyme was recently found to be active in the aerobic vinyl chloride (VC) and ethene assimilation pathways of Mycobacterium strain JS60. In the present study, EaCoMT activity and genes were investigated in 10 different mycobacteria isolated on VC or ethene from diverse environmental samples. In all cases, epoxyethane metabolism in cell extracts was dependent on CoM, with average specific activities of EaCoMT between 380 and 2,910 nmol/min/mg of protein. PCR with primers based on conserved regions of EaCoMT genes from Mycobacterium strain JS60 and the propene oxidizers Xanthobacter strain Py2 and Rhodococcus strain B-276 yielded fragments (834 bp) of EaCoMT genes from all of the VC- and ethene-assimilating isolates. The Mycobacterium EaCoMT genes form a distinct cluster and are more closely related to the EaCoMT of Rhodococcus strain B-276 than that of Xanthobacter strain Py2. The incongruence of the EaCoMT and 16S rRNA gene trees and the fact that isolates from geographically distant locations possessed almost identical EaCoMT genes suggest that lateral transfer of EaCoMT among the Mycobacterium strains has occurred. Pulsed-field gel electrophoresis revealed large linear plasmids (110 to 330 kb) in all of the VC-degrading strains. In Southern blotting experiments, the strain JS60 EaCoMT gene hybridized to many of the plasmids. The CoM-mediated pathway of epoxide metabolism appears to be universal in alkene-assimilating mycobacteria, possibly because of plasmid-mediated lateral gene transfer.     

Bioremediation of soluble heavy metals with recombinant Caulobacter crescentus

To achieve one-step separation of heavy metal ions from contaminated water, we have developed a novel bioremediation technology based on self-immobilization of the Caulobacter crescentus recombinant strain JS4022/p723-6H, which overexpresses hexahistidine peptide on the surface of the bacterial cells and serves as a whole-cell adsorbent for dissolved heavy metals. Biofilms formed by JS4022/p723-6H are effective at retaining cadmium from bacterial growth media or environmental water samples. Here we provide additional experiment data discussing the application potential of this new technology. Supplementation of calcium to the growth media produced robust JS4022/p723-6H cells by alleviating their sensitivity to chelators. After growth in the presence of 0.3% CaCl(2)·2H(2)O, double the amount of JS4022/p723-6H cells survived the treatment with 2 mM EDTA. Free cells of JS4022/p723-6H effectively sequestered 51% of the total cadmium from a Lake Erie water sample at pH 5.4, compared to 37% retrieved by the control strain. Similar levels of adsorption were observed at pH 4.2 as well. Cells of JS4022/p723-6H were tolerant of acid treatment for 90 min at pH ≥1.1 or 120 min at pH ≥2.5, which provides an avenue for the convenient regeneration of the bacterial cells metal-binding capacity with acidic solutions. Designs of possible bioreactors and an operation system are also presented.     

Extending the alkene substrate range of vinyl chloride utilizing Nocardioides sp. strain JS614 with ethene oxide

Nocardioides sp. strain JS614 grows on the C₂ alkenes ethene (Eth), vinyl chloride, and vinyl fluoride as sole carbon sources. The presence of 400–800 μM ethene oxide (EtO) extended the growth substrate range to propene (C₃) and butene (C₄). Propene-dependent growth of JS614 was CO₂ dependent and was prevented by the carboxylase/reductase inhibitor 2-bromoethanesulfonic acid, sodium salt (BES), while growth on Eth was not CO₂ dependent or BES sensitive. Although unable to promote growth, both propene and propene oxide (PrO)-induced expression of the genes encoding the alpha subunit of alkene monooxygenase (etnC) and epoxyethane CoM transferase (etnE) to similar levels as did Eth and EtO. Propene was transformed by Eth-grown and propene-grown/EtO-induced JS614 to PrO at a rate 4.2 times faster than PrO was consumed. As a result PrO accumulated in growth medium to 900 μM during EtO-induced growth on propene. PrO (50–100 μM) exerted inhibitory effects on growth of JS614 on both acetate and Eth, and on EtO-induced growth on Eth. However, higher EtO concentrations (300–400 μM) overcame the negative effects of PrO on Eth-dependent growth.     

Quantitative PCR confirms purity of strain GT, a novel trichloroethene-to-ethene-respiring Dehalococcoides isolate

A novel Dehalococcoides isolate capable of metabolic trichloroethene (TCE)-to-ethene reductive dechlorination was obtained from contaminated aquifer material. Growth studies and 16S rRNA gene-targeted analyses suggested culture purity; however, the careful quantitative analysis of Dehalococcoides 16S rRNA gene and chloroethene reductive dehalogenase gene (i.e., vcrA, tceA, and bvcA) copy numbers revealed that the culture consisted of multiple, distinct Dehalococcoides organisms. Subsequent transfers, along with quantitative PCR monitoring, yielded isolate GT, possessing only vcrA. These findings suggest that commonly used qualitative 16S rRNA gene-based procedures are insufficient to verify purity of Dehalococcoides cultures. Phylogenetic analysis revealed that strain GT is affiliated with the Pinellas group of the Dehalococcoides cluster and shares 100% 16S rRNA gene sequence identity with two other Dehalococcoides isolates, strain FL2 and strain CBDB1. The new isolate is distinct, as it respires the priority pollutants TCE, cis-1,2-dichloroethene (cis-DCE), 1,1-dichloroethene (1,1-DCE), and vinyl chloride (VC), thereby producing innocuous ethene and inorganic chloride. Strain GT dechlorinated TCE, cis-DCE, 1,1-DCE, and VC to ethene at rates up to 40, 41, 62, and 127 micromol liter-1 day-1, respectively, but failed to dechlorinate PCE. Hydrogen was the required electron donor, which was depleted to a consumption threshold concentration of 0.76+/-0.13 nM with VC as the electron acceptor. In contrast to the known TCE dechlorinating isolates, strain GT dechlorinated TCE to ethene with very little formation of chlorinated intermediates, suggesting that this type of organism avoids the commonly observed accumulation of cis-DCE and VC during TCE-to-ethene dechlorination.     

Bioaugmentation potential of a vinyl chloride-assimilating Mycobacterium sp., isolated from a chloroethene-contaminated aquifer

An aerobic bacterium, Mycobacterium sp. strain TRW-2 that assimilated vinyl chloride (VC) or ethene (ETH) as the sole carbon source was isolated from a chloroethene-degrading enrichment culture. The strain TRW-2 also degraded cis-dichloroethene (cis-DCE) in mineral salts medium, but only when VC was present as the primary carbon source. However, no degradation of trans-dichloroethene or trichloroethene occurred in either the presence or absence of added VC. The measured growth yield values were 6.53 and 14.1g protein/mol of VC and ETH utilized, respectively. Inoculation by strain TRW-2 in microcosms prepared with aquifer samples resulted in rapid degradation of VC, whereas native bacteria degraded negligible amounts of VC within the same time period, thus suggesting bioaugmentation potential of the isolate. Phylogenetic analysis of the 16S rDNA sequence of the isolate revealed 98% sequence similarity to the members of the genus Mycobacterium. In summary, the isolate's ability to degrade VC, cis-DCE, and ETH and also its ability to survive and degrade VC in the presence of other microorganisms is relevant to the remediation of VC-impacted aquifers.     

Discrimination of multiple Dehalococcoides strains in a trichloroethene enrichment by quantification of their reductive dehalogenase genes

While many anaerobic microbial communities are capable of reductively dechlorinating tetrachloroethene (PCE) and trichloroethene (TCE) to dichloroethene (DCE), vinyl chloride (VC), and finally ethene, the accumulation of the highly toxic intermediates, cis-DCE (cDCE) and VC, presents a challenge for bioremediation processes. Members of the genus Dehalococcoides are apparently solely responsible for dechlorination beyond DCE, but isolates of Dehalococcoides each metabolize only a subset of PCE dechlorination intermediates and the interactions among distinct Dehalococcoides strains that result in complete dechlorination are not well understood. Here we apply quantitative PCR to 16S rRNA and reductase gene sequences to discriminate and track Dehalococcoides strains in a TCE enrichment derived from soil taken from the Alameda Naval Air Station (ANAS) using a four-gene plasmid standard. This standard increased experimental accuracy such that 16S rRNA and summed reductase gene copy numbers matched to within 10%. The ANAS culture was found to contain only a single Dehalococcoides 16S rRNA gene sequence, matching that of D. ethenogenes 195, but both the vcrA and tceA reductive dehalogenase genes. Quantities of these two genes in the enrichment summed to the quantity of the Dehalococcoides 16S rRNA gene. Further, between ANAS subcultures enriched on TCE, cDCE, or VC, the relative copy number of the two dehalogenases shifted 14-fold, indicating that the genes are present in two different Dehalococcoides strains. Comparison of cell yields in VC-, cDCE-, and TCE-enriched subcultures suggests that the tceA-containing strain is responsible for nearly all of the TCE and cDCE metabolism in ANAS, whereas the vcrA-containing strain is responsible for all of the VC metabolism.     

Influence of vitamin B12 and cocultures on the growth of Dehalococcoides isolates in defined medium

Bacteria belonging to the genus Dehalococcoides play a key role in the complete detoxification of chloroethenes as these organisms are the only microbes known to be capable of dechlorination beyond dichloroethenes to vinyl chloride (VC) and ethene. However, Dehalococcoides strains usually grow slowly with a doubling time of 1 to 2 days and have complex nutritional requirements. Here we describe the growth of Dehalococcoides ethenogenes 195 in a defined mineral salts medium, improved growth of strain 195 when the medium was amended with high concentrations of vitamin B(12), and a strategy for maintaining Dehalococcoides strains on lactate by growing them in consortia. Although strain 195 could grow in defined medium spiked with approximately 0.5 mM trichloroethene (TCE) and 0.001 mg/liter vitamin B(12), the TCE dechlorination and cellular growth rates doubled when the vitamin B(12) concentration was increased 25-fold to 0.025 mg/liter. In addition, the final ratios of ethene to VC increased when the higher vitamin concentration was used, which reflected the key role that cobalamin plays in dechlorination reactions. No further improvement in dechlorination or growth was observed when the vitamin B(12) concentration was increased to more than 0.025 mg/liter. In defined consortia containing strain 195 along with Desulfovibrio desulfuricans and/or Acetobacterium woodii and containing lactate as the electron donor, tetrachloroethene ( approximately 0.4 mM) was completely dechlorinated to VC and ethene and there was concomitant growth of Dehalococcoides cells. In the cultures that also contained D. desulfuricans and/or A. woodii, strain 195 cells grew to densities that were 1.5 times greater than the densities obtained when the isolate was grown alone. The ratio of ethene to VC was highest in the presence of A. woodii, an organism that generates cobalamin de novo during metabolism. These findings demonstrate that the growth of D. ethenogenes strain 195 in defined medium can be optimized by providing high concentrations of vitamin B(12) and that this strain can be grown to higher densities in cocultures with fermenters that convert lactate to generate the required hydrogen and acetate and that may enhance the availability of vitamin B(12).