Phylogenetic and Kinetic Diversity of Aerobic Vinyl Chloride-Assimilating Bacteria from Contaminated Sites

Abstract

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...

Evaluating the fate of chlorinated ethenes in streambed sediments by combining stable isotope, geochemical and microbial methods

“…The DNA samples were also subjected to PCR with specific primers for the EaCoMT gene (CoM-F1L and CoM-R2E) which codes for the enzyme epoxyalkane:coenzyme M transferase that is involved in the aerobic degradation of VC and ethene (Coleman and Spain, 2003b;Danko et al, 2006). DNA from Nocardioides sp JS 614 (Coleman et al, 2002) and from the KB-1 mixed culture (SiREM, Guelph, ON, Canada) was used as positive control for the EaCoMT gene and Dehalococcoides, respectively. In order to increase the sensitivity of molecular detection, the PCR amplification process was repeated twice whereby a ten-fold dilution of the previous PCR product was used as template for the following PCR with the same primer set.…”

Hydrocarbon monooxygenase in Mycobacterium: recombinant expression of a member of the ammonia monooxygenase superfamily

“…Peak areas were converted using the standard curve to amounts expressed as mmoles per bottle. Protein was quantified by an ultraviolet absorption method (Kalb and Bernlohr, 1977) following hot alkaline lysis of the cells (Coleman et al, 2002a). Chloride analysis was done via a colorimetric assay (Coleman et al, 2002b).…”

“…Construction, screening and sequencing of fosmid libraries High molecular weight genomic DNA from strain NBB4 was extracted and purified by a previously described method (Coleman et al, 2002a), then a fosmid library using the pCC1FOS vector was prepared according to the manufacturer's instructions (Epicentre Biotechnologies). Clones were screened for the presence of SDIMOs by PCR as described previously , and one clone containing an SDIMO similar to soluble methane monooxygenase was sequenced on both strands by a primer-walking strategy using the Sanger sequencing method at the Australian Genome Research Facility (Westmead node).…”

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

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Aerobic bacteria that grow on ethene and vinyl chloride (VC) are widely distributed in the environment and have attracted interest because of their potential applications in bioremediation and biocatalysis (5,6,11,12,32,33). The first step in ethene and VC assimilation is known to be a monooxygenase reaction yielding epoxyethane from ethene (5, 7) and chlorooxirane from VC (12, 33), but the downstream pathways are not well understood.

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