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

Abstract

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...

Carbon and Chlorine Isotope Fractionation during Aerobic Oxidation and Reductive Dechlorination of Vinyl Chloride and cis-1,2-Dichloroethene

“…Aerobic VC oxidation experiments used the Nocardioides strain JS614 (30), and aerobic cDCE experiments used the -Proteobacterium strain JS666 (31). Both strains were kindly provided by Dr. Spain's laboratory (Georgia Institute of Technology, Atlanta, GA).…”

“…Both strains were kindly provided by Dr. Spain's laboratory (Georgia Institute of Technology, Atlanta, GA). The culture medium was prepared as described elsewhere (30,31). The stock cultures were kept in 250 mL glass bottles with 100 mL headspace and capped with Mininert-Valves (Vici Precision Sampling, Baton Rouge, LA).…”

Mutagenesis of the hydrocarbon monooxygenase indicates a metal centre in subunit-C, and not subunit-B, is essential for copper-containing membrane monooxygenase activity

“…Substrate depletion was monitored by further headspace sampling over 6 h and corrected for substrate loss due to headspace removal. Degradation rates were calculated using the linear section of depletion curves and converted to apparent specific activity (nmol substrate min 21 mg 21 ) by dividing (Coleman et al, 2002;. The cell density in each vial was measured after assay completion by sampling 100 ml of the cell suspension, diluting to 10 21 in KP and measuring OD 600 .…”

“…E. coli was grown aerobically at 37 uC in LB (Sambrook & Russell, 2001). Mycobacterium smegmatis was grown aerobically at 30 uC in TSG [3 g trypticase soy broth (Oxoid) l 21 , 1 % (w/v) glucose] or minimal MSM medium (Coleman et al, 2002) supplemented with 20 mM glucose. Kanamycin (Km) was added at 50 mg ml 21 for E. coli and 20 mg ml 21 for mc 2 -155 cultures for vector maintenance.…”

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

“…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). Epoxides were detected using a nitrobenzylpyridine assay based on the protocol of Guengerich et al.…”