Showing papers by "Dick B. Janssen published in 1989"

Degradation of chlorinated aliphatic hydrocarbons by Methylosinus trichosporium OB3b expressing soluble methane monooxygenase.

Abstract: Degradation of trichloroethylene (TCE) by the methanotrophic bacterium Methylosinus trichosporium OB3b was studied by using cells grown in continuous culture. TCE degradation was a strictly cometabolic process, requiring the presence of a cosubstrate, preferably formate, and oxygen. M. trichosporium OB3b cells degraded TCE only when grown under copper limitation and when the soluble methane monooxygenase was derepressed. During TCE degradation, nearly total dechlorination occurred, as indicated by the production of inorganic chloride, and only traces of 2,2,2-trichloroethanol and trichloroacetaldehyde were produced. TCE degradation proceeded according to first-order kinetics from 0.1 to 0.0002 mM TCE with a rate constant of 2.14 ml min-1 mg of cells-1. TCE concentrations above 0.2 mM inhibited degradation in cell suspensions of 0.42 mg of cells ml-1. Other chlorinated aliphatics were also degraded by M. trichosporium OB3b. Dichloromethane, chloroform, 1,1-dichloroethane, and 1,2-dichloroethane were completely degraded, with the release of stoichiometric amounts of chloride. trans-1,2-Dichloroethylene, cis-1,2-dichloroethylene, and 1,2-dichloropropane were completely converted, but not all the chloride was released because of the formation of chlorinated intermediates, e.g., trans-2,3-dichlorooxirane, cis-2,3-dichlorooxirane, and 2,3-dichloropropanol, respectively. 1,1,1-Trichloroethane, 1,1-dichloroethylene, and 1,3-dichloropropylene were incompletely converted, and the first compound yielded 2,2,2-trichloroethanol as a chlorinated intermediate. The two perchlorinated compounds tested, carbon tetrachloride and tetrachloroethylene, were not converted.

Cloning of 1,2-dichloroethane degradation genes of Xanthobacter autotrophicus GJ10 and expression and sequencing of the dhlA gene.

Abstract: A gene bank from the chlorinated hydrocarbon-degrading bacterium Xanthobacter autotrophicus GJ10 was prepared in the broad-host-range cosmid vector pLAFR1. By using mutants impaired in dichloroethane utilization and strains lacking dehalogenase activities, several genes involved in 1,2-dichloroethane metabolism were isolated. The haloalkane dehalogenase gene dhlA was subcloned, and it was efficiently expressed from its own constitutive promoter in strains of a Pseudomonas sp., Escherichia coli, and a Xanthobacter sp. at levels up to 30% of the total soluble cellular protein. A 3-kilobase-pair BamHI DNA fragment on which the dhlA gene is localized was sequenced. The haloalkane dehalogenase gene was identified by the known N-terminal amino acid sequence of its product and found to encode a 310-amino-acid protein of molecular weight 35,143. Upstream of the dehalogenase gene, a good ribosome-binding site and two consensus E. coli promoter sequences were present.

Degradation of Epichlorohydrin and Halohydrins by Bacterial Cultures Isolated from Freshwater Sediment

Abstract: SUMMARY: Three bacterial cultures, the Gram-negative strain AD1 and the Gram-positive strains AD2 and AD3, were isolated from freshwater sediment after enrichment with epichlorohydrin as sole carbon source. In batch cultures of strain AD1 and strain AD3, epichlorohydrin was rapidly degraded to 3-chloro-l,2-propanediol. Crude extracts of strain AD1 contained epoxide hydrolase activity towards epichlorohydrin, epibromohydrin, glycidol and propylene oxide as substrates. In contrast, strain AD2 did not actively convert epichlorohydrin but utilized 3-chloro-l,2-propanediol produced by slow chemical hydrolysis. No epichlorohydrin epoxide hydrolase was found in extracts of this organism. Crude extracts of strains AD1 and AD2 dehalogenated a number of mono- and dihalogenated alcohols and ketones, such as 1,3-dichloro-2-propanol, 3-chloro-l,2-propanediol, l-chloro-2-propanol, l,3-dibromo-2-propanol, chloro-acetone and 1,3-dichloroacetone. Dehalogenation yielded epoxides as products. The results suggest that epichlorohydrin is converted by strain AD1 via 3-chloro-l,2-propanediol and glycidol by the action of an epoxide hydrolase and a dehalogenase, respectively. The same route for dehalogenation proceeds in strain AD2, which, however, is dependent on chemical hydrolysis of epichlorohydrin rather than enzymic conversion.

Degradation of chlorinated and non-chlorinated aromatic solvents in soil suspensions by pure bacterial cultures

Abstract: Several strains that utilize aromatic solvents were isolated and tested for their ability to degrade chlorinated and non-chlorinated aromatic hydrocarbons. The effect of inoculation with pure bacterial cultures on the degradation of benzene, toluene, o-, m- and p-xylene, chlorobenzene o-dichlorobenzene and 1,3,5-trichlorobenzene in soil slurries was studied. The compounds for which organisms were added were rapidly degraded. Without inoculation, however, degradation of benzene, toluene, m- and p-xylene and chlorobenzene was slow, while o-xylene and o-dichlorobenzene were only slightly degraded. The results showed that degradation was due to growth of the inoculated cells using the aromatic compounds as sources of carbon and energy. Addition of activated sludge did not stimulate degradation. The degradation rate of aromatic solvents by the added bacteria in soil slurries was similar or higher than that observed in liquid cultures of the same organisms.