Environ. Sci. Technol. 1997, 31, 2292-2297
Microbial Reduction of Vitamin B12 by Shewanella alga Strain BrY with Subsequent Transformation of Carbon Tetrachloride D A R L A J . W O R K M A N , * ,†,‡ SANDRA L. WOODS,‡ YURI A. GORBY,† JIM K. FREDRICKSON,† AND M I C H A E L J . T R U E X †,‡ Environmental Microbiology Group Battelle PNNL, P.O. Box 999, Richland, Washington 99352, and Department of Civil, Construction, and Environmental Engineering, 202 Apperson Hall, Oregon State University, Corvallis, Oregon 97331-2302
The ability of a metal-reducing bacterium to microbially reduce vitamin B12 was determined to expand our understanding of the role vitamin B12 plays in the transformation of halogenated compounds in microbial systems. The subsequent transformation of chlorinated methanes catalyzed by this microbially-reduced vitamin B12 was then evaluated. When incubated in the presence of Shewanella alga strain BrY and an electron donor, the microbial reduction of vitamin B12a to B12r was observed as a shift in the vitamin B12 spectrum. In treatments containing vitamin B12 and an electron donor but without BrY, the predominant species was vitamin B12a. The introduction of BrY into the system resulted in the production of vitamin B12r. The transformation of carbon tetrachloride (CT), chloroform (CF), and dichloromethane (DCM) was examined in batch systems containing vitamin B12, Shewanella alga strain BrY, and an electron donor. Transformation of both CT and CF was observed, while no significant change in the DCM concentration was detected. Carbon monoxide was the major product of CT transformation. No significant transformation of CT or CF was detected when vitamin B12 was omitted from the system. This work demonstrates that a metal-reducing bacterium, with no apparent ability to transform CT or CF directly, mediates the reduction of vitamin B12, which in turn catalyzes the transformation of CT.
Introduction The abiotic reductive dechlorination of halogenated alkanes and alkenes using various corrinoids, porphyrins, and/or cofactors coupled with a chemical reductant has been studied extensively (1-12). Perhaps the most frequently used corrin, for studying corrin-catalyzed reductive dechlorination, is vitamin B12. Vitamin B12 is a corrin ring containing a cobalt atom at the center. When hydrated, the cyanide group within cyanocobalamin is replaced with water to form aquocob(III)alamin, otherwise known as vitamin B12a. This can be reduced by the addition of one electron to form vitamin B12r (cob(II)alamin) or two electrons to form vitamin B12s (cob(I)alamin). Using values reported by Lexa and Saveant (13), * Corresponding author telephone: (509) 376-1520; fax: (509) 3756666; e-mail:
[email protected]. † Battelle PNNL. ‡ Oregon State University. § Present address: Bioprocessing Group, Battelle PNNL.
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the standard reduction potentials for each step are +0.2 V for B12a f B12r and -0.61 V for B12r f B12s at pH 7 vs the standard hydrogen electrode (SHE). In these corrin-catalyzed dechlorination systems, vitamin B12r and B12s have been identified as the catalyst for the transformation of halogenated hydrocarbons while the unreduced form, vitamin B12a, is nonreactive toward any of the compounds tested to date. Lewis et al. (10) recently demonstrated that product distribution from the transformation of CT by various cobaltcontaining corrins was dependent on the corrin’s valence state and the type of reductant used in the system. When Ti(III) was used, producing a Co(I) corrin, the major products included methane and chloromethane as a result of hydrogenolysis of the CT. Product distribution was not only different for the Co(I) and Co(II) systems, it was also different when either dithiothreitol or a sulfide/cysteine reductant was used, even though both produced a Co(II) corrin. When dithiothreitol was used as the reductant, the major products identified were dichloromethane, carbon monoxide, and formate; while chloroform, carbon disulfide, 2-oxothiazolidine carboxylic acid, and 2-thioxo-4-thiazolidinecarboxylic acid were observed in the sulfide/cysteine reduced system. Several consortia as well as pure cultures of microorganisms, ranging from denitrifiers to methanogens, can reductively dechlorinate CT (14-18). The mechanisms involved in these transformations are generally not well known. However, both enzymes and various co-factors have been implicated as important participants in the reductive dechlorination of CT (15, 16, 18, 19). Investigators have also noted an enhanced rate of reductive dechlorination by microbial consortia when vitamin B12 is added to the system (2, 5, 7, 11). Hashsham et al. (7) observed a 10-fold increase in the rate of CT transformation when vitamin B12 was added to their anaerobic enrichment culture that was capable of growth on dichloromethane as the sole organic carbon and energy source. In addition, a shift in product distribution from CS2 (21%), CO2 (21%), and CF (17%) to CS2 (11%), CO2 (59%), and CF (
