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Appl. Environ. Microbiol. doi:10.1128/AEM.02574-06
Copyright (c) 2007, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved.

Influence of Vitamin B₁₂ and Co-cultures on the Growth of Dehalococcoides Isolates in Defined Medium

Department of Civil and Environmental Engineering. University of California at Berkeley, Berkeley, CA 94720-1710; Earth Sciences Division, Lawrence Berkeley National Laboratory, 1 Cyclotron Rd., Berkeley, CA 94720, Division of Environmental Science and Engineering, National University of Singapore, Singapore 117576

^* To whom correspondence should be addressed. Email: alvarez{at}ce.berkeley.edu.

	Abstract

Bacteria of the genus Dehalococcoides play a key role in the complete chloroethene detoxification processes as they are the only microbes known to be capable of dechlorination beyond dichloroethenes (DCEs) to vinyl chloride (VC) and ethene. However, Dehalococcoides cells usually grow slowly with a doubling time of 1 to 2 days and have complex nutritional requirements. Here we describe the growth of D. ethenogenes 195 in defined mineral salts medium, demonstrate the improved growth of strain 195 when amended with high vitamin B₁₂ concentrations, and describe a strategy for maintaining Dehalococcoides strains on lactate by growing them in constructed consortia. Although strain 195 was capable of growth in defined medium spiked with 0.5 mM TCE and 0.001 mg/L vitamin B₁₂, TCE dechlorination and cellular growth rates doubled when B₁₂ was increased 25 fold to 0.025 mg/L. In addition, final ratios of ethene to VC increased in the higher vitamin condition, reflective of the key role that cobalamin plays in dechlorination reactions. No further dechlorination or growth improvement was observed when vitamin B₁₂ concentrations were increased above 0.025 mg/L. In defined consortia containing strain 195, Desulfovibrio desulfuricans, and/or Acetobacterium woodii, and provided lactate as the electron donor, tetrachloroethene (PCE, 0.4 mM) was completely dechlorinated to VC and ethene with concomitant growth of Dehalococcoides cells. Strain 195 cells grew to densities that were 1.5 times greater in the co- and tri- cultures than 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 its metabolic process. These findings demonstrate that growth of D. ethenogenes strain 195 in defined medium can be optimized by providing high concentrations of vitamin B₁₂ and that this strain can be grown to higher densities in co- and tri- cultures with fermenters that convert lactate to generate required hydrogen and acetate and that may enhance the availability of vitamin B₁₂.

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