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The metabolism of dichloromethane by Dehalobacterium formicoaceticum in cell suspensions and crude cell extracts was investigated. The organism is a strictly anaerobic gram-positive bacterium that utilizes exclusively dichloromethane as a growth substrate and ferments this compound to formate and acetate in a molar ratio of 2:1. When [13C]dichloromethane was degraded by cell suspensions, formate, the methyl group of acetate, and minor amounts of methanol were labeled, but there was no nuclear magnetic resonance signal corresponding to the carboxyl group of acetate. This finding and previously established carbon and electron balances suggested that dichloromethane was converted to methylene tetrahydrofolate, of which two-thirds was oxidized to formate while one-third gave rise to acetate by incorporation of CO2 from the medium in the acetyl coenzyme A synthase reaction. When crude desalted extracts were incubated in the presence of dichloromethane, tetrahydrofolate, ATP, methyl viologen, and molecular hydrogen, dichloromethane and tetrahydrofolate were consumed, with the concomitant formation of stoichiometric amounts of methylene tetrahydrofolate. The in vitro transfer of the methylene group of dichloromethane onto tetrahydrofolate required substoichiometric amounts of ATP. The reaction was inhibited in a light-reversible fashion by 20 μM propyl iodide, thus suggesting involvement of a Co(I) corrinoid in the anoxic dehalogenation of dichloromethane.D. formicoaceticum exhibited normal growth with 0.8 mM sodium in the medium, and crude extracts contained ATPase activity that was partially inhibited byN,N′-dicyclohexylcarbodiimide and azide. During growth with dichloromethane, the organism thus may conserve energy not only by substrate-level phosphorylation but also by a chemiosmotic mechanism involving a sodium-independent F0F1-type ATP synthase.

Alkyl-and arylsulfonates were tested as sole added sources of sulfur for the growth of enrichment cultures under strictly anaerobic denitrifying or fermentative conditions. Cultures that utilized taurine, ethylsulfonate, the dyestuffs orange II and acid red I, tolylsulfonate, 2-(4-sulfophenyl)butyrate (SPB), a dialkyltetralinesulfonate, and 1-(4-sulfophenyl)octane were readily obtained. We chose to work with the simple aromatic compounds and isolated a fermentative bacterium, strain EV4, which utilized SPB as the sole added source of sulfur in glucose-mineral medium. The organism was identified as a Clostridium sp. related to Clostridium beijerinckii. Clostridium sp. strain EV4 utilized seven of seven tested arylsulfonates quantitatively. The growth yield was about 3 kg of protein per mol of sulfur, whether sulfonate or sulfate was utilized. A major product specific to each sulfonate could be observed. Although no product was identified, the existence of anaerobic desulfonation has been established.

A strictly anaerobic two-component culture able to grow exponentially with a doubling time of 20 h on a medium containing dichloromethane as the carbon and energy source was characterized. On a medium without sulfate, we observed (per mol of dichloromethane) a mass balance of 2 mol of chloride, 0.26 mol of acetate, 0.05 mol of formate, and 0.25 mol of carbon in biomass. One component of the culture, strain DMB, was identified by a 16S ribosomal DNA analysis as a Desulfovibrio sp. The other component, the gram-positive organism strain DMC, could not be isolated. It was possible, however, to associate strain DMC on a medium containing dichloromethane in a coculture with Acetobacterium woodii or Methanospirillum hungatei. Coculture of strain DMC with the Archaeon M. hungatei allowed us to specifically amplify by PCR the 16S rRNA gene of strain DMC. A phylogenetic analysis of the 16S ribosomal DNA sequence revealed that this organism groups within the radiation of the Clostridium-Bacillus subphylum and exhibits the highest levels of sequence similarity (89%) with Desulfotomaculum orientis and Desulfitobacterium dehalogenans. Since the novel organism strain DMC was able to grow acetogenically with dichloromethane when it was associated with one of three metabolically different partners and since, in contrast to strain DMB, strain DMC contained carbon monoxide dehydrogenase activity, this bacterium is responsible for both the dehalogenation of dichloromethane and the acetogenesis observed in the original two-component culture. The obligatory dependence of strain DMC on a partner during growth with dichloromethane is thought to stem from the need for a growth factor produced by the associated organism.

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