Isolation and characterization of Desulfovibrio dechloracetivorans sp. nov., a marine dechlorinating bacterium growing by coupling the oxidation of acetate to the reductive dechlorination of 2-chlorophenol. Appl Environ Microbiol 66

Abstract

Strain SF3, a gram-negative, anaerobic, motile, short curved rod that grows by coupling the reductive dechlorination of 2-chlorophenol (2-CP) to the oxidation of acetate, was isolated from San Francisco Bay sediment. Strain SF3 grew at concentrations of NaCl ranging from 0.16 to 2.5%, but concentrations of KCl above 0.32% inhibited growth. The isolate used acetate, fumarate, lactate, propionate, pyruvate, alanine, and ethanol as electron donors for growth coupled to reductive dechlorination. Among the halogenated aromatic compounds tested, only the ortho position of chlorophenols was reductively dechlorinated, and additional chlorines at other positions blocked ortho dechlorination. Sulfate, sulfite, thiosulfate, and nitrate were also used as electron acceptors for growth. The optimal temperature for growth was 30°C, and no growth or dechlorination activity was observed at 37°C. Growth by reductive dechlorination was revealed by a growth yield of about 1 g of protein per mol of 2-CP dechlorinated, and about 2.7 g of protein per mole of 2,6-dichlorophenol dechlorinated. The physiological features and 16S ribosomal DNA sequence suggest that the organism is a novel species of the genus Desulfovibrio and which we have designated Desulfovibrio dechloracetivorans. The unusual physiological feature of this strain is that it uses acetate as an electron donor and carbon source for growth with 2-CP but not with sulfate. Substantial amounts of halogenated aromatic compounds have been released to the environment and many of them have accumulated in groundwater and sediments To date, much of the dehalogenation research has been directed toward understanding the fate and behavior of halogenated pollutants in freshwater sediments, soils, and sludges. Comparatively little is known about the fates of these pollutants or dehalogenating organisms from the marine environment despite the fact that marine biota produce a remarkable array of halogenated compounds (12). For example, studies by King (15) indicate that 2,4-dibromophenol occurred at concentrations of up to several hundred micromolar in hemichordate burrow walls and that this chemical was dehalogenated in these sediments under anaerobic conditions. This result suggested that bacterial populations from some marine habitats may have developed enzymatic capabilities to degrade these naturally occurring organohalides. Hence, marine sources may reveal further diversity of dehalogenating microorganisms. Here, we describe enrichment, isolation, and characterization of a novel marine bacterium capable of growth in a synthetic seawater medium on 2-CP and acetate. This new isolate dechlorinates ortho-chlorophenol, producing phenol as a product. Phenotypic and 16S ribosomal DNA (rDNA) phylogenetic studies indicate that the organism belongs to the Desulfovibrio group of the sulfate-reducing bacteria. To our knowledge, this is the first member of the Desulfovibrio group that is capable of oxidizing acetate. MATERIALS AND METHODS Media and growth conditions. Cultures were grown in 160-ml serum bottles with 50 or 100 ml of anaerobic synthetic seawater medium or in 30-ml anaerobic culture tubes with 20 ml of medium. The medium was modified from standard seawater media to remove sulfate so that dechlorinators rather than sulfate reducers could be enriched, and to achieve an Na ϩ concentration of 0.46 M, which approximates that of seawater. It contained the following mineral salts (in grams/liter): NaCl, 25; MgCl 2 , 1.4; KH 2 PO 4 , 0.2; NH 4 Cl, 0.3; KCl, 0.5; and CaCl 2 , 0.1. A trace element solution was added to give the following final concentrations (in milligrams/liter): MnCl 2 ⅐ 6H 2 O, 5; H 3 BO 3 , 0.5; ZnCl 2 , 0.5; CoCl 2 ⅐ 6H 2 O, 0.5; NiSO 4 ⅐ 6H 2 O, 0.5; CuCl 2 ⅐ 2H 2 O, 0.3; and NaMoO 4 ⅐ 2H 2 O, 0.1. In addition, the medium contained 0.003 mg of NaSeO 3 and 0.008 mg of Na 2 WO 4 per liter and 10 mg of resazurin per liter. The medium was boiled under oxygen-free N 2 and cooled to room temperature under N 2 -CO 2 (95:5). Na 2 S (as a reductant) and NaHCO 3 were then added to final concentrations of 1 and 30 mM, respectively, and the pH of the medium was adjusted to 7.3 to 7.5 by varying the CO 2 concentration in the headspace. The medium was dispensed into N 2 -CO 2 -flushed serum bottles or culture tubes capped with butyl rubber stoppers and sterilized by autoclaving. The sterile medium was amended with an anaerobic sterile Wolin vitamin solution (34) plus thiamine, 1,4-naphthoquinone