Isolation and partial characterization of aClostridium species transforming para-hydroxybenzoate and 3,4-dihydroxybenzoate and producing phenols as the final transformation products

Abstract: Organisms present in methanogenic freshwater lake sediments from the vicinity of Athens, Georgia, were adapted to mineralize 2,4-dichlorophenol. Repeated addition of 0.5 to 2.7 mmol/liter of phenol, and later of 0.5–6.2 mmol/liter p-hydroxybenzoate (p-OHB), to such enrichments led to the conversion of p-OHB to phenol at a rate of up to 100 mmol p-OHB per liter per day. Subsequently, a spore-forming, obligately anaerobic bacterium, strain JW/Z-1, was isolated which transformed p-OHB to phenol and 3,4-dihydroxybenzoate (3,4-OHB) to catechol (1,2-dihydroxybenzene) stoichiometrically without further metabolism of the phenols. The strain did not transform benzoate, 4-chlorophenol, 2,4-dichlorophenol, 4-chlorobenzoate, o- and m-hydroxybenzoate, 2,4- and 3,5-dihydroxybenzoate, 2,3,4- and 3,4,5-trihydroxybenzoate, or 4-aminobenzoate. Yeast extract was required for growth of strain JW/Z-1 and only high concentrations of casein hydrolysate or tryptone could substitute it, to some extent. Except for sodium acetate, and some amino acids together with a 20-fold increased concentration of vitamins, no single carbohydrate or defined organic compound has been found to support growth of this strain in the presence (or in the absence) of 0.2 to 0.5% (w/v) yeast extract. The fermentation products during growth on yeast extract indicated that the metabolism of amino acid degradation was the major source for growth. The decarboxylating activity was inducible by p-OHB for the decarboxylation of p-OHB, and at a lower rate for 3,4-OHB, and by 3,4-OHB only for 3,4-OHB, suggesting that two different enzyme systems exist. The addition of the aromatic amino acids phenol or benzoate did not induce the decarboxylation activity in cultures growing with yeast extract. Growth was observed at temperatures ranging from 12–41C (T_opt, 33–34C) and at pH-values ranging from 6.0–10.0 (pH_opt, 7.2–8.2). The shortest doubling time observed for strain JW/Z-1 was 3.2 hours.