DL-Tetrahydrofolate (THF) and ATP were necessary for the anaerobic 0-demethylation of phenylmethylethers in cell extracts of the type strain (ATCC 29683) of the homoacetogen Acetobacterium woodii. The reactants for this enzymatic activity have not been previously demonstrated in any system, nor has the mediating enzyme been studied. An assay using reaction mixtures containing 1 mM THF, 2 mM ATP, and 2 mM hydroferulate (i.e., 4-hydroxy,3-methoxyphenylpropionate) was developed and was performed under stringent anaerobic conditions. Pyridine nucleotides and several other possible cofactors were tested but had no effect on the activity. After centrifugation of disrupted cells at 27,000 x g, the activity was found primarily in the supernatant, which had a specific activity of 14.2 + 0.5 nmol/min/mg of protein. At saturating levels of each of the other two substrates, apparent Km values for the variable substrate were 0.65 mM hydroferulate, 0.27 mM ATP, and 0.17 mM THF. Activity was significantly decreased when extract was preincubated at 60°C and was completely lost after preincubation in air for 30 min. Thus, the soluble anaerobic 0-demethylating enzyme system of A. woodii is oxygen sensitive. The THF-and ATP-dependent activity measurable in the soluble fraction of cell extracts constituted about 34% of the activity seen with intact cells.

Bromoxynil (3,5-dibromo-4-hydroxybenzonitrile) is a halogenated aromatic nitrile herbicide used on a variety of crops for the postemergence control of annual broad-leaved weeds. The anaerobic biodegradability of bromoxynil and its aerobic transformation product, 3,5-dibromo-4-hydroxybenzoate, were examined in enrichment cultures established with anaerobic sediment under denitrifying, Fe(III)-reducing, sulfidogenic, and methanogenic conditions. Bromoxynil (100 microM) was depleted in 20 to 30 d in the methanogenic, sulfidogenic, and Fe(IIi)-reducing enrichments but was stable under denitrifying conditions. The 3,5-dibromo-4-hydroxybenzoate (100 microM) was depleted within 20 to 35 d under all four anaerobic conditions. Both compounds were stable in sterile controls. Bromoxynil and 3,5-dibromo-4-hydroxybenzoate were readily utilized upon respiking of the cultures. During utilization of bromoxynil, stoichiometric release of bromide was observed with transient accumulation of metabolites identified as bromocyanophenol, cyanophenol, and phenol. Bromoxynil heptanoate and octanoate were rapidly hydrolyzed to bromoxynil, which was further degraded. These results indicate that bromoxynil and 3,5-dibromo-4-hydroxybenzoate are degraded under different anaerobic conditions. Anaerobic degradation of bromoxynil proceeds via reductive debromination to 4-cyanophenol, which is further transformed to phenol and can ultimately be degraded to carbon dioxide.

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