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From various oxic or anoxic habitats several strains of bacteria were isolated which in the absence of molecular oxygen oxidized phenol to CO2 with nitrate as the terminal electron acceptor. All strains grew in defined mineral salts medium; two of them were further characterized. The bacteria were facultatively anaerobic Gram-negative rods; metabolism was strictly oxidative with molecular oxygen, nitrate, or nitrite as electron acceptor. The isolates were tentatively identified as pseudomonads. Besides phenol many other benzene derivatives like cresols or aromatic acids were anaerobically oxidized in the presence of nitrate. While benzoate or 4-hydroxybenzoate was degraded both anaerobically and aerobically, phenol was oxidized under anaerobic conditions only. Reduced alicyclic compounds were not degraded. Preliminary evidence is presented that the first reaction in anaerobic phenol oxidation is phenol carboxylation to 4-hydroxybenzoate.

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The initial reactions in anaerobic metabolism of methylphenols (cresols) and dimethylphenols were studied with denitrifying bacteria. A newly isolated strain, possibly a Paracoccus sp., was able to grow on o- or p-cresol as sole organic substrate with a generation time of 11 h; o- or p-cresol was completely oxidized to CO2 with nitrate being reduced to N2. A denitrifying Pseudomonas-like strain oxidized m- or -p-cresol as the sole organic growth substrate completely to CO2 with a generation time of 14 h. Demonstration of intermediates and/or in vitro measurement of enzyme activities suggest the following enzymatic steps: (1) p-Cresol was metabolized by both strains via benzoyl-CoA as central intermediate as follows: p-cresol----4-OH-benzaldehyde----4-OH-benzoate----4-OH-benzoyl-CoA----be nzoyl-CoA. Oxidation of the methyl group to 4-OH-benzaldehyde was catalyzed by p-cresol methylhydroxylase. After oxidation of the aldehyde to 4-OH-benzoate, 4-OH-benzoyl-CoA is formed by 4-OH-benzoyl-CoA synthetase; subsequent reductive dehydroxylation of 4-OH-benzoyl-CoA to benzoyl-CoA is catalyzed by 4-OH-benzoyl-CoA reductase (dehydroxylating). (2) o-Cresol was metabolized in the Paracoccus-like strain via 3-CH3-benzoyl-CoA as central intermediate as follows: o-cresol----4-OH-3-CH3-benzoate----4-OH-3-CH3-benzoyl-CoA----3-CH3-benzo yl-CoA. The following enzymes were demonstrated: (a) An enzyme catalyzing an isototope exchange reaction between 14CO2 and the carboxyl of 4-OH-3-CH3-benzoate; this activity is thought to be a partial reaction catalyzed by an o-cresol carboxylase. (b) 4-OH-3-CH3-benzoyl-CoA synthetase (AMP-forming) activating the carboxylation product 4-OH-3-CH3-benzoate to its coenzyme A thioester. (c) 4-OH-3-CH3-benzoyl-CoA reductase (dehydroxylating) catalyzing the reductive dehydroxylation of the 4-hydroxyl group with reduced benzyl viologen as electron donor to yield 3-CH3-benzoyl-CoA. This thioester may also be formed by action of a coenzyme A ligase when 3-CH3-benzoate is metabolized. 2,4-Dimethylphenol was metabolized via 4-OH-3-CH3-benzoate and further to 3-CH3-benzoyl-CoA. (3) The initial reactions of anaerobic metabolism of m-cresol in the Pseudomonas-like strain were not resolved. No indication for the oxidation of the methyl group nor for the carboxylation of m-cresol was found. In contrast, 2,4- and 3,4-dimethylphenol were oxidized to 4-OH-3-CH3- and 4-OH-2-CH3-benzoate, respectively, probably initiated by p-cresol methylhydroxylase; however, these compounds were not metabolized further.

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