Washed cell suspensions of Pseudomonas putida, grown with toluene as sole source of carbon, oxidized benzene, toluene, and ethylbenzene, at equal rates. Cell extracts were used to study benzene degradation and were shown to require nicotinamide-adenine dinucleotide, ethanol, ferrous ions, and L-cysteine for maximal activity. The benzene oxygenase system

Pseudomonas putida, grown with toluene as sole source of carbon, oxidized chloro-, bromo-, iodo-, and fluorobenzenes to their respective 3-halogenated catechol derivatives. The dihydroxy compounds from the first three substrates above were identified by isolation and comparison with synthetic compounds. The investigation of the metabolism of ^-chlorotoluene resulted in the isolation of two compounds which have Our continued interest in the mechanism of oxygen fixation into the aromatic nucleus led us to try to isolate the early intermediates in the degradation of several aromatic compounds. Since both toluene and benzene are metabolized at an extremely rapid rate by Pseudomonas putida (Gibson et al., 1968), with little accumulation of reaction products, it seemed probable that cooxidation (Leadbetter and Foster, 1960; Davis and Raymond, 1961) might be of use in our studies. Halogenated aromatic derivatives were chosen as potential partially metabolizable intermediates. The presence of a halogen atom seems to greatly reduce the biodegradability of many aromatic compounds (Alexander and Lustigman, 1966;Ichihara et al., 1962).This paper describes the isolation and identification of the products formed from chloro-, bromo-, iodo-, and fluorobenzenes and also p-chlorotoluene when these substrates are incubated with cells of P. putida, grown with toluene as the sole source of carbon. Materials and Methods1Organism and Growth Conditions. P. putida was grown in media whose composition has been described previously (Gibson et al., 1968).

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