ISOLATION, CHARACTERIZATION, AND DISTRIBUTION OF DENITRIFYING TOLUENE DEGRADERS FROM A VARIETY OF HABITATS

Enrichments capable of toluene degradation under O-2,-free denitrifying conditions were established with diverse inocula including agricultural soils, compost, aquifer material, and contaminated sail samples from different geographic regions of the world. Successful enrichment was strongly dependent on the initial use of relatively low toluene concentrations, typically 5 ppm. From the enrichments showing positive activity for toluene degradation, 10 bacterial isolates were obtained. Fingerprints generated by PCR-amplified DNA, with repetitive extragenic palindromic sequence primers, showed that eight of these isolates were different. Under aerobic conditions, all eight isolates degraded toluene, five degraded ethylbenzene, three consumed benzene, and one degraded chlorobenzene. meta-Xylene was the only other substrate used anaerobically and was used by only one isolate. All isolates were motile gram-negative rods, produced N-2 from denitrification, and did not hydrolyze starch. All strains but one fixed nitrogen as judged by ethylene production from acetylene, but only four strains hybridized to the nifHDK genes. All strains appeared to have heme nitrite reductase since their DNA hybridized to the heme (nirS) but not to the Cu (nirU) genes. Five strains hybridized to a toluene ortho-hydroxylase catabolic probe, and two of those also hybridized to a toluene meta-hydroxylase probe. Partial sequences of the 16S rRNA genes of all isolates showed substantial similarity to 16S rRNA sequences of Azoarcus sp. Physiological, morphological, fatty acid, and 16S rRNA analyses indicated that these strains were closely related to each other and that they belong to the genus Azoarcus. The activity and isolation of at least one toluene-degrading denitrifier from the majority of the habitat types studied suggest that microbes with the capacity to grow anaerobically on toluene are common in nature.