Diversity of bacteria capable of degrading halobenzoates under denitrifying conditions

Halobenzoate degradation was observed in denitrifying enrichment cultures established with sediments or soils from various geographical and ecological locations. Halobenzoate degrading denitrifying bacteria was isolated from enrichment cultures utilizing 2-fluorobenzoate, 4-fluorobenzoate, 3-chlorobenzoate and 3-bromobenzoate. Each isolate could grow on the selected halobenzoate with nitrate as the terminal electron acceptor and had distinctive substrate specificity. The isolates were identified and classified on the basis of 16S rRNA gene analysis and their cellular fatty acid profiles. They were placed in nine genera belonging to either the alpha-, beta-, or gamma-branch of the Proteobacteria, namely, Acidovorax, Azoarcus, Bradyrhizobium, Ochrobactrum, Paracoccus, Pseudomonas, Mesorhizobium, Ensifer , and Thauera.; Halobenzoate degrading denitrifying bacteria assigned to the genera Azoarcus and Thauera in the beta-branch of the Proteobacteria were studied further to examine the species diversity with morphology, phenotypic characterization, cellular fatty acid analysis, 16S rRNA gene analysis, ribotyping and DNA-DNA hybridization. On the basis of polyphasic analysis, the studied strains clustered into two groups closely related to the species Azoarcus tolulyticus and Thauera aromatica. In addition, isolates assigned to the genus Ochrobactrum in the alpha-branch of the Proteobacteria were classified further with fatty acids analysis, 16S rRNA gene analysis and DNA-DNA hybridization. At least two novel species in the genus Ochrobactrum can be proposed among the studied isolates.; 4-Chlorobenzoate and 4-bromobenzoate were readily degraded by denitrifying enrichment cultures. However, repeated attempts to isolate 4-chlorobenzoate and 4 brombenzoate degrading pure cultures failed, although Microbial community analyses were performed to characterize the 4-chlorobenzoate and 4-bromobenzoate degrading consortia with terminal restriction fragment length polymorphism. (TRFLP) and cloning of 16S rRNA genes. Interestingly, two phylotypes were identified as major components of the 4-chlorobenzoate and 4-bromobenzoate degrading consortia regardless of their different geographical and ecological sources. One of the phylotypes was closely related to the species Thauera aromatica, while the other phylotype was distantly related to Ralstonia solanacearum and in the beta-subunit of the Proteobacteria.; In conclusion, this study demonstrates that the ability to utilize different halobenzoates under denitrifying conditions is ubiquitously distributed in the Proteobacteria and that these bacteria are widely spread in soils and sediments. Taxonomic diversity of halobenzoate degrading denitrifiers was observed regardless of specific substrates, geographical and ecological sources. However, the genera Azoarcus and Thauera can be considered as important taxa for halobenzoate degradation under denitrifying conditions.