Two Gram-negative, rod-shaped, non-spore-forming bacterial strains were isolated from a hexane-treated, full-scale biofilter from an oil mill. The strains were cultivated with hexane as the sole carbon source. One strain, MN154.3(T), showed a fatty acid profile that contained 16 : 0, 18 : 1cis11 and 19 : 0 cyclo11-12 as major compounds, while the second strain, isolate MN28(T), contained 14 : 0 3-OH, 16 : 0 and 18 : 1cis11 as the predominant fatty acids. On the basis of almost-complete 16S rRNA gene sequences, both strains could be allocated to the Nevskia branch of the class Gammaproteobacteria. The sequence similarities for strains MN154.3(T) and MN28(T) with respect to the most closely related type strains of this branch were 90.5 and 94.1 %, respectively. The sequence similarity between strains MN154.3(T) and MN28(T) was 90.6 %. The DNA G+C content of strain MN154.3(T) was 62.8 mol% and that for strain MN28(T) was 64.9 mol%. Both strains possessed ubiquinone-8 as the major quinone. On the basis of the 16S rRNA gene sequences of these two new isolates and several phenotypic differences exhibited with respect to known species of the Nevskia branch, strains MN154.3(T) and MN28(T) represent two novel genera and species, for which the names Alkanibacter difficilis gen. nov., sp. nov. and Singularimonas variicoloris gen. nov., sp. nov. are proposed. The type strain of Alkanibacter difficilis gen. nov., sp. nov. is MN154.3(T) (=DSM 14804(T)=LMG 22842(T)) and that of Singularimonas variicoloris gen. nov., sp. nov. is MN28(T) (=DSM 15731(T)=LMG 22844(T)).
The hexane-degrading bacterial community of a biofilter was characterised by a combination of stable isotope-based phospholipid fatty acid analyses, fluorescence in situ hybridisation and cultivation. About 70 bacterial strains were isolated from a full-scale biofilter used for treatment of hexane containing waste gas of an oil mill. The isolation approach led to 16 bacterial groups, which were identified as members of the Alpha-, Beta- and Gammaproteobacteria, Actinobacteria and Firmicutes. Three groups showed good growth on hexane as the sole source of carbon. These groups were allocated to the genera Gordonia and Sphingomonas and to the Nevskia-branch of the Gammaproteobacteria. Actively degrading populations in the filter material were characterised by incubation of filter material samples with deuterated hexane and subsequent phospholipid fatty acid analysis. Significant labelling of the fatty acids 16:1 cis10, 18:1 cis9 and 18:0 10methyl affiliated the hexane-degrading activity of the biofilter with the isolates of the genus Gordonia. In vitro growth on hexane and in situ labelling of characteristic fatty acids confirmed the central role of these organisms in the hexane degradation within the full-scale biofilter.
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