Development of specific oligonucleotide probes for the identification and in situ detection of hydrocarbon‐degrading Alcanivorax strains

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Abstract
The genus Alcanivorax comprises diverse hydrocarbon‐degrading marine bacteria. Novel 16S rRNA‐targeted oligonucleotide DNA probes (ALV735 and ALV735‐b) were developed to quantify two subgroups of the Alcanivorax/Fundibacter group by fluorescence in situ hybridization (FISH), and the conditions for the single‐mismatch discrimination of the probes were optimized. The specificity of the probes was improved further using a singly mismatched oligonucleotide as a competitor. The growth of Alcanivorax cells in crude oil‐contaminated sea water under the biostimulation condition was investigated by FISH with the probe ALV735, which targeted the main cluster of the Alcanivorax/Fundibacter group. The size of the Alcanivorax population increased with increasing incubation time and accounted for 91% of the 4′,6‐diamidino‐2‐phenylindole (DAPI) count after incubation for 2 weeks. The probes developed in this study are useful for detecting Alcanivorax populations in petroleum hydrocarbon‐degrading microbial consortia.