16S rRNA-Based Molecular Identification of Indigenous Hydrocarbon-Associated Bacteria from Crude Oil-Contaminated Soil in Kirkuk, Iraq
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Petroleum hydrocarbon contamination can alter soil physicochemical conditions and select for microbial populations with potential relevance to natural attenuation and bioremediation. This study aimed to isolate and molecularly identify culturable indigenous bacteria from crude oil-contaminated soil collected near the North Oil Company facilities in Kirkuk, northern Iraq. The contaminated soil was characterized by visible petroleum contamination, alkaline pH, high organic carbon, and high total petroleum hydrocarbon concentration. Bacterial isolates were cultured, genomic DNA was extracted using a commercial DNA extraction kit, and the 16S rRNA gene was amplified by PCR. PCR products were verified by agarose gel electrophoresis and sequenced by Sanger sequencing. BLASTn analysis identified five bacterial isolates affiliated with Bacillus subtilis, Stutzerimonas balearica, Ochrobactrum sp., Alcaligenes faecalis, and a putative Ralstonia sp. The closest BLAST matches showed sequence identities ranging from 94.70% to 100.00%. Phylogenetic analysis supported the placement of the isolates within their respective bacterial lineages. The occurrence of these culturable taxa in highly hydrocarbon-contaminated soil suggests the presence of indigenous hydrocarbon-associated bacteria that may be relevant for future bioremediation studies. However, because hydrocarbon degradation activity was not directly tested, the isolates should be considered hydrocarbon-associated rather than confirmed hydrocarbon-degrading bacteria.
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