Biochemical and PCR-based identification of Hydrocarbonoclastic Bacteria isolated from spent engine oil polluted soil

Adeleye,A.O.,Yerima,M.B.Nkereuwem,M.E.Onasanya,G.O.,Onokebhagbe,V.O.,Bate,G.B and Raji M.

Keywords: Spent engine oil, soil, bacterial isolates, biochemical tests and nucleotide similarity index


This study was conducted to isolate and identify spent engine oil (SEO) utilizing bacteria from mechanic village, Dutse. Test tubes containing one (1) mL of the supernatant derived from the mixture of one (1) gram of SEO polluted soil and nine (9) mL sterile distilled water supplemented with mineral salt medium and overlaid with 0.5 mL of SEO were incubated at 37.3 oC for four (4) days. Bacterial growth in form of turbidity was sub-cultured into test tubes containing Trypticase Soy broth and Trypticase Soy agar, incubated at 37.3 oC for 48 hours. Developed colonies were observed and recorded. Six (6) distinct colonies were subsequently picked and transferred to glass slides to form smears for Gram staining. Biochemical tests based on morphological attributes of the bacterial isolates were conducted for its identification and further confirmed using molecular technique. Gram positive cocci in clusters, gram positive rods with squared ends, gram negative rods, gram negative rods in chains, gram positive cocci in chains and gram positive cocci bacteria were identified under the microscope. Results of the biochemical tests indicated Staphylococcus aureus, Bacillus cereus, Pseudomonas aeruginosa, Alcaligenes faecalis, Streptococcus pyogenes and Enterococcus faecalis as the isolated bacteria. Nucleotide similarity index (NSI) results generated showed 70.71% – 96.55% between the nucleotide sequences of all the bacteria isolated in this study when compared against the National Center for Biotechnology Information (NCBI) database. Owing to the results obtained in this study, the method employed for the isolation of hydrocarbonoclastic bacteria can therefore be recommended for subsequent isolation of such bacteria that can be employed in achieving bioremediation of hydrocarbon impacted environments.