S.N. GAWANDE AND D.G. MULATKAR
Abstract
Soil contamination by petroleum hydrocarbons, particularly used engine oil, poses a major environmental challenge due to the persistence of polycyclic aromatic hydrocarbons (PAHs) and heavy metals. Indigenous microorganisms possessing hydrocarbon-degrading abilities offer a sustainable and eco-friendly alternative for remediation. Understanding their physiological, biochemical, and molecular characteristics is essential for designing effective bioremediation strategies. The present study aimed to isolate, characterize, and identify bacteria from engine-oil contaminated soil and to assess their ability to utilize used engine oil as a sole carbon source. Soil samples were collected from automobile workshop sites in Akola, Maharashtra, India. Enrichment was performed using mineral salt medium (MSM) supplemented with used engine oil. Distinct colonies obtained on MSM agar were purified and labeled as EO1âEO4. Morphological characterization included colony features and Gram staining. Biochemical profiling was performed using IMViC tests and sugar fermentation assays. Molecular identification was carried out by extracting genomic DNA using the CTAB method, followed by PCR amplification and sequencing of the 16S rRNA gene. BLAST analysis was used for species-level identification. Four bacterial isolates (EO1âEO4) were successfully obtained from engine oil-contaminated soil using MSM, each showing distinct colony features. Gram staining revealed EO1 and EO4 as Gram-negative rods, while EO2 and EO3 were Gram-positive rods. Biochemical tests differentiated the isolates based on sugar fermentation and IMViC profiles, enabling their preliminary identification. MS agar photoplates confirmed the growth of hydrocarbon-utilizing bacteria, and Gram-stained images verified their purity. High-quality genomic DNA was extracted from EO1 and EO2, and ~1200 bp 16S rRNA fragments were successfully amplified. BLAST analysis identified EO1 as Priestia megaterium (98.09% identity) and EO2 as Bacillus badius, confirming their molecular identity and potential for hydrocarbon degradation. This study demonstrates the presence of efficient hydrocarbon-degrading bacteria in used engine-oil- contaminated soils. The identification of Priestia megaterium as a dominant degrader highlights its relevance in bioremediation applications due to its resilience, enzymatic versatility, and ability to metabolize complex hydrocarbons. The findings provide valuable insights into native soil microbial communities and their application in eco-friendly remediation strategies aimed at restoring petroleum- polluted environments.