ABSTRACT

The evaluation of the physicochemical parameters, biosurfactant production and metagenomic analysis of hydrocarbon polluted soil in Opuama, Ogini, Oteghele, Transforcados pipeline and Transforcados right of way sites located at Isoko North, Warri North and South LGA of Delta State Nigeria, was carried out using standard microbiological and physicochemical methods. Hydrocarbon utilizing bacteria (HUB), total heterotrophic bacterial count (THB), physicochemical analysis including heavy metals, screening and characterization, optimization of biosurfactant using response surface methodology (RSM) and metagenomic analysis were all carried out.  The DNA of the microorganisms from the soil samples were extracted using ZymoDNA extraction kit, amplified and subjected to next generational sequencing (NGS) on Pacbio SMR sequencing platform. Following NGS, gene calling was performed using freg gene scan and the resulting metagenome were then functionally annotated onto two pipelines namely: cluster of orthology group (COG) and Kyoto encyclopedia of genes and genomes (KEGG) for functional gene analysis. The bacteria screened and characterized for biosurfactant production were Escherichia coli, Bacillus subtilis, Enterobacter aerogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, Providencia stuartii and Burkholderia pseudomallei. All of the screened bacteria, Bacillus subtilis indicated high level of biosurfactant production and was subsequently subjected to optimization for biosurfactant production. The emulsification index (E1%) due to change in temperature and pH on glucose/KN0₃ is 77.28, sucrose/KN0₃ is 59.80 and spent vegetable oil/KN0₃ is 57.16. The  mean value for hydrocarbon utilizing bacteria and total heterotrophic bacterial count were 1.5×10⁵ cfu/g and 3.76×10⁸ cfu/g for Opuama flow station, 5.8×10⁴ cfu/g and 2.60×10⁸ cfu/g for Ogini flow station, 2.80×10⁵ cfu/g and 2.55×10⁸ cfu/g for Oteghele river spill, 4.2×10⁴ cfu/g and 3.04×10⁸ cfu/g for Transforcados pipeline spill and 1.72×10⁵ cfu/g and 2.75×10⁸ cfu/g for Transfoecados right of way spill. Oteghele river spill had the highest concentration of crude oil utilization bacteria 2.80×10⁵ cfu/g followed by Transforcados right of way spill 1.72×10⁵ cfu/g while Opuama flow station had the highest concentration of heterotrophic bacteria 3.76×10⁸ cfu/g. The performance of the pathogen for hemolytic reaction on blood agar, oil spreading on glass slide, blue plate agar reaction, emulsification index and lypolytic enzyme reaction indicated that they were significant at (p<0.05). There was significant difference in the physicochemical parameters at (p<0.05). The mean values of the pH range from 4.15^d±0.1-5.75^b±0.1, electrical conductivity 1179.61^e±7.51- 2079.55^b±1.45. Some of the heavy metals were higher in value when compared with the WHO standard. The structural metagenomics revealed eight top phyla; Proteobacteria, Actinobacteriota, Acidobacteriota, Bacteroidota, Planctomycetota, Firmicutes and Cloroflexi. The functional gene analysis revealed Carbohydrate metabolism, energy metaboilism, environmental information processing, degradation genes and various protein classes in the samples as contained in the KEGG and COG pipelines. Giving the findings of this study microorganisms found in petroleum hydrocarbon polluted soil were able to produce biosurfactants which helped in biodegradation.