Research Article Open Access Ogbulie and Nwaokorie, J Ecosys Ecograph 2016, S5 DOI: 10.4172/2157-7625.S5-002 Research Article Open Access Journal of Ecosystem & Ecography ISSN: 2157-7625 J o u r n a l o f E c o s y s t e m & E c o g r a p h y J Ecosyst Ecogr Global Climate Change ISSN:2157-7625 JEE, an open access journal Keywords: Microbes; Diversity; Degradative genes; Crude oil; Agricultural soil; Sequence analysis Introduction Incidence of environmental pollution due to high rate of petroleum related activities in Nigeria and other oil producing areas of the world has been associated with frequent oil spills, especially through oil wells blow out, tanker accidents and bunkering. Disasters arising from such incidence results in the discharge of crude oil into the environment afecting both soil, air and water bodies. Tis threatens human health and that of organisms that are dependent on soil. Soil contains a variety of microorganisms including bacteria that can be found in any natural ecosystem. Microbial survival in polluted soil depends on intrinsic biochemical and structural properties, physiological and genetic adaptation including morphological changes of cells as well as environmental modifcations [1]. Over the years, isolation and identifcation of hydrocarbon-degrading microorganisms have been carried out using isolation techniques. Previous studies on population dynamics showed that bacteria genera such as Pseudomonas, Bacillus, Brevibacterium, Corynebacterium, Acinetobacter and Mycobacterium are potential organisms for hydrocarbon degradation [2-4]. Shi et al. [5] compared culture-based diversity of agricultural soil communities with diversity obtained by molecular means and found that molecular methods revealed a much higher bacterial diversity than classical isolation techniques. A variety of molecular methods have been developed to assay the presence of micro-organisms in soil. Most recently, the method of choice to determine what micro-organisms are present in environmental sample is to amplify the conserved small subunit rRNA gene; where DNA is isolated from the soil using bead beating and Polymerase Chain Reaction (PCR) with universal or gene-specifc primers used to amplify the specifc gene from the sample. Tis study looked at the diversity of microorganisms persistent in agricultural soil sample polluted with 100 ml of 100% Nigerian Bonny light crude oil and lef for four years with a view to ascertain the presence of microbes with probable degradative gene for crude oil degradation which can be harnessed for the creation of superbugs for faster clean up opertaions and to confrm similarities in microbial identities. Material and Method Procurement of samples Te crude oil used was bonny light Crude and was collected with sterile containers from Akiri in Oguta, Imo State, Nigeria. Te agricultural soil subjected to pollution was obtained from Federal University of Technology Owerri (FUTO) farm land using surface sterilized soil auger at the depth of 15-30 cm. Treatment of test soil sample Surface sterilized plastic pot with no drainage holes was flled with 450 g of soil. Tereafer, 100 ml of crude oil was used to pollute the soil and 300 ml of sterile water added biweekly following modifed method *Corresponding author: Ogbulie TE, Department of Biotechnology, School of Science, Federal University of Technology Owerri (FUTO) Imo State, Nigeria, Tel: +2348035472379; E-mail:ogbulie_toochi@yahoo.com Received August 15, 2015; Accepted February 09, 2016; Published February 17, 2016 Citation: Ogbulie TE, Nwaokorie FO (2016) Molecular Diversity of Microbes with Probable Degradative Genes in Agricultural Soil Contaminated with Bonny Light Crude Oil. J Ecosys Ecograph S5: 002. doi: 10.4172/2157-7625.S5-002 Copyright: © 2016 Ogbulie TE, et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Abstract This study looked at the diversity of microorganisms persistent in agricultural soil sample polluted with 100 ml of 100% Nigerian Bonny light crude oil left for four years. DNA from crude oil polluted agricultural soil sample was extraction using ZYMO soil DNA extraction Kit. DNA sequencing was performed by Next Generation Sequencing Technique [NGST] using automated PCR cycle- Genome Sequencer™ FLX System from 454 Life Sciences™ and Roche Applied. Sequence analysis and alignment was performed using Vecton NTI suite 9 (InforMax, Inc.). The resulting nucleotide sequences were compared to sequences obtained from GenBank by BLASTx analysis using CLO Bio software as well as BLASTn using NCBI. Molecular Identities of microbial community was obtained by creating different dendrograms. Gene sequencing carried out read 513 different nucleotide sequences. Seven phyla with 47 corresponding culture-dependent species and 169 culture-independent bacteria clone were obtained. The resultant tree showed cladogram of proteobacteria ( b and g - proteobacteria), bacteria/enterobacteria, frmicutes, plantomycetes, acidobacteria group/ fbrobacteres, Bacteriodetes/chlorobi Actinobacteria/high G + C and chlorifexi phyla. Furher taxonomical classifcation was carried out with reads of suffcient Q scores (> q30) and lengths and a total of 420 read count of top kingdom classifcation of 100% bacteria kingdom was obtained. Proteobacteria phyla of class betaproteobacteria, order Burkholderiales and family Comamonadaceae had the highest read count with percentage diversity of 57.14%, 53.81%, 53.81 and 53.57% respectively. The nucleotide sequences with no hit (208) was sent to Genbank for asigning of ascension number. The detection of these diverse organisms from crude oil polluted agricultural soil left for four years, depict that the organism probably, have degradative genes which aided their survival. Molecular Diversity of Microbes with Probable Degradative Genes in Agricultural Soil Contaminated with Bonny Light Crude Oil Ogbulie TE 1 * and Nwaokorie FO 2 1 Department of Biotechnology, School of Science, Federal University of Technology Owerri (FUTO), Imo State, Nigeria 2 Molecular biology and Biotechnology Division, Nigerian Institute for Medical Research [NIMR], Yaba Lagos State, Nigeria