PLASMID ANALYSIS OF HYDROCARBON UTILIZING MICROORGANISMS IN ESIT EKET L.G.A- AKWA IBOM STATE

CHAPTER ONE
INTRODUCTION
1.1 Background of the Study
Crude oil is a mixture of compounds such as organic & inorganic including aliphatic and aromatic hydrocarbon. (Edema,2012). Soils which are contaminated by hydrocarbons expose an extensive damage to local ecosystems as accumulation of pollutants in animals and plants tissues can cause death or mutation (Alvarez and Vogel 1991). We need to prevent from expansion of contamination and also clean up the contaminated areas. Various methods, including bioremediation, can be used for this purpose (Kristanti et al. 2011). The ability of microorganisms to utilize hydrocarbons in oil-contaminated environments has been documented. (Adekunle and Adebambo 2007, Atlas 1981,Ashraf and Ali 2006). Bacteria are usually the dominant degraders in aquatic system and can utilize the most recalcitrant petroleum hydrocarbon, but, Several fungi have been found to exhibit greater hydrocarbon biodegradability than bacteria (Cemiglia and Perry 1973). The advantages associated with fungal bioremediation lay primarily in versatility of the technology and its cost efficiency as compared to other remediation technologies such as incineration, thermal desorption and extraction. (George-Okafor 2009). The use of fungi is expected to be relatively economical as they can be grown on a number of inexpensive agricultural or forest wastes such as corncobs and sawdust. Moreover, their utilization is a gentle non-aggressive approach. Species of many fugal genera are known to metabolize hydrocarbons and thrive in oil-contaminated sites and the most common fungi which have been recorded as biodegraders belong to the genera: Alternaria, Amorphoteca, Aspergillus, Candida, Cephalosporium, Cladosporium,Fusarium, Geotrichum, Graphium, Mucor, Paecilomyces, Penicillium, Rhizopus, Rhodotorula, Sphaeropsidales, Talaromyces and Trichoderma. (Dawoodi and Madani 2015).
Niger Delta Region of Nigeria is one of the top countries in the world with huge oil and gas reserve. The exploration of this crude resource gives rise to hydrocarbon pollution through routine oil operation, vehicular accident and illegal petroleum refining activities. The latter is currently the leading source of oil pollution in the region and the likelihood of putting a stop to this act is very slim because of the economic benefits enjoyed by the perpetrators and their collaborators. Oil spillage in an oil producing community is inevitable and the Niger Delta is a classical example.
Oil products are continually used as a main source of energy in industry and daily life. Release of hydrocarbons into the environment, whether accidentally or due to anthropogenic activities, is a major cause of water and soil contamination. As dependency to oil is increasing, the allied problems are becoming more and more cumbersome. Soils which are contaminated by hydrocarbons expose an extensive damage to local ecosystems as accumulation of pollutants in animals and plants tissues can cause death or mutation. We need to prevent our environments from expansion of contamination and also clean up the contaminated areas. Various methods, including bioremediation, can be used for this purpose. The ability of microorganisms to utilize hydrocarbons in oil-contaminated environments has been documented revealing the fact that several fungi have been found to exhibit greater hydrocarbon biodegradability than bacteria thereby expanding our specs to microrganisms as a whole.
Plasmid is a small DNA molecule found mainly in bacteria that is physically separated from a double stranded chromosomal DNA and can replicate independently. (Actis et al., 2009). Plasmids are mobile genetic elements capable of independent replication from host chromosome. Furthermore, they play prominent roles in prokaryote evolution and can be transferred vertically and horizontally to other prokaryotes (Obayori and Salam, 2010; Koonin and Wolf, 2008). There is a proposition that the development of the resistant or degradative population in a polluted site can lead to gene transfers via transposons, plasmids and possibly spontaneous mutants (Actiset al., 1999).
Their host cell for its survival does not usually require plasmids. Instead, they carry genes that confer a selective/specific advantage on their host, such as resistance to heavy metals, toxic substances, antibiotic or resistance to naturally made antibiotics by other organisms (Actis et al., 1999, Anna et al., 2008).Many bacterial strains have genetic determinants of resistance or degradative abilities to pollutants. These determinants are often found on plasmids, chromosomes and transposons. These degradative abilities occur in high frequencies with much greater quantitative prominence after pollution(Shuklaet al., 2006). Loss of plasmid does not lead to loss of degradative ability (Akpeet al., 2013). Furthermore, due to their stable inheritance, chromosomes are favoured location for functioning essential genes (Tazzyman and Bonhoeffer, 2014). Plasmid analysis therefore involves the use of DNA constituents found in microorganisms to characterize and analyse their hydrocarbon utilization effects in contaminated environments.
Statement of the Problem
The life activity of hydrocarbon utilizing micro-organisms occuring in soil samples has significant influence on its chemical composition and physical- chemical properties, and as a result often changes its biological content. This changes often causes damage to the soil nature due to the hydrocarbon content of the soil thereby attracting micro-organisms which utilize them in their elastic form. Micro-organisms in turn pose risks, multiply and become uncontrollable in those contaminated areas.
Aim and Objectives
The main aim of this work is to isolate crude oil utilizing/degrading bacteria and profile their plasmid for alkane monogenase gene.
The objectives include:
To determine the total hydrocarbon heterotrophic counts.
To extract the genomic and plasmid DNA from bacteria isolates.
To isolate, characterize the crude oil utilizing bacteria (HUB and HUF).
Gel electrophoresis of the plasmid DNA.

Justification of the study
Soil samples contaminated with hydrocarbons examined from the river in Esit Eket will possibly harbor bacteria of possible biodegradation importance(Kastner,1994). Per this research, the following bacterial species: Pseudomonas spp., Bacillus spp., Staphylococcus spp. and Streptococcus spp. will be isolated by plasmid analysis and found to be able to utilize and/or degrade spent engine oil as energy source. It is, however, evident from the outcome of this study that hydrocarbon utilizing and/or degrading microorganisms could readily be isolated from oil contaminated soil samples in the river and possibly used for bioremediation of hydrocarbon polluted soil.