Bio-refining of crude oil using microorganisms isolated from petroleum refinery waste water

M.Tech. (Biotechnology)Abstract: The petroleum industry contributes significantly in greenhouse gas (GHG) emissions such as carbon dioxide which amounts to 97.67% of the total GHG emission during production while methane contributes about 2.25% and nitrous oxide contributes about 0.08%. Greenhouse gases are a major cause for drastic climate changes facing the world today. The effects of climate change are quite visible as the world is experiencing an increase in the average global temperature, accelerated melting of icebergs during summer and unusual precipitation patterns. These effects of climate change may affect the ecosystems as their effects are life threating. The aim of this study was to isolate and identify microorganisms that inhabit petroleum refinery waste water and to assess their ability to hydrolyse complex hydrocarbons present in crude oil to simpler hydrocarbons found in refined petroleum. Five bacterial strains were isolated using serial dilutions, spread and streaked plate techniques. Colonies were selected based on colony morphology and Gram stain characteristics. Characterization of bacteria was accomplished by 16S rRNA gene sequencing. According to sequencing data, the isolates were identified as Bacillus thuringiensis, Staphylococcus warneri, Citrobacter amalonaticus, Acinetobacter tandoii and Bacillus sp. These microorganisms were inoculated in minimal salt media containing 5% of crude oil at pH 7 and temperature 30oC at 140 rpm in a shaking incubator in order to study microbial degradation patterns.. The biodegradation studies were carried over a period of five days and a volume of 1mL of sample was aseptically drawn from the flasks on each day of the incubation period. Bacterial cells were removed using 0.22 μm filters. The breakdown products of crude oil were solubilized by adding 1ml of chloroform into 1mL of the sample. Biodegradation products were analysed by GCxGC-ToF-MS and show that all the isolates were capable of hydrolysing the crude oil. A. tandoii was the most efficient microorganism amongst the 4 isolates as it was the only isolate that was able to transform high molecular weight hydrocarbons (C30 and above) and produced significant amounts of new compounds whereas C. amalonaticus and S. warneri could only transform short and medium chain hydrocarbons (C1-C20), B. thuringiensis was able to transform compounds with chain lengths ranging between C1 and C30. From these observations A. tandoii and B. thuringiensis would be more applicable in bio-refining of crude oil as they are able to transform medium and heavy molecular weight hydrocarbons into short chain hydrocarbons which are normally found in refined petroleum hydrocarbons such as gasoline, kerosene and diesel fuel