Screening, Isolation And Characterization Of Hydrocarbonoclastic Bacteria From Oil Contaminated Soils

Abstract

Petroleum and its products continue to serve as a principle source of energy for industries and daily life. However, their release into the environment is a worldwide concern since some products are acutely toxic or possess mutagenic, teratogenic and carcinogenic properties. Several oil disposal methods have been applied over time with bioremediation emerging as the most promising technology. It takes advantage of the versatility of soil microbes to degrade hydrocarbon contaminants. Unlike conventional disposal methods, bioremediation is an environmentally friendly and cost effective method that simulates natural processes for complete degradation of hydrocarbons into innocuous compounds. This study focused on isolation, morphological and biochemical characterization as well as molecular identification of bacteria possessing hydrocarbon-degrading properties. The study also aimed at optimizing appropriate culture conditions for the isolates as well as screening for alkane hydroxylase enzyme. Isolation of hydrocarbon degrading microbes from soils polluted with used motor oil around Ngara, Nairobi-Kenya was carried out using Bushnell Haas media supplemented with used engine oil. The isolates were screened for ability to utilize heating oil, hexane, octane, toluene and diesel oil hydrocarbons. Characterization of the isolates was carried out by performing Gram’s iodine and potassium hydroxide as well as starch, catalase and carbohydrate fermentation tests. The isolates were also identified through PCR amplification and sequencing of 16S rDNA gene and comparison of obtained sequences with those retrieved from Genbank database. Optimization of culture conditions of three efficient degraders was performed using diesel oil and cellular growth monitored through biomass determination. Hydrocarbon analysis was performed using GC-MS following culturing in diesel oil. Alkane hydroxylase (alkB) gene was amplified using alk-3F and alk-3R primer pair. Among 21 microbes isolated, nine were selected based on their ability to utilize the hydrocarbons and characterized. The isolates were observed to mineralize heating oil, hexane, octane and toluene as well as diesel oil. PCR amplification of 16S rDNA gene revealed that the nine isolates belong to six different genera; Pseudomonas, Acinetobacter, Klebsiella, Enterobacter, Salmonella and Ochrobactrum. Based on their ability to degrade the hydrocarbons, three isolates were selected and their growth conditions optimized. Optimum degradation of diesel oil was recorded at <1 % substrate concentration, pH 7, temperature of 37 0C and using yeast extract as a nitrogen source. GC-MS analysis of diesel oil degradation demonstrated that the isolates were capable of readily degrading linear, branched, cyclic and isoprenoid alkanes as well as aromatic hydrocarbons with fatty acids, aldehydes and alcohols produced as intermediate metabolites. Isolate 1C was identified as the most efficient hydrocarbon degrader based on utilization of the different hydrocarbons tested. Its alkane hydroxylase gene was successfully amplified indicating the isolate’s potential catabolic capability in degrading alkanes. Overall, the characterized bacterial isolates may constitute potential candidates for biotechnological application in environmental cleanup of petroleum contaminants.