Isolation and determination of biotechnological potentials of petroleum and hydrocarbon degrading bacteria

Petroleum is the most common environmental pollutant in the world. Oil pollutants pose a serious threat to fisheries, marine habitats, halobionts and human health, and destroy the ecological balance that can last for years or even decades. In this study, by isolating bacteria from oil-contaminated areas in the Batman region, these oil-degrading bacteria were identified by performing morphological, physiological, biochemical and phylogenetic analyzes on the varieties. In addition, studies were carried out on the capacity of these bacteria to use crude oil and various hydrocarbons as energy sources, their ability to break down hydrocarbons, and the determination of genes involved in biodegradation. Eight bacteria belonging to Pseudomonas, Enterobacter, Delftia, Acinetobacter genera were isolated from oil-contaminated areas of Batman oil fields. Phenotypic analyzes of these isolated bacteria were carried out and according to the 16S rRNA gene sequence analysis, the bacteria designated as D7, D8, D9, D10, D11, D12, E1 and E2 were found to be similar as 100% to Pseudomonas aeruginosa, 100% to Enterobacter ludwigii, 100% to Delftia tsuruhatensis, 100% to Pseudomonas putida, 99,93% to Acinetobacter pittii, 100% to Pseudomonas rhodesiae, 100% to Acinetobacter pittii and 100% to Acinetobacter calcoaceticus, respectively. It has been determined that all bacterial strains studied use crude oil as a carbon and energy source. It has been observed that bacteria grow well from aliphatic hydrocarbons of different lengths, only in long chain n-alkanes (pentadecane, hexadecane) and decane. Among the long chain alkanes tested, hexadecane was the most preferred by bacteria overall. After five days of incubation, GC-MS analysis is performed and the order of crude oil degradation capacity by bacteria from high to low were as follows; D11, D8, D12, D10, D9, E2 and E1 degraded 43%, 27%, 21%, 18%, 12%, 12% and 9%, respectively. After three days of incubation, the rate of degradation of 1% alkane hydrocarbons by bacteria was determined by GC-MS analysis. It has been determined that 1% hexadecane decomposes approximately 65% by D9, 70% by D11, 38% by D12 and 76% by E1. Pentadecane was found to be decomposed 3% by D7, 29% by D8, and 48% by E2. In addition, as a result of three days of incubation, the rate of bacterial degradation of hydrocarbon mixtures (total 1%) consisting of hexadecane and pentadecane, singly or as a mixture, was determined. While individually D8, D11 and E2 degraded 31%, 43% and 53% respectively, in mixtures biodegradation of hydrocarbon mixtures was as high as 60% by D8+D11, 49% by D11+E2, 61% by D8+E2 and 72% by D8+D11+E2. The results of the sequence analysis of the alkM gene of PCR products obtained using the first primer (primer 1) designed for D11 and E2 strains showed 93,06% and 92,72% alkM gene similarity to that of Acinetobacter sp. BUU8 (A6N7F9), respectively. The sequence analysis of the alkb gene PCR products using the other primer (primer 3) designed for D11and E2 strains showed 94,48% and %95,52% alkB gene similarity to that of Psudomonas citronellolis (A0A024A3Z2) and Pseudomonas mendocina (A4XPE8), respectively.