Evaluation of the use of amplified 16S rRNA gene-restriction fragment length polymorphism analysis to detect Enterobacter cloacae and Bacillus licheniformis for microbial enhanced oil recovery field pilot

Evaluation of effectiveness of restriction fragment length polymorphism (RF LP) analysis of the 16S rRNA gene of microorganisms injected into an oil re servoir, for monitoring their levels over time, was conducted. Two microorg anisms, Enterobacter cloacae TRC-322 and Bacillus licheniformis TRC-18-2-a, were focused in this. paper among the microorganisms selected for injectio n, and gene fragments of the 16S rRNA gene of these microorganisms were amp lified by polymerase chain reaction (PCR), using one set of universal prime rs. Samples of the reservoir brine and reservoir rock were obtained; the mi croorganisms inhabiting in the reservoir were isolated from these samples, and the 16S rRNA gene of these microorganisms war; amplified, condition rem aining the same. RFLP analysis was performed on the 16S rRNA gene of each o f those microorganisms, using restriction endonucleases Hha1, MspI, AluI an d TaqI as necessary. Comparison of the resultant rRNA gene fragments, demon strated that closely-related species displaying RFLP profile similar to tha t of E, cloacae TRC-322 or B, licheniformis TRC-18-2-a were not among the m icroorganisms isolated from the reservoir. PCR-RFLP analysis of the 16S rRN A gene, using the protocol presented in this paper, is effective to detect the presence of appropriate injecting microorganisms. This method was also effective for studying microorganisms isolated from the reservoir, which ha ve the ability to grow on a molasses.