[Isolation of functional bacteria guided by PCR-DGGE technology from high temperature petroleum reservoirs].

ABSTRACT

It is a brand-new method to isolate functional bacteria from high temperature petroleum reservoirs according to the sequence information obtained from PCR-DGGE patterns. Three-set primers of 16S rDNA high variable region, V3, V8, V9, were compared. The results showed that more microbial diversity information could be obtained from the PCR product of V9 region. Sequence analysis indicated that the dominant bacteria in the petroleum reservoir had high sequence similarity with bacteria from alpha, beta, gamma-Proteobacterias and Bacilli from the GenBank database. According to the sequences information, multi-cultivation technology including enrichment cultivation, special cultivation and direct cultivation methods were employed, and finally, five strains (three strains by traditional methods) were isolated from oil-water samples. Among them, three thermophilic hydrocarbon-degrading bacteria, which belonged to Bacillus sp., Geobacillus sp. and Petrobacter sp., respectively, could grow well under 55 degrees C in obligate anaerobic condition. The crude oil could be utilized by these strains with the degradation rate of 56.5%, 70.01% and 31.78% respectively along with the viscosity reduction rate of 40%, 54.55% and 29.09%, meanwhile the solidify points of crude oil were reduced by 3.7, 5.2 and 3.1 degrees C. Therefore, the combination of sequence information from PCR-DGGE and altering cultivation conditions is an available novel method to isolate more functional microorganisms which could be utilized for microbial enhanced oil recovery.