Molecular Microbial Ecology Study Of Oil Reservior And Simulated Oil Degrading System

Most of reservoirs in the world have entered the secondary recovery after decades of exploitation, in which water flooding plays a significant role. Water-flooding dramatically improves the efficiency of oil recovery. However, exogenous microorganisms and dissolved oxygen which may affect the microbial structure will enter the reservoir environment flowed with flooded water. For long-term water-flooding reservoirs, whether microbial structure of reservoir will assimilate by that in injection water is still not clear. Otherwise, more than half of crude oil still left in the reservoirs even after the exploit by using various oil recovery methods. Previous studies have proved the potential of conversion of the residual oil into methane by microorganisms, the study of microbial community in reservoir has an important significance on resolving the conflicts of crude oil supply and extending demand.In this study, Gudao petroleum reservoir, which has been water-flooded for over 30 years, was selected. Using denaturing gradient gel electrophoresis (DGGE), 16S rRNA gene clone library and pyrosequencing, we compared the microbial communities of injection well with that of production well. The results showed that microbial composition between injection well and production well were significantly different (P<0.05). It indicated that microbial composition of production well and injection well were not converged during long-term water flooding. Clone library result showed that 96.3% of bacterial operational taxonomic units (OTUs) and 70.6% of archaeal OTUs in injected water cannot be detected from production well. Similar results were obtained by pyrosequencing, more than 80% of OTUs in the injection water, including two of the most predominant OTUs, were difficult to detect in the production wells. UniFrac significance analysis showed that bacterial structures in production wells with the same oil-bearing strata has no significant difference (P>0.05), but bacterial structures were significantly different in the production wells with different oil-bearing strata (P<0.05). This suggests that microbial composition of production well may be closely related with the stratigraphic structure of itself.In the second part, we compared the microbial community structures of three working blocks of Shengli petroleum reservoir (Chengdong, zhansan and Gudao) using bar-coded pyrosequencing. 21 bacterial phyla and 2 archaeal phyla, together with 14.2% bacterial and 26.8% archaeal unclassified sequences were detected from more than 30,000 sequences. Phylogenetic analysis showed that predominant phylotypes in Shengli petroleum reservoir were Proteobacteria, Firmicutes, Bacteroidetes, and methnogens, which were considered as common type of microorganisms in petroleum reservoir. Except that, we also found Aquificae and Fusobacteria and other rarely reported bacteria in oil reservoir environment. UniFrac significance test indicated that bacterial community structure of three working block has no significant difference (P>0.05), but opposite result was found in archaea (P<0.05). Canonical Correspondence Analysis (CCA) showed stronger effect of temperature than mineralization on microbial community, SO42- has greater effect on bacterial community than other ions, but HCO3- has greater effect on archaeal community than others.Finally, we constructed high temperature sulfate reducing and methanogenic anaerobic crude oil degrading systems, investigated the degradation of crude oil and microbial composition of two systems for period of 540 days incubation. The result showed that aromatic hydrocarbon of crude oil was degraded under both conditions and the degradation rate under methanogenic condition was higher than sulfate reducing condition at 60℃. Both DGGE and bar-coded pyrosequencing results showed that the microbial community structures in two systems were different although using the same inoculums, indicating that sulfate has great impact on microbial composition during petroleum degradation process. But in the same culture condition, the structure of archaea changed greater than bacteria over time. Phylogenetic analysis showed that fermentative bacteria, sulfate-reducing bacteria and syntrophic bacteria were detected from both systems. Moreover, there exist two pathways for methane formation in these systems, acetate fermentation and H₂/CO₂ reduction, and the dominant pathway was H₂/CO₂ reduction.In summary, in this study, using both traditional molecular biological methods and pyrosequencing, we proved that microorganisms in the injection well are not the main factor that affects the endogenous ecology although it suffered long time water flooding. In the same oil field, the microbial communities of different working block were different, and the influence of temperature is greater than salinity. Special exogenous microbial community can degrade petroleum hydrocarbons in both sulfate reducing and methanogenic conditions at 60℃, and the degradation rate of aromatic hydrocarbon in methanogenic condition is fast than sulfate reducing condition.