Characterization Of Microbial Communities And Their Function Involved In Methanogenic Alkane Degradation

Research on anaerobic degradation of petroleum hydrocarbon is currently an important direction, which is of great importance in theoretical study as well as industrial application in remidation of hydrocarbon contamination and recovery of gasification of residual oil.In this thesis, the microbial communities in the hydrocarbon-impacted environments and their evolutions features when incubated with alkanes under different electron acceptors were studied by molecular biological methods. Based on these, the methanogenic alkanes-degrading microcosms were constructed and the mechanism of anaerobic alkane degradation as well as methanogenic consortia were analyzed.The clone libraries used bacterial 16S rRNA gene-based approaches to identify the dominant bacteria present in 7 different petroleum reservoirs and the data showed that bacterial sequences affiliated with the y-Proteobacteria were the most frequently detected sequence type (37% of sequences) and identified in all 7 bacterial 16S rRNA libraries. Firmiucutes were detected at the second highest frequency (20% of sequences) and identified in 6 out of the 7 libraries included 2 of high-temprature petroleum reservoirs. The canonical correspondence analysis (CCA) of the distribution of bacterial communities and the environmental variables of the petroleum reservoirs indicated that the bacterial type was effected by temperature, concentration of Cl- and mineralization degree. Analysis of 6 archaeal 16S rRNA clone libraries showed that most of sequences affiliated with methanogens and CO2-reducing methanogens being most commonly encountered among studied of petroleum reservoirs archaea as well as all the methanogens show temperature dependent growth and activity. The canonical correspondence analysis (CCA) of the distribution of archaeal communities and the environmental variables of the petroleum reservoirs indicated that most of the aechaeal type had positive correlation with temperature. In addition, analysis of 1 bacterial 16S rRNA clone libraries retrieved from the subsurface hydrocarbon-impacted environments (oily sludge) and the data showed that?-Proteobacteria and Chloroflexi were the dominant detected sequence type, accounted for 47% and 28% of the total clone library, respectively. Analysis of archaeal 16S rRNA clone libraries showed that methytrophic and CO2-reducing methanogens reprensented by the genus Methanomethylovorans and Methanolinea, respectively were detected in oily sludge. Furthermore, catabolic genes encoding benzylsuccinate synthase (bssA) were detected in 1 petroleum reservoir and genes encoding alkylsuccinate synthase (assA) were detected in 3 petroleum reservoir.An alkane-degrading enrichment cultures from production water were established under methanogenic, sulfate-reducing and denitrifying conditions. GC-MS analysis of the n-alkanes (C15-C20) profile revealed that concentrations of all alkanes were reduced in the three active enrichment cultures incubated at 37?and about 77?mol of net methane were produced from the methanogenic cultures after one year of incubation. Phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichment microcosms indicated that?-Proteobacteria,?-Proteobacteria,?-Proteobacteria and Bacteroidetes were identified in the nitrate-reducing n-alkanes degrading enrichment; Actinobacteria and Nitrospira were recovered in sulfate-reducing and methanogenic n-alkanes degrading enrichment while Actinobacteria was the most abundant in methanogenic n-alkanes degrading enrichment and Nitrospira were the most abundant in sulfate-reducing n-alkanes degrading enrichment. These results showed that electron acceptors have a strong influence on the microbial community composition. Gene encoding the alkylsuccinate synthase (assA) and methyl coenzyme-M reductase (mcrA) were amplified and the results suggested that fumarate addition and methanogenisis were involved in the degradation of n-alkanes.An alkanes-degrading methanogenic enrichment culture from production water was established and analyzed. Results showed that a net 538 mmol of methane higher than the controls were produced over 274 days of incubation in microcosms amended with alkanes at 37?. A decrease in the alkanes profile was observed by GC-MS analyzed and the methane percent of the obseverd to theoretical production was 5.7%. Phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichment microcosms indicated that the bacterial clone library was composed of sequences affiliated with the Firmicutes, Proteobacteria, Deferribacteres, and Bacteroidetes. The archaeal phylotypes were mostly related to members of the orders Methanosarcinales and Methanobacteriales.An active methanogenic n-alkane (C15-C20) degrading microbial consortium enriched from oily sludge was successfully established for over a total of 1000 days of incubation at 37?. Total genomic DNAs were extracted from three samples including the original oil sludge, the sludge incubated for 500-days under methanogenic condition without any addition of carbon sources, and the enrichment cultures after exhaustion of the indigenous carbon from the sludge and amended with n-alkanes and incubated for another 500 days. Phylogenetic diversity of microbial communities of the three samples was assayed by amplification of 16S rRNA gene. At the same time, catabolic genes encoding benzyl succinate synthase (bssA) were detected only in the sludge incubated for 500-days under methanogenic condition without any addition of carbon sources, whereas genes encoding alkyl succinate synthase (assA) could only be detected in the enrichment cultures after exhaustion of the indigenous carbon from the sludge and amended with n-alkanes and incubated for another 500 days. These results provide important evidence in that carbon source available had a profound influence on microbial community composition at different stages; resulting in a shift from methanogenic aromatic hydrocarbon degradation to an alkane-degrading community.Through studing the microbial communities and their evolutions features when incubated with alkanes under different electron acceptors as well as the mechanism of anaerobic alkane degradation derived from representative hydrocarbon-impacted environments of different temperatures, the knowledge of the regulation of the anaerobic alkane degradation was enriched, which play an important role in theoretical study and industrial application in remidation of hydrocarbon contamination and recovery of gasification of residual oil.