Microbial Diversity In Crude Oil-associated Environments And Thermophilic Hydrocarbon-dependent Methanogenesis From Production Water Of High Temperature Petroleum Reservoir

The anaerobic biodegradation of alkanes, which has now been documented to occur in anoxic habitats, is of great importance for the oil industry. Today, this process remains an exciting area of investigation to understand the factors that govern the biodegradation of oil in deep-subsurface reservoirs, and for the bioremediation of oil-contaminated environments. Over the past two decades, significant advances have been made in the understanding of the anaerobic biodegradability of alkanes in terms of the microorganisms involved and the biochemical pathways. Alkanes can be used as sole carbon and energy sources by different physiological groups of microorganisms grown under the reducing conditions prevailing in anoxic environments.In this study, samples collected from high-temperature oil reservoir were used to establish methanogenic enrichment cultures capable of utilizing crude oil components. After long term incubations with subsequent production of methane, the methanogenic enrichment cultures were analyzed by means of 16S rRNA gene clone library. Results indicated that production water incubated with a mixture of long chain rc-alkanes generated approximately 400μmol of methane above the control incubations. Stoichiometric estimation showed that alkane-dependent methanogenesis accounted for about 19.8% of the total of the total amount of methane predicted. Phylogenetic analysis of the methanogenic population revealed that the bacterial community was dominated by members of the Firmicutes closely related to the genus Coprothermobacter. Other prominent constituents were bacterial clones affiliating with the syntrophic acetogen Moorella thermoacetica as well as members of the Thermotoga. Also represented were phylotypes related to Thermodesulfovibrio (Nitrospira), Actinobacteria, Candidate Division OP8 and several others without cultured representative. The archaeal community was predominantly represented by CO2-reducing methanogens in addition to phylotypes clustering with the sulfate-reducer, hyperthermophilic archeon Archaeoglobus sp. Hydrogen was occasionally detected together with methane. Analysis of volatile fatty acids resulted in the detection of acetate and formate. Besides, for the first time, genetic biomarkers associated with the initial activation of alkanes under anaerobic conditions were detected in thermophilic alkane-degrading methanogenic enrichment. In a parallel study, control incubations that were not supplemented with alkanes were used to established crude oil-degrading methanogenic consortium. Following a lag phase of about 45 days, methane started to be produced in the transferred incubations with oil. Methane accumulated in the headspace to reach 135.30μmol at day 550. This amount corresponds to 14.6% of theoretical predicted methane if the mass of n-alkanes (C9-C33) consumed was totally converted to methane. No obvious methane was observed in the control incubations without oil. This observation indicates that there was almost no organic carbon left in the primary methanogenic enrichment. Low concentrations of hydrogen were detected in the gas phase. Acetate, formate, propionate and butyrate were detected from the analysis of organic acids. Phylogenetic analysis revealed that the bacterial population in the oil-degrading enrichment was essentially represented by members of the Chloroflexi, Thermotoga, Actinobacteria, Nitrospira and Firmicutes, whereas the archaeal community was dominated by phylotypes related to the phylum Euryarchaeota that essentially included Archaeoglobus-like members as well as Methanothermobacter spp. Results suggested that oil alkanes were principally degraded to acetate coupled to syntrophic acetate oxidation and methanogenesis from CO2-reduction.The work presented in this thesis is of fundamental interest for the understanding of the anaerobic processes associated with high-temperature petroleum reservoir. In addition, it has demonstrated that genetic biomarkers of anaerobic hydrocarbon degradation that have been identified through laboratory studies enrich the database of existing genes encoding the alkylsuccinate synthase. These biomarkers can also be applied for evaluation of potential in-situ anaerobic degradation of petroleum hydrocarbons in high-temperature oil reservoirs as well as in oil contaminated sites.