| Research on methanogenic biodegradation of petroleum hydrocarbons plays an important role in understanding the anaerobic biodegradation processes of petroleum hydrocarbons in subsurface oil reservoirs, microbial remediation of oil-contaminated environments and residual oil conversion to natural gas. Presently, research on methanogenic biodegradation of hydrocarbons is mostly based on laboratory enriched cultures. However, reports on microbial community composition and dynamics following long-term incubation are rather scarce. In the present study, two n-alkane-dependent methanogenic consortia incubated with different inoculums (oil reservoir production water and activated oily sludge) were successfully established. Using molecular and chemical tools, community composition, functional genes and metabolites of the two consortia were analyzed; community dynamics and putative mechanisms involved were also discussed.A two-step synthesis of specifically deuterated n-alkanes from carboxylic acids was described. Through H/D exchange and Kolbe electrolysis,8,8,9,9-d4-hexadecane was prepared from nonanoic acid under optimum reaction conditions. The synthetic product can be used as a model substrate to provide information about the transformation mechanisms involved in anaerobic biodegradation of hydrocarbons. This method might provide an alternative route for the preparation of specifically deuterated alkanes in which deuterium atoms are located at two adjacent carbons of the alkane's carbon chain.The n-alkane-dependent methanogenic consortium enriched from thermophilic oilfield fluid incubated at 55℃with a mixture of long chain n-alkanes (C15-C20) was characterized. Following 749 days of incubation, biodegradation of n-alkanes was observed as methane production in the alkanes-amended methanogenic enrichment reached 141μmol above the controls, corresponding to 17.1% of theoretically predicted. GC-MS analysis confirmed the presence of putative downstream metabolites, including linear fatty acids, volatile fatty acids and fumarate, probably from the anaerobic biodegradation of n-alkanes and indicating an incomplete conversion of the n-alkanes to methane.16S rRNA gene clone libraries showed that the alkanes-dependent community was dynamic during the incubation period. The dominant bacterial species were affiliated with Firmicutes members clustering with thermophilic syntrophic bacteria of the genera Moorella sp. and Gelria sp. The archaeal community was predominantly represented by members of the phyla Crenarchaeotes and hydrogenotrophic methanogens. On the other hand, PCR amplification for detection of functional genes encoding the alkylsuccinate synthase a-subunit (assA) in the enrichment cultures resulted positively. Interestingly, the appearance of a new assA gene sequence identified on day 749 supported the establishment of a functioning microbial species in the enrichment. Therefore, the biochemical degradation pathways that could have been involved in the anaerobic conversion of n-alkanes to methane were proposed. n-Alkanes were most likely activated by addition to fumarate and subsequently biodegraded via fatty acids by members of the Firmicutes and Crenarchaeotes into formate, acetate, H2 and CO2 which in turn were consumed by acetoclastic and hydrogenotrophic methanogens into methane, among which CO2-reducing methanogenesis was the dominat pathway.The n-alkane-dependent methanogenic consortium enriched from activated oily sludge incubated at 37℃with a mixture of long chain n-alkanes (C15-C20) was analyzed. In the alkane-amended enrichment cultures,37.3% amount of n-alkanes was consumed and 314μmol of methane was produced. Molecular analysis showed that the alkanes-dependent community was dynamic during incubation. The dominant bacterial species were affiliated with Clostridia,Actinobacteria,Synergistaceae and Chloroflexi, while the dominant archaeal species were acetoclastic methanogens. The transferred cultures incubated with different n-alkanes were all observed with methane production and n-alkanes biodegradation. Especially in the transferred cultures incubated with 8,8,9,9-d4-hexadecane,107μmol of methane above the controls was produced, corresponding to 83.6% of the theoretically predicted, indicating that the 37℃activated oily sludge consortium was capable of converting n-alkanes to methane. The assA-like genes were positively amplified in all the cultures in accordance with the identification of Deltaproteobacteria phylotypes. After 660 days of incubation, all the bacteria phylotypes of transferred cultures were dominated by Chloroflexi,Synergistaceae,Actinobacteria and Deltaproteobacteria, and the archaeal community was predominantly represented by acetoclastic methanogens, except that hydrogenotrophic methanogens dominanted the transferred cultures incubated with 8,8,9,9-d4-hexadecane. In the transferred cultures incubated with 8,8,9,9-d4-hexadecane, n-alkanes were most likely activated by addition to fumarate, and subsequently syntrophically and fermentatively biodegraded into acetate which in turn was consumed by acetoclastic and hydrogenotrophic methanogens into methane, among which CO2-reducing methanogenesis was the dominant way.The analysis of the two n-alkane-dependent methanogenic consortia mentioned above showed that, there were some differences between the microbial community compositions. This suggested that different sampling sources, substrates and culture conditions will have an important influence on community structure, and probably on the conversion capability of n-alkanes to methane. The present study have enriched the microbial diversity of anaerobic hydrocarbons biodegradation, and enhanced the understanding of n-alkane-dependent methanogenic community dynamics and functional genes. It also plays an important role in understanding the mechanisms involved, screening for functional microbes, and monitoring microbial activities in oil-rich ecosystems.
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