Microbial Communities Composition And Dynamics In Methanogenic Long Chain N-Alkanes Degrading Enrichment Cultures

As an important fraction of petroleum hydrocarbons, alkanes have been shown to be preferentially consumed by microorganisms under methanogenic conditions. Methanogenic conversion of alkane has implication for oil recovery from marginal petroleum reservoirs and for the bioremediation oil-contaminated environments. The objective of the present study was the establishment of long chain alkane-dependent methanogenic enrichment cultures derived from oil reservoir production fluids and from oily sludge. Molecular tools were used to reveal the microbial communities composition and dynamics in the methanogenic enrichment cultures as well as the biochemical mechanisms likely involved in the methanogenic conversion of alkanes. This study is of a fundamental interest for the understanding of complex microbial associations involved in energy recovery from oil reservoirs via residual oil conversion to methane gas.In the present study, high temperature oil reservoir fluids were used as inoculums for the preparation of methanogenic enrichment cultures incubated at 55℃with long chain n-alkanes (C15-C20).Following a lag phase of approximately five months, higher amounts of methane were formed in the cultures amended with n-alkanes reaching an average of 141μmol at day 749. Samples withdrawn at different incubations time from the n-alkanes-amended methanogenic cultures were subjected to molecular phylogenetic analysis with respect to 16S rRNA gene clone libraries construction. Results shows that the bacterial clones'library was predominantly composed by members of the Firmicutes respectively related to the genera Moorella and Gelria. At day 154, the bacterial clones'library was dominated (-79% of all bacterial clones) by Moorella spp. whereas at the end of the incubation period on day 749, Moorella spp. decreased to reach approximately 44% of all bacterial clones and Gelria spp. emerged to represent the most abundant (53%) bacterial constituents within the library. The archaeal clones'library was, at earlier incubations stage (day 154), mainly composed by thermophilic methylotrophic methanogens affiliated with the genus Methanomethylovorans and unclassified members of the phylum Euryarchaeota. A dynamic shift in the archaeal community was observed on day 749 with the emergence of Thermoprotei-like organisms within the phylum Crenarchaeota and CO2-reducing methanogens closely related to Methanothermobacter spp. Most of the above described organisms are thermophilic anaerobes with fermentative and/or syntrophic capabilities. Interestingly, clone libraries analysis revealed that specific microorganisms that were not detected in the control incubations were exclusively enriched in the n-alkanes-amended methanogenic cultures. In addition, genes encoding the candidate alkylsuccinate synthase a-subunit (assA) were detected in the genomic DNAs retrieved from the n-alkanes-amended thermophilic methanogenic cultures with two clones (detected at day 749) highly similar to assAl from the well characterized anaerobe, alkanes degrader Desulfatibacillum alkenivorans AK-01. Results show that alkanes were most likely activated by addition to fumarate and subsequently biodegraded by members of the Firmicutes and Crenarchaeotes into methanogenic precursors (acetate, formate and H2). Acetate would have undergone syntrophic oxidation to H2 and CO2 followed by methanogenesis from CO2-reduction.On the other hand, mesophilic (37℃) methanogenic enrichment cultures established from oil refinery sludge and supplemented with the n-alkanes mixture described above was also investigated. Methane was quickly formed in the headspace of the serum bottles before remaining almost constant at 300μmol after 152 days of incubation. Molecular phylogenetic analyses showed that the microbial communities, in which majority of constituent's members were almost anaerobes thriving at mesophilic temperatures, were highly rich and diverse. The bacterial clones'libraries were essentially composed by fermentative and syntrophic microorganisms related to the Actinobacteria, Bacteroidetes, Chloroflexi, Firmicutes, Proteobacteria and Synergistetes. Microorganisms such as Acidaminobacter sp., Syntrophomonas sp., Veillonellaceae sp., Desulfomicrobium sp. and Anaerobaculum sp. that were not identified at earlier incubations stage (day 152) were exclusively detected in the n-alkanes-amended methanogenic cultures at day 850. The archaeal clones'libraries were, at day 152, predominantly composed by strictly acetoclastic methanogens of the genus Methanosaeta and some members affiliated to hyperthermophilic sulfidogenic archaea of the genus Archaeoglobus. More than 800 days later,90% of archaeal clones were related to Methanosaeta sp. and a few members clustering with the hydrogenotrophic Methanospirillum sp. In the methanogenic cultures supplemented with n-alkanes, a fragment of assA gene closely related to assAl from Desulfatibacillum alkenivorans AK-01 was exclusevily detected whereas the majority of assA genes found in other samples were mostly related to assA genes from oil-contaminated environments. Results show that methane generated in the setting was a result of the cooperation between fermentative bacteria, syntrophic bacteria and aceticlastic methanogens.