Metabolic capability and in situ activity of microorganisms in an oil reservoir

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Methanogenic Microbial Community Composition of Oily Sludge and Its Enrichment Amended with Alkanes Incubated for Over 500 Days

Methanogenic microbial community is responsive to the availability of hydrocarbons and such information is critical for the assessment of hydrocarbon degradation in remediation and also in biologically enhanced recovery of energy from non-producing oil reserves. In this study, methanogenic enrichment cultures from oily sludge amended with n-alkanes (C 15-C 20) showed a development of active methanogenic alkanes-degrading consortium for over a total of 1000 days of incubation at 37°C. Total genomic DNAs were extracted from three types of samples, the original oily sludge (OS), the sludge after incubation for 500 days under methanogenic condition without any external carbon addition (EC), and the enrichment culture from the EC amended with n-alkanes (ET) incubated for another 500 days. The phylogenetic diversities of microbial communities of the three samples were analyzed by PCR amplification of partial 16S rRNA genes. The catabolic genes encoding benzylsuccinate synthase (bssA) and alkylsuccinate synthase (assA) were also examined by PCR amplification. These results provide important evidence in that microbial populations in an oily sludge shifted from methanogenic aromatic compounds degrading communities to potential methanogenic alkane-degrading communities when the enrichment was supplemented with n-alkanes and incubated under anaerobic conditions. © 2012 Copyright Taylor and Francis Group, LLC.link_to_subscribed_fulltex

Microbial communities involved in anaerobic degradation of alkanes

Saturated hydrocarbons are quantitatively the most abundant fraction among all petroleum hydrocarbons. Significant advances have been made in the understanding of the anaerobic biodegradability of alkanes in terms of the microorganisms involved and the biochemical pathways over the past two decades. They can be used as carbon and energy sources by diverse physiological groups of microorganisms (isolates or consortia) grown under chlorate-reducing, nitrate-reducing, sufidogenic or methanogenic conditions. Two general biochemical mechanisms have been proposed for the initial activation of alkanes including addition of fumarate and carboxylation. However, glycyl radical enzymes dependent fumarate addition which yields alkyl-substituted succinate appear to be the most commonly shared mechanism for the anaerobic attack of alkanes under various redox conditions by phylogenetically diverse microorganisms. The genes encoding the candidate alkylsuccinate synthase have been recently described in alkane-degrading sulfate- and nitrate-reducers as well as in hydrocarbon-rich environments. Alternative mechanisms may also be available depending on the alkane-degrading microbial community and electron acceptors utilized. © 2010 Elsevier Ltd.link_to_subscribed_fulltex

Analyses of n-alkanes degrading community dynamics of a high-temperature methanogenic consortium enriched from production water of a petroleum reservoir by a combination of molecular techniques

Despite the knowledge on anaerobic degradation of hydrocarbons and signature metabolites in the oil reservoirs, little is known about the functioning microbes and the related biochemical pathways involved, especially about the methanogenic communities. In the present study, a methanogenic consortium enriched from high-temperature oil reservoir production water and incubated at 55 °C with a mixture of long chain n-alkanes (C 15-C 20) as the sole carbon and energy sources was characterized. Biodegradation of n-alkanes was observed as methane production in the alkanes-amended methanogenic enrichment reached 141.47 μmol above the controls after 749 days of incubation, corresponding to 17 % of the theoretical total. GC-MS analysis confirmed the presence of putative downstream metabolites probably from the anaerobic biodegradation of n-alkanes and indicating an incomplete conversion of the n-alkanes to methane. Enrichment cultures taken at different incubation times were subjected to microbial community analysis. Both 16S rRNA gene clone libraries and DGGE profiles showed that alkanes-degrading community was dynamic during incubation. The dominant bacterial species in the enrichment cultures were affiliated with Firmicutes members clustering with thermophilic syntrophic bacteria of the genera Moorella sp. and Gelria sp. Other represented within the bacterial community were members of the Leptospiraceae, Thermodesulfobiaceae, Thermotogaceae, Chloroflexi, Bacteroidetes and Candidate Division OP1. The archaeal community was predominantly represented by members of the phyla Crenarchaeota and Euryarchaeota. Corresponding sequences within the Euryarchaeota were associated with methanogens clustering with orders Methanomicrobiales, Methanosarcinales and Methanobacteriales. On the other hand, PCR amplification for detection of functional genes encoding the alkylsuccinate synthase α-subunit (assA) was positive in the enrichment cultures. Moreover, the appearance of a new assA gene sequence identified in day 749 supported the establishment of a functioning microbial species in the enrichment. Our results indicate that n-alkanes are converted to methane slowly by a microbial community enriched from oilfield production water and fumarate addition is most likely the initial activation step of n-alkanes degradation under thermophilic methanogenic conditions. © Springer Science+Business Media, LLC 2012.link_to_subscribed_fulltex

Characterization of an alkane-degrading methanogenic enrichment culture from production water of an oil reservoir after 274 days of incubation

Oil reservoirs represent special habitats for the activity of anaerobic microbial communities in the transformation of organic compounds. To understand the function of microbial communities in oil reservoirs under anaerobic conditions, an alkane-degrading methanogenic enrichment culture was established and analyzed. Results showed that a net 538 μmol of methane higher than the controls were produced over 274 days of incubation in microcosms amended with alkanes and a decrease in the alkanes profile was also observed. Phylogenetic analysis of 16S rRNA gene sequences retrieved from the enrichment microcosms indicated that the archaeal phylotypes were mostly related to members of the orders Methanobacteriales and Methanosarcinales. The bacterial clone library was composed of sequences affiliated with the Firmicutes, Proteobacteria, Deferribacteres, and Bacteroidetes. However, most of the bacterial clones retrieved from the enrichment cultures showed low similarity to 16S rRNA gene sequences of the cultured members, indicating that the enrichment cultures contained novel bacterial species. Though alkane-degrading methanogenic enrichment consortium has rarely been reported from petroleum reservoirs, our results indicated that oilfield production water harbors a microbial community capable of syntrophic conversion of n-alkanes to methane, which sheds light on the bio-utilization of marginal oil reservoirs for enhanced energy recovery. © 2011 Elsevier Ltd.link_to_subscribed_fulltex

Analysis of alkane-dependent methanogenic community derived from production water of a high-temperature petroleum reservoir

Microbial assemblage in an n-alkanes-dependent thermophilic methanogenic enrichment cultures derived from production waters of a high-temperature petroleum reservoir was investigated in this study. Substantially higher amounts of methane were generated from the enrichment cultures incubated at 55 °C for 528 days with a mixture of long-chain n-alkanes (C 15-C 20). Stoichiometric estimation showed that alkanes-dependent methanogenesis accounted for about 19.8% of the total amount of methane expected. Hydrogen was occasionally detected together with methane in the gas phase of the cultures. Chemical analysis of the liquid cultures resulted only in low concentrations of acetate and formate. Phylogenetic analysis of the enrichment revealed the presence of several bacterial taxa related to Firmicutes, Thermodesulfobiaceae, Thermotogaceae, Nitrospiraceae, Dictyoglomaceae, Candidate division OP8 and others without close cultured representatives, and Archaea predominantly related to uncultured members in the order Archaeoglobales and CO 2-reducing methanogens. Screening of genomic DNA retrieved from the alkanesamended enrichment cultures also suggested the presence of new alkylsuccinate synthase alpha-subunit (assA) homologues. These findings suggest the presence of poorly characterized (putative) anaerobic n-alkanes degraders in the thermophilic methanogenic enrichment cultures. Our results indicate that methanogenesis of alkanes under thermophilic condition is likely to proceed via syntrophic acetate and/or formate oxidation linked with hydrogenotrophic methanogenesis. © Springer-Verlag 2012.link_to_subscribed_fulltex

The use of chlorate, nitrate, and perchlorate to promote crude oil mineralization in salt marsh sediments

Due to the high volume of crude oil released by the Deepwater Horizon oil spill, the salt marshes along the gulf coast were contaminated with crude oil. Biodegradation of crude oil in salt marshes is primarily limited by oxygen availability due to the high organic carbon content of the soil, high flux rate of S(2-), and saturated conditions. Chlorate, nitrate, and perchlorate were evaluated for use as electron acceptors in comparison to oxygen by comparing oil transformation and mineralization in mesocosms consisting of oiled salt marsh sediment from an area impacted by the BP Horizon oil spill. Mineralization rates were determined by measuring CO2 production and δ (13)C of the produced CO2 and compared to transformation evaluated by measuring the alkane/hopane ratios over a 4-month period. Total alkane/hopane ratios decreased (~55-70 %) for all treatments in the following relative order: aerated ≈ chlorate \u3e nitrate \u3e perchlorate. Total CO2 produced was similar between treatments ranging from 550-700 mg CO2-C. The δ (13)C-CO2 values generally ranged between the indigenous carbon and oil values (-17 and -27‰, respectively). Oil mineralization was greatest for the aerated treatments and least for the perchlorate amended. Our results indicate that chlorate has a similar potential as oxygen to support oil mineralization in contaminated salt marshes, but nitrate and perchlorate were less effective. The use of chlorate as a means to promote oil mineralization in situ may be a promising means to remediate contaminated salt marshes while preventing unwanted secondary impacts related to nutrient management as in the case of nitrate amendments