Dongshan An

Metagenomics of Hydrocarbon Resource Environments Indicates Aerobic Taxa and Genes to be Unexpectedly Common

Oil in subsurface reservoirs is biodegraded by resident microbial communities. Water-mediated, anaerobic conversion of hydrocarbons to methane and CO2, catalyzed by syntrophic bacteria and methanogenic archaea, is thought to be one of the dominant processes. We compared 160 microbial community compositions in ten hydrocarbon resource environments (HREs) and sequenced twelve metagenomes to characterize their metabolic potential. Although anaerobic communities were common, cores from oil sands and coal beds had unexpectedly high proportions of aerobic hydrocarbon-degrading bacteria. Likewise, most metagenomes had high proportions of genes for enzymes involved in aerobic hydrocarbon metabolism. Hence, although HREs may have been strictly anaerobic and typically methanogenic for much of their history, this may not hold today for coal beds and for the Alberta oil sands, one of the largest remaining oil reservoirs in the world. This finding may influence strategies to recover energy or chemicals from these HREs by in situ microbial processes.

Roles of Thermophiles and Fungi in Bitumen Degradation in Mostly Cold Oil Sands Outcrops

ABSTRACT Oil sands are surface exposed in river valley outcrops in northeastern Alberta, where flat slabs (tablets) of weathered, bitumen-saturated sandstone can be retrieved from outcrop cliffs or from riverbeds. Although the average yearly surface temperature of this region is low (0.7°C), we found that the temperatures of the exposed surfaces of outcrop cliffs reached 55 to 60°C on sunny summer days, with daily maxima being 27 to 31°C. Analysis of the cooccurrence of taxa derived from pyrosequencing of 16S/18S rRNA genes indicated that an aerobic microbial network of fungi and hydrocarbon-, methane-, or acetate-oxidizing heterotrophic bacteria was present in all cliff tablets. Metagenomic analyses indicated an elevated presence of fungal cytochrome P450 monooxygenases in these samples. This network was distinct from the heterotrophic community found in riverbeds, which included fewer fungi. A subset of cliff tablets had a network of anaerobic and/or thermophilic taxa, including methanogens, Firmicutes, and Thermotogae, in the center. Long-term aerobic incubation of outcrop samples at 55°C gave a thermophilic microbial community. Analysis of residual bitumen with a Fourier transform ion cyclotron resonance mass spectrometer indicated that aerobic degradation proceeded at 55°C but not at 4°C. Little anaerobic degradation was observed. These results indicate that bitumen degradation on outcrop surfaces is a largely aerobic process with a minor anaerobic contribution and is catalyzed by a consortium of bacteria and fungi. Bitumen degradation is stimulated by periodic high temperatures on outcrop cliffs, which cause significant decreases in bitumen viscosity.

Metagenomic analysis indicates Epsilonproteobacteria as a potential cause of microbial corrosion in pipelines injected with bisulfite

Sodium bisulfite (SBS) is used as an oxygen scavenger to decrease corrosion in pipelines transporting brackish subsurface water used in the production of bitumen by steam-assisted gravity drainage. Sequencing 16S rRNA gene amplicons has indicated that SBS addition increased the fraction of the sulfate-reducing bacteria (SRB) Desulfomicrobium, as well as of Desulfocapsa, which can also grow by disproportionating sulfite into sulfide, sulfur, and sulfate. SRB use cathodic H2, formed by reduction of aqueous protons at the iron surface, or use low potential electrons from iron and aqueous protons directly for sulfate reduction. In order to reveal the effects of SBS treatment in more detail, metagenomic analysis was performed with pipe-associated solids (PAS) scraped from a pipe section upstream (PAS-616P) and downstream (PAS-821TP) of the SBS injection point. A major SBS-induced change in microbial community composition and in affiliated hynL genes for the large subunit of [NiFe] hydrogenase was the appearance of sulfur-metabolizing Epsilonproteobacteria of the genera Sulfuricurvum and Sulfurovum. These are chemolithotrophs, which oxidize sulfide or sulfur with O2 or reduce sulfur with H2. Because O2 was absent, this class likely catalyzed reduction of sulfur (S0) originating from the metabolism of bisulfite with cathodic H2 (or low potential electrons and aqueous protons) originating from the corrosion of steel (Fe0). Overall this accelerates reaction of of S0 and Fe0 to form FeS, making this class a potentially powerful contributor to microbial corrosion. The PAS-821TP metagenome also had increased fractions of Deltaproteobacteria including the SRB Desulfomicrobium and Desulfocapsa. Altogether, SBS increased the fraction of hydrogen-utilizing Delta- and Epsilonproteobacteria in brackish-water-transporting pipelines, potentially stimulating anaerobic pipeline corrosion if dosed in excess of the intended oxygen scavenger function.

Biocide-mediated corrosion of coiled tubing.

Coiled tubing corrosion was investigated for 16 field water samples (S5 to S20) from a Canadian shale gas field. Weight loss corrosion rates of carbon steel beads incubated with these field water samples averaged 0.2 mm/yr, but injection water sample S19 had 1.25±0.07 mm/yr. S19 had a most probable number of zero acid-producing bacteria and incubation of S19 with carbon steel beads or coupons did not lead to big changes in microbial community composition. In contrast other field water samples had most probable numbers of APB of 102/mL to 107/mL and incubation of these field water samples with carbon steel beads or coupons often gave large changes in microbial community composition. HPLC analysis indicated that all field water samples had elevated concentrations of bromide (average 1.6 mM), which may be derived from bronopol, which was used as a biocide. S19 had the highest bromide concentration (4.2 mM) and was the only water sample with a high concentration of active bronopol (13.8 mM, 2760 ppm). Corrosion rates increased linearly with bronopol concentration, as determined by weight loss of carbon steel beads, for experiments with S19, with filtered S19 and with bronopol dissolved in defined medium. This indicated that the high corrosion rate found for S19 was due to its high bronopol concentration. The corrosion rate of coiled tubing coupons also increased linearly with bronopol concentration as determined by electrochemical methods. Profilometry measurements also showed formation of multiple pits on the surface of coiled tubing coupon with an average pit depth of 60 μm after 1 week of incubation with 1 mM bronopol. At the recommended dosage of 100 ppm the corrosiveness of bronopol towards carbon steel beads was modest (0.011 mm/yr). Higher concentrations, resulting if biocide is added repeatedly as commonly done in shale gas operations, are more corrosive and should be avoided. Overdosing may be avoided by assaying the presence of residual biocide by HPLC, rather than by assaying the presence of residual surviving bacteria.

Use of acetate, propionate and butyrate for reduction of nitrate and sulfate and methanogenesis in microcosms and bioreactors simulating an oil reservoir

ABSTRACT Acetate, propionate, and butyrate (volatile fatty acids [VFA]) occur in oil field waters and are frequently used for microbial growth of oil field consortia. We determined the kinetics of use of these VFA components (3 mM each) by an anaerobic oil field consortium in microcosms containing 2 mM sulfate and 0, 4, 6, 8, or 13 mM nitrate. Nitrate was reduced first, with a preference for acetate and propionate. Sulfate reduction then proceeded with propionate (but not butyrate) as the electron donor, whereas the fermentation of butyrate (but not propionate) was associated with methanogenesis. Microbial community analyses indicated that Paracoccus and Thauera (Paracoccus-Thauera), Desulfobulbus, and Syntrophomonas-Methanobacterium were the dominant taxa whose members catalyzed these three processes. Most-probable-number assays showed the presence of up to 107/ml of propionate-oxidizing sulfate-reducing bacteria (SRB) in waters from the Medicine Hat Glauconitic C field. Bioreactors with the same concentrations of sulfate and VFA responded similarly to increasing concentrations of injected nitrate as observed in the microcosms: sulfide formation was prevented by adding approximately 80% of the nitrate dose needed to completely oxidize VFA to CO2 in both. Thus, this work has demonstrated that simple time-dependent observations of the use of acetate, propionate, and butyrate for nitrate reduction, sulfate reduction, and methanogenesis in microcosms are a good proxy for these processes in bioreactors, monitoring of which is more complex. IMPORTANCE Oil field volatile fatty acids acetate, propionate, and butyrate were specifically used for nitrate reduction, sulfate reduction, and methanogenic fermentation. Time-dependent analyses of microcosms served as a good proxy for these processes in a bioreactor, mimicking a sulfide-producing (souring) oil reservoir: 80% of the nitrate dose required to oxidize volatile fatty acids to CO2 was needed to prevent souring in both. Our data also suggest that propionate is a good substrate to enumerate oil field SRB.

Control of microbial sulfide production by limiting sulfate dispersal in a water-injected oil field

Oil production by water injection often involves the use of makeup water to replace produced oil. Sulfate in makeup water is reduced by sulfate-reducing bacteria to sulfide, a process referred to as souring. In the MHGC field souring was caused by using makeup water with 4mM (384ppm) sulfate. Mixing with sulfate-free produced water gave injection water with 0.8mM sulfate. This was amended with nitrate to limit souring and was then distributed fieldwide. The start-up of an enhanced-oil-recovery pilot caused all sulfate-containing makeup water to be used for dissolution of polymer, which was then injected into a limited region of the field. Produced water from this pilot contained 10% of the injected sulfate concentration as sulfide, but was free of sulfate. Its use as makeup water in the main water plant of the field caused injection water sulfate to drop to zero. This in turn strongly decreased produced sulfide concentrations throughout the field and allowed a decreased injection of nitrate. The decreased injection of sulfate and nitrate caused major changes in the microbial community of produced waters. Limiting sulfate dispersal into a reservoir, which acts as a sulfate-removing biofilter, is thus a powerful method to decrease souring.