Akhil Agrawal

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.

Acetate Production from Oil under Sulfate-Reducing Conditions in Bioreactors Injected with Sulfate and Nitrate

ABSTRACT Oil production by water injection can cause souring in which sulfate in the injection water is reduced to sulfide by resident sulfate-reducing bacteria (SRB). Sulfate (2 mM) in medium injected at a rate of 1 pore volume per day into upflow bioreactors containing residual heavy oil from the Medicine Hat Glauconitic C field was nearly completely reduced to sulfide, and this was associated with the generation of 3 to 4 mM acetate. Inclusion of 4 mM nitrate inhibited souring for 60 days, after which complete sulfate reduction and associated acetate production were once again observed. Sulfate reduction was permanently inhibited when 100 mM nitrate was injected by the nitrite formed under these conditions. Pulsed injection of 4 or 100 mM nitrate inhibited sulfate reduction temporarily. Sulfate reduction resumed once nitrate injection was stopped and was associated with the production of acetate in all cases. The stoichiometry of acetate formation (3 to 4 mM formed per 2 mM sulfate reduced) is consistent with a mechanism in which oil alkanes and water are metabolized to acetate and hydrogen by fermentative and syntrophic bacteria (K. Zengler et al., Nature 401:266–269, 1999), with the hydrogen being used by SRB to reduce sulfate to sulfide. In support of this model, microbial community analyses by pyrosequencing indicated SRB of the genus Desulfovibrio, which use hydrogen but not acetate as an electron donor for sulfate reduction, to be a major community component. The model explains the high concentrations of acetate that are sometimes found in waters produced from water-injected oil fields.

Toluene Depletion in Produced Oil Contributes to Souring Control in a Field Subjected to Nitrate Injection

Souring in the Medicine Hat Glauconitic C field, which has a low bottom-hole temperature (30 °C), results from the presence of 0.8 mM sulfate in the injection water. Inclusion of 2 mM nitrate to decrease souring results in zones of nitrate-reduction, sulfate-reduction, and methanogenesis along the injection water flow path. Microbial community analysis by pyrosequencing indicated dominant community members in each of these zones. Nitrate breakthrough was observed in 2-PW, a major water- and sulfide-producing well, after 4 years of injection. Sulfide concentrations at four other production wells (PWs) also reached zero, causing the average sulfide concentration in 14 PWs to decrease significantly. Interestingly, oil produced by 2-PW was depleted of toluene, the preferred electron donor for nitrate reduction. 2-PW and other PWs with zero sulfide produced 95% water and 5% oil. At 2 mM nitrate and 5 mM toluene, respectively, this represents an excess of electron acceptor over electron donor. Hence, continuous nitrate injection can change the composition of produced oil and nitrate breakthrough is expected first in PWs with a low oil to water ratio, because oil from these wells is treated on average with more nitrate than is oil from PWs with a high oil to water ratio.

In situ detection of anaerobic alkane metabolites in subsurface environments

Alkanes comprise a substantial fraction of crude oil and refined fuels. As such, they are prevalent within deep subsurface fossil fuel deposits and in shallow subsurface environments such as aquifers that are contaminated with hydrocarbons. These environments are typically anaerobic, and host diverse microbial communities that can potentially use alkanes as substrates. Anaerobic alkane biodegradation has been reported to occur under nitrate-reducing, sulfate-reducing, and methanogenic conditions. Elucidating the pathways of anaerobic alkane metabolism has been of interest in order to understand how microbes can be used to remediate contaminated sites. Alkane activation primarily occurs by addition to fumarate, yielding alkylsuccinates, unique anaerobic metabolites that can be used to indicate in situ anaerobic alkane metabolism. These metabolites have been detected in hydrocarbon-contaminated shallow aquifers, offering strong evidence for intrinsic anaerobic bioremediation. Recently, studies have also revealed that alkylsuccinates are present in oil and coal seam production waters, indicating that anaerobic microbial communities can utilize alkanes in these deeper subsurface environments. In many crude oil reservoirs, the in situ anaerobic metabolism of hydrocarbons such as alkanes may be contributing to modern-day detrimental effects such as oilfield souring, or may lead to more beneficial technologies such as enhanced energy recovery from mature oilfields. In this review, we briefly describe the key metabolic pathways for anaerobic alkane (including n-alkanes, isoalkanes, and cyclic alkanes) metabolism and highlight several field reports wherein alkylsuccinates have provided evidence for anaerobic in situ alkane metabolism in shallow and deep subsurface environments.

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.