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

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.

Methods for recovery of microorganisms and intact microbial polar lipids from oil-water mixtures: laboratory experiments and natural well-head fluids.

Most of the world's remaining petroleum resource has been altered by in-reservoir biodegradation which adversely impacts oil quality and production, ultimately making heavy oil. Analysis of the microorganisms in produced reservoir fluid samples is a route to characterization of subsurface biomes and a better understanding of the resident and living microorganisms in petroleum reservoirs. The major challenges of sample contamination with surface biota, low abundances of microorganisms in subsurface samples, and viscous emulsions produced from biodegraded heavy oil reservoirs are addressed here in a new analytical method for intact polar lipids (IPL) as taxonomic indicators in petroleum reservoirs. We have evaluated the extent to which microbial cells are removed from the free water phase during reservoir fluid phase separation by analysis of model reservoir fluids spiked with microbial cells and have used the resultant methodologies to analyze natural well-head fluids from the Western Canada Sedimentary Basin (WCSB). Analysis of intact polar membrane lipids of microorganisms using liquid chromatography-mass spectrometry (LC-MS) techniques revealed that more than half of the total number of microorganisms can be recovered from oil-water mixtures. A newly developed oil/water separator allowed for filtering of large volumes of water quickly while in the field, which reduced the chances of contamination and alterations to the composition of the subsurface microbial community after sample collection. This method makes the analysis of IPLs (or indirectly microorganisms) from well-head fluids collected in remote field settings possible and reliable. To the best of our knowledge this is the first time that IPLs have been detected in well-head oil-water mixtures.

Competitive, microbially-mediated reduction of nitrate with sulfide and aromatic oil components in a low-temperature, western Canadian oil reservoir.

Fields from which oil is produced by injection of sulfate-bearing water often exhibit an increase in sulfide concentration with time (souring). Nitrate added to the injection water lowers the sulfide concentration by the action of sulfide-oxidizing, nitrate-reducing bacteria (SO-NRB). However, the injected nitrate can also be reduced with oil organics by heterotrophic NRB (hNRB). Aqueous volatile fatty acids (VFAs; a mixture of acetate, propionate, and butyrate) are considered important electron donors in this regard. Injection and produced waters from a western Canadian oil field with a low in situ reservoir temperature (30 degrees C) had only 0.1-0.2 mM VFAs. Amendment of these waters with nitrate gave therefore only partial reduction. More nitrate was reduced when 2% (v/v) oil was added, with light oil giving more reduction than heavy oil. GC-MS analysis of in vitro degraded oils and electron balance considerations indicated that toluene served as the primary electron donor for nitrate reduction. The differences in the extent of nitrate reduction were thus related to the toluene content of the light and heavy oil (30 and 5 mM, respectively). Reduction of nitrate with sulfide by SO-NRB always preceded that with oil organics by hNRB, even though microbially catalyzed kinetics with either electron donor were similar. Inhibition of hNRB by sulfide is responsible for this phenomenon. Injected nitrate will thus initially be reduced with sulfide through the action of SO-NRB. However, once sulfide has been eliminated from the near-injection wellbore region, oil organics will be targeted by the action of hNRB. Hence, despite the kinetic advantage of SO-NRB, the nitrate dose required to eliminate sulfide from a reservoir depends on the concentration of hNRB-degradable oil organics, with toluene being the most important in the field under study. Because the toluene concentration is lower in heavy oilthan in light oil, nitrate injection into a heavy-oil-producing field of low temperature is more likely to succeed in containing souring.

Biological activity in the deep subsurface and the origin of heavy oil.

At temperatures up to about 80 degrees C, petroleum in subsurface reservoirs is often biologically degraded, over geological timescales, by microorganisms that destroy hydrocarbons and other components to produce altered, denser 'heavy oils'. This temperature threshold for hydrocarbon biodegradation might represent the maximum temperature boundary for life in the deep nutrient-depleted Earth. Most of the world's oil was biodegraded under anaerobic conditions, with methane, a valuable commodity, often being a major by-product, which suggests alternative approaches to recovering the world's vast heavy oil resource that otherwise will remain largely unproduced.

The microbiology of hydrocarbon degradation in subsurface petroleum reservoirs: perspectives and prospects.

The majority of the Earth's petroleum resource is partly biodegraded. This is of considerable practical significance and can limit economic exploitation of petroleum reserves and lead to problems during petroleum production. Knowledge of the microorganisms present in petroleum reservoirs, their physiological properties and the biochemical potential for hydrocarbon degradation benefits successful petroleum exploration. Anaerobic conditions prevail in petroleum reservoirs and biological hydrocarbon degradation is apparently inhibited at temperatures above 80-90 degrees C. We summarise available knowledge and conjecture on the dominant biological processes active during subsurface petroleum biodegradation.