Anaerobic microbial communities and their potential for bioenergy production in heavily biodegraded petroleum reservoirs.

Most of the oil in low temperature, non-uplifted reservoirs is biodegraded due to millions of years of microbial activity, including via methanogenesis from crude oil. To evaluate stimulating additional methanogenesis in already heavily biodegraded oil reservoirs, oil sands samples were amended with nutrients and electron acceptors, but oil sands bitumen was the only organic substrate. Methane production was monitored for over 3000 days. Methanogenesis was observed in duplicate microcosms that were unamended, amended with sulfate or that were initially oxic, however methanogenesis was not observed in nitrate-amended controls. The highest rate of methane production was 0.15 µmol CH4 g-1 oil d-1 , orders of magnitude lower than other reports of methanogenesis from lighter crude oils. Methanogenic Archaea and several potential syntrophic bacterial partners were detected following the incubations. GC-MS and FTICR-MS revealed no significant bitumen alteration for any specific compound or compound class, suggesting that the very slow methanogenesis observed was coupled to bitumen biodegradation in an unspecific manner. After 3000 days, methanogenic communities were amended with benzoate resulting in methanogenesis rates that were 110-fold greater. This suggests that oil-to-methane conversion is limited by the recalcitrant nature of oil sands bitumen, not the microbial communities resident in heavy oil reservoirs.

On-line desalting of crude oil in the source region of a Fourier transform ion cyclotron resonance mass spectrometer.

The presence of dissolved metal ions in waters associated with crude oils has many negative implications for the transport, processing, and refining of petroleum. In addition, mass spectrometric analysis of sodium containing crude oil samples suffers from ionization suppression, unwanted adduct formation, and an increase in the complexity of data analysis. Here, we describe a method for the reduction/elimination of these adverse effects by modification of the source region gas-inlet system of a 12 T Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer. Several acids were examined as part of this study, with the most suitable for on-line desalting found to have both high vapor pressure and low pK(a); 12.1 M HCl showed the strongest desalting effect for crude oil samples with a sodium removal index (SRI) of 88%-100% ± 7% for the NaOS compound class. In comparison, a SRI of only 38% ± 9% was observed for a H2O/toluene solution-phase extraction of oil 1. These results clearly demonstrate the increased efficacy of pseudo-vapor phase desalting with the additional advantages that initial sample solution conditions are preserved and no sample preparation is required prior to analysis.

Massive dominance of Epsilonproteobacteria in formation waters from a Canadian oil sands reservoir containing severely biodegraded oil.

The subsurface microbiology of an Athabasca oil sands reservoir in western Canada containing severely biodegraded oil was investigated by combining 16S rRNA gene- and polar lipid-based analyses of reservoir formation water with geochemical analyses of the crude oil and formation water. Biomass was filtered from formation water, DNA was extracted using two different methods, and 16S rRNA gene fragments were amplified with several different primer pairs prior to cloning and sequencing or community fingerprinting by denaturing gradient gel electrophoresis (DGGE). Similar results were obtained irrespective of the DNA extraction method or primers used. Archaeal libraries were dominated by Methanomicrobiales (410 of 414 total sequences formed a dominant phylotype affiliated with a Methanoregula sp.), consistent with the proposed dominant role of CO(2) -reducing methanogens in crude oil biodegradation. In two bacterial 16S rRNA clone libraries generated with different primer pairs, > 99% and 100% of the sequences were affiliated with Epsilonproteobacteria (n = 382 and 72 total clones respectively). This massive dominance of Epsilonproteobacteria sequences was again obtained in a third library (99% of sequences; n = 96 clones) using a third universal bacterial primer pair (inosine-341f and 1492r). Sequencing of bands from DGGE profiles and intact polar lipid analyses were in accordance with the bacterial clone library results. Epsilonproteobacterial OTUs were affiliated with Sulfuricurvum, Arcobacter and Sulfurospirillum spp. detected in other oil field habitats. The dominant organism revealed by the bacterial libraries (87% of all sequences) is a close relative of Sulfuricurvum kujiense - an organism capable of oxidizing reduced sulfur compounds in crude oil. Geochemical analysis of organic extracts from bitumen at different reservoir depths down to the oil water transition zone of these oil sands indicated active biodegradation of dibenzothiophenes, and stable sulfur isotope ratios for elemental sulfur and sulfate in formation waters were indicative of anaerobic oxidation of sulfur compounds. Microbial desulfurization of crude oil may be an important metabolism for Epsilonproteobacteria indigenous to oil reservoirs with elevated sulfur content and may explain their prevalence in formation waters from highly biodegraded petroleum systems.

Thermodynamic constraints on methanogenic crude oil biodegradation.

Methanogenic degradation of crude oil hydrocarbons is an important process in subsurface petroleum reservoirs and anoxic environments contaminated with petroleum. There are several possible routes whereby hydrocarbons may be converted to methane: (i) complete oxidation of alkanes to H2 and CO2, linked to methanogenesis from CO2 reduction; (ii) oxidation of alkanes to acetate and H2, linked to acetoclastic methanogenesis and CO2 reduction; (iii) oxidation of alkanes to acetate and H2, linked to syntrophic acetate oxidation and methanogenesis from CO2 reduction; (iv) oxidation of alkanes to acetate alone, linked to acetoclastic methanogenesis and (v) oxidation of alkanes to acetate alone, linked to syntrophic acetate oxidation and methanogenesis from CO2 reduction. We have developed the concept of a 'window of opportunity' to evaluate the range of conditions under which each route is thermodynamically feasible. On this basis the largest window of opportunity is presented by the oxidation of alkanes to acetate alone, linked to acetoclastic methanogenesis. This contradicts field-based evidence that indicates that in petroleum rich environments acetoclastic methanogenesis is inhibited and that methanogenic CO2 reduction is the predominant methanogenic process. Our analysis demonstrates that under those biological constraints oxidation of alkanes to acetate and H2, linked to syntrophic acetate oxidation and methanogenesis from CO2 reduction offers a greater window of opportunity than complete oxidation of alkanes to H2 and CO2 linked to methanogenic CO2 reduction, and hence is the process most likely to occur.