Aerobic biodegradation of cycloalkanes in non-aqueous extracted oil sands tailings.

Management of growing volumes of fluid fine tailings (FFT) is a significant challenge for oil sands industry. A potential alternative non-aqueous solvent extraction (NAE) process uses cycloalkane solvent such as cyclohexane or cyclopentane with very little water and generates smaller volumes of 'dry' solids (NAES) with residual solvent. Here we investigate remediation of NAES in a simulated bench-scale upland reclamation scenario. In the first study, microcosms with nutrient medium plus FFT as inoculum were amended with cyclohexane and incubated for ∼1 year, monitoring for cyclohexane biodegradation under aerobic conditions. Biodegradation of cyclohexane occurred under aerobic conditions with no metabolic intermediates detected. A second study using NAES mixed with FFT spiked with cyclohexane and cyclopentane, with or without additional nutrients (nitrogen and phosphorus), showed complete and rapid aerobic biodegradation of both cycloalkanes in NAES inoculated with FFT and supplemented with nutrients. 16S rRNA gene sequencing revealed dominance of Rhodoferax and members of Burkholderiaceae during aerobic cyclohexane biodegradation in FFT, and Hydrogenophaga, Acidovorax, Defluviimonas and members of Porticoccaceae during aerobic biodegradation of cyclohexane and cyclopentane in NAES inoculated with FFT and supplemented with nutrients. The findings indicate that biodegradation of cycloalkanes from NAES is possible under aerobic condition, which will contribute to the successful reclamation of oil sands tailings for land closure.

Process optimization for simultaneous production of cellulase, xylanase and ligninase by Saccharomyces cerevisiae SCPW 17 under solid state fermentation using Box-Behnken experimental design.

Multienzyme complex has attracted increased attention in biofuel technology. They offer solutions to effective degradation of complex plant material into fermentable sugars. Microorganisms, especially bacteria and fungi, are well studied for their ability to produce enzymes complex unlike yeast. Yeast strain isolated from mushroom farm was studied for simultaneous production of cellulase, xylanase and ligninase enzymes using lignocellulose waste as substrates. A response surface methodology (RSM) involving Box-Behnken design (BBD) was used to investigate interaction between variables (moisture content, inoculum size, initial pH, incubation time) that affect enzyme production. Crude filtrate was partially purified and characterised. Yeast strain identified as Saccharomyces cerevisiae SCPW 17 was finally studied. Evaluation of lignocellulose waste for enzyme complex production revealed corn cob to be most effective substrate for cellulase, xylanase and ligninase production with enzyme activity of 17.63 ± 1.45 U/gds, 29.35 ± 1.67 U/gds and 150.75 ± 2.01 µmol/min respectively. Time course study showed maximum enzyme complex production was obtained by day 6 with cellulase activity of 12.5 U/gds, xylanase 48.3 U/gds and ligninase 90.8 µmol/min. Using RSM involving BBD, maximum enzyme activity was found to be 19.51 ± 0.32 U/gds, 56.86 ± 0.38 U/gds, 408.17 ± 1.04 µmol/min for cellulaase, xylanase and ligninase respectively. The developed models were highly significant at probability level of P = 0.0001 and multiple correlation co-efficient (R2) was 0.9563 for cellulase, 0.9532 for xylanase and 0.9780 for ligninase. Enzyme complex was stable at varying pH and temperature conditions. Saccharomyces cerevisiae (SCPW 17) studied produced enzyme complex which can be used for bioconversion of biomass to value-added chemicals.

Comparison of Nitrate and Perchlorate in Controlling Sulfidogenesis in Heavy Oil-Containing Bioreactors.

Control of microbial reduction of sulfate to sulfide in oil reservoirs (a process referred to as souring) with nitrate has been researched extensively. Nitrate is reduced to nitrite, which is a strong inhibitor of sulfate-reducing bacteria (SRB). Perchlorate has been proposed as an alternative souring control agent. It is reduced to chlorate (ClO3 -) and chlorite (ClO2 -), which is dismutated to chloride and O2. These can react with sulfide to form sulfur. Chlorite is also highly biocidal. Here we compared the effectiveness of perchlorate and nitrate in inhibiting SRB activity in medium containing heavy oil from the Medicine Hat Glauconitic C (MHGC) field, which has a low reservoir temperature and is injected with nitrate to control souring. Using acetate, propionate and butyrate as electron donors, perchlorate-reducing bacteria (PRB) were obtained in enrichment culture and perchlorate-reducing Magnetospirillum spp. were isolated from MHGC produced waters. In batch experiments with MHGC oil as the electron donor, nitrate was reduced to nitrite and inhibited sulfate reduction. However, perchlorate was not reduced and did not inhibit sulfate reduction in these incubations. Bioreactor experiments were conducted with sand-packed glass columns, containing MHGC oil and inoculated with an oil-grown mesophilic SRB enrichment. Once active souring (reduction of 2 mM sulfate to sulfide) was observed, these were treated with nitrate and/or perchlorate. As in the batch experiments, 4 mM nitrate completely inhibited sulfide production, while partial inhibition occurred with 1 and 2 mM nitrate, but injection of 4 mM perchlorate did not inhibit sulfate reduction and perchlorate was not reduced. The enriched and isolated PRB were unable to use heavy oil components, like alkylbenzenes, which were readily used by nitrate-reducing bacteria. Hence perchlorate, injected into a low temperature heavy oil reservoir like the MHGC, may not be reduced to toxic intermediates making nitrate a preferable souring control agent.

Synergy of Sodium Nitroprusside and Nitrate in Inhibiting the Activity of Sulfate Reducing Bacteria in Oil-Containing Bioreactors.

Sodium nitroprusside (SNP) disrupts microbial biofilms through the release of nitric oxide (NO). The actions of SNP on bacteria have been mostly limited to the genera Pseudomonas, Clostridium, and Bacillus. There are no reports of its biocidal action on sulfate-reducing bacteria (SRB), which couple the reduction of sulfate to sulfide with the oxidation of organic electron donors. Here, we report the inhibition and kill of SRB by low SNP concentrations [0.05 mM (15 ppm)] depending on biomass concentration. Chemical reaction of SNP with sulfide did not compromise its efficacy. SNP was more effective than five biocides commonly used to control SRB. Souring, the SRB activity in oil reservoirs, is often controlled by injection of nitrate. Control of SRB-mediated souring in oil-containing bioreactors was inhibited by 4 mM (340 ppm) of sodium nitrate, but required only 0.05 mM (15 ppm) of SNP. Interestingly, nitrate and SNP were found to be highly synergistic with 0.003 mM (1 ppm) of SNP and 1 mM (85 ppm) of sodium nitrate being sufficient in inhibiting souring. Hence, using SNP as an additive may greatly increase the efficacy of nitrate injection in oil reservoirs.

Effect of Thermophilic Nitrate Reduction on Sulfide Production in High Temperature Oil Reservoir Samples.

Oil fields can experience souring, the reduction of sulfate to sulfide by sulfate-reducing microorganisms. At the Terra Nova oil field near Canada's east coast, with a reservoir temperature of 95°C, souring was indicated by increased hydrogen sulfide in produced waters (PW). Microbial community analysis by 16S rRNA gene sequencing showed the hyperthermophilic sulfate-reducing archaeon Archaeoglobus in Terra Nova PWs. Growth enrichments in sulfate-containing media at 55-70°C with lactate or volatile fatty acids yielded the thermophilic sulfate-reducing bacterium (SRB) Desulfotomaculum. Enrichments at 30-45°C in nitrate-containing media indicated the presence of mesophilic nitrate-reducing bacteria (NRB), which reduce nitrate without accumulation of nitrite, likely to N2. Thermophilic NRB (tNRB) of the genera Marinobacter and Geobacillus were detected and isolated at 30-50°C and 40-65°C, respectively, and only reduced nitrate to nitrite. Added nitrite strongly inhibited the isolated thermophilic SRB (tSRB) and tNRB and SRB could not be maintained in co-culture. Inhibition of tSRB by nitrate in batch and continuous cultures required inoculation with tNRB. The results suggest that nitrate injected into Terra Nova is reduced to N2 at temperatures up to 45°C but to nitrite only in zones from 45 to 65°C. Since the hotter zones of the reservoir (65-80°C) are inhabited by thermophilic and hyperthermophilic sulfate reducers, souring at these temperatures might be prevented by nitrite production if nitrate-reducing zones of the system could be maintained at 45-65°C.

Implications of Limited Thermophilicity of Nitrite Reduction for Control of Sulfide Production in Oil Reservoirs.

UNLABELLED: Nitrate reduction to nitrite in oil fields appears to be more thermophilic than the subsequent reduction of nitrite. Concentrated microbial consortia from oil fields reduced both nitrate and nitrite at 40 and 45°C but only nitrate at and above 50°C. The abundance of the nirS gene correlated with mesophilic nitrite reduction activity. Thauera and Pseudomonas were the dominant mesophilic nitrate-reducing bacteria (mNRB), whereas Petrobacter and Geobacillus were the dominant thermophilic NRB (tNRB) in these consortia. The mNRB Thauera sp. strain TK001, isolated in this study, reduced nitrate and nitrite at 40 and 45°C but not at 50°C, whereas the tNRB Petrobacter sp. strain TK002 and Geobacillus sp. strain TK003 reduced nitrate to nitrite but did not reduce nitrite further from 50 to 70°C. Testing of 12 deposited pure cultures of tNRB with 4 electron donors indicated reduction of nitrate in 40 of 48 and reduction of nitrite in only 9 of 48 incubations. Nitrate is injected into high-temperature oil fields to prevent sulfide formation (souring) by sulfate-reducing bacteria (SRB), which are strongly inhibited by nitrite. Injection of cold seawater to produce oil creates mesothermic zones. Our results suggest that preventing the temperature of these zones from dropping below 50°C will limit the reduction of nitrite, allowing more effective souring control. IMPORTANCE: Nitrite can accumulate at temperatures of 50 to 70°C, because nitrate reduction extends to higher temperatures than the subsequent reduction of nitrite. This is important for understanding the fundamentals of thermophilicity and for the control of souring in oil fields catalyzed by SRB, which are strongly inhibited by nitrite.