Long-Term Incubation Reveals Methanogenic Biodegradation of C5 and C6 iso-Alkanes in Oil Sands Tailings.

iso-Alkanes are major components of petroleum and have been considered recalcitrant to biodegradation under methanogenic conditions. However, indigenous microbes in oil sands tailings ponds exposed to solvents rich in 2-methylbutane, 2-methylpentane, 3-methylpentane, n-pentane, and n-hexane produce methane in situ. We incubated defined mixtures of iso- or n-alkanes with mature fine tailings from two tailings ponds of different ages historically exposed to different solvents: one, ~10 years old, receiving C5-C6 paraffins and the other, ~35 years old, receiving naphtha. A lengthy incubation (>6 years) revealed iso-alkane biodegradation after lag phases of 900-1800 and ~280 days, respectively, before the onset of methanogenesis, although lag phases were shorter with n-alkanes (~650-1675 and ~170 days, respectively). 2-Methylpentane and both n-alkanes were completely depleted during ~2400 days of incubation, whereas 2-methylbutane and 3-methylpentane were partially depleted only during active degradation of 2-methylpentane, suggesting co-metabolism. In both cases, pyrotag sequencing of 16S rRNA genes showed codominance of Peptococcaceae with acetoclastic (Methanosaeta) and hydrogenotrophic (Methanoregula and Methanolinea) methanogens. These observations are important for predicting long-term greenhouse-gas emissions from oil sands tailings ponds and extend the known range of hydrocarbons susceptible to methanogenic biodegradation in petroleum-impacted anaerobic environments.

Reduction of Fe(III) oxides by phylogenetically and physiologically diverse thermophilic methanogens.

Three thermophilic methanogens (Methanothermobacter thermautotrophicus, Methanosaeta thermophila, and Methanosarcina thermophila) were investigated for their ability to reduce poorly crystalline Fe(III) oxides (ferrihydrite) and the inhibitory effects of ferrihydrite on their methanogenesis. This study demonstrated that Fe(II) generation from ferrihydrite occurs in the cultures of the three thermophilic methanogens only when H2 was supplied as the source of reducing equivalents, even in the cultures of Mst. thermophila that do not grow on and produce CH4 from H2/CO2. While supplementation of ferrihydrite resulted in complete inhibition or suppression of methanogenesis by the thermophilic methanogens, ferrihydrite reduction by the methanogens at least partially alleviates the inhibitory effects. Microscopic and crystallographic analyses on the ferrihydrite-reducing Msr. thermophila cultures exhibited generation of magnetite on its cell surfaces through partial reduction of ferrihydrite. These findings suggest that at least certain thermophilic methanogens have the ability to extracellularly transfer electrons to insoluble Fe(III) compounds, affecting their methanogenic activities, which would in turn have significant impacts on materials and energy cycles in thermophilic anoxic environments.

Relation between the activity of anaerobic microbial populations in oil sands tailings ponds and the sedimentation of tailings.

Oil sands tailings ponds contain a variety of anaerobic microbes, including methanogens, sulfate- and nitrate-reducing bacteria. Methanogenic activity in samples from a tailings pond and its input streams was higher with trimethylamine (TMA) than with acetate. Methanogens closely affiliated to Methanomethylovorans hollandica were found in the TMA enrichments. Tailings sedimentation increased with methanogenic activity, irrespective whether TMA or acetate was used to stimulate methanogenesis. Increased sedimentation of autoclaved tailings was observed with added pure cultures under methanogenic, as well as under nitrate-reducing conditions, but not under sulfate-reducing conditions. Scanning electron microscopy and energy-dispersive X-ray spectroscopy indicated the presence of microbes and of extracellular polymeric substances in tailings particle aggregates, especially under methanogenic and nitrate-reducing conditions. Hence different classes of microorganisms growing in tailings ponds contribute to increased tailings aggregation and sedimentation. Because addition of nitrate is known to lower methane production by methanogenic consortia, these observations offer the potential to combine lower methane emissions with improved microbially-induced tailings sedimentation.

Anaerobic methanethiol degradation and methanogenic community analysis in an alkaline (pH 10) biological process for liquefied petroleum gas desulfurization.

Anaerobic methanethiol (MT) degradation by mesophilic (30 degrees C) alkaliphilic (pH 10) communities was studied in a lab-scale Upflow Anaerobic Sludge Bed (UASB) reactor inoculated with a mixture of sediments from the Wadden Sea (The Netherlands), Soap Lake (Central Washington), and Russian soda lakes. MT degradation started after 32 days of incubation. During the first 252 days, complete degradation was achieved till a volumetric loading rate of 7.5 mmol MT/L/day, and sulfide, methane, and carbon dioxide were the main reaction products. Temporary inhibition of MT degradation occurred after MT peak loads and in the presence of dimethyl disulfide (DMDS), which is the autooxidation product of MT. From day 252 onwards, methanol was dosed to the reactor as co-substrate at a loading rate of 3-6 mmol/L/day to stimulate growth of methylotrophic methanogens. Methanol was completely degraded and also a complete MT degradation was achieved till a volumetric loading rate of 13 mmol MT/L/day (0.77 mmol MT/gVSS/day). However, from day 354 till the end of the experimental run (day 365), acetate was formed and MT was not completely degraded anymore, indicating that methanol-degrading homoacetogenic bacteria had partially outcompeted the methanogenic MT-degrading archea. The archeal community in the reactor sludge was analyzed by DGGE and sequencing of 16S rRNA genes. The methanogenic archea responsible for the degradation of MT in the reactor were related to Methanolobus oregonensis. A pure culture, named strain SODA, was obtained by serial dilutions in medium containing both trimethyl amine and dimethyl sulfide (DMS). Strain SODA degraded MT, DMS, trimethyl amine, and methanol. Flow sheet simulations revealed that for sufficient MT removal from liquefied petroleum gas, the extraction and biological degradation process should be operated above pH 9.

Metabolite and enzyme profiles of glycogen metabolism in Methanococcoides methylutens.

When a buffered anaerobic cell suspension of Methanococcoides methylutens was maintained under methanol-limited conditions, intracellular glycogen and hexose phosphates were consumed rapidly and a very small amount of methane formed at 4 h of a starvation period. When methanol was supplemented after a total of 20 h of starvation, a reverse pattern was observed: the glycogen level and the hexose phosphate pool increased, and formation of methane took place after a lag period of 90 min. A considerable amount of methane was formed in 120 min after its detection with a rate of 0.18 micromol mg(-1) protein min(-1). When methane formation decreased after 270 min of incubation and finally came to a halt, probably due to complete assimilation of supplemented methanol, the levels of glycogen and hexose monophosphates decreased once again. However fructose 1,6-diphosphate levels showed a continuous increase even after exhaustion of methane formation. In contrast to the hexose phosphate pool, levels of other metabolites showed a small increase after addition of methanol. The enzyme profile of glycogen metabolism showed relatively high levels of triose phosphate isomerase. Glyceraldehyde 3-phosphate dehydrogenase reacted with NADPH with a three-fold higher activity as compared to that with NADH.

Coenzyme Q10 in vesicles composed of archaeal ether lipids or conventional lipids enhances the immuno-adjuvanticity to encapsulated protein.

Cellular accumulation, tissue distribution, and immuno-adjuvanticity were evaluated for liposomal CoQ10 prepared from either distearoylphosphatidylcholine:dicetylphosphate:cholesterol (4:1:5, mol. ratio) (conventional liposomes) or from the total polar lipids of the archaeon Methanosarcina mazei (archaeosomes). Liposomal CoQ10 vesicles of approximately 100 nm diameter, containing up to 179 mumol of CoQ10 per mg of lipid have been evaluated using J774A.1 macrophages and Balb/c mice. Archaeosomes uptake by J774A.1 macrophages was better than with the conventional liposome, and the incorporation of CoQ10 enhanced the uptake of both lipid vesicle types. All vesicle types were detected in the liver and spleen of mice (4-27% of injected dose) within 3 h of intraperitoneal injection. Moreover, incorporation of CoQ10 into lipid vesicles enhanced the immuno-adjuvanticity of both conventional liposomes and archaeosomes, to achieve approximately a doubling in the titres of BSA-specific antibody in sera to 169 and 430 micrograms ml-1, respectively. Increases in IgG1 and IgG2a/2b accounted for most of the CoQ10-induced increases in anti-BSA titres. These results are rationalized on the basis of surface hydrophobicity and opsonization changes induced by the presence of CoQ10 in vesicles. We suggest that liposomal CoQ10 has potential as a new generation of vaccine delivery system to enhance the immune response. Its use as a novel delivery system may be particularly effective under pathological conditions where the occurrence of an oxidative stress condition significantly impairs the immune system functions.