A thermophilic spore-forming bacterium (strain AMP) was isolated from a thermophilic methanogenic bioreactor that was fed with cobalt-deprived synthetic medium containing methanol as substrate. 16S rRNA gene analysis revealed that strain AMP was closely related to the acetogenic bacterium Moorella thermoacetica DSM 521(T) (98.3% sequence similarity). DNA-DNA hybridization showed 75.2 +/- 4.7% similarity to M. thermoacetica DSM 521(T), suggesting that strain AMP is a M. thermoacetica strain. Strain AMP has a unique one-carbon metabolism compared to other Moorella species. In media without cobalt growth of strain AMP on methanol was only sustained in coculture with a hydrogen-consuming methanogen, while in media with cobalt it grew acetogenically in the absence of the methanogen. Addition of thiosulfate led to sulfide formation and less acetate formation. Growth of strain AMP with CO resulted in the formation of hydrogen as the main product, while other CO-utilizing Moorella strains produce acetate as product. Formate supported growth only in the presence of thiosulfate or in coculture with the methanogen. Strain AMP did not grow with H(2)/CO(2), unlike M. thermoacetica (DSM 521(T)). The lack of growth with H(2)/CO(2) likely is due to the absence of cytochrome b in strain AMP.
A 5.1 L laboratory scale upflow anaerobic sludge bed (UASB) reactor was operated at 55 degrees C over 130 days in order to investigate the feasibility of treating methanol-containing wastewater under thermophilic conditions, focussing on start-up and process stability. Batch assays were conducted to elucidate the most probable pathway for methanol conversion. The results demonstrated a good performance, with a chemical oxygen demand (COD) removal averaging 82% throughout the experiment. No significant VFA accumulation was detected in the effluent, even with bicarbonate concentration exceeding 20 mM. Acetate was the main component of the VFA at relatively low organic loading rates (OLR). At high OLR, the main components were propionate and butyrate. Reactor performance was hardly affected when the system was exposed to non-optimal conditions, i.e., temperature drop, overloading and no feeding. Good thermophilic granular sludge was retained in the reactor. Washout of biomass was not severe during the experiment. From the pathway analysis it could be concluded that indirect pathways play an important role in the methanol degradation by the cultivated consortia.
The importance of cobalt on the anaerobic conversion of methanol under thermophilic conditions was studied in three parallel lab-scale UASB-reactors and in cobalt-limited enriched cultures. Reactors R1, R2, and R3 were fed with methanol in a bicarbonate-buffered medium, supplied with iron and macronutrients: in R1 all metals were supplied (control), R2 was cobalt deprived, and in R3 all metals were deprived. In the 136 days of continuous experiment, a drop in performance was observed over the last 30 days. Particularly in R3, both methanol removal and methane formation dropped by 7.1% and 13.7%, respectively, compared to the control reactor, R1. When the medium was cobalt-deprived, acetate was not produced and, as a consequence, the enriched consortium lost its capacity to degrade acetate, indicating that the acetotrophic microorganisms were washed out. The addition of 0.5 microM of cobalt to a cobalt-deprived enrichment culture led to acetate accumulation. The results obtained in this study indicate that the mixed consortium requires a proper amount of cobalt, and its addition to a concentration of 0.1 microM leads to the highest methanol conversion rate, with methane as the sole end product from methanol.
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