Effect of temperature on microbial community of a glucose-degrading methanogenic consortium under hyperthermophilic chemostat cultivation.

We continuously fed an anaerobic chemostat with synthetic wastewater containing glucose as the sole source of carbon and energy to study the effects of temperature on the microbial community under hyperthermophilic (65-80 degrees C) conditions. Methane was produced normally up to 77.5 degrees C at a dilution rate of 0.025 d(-1). However, the concentration of microorganisms and the rate of gas production decreased with increasing operation temperature. The microbial community in the chemostat at various temperatures was analyzed based on the 16S rRNA gene using molecular biological techniques including clone library analysis and denaturing gradient gel electrophoresis (DGGE). Aceticlastic methanogens related to Methanosarcina thermophila were detected at 65 degrees C and hydrogenotrophilic methanogens related to Methanothermobacter thermautotrophicus were the dominant methanogens between 70 degrees C to 77.5 degrees C. Bacteria related to Clostridium stercorarium and Thermoanaerobacter subterraneus comprised the dominant glucose-fermenting bacteria at temperatures of 65 degrees C and above, respectively. Bacteria related to Thermacetogenium phaeum and to Tepidiphilus margaritifer and Petrobacter succinatimandens were the dominant acetate-oxidizing bacteria at 70 degrees C and at 75-77.5 degrees C, respectively. The results suggested that, at temperatures of 70 degrees C and above, methane production via the aceticlastic pathway was negligible and indirect methanogenesis from acetate was dominant. Since acetate oxidation is a rate limiting step and a higher temperature favors the hydrolysis and acid formation, a two stage fermentation process, acidogenic and methanogenic fermentation stages operated under different temperatures, should be more suitable for the thermophilic anaerobic treatment at temperatures above 65 degrees C.

[1]  A. Stams,et al.  Enrichment and Detection of Microorganisms Involved in Direct and Indirect Methanogenesis from Methanol in an Anaerobic Thermophilic Bioreactor , 2005, Microbial Ecology.

[2]  Yanhe Ma,et al.  Thermoanaerobacter tengcongensis sp. nov., a novel anaerobic, saccharolytic, thermophilic bacterium isolated from a hot spring in Tengcong, China. , 2001, International journal of systematic and evolutionary microbiology.

[3]  Irini Angelidaki,et al.  Anaerobic thermophilic digestion of manure at different ammonia loads: Effect of temperature , 1994 .

[4]  G. Lettinga,et al.  Anaerobic digestion and wastewater treatment systems , 2004, Antonie van Leeuwenhoek.

[5]  J. V. Lier,et al.  Limitations of thermophilic anaerobic wastewater treatment and the consequences for process design , 2004, Antonie van Leeuwenhoek.

[6]  L. Forney,et al.  Distribution of bacterioplankton in meromictic Lake Saelenvannet, as determined by denaturing gradient gel electrophoresis of PCR-amplified gene fragments coding for 16S rRNA , 1997, Applied and environmental microbiology.

[7]  B. Patel,et al.  Transfer of thermobacteroides leptospartum and Clostridium thermolacticum as Clostridium stercorarium subsp. leptospartum subsp. thermolacticum subsp. nov., comb. nov. and C. stercorarium subsp. thermolacticum subsp. nov., comb. nov. , 2001, International journal of systematic and evolutionary microbiology.

[8]  M. J. Park,et al.  Symbiobacterium thermophilum gen. nov., sp. nov., a symbiotic thermophile that depends on co-culture with a Bacillus strain for growth. , 2000, International journal of systematic and evolutionary microbiology.

[9]  James R. Cole,et al.  The Ribosomal Database Project (RDP-II): sequences and tools for high-throughput rRNA analysis , 2004, Nucleic Acids Res..

[10]  K. Kida,et al.  Effect of dilution rate on structure of a mesophilic acetate-degrading methanogenic community during continuous cultivation. , 2003, Journal of bioscience and bioengineering.

[11]  E. Mikami,et al.  Isolation and characterization of a thermophilic benzoate-degrading, sulfate-reducing bacterium, Desulfotomaculum thermobenzoicum sp. nov. , 1991, Archives of Microbiology.

[12]  James G. Ferry,et al.  Methanogenesis : Ecology, Physiology, Biochemistry and Genetics , 1994 .

[13]  B. Patel,et al.  Petrobacter succinatimandens gen. nov., sp. nov., a moderately thermophilic, nitrate-reducing bacterium isolated from an Australian oil well. , 2004, International journal of systematic and evolutionary microbiology.

[14]  Y. Kamagata,et al.  Pelotomaculum thermopropionicum gen. nov., sp. nov., an anaerobic, thermophilic, syntrophic propionate-oxidizing bacterium. , 2002, International journal of systematic and evolutionary microbiology.

[15]  Birgitte Kiær Ahring,et al.  Status on science and application of thermophilic anaerobic digestion , 1994 .

[16]  K. Kida,et al.  Microbial community analysis of mesophilic anaerobic protein degradation process using bovine serum albumin (BSA)-fed continuous cultivation. , 2005, Journal of bioscience and bioengineering.

[17]  K Kida,et al.  Influence of Ni2+ and Co2+ on methanogenic activity and the amounts of coenzymes involved in methanogenesis. , 2001, Journal of bioscience and bioengineering.

[18]  J. Zeikus,et al.  Methanobacterium thermoautotrophicus sp. n., an Anaerobic, Autotrophic, Extreme Thermophile , 1972, Journal of bacteriology.

[19]  A. Maruyama,et al.  Thermanaeromonas toyohensis gen. nov., sp. nov., a novel thermophilic anaerobe isolated from a subterranean vein in the Toyoha Mines. , 2002, International journal of systematic and evolutionary microbiology.

[20]  J. Felsenstein CONFIDENCE LIMITS ON PHYLOGENIES: AN APPROACH USING THE BOOTSTRAP , 1985, Evolution; international journal of organic evolution.

[21]  A. Nozhevnikova,et al.  Anaerobic manure treatment under extreme temperature conditions , 1999 .

[22]  J. Thompson,et al.  The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools. , 1997, Nucleic acids research.

[23]  B. Ahring,et al.  Effect of temperature increase from 55 to 65 degrees C on performance and microbial population dynamics of an anaerobic reactor treating cattle manure. , 2001, Water research.

[24]  C. Manaia,et al.  Tepidiphilus margaritifer gen. nov., sp. nov., isolated from a thermophilic aerobic digester. , 2003, International journal of systematic and evolutionary microbiology.

[25]  P. Scherer,et al.  Development of a methanogenic process to degrade exhaustively the organic fraction of municipal "grey waste" under thermophilic and hyperthermophilic conditions. , 2000, Water science and technology : a journal of the International Association on Water Pollution Research.

[26]  B. Patel,et al.  Thermoanaerobacter subterraneus sp. nov., a novel thermophile isolated from oilfield water. , 2000, International journal of systematic and evolutionary microbiology.

[27]  Y. Nodasaka,et al.  Bacillus asahii sp. nov., a novel bacterium isolated from soil with the ability to deodorize the bad smell generated from short-chain fatty acids. , 2004, International journal of systematic and evolutionary microbiology.

[28]  W. Whitman,et al.  Diversity and Taxonomy of Methanogens , 1993 .

[29]  Y. R. Chen,et al.  Effect of Temperature and Retention Time on Methane Production from Beef Cattle Waste , 1980, Applied and environmental microbiology.

[30]  Y. Kamagata,et al.  Thermacetogenium phaeum gen. nov., sp. nov., a strictly anaerobic, thermophilic, syntrophic acetate-oxidizing bacterium. , 2000, International journal of systematic and evolutionary microbiology.

[31]  K. Kida,et al.  The effects of micro-aeration on the phylogenetic diversity of microorganisms in a thermophilic anaerobic municipal solid-waste digester. , 2004, Water research.

[32]  S. Zinder,et al.  NOTES: Methanosarcina thermophila sp. nov., a Thermophilic, Acetotrophic, Methane-Producing Bacterium , 1985 .

[33]  Sudhir Kumar,et al.  MEGA3: Integrated software for Molecular Evolutionary Genetics Analysis and sequence alignment , 2004, Briefings Bioinform..