Structure and function of the methanogenic archaeal community in stable cellulose-degrading enrichment cultures at two different temperatures (15 and30 degrees C)

Methanogenic cultures were enriched from an air-dried rice field soil and i ncubated under anaerobic conditions at 30 degrees C with cellulose as subst rate (ET1). The culture was then transferred and further incubated at eithe r 15 degrees C (E15) or 30 degrees C (E30), to establish stable cultures th at methanogenically degrade cellulose. After five transfers, the rates of C H4 production became reproducible. At 30 degrees C, CH4 production rates we re (mean +/- S.D.) 15.2 +/- 0.7 nmol h(-1) ml(-1) culture for the next 16 t ransfers and at 15 degrees C, they were 0.38 +/- 0.07 nmol h(-1) ml(-1) for the next six transfers. When E30 was assayed at temperatures between 5-50 degrees C, CH4 production rates increased with the temperature, reached a m aximum at 40 degrees C and then decreased. The same temperature optimum was observed in E15, but with a lower maximum CH4 production rate. The apparen t activation energies of CH4 production were similar (about 120 kJ mol(-1)) for the cultures at 15 and 30 degrees C. Methanogenesis was not limited by acetate which was >4 mM at the beginning of the assay. The structure of th e archaeal community was analyzed by molecular techniques. Total DNA was ex tracted from the microbial cultures before the transfer to different temper atures (ET1) and afterwards (E15, E30). The archaeal small subunit (SSU) ri bosomal RNA-encoding genes (rDNA) of these DNA samples were amplified by PC R with archaeal-specific primers and characterized by terminal restriction fragment length polymorphism (T-RFLP). After obtaining a constant T-RFLP pa ttern in the cultural transfers at 15 and 30 degrees C, the PCR amplicons w ere used for the generation of clone libraries. Representative rDNA clones (n = 10 for each type of culture) were characterized by T-RFLP and sequence analysis. In the primary culture (ET1), the archaeal community was dominat ed by clones representing 'rice cluster I', a novel lineage of methanogenic Euryarchaeota. However, further transfers resulted in the dominance of Met hanosarcinaceae and Methanosaetaceae at 30 and 15 degrees C, respectively. This dominance was confirmed by fluorescence in situ hybridization (FISH) o f archaeal cells. Obviously, different archaeal communities were establishe d at the two different temperatures, but their activities nevertheless exhi bited similar temperature optima. (C) 1999 Federation of European Microbiol ogical Societies. Published by Elsevier Science B.V. All rights reserved.