Structure and function of the methanogenic archaeal community in stable cellulose-degrading enrichment cultures at two different temperatures (15 and 30°C)

• Published in: FEMS microbiology ecology 1999-12, Vol.30 (4), p.313-326
• Main Authors: Chin, Kuk-Jeong, Lukow, Thomas, Stubner, Stephan, Conrad, Ralf
• Format: Article
• Language: English
• Subjects: Acetic acid; Anaerobic conditions; Autosomal dominant inheritance; Cell culture; Cellulose; CH4 production; Deoxyribonucleic acid; DNA; Ecology; Fluorescence; Fluorescence in situ hybridization; Fluorescent in situ hybridization; Methane; Methanogenesis; Microbiology; Microorganisms; Phylogenetic tree; Polymerase chain reaction; Polymorphism; Restriction fragment length polymorphism; Ribosomal RNA; Rice field soil; rRNA; Sequence analysis; Structure-function relationships; Substrates; Temperature; Temperature optimum; T‐RFLP
• ISSN: 0168-6496; 1574-6941
• DOI: 10.1111/j.1574-6941.1999.tb00659.x

Abstract Methanogenic cultures were enriched from an air-dried rice field soil and incubated under anaerobic conditions at 30°C with cellulose as substrate (ET1). The culture was then transferred and further incubated at either 15°C (E15) or 30°C (E30), to establish stable cultures that methanogenically degrade cellulose. After five transfers, the rates of CH4 production became reproducible. At 30°C, CH4 production rates were (mean±S.D.) 15.2±0.7 nmol h−1 ml−1 culture for the next 16 transfers and at 15°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°C, CH4 production rates increased with the temperature, reached a maximum at 40°C and then decreased. The same temperature optimum was observed in E15, but with a lower maximum CH4 production rate. The apparent activation energies of CH4 production were similar (about 120 kJ mol−1) for the cultures at 15 and 30°C. Methanogenesis was not limited by acetate which was >4 mM at the beginning of the assay. The structure of the archaeal community was analyzed by molecular techniques. Total DNA was extracted from the microbial cultures before the transfer to different temperatures (ET1) and afterwards (E15, E30). The archaeal small subunit (SSU) ribosomal RNA-encoding genes (rDNA) of these DNA samples were amplified by PCR with archaeal-specific primers and characterized by terminal restriction fragment length polymorphism (T-RFLP). After obtaining a constant T-RFLP pattern in the cultural transfers at 15 and 30°C, the PCR amplicons were 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 dominated by clones representing ‘rice cluster I’, a novel lineage of methanogenic Euryarchaeota. However, further transfers resulted in the dominance of Methanosarcinaceae and Methanosaetaceae at 30 and 15°C, respectively. This dominance was confirmed by fluorescence in situ hybridization (FISH) of archaeal cells. Obviously, different archaeal communities were established at the two different temperatures, but their activities nevertheless exhibited similar temperature optima.