Notes: The aim of this study was to examine whether the terminal restriction fragment length polymorphism (T-RFLP) analysis represents an appropriate technique for monitoring highly diverse soil bacterial communities, i.e. to assess spatial and/or temporal effects on bacterial community structure. The T-RFLP method, a recently described fingerprinting technique, is based on terminal restriction fragment length polymorphisms between distinct small-subunit rRNA gene sequence types. This technique permits an automated quantification of the fluorescence signal intensities of the individual terminal restriction fragments (T-RFs) in a given community fingerprint pattern. The indigenous bacterial communities of three soil plots located within an agricultural field of 110 m2 were compared. The first site was planted with non-transgenic potato plants, while the other two were planted with transgenic GUS and Barnase/Barstar potato plants, respectively. Once prior to planting and three times after planting, seven parallel samples were taken from each of the three soil plots. The T-RFLP analysis resulted in very complex but highly reproducible community fingerprint patterns. The percentage abundance values of defined T-RFs were calculated for the seven parallel samples of the respective soil plot. A multivariate analysis of variance was used to test T-RFLP data sets for significant differences. The statistical treatments clearly revealed spatial and temporal effects, as well as space×time interaction effects, on the structural composition of the bacterial communities. T-RFs which showed the highest correlations to the discriminant factors were not those T-RFs which showed the largest single variations between the seven-sample means of individual plots. In summary, the T-RFLP technique, although a polymerase chain reaction-based method, proved to be a suitable technique for monitoring highly diverse soil microbial communities for changes over space and/or time.

Notes: 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.