Charakterisierung unkultivierter Archaea aus Böden

Due to the development of molecular techniques many new lineages of Euryarchaeota and Crenarchaeota from soils have been detected. However, the phenotypes of the microorganisms belonging to these lineages are widely unknown, since isolates are lacking. The goal of this study was to get new information on these uncultured Archaea. The enrichment of Rice Cluster (RC-) III-Archaea (Thermoplasmatales relatives) from two anoxic soils of the River Waal was carried out in an anoxic mineral medium with yeast extract as complex substrate. The successful enrichment of these Euryarchaeota was shown by the T-RFLP method and the phylogenetic analysis of clone sequences. Using the real time-PCR-method it was possible to follow the growth of the RC-III-Archaea within the microbial community. The slowly growing microorganisms (doubling time approx. 3 days) showed a high abundance within Archaea (up to 60%). Due to of the high background of different Bacteria (1 billion 16S rDNA target molecules per ml) the relative ratio of RC-III constituted only approximately 0.1% of the whole microbial community. However, the phenotype of the RC-III Archaea could be described. They were characterized as anaerobic, heterotrophic, mesophilic and neutrophilic microorganisms, being supposedly involved in the degradation of oligopeptides. After further enrichment of RC-III-Archaea with peptone as substrate, the shape could be described as coccoid with the help of fluorescence in situ hybridization. Furthermore the abundance as well as the phenotype of methanogenic RC-I-Archaea were investigated in an enrichment culture obtained from rice field soil (Lueders et al., 2001). The ratio of these Euryarchaeota within the microbial community represented approx. 50%. These RC-I methanogens were thermophil and hydrogenotrophic, in edition growing on format. Beside the close relation to the acetoclastic Methanosarcinales, RC-I Archaea were not able to thrive on acetate. For the first time it succeeded to singularize RC-I colonies from solid medium. The RC-I Archaea were only isolated together with a characteristic accompanied bacterial flora. The detection of the coenzym F420, which occurs in the methanogenic pathway, was shown by means of the autofluorescence of the rod shaped cells. The molecular investigation of the depth profile of a forest soil led to the detection of uncultured Euryarchaeota and Crenarchaeota. Sequences of the Euryarchaeota were closest related with the Thermoplasmatales, whereas the Crenarchaeota clustered together with Group 1.1b and 1.1c (described before by DeLong, 1998). In all soil layers the dominance of Crenarchaeota (most notably Group 1.1c) within Archaea was shown by T-RFLP analysis. The amount of Archaea and Bacteria was determined with the help of the real time-PCR-method. The number of Archaea hardly changed with depth, but their relative ratio of the whole microbial community increased. In comparison with previous soil studies the investigated forest soil showed a very high abundance of 60% of Archaea.