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Kemnitz, D; Kolb, S; Conrad, R: Enrichment of Rice Cluster III Archaea from Soil and Phenotypic Characterization Prior to Isolation by using Real-Time PCR, Environmental Microbiology, 7(4), 553-565 (2005), doi:10.1111/j.1462-2920.2004.00723.x
A so far uncultured Euryarchaeote was enriched from anoxic soil and phenotypically characterized prior to isolation. The microorganism is related to the Thermoplasmatales and belongs to Rice Cluster III (RC-III). Enrichment resulted in a mixed methanogenic community of Bacteria and Archaea, the latter consisting of up to 60% of RC-III. The 16S rRNA gene sequence of the enriched RC-III archaeon was similar to that detected in the soil inoculum. The enrichment culture utilized yeast extract by transiently accumulating acetate as major fermentation product, which was subsequently converted to methane. The abundance of RC-III within the enrichment culture was quantified by terminal restriction fragment length polymorphism and real time PCR. Despite the about 1000-fold lower 16S rRNA gene numbers of RC-III in comparison to bacterial sequences, we were able to estimate growth parameters and physiological properties. The RC-III archaeon grew optimally at temperatures between 20 and 30°C and neutral pH (around 7) using yeast extract, meat extract, peptone or tryptone under anaerobic conditions. Doubling time was on the order of 3 days. No proliferation was detected on carbohydrates, amino acids, fatty acids, glycerol, alcohols, aromatic compounds, purine and pyrimidine bases or pyruvate. Oxygen, nitrate, ferric iron and sulfate did not support growth on yeast extract. RC-III was hardly affected by the antibiotics ampicillin, kanamycin and streptomycin. These findings suggest that the enriched representatives of RC-III are mesophilic anaerobic Archaea, which can grow heterotrophically on peptides.
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