Phenotypic characterization of Rice Cluster III archaea without prior isolation by applying quantitative polymerase chain reaction to an enrichment culture

A so far uncultured member of the Euryarchaeota was enriched from an anoxic riparian soil and phenotypically characterized using quantitative polymerase chain reaction (qPCR; 'real-time PCR'). The microorganism is related to the Thermoplasmatales and belongs to Rice Cluster III (RC-III). Enrichment cultures utilized yeast extract (YE) by transiently accumulating acetate as major fermentation product, which was subsequently converted to methane. The abundance of RC-III archaea within the enrichment cultures was quantified by analysis of the terminal restriction fragment length polymorphism (T-RFLP) and by qPCR. We developed qPCR assays targeting the 16S rRNA genes (16S rDNA) specific for RC-III as well as for the Archaea in general. The enrichment cultures consisted of a mixed methanogenic community of Bacteria and Archaea, the latter consisting of up to 60% of members of RC-III. The other archaea belonged to Methanosarcinaceae, Methanomicrobiaceae and Methanobacteriaceae. The enriched RC-III archaea were represented by two sequences (LL25A, LL37A) that were highly similar to each other and to those detected in the soil inoculum (> 98% similarity). However, the 16S rDNA copy numbers of RC-III archaea were about 1000-fold lower than those of Bacteria. Nevertheless, we were able to estimate growth parameters and physiological properties of one of the enriched RC-III archaea (LL25A) by measuring the increase of 16S rDNA copy numbers specific for this group under different growth conditions. The enriched RC-III archaeon grew optimally at temperatures between 20 and 30 degrees C and neutral pH using YE, meat extract, peptone or tryptone under anoxic conditions. Doubling time was approximately 3 days. No proliferation was detected on carbohydrates, amino acids, fatty acids, glycerol, alcohols, aromatic compounds, purine and pyrimidine bases or pyruvate. Various exogenous electron acceptors (e.g. ferric iron, S(0)) did not support growth on YE. Proliferation of the enriched RC-III archaeon was hardly affected by the antibiotics ampicillin, kanamycin and streptomycin. These findings suggest that the enriched archaeon is a mesophilic anaerobe, which can grow heterotrophically on peptides. Further enrichment on peptone and kanamycin eventually allowed the microscopic detection of coccoid cells stained by fluorescence in situ hybridization (FISH).