Construction and estimation of a novel isolation method for thermophilic archaea from hot springs.

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We have developed a new isolation method for thermophilic archaea, in which diluted hot spring samples were distributed according to the MPN (most probable number) method and incubated under various growth conditions, followed by detection of microorganisms in grown cultures by means of PCR using specific primers for.certain genera of the thermophilic archaea. Otherwise, the microorganisms were tentative assigned by the partial 16S rDNA sequencing.From a sample of solfataric soil at Ohwakudani (90℃, pH 2.0), strains related to Sulfurisphaera (under air, at 85℃, pH 2.0), Acidianus and Stygiolobus (H_2-CO_2, 70℃, pH 4.0) were detected. From another sample of solfataric soil at Ohwakudani (60℃, pH 1.4), Acidianus were grown un der H_2-CO_2, at 70℃, pH 4.0. Under air, at 70℃, pH5.0, novel member of Sulfolobales were detected in addition to Acidianus, Sulfurisphaera, Acidianus brierlyei, and Sulfolobus hakonensis. Furthermore, Picrophilus and a novel Thermoplasmatales genus were detected under air, at 55℃, pH 2.5. From hot spring water collected at Soun-zan Onsen (40℃, pH 2.4),Sulfurisphaera and Metallosphaera were cultivated at 85℃ and 70℃, respectively, under air, at pH5.0.Thus, it would be noteworthy that the above mentioned procedure allowed to isolate the thermophilic archaea that have never been isolated before from the Hakone hot springs such as strains of Stygiolobus and the new Sulfolobales genus.