Isolation of the anaerobic thermoacidophilic crenarchaeote Acidilobus saccharovorans sp. nov. and proposal of Acidilobales ord. nov., including Acidilobaceae fam. nov. and Caldisphaeraceae fam. nov.

An anaerobic acidophilic, hyperthermophilic archaeon, designated strain 345-15(T), was isolated from an acidic hot spring of Kamchatka (Russia). Cells of strain 345-15(T) were regular or irregular cocci, 1-2 mum in diameter, with flagella. Strain 345-15(T) grew optimally at 80-85 degrees C and pH 3.5-4.0 and fermented a wide range of carbohydrates, including polysaccharides. Acetate, ethanol and lactate were the fermentation products. Growth was stimulated by elemental sulfur and thiosulfate, which were reduced to hydrogen sulfide. The G+C content of the DNA was 54.5 mol%. The 16S rRNA gene sequence analysis indicated that strain 345-15(T) belonged to the genus Acidilobus. The level of DNA-DNA hybridization between strain 345-15(T) and Acidilobus aceticus 1904(T) was 61 %. Thus, strain 345-15(T) was considered as representing a novel species of the genus Acidilobus, with the name Acidilobus saccharovorans sp. nov. (type strain, 345-15(T)=DSM 16705(T)=VKM B-2471(T)), which shared the main morphological and physiological properties of the genus but differed by the presence of flagella and the spectrum of substrates utilized. Phylogenetic analysis showed that the genus Acidilobus, with its species Acidilobus aceticus, Acidilobus saccharovorans sp. nov. and 'Acidilobus sulfurireducens', and the genus Caldisphaera, represented by Caldisphaera lagunensis and 'Caldisphaera draconis', formed a separate cluster that adjoins the cluster formed by the species of the order Desulfurococcales. Members of the Acidilobus-Caldisphaera cluster are thermophilic, organotrophic anaerobic cocci that can be distinguished from all species of the order Desulfurococcales on the basis of acidophily. Based on these considerations, we propose a new family, Acidilobaceae fam. nov., to accommodate the subcluster of hyperthermophiles represented by the genus Acidilobus, a new family, Caldisphaeraceae fam. nov., for the subcluster of extreme thermophiles represented by the genus Caldisphaera, and a new order, Acidilobales ord. nov., to accommodate the two new families.

Thermoanaerobacterium aciditolerans sp. nov., a moderate thermoacidophile from a Kamchatka hot spring.

An anaerobic, moderately thermoacidophilic bacterium, strain 761-119T, was isolated from an acidic hot spring in the Orange Field of the Uzon Caldera (Kamchatka, far-eastern Russia). Cells were spore-forming, Gram-positive rods, possessing one polar flagellum. Growth of strain 761-119T was observed between 37 and 68 degrees C and in the pH(20 degrees C) range 3.2-7.1. No growth was observed within 5 days of incubation at or below 35 degrees C and at or above 70 degrees C, as well as at or below pH(20 degrees C) 2.8 and at or above pH(20 degrees C) 7.5. The optimal temperature and pH(20 degrees C) for growth were 55 degrees C and pH(20 degrees C) 5.7, respectively. A wide range of carbohydrates and polysaccharides were fermented, as well as peptides and proteinaceous substrates. The main products of glucose fermentation were acetate, ethanol, lactate, H2 and CO2. The DNA G+C content was 34 (+/-0.5) mol%. 16S rRNA gene sequence analysis indicated that strain 761-119T belonged to the genus Thermoanaerobacterium. The level of 16S rRNA gene sequence similarity with other Thermoanaerobacterium species was 86.5-97.8 %, with the only moderately acidophilic member of this genus, Thermoanaerobacterium aotearoense, being one of its closest relatives. DNA-DNA hybridization with T. aotearoense showed 33 % relatedness. Thus, morphological (one polar flagellum) and physiological characteristics (lower pH limit of growth at pH(20 degrees C) 3.2 compared with T. aotearoense) and 16S rRNA gene sequence analyses revealed that strain 761-119T represents a novel species in the genus Thermoanaerobacterium, for which the name Thermoanaerobacterium aciditolerans sp. nov. is proposed, with the type strain 761-119T (=DSM 16487T=VKM B-2363T).

Acidilobus aceticus gen. nov., sp. nov., a novel anaerobic thermoacidophilic archaeon from continental hot vents in Kamchatka.

New thermoacidophilic organisms that were able to grow anaerobically on starch were isolated from the acidic hot springs of Kamchatka. Strain 1904T, isolated from a hot spring of the Moutnovski volcano, was characterized in detail. Its cells were regular or irregular cocci that were 1-2 microm in diameter, non-motile, and had a cell envelope consisting of one layer of subunits. The new organism was a hyperthermophile, growing in the temperature range 60-92 degrees C (with an optimum at 85 degrees C), an acidophile, having the pH range for growth of 2.0-6.0 (with an optimum at 3.8), and an obligate anaerobe. It fermented starch, forming acetate as the main growth product. Other growth substrates were yeast extract, beef extract and soya extract. Growth on yeast extract, beef extract and soya extract was stimulated by elemental sulfur, which was reduced to H2S. Acetate, arabinose, cellulose, formate, fructose, galactose, glucose, glycine, guar gum, lichenan, malate, maltose, methanol, pectin, pyruvate, propionate, xylan, xylose or a mixture of amino acids failed to support growth both in the presence and the absence of sulfur. When starch was used as the growth substrate, yeast extract (100 mg l(-1)) was required as a growth factor. The G+C content of the DNA was found to be 53.8 mol%. Comparison of the complete 16S rDNA sequence with databases revealed that the new isolate belonged to the kingdom Crenarchaeota. It was not closely related to any described genera (showing sequence similarity below 90.8%) and formed a separate branch of the Crenarchaeota. On the basis of physiological differences and rRNA sequence data, a new genus--Acidilobus--is proposed, the type species being Acidilobus aceticus strain 1904T (= DSM 11585T).