Nanobdella aerobiophila gen. nov., sp. nov., a thermoacidophilic, obligate ectosymbiotic archaeon, and proposal of Nanobdellaceae fam. nov., Nanobdellales ord. nov. and Nanobdellia class. nov.

A co-culture of a novel thermoacidophilic, obligate symbiotic archaeon, designated as strain MJ1T, with its specific host archaeon Metallosphaera sedula strain MJ1HA was obtained from a terrestrial hot spring in Japan. Strain MJ1T grew in the co-culture under aerobic conditions. Coccoid cells of strain MJ1T were 200-500 nm in diameter, and attached to the MJ1HA cells in the co-culture. The ranges and optima of the growth temperature and pH of strain MJ1T in the co-culture were 60-75 °C (optimum, 65-70 °C) and pH 1.0-4.0 (optimum, pH 2.5), respectively. Core lipids of dialkyl glycerol tetraethers (GDGT)-3 and GDGT-4 were highly abundant in MJ1T cells concentrated from the co-culture. Strain MJ1T has a small genome (0.67 Mbp) lacking genes for biosynthesis of essential biomolecules, such as nucleotides, lipids and ATP. The genomic DNA G+C content was 24.9 mol%. The 16S rRNA gene sequence of strain MJ1T was most closely related to that of the cultivated species, 'Nanopusillus acidilobi' strain N7A (85.8 % similarity). Based on phylogenetic and physiological characteristics, we propose the name Nanobdella aerobiophila gen. nov., sp. nov. to accommodate the strain MJ1T (=JCM 33616T=DSM 111728T). In addition, we propose the names Nanobdellaceae fam. nov., Nanobdellales ord. nov., and Nanobdellia class. nov. to accommodate the novel genus.

Conexivisphaera calida gen. nov., sp. nov., a thermophilic sulfur- and iron-reducing archaeon, and proposal of Conexivisphaeraceae fam. nov., Conexivisphaerales ord. nov., and Conexivisphaeria class. nov. in the phylum Thaumarchaeota.

A thermoacidophilic, anaerobic, and iron- and sulfur-reducing archaeon, strain NAS-02T, was isolated from a terrestrial hot spring in Japan, as previously reported. This organism is the first non-ammonia-oxidizing isolate in the phylum Thaumarchaeota. Here, we propose Conexivisphaera calida gen. nov., sp. nov. to accommodate this strain. The type strain of the type species is NAS-02T (=JCM 31663T=DSM 105898T). The values of 16S rRNA gene similarity and average amino acid identity between NAS-02T and its closest relatives are <86 and <42 %, respectively. Based on the phylogeny and physiology, we propose the family Conexivisphaeraceae fam. nov., the order Conexivisphaerales ord. nov. and the class Conexivisphaeria class. nov. to accommodate the novel genus.

Sulfuracidifex tepidarius gen. nov., sp. nov. and transfer of Sulfolobus metallicus Huber and Stetter 1992 to the genus Sulfuracidifex as Sulfuracidifex metallicus comb. nov.

Two novel, strictly aerobic, sulfur-dependent, thermoacidophilic strains, IC-006T and IC-007, were isolated from a solfataric field at Hakone Ohwaku-dani, Kanagawa, Japan. Cells of the two strains were irregular cocci with a diameter of 1.0-1.8 µm. They were strict aerobes and grew in a temperature range between 45 and 69 °C (optimally at 65 °C) and a pH range between 0.4 and 5.5 (optimally at pH 3.5). They required sulfur or a reduced sulfur compound, and sulfur was oxidized to sulfate. They grew autotrophically or mixotrophically utilizing several sugars and complex organic substances as carbon sources. The DNA G+C content was 42.4 mol%. A comparison of the 16S rRNA gene sequences among members of the order Sulfolobales indicated that they were closely related to Sulfolobus metallicus, forming an independent lineage within this order. The two isolates and Sulfolobus metallicus were also diffentiated based on their phenotypic properties from the other members of the order Sulfolobales. Detailed comparisons of the phenotypic properties and DNA-DNA hybridization study illustrated that the two isolates belong to a species different from Sulfolobus metallicus. On the basis of the phylogenetic and phenotypic comparisons, we propose a new genus and species, Sulfuracidifex tepidarius gen. nov., sp. nov. to accommodate strains IC-006T and IC-007. The type strain of Sulfuracidifex tepidarius is IC-006T (=JCM 16833T=DSM 104736T). In addition, Sulfolobus metallicus should be transferred to the new genus as Sulfuracidifex metallicus comb. nov.: the type strain is Kra23T (=DSM 6482T=JCM 9184T=NBRC 15436T).

Bioethanol production from steam-exploded rice husk by recombinant Escherichia coli KO11.

Rice husk is one of the most abundant types of lignocellulosic biomass. Because of its significant amount of sugars, such as cellulose and hemicellulose, it can be used for the production of biofuels such as bioethanol. However, the complex structure of lignocellulosic biomass, consisting of cellulose, hemicellulose and lignin, is resistant to degradation, which limits biomass utilization for ethanol production. The protection of cellulose by lignin contributes to the recalcitrance of lignocelluloses to hydrolysis. Therefore, we conducted steam-explosion treatment as pretreatment of rice husk. However, recombinant Escherichia coli KO11 did not ferment the reducing sugar solution obtained by enzymatic saccharification of steam-exploded rice husk. When the steam-exploded rice husk was washed with hot water to remove inhibitory substances and M9 medium (without glucose) was used as a fermentation medium, E. coli KO11 completely fermented the reducing sugar solution obtained by enzymatic saccharification of hot water washing-treated steam-exploded rice husk to ethanol. We report here the efficient production of bioethanol using steam-exploded rice husk.

Athalassotoga saccharophila gen. nov., sp. nov., isolated from an acidic terrestrial hot spring, and proposal of Mesoaciditogales ord. nov. and Mesoaciditogaceae fam. nov. in the phylum Thermotogae.

A novel moderately thermophilic, weakly acidophilic, heterotrophic, anaerobic, short-rod bacterium having an outer sheath-like structure (toga) was isolated from a low-salt acidic terrestrial hot spring in Oku-Shiobara, Tochigi, Japan. The strain, designated NAS-01T, grew between 30 and 60 °C (optimum 55 °C), and at pH 4.5 and 7.5 (optimum pH 5.5 to 6.0) and could not grow in media with ≥ 1 % NaCl (optimum 0 % NaCl). It utilized Fe(III), thiosulfate or l-cystine as electron acceptor for growth, and yeast extract, peptone or a variety of sugars as carbon and energy sources. The major cellular fatty acid was C16 : 0, and no lipoquinone was detected. Strain NAS-01T contained phospholipids and glycolipids, but not aminolipids. The DNA G+C content was 41.1 mol%. Phylogenetic analysis based on the 16S rRNA gene sequence revealed that the strain was included in the phylum Thermotogae, and was closely related to Mesoaciditoga lauensis. The 16S rRNA gene sequence similarity between them was 90 %, and they were deeply branched off the rest of the known Thermotogae species. On the basis of the phylogenetic and ecophysiological properties, strain NAS-01T represents a novel species of a new genus in the phylum Thermotogae, for which we propose the name Athalassotoga saccharophila gen. nov., sp. nov. The type strain of the type species is NAS-01T ( = JCM 19762T = DSM 28954T). In addition, we propose a new order and family, Mesoaciditogales ord. nov. and Mesoaciditogaceae fam. nov., respectively, to accommodate the novel genus and the closely related genus Mesoaciditoga.

Gene analysis, expression, and characterization of an intracellular α-amylase from the extremely halophilic archaeon Haloarcula japonica.

Haloarcula japonica is an extremely halophilic archaeon that requires high concentrations of NaCl to grow. Recently the draft genome sequence of Ha. japonica was determined, and a gene encoding an α-amylase, malA, was identified. The deduced amino acid sequence of MalA, consisting of 663 amino acids, showed homology to α-amylase family enzymes. The sequence did not contain a secretion signal sequence, indicating that the protein is a cytoplasmic enzyme. Transcription of the malA gene was confirmed by reverse transcription (RT)-PCR, and the transcription start site was determined by a 5'-RACE experiment. The malA gene was cloned and expressed in Ha. japonica. The recombinant MalA was purified and characterized. MalA required a high concentration of NaCl for starch-hydrolyzing activity. It showed higher activity on soluble starch, amylose, and amylopectin, and lower activity on glycogen.

Aciditerrimonas ferrireducens gen. nov., sp. nov., an iron-reducing thermoacidophilic actinobacterium isolated from a solfataric field.

An iron-reducing, moderately thermophilic, acidophilic actinobacterium, strain IC-180(T), isolated from a solfataric field in Hakone, Japan, was subjected to polyphasic taxonomic analysis. Strain IC-180(T) was a motile, short rod-shaped, Gram-positive bacterium that was able to grow at temperatures of 35-58 °C (optimally at 50 °C) and at pH 2.0-4.5 (optimally at pH 3.0). The strain grew aerobically and heterotrophically. It also grew anaerobically or autotrophically by dissimilatory reduction of ferric iron. No oxidation of ferrous iron was observed. Major cellular fatty acids detected were iso-C(16 : 0), anteiso-C(17 : 0) and iso-C(18 : 0); the major menaquinone was MK-9(H(8)). Phosphatidyl-N-methylethanolamine and an unknown ninhydrin-positive phosphoglycolipid were detected. The total DNA G+C content was 74.1 mol%. 16S rRNA gene sequence comparisons revealed that strain IC-180(T) was a member of the order Acidimicrobiales and clustered coherently with uncultured actinobacteria from a geothermal site and a bioreactor operated under moderately thermophilic conditions. This cluster could be distinguished from the two other clusters comprising the families of this order, Acidimicrobiaceae and Iamiaceae, respectively. Based on the properties of strain IC-180(T) determined in this polyphasic taxonomic study, this strain represents a novel species in a new genus in the order Acidimicrobiales, for which the name Aciditerrimonas ferrireducens gen. nov., sp. nov. is proposed; the type strain is IC-180(T) ( = JCM 15389(T)  = DSM 45281(T)).

Isolation and characterization of a thermophilic sulfur- and iron-reducing thaumarchaeote from a terrestrial acidic hot spring.

A deep-branching clade of Thaumarchaeota, conventionally called Terrestrial hot spring creanarchaeotic group (THSCG), is a missing link between thaumarchaeotic ammonia oxidizers and the deeper-branching non-ammonia oxidizers, such as Crenarchaeota and Candidatus Korarchaeota. Here, we report isolation of the first cultivated representative from the THSCG, named as NAS-02. Physiological characterization demonstrated that the isolate was a thermoacidophilic, sulfur- and iron-reducing organoheterotroph, which was supported by gene contents encoded in its complete genome. There was no evidence for ammonia oxidation by the isolate. Members in THSCG are likely thermophiles, and may play roles in degrading cell debris as a scavenger and in biogeochemical cycling of sulfur and iron in the hot environments, as suggested by the physiological characteristics of the isolate and the geographical distribution of the 16S rRNA gene sequences of THSCG in terrestrial hot springs and marine hydrothermal fields. Phylogenetic analysis suggests that the THSCG lineage represented by NAS-02 has gained the ability of sulfur reduction via horizontal gene transfer. Based on the phylogeny and physiology, we propose the name Conexivisphaera calidus gen. nov., sp. nov. to accommodate the isolate.

Identification of carotenoids from the extremely halophilic archaeon Haloarcula japonica.

The carotenoids produced by extremely halophilic archaeon Haloarcula japonica were extracted and identified by their chemical, chromatographic, and spectroscopic characteristics (UV-Vis and mass spectrometry). The composition (mol%) was 68.1% bacterioruberin, 22.5% monoanhydrobacterioruberin, 9.3% bisanhydrobacterioruberin, <0.1% isopentenyldehydrorhodopin, and trace amounts of lycopene and phytoene. The in vitro scavenging capacity of a carotenoid, bacterioruberin, extracted from Haloarcula japonica cells against 1,1-diphenyl-2-picrylhydrazyl (DPPH) radicals was evaluated. The antioxidant capacity of bacterioruberin was much higher than that of ß -carotene.

Thermogymnomonas acidicola gen. nov., sp. nov., a novel thermoacidophilic, cell wall-less archaeon in the order Thermoplasmatales, isolated from a solfataric soil in Hakone, Japan.

A novel thermoacidophilic, cell wall-less archaeon, strain IC-189T, was isolated from a solfataric field in Ohwaku-dani, Hakone, Japan. The cells were irregular cocci, sometimes lobed, club-shaped or catenated, and were highly variable in size, ranging from 0.8 to 8.0 microm in diameter. The strain grew at temperatures in the range 38-68 degrees C (optimally at 60 degrees C) and at pH 1.8-4.0 (optimally at around pH 3.0). Strain IC-189T was obligately aerobic and heterotrophic, requiring yeast extract for growth. Yeast extract, glucose and mannose served as carbon and energy sources. The polar lipids consisted mainly of cyclic or acyclic glycerol-bisdiphytanyl-glycerol tetraethers, and the predominant quinone was a menaquinone with seven isoprenoid units (MK-7). The G+C content of total DNA was 56.1 mol%. 16S rRNA gene sequence analysis revealed that strain IC-189T was a member of the order Thermoplasmatales, but diverged from the hitherto known species of the genera Thermoplasma, Picrophilus and Ferroplasma (86.2-91.0% sequence similarity). These phenotypic and phylogenetic properties clearly support a separate taxonomic status for this strain. Therefore, strain IC-189T represents a novel genus (order Thermoplasmatales) and species, for which the name Thermogymnomonas acidicola gen. nov., sp. nov. is proposed, with type strain IC-189T (=JCM 13583T=DSM 18835T).

Natronolimnobius baerhuensis gen. nov., sp. nov. and Natronolimnobius innermongolicus sp. nov., novel haloalkaliphilic archaea isolated from soda lakes in Inner Mongolia, China.

Three novel isolates of haloalkaliphilic archaea, strains IHC-005T, IHC-010, and N-1311T, from soda lakes in Inner Mongolia, China, were characterized to elucidate their taxonomic positions. The three strains were aerobic, Gram-negative chemoorganotrophs growing optimally at 37-45 degrees C, pH 9.0-9.5, and 15-20% NaCl. Cells of strains IHC-005T/IHC-010 were motile rods, while those of strain N-1311T were non-motile pleomorphic flats or cocci. The three strains contained diphytanyl and phytanyl-sesterterpanyl diether derivatives of phosphatidylglycerol and phosphatidylglycerophosphate methyl ester. No glycolipids were detected. On phylogenetic analysis of 16S rRNA gene sequences, they formed an independent cluster in the Natro group of the family Halobacteriaceae. Comparison of their morphological, physiological, and biochemical properties, DNA G + C content and 16S rRNA gene sequences, and DNA-DNA hybridization study support the view that strains IHC-005T/IHC-010 and strain N-1311T represent separate species. Therefore, we propose Natronolimnobius baerhuensis gen. nov., sp. nov. for strains IHC-005T (=CGMCC 1.3597T =JCM 12253T)/IHC-010 (=CGMCC 1.3598 = JCM 12254) and Natronolimnobius innermongolicus sp. nov. for N-1311T (=CGMCC 1.2124T =JCM 12255T).