Isolation And Polyphasic Taxonomy Studies Of Thermophilic Bacterium From Changbai Mountains' Hot Springs

For Thermophilic microorganism and enzymes, the research on them can not only develop various novel applications on biological engineering industry, but also reveal the essence of life i.e. heat-genesia theory. Changbai mountains exhibit an aechaeo-ecological system because of its vulcanian geology and special geography environment, with many hot springs existing redundant thermophilic microorganisms and gene resource. This paper has investigated the diversity and evolution relationship of cultured thermophilic microorganism from Changbai mountains'hot springs. Several novel hyper-thermophilic species have been isolated.Changbian mountains'hot springs are neutral (pH of water, ~7.2) and rich of metal ion and acid radical. Based on this characteristic various mediums of thermophilc microorganisms are chosen for cultivation to enrich the microorganisms of samples from hot springs. By the classic culture method and gradient dilution isolation method, eight thermophilic strains are obtained, named as CBS-2 to CBS-9.To determine the taxonomy position of the newly isolated thermophilic bacterium in evolution history, 16S rRNA sequence homologous alignment and phylogeny analysis are performed between them and related species. The result shows that they belong to four genera from different three families, indicating the extensive microorganism diversity of Changbai mountains'hot springs. CBS-2, CBS-3, CBS-4 and CBS-8 belong to Thermotog, which is located at the foot of cladogram of three domains for life and is the most thermophilic branch in bacterium domain. It is suggested that members of Thermotoga are close to aechaeo-life. 16S rRNA sequence homology between CBS-4 strain and other species of Fervidobacterium genus are 90-97%. 16S rRNA sequence homology are 99-99.8% homology among CBS-2, CBS-3, CBS-8 and hyperthermophilic bacterium Fervidobacterium changbaicum CBS-1T identified by our lab, and they are in the same species level. 16S rRNA sequence homology between CBS-5 and CBS-6 are 99.8 %, with 95-98 % homology between them and species of Anoxybacillus. 16S rRNA sequence alignment of CBS-7 shows that the homology between it and species of Brevibacillus are 97-99 %. The data indicates that two strains (CBS-4 and CBS-5) of the eight newly isolated thermophilic strains are potential new species, and that they belong to Thermotoga and Bacillus family respectively.As indicated by the result of 16S rRNA sequence homologous alignment, strain CBS-4 shows low homology (90-97%) with other species of Fervidobacterium genus. To verify the taxonomy position of strain CBS-4, polyphasic taxonomy methods have been performed on genotype, phenotype and chemical taxonomy. The physiological and biology chemical characteristics of strain CBS-4 are shown as following:It is Gram-negative, non-sporulating, rod shaped and generally occurrs singly or in pairs. Cells size is 0.5-0.6×2-4μm. Most of the cells form a spheroid structure"toga"at the end. Growth occurs at 40-80°C (optimum 70°C), at pH 6.5-8.2 (optimum 7.5), at the NaCl concentration less than 6.0 g/L. The generation time at optimum growth condition is 124 min. It grows on a wide range of carbon sources, including glucose, mannose, strach, arabinose, lactose, rhamnose, maltose, galactose, sucrose, D-xylose, fructose, cellobiose, cellulose, CMC-Na, L-aspartate, pyruvate and so on. Sulphur powder can stimulate the growth. Growth is inhibited by 10μg/ml kanamycin, ampicillin, tetracycline and carbenicillin respectively, and 100μg/ml tetracycline and carbenicillin respectively. GC content of genome DNA of strain CBS-4 measured by T_m method is 27.25±0.50 mol%, which is the lowest among the species of Fervidobacterium genus. 16S rRNA homology and phylogeny analysis show that strain CBS-4 is relative to the genera Fervidobacterium, Thermotoga and Thermosipho in Thermotoga family, especially to the type stain of Fervidobacterium genus F. nodosum. Strain CBS-4 and F. nodosum locate at the same branch of cladogram, with 97.6% homology between their 16S rRNA sequence. Fatty acids profile of strain CBS-4~T, such as the most dominant fatty acid C16: 0 as species of Fervidobacterium, supported the assignment of this strain to the genus Fervidobacterium. However, there was markedly divergence in the content of the major fatty acid. DNA-DNA hybridization between strain CBS-4 and four species of Fervidobacterium shows DNA homology lower than 70% (F. nodosum, 44.88%; F. changbaicum, 51.22%; F. pennivarons and F. islandicum,≤5%). Based on the data of polyphasic taxonomy, we propose to assign strain CBS-4T to a novle species of the genus Fervidobacterium, for which we propose the name Fervidobacterium tianchisum sp. nov.. The type strain is strain CBS-4~T (=JCM14752~T).