The Study On Isolation, Purification And Identification Of Five Novel Species Of Bacillus

Bacillus are a kind of aerobic or facultative anaerobic bacteria, which can produce spores that are dormant and resistant forms of the cells that can survive more stressful environmental conditions. Bacillus contain many special functions, not only the degradation of crude oil, decomposition of insoluble compounds containing potassium or phosphorus, prevention and control of plant diseases, but also have a very extensive application in agriculture, industry, medicine and other fields.Therefore, it has a very important scientific significance to enrich the resources and research the application of Bacillus.Five samples were mainly collected from paddy soil and forest soil from Guangdong province and Hainan province in South China. Isolation was carried out using two different separation methods(serially diluted and spread on medium agar after incubation in enriched system & serially diluted and spread on medium agar directly) under different cultivated temperatures. In this experiment, about 200 strains of Bacillus were isolated. Five novel candidate species(C-89 T, GSS03 T, DX-4T,GD01 T and GSS02T) were selected from these strains and they were identified based on the study of phylogenetic, chemotaxonomic, and phenotypic analysis. As a result,this study would provide some help for the further research of Bacillus. The main conclusions of this stduy are summarized as follows:(1) The species of Bacillus isolated from two different methods and different temperature conditions are different.(2) Three bacillus, C-89 T, GSS03 T and DX-4T were isolated from a paddy soil sample, a forest soil sample and a sludge sample for bacterial isolation obtained from the electrochemically active biofilm, respectively.(A) Phylogenetic analyses based on 16 S r RNA gene sequences showed that strain C-89 T was most closely related to the type strains of Bacillus vietnamensis JCM 11124T(98.8 %),Bacillus aquimaris JCM 11545T(98.6 %) and Bacillus marisflavi JCM 11544T(98.5%). The DNA-DNA relatedness values of strain C-89 T with closest relativeswere all below enough for the 70% threshold accepted for species delineation,indicating that strain C-89 T represents a separate genomic species. Therefore, on the basis of phylogenetic, chemotaxonomic, and phenotypic results, strain C-89 T represents a novel species within the genus Bacillus, for which the name Bacillus haikouensis sp. nov., is proposed.(B) Phylogenetic analyses based on 16 S r RNA gene sequences indicated that strain GSS03 Twas most closely related to the type strains of B. psychrosaccharolyticus DSM 6T, B. muralis DSM 16288 T, B. asahii JCM 12112 T, B. simplex DSM 1321 T and B. frigoritolerans DSM 8801 T. The DNA-DNA reassociation values of strain GSS03 T with closest relatives were low enough for strain GSS03 T to be classified as a member of a separate genomic species. Therefore, on the basis of phylogenetic, chemotaxonomic, and phenotypic results, strain GSS03 T represents a novel species of the genus Bacillus,for which the name Bacillus huizhouensis sp.nov. is proposed.(C) The 16 S r RNA sequence of DX-4T showed the highest similarity with Bacillus aeolius 4-1T(95.4 %). On the basis of the results of physiological and biochemical tests, its phenotypic, phylogenetic properties and chemotaxonomic analysis, it can be firmly concluded that strain DX-4T represents a novel species of Bacillus, for which the name Bacillus borbori sp. nov. is proposed.(3) Two bacillus, GD01 T and GSS02 T were isolated from a mangrove forest soil sample and a forest soil sample, respectively.(A) The 16 S r RNA sequence of GD01 Tshowed the highest similarity with Oceanobacillus profundus DSM18246T(96.4 %). Because of the low 16 S r RNA gene sequence similarity to its closest relatives, other phenotypic and chemotaxonomic differences, and phylogenetic properties, it can be firmly concluded that strain GD01 T is considered to represent a novel species of Oceanobacillus for which the name Oceanobacillus halophilum sp. nov. is proposed.(B) Phylogenetic analyses based on 16 S r RNA gene sequences indicated that strain GSS02 T was most closely related to the type strains of Paenibacillus terrigena JCM 21741T(98.1 %).DNA-DNA relatedness value between strain GSS02 T and Paenibacillus terrigena JCM 21741 T was 58.8±0.5 %, which is well below the 70 % threshold accepted for species delineation. The phylogenetic, chemotaxonomic, and phenotypic results clearly demonstrated that strain GSS02 T belongs to the genus Paenibacillus and represents a novel species for which the name Paenibacillus guangzhouensis sp. nov. is proposed. In addition, according to the study about theHS- and Fe(III)-reducing ability of strain GSS02 T, it can be concluded that strain GSS02 T was capable of reducing both humic substances and FH under anaerobic conditions.