Isolation And Phylogenetic Taxonomy Of Halophilic And Halotolerant Microorganisms

In this thesis, a brief summary has been made about the classification of halophilic microorganisms and halophilic mechanism. The advance classification of halophilic microorganisms and main methods applied to polyphasic taxonomy have also been stated. The aim on research of halophilic microorganisms has been introduced.Forty-eight halophilic and halotolerant strains were isolated from thirty-five samples in salt pool at Cangzhuo and in salt lakes of Hanghua port , which included forty-six actinomycetes and two fungi. In this study, Actinomycetes isolated have been analyzed using polyphasic taxonomy technology including morphological and physiological tests, analysis of chemotaxonomy, DNA G+C content and 16S rDNA/RNA sequencing. In addition, two fungi isolated have been done on morphological, physiological tests, DNA G+C content, 18S rDNA/RNA sequencing and life cycle.Morphological studies indicated that most strains' taxonomy status were hard to determine only by morphological characteristics. Most of the new isolations were moderately halophiles. They grew best in 5-10 % NaCl condition. They also could bear high alkaline condition and grow well at pH8.0-9.0. Diaminopimelic acid in the cell walls of actimomycetes was most meso-DAP. Main menaquinones were MK-9(H6),MK-9(H8) of Nocardiopsis .Strain 10001 was MK-9(H2). Physiological and chemical tests showed halophilic actinomycetes can utilize more carbon and produce starch enzyme and decompose casin.DNA G+C mol% and 16S rDNA sequence analysis were determined. DNA G+C mol% of strains matched to their genera. The phylogenetic relationship of eleven strains were revealed by phylogenetic analysis. Strain 10001 was belong to Cellulomonadaceae. The sequence homology with type strains of this family was 93.23%. It formed an indepent branch. The sequences difference between other species was on genus level. So it maybe a new genus. Strain 10035 belonged to Streptomyces. The sequencehomology with type strains of this genus was 97.54%. Strain 10002 belong to Nocardiopsis , the sequence homology with N.lucentensis was 98.51%. Strain 10010 belong to Nocardiopsis, the sequence homology with N.lucentensis was 98.80%.The two strains were similar to N.lucentensis. But they were different in colony colour, sugar of whole cell, Menaquinones characteristics and DNA G+C mol%. So they are maybe different species. Strain 10030 was similar to N.lucentensis with 98.94%. Strain 10036 was similar to N.lucentensis with 98.94%. Their sequence were similar with 100%, But they were different in sugar of whole cell, Menaquinones characteristics and DNA G+C mol%. So they were maybe different species. Strain 10021 was similar to N.antarctica. Strain 60002 was similar to N.antarctica. Their sequence homology was 99.86%. Strain 60006 was similar to N.antarctica. Strain 10011 and Strain 10019 were belong to Nocardiopsis.Strain 05001 was a extremely halophilic fungus.Its spores were sphericity with rough surfaces and its diameter was about 7-8um. It grew on GT medium with NaCl concentrations between 5 and 32%. But it grew optimally at. 15-20% NaCl (wt/vol). It grew best at 7.0. G+C content of strain 05001 was 65.85%. Phylogenetic analysis showed that strain 05001 was belong to Trichocomaceae. Strain 05001 has 98.91% similarity with it's nearest neighbor Eupenicillium javanicum. Phylogenetic tree showed strain 05001 was obviously distinguished from other genera of the family Trichocomaceae. Therefore, strain 05001 was probably a new taxon. Strain 05001was proposed as Streptosporomy halophila gen. nov. sp. nov. No sexual phase had been found. So strain 05001was temporarily belong to fungi imperfecti. Strain 05002 was another halotolerant fungus. It grew in 5-10 % NaCl condition but best at 0-5%.It optimally grew at pH 7.0 and 4-10℃ .So it belong to Psychrophiles.On the basis of the above results of polyphasic taxonomy, the phylogenetic structure and classification of unknown strains were exactly defined.Strain 10001 was a new genus of Cellulomonadaceae. Strain 10035 was a n...