High Throughput Culturing And Polyphasic Taxonomy Of Marine Bacteria And Mining Of Enteromorpha Prolifera Cellulose Degrading Enzymes

The ocean makes up 71%of the earth’s surface area,containing huge marine microbial resources.Marine microorganisms have diverse and complex physiological and metabolic characteristics and important ecological significance and huge application potential.However,due to the limitations of isolation and culture methods,the majority of marine microorganisms presently can not be cultured.The high-throughput dilution-to-extinction culturing method can improve the culturability of marine microorganisms and hence obtain marine microbial strains with more diversity and representativeness for further studying their properties and ecological roles.In recent years,Enteromorpha prolifera blooms often occur in the offshore areas of Qingdao city,China,causing ecological disasters.However,due to its rapid growth,Enteromorpha prolifera has become a rich and economic marine biomass resource Mining Enteromorpha prolifera cellulose degrading enzymes and developing novel tool enzymes for algae processing so as to efficiently degrade cellulose in Enteromorpha prolifera is of high importance for exploiting Enteromorpha prolifera biomass resources and relieving offshore environmental pressureIn this thesis,marine bacterial strains were firstly isolated by using the high-throughput dilution-to-extinction culturing method from surface seawater samples collected from offshore of Qingdao city,China and diversity of the isolated strains was analyzed;two of the isolated bacterial strains were selected for taxonomic characterization using a polyphasic approach to determine their taxonomic positions.Secondly,from the genomes of three Enteromorpha prolifera degrading bacterial strains,12 potential cellulase were mined,and the abilities of these enzymes to degrade(hydrolyze)Enteromorpha prolifera cellulose were determined through heterologous expression and enzyme activity determination.The specific research results of this thesis are as follows:1.Isolation of marine bacteria by high-throughput dilution-to-extinctlion culturing method.A total of 111 bacterial strains were isolated using the high-throughput dilution-to-extinction culturing method from surface seawater samples collected from three sites in Taiping cape,Tianheng island and Aoshan bay,of Qingdao city,China Their 16S rRNA gene sequences were further determine.γ-proteobacteria were found to account for 71.17%and α-proteobacteria for 25.23%of all isolated strains.These strains belonged to 22 genera and two clades,with the dominant genus being Pseudoalteromonas(48.65%).There were 12 strains accounting for 10.8%of the total isolated strains were likely to represent new species of bacteria with 16S rRNA gene sequence similarity with known bacterial species lower than 98.0%.In addition,four oligotrophic strains were obtained,of which three belonged to the SAR92 clade,one belonged to the OM43 cladeStrains SM1969T and SM1979T were two of the strains isolated using the high-throughput dilution-to-extinction and were found to share 97.0%16S rRNA gene sequence similarity with each other and<98.0%highest 16S rRNA gene sequence similarity with known species,they were therefore selected for further polyphasic characterization to determine their taxonomic positions.Strains SM1969T and SMvi79T were oval-shaped,non-flagellated,motile and strictly aerobic gram-negative bacteria.They did not reduce nitrate to nitrite and were both oxidase-and catalase-positive.Strain SM1969T grew at 10-37℃(optimum,25-30℃),pH 6.5-10.0(optimum,pH 6.5-7.0)and with 1-6.5%(w/v)NaCl(optimum,2.5%)Strain SM1979T grew at 15-37℃(optimum,25-30℃),pH 6.0-10.0(optimum,pH 7.0)and with 1-6%(w/v)NaCl(optimum,3%).Their predominantpolar lipids included phosphatidylglycerol,phosphatidylcholine,one aminolipid and one unidentified lipid;their major fatty acids were summed feature 8(C18:1 ω7c and/or C18:1 ω6c)with their sole respiratory quinone being ubiquinone 10.The genomic DNA G+C content of strains SM1969T and SM1979T were 56.4 and 57.5 mol%.In the phylogenetic tree based on the single-copy orthologous clusters sequences,strains SM1969T and SM1979T clustered with known species of the genus Tritonibacter and formed a separate branch together with Tritonibacter ulvae.Based on the phenotypic chemotaxonomic and phylogenetic characterization for strains SM169T and SM1979T,they represelt two new species of the genus Tritonibacter.The names Tritonibacter aquimaris sp.nov.and Tritonibacter litoralis sp.nov.are proposed for the two new species,with type strains being SM1969T(=MCCC 1K04320T=KCTC 72843 T)and SM1979T(=MCCC 1K04321T=KCTC 72842T),respectively.2.Mining of Enteromorpha prolifera cellulose degrading enzymesA total of 12 potential cellulase(including enzymes 1-2,1-3,1-4,1-5,3-1,3-8,3-9)were minded from genomes of three Enteromorpha prolifera dedrading strains including A lteromonas sp.HT-1,Photobacterium sp.HT-2 and Echinicola sp.HT-3.These potential cellulases were further heterologously expressed in E.coli.Respectively with CMC-Na,Avicel and p-nitrophenyl-β-D-glucoside(pNPG)as substrates,the enzyme activity determination experiments found that enzymes 1-4,1-5,3-1,3-8 and 3-9 were be endoglucanases,enzymes 1-2 and 1-3 were exoglucanases,and enzymes 2-1,3-2,3-3,3-5 and 3-6 wereβ-glucanases.With Enteromorpha prolifera cellulose as substrate,endoglucanases 1-4,1-5,3-1,3-8,3-9 and exoglucanases 1-2,1-3 were found to have the ability to degrade(hydrolyze)Enteromorpha prolifera cellulose with enzyme 3-8 showing the highest activity.