Studies Of Cultivated Marine Bacterial Diversities In Two Specific Ecological Environments In Qingdao Coastal Area And Taxonomic Analysis Of Three Novel Bacteria

The ocean, which contains abundant microbial resources, is an essential part of thelife system on earth and is important for achieving sustainable development. Marinemicroorganisms play a key role in maintaining the diversity of marine ecosystem. Thereare considerable diversities in terms of microbial taxa, metabolic pathways and products,which indicate they possess rich novel resources. It has been showed that, with thedevelopment of scientific technology and economy, the marine living resources and theecosystem are seriously destroyed by unreasonable exploitation of marine resources.Therefore, in order to exploit the marine microbial resources comprehensively andsustainably, as well as avoid excessive waste, studies on genetic characteristics,physiological features and metabolites of marine microorganisms seem to be of greattheoretical significance and practical value.In this thesis, cultivated marine bacterial diversities have been studied in twospecific ecological environments in Qingdao coastal area, and taxomic positons of threenovel bacteria have been analysed on the basis of phenotypic, chemotaxonomic andphylogenetic distinctiveness.To study the bacterial diversity of involved in biofilm, epiphytic microorganismswere isolated from4large seaweeds, i.e., Gloiosiphoniaelin Berkeley, Undariapinnatifida, Colpomenia sinuosa, and Enteromorpha prolifra, collected from theoffshore sea water of Qingdao where the massive green algae （Enteromorpha prolifera）bloom broke out in June2008.99strains were isolated from these seaweeds by usingfour types of culture media, i.e.,2216E, marine R2A, TCBS and marine agar. TheProteobacteria is the dominant Phylum in terms of quantity and species according tothe analysis of16S rDNA sequences, while most of the strains belong to the class of Gamaproteobacteria. There are94strains belong to the Gammaproteobacteria,3strainsbelong to the Alphaproteobacteria,1strain belongs to the Actinobacteria and1strainbelongs to the Firmicutes. There are three genera, Pseudoalteromonas （22）, Vibrio （20）and Halomonas （15）, taking the hightest possession of abundance ratio. And theremaining strains belong to Alcanivorax （9）, Enterobacter （8）, Pseudomonas （7）,Aeromonas （5）, Alteromonas （4）, Escherichia （1）, Marinomonas （1）, Psychromonas （1）,Photobacterium （1）, Pseudoruegeria （1）, Erythrobacter （1）, Loktanella （1）, Bacillus （1）and Curtobacterium （1）. This study showed that there was a rich diversity on thepopulation of marine epiphytic bacterica in the period of green tide outbreak.Nevertheless, the dominant flora and community structure were different from thenormal period of the local ecosystem.Conventional cultivation-based method was used to investigate the marine bacterialdiversity from bottom seawater samples of the amphioxus breeding site in Qingdaocoast.33representative strains were selected to be classified from all of the213strains.16S rDNA sequence analysis showed that Gammaproteobacteria take the major role ofmarine bacteria in this region, including Vibrio （13）, Halomonas （7）, Pseudomonas （4）,Pseudoalteromonas （3）, Psychromonas （1），Marinomonas （1），Stenotrophomonas （1）.And another2strains belong to the Phaeobacter of Alphaproteobacteria.1strainbelongs to the Salinicoccus of Firmicutes.A Gram-negative, non-spore-forming, catalase-and oxidase-positive, strictlyaerobic, curved-rod shaped bacterium with polar flagellum, designated strain JYr2^T,was isolated from a sediment sample of an amphioxus breeding zone in the coastalregion of Qingdao, China. The organism grew optimally at37C, pH8-9and in thepresence of5-6%（w/v） NaCl. It contained isoprenoid quinine8（Q-8,100%） as thepredominant isoprenoid quinine. The major fatty acids were C16:0（30.2%） and C16:1ω7cand/or C16:1ω6c （34.6%）. Phosphatidylethanolamine （PE）, phosphatidylglycerol （PG）,aminophospholipid （PN） and two kinds of aminolipid （AL1, AL2） were the majorconstituents of the phospholipids. A phylogenetic analysis based on16S rRNA genesequences indicated that strain JYr2^Tformed a distinct evolutionary lineage within thefamily Colwelliaceae of the class γ-Proteobacteria. It showed <95.0%sequence similarities to all published species of the family, except95.2%to Thalassomonasviridans DSM13754Tand95.02%to Colwellia polaris537T. The G+C content of theDNA was42.7mol%. On the basis of the polyphasic taxonomic study, strain JYr2T（=CGMCC1.10794T=JCM17549T） was considered to represent a novel genus andspecies in γ-Proteobacteria, for which the name Litorilituus sediminis gen. nov. sp. nov.was proposed.A novel Gram-negative, strictly aerobic, motile with peritrichous flagella bacterium,designated JYr12^T, was isolated from sediment of an amphioxus breeding zone in thecoastal region of Qingdao, China. Strain JYr12^Tgrew optimally at28C, pH7and in thepresence of2-3%（w/v） NaCl. The major fatty acids were C16:0（28.3%）, and C16:1ω7cand/or C16:1ω6c （34.8%）. It contained Q-8（90%） and Q-7（7%） as the major isoprenoidquinine. Phosphatidylglycerol （PG）, phosphatidylethanolamine （PE） were the majorconstituents of the phospholipids, following with a small amount ofdiphosphatidylglycerol （DPG）, an unknown phospholipid （PL1） and an unknownglycolipid （GL1）. The G+C content of the DNA was42.3mol%. A phylogeneticanalysis based on16S rRNA gene sequences demonstrated that strain JYr12^Twasunique, showing <95.0%sequence similarities to all published species, i.e.,91.2%toThalassolituus oleivorans,91.0%to Oceanospirillum linum,90.6%to Spongiispiranorvegica,90.5%to Oceaniserpentilla haliotis,90.4%to Oleispira Antarctica,90.4%to Microbulbifer donghaiensis,89.8%to Microbulbifer hydrolyticus. It indicated thatthis isolate formed a distinct evolutionary lineage within the family Oceanospirillaceaeof the class γ-Proteobacteria. On the basis of the polyphasic taxonomic study, strainJYr12^Twas considered to represent a novel genus and species in γ-Proteobacteria, forwhich the name Litoribacillus peritrichatus gen. nov. sp. nov. was proposed. The typestrain was JYr12^T（=CGMCC1.10796^T=JCM17551^T）.A novel Gram-negative, non-spore-forming, strictly aerobic, irregular-rod shapedbacterium without flagellum, designated strain H94^T, was isolated with thehigh-throughput cultivation method from a bottom sea water sample of an amphioxusbreeding zone in the coastal region of Qingdao, China. The organism can grow at4C,16C,28C,37C, pH6-10and in the presence of1-12%（w/v） NaCl. The major fattyacids were C_18:1ω9c （28.18%）,C16:0（22.18%）and C16:1ω9c（15.42%）. Aphylogenetic analysis based on16S rRNA gene sequences indicated that strain H94^T formed a distinct evolutionary lineage within the genus Hahella of the classγ-Proteobacteria. It showed <97.0%sequence similarities to all published species, i.e.,95.89%to Hahella antarctica,92.89%to Hahella chejuensis,92.18%to Hahellaganghwensis. The G+C content of the DNA was56.2mol%. It contained Q-9（91%）and Q-8（9%） as the major isoprenoid quinine. On the basis of the polyphasictaxonomic study, strain H94^T（=CGMCC1.10794^T=JCM17549^T） was considered torepresent a novel species of the genus Hahella, for which the name Hahellaqingdaonensis sp. nov. was proposed.The novel bacteria JYr2^Tand H94^Tare capabale of producing urease, which is ahighly effective urea decomposition catalyst, involved in the biological nitrogenrecycling. Therefore, these two strains possess potential value for biologicalengineering applications.