Cultivation Of Deep-sea Microorganisms From The South China Sea And Taxonomic Analysis Of Four Novel Bacteria

The marine environment provides extremely various habitats and survivalenvironments for microorganisms, and is the largest ecosystem on the earth. There arehuge numbers and diverse kinds of marine microbes, and it is estimated that thenumber of the prokaryote in the ocean is approximately1029. As the microbialresources are so ample, the basic research of diversity of microbes shows anextremely significant role in the field of microbial study. The study of microbialdiversity using molecular biology techniques has been developing rapidly. Meanwhilethe cultivation method is lagging behind, which is not conducive for further study ofmicrobial physiological and biochemical characteristics, as well as the developmentand utilization of microbial resources more efficiently.Phylogenetic diversity of cultivable bacteria from the deep-sea in the South ChinaSea was studied in this thesis. And sea trial of microencapsulation and high-througputcultivation was carried out. Besides, three novel species isolated from the SouthPacific Gyre and one novel species isolated from an amphioxus breeding zone in thecoastal region of the Yellow Sea were studied using ployphasic approach.403bacterial strains were isolated from2216E plates and marine R2A plates. The16S rDNA of392strains were sequenced. Phlogenetic analysis of392bacterialstrains showed that bacterial phylotypes from the deep-sea in the South China Seawere made up of four phyla of bacteria, i.e. Proteobacteria, which includesα-Proteobacteria and γ-Proteobacteria, Actinobacteria, Firmicutes and Bacteroidetes.α-Proteobacteria is the dominant group both in variety and number of strains.α-Proteobacteria has20genera,25species, a total of223strains, accounting for56.9%of the total. γ-Proteobacteria has7genera,11species, a total of44strains,accounting for11.2%of the total. Actinobacteria has7genera,7species, a total of114strains, accounting for29.1%of the total. Bacteroidetes has6genera,7species, a total of9strains. Firmicutes has2genera,2species, a total of2strains. There were7dominant genera which form76.02%of the total, such as Aeromicrobium,Erythrobacter, Pelagibaca, Roseivivax, Alcanivorax, Sphingopyxis, Nocardioides, and12dominant species which form70.66%of the total, such as Aeromicrobiumerythreum, Erythrobacter flavus, Pelagibaca bermudensis, Alcanivorax borkumensis,Sphingopyxis baekryungensis, Roseivivax halotolerans, Nocardioides marinus,Roseivivax lentus.Sea trial of microencapsulation and high-througput cultivation was carried out.The results indicated that the microbeads meeting the requirements of sorting can beobtained using simplified microencapsulation technique on the ship; however,high-throughput culture device still needs further improvement in order to meet allaspects of experimental requirements on the sea.Three novel marine bacteria belonging to Aquimarina isolated from seawater ofSouth Pacific Gyre were studied using ployphasic approach. They are designatedSW024T，SW150Tand XH134Trespectively. Cells of SW024Tare strictly aerobic,long rod-shaped,0.3μm in width and3.0-66.0μm in length. It requires sea salt forgrowth, and produces pigment with maximum absorption at453nm and479nm.Flexirubin-type pigments are not formed. The major respiratory quinone ismenaquinone6(MK-6). The dominant cellular fatty acids are iso-C15:0(34.8%),iso-C17:03-OH (15.9%), iso-C15:1G (10.5%), C16:010-methyl and/or iso-C17:1ω9c(9.8%) and iso-C15:03-OH (7.5%). The polar lipid compositions of strain SW024Twere PE, three unknown aminolipids (AL1-AL3) and five unknown polar lipids(L1-L5). Phylogenetic analyses based on16S rRNA gene sequences showed that theSW024Tbelonged to Aquimarina and was most closely related to Aquimarinamuelleri KMM6020Twith96.1%sequence similarity. Therefore, strain SW024Twasconsidered to represent a novel species in the Aquimarina, for which the nameAquimarina longa sp. nov. is proposed. Cells of SW150Tare strictly aerobic,rod-shaped,0.2μm in width and2.5-4.5μm in length, and a layer of extracellularsecretion can be observed. It requires sea salt for growth, and produces pigment withmaximum absorption at477nm. Flexirubin-type pigments are not formed. The dominant cellular fatty acids are iso-C15:0(19.6%), iso-C17:03-OH (13.8%), iso-C15:1G(10.7%), C16:1ω6c and/or C16:1ω7c (8.3%) and C16:010-methyl and/or iso-C17:1ω9c(7.7%). The polar lipid compositions of strain SW150Twere PE, one unknownaminolipids (AL) and three unknown polar lipids (L1-L3). Phylogenetic analysesbased on16S rRNA gene sequences showed that the SW150Tbelonged toAquimarina and was most closely related to Aquimarina macrocephali JAMB N27Twith97.8%sequence similarity. Therefore, strain SW150Twas considered torepresent a novel species in the Aquimarina, for which the name Aquimarina pacificasp. nov. is proposed. Cells of XH134Tare strictly aerobic, long rod-shaped,0.5μm inwidth and5.4-77.8μm in length. It requires sea salt for growth. Produces pigmentwith maximum absorption at473nm. Flexirubin-type pigments are formed. The majorrespiratory quinone is MK-6. The dominant cellular fatty acids are iso-C15:0(33.5%),iso-C15:1G (12.6%), C16:1ω6c and/or C16:1ω7c (11.7%), iso-C17:03-OH (8.6%),iso-C15:03-OH (7.3%), C16:010-methyl and/or iso-C17:1ω9c (7.0%). The polar lipidcompositions of strain XH134Twere PE, one unknown aminolipids (AL) and threeunknown polar lipids (L1-L3). Phylogenetic analyses based on16S rRNA genesequences showed that the XH134Tbelonged to Aquimarina and was most closelyrelated to Aquimarina macrocephali JAMB N27Twith96.9%sequence similarity.Therefore, strain XH134Twas considered to represent a novel species in theAquimarina, for which the name Aquimarina megaterium sp. nov. is proposed.A novel marine bacterium, designated strain H61T, was isolated with thehigh-throughput cultivation method from a bottom sea water sample of an amphioxusbreeding zone in the coastal region of Qingdao, China. Cells of H61Tare strictlyaerobic, short rod-shaped,0.72μm in width and1.25μm in length. The dominantcellular fatty acids are C16:1ω6c and/or C16:1ω7c (40.9%), C16:0(20.3%), C18:1ω7c(18.8%). The result of16S rRNA gene sequences similarity showed that the H61Twasmost closely related to Thalassolituus oleivorans MIL-1T(96.4%) and Oceanobacterkriegii IFO15467T(96.3%)..On the basis of Phenotypic and phylogenetic evidences,strain H61Twas preliminary considered to represent a novel species in the Aquimarina, for which the name Aquimarina megaterium sp. nov. is proposed.