The Establishment And Application Of High-throughtput Cultivating And Sorting Method For Marine Microorganisms

It is widely believed that less than 1% microbial cells from marine environments have been cultivated. It has become a bottle-neck of marine microbiology to acquire more microorganism pure cultures efficiently from marine environments. Researches have showed that standard plating method is not suitable for cultivating most of the marine microorganisms. Therefore, one of the main directions of marine microbiology is finding ways to increase the microorganism cultivability. To establish a new microorganism cultivation and isolation technique, three parts were developed in this dissertation, which are preparation of microbeads, establishment and applations of high-throughput cultivation and isolation technique and taxonomic analysis of a novel marine bacterium.Based on the application of microencapsulation of viable cells, three microbead preparation methods were established, which are mechanical stirring emulsification method, coflowing method and membrane emulsification method. Uniform-sized microbeads from different natural polymer materials (e.g. alginate, agarose and gellan gum) could be produced by all the three methods. The preparation procedures are simple and mild, which are suitable for microencapsulation of viable cells. Moreover, microbead preparation is a technique of various other applications.On the basis of the built microbead preparation methods, a high-throughput cultivation and isolation technique was established. The technique includes the following steps: first, after sample collection, microorganisms from environments are concentrated by filtration and centrifugation; second, the concentrated cells are encapsulated into microbeads; third, the encapsulated cells are transferred into chromatography column and cultivating under flowing seawater; fourth, colony-forming microbeads are sorted by flow cytometer into 96-well microtiter plates filled with enrichment medium; at last, the isolations are preserved and analysed. The feasibility of the method is confirmed by microencapsulation, cultivation, sorting and identification of a pure culture and two marine samples. By using three self-designed methods suitable for viable cell microencapsulation, in especial coflowing method and membrane method, the microorganism encapsulating condition is improved and the cell viabilities are well protected; excepting for agarose, alginate and gellan gum are also employed as encapsulating materials to increase microorganism cultivability; furthermore, a higher cell numbers is encapsulated, and this procedure will increase microorganism communication and hopefully increase the cultivability.A sediment sample from the Arctic Ocean and seawater and sediment samples from an amphioxus breeding zone in Qingdao were studied by using the high-throughput method and standard plating method. A total of 500 strains were isolated and preserved from high-throughput method, in which 350 isolates from arctic sediment and 150 isolates from the seawater of amphioxus breeding zone. A total of 254 strains were isolated and preserved from standard plating method, in which 213 isolates from seawater and 37 isolates from sediment.The 16S rRNA gene sequencing results of 47 arctic-derived isolates showed that these isolates belonged to six taxonomic groups, which were Alphaproteobacteria (19), Betaproteobacteria (2), Gammaproteobacteria (17), Actinobacteria (2), Firmicutes (4) and Bacteroidetes (3), and were from 21 genera. Six strains had 16S rRNA gene similarities lower than 97%, which all came from Paracoccus.80 isolates derived from the seawater of amphioxus breeding zone were selected for sequencing and phylogenetic analysis. These strains belonged to 7 taxonomic groups: Alphaproteobacteria (26), Gammaproteobacteria (35), Bacteroidetes (9), Actinobacteria (4), Firmicutes (4) and the rarely cultivated Epsilonproteobacteria (1) and Verrucomicrobia (1), belonging to 41 genera. 13 16S rRNA gene sequence similarities values were lower than 97%. In the 70 cultures derived from seawater based on plating method, 57 isolates were from Gammaproteobacteria, 6 belonged to Alphaproteobacteria, 2 isolates were identified as Bacteroidetes, and 5 isolates were members of the phylum Firmicutes, belonging to 26 genera. 3 16S rRNA gene sequences with similarities lower than 97% was found. The 16S rRNA gene sequencing results of 37 sediment-derived plating isolates showed that these isolates belonged to four taxonomic groups, which were Alphaproteobacteria (5), Gammaproteobacteria (27), Firmicutes (2) and Bacteroidetes (3), belonging to 22 genera. 8 isolates had 16S rRNA gene sequence similarities lower than 97%.The comparison results between the plating-derived isolates and the high-throughput-derived isolates demonstrated that the latter method enabled the cultivation of bacteria with more diversity and more formerly uncultured strains, including the rarely cultivated Epsilonproteobacteria and Verrucomicrobia. A higher cultivation and isolation efficiency was achieved.A Gram-negative, rod, aerobic, facultatively anaerobic bacterium with peritrichous flagella, designated strain JYr13~T, was isolated from coastal sediment of an amphioxus breeding zone located in Qingdao, China. The strain reduced selenite to elemental selenium (Se0) and also reduced iron(III) oxyhydroxide, iron(III) citrate. This organism grew optimally at pH 7.0-8.0 and in presence of 3% (w/v) NaCl. Strain JYr13~T contained menaquinones (MK-7) and ubiquinones (Q-7 and Q-8) as the predominant isoprenoid quinones, and C16:0 (23.92%) as the major fatty acid. The DNA G+C content of strain JYr13~T was 59.1 mol%. A phylogenetic analysis based on 16S rRNA and gyrB gene sequences showed that the closest phylogenetic neighbours of JYr13~T were F. kyonanensis DSM 18153T and F. marina DSM 16917~T with sequence similarities of 96.0% and 81.2%, respectively. Based on the phenotypic, chemotaxonomic and phylogenetic distinctiveness, strain JYr13~T was considered to represent a novel species of the genus Ferrimonas, for which the name Ferrimonas sediminum sp. nov. is proposed. The type strain is JYr13~T (=DSM 23317~T=GU391222~T=JCM 17552~T).