Enrichment Culture Of Marine Bacteroidetes And Classification And Identification Of Novel Bacteria Strains

Associated bacteria were isolated from sediment samples of Xiaoshi Island and Golden Beach collected from the coast of Weihai, and sediment samples of aquiculture area and Zhangzhou mangrove collected from the coast of Fujian. They were enriched by using conventional culture-dependent method and investigated by using phylogenetic analysis based on16S rRNA gene sequence comparisons.117and135isolates were sediment samples of Xiaoshi Island and Golden Beach on marine agar2216(Difco) and TSA, respectively.15and15isolates were from sediment samples of aquiculture area and Zhangzhou mangrove on marine agar2216(Difco). Based on morphological characters, we selected97strains from sediment samples of Xiaoshi Island,111strains from sediment samples of Golden Beach,11strains from sediment samples of aquiculture area and11strains from sediment samples of Zhangzhou mangrove for molecular phylogenetic study using16S rRNA gene sequences. The results showed that97isolates from sediment samples of Xiaoshi Island were distributed in42genera of25families in four major phylogenetic groups (Aclinobacteria, Bacteroidetes, Firmicutes,and Proteobacteria). The lower similarity of16S rRNA gene sequence suggested that at least4strains (FX10, FX11, FX12, XM4) represented new species.111isolates from sediment samples of Golden Beach were distributed in51genera of31families in four major phylogenetic groups (Actinobacteria, Bacteroidetes, Firmicutes and Proteobacteria). The isolates FH2, FH4, FH5, FH6, FH7, FH8, KH2may represent new species.11isolates from sediment samples of aquiculture area were distributed in four genera of three families in two major phylogenetic groups(Bacteroidetes, Proteobacteria).11isolates from sediment samples of Zhangzhou mangrove were distributed in seven genera of four families in four major phylogenetic groups(Actinobacteria, Bacteroidetes, Firmicutes, Proteobacteria). After enrichment culture for3weeks, new species suspected of Bacteroidetes isolated on agar2216(Difco) were the dominant strains. The above results showed there were abundant species diversity and phylogenetic diversity of bacteria isolated from the sediments of the coast of Weihai and Fujian.During our study of the microbial diversity associated with the sediment samples of Golden Beach, strain FH5T is supposed to be a novel species in the class Bacteroidia. The study of morphological, physiological, biochemical characteristics showed that FH5T was Gram-negative, facultatively anaerobic, heterotrophic, non-flagellated and formed light pink colonies on marine agar2216(Difco). The optimum growth of the strain was observed at28-33℃and pH6.5-7.5, with1-5%(w/v) NaCl. The DNA (G+C) content of strain FH5T was44.2mol%. Strain FH5T possessed anteiso-C15:0,iso-C15:0, C17:1ω6c, anteiso-C17:0, Sum In Feature4(anteiso B/iso I-C17:1), C17:02-OH, iso-C16:03-OH, Sum In Feature1(C13:03-OH/,5:1i H) as the major cellular fatty acids. The major polar lipids were Phosphatidylethanolamine, Aminolipid. Phospholipid and Lipids. Based on the study, strain FH5T should be identificated as a novel species in a new genus and a new family, for which the name Draconibaclerium orientalis gen. nov., sp. nov. is proposed. Proposal for designation of the Draconibacleriaceae fam. nov. is also presented. The type strain is FH5T (=DSM25947T=CICC10585T). The GenBank accession number for the16S rRNA gene sequence of Draconibacterium orientalis FH5T is JQ683778.According to the same methods for identification, the strains HY2and HY3were studied. The strain HY2was Gram-negative, oxidase-negative, facultatively anaerobic, rod-shaped cell and non-flagellated. The optimum growth of strain HY2T was observed at28-33℃and pH7.2-8.4with0.5-4%(w/v) NaCl. The dominant fatty acids were iso-C15:0, C15:0, C15:1ω6c, iso-C13:0, iso-C15:03-OH, iso-C17:03-OH, anteiso-C15:0, C15:03-OH, C16:03-OH, C16:0.The DNA (G+C) content was45.9mol%. Phylogenetic analysis based on the16S rRNA gene sequences showed that strain HY2T was a member of the family Marinilabiliaceae. It formed a distinct phyletic line with less than91%sequence identity to any species within previously recognized genera. On the basis of the study, strain HY2T should represent a novel genus and species, for which the name Flavilabilia marina gen. nov. sp. nov. is proposed, with strain HY2T (=DSM24566T=CICC10658T) as the type strain. The GenBank accession number for the16S rRNA gene sequence of Flavilabilia marina HY2T is JQ683774.Strain HY3T was Gram-negative, non-motile, straight or curved rods. The optimum growth occurred in the temperature range30-35℃and pH range6.5-8.0with0.5-3%(w/v) NaCl. Strain HY3T had MK-7as the major menaquinone and iso-C15:0, C15:0, iso-C15:03-OH,C15:1ω6c, anteiso-C15:0, iso-C17:03-OH, C15:03-OH, iso-C16:03-OH. C17:1ω6c, iso-C13:0, iso-C14:0and C16:03-OH as major fatty acids. The genomic DNA (G+C) content was45.5mol%. Phylogenetic analysis based on16S rRNA gene homology showed that strain HY3T should be identificated as a novel species within the genus Flavilabilia, for which the name Flavilabilia weihaiensis sp. nov. is proposed. The type strain is HY3T (=DSM24567T). The GenBank accession number for the16S rRNA gene sequence of Flavilabilia weihaiensis HY3T is JQ683775.Cytophaga fermentans remains to be reclassified. Cytophaga fermentans was a novel genus of the family Marinilabiliaceae based on the analysis of16S rDNA sequence and biochemical characteristics.