Enrichment Culture And Isolation Of Bacteria From Marine Sediment And Polyphasic Taxonomy Analysis Of Three Novel Bacteroidetes Strains

The marine sediments near coast, which contain abundant microbial diversity, may exist in enormous number of novel microorganisms. Therefore, there is an immense potential research and development and innovation space. In this study, we established and improved the enrichment cultivation method in our laboratory, and a coastal sediment sample collected from Weihai was implemented. Cultivation and Roche454high-throughput sequencing technology approaches were simultaneously used to study the culture-dependent and independent microbial diversity in the process of enrichment culture, the bacterial communities structure succession were monitored. Analyzed the results, we discussed the feasibility and effectiveness of this enrichment cultivation method. In addition, three novel Bacteroidetes strains were isolated from the shark gill homogenate and other sediments by using the same approach, characterized using a polyphasic approach to elucidate their taxonomic status, and further enriched the bacterium resources of Bacteroidetes.For the research of bacterial richness and communities structure, the available information by Roche454FLX high-throughput sequencing technology was much more than that by traditional cultivation method. The dominant bacterial communities detected by the two methods were not completely consistent, indicating the entire species in the sample could not be fully represented by the cultivable bacteria. However, both results showed that the bacterial richness and diversity in the enrichment habitat were gradually reduced along with the extension of enriched time. The enrichment cultivation method was not conducive to the enrichment of the bacteria within a-proteobacteria, y-proteobacteria, Cyanobacteria, Chloroflexi, but especially promotes the enrichment of Bacteroidetes, and the enrichment of Firmicutes, Fusobacteria and other part of δ-proteobacteria were also obvious. Data in this study suggested that high abundance of new microbial resources deserved to further understanding and development in the marine sediments near coast. At the same time, we provide an effective enrichment cultivation method to isolate and culture more Bacteroidetes novel strains.A novel marine bacterium, designated as SS12T, was isolated from shark gill homogenate. Comparative analysis of16S rRNA gene sequences demonstrated that the novel isolate showed the highest sequence similarity (94.68%) to Saccharicrinis fermentans. A phylogenetic tree, based on16S rRNA gene sequences, showed that strain SS12T and Saccharicrinis fermentans formed a distinct cluster within the family Marinilabiliaceae. Gram-stain-negative rods, facultative anaerobic, gliding, yellow pigmented and require NaCl for growth. Catalase-positive and oxidase-negative. Optimal growth occurred at28-30℃, pH7.0-7.5, and in the presence of2-4%(w/v) of NaCl. The DNA G+C content was40.0mol%. The strain contained MK-7as the prevailing menaquinones; iso-C15:0and anteiso-C15:0as the major cellular fatty acids; and phosphatidylethanolamine and an unknown lipid as the predominant polar lipid. On the basis of polyphasic taxonomy traits, strain SS12T (=CICC10590T=DSM27040T) represents a novel species of genus Saccharicrinis, for which the name Saccharicrinis carchari sp.nov.is proposed.A novel marine strain, designated as Y11T, was isolated from marine sediment at Weihai in China. Comparative analysis of16S rRNA gene sequences demonstrated that the novel isolate showed the highest sequence similarity (93.4%) to Saccharicrinis fermentans and (93.0%) to strain SS12T. Gram-stain-negative rod, non-endospore-forming, yellow pigmented and slightly agar-hydrolysing. Facultative anaerobic, gliding, require NaCl for growth. Catalase-negative and oxidase-positive. Optimal growth occurred at28-30℃, pH7.0-7.5, and in the presence of2-3%(w/v) of NaCl. The DNA G+C content was34.4mol%. The strain contained MK-7as the prevailing menaquinones; iso-C15:0, anteiso-C15:0and C15:1ω6c as the major cellular fatty acids; and phosphatidylethanolamine and two unknown lipids as the predominant polar lipid. On the basis of polyphasic taxonomy traits, strain Y11T (= CICC10835T=DSM T) represents a novel species of a novel genus, for which the name Labilibacter marina gen. nov., sp. nov. is proposed.A novel bacterium, designated G22T, was isolated from sediment of Gahai, a saltwater lake in Qinghai, China. Another strain G312, isolated from the same sediment but different enrichment cultivation time period, showed99.0%sequence similarity, was obtained as the paralleled strain. Phylogenetic analysis based on16S rRNA sequences demonstrated that the novel isolates showed the highest sequence similarity (90.04%) to Draconibacterium orientalis. Gram-stain-negative, facultative anaerobic, non-motile, pink or red. Positive for catalase and oxidase. Require NaCl for growth. Optimal growth occurred at33-35℃, pH7.0-7.5, and2-4%(w/v) of NaCl. The major polar lipids are phosphatidylethanolamine and two unknown lipids. The predominant cellular fatty acids are iso-C15:0, anteiso-C15:0and iso-C17:03OH and MK-7is the main respiratory quinine. The DNA G+C content was40.0mol%. On the basis of phenotypic, chemotaxonomic and phylogenetic evidence observed, we describe a new species of a novel genus within family Draconibacteriaceae represented by strain G22T (=CICC10587T=DSM27063T), for which we propose the name Tangfeifania gahaiensis gen. nov. sp. nov..Based on the phylogenetic analysis class Bacteroidia, we propose a novel order, for which we recommend the name Marinilabiliales, to contain family Marinilabiliaceae, famiy Draconibacteriaceae, family Prolixibacteraceae and genus Marinifilum, which appeared ambiguous affilitation with recognized families. So that, the class Bacteroidia would be divided into two orders, Bacteroidales and Marinilabiliales.