Isolation Of Marine Sponge-associated Bacteria And Identification Of Strain HB09009, HB09012

Marine sponges, a kind of marine benthic filter-feeding animals, have contained a vast reservoir of microorgnisms that are vital for the survival of themselves or for human exploration due to their unique filter-feeding system. The marine sponge diversity is enormous and the biomass is huge in the South China sea. For discovering new species of symbiotic and epiphyte microorganisms in marine sponges, this paper focused on the effect of culture time, medium, marine sponge extract, inorganic nitrogen and phosphorus, secondary isolation method on the culturable ability of sponge-associated bacteria, and based on the principle of polyphasic taxonomy, a systemaric study was conducted on the taxonomy of strain HB09009 and HB09012. The results were as follows.It was found that formations of the amounts of colony and the varieties were rapid at the first week, however, after one week the amounts and varieties increased more slowly but new colonies still formed.The results showed that, medium composition had significant effect on the isolation of bateria. We noticed that the R2A medium could obtain the most colonies and varieties between 2216E and R2A agar. Also, R2A medium added marine sponge extract, inorganic nitrogen and inorganic phosphorus was beneficial for isolation.Our study established secondary-isolation method for the first time. It based on the fact that the colonies formed on the common medium were seperated firstly, and then the colonies which could not be seen by naked eyes were transplanted to other media on which they could grow to seen. This method used 2216E as the first separation media, and then used SRA as the second separation media. Eighteen strains were isolated by this method.By 16S rDNA sequencing and blast analysis, strain HB09009 had the highest sequence similarity with Bacillus carboniphilus JCM9731T (AB021182) and the similarity value was only 97.1%, strain HB09011 had the highest sequence similarity with Coxiella burnetii (AY342037) and the similarity value was 95.4%, strain HB09012 had the highest sequence similarity with Planctomyces maris DSM8797T(NR025327)and the similarity value was 97.4 %. The 16S rDNA sequences were registered in GenBank and the accession numbers were GU263833、GU319886 and GU263834.The polyphasic classification studies of two strains were conducted. The results showed that strain HB09009 had typical characteristics of genus Bacillus shared a high homology with Bacillus carboniphilus, Strain HB09012 had typical characteristics of genus Planctomyces shared a high homology with Planctomyces maris. However, there were obvious differences between phenotypic and chemotaxonomic characterization. Results indicated that HB09009 might be a novel Bacillus speices and preliminarily identified as Bacillus sp. HB09009, HB09012 might be a novel Planctomyces one and identified as Planctomyces sp. HB09012.