Gel Microbead Cultivation with a Subenrichment Procedure Can Yield Better Bacterial Cultivability from a Seawater Sample than Standard Plating Method

A gel microbead (GMD) cultivation method was employed to cultivate microorganisms from an amphioxus breeding zone in Qingdao, P. R. China. The culture results were compared with those by standard plating method. In the GMD-based method, the microcolony-forming GMDs were sorted by fluorescence-activated cell sorting (FACS). To further get pure cultures, a subsequent enrichment culture and a streaking purification procedure were conducted on marine R2A medium. Eighty bacterial strains isolated by the GMD-based method were randomly selected for sequencing. These isolates belonged to Alphaproteobacteria (33%), Gammaproteobacteria (44%), Bacteroidetes (11%), Actinobacteria (5%), Firmicutes (5%), Epsilonproteobacteria (1%), and Verrucomicrobia (1%), the last two groups being usually difficult to culture. The 16S rRNA gene sequences revealed a diverse community with 91.1%-100% of the bacterial rRNAs similarities. Thirteen strains were sharing 16S rRNA gene sequence which was less than 97% similar to any other rRNA genes currently deposited in TYP16S database. Seventy isolates derived from the standard plating method fell into 4 different taxonomic groups: Alphaproteobacteria (9%), Gammaproteobacteria (81%), Bacteroidetes (7%) and Firmicutes (3%) with a 16S rRNA gene sequence similarities between 95.8%-100%, in which only 3 strains were sharing 16S rRNA gene sequence of less than 97%. The results indicated that the GMD-based method with subenrichment culture yielded more taxonomic groups and more novel microbial strains, including members of previously rarely cultured groups, when compared with the standard plating method, and that this method markedly improved the bacterial cultivability.

Prospecting Exopolysaccharides Produced by Selected Bacteria Associated with Marine Organisms for Biotechnological Applications

In this study, bacteria associated with marine organisms were screened for the production of exopolysaccharides（EPSs） on MY media containing sea salts（2.5%-10%）. Three selected isolates were identified as Alteromonas sp. PRIM-21, Nitratireductor sp. PRIM-24 and Enterobacter sp. PRIM-26 using 16 S r RNA gene sequencing. Optimization of the growth and EPS production kinetics in relation to incubation time were assessed. The purified EPS yield was 590, 650 and 540 mg·L-1 culture media respectively in Alteromonas sp. PRIM-21, Nitratireductor sp. PRIM-24 and Enterobacter sp. PRIM-26. Biochemical and FTIR analyses revealed the presence of biologically important functional groups in the EPS produced by all the three isolates. The EPS produced by Nitratireductor sp. PRIM-24 and Alteromonas sp. PRIM-21 showed 2.0% sulfate content. These bacterial EPS also showed antioxidant and emulsifying activities and the EPS produced by Enterobacter sp.PRIM-26 showed significantly higher antioxidant activities in terms of superoxide（IC50 0.33 mg·mL -1） and DPPH（IC50 0.44 mg·mL -1） radical scavenging. It also showed higher emulsifying activities against selected hydrophobic substrates with EI24 values above 60%. From the results of the study, it can be concluded that the isolated bacteria produce EPS that can be investigated in detail for biotechnological applications.