The Establishment Of High-Throughput Methods For Culturing Marine Microorganisms And Marine Bacterial Diversity In Qingdao Coast

Only a tiny amount of microorganisms on the earth can be cultured so far. To culture the previously uncultured organisms will enhance our understanding of microbial physiology and metabolic adaptation and will provide new sources of microbial metabolites. In recent years, some new culturing methods have been developed. For example, (1) adding signal compounds to the culture media; (2) dilution cultivation method; (3) mimic the natural environment of microorganisms; (4) high-throughput cultivation method. Out of these 4 methods, the high-throughput cultivation method is most promising. It bases on the combination of a single cell encapsulation procedure with flow cytometry that enables cells to grow with nutrients that are present at environmental concentrations. Microdroplets separate microorganisms from each other while still allowing the free flow of metabolites and signaling molecules between different microcolonies. The establishment of high- throughput cultivation method will culture the uncultured microorganisms. It will have a great meaning to the research of filtrating microbial metabolites from microorganisms. This technology can be applied to samples from several different environments, including seawater and soil. At present, there is no report about the high throughput cultivation methods in our country.This research has established the high- throughput cultivation method for isolating and cultivating the marine microorganisms. It consists of four parts. (1) Encapsulation of cells in gel microdroplets. Mixing the bacterial with the gel and making the microdroplets with RW20D2M beater whose diameter is 50μm. Approximately, 10% of the microdroplets are occupied by cells. (2) Culturing technology. The microdroplets are dispensed into sterile chromatography column which contains 25 ml media. Columns are equipped with two sets of filter membranes (0.1μm at the inlet of the column and 8μm at the outlet). The filters prevent free-living cells contaminating the media reservoir and retain microdroplets in the column while allowing free-living cells to be washed out. Media used for incubation of marine samples is sterile seawater. Media is pumped through the column at a flow of 10ml/h. (3) Microdroplets containing colonies are separated from free-living cells and empty microdroplets by using the flow cytometer. Dropping the microdroplets into the 96-well microtiter plates which contains rich organic medium. (4) Phylogenetic analysis of the isolates. Genomic DNA from bacteria isolates is extracted, and 16SrDNA is amplified by PCR with 16SrDNA universal primers. The partial 16SrDNA sequences of the bacterial isolates are compared in the GenBank by the BLAST search. This technique combines encapsulation of cells in gel microdroplets for massively parallel microbial cultivation under low nutrient flux conditions, followed by flow cytometry to detect microdroplets containing microcolonies.Water sample was collected in the yellow sea. The abundance of the cultivable bacteria was3.7×104 CFU /ml .600 aerobic heterotrophic bacterial isolates were recovered through the high-throughput cultivation method. Analysis of the 16S rDNA sequences of 32 representative isolates placed them into two phylums, i.e. Proteobacteria (31), Firmicutes (1), The predominant bacteria were members of Proteobacteria, particularly the Gammaproteobacteria. In this research, we have established the high throughput cultivation technology and successfully cultured some marine microorganisms. It is the first time to establish this technology in our country. The massive green algae (Enteromorpha prolifera) bloom in the coastal region of Qingdao, China between May and July 2008 prior to the sailing competition of the 29th Olympic Games has raised great concerns on environmental deterioration. To assess the effects of massive algae blooms on the marine microbial diversity, the phylogenetic diversity of the cultivable bacterial community isolated from the coastal water of Qingdao during the massive green algae blooms was studied, and was compared with those of previous years. The abundance of the cultivable bacteria was 4.7～6.5×104 CFU/ml in 3 different sea water samples at the middle stage (from the end of June to the beginning of July 2008) of the bloom, and 180 aerobic heterotrophic bacterial isolates were recovered by using four culture media. Analysis of the 16S rDNA sequences of 63 representative isolates placed them into four phylums, i.e. Proteobacteria (45), Firmicutes (10), Actinobacteria (6) and Bacteroidetes (2).The predominant bacteria were members of Proteobacteria, particularly the Gammaproteobacteria. The majority of the isolates were strains of known species, and 6 isolates may represent new species in the genus Nautella, Pseudoruegeria, Pseudoalteromonas and Alteromonas of phylum Proteobacteria, and Salinicoccus of phylum Firmicutes. Five genera of AlphaProteobacteria belongs to the Roseobacter lineage: Loktanella, Jannaschia, Nautella, Phaeobacter and Pseudoruegeria and the Roseobacters have been found to be associated with marine phytoplankton. There are no Roseobacter group in the coastal of Qingdao before. The other dominant genus were quite different from those previous years when no massive green algae blooms occurred. To our knowledge, it is for the first time that the bacterial diversity during a massive green algae bloom was studied.