Molecular Analysis Of Bacterial Diversity In The Sediment Of Shrimps Culture Environment And Research On Some Optimization Methods For Culturable Bacteria

Less than 1% of the microorganisms have be cultured and isolated by traditional pure culture technique, most of them in nature remain uncultured. Current researches have focused on the development of new culture techniques. In this research, we improved the pure culture technique, and the denaturing gradient gel electrophoresis technique was applied to study the bacterial diversity variation of the enriched samples to evaluate the improved pure culture technique. In the same time, we incised the dominant intensity bands in DGGE patterns of surface sediments of shrimp pond and the enrichment samples cultured by solid media, after cloning, sequencing, the phylogenetic relationship of the bacteria was analyzed. The main results were as follows:1. Bacterial diversity of sediment in shrimp pond was analyzed by using PCR-DGGE technique, the Shannon-wiener index (H) is 3.4971, the Richness (S) is 41 and the Evenness (EH) is 0.94171; 17 dominant bands were excised from DGGE profile of surface sediment bacteria in shrimp pond and the V3 region of 16S rRNA gene was cloned and sequenced followed by phylogenetical analysis. The results showed that these 17 bands represented bacteria from proteobacteria (includingα-proteobacteria,γ-proteobacteria,δ-proteobacteria andε-proteobacteria), Chloroflexi, Actinobacteria, Firmicutes, Bacteroidetes and the unknown species. There are 15 bands belong to uncultured bacteria.2. 2 kinds of inoculation mode (designated asⅠandⅡ), 2 kinds of cultivate mode (designated as Y and R) and 8 kinds of liquid media (designated as 2216E, D2216E, VL55, DNB, HM1, HM2, DR2A and R2A) were applied to the enrichment of bacteria from shrimp pond sediments for 2 weeks. The effect of different inoculation mode, cultivate mode and media was evaluated by DGGE and bacterial diversity index. The results showed thatⅠY andⅡR mode could be applied to most of the liquid media, and low nutrient media got more DGGE bands and higher bacterial diversity than that of the high nutrient media.3. 16 kinds of solid media (designated as A2216E, AD2216E, AVL55, ADNB, AHM1, AHM2, ADR2A, AR2A and G2216E, GD2216E, GVL55, GDNB, GHM1, GHM2, GDR2A, GR2A, respectivelly) were applied to the enrichment of bacteria from shrimp pond sediments for 12 weeks. The colony counting results showed that the colony number on AVL55, GVL55, GDNB, GHM2 and GDR2A media in 2 weeks and 12 weeks cultivation increased obviously. Dominant bands excised from the DGGE profile of enrichment samples were sequenced. The results revealed that the 19 characteristic bands represent bacteria from proteobacteria (includingγ-proteobacteria,α-proteobacterium andε-proteobacteria), Firmicutes, marine bacteria and unknown bacteria. There are 9 bands belong to uncultured bacteria.4. As a new type of bacterial exopolysacchsride (EPS), Gellan gum can be used as a substitute for agar, because of its few dosage, high gelling quality diaphaneity and stability, et al.. In our experiment, we compared the culture effects of 8 kinds of media gelled respectively with gellan gum and agar, the results showed that the media gelled by gellan gum had better effect in a higher value in the index of H, S and EH, and GDNB and GDR2A media got the highest index value.