| Due to the current tendence and focus on the research of the microbial role playing in aquiculture system, the variability of bacterial community composition with the culturing and spacial distribution in the culture pond and water source were investigated using a culture-independent method of 16S rDNA PCR-DGGE fingerprinting, which overcome the cultivation-based limitations, and that of physic-chemical factors and bacterial number was carried out by traditional cultivation methods. Clonging and sequencing of PCR-amplified 16SrRNA gene fragments excised from the dominant intensity bands in DGGE patterns of sediments can help us to understand the ecological function of the bacteria, and to get beneficial functional bacteria, useful and potential for probiotics.The main results are as follows:1. Optimization of a special method for total DNA extraction from the pond sediment. TENP was used to remove humic acids from sediments before cell lysis in this study, then SDS was combined with lysozyme in treatments. As a result, the recovery ratio was high, reaching more than 90%. DNA yields ranged from 2 to 20μg per gram wet sediment,with the size over 23 kb,which could be used directly for PCR amplification and restriction enzyme digestion without extensive purification. Therefore, this is a simple, reliable protocal to extract DNA from the shrimp pond sediments.2. Establishment and optimization of the PCR-DGGE method system. The 16S rRNA gene (V3 region) was amplified using the primers (GC-341F and 517R) which were specific for most bacteria and then the PCR products ,about 200bp long were separated in 8% polyacrylamide gels with a denaturant (where 100% denaturant contains 7M urea and 40% formamide) gradient from 40% to 60%. Analysis of the DGGE profiles showed the information of bacteria diversity in the water and sediments.3. A series of water and sediment samples were collected from the new and old shrimp ponds from October 2003 to May 2004 in Quanzhou bay. The dynamic change of physic-chemical factors, bacteria abundance was investigated to acquire their interaction during the culture processes. The result showed that the physic-chemical factors fluctuated erratically in the culture processes, but change within water quality demand for shrimp cultured healthily. Total bacterial number in the sediments increased gradually and was closely correlated with the TOC. Number of heterotrophic bacteria and Vibrio decreased gradually and was not correlated with the temperature, DO, TN, PO4-P.4. Bacterial diversity in the pond water and sediments was abundant. The bacterial genus composition was relative lower before culturing and at the beginning stage of shrimp culturing, increased gradually since middle stage of culturing, and decreased gradually at the end of culturing. The variability in bacterial community composition fluctuated erratically in the culture processes, which was not correlated with the change of heterotrophic bacterial number and Vibrio. Bacterial diversity in the sediments was higher than that in the water and the variation of bacterial community composition in the sediments was lower than that in the water.5. Sequencing of dominant bands excised from the DGGE patterns revealed that the most common bacteria in the sediments included members ofγ- proteobacteria,δ-proteobacteria, Bacteroidetes, Chloroflexi, Actinobacteria, Firmicutes and uncultured bacteria.These bacteria may play an important role in material and energy flow.6. Spacial distribution of bacterial abundance and bacterial diversity among the pond and water source were compared using traditional cultivation methods and the PCR-DGGE technique. The results revealed that the heterotrophy bacteria and Vibrio number in water source was the largest, but the bacterial diversity of the new pond was the most abundant. The difference of bacterial community composition between them was obvious. It is probably related to metabolic adjustments.
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