The Study Of Some Functional Microbes In Shrimp And Crab Mix-Culturing Ponds

There is an urgent need in aquaculture to develop microbial control strategies, since disease outbreaks are recognized as important constraints to aquaculture production and trade and since antibiotic resistance has become a matter of growing concern.In attempt to bioremedy aquaculture shrimp farming environments, one of the alternatives to antimicrobials in disease control could be the use of probiotic bacteria as microbial control agents. Being lack of validity, pertinene and obvious dominance of probiotic bacteria exsiting in the shrimp farming, this study is conducted to isolate and screen some functional microbes which can degrade the protein and starch of remaining feeds in the shrimp and crab mix-culturing environment by their extracellular enzyme producing activity on the basis of the indigenous bacterial flora in shrimp and crab mix-culturing ponds having been investigated, and those predominant functional microbes were further studied using a culture-independent method of 16S rDNA PCR-DGGE. The main results are as the follows:1. The number of culturable heterotrophic bacteria including amylase-degrading bacteria(AB), organic phosphate-degrading bacteria (OPB),inorgan phosphate-solublzing bacteria (IPB) , chitin-degrading bacteria(CLB) ,lipin-degrading bacteria(LB), cellulose-degrading bacteria (CB),sulfur-oxidizing bacteria (SOB) and Vibrio (VB) in the water and sediments of a new-built and a fifteen-year old shrimp and crab mix-culturing pond in the East Sea of Quan Zhou were estimated by using the plate counting methods from October 2003 to May 2004. The correlation between culturable heterotrophic bacteria and other functional microbes was studied. The number of culturable heterotrophic bacteria showed high fluctuation in the water as well as in sediments of culturing ponds in the whole farming periods. The same was the case for other functional microbes and Vibrio. The number of functional microbes in the water of the old culturing ponds was larger than that of the new ponds,but the number of functional microbes in the sediments of the new culturing ponds was larger than that of the old one. In both of the culturing ponds, the number of functional microbes in the sediments was larger than that of the water by two or one order of magnitude. Statistical analysis indicated that AB, OPB, LB and VB were positively correlated with HB, while CLB, CB and SOB had no obvious correlation with HB.2. The producing conditions and the properties of extracellular amylase and protease of the six strains of marine bacteria isolated from the sediments of shrimp and crab mix-culturing ponds by the selected media were studied. Among the six strains obtained, Pseudomonas sp. strain SL-9, Arthrobacter sp. strain S-32 and Bacillus sp. strain OP -8 had higher amylase activity. The optimum temperature of the amylase activity of the three strains is all 50℃, and optimum pH of each strain is respectively 9.5, 8.0, 9.0. The other three strains of bacteria including Bacillus sp. strain NOPL-2, Serratia sp. strain SL-1 and Serratia sp. strain O-19 showed higher protease activity. The optimum temperature and pH of the protease activity of the three strains are respectively 60℃, 50℃, 40℃and pH 8.5, 7.0, 8.5. With the exception of OP-8 and NOPL-2, the enzyme protein of the other four strains is sensitive to heat.3. The 16S rRNA gene of the six marine bacteria strains were cloned and sequenced. The phylogenetic analysis demonstrated that the six marine bacteria strains belong to genera Bacillus, Pseudomona, Arthrobacter and Serratia, respectively.4. Pseudomonas sp. strain SL-9, Serratia sp. strain SL-1 and Serratia sp. strain O-19 can also elaborate lipase activity; Bacillus sp. strain NOPL-2 can produce amylase and phosphatease; Bacillus sp. strain OP-8 and Serratia sp. strain S-32 can also excrete protease. The results indicated that all strains could produce several extracellular enzymes and should have lay a foundation for the further screening of the probiotics candidate strains of complex enzymes to some extent.5. Total community DNA and strains DNA were extracted and puried.The V3 variable region of 16S rRNA gene was then amplified by PCR with bacterial primers and analyzed by denaturing gradient gel electrophoresis(DGGE), Comparaison of the DGGE profilings of the isolated strains and the samples showed that the isolated bacteria are not the predominant species in the community. Probably due to the fact that the strains best adapted to the enriched environmental conditions were isolated and cultured.