Molecular Analysis Of Microbial Diversity In Shrimp Culture Environment And Study And Application Of Probiotics For Shrimp Aquaculture

The probiotics recently used in aquaculture have the deficiency of short effect time and no obvious dominance in the environment.This study aimed at exploring the application potential of spore-producing bacteria to shrimp aquaculture based on the microecological theory and the good characteristics of Bacillus used as probiotics in the previous works.Many spore-producing bacteria were therefore isolated from shrimp aquaculture environment and shrimp intestinal.Their abilities to remove the feeding remains in the aquaculture were evaluated by determining the activity of producing extracellular digestive enzymes(amylase,protease and lipase).Then,a potential probiotic Bacillus licheniformis strain S-40 was tested as an additive for improving the shrimp aquaculture.In addition,the bacterial compositions of shrimp aquaculture systems were investigated in the study.The main results were as follows:1.The bacterial compositions in the shrimp culturing environment were characterized by analyzing three 16S rDNA clone libraries constructed from the intestines of shrimp,water and surface sediment of the shrimp pond respectively. The RFLP analysis and sequence alignment of 16SrDNA libraries showed that intestinal microflora in shrimp belonged to Proteobacteria(98.7%)and Firmicute(1.3%)phylogenetically,of whichγ-Proteobacteria was dominant (97.4%)compared toα-Proteobacteria(1.3%);The bacteria in shrimp pond water mainly consisted of Proteobacteria(24.1%)and Bacteroidetes(17.7%),withα-Proteobacteria 21.5%;The bacterial diversity was much higher in the surface sediment of the shrimp pond with the proportion of 52.3%Proteobacteria,9.3% Bacteroidetes,4.7%Planctomycete,1.9%Firmicute and 0.9%Actino-bacteria. However,it deserved to mention that in the water and sediment sample, approximately 55.7%and 23.4%of representative sequences were PCR-amplified from unicellular algae,probably derived from the organelle.2.One hundred and ninety-one spore-producing bacteria were isolated from shrimp intestines and the sediments of shrimp pond.Based on their morphology and results of RFLP analysis,70 of them were chosen for physiological and biochemical tests as well as 16S rRNA identification.The results indicated that these strains were grouped into four genera Brevibacillus,Sporolactobacillus, Paenibacillus,Bacillus.Thirteen species of genus Bacillus were found out, including B.circulans,B.lentus,B.megaterium,B.sphaericus,B.horis,B.mycoides, B.pantothenticus,B.pumilis,B.firmus,B.fusiformis,B.licheniformis,B.coagulans, B.subtilis.3.Nine strains,with high efficiency of producing amylase,protease and lipase,had been screened out from those spore-producing bacteria by selective media.Their conditions of enzyme-producing were optimized and the activities of crude enzymes were determined.The results showed that most of them had high enzyme activities in the certain condition of temperature,pH value and salinity.Among them,one strain,designated as Bacillus licheniformis S-40,was chosen as potential probiotic for shrimp culture experiment according to its high and stable activities of digestive enzymes.4.An experiment was conducted with the strain Bacillus licheniformis S-40 as a supplement,to investigate its effects on the intestinal microflora,and the immune response of the white shrimp,Litopenaeus vannamei.Shrimp were fed in four tanks for 40 days with different water conditions,control(non-supplemented),and water supplemented with B.licheniformis S-40 at 10³ cfu·mL^-1,10⁴ cfu·mL^-1,and 10⁵ cfu·mL^-1respectively.The results indicated that compared to the control group, the total bacterial counts in the shrimp intestine remained constant,while Vibrio sp.numbers decreased significantly,and the total haemocyte count(THCs)and PO activity in the shrimps increased significantly,SOD activity increased significantly only when water was supplemented with B.licheniformis S-40 at 10⁵ cfu·mL^-1.However,the protein contents were not remarkably different between the experimental and control groups.5.Finally,the spores of B.licheniformis S-40 were embedded in microporous starch to make microcapsule.By analyzing multiple factors,the optimal embedding conditions were estimated as follows:pH 7.5,temperature 30℃,absorption time 1.5h.