Development And Application Of Compound Functional Microecological Preparation For Prawn Aquaculture

With the rapid development of shrimp aquaculture industry in China,the deterioration of water quality in the aquaculture environment and the frequent occurrence of shrimp diseases have become problems that cannot be ignored.Among them,excessive ammonia nitrogen in the aquaculture water and the flooding of bacterial vibrio disease are the two major problems affecting shrimp aquaculture.As a new and harmless biological agent,aquatic micro-ecological preparation can improve water quality,inhibit aquatic diseases,improve immunity of aquatic animals and maintain micro-ecological balance when applied to aquaculture water bodies.At present the existing probiotics function on market tend to be more generic,professionalism is poorer,therefore,this study aimed at blood ammonia and pathogenic vibrio,through screening and domestication development of both the above two kinds of special ability of microbial bacteria,in order to preparation of targeted and professional in shrimp farming stronger composite functional probiotics.A dominant pathogenic Vibrio Vf-1 strain was isolated from the pathogenic prawn samples from the shrimp farms in fuqing area,and the Vf-1 was identified as Vibrio harveyi by 16S rDNA molecular identification method,indicating that the main pathogenic Vibrio in the farms was Vibrio harveyi.Using 49 bacillus strains preserved in the laboratory as screening strains,20 strains with strong capacity of reducing ammonia nitrogen were obtained through the detection of ammonia nitrogen degradation.On this basis,two strains FS017 and FS037 with strong anti-vibrio effects were screened from 20 strains of vibrio harveyi Vf-1 as the indicator bacteria,which possessed good properties of reducing ammonia nitrogen and anti-vibrio.The strain FS017 was identified as Bacillus velezensis and the strain FS037 as Bacillus subtilis by morphological,physiological and biochemical tests and 16S rDNA molecular identification.The high-yielding fermentation process of strain FS017 and strain FS037 was optimized to obtain the maximum bacterial biomass and antimicrobial metabolites.By single factor experiment and box-behnken response surface design optimization,the optimal medium composition for the fermentation of strain FS017 was determined:sucrose 7.5 g/L,yeast extract 24.38 g/L,FeSO₄·7H₂O 0.31 g/L,（NH₄）₂SO₄ 1.0 g/L,KH₂PO₄ 2 g/L.The best culture conditions for:quantity of 2.62%,33℃temperature,rotating speed of 180 r/min,the initial pH 6.5,45.18 mL fluid volume（250 mL bottle）triangle;The inhibitory zone diameter of the final fermentation broth was 25.04 mm,and the biomass was up to 1.19×10¹00 CFU/mL.The optimum medium components for FS037fermentation were glucose 8.01 g/L,peptone 20.10 g/L,NH₄NO₃ 1.0 g/L,Al₂（SO₄）₃ 0.2g/L.The best culture conditions as follows:the quantity of 2.5%,temperature 30℃,the speed of 195 r/min,initial pH 7.5,45.18 mL fluid volume（250 mL bottle）,increase the antibacterial circle diameter of 27.67 mm,biological bacteria quantity can reach 8.93×10⁹CFU/mL.Ten different carrier material are investigated the effect of water pH value,adsorption effect of ammonia nitrogen,the bacteria immobilization of ammonia nitrogen effect after four characteristics,such as screening of comprehensive conditions best carrier material is corn cob powder,its influence on water pH value is not big,bibulous rate is 379%,the adsorption of ammonia nitrogen in water at a rate of 17.5%,strain FS017 immobilized on ammonia nitrogen removal rate was 65.25%.Four different methods were designed to prepare solid microecologicals,and the bacterial activity in the preparation was regularly detected.The results showed that the microecologicals prepared by freeze-drying method could reach up to 10¹11 CFU/g by mixing the fermentation broth of FS017 and FS037 strains,centrifugation and resuspension with seawater,and immobilization of the bacterial suspension with corncob powder.The microecological preparation was applied to the simulated aquaculture ammonia nitrogen wastewater,and the ammonia nitrogen degradation rate was up to 50.12%.The results showed that the microecological agent could inhibit the growth of vibrio effectively at the initial fermentation stage of vibrio culture.In the logarithmic phase of vibrio fermentation,the amount of vibrio was no longer increased or even decreased,indicating that the microecological preparation had a good arc resistance effect.