Application Of Bacillus Firmus PC465in Shrimp Culture And The Evalution Of Effects On Growth, Disease Resistance Of L.vannamei

The research aimed to the extensive implication of a commensal bacteria inthe intestine of shrimps in the shrimp aquaculture and the evaluation of theeffects on L.vannamei. The research can be divided into four sections:Firstly, we researched the optimal culturing parameters for Bacillus firmusPC465to culture this probiotic in large scale, in order to implicate the probioticfor better practice of shrimp aquaculture. We got the growth curve via themeasurement of the absorbance for bacterial suspension at600nm at certaintimes in turns; we also got the optimal culture medium and conditions, as well asthe optimal inoculum volume, which were obtained via the measurement ofabsorbance for bacterial at600nm at some turns in turns. The factors consideredwere types of water, carbon source, nitrogen source, temperatures, inorganic salts,pH and inoculum volume. Finally, we got the optimized medium, with theformula of5g peptone,1g yeast extract,0.05gFeSO4and1L running water; gotthe optimized culture conditions at a temperature range of28-37℃and a initialpH at6with the optimal inoculum volume at4%. The next, the fermentation wasconducted using5L and50L capacity of fermenters in simple and large-scalemanner, and the suspension was centrifuged at4℃，7,500×g to collect theprecipitation, later the bacterial precipitation was stored at-80℃until used. Toget the feeds with PC465supplement with different adding dose, the bacterialprecipitation was:1). diluted properly to incorporated into the powers of feedmaterial with respective volumes to get feeds in the lab;2). freeze-dried toincorporated into the powers of feed material with respective quality to get feeds in factory;3) diluted properly to incorporated into feed particles with repectivevolume to get feeds packaged by fish oil and sodium alginate.As the advantage of resistance to extreme environment factors in sporeformer and the benefits in shrimp aquaculture were quite obvious, probiotics ofBacillus as feed additive can be of great value to be applied during the period ofvarious epidemic disease spreading all over the world. Bacillus PC465, whichwas obtain from the shrimp, is an indigenous and as a spore-former. We hadresearched the simple and commercial conditions for fermenting Bacillus PC465in larger scale for the practice in shrimp culturing. We freeze-dried theconcentrated bacterial liquid after fermentation to fit for the processing in factoryor added the liquid to the materials to produce feeds simply in our lab. Thisresearch based on the effects of Bacillus. firmus PC465to a key shrimp–L.vannamei for the evaluation of growth promoting and immune stimulation. Theconcentrate B.firmus PC465was mixed directly with feeds to get the granules, inwhich series adding doses of10⁶,10⁸,10¹⁰CFU/g was made to investigate theeffect of different feeding doses on the growth rate and STAT gene expression ofL.vannamei. The concentrate B.firmus PC465was freeze-dried and mixed withfeeds to get granules at an adding dose of107CFU/g to study the effect listedabove, with a setting-up of continuous feeding, interval “4+3”, interval”1+1”. Itindicated during an period of35days that10⁶,10⁸,10¹⁰CFU/g PC465addingdoses improved the growth rate of L.vannamei significantly（P<0.05）, although10¹⁰CFU/g feeding dose had a better effect of increasing growth rate and STATgene expression than106and108CFU/g, but10⁶CFU/g feeding dose, but10⁶CFU/g adding dose had a raio of5.19×10^‐7CFU between the effect of growth‐promoting and adding dose in a comprehensive evaluation, which is great better than others; continuous feeding, interval”4+3” and interval”1+1” increased growth rate and STAT gene expression level significantly（P<0.05）, but continuous feeding had the best effect.As the effects of middle capacity of culture water had been indentified, alarge capacity of30m3will be tested for the growth rate, survival, digestiveenzymes and the structures of interior surface of mid-gut. As the WSSV infection during the experiment period, the resistance of WSSV could be expressed by thesurvivals. It showed that the administration of PC465had a significant effects ofpromoting growth, survival, activities of digestive enzymes and changing theinterior surface of mid-gut. With the effects listed above, PC465can be betterused in practices of shrimp aquaculture.The short-term effect of PC465on the immune status of L.vannamei wasevaluated by the measure of the expression level of immune-related genes, suchas proPO，SP，Crustin，Penaeidin，lysozyme and Dicer-1. The hemolymph ofshrimps was collected and mixed with anticoagulant and RNAlater solution andthen transfer at-80℃. The outcomes showed great effects of PC465administration to the immune promoting of L.vannamei.