The Application Of Flocculent Producing Microbial In The Culture Of Shrimp (Litopenaeus Vannamei)

Understanding the microbial community structure in shrimp ponds is helpful torealize the status of shrimp health. To keep the probiotics in high concentration inpond of shrimp, if the aquaculture water microbial communities could be regulatedartificial, can improve culture benifit. In this experiment water samples werecollected in Litopenaeus vannamei culture ponds from Wenzhou, Zhejiang Province,Sheyang, Jiangsu Province and Changyi, Shandong Province, to investigate themicrobial community. Fifteen cultivable bacteria have been isolated and identifiedby16S rDNA method. Preliminary results showed that bacteria isolated from shrimpponds in Wenzhou consisted of Acinetobacter baumannii, Micrococcus luteus,Paracoccus sp., Acinetobacter sp., Kurthia gibsonii and Staphylococcus sp.. Bacteriaisolated from shrimp ponds in Sheyang consisted of Vibrio sp., Rheinheimeraaquimaris, Shewanella sp., Halobacillus sp., Pseudomonas sp.and Brachybacteriumsp..Bacteria isolated from shrimp ponds in Changyi consisted of Pseudoalteromonassp., Alteromonas litorea and Vibrio sp.. This survey demonstrates the diversity ofthe culturable bacterial in water column of shrimp ponds, and enriches informationof bacteria in shrimp culture.Bio-floc technology has great development potential and value of promotion inaquaculture. Some studies have been carried out in the aspects of the formationmechanism, control technology and the application of bio-floc in many countries. Inthis study, bio-flocs were achieved by artificial culture in natural marine water. Inorder to investigate the active ingredients in the microbial flocs, molecular weight,content of total polysaccharide and amino acid in the microbial flocs were analyzedby methods of gel permeation chromatography, Phenol sulfuric acid colorimetry andautomatic analyzer for amino acid respectively. The results of preliminarybiochemical analysis showed that the average molecular weight of extractive from supernatant of microbial flocs was about213281Da, the polysaccharide and aminoacid content of sediment from microbial flocs was29.57%and12.63%respectively.Further study was conducted to investigate the function of microbial flocs toenhance the immune activity of shrimp. Microbial flocs were added in theLitopenaeus vannamei diet at a rate of0%,0.02%,0.1%,0.5%, and2.5%. Activity ofantibacterial, lysozyme and phenoloxidase （PO） in serum of shrimp were analyzedafter14days of feeding trial. Challenge test was carried out to evaluate the effect ofmicrobial flocs on improving the ability of shrimp against virulent bacterium ofVibrio Harveyi after30days of feeding trial. Results show that activity ofantibacterial and lysozyme in serum of shrimp fed diet containing microbial flocs at arate2.5%were higher than that in other groups （P<0.05）. PO activity in serum ofshrimp fed diet containing microbial flocs at rates0.5%and2.5%were higher thanthat in other groups（P<0.05）. Challenge test show that cumulative mortality of shrimpfed diet containing microbial flocs at a rate of0.1%was the lowest than that in othergroups（P<0.05）. The present results suggested that microbial flocs can be utilized byshrimp and can improve the ability of non-specific immunity of shrimp.In order to further study the composition of bacteria in the bio-floc, culturablebacteria in bio-floc were isolated. Identification method of bacteria mainly adoptedthe16S rDNA sequcing as well as bacterial whole fatty acid identification method.Bacteria in this bio-floc are comprised of Vibrio-fluvialis/furnissii, Vibrio-gazogenes,Halomonas taeanensis, Bowmanella denitrificans, and Vibrio natriegens. Researchon nitrification, denitrification and remove nitrite characteristics of bacteriaBowmanella denitrificans were conducted. The results show that bacteria have greatcapacity of utilization of nitrogen. And pH7.0, C/N15, salinity20is the bestconditions for the denitrification of bacteria.The bacteria Bowmanella denitrificans flocculate activity is up to79%, andhave a good vitality of flocculation. The affect of the heavy metal ions of Cu²⁺, Fe3+,Ni²⁺on the vitality of flocculation were analysied. The results showed that the threeheavy metal ions could inhibition of vitality of flocculation of the bacteria in different degrees. Fe3+has the ability to inhibite of the most significant in forty eighthours, flocculation rate of added Cu²⁺group have a decreasing trend after24hours,but other groups are growing trend at the same time. Different concentrations ofbacteria were injected in the shrimp, and computed for median lethal dose （LD50） ofshrimp was2.25×10⁸cfu/mL. This value far higher than the LD50value of shrimpcommon pathogens. Under laboratory conditions farming shrimp, and a certainconcentration of bacteria is added in water bodies, and seted different concentrationof sucrose. The plus bacteria and plus sucrose8mg/L simultaneously group have thehighest survival rate of shrimp, and value of ammonia nitrogen less than the valueother groups after fourteen days experiment.