Analysis Of Community Structure And Function Of The Culturable Protease-produced Bacteria In Reared Shrimp System Of Hainan And The ERIC-PCR Classification Of The Dominant Vibrio Species

With the development of aquaculture,eutrophication of water bodies has become growingly serious and has an important factor hindering the sustainable development of aquaculture.Protease which can hydrolyze the peptide chain of a protein molecule catalyzes the decomposition of organic protein macromolecules.At present,most of the bacteria in the culture system are known to have the ability to produce extracellular proteases,but the the diversity of the bacteria flora and their ecological functions have not been clearly clarified yet.This study intends to use the combination of traditional plate culture method,16S rDNA identification method,and Biolog automatic microbial identification for the dominant species to clarify the protease-produced bacteria in culture water,sediment,and probiotics product from Wenchang,Hainan.Then,the analysis of their abundance was carried out.To determines the ecological function of the flora structure,we explored the correlation between the major water quality index,such as ammonia nitrogen,nitrite nitrogen,nitrate nitrogen,and bacterial community structure,which not only reveals the diversity of the protease-produced flora structure in the system,but also provides the strain resources for the development of protease-produced microbial preparations suitable for shrimp culture systems,and lays a foundation for realizing green ecological breeding of shrimp.A total of 525 protease-produced bacterial isolates were obtained from water samples of WenChang with screening medium.The abundance of cultivable protease-produced bacteria was ranging from 6.58×102 to 2.10×103 CFU/mL of 7 water samples.Analysis based on the sequences of 16S rDNA suggested that the isolates belonged to 16 genera from 3 phyla including Proteobacteria(71.6%),Firmicutes(28%),and Actinobacteria(0.38%).Vibrio(61.7%)and Bacillus(24.2%)were predominant genera.Vibrio owensii(24.7%)and Bacillus hwajinpoensis(65.4%)were predominant species.The result of the extracelluar enzymatic activities showed that all strains have the ability to hydrolyze casein,and more than 38.1%strains have high hydrolytic ability,most of them were Vibrio and Bacillus.With the same method,664 protease-producted bacterial isolates were obtained from sediment samples in 7 sites and the abundance of cultivable protease-produced bacteria was ranging from 2.05 x 104 to 2.85×105 CFU/g of 7 sediment samples.They were belonged to 20 genera from 3 different phyla including Firmicutes(91.57%),Proteobacteria(7.23%),and Actinobacteria(1.2%).There were 3 dominant genera such as genera Bacillus(60.6%),Exiguobacterium(28.6%),and Halobacillus(10.3%)which were the typical dominant species.In addition,the abundance and predominant bacteria varied in different waters and sediments.The result of the extracelluar enzymatic activities showed that all strains have the ability to hydrolyze casein,and more than 46.2%strains have high hydrolytic ability,most of them were belonged to Bacillus.67 protease-produced bacterial isolates obtained from probiotics were B.subtilis and B paralicheniformis and the result showed that these strains have a lower hydrolytic ability than that in aquaculture water and sediment,indicating that Bacillus origin from probiotics can not colonize suscessfully.In conclusion,the diversity and community strucure of cultivable protease-produced bacteria in the water and sediment in reared shrimp system of Wenchang were abundant and complex,Bacillus and Vibrio were the dominant native bacteria.The correlation analysis between bacterial flora structure and the water quality index showed that the total number of bacteria in the water had a significant effect on the ammonia nitrogen content in the water.As the total number of bacteria in the water increased,the ammonia nitrogen decreased first and then increased.The number of protease producing bacteria in the sediment had a significant effect on nitrite nitrogen.With the increase of protease-producing bacteria in the sediment,the nitrite nitrogen decreased first and then increased.The amount of protease producing bacteria in water body has a significant effect on the content of nitrate nitrogen.With the increase of proteolytic bacteria in water,nitrate nitrogen decreased first and then increased.Therefore,bacterial flora structure plays an important role in the circulation of substances such as ammonia nitrogen,nitrite nitrogen,and nitrate nitrogen.80 strains of the dominant species V.owensii in water were classified by using ERIC-PCR method.All strains showed clear and distinguishable polymorphic fingerprinting map by ERIC-PCR,which indicating that this techniche can be used as a reliable method of genetic diversity analysis of V.owensii.The ERIC-PCR fingerprints of these strains provide important basic data for future epidemiological investigations and identification of indigenous strains.Cluster analysis of ERIC-PCR fingerprints revealed that 80 strains were classified into 4 groups with a similarity coefficient of 0.58,among which the IVⅣ and Ⅲ strains have the largest number and were distributed in multiple shrimp ponds.The number of the Ⅱ and Ⅰ strains was relatively small,and the source was relatively single,which indicating that the V.owensii in different shrimp ponds in this area have a similar genetic background and there may be a risk of outbreak of waterborne of Vibriosis,16Sr DNA analysis showed that the V.owensii isolated in this study was an indigenous strain,which provided excellent bacterial resources for the development of probiotic preparations for shrimp culture in Hainan.