Analysis On The Diversity Of Bacillus In Two Different Reared Shrimp System In Hainan

In recent years,Bacillus has been widely used in shrimp farming in Hainan in response to aquaculture environment problems such as eutrophication of rearing water and the widespread existence of antimicrobial resistant pathogenic bacteria.However,the diversity of Bacillus in the shrimp farming system of Hainan,the survival status and the effect of administrated Bacillus in the environment are still unclear;the impact of adding a large number of carbon sources on the microbial flora during the cultivation process is also unclear.To this end,this study isolated,screened and identified Bacillus in two different shrimp farming systems in Changjiang and Wenchang,Hainan,and screened for amylase-producing strains.The results showed that:Six artificial concrete ponds at Changjiang aquaculture farm were chosen for this study,with high degree of manual intervention(a large amount of probiotic preparations and carbon sources are added)and good water quality.The cultivable Bacillus in rearing water of Changjiang was 1.3×10~3～2.11×10~4 CFU/m L,and the concentration of Vibrio spp was 0～9.00×10~3 CFU/m L.A total of 266 strains of Bacillus were isolated from the rearing water in Changjiang.16S r DNA sequence comparison results showed that these 266 strains of Bacillus belonged to the genus Bacillus and Halobacillus,of which B.paralicheniformis is the dominant species,with a total of 251 strains;RAPD-PCR analysis results showed that the similarity coefficient of B.paralicheniformis is from 0.056 to 1,when the similarity coefficient is 0.89,all strains can be divided into 13 categories,in addition,different genotypes of B.paralicheniformis may differ in their ability to inhibit Vibrio.The screening of the amylase producing capacity of 266 strains of Bacillus showed that a total of 248strains have the ability to produce amylase,and there was a wide diversity of enzyme producing activities.The shrimp aquaculture in Wenchang was with low artificial intervention(rarely added probiotic preparation and carbon source)and poor water quality,the concentration of Bacillus in rearing water of Wenchang farm was 9.50×10~1～1.64×10~3CFU/m L,and the concentration of Vibrio spp.was(2.37～5.98)×10~3 CFU/m L.The fitted regression equation between the concentration of Bacillus and Vibrio was y=0.0034x~2-8.4465x+7545.5(x:the concentration of Bacillus)(p<0.05),indicating that with the concentration of Bacillus increasing in water,the concentration of Vibrio in water first decreased and then increased.A total of 557 strains of Bacillus were isolated from cultured water and 16S r DNA sequence alignment results showed that the 557 strains of Bacillus belong to seven genera including Bacillus(63%),Halobacillus(33%),and Thalassobacillus(3.6%)and so on,among them,Bacillus hwajinpoensis(58.2%)and Halobacillus trueperi(59.1%)are the dominant species in Bacillus and Halobacillus with the effect of reducing ammonia nitrogen and inhibiting the growth of Vibrio,respectively.The results of RAPD-PCR analysis showed that the similarity coefficient of B.hwajinpoensis is between 0.62 and 1,when the similarity coefficient is 0.81,all strains can be divided into 18 categories;the similarity coefficient of H.trueperi is between 0.72～1,when the similarity coefficient is 0.86,all strains can be divided into 18 categories.A total of 594 Bacillus strains were obtained from Wenchang sediments,which belonged to nine genera including Bacillus(73%),Halobacillus(22%),Thalassobacillus(3.5%)and so on,and B.hwajinpoensis and H.trueperi are the dominant species in Bacillus and Halobacillus,accounting for 28%and 30.8%,respectively.The results of RAPD-PCR analysis showed that the similarity coefficient of B.hwajinpoensis is between 0.71and 1,when the similarity coefficient is 0.81,all strains can be divided into 16categories;the similarity coefficient of H.trueperi is between 0.78～1,when the similarity coefficient is 0.86,all strains can be divided into 11 categories.A total of1151 strains of Bacillus in Wenchang water samples and sediments were screened for amylase-producing ability.A total of 651 strains had amylase-producing ability,among them,B.hwajinpoensis and H.trueperi in water samples had strong enzyme-producing ability,and B.oryzaecorticis and H.trueperi in sediment had strong enzyme-producing ability.Three kinds of Bacillus were screened from Changjiang probiotic preparations,namely B.patagoniensis,B.subtilis,and Paenibacillus glucanolyticus;and three types of Bacillus were screened out from two probiotic preparations in Wenchang,namely B.subtilis,B.paralicheniformis,and B.tequilensis.After RAPD typing of the selected B.paralicheniformis,the genotype was found to be the same as the B.paralicheniformis genotype in Changjiang B4 breeding pond.This shows that different genetic strains have different environmental adaptability,therefore,according to different farming environments,the precise development of Bacillus preparations is the key to fully and steadily exert the ecological functions of such preparations.In short,the artificial addition of Bacillus in two farming modes has not been successfully colonized in the rearing water,which indicated that the water quality improvement microbial preparation for the shrimp farming system still needs to be improved.The cultured water with high manual intervention had a high concentration of Bacillus,and a low diversity within dominant strains,but good water quality,therefore,we speculate that artificial selection and administration of a few types of suitable indigenous strains can make it a dominant strain in shrimp culture systems and can effectively promote the material circulation of the system,which points the way for the selection of beneficial bacteria for water quality improvement.The concentration of Bacillus in the cultured water of shrimps with low manual intervention is low and the diversity of species is high,but the water quality is poor.This shows that there are abundant Bacillus resources naturally in the shrimp farming system of Hainan,therefore,it is completely feasible to dig deep into the resources of Bacillus spp.in Hainan and develop special water quality improved Bacillus preparations for shrimp culture systems.