Screening And Application Of Probiotics Based On The Purification Of Sea Cucumber Culture Water

The intensive high-density aquaculture of sea cucumber culture results in increasing pollution of aquaculture water.Microecological agents are widely used in the aquaculture industry because of their practicability,low cost,large profit,easy operation and difficulty in forming secondary pollution.However,there are still some problems,such as low specificity,unsuitability for marine environment,low number of living cells and unsuitable compatibility of strains.In this study,the probiotics adapted to the marine environment were enriched and screened,and their growth characteristics,inhibition of pathogenic flora dominated by Vibrio and their ability to purify aquaculture water were studied.According to the characteristics of each strain,the artificial flora was constructed,and new microecological agents were developed to reduce the occurrence of sea cucumber skin rot and solve the water pollution problem in the process of sea cucumber culture.Firstly,8 strains of probiotics were screened from liquid samples of A,B,and C and sediment samples from sea cucumber culture ponds.According to the physiological and biochemical experiments and 16S rRNA gene sequencing,the strains were classified as Lactobacillus buchneri(strain Y1),Acetobacter pasteurianus(strain Y2),Sphingomonas sp.(strain Y3),Lactobacillus casei(strain Y4),Bacillus subtilis(strain Y5),Bacillus velezensis(strain Y6),Kerstersia gyiorum(strain Y7)and Pediococcus pentosaceus(strain Y8).The cell density and viable cell count of 8 strains and a marine strain of Bacillus subtilis DL11-11 in basic and simplified seawater medium were compared,and the growth kinetics were analyzed.It was found that Y2 cannot grow well in seawater medium.Other strains grew much better in seawater medium,especially Y3,Y4,Y5 and Y6.Secondly,the pathogenic flora was enriched and screened by TCBS selective medium from the water samples collected from sea cucumber culture ponds after high temperature explosion.It was found that the flora was mainly composed by Vibrio alginolyticus and Vibrio parahaemolyticus,two kinds of pathogens in China seas for common sea cucumber skin rot.The strains of Y2,Y4,Y5,Y6,and Y8,and DL11-11 all had an inhibitory effect on the pathogenic flora determined by the paper method.Subsequently,the ability of strains to purify water was examined.Among the strains cultured in basic medium,Kerstersia gyiorum Y7 had the highest nitrite nitrogen removal efficiency of 2.62 mg/L·d·10~8 cfu,Bacillus subtilis DL11-11 had the highest ammonia nitrogen removal efficiency of 0.11 mg/L·d·10~8 cfu,and Kerstersia gyiorum Y7 showed the highest COD removal rate.Among the probiotics cultured in simplified seawater medium,Sphingomonas sp.Y3 displayed the highest efficiency in nitrite nitrogen removal and ammonia nitrogen removal,which were 3.82 mg/L·d·10~8 cfu and 0.37 mg/L·d·10~8 cfu,respectively,and COD removal rate was also high.Finally,different combinations of strains were designed according to previous studies on growth characteristics in the media,interactions among strains,inhibition on pathogenic bacteria and the removal on ammonia nitrogen,nitrite nitrogen,COD and turbidity.Among the combinations based on the results from basic medium,complex 4(Y1:Y3:Y4:Y5:DL11-11=5:3:3:2:1)can effectively reduce ammonia nitrogen,nitrite nitrogen and COD after inoculation the culture broth from basic medium,and the nitrite nitrogen removal efficiency and removal rate of ammonia nitrogen were the highest.The removal efficiency of nitrite nitrogen can reach 3.35 mg/L·d·10~8 cfu,which was 27.86%higher than the highest one achieved from individual strain treatment.The removal efficiency of ammonia nitrogen can reach 0.14 mg/L·d·10~8 cfu,27.27%higher than the highest one treated by single strain.Among the combinations based on the results from seawater medium,complex B(Y1:Y3:Y4:DL11-11=3:1:1:5)was effective in degrading nitrite and COD,while complex A(Y3:Y4:Y5:Y6:Y7=2:5:3:3:5)was effective in nitrite and ammonia nitrogen removal.The difference between combination A and B in nitrite nitrogen removal was not significant.The average removal efficiency of nitrite nitrogen by combination B and A treatment in 5 days was 4.26 mg/L·d·10~8 cfu,which was 11.52%higher than the highest one achieved by single strain treatment and 27.16%higher than Complex 4.The removal efficiency of ammonia nitrogen by combination A can reach 0.40 mg/L·d·10~8 cfu,which was 8.10%higher than the highest removal efficiency achieved by single bacteria treatment and 1.86 times higher than Complex 4.The results indicate that probiotics adaptive in seawater is much more promising in water purification than those culture in fresh water.