Study On Antibacterial Characterization Of Bacillus Velezensis DY-6 And Its Influence On The Growth And Immunity Activities Of Apostichopus Japonicus And The Mocrobiota In The Culture Pond

1.Screening and characteristic of Bacillus velezensis derived from ponds for sea cucumber?Apostichopus japonicus?For the purpose of screening local probiotic strains,bacteria in water and sedime nt samplings from cultured ponds of sea cucumber Apostichopus japonicus were isolated,in which 66 kinds of cultivable bacterial strains were gained in Dongying in May 2014.Firstly,bacterial antagonistic experiments were carried out by using Pseudoalteromonas nigrifaciens,Vibrio splendidus,Vibrio parahaemolyticus,and Vibrio alginolyticus,which were main pathogenic bacteria of‘skin ulcer syndrome'for Apostichopus japonicus.Secondly,used selective medium to test the production of amylase and protease activities of the isolated strains,and obtained a potential probiotic strain DY-6.Then,it's physiological and biochemical experiments,similarity analysis of 16S rDNA sequences and the growth characteristics have been conducted,as well as the safety for the Apostichopus japonicus.The results revealed that DY-6 had good inhibition to P.nigrifaciens,V.splendidus,V.parahaemolyticus and V.alginolyticus,in which the diameter of inhibition zone was 24 mm,22 mm,27 mm and 37 mm,respectively.Meanwhile,the diameter of amylase and protease hydrolysis were 28 mm and 20 mm.A safe experiment had been fulfilled to test the safety of DY-6.During the experiment period,either bathing group or feeding group,sea cucumbers were in good conditions without fal ing ill or dying.Based on the physiological and biochemical experiments and similarity analysis of 16S rDNA sequences,the strain DY-6 had 99%similarity to Bacillus velezensis CP015911.Thus,it was preliminarily identified as Bacillus velezensis.Comparing its growth characteristics to the 4 kinds of bacterial strains isolated from commercial probiotic products.We found that DY-6 had better growing under the temperature 20 to 35?as well as the salinity 0 to 35,which were superior to other 4strains mentioned above.Moreover,DY-6 got into the logarithmic period after 4 h cultivation,and got into the growth summit after 10 h.In summary,the strain DY-6 has superb inhibition to the pathogenic bacteria for sea cucumber,rapid growth,as well as rycheme and euryhaline features.Therefore,DY-6 could not only decrease the disease occurrence,but also suitable applying to high temperature in summer and the great fluctuation of salinity in Yelow River Estuary.Thus,the strain DY-6 has a good commercial development potential.2.Analysis of influences of Bacillus velezensis DY-6 addition on the growth,enzyme activity and gut microbiota of Apostichopus japonicus and their correlationThis experiment aims to figure out the influences on the relative growth rate,and non-specific immune activities of coelom fluid of Apostichopus japonicus after using Bacillus velezensis DY-6 in different concentration,and obtains the optimum concentration.Then by conducting high-through-put sequencing analysis technology,this experiment also analyzed the influences on the gut microbiota of Apostichopus japonicus at the optimum concentration of Bacillus velezensis DY-6 at the experimental period of 49 d.The result turned out that,the relative growth rates of experimental groups which had been fed with Bacillus velezensis DY-6 were all higher than the control groups,and the experimenta l group of 1×10³ CFU/mL achieved the highest growth rate,that was 25.3%.The non-specific immune activities of the experimental groups?ACP,AKP,SOD,LZM?demonstrated promotions of different degrees,and when the concentration was 1×10³CFU/mL or 1×10⁴ CFU/mL,the non-specific immune activity of coelom fluid of Apostichopus japonicus had been promoted to a considerable extent,reaching its crest time at 21 d or 28 d.In conclusion,the optimum concentration of Bacillus velezensis DY-6 is 1×10³ CFU/mL.By taking 16S rDNA sequencing analysis,this study also investigated the influences towards gut microbiota with the concentration of 1×10³CFU/mL to find out the changing process of it with the involvement of Bacillus velezensis DY-6.Taking 28 d as the dividing line,the gut microbiota could be obviously divided into two groups,with its two peaks of richness and diversity at 14 d and 21 d respectively.According to the research,there was an obvious correlation between the non-specific immune activities and some gut bacteria after tusing Bacillus velezensis DY-6,and mainly focused on the phylum of Firmicutes,Proteobacteria,and Bacteroidetes.This conclusio n provides the theoretical foundation of using probiotics in the farming of Apostichopus japonicus.3.The influence of Bacillus velezensis DY-6 on the Apostichopus japonicus ponds water and the water microbiota.This study investigated the influence of Bacillus velezensis DY-6 on the Apostichopus japonicus pond water,and analysis the change of water microbiota by high throughp ut sequencing technology during the experiment time of 55 days.The results showed that the sea cucumbers in the pond that used Bacillus velezensis DY-6 had a significa nt improve in SGR and WGR to the control group,up to 112%,1.36%/d,respectively;the use of Bacillus velezensis DY-6 could decrease the content of NO₂-N,COD and NH₃-N,although there was a increase trend during the last period of the experiment,it still lower than the control group.By the analysis of high throughput sequencing technology,it showed that,the microbiota of the water samples in the pond hadn't using Bacillus velezensis DY-6 were classified into the same cluster,while the microbiota of the water samples in the pond using Bacillus velezensis DY-6 were classified into the same cluster;by analysis the PCoA results,we found that the microbiota of the water samples which hadn't use the Bacillus velezensis DY-6 was significantly different to the samples that used Bacillus velezensis DY-6,it is revealed that the use of Bacillus velezensis DY-6changed the water microbiota of the pond;the Ace index and Chao1 index,Shannon index presented an increasing trend,it suggested that the riches and diversity of the sea cucumber pond water was increasing and upped to the highest in the W4 group;moreover,analyzing the unifrac of water samples in different time,it suggested that the dominant bacteria in 5 groups had a significantly different?p<0.05?.4.Analysis of the characteristics of extracellular antibacterial proteins of Bacillus velezensis DY-6 and the whole-genome sequence of DY-6.This experiment used glass covering method to make extracellular and intracellular products of DY-6.And using Vibrio alginolyticus as indicator,detected the antibacter ia l activities of extracellular and intracellular products,respectively,it turned out that either extracel ular and intracellular products had antibacterial activities to V.alginolyticus,moreover,the antibacterial activities extracel ular products was better than the intracellular products.In the meantime,the experiment of antibacterial activities of different time's extracel ular products was conducted,it showed that the extracellular products in 24⁷2h were better than the other time's;the extracellular products was precipitated by saturated ammonium sulfate,and the results showed that when the concentration were in 60⁸0%,80%,we had a better ntibacterial activities to V.alginolyticus.Based on this,we conducted the whole-genome sequence of DY-6.After analyzing the sequence data,we found that the DY-6 was 3.79 M,had 3858 ORFs,making89.1%of the total genome.Compared in the COG database,there were 2487 genes classification information could be found,of which 2232 could be clearly to find the protein function classification,255 for the unknown function protein.In the GO database comparison results showed that the genes and gene product properties were mainly concentrated in cels,enzyme catalysis and metabolic process,CAZy database annotation results showed that the enzyme genes DY-6 major such as Glycosyl Transferases and Glycoside Hydrolases and other four families,through experiments and analysis indicate that DY-6 has great development potential.