Effects Of Different Probiotic On The Microbial Community In The Culture Water And The Intestine Of Cea Cucumber(Apostichopus Japonicus)

This study presents a research of the effect of different probiotics on the bacterialcommunity in aquatic water and the intestine of cea cucumber by combinationof traditional method and PCR-DGGE, providing the theory basis for the rational useof probiotics in cea cucumber and related research. Get the results were as follows:1Effect of Bacillus cereus BC-1on the growth and microbial communitystructure in water and intestine of sea cucumberAn8-week trial was conducted to study effects of Bacillus cereus on the growth ofsea cucumber （Apostichopus japonicus） and microbial community structure in thewater and sea cucumber’s intestine by combination of traditional methodand PCR-DGGE. Sea cucumbers were cultured in25aquaria and divided into fivegroups including three treatments and two controls. B. cereus was applied into thewater with the doses of10⁶,10⁸and10¹⁰cfu/m³every7days after water exchange（BC6，BC8and BC10, respectively）. Those with no B. cereus application were as thecontrols, in which water was exchanged every3days （CT1） or7days （CT2）,respectively. The results showed that the specific growth rates of sea cucumber inBC6and BC10were significantly higher than the other groups （P<0.05）, and theindividual differences of sea cucumber were significantly lower than the controlgroup （P<0.05）.With the traditional2216E medium coated method, B. cereus coloniescould only be found in the water of BC6，BC8and BC10on the third day when B.cereus was spilled and disappeared on the7th day; and all groups had found B. cereusin the intestine feces samples in the end of trial. The results from the analysis ofPCR-DGGE showed that α-Proteobacteria, γ-Proteobacteria, δ-Proteobacteria,Flavobacteria, Bacteroidetes and Verrucomicrobiae were dominant in the water, whileα-Proteobacteria, γ-Proteobacteria, δ-Proteobacteria, Flavobacteria, Bacteroidetes andBacilli were dominant in the intestine of sea cucumber. The relative content of Flavobacteria and Bacteroidetes in the intestine of sea cucumber had decreased to acertain extent, and Bacilli had accounted for certain proportion of microbialcommunities in all groups, even in BC6with the relative content reaching to23.3%compared to CT2, while γ-Proteobacteria which is the dominant and fixed bacteriumto sea cucumber had disappeared. These results suggested that B. cereus spilling intowater have significant influences on microbial communities and species, even toabundance of culture water and intestine of sea cucumber. The suitable spillingconcentrations of B. cereus could effectively enhance the growth rate of sea cucumberand reduce the individual differences of sea cucumber.2. Effect of a composite Bacillus on the growth and microbial communitystructure in water and intestine of sea cucumberAn8-week trial was conducted to study effects of a composite Bacillus onthe growth of sea cucumber （Apostichopus japonicus） and microbial communitystructure in the water and sea cucumber’s intestine by combinationof traditional method and PCR-DGGE. Sea cucumbers were cultured in25aquariaand divided into five groups including three treatments and two controls. Thecomposite Bacillus，which contains Bacillus subtilis, B. cereus and B. licheniformis,was applied into the water with the doses of10⁶,10⁸and10¹⁰cfu/m³every7daysafter water exchange （WSR6, WSR8and WSR10, respectively）. Those with nocomposite Bacillus application were as the controls, in which water was exchangedevery3days （CT1） or7days （CT2）, respectively. The results showed that the specificgrowth rates of sea cucumber in WSR6and WSR10were significantly higherthan those in WSR8and the controls （P<0.05）, and the weight coefficients of seacucumber were significantly lower than those in the controls （P<0.05）.With the traditional2216E medium coated method, B. cereus colonies could only befound in the water of WSR10group on the third day when the Bacillus was spilledand disappeared on the7th day; and all groups had found B. cereus in the intestinefeces samples in the end of trial. The results from the analysis of PCR-DGGE showedthat α-Proteobacteria, γ-Proteobacteria, δ-Proteobacteria, Flavobacteria, Bacteroidetesand Verrucomicrobiae were dominant in the water, while α-Proteobacteria, γ-Proteobacteria, δ-Proteobacteria, Flavobacteria, Bacteroidetes and Bacilli weredominant in the intestine of sea cucumber. Bacilli were the most dominant microflorain the intestine of sea cucumber and increased up to35.5%in relative abundance afterthe application of the composite Bacillus. Compared to CT2, the relative abundanceof γ-Proteobacteria increased to some extent as well, while those of the other bacteriadecreased. The results in present study demonstrated that the composite Bacillusapplied into water had significant influences on microbial community structure inboth the water and sea cucumber’s intestine. The composite Bacillus in moderateapplication dose could effectively enhance the growth rate of sea cucumber andreduce the individual differences between sea cucumbers.3. Effect of five Probiotics on microbial community structure of water andthe growth，survival of sea cucumberA8-week feeding trial was conducted the effect of probiotics spilling into water onmicrobial community structure of water and the growth，survival of sea cucumberby combination of traditional method and PCR-DGGE. This five probiotics wereNitrifying bacteria，Bacillus，Photosynthetic bacteria，Effective Microorganisms （aproduct of Shandong Liuhe Group） and Paracoccus sp, and respectively named S1，S2，S3，S4and S5with its spilling concentrations at10¹⁰cfu/m³ï¼ŒThe groupwithout spilling probiotics was control2（CT2）. The water of S1，S2，S3，S4，S5andCT2were exchanged every7-day. Those with no probiotics application were as thecontrols, in which water was exchanged every3days （CT1） or7days （CT2）,respectively. The results showed that the growth rate of sea cucumber of all groupexcepting the S4group after spilling probiotics into water were higher than thegrowth rates of CT2. The S5group showed the best growth performance, and wassignificantly higher than CT2（P<0.05）. The growth rate of sea cucumber in all groupswere significantly higher than the growth rate of CT1（P<0.05）. There was nodifference of survival rate of sea cucumber among all groups. The total heterotrophicbacteria in each group was significantly lower than those of CT2（P<0.05）, whoseheterotrophic bacteria was significantly higher than CT1（P<0.05）. The analysis ofPCR-DGGE showed that the microbial community contained α-Proteobacteria， β-Proteobacteria，γ-Proteobacteria， Flavobacteria and Bacteroidetes. Flavobacteriawas dominant in the culture water of CT1，CT2and S1, and the dominant microflorain the culture water of S3，S4and S5. These results suggested that this Paracoccus spwith spilling concentrations at10¹⁰cfu/m³into water have significant influences ongrowth performance of sea cucumber; this five bacterium spilled into water couldeffectively change microbial communities and species of the culture water; moreover,Probiotics could have suppressed the growth of heterotrophic bacteria.