| The strain SC02with denitrification activity was isolated and identified as Bacillus subtilis through16S rDNA sequence analysis. The present study was designed to evaluate the removal efficiency of ammonia nitrogen and nitrite nitrogen under different nitrogen sources, initial ammonia-nitrogen and nitrite-nitrogen concentrations, pH, temperatures and DO. The results showed that the optimal conditions for SC02to remove nitrogen were: nitrogen source was ammonia nitrogen or nitrate nitrogen, initial ammonia and nitrite nitrogen concentration was10mg/L and10-30mg/L, respectively, rotation speed was0-50rpm, pH was7-9, and the temperature was25-37℃. We also evaluated the effects and mechanisms of strain SC02(lx108cfu/L) and complex bacteria (Bacillus subtilis SC02:Pseudomonas stutzeri F1M=1:1,2x108cfu/L) on water quality in grass carp culture. The results showed that Bacillus subtilis SC02and complex bacteria added into grass carp culture water could significantly reduce nitrogen compounds levels (ammonia, nitrite nitrogen, nitrate nitrogen, total inorganic nitrogen and total nitrogen, etc.) in water over an extended period, and improve water quality.454-pyrosequencing technology was applied to evaluate the effects of probiotics on microbial community structure in grass carp farming environment. The results showed that:(1) SC02could increase the bacterial richness in grass carp culture water. Compared with the control, the proportions of phylum proteobacteria and firmicutes were decreased in SC02group, while bacteroidetes and actinobacteria were increased. For proteobacteria, alphaproteobacteria and betaproteobacteria in SC02treatment were increased, while deltaproteobacteria and gammaproteobacteria were decreased, compared with control. To further compare the microbial communities of the two samples from the control and treatment groups, all-against-all comparison was conducted by using the MEGAN software. The results showed that the relative abundance from the phylum to the genus in the two samples was different, especially at the level of genus.(2) Compared with the control, firmicutes and bacteroidetes were increased, while verrucomicrobia and proteobacteria were decreased in F1M group on the6th d; On the12th d, firmicutes, verrucomicrobia and bacteroidetes were decreased, while actinobacteria and proteobacteria were increased. Samples compared by MEGAN suggested that the relative abundance from the phylum to the genus in the control and treatment samples on the6th d and12th d was different. Not only there are shared bacteria for both groups, but also has their specific strains for each group at different time point.(3) Compared with the control, complex bacteria could increase the level of proteobacteria, bacteroidetes, actinobacteria and firmicutes on the6th d and decrease the relative abundance of cyanobacteria and verrucomicrobia; increase the relative abundance of cyanobacteria, bacteroidetes and firmicutes on the15th d, and decrease proteobacteria, actinobacteria and verrucomicrobia. In addition, based on oxygen requirement, metabolism and biotic habitat, the relative abundance of aerobic bacteria, anaerobic bacteria, ammonia oxidizer bacteria, denitrifying bacteria, nitrite reducer bacteria, free living bacteria, and symbiotic bacteria were increased in complex bacteria group on the6th d (P<0.05). There was no differences between the two groups on the15th d (P>0.05).(4) Complex bacteria added in to water could decrease the bacterial richness in the sediments of grass carp culture, decrease proteobacteria and fusobacteria, while increase bacteroidetes, firmicutes and cyanobacteria. Based on oxygen requirement, metabolism and biotic habitat, ammonia oxidizer bacteria and free living bacteria were significantly increased in complex bacteria (P<0.05), while nitrite reducer bacteria was significantly decreased (P>0.05).The study was designed to evaluate the effect of complex bacteria on immunity, antioxidant activities and gut contents microbial community structure of grass carp. The results showed that complex bacteria added into water could increase the expression of MHCI and TLR3in liver, as well as IgM, TLR3and TLR7in mucosa, while decrease the gene expression of MyDd88and TLR7in mucosa, as well as TNF-a, IL-8and TGF-p in liver. Compared with the control, the levels of total antioxidant activity (T-AOC), superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) in the mucosa were significantly increased (P<0.05). There was no difference about maleic dialdehyde (MDA) in mucosa and antioxidant activities in liver between two groups. The results of454sequencing showed that proteobacteria, cyanobacteria, planctomycetes, verrucomicrobia, firmicutes and actinobacteria were dominant in gut contents of grass carp. Compared with the control, the relative abundances of cyanobacteria, planctomycetes, verrucomicrobia, firmicutes and actinobacteria were increased, while proteobacteria was decreased. Samples from control and treatment groups were compared by MEGAN showed that the relative abundance from the phylum to the genus in the two groups were differences. At the genus level, there were some shared bacteria for two groups, such as Lactococcus, Anoaerostipes, Faecalibacterium, etc and some specific bacteria for each group, for example, Sphingobacterium, Enterococcus and Desulfobacca resisted only in control group, while Acfipia, Thermomonas and Nannocystis only in complex group. As above, the results indicated that complex bacteria as water additive could enhance the immunity and antioxidant activities, improve the gut healthy, reduce stress and inflammatory response of Grass carp.In conclusion, probiotics added into water can improve aquaculture water quality under certain conditions, and its mechanism is that probiotics have denitrification capabilities; Probiotics can change the microbial structure of water and sediment; Probiotics can improve the intestinal microflora, enhance immune function, reduce stress, and improve fish health. |
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