| In recent years,there are some problems in turtle breeding,such as poor water quality and low feeding efficiency.Bio-floc technology is applied to solve the problem of excessive inorganic nitrogen and frequent changes in water body in aquaculture.Adding probiotics is also a common means to help purify water quality,reduce disease bacteria and improve the immunity of aquaculture.However,while blindly adding carbon sources to improve water quality,a large number of bacteria grow in water,which brings certain risks to aquaculture.Microorganism is an important component unit in water,and the change of microbial community structure affects the function of biological flocs.The purpose of this study was to add probiotics to the biological floc technology to make it colonized in water and become the main flora of flocs,so as to enhance the ability of biological flocs to purify water quality,reduce the proportion of pathogenic bacteria and improve the efficiency of aquaculture.Firstly,the ability of inorganic nitrogen transformation and utilization of 15 species of yeast,Bacillus and Lactobacillus was determined,and Candida utilis which can effectively transform and utilize three kinds of inorganic nitrogen was screened.Then it was found that the optimal C/N ratio for the conversion of ammonia nitrogen and nitrate nitrogen was 15,the optimal C/N ratio for the conversion of nitrite nitrogen was 12.5,the optimal C/N ratio for the conversion of nitrate nitrogen was 15,and the optimal rotation speed for the conversion of three inorganic nitrogen was 150 rpm,namely,dissolved oxygen was 8.69 mg/L.Under the optimum conditions,Candida utilis could assimilate three kinds of inorganic nitrogen,and the absorption and transformation utilization law of Candida utilis when inorganic nitrogen was mixed was explored.It was found that the preference of inorganic nitrogen utilization of Candida utilis was: ammonia nitrogen > nitrite ≥ nitrate nitrogen.Secondly,the screened Candida utilis was applied to the biofloc model of softshelled turtle,and it was found that the optimal C/N ratio of Candida utilis was 15,and the most Candida utilis was found in water.Adding Candida utilis in water can not only effectively reduce the content of ammonia nitrogen and nitrite nitrogen in water,effectively convert inorganic nitrogen into bacterial protein,but also improve the flora of water,reduce the number of harmful bacteria such as Aeromonas and Enterobacter,and increase the number of Bacteroides.The effects of C/N ratio and Candida utilis on the transformation of inorganic nitrogen in turtle aquaculture water were explained by the determination of three-state nitrogen and q PCR results of nitrogen cycle genes.It was found that Candida utilis could promote the transformation of nitrite nitrogen by bacteria in water,but the addition of Candida utilis at C/N = 20 and 15 could lead to low denitrification of nitrate nitrogen.At the same time,16 s high-throughput technology was used to explore the bacterial community structure under this mode.Through the analysis of bacterial community distribution,it was found that the dominant bacteria in each group were Proteobacteria,and the proportion increased with the increase of carbon nitrogen ratio.By ternary phase diagram analysis,it was found that when C/N ratio was 10,the main flora tended to Actinomycetes,and when C/N ratio increased to 15 and 20,the main flora tended to Proteobacteria.By analyzing the nitrogen factor and flora in the environment,it was found that the nitrogen factor with the largest influencing factor was total nitrogen.Then FAPROTAX function was predicted,and it was found that the low C/N ratio level could better transform nitrate nitrogen,and Candida utilis could reduce the function of animal parasites and symbionts of bacteria in water.It was found that increasing C/N ratio could improve water quality and reduce feed coefficient of turtle,and adding Candida utilis liquid could also help to reduce feed coefficient.The verification of inhibition zone and growth inhibition experiment showed that the live Candida utilis strain could inhibit the growth of Aeromonas hydrophila.Then,PMA-qPCR technology was used to explore the number of live bacteria in the solid-state fermentation process of three yeasts,and it was found that different species had different effects on the number of live yeasts.Considering the significant improvement of water quality by Candida utilis,Candida utilis was selected as the main fermentation strain in this study,and it was expected to feed fermented feed to improve water quality.PMA-q PCR showed that the species of lactic acid bacteria had the greatest increase in the number of viable Candida utilis.Then,the effects of different single lactic acid bacteria and their combinations on the number of viable Candida utilis were investigated to determine that Lactobacillus plantarum and Bifidobacterium lactis could increase the number of viable Candida utilis.Finally,the effects of trace metal ions,calcium carbonate and protease addition in solid-state fermentation feed on the number of viable Candida utilis were investigated,and it was found that these additives could lead to a decrease in the number of viable Candida utilis.Considering the quality of fermentation products and aquaculture applications,the combination of strains in the fermentation feed of Candida utilis was determined as follows: Candida utilis,Lactobacillus plantarum,Bifidobacterium lactis,Bacillus subtilis and Bacillus natto.Then,with small peptides as the optimization index,the fermentation conditions were optimized by response surface methodology.The optimized process was as follows: the ratio of soybean residue,soybean meal and secondary powder was 4:5:1,and the combination of strains was the above.The inoculation amount of strains was15 %(V/W),the addition of protease was 1.35 %(W/W),and the water content was51.6 %(V/W).The fermentation was carried out in one-step package anaerobic fermentation at 33.9 ℃ for 7 days.Under this fermentation condition,the content of small peptide in fermented soybean dregs soybean meal feed increased from 7.35 %to 36.54 %.SDS-PAGE showed that most of the large molecular weight proteins were degraded and basically transformed into small molecular proteins and peptides below27 k Da.In order to consider the feasibility of replacing the full-priced fish meal,the mixture ratio was designed to explore the optimal addition ratio.Considering the practical application,the proportion of different amounts of starch and binder was explored under the replacement amount of 25 % and 50 % of fermented feed.The results showed that 0.5 % xanthan gum was added to the 25 % group of biological feed replacement,2.5 % α-starch and 0.5 % xanthan gum were added to the 50%group of biological feed replacement,and the finished feed products after addition could meet the standard of fishery feed.Finally,in order to explore the feasibility of replacing fish meal with Candida utilis feed,the feeding experiments of 25 % and 50% replacing fish meal were conducted.It was found that the feed coefficient of the group with 50 % substitution increased significantly.Therefore,25 % replacement of fish meal group was adopted to continue feeding,and the measured inorganic nitrogen in water was lower than that in fish meal group.And turtle blood routine test found that biological feed partial replacement of fish meal in 25 % group of blood routine normal,more red blood cells.The determination of physiological and biochemical indexes showed that glutamic pyruvic transaminase and glutamic oxalacetic transaminase in the biological feed group were lower than those in the fish meal group,but the serum GLU content was higher than that in the control group.The total antioxidant capacity,alkaline phosphatase and lysozyme activities of the bio-feed group were higher than those of the fish meal group in the determination of serum antioxidant immune indexes of turtle.Small intestine slices showed that bio-feed could significantly improve the ratio of small intestine villi and villi height to crypt depth,but there was obvious villi fragmentation in bio-feed groups.This experiment provides a reference for the partial substitution of fish meal full-value protein source by Candida utilis fermentation feed.To sum up,the scheme of replacing 25 % fish meal by Candida utilis fermented feed has good overall benefit.In summary,Candida utilis with high utilization of inorganic nitrogen was screened in this paper,and the optimum C/N ratio was 15 in turtle aquaculture water,which can help purify water,optimize microbial community structure and inhibit the growth of harmful bacteria,such as Aeromonas hydrophila.And through the study of fermentation flora and process optimization of preparation of Candida utilis fermented feed,replace fish meal feed found not only can effectively improve water quality,but also help to improve the intestines of turtle,improve the immune index of turtle.This study provides a reference for the addition of Candida utilis combined with carbon sources to aquaculture,and provides theoretical guidance for building an ecosystem with efficient nitrogen recycling. |
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