Development Of Fermented Feed Of Tegillarca Granosa And Its Applicantion In Shellfish And Algae Systems

Due to the lack of inexpensive,safe and efficient carbon sources,nitrogen sources and minerals,live microalgae produce low,which directly affects the growth performance of the Tegillarca granosa.Therefore,it is urgent to develop safe,efficient,and inexpensive aquatic feed for the long-term growth of microalgae and Tegillarca granosa.Microalgae growth rates are thought to be sped up by performing mixotrophic cultivation,employing organic nitrogen as nitrogen sources,and adding minerals.These three essential nutrients are abundant in fermented feed,but different algae species have varying rates of assimilation of various organic carbon,organic nitrogen,and mineral sources.Particularly few investigations have been done on shellfish-preferred diatoms like Chaetoceros muelleri and chrysophytes like Isochrysis zhanjiangensis.In this study,the optimal nutrient factors for the growth of Chaetoceros muelleri and Isochrysis zhanjiangensis were selected to prepare fermented feeds of Tegillarca granosa.Firstly,glucose,ethanol,lactic acid,acetic acid,citric acid,tartaric acid,malic acid,succinic acid,formic acid,and propionic acid,which are commonly found in fermented feed products,are used as organic carbon sources under the mixotrophic cultivation of Chaetoceros muelleri and Isochrysis zhanjiangensis.Results indicated that 0.5 g/L acetic acid was optimal for growth in Chaetoceros muelleri and Isochrysis zhanjiangensis.At this time,the algal cell densities of Chaetoceros muelleri and Isochrysis zhanjiangensis were 1.23×10~7CFU/m L and 1.70×10~7CFU/m L,respectively,which were 2.10 and 2.13 times higher than those of autotrophic culture group.Based on this,the response surface experiment was used to optimize the organic acid composite ratio,and it was discovered that Chaetoceros muelleri could grow to a maximum of1.41×10~7CFU/m L when cultured with acetic acid 0.599 g/L,citric acid 0.492 g/L,and succinic acid 0.208 g/L.When Isochrysis zhanjiangensis was cultured with acetic acid 0.400 g/L,tartaric acid 0.512 g/L,and formic acid 0.206 g/L,the algal cell density increased to a maximum of 2.07×10~7CFU/m L.Therefore,the compound organic acid is more effective than the single organic acid in Chaetoceros muelleri and Isochrysis zhanjiangensis cultures.Then,17 free amino acids and small peptides(glutathione,soybean peptides,and wheat oligopeptides)commonly found in fermented feed were used as nitrogen sources for the growth of Chaetoceros muelleri and Isochrysis zhanjiangensis.Chaetoceros muelleri and Isochrysis zhanjiangensis could utilize multiple amino acids and small peptides for growth,according to the findings.When N=882μmol/L arginine and lysine were used as nitrogen sources,Chaetoceros muelleri and Isochrysis zhanjiangensis had the highest algae cell density,with 6.05×10~6CFU/m L and 7.15×10~6CFU/m L,respectively,significantly higher than the inorganic nitrogen group.Using small peptides as nitrogen sources for the first time,the algal cell density of Chaetoceros muelleri and Isochrysis zhanjiangensis attained its maximum when 0.15 g/L glutathione was added.They were 6.85×10~6CFU/m L and 9.55×10~6CFU/m L,which were 2.28and 2.55 times greater than those in the no-nitrogen group,respectively.Subsequently,chelated minerals are used as nutritional supplements,the effects of inorganic,organic,and chelated iron,copper,zinc,manganese,and calcium sources,as well as silicon sources closely related to diatoms,on the cultivation of Chaetoceros muelleri and Isochrysis zhanjiangensis be explored.Using ferrous lactate as the iron source,copper glycinate as the copper source,calcium aspartate as the calcium source,zinc lactate as the zinc source,and manganese acetate as the manganese source produced the greatest growth effect for Chaetoceros muelleri.The optimal growth effect of Isochrysis zhanjiangensis is achieved with ferric chloride as the iron source,copper glycine as the copper source,calcium aspartate as the calcium source,zinc sulfate as the zinc source,and manganese chloride as the manganese source.The best silicon source for the proliferation of Chaetoceros muelleri and Isochrysis zhanjiangensis is sodium silicate with Si=105umol/L.Based on the above research on the nutritional factors of Chaetoceros muelleri and Isochrysis zhanjiangensis,we finally used N source:C source=1:9 in feed raw materials,Lactobacillus plantarum,Bifidobacterium lactis,Lactobacillus buchneri,Candida utilis,and Bacillus subtilis as fermentation strains,in which lactic acid bacteria:yeast:bacillus=3:2:1,the content of keratinase and acid protease is 1.185%,sodium sulfite content is 0.076%,and fermented time is 9 days.Under this optimal condition,the content of small peptides in fermented feed reached 36.96%,the content of acetic acid increased from 0.47 mg/g to 36.52mg/g,and the total free amino acid content increased from 5.02 mg/g to 29.18 mg/g.Adding fermented feed to the microalgae culture medium,it was found that the algal cell density of Chaetoceros muelleri and Isochrysis zhanjiangensis reached 1.50×10~7CFU/m L and 2.10×10~7CFU/m L,respectively,2.56 and 2.63 times higher than that of ordinary F/2 culture medium,and both were higher than those of the group with only acetic acid added.Finally,the fermented feed was applied to Tegillarca granosa culture.First,the survival rate of the Tegillarca granosa was taken as the index,and the growth conditions of the Tegillarca granosa were optimized as the salinity of 20 ppt,oxygen duration of 20h,and mixed algae ratio of 1:1.After that,microalgae,fermented feed and shellfish powder were fed into Tegillarca granosa with a single/mixed match.The results indicated that there were no significant differences in survival rate,daily gain rate,or ammonia nitrogen content in water quality between the fermented feed and half the amount of microalgae feeding group and the microalgae feeding group alone.In conclusion,acetic acid had the best effect on Chaetoceros muelleri and Isochrysis zhanjiangensis among the organic carbon sources,and fermented feed rich in acetic acid,amino acids,and small peptides had a better growth effect on Chaetoceros muelleri and Isochrysis zhanjiangensis than the single acetic acid group.Fermented feed promoted the growth of Tegillarca granosa by increasing the production of microalgae,while simultaneously reducing the risk of water quality degradation due to overfeeding and feeding quantity of single-cell microalgae in the culture of Tegillarca granosa.This study is of great importance for the selection of nutritional supplements to increase the yield of Chaetoceros muelleri and Isochrysis zhanjiangensis,and it serves as a reference for the future development of safe,effective,and affordable aquatic fertilizers.