The Effect Of Chitosan As A Flocculation Additive On The Physiological Indexes And Biological Flocculation Performance Of Tilapia In Raw Flocculation Culture

As a commonly used feed additive for aquatic products,chitosan has been proven to be used as a flocculant,immunologically active agent and bacteriostatic agent.In this study,chitosan was used as a biological floc additive to investigate the effects of chitosan on water quality indicators,floc performance,microbial structure,and non-specific immunity of cultured objects.This article is mainly divided into 5parts.1.The effect of chitosan on the nitrogen conversion capacity of bioflocculation and its degradation law in bioflocculationIn order to verify whether chitosan can be used as a carbon source to affect the nitrogen conversion capacity,this experiment is divided into four groups of nitrification conditions and assimilation conditions.The addition of glucose is the assimilation condition,and the non-addition is the nitrification condition.All groups are charged at 10 mg/add 0.42g of ammonium chloride crystals to the total ammonia nitrogen（TAN）of L:blank control group,no chitosan or glucose added;glucose added group,according to the ratio of dissolved organic carbon to TAN（DOC/TAN）add 6.2g glucose monohydrate for 20;add chitosan group,only add 25g chitosan;add chitosan and glucose group,add 6.2g glucose and 25g chitosan according to DOC/TAN.The results showed that under nitrification conditions,the TAN removal rates of the blank control group and the chitosan added group reached 93.70%and99.25%,respectively;under the assimilation conditions it reached 99.3%and 97.66%.The results showed that under the two conditions,the addition of chitosan had no significant effect on the nitrogen conversion ability of the flocs in a short time.In order to explore the degradation law of chitosan in biological flocculation,this experiment was divided into four groups.The blank control group did not add chitosan,and the other three groups added 20 g,40 g and 60 g chitosan.The TAN of the experimental group supplemented with chitosan increased rapidly,reaching the highest point at 24 h,which were 0.11 mg/L,0.36 mg/L,0.42 mg/L and 0.49 mg/L,respectively.There was a significant difference between all groups（P<0.05）.Similar to TAN,there was a significant difference between the highest values of nitrite nitrogen（NO₂^--N）in all groups（P<0.05）.Nitrate nitrogen（NO₃^--N）showed an overall upward trend throughout the experiment.At the end of the experiment,the four groups were 77.68 mg/L,92.90 mg/L,106.33 mg/L,and 121.45 mg/L.Significant difference（P<0.05）.The phosphate(PO₄^3-)of the blank group showed an upward trend throughout the experiment.The PO₄^3-of the chitosan group first decreased and then increased,reaching the lowest point at 84 hours,respectively 5.56mg/L,5.10 mg/L and 4.47 mg/L,there was a significant difference between the three groups（P<0.05）.The experimental results of this part show that:chitosan can be degraded in biological flocculation,and the degradation of chitosan will have a significant impact on water quality indicators,NO₃^--N,total nitrogen（TN）and dissolved total nitrogen（Dissolved total）.nitrogen,TDN)significantly increased,PO₄^3-significantly decreased,carbon to nitrogen ratio（C/N）and crude protein（Crude protein）were significantly increased,crude fat（Crude fat）was significantly reduced,chitosan was added The effect of the amount on the nutritional indicators is basically insignificant.2.The effect of adding chitosan to the biological flocculation breeding system on the breeding effectIn order to explore the application effect of chitosan as a floc additive in actual aquaculture production,this experiment tested the changes in water quality indicators,floc indicators,fish body indicators,and system nitrogen and phosphorus budgets.The experiment was divided into four groups.The blank control group only added glucose,and the other groups added chitosan at the proportions of 5%,10%and 15%of the dry weight of the flocs.The cultivation period was 60 days.At the end of the experiment,the NO₃^--N,NO₂^--N,TAN and PO₄^3-indexes of all groups had no significant difference（P>0.05）.The TN of all groups continued to increase throughout the experiment.At the end of the experiment,the highest TN values of the four groups were 217.80 mg/L,232.17 mg/L,256.77 mg/L,and 275.77mg/L.There were significant differences among the four groups.（P<0.05）.During the whole experiment,the TDN of the four groups showed a rising trend,and there were significant differences among the four groups（P<0.05）.Vibrio（Vibrio）was in the trend of increasing first and then decreasing continuously throughout the experiment.The lowest values of each group at the end of the experiment were4083 cfu/ml,2767 cfu/ml,2233 cfu/ml and 433 cfu/ml,respectively.There were significant differences among the four groups（P<0.05）.The addition of chitosan had no significant effect on C/N,crude protein,crude ash and fatty acids（P>0.05）.The content of methionine（Met）in the chitosan group was significantly higher than that of the blank control group（P>0.05）.<0.05),the crude fat content of 15%chitosan group was significantly higher than other groups（P<0.05）.The addition of chitosan has no significant effect on the growth performance and survival rate of tilapia（P>0.05）,and has no significant effect on the nitrogen and phosphorus budget and nitrogen and phosphorus utilization of the biological flocculation culture system（P>0.05）,and it has no significant effect on the whole fish.And muscle C/N,crude protein,water,crude fat and amino acid（Amino acid）had no significant effect（P>0.05）,but had a significant effect on the crude ash content of the whole fish（P<0.05）.The addition of chitosan can significantly affect the antioxidant capacity of tilapia,and the maximum superoxide dismutase（SOD）in serum can reach136.36±2.09 U/m L.The addition of chitosan can significantly affect the non-specific immune indicators of tilapia,making the serum nitric oxide（NO）and alkaline phosphatase（ALP）up to 25.81±0.27μmol/L and 7.01±0.07 U/100m L,so that the maximum nitric oxide（NO）,alkaline phosphatase（ALP）and lysozyme（LZM）in the liver can reach 1.20±0.03μmol/g,19.06±0.19 U/g and 0.42±0.02 U/mg,respectively.This experimental study shows that adding a small amount of chitosan to the biological flocculation aquaculture system can have a significant impact on the flocs and cultured objects.These effects include the nutritional indicators of the flocs,the content of vibrio and the non-specific immunity of tilapia,etc.The application of chitosan in biological flocculation has practical significance.3.The influence of chitosan on the nitrogen conversion capacity of settleable and non-settled flocs and the particle size of the flocsIn order to study the effect of chitosan on biological flocculation in more detail,the nitrogen conversion capacity of the settleable and non-settable flocs and the particle size of the flocs were studied.This experiment uses the flocs from the culture experiment,and the grouping is the same as that of the culture experiment.All reactors are added with 0.42g ammonium chloride crystals at 10 mg/L of TAN,and6.2g of glucose is added at DOC/TAN of 20 under assimilation treatment conditions.Do not add under nitrification conditions.Under the conditions of assimilation,there was no significant difference in the removal rate of TAN of settleable flocs in all groups（P>0.05）.Under nitrification conditions,there was no significant difference in the TAN removal rate of settleable flocs between the blank control group and the 10%chitosan added group（P>0.05）.Under the conditions of assimilation,the TAN removal rates of the four groups of non-sediable flocs reached 7.32±0.15 mg TAN/（g TSS·h）,3.30±0.11 mg TAN/（g TSS·h）,3.22±0.14 mg TAN/（g TSS·h）and13.44±0.39 mg TAN/（g TSS·h）,the TAN removal rate of the 15%chitosan group was significantly higher than that of the other groups（P<0.05）.Under nitrification conditions,the TAN removal rates of the four groups of non-settling flocs reached1.55±0.21 mg TAN/（g TSS·h）,1.85±0.31 mg TAN/（g TSS·h）,1.82±0.16 mg TAN/（g TSS·h）and 7.70±0.23 mg TAN/（g TSS·h）,the TAN removal rate of the 15%chitosan group was significantly higher than that of the other groups（P<0.05）.In the>50μm floc group,the 125.0-500.0μm flocs of the chitosan group were significantly reduced compared with the blank group（P<0.05）.In the<50μm floc group,the 16.0-32.0μm particle size flocs of the 15%chitosan group were significantly increased compared with other groups（P<0.05）,while the 63.0-125.0μm particle size flocs were compared The other groups decreased significantly（P<0.05）.There was no significant difference in the crude fat content and crude ash content of all groups（P>0.05）.The types of fatty acids in the chitosan added group increased compared to the blank control group,and the met content of the chitosan added group was significantly higher than that of the blank control group.（P<0.05）.The results show that the addition of chitosan can significantly increase the TAN removal rate of non-settling flocs,and can significantly reduce the particle size of the flocs.4.The effect of chitosan on the structure of bioflocculation flora,gene expression and fish intestinal flora structureIn order to better understand the impact of chitosan on biological flocculation,this experiment uses the floccules from the breeding experiment,and the grouping is the same as that of the breeding experiment.Settling flocs were detected through metagenomics analysis,macrotranscriptomics analysis and 16s DNA sequencing analysis.The structure and function of microbial clusters,non-sinkable flocs and fish intestines.Among the initial flocs,the most abundant metagenes and macrotranscripts were Proteobacteria,which were 46.85%and 48.36%,respectively.The main bacteria phyla with the highest%RNA/%DNA value was Planctomycetes.The value is 2.78.As the amount of chitosan increased,the%RNA/%DNA value of Proteobacteria and Mucoromycota gradually increased,while the%RNA/%DNA value of Verrucomicrobia gradually increased.Decrease;the value of%RNA/%DNA of Chytridiomycota in the chitosan group was significantly lower than that of the blank control group.The abundance of Fusobacteria in the intestine of fish in the 10%chitosan group was significantly higher than that in the other groups（P<0.05）.The results showed that in the classification level of phylum,class,order,family,genus,and species in biological flocs,the flora structure in metatranscriptomics and metagenomics was significantly different.The results show that chitosan has a significant effect on the settleable flocs,non-settled flocs and the microbial structure of the fish intestines,and chitosan with different addition amounts can have a significant effect.