Effects And Mechanism Of Cd(2+) On Biological Nitrogen Removal Enzymes And Functional Genes In Livestock And Poultry Wastewater

Livestock and poultry wastewater has become an important source of pollution.As heavy metals are widely used as feed additives in livestock and poultry industry,high content of heavy metals is an important feature of livestock and poultry wastewater.As a heavy metal with a high detection rate in aquaculture wastewater,Cd²⁺will cause serious damage to biological nitrogen removal system.However,the current research is limited to its impact on biological nitrogen removal performance.The concentration gradient of the research is not comprehensive enough,and the mechanism has not been deeply explored at the molecular biological level.The changes of enzymes and functional genes affect the biological nitrogen removal process.Therefore,by adding different concentrations of Cd²⁺（0 mg/L,0.5 mg/L,1 mg/L,3 mg/L,5 mg/L,10 mg/L,20 mg/L,40 mg/L,80 mg/L）to the SBR reactor,Chemical methods and molecular docking simulation techniques were used to analyze the effects of Cd²⁺on the activity and structure of biological nitrogen removal enzymes.q PCR was used to determine the effects of Cd²⁺on functional genes of nitrogen transformation in biological nitrogen removal systems.Correlation analysis,redundancy analysis and principal coordinate analysis were used to analyze the relationship between functional genes and biological nitrogen removal under Cd²⁺stress.The main conclusions are as follows:（1）The effect of Cd²⁺on the performance of biological nitrogen removal was characterized by measuring the changes of various forms of nitrogen.When the concentration of Cd²⁺is below 5 mg/L,the removal of ammonia has no significant effect,while the accumulation of nitrite nitrogen increases.When the concentration of Cd²⁺is greater than 5 mg/L,the accumulation of nitrite nitrogen decreases,while the concentration of ammonia in effluent increases,indicating that low concentration of Cd²⁺will first inhibit the nitrite oxidation process,while high concentration of Cd²⁺will significantly inhibit the ammonia process.The concentration of effluent COD and aerobic terminal nitrate increased with the increase of Cd²⁺concentration.（2）The activity of biological nitrogen removal enzymes was determined by chemical method.Cd²⁺can significantly inhibit the activity of biological nitrogen removal enzymes.When the concentration of Cd²⁺was greater than 10 mg/L,ammonia monooxygenase was severely inhibited.Linear regression analysis showed that ammonia monooxygenase had a significant linear relationship with the amount of ammonia oxidation,R²=0.93.The activity of nitrite oxidase began to decline when the concentration of Cd²⁺was 1 mg/L.Linear regression analysis showed that nitrite oxidase had a linear correlation with nitrification,R²=0.76.The activities of nitrate reductase and nitrite reductase began to decline when the concentration of Cd²⁺was 1 mg/L,and their activities decreased to the lowest when the concentration of Cd²⁺was 80 mg/L and 40 mg/L,respectively.Linear regression analysis showed that nitrate reductase and nitrite reductase had a significant linear relationship with the denitrification capacity.R² values were 0.76 and 0.89.（3）Molecular docking simulation was used to analyze the effect of Cd²⁺on the structure of biological nitrogen removal enzymes.Molecular docking simulation results show that ammonia monooxygenase,nitrite oxidase,nitrate reductase and nitrite reductase can spontaneously bind with Cd²⁺.Cd²⁺combines with amino acid residues of biological nitrogen removal enzymes through metal complex bond to form metal-protein complex,which destroys protein conformation of enzyme and affects protein function.And block the substrate into the active center of the enzyme channel,so that the enzyme activity is reduced.（4）The abundance of nitrogen transformation functional genes was determined by q PCR.The results showed that the abundance of functional genes of nitrogen conversion decreased with the increase of Cd²⁺concentration.When the concentration of Cd²⁺was 0.5mg/L,the abundance of amo A was inhibited.When Cd²⁺concentration was 3 mg/L,the abundance of nos Z began to decline,and when Cd²⁺concentration was 5 mg/L,the abundance of 16S r RNA,nar G,nir K and nir S began to decline.When the Cd²⁺concentration was 10 mg/L,the abundance of nap A began to decrease.The abundance of nxr A and nor B decreased when the concentration of Cd²⁺was 40 mg/L.nos Z was the most highly inhibited gene and nor B was the least inhibited gene under Cd²⁺stress.（5）Statistical methods were used to study the relationship between functional genes and biological nitrogen removal under Cd²⁺stress.Correlation analysis showed that 16S r RNA and aerobic terminal nitrate were positively correlated with all functional genes under Cd²⁺stress,while effluent ammonia and effluent COD were negatively correlated with all functional genes.Redundancy analysis showed that aerobic terminal nitrate,effluent nitrate and Cd²⁺were the chemical indexes with the highest interpretation rate,and had significant correlation with functional genes.Principal coordinate analysis showed that there were significant differences in functional genes under the action of low,medium and high concentrations of Cd²⁺.The analysis of functional gene network showed that the network complexity of functional genes decreased under the action of medium concentration of Cd²⁺,while the network complexity of functional genes increased significantly under the action of high concentration of Cd²⁺.（6）Linear regression method was used to analyze the key driving factors of functional genes under Cd²⁺stress.nxr A/amo A has a significant linear relationship with the concentration of ammonia nitrogen in effluent.nxr A/amo A is the most important driving factor for the removal of ammonia nitrogen.The nxr A/bacterial 16S r RNA had a significant linear relationship with the nitrate nitrogen concentration in effluent when the three highest concentrations were removed.nxr A/bacterial 16S r RNA was an important driving factor for nitrous nitrogen accumulation.There is a significant linear relationship between nos Z/nar G and nitrate removal in denitrification process.nos Z/nar G is an important driving factor for nitrate removal in denitrification process.Figure[42]Table[11]Reference[120]...