| As a typical synthetic sulfonamide drug,sulfamethoxazole has the advantages of broad antibacterial spectrum,stable property and simple use,so it has been widely used in intensive livestock and poultry breeding.A large amount of residual sulfamethoxazole will enter the sewage treatment system with aquaculture wastewater,and biological phosphorus removal is the main method of phosphorus removal in the sewage treatment process.The biological toxicity of sulfamethoxazole will affect the biological phosphorus removal process,making the wastewater treatment effect worse.At present,there are few systematic studies on the effect of sulfamethoxazole on the intracellular polymer and enzyme activity in the biological phosphorus removal process,so it is of great significance to explore the effect and mechanism of sulfamethoxazole on the biological phosphorus removal process of aquaculture wastewater.The experiment first added different concentrations of sulfamethoxazole(0 mg/L,0.05mg/L,0.1 mg/L,1 mg/L,5 mg/L,10 mg/L,20 mg/L,40 mg/L,60 mg/L)to the SBR reactor to study its effect on the performance of the biological phosphorus removal reactor,and analyzed the effect of different concentrations of sulfamethoxazole on the metabolism and structure of the main substances in the microbial cell during the phosphorus removal process using chemical method and Fourier transform infrared spectroscopy technology,The effects of sulfamethoxazole on the activities of major functional enzymes(PPX,PPK,ADK,ACP,ALP)and their interaction modes were studied by chemical methods and molecular docking techniques.The structural equation model was constructed to systematically analyze the interaction between the activities of major functional enzymes,intracellular substances synthesis and metabolism and phosphorus removal rate in the biological phosphorus removal process under the action of sulfamethoxazole.The main conclusions of the study are as follows:(1)A SBR reactor was constructed to study the effect of sulfamethoxazole on its pollutant removal.When the concentration of sulfamethoxazole was 0.05 mg / L and0.1 mg / L,the concentration of COD and orthophosphate in the effluent of each reactor was consistent with that of the blank group.When the concentration of sulfamethoxazole reached 1 mg / L,the concentration of COD and orthophosphate in the effluent significantly increased.With the continuous increase of the concentration of sulfamethoxazole,the concentration of COD and orthophosphate in the effluent significantly increased.It shows that 1 mg / L sulfamethoxazole has inhibitory effect on biological phosphorus removal performance,and the inhibitory effect increases with the increase of concentration.(2)The effects of different concentrations of sulfamethoxazole on nutrient utilization of microorganisms in typical cycles were studied by chemical method.The results showed that 0.05 mg / L and 0.1 mg / L sulfamethoxazole had no significant effect on COD consumption and phosphorus release and absorption.As the subsequent concentration increases to more than 1 mg / L,the utilization of organic matter in the anaerobic stage will be significantly inhibited,and the ability of anaerobic phosphorus release and aerobic phosphorus absorption of phosphorus accumulating bacteria will be inhibited,and the inhibition effect is dose-dependent.(3)The effects of sulfamethoxazole on the synthesis and metabolism of intracellular polymers in typical cycles were studied by chemical methods.The results showed that sulfamethoxazole 0.05 mg/L and 0.1 mg/L did not affect the synthesis and metabolism of intracellular poly-P,PHB and glycogen.When the concentration of sulfamethoxazole was more than 1 mg/L,the synthesis and degradation of intracellular poly-P and PHB were significantly inhibited in the typical cycle,while the metabolism of glycogen was significantly increased.It can be concluded from the analysis of stoichiometric ratio,The increase of sulfamethoxazole concentration will make the metabolism in the reactor change from phosphorus accumulating bacteria to glycan bacteria.(4)Fourier transform infrared spectrum analysis showed that sulfamethoxazole0.05 mg/L and 0.1 mg/L did not change the structure of microbial intracellular polymers.When the concentration of sulfamethoxazole was higher than 1 mg/L,the absorption peak intensity at 939 cm-1 in the poly-P infrared spectrogram changed,and the content of P-O-P in the cell gradually decreased,indicating that the length of poly-P chain became shorter;The absorption peak of C=O bond at 1724cm-1 in the infrared spectrum of PHB shifted to the left,indicating the presence of PHV polymer in the cell,which also showed that the abundance of intracellular glycan bacteria increased with the increase of sulfamethoxazole concentration;In the infrared spectrum of glycogen,the transmissivity of about 914cm-1 is getting bigger and bigger,while the transmissivity of about 836cm-1 is getting smaller and smaller.The connection mode of glycogen is from α-Glycoside orientation β-The trend of glycosidic bond conversion.(5)The effects of different concentrations of sulfamethoxazole on the activities of main functional enzymes of biological phosphorus removal were studied by chemical method.The results showed that the activity changes of PPK,PPX,ADK,ACP,ALP were not the same under the effect of different concentrations of sulfamethoxazole.The activities of PPK,ACP and ALP reached the highest in aerobic stage,while PPX and ADK reached the highest in anaerobic stage.Sulfamethoxazole inhibited the activities of five functional enzymes in a concentration dependent manner.When the concentration of sulfamethoxazole was 0.05 mg / L and 0.1 mg / L,there was almost no inhibitory effect on the five phosphorus removal enzymes.When the concentration of sulfamethoxazole increased to 60 mg / L,the relative inhibition rates of sulfamethoxazole on PPK,PPX,ADK,ACP and ALP reached 28.03%,26.89%,24.51%,21.77% and 21.65%.(6)The binding mode of sulfamethoxazole with phosphorus removal enzyme was studied by molecular docking technology.The results showed that the order of inhibition of sulfamethoxazole on five major phosphorus removal enzymes was PPK>PPX>ADK>ALP>ACP.Sulfamethoxazole will combine with the amino acid residues at the active sites of five functional enzyme molecules through hydrogen bonds,hydrophobic bonds,ionic bonds and a small amount of halogenated bonds to form a stable complex,thus changing the spatial conformation of the enzyme molecules and reducing the enzyme activity.The reason for the strong inhibition of PPK,PPX and ADK is that it will produce more chemical bonds with the amino acid residues in the molecular active centers of the three functional enzymes,and will produce unfavorable binding forces with the amino acids such as glutamic acid,aspartic acid and alanine in the active sites of ACP and ALP,resulting in relatively high binding energy and relatively low inhibition.(7)A structural equation model was constructed to illustrate the relationship between the main functional enzyme activities,intracellular substance metabolism and phosphorus removal rate in the biological phosphorus removal process under the action of sulfamethoxazole.The results showed that the activities of PPX and ADK in anaerobic stage significantly affected the degradation of polyphosphate,and the activity of PPK in aerobic stage had a significant positive effect on the formation of polyphosphate,and the activities of ACP and ALP were significantly positively correlated with the amount of aerobic phosphorus uptake.And the metabolism of intracellular polymers will significantly affect the release and absorption of phosphorus and the phosphorus removal rate of the system.Figure [33] Table [22] Reference [147]... |
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