Effects Of Ag NPs On Nitrogen Metabolism And N₂O Production In Biofilm

For the widespread use of nanomaterials and inevitably entering the sewage treatment system during preparation,use,waste,and recycling,Ag NPs will enter the wastewater more and more as one of the most widely used nanomaterials.It has been confirmed that the antibacterial properties of Ag NPs could adversely affect the functional microorganisms in sewage biological treatment.Moreover,SBBR is commonly used in biological treatment in sewage treatment plants,and has an advantage in nitrogen removal.In addition,as a kind of strong greenhouse gas,N₂O can be produced in the process of denitrification,and the effect of Ag NPs on N₂O release during denitrification is rarely studied.In view of the problems above,this paper mainly explored the effects of short and long-term exposure of Ag NPs on nitrogen removal and N₂O production in biofilm.In the experiments,the effects of Ag NPs（20μg/L,100μg/L,1mg/L,5mg/L）on nitrogen removal and N2O production in SBBR were studied.In order to study the toxicity source of Ag NPs,20μg/L Ag⁺was set up to compare with Ag NPs,and the exposure experiment lasted for 81 days.In the paper,the effects of Ag NPs and Ag⁺on the microbial activity in biofilms were studied.It has been found that the destruction to cell integrity was related to the accumulation of Ag NPs and silver ions in biofilms through the release of lactate dehydrogenase.Regarding the effect of Ag NPs on extracellular polymer,it has been found that 5mg/L Ag NPs significantly stimulated the secretion of extracellular protein and extracellular polysaccharide,and there was not significant effect under exposure of other concentrations.It indicated that the acute toxicity of 5mg/L Ag NPs caused the self-protection mechanism of microbes in biofilm.The extracellular protein and extracellular polysaccharide contents were significantly inhibited during long-term exposure of 5 mg/L Ag NPs,indicating that the accumulation of Ag NPs in the biofilm caused a decrease in microbial metabolic capacity and a large number of cells death.By monitoring of the denitrification performance and N₂O emission of the SBBRs,it has been found that the short-term exposure of 20μg/L-5mg/L Ag NPs had not obvious acute influence on the nitrogen metabolic process in SBBR.After long-term exposure,5mg/L Ag NPs inhibited the TN and COD removal ability of SBBR,and the removal rate decreased by 23.78%and 11.04%,the ammonia nitrogen removal rate decreased by 25.71%.The rate of ammonia nitrogen metabolism decreased with increasing exposure concentration in the cycle after long-term exposure.In addition,the short-term exposure of Ag NPs significantly promoted the release of N₂O,and the acute promotion effect of 100μg/L Ag NPs group was the most significant increased by41.04%.After the long-term exposure,the promotion of N₂O release in biofilms gradually decreased under 20μg/L-1mg/L Ag NPs and 20μg/L silver ion,and it was inhibited at the end of the experiment,decreased by 9.02%,18.73%,22.99%and23.26%,respectively,compared with the control group.However,the release of N₂O increased by 67.54%under 5 mg/L Ag NPs.Through the N₂O stable isotope isomer technology,it has been found that the N₂O formation pathway by nitrite reduction were inhibited after long-term exposure with20μg/L-1mg/L Ag NPs and 20μg/L silver ion,thus reducing the amount of N₂O production.However,after long-term exposure of 5 mg/L of Ag NPs,the pathway of N₂O production by nitrite reduction was greatly promoted.From the DO microenvironment in biofilm,the depth of oxygen diffusion gradually increased under the long-term exposure of 20μg/L-1mg/L Ag NPs and 20μg/L silver ion,and the anaerobic region decreased,but the oxygen diffusion range reaches a minimum of 230μm under 5mg/L.It could be seen from the N₂O microenvironment that the long-term contact of 20μg/L-1mg/L Ag NPs and 20μg/L silver ion greatly reduced the N₂O concentration in the anaerobic and low DO regions below 200μm in the biofilm,and N₂O was mainly produced in the aerobic surface in the biofilm,and the production of N₂O in the biofilm of 5 mg/L was mainly in the anaerobic region below 200μm.By analyzing the microbial population structure and functional enzyme gene in biofilms after long-term exposure of 20μg/L-5mg/L Ag NPs,the abundance of ammonia oxidizing bacteria Nitrosomonas and the expression of nitrogen monooxygenase gene in biofilms of each contact group decreased compared with the control.The expression of ammonia monooxygenase in each group decreased by 61.66%-82.56%.Six denitrifying bacteria were detected in the experiment,which gave different responses to the long-term exposure of Ag NPs.Azoarcus,Arenimonas and Brevundimonas reached the maximum abundance under the 5 mg/L,indicating that the accumulation of Ag NPs promoted the metabolic processes and bacterial proliferation of these denitrifying species.The abundance of Meganema and Thauera related to EPS secretion reached the lowest value under 5 mg/L Ag NPs,and the abundance of Flavobacterium that hydrolyzed complex organics was significantly increased under 5 mg/L Ag NPs.Through the expression of related functional enzyme genes,it was found that the expression of nitric oxide reductase[NAD（P）⁺]and cytochrome c that directly related to N₂O production was inhibited under long-term exposure of 20μg/L-1 mg/L Ag NPs and silver ions,and the expression of the nitrous oxide reductase gene was up-regulated after long-term exposure of Ag NPs and silver ions.The surface morphology of the biofilm was observed by scanning electron microscopy.It was found that the surface structure of the biofilm samples of the control and under long-term exposure of 20μg/L,100μg/L,1mg/L Ag NPs and 20μg/L silver ion were dense and firm,and existed large number of filamentous bacteria.However,the number of filamentous bacteria in the 5mg/L biofilm samples was greatly reduced,and the structure was much looser.Based on the results of various test items,the effects of 20μg/L silver ion on biofilm activity,denitrification performance and N₂O production were similar to that of 1mg/L,indicating that the toxicity of the same concentration of silver ion is stronger than Ag NPs on the biofilm.