Study On The Effect Of Copper Oxide Nanoparticles On Soil Denitrification

Due to their unique antifungal properties,copper oxide nanoparticles（CuO NPs）are used on antimicrobial,herbicide and insecticides in agricultural systems.Widely applications of CuO NPs can lead to elevated concentrations of these metal oxides in surface soils,which threatens ecosystems and human health.Nitrogen is an essential element for the growth of plants and microorganisms,however,the efficiency of nitrogen fertilizer is only 21-36%,and the remaining nitrogen will accumulate in the environment,causing a series of environmental problems,such as excess soil fertility,desertification of soil,water eutrophication and so on.Denitrification is a biological reduction process that reduces nitrate（NO₃^-）to nitrous oxide（N₂O）,and then further reduces to N₂ to release from the soil,to maintain the balance of ecosystem nitrogen recycle.During denitrification process,N₂O is a key atmospheric greenhouse gas,which has 298 times stronger greenhouse potential than CO₂,contributes to global warming and the destruction of stratospheric ozone.The denitrification process is the main source of N₂O emission,and soil contributes 59%of global N₂O release.Therefore,attention should be paid to the environmental effects of soil denitrification and its related N₂O emission.Previous studies have shown that CuO NPs will affect the soil microbial community structure,but it is unclear whether CuO NPs will affect the soil microbial denitrification process and its mechanism.In this experiment,use routine indicators（nitrate,nitrite,nitrous oxide concentrations）at the macro level to prove the effect of CuO NPs on the denitrification process;then use the change of electron transfer system activity（ETSA）,denitrifying reductase activity,ROS production,denitrification function genes abundances and microbial community composition to explain the effect of CuO NPs on the denitrification process.（1）To study the effects of CuO NPs on the denitrification process during 60 hours.The results showed that CuO NPs significantly inhibited the denitrification process,leading to an increase in the concentration of NO₃^-and NO₂^-and a reduction in the N₂O emission rates.The results showed that compared with the control,when CuO NPs increased to 500 mg kg^-1,the NO₃^-concentration increased by 11 times,while the N₂O emission rates decrease by 10.2-24.1%.In order to investigate the cause of the effect of CuO NPs on denitrification,we measured the concentrations of NO₃^-and N₂O after adding Cu ions released from CuO NPs.The results showed that copper ions significantly inhibited the removal of NO₃^-and the N₂O emission rates during denitrification,and the effect was basically the same as that of CuO NPs.It can be considered that Cu ion release is,in the main,responsible for the toxic effects of CuO NPs on microbial denitrification.（2）In this study,the mechanism of CuO NPs on denitrification process was investigated from the electron transfer system activity（ETSA）,denitrification enzyme activity and ROS production.The experimental results showed that ETSA was significantly inhibited under the treatment of CuO NPs,which reduced by 23.8-52.4%compared with the control.Enzyme activity data showed nitrate reductase and nitric oxide reductase activities decreased by 21.1-42.1%and 10.3-16.3%,respectively.This suggests that the decrease in ETSA and enzyme activity may be the reason for the inhibitory effect of CuO NPs on denitrification.ROS analysis showed that there was no significant change in ROS production in each treatment group,and ROS was not the cause of CuO NPs affecting denitrification.（3）We measured changes in soil community composition and denitrification function gene abundances.The results showed that the relative abundances of five key functional genes for denitrification,nar G,nir K/nir S,nor B and nos Z were affected to varying degrees after the application of different concentrations of CuO NPs.Among them,the nar G and nir S genes were most significantly inhibited and decreased from 5.2%to 33.3%and from 15.8%to 61.8%,respectively.After CuO NPs application,the microbial community changes.Firmicutes was the most sensitive to CuO NPs and its levels decreased,butβ-Proteobacteria andα-Proteobacteria levels increased.Bacillus and Clostridium potentially contribute to denitrification in natural ecosystems,the relative abundances of Bacillus and Clostridium had an obvious decrease after CuO NPs application.The results showed that the application of CuO NPs could reduce the abundance of soil denitrification functional genes,while lead to negative effects on soil microbial diversity and community structure.