Study On The Regulation Mechanism Of Microbial Enzymes On N₂O Production During Biological Nitrogen Removal From Wastewater

In biological nitrogen removal process of wastewater treatment plants（WWTPs）,it usually produced a large amount of nitrous oxide（N₂O）which has a strong greenhouse effect.Therefore,it is imperative to reduce N₂O emissions in the WWTPs under the“Double-Carbon”strategies.The premise of reducing N₂O emission is to master the law and mechanism of N₂O production.In the biological nitrogen removal process,the conversion process of nitrogen compounds is catalyzed by various enzymes.Therefore,identifying the regulation of these enzymes is a key to understand the law and mechanism of N₂O production,and a key to exploring how different environmental factors affect the denitrification products and processes.However,at present,there is still a lack of systematic understanding of these at home and abroad.To reveal the enzymatic regulation mechanism of microorganisms on N₂O generation in the biological nitrogen removal process of wastewater,this thesis firstly established the extraction and determination methods of the key enzymes（nitric oxide reductase（Nor）and nitrous oxide reductase（N₂OR））in the denitrification process.Secondly,in A/O SBR with different COD/TN and aeration intensity and in anoxic SBR with different COD/TN,by analyzing the activity of nitrate reductase（Nar）,nitrite reductase（Nir）,Nor and N₂OR,the main production process and main consumption process of N₂O in the aerobic phase and the anoxic phase were identified.The mechanism of microorganisms regulating N₂O production by various enzymes was revealed.The feasible measures to reduce N₂O emissions had been found.A concise and feasible index that can predict and evaluate the potential of N₂O production had been explored.Finally,the activities of Nar,Nir,Nor and N₂OR and the N₂O production in three WWTPs（AMAO process,Orbal oxidation ditch process,and A/A/O process）in Xi’an were measured.Combined with the operating conditions of the WWTPs,from the perspective of denitrifying enzyme activity,the reasons of N₂O production in the three WWTPs were analyzed.The main results of this paper are as follows:（1）Taking into account breaking cell method,amount of lysate added,the ratio of electron acceptor to electron donor in the enzyme activity determination and the enzyme catalyzed time,the parameters of the existing method were optimized,and a method system for the extraction and activity determination of Nor and N₂OR in activated sludge was established.Results show that the optimum parameters of Nor extraction by ultrasonic fragmentation were at ultrasonic treatment times of 100,ultrasonic intensity of 500 W,and amount of lysate addition of 0.1 m L.The optimum parameters of Nor extraction by low temperature and high-pressure fragmentation were breaking treatment times of 4,breaking pressure of 50 Mpa,and amount of lysate addition of 0.1 m L.The optimum parameters of N₂OR extraction by ultrasonic fragmentation were ultrasonic treatment times of 60,ultrasonic intensity of 400 W and no lysate addition.In the Nor activity determination method,the end time for activity determination of Nor was 15min.In the N₂OR activity determination method,the concentration of Na₂S₂O₄（electron donor）was150 m M,and the end time for activity determination of N₂OR was 30 min.（2）The N₂O production pathway and the dynamic changes of Nir,Nor and N₂OR activities in the aerobic phase of the A/O SBR reactor with different organic load and aeration intensity were analyzed.Results show that in the aerobic phase of A/O SBR,the main production process and main consumption process of N₂O occurred in different types of bacteria and metabolism.The accumulation of N₂O was mainly due to denitrification by nitrifiers,while the consumption（decomposition）of N₂O was mainly denitrification by heterotrophic bacteria.This is very different from the general understanding that nitrifiers do not perform denitrification in an aerobic condition and N₂O mainly comes from anoxic denitrification.At the same time,the activities of Nor were much higher than that of N₂OR,which led to the lower consumption rates of N₂O by heterotrophic denitrifiers than the production rate of N₂O by nitrifiers in the aerobic condition.This paper believes that this is the main reason for N₂O accumulation under aerobic conditions.The Nir activity could reflect the intensity of N₂O production in the aerobic（nitrification）stage and could be used as an indicator for predicting N₂O production.（3）The dynamic changes of the activities of all denitrifying enzymes under different organic loads were analyzed.Results show that the activities of Nar,Nir,Nor and N₂OR increased with the increase of the influent COD/TN of the reactor.However,the increase in organic load promoted the activity of different enzymes differently（Nar>Nir>Nor>N₂OR）.Compared with Nar and Nir,the catalytic processes of Nor and N₂OR were more susceptible to the influence of the substrate concentration and the content of internal and external carbon sources.Nor activity was sufficient to rapidly reduce the denitrification intermediate nitric oxide（NO）when the electron donor and substrate（NO）were sufficient.On the contrary,Nor reduced to a relatively lower catalytic activity.The activities of N₂OR were generally weaker than that of other denitrifying enzymes.Especially when electron donor was limited,N₂OR was more susceptible to the insufficient electron donor than Nor,which in turn led to a decrease in its activity.SBR with COD/TN≤4.9 would produce more N₂O.From the model of the enzyme activities（Nor and N₂OR）and the total concentrations of N₂O,when COD/TN≤2.5,the reason for the N₂O production was that the N₂OR activity was inhibited due to its inability to acquire enough electrons.When 2.5<COD/TN≤4.9,the reason for the N₂O production was that the N₂OR activities were lower than the Nor activities.（4）The catalytic activities of denitrifying enzymes of activated sludge in three typical WWTPs（AMAO process,Orbal oxidation ditch process,and A/A/O process）in Xi’an were measured.Results show that the catalytic activities of Nor and N₂OR were much greater than those of Nar and Nir,and activated sludge in these three WWTPs had strong reduction ability of NO and N₂O.In AMAO process,N₂O in anoxic pool was mainly produced by denitrification process,and N₂O in aerobic pool was mainly produced by the incomplete oxidation process of NH₂OH to NO₂^-.In Orbal oxidation ditch process,both incomplete oxidation process of NH₂OH to NO₂^-and denitrification process produces N₂O,and the former was dominant.In A/A/O process,N₂O was mainly produced and dissipated in aerobic pool.The main sources of N₂O were incomplete oxidation process of NH₂OH to NO₂^-and denitrification process.