N₂O Production Processes And The Abundance And Expression Of Relative Functional Genes In Calcareous Fluvo Aquic Soil

Better understanding of N₂O production processes and its mechanism can help to propose effective mitigation measures.There were many researches about N₂O emission fluxes,abundance of ammonium archaea?AOA?and bacteria?AOB?in different soils,however,few studies related N₂O emission in the field,abundance of nitrify-denitrify functional genes in situ soil sample and soil substrate together.The same research status is in calcareous fluvo aquic soil of North China Plain.To know better about N₂O emission processes and mechanisms in this calcareous fluvo aquic soil,our research was based on a long-term experiment,measured the N₂O fluxes in the field,sampled soil in situ and extracted soil total DNA,quantified the abundance of related functional genes including 16S rRNA,amoA gene encoding ammonia monooxygenase,narG gene encoding nitrate reductase,nirS/K encoding nitrite reductase and nosZ gene encoding N₂O reductase,analysed the linkage between N₂O fluxes,the annual N₂O emission,soil properties and abundance of functional genes to explain the strong N₂O emission after fertilization event and small N₂O pulses after rainfall or irrigation.To further explain the N₂O emission in the soil and find the main controlling factors,we set up an incubation experiment,added different rates and sources of nitrogen and carbon into the soil,incubated with Robot System for 336 h in the dark.During the incubation,we monitored the O₂,CO₂,NO,N₂O and N₂ kinetics in the vial;we also set parallel treatments for soil sampling,measured soil NH4+-N,NO₂--N,NO3--N of the soil at five points of the incubation,also,we extracted soil RNA,did reverse transcription PCR and did Real-time PCR of 16S rRNA,amoA,narG,nirS/K and nosZ gene,trying to figure out the kinetics of the nitrogen transformation in the soil and functional gene transcript under O₂ concentration transition during the incubation.The overall research is to further explore the processes of N₂O production and its main controlling factors.The main results are as follows.1.Long-term urea and/or manure application significantly increased the number of AOB in the soil,the gene copy per dry soil was in the range from 1.4e+7 to 1.9e+7,fertilization event increased the AOB number in fertilized treatments?Nopt:Improved Nmin test treatment;CNopt:Improved Nmin test treatment with straw returned;CM:cattle manure supplementary applied nitrogen based on the nitrogen balance calculation with straw returned?,increased by 14.7,124.5 and 107.6%respectively compared with N0 treatment,which induced the strong N₂O emission after the fertilization event.Long-term straw returned and manure application increased the abundance of denitrifiers,especially for the CM treatment;there was significant positive relationship between the annual N₂O emission and the abundance of the denitrifying functional genes?narG,nirS and nirK?,which meant that the abundance of these functional genes was linked to denitrification when conditions were proper.However,nosZ gene copies also increased,its effect of reducing N₂O to N₂ was partly offset by increased N₂O emission by enhanced narG,nirS and nirK genes.Strong N₂O peak after fertilization event and small N₂O pulses after strong rainfall or irrigation both contributed to the annual N₂O emission.2.When the initial O₂ concentration was 21%and 3%,the ammonium addition treatments?AS60 and AS200?enhanced soil ammonium oxidation and the O₂ consume rate,increased the amoA mRNA transcription,and produced N₂O with NO₂-accumulated,with the continuous consumption of O₂ lower to 2%,denitrification gene transcription started to increase which induced more N₂O emission;When the initial O₂ concentration was 0%,denitrification happened in the soil,however,if the NH4+ or NO3-concentration was very high it kind of inhibited soil denitrification;The C2H2 depressed soil ammonium oxidation,significantly decrased N₂O emission and avoided NO₂-accumulation under 21%and 0%initial O₂ concentrations,however,it did not block the transformation from NH4+ to NO3-in the soil but slowed down the transformation rate;Glucose addition increased the nosZ gene mRNA transcript number when the initial O₂ concentration was 21%and 3%which resulted in significant high N₂ production;Under aerobic conditions?21%-3%?,if the NH4+ concentration in the soil was low,even NO3-concentration was high there was no NO3-reduced and little N₂O produced,while under the anerobic condition,NH4+ stayed stable in the soil.In the middle and late stages of the incubation,soil NH4+ concentration increased and NO3-decreased meanwhile indicated DNRA?Dissimilatory Nitrate Reduction to Ammonium?happened in some fertilized treatments.3.With the consumption of O₂,the ratio of N₂O/?NO+N₂O+N₂?showed a general upward trend.The ratio of N₂O/?NO+N₂O+N₂?of ammonium addition treatments?AS60 and AS200?was between 28%and 62%,and it was higher when the initial O₂ concentration was 3%than 21%,carbon addition enhanced the transformation of N₂O to N₂ and decreased the he ratio of N₂O/?NO+N₂O+N₂?to 1.7-3.5%.There was significant negative relationship between O₂ concentration and NO,N₂O,N₂ concentration and the ratio of N₂O/?NO+N₂O+N₂??P<0.01?,and significant negative relationship between O₂ concentration and transcript number of nirS and nosZ gene?P<0.01?.4.The structure of soil microorganism changed under O₂ transition.However,as the abundance of nitrifier-denitrifier was quite low in the soil,there was no obvious alternation in the soil microbiota at the phylum level.In summary,in this calcareous low carbon soil,long-term fertilization enlarged the abundance of soil nitrify and denitrify gene.Fertilization event activated the amoA gene for nitrification which resulted in high N₂O emission,decreased O₂ concentration activated genes for nitrify denitrification or denitrification.Our study explored the relationship between O₂ concentration and kinetics of gaseous production during nitrification and denitrification,soil N transformation and gene transcription kinetics primarily.And we found some significant relationship between O₂ concentration and gaseous products and transcript number of relative functional genes.However,It was not enough to only consider O₂ concentration,the gaseous products and gene transcription were controlled by both O₂ and soil substrate.In this kind of soil,use nitrification inhibitor to avoid creating high NH4+ concentration in the soil when fertilizing,which will not lead to strong ammonium oxidation and strong O₂ consumption,then the following chain reaction could not happen in anerobic condition is an efficient way to mitigate N₂O emission.