| Denitrification is an important part of the conversion of reactive nitrogen into inert nitrogen(N2)in the biogeochemical cycle of nitrogen,so it is of great significance for maintaining the nitrogen balance of ecosystems.As an intermediate product of denitrification,nitrous oxide(N2O)is an important greenhouse gas whose metabolites could destroy the ozone layer and closely related to global changes.During the growth of rices,the soil is in a flooded anaerobic environment favorable for denitrification for a long time.The nitrogen loss caused by denitrification is one of the main ways of nitrogen loss in paddy fields.Therefore,studying the denitrification capacity of rice soil and its emission law of gaseous products,and clarifying its influencing factors would be of great significance for improving nitrogen use efficiency and reducing active nitrogen emissions in paddy fields.However,due to the extremely high concentration of N2 in the atmosphere,which seriously affects the accurate determination of the N2 concentration of denitrification end products,it is urgent to establish a method for accurately determining the concentration of N2,which provides technical support for understanding the denitrification and product composition of paddy soil.Based on the stable isotope ratio mass spectrometer(IRMS),a mass spectrometry method for simultaneous determination the concentration and 15N abundance of N2 in samples was established in this sduty.Based on this method,the laboratory incubation experiments were carried out by using 15N stable isotope labeling technique.The denitrification rates and gaseous product emission of paddy soils under different conditions and their influencing factors were studied.In view of the difficulty in accurately determining the concentration of denitrification end product N2,this study established a stable isotope ratio mass spectrometer analysis method for simultaneous determination of N2 concentrations and 15N abundance.The standard operating curves for N2 concentrations of both 15N natural abundance and low abundance(atom%?0.5%)samples were drawn by using the IRMS measurement results.The results showed that,the concentration of N2 in the samples could be accurately determined according to the injection volumes and standard working curves of the gas samples.The injection volumes of the samples must be strictly controlled,so that the ion current intensity of the gas sample m/z 28 could be above higher than 600 mV,which could ensure the stability and accuracy of 15N abundance measurement results of the samples.It should be noted that the operating parameters of mass spectrometer would be changed after shutting down and restarting or changing the carrier gas flow rate and column temperature.The measurement result of the m/z 28 ion current intensity of N2 would also change,which could obviously affect the N2 standard working curves.When IRMS measures N2 samples with high 15N abundance(such as atom%>10%),the results calculated by the instrumental analysis software tend to be highly biased,which seriously affects the accurate determination of 15N abundance of N2 samples.In response to this problem,a series of high-abundance 15N2 standard samples of 10-99.14 atom%were prepared for mass spectrometry,and three formulas for calculating 15N2 abundance were derived.The results showed that the 15N abundance of high abundance N2 samples was mainly depended on the ion current intensity of m/z 30.In the calculation of high abundance 15N2 samples,the results obtained by using the peak area of m/z 28,29 and 30 were more accurate than those calculated by peak height.The 15N high abundance of 15N2 was calculated by three different formulas,and the accurate 15N abundance was finally obtained.The artificial calculation method for accurate determination of high abundance 15N2 samples by IRMS was preliminarily established.Based on the mass spectrometry method for simultaneous determination of N2 concentration and 15N abundance,six kinds of paddy soils with different properties were collected in Longhushan(LHS),Yingtan(YT),Jiangxi province,Yanting(SC),Sichuan province,and Huai'an(HA),Jurong(JR),Yixing(YX),Jiangsu province,respectively,and the laboratory incubation experiments were carried out by using 15N stable isotope labeling technique.The denitrification rates and gaseous product emission of paddy soils under different conditions and their influencing factors were studied.The 15N tracer technique and simultaneous determination of sample N2 concentration and 15N abundance method were used to study the denitrification capacity and product composition ratio of paddy soils under strict anaerobic conditions(flooding,headspace N2 filling)and the relationship with soil properties.The results showed that the N2O emission rates of the tested paddy soils ranged from 0.0163 to 0.1596 mg N kg-1 h-1.Among which,the N2O emission rates of YT and HA paddy soils were significantly higher than other soils,followed by YX paddy soil,while the LHS,JR and SC paddy soils had lower N2O emission rates.The N2 emission rates varied from 0.1621-0.4974 mg N kg-1h-1.Among which,the alkaline paddy soil SC was the highest,which was significantly higher than other soils,and the acid paddy soil LHS was the lowest.The N2 emission rates of all tested soils were significantly higher than that of N2O,indicating that N2 was the main product of denitrification of paddy soils studied.The ratio of denitrified gaseous products 15N2/15N2O was the highest in alkaline SC paddy soil,reaching 28.5,which was significantly higher than the other five tested soils;followed by JR and LHS paddy soils,while alkaline HA paddy soil has the lowest 15N2/15N2O ratio.In this study,the denitrification rate was expressed by the sum of the emission rates of denitrified gaseous products N2 and N2O.The denitrification rates ranged from 0.18 to 0.52 mg N kg-1 h-1,which were significantly different between the tested paddy soils.The alkaline paddy soil SC was the highest,significantly higher than other soils;the acid paddy soil LHS was the lowest,indicating that alkaline soil conditions were more favorable for denitrification.Correlation analysis showed that there was a significant positive correlation between 15N2/15N2O and soil pH(P<0.05)and C/N(P<0.05)except for HA paddy soil.The gene abundance of nirK and nirS increased with the increase of soil pH and C/N,while the gene abundance of nosZ decreased with the increase of soil pH and C/N.The contribution of the unit number of nosZ gene to N2 emission rate was significantly positively correlated with soil pH(P<0.01)and C/N(P<0.05),indicating that soil pH and C/N were the important factors affecting the denitrification of paddy soils,especially affecting the ratio of N2/N2O in gaseous products.Due to the characteristics of alternating wet and dry during the growth of rice,the soil water content varies greatly.Therefore,this paper further studied the denitrification rates and the proportion of gaseous products in paddy soils under different water content conditions.Four soil moisture conditions were set,which were 60%WHC,80%WHC,100%WHC,and water and soil ratio 1:1 flooding(culture flask headspace air filling,culture temperature 25?),respectively.The results showed that the emission rates of N2O and N2 changed significantly with the change of soil moisture content(P<0.05).Under different water treatment conditions,the N2O emission rates of alkaline paddy soil SC changed little,ranging from 0.010-0.012 mg N kg-1h-1,with no significant difference;the acid paddy soil LHS had obvious N2O emissions only under flooding condition;when YT paddy soil was at 100%WHC;the N2O emission rates reached the maximum value,but after further increasing the soil moisture content to the flooding condition,the N2O emission rates decreased;the N2O emission rates of other three tested soils increased with the increase of water content.Under the aerobic condition of 60%WHC,there was no obvious N2 emission in the six tested soils;under the condition of 80%WHC,except for the acid paddy soil LHS,the other five tested soils all had N2 emission,among which the N2 emission rate of SC paddy soil was the highest,reaching 0.249 mg N kg-h-1,followed by YX and HA paddy soils,and the other three tested soils were lower.LHS paddy soil began to have N2 emissions when the moisture content was 100%WHC.Once N2 was produced in each test soil,the N2 emission rates increased with the soil moisture content increased(P<0.05).When the soil moisture content was 60%WHC,the 15N2/15N2O ratios of the six tested soils were close to zero;when the soil water content increased to 80%WHC,the 15N2/15N2O ratio of SC paddy soil increased significantly,reaching 19.68,which was significantly higher than other soils;when the soil water content reached 100%WHC,the 15N2/15N2O ratio continued to increase,and the 15N2/15N2O ratio of SC paddy soil was still the highest,at 26.97,which was still significantly higher than other soils;when the soil water content reached the water-soil ratio of 1:1 flooding condition,the 15N2/15N2O ratios of the six tested soils reached the maximum,and the alkaline paddy soil SC still had the highest ratio,which was 31.85.The denitrification rates,15N2/15N2O ratios and 15N gas loss increased with the increase of soil moisture content.The results of the first two parts indicated that the denitrification rate and the gaseous composition of N2O and N2 in alkaline paddy soil HA were very different from those of alkaline paddy soil SC,which is presumably due to NO3--N concentration of HA paddy soil(52 mg N kg-1)was much higher than that of SC(1.7 mg N kg-1).In order to verify the effect of NO3--N substrate concentration on denitrification of paddy soil,acid paddy soil YT and alkaline paddy soil HA were selected.15N labeling and anaerobic culture experiments were carried out under laboratory conditions.Five concentrations of NO3--N were set(10,20,50,70 and 100 mg N kg-1)to study the effect of NO3--N substrate concentrations on denitrification rates,gaseous product composition and 15N gas loss.The results showed that the N2O emission rates of the two tested paddy soils both increased with the increase of NO3--N substrate,and the N2O emission rate of acid paddy soil YT was always higher than that of alkaline paddy soil HA.With the increase of NO3--N substrate,the N2 emission rates of the two tested soils increased first and then decreased.The N2 emission rate of HA paddy soil was always higher than that of acid YT paddy soil.The ratio of 15N2/15N2O in both paddy soils decreased with the increase of NO3--N substrate,indicating that high NO3--N concentration could inhibit the conversion of N2O to N2.The denitrification rates of the two tested soils increased first and then decreased with the increase of NO3--N substrate.The results of this study indicate that NO3--N concentration was an important factor affecting soil denitrification and its gaseous product composition.In conclusion,this study established a new method for the determination of N2 concentration and 15N abundance using a stable isotope mass spectrometer,which could simultaneously obtain the concentration and 15N abundance of soil denitrification end product N2.The problem of inaccurate determination of high abundance N2 sample by mass spectrometer was solved,which could support for the study of soil denitrification by I5N tracer method.The denitrification rates,proportion of gaseous products and their influencing factors of paddy soils were studied by using the newly established measurement method.The results showed that under strict anaerobic conditions(flooding,headspace N2 filling),the N2 emission rates of all tested soils were significantly higher than that of N2O5 indicating that N2 was the main product of denitrification of paddy soil.Soil pH and C/N were important factors affecting the denitrification of paddy soils,especially the ratio of N2/N2O in gaseous products.The denitrification rates,15N2/15N2O ratios and 15N gas loss increased with the increase of soil moisture content,indicating that soil water content also had significant effect on soil denitrification and its product composition.The N2O emission rates of paddy soils increased with the increase of NO3--N substrate,while the N2 emission rates increased first and then decreased.The ratio of 15N2/15N2O increased with NO3--N substrate increase,indicating that high NO3--N concentration could inhibit the conversion of N2O to N2.The soil denitrification rates increased first and then decreased with the increase of NO3--N substrate.These results indicated that the concentration of NO3--N also significantly affected soil denitrification and its proportion of gaseous products.The results of this study had important scientific significance for understanding the denitrification process of rice soil and the proportion of gaseous products,which could provide data support and scientific basis for reducing the loss of denitrifying nitrogen in paddy fields and reducing the proportion of N2O emissions. |
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