Evaluation method for the contribution of the life and death cycle of ammonia oxidizing microorganisms to soil carbon neutrality

Abstract

The present invention relates to the field of environmental carbon neutrality analysis methods, and specifically discloses a method for evaluating the contribution of ammonia oxidizing microorganisms to soil carbon neutrality throughout their life cycle, including DNA-SIP microcosm cultivation stage I and microcosm cultivation stage II of organic carbon degradation, and evaluates the contribution of ammonia oxidizing microorganisms to soil carbon neutrality throughout their life cycle based on their contribution to organic carbon, lethality, net greenhouse gas CO2 emissions, and carbon neutrality contribution rate. The present invention found that 5.28% of the organic carbon fixed in the reproduction of active cells of ammonia oxidizing microorganisms degraded into CO2 emissions after cell death; throughout the entire life cycle of ammonia oxidizing microorganisms, soils with long-term high-intensity nitrogen fertilizer input increased net greenhouse gas CO2 emissions by 3 times, while the contribution rate to carbon neutrality was reduced by 2 times. The role of the entire life cycle of ammonia oxidizing microorganisms in carbon neutrality should be considered, so as to better understand the global greenhouse gas emission potential.

Description

Technical Field

[0001] The present invention relates to the field of environmental carbon neutrality analysis methods, and in particular to a method for evaluating the contribution of the life and death cycle of ammonia oxidizing microorganisms to soil carbon neutrality. Background Art

[0002] Carbon neutrality is a term for energy conservation and emission reduction. Carbon neutrality refers to the total amount of carbon dioxide or greenhouse gas emissions directly or indirectly generated by a country, enterprise, product, activity or individual within a certain period of time. Through afforestation, energy conservation and emission reduction, etc., the carbon dioxide or greenhouse gas emissions generated by itself can be offset to achieve positive and negative offsets and achieve relative "zero emissions".

[0003] Nitrifying microorganisms are chemoautotrophic organisms that use CO2 as a carbon source and fix it in the soil organic carbon (SOC) pool. This partial C input can offset part of the greenhouse gas N2O produced during nitrification. And it has been confirmed that the offset rate of N2O emission / CO2 fixation is as high as 4.72% in grassland soils. However, the expected large-scale aging and senescence of microorganisms may have occurred, and the death of microorganisms will produce a large amount of microbial-derived organic carbon. It is estimated that microbial residues contribute more than half of the soil organic carbon. However, microbial metabolites and residues can also be decomposed into carbon dioxide. Although there have been a large number of reports on the autotrophy of soil microorganisms, there are few studies on chemoautotrophic nitrifying microorganisms in soil, and no trade-off between the life and death of ammonia-oxidizing microorganisms has been considered, and no one has even paid attention to its impact on the net emission of greenhouse gas CO2.

[0004] Long-term fertilization can lead to large amounts of N2O emissions by increasing autotrophic nitrification. However, due to the neglect of the role of nitrifying bacteria in soil microorganisms, 13 Due to the fixation of CO2, the net production of greenhouse gases may be overestimated, resulting in inaccurate assessment of the carbon neutrality effect. Summary of the invention

[0005] In order to study the contribution of the whole life cycle of ammonia oxidizing microorganisms to organic carbon, the impact of active microbial expression and net emission of greenhouse gas CO2, so as to better understand the global greenhouse gas warming potential, the present invention provides an evaluation method for the contribution of the whole life cycle of ammonia oxidizing microorganisms to soil carbon neutrality. The method of the present invention can evaluate the contribution of active ammonia oxidizing microorganisms AOB and AOA to organic carbon during microbial reproduction. 13 The contribution of net C-SOC production can also quantify the mortality rate of ammonia oxidizing microorganisms, the net greenhouse gas CO2 emissions generated during their entire life cycle, and the contribution of ammonia oxidizing microorganisms to carbon neutrality during their entire life cycle. Combined with the results of soils with different fertilizations, fertilization strategies to reduce greenhouse gas emissions can be provided.

[0006] The present invention provides a method for evaluating the contribution of ammonia oxidizing microorganisms to soil carbon neutralization throughout their life cycle, comprising:

[0007] DNA-SIP microcosm cultivation phase I: After the cultivation, the content of N2O, the content of soil organic carbon (SOC), and the content of soil organic carbon (SOC) were detected. 13 C atomic percentage; by extracting the total DNA of the soil after the cultivation, detecting the gene abundance of ammonia oxidizing microorganisms, and analyzing the increase in active ammonia oxidizing microbial cells;

[0008] Microcosm cultivation phase II of organic carbon degradation, after the cultivation, the CO2 content and CO2 13 C atomic percentage;

[0009] The contribution of ammonia oxidizing microorganisms to organic carbon is calculated using the obtained increase in active ammonia oxidizing microbial cells;

[0010] Soil organic carbon (SOC) content and organic carbon (SOC) at the end of DNA-SIP microcosm cultivation phase I 13 C atomic percentage, and the CO2 content and CO2 content measured after the end of the microcosm cultivation phase II of organic carbon degradation 13 The lethality was calculated by the atomic percentage of C;

[0011] The N2O content and soil organic carbon (SOC) content were measured after the end of the DNA-SIP microcosm cultivation phase I. 13 The atomic percentage of C and the content of soil organic carbon (SOC), as well as the content of CO2 and CO2 measured after the end of the cultivation of the microcosm cultivation phase II of organic carbon degradation 13 The net greenhouse gas CO2 emissions were calculated by C atomic percentage;

[0012] Soil organic carbon (SOC) measured after the end of DNA-SIP microcosm cultivation phase I 13 The atomic percentage of C and the content of soil organic carbon (SOC), as well as the content of CO2 and the CO2 concentration measured after the end of the microcosm cultivation phase II of organic carbon degradation 13 The carbon neutrality contribution rate was calculated by calculating the C atomic percentage;

[0013] The impact of the entire life cycle of ammonia oxidizing microorganisms on soil carbon neutrality was evaluated based on their contribution to organic carbon, mortality rate, net greenhouse gas CO2 emissions and carbon neutrality contribution rate.

[0014] Furthermore, the contribution of active ammonia-oxidizing microorganisms to organic carbon varies with soil type, and the lethality of ammonia-oxidizing microorganisms is inversely proportional to their contribution to organic matter;

[0015] The greater the net greenhouse gas CO2 emissions, the higher the greenhouse gas emissions produced by this type of soil. Fertilization strategies for reducing greenhouse gas emissions in the soil can be provided based on the carbon neutrality contribution rate.

[0016] Further, the contribution of the ammonia oxidizing microorganisms to organic carbon is the sum of the contribution of active AOB to organic carbon and the contribution of active AOA to organic carbon;

[0017] Contribution of active AOB to organic carbon = active AOB cell mass / (active AOB + active AOA cell mass sum)* 13 C-SOC I ;

[0018] Contribution of active AOA to organic carbon = active AOA cell mass / (active AOB + active AOA cell mass sum)* 13 C-SOC I ;

[0019] Active cell mass = number of active cells * cell mass;

[0020] In the formula, 13 C-SOC I Representation of organic carbon in DNA-SIP microcosm culture stage I 13 Net C-SOC production, active cell number represents the increase in active ammonia-oxidizing microbial cells in DNA-SIP microcosm culture phase I, and the cell numbers of ammonia-oxidizing microorganisms are AOA (10.2 fg protein per cell) and AOB (120 fg protein per cell), respectively;

[0021] Organic Carbon 13 The net production of C-SOC is determined by the soil organic carbon SOC 13 The C atomic percentage and soil organic carbon SOC content were calculated using the following formula:

[0022] 13 C-SOC I =SOC I *(SOC- 13 C ％I -1.08) / 100

[0023] In the formula, 13 C-SOC I Representation of organic carbon in DNA-SIP microcosm culture stage I 13 C-SOC net generation, SOC I represents the soil organic carbon content in DNA-SIP microcosm incubation stage I, SOC- 13 C ％1 Figure 2 shows the soil organic carbon (SOC) of DNA-SIP microcosm incubation stage I.13 C atomic percentage.

[0024] Furthermore, the lethality rate is used to evaluate the organic carbon loss caused by the death of ammonia-oxidizing microbial cells, and the calculation formula is as follows:

[0025] Mortality rate = ( 13 C-CO2 II ) / ( 13 C-SOC I )*100

[0026] In the formula, 13 C-CO2 II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13 C - net CO2 emissions, 13 C-SOC I Representation of new organic carbon in DNA-SIP microcosm cultivation stage I 13 C-SOC net generation;

[0027] Phase II of the Microcosm Cultivation of Organic Carbon Degradation 13 The calculation formula of C-CO2 net emission is as follows:

[0028] 13 C-CO2 II =CO 2II *(CO2- 13 C ％II -1.08) / 100

[0029] In the formula, 13 C-CO2 II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13 C-CO2 net emissions, CO 2II The CO2 content after the end of the microcosm cultivation phase II of organic carbon degradation, CO2- 13 C ％II The CO2 in the air after the microcosm cultivation phase II indicates organic carbon degradation 13 C atomic percentage.

[0030] Furthermore, the net greenhouse gas CO2 emissions are calculated as follows:

[0031] Net greenhouse gas CO2 emissions = (CO2-equivalent I )-( 13 C-CO2-fixed I )+( 13 C-CO2 II )

[0032] In the formula, CO2-equivalent I represents the equivalent CO2 emission from N2O in stage I of DNA-SIP microcosm cultivation. This calculation is based on the fact that the warming potential of N2O is 273 times higher than that of CO2 (IPCC, 2021); 13 C-CO2-fixed I Indicates that DNA-SIP microcosm culture stage I comes from organic carbon 13 C-SOC net generation equivalent 13 C-CO2 fixation, which is calculated based on the conversion of SOC and CO2 having the same C molar number; 13 C-CO2 II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13 C - net CO2 emissions.

[0033] Furthermore, the organic carbon from DNA-SIP microcosms in stage I was used to culture 13 The equivalent amount of CO2 fixation of the net production of C-SOC minus the organic carbon degradation of the microcosms during the cultivation phase II 13 The value after C-CO2 net emission is divided by the equivalent CO2 emission from N2O as carbon neutrality, which is used to evaluate the contribution of ammonia oxidizing microorganisms to carbon neutrality throughout their life cycle. The calculation formula is as follows:

[0034] Carbon neutrality contribution rate = [( 13 C-CO2-fixed I )-( 13 C-CO2 II )] / net greenhouse gas CO2 emissions*100

[0035] Among them, net greenhouse gas CO2 emissions = (CO2-equivalent I )-( 13 C-CO2-fixed I )+( 13 C-CO2 II )

[0036] In the formula, 13 C-CO2-fixed I Indicates that DNA-SIP microcosm culture stage I comes from organic carbon 13 C-SOC net generation equivalent 13 C-CO2 fixation, which is calculated based on the conversion of SOC and CO2 having the same C molar number; 13 C-CO2 II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13C-CO2 net emissions; CO2-equivalent I Represents the equivalent CO2 emission from N2O during stage I of DNA-SIP microcosm cultivation.

[0037] Furthermore, the CO2 fixation amount in the DNA-SIP microcosm cultivation stage I is equal to the organic carbon in the DNA-SIP microcosm cultivation stage I. 13 The net generation of C-SOC is calculated as follows:

[0038] 13 C-CO2-fixed I = 13 C-SOC

[0039] In the formula, 13 C-CO2-fixed I Indicates that DNA-SIP microcosm culture stage I comes from organic carbon 13 C-SOC net generation equivalent 13 The C-CO2 fixation amount is calculated based on the fact that SOC and CO2 have the same C molar number.

[0040] Furthermore, the DNA-SIP microcosm cultivation stage I process is: pre-cultivating the soil sample at 30-40% field water holding capacity (30-40% WHC), 25-30°C, and dark conditions for 7-14 days, the purpose is to restore microbial activity, reduce the amount of CO2 emitted by soil respiration, and prevent the 5% 13 The dilution of CO2 increases the success rate of labeling. 13 CO2 labeling, 12 CO2 was used as the control group, urea-N source was added, soil moisture content was adjusted to 60% WHC, and dark cultivation was continued at 25-30°C for 28-30 days.

[0041] Furthermore, the amount of urea-N source added is 300-400 μg N g -1 dws.

[0042] The process of the microcosm cultivation stage II for organic carbon degradation is as follows: taking a soil sample after the DNA-SIP microcosm cultivation stage I, adjusting the soil moisture content to 60% WHC, injecting synthetic air 79% N2+21% O2, and cultivating in the dark at 25-30°C for 28-30 days.

[0043] Compared with the prior art, the present invention has the following beneficial effects:

[0044] 1. The present invention provides a method for evaluating the contribution of ammonia oxidizing microorganisms to soil carbon neutrality during their entire life cycle, including: (1) the contribution of microbial biomass to soil organic carbon (SOC) and the amount of N2O produced by fixing CO2 during the rapid reproduction of ammonia oxidizing microorganisms (first stage); (2) the amount of CO2 produced by the degradation of ammonia oxidizing microorganisms by heterotrophic microorganisms after the death of ammonia oxidizing microorganisms, and the evaluation of organic carbon loss caused by microbial cell death (second stage). Through two-stage experiments, the present invention found that 5.28% of the organic carbon fixed during the reproduction of active cells of ammonia oxidizing microorganisms was degraded into CO2 emissions after cell death; throughout the entire life cycle of ammonia oxidizing microorganisms, soils with long-term high-intensity nitrogen fertilizer input increased net greenhouse gas CO2 emissions by 3 times, while the contribution to carbon neutrality was reduced by 2 times. In the future, the role of ammonia oxidation or other soil microorganisms in carbon neutrality during their entire life cycle should be considered to better understand the global greenhouse gas emission potential.

[0045] 2. The method for evaluating the contribution of the whole life cycle of ammonia oxidizing microorganisms to soil carbon neutrality provided by the present invention can use the contribution of microbial biomass increased by the reproduction of ammonia oxidizing microorganisms to organic carbon, the offset of its contribution to N2O emissions, the contribution rate of active ammonia oxidizing microorganisms to organic carbon in different soils, the mortality rate caused by the death of ammonia oxidizing microorganisms, and the contribution of the whole life cycle to carbon neutrality as evaluation criteria to evaluate the contribution of the whole life cycle of ammonia oxidizing microorganisms to the soil carbon cycle. This solves the problem that the net production of greenhouse gases may be overestimated, and provides the possibility of fertilization strategies to reduce greenhouse gas emissions in the future.

[0046] 3. The present invention provides a method for evaluating the contribution of the life and death cycle of ammonia oxidizing microorganisms to soil carbon neutralization. It is found that in the process of soil autotrophic ammonia oxidation, an average of 3.17% of the newly generated biogenic organic carbon (average 20.8 μg 13 C g -1 ) is lost in the form of CO2 emissions. By injecting 13 After culturing with CO2 and urea for 28 days (the first stage), it was found that the active ammonia oxidizing microorganisms in the soil without fertilizer (CK) and the soil with chemical fertilizer and organic fertilizer (NPKM) were both ammonia oxidizing archaea (AOA) and ammonia oxidizing bacteria (AOB), while the active ammonia oxidizing microorganisms in the soil with chemical fertilizer alone (NPK) were only AOB. The former offset the N2O emissions through CO2 fixation (reaching 34.0%) more than the latter. Excluding this part of the overestimated greenhouse gas emissions, the net greenhouse gas emissions of the three soils were 155.9, 393.5 and 421.1 μg CO2 g -1 The soil with active nitrifying microorganisms was further cultured under suitable water conditions for 30 days, and the top of the culture bottle was 13 The net emission flux of C-CO2 was used to evaluate the death of active cells. 135.28%, 3.18% and 1.86% of C-CO2 were emitted. Throughout the life cycle of ammonia oxidizing microorganisms, soils with long-term high-intensity nitrogen fertilizer input increased net greenhouse gas CO2 emissions by 3 times and reduced their contribution to carbon neutrality by 2 times. In summary, the role of ammonia oxidizing microorganisms in soil carbon neutrality throughout their life cycle should be considered to better understand the global greenhouse gas emission potential. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1 The soil nitrate nitrogen NO before and after the DNA-SIP microcosm cultivation stage I of the present invention x -1 Content, ammonium nitrogen content and organic carbon 13 Changes in net C-SOC generation;

[0049] In the figure, A is the addition of CK, NPK and NPKM soil 12 CO2( 12 C-) or 13 CO2( 13 C-) Changes in soil nitrate nitrogen concentration (nitrification activity) on days 0 and 28 of gas and urea incubation;

[0050] B is added to CK, NPK and NPKM soils 12 CO2( 12 C-) or 13 CO2( 13 C-) Changes in soil ammonium nitrogen content on the 0th and 28th days of gas and urea incubation;

[0051] C is added to CK, NPK and NPKM soils 12 CO2( 12 C-) or 13 CO2( 13 C-) Soil organic carbon on days 0 and 28 of gas and urea incubation 13 Changes in net C-SOC production.

[0052] Figure 2 The changes in soil microbial gene abundance before and after stage I of the DNA-SIP microcosm cultivation of the present invention;

[0053] A is added to CK, NPK and NPKM soils 12CO2( 12 C-) or 13 CO2( 13 C-) Changes in amoA gene abundance of soil AOB on days 0 and 28 of gas and urea incubation;

[0054] B is added to CK, NPK and NPKM soils 12 CO2( 12 C-) or 13 CO2( 13 C-) Changes in soil AOA amoA gene abundance on days 0 and 28 of gas and urea incubation.

[0055] Figure 3 The active cell growth of AOB and AOA after the completion of the DNA-SIP microcosm culture phase I of the present invention, and 13 C enrichment and organic carbon 13 Contribution of net C-SOC generation;

[0056] In the figure, A is the addition of CK, NPK and NPKM soil 12 CO2( 12 C-) or 13 CO2( 13 C-) Quantitative distribution of amoA gene abundance of AOB in each layer of DNA on the buoyant density gradient after 28 days of gas and urea culture;

[0057] B is added to CK, NPK and NPKM soils 12 CO2( 12 C-) or 13 CO2( 13 C-) Quantitative distribution of amoA gene abundance of AOA in each layer of DNA on the buoyant density gradient after 28 days of gas and urea culture;

[0058] C is added to CK, NPK and NPKM soils 12 CO2( 12 C-) or 13 CO2( 13 C-) Gas and urea cultured for 28 days based on high-throughput sequencing of 16S rRNA gene 13 Enrichment of AOB and AOA in C-labeled DNA;

[0059] D is added to CK, NPK and NPKM soils 12 CO2( 12 C-) or 13 CO2( 13 C-) Ammonia oxidizing microorganisms after 28 days of incubation with gas and urea 13 C labeled AOB and AOA in cells13 Contribution of C-SOC net production; D-1 is the addition of CK, NPK and NPKM soils 12 CO2( 12 C-) or 13 CO2( 13 C-) Soil organic carbon (SOC) in soil acidified and non-acidified treatments after 28 days of gas and urea incubation 13 C atomic percentage changes.

[0060] Figure 4 The changes of various indicators after the end of the microcosm cultivation stage II of the organic carbon degradation of the present invention;

[0061] In the figure, A shows the changes in CO2 content in CK, NPK and NPKM soils after the end of microcosm stage II cultivation; A-1 shows the changes in CO2 13 Changes in C atomic percentage;

[0062] B is CK, NPK and NPKM soil 13 Changes in net C-CO2 emissions;

[0063] C is the change in the lethality of ammonia-oxidizing microorganisms;

[0064] The calculation method is CK, NPK and NPKM soil in microcosm stage II 13 C-CO2 net emission and organic carbon in microcosm stage I ( 13 C-SOC) net production ratio;

[0065] D is the change in net greenhouse gas CO2 emissions from CK, NPK, and NPKM soils;

[0066] The calculation method is to subtract the equivalent CO2 emission from N2O in microcosm stage I from the CO2 emission from organic carbon in culture stage I. 13 Equivalent CO2 fixation plus organic carbon degradation in microcosm cultivation phase II of C-SOC net production 13 C-CO2 net emission is used as net greenhouse gas CO2 emission to evaluate the net greenhouse gas CO2 emission of ammonia oxidizing microorganisms throughout their life cycle. Three replicates of each treatment were used to calculate the average value, and different letters above the bar graph indicate significant differences (p<0.05). DETAILED DESCRIPTION

[0067] The specific embodiments of the present invention are described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. Based on the embodiments in the present invention, all other embodiments obtained by ordinary technicians in this field without creative work belong to the scope of protection of the present invention. The experimental methods described in the embodiments of the present invention are conventional methods unless otherwise specified, and the materials, reagents, etc. used in the following embodiments can be obtained from commercial channels unless otherwise specified.

[0068] Example 1: Method for evaluating the contribution of the life and death cycle of ammonia oxidizing microorganisms to soil carbon neutralization.

[0069] 1. Site Description and Soil Sampling

[0070] Soil samples (soil samples) were collected from the long-term experimental site of Jilin Academy of Agricultural Sciences (124°48′36″E, 43°30′24″N), located in Gongzhuling, Jilin Province, Northeast China. The area has a temperate semi-humid continental monsoon climate, with an average annual temperature of 5-6°C, precipitation of 500-600 mm, frost-free period of 125-140 days, and an altitude of 200 m. The soil is classified as black soil (FAO classification), developed in the Quaternary loess-like sediment parent material, and is a dryland rain-fed agriculture without irrigation. The experimental site was established in a monoculture corn field in 1987, and the soil organic matter was 29.7 g kg -1 Total nitrogen 1.62 g kg -1 , total phosphorus 0.86gkg -1 , pH7.6.

[0071] The present invention selected three treatments: no fertilizer (CK), chemical fertilizer alone (NPK), and chemical fertilizer and organic fertilizer combination (NPKM). In the past 32 years (1987-2019), 150 kg hm2 of fertilizer was applied to the field plots each year. -2 Urea-N, 75kg hm -2 Phosphoric acid and 75kg hm -2 Potassium oxide. Organic fertilizer is pig manure (equivalent to 115kg hm -2 Urea-N) was applied to the surface once after the harvest in early autumn of the previous year. After the plants were harvested in October 2019, the five-point method was used to collect soil samples from the surface layer of 0-20 cm, which were mixed and divided into three parts for microcosm culture experiments, soil chemical properties analysis, and microbial molecular analysis. For microcosm culture soil samples, the collected fresh soil samples need to be air-dried to a moisture content of about 15% (w / w), sieved through a 2mm sieve, and stored in a refrigerator at 4°C.

[0072] 2. Stable isotope nucleic acid probe (DNA-SIP) microcosm culture to detect the impact of the entire life cycle of ammonia oxidizing microorganisms on carbon neutrality

[0073] 1. The first stage: stable isotope nucleic acid probe (DNA-SIP) microcosm cultivation (referred to as DNA-SIP microcosm cultivation stage I) and soil sample treatment after cultivation.

[0074] (1) Weigh 6 g (dry weight) of fresh soil of CK, NPK and NPKM as soil samples, place them in 120 mL serum bottles, and pre-cultivate them in the dark in an incubator at 28°C for 10 days under the condition of 40% field water holding capacity (40% WHC) to restore microbial activity and reduce the amount of CO2 emitted by soil respiration to prevent the occurrence of 5% 13 CO2 dilution, thereby increasing the success rate of labeling. Formal culture experiment setting 5% (v / v) 13 CO2 treatment group, 5% 12 CO2 was used as the control group, and each treatment was repeated 4 times. Only CK soil was used for the control group experiment. 400 μg N g -1 The urea-N source was added, the soil moisture content was adjusted to 60% WHC, the bottle was sealed with a butyl rubber stopper, CO2 was injected into the bottle, and the bottle was cultured in the dark at 28°C in an incubator for 28 days.

[0075] (2) After the incubation period, the top air was collected from each bottle to measure the N2O content, which was converted into an equivalent amount of CO2 emissions (or CO2 production, CO2 content). Considering that the greenhouse gas warming potential of N2O is 273 times that of CO2, it is used to analyze the total greenhouse gas emissions generated by the reproduction of ammonia oxidizing microorganisms.

[0076] (3) After the first stage of cultivation, the content of soil organic carbon (SOC) and the 13 C atomic percentage, calculate soil organic carbon ( 13 C-CO2) net production; analyze the contribution of CO2 fixed by rapid reproduction of ammonia oxidizing microorganisms as microbial biomass to soil SOC; combine the CO2 emission results in (1) to calculate the ability of the organic carbon fixed by ammonia oxidizing microorganisms to offset CO2 emissions.

[0077] (4) After the first stage of cultivation, the total soil DNA was extracted and stratified by ultra-high-speed density gradient centrifugation (SIP stratification) to obtain 15 stratified DNAs. The amoA functional gene abundance of ammonia oxidizing bacteria (AOB) and ammonia oxidizing archaea (AOA) in the total soil DNA and stratified DNA was quantified, and the active ammonia oxidizing microorganisms in the soil and the cell increase produced by their reproduction were analyzed; at the same time, based on the molecular weight of AOB and AOA themselves, their contribution to the increase in net organic carbon produced by their own reproduction was evaluated; after the first stage of cultivation, the soil samples in the bottles were destructively sampled, and 1 g of each soil sample was immediately stored at -20°C for microbial molecular analysis.

[0078] 3. Microcosm cultivation phase II (second phase) of organic carbon degradation: Based on the soil samples that have fixed organic carbon (SOC) in the first phase, the microcosm was further cultivated for 30 days.

[0079] 1. After the first stage of cultivation, the soil samples were treated with acidification and non-acidification, and the soil organic carbon content and soil organic carbon of the two treatments were compared. 13 C atomic percentage ( 13 C abundance) changes to assess whether the fixed organic carbon in the first stage has a contribution from inorganic carbon.

[0080] 2. Acidification process: The acidification process is to wash the soil three times with 0.5M HCl and then rinse with ultrapure water. 3g of each soil sample was vacuum freeze-dried for 12 hours and then sieved through a 0.15mm mesh. Subsequently, two samples were packed in a tin boat for acidification and non-acidification treatment (i.e., no treatment was done). Finally, the content of organic carbon (SOC) in the soil (acidified and non-acidified soil) and the organic carbon (SOC) were determined by an elemental analyzer (multi N / C 3100, Analytik Jena, Germany) and a stable isotope ratio mass spectrometer (EA-IRMS, Thermo Fisher Scientific, USA). 13 C atomic percentage. According to the organic carbon (SOC) during the culture period 13 C atomic percentage enrichment, calculate the organic carbon in the soil ( 13 C-SOC), which is the amount of CO2 fixed by the growth of ammonia-oxidizing microorganisms during nitrification.

[0081] The results showed that compared with non-acidification treatment, acidification treatment had a significant effect on organic carbon (SOC). 13 C atomic percentage ( 13 C atomic abundance) and SOC content, indicating that there is no contribution of inorganic carbon to soil organic carbon, so the ammonia oxidizing microorganisms 13 C biomass comes entirely from the fixed 13 C-SOC. Therefore, in the second stage of microbial culture, 13 C-labeled soil without acidification treatment was used as the research object.

[0082] The remaining 2 g of soil was extracted with 2M KCl to determine the ammonium nitrogen (NH4 + ) and nitrate (NO x - ) content. x - ) concentration increase to estimate the net nitrification rate of the soil.

[0083] 3. The second stage: Microcosm cultivation stage II for organic carbon degradation (microcosm cultivation stage II)

[0084] The second stage selects the 13 C-treated soil (soil not treated with acidification) was the research object. About 0.5 g of soil was weighed and placed in a 20 ml serum bottle. After adjusting the soil moisture content to 60% WHC, the bottle was sealed with a butyl rubber stopper. The culture bottle was further vacuumed and aired three times and injected with synthetic air (79% N2 + 21% O2) after the last vacuuming. It was then cultured in the dark in an incubator at 28°C for 30 days. After the culture was completed, the CO2 content in the air at the top of each culture bottle and the CO2 13 C atomic percentage.

[0085] 4. Determination of soil properties, nitrification rate, N2O and CO2 production.

[0086] The basic chemical properties of the in-situ soil were determined. The soil and deionized water were mixed in a mass ratio of 1:2.5 to prepare a soil suspension for determining the soil pH. The total carbon and total nitrogen content in the soil was determined using an elemental analyzer (multi N / C3100, AnalytikJena, Germany). The exchangeable ammonium nitrogen (NH4 + ) and nitrate nitrogen (NO x - ) concentrations were measured by a continuous flow analyzer (SanCC System, Skalar, The Netherlands). 13 The atomic percentage of C was determined using a stable isotope ratio mass spectrometer (EA-IRMS, Thermo Fisher Scientific, USA). The contents of N2O and CO2 in the head air were determined by gas chromatography.

[0087] 4. The contribution of the entire life cycle of ammonia oxidizing microorganisms to soil carbon neutrality was evaluated based on the contribution of organic carbon, mortality rate, net greenhouse gas CO2 emissions and carbon neutrality contribution during the entire life cycle of ammonia oxidizing microorganisms during the first stage (stage I) and the second stage (stage II) of the reproduction and death of ammonia oxidizing microorganisms, as follows:

[0088] The contribution of ammonia oxidizing microorganisms to organic carbon is calculated using the obtained increase in active ammonia oxidizing microbial cells;

[0089] Soil organic carbon (SOC) content and organic carbon (SOC) at the end of DNA-SIP microcosm cultivation phase I 13 C atomic percentage, and the CO2 content and CO2 content measured after the end of the microcosm cultivation phase II of organic carbon degradation 13The lethality was calculated by the atomic percentage of C;

[0090] The N2O content and soil organic carbon (SOC) content were measured after the end of the DNA-SIP microcosm cultivation phase I. 13 The atomic percentage of C and the content of soil organic carbon (SOC), as well as the content of CO2 and CO2 measured after the end of the cultivation of the microcosm cultivation phase II of organic carbon degradation 13 The net greenhouse gas CO2 emissions were calculated by C atomic percentage;

[0091] Soil organic carbon (SOC) measured after the end of DNA-SIP microcosm cultivation phase I 13 The atomic percentage of C and the content of soil organic carbon (SOC), as well as the content of CO2 and CO2 measured after the end of the cultivation of the microcosm cultivation phase II of organic carbon degradation 13 The carbon neutrality contribution rate was calculated by calculating the C atomic percentage;

[0092] (1) The active cell mass ratio of ammonia oxidizing microorganisms multiplied by the organic carbon 13 The net production of C-SOC is calculated as the contribution of ammonia oxidizing microorganisms to organic carbon. The contribution of ammonia oxidizing microorganisms to organic carbon includes the contribution of active AOB to organic carbon and the contribution of active AOA to organic carbon. The specific calculation formula is as follows:

[0093] Contribution of active AOB to organic carbon = active AOB cell mass / (active AOB cell mass + active AOA cell mass)* 13 C-SOC I

[0094] Contribution of active AOA to organic carbon = active AOA cell mass / (active AOB + active AOA cell mass sum)* 13 C-SOC I

[0095] Active cell mass = number of active cells * cell mass

[0096] In the formula, 13 C-SOC I Representation of organic carbon in DNA-SIP microcosm culture stage I 13 The net production of C-SOC; the number of active cells represents the increase in active ammonia oxidizing microbial cells (including AOA and AOB) in the DNA-SIP microcosm culture phase I. The number of ammonia oxidizing microorganisms includes two types: AOA (10.2 fg protein per cell) and AOB (120 fg protein per cell).

[0097] (2) Microcosm cultivation stage II based on organic carbon degradation 13C-CO2 net emission and organic carbon in DNA-SIP microcosm cultivation stage I 13 The result of the ratio of C-SOC net production is used as the lethality to evaluate the organic carbon loss caused by the death of ammonia oxidizing microbial cells. The calculation formula is as follows:

[0098] Mortality rate = ( 13 C-CO2 II ) / ( 13 C-SOC I )*100

[0099] In the formula, 13 C-CO2 II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13 C - net CO2 emissions, 13 C-SOC I Representation of organic carbon in DNA-SIP microcosm culture stage I 13 C-SOC net generation.

[0100] Phase II of the Microcosm Cultivation of Organic Carbon Degradation 13 The calculation formula of C-CO2 net emission is as follows:

[0101] 13 C-CO2 II =CO 2II *(CO2- 13 C ％II -1.08) / 100

[0102] In the formula, 13 C-CO2 II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13 C-CO2 net emissions, CO 2II The CO2 content after the end of the microcosm cultivation phase II of organic carbon degradation, CO2- 13 C ％II The CO2 in the air after the microcosm cultivation phase II indicates organic carbon degradation 13 C atomic percentage;

[0103] Organic Carbon 13 The net production of C-SOC is determined by the soil organic carbon SOC 13 The C atomic percentage and soil organic carbon SOC content were calculated using the following formula:

[0104] 13 C-SOC I =SOC I *(SOC- 13 C ％I-1.08) / 100

[0105] In the formula, 13 C-SOC I Representation of organic carbon in DNA-SIP microcosm culture stage I 13 C-SOC net generation, SOC I represents the soil organic carbon content in DNA-SIP microcosm incubation stage I, SOC- 13 C ％1 Figure 2 shows the soil organic carbon (SOC) of DNA-SIP microcosm incubation stage I. 13 C atomic percentage.

[0106] (3) The equivalent CO2 emission from N2O in DNA-SIP microcosm culture phase I minus the CO2 emission from organic carbon in culture phase I 13 Equivalent CO2 fixation plus organic carbon degradation in microcosm cultivation phase II of C-SOC net production 13 C-CO2 net emissions are used as net greenhouse gas CO2 emissions to evaluate the net greenhouse gas CO2 emissions of ammonia oxidizing microorganisms throughout their life cycle. The calculation formula is as follows:

[0107] Net greenhouse gas CO2 emissions = (CO2-equivalent I )-( 13 C-CO2-fixed I )+( 13 C-CO2 II )

[0108] In the formula, CO2-equivalent I represents the equivalent CO2 emission from N2O during the DNA-SIP microcosm cultivation phase I, which is calculated based on the fact that the warming potential of N2O is 273 times higher than that of CO2; 13 C-CO2-fixed I Indicates that DNA-SIP microcosm culture stage I comes from organic carbon 13 C-SOC net generation equivalent 13 C-CO2 fixation, which is calculated based on the conversion of SOC and CO2 having the same C molar number; 13 C-CO2 II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13 C - net CO2 emissions.

[0109] (4) Organic carbon from DNA-SIP microcosm culture phase I 13 The equivalent amount of CO2 fixation of the net production of C-SOC minus the organic carbon degradation of the microcosms during the cultivation phase II13 The value after C-CO2 net emission is divided by the equivalent CO2 emission from N2O as carbon neutrality, which is used to evaluate the contribution of ammonia oxidizing microorganisms to carbon neutrality throughout their life cycle. The calculation formula is as follows:

[0110] Carbon neutrality contribution rate = [( 13 C-CO2-fixed I )-( 13 C-CO2 II )] / net greenhouse gas CO2 emissions*100

[0111] Among them, net greenhouse gas CO2 emissions = (CO2-equivalent I )-( 13 C-CO2-fixed I )+( 13 C-CO2 II )

[0112] In the formula, 13 C-CO2-fixed I Indicates that DNA-SIP microcosm culture stage I comes from organic carbon 13 C-SOC net generation equivalent 13 C-CO2 fixation, which is calculated based on the conversion of SOC and CO2 having the same C molar number; 13 C-CO2 II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13 C-CO2 net emissions; CO2-equivalent I Represents the equivalent CO2 emission from N2O during stage I of DNA-SIP microcosm cultivation.

[0113] The amount of CO2 fixed in the DNA-SIP microcosm cultivation stage I is equal to the amount of organic carbon in the DNA-SIP microcosm cultivation stage I. 13 The net generation of C-SOC is calculated as follows:

[0114] 13 C-CO2-fixed I = 13 C-SOC

[0115] In the formula, 13 C-CO2-fixed I Indicates that DNA-SIP microcosm culture stage I comes from organic carbon 13 C-SOC net generation equivalent 13 The C-CO2 fixation amount is calculated based on the fact that SOC and CO2 have the same C molar number.

[0116] Organic Carbon 13 The higher the net C-SOC production, the higher the soil quality. The contribution rate of active ammonia oxidizing microorganisms to organic carbon varies depending on the soil type. The mortality rate of ammonia oxidizing microorganisms is inversely proportional to their contribution to organic matter. The greater the net greenhouse gas CO2 emissions, the higher the greenhouse gas emissions generated by this type of fertilized soil. Fertilization strategies to reduce greenhouse gas emissions can be provided based on the carbon neutrality contribution rate.

[0117] Ultimately, the increase in microbial growth and the activity of ammonia oxidizing microorganisms on organic carbon 13 The contribution of net C-SOC production, the mortality rate caused by the death of ammonia oxidizing microorganisms, the net greenhouse gas CO2 emissions, and the contribution rate of the entire life cycle of ammonia oxidizing microorganisms to carbon neutrality were used as evaluation criteria to evaluate the contribution of the entire life cycle of ammonia oxidizing microorganisms to the soil carbon cycle.

[0118] V. Results Analysis

[0119] 1. Changes of various indicators after DNA-SIP microcosm cultivation stage I (first stage)

[0120] The results are as follows Figure 1 As shown in the figure, after the first stage of cultivation, the soil nitrate nitrogen NO x -1 The content increased significantly ( Figure 1 of A), while the ammonium nitrogen content was significantly reduced ( Figure 1 The net nitrification rates of CK, NPK, and NPKM soils were 9.96, 9.89, and 10.8 μg N g, respectively. -1 day -1 (As shown in Table 1), it shows that aerobic nitrifying microorganisms play a leading role in soil ammonia oxidation. Soil organic carbon ( 13 The net production of C-SOC was 1.82, 1.14 and 1.84 μmol in CK, NPK and NPKM soils, respectively. 13 C g -1 ( Figure 1 C), which is equivalent to CO2 fixation of 1.82, 1.14 and 1.84 μmol respectively. 13 C g -1 (Table 2), indicating that the amount of SOC produced by microbial assimilation in the CK soil was the same as that in the NPKM soil. 13 The net production of C-SOC and the emission of greenhouse gas N2O showed a downward trend: NPKM (41.8 nmol g -1 )>NPK(36.9nmol g -1 )>CK(19.6nmol g -1). Based on the fact that the warming effect of N2O within 100 years is 273 times that of CO2, the N2O emissions in NPKM, NPK and CK soils are equivalent to CO2 emissions of 11.4, 10.1 and 5.36 μmol g -1 (Table 2), which means that long-term fertilization leads to higher greenhouse gas emissions, especially in soils with high soil organic carbon content (Table 1). Therefore, the contribution of organic carbon to the growth of ammonia oxidizing microorganisms in the first stage offset the greenhouse effect of N2O emissions by 34.0% ± 2.68%, 16.2% ± 0.45% and 11.3% ± 1.36% in CK, NPKM and NPK soils, respectively (Table 2). This indicates that urea application stimulated the increase of active ammonia oxidizing bacteria, making CK soil have the highest offsetting capacity.

[0121] Nitrification and autotrophic ammonia oxidation increased the abundance of AOB in all soils (CK, NPK, and NPKM). Figure 2 A), while the abundance of AOA increased only in CK and NPKM soils ( Figure 2 B). The AOA / AOB abundance ratios of CK, NPK, and NPKM soils on day 0 were 613, 71.0, and 108 times, respectively (Table 3), which were significantly higher than the ratios on day 28 (CK 2.77 times, NPK 0.28 times, and NPKM 1.24 times) (Table 3), indicating that urea application led to a sharp decrease in AOA abundance. Accordingly, a large number of AOB and AOA cells were enriched 13 C, reflecting the ammonia oxidizing bacteria by using DNA-SIP 13 Distribution and abundance of C-labeled biomass ( Figure 3 A and B, Table 3). Based on 16S rRNA gene classification, 13 C-group 3a-associated AOB and 1.1b-Nitrosophaera AOA 13 C-labeled “heavy” DNA accounted for 4.7%-49.6% of the total microbial abundance ( Figure 3C, Table 4), indicating that ammonia oxidizers play a major role in CO2 fixation. Nitrosospira cluster 3a in AOB and group 1.1b-Nitrosophaera lineage in AOA are the main drivers of nitrification, indicating that nitrification in these soils has different active ammonia oxidizers and long-term field fertilization is conducive to the functional stability of active ammonia oxidizers. Calculations of cell-specific activities further support that AOB and AOA dominate CO2 fixation and N2O emissions in CK and NPKM soils, while only AOB plays a greater role in NPK soils (Table 5). These results indicate that the active proliferation of AOB cells is associated with most of the N2O emissions in long-term fertilized soils, while the growth and activity of AOA in non-fertilized CK soils are greatly stimulated, resulting in the lowest N2O emissions from the soil.

[0122] Table 1 Soil chemical properties and net nitrification rate of soil after incubation

[0123]

[0124] Note: Different letters (a, b, c) in each column indicate significant differences within the same treatment.

[0125] Table 2 13 Carbon and nitrogen transformations in soils treated with CO2 and urea during 28-day incubation

[0126]

[0127] Note: The values ​​are the average of three measurements (± standard deviation). * refers to the added mark 13 Soil treated with C-CO2 and urea. Different letters (a, b, c) in each column indicate significant differences within the same treatment; N2O emissions after cultivation ends; θThe global warming potential of N2O is estimated to be 273 times that of CO2 on a 100-year time scale; ξCO2 fixation is estimated by the molar exchange of carbon and CO2 into net soil organic matter. 13 C input; Represents the global warming potential of CO2 fixation in different soils to offset N2O emissions after cultivation.

[0128] Table 3. AOB and AOA labeled in SIP picocosm culture after 28 days of culture

[0129]

[0130] Note: * refers to soil treated with labeled 13C-CO2 and urea. Values ​​are the means (± standard deviation) of three measurements; The abundance ratio of AOA to AOB in SIP microcosms cultured with urea on days 0 and 28; represents the ratio of gene copy number in ‘heavy’ layers 5, 6 and 7 (HF) DNA to the sum of gene copy number in all 15 DNA ladder fractions; represents the bacterial amoA and archaeal amoA gene copy numbers in the ‘heavy’ layers 5, 6 and 7 (HF) DNA in each treatment; The number of 13C-labeled cells was estimated by dividing the number of 13C-labeled amoA gene copies by 2.5 (AOB) and 1.0 (AOA), respectively. This analysis is based on the fact that AOB cells usually carry two to three highly similar amoA gene copies, while AOA cells usually contain only one amoA gene copy.

[0131] 2. It is worth noting that the newly fixed 13 The amount of CO2 comes entirely from the biomass of ammonia-oxidizing microorganisms-C, since no inorganic 13 CO2. This is achieved by acidifying and non-acidifying the soils, and then 13 There is no significant difference in the C atomic percentage to confirm ( Figure 3 However, due to the huge difference in cell biomass between AOA (10.2 fg protein per cell of SCM1) and AOB (120 fg protein per cell of N. europaea), the AOB cells 13 C cell biomass 13 The contribution of the net production of C-SOC (CK, NPK and NPKM were 1.11, 1.14 and 1.71 μmol, respectively) 13 C g -1 ) was greater than AOA (CK, NPK and NPKM were 0.71, 0 and 0.14 μmol, respectively 13 C g -1 )( Figure 3 D). These findings suggest that in these soils, AOB play a dominant role in CO2 fixation through growth via the ammonia oxidation process.

[0132] Table 4 After 28 days of cultivation (microcosm stage I) 13 Relative abundance of autotrophic microorganisms in C-labeled DNA

[0133] Taxonomy <![CDATA[ 13 CO2+CK(%)]]> <![CDATA[ 13 CO2+NPK(%)]]> <![CDATA[ 13 CO2+NPKM(%)]]> Candidatus Nitrosopumilus 0 0 0.01 Nitrososphaera 37.6* 0.14* 1.17* Nitrososphaeraceae 0 0 0.14* Nitrosospira 12.0* 20.7* 3.53* Nitrosomanas 0.30* 0.06 0.12* Brevundimonas 0.02 0.12* 0.09 Nitrospira 5.12* 35.4* 2.36* Solirubrobacter 0.75* 0.25* 1.49* Streptomyces 0.92* 0.52* 1.60* Cupriavidus 0.25* 0.23* 0.13 Rhodanobacter 0.17* 0.62* 1.16* Blastococcus 1.87* 0.93* 2.61* Agromyces 0.02 0.02 0.27* Microbacterium 0 0.09 0.25*

[0134] Note: * indicates the relative abundance of the enriched microorganisms is greater than 0.1%.

[0135] Table 5 Cell-specific activities of AOA and AOB after 28 days of SIP microcosm culture

[0136]

[0137] Note: * indicates the marked 13 C-CO2 and urea-added soil. Values ​​are means (± standard deviation) of three measurements;

[0138] Net soil organic carbon 13 C accumulation rate Net soil organic carbon 13 The C accumulation rate was calculated from the soil organic carbon content during the 28-day incubation period. 13 C is calculated by the net increase; § N2O production measured after 28 days of cultivation;

[0139] By converting soil organic carbon into 13 The net increase in C divided by 13 This calculation assumes that each cell has the same activity and that 13 C biomass synthesis is entirely derived from either AOB or AOA. The carbon content of a typical pure culture of AOB cells is estimated to be 8.67-92.9 femto mol C cell -1 within the scope of

[0140] Estimated as the soil organic carbon content during the 28-day incubation period 13 The rate at which C increases;

[0141] The N2O production rate is determined by 13 It is calculated by dividing the number of active AOB or AOA cells obtained in the C-labeled DNA isolation by the amount of N2O produced. This calculation assumes that the activity of each cell is the same and that the N2O produced comes entirely from either AOB or AOA;

[0142] θ Through organic carbon 13 The equivalent amount of CO2 fixation of the net C-SOC production minus 13 C-CO2 net emissions divided by net greenhouse gas CO2 emissions

[0143] 4. Microcosm Stage II Cultivation

[0144] After further incubation of the labeled soil at 60% water content for 30 days, it was found that 12 Compared with the C-CK control treatment, CO2 13The atomic percentage and content of C showed a significant downward trend: CK>NPKM>NPK( Figure 4 A), for example 13 The atomic percentages of C in CK, NPKM, and NPK soils were 3.12±0.01%, 1.80±0.07%, and 1.64±0.12%, respectively. Figure 4 A-1). 13 The net C-CO2 emission was highest in CK soil (0.10 ± 0.003 μmol 13 C g -1 ), followed by NPKM (0.06±0.005μmol 13 C g -1 ) and NPK (0.02±0.003μmol 13 C g -1 )( Figure 4 B) Then, by 13 CO2 and fixed 13 The mortality rate of ammonia oxidizing microorganisms was estimated by the ratio of C-SOC. The results showed that the mortality rates in CK, NPKM and NPK soils were 5.28%±0.38%, 3.18%±0.21% and 1.86%±0.42%, respectively. Figure 4 After offsetting the CO2 fixation in the micro-ecosystem in the first stage and calculating the CO2 emission in the micro-ecosystem in the second stage, the net emission of greenhouse gas CO2 from CK soil reached 3.64±0.43μmol CO2 g -1 , NPK soil reached 8.96±0.13μmol CO2 g -1 , NPKM soil reached 9.63±0.40μmol CO2 g -1 ( Figure 4 D). The contribution rates of ammonia oxidizing microorganisms to carbon neutralization during their entire life cycle were 47.7%, 12.5% ​​and 18.6% respectively (Table 5). These findings indicate that the life and death process of ammonia oxidizing bacteria is an important pathway for soil carbon neutralization.

[0145] The present invention obtained the contribution of organic matter produced by the whole life cycle of ammonia oxidation in CK, NPK and NPKM soils from the three different fertilized soils in the experiment: 1.82, 1.14 and 1.84 μmol 13 C g -1 ; Effects of ammonia oxidizing microbial cell proliferation on organic carbon 13The contributions of net C-SOC production were 60.9% AOB+39.1% AOA, 100% AOB and 92.5% AOB+7.5% AOA, respectively. The mortality rates of ammonia oxidizing microorganisms were 5.28%, 1.86% and 3.18%, respectively. The net greenhouse gas CO2 emissions were 3.64, 8.96 and 9.63 μmol CO2 g -1 ; The contribution rates of ammonia oxidizing microorganisms to carbon neutrality during their entire life cycle were 47.7%, 12.5% ​​and 18.6%, respectively. These results indicate that NPKM soils with long-term application of organic and inorganic fertilizers will increase soil organic matter and improve soil quality compared to NPK soils with single application of chemical fertilizers, but the mortality rate of ammonia oxidizing microorganisms in the soil is the opposite; at the same time, the net greenhouse gas CO2 emissions caused by long-term fertilization increased by 3 times compared to unfertilized soils, while the contribution rate of ammonia oxidizing microorganisms to carbon neutrality during their entire life cycle decreased by 2 times.

[0146] In summary, the role of ammonia oxidizing microorganisms in soil carbon neutralization during their entire life cycle should be considered to avoid overestimating net greenhouse gas emissions. A large amount of CO2 is fixed as organic carbon by ammonia oxidizing microorganisms. Although it is quickly excreted by microbial respiration, it takes a long time for the new fixed carbon to be fully mineralized. However, it should be emphasized that the measurement of CO2 fixation flux in the present invention represents the physiological activities of all organisms that can fix CO2, including non-nitrifying bacteria. Although some unknown CO2-fixing organisms may have been overlooked, ammonia oxidizing microorganisms in labeled soils were highest in CK, NPK, and NPKM soils compared to other autotrophic organisms (Table 3). Therefore, net greenhouse gas emissions remain unchanged, and in the future, these active microorganisms and their relationship should be clarified through in situ stable isotope labeling combined with nano-SIMS (secondary ion beam mass spectrometry) imaging or metagenomic sequencing. 13 Functional metabolic pathways related to CO2 fixation.

[0147] It should be noted that all sequences of the present invention have been submitted to the SRA database of NCBI, and the accession code is PRJNA938375.

[0148] It should also be noted that the organic carbon (SOC) in the present invention has the following representations: organic carbon, SOC, organic carbon SOC; organic carbon ( 13 There are several ways to express the net production of organic carbon (C-SOC): 13 C-SOC net production, organic carbon ( 13 C-SOC) net generation.

[0149] Although the preferred embodiments of the present invention have been described, those skilled in the art may make other changes and modifications to these embodiments once they have learned the basic creative concept. Therefore, the appended claims are intended to be interpreted as including the preferred embodiments and all changes and modifications that fall within the scope of the present invention.

[0150] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A method for evaluating the contribution of the entire life cycle of ammonia oxidizing microorganisms to soil carbon neutrality, characterized in that: include: DNA-SIP microcosm cultivation stage I, the DNA-SIP microcosm cultivation stage I process is: soil samples are pre-cultured at 30-40% field water holding capacity (30-40% WHC), 25-30°C, dark conditions for 7-14 days, and then 13 CO2 labeling, 12 CO2 was used as the control group, and 300-400 μg N g –1 dws Urea-N source, adjust soil water content to 60% WHC, and continue dark cultivation at 25-30℃ for 28-30 days; After the incubation, the content of N2O, the content of soil organic carbon SOC, and the content of soil organic carbon SOC were detected. 13 C atomic percentage; by extracting the total DNA of the soil after the cultivation, detecting the gene abundance of ammonia oxidizing microorganisms, and analyzing the increase in active ammonia oxidizing microbial cells; The microcosm cultivation stage II of organic carbon degradation, the process of the microcosm cultivation stage II of organic carbon degradation is: taking soil samples after the DNA-SIP microcosm cultivation stage I, adjusting the soil moisture content to 60%, injecting synthetic air 79% N2 + 21% O2, and cultivating in the dark at 25-30°C for 28-30 days; after the cultivation, the CO2 content and CO2 13 C atomic percentage; The contribution of ammonia oxidizing microorganisms to organic carbon is calculated using the obtained increase in active ammonia oxidizing microbial cells; The contribution of the ammonia oxidizing microorganisms to organic carbon is the sum of the contribution of active AOB to organic carbon and the contribution of active AOA to organic carbon; Contribution of active AOB to organic carbon = active AOB cell mass / (active AOB + active AOA cell mass sum)* 13 C-SOC I ; Contribution of active AOA to organic carbon = active AOA cell mass / (active AOB + active AOA cell mass sum)* 13 C-SOC I ; Active cell mass = number of active cells * cell mass; In the formula, 13 C-SOC I Representation of organic carbon in DNA-SIP microcosm culture stage I 13 The net production of C-SOC and the number of active cells indicate the increase in the number of active ammonia-oxidizing microbial cells in the DNA-SIP microcosm culture phase I, and the cell numbers of ammonia-oxidizing microorganisms are AOA and AOB, respectively; Organic Carbon 13 The net production of C-SOC is determined by the soil organic carbon SOC 13 The C atomic percentage and soil organic carbon SOC content were calculated using the following formula: 13 C-SOC I =SOC I *(SOC- 13 C %I (-1.08) / 100 In the formula, 13 C-SOC I Representation of organic carbon in DNA-SIP microcosm culture stage I 13 C-SOC net generation, SOC I represents the soil organic carbon content in DNA-SIP microcosm incubation stage I, SOC- 13 C %1 Figure 2 shows the soil organic carbon (SOC) of DNA-SIP microcosm incubation stage I. 13 C atomic percentage; Soil organic carbon (SOC) content and organic carbon (SOC) at the end of DNA-SIP microcosm cultivation phase I 13 C atomic percentage, and the CO2 content and CO2 content measured after the end of the microcosm cultivation phase II of organic carbon degradation 13 The lethality is calculated by the atomic percentage of C; the lethality is used to evaluate the organic carbon loss caused by the death of ammonia-oxidizing microbial cells, and the calculation formula is as follows: Fatality rate = ( 13 C-CO 2II ) / ( 13 C-SOC I )*100 In the formula, 13 C-CO 2II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13 C - net CO2 emissions, 13 C-SOC I Representation of organic carbon in DNA-SIP microcosm culture stage I 13 C-SOC net generation; Phase II of the Microcosm Cultivation of Organic Carbon Degradation 13 The calculation formula of C-CO2 net emission is as follows: 13 C-CO 2 II = WHAT 2II *(CO2- 13 C %II -1.08) / 100 In the formula, 13 C-CO 2 II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13 C-CO2 net emissions, CO 2II The CO2 content after the end of the microcosm cultivation phase II of organic carbon degradation, CO2- 13 C %II The CO2 in the air after the microcosm cultivation phase II indicates organic carbon degradation 13 C atomic percentage; The N2O content and soil organic carbon (SOC) content were measured after the end of the DNA-SIP microcosm cultivation phase I. 13 The atomic percentage of C and the content of soil organic carbon (SOC), as well as the content of CO2 and CO2 measured after the end of the cultivation of the microcosm cultivation phase II of organic carbon degradation 13 The net greenhouse gas CO2 emissions are calculated based on the C atomic percentage; the calculation formula for the net greenhouse gas CO2 emissions method is as follows: Net greenhouse gas CO2 emissions = (CO2-equivalent I )-( 13 C-CO2-fixed I )+( 13 C-CO 2II ) In the formula, CO2-equivalent I represents the equivalent CO2 emission from N2O during the DNA-SIP microcosm cultivation phase I, which is calculated based on the fact that the warming potential of N2O is 273 times higher than that of CO2; 13 C-CO2-fixed I Indicates that DNA-SIP microcosm culture stage I comes from organic carbon 13 C-SOC net generation equivalent 13 C-CO2 fixation, which is calculated based on the conversion of SOC and CO2 having the same C molar number; 13 C-CO 2II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13 C - net CO2 emissions; Organic carbon from DNA-SIP microcosms during stage I 13 Equivalent CO2 fixation of net C-SOC production minus organic carbon degradation in microcosm cultivation phase II 13 The value after C-CO2 net emission is divided by the equivalent CO2 emission from N2O as carbon neutrality, which is used to evaluate the contribution of ammonia oxidizing microorganisms to carbon neutrality throughout their life cycle. The calculation formula is as follows: Carbon neutrality contribution rate = [( 13 C-CO2-fixed I )-( 13 C-CO 2II ) ] / net greenhouse gas CO2 emissions*100 Among them, net greenhouse gas CO2 emissions = (CO2-equivalent I )-( 13 C-CO2-fixed I )+( 13 C-CO 2II ) In the formula, 13 C-CO2-fixed I Indicates that DNA-SIP microcosm culture stage I comes from organic carbon 13 C-SOC net generation equivalent 13 C-CO2 fixation, which is calculated based on the conversion of SOC and CO2 having the same C molar number; 13 C-CO 2II Figure 2 shows the microcosm culture stage II for organic carbon degradation 13 C-CO2 net emissions; CO2-equivalent I represents the equivalent CO2 emission from N2O in DNA-SIP microcosm culture stage I; The amount of CO2 fixed in the DNA-SIP microcosm cultivation stage I is equal to the amount of organic carbon in the DNA-SIP microcosm cultivation stage I. 13 The calculation formula for C-SOC net generation is as follows: 13 C-CO2-fixed I = 13 C-SOC In the formula, 13 C-CO2-fixed I Indicates that DNA-SIP microcosm culture stage I comes from organic carbon 13 C-SOC net generation equivalent 13 C-CO2 fixation amount, calculated based on the fact that SOC and CO2 have the same C molar number; The impact of the entire life cycle of ammonia oxidizing microorganisms on soil carbon neutrality was evaluated based on their contribution to organic carbon, mortality rate, net greenhouse gas CO2 emissions and carbon neutrality contribution rate.

2. The method for evaluating the contribution of the entire life cycle of ammonia oxidizing microorganisms to soil carbon neutralization according to claim 1, characterized in that: The contribution rate of active ammonia oxidizing microorganisms to organic carbon varies with soil types, and the lethality of ammonia oxidizing microorganisms is inversely proportional to their contribution to organic matter; The greater the net greenhouse gas CO2 emissions, the higher the greenhouse gas emissions produced by this type of soil. Soil fertilization strategies to reduce greenhouse gas emissions can be provided based on the carbon neutrality contribution rate.

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