The contributions of ammonia oxidizing bacteria and archaea to nitrification-dependent N
N 2 O is a trace greenhouse gas with 265 times the warming potential of CO 2 (on a 100-year scale) in the atmosphere and acts to deplete stratospheric ozone . The global concentration of N 2 O in the atmosphere was 328.8 ppb in 2015 with a 21% increase since the industrial revolution . The sustained emissions at the current rate will result to increase by another 18% until 2030 from current projections indicate . Fertilized agricultural soil is a hot spot for emissions substantial N 2 O to atmosphere, accounting for about 60% of global atmospheric N 2 O emissions . However, increasing demand for food, animal husbandry and biomass energy will inevitably accompany the continuous application of chemical fertilizers. Therefore, it is particularly important to understand the mechanism responsible for N 2 O production from fertilized soil to find optimal measures for regulating N 2 O emissions for sustainable agriculture. In agro-ecosystems, N 2 O is produced by nitrification and denitrification driven by soil microorganisms , , and some abiotic processes also contribute a small part of it . Nitrification is a main N 2 O production process under a suitable soil oxygen content. Ammonia oxidation (i.e. NH 3 being oxidized to NO 2 − , via the intermediate product NH 2 OH) is considered to be the primary and rate-limiting step of nitrification. Both ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) have the genetic potential for ammonia oxidation which cause production of N 2 O , . AOB could produce N 2 O which acts as the intermediate by the incomplete NH 2 OH oxidation to NO , or by regulating nitrifier denitrification of reduction NO 2 − to NO and N 2 O . In contrast, N 2 O produced by AOA attributes to multiple processes conventionally detected in pure and enrichment cultures, but there was no clear evidence to date for supporting production of N 2 O by AOA enzymatic catalytic reaction in soils . In addition, the newly discovered and widespread complete ammonia oxidizers (comammox) which act as one of ammonia-oxidizing functional guilds constitute remains uncertainty of influence on N 2 O emission, though some studies suggested it playing a minor role than AOB , . Nitrification inhibitors have been widely applied in agricultural soils to reduce transformation of NH 4 + -N to NO 3 – N, thus improving N use efficiency . One of the key functional enzymes in both AOA and AOB ammonia oxidizers is ammonia monooxygenase (AMO) which directly catalyzed the process of N 2 O synthesis. Acetylene (C 2 H 2 ) is a non-selective nitrification inhibitor for AMO, thus inhibiting ammonia oxidation of both AOA and AOB at a low concentration (0.1–10 Pa) , . Meanwhile, 1-octyne is a selective inhibitor that can inhibit AOB activity but not AOA in soils, and it can be used to distinguish the relative contribution of AOB and AOA to nitrification – . Substantial studies have found that many factors including soil types and environmental factors determine the abundance, activities and relative contribution to N 2 O emission of AOA and AOB, especially the soil pH and inorganic nitrogen (N) supply – . For example, the abundance and activity of AOB increased in ammonium concentration-rich soils, whereas AOA act as not affected or inhibited , . In unfertilized or acidic soils, abundance and activity of AOA are much higher than those of AOB , . Wang et al. reported that nitrogen fertiliser-induced N 2 O emissions are attributed 70.5 ~ 78.1% by AOB and 18.7 ~ 19.7% by AOA using the method of inhibitors both in acidic (pH = 6) and alkaline (pH = 8) arable soils of China. Similarly, using the method of inhibitors, Yang et al. found that AOB was the key microbial player in alkaline soil which contributing about 85% of nitrification-related N 2 O, while 78% of nitrification-related N 2 O was contributed by AOA in acidic soil. In addition, the relative contribution of AOB and AOA to N 2 O emissions was also regulated by type and amount of applied synthetic Hink et al. found that high ammonia addition stimulated the production of N 2 O from AOB, but AOA dominance during low ammonium supply. However, Fu et al. illustrated that the relative contribution of AOB to N 2 O emissions in the treatment of no N applied was larger than the treatment of ammonium-N addition in both acidic (pH = 5.5) and alkaline (pH = 7.9) soils and treatment of urea-N addition in alkaline soil. Numerous studies have investigated the relative contribution and influence factors of AOA and AOB to N 2 O emission, but no consensus exists regarding the mechanisms for explaining the diversities because of complex mechanism of abundance change and N 2 O yields of AOA and AOB accompanied by spatial and temporal heterogeneity of environmental conditions and soil properties. Owing to the considerable N 2 O emission source caused by the heavy N fertilizer application on farmland in purple soil in mountain area of the Upper Yangtze River watershed , , it is imperative to put forward specific measures to alleviate N 2 O emission for achieving the strategic goal of “carbon reduction” in this region. Therefore, we conducted a microcosm incubation experiment with two contrasting purple soils, using newly developed inhibitor 1-octyne and molecular biology method to assess the factors affecting N 2 O productions, yield and gene abundance associated with AOA and AOB in different soils. The objective is to acquire a more profound understanding of the mechanism of ammonia oxidation process and promote the development of low-emission technology in agriculture. 

Soil sampling In Sep 2020, two test surface soil samples were collected from a long-term fertilization experiment plots (5 m × 1.5 m, triplicate plots of each test soil) at Yanting Agro-Ecological Station of Purple Soil, Chinese Academy of Sciences (N 31°16′, E 105°28′ ), located in the central Sichuan Basin, upper Yangtze River, China. The average temperature was 17.3 °C and the annual mean precipitation was 863 mm of which approximately 70% occurs from May to September at this site. The cropping system is summer maize-winter wheat rotation there and N–P 2 O 5 –K 2 O was applied at 150–90–36 kg ha −1 for maize and 130–90–36 kg ha −1 for wheat, respectively. Two test soils including a neutral (pH = 6.75 and named as SX below) and an alkaline (pH = 8.35 and named as PL below) were formulated from the similar parental bedrock of purplish sandstone with different weathering degree and it is less than 50 years since the soils formation , . They are the predominant soil types in hilly areas of the Upper Yangtze River watershed where was an important grain-producing area in southwest China and feeding more than 10% of the Chinese population. Two test surface soils (0–15 cm) were collected using a soil auger and triplicate cores, which were along the longitudinal center line of the plots with a two meters interval, were pooled and homogenized for each plot. All soils were sieved through a 2 mm sieve after removing plant roots and debris and then divided into two parts. One part was used to measure soil water content and basic physical and chemical properties, the remained part of the soil was stored at 4 °C until the incubation experiment. Some basic physical and chemical properties of the two soils were shown in Table . Microcosm experiment To distinguish the relative contribution of different ammonia oxidation processes to N 2 O emission, we employed acetylene and 1-octyne as selective inhibitors to block the interaction of the ammonia oxidizers (i.e. AOB and AOA). The incubation experiments were conducted in 250-ml serum bottles with a butyl rubber stopper containing 18 g of soils (dry weight). The fresh soils were pre-incubated at 25 °C for 7 days to stabilize soil microbial activities in 250-ml serum bottles. After pre-incubation, soil was adjusted to 60% WFPS following amendment with sterilized water only (control, no N addition) or inorganic nitrogen solution (100 mg NH 4 Cl-N or KNO 3 -N g −1 soil dw ). Then, the bottles were covered with lids and some pumped out air have replaced with pre-prepared ammonia oxidizer inhibitors acetylene (Ace, 0.01%, v/v) or 1-octyne (Oct, 5 μM aqueous, following Taylor et al. ). In total, nine treatments with three replicates were conducted as follows: N-free (no N and no inhibitors) N-free + Ace (no N and 0.01% acetylene) N-free + Oct (no N and 5 μM 1-octyne) NH 4 + (100 mg g −1 NH 4 Cl-N and no inhibitors) NH 4 + + Ace (100 mg g −1 NH 4 Cl-N and 0.01% acetylene) NH 4 + + Oct (100 mg g −1 NH 4 Cl-N and 5 μM 1-octyne) NO 3 − (100 mg g −1 KNO 3 -N and no inhibitors) NO 3 − + Ace (100 mg g −1 KNO 3 -N and 0.01% acetylene) NO 3 − + Oct (100 mg g −1 KNO 3 -N and 5 μM 1-octyne) All treatments conducted at 25 °C for 21 days. During this period, oxic conditions were maintained by aerated every 2 days and re-establishing the inhibition environment by addition acetylene (0.01% v/v) and 1-octyne (5 μM aqueous). The N 2 O emission from soils without inhibitors was contributed by nitrification (including contributions of AOB and AOA), denitrification and abiotic processes. Acetylene could inhibit ammonia oxidation both of AOA and AOB, so the N 2 O emission from AOA plus AOB was calculated by subtracting N 2 O emission in the “NH 4 + + Ace” (“NO 3 − + Ace” or “Ace”) treatment from values measured in the “NH 4 + ” (“NO 3 − ” or “N-free”) treatment. Because of 1-Octyne specifically inhibits AOB growth only, the N 2 O emission from AOA was calculated by subtracting N 2 O emission in the “NH 4 + + Ace” (“NO 3 − + Ace” or “Ace”) treatment from values measured in the “NH 4 + + Oct” (“NO 3 − + Oct” or “Oct”) treatment. N 2 O emission from AOB was calculated by subtracting AOA from values of AOA plus AOB. N 2 O and soil sampling The 20 ml headspace gas samples were collected at 0, 1, 2, 3, 5, 7, 11, 14, 18 and 21 days by syringe (with a triple valve) during the whole incubation and N 2 O emission concentrate was determined with a gas chromatograph which equipped with a 63Ni electron capture detector for N 2 O concentrations (Agilent 7890B, USA). The gas measurement was calibrated using a known concentration of mixed gas (440 ppb N 2 O in mixed standard gas). The incubated soils were destructively sampled at 0, 7, 14 and 21 days. Soil samples were divided into two parts, a part stored at 4 °C for measuring soil contents of NH 4 + -N and NO 3 − -N; and another portion was kept at − 80 °C for DNA extraction. Soil contents of NH 4 + -N and NO 3 – N were extracted by 2 M KCl solution (soil: solution = 1:5 w/v), and then were filtered through 0.45 m filter membrane after shaking for 1 h. Extracts were analyzed by a continuous flow analyzer (Auto Analyzer 3, SEAL Analytical, Germany). DNA extraction and quantitive PCR (qPCR) analyses The soil samples collected before incubation and after incubating 21 days were used to extract DNA because the fluxes of N 2 O emission have stabilized after incubating 21 days. According to the manufacturer's instructions, 0.5 g wet soil was used to extract DNA by using DNeasy PowerSoil DNA Isolation kit (QIAGEN, Germany). The length of extracted DNA was checked by 1% agarose gel electrophoresis and the concentration and qualification were measured by NanoDrop ND-1000 spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA). And the ratio of A 260/280 and A 260/230 were in the range of 1.5–1.9 and 0.7–1.0, respectively. Purified DNA concentrations varied from 10.8 to 36.8 ng/μL. Soil DNA samples were stored at − 80 °C for quantitative PCR of amoA genes analyses. AOB and AOA amoA genes of all treatments with three biological replicates were amplified and quantified using ABI 9700 real-time quantitative fluorescence PCR (Applied Biosystem, America); The sequence of AOB amoA amplified primers were amoA -1F (5'-GGGGTTTCTACTGGTGGT-3')/ amoA -2R (5'-CCCCTCKGSAAAGCCTTCTTC-3') while ArchamoAF (5'-TAATGGTCTGGCTTAGACG-3')/ArchamoAR (5'-GCGGCCATCCATCTGTATGT-3') were used to amplify and quantify AOA amoA gene. Each 20-μl reaction system contained 10 μl GoTaq qPCR Master Mix (SYBR Premix Ex TaqTM), 0.5 μl of each primer (10 mM), 2 μl tenfold diluted DNA template and 7 μl sterilised pure water. The amplified reaction conditions of AOB and AOA were as follows: initial denaturation at 95 °C for 3 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 34 s and extension at 72 °C for 32 s, and extension at 72 °C for 5 min for data collection. The standard curves which were used to quantify the abundance of AOA and AOB amoA gene were obtained by ten-fold serial dilution of AOA and AOB plasmid DNA with known concentration (five points form 10 –3 ~ 10 –7 in this study). The melting curve analyses which were used to check the specificity of amplification products showed that amplification efficiencies of AOB and AOA amoA ranged from 92 to 98%, along with correlation coefficient (R 2 ) of standard curves was 0.994 and 0.998 for AOB and AOA amoA genes, respectively. Calculations and statistical analysis N 2 O fluxes were calculated using Eq. ( ): 1 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$${} = }_{0} }}{{{} + {}_{0} }}  }}{{{}_{0} }}  }}{{}}  }}}{{{}}}  24  {}$$ F = T 0 T + T 0 × V V 0 × M m × dc dt × 24 × K where F (ng N g −1 d −1 ) is the N 2 O emission rate; T 0 (237 K) is the temperature at standard atmospheric state; T (°C) is the air temperature within the serum bottles; V (L) is the volume of the headspace; V 0 (22.41 × 10 –3 m 3 ) is the molar volume at standard atmospheric state; M (28 g mol −1 ) is the molecular weight of N in N 2 O molecular; m (18 g) is the weight of oven-dried soil in the serum bottles; dc/dt is the change of N 2 O concentration (c) per unit interval (t); 24 is the number of hours in a day and K is the dimensional conversion coefficient. The relative contributions of AOA and AOB to nitrification-driven N 2 O emission were calculated using Eqs. ( ) ~ ( ): 2 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$${}_{{2}} {}( {{}} )( \% ) = }_{2} {}}}{{{}_{2} {}}}$$ N 2 O AOA % = N 2 O emission by AOA total of N 2 O emission 3 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$${}_{{2}} {}( {{}} )( \% ) = }_{2} {}}}{{{}_{2} {}}}$$ N 2 O AOB % = N 2 O emission by AOB total of N 2 O emission 4 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$${}_{{2}} {}( {{}} )( \% ) = }_{2} {}}}{{{}_{2} {}}}$$ N 2 O Others % = N 2 O emission by Others total of N 2 O emission where “total of N 2 O emission” was the cumulative N 2 O production of treatment without inhibitors addition after 21 days of incubation; “N 2 O emission by Others” was the cumulative N 2 O production of other processes after 21 days of incubation. N 2 O yield for AOA, AOB and others was calculated using Eq. ( ): 5 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$${}_{{2}} {}\,\,{}_{{( {} )}} = }_{2} {}_{{( {} )}} }}{{{}_{3}^{ - } {}}}$$ N 2 O yield x = N 2 O emission x NO 3 - produced where x is AOA, AOB and others, “N 2 O emission (x) ” and “NO 3 − produced” are the cumulative N 2 O and nitrate over the whole 21 days for incubation, the unit of “N 2 O emission (x) ” and “NO 3 – N produced” is mg N kg −1 . The statistical analysis was performed using SPSS 24.0 software (SPSS Inc., USA) and data in this study expressed as a mean ± standard error. Differences among different treatments were tested by ANOVA after Tukey's multiple range test by the least significant difference at the 5% level. Independent-samples t-test was performed for the statistical analysis of N 2 O yield between two soils. Pearson’s correlation between cumulative N 2 O emissions and AOB or AOA amoA gene copies were calculated. Figures were made using Origin 9.4 software (Origin Lab Corporation, Northampton, USA). 

In Sep 2020, two test surface soil samples were collected from a long-term fertilization experiment plots (5 m × 1.5 m, triplicate plots of each test soil) at Yanting Agro-Ecological Station of Purple Soil, Chinese Academy of Sciences (N 31°16′, E 105°28′ ), located in the central Sichuan Basin, upper Yangtze River, China. The average temperature was 17.3 °C and the annual mean precipitation was 863 mm of which approximately 70% occurs from May to September at this site. The cropping system is summer maize-winter wheat rotation there and N–P 2 O 5 –K 2 O was applied at 150–90–36 kg ha −1 for maize and 130–90–36 kg ha −1 for wheat, respectively. Two test soils including a neutral (pH = 6.75 and named as SX below) and an alkaline (pH = 8.35 and named as PL below) were formulated from the similar parental bedrock of purplish sandstone with different weathering degree and it is less than 50 years since the soils formation , . They are the predominant soil types in hilly areas of the Upper Yangtze River watershed where was an important grain-producing area in southwest China and feeding more than 10% of the Chinese population. Two test surface soils (0–15 cm) were collected using a soil auger and triplicate cores, which were along the longitudinal center line of the plots with a two meters interval, were pooled and homogenized for each plot. All soils were sieved through a 2 mm sieve after removing plant roots and debris and then divided into two parts. One part was used to measure soil water content and basic physical and chemical properties, the remained part of the soil was stored at 4 °C until the incubation experiment. Some basic physical and chemical properties of the two soils were shown in Table . 

To distinguish the relative contribution of different ammonia oxidation processes to N 2 O emission, we employed acetylene and 1-octyne as selective inhibitors to block the interaction of the ammonia oxidizers (i.e. AOB and AOA). The incubation experiments were conducted in 250-ml serum bottles with a butyl rubber stopper containing 18 g of soils (dry weight). The fresh soils were pre-incubated at 25 °C for 7 days to stabilize soil microbial activities in 250-ml serum bottles. After pre-incubation, soil was adjusted to 60% WFPS following amendment with sterilized water only (control, no N addition) or inorganic nitrogen solution (100 mg NH 4 Cl-N or KNO 3 -N g −1 soil dw ). Then, the bottles were covered with lids and some pumped out air have replaced with pre-prepared ammonia oxidizer inhibitors acetylene (Ace, 0.01%, v/v) or 1-octyne (Oct, 5 μM aqueous, following Taylor et al. ). In total, nine treatments with three replicates were conducted as follows: N-free (no N and no inhibitors) N-free + Ace (no N and 0.01% acetylene) N-free + Oct (no N and 5 μM 1-octyne) NH 4 + (100 mg g −1 NH 4 Cl-N and no inhibitors) NH 4 + + Ace (100 mg g −1 NH 4 Cl-N and 0.01% acetylene) NH 4 + + Oct (100 mg g −1 NH 4 Cl-N and 5 μM 1-octyne) NO 3 − (100 mg g −1 KNO 3 -N and no inhibitors) NO 3 − + Ace (100 mg g −1 KNO 3 -N and 0.01% acetylene) NO 3 − + Oct (100 mg g −1 KNO 3 -N and 5 μM 1-octyne) All treatments conducted at 25 °C for 21 days. During this period, oxic conditions were maintained by aerated every 2 days and re-establishing the inhibition environment by addition acetylene (0.01% v/v) and 1-octyne (5 μM aqueous). The N 2 O emission from soils without inhibitors was contributed by nitrification (including contributions of AOB and AOA), denitrification and abiotic processes. Acetylene could inhibit ammonia oxidation both of AOA and AOB, so the N 2 O emission from AOA plus AOB was calculated by subtracting N 2 O emission in the “NH 4 + + Ace” (“NO 3 − + Ace” or “Ace”) treatment from values measured in the “NH 4 + ” (“NO 3 − ” or “N-free”) treatment. Because of 1-Octyne specifically inhibits AOB growth only, the N 2 O emission from AOA was calculated by subtracting N 2 O emission in the “NH 4 + + Ace” (“NO 3 − + Ace” or “Ace”) treatment from values measured in the “NH 4 + + Oct” (“NO 3 − + Oct” or “Oct”) treatment. N 2 O emission from AOB was calculated by subtracting AOA from values of AOA plus AOB. 

2 O and soil sampling The 20 ml headspace gas samples were collected at 0, 1, 2, 3, 5, 7, 11, 14, 18 and 21 days by syringe (with a triple valve) during the whole incubation and N 2 O emission concentrate was determined with a gas chromatograph which equipped with a 63Ni electron capture detector for N 2 O concentrations (Agilent 7890B, USA). The gas measurement was calibrated using a known concentration of mixed gas (440 ppb N 2 O in mixed standard gas). The incubated soils were destructively sampled at 0, 7, 14 and 21 days. Soil samples were divided into two parts, a part stored at 4 °C for measuring soil contents of NH 4 + -N and NO 3 − -N; and another portion was kept at − 80 °C for DNA extraction. Soil contents of NH 4 + -N and NO 3 – N were extracted by 2 M KCl solution (soil: solution = 1:5 w/v), and then were filtered through 0.45 m filter membrane after shaking for 1 h. Extracts were analyzed by a continuous flow analyzer (Auto Analyzer 3, SEAL Analytical, Germany). 

The soil samples collected before incubation and after incubating 21 days were used to extract DNA because the fluxes of N 2 O emission have stabilized after incubating 21 days. According to the manufacturer's instructions, 0.5 g wet soil was used to extract DNA by using DNeasy PowerSoil DNA Isolation kit (QIAGEN, Germany). The length of extracted DNA was checked by 1% agarose gel electrophoresis and the concentration and qualification were measured by NanoDrop ND-1000 spectrophotometer (Nano Drop Technologies, Wilmington, DE, USA). And the ratio of A 260/280 and A 260/230 were in the range of 1.5–1.9 and 0.7–1.0, respectively. Purified DNA concentrations varied from 10.8 to 36.8 ng/μL. Soil DNA samples were stored at − 80 °C for quantitative PCR of amoA genes analyses. AOB and AOA amoA genes of all treatments with three biological replicates were amplified and quantified using ABI 9700 real-time quantitative fluorescence PCR (Applied Biosystem, America); The sequence of AOB amoA amplified primers were amoA -1F (5'-GGGGTTTCTACTGGTGGT-3')/ amoA -2R (5'-CCCCTCKGSAAAGCCTTCTTC-3') while ArchamoAF (5'-TAATGGTCTGGCTTAGACG-3')/ArchamoAR (5'-GCGGCCATCCATCTGTATGT-3') were used to amplify and quantify AOA amoA gene. Each 20-μl reaction system contained 10 μl GoTaq qPCR Master Mix (SYBR Premix Ex TaqTM), 0.5 μl of each primer (10 mM), 2 μl tenfold diluted DNA template and 7 μl sterilised pure water. The amplified reaction conditions of AOB and AOA were as follows: initial denaturation at 95 °C for 3 min, 40 cycles of denaturation at 95 °C for 30 s, annealing at 55 °C for 34 s and extension at 72 °C for 32 s, and extension at 72 °C for 5 min for data collection. The standard curves which were used to quantify the abundance of AOA and AOB amoA gene were obtained by ten-fold serial dilution of AOA and AOB plasmid DNA with known concentration (five points form 10 –3 ~ 10 –7 in this study). The melting curve analyses which were used to check the specificity of amplification products showed that amplification efficiencies of AOB and AOA amoA ranged from 92 to 98%, along with correlation coefficient (R 2 ) of standard curves was 0.994 and 0.998 for AOB and AOA amoA genes, respectively. 

N 2 O fluxes were calculated using Eq. ( ): 1 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$${} = }_{0} }}{{{} + {}_{0} }}  }}{{{}_{0} }}  }}{{}}  }}}{{{}}}  24  {}$$ F = T 0 T + T 0 × V V 0 × M m × dc dt × 24 × K where F (ng N g −1 d −1 ) is the N 2 O emission rate; T 0 (237 K) is the temperature at standard atmospheric state; T (°C) is the air temperature within the serum bottles; V (L) is the volume of the headspace; V 0 (22.41 × 10 –3 m 3 ) is the molar volume at standard atmospheric state; M (28 g mol −1 ) is the molecular weight of N in N 2 O molecular; m (18 g) is the weight of oven-dried soil in the serum bottles; dc/dt is the change of N 2 O concentration (c) per unit interval (t); 24 is the number of hours in a day and K is the dimensional conversion coefficient. The relative contributions of AOA and AOB to nitrification-driven N 2 O emission were calculated using Eqs. ( ) ~ ( ): 2 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$${}_{{2}} {}( {{}} )( \% ) = }_{2} {}}}{{{}_{2} {}}}$$ N 2 O AOA % = N 2 O emission by AOA total of N 2 O emission 3 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$${}_{{2}} {}( {{}} )( \% ) = }_{2} {}}}{{{}_{2} {}}}$$ N 2 O AOB % = N 2 O emission by AOB total of N 2 O emission 4 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$${}_{{2}} {}( {{}} )( \% ) = }_{2} {}}}{{{}_{2} {}}}$$ N 2 O Others % = N 2 O emission by Others total of N 2 O emission where “total of N 2 O emission” was the cumulative N 2 O production of treatment without inhibitors addition after 21 days of incubation; “N 2 O emission by Others” was the cumulative N 2 O production of other processes after 21 days of incubation. N 2 O yield for AOA, AOB and others was calculated using Eq. ( ): 5 [12pt]{minimal}
				
				 
				 
				 
				
				
				
				
				$${}_{{2}} {}\,\,{}_{{( {} )}} = }_{2} {}_{{( {} )}} }}{{{}_{3}^{ - } {}}}$$ N 2 O yield x = N 2 O emission x NO 3 - produced where x is AOA, AOB and others, “N 2 O emission (x) ” and “NO 3 − produced” are the cumulative N 2 O and nitrate over the whole 21 days for incubation, the unit of “N 2 O emission (x) ” and “NO 3 – N produced” is mg N kg −1 . The statistical analysis was performed using SPSS 24.0 software (SPSS Inc., USA) and data in this study expressed as a mean ± standard error. Differences among different treatments were tested by ANOVA after Tukey's multiple range test by the least significant difference at the 5% level. Independent-samples t-test was performed for the statistical analysis of N 2 O yield between two soils. Pearson’s correlation between cumulative N 2 O emissions and AOB or AOA amoA gene copies were calculated. Figures were made using Origin 9.4 software (Origin Lab Corporation, Northampton, USA). 

Dynamics of soil mineral N concentration The exchangeable NH 4 + -N concentration of the “NH 4 + ” treatment decreased rapidly from a value of 104 mg/kg to a value of 41 mg/kg and 107 mg/kg to 31 mg/kg in SX and PL soil at the first week, respectively. And the rate of decrease became slow in the following two weeks (Fig. a 2 , b 2 ). Contrasting with the “NH 4 + ” treatment, 1-octyne inhibited the decrease of exchangeable NH 4 + –N concentration effectively which showed a slower decrease rate in both of the two soils. As for “NH 4 + + Ace” treatments, the conversion of NH 4 + was completely inhibited, and no obvious decreasing trend was detected. Treatments without NH 4 + addition maintained a low level of exchangeable NH 4 + -N concentration throughout the whole incubation, and there was no significant difference in these treatments whether inhibitors were applied or not ( P > 0.05; Fig. a 1 , a 3 , b 1 , b 3 ). The exchangeable NO 3 – N concentrations correspondingly increased by the decreased of exchangeable NH 4 + -N concentrations in the “NH 4 + ” treatments (Fig. c 2 , d 2 ). As expected, 1-octyne present partly inhibited exchangeable NO 3 – N formation in the “NH 4 + + Oct” treatments and acetylene almost completely inhibited the transformation of NH 4 + -N to NO 3 – N in the “NH 4 + + Ace” treatments in the two soils. As for the treatments without NH 4 + addition, there is no obvious difference regardless of the inhibitors were applied or not ( P > 0.05; Fig. c 1 , c 3 , d 1 , d 3 ). N 2 O emission fluxes and accumulative N 2 O emissions The change trends of N 2 O fluxes varied with soil type. The treatments which addition NH 4 + alone show a distinct N 2 O peak (7 and 33 ng N g −1 d −1 for SX and PL experiment soil, respectively) at the first day of incubation, and quickly decrease at the following days (Fig. a 2 , b 2 ). Acetylene addition has a significant effect in decreasing N 2 O production in the “NH 4 + + Ace” treatment of the two soils throughout the whole incubation, indicating that AOA plus AOB contribute more N 2 O emissions than the abiotic and denitrification processes at aerobic and 60% WFPS experiment conditions. Relative to the acetylene addition, 1-octyne has a similar but slighter inhibition to N 2 O emissions in the “NH 4 + + Oct” of the two soils, showing that different nitrification inhibitors had the selective inhibitory effects in alleviating N 2 O emissions as expected (Fig. a 2 , b 2 ). Except for the NH 4 + addition treatments, the results also reveal that treatments which with or without NO 3 – N show no significant difference in N 2 O emissions no matter the inhibitors present or not during the incubation period of 21 days ( P > 0.05). And there is no emission peaks in these treatments without NH 4 + addition and the fluxes show a periodic fluctuation until the end of incubation (Fig. a 1 , a 3 , b 1 , b 3 ). The accumulated N 2 O emissions varied with the soil type, types of nitrogen fertilizer and inhibitors (Fig. c 1 ~ c 3 , d 1 ~ d 3 ). In the “NH 4 + ” treatments, N 2 O accumulated emission which significantly greater than the rest treatments, reached 14 and 45 μg N kg −1 dry soil for SX and PL experiment soil at the end of incubation, respectively ( P < 0.05; Table ). When NH 4 + addition was employed with 1-octyne, N 2 O accumulated emissions were dramatically decreased by 24.1% and 72.0% for SX and PL experiment soil, respectively, and the more intense inhibition by the acetylene addition which was reduced by 80.3% and 92.4% relative to the “NH 4 + ” treatments. As for the NO 3 − addition and no nitrogen fertilizer addition treatments, with or without an inhibitor, there was no significant difference for the two test soils at the end of incubation ( P > 0.05; Table ). The yields and relative contributions to N 2 O emission of AOB and AOA The N 2 O yield (%) was defined as the rate of N 2 O production per unit nitrate content in soil over the whole 21 days for incubation which calculated by Eq. ( ). The results showed that the yield of N 2 O in different ammonia oxidation processes changed with the soil type under the condition of adding NH 4 + (Fig. ). The N 2 O yield of AOB in PL soil was 0.22% which was significantly higher than that of SX (0.03%) ( P < 0.01), and similar significant differences were found in the N 2 O yields induced by AOA ( P < 0.05). However, for other processes (abiotic-induced or denitrification-induced), the N 2 O yield of SX soil was significantly higher than that of the PL soil ( P < 0.05). Under the same soil conditions with NH 4 + addition, the yield of N 2 O changed with different induction processes: in PL soils, the N 2 O yield induced by AOB was significantly higher than that induced by AOA and the other processes ( P < 0.01); But in SX soils, the N 2 O yield induced by other processes was significantly higher than that induced by AOA and AOB ( P < 0.01). The relative contribution of AOA and AOB to N 2 O production varied significantly from the soil types with NH 4 + amendment in the both of soils (Fig. ). The fractions of N 2 O accumulate production of octyne-sensitive (AOB) were much higher than the octyne-resistent (AOA) in the PL soils. And for SX soil, the fraction of AOB production was slightly higher than the AOA and the others. In details, the fractions of N 2 O emission were 36.1%, 33.2% and 30.7% for AOB, AOA and others process in SX soil, respectively. The contributions of relevant AOB, AOA and others are 70.8%, 21.4% and 8.9% in PL soil, respectively. Abundance of AOA and AOB amoA genes At the start of incubation, the AOB amoA genes abundance were 1.65 × 10 5 and 1.62 × 10 6 copies g −1 dry soil in SX and PL soil, respectively (Fig. a, b) and different treatments have the equal abundance at the initiate of incubation. The NH 4 + amendment significantly stimulated the increase of the AOB amoA genes, reaching at 3.38 × 10 5 and 3.58 × 10 6 copies g −1 in SX and PL soil at the day 21 of incubation, respectively ( P < 0.01; Fig. a, b). There is no obvious difference of AOB amoA genes abundance among the other treatments throughout the incubation regardless of nitrogenous fertilizer and inhibitors were applied or not in both of the two soils. As for AOA amoA genes, the AOA amoA genes abundance was 1.23 × 10 7 copies g −1 and 8.35 × 10 6 copies g −1 dry soil in SX and PL soil at the initiate of incubation, respectively. When amendment with ammonia and 1-octyne, the abundance of AOA amoA genes increased observably than the other treatments in the two soils ( P < 0.01; Fig. c, d). Presence of 1-octyne which was selected as inhibitor of AOB did not show an effective suppression to growth of AOB amoA genes, on the contrary, acted as a positive stimulate to the abundance of AOA amoA genes in the two test soils. Treatments with water or NO 3 - amendment which were applied with inhibitors or not did not change AOA amoA genes abundance significantly during the whole incubation ( P > 0.05). 

The exchangeable NH 4 + -N concentration of the “NH 4 + ” treatment decreased rapidly from a value of 104 mg/kg to a value of 41 mg/kg and 107 mg/kg to 31 mg/kg in SX and PL soil at the first week, respectively. And the rate of decrease became slow in the following two weeks (Fig. a 2 , b 2 ). Contrasting with the “NH 4 + ” treatment, 1-octyne inhibited the decrease of exchangeable NH 4 + –N concentration effectively which showed a slower decrease rate in both of the two soils. As for “NH 4 + + Ace” treatments, the conversion of NH 4 + was completely inhibited, and no obvious decreasing trend was detected. Treatments without NH 4 + addition maintained a low level of exchangeable NH 4 + -N concentration throughout the whole incubation, and there was no significant difference in these treatments whether inhibitors were applied or not ( P > 0.05; Fig. a 1 , a 3 , b 1 , b 3 ). The exchangeable NO 3 – N concentrations correspondingly increased by the decreased of exchangeable NH 4 + -N concentrations in the “NH 4 + ” treatments (Fig. c 2 , d 2 ). As expected, 1-octyne present partly inhibited exchangeable NO 3 – N formation in the “NH 4 + + Oct” treatments and acetylene almost completely inhibited the transformation of NH 4 + -N to NO 3 – N in the “NH 4 + + Ace” treatments in the two soils. As for the treatments without NH 4 + addition, there is no obvious difference regardless of the inhibitors were applied or not ( P > 0.05; Fig. c 1 , c 3 , d 1 , d 3 ). 

2 O emission fluxes and accumulative N 2 O emissions The change trends of N 2 O fluxes varied with soil type. The treatments which addition NH 4 + alone show a distinct N 2 O peak (7 and 33 ng N g −1 d −1 for SX and PL experiment soil, respectively) at the first day of incubation, and quickly decrease at the following days (Fig. a 2 , b 2 ). Acetylene addition has a significant effect in decreasing N 2 O production in the “NH 4 + + Ace” treatment of the two soils throughout the whole incubation, indicating that AOA plus AOB contribute more N 2 O emissions than the abiotic and denitrification processes at aerobic and 60% WFPS experiment conditions. Relative to the acetylene addition, 1-octyne has a similar but slighter inhibition to N 2 O emissions in the “NH 4 + + Oct” of the two soils, showing that different nitrification inhibitors had the selective inhibitory effects in alleviating N 2 O emissions as expected (Fig. a 2 , b 2 ). Except for the NH 4 + addition treatments, the results also reveal that treatments which with or without NO 3 – N show no significant difference in N 2 O emissions no matter the inhibitors present or not during the incubation period of 21 days ( P > 0.05). And there is no emission peaks in these treatments without NH 4 + addition and the fluxes show a periodic fluctuation until the end of incubation (Fig. a 1 , a 3 , b 1 , b 3 ). The accumulated N 2 O emissions varied with the soil type, types of nitrogen fertilizer and inhibitors (Fig. c 1 ~ c 3 , d 1 ~ d 3 ). In the “NH 4 + ” treatments, N 2 O accumulated emission which significantly greater than the rest treatments, reached 14 and 45 μg N kg −1 dry soil for SX and PL experiment soil at the end of incubation, respectively ( P < 0.05; Table ). When NH 4 + addition was employed with 1-octyne, N 2 O accumulated emissions were dramatically decreased by 24.1% and 72.0% for SX and PL experiment soil, respectively, and the more intense inhibition by the acetylene addition which was reduced by 80.3% and 92.4% relative to the “NH 4 + ” treatments. As for the NO 3 − addition and no nitrogen fertilizer addition treatments, with or without an inhibitor, there was no significant difference for the two test soils at the end of incubation ( P > 0.05; Table ). 

2 O emission of AOB and AOA The N 2 O yield (%) was defined as the rate of N 2 O production per unit nitrate content in soil over the whole 21 days for incubation which calculated by Eq. ( ). The results showed that the yield of N 2 O in different ammonia oxidation processes changed with the soil type under the condition of adding NH 4 + (Fig. ). The N 2 O yield of AOB in PL soil was 0.22% which was significantly higher than that of SX (0.03%) ( P < 0.01), and similar significant differences were found in the N 2 O yields induced by AOA ( P < 0.05). However, for other processes (abiotic-induced or denitrification-induced), the N 2 O yield of SX soil was significantly higher than that of the PL soil ( P < 0.05). Under the same soil conditions with NH 4 + addition, the yield of N 2 O changed with different induction processes: in PL soils, the N 2 O yield induced by AOB was significantly higher than that induced by AOA and the other processes ( P < 0.01); But in SX soils, the N 2 O yield induced by other processes was significantly higher than that induced by AOA and AOB ( P < 0.01). The relative contribution of AOA and AOB to N 2 O production varied significantly from the soil types with NH 4 + amendment in the both of soils (Fig. ). The fractions of N 2 O accumulate production of octyne-sensitive (AOB) were much higher than the octyne-resistent (AOA) in the PL soils. And for SX soil, the fraction of AOB production was slightly higher than the AOA and the others. In details, the fractions of N 2 O emission were 36.1%, 33.2% and 30.7% for AOB, AOA and others process in SX soil, respectively. The contributions of relevant AOB, AOA and others are 70.8%, 21.4% and 8.9% in PL soil, respectively. 

amoA genes At the start of incubation, the AOB amoA genes abundance were 1.65 × 10 5 and 1.62 × 10 6 copies g −1 dry soil in SX and PL soil, respectively (Fig. a, b) and different treatments have the equal abundance at the initiate of incubation. The NH 4 + amendment significantly stimulated the increase of the AOB amoA genes, reaching at 3.38 × 10 5 and 3.58 × 10 6 copies g −1 in SX and PL soil at the day 21 of incubation, respectively ( P < 0.01; Fig. a, b). There is no obvious difference of AOB amoA genes abundance among the other treatments throughout the incubation regardless of nitrogenous fertilizer and inhibitors were applied or not in both of the two soils. As for AOA amoA genes, the AOA amoA genes abundance was 1.23 × 10 7 copies g −1 and 8.35 × 10 6 copies g −1 dry soil in SX and PL soil at the initiate of incubation, respectively. When amendment with ammonia and 1-octyne, the abundance of AOA amoA genes increased observably than the other treatments in the two soils ( P < 0.01; Fig. c, d). Presence of 1-octyne which was selected as inhibitor of AOB did not show an effective suppression to growth of AOB amoA genes, on the contrary, acted as a positive stimulate to the abundance of AOA amoA genes in the two test soils. Treatments with water or NO 3 - amendment which were applied with inhibitors or not did not change AOA amoA genes abundance significantly during the whole incubation ( P > 0.05). 

The abiotic processes and biotic processes including nitrification, denitrification are considered to be primary processes that produce N 2 O in arable soils from numerous reports , , . This is first to distinguish the relative contribution of ammonia-oxidizing and N 2 O yield resulting from AOA and AOB in different pH of purple soil, southwest of China, using 1-octyne to specifically inhibit AOB. In this study, we verified that N 2 O emissions from the two tested agricultural soil were driven by nitrification and non-negligible others processes based on the following points: (1) NH 4 + -N rapidly transformed into NO 3 – N in the NH 4 + -N addition treatments and resulted in much higher N 2 O production than NO 3 – N addition treatments in which the NO 3 – N remained stable in during the incubation (Fig. ); (2) both of test soils were performed under aerobic and 60% WFPS conditions which are optimal N 2 O production via ammonia oxidation according to previous studies6. And N 2 O emissions from heterotrophic denitrifiers were negligible ; (3) Nevertheless, N 2 O accumulated slowly but non-negligible in acetylene treated microcosms when NH 3 oxidizer including AOA and AOB growth and activity were inhibited indicating that others processes–induced (heterotrophic nitrification and abiotic processes, etc.) N 2 O emission also hold an assignable contribution, especially in the neutral soils where others processes contribute 30.7% to the gross N 2 O emission in the incubation (Table and Fig. ). Numerous studies have confirmed that N 2 O emissions in arable soils were regulated by multiple environmental factors such as soil moisture, soil pH and management factors including N application, plowing and other soil management treatments – . Soil pH is a key parameter that controls the abundance change of AOA and AOB which influence N 2 O production . In our study, AOB contributed more accumulated N 2 O emissions than AOA in alkaline soil at the condition of NH 4 + -N addition which indicated AOB dominated the nitration process in alkaline soil, while there is no significant difference observe in neutral soil where both AOA and AOB contributed nearly a third of gross N 2 O emissions (Fig. and Table ). We found that AOB played a more important role than AOA in ammonia oxidation in a high-pH soil, supporting previous reports , , , . One possible explanation is that soils with higher pH accelerate the rate transformation of NH 4 + –N to availability NH 3 which affected population and activity of ammonia oxidizers , while bacterial growth would possibly have been impeded in low soil pH . Clearly, tested soils with NH 4 + stimulated N 2 O production both by AOA and AOB to varying degrees with respect to the control (Fig. and Table ). And these results were also confirmed by the markedly increased amoA gene abundance of AOA and AOB at the end of incubation (Fig. ). At present, the best explanation for different growth and activities of AOA and AOB in soils is a significantly different affinity for NH 3 – . For example, soils with high NH 4 + -N concentrate is conducive to the growth of AOB , , whereas AOA activity which is favored by low NH 4 + –N soil may be restricted or not affected in this condition of high NH 4 + –N , . In our study, the AOA amoA abundance in the control of two test soil increased but not significantly, indicating that AOA could grow using organic N in low ammonia fertility status soils , . Unexpectedly, we found that AOA also grew in this condition where high NH 4 + -N concentration and AOB was inhibited by 1-octyne because the significant increase of AOA amoA abundance proved this point which was different from several previous studies (Fig. c, d) , . And this result was in line with a recent report that there was a direct competition between AOA and AOB for NH 3 under high NH 4 + -N concentration when AOB was inhibited by 1-octyne, whereas AOA growth continued and AOB growth ceased when NH 4 + -N became undetectable , . When NH 4 + –N supplied as fertilizer, AOB dominated NH 3 oxidation process and N 2 O yield ranged from 0.03 to 0.22%, which varied with soil type (Fig. ). In neutral soil, the N 2 O yield induced by AOB (~ 0.03%) fell within the range values (0.02–0.09%) which were derived from soil-slurries with NH 4 + amended experiment . While in alkaline soil, the higher N 2 O yield of AOB (0.22%) which similar to the results of the pure culture of soil Nitrosospira lineage which predominate in soil AOB communities . As for AOA, when addition of NH 4 + –N, N 2 O yield are 0.02% ~ 0.04% in the two test soils, and these yields are similar to values of 0.035% reported previously by Hink et al. and slightly lower than those of cultivated soil AOA (0.08% ~ 0.23%) , , . These results suggested that AOB might have a higher N 2 O yield than AOA in the process of producing N 2 O in both alkaline and neutral soils. The higher N 2 O yield for AOB than AOA could be explained by the current acknowledgment: there were two enzymatic mechanisms for N 2 O production in AOB (i.e. nitrifier denitrification and incomplete NH 2 OH oxidation), while AOA appeared to lack a known NO reductase which was a key enzyme of reducing NO to N 2 O – . Therefore, N 2 O production of AOB was known as a biotic process, whereas inducing by AOA more liked a biotic and abiotic hybrid formation . In addition, the method of nitrification inhibitor addition has limitations on the effectiveness to distinguish relative N 2 O emission of AOA and AOB . 1-octyne is an effective selective inhibitor of AOB activity and amoA abundance in test soils, and we found that AOB holds the leading role to soil N 2 O production in NH 4 + -N addition treatments although AOA amoA abundance showing several times of AOB in both of two soils (Figs. and ). Acetylene acted as a non-selective inhibitor which was used to block the activity of both AOA and AOB biotic ammonia oxidation, while the accumulated N 2 O production by others processes (heterotrophic nitrification and abiotic processes, etc.) contributed a remarkable scale to gross of accumulated N 2 O production (Fig. ). Additional investigations like isotope labeling should be carried in the future to reveal the underlying mechanism of other processes leading to N 2 O. At present, nitrification inhibitors , slow-release fertilizers – , appropriate timing of fertilizer application , and no-till , were considered to be the main strategies to increase fertilizer use efficiency and inhibit N 2 O emission. Our results evaluated the consequences of specialization of ammonia oxidation accompanied by varied N 2 O yield of AOA and AOB which provide a potential strategy for the alleviation of N 2 O emissions in purple soils in hilly areas of upper Yangtze River, China. N 2 O accumulative production and N 2 O yield (especially in AOB) significantly increase with the rising pH of soils under aerobic conditions indicated a reduction in pH could be a potential way to decrease N 2 O emission. In addition, the application of a moderate nitrification inhibitor could both alleviate the N 2 O emission and reduce the risk of nitrate leaching in this area, and this viewpoint was supported by a recent review . In all, measures that prevent ammonia oxidization directly or change specialization causing the increased dominance of NH 3 oxidation by AOA indirectly will decrease the N 2 O production in this area. Meanwhile, crop yield, measures feasibility and cost should be considered with environmental due diligence which was derived from reducing N 2 O emission. 

In conclusion, we explored the relative contribution of ammonia-oxidizing bacteria and archaea to N 2 O emission using a selected inhibitor of AOB in purple soils with different pH values. Results demonstrated that ammonia oxidation was dominated by AOB rather than AOA under the 60% WHC soil moisture and aerobic condition in both neutral and alkaline soils. NH 4 + -N supply significantly increased N 2 O production of AOB, while AOA-related N 2 O production also increased when AOB activity was inhibited in this condition. pH act as a key factor to mediate the abundance change of AOA and AOB, and N 2 O production varied with different soils of pH. These results may help inform the development of N 2 O reduction strategies in the future. 

Supplementary Information 1. Supplementary Information 2. 

