The contributions of ammonia oxidizing bacteria and archaea to nitrification

Scientific Reports volume 12, Article number: 19928 (2022) Cite this article

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Nitrification is believed to be one of the primary processes of N2O emission in the agroecological system, which is controlled by soil microbes and mainly regulated by soil pH, oxygen content and NH4+ availability. Previous studies have proved that the relative contributions of ammonia oxidizing bacteria (AOB) and archaea (AOA) to N2O production were varied with soil pH, however, there is still no consensus on the regulating mechanism of nitrification-derived N2O production by soil pH. In this study, 1-octyne (a selective inhibitor of AOB) and acetylene (an inhibitor of AOB and AOA) were used in a microcosm incubation experiment to differentiate the relative contribution of AOA and AOB to N2O emissions in a neutral (pH = 6.75) and an alkaline (pH = 8.35) soils. We found that the amendment of ammonium (NH4+) observably stimulated the production of both AOA and AOB-related N2O and increased the ammonia monooxygenase (AMO) gene abundances of AOA and AOB in the two test soils. Among which, AOB dominated the process of ammonia oxidation in the alkaline soil, contributing 70.8% of N2O production derived from nitrification. By contrast, the contribution of AOA and AOB accounted for about one-third of nitrification-related N2O in acidic soil, respectively. The results indicated that pH was a key factor to change abundance and activity of AOA and AOB, which led to the differentiation of derivation of N2O production in purple soils. We speculate that both NH4+ content and soil pH mediated specialization of ammonia-oxidizing microorganisms together; and both specialization results and N2O yield led to the different N2O emission characteristics in purple soils. These results may help inform the development of N2O reduction strategies in the future.

N2O is a trace greenhouse gas with 265 times the warming potential of CO2 (on a 100-year scale) in the atmosphere and acts to deplete stratospheric ozone1. The global concentration of N2O in the atmosphere was 328.8 ppb in 2015 with a 21% increase since the industrial revolution2. The sustained emissions at the current rate will result to increase by another 18% until 2030 from current projections indicate3. Fertilized agricultural soil is a hot spot for emissions substantial N2O to atmosphere, accounting for about 60% of global atmospheric N2O emissions4. 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 N2O production from fertilized soil to find optimal measures for regulating N2O emissions for sustainable agriculture.

In agro-ecosystems, N2O is produced by nitrification and denitrification driven by soil microorganisms5,6, and some abiotic processes also contribute a small part of it7. Nitrification is a main N2O production process under a suitable soil oxygen content. Ammonia oxidation (i.e. NH3 being oxidized to NO2−, via the intermediate product NH2OH) 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 N2O8,9. AOB could produce N2O which acts as the intermediate by the incomplete NH2OH oxidation to NO10, or by regulating nitrifier denitrification of reduction NO2− to NO and N2O11. In contrast, N2O 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 N2O by AOA enzymatic catalytic reaction in soils12. 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 N2O emission, though some studies suggested it playing a minor role than AOB13,14.

Nitrification inhibitors have been widely applied in agricultural soils to reduce transformation of NH4+-N to NO3–N, thus improving N use efficiency15. One of the key functional enzymes in both AOA and AOB ammonia oxidizers is ammonia monooxygenase (AMO) which directly catalyzed the process of N2O synthesis. Acetylene (C2H2) 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)16,17. 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 nitrification18,19,20,21.

Substantial studies have found that many factors including soil types and environmental factors determine the abundance, activities and relative contribution to N2O emission of AOA and AOB, especially the soil pH and inorganic nitrogen (N) supply22,23,24. For example, the abundance and activity of AOB increased in ammonium concentration-rich soils, whereas AOA act as not affected or inhibited16,18. In unfertilized or acidic soils, abundance and activity of AOA are much higher than those of AOB25,26. Wang et al.21 reported that nitrogen fertiliser-induced N2O 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.27 found that AOB was the key microbial player in alkaline soil which contributing about 85% of nitrification-related N2O, while 78% of nitrification-related N2O was contributed by AOA in acidic soil. In addition, the relative contribution of AOB and AOA to N2O emissions was also regulated by type and amount of applied synthetic Hink et al.24 found that high ammonia addition stimulated the production of N2O from AOB, but AOA dominance during low ammonium supply. However, Fu et al.28 illustrated that the relative contribution of AOB to N2O 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 N2O emission, but no consensus exists regarding the mechanisms for explaining the diversities because of complex mechanism of abundance change and N2O yields of AOA and AOB accompanied by spatial and temporal heterogeneity of environmental conditions and soil properties. Owing to the considerable N2O emission source caused by the heavy N fertilizer application on farmland in purple soil in mountain area of the Upper Yangtze River watershed29,30, it is imperative to put forward specific measures to alleviate N2O 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 N2O 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.

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–P2O5–K2O 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 formation31,32. 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 1.

To distinguish the relative contribution of different ammonia oxidation processes to N2O 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 NH4Cl-N or KNO3-N g−1 soildw). 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.20). 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)

NH4+ (100 mg g−1 NH4Cl-N and no inhibitors)

NH4+  + Ace (100 mg g−1 NH4Cl-N and 0.01% acetylene)

NH4+  + Oct (100 mg g−1 NH4Cl-N and 5 μM 1-octyne)

NO3− (100 mg g−1 KNO3-N and no inhibitors)

NO3− + Ace (100 mg g−1 KNO3-N and 0.01% acetylene)

NO3− + Oct (100 mg g−1 KNO3-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 N2O 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 N2O emission from AOA plus AOB was calculated by subtracting N2O emission in the "NH4+  + Ace" ("NO3− + Ace" or "Ace") treatment from values measured in the "NH4+" ("NO3− " or "N-free") treatment. Because of 1-Octyne specifically inhibits AOB growth only, the N2O emission from AOA was calculated by subtracting N2O emission in the "NH4+ + Ace" ("NO3− + Ace" or "Ace") treatment from values measured in the "NH4+ + Oct" ("NO3− + Oct" or "Oct") treatment. N2O emission from AOB was calculated by subtracting AOA from values of AOA plus AOB.

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 N2O emission concentrate was determined with a gas chromatograph which equipped with a 63Ni electron capture detector for N2O concentrations (Agilent 7890B, USA). The gas measurement was calibrated using a known concentration of mixed gas (440 ppb N2O 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 NH4+-N and NO3−-N; and another portion was kept at − 80 °C for DNA extraction. Soil contents of NH4+-N and NO3–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 N2O 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 A260/280 and A260/230 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')33 while ArchamoAF (5'-TAATGGTCTGGCTTAGACG-3')/ArchamoAR (5'-GCGGCCATCCATCTGTATGT-3')34 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 (R2) of standard curves was 0.994 and 0.998 for AOB and AOA amoA genes, respectively.

N2O fluxes were calculated using Eq. (1):

where F (ng N g−1 d−1) is the N2O emission rate; T0 (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; V0 (22.41 × 10–3 m3) is the molar volume at standard atmospheric state; M (28 g mol−1) is the molecular weight of N in N2O molecular; m (18 g) is the weight of oven-dried soil in the serum bottles; dc/dt is the change of N2O 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 N2O emission were calculated using Eqs. (2) ~ (4):

where "total of N2O emission" was the cumulative N2O production of treatment without inhibitors addition after 21 days of incubation; "N2O emission by Others" was the cumulative N2O production of other processes after 21 days of incubation.

N2O yield for AOA, AOB and others was calculated using Eq. (5):

where x is AOA, AOB and others, "N2O emission(x)" and "NO3− produced" are the cumulative N2O and nitrate over the whole 21 days for incubation, the unit of "N2O emission(x)" and "NO3–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 N2O yield between two soils. Pearson's correlation between cumulative N2O emissions and AOB or AOA amoA gene copies were calculated. Figures were made using Origin 9.4 software (Origin Lab Corporation, Northampton, USA).

The exchangeable NH4+-N concentration of the "NH4+" 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. 1a2, b2). Contrasting with the "NH4+" treatment, 1-octyne inhibited the decrease of exchangeable NH4+–N concentration effectively which showed a slower decrease rate in both of the two soils. As for "NH4+  + Ace" treatments, the conversion of NH4+ was completely inhibited, and no obvious decreasing trend was detected. Treatments without NH4+ addition maintained a low level of exchangeable NH4+-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. 1a1, a3, b1, b3).

The exchangeable NO3–N concentrations correspondingly increased by the decreased of exchangeable NH4+-N concentrations in the "NH4+" treatments (Fig. 1c2, d2). As expected, 1-octyne present partly inhibited exchangeable NO3–N formation in the "NH4+  + Oct" treatments and acetylene almost completely inhibited the transformation of NH4+-N to NO3–N in the "NH4+  + Ace" treatments in the two soils. As for the treatments without NH4+ addition, there is no obvious difference regardless of the inhibitors were applied or not (P > 0.05; Fig. 1c1, c3, d1, d3).

The change trends of N2O fluxes varied with soil type. The treatments which addition NH4+ alone show a distinct N2O peak (7 and 33 ng N g−1d−1 for SX and PL experiment soil, respectively) at the first day of incubation, and quickly decrease at the following days (Fig. 2a2, b2). Acetylene addition has a significant effect in decreasing N2O production in the "NH4+  + Ace" treatment of the two soils throughout the whole incubation, indicating that AOA plus AOB contribute more N2O 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 N2O emissions in the "NH4+  + Oct" of the two soils, showing that different nitrification inhibitors had the selective inhibitory effects in alleviating N2O emissions as expected (Fig. 2a2, b2). Except for the NH4+ addition treatments, the results also reveal that treatments which with or without NO3–N show no significant difference in N2O 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 NH4+ addition and the fluxes show a periodic fluctuation until the end of incubation (Fig. 2a1, a3, b1, b3).

The accumulated N2O emissions varied with the soil type, types of nitrogen fertilizer and inhibitors (Fig. 2c1 ~ c3, d1 ~ d3). In the "NH4+" treatments, N2O 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 S1). When NH4+ addition was employed with 1-octyne, N2O 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 "NH4+" treatments. As for the NO3− 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 S1).

The N2O yield (%) was defined as the rate of N2O production per unit nitrate content in soil over the whole 21 days for incubation which calculated by Eq. (5). The results showed that the yield of N2O in different ammonia oxidation processes changed with the soil type under the condition of adding NH4+ (Fig. 3). The N2O 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 N2O yields induced by AOA (P < 0.05). However, for other processes (abiotic-induced or denitrification-induced), the N2O yield of SX soil was significantly higher than that of the PL soil (P < 0.05). Under the same soil conditions with NH4+ addition, the yield of N2O changed with different induction processes: in PL soils, the N2O yield induced by AOB was significantly higher than that induced by AOA and the other processes (P < 0.01); But in SX soils, the N2O 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 N2O production varied significantly from the soil types with NH4+ amendment in the both of soils (Fig. 4). The fractions of N2O 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 N2O 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.

At the start of incubation, the AOB amoA genes abundance were 1.65 × 105 and 1.62 × 106 copies g−1 dry soil in SX and PL soil, respectively (Fig. 5a, b) and different treatments have the equal abundance at the initiate of incubation. The NH4+ amendment significantly stimulated the increase of the AOB amoA genes, reaching at 3.38 × 105 and 3.58 × 106 copies g−1 in SX and PL soil at the day 21 of incubation, respectively (P < 0.01; Fig. 5a, 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 × 107copies g−1 and 8.35 × 106 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. 5c, 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 NO3- 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 N2O in arable soils from numerous reports7,35,36. This is first to distinguish the relative contribution of ammonia-oxidizing and N2O 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 N2O emissions from the two tested agricultural soil were driven by nitrification and non-negligible others processes based on the following points: (1) NH4+-N rapidly transformed into NO3–N in the NH4+-N addition treatments and resulted in much higher N2O production than NO3–N addition treatments in which the NO3–N remained stable in during the incubation (Fig. 1); (2) both of test soils were performed under aerobic and 60% WFPS conditions which are optimal N2O production via ammonia oxidation according to previous studies6. And N2O emissions from heterotrophic denitrifiers were negligible24; (3) Nevertheless, N2O accumulated slowly but non-negligible in acetylene treated microcosms when NH3 oxidizer including AOA and AOB growth and activity were inhibited indicating that others processes–induced (heterotrophic nitrification and abiotic processes, etc.) N2O emission also hold an assignable contribution, especially in the neutral soils where others processes contribute 30.7% to the gross N2O emission in the incubation (Table S1 and Fig. 4).

The dynamics NH4+-N content (a1 ~ a3, b1 ~ b3) and NO3−–N content (c1 ~ c3, d1 ~ d3) with different N fertilizers (N-free, ammonium-N, nitrate–N) in combination with air (no inhibitors), acetylene and 1-octyne during incubation of SX and PL soil, error bars represent standard errors of three biological replicates.

Numerous studies have confirmed that N2O 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 treatments37,38,39. Soil pH is a key parameter that controls the abundance change of AOA and AOB which influence N2O production40. In our study, AOB contributed more accumulated N2O emissions than AOA in alkaline soil at the condition of NH4+-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 N2O emissions (Fig. 4 and Table S1). We found that AOB played a more important role than AOA in ammonia oxidation in a high-pH soil, supporting previous reports21,22,41,42. One possible explanation is that soils with higher pH accelerate the rate transformation of NH4+–N to availability NH3 which affected population and activity of ammonia oxidizers27, while bacterial growth would possibly have been impeded in low soil pH43.

Clearly, tested soils with NH4+ stimulated N2O production both by AOA and AOB to varying degrees with respect to the control (Fig. 2 and Table S1). And these results were also confirmed by the markedly increased amoA gene abundance of AOA and AOB at the end of incubation (Fig. 5). At present, the best explanation for different growth and activities of AOA and AOB in soils is a significantly different affinity for NH344,45,46. For example, soils with high NH4+-N concentrate is conducive to the growth of AOB16,20, whereas AOA activity which is favored by low NH4+–N soil may be restricted or not affected in this condition of high NH4+–N 47,48. 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 soils22,49. Unexpectedly, we found that AOA also grew in this condition where high NH4+-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. 5c, d)21,28. And this result was in line with a recent report that there was a direct competition between AOA and AOB for NH3 under high NH4+-N concentration when AOB was inhibited by 1-octyne, whereas AOA growth continued and AOB growth ceased when NH4+ -N became undetectable24,50.

The N2O emission fluxes and accumulated fluxes with different N fertilizers (N-free, ammonium-N, nitrate–N) in combination with air (no inhibitors), acetylene and 1-octyne after 21 days incubation in SX (a and c) and PL (b and d) soil, error bars represent standard errors of three biological replicates.

When NH4+–N supplied as fertilizer, AOB dominated NH3 oxidation process and N2O yield ranged from 0.03 to 0.22%, which varied with soil type (Fig. 3). In neutral soil, the N2O yield induced by AOB (~ 0.03%) fell within the range values (0.02–0.09%) which were derived from soil-slurries with NH4+ amended experiment51. While in alkaline soil, the higher N2O yield of AOB (0.22%) which similar to the results of the pure culture of soil Nitrosospira lineage52 which predominate in soil AOB communities53. As for AOA, when addition of NH4+–N, N2O 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.50 and slightly lower than those of cultivated soil AOA (0.08% ~ 0.23%)12,54,55. These results suggested that AOB might have a higher N2O yield than AOA in the process of producing N2O in both alkaline and neutral soils. The higher N2O yield for AOB than AOA could be explained by the current acknowledgment: there were two enzymatic mechanisms for N2O production in AOB (i.e. nitrifier denitrification and incomplete NH2OH oxidation), while AOA appeared to lack a known NO reductase which was a key enzyme of reducing NO to N2O56,57,58. Therefore, N2O production of AOB was known as a biotic process, whereas inducing by AOA more liked a biotic and abiotic hybrid formation59.

The yield of N2O associated with ammonia oxidation in NH4+-amended treatments; Different letters above the bars denote significant difference and the same letters denote no significant difference and error bars represent standard errors of three biological replicates.

In addition, the method of nitrification inhibitor addition has limitations on the effectiveness to distinguish relative N2O emission of AOA and AOB21. 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 N2O production in NH4+-N addition treatments although AOA amoA abundance showing several times of AOB in both of two soils (Figs. 4 and 5). 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 N2O production by others processes (heterotrophic nitrification and abiotic processes, etc.) contributed a remarkable scale to gross of accumulated N2O production (Fig. 4). Additional investigations like isotope labeling should be carried in the future to reveal the underlying mechanism of other processes leading to N2O.

The relative contributions to N2O production of AOA and AOB from two soils with ammonia amendment treatments after 21 days incubation. Error bars represent standard errors of three biological replicates.

The AOB and AOA amoA genes abundance at 0 and 21 days of incubation in SX (a and c) and PL (b and d) soils; Different letters above the bars denote significant difference and the same letters denote no significant difference and error bars represent standard errors of three biological replicates.

At present, nitrification inhibitors60, slow-release fertilizers61,62,63, appropriate timing of fertilizer application61,64 and no-till65,66 were considered to be the main strategies to increase fertilizer use efficiency and inhibit N2O emission. Our results evaluated the consequences of specialization of ammonia oxidation accompanied by varied N2O yield of AOA and AOB which provide a potential strategy for the alleviation of N2O emissions in purple soils in hilly areas of upper Yangtze River, China. N2O accumulative production and N2O 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 N2O emission. In addition, the application of a moderate nitrification inhibitor could both alleviate the N2O emission and reduce the risk of nitrate leaching in this area, and this viewpoint was supported by a recent review67. In all, measures that prevent ammonia oxidization directly or change specialization causing the increased dominance of NH3 oxidation by AOA indirectly will decrease the N2O production in this area. Meanwhile, crop yield, measures feasibility and cost should be considered with environmental due diligence which was derived from reducing N2O emission.

In conclusion, we explored the relative contribution of ammonia-oxidizing bacteria and archaea to N2O 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. NH4+-N supply significantly increased N2O production of AOB, while AOA-related N2O 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 N2O production varied with different soils of pH. These results may help inform the development of N2O reduction strategies in the future.

All data generated or analysed during this study are included in this published article and its supplementary information files.

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This research was supported by the Key Project of National Science Foundation of China (U20A20107) and the National Natural Science Foundation of China (41301266).

Key Laboratory of Mountain Surface Processes and Ecological Regulation, Institute of Mountain Hazards and Environment, Chinese Academy of Sciences, #9, Block 4, Renminnanlu Road, Chengdu, 610041, Sichuan, China

Lei Hu, Zhixin Dong & Bo Zhu

University of Chinese Academy of Sciences, Beijing, 100049, China

Lei Hu

Institute of Geographic Sciences and Natural Resources Research, Chinese Academy of Sciences, Beijing, 100101, China

Zheng Wang

Chengdu University of Technology, Chengdu, 610059, China

Liwei Xiao

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L.H. and B.Z. designed the experiments. L.H., Z.W. and L.X. participated in acquisition and analysis of data for the work. L.H. wrote the manuscript. Z.D. and B.Z. revised it critically for important intellectual content. All authors approved the submission.

Correspondence to Bo Zhu.

The authors declare no competing interests.

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Hu, L., Dong, Z., Wang, Z. et al. The contributions of ammonia oxidizing bacteria and archaea to nitrification-dependent N2O emission in alkaline and neutral purple soils. Sci Rep 12, 19928 (2022). https://doi.org/10.1038/s41598-022-23084-1

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Received: 14 June 2022

Accepted: 25 October 2022

Published: 19 November 2022

DOI: https://doi.org/10.1038/s41598-022-23084-1

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