The enhanced nitrous oxide emissions are driven by prokaryotic ammonia oxidizers and nirK-denitrifiers under various combinations of nitrogen and rice straw in a paddy soil

Abstract

Nitrous oxide (N₂O), a potent greenhouse gas, contributes significantly to global warming, with agricultural soils being a major source due to intensified nitrogen fertilizer use. While straw incorporation is widely adopted to enhance soil organic carbon sequestration and nutrient retention, its impact on N₂O emissions remains controversial. This study investigated the effects of various combinations of rice straw and nitrogen fertilizer on N₂O emissions in a paddy soil through a controlled microcosm experiment. We monitored CO₂ and N₂O fluxes, ammonium and nitrate dynamics, the activities of key extracellular enzymes involved in carbon and nitrogen cycling, and the abundances of N₂O-related microbial guilds. Results showed that straw amendment stimulated microbial activity and enhanced CO₂ emissions, whereas nitrogen addition suppressed heterotrophic respiration. Both straw and nitrogen amendments significantly increased N₂O emissions, with a synergistic effect observed under combined applications. N₂O emissions were primarily driven by nitrogen amendment and exhibited significant positive correlations with the abundances of ammonia-oxidizing bacteria (AOB), complete ammonia oxidizers clade A (comammox clade A), nirK-denitrifiers, and fungal denitrifiers; moreover, random forest modeling revealed that the abundances of AOB, comammox clade A, nirK-denitrifiers accounted for a substantial portion of the variation in cumulative N₂O emissions. Additionally, partial least squares path modeling (PLS-PM) identified a hierarchical regulatory cascade involving nitrogen availability, microbial community dynamics, and enzyme activity as the key mechanisms governing N₂O fluxes. Overall, these findings underscore the critical roles of prokaryotic ammonia oxidizers and nirK-denitrifiers in modulating N₂O emissions and provide valuable insights for developing field management strategies to mitigate greenhouse gas emissions from agricultural soils receiving straw amendment.

Graphical abstract

Keywords

nitrous oxide / straw / nitrogen / AOB / nirK-denitrifiers

Highlight

	● Straw and nitrogen amendments synergistically enhance N2O emissions from paddy soils.
	● Prokaryotic ammonia oxidizers and nirK -denitrifiers emerge as key drivers of enhanced N2O emissions.
	● Nitrogen substrate availability governs N2O emissions under straw amendments through a hierarchical regulatory cascade.
	● Distinct niche differentiation among functionally similar N2O-related guilds shapes emission patterns.

References

[1] Abalos, D., Recous, S., Butterbach-Bahl, K., De Notaris, C., Rittl, T.F., Topp, C.F.E., Petersen, S.O., Hansen, S., Bleken, M.A., Rees, R.M., Olesen, J.E., 2022. A review and meta-analysis of mitigation measures for nitrous oxide emissions from crop residues. Science of the Total Environment828, 154388.

[2] Aldossari, N., Ishii, S., 2021. Fungal denitrification revisited – recent advancements and future opportunities. Soil Biology and Biochemistry157, 108250.

[3] Archer, E., 2025. rfPermute: estimate permutation p-values for random forest importance metrics. R package version 2.5.4 [Online].

[4] Assémien, F.L., Cantarel, A.A.M., Florio, A., Lerondelle, C., Pommier, T., Gonnety, J.T., Le Roux, X., 2019. Different groups of nitrite-reducers and N₂O-reducers have distinct ecological niches and functional roles in west African cultivated soils. Soil Biology and Biochemistry129, 39–47.

[5] Bateman, E.J., Baggs, E.M., 2005. Contributions of nitrification and denitrification to N₂O emissions from soils at different water-filled pore space. Biology and Fertility of Soils41, 379–388.

[6] Bösch, Y., Pold, G., Saghaï, A., Karlsson, M., Jones, C.M., Hallin, S., 2023. Distribution and environmental drivers of fungal denitrifiers in global soils. Microbiology Spectrum11, e00061–23.

[7] Chen, H.H., Li, X.C., Hu, F., Shi, W., 2013. Soil nitrous oxide emissions following crop residue addition: a meta-analysis. Global Change Biology19, 2956–2964.

[8] Chen, Z., Luo, X.Q., Hu, R.G., Wu, M.N., Wu, J.S., Wei, W.X., 2010. Impact of long-term fertilization on the composition of denitrifier communities based on nitrite reductase analyses in a paddy soil. Microbial Ecology60, 850–861.

[9] Chen, Z.X., Tu, X.S., Meng, H., Chen, C., Chen, Y.J., Elrys, A.S., Cheng, Y., Zhang, J.B., Cai, Z.C., 2021. Microbial process-oriented understanding of stimulation of soil N₂O emission following the input of organic materials. Environmental Pollution284, 117176.

[10] Cleveland, C.C., Liptzin, D., 2007. C:N:P stoichiometry in soil: is there a “redfield ratio” for the microbial biomass. Biogeochemistry85, 235–252.

[11] Crenshaw, C.L., Lauber, C., Sinsabaugh, R.L., Stavely, L.K., 2008. Fungal control of nitrous oxide production in semiarid grassland. Biogeochemistry87, 17–27.

[12] Daims, H., Lebedeva, E.V., Pjevac, P., Han, P., Herbold, C., Albertsen, M., Jehmlich, N., Palatinszky, M., Vierheilig, J., Bulaev, A., Kirkegaard, R.H., von Bergen, M., Rattei, T., Bendinger, B., Nielsen, P.H., Wagner, M., 2015. Complete nitrification by Nitrospira bacteria. Nature528, 504–509.

[13] Daims, H., Lücker, S., Wagner, M., 2016. A new perspective on microbes formerly known as nitrite-oxidizing bacteria. Trends in Microbiology24, 699–712.

[14] Dong, W.L., Li, X., Wang, E.Z., Liu, X.D., Wang, M., Song, A.L., Yin, H.Q., Fan, F.L., 2021. Linking microbial taxa and the effect of mineral nitrogen forms on residue decomposition at the early stage in arable soil by DNA-qSIP. Geoderma400, 115127.

[15] German, D.P., Weintraub, M.N., Grandy, A.S., Lauber, C.L., Rinkes, Z.L., Allison, S.D., 2011. Optimization of hydrolytic and oxidative enzyme methods for ecosystem studies. Soil Biology and Biochemistry43, 1387–1397.

[16] Graf, D.R.H., Jones, C.M., Hallin, S., 2014. Intergenomic comparisons highlight modularity of the denitrification pathway and underpin the importance of community structure for N₂O emissions. PLoS One9, e114118.

[17] Hallin, S., Philippot, L., Löffler, F.E., Sanford, R.A., Jones, C.M., 2018. Genomics and ecology of novel N₂O-reducing microorganisms. Trends in Microbiology26, 43–55.

[18] Hink, L., Nicol, G.W., Prosser, J.I., 2017. Archaea produce lower yields of N₂O than bacteria during aerobic ammonia oxidation in soil. Environmental Microbiology19, 4829–4837.

[19] Islam, M.U., Guo, Z.C., Jiang, F.H., Peng, X.H., 2022. Does straw return increase crop yield in the wheat-maize cropping system in China? A meta-analysis. Field Crops Research279, 108447.

[20] Ji, C., Wang, J.D., Sun, Y.X., Xu, C., Zhou, J., Zhong, Y.H., Ning, Y.W., Zhang, H., Zhang, Y.C., Chen, Y.L., 2024. Wheat straw and microbial inoculants have an additive effect on N₂O emissions by changing microbial functional groups. European Journal of Soil Science75, e13494.

[21] Jiang, M.D., Yang, N.P., Zhao, J.S., Shaaban, M., Hu, R.G., 2021. Crop straw incorporation mediates the impacts of soil aggregate size on greenhouse gas emissions. Geoderma401, 115342.

[22] Jones, C.M., Graf, D.R.H., Bru, D., Philippot, L., Hallin, S., 2013. The unaccounted yet abundant nitrous oxide-reducing microbial community: a potential nitrous oxide sink. The ISME Journal7, 417–426.

[23] Kits, K.D., Jung, M.Y., Vierheilig, J., Pjevac, P., Sedlacek, C.J., Liu, S.R., Herbold, C., Stein, L.Y., Richter, A., Wissel, H., Brüggemann, N., Wagner, M., Daims, H., 2019. Low yield and abiotic origin of N₂O formed by the complete nitrifier Nitrospira inopinata. Nature Communications10, 1836.

[24] Kuypers, M.M.M., Marchant, H.K., Kartal, B., 2018. The microbial nitrogen-cycling network. Nature Reviews Microbiology16, 263–276.

[25] Lenth, R.V., 2025. emmeans: estimated marginal means, aka Least-squares means. R package version 1.11.0 [Online].

[26] Li, C.Y., He, Z.Y., Hu, H.W., He, J.Z., 2023. Niche specialization of comammox Nitrospira in terrestrial ecosystems: oligotrophic or copiotrophic. Critical Reviews in Environmental Science and Technology53, 161–176.

[27] Liang, L.L., Eberwein, J.R., Allsman, L.A., Grantz, D.A., Jenerette, G.D., 2015. Regulation of CO₂ and N₂O fluxes by coupled carbon and nitrogen availability. Environmental Research Letters10, 034008.

[28] Liaw, A., Wiener, M., 2002. Classification and regression by randomForest. R News2, 18–22.

[29] Lin, J.T., Xu, Z.Y., Xue, Y.H., Sun, R.H., Yang, R.G., Cao, X.X., Li, H., Shao, Q., Lou, Y.H., Wang, H., Yang, Q.G., Pan, H., Zhuge, Y.P., 2023. N₂O emissions from soils under short-term straw return in a wheat-corn rotation system are associated with changes in the abundance of functional microbes. Agriculture, Ecosystems & Environment341, 108217.

[30] Liu, C., Lu, M., Cui, J., Li, B., Fang, C.M., 2014. Effects of straw carbon input on carbon dynamics in agricultural soils: a meta-analysis. Global Change Biology20, 1366–1381.

[31] Ma, J., Ma, E.D., Xu, H., Yagi, K., Cai, Z.C., 2009. Wheat straw management affects CH₄ and N₂O emissions from rice fields. Soil Biology and Biochemistry41, 1022–1028.

[32] Ma, W.B., Jiang, S.J., Assemien, F., Qin, M.S., Ma, B.B., Xie, Z., Liu, Y.J., Feng, H.Y., Du, G.Z., Ma, X.J., Le Roux, X., 2016. Response of microbial functional groups involved in soil N cycle to N, P and NP fertilization in Tibetan alpine meadows. Soil Biology and Biochemistry101, 195–206.

[33] Miller, M.N., Zebarth, B.J., Dandie, C.E., Burton, D.L., Goyer, C., Trevors, J.T., 2008. Crop residue influence on denitrification, N₂O emissions and denitrifier community abundance in soil. Soil Biology and Biochemistry40, 2553–2562.

[34] Mooshammer, M., Wanek, W., Hämmerle, I., Fuchslueger, L., Hofhansl, F., Knoltsch, A., Schnecker, J., Takriti, M., Watzka, M., Wild, B., Keiblinger, K.M., Zechmeister-Boltenstern, S., Richter, A., 2014. Adjustment of microbial nitrogen use efficiency to carbon:nitrogen imbalances regulates soil nitrogen cycling. Nature Communications5, 3694.

[35] Mooshammer, M., Wanek, W., Schnecker, J., Wild, B., Leitner, S., Hofhansl, F., Blöchl, A., Hämmerle, I., Frank, A.H., Fuchslueger, L., Keiblinger, K.M., Zechmeister-Boltenstern, S., Richter, A., 2012. Stoichiometric controls of nitrogen and phosphorus cycling in decomposing beech leaf litter. Ecology93, 770–782.

[36] Muhammad, I., Sainju, U.M., Zhao, F.Z., Khan, A., Ghimire, R., Fu, X., Wang, J., 2019. Regulation of soil CO₂ and N₂O emissions by cover crops: a meta-analysis. Soil and Tillage Research192, 103–112.

[37] Ouyang, Y., Evans, S.E., Friesen, M.L., Tiemann, L.K., 2018. Effect of nitrogen fertilization on the abundance of nitrogen cycling genes in agricultural soils: a meta-analysis of field studies. Soil Biology and Biochemistry127, 71–78.

[38] Palomo, A., Pedersen, A.G., Fowler, S.J., Dechesne, A., Sicheritz-Pontén, T., Smets, B.F., 2018. Comparative genomics sheds light on niche differentiation and the evolutionary history of comammox Nitrospira. The ISME Journal12, 1779–1793.

[39] Paustian, K., Lehmann, J., Ogle, S., Reay, D., Robertson, G.P., Smith, P., 2016. Climate-smart soils. Nature532, 49–57.

[40] Philippot, L., Hallin, S., Schloter, M., 2007. Ecology of denitrifying prokaryotes in agricultural soil. Advances in Agronomy96, 249–305.

[41] Pold, G., Bonilla-Rosso, G., Saghaï, A., Strous, M., Jones, C.M., Hallin, S., 2024. Phylogenetics and environmental distribution of nitric oxide-forming nitrite reductases reveal their distinct functional and ecological roles. ISME Communications4, ycae020.

[42] Prosser, J.I., Hink, L., Gubry-Rangin, C., Nicol, G.W., 2020. Nitrous oxide production by ammonia oxidizers: physiological diversity, niche differentiation and potential mitigation strategies. Global Change Biology26, 103–118.

[43] Ravishankara, A.R., Daniel, J.S., Portmann, R.W., 2009. Nitrous oxide (N₂O): the dominant ozone-depleting substance emitted in the 21st century. Science326, 123–125.

[44] Sanchez, G., Trinchera, L., Russolillo, G., 2015. plspm: tools for partial least squares path modeling (PLS-PM). R package version 0.4.9 [Online].

[45] Senbayram, M., Well, R., Bol, R., Chadwick, D.R., Jones, D.L., Wu, D., 2018. Interaction of straw amendment and soil NO₃⁻ content controls fungal denitrification and denitrification product stoichiometry in a sandy soil. Soil Biology and Biochemistry126, 204–212.

[46] Shan, J., Yan, X.Y., 2013. Effects of crop residue returning on nitrous oxide emissions in agricultural soils. Atmospheric Environment71, 170–175.

[47] Tan, C., Yin, C., Li, W.J., Fan, X.P., Jiang, Y.S., Liang, Y.C., 2022. Comammox Nitrospira play a minor role in N₂O emissions from an alkaline arable soil. Soil Biology and Biochemistry171, 108720.

[48] Tian, H.Q., Xu, R.T., Canadell, J.G., Thompson, R.L., Winiwarter, W., Suntharalingam, P., Davidson, E.A., Ciais, P., Jackson, R.B., Janssens-Maenhout, G., Prather, M.J., Regnier, P., Pan, N.Q., Pan, S.F., Peters, G.P., Shi, H., Tubiello, F.N., Zaehle, S., Zhou, F., Arneth, A., Battaglia, G., Berthet, S., Bopp, L., Bouwman, A.F., Buitenhuis, E.T., Chang, J.F., Chipperfield, M.P., Dangal, S.R.S., Dlugokencky, E., Elkins, J.W., Eyre, B.D., Fu, B.J., Hall, B., Ito, A., Joos, F., Krummel, P.B., Landolfi, A., Laruelle, G.G., Lauerwald, R., Li, W., Lienert, S., Maavara, T., MacLeod, M., Millet, D.B., Olin, S., Patra, P.K., Prinn, R.G., Raymond, P.A., Ruiz, D.J., van der Werf, G.R., Vuichard, N., Wang, J.J., Weiss, R.F., Wells, K.C., Wilson, C., Yang, J., Yao, Y.Z., 2020. A comprehensive quantification of global nitrous oxide sources and sinks. Nature586, 248–256.

[49] Wallenstein, M.D., Myrold, D.D., Firestone, M., Voytek, M., 2006. Environmental controls on denitrifying communities and denitrification rates: insights from molecular methods. Ecological Applications16, 2143–2152.

[50] Wang, J.C., Wang, J.L., Rhodes, G., He, J.Z., Ge, Y., 2019. Adaptive responses of comammox Nitrospira and canonical ammonia oxidizers to long-term fertilizations: implications for the relative contributions of different ammonia oxidizers to soil nitrogen cycling. Science of the Total Environment668, 224–233.

[51] Wang, R., Pan, Z.L., Liu, Y., Yao, Z.S., Wang, J., Zheng, X.H., Zhang, C., Ju, X.T., Wei, H.H., Butterbach-Bahl, K., 2022. Full straw incorporation into a calcareous soil increased N₂O emission despite more N₂O being reduced to N₂ in the winter crop season. Agriculture, Ecosystems & Environment335, 108007.

[52] Wang, S.P., Zhai, L.M., Guo, S.F., Zhang, F.L., Hua, L.L., Liu, H.B., 2023. Returned straw reduces nitrogen runoff loss by influencing nitrification process through modulating soil C:N of different paddy systems. Agriculture, Ecosystems & Environment354, 108438.

[53] Wei, H.H., Li, Y., Zhu, K., Ju, X.T., Wu, D., 2024. The divergent role of straw return in soil O₂ dynamics elucidates its confounding effect on soil N₂O emission. Soil Biology and Biochemistry199, 109620.

[54] Wei, W., Isobe, K., Shiratori, Y., Nishizawa, T., Ohte, N., Otsuka, S., Senoo, K., 2014. N₂O emission from cropland field soil through fungal denitrification after surface applications of organic fertilizer. Soil Biology and Biochemistry69, 157–167.

[55] Wu, D., Senbayram, M., Well, R., Brüggemann, N., Pfeiffer, B., Loick, N., Stempfhuber, B., Dittert, K., Bol, R., 2017. Nitrification inhibitors mitigate N₂O emissions more effectively under straw-induced conditions favoring denitrification. Soil Biology and Biochemistry104, 197–207.

[56] Xia, L.L., Lam, S.K., Wolf, B., Kiese, R., Chen, D.L., Butterbach-Bahl, K., 2018. Trade-offs between soil carbon sequestration and reactive nitrogen losses under straw return in global agroecosystems. Global Change Biology24, 5919–5932.

[57] Xie, Z., Le Roux, X., Wang, C.P., Gu, Z.K., An, M., Nan, H.Y., Chen, B.Z., Li, F., Liu, Y.J., Du, G.Z., Feng, H.Y., Ma, X.J., 2014. Identifying response groups of soil nitrifiers and denitrifiers to grazing and associated soil environmental drivers in Tibetan alpine meadows. Soil Biology and Biochemistry77, 89–99.

[58] Xu, C., Han, X., Zhuge, Y.P., Xiao, G.M., Ni, B., Xu, X.C., Meng, F.Q., 2021. Crop straw incorporation alleviates overall fertilizer-N losses and mitigates N₂O emissions per unit applied N from intensively farmed soils: an in situ ¹⁵N tracing study. Science of the Total Environment764, 142884.

[59] Yang, Y.J., Liu, H.X., Chen, Y., Wu, L.J., Huang, G., Lv, J.L., 2024. Soil nitrogen cycling gene abundances in response to organic amendments: a meta-analysis. Science of the Total Environment921, 171048.

[60] Yin, C., Fan, F.L., Song, A.L., Fan, X.P., Ding, H., Ran, W., Qiu, H.Z., Liang, Y.C., 2017. The response patterns of community traits of N₂O emission-related functional guilds to temperature across different arable soils under inorganic fertilization. Soil Biology and Biochemistry108, 65–77.

[61] Yin, C., Fan, X.P., Chen, H., Jiang, Y.S., Ye, M.J., Yan, G.C., Peng, H.Y., Wakelin, S.A., Liang, Y.C., 2021. 3, 4-Dimethylpyrazole phosphate is an effective and specific inhibitor of soil ammonia-oxidizing bacteria. Biology and Fertility of Soils57, 753–766.

[62] Yoshida, M., Ishii, S., Otsuka, S., Senoo, K., 2010. nirK-Harboring denitrifiers are more responsive to denitrification- inducing conditions in rice Paddy soil than nirS-harboring bacteria. Microbes and Environments25, 45–48.

[63] Zhang, X., Liang, Q., Wang, G.Y., Zhang, H.W., Zhang, A.J., Tan, Y.C., Bol, R., 2023. Incorporating straw into intensively farmed cropland soil can reduce N₂O emission via inhibition of nitrification and denitrification pathways. Journal of Environmental Management342, 118115.

[64] Zhu, K., Ye, X., Ran, H.Y., Zhang, P.X., Wang, G., 2022. Contrasting effects of straw and biochar on microscale heterogeneity of soil O₂ and pH: implication for N₂O emissions. Soil Biology and Biochemistry166, 108564.

RIGHTS & PERMISSIONS

Higher Education Press