Long-term nitrogen and phosphorus addition enhances soil organic carbon stabilization by strengthening mineral-organic interactions in a temperate grassland

Abstract

Temperate grasslands represent a major component of the global carbon cycle, serving as significant carbon sinks. Much of this carbon storage occurs in the form of soil organic carbon (SOC). Microbial necromass carbon (MNC) is considered to be a major contributor to SOC in temperate grasslands. However, it remains unclear how nitrogen (N) and/or phosphorus (P) enrichment influence the formation and stabilization of mineral-associated organic carbon (MAOC) via MNC turnover and mineral-organic interactions. Here, based on a 12-year in situ nutrient addition experiment, we found that the simultaneous addition of N and P significantly increased MAOC. Although N and P addition reduced MNC, the retained high proportion of MNC within SOC and the decreased C/N ratio of mineral-associated organic matter (MAOM) directly demonstrate MNC dominance in MAOC formation. Our findings reveal that MNC is more readily enriched in fine-sized minerals (0–20 µm, fine silt and clay), facilitating the formation of stable MAOC. As finer fractions approach their capacity to associate with organic matter, coarser silt (20–50 µm) may represent a potential secondary sink for MNC accumulation, as indicated by their positive correlation with the proportion of MNC in SOC. This process is likely facilitated by nutrient-driven increases in ion concentrations and enhanced mineral-organic binding through metal oxide and cation complexation. Altogether, these results contribute to understanding how nutrient enrichment alters the dynamics of polyvalent cations and subsequently influences MAOC formation, highlighting the importance of mineral-organic interactions in promoting carbon sequestration and stabilization within temperate grassland ecosystems.

Graphical abstract

Keywords

microbial necromass carbon / polyvalent cations / mineral-associated organic carbon / fine soil particles / nutrient fertilization / temperate grasslands

Highlight

	● N and/or P additions decreased soil microbial necromass C (MNC) and MNC/SOC.
	● MAOC formation is not limited by the availability of organic precursors.
	● NP addition significantly increased soil MAOC and MAOC/SOC.
	● The increase of MAOC is due to enhanced Ca2+ bridging and Fe/Al oxide complexation.

References

[1] Angst, G., Mueller, K.E., Kögel-Knabner, I., Freeman, K.H., Mueller, C.W., 2017. Aggregation controls the stability of lignin and lipids in clay-sized particulate and mineral associated organic matter. Biogeochemistry132, 307–324.

[2] Angst, G., Mueller, K.E., Nierop, K.G.J., Simpson, M.J., 2021. Plant- or microbial-derived? A review on the molecular composition of stabilized soil organic matter. Soil Biology and Biochemistry156, 108189.

[3] Bao, S.D., 2000. Soil and Agricultural Chemistry Analysis. 3rd ed. Beijing: China Agricultural Press.

[4] Begill, N., Don, A., Poeplau, C., 2023. No detectable upper limit of mineral-associated organic carbon in temperate agricultural soils. Global Change Biology29, 4662–4669.

[5] Borer, E.T., Seabloom, E.W., Gruner, D.S., Harpole, W.S., Hillebrand, H., Lind, E.M., Adler, P.B., Alberti, J., Anderson, T.M., Bakker, J.D., Biederman, L., Blumenthal, D., Brown, C.S., Brudvig, L.A., Buckley, Y.M., Cadotte, M., Chu, C.J., Cleland, E.E., Crawley, M.J., Daleo, P., Damschen, E.I., Davies, K.F., DeCrappeo, N.M., Du, G.Z., Firn, J., Hautier, Y., Heckman, R.W., Hector, A., HilleRisLambers, J., Iribarne, O., Klein, J.A., Knops, J.M.H., La Pierre, K.J., Leakey, A.D.B., Li, W., MacDougall, A.S., McCulley, R.L., Melbourne, B.A., Mitchell, C.E., Moore, J.L., Mortensen, B., O'Halloran, L.R., Orrock, J.L., Pascual, J., Prober, S.M., Pyke, D.A., Risch, A.C., Schuetz, M., Smith, M.D., Stevens, C.J., Sullivan, L.L., Williams, R.J., Wragg, P.D., Wright, J.P., Yang, L.H., 2014. Herbivores and nutrients control grassland plant diversity via light limitation. Nature508, 517–520.

[6] Bossio, D.A., Scow, K.M., Gunapala, N., Graham, K.J., 1998. Determinants of soil microbial communities: effects of agricultural management, season, and soil type on phospholipid fatty acid profiles. Microbial Ecology36, 1–12.

[7] Bowman, W.D., Cleveland, C.C., Halada, Ĺ., Hreško, J., Baron, J.S., 2008. Negative impact of nitrogen deposition on soil buffering capacity. Nature Geoscience1, 767–770.

[8] Chang, Y., Sokol, N.W., van Groenigen, K.J., Bradford, M.A., Ji, D.C., Crowther, T.W., Liang, C., Luo, Y.Q., Kuzyakov, Y., Wang, J.K., Ding, F., 2024. A stoichiometric approach to estimate sources of mineral-associated soil organic matter. Global Change Biology30, e17092.

[9] Chen, J., Luo, Y.Q., van Groenigen, K.J., Hungate, B.A., Cao, J.J., Zhou, X.H., Wang, R.W., 2018. A keystone microbial enzyme for nitrogen control of soil carbon storage. Science Advances4, eaaq1689.

[10] Chen, J.G., Xiao, W., Zheng, C.Y., Zhu, B., 2020. Nitrogen addition has contrasting effects on particulate and mineral-associated soil organic carbon in a subtropical forest. Soil Biology and Biochemistry142, 107708.

[11] Chen, Y., Liu, X., Hou, Y.H., Zhou, S.R., Zhu, B., 2021. Particulate organic carbon is more vulnerable to nitrogen addition than mineral-associated organic carbon in soil of an alpine meadow. Plant and Soil458, 93–103.

[12] Chen, Y.Z., Sun, Z.G., Qin, Z.H., Propastin, P., Wang, W., Li, J.L., Ruan, H.H., 2017. Modeling the regional grazing impact on vegetation carbon sequestration ability in Temperate Eurasian Steppe. Journal of Integrative Agriculture16, 2323–2336.

[13] Cotrufo, M.F., Lavallee, J.M., 2022. Soil organic matter formation, persistence, and functioning: a synthesis of current understanding to inform its conservation and regeneration. Advances in Agronomy172, 1–66.

[14] Cotrufo, M.F., Ranalli, M.G., Haddix, M.L., Six, J., Lugato, E., 2019. Soil carbon storage informed by particulate and mineral-associated organic matter. Nature Geoscience12, 989–994.

[15] Cui, Y.X., Wang, X., Zhang, X.C., Ju, W.L., Duan, C.J., Guo, X.B., Wang, Y.Q., Fang, L.C., 2020. Soil moisture mediates microbial carbon and phosphorus metabolism during vegetation succession in a semiarid region. Soil Biology and Biochemistry147, 107814.

[16] Fan, L.J., Xue, Y.W., Wu, D.H., Xu, M.C., Li, A.D., Zhang, B.X., Mo, J.M., Zheng, M.H., 2024. Long-term nitrogen and phosphorus addition have stronger negative effects on microbial residual carbon in subsoils than topsoils in subtropical forests. Global Change Biology30, e17210.

[17] Fang, K., Qin, S.Q., Chen, L.Y., Zhang, Q.W., Yang, Y.H., 2019. Al/Fe mineral controls on soil organic carbon stock across Tibetan alpine grasslands. Journal of Geophysical Research: Biogeosciences124, 247–259.

[18] Feng, W.T., Plante, A.F., Six, J., 2013. Improving estimates of maximal organic carbon stabilization by fine soil particles. Biogeochemistry112, 81–93.

[19] Fernández-Catinot, F., Pestoni, S., Gallardo, N., Vaieretti, M.V., Pérez Harguindeguy, N., 2023. No detectable upper limit when predicting soil mineral-associated organic carbon stabilization capacity in temperate grassland of Central Argentina mountains. Geoderma Regional35, e00722.

[20] Fierer, N., Bradford, M.A., Jackson, R.B., 2007. Toward an ecological classification of soil bacteria. Ecology88, 1354–1364.

[21] Georgiou, K., Jackson, R.B., Vindušková, O., Abramoff, R.Z., Ahlström, A., Feng, W.T., Harden, J.W., Pellegrini, A.F.A., Polley, H.W., Soong, J.L., Riley, W.J., Torn, M.S., 2022. Global stocks and capacity of mineral-associated soil organic carbon. Nature Communications13, 3797.

[22] Gong, J.R., Dong, X.D., Li, X.B., Yue, K.X., Shi, J.Y., Song, L.Y., Zhang, Z.H., Zhang, W.Y., Li, Y., 2023. Phosphorus fertilization affects litter quality and enzyme activity in a semiarid grassland. Plant and Soil492, 91–108.

[23] Han, L.F., Sun, K., Jin, J., Xing, B.S., 2016. Some concepts of soil organic carbon characteristics and mineral interaction from a review of literature. Soil Biology and Biochemistry94, 107–121.

[24] Hansen, P.M., Even, R., King, A.E., Lavallee, J., Schipanski, M., Cotrufo, M.F., 2024. Distinct, direct and climate-mediated environmental controls on global particulate and mineral-associated organic carbon storage. Global Change Biology30, e17080.

[25] Hassink, J., 1997. The capacity of soils to preserve organic C and N by their association with clay and silt particles. Plant and Soil191, 77–87.

[26] He, M., Fang, K., Chen, L.Y., Feng, X.H., Qin, S.Q., Kou, D., He, H.B., Liang, C., Yang, Y.H., 2022. Depth-dependent drivers of soil microbial necromass carbon across Tibetan alpine grasslands. Global Change Biology28, 936–949.

[27] Hu, J.X., Du, M.L., Chen, J., Tie, L.H., Zhou, S.X., Buckeridge, K.M., Cornelissen, J.H.C., Huang, C.D., Kuzyakov, Y., 2023. Microbial necromass under global change and implications for soil organic matter. Global Change Biology29, 3503–3515.

[28] Kang, J., Qu, C.C., Chen, W.L., Cai, P., Chen, C.R., Huang, Q.Y., 2024. Organo-organic interactions dominantly drive soil organic carbon accrual. Global Change Biology30, e17147.

[29] Kleber, M., Eusterhues, K., Keiluweit, M., Mikutta, C., Mikutta, R., Nico, P.S., 2015. Mineral-organic associations: formation, properties, and relevance in soil environments. Advances in Agronomy130, 1–140.

[30] Lavallee, J.M., Soong, J.L., Cotrufo, M.F., 2020. Conceptualizing soil organic matter into particulate and mineral-associated forms to address global change in the 21st century. Global Change Biology26, 261–273.

[31] Lefcheck, J.S., 2016. piecewiseSEM: piecewise structural equation modelling in R for ecology, evolution, and systematics. Methods in Ecology and Evolution7, 573–579.

[32] Li, N., Du, H.B., Li, M.H., Na, R.S., Dong, R.K., He, H.S., Zong, S.W., Huang, L.R., Wu, Z.F., 2023a. Deyeuxia angustifolia upward migration and nitrogen deposition change soil microbial community structure in an alpine tundra. Soil Biology and Biochemistry180, 109009.

[33] Li, Y.F., Zhang, X., Wang, B.Y., Wu, X.D., Wang, Z.J., Liu, L.C., Yang, H.T., 2023b. Revegetation promotes soil mineral-associated organic carbon sequestration and soil carbon stability in the Tengger Desert, northern China. Soil Biology and Biochemistry185, 109155.

[34] Li, Z., Duan, X., Guo, X.B., Gao, W., Li, Y., Zhou, P., Zhu, Q.H., O'Donnell, A.G., Dai, K., Wu, J.S., 2024. Microbial metabolic capacity regulates the accrual of mineral-associated organic carbon in subtropical paddy soils. Soil Biology and Biochemistry195, 109457.

[35] Liang, C., Amelung, W., Lehmann, J., Kästner, M., 2019. Quantitative assessment of microbial necromass contribution to soil organic matter. Global Change Biology25, 3578–3590.

[36] Liang, C., Duncan, D.S., Balser, T.C., Tiedje, J.M., Jackson, R.D., 2013. Soil microbial residue storage linked to soil legacy under biofuel cropping systems in southern Wisconsin, USA. Soil Biology and Biochemistry57, 939–942.

[37] Liang, C., Kao-Kniffin, J., Sanford, G.R., Wickings, K., Balser, T.C., Jackson, R.D., 2016. Microorganisms and their residues under restored perennial grassland communities of varying diversity. Soil Biology and Biochemistry103, 192–200.

[38] Liang, C., Schimel, J.P., Jastrow, J.D., 2017. The importance of anabolism in microbial control over soil carbon storage. Nature Microbiology2, 17105.

[39] Lützow, M.V., Kögel-Knabner, I., Ekschmitt, K., Matzner, E., Guggenberger, G., Marschner, B., Flessa, H., 2006. Stabilization of organic matter in temperate soils: mechanisms and their relevance under different soil conditions-a review. European Journal of Soil Science57, 426–445.

[40] Ma, H.Z., Mo, L.D., Crowther, T.W., Maynard, D.S., van den Hoogen, J., Stocker, B.D., Terrer, C., Zohner, C.M., 2021. The global distribution and environmental drivers of aboveground versus belowground plant biomass. Nature Ecology & Evolution5, 1110–1122.

[41] Ma, T., Yang, Z.Y., Shi, B.W., Gao, W.J., Li, Y.F., Zhu, J.X., He, J.S., 2023. Phosphorus supply suppressed microbial necromass but stimulated plant lignin phenols accumulation in soils of alpine grassland on the Tibetan Plateau. Geoderma431, 116376.

[42] Ma, T., Zhu, S.S., Wang, Z.H., Chen, D.M., Dai, G.H., Feng, B.W., Su, X.Y., Hu, H.F., Li, K.H., Han, W.X., Liang, C., Bai, Y.F., Feng, X.J., 2018. Divergent accumulation of microbial necromass and plant lignin components in grassland soils. Nature Communications9, 3480.

[43] Mikutta, R., Zang, U., Chorover, J., Haumaier, L., Kalbitz, K., 2011. Stabilization of extracellular polymeric substances (Bacillus subtilis) by adsorption to and coprecipitation with Al forms. Geochimica et Cosmochimica Acta75, 3135–3154.

[44] Moens, C., Smolders, E., 2021. Suwannee River Natural Organic Matter concentrations affect the size and phosphate uptake of colloids formed by iron oxidation. Geochimica et Cosmochimica Acta312, 375–391.

[45] Moore, O.W., Curti, L., Woulds, C., Bradley, J.A., Babakhani, P., Mills, B.J.W., Homoky, W.B., Xiao, K.Q., Bray, A.W., Fisher, B.J., Kazemian, M., Kaulich, B., Dale, A.W., Peacock, C.L., 2023. Long-term organic carbon preservation enhanced by iron and manganese. Nature621, 312–317.

[46] Morrissey, E.M., Kane, J., Tripathi, B.M., Rion, M.S.I., Hungate, B.A., Franklin, R., Walter, C., Sulman, B., Brzostek, E., 2023. Carbon acquisition ecological strategies to connect soil microbial biodiversity and carbon cycling. Soil Biology and Biochemistry177, 108893.

[47] Ni, J., 2002. Carbon storage in grasslands of China. Journal of Arid Environments50, 205–218.

[48] Prommer, J., Walker, T.W.N., Wanek, W., Braun, J., Zezula, D., Hu, Y.T., Hofhansl, F., Richter, A., 2020. Increased microbial growth, biomass, and turnover drive soil organic carbon accumulation at higher plant diversity. Global Change Biology26, 669–681.

[49] Qafoku, O., Battu, A.K., Varga, T., Marcus, M.A., O'Callahan, B., Zhao, Q., Mergelsberg, S.T., Kew, W.R., Loring, J.S., Qafoku, N.P., Leichty, S.I., 2023. Chemical composition, coordination, and stability of Ca-organic associations in the presence of dissolving calcite. Environmental Science: Nano10, 1504–1517.

[50] R Core Team, 2022. R: a Language and Environment for Statistical Computing. R Foundation for Statistical Computing, Vienna, Austria. Available at the website of R-project.

[51] Riggs, C.E., Hobbie, S.E., 2016. Mechanisms driving the soil organic matter decomposition response to nitrogen enrichment in grassland soils. Soil Biology and Biochemistry99, 54–65.

[52] Rowley, M.C., Grand, S., Verrecchia, É.P., 2018. Calcium-mediated stabilisation of soil organic carbon. Biogeochemistry137, 27–49.

[53] Sinsabaugh, R.L., Follstad Shah, J.J., 2012. Ecoenzymatic stoichiometry and ecological theory. Annual Review of Ecology, Evolution, and Systematics43, 313–343.

[54] Six, J., Conant, R.T., Paul, E.A., Paustian, K., 2002. Stabilization mechanisms of soil organic matter: implications for C-saturation of soils. Plant and Soil241, 155–176.

[55] Six, J., Doetterl, S., Laub, M., Müller, C.R., Van de Broek, M., 2024. The six rights of how and when to test for soil C saturation. Soil10, 275–279.

[56] Sowers, T.D., Adhikari, D., Wang, J., Yang, Y., Sparks, D.L., 2018. Spatial associations and chemical composition of organic carbon sequestered in Fe, Ca, and organic carbon ternary systems. Environmental Science & Technology52, 6936–6944.

[57] Sun, T., Mao, X.L., Han, K.F., Wang, X.J., Cheng, Q., Liu, X., Zhou, J.J., Ma, Q.X., Ni, Z.H., Wu, L.H., 2023. Nitrogen addition increased soil particulate organic carbon via plant carbon input whereas reduced mineral-associated organic carbon through attenuating mineral protection in agroecosystem. Science of the Total Environment899, 165705.

[58] Viscarra Rossel, R.A., Webster, R., Zhang, M., Shen, Z., Dixon, K., Wang, Y.P., Walden, L., 2024. How much organic carbon could the soil store? The carbon sequestration potential of Australian soil. Global Change Biology30, e17053.

[59] Wang, B.R., An, S.S., Liang, C., Liu, Y., Kuzyakov, Y., 2021. Microbial necromass as the source of soil organic carbon in global ecosystems. Soil Biology and Biochemistry162, 108422.

[60] Wang, B.R., Huang, Y.M., Li, N., Yao, H.J., Yang, E., Soromotin, A.V., Kuzyakov, Y., Cheptsov, V., Yang, Y., An, S.S., 2022. Initial soil formation by biocrusts: nitrogen demand and clay protection control microbial necromass accrual and recycling. Soil Biology and Biochemistry167, 108607.

[61] Wang, H., Tang, S., Han, S., Li, M., Cheng, W.L., Bu, R.Y., Cao, W.D., Wu, J., 2023. Effects of long-term substitution of chemical fertilizer with Chinese milk vetch on soil phosphorus availability and leaching risk in the double rice systems of eastern China. Field Crops Research302, 109047.

[62] Warren, C., 2021. What are the products of enzymatic cleavage of organic N. Soil Biology and Biochemistry154, 108152.

[63] Xu, C.H., Xu, X., Peñuelas, J., Sardans, J., Reich, P., Chen, H.Y.H., Luo, Y.Q., Zou, X.M., Fan, W., Ju, C.H., Lin, M.Y., Cui, J., Liu, W.F., Chen, X.C., Wang, J.J., 2025. Soil pH-dependent nitrogen stimulation of plant biomass: magnesium and calcium as key constraints. New Phytologist246, 936–946.

[64] Xu, X.F., Thornton, P.E., Post, W.M., 2013. A global analysis of soil microbial biomass carbon, nitrogen and phosphorus in terrestrial ecosystems. Global Ecology and Biogeography22, 737–749.

[65] Yang, Y., Chen, X.L., Liu, L.X., Li, T., Dou, Y.X., Qiao, J.B., Wang, Y.Q., An, S.S., Chang, S.X., 2022a. Nitrogen fertilization weakens the linkage between soil carbon and microbial diversity: a global meta-analysis. Global Change Biology28, 6446–6461.

[66] Yang, Y.L., Xie, H.T., Mao, Z., Bao, X.L., He, H.B., Zhang, X.D., Liang, C., 2022b. Fungi determine increased soil organic carbon more than bacteria through their necromass inputs in conservation tillage croplands. Soil Biology and Biochemistry167, 108587.

[67] Ye, C.L., Chen, D.M., Hall, S.J., Pan, S., Yan, X.B., Bai, T.S., Guo, H., Zhang, Y., Bai, Y.F., Hu, S.J., 2018. Reconciling multiple impacts of nitrogen enrichment on soil carbon: plant, microbial and geochemical controls. Ecology Letters21, 1162–1173.

[68] Yuan, X., Qin, W.K., Xu, H., Zhang, Z.H., Zhou, H.K., Zhu, B., 2020. Sensitivity of soil carbon dynamics to nitrogen and phosphorus enrichment in an alpine meadow. Soil Biology and Biochemistry150, 107984.

[69] Yuan, Y., Li, Y., Mou, Z.J., Kuang, L.H., Wu, W.J., Zhang, J., Wang, F.M., Hui, D.F., Peñuelas, J., Sardans, J., Lambers, H., Wang, J., Kuang, Y.W., Li, Z.A., Liu, Z.F., 2021. Phosphorus addition decreases microbial residual contribution to soil organic carbon pool in a tropical coastal forest. Global Change Biology27, 454–466.

[70] Yuan, Z.Y., Chen, H.Y.H., 2009. Global trends in senesced-leaf nitrogen and phosphorus. Global Ecology and Biogeography18, 532–542.

[71] Zhang, H., Jiang, N., Wang, H., Zhang, S.Y., Zhao, J.N., Liu, H.M., Zhang, H.F., Yang, D.L., 2025. Importance of plant community composition and aboveground biomass in shaping microbial communities following long-term nitrogen and phosphorus addition in a temperate steppe ecosystem. Plant and Soil509, 543–560.

[72] Zhang, X.D., Amelung, W., 1996. Gas chromatographic determination of muramic acid, glucosamine, mannosamine, and galactosamine in soils. Soil Biology and Biochemistry28, 1201–1206.

[73] Zhang, X.Y., Jia, J., Chen, L.T., Chu, H.Y., He, J.S., Zhang, Y.J., Feng, X.J., 2021. Aridity and NPP constrain contribution of microbial necromass to soil organic carbon in the Qinghai-Tibet alpine grasslands. Soil Biology and Biochemistry156, 108213.

[74] Zhang, Y.Y., Zheng, N.G., Wang, J., Yao, H.Y., Qiu, Q.F., Chapman, S.J., 2019. High turnover rate of free phospholipids in soil confirms the classic hypothesis of PLFA methodology. Soil Biology and Biochemistry135, 323–330.

[75] Zheng, Z., Ma, P.F., 2018. Changes in above and belowground traits of a rhizome clonal plant explain its predominance under nitrogen addition. Plant and Soil432, 415–424.

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