Microbial processing drives humification of dissolved organic matter under long-term biochar application in agricultural soil

Abstract

Biochar application alters soil dissolved organic matter (DOM) composition; however, how microbial processing modifies soil DOM composition through its linkage with soil extracellular enzyme activities (EEA), and whether this process shifts under long-term biochar application, remains unclear. Soil samples were collected in 2021 and 2023 from a wheat-soybean rotation field amended with biochar in China to investigate how biochar regulates soil DOM composition in short-term (STE) and long-term (LTE) field experiments. By integrating fluorescence spectrometry characteristics of water-extracted DOM and soil EEA, parallel factor analysis, correlation analysis, random forest modelling, and structural equation model (SEM) were employed to investigate the effects of different biochar treatments on soil DOM composition. Results showed that biochar significantly increased soil organic carbon (SOC) content without stimulating soil respiration in the short term, while having little effect on DOC content. Soil DOM in STE was dominated by humic-like components, which showed a weak linkage to microbial activity, while biochar-derived DOM dominated the soluble low-aromatic inputs. In LTE, soil DOM composition shifted toward microbially derived humic acid-like components and substances with high aromaticity and high molecular weight following biochar treatment, accompanied by stronger associations between DOM fluorescence characteristics, microbial activity, and EEA, indicating a transition from input-driven DOM regulation to microbially mediated processing. Notably, the coupling between DOM fractions and N-acquiring enzyme activities suggests that biochar indirectly promoted microbial processing by enhancing nutrient acquisition capacity rather than by directly stimulating microbial biomass. Overall, our findings reveal a time-dependent transition in soil DOM composition following biochar amendment, highlighting that the long-term carbon sequestration effects of biochar are governed not only by its intrinsic stability but also by microbially regulated DOM transformation.

Graphical Abstract

Keywords

Biochar / Dissolved organic matter composition / Pyrolyzed biochar-derived DOM (PyDOM) / Fluorescence spectroscopy / Extracellular enzyme activity

Highlight

•	Soil extracellular enzyme activities were closely associated with DOM fluorescence characteristics.
•	In the short term, soil DOM composition was primarily driven by biochar-derived aromatic inputs.
•	In the long term, microbial processing became the dominant regulator of DOM transformation.
•	Nutrient-acquiring enzymes regulated microbial processing during long-term DOM transformation.

References

[1] Allison SD, Vitousek PM. Responses of extracellular enzymes to simple and complex nutrient inputs. Soil Biol Biochem, 2005, 37: 937-944.

[2] Bastidas Navarro M. Large differences in bacterial community composition of nearby shallow lakes surrounded by Nothofagus pumilio forest in Patagonia (Argentina). J Plankton Res, 2022, 44: 350-364.

[3] Bolan NS, Adriano DC, Kunhikrishnan A. Sparks DL. Dissolved organic matter: biogeochemistry, dynamics, and environmental significance in soils. Advances in agronomy, 2011. San Diego, Academic Press: 1-75vol 110

[4] Bostick KW, Zimmerman AR, Wozniak AS, et al.. Production and composition of pyrogenic dissolved organic matter from a logical series of laboratory-generated chars. Front Earth Sci, 2018, 6: 43.

[5] Burns RG, Deforest JL, Marxsen J, et al.. Soil enzymes in a changing environment: current knowledge and future directions. Soil Biol Biochem, 2013, 58: 216-234.

[6] Chagas JKM, De Figueiredo CC, Ramos MLG. Biochar increases soil carbon pools: evidence from a global meta-analysis. J Environ Manag, 2022, 305: 114403.

[7] Chen ML, Kim SH, Jung HJ, et al.. Dynamics of dissolved organic matter in riverine sediments affected by weir impoundments: production, benthic flux, and environmental implications. Water Res, 2017, 121: 150-161.

[8] Chen ST, Xia X, Feng X, et al.. Short-term crop residue amendments altered the chemodiversity and thermodynamic stability of dissolvable organic matter in paddy soil. Eur J Soil Sci, 2024, 75: e70027.

[9] Chen W, Westerhoff P, Leenheer JA, et al.. Fluorescence excitation–emission matrix regional integration to quantify spectra for dissolved organic matter. Environ Sci Technol, 2003, 37: 5701-5710.

[10] Chen Y, Du Z, Weng Z, et al.. Formation of soil organic carbon pool is regulated by the structure of dissolved organic matter and microbial carbon pump efficacy: a decadal study comparing different carbon management strategies. Glob Change Biol, 2023, 29: 5445-5459.

[11] Clough TJ, Condron LM. Biochar and the nitrogen cycle: Introduction. J Environ Qual, 2010, 39: 1218-1223.

[12] Coble PG, Green SA, Blough NV, et al.. Characterization of dissolved organic matter in the Black Sea by fluorescence spectroscopy. Nature, 1990, 348: 432-435.

[13] Coble PG. Characterization of marine and terrestrial dom in seawater using excitation–emission matrix spectroscopy. Mar Chem, 1996, 51: 325-346.

[14] Cory RM, Mcknight DM. Fluorescence spectroscopy reveals ubiquitous presence of oxidized and reduced quinones in dissolved organic matter. Environ Sci Technol, 2005, 39: 8142-8149.

[15] Cotrufo MF, Soong JL, Horton AJ, et al.. Formation of soil organic matter via biochemical and physical pathways of litter mass loss. Nat Geosci, 2015, 8: 776-779.

[16] Fellman JB, Hood E, Spencer RGM. Fluorescence spectroscopy opens new windows into dissolved organic matter dynamics in freshwater ecosystems: A review. Limnol Oceanogr, 2010, 55: 2452-2462.

[17] Feng Z, Fan Z, Song H, et al.. Biochar induced changes of soil dissolved organic matter: The release and adsorption of dissolved organic matter by biochar and soil. Sci Total Environ, 2021, 783: 147091.

[18] Fierer N, Schimel JP. Effects of drying–rewetting frequency on soil carbon and nitrogen transformations. Soil Biol Biochem, 2002, 34: 777-787.

[19] Freeman EC, Emilson EJS, Dittmar T, et al.. Universal microbial reworking of dissolved organic matter along environmental gradients. Nat Commun, 2024, 15: 187.

[20] Gunapala N, Venette RC, Ferris H, et al.. Effects of soil management history on the rate of organic matter decomposition. Soil Biol Biochem, 1998, 30: 1917-1927.

[21] Huang S, Zhu X, Fang J, et al.. Pyrolysis temperature dependent effects of biochar on shifting fluorescence spectrum characteristics of soil dissolved organic matter under warming. Sci Total Environ, 2023, 892: 164656.

[22] Hudson N, Baker A, Reynolds D. Fluorescence analysis of dissolved organic matter in natural, waste and polluted waters—a review. River Res Appl, 2007, 23: 631-649.

[23] Huguet A, Vacher L, Relexans S, et al.. Properties of fluorescent dissolved organic matter in the Gironde estuary. Org Geochem, 2009, 40: 706-719.

[24] Ippolito JA, Cui LQ, Kammann C, et al.. Feedstock choice, pyrolysis temperature and type influence biochar characteristics: a comprehensive meta-data analysis review. Biochar, 2020, 2: 421-438.

[25] Ishii SKL, Boyer TH. Behavior of reoccurring parafac components in fluorescent dissolved organic matter in natural and engineered systems: a critical review. Environ Sci Technol, 2012, 46: 2006-2017.

[26] Jiang H, Han S, Zhang H, et al.. Calvin cycle driven autotrophic CO₂-fixation traits and autotrophic microbial communities in paddy (anthrosol) and upland (vertisol) soils: rhizosphere effects and impacts of biochar. Biochar, 2025, 7: 118.

[27] Jiao N, Herndl GJ, Hansell DA, et al.. Microbial production of recalcitrant dissolved organic matter: long-term carbon storage in the global ocean. Nat Rev Microbiol, 2010, 8: 593-599.

[28] Jones DL, Owen AG, Farrar JF. Simple method to enable the high resolution determination of total free amino acids in soil solutions and soil extracts. Soil Biol Biochem, 2002, 34: 1893-1902.

[29] Joseph SD, Camps-Arbestain M, Lin Y, et al.. An investigation into the reactions of biochar in soil. Aust J Soil Res, 2010, 48: 501-515.

[30] Kaiser K, Kalbitz K. Cycling downwards—dissolved organic matter in soils. Soil Biol Biochem, 2012, 52: 29-32.

[31] Kalbitz K, Solinger S, Park J-H, et al.. Controls on the dynamics of dissolved organic matter in soils: a review. Soil Sci, 2000, 165: 277-304.

[32] Kalu S, Seppänen A, Mganga KZ, et al.. Biochar reduced the mineralization of native and added soil organic carbon: evidence of negative priming and enhanced microbial carbon use efficiency. Biochar, 2024, 6. ArticleID: 7

[33] Kleber M, Sollins P, Sutton R. A conceptual model of organo-mineral interactions in soils: self-assembly of organic molecular fragments into zonal structures on mineral surfaces. Biogeochemistry, 2007, 85: 9-24.

[34] Kuzyakov Y. Priming effects: interactions between living and dead organic matter. Soil Biol Biochem, 2010, 42: 1363-1371.

[35] Lehmann J, Rillig MC, Thies J, et al.. Biochar effects on soil biota—a review. Soil Biol Biochem, 2011, 43: 1812-1836.

[36] Lehmann J, Kleber M. The contentious nature of soil organic matter. Nature, 2015, 528: 60-68.

[37] Li YF, Hu SD, Chen JH, et al.. Effects of biochar application in forest ecosystems on soil properties and greenhouse gas emissions: a review. J Soils Sed, 2018, 18: 546-563.

[38] Liang C, Schimel JP, Jastrow JD. The importance of anabolism in microbial control over soil carbon storage. Nat Microbiol, 2017, 2: 17105.

[39] Liu C, Wang H, Li P, et al.. Biochar's impact on dissolved organic matter (DOM) export from a cropland soil during natural rainfalls. Sci Total Environ, 2019, 650: 1988-1995.

[40] Luo L, Wang J, Lv J, et al.. Carbon sequestration strategies in soil using biochar: advances, challenges, and opportunities. Environ Sci Technol, 2023, 57: 11357-11372.

[41] Mcknight DM, Boyer EW, Westerhoff PK, et al.. Spectrofluorometric characterization of dissolved organic matter for indication of precursor organic material and aromaticity. Limnol Oceanogr, 2001, 46: 38-48.

[42] Mooshammer M, Wanek W, Hämmerle I, et al.. Adjustment of microbial nitrogen use efficiency to carbon:nitrogen imbalances regulates soil nitrogen cycling. Nat Commun, 2014, 5: 3694.

[43] Murphy KR, Stedmon CA, Graeber D, et al.. Fluorescence spectroscopy and multi-way techniques. Parafac Anal Methods, 2013, 5: 6557-6566.

[44] Musadji NY, Lemée L, Caner L, et al.. Spectral characteristics of soil dissolved organic matter: long-term effects of exogenous organic matter on soil organic matter and spatial-temporal changes. Chemosphere, 2020, 240: 124808.

[45] Nguyen TTN, Xu CY, Tahmasbian I, et al.. Effects of biochar on soil available inorganic nitrogen: a review and meta-analysis. Geoderma, 2017, 288: 79-96.

[46] Ohno T. Fluorescence inner-filtering correction for determining the humification index of dissolved organic matter. Environ Sci Technol, 2002, 36: 742-746.

[47] Painter SC, Lapworth DJ, Woodward EMS, et al.. Terrestrial dissolved organic matter distribution in the North Sea. Sci Total Environ, 2018, 630: 630-647.

[48] Parlanti E, Wörz K, Geoffroy L, et al.. Dissolved organic matter fluorescence spectroscopy as a tool to estimate biological activity in a coastal zone submitted to anthropogenic inputs. Org Geochem, 2000, 31: 1765-1781.

[49] Peng N, Wang K, Tu N, et al.. Fluorescence regional integration combined with parallel factor analysis to quantify fluorescencent spectra for dissolved organic matter released from manure biochars. RSC Adv, 2020, 10: 31502-31510.

[50] Quan G, Fan Q, Zimmerman AR, et al.. Effects of laboratory biotic aging on the characteristics of biochar and its water-soluble organic products. J Hazard Mater, 2020, 382: 121071.

[51] Rombola AG, Torri C, Vassura I, et al.. Effect of biochar amendment on organic matter and dissolved organic matter composition of agricultural soils from a two-year field experiment. Sci Total Environ, 2022, 812: 151422.

[52] Roth V-N, Lange M, Simon C, et al.. Persistence of dissolved organic matter explained by molecular changes during its passage through soil. Nat Geosci, 2019, 12: 755-761.

[53] Singh BP, Cowie AL, Smernik RJ. Biochar carbon stability in a clayey soil as a function of feedstock and pyrolysis temperature. Environ Sci Technol, 2012, 46: 11770-11778.

[54] Sinsabaugh RL, Lauber CL, Weintraub MN, et al.. Stoichiometry of soil enzyme activity at global scale. Ecol Lett, 2008, 11: 1252-1264.

[55] Sinsabaugh RL, Hill BH, Follstad Shah JJ. Ecoenzymatic stoichiometry of microbial organic nutrient acquisition in soil and sediment. Nature, 2009, 462: 795-798.

[56] Smebye A, Ailing V, Vogt RD, et al.. Biochar amendment to soil changes dissolved organic matter content and composition. Chemosphere, 2016, 142: 100-105.

[57] Song W, Qi R, Liu X, et al.. Distinct linkages of microbial communities and enzymatic activities with labile and recalcitrant soil organic carbon in urban wetlands. Appl Soil Ecol, 2026, 218: 106742.

[58] Sun H, Wang Q-X, Liu N, et al.. Effects of different leaf litters on the physicochemical properties and bacterial communities in Panax ginseng-growing soil. Appl Soil Ecol, 2017, 111: 17-24.

[59] Sun Y, Xiong X, He M, et al.. Roles of biochar-derived dissolved organic matter in soil amendment and environmental remediation: a critical review. Chem Eng J, 2021, 424: 130387.

[60] Tang G, Li B, Zhang B, et al.. Temperature effects on microbial dissolved organic matter metabolisms: linking size fractions, fluorescent compositions, and functional groups. Sci Total Environ, 2023, 864: 161175.

[61] Tian L, Dell E, Shi W. Chemical composition of dissolved organic matter in agroecosystems: correlations with soil enzyme activity and carbon and nitrogen mineralization. Appl Soil Ecol, 2010, 46: 426-435.

[62] Vance ED, Brookes PC, Jenkinson DS. An extraction method for measuring soil microbial biomass C. Soil Biol Biochem, 1987, 19: 703-707.

[63] Wang G, Tang X, Zhang Q, et al.. The relationship between soil organic matter composition and soil enzymes activities in various land use types in the upper watershed of Danjiangkou reservoir in China. Land Degrad Dev, 2025, 36: 2557-2570.

[64] Yamashita Y, Maie N, Briceño H, et al.. Optical characterization of dissolved organic matter in tropical rivers of the Guayana shield, Venezuela. J Geophys Res Biogeosci, 2010, 115: G00f10.

[65] Yang W, Shang J, Li B, et al.. Surface and colloid properties of biochar and implications for transport in porous media. Crit Rev Environ Sci Technol, 2020, 50: 2484-2522.

[66] Zhang AF, Zhou X, Li M, et al.. Impacts of biochar addition on soil dissolved organic matter characteristics in a wheat-maize rotation system in Loess Plateau of China. Chemosphere, 2017, 186: 986-993.

[67] Zhang XY, Su C, Liu XY, et al.. Periodical changes of dissolved organic matter (DOM) properties induced by biochar application and its impact on downward migration of heavy metals under flood conditions. J Clean Prod, 2020, 275: 123787.

[68] Zhu X, Chen B, Zhu L, et al.. Effects and mechanisms of biochar-microbe interactions in soil improvement and pollution remediation: a review. Environ Pollut, 2017, 227: 98-115.

[69] Zhu X, Mao L, Chen B. Driving forces linking microbial community structure and functions to enhanced carbon stability in biochar-amended soil. Environ Int, 2019, 133: 105211.

Funding

National Natural Science Foundation of China(42177107)

Natural Science Foundation of Anhui Province, China (2108085MC85)

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The Author(s)