Biochar orchestrates coordinated soil-microbe-metabolite responses in acidifying paddy soils: evidence from a 5-year field study

Abstract

Biochar is increasingly recognized for its capacity to remediate acidifying soils, but the mechanisms through which it achieves long-term effects remain poorly understood. This five-year field study examined how biochar’s effects on soil chemistry propagate through biological systems to reshape soil function. We conducted a randomized field experiment comparing three biochar application rates (4.5, 11.25, and 22.5 t ha^–1) with lime and swine manure in an acidic paddy soil. Integrated soil microbiome, metagenomic, metaviromic, and metabolomic analyses assessed how amendments altered soil properties and their associations with microbial communities and metabolic functions. All amendments alleviated acidification (pH increased from 5.5 to 6.4) and reduced exchangeable aluminum (from 12.5 to 3.5 mg kg^–1). High-dose biochar (22.5 t ha⁻¹) initiated a mechanistic cascade absent under traditional amendments: improved soil chemistry drove restructuring of prokaryotic and viral communities toward nutrient-cycling phenotypes (enriching Chloroflexi, Planctomycetota, Algavirales, and Crassvirales), which in turn reshaped metagenomic functions and soil metabolite profiles. Specifically, biochar elevated genes related to nutrient exchange and cell–cell interactions while enriching lipids and terpenoids that support plant growth and long-term carbon stabilization. This coordinated restructuring of soil chemistry, microbial communities, and metabolic function did not occur under lime or manure. The findings demonstrate that biochar’s long-term superiority emerges from orchestrating sequential changes across the soil-microbe-metabolite system as an integrated whole. This mechanistic understanding provides novel insights for deploying biochar as an ecosystem restoration tool in acidifying agricultural systems.

Graphical Abstract

Keywords

Biochar / Mechanistic cascade / Soil-microbe-metabolite interface / Acidic soil remediation / Microbial communities / Metabolomics

References

[1] Abarenkov K, Nilsson RH, Larsson K-H, Taylor AFS, May TW, Frøslev TG, Pawlowska J, Lindahl B, Põldmaa K, Truong C, Vu D, Hosoya T, Niskanen T, Piirmann T, Ivanov F, Zirk A, Peterson M, Cheeke TE, Ishigami Y, Jansson AT, Jeppesen TS, Kristiansson E, Mikryukov V, Miller JT, Oono R, Ossandon FJ, Paupério J, Saar I, Schigel D, Suija A, Tedersoo L, Kõljalg U. The UNITE database for molecular identification and taxonomic communication of fungi and other eukaryotes: sequences, taxa and classifications reconsidered. Nucleic Acids Res. 2024, 52(D1): D791-D797.

[2] Abisado RG, Benomar S, Klaus JR, Dandekar AA, Chandler JR. Bacterial quorum sensing and microbial community interactions. Mbio. 2018, 9(3. 10.1128/mbio.02331-17

[3] Alseekh S, Aharoni A, Brotman Y, Contrepois K, D’Auria J, Ewald J, Ewald JC, Fraser PD, Giavalisco P, Hall RD, Heinemann M, Link H, Luo J, Neumann S, Nielsen J, Perez de Souza L, Saito K, Sauer U, Schroeder FC, Schuster S, Siuzdak G, Skirycz A, Sumner LW, Snyder MP, Tang H, Tohge T, Wang Y, Wen W, Wu S, Xu G, Zamboni N, Fernie AR. Mass spectrometry-based metabolomics: a guide for annotation, quantification and best reporting practices. Nat Methods. 2021, 187): 747-756.

[4] Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990, 215(3): 403-410.

[5] Annabi M, Le Bissonnais Y, Le Villio-Poitrenaud M, Houot S. Improvement of soil aggregate stability by repeated applications of organic amendments to a cultivated silty loam soil. Agric Ecosyst Environ. 2011, 144(1): 382-389.

[6] Bai N, Zhang H, Zhou S, Sun H, Zhao Y, Zheng X, Li S, Zhang J, Lv W. Long-term effects of straw return and straw-derived biochar amendment on bacterial communities in soil aggregates. Sci Rep. 2020, 10(1): 7891.

[7] Biddle JF, Fitz-Gibbon S, Schuster SC, Brenchley JE, House CH. Metagenomic signatures of the Peru Margin subseafloor biosphere show a genetically distinct environment. Proc Natl Acad Sci USA. 2008, 1053010583-10588.

[8] Biederman LA, Harpole WS. Biochar and its effects on plant productivity and nutrient cycling: a meta-analysis. GCB Bioenergy. 2013, 5(2): 202-214.

[9] Buchfink B, Xie C, Huson DH. Fast and sensitive protein alignment using DIAMOND. Nat Methods. 2015, 12(1): 59-60.

[10] Bunemann EK, Bongiorno G, Bai Z, Creamer RE, De Deyn G, de Goe R, Fleskens L, Geissen V, Kuyper TW, Mader P, Pulleman M, Sukkel W, van Groenigen JW, Brussaard L. Soil quality-a critical review. Soil Biol Biochem. 2018, 120: 105-125.

[11] Callahan BJ, McMurdie PJ, Rosen MJ, Han AW, Johnson AJA, Holmes SP. DADA2: High-resolution sample inference from Illumina amplicon data. Nat Methods. 2016, 13(7): 581-583.

[12] Chen S. Ultrafast one‐pass FASTQ data preprocessing, quality control, and deduplication using fastp. iMeta. 2023, 2(2. e107

[13] Chen W, Meng J, Han X, Lan Y, Zhang W. Past, present, and future of biochar. Biochar. 2019, 11): 75-87.

[14] Chen D, Wang X, Carrión VJ, Yin S, Yue Z, Liao Y, Dong Y, Li X. Acidic amelioration of soil amendments improves soil health by impacting rhizosphere microbial assemblies. Soil Biol Biochem. 2022, 167. 108599

[15] Clapham DE. Calcium signaling. Cell. 2007, 131(6): 1047-1058.

[16] Cui X, Yuan J, Yang X, Wei C, Bi Y, Sun Q, Meng J, Han X. Biochar application alters soil metabolites and nitrogen cycle-related microorganisms in a soybean continuous cropping system. Sci Total Environ. 2024, 917. 170522

[17] Dai Z, Xiong X, Zhu H, Xu H, Leng P, Li J, Tang C, Xu J. Association of biochar properties with changes in soil bacterial, fungal and fauna communities and nutrient cycling processes. Biochar. 2021, 3(3): 239-254.

[18] Ding Y, Liu Y, Liu S, Li Z, Tan X, Huang X, Zeng G, Zhou L, Zheng B. Biochar to improve soil fertility. A review. Agron Sustain Dev. 2016, 36(2. 36

[19] Dixon P. VEGAN, a package of R functions for community ecology. J Veg Sci. 2003, 146): 927-930.

[20] Dodor DE, Tabatabai MA. A simple alkaline hydrolysis method for estimating nitrogen mineralization potential of soils. West Afr J Appl Ecol. 2019, 27(2): 16-31.

[21] Dondjou DT, Diedhiou AG, Mbodj D, Mofini M-T, Pignoly S, Ndiaye C, Diedhiou I, Assigbetse K, Manneh B, Laplaze L, Kane A. Rice developmental stages modulate rhizosphere bacteria and archaea co-occurrence and sensitivity to long-term inorganic fertilization in a West African Sahelian agro-ecosystem. Environ Microbiome. 2023, 18(1. 42

[22] Edgar RC. Search and clustering orders of magnitude faster than BLAST. Bioinformatics. 2010, 2619): 2460-2461.

[23] El-Naggar A, Lee SS, Rinklebe J, Farooq M, Song H, Sarmah AK, Zimmerman AR, Ahmad M, Shaheen SM, Ok YS. Biochar application to low fertility soils: a review of current status, and future prospects. Geoderma. 2019, 337: 536-554.

[24] Ferreira TR, Pires LF, Wildenschild D, Brinatti AM, Borges JAR, Auler AC, dos Reis AMH. Lime application effects on soil aggregate properties: use of the mean weight diameter and synchrotron-based X-ray μCT techniques. Geoderma. 2019, 338: 585-596.

[25] Fierer N, Bradford MA, Jackson RB. Toward an ecological classification of soil bacteria. Ecology. 2007, 88(6): 1354-1364.

[26] Fierer N, Wood SA, de Bueno Mesquita CP. How microbes can, and cannot, be used to assess soil health. Soil Biol Biochem. 2021, 153. 108111

[27] Frimpong KA, Owusu S, Darko RO, Hanyabui E, Abbey ANA, Tetteh DA. Effect of biochar application rates on soil properties and growth of amaranthus caudatus. Discover Agriculture. 2025, 3(1): 1-17.

[28] Fu Y, de Jonge LW, Moldrup P, Paradelo M, Arthur E. Improvements in soil physical properties after long-term manure addition depend on soil and crop type. Geoderma. 2022, 425. 116062

[29] Fu Q, Qiu YB, Zhao J, Li J, Xie S, Liao Q, Fu X, Huang Y, Yao Z, Dai Z, Qiu YP, Yang Y, Li F, Chen H. Monotonic trends of soil microbiomes, metagenomic and metabolomic functioning across ecosystems along water gradients in the Altai region, northwestern China. Sci Total Environ. 2024, 912. 169351

[30] Gao M, Yang J, Liu C, Gu B, Han M, Li J, Li N, Liu N, An N, Dai J, Liu X, Han X. Effects of long-term biochar and biochar-based fertilizer application on brown earth soil bacterial communities. Agric Ecosyst Environ. 2021, 309. 107285

[31] Gardes M, Bruns TD. ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol. 1993, 2(2): 113-118.

[32] Guo J, Bolduc B, Zayed AA, Varsani A, Dominguez-Huerta G, Delmont TO, Pratama AA, Gazitúa MC, Vik D, Sullivan MB, Roux S. VirSorter2: a multi-classifier, expert-guided approach to detect diverse DNA and RNA viruses. Microbiome. 2021, 9(1. 37

[33] Han T, Li D, Liu K, Huang J, Zhang L, Liu S, Shah A, Liu L, Feng G, Zhang H. Soil potassium regulation by initial K level and acidification degree when subjected to liming: a meta-analysis and long-term field experiment. CATENA. 2023, 232. 107408

[34] Hartmann M, Six J. Soil structure and microbiome functions in agroecosystems. Nat Rev Earth Env. 2023, 41): 4-18.

[35] Hossain MZ, Bahar MM, Sarkar B, Donne SW, Ok YS, Palansooriya KN, Kirkham MB, Chowdhury S, Bolan N. Biochar and its importance on nutrient dynamics in soil and plant. Biochar. 2020, 2(4): 379-420.

[36] Hu M, Xiang Y, Lu J. Effects of lime application rates on soil acidity and barley seeding growth in acidic soils. Sci Agric Sin. 2016, 49(20): 3896-3903.

[37] Hu Y, Cheng H, Tao S. The challenges and solutions for cadmium-contaminated rice in China: a critical review. Environ Int. 2016, 92: 515-532.

[38] Hu X, Gu H, Liu J, Wei D, Zhu P, Cui X, Zhou B, Chen X, Jin J, Liu X, Wang G. Metagenomics reveals divergent functional profiles of soil carbon and nitrogen cycling under long-term addition of chemical and organic fertilizers in the black soil region. Geoderma. 2022, 418. 115846

[39] Huang Y, Shi W, Fu Q, Qiu YB, Zhao J, Li J, Lyu Q, Yang X, Xiong J, Wang W, Chang R, Yao Z, Dai Z, Qiu YP, Chen H. Soil development following glacier retreat shapes metagenomic and metabolomic functioning associated with asynchronous C and N accumulation. Sci Total Environ. 2023, 892. 164405

[40] Hyatt D, Chen G-L, LoCascio PF, Land ML, Larimer FW, Hauser LJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics. 2010, 11(1. 119

[41] Karaca A, Cetin SC, Turgay OC, Kizilkaya R. Effects of heavy metals on soil enzyme activities. Soil heavy metals. 2010, Berlin, Heidelberg, Springer237262.

[42] Keiluweit M, Wanzek T, Kleber M, Nico P, Fendorf S. Anaerobic microsites have an unaccounted role in soil carbon stabilization. Nat Commun. 2017, 8: 1771.

[43] Kembel SW, Cowan PD, Helmus MR, Cornwell WK, Morlon H, Ackerly DD, Blomberg SP, Webb CO. Picante: R tools for integrating phylogenies and ecology. Bioinformatics. 2010, 2611): 1463-1464.

[44] Kerner P, Struhs E, Mirkouei A, Aho K, Lohse KA, Dungan RS, You Y. Microbial responses to biochar soil amendment and influential factors: a three-level meta-analysis. Environ Sci Technol. 2023, 574819838-19848.

[45] Kuzyakov Y, Mason-Jones K. Viruses in soil: nano-scale undead drivers of microbial life, biogeochemical turnover and ecosystem functions. Soil Biol Biochem. 2018, 127: 305-317.

[46] Lauricella D, Butterly CR, Clark GJ, Sale PWG, Li G, Tang C. Effectiveness of innovative organic amendments in acid soils depends on their ability to supply P and alleviate Al and Mn toxicity in plants. J Soil Sediment. 2020, 20(11): 3951-3962.

[47] Lehmann J. A handful of carbon. Nature. 2007, 447(7141): 143-144.

[48] Lehmann J, Bossio DA, Kögel-Knabner I, Rillig MC. The concept and future prospects of soil health. Nat Rev Earth Environ. 2020, 1(10): 544-553.

[49] Levasseur A, Drula E, Lombard V, Coutinho PM, Henrissat B. Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes. Biotechnol Biofuels. 2013, 6(1. 41

[50] Li D, Luo R, Liu C-M, Leung C-M, Ting H-F, Sadakane K, Yamashita H, Lam T-W. MEGAHIT v1.0: a fast and scalable metagenome assembler driven by advanced methodologies and community practices. Methods. 2016, 102: 3-11.

[51] Li X-M, Chen Q-L, He C, Shi Q, Chen S-C, Reid BJ, Zhu Y-G, Sun G-X. Organic carbon amendments affect the chemodiversity of soil dissolved organic matter and its associations with soil microbial communities. Environ Sci Technol. 2019, 531): 50-59.

[52] Li X, Chen D, Carrión VJ, Revillini D, Yin S, Dong Y, Zhang T, Wang X, Delgado-Baquerizo M. Acidification suppresses the natural capacity of soil microbiome to fight pathogenic Fusarium infections. Nat Commun. 2023, 14(1): 5090.

[53] Liu J, Shu A, Song W, Shi W, Li M, Zhang W, Li Z, Liu G, Yuan F, Zhang S, Liu Z, Gao Z. Long-term organic fertilizer substitution increases rice yield by improving soil properties and regulating soil bacteria. Geoderma. 2021, 404. 115287

[54] Liu X, Liu H, Zhang Y, Liu C, Liu Y, Li Z, Zhang M. Organic amendments alter microbiota assembly to stimulate soil metabolism for improving soil quality in wheat-maize rotation system. J Environ Manage. 2023, 339. 117927

[55] Lombard V, Bernard T, Rancurel C, Brumer H, Coutinho PM, Henrissat B. A hierarchical classification of polysaccharide lyases for glycogenomics. Biochem J. 2010, 4323437-444.

[56] Louca S, Polz MF, Mazel F, Albright MBN, Huber JA, O’Connor MI, Ackermann M, Hahn AS, Srivastava DS, Crowe SA, Doebeli M, Parfrey LW. Function and functional redundancy in microbial systems. Nat Ecol Evol. 2018, 2(6): 936-943.

[57] Murphy J, Riley JP. A modified single solution method for the determination of phosphate in natural waters. Anal Chim Acta. 1962, 27: 31-36.

[58] Nayfach S, Camargo AP, Schulz F, Eloe-Fadrosh E, Roux S, Kyrpides NC. Checkv assesses the quality and completeness of metagenome-assembled viral genomes. Nat Biotechnol. 2021, 39(5): 578-585.

[59] Neira G, Vergara E, Holmes DS. Genome-guided prediction of acid resistance mechanisms in acidophilic methanotrophs of phylogenetically deep-rooted Verrucomicrobia isolated from geothermal environments. Front Microbiol. 2022.

[60] Nicholson FA, Chambers BJ, Williams JR, Unwin RJ. Heavy metal contents of livestock feeds and animal manures in England and Wales. Bioresource Technol. 1999, 70(1): 23-31.

[61] Palansooriya KN, Wong JTF, Hashimoto Y, Huang L, Rinklebe J, Chang SX, Bolan N, Wang H, Ok YS. Response of microbial communities to biochar-amended soils: a critical review. Biochar. 2019, 1(1): 3-22.

[62] Pasternak K, Kocot J, Horecka A. Biochemistry of magnesium. J Elem. 2010, 15(3): 601-616.

[63] Ren C, Zhang X, Zhang S, Wang JY, Xu M, Guo Y, Wang J, Han X, Zhao F, Yang G, Doughty R. Altered microbial CAZyme families indicated dead biomass decomposition following afforestation. Soil Biol Biochem. 2021, 160. 108362

[64] Santos-Medellin C, Zinke LA, ter Horst AM, Gelardi DL, Parikh SJ, Emerson JB. Viromes outperform total metagenomes in revealing the spatiotemporal patterns of agricultural soil viral communities. ISME J. 2021, 15(7): 1956-1970.

[65] Santos-Medellín C, Estera-Molina K, Yuan M, Pett-Ridge J, Firestone MK, Emerson JB. Spatial turnover of soil viral populations and genotypes overlain by cohesive responses to moisture in grasslands. Proc Natl Acad Sci. 2022, 119(45. e2209132119

[66] Shang J, Sun Y. CHERRY: a computational method for accurate prediction of virus-prokaryotic interactions using a graph encoder-decoder model. Brief Bioinform. 2022, 235. bbac182

[67] Singh H, Northup BK, Rice CW, Prasad PVV. Biochar applications influence soil physical and chemical properties, microbial diversity, and crop productivity: a meta-analysis. Biochar. 2022, 41. 8

[68] Stegen JC, Lin X, Fredrickson JK, Chen X, Kennedy DW, Murray CJ, Rockhold ML, Konopka A. Quantifying community assembly processes and identifying features that impose them. ISME J. 2013, 711): 2069-2079.

[69] Steinegger M, Söding J. Clustering huge protein sequence sets in linear time. Nat Commun. 2018, 9(1. 2542

[70] Sun Q, Zhang Q, Huang Z, Wei C, Li Y, Xu H. Effect of organic fertilizer application on microbial community regulation and pollutant accumulation in typical red soil in South China. Agronomy. 2024, 149. 2150

[71] Trivedi P, Leach JE, Tringe SG, Sa T, Singh BK. Plant-microbiome interactions: from community assembly to plant health. Nat Rev Microbiol. 2020, 1811): 607-621.

[72] Tyc O, Song C, Dickschat JS, Vos M, Garbeva P. The ecological role of volatile and soluble secondary metabolites produced by soil bacteria. Trends Microbiol. 2017, 25(4): 280-292.

[73] Urgessa TG. Effect of different source and rates of biochar application on selected physic-chemical properties of acidic soil in western ethiopia. Modern Chemistry. 2021, 9(4): 77-82.

[74] Uchimiya M, Chang S, Klasson KT. Screening biochars for heavy metal retention in soil: role of oxygen functional groups. J Hazard Mater. 2011, 190(1): 432-441.

[75] Wang J, Wang S. Preparation, modification and environmental application of biochar: a review. J Clean Prod. 2019, 227: 1002-1022.

[76] Wang J, Appidi MR, Burdick LH, Abraham PE, Hettich RL, Pelletier DA, Doktycz MJ. Formation of a constructed microbial community in a nutrient-rich environment indicates bacterial interspecific competition. mSystems. 2024, 9. e00006-24

[77] Wei X, Ge T, Wu C, Wang S, Mason-Jones K, Li Y, Zhu Z, Hu Y, Liang C, Shen J, Wu J, Kuzyakov Y. T4–like phages reveal the potential role of viruses in soil organic matter mineralization. Environmental Science & Technology. 2021, 55(9): 6440-6448.

[78] Wen T, Xie P, Yang S, Niu G, Liu X, Ding Z, Xue C, Liu Y-X, Shen Q, Yuan J. ggClusterNet: an R package for microbiome network analysis and modularity-based multiple network layouts. iMeta. 2022, 1(3. e32

[79] Withers E, Hill PW, Chadwick DR, Jones DL. Use of untargeted metabolomics for assessing soil quality and microbial function. Soil Biol Biochem. 2020, 143. 107758

[80] Wong JWC, Fang M. Effects of lime addition on sewage sludge composting process. Water Res. 2000, 3415): 3691-3698.

[81] Wu H, Wan S, Ruan C, Niu X, Chen G, Liu Y, Zhu K, Schulin R, Wang G. Phage-bacterium interactions and nutrient availability can shape C and N retention in microbial biomass. Eur J Soil Sci. 2022, 73(4. e13296

[82] Yao Y, Gao B, Zhang M, Inyang M, Zimmerman AR. Effect of biochar amendment on sorption and leaching of nitrate, ammonium, and phosphate in a sandy soil. Chemosphere. 2012, 8911): 1467-1471.

[83] Yuan J-H, Xu R-K, Zhang H. The forms of alkalis in the biochar produced from crop residues at different temperatures. Bioresour Technol. 2011, 102(3): 3488-3497.

[84] Zhang M, Zhang L, Riaz M, Xia H, Jiang C. Biochar amendment improved fruit quality and soil properties and microbial communities at different depths in citrus production. J Clean Prod. 2021, 292. 126062

[85] Zhao J, Qiu YB, Yi F, Li J, Wang X, Fu Q, Fu X, Yao Z, Dai Z, Qiu YP, Chen H. Biochar dose-dependent impacts on soil bacterial and fungal diversity across the globe. Sci Total Environ. 2024, 930. 172509

[86] Zheng X, Xu W, Dong J, Yang T, Shangguan Z, Qu J, Li X, Tan X. The effects of biochar and its applications in the microbial remediation of contaminated soil: a review. J Hazard Mater. 2022, 438. 129557

[87] Zhong Y, Yan W, Wang R, Wang W, Shangguan Z. Decreased occurrence of carbon cycle functions in microbial communities along with long-term secondary succession. Soil Biol Biochem. 2018, 123: 207-217.

[88] Zhou G, Chen L, Zhang C, Ma D, Zhang J. Bacteria-virus interactions are more crucial in soil organic carbon storage than iron protection in biochar-amended paddy soils. Environ Sci Technol. 2023, 57(48): 19713-19722.

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

Funding

National Natural Science Foundation of China(42277282)

the Public Welfare Technology Application Research Project of Zhejiang Province(LGF21D010002)

Basic and Applied Basic Research Foundation of Guangdong Province(2022A1515010861)

Shenzhen Science and Technology Program(JCYJ20220530150201003)

RIGHTS & PERMISSIONS

The Author(s)