Exfoliated biochar activates soil microbial metabolism and demonstrates immobilization potential toward toxic metals and metalloids

Abstract

Biochar is widely recognized for its potential to enhance soil microbial activity and immobilize toxic heavy metals. However, its large-scale adoption is limited by inconsistent performance and high market costs (320 to 800 € m⁻³). Here, we present a cost-competitive strategy for the industrial-scale production of exfoliated biochar from phytowaste feedstock and its robust validation in soil–microbial systems. Exfoliated biochar was continuously manufactured at pilot-to-industrial scale and evaluated across multiple independent trials using representative agricultural soils. Its performance was systematically benchmarked against virgin biochar in terms of microbial metabolic activation and immobilization of environmentally relevant heavy metals (Cu²⁺, Cr⁶⁺, and As³⁺). Across all validation sets, exfoliated biochar consistently promoted higher microbial metabolic activity and superior metal adsorption efficiency, demonstrating both reproducibility and process robustness. These enhancements are attributed to increased surface area, optimized pore architecture, and improved accessibility of reactive functional groups introduced during exfoliation. Molecular dynamics simulations combined with radial distribution function analyses revealed distinct adsorption mechanisms, including preferential interactions of hydroxyl (–OH) groups with Cr⁶⁺ ions and pyridinic nitrogen sites with Cu²⁺ ions. Complementary spectroscopic analyses further identified aliphatic hydrocarbons, aromatic domains, aromatic C=C bonds, and hydrogen-bonded –OH groups as major contributors to adsorption performance. Overall, this study demonstrates that industrially scalable exfoliation, coupled with targeted structural and functional optimization, enables reproducible enhancement of biochar–microbe–metal interactions. This approach provides a robust, systems-oriented pathway for sustainable soil remediation and biomanufacturing-relevant environmental applications.

Keywords

Soil microbiota / Biochar / Heavy metals / Bioeconomy

References

[1] Berendsen HJ, van der Spoel D, van Drunen R. GROMACS: a message-passing parallel molecular dynamics implementation. Comput Phys Commun, 1995, 91(1–3): 43-56

[2] Bernas J, Bernasova T, Gerstberger P, Moudry J, Konvalina P, Moudry JJr. Cup plant, an altervirgin to conventional silage from a LCA perspective. Int J Life Cycle Assessm, 2021, 26(2): 311-326

[3] Bogusz A, Oleszczuk P, Dobrowolski R. Application of laboratory prepared and commercially available biochars to adsorption of cadmium, copper and zinc ions from water. Bioresource Technol, 2015, 196: 540-549

[4] Brabenec T, Marouskova A, Zoubek T, Filip M. Residues from water precipitation via ferric hydroxide threaten soil fertility. Sustainability, 2021, 13(8): 4327

[5] Chi J, Liu H. Effects of biochars derived from different pyrolysis temperatures on growth of Vallisneria spiralis and dissipation of polycyclic aromatic hydrocarbons in sediments. Ecol Eng, 2016, 93: 199-206

[6] Cui S, Zhang R, Peng Y, Gao X, Li Z, Fan B, Guo L. New insights into ball milling effects on MgAl-LDHs exfoliation on biochar support: a case study for cadmium adsorption. J Hazard Mater, 2021, 416 Article ID: 126258

[7] Darden T, York D, Pedersen L. Particle mesh Ewald: An N⋅ log (N) method for Ewald sums in large systems. J Chem Phys, 1993, 98(12): 10089-10092

[8] Dong X, Guan T, Li G, Lin Q, Zhao X. Long-term effects of biochar amount on the content and composition of organic matter in soil aggregates under field conditions. J Soils Sediments, 2016, 16: 1481-1497

[9] Doušová B, Bedrnová E, Maxová K, Lhotka M, Pilař L, Koloušek D, Pohořelý M. Biochar control of water regime and adsorption rate in soils. Appl Sci, 2025, 15(17): 9392

[10] Ezzat AO, Iwuozor KO, Al-Dhayan D, Al-Lohedan HA, Micheal KT, Emenike EC, Adeniyi AG. Effect of different exfoliation routes on the properties of chicken feather and elephant grass hybrid biochar. Biofuels, Bioprod Biorefin, 2024, 18(5): 1511-1525

[11] Genovese M, Jiang J, Lian K, Holm N. High capacitive performance of exfoliated biochar nanosheets from biomass waste corn cob. J Mater Chem A, 2015, 3(6): 2903-2913

[12] Ghorbani M, Amirahmadi E, Neugschwandtner RW, Konvalina P, Kopecky M, Moudry J, Murindangabo YT. The impact of pyrolysis temperature on biochar properties and its effects on soil hydrological properties. Sustainability, 2022, 14(22): Article ID: 14722

[13] Godlewska P, Ok YS, Oleszczuk P. The dark side of black gold: ecotoxicological aspects of biochar and biochar-amended soils. J Hazard Mater, 2021, 403 Article ID: 123833

[14] Goswami R, Deb P, Thakur R, Sarma KP, Basumallick A. Removal of As (III) from aqueous solution using functionalized ultrafine iron oxide nanoparticles. Sep Sci Technol, 2011, 46(6): 1017-1022

[15] Haskova S. Holistic assessment and ethical disputation on a new trend in solid biofuels. Sci Eng Ethics, 2017, 23(2): 509-519

[16] Humphrey W, Dalke A, Schulten K. VMD: visual molecular dynamics. J Mol Graph, 1996, 14(1): 33-38

[17] Iacomino G, Idbella M, di Costanzo L, Amoroso G, Allevato E, Abd-ElGawad AM, Bonanomi G. Biochar aging, soil microbiota and chemistry of charcoal kilns in Mediterranean forests. Biochar, 2024, 6(1): Article ID: 82

[18] Jacka L, Trakal L, Ourednicek P, Pohorely M, Sipek V. Biochar presence in soil significantly decreased saturated hydraulic conductivity due to swelling. Soil Tillage Res, 2018, 184: 181-185

[19] Kopecky M, Kolar L, Konvalina P, Strunecky O, Teodorescu F, Mraz P, Bucur D. Modified biochar—a tool for wastewater treatment. Energies, 2020, 13(20): Article ID: 5270

[20] Kopecky M, Peterka J, Kolar L, Konvalina P, Marousek J, Vachalova R, Tran DK. Influence of selected maize cultivation technologies on changes in the labile fraction of soil organic matter sandy-loam cambisol soil structure. Soil Tillage Res, 2021, 207 Article ID: 104865

[21] Kumar R, Patel M, Singh P, Bundschuh J, Pittman CUJr, Trakal L, Mohan D. Emerging technologies for arsenic removal from drinking water in rural and peri–urban areas: methods, experience from, and options for Latin America. Sci Total Environ, 2019, 694 Article ID: 133427

[22] Li TT, Wang K, Sueyoshi T, Wang DD. ESG: research progress and future prospects. Sustainability, 2021, 13(21): Article ID: 11663

[23] Li F, Shen K, Long X, Wen J, Xie X, Zeng X, Liang Y, Wei Y, Lin Z, Huang W, Zhong R. Preparation and characterization of biochars from Eichornia crassipes for cadmium removal in aqueous solutions. PLoS ONE, 2016, 11(2): Article ID: e0148132

[24] Lindahl E, Hess B, Van Der Spoel D. GROMACS 3.0: a package for molecular simulation and trajectory analysis. J Mol Model, 2001, 7: 306-317

[25] Lu K, Yang X, Gielen G, Bolan N, Ok YS, Niazi N, Xu S, Yuan G, Chen X, Zhang X, Liu D, Song Z, Liu X, Wang H. Effect of bamboo and rice straw biochars on the mobility and redistribution of heavy metals (Cd, Cu, Pb and Zn) in contaminated soil. J Environ Manage, 2017, 186: 285-292

[26] Mardoyan A, Braun P. Analysis of Czech subsidies for solid biofuels. Int J Green Energy, 2015, 12(4): 405-408

[27] Mark P, Nilsson L. Structure and dynamics of the TIP3P, SPC, and SPC/E water models at 298 K. J Phys Chem A, 2001, 105(43): 9954-9960

[28] Marousek J, Marouskova A, Gavurova B, Minofar B. Techno–economic considerations on cement substitute obtained from waste refining. J Clean Prod, 2023, 412 Article ID: 137326

[29] Marousek J, Gavurova B, Marouskova A. Cost breakdown indicates that biochar production from microalgae in Central Europe requires innovative cultivation procedures. Energy Nexus, 2024, 16 Article ID: 100335

[30] Marousek J, Gavurova B, Marouskova A, Minofar B. Techno-economic and environmental considerations of digestate oil enrichment via Aluminum oxide nanoparticles from beverage packaging waste. Renew Energy, 2025

[31] Marousek J, Zakova K. Techno-economic perspective on the use of pyrolysis oil from digestate in spark-ignition engines. Aircraft Eng Aerosp Technol 2025.

[32] Minofar B, Milcic N, Marousek J, Gavurova B, Marouskova A. Understanding the molecular mechanisms of interactions between biochar and denitrifiers in N₂O emissions reduction: pathway to more economical and sustainable fertilizers. Soil till Res, 2025, 248 Article ID: 106405

[33] Mitchell K, Beesley L, Sipek V, Trakal L. Biochar and its potential to increase water, trace element, and nutrient retention in soils. In: Biochar in Agriculture for Achieving Sustainable Development Goals (pp. 25–33). Academic Press, 2022.

[34] Oleszczuk P, Josko I, Kusmierz M. Biochar properties regarding to contaminants content and ecotoxicological assessment. J Hazard Mater, 2013, 260: 375-382

[35] Panwar NL, Pawar A. Influence of activation conditions on the physicochemical properties of activated biochar: a review. Biomass Convers Biorefinery, 2020

[36] Rovira P, Vallejo VR. Labile and recalcitrant pools of carbon and nitrogen in organic matter decomposing at different depths in soil: an acid hydrolysis approach. Geoderma, 2002, 107(1-2): 109-141

[37] Roy S, Kumar U, Bhattacharyya P. Synthesis and characterization of exfoliated biochar from four agricultural feedstock. Environ Sci Pollut Res, 2019, 26(7): 7272-7276

[38] Saliu OD, Adeniyi AG, Mamo M, Ndungu P, Ramontja J. Microwave exfoliation of a biochar obtained from updraft retort carbonization for supercapacitor fabrication. Electrochem Commun, 2022, 139 Article ID: 107308

[39] Schaftenaar G, Vlieg E, Vriend G. Molden 2.0: quantum chemistry meets proteins. J Comput Aided Mol des, 2017, 31: 789-800

[40] Schaftenaar G, Noordik JH. Molden: a pre-and post-processing program for molecular and electronic structures. J Comput Aided Mol des, 2000, 14: 123-134

[41] Seyedsadr S, Sipek V, Jacka L, Snehota M, Beesley L, Pohorely M, Trakal L. Biochar considerably increases the easily available water and nutrient content in low-organic soils amended with compost and manure. Chemosphere, 2022, 293 Article ID: 133586

[42] Shokrollahi A, Alizadeh A, Malekhosseini Z, Ranjbar M. Removal of bromocresol green from aqueous solution via adsorption on Ziziphus nummularia as a new, natural, and low-cost adsorbent: kinetic and thermodynamic study of removal process. J Chem Eng Data, 2011, 56(10): 3738-3746

[43] Stehel V, Marouskova A, Kolar L. Techno-economic analysis of fermentation residues management places a question mark against current practices. Energy Sources A, 2018, 40(6): 721-726

[44] Stehel V, Marouskova A, Kolar L, Strunecky O, Shreedhar S. Advances in dry fermentation extends biowaste management possibilities. Energy Sources A, 2020, 42(2): 212-218

[45] Strunecky O, Shreedhar S, Kolar L, Marouskova A. Changes in soil water retention following biochar amendment. Energy Sources Part A Util Environ Eff, 2021

[46] Sun Y, Gao B, Yao Y, Fang J, Zhang M, Zhou Y, Chen H, Yang L. Effects of feedstock type, production method, and pyrolysis temperature on biochar and hydrochar properties. Chem Eng J, 2014, 240: 574-578

[47] Trakal L, Veselska V, Safarik I, Vitkova M, Cihalova S, Komarek M. Lead and cadmium sorption mechanisms on magnetically modified biochars. Bioresour Technol, 2016, 203: 318-324

[48] Trakal L, Raya-Moreno I, Mitchell K, Beesley L. Stabilization of metal (loid) s in two contaminated agricultural soils: comparing biochar to its non-pyrolysed source material. Chemosphere, 2017, 181: 150-159

[49] Trakal L, Michálková Z, Beesley L, Vítková M, Ouředníček P, Barceló AP, Komárek M. AMOchar: amorphous manganese oxide coating of biochar improves its efficiency at removing metal (loid) s from aqueous solutions. Sci Total Environ, 2018, 625: 71-78

[50] Uchimiya M, Wartelle LH, Klasson KT, Fortier CA, Lima IM. Influence of pyrolysis temperature on biochar property and function as a heavy metal sorbent in soil. J Agric Food Chem, 2011, 59(6): 2501-2510

[51] Van Der Spoel D, Lindahl E, Hess B, Groenhof G, Mark AE, Berendsen HJ. GROMACS: fast, flexible, and free. J Comput Chem, 2005, 26(16): 1701-1718

[52] Vochozka M, Marouskova A, Vachal J, Strakova J. Biochar pricing hampers biochar farming. Clean Technol Environ Policy, 2016, 18(4): 1225-1231

[53] Vochozka M, Marouskova A, Vachal J, Strakova J. Advances in economically driven optimization of processing of biosolids from sewage sludge. Energy Sources Part A Util Environ Eff, 2016, 38(16): 2413-2417

[54] Vochozka M, Marouskova A. Assessment of shockwave pretreatment in biomass processing. Energy Sour Part a Recov Util Environ Effects, 2017, 39(11): 1195-1199

[55] Vochozka M, Marouskova A. Valuation of new inhibitors detection method. Waste Biomass Valoriz, 2018, 9(7): 1243-1246

[56] Wang J, Wolf RM, Caldwell JW, Kollman PA, Case DA. Development and testing of a general Amber force field. J Comput Chem, 2004, 25(9): 1157-1174

[57] Wang J, Wang W, Kollman PA, Case DA. Automatic atom type and bond type perception in molecular mechanical calculations. J Mol Graph Model, 2006, 25(2): 247-260

[58] Wen E, Yang X, Chen H, Shaheen SM, Sarkar B, Xu S, Wang H. Iron-modified biochar and water management regime-induced changes in plant growth, enzyme activities, and phytoavailability of arsenic, cadmium and lead in a paddy soil. J Hazard Mater, 2021, 407 Article ID: 124344

[59] Zhang X, Li Y, Wang H, et al. One-step method for preparing magnetic magnesium–iron layered double hydroxide (LDH)–biochar composite for efficient removal of heavy metals from water. Chem Eng J 2025;172186.

[60] Zielinska A, Oleszczuk P, Charmas B, Skubiszewska-Zieba J, Pasieczna-Patkowska S. Effect of sewage sludge properties on the biochar characteristic. J Anal Appl Pyrolysis, 2015, 112: 201-213

Funding

Technical University of Kosice

RIGHTS & PERMISSIONS

The Author(s)