Manganese-Loaded Biochar Supercharges Fungal Humification of Crop Waste

In a 35-day solid-state fermentation experiment using wheat straw, the manganese-modified biochar, or MBC, increased lignin degradation by 93.4 percent, humic acid content by 47.7 percent, and the degree of polymerization of humic material by 112.4 percent compared with a control receiving fungi alone.

White-rot fungi secrete lignin peroxidase, manganese peroxidase, and laccase, enzymes that together dismantle the tough aromatic scaffolding of lignin. Their practical use, however, has been limited by fluctuating ligninolytic activity and incomplete substrate decomposition during bioreactor operation.

To build the amendment, the team loaded Mn(II) onto bamboo-derived biochar by impregnation. Characterization confirmed that manganese had bound to the biochar surface, reaching a total manganese content of 11.61 percent, roughly 89.1 percent of it in the Mn(II) form. Compared with unmodified biochar, the modified material showed slightly lower pH, higher electrical conductivity, and increases in both specific surface area and average pore size. The fermentation setup used 10 grams of wheat straw in flasks, with the carbon-to-nitrogen ratio adjusted to 20 and moisture held at 60 percent. Four treatments ran in parallel: fungi alone as the control, modified biochar without fungi to test abiotic effects, fungi plus pristine biochar, and fungi plus manganese-modified biochar, each at a 5 percent addition rate. Sampling occurred on days 7, 14, 21, 28, and 35, with three replicates per treatment.

By day 35, straw in the manganese-modified treatment had fragmented extensively into fine debris and fibrous structures, and scanning electron microscopy revealed surfaces transformed from smooth and compact to roughened textures studded with irregular pores, deep cavities, and dissociated fiber bundles. Fourier transform infrared spectroscopy showed attenuated peaks associated with aliphatic chain disruption, beta-glycosidic bond cleavage, hemicellulose acetyl dissociation, and lignin side-chain cleavage. X-ray diffraction recorded a 14.5 percent drop in cellulose crystallinity in the modified biochar treatment, roughly triple the 5.0 percent decrease seen in the fungal control, indicating deeper disruption of the crystalline hydrogen-bond network. Solid-state carbon-13 nuclear magnetic resonance confirmed weakened signals across diagnostic polysaccharide and lignin regions. Degradation rates of lignin, cellulose, and hemicellulose rose by 93.4, 77.0, and 30.9 percent respectively over the control, exceeding the gains of 53.7, 57.0, and 20.48 percent achieved with pristine biochar.

Humic substance and humic acid contents climbed throughout fermentation while fulvic acid, a low-molecular-weight transitional fraction, declined, consistent with its conversion into heavier, more aromatic humic acid through polycondensation with quinones, polyphenols, and amino acids. On day 35, the manganese-modified treatment recorded the highest humic substance and humic acid levels, 15.4 percent and 47.7 percent above the control, and the lowest fulvic acid content. The humic acid to fulvic acid ratio, an index of polymerization, rose 42.8 percent with pristine biochar and 112.4 percent with the modified material. Three-dimensional fluorescence spectroscopy tracked the shift molecularly: protein-like fluorescence dominated early, but by day 14 the modified treatment already showed intensified fulvic-like, soluble microbial byproduct, and humic acid-like signals, a transition other treatments did not display until day 28. The humification index peaked in the modified treatment, alongside elevated biological index values signaling stronger fungal metabolic activity.

To see what was happening at the molecular scale, the researchers turned to Fourier transform ion cyclotron resonance mass spectrometry, a technique capable of resolving the elemental formulas of thousands of dissolved organic matter compounds. Dissolved organic matter, the most reactive fraction in solid-state fermentation, serves as both substrate and intermediate in microbial metabolism. Formula assignments placed DOM chiefly in CHO compounds, followed by CHON, CHOS, and CHONS classes. Over fermentation, nitrogen- and sulfur-containing compounds declined while CHO compounds rose, driven largely by lignin-like and tannin-like substances. Tannin-like abundance jumped from an initial 8 percent to 23 percent in the control and 22 percent in the modified treatment, laying groundwork for phenolic oxidation-polymerization. Weighted average molecular indices told a coherent story: hydrogen-to-carbon ratios fell while oxygen-to-carbon ratios, double bond equivalents, and modified aromaticity climbed, marking rising unsaturation, aromaticity, and oxidation. A rapid early decline in H/C paired with rising O/C suggested dehydrogenation and cyclization consistent with lignin oxidative dehydrogenation toward quinone structures.

The team then classified molecules as precursors, which disappeared, resistant compounds, which persisted, and products, which newly formed. Precursors clustered at low oxygen-to-carbon and high hydrogen-to-carbon regions, while products concentrated where oxygen was high and hydrogen low, implying fungal-driven evolution toward stable humic structures through dehydrogenation, condensation, and side-chain oxidation. A random forest machine learning model, interpreted through SHAP values, identified 15 molecular features predicting reactivity. Across treatments, molecules rich in nitrogen and sulfur, high in double bond equivalent minus oxygen, and low in oxygen-to-carbon ratio proved most reactive, indicating that humification selectively consumes heteroatom-rich, reduced aromatic precursors rather than proceeding randomly. Class-specific patterns followed: lignin-like molecules with low oxidation and high unsaturation were primed for oxidative activation into aromatic backbones, tannin-like polyphenols were readily oxidized into quinone-like intermediates, and protein-like molecules with high hydrogen and nitrogen content served as both substrates and nitrogen donors for condensation.

Mass difference analysis across 24 reaction types quantified the transformation pathways. The modified biochar treatment showed significantly higher overall DOM reactivity than the control. Within it, deamination and desulfurization reactions were most active at 28.79 percent, followed by delignification at 24.1 percent and dealkylation at 17.06 percent, with deamination peaking during the first 14 days. Oxidative deamination likely released amino-containing intermediates and restructured carbon skeletons, while delignification released methoxy groups and monophenolic derivatives such as phenol, guaiacol, and syringol, recognized structural precursors of humification. Notably, the Maillard reaction pathway, in which dicarbonyl compounds polymerize with amino acids, contributed only 8.86 percent in the modified treatment against 17.02 percent in the control. The authors attribute this shift to the abundance of polyphenolic precursors generated by enhanced lignocellulose degradation, which strengthened the polyphenol-dominated humification route at the expense of the Maillard route.

Fungal biomass in the modified treatment reached 2.82 times that of the control by day 35, with biofilm entering exponential growth after day 14, and straw surfaces fully colonized by mycelia. Activities of lignin peroxidase, manganese peroxidase, and laccase, along with hydroxyl radical production, all ranked modified biochar above pristine biochar above control. The elevated hydroxyl radical level likely reflects both fungal metabolism supplying hydrogen peroxide and Mn(II) reacting with it through Fenton-like reactions, a non-enzymatic oxidative route that complements enzymatic attack. Transcriptomics on day 21 identified 451 upregulated and 354 downregulated genes versus the control, including 37 differentially expressed CAZyme genes, mostly glycoside hydrolases and auxiliary activity enzymes such as lignin and manganese peroxidases and lytic polysaccharide monooxygenases. KEGG enrichment pointed to carbohydrate metabolism, amino acid metabolism, and quinone compound biosynthesis. Metabolomics on day 35 found 314 differential metabolites, 183 upregulated, including phenolic compounds, phenolic acids, glycosides, organic acids, and amino acids, with 39 enhanced pathways supporting precursor generation.

The authors are careful about the inferential limits of their framework. Machine learning results reflect predictive associations rather than causal proof, and the proposed deamination, dealkylation, and delignification pathways were inferred from molecular formulas and exact mass-difference matching rather than directly verified structural conversions, leaving open the possibility of false positives in mass-association analysis. They recommend stable isotope labeling and equivalent free-Mn(II) treatments in future work to separate the contributions of the biochar matrix, manganese species, and fungal metabolism. Practical questions also remain: manganese release, migration, and leaching in soils cannot be neglected, and the optimal application rate in real composting will depend on amendment cost, manganese loading, and substrate heterogeneity.

Subject of Research: How manganese-modified biochar enhances white-rot fungi-mediated lignocellulose humification

Article Title: Manganese-modified biochar enhances white-rot fungi-mediated lignocellulose humification: insights from molecular transformation and multi-omics analyses

Article References: Wang, F., Zhang, L., Zhou, F., Wang, W., Su, Y., Zhou, D., Hong, S., Zhan, M., & Xie, B. (2026). Manganese-modified biochar enhances white-rot fungi-mediated lignocellulose humification: insights from molecular transformation and multi-omics analyses. Biochar, 8(1), Article 140. https://doi.org/10.1007/s42773-026-00659-z

Image Credits: AI Generated

DOI: 10.1007/s42773-026-00659-z

Keywords: biochar, manganese, white-rot fungi, lignocellulose, humification, humic substances, solid-state fermentation, FT-ICR MS, transcriptomics, metabolomics, soil carbon, wheat straw