A lignin-derived material improves plant nutrient bioavailability and growth through its metal chelating capacity

The study addresses two interconnected challenges: the low valorization of industrial lignin from lignocellulosic biorefineries and widespread micronutrient deficiencies (notably iron) in crops grown on alkaline soils. Industrial lignin, especially sulfuric acid lignin (SAL) generated during sulfuric acid hydrolysis, is highly condensed, poorly soluble, and largely burned, contributing substantially to CO2 emissions and undermining the sustainability of biorefineries. Concurrently, hidden hunger affects about one third of the global population due to low bioavailability of micronutrients like Fe, Ca, and Zn in staple crops, exacerbated by alkaline soils (25–40% of arable land), unbalanced fertilizer use, and rising CO2. Synthetic chelators such as EDTA can increase metal bioavailability but are costly and non-biodegradable, posing environmental risks. Lignin-derived materials and humic substances possess functional groups (phenolic OH, carbonyl, aliphatic OH) capable of metal chelation and could offer a sustainable alternative. The study’s purpose is to convert SAL into a water-soluble lignin material (HSAL) with enhanced metal-chelating functionality and to test whether HSAL improves nutrient bioavailability and plant growth across species, with a focus on iron, and to benchmark HSAL’s efficacy against EDTA.

The paper situates the work within: (1) biorefinery and lignin valorization efforts, noting current technologies (pyrolysis, hydrogenolysis, oxidation) and products (carbon fibers, bioplastics, fuels, commodity chemicals), yet only ~2% of industrial lignin is valorized; (2) agronomic and nutritional challenges of micronutrient malnutrition and iron deficiency in alkaline soils; (3) the role and drawbacks of synthetic chelators (EDTA, EDDHA, DTPA) including cost and persistence; (4) evidence that lignin-derived compounds constitute major inputs to humic substances, which act as natural chelators enhancing metal availability; (5) prior uses of lignosulfonates (e.g., lignosulfonate-iron) and humic substances improving plant nutrient uptake. The review highlights a gap: industrial lignin addition to fertilizers has been underexplored despite lignin’s chelating potential, and no studies have leveraged hydrothermally solubilized SAL as a plant growth-promoting chelator.

HSAL synthesis: High-purity SAL (simulating industrial SAL) was prepared from Japanese cedar via Klason sulfuric acid hydrolysis. A mixture of SAL (500 mg), NaOH (500 mg), and water (15 mL) was sealed in a stainless-steel tube and hydrothermally treated at 280 °C for 2 h. The reaction mixture was filtered, dialyzed through a 3500 Da MWCO cellulose membrane to remove inorganics, and lyophilized to yield HSAL powder, soluble in water at ~3 g/100 mL. Chemical characterization: Average molecular weight was assessed (SAL 57 kDa; HSAL 7.5 kDa). FT-IR (400–4000 cm−1) compared functional groups of SAL vs HSAL. Methoxy content was quantified by HI demethylation to CH3I and GC-FID. Total hydroxyls were quantified via acetylation and titration; distribution of phenolic vs aliphatic hydroxyls in HSAL was determined by 1H NMR of acetylated HSAL; phenolic OH in SAL was quantified by selective ammonolysis. Functional group counts per 100 C9 units were computed from quantitative analyses and degree of polymerization. Chelation analyses: HSAL was complexed with FeSO4, FeCl3, or CaCl2 (0.2 g HSAL in 20 mL water, add 0.1 M metal salt, stir 6 h, dialyze, lyophilize). Elemental composition and valence states were examined by XPS (Al Kα; Fe 2p and Ca 2p binding energies). FT-IR assessed shifts/bands associated with O–H and C–O (guaiacol) upon chelation. Chelation capacity was quantified by complexometric titration: Fe3+ with 0.01 M NH4Fe(SO4)2 using sulfosalicylic acid indicator; Ca2+ with 0.2 M calcium acetate under ammonia buffer using mixed dye indicator; capacities computed from titrant consumption. XRPD and TEM-EDS assessed crystallinity and morphology of HSAL and HSAL–FeCl3 complexes. Plant growth assays: Rice (Oryza sativa L. japonica cv. Nipponbare; also Taichung 65, Kasalath) were grown hydroponically in water with HSAL at 0, 0.01%, 0.05%, 0.1% (w/w) under long-day conditions for 14 d; root/shoot lengths and biomass measured. Time-course growth curves compared control vs 0.05% HSAL. Corn (Zea mays) seedlings were grown in soil and watered with control, 0.4% fertilizer, 0.4% HSAL, or both, every 2 d for 14 d; root/shoot metrics recorded. Iron-specific assays used IRRI hydroponics with iron-sufficient (36 μM EDTA-Fe(II)) or iron-deficient (removed EDTA-Fe(II)) media ±0.05% HSAL for 10 d; lengths measured; tissues dried for elemental analysis. Arabidopsis thaliana Col-0 and mutants (opt3-2, irt1-1, fro2) were grown on 1/2 MS agar ±Fe, ±0.05% HSAL; EDTA-Na2 dose–response (0–1300 μM) under ±Fe conditions performed. Cellular assays: Root cross-sections (Cryo-SEM) measured total cell numbers, radial diameters, and mature zone cell lengths after 7 d ±0.05% HSAL. EdU incorporation (5 μM, 2 h pulse) and confocal imaging assessed meristematic DNA synthesis at multiple time points up to 8 d. Transcriptomics: RNA-seq on rice root tips (5 mm) after 12 h ±0.05% HSAL; libraries (Illumina NovaSeq), alignment to O. sativa Nipponbare v4 (TopHat v2.0.9), expression quantification (FPKM, HTSeq), differential expression (DESeq2; FDR ≤0.05; |log2FC| ≥1), GO enrichment (clusterProfiler) using AgriGO/GO.db annotations. qRT-PCR validated iron transporter gene expression (six biological replicates). Elemental analyses: HSAL digest (HNO3/H2O2) quantified K, Ca, Mg, Fe, Cu, Zn by ICP-MS. Plant tissues were washed (10 mM EDTA-Na2), rinsed, dried, digested in HNO3 at high temperature; Ca, Mg, Fe, Cu, Zn quantified by MP-AES. Background metal contributions from HSAL to media quantified (Supplementary Tables). Economic assessment: Energy and reagent costs estimated for laboratory-scale and projected industrial-scale HSAL production, factoring NaOH usage (10% loss), specific heat capacities, and standard coal energy equivalence; yields assumed at 46.7%.

• Hydrothermal conversion produced a fully water-soluble lignin material (HSAL) from SAL with high yield (46.7% of SAL) and solubility (~3 g/100 mL). Average molecular weight decreased from 57 kDa (SAL) to 7.5 kDa (HSAL).
• Functional group transformations: Methoxy content decreased from 5.91 to 1.91 mmol g−1; total phenolic hydroxyls increased from 2.20 to 5.42 mmol g−1, while aliphatic hydroxyls decreased. Per 100 phenylpropane units, methoxy groups declined (SAL 0.98–1.08 to HSAL 0.32–0.35), phenolic OH increased (SAL 0.36–0.40 to HSAL 0.90–0.99), exceeding softwood lignin (0.15–0.30).
• Chelation evidence: XPS showed Fe 2p and Ca 2p signals in HSAL–Fe and HSAL–Ca complexes with shifted binding energies consistent with chelation. FT-IR exhibited O–H band shifts (e.g., 3360 cm−1 to 3320/3410 cm−1 with Fe salts; to 3400 cm−1 with CaCl2) and emergence of Fe–O (~665 cm−1) and guaiacol C–O (~1220 cm−1) bands, implicating phenolic OH in chelation. XRPD and TEM-EDS supported amorphous complexes (no crystalline inorganic phases).
• Chelation capacity (complexometric titration): HSAL chelated Fe3+ at 101.9 mg g−1 vs EDTA 138.8 mg g−1 (p=9.3E−04); HSAL chelated Ca2+ at 69.7 mg g−1 vs EDTA 173.8 mg g−1 (p=3.0E−05). On a molar basis, given HSAL’s higher MW (7.5 kDa), equimolar HSAL can bind more metals than EDTA.
• Plant growth promotion: In rice (Nipponbare) grown in water, HSAL increased root length up to 2.39-fold and root biomass 1.30-fold at 0.05% HSAL (14 d), with concomitant shoot length and biomass increases; growth advantages emerged by day 3 and persisted beyond day 7 when controls stagnated. Similar effects were observed in Taichung 65 and Kasalath rice. In corn grown in soil, root length increased 2.56-fold with 0.4% HSAL, exceeding 0.4% fertilizer alone (1.41-fold); combining HSAL + fertilizer yielded 2.80-fold increases; shoot metrics also improved.
• Cellular basis: HSAL (0.05%) increased total cell numbers (1.42-fold, p=6.8E−06) and radial cell diameters in rice roots without significantly altering mature cell length; EdU labeling indicated sustained meristematic proliferation with HSAL compared to controls by day 8.
• Transcriptomics: 956 genes up- and 1112 down-regulated after 12 h HSAL in rice root tips (FDR ≤0.05, |log2FC| ≥1). GO terms enriched included cell wall/lignin metabolism, fatty acid and carbohydrate biosynthesis, transmembrane transport, oxygen transport, nicotianamine biosynthesis; heme binding and cation binding were prominent molecular functions. Iron transport genes were induced (validated by qRT-PCR).
• Iron bioavailability: Under IRRI hydroponics, 0.05% HSAL increased rice root length 2.84-fold and shoot length 1.82-fold under Fe deficiency; roots with HSAL under −Fe exceeded +Fe controls. Fe content increased in roots (1.57×) and shoots (1.17×) under −Fe with HSAL; under +Fe, Fe increased 2.12× (roots) and 2.33× (shoots). HSAL also increased Ca and Cu in both organs and Mg/Zn in roots. Trace metals contributed by HSAL to media were negligible relative to media concentrations, confirming enhanced bioavailability rather than supplementation.
• Mechanism and comparators in Arabidopsis: 0.05% HSAL rescued chlorosis (chlorophyll +31%) and tripled root length (3.08×) under −Fe. HSAL alleviated Ca and Mg deficiency phenotypes to a lesser extent. In mutants, HSAL rescued opt3-2 but not irt1-1 or fro2, indicating dependence on FRO2-mediated ferric reduction and IRT1 ferrous uptake—consistent with HSAL acting as a ferric chelator. EDTA exhibited a similar rescue pattern across genotypes and conditions, indicating comparable functional mode.
• Economics and sustainability: Estimated production cost for HSAL is USD 329–430 per ton at lab scale and USD 139–213 per ton at industrial scale, far below synthetic chelators (USD 3,500–350,000 per ton) and lignosulfonates (USD 495–850 per ton). Deploying HSAL at 0.05% over iron-deficient lands could utilize a large fraction of industrial lignin and avoid substantial CO2 emissions otherwise produced by lignin burning.

The findings demonstrate that hydrothermal conversion of industrial SAL yields a water-soluble lignin fraction (HSAL) enriched in phenolic hydroxyl groups that confers substantive metal-chelating capability. This structural shift underpins HSAL’s ability to chelate ferric iron and other cations, improving nutrient mobility and availability at the root–soil/medium interface. In both monocots (rice, Strategy II) and dicots (Arabidopsis, Strategy I), HSAL enhanced root meristem activity and overall growth under nutrient-poor and iron-deficient conditions. Genetic evidence in Arabidopsis (requirement for FRO2 and IRT1) indicates that HSAL functions as a ferric iron chelator akin to EDTA: ferric iron chelated by HSAL is reduced extracellularly and transported as ferrous iron into root cells. In rice, HSAL likely enhances ferric mobility and apoplastic availability, with subsequent uptake as phytosiderophore–Fe complexes. By matching EDTA’s efficacy in improving iron bioavailability while being derived from lignin and expected to be biodegradable similar to humic substances, HSAL offers an environmentally preferable and cost-effective chelator. Beyond iron, HSAL increased availability of Ca, Mg, Zn, and Cu, suggesting broader utility to mitigate multiple micronutrient limitations. Economically, HSAL production is inexpensive and scalable, potentially transforming a waste liability into a high-demand agricultural input while reducing CO2 emissions from lignin combustion and potentially enhancing soil carbon sequestration by promoting root growth.

This work introduces HSAL, a hydrothermally derived, water-soluble lignin material with increased phenolic hydroxyl content and robust metal-chelating capacity. HSAL enhances metal nutrient bioavailability—especially iron—and promotes root proliferation, root length, and biomass across monocot and dicot species, performing comparably to EDTA via a ferric chelation mechanism requiring FRO2 and IRT1. The approach valorizes industrial lignin into a low-cost, potentially biodegradable agricultural chelator with environmental and economic benefits, offering a pathway to improve crop nutrition and sustainability. Potential future directions indicated by the study include: mechanistic dissection of HSAL–iron dynamics in Strategy II plants (e.g., release and uptake routes relative to phytosiderophores), optimization of HSAL formulation and application rates across crops and soils (including alkaline and micronutrient-deficient soils), assessment of long-term environmental fate and biodegradation in field conditions, evaluation of interactions with other soil metals and nutrients, and scaling/optimization of industrial HSAL production from diverse lignin feedstocks.