Production of Bio-derived Humic Acid from Lignocellulosic Wastes Using Trichoderma harzianum and Evaluating its Structural and Antifungal Properties effect on Wheat on Wheat Growth

Introduction
Humic substances are essential organic components of soil that significantly influence nutrient availability, microbial activity, and soil aggregation. Conventional humic acids are mainly extracted from mineral sources such as leonardite, which are nonrenewable and environmentally limiting. Consequently, interest has grown in bio-derived humic substances produced from agricultural residues via microbial fermentation. Among microorganisms, Trichoderma harzianum is recognized for its lignocellulolytic and biocontrol capabilities, making it an ideal candidate for converting lignocellulosic wastes into biologically active humic-like substances. The present study was designed to produce bio-humic acid through solid-state fermentation of agricultural residues using T. harzianum, characterize its chemical and structural features, and evaluate its antifungal and growth-promoting effects on wheat under greenhouse conditions.
 
Materials and Methods
The fermentation was carried out in a 500-liter polyethylene solid-state bioreactor equipped with a centrifugal blower for aeration and sensors for temperature and oxygen monitoring. The lignocellulosic substrate consisted of chopped wheat straw (40–50 mm) adjusted to 60% moisture. Pasteurization was performed using steam at 80 °C for 60–90 minutes to minimize microbial contamination. The substrate was supplemented with 1% molasses (Brix 60) to provide easily available carbon. The fungal inoculum, identified as T. harzianum based on β-tubulin gene sequencing (accession no. PV176108), was introduced, and the fermentation proceeded at 28–30 °C for 8–10 days. After completion, humic substances were extracted using 4% potassium hydroxide, followed by multistage filtration and centrifugation. The filtrate was sterilized for further analysis. Structural and chemical characterization involved Fourier transform infrared spectroscopy, ultraviolet–visible spectrophotometry, field emission scanning electron microscopy, gas chromatography–mass spectrometry, and liquid chromatography–mass spectrometry. The antifungal effect of the bio-humic extract was tested against Fusarium graminearum and Rhizoctonia solani using the well diffusion method, while the plant growth response was evaluated in a greenhouse pot experiment under artificial infection with Fusarium. Enzymatic activity was determined on carboxymethyl cellulose agar to verify cellulase production by T. harzianum.
 
Results
The FTIR spectrum of the bio-humic acid exhibited characteristic absorption bands at approximately 3400 cm⁻¹ (O–H stretching), 1720 cm⁻¹ (C=O stretching of carboxylic and carbonyl groups), and 1100–600 cm⁻¹ (C–O and C–H bending). The higher intensity of the carbonyl band relative to the commercial humic acid indicated greater abundance of oxygen-containing functional groups and a more reactive molecular structure. UV–Vis analysis showed a higher E4/E6 ratio, suggesting lower aromaticity and higher biological reactivity. FE-SEM micrographs obtained at two magnifications revealed a layered, porous morphology with cellulose nanocrystals between 30 and 100 nm. The nanostructure reflected partial enzymatic degradation of lignocellulosic fibers during fermentation, increasing surface area and adsorption capacity. Such morphology implies enhanced ion exchange and improved water-holding potential in soil. Chromatographic analyses confirmed the presence of aromatic esters, phenolic compounds, and organic acids derived from fungal metabolism. The methanolic extract contained Benzenpropanoic acid, 3,5-bis(1,1-dimethylethyl)-4-hydroxy-, methyl ester, while the ethyl acetate extract predominantly contained 1,4-Benzenedicarboxylic acid, bis(2-ethylhexyl) ester. These compounds suggested partial esterification reactions that increased structural stability of the humic matrix. LC–MS analysis identified a molecular ion at m/z = 351.27 corresponding to Viridin, a well-known antifungal metabolite from T. harzianum. The well diffusion assay demonstrated inhibition zones of 2.3 ± 0.10 mm for F. graminearum and 1.30 ± 0.19 mm for R. solani in response to the 1% bio-humic extract, confirming antifungal potential. No inhibition was observed in the control. In the greenhouse experiment, wheat grown in infected soil without fertilizer had only 71% germination, whereas bio-humic treatment restored germination to 100%, comparable to healthy control plants. Mean shoot length (31.2 ± 1.4 cm) and dry weight (2.35 ± 0.15 g) in the bio-humic treatment were not significantly different from those in uninfected controls, while plants treated with commercial humic acid showed intermediate growth responses.
Discussion
The structural features observed in FTIR and UV–Vis analyses suggest that the biological fermentation pathway generated humic substances with higher oxygenated functionality and reduced aromatic condensation compared with mineral-derived humic acids. The porous nanostructure visible in FE-SEM images is characteristic of biologically formed humic matter and supports improved ion exchange, water retention, and microbial colonization in soil. The presence of esterified and phenolic derivatives detected by GC–MS indicates biochemical modification of lignin intermediates during fermentation, while the detection of Viridin reveals integration of antifungal metabolites into the humic matrix. The bio-humic product thus acts as a multifunctional material, combining physicochemical soil amendment properties with biological control potential. The fungal secondary metabolites contribute to suppression of soilborne pathogens, whereas the oxygen-rich humic structure enhances nutrient availability and promotes root development. The positive greenhouse results demonstrate that bio-humic application mitigates disease stress and maintains plant vigor without chemical fungicides. The combination of humification and biocontrol mechanisms in a single process reflects an emerging approach to biofertilizer development. By coupling organic matter recycling with microbial metabolism, this method provides a route to renewable, environmentally friendly soil conditioners that replace costly and ecologically burdensome mineral humic acids.
 
Conclusion
This study demonstrated that Trichoderma harzianum can effectively transform lignocellulosic wastes into a bio-humic acid with reactive functional groups, nanostructured morphology, and antifungal bioactivity. The product inhibited major soilborne pathogens and enhanced wheat growth under infected conditions, functioning simultaneously as a soil conditioner and a biocontrol agent. The integration of structural stability, biological activity, and sustainability makes bio-humic production via fungal fermentation a promising strategy for advancing environmentally compatible agriculture.
 
Acknowledgements
This research was conducted at the Agricultural Biotechnology Laboratory, Department of Plant Production and Genetics, Faculty of Agriculture, Bu-Ali Sina University, with financial support from Mazo Gene Razak Kala Company.