Edible fungi are globally recognized as a critical nutritional resource, with annual production exceeding 11 million tons. They provide essential proteins, fibers, vitamins and minerals along with antioxidants that promote health (Anand et al., 2025). Oyster mushrooms (Pleurotus spp.) are especially favored for cultivation due to their high yield potential, rapid growth and ability to thrive on inexpensive waste. These species colonize agro-waste by secreting a powerful range of enzymes, such as cellulase, xylanase, laccase and peroxidases, which transform complex plant fibers into useful substrate (Muswati et al., 2021).
       
Beyond their role in substrate colonisation, the same enzymatic machinery makes cultivated mushrooms a source of valuable biochemicals once fruiting is complete. Cellulase produced during growth has major applications in biotechnology and biofuel production (Huang et al., 2024; Rahman et al., 2018). Furthermore, fungal cell walls contain chitin, which can be converted into chitosan (Pantić et al., 2023). Fungal-derived chitosan is vegan-friendly, allergen-free and possesses functional characteristics comparable to those of marine sources (Jayasree, 2020; Ayser et al., 2024), positioning spent mushroom biomass as a feedstock for both enzymes and biopolymers, rather than only a food product.
       
Sugarcane bagasse is a byproduct of the sugar industry, rich in cellulose and hemicellulose. Large quantities often remain underutilised, leading to environmental pollution. Using bagasse as a mushroom substrate offers a sustainable management strategy (Kumar, 2025). Recent research shows that substrates containing 80% sugarcane bagasse significantly improve the yield of Pleurotus ostreatus compared to traditional substrates (Karpagavalli et al., 2023) and pre-treatment/disinfection choice strongly influences how well a bagasse-based substrate performs (Nanje and Manvi, 2019). This approach generates high-quality protein and reduces environmental impact (Thakur, 2024; Devi et al., 2023).
       
However, most of this literature evaluates either mushroom yield or by-product recovery (enzyme or chitosan) separately; the potential to recover high-value bioactive compounds within the same cultivation cycle that produces the edible mushroom remains comparatively unexplored. We hypothesised that a single sugarcane-bagasse substrate, without additional processing steps beyond standard cultivation, could support the simultaneous, quantifiable recovery of mushroom biomass, cellulase and fungal chitosan. This study aimed to evaluate a two-step method for cultivating Pleurotus ostreatus using sugarcane bagasse, with the specific objectives of (i) quantifying mushroom yield and biological efficiency and (ii) recovering and characterising cellulase enzyme and fungal chitosan from the same fruiting cycle.

The study was carried out at Krishna Institute of Science and Technology, Karad, Maharashtra, India, between June 2024 and January 2026. Pleurotus ostreatus was used as the test fungus throughout.
 
Materials and reagents
 
Fresh sugarcane bagasse was procured from a local sugar mill and wheat bran was purchased from a local grain supplier for use as a nitrogen-rich supplement. Certified spawn of Pleurotus ostreatus was obtained from a registered mushroom spawn laboratory. Analytical-grade sodium hydroxide, hydrochloric acid, sodium phosphate (mono- and di-basic), carboxymethyl cellulose (CMC), Congo red dye, 3,5-dinitrosalicylic acid (DNSA), sodium potassium tartrate and D-glucose (used to prepare the standard curve) were procured from standard laboratory chemical suppliers. All solutions were prepared in double-distilled water unless stated otherwise. Polypropylene cultivation bags, a UV-sterilised inoculation chamber, a hot-air oven, an autoclave, a centrifuge, a water bath, a UV-Visible spectrophotometer and an FTIR spectrometer equipped with an attenuated total reflectance (ATR) accessory were used across the different stages of the experiment.
 
Substrate preparation and spawning
 
Sugarcane bagasse obtained from local mills was air-dried to roughly 10% moisture content, diced and supplemented with wheat bran to achieve an optimal C/N ratio of 80:20. The substrate was pasteurized at 70°C for 2 hours to eliminate competitive microorganisms (Grimm et al., 2024). After cooling, it was inoculated with 5-10% (w/w) grain spawn of Pleurotus ostreatus and incubated at 25°C until complete mycelial colonization (10-14 days). Fruiting occurred in a ventilated chamber (85-90% RH) under low light. Mature fruit bodies (first flush) were harvested to determine fresh yield and biological efficiency (fresh weight/dry substrate) (Kelemu, 2026) (Fig 1).


 
Cultivation monitoring and yield determination
 
Bags were examined every alternate day for the extent of mycelial spread, presence of contaminant moulds and any discoloration of the substrate. Bags showing visible contamination were discarded from further analysis. Once colonization was complete, bags were transferred to the fruiting chamber, where relative humidity was maintained through periodic misting and gentle ventilation was provided to prevent accumulation of carbon dioxide, which can otherwise cause elongated, deformed stipes. Pinhead formation was recorded as the day on which visible primordia first appeared on the bag surface and the interval between spawning and first harvest was recorded as the cropping cycle length. Fresh yield was recorded as the cumulative weight of all fruit bodies harvested from the first flush per bag and biological efficiency (BE) was calculated using the standard relationship:

 

 

This parameter is widely used in mushroom cultivation studies because it normalises yield against the amount of substrate consumed, allowing direct comparison across studies that use different bag sizes or substrate quantities.
 
Enzyme extraction and assay
 
Harvested mushrooms (300 g) were homogenized in 100 mL ice-cold phosphate buffer (0.1 M, pH 7, 4°C). The filtrate was centrifuged at 5,000 rpm for 10 min to obtain the crude enzyme supernatant. Qualitative cellulase activity was screened on carboxymethyl cellulose (CMC) agar plates via Congo red staining (Banni, 2021) (Fig 2). Quantitative activity was determined using the dinitrosalicylic acid (DNSA) method; reaction mixtures containing CMC and crude extract were incubated at 37°C for 1 hour (Rahman et al., 2018). Reducing sugar concentration was measured at 540 nm against a glucose standard curve (0-1.0 mg/mL). For this qualitative screen, plates were flooded with 1 mg/mL Congo red solution for 15 minutes, destained with 1 M NaCl for a further 15 minutes and examined for a pale halo against the red-stained background, which indicates local hydrolysis of the CMC substrate by secreted cellulase. All absorbance readings were corrected against a reagent blank and each of the reagent control, substrate control, enzyme control and test conditions was run in parallel so that the contribution of the enzyme extract itself, independent of substrate hydrolysis, could be distinguished from true catalytic activity.


 
Chitosan recovery and analysis
 
Residual mushroom biomass was processed for chitosan recovery following established protocols (Ayser et al., 2024; Jayasree, 2020). Cell walls were deproteinised in 1M NaOH (80°C, 3 hrs) and demineralised in 1M HCL. Deacetylation was performed using 50% NaOH at 100°C for 2 hours. The resulting chitosan was washed to neutral pH, dried and weighed. Structural identity was confirmed through Fourier-transform infrared (FTIR) spectroscopy, monitoring characteristic bands such as N-H bending at 1595 cm^-1. All experiments were performed in triplicate to ensure statistical robustness. The degree of deacetylation (DD%) of the recovered chitosan, which describes the proportion of acetyl groups removed from the parent chitin backbone and strongly influences solubility and bioactivity, was estimated from the FTIR spectrum using the baseline absorbance ratio method, comparing the amide band near 1655 cm^-1 against the hydroxyl reference band near 3450 cm^-1. Chitosan yield was expressed as a percentage of the dry residual biomass remaining after enzyme extraction and all recovery steps were performed on biomass pooled across replicate bags to obtain sufficient material for structural analysis.
 
Statistical analysis
 
All quantitative measurements (fresh yield, biological efficiency, absorbance readings and chitosan yield) were obtained from three independent replicate bags/extractions and are expressed as the mean of triplicate determinations, with the corresponding range reported alongside each value in the results. Absorbance values were converted to reducing-sugar equivalents using a glucose standard curve prepared in the same assay run to minimise inter-day variability.

Mushroom yield and biological efficiency
 
The Pleurotus ostreatus successfully colonized the sugarcane bagasse within 12 days and produced healthy fruit bodies. The fresh yield ranged from 650-800 g/kg of dry substrate, which resulted in a biological efficiency of 75-80%. These high productivity levels confirmed that the carbon found in the lignocellulosic composition of sugarcane bagasse provided an excellent environment for oyster mushroom growth. The performance of the Pleurotus spp. in this study aligned with previous findings that bagasse-based materials are more productive than many traditional growth media (Kelemu 2026; Jayasree, 2020). This high efficiency was likely due to the nutrient-rich composition of the sugarcane waste, which acted as an ideal energy source (Karpagavalli et al., 2023). To place these results in context, Table 1 below compares the biological efficiency obtained in the present study with values reported for Pleurotus species cultivated on other lignocellulosic residues. The comparison indicates that sugarcane bagasse performs favourably relative to several conventional substrates, which supports its suitability as a low-cost growth medium for oyster mushroom cultivation.


 
Nutritional relevance of the harvested biomass
 
Beyond the biological efficiency values discussed above, it is worth situating the harvested Pleurotus ostreatus fruit bodies within the broader nutritional profile reported for the species in the literature. Oyster mushrooms are widely reported to be a good source of protein and dietary fibre on a dry-weight basis, to be naturally low in fat and to contain B-group vitamins, potassium and antioxidant compounds including phenolics and ergothioneine (Anand et al., 2025). While detailed proximate and micronutrient analysis of the fruit bodies produced in this study was outside the scope of the present work, the consistency of yield and morphology observed here suggests that the bagasse-based substrate supported normal fruit body development and there is no indication in the cultivation record of nutrient deficiency symptoms such as thin, pale, or malformed fruit bodies that are sometimes reported when substrates are severely nitrogen-limited. This observation is consistent with the C/N ratio adjustment performed during substrate formulation (Section 2.2) and supports the suitability of wheat-bran-supplemented bagasse as a substrate that meets the nutritional requirements of the fungus itself during fruiting.
 
Enzymatic activity
 
Qualitative tests using CMC-agar plates showed clear hydrolysis zones where the substrate was broken down, confirming that the fungi produced active cellulase enzyme (Fig 3).  


       
The DNSA assay showed a significant increase in absorbance (OD at 540 nm) in test samples (0.52-0.55) compared to the control (0.50), as shown in Table 2. This change indicated a glucose release of 0.1-0.15 mg/mL. The measured cellulase activity of the crude, unconcentrated extract was approximately 0.005 U/mL. Although this titer was low compared to commercial hyper-producing industrial strains, it successfully demonstrated that the 32-37% cellulose content in the sugarcane bagasse functioned as an effective substrate to induce cellulase expression in Pleurotus spp (Salomão et al., 2019; Khalil et al., 2011). Similarly, the successful production of enzymes supported in recent research suggests that bagasse is a potential inducer for cellulase (Huang et al., 2024; Banni, 2021). Although the activity levels were slightly below those of industrial strains, they were sufficient to demonstrate the potential of this substrate for enzyme recovery (Rahman et al., 2018).


       
A higher titre in the case of industrial cellulase production can be correlated to the use of specialized, hyper-secreting micro-fungal strains, which are usually genetically modified (e.g. Trichoderma reesei or Aspergillus niger). Contrary to this, Pleurotus spp. are macro-fungi which perform biomass proliferation and fruiting body development rather than the hyper-secretion of extracellular hydrolases during solid-state cultivation. Here, genetic modification can enhance the potential of these fungi, allowing them to be successfully utilized in industrial enzyme applications (Lagunes-Reyes et al., 2023; (Lagunes-Reyes et al., 2023).
                  
Chitosan recovery and characterisation
 
The study recovered chitosan from the residual fungal biomass at a rate of approximately 1.1-2.0% (w/w) of the dry weight. The identity of this biopolymer was verified via FTIR analysis, which showed specific chemical markers at 1590 cm^-1 (NH₂ bending) and 1080 cm^-1 (C-O stretching). This confirmed the presence of the expected amino and carbon-oxygen groups within the recovered material. In addition to the diagnostic amino and carbon-oxygen bands highlighted above, chitosan spectra reported in the literature typically also display a broad O-H/N-H stretching envelope near 3200-3400 cm^-1, a C-H stretching band near 2870-2920 cm^-1 and residual amide I and amide II bands near 1650 and 1550 cm^-1 that reflect incomplete deacetylation of the parent chitin. The presence of these expected functional groups, together with the absence of major spectral features inconsistent with a chitin-glucan-derived polymer, supports the identification of the recovered material as chitosan rather than an unconverted chitin-glucan complex or a non-specific proteinaceous residue. A summary of the key FTIR band assignments used to confirm chitosan identity is provided in Table 3.


       
The chitosan yields (1.1-2.0% w/w of dry biomass) observed in this experiment were also consistent with reported values for other fungi, such as Agaricus and Aspergillus, when grown on agricultural waste (Pantić et al., 2023; Jayasree, 2020). Although this yield was lower than traditional commercial sources, like marine shellfish waste, which typically yields 15-20% crude chitosan, the advantage of fungal extraction is in providing a predictable, season-independent production cycle free from heavy metal contamination (Huq et al., 2022; Gîjiu et al., 2022). By producing both food and industrial polymers from a single source of waste, this study demonstrated an effective way to maximize the value of agricultural by-products (Kumar, 2025). This combined approach offers a practical path toward more sustainable waste management in the agricultural sector. The observed trends confirm that high-cellulose feedstocks like bagasse enhanced both fungal productivity and enzyme yield (Devi et al., 2023; Kelemu, 2026).  Although the extraction processes for enzyme and chitosan were not optimized in this proof of concept, the feasibility of this integrated biorefinery model was evident. Future research focusing on solid-state fermentation and enzymatic pre-treatment could further enhance the recovery of these bio-products (Taneja et al., 2023).
 
Economic and environmental significance
 
From a waste-management perspective, diverting sugarcane bagasse into mushroom cultivation reduces the volume of residue left for open-field disposal or uncontrolled burning, both of which are associated with particulate air pollution and loss of biomass carbon. From an economic perspective, the same batch of substrate is shown here to yield three potentially marketable outputs- fresh mushrooms for direct food use, a crude cellulase fraction that could be applied in textile, detergent, biofuel, or animal-feed processing after further purification and a chitosan fraction with potential application in food packaging, agriculture and water treatment. Even though the crude extracts obtained in this study were not purified to commercial specifications, the demonstration that all three products can be recovered sequentially from a single low-cost feedstock strengthens the economic case for treating spent mushroom substrate as a resource rather than a waste stream, particularly for smallholder or cooperative-scale mushroom producers who already generate large volumes of spent substrate as a by-product of cultivation.
 
Limitations of the study
 
This study has several limitations that should be considered when interpreting the results. The extraction protocol relied on a crude, unconcentrated washing and filtration process without downstream concentration steps such as ultrafiltration or precipitation; hence, future work must focus on optimizing fermentation parameters specific to enzyme production and incorporating concentration protocols to achieve industrially viable enzyme titers. Cellulase activity was evaluated using a single time-point endpoint assay rather than a full time-course, which limited the ability to comment on enzyme kinetics or stability. Chitosan was characterised structurally by FTIR but was not subjected to complementary analyses such as nuclear magnetic resonance spectroscopy and gel permeation chromatography for molecular weight, all of which would strengthen confidence in the reported estimates. Finally, the experiment was conducted using a single flush of a single Pleurotus ostreatus strain under one set of environmental conditions, so the generalisability of the yield and biological efficiency figures to other strains, climates, or scales of production remains to be established.
 
Applications of recovered cellulase and chitosan
 
The crude cellulase fraction recovered in this study belongs to a class of enzymes with wide industrial relevance. Cellulases are used commercially in the textile industry for bio-polishing and stone-washing of denim, in the detergent industry to improve fabric softness and colour retention, in the pulp and paper industry to reduce fibre coarseness, in animal feed formulation to improve digestibility of fibrous feed and in the biofuel industry to hydrolyse lignocellulosic biomass into fermentable sugars ahead of bioethanol production. Because these applications generally tolerate a crude or partially purified enzyme preparation rather than requiring pharmaceutical-grade purity, cellulase recovered from spent mushroom substrate is a realistic candidate for at least some of these lower-specification uses, provided activity can be increased through the process optimisation discussed above.
       
Chitosan, similarly, already has an established and growing set of commercial applications that would be compatible with a fungal source. In the food industry, chitosan is used as an edible antimicrobial coating that extends the shelf life of fruit, vegetables and meat products and as a clarifying and flocculating agent in beverage processing. In agriculture, chitosan is applied as a seed treatment and foliar spray that can elicit plant defence responses against fungal and bacterial pathogens, reducing reliance on synthetic fungicides. In water treatment, its cationic charge allows it to act as a natural flocculant for removing suspended solids, heavy metals and dyes from wastewater. In biomedicine, chitosan-based hydrogels, films and nanoparticles are being investigated for wound dressings, drug delivery systems and tissue engineering scaffolds, owing to the polymer’s biocompatibility and biodegradability. The fungal origin of the chitosan recovered in this study is particularly relevant to the food, agricultural and water-treatment applications, where the vegan, allergen-free and consistent-supply advantages of fungal chitosan over crustacean-derived material are most directly valuable.

Building on the feasibility demonstrated here, several directions merit further investigation. Solid-state fermentation approaches, in which the substrate is deliberately held under conditions that favour extracellular enzyme secretion before fruiting is allowed to proceed, could be explored as a means of increasing cellulase titres without sacrificing mushroom yield. Partial purification steps such as ammonium sulfate precipitation, ultrafiltration, or ion-exchange chromatography could be applied to the crude enzyme extract to generate a more concentrated and better-characterised product suitable for downstream application testing. For the chitosan fraction, future work should quantify the degree of deacetylation and molecular weight directly, evaluate functional properties such as solubility, viscosity and antimicrobial activity and compare these properties systematically against commercial shellfish-derived chitosan. Finally, a full techno-economic and life-cycle assessment comparing this integrated biorefinery approach against conventional single-product mushroom cultivation would help clarify the conditions under which the additional processing steps for enzyme and chitosan recovery are economically justified.

This study confirmed that sugarcane bagasse served as an effective substrate for the cultivation of Pleurotus mushrooms with the recovery of cellulase enzyme and fungal chitosan. The research demonstrated a successful way to turn waste into a substrate (resource). Scaling this approach, together with process optimisation and characterisation, could allow mushroom growers to capture additional value from substrate that is currently discarded after a single harvest cycle. This could contribute both to reduced agricultural waste burden and to more diversified income streams within a circular bioeconomy framework.

The authors gratefully acknowledge the laboratory facilities and technical support provided for this research.
 
Author contributions
 
All authors contributed to the conception and design of the study, performed the cultivation, enzymatic and chitosan-extraction experiments, analysed the resulting data and contributed to drafting and revising the manuscript. All authors read and approved the final version of the manuscript.

The authors declare that they have no conflict of interest.