Hydrogel composites incorporating PVA, DAC, and mycelia from Pleurotus eryngii (PVA/DAC/E), Ganoderma boninense (PVA/DAC/L), and Pleurotus ostreatus (PVA/DAC/O) were successfully fabricated. All fabricated hydrogels, including those with mycelial reinforcement, exhibited excellent elasticity upon initial handling, capable of being stretched and reforming to their original shape. This indicated robust intermolecular interactions, likely enhanced by the presence of DAC and the mycelial networks The general physical characteristics aligned with previous reports [49]. While subsequent analyses revealed species-specific impacts, initial observations suggested successful composite formation across all mycelial types. While initial visual assessments and broad spectroscopic features (FTIR, XRD) suggested successful composite formation with common polymeric backbones across all mycelial types, subsequent detailed thermal, mechanical, and physico-chemical analyses (discussed below) revealed distinct species-specific impacts on the composite properties.

Fig. 5 presents FTIR spectra of DAC–PVA and MBC samples. All characteristic curves exhibited similar features, indicating the presence of common functional groups across the composites. Notably, peaks were observed at wavenumbers of 3300 cm⁻¹ and 2921 cm⁻¹, which are attributed to O–H stretching and C–H stretching vibrations, respectively. The presence of hydroxyl groups suggests that the hydrogel composites can effectively adhere to water molecules through hydrogen bonding. Subsequent peaks at 1726 cm⁻¹ and 1568 cm⁻¹ were identified, likely due to the presence of C–O stretching vibrations. These peaks correspond to the carbonyl groups located on the side chains of DAC, confirming successful oxidation of cellulose. Additionally, peaks observed at 1133 cm⁻¹ and 842 cm⁻¹ correspond to C–O–C stretching vibrations. The peak at 842 cm⁻¹ is typically associated with the presence of hemicellulose, which is the most easily degraded component of fungal cell walls, as suggested by Liu et al. (2019) [54]. These characteristic peaks align with previous findings reported by Chen et al. (2023), further validating the structural integrity of the composites [55]. Furthermore, the integration of three mycelial types—Ganoderma boninense, Pleurotus eryngii and Pleurotus ostreatus—into the hydrogel composite resulted in the emergence of a distinct absorption peak at 1373 cm⁻¹. This peak is indicative of N–H stretching vibrations, which is likely attributable to the presence of chitin within the mycelial structure [33,56].

The incorporation of chitin not only enhances the mechanical properties of the composite but also contributes to its biocompatibility and structural stability. Overall, the FTIR analysis confirms the successful formation of DAC and its effective integration into the PVA matrix, as well as the incorporation of mycelial components. The presence of aldehyde and chitin functional groups plays a crucial role in enhancing the composite's mechanical strength and water retention capabilities, making them suitable for various structural and functional applications.

XRD patterns (Fig. 6) for PVA, DAC, PVA/DAC, and MBCs revealed that all PVA-containing samples exhibited a prominent broad peak at 2θ = 19.5°, characteristic of PVA's semi-crystalline structure [57]. The incorporation of DAC and mycelium did not significantly alter this primary PVA peak, suggesting the mycelium was well-integrated within the matrix without forming large, distinct crystalline domains or substantially changing the bulk PVA crystallinity, likely due to the relatively small amount of mycelium [58].

Fig. 7 illustrates the thermal decomposition behavior of DAC-PVA and MBCs. This analysis enabled the assessment of the thermal resistance properties of the samples when exposed to elevated temperatures. For comparative purposes, pristine DAC and PVA-based hydrogel composites were also included. All samples exhibited similar thermal decomposition profiles, characterized by three distinct regions upon application of external heat. In the temperature range from room temperature to 200 °C, approximately 10 wt% loss was observed, likely attributable to the evaporation of water. This indicates that hydrogel composites have an inherent affinity for water molecules, facilitating hydrogen bond interactions. To avoid degradation, and bacterial damage, it is recommended that the hydrogels be stored at low temperatures, consistent with the findings of Cesar et al. (2023) [59].

Between 200 °C and 500 °C, a broad region of weight loss was observed, corresponding to the pyrolysis of organic structures within the composites. The hydrogel composites, primarily composed of organic components, undergo thermal degradation, resulting in the formation of volatile gases such as CO₂ and NO_x. At temperatures above 500 °C, only about 10 wt% loss was observed, attributed to the formation of char and residual materials, as reported by Nigmatullin et al. (2004) [60]. These results indicate that the composites possess considerable thermal stability, making them suitable for applications requiring resistance to high temperatures.

Fig. 8 illustrates the thermal properties, including melting (T_m) and crystallization (T_c) behaviors, of various composite materials. A sharp endothermic melting peak is observed for the DAC and PVA-based composites with added mycelium, occurring at approximately 140 °C, with enthalpy changes (ΔH) of around 124 J/g for Pleurotus eryngii, 118 J/g for Ganoderma boninense, and 143 J/g for Pleurotus ostreatus. These results show minor variations between the mycelium species. Furthermore, all mycelium-enhanced composites exhibit pronounced exothermic crystallization peaks at approximately 380 °C, with enthalpy changes of around 171 J/g, 172 J/g, and 256 J/g, respectively. This behavior is attributed to the specific mycelium species and the pre-treatment of the composite material in potato dextrose broth (PDB), a culture medium used prior to mycelium inoculation. In contrast, the original composite material (PVA/DAC) shows two distinct endothermic melting peaks at 73.96 °C and 180.27 °C, with enthalpy changes of 87.70 J/g and 30.63 J/g, respectively, indicating weaker mechanical properties compared to the mycelium-enhanced composites.

In addition, Fig. 9 (left) presents the bendable characteristics of DAC-PVA and MBCs. All samples containing the three types of mycelia—Ganoderma boninense, Pleurotus eryngii, and Pleurotus ostreatus—demonstrated excellent bendable behavior under applied external loads. This flexibility suggests that these composites have potential for various applications, aligning with the previous works of Antinori et al. (2020, 2021) [61,62]. Correspondingly, scanning electron microscopy (SEM) revealed the morphological properties (Fig. 9, right panels). The PVA/DAC control (Fig. 9a, right panel) showed a relatively dense structure, while the mycelium-based composites (Fig. 9c–e, right panels) clearly displayed an interwoven network of fungal hyphae integrated within the polymer matrix, confirming effective mycelial colonization. The microstructure of the composites revealed an oriented arrangement of DAC fibers, which were randomly distributed within the composite network. The interface between fibers were introduced due to the repulsive forces of hydroxyl groups, acting as pendent groups alongside the cellulose chains. This morphological configuration is consistent with previous studies by Nigmatullin et al. (2004), confirming the effective integration of DAC and mycelial networks within the PVA matrix [60]. The random distribution and interfacial bonding contribute to the composite's mechanical strength and structural integrity, enhancing its suitability for practical application.

The mechanical properties of DAC-PVA and MBCs were evaluated using a Universal Testing Machine (UTM), as reported in Fig. 10. This instrument facilitated the determination of tensile strength and elongation at break of the composite. It was observed that the incorporation of mycelium into the DAC-PVA matrix significantly enhanced both tensile strength and elongation at break. The mycelium acts as a reinforcing agent, forming strong bonds at the interface between DAC and polyvinyl alcohol, thereby improving the composite's mechanical integrity. Specifically, the composite containing Pleurotus eryngii, exhibited the highest tensile strength, while the composite with Ganoderma boninense demonstrated the maximum elongation at break. These results indicate that different mycelial species contribute uniquely to the mechanical properties of the composites, likely due to variations in their structural networks and bonding capabilities. The enhanced tensile strength suggests improved load-bearing capacity, whereas increased elongation at break indicates greater flexibility and ductility of the composites. These findings are consistent with the results reported Appels et al. (2018), who demonstrated the beneficial effects of mycelial integration on the mechanical performance of composite materials. The observed differences in mechanical performance among the MBCs can be attributed to the distinct morphological characteristics of the fungal species [34]. Pleurotus eryngii demonstrated the highest tensile strength due to its thick, densely packed hyphae, which form a rigid and well-aligned network within the DAC–PVA matrix. This structural organization enhances interfacial adhesion and facilitates efficient stress transfer, thereby increasing the load-bearing capacity of the composite. In contrast, Ganoderma boninense exhibited the greatest elongation at break, likely due to its more flexible, branched hyphal architecture and the production of extracellular polysaccharides (EPS). These features contribute to a more extensible composite structure that allows greater deformation under stress before failure. Thus, while P. eryngii reinforces the composite through stiffness and strength, G. boninense enhances ductility and flexibility, demonstrating the species-specific tuning potential of fungal mycelia in bio-based composite design [34,35].

Fig. 11 exhibits the physico-chemical properties of DAC-PVA and MBCs, focusing on water-swelling behavior, equilibrium water content (EWC), and gel fraction. The evaluations were conducted over a 24-h period to comprehensively assess the composites' interactions with water. In the swelling behavior assessment, the pristine DAC-PVA composite exhibited a high swelling ratio of approximately 800%, indicating a substantial capacity to absorb water. In contrast, all MBCs demonstrated significantly lower swelling ratios, ranging between 200% and 300%. This reduction in swelling capacity can be attributed to the incorporation of mycelium, which provides additional dimensional stability and restricts excessive water uptake. Over the investigation period, a slight increase in the swelling ratio was observed in MBCs, likely due to gradual infiltration of water molecules into the composite network. This behavior underscores the role of mycelium in modulating the hydrogel's swelling properties, enhancing its suitability for applications where controlled water absorption is essential. Consistent with the swelling behavior results, the equilibrium water content (EWC) of the composites showed a similar trend. The pristine DAC-PVA composite maintained a high EWC of 90%, reflecting its high-water retention capability. Conversely, MBCs exhibited a reduced EWC ranging from 40% to 60%. The decrease in EWC with mycelium incorporation suggests enhanced structural integrity and reduced free water availability within the composite network. This reduction is beneficial for applications requiring stable water content and minimized swelling, thereby preventing potential degradation or mechanical instability. The gel fraction analysis revealed that all samples, including both pristine DAC-PVA and MBCs, maintained a high gel fraction between 80% and 90 wt%.

This high gel fraction indicates effective crosslinking within the hydrogel matrix, ensuring that only 10 wt% of water remains within the composite network. The presence of mycelium did not adversely affect the gel fraction, suggesting that the integration of mycelial networks does not compromise the crosslinking efficiency of the hydrogel. Instead, it likely contributes to a more robust and interconnected network, enhancing the composite's mechanical and structural properties. The incorporation of mycelium plays a crucial role in enhancing the composite properties by forming chemical bonds between DAC and PVA throughout the hydrogel network [26]. The mycelial networks act as reinforcing agents, providing additional crosslinking points that improve dimensional stability and mechanical strength. This synergistic interaction not only reduces excessive swelling and water content but also maintains a high gel fraction, ensuring the composite's structural integrity and durability. These findings are consistent with previous studies reported by Appels et al. (2018), which demonstrated that the integration of mycelial networks into polymeric matrices significantly enhances mechanical and physico-chemical properties [34]. The observed trends in swelling behavior, EWC, and gel fraction align with the established benefits of mycelial reinforcement in composite materials, validating the effectiveness of mycelium as a functional additive in hydrogel-based composites.

In addition, the MBCs were degraded within 3 to 4 weeks with a 10–25% degradation rate. The rate was superior to pristine PVA/DAC because mycelium was considered as a naturally occurring organic matter that microorganisms can effortlessly consume and break down. Conversely, PVA/DAC is also biodegradable. It requires specific environmental conditions alongside some microorganism actions to degrade. The biodegradability of the composites in PBS was monitored over 28 days (Fig. 11d). The pristine PVA/DAC composite exhibited minimal degradation, with approximately 5% weight loss by day 28. In contrast, all mycelium-based composites (MBCs) demonstrated significantly higher degradation. Specifically, by day 28, composites reinforced with P. eryngii (PVA/DAC/E), G. boninense (PVA/DAC/L), and P. ostreatus (PVA/DAC/O) showed weight losses in the range of 20–35%, with distinct degradation profiles for each species. The PVA/DAC/PDB control also showed considerable degradation, greater than PVA/DAC alone. These findings suggest that mycelial incorporation enhances the susceptibility of the composites to breakdown in PBS, indicating improved biodegradability compared to the PVA/DAC matrix alone.

The incorporation of mycelium into the DAC–PVA composites significantly increased the water contact angle, shifting surface characteristics from hydrophilic to distinctly hydrophobic (Fig. 12). This transformation is primarily due to the hydrophobic nature of fungal mycelia, particularly resulting from the lipid-rich components and chitinous structures of fungal cell walls. Hydrophobic surface modification enhances moisture resistance, minimizes water uptake, and thereby substantially improves dimensional stability and long-term durability. Such surface properties are highly relevant in industrial applications where water-resistance is crucial, including sustainable packaging materials, biodegradable construction components, and outdoor environmental sensors. Environmentally, this reduced moisture sensitivity also extends the lifetime of materials in humid conditions, lowering replacement rates and waste generation, thus supporting sustainable material utilization and circular economy practices.

In the case of composites without mycelium, the contact angle was measured to be 48.40°, indicating hydrophilic characteristics. This low contact angle suggests that the pristine DAC-PVA composites readily adhere to water molecules through hydrogen bonding, facilitating excellent water uptake and interaction. Conversely, the incorporation of mycelium into the composite matrix resulted in a significant increase in the contact angle, ranging from 85° to 110°. This shift towards higher contact angles indicates a transition from hydrophilic to hydrophobic behavior, imparting the composites with water-resistant properties. This transformation is attributed to the mycelium's cell membrane, which features a lipid bilayer known for its hydrophobic characteristics. The enhanced wettability and surface modulation observed at the membrane surface and the mycelium-membrane interface indicate improved hydrophilicity. Notably, the membrane-mycelium interface exhibited a hydrophilic character, while the opposing hydrophobic side was characterized by a densely packed layer of fibrous hyphae [30,44]. The enhanced hydrophobicity is attributed to the presence of mycelial networks, which are likely to introduce hydrophobic functional groups and disrupt the hydrogen bonding network within the hydrogel matrix.

This modification in surface properties not only reduces water absorption but also enhances the durability and stability of the composites in aqueous environments. The observed increase in contact angle upon mycelium incorporation aligns with previous studies by Antinori et al. [61,62], who demonstrated that mycelial integration can effectively modulate the surface wettability of polymeric composites, thereby broadening their application potential in areas requiring moisture resistance and structural integrity. Overall, the contact angle measurements confirm that mycelium incorporation plays a pivotal role in tailoring the surface properties of DAC–PVA composites, enhancing their suitability for diverse applications ranging from biomedical devices to environmental sensors. Furthermore, the transition from hydrophilic to hydrophobic behavior following mycelium incorporation into the DAC–PVA matrix is attributed to both biochemical alterations and microstructural transformations within the composite. Fungal mycelia inherently contain chitin, hydrophobic proteins, and lipids—components with low surface energy that naturally repel water [33,62,63]. When introduced into the DAC–PVA network, these hydrophobic biomolecules partially mask or replace the polymer's hydrophilic hydroxyl and aldehyde groups, thereby reducing hydrogen bonding capacity with water molecules. Simultaneously, the formation of an entangled hyphal network contributes to increased surface roughness and porosity at both the micro and nanoscale levels [64]. This textured surface promotes the entrapment of air pockets between the water droplets and the material, giving rise to the Cassie–Baxter state, in which the liquid rests on a composite interface of solid and air, effectively minimizing the solid–liquid contact area and increasing the water contact angle [65].

Additionally, during active mycelial growth, fungi excrete extracellular polymeric substances (EPS) comprising glycoproteins, polysaccharides, and fatty acids that further lower the surface energy and introduce a hydrophobic barrier. These EPS layers protect fungal colonies and coat the surrounding material, leading to enhanced surface water resistance. Importantly, the hydrophobicity is not solely a result of surface chemistry but also a function of the hierarchical structure and orientation of the fungal hyphae, which create physical barriers to water penetration. These combined effects—chemical masking of hydrophilic groups, structural roughness, EPS deposition, and the Cassie–Baxter effect—synergistically transform the initially hydrophilic DAC–PVA surface into one that exhibits robust hydrophobicity. This shift plays a critical role in improving the composite's dimensional stability, moisture resistance, and long-term durability under humid or wet conditions.

Fig. 13 indicates the surface roughness of DAC-PVA and MBCs; the presence of three types of mycelia—Ganoderma boninense, Pleurotus eryngii, and Pleurotus ostreatus—demonstrated covering the PVA-DAC surface; hence, the surface roughness increased significantly due to the clear difference in height variation and surface structural irregularities compared to PVA/DAC and PVA/DAC/PDB. It exhibited peaks and valleys distributed in multiple directions, indicating a higher roughness amplitude. The amplitude range of MBCs was −4 to 4 μm, whereas that of PVA/DAC and PVA/DAC/PDB was −0.2 to 0.2 μm. The increase in surface roughness observed in the MBCs composites can be interpreted through the Cassie–Baxter theory. According to this model, the presence of hierarchical micro- and nano-structures on a surface can effectively trap air within surface grooves, especially when combined with low surface energy. This entrapment of air pockets significantly reduced the effective solid-liquid contact area, thereby elevating the water contact angle and diminishing the material's overall wettability [30,66]. Consequently, the more complex and irregular surface topography introduced by the mycelium was more conducive to establishing a Cassie–Baxter state compared to the PVA/DAC system. This structural modification promoted a synergistic effect that enhanced the composite's hydrophobicity and water resistance, provided that the surface was concurrently characterized by chemical functional groups with low surface energy.

We further interpret the observed increases in apparent hydrophobicity by considering hydrophobins—amphipathic fungal proteins that self-assemble at interfaces and are known to render hyphae and colonized surfaces water-repellent—alongside previously discussed contributors such as chitin, lipids/extracellular polymeric substances (EPS), and Cassie–Baxter surface-roughness effects. In our system, species-dependent deposition of hydrophobins on (or within) the DAC–PVA surface could synergize with topographical changes from mycelial growth to elevate contact angles and reduce wetting. To disentangle these mechanisms, future work will quantify hydrophobin signatures and their functional impact via (i) partitioning SDS-soluble vs. SDS-insoluble protein fractions and (iii) contact-angle hysteresis measurements before/after protease treatment, thereby directly testing the contribution of proteinaceous surface assemblies relative to polysaccharide/lipid components and roughness. We note the darkened/black coloration of the composites after growth-stop and drying, which likely reflects a combination of medium residues (e.g., PDB components concentrated or thermally modified during processing) and fungal pigments/metabolites deposited at the surface. The relevance of color is application-dependent: for biomedical/food-adjacent uses, we recommend leachables profiling (UV–Vis of rinsates; targeted LC–MS for pigment/metabolite signatures) together with cytocompatibility screening; for packaging/industrial uses, color is typically secondary to moisture/mechanical performance, though consistency can be improved through strain and medium control and optional post-treatments (e.g., mild bleaching or surface sealing) to stabilize appearance under handling and moisture-cycling conditions.

In terms of scalability and commercialization, the DAC–PVA platform is compatible with established casting/curing workflows (batch trays or roll-to-roll lines) and freeze-drying where porosity is desired, with mycelial growth staged post-cure in controlled humidity/temperature chambers. The matrix tolerates steam/thermal sterilization cycles commonly used in bioprocessing, and growth-stop plus drying can be integrated into continuous ovens. Quality assurance can leverage routine monitoring of the degree of oxidation (DAC), gel fraction, residual aldehyde content, moisture uptake, and mechanical benchmarks to ensure batch-to-batch consistency. Cost and sustainability considerations include the price and availability of cellulose feedstock, periodate use and recovery/regeneration, and PVA grade, with periodate recycling and water reuse reducing the environmental footprint. Near-term applications include water-resistant flexible films, biodegradable packaging substrates, and sensor/biomaterial supports where moderate strength, tunable hydrophobicity, and bio-integration are advantageous. Key risks—such as variability in fungal morphology and pigmentation—can be mitigated via strain selection, tight control of growth parameters, and optional surface sealing to enhance colorfastness and stability under moisture cycling.