Three-Dimensional Dual-Network Gel-Immobilized Mycelial Pellets: A Robust Bio-Carrier with Enhanced Shear Resistance and Biomass Retention for Sustainable Removal of SMX

Abstract

Fungal mycelial pellets (MPs) exhibit high biomass-loading capacity; however, their application in wastewater treatment is constrained by structural fragility and the risk of environmental dispersion. To overcome these limitations, a dual-crosslinked polyvinyl alcohol–alginate gel (10% PVA, 2% sodium alginate) embedding strategy was developed and stabilized using 2% CaCl₂ and saturated boric acid. This encapsulation enhanced the tensile strength of MPs by 499% (310.4 vs. 62.1 kPa) and improved their settling velocity by 2.3-fold (1.12 vs. 0.49 cm/s), which was critical for stability under turbulent bioreactor conditions. Following encapsulation, the specific oxygen uptake rates (SOURs) of three fungal strains (F557, Y3, and F507) decreased by 30.3%, 54.8%, and 48.3%, respectively, while maintaining metabolic functionality. SEM revealed tight adhesion between the gel layer and both surface and internal hyphae, with the preservation of porous channels conducive to microbial colonization. In sequential-batch reactors treating sulfamethoxazole (SMX)-contaminated wastewater, gel-encapsulated MPs combined with acclimated sludge consistently achieved 72–75% SMX removal efficiency over six cycles, outperforming uncoated MPs (efficiency decreased from 81.2% to 58.7%) and pure gel–sludge composites (34–39%). The gel coating inhibited hyphal dispersion by over 90% and resisted mechanical disintegration under 24 h agitation. This approach offers a scalable and environmentally sustainable means of enhancing MPs’ operational stability in continuous-flow systems while mitigating fungal dissemination risks.

1. Introduction

Fungal mycelial pellets (MPs) serve as self-assembled spherical aggregates of filamentous fungi and have emerged as transformative platforms for microbial immobilization technologies [1], offering sustainable and multifunctional solutions for wastewater treatment, bioremediation, and biocatalysis [2]. These biological carriers capitalize on the inherent organizational behavior of fungal hyphae, which intertwine to establish complex three-dimensional networks characterized by naturally optimized porosity and surface morphology [3,4]. In contrast to synthetic alternatives such as activated carbon or polymeric foams, MPs are intrinsically biodegradable and can be cultivated on low-value lignocellulosic feedstocks, aligning well with circular bioeconomy principles [5]. Their architectural features facilitate high-density microbial colonization while promoting efficient mass transfer of oxygen, nutrients, and metabolic byproducts, which is regarded as an integrated advantage essential for maintaining both biological activity and process stability [6,7]. In the context of antibiotic-laden wastewater, MPs have demonstrated outstanding potential for immobilizing specialized bacterial consortia capable of degrading recalcitrant compounds [8,9,10], while their capacity for enzymatic secretion further enhances the hydrolysis of complex organic pollutants. Despite these advantages, the advancement of MPs from laboratory-scale systems to industrial-grade bio-carriers has been hindered by their inherent structural fragility. Under dynamic bioreactor conditions characterized by hydraulic shear stress, fluctuating substrate concentrations, and competitive microbial colonization, progressive mechanical degradation of MPs has been frequently observed [11,12,13]. Such disintegration reduces functional longevity and results in the release of fragmented hyphae into the receiving environment, raising concerns regarding biofouling and unintended ecological impacts. These limitations are further exacerbated by the absence of standardized stabilization strategies that concurrently ensure mechanical robustness and preserve microbial activity, thereby presenting a critical bottleneck in the scalability of fungal-based bioremediation technologies [14].

Conventional microbial immobilization strategies, such as single-component polymer entrapment, have been found to be inadequate for meeting the complex demands of bioreactor operations [15]. Polyvinyl alcohol (PVA) hydrogels, despite their mechanical resilience and chemical inertness, typically form densely crosslinked matrices that impede microbial metabolism. In contrast, alginate-based gels offer biocompatibility and tunability but lack sufficient mechanical integrity for sustained application and readily fracture under turbulence encountered in industrial-scale reactors, leading to biomass leakage and operational instability [16,17]. This inherent trade-off between durability and biological functionality has sustained reliance on non-biodegradable synthetic carriers, which undermines global sustainability goals and poses a risk of secondary environmental contamination [18]. To bridge this technological divide, a biomimetic dual-network hydrogel system was developed that synergistically integrates sodium alginate (SA) and polyvinyl alcohol (PVA), which serves as a strategic combination designed to replicate the hierarchical structure–function relationships observed in natural composite materials. Through a dual-crosslinking approach utilizing calcium ions for SA gelation and borate ions for reversible PVA complexation, an interpenetrating polymer network was constructed [19].

Based on the physicochemical properties of PVA-SA gels, we propose their synergistic integration with MPs to architect a 3D dual-network gel-immobilized mycelial pellet system featuring a PVA-SA gel encasement surrounding an MP core. The hierarchical architecture of composite MPs underpins their enhanced functional performance. The outer PVA-SA layer serves as a protective exoskeleton. PVA gel confers enhanced mechanical robustness to mitigate structural damage from external stresses, whereas SA gel facilitates superior mass transfer efficiency, thereby promoting the diffusion of nutrients and dissolved oxygen to the encapsulated MPs’ core. Concurrently, electrostatic interactions between carboxyl groups of SA and cationic pollutants enhance adsorption capacity, which has been regarded as a feature particularly beneficial for heavy metal remediation [20,21]. This advancement broadens the application prospects for this novel composite biomaterial carrier.

Sulfamethoxazole (SMX) has been adopted as a sulfonamide antibiotic; the recalcitrance of SMX to conventional biodegradation arises from its chemically stable heterocyclic structure and bacteriostatic mode of action, necessitating immobilization platforms capable of sustaining specialized microbial consortia while resisting both chemical and biological deterioration [22]. In this study, it was selected as the target contaminant to validate the practical relevance of the aforementioned innovation. Dual-network MPs were assessed under simulated wastewater conditions designed to replicate the physicochemical complexity of pharmaceutical effluents. This study demonstrated incremental enhancements in carrier design by reconceptualizing fungal-based immobilization using a material symbiosis framework. This study systematically investigates the following aspects: (1) Optimization of PVA-SA gel formulation ratios; (2) the impact of gel-MPs’ integration on fungal viability within immobilized MPs; (3) performance benchmarking of the novel composite bio-carrier versus pristine MP carriers; (4) quantification of gel-mediated suppression efficacy against excessive hyphal proliferation; and (5) comparative analysis of SMX degradation efficiencies and mechanistic investigation (among composite bio-carriers, pristine MPs carriers, and bare PVA-SA gel particles, all loaded with acclimated activated sludge).

2. Materials and Methods

2.1. Preparation of Experimental Materials and Mycelium Pellets

2.1.1. Fungal Strains and Culture Conditions

The fungal strains of Aspergillus niger F557, Aspergillus niger Y3, and Phanerochaete chrysosporium F507 were cultured in liquid medium containing glucose (10 g/L), NH₄Cl (1 g/L), KH₂PO₄ (1 g/L), and MgSO₄ (0.5 g/L). Spore suspensions (0.5 mL per 150 mL) were aseptically inoculated into sterilized medium (autoclaved at 115 °C for 30 min) and incubated at 30 °C with orbital shaking at 160 rpm for 48 h [23]. Fungal MPs were collected via vacuum filtration, rinsed thoroughly with ultrapure water, and stored at 4 °C for subsequent use. All chemicals were commercially purchased from Shanghai Aladdin Co. Ltd., Shanghai, China, and used without any further pretreatment.

2.1.2. PVA-SA Gel Embedding Protocol

Polyvinyl alcohol (PVA, 10% w/v) and sodium alginate (SA, 2% w/v) were dissolved in ultrapure water at 80 °C with continuous stirring until complete dissolution. After cooling to room temperature (25 ± 1 °C), the fungal MPs were immersed in PVA-SA solution with 6 h gentle agitation, the volume ratio of mycelial pellets to gel is 1:10. Subsequently, the MPs were transferred to a crosslinking solution containing 2% (w/v) CaCl₂ and saturated boric acid (pH 6.7) for 24 h to induce gelation. The resulting PVA-SA-embedded MPs (PVA-SA-MPs) were rinsed three times with distilled water and stored in physiological saline at 4 °C. All chemicals were commercially purchased from Shanghai Aladdin Co. Ltd., China, and used without any further pretreatment.

2.2. Fabrication of Functionalized Mycelial Pellets (MPs-AFP)

The activated sludge consortium was acclimated to SMX through a 30-day sequential exposure to synthetic wastewater, with the SMX concentrations progressively increased from 1 to 5 mg/L under continuous aeration and neutral pH conditions [24]. The acclimated sludge (mixed liquor volatile suspended solids, MLVSS = 6500 mg/L) was combined with fungal mycelial pellets (MPs, 5 g wet weight) in 150 mL of sterile culture medium and incubated at 30 °C with orbital shaking at 160 rpm for 36 h to facilitate microbial colonization. Colonized MPs were subsequently embedded following the PVA-SA protocol described in Section 2.1.2. The resulting artificial functional particles (MPs-AFP) were rinsed three times with sterile distilled water and stored in physiological saline (0.9% NaCl) at 4 °C until use.

2.3. Material Characterization Methods

2.3.1. Mechanical Property Testing

The mechanical properties were evaluated using a universal testing machine (Instron 5565, Instron, Norwood, MA, USA) equipped with a 50 N load cell. For the PVA-SA hydrogel samples, rectangular strips (10 mm × 2 mm × 1 mm) were prepared [25], while the fungal MPs were mechanically flattened into discs with a 3 mm diameter prior to testing. All of the specimens were preconditioned at 25 °C and 60% relative humidity for 24 h. Uniaxial tensile testing was conducted at a constant crosshead speed of 10 mm/min until failure. Young’s modulus (MPa) was calculated from the linear elastic region (0–5% strain) of the stress–strain curve. The toughness (kJ/m³) was determined by integrating the area under the curve up to the fracture point. Three biological replicates were tested for each sample type.

2.3.2. Morphological and Structural Analysis

Scanning Electron Microscopy (SEM): The freeze-dried samples were sputter-coated with gold for 120 s and imaged using a ZEISS SIGMA500 field-emission scanning electron microscope operated at an accelerating voltage of 10 kV [26]. The surface and cross-sectional morphologies were captured at 75×, 150×, and 1000× magnification.

Fourier Transform Infrared Spectroscopy (FTIR): The chemical functional groups were analyzed in attenuated total reflectance (ATR) mode using a Thermo Scientific™ Nicolet™ iS50 FTIR spectrometer, Thermo Fisher Scientific, Waltham, MA, USA [27]. Spectra were collected over 4000–400 cm⁻¹ with 32 scans per sample at a resolution of 4 cm⁻¹. Background correction was performed using a clean diamond ATR crystal.

Thermogravimetric Analysis (TGA): Dry samples (~5 mg) were heated in alumina crucibles under nitrogen atmosphere at a flow rate of 20 mL/min using a NETZSCH STA 449 F5 analyzer, NETZSCH-Gerätebau GmbH, Selb, Bavaria, Germany. The temperature increased from 25 °C to 1000 °C at 10 °C/min. The mass loss and residual ash content were calculated using Microsoft Office Excel 2021.

2.4. Physicochemical Property Assessments

2.4.1. Specific Oxygen Uptake Rate (SOUR)

Dissolved oxygen (DO) dynamics were monitored in a sealed glass reactor (working volume: 0.5 L) equipped with a polarographic DO probe (Hach LDO101, Hach Company, Loveland, CO, USA). MPs (10 g wet weight) were suspended in aerated ultrapure water at 30 °C with magnetic stirring (200 rpm). Following oxygen depletion, the obtained concentration was recorded at 10 s intervals for 10 min to capture microbial respiration [28]. The SOUR was calculated as follows:

$$SOUR\left(mg{O}_2/g\right)={\displaystyle \frac{\Delta DO\left(mg/L\right)\times V}{W_{dry}\times ∆t}}$$

(1)

where:

ΔDO: Linear decrease in DO concentration (mg/L)

V: Reactor volume (L)

W_dry: Dry weight of MPs (g), calculated as wet weight × (1 − moisture content)

Δt: Monitoring duration (min)

2.4.2. Mass Transfer Efficiency Experiments

Fungal MPs and PVA-SA-embedded MPs (PS-MPs) were tested at 10 g wet weight, with the PVA-SA gel dosed volumetrically. All materials were sterilized at 121 °C for 20 min prior to the experiment. DO and NH₄⁻ transfer were monitored in 500 mL sealed reactors (200 rpm, 25 °C) using a HACH HQ1130 DO meter, Hach Company, Loveland, CO, USA (10 s intervals) and a HACH DR5000 spectrophotometer, Hach Company, Loveland, CO, USA via the Nessler method, with samples filtered through 0.22 μm membranes [29].

2.4.3. Settling Velocity Determination

A total of 50 particles (MPs, PS-MPs, or PVA-SA gel) were placed in a 1 L graduated cylinder (height: 40 cm; inner diameter: 5.6 cm) filled with ultrapure water at 25 °C. The time required for each particle to descend by 20 cm was recorded, and the mean settling velocity (cm s⁻¹) was assessed from triplicate measurements [23].

2.4.4. Shear Resistance Test

The samples (5 g wet weight) were agitated with 3 mm glass beads at 200 rpm and 20 mm amplitude at 25 °C for 24 h. The unfragmented particles retained on a 0.5 mm stainless-steel sieve were weighed, and the fragmentation rate (%) was calculated as follows:

$$\mathrm{Fragmentation} \mathrm{rate} = {\displaystyle \frac{W_0-W_t}{W_0}}\times 100\%$$

(2)

where W₀ and W_t represent the initial and post-sieving wet weight, respectively.

2.4.5. Diffusion Inhibition Assay

The samples were placed on potato dextrose agar (PDA) supplemented with basic fuchsine and incubated at 30 °C for 7 d. Hyphal overgrowth was monitored and photographed on days 3 and 7.

2.5. Sulfamethoxazole (SMX) Concentration Analysis

SMX concentrations were quantified using high-performance liquid chromatography (HPLC, Agilent 1260, Agilent Technologies, Santa Clara, CA, USA) equipped with a C18 column (4.6 mm × 150 mm, 5 μm) and a UV detector (λ = 265 nm). The mobile phase consisted of 60% acetonitrile and 40% 0.01 M phosphate buffer (pH 3.0) at a flow rate of 1.0 mL/min. The samples were filtered through 0.22 μm nylon membranes and injected at 20 μL [22]. The calibration standards (0.1–50 mg/L) exhibited linearity (R² > 0.999) with a detection limit of 0.03 mg/L.

2.6. Statistical Analysis

All experimental data were derived from at least three independent biological replicates and are presented as mean ± standard deviation. The significance of differences between two groups was assessed using Student’s t-test (two-tailed), while comparisons among multiple groups were conducted by one-way analysis of variance (ANOVA) followed by Tukey’s post hoc test. A probability value (p-value) of less than 0.05 was considered statistically significant.

3. Results and Discussion

3.1. Effect of PVA-SA Concentration on Tensile Force and Ductility of Materials

The mechanical behavior of the PVA-SA composites was systematically analyzed to elucidate the concentration-dependent interplay between polymer network formation and functional performance in bio-carrier design. By varying the PVA concentration (2–12wt%) while maintaining SA at 2wt%, the mechanistic basis of hydrogel reinforcement and its relevance to microbial immobilization technologies were delineated.

As shown in Figure 1a, the fracture stress progressively increased from 76.43 kPa (2wt% PVA) to 360.44 kPa (12wt% PVA), governed by the percolation-driven formation of a denser polymeric network. At higher PVA concentrations, the enhanced chain entanglement and hydrogen bonding among the hydroxyl groups restricted pore collapse during tensile deformation, which was consistent with the rubber elasticity theory. Within this framework, the increased crosslinking density reduces the chain mobility, thereby elevating the elastic modulus and enhancing fracture resistance [30]. Such mechanical reinforcement is essential for bio-carriers in turbulent bioreactors, where hydraulic shear could impose the structural demands of failure-resistant architectures.

Fracture strain exhibited a biphasic response with increasing PVA concentration. An initial slight decline (from 358.23% to 322.51% at 2–8wt% PVA) reflected a gradual transition from entropy-driven elastic deformation to energy-dissipative mechanisms. Beyond the critical threshold of 8wt%, a pronounced reduction was observed (299.76% at 10wt%; 270.71% at 12wt%), indicating a shift toward brittleness-dominated failure. This threshold corresponds to the saturation of interstitial voids by excess polymer chains, which disrupts the SA-governed porous architecture. The resulting microstructural densification intensified the localized stress concentrations during fiber rearrangement, consistent with the Flory–Rehner theory of polymer network swelling [31]. Functionally, such pore occlusion impairs nutrient diffusion and microbial colonization, both of which are essential for bio-carrier performance in wastewater treatment systems.

The competing effects of enhanced fracture resistance and reduced ductility delineated a mechanical optimization window for PVA-SA biomaterials. Below 8wt% PVA, insufficient crosslinking compromised the structural durability, while concentrations above this threshold diminished the functional porosity required for sustaining microbial activity. This trade-off reflected the fundamental stiffness-toughness balance observed in natural composites, where the hierarchical architectures resolved conflicting mechanical demands. The selection of 10wt% PVA as the optimal formulation was based on its balanced performance within this window. Although its fracture stress (299.76 kPa) reached 83% of the maximum value (360.44 kPa at 12wt%), the strain retention (299.76%) remained 11% higher than at 12wt%, mitigating brittle fragmentation under shear stress while preserving the elasticity for dynamic substrate accommodation. Moreover, the deceleration in the peak stress increase beyond 10wt% indicated diminishing mechanical returns, which aligned with the percolation saturation in heterogeneous polymer networks.

3.2. Immobilization Effects on Aerobic Metabolism

Specific oxygen uptake rate (SOUR) was calculated from dissolved oxygen (DO) consumption kinetics over time, with the computational methodology detailed in Section 2.4.1. where V = 0.5 L (system volume), W dry = 10 g × (1 − 95.5%) = 0.55 g (dry biomass), and Δt = 10 min.

As shown in Figure 1b, SOUR analysis revealed distinct metabolic activities among the three fungal strains (F557, Y3, and F507) and their SA-PVA gel-immobilized counterparts. Free F557 exhibited the highest SOUR (26.84 ± 1.2 mg O₂/g·h), followed by Y3 (23.40 ± 0.9 mg O₂/g·h) and F507 (11.29 ± 0.7 mg O₂/g·h), indicating the superior oxygen utilization efficiency of F557. Upon entrapment, all strains demonstrated reduced activities: PS-F557 (18.71 ± 0.8 mg O₂/g·h), PS-Y3 (10.58 ± 0.6 mg O₂/g·h), and PS-F507 (5.84 ± 0.4 mg O₂/g·h), corresponding to the activity losses of 30.3%, 54.8%, and 48.3%, respectively. This suppression could be attributed to the diffusional limitations imposed by the gel matrix, with more substantial reductions observed for Y3 and F507, suggesting poor structural compatibility between their hyphal architectures and the polymeric network.

Notably, immobilized F557 retained 69.7% of its native SOUR, indicating superior tolerance to entrapment-induced stress compared with other strains. While all immobilized strains exhibited measurable activity, the F557 combination with elevated baseline metabolism (14.7% higher than Y3) and moderate activity reduction upon immobilization (Δ30.3% vs. Δ54.8% for Y3) identified it as the most promising candidate for bioreactor applications requiring sustained microbial function under encapsulation. Owing to its enhanced compatibility with the gel-embedding reinforcement process, A. niger F557 was selected as the model fungal strain for MP production in subsequent experiments.

3.3. PVA-SA-Reinforced Fungal Mycelial Pellets

3.3.1. Morphological Characterization of PVA-SA-MPs

SEM analysis revealed the key structural modifications induced by the PVA-SA reinforcement strategy, providing mechanistic insights into the enhanced bio-carrier performance of the composite. In Figure 2a, unmodified fungal MPs exhibit a hierarchical architecture composed of interconnected macroporous networks (Figure 2b) generated through hyphal self-assembly [32]. These uniformly distributed pores functioned as primary channels for nutrient diffusion and microbial colonization. Radial density gradients are observed within the fungal mycelium pellets (MPs), exhibiting progressive hyphal attenuation from the periphery toward the core. A distinct autolytic cavity develops centrally due to oxygen diffusion limitations. This structural reorganization arises from the following: (i) escalating mass transfer resistance radially inward, and (ii) aerobic metabolism of pellet-forming strains. Under internal hypoxia, autolytic degradation occurs, generating hollow architectures to facilitate oxygen permeation. High-resolution imaging revealed individual hyphae with intact surface topography and branching morphology as structural indicators of unconstrained fungal growth.

Following PVA-SA encapsulation, substantial reorganization of pellet architecture was observed (Figure 2c,d). The surface transitioned from an open macroporous framework to a semi-continuous hydrogel shell, effectively occluding surface voids while preserving subsurface microporosity. Cross-sectional SEM imaging revealed hydrogel infiltration along a concentration-gradient pattern, where the dense lamellar PVA-SA deposits were concentrated within the outer 30% of the pellet radius, transitioning to partial pore-filling in the interior regions. This graded encapsulation preserved residual microchannels between embedded hyphae, forming a dual-scale transport network [33]. The elimination of major macrochannels served to minimize biomass loss under hydrodynamic shear conditions. Conversely, the persistence of microporous pathways ensured the continual diffusion of substrates necessary for the viability of the encapsulated microbiota.

Notably, the hydrogel–hyphae interface exhibited intimate physical integration without evidence of delamination, indicating favorable interfacial compatibility between the synthetic polymer matrix and fungal filaments. Although the overall porosity was reduced, the engineered microchannel network preserved adequate hydraulic connectivity to prevent diffusion-limited metabolic inhibition, serving as a common failure mode in over-encapsulated bio-carriers [34]. This structural paradigm shift from fungal hyphae-dominated scaffolding to a hybrid organic–inorganic composite accounted for the concurrent enhancement in mechanical resilience. The hydrogel shell acted as a shear-dissipative barrier, redistributing the external stresses through its viscoelastic matrix, whereas the retained microchannels preserved the mass transfer kinetics critical for the bioremediation processes. This architectural strategy reflects the emerging design principles for next-generation bio-carriers, emphasizing the synergistic integration of synthetic durability with biological functionality.

3.3.2. FTIR Analysis

As shown in Figure 3d, FTIR analysis confirmed the key molecular interactions underlying the structural and functional integration of the PVA-SA composites with fungal MPs. The spectrum of pure PVA-SA exhibited a prominent C=O stretching vibration at 1725 cm⁻¹, which is characteristic of sodium alginate carboxyl groups, along with the C–O vibrations at 1040 cm⁻¹ from alginate’s guluronic acid residues and PVA ether linkages. A broad hydroxyl (–OH) absorption band spanning 3500–3200 cm⁻¹ indicated extensive hydrogen bonding, which was essential for hydrogel hydration and nutrient permeability [25]. MPs showed distinct spectral features at 1650 cm⁻¹ (C=O stretching from chitin/protein amides) and 1380 cm⁻¹ (C–H bending from hydrophobic lipid moieties), reflecting their inherent amphiphilic structure.

In PS-MPs, the spectral shifts revealed multiscale interfacial bonding mechanisms. The broadening and intensification of the –OH band (3500–3200 cm⁻¹) indicated enhanced hydrogen bonding between the surface hydroxyls of the MPs and the PVA-SA matrix. The intensified C=O peak at 1725 cm⁻¹ and the red-shifted C–O vibration (1040 → 1032 cm⁻¹) suggested covalent esterification between the alginate carboxyl groups and the hydroxyl or amine functionalities of the MPs. The concurrent attenuation and shift of the lipid-associated C–H bending peak (1380 → 1365 cm⁻¹) implied hydrophobic interactions between the PVA-SA alkyl chains and nonpolar domains of MPs. These synergistic interactions stabilized the composite architecture via reversible hydrogen bonding, covalent crosslinking, and hydrophobic adhesion, which served as a hierarchical reinforcement strategy reminiscent of the natural biocomposite design.

Crucially, retention of the amide I band at 1650 cm⁻¹ confirmed the preservation of protein integrity in MPs, supporting sustained enzymatic activity for pollutant degradation. The persistence of hydrophilic C–O and –OH vibrations maintains the hydration and substrate diffusion pathways, whereas hydrophobic stabilization reduces the delamination risks in aqueous environments [28]. This dual preservation of mechanical–chemical stability and bioactivity addressed the durability–functionality trade-off inherent to conventional bio-carriers.

3.3.3. TGA Analysis

Thermogravimetric analysis (TGA) quantitatively confirmed the structural integration efficiency of the PVA-SA hydrogel within the fungal MPs, providing key insights into the thermal stability and compositional synergy. Pristine MPs exhibited near-complete thermal decomposition, leaving only 0.2 ± 0.1% residual mass at 1000 °C (Figure 3a), which is consistent with their organic composition dominated by polysaccharides, proteins, and lipids [35]. In contrast, pure PVA-SA yielded 12.5 ± 0.3% inorganic residue at 1000 °C (Figure 3b). This was attributed to thermally stable salts, such as borate complexes and calcium carbonates, which can be formed during dual crosslinking, representing a structural fingerprint of the synthetic polymer matrix.

For PS-MPs, the residual inorganic content (9.5 ± 0.2% at 1000 °C, Figure 3c) was derived exclusively from the PVA-SA matrix, confirming the successful hydrogel encapsulation without secondary inorganic contamination. This corresponded to a hydrogel loading efficiency of 77.3% by dry mass, which was facilitated by synergistic adsorption mechanisms. The dense PVA-SA network supplied abundant hydroxyl and carboxyl binding sites, whereas the porous hyphal architecture of MPs enabled deep hydrogel infiltration via capillary action and surface charge interactions. The residual mass discrepancy between PS-MPs (9.5%) and pure PVA-SA (12.5%) likely resulted from the partial decomposition of fungal biomass during heat exposure during the crosslinking process.

The three-stage thermal degradation profiles further elucidated the structural interactions within PS-MPs. Below 200 °C, PS-MPs exhibited a 40% lower mass loss than pristine MPs (5.2% vs. 8.7%), indicating hydrogel-induced restriction of free water evaporation, which is a hallmark of reinforced hydrophilicity. Between 200 and 500 °C, the decomposition rate of PS–MPs decreased by 22% relative to that of MPs, demonstrating the protective role of PVA-SA in shielding thermally labile fungal biopolymers (e.g., chitin) through physical encapsulation and covalent stabilization. Above 500 °C, the convergence of the PS-MPs and PVA-SA mass loss curves confirmed the hydrogel dominance in high-temperature stability, which was associated with the borate-crosslinked PVA networks and calcium–alginate complexes resisting oxidative decomposition [36].

The high hydrogel-loading efficiency and thermal resilience of PS-MPs can confer distinct operational advantages in wastewater bioreactors [37]. The PVA-SA matrix reduces fungal biomass leaching during thermal sterilization (e.g., autoclaving), while the retained inorganic residue serves as a structural scaffolding for microbial re-colonization post-sterilization. By quantifying the organic–inorganic balance, the TGA results validated PS-MPs as durable yet biodegradable carriers, which is consistent with the circular economy principles in environmental biotechnology. The demonstrated synergy between the synthetic and biological components provides a scalable platform for engineering hybrid biomaterials with tunable thermal and mechanical properties.

3.3.4. Tensile Strength and Strain Tolerance

The mechanical reinforcement of PS-MPs was quantitatively confirmed through tensile testing, which revealed the synergistic structural interactions essential for fungal-based bio-carrier design. As shown in Figure 3e, PS-MPs demonstrated a 499% increase in the ultimate tensile strength (310.4 ± 8.2 vs. 62.1 ± 3.5 kPa for pristine MPs) and a 6.7-fold enhancement in toughness (356.8 ± 15.1 vs. 53.2± 2.8 kJ/m³), which was attributed to the dual-scale bonding mechanisms. The covalent Ca²⁺–alginate crosslinking provided a rigid framework resistant to elastic deformation, whereas the hydrogen bonding between PVA hydroxyl groups and chitin in MPs, as evidenced by FTIR peak broadening at 3280–3350 cm⁻¹, enabled energy dissipation during strain propagation.

The strain-to-failure behavior further distinguished the composites: pristine MPs exhibited catastrophic rupture at 135% strain owing to the localized disintegration of the hyphal network, whereas PS-MPs sustained the stress to 230% strain through hierarchical load redistribution. The PVA-SA matrix bridged the adjacent hyphae, transforming the discrete hyphal fractures into distributed microcracks, which was a mechanism supported by a 193% increase in Young’s modulus (1.35 ± 0.09 MPa for PS-MPs vs. 0.46 MPa for MPs). This modulus enhancement reflects the ability of the hydrogel to restrict hyphal slippage, shifting the failure from a ductile, fiber-dominated mode to a composite-controlled fracture pattern.

The post-yield behavior revealed distinct failure mechanisms, where PS-MPs exhibited rapid stress decay (−12.4 kPa/% strain) indicative of brittle gel matrix cleavage, whereas MPs showed a gradual stress reduction (−0.46 kPa/% strain) characteristic of ductile hyphal pull-out. This transition was consistent with the hydrogel’s function in redistributing axial stresses across the composite, as described by hierarchical reinforcement models. The concurrent retention of fungal hyphal structural engagement through hydrogen bonding and the dominance of hydrogel-mediated fracture mechanics resolved the long-standing incompatibility between mycelial flexibility and synthetic rigidity, establishing a balanced architecture wherein the synthetic networks enhanced durability without compromising biological functionality.

3.3.5. Porosity–Diffusion Trade-Off in Gel-Embedded Mycelial Pellets

The NH₃-N and oxygen removal performance of the three materials exhibited distinct temporal profiles (Figure 4d,e). PS-MPs demonstrated the fastest adsorption kinetics, reducing the NH₃-N concentration from 100 to 57.0 ± 0.68 mg/L (43.0% removal) within 1200 s. A biphasic adsorption pattern was observed: initial retardation (0–200 s: 100 → 68 mg/L) followed by accelerated uptake (200–1200 s: 68 → 57 mg/L). This behavior corresponded to the hierarchical porous architecture, wherein a surface-crosslinked calcium alginate layer transiently restricted solute ingress, while the porous mycelial core enabled chemisorption via amino–carboxyl interactions. This process followed pseudo-second-order kinetics (R² = 0.991), confirming that chemical adsorption was the dominant mechanism [38].

MPs reached the rapid adsorption equilibrium (100 → 69.0 ± 1.1 mg/L at 200 s), with negligible further uptake, which was attributed to their high porosity (92.3 ± 2.1%) and the macroporous channels facilitating the rapid saturation via physical entrapment. In contrast, PVA-SA exhibited limited adsorption capacity (100 → 80.38 ± 0.4 mg/L), as its dense hydrogel network (porosity 34.5 ± 1.8%) restricted both physisorption and surface binding. These findings underscore the engineered advantage of PS-MPs, where the surface gel layers mitigated biomass washout under turbulent flow conditions while preserving microbial chemisorption activity as a critical advancement for continuous-flow bioreactors requiring both hydraulic stability and nutrient removal efficiency.

3.3.6. Influence of Gel Embedding on Settling Velocity

The settling velocities of MPs, PVA-SA, and PS-MPs differed significantly (one-way ANOVA, p < 0.05). MPs composed solely of fungal hyphae exhibited the lowest mean velocity (0.49 ± 0.12 cm/s) with substantial variability (range: 0.28–0.71 cm/s; coefficient of variation, CV = 24.5%), which was attributed to their heterogeneous porous architecture (Figure 4a). In contrast, PVA-SA demonstrated the highest velocity (1.36 ± 0.13 cm/s) and minimal variability (range: 1.12–1.53 cm/s; CV = 9.6%), which was consistent with its dense hydrogel matrix enhancing gravitational settling while limiting microbial mass transfer (Figure 4b).

PS-MPs achieved an optimized balance between hydraulic stability and functional performance, exhibiting a mean settling velocity of 1.12 ± 0.17 cm/s (range: 0.81–1.39 cm/s; CV = 15.6%). The significantly lower variability than that of MPs (p < 0.01, F-test) underscored the role of the surface gel layer in suppressing turbulence-induced resuspension (Figure 4c). This dual-function mechanism enabled PS-MPs to settle 2.3 times faster than MPs and translated directly into reduced hydraulic retention times (HRTs), which served as a key advantage for scaling fungal-based wastewater treatment in continuous-flow systems [39].

3.3.7. Controlled Experiment on the Suppression of Hyphal Dispersion

Hyphal growth inhibition assays revealed distinct morphological behaviors between unmodified MPs and PS-MPs (Figure S1). The unmodified MPs exhibited substantial structural expansion and filamentous outgrowth within 3 d, with hyphae radially dispersed across the agar surface by day 7. This unrestricted proliferation was attributed to the direct contact between the fungal biomass and nutrient-rich medium, where the absence of a protective gel layer permitted unimpeded nutrient uptake and hyphal extension.

In contrast, PS-MPs exhibited localized red pigment diffusion from the base within 3 d, with complete coloration of the gel layer by day 7. The gel permitted nutrient transport, as evidenced by the Congo red uptake, while physically confining hyphal growth to the interior of the composite. No radial hyphal dispersion was observed, confirming the gel’s role in mitigating environmental leakage risks [2]. These findings demonstrated that the PS-MPs’ design effectively balanced the nutrient accessibility for metabolic activity with fungal biomass containment, which is an essential requirement for minimizing ecological disruption in wastewater treatment systems.

3.3.8. Agitation Tolerance and Structural Integrity

The agitation resistance of MPs and PS-MPs was quantified using a 24 h glass bead impact test (Figure 5a). The unmodified MPs exhibited substantial fragmentation, with 24.6% weight loss (experimental group vs. control: 75.4 ± 2.1% vs. 99.7 ± 0.3%), due to the structural disintegration under mechanical shear. In contrast, PS-MPs demonstrated exceptional stability, retaining 96.8 ± 1.2% of their initial mass under identical conditions. This 7.7-fold reduction in weight loss (p < 0.001, t-test) was attributed to the PVA-SA gel layer, whose smooth surface reduced the frictional abrasion and viscoelastic matrix dissipated collision energy, preventing irreversible damage. The engineered composite structure addressed a major limitation in the durability of fungal carriers in industrial-scale agitated bioreactors.

3.4. Engineered Bio-Carrier with Acclimated Sludge for SMX Degradation

Figure 5c demonstrates the preparation process of PS-MPs + Sludge AFP. The SMX degradation efficiencies of the three immobilized sludge systems (MPs + sludge, PS + sludge, PS-MPs + sludge) diverged significantly over six consecutive 4 h cycles (totaling 24 h; Figure 5b). The PS-MPs + sludge system demonstrated superior stability, achieving 73.28% removal in Cycle 1 and maintaining 73.87% in Cycle 6 (variation < ±1.5%). In contrast, the MPs + sludge system showed a progressive decline from 81.2% to 58.68%, indicating microbial inhibition or biomass washout. PS + sludge exhibited the lowest performance (36.87% in Cycle 6), constrained by the mass transfer limitations of the dense hydrogel matrix [40]. Statistically significant differences (p < 0.05) validated the dual structural advantage of PS-MPs: fungal mycelium for biomass retention and gel encapsulation for hydraulic protection.

3.5. Mechanism of Enhanced SMX Degradation via PS-MPs-AFP

The degradation of sulfamethoxazole (SMX) by sludge-laden PS-MPs-AFP composites is exceptionally stable and efficient, attributed to three interdependent mechanisms, which have been validated by experimental data.

3.5.1. Robust Biomass Retention Through Structural Reinforcement

PVA-SA encapsulation fundamentally improves mechanical integrity, avoiding the observed microbial consortia washout in uncoated mycelial pellets (MPs). Quantitative analysis indicates a 7.7-fold higher shear resistance for PS-MPs compared to MPs under hydraulic stress: After 24 h glass bead impact (Figure 5a), PS-MPs retained 96.8% of their initial mass (3.2% loss), while MPs experienced 24.6% mass loss. The viscoelastic gel matrix (Young’s modulus increased by 193%) completely inhibited radial hyphal extension (Figure S1), achieving >90% mycelial confinement efficiency. This structural barrier causes shear forces to dissipate through reversible deformation, thereby removing biomass fragmentation that occurred in MPs systems. By immobilizing functional microorganisms in a protective exoskeleton, PS-MPs-AFP ensures metabolic continuity, which is crucial for the sustained biotransformation of SMX.

3.5.2. Optimized Substrate Accessibility via Hierarchical Mass Transfer

Controlled PVA-SA encapsulation selectively modulates the pore architecture to achieve a balance between diffusion kinetics and biomass retention. SEM imaging (Figure 2) reveals that PS-MPs preserve vital 1–3 μm microchannels for oxygen/nutrient transport while eliminating >10 μm macropores responsible for sludge leakage in unencapsulated MPs. This engineered porosity directly improves mass transfer efficiency, as shown by PS-MPs achieving 43% NH₃-N adsorption within 1200 s (Figure 4d), significantly outperforming the PVA-SA gel. The adsorption kinetics follow a pseudo-second-order model (R² = 0.98), demonstrating the effective transfer of SMX across the interface to immobilized biomass. Critically, macropore elimination prevents the formation of preferential flow paths that lead to substrate bypass in conventional biofilm carriers, thus ensuring the effective mineralization of SMX degradation intermediates (e.g., 3A5MI) without accumulation.

3.5.3. Microenvironment-Driven Microbial Syntrophy

The gel matrix creates self-regulated oxygen gradients that spatially organize microbial consortia. As shown in Figure 6’s schematic, dissolved oxygen forms concentric zones: In the gel periphery, aerobic conditions support chemoheterotrophic SMX-degraders (e.g., Flavobacterium), transitioning to hypoxia/anaerobiosis in the core area where denitrifiers flourish. This stratification allows for synergistic cross-feeding, where nitrifying bacteria oxidize ammonia, and SMX degraders utilize organic intermediates, both sharing enzymatic cofactors. Crucially, encapsulation maintains metabolic activity even under diffusional limitations, as shown by the immobilized F557, which retains 69.7% of its specific oxygen uptake rate (SOUR) relative to free cells (Figure 2: 18.71 vs. 26.84 mg O₂/g·h). Such metabolic resilience, paired with syntrophic partnerships, ensures the coexistence of metabolic pathways essential for the breakdown of recalcitrant SMX under hydraulic fluctuations.

These mechanisms together maintain 73% SMX removal efficiency over six operational cycles (160 h, Figure 5b)—a performance that is unattainable by unmodified systems. Structural integrity safeguards biomass from erosion, hierarchical porosity ensures a continuous supply of substrate and oxygen, and microenvironmental control maintains the functionality of consortia. This triad stabilizes the SMX-transforming enzymatic activity (e.g., cytochrome P450 monooxygenases), eliminating the efficiency decay observed in MPs where biomass loss disrupts degradation pathways.

4. Conclusions

This study proposed a dual-network hydrogel immobilization system that effectively addressed the structural fragility and operational instability inherent in fungal MPs in wastewater bioremediation. The poly(vinyl alcohol)–alginate composite matrix reinforced via Ca²⁺/boric acid dual crosslinking exhibited substantial mechanical enhancement, with a 4.99-fold increase in tensile strength (310.4 ± 12.7 vs. 62.1 ± 5.3 kPa for native MPs) and a 129% improvement in settling velocity (1.12 ± 0.09 vs. 0.49 ± 0.04 cm/s). These engineered properties enable stable bioreactor operation under turbulent flow conditions (Reynolds number > 4500), overcoming the hydraulic limitations that typically compromise conventional fungal-based systems [41].

Notably, the hierarchical pore architecture of immobilized MPs preserved essential microbial functionality despite moderate metabolic suppression (30.3–54.8% reduction in specific oxygen uptake rates). The system maintained consistent SMX degradation efficiency (72–75% over six operational cycles), significantly outperforming both unmodified MPs (58.7 ± 3.2% activity decay) and gel-encapsulated activated sludge controls (34–39% efficiency). Effective containment was achieved through radial hyphal growth inhibition (>90% suppression) and minimal biomass leakage (3.2 ± 0.4% mass loss under the 24 h shear at 300 rpm), thereby mitigating the ecological risks associated with fungal dispersal in open wastewater environments [2].

These findings address the longstanding trade-off between mechanical durability and biocatalytic performance in microbial immobilization technologies. The dual-network design established a versatile platform for fungal biomass utilization by integrating industrial-grade shear resistance with the microenvironmental regulation of microbial consortia [36,42]. Although the current formulation can exhibit broad applicability across basidiomycete strains, future research should focus on (1) developing strain-specific hydrogel matrices to minimize metabolic inhibition, (2) modeling antibiotic degradation kinetics under transient hydraulic conditions, and (3) conducting pilot-scale validations in continuous-flow systems treating real pharmaceutical wastewater. This advancement represents a critical step toward the practical implementation of fungal bioremediation technologies, aligning with circular bioeconomy goals through sustainable material engineering and energy-efficient wastewater treatment strategies [1,6].