From Biomass to Biofabrication: Advances in Substrate Treatment Technologies for Fungal Mycelium Composites

Highlights

What are the main findings?

• Substrate treatment critically determines hyphal colonization, microstructural uniformity, and the resulting mechanical performance of mycelium-based composites (MBCs).
• Optimized physical, chemical, biological, and hybrid pretreatments enhance digestibility, interfacial bonding, and composite strength; however, over- or under-treatment negatively affects performance.
• Scalability, reproducibility, moisture sensitivity, and durability remain key technical challenges limiting industrial adoption.

What are the implications of the main findings?

• Substrate engineering must be treated as a core material design parameter to achieve consistent and predictable MBC properties.
• Standardized and energy-efficient pretreatment protocols are essential for regulatory compliance and sustainable large-scale manufacturing.
• Integrating advanced monitoring and modeling tools can improve reproducibility and accelerate the commercialization of MBC technologies.

Abstract

Mycelium-based composites (MBCs) have emerged as promising biofabricated materials that align with circular economy and clean technology goals by utilizing fungal networks to transform lignocellulosic residues into functional, biodegradable composites. Despite the MBC’s potentials, the intrinsic nature of the fungal strain, substrate physico-chemical composition and engineering property variability remain significant hurdles that should be critically surmounted. Substrate treatment is central to determining growth kinetics, microstructural uniformity, and mechanical performance in MBC production. This review highlights recent advancements in physical, chemical, biological, and hybrid pretreatment methods, including comminution, pasteurization, alkali hydrolysis, enzymatic conditioning, microwave-assisted hydrolysis, ultrasound pretreatment, steam explosion, plasma activation, and irradiation. These technologies collectively enhance substrate digestibility, aeration, and permeability while reducing contamination. Optimization parameters—temperature, pH, C:N ratio, moisture content, particle size, porosity, and aeration—are examined as critical process levers influencing hyphal density, bonding efficiency, and composite uniformity. Evidence suggests that properly engineered substrate treatments accelerate colonization, strengthen hyphal networks, and significantly improve compressive, tensile, and flexural material properties. The review discusses emerging process control tools such as AI-assisted modeling, micro-CT porosity analysis, and sensor-integrated bioreactors that enable reproducible and energy-efficient fabrication. Collectively, the findings position substrate engineering as a foundational technology for scaling high-performance mycelium composites and advancing sustainable material innovation.

1. Introduction

A compelling reason for biofabricated materials is changing the paradigm of composite manufacture from synthetic binding to biological self-assembling via fungal mycelium. By immersing fungal hyphal networks in lignocellulosic or organic substrates, mycelium-based composites (MBCs) produce materials that are biodegradable, lightweight, and moldable. This synergy not only offers an attractive route toward sustainable materials, but also addresses escalating concerns over plastic waste management, resource depletion, and the environmental footprints of traditional polymer composites [1].

In the pursuit of circular bioeconomy strategies, substrate pretreatment has emerged as a crucial factor for optimizing mycelial growth and enhancing composite performance. The pre-treatment procedure affects hyphal colonization, nutrient availability, and binding strength. It also serves as a conduit for precise regulation of porosity, density, and mechanical properties. Minor fluctuations in particle size, moisture content, sterilization protocols, or biochemical alterations might result in substantial differences in material outcomes. This highlights the significance of a methodical understanding of substrate conditioning. Recent studies indicate that extensive chemical or thermal pretreatment can improve digestibility but may lead to nutrient loss or the development of hazardous byproducts. In addition, insufficient preparation of the substrate also hinders fungal infiltration and reduces composite integrity [2,3].

This review examines the advancements in fungal mycelium substrate treatment technologies across five interconnected domains. Firstly, it highlights the fundamentals of substrate technology, discussing the criteria for substrate selection, preconditioning, and sterilization. Secondly, it emphasizes modern technological innovations such as microwave-assisted, steam explosion, and plasma treatment procedures. Third, it discusses optimization parameters and process control. This emphasizes aspects such as temperature, pH, particle size, and moisture content that directly influence fungal colonization and mechanical integrity. The fourth part analyzes the influence of substrate treatment on mycelium proliferation and composite characteristics. This demonstrates how regulated processing can enhance hyphal density, structural consistency, and mechanical integrity. The last part reviews the market and social acceptability of the MBCs.

In fungal biology and materials engineering, substrate treatment is more than a preliminary step. It is also a key design variable. It strongly influences the feasibility, reliability, and performance of MBCs. This work positions substrate engineering as a fundamental technology for future bio-derived composites. It does so by examining physical, chemical, and biological treatment methods in detail. It then relates these treatments to material properties and application requirements. The goal is to support researchers, practitioners, and industry stakeholders. It aims to help them develop effective pathways for converting waste biomass into valuable, sustainable mycelium-based products. Figure 1 presents an integrated schematic of the overall MBC production pathway from biomass as feedstock to the final composite.

2. Overview of Substrate Treatment Techniques

The substrate used for cultivating fungal mycelium serves as both a nutritional medium and a structural matrix for mycelium-based composites (MBCs). Its preparation directly affects mycelial colonization, mechanical strength, water retention, and biodegradability. The goal of substrate treatment is to optimize physicochemical and biological characteristics. Doing this will promote rapid hyphal penetration, effective binding, and minimal contamination. These treatments are broadly classified into physical, chemical, and biological methods—each offering unique advantages and limitations.

2.1. Physical Treatments

The typical sequence of physical substrate pretreatment is illustrated in Figure 2. Physical treatments are the most common and often the first step in substrate preparation. They are primarily used to modify particle size, sterilize the material, and enhance surface accessibility to prevent fungal colonization.

Table 1. Comparative overview of substrate treatment techniques.

Treatment Type	Example Methods	Advantages	Limitations	References
Physical	Milling, autoclaving, and pasteurization	Simple, effective sterilization; improves surface area	Energy-intensive; nutrient degradation	[4,5]
Chemical	NaOH, H2SO4, H2O2, ozone	Enhances cellulose accessibility; improves fungal binding	Toxicity, pH imbalance, waste disposal issues	[6]
Biological	Enzymatic pre-digestion, microbial co-culture	Eco-friendly; improves bioavailability	Slower; needs process control	[2,3]

summarizes the comparative overview of substrate treatment techniques.

2.1.1. Comminution (Milling and Grinding)

Reducing substrate particle size through milling or grinding increases the surface area-to-volume ratio. It also facilitates mycelial attachment and enzymatic degradation. Fine particles enhance compactness and binding within the composite matrix [4]. However, excessively fine milling may reduce porosity, limit aeration, and impede oxygen transfer—key parameters for aerobic fungal metabolism [2]. Studies on Pleurotus ostreatus and Ganoderma lucidum have shown optimal colonization with substrate particle sizes between 1 and 5 mm, balancing aeration and nutrient accessibility [3].

2.1.2. Thermal Treatments (Autoclaving, Pasteurization, and Drying)

Thermal sterilization eliminates microbial competitors and partially softens lignin, increasing substrate permeability. Autoclaving (121 °C for 15–30 min) provides near-complete sterilization but may degrade fermentable sugars or produce inhibitory compounds such as furfural [1]. Pasteurization (60–80 °C for 1–2 h), though less rigorous, preserves more nutrients and is preferred in sustainable, low-energy setups. For example, Schizophyllum commune and Lentinus sajor-caju show better hyphal growth on pasteurized straw than on autoclaved substrates due to preserved polysaccharides and lower sugar degradation [7]. Drying post-treatment stabilizes the substrate, reduces moisture heterogeneity, and prevents microbial regrowth before inoculation.

2.1.3. Mechanical Densification and Sieving

Controlling substrate density is crucial for consistent porosity and air diffusion. Mechanical densification (e.g., pelletizing) ensures uniform packing during molding, enhancing structural stability of the composite [6]. Sieving helps maintain consistent particle size, improving reproducibility in growth experiments. A uniform substrate allows predictable gas exchange and mechanical performance, which are key parameters in MBC engineering [2].

2.2. Chemical Treatments

Chemical treatments change the chemical composition of lignocellulosic substrates to improve digestibility and surface reactivity. These processes typically aim to remove or alter lignin and hemicellulose, which act as barriers to enzymatic action and fungal colonization.

2.2.1. Alkali and Acid Hydrolysis

Alkali treatments commonly use sodium hydroxide (NaOH) or potassium hydroxide (KOH). These treatments disrupt ester linkages in lignin and hemicellulose, which enhances cellulose exposure and improves water retention capacity [5]. For instance, treating corn stover with 2–4% NaOH improved Pleurotus eryngii colonization rates by up to 40% [3]. Acid hydrolysis, typically using sulfuric (H₂SO₄) or hydrochloric acid (HCl), depolymerizes hemicellulose and enhances nutrient release. However, both alkali and acid treatments risk pH shifts, corrosion of equipment, and the formation of toxic degradation byproducts [2]. Thus, post-treatment neutralization and washing steps are necessary to ensure mycelial compatibility.

2.2.2. Oxidative Treatments and Photocatalysis

Oxidative treatments include hydrogen peroxide (H₂O₂), ozone (O₃), and use of peracetic acid. These treatments activate the substrate surface by creating carboxyl and hydroxyl groups that promote mycelial adhesion [7]. H₂O₂-based pretreatments are particularly effective for white-rot fungi due to their synergy with fungal peroxidase systems. Nonetheless, prolonged exposure or excessive concentrations can cause cellulose oxidation and weaken the structural matrix. While chemical methods effectively enhance substrate digestibility, they often pose environmental and cost challenges, as shown in Table 1. They require strict process control, safety measures, and post-treatment handling to prevent contamination or toxicity. Increasingly, researchers are moving toward milder, eco-friendly chemical protocols integrated with enzymatic treatments or hydrothermal processes to reduce environmental impact [6].

2.3. Biological Treatments

Biological treatments represent the most sustainable and environmentally friendly approach, using microbial or enzymatic agents to pre-digest the substrate before fungal inoculation. This process simulates natural lignocellulose decomposition while enhancing nutrient accessibility and reducing the need for chemical reagents.

2.3.1. Enzymatic Pre-Digestion

Enzymatic pre-digestion has emerged as an effective biological strategy for enhancing substrate suitability in mycelium-based composite production. Targeted hydrolytic and oxidative enzymes such as cellulases, xylanases, laccases, and manganese peroxidases can be applied to the substrate. These enzymes partially depolymerize lignocellulosic matrices before fungal inoculation. This controlled alteration reduces structural recalcitrance and increases substrate porosity. It also exposes cellulose microfibrils and hemicellulosic fractions, improving accessibility for fungal enzymes during subsequent colonization [8,9]. In parallel, enzymatic cleavage releases soluble oligosaccharides and low-molecular-weight phenolics. These compounds act as readily available carbon sources that stimulate early hyphal extension and metabolic activity [10].

Oxidative enzymes such as laccase and manganese peroxidase are particularly valuable for selectively modifying lignin. This occurs without extensive carbohydrate loss, thereby preserving the fibrous scaffold necessary for mechanical integrity [11,12]. For example, wheat straw was pre-treated with laccase and manganese peroxidase. This pre-treatment enhanced Ganoderma lucidum mycelial growth rates by approximately 32% compared to untreated substrates [13]. This improvement was attributed to better enzyme–substrate synergy and a reduced colonization lag time. Beyond accelerating growth, enzymatic pre-digestion can promote more uniform hyphal penetration and interparticle bonding. This ultimately contributes to composites with greater homogeneity and more consistent mechanical properties. When optimized, this low-energy, biologically compatible approach aligns well with cleaner production principles for scalable mycelium composite manufacturing.

2.3.2. Microbial Co-Culturing

Microbial co-culturing represents a biologically inspired strategy for improving substrate readiness in mycelium-based composite fabrication. In this approach, selected non-competitive microorganisms—such as Bacillus subtilis or Trichoderma reesei—are introduced during a controlled preconditioning phase to modify lignocellulosic substrates before fungal inoculation. These organisms secrete a broad spectrum of extracellular enzymes, including cellulases, hemicellulases, and lignin-modifying enzymes. The secreted enzymes partially depolymerize complex plant polymers and increase substrate porosity. Simultaneously, microbial metabolism generates low-molecular-weight sugars, organic acids, and growth-promoting metabolites. These enhance nutrient bioavailability and stimulate early mycelial development [14,15].

An additional advantage of co-culturing lies in its inherent biocontrol capacity. Certain bacterial and fungal species produce antimicrobial compounds that suppress opportunistic contaminants. Co-culturing also reduces reliance on intensive sterilization or chemical additives.

Moreover, microbial co-culturing can rebalance carbon-to-nitrogen ratios and micro-nutrient availability. This facility minimizes the need for external nutrient supplementation and improves process sustainability. The method also effectively mimics natural composting and soil conditioning processes, but under controlled conditions tailored to favor mycelial colonization and uniform hyphal penetration [11]. When carefully optimized to avoid competitive inhibition, microbial co-culturing offers a low-energy, ecologically aligned pathway for enhancing substrate performance and composite consistency.

2.3.3. Advantages and Limitations

Biological treatments are low-cost, environmentally benign, and compatible with large-scale bioprocessing. However, they require precise control of temperature, pH, and microbial population dynamics to prevent over-degradation or contamination. The slower processing time compared to chemical methods remains a constraint for industrial scalability [16].

Collectively, substrate treatment techniques form the foundation for optimizing mycelium-based composite production. Physical treatments enhance accessibility and sterilization, chemical methods improve digestibility and surface reactivity, and biological treatments introduce sustainability and eco-efficiency [17]. The ideal strategy often integrates multiple methods—for instance, mild alkali pretreatment followed by enzymatic conditioning—to maximize fungal colonization and material performance [18]. A schematic classification of substrate treatment approaches is shown in Figure 3.

Future research should focus on developing integrated hybrid treatment systems. These will combine energy-efficient physical techniques with biologically selective processes, while minimizing chemical residues. Emerging technologies such as microwave-assisted hydrothermal pretreatment, plasma surface activation, and enzyme immobilization show great promise. These have the potential to achieve reproducible, cost-effective, and environmentally sustainable substrate conditioning for next-generation mycelium-based materials.

3. Recent Technological Advancements in Substrate Treatment

3.1. Microwave-Assisted Hydrolysis

Microwave-assisted pretreatment has emerged as an attractive route to disrupt the recalcitrant lignocellulosic matrix. It offers rapid, volumetric heating that can reduce process times and energy inputs relative to conventional thermal methods [19]. Microwaves interact directly with polar molecules to produce localized heating. The produced heat accelerates hydrolytic cleavage of hemicellulose and partial delignification when combined with chemical agents (alkali or dilute acids) or green solvents. For mycelium applications, the primary benefits are increased cellulose accessibility and enhanced surface area for fungal enzyme action. Recent work demonstrates that microwave-assisted alkali treatments (e.g., KOH combined with microwave exposure) significantly increase the enzymatic digestibility of sawdust and agricultural residues. This yields higher sugar release and improves downstream fungal growth compared with conventional heating at the same nominal temperature [20,21].

However, process optimization is critical: excessive microwave power or prolonged exposure can cause sugar degradation (furfural formation) and generate inhibitory by-products that reduce fungal vigor. Sequential treatment regimes, short microwave pulses combined with mild alkali, have been proposed to balance disruption and preservation of fermentable oligomers [22,23]. Additionally, microwave pretreatment scales differently from conventional steam processes. While small-batch microwave reactors are readily implemented in lab settings and pilot lines, large-scale continuous microwave systems require specialized reactor design and significant capital investment. Energy analyses in biomass conversion contexts indicate potential for reduced overall energy use when microwave systems are tightly integrated with enzymatic hydrolysis. However, the environmental and economic advantages depend on feedstock moisture content and the availability of low-cost electricity [24,25]. For mycelium composite manufacture, microwave pretreatment gives a fast way to condition substrates before inoculation. It also ensures the process parameters are tuned to avoid chemical damage and guarantee microbial compatibility.

Key recent studies: Ethaib, 2024 and Venegas-Vasconez et al., 2025 [26,27] reported improved enzymatic yields and reduced processing times using microwave-assisted alkaline protocols; Xu et al., 2025 [28] applied microwave-assisted deep eutectic solvent pretreatment for lignin-containing residues with promising results for downstream material use.

3.2. Ultrasound (Sonication) Pretreatment

Ultrasound (high-intensity, low-frequency sonication) is another green pretreatment that mechanically and chemically alters biomass. It does this via cavitation—formation and violent collapse of microbubbles—that produces intense local shear and microjetting. Cavitational forces disrupt cell walls, increase surface area, and can enhance the porosity and wettability of lignocellulosic particles. It also increases the enzyme accessibility and facilitates microbial colonization [10]. In combination with mild alkali or enzymatic treatment, ultrasound has been shown to improve hemicellulose solubilization and increase yields of oligosaccharides. Importantly, treated substrates often exhibit faster initial colonization rates and more homogeneous hyphal penetration, likely due to reduced physical barriers [10,29].

Practical advantages of ultrasound include the ability to operate at near-ambient bulk temperatures, compatibility with aqueous systems, and relative ease of integration into continuous flow reactors. Laboratory studies indicate that ultrasound preconditioning can reduce the dose and duration of chemical reagents required for effective pretreatment. For instance, lower alkali concentrations combined with sonication achieve similar delignification to higher chemical loads without sonication [10]. Limitations include equipment erosion, the need for energy for sonicators, and non-uniform effects in large piles of biomass. The non-uniform effects can be surmounted if flow-through or agitation systems are used. Recent advances—such as coupling ultrasound with electrochemical or ionic liquid treatments—show further promise for tailored fractionation without harsh chemicals [30].

For MBC fabrication, ultrasound is attractive because it can selectively loosen bonds and open pore networks without removing the entire structural polymers. This preserves sufficient particle structure for mechanical interlocking while enabling hyphal ingress. Optimization work remains needed to define frequency, intensity, and exposure time parameters specific to typical MBC feedstocks (sawdust, Dried Distiller’s Grains with Solubles (DDGS), straw) and target fungal species.

3.3. Steam Explosion and Hydrothermal Pretreatment

Steam explosion (SE) and related hydrothermal pretreatments (including autohydrolysis and subcritical water processing) are perhaps the most industrially mature, eco-friendly methods for lignocellulosic deconstruction [31]. SE uses high-pressure saturated steam (often 160–240 °C) followed by rapid decompression that explosively separates cell wall components. This triggers an increasing porosity and partially solubilizes hemicellulose and some lignin fractions [32,33]. Compared with chemical treatments, SE is attractive for large-scale operations because it uses only steam and avoids corrosive reagents. It also preserves lignin fractions that can be valorized or left to contribute stiffness in composite contexts. In mycelium composite work, steam-exploded substrates often demonstrate faster colonization and improved bonding due to the exposure of cellulose fibrils and the increased surface area [33].

Studies specifically using mushroom substrate residues found that substrates supported robust mycelial networks and yielded composites with improved compressive properties and reduced voids [33]. Hydrothermal processes are tunable: temperature and residence time can be adjusted to balance hemicellulose removal with retention of useful structural polymers. SE does have drawbacks. Capital costs for continuous steam explosion reactors can be high, and the process consumes large amounts of thermal energy. However, coupling the system to waste heat or biomass boilers can reduce the energy footprint in integrated facilities.

Recent literature [32,33] reinforces SE’s role as a scalable, lower-chemical-input pretreatment well-suited for agricultural residues destined for mycelial growth. SE is particularly compelling in regional biorefinery contexts where feedstocks are bulky and abundant and where downstream uses (MBCs, biochar, lignin coproducts) can absorb heterogeneous streams.

3.4. Plasma Sterilization and Surface Activation

Nonthermal or cold plasma technologies are increasingly explored as advanced alternatives to conventional thermal sterilization for biomass processing. Unlike autoclaving, plasma-based treatments do not rely on high temperature and pressure. Autoclaving can also cause carbohydrate degradation, lignin condensation, or Maillard reactions in lignocellulosic substrates. In contrast, plasma-based treatments achieve effective microbial inactivation without bulk heating or liquid immersion. Plasma generates a complex mixture of reactive oxygen and nitrogen species (ROS/RNS), ultraviolet photons, and charged particles. These species and particles disrupt microbial membranes, denature proteins, and damage nucleic acids while largely preserving the native chemical composition of the substrate [19,34].

Recent advances in plasma reactor design and process control have improved treatment uniformity, scalability, and energy efficiency. These advances make plasma sterilization increasingly compatible with continuous and low-energy manufacturing workflows. For mycelium-based composite (MBC) production, plasma treatment offers two distinct and synergistic advantages over traditional autoclaving. First, it enables rapid decontamination of substrates and spawn without exposing them to thermal stress. This supports more consistent fungal colonization. Second, plasma-induced surface activation introduces oxygen-containing functional groups (e.g., hydroxyl, carbonyl, and carboxyl moieties) and increases surface wettability. These features can enhance hyphal attachment, enzymatic accessibility, and interfacial bonding between mycelium and substrate particles [35].

Summarily, these attributes position plasma sterilization not only as a contamination-control strategy but also as a surface engineering tool capable of improving biological efficiency and material performance. By reducing energy demand, minimizing chemical alteration, and enhancing hypha–substrate interactions, plasma-based processing addresses several limitations associated with autoclaving and aligns with cleaner production principles for scalable MBC manufacturing [36].

3.5. Irradiation (Gamma, UV) for Decontamination and Activation

Irradiation—using gamma rays, electron beam, or high-intensity UV—has long been used for sterilizing medical devices and treating food. For lignocellulosic substrates, irradiation can decontaminate and, in some cases, alter polymer crystallinity, aiding enzymatic access. Low doses of gamma irradiation have been shown to reduce microbial load on biomass and can reduce the need for chemical sterilants. However, irradiation can also cause crosslinking or increase cellulose crystallinity at certain doses, which may hinder enzymatic hydrolysis if not carefully controlled [37].

UV irradiation (surface-limited) can be applied in spawn rooms and on exposed surfaces to reduce bioburden. The penetration limit of this irradiation source makes it less effective for packed or dense substrates. Gamma and electron beam irradiation are effective for bulk decontamination but require expensive infrastructure and regulatory oversight [38]. For MBC production, irradiation is a feasible option where regulatory frameworks permit and where the cost can be justified. This approach is relevant to high-value biomedical or food-contact products. However, it may be less suitable for low-margin packaging materials, where lower-cost pretreatments such as pasteurization or steam explosion are sufficient [39].

3.6. Comparative Efficiency, Scalability, and Environmental Footprint

When comparing these technologies (Table 2), a few encompassing themes emerge. Steam explosion and hydrothermal methods are among the most scalable and relatively low in chemical footprint. They typically require moderate thermal energy inputs in the range of ~0.8–1.2 MJ/kg dry biomass, particularly when integrated with waste-heat recovery systems. In contrast, microwave and ultrasound offer rapid, tunable, and often more energy-efficient pretreatments for smaller feedstock batches. Both are also suitable when integration with enzymatic hydrolysis is desired, with reported specific energy demands that can be 30–60% lower than conventional thermal methods [27,32]. Plasma and irradiation excel in sterilization and surface activation but carry higher capital and energy costs and are surface-limited [34,37]. Ultrasound and microwave techniques are amenable to hybrid systems (e.g., ultrasound + mild alkali; microwave + deep eutectic solvents) that reduce chemical usage while achieving high digestibility [10,40].

From an environmental perspective, methods that minimize harsh chemicals (steam explosion, ultrasound, plasma, microwave with green solvents) are preferable, but true sustainability depends on system boundaries, energy sources, and integration with heat recovery or cogeneration [19,27]. Recent life cycle assessments of mycelium-based materials suggest greenhouse gas emissions in the range of approximately 0.5–1.3 kg CO₂-eq per kg of material, which is substantially lower than many petrochemical foams and some wood-based composites, though results vary with feedstock, scale, and energy mix [41,42]

The feasibility of the economy, likewise, depends on product value. High-value MBCs for niche applications (biomedical scaffolds, specialty insulation) can absorb higher pretreatment costs (irradiation or plasma finishing). In contrast, commodity packaging applications demand low-cost and high-throughput pretreatment (steam explosion, mild pasteurization, or low-energy microwave).

In practice, the most promising direction is integrated, feedstock-specific pretreatment strategies that combine gentle physical or biological conditioning with targeted advanced treatments (e.g., microwave pulses or plasma finishing) to yield reproducible, high-performance substrates for mycelial growth while keeping environmental and economic costs manageable. Continued work is required to quantify life-cycle impacts more consistently, optimize reactor designs for scale, and develop standard process metrics tailored specifically to MBC production. Table 2 summarizes recent fungal substrate treatment technologies.

Table 2. Comparative summary of the recent fungal substrate treatment technologies.

Treatment Technique	Advantages	Limitations	Industrial Potential	Environmental Impact	Applications in MBCs	Source
Microwave-assisted hydrolysis	Fast heating; reduced energy use; uniform treatment; shorter incubation time	Possible sugar degradation and inhibitor formation at high power; reactor cost	Moderate—scalable with continuous systems, best for small-to-medium biorefineries	Moderate—minimal chemicals but electricity-dependent	Enhances growth rate, density, and uniformity of MBCs using DDGS, sawdust, or straw	[27,40]
Ultrasound (sonication)	Operates at mild temperatures; enhances enzymatic hydrolysis; lowers chemical use	Non-uniform treatment in dense biomass, equipment wear, and high energy demand	High for slurry or continuous systems; limited for dry materials	Low—minimal chemicals, moderate energy	Enhances initial mycelial colonization; improves moisture retention and nutrient transfer	[10,30,43]
Steam explosion/Hydrothermal pretreatment	Chemical-free; scalable; improves porosity and fungal access	High steam energy demand; sugar degradation; variability between batches	Very high—industrially proven in biomass biorefineries	Moderate—recyclable steam systems possible	Suitable for large-scale agricultural residues in MBCs	[32,33]
Plasma sterilization/activation	No chemicals; low temperature; rapid sterilization; surface functionalization	Surface-limited effects; high energy cost; needs specialized systems	Moderate—feasible for thin or conveyor-based treatment	Low—no waste or chemical residue	Surface activation and sterilization for improved fungal adhesion	[19,34,36]
Irradiation (gamma, UV, e-beam)	Effective sterilization; dry process; penetrates dense substrates	High capital and safety cost; may overharden cellulose	Moderate—scalable at centralized irradiation facilities	Low chemical impact but energy-intensive	Used for sterile, high-value MBCs in biomedical or packaging sectors	[37]

Table 2. Comparative summary of the recent fungal substrate treatment technologies.

Treatment Technique	Advantages	Limitations	Industrial Potential	Environmental Impact	Applications in MBCs	Source
Microwave-assisted hydrolysis	Fast heating; reduced energy use; uniform treatment; shorter incubation time	Possible sugar degradation and inhibitor formation at high power; reactor cost	Moderate—scalable with continuous systems, best for small-to-medium biorefineries	Moderate—minimal chemicals but electricity-dependent	Enhances growth rate, density, and uniformity of MBCs using DDGS, sawdust, or straw	[27,40]
Ultrasound (sonication)	Operates at mild temperatures; enhances enzymatic hydrolysis; lowers chemical use	Non-uniform treatment in dense biomass, equipment wear, and high energy demand	High for slurry or continuous systems; limited for dry materials	Low—minimal chemicals, moderate energy	Enhances initial mycelial colonization; improves moisture retention and nutrient transfer	[10,30,43]
Steam explosion/Hydrothermal pretreatment	Chemical-free; scalable; improves porosity and fungal access	High steam energy demand; sugar degradation; variability between batches	Very high—industrially proven in biomass biorefineries	Moderate—recyclable steam systems possible	Suitable for large-scale agricultural residues in MBCs	[32,33]
Plasma sterilization/activation	No chemicals; low temperature; rapid sterilization; surface functionalization	Surface-limited effects; high energy cost; needs specialized systems	Moderate—feasible for thin or conveyor-based treatment	Low—no waste or chemical residue	Surface activation and sterilization for improved fungal adhesion	[19,34,36]
Irradiation (gamma, UV, e-beam)	Effective sterilization; dry process; penetrates dense substrates	High capital and safety cost; may overharden cellulose	Moderate—scalable at centralized irradiation facilities	Low chemical impact but energy-intensive	Used for sterile, high-value MBCs in biomedical or packaging sectors	[37]

4. Optimization Parameters and Process Control

4.1. Temperature and pH Regulation

Temperature and pH are two of the most important yet interdependent environmental factors influencing fungal physiology, enzyme secretion, and substrate digestibility in the production of MBCs. Their regulation is crucial not only for maximizing mycelial growth rates but also for ensuring the consistency and mechanical performance of the final bio-composite material. While temperature directly affects metabolic rate and enzymatic kinetics, pH modulates enzyme stability, nutrient solubility, and the physicochemical conditions that govern lignocellulosic degradation. Optimal control of both parameters enhances substrate colonization, structural binding between hyphae and substrate particles, and the formation of a cohesive mycelial matrix [13,25,44]. The subsequent subsections will discuss the effects of temperature and pH on the growth of mycelium.

4.1.1. Temperature Effects on Mycelial Growth

Temperature has a significant impact on fungal enzymatic activity and hyphal extension rates. Most white-rot Basidiomycetes used in mycelium composite production, such as Pleurotus ostreatus, Ganoderma lucidum, Trametes versicolor, and Schizophyllum commune, exhibit optimal mycelial growth between 20 °C and 28 °C, though this range can vary slightly by strain and substrate composition [3,45]. At temperatures below this range, metabolic activity declines due to the reduced fluidity of cell membranes and slower enzyme kinetics, leading to delayed colonization. Conversely, higher temperatures (>30 °C) can result in heat stress, denaturation of extracellular enzymes, and inhibition of growth. In extreme cases, elevated temperatures stimulate sporulation rather than vegetative growth, compromising the uniformity of the composite [46].

The influence of temperature on enzymatic activity is particularly evident in lignocellulose degradation. Laccases, peroxidases, and cellulases—key enzymes in lignin and cellulose hydrolysis—display optimal catalytic activity around 25–30 °C. When temperatures rise beyond 35 °C, enzyme stability declines significantly, and the synergistic action between oxidative and hydrolytic enzymes is disrupted [47]. Furthermore, high temperatures favor bacterial contaminants that outcompete fungi for available nutrients, resulting in poor substrate colonization and lower tensile strength in the final composite. Controlled thermal environments are therefore essential. Recent studies demonstrate that temperature fluctuations greater than ±2 °C can cause up to a 20% reduction in compressive strength in MBCs, underscoring the importance of precise environmental regulation [48].

4.1.2. Technological Control and Monitoring

In laboratory and pilot-scale operations, thermostatically controlled incubators or growth chambers maintain stable temperatures. In contrast, the industrial-scale production employs automated climate control systems that regulate both temperature and humidity. The integration of Internet of Things (IoT)-based sensors—including distributed temperature probes, humidity sensors, oxygen sensors, and CO₂ monitors—has improved environmental monitoring in MBC production. These sensors enable continuous real-time tracking of microenvironmental conditions within and around the substrate [3]. In practice, such sensor networks can be connected to commercial IoT platforms such as Siemens MindSphere, Schneider Electric EcoStruxure, Microsoft Azure IoT Hub, or Amazon Web Services (AWS) IoT Core. The platforms can then facilitate data acquisition, visualization, and automated process control. The platforms also enable closed-loop feedback systems that automatically adjust heating, humidification, ventilation, or aeration to maintain uniform and optimal growth conditions [49,50].

Thermographic imaging and embedded thermocouples have been used to detect local “hotspots” generated by metabolic heat during active fungal growth. This phenomenon is particularly relevant in large molds where internal heat buildup can inhibit deeper colonization [45]. Adaptive control systems that adjust airflow or external cooling in response to such hotspots are now being developed to maintain thermodynamic balance across large substrate volumes.

Beyond reactive control, artificial intelligence (AI) and machine learning (ML) approaches are beginning to play a predictive role in MBC production. Data from IoT sensor networks can be used to train models that forecast temperature evolution, moisture redistribution, and oxygen depletion within growing composites. Such models can support predictive control strategies, allowing operators to pre-emptively adjust aeration, humidity, or cooling before conditions become suboptimal. In advanced implementations, digital twins of bioreactors or growth chambers are being explored, where real-time sensor data are integrated with computational models to simulate mycelial growth dynamics and optimize process parameters at scale.

Together, IoT-enabled sensing and AI-driven analytics represent a shift from static environmental control toward data-driven, adaptive, and scalable manufacturing of mycelium-based composites.

4.1.3. pH Influence on Enzymatic Activity and Fungal Metabolism

The pH of the substrate governs enzyme function, ion solubility, and nutrient uptake in fungi. Most white-rot fungi prefer slightly acidic to neutral pH values (5.5–7.0) for optimal growth and enzymatic activity. The influence of pH is particularly critical for ligninolytic enzymes, which are often secreted in acidic environments but lose activity at pH extremes. For example, laccase activity in Ganoderma lucidum peaks around pH 5.5–6.0, whereas cellulases perform best near pH 6.5–7.0 [47]. Consequently, maintaining a near-neutral pH enables a synergistic balance between lignin degradation and cellulose hydrolysis—key to producing substrates that are both porous and structurally cohesive.

Substrate pH also changes dynamically during fungal colonization. As fungi metabolize carbohydrates and proteins, organic acids (such as oxalic and citric acid) are secreted, gradu-ally lowering the pH. Without buffering, this acidification can inhibit further enzyme activity and slow mycelial expansion [3]. To mitigate these effects, buffering agents such as calcium carbonate (CaCO₃) or phosphate buffers are incorporated into substrates to stabilize pH levels. In long-term incubations, periodic measurement using pH probes or colorimetric indicators provides feedback for adaptive control of nutrient feeding or gas exchange [51].

4.2. Interactions Between Temperature and pH

Temperature and pH do not act independently; their interaction influences both enzyme conformation and metabolic flux. Enzyme stability often follows a bell-shaped relationship with temperature and pH—small deviations from the optimum can exponentially decrease catalytic efficiency. For instance, cellulases from Pleurotus ostreatus maintain high activity only within a narrow window around 25 °C and pH 6.0 [46]. In combined studies, fungal cultures exposed to both suboptimal temperature and pH conditions exhibited reduced enzyme se-cretion rates and irregular hyphal morphology, resulting in composites with heterogeneous texture and poor mechanical strength [48].

Moreover, pH shifts can influence temperature tolerance by modifying enzyme proto-nation states, while high temperatures accelerate acid accumulation in the substrate, intensi-fying pH drops. Such feedback loops make co-regulation essential. Advanced bioprocessing strategies now use predictive modeling to correlate thermal and pH parameters with fungal respiration rate, CO₂ evolution, and oxygen uptake, enabling fine-tuned control of metabolic state [45]

4.3. Optimization Approaches

Optimizing temperature and pH begins with selecting fungal strains suited to the in-tended substrate and processing environment. Screening studies often employ factorial ex-perimental designs (response surface methodology) to quantify the interactive effects of both variables on biomass yield, enzyme activity, and mechanical strength. Once optimal condi-tions are identified, real-time control systems maintain parameters within the desired range using feedback from embedded sensors. Adaptive algorithms in bio-incubators now auto-matically adjust heating, ventilation, and nutrient feeding based on temperature–pH correla-tions, ensuring reproducibility and energy efficiency [3].

Emerging approaches also explore strain engineering and acclimatization to extend tol-erance ranges. For example, directed evolution of Trametes versicolor has produced strains with stable ligninolytic activity at temperatures up to 32 °C, reducing the need for strict climate control [51]. Similarly, modifying substrate chemistry—through lime treatment or organic buffering—can maintain near-neutral pH even under high metabolic load. These innovations are particularly useful for scaling up MBC production in environments where ambient tem-perature fluctuations are unavoidable.

Temperature and pH regulation are foundational to both the biological and engineering aspects of MBC production. While early studies treated them as fixed incubation conditions, modern process engineering views them as dynamic, controllable variables that interact with fungal metabolism. Maintaining optimal conditions (20 –28°C, pH 5.5–7.0) enhances enzymatic synergy, accelerates colonization, and improves the uniformity and strength of mycelium composites. Future advancements are likely to focus on sensor-integrated bioreactors, adap-tive control algorithms, and thermo-tolerant fungal strains, ensuring that substrate treatment processes remain both efficient and resilient to environmental variability.

4.4. Carbon-to-Nitrogen Ratio Adjustment

The carbon-to-nitrogen (C:N) ratio of a substrate is one of the most influential biochemical parameters governing fungal metabolism, enzymatic secretion, and mycelial morphology during the production of mycelium-based composites (MBCs) [52]. It represents the balance between carbon, which serves primarily as an energy source and structural component, and nitrogen, which is crucial for protein synthesis, nucleic acid formation, and enzymatic activity. An optimal C:N ratio ensures that the fungus maintains a steady rate of mycelial growth without entering premature sporulation or nutrient depletion phases [5,47,53]. Inappropriate C:N ratios can lead to poor colonization, uneven substrate binding, or reduced mechanical performance in the resulting bio-composites.

4.4.1. Role of Carbon and Nitrogen in Mycelial Growth

Fungi metabolize carbon mainly from lignocellulosic polysaccharides such as cellulose and hemicellulose, which provide both structural material and energy through glycolysis and the Tricarboxylic acid (TCA) cycle. Nitrogen, on the other hand, is assimilated from proteins, amino acids, urea, or inorganic compounds (e.g., ammonium salts, nitrates), supporting the biosynthesis of enzymes, chitin, and other cellular macromolecules. The balance between these elements affects whether the fungus prioritizes biomass accumulation or secondary metabolism [51].

In mycelium composite production, a C:N ratio between 20:1 and 35:1 is often considered optimal for most white-rot fungi such as Pleurotus ostreatus, Ganoderma lucidum, and Trametes versicolor [47,54]. Substrates with a very high C:N ratio (above 50:1), such as straw or sawdust, provide abundant carbon but insufficient nitrogen, resulting in slow mycelial colonization and weak interparticle bonding. Conversely, excessively low C:N ratios (below 15:1), as seen in protein-rich residues like soybean meal or manure, can cause excessive vegetative growth and microbial contamination due to nitrogen-induced acidification and bacterial proliferation [55,56].

Maintaining an appropriate balance, therefore, allows fungi to invest energy efficiently in extracellular enzyme production for lignocellulose degradation and matrix formation—both essential for composite performance.

4.4.2. C:N Ratio and Enzymatic Activity

The C:N ratio directly affects the expression of ligninolytic and cellulolytic enzymes. White-rot fungi typically upregulate lignin-degrading enzymes (laccases, manganese peroxidases, and lignin peroxidases) under nitrogen-limited conditions, as nitrogen scarcity triggers secondary metabolism [51]. This leads to enhanced lignin breakdown and greater accessibility of cellulose fibers for colonization. In contrast, nitrogen-rich conditions suppress ligninolytic activity but favor rapid biomass accumulation, yielding dense yet mechanically weaker composites [3].

For example, Pleurotus ostreatus grown on rice straw (C:N ≈ 35:1) exhibits strong lignin degradation and uniform matrix formation, whereas the same fungus on corn husk supplemented with urea (C:N ≈ 15:1) produces more biomass but exhibits poor tensile strength due to insufficient crosslinking [48]. Thus, controlling the C:N ratio serves as an indirect but powerful lever to optimize the chemical and structural attributes of MBCs.

4.4.3. Substrate Composition and C:N Ratio Modulation

Lignocellulosic substrates used in mycelium composites—such as sawdust, wheat straw, corn stover, and spent grains—are typically high in carbon but deficient in nitrogen. To adjust the C:N ratio, substrates are often supplemented with nitrogen sources like bran, soybean meal, urea, or ammonium sulfate. Studies show that supplementing sawdust with 5–10% wheat bran can reduce the C:N ratio from 70:1 to about 30:1, resulting in a 40–50% increase in colonization rate and significantly improved composite density.

However, supplementation strategies must be carefully balanced. Excess nitrogen not only alters the pH of the substrate, but can also favor bacterial competition. A study by Madusanka et al. (2024) found that substrates enriched with urea at concentrations exceeding 2% (w/w) led to microbial contamination and reduced mechanical stability of Ganoderma composites [46]. Therefore, integrating organic nitrogen supplements (like bran or corn meal) tends to be safer and more sustainable than inorganic options, as these provide both structural carbohydrates and trace minerals that aid fungal metabolism.

4.4.4. Monitoring and Process Control

Quantifying the C:N ratio in substrates involves elemental analysis using techniques such as CHN analyzers or colorimetric assays. During the colonization phase, fungi dynamically alter this ratio through metabolic processes. Carbon is oxidized to CO₂, while nitrogen is assimilated into biomass, meaning the substrate’s effective C:N ratio declines over time [54,57]. Real-time monitoring using gas sensors and spectroscopic tools now allows researchers to estimate nutrient consumption patterns and adjust feed formulations accordingly.

Modern process control systems integrate AI-assisted feedback mechanisms that correlate carbon and nitrogen depletion rates with environmental parameters such as temperature and moisture content [3]. Machine learning models can predict when the substrate is approaching nutrient limitation and trigger supplementation events—ensuring consistent mycelial growth and uniform composite properties. This technology-driven approach is particularly relevant for large-scale operations where batch-to-batch variability poses a significant challenge.

4.4.5. Optimization Strategies in Practice

Optimization of the C:N ratio is often achieved experimentally through response surface methodology (RSM) or design of experiments (DoE) approaches. These techniques model the interactive effects of the C:N ratio with other variables, such as temperature, moisture, and pH, to identify conditions that yield maximum biomass and the desired material properties. For instance, Aiduang et al. (2024) found that an optimized combination of C:N = 28:1, temperature = 26 °C, and moisture = 60% resulted in composites with the highest compressive strength (≈0.75 MPa) and lowest water absorption [47].

Pre-treatment methods can also indirectly affect the C:N ratio by modifying substrate chemistry. Thermal or oxidative treatments may volatilize carbon compounds or solubilize hemicellulose, thus lowering available carbon content. Biological pre-digestion using nitrogen-fixing microbes (e.g., Azotobacter or Rhizobium spp.) is an emerging technique that enriches nitrogen naturally without additives. Such biological enhancement aligns with circular economy principles by minimizing chemical inputs and leveraging symbiotic microbial processes [45].

4.4.6. Implications for Composite Performance

The mechanical and physical performance of mycelium composites is tightly linked to the metabolic state of the fungus, which in turn depends on nutrient balance. Composites grown under nitrogen-limited but carbon-rich conditions often exhibit better stiffness and dimensional stability due to higher chitin-to-protein ratios in the cell wall [48]. These structural polymers act as reinforcing agents within the composite, improving compressive and tensile strength. Conversely, nitrogen-excess conditions may yield composites with high moisture content and lower density, leading to poor water resistance and faster biodegradation.

Furthermore, the C:N ratio influences not only physical attributes but also functional properties like hydrophobicity, conductivity, and thermal insulation. Adjusting nutrient balance can modify the proportion of melanin, glucan, and hydrophobic proteins in the mycelial matrix. This step is important for designing composites for specific applications such as packaging, insulation, or wearable electronics [52].

4.4.7. Environmental and Sustainability Considerations

Optimizing the C:N ratio also has sustainability implications. Using nitrogen-rich agricultural residues, such as distillers’ grains or legume husks, can minimize the need for synthetic nitrogen supplementation and enhance waste valorization. By fine-tuning C:N balance using locally available biomass, the environmental footprint of MBC production can be significantly reduced. Studies indicate that substrate optimization can lower total process energy by up to 25% due to faster colonization and reduced incubation times [3].

In summary, maintaining an optimal C:N ratio is a cornerstone of efficient MBC production. It governs enzyme expression, metabolic balance, structural integrity, and ultimately the functionality of the composite. While an ideal range of 20–35:1 suits most white-rot fungi, the precise ratio should be empirically tailored to the substrate, fungal strain, and production environment. The integration of modern analytical tools and AI-assisted process control represents a paradigm shift from static to adaptive optimization, paving the way for reproducible, sustainable, and high-performance mycelium-based materials.

4.5. Particle Size and Porosity Optimization

Particle size and porosity are fundamental physical determinants of substrate performance in fungal mycelium cultivation and composite fabrication. They control critical parameters such as air diffusion, moisture retention, nutrient accessibility, and hyphal penetration. These parameters collectively define the mechanical integrity, density, and homogeneity of mycelium-based composites (MBCs). Substrate particle size influences not only the microstructure of the developing mycelial network but also the physicochemical properties of the resulting bio-composite. As mycelium grows, it colonizes and binds substrate particles into a cohesive matrix. Therefore, understanding and optimizing particle size and porosity are essential for producing high-quality, reproducible, and functional materials.

4.5.1. Fundamental Concepts of Particle Size and Porosity

The relationship between particle size and fungal colonization is governed by surface area and pore architecture. Smaller particles provide a higher specific surface area for hyphal attachment and enzymatic degradation. However, particles of smaller sizes can cause reduced macro-porosity, impede gas exchange, and lead to localized oxygen depletion. Conversely, larger particles enhance aeration but reduce the available surface area for fungal adhesion and enzymatic access [4,47]. Thus, achieving an optimal balance between these extremes is key to maintaining both efficient colonization and structural stability.

Porosity, defined as the fraction of void volume within a substrate or composite, dictates the diffusion of gases (O₂, CO₂) and water vapor during colonization. High porosity facilitates oxygen penetration essential for aerobic metabolism, whereas excessively low porosity induces anaerobic conditions that stunt growth and favor contamination. The target porosity for most white-rot fungal substrates lies between 65% and 80%, depending on the fungal strain and substrate type [58].

4.5.2. Influence on Mycelial Growth Kinetics

Empirical evidence suggests that substrate particle size profoundly affects fungal growth kinetics. Studies have shown that Pleurotus ostreatus and Ganoderma lucidum exhibit maximum radial growth rates on substrates with medium-sized particles—typically 1–3 mm [48]. When substrates are ground too finely (<0.5 mm), the compaction limits air permeability, reducing mycelial respiration and enzymatic activity. On the other hand, coarse particles (>5 mm) produce non-uniform colonization and weak interparticle bonding, leading to heterogeneous mechanical performance in the final composite [51].

Aiduang and his team recently demonstrated that Schizophyllum commune composites produced from rice husk milled to 2 mm exhibited 45% faster colonization and 30% higher compressive strength compared to coarser fractions (4–6 mm) [47]. This finding supports the notion that medium particle size facilitates an optimal trade-off between surface availability and airflow. Moreover, smaller particles often require less incubation time, enabling higher production throughput in industrial setups [47].

Porosity directly correlates with fungal oxygen demand. White-rot fungi are obligate aerobes that rely on oxidative enzymatic systems (laccases, peroxidases) to degrade lignin and cellulose. Low substrate porosity reduces oxygen diffusion, leading to incomplete degradation and uneven hyphal distribution. Controlled porosity also prevents internal heat buildup, as active fungal metabolism generates considerable heat, which can inhibit growth if not properly dissipated [58].

4.5.3. Mechanical Implications for Composite Properties

The particle size and pore architecture of the substrate strongly influence the mechanical behavior of the resulting mycelium composite. Finer substrates produce denser, more homogeneous composites with smooth surfaces and higher compressive strength, while coarse substrates create lightweight, porous composites better suited for insulation or packaging [3]. For example, Fritz et al. [48] reported that composites from fine sawdust (particle size 1 mm) had a compressive strength of 0.72 MPa and density of 0.28 g/cm³, compared to 0.42 MPa and 0.21 g/cm³ for those from coarser particles [48]. The denser network in fine substrates enhances load-bearing capacity due to improved bonding and reduced voids. However, such dense matrices often exhibit lower sound absorption and thermal insulation because smaller pores limit acoustic damping and thermal barrier effects [48].

Porosity optimization is also crucial for determining water absorption behavior. Highly porous composites tend to absorb more moisture, reducing dimensional stability and increasing biodegradability. Consequently, the intended application of MBCs dictates the preferred particle size and porosity: finer and denser composites are ideal for structural applications, while coarse and porous composites perform better in packaging or thermal insulation contexts [47].

4.5.4. Pre-Treatment and Processing Effects

Physical pre-treatments such as milling, grinding, and sieving not only reduce particle size but also alter surface chemistry and accessibility. Mechanical comminution disrupts lignin-cellulose linkages, enhancing enzyme-substrate interactions and facilitating mycelial attachment. However, excessive grinding can generate fine dust that reduces porosity and increases compaction, requiring controlled processing parameters.

Thermal and hydrothermal pre-treatments can further modify substrate porosity by removing hemicellulose and softening lignin, resulting in more open structures. Steam explosion, for instance, increases accessible pore volume and enhances mycelial infiltration [59]. Similarly, alkali treatment can etch particle surfaces, increasing roughness and improving fungal adhesion.

Recent advances in 3D substrate engineering use computational modeling and additive manufacturing to create substrates with tailored porosity gradients and particle size distributions [60]. These techniques allow for designing hierarchical structures that balance strength and permeability, thus achieving functional composites with spatially optimized growth environments.

4.5.5. Analytical Techniques for Measuring Particle Size and Porosity

Quantitative assessment of substrate particle size and porosity is critical for reproducible research and industrial scaling. Laser diffraction and sieve analysis are standard for determining particle size distributions, while gas adsorption (Brunauer-Emmett-Teller (BET) method) and mercury intrusion porosimetry are used to measure total porosity and pore size distribution.

Micro-computed tomography (micro-CT) has become a powerful non-destructive tool for visualizing internal pore networks in MBCs, offering 3D reconstructions that correlate porosity with mechanical performance. Studies employing micro-CT have shown that porosity values exceeding 80% often correspond to lower mechanical strength but improved acoustic absorption [61].

Combining imaging and computational fluid dynamics (CFD) enables the simulation of airflow and moisture diffusion within substrates, allowing for the optimization of particle size and pore geometry before physical production. Such integrated modeling approaches are pivotal for scaling up MBC manufacturing, where process variability must be minimized.

4.5.6. Interactions with Other Growth Parameters and Optimization

Particle size interacts dynamically with other cultivation parameters—especially moisture, aeration, and temperature. Fine substrates retain more water, which can cause local anaerobiosis if not properly ventilated. Therefore, controlling aeration and mixing frequency is essential to maintaining oxygen levels within compacted matrices [51].

Moreover, fine substrates exhibit faster temperature increases during colonization due to limited heat dissipation, necessitating tighter thermal control. Temperature gradients in large-scale reactors can lead to uneven growth and structural defects. Modern bioreactors address this challenge by using controlled aeration channels or mixing arms to maintain uniform gas and heat distribution throughout the substrate block.

Optimization of particle size and porosity can be achieved experimentally through factorial design or response surface methodology (RSM). Such approaches identify the interactions between physical and biological variables, helping to pinpoint particle size ranges that maximize growth rate and mechanical performance.

AI-driven predictive models are increasingly being adopted to correlate morphological data such as particle size, porosity, and surface roughness with material behavior. These models link such features to mechanical and biological outputs, including tensile strength and colonization speed [3]. They also reduce trial-and-error experimentation and enhance process reproducibility in commercial settings.

Furthermore, adaptive manufacturing systems can adjust milling intensity and sieving mesh size based on real-time feedback from fungal growth monitoring, exemplifying the integration of industrial principles into mycelium material fabrication.

4.5.7. Sustainability and Energy Considerations

From a sustainability perspective, optimizing particle size and porosity minimizes energy inputs by reducing colonization time and material waste. Mechanical treatments like grinding are energy-intensive; hence, balancing the energy cost with growth efficiency is crucial. Studies show that intermediate particle sizes (1–3 mm) offer a favorable compromise, providing both efficient colonization and reduced power consumption [61].

The use of agricultural residues with naturally variable particle sizes (e.g., corn stalks or wheat straw) can be managed through selective sieving instead of excessive milling, lowering both energy demand and CO₂ emissions. Thus, the optimization of particle size and porosity is not only a biological or mechanical concern but also an environmental and economic one.

Optimizing substrate particle size and porosity is central to advancing fungal mycelium-based composite technology. The interplay between surface area, gas exchange, and mechanical cohesion determines both biological efficiency and material performance. Medium-sized particles (1–3 mm) with porosity between 65% and 80% generally provide the best balance for most white-rot fungi, though fine-tuning remains species and application-dependent. Emerging tools like micro-CT, CFD modeling, and AI-driven control systems are transforming how these parameters are understood and regulated. Ultimately, strategic management of particle size and porosity enhances not only the quality and consistency of MBCs but also their sustainability and scalability within bio-based manufacturing industries. Below is the table (

Table 3. Factors influencing the MBCs’ performance.

Parameter	Optimal Range	Control Methods	Effect on Mycelium Growth	Ref
Temperature	20–28 °C (species dependent)	Thermocouples, thermostatic incubators	Regulates enzyme activity and colonization rate	[3,45]
pH	5.5–7.0	pH meter, buffer agents (CaCO3)	Ensures enzyme stability and nutrient solubility	[51]
Moisture Content	55–65%	Gravimetric, IR sensors, automated humidifiers	Promotes enzyme diffusion and aeration balance	[47,48]
C:N Ratio	20:1–60:1	Elemental analysis, feedstock blending	Balances mycelial metabolism and mechanical strength	[51]
Particle Size	1–5 mm	Sieving, image-based granulometry	Balances porosity and density	[6]
Aeration	0.5–2 L air/min/kg substrate	CO2/O2 sensors, forced ventilation	Prevents anaerobic zones and supports aerobic metabolism	[45]
Sterility	Pasteurization or Autoclaving	Temperature/time monitoring, microbial plating	Reduces contamination, ensures uniform growth	[32,47]

) summarizing the key factors influencing the MBCs’ performance.

4.6. Aeration and Mixing Control

Aeration and mixing are pivotal in determining the physiological performance of fungi during substrate colonization and the final properties of mycelium-based composites (MBCs). Fungal metabolism, particularly for aerobic species such as Pleurotus ostreatus, Ganoderma lucidum, and Schizophyllum commune, depends heavily on adequate oxygen supply and carbon dioxide removal. Equally, controlled mixing ensures even distribution of nutrients, inoculum, and moisture throughout the substrate, mitigating the risk of anaerobic microzones and microbial contamination. Inadequate aeration can restrict mycelial growth, alter metabolic pathways, and impair enzymatic secretion, whereas excessive aeration or agitation can mechanically damage delicate hyphae, leading to fragmentation and reduced colonization efficiency [47]. Hence, maintaining an optimal balance between gas exchange and physical integrity is critical for scaling MBC production efficiently and sustainably.

4.6.1. Fundamentals of Aeration Dynamics in Mycelial Systems

Aeration involves the controlled delivery of oxygen and removal of carbon dioxide generated by fungal respiration. Oxygen plays a vital role in oxidative enzyme systems, such as laccases and peroxidases, that degrade lignocellulosic materials [3]. The stoichiometric oxygen demand for fungal metabolism is high. However, mycelial mats tend to form dense interwoven structures that limit gaseous diffusion. The result is a steep oxygen gradient across the substrate depth, often leading to anoxic zones in the interior if gas exchange is not adequately maintained.

To mitigate this, substrate porosity and bulk density must be optimized in conjunction with active or passive aeration strategies. Porous substrates permit passive diffusion, while denser materials may require forced aeration through perforated trays, tubes, or aeration channels. Studies in solid-state fermentation bioreactors indicate that oxygen diffusivity decreases exponentially with increasing moisture and compaction; thus, coupling aeration with moisture management becomes necessary [62,63]. The ideal aeration rate depends on fungal strain, substrate type, and bioreactor configuration. For white-rot fungi, airflows of 0.2–0.5 L/min/kg dry substrate are typically sufficient to maintain aerobic metabolism [59]. However, too high an aeration rate can dry the substrate surface and lower humidity below the optimal range (60–70%), impeding hyphal expansion.

4.6.2. Role of Mixing and Substrate Homogeneity

Mixing promotes uniform distribution of fungal inoculum, nutrients, and moisture. Without proper mixing, inoculated regions may overgrow while others remain uncolonized, resulting in non-uniform composite density and weak mechanical zones. Mechanical or manual mixing at inoculation is standard in small-scale production, whereas in industrial settings, continuous or intermittent mixing systems are employed.

Mixing intensity is a crucial parameter: low mixing rates may allow stratification and uneven gas flow, while excessive agitation can shear hyphae, breaking them into fragments incapable of bridging substrate particles [48]. Studies have shown that moderate mixing frequencies—every 24–48 h during early colonization—improve uniformity without compromising mycelial structure [3]. Han et al. observed that Pleurotus florida cultivated in a 10 L stirred bioreactor achieved 35% higher biomass yield and 25% higher lignin degradation efficiency when intermittent mixing was implemented compared to static conditions. Moreover, mixing accelerates heat dissipation, preventing localized overheating due to fungal metabolic heat—a critical factor in solid-state systems where temperature control is difficult [51].

4.6.3. Bioreactor Design and Aeration Strategies

Recent technological developments have introduced multiple aeration configurations for MBC cultivation, including tray systems, packed-bed reactors, and fluidized-bed bioreactors. Each design carries distinct trade-offs in aeration efficiency, contamination control, and energy cost. These are briefly discussed below:

(a) Tray-based systems (common in mushroom cultivation) rely primarily on passive diffusion aided by periodic air circulation. They are simple and cost-effective but exhibit limited scalability due to uneven gas flow in deeper substrates. Packed-bed bioreactors, on the other hand, allow forced aeration through perforated channels, facilitating uniform oxygen distribution and CO₂ removal. However, their design must avoid excessive pressure drops and drying [43].

(b) Fluidized-bed and rotary-drum bioreactors represent advanced designs capable of continuous mixing and aeration. In these systems, air is introduced from below (in fluidized beds) or the substrate is tumbled (in drum reactors), improving gas–solid contact and homogeneity [47,64]. However, several studies report that excessive agitation or improperly controlled aeration can cause shear damage, uneven drying, or reduced biological performance, so process parameters must be carefully controlled [47,64].

(c) Air-lift systems: This is a new design that has the facility which circulates air vertically through a column reactor, using the buoyant force of bubbles to mix the substrate without mechanical agitation. This innovation minimizes shear stress on hyphae and is particularly suited for delicate fungal species like Ganoderma lucidum [51]. A comparative schematic of major aeration and mixing configurations is presented in Figure 4.

4.6.4. Environmental and Process Control

Aeration also influences substrate temperature, moisture, and pH—three parameters tightly coupled to fungal metabolism. Mycelial respiration produces heat; without sufficient ventilation, substrate temperatures can exceed 35 °C, inhibiting growth. Thus, temperature and aeration must be co-regulated. Automated bioreactors now use feedback sensors to adjust airflow based on oxygen consumption and CO₂ concentration [48].

Humidity control is equally vital. Excessive airflow can desiccate the surface layer, causing mycelial dieback. To counteract this, humidified air streams or misting systems are often incorporated. Optimal relative humidity (RH) levels range between 60% and 75% for most Basidiomycetes. Maintaining this range ensures high enzymatic activity and continuous hyphal expansion [47]. Figure 5 schematically illustrates the coupling between aeration, temperature, and fungal metabolism in controlled bioreactors.

pH regulation is indirectly affected by aeration, as oxygen influences the rate of organic acid production and nitrogen metabolism. Proper aeration prevents the buildup of acidic intermediates, stabilizing substrate pH near the optimum (5.5–6.5) for enzymatic performance.

4.6.5. Modeling and Computational Approaches

To optimize aeration and mixing efficiency, computational fluid dynamics (CFD) and artificial intelligence (AI)-based process control systems are increasingly adopted. CFD simulations provide insights into airflow distribution, heat transfer, and gas exchange within complex reactor geometries. Shen et al. [59] utilized CFD modeling to design an aeration layout that achieved a uniform oxygen profile across large-scale packed beds, improving growth uniformity by 18%.

Machine learning algorithms are also being used to predict growth kinetics under varying aeration regimes. By integrating oxygen uptake rate (OUR) and CO₂ evolution rate (CER) data, predictive models can dynamically adjust aeration intensity, thereby optimizing energy consumption and maintaining consistent growth conditions [3]. Such data-driven process control is key for scaling MBC production within smart biomanufacturing frameworks.

4.6.6. Aeration–Contamination Interactions, Comparative Studies and Performance Outcomes

Aeration can influence microbial contamination dynamics. Although oxygen supports beneficial fungal metabolism, it can also encourage aerobic contaminants such as Trichoderma spp. or bacteria. Therefore, air-filtration and sterilization (e.g., High-Efficiency Particulate Air (HEPA) filters and UV-C units) are critical for supplying clean air streams and reducing contamination risk [65].

Mixing also presents contamination challenges; improper cleaning or overmixing can disperse contaminants throughout the substrate. Thus, aseptic design principles, including smooth interior surfaces and automated cleaning-in-place (CIP) systems, are recommended for industrial reactors.

Several studies have compared static and dynamic aeration regimes to determine their effects on mechanical and biological outcomes. In one investigation, Fritz et al. (2025) found that composites produced under controlled aeration exhibited 30% greater compressive strength and 22% less variability than non-aerated controls [48]. Similarly, Dong et al. (2025) reported enhanced tensile strength (0.71 MPa vs. 0.53 MPa) in mycelium composites produced with intermittent aeration [3].

Furthermore, aerated systems demonstrate improved homogeneity and reduced void formation—key factors for structural applications. However, increased equipment costs and energy demands must be weighed against quality gains. Recent life-cycle analyses show that optimized aeration contributes less than 10% to total production energy when properly managed [65].

Future research should emphasize adaptive aeration control systems that synchronize gas flow with fungal metabolic state. Integrating biosensors that measure oxygen uptake, CO₂ emission, and moisture gradients in real time will allow dynamic, energy-efficient adjustments. Moreover, sustainable aeration using renewable energy sources, such as solar-powered air compressors or passive wind-driven systems, could further reduce the environmental footprint of large-scale MBC production.

Mixing strategies are also evolving toward non-invasive agitation using acoustic or pneumatic pulsation, reducing hyphal damage while maintaining uniformity. Exploring symbiotic co-cultures of fungi and beneficial bacteria that naturally enhance aeration through metabolic gas exchange could offer biological alternatives to mechanical systems.

Aeration and mixing are indispensable in fungal substrate engineering, influencing biological growth, composite homogeneity, and mechanical performance. The transition from static, low-control setups to intelligent bioreactors capable of precise airflow and mixing regulation marks a major step in scaling mycelium-based material production. By integrating modeling tools, environmental sensors, and automation, future aeration systems will not only maximize productivity but also minimize energy consumption and contamination risk—making mycelium composites more viable for industrial adoption.

Summarily, aeration and mixing function as the physiological “control knobs” that translate fungal biology into reproducible material performance in mycelium-based composites. In practical application scenarios—from tray-based pilot systems to industrial packed-bed and rotary-drum reactors—well-regulated oxygen supply, moisture retention, and gentle substrate redistribution enable uniform mycelial colonization, consistent enzymatic activity, and effective heat dissipation. These conditions directly manifest as improved composite homogeneity, reduced void formation, and enhanced mechanical reliability, which are essential for meeting performance standards in packaging, insulation, and structural panels. Conversely, insufficient aeration or poorly tuned mixing leads to oxygen gradients, localized overheating, and structural weak points that undermine scalability and product consistency.

Recent advances in bioreactor design, sensor-based feedback control, and computational modeling now allow aeration and mixing to be dynamically matched to fungal metabolic demand, reducing energy consumption while improving material quality. From an industrial perspective, this integrated control framework bridges the gap between laboratory optimization and large-scale, low-impact manufacturing. By aligning biological requirements with process engineering and automation, aeration and mixing strategies emerge not merely as operational variables, but as enabling technologies for the reliable, energy-efficient, and commercially viable production of mycelium-based composites

5. The Impact of Substrate Treatment on Mycelium Growth and Composite Properties

The properties of mycelium-based composites (MBCs) are fundamentally linked to the substrate preparation and processing regime. The substrate acts as both a nutrient reservoir and the primary structural filler, while the fungal hyphal network binds, infiltrates, and cements the particles into a coherent matrix [66]. Consequently, substrate treatment—thermal, physical, chemical, and biological—plays a crucial role in determining hyphal density, structural uniformity, and mechanical strength of the resulting composite material. Controlled substrate processing not only improves the bioactivity of the growth medium but also enhances interfacial adhesion, porosity control, and network continuity, all of which are critical determinants of composite performance [66,67,68]. In this section, we examine how substrate treatment influences each of these three parameters and discuss how they translate into improved mechanical and structural outcomes. Figure 6 below summarizes the process–structure–property relationships governing MBC performance.

5.1. Substrate Treatment Influence on Hyphal Density

Hyphal density refers to the number, branching frequency, and volume fraction of fungal hyphae within the colonized substrate. A higher density indicates more continuous load paths, greater inter-particle connectivity, and superior mechanical integrity [69,70,71].

5.1.1. Thermal Pretreatment

The decision between pasteurization and sterilization significantly affects colonization dynamics. Pasteurized substrates retain residual microbial communities and biochemical cues that can stimulate fungal branching, potentially resulting in a denser mycelial network than those fully sterilized [58]. Sterilized substrates, while yielding faster colonization rates, often produce slightly less intricate hyphal networks due to the absence of microbial stimuli [72,73].

5.1.2. Physical Optimization

Particle size distribution and surface area have a profound influence on fungal attachment and colonization. Fine particles offer higher surface area for hyphal attachment, but excessive fineness reduces aeration, leading to oxygen limitation and internal stalling of growth [74]. Optimal mixtures of fine (<1 mm) and medium (1–3 mm) particle sizes promote balanced pore structure and nutrient diffusion, supporting denser and more uniform hyphal growth. Aiduang et al. [4] demonstrated that bimodal particle size distributions yielded stronger and denser mycelium matrices in lignocellulosic substrates.

5.1.3. Chemical Pretreatment

Mild alkaline or enzymatic pretreatments remove lignin, waxes, and inhibitory extractives, thereby enhancing substrate permeability and fungal enzyme activity [75]. This process enhances nutrient accessibility and promotes thicker, more robust hyphal mats. For instance, NaOH-treated substrates have been shown to support higher hyphal infiltration and greater binding efficiency compared to untreated controls [76].

5.1.4. Biological and Biotechnological Enhancements

Using actively growing pre-colonized spawn reduces lag time and increases initial inoculum density, both of which correlate positively with network formation. Moreover, genetic or strain-level optimization—such as selecting fungi with high hydrophobin expression—can enhance hyphal branching and surface adhesion [77]. Collectively, these treatments enhance filament continuity and reduce voids, resulting in a more compact and mechanically resilient matrix.

5.2. Enhancement of Structural Uniformity

Uniform colonization and consistent microstructure are crucial for ensuring reliable load distribution and optimal mechanical performance. Heterogeneous colonization, such as incomplete core infiltration or surface densification, often leads to weak zones and unpredictable behavior [68]. These can be achieved through:

(a)

Inoculum Distribution and Mixing

Even dispersion of inoculum throughout the substrate ensures simultaneous colonization fronts and minimizes the formation of uncolonized pockets. Jinanukul et al. found that pre-colonized spawn mixed homogeneously into the substrate resulted in more uniform internal growth and significantly improved mechanical consistency [76].

(b)

Controlled Aeration and Moisture Content

Substrate treatments must ensure adequate aeration and a controlled moisture gradient (typically 50–70% wet basis) to maintain uniform growth conditions [67]. Techniques such as inserting aeration channels and optimizing packing density improve oxygen penetration into core regions, thus preventing underdeveloped zones [66].

(c)

Particle Gradation and Packing Density

Densely packed substrates reduce large voids and enhance hyphal bridging between particles. Ref. [66] observed that higher volumetric packing fractions yielded stronger composites due to more uniform network formation. Similarly, Aiduang et al. (2022) reported that an optimal gradation of particle size resulted in composites with higher compressive strength [4].

(d)

Post-Growth Consolidation

After full colonization, post-growth densification through mechanical pressing or hot-pressing enhances composite homogeneity and reduces residual voids. However, excessive compression can damage hyphal bonds, so a balance must be maintained [67]. Controlled densification ensures consistent stiffness and density across the entire material, improving reproducibility and scalability. Figure 7 illustrates the post-growth densification pathway used to improve structural uniformity.

6. Improvements in Mechanical Strength

Mechanical strength—typically measured through compressive, flexural, and tensile tests—serves as the ultimate indicator of composite performance. Substrate treatments influence this through changes in hyphal density, structural uniformity, and interfacial bonding.

6.1. Mechanisms of Strength Improvement

High hyphal density improves stress transfer between substrate particles and distributes load more evenly [61]. Structural uniformity eliminates weak zones and minimizes the initiation of premature fractures. Chemical pretreatments enhance interfacial bonding by increasing available hydroxyl sites for hyphal adhesion [78]. Additionally, densification increases particle contact, reduces porosity, and strengthens the overall structure [66]. For instance, bacterial cellulose reinforcement within mycelium matrices has been shown to increase internal bond strength by up to eightfold compared to untreated composites [68].

Substrate stiffness also plays a significant role; stiffer substrates promote more rapid and directed surface colonization, producing denser and more uniform mycelial networks [79]. This observation aligns with mechanical–biological coupling principles observed in mycelial growth kinetics.

6.2. Empirical Evidence

Comparative studies on various lignocellulosic residues indicate that MBC mechanical performance depends more on substrate morphology (particle size, compaction) than on chemical composition alone [4]. Jinanukul et al., [76] demonstrated that composites made with reduced corn husk ratios exhibited a higher modulus of elasticity and density, whereas higher husk content increased bending strength but also elevated water absorption. These results emphasize the trade-off between stiffness, toughness, and hydrophilicity in MBC optimization [76].

6.3. Processing Guidelines

Practical processing recommendations include:

• Chemical: Mild alkaline washing (0.5–1% NaOH) to remove extractives, followed by thorough rinsing.
• Physical: Bimodal particle size distribution for optimal contact and porosity.
• Thermal: Pasteurization preferred over sterilization when contamination control is feasible.
• Biological: Even inoculum distribution and moisture/aeration control during growth.
• Mechanical: Controlled densification post-growth to maximize modulus without crushing hyphal bonds.

Finally, correlating hyphal density (via Scanning Electron Microscopy (SEM) or micro-CT) with mechanical testing results is essential to establish process–structure–property relationships [68].

7. Social and Market Acceptability of Mycelium-Based Composites

7.1. Public and Professional Perceptions

Mycelium-based composites’ social acceptability is determined by a combination of ecological appeal, novelty, esthetics, and risk perception. Early research reveals that MBCs are not yet generally recognized by architecture and design professionals, but they have a latent positive reception when encountered. Bonenberg et al., 2023 and Lewandowska et al., 2024 surveyed professional architects and interior designers and discovered that only about 56% of respondents were aware of MBCs before the study; however, once shown examples, up to 90% found the material visually appealing (despite some esthetic reservations) and many were willing to use MBCs in professional contexts (though less so in their personal spaces) [80,81]. This “double standard” underscores both curiosity and uncertainty surrounding the material’s unfamiliar biological origin and appearance in conventional built environments [81].

Consumer studies support professional findings: architecture students gave MBCs generally positive or neutral ratings for interior design and furniture applications. The rating emphasized environmental friendliness and originality of MBCs, even though they ranked traditional materials higher in perceived esthetic fit [82]. Respondents also connected MBCs with eco-friendly solutions but were hesitant to use them in their personal environments, indicating a gap between conceptual environmental support and personal adoption intent [81]. Part of this hesitancy stems from cultural connotations with fungi. MBCs’ biological origins can elicit underlying fear or aversion, anchored in feelings of mold and contamination, despite thermal deactivation rendering the final products inert [83].

7.2. Market Emergence: Early Commercialization and Applications

The market acceptability of MBCs is advancing in niche sustainable sectors, particularly where ecological narratives resonate strongly with consumer values and companies seek uniqueness/shift from petrochemical materials. While broad retail markets remain nascent due to cost, production scalability, and unfamiliarity, specific segments show tangible interest:

• Sustainable Packaging and Insulation: Academic and corporate partnerships in Asia, North America, and Europe are developing MBCs as biodegradable packaging alternatives to Expanded Polystyrene (EPS) and plastic foams, intending to reduce landfill waste and greenhouse gas emissions associated with petroleum-derived materials [84].
• Eco + Design Products: Firms and startups in Europe and North America have marketed mycelium products as acoustic panels, interior fittings, and decorative elements, leveraging their long-term viability and distinct esthetic as selling points for ecologically aware designers and consumers. Although not yet popular, such offerings indicate first market niches where ecological benefits exceed unfamiliarity or cost constraints [82,85].
• High-End and Emerging Applications: Beyond structural uses, research into wearable bio-materials, fungal electronics, and reactive biosensing systems suggests that MBC market potential extends into innovative technology sectors, albeit currently at the laboratory or prototype level [85].

7.3. Barriers to Wider Adoption

Despite positive signals, several market and social barriers slow acceptance:

• Awareness and Education Gaps: Limited exposure among experts and consumers results in limited familiarity, which inhibits the desire to adopt MBCs for everyday use [81].
• Esthetics and Material Expectations: MBCs’ natural, porous, and uneven appearance may contrast with established expectations for surface uniformity, color consistency, and perceived longevity, particularly in mainstream furniture and architectural goods [80].
• Price and Scale: Currently, production costs and a lack of automated, large-scale manufacturing techniques restrict MBCs in the premium or artisanal segments, limiting consumer access. Cost differences with conventional plastics or timber composites continue to be a challenge for general market uptake [82].
• Regulatory and Standards Gaps: Innovative materials frequently encounter tardy regulatory acceptance, with building codes, safety requirements, and industry certifications falling behind material innovation. Without defined performance benchmarks, architects and builders may be hesitant to specify MBCs, especially in load-bearing or safety-critical applications [84].

7.4. Cultural and Regional Variation

The acceptance of bio-based materials, such as MBCs, varies by cultural and regional context. In areas with stronger traditions of natural architecture, ecological design, or artisan skill, MBCs may be more readily accepted as alternatives to industrially manufactured materials. In contrast, in markets that value high-performance synthetic materials, the biological character of MBCs may be viewed skeptically in the absence of strong proof of long-term reliability. Cross-cultural perception research in design and sustainability domains shows how ecological narratives connect differently with consumer values and esthetic preferences, altering adoption trajectories [81].

7.5. Outlook: Education, Exposure, and Market Evolution

Overall, the literature indicates that the social and market acceptability of MBCs is developing and evolving (

Table 4. Application and social acceptance of MBCs.

Sector/Application	Context	Key Focus	Market/Social Status	Representative Source
Sustainable Packaging	India (IIT Madras research)	Biodegradable packaging from agricultural waste	Early commercialization, startup formation	[84]
Architecture and Interiors	Europe (architect surveys)	Perception, esthetic acceptance in design	Mixed awareness; positive receptivity with exposure	[81]
Furniture and Bio-Design	University settings	Consumer “likability” studies	Emerging interest; ecology valued	[83]
Art and Sustainable Design	Global design literature	Eco art, prototypes, exhibitions	Niche adoption; esthetic experimentation	[82]
Emerging Tech (Bio-electronics)	Research labs	Sensing, fungal electronics, smart materials	Lab-level prototypes; future potential	[82,83,86]

). Early adopters in sustainable design, eco-fashion, and green architecture drive interest, while educational activities and visible projects raise awareness and perceived validity. As material qualities improve and standardized performance data gathered, acceptability may increase. Progress toward commercialization will most likely be dependent on coordinated efforts in performance benchmarking, design education, storytelling about environmental value, and improved production economics.

At the social level, as younger generations become more environmentally conscious and receptive to innovative biomaterials, acceptance of MBCs may increase—especially when combined with design interventions that address esthetic expectations and dispel myths about fungus-based materials.

8. Conclusions

Substrate treatment represents the primary engineering determinant governing the microstructural development and macroscopic performance of mycelium-based composites. Precise control of thermal, physical, chemical, and biological parameters directly influences hyphal density, interfacial adhesion, and structural uniformity, thereby defining the mechanical strength, dimensional stability, and functional reliability of these materials. Although extensive laboratory studies have demonstrated the tunability of MBC properties through substrate optimization, significant challenges related to scalability, reproducibility, and long-term durability continue to limit their broader technological and commercial adoption.

These challenges critically affect acceptability across industrial, regulatory, and market domains. Variability in substrate preparation and fungal colonization can result in inconsistent mechanical performance and moisture sensitivity, undermining compliance with standardized testing protocols and building or packaging regulations. Energy-intensive sterilization requirements and labor-driven fabrication workflows further elevate production costs, eroding the environmental and economic advantages that motivate the transition from petroleum-based materials. Additionally, uncertainties surrounding aging behavior, biodegradation kinetics, and biological variability reduce confidence among manufacturers and end users who require predictable, certifiable materials for large-scale deployment.

Future research must therefore move beyond empirical optimization toward integrated, scalable substrate treatment strategies that balance biological efficiency with industrial feasibility. Priorities include systematic evaluation of species–substrate compatibility, development of low-energy pasteurization and densification protocols, and molecular-level understanding of hypha–substrate adhesion mechanisms governing load transfer and durability. Emerging tools such as in situ three-dimensional imaging and machine-learning-assisted process optimization offer powerful opportunities to enhance reproducibility and accelerate material design. Collectively, these advances are essential for positioning mycelium-based composites as reliable, scalable, and competitive sustainable materials in real-world applications.