Mycelium-Based Composites Derived from Lignocellulosic Residual By-Products: An Insight into Their Physico-Mechanical Properties and Biodegradation Profile

Abstract

The bio-fabrication of sustainable mycelium-based composites (MBCs) from renewable plant by-products offers a promising approach to reducing resource depletion and supporting the transition to a circular economy. In this research, MBCs were obtained by cultivating Ganoderma resinaceum GA1M on essential oils and agricultural by-products: hexane-extracted rose flowers (HERF), steam-distilled lavender straw (SDLS), wheat straw (WS), and pine sawdust (PS), used as single or mixed substrates. The basic physical and mechanical properties revealed that MBCs perform comparably to high-efficiency thermal insulating and conventional construction materials. The relatively low apparent density, ranging from 110 kg/m³ for WS-based to 250 kg/m³ for HERF-based composites, results in thermal conductivity values between 0.043 W/mK and 0.054 W/mK. Compression stress (40–180 kPa at 10% deformation) also revealed the good performance of the composites. The MBCs had high water absorption due to open porosity, necessitating further optimization to reduce hydrophilicity and meet intended use requirements. An aerobic biodegradation test using respirometry indicated ongoing microbial decomposition for all tested bio-composites. Notably, composites from mixed HERF and WS (50:50) showed the most rapid degradation, achieving over 46% of theoretical oxygen consumption for complete mineralization. The practical applications of MBCs depend on achieving a balance between biodegradability and stability.

1. Introduction

The growing environmental pollution caused by human activities and the rapid depletion of natural resources have made the bio-fabrication of sustainable composite materials a key focus in scientific research. Producing materials from renewable plant biomass represents an encouraging approach to reducing resource depletion and supporting the shift towards a circular economy [1,2]. In addition, a more than threefold increase in agricultural production and processing over the past five decades has resulted in the generation of a substantial number of agricultural by-products and wastes [3]. Falade [4] reports that global crop production generates more than 5 × 10⁹ t of agricultural waste daily. Most of these agricultural wastes are neither recycled nor repurposed but instead are burned or discarded, leading to secondary pollution or uncontrolled accumulation in the environment, both of which contribute to serious ecological issues [5,6]. According to the World Health Organization (WHO), one of the largest sources of ambient air pollution is the smog caused by agricultural waste burning [7].

Conversely, agricultural crop by-products and waste contain abundant cellulose, hemicellulose, and lignin. Coupled with the lignocellulose degradation potential of certain fungi, they could become essential elements in the bio-fabrication of cutting-edge bio-materials, boasting a minimal carbon footprint. This innovation holds promise for substantial reductions in detrimental environmental effects and economic losses [8,9]. The approach aligns closely with the United Nations’ 2050 Agenda, which emphasizes the urgent need to conserve natural resources and achieve climate neutrality by 2050 as key economic goals. Within this framework, the agricultural sector plays a crucial role in advancing the Sustainable Development Goals (SDGs), by ensuring access to nutritious foods, promoting circular agricultural systems, and reducing waste across the supply chain, as outlined by the Food and Agriculture Organization (FAO).

Therefore, in recent years, there has been a growing exploration into the potential of diverse lignocellulosic wastes sourced from agriculture and forestry. Many studies have been searching for effective methods for utilizing them as feedstock, stimulating the vegetative growth of higher fungi. Under controlled solid-state cultivation, fungal mycelium binds the pieces of waste and produces functional mycelium-based composites (MBCs) without energy input and zero-byproducts generated [10]. Here, the fungal mycelium acts as a matrix, while the lignocellulose fibers reinforce the resulting bio-materials [11,12,13,14]. Wheat straw and pine sawdust as well as rose flower and lavender straw by-products are important feedstocks for mycelium-based composite production due to their abundance, lignocellulosic composition, and favorable physical properties. Rose flower and lavender straw by-products are typically treated as waste, with most either incinerated for energy recovery or discarded near distilleries—practices that pose significant environmental risks. Classified as lignocellulosic waste due to their high content of lignin, cellulose, hemicellulose, pectic carbohydrates, and polyphenols, these by-products hold valuable biological compounds. Innovative “self-growing” technologies can harness this biomass to produce value-added, sustainable materials with positive social and economic impacts [11].

MBCs are innovative, 100% natural cellular materials that completely change the way materials are produced. Mycelium-based composites have the potential to support the emerging “green” economy by replacing many petroleum-based products and transforming lignocellulosic waste into value-added, biodegradable materials that do not disrupt ecological cycles. This is one of the most environmentally friendly mechanisms for recycling agricultural by-products and at the same time to obtain value-added products with the realistic potential to replace many synthetic materials [11,15]. The achieving of unique self-grown materials is based on a revolutionary approach in materials science—“Materials that are grown are better than manufactured” [12,13,16]. The structural and macroscopic properties of mycelium-based bio-composites are highly influenced by the fungal species used, the type and chemical makeup of the substrates, and the conditions during cultivation and post-cultivation processing. These factors collectively determine whether the material is suited for non-structural, semi-structural, or specialized applications. Their low density, thermal conductivity and flammability, and high acoustic absorption make them potential substitutes for foams, plastics, and wood products used for packaging or insulation.

Numerous studies have stated that various mushrooms, belonging mostly to the Trametes, Ganoderma, and Pleurotus genera, can be cultivated on a range of lignocellulose waste, including wood chips, cottonseed hulls, corn cobs, sawdust, wheat and rice straw, coconut powder, and by-products from rose flowers and lavender straw, due to their enzyme systems and high colonization rates, turning them into mycelium-based bio-materials [2,11,12,14,17,18,19,20,21,22,23,24].

The physical, mechanical, and structural properties of the MBCs are key characteristics that direct them toward non-structural, semi-structural, or specific applications [2,9,11,12,13,19,24,25,26,27,28]. The characteristics of MBCs strongly depend on the type of the fungal species and substrates, as well as cultivation conditions and post-cultivation processing.

Even a small deviation in the above parameters could result in obtaining bio-materials with different properties and functional aspects [10]. Numerous studies focus on investigating the physical and mechanical characteristics of MBCs derived from various agricultural substrates and fungal strains, as these properties are essential for their utilization in engineering applications. The apparent density of MBCs is a critical factor that affects mechanical properties, water absorption, and thermal conductivity, determining the overall competitiveness and suitability of these materials. Studies show a wide range of apparent densities (25–954 kg/m³), influenced by the type of natural lignocellulose substrate and fungal colonization levels [9]. High water absorption of MBCs, attributed to high open porosity, poses a challenge to their effective applications [29]. Thermal conductivity varies between 0.025 and 0.105 W/mK, with lower values indicating better insulation [25,30,31,32,33,34]. Compressive strength, ranging from 29 to 567 kPa, is pivotal for functional mycelium bio-materials, dependent on substrate type and fungal strain [29,35,36,37,38].

In contrast to the widely studied physical and mechanical properties, the biodegradability of MBCs remains a relatively neglected field of research despite many statements about their biodegradability. Biodegradation of hemp MBCs and wood MBCs by composting has been carried out by Zimele et al. [39]. The results show that the mass loss of hemp and wood MBCs after 12 weeks of biodegradation was above 70%. In order to deliver a novel proof of concept of the material’s biodegradability, a modified soil burial test for ISO 20200 [40] has been used by van Wylick et al. [41]. The weight loss of MBCs obtained from T. versicolor, G. resinaceum, beech wood, and hemp increases after 16 days of incubation in the soil and reaches final weight loss values within the range of 19.06–43.03%. When hardwood chips and hemp are used as substrates, the weight loss reaches 70% after 12 days of incubation, which is in accordance with the claims that many factors influence the disintegration of MBCs. Beyond simple disintegration, degradation of these materials can also enable resource recovery at the end of their life cycle. For example, using MBCs in anaerobic digestion may recover a certain amount of energy through biogas production [42].

Although mycelium-based materials typically incorporate natural fibers known for their biodegradability, their actual biodegradability remains unquantified [9,26,41,43]. Furthermore, there is currently no standardized method or protocol for the evaluation of different MBCs, as they significantly differ in nature and application [8,44].

Each particular case has to be evaluated taking into account the balance between the stability of bio-composites and their decomposition dynamics. Biodegradability testing typically determines the biological (often microbial) utilization of a material by measuring oxygen consumption or CO₂ production during the degradation process. According to OECD guidelines, these tests are carried out under strictly controlled conditions to ensure reproducibility and comparability across different laboratories [45]. For example, the microbial inoculum is first acclimated under standardized conditions, and the test system is rigorously maintained for parameters such as pH, temperature, and nutrient levels. Blank controls are included to account for background microbial activity, while positive controls help confirm that the inoculum is active and capable of degrading readily biodegradable compounds. The experimental data—whether it be oxygen uptake or CO₂ evolution—is compared to the theoretical values calculated from the compound’s chemical structure and stoichiometry. This quantitative approach not only provides a measure of the biodegradation extent but also serves as a critical parameter in environmental risk assessments and regulatory decision-making.

This research focuses on the experimental investigation of producing and characterizing the physico-mechanical properties and biodegradation profile of MBCs, utilizing lignocellulosic residues as raw materials and the mycelium of Ganoderma resinaceum as a binding agent.

2. Materials and Methods

2.1. Substrates and Mushroom

Hexane-extracted rose flowers (Rose damascene Mill.) (HERF), steam-distilled lavender straw (Lavandula angustifolia Mill.) (SDLS), wheat straw (WS) and pine sawdust (PS) were used as feeding substrates for macrofungal strain cultivation and obtaining MBCs. The WS (Figure 1a) and PS (Figure 1b) were sourced from agricultural areas near Plovdiv, Bulgaria, and stored at room temperature. HERF (Figure 1c) and SDLS (Figure 1d) crop (2020) were provided by the company Galen-N, based at Zelenikovo distillery, Brezovo region, Bulgaria). The SDLS was collected from the area near the distillery and was immediately air-dried at 40 °C as the HERF was first stored at −18 °C and dried before use. The all studied substrates were milled and sieved (particle size 1–5 mm).

The macrofungal strain used in this study was Ganoderma resinaceum GA1M, which is a part of the fungal collection of the Department of Microbiology and Biotechnology, University of Food Technologies, Plovdiv.

2.2. Solid-State Cultivation of G. resinaceum for MBCs Acquisition

Six feeding substrates, namely HERF, 100%; SDLS, 100%; WS, 100%; HERF, 50%:WS, 50%; SDLS, 50%:WS, 50%; and WS, 50%:PS, 50% were used for the solid-state cultivation of G. resinaceum. The obtaining of the MBCs was performed according to Angelova et al. [11] with some modifications.

Briefly, the feeding substrates were first moisturized to a final humidity of 65–75% with a solution containing (g/L): MgSO₄—0.5, KH₂PO₄—0.5, K₂HPO₄—1.0, peptone—2.0, yeast extract—2.0. Then, 0.1% CaCO₃ was added, and the substrates were well mixed and transferred to autoclavable mushroom growing bags (SacO₂, Belgium) (100 × 350 mm) and sterilized at 121 °C for 45 min to render the substrate inert. The vegetative inoculum, in the form of pellets (10% w/w), was aseptically mixed with the substrate and left to grow at 26 °C for 8 days until so-called “pre-grown” substrates formation. To achieve the desired shape and dimensions of MBCs necessary for subsequent physical, mechanical, and biodegradation studies, 4% wheat flour was added to the „pre-grown“ substrates and transferred into 3D-printed molds (55 × 55 × 55 mm and 200 × 200 × 55 mm); cultivation continued at 26 °C and 95% humidity in a Nüve climatic chamber (Nüve, Ankara, Turkey) for 8 days. Further, the samples were de-molded and put back into an incubation chamber for another 7 days to allow for the full development of the external mycelium skin. The ready samples were dried in a drying oven SLW 32 (POL-EKO-APARATURA, Wodzisław Śląski, Poland) at 60 °C for 8 h, and specimens with approximate sizes 50 × 50 × 50 mm and 150 × 150 × 50 mm were obtained and used for further tests.

2.3. Physical and Mechanical Characterization of MBCs

2.3.1. Apparent Density

The apparent density of materials is considered one of the main parameters, allowing for the prediction of other significant characteristics (porosity, water absorption, strength, thermal conductivity, etc.) and is a good indicator of mechanical properties [36,43,46]. The density, and especially the porosity, of the MBCs depends on many parameters, among which are the chemical composition, particle size, and distribution of the used substrate together with the mycelium growth and interaction between the components. The apparent density was determined according to EN ISO 29470:2020 [47] in a dry stage on samples with a parallelogram shape, consisting of measurements of the sample’s mass (precision of 0.01 g) and size (by a caliper with a precision of 0.01 mm). The apparent density of each sample was calculated by the formula:

$${\rho }_a={\displaystyle \frac{m_d }{a·b·h}}$$

(1)

where ρ_a is the apparent dry density, in kg/m³; m_d is the mass in dry condition, in kg; and a, b, h are the dimensions (length, width, and height) of the samples in dry condition, in m.

2.3.2. Water Absorption

Water absorption is another very important parameter that influences the quality, durability, and application range of a mycelium-based bio-composite [29]. The capillary absorption, short-term water absorption, and long-term water absorption of the HERF/SDLS-based mycelium bio-composites were evaluated.

The capillary absorption was measured by a partial immersion (1 cm of the height of samples) in water at +20 ± 3 °C. The samples were placed on a grid, allowing free water circulation beneath the sample’s surface. The water level was maintained constant for 24 h. After removal from the water, the surface was wiped off with a moist cloth, and the mass of each sample was then measured. The calculations were made according to Formula (2):

$$W_c={\displaystyle \frac{{m_{w,c}-m}_d }{a·b}}$$

(2)

where W_c is the capillary absorption per unit area for 24 h, in kg/m²; m_d is the mass in dry condition, in kg; m_w,c is the mass in wet condition after 24 h of partial immersion in water, in kg; and a, b are the width and length of the samples, in m.

Water absorption was measured after short-term (1 day) and long-term (28 days) immersion in water. The short- and long-term water absorption was measured according to EN ISO 29767:2019 [48] and EN ISO 16535:2019 [49], respectively.

The samples were placed between 2 grids and covered by 1 cm of water at +20 ± 3 °C. The bottom grid allowed free circulation of water, while the upper grid served as support for additional weights on the samples in order to avoid their emergence. After removal from the water, the surface was wiped off with a moist cloth, and the mass of each sample was then measured.

The water absorption was calculated as follows:

$${W^m}_{a,1d}={\displaystyle \frac{{m_{w,1}-m}_d }{m_d}}·100$$

(3)

$${W^v}_{a,28d}={\displaystyle \frac{{{(m}_{w,28}-m}_d)/{\rho }_w }{a·b·h}}·100$$

(4)

where W^m_a,1d is the short-term water absorption after 1 day of full immersion in water, expressed per unit mass, in % wt; W^v_a,28d is the long-term water absorption after 28 days of full immersion in water, expressed per unit volume, in % vol.; m_d is the mass in dry condition, in kg; m_w,1d is the mass in wet condition after 1 day of full immersion in water, in kg; m_w,28d is the mass in wet condition after 28 days of full immersion in water, in kg; a, b, h are the dimensions (length, width, and height) of the samples in dry condition, in m and ρ_w is the density of water at +20 °C, i.e., 997 kg/m³.

2.3.3. Compressive Resistance at 10% Deformation

The behavior during compression is important to define the appropriate fields of application of MBCs; for instance, whether they can bear some loads (in flooring) and whether they are compressed too much under normal use, which might harm the insulation behavior.

Since in the preliminary studies of compressive behavior of MBCs [11] no fracture was observed, the behavior under compression is characterized by the compressive stress corresponding to 10% of longitudinal strain, i.e., compressive stress at 10% relative deformation, performed according to EN 826:2013 [50]. In order to create smooth and parallel loading surfaces, a preliminary treatment by a thin layer of cement paste was applied and the testing was performed after 48 h. Three samples of approximate size 50 × 50 × 50 mm were used for each of the bio-composites.

The compressive stress at 10% relative deformation was calculated as follows:

$${\sigma }_{10}={\displaystyle \frac{F_{10}}{a·b}}$$

(5)

where σ₁₀ is the compressive stress at 10% deformation, in MPa; F₁₀ is the load at which the test specimen reaches the required longitudinal deformation of 10%, in N and a, b are the width and length of the samples, in mm.

2.3.4. Thermal Conductivity

Steady-state heat transfer properties may be measured by a number of standardized test methods. Taking into consideration the expected low thermal conductivity of MBCs, the EN 12667:2004 [51] method was applied to measure the coefficient of thermal conductivity (λ). The method is based on the so-called guarded hot plate at a temperature difference of 10 °C. A specimen of size 150 × 150 × 50 mm was placed between the hot and the cold plates and centered below the temperature gauges metering area of 100 × 100 mm. An additional lateral insulation of EPS was provided to complete the measurements space of 300 × 300 mm. The coefficient of thermal conductivity λ (W/m·K) was calculated as follows:

$$\lambda ={\displaystyle \frac{\Phi ·d}{A·\left(T_1-T_2\right)}}$$

(6)

where Φ is the average power supplied to the metering section of the heating unit, T₁ is the average specimen’s hot side temperature (+20 °C), T₂ is the average specimen’s cold side temperature (+10 °C), A is the metering area (0.0001 m²), and d is the average specimen’s thickness (ca. 50 mm).

2.4. Biodegradation of MBCs

For the evaluation of the biodegradability of the bio-composite materials, the concept of the 301F method developed and approved by the OECD was used after partial modification according to the sample’s specifics and the objectives of this study. Namely, this includes the complex and non-defined organic content of the samples and the requirement to investigate the behavior of the bio-composites in terms of decomposition in a soil environment.

OECD 301F is an aerobic biodegradation test that utilizes respirometry as an indication of oxygen consumption and ongoing biodegradation. It aims at screening biodegradable materials within 28 days of degradation by activated sludge collected from WWTP. Among the group of 301 methods, it is the one suitable for testing poorly soluble and non-soluble materials such as the samples that are the subject of this study.

The degradation percentage is calculated as the ratio of oxygen consumption to the theoretical oxygen demand (ThOD). The chemical formula of the test substance and its purity may be known to calculate the ThOD. If the ThOD cannot be obtained, the chemical oxygen demand (COD) may be experimentally determined and used as an alternative. The validation of the results needs a blank control containing only the inoculum (which, in this study, was a mixed culture of aerobic soil bacteria) without any organic medium for evaluation of the autolysis contribution to the test results measured.

2.4.1. COD

The theoretical oxygen demand was determined by measuring the COD of 500 mg/L suspensions of each tested bio-composite in mineral medium (the medium composition is prepared according to OECD Guidelines [45]. The method was performed by Lange DR 3900 Spectrophotometer using HACH Lange LCK114 cuvette tests.

2.4.2. Microbial Culture Enrichment and Inoculum Preparation

To obtain an active microbial consortium for the biodegradability tests, the initial soil microbial culture was enriched under aerobic conditions. The enrichment process was carried out by cultivating soil samples at 28 °C on Luria–Bertani (LB) agar medium. The composition of the medium was as follows (g/L): NaCl—5, Tryptone—10, Yeast Extract—5, and Glucose—5. These conditions promoted the growth of a diverse population of heterotrophic microorganisms. After sufficient biomass development, the microbial cells were carefully harvested from the agar medium surface and re-suspended into a sterile mineral medium. This suspension served as the inoculum for the biodegradability experiments. The microbial concentration in the test medium was adjusted to approximately 10⁴ CFU/mL to ensure consistent and reproducible microbial activity during the tests.

2.4.3. Biodegradation Tests and Interpretation of the Results

For measuring the oxygen consumption during the biodegradation of the tested materials OxiDirect aparatus produced by Lovibond was used. A total of 160 mL of bio-composite-containing suspensions were loaded into the test bottles according to the expected oxygen demand levels.

The consumption of oxygen for oxidation of the bio-composites is measured by the pressure difference within a closed system (respirometric biological oxygen demand, BOD). The process dynamics were followed for 28 and 56 days. Two control samples are used, namely an abiotic control containing only mineral medium and a blank sample containing mineral medium with suspended microbial culture for the evaluation of the oxygen consumed for autolysis.

The values obtained within the 28 days of incubation are used for comparison of the bio-biodegradability rates among the samples tested. The values for the 56 days of oxygen consumption are used to determine the bio-biodegradability level as a percentage of the ThOD.

2.5. Statistical Analysis

All the experiments were conducted in triplicate and the values were expressed as mean ± SD. Statistical significance was detected by analysis of variance (ANOVA, Tukey’s test; the value of p < 0.05 indicated statistical difference).

3. Results and Discussion

3.1. Characterization of the Mycelium-Based Composites

HERF, SDLS, WS, and PS were chosen as substrates for this study primarily because of their widespread availability globally, particularly in Bulgaria, where they are significant by-products of the essential oil and agricultural industries. Despite their potential value, waste plant biomass is often overlooked and discarded. G. resinaceum GA1M, isolated from Bulgaria, demonstrates favorable growth kinetics when colonizing various lignocellulosic substrates, resulting in the formation of MBCs. Visually, all obtained mycelium composites exhibited a surface texture that could be characterized as smooth and velvety to the touch and have good dimensional stability (Figure 2).

The complete coverage of the composite by aerial mycelium and the dense network of hyphae in the material’s center significantly affect its physical and mechanical properties. An increased closed (not accessible to water) porosity of MBCs was targeted in order to achieve lower density and thermal conductivity than in our previous study [11], while ensuring minimum compressive strength and keeping water absorption within certain limits.

In order to be able to compare the physical and mechanical characteristics of MBCs with those of thermal insulating products for building applications, the characterization methodology applied was based on standardized methods, adapted to the MBC samples’ peculiarities (shape, size, number).

There were structural differences between the core and the near-surface zones of the samples, due to the mycelium “coating” of the near-surface zones. The testing was performed on intact samples because the removal of the coating (e.g., by cutting or sawing) might lead to different types of degradation of different MBCs. Thus, the results reflected the overall behavior but were highly influenced by the surface/volume ratio. In case the bio-composites are to be used in applications where the edges need to be trimmed or the surface layer removed, the tests must be carried out on the relevant representative samples.

The results from the testing of the physical and mechanical properties of MBCs are summarized in

Table 1. Physical and mechanical properties of the MBCs with different substrates.

MBCs	Apparent Density, ρa	Compressive Resistance at 10% Deformation, σ10	Capillary Absorption, Wc	Short-Term Water Absorption, Wma,1d	Long-Term Water Absorption, Wva,28d	Coefficient of Thermal Conductivity, λ
	kg/m3	kPa	[kg/m2]	%wt.	% vol.	W/mK
HERF	246 ± 23.8	176 ± 21.2	4.79 ± 0.431	123 ± 11.1	59.3 ± 5.34	0.054 ± 0.0043
SDLS	184 ± 22.4	125 ± 22.3	5.63 ± 0.620	148 ± 16.3	50.4 ± 4.54	0.049 ± 0.0049
WS	110 ± 2.6	104 ± 12.1	1.83 ± 0.037	197 ± 5.9	52.8 ± 1.59	0.043 ± 0.0017
HERF: WS	114 ± 4.8	49 ± 6.9	2.85 ± 0.114	242 ± 12.1	61.5 ± 3.07	0.043 ± 0.0026
SDLS:WS	122 ± 3.0	39 ± 3.2	1.44 ± 0.029	148 ± 4.4	47.3 ± 1.89	0.045 ± 0.0018
WS: PS	125 ± 8.1	54 ± 4.4	4.17 ± 0.292	186 ± 14.9	49.2 ± 3.44	0.045 ± 0.0036

.

The apparent density of the studied MBCs varies between 110 and 250 kg/m³, thus being close to many thermal insulating materials used in buildings, such as rigid and semi-rigid mineral wool boards, foam glass, mineral insulation boards, etc. Compared to conventional thermal insulating materials, such as EPS, XPS, PUR, or PIR, having densities from 15 to 50 kg/m³, the apparent density of MBCs is higher [10,33,52,53,54].

However, the mycelium-based binder seems to be a much more efficient binder in terms of thermal insulation than other binders, usually used with natural particles such as lime, gypsum, and cement (e.g., in particle boards, hempcrete, etc.), because the pure mycelium itself has very low density of 0.03–0.05 g/cm³ [14].

The thermal conductivity of all studied MBCs characterizes them as very efficient, being below 0.06 W/mK, while the λ-value of the composites mentioned above is usually in the range of 0.06–0.12 W/mK, indicating thermal insulating materials with medium efficiency.

The presented results on thermal conductivity correlate very well with the forecasts made in our previous study [11], those reported by Jones et al. [43], and Elsacker et al. [29]. They also show a good relationship with the apparent density of MBCs, just as with the majority of thermal insulating materials—Figure 3. The lower apparent density of the composite with 100% WS, when mixed with HERF, also determines a lower thermal conductivity of about 0.043 W/m.K. When WS is mixed with SDLS and PS, this leads to a certain increase in the apparent density (by 11% to 13%) and a marginal increase in λ to 0.045 W/mK. The composites based on 100% HERF and 100% SDLS have significantly higher densities (123% and 67%, respectively), but the thermal conductivity does not increase proportionally and is 0.054 W/m.K and 0.049 W/mK, respectively.

At the same time, the composites with 100% HERF and 100% SDLS have the best mechanical performance. The compressive resistance at 10% deformation is well above 100 kPa (up to 176 MPa), which would allow for use as thermal floor/roof insulation, as well as impact-born noise insulation. The porosity of those composites is relatively coarse because they have the highest capillary absorption, but the lowest short-term absorption after 24 h of immersion in water. Usually, the short-term water absorption is considered representative of finer porosity.

As it has been observed during our preliminary studies [11], the cross-section of the SDLS-based mycelium composites showed the presence of more macro-pores, allowing a larger amount of hyphae inside the material. The structure of the HERF-based bio-composites was more compact and tougher, especially in the core of samples, leading to a higher apparent density of the mycelium HERF-based bio-composites.

All results on the compressive resistance at 10% deformation fall within the limits of Amstivslaski et al. [35], between 29 and 567 kPa, specified for different mycelium-based bio-composites. Nava et al. [55] stated that the compressive strength of mycelium-based bio-composites was lower than that of most EPS categories; however, our results did not confirm this and actually demonstrated very close superior performance to EPS—at 100% HERF and 100% SDLS. When mixed substrate was used, the σ₁₀ was still above 30 kPa, which is the most reported value for EPS wall thermal insulation.

The highest capillary absorption is measured within 100% SDLS composite, followed by 100% HERF composite. It is determined not only by the open voids but also from the water suction by the used HERF and SDLS. Modifying porosity by adding 50% of WS to both HERF and SDLS substrates significantly reduces (by 40% and 75%, respectively) the capillary absorption of composites. The results obtained for these composites and the 100% WS composite are close to those reported by Elsacker et al. [29] for mycelium-chopped hemp, flax- and straw-based bio-composites, having capillary absorption between 2 and 3.8 kg/m². Adding 50% of PS to the WS composite leads to more than a double increase in the capillary absorption of the composite.

The short-term water absorption (after 1 day of immersion in water) is representative of the finer porosity of materials. The results on water absorption after one day of full immersion vary among the different composites between ca. 120 %wt and ca. 250 %wt, being lower for 100%HERF and SDLS-based composites and higher for WS-containing composites. It seems that the combination of different substrates is able to convert the course to a finer porosity, without reduction in the absorption capacity as a whole.

The long-term water absorption, very often used for assessing the durability aspects of thermal insulating materials, seems to be the less sensitive parameter to distinguish the different MBCs because it varies roughly between 60%vol. for HERF-containing composites and 50% for other composites. Despite the significantly reduced (by more than 50%) apparent density of 100% HERF composite, i.e., increased overall porosity, compared to the MBC in our previous research [11], the long-term water absorption remains the same (around 60%vol). The water absorption of 100%SLDS composite is even reduced from ca.85%vol. to ca.50%vol. Still, the long-term water absorption can be assessed as quite high, which reveals that all MBCs have up to 60% open porosity, accessible for water. Long-term water absorption of conventional thermal insulating material is much lower—between 0.2%vol. for extruded polystyrene (XPS) to 2.8%vol. for EPS, after 2 months of immersion [56]. However, the water absorption of the bio-composites could be significantly modified, because it is not only due to the open (accessible to water) porosity but also to the water suction by the used substrate. The behavior of mycelium-based bio-composites during water immersion strongly depends on the hydrophobic properties of the fungal mycelium [26,36] and the hydrophilicity of lignocellulose fibers [57]. The hydrophobicity of basidiomycetes is mainly a result of the content of a low-weight protein, called hydrophobin. This protein was also reported to affect the process of adhesion of lignocellulose particles [12,26]. To limit the hydrophilicity of the lignocellulose fibers and reduce the water absorbance of the mycelium HERF/SDLS-based bio-composites, further investigations are needed in order to obtain well-developed mycelium, fully enveloped substrate particles together with an intact outer mycelium layer that covers the entire bio-composite. Another option would be one that was reported to considerably decrease the water absorbance [55].

Unless modified on the surface or in volume (e.g., by reducing open porosity and water absorbance of mycelium and substrate), the MBCs would not be recommended for external thermal insulation because the high-water content would reduce the insulation properties, making the composites vulnerable to frost and to biological degradation. Further testing shall clarify the impact of water absorption on the bio-composite swelling and the mycelium changes.

Thus, the present findings confirm that by optimizing the porous structure of MBCs, different physical and mechanical performances can be achieved according to the composites’ application requirements.

3.2. Biodegradation Profile

Biodegradation profiling typically begins with determining the theoretical oxygen demand (ThOD), calculated stoichiometrically based on the substrate’s known chemical composition. However, for complex substrates—where the composition is either unknown or variable—the stoichiometric approach becomes impractical. In such cases, the chemical oxygen demand (COD) is determined experimentally, offering a realistic measure of the oxygen required to oxidize all oxidizable compounds in the sample.

Table 2. Theoretical oxygen demand for the tested samples.

Sample	COD of 500 mg/L Suspension (mgO2/dm3)	Theoretical Oxygen Demand per Gram Material (g/g)
HERF	666 (±43)	1.332
SDLS	552(±26)	1.104
WS	584(±37)	1.168
HERF:WS	414 (±17)	0.828
SDLS:WS	440 (±11)	0.88
WS: PS	808 (±44)	1.616

presents the results of the analysis, and the COD values obtained. The COD of the tested suspensions is also interpreted as specific oxygen demand in grams of O₂ per gram of material.

As expected, the results obtained show that the oxidation of the materials studied varies significantly among the different samples, which is probably due to their different composition.

For a comprehensive biodegradability assessment, these data serve as a baseline for comparing with the biological oxygen consumption observed during simulated degradation processes (Figure 4).

The degradation dynamics over the initial 28 days reveal distinct behaviors among the samples. All samples exhibited elevated oxygen consumption compared to the abiotic control, indicating active microbial decomposition of the bio-composite materials. Notably, the HERF:WS sample demonstrated the most rapid degradation rate under the test conditions, while the HERF-only samples showed the slowest rate, with oxygen consumption comparable to the control by day 22.

Over the extended 56-day period, the oxygen consumption data indicate that, following an initial phase of rapid degradation, the process continued with similar trends (

Table 3. Oxygen consumption during the biodegradation tests for 28- and 56-day incubation periods and the decomposition degree reached compared to ThOD.

Sample Period for Degradation	HERF	SDLS	WS	HERF: WS	SDLS: WS	WS: PS	BC	AC
28 days	72	125	113	161	114	106	57	18
56 days	99	149	141	192	133	122	49	18
Oxygen consumption as % of the ThOD *	14.9	27.1	24.1	46.4	30.2	15.1	-	-

* for the calculation of the oxygen consumption for biological degradation, the amount of oxygen consumed by the BC (Blank control) is not taken into account, based on the assumption that microbial autolysis, a process, has not yet taken place at this stage of the degradation process.

). In terms of degradation extent, the HERF: WS sample achieved over 46 percent of the theoretical oxygen consumption necessary for complete mineralization, whereas the worst-performing samples (HERF-only and WS:PS samples) reached only about 15% of the ThOD.

The observed behavior of the tested mycelium-based materials can be attributed to their specific composition, the nature of the raw materials employed, and the processing they undergo before being utilized as components of the bio-composites. For instance, the rapid oxygen consumption observed in the HERF:WS sample suggests that incorporating certain components (like WS) can enhance microbial activity and accelerate degradation. In contrast, the slower degradation seen in the HERF-only samples implies that these formulations may be more stable, due to the presence of specific chemical compounds, such as essential oils or polyphenols [11,58], known to inhibit microbial activity [59].

Ultimately, these insights are critical when designing bio-composites. They highlight the need to strike a balance between biodegradability and stability, ensuring that materials perform effectively during their intended use while still being capable of environmental breakdown once their service life is over.

4. Conclusions

This research highlights the significant potential of MBCs as sustainable, high-performance alternatives to conventional materials. By utilizing renewable agricultural and essential oil by-products, we demonstrated that MBCs can achieve competitive thermal insulation and mechanical properties while maintaining biodegradability, thus supporting the principles of a circular economy. Although challenges such as high water absorption remain, the promising balance between structural stability and environmental responsibility positions MBCs as a transformative solution for sustainable construction and material innovation. Moreover, due to their high porosity and biodegradability, these composites show great promise as support for the immobilization of carbonic anhydrase, offering an innovative pathway for direct carbon capture technologies.