Novel mycelium-based biocomposites from nutshell particles produced through a bioprocess engineering approach combining liquid- and solid-state fermentation

Novel mycelium-based biocomposites from nutshell particles produced through a bioprocess 1 engineering approach combining liquidand solid-state fermentation 2 Štěpán Hýsek1,2,*, Paraskevi Charalambous3, Christoph Preimesberger2,4, Luboš Prokůpek5, 3 Notburga Gierlinger3,*, Rupert Wimmer2,6 4 1 - Faculty of Forestry and Wood Sciences, Czech University of Life Science Prague, Kamýcká 5 129, Prague, 165 00, Czech Republic 6 2 - BOKU University, Institute of Wood Technology and Renewable Materials, Department of 7 Natural Sciences and Sustainable Resources, Konrad-Lorenz-Straße 24, 3430 Tulln, Austria 8 3 - BOKU University, Institute of Biophysics, Department of Natural Sciences and Sustainable 9 Resources, Muthgasse 11, 1190 Vienna, Austria 10 4 - Wood K plus - Competence Center for Wood Composites & Wood Chemistry, 11 Kompetenzzentrum Holz GmbH, Altenberger Straße 69, A-4040 Linz, Austria 12 5 - Institute of Chemistry and Technology of Macromolecular Materials, Faculty of Chemical 13 Technology, University of Pardubice, Studentská 573, 532 10 Pardubice, Czech Republic 14 6 - Department of Wood Science and Technology, Faculty of Forestry and Wood Technology, 15 Mendel University in Brno, Zemědělská 1, 613 00 Brno, Czech Republic 16 *Corresponding author: burgi.gierli[email protected].at; hyse[email protected].cz 17 Abstract 18 This study presents the development and characterisation of mycelium-based biocomposites 19 (MBBs) reinforced with grinded walnut and pistachio nutshells. A combined liquidand solid20 state fermentation process employing Ganoderma sessile was used to bind the lignocellulosic 21 particles without synthetic adhesives. The influence of nut species and glycerol treatment on 22 composite properties was studied through dynamic mechanical analysis (DMA), Shore A 23 hardness, dynamic vapour sorption (DVS), Fourier-transform infrared spectroscopy (FTIR), 24 simultaneous thermal analysis (STA), and scanning electron microscopy (SEM). Results 25 showed that walnut-based MBBs exhibited higher stiffness and Shore A hardness compared 26 to pistachio-based variants, with the glycerol-treated walnut MBB reaching the highest 27 mechanical performance. DVS analysis confirmed reduced moisture uptake and hysteresis in 28 glycerol-treated samples. SEM revealed a dense hyphal network interconnecting nutshell 29 particles and a surface fungal skin. FTIR spectra indicated compositional differences related 30 to lignin and chitin content, while STA revealed typical thermal decomposition profiles of 31 lignocellulosic composites with additional degradation stages attributed to glycerol. Results 32 showed that walnut-based MBBs exhibited higher stiffness and Shore A hardness compared 33 to pistachio-based variants, with the glycerol-treated walnut MBB reaching the highest 34 mechanical performance. Its Shore A hardness was 71.4, compared to 63.8 in the untreated 35 pistachio MBB. DVS analysis confirmed reduced equilibrium moisture content in glycerol36 treated samples and a decrease in hysteresis. STA showed a residual mass of 25.6% and 37 main DTG peaks at 300.6 °C and 354.6 °C for the glycerol-treated walnut MBB, compared to 38 29.9% residual mass and main DTG peaks at 281.5 °C and 335.1 °C for the untreated pistachio 39 variant. FTIR spectra indicated higher lignin-associated absorbance in walnut composites, 40 consistent with their mechanical performance. SEM revealed a dense hyphal network 41 interconnecting the nutshell particles and a surface fungal skin, with no structural differences 42 observed due to glycerol treatment. 43 Keywords: composite material, mycelium, walnut, pistachio, fermentation 44 1. Introduction 45 Mycelium-based biocomposites (MBB) are composite materials from lignocellulosic 46 (LC) raw materials bonded with fungal fibres. As mycelium is used in order to interconnect the 47 LC particles, no adhesives are used for bonding the composite material (Muiruri et al. 2023). 48 Mycelium, the living organism, can be understood as a big advantage in the process of material 49 production. The living mycelium in certain stages of material production or material use 50 provides a possibility of material partial regeneration during MBB production and even potential 51 regeneration of damaged mycelium during product use. These abilities can finally prolong 52 materials' life cycle and significantly influence its functional properties (Elsacker et al. 2021; 53 Sandak 2023). 54 LC raw material used for MBB production is mostly wood or straw and represents the 55 main substrate component for MBB (Alaneme et al. 2023). Additionally, agricultural residues 56 from cotton production, hemp straw and seeds, flax shives, kenaf fibres, sugarcane bagasse 57 or rice husks were successfully used in substrates for MBB production since they are rich in 58 cellulose, lignin or lower molecular-weight sugars (Sydor et al. 2021). These agricultural 59 residues are also commonly utilised as reinforcing fillers or matrix components in other types 60 of biocomposites (Hýsek et al. 2016; Gajdačová et al. 2018; Němec et al. 2025). Other valuable 61 sources of LC biomass are nutshell particles, which have been previously used for the 62 production of various types of composites (Sutivisedsak et al. 2012; Dong et al. 2017; Zhang 63 and Yu 2022; Sahin et al. 2024), but not yet for the production of MBB. Nutshells are not only 64 composed of fibres, but also thick-walled lignified sclerenchyma cells of different shape (Huss 65 et al. 2020; Huss and Gierlinger 2021). Among the different nutshells, walnut and pistachio 66 shells stand out by being solely composed of polylobate puzzle cells (Antreich et al. 2019, Xiao 67 et al. 2021). 68 Solid-state fermentation is widely used to manufacture MBB. This method is based on 69 LC substrate colonisation with fungal mycelium. To produce particles or fibres for substrates, 70 LC raw materials have to be disintegrated, and the substrate is further hydrated with water and 71 sterilised or pasteurised, usually with heat. After this heat treatment, the substrate is ready to 72 be inoculated by the selected fungus. Fungi used for MBB production are wood-decaying fungi 73 that are able to decompose lignin, cellulose, and hemicelluloses. Therefore, LC substrate 74 serves as a source of nutrition for fungus and provides it with enough nutrients to build a 75 network of fungal fibres that interconnect the particles. When the mycelium colonizes the 76 substrate, the LC substrate is, however, not fully degraded and, therefore, provides structural 77 stability to the MBB (Attias et al. 2020; Elsacker et al. 2020; Sydor et al. 2021). The properties 78 of MBB are substantially affected by the fungus used (Appels et al. 2019). The resulting MBB 79 properties are, however, not the only criteria used for the selection of fungus. Growth rate, 80 resistance to contamination and undemanding growth conditions have also to be considered. 81 The most suitable fungal species for the production of MBB, therefore, belong to the Pleurotus 82 genus, followed by Ganoderma and Trametes (Aiduang et al. 2022; Cerimi et al. 2019). 83 This production method based on solid-state fermentation provides MBB with porous 84 structure characterised by density ranging mostly from 110 kg/m3 to 330 kg/m3, thermal 85 conductivity comparable to the commonly used thermal insulation materials (0.05 - 0.07 86 W/m.K) and great acoustic absorption properties. However, the composites are soft, 87 characterised by low mechanical properties and high water absorption (Alaneme et al. 2023; 88 Madusanka et al. 2024). For instance, MBB produced by the mycelium of P. ostreatus grown 89 on straw substrate reached a compressive strength of 0.02 MPa; when grown on the substrate 90 from oak sawdust, the compressive strength was 0.15 MPa (Ghazvinian et al. 2019). The 91 bending strength of MBB produced from wood particles enriched with cellulose nanofibers 92 (CNF) was 3.5 MPa (Sun et al. 2019) and the tensile strength of MBB from wood sawdust was 93 reported to be from 0.05 MPa to 0.18 MPa, whereas the composites grown on straw reached 94 substantially lower tensile strength (Jones et al. 2020). All studies clearly confirmed that the 95 mechanical properties are influenced mainly by MBB density, size of particles used and fungal 96 strain (Sydor et al. 2021; Alaneme et al. 2023). 97 The water absorption rate of MBB ranges from 40% to 580% and is affected by 98 substrate composition, manufacturing parameters, and method of measurement (Attias et al. 99 2020; Madusanka et al. 2024). Besides this, the surface of MBB strongly affects the water 100 absorption rate. In the case that strong mycelium, called fungal skin, is formed on the surface 101 of the sample, the water absorption dramatically decreases. However, given the hydrophilic 102 properties of LC substrates used for MBB production, the bulk of not modified MBB is always 103 reported to be hydrophilic, which also determines MBB durability and biodegradability (Hýsek 104 et al. 2023; Madusanka et al. 2024). 105 Liquid-state fermentation has already been employed in order to produce pure 106 mycelium (fungal skin). As the fungal skin is grown on the surface of the liquid culture, the 107 advantage of this method is that it enables the production of pure mycelium without mixing the 108 mycelium with LC substrate (Appels et al. 2018; Appels et al. 2020; Sayfutdinova et al. 2023). 109 However, no approach has been reported in scientific papers so far where liquid-state 110 fermentation is used to bond LC particles with mycelium and subsequently bond a composite. 111 The objective of this study was to develop MBB from pistachio and walnut nutshell 112 particles using an innovative method composed of a combination of solidand liquid-state 113 fermentation. We hypothesise that the used production method provides the novel MBB with 114 a nature-inspired structure, resulting in MBB with unique properties compared to the current 115 state of the art. 116 2. Materials and Methods 117 2.1 Materials 118 Pistachio (Pistacia vera) and walnut (Juglans regia) nutshell particles with a particle 119 size lower than 100 micrometres were used in order to produce mycelium-based 120 biocomposites and the selected strain was Ganoderma sessile (“North Blair”, Terrestrial Fungi, 121 USA). Malt extract (ME) was purchased from Carl Roth GmbH + Co. KG, Germany, and a 2% 122 water solution of ME (2%ME) was used in order to produce liquid culture as well as liquid 123 inoculum. Glycerol (≥99 %) was purchased from Carl Roth GmbH + Co. KG, Germany. Malt 124 extract agar (MAE) was purchased from Carl Roth GmbH + Co. KG, Germany and was used 125 in order to cultivate the cultures on agar placed in Petri dishes. 126 2.2 Methods 127 MBB production 128 Five squares (5 mm x 5 mm in dimensions) of mycelium grown on MEA were shaken 129 (150 rpm) with 200 ml 2%ME at 22°C for 13 days in order to prepare the liquid inoculum of G. 130 sessile. Liquid cultures were prepared by inoculation of 200 ml 2%ME by 40 ml of inoculum, 131 and the cultures were cultivated at 20°C for 7 days by constant shaking at 150 rpm. After 7 132 days, 5% (dry weight/volume) of grinded nutshells were added to the liquid culture and shaken 133 at the same condition for another 4 days. After this period, 40 g of the culture was filtered using 134 a polyethylene (PE) filter with pores 29 μm in diameter, the pressure during filtration was 20 135 mbar. The filter cake was placed on a petri dish and lightly covered in order to allow air to enter 136 the petri dish and enable further metabolic activity of the mycelium. This experiment design 137 enabled an additional 7-day-long solid-state fermentation, after which the growing composite 138 was turned over in order to allow the mycelium to grow properly on both sides. After another 7 139 days, the composites were fully grown, and the growth was terminated by vacuum drying at 140 70°C and 700 mbar for 2 days. In order to determine the dry mass of the produced composites, 141 all samples were oven-dried at 103°C. This procedure resulted in the production of particle142 based composites bonded with mycelium non-woven structure; the weight per unit area of the 143 resulting composites from walnut shell particles was 1035 g/m2, composites from pistachio 144 nutshells reached 935 g/m2 and reference without nutshell particles 230 g/m2. The mycelium145 based biocomposites were treated with glycerol. Glycerol treatment was conducted as 146 immersion of the samples in 20% water solution of glycerol for 96 hours at 20°C. The list of 147 produced variants is provided in Table 1. 148 Table 1 Variants of mycelium-based biocomposites 149 Variant Nutshell Treatment W0 Walnut - WG Walnut Glycerol P0 Pistachio - PG Pistachio Glycerol R0 - - RG - Glycerol 150 151 Dynamic Vapour Sorption (DVS) 152 Sorption/desorption isotherms were determined using a DVS Advantage apparatus (Surface 153 Measurement Systems Ltd., London, United Kingdom). All measurements were conducted at 154 a constant temperature of 25 °C. The measurement program is illustrated in Figure 1A. Briefly, 155 the samples were initially pre-dried at 0% relative humidity (RH) for 9 hours. Following this, the 156 RH was increased in 20% increments, each maintained for 3 hours, until reaching 100% RH. 157 The desorption cycle followed the same steps in reverse, decreasing the RH in 20% 158 increments from 100% back to 0%. 159 160 Figure 1 The measurement program for sorption/desorption isotherms determination (A), the 161 measurement program used in STA experiments (B) 162 Dynamic Mechanical Analysis 163 Dynamic mechanical analysis (DMA) was conducted using DMA 303 Explexor (Netzsch, Selb, 164 Germany). The samples were tested using a pressure mode, the frequency was 1 Hz, the 165 dynamic deformation was 0.03 mm, the dynamic force was 10 N, and the static force was 13 166 N. The tested temperature interval was from 20°C to 230°C with an increment of 3°C/min. The 167 tested MBB samples had dimensions of 5 mm × 5 mm × 1 mm (length × width × thickness). In 168 contrast, the reference samples R0 and RG were significantly thinner, and it was not feasible 169 to test a single layer of these materials. Therefore, these samples for DMA were prepared by 170 stacking five layers of R0 and RG to achieve comparable thickness. It should be noted that 171 this layered structure differs from the structure of the MBB samples containing nutshell 172 particles. This structural difference must be taken into account when interpreting the DMA 173 results. 174 Shore Hardness 175 Shore hardness was measured using a Shore durometer (PTC Instruments, USA), in 176 accordance with the EN ISO 868. Shore A method with a flattened tip of the indenter was 177 employed. Although the Shore A method is primarily intended for testing flexible materials, it 178 was employed in this study because the produced composites were brittle, and alternative 179 hardness testing methods led to material chipping. Each variant was measured 10 times. 180 Simultaneous Thermal Analysis (STA) 181 The thermal decomposition behaviour of the material was analysed by simultaneous thermal 182 analysis (STA) using a Netzsch STA 409 PG under pyrolytic conditions with a nitrogen flow of 183 60 mL/min. Prior to measurement, the sample was ground to a particle size below 0.5 µm using 184 a Retsch Ultra Centrifugal Mill ZM 200. The analysis was performed in two steps. In the first 185 step, the sample was heated to 105 °C, followed by a 10-minute isothermal drying phase. 186 Afterward, the sample was cooled to 20 °C using liquid nitrogen. In the second step, the 187 material was heated to 600 °C at a heating rate of 10 K/min. An illustration of the measurement 188 program can be found in Figure 1B. 189 Fourier-Transform Infrared Spectroscopy (FT-IR) 190 To conclude on differences in the chemical composition of the films, FTIR spectra were 191 acquired with 32 scans using an ATR-unit attached to an FT-IR spectrometer (Vertex 70, 192 Bruker, Billerica, USA). The dry samples were milled to have a homogeneous mixture and four 193 spectra measured, averaged and cut to the most relevant wavenumber range from 1800 cm-1 194 to 850 cm-1 using OPUS 7.5 software (Bruker, USA). 195 196 197 Scanning Electron Microscopy (SEM) 198 SEM was performed to examine the architecture of the composites. The samples were gold 199 sputtered (4nm) with a sputter coater (LEICA EM SCD005). High-vacuum secondary electron 200 imaging was performed using an Apreo VS SEM (Thermo Scientific, The Netherlands) at 5 and 201 10 kV and 0.10 nA current, using the OptiPlan use case, detector T1, and mode A+B. 202 3. Results and discussion 203 Dynamic Vapour Sorption 204 The untreated MBB from walnut and pistachio nutshells exhibited sorption and desorption 205 behaviour comparable to that of conventional wood-based composites (Wu 1999). The highest 206 sorption hysteresis was observed in the untreated reference samples, while the lowest 207 hysteresis was found in glycerol-treated MBB made from pistachio nutshells (Figure 2A-B). 208 Glycerol-treated samples generally reached a lower equilibrium moisture content than their 209 untreated counterparts. Although this observation does not fully correspond with our STA 210 results (presented later in this manuscript), it should be emphasised that the DVS method 211 offers higher precision and is specifically designed for the determination of this characteristic. 212 Furthermore, the glycerol treatment led to a flattening of the sorption/desorption isotherms, 213 suggesting a modified interaction between the material and ambient humidity caused by the 214 blocking of free hydroxyl groups, which corresponds to already published studies (Essoua et 215 al. 2016; Appels et al. 2020). A negative change in mass during desorption was observed in 216 the glycerol-treated samples. This behaviour is caused by residual ME in the MBB samples 217 and can be explained by hydrate formation (Scholl and Schmidt 2014). 218 219 220 304 Figure 4 TG, DTG and DSC curves of the mycelium-based composites 305 Fourier-Transform Infrared Spectroscopy 306 The FT-IR spectra of the reference pure fungi materials (RO, RG) showed bands attributed to 307 ß-chitin: the strong amide I band at 1645 cm-1 and high intensity skeletal C-O-C stretching 308 bands around 1032 cm-1 (Fig. 5, black spectra) (Kumirska et al. 2010). Typical glycerol bands 309 are found in none of the variants (RG, PG, WG), explained by the fact that it was just a very 310 thin outer layer. In all walnut and pistachio composites the bands related to chitin were mixed 311 with typical bands for ligno-cellulosic materials. An acetyl group (C=O vibration) attributed to 312 hemicelluloses was found at 1733 cm-1 and an aromatic stretching vibration typical for lignin 313 around 1505 cm-1 (Faix 1991). Based on the band intensity of the latter one we can conclude 314 on lower lignin content in pistachio (P0, PG, blue spectra) than in walnut shell composites 315 (WO, WG). This is in agreement with former spectroscopic results (Xiao et al. 2021) and 316 chemical analysis pointing up to 50-% lignin of the total weight of the fruit (Zhao et al. 2019, 317 Morales et al. 2022). The higher lignin content in walnut shell composites could explain the 318 higher shore hardness. 319 320 Figure 5 FT-IR spectra of MBB variants 321 Scanning Electron Microscopy 322 SEM confirmed the hypothesis that filtration of the liquid culture, consisting of mycelium and 323 grinded nutshells in malt extract, resulted in a composite structure formed by lignocellulosic 324 particles interconnected with fungal hyphae (Figure 6A–D). On the surfaces of the composites 325 that were exposed to air, a continuous fungal skin was developed (Figure 6B). As previously 326 reported, the fungal skin can provide additional functions, as regular coating of materials 327 (Appels et al. 2020; Hýsek et al. 2023). Grinded walnut and pistachio shells were visibly bound 328 together by hyphae, forming a porous and interconnected network (Figure 6A, C, D). The 329 dense presence of fungal material is consistent with FTIR results indicating a high chitin and 330 polysaccharide content. No significant structural differences were observed using SEM 331 between untreated and glycerol-treated samples, suggesting that the glycerol treatment did 332 not affect the morphology of the composite at the microscale. 333 334 Figure 6 Structure of created composites; cross section of PG sample (A), cross section of 335 the surface of PG sample (B), structure of breached W0 sample (C), cross section of WG 336 sample (D). 337 4. Conclusion 338 This study demonstrated the fabrication of MBBs reinforced with grinded walnut and pistachio 339 nutshells using a combination of liquidand solid-state fermentation. The applied process 340 enabled effective binding of lignocellulosic particles by Ganoderma sessile without synthetic 341 adhesives, resulting in self-binding composite structures. The type of nutshell filler and post342 treatment with glycerol significantly influenced the properties of the materials. Walnut-based 343 MBBs outperformed pistachio-based variants in mechanical performance, with the glycerol344 treated walnut composite achieving the highest Shore A hardness and exhibiting the best 345 stiffness and thermal stability characteristics. Moisture-related properties were also improved. 346 DVS measurements showed that glycerol-treated MBBs had a lower equilibrium moisture 347 content and a reduction in sorption hysteresis. FTIR spectra confirmed compositional 348 differences, particularly higher lignin signals in walnut-based samples and strong chitin-related 349 bands across all variants. SEM imaging revealed a uniform composite morphology, with no 350 structural degradation due to glycerol treatment, and the presence of a fungal skin layer on 351 surfaces exposed to air. These results demonstrate that nutshells are viable fillers for MBBs 352 and that biotechnological approaches, such as combined-state fermentation, can be applied 353 to engineer mechanically reinforced bio-composites for sustainable applications. 354 Acknowledgement: This research was supported by HORIZON-MSCA-2022-PF-01-01, grant 355 agreement ID: 101105443, project acronym: LignoMBB and ERC-POC grant agreement ID: 356 101113395, project acronym PUZZLE MATERIALS. 357 Data Availability Statement: Data can be downloaded at the following link: 358 https://doi.org/10.5281/zenodo.17539612 359 Declaration of generative AI and AI-assisted technologies in the manuscript preparation 360 process: During the preparation of this work, the authors used Google Translate, Grammarly 361 and ChatGPT to refine the stylistics of the English language. 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