Pure mycelium materials can be produced by drying a mycelium pulp obtained from liquid shaken cultures. Monokaryons of S. commune were so far preferred to produce such materials because of their high biomass production and their uniform materials due to the absence of (early stages of) fruiting bodies under any growth conditions. The presence of a second nucleus in dikaryotic hyphal compartments, however, enhances phenotypic stability (Clark and Anderson 2004; Kües 2000), masking recessive mutations that negatively impact biomass production and material properties. We here compared properties of pure mycelium materials from four dikaryotic strains of S. commune with those of the 4–39 monokaryon that is commonly used in mycelium material production (Appels et al. 2018;2020; d’Errico et al. 2024; 2025ab; van den Brandhof et al. 2024). Strain and medium composition determined biomass productivity and material properties. For instance, sheets displayed a σ of up to 47 MPa.

Results obtained in three media show that dikaryons can produce a biomass similar to that of a monokaryon. Monokaryon 4–39 was in the group of strains producing most biomass. High biomass of this strain was obtained in SCMM and PM. Dikaryon 176 produced a similar biomass as 4–39 in SCMM, while strains 139, 351, and H4-8 did so in PM. Lowest biomass was generally obtained in MM-N medium. This is probably explained by the presence of ammonium instead of asparagine without the addition of extra phosphate buffer to maintain the pH of the medium around 5.

The mycelial sheets of 4–39 grown in SCMM had a σ of 7.77 MPa, an ε of 1.89%, and an E of 0.61 GPa. Similar results were obtained when using MM-N and PM, showing that medium composition in this case had no effect on material properties of 4–39. In general, the medium had a low effect on mechanical properties of the different strains. Growth of strain 139 and H4-8 on PM resulted in a lower σ when compared to SCMM and SCMM and MM-N, respectively, while growth of strain 351 in PM resulted in a higher σ when compared to SCMM. E was only lower in the case of H4-8 on PM when compared to MM-N, while ε was lower on PM and MM-N when compared to SCMM in the case of strains 139 and H4-8. Data suggest that PM has the lowest performance, especially compared to SCMM. This may be due to the nitrogen source (asparagine in SCMM versus ammonium in MM-N and PM) and phosphate content. More detailed future studies should assess whether the nitrogen source and phosphate indeed impact the mechanical properties of pure mycelium materials.

Properties of the materials resulting from drying the mycelium of liquid shaken cultures of the S. commune 4–39 monokaryon were not very different from that of dikaryons of Ganoderma resinaceum and Trametes betulina (van den Brandhof et al. 2025). By contrast, we here showed that the S. commune dikaryons outperformed the monokaryon’s mechanical properties, producing 2.5- to 10-fold stronger (σ), up to 5.4-fold stiffer materials (E), and, in some cases, up to 5-fold more ductile materials (ε). The materials of strain 139 grown in SCMM and strain 351 grown in PM were the most performative, with σ values of 47.29 MPa and 47.16 MPa, ε values of 6.51% and 4.78% and E values of 1.73 GPa and 1.67 GPa, respectively. The strongest reported untreated pure mycelium material so far (σ = 40.4 MPa) was obtained by growing the S. commune sc3 deletion mutant in a liquid static medium in the presence of light and high CO2 (Appels et al. 2018). Therefore, the materials obtained from strain 139 and 351 grown in SCMM and PM, respectively, are the strongest untreated mycelium materials produced so far. Future studies should reveal whether these differences are also found after chemical (d’Errico et al. 2024) or physical treatment (Sinha et al. 2025). In the latter case, materials were obtained with a σ of 67.6 MPa. This was achieved by passing mycelium through a three-roll mill. First, 1400 and 700 μm gaps were used between the rolls, followed by a second pass through 400 and 200 μm gaps. The resulting homogenous dispersion was dried into a film at high RH to enable regrowth during the drying process.

Our results indicate that the higher strength of the mycelium materials of the dikaryons is due to, at least partly, a high percentage of KOH-soluble cell wall components as well as a low sc3 expression. The sc3 gene is highly expressed during formation of aerial hyphae both in monokaryons and dikaryons of S. commune but expression is much lower when fruiting bodies are formed (Wösten and Wessels 2006). Deletion of the sc3 gene in a S. commune monokaryon increases σ as a result of increased ρ of the mycelium material (Appels et al. 2018). This increased ρ is the result of collapse of air voids in the materials that are normally stabilized by the SC3 hydrophobin in the wild-type. The lower expression of sc3 in the dikaryons used in our study would therefore also result in collapse of air voids and thereby increase ρ compared to the monokaryon, in turn, causing the higher σ. Reduced expression of sc3 may also reduce the surface hydrophobicity of the mycelium material caused by self-assembly of this hydrophobin at the mycelium-air interface when the mycelium is dried into a sheet (Wösten et al., 1993).

The higher strength of the dikaryon materials may also be caused by the cell wall composition of the dikaryotic mycelium compared to that of the 4–39 monokaryon. Water-soluble mycelium material, mainly consisting of β-(1,3)(1,6)‐glucan (i.e. schizophyllan), protein, and cytoplasmic material is being removed by washing the cell walls with water (d’Errico et al. 2024; Ehren et al. 2020; Wessels et al. 1972). The percentage of water-insoluble material in the mycelium, corresponding to the water-extracted cell walls and consisting of both KOH-soluble and KOH-insoluble components (Safeer et al. 2023; Kleijburg et al. 2023), was a strong positive predictor of σ. In fact, a strong positive relationship was also found between the amount of KOH-soluble cell wall components and σ. This, together with the absence of a relationship between the KOH-insoluble fraction and σ, suggests a major role of KOH-soluble cell wall components in determining material strength. To illustrate this, the 4–39 material produced from PM-grown mycelium had a low σ (4.62 MPa) and 29.5% KOH-soluble material, while the material of 139 and 351 resulting from mycelium grown in SCMM and PM, respectively, had a σ of 47.29 and 47.16 MPa and 59.3% and 57.8% KOH-soluble cell wall content. How could the KOH-soluble material determine material strength? The KOH-soluble cell wall material mainly consists of the cell wall proteins (d’Errico et al., 2025a), an unknown polysaccharide X, and mannan, as well as part of the β‐(1,3)(1,6)‐glucan and α-(1–3)-glucan (Ehren et al., 2020). The KOH-insoluble material contains the other part of the latter two molecules, which may be in a different conformation compared to their KOH-soluble counterparts, as well as β‐(1,3)‐glucan and chitin (Ehren et al., 2020). The molecules in the KOH-soluble fraction, at least part of them, could have a higher propensity to form inter-molecular hydrogen bonds than the molecules found in the KOH-insoluble cell wall fraction and could thereby more effectively bind cell walls within the mycelium material together.

Interestingly, the KOH-soluble fraction also displays the strongest positive relationship with ε among all material components. By contrast, large amounts of KOH-insoluble cell wall components, consisting of chitin, α- and β-(1,3)-glucans, β-(1,3)-(1,6)-glucan, and fucan (Ehren et al. 2020) have a negative impact on ε and have no impact on σ. It has been shown that alkali-extracted chitin-glucan complexes that are present in the rigid backbone of the fungal cell wall yield materials with high tensile strength (Nawawi et al. 2019). The present data suggest that a large percentage of these molecules in untreated mycelium materials confers rigidity rather than strength, rendering the materials fragile. It is worth noting that material strength may not solely stem from the individual material components but could also arise from the architectural cell wall structure. Differences in this may, for instance, result in increased ρ (Appels et al. 2018).

Together, we here showed that S. commune dikaryons offer a phenotypically stable platform that yield stronger, stiffer, and more ductile materials compared to monokaryons. The properties of the dikaryon materials are similar to polymers, while the monokaryon 4–39 sheets are classified as a natural material when comparing the density and the stiffness of the material. These differences in material properties will favour either the monokaryon or the dikaryons for specific applications. Future studies should assess whether it is possible to obtain synergistic effects in changing the material’s strength and ductility by making combinations of physical and chemical treatments as well as genetic modification to reduce for instance sc3 expression and/or increase the amount of KOH insoluble material in the cell wall.