Impact of malt concentration in solid substrate on mycelial growth and network connectivity in Ganoderma species

With its distinctive material properties, fungal mycelium has emerged as an innovative material with a diverse array of applications across various industries. This study focuses on how the growth strategies of wood fungi adapt to nutrient availability. The effect of malt extract concentration in the growth medium on radial growth kinetics, morphology, mycelium network connectivity, and mechanical characteristics of mycelium from two Ganoderma species were investigated. While an evident pattern of radial growth rate enhancement with malt concentrations was not apparent, there was a discernible trend towards denser mycelium network characteristics as revealed by spectrophotometry. Increased malt extract contents corresponded to elevated optical density measurements and were visually confirmed by denser mycelium networks in photographic images. Investigating the mechanical characteristics of mycelium cultivated on varying solid substrate concentrations, the Young’s modulus exhibited a substantial difference between mycelium grown on 5 wt% malt substrate and samples cultivated on 2 wt% and 0.4 wt% malt substrates. The obtained results represent a new understanding of how malt availability influences mycelial growth of two Ganoderma species, a crucial insight for potentially refining mycelium cultivation across diverse applications, including meat alternatives, smart building materials, and alternative leather.


Edibility of Ganoderma lucidum
Figure S1 depicts a food product distributed in China, containing Ganoderma lucidum.Apart from promising results in preliminary experiments and presence in literature, Ganoderma lucidum was included in this study due to its established edibility 1 (Fig. 1)
Figure S2 shows the preliminary results of growth kinetics of the three fungal species Ganoderma lucidum, Ganoderma sessile and Pleurotus ostreatus var florida on SMA over 7 days.From the depicted fits in the growth curves, it is apparent that the fungi exhibit linear growth behavior from days 3 through 7.There is no data point from day 7 for Ganoderma sessile since all samples had already fully grown to the edge of the petri dish.
Figure S3 shows the results of additional kinetics measurements of G.sessile and G.lucidum with substrate containing 2 wt% agar, 0.2 wt% yeast extract, and malt concentrations ranging from 2 wt% to 8 wt%.Extensional growth was measured every 24 hours over 3-4 days.Growth was measured until the mycelium culture had reached the edge of the agar plate, which occurred after 5 days for G.sessile and 6 days for G.lucidum.

Optical Density Measurement
Figure S4 shows representative heatmaps of absorbance measurements, where the signal's dispersion is quantified over one well.At each of the 144 locations, which were organized as a grid of 12 x 12 squares, the individual absorbance values within one well were quantified.The signal is evenly distributed, with lower values near the well borders-which correspond to the center's highly dense mycelium inoculum-and decreasing density near the walls, which represents the expansion of the mycelium over time.Figure S5 shows the average (n=6) OD values of the wells inoculated with Ganoderma lucidum (a) and Ganoderma sessile (b), measured every 24 hours in-between incubation at 30°C and 80 % RH.The x-axis displays the growth distance [cm] by plotting lines of successive data points from the center (inoculation point) to the edge of the well.Hence, it is important to note that the data points at 1.4 mm growth distance refer to the mycelium inoculation point, which naturally displays a relatively high density from day 0 on.For the quantification of mycelium density in Fig. 2, the integrals of the OD curves after 7 days were compared.

Mechanical analysis
As mentioned in the main text, there are many factors during the preparation for the mechanical analysis of mycelium materials that can influence the resulting characterization.The process phases are summarized in Fig. S6 along with the variables that are impacted by each step.First, the phenotype of fungi used as well as the substrate composition affect mycelium water  uptake, growth speed, density, and composition 9,10 .Next, the inoculation condition (inoculum density) has an influence on growth rate and yield 11 .Further, the time of incubation as well as the accuracy of growth conditions account for the mycelium yield.In particular, it can not be discounted that the removal of the mycelium from the substrate has an impact on mycelium structure.Further, proper storage of mycelium samples pre-measuring is needed to avoid mycelium drying out.In addition, pure mycelium samples are very fragile and must be handled with great care when mounting into the tensile geometry to avoid breakage of material.Similar issues occurred in earlier research using tensile tests to study mycelium materials 12,13 .Finally, as shown in Fig. S7, various post-processing processes of the mycelium samples determine numerous attributes, such as water absorption and network density, impacting its mechanical properties 9,10,12,13 .
To facilitate data comparison, in figure S7, we compare moduli of pure mycelial materials, while neglecting data on mycelium-composite specimens.The sample preparation for mechanical assessment in this study was based on a study by Haneef et al. (2017)  9 .The measured elastic moduli of the two chosen Ganoderma strains are in the same size range (10 -1 -10 1 MPa) as in the reported study 9 .Another technique of sample preparation described in the literature involves hydrolyzing the obtained mycelium mats before mechanical measurement.Obtained values using this sample preparation are slightly higher (10 0 -10 2 MPa).However, it is important to keep in mind, that the water uptake, which affects the final Young's modulus, can itself vary depending on the characteristics of the substrate 9,10 .Further, mycelium mats have been pressed for assessment after drying (green), which  resulted in elastic moduli of a similar size range as hydrolysis.Pressing of mycelium material results in increased material density, which improves the strength of the material 14 .Finally, some measurements were done with mycelium samples that had been layered before drying, which resulted in higher material density and thus, higher elastic moduli.

Figure S1 .
Figure S1.Commercialized canned product of Lingzhi mushroom, i.e.Ganoderma lucidum manufactured in China and sold in Europe.

Figure S3 .
Figure S3.Growth kinetics of a) G.sessile and b) G.lucidum on agar media with malt concentrations from 2 wt% to 8 wt%.

Figure S4 .
Figure S4.Optical densities of Ganoderma lucidum and Ganoderma sessile mycelium cultures measured in 12 x 12 square per well at an absorbance of 595 nm.

Figure S5 .
Figure S5.Effect of nutritional conditions on growth speed and density shown via absorbance.Absorbance values at 595 nm of Ganoderma lucidum on LMA (a), SMA (b), and HMA (c); similarly, for Ganoderma sessile on LMA (d), SMA (e), and HMA (f).

Figure S6 .
Figure S6.Process flow chart showing the processing steps of mycelium sample preparation for mechanical characterization including a list of affected parameters below 9, 10, 12, 13 .Created with BioRender.com

Table S1 .
Ganoderma lucidum manufactured in China and sold in Europe.Fungal species used in this study including abbreviation and supplier.