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

Methods MBB production Five squares (5 mm x 5 mm in dimensions) of mycelium grown on MEA were shaken (150 rpm) with 200 ml 2%ME at 22°C for 13 days in order to prepare the liquid inoculum of G. sessile. Liquid cultures were prepared by inoculation of 200 ml 2%ME by 40 ml of inoculum, and the cultures were cultivated at 20°C for 7 days by constant shaking at 150 rpm. After 7 days, 5% (dry weight/volume) of pulverised nutshells were added to the liquid culture and shaken at the same condition for another 4 days. After this period, 40 g of the culture was filtered using a polyethylene (PE) filter with pores 29 μm in diameter, the pressure during filtration was 20 mbar. The filter cake was placed on a petri dish and lightly covered in order to allow air to enter the petri dish and enable further metabolic activity of the mycelium. This experiment design enabled an additional 7-day-long solid-state fermentation, after which the growing composite was turned over in order to allow the mycelium to grow properly on both sides. After another 7 days, the composites were fully grown, and the growth was terminated by vacuum drying at 70°C and 700 mbar for 2 days. In order to determine the dry mass of the produced composites, all samples were oven-dried at 103°C. This procedure resulted in the production of particle-based composites bonded with mycelium non-woven structure; the weight per unit area of the resulting composites from walnut shell particles was 1035 g/m2, composites from pistachio nutshells reached 935 g/m2 and reference without nutshell particles 230 g/m2. The mycelium-based biocomposites were treated with glycerol. Glycerol treatment was conducted as immersion of the samples in 20% water solution of glycerol for 96 hours at 20°C. The list of produced variants is provided in Table 1. Table 1 Variants of mycelium-based biocomposites Variant Nutshell Treatment W0 Walnut - WG Walnut Glycerol P0 Pistachio - PG Pistachio Glycerol R0 - - RG - Glycerol Dynamic Vapour Sorption (DVS) Sorption/desorption isotherms were determined using a DVS Advantage apparatus (Surface Measurement Systems Ltd., London, United Kingdom). All measurements were conducted at a constant temperature of 25 °C. The measurement program is illustrated in Figure 1. Briefly, the samples were initially pre-dried at 0% relative humidity (RH) for 9 hours. Following this, the RH was increased in 20% increments, each maintained for 3 hours, until reaching 100% RH. The desorption cycle followed the same steps in reverse, decreasing the RH in 20% increments from 100% back to 0%. Figure 1 The measurement program for sorption/desorption isotherms determination Dynamic Mechanical Analysis Dynamic mechanical analysis (DMA) was conducted using DMA 303 Explexor (Netzsch, Selb, Germany). The samples were tested using a pressure mode, the frequency was 1 Hz, the dynamic deformation was 0.03 mm, the dynamic force was 10 N, and the static force was 13 N. The tested temperature interval was from 20°C to 230°C with an increment of 3°C/min. The tested MBB samples had dimensions of 5 mm × 5 mm × 1 mm (length × width × thickness). In contrast, the reference samples R0 and RG were significantly thinner, and it was not feasible to test a single layer of these materials. Therefore, these samples for DMA were prepared by stacking five layers of R0 and RG to achieve comparable thickness. It should be noted that this layered structure differs from the structure of the MBB samples containing nutshell particles. This structural difference must be taken into account when interpreting the DMA results. Shore Hardness Shore hardness was measured using a Shore durometer (PTC Instruments, USA), in accordance with the EN ISO 868. Shore A method with a flattened tip of the indenter was employed. Although the Shore A method is primarily intended for testing flexible materials, it was employed in this study because the produced composites were brittle, and alternative hardness testing methods led to material chipping. Each variant was measured 10 times. Simultaneous Thermal Analysis (STA) The thermal decomposition behaviour of the material was analysed by simultaneous thermal analysis (STA) using a Netzsch STA 409 PG under pyrolytic conditions with a nitrogen flow of 60 mL/min. Prior to measurement, the sample was ground to a particle size below 0.5 µm using a Retsch Ultra Centrifugal Mill ZM 200. The analysis was performed in two steps. In the first step, the sample was heated to 105 °C, followed by a 10-minute isothermal drying phase. Afterward, the sample was cooled to 20 °C using liquid nitrogen. In the second step, the material was heated to 600 °C at a heating rate of 10 K/min. An illustration of the measurement program can be found in Figure 2. 0 10 20 30 40 50 60 70 80 90 100 39 40 41 42 43 44 45 46 47 0 500 1000 1500 2000 2500 Target RH (%) Mass (mg) Time (min) Mass Target RH Figure 2 Measurement program used in STA experiments Fourier-Transform Infrared Spectroscopy (FT-IR) To conclude on differences in the chemical composition of the films, FTIR spectra were acquired with 32 scans using an ATR-unit attached to an FT-IR spectrometer (Vertex 70, Bruker, Billerica, USA). The dry samples were milled to have a homogeneous mixture and four spectra measured, averaged and cut to the most relevant wavenumber range from 1800 cm-1 to 850 cm-1 using OPUS 7.5 software (Bruker, USA).