3.2.3. Mycelium-Based Composite Fabrication Substrate Inoculation

Hemp hurds were collected from a local wood mill called Bafa GmbH (Malsch, Germany) and mixed with wheat bran to enhance the growth of mycelium, while calcium sulfate (CaSO4) was added in a dry condition to adjust the pH of the mixture to the desirable threshold of 5 to 6, suitable for mycelium growth. The mixture was then blended with 60 wt% (weight percentage) of water, and eventually sterilized at 121 ◦C for 60 min. Subsequently, the mixture was cooled to room temperature before it could be inoculated with the selected *G. lucidum* grain spawn. Once cooled down, it was mixed with 1 wt% of colonized mycelium spawn. The colonized substrate was eventually left in the incubation room at 26–28 ◦C with 70–80% humidity for two weeks to develop the full mycelium network.

## Molding and Sample Preparation

After two weeks of substrate colonization in the incubation room, the samples were taken out and transferred into molds prepared for compression, pull-out, and flexural tests. Following the filling of the molds with colonized substrates, they were transferred to the incubation room with similar conditions to the previous phase. The molds were kept there for an additional 3–6 days until the mycelium network was observed to have covered the substrate surface. Then, the mold was removed, and the samples were left in the incubation

room for another 3–5 days to expedite the growth of the mycelium network inside and on the surface of the samples with better aeration. The samples´ preparation process and the final mycelium-based composites for each type of test are shown in Figure 5. *Biomimetics* **2022**, *7*, x FOR PEER REVIEW 8 of 21

**Figure 5.** Mycelium composite samples' production process: (**a**) Compressive strength test cube; (**b**) One-side single veneer pull-out test cube; (**c**) Two-side, middle overlapped veneer with and without welding reinforced cube; (**d**) Lightweight block with and without low- and high-density lattices; (**e**) Pressed board with and without low- and high-density lattices. **Figure 5.** Mycelium composite samples' production process: (**a**) Compressive strength test cube; (**b**) One-side single veneer pull-out test cube; (**c**) Two-side, middle overlapped veneer with and without welding reinforced cube; (**d**) Lightweight block with and without low- and high-density lattices; (**e**) Pressed board with and without low- and high-density lattices.

For compression and pull-out tests, we prepared cubes of 5 × 5 × 5 cm<sup>3</sup> . A moistened and sterilized maple veneer strip with a length, width and thickness of about 16 cm, 1.2 cm, and 0.05 cm, respectively, was then placed in the center of the mold, parallel to one mold side for the pull-out tests. We prepared three types of pull-out samples: a series with a single veneer strip penetration of up to 75% of the height of the cubes, samples with unwelded overlapped veneer extending from both sides of the cubes, and lastly samples with overlapped and welded veneer. For the last two series of the above-mentioned samples, we placed the overlapped section of the veneer strips in the center of the cubes. The tests aimed to determine the interfacial shear strength between the mycelium matrix and veneer, and to evaluate the bonding mechanism that was developed at the interface of the For compression and pull-out tests, we prepared cubes of 5 <sup>×</sup> <sup>5</sup> <sup>×</sup> 5 cm<sup>3</sup> . A moistened and sterilized maple veneer strip with a length, width and thickness of about 16 cm, 1.2 cm, and 0.05 cm, respectively, was then placed in the center of the mold, parallel to one mold side for the pull-out tests. We prepared three types of pull-out samples: a series with a single veneer strip penetration of up to 75% of the height of the cubes, samples with unwelded overlapped veneer extending from both sides of the cubes, and lastly samples with overlapped and welded veneer. For the last two series of the above-mentioned samples, we placed the overlapped section of the veneer strips in the center of the cubes. The tests aimed to determine the interfacial shear strength between the mycelium matrix

> Flexural samples were prepared in molds of 19 cm × 8 cm × 7 cm, with and without veneer lattices (Figure 5d,e). First, we filled up half of the height of the mold with the

veneer and mycelium matrix.

and veneer, and to evaluate the bonding mechanism that was developed at the interface of the veneer and mycelium matrix.

Flexural samples were prepared in molds of 19 cm × 8 cm × 7 cm, with and without veneer lattices (Figure 5d,e). First, we filled up half of the height of the mold with the colonized substrates before placing a veneer lattice, and afterwards finished filling up the rest of the mold with substrate. It was ensured that the density of all the samples would stay the same throughout the sample preparation. We used two types of veneer lattices for this study: high- and low-density lattices. Given the lack of prior research on the use of veneer reinforcement for mycelium-based composite materials, the size of the samples was chosen to suit the available testing facilities, while necessary references to ASTM and European standards were made. For comparison purposes, we prepared a series of flexural test samples with two layers of low-density veneer lattices embedded: one lattice at the top and one at the bottom of the molds with a 10 mm distance from the surface of the substrate. Further details are provided in Section 3.2.4.

### Post-Processing

Once the growth cycle was completed, the samples were transferred to a drying oven and kept there at a temperature range of 60–70 ◦C for 2–3 days. The samples were weighed regularly during this period to ensure their weight was stabilized. When no change was observed, they were removed from the oven, and their final density was measured.

Compression and pull-out test samples were directly tested after drying, while the flexural test samples with and without veneer lattices were prepared for an additional pressing process to produce dense mycelium-based composites (DMC) as per the procedure explained in an earlier study [11]. The flexural test samples were placed in a hot press compression molding machine and pressed at a temperature of 120 ◦C, with the pressure set to 10 MPa for a duration of 15 minutes. The compressed samples were then moved to an oven with a temperature of 40 ◦C for 12 to 24 h to adjust to the room temperature and avoid any thermal stress shock within the samples.

### 3.2.4. Testing

### Tensile Tests

Ten maple veneer strips were tested in order to determine the tensile strength of the reinforcement material used for this study. Each end of the veneer strip was fixed to the grip of a UTM with a 30 kN HBM load cell attached, and pulled by applying 1 N with a loading rate of 10 mm/min.

Similarly, the weld strength was also investigated through tensile tests. Two maple veneer strips that were 10 cm long were overlapped along their grains (in the same axis) and on the end points with an area of 1.2 cm × 3 cm, in which the 1.8 cm from the center of the overlap was welded. Twenty samples were prepared, and the ends were fixed to the grip of the testing machine with the same setup and pulled apart by applying 1 N with a loading rate of 10 mm/min.

A testing standard specific to wood veneers was not found. However, for the climate conditions of the testing, there are numerous standards, such as DIN 52377 (Testing of plywood—Determination of modulus of elasticity in tension and of tensile strength), DIN EN 302-x and DIN EN 205 (Adhesives—Wood adhesives for non-structural applications— Determination of tensile shear strength of lap joints) for wood, and DIN EN ISO 291 (Plastics— Standard atmospheres for conditioning and testing) for plastics with very similar conditions that can be considered as a baseline. Therefore, the climate recommendations from these standards were taken as the reference and the tests were carried out in circa 23 ◦C and at 50% humidity. While the welded samples were stored in 20 ◦C and at 60–65% humidity prior testing, single veneer strips were tested immediately in the recommended testing room conditions. The test setup was designed as per recommendations given by the DIN EN 205 testing standard, while the sample shape had to be adapted due to the material restrictions. Other specifications, such as clamping length and temperature, were followed.

Since almost all the welded samples demonstrated material failure rather than joint failure (see Section 4.2.1), tensile strength was evaluated instead of shear strength. The following formula was used for the calculation:

$$
\sigma\_t = \frac{F\_{\text{max}}}{bt} \tag{1}
$$

where *Fmax* stands for the maximum load in N measured by the UTM at the failure, and *b* and *t* represent the specimen width and thickness in mm, respectively.

### Compression Tests

The compression test samples had an average density of 145 kg/m<sup>3</sup> , which places them in the range of flexible polyurethane foam products, due to their soft texture and low density. Given the lack of standard testing methods for lightweight mycelium-based materials, ASTM D3574:2017 (Standard Test Methods for Flexible Cellular Materials—Slab, Bonded, and Molded Urethane Foams), which is a commonly used standard for testing flexible cellular foam materials, was used as the reference for the testing and evaluation of the compressive properties. The samples were tested using a Universal Testing Machine (UTM) with a 5 kN HBM load cell (HBK, Germany). The cubes were compressed at a rate of 0.5 mm/min and tested until failure. The compressive strength and elastic modulus were calculated using the following formula:

$$
\sigma\_{\mathcal{C}} = \frac{F\_{\text{max}}}{bd} \tag{2}
$$

where *Fmax* is the failure load in N recorded during the test, and *b* and *d* are the width and depth of the cubes in mm, respectively. The elastic modulus was calculated using the slope of the stress–strain curves obtained from each individual test.

## Pull-Out Tests

The bond between the veneer and mycelium matrix can be assessed with the pull-out tests, similar to the methods employed to measure the reinforcement-concrete matrix bond in steel-reinforced concrete elements. There are multiple testing standards with similar scenarios where the reinforcement (veneer strips in this study) is embedded in concrete. Using a UTM, the reinforcement is pulled out by applying a tension force with a defined loading rate, while the sample is restrained to avoid its movement. However, a testing standard for the exact type of material combination of timber and mycelium does not yet exist, since it is a novel composite material. Therefore, testing standards for other materials had to be followed. We selected the procedures explained in RILEM technical recommendation (RC6 Bond test for reinforcement steel. 2. Pull-out test) and ASTM D7913:2020 (Standard Test Method for Bond Strength of Fiber-Reinforced Polymer Matrix Composite Bars to Concrete by Pullout Testing) as the most relevant standards for this study. Thus, we designed the test setup as per the recommendations given by these two testing standards and made the necessary modifications to suit the available testing machines.

The interfacial shear strength (IFSS), or the bond strength, was measured by using a 5kN HBM load cell attached to a Universal Testing Machine (UTM). As explained earlier, three types of pull-out tests were performed in this study. For the pull-out tests where the veneer strip was extended only from one side of the cubes, the veneer strip was fixed to the grip of the tensile test setup and was pulled out on the fixed end. It was ensured that the cube could be held in place to prevent any movement or slipping during the tests. Subsequently, the IFSS was measured using the following formula:

$$
\pi = \frac{F\_p}{2l(t+b)}\tag{3}
$$

where *F<sup>p</sup>* is the pull-out force measured by the machine in N; *t* and *b* are the veneer thickness and width in mm, respectively; and *l* is the embedded veneer length (75% of the cube height) in the mycelium matrix in mm.

In the case of welded and unwelded overlapped veneers, which extended from the two sides of the pull-out samples, a similar overlapped area of 1.2 cm × 3 cm was used to compare the bonding properties. The veneers were overlapped and then embedded within the mycelium matrix and the two free ends were pulled out with the help of the UTM tensile grips from both sides.

### Flexural Tests

To evaluate the flexural capacity of the samples, the recommendations of ASTM D1037:2020 (Standard Test Methods for Evaluating Properties of Wood-Base Fiber and Particle Panel Materials) were adopted. A three-point flexural test was used to find the modulus of rupture and to evaluate the flexural properties, including the elastic modulus in flexure. The support span was set to 140 mm, and a loading rate of 1 mm/min was used for the testing. The lightweight blocks and dense boards with and without veneer lattices were each tested for their flexural properties. The Modulus of Rupture (*MOR*) was calculated using the following formula, while the elastic modulus in flexure was calculated using the stress–strain curves obtained for each sample from the UTM:

$$\text{MOR} = \frac{\text{3LF}\_{\text{max}}}{2bt^2} \tag{4}$$

where *Fmax* stands for the maximum load in N measured by the UTM at failure, and *b*, *t* and *L* represent the specimen width, thickness, and distance between the support points in mm, respectively. *L* was set to 140 mm for all the samples, given the size of the specimens and the available testing machines. It should be noted that the lightweight blocks and the dense boards had a final size of 18.5 cm × 7.5 cm × 6.5 cm after drying and pressing.
