*3.3. Preparing Samples for Mechanical Test*

The formworks for this experiment were made of cardboard covered with plastic tapes to avoid the hyphae feeding on the cardboard and decrease the cardboard's humidity level by capillary action. All formworks were cubes of 5 cm in length. The materials have been moved to the formworks after the first growth phase in the bags. We filled the formworks in three steps and hand-pressed the materials at each step. To control the conditions of similar samples for later experiments, the amount of material added in each step to the formwork was controlled by weight. For example, while all the formworks that were filled for samples Aij weighed 120 g in total, each formwork was filled in three stages, adding 40 g of the material at each stage. Finally, the formworks were placed in the same room for the second phase of the growth process.

Following the growth phase, the samples were unmolded and placed in the oven for 48 h at 92 ◦C. After this heating process, almost all samples lost about two-thirds of their weight, showing that they were thoroughly dried and ceased the growth process. The cubic samples were then moved to the lab for the mechanical tests.

### *3.4. Mechanical Tests 3.4. Mechanical Tests*

The mechanical tests were performed on an MTS machine (Figure 3). ASTM C109 standard procedure requires testing three samples of each treatment. Therefore, for each treatment, we have created three samples. Each sample has been compressed to 80% of its initial height with a 0.05 mm per second rate to study its behavior under compression. We considered material strength as the stress in which material collapsed (the peak stress in stress-strain diagrams when a peak occurs) or the stress at the 10% strain, whichever comes first. This paper reports the average result of each sample group when the difference is less than 8.7%. The mechanical tests were performed on an MTS machine (Figure 3). ASTM C109 standard procedure requires testing three samples of each treatment. Therefore, for each treatment, we have created three samples. Each sample has been compressed to 80% of its initial height with a 0.05 mm per second rate to study its behavior under compression. We considered material strength as the stress in which material collapsed (the peak stress in stress-strain diagrams when a peak occurs) or the stress at the 10% strain, whichever comes first. This paper reports the average result of each sample group when the difference is less than 8.7%.

These different substrate mixtures and timeframes made 35 different treatments. The treatments are coded as Xij: X indicates the substrate mixture used for the cultivation, and *i* and *j* indicate the cultivation time (weeks) in bags and formworks. The details about the

The formworks for this experiment were made of cardboard covered with plastic tapes to avoid the hyphae feeding on the cardboard and decrease the cardboard's humidity level by capillary action. All formworks were cubes of 5 cm in length. The materials have been moved to the formworks after the first growth phase in the bags. We filled the formworks in three steps and hand-pressed the materials at each step. To control the conditions of similar samples for later experiments, the amount of material added in each step to the formwork was controlled by weight. For example, while all the formworks that were filled for samples Aij weighed 120 g in total, each formwork was filled in three stages, adding 40 g of the material at each stage. Finally, the formworks were placed in the same

Following the growth phase, the samples were unmolded and placed in the oven for 48 h at 92 °C. After this heating process, almost all samples lost about two-thirds of their weight, showing that they were thoroughly dried and ceased the growth process. The

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 6 of 15

total and partial duration of the treatments are shown in Table A1.

cubic samples were then moved to the lab for the mechanical tests.

*3.3. Preparing Samples for Mechanical Test* 

room for the second phase of the growth process.

**Figure 3.** Mechanical tests were performed on MBC sample C33 with a 0.05 mm per second rate with the MTS machine (Figures are taken at 15-second intervals). **Figure 3.** Mechanical tests were performed on MBC sample C<sup>33</sup> with a 0.05 mm per second rate with the MTS machine (Figures are taken at 15-s intervals).

For the treatments X24, X33, and X42, the Correlated Solutions VIC-3D Digital Image Correlation (DIC) system was also used to enable a more detailed study of MBC behavior under compression (Figure 4). In this system, a setup with two (or more) cameras captures images from the samples while they are reacting to an external force or stimulus. The samples are prepared with speckle points, lights, or other readable signs. After the experiment is conducted, the images from the cameras are correlated to present the alterations For the treatments X24, X33, and X42, the Correlated Solutions VIC-3D Digital Image Correlation (DIC) system was also used to enable a more detailed study of MBC behavior under compression (Figure 4). In this system, a setup with two (or more) cameras captures images from the samples while they are reacting to an external force or stimulus. The samples are prepared with speckle points, lights, or other readable signs. After the experiment is conducted, the images from the cameras are correlated to present the alterations of the samples throughout the process. The DIC shows the exact deformations of the sample and enables studying the quantitative and qualitative mechanical behavior. *Biomimetics* **2022**, *7*, x FOR PEER REVIEW 7 of 15 of the samples throughout the process. The DIC shows the exact deformations of the sample and enables studying the quantitative and qualitative mechanical behavior.

**Figure 4.** (**A**) The Digital Image Correlation (DIC) system and (**B**) its scheme. DIC includes two cameras that capture the alterations of the sample while under compression and correlate the im-**Figure 4.** (**A**) The Digital Image Correlation (DIC) system and (**B**) its scheme. DIC includes two cameras that capture the alterations of the sample while under compression and correlate the images.

### ages. **4. Results and Discussion**

**4. Results and Discussion**  *4.1. The Effects of Sawdust to Straw Ratio in Substrate Mixtures on Compressive Strength*

**Table 2.** Compressive strength of different treatments (kPa).

strates that include both straw and sawdust is negligible.

*4.2. The Effects of Total Growth Time on Compressive Strength* 

*4.1. The Effects of Sawdust to Straw Ratio in Substrate Mixtures on Compressive Strength*  Five substrate mixtures (A, B, C, D, and E) with different ratios of sawdust and straw have been prepared (Table 1). Each mixture has been subjected to seven different differential growth times, resulting in 35 treatments. Table 2 shows the results of the mechanical tests for all 35 treatments. The treatments with only sawdust content (A) showed the best Five substrate mixtures (A, B, C, D, and E) with different ratios of sawdust and straw have been prepared (Table 1). Each mixture has been subjected to seven different differential growth times, resulting in 35 treatments. Table 2 shows the results of the mechanical tests for all 35 treatments. The treatments with only sawdust content (A) showed the best mechanical behavior. This result is in line with the literature [50,57]. While the treatments

> mechanical behavior. This result is in line with the literature [50,57]. While the treatments with 1 to 1 sawdust to the straw ratio (E) showed the weakest mechanical behavior and

> The test results show that stronger substrates with more lignin content, such as sawdust, result in MBC with better compressive strengths, while weaker substrates, such as straw, result in MBC with weaker compressive strengths. There is a direct correlation between the density of the substrate, the density of MBC, and the mechanical strength of the material. However, the difference in the mechanical strength of MBC cultivated with sub-

> Substrate mixtures have been grown for five (X23, X32), six (X24, X33, X42), and seven (X34, X43) weeks. According to the literature [25], the longer growth time causes more organic substrate degradation, which means less substrate and more hyphal structures. Since most of the compressive strength of MBC is from the substrates, longer growth times result in less compressive strength. The results from our mechanical tests are also in line with the literature [7,25]. Figure 5 shows the average compressive strength for each substrate mixture (A, B, C, D, and E) grown for five, six, and seven weeks. For each substrate mixture, the compressive strength decreases by increasing the total cultivation time.

 X23 X24 X32 X33 X34 X42 X43 A 498 416 330 360 303 325 288 B 187 168 107 143 97 69 71 C 177 159 118 103 82 65 62 D 192 142 62 95 74 58 73 E 116 135 34 68 75 39 58

ratios of 1 to 2 (D), 1 to 3 (C), and 1 to 7 (B) exhibited negligible differences.

 **Treatments** 

**Substrate Mixtures** 

with 1 to 1 sawdust to the straw ratio (E) showed the weakest mechanical behavior and lowest compressive strength, the other three substrate mixtures with sawdust to straw ratios of 1 to 2 (D), 1 to 3 (C), and 1 to 7 (B) exhibited negligible differences.


**Table 2.** Compressive strength of different treatments (kPa).

The test results show that stronger substrates with more lignin content, such as sawdust, result in MBC with better compressive strengths, while weaker substrates, such as straw, result in MBC with weaker compressive strengths. There is a direct correlation between the density of the substrate, the density of MBC, and the mechanical strength of the material. However, the difference in the mechanical strength of MBC cultivated with substrates that include both straw and sawdust is negligible.
