3.4.1. Compression Testing

Of the 40 specimens grown with *Pleurotus ostreatus* (first batch) for compression tests, 26 were useable for testing due to the removal of contaminated specimens throughout all experimental groups. In this regard, the sample size of each experimental group was reduced from five to three or four depending on the group. By comparison, for the second batch, all mycelium-based materials grown from mixture S were usable (sample size of 10), but none from mixture L. Therefore, the comparison between both mixtures could not be performed for the *Coprinus comatus* species. Young's modulus of each sample is shown in Table 2. No specimen experienced sudden failure, as they were all compressed throughout the entirety of the test (i.e., until a strain of 0.75 or a load of 10 kN was reached). The compressive Young's moduli were calculated following three methods. As stated in Section 2.6.1, the method considering the linear slope between a strain of 0.19 to 0.21 was selected for this analysis (bold in Table 2). The compressive Young's modulus ranged from 0.15 MPa (PO\_LNCB) to 4.55 MPa (PO\_SCB). A positive correlation between the density of specimens and their compressive Young's modulus was observed for all samples, except one (PO\_SNCD). There was no significant difference between the compressive Young's modulus of the materials grown from *Pleurotus ostreatus* and *Coprinus comatus*.

**Table 1.** This table shows the names of the various samples produced in this study. The fungal species was added in front of these names: PO\_ for *Pleurotus ostreatus* (e.g., PO\_SNCD), and CC\_ for *Coprinus comatus* (e.g., CC\_SNCD).


**Table 2.** This table shows relative humidity before testing, density, and Young's moduli of the samples for the three different calculation techniques. Acronyms for each sample are labeled in Table 1.


In our first batch grown from *Pleurotus ostreatus*, the compressive Young's moduli of the samples ranged from 0.15 to 4.55 MPa. Specimens (PO\_LNCB) made from mixture

L (substrate without fine particles) that were non-compacted and baked resulted in the lowest average Young's modulus at 0.15 MPa for a density of 136.22 kg/m<sup>3</sup> . Specimens (PO\_SCB) made from mixture S (substrate with fine particles) that were compacted and baked resulted in the highest average Young's modulus at 4.55 MPa. These specimens had a mean density of 283.07 kg/m<sup>3</sup> . Overall, the average Young's moduli of the samples made from mixture S ranged from 0.29 to 4.55 MPa. The Young's moduli and stress– strain curves of the various samples grown from *Pleurotus ostreatus* show multiple trends (Figures 3 and 4): first, there was a positive correlation between the density of the samples and their compressive Young's moduli. Only one sample (PO\_SNCD) of eight did not follow this correlation. Second, pre-compacting the myceliated specimens before the compression testing increased their Young's moduli as the four highest moduli were extracted from pre-compacted samples. Third, for the same post-growth treatments, specimens made out of mixture S (with fine particles) had higher Young's moduli than those from mixture L (without fine particles). Fourth, the baked materials had a higher Young's moduli than dried ones in three cases of four. The exception was the Young's modulus of LNCD being higher than that of LNCB. Finally, the mean relative humidity of the samples before testing was 8.82 ± 1.23%. The low variation in the relative humidity of the samples, especially due to the lack of correlation with the Young's moduli, did not likely affect the results of these compressive tests. The difference in relative humidity between samples is likely related to the post-growth treatment used. Baking resulted in a lower relative humidity than drying for 3 samples out of 4. However, the standard deviations show variations within each sample. Therefore, differences in relative humidity could also have emerged from variations in material heterogeneity (i.e., amount of mycelium growth, and distribution and orientation of particles). Since the weight of fruiting bodies harvested on each specimen was collected, the impact of fruiting body formation on mechanical performance of the composite material could be addressed. However, these results should be considered with care as they emerged from a study that was not designed for addressing such impact. Larger amounts of fruiting bodies harvested led to slightly lower Young's modulus in five of eight samples.

In the second batch grown from *Coprinus comatus*, the Young's moduli of specimens made from mixture S (substrate with fine particles) ranged from 0.58 to 3.69 MPa. Specimens (CC\_SCB) that were compacted and baked had the highest average Young's modulus at 3.69 MPa with a mean density of 270.21 kg/m<sup>3</sup> . Similar trends were observed in compression tests of materials grown from both species (Figures 3 and 4). There was a positive correlation between the samples' densities and their compressive Young's moduli. Compacted samples had higher Young's moduli than non-compacted ones. In both cases, baked materials showed a higher Young's modulus than dried ones. For non-compacted specimens, the difference between baking and drying was small. For compacted specimens, similar to the previous batch, baking resulted in higher Young's moduli than drying. However, the humidity of the baked samples was slightly lower than that of the dried samples. The mean relative humidity of the samples before testing was 5.86 ± 0.97%. The high sample size (i.e., 10) increases the validity of these results compared to those of the previous batch.

The study of materials' hysteresis showed that material height after test 2 was slightly lower than that after test 1 (Table 3 and Figure 5). Therefore, the material was still compressible after the first test (i.e., strain of 0.75 or 10 kN reached). In terms of dilatation, specimens did not dilate back to their initial height prior to testing (Table 3). During the first test, most uncompacted specimens reached the 0.75 strain limit first, while most of the pre-compacted ones reached the 10 kN limit first. For the second test, the maximal load of 10 kN was reached before 0.75 strain for all specimens. After the second test, most of the dilatation occurred within the first hour. Some dilatation was already observed 5 s after load removal (Figure 5). However, dilatation still occurred beyond one hour after compressive testing. A week after testing, pre-compacted specimens dilated back to a height closer to their initial height, in comparison to uncompacted specimens. In conclusion, both elastic and plastic

deformations occurred in mycelium-based materials as all specimens experienced some dilatation after testing.

### 3.4.2. Bending Testing

For bending tests, 17 of 48 specimens grown with *Pleurotus ostreatus* (first batch) were useable for testing. Due to high levels of contamination throughout growth, extra specimens, grown from leftover mixtures in different molds, had to be cut down and used for bending testing. However, these extra specimens were not grown in the same environments nor processed along the same timeline. They were marked with the letter "I" to separate them from the regular bending specimens (e.g., PO\_ILNCB and PO\_ISCB). Therefore, the sample size of available specimens for testing ranged from 0 to 6 when considering both original and extra specimens separately (Table 4). For materials grown with *Coprinus comatus*, 52 of 54 mycelium-based materials grown from mixture S were usable, leading to sample sizes ranging from 11 to 15. Similar to specimens tested in compression, no specimens from mixture L were usable for bending testing. The comparison between both mixtures was again only possible for the *Pleurotus ostreatus* species. The elastic moduli of all samples are shown in Table 4 and Figure 6. No significant difference was observed between the elastic moduli of samples grown from the two fungal species. Each wooden mold contained six specimens, which were cut prior to testing. The edges of myceliated panels often exhibited more mycelial growth than their inside. Therefore, specimens on the edge of the panels were tracked. Yet, no significant difference in the elastic modulus was observed between edge and central specimens across all samples. *Biomimetics* **2022**, *7*, x FOR PEER REVIEW 16 of 36 PO\_SNCB 3 6.50 ± 0.32 186.13 ± 5.46 0.75 ± 0.13 **0.51 ± 0.12** 2.75 ± 0.52 PO\_SCD 3 10.40 ± 1.10 258.93 ± 7.17 2.45 ± 0.34 **2.31 ± 0.13** 3.63 ± 0.14 PO\_SCB 3 8.80 ± 1.09 283.07 ± 30.33 2.36 ± 0.79 **4.55 ± 2.29** 4.56 ± 0.73 PO\_LNCD 3 7.78 ± 0.52 156.83 ± 5.45 0.44 ± 0.11 **0.36 ± 0.11** 2.08 ± 0.55 PO\_LNCB 3 8.17 ± 1.12 136.22 ± 2.94 0.23 ± 0.01 **0.15 ± 0.03** 1.41 ± 0.16 PO\_LCD 3 10.67 ± 0.34 188.88 ± 3.69 1.22 ± 0.30 **1.08 ± 0.28** 2.72 ± 0.29 PO\_LCB 4 9.40 ± 0.90 217.46 ± 17.26 1.58 ± 0.24 **1.40 ± 0.22** 3.24 ± 0.21 CC\_SNCD 10 6.51 ± 0.23 167.69 ± 3.83 0.57 ± 0.06 **0.61 ± 0.07** 3.17 ± 0.04 CC\_SNCB 10 6.05 ± 0.23 175.79 ± 3.25 0.72 ± 0.07 **0.78 ± 0.12** 3.21 ± 0.05 CC\_SCD 10 6.36 ± 0.45 264.55 ± 8.90 2.98 ± 0.30 **3.24 ± 0.25** 4.42 ± 0.17 CC\_SCB 10 4.52 ± 0.97 270.21 ± 5.65 3.80 ± 0.17 **3.93 ± 0.30** 4.87 ± 0.17 CC\_LNCD 0 / / / **/** / CC\_LNCB 0 / / / **/** / CC\_LCD 0 / / / **/** / CC\_LCB 0 / / / **/** /

**Figure 3.** This figure shows the mean stress–strain curves for each sample of mycelium-based materials. The stress required to compress the material (i.e., increasing strain) increases linearly then exponentially. Due to the high variability in the stress–strain slope, the Young's modulus was calculated from 3 different strains as shown in Table 2. The Young's modulus used for comparison was calculated from 0.19 to 0.21 strain, to represent the slope of the stress/strain curve at 0.20 strain. "PO" stands for samples grown from *Pleurotus ostreatus*, and "CC" for *Coprinus comatus*. Acronyms for each sample are labeled in Table 1. **Figure 3.** This figure shows the mean stress–strain curves for each sample of mycelium-based materials. The stress required to compress the material (i.e., increasing strain) increases linearly then exponentially. Due to the high variability in the stress–strain slope, the Young's modulus was calculated from 3 different strains as shown in Table 2. The Young's modulus used for comparison was calculated from 0.19 to 0.21 strain, to represent the slope of the stress/strain curve at 0.20 strain. "PO" stands for samples grown from *Pleurotus ostreatus*, and "CC" for *Coprinus comatus*. Acronyms for each sample are labeled in Table 1.

**Figure 4.** This figure shows the mean Young's moduli of the samples in relation to their mean density before compression testing. Young's moduli represented in this figure were calculated from 0.19 to 0.21 strain as it is closest to the traditional calculation of the Young's modulus. "PO" stands for samples grown from *Pleurotus ostreatus*, and "CC" for *Coprinus comatus*. Acronyms for each sample are labeled in Table 1. **Figure 4.** This figure shows the mean Young's moduli of the samples in relation to their mean density before compression testing. Young's moduli represented in this figure were calculated from 0.19 to 0.21 strain as it is closest to the traditional calculation of the Young's modulus. "PO" stands for samples grown from *Pleurotus ostreatus*, and "CC" for *Coprinus comatus*. Acronyms for each sample are labeled in Table 1.

slightly lower than that after test 1 (Table 3 and Figure 5). Therefore, the material was still compressible after the first test (i.e., strain of 0.75 or 10 kN reached). In terms of dilatation, specimens did not dilate back to their initial height prior to testing (Table 3). During the first test, most uncompacted specimens reached the 0.75 strain limit first, while most of the pre-compacted ones reached the 10 kN limit first. For the second test, the maximal **Table 3.** This table shows height deformation or strain of samples over time throughout the mechanical testing process (i.e., before the first test, immediately after the first test, before the second test (which equaled 9 min after the end of the first test), immediately after the second test, one hour after the second test, and one week after the test). Acronyms for each sample are labeled in Table 1.

The study of materials' hysteresis showed that material height after test 2 was


PO\_SNCD 0.00 75.00 50.44 77.87 51.50 47.88 PO\_SNCB 0.00 73.81 48.20 76.61 47.35 42.32

PO\_SCD 0.00 50.39 21.01 51.94 21.95 18.06 PO\_SCB 0.00 47.17 20.31 48.57 20.47 16.04 PO\_LNCD 0.00 75.02 / / 45.51 38.44

PO\_LCB 0.00 59.25 29.68 60.71 30.52 27.68

(**d**) (**e**) (**f**)

**Figure 5.** This figure shows the deformation of a specimen throughout the compression test: (**a**) beginning of the test; (**b**) end of the first test where a compressive strain of 0.75 was reached; (**c**) 5 s after removing the load from the first test; (**d**) 10 min after removing the load from the first test which equals the beginning of the second test; (**e**) end of the second test where a compressive strain of 0.62 and a load of 10 kN were reached; (**f**) 5 s after removing the load from the second test. 3.4.2. Bending Testing **Figure 5.** This figure shows the deformation of a specimen throughout the compression test: (**a**) beginning of the test; (**b**) end of the first test where a compressive strain of 0.75 was reached; (**c**) 5 s after removing the load from the first test; (**d**) 10 min after removing the load from the first test which equals the beginning of the second test; (**e**) end of the second test where a compressive strain of 0.62 and a load of 10 kN were reached; (**f**) 5 s after removing the load from the second test.

For bending tests, 17 of 48 specimens grown with *Pleurotus ostreatus* (first batch) were useable for testing. Due to high levels of contamination throughout growth, extra specimens, grown from leftover mixtures in different molds, had to be cut down and used for bending testing. However, these extra specimens were not grown in the same environments nor processed along the same timeline. They were marked with the letter "I" to separate them from the regular bending specimens (e.g., PO\_ILNCB and PO\_ISCB). Therefore, the sample size of available specimens for testing ranged from 0 to 6 when considering both original and extra specimens separately (Table 4). For materials **Table 4.** This table shows relative humidity before testing, density, and the modulus of elasticity calculated by the Instron and from the ASTM D1037 formula (bold). It also presents the maximum load sustained by all samples, the distance between the center of the specimen and the fracture location (i.e., fracture offset from center), and the angle of that fracture in comparison to the axis of the bending supports. The letter "I" in the sample name means that the corresponding specimens were grown as an extra under different growth environments, mold sizes, and timelines. Acronyms for each sample are labeled in Table 1.



**Figure 6.** This figure shows the modulus of elasticity of samples in relation to their density prior to testing. "PO" stands for samples grown from *Pleurotus ostreatus* and the added "\_I" means that corresponding samples were grown as extras under different growth environments, mold sizes, and timelines. "CC" refers to *Coprinus comatus* samples. Acronyms for each sample are labeled in Table 1. **Figure 6.** This figure shows the modulus of elasticity of samples in relation to their density prior to testing. "PO" stands for samples grown from *Pleurotus ostreatus* and the added "\_I" means that corresponding samples were grown as extras under different growth environments, mold sizes, and timelines. "CC" refers to *Coprinus comatus* samples. Acronyms for each sample are labeled in Table 1.

**Figure 7.** This figure shows the deformation of a PO\_LNCB specimen throughout the bending test: (**a**) beginning of the test; (**b**) after 6 min and 25 s; (**c**) after 13 min (just before the end of the test). (**d**) shows the asymmetric deformation of a different specimen from the PO\_LCD sample after 3 min.

(**a**) (**b**)

(**c**) (**d**)

For specimens from the first batch (grown with *Pleurotus ostreatus*) tested in bending, contradicting results were observed. However, specimens grown as extras were used due to high levels of contamination and resulting low sample size. Since they were grown in molds of a different size and processed on a delayed timeline, results should be analyzed with care. Therefore, data comparison between original specimens is more reliable than comparison between specimens grown as extras. Comparisons between original and extra specimens are only shown when the variable addressed cannot be analyzed between original or extra specimens. For instance, specimens (PO\_ILCB) made from mixture L (substrate without fine particles), which were compacted and baked, resulted in the highest average elastic modulus at 106.08 <sup>±</sup> 40.65 MPa for a density of 286.67 kg/m<sup>3</sup> . In comparison, the rest of the samples ranged from 0.40 MPa (PO\_ISCB) to 22.39 MPa (PO\_SCD). One specimen (PO\_ISCB) made from mixture S (substrate with fine particles), which was compacted and baked, resulted in the lowest average elastic modulus at 0.40 MPa for a density of 160.60 kg/m<sup>3</sup> . This result is in contradiction with the trends observed across other samples and may be due to the different process used since this panel was grown as an extra from leftover mixture. In terms of variables, mixture S resulted in higher elastic moduli than mixture L in two samples out of two (PO\_NCB and PO\_CD) for the original specimens; and three samples out of four (PO\_INCB, PO\_INCD, PO\_ICD and PO\_ICB) for panels grown as extra. Drying resulted in higher moduli of elasticity than baking in one sample out of one (PO\_LNC) for original specimens; one sample out of two (PO\_ILNC and PO\_ISNC) for panels grown as extra; and one sample out of two (PO\_LC/PO\_ILC and PO\_SC/PO\_ISC) when comparing original and extra specimens. Compacting led to higher moduli of elasticity in one sample out of one (PO\_LD) for original specimens; one sample out of two (PO\_ISB and PO\_ILB) for panels grown as extra; and one sample out of one (PO\_SD/PO\_ISD) when comparing original and extra specimens. Such results should be interpreted with care since sample sizes ranged from one to six specimens. In conclusion, variables that led to higher elastic moduli were mixture S and compaction. The difference between the effects of drying or baking was less pronounced. For each sample tested under bending, specimens (i.e., that received the same treatments) were grown in the same mold. The quantity of fruiting bodies harvested was recorded for each mold. Therefore, no data analysis on fruiting bodies' effect on bending properties could be performed.

In the second batch, elastic moduli of mycelium-based samples made of *Coprinus comatus* grown on mixture S (with fine particles) ranged from 1.16 to 48.39 MPa. Specimens (CC\_SNCB) that were not compacted and baked resulted in the lowest average elastic modulus at 1.16 <sup>±</sup> 0.54 MPa for an average density of 132.49 kg/m<sup>3</sup> . Specimens (CC\_SCB) that were compacted and baked resulted in the highest average elastic modulus at 48.39 <sup>±</sup> 23.37 MPa for an average density of 291.66 kg/m<sup>3</sup> . In both cases, compacted specimens had substantially higher moduli of elasticity than non-compacted equivalents. Baking resulted in a higher modulus of elasticity than drying only for compacted specimens. This increase in modulus due to baking can partially be explained by an increase in density for these baked specimens. For non-compacted specimens, the process of baking or drying did not have a significant effect on their elastic modulus.

For all specimens, the location of failure (i.e., fracture) under the bending load was recorded (Table 4 and Figure A1). In a three-point bending test, failure should occur in the middle of the specimen. This fracture should also be parallel to the supports bending the specimen, so the angle between the axis of the fracture and the axis of the supports (i.e., its width as the supports were parallel to the width of the specimens) was calculated (Figure A1). Fracture location varied throughout all samples grown from both species (Figure 7), with no consistent trend observed (Table 4). In specimens grown from *Pleurotus ostreatus*, fracture angle heavily varied in comparison to the ideal failure scenario, ranging from 2.19◦ (PO\_SNCB) to 13.35◦ (PO\_LNCD) (Table 4). By comparison, specimens grown from *Coprinus comatus* failed at an angle close to the ideal scenario as fracture angle ranged from 0.51◦ (CC\_SNCD) to 0.67◦ (CC\_SCB). Within compacted specimens grown from *Coprinus comatus*, there was a decrease in modulus of elasticity as

fracture happened further away from the specimen's center, especially in baked specimens (CC\_SCB) (Figure 8). Therefore, location and angle of failure can be indicators of decreasing resistance under bending. testing. "PO" stands for samples grown from *Pleurotus ostreatus* and the added "\_I" means that corresponding samples were grown as extras under different growth environments, mold sizes, and timelines. "CC" refers to *Coprinus comatus* samples. Acronyms for each sample are labeled in Table 1.

**Figure 6.** This figure shows the modulus of elasticity of samples in relation to their density prior to

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 21 of 36

(**c**) (**d**)

**4. Discussion**  *4.1. Mycelial Growth* 

**Figure 7.** This figure shows the deformation of a PO\_LNCB specimen throughout the bending test: (**a**) beginning of the test; (**b**) after 6 min and 25 s; (**c**) after 13 min (just before the end of the test). (**d**) shows the asymmetric deformation of a different specimen from the PO\_LCD sample after 3 min. **Figure 7.** This figure shows the deformation of a PO\_LNCB specimen throughout the bending test: (**a**) beginning of the test; (**b**) after 6 min and 25 s; (**c**) after 13 min (just before the end of the test). (**d**) shows the asymmetric deformation of a different specimen from the PO\_LCD sample after 3 min. *Biomimetics* **2022**, *7*, x FOR PEER REVIEW 22 of 36

**Figure 8.** This figure shows elastic moduli calculated from the ASTM D1037 formula in relation to the distance between the fracture and the center of the specimen, grown from *Coprinus comatus*: (**a**) compacted samples; (**b**) uncompacted samples. For the compacted and baked non-compacted samples, elastic modulus decreases as fracture happens further away from the ideal scenario. Acronyms for each sample are labeled in Table 1. **Figure 8.** This figure shows elastic moduli calculated from the ASTM D1037 formula in relation to the distance between the fracture and the center of the specimen, grown from *Coprinus comatus*: (**a**) compacted samples; (**b**) uncompacted samples. For the compacted and baked non-compacted samples, elastic modulus decreases as fracture happens further away from the ideal scenario. Acronyms for each sample are labeled in Table 1.

The mycelium-based materials recipe and processes impact the growth of contami-

Furthermore, the growth of contaminants competes with the growth of the inoculated fungal mycelium species. Existing research projects have shown variations in growth time ranging from 6 days up to months [7,13,34,35]. In our case, access to the

batches grown in this study, the substrate was either sterilized (batch 1) or pasteurized (batch 2). Pasteurization is known to kill pests and competitors while minimizing the loss of beneficial micro-organisms [59]. In this regard, pasteurization resulted in lower contamination levels during mycelium growth over the substrate than sterilization [5]. In our study, medium levels of contamination were observed throughout all specimens during mycelium growth over sterilized substrate (i.e., both mixtures of batch 1). By comparison, specimens grown with pasteurized substrate (i.e., batch 2) were either fully contaminated (i.e., those grown with mixture L) or only displayed nil to very low amounts of contamination (i.e., those grown with mixture S containing fine particles). Since no significant difference in contamination was observed between specimens grown with both mixtures in batch 1, and mixtures with fine particles are harder to sterilize or pasteurize than those without, it seems unlikely that the mixture was the cause of the contamination levels. The variation in contamination levels between mixtures of batch 2 was most probably due to the success of the pasteurization process. Even if both sterilization/pasteurization processes were conducted on two different fungal species, pasteurization seems more effective as it can inhibit all contamination if successful [5]. The absence of contamination increases the process' potential for replication. In conclusion, future research should prior-

itize pasteurization of the substrate.

4.1.1. Reducing Contamination and Promote Mycelial Growth
