*3.3. Measuring the Results*

The mushroom morphology was documented at the end of day 27, through photography (Fujifilm X-T2 with 80 mm lens), microscopy (Dino-Lite digital microscope at 70× magnification) and 3D scanning (EinScan-SE desktop scanner). These tools helped to analyze the overall mushroom forms by allowing for the digital measurement of dimensions of the caps and stalks [3].

The biggest mushroom from each replicate was selected as the most mature specimen (Figure 4). Measurements were made digitally using Rhinoceros 3D due to the difficulty in measuring delicate mushrooms of a small size. *Biomimetics* **2022**, *7*, x FOR PEER REVIEW 8 of 18

**Figure 4.** The locations of the measurement points. **Figure 4.** The locations of the measurement points.

**4. Results**  The location of the measurement points for each specimen were standardized as follows:

	- 1. Drawing a line between the lowest and highest point on the cap edge.
	- 2. Measuring the angle between this line and the x-axis (parallel to the ground).

### **4. Results**

### *4.1. The Results of the Humidity Experiment*

In line with the previous study, humidity influences the curvature of cap edges and the stalks [3]. The replicates grown in the in-between conditions exhibit in-between morphologies. As we know from the humidity experiment, cap edge and stalk curvature increase with the increase in humidity level, as seen in Figure 5 and Table 2.

**Figure 5.** The results of the humidity experiment, front (column 1), side (column 2) and detailed (column 3). **Figure 5.** The results of the humidity experiment, front (**column 1**), side (**column 2**) and detailed (**column 3**).



Averge sprout number 2 4.3 4 3.3

However, the cap sizes seem smaller in 80% humidity than the mushrooms grown in 75% humidity. This could be because the two outlier mushrooms grew bigger than expected and raised the average value, although this hypothesis needs to be validated by a bigger sample size. The texture of stalks and the depth of gills are qualitative results, and it can be observed from Figure 6 that in 80% humidity, gills are shallower, and stipes are hairier than 75%. However, the cap sizes seem smaller in 80% humidity than the mushrooms grown in 75% humidity. This could be because the two outlier mushrooms grew bigger than expected and raised the average value, although this hypothesis needs to be validated by a bigger sample size. The texture of stalks and the depth of gills are qualitative results, and it can be observed from Figure 6 that in 80% humidity, gills are shallower, and stipes are hairier than 75%.

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**Figure 6.** The comparison of gills and stipe under different humilities; images are captured using a Dino-Lite digital microscope at 70× magnification. **Figure 6.** The comparison of gills and stipe under different humilities; images are captured using a Dino-Lite digital microscope at 70× magnification.

### *4.2. The Results of the CO2 Experiment 4.2. The Results of the CO<sup>2</sup> Experiment*

Altering CO2 levels has a significant effect on the cap size [3]. High CO2 decreases the cap size and inhibits mushroom maturation. As seen in Figure 7, although there are many Altering CO<sup>2</sup> levels has a significant effect on the cap size [3]. High CO<sup>2</sup> decreases the cap size and inhibits mushroom maturation. As seen in Figure 7, although there are many sprouts, they elongate without cap formation. Their stalks get longer up to a certain

sprouts, they elongate without cap formation. Their stalks get longer up to a certain level, as seen in Table 3. However, after a certain level (somewhere between 5000 ppm and 3000

ppm) the stalk length starts to decrease due to the high CO2 level.

Cap

Stalk

level, as seen in Table 3. However, after a certain level (somewhere between 5000 ppm and 3000 ppm) the stalk length starts to decrease due to the high CO<sup>2</sup> level. Average curv. 72.7 ± 5.3 41 ± 1.2 56.3 ± 3.3 Averge sprout number 8.3 6 6.6

**5000 ppm 3000 ppm 1000 ppm Rep-1A Rep-2A Rep-3A Rep-1B Rep-2B Rep-3B Rep-1C Rep-2C Rep-3C** 

Size (cm) 0.4 0.2 0.1 1.3 0.8 1 2.4 2.3 2.5

Average size 0.2 ± 0.1 1 ± 0.2 2.4 ± 0.1 Curvature(degree) 31 2 5 41 53 40 8 7 9 Average curv. 12.7 ± 11.8 44.7 ± 5.3 8 ± 0.8

Average length 2.3 ± 1.5 4.2 ± 0.3 2 ± 0.1 Curvature(degree) 66 79 73 44 41 39 53 61 55

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**Table 3.** The measurements of the CO2 experiment.

**Figure 7.** The results of the CO2 experiment, front (column 1), side (column 2), and detailed photos (column 3). **Figure 7.** The results of the CO<sup>2</sup> experiment, front (**column 1**), side (**column 2**), and detailed photos (**column 3**).


**Table 3.** The measurements of the CO<sup>2</sup> experiment.

### *4.3. The Results of the Gravity Experiment*

As seen in Figure 8, there was a tendency for the fruiting body to grow vertically so the mushroom caps tended towards being parallel with the horizontal plane (Table 4). This led to the stalks being bent from underneath the cap, as they grow away from the tilted plane of the tile towards a vertical direction. As seen in Figure 9, the mushrooms grown at 5000 (high) and 2000 (low) ppm of CO<sup>2</sup> presented the same behavior in terms of orientation.

In summary, gravity affects the orientation of the caps, which leads the stalks to curve

**90° 135° 180° Rep-1A Rep-2A Rep-3A Rep-1B Rep-2B Rep-3B Rep-1C Rep-2C Rep-3C** 

accordingly but there is no significant impact on the size of the mushrooms.

**Table 4.** The measurements of the gravity experiment in high CO2.

Cap Curvature(degree) 6 12 0 61 35 16 44 19 35 Average curv. 6 ± 2.4 37.3 ± 10.6 32.7 ± 10.2

Average curv. 50.7 ± 3.7 34.7 ± 6.6 19.7 ± 10.5

**Figure 8.** The results of gravity experiment at high CO2 (5000 ppm), front (column 1), side (column 2), and detailed (column 3) photos. **Figure 8.** The results of gravity experiment at high CO<sup>2</sup> (5000 ppm), front (**column 1**), side (**column 2**), and detailed (**column 3**) photos.


**Table 4.** The measurements of the gravity experiment in high CO<sup>2</sup> .

In summary, gravity affects the orientation of the caps, which leads the stalks to curve accordingly but there is no significant impact on the size of the mushrooms.

### *4.4. The Results of the Substrate Amount Experiment*

When the mushroom sizes and the number of sprouts is compared, as seen in Figure 10, an increase in cap size, stalk length and sprout number can be observed as the substrate amount increases (see Table 5). The reason for mushroom stalks growing with different curvatures is that they curled as they came out of the hole in the foil wrap.

The variable that has the most effect on the overall size of the mushrooms is the amount of substrates. Although an increase in humidity enlarges them to some extent, the substrate amount is the main determinant. Without sufficient substrates, the mushrooms cannot reach their maturity.

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**Figure 9.** The results of gravity experiment at low CO2 (200 ppm), front (column 1), side (column 2), and detailed (column 3) photos. **Figure 9.** The results of gravity experiment at low CO<sup>2</sup> (200 ppm), front (**column 1**), side (**column 2**), and detailed (**column 3**) photos. *Biomimetics* **2022**, *7*, x FOR PEER REVIEW 14 of 18

**Figure 10.** The results of the substrate amount experiment, side (column 1), top (column 2), and detailed (column 3) photos. **Figure 10.** The results of the substrate amount experiment, side (**column 1**), top (**column 2**), and detailed (**column 3**) photos.

The experiments showed that the effect of different variables is often connected to similar affects in terms of mushroom morphology. For example, high humidity and sub-

one affect. For example, humidity also affects curvature. Despite this, there is a fairly predictable relationship between each of the environmental parameters described in the experiments. Cap curvature is related to humidity, cap size is related to CO2, stalk bend is related to gravity, and overall mushroom size is related to substrate amount, as seen in

**5. Discussion** 

Figure 11.


**Table 5.** The measurements of the substrate amount.

The samples grown in-between conditions exhibit in-between morphologies. As we know from the previous substrate amount experiment, the size of cap edges and the stalks get bigger as the substrate amount increases. All the curvature measurements in the 120 g mixture are somewhere between the 80 g and 160 g substrate amount. Although the sprout number is similar to the 80 g sample, it is less than the 160 g sample.
