• Low cost

an SD card loaded on the Arduino.

Three heat-resistant colonization bags (candy bags) with identical substrates were linked for the testing process. A specially designed CC unit connected these bags in a chain. The main purpose of these connectors was to achieve better environmental control in the bags, while yielding the removal of desired bags without contaminating the system. An air blower with a 100 mm air outlet and a DC motor of about 100 W accommodated in the chain circulated the air through bags to ventilate the heating caused by the fermentation process. This motor blows 15 s of air per 3 min to circulate the air held inside the candy bags connected in chains through CCs. Meanwhile, a central humidifier mechanism feeds the system via silicon pipes connected to CCs with steam to keep the system in the range of 60–80% moisture, responsively to sensor readings. The relative humidity was adjusted simultaneously with the data collected from humidity sensors located within the CCs. In this study, Arduino Mega was used as a microcontroller to process the data gathered from sensors (DHT11) to maintain the relative humidity and air circulation at a desired level inside the bags by controlling air blowers and humidifier mechanisms, with the help of DC relays. The whole setup was constructed in a room with an air conditioner to cool the place to desired production temperatures between 25 and 28 ◦C. The temperature levels inside the bags were constantly monitored and recorded on an SD card loaded on the Arduino. **Figure 2.** MCB—3 Unit chain scheme. The humidifier mechanism consists of an air blower with a 75 mm air outlet and a DC motor of about 60 W, a sterilized humidifier with a 4 L water capacity, and pipes. The

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 5 of 12

fermentation process. This motor blows 15 secs of air per 3 min to circulate the air held inside the candy bags connected in chains through CCs. Meanwhile, a central humidifier mechanism feeds the system via silicon pipes connected to CCs with steam to keep the system in the range of 60-80% moisture, responsively to sensor readings. The relative humidity was adjusted simultaneously with the data collected from humidity sensors located within the CCs. In this study, Arduino Mega was used as a microcontroller to process the data gathered from sensors (DHT11) to maintain the relative humidity and air circulation at a desired level inside the bags by controlling air blowers and humidifier mechanisms, with the help of DC relays. The whole setup was constructed in a room with an air conditioner to cool the place to desired production temperatures between 25 and 28°C. The temperature levels inside the bags were constantly monitored and recorded on

The humidifier mechanism consists of an air blower with a 75 mm air outlet and a DC motor of about 60 W, a sterilized humidifier with a 4 L water capacity, and pipes. The working principle of the humidifier is simply that an air blower is connected to the tank's top level, forcing moisture to circulate inside the colonization bags. This process is controlled with the microcontroller. Figures 3 and 4 illustrate a simplified block diagram of the electrical and mechanical system of the MCB and the humidifier. working principle of the humidifier is simply that an air blower is connected to the tank's top level, forcing moisture to circulate inside the colonization bags. This process is controlled with the microcontroller. Figures 3 and 4 illustrate a simplified block diagram of the electrical and mechanical system of the MCB and the humidifier.

**Figure 3.** Simplified block diagram of MCB. **Figure 3.** Simplified block diagram of MCB.

The MCB system can be considered an example of lean entrepreneurship to prevent contamination with its modularity. The most important part of this system is the CC apparatus and the filters between this apparatus. Unlike traditional bag systems, the additional cost of filters is \$1.80 per bag.

**Figure 4.** The simplified electrical and mechanical system of the MCB and the humidifier: (1) humidity/temperature sensor, (2) CC, (3) air filter, (4) solid substrate, (5) hose pipe, (6) Arduino, (7) 100 W air blower, (8) vapor chamber, (9) 50 W air blower. **Figure 4.** The simplified electrical and mechanical system of the MCB and the humidifier: (1) humidity/temperature sensor, (2) CC, (3) air filter, (4) solid substrate, (5) hose pipe, (6) Arduino, (7) 100 W air blower, (8) vapor chamber, (9) 50 W air blower.

### The MCB system can be considered an example of lean entrepreneurship to prevent *2.3. Fungal Biomass Production Experiments*

contamination with its modularity. The most important part of this system is the CC apparatus and the filters between this apparatus. Unlike traditional bag systems, the additional cost of filters is \$1.80 per bag. *2.3. Fungal Biomass Production Experiments* The efficiency of the MCB prototype was examined by colonizing a pure culture of *G. lucidum* from the author's previous research [24]. White-rot fungi, *G. lucidum*, with easy colonization and popular in academic research, was chosen as the control culture for the repetition of the tests. The zeolite was used as an inert support material to absorb the nutrients and minerals required for mycelium production and to be used at optimum pH levels [17,25]. Zeolite obtained from volcanic rocks is a mixed mineral-salt-medium solution as a nutrient broth, used as the substrate to achieve fast colonization and easy deter-The efficiency of the MCB prototype was examined by colonizing a pure culture of *G. lucidum* from the author's previous research [24]. White-rot fungi, *G. lucidum*, with easy colonization and popular in academic research, was chosen as the control culture for the repetition of the tests. The zeolite was used as an inert support material to absorb the nutrients and minerals required for mycelium production and to be used at optimum pH levels [17,25]. Zeolite obtained from volcanic rocks is a mixed mineral-salt-medium solution as a nutrient broth, used as the substrate to achieve fast colonization and easy determination of the biomass amount. A pure culture of fungus was inoculated on the substrate for colonization. As can be expected from inorganic zeolite, its mass did not change during colonization, while fungi increased their mass by using nutrients. The dry mass obtained at the end of the incubation was proportional to the biomass of the fungus. The following equation was used in the calculations:

$$\mathbf{m}\_{\text{(final dry weight)}} = \mathbf{m}\_{\text{(2codeite dry)}} + \mathbf{m}\_{\text{(myvelium biomass dry)}} \tag{1}$$

### colonization, while fungi increased their mass by using nutrients. The dry mass obtained *2.4. Contamination Spread Pilot Experiment*

at the end of the incubation was proportional to the biomass of the fungus. The following equation was used in the calculations: m(final dry weight) = m(zeolite dry) + m(mycelium biomass dry) (1) *2.4. Contamination Spread Pilot Experiment* In the MCB, a three-bag experiment was designed to measure the effect of the filter system and the contamination spread between the bags. The first and third bags were prepared under sterile conditions, and *G. lucidum* was inoculated. The second bag, which had filtered connections with other bags, was prepared under non-sterile conditions and was kept in the solid-state fermentation laboratory for half an hour before being included in the MCB. The bags were then connected to the MCB and supplied with humidity and air.

In the MCB, a three-bag experiment was designed to measure the effect of the filter

### system and the contamination spread between the bags. The first and third bags were **3. Results and Discussion**

air.

### prepared under sterile conditions, and *G. lucidum* was inoculated. The second bag, which *3.1. Contamination Control Experiments*

had filtered connections with other bags, was prepared under non-sterile conditions and was kept in the solid-state fermentation laboratory for half an hour before being included in the MCB. The bags were then connected to the MCB and supplied with humidity and **3. Results and Discussion** *3.1. Contamination Control Experiments* In the three-bag experiment, the second bag in the middle was kept in a non-sterile area and added to the system to control the spread of contamination between modular systems. Three observations were made on the 3rd, 5th, and 7th days (Figure 5). Visual inspection shows apparent white hyphae (*G. lucidum*) colonizing the first and the third In the three-bag experiment, the second bag in the middle was kept in a non-sterile area and added to the system to control the spread of contamination between modular systems. Three observations were made on the 3rd, 5th, and 7th days (Figure 5). Visual inspection shows apparent white hyphae (*G. lucidum*) colonizing the first and the third bags without any contamination; however, an unknown microorganism (orange-pink color) grew predominantly in the non-sterile bag (middle unit). The contamination in the second bag did not spread to other bags, indicating that the modularity of the filtration system and the developed MCB system were successful. If the filtration system had been inadequate and the contamination had spread to bags one and three, it would have also inhibited the mycelial production of *G. lucidum*. On the other hand, when Figure 5 is examined, the development of *G. lucidum* in bags one and three on the 5th and 7th days, and the

contamination in the 2nd bag, can be clearly distinguished in terms of mycelium production and color. This shows that the filtration between modules successfully prevented the spread of contamination as intended (Figure 6). duction and color. This shows that the filtration between modules successfully prevented the spread of contamination as intended (Figure 6). the spread of contamination as intended (Figure 6).

bags without any contamination; however, an unknown microorganism (orange-pink color) grew predominantly in the non-sterile bag (middle unit). The contamination in the second bag did not spread to other bags, indicating that the modularity of the filtration system and the developed MCB system were successful. If the filtration system had been inadequate and the contamination had spread to bags one and three, it would have also inhibited the mycelial production of *G. lucidum*. On the other hand, when Figure 5 is examined, the development of *G. lucidum* in bags one and three on the 5th and 7thdays, and the contamination in the 2nd bag, can be clearly distinguished in terms of mycelium pro-

bags without any contamination; however, an unknown microorganism (orange-pink color) grew predominantly in the non-sterile bag (middle unit). The contamination in the second bag did not spread to other bags, indicating that the modularity of the filtration system and the developed MCB system were successful. If the filtration system had been inadequate and the contamination had spread to bags one and three, it would have also inhibited the mycelial production of *G. lucidum*. On the other hand, when Figure 5 is examined, the development of *G. lucidum* in bags one and three on the 5th and 7thdays, and the contamination in the 2nd bag, can be clearly distinguished in terms of mycelium production and color. This shows that the filtration between modules successfully prevented

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 7 of 12

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 7 of 12

**Figure 5.** MCB contamination control experiments. **Figure 5.** MCB contamination control experiments.

**Figure 6.** MCB contamination control experiment setup: 1, humidity/temperature sensor; 2, CC; 3, air filter; 4, solid substrate; 5, hose pipe; 6, Arduino). **Figure 6.** MCB contamination control experiment setup: 1, humidity/temperature sensor; 2, CC; 3, air filter; 4, solid substrate; 5, hose pipe; 6, Arduino). **Figure 6.** MCB contamination control experiment setup: 1, humidity/temperature sensor; 2, CC; 3, air filter; 4, solid substrate; 5, hose pipe; 6, Arduino).

As a result of the biomass production, 20.7 g and 11.3 g of mycelium production was observed in the bags (the 1st and 3rd bags, respectively). The 3rd bag did not obtain sufficient moisture due to water accumulation inside the humidifier's silicon tube. A biomass As a result of the biomass production, 20.7 g and 11.3 g of mycelium production was observed in the bags (the 1st and 3rd bags, respectively). The 3rd bag did not obtain sufficient moisture due to water accumulation inside the humidifier's silicon tube. A biomass production of 55.7 g was observed in the contaminated bag (Table 3). As a result of the biomass production, 20.7 g and 11.3 g of mycelium production was observed in the bags (the 1st and 3rd bags, respectively). The 3rd bag did not obtain sufficient moisture due to water accumulation inside the humidifier's silicon tube. A biomass production of 55.7 g was observed in the contaminated bag (Table 3).

production of 55.7 g was observed in the contaminated bag (Table 3).

