*3.2. Modular Chain Bioreactor System Results*

Mycelium development as a result of biomass production in the MCB is given below (Figure 7). As a result of the experiment, it was seen that mycelium production was rela-

tively rapid with a high humidity and high air supply. In environments where the amount of free water is high, the mycelium formation cannot increase, and the microorganism loses its viability. Because solid culture fermentation occurs in static conditions, if there is excess water in the environment, oxygen cannot be included in the substrate as there is no mixing as in submerged fermentation. Therefore, the dissolved oxygen contained in the excess water is consumed in a short time, and anaerobic conditions are formed. As a result, the diffusion of O2, which is needed for mycelium production, is prevented [21]. It should be taken into account in future studies that anaerobic conditions may have prevented aerobic contaminants. The main issue to be considered here is the O2-CO<sup>2</sup> diffusion between the biomass and the substrate. In the presence of free water, this diffusion is restricted and inhibits aerobic microbial growth. In this case, the mycelium cannot reach the oxygen it needs, and microbial growth remains weak. However, because the moisture in the 3rd bag was too high, free water accumulation occurred in the environment. While the amount of free water was 0 mL in the inoculum fluid at the beginning of the experiment, approximately 25 mL of water accumulation was measured at the end. In environments where the amount of free water is high, the mycelium cannot grow, and the microorganism loses its viability. Therefore, the moisture supply to the environment during fermentation must be optimized so that it is neither too low nor too high to cause free water accumulation. Compared to conventional bags, a more successful result was obtained with the mycelial biomass developed in the MCB candy bag. Mycelial production in the moisture- and air-fed MCB was three times higher than that in the candy bag and mycelial biomass formation was 3.5 times higher than in conventional bag systems (Table 4). tively rapid with a high humidity and high air supply. In environments where the amount of free water is high, the mycelium formation cannot increase, and the microorganism loses its viability. Because solid culture fermentation occurs in static conditions, if there is excess water in the environment, oxygen cannot be included in the substrate as there is no mixing as in submerged fermentation. Therefore, the dissolved oxygen contained in the excess water is consumed in a short time, and anaerobic conditions are formed. As a result, the diffusion of O2, which is needed for mycelium production, is prevented [21]. It should be taken into account in future studies that anaerobic conditions may have prevented aerobic contaminants. The main issue to be considered here is the O2-CO<sup>2</sup> diffusion between the biomass and the substrate. In the presence of free water, this diffusion is restricted and inhibits aerobic microbial growth. In this case, the mycelium cannot reach the oxygen it needs, and microbial growth remains weak. However, because the moisture in the 3rd bag was too high, free water accumulation occurred in the environment. While the amount of free water was 0 mL in the inoculum fluid at the beginning of the experiment, approximately 25 mL of water accumulation was measured at the end. In environments where the amount of free water is high, the mycelium cannot grow, and the microorganism loses its viability. Therefore, the moisture supply to the environment during fermentation must be optimized so that it is neither too low nor too high to cause free water accumulation. Compared to conventional bags, a more successful result was obtained with the mycelial biomass developed in the MCB candy bag. Mycelial production in the moisture- and air-fed MCB was three times higher than that in the candy bag and mycelial biomass formation was 3.5 times higher than in conventional bag systems (Table 4).

**Bag Final Weight (g) Final Dry Weight (g) Zeolite (g) Biomass (g)** 264.4 220.7 200 20.7 285.2 255.7 200 55.7 255.4 211.3 200 11.3

Mycelium development as a result of biomass production in the MCB is given below (Figure 7). As a result of the experiment, it was seen that mycelium production was rela-

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 8 of 12

**Table 3.** Contaminant control experiment assay.

*3.2. Modular Chain Bioreactor System Results*

**Figure 7. Figure 7.**  MCB setup for biomass experiments. MCB setup for biomass experiments.

According to the results, the conditions provided for mycelium production during the 7 days showed a positive effect. The provided air supply and humidification were shown to provide early microbial activation. In addition, the fact that mixing can be achieved manually accelerates the growth of microorganisms that are activated early. However, during the experiment, it was observed that water accumulation occurred in the second bag. This negative situation can prevent mycelial production in solid-state fermentation. For this reason, as a result of the obtained biomass, the production in the 2nd bag was lower than in the other bags (Figure 8).


Control candy bag 261.3 209.7 200 9.7 Control traditional bag 248.2 206.1 200 6.1

According to the results, the conditions provided for mycelium production during the

**Bag Final Weight Final Dry Weight Zeolite Biomass** 263.88 221.88 200 21.88 251.1 217.4 200 17.4 271.5 220.3 200 20.3

**Table 4.** MCB experiment biomass assay. 7 days showed a positive effect. The provided air supply and humidification were shown

**Table 4.** MCB experiment biomass assay.

*Biomimetics* **2022**, *7*, x FOR PEER REVIEW 9 of 12

Another outcome of the experiments can be derived as follows: the design of the MCB, yields the potential to grow different kinds of organisms without affecting or contaminating each other directly. This way, different stages of mycelium development can be kept together, and the faster colonies can be harvested easily. Another outcome of the MCB would be the ability to multi-use. For example, while one bag houses spore-producing fruiting of medicinal *G. lucidum*, the neighbor bag can be a resident for *T. versicolor*, produced for enzyme subtraction. This chain can be continued for any use requiring similar Another outcome of the experiments can be derived as follows: the design of the MCB, yields the potential to grow different kinds of organisms without affecting or contaminating each other directly. This way, different stages of mycelium development can be kept together, and the faster colonies can be harvested easily. Another outcome of the MCB would be the ability to multi-use. For example, while one bag houses spore-producing fruiting of medicinal *G. lucidum*, the neighbor bag can be a resident for *T. versicolor*, produced for enzyme subtraction. This chain can be continued for any use requiring similar environmental conditions, ranging from commercial to personal use.

### environmental conditions, ranging from commercial to personal use. **4. Conclusions**

**4. Conclusions** In this study, a modular bioreactor chain system was produced to eliminate the disadvantages of bag systems frequently used in solid culture fermentation. In a modular system, parameters, such as environment control, sensor systems, and modular connectivity, were discussed, and a particular connection system named CC was developed to connect the modules. The MCB is constituted of connected modules and is a modular In this study, a modular bioreactor chain system was produced to eliminate the disadvantages of bag systems frequently used in solid culture fermentation. In a modular system, parameters, such as environment control, sensor systems, and modular connectivity, were discussed, and a particular connection system named CC was developed to connect the modules. The MCB is constituted of connected modules and is a modular chain bioreactor prototyped to test the efficiency of the setup.

chain bioreactor prototyped to test the efficiency of the setup. Within the scope of the study, the prototype produced with three bag modules with a two-filtered opening contained equivalent solid culture samples connected by CCs. There was a humidity/temperature sensor and a hose-pipe connection in the CCs. In addition, air blowers were added to provide air and humidity flow to the chamber, and these systems were connected with Arduino to provide environmental control. A double-sided filtration system was developed to minimize the risk of contamination between the CCs and bags to provide humidity control. Typical bag containers mostly have one hole for air circulation, and in the case of mass production scenarios, each bag is vulnerable during operation times. However, the double-sided filtration system fixes this vulnerability by connecting all openings in one filtered line for airflow. It allows the chosen unit to be removed without disturbing the rest of the chain.

Biomass production and contamination spread experiments were performed to show that the system works effectively in this study. In the pilot contamination experiments, the second module was left in a non-sterile environment, and the development of all three samples was observed. As a result of the 7-day mycelium-development observation in all modules, when contamination was observed in the second module as expected, the absence of contamination in other modules supported the system's efficiency.

According to the biomass production trial experiments, it has been predicted that the ventilation and humidification system designed in the MCB could be more effective than traditional bag systems. Furthermore, the system's modularity offers the potential to work with different microorganisms simultaneously, the option of harvesting at different times in the same production, and the loss of a single bag rather than the whole production in case of possible contamination. The results of the experiments and observations through the process revealed these advantages of the current MCB prototype in solid-state fermentation:


However, there are still some issues with the system that need improvement. The accumulation of water in the feed hoses during humidification in the experiments and the flow of this accumulated water to the environment cause the risk of preventing mycelium production. The length of the silicon tubing should be adjusted to prevent water accumulation in further tests. In addition, the air blowers' response sequences and the optimization of the sensor reading times are required. The air blower's power capacity should be adjusted according to the filter's number and resistance to the direct blow of the wind. In addition, another factor to be considered in future studies is the determination of how long the prepared system can be used by calculating the efficiency in terms of gram biomass/gram substrate. This study used solid support materials to compare the MCB system and traditional bag systems. However, using a solid substrate instead of a solid support material will increase the yield for more extended use. Finally, the MCB needs more space than the standard bag containers, trained personnel, and the risk of damage to sensitive system elements due to moisture and disinfection chemicals.

**Author Contributions:** Conceptualization, O.K. and B.Ç.; methodology, O.K., B.Ç., T.D.A.A. and E.E.H.; software, O.K.; validation, O.K., B.Ç. and A.A.; formal analysis, O.K., B.Ç. and A.A.; resources, O.K., T.D.A.A. and E.E.H.; data curation, O.K. and B.Ç.; writing—original draft preparation, O.K., B.Ç., ˙I.K., T.D.A.A. and E.E.H.; writing—review and editing, O.K., B.Ç., ˙I.K., T.D.A.A. and E.E.H.; visualization, O.K. and ˙I.K.; supervision, T.D.A.A. and E.E.H.; project administration, O.K. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Data Availability Statement:** Data are available upon request.

**Acknowledgments:** O. Kırdök and B. Çetinta¸s would like to acknowledge The Scientific and Technological Research Council of Turkey (TUBITAK-BIGG) for the grant (no: 2211182), which is partially related to this study.

**Conflicts of Interest:** The authors declare no conflict of interest.
