**1. Introduction**

Today, mycelium growing plays an important role in mushroom production and various biotechnological studies. In recent years, bag containers have been investigated as promising tools for mycelium development, going beyond traditional tray-type bioreactors [1]. According to research, although a limited gas exchange and a limited heat transfer are observed in bag systems, this provides an ideal environment for mycelium production due to low moisture loss [2]. Since the beginning of the use of bag containers, studies have been carried out on edible mushrooms, such as *Ganoderma lucidum* [3,4], *Pleurotus ostreatus* [5,6], *Trametes versicolor* [7,8], and *Agaricus bisporus* [9,10]. As a result of these studies, bag systems are used for mushroom growing in many countries for academic and commercial purposes [11]. Such systems can be divided into two groups depending on the sterility. Commercial bag cultivator systems mostly use plastic bags with needle holes, or even straw sacks, that are often vulnerable to contamination risks. More recently, microholed plastic bags have become popular. However, the risk of contamination continues due to the humidity and perspiration on the plastic surface. Therefore, mushroom farmers mostly prefer pasteurizing the substrate before inoculation to ensure that the fungus is the dominant organism in production. On the other hand, in academic studies, sterile conditions are mainly created to provide controlled conditions, and cultivation is carried out with the selected pure microorganism. To achieve that, bags to be used in such systems should be able to be effectively sterilized. In most cases, the preferred sterilization method is high-pressure steam heated using an autoclave. These bags must be made of a hightemperature-resistant material, such as polypropylene, to be autoclaved. The sterilization of the substrate usually requires a temperature of 121 ◦C, and the bag used in the system must also be resistant to high temperatures.

**Citation:** Kırdök, O.; Çetinta¸s, B.; Atay, A.; Kale, ˙I.; Akyol Altun, T.D.; Hame¸s, E.E. A Modular Chain Bioreactor Design for Fungal Productions. *Biomimetics* **2022**, *7*, 179. https://doi.org/10.3390/ biomimetics7040179

Academic Editor: Stanislav N. Gorb

Received: 16 July 2022 Accepted: 24 October 2022 Published: 27 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

Bag systems are also used for mushroom cultivation and microalgae systems. It is used worldwide in microalgae cultivation in liquid media due to its easy accessibility, low cost, and ability to be ordered in desired volumes [12]. In addition, it has application areas, such as biomass production, biofuel production, and wastewater treatment [13,14]. Accordingly, bag systems are frequently used for commercial and academic purposes due to their rapid growth and high productivity, easy homogenization, cheapness, easy addition to the nutrient medium, ease of work, easy cleaning, easy detection, and the intervention of possible contamination [15]. Using heat-resistant plastic bags as containers during production is common in industrial mushroom production. Specifically, micro-holed plastic bags have become popular among *P. ostreatus* producers. However, bag systems have some disadvantages compared to conventional tray bioreactors due to their limited gas exchange, insufficient air circulation, inability to remove heat caused by microbial growth, and limited heat transfer [16]. Therefore, bigger-scale mushroom production facilities mostly prefer tray-type bioreactors, and in some cases, a mixed use of bag systems and tray reactors is chosen. Tray systems are used in defined spaces (rooms, tents, greenhouses, etc.) with climate control, and they require vast space and expensive equipment. However, according to tray bioreactors, bag systems provide an advantage when limited air circulation and heat exchange issues are fixed (Table 1).

**Table 1.** Comparison of bag system and tray bioreactor.


Conclusively, as stated, bag systems are relatively sufficient for biomass production on a laboratory scale or on a large scale. However, their most important disadvantage is their insufficient air circulation, which reduces production efficiency in biomass growth. Their advantages, such as ventilation, mixing, and moisture retention, make them an efficient tool for solid-state bioreactor designs for controlled mass production. However, such systems lack air circulation, controlled sterility, and humidity control.

In this study, a modular bioreactor was developed to eliminate the weaknesses of bag bioreactors, especially for the three main properties (air circulation, controlled sterility, and humidity control). This bioreactor, named in the study as the modular chain bioreactor (MCB), contains candy-bag modules connected with chain connectors (CCs). The candy bags are pouches with both sides open. A cotton filter was installed on both parts of the bag for high air circulation, and both cotton filters were fixed with a cardboard cup. Finally, this module was duplicated and connected by CCs to form the MCB.

To examine the effect of the developed modular bioreactor on the production of fungi in solid-state cultures, biomass production was investigated using *G. lucidum*. In addition, the prevention of the spread of contamination, which is the main risk factor in mushroom cultivation and causes great losses, was examined.

### **2. Materials and Methods**

### *2.1. Substrates and Microorganism*

*G. lucidum*, one of the most studied species of white-rot Basidiomycetes, was used for the pilot tests of this study. The fungus was maintained on malt extract agar (MEA) slants at 4 ◦C. In a different ongoing study by the authors, inoculum production was optimized for pH, time, and an initial amount of inoculum. Accordingly, inoculum production was prepared using malt extract broth (MEB) (pH 5) by incubation at 28 ◦C and 200 rpm for 11 days. After incubation, the inoculum (Figure 1a) was used as 1 mL/5 g of dry substrate. (right).

compared to the bag system (data not shown).

"candy"-like bag container increased the colonization speed of the fungus (Figure 1). Enzymes are one of the most important products produced in solid-state fermentation. Microorganism growth in solid-state fermentation also affects enzyme production [21]. Culture conditions (pH, temperature, humidity, etc.) should be at optimum levels for the microbial used for rapid microbial growth [22]. However, one of the most fundamental limitations of enzyme production in solid-state fermentation is the measurement accuracy of the enzyme obtained. This may be due to the extraction method, the difference in the substrate used in the enzyme determination, and the fermentation parameters [23]. The developed candy-bag system provides better air circulation compared to traditional bag systems. The two-mouth candy-bag system proposed in the author's ongoing solid-state fermentation and enzyme production study was compared with the traditional single-mouth

**Figure 1.** (**a**) Pellets (bottom the flask); (**b**) cotton-filtered bag container; (**c**) Candy-bag bioreactor **Figure 1.** (**a**) Pellets (bottom the flask); (**b**) cotton-filtered bag container; (**c**) Candy-bag bioreactor (right).

This study used 45 × 50 cm polypropylene bags as candy-bag bioreactor systems for biomass production. Cotton was used as an air filter and cardboard cups (with the bottoms cut off) were used as scaffolding to keep the cotton stable. However, fixed humidity control and problems in air circulation still occurred due to the crumpled structure of plastic bags and sterility limits. To solve these problems, the MCB was generated using separable, filtered, interlocking Chain Connectors (CCs) between the candy bags (Figure 2). It was designed in Fusion 360, and prototypes were de-In order to see the effect of the system designed for biomass production, three candy bags were prepared for the MCB. One candy bag and one traditional bag were prepared as a control group. Both bags were filled with 200 g of zeolite support material and were enriched with a liquid solution containing mineral salts and nutrients (Table 2). Zeolite is an inert support material, consisting of SiO4 and/or AlO4, and is ideal for mycelium production in solid-state fermentation, thanks to the large voids in its structure [17]. When zeolite reaches the ideal humidity with a liquid solution containing nutrients, it has a structure that triggers mycelium production and keeps the pH value of the microorganism constant [18]. Therefore, this solution is also used as a moistening liquid to trigger microorganism growth.

veloped by 3D printing. In order to test the design proposal, a system prototype was in-**Table 2.** Nutrient-mineral salt solution.


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 *2.2. Modular Chain Bioreactor (MCB) Prototype & Arduino Setup for Environmental Control*

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 Solid-state bioreactors are an engineering and design issue for cultivating these organisms for research and industrial purposes. Although there are many different types of bioreactors, the main design principles of solid-state bioreactors focus on several points. These designs should be able to:

	- Carry on solutions for ventilation and mixing required by the production process,
	- Block any organism from entering inside to prevent contamination and keep colonized organisms inside to expel any harmful effect that the organism can cause,
	- Be produced by a durable and corrosion-resistant material, which should also not cause any toxic effect on colonized organisms-,
	- Allow sampling and observation,

Traditionally used bag containers are temperature-resistant bags with a substrate added. The required air is supplied to the mouth of the bag with a cotton filter supported by a cardboard neck collar. Thus, while the containers can reach the air needed by the microorganisms, the substrate bed is protected from contamination. In order to enhance the properties of air circulation, controlled sterility, and humidity, a secondary cotton-filtered opening on the opposite side were examined first. It was observed that this "candy"-like bag container increased the colonization speed of the fungus (Figure 1). Enzymes are one of the most important products produced in solid-state fermentation. Microorganism growth in solid-state fermentation also affects enzyme production [21]. Culture conditions (pH, temperature, humidity, etc.) should be at optimum levels for the microbial used for rapid microbial growth [22]. However, one of the most fundamental limitations of enzyme production in solid-state fermentation is the measurement accuracy of the enzyme obtained. This may be due to the extraction method, the difference in the substrate used in the enzyme determination, and the fermentation parameters [23]. The developed candy-bag system provides better air circulation compared to traditional bag systems. The twomouth candy-bag system proposed in the author's ongoing solid-state fermentation and enzyme production study was compared with the traditional single-mouth bag system, and 1.8 times higher enzyme activity was observed in the candy-bag system compared to the bag system (data not shown). *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 lo-

This study used 45 × 50 cm polypropylene bags as candy-bag bioreactor systems for biomass production. Cotton was used as an air filter and cardboard cups (with the bottoms cut off) were used as scaffolding to keep the cotton stable. cated 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

However, fixed humidity control and problems in air circulation still occurred due to the crumpled structure of plastic bags and sterility limits. To solve these problems, the MCB was generated using separable, filtered, interlocking Chain Connectors (CCs) between the candy bags (Figure 2). It was designed in Fusion 360, and prototypes were developed by 3D printing. In order to test the design proposal, a system prototype was installed for 7 days and observed daily. 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. **Figure 2.** MCB—3 Unit chain scheme.

The proposed bioreactor aims to offer an improvement on the following qualities:

The humidifier mechanism consists of an air blower with a 75 mm air outlet and a • Humidity-controlled environment

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


the electrical and mechanical system of the MCB and the humidifier.

trolled with the microcontroller. Figures 3 and 4 illustrate a simplified block diagram of • Sterile conditions
