In order to accomplish our project’s ultimate goal, we need to produce bacterial cellulose in large scale because it is the essential raw material for our artificial leather synthesis. Previously, iGEM 2014 Imperial proposed a way to ferment G. xylinus in large scale by placing 61 trays of G. xylinus and yeast co-culture in the laboratory (Figure 1). However, although they achieved mass production, this method is not practical for us due to the limited space. Meanwhile, their static cultivation will cause a lack of oxygen supplement for G. xylinus, which inhibits its growth, leading to a significant decrease in production rate. To improve the efficiency of the production method, we designed our hardware, the Rotary Disc Reactor (RDR).
G. xylinus is aerobe microorganism so it can only produce bacterial cellulose at the surface of the medium which have abundant oxygen1. When using static culture, G. xylinus will die of the deficit of oxygen after the medium’s surface is covered with cellulose and lead to a decrease in yield2. The advantage of rotary disc reactor is that the rotation of the plate enables G. xylinus at the bottom of the culture medium to contact with oxygen, so that almost all areas of the culture system could become the active layer where production of cellulose taken place. In addition, once cellulose is produced, it will be winded up by the mesh, and there will be no oxygen shortage without cellulose covering the surface of the medium (Figure 2).
The hardware is made of two parts. The circuit system is placed in a small acrylic box. And the culture tank is connected to the circuit box with an axle and a connector. During cultivation, the circuit system and the culture tank are connected. The DC motor of the circuit system drives the axle in the culture tank to rotate under a given speed. While the incubator is being sterilized, the circuit system and the incubator can be disassembled into two parts. The culture tank can be put into the autoclave sterilizer for sterilization. Since the circuit does not have any contact with the culturing environment, it does not need to be sterilized, so the experimental process is simplified.
The circuit box is designed to protect the circuit from laboratory reagent (Figure 4). The circuit box can be opened by the side board, which is connected to the bottom with hinges. Inside the box is the electrical system. All parts of circuits use 12 voltage direct current to ensure that the hardware can operate under laboratory conditions. After 220V AC is connected to the power box, it is converted into 12V DC by the power box and supplied to the whole electrical system. The electrical system consists of a fan and PWM DC motor driving board. The fan prevents the circuit from overheating. The 12V PWM motor driving board is connected to the motor and a potentiometer. The motor driving board can moderate the speed of DC motor by adjusting the PWM wave through the external potentiometer (Figure 5). There are switches in the circuits where DC is converted to AC and where motor is used to ensure the safety of electrical system.
The other part, the incubator tank, is made of six glass boards that stick together with silicon grease, which is waterproof and airtight. Two side boards were designed with holes for axle to pass through. The top plate is fixed with the side boards by hinges. Metal meshes are fixed on the axle. As the axle rotates, the metal mesh can wind the cellulose in the liquid medium (Figure 6).
Our team members and members from KEYSTONE_A constructed the hardware together. Two teams jointly completed the initial construction work and completed the hardware that can be basically operated (Figure 7). We came up with the idea of RDR and mainly focused on the design of electrical system. Students from KEYSTONE_A helped to do the user test and helped us troubleshooting the problems of PWM driving board. Both of our teams are looking forward to further cooperation in the next phase in 2021.
We have finished the assembly of the entire hardware and the idling test proved that it could work well more than 24 hours. The potentiometer could moderate the speed of motor well to fit the production rate. The temperature of electrical system raised to 48℃ after 24 hours of test but is proved to be controlled at this temperature.
Due to the COVID-19 pandemic, our experiments were limited and we could only finish the theory part of our project. Thus, the Rotary Disc Reactor will be put into practical usage in the second phase of our project next year. More heat dissipation methods shall be implemented in the electrical system to cool down the electrical system to reinforce the safety. Slot can be designed on the axle to stabilize the metal mesh.
1. Liu, M. et al. Enhanced bacterial cellulose production by Gluconacetobacter xylinus via expression of Vitreoscilla hemoglobin and oxygen tension regulation. Applied Microbiology and Biotechnology 102, 1155-1165, doi:10.1007/s00253-017-8680-z (2018).
2. Pae, N. Rotary discs reactor for enhanced production of microbial cellulose. Tp Chemical Technology (2009).