The 5-L fermenters in laboratories and large fermenters used in industrial production are not only large in volume, but also costly to install, expensive to operate, and inconvenient to use. Therefore, they are not suitable for high school students to learn fermentation experiments. Designing a small, low-cost, easy-to-operate fermenter for high school students to use in fermentation experiments is of great significance. This design not only allows us to learn basic knowledge related to fermenters and conduct fermentation experiments but also helps develop our hands-on design skills.

- Introduction of our hardware -

·The designed small fermenter should have the basic functions of a 5-L fermenter. In operating this fermenter, a more convenient temperature-controlled magnetic stirrer can be used to simulate the temperature control and stirring functions of a 5-L fermenter. A small air pump, in conjunction with a circulation timer switch, can be used to control aeration. Acid and alkali flow controllers can be used to adjust and control the pH, and a circulation controller can be employed to control the glucose feeding rate.

The tank selected is a 2L glass bottle with three openings. The middle opening is connected to a pH electrode, while the lid on the left side has three holes: one connected to an air inlet, one for adjusting the pH with acid or alkali, and the other for a feed port. Below the tank is a temperature-controlled magnetic stirrer, which allows the temperature to be set independently. The magnetic stirrer works in conjunction with the stirrer rotor inside the tank to achieve liquid agitation. On the right side of the tank is the control device, which controls the timing of air input, acid/alkali input, and glucose addition through a cyclic timer switch. By adding polytetrafluoroethylene (PTFE) tubes to the controller, one side is connected to the glass tank, and the other side is connected to the bottles of acid, alkali, or glucose. This ensures that acid, alkali, and glucose can be added to the tank under sterile conditions. During the fermentation process, the pH value is monitored in real-time on the display. By adjusting the speed of the acid and alkali pumps and setting the timer, the pH can be controlled. In the entire fermentation device, the control unit plays an important role in regulation.
Below are the design of the controler:

· User Manual

At the same time, we wrote a user manual to help iGEMer to use this bioreactor easier in the future. This PDF document provides a comprehensive guide on the installation and operation of a handcrafted fermenter. It covers various aspects, including the setup and usage of different components such as the pH and dissolved oxygen electrodes, air pump, peristaltic pumps for adding acid, alkaline, and antifoam agents, and controllers for mechanical stirring, temperature, and air circulation. The manual also includes safety precautions, step-by-step instructions for installing PTFE tubes, stainless steel lids, and the fermenter's body, as well as guidelines for configuring various controllers and emergency stop mechanisms. Additionally, it offers details on maintenance, calibration, and troubleshooting to ensure optimal performance and safe operation.

· Cost structure:

Additionally, we calculated the total cost of developing this fermenter system. The expenses for all the materials amounted to 3,003 RMB, which is less than 400 USD. This budget enabled us to successfully construct a fermenter setup suitable for use in a garage laboratory environment.

- Fermentation Results -

We used this bioreator to conduct a fermentation using strain DT-D9. After 26 h fermantation, the final tagatose concentration reached 1083.01 mg/L after 26h fermentation. Which proved it is a very useful and powerful tool for E.coli fermentation.

- Bioreactor Opitimization -

In the actual operation of our fermentation experiment, we found that there is room for improvement and optimization of this bioreactor device. Through further optimization and adjustments, we believe that our fermentation device can fully utilize its advantages for microbial fermentation experiments conducted by high school teams.

1. Temperature control

During the actual operation, we found that the temperature control of the magnetic stirrer was not ideal. The temperature of the microorganisms inside the tank did not reach the level displayed on the control monitor because it only made contact with the bottom of the tank. Therefore, in future optimizations, we aim to improve the accuracy of temperature control by placing the tank in a thermostatic water bath with magnetic stirring. The constant temperature control of the water bath is highly stable, and the water level can reach about the middle of the tank, providing a much more effective temperature regulation.

2. Waterproofness

There is no protective shield placed on the right-side display of the controller, which may result in water entering and damaging the display when it comes into contact with water. In future optimizations, we plan to enhance the hardware’s waterproofing by using 3D printing technology to create a protective cover for the display screen. This will ensure that the screen remains viewable while providing protection.

3. Air supply

In the later stages of the fermentation experiment, we observed that as the biomass increased, the air supplied through the controller's air inlet could no longer meet the oxygen demand of the dense microbial culture, and the bubbles introduced were uneven. In the future, we intend to improve the uniformity of the bubbles and increase the air supply to meet the oxygen needs of the dense microbial population.

4. Automated pH control

Currently, we use a pH detector to monitor the pH levels inside the bioreactor and manually control the pump to add acid or alkali as needed. In the future, we plan to automate this process using an Arduino. We have developed a PID control program that adjusts two digital output ports based on signals from a pH electrode. Output Port 1 will regulate the acid pump, while Output Port 2 will manage the base pump.

· Hardware Connections

- pH electrode signal connected to the Arduino analog input pin A0.
- Acid pump connected to digital output pin D3.
- Base pump connected to digital output pin D4.

· Program Code


                                #include 

                                    // Pin definitions
                                    const int pHInputPin = A0;   // pH electrode signal input pin
                                    const int acidPumpPin = 3;   // Acid pump output pin
                                    const int basePumpPin = 4;   // Base pump output pin
                                    
                                    // PID related variables
                                    double Setpoint, Input, Output;
                                    double Kp = 2.0, Ki = 5.0, Kd = 1.0;  // PID parameters
                                    PID myPID(&Input, &Output, &Setpoint, Kp, Ki, Kd, DIRECT);
                                    
                                    void setup() {
                                      // Initialize serial communication
                                      Serial.begin(9600);
                                      
                                      // Set input and output pins
                                      pinMode(pHInputPin, INPUT);
                                      pinMode(acidPumpPin, OUTPUT);
                                      pinMode(basePumpPin, OUTPUT);
                                    
                                      // Initial setpoint for pH value
                                      Setpoint = 7.0;
                                    
                                      // Initialize PID control
                                      myPID.SetMode(AUTOMATIC);
                                    }
                                    
                                    void loop() {
                                      // Read the pH electrode signal and convert it to the actual pH value
                                      int sensorValue = analogRead(pHInputPin);
                                      Input = map(sensorValue, 0, 1023, 0, 14);  // Assuming a pH range of 0 to 14
                                      
                                      // Compute PID output
                                      myPID.Compute();
                                      
                                      // Control acid pump and base pump based on PID output
                                      if (Output > 0) {
                                        // pH is too high, activate the acid pump
                                        digitalWrite(acidPumpPin, HIGH);
                                        digitalWrite(basePumpPin, LOW);
                                      } else if (Output < 0) {
                                        // pH is too low, activate the base pump
                                        digitalWrite(acidPumpPin, LOW);
                                        digitalWrite(basePumpPin, HIGH);
                                      } else {
                                        // pH is near the setpoint, turn off all pumps
                                        digitalWrite(acidPumpPin, LOW);
                                        digitalWrite(basePumpPin, LOW);
                                      }
                                    
                                      // Print debug information
                                      Serial.print("pH Value: ");
                                      Serial.print(Input);
                                      Serial.print(" | Output: ");
                                      Serial.println(Output);
                                    
                                      // Delay for a while to avoid rapid operation
                                      delay(1000);
                                    }

· Code Explanation

pH Electrode Signal Reading: The pH electrode signal is read from the A0 port and converted to the actual pH value using the map function.
PID Control: The PID_v1.h library is used to implement PID control. It calculates a control output value (Output) based on the setpoint pH value (Setpoint) and the current measured pH value (Input).
Pump Control Logic:
- If Output is positive, the pH value is above the setpoint, so the acid pump is activated (and the base pump is turned off).
- If Output is negative, the pH value is below the setpoint, so the base pump is activated (and the acid pump is turned off).
- If Output is zero, the pH value is close to the setpoint, and both pumps are turned off.
Debug Information Output: The current pH value and PID output value are printed to the serial monitor for debugging purposes.

You can adjust the map function in the code based on the actual characteristics of your pH electrode signal to ensure accurate pH value calculations. If needed, you can fine-tune the PID parameters by adjusting the Kp, Ki, and Kd values to optimize the control performance.

- Conclution -

We successfully constructed and implemented a small-scale bioreactor designed for educational purposes, particularly suitable for high school students learning fermentation experiments. Our bioreactor mimics the essential functions of a standard 5-liter fermenter but at a reduced cost and size, making it accessible and easy to use in smaller laboratory settings, such as a garage lab.
This bioreactor has been applied effectively in a fermentation experiment using E. coli strain DT-D9, achieving a final tagatose concentration of 1083.01 mg/L after 26 hours, demonstrating its potential as a powerful tool for microbial fermentation. Meanwhile, based on our operational experience, we have identified several areas for optimization, including temperature control, waterproofing, air supply and automated pH control. These proposed optimizations will further enhance the efficiency and functionality of the bioreactor, making it an even more valuable tool for future iGEM teams.

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Menu

⚪ Background

⚪ Introduction of our hardware

⚪ Fermentation Results

⚪ Bioreactor Opitimization

⚪ Conclusion