Contribution

Figure 1. iGEM at Hopkins 2021 Frugal Bioreactor Original Model

Parts List

Original Parts List

General

• Mason Jar - $7.80
• Stirrer Motor - $17.95
• DFRobot pH probe - $65.55
• DFRobot water temperature sensor - $8.08
• Silicone tubing (large) - $13.32
• Silicone tubing (small) - $8.82
• Silicone heating pad - $8.99
• Power supply - $13.85

Photometer

• 16x2 LCD - $9.95
• I2C LCD Backpack - $9.95
• Pasteur Pipette - $0.11
• 600 nm LED - $0.12
• Stepper Motor - $16.85
• Miscellaneous Nuts/Screws - $26.69

Total: $208.03

New Parts List

General

• Mason Jar - $7.80
• Styrofoam Box - Free
• Stirrer Motor - $17.95
• DFRobot pH probe - $65.55
• DFRobot water temperature sensor - $8.08
• Silicone tubing - $8.82
• Silicone heating pad - $8.99
• Power supply - $13.85
• Sparge - $25.50
• UNF to NPT Adapter - $7.70
• Yor-Lok NPT Fitting - $16.87
• Yor-Lok to Flex Tubing Adapter (6) - $86.28
• 1/4 inch stainless steel tube - $17.84
• 1/4 inch Peristaltic Tube - $23.25

OD600 Measurement

• OPT3002 optical density sensor - $9.99

Peristaltic Pump

• Stepper Motor - $16.85
• Pump Casing (Custom printed) - $0.10

Control Electronics

• RAMPS 1.4 Board - $9.39
• Arduino Mega - $48.40
• Microcontroller board (RP2040) - $7.50

Total: $308.48

Figure 2. iGEM at Hopkins 2023 Frugal Bioreactor New Model

Figure 3. 2D Diagram Setup of the Bioreactor

Procedure

Assembly Key

1. Mason Jar
2. Styrofoam Box
3. Stirrer Motor
4. DFRobot pH Probe
5. Water Temperature Sensor
6. OPT3002 OD600 Sensor
7. Silicone Heating Pad
8. Separate Beakers for DFRobot pH Probe and Water Temperature Sensor
9. Silicone Tubing
10. Sparge
11. Magnetic Stir Bar (Optional)
12. RAMPS 1.4 Board
13. Arduino Mega
14. Microcontroller Board (RP2040)
15. Yor-Lok to Flex Tubing Adapters
16. Power Supply
17. Stepper Motor for Peristaltic Pump
18. Custom-printed Pump Casing
19. 1/4 inch Peristaltic Tube

Building Procedure

1. Clean a Mason jar and place it in a Styrofoam box for insulation.
2. Mount a Stirrer Motor on the lid of a Mason jar, ensuring it is centrally aligned.
3. Attach a Silicone heating pad to the bottom of a Mason jar, inside the Styrofoam box.
4. Prepare separate beakers for the DFRobot pH probe and water temperature sensor, attach tubes and fill them with the necessary fluid.
5. Place the DFRobot pH probe and water temperature sensor in their respective beakers.
6. Connect Silicone tubing to a Sparge and secure the tubing using the adapters.
7. Connect a power supply to the Stirrer Motor and Silicone heating pad.
8. Assemble a peristaltic pump using a Stepper Motor and the custom-printed Pump Casing. Attach a 1/4 inch Peristaltic Tube to the pump.
9. Connect a RAMPS 1.4 Board and an Arduino Mega. Attach the Microcontroller board (RP2040) for control.
10. Open the GitHub repo and run code on the Arduino IDE for sensor reading and motor control.
11. Alternatively, if an LCD display is available, run the code to output pH, temp, & OD600 to the LCD.

Testing Procedure

1. Check all connections and make sure the power is off before starting.
2. Fill the Mason jar with a sterilized growth medium appropriate for the bacteria you are cultivating.
3. Start the Stirrer Motor and observe its rotation to ensure it is stable and aligned.
4. Power on the Silicone heating pad and monitor the temperature until it stabilizes at the desired set point.
5. Submerge the DFRobot pH probe and water temperature sensor in their respective beakers filled with calibration solutions. Verify the sensor readings are accurate.
6. Run the peristaltic pump to transfer a small amount of fluid into a test container.
7. Power up the RAMPS 1.4 Board, Arduino Mega, and the RP2040 microcontroller.
8. Execute a simple control program to adjust the Stirrer Motor's speed and heating pad's temperature. Verify the changes through observations and sensor readings.
9. Run a complete cycle of the bioreactor in a controlled environment to ensure all parts function together cohesively.
10. Review the data logs to confirm that the system is recording all necessary data with reasonable output.

Results

Old Design

Figure 1 shows a prototype demonstration of the original bioreactor model as it was designed for iGEM 2021. The 2021 bioreactor model was successful in that it made many improvements to the 2018 open source bioreactor, particularly in PCB design and easy assembly. However, though successful in many aspects, the system showed several areas that called for refinement. Firstly, the model relied on an obsolete analog light sensor which was quite limited in adaptability. In the 2023 model, we replaced this with an I2C digital light sensor, which processes internal gain compensation, thus allowing for necessary calibrations and increased accuracy. Secondly, another issue of the 2021 design was lack of autoclavability due to size and material. The original model components were printed in PETG (to make it easily sterilizable in 70% EtOH) and also contained some non-autoclavable parts. With the new model, we have transitioned to a majority of stainless steel parts. These parts can easily be assembled without any sealants or tape thanks to the compression fittings, which drastically improves the model's ability to be sterilized. Another issue was with lack of proper aeration in the model, which is extremely important to promote bacterial growth and achieving the best performance possible. Our initial thought was to use an airstone typically used in hydroponic systems for plant aeration. Upon further research, we decided to integrate a stainless steel sparge into our design. The sparge promotes more efficient oxygen dissolution in the medium by increasing surface area and adds greater resistance to corrosion due to the material. Lastly, the 2021 model’s firmware was quite rigid and was not easily adapted to support various growth protocols for different organisms. In the 2023 design, we addressed this issue by integrating Marlin, an existing open-source 3D-printer software. Marlin provides a highly flexible and configurable platform that allows any GCODE controller to easily control the bioreactor. Furthermore, we recognized that many elements of the PCB in the 2021 design could be substituted with more accessible and cost-effective Commercial Off-The-Shelf (COTS) components. This was a key factor in our decision to incorporate the RAMPS-1.4 (3D-printer) control board, I2C interface boards, and the RP2040 microcontroller into our new design. This shift not only will reduce the overall cost and complexity of the system but also will enhance its user-friendliness, accessibility, and adaptability, compared to the 2021 design.

Figure 4. Setup of the RAMPS-1.4 Board and Arduino Mega

New Design

Marlin Integration

A significant limitation of the 2021 model was a lack of a user-friendly software for proficient control over bioreactor parameters such as pH and temperature. As we brainstormed ways to create an intuitive LCD display software and improve hardware, we made an interesting observation: the basic operating principles of bioreactors closely resemble those of 3D printers. For instance, a 3D printer utilizes heated extruders/beds to maintain ideal printing conditions, similar to how bioreactors use heat to effectively cultivate media. Similarly, a 3D printer's extruder depositing plastic can be compared to our bioreactor's method of dispensing media. This led us to conclude that it would be easier to enhance and adapt an established 3D printer software Marlin [1] (Github) to change bioreactor parameters (mirroring how a 3D printer can change bed/extruder temperatures via LCD interface) rather than develop from scratch. Building on this approach, we integrated the use of the RAMPS 1.4 board, a widely used open-source 3D printer controller, and the Arduino Mega microcontroller board. The RAMPS 1.4 and Arduino Mega provide a robust platform to easily control and adjust the bioreactor's parameters; the setup can be seen in Figure 5. Building on the integration of the RAMPS 1.4 board and Arduino Mega, the importance of precision temperature measurements was recognized within the bioreactor. Accurate temperature readings are pivotal in ensuring optimal growth conditions for bacteria. To address this, we incorporated the DallasTemperature Library [2] into our adapted Marlin software. The DallasTemperature Library is designed to work seamlessly with the temperature sensors, providing reliable and precise temperature readings. By integrating this library with Marlin, we could easily monitor and control the internal temperature of the bioreactor, all while maintaining the user-friendly interface provided by Marlin. This allowed us to achieve a synergy between the established capabilities of 3D printing software and the specific needs of our bioreactor, ensuring efficient and effective control over critical growth parameters. The Marlin code edits can be seen in reference [3] on Github.

Figure 5. Marlin integrated with DallasTemperature reading Water Temperature

OD600 Improved Integration

Optical density at 600nm (OD600) is a critical parameter for monitoring bacterial growth within the bioreactor, as it gives an indication of the bacterial cell concentration in the culture medium. In the context of Caddisfly, OD600 is crucial as it guides the optimization of silk protein production conditions, as the bacteria's growth phase significantly impacts silk protein synthesis. By closely monitoring this parameter, we can ensure that bacteria are harvested during their most productive phase, which will maximize our caddisfly silk protein yield. In our 2021 design, a photometer was used, which included LCDs, LEDs, and a pipette for taking samples. However, this system required manual operation and constant monitoring. In the 2023 update, we replaced the photometer with an OPT3002 optical density sensor, which can transmit OD600 data directly to the RP2040 microcontroller in real-time. The microcontroller board interprets this data and sends necessary GCODE commands to the RAMPS 1.4 stepper motor control board. The board then adjusts the bioreactor's conditions accordingly, allowing the bioreactor to autonomously respond to changes in bacterial density, or through the Marlin interface. Overall, this has automated the monitoring process and reduced the need for continuous manual oversight. This not only enhances the accuracy and consistency of the readings, but it significantly advances the efficiency and reliability of our bioreactor design.

Figure 6. OPT3002 Optical Density Sensor Photo

CAD Improvements

Figure 7. 3D Printed Housing of an LCD Display for ease of monitoring

Figure 8. 3D Lid of Bioreactor with flexible, modular openings for outlets

Figure 9. 3D Printed stepper motor housing with intake and outlet holes for peristaltic pumping of buffer

Contributions to Future Teams

The advancements undertaken in this project lay the groundwork for future teams looking to further innovation and efficiency in frugal biomedical equipment. By addressing past limitations and introducing new solutions, we have showcased the importance of iterative design and the value of building upon established knowledge. Our emphasis on adaptability, user-friendliness, and sterility provides a clear direction for those wishing to prioritize essential functional attributes. By harnessing existing software solutions like Marlin and novelly integrating different sensor types into our design, we underscored the potential of repurposing and adapting existing platforms for novel challenges and building upon what others have started. This approach not only streamlines the development process but also broadens accessibility for users, hopefully allowing for more wider adoption of this type of technology in the future.

Collaboration

As part of our efforts to improve bioreactor design and brainstorm for new ideas, iGEM at Hopkins partnered with the Open Bioeconomy Lab at the University of Cambridge. This collaboration provided a unique opportunity for us to join a global community of researchers and engineers focused on their own bioreactor development and the common goal of an accessible yet frugal bioreactor. During our virtual meetings, we engaged in detailed discussions about design considerations, control systems, and sensor technologies with professionals and students from the UK, the Philippines, Argentina, and all around the world. These interactions enriched our project by exposing us to different approaches and solutions to common problems that we were facing. One significant outcome was a new 3D-printed lid design for our bioreactor on the mason jar, which was partially developed through collaborative brainstorming with the group.

References

[1] Marlin Firmware. (n.d.). Marlin Firmware Repository. GitHub. Retrieved from https://github.com/MarlinFirmware/Marlin
[2] DallasTemperature Library. (n.d.). Arduino Temperature Control Library. GitHub. Retrieved from https://github.com/milesburton/Arduino-Temperature-Control-Library
[3] Marlin Code Edits. (n.d.) Marlin Fork. Github. Retrieved from https://github.com/elrobitaille/Marlin