A problem to solve: Small Scale BioReactor Cost

Sometimes Research and Development Labs can develop excellent heterologous biosynthetic pathways at lab scale (microtiter plates, petri dishes and Erlenmeyer in shakers), but those systems are not capable of monitoring all parameters online, nor simulate all conditions of a bigger scale reactors [1].

To simulate real production of the engineered cell and optimize throughput, most labs test those cells on Bench-top bioreactors capable of measuring online data, simulate environment conditions, e.g., fed batch or pH control. Sometimes, those labs do not have enough resources to acquire these systems.

HARDWARE: Open-Source Modular Bioreactor

We created a low-cost bioreactor with the goal of using it to test the production of our genetically modified Lactobacillus acidophilus on a larger scale. We wanted to define the best parameters for Lactobacillus growth because we would need to increase its production efficiency to achieve lower and affordable prices for those who suffer most with helminths to implement our biodrug solution. However, we faced some problems. The first was the lack of time, because it took 1 month for our gene synthesis to arrive in Brazil and we were unable to receive our iGEM Kits due to bureaucratic problems with government agencies. The second was the geographical distance, because our team member Vitor, who assembled the bioreactor, works 600km away from our headquarters laboratories. Therefore, we first tested the bioreactor with other microorganisms, using partner laboratories in the university where he works to test and validate its functionality and flexibility. We plan to test the bioreactor with L. acidophilus in the future.

The result was: we developed an open-source Bioreactor, capable of working as Batch, Fed-Batch, PhotoBioReactor and more. The system is modular, and the user can add only the specific needed items, optimizing resources.

The building materials were carefully selected by the cost and by how easy it is to acquire it.

Below, there is a demonstration of the reactor with a Saccharomyces cerevisiae fermentation of molasses:

It can also work as Photobioreactor, as shown below:

The user interface allows the addition of sensors, the reading of online real time data and the activation and deactivation of actuators, everything through a web interface. There is a print of the interface below:

Parts

• Stainless steel straws;
• GLS 80 glass 1L or 500 mL as vessel (alternatively, a mason jar can be used);
• 3D printed parts;
• Polymerizable silicon rubber;
• Raspberry Pi 3 or better;
• Prototyping Breakout PCB Shield Hat for Raspberry Pi;
• A pH kit for Arduino + ADS1115 ADC + logic level shifter. Alternatively, a pH kit for Raspberry can be used directly;
• APDS 9960 Color Sensor;
• LED white light strips for color sensing and for photobioreactor configuration;
• DS18B20 temperature sensor;
• 12V power source;
• 12V to 5V Voltage converter;
• 400W DC Mosfet module;
• 2 m Silicone Tube 2 mm ID x 4 mm OD or aquarium silicone tubing;
• Peristaltic pump.

Raspberry Pi

400W DC Mosfet module

Your laboratory will need to have:

To assemble the bioreactor and to cultivate microorganisms in it, your lab will need:

• An autoclave;
• A laminar flow cabinet;
• A magnetic stirrer;
• 3D printer;

Cost

The average price of a used benchtop bioreactor is about $10.000 while a new one is around $40.000. On the other hand, a photobioreactor can cost up to $150.000. The table below show the cost of the parts in our developed bioreactor. It does not include any consumable parts you may need, eg, sterile filters.

Tests and Results

Unfortunately, due to lack of time and geographical distance -Vitor Marchesan, who assembled the Hardware, lives in a different city from where ProChi WetLabs are located, we are 600km apart-, we were unable to test the bioreactor with our genetically modified Lactobacillus acidophilus. However, we were able to test the bioreactor functionalities with another microorganisms, obtained from partner laboratory in his University.

Experiments were made in triplicate, using the microalgae Chlamydomonas reinhardtii, since it is a model organism. Those tests were performed in a room with always on air-conditioning set to 25°C. The strain used was a wildtype one CC1690 and the reactor configuration was a single batch PBR with 500mL working volume. Strain was grown in Erlenmeyer flasks containing 100 mL of TAP media [2] on an orbital shaker at 110 RPM, and under constant light (100 μmol photons.m^-2.s^-1) for 3 days at room temperature, in which an inoculation of 5mL of this culture was added to the reactor containing 500mL of TAP. A sample was daily taken from days zero to five, and an extra sample was taken on day 7 to verify reactor stability. Results are shown below:

Bioreactor Manual of Instructions

We created a Manual with instructions to help others to assemble our Low-cost bioreactor. We wrote this document because we want to democratize science and access to laboratory equipment. As we showed in this page, bioreactors in the market have exorbitant prices that do not match the economic reality of most laboratories. Click here to access it.

We also provided the archives needed to print the parts using a 3D printer. Click here to access it.

References

[1] Formenti, L. R.; et al. Challenges in industrial fermentation technology research. Biotechnology Journal, v. 9, n. 6, p. 727–738, jun. 2014.

[2] Gorman, D.S.; Levine, R.P. Cytochrome f and plastocyanin: their sequence in the photosynthetic electron transport chain of Chlamydomonas reinhardi. Proceedings of the National Academy of Sciences of the United States of America, 54, 6, p. 1665-1669, 1965). Available on: https://doi.org/10.1073/pnas.54.6.1665.

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