HARDWARE

Introduction

After discussing our project’s goals and future opportunities with a microbiologist he insisted that we test our system in a bioreactor. One thing is our system working in a flask but it's not given that the same system would work on a bigger scale. First step of proving our validity in a high volume system is to test the process in a lab-grade bioreactor. These tests would provide data on how to scale up our systems and to prove that our glue could be mass produced.

Implementation

A laboratory-scale bioreactor is costly, leading us to consider constructing one ourselves, with the intention that our plans could be beneficial to other teams. Initially, we outlined our objectives, specifying the desired functionalities of our reactor. We aimed for full automation to minimize human error, which could compromise data integrity. Bioreactors typically incorporate various sensors and components, so certain parts were identified as essential for the goal:

Temperature sensor: used for monitoring the temperature of the bioreactor, ensuring that the temperature is kept at the optimal level for the bacteria to grow.
Heating element: used to heat the bioreactor when the temperature is too low.
pH sensor: used for monitoring the pH of the bioreactor, allowing us to adjust pH levels if necessary.
Mixing impeller: used to continuously stir the contents of the vessel, ensuring homogenous mixing and therefore providing the cells with substantial oxygenation of the medium.
Turbidity sensor: used instead of Optical Density (OD) sensor to measure the growth of the bacteria.

A diagram of the main components of the bioreactor can be seen below (Fig. 1):

**Fig. 1.** Main bioreactor components diagram

For the container a glass jar with an aluminum lid was used, an aluminum lid was chosen because it is easy to modify and doesn’t require complex tools. All of the parts and sensors were fixed to the lid, this design choice was made in order to allow sterilization. The bioreactor must be sterilized after each use; however, the sensors and electronic components cannot be autoclaved. The sterilization process comprises two steps: first, the lid and base are separated, after which the base is placed into the autoclave. Lid components were sealed and waterproofed using a silicone based sealant. This allowed us to submerge the lid with all the components into 70% isopropanol solution, which would effectively sterilize it. Holes were drilled in the lid according to precise measurements for each component that needed to be inserted into the reactor. Each part was enclosed, sealing the bioreactor from the outside and reducing the risk of contamination. All the electronic components were fixed in a box separate from the bioreactor.

The full list of components required for building the bioreactor, including their prices, is shown in Table 1:

Product Name	Price (EUR)
Arduino UNO R4 WiFi	45.80
Gravity: Screw Shield V2 for Arduino	13.30
Arduino wires female-to-female 20 units	3.80
1.5l glass jar	4.00
Gravity: Analog Industrial pH Sensor	86.30
MAX6675 Temperature Sensor with Thermocouple Cable	13.10
Ceramic Heater for Reprap 3D Printer 12V 40W	3.90
1-channel relay module - R1 - 5V - 10A/250V - for Arduino	2.90
Power supply GPX ZTD-1250 12V/5A - DC	9.57
Engine 25Dx56L 227:1 12V 33RPM replacement Pololu 3233	17.10
L298 H-Bridge Dual Motor Driver	6.00
Mixer with plastic propeller blade	7.26
Gravity: Analog Turbidity Sensor for Arduino	16.60
Jaw Spider Shaft Coupling for Stepper Motor 5x8mm	5.50
10mm Kapton Tape	4.70
Sanitary silicone sealant MAKROFLEX SA102, colorless, 300ml	4.79
Aerosol paint Maston Special, waterproof, black, 0.5l	10.61
Wire heat shrink tubing	0.97
Total	256.20

Table 1. Bioreactor components and prices

The bioreactor in action is shown in the picture below (Fig. 2.). The electronics of the bioreactor is placed inside the box on the left, protecting it from any external damage, also making the setup more visually appealing.

Measuring bacterial growth

Our first idea was to use an OD meter to determine the optical density of our medium. But that plan fell through when we saw the prices of OD meters and the fact that OD meters are not directly compatible with the Arduino interface. Our bioreactor is designed with accessibility in mind: we aimed to create a foundation for other teams by providing simple instructions for building an affordable, lab-grade bioreactor. For this reason, we opted not to use OD meters and after scouring the web for alternatives, we found turbidity sensors. Turbidity is a measurement of how much sediment is in a liquid. This technology is not often used in biology as a way to determine bacterial growth. Finding a workaround was not easy but we came up with an idea of making a calibration curve [1]. Measuring turbidity and OD at the same time increments gave us enough data to make a calibration curve. Plugging in the turbidity value into a simple formula (y = mx + k) gave us the corresponding OD. This process was automated to deter human error (more about that in Software wiki page).

Electronics

For the "brain" of the bioreactor, we decided to use the Arduino Uno R4 WiFi. It is a versatile, open-source microcontroller with built-in Wi-Fi capabilities, making it ideal for remote monitoring and control (more about that in Software wiki page). The Arduino platform is highly favored in prototyping because of its simplicity, flexibility, and large community support. Additionally, the Wi-Fi functionality allows seamless integration with cloud-based services and data logging systems, which are crucial for real-time data monitoring. One of its most important features is the ability to interface with various sensors, making it highly adaptable to the bioreactor’s requirements, such as temperature, pH, and turbidity sensors. Furthermore, its cost-effectiveness and ease of use make it an attractive choice for both academic and industrial applications.

All the system's electronic components, connected to the Arduino, are displayed in the schematic (Fig. 3). The turbidity and pH sensors are connected to Arduino's analog pins, while the temperature sensor, DC motor control module and heater's relay are connected to the digital pins. The DC motor and heater is powered by 12V external power supply, while other devices - by 5V power supply provided by the Arduino microcontroller.

**Fig. 3.** Bioreactor electronics schematic

A more detailed instruction on how to build the bioreactor and connect the electronics together can be found the document provided below (Fig. 4):

Fig. 4. Bioreactor DIY instructions

Engineering

Design

In an effort to automate and optimize part of our wet lab workflow, we designed and built a laboratory-scale bioreactor from scratch. The bioreactor includes essential components for monitoring and maintaining bacterial growth, such as sensors for temperature, pH, and turbidity, as well as an impeller and a heater. In addition to that, we also developed a remote interface for the bioreactor to make bacteria monitoring less time consuming.

Build

We built everything by hand using widely available supplies, so it would be easily recreatable. Used a 0,5 liter jar with a metal lid. Every component that had to be inserted into the bioreactor was measured and according to the measurements holes were made in the lid.

Test

Firstly we tested our reactor with water. Our goal was to test each sensor's functionality and if our software was working correctly. The test came in handy and we realized that our reactor had problems keeping the temperature consistent. We came to a conclusion that our heat sensor and heater were too close and the heater interfered with the sensor's readings. Also the heating element was poorly insulated, which caused it to leak electricity.

Learn

Each component on its own operated as expected
Our software also worked as intended
The heater and temperature sensor need to be separated for more accurate measurements
Also we suspect the heating element may be leaking electricity, based on irregular readings from the temperature sensor

Design

The main design stayed consistent, we solely changed the position of the heating element. And insulated the heating element.

Build

We took our lid components out and made a new hole. Heating sensor was not mowed but the heater now occupied the freshly made hole. Previous and now unoccupied hole was sealed using a silicone based sealant.

Test

For our second test we used water again, the only difference being the time span it was left in the reactor. This was our stress test so the water spent a total 24h in the reactor with all the hardware beneath the water's surface. We chose a longer time span because our systems produce ring formations after 24h. The test provided crucial information. Firstly we learned that our turbidity sensor had a small leak in it and over time it accumulated moisture inside the sensor. Water damaged the sensor beyond repair, so we had to buy a new one. Secondly we observed that our temperature sensor had a screw that was not stainless steel so it rusted immediately. And lastly the impeller rod was cut and grinded down so it fit the connector to the motor. The grinded down part was also corroded because we had removed the protective layer.

Learn

Our turbidity sensor needs to be waterproofed
Screw on the heat sensor need to be removed or insulated
Also the impeller rod needs to be covered with a protective layer so that it won't rust
Fixed the problems with temperature readings

Design

Overall design stayed unaltered but separate components were tweaked.

Build

We used a silicon based sealant for our new turbidity sensor and waterproofed it. Screw on our temperature sensor was the least of our problems, we just removed it. And the final problem of our previous design was our rusty impeller rod. We used a rubber based coating spray, after multiple coats the rod had no contact with water and was successfully sealed.

Test

Third test evaluated our sterilization method. It was run with 600 milliliters of LB medium without antibiotics and the conditions (37 celsius, X rpm’s and neutral pH) that we plan to grow our bacteria in. The bioreactor was left working for 3 hours straight then we turned it off and left it overnight at room temperature. This test came out positive because the LB medium did not get contaminated.

**Fig. 5.** Bioreactor temperature sensor

But a problem occurred. We observed that our bioreactors sensors were too high for the volume of LB we used and some of the sensors were right at the liquids surface.

Learn

Need to lower our sensors for the bioreactor to be adaptable for a wider range of usable volume
Our sterilization method has been proven effective

Design

Based on insights gained from the previous version, we identified that the components were positioned too far from the center of the lid. To address this, a new lid was acquired, and the components were repositioned closer to the center, away from the edges. Additionally, the height was adjusted to optimize the bioreactor for lower culture volumes.

Build

All the components were removed from the old lid and transferred to a new one. On the new lid the hole positions were remeasured to accommodate the space better. After the adjustments were made we resealed everything for it to be isolated.

Test

The fourth test involved bacterial cultures, during which we attempted protein expression. The purpose of this test was to confirm that the bacterial growth rate in our bioreactor matched the growth rate typically observed in a flask. Due to a delay in the arrival of the turbidity sensor, growth measurements were taken manually using a spectrophotometer. Additionally, the IPTG solution was manually added once the bacteria reached an optical density (OD) of 0.5.

Learn

Determined that the pH meter is not properly calibrated
Additionally, a software malfunction causes the impeller to shut down after three hours of operation.
This test successfully validated the effectiveness of our sterilization protocol.

Validation

To prove our bioreactors functionality we performed experiments with E. coli that has our full CB2 system of plasmids (read more about that in Design). During the first experiment we still did not have our turbidity sensor, so the optical density (OD) was measured manually with a spectrophotometer. The test was done using 700 ml of LB medium and 10 ml of overnight bacteria which had the OD of 6.47.