Introduction

With MEtaPhos, this year's project aims to use an optogenetic switch to control the binding and release of phosphate to a phosphate binding protein (PBP). Since our goal is the use of MEtaPhos in larger applications, a reliable way to illuminate the system is needed. As the illumination of biological processes in bioreactors is rarely performed, most projects use external LED-systems to illuminate the reactors. Therefore, issues such as light-absorption, -reflection and -refraction compromise light-dependent reactions. That is why we were interested in developing an illumination system which can be used inside of the reactor and thus minimizes the loss of light mentioned above. Following this idea, we focused on constructing a new illumination system for bioreactors, namely an illuminating stirrer.

Lightswirler

As we decided to use optogenetic switches in this year's project, we visited Professor Rother, Dr. Krauss and Dr. Drepper at the FZ Jülich, who are experts in the application of optogenetic switches and photosensitizers. We based our hardware project on their approaches to provide blue light to biological reactions and reaction cascades. As they told us about their illumination from outside the reaction vessel and the problems caused by that construction, we wanted to improve the illumination process with our hardware project. Therefore, we talked to Prof. Dr. Wolfgang Wiechert from the FZ Jülich and Katharina Sauer from the AVT institute at RWTH Aachen to figure out which parts of a bioreactor could be used to provide a light source. We concluded that a light module reaching into the reaction vessel could reduce the loss of light and in that way improve the illumination process. At this point, we had to ask ourselves how interferences between the light module and the stirrer, which is needed for all larger bioreactors, could be prevented. We figured that the easiest way to prevent any unwanted interferences would be to use the stirrer itself for illumination. That is how we came to the idea to build the Lightswirler.

We thought about incorporating LEDs into various materials commonly used for bioreactor stirrers, such as stainless steel or epoxy resins. But after talking to Stefan Taubert and Udo Kosfeld from the AVT institute at RWTH Aachen, we had to reconsider our choice of material. Hearing their ideas and suggestions put us onto the path of using acrylic glass as the stirrer’s basic material. Acrylic glass conducts light through its entire length when illuminated at one point. If the acrylic glass is roughened with sandpaper, for example, it diffuses the light over the entire roughened surface. Therefore, we are using a stirrer made of acrylic glass, which is roughened at the parts that reach into the reaction medium. The upper section is used for illumination and connects the engine to the stirrer. By realizing the stirrer’s illumination in this way, we also keep it easy enough that it is replicable and can be used in DIY-approaches or table-top-reactors. It also allows the implementation of different stirrer geometries. In addition, the light origin is moved out of the reaction vessel so that the LEDs no longer heat up the reaction medium and no electrical energy enters the vessel.

Figure 1: Construction of the Lightswirler

Figure 2: Construction of the illuminated Lightswirler

Figure 3: Lightswirler in the reactor

For the construction of the Lightswirler, we used a student's garage (Studiwerkstatt Aachen). We cut the acrylic glass rod in the right length, generated the rough surface, and fixed the blades. For an optimized light transfer it is important to polish the upper end of the stirrer, so there is no reflection. To combine the lens needed for focusing and the LED, we built a metal construction. This enables the user to change the LED, and thus use different wavelengths for illumination. At the same time, this guarantees the focusing of the light through the lens to the stirrer. The LEDs are cooled by cooling units stuck at the back of the LED. As a top for our little reactor, we used a metal plate. The plate is fixed to four bases and holds the engine and the construction with the LED. For that, we used screw clamps. We drilled holes through the plate to fixate the stirrer by using a flange bearing. The translation from the engine to the stirrer is realized by two gearwheels, so we could build it in a 90-degree angle. The top of the stirrer must be free for illumination.

This is how to use the Lightswirler:

Combine the right LED to the construct
Choose the right velocity for stirring
Connect the LED to the cables
Connect the Arduino to the program and power
Start the stirring and illumination

Outlook

We managed to build an illuminable stirrer for simple bioreactors with small volume. The stirring mechanism can be precisely controlled using a Arduino and can be illuminated using the provided LEDs. But there is still plenty of room for improvement. Starting with the design of the motoric section and the LED-section, the mechanical structure could be optimized. Adding to that, a control interface could be added to regulate the stirrer and the LEDs more precisely. The LEDs could also be optimized, for example by using full spectrum covering LEDs providing light in all wavelengths. These could be applied for a wide range of biological processes. Furthermore, an improved method of cooling would surely be useful. The stirrer itself could also be optimized, for example by targeting the basic materials or the connection of the stirrer's agitator and blades.

Those implementations would make the Lightswirler easier to handle and would improve its durability, especially in chemically harsh reaction media. Additionally, the performance of the entire system could be improved, which would allow opportunities for scale-up.

Downloads

Here you can download the schematics and the list of the used parts.

Parts list

Schematic of the shaft

Schematic of the stirring blades

Schematic of the frame plate

Contribution