Overview
From an engineer's eyes, "what is a bioreactor?" We believe it's just a condition that you create for microorganisms which may be sometimes paradise and at other times hell.
So what do we need to make it? Well, the answer is a lot of electronics and some real good brainstorming sessions. So we planned and realised our very own bioreactor which is aerating, pushing and pulling out fluids, stirring, heating and also checking the concentration of cells. The bioreactor system is assembled from multiple devices that were optimized independently. The individual components can potentially be used to construct other setups as well.
Key Achievements
- design and characterization of DIY peristaltic pumps with a dynamic range of 0.5-1400 ml/h
- development of a semi-continuous OD measurement unit
- calibrated magnetic stirring from 100-1400 rpm
- integrated circuit for controlling three pumps and stirring
- designed DIY constructions to hold circuit, pumps and bioreactor setup
- parallelization of multiple reactor control units
- characterization of DIY chemostat bioreactors
Pumps
We built upon a design from thingyverse [1] that uses a 4-wired NEMA 17 stepper motor with 1.8 °/step to construct a peristaltic pump. The original design however did not fit our requirement of sterile assembly, because the tubing was pushed through a hole in a 3D-printed structure. Using Autodesk Inventor, we designed a different geometry to accomodate for rapid prototyping and sterile assembly.
Principle
For our bioreactor we need a peristaltic pump because it is necessary to autoclave the tubes. This can be acheived if the pumps can be assembles and disassembled quickly. Thus we have build pumps with different layers and a 3D structure that can be partly disassembled so as to push the tube into it. The tube gets pressed between the 3 D structure and the walls thus generating the required amount of pressure used to push liquid from one side to another.
A detailed construction manual is available at pumps.
Stirrer
- We use a DC motor and a 3D printed structure holding 4 magnets as our stirrer. The Voltage to the circuit is controlled using Analog output values thus controlling the speed of the stirrer. The magnets are placed in such a way so that half of the structure is north and the other half is south. This increases the area of magnetism thus holding the stirrer firmly into its magnetic pull.
Contruction Manual can be found on stirrer.
Continous OD Device
How can we find out if there is really bacteria growing in our reactor? The common way is doing OD measurements. But if there is only 10 ml of reactor volume, every measurement would disturb the conditions drastically and thereby influence the experiment itself.
It is most important that the samples are taken sterile. Furthermore the OD should be measured automatically so that the experiment can run without the need of a person taking samples. Finally the device to measure to OD has to be cheap and easy to assemble while having a precision to compete with commercial spectrometers.
Measurement Principle
Light with a wavelength of 605 nm is emitted by a LED and shines through the medium. On the other side we have a phototransister to measure the intensity of the light reaching through the sample, therefore we use a TSL 235 R, a special transitor converting light to frequency.
We have developed on iGEM Aachen 2014 OD device measurement principle to get live data, and built using optimum materials, along with use of calibration file to get as precise value as possible.
To keep the whole measurement sterile, the medium with the bacteria flows through silicon tubes and a transparent glass structure through the device without getting in contact with the surrounding air, while the device itself is mounted around this tube.
A detailed construction manual is available at biomass sensor.
To test if our continuous OD device can also be used to monitor batch fermentations, we set up a simple batch culture. We used a preculture of #RP1O# and M9 medium with 40 mM glucose that was grown in a stirred flask. The continuous OD sensor was attached right above the flask and a pump was used to cycle culture from the vessel through the sensor and back into the vessel. This setup was far away from perfect from a bioprocess perspective, but the goal of this experiment was testing the OD sensor and not the generation of biologically relevant samples.
Assembly
The assembly of the bioreactor electronics was the biggest challenge that we faced. Each device had a pin protocol which was maintained and tagged to the controling firmware. The assembled final firmware had to be checked and rechecked to compromise between each of the seperated devices. Finally we came up with idea of not using delays and using counters in our firmware to make it look easy and compatible to all components.
Outlook
Since in the current version of our project all digital ports of the Arduino Uno are in use, for future designs it will be necessary to change to a bigger controller, for example an Arduino Mega.
Then it will be possible to include further analysis units. We allready took some steps towards an offgas analysis with CO2 and O2 sensors, and CHx analysis would be interesting for many experiments. An integration of these components would be very easyEven more we cooperated with a local community lab, the Technik Garage, who developed a pumping and heating system for the water bath for us. To include this in the whole
References
- ↑ http://www.thingiverse.com/thing:642192