The goal of our project is to develop new bioreactors that are easier and lower cost to operate. Bioreactors are used by the biotechnology industry to grow cells. Typically, these cells have been genetically modified to produce a chemical, material, food, or therapeutic. During many conversations with biotech industry researchers, we kept hearing the same problem: that operating bioreactors are time consuming and expensive. The end goal of our project is to make bioreactor experiments considerably more automated.
For our affiliate residency at Autodesk Pier 9, we have built a web controlled, 3D printed bioreactor. In the future, we envision creating an tabletop array of these bioreactors that are operated by a robotic gantry.
The present prototype contains a number of parts that work in concert:
- A polycarbonate vessel composed of two 3D printed parts and a polycarbonate tube
- pH, temperature, and DO probes that analyze the cells growing in the bioreactor.
- A motor that spins an impeller, agitating the growing cell culture.
- A heating & cooling system that can heat the cells up to a specific temperature and cool the cells when they produce heat.
- A quad-syringe pump feeding system that pumps nutrients and energy sources (sugar) into the vessel.
- An an air pump and a control system that sparges (aka, bubbles) air into the vessel.
- A raspberry pi computer than reads the sensors and controls the feedback loops.
The design and creation of some of these components will be covered in individual instructables.
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Step 1: Why Build Bioreactors?
Bioreactors are used by biotechnology companies to make all sorts of incredible materials.
For example, companies here in the bay area have designed microbes to produce:
- New materials such as Spider Silk (no spiders required!)
- Hen-free egg white proteins
- Cow-free milk proteins
- Gelatin without horse hooves
- Speciality chemicals
- Enzymes used in detergent
Bioreactors are used for the actual production of these materials and chemicals in a process that is typically called fermentation. Bioreactors used in large production scale fermentation can be as large as 1,000,000 liters big - the size of a multistory building! During development, companies approximate the environment of these large scale reactors using bench top bioreactors that are about 1/2 to 2L in volume.
Step 2: Problem: Operation of Traditional Bioreactors Is Very Expensive
However, the operation of traditional, bench-scale bioreactors tends to be highly manual and expensive. We've heard repeatedly from researchers that they would like to run many more experiments bioreactors, if they could.
Step 3: Solution: Fermentation Cloud Service
We think we can enable researchers to run many more bioreactor experiments by creating an automated facility operated by users on the web. Just like a computer engineer can spin up thousands of cloud-based computing cores to run their code, we think a synthetic biologist should be able to quickly and easily test their strains.
We think it's possible to design a system like the diagram above, where a synthetic biologist sends their custom microbes (strains) to our facility. From there a robot begins processing and nurturing their yeast or bacteria, getting the cells ready for larger-scale work. Meanwhile, as the vials of cells are being sent to our "fermentation farm," a scientist will design their experiment via our web interface. Using custom logic and their understanding of fermentation principles, scientists can pick parameters for things like stirring speed, pH setpoints and oxygenation levels. Chemicals can also be programmed in to be added at critical points in the process -- for instance, some microbes have, in their DNA, a programmable "switch" that, when flipped, activates the production of a target protein. These "inducing agents" need to be added at the right time so the cells are sufficiently viable to begin their work.
Once the experimental protocol is designed and the strains have arrived, the robotic facility begins its work. A number of bioreactors will be reserved for the experiment and the target strain will be added to the system. The control logic designed by the fermentation scientist will take effect, guiding the bioreactors' actuators through their work.
As the system is working, our auto-sampler will be doing it's own independent thing! Once every few hours, a small volume of liquid will be pulled out of each bioreactor and analyzed by other instruments in greater detail. This can show a lot of things -- the amount of sugar remaining in the system, the amount of protein produced, the number of viable cells..the list goes on! The auto-sampler will be an important part of the system. Some of these samples will be mailed back to the scientists for further analysis in their labs.
To learn more about the synthetic biology processes described above, check out these excellent MOOCs:
Step 4: Design Process
Our design process started by visiting companies in the bay are that use bioreactors. We observed fermentation engineers and technicians use the equipment to understand their technical requirements.
Based upon this, we started with a basic block diagram of components and subsystems required:
- A vessel between 1/4 to 1L in size
- Heating and cooling capabilities
- Agitation between 0 - 1000 RPM
- Online DO, pH, and temperature sensors
- Air sparging between 0 - 1 vessel volumes per minute (i.e. 1 vvm = 1 L / min, in a 1L vessel)
- Multiple feed lines for sugar and acid/base addition.
- Auto-sampling the vessel by a liquid handling systems.
From this list of system requirements, we then moved to designing the hardware in CAD, the electronics that controlled the pumps and read sensors, and developed the web application that allowed users to design processes and send them to the bioreactor.
A simple CAD render is shown in the attach photo.
Step 5: Components of Our Prototype
Our prototype has many builtin sensors and actuators. All of these work together via custom software to create a controlled environment for growing cells. Let's go from left to right in the picture above!
The vessel assembly is on the far left. It consists of a 250mL volume reaction vessel. It's 3D printed polycarbonate, made on a Fortus 450mc and has builtin air channels to support oxygenation. A lot of careful design went into this to create channels that do not require support material.
A 150W motor stirs the vessel with rushton impellers, creating a turbulent environment for efficient mixing, oxygenation and heat transfer. In the vessel are also three probes: temperature, pH and dissolved oxygen sensors, all made by Atlas Scientific.
Hanging off the edge of the vessel is a solid state heating / cooling device, a peltier module + heatsink and clamping device. Above the vessel is a sparging system -- a pump that moves air through a sterilizing filter and then into the vessel. The air also passes over a mass flow sensor to gauge the precise amount of air added to the system.
The core of the electronics consists of a Raspberry Pi 3 and Teensy 3.2. The Pi communicates to the cloud system, receiving control parameters and sending back sensor measurements. The teensy controls any realtime processing, needed for generating PWM signals for instance. The Atlas probes communicate directly to the Pi through i2c and the teensy and the Pi communicate over USB / Serial.
Our power electronics are on the far right -- a 350W 12V supply powers most things easily, the Victor SP ESC controls the motor speed and a small current sensor tracks the input power to the motor.
Step 6: Next Steps: Building an Auto-sampler
Above are some renders of our auto-sampler. The sampling system will be a very important part of an automated bioreactor facility. With each sample, a scientist can do a deep dive on a snapshot of their experiment. Analysis tools like gas and liquid chromatography and mass spectrometry can be applied to learn more about the behavior and dynamics of their cells.The autosampler will pay a visit to each bioreactor and open an air-tight sample port. A pipette tube will draw out some liquid and transfer it to another container in the system.
Our auto-sampler is built around parts from Open Builds, an awesome group that provides equipment for all types of CNC machines. The sampler is powered by stepper-driven belt-drives in the X- and Y-axes, and a lead screw in the Z-axis.