Team:Harvard/Bioreactor
Bioreactor
Introduction
There are currently two main options for synthesizing substances in bacterial culture: shaker flasks and bioreactors. These two options appeal to research scientists looking for small yields and for industrial applications seeking very high yields, respectively. Shaker flasks are ubiquitous in bacterial laboratories, but bioreactors cost tens of thousands of dollars to purchase and many thousands more to maintain. Thus they are largely inaccessible to research scientists who are hoping to bring their biological manufacturing processes to larger scales. Shaker flasks offer very limited yields for biologically manufactured substances while bioreactors offer high enough yields to sustain some industrial applications today. However, researchers who seek to develop new biological manufacturing processes face difficulty accessing tools to make the jump from shaker flask yields up to industrial yields. There is a need for an intermediate level solution that allows researchers to reach higher yields without the high cost of existing high-yield solutions.
We have designed a bioreactor that can be cheaply built while providing a cell culture environment that produces yields higher than the shaker flask can achieve.
Design
Shaker Flasks are partially filled with growth media, cells, and any inducers or promoters necessary for the desired process. These flasks are placed in incubators and rotated at a set speed. The limiting nutrient in these processes is often oxygen. The aeration achieved by shaking the culture in insufficient to sustain high cell densities and high production rates.[2] Other environmental gradients in the shaker flask (e.g. temperature and pH) also inhibit the yield.[3] Figure 1 depicts how oxygen depletion corresponds to E. coli entering stationary phase, limiting protein production.
Fig. 1 Measurement on OD, pH and dissolved oxygen concentration in a shaker flask at 37 C and 180 RPM.
The instantaneous dips in the oxygen curve represent momentary pauses in agitation to allow sampling. Taken from [3].
The instantaneous dips in the oxygen curve represent momentary pauses in agitation to allow sampling. Taken from [3].
On the other hand, industrial bioreactors include various sensors and actuators that are able to rigorously control those environmental factors, including aeration. In doing so, the produce a cell culture environment that is more conducive to high-yields. Figure 2 depicts how a bioreactor can control oxygen saturation. Via aeration and stirring.
Fig. 2 Measurement of dissolved oxygen saturation in a stirred tank bioreactor (time scale is in hours) taken from [4].
In order to to produce an intermediate solution, we have sacrificed some of the precision and thoroughness of existing bioreactors by eliminating expensive sensors and incorporating 3D printing to build our bioreactor. The key parameters that we chose to optimize are aeration and stirring.
There are many types of industrial bioreactors available. They vary in terms of how the modulate the culture environment (e.g. adding fluid, removing fluid, agitating the culture). We have chosen to build a stirred-batch bioreactor which means that once the culture is started, no liquid is added or removed, and the culture is stirred by spinning blades called impellers.
Fig. 3 Diagram of a stirred tank bioreactor. Taken from [5].
Individual parts
Internal connections and assembled lid
Complete bioreactor
Results
Unfortunately, when we attempted to culture cells in the bioreactor, we found that the cells struggled to reach and sustain the logarithmic phase of growth. We hypothesize that this is because the sealant that we used to waterproof the culture chamber was toxic to the cells. In the future, using a non-toxic sealant could promote better growth.
References
[1]John A. Ryan. Growing Cells: A Simple Guide to Small Volume Cell Culture Scale-Up. Growing Cells: A Simple Guide to Small Volume Cell Culture Scale-Up, www.corning.com/media/worldwide/cls/documents/cc_scale_up_guide.pdf
[2]Bruecher, Daniel. “Evolution of Shake Flask Technology.” GEN, Genetic Engineering & Biotechnology News, 15 Sept. 2015, www.genengnews.com/gen-articles/evolution-of-shake-flask-technology/5579.
[3]Vasala, Antti, et al. “A new wireless system for decentralised measurement of physiological parameters from shake flasks.” Microbial Cell Factories, vol. 5, no. 1, Feb. 2006, p. 8., doi:10.1186/1475-2859-5-8.
[4]Bruecher, Daniel. “Evolution of Shake Flask Technology.” GEN, Genetic Engineering & Biotechnology News, 15 Sept. 2015, www.genengnews.com/gen-articles/evolution-of-shake-flask-technology/5579.
[5]“Bioreactor.” Wikipedia, Wikimedia Foundation, 11 Oct. 2017, en.wikipedia.org/wiki/Bioreactor.
