To validate the CB2A bioreactor, we ran growth curves in our bioreactor and compared it to traditional methods that used culture flasks and shakers. We used the 30 °C temperature-controlled room and sampled using optical density at 600 nm at a frequency of 1 hour. Ultimately, we found that we were able to reach exponential phase quicker than wet lab, but heating issues stunted the curve later.
Wet Lab Validation of CB2A Bioreactor Performance
Validation Objective
A key requirement for any novel bioreactor is the ability to sustain cell growth at a comparable or improved level to conventional flask-based methods. For this project, the validation process was designed to determine whether the CB2A bioreactor could reliably culture Caulobacter crescentus. Validation was achieved by running the bioreactor with live cultures, measuring growth over time, and comparing results against a conventional growth curve prepared by both the dry lab team and wet lab team.
This demonstrates that not only is the device functional, but also that it could support cell development comparable to standard benchmarks. Additionally, by comparing the growth curves produced between different means of agitation, the optimal impeller design can be quantitatively evaluated. These comparisons will allow further iterations of the bioreactor to support Caulobacter crescentus growth at improved growth rates, acting as a framework for continued optimization.
Experimental Approach
Before testing could be performed, basic microbiology materials and equipment were gathered. This ensured both sterility during culture preparation and accuracy during measurement. Standard Good Laboratory Practices (GLP) were followed by ensuring adequate training and preparation, while integrating the functionalities of the bioreactor. The steps below outline the workflow used for preparing, inoculating, and monitoring cell growth.
Materials
Procedure
Results
Two iterations of the bioreactor were tested. The first design was evaluated to establish baseline performance, followed by design improvements to enhance usability and consistent agitation. Validation of the second design demonstrated successful growth comparable to standard methods, providing confidence in the utility of the device.
Mark 1 Results:
The Mark 1 bioreactor served as the initial prototype to test whether a simple vessel with integrated agitation could sustain Caulobacter crescentus growth. Its purpose was to establish proof of concept and identify design limitations that could be addressed in future iterations.
Figure 1. Growth Curve in Mark 1 CB2A Bioreactor at OD 600 nm. Figure 2. Normalized Growth Curve in Mark 1 CB2A Bioreactor at OD 600 nm.
The growth curve produced from the Mark 1 CB2A Bioreactor was not able to match that of conventional growth curves. Without the characteristic exponential shape or verification from the logarithmic graph, it suggests that growth occurred much more slowly than that of the standard conditions, and rapid growth has yet to be observed.
Learning Phase
During testing of the Mark 1 bioreactor, several practical limitations were identified that affected both culture handling and system reliability. The most significant issue was the sampling process. To withdraw culture for OD measurements, several components of the bioreactor had to be removed before reaching the culture vessel. This not only increased the risk of contamination but also made the procedure time-consuming and not user-friendly.
Mechanical reliability was also a concern: the internal tubing frequently interfered with the impeller during operation, leading to tangling that disrupted mixing and sometimes caused the tubing to detach. Alongside that, the press-fit tolerances between the impeller shaft and motor casing were not sufficiently secure, leading to the impeller loosening or falling off. These issues collectively reduced the consistency of agitation, which was likely a contributing factor to the weaker growth curve observed in Mark 1.
Mark 2 Results:
Figure 3. Mark 2 Bioreactor Design in Use
Based on user feedback and results from Mark 1, modifications were made to Mark 2 to enhance user experience, provide a more seamless sampling procedure, and require fewer interventions to ensure functionality. The following changes were made:
New sampling tubing
Silicon tubing was fed into the vessel, with one end submerged in the culture, while the other end is easily accessible from the exterior during sampling. A sterile luer lock syringe is used to withdraw ~ 1mL at each hour to record OD.
Improved agitation attachment
By fixing the slight tolerances and press-fits of the motor casing and impeller shaft, a more secure attachment was created. This minimized the chances of the impeller becoming loose and detaching from the motor shaft, thus improving the consistency of agitation in the bioreactor.
Organization of internal tubing
Replacing the sparger and silicon tubing, a copper tube with silicon tubing as an external sleeve was used for aeration. Providing the same effects required for Caulobacter crescentus, this alteration would no longer risk tangling with the impeller. As the design pushes tubing to stay near the walls, it does not interfere within the radius of the impeller.
Figure 4. Growth Curve in Mark 2 CB2A Bioreactor at OD 600nm. Figure 5. Normalized Growth Curve in Mark 2 CB2A Bioreactor at OD 600nm.
In comparison to the growth curves produced from Mark 1, there is a sharp exponential increase in OD600, corresponding to the plot of conventional growth curves. Similarly, the logarithmic plots show a well-defined region consistent with exponential growth. This suggests that the design modifications made to the bioreactor improved biological performance, enabling the bioreactor to match conventional growth kinetics and validating its effectiveness as a functional culture system.
Interpretations
The validation experiments showed that the first design of the CB2A bioreactor did not reproduce the conventional growth curve of Caulobacter crescentus. Growth was sustained, but the rate and overall culture density were below the expected benchmark, indicating that mixing and aeration were insufficient.
In contrast, the second design demonstrated growth curves that matched, and in some cases slightly exceeded, those obtained through flask-based culture. These results indicated that the modifications introduced between the first and second designs improved both mixing reliability and overall usability, leading to reproducible culture performance.
Overall, the results demonstrate that the CB2A bioreactor is capable of supporting microbial growth at levels comparable to conventional methods. Importantly, the experiments establish a validated baseline for future testing. While current results confirm equivalence, additional work will be required to determine whether specific impeller features can further enhance growth beyond the curve established as of now.
Integrated Human Practices
We reached out to members of academia and industry and was introduced to John Collins, who has a wealth of experience working with Caulobacter, space bioreactors, and their applications.
The integration of monitoring tools is great to see. The more data points that can be linked to the growth of the Caulobacter, the better, as it will make it easier to optimize the culture conditions to maximize cementation.
John helped us with our CB2A Bioreactor design and gave us feedback on our completed Mark 2. He helped provide insights into how our bioreactors could be better tuned for space use and exploration. His insights also helped us plan our future steps, addressing potential issues in our future projects like biofilm formation.
John Collins
Former Life Support System Engineer at the European Space Agency and PhD student in synthetic microbiology at UBC
Outcomes
Specifically, he told us about scaling up our bioreactor systems and improving resource usage and management in space environments:
Caulobacter biofilm could be reduced by chemical treatment, genetic modification to lower holdfast activity, or designing a removable internal matrix to control areas of growth and simplify harvesting.
The system could potentially be retrofitted with the ability to remove culture when it reaches a pre-specified density and then add new media. This type of semi-continuous system would allow for constant growth of the culture, moving the process from batch production toward the continuous production needed for a larger-scale application.
Using dry ice as a CO₂ source for cyanobacteria growth is clever. Being able to leverage the same bioreactor for both cyanobacteria and Caulobacter is advantageous in a space setting. On orbit, systems should be simplified and made as robust as possible, since you may have someone with limited microbiology training operating them.
It might also be worth thinking about integrating nutrient cycling between the two organisms. The oxygen produced by the cyanobacteria could be used by the Caulobacter, for instance. Creating a more closed-loop system where they support each other could reduce the total amount of consumables needed.
Maintaining cleanliness in zero-g is essential and challenging. It’s a good move to modularize the bioreactor to ensure that parts can be easily cleaned or replaced, addressing the unique sterilization challenges in space.
Thinking about how the Caulobacter cells will be harvested from the bioreactor and then applied to create the cement is a key next step. Integrating this application process with the bioreactor would be crucial for developing a more automated, continuous system rather than a manual, batch one.
