The CB2A Bioreactor explores the optimization of Caulobacter crescentus given its cellular properties and growth factors. The research questions that we hope to answer with this project is how agitation methods affect CB2A, specifically impeller design and how we can reduce shear stress while optimizing cell growth.
Research and Design
Background
Bioreactors provide a stable and controlled environment for cell culturing, enabling higher productivity and more efficient use of resources. Their ability to regulate factors such as temperature, pH, agitation, and aeration makes them indispensable tools in both research and industrial applications. As part of UBC iGEM’s 2025 project, we will be growing Caulobactor crescentus CB2A strain, a bacteria that demonstrates strong surface adhesion properties. This unique trait allows it to cement mine tailings and further solidify them. This can reduce waste leaching into the environment in liquid form. To achieve reliable and scalable bacterial growth, we designed a customized CB2A bioreactor. The central design challenge is that CB2A is sensitive to shear stress and prone to producing adhesive biofilms while also requiring efficient nutrient and oxygen delivery. Our focus is on evaluating which agitation method could maximize biomass production while also monitoring other design factors.
Literature Research
Surface Adhesion
Each species of bactecteria has their unique shape that may provide specific advantages for their growth. Persat et al.([1]) investigated the curious curved shape of Caulobacter crescentus, demonstrating that the curvature allows the bacteria to anchor on surfaces in flow or dynamic environments. Additionally, it is observed that Caulobacter forms surface colonization under a wall shear stress of 0.23 Pa, but begins losing attachment at shear stress greater than 2 Pa as the cells lose their curvature.
Lawler and Brun ([2]) suggests that this property of Caulobacter helps to improve nutrient uptake. As the bacteria body elevates, nutrients enter through the outer membrane and are captured by high-affinity proteins. The increase in cellular surface-to-volume ratio also helps them to thrive in nutrient-limiting environments.
Bioremediation Application
Excessive amounts of heavy metals released from human industrialization and extraction threatens our environment. Caulobacter is becoming of increasing interest in bioremediation because it is nonpathogenic, easy to cultivate with limited nutrients, and naturally resistant to accumulated heavy metals. The cell’s outermost surface displays heavy-metal-binding peptides, which absorbs heavy metal ions while minimizing toxicity to the cell([3]).
Optimal Growth Environment
Through a comprehensive review of caulobacter research, we have identified Caulobacter’s key biological properties and ideal growth environment. It is found that, in complex media, none of the Caulobacter strains are able to grow anaerobically, their growth halted due to the accumulation of unreduced nitrite. This suggests the importance of O2 presence in the growth of Caulobacter.
Moreover, under the temperature of 30C, growth is observed to occur over the range of pH 6.1 to 7.8. Cultures of Caulobacter also grow the most rapid at 30C. Lower temperatures mean slower growths for Caulobacter; and for some strains, temperatures over 30 could also hinder their culture growth. Therefore we will use a 30 C room to provide a stable temperature environment and heating for our culture.
Common CB2A Bioreactor Designs
Immobilized microbial bioreactors are commonly used for growing Caulobacter bacteria.
Bai et al.([4]) suggested that the following factors play a major role in determining the choice of bioreactor for immobilized cells:
The engineering aspects of the bioreactors are influenced by the way cells are immobilized;
Physiological properties of cultured cells, as each species requires distinct culturing environments;
Hydrodynamic and mass transfer performances of the bioreactors. We reviewed several representative immobilized cell bioreactor designs discussed in the paper (3 figures below are from [4]).
Stirred Tank
Stirred tank bioreactors are commonly used by cell immobilized by gel entrapment, and can be operated at either batch or continuous mode. Under continuous mode, fresh medium are constantly supplied at a higher concentration to avoid nutrient depletion.
Figure 1. Stirred-tank bioreactor design with different methods of medium feeding.
Airlift bioreactors are found to be especially ideal for culturing immobilized cells under a large scale. By sparging air into the medium at the bottom of the vessel, both aeration and agitation are achieved. The generated vertical fluid flow both improves gas transfer and balances shear forces.
Figure 2. Airlift bioreactor design with internal tube (left) and external loop (right).
Cells are separated from the liquid phase by a membrane, which nutrients diffuse through. Recirculation of medium is often required to avoid nutrient gradients or waste buildup. To do this, the medium are pumped around quickly to achieve a near-well-mixed state.
Figure 3. Membrane bioreactor design with demonstration of medium recirculation.
Based on the preliminary research, we have identified some of the central user needs that a bioreactor should address, focusing on functionality, user experience, and design efficiency.
Modular and compact
Reliable biomass production
Cost efficient
Easy monitoring and controlling
Low maintenance
Power efficient
Prolongs the bacteria’s exponential growth phase
Effective agitation
CB2A Bioreactor Engineering Requirements
Table 1. Must-meet Design Requirements for Building an Effective CB2A Bioreactor
Requirement
Justification
Shear stress cannot exceed 2 Pascal
Caulobacter begins significantly losing attachment probabilities as cell shape changes at greater than 2 Pa, discouraging surface colonization.([1])
Sufficient Oxygen supply
Caulobacter is strictly aerobic. Research shows that under anaerobic culture conditions, bacterial growth is often halted due to nitrate accumulation ([7])
Static aeration is avoided
Because oxygen penetration into static culture media is limited to ~1 mm from the surface, the bulk of the medium quickly becomes oxygen-poor, preventing biomass production.([8])
Temperature is maintained at 30 - 37°C
Optimal growth conditions identified for Caulobacter.([9])
pH is within the range of 6.5 - 7.2
Literature shows that growth occurs at 6.1 to 7.2 pH, and the optimal pH is 6.5 ([7]).
The parts are autoclavable or sterilizable
To maintain aseptic conditions and eliminate any factors that may influence bacterial growth.
The assembled bioreactor is airtight and leak-free
Prevents contamination and ensures gas control.
Optical density matches Wetlab benchmark
A quantitative proof that our bioreactor is working effectively. The bioreactor would need further iterations if less cells are grown than under bench conditions.
Concept Generation
Based on considerations on available supplies, design complexity, and effectiveness, we decided to build a stirred-tank CB2A bioreactor. We analyzed the most suited method for heating, aeration, and agitation.
Table 2. Considerations on Heating Method
Heating Method
Positives
Negatives
Environmental Temperature Control
Consistent, uniform, and power efficient
Fixed location, difficult to monitor in-person
External heating device
Consistent heat supply. Easy to setup and monitor
May not be uniformly distributed
Internal heating coil
Direct heating element for culture. Can create relatively uniform distributions of heat
Creates localized heat gradients
Table 3. Considerations on Aeration Method
Aeration Method
Positives
Negatives
Bubbling via Pipette tip
Cost effective and simple to implement
Non uniform gas distribution throughout the culture medium; irregular bubbling patterns and therefore small scale
Membrane aeration
Efficient Gas transfer; can customize oxygen path/distribution area
Requires high concentration of gas to efficiently diffuse oxygen
Surface Aeration
Low cost and maintenance
Concentrated at surface, nonuniform distribution
Table 4. Considerations on Agitation Method
Agitation Method
Positives
Negatives
magnetic stirring
Low shear stress; easy monitoring
May not be suitable for large scale
paddle impellers
Gentle mixing at controlled rate, good for shear sensitive cells
Biofilm disruption
Rushton Turbine
Slightly more rapid mixing then paddle
High shear stress
airlift reactors
Little mechanical stress; combining aeration and agitation in one module
Less mechanical mixing, may not be uniform
Pipetting Agitation
Good mixing for small scale culturing
High variability, no continuous mixing, mostly manual
We also developed different weighted scorings to help determine the optimal method.
Figure 4. Weighted scoring for temperature control, aeration, and agitation. After considering the scoring results, component compatibility, and overall design feasibility, we developed our Mark I bioreactor concept. We selected water bath as a cost-effective and easy-to-set-up method for temperature control and sparging as the aeration approach. For agitation, we designed three different types of impellers, which will be further evaluated through a factorial experiment design.
Materials
After we finished generating possible design concepts, we moved on to determining the materials required for our build plan. We are aiming to keep materials accessible and affordable, in line with our team’s 2024 bioreactor project.
Table 5. Essential Building Materials for CB2A Bioreactor
Items
Description
Quantity
Mason Jar
Bioreactor vessel, autoclavable and relatively durable
1
Impellers
Different impeller designs to research the optimal agitation method for culturing CB2A
3 designs, 1 of each (and 1 more as backup)
NEMA-17 Motor
Agitation
1
Magnetic Stirrer
Serves as the control of our factorial experiment design of agitation
1
12 V Peristaltic Pump
Oxygen pumper
1
Air tubing (0.25 in)
Oxygen and liquid input/output
2 metres
Sparger
Improving Aeration by decreasing volume to surface area ratio
1
Water Bath (alternative: temperature controlled room)
Temperature control
1
Temp Sensor
Monitor temperature
1
Seals / Parafilms
Ensure the bioreactor is fully sealed. This allows the vessel to remain sterile and prevents contamination
Excess
Nutrients
Media, PYE broth
150 mL per experiment
CAD Components(PLA)
Base/support structure
1 kg / excess
Circuitry
Circuitry/power supply
1 x 4-conductor, 12 V 2A wall power adapter, 1x barrel jack connector, breadboard
Arduino
Microcontroller
1
DRV8825 Motor Driver
Controls NEMA 17 stepper motor
1
Computer-Aided Designs
MK. 1
Figure 5. Assembly of the CB2A bioreactor support structure and electronics holders. A multipurpose grid was designed to house all electronic components, preventing contact with water. A 3D-printed mount (left corner) secures the peristaltic pump, the holder (centre) supports the NEMA-17 stepper motor, and two trays hold the microcontrollers. Bottom extrusions allow the parts to slide onto the grid and lock into place.
1. Persat A, Stone HA, Gitai Z. The curved shape of Caulobacter crescentus enhances surface colonization in flow. Nature communications [Internet]. 2014 May 8 [cited 2025 Feb 20];5:3824. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC4104588/
2. Lawler ML, Brun YV. Advantages and mechanisms of polarity and cell shape determination in Caulobacter crescentus. Curr Opin Microbiol [Internet]. 2007 Dec [cited 2025 Oct 1];10(6):630—7. Available from: https://pmc.ncbi.nlm.nih.gov/articles/PMC2175029/
3. Xu Z, Lei Y, Patel J. Bioremediation of soluble heavy metals with recombinant Caulobacter crescentus. Bioeng Bugs [Internet]. 2010;1(3):207—12. Available from: https://www.ncbi.nlm.nih.gov/pubmed/21326927
7. Poindexter JS. BIOLOGICAL PROPERTIES AND CLASSIFICATION OF THE CAULOBACTER GROUP. Bacteriol Rev [Internet]. 1964 Sept;28(3):231—95. Available from: https://www.ncbi.nlm.nih.gov/pubmed/14220656
8. Somerville GA, Proctor RA. Cultivation conditions and the diffusion of oxygen into culture media: The rationale for the flask-to-medium ratio in microbiology. BMC Microbiology [Internet]. 2013 Jan 16 [cited 2025 Oct 2];13(1):9. Available from: https://doi.org/10.1186/1471-2180-13-9
9. Mazzon RR, Lang EAS, Braz VS, Marques MV. Characterization of Caulobacter crescentus response to low temperature and identification of genes involved in freezing resistance. FEMS Microbiol Lett [Internet]. 2008 Nov;288(2):178—85. Available from: https://www.ncbi.nlm.nih.gov/pubmed/18801049
