Team:Calgary/Metal Separation Hardware
Laboratory Scale Bioreactor
Although we are currently focused on a proof-of-concept, our ultimate goal is to implement our LanM-based system of REE recovery on an industrial scale. Since the production of LanM is a limiting factor when it comes to the scalability of our system, we initially looked into potential laboratory-scale bioreactors that can be implemented in our research for upscaling production of LanM. However, we quickly realized that commercial bioreactors are completely unaffordable for smaller academic laboratories such as ours [1]. This inspired us to design LanHome, an affordable laboratory-scale bioreactor that utilizes materials commonly found in standard laboratories or can be easily purchased and assembled. LanHome serves itself as a platform for researching the large-scale cultivation of the transformed E. coli. The primary considerations of the design are of the following:
1. Cost
2. Aseptic Culturing
3. Sterilizability
4. Mixing Capability
5. Capacity for sampling and monitoring
Other design parameters that were considered were adaptability for scaling up, and automated sampling for consistent monitoring. The bioreactor scheme that is implemented is a stirred tank fed-batch system that allows for a simple and low-cost prototype to be developed and iterated upon.
LanHome Construction
MATERIALS AND COSTS
Table 1. Material purchased for construction of LanHome
Table 2. Material present in lab and used for assembly
With one of the main design criteria being cost, we were able to purchase all the material components that we required at $156.93. While the screw cap, media bottle, and syringe hardware aspects were already present in our given lab, understanding that this may not be the case for other labs or researchers may add upwards of $200 to the cost considering the market value of the three parts. However, all the materials used to construct the reactor are common and thus for another laboratory setting, the additional cost may be offset by already having other components that we did not have. Overall, with the total cost being in the $400 range, this is still significantly lower than the cost of commercial bioreactors.
BIOREACTOR ASSEMBLY
The following figure demonstrates a diagram and photographed representation of the bioreactor assembly.
The vessel (10) shown in the figure is a 250mL media bottle for the given prototype. For the upscaling process of the bioreactor, a major consideration would be a higher volume than conventional shaker flasks used, but for the initial prototyping, the given media bottle was chosen based on availability in the lab for experimentation. However, the same given bioreactor set up and design process can be applied by switching the vessel to a higher volume vessel for upscaling purposes. Media bottles are also autoclavable which enables reuse by sterilization.
Figure 1: Bioreactor Assembly setup diagram
Air-in: Starting from the component labeled 1, this is an adjustable air pump unit that allows for the air to be pumped into the bioreactor at a controlled rate. The air pump unit is then connected to a low-cost air filter system, (2) a syringe packed with cotton that allows for the air pump to filter before entering the bioreactor. Next, the filtered air flows through the silicon tubing attached to a metal rod that enters through one of the three connectors designed on the cap (3). The cap is made of polypropylene which is an autoclavable material that adheres to the design consideration of sterilizability of the unit. There are three connectors that lead into the vessel unit with one for air in, one for air out, and one for sampling. The hole for air in allows the steel rod that is attached to the air pump and filters by silicon tubing to enter into the vessel into the culture media. The end of the air-in steel rod is then attached to an aeration stone that allows for sufficient oxygenation for bacterial growth.
Air-out:In order to allow for sufficient gas exchange, another port on the cap allows for the entrance of a steel rod and silicone tubing system that allows for airflow out (8), and the air out can then be sanitized through either putting end of the silicon tubing into a beaker full of bleach solution (9) or alternatively the bioreactor can be placed under a fume hood.
Figure 2: Bioreactor set up with air in and outflow
Sampling: As for meeting the design consideration of having the capacity for sampling and monitoring the conditions within the vessel, the third connector that leads into the vessel allows for tubing to enter into the bioreactor content (5). For a fed-batch system, continuously sampling and monitoring the bioreactor content is a challenge as it has to be done manually. In order to automate the sampling process, we designed a sampling wheel (7) using an Arduino microcontroller. A 5-volt DC stepper motor was used as the main rotation motor for the wheel. The sampling wheel moves at a regular interval, and a peristaltic pump (6) is activated to transfer liquid from the bioreactor content into the wheel’s microcentrifuge tubes (placed in holes surrounding the wheel as shown in the image). The stepper motor was connected to the Arduino board via a ULN2003 motor driver, which protects the microcontroller from damages potentially caused by back emf from the stepper motor[2]. Future revisions of this circuit design will include the installation of a 12VDC peristaltic pump motor to automate the liquid transfer from the bioreactor container into the sampling wheel’s microcentrifuge tubes. The image provided shows the sampling wheel prototype.
Figure 3. Sampling wheel and Arduino set up
PERFORMANCE EVALUATION
In order to evaluate the performance of LanHome, it needed to first demonstrate the ability for aseptic culturing. To test this, we brought the assembled LanHome into the lab to see if it can cultivate transformed E. coli successfully through measuring cell density at OD600 at hour intervals. To cultivate the bacteria, we transferred 120mL of ampicillin LB and inoculated it with a single transformed colony. To maintain the temperature at 37C for the conditions of bacteria growth, rather than using a hot plate we used a heated water bath for more uniform heat distribution in the vessel. However, one advantage of using a hot plate would be the ability to use a magnetic stir bar for mixing capabilities. Due to the size of the vessel and volume of the content, we assumed the bubbling due to the aeration stone in the vessel being sufficient for mixing. However, in an upscaled vessel volume, a hot plate as a method of temperature control may be more versatile as it allows for mixing capacity with a magnetic stir bar as well as temperature control. 
Figure 4: Ampicillin LB broth with inoculated transformed E. coli
With the experimental set-up, we allowed the bacteria to cultivate over the span out of 8 hours and took OD measurements at every hour getting the following results.
Figure 5: Cell density at OD600 as a function of time
The graphed results in figure 4 demonstrates successful cultivation of the transformed E. coli, showing the lag, log, parts of the stationary phase of the bacterial growth curve. Based on the OD measurement results, we calculated a doubling time of 35.6 mins, and at hour 7, the maximum cell density reached 0.87. Conventional methods of bacterial cultivation in labs involve the use of shaker flasks. When comparing the results with literature values for shaker flasks as a method of E. coli cultivation in LB, the shaker flasks demonstrate an advantage with a lower steady-state doubling time of 20 mins [3]. While one consideration when making this comparison is that the LB we used was ampicillin LB which could have hindered the bacteria growth due to having to grow against antibiotics. The use of ampicillin in the broth is used to isolate antibiotic-resistant strains easily, but it poses resistance for the bacteria to grow against. However, with this factor in mind, the doubling time and the saturation OD from the initial prototype of LanHome is still at disadvantage and thus iterations to the design must be made in order to optimize growth to have a competitive advantage over conventional shaker flasks.
FUTURE DIRECTIONS
While we have established that the bioreactor was able to cultivate the transformed E. coli, we plan on comparing how well it is able to support this cultivation relative to conventional shaker flasks experimentally. The results of this comparison will provide a basis for reiterative development and optimization. To do so, we will perform temperature testing to correlate the water bath settings with the actual temperature of the bioreactor content and to determine if there are existing temperature gradients inside the reactor. Additionally, one crucial factor to be controlled with bioreactors is the pH of the culture media. Over time, the pH decreases as a result of the accumulation of metabolic byproducts involved in microbial cultivation. This pH decrease is not conducive for further cultivation and thus will inhibit proliferation. Hence, we plan on automating a pH monitor and complementing it with a pH controller through the addition of a basic solution. After the scheme has been set up, we will perform growth curve experiments to determine the settings that will provide the highest specific growth rate.
REFERENCES
[1] Theodore CM, Loveridge ST, Crews MS, Lorig‐Roach N, Crews P. Design and implementation of an affordable laboratory‐scale bioreactor for the production of Microbial Natural Products. Engineering Reports. 2019;1(4). doi:10.1002/eng2.12059
[2] Nasir SZ. Introduction to ULN2003. Theengineeringprojects.com. 2017 Jun 9 [accessed 2021 Oct 20]. https://www.theengineeringprojects.com/2017/06/introduction-to-uln2003.html
[3] Sezonov G, Joseleau-Petit Danièle, D'Ari R. Escherichia coli physiology in Luria-Bertani broth. Journal of Bacteriology. 2007;189(23):8746–8749. doi:10.1128/jb.01368-07
