UTEX 2973 Bioreactor: Validation

Much like the CB2A Bioreactor, we validated the bioreactor by running growth curves and comparing it to traditional culture flasks. A key difference between these methods is the addition of sub-surface CO2 aeration. We utilized the CO2 chamber and pumped in CO2 through a thin copper tube. Overall, the growth curved reached higher optical densities quicker than wet lab methods.

Wet Lab Validation

Comparing Bioreactor Performance Against Conventional Culture Growth Curves

To show that the UTEX bioreactor improves the growth rate efficiency of cyanobacteria, growth curves will be run with each successive DBTL cycle iteration of the cyanobacteria bioreactor. The best growth curve done with each iteration, as well as the growth data that has been done in our team’s wet lab, will be plotted together.

The most important part to look for in this comparison is the amount of time that each growth curve follows an exponential curve. It is the goal of each bioreactor iteration to extend the length of this section of the growth curve as much as possible, as this time is where the majority of bacterial growth comes from. Validation of this bioreactor requires that a mark of the bioreactor can grow bacteria as quickly as possible, and therefore, the goal is to extend this growth time for as long as possible.

In doing this, there are a few considerations that the bioreactor will take that wet lab experiments fail to consider. Aeration and agitation methods are two parts of growth curves that are not optimized for in wet lab experiments, which our bioreactor will consider. The main variable that will be considered to reduce growth curve time as much as possible when compared to standard wet lab practice will be the lighting conditions that UTEX will be subject to during growth.

The method to expose a controlled wavelength and intensity of light onto the bioreactor is another consideration to make. There are multiple feasible options for lighting to give reasonable and customizable results during testing. LED lighting systems were chosen as the lighting system for our bioreactor iterations due to their simplicity to work with and customize, allowing for effective testing of wavelength and intensity. Furthermore, this type of light is the one that would most effectively allow for consistent lighting on any part of the bioreactor, as it is easier to access smaller LEDs. This means that each one can be spaced evenly, resulting in a more even spread of light on the edges of the bioreactor than alternative options.

Alternative light sources were considered for testing but ultimately dropped for a variety of reasons. Using incandescent light bulbs was a consideration, as was fluorescent lighting sources. The reduction in versatility of these sources and the amount of heat that is generated from incandescent lighting systems were both reasons that these were dropped from design considerations. Natural light was also a consideration; however, this was dropped quickly due to the inconsistency of the amount of light that the bioreactor would be exposed to. A more comprehensive list of considerations can be seen below:

Table 1: Light types and their considerations in the experiment

Category	Incandescent	Fluorescent	LED	Natural
Mechanism	Heated filament that converts electrical energy to heat to light	Electric current that excites mercury vapour or exterior phosphor coating converts UV light to visible light	Causes electrons to release light energy and produces photons through semiconductor materials	Sunlight
Implementation	Incandescent lightbulb lamp in light-controlled environment	Laboratory light in rooms	LED strips or panels	5-closed face box with open face towards window
Pros	Longer wavelengths of light, low cost, easily accessible	Significant longer lifespan, shorter wavelengths of light	Easily adjustable to other wavelengths, low heat byproduct, customizable geometry	No cost, no power consumption required
Cons	Low luminous efficacy/high energy costs. Significant heat byproducts and can alter culture growth environment	Contains mercury and may need multiple sources to ensure even light transmittance	High cost, quality fluctuations	Could be inconsistent due to weather factors and lack of light during night hours. difficult to implement while following lab biosafety guidelines.

Based on this analyses, we concluded that a strip of LEDs can be used for each mark of the UTEX bioreactor to control light wavelength and intensity the best.

Investigating Optimal Wavelength for UTEX 2973 Culture

Different expressions of light onto the surface of our bioreactors will enhance the ability of UTEX to grow. Our bioreactor light optimization testing focuses on two main categories to determine which light level to use: wavelength and intensity. Studies show that exposing cyanobacteria to different wavelengths of light will affect the growth potential of the cells in different ways ([1]).

In the case of a strain of cyanobacteria called Navicula pelliculosa, research has found that this bacterium has the greatest ability to absorb light at roughly 450nm ([2]). Furthermore, this strain of cyanobacteria has another peak for light absorbance around 680nm.

Based on this information, we know that adjusting the light wavelength that shines on our UTEX strain could alter its growth efficiency. This is one of the two main ways to change the type of light the bioreactor is exposed to, and the first variable to optimize for growth.

To show that changing the wavelength of the light being exposed to UTEX cultures is an effective way to increase growth speed, the bioreactor is placed into a controlled light environement for the experiment. This container prevents exterior light from making direct contact with the bioreactor, and an LED system was wrapped around the culture to display a uniform amount of red light from all directions. This was done due to there being better absorption of light form UTEX at this light wavelength, and the amound of light that was directed into it was enough to remove any spots on the growth media where light would not reach.

To show that a given light wavelength can accelerate the growth of UTEX, the light absorbance of our UTEX growth media was measured at 750 nm. This is the wavelength that was measured in some previous wet lab experiments, and it will therefore be effective in creating a comparison between bioreactor growth and the standard procedure.

Setting up these growth curves requires that the bioreactor be partly disassembled, allowing for the main jar to be cleaned in an autoclave, and the rest of the pieces to be chemically sterilized if they are not going to come into contact with any living matter for the duration of the growth curves being run on them.

Once this has been completed, and the autoclaved parts have had a chance to cool, the bioreactor is reassembled, including a magnetic stir bar that is added, and the liquid cell media for UTEX is added to the main chamber of the bioreactor. Beyond this, all electrical parts that need power are plugged into a power source. The glass jar of the bioreactor is then lifted over a magnetic stirrer, which is turned on. For these growth curves, samples are taken every 3 hours until the exponential growth phase of the curve has ended.

To help determine the effectiveness of using alternative lighting methods to grow UTEX, the first mark of the bioreactor is used to determine growth conditions with standard lab lighting, and the second mark will attempt to determine how using red LED will improve growth speed in comparison.

Investigating Optimal Light Intensity for UTEX 2973 Culture

There is more than one factor that should be considered when determining how light should be utilized to increase the productivity of these bioreactors. The other major category that can be adjusted for in these experiments is the intensity of the light being used.

Multiple light intensities must be selected to run growth curves to find which one will work the best for our bioreactor designs. Based on the literature, it has been found that UTEX was around 261 $μmol/m^2s$ before accounting for light attenuation ([3]).

Within the growth curves that are being completed for this project, the light display being used will be getting operated at 100% power to ensure that the UTEX cultures are effectively recieving enough light to grow. The effectiveness of the light intensity was measured during the same growth curve where the wavelength was being tested. That is, red wavelength and max light intensity were tested with the same growth curve.

The LED light strips being used as lighting were wrapped around the outside of the bioreactor five times, with ~1cm of space between each light on the strip and the edge of the jar being used to contain the cell media.

Setting up the growth curve here is done using the same process as was done with the first mark of the bioreactor. Now using the second iteration of the bioreactor, it is much easier to move, and additionally, it allows easier implementation of the lighting systems when compared to the first iteration. Any parts that are able to be easily disassembled were autoclaved to sterilize them once again, and other parts that do not come into contact with any growth media during the growth curve are cleaned chemically once more.

Once again, the optimized liquid cell media was added to the bioreactor, alongside a magnetic stir bar, and the growth curve was begun once power was able to be effectively added to all segments of the bioreactor requiring it. For this growth curve, cell samples are once again taken every 3 hours until the exponential section of the growth curve has ended. The amount of growth of the bacteria is measured using the samples that are taken periodically. Furthermore, the bioreactor was blocked from the lights being used in the lab to isolate that vairable from the experiments being run.

Growth Curves

For the Mk2 growth curve, a sample of the absorbance of the growth culture was taken occasionally throughout the course of 2 days to determine roughly where the lag, exponential, and stationary phases of growth. In each of the below figures, the time at which a sample was taken is plotted on the x-axis, and the absorbance of the growth medium on a linear and logarithmic scale.

Results

From the figures above, we can see that the total growth of UTEX within the bioreactor is roughly 6.1 times. Below is a growth curve that was completed by our wet lab previously to use as a comparison:

The UTEX Mk2 bioreactor was able to achieve a maximum growth that was 6.1 times that of the starting culture. The wet lab growth curve that was completed allowed for a final growth ratio of 6.9 times their starting culture. It is also worth noting that the absorbances that were recorded in wet lab measurements were significantly higher than those found in Mk2 bioreactor testing.

Taking note of the amount of time that each growth curve took, it can be seen that the growth that was achieved by the UTEX bioreactor reached a maximum concentration 40 hours into the growth curve. The wet lab growth curve reached its maximum absorbance after 127 hours. However, it should be noted that the growth curve done here slowed down significantly after the measurement taken at 52 hours, where a growth ratio, when compared to the initial condition, was 4.7 times higher absorbance.

‌