UTEX 2973 Bioreactor: Requirements + Design

The bioreactor for UTEX 2973 strain is different from our previous bioreactors in the way it requires carbon dioxide delivery and light. Literature has shown that these two factors are critical in cellular growth and therefore their delivery system is a priority in the design. This page will outline how we will design these delivery systems.

Photoautotrophic Bacteria and Their Growth Conditions

Overview of UTEX 2973 and its Growth Conditions

Synechococcus elongatus strain UTEX 2973 grows anaerobically through photosynthesis. Light energy, water, and CO2 are the three critical factors promoting its growth. Cyanobacteria are of increasing commercial interest due to their autotrophic nature and the ability to consume CO2 and produce O2.

In addition to lighting and aeration, ensuring a thorough agitation, stable temperature and pH is also necessary. Cui et al. ([1]) reported that in a shaking incubator at 130 rpm at 37°C, wild-type and engineered strains grown in BG11 medium of 7.5 pH under a light intensity of approximately 200 or 500 μmol photons·m⁻²·s⁻¹ with CO2 from air were able to thrive. Yu et al.([2]) assessed that the shortest doubling time of Syn2973 is 1.9 hrs when cultured in BG11 medium at 41°C under continuous 500 μmoles photons·m⁻²·s⁻¹ white light with 3% CO2. Overall, the two studies show that cyanobacteria exhibit more rapid growth under warmer temperatures (≥ 37°C), a light intensity of 500 μmoles photons·m⁻²·s⁻¹, proper agitation, and sufficient CO2 supply.

Light

Light is observed to directly influence UTEX biomass production. While most photoautotrophic microbes thrive under natural sunlight, it is not the most ideal for large scale production. The weather, season, and even geographic location may cause the light intensity to vary. Artificial lights are often preferred for culturing photosynthetic bacteria, as they provide stable and uniform illumination, and can penetrate the culture volume more effectively than natural lights([3]). Incandescent, fluorescent, and LED lights are all some choices to consider. While all of blue, red, and white lights are used in various experiments for cyanobacteria biomass production, it is found that highest growth rate occurs at red LED and highest pigment yield occurs at blue LED ([4]). When the biomass density reaches 1 g·L$^{-1}$ to 3 g·L$^{-1}$, cyanobacterial growth is hindered due to significant light attenuation, even if excess nutrients are present. It has also been found that extremely high light intensities can result in photoinhibition ([5]). Research also suggests that 12 hour light : 12 hour darkness best promote UTEX growth ([6]).

Blue Green 11 Medium

BG-11 medium is commonly used for culturing cyanobacteria. It contains a mix of macronutrients (carbon, nitrogen, phosphorus, etc.) and trace elements (zinc, vitamins, etc.) that support microalgal growth. BG-11 is a widely preferred growth medium because it promotes the growth of various freshwater microalgae, including cyanobacteria ([7]). Biomass production is generally higher in BG-11, as it supplies cells with essential nutrients for optimal growth.

CO2

Cyanobacterial strains respond differently to rising CO₂ levels due to variations in their respective cellular properties, though most strains perform better under elevated CO₂. Qiu and Gao found that, compared to algae grown under 350 ppm CO₂, growth rates increased by 52—77% under 750 ppm CO₂, with photosynthetic efficiency and dark respiration per unit of Chl a also improving ([8]). Using a sparger to increase the gas—liquid contact surface area and decrease volume to surface area ratio is generally ideal, as it enhances aeration and promotes more efficient CO₂ delivery and diffusion.

Photoautotrophic Bioreactors

Chanquia et al. ([3]) categorizes photobioreactors (PBRs) into two types, conventional and unconventional. Conventional PBRs include stirred-tank, tubular, flat panel designs and more, while the unconventional ones feature pyramid, nature-inspired, and hybrid designs. We will highlight the most representative and commonly used bioreactor designs from the paper.

Stirred Tank PBRs

• Stirred tank photobioreactors (PBRs) use impellers for mechanical agitation, sometimes aided by baffles, to achieve efficient heat and mass transfer as well as uniform nutrient distribution. Their simple design makes them easy to modify for different processes. However, their main drawback is a low surface-to-volume ratio, which limits light availability, and excessive agitation can cause shear stress that damages cells. While internal illumination has been proposed to improve light capture, stirred tank PBRs remain energy-intensive and unsuitable for large-scale photobioprocesses. Despite this, they are widely used in labs and industry for their operational flexibility. They have also been applied in continuous and fed-batch modes, demonstrating potential for efficient bioprocessing.

Tubular PBRs

• Tubular photobioreactors are among the most common types and come in a wide variety, including vertical, horizontal, near-horizontal, and spiral/helical forms. Unlike stirred tanks, tubular PBRs rely on pumps or other aeration systems for mixing and gas exchange. Vertical designs are often categorized as bubble-column or airlift. Bubble-column bioreactors, as their name implies, achieve aeration and mixing by bubbling air from the bottom of the column. Airlift bioreactors, on the other hand, feature a riser and downcomer compartment: as air enters from the bottom, the gassed medium rises through the riser while the degassed medium circulates downward through the downcomer. Their large illuminated surface area makes tubular PBRs well suited for outdoor mass cultivation. Their designs are also relatively simple and cost-effective. Horizontal tubular PBRs, in particular, provide a high surface-to-volume ratio and minimize shading, though they can also increase the risk of photoinhibition under excessive light.

Flat Panel PBRs

• Flat-panel PBRs consist of two parallel transparent panels forming a rectangular channel, maximizing light exposure per volume. Researchers have tried coloring the medium and coating the panels to optimize the incident light. Panel alignment can vary (horizontal, vertical, V-shaped, inclined, or accordion) to balance photosynthetic efficiency, space, and the risk of photoinhibition. Mixing is achieved through air bubbling, often using a sparger. Flat panel PBRs remain mostly limited to small-scale production due to the need of additional compartments and support materials when scaling‐up.

Despite their variety, current photobioreactor (PBR) designs face several fundamental limitations that constrain their scalability and efficiency. A recurring challenge across all types is the trade-off between light availability and system design. Tubular and flat-panel PBRs maximize illumination through large surface areas, but this also increases the risk of photoinhibition under high light intensity and creates difficulties in maintaining uniform light distribution as cultures scale up. Stirred-tank PBRs, while excellent for mixing and control of heat and mass transfer, suffer from poor surface-to-volume ratios, which limit light penetration and biomass productivity. Different designs from [3] are shown in Figure 8.

Takeaways from Our Literature Review

From a product design perspective, these are the identified design needs:

• Cost efficient
• Power efficient
• Easy monitoring and controlling
• Low maintenance
• Reliable biomass production
• Prolongs the bacteria’s exponential growth phase
• Modular and compact

Translating Design Needs into Engineering Requirements

Table 1. Must-meet Design Requirements for Building an Effective UTEX Bioreactor

Requirements	Justifications
CO₂ supply is sufficient	Cyanobacteria grows anaerobically.
Effective CO₂ diffusion is ensured	Based on wet lab growth feedback, it is important to make sure that enough CO₂ diffuses into the culture.
pH is maintained at around 7.5	Optimal growth conditions identified for cyanobacteria.
Light levels are adequately delivered	Light is a critical growth factor for cyanobacteria
Extremely high lighting intensities are avoided	Could cause photoinhibition([3]).
Constant agitation throughout	To evenly distribute nutrients and allow light permeation.
The parts are autoclavable or sterilizable	To maintain aseptic conditions and eliminate any factors that may influence or contaminate bacterial growth.
The assembled bioreactor is airtight and leak-free	Prevents contamination and ensures gas control.

Determining Optimal Light Settings for a UTEX 2973 Bioreactor

Our research question: What is the optimal method of light delivery that allows maximum cyanobacteria biomass growth?

After researching the optimal growth conditions for UTEX 2973, we identified lighting as the most critical factor. Our literature review shows that light intensity, wavelength, and uniformity must all be carefully considered to ensure consistent and reliable biomass production.

We will try to assess the performance of four lighting types: natural sunlight, incandescent, fluorescent, and LED wavelengths. We will be using evaluation criteria that considers functionality, impact on biomass growth, cost, and power efficiency.

Table 2. Qualitative Assessment on the Performance of Each Method

Evaluation Criteria	Natural Light	Incandescent	Fluorescent	LED
Brightness	Brightness varies greatly depending on the weather and time (may be lowered, but cannot be amplilfied).	Cannot be dimmed or brightened.	Can be dimmed but gives possibility of flickering (inconsistency).	Can be dimed and brightened while producing consistent light.
Cost	Sunlight is easily accessible without any cost.	Lowest inital cost of installing the light source, but may need to be replaced due to a short lifespan.	Moderate cost of installation with a moderate lifespan.	Highest initial cost of installing the light source, but has the longest lifespan.
Power Efficiency	There should not be any power associated with using natural light. No cost, so 100% efficent.	Requires the most power to run. Needs around 40W per 400 - 500 lumens. [9]	Relatively more efficient than incandescents.Requires 8 - 12W to produce 400 - 500 lumens. [9]	The most efficient out of all artificial lights. 6 - 7W to produce 400 - 500 lumens. [9]
Excess Heat Produced	Varies depending on the temperature.	Due to the mechanism of light emission, there is a high output of heat (90%). Could affect the desired and set temperature of the culture, as the light source would additionally be a heat source.	About 10-30% of energy released is heat energy. The effect may be negligible as growth rate is often found to increase with temperature. But specifics might require further experimentation.	Only about 5% of its energy is released as heat and it is only at the junction of intersection. Would not have significant influences on growth rate.
Growth Efficiency	Although the cost related to using natural light is low, the growth rate of cyanobacteria in outdoor photobioreactors is found to be lower than using artificial lighting. Not applicable for large scale production.	Less efficient for growth compared to fluorescent and LED since only a small parts of the emitted light is used by cells. [10]	No measurable difference was experimentally found supporting the idea that LED or fluorescent light affect growth rates, or inhibition of growth.	No measurable difference was experimentally found supporting the idea that LED or fluorescent light affect growth rates, or inhibition of growth.
Customizability	Very low customizability and consistency (due to lack of control). Inconsistencies come from our inability to control the maximum amount of available sunlight per day due day day cycles and weather.	Better customizability than natural light, but still not ideal due to its short life span.	Can use filtered to change the lighting colour, but may affect the brightness of the light	Highest level of customizability with flexible selections of light wavelength (colour).

Table 3. Determining the Optimal Lighting Mechanism Through Weighted Scoring

Category	Weighting	Natural Light	Incandescent	Fluorscent	LED
Brightness	0.25	4	0	9	10
Cost	0.10	10	8	6	3
Power Efficiency	0.15	10	3	5	9
Excess Heat Produced	0.05	6	2	7	9
Growth Efficiency	0.05	5	7	10	10
Customizability	0.40	0	4	9	10
TOTAL	1	4.05	3.3	8.05	9.1

LED lights scored the highest out of all options; a factorial experiment design will also be conducted to determine the most effective lighting method based on bacterial growth curves.

We also assessed different agitation and aeration methods:

Agitation:

• enhances light penetration
• breaks boundary layers for improved mass transfer
• improves temperature distribution

Mixing is proven to be more effective than pure diffusion for increasing growth rate. Turbulent stirring, especially, ensures more reliable chorlophyll and phycocyanin production than orbital shaking. We also do not want to have excessive agitation, as additional shear stress can reduce impose damage to cells. Less than 30 mpa in an agitation bioreactor is found to improve growth rate by breaking cell colonies while avoiding excessive shear. Considering both effectiveness and simplicity, we eliminated impeller stirring and baffles, choosing magnetic stirring instead as they can be used easily and will achieve optimal rpm([11]).

Aeration:

High intensity of aeration (330 bubble /min) gives high biomass density. According to Wetlab feedback, limited carbon dioxide transport likely leads to slow UTEX growth rate. Between the options of bubbling through pipettes, membrane aeration and bubbling through spargers, we decided to proceed with sparging as it ensures higher aeration intensity([12]). A CO₂ supply apparatus is determined to be necessary for aeration sufficiency.

Sketching

Below are the initial design sketches we developed based on our design requirements:

After careful consideration, we chose to proceed with design similar to the C-sketch in Figure 6 because it offered the best balance of effectiveness and feasibility. It also featured many standard components that we already had in the lab, allowing for a faster initial build and easier replication to be adopted by other teams.

How does this translate to our Space Mission

As a part of the space application of UBC iGEM’s 2025 project, we plan to cement the engineered cyanobacteria with martian regolith to produce living building materials in space. The hardware team’s central goal is to design an adaptable device that realize resource-efficient biomanufacturing on Earth and beyond. The autotrophic nature of cyanobacteria generates large quantities of cells with minimal resource input. The bioreactor further promises fast growth and high production rates, ensuring sufficient bacterial biomass for producing bio-ink used in biobricks printing .

Biomass production data from the UTEX 2973 bioreactor serves as a reference for understanding bacterial performance under Earth-like conditions. By comparing these results with those obtained from low-gravity bioreactors, we can gain clearer insights into how space-like environments influence cell behavior, biomass production rates, and related growth characteristics.