UTEX 2973 Bioreactor: Build

This page outlines the UTEX Bioreactor build plan and how we implemented our critical CO2 and light delivery systems. Similar to the CB2A Bioreactor, we printed custom parts from polylactic acid (PLA) and tested early iterations on an Arduino microcontroller. Our CO2 delivery system consisted of a water-drip mechanism that evaporated dry ice and pumped into the system. Light was controlled using LED strips and interwoven around Mark 2.

Overview of Our Engineering Process

We conducted comprehensive literature review on the cellular properties of cyanobacteria, analyzing experiments that culture this specific strain. We mainly focused on the influence of lights, agitation, and aeration on cyanobacteria biomass production. Using the takeaways from the literature, we then established our design needs, using weighted scorings and collaborative sketches to find an optimal design.

Computer-aided designs are generated and 3D-printed during our build phase. We developed a few comprehensive virtual prototypes before moving on to physical prototyping. The parts are assembled and circuits are built during this phase.

The bioreactor is put to use during the test phase. UTEX 2973 cells are cultured using the bioreactor. Growth curves are created to evaluate the bioreactor’s performance.

Based on our wet lab validation results and iHP feedback, we reflect on the what we did well and what still needs improvements. We then iterate through our DBTL cycle to further refine our design.

C-sketch of bioreactor design — Figure 1. Comprehensive bioreactor design C-sketch. Among the various C-sketches we developed, this design was ultimately selected for further development in our build plan.

Build Plan

Overview of Materials

Table 3. Bill of Materials Needed for Bioreactor Building

Items	Description	Quantity
Computer aided design models	Bioreactor support & electronics holder	Print all the designs
Large Glass Mason Jar	Vessel	1
BG11 Medium	Nutrients for optimal growth
Parafilms/seals	Secure the vessel to maintain a closed environment	As many as needed
LED Strips	Determine optimal light delivery mechanism	1 roll
Incandescent Light	Determine optimal light delivery mechanism	1
Fluorescent Light	Determine optimal light delivery mechanism	1
Natural Light	Determine optimal light delivery mechanism	1
Air tubing	As air inlet/outlet	1/4”, as many as needed
Air filters	For sterilization	1
CO₂ supply chamber	CO₂ supply	1, customized
Peristaltic pump	For aeration	2
Sparger	For aeration	1
Magnetic stirring bar & plate	For agitation	1 set
Temperature measurement probe	To measure the temperature data	1

Computer Aided Design

As an improvement from Mark 1, a base support is added to group all components into one piece for easier transportation. A mesh-like holder was additionally designed for lighting implementation.

Carbon Dioxide Supply Apparatus

A holder was designed for the CO2 chamber to allow water to drip steadily onto the dry ice, reducing the need for frequent refilling and extending the duration of evaporation. It is designed to have a funnel be placed in the central opening to guide the water, with filter paper and course sand to reduce water flow while several small invisible holes around its rim prevent pressure buildup from evaporating gas.

Validation of Carbon Dioxide Output

To validate that the built apparatus is able to capture the created carbon dioxide gas and provide it at the output, we submerged the output hose into a solution of calcium hydroxide ( $Ca(OH)_2$ ), created by mixing 2.40g calcium chloride dihydrate ( $CaCl_2 \cdot 2H_2O$ ) dissolved into 100mL of distilled water, with 1.31g $NaOH$ dissolved into 100mL of distilled water. The resulting solution of saturated calcium hydroxide was throughly mixed and set to rest for 15 minutes to allow the precipitate to settle. The saturated solution was then decanted into a funnel with filter paper to remove the remaining precipitate suspended in the solution to clarify it.

Calcium hydroxide is only slightly soluble in water (1.2 g/L @ 25 °C) and its dissolved form is commonly called limewater. Upon contact with carbon dioxide, it is undergoes a reaction to form calcium carbonate ( $CaCO_3$ ).

$Ca(OH)_2 + CO_2 \rightarrow CaCO_3 + H_2O$

Calcium carbonate is only sparingly soluable in water (0.013 g/L @ 25 °C). This will immediately precipitate in the saturated solution and will lead to a milky appearance. The onset of turbidity allows for visible validation that the output gas contains $CO_2$ .

Electrical Circuit

Circuit diagram of mk.1 cyanobacteria bioreactor — Figure 3. Circuit diagram of mark 1 cyanobacteria bioreactor

Assembly of parts

Obtain 3 pieces of tubing, where 2 pieces must fit inside the vessel, and the other one will be connected to the exterior.
Get two syringe filters. For air inlet, take two pieces of tubing and push each onto either side of the filter ports, then wrap the connection spot with parafilm. For air outlet, push one side of the port onto the tubing, then wrap with parafilm.
1. Inlet: one end of the tubing connects to a sparger head at the bottom of the bioreactor vessel, while the other end connects to the peristaltic pump on the outside.
2. Outlet: a short segment of tubing that goes from the air space inside the bioreactor to the outside
Attach the temperature probe to the temp sensor of the microcontroller by connecting the 2.54mm 3-pin connectors. Feed the sensor into the vessel through one of the 1/4 inch holes.
Put in the magnetic stir bar.
Add in media and engineered cyanobacteria.
Secure the lid onto the vessel by using parafilm around the lid and edges, tightening with the lid rim.
Put in the stopper in the central 1/2 inch hole; remove the stopper and use a pipette to take samples. Note: autoclave and sterile all non-electrical components prior to use, maintain an aseptic environment once media and bacteria are in the vessel.

Design History Files

For a detailed and comprehensive record of the engineering process behind the cyanobacteria bioreactor, please refer to the design history file provided below.

Cyanobacteria Bioreactor DHF.pdf