Building and Operating a Low-Cost Elevated Carbon Dioxide Growth Chamber to Evaluate Microgreen Physiology under Spaceflight-Relevant CO₂ Levels

We are interested in how extreme carbon dioxide levels at 3000 parts per million affect plant growth and nutrition. We wanted to develop a low-cost, extreme CO2 chamber. Because currently, few growth chambers can maintain extreme CO2 levels.

To begin, install and turn on the equipment required for the procedure. Wipe all non-electronic surfaces of the chamber, including the walls, ceiling and door with SA-20 disinfectant prepared at 7.8 milliliters per liter of water. Leave the chamber door open and allow the chamber to air-dry overnight.

Prepare 0.25x MS basal salt media without auger by dissolving 4.3 grams of MS salts and one gram of 2-ethanesulfonic acid in one liter of deionized water. Use One Molar Potassium Hydroxide to adjust the media pH to 5.7. Dilute the solution to a final volume of four liters and dispense 700 milliliter aliquots into one-liter bottles.

Then fill autoclavable bottles with distilled or deionized water for seed sterilization and rinsing. Autoclave the MS media and the prepared water using a liquid cycle for 30 minutes. Wrap the hydroponic boxes, tools and containers in aluminum foil.

Autoclave the wrapped materials on a dry cycle with a 30-minute dry time. And perform the next steps in a laminar flow hood. Inside the hood, add radish seeds to a 2%bleach solution, containing 0.05%Triton X-100 and shake the seed suspension gently for five minutes at room temperature.

Decant the bleach solution from the seeds and rinse the seeds three times with sterile water. After rinsing, soak the seeds overnight in sterile water to promote uniform germination. Next, fill each hydroponic box with sterile 0.25x MS medium, until the felt surface is covered with approximately one to two millimeters of liquid.

Verify that no air gap exists between the felt and the liquid. Using sterile forceps, place up to 30 radish seeds onto the felt in each hydroponic box. Press the seeds gently into the felt with the forceps and ensure that the seeds are centered above the tip holes on the insert.

Cover each planted hydroponic box with cling wrap or sterile lids to maintain humidity during germination. Set the primary carbon dioxide regulator to approximately 10 pounds per square inch. Now, slowly open the main valve on the carbon dioxide tank.

Adjust the secondary step-down regulator between one and three pounds per square inch. Turn on the benchtop incubator chambers and set the internal temperature to 23 degrees Celsius. Verify that the internal fan is functioning and ensure that the fan is unobstructed to maintain proper air circulation.

Next, open the Control_chamber_JoVE. py code in Thonny. Confirm the carbon dioxide in light settings.

Run the program. Then verify that the solenoids, sensors and lights respond properly. Once the environment is stable, place the covered hydroponic boxes inside each chamber.

Close the chamber door. And confirm that the carbon dioxide concentration returns to the set point within three to five minutes. Cover the chamber door panels with aluminum foil or blackout film to prevent light interference.

Flip open the cover to check the plants daily and readhere the cover with tape after inspection. Monitor the chamber readings one to three times daily using RealVNC, or locally through a high-definition multimedia interface display. Allow the seeds to germinate with the humidity covers in place for three to four days, until cot lead in emergence is visible.

Remove the humidity covers, once most seedling roots have penetrated the felt substrate. Respond promptly to any email alerts generated by the system and record any anomalies or manual interventions. Continue the incubation, until the plant growth reaches the desired stage.

At the end of the growth cycle, open the chamber door and remove the plants from the chamber. Process the plants is required for the experimental goals such as biomass, chlorophyll or nutrient analysis. Stop the Control script and power down the benchtop incubator and lights.

Rename the Chamber_data_csv environmental data file and copy the renamed file to a flash drive. Remove condensate from the chamber interior and wipe the non-electronic interior surfaces with 70%ethanol. Leave the chamber door open and run the air pump until the chamber and tubing are dry.

Finally, turn off the power strips and close the carbon dioxide tank valve to prevent leakage and conserve gas. Radish microgreens reach typical development 10 days after planting. The carbon dioxide regulation system, maintained the target concentrations for the ambient and extreme carbon dioxide conditions.

Mean carbon dioxide concentrations during radish experiments were 618 parts per million for the ambient treatment and 3061 parts per million for the extreme condition treatment. Carbon dioxide levels in the radish chambers became more variable during the daytime as plant mass increased, requiring frequent injections to maintain concentrations. Nighttime plant respiration, increased carbon dioxide concentrations in the ambient radish chamber to peaks above 1, 200 parts per million.

Carbon dioxide levels declined to near 400 parts per million shortly after the light period began in the ambient chamber, indicating rapid daytime uptake. Chamber temperatures remained near 25 degrees Celsius during the daytime and close to the 23 degrees Celsius set point during the night in both the conditions. Relative humidity increased gradually over the course of the radish experiment as plant transpiration increased.

Air pressure remained constant and nearly identical between the ambient and extreme carbon dioxide treatments throughout the experiment. Our method could be used by researchers interested in space biology, microgreen physiology, or plant response to elevated carbon dioxide. Attention to sterility and sanitation is critical, while performing this protocol to prevent disease.

The chambers should be checked regularly to ensure proper functioning. These chambers could be used to investigate how a range of carbon dioxide concentrations affect the physiology of other small plants.