Name: Building and Operating a Low-Cost Elevated Carbon Dioxide Growth Chamber to Evaluate Microgreen Physiology under Spaceflight-Relevant CO2 Levels (Video) | JoVE Journal
Uploaded: 2026-04-17T12:40:00.000+00:00
Duration: 9 min 2 s
Description: Learn to build a low-cost elevated CO2 growth chamber for microgreen physiology research. This protocol enables stable CO2 levels above 3,000 ppm to simulate spaceflight conditions using radish and amaranth.

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We would like to thank undergraduate research assistants Makayla Destafino, Emily Dettmer, and Ellie Saltier for their help with preparing media, sterilizing seeds, and measuring plants. Thanks to the technical staff of the NCSU Phytotron for tools, supplies, and advice. This research was funded by NASA grant number (80NSSC23K0344) and an undergraduate NC Space Grant fellowship awarded to Samantha Rueckeis.

<ol><li>De Micco, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+and+microbial+science+and+technology+as+cornerstones+to+Bioregenerative+Life+Support+Systems+in+space%5BTitle%5D)+AND+%22Npj+Microgravity%22%5BJournal%5D)">Plant and microbial science and technology as cornerstones to Bioregenerative Life Support Systems in space.</a> Npj Microgravity. 9, 69(2023).</li>
<li>Georgescu, M. R., Meslem, A., Nastase, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Accumulation+and+spatial+distribution+of+CO2+in+the+astronaut%27s+crew+quarters+on+the+International+Space+Station%5BTitle%5D)+AND+%22Build+Environ%22%5BJournal%5D)">Accumulation and spatial distribution of CO2 in the astronaut's crew quarters on the International Space Station.</a> Build Environ. 185, 107278(2020).</li>
<li>Boles, H. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((build+and+testing+of+hardware+to+safely+harvest+microgreens+in+microgravity%5BTitle%5D)+AND+%22Gravitat+Space+Res%22%5BJournal%5D)">build and testing of hardware to safely harvest microgreens in microgravity.</a> Gravitat Space Res. 11 (1), 1-14 (2023).</li>
<li>Poulet, L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Large-Scale+crop+production+for+the+Moon+and+Mars%3A+Current+gaps+and+future+perspectives%5BTitle%5D)+AND+%22Front+Astron+Space+Sci%22%5BJournal%5D)">Large-Scale crop production for the Moon and Mars: Current gaps and future perspectives.</a> Front Astron Space Sci. 8, 733944(2022).</li>
<li>Cooper, M., Perchonok, M., Douglas, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Initial+assessment+of+the+nutritional+quality+of+the+space+food+system+over+three+years+of+ambient+storage%5BTitle%5D)+AND+%22Npj+Microgravity%22%5BJournal%5D)">Initial assessment of the nutritional quality of the space food system over three years of ambient storage.</a> Npj Microgravity. 3, 17(2017).</li>
<li>Poorter, H., Navas, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Plant+growth+and+competition+at+elevated+CO2%3A+On+winners%2C+losers+and+functional+groups%5BTitle%5D)+AND+%22New+Phytol%22%5BJournal%5D)">Plant growth and competition at elevated CO2: On winners, losers and functional groups.</a> New Phytol. 157 (2), 177-198 (2003).</li>
<li>Prior, S. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+elevated+atmospheric+CO2+effects+on+plant+growth+and+water+relations%3A+Implications+for+horticulture%5BTitle%5D)+AND+%22Hortscience%22%5BJournal%5D)">A review of elevated atmospheric CO2 effects on plant growth and water relations: Implications for horticulture.</a> Hortscience. 46 (2), 15-162 (2011).</li>
<li>Dong, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Effects+of+elevated+CO2+on+nutritional+quality+of+vegetables%3A+A+review%5BTitle%5D)+AND+%22Front+Plant+Sci%22%5BJournal%5D)">Effects of elevated CO2 on nutritional quality of vegetables: A review.</a> Front Plant Sci. 9, 924(2018).</li>
<li>Burgner, S. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Growth+and+photosynthetic+responses+of+Chinese+cabbage+%28Brassica+rapa+L.+cv.+Tokyo+Bekana%29+to+continuously+elevated+carbon+dioxide+in+a+simulated+Space+Station+%26%2334%3BVeggie%26%2334%3B+crop-production+environment%5BTitle%5D)+AND+%22Life+Sci+Space+Res%22%5BJournal%5D)">Growth and photosynthetic responses of Chinese cabbage (Brassica rapa L. cv. Tokyo Bekana) to continuously elevated carbon dioxide in a simulated Space Station &#34;Veggie&#34; crop-production environment.</a> Life Sci Space Res. 27, 83-88 (2020).</li>
<li>Levine, L. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Physiologic+and+metabolic+responses+of+wheat+seedlings+to+elevated+and+super-elevated+carbon+dioxide%5BTitle%5D)+AND+%22Adv+Space+Res%22%5BJournal%5D)">Physiologic and metabolic responses of wheat seedlings to elevated and super-elevated carbon dioxide.</a> Adv Space Res. 42 (12), 1917-1928 (2008).</li>
</ol>

None of the authors has conflicts of interest related to the work described above.

This protocol provides a reproducible and cost-effective method for growing plants under extreme CO2 (exCO2) concentrations of approximately 3,000 ppm, comparable to those found on the International Space Station2. The system modifies a benchtop incubator, combining SBC-based environmental control with a passive hydroponic growth system to support plant growth for up to two weeks without manual watering or opening the chamber. The approach bridges a critical gap in resources ...

Plants will play an essential role in sustaining human life during long-duration space missions by contributing to food production, meeting nutritional needs, and promoting psychological well-being1. However, the atmospheric composition of a crewed spacecraft or envisioned future habitats differs substantially from that on Earth. The International Space Station (ISS) routinely experiences CO2 levels above 3,000 parts per million (ppm), nearly an order of magnitude higher than ambient terrestrial concentrations2. Levels on the ISS have reached as high as 6,500 ppm. These extreme CO2 (exCO2) levels can influence plant physiology, morphology, and nutrient composition. Yet these exCO2 levels are outside the range of most elevated CO2 experiments, which focus on CO2 ranges between 700 ppm and 1500 ppm, and, indeed, outside of the range of what most terrestrial plant growth chambers can maintain. Consequently, the effects of such extreme conditions remain poorly characterized. Understanding plant responses to spaceflight-relevant exCO2 levels is, therefore, critical to ensuring the success of space-based agriculture and bioregenerative life-support systems.
Microgreens have recently emerged as promising candidate crops for astronaut nutrition due to their rapid growth, small footprint, and high nutrient density3,4. These short-cycle crops can provide fresh vitamins and antioxidants that degrade over time in stored spaceflight rations. Currently formulated astronaut meals show substantial nutrient loss during long-duration storage. When measured after three years of storage at room temperature, vitamin C and vitamin B1 drop below recommended levels, and vitamins A and B6 also exhibit moderate decreases5. Access to fresh, nutrient-rich microgreens could therefore help maintain crew health on missions lasting several years. Previous studies under moderately increased CO2 levels (800-1,000 ppm) have shown that plants tend to accumulate more carbon-rich compounds, including carbohydrates, flavonoids, and vitamin C, while reducing nitrogen-rich compounds such as proteins6,7. Other studies have observed reduced seed set, shifts in photosynthetic enzyme expression, and changes in elemental composition, particularly reductions in iron, zinc, and magnesium8. These compositional shifts suggest that carbon availability (as CO2) alters the balance between carbon and nitrogen, and, consequently, primary and secondary metabolism. Thus, increased CO2 levels may have implications for crop nutritional value, a critical need for astronaut health on long-duration space missions.
Far less is known about how plants respond to exCO2 levels encountered in spacecraft environments, which can exceed 3,000 ppm on the ISS. Spaceflight and ground-based analog studies have reported growth abnormalities under these conditions, including reduced leaf expansion, chlorosis, and altered growth rates2,9,10. For example, Brassica rapa grown aboard the ISS displayed yellow leaves with reduced size, which were attributed to exCO2 levels rather than microgravity effects9. These observations underscore that plant responses to exCO2 levels are not merely an extrapolation of the effects of moderately elevated CO2 but rather suggest distinct physiological and metabolic disruptions. However, few terrestrial growth systems can maintain stable exCO2 levels, making it challenging to consistently replicate spacecraft conditions or to test plant genotypic variation under such extreme atmospheres.
To address this limitation, we developed a small, low-cost chamber capable of maintaining consistent, programmable exCO2 levels that simulate spacecraft atmospheres (Figure 1). This system is constructed by modifying a benchtop incubator and integrating sensors and solenoid valves, which are controlled by an inexpensive single-board computer (SBC) with programmable pins. The design enables automated CO2 regulation and continuous monitoring of environmental parameters, including temperature, humidity, and pressure. To prevent rapid CO2 depletion caused by frequent door openings, we incorporated a passive hydroponic wicking system that supports microgreen growth for up to 14 days without supplemental watering. This closed setup minimizes disturbances while providing a consistent atmosphere.
Here, we describe a detailed, reproducible protocol for constructing and operating an exCO2 growth chamber and for cultivating microgreens under controlled, spaceflight-relevant atmospheric conditions. The protocol includes steps for installing airtight cable and gas pass-throughs, a CO2 injection and ventilation system, connecting sensors to the SBC, programming the exCO2 control code, and assembling the passive hydroponic wicking system. Using this setup, we demonstrate the successful growth of radish (Raphanus sativus, a C3 plant) and amaranth (Amaranthus cruentus, a C4 plant) microgreens under both ambient and ISS-like exCO2 conditions. The platform offers an affordable and scalable approach for studying plant responses to extreme atmospheric environments and to prepare experiments for spaceflight.

building and operating a low-cost elevated carbon dioxide growth chamber to evaluate microgreen physiology under spaceflight-relevant co2 levels

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

Carbon dioxide (CO2) levels aboard crewed spacecraft routinely exceed 3,000 ppm, significantly higher than atmospheric concentrations on Earth. Sustained exposure to extreme CO2 (exCO2) at levels above 3,000 ppm can alter plant growth, physiology, and nutritional composition. Yet, few laboratory systems can reliably reproduce these exCO2 concentrations. This protocol describes an accessible and low-cost method for constructing a benchtop growth chamber capable of maintaining stable CO2 levels above 3,000 ppm for plant experiments.
The chamber is created by modifying a benchtop incubator to include airtight cable and gas fittings, an automated CO2 delivery and venting system, and integrated temperature, humidity, and pressure sensors. The system is controlled by an inexpensive single-board computer, using open-source Python code to maintain a target CO2 concentration by regulating solenoids that inject CO2 or vent the chamber in response to system readings. A custom passive wicking box sustains plant growth without requiring active watering, reducing the need to open the chamber and minimizing fluctuations in CO2 levels.
Using this system, we successfully grew two microgreen species, radish (Raphanus sativus, a C3 plant) and amaranth (Amaranthus cruentus, a C4 plant), under &#34;ambient CO2&#34; (minimum 400 ppm) and &#34;ISS-like exCO2&#34; (minimum 3,000 ppm) conditions. The chambers maintained stable CO2 levels, temperature, and humidity throughout the 10&#8211;14-day growth cycles. This reproducible, spaceflight-relevant CO2 system provides an accessible tool for studying plant responses to extreme atmospheric environments and for preparing experiments for spaceflight.

Two benchtop incubators were successfully modified to maintain programmable CO2 concentrations suitable for microgreen growth experiments. For each experimental run, one chamber was programmed to maintain "ISS-like exCO2" conditions (minimum 3000 ppm), and the other to "indoor ambient CO2" conditions (minimum 400 ppm). We grew radish and amaranth microgreens for 10 and 14 days, respectively (Figure 13). Each treatment was replicated six times, alternating whi...

This protocol describes how to construct and operate a small, low-cost growth chamber capable of maintaining extreme carbon dioxide (exCO2) levels for plant experiments. The chamber enables reproducible studies of microgreen physiology under spaceflight-relevant atmospheric conditions, using a single-board computer-based monitoring and control system and a passive hydroponic wicking system.

Carbon dioxide (CO2) levels aboard crewed spacecraft routinely exceed 3,000 ppm, significantly higher than atmospheric concentrations on Earth. Sustained ...

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.

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Learn to build a low-cost elevated CO2 growth chamber for microgreen physiology research. This protocol enables stable CO2 levels above 3,000 ppm to simulate spaceflight conditions using radish and amaranth.

Building and Operating a Low-Cost Elevated Carbon Dioxide Growth Chamber to Evaluate Microgreen Physiology under Spaceflight-Relevant CO2 Levels (Video) | JoVE Journal

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

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