Building and Operating a Low-Cost Elevated Carbon Dioxide Growth Chamber to Evaluate Microgreen Physiology under Spaceflight-Relevant CO<sub>2</sub> 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 "ambient CO2" (minimum 400 ppm) and "ISS-like exCO2" (minimum 3,000 ppm) conditions. The chambers maintained stable CO2 levels, temperature, and humidity throughout the 10-14-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.