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

Summary

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.

Abstract

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.

Introduction

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 density^3,4. 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 proteins^6,7. 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 rates^2,9,10. 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.

Protocol

1. Installing cables and sensors through the back of the benchtop incubator and sealing with silicone sealant (Figure 2)

1. Identify two locations on the back of the chamber free from wiring, electrical circuitry, or fan components (See cable gland locations in Figure 1B for an example). Drill two holes at these locations slightly larger than the external diameter of the cable glands.
2. Using approximately 10 mm (⅜ inch) diameter cable glands, wrap the threads of each gland with plumbing tape. Screw one gland onto a 20 mm outer diameter (OD), 2.5 cm long stainless steel pipe fitting (female ⅜ inch National Pipe Thread (NPT)-threaded). Insert through the drilled hole from the exterior wall of the chamber and attach another gland from the interior side (Figure 3).
   NOTE: This configuration enables an airtight pass through the thick chamber insulation.
3. Ensure the rubber washers of the glands are tightly secured on both sides of the wall, then apply silicone sealant around the points of contact with the chamber walls of each cable gland to seal any air gaps.
   NOTE: All chamber openings must be airtight to reduce CO₂ leakage.
4. Run a four-stranded data cable through each cable gland, leaving enough length to connect the SBC to the sensors.
5. Tighten the interior cable gland gasket to ensure an airtight seal. Loosen and adjust the cable as needed to provide more slack for sensor positioning.
   NOTE: Label each wire strand to ensure correct sensor connections. Although soldering the wires to the sensors is sufficient, jumper plugs, screw terminals, or other quick-connect options facilitate wire replacement and connection to the SBC pins.
6. Connect the temperature, humidity, and air pressure sensors (Figure 2).
   1. Attach the wires from one four-stranded data cable to a temperature/humidity sensor, then connect the air pressure sensor to the temperature sensor using 4-pin connectors.
   2. Connect the other end of the data cable to the SBC as follows: PWR to a 3.3 V power pin, GND to a ground pin, SDA to GPIO 2 (I2C_SDA1), and SCL to GPIO 3 (I2C_SCL1).
   3. Mount both sensors to the upper chamber wall using interlocking adhesive strips to allow easy removal for calibration or cleaning (Figure 4).
7. Connect and position the CO₂ sensor.
   1. Connect the CO₂ sensor to the second four-stranded data cable.
   2. Connect the other end to the SBC as follows: PWR to a 5V power pin, GND to a ground pin, RxD to GPIO 14 (UART_TxD), TxD to GPIO 15 (UART_RxD).
   3. Mount the CO₂ sensor on the upper back wall or place securely on top of the light shelf using Dual Lock strips.
8. (Optional, but recommended) Connect the SBC power supply to a constantly charging backup battery to prevent restarts and ensure continuous sensor data logging.

2. Constructing the CO₂ handling system (Figure 5)

NOTE: The CO₂ handling system regulates gas concentration inside the chamber. When CO₂ levels drop below the set point, the SBC triggers a solenoid valve to release short bursts of CO₂ into the chamber. When CO₂ levels exceed the set point, the vent solenoids open briefly to exhaust the air from the chamber. A 2.75 L/min diaphragm air pump circulates air through the system.

1. Install bulkhead fittings.
   1. Drill two additional holes in the back chamber wall (approximately 14 mm) for the CO₂ input and air outlet lines, slightly larger than the widest part of the fitting used in step 2.1.3.
   2. Modify a bulkhead fitting by cutting the hose barb off on the threaded side (Figure 6). Attach a metal and rubber washer and wrap the threads with plumbers tape.
   3. Screw a 3 cm, 8 mm ID threaded pipe fitting to the end of the modified bulkhead fitting to extend through the chamber wall and insulation (Figure 6).
   4. Seal the modified bulkhead fittings in place with washers on each side and apply an additional coating of silicone sealant. Screw tightly to create an airtight seal.
2. Connect tubing, solenoids, filter, and air pump using flexible, aquarium-grade tubing (4.8 mm ID, 6.35 mm OD) (Figure 7).
   1. For the CO₂ input line, connect components with the tubing as follows: Flow meter -> CO₂ solenoid valve -> T-connector -> T-connector -> exterior side of the CO₂ input bulkhead. Attach the first T-connector to the pump outlet. Attach the second T-connector to a vent solenoid (Figure 5).
   2. Connect the exterior side of the air outlet bulkhead fitting as follows: air outlet -> T-connector -> in-line air filter -> air pump input. Attach another vent solenoid to this T-connector (Figure 5).
   3. Inside the chamber, attach a ~15 cm length of tubing to the CO₂ input side, and a ~30 cm length of tubing to the air outlet side.
   4. Seal connections with silicone sealant.
3. Install luer-lock fittings. (Figure 7)
   1. Attach barbed leur-lock fittings at key tubing junctions to allow easy adjustment and component replacement.
   2. Seal each hose barb connection with silicone to reduce leaks.
      NOTE: Solenoids typically wear out after several months. Luer-lock fittings allow easy replacement and eliminate the need to shorten tubing during replacement. Therefore, adding these fittings at the solenoid junctions is strongly recommended.
4. Connect the CO₂ supply and regulators.
   NOTE: Use 6.4 mm (¼ inch) polyethylene tubing between the tank regulators and the flow meter.
   1. With a wrench, attach the primary regulator to the CO₂ tank (set regulator at 10 psi), then connect as follows: primary regulator -> threaded push-to-fit connector -> polyethylene tubing -> (optional) push-to-fit T-connector (for multiple chambers) -> polyethylene tubing -> threaded push-to-fit connector -> step-down regulator (set regulator at 1–3 psi) -> threaded push-to-fit connector -> polyethylene tubing -> flow meter (Figure 8).
      NOTE: Multiple chambers can be run from a single 22 kg (50 lb) CO₂ tank using optional push-to-fit T-connectors between step-down regulators for each chamber (Figure 8).
5. Connect solenoids to the SBC-controlled relays.
   1. Connect the CO₂ input solenoid (+) to the positive terminal of a 5 V power supply, and the solenoid (-) lead to terminal 3 of the CO₂ relay. Connect terminal 4 of the CO₂ relay to the ground lead of the power supply. Connect the CO₂ relay terminals 1 and 2 to GPIO 12 and a ground on the SBC, respectively.
   2. Connect both vent solenoids similarly: each solenoid (+) lead to the 5 V power supply (+) and each (-) lead to terminal 3 on the vent relay. Connect terminal 4 of that relay to the power supply ground, and the vent relay terminals 1 and 2 to GPIO 17 and the ground on the SBC.
   3. Label each relay (e.g., "CO₂ input," "Vent") to aid in troubleshooting.
      NOTE: Solenoids can share a single 5 V power supply if wired in parallel. Adjust the control code if using different GPIO pins.

3. Building the grow lights

1. Select compact light-emitting diodes (LED) strips protected by a silicone coating, offering a light spectrum that is suitable for plant growth and low heat output.
   NOTE: The custom grow lights we used deliver up to 550 µmol·m^-2·s^-1 of photosynthetically active radiation (PAR) at the height of the surface of the hydroponic grow boxes (the lowest height reached by the germinating seedlings). The lights consist of 24-V COB LED strips with alternating 2700K and 6500K diodes.
2. Prepare LED strips.
   1. Cut six strips of LEDs (approximately 25 cm) at the manufacturer-indicated junctions.
   2. Use a razor blade to remove the silicone coating from the copper pads at each end of every strip (Figure 9).
      CAUTION: Use care to avoid personal injury or damage to the LED strip. Work on a cutting board.
   3. Apply a small amount of solder to each copper pad, connecting the light strips in series using 3.5 cm pieces of 22-G stranded-core wire (+ to + and ground to ground) (Figure 9).
   4. Solder two 20–30 cm pieces of wire to one end of the array at each copper pad to connect to the power source.
      CAUTION: Use appropriate eye protection and solder under a fume hood or in a well-ventilated area. Work on a heat-resistant mat.
3. Mount the lights.
   1. Position the LED array to the underside of the upper shelf inside the chamber for even illumination across the growth area (Figure 10).
   2. Peel off the adhesive backing on the lights and press the strips firmly into place (Figure 10).
   3. Coat exposed pads and wires with electrical-grade silicone to prevent corrosion and electrical shock.
   4. Once the silicone is dry, position the light shelf on the chamber's uppermost built-in shelf bracket.
4. Connect the LED strips to power and control systems (Figure 2).
   1. Route two additional 22-G wires through one of the cable glands. Cut these long enough to connect the lights to the power supply and relay. Connect these to the long wires on one of the LED strips with a barrel jack or electrical quick-connect.
   2. Connect the LED array (+) to the 24 V power supply positive terminal, the LED (-) to terminal 2 of the light relay, and the light relay's terminal 1 to the power supply's negative terminal (Figure 2).
   3. Connect terminal 3 of the light relay to the SBC GPIO 21 and terminal 4 of the relay to a GND pin on the SBC (Figure 11).
5. Secure long light wires with adhesive clips to the chamber wall to prevent strain on soldered joints.
6. Verify the light intensity is as desired at the plant surface using a PAR meter. Measure in 4–5 locations at the height of the hydroponic grow system boxes.

4. Installing software for chamber control and alarm system onto an SBC

1. Download the chamber control code (Control_chamber_JoVE.py, Supplementary File 1) and save it in a designated folder on the SBC (e.g., /home/pi/chamber/ ). See https://www.raspberrypi.org/ for details on the SBC's initial configuration and how to navigate its operating system.
2. Install required libraries listed at the top of the Control_chamber_JoVE.py script (serial, datetime, csv, board, etc.) and the libraries for each sensor. See https://projects.raspberrypi.org/en/projects/generic-python-installing-with-pip for details on installing libraries on the SBC.
3. Open Control_Chamber.py in Thonny (or similar Python IDE) to configure sensor and relay parameters. Update the pin assignments in the top section of the Control_chamber_JoVE.py script to match the wiring of the temperature/humidity, pressure, and CO₂ relay connections established in Sections 1 and 2 (If wired as suggested, no changes are needed). See https://thonny.org/ for details about using Thonny.
4. Define the minimum CO₂ level set point and set parameters for photoperiod, temperature, and alarm thresholds within the configuration block at the top of the Control_chamber_JoVE.py script. Save the file.
   NOTE: All set points can be modified by editing the configuration block without requiring a software reinstall.
5. Set up the e-mail-based alarm system.
   1. Create a dedicated email account (e.g., MyPiAlarm@gmail.com) for automated alerts.
   2. Enter the credentials and recipient addresses in the "CO2 Alarm" and "Temperature Alarm" sections of the Control_Chamber.py script and save.
      CAUTION: Do not use personal or professional email address to send messages. Use a dedicated account to protect security credentials and use an app-specific password.
6. Test chamber operation.
   1. Click the Run icon in Thonny to execute the code.
   2. Confirm sensors report valid readings and relays toggle correctly when CO₂ levels change.
   3. Simulate low and high CO₂ and temperature conditions by changing the flow and chamber settings. Ensure that automatic injection and venting occur and that email alarms are triggered.
   4. If CO₂ readings fluctuate, check for leaks around cable glands or bulkhead fittings and reseal with silicone sealant as needed.
7. Enable remote monitoring
   NOTE: While the SBC can run "headless," connecting a monitor via HDMI simplifies troubleshooting.
   1. Install the RealVNC Viewer or similar remote-desktop application on a device connected to the same network as the SBC. See https://www.realvnc.com/en/connect/download/viewer/raspberrypi/ for instructions on how to install RealVNC.
   2. Enable VNC Server on the SBC and confirm connectivity. See https://help.realvnc.com/hc/en-us/articles/360002249917-RealVNC-Connect-and-Raspberry-Pi#setting-up-your-raspberry-pi-0-0 for instructions on enabling VNC Server.
   3. Use the remote interface to monitor status, adjust settings, or restart the program.

5. Constructing a passive hydroponic wicking system (Figure 12)

NOTE: Each passive hydroponic wicking system uses a sterile 1,000 µL pipette tip box with a removable insert fitted with a fringed felt wick, which draws nutrient solution from the lower reservoir to the seeds.

1. Prepare the box housing.
   1. Select an empty 1,000 µL pipette tip box with a removable insert.
   2. Using a Dremel tool, rotary saw, or a drill and coping saw, cut a rectangular opening ~ 11 cm by 7 cm in the top lid.
2. Cut and prepare the felt wick.
   1. Cut bamboo felt into 34 cm 7.5 cm strips. Three tip-holder inserts can serve as a template.
   2. Cut eight evenly spaced 14 cm fringes along each long edge of the felt strip.
   3. Thread each fringe through the row of holes at the short ends of the tip-holder insert using a pair of forceps so the fringes hang downward and the uncut center remains flat on top.
   4. Ensure fringes reach the lower reservoir when the insert is in the box. Store boxes dry until use.

6. Preparing materials for the microgreen experiment

1. Sanitize the chamber by wiping all non-electronic surfaces (walls, ceiling, door, etc.) with SA-20 disinfectant (7.8 mL/L water). Allow the chamber to air dry overnight with the door open.
   NOTE: This step is crucial to reduce the risk of pathogens that grow in humid environments.
2. Prepare 0.25x Murashige and Skoog (MS) basal salt media without agar. Dissolve 4.3 g of MS salts and 1 g of 2-(N-morpholino)ethanesulfonic acid (MES) in 1 L of deionized (DI) water, and adjust pH to 5.7 with 1M KOH solution. Dilute to 4 L. Dispense 700 mL aliquots (enough for one hydroponic box) into 1-L bottles.
3. Fill autoclavable bottles with distilled or deionized water for seed sterilization and rinsing.
4. Autoclave both the MS media and water using a liquid cycle for 30 min.
5. Wrap hydroponic boxes, tools, and containers in aluminum foil. Autoclave on a dry cycle with a 30-min dry time, and keep materials wrapped until placed in a laminar flow hood.
   NOTE: Steps 6.6–6.8 must be carried out in a laminar flow hood to prevent contamination.
6. Sterilize seeds
   NOTE: This protocol will need to be adapted depending on the plant species. The following describes a protocol that works for radish and amaranth seeds.
   CAUTION: Use gloves, eye protection, and a lab coat while working with bleach solution.
   1. In a laminar flow hood, add seeds to a 2% bleach solution containing 0.05% Triton X-100.
   2. Shake gently for 5 min at room temperature.
   3. Decant the bleach solution and rinse three times with sterile water.
   4. After rinsing, soak seeds overnight in sterile water to promote uniform germination.
      NOTE: For this study, 8 mL of 7.5% bleach, 22 mL of DI H₂O, and 15 µL of Triton X-100 surfactant in a 50 mL centrifuge tube were sufficient for sterilizing four boxes of radish or amaranth microgreens.
7. Fill hydroponic boxes and plant seeds in a laminar flow hood.
   1. Fill each hydroponic box with sterile 0.25 MS medium until the felt surface is covered with a mm or two of liquid (~650 mL). Verify that no air gap exists between the felt and the liquid; gaps will reduce seedling establishment.
   2. Using sterile forceps, plant up to 60 amaranth or 30 radish seeds per box. Press the seeds gently into the felt with forceps, ensuring they are centered above the tip holes on the insert.
   3. Cover each planted box with cling wrap or sterile lids to maintain humidity during germination. Once covered, boxes can be removed from the sterile hood.

7. Running the microgreen experiment

NOTE: Operate at least two chambers simultaneously, one under exCO₂ and the other under ambient conditions as a control. Alternate treatments between chambers across replicates to minimize chamber bias. Randomly assign (using a coin flip or random number generator) a treatment to each chamber. For the following experiment, swap the treatments to different chambers.

1. Set the primary CO₂ regulator to approximately 10 psi and the secondary "step-down" regulator(s) to 1–3 psi. Open the main valve on the CO₂ tank slowly.
   CAUTION: Air pressures above 5 psi will damage the solenoids. Do not adjust the "step down" regulator above 4 psi.
2. Start the chamber and verify functionality.
   1. Turn on the benchtop incubator chambers and set the internal temperature to 23 °C or the desired temperature.
   2. Verify that the internal fan is functioning and unobstructed to ensure air circulation. Ensure each chamber unit equilibrates to within 2 °C of the set temperature before planting.
3. Open Control_chamber_JoVE.py code in Thonny, confirm the CO₂ and light settings (section 4.7), then run the program. Verify solenoids, sensors, and lights respond properly. Save the file and execute the control program by hitting the Run icon.
4. Load and germinate planted boxes.
   1. Once the environment is stable, place the covered hydroponic boxes inside each chamber. Close the door and confirm that the CO₂ concentrations return to the set points within 3–5 min.
   2. Allow the seeds to germinate with the humidity covers in place for 3–4 days until cotyledon emergence is visible, then remove the humidity covers once most seedling roots have penetrated the felt substrate.
      NOTE: If using species other than radish or amaranth, adjust germination duration as needed to ensure consistent root penetration into the felt surface before uncovering.
5. Maintain and monitor chamber conditions.
   1. Keep the chambers closed throughout the experiment to maintain stable CO₂ levels.
   2. Cover door panels with aluminum foil or blackout film to prevent light interference. Flip open the foil daily to check the plant's status and re-adhere the cover with tape.
   3. Monitor readings one to three times daily using RealVNC or locally via an HDMI display.
   4. Respond promptly to any email alerts and record anomalies or manual interventions.
      CAUTION: Ambient temperatures below the chamber set point may cause condensation in the tubing. If condensation occurs, dry and reseal lines and fittings before restarting to prevent airflow obstructions. Close CO₂ tank valve before opening lines.
6. Harvest plants and collect data.
   1. At the end of the growth cycle, open the chamber and remove and process plants as needed for experimental goals (e.g., biomass, chlorophyll, or nutrient analysis).
   2. Stop the control script and power down the benchtop incubator and lights.
   3. Rename chamber_data.csv, the environmental data file (e.g., Date_Species_3000vs400ppm.csv), and copy it to a flash drive.
      CAUTION: The program logs data to the chamber_data.csv file in the home directory every minute. Always rename this file when saving to avoid amending previous datasets.
   4. Document any deviations or equipment malfunctions during the run.
7. Post-experiment cleaning.
   1. Remove condensate and wipe non-electronic interior surfaces with 70% ethanol.
   2. Leave the chamber door open and run the air pump until the chamber and tubing are dry.
   3. Turn off power strips and close the CO₂ tank valve to prevent leakage and conserve gas.

Results

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...

Discussion

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 ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
1/4 inch polyethylene tubing	Lowes	https://www.lowes.com/pd/EZ- FLO-1-4-in-Inner-Diameter-x-25 -ft-Polyethylene-Tubing/1000180497	Use to attach CO2 tank regulator to step down regulators.
2-(N-morpholino)ethanesulfonic acid (MES)	Fisher Scientific	BP300-100	Buffering agent in fertilizer solution.
24 V Power supply for lights	Mean Well	LPV-35-24	Use power supply for lights depending on light specs.
25 mL graduated cylinder	Cole Parmer	UX-34502-70	To measure liquids for seed sterilization solution.
250 mL glass graduated cylinder - autoclave to sterilize	Fisher Scientific	https://www.fishersci.com/shop/ products/pyrex-cylinders-single -white-enamel-metric-graduatio ns-18/085524D	For measuring MS media when loading hydroponic boxes. Brand doesn't matter.
388 Electrical grade silicon sealant	American Sealants, Inc	https://meridianadhesives. com/products/asi-388/	Used to insulate lights and seal chamber. Other brands are fine, but make sure it's electrical grade if used on lights.  Non-electrical grade is fine to use on the rest of chamber.
70% Ethanol	Avantor	71002-508	Used for sanitizing hood and growth chamber.
8K diaphram pump - 110 V/60 Hz version	Boxer	8K	Used to circulate air between chamber, CO2 input, and ventilation system. $120 at time of purchase.
AC cord for  pump	GE	https://www.homedepot.com /p/GE-8-ft-Replacement-Cord -Set-with-Polarized-Plug-on- 1-End-Black-54435/203735885	Brand and company do not matter
Adafruit LPS35HW Water Resistant Pressure Sensor	Adafruit	Product #:4258	Used to measure air pressure.
Adafruit Sensirion SHT40 Precision Temperature and Humidity Sensor	Adafruit	Product #:4885	Used to measure temperature and humidity.
Adjustable wrenches	Ace Hardware	https://www.acehardware.com /departments/tools/hand-tools/ wrenches/2299329	Used to attach regulators
Aluminum foil	Costco	https://www.costco.com/p/-/ kirkland-signature-reynolds- foodservice-foil-12-in-x-100 0-ft/100116721?langId=-1	For covering containers during autoclaving.
Aquarium tubing - 1/4" OD, 3/16" ID	Amazon	https://www.amazon.com/dp/B0DLNH81LJ/	Use to for conveying CO2 into chamber
Assorted aquarium tubing connectors - polypropylene	Amazon	https://www.amazon.com/AQU ANEAT-Aquarium-Airline-Conn ector-Plastic/dp/B08LMZ86QC/	Used to split and connect aquarium tubing
Assorted machine screws	Amazon	https://www.amazon.com/AETTL- Assortment-Stainless-Assorted- Phillips/dp/B0B428NP2L/ref=sr_ 1_2_sspa?crid=FQ8XLSW2WZE6 &keywords=assorted%2Bmetric% 2Bbolts%2Band%2Bnuts&qid=16 92727621&s=industrial&sprefix= assorted%2Bmetri%2Cindustria l%2C159&sr=1-2-spons&sp_cs d=d2lkZ2V0TmFtZT1zcF9hdGY&th=1	Used to attach components to peg board.
Backup battery with USB output	Voltaic Systems	V25	Used to power pi's to maintain power during brief power outages.  Any USB battery that allows for pass-through charging will work.
Bamboo felt microgreens grow mat roll (10" x 100 ft)	VegBed	https://www.vegbed.com/products /vegbed-bamboo-microgreens-roll-10in-x-100ft	Used for grow box wick.
Barrel Jack connectors - 10 pack	Amazon	https://www.amazon.com/dp/B079RBL339/	Used to connect electrical components to power.  Exact brand doesn't matter.
Beaker - 2 L	Fisher	CLS10002L	Used for making media.  You will need two to make 4 L total.
Benchmark Scientific H2200-HC MyTemp Mini Incubator	Benchmark Scientific	H2200-HC	Chamber modified for this protocol. Make sure to get the version with both heat and cooling control. $850 at time of purchase.
Bleach - 7.5%	Chlorox	https://www.clorox.com/ products/clorox-performance -bleach-with-cloromax/	Used for sterilize seeds.  Brand doesn't matter, but get a verision with no perfumes or pouring agents.
Bulkhead fitting - 3/16 inch	ANPTGHT	https://www.amazon.com/AN PTGHT-Thru-Bulk-Bulkhead-F ittings-Aquarium/dp/B08SCDC28R/	Used to pass tubing throught chamber
Cafeteria tray	Amazon	https://www.amazon.com/Carlisl e-CT121605-Caf%C3%A9-Stan dard-Cafeteria/dp/B008BLINFE/	A tray was placed under each chamber to catch condensation that leaked out when opened.  Exact brand doesn't matter.  It just needs to be larger than the footprint of the chamber, but not obsruct the door.
Centrifuge Tube - 50 mL, sterile	Fisher Scientific	339653	Used to contain seeds during and after sterilization.
CO2 alarm (optional)	Amazon	https://www.amazon.com/ dp/B09MRX4F12/	Optional, but recommended to detect external leaks in the CO2 system.  Prevents unsafe levels of CO2 in chamber room.
CO2 Regulator  - low pressure	Amazon	https://www.amazon.com/gp/ aw/d/B01BPQDG62	Exact brand does not matter.  But should be able to reduce pressure somewhere between 1 and 4 psi.
CO2 tank - 50 lb	AirGas	https://www.airgas.com/product/ Gases/Carbon-Dioxide/p/CD%2050S	A smaller tank than this could probably be used.  It just would need to be replaced more often.
Crimper	Grainger	806FA9	To attach crimp connectors to wires
Digital pressure guage (optional)	Amazon	https://www.amazon.com/Pressure -Connector-Protector-Uharbour-0- 200psi/dp/B0783PYQ27	To read pressure setting of low-pressure step-down regulator.  Requires AAA battery. Alternatively, you can use a non-powered analogue guage.
Dissecting forceps - autoclave to sterilize	Fisher Scientific	13-812-39	To manipulate sterilized seeds.  Brand doesn't matter.
Dual lock interlocking adhesive strips	3M	SJ3560	Used to attach sensors to side of chamber.
Empty 1000 µL pipette tip boxes	Genesee	24-160R	Use a dremel to remove most of the top rectangle of the lid.  Brand doesn't matter, but some may hold more liquid than others.  Newer Genesee boxes hold less liquid than older ones.
Fork Terminal crimp connectors - 22-16 AWG	Amazon	https://www.amazon.com/Baomain -Insulated-Terminal-22-18-Gauge/ dp/B01B17DL0G/	Used to connect wires to relays.  Exact brand doesn't matter.
Glass bottle - 1 L	Millipore Sigma	CLS13951L-1EA	Used to contain aliquots of sterile 0.25x MS media.
Harris CO2 tank regulator	Grainger	5KZ48	Set at 10 psi.  Note this regulator is separate from the second "step down" regulator.
HDMI cable to micro HDMI cable (optional)	Amazon	https://www.amazon.com/KELink- Aluminum-Compatible-Camcorde r-Raspberry/dp/B0C3LR54C3/	(Optional) To connect pi to HDMI monitor to view senor readings
Heat gun	Amazon	https://www.amazon.com/MAXXH EAT-Handheld-Embossing-Wrapping -MF300L/dp/B09P66LLMW/	For melting heat shrink in place to insulate wires. Brand does not matter.
Heat shrink	Amazon	https://www.amazon.com/560PCS- Heat-Shrink-Tubing-Eventronic/dp/ B072PCQ2LW/ref=sr_1_4?crid=21 4VBYLN60TGD&keywords=heat+ shrink+tubing&qid=1685586705&s prefix=heat+sh%2Caps%2C143&sr=8-4	Needed to insulate wires. Exact brand doesn't matter.  We recommend getting a variety pack of different diameters.
Inline filter to protect pump	Grainger	794F21	Mount upstream of the diaphram pump to protect it from debris and condensation
Jumper cords	Amazon	https://www.amazon.com/Elegoo- EL-CP-004-Multicolored-Breadbo ard-arduino/dp/B01EV70C78/ref= sr_1_1?keywords=raspberry+pi+j umper+wires&qid=1685586340&sr=8-1	Needed to connect items to pi.  Exact brand doesn't matter.
K30 FR CO2 Sensor	Sensair	030-8-0010	Used to detect CO2 levels in the chamber. $60 at time of purchase.
Lab tape	Millipore Sigma	BAF134620300	For labeling containers and adhering foil to door of chamber
LED Strip - COB, 24 V	Super Bright LEDs	https://www.superbrightleds.com /led-strips-and-bars/cob-series-l ed-strip-and-tape-lights/5m-tuna ble-white-cob-led-strip-light-cob -series-led-tape-light-2700k-6500 k-ip20-24v	Use for custom grow light.  Other type of white LED strips would likely work fine, but use a PAR meter to adjust density of strips to get desired light intensity.
Luer Lock to 3/16 hose barb fittings - polypropylene - female	Amazon	https://www.amazon.com/Femal e-Luer-Lock-4-8mm-Hose/dp/B08GQDBY46/	Used to attach frequently-replaced parts to tubing.
Luer Lock to 3/16 hose barb fittings - polypropylene - male	Amazon	https://www.amazon.com /Syringe-Adapter-Male-Lue r-Inch-Luer-Connector-Kit/d p/B0C1FV8NGL/	Used to attach frequently-replaced parts to tubing.
Magnetic stir bar (optional)	Millipore Sigma	Z282456	Used with stir plate to mix MS media. Can alternatively use a spoon.
Magnetic stir plate (optional)	Fisher	S194615	Used to disolve salts in media.  Can also use a spoon.
MS Salts	Caisson	MSP01	Used as hydroponic solution. Dilute to 0.25 x.
Needle-nosed plyers	Grainger	2078216	Used to hold wires when soldering
Nitrile Gloves	Genesee	https://www.geneseesci.com/ product/x-gen-nitrile-gloves- powder-free/	To protect hands when working with bleach and for sterile technique. Brand does not matter.
O2 Flow meter	JIAWANSHUN	https://www.amazon.com/JIAW ANSHUN-0-1-1-5LPM-Flowmeter -Connector-Conectrator/dp/B01N0UWZ2T	Used to control CO2 flow into the chamber.
Pipettor - 20 µL and tips	Millipore Sigma	EP3124000040-1EA	To measure TritonX for seed sterilization.  Brand doesn't matter.
Plastic cling wrap	Costco	https://www.costco.com/p/-/kirkl and-signature-stretch-tite-plasti c-food-wrap-12-in-x-3000-ft/100300513	Used to cover boxes while seeds are germinating to increase humidity and improve plant establishment.
Plastic Peg board (19"D x 5"W x 12"H)	Amazon	https://www.amazon.com/SWAN LAKE-Plastic-Pegboard-Panels -Garage/dp/B0BGS3C41N/	Components, wires, and tubing were attached to pegboard to faciliate easy moving and organization.  Exact brand doesn't matter.
Power supply with barrel jack connector - 5V 2A	Amazon	https://www.amazon.com/Acloro l-100-240V-Universal-Converter -Transformer/dp/B099RBBTWY /ref=sr_1_4?crid=XKSME1C2 86OE&keywords=5v%2Bpower %2Bsupply&qid=1683217539& s=hi&sprefix=5v%2Bpower%2 Bsupply%2Ctools%2C93&sr= 1-4&th=1	Used to power solenoids. Exact brand doesn't matter.
Precision digital scale	Fisher	FB30786159	Used for measuring MS salts and MES.
Push-to-connect 1/4 inch to 1/4 inch NPT	Amazon	https://www.amazon.com/CE KER-Connect-Fittings-Pneu matic-Connectors/dp/B07XBHYP3B/	Used to connect 1/4 inch tubing to tank regulator
Push-to-connect elbow to 3/8 inch NPT	Amazon	https://www.amazon.com/Q olekog-Pneumatic-Connec t-PL-3-8-N2/dp/B0B59JXMRW/	Used to connect 1/4 inch tubing to step down regulator (could alternatively use a straight connector, depending on your setup configuration)
Push-to-connect T-connector - 1/4 inch	Amazon	https://www.amazon.com /dp/B07TV3JS9Q/	(optional) use if splitting 1/4 inch line from CO2 tank between two chambers.
Quick connectors for large wire	XHF	B086J49D1J	Optional, but useful for temporarily splicing wires.
Raspberry Pi 4 2 GB Starter Kit - 32 GB	Canakit	PI4-2GB-STR32F-C4-BLK	Controls sensors and relays.  $110 at time of purchase for kit.
Raspberry Pi OS	Raspberry Pi Foundation	https://www.raspberrypi. com/software/	Operating system for pi.
RealVNC Viewer	RealVNC	https://www.realvnc.com/en/ connect/download/viewer/raspberrypi/	Used for remote monitoring the pi readouts.
Red Garnet Amaranth seeds	Mary's Heirloom Seeds	https://www.marysheirloomseeds .com/products/red-garnet-amaranth	Make sure seeds are from a recent seed lot.
Red King F1 Radish seeds	Johnny's Seeds	https://www.johnnyseeds.com/ vegetables/radishes/daikon-ko rean-radishes/red-king-2-f1-ra dish-seed-4173.html	Make sure seeds are from a recent seed lot.
Relays	uxcell	ASH-10 DD	Controls solenoids and light power supply.
SA-20 Disinfectant	Southern Ag	#10501	Fungicidal cleaner for growth chamber.  Avoid spraying on electronic parts.
Safety glasses	Fisher Scientific	19-130-2089	Recommended for eye protection. Brand does not matter.
Screwdriver - multiple bits	Grainger	6XW12	Used to take apart chamber and screw items together during construction.
Silicone mat	Amazon	https://www.amazon.com/K aisi-Insulation-Soldering-Ma intenance-Electronics/dp/B073RFB6BX/	(Optional, but recommended) To protect work surfaces from heat when soldering
Small monitor with HDMI hookup (optional)	Amazon	https://www.amazon.com/LON CEVON-7-Raspberry-1024X6 00-Speakers-Earphone/dp/B06XQJVXHL/	Optional, but sometimes helpful when setting up or monitoring a pi. We recommend using an existining HDMI computer monitor you may already have, but provide a link for an inexpensive option, if you need one.
Small screwdriver set	Amazon	https://www.amazon.com/Screw driver-FIXITOK-Screwdrivers-Pe ntalobe-Different/dp/B093372MGD/	Used for uncrewing small screws
Socket Headers for Raspberry Pi Pico	Adafruit	Product #:5583	Optional to use instead of jumper cords.
Solder - lead free, rosin core	Amazon	https://www.amazon.com/Dia0 -032in-0-11lb-Precision-Electr onics-Soldering/dp/B07Q167J 98/ref=sr_1_5?crid=17RNDW GBX6X5O&keywords=lead+f ree+solder&qid=1692729157 &sprefix=lead+free+solder% 2Caps%2C160&sr=8-5	Needed to attach wires to lights. Any lead-free rosin core solder works for this.
Soldering iron	Amazon	https://www.amazon.com/Wel ler-120V-Soldering-Iron-Halo/ dp/B0962SX47X/ref=sr_1_4?c rid=NE9BVDKLETUL&keyw ords=weller+soldering+iron&qi d=1692727772&s=industrial&s prefix=weller+soldiering+iron% 2Cindustrial%2C190&sr=1-4	Needed to attach wire to lights.
Stainless steel pipe fitting - 20 mm OD, 16 mm ID, 2.5 cm long (3/8 inch x 3/8 inch female NPT thread)	Amazon	https://www.amazon.com/ gp/aw/d/B08SCL8VRS	Used to extend cable glands through box insulation
Stainless steel pipe fitting - 8 mm ID/12 mm OD, 3 cm long (M8 female threaded both sides)	Amazon	https://www.amazon.com/ gp/aw/d/B09ZS17WYB	Used to extend bulkhead fitting through box insulation
STEMMA QT / Qwiic JST SH 4-Pin Cable - 50mm Long	Adafruit	Product #: 4399	Used to connect sensors to each other.
STEMMA QT / Qwiic JST SH 4-pin to Premium Male Headers Cable - 150mm Long	Adafruit	Product #: 4209	Used to connect sensors to pi.
Sterile DI water	Produced on site	Produced on site	Autoclave DI water at L30 cycle.
Surge Protector/Power strip	Amazon	https://www.amazon.com/GE- Outlet-Protector-Extension- 14092/dp/B00DOMYL24/	Use to protect chamber components from power surges.
Teflon plumbing tape	Amazon	https://www.amazon.com/ Inches-Plumbers-Plumbing- Plumber-Sealing/dp/B091913Z7F/	To seal threads of connectors
Thonny (Python IDE) - should come pre-installed on pi	Thonny	https://thonny.org/	Used to edit and run chamber control software.
TritonX surfactant	Fisher Scientific	https://www.fishersci.com/ shop/products/triton-x-100 -thermo-scientific/AAA16046AE	To improve coating of seeds with bleach water during surface sterilization.
uxcell Miniature Solenoid Valve 2 Way Normally Closed DC5V 0.22A Water Air Solenoid Valve	uxcell	a19051000ux0811	Three needed for chamber and we recommend having a few spares on hand.
Waterproof cable pass-through pack	MAKERELE	‎NPT(38)Box4A-B	Used to create an air tight pass through for wires into the chamber. Used 3/8 inch cable glands.
Wire - stranded, silicone-insulated, 22 G	Amazon	https://www.amazon.com/Fermerry -Stranded-Electric-Tinned-Copper/ dp/B089CQHRDT/ref=sr_1_1? crid=PEFEI7BQBPNB&keywords =22%2Bgauge%2Bwire&qid=168 5586908&sprefix=22%2Bguage% 2Bwire%2Caps%2C144&sr=8-1&th=1	Needed to connect items to pi.  Exact brand doesn't matter.
Wire stripper	Amazon	https://www.amazon.com/Southwire -Equipment-S1626STR-Stranded-Stripping/dp/B00LQMRS7O/	Used to cut and strip insulation of wires for lights and other electrical connections
Zipties	Amazon	https://www.amazon.com/HAVE -ME-TD-Cable-Ties/dp/B08TVLYB3Q/	Used to organizer wires and attach tubing to solenoids. Exact brand doesn't matter, but need a variety of small sizes.