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.
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Method Article
Building and Operating a Low-Cost Elevated Carbon Dioxide Growth Chamber to Evaluate Microgreen Physiology under Spaceflight-Relevant CO2 Levels
In This Article
Summary
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....
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Protocol
1. Installing cables and sensors through the back of the benchtop incubator and sealing with silicone sealant (Figure 2)
- 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.
- 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 lon....
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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.......
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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 .......
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Disclosures
None of the authors has conflicts of interest related to the work described above.
Acknowledgements
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.
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Materials
| 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. |
References
- De Micco, V., et al. Plant and microbial science and technology as cornerstones to Bioregenerative Life Support Systems in space. Npj Microgravity. 9, 69(2023).
- Georgescu, M. R., Meslem, A., Nastase, I.
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