Design of a Low-Cost Open-Top Chamber Facility for the Investigation of the Effects of Elevated Carbon Dioxide Levels on Plant Growth

: Open-top chambers (OTCs) consist of semi-open enclosures used to investigate the impact of elevated carbon dioxide [CO 2 ] on crops and larger plant communities. OTCs have lower operational costs than alternatives such as controlled environment cabinets and Free Air Carbon Dioxide Enrichment (FACE). A low-cost design is presented for an OTC with a surface area of 1.2 m 2 and a target elevated CO 2 concentration [CO 2 ] of 650 µ mol mol − 1 adequate for trials involving cereals or grain legumes. The elevated CO 2 chambers maintained an average concentration ± standard deviation of 652 ± 37 µ mol mol − 1 despite wind and air turbulences, in comparison to 407 ± 10 µ mol mol − 1 for non-enriched chambers. Relative to ambient (non-chamber) conditions, plants in the chambers were exposed to slightly warmer conditions (2.3 ◦ C in daylight hours; 0.6 ◦ C during night environment). The materials’ cost for constructing the chambers was USD 560 per chamber, while the CO 2 control system for four chambers dedicated to CO 2 -enriched conditions cost USD 5388. To maintain the concentration of 650 µ mol mol − 1 during daylight hours, each chamber consumed 1.38 L min − 1 of CO 2 . This means that a size G CO 2 cylinder was consumed in 8–9 days in the operation of two chambers (at USD 40).


Introduction
The study of plant responses to changed atmospheric composition can be conducted using leaf cuvettes for the consideration of short-term effects on leaf physiology prolonged for minutes or a few hours.For the study of a whole plant and plant community (crop) responses observed in the medium-term, including weeks to several months, larger cuvettes are required.This can be accomplished using controlled environment cabinets, also known as phytotrons, which involve an artificial environment with a high level of control of the temperature, humidity, light levels and gas levels, but these involve high capital and maintenance costs [1].
An alternative is the Free Air CO 2 Enrichment (FACE) design [2].This design could be used for any gas, but it has achieved some popularity in CO 2 enhancement research.The FACE design involves modification of the atmosphere of an open (wall-less) area of a crop or native ecosystem providing insights on plants' phycological responses that are noticeable in the long term, typically from one to more years.A pipe system is used for distribution within a typically circular area, with a control system adjusting gas (CO 2 ) release in response to wind speed and direction and consequent ambient CO 2 levels.The wall-less nature of these systems is attractive in terms of maintaining ambient conditions other than the gas of interest; however, their CO 2 use is high.Also, the control of CO 2 levels could be compromised in windy conditions.For instance, Allen et al. [3] reported that fluctuations in FACE experiments can be >200 ± 50 µmol mol −1 above the target [CO 2 ], although some designs showed that concentrations can be maintained at or higher than 90% of the target for 93-98% of the time [4].
OTCs are semi-open framed structures designed to maintain the desired concentration of gaseous pollutants and represent a compromise between cost and environmental modification [5,6].An area of ground is enclosed by greenhouse film or transparent sheeting, with the top left open to the atmosphere.A concentrated gas source is ducted into a mixing chamber, mixed with ambient air, and the mixed gas is then ducted into the base of the OTC.The gas concentration inside the open cuvette is usually monitored to control how much gas is delivered.For elevated CO 2 studies, the source of CO 2 is typically a cylinder of compressed pure CO 2 , which is diluted to the desired concentration with ambient air before introduction to the cuvette.
The chamber size is varied, from small conical units enclosing single plants to multiple plants and larger plant communities enclosed in structures having >10 m diameter [5].In the context of climate change, OTCs represent a valuable tool for studying the effects of elevated CO 2 on plants, but assessing their long-term sustainability and scalability is crucial.The advantage of using OTCs relies on their sustainability due to both low environmental impact in terms of carbon footprint and costs, including reasonable initial investments and ongoing operational expenses.
Moreover, OTCs can be effectively expanded from small-scale experiments to larger ecosystems or broader research applications involving growth environment manipulation, such as, for instance, nitrogen sink and source experiments [19].However, while on one hand, larger structures with wider openings allow for studying the long-term impacts of elevated CO 2 on trees and forest ecosystems, on the other hand, they are more exposed to wind interference, which consequently makes it difficult to maintain the gas concentration close to the target involving higher capital and operational costs [20,21].
The chamber can also cause a heating effect, mitigated by the high volume of air passing through the ventilation system.However, it has been noted that the temperature inside OTCs dedicated to temperature-manipulation experiments is uniform across vertical and horizontal space [22].
With CO 2 levels currently rising at ~2.17 µmol mol −1 per annum [23], there is a wide interest in undertaking studies to anticipate the impact of this change on crop performance.Such studies need to be undertaken at the whole plant/crop level to evaluate not only the effect of elevated CO 2 on leaf-level photosynthetic rates but also on plant functions such as assimilate partitioning between organs.In our case, interest in the effect of elevated CO 2 on peanut growth in the context of a 'graze and grain' strategy, i.e., off-take of a hay crop before flowering and pod set, initiated our interest in the use of an OTC design.
The present study outlines the design, specifications, and performance of an opentop chamber (OTC) of 1.2 m 2 area tailored specifically for investigating the impact of elevated CO 2 levels on smaller broadacre crops.This study builds upon the designs implemented in previous research, refining and adapting these earlier models to better suit the specific requirements of assessing CO 2 effects under controlled environmental conditions, particularly Messerli et al. [15].
The key point of our approach lies in presenting a cost-effective open-top chamber (OTC) design.These modifications not only aim to enhance the accuracy and reliability of the results but also provide critical insights into the effects of increased CO 2 concentrations on crop growth and development in smaller agricultural settings.This practical, economical solution allows for detailed observation and analysis, ensuring that our findings are both accessible and applicable to a wide range of agricultural research scenarios.
Although this facility was specifically designed to conduct CO 2 enrichment experiments, the design provides substantial versatility to accommodate studies involving a range of gaseous pollutants.For instance, the chambers can be suited for investigating the impacts of nitrogenous compounds such as ammonia (NH 4+ ), commonly emitted from agricultural practices and livestock operations, as well as sodium fluoride, which often originates from industrial processes including aluminium production and phosphate ore processing.
Additionally, the facility could be adapted for experiments with ozone, a pollutant produced when nitrogen oxides (NOx) from vehicle exhaust and industrial emissions reacting with sunlight.To facilitate ozone research, significant modifications would be necessary, such as the application of PTFE coatings to protect the structural integrity of the chamber from ozone's highly reactive nature.

OTC Design
The design goals included the following: (i) a chamber large enough to produce a crop of peanuts, allowing sampling of 'nonedge' plants.(ii) a chamber that is sufficiently low-cost and operationally easy to build to allow multiple units to be built and replicated in experimental designs.(iii) imposition of a 650 µmol mol −1 elevated [CO 2 ] treatment.
The chambers were designed to enclose a ground area of 1.25 m 2 (Figure 1a).A raised garden bed of 1.25 m diameter was used to facilitate the use of farm soil and ease of operator access to the crop (Figure 1b) The enclosing octagonal chamber was 1.2 m high, with an open conical top (frustrum) to reduce the amount of wind-induced turbulence within the chamber, extending the suggestion of Drake et al. [9].CO 2 sensors were installed within the chambers, with feedback to a CO 2 control system operating with a set point of 650 ± 50 µmol mol −1 , with a final control element of a valve on the CO 2 input line.The CO 2 source was bottled pressurized pure CO 2 (BOC, NSW, Sydney, Australia).
A VKM 100 Centrifugal Inline blower, (Fanco, Melbourne, VIC, Australia) was used to deliver a relatively high flow rate into the chamber, causing an even CO 2 concentration distribution within the chamber compared to the injection of pure CO 2 and to mitigate temperature increase inside the OTC due to a chamber greenhouse effect [24].
Air intake to the blower consisted of the regulated pure CO 2 feed from the bottled pressurized pure CO 2 , ambient air, and recycled chamber air from pick-up bottoms around the base of the frustrum.The opening at the bottom of the mixing box allowed the supply of fresh ambient air at the flow rate of 0.047 m 3 s −1 , while the backflow pipe connected to the box allowed the recycling of CO 2 air mix at the rate of 0.028 m 3 s −1 (as measured using a thermo-anemometer Protech, model QM1646, Electus Distribution, Sydney, Australia).Hence, approximately 35% of the CO 2 air mix was recycled into the system through the backflow piping.

Replication
Replication is required in any experimental evaluation.In the case of OTC work, the replication of chambers is required at the chamber level.However, the chamber itself may impact the plant growing environment, and so, a treatment involving plants grown in the raised bed without a chamber is recommended.At least three replicate units are recommended, giving a minimum build of nine units, i.e., three beds without chambers, three beds with chambers and ventilation operating at ambient CO 2 , and three beds with chambers and chambers operating at elevated CO 2 , placed in a random arrangement.In our build, four replicate units were constructed for each of the three treatments (Figure 2).Air intake to the blower consisted of the regulated pure CO2 feed from the bottled pressurized pure CO2, ambient air, and recycled chamber air from pick-up bottoms around the base of the frustrum.The opening at the bottom of the mixing box allowed the supply of fresh ambient air at the flow rate of 0.047 m 3 s −1 , while the backflow pipe connected to the box allowed the recycling of CO2 air mix at the rate of 0.028 m 3 s −1 (as measured using a thermo-anemometer Protech, model QM1646, Electus Distribution, Sydney, Australia).Hence, approximately 35% of the CO2 air mix was recycled into the system through the backflow piping.Replication is required in any experimental evaluation.In the case of OTC work, the replication of chambers is required at the chamber level.However, the chamber itself may impact the plant growing environment, and so, a treatment involving plants grown in the raised bed without a chamber is recommended.At least three replicate units are recommended, giving a minimum build of nine units, i.e., three beds without chambers, three beds with chambers and ventilation operating at ambient CO2, and three beds with chambers and chambers operating at elevated CO2, placed in a random arrangement.In our build, four replicate units were constructed for each of the three treatments (Figure 2).

Bill of Materials
A Bill of Materials is presented in Table 1, describing the costs for the OTC framing, ventilation, and CO2 regulation systems and irrigation.

Bill of Materials
A Bill of Materials is presented in Table 1, describing the costs for the OTC framing, ventilation, and CO 2 regulation systems and irrigation.

Garden Beds
The garden beds (125 cm diameter × 72.5 cm height; from Plastic Forests, North Albury, New South Wales, Australia) were manufactured from a stabilized UV-resistant recycled plastic material (Simply Cups recycling program).Each garden bed was divided into two halves by inserting a plastic board, to allow for sub-plot treatments, e.g., of water or fertilization.Each bed was filled with 1.0 m 3 of agronomically relevant soil, in this case, a ferrosol (according to the Australian soil classification), acquired from a farm located in Rossmoya, Queensland.The soil was excavated from the horizons between A1 and B2 (0-600 mm).

Chamber
The construction of each chamber was based on eight 3.6 cm square, 180 cm long recycled-plastic pickets (Plastic Forests, North Albury, NSW, Australia).These uprights were placed in an octagonal pattern against the inside wall of the garden bed ring, driven through the garden bed soil to ground level, and fixed to the bed using 3 cm hex-head screws (Figure 1b).The posts were connected at their tops with a polyethylene (PE) pipe ring (392 cm circumference, 3 cm diam), providing more rigidity to the structure, and a truncated conical frame was then mounted onto this structure.
A glasshouse low-density polyethylene (LPDE) film of 180 µm thickness and >91% light transmission (Hortiplus180, from Redpath, Bendigo, VIC, Australia) was placed around the frame and fixed with double-sided clear mounting tape.As specified by the manufacturer, the plastic film contains surface tension modifiers to prevent condensation and dirt accumulation that could degrade light transmission and is UV-stabilized, with a 30-month warranty provided.An opening allowed operator access into the OTC.Initially, 'door' closing was facilitated by adhesive "hook and loop" strips with double-sided tape, applied on an opening flap of plastic film and on an upright frame.However, this arrangement was not robust.A simple hinged door frame (approx.100 × 45 cm) was constructed of PVC angle building molding (2.5 × 2.5 cm).

CO 2 Injection System
Bottled CO 2 (size G, cylinder water capacity 50 L containing 31 kg CO 2 , or 18 m 3 at 25 • C) and 1 atm Industrial Grade (gas code 081, >99.9% CO 2 , initial pressure 20 MPa, 2.0 Sm 3 at 15 • C and 101.3 kPa) was used.Outlet pressure was set at 90-100 KPa (12-14 psi) using a CO 2 regulator (BOC 801325, NSW, Sydney, Australia).The regulator outlet was fitted with a dual split connector on which two flowmeters (BOC 10521, NSW, Sydney, Australia) set at 1 L min −1 were installed.Plastic tubing (65 mm diameter) was used in the connection of the flowmeter output and the solenoid inlet and in the connection of the solenoid outlet to the mixing box of the respective OTC chamber.
The gas mixing box consisted of a plastic container (60 × 40 × 35 cm) with an opening of 5 × 5 cm width cut into the base of the container to allow air entry.A neoprene adhesive foam tape was placed along the top edges of the box to affect a seal with the lid.An electric blower (73 W, 270 m 3 h −1 , 2830 r min −1 , Figure 3) (VKM 100, Fanco) was mounted inside the container with the intake facing the internal area of the mixing box which was employed to propel CO 2 through a lay-flat aluminium/plastic flexible ventilation ducting (~50 cm in length; 10 cm diam) connecting the blower to the chamber with a PVC dual split coil (10 cm diam), and finally, a ducting made of the same material (400 cm long; 10 cm diam) was connected to both ends and placed along the OTC circumference to serve as a plenum.
The plenum was perforated at regular intervals (~5 perforations of 22 mm diameter per linear meter), and a plastic irrigation barbed plug having 19 mm internal diameter was placed in each hole as a nozzle and fixed with reinforced insulation tape.The decision to add a nozzle instead of just leaving an open perforation in the ducting was justified by several considerations.Having outlets in each perforation ensures a steady distribution of the air mix, allowing for consistent levels of gas injection.Additionally, the angle of each nozzle can be adjusted as needed, which allows for a more targeted dispersal of CO 2 in the OTC environment.Lastly, this setup prevents the perforation from enlarging with time and prevents water entering the plenum and, consequently, into the mixing box.
To improve CO 2 level stability during windy periods, a corrugated PVC pipe (50 mm diameter) (Vinidex, Sydney, NSW, Australia) was installed around the chamber's top opening, with a connection to the gas mixing box.This allowed a partial capture of exiting gases, recycling into the air mix cycle.This backflow pipe was perforated at 10 cm intervals with 19 mm diameter holes and equipped with nozzles for the plenum.To improve CO2 level stability during windy periods, a corrugated PVC pipe (50 mm diameter) (Vinidex, Sydney, NSW, Australia) was installed around the chamber's top opening, with a connection to the gas mixing box.This allowed a partial capture of exiting gases, recycling into the air mix cycle.This backflow pipe was perforated at 10 cm intervals with 19 mm diameter holes and equipped with nozzles for the plenum.
The CO2 sensors (GMP252, Vaisala, Helsinki, Finland) were mounted inside the four elevated CO2 chambers.The sensors (Figure 4) were connected to two control systems (Indigo520, Vaisala, Helsinki, Finland).The CO2 controllers were programmed to log the CO2 concentration data at two-minute intervals, with data downloaded through a LAN linked to the Vaisala web interface and saved as a .csvfile.The transmitters also activated solenoid valves (Bürkert 6013 G1/4 24V) to regulate CO2 release on a set point of 650 µmol mol −1 , with the hysteresis value set at 150 µmol mol −1 .The value of 150 µmol mol −1 was chosen with the purpose of avoiding short cycling of the system which can cause wear and tear of the solenoid components and over-heating.Therefore, the solenoids were switched off automatically when the internal [CO2] exceeded 800 µmol mol −1 and switched on when dropping below 500 µmol mol −1 .The CO 2 sensors (GMP252, Vaisala, Helsinki, Finland) were mounted inside the four elevated CO 2 chambers.The sensors (Figure 4) were connected to two control systems (Indigo520, Vaisala, Helsinki, Finland).The CO 2 controllers were programmed to log the CO 2 concentration data at two-minute intervals, with data downloaded through a LAN linked to the Vaisala web interface and saved as a .csvfile.The transmitters also activated solenoid valves (Bürkert 6013 G1/4 24V) to regulate CO 2 release on a set point of 650 µmol mol −1 , with the hysteresis value set at 150 µmol mol −1 .The value of 150 µmol mol −1 was chosen with the purpose of avoiding short cycling of the system which can cause wear and tear of the solenoid components and over-heating.Therefore, the solenoids were switched off automatically when the internal [CO 2 ] exceeded 800 µmol mol −1 and switched on when dropping below 500 µmol mol −1 .

Set up and Run
In our experience, the operation of the CO2 dosing chain to the OTCs can be troublefree, although maintenance of and access to a stock of spare components (blower, sensors,

Set up and Run
In our experience, the operation of the CO 2 dosing chain to the OTCs can be troublefree, although maintenance of and access to a stock of spare components (blower, sensors, valves) is recommended, as for any equipment.The primary operational issue is allowance for purchasing and delivery time of CO 2 cylinders.Longer-term (over several years) maintenance includes the replacement of the plastic sheeting, yearly calibration of sensors, and checking of electrical components, e.g., blower.
The gas supply to the four OTCs was carried out using two 31 kg gas cylinders which were monitored daily for timely replacement, as needed, to avoid CO 2 enrichment gaps.The size of the cylinder was relatively easy to handle; however, gas supply involved manual replacement of cylinders, which is time-consuming and labour-intensive.Such limitations could be overcome by using an automatic electronic switching system able to open the valves of another couple of cylinders, by using high-capacity tanks or by producing CO 2 on-site.Also, it is recommended to progressively raise the CO 2 sensors within each chamber to a constant distance above crop canopy height throughout the growing season, to ensure that the foliage does not cover the sensor.
The use of soil moisture probes, not discussed in this report, is recommended for agronomic studies, given the biomass and water use efficiency (WUE) differences, and thus water use, of plants between treatments.

Maintenance
The ducting used for the plenum and the clear plastic sheeting are affected by the UV component of solar radiation, with degradation obvious after 12 months in the harsh tropical location of the current experiment.Metal ducting would be resistant to UV and extreme weather conditions but would result in higher cost.Annual replacement is the alternative.The chamber plastic film required cleaning with a soapy solution to remove soil dust, insects, and bird droppings.However, the light transmissibility of the clean plastic decreased to 71% after 30 months, presumably due to UV damage, and replacement (nominally at 2 years) is recommended.

Troubleshooting
During severe precipitation events, the CO 2 control system showed low input signal from the CO 2 probes, Vaisala GMP 252, due to condensation on the optical surface of the sensor.The issue was solved by letting the sensor dry for some hours.In some situations, it was required to remove the filter for drying (using a compressed air instrument followed by placement in a sealed container with dry silica for 12 h).
During the trials conducted on peanuts from 2021 to 2024, it was observed that crops inside OTCs were slightly more prone to pests and diseases with mild infestations of Cowpea aphids (Aphis craccivora) and peanut mites (Paraplonobia spp.) Finally, in our experimental trials, we observed that crops cultivated in garden beds without OTC tended to grow beyond the bed edges, benefiting from unrestricted space.To mitigate the edge effect and maintain standardized growth conditions, we suggest using guard plants planted in the outer areas of the beds and regularly clipping overgrown branches during the season.These guard plants must not be considered during in-season measurements and must be discarded at harvest.Alternatively, we suggest installing short barriers, e.g., plastic or metal mesh, around the garden beds to contain the plants, especially if using crops with a decumbent or prostrate habitus like peanuts.

Airflow
Airflow direction was visualized using a non-toxic smoke-emitting tool (Smoke-Pen, Bjornax, Sweden), which is used for testing airflow in industrial ventilation systems.
Airflow speed and temperature were recorded for each point with a handheld thermoanemometer (Protech, model QM1646, Electus distribution, Sydney, Australia).CO 2 concentration was measured using a CO 2 probe, Vaisala GMP 252.Measurements were taken at regular intervals (20 cm) from ground level to above the open chamber top, both along the side wall and in the centre of the OTC.Measurements were conducted during a calm period and a period with a constant (2.78 m s −1 at time of measurement) wind.Measurements were also made of a chamber without the open conical collar top on the day of constant wind.
Chamber CO 2 concentrations were not different (p ≤ 0.05) in the calm and windy period.CO 2 concentration profiles and airflow patterns were quite different for an OTC without the conical collar, with much less uniformity in CO 2 concentration within the chamber (Figure 5).Air from the ventilation ducting flowed in horizontally at the base of the chamber, then tended to flow upwards through the centre part of the chamber and out into the general atmosphere.Without the collar, a downward draught along the inner wall of the chamber brought atmospheric air into the OTC, lowering CO 2 concentration (Figure 5a).This did not occur with the collar installed, providing more consistent elevated CO 2 levels within the OTC (Figure 5b).The position of the CO2 control system sensor was considered.The target concentration of 650 µmol mol −1 was maintained at positions up to 60 cm from ground level, which exceeds the canopy height expected for this crop.A sensor height of between 30 and 45 cm was adopted, a choice that was driven by the optimal performance of the system to maintain the target concentration enhancing the accuracy in monitoring the CO2 levels (Figure 6).
The CO2 target was not maintained when the control sensor was positioned at 90 cm above ground level, due to the dilution of CO2 in the chamber air with ambient air (Figure 7).In this circumstance, the control system attempts to maintain the set point, resulting in The position of the CO 2 control system sensor was considered.The target concentration of 650 µmol mol −1 was maintained at positions up to 60 cm from ground level, which exceeds the canopy height expected for this crop.A sensor height of between 30 and 45 cm was adopted, a choice that was driven by the optimal performance of the system to maintain the target concentration enhancing the accuracy in monitoring the CO 2 levels (Figure 6).The suction in the return line situated on the inside of the base of the plenum was visually determined using a smoke pen (Figure 8).The suction strength was greatest in the inlets located in the proximity (30° to either side) of the junction with the main pipe, medium at 30°-120° either side of the junction, and low in the range 120°-180° on either side of the junction.The CO 2 target was not maintained when the control sensor was positioned at 90 cm above ground level, due to the dilution of CO 2 in the chamber air with ambient air (Figure 7).In this circumstance, the control system attempts to maintain the set point, resulting in excess CO 2 levels within the crop.The suction in the return line situated on the inside of the base of the plenum was visually determined using a smoke pen (Figure 8).The suction strength was greatest in the inlets located in the proximity (30° to either side) of the junction with the main pipe, medium at 30°-120° either side of the junction, and low in the range 120°-180° on either side of the junction.The suction in the return line situated on the inside of the base of the plenum was visually determined using a smoke pen (Figure 8).The suction strength was greatest in the inlets located in the proximity (30 • to either side) of the junction with the main pipe, medium at 30 • -120 • either side of the junction, and low in the range 120 • -180 • on either side of the junction.

Air Temperature
OTC internal temperature was recorded at hourly intervals with temperature dataloggers positioned above canopy height (45 cm) next to the CO2 probes.There was no difference in day time (05:00-18:00) temperature averages for OTC operated at ambient and elevated CO2 levels.However, these chambers were warmer (with approximately 2.3 °C higher temperature) than the no-chamber control plots (Figure 9).The within-OTC and control temperatures gap was narrower in cloudy or rainy days.The night air temperature (19:00-4:30) was increased by 0.6 °C inside the OTCs (23.99 °C), compared to unchambered (control) plots' temperature (control), with an average value of 23.40 °C.These temperature variations could alter biological processes such as plant growth, metabolic rates, and phenological events.OTCs also modify other aspects of the microclimate, including humidity, light exposure, and wind flow.Such differences, particularly between day and night, introduce additional variability that may not accurately reflect natural conditions, thereby challenging the ecological validity and reproducibility of the results across different settings.To mitigate these effects, it is recommended to standardize OTC design, accurately monitor environmental conditions, and employ ambient OTC for direct comparison, while unchambered plots can serve as a reference for current environmental conditions.This "chamber effect" can be easily detected by investigating changes in

Air Temperature
OTC internal temperature was recorded at hourly intervals with temperature dataloggers positioned above canopy height (45 cm) next to the CO 2 probes.There was no difference in day time (05:00-18:00) temperature averages for OTC operated at ambient and elevated CO 2 levels.However, these chambers were warmer (with approximately 2.3 • C higher temperature) than the no-chamber control plots (Figure 9).The within-OTC and control temperatures gap was narrower in cloudy or rainy days.The night air temperature (19:00-4:30) was increased by 0.6 • C inside the OTCs (23.99 • C), compared to unchambered (control) plots' temperature (control), with an average value of 23.40 • C.These temperature variations could alter biological processes such as plant growth, metabolic rates, and phenological events.OTCs also modify other aspects of the microclimate, including humidity, light exposure, and wind flow.Such differences, particularly between day and night, introduce additional variability that may not accurately reflect natural conditions, thereby challenging the ecological validity and reproducibility of the results across different settings.To mitigate these effects, it is recommended to standardize OTC design, accurately monitor environmental conditions, and employ ambient OTC for direct comparison, while unchambered plots can serve as a reference for current environmental conditions.This "chamber effect" can be easily detected by investigating changes in morphological traits on plants such as, for instance, flower count, fresh weight measurements, and physiological parameters such as photosynthetic rates and stomatal conductance [25].morphological traits on plants such as, for instance, flower count, fresh weight measurements, and physiological parameters such as photosynthetic rates and stomatal conductance [25].When the ventilation system of an OTC was turned off at the start of a day, the average air temperature through the monitored day was higher by 0.7 °C within the chamber without ventilation than the chamber with ventilation (Figure 10).When the ventilation system of an OTC was turned off at the start of a day, the average air temperature through the monitored day was higher by 0.7 • C within the chamber without ventilation than the chamber with ventilation (Figure 10).morphological traits on plants such as, for instance, flower count, fresh weight measurements, and physiological parameters such as photosynthetic rates and stomatal conductance [25].When the ventilation system of an OTC was turned off at the start of a day, the average air temperature through the monitored day was higher by 0.7 °C within the chamber without ventilation than the chamber with ventilation (Figure 10).

System CO 2 Consumption
With CO 2 injected to maintain a concentration of 650 µmol mol −1 , CO 2 consumption was, on average, 3.15 L min −1 (0.22 m 3 h −1 ) per chamber for the non-collared OTC design, operated 24 h per day.Consumption was decreased to 2.67 L min −1 (0.16 m 3 h −1 ) when the system was operated during daylight hours (0530-1800) only.With the installation of the conical collar and recirculation of a portion of the airflow, consumption was decreased to 1.38 L min −1 (0.09 m 3 h −1 ) per chamber, requiring the replacement of a 31 kg CO 2 cylinder replacement every 8-9 days for the operation of four chambers.
A single size G compressed CO 2 bottle contains 31 kg CO 2 , or 704 moles CO 2 , which, at 25.4 L mole −1 , represents 17,881 L at 1 atm.This source can be diluted with ambient air to produce approximately 17,881/0.00065= 27.5 M L of mixture, or 27,509 m 3 (×2 = approx.60 ML or 60,000 m 3 of gas for 650 µmol m −2 s −1 ).With the blower operating at 270 m 3 h −1 , one bottle should, therefore, support operation for 60,000/270 = 300 h for a single chamber or 75 h for four chambers.Allowing for 35% recirculation of chamber air, the operation should be extended to 105 h for four chambers.With operation at 12 h a day, it should operate for approximately 9 days, consistent with observed usage.
The capital costs, OTC specifications, CO 2 consumption rates, temperature, and overall performance results are reposted in Table 3.The OTCs performance is compared to the findings presented by Messerli at al. [15], providing a comprehensive analysis of how our current results align or differed with the previous studies.

Plant Performance
Commercial peanut plants (Arachis hypogaea L., cultivar Sutherland) were planted in December 2021 into the 12 beds, including four OTC with 650 µmol mol −1 CO 2 (EC), four OTC with ambient CO 2 levels (AC), and four control beds.The injection of CO 2 to the four chambers (EC) started when the first leaves were fully developed (V-3) [26], and injection was terminated at harvest, 30 days after planting, when the peanuts reached the flowering stage (R1).The chamber's internal temperature was monitored with temperature probes (HOBO dataloggers, Bourne, MA, USA).Leaf gas exchange parameters were measured on one per plot of the last fully expanded young leaflet at the canopy top, one day before harvest, using a portable photosynthesis system (LI-COR 6800, LI-COR Inc., Lincoln, NE, USA).Measurements were conducted with a solar photosynthetic photon flux density (PPFD) manually set at 2000 µmol mol −1 .Humidity reference was set at 60%, fan speed at 8000 rpm, air flow at 500 mmol −1 , and cuvette temperature at 30 • C. The cuvette [CO 2 ] was adjusted based on the growth condition: 400 µmol mol −1 for AC OTC and 650 µmol mol −1 for EC OTC.Measurements were taken between 9:00-11:00 a.m. and 14:00-16:00 p.m.The intrinsic water use efficiency (WUE) was calculated as a ratio between net CO 2 assimilation rates and the transpiration.The crop performance results are reported in Table 4.Over the 30-day growth period, the accumulated above-ground biomass, measured as the dry weight of 10 plants per chamber, was similar between the control (no chamber) and the OTC with ambient CO 2 levels (AC), with values of 61.4 g (no chamber) compared to 60.9 g for AC.In contrast, the accumulated above-ground biomass was ~40% greater in the OTC with elevated CO 2 (EC) compared to the chambers with ambient CO 2 ) (p = 0.0060) (Table 4).Similarly, net CO 2 assimilation rates were greater for EC-grown peanuts compared to peanuts grown under ambient control and AC.This difference, however, was not significant.In contrast, intrinsic WUE was greater for EC-grown peanuts compared to control and AC-grown peanuts, and this difference was significant (p = 0.0024).These results are in close agreement with previous studies reporting the stimulation of biomass, photosynthesis, and water use efficiency of crops when grown under elevated CO 2 [27].

Conclusions
The design of an open-top chamber facility described in this research effectively showcases the impact of elevated CO 2 levels on crops, combining precision with cost effectiveness for a sustainable approach to agricultural research.Details of its construction and the results provided for the facility evaluation show that it represents a viable and practicable alternative to costly methods of ascertaining the effects of CO 2 on plants.
The test performed on the OTCs and CO 2 concentration control equipment highlighted the system's efficacy in maintaining the internal target concentration throughout the trial, which, for this research, was set to 650 ± 50 µmol mol −1 .Additionally, recycled chamber air was used, reducing overall CO 2 usage from 2.68 min −1 to 1.38 L min −1 .
The daily average CO 2 consumption observed during daylight hours injection in this study was 1.56 kg m −2 .Some adjustments and system fine-tuning were required to improve CO 2 consumption rates and limit wind interference.Simple modifications implemented to the OTC structure, such as partially modifying the opening with a conical top opening (frustum), resulted in effective measures for mitigating the issues caused by wind and improving the system's functioning.However, the use of a conical frame for the frustrum could lead to changes in the indoor-outdoor continuum as a trade-off, modifying the amount of received rainfall.However, such an issue can be overcome with automated irrigation systems set

Figure 1 .
Figure 1.Open-top chamber (OTC) design, with octagonal framing mounted on a raised garden bed (a).Pure CO2 is delivered at a controlled rate into a mixing box equipped with an industrial blower, which delivers the mixed gas to a perforated duct installed around the OTC circumference.(b) An OTC under fabrication where the framing and the garden bed are visible.(c) OTC showing connection to ventilation system.(d) Perforated back flow pipe inside the OTC.The original design document is in the Supplementary Materials.

Figure 1 .
Figure 1.Open-top chamber (OTC) design, with octagonal framing mounted on a raised garden bed (a).Pure CO 2 is delivered at a controlled rate into a mixing box equipped with an industrial blower, which delivers the mixed gas to a perforated duct installed around the OTC circumference.(b) An OTC under fabrication where the framing and the garden bed are visible.(c) OTC showing connection to ventilation system.(d) Perforated back flow pipe inside the OTC.The original design document is in the Supplementary Materials.

Figure 2 .
Figure 2. Overview of the experimental site, featuring the main components of the facility.The gas cylinders supplying carbon dioxide stand alongside the CO2 control system, with distinct octagonalshaped OTCs and control plots without chambers.

Figure 2 .
Figure 2. Overview of the experimental site, featuring the main components of the facility.The gas cylinders supplying carbon dioxide stand alongside the CO 2 control system, with distinct octagonalshaped OTCs and control plots without chambers.

Hardware 2024, 2 ,Figure 3 .
Figure 3. Representation of the Open-top chamber ventilation system which included (a) an industrial blower, (b) mixing box with inside fan used to propel the CO2/ambient air mix into the chamber.

Figure 3 .
Figure 3. Representation of the Open-top chamber ventilation system which included (a) an industrial blower, (b) mixing box with inside fan used to propel the CO 2 /ambient air mix into the chamber.

Hardware 2024, 2 ,Figure 4 .
Figure 4.The internal CO2 concentration was monitored using two CO2 system controllers Indigo 520 Vaisala (a); a CO2 probe protected by a weather shield was installed inside each open-top chamber dedicated to the CO2-enriched conditions (b).

Figure 4 .
Figure 4.The internal CO 2 concentration was monitored using two CO 2 system controllers Indigo 520 Vaisala (a); a CO 2 probe protected by a weather shield was installed inside each open-top chamber dedicated to the CO 2 -enriched conditions (b).

Figure 5 .
Figure 5. Airflow direction and speed, CO2 concentration, and temperature within (a) OTC without a conical collar and (b) with a collar.Measurements were conducted with a crop (canopy height = ~35 cm) planted in the beds and placing a control sensor at 45 cm from ground level.The original design document is in the Supplementary Materials.

Figure 5 .
Figure 5. Airflow direction and speed, CO 2 concentration, and temperature within (a) OTC without a conical collar and (b) with a collar.Measurements were conducted with a crop (canopy height = ~35 cm) planted in the beds and placing a control sensor at 45 cm from ground level.The original design document is in the Supplementary Materials.

Figure 6 .
Figure 6.Average [CO2] as measured on blank soil with no crop planted in the beds when the sensor was positioned at different heights from the soil level in an OTC with conical collar.Bars represent and standard deviation on measurements made over 3 h at 2 min intervals, i.e., n = 90.The red line represents the target concentration.

Figure 7 .
Figure 7.Comparison of system's performance when CO2 sensor was installed at 30 cm (a) and 90 cm (b) from the ground level.

Figure 6 .
Figure 6.Average [CO 2 ] as measured on blank soil with no crop planted in the beds when the sensor was positioned at different heights from the soil level in an OTC with conical collar.Bars represent and standard deviation on measurements made over 3 h at 2 min intervals, i.e., n = 90.The red line represents the target concentration.

Hardware 2024, 2 , 12 Figure 6 .
Figure 6.Average [CO2] as measured on blank soil with no crop planted in the beds when the sensor was positioned at different heights from the soil level in an OTC with conical collar.Bars represent and standard deviation on measurements made over 3 h at 2 min intervals, i.e., n = 90.The red line represents the target concentration.

Figure 7 .
Figure 7.Comparison of system's performance when CO2 sensor was installed at 30 cm (a) and 90 cm (b) from the ground level.

Figure 7 .
Figure 7.Comparison of system's performance when CO 2 sensor was installed at 30 cm (a) and 90 cm (b) from the ground level.

Hardware 2024, 2 ,Figure 8 .
Figure 8. Smoke test conducted on the backflow pipe inlets installed around the OTC circumference in the upper area.(a) The negative pressure formed in the mixing box generated a suction force which was maximum near the inlets located in proximal distance to the main backflow pipe; (b) and (c) The air suction reduced its effectiveness for the inlets located between 30°-120° and 240°-300° from the main pipe (0°).(d) The suction strength was minimal in the furthermost inlets to the main pipe (120°-240°).

Figure 8 .
Figure 8. Smoke test conducted on the backflow pipe inlets installed around the OTC circumference in the upper area.(a) The negative pressure formed in the mixing box generated a suction force which was maximum near the inlets located in proximal distance to the main backflow pipe; (b) and (c) The air suction reduced its effectiveness for the inlets located between 30 • -120 • and 240 • -300 • from the main pipe (0 • ).(d) The suction strength was minimal in the furthermost inlets to the main pipe (120 • -240 • ).

Figure 9 .
Figure 9. Average daily day-time air temperature in OTCs with ambient (AC) and elevated (EC) CO2 levels, and for the control plots without chambers.

Figure 10 .
Figure 10.OTC differential air temperature through a day calculated as the difference between temperatures recorded with blower on and off.

Figure 9 .
Figure 9. Average daily day-time air temperature in OTCs with ambient (AC) and elevated (EC) CO 2 levels, and for the control plots without chambers.

Figure 9 .
Figure 9. Average daily day-time air temperature in OTCs with ambient (AC) and elevated (EC) CO2 levels, and for the control plots without chambers.

Figure 10 .
Figure10.OTC differential air temperature through a day calculated as the difference between temperatures recorded with blower on and off.Figure10.OTC differential air temperature through a day calculated as the difference between temperatures recorded with blower on and off.

Figure 10 .
Figure10.OTC differential air temperature through a day calculated as the difference between temperatures recorded with blower on and off.Figure10.OTC differential air temperature through a day calculated as the difference between temperatures recorded with blower on and off.

Table 1 .
Items used for the fabrication of one OTC with cost description for installing a CO2 system for four elevated CO2 OTCs, four ambient CO2 OTCs, and four control plots.Costs are reported in

Table 1 .
Items used for the fabrication of one OTC with cost description for installing a CO 2 system for four elevated CO 2 OTCs, four ambient CO 2 OTCs, and four control plots.Costs are reported in USD as per 2021.

Table 2 .
Percentage of observations in each of three CO2 concentration ranges.The set point was 650 µmol mol −1 .

Table 2 .
Percentage of observations in each of three CO 2 concentration ranges.The set point was 650 µmol mol −1 .

Table 3 .
[15]ary of the OTC specifications and comparison with Messerli et al.[15]OTC design.