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

Abstract

Open-top chambers (OTCs) consist of semi-open enclosures used to investigate the impact of elevated carbon dioxide [CO₂] 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 m² and a target elevated CO₂ concentration [CO₂] of 650 µmol mol⁻¹ adequate for trials involving cereals or grain legumes. The elevated CO₂ chambers maintained an average concentration ± standard deviation of 652 ± 37 µmol mol⁻¹ despite wind and air turbulences, in comparison to 407 ± 10 µmol 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 CO₂ control system for four chambers dedicated to CO₂-enriched conditions cost USD 5388. To maintain the concentration of 650 µmol mol⁻¹ during daylight hours, each chamber consumed 1.38 L min⁻¹ of CO₂. This means that a size G CO₂ cylinder was consumed in 8–9 days in the operation of two chambers (at USD 40).

1. 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 CO₂ Enrichment (FACE) design [2]. This design could be used for any gas, but it has achieved some popularity in CO₂ 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 (CO₂) release in response to wind speed and direction and consequent ambient CO₂ levels. The wall-less nature of these systems is attractive in terms of maintaining ambient conditions other than the gas of interest; however, their CO₂ use is high. Also, the control of CO₂ 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 [CO₂], 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 CO₂ studies, the source of CO₂ is typically a cylinder of compressed pure CO₂, which is diluted to the desired concentration with ambient air before introduction to the cuvette.

Early examples of studies employing OTCs include investigations on the impact of air pollution on plants, i.e., the effect of ozone (O₃) on tobacco [7] and the effect of elevated CO₂ [8,9,10]. OTC designs generally comprise a framework support for a transparent cover, e.g., polyvinyl chloride, plexiglass, or LDPE glasshouse film, forming an open-topped chamber. The chambers are typically cylindrical [11,12,13], although square or rectangular [14], hexagonal [15,16], and octagonal [17,18] shapes have been used.

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 CO₂ 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 CO₂ 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 CO₂ 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 CO₂ 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 CO₂ 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 open-top chamber (OTC) of 1.2 m² area tailored specifically for investigating the impact of elevated CO₂ 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 CO₂ 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 CO₂ 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 CO₂ 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 (NH⁴⁺), 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.

2. Design

2.1. OTC Design

The design goals included the following:

(i)

a chamber large enough to produce a crop of peanuts, allowing sampling of ‘non-edge’ 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 [CO₂] treatment.

The chambers were designed to enclose a ground area of 1.25 m² (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].

CO₂ sensors were installed within the chambers, with feedback to a CO₂ control system operating with a set point of 650 ± 50 µmol mol⁻¹, with a final control element of a valve on the CO₂ input line. The CO₂ source was bottled pressurized pure CO₂ (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 CO₂ concentration distribution within the chamber compared to the injection of pure CO₂ 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 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 m³ s⁻¹, while the backflow pipe connected to the box allowed the recycling of CO₂ air mix at the rate of 0.028 m³ 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.

2.2. 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 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).

3. Build Instructions

3.1. Bill of Materials

A Bill of Materials is presented in

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 USD as per 2021.

Item	Brand/Model	Specifications/Description	Cost	Units	Total
OTC Framing and Ventilation System
Garden beds	Plastic Forests	Diameter: 125 cm; height 72.5 cm; thickness: 5 mm; length 400 cm	118.80	1	118.80
Plastic posts	Plastic Forests	Section: 3.8 × 3.8 cm; length; 180 cm	7.20	8	57.60
Poly pipe	Holman	Length: 500 cm per OTC; diameter: 2.5 cm	7.20	1	7.20
Greenhouse LDPE film	Redpath, Hortiplus180	Light transmission >91%; thickness: 180 µm	36.00	1	36.00
Mounting tape	Permastik	Length used: 800 cm; width: 2.4 cm	7.20	2	14.40
Mixing box	Inabox	Size: 60 × 39 × 39 cm; capacity: 55 L	10.18	1	10.18
Box sealant	Moroday	Length used: 200 cm	5.03	1	5.03
Blower	Fanco VKM 100	Voltage 220/240, capacity 270 m3 h−1, 2830 r min−1	151.20	1	151.20
Ducting	Fanco	Length: 4 m; diameter: 10 cm	18.72	1	18.72
Plugs (nozzles)	K-Rain	Internal diameter: 1.9 cm	0.27	40	10.66
T-joint drain coil	Vinidex	Diameter: 10 cm	6.31	1	6.31
PVC connector	Vinidex	Diameter: 10 cm	2.87	2	5.75
Duct clamps	Pacific Air	Diameter: 12.5 cm	2.95	2	5.90
Screws	Buildex	Length: 3–5 cm	NA	50	15.84
Angle molding	Brutus	Size: 25 × 25; thickness: 2.5 mm	4.64	2	9.29
Backflow pipe	Vinidex	Length used: 600 cm; diameter: 5 cm	15.53	1	15.53
Consumables	NA	Cable ties, wire, tape, silicone sealant, etc.	NA	NA	72.00
			Subtotal		560.41
Irrigation for an OTC (Inside OTC only)
Poly pipe	Holman	Length used: 300 cm	1.94	1	1.94
Barbed elbow	Pope	Diameter: 1.9 cm	0.92	2	1.84
T-joint	Pope	Diameter: 1.9 cm	0.78	1	0.78
Inline tap	Pope	Diameter: 1.9 cm	5.58	1	5.58
End plug	Pope	Diameter: 1.9 cm	0.57	3	1.71
Drippers	Netafim	Flow: 0.5 L h−1	0.35	30	10.58
			Subtotal		22.43
CO2 regulation for four units
CO2 probe	GMP252 CO2 Vaisala	Operating temperature: −40/+60 °C	607.00	4	2428.00
CO2 transmitter *	Indigo 520 Vaisala	M12 5-pin cable; RJ45-ethernet cable; built-in web server	1179.00	2	2358.00
Solenoid	Bürkert 6013	Voltage: 24 V; 2 ports; 1/4 in G; flow factor: 0.23 m3 h−1	92.16	1	92.16
Gas regulator *	BOC CO2 Regulator	Inlet: type 30; outlet: Side 5/8” UNF RH; outlet pressure 400 kPa	164.95	2	330.00
Other/Not Classified	NA	Fittings, connectors, tubing, clamps, etc.	NA	NA	180.00
			Subtotal		5388.16
Cost for 4 elevated CO2 units.					USD 7719
Cost for 4 ambient CO2 units.					USD 2331
Cost for 4 control CO2 units.					USD 565
Total OTC facility cost					USD 10,615

Note. * Served two OTCs. NA: not applicable

, describing the costs for the OTC framing, ventilation, and CO₂ regulation systems and irrigation.

3.2. 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 m³ 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).

3.3. 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).

3.4. CO₂ Injection System

Bottled CO₂ (size G, cylinder water capacity 50 L containing 31 kg CO₂, or 18 m³ at 25 °C) and 1 atm Industrial Grade (gas code 081, >99.9% CO₂, initial pressure 20 MPa, 2.0 Sm³ at 15 °C and 101.3 kPa) was used. Outlet pressure was set at 90–100 KPa (12–14 psi) using a CO₂ 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 m³ 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 CO₂ 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 CO₂ 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 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 .csv file. 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.

4. Operating Instructions

4.1. Set up and Run

In our experience, the operation of the CO₂ dosing chain to the OTCs can be trouble-free, 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 CO₂ 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 CO₂ 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 CO₂ on-site.

Also, it is recommended to progressively raise the CO₂ 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.

4.2. 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.

4.3. Troubleshooting

During severe precipitation events, the CO₂ control system showed low input signal from the CO₂ 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.

5. Validation

5.1. 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 thermo-anemometer (Protech, model QM1646, Electus distribution, Sydney, Australia). CO₂ concentration was measured using a CO₂ 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 CO₂ concentrations were not different (p ≤ 0.05) in the calm and windy period. CO₂ concentration profiles and airflow patterns were quite different for an OTC without the conical collar, with much less uniformity in CO₂ 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 CO₂ concentration (Figure 5a). This did not occur with the collar installed, providing more consistent elevated CO₂ 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 excess CO₂ 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.

5.2. CO₂ Concentration

CO₂ measurements were acquired at 2 min intervals over a 12 h period. The concentration inside the chamber was within 50 µmol mol⁻¹ (±8%) of the target 650 µmol mol⁻¹ in 43% of observations, within 100 µmol mol⁻¹ (±15%) of the target in 79% of observations, and within 150 µmol mol⁻¹ (±23%) of the target in 92% of observations (

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

CO2 Concentration Range (µmol mol−1)	Range as % of Set Point	% of Observations
±50	±8	43
±100	±15	79
±150	±23	92

).

5.3. 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 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).

5.4. System CO₂ Consumption

With CO₂ injected to maintain a concentration of 650 µmol mol⁻¹, CO₂ consumption was, on average, 3.15 L min⁻¹ (0.22 m³ 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 m³ 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 m³ h⁻¹) per chamber, requiring the replacement of a 31 kg CO₂ cylinder replacement every 8–9 days for the operation of four chambers.

A single size G compressed CO₂ bottle contains 31 kg CO₂, or 704 moles CO₂, 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 m³ (×2 = approx. 60 ML or 60,000 m³ of gas for 650 µmol m⁻² s⁻¹). With the blower operating at 270 m³ 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, CO₂ consumption rates, temperature, and overall performance results are reposted in

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

Attribute	This Study	Messerli et al. [15]
Capital cost (USD per 12 units)	USD 10,615 for 12 units.	USD 14,000 for eight units.
Ground area per chamber	1.25 m2	1.2 m2
Chamber volume	1.5 m3	1.2 m3
CO2 usage	×2 31 kg CO2 cylinders every 8–9 days (daylight hours injection) to maintain 650 µmol mol−1 target in 4 OTC. Average daily consumption of 0.99 kg/day/chamber at the rate of 1.38 L min−1	22.68 kg CO2 every 4–7 days to maintain 600 µmol mol−1 4 OTCs, 5.5 kg/5 day = 1.1 kg/day/chamber Average daily CO2 consumption of 3.0 kg m−2 of elevated CO2 area (design to mix fresh air and recirculated air in 5:1 ratio)
Air flow into chamber	Rate of the fresh ambient air taken inside the mixing box: 0.047 m3 s−1; backflow: CO2/air mix recycling rate: 0.028 m3 s−1.	5 air changes per minute 5.66 m3/min−1
CO2 control	79% of observations within 100 µmol mol−1 ±15% to target 650 µmol mol−1	93% of the time within ±20% (i.e., 120 µmol mol−1) of the targeted 600 µmol mol−1 CO2, based on 10 min averages
Temperature	OTCs warmer than unchambered plots (control) by 2.3 °C (30-day measurements) during daylight h. Night temperatures were 0.6 °C in OTC over control plots. OTCs warmer when ventilation system was turned off by 0.7 °C	OTCs 0.7 °C warmer than control plot similar to climate change prediction (seasonal measurements). Midday temperature increase 1.03 °C; night temperature increases 0.43 °C

. 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.

5.5. 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 CO₂ (EC), four OTC with ambient CO₂ levels (AC), and four control beds. The injection of CO₂ 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 [CO₂] 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 CO₂ assimilation rates and the transpiration. The crop performance results are reported in

Table 4. Peanut crop parameters after 30 days of growth in (a) no chamber, (b) OTC with ambient CO₂ levels; (c) OTC with 650 µmol m⁻² s⁻¹ CO₂. Means ± SD, n = 4 chambers per each treatment.

Attribute	No Chamber (Control)	OTC with Ambient (~400 µmol mol−1) CO2 Levels (AC)	OTC with 650 µmol mol−1 CO2 (EC)	p Value
Above ground dry biomass (g) per chamber (of 10 plants)	61.4 ± 8.9 b	60.9 ± 12.1 b	87.8 ± 24.8 a	0.0060
Net CO2 assimilation rate (µmol m−2 s−1)	32.2 ± 3.9 a	29.1 ± 3.5 a	35.1 ± 8.2 a	ns
Intrinsic WUE (µmol CO2 mol−1 H2O)	3.2 ± 0.1 b	3.1 ± 0.1 b	4.4 ± 0.3 a	0.0024
PAR at ground level (% of Control)	-	76	76	-

Note. Same superscript letters within the row indicate no statistical difference between groups according to Tukey’s post hoc test.

.

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 CO₂ 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 CO₂ (EC) compared to the chambers with ambient CO₂) (p = 0.0060) (Table 4). Similarly, net CO₂ 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 CO₂ [27].

6. Conclusions

The design of an open-top chamber facility described in this research effectively showcases the impact of elevated CO₂ 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 CO₂ on plants.

The test performed on the OTCs and CO₂ 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 CO₂ usage from 2.68 min⁻¹ to 1.38 L min⁻¹.

The daily average CO₂ 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 CO₂ 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 to release the amount of water according to the crop need, geographical location, and soil characteristics. A potential limitation that could affect the interpretation of the effects of elevated CO₂ is the increased temperature inside the chambers, which could confound the effects attributed solely to CO₂. As a mitigation measure, each chamber ventilation system was equipped with a blower which effectively contributed to reduce temperature through the high-volume air produced. Furthermore, the facility included OTCs with ambient CO₂ levels for direct comparison and “control plots” without chambers which are recommended to reflect current CO₂ and environmental conditions accurately.