Experimental design.

In the current study, CO₂ was the main independent variable, and split-plot was the experimental design. CO₂ was injected into the entire growth chamber, making it the main plot with four simultaneous PPFD treatments as subplots. Two replicates of all treatment combinations with subplots were conducted over time. The first replicate included CO₂ concentrations of 400, 800, 1200, and 1600 µmol·mol⁻¹, followed by a second replicate with the same CO₂ concentrations.

Environmental settings.

Experiments were conducted in a single walk-in growth chamber (EGC, Chagrin Falls, OH, USA), with experimental replications conducted over time. Air temperature was set to 23/21 ± 2 °C during light/dark periods, respectively. Relative humidity was maintained at 70% ± 5%. CO₂ injection/maintenance began on day 8 after sowing seeds, when the first set of true leaves had emerged. The CO₂ set point was 400, 800, 1200, or 1600 µmol·mol⁻¹ during the photoperiod and 400 µmol·mol⁻¹ during the dark period. Although 400 µmol·mol⁻¹ was the near-ambient setpoint, 800 µmol·mol⁻¹ is the industry standard; 1200 and 1600 µmol·mol⁻¹ are elevated CO₂ concentrations that were maintained for treatment comparisons.

Sole-source-lighting-system adjustments.

Four height-adjustable LED fixtures (Biomass Production System for Education; ORBITEC/Sierra Space Corporation, Madison, WI, USA) were mounted on wire-mesh benches in the walk-in growth chamber. Each fixture included three channels of blue, green, and red wavebands with peak emission wavelengths of 448 nm, 530 nm, and 627 nm, respectively. Photoperiod was adjusted to 16 h from 0600 HR to 2200 HR daily. The vertical distance between photon-emitting surface and cropping surface was set to 15 cm (5.9 inches). Using a spectroradiometer (LI-180 Spectrometer, LI-COR Bioscience, Lincoln, NE, USA), target PPFD and spectral composition were determined as the average of four corner points and one central point under each fixture. At each given CO₂ concentration, PPFD was set to 200 µmol·m⁻²·s⁻¹ during the first 4 d after sowing seeds, with a spectral composition of 82% red, 9% blue, and 9% green. On day 4, using the same spectral composition, PPFD treatments of 200, 300, 400, and 500 µmol·m⁻²·s⁻¹ were randomly assigned to the four LED fixtures. Each fixture was equipped with a power/energy meter (Poniie, PN-2000, accuracy-class 1.0 with 0.01 W, 0.01 V, and 0.001A resolution), and cumulative energy consumption (kWh) was recorded daily and at the end of the cropping cycle.

Plant material and substrate.

Red Oakleaf lettuce cv. Rouxai (Rijk Zwaan, De Lier, Netherlands) was used as the test model crop for this study. Plants were grown in 36-cell trays (CN-IKN-606, Greenhouse Megastore, Danville, IL, USA) placed within a propagation tray (25.4 × 50.8 cm) filled with a 50:50 (v/v) coco coir/perlite substrate at a planting density of 279 plants/m². Translucent domes were placed over trays to promote uniform germination immediately after sowing seeds. Two layers of wicking fiber (Cap-Mat II, Hummert International, Earth City, MO, USA) were placed in the bottom of the trays to ensure uniform nutrient-solution delivery to each cell. Initially, trays were overhead-irrigated with 1500 mL of tap water, ensuring adequate, uniform substrate hydration. A commercial fertilizer solution containing macro and micro elements (Fancy Lettuce, AmHydro, Arcata, CA, USA) was dissolved in reverse osmosis water to reach an electrical conductivity (EC) of 1.2 to 1.4 mS·cm⁻¹ and a pH of 5.6 to 5.8, measured with a portable EC/pH meter (HI 9813-6 pH/EC/TDS/C, Hanna Instrument GroChek, Woonsocket, RI, USA). Bottom fertigation with upward capillary wicking started on day 4 after sowing seeds, at which time domes were removed. Four hundred milliliters of fertilizer solution were then added to one corner of each tray every other day. Foliar canopy closure coincided with harvest 17 d after sowing seeds. Nine plants from each treatment were randomly selected for nondestructive image analyses of pigmentation and chlorophyll concentration. The largest leaf of each plant was clamped for chlorophyll measurement (MC-100 Chlorophyll Concentration Meter, Apogee Instruments, Logan, UT, USA). Overhead photographic images were used for pigmentation analysis using “png” package within R-studio statistical software (R Foundation, Vienna, Austria) to calculate L*a*b values defined by the International Commission on Illumination. The greater the a* value, the redder the leaf, whereas the smaller the a* value, the greener the leaf. The same methodology was used by Kelly and Runkle (2023a, 2023b).

Shoot fresh mass of individual plants was measured immediately upon harvest (PL602E, Mettler-Toledo, Columbus, OH, USA). Leaf area was subsequently measured using a leaf area meter (LI-3100C, LI-COR Bioscience), after which individual plant tissues were bagged and oven-dried for 4 d (Isotemp 180L oven; Thermo Fisher Scientific, Waltham, MA, USA) before shoot dry mass was measured.

Statistical analysis.

Growth data within a given CO₂ concentration were pooled for each PPFD tested. Using statistical software (version 9.4, SAS Institute Inc., Cary, NC, USA), data were subjected to linear-mixed effect model analysis. P values were determined using least square means, and α < 0.05 was used to determine differences between means, where appropriate.

EUE calculations.

Although measured parameters were expressed on a “per-plant” basis, EUE was expressed as total shoot fresh mass produced in a given tray under each PPFD tested. Cumulative energy (kWh) associated with the LED lighting system for that tray was recorded over the entire cropping cycle, and EUE was calculated based on the following formula:$$\text{Energy}\text{use\hspace{0.17em}efficiency}\hspace{0.17em}=\frac{\text{ Total shoot fresh mass }(\text{g})}{\text{Cumulative energy expended for LED lighting }(\text{kWh})}$$

Cropping cycle estimation.

Response to PPFD and CO₂ concentration: shoot fresh mass.

Within each CO₂ concentration tested, increasing PPFD increased shoot fresh/dry mass, indicating that PPFD was limiting to crop productivity within the range tested. At 400 µmol·mol⁻¹ CO₂, the highest PPFD of 500 µmol·m⁻²·s⁻¹ yielded 73%, 45%, and 14% higher shoot fresh mass than did 200, 300, and 400 µmol·m⁻²·s⁻¹, respectively (Fig. 1A). At 800 µmol·mol⁻¹ CO₂, growth increased robustly between PPFDs of 200, 300, and 400 µmol·m⁻²·s⁻¹, but began to taper off between 400 and 500 µmol·m⁻²·s⁻¹. No significant difference was noted in shoot fresh mass between PPFDs of 400 and 500 µmol·m⁻²·s⁻¹, although shoot fresh mass was 41% and 19% higher under the highest PPFD compared with 200 and 300 µmol·m⁻²·s⁻¹, respectively (Fig. 1A). Unlike 800 µmol·mol⁻¹ CO₂, at 1200 or 1600 µmol·mol⁻¹ CO₂, shoot fresh mass at 500 µmol·m⁻²·s⁻¹ was 17% and 13% higher than at 400 µmol·m⁻²·s⁻¹, respectively (Fig. 1A). At 1200 µmol·mol⁻¹ CO₂, shoot fresh mass was 36%, 22%, and 17% higher when 500 µmol·m⁻²·s⁻¹ was used compared with 200, 300, and 400 µmol·m⁻²·s⁻¹, respectively. A similar pattern was measured at 1600 µmol·mol⁻¹ CO₂, at which shoot fresh mass was 31%, 24%, and 13% higher for plants grown under the highest PPFD of 500 µmol·m⁻²·s⁻¹, compared with 200, 300, and 400 µmol·m⁻²·s⁻¹, respectively (Fig. 1A). Overall, among all 16 combinations tested, the highest shoot fresh mass occurred under the combination of 1200 µmol·mol⁻¹ of CO₂ and 500 µmol·m⁻²·s⁻¹ of PPFD (Fig. 1A).

Response to PPFD and CO₂ concentration: shoot dry mass.

At 400 µmol·mol⁻¹ CO_2, shoot dry mass of young lettuce plants grown under 500 µmol·m⁻²·s⁻¹ was 74%, 45%, and 10% higher than that achieved at 200, 300, and 400 µmol·m⁻²·s⁻¹, respectively (Fig. 1B). Likewise, at 800 µmol·mol⁻¹ CO₂, shoot dry mass at 500 µmol·m⁻²·s⁻¹ was 18%, 13%, and 10% higher than at 200, 300, and 400 µmol·m⁻²·s⁻¹, respectively (Fig. 1B). A slightly different trend was measured at 1200 µmol·mol⁻¹ CO₂, for which shoot dry mass was similar for 300 and 400 µmol·m⁻²·s⁻¹. Shoot dry mass at 500 µmol·m⁻²·s⁻¹ was 74%, 35%, and 28% higher than that at 200, 300, and 400 µmol·m⁻²·s⁻¹, respectively (Fig. 2B). At 1600 µmol·mol⁻¹, shoot dry mass under 500 µmol·m⁻²·s⁻¹ was 58%, 33%, and 12% higher than at 200, 300, and 400 µmol·m⁻²·s⁻¹, respectively (Fig. 1B). Similar to fresh mass, the highest shoot dry mass occurred at the combination of 1200 µmol·mol⁻¹ CO₂ and PPFD of 500 µmol·m⁻²·s⁻¹ among all 16 combinations tested.

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Response to PPFD and CO₂ concentration: leaf area.

Leaf area typically increased with increasing PPFD within each CO₂ concentration tested (Fig. 1C). For instance, at 400 µmol·mol⁻¹ CO₂, plants grown under a PPFD of 500 µmol·m⁻²·s⁻¹ had 95%, 37%, and 10% larger leaves compared with 200, 300, and 400 µmol·m⁻²·s⁻¹, respectively (Fig. 1C), whereas at 800 µmol·mol⁻¹ CO₂, no significant difference occurred for leaf area between 400 and 500 µmol·m⁻²·s⁻¹. Plants grown at 500 µmol·m⁻²·s⁻¹ had 41% and 9% larger leaves than those under 200 and 300 µmol·m⁻²·s⁻¹, respectively (Fig. 1C). At 1200 µmol·mol⁻¹ CO₂, plants grown at a PPFD of 500 µmol·m⁻²·s⁻¹ had 53% larger leaves than those grown under 200 µmol·m⁻²·s⁻¹, whereas leaf area for PPFDs of 300 and 400 µmol·m⁻²·s⁻¹ trended in-between those extremes (Fig. 1C). Similarly, at 1600 µmol·mol⁻¹ CO₂, plants grown under a PPFD of 200 µmol·m⁻²·s⁻¹ had 34% smaller leaves compared with at 500 µmol·m⁻²·s⁻¹, whereas 300 and 400 µmol·m⁻²·s⁻¹ trended in-between (Fig. 1C).

Response to PPFD and CO₂ concentration: specific leaf area.

Specific leaf area followed slightly different trends across tested CO₂ concentrations. Higher values of specific leaf area indicating degree of leaf thinness occurred at the lowest PPFD of 200 µmol·m⁻²·s⁻¹, whereas trends were slightly different at each given CO₂ concentration. For example, at 400 µmol·mol⁻¹ CO₂, the thinnest leaves occurred for plants grown under 200 µmol·m⁻²·s⁻¹, whereas no significant differences occurred among all other tested PPFDs (Fig. 1D). At the elevated 800, 1200, and 1600 µmol·mol⁻¹ CO₂ concentrations_, leaf thinness was similar for plants grown under 200 and 300 µmol·m⁻²·s⁻¹, and significantly lower from higher PPFDs of 400 and 500 µmol·m⁻²·s⁻¹ (Fig. 1D).

Response to PPFD and CO₂ concentration: pigmentation development.

Consistent with growth parameters, the highest value of a* occurred at 1200 µmol·mol⁻¹ CO₂ and 500 µmol·m⁻²·s⁻¹ PPFD. Plants grown at the lowest PPFD of 200 µmol·m⁻²·s⁻¹ had the least pigmentation, whereas young lettuce plants grown at 500 µmol·m⁻²·s⁻¹ PPFD had the most pigmentation across all CO₂ concentrations (Fig. 2A). No significant differences in pigmentation were measured for plants grown under PPFDs of 400 and 500 µmol·m⁻²·s⁻¹ across CO₂ concentrations, whereas the difference was significant between PPFDs of 300 and 200 µmol·m⁻²·s⁻¹ (Fig. 2A).

Response to PPFD and CO₂ concentration: chlorophyll concentration.

Both CO₂ concentration and PPFD were significant as main effects (P values = 0.034 and <0.0001, respectively). However, the CO₂ × PPFD interaction was not significant, suggesting that chlorophyll concentration was a function of increasing PPFD (Fig. 2B). The highest chlorophyll concentration occurred at the highest CO₂ concentration of 1600 µmol·mol⁻¹ and the highest PPFD of 500 µmol·m⁻²·s⁻¹ (Fig. 2B), whereas the lowest chlorophyll concentration occurred at the lowest PPFD of 200 µmol·m⁻²·s⁻¹, with PPFDs of 300 and 400 trending in-between the two extremes (Fig. 2B).

Energy-use efficiency.

Cumulative energy for LED lighting recorded for 17-d cropping cycles was 19, 24, 29, and 36 kWh for PPFDs of 200, 300, 400, and 500 µmol·m⁻²·s⁻¹, respectively. An EUE of 1.82 g kWh⁻¹ was found for the industry-standard combination of 200 µmol·m⁻²·s⁻¹ PPFD × 800 µmol·mol⁻¹ CO₂. However, under PPFDs of 300, 400, or 500 µmol·m⁻²·s⁻¹ at the same 800 µmol·mol⁻¹ CO₂ concentration, EUE declined to 1.69, 1.58, and 1.35 g kWh⁻¹, respectively. At an even higher CO₂ concentration of 1200 µmol·mol⁻¹, EUE at 200 µmol·m⁻²·s⁻¹ PAR was 2.00 g kWh⁻¹, but declined to 1.76, 1.51, and 1.43 g kWh⁻¹ at the same increasing PPFDs. Thus, within a given CO₂ concentration, increasing PPFD was accompanied by progressively diminishing returns for gains in crop biomass. At the near-ambient CO₂ concentration of 400 µmol·mol⁻¹, the degree of diminishment was less steep with an average slope of −0.04, while the average slope was −0.156, −0.19, and −0.20 at CO₂ concentrations of 800, 1200, and 1600 µmol·mol⁻¹, respectively (Fig. 3).

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Cropping-cycle estimation.

At the industry-standard CO₂ concentration of 800 µmol·mol⁻¹, shoot fresh mass of a baby-stage crop grown at a PPFD of 500 µmol·m⁻²·s⁻¹ was 1.4-fold higher than that of a crop grown at 200 µmol·m⁻²·s⁻¹. The feasibility of shortening a cropping cycle at higher PPFD can be estimated:$$\text{cropping cycle at PPFD of }500\hspace{0.17em}= \frac{\text{cropping cycle at PPFD of }200}{1.4} =\frac{17}{1.4}=\text{12\hspace{0.17em}}\left(\text{days}\right)$$

Thus, $$\text{Annual number of cropping cycles }\text{at PPFD of }500\hspace{0.17em}=\hspace{0.17em}\frac{365}{12}\approx \text{3}0$$

Shoot fresh mass of plants grown at a PPFD of 400 µmol·m⁻²·s⁻¹ was 1.32-fold higher than that at 200 µmol·m⁻²·s⁻¹. Thus,$$\text{Cropping cycle at PPFD of }400=\hspace{0.17em}\frac{\text{cropping cycle at PPFD of }200}{1.32}=\frac{17}{1.32}=\text{12}.\text{8\hspace{0.17em}}\left(\text{days}\right)$$

Thus, $$\text{Annual number of cropping cycles }\text{at PPFD of }400\hspace{0.17em}=\hspace{0.17em}\frac{365}{12.8}\approx \text{28}$$

Shoot fresh mass produced at a PPFD of 300 µmol·m⁻²·s⁻¹ was 1.17-fold higher than that at 200 µmol·m⁻²·s⁻¹. Cropping cycle at $\text{PPFD}\text{of}300\hspace{0.17em}=\mathrm{ }\frac{\text{cropping}\text{cycle}\text{at}\text{PPFD}\text{of}200}{1.17}\hspace{0.17em}=\hspace{0.17em}\frac{17}{1.17}=\text{14}.\text{5 }\left(\text{days}\right)$

Thus,$$\text{Annual number of cropping cycles }\text{at PPFD of }300\hspace{0.17em}=\hspace{0.17em}\frac{365}{14.5}\hspace{0.17em}=\hspace{0.17em}\text{25}$$