Determination of optimal daily light integral (DLI) for indoor cultivation of iceberg lettuce in an indigenous vertical hydroponic system

The indoor cultivation of lettuce in a vertical hydroponic system (VHS) under artificial lighting is an energy-intensive process incurring a high energy cost. This study determines the optimal daily light integral (DLI) as a function of photoperiod on the physiological, morphological, and nutritional parameters, as well as the resource use efficiency of iceberg lettuce (cv. Glendana) grown in an indoor VHS. Seedlings were grown in a photoperiod of 12 h, 16 h, and 20 h with a photosynthetic photon flux density (PPFD) of 200 µmol m−2 s−1 using white LED lights. The results obtained were compared with VHS without artificial lights inside the greenhouse. The DLI values for 12 h, 16 h, and 20 h were 8.64, 11.5, and 14.4 mol m−2 day−1, respectively. The shoot fresh weight at harvest increased from 275.5 to 393 g as the DLI increased from 8.64 to 11.5 mol m−2 day−1. DLI of 14.4 mol m−2 day−1 had a negative impact on fresh weight, dry weight, and leaf area. The transition from VHS without artificial lights to VHS with artificial lights resulted in a 60% increase in fresh weight. Significantly higher water use efficiency of 71 g FW/L and energy use efficiency of 206.31 g FW/kWh were observed under a DLI of 11.5 mol m−2 day−1. The study recommends an optimal DLI of 11.5 mol m−2 day−1 for iceberg lettuce grown in an indoor vertical hydroponic system.

www.nature.com/scientificreports/ to increase the production and productivity of lettuce in India using modern intensive cultivation techniques like vertical farming. Light is one of the most significant environmental variables in vertical farming that influences plant development and governs plant activity based on light quantity, quality, and direction 12,13 . Light can be naturally emitted by the sun, undergo spectrum transformation, or even be redirected. The primary energy source for traditional crop cultivation is sunlight, and the light that plants intercept in a natural setting fluctuates and is complex 14 . During cloudy days and especially in winters plants receive much less light than that required for its normal growth and development. Artificial grow lights can be used as supplemental light when natural sunshine is inadequate, and grow lights are the only source of light for vertical indoor farms with artificial lights 15,16 . Artificial lighting generates radiation that serves as both an energy source for photosynthesis and a biological signal that can modify plant morphology and quality. For effective, year-round indoor production of high-yield crops, it is important to maximize plant growth under artificial lights while simultaneously reducing expenses [17][18][19] . LEDs have the highest photosynthetically active radiation (PAR) efficiency of all the artificial light sources. They also allow for the optimal level of irradiance to be achieved in terms of biomass output and overall energy costs 20 . Previous studies 21,22 have shown that plants grown under a combination of red and blue LEDs look reddish, which makes it hard to identify the signs of disease and growth problems. There were no significant changes in all the physiological and yield characteristics of lettuce plants between white LEDs and cool white fluorescent bulbs. However, white LEDs consumed less electricity, indicating that white LEDs might effectively replace classic fluorescent bulbs 23,24 .
Adequate amount of light can encourage photosynthetic activity and increase biomass production, but too much light will lead to phenomenon of photoprotection and photodamage in plants 25 . Daily light integral (DLI) is the total amount of photosynthetically active radiation (400-700 nm) delivered to plants in a 24-h period. It is a function of photoperiod and light intensity (PPFD) and indicates the amount of light needed for plant growth and development. The DLI is a crucial environmental factor for plants and demonstrates a stronger association with biomass accumulation, plant growth [26][27][28][29] , nutritional quality 30 , and daily irrigation amount 31 . However, increasing DLI deteriorated visual appearance and reduced the quantum photosynthetic yield of PSII 32 , and light use efficiency 30 . There were no significant increases in the total fresh weight and total phenolic content of sweet basil when the DLI was increased above 16.5 mol m -2 day -112 . Gent 33 , studied the effect of daily sunlight integral on the concentration of secondary metabolites in hydroponically grown butterhead lettuce. The study suggested that lettuce should be harvested in the afternoon after growth under high light to get maximum nutritional quality. Runkle 34 suggested a minimum DLI of 12-14 mol m -2 day -1 for greenhouse lettuce production. Cui et al. 29 recommended a DLI of 6.35 mol m -2 day -1 for cucumber plug seedlings that could be achieved by using PPFD of 110-125 µmol m −2 s −1 and a photoperiod of 14-16 h. For indoor cultivation of red-leaf lettuce plants, a minimum DLI of 6.5 to 9.7 was suggested 35 . DLI requirement for lettuce ranges between 12 and 16 mol m -2 day -1 , however their value varies among species and cultivars 17,22,26,36 .
Increasing PPFD has a positive effect on the rate of photosynthesis of plants up to a certain intensity at which the maximum rate of photosynthesis occurs. Further increase in PPFD results in decreased light use efficiency and plant growth 37 . This is because photoprotective activities cause absorbed light energy to be converted into heat rather than allowing it to be utilized for electron transport in the light reactions that occur during photosynthesis 38 . Many researchers have demonstrated that PPFD in the range of 150-300 µmol m −2 s −1 is optimal for cultivation of lettuce under artificial lights 26,[39][40][41][42] or even above 300 µmol m −2 s −143-45 . Because of the higher energy requirement, light intensities greater than 300 are generally not recommended 46 . While adding the extra photons by increasing PPFD has diminishing marginal gains, increasing photoperiod is another strategy to augment DLI and gross photosynthesis 17,47,48 .
Photoperiod is an important environmental factor regulating plant growth and development. Many plants species sense daylength to determine when to transition from vegetative growth to reproductive development. Photoperiod can influence the quantity of light plants experience in a day, the entrainment of their circadian rhythm, and the crucial dark period necessary for flowering in photoperiodically sensitive plants 49,50 . Many studies have demonstrated that plants grown under the same DLI and increasing photoperiod at low PPFD resulted in more photosynthetic activity and biomass production 38,51,52 . Additionally, Arabidopsis 53 (Lepisto et al., 2009) and lettuce 54 were proven to have an increase in their biomass output when exposed to continuous light. continuous lighting also increased dry and fresh shoot biomass and plant growth of lettuce (cv. Yidali) under red and blue LEDs 14 . Similar results were obtained for Nasturtium (Tropaeolum majus L.) plants 55 . However, in another study 17 found that extending photoperiod from 16 to 24 h day −1 at DLI of 10.4 mol m −2 day −1 did not increase the dry weight of lettuce (cv. Rouxai) significantly. Additionally, CL had a detrimental impact on the rate of photosynthesis in leaves as well as the total dry weight of tomato plants 56 . It was also reported that CL has a negative impact on the fresh weight and dry weight of common ice plant 48 . Hence, the effect of photoperiod varies according to the type of crop as well as their cultivars (Supplementary Table 1).
It is important to emphasize that the plant density used in this study was in line with commercial cultivation of lettuce. This is because high-density plant spacing can result in light competition, which is not accounted for in studies that use a single plant per pot design. Therefore, it is crucial to consider high-density plant spacing to accurately evaluate the impact of light competition on plant growth and development. Both the amount of light a crop captures and how effectively it uses that light to produce biomass must be taken into account in order to accurately evaluate crop growth and productivity. One of the major disadvantages of vertical farming with artificial lights is higher electricity costs. The number of hours of light provided to plants is directly related to the cost of electricity. Hence, more research needs to be done on DLI strategies offered by sole-source white LEDs, taking energy consumption into consideration. Our study adds to the existing literature on DLI and lettuce production by assessing the impact of different DLI regimes on not only growth and yield but also nutritional quality and resource use efficiency of lettuce grown in greenhouse-based VHS, providing a more comprehensive www.nature.com/scientificreports/ understanding of DLI's impact on lettuce production. Furthermore, third study compares lettuce growth under different DLI regimes provided by artificial LED lights to that of natural sunlight inside a greenhouse, allowing us to identify the optimal DLI regime for lettuce production and provide insights into the relative advantages of artificial lights in comparison to natural sunlight, which was not done before. To our knowledge, no study has examined the response of iceberg lettuce to varying DLIs using sole-source white LEDs. Very few researchers have studied the impact of DLI by varying photoperiod and keeping the PPFD constant. Therefore, the aim of our study was to study the effect of photoperiod/DLI on crop growth, photosynthesis, and quality parameters and to deter mine the optimal photoperiod/DLI for iceberg lettuce in order to optimize plant growth while taking resource use efficiency into account. We hypothesized that a higher DLI would lead to higher biomass accumulation and nutritional quality. We tested the hypotheses with three DLI treatments obtained by varying the photoperiod.

Results
Fresh weight. The shoot and root fresh weight of lettuce grown in vertical hydroponic system with artificial lights were significantly affected (p < 0.05) by DLI at all sampling stages except the root FW at 20 DAT (Fig. 1   Leaf area. Leaf area of an iceberg lettuce grown in indoor hydroponic system showed a distinct response to DLI/photoperiod. Leaf area increased by 43% with increase in DLI from 8.64 to 11.5 mol m −2 day −1 (Fig. 3A), further increment in DLI to 14.4 mol m −2 day −1 , slightly decreased the leaf area (3%). However, the decrease in leaf area at 14.4 mol m −2 day −1 DLI was not statistically significant. No significant difference in all the treatments were found at 20 DAT. Also, the 8.64 mol m −2 day −1 DLI had 12% more leaf area compared to control treatment but the difference was not statistically significant.
Specific leaf area. DLI/Photoperiod did not significantly (p < 0.05) affect the specific leaf area of lettuce at the time of harvest. There was no consistent trend over the course of time between the photoperiod and specific leaf area (Fig. 3B). The specific leaf area for 8.64, 11.5 and 14.4 mol m −2 day −1 DLI treatments were not substantially different at all the sampling stages.

Number of leaves.
The number of leaves were significantly (p < 0.05) higher in the case of 11.5 mol m −2 day −1 DLI treatment compared to control treatment at the time of harvesting (Fig. 4A). The 11.5 mol m −2 day −1 DLI had the highest average number of leaves (24), followed by 8.64 (22), 14.4 (21), and control (19). There was no significant difference between all DLIs/ photoperiods at 10 DAT. It was also found that the DLI provided by artificial lights (8.64, 11.5, 14.4) have not substantially affected the number of leaves.

Root shoot ratio.
A decreasing trend was observed in root shoot ratio at each successive sampling stage The response curve for transpiration rate (Fig. 5B) showed a continuous increasing trend with increasing PPFD unlike Pr, where it was decreased after increasing PPFD from 1000 µmol m −2 s −1 to 1200 µmol m −2 s −1 . Augmenting PPFD from 400 to 600 µmol m −2 s −1 , increased the transpiration rate by 28%. However, further increase in PPFD to 800 and to 1000 µmol m −2 s −1 resulted in 14% and 11% respectively. For analyzing leaf gas exchange parameters, the control treatment which consisted of a three-layer VHS without artificial lights, was divided into top, middle, and bottom layer. We have studied the response of leaf gas exchange parameters per layer in control treatment consist of a three-layer VHS without artificial lights which was then compared with the VHS with artificial lights. We observed, for control condition that the photosynthetic activity of lettuce decreased substantially from top layer to bottom layer by 70% (Table 1). Increasing DLI from 8.64 to 11.5 mol m −2 day −1 , increased the photosynthetic activity by 22%. However, no significant difference was observed between 11.5 and 14.4 mol m −2 day −1 . Water use efficiency (Pn/E) was also found to be decrease from top layer to bottom in control treatment. The highest WUE was observed under a DLI treatment of 11.5 mol m −2 day −1 corresponding to a photoperiod of 16 h.
Qualitative parameters. All the qualitative parameters of lettuce were varied widely in response to changing photoperiod. Total phenolic content of 11.5 mol m −2 day −1 DLI (15.27 ± 0.49 mg GAE/100 g of FW) was significantly (p < 0.05) higher compared to 14.4 and control (Table 2). Total phenols were 2.5% higher in the 11.5 mol m −2 day −1 DLI than in the 8.64 mol m −2 day −1 DLI, which was not statistically significant. Additionally, total phenols in all the photoperiod treatments provided by artificial lights were substantially higher than that of control. The photoperiod had no effect on the antioxidant capacity of lettuce grown under artificial lights. However, it was significantly (p < 0.05) higher compared to control. The highest antioxidant capacity (8.48 ± 0.17 µmol Trolox/g FW) was observed under 11.5 mol m −2 day −1 DLI followed by 14.4 mol m −2 day −1 (8.05 ± 0.14 µmol Trolox/g FW) DLI. The antioxidant capacity of 11.5 mol m −2 day −1 DLI was 29.70% higher than the control. Vitamin C content of iceberg lettuce increased with increasing DLI from 11.5 to14.4 mol m −2 day −1 . However, there was no significant difference between8.64 and 11.5 mol m −2 day −1 DLI. Vitamin C content was found to be highest under14.4 (14.38 mg/100 g) period whereas the lowest was observed in case of control treatment (12.10 mg/100 g)0.14.4 mol m −2 day −1 DLI had 19% more vitamin C content than the control. The physiological parameters like leaf length, root length and plant height were also measured at time of harvest. Leaf length was not affected photoperiod of artificial LED lights but it was significantly higher compared to control. Root length   Bars represent means ± standard error. A significant (p < 0.05) variation in means is denoted by different letters according to Tukey's HSD test.  www.nature.com/scientificreports/ was observed at for the combination of 200 µmol m −2 day −1 and a photoperiod of 16 h ((DLI = 11.5 mol m −2 day −1 ) using four different LED lights provided at seedling stage. In our study, we found the similar trend at harvesting stage where the LED light treatment was provided after transplanting. The CO 2 concentration maintained by Yan et al. 22 was almost double (800 ± 50 μmol·mol −1 ) than our study. They tested the effects of two different photoperiods (14 and 16 h) and two different levels of PPFD (200 and 250 µmol m −2 day −1 ) while exposing the lettuce seedlings to four different light quality conditions. Dry biomass of lettuce increased substantially as DLI increased from 8.64 to 11.5 mol m −2 day −1 . Increase in dry weight was associated with increased leaf area of lettuce. However, further increment in DLI to14.4 mol m −2 day −1 decreased the fresh weight substantially demonstrating that an excessive amount of DLI had a negative impact on the accumulation of carbohydrates and even caused a reduction in the accumulation of dry weight 30,35,66 . There was no significant difference in dry weight of shoot and root between 11.5 and 20 h photoperiod. These results were in line with previous studies 26,64 . Increasing DLI from 11.5 to 14.4 mol m −2 day −1 decreased fresh weight and dry weight of leaves by 10% and 23% respectively. This may be explained by Photoinhibition which is defined by Xu and Shen 65 , as a light-induced decrease in photosynthetic efficiency that occurs when plants receive more light energy than they require for photosynthesis. Furthermore, the closure of stomatal pores and the subsequent reduction in gas exchange may account for the inferior performance of 20 h photoperiod 67 (DLI = 14.4 mol m −2 day −1 ). It was previously demonstrated that the fresh weight of green and red lettuce did not vary substantially in response to DLI levels ranging from 13 to 26 mol m −2 day −125 . Leaf area of lettuce was increased by 43% with increase in DLI from 8.64 to 11.5 mol m −2 day −1 under LED lights. Similar results were obtained by Refs. 62 www.nature.com/scientificreports/ lamps to study the impact of various combinations of photoperiod and PPFD on leaf lettuce. Carvalho et al. 70 , observed that the total phenolic content in leaves of sweet potato was higher at 16 h photoperiod compared to 8 h photoperiod with PPFD of 150 µmol m −2 day −1 . Antioxidants found in nutrient-dense plant foods, such as fruits and vegetables, are crucial for lowering the risk of oxidative stress-related chronic diseases 71,72 . Ali et al. 73 observed that the levels of antioxidant activity in swiss chard, red beet, green amaranth, red amaranth and red spinach were found to be higher at a photoperiod of 12 h. Antioxidant capacity of lettuce was found to be highest under a combination of 12 h photoperiod and half strength of nutrient solution 74 . Cho et al. 69 , demonstrated that the antioxidant capacity of green leaf lettuce increased up to 20 h and then decreased at 24 h. In our study, we observed that the antioxidant capacity was increased up to A DLI of 11.5 mol m −2 day −1 and then decreased at 14.4 mol m −2 day −1 . Antioxidant activity has been demonstrated by about 70% of the phenolic substances found in lettuce 75 , suggesting that the trends in total phenolic content and antioxidant level in this study may be similar to each other. Vitamin C content of iceberg lettuce increased with increasing DLI. Several other researchers reported similar trend 26,76 .
Effect of photoperiod/DLI on resource use efficiency of lettuce grown in vertical hydroponic system. The higher energy demand is one of the barriers in the widespread adoption of indoor vertical farming with artificial lights. Hence, increasing resource use efficiency helps to reduce costs and improve the system's overall sustainability. It can also assist in ensuring that the plants receive a sufficient amount of resources, which can improve their growth and yield. By optimizing resource use efficiency, the productivity as well as profitability of indoor hydroponic system can be increased while, also reducing their environmental impact. In the present study, WUE of lettuce was increased with increase in DLI up to 11.5 mol m −2 day −1 and then decreased thereafter. The highest WUE of 71 g FW/lit was observed at DLI of 11.52 mol m −2 day −1 . Our findings were in line with Pennisi et al. 77 but with higher DLI, where they observed that the WUE of butterhead lettuce (cv. Rebelina) was increased successively by increasing PPFD from 100 µmol m −2 day −1 (DLI = 5.8 mol m −2 day −1 ) to 250 µmol m −2 day −1 (DLI = 14.4 mol m −2 day −1 ). Increasing PPFD more than 250 µmol m −2 s −1 , resulted in a slight decrease in WUE. They observed the highest WUE of 60 g FW/lit, which is less than our findings. Further, Pennisi et al. 42 , observed that changes in photoperiod had no impact on WUE of butterhead lettuce (cv. Rebelina). WUE observed in the present study is far better than the WUE of lettuce reported in traditional open field and greenhouse condition. Barbosa et al. 78 reported WUE of lettuce in open field and greenhouse hydroponic system as 4 g FW/lit and 20 g FW/lit respectively. In the present study, energy requirement increased with increase in DLI. The maximum EUE of 206.31 g FW/ kWh was observed under a DLI of 11.5 mol m −2 day −1 . Zhang et al. 26 demonstrated that the EUE of lettuce was increased from 15.9 to 40.6 g FW/kWh by shifting from fluorescent lights to LED lights. Yan et al. 79 studied the effect of combination of various LEDs on EUE of green and purple leaf lettuce. They found that the maximum EUE of 80 g FW/kWh was observed under white LED lights. Pennisi et al. 42 revealed that the EUE of butterhead lettuce decreased with increase in photoperiod from 16 to 24 h at PPFD of 250 µmol m −2 s −1 under blue and red LEDs. The maximum EUE of 138 g FW/kWh was observed under 16 h photoperiod corresponding to a DLI of 14.4 mol m −2 day −1 . Improvements in the intrinsic characteristics of the light source, particularly the light's energy usage and photosynthetic photon efficacy, are primarily responsible for the significant increases in EUE observed in the present study 80 . The photosynthetic photon efficacy of LED lights used in our study was 2.2 µmol/J which was more than 1.52 µmol/J used by Pennisi et al. 42 .

Conclusion
It is essential to comprehend the response of plants to varying lighting conditions in order to determine the optimal light recipe. To ensure optimal plant growth under artificial lighting, the relationship between PPFD, photoperiod, DLI, and resource use efficiency should be considered when developing an optimal light control algorithm. Growers often wants to know the minimum PPFD and duration for optimal growth of crops to reduce the cost of cultivation in addition to resource use efficiency. This is the first study to demonstrate the response of an iceberg lettuce grown in vertical hydroponic system to various DLI obtained by altering photoperiod. Our study demonstrated that altering DLI by modifying photoperiod had a substantial effect on the growth and development of iceberg lettuce. We observed that increasing DLI by changing photoperiod from 8.64 (DLI = 8.64 mol m −2 day −1 ) to 16 h (DLI = 14.4 mol m −2 day −1 ) resulted in significant increase in carbohydrate accumulation, leaf area, and resource use efficiency of an iceberg lettuce, while 20 h photoperiod (DLI = 14.4 mol m −2 day −1 ) resulted in marginal decrease in physiological and morphological parameters and a significant decrease in resource use efficiency. Thus, from our study, we can conclude that the optimal growth, nutritional quality, and resource use efficiency of an iceberg lettuce was achieved at a photoperiod of 16 h corresponding to a DLI of 11.5 mol m −2 day −1 and a PPFD of 200 µmol m −2 s −1 under white LED lights. The study has demonstrated a practical light management strategy for growing iceberg lettuce under indoor vertical hydroponic system using minimal resources. Our findings suggest that determining an optimal DLI regime for lettuce production, farmers could potentially reduce lighting energy costs while also improving the nutritional quality of crop, which could have far-reaching implications for sustainable agriculture.
The present study revealed that the nutritional quality of lettuce improved up to threshold DLI, after which it decreased. Whereas in case of antioxidant content no significant change was observed under all DLI treatments. The study demonstrates that by manipulating the lighting conditions within the optimal range, growers can achieve higher resource use efficiency in VHS. Increasing the DLI within a specific range resulted in significant improvements in carbohydrate accumulation, leaf area, and resource use efficiency of the lettuce. This study has demonstrated a cost-effective light management strategy for indoor cultivation of iceberg lettuce in greenhouse based indoor VHS. The electrical conductivity (EC) of the nutrient solution was maintained between 1.2 and 2 dS/m using water and nutrient solution. The pH of the nutrient solution was maintained at 5.5-6.5, using acidic (1 M, H 2 SO 4 ) and basic (1 M, KOH) solutions. Fresh nutrient solution was added to the reservoir when the water level reached near the pump at the bottom. The nutrient solution was renewed every 2 weeks after transplantation. The mean temperature (mean ± SD) and relative humidity inside the chamber were 20.1 ± 3.3 °C and 69.61 ± 7.6%, respectively (with ambient CO 2 concentration). The methods employed in this study were conducted in strict adherence to the relevant guidelines and regulations set forth by relevant national and international guidelines, ensuring the scientific integrity and ethical soundness of the research presented in this report.
Light treatments. The artificial light was provided by white LED tubes (Model-HYGL8, spectrum N × 4, 17 Watt, Nexel Tech Pvt. Ltd., India) with R:B ratio of 2.6. Details of the light spectrum are shown in Fig. 7. Four white LEDs were mounted uniformly on top of each layer. A distance of 15 cm was maintained between each LED to get uniform PPFD. A quantum sensor LI-190 (Li-Cor, Lincoln, Nebraska, USA) was used to measure the photosynthetic photon flux density (PPFD) over the plant canopy. The PPFD output of LED tubes was maintained at 200 ± 10 µmol m −2 s −1 . The photoperiods of 12, 16, and 20 h were provided by connecting LED tubes to a timer with a corresponding DLI value of 8.64, 11.5 and 14.4 mol m −2 day −1 , respectively (Table 3). Calculations  www.nature.com/scientificreports/ for the Daily Light Integrals (DLI) were made by multiplying the PPFD (in µmol m −2 s −1 ) by the photoperiod (in seconds), and the results were given as mol m −2 day −1 . The growth of plants under artificial light was compared with the plants grown in a vertical hydroponic system (NFT) under sunlight in the greenhouse condition. The daily variation of DLI (in mol m −2 day −1 ) received from sunlight is depicted in Fig. 8. DLI was computed by using solar radiation data from automatic weather station located inside greenhouse The solar radiation data in W m −2 was converted to PPFD in µmol m −2 s −1 by using a conversion factor of 2.06 57 .
Growth measurements. Plant morphological parameters. The growth measurements were taken from three plants of each treatment at an interval of 10 days after transplanting. The following parameters were measured from sampled plants: Fresh and dry weight of shoot and root was measured at 10 days interval from transplanting to harvest using an electronic weighing balance. The harvested plants were dried in an oven at 60 °C for 72 h and then the measurements of dry weight of root and shoot were taken. Number of leaves per plant (length more than 2 cm) were counted and recorded. The leaf area was measured using LI-3100C leaf area meter (LI-COR Inc., Lincoln, Nebraska). The Specific Leaf Area (SLA) was determined by taking the ratio of leaf area of the plant to its dry weight.
Resource use efficiency. The total water use per treatment was computed separately and the water use efficiency was calculated from the ratio of plant fresh weight to the irrigation water used by plants expressed in g FW L −1 H 2 O. The electric energy use efficiency was calculated from the ratio of plant fresh weight at harvest to the total electricity consumption of LEDs expressed as g FW kWh −1 and g DW kWh −1 . Qualitative parameters. Total phenols. The total phenolic content of fresh lettuce samples was determined according to Ainsworth and Gillespie 58 . 1 g of fresh lettuce samples from each treatment were homogenized in 20 ml ethanol (80%). The crushed samples were centrifuged at 4 °C for 20 min at 10,000 rpm. An aliquot of 0.1 ml was mixed with 0.5 ml Folin-Ciocalteu reagent and left for 5 min. Following the addition of 2 mL of 20% Na 2 CO 3 , the remaining volume was brought up to 20 mL with 80% ethanol. One-centimeter cuvette was used in a spectrophotometer (Spectra Max M2, Molecular Devices, USA) to measure absorbance at 765 nm.
Antioxidant activity. A test for a DPPH free-radical scavenging effect was carried out according to Mensor and others 59 . A fresh sample of lettuce of 1 g was taken and crushed in 20 ml Methanol. The samples were centrifuged at 4 °C for 20 min at 10,000 rpm. A mixture of 1 mL of extract solution and 3.9 mL of DPPH was kept in the dark for 30 min. A UV-VIS Spectrophotometer (Spectra Max M2, Molecular Devices, USA) was used to record the absorbance of the reaction mixture at the 517 nm wavelength. The percentage of DPPH inhibition was computed as follows: where A 1 = Sample absorbance reading at 517 nm, A 0 = Blank absorbance reading at 517 nm, Methanol (95%) was used as a blank. The results were presented in µmol Trolox/g FW.
Scavenging effect (% DPPH inhibition) = 1 − (A 1 /A 0 )] × 100, www.nature.com/scientificreports/ Ascorbic acid. The volumetric approach with 2, 6-dichlorophenol-indophenol dye was utilized for the determination of ascorbic acid 60 . 2 g of lettuce sample was crushed in 100 mL Metaphosphoric acid (HPO 3 ) solution of 3% concentration. Following that, the sample was filtered through Whatman No. 1 filter paper. 10 mL of filtered solution was transferred to a conical flask and titrated with a dye until a pink hue appeared. The readings were noted and the results were expressed in mg per 100 g.

Experimental design and statistical analysis
The experiment was designed with a Completely Randomized Design (CRD) with three replications. Each layer of the vertical hydroponic system contained four NFT channels. Each NFT channel was treated as a separate replication. The plants were destructively sampled at the desired intervals (10, 20, 30 and harvest) and growth parameters were recorded. Three plants were sampled from each treatment to assess growth characteristics. The data obtained was analyzed by using SPSS software (International Business Machines Corporation, Chicago, United States) to measure the significance of variance among different parameters in three photoperiods. The univariate analysis was performed using Tukey's honest significance difference (HSD) test at p < 0.05 was used to measure significant difference in means among treatments. The ANOVA tables obtained for each parameter are presented in a supplementary data file (Supplementary information).

Data availability
The datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. www.nature.com/scientificreports/ Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/.