ORIGINAL RESEARCH published: 13 May 2020 doi: 10.3389/fpls.2020.00537 Air Distribution in a Fully-Closed Higher Plant Growth Chamber Impacts Crop Performance of Hydroponically-Grown Lettuce Enrique Peiro 1,2* † , Antonio Pannico 3† , Sebastian George Colleoni 4 , Lorenzo Bucchieri 4 , Youssef Rouphael 3 , Stefania De Pascale 3 , Roberta Paradiso 3 and Francesc Gòdia 1,2 1 MELiSSA Pilot Plant – Claude Chipaux Laboratory, Universitat Autònoma de Barcelona, Barcelona, Spain, 2 Centre d’Estudis I Recerca Espacials, Institut d’Estudis Espacials de Catalunya, Universitat Autònoma de Barcelona, Barcelona, Spain, 3 Department of Agricultural Sciences, University of Naples Federico II, Portici, Italy, 4 EnginSoft S.p.A., Bergamo, Italy The MELiSSA Pilot Plant (MPP) is testing in terrestrial conditions regenerative life support technologies for human exploration in Space. One of its components is a controlled Higher Plant Chamber (HPC) accommodating hydroponic plant cultures. It Edited by: consists of a 9 m3 single closed growth chamber providing adequate environmental Ann Cuypers, Hasselt University, Belgium conditions for growing plants, enabling the production of food, water and oxygen for Reviewed by: the crew. A critical aspect for a reliable HPC performance is to achieve homogeneous Thorsten Rocksch, air distribution. The initial experiment carried out in the MPP with lettuce as salad crop, Humboldt University of Berlin, showed uneven plant growth throughout the HPC, which was attributed to inadequate Germany Andreas M. Savvides, air distribution due to non-homogeneous air velocity profile along the inlet-vents. After Agricultural Research Institute a detailed computational fluid dynamics (CFD) analysis, the heating, ventilation, and of Cyprus, Cyprus air conditioning subsystem of the HPC was upgraded and a new experiment was *Correspondence: Enrique Peiro carried out in optimized air flow conditions. Nine-day seedlings of lettuce cultivar “Grand Enrique.Peiro@uab.cat Rapids” were transplanted into the HPC and harvested at the end of the growing cycle, † These authors share first authorship where shoot fresh weight, dry biomass, and shoot mineral composition were analyzed. During the experiment, the environmental control system performed remarkably well Specialty section: This article was submitted to based on the biometric measurements as well as the mineral composition leading to Plant Abiotic Stress, a vast homogeneous growth. Overall, the results demonstrated the beneficial effect of a section of the journal an adequate air distribution system in HPCs and the effectiveness of CFD-analysis to Frontiers in Plant Science design properly the gas distribution. The obtained results are of high relevance for life Received: 13 January 2020 Accepted: 08 April 2020 support systems in space involving plants growth. Published: 13 May 2020 Keywords: closed loop life support system, computational fluid dynamics, higher plant characterization, Citation: hydroponics, Lactuca sativa L., mineral composition Peiro E, Pannico A, Colleoni SG, Bucchieri L, Rouphael Y, De Pascale S, Paradiso R and Gòdia F (2020) Air Distribution in a INTRODUCTION Fully-Closed Higher Plant Growth Chamber Impacts Crop Performance Controlled ecological life support systems (CELSSs) or bio-regenerative life support systems of Hydroponically-Grown Lettuce. (BLSSs) have been defined as systems that guarantee human life for long-term in space Front. Plant Sci. 11:537. environments, being able to provide the necessary food sources (Guo et al., 2017). Their main doi: 10.3389/fpls.2020.00537 objective is to provide the crew with food, oxygen and water without the need of continuous Frontiers in Plant Science | www.frontiersin.org 1 May 2020 | Volume 11 | Article 537 Peiro et al. Airflow Impacting Lettuce Crop Performance resupplying from Earth, therefore recovering resources from the In order to obtain an air distribution field inside the waste generated by the crew (Zabel et al., 2016). plant growth region, the internal air circulation was simulated MELiSSA (Micro-Ecological Life-Support System Alternative) using computational fluid dynamics (CFD) models. Over the project was conceived for the development of a regenerative life- past few years, CFD has proven to be a useful tool to support system for long-term space missions (Mergeay et al., estimate air velocity in a reliable and accurate way, improving 1988). Micro-Ecological Life-Support System Alternative project airflow mal-distributions and asymmetric airflow in closed has five major compartments colonized by (i) thermophilic environments (Martins et al., 2014; Di Perta et al., 2016; Lee anoxygenic bacteria, (ii) photo-heterotrophic bacteria, (iii) et al., 2018). In particular, three-dimensional CFD analyses have nitrifying bacteria, (iv) photosynthetic bacteria and higher plants, been successfully used to predict and improve the air profiles and (v) the crew, respectively (Gòdia et al., 2004; Lasseur et al., surrounding the growing crops (Zhang et al., 2016). 2010). The Higher Plants Compartment or Chamber (HPC) is an In order to optimize the input/output of the MELiSSA important component of the MELiSSA loop (Favreau et al., 2005; Higher Plant compartment, an appropriate crop selection is Waters et al., 2005). The complete MELiSSA loop is demonstrated necessary. Hitherto, various species such as cereals, fruit, tubers in the MELiSSA Pilot Plant (MPP), at terrestrial conditions and and leafy vegetables have been tested as potential candidates using rats to mock-up the respiration of the crew. The sizing, for food production in space (Wolff et al., 2014). The main design, and construction of the different compartments of this selection criteria for these species were their adaptability Pilot Plant has been performed for a final demonstration target based on environmental constraints; therefore, plant size, light of providing the oxygen required for one human and 20–40% of requirements, harvest index (HI), as well as nutritional value are the required food. considered fundamental aspects for the crop selection (Chunxiao The use of HPC within BLSSs has a double objective: (i) to and Hong, 2008; Kyriacou et al., 2016, 2017; Wheeler, 2017). provide atmosphere regeneration for the respiration and also (ii) Salad crops have a low water uptake/transpiration ratio, short to supply fresh food for the crew, which constitutes not only a key growing cycle, very high HI and require little crew commitment point for nutrition but also encompasses beneficial impact on the for cultivation. Lettuce is a ubiquitous species among the crops crew psychological and overall health status (Koga and Iwasaki, suggested for life support systems as candidate salad crops 2013). A detailed review of the agricultural systems developed for for near-term missions such as tomato, radish, spinach, chard, space addressing the constraints and needs for the improvement and carrot (Wheeler, 2002). Furthermore, it is a very good of these systems has been recently published by Wheeler (2017). candidate in terms of space/time efficiency, light/energy use Among these factors, the advances in hydroponics and in efficiency, HI and handling time, as well as marking the highest illumination by the use of efficient Light Emitting Diode (LED) score among the selected crops to be cultivated in the Future are highlighted. Moreover, the use of fully controlled plant Exploration Greenhouse (FEG) at Neumayer Station III and chambers is an utmost need for a consistent and reproducible in the International Standard Payload Rack (ISPR) on the food and oxygen production (Haeuplik-Meusburger et al., 2014). International Space Station (ISS; Dueck et al., 2016). The most relevant environmental variables (air temperature, Starting from the above considerations, the aim of the humidity, light, air flow, pressure, atmosphere gas composition, current fully controlled experiments was to assess the effects etc.) have to be controlled in an HPC to sustain optimal plant of an improved airflow distribution in the HPC on growth growth, this being achieved only if plants are cultivated in isolated homogeneity, agronomical performance, and mineral profiling of chambers (Lasseur et al., 2010). hydroponically-grown lettuce plants. One of the main driving factors for the design of the HVAC system of a HPC is to obtain maximal gas exchange rates, which is essential for plant photosynthesis and consequently for plant MATERIALS AND METHODS growth. In order to achieve this objective, the air velocity inside the canopy should be above 0.2 m s−1 (Kitaya et al., 2004) and the Higher Plant Chamber Description air current speed above the canopy should be more than 1.0 m s−1 The HPC of the MPP was manufactured by the Controlled to obtain maximal gas exchange rates (Kitaya et al., 2003). Air Environment Systems Research Facility (CESRF – University of flow is important not only for promoting plant growth, but also Guelph, Ontario, Canada) in collaboration with the company for water purification via plant transpiration and maintaining Angstrom Engineering Inc. (Ontario, Canada). It is a closed healthy conditions of the crops. Lee et al. (2013) found a chamber of the following dimensions: 5.0 m × 1.0 m × 1.2 m clear decrease in tipburn symptoms on lettuce cultivars leaves (L × W × H), providing a total volume of 6.9 m3 for the crops’ in a closed plant factory, using pre-screened tipburn-sensitive growth and a growing surface of around 5 m2 . It is composed of cultivars, when horizontal air velocity was set to 0.28 m s−1 or three modules (A, B, and C) assembled longitudinally (Figure 1). higher, validating the positive effect of air turbulence to prevent The growing area can host 20 trays. Each tray consisted of five tipburn. On the other hand, in the same work, a higher air plants (n = 100 lettuce plants). For the introduction of new velocity (1.04 m s−1 ) caused a decrease in plant growth. trays and the harvest of already grown plants, two airlocks of Accordingly, the internal air recirculation in the HPC of 0.69 m3 each were installed at both ends (A and C) of the the MPP was set to an air exchange rate that according to chamber (Figure 1). the design would provide an air velocity inside the canopy of Air recirculation was provided by a centrifugal blower about 0.3 m s−1 . (Centrifugal Exhauster Delhi 410, Canarm, Brockville, Ontario, Frontiers in Plant Science | www.frontiersin.org 2 May 2020 | Volume 11 | Article 537 Peiro et al. Airflow Impacting Lettuce Crop Performance FIGURE 1 | Schematic top view of the growth chamber with plants location and trays numbering (A) and hardware representation of the Higher Plant Compartment of MELiSSA Pilot Plant Facilities (B). Canada), controlled by a VFD motor with a controller of reach the proper temperature in the chamber. An additional 0–50 Hz (SMVector, Lenze, Barcelona, Spain). The airflow was RH/T sensor was located in the air distribution plenum, and measured in the HVAC plenum by means of an air velocity sensor additional thermistors (2–3 per module) were provided along the (8455 TSI, Shoreview, MN, United States). From the plenum, chamber. The generated condensates were collected in a stainless the air was distributed in the chamber through nine louvers steel 4 L condensate collecting tank and then transferred to the with adjustable slats, and was recovered again at the bottom hydroponics nutrient tank. The lighting was provided by six through perforated baffle plates under the trays (Figure 2). lamps 600 W High pressure sodium (HPS) (HSE 600, P.L. Light Three PTFE bags of 100 L volume each (Keika Ventures, LLC, Systems, Beamsville, Ontario, Canada), two per each module, and Chapel Hill, NC, United States), located behind each module, three 400 W Metal Halide (LMH) lamps (MH-400, P.L. Light allowed atmospheric pressure compensation. Relative humidity Systems, Beamsville, Ontario, Canada), one per module, located and temperature were controlled in the chamber based on at the roof of the growing area that presents one glass window the measurement provided by the humidity and temperature per module. For each module, two dedicated fans (MC2483, sensors and the action of the dedicated heat exchangers (Delhi Comair Rotron, Richardson, TX, United States) were installed CW15, Canarm, Brockville, Ontario, Canada). The first one was to remove the generated heat (keeping the temperature in the controlled by cooling water in order to reduce the excess of lamps loft below 40◦ C). The gas composition inside the chamber humidity, and the second one was supplied by hot water to was analyzed by an O2 /CO2 gas analyzer (600 Series, California Frontiers in Plant Science | www.frontiersin.org 3 May 2020 | Volume 11 | Article 537 Peiro et al. Airflow Impacting Lettuce Crop Performance FIGURE 2 | Perspective view of the Higher Plant Compartment, highlighting the air circulation system and components and showing the airflow fluxes (red arrows). Analytical, Orange, United States), and CO2 concentration was the airflow encounters different local flow regimes, the Gamma- controlled by the injection of CO2 until reaching the desired level. Theta transitional laminar-turbulent model was implemented. In The flow was measured by a mass flow controller (EW-32907-67, this way and based on the local Reynolds number, the flow can Cole-Parmer, Vernon Hills, IL, United States), allowing the switch from fully turbulent (e.g., in the blower region) to laminar calculation of the cumulated CO2 volume injected. (e.g., close to the growing crops). Due to the low temperature The hydroponic loop included a 200 L main nutrient reservoir variations, air was considered incompressible. As boundary (Central Plastics, Irvine, CA, United States), an irrigation pump condition, the models imposed a fix value of recirculating mass with a flow of 0–200 L min−1 (Penta KB Drive, Emerson, St. flow rate, equal to the measured experimental value with nominal Louis, MO, United States) and a distribution piping composed blower speed (0.77 kg s−1 ). of four branches that supply the nutrient flow to all the trays Firstly, the CFD study had the objective of replicating the through 20 spigots. The hydroponic total flow was measured airflow conditions with suboptimal air distribution. Once the by a 0–37 L min−1 flow sensor (RFO-2500, Gems, Plainville, air velocity field was obtained in the overall fluid domain, an CT, United States). In a bypass of the main pipe, pH and optimization study was performed to balance the air pattern at electrical conductivity (EC) sensors were fixed. The nutrient growing plants height. In order to identify the best solution, solution pH and EC were measured by a pH Sensor (HI 8614, the objective was to minimize the standard deviation of the air Hanna Instruments, Villafranca, Padovana, Italy) and an EC velocity in the region of interest. sensor (HI 7638, Hanna Instruments, Villafranca, Padovana, All 3D CFD models were carried out using identical boundary Italy), respectively. The pH control was performed by the PLC conditions, in order to compare the internal air distribution at the according the desired set point through the addition of acid same operating point. (0.5 M HNO3 ) and base (0.5 M KOH) that were stored in dedicated 10-L tanks. Two concentrated solutions (Stock A and Plant Material and Growth Chamber B) were stored also in dedicated 10-L tanks and were injected Conditions into the loop in order to control the EC according to the EC About 200 lettuce seeds (Lactuca sativa L. cultivar ‘Grand set point. The nutrient tank and trays were manufactured from Rapids’, Stokes Seeds Ltd.) were first sanitized with 5% polypropylene, as well as the acid, base, nutrient A and nutrient B sodium hypochlorite solution for 15 min and after rinsing tanks (Nalgene, Rochester, NY), while the main collector and the with demineralized water they were dispersed onto previously pipelines were manufactured from stainless steel (316-L). autoclaved dark absorbent paper, moist with demineralized water and incubated at room temperature under indirect Computational Fluid Dynamics Models lighting. After 48 h, seeds having about 1 cm radicle were The numerical models used to simulate the air distribution inside transferred onto small rockwool cubes (Grodan AO 36/40 the plant growth chamber were generated using commercial CFD 6/15W), previously autoclaved and soaked with the nutrient software (CFX, ANSYS Inc., Canonsburg, PA, United States). All solution, and then they were incubated at 24◦ C in a dedicated the analyses were performed in steady-state conditions. Since nursery according to a light/dark regime of 16/8 h and a Frontiers in Plant Science | www.frontiersin.org 4 May 2020 | Volume 11 | Article 537 Peiro et al. Airflow Impacting Lettuce Crop Performance FIGURE 3 | Computational Fluid Dynamics (CFD) analysis of the airspeed at the canopy level along the Higher Plant Chamber (HPC) surface and average airspeed at the canopy level per tray in Test 1 (A,C, respectively) and Test 2 (B,D, respectively). light intensity of 200 ± 50 µmol m−2 s−1 . After seven starting from 100 times concentrated Stock A (Ca(NO3 )2 · days, selected seedlings (homogenous second true leaf stage) 4H2 O, FeCl3 · 6H2 0 and Na · EDTA) and Stock B (rest of the were transplanted into large sterilized rockwool cubes (Grodan components) solutions, while the nutrient solution pH and EC Delta 4G 42/40) presoaked with the nutrient solution, and were automatically managed by the control system maintaining then placed on the trays of HPC. Five seedlings were placed a set point of 5.9 ± 0.1 and 1.9 ± 0.05 dS m−1 , respectively. on each tray, with a total of 20 trays placed into the The nutrient solution was completely replaced every 2 weeks. HPC (100 plants in total). Plants in the HPC were grown The harvesting of all the plants was performed at 19 and according to a light/dark regime of 16/8 h with an intensity 28 days after transplanting (DAT) in the HPC for Test 1 and of 460 ± 60 µmol m−2 s−1 , while temperature and relative Test 2, respectively. humidity were regulated at 26/20◦ C and 50/70%, respectively. The experiment was carried out with a CO2 concentration Biomass Determination and Leaf Mineral set at 1000 ppm. Analysis Lettuce plants were cultivated in a Nutrient Film Technique At the end of each test, all plants were harvested and separated (NFT) growing system (closed loop hydroponic system). The into edible and inedible fractions on a plant basis. Shoot fresh trays were 80 cm long, 15 cm wide and 8 cm deep, having weight per plant was determined immediately after chamber an inclination of 1%. Each tray was covered with perforated opening. Shoot and roots of each plant were oven dried at 70◦ C stainless steel covers to avoid the algae proliferation and to limit for three days, until reaching a constant weight and then weighed the nutrient solution evaporation. The flow rate of the nutrient for dry biomass determination. solution was set at 1.5 L min−1 , supplied at the top end of each The relative growth rate (RGR) was calculated based on the tray, while the excess was gathered in the general collector and following formula described by Hunt et al. (2002): subsequently in the reservoir tank. The composition of the nutrient solution was as follows: RGR = (loge W2 − loge W1 )/(t2 − t1 ) 3.62 mM Ca(NO3 )2 · 4H2 O, 0.08 mM FeCl3 · 6H2 0, 0.1 mM Na · EDTA, 1 mM MgSO4 · 7H2 O, 5 mM KNO3 , 1.5 mM NH4 H2 PO4 , where t is time (t 1 transplant day; t 2 harvest day), W 1 shoot dry 1 mM (NH4 )2 SO4 , 0.02 mM H3 BO3 , 5 µM MnSO4 · H2 O, weight at transplant and W 2 shoot dry weight at harvest. 3.5 µM ZnSO4 · 7H2 O, 0.8 µM CuSO4 · 5H2 O, and 0.5 µM Determinations of morphological traits: plant and roots dry H2 MoO4 (85% MoO3 ). The nutrient solution was prepared weight, fresh and dry shoot weight were conducted at harvest Frontiers in Plant Science | www.frontiersin.org 5 May 2020 | Volume 11 | Article 537 Peiro et al. Airflow Impacting Lettuce Crop Performance on all five plants of the experimental unit (i.e., tray) and in order to guarantee uniform plant growth throughout the were averaged to produce the replicate mean (n = 20). The overall cultivation area of the HPC. Both tests shared the same dry biomass produced from these lettuce plants was further methodology in terms of plant material, growth conditions and used as an aggregate replicate sample for analysis of mineral biomass analysis as described above, as well as the plant harvest composition (n = 20). scheduled at 28 DAT. However, during Test 1, following the Dried leaf samples were ground separately with an daily routine visual inspections, an evident heterogeneity of electrical mill to an 841 µm screen, then 0.25 g of the plant size was noted starting from the beginning of the third dried tissues were analyzed for mineral content by ion week after transplanting. This lack of homogeneity showed chromatography: P, K, Ca, and Mg as described in detail a different plant growth stage between the HPC modules, by Rouphael et al. (2017) and Kyriacou et al. (2019). in particular all plants in the growth chamber were at the Nitrogen (total N) concentration in the shoot tissues was phenological phase of head development (Stage 4) according determined on 1 g of dried samples by Kjeldahl method to Biologische Bundesanstalt, Bundessortenamt and CHemical (Bremner, 1965). industry (BBCH) scale for leaf vegetables (Meier, 2001), but about 70% of lettuce plants had already reached the full maturity (Code 49 of BBCH-scale). Therefore, in order to avoid the RESULTS AND DISCUSSION early achievement of the phenological flowering phase (Code 50 of BBCH-scale), the experiment was terminated earlier than Test Rationale expected and the lettuce plants were harvested at 19 DAT to The two tests conducted in our study reflect the need to have further analyze potential causes of the visually evident non- a proper distribution of the gas phase in a plant chamber homogeneous growth. Thus, it was identified that there was a TABLE 1 | Plant dry weight, roots dry weight, shoot dry and fresh weight, harvest index, relative growth rate (RGR), shoot dry weight standard deviation (SD) and shoot dry weight standard deviation percentage of hydroponically-grown lettuce plants in Test 1 and Test 2. Test Plant dry Roots dry Shoot dry Shoot fresh Harvest index RGR Shoot dry Shoot dry weight weight weight weight (mg mg−1 day−1 ) weight (SD) weight (SD%) (g plant−1 ) (g plant−1 ) (g plant−1 ) (g plant−1 ) Test 1 5.13 ± 0.26 0.73 ± 0.04 4.40 ± 0.23 87.42 ± 4.61 0.86 ± 0.001 0.089 ± 0.003 1.01 23.01 Test 2 12.68 ± 0.35 1.05 ± 0.05 11.63 ± 0.33 278.61 ± 6.19 0.92 ± 0.003 0.083 ± 0.001 1.46 12.59 Student’s *** *** *** *** *** ns – – t-test All data are expressed as mean ± standard error, n = 20. ns, *** Non-significant or significant at P ≤ 0.001, respectively. SD: standard deviation. FIGURE 4 | Evolution of lettuce plants growth over the weeks (A, B, C and D, respectively for 7, 14, 21, and 28 DAT) during Test 2, captured by one of the internal cameras of the HPC. Frontiers in Plant Science | www.frontiersin.org 6 May 2020 | Volume 11 | Article 537 Peiro et al. Airflow Impacting Lettuce Crop Performance FIGURE 5 | 3D plant dry biomass distribution (n = 100) and shoot dry weight distribution (n = 20) along the Higher Plant Chamber (HPC) in Test 1 (A,C, respectively) and Test 2 (B,D, respectively). lack of homogeneous distribution of the gas phase in the chamber trays (about 0.35 and 0.6–0.7 m s−1 in the side and central as well as not appropriate circulation regime. Consequently, trays, respectively). CFD tools were used to guide the design of the hardware In order to improve the chamber homogeneity for the gas modifications in order to solve this malfunction. After the phase, the CFD analysis guided changes in several elements of corresponding changes in the HPC, Test 2 was conducted in the heating, ventilation and air conditioning subsystem of the which a higher homogeneity of growth compared to Test 1 chamber, as follows: was detected and all the plants in the chamber reached the full maturity (Code 49 of BBCH-scale) at 28 DAT as scheduled in our a) The plenum region was modified by the insertion of a test plan protocol. deflector, a stainless steel perforated baffle designed in order to deviate the air flow from the central region and obtain a more uniform air distribution at the plenum exit. CFD Analysis and Technical b) In order to achieve control on the supply air flow to the Modifications Performed in the Airflow growing region, new dampers were installed connecting System of the Plant Chamber the plenum region to the chamber. Such components are CFD analyses enabled the determination of the complete fluid adjustable, able to modify their open area to allow more dynamics field inside the plant growth chamber, studying or less crossing air flow. Nine stainless steel dampers the suboptimal air distribution of the original configuration were manufactured with dimensions 350 mm × 450 mm, of the chamber. The results assessment was primarily based guaranteeing the flexibility of the air distribution system. on the investigation of the air velocity magnitude at plant c) In the return air system, the baffle panels’ configuration height, in order to have complete information regarding the was modified. The arrangement of the baffle panels environment in which the crop was grown. With suboptimal was based on the original configuration, considered not air distribution (Test 1), the air velocity was significantly adequate for a sufficient return air uniformity. The baffle higher in the trays located in the central part of the chamber, panels’ configuration was redesigned, with different hole where the magnitude approached 1.0 m s−1 (Figure 3A). patterns on the baffles. The air velocity was averaged for the five plants per tray d) Finally, in the original configuration the baffle panels were (Figure 3C). Particularly, the central trays (tray no. 7 to tray not positioned against the side walls, leaving open gaps no. 14) had almost the double velocity value of that at the side where air could bypass the panels. In order to force the air Frontiers in Plant Science | www.frontiersin.org 7 May 2020 | Volume 11 | Article 537 Peiro et al. Airflow Impacting Lettuce Crop Performance TABLE 2 | Comparison of shoot mineral copmposition of hydroponically-grown lettuce plants coming from Test 1 and Test 2 with scientific literature. Test N (g 100 P (g 100 K (g 100 Ca (g 100 Mg (g 100 g−1 dw) g−1 dw) g−1 dw) g−1 dw) g−1 dw) Test 1 5.2 0.9 7.3 0.9 0.2 Test 2 5.8 0.8 6.9 0.6 0.2 Wheeler et al. 4.8 0.4 17.0 0.9 0.3 (1994) McKeehen et al. 4.5 0.6 8.2 0.6 0.2 (1996) Masot Mata (2007) 5.5 1.0 7.9 1.2 0.3 Rouphael et al. – 0.5 6.5 0.8 0.3 (2019) Test 1 and Test 2 data are expressed as mean, n = 20. Dw, dry weight basis. about 25% lower than the reference values obtained from several tests performed under comparable environmental conditions and crop growth duration (Hanford, 2006; Wheeler and Sager, 2006). The harvest in Test 2 occurred 9 days later compared to Test 1, and the fresh and dry weight per plant were obviously higher in FIGURE 6 | Regression between the air flow velocity at the different trays the second test, but in any case the shoot dry biomass in Test 2 position (n = 20) of the Higher Plant Chamber (HPC) and the respective shoot was about 25% higher than that obtained by Wheeler et al. (1994) dry weight (n = 20) in Test 1 and Test 2. after 28 DAT on a different cultivar, but in completely comparable environmental conditions. Regarding the distribution of the shoot dry weight along flow through the baffle panels, 10 additional stainless steel the HPC, the value of the standard deviation has considerably components were designed and manufactured closing the decreased in Test 2 with respect to Test 1 (12.59 and 23.01, side gaps. respectively; Table 1). Plant dry biomass and shoot dry weight With these changes, CFD models were predicting an improved distribution along the growth chamber in both tests are presented balance throughout the 20 trays with an average air velocity in Figure 5. Comparing the distribution of plant growth along of 0.34 m s−1 (Figures 3B,D), corroborating that in the the chamber in the two tests, a clear lack of uniformity was improved configuration, the air velocity increased its evenness noted in Test 1, both in terms of shoot and total dry biomass at plant height, with the same overall flow rate (averaged (Figures 5A,C). An evident decrease in shoot dry weight was for the five plants per tray, Figure 3D). Once the changes observed starting the tray no. 8 until the tray no. 14. In particular, in the chamber had been implemented, a new test (Test a 33.5% average reduction of the shoot dry weight in the seven 2) was performed. central trays was recorded with respect to the plants positioned on the lateral trays of the HPC (Figure 5C). Test 2 clearly shows a Assessment of Plant Biomass better uniformity of plant dry biomass distribution and a greater homogeneity of the average shoot dry weight per tray along Distribution Along the HPC and Shoot the growth chamber (Figures 5B,D). In addition, the lettuce Mineral Composition in the Two Plant plants of Test 2 recorded a HI of 0.92, in agreement with data Cultivation Tests reported by Wheeler and Sager (2006) in several experiments of The growth parameters measured at harvest in both Tests 1 and 2 lettuce grown in a fully-closed growth chamber in comparable are reported in Table 1. Considering the different duration of the conditions. Instead, the significantly lower HI obtained in the two tests, in Test 1 the lettuce plants were harvested 19 DAT, while first experiment (0.86), denotes a stress condition at the canopy in Test 2 the harvest occurs 28 DAT (Figure 4); plant roots and level leading to an imbalance of the shoot to roots ratio. An shoot fresh and dry weight at harvest were significantly higher appropriate air circulation inside a growth chamber promotes in the second test compared to the first one (Table 1). On the growth and maximizes gas exchange rates (Kitaya et al., 1998). other hand, the RGR was not statistically different in the two tests, In Test 1, an air velocity higher than 0.5 m s−1 at the level this result can be explained considering that the plants in both of the central trays of the HPC (Figure 3C) has resulted in tests were harvested during the same phenological phase of head a reduction of the shoot dry weight in correspondence of the development (Stage 4 of BBCH-scale) and therefore long before same positions of the chamber (Figure 5C). This result was the stationary growth phase was reached which coincides with statistically confirmed by the significant inverse linear regression the beginning of the flowering phase (Code 50 of BBCH-scale; (R2 = 0.62) existing between the air flow at the different trays Meier, 2001). However, the HI was significantly higher in Test 2 position of the chamber and the respective shoot dry weight of than in Test 1 (Table 1), while the plant biomass in Test 1 was Test 1 plants (Figure 6). On the contrary, in Test 2 the poor Frontiers in Plant Science | www.frontiersin.org 8 May 2020 | Volume 11 | Article 537 Peiro et al. Airflow Impacting Lettuce Crop Performance correlation, confirmed by the low value of the coefficient of fresh biomass, according to a highly significant positive quadratic determination (R2 = 0.02), indicated that the shoot dry weight correlation (El-Nakhel et al., 2019). Regardless the different air was not affected by the air velocity inside the chamber (Figure 6). velocity conditions of the two tests, the shoot mineral content These findings comply with what was found in the work of Lee appears to be almost in line with the literature data referring to et al. (2013), in which the lettuce cultivar “Dambaesangchuesse” the lettuce cultivation in closed or semi-closed growth chambers observed a significant decrease in leaf area and shoot fresh (Table 2). The only notable difference was observed in the work weight at an air velocity greater than 0.55 m s−1 . Likewise, in of Wheeler et al. (1994), in which higher potassium content and another work carried out in a plant factory on “Greenwave” a lower concentration of phosphorus are reported, probably due lettuce cultivar, a reduction in the dry weight of the shoots was to different growing conditions rather than to genetic factors. observed at an air velocity greater than 0.9 m s−1 (Nishikawa et al., 2013). Kitaya et al. (2003) found in a work with sweet potato, that the net photosynthetic rate and the transpiration rate CONCLUSION increased significantly as the air velocity increased from 0.01 to 0.2 m s−1 , but further increases of air speed up to 1.0 m s−1 The successful cultivation of higher plants in a growth chamber is gradually increased transpiration rate, while the photosynthetic strongly related to the precise control of environmental variables. rate remained rather constant. Moreover, in a similar work with To sustain optimal plant growth, it is essential to maximize tomato, a decrease in the photosynthetic rate was found following gas exchange rates especially in a fully closed growth chamber. an increase in air velocity from 0.4 to 0.8 m s−1 (Kitaya et al., One of the main factors that strongly affects the gas exchanges 2004). In lettuce, on the other hand, it was observed that an and consequently the plant growth is the air velocity at the air velocity of 0.7 m s−1 caused water stress followed by the canopy level. Furthermore, the movement of air is important reduction of stomatal conductance (Shibata et al., 1995). Based not only to enhance plant growth, but also for effective water on these findings and the results from Test 2, we can deduce recycling through plant transpiration in human life support that in a closed growth chamber, a homogeneous air velocity systems. Especially in limiting conditions, such as space outposts, between 0.3 and 0.5 m s−1 enables to optimize gas exchange it is important to ensure a homogeneous distribution of biomass rates and consequently promotes lettuce plants growth and more along the growth chamber, in order to achieve efficient use of importantly plants growth uniformity in the chamber. resources. The results of this study indicate that air velocities Shoot mineral composition of both harvests are presented in between 0.3 and 0.5 m s−1 maximizes biomass production in Table 2. Among the minerals analyzed, K was by far the most the HPC, while air speed values above 0.6 m s−1 can depress the abundant, regardless of the two tests, followed by N, P, Ca, and lettuce plant growth. Finally, a homogeneous air circulation at the Mg. The nitrogen content was higher in the second test, reaching canopy level guarantees uniform distribution of shoot biomass values of 5.8 g 100 g−1 dw compared to 5.2 g 100 g−1 dw in along the growth chamber. Test 1. Contrarily, the concentration of potassium and calcium had slightly decreased in Test 2 with respect to Test 1, where potassium content reached 7.3 and 6.9 g 100 g−1 dw and calcium DATA AVAILABILITY STATEMENT content reached 0.9 and 0.6 g 100 g−1 dw, in Test 1 and 2, respectively. However, phosphorus and magnesium content was The datasets generated for this study are available on request to instead fully comparable in both tests. the corresponding author. The reduction of K and Ca content recorded in Test 2 compared to Test 1 could be a result of a higher transpiration rate that occurred in the first test following the higher air velocity AUTHOR CONTRIBUTIONS measured in the central position of the HPC. In fact, the increase in transpiration rate promotes the transport of macronutrients EP, AP, and FG coordinated the whole project, provided the toward the leaves; in particular Goto and Takakura (1992) had intellectual input and set up the experiment. EP and FG found in several works on lettuce, an increase in calcium foliar conducted the Test 1 and AP the Test 2 and wrote the content following the increase in air speed at canopy level. Introduction and a section of the Materials and Methods. Conversely, the increase in shoot nitrogen content in Test 2 SC and LB wrote the parts relative to CFD models. AP and compared to Test 1 can be related to the growing stage, as well YR were involved in writing agronomical data analysis and as to the size of the plant. Indeed, in a test carried out in an data interpretation. AP wrote the parts relative to the lettuce open-gas-exchange growth chamber on two butterhead lettuce crop performance. EP, AP, YR, and FG critically revised the cultivars, it has been observed that the shoot nitrogen content final draft of the manuscript. All authors approved the final during the growing period increased with the increase of the plant draft preparation. REFERENCES Chunxiao, X., and Hong, L. (2008). Crop candidates for the bioregenerative life support systems in China. Acta Astronaut. 63, 1076–1080. doi: 10.1016/j. Bremner, J. M. (1965). “Total nitrogen,” in Methods of Soil Analysis, eds C. A. actaastro.2008.02.003 Black, D. D. Evans, I. L. White, L. E. Ensminger, and F. E. Clark (Madison, WI: Di Perta, E. S., Agizza, M. A., Sorrentino, G., Boccia, L., and Pindozzi, American Society of Agronomy), 1149–1178. doi: 10.2134/agronmonogr9.2. S. (2016). Study of aerodynamic performances of different wind tunnel 2ed.c32 configurations and air inlet velocities, using computational fluid dynamics Frontiers in Plant Science | www.frontiersin.org 9 May 2020 | Volume 11 | Article 537 Peiro et al. Airflow Impacting Lettuce Crop Performance (CFD). Comp. Elect. Agric. 125, 137–148. doi: 10.1016/j.compag.2016. Masot Mata, A. (2007). Engineering Photosynthetic Systems For Bioregenerative Life 05.007 Support. Ph. D. thesis, Universitat Autònoma de Barcelona, Bellaterra. Dueck, T., Kempkes, F., Esther, M., and Cecilia, S. (2016). “Choosing crops for McKeehen, J. D., Smart, D. J., Mackowiak, C. L., Wheeler, R. M., and cultivation in space,” in Proceedings of the 46th International Conference on Nielsen, S. S. (1996). Effect of CO2 levels on nutrient content of Environmental Systems, Vienna. lettuce and radish. Ad. Space Res. 18, 85–92. doi: 10.1016/0273-1177(95) El-Nakhel, C., Giordano, M., Pannico, A., Carillo, P., Fusco, G. M., De Pascale, 00864-b S., et al. (2019). Cultivar-specific performance and qualitative descriptors Meier, U. (2001). Growth Stages Of Mono- And Dicotyledonous Plants. Bbch for butterhead Salanova lettuce produced in closed soilless cultivation as a Monograph. German Federal Biological Research Centre For Agriculture And candidate salad crop for human life support in space. Life 9:61. doi: 10.3390/ Forestry. Hoboken, NJ: Blackwell Science. life9030061 Mergeay, M., Verstraete, W., Dubertret, G., Lefort-Tran, M., Chipaux, C., and Favreau, M., Ordonez, L., Rodriguez, A., and Waters, G. (2005). “Application of Binot, R. A. (1988). “MELiSSA’—A micro-organisms-based model for ‘CELSS’ non-rectangular hyperbola model to the lettuce and beet crops,” in Proceedings development,” in Proceedings at the 3rd European Symposium on Space Thermal of the G. 05ICES-23, 35th ICES, Rome. Control Life Support Systems Noordwijk, Piscataway, NJ. Gòdia, F., Albiol, J., Perez, J., Creus, N., Cabello, F., Montras, A., et al. (2004). The Nishikawa, T., Fukuda, H., and Murase, H. (2013). Effects of airflow for lettuce MELISSA pilot plant facility as an integration test-bed for advanced life support growth in the plant factory with an electric turntable. IFAC Proc. 46, 270–273. systems. Adv. Space Res. 34, 1483–1493. doi: 10.1016/j.asr.2003.08.038 doi: 10.3182/20130327-3-jp-3017.00062 Goto, E., and Takakura, T. (1992). Prevention of lettuce tipburn by supplying air to Rouphael, Y., Colla, G., Giordano, M., El-Nakhel, C., Kyriacou, M. C., and De inner leaves. Trans. ASAE 35, 641–645. doi: 10.13031/2013.28644 Pascale, S. (2017). Foliar applications of a legume-derived protein hydrolysate Guo, S. S., Mao, R. X., Zhang, L. L., Tang, Y. K., and Li, Y. H. (2017). Progress and elicit dose-dependent increases of growth, leaf mineral composition, yield and prospect of research on controlled ecological life support technique. Reach 6, fruit quality in two greenhouse tomato cultivars. Sci. Hortic. 226, 353–360. 1–10. doi: 10.1016/j.reach.2017.06.002 doi: 10.1016/j.scienta.2017.09.007 Haeuplik-Meusburger, S., Paterson, C., Schubert, D., and Zabel, P. (2014). Rouphael, Y., Petropoulos, S. A., El Nakhel, C., Pannico, A., Kyriacou, Greenhouses and their humanizing synergies. Acta Astronaut. 96, 138–150. M. C., Giordano, M., et al. (2019). Reducing energy requirements in doi: 10.1016/j.actaastro.2013.11.031 future bioregenerative life support systems (BLSSs): performance and Hanford, A. J. (2006). Advanced Life Support Baseline Values And Assumptions bioactive composition of diverse lettuce genotypes grown under optimal and Document. Technical Reports, 3. Available online at: http://docs.lib.purdue.edu/ suboptimal light conditions. Front. Plant Sci. 10:1305. doi: 10.3389/fpls.2019. nasatr/3 (accessed October 10, 2019). 01305 Hunt, R., Causton, D. R., Shipley, B., and Askew, A. P. (2002). A modern tool for Shibata, T., Iwao, K., and Takano, T. (1995). Effect of vertical air flowing on classical plant growth analysis. Ann. Bot. 90, 485–488. doi: 10.1093/aob/mcf214 lettuce growing in a plant factory. Green Environ. Control Autom. 399, 175–182. Kitaya, Y., Shibuya, T., Kozai, T., and Kubota, C. (1998). Effects of light doi: 10.17660/actahortic.1995.399.20 intensity and air velocity on air temperature, water vapor pressure, and CO2 Waters, G., Zheng, Y., Gidzinski, D., and Dixon, M. (2005). Empirical Relationships concentration inside a plant canopy under an artificial lighting condition. Life Between Light Intensity And Crop Net Carbon Exchange Rate At The Leaf and Supp. Bios. Sci. 5, 199–203. FULL CANOPY SCALE: Towards the Integration Of A Higher Plant Chamber In Kitaya, Y., Shibuya, T., Yoshida, M., and Kiyota, M. (2004). Effects of air velocity The MELiSSA Loop, - 05ICES-290, 35th ICES. Guelph: University of Guelph. on photosynthesis of plant canopies under elevated CO2 levels in a plant culture Wheeler, R. M. (2002). “Horticulture for mars,” in Proceedings of the XXVI system. Adv. Space Res. 34, 1466–1469. doi: 10.1016/j.asr.2003.08.031 International Horticultural Congress: Horticulture, Art And Science For Life- Kitaya, Y., Tsuruyama, J., Shibuya, T., Yoshida, M., and Kiyota, M. (2003). Effects Colloquia Presentations, Toronto, ON. of air current speed on gas exchange in plant leaves and plant canopies. Adv. Wheeler, R. M. (2017). Agriculture for space: people and places paving the way. Space Res. 31, 177–182. doi: 10.1016/s0273-1177(02)00747-0 Open Agric. 2, 14–32. doi: 10.1515/opag-2017-0002 Koga, K., and Iwasaki, Y. (2013). Psychological and physiological effect in humans Wheeler, R. M., Mackowiak, C. L., Sager, J. C., Yorio, N. C., Knott, W. M., and of touching plant foliage - using the semantic differential method and cerebral Berry, W. L. (1994). Growth and gas exchange by lettuce stands in a closed, activity as indicators. J. Physiol. Anthropol. 32, 1–9. doi: 10.1186/1880-6805-32- controlled environment. J. Amer. Soc. Hortic. Sci. 119, 610–615. doi: 10.21273/ 7 jashs.119.3.610 Kyriacou, M. C., De Pascale, S., Kyratzis, A., and Rouphael, Y. (2017). Microgreens Wheeler, R. M., and Sager, J. C. (2006). Crop Production For Advanced Life Support as a component of space life support systems: a cornucopia of functional food. Systems. Technical Reports, 1. Available online at: http://docs.lib.purdue.edu/ Front. Plant Sci. 8:1587. doi: 10.3389/fpls.2017.01587 nasatr/1 (accessed November 8, 2019). Kyriacou, M. C., El-Nakhel, C., Graziani, G., Pannico, A., Soteriou, G. A., Wolff, S., Coelho, L., Karoliussen, I., and Jost, A. I. (2014). Effects of the Giordano, M., et al. (2019). Functional quality in novel food sources: extraterrestrial environment on plants: recommendations for future space genotypic variation in the nutritive and phytochemical composition of thirteen experiments for the MELiSSA higher plant compartment. Life 4, 189–204. doi: microgreens species. Food Chem. 277, 107–118. doi: 10.1016/j.foodchem.2018. 10.3390/life4020189 10.098 Zabel, P., Bamsey, M., Schubert, D., and Tajmar, M. (2016). Review and analysis Kyriacou, M. C., Rouphael, Y., Di Gioia, F., Kyratzis, A., Serio, F., Renna, M., et al. of over 40 years of space plant growth systems. Life Sci. Space Res. 10, 1–16. (2016). Micro-scale vegetable production and the rise of microgreens. Trends doi: 10.1016/j.lssr.2016.06.004 Food Sci. Technol. 57, 103–115. doi: 10.1016/j.tifs.2016.09.005 Zhang, Y., Kacira, M., and An, L. (2016). A CFD study on improving air flow Lasseur, C., Brunet, J., de Weever, H., Dixon, M., Dussap, G., Godia, F., et al. (2010). uniformity in indoor plant factory system. Biosyst. Engin. 147, 193–205. doi: MELiSSA: the European Project of closed life support system. Gravitat. Space 10.1016/j.biosystemseng.2016.04.012 Biol. 23, 3–12. Lee, J. G., Choi, C. S., Jang, Y. A., Jang, S. W., Lee, S. G., and Um, Y. C. (2013). Conflict of Interest: The authors declare that the research was conducted in the Effects of air temperature and air flow rate control on the tipburn occurrence absence of any commercial or financial relationships that could be construed as a of leaf lettuce in a closed-type plant factory system. Hortic. Environ. Biotechnol. potential conflict of interest. 54, 303–310. doi: 10.1007/s13580-013-0031-0 Lee, M. S., Li, Z., Ling, J., and Aute, V. (2018). A CFD assisted segmented control Copyright © 2020 Peiro, Pannico, Colleoni, Bucchieri, Rouphael, De Pascale, volume based heat exchanger model for simulation of air-to-refrigerant heat Paradiso and Gòdia. This is an open-access article distributed under the terms exchanger with air flow mal-distribution. Appl. Thermal Eng. 131, 230–243. of the Creative Commons Attribution License (CC BY). The use, distribution or doi: 10.1016/j.applthermaleng.2017.11.094 reproduction in other forums is permitted, provided the original author(s) and the Martins, N. M., Carrico, N. J., Ramos, H. M., and Covas, D. I. (2014). Velocity- copyright owner(s) are credited and that the original publication in this journal distribution in pressurized pipe flow using CFD: accuracy and mesh analysis. is cited, in accordance with accepted academic practice. No use, distribution or Comp. Flui. 105, 218–230. doi: 10.1016/j.compfluid.2014.09.031 reproduction is permitted which does not comply with these terms. Frontiers in Plant Science | www.frontiersin.org 10 May 2020 | Volume 11 | Article 537