Comparison of Different Temperature Control Systems in Tropical-Adapted Greenhouses for Green Romaine Lettuce Production

Abstract

The cultivation of lettuce in greenhouses is negatively impacted by high temperatures, especially in hot climates. Therefore, developing an efficient method to regulate the internal temperature of greenhouses is essential to sustain crop productivity throughout the year. This study intends to investigate differential temperature control systems for green romaine lettuce production in the greenhouse. The experiment was conducted in a completely randomized design (CRD) with five replications. The four treatments consisted of (1) control, (2) Fogging + ventilation fan, (3) Fogging + shading, and (4) Fogging + ventilation fan + shading. The different temperature control systems influenced the growth and yield parameters, in which shading operation appears to cause reductions in photosynthetic rate, leading to declines of marketable fresh weight. However, the operation of the fogging and ventilation fan was considered the appropriate method for improving indoor air temperature since this resulted in higher growth and yield and a greater sensory evaluation. Maintaining VPD values between 1.62 and 1.76 kPa and controlling light intensity within the 530–700 mol m⁻² s⁻¹ range were recommended to ensure a consistent lettuce yield of 65–82 g plant⁻¹ throughout the year.

1. Introduction

Climate change and global warming are serious issues that are impacting many aspects of plant production. It is crucial that production methods are developed to be sustainable, year-round, and applicable under severe weather conditions, in order to be able to feed the world population [1,2]. Current production is primarily performed outdoors and in greenhouse facilities, both of which can be directly affected by outdoor temperature. With average surface temperatures on Earth rising by as much as 1.3 to 3.5 °C per year [3], viable innovative greenhouse solutions are becoming indispensable, especially temperature-controlled greenhouses (GHs).

It is well known that greenhouse ambient temperatures can be higher than external ambient temperatures, especially during summer, due to the properties of the construction material or insufficient ventilation [4]. Accordingly, several studies focused on suitable greenhouse microclimate control (e.g., temperature, humidity, and light quantity) by using various mechanical technologies, depending on plant types, environmental conditions, operational ease, and economic viability [5,6]. However, recent research outcomes are mostly limited to optimal root temperature or suitable light quantity and quality, using artificial lighting in hydroponics systems [7,8,9,10]. For example, Hooks et al. [11] found improved shoot fresh weight when roots were subjected to nutrient solution cooling during the summer.

Plant production under artificially controlled conditions, such as in indoor vertical farming systems or plant factories, is increasingly being used commercially. Such circumstances have led to studies on the economic feasibility of plant production in plant factories and under artificial lighting conditions [12]. While some researchers guarantee the optimistic prospect of plant factories with artificial lighting [12,13], several scientific papers report that vegetable production in plant factories has no economic advantage over greenhouse production [14,15].

Lettuce (Lactuca sativa L.) is a leafy vegetable mainly cultivated in the greenhouse, ranking as highly productive and economically valuable among vegetables grown worldwide [16,17]. The environmental conditions affect lettuce plants’ growth and quality (phytochemical compounds, sensory test) [9,18]. Recent studies have discussed widely the effect of temperature on lettuce growth both directly (leaf and air temperature) and indirectly (photosynthesis, absorption, and transportation of water and plant nutrition through the roots) [9,17,19]. It is well established that high day/night temperature conditions lead to a decrease in polyphenols (quercetin, chlorogenic acid) and flavonoid contents, consequently increasing tipburn occurrence and bitterness, as well as accelerating the premature bolting and resulting in reduced marketability [20,21,22]. Several reviewed experiments conducted in plant factories and controlled environments with artificial lighting show that the optimal air temperature management should be 22–25 °C and 18–20 °C day and night, respectively [9]. Jones [23] also revealed that the optimal temperatures for lettuce production in greenhouses range from 17–28 °C in the day to 3–12 °C in the night periods. About the other microenvironment (relative air humidity, light intensity, air velocity, and CO₂ concentration) that drastically affect the physiological processes related to plant growth, Ahmed et al. [9] reviewed much of the literature. They summarized that the optimal ranges in the relative air humidity and light intensity for lettuce production in the controlled environments with artificial lighting were 70–80% and 200–250 µmol m⁻² s⁻¹, respectively. However, Zhou et al. [17] revealed that the optimal light intensity under the high-temperature condition (30 °C) ranged from 500–600 µmol m⁻² s⁻¹.

Recently, there has been widespread investigation into greenhouse cooling technologies due to the need for air cooling to prevent overheating while also considering the balance between yield and related costs [5]. Generally, natural ventilation can be considered a passive cooling system effective for greenhouse cooling applications in hot climates, dependent on greenhouse design and direction [6,9]. Nonetheless, forced ventilation, including an electrically operated ventilation fan, has been reported to gain a homogeneous air distribution inside the greenhouse, and some studies resulted in heat removal and relative humidity control [24]. The fogging systems are also commonly used in commercial greenhouses to provide pressuring and spraying water through small nozzles, cooling the ambient air inside the greenhouse by humidifying [6]. In addition, several studies have been conducted to explore the effect of shading on greenhouse microclimates, which reported that shading has reduced indoor air temperature via decreasing transmitted solar radiation [25,26]. This system is more effective when combined with other cooling techniques (e.g., ventilation and evaporative cooling) [9]. Since manipulated variables in temperature control systems have an influence on the internal ambient temperature of the greenhouse that is one of the primary environmental factors affecting the yield and yield quality of lettuce. To achieve the optimal temperature control system for lettuce production in an individual greenhouse, this study aimed to compare different temperature control system combinations to improve lettuce production in greenhouses.

2. Materials and Methods

2.1. Experimental Designs and Management of Temperature Control System

The experimental arrangement was a completely randomized design (CRD) with five replicates. The treatments were four different greenhouse temperature control systems that consisted of T1: control (no temperature control system), T2: fogging + ventilation fan, T3: fogging + shading, and T4: fogging + ventilation fan + shading. Trails were carried out in three consecutive cycles from September to November 2022 at the Agricultural Technology Farming Center greenhouse, Thammasat University, Thailand (Latitude: 14.074191, Longitude: 100.609026).

The greenhouses were built with a steel frame with a size 32 mesh net that covered the greenhouse including a side wall and roof, which have a high natural ventilation capacity, resulting in a climate closely coupled to the outdoor climate. The roofs are covered with multifunctional film (MultiTech, MTEC, NSTDA, Pathum Thani, Thailand), which can have high UV filtration and high NIR reflection. (Figure 1).

Three temperature control systems, namely fogging, ventilation fan, and shading, were operated in the greenhouse from transplanting until the end of each harvesting. All temperature control systems underwent a preliminary test to find the optimal operating time of each system that could maintain the average indoor temperature in the daytime (10:00–15:00 h) near 30 ± 5 °C and a difference of less than 2 °C between the outside and inside of the greenhouse. The fogging system consisted of double water filters to prevent nozzle clogging, a water reservoir tank, a water pressure pump (2.8 bar), a pressure regulator, and fog-generating nozzles (4-way fogging nozzle with 0.6 mm nozzle size). The fogging nozzles were uniformly located at each line with 2.0 m nozzle spacing. The fogging system was automatically electrically operated from 10:00 to 15:00 h, which provided the average relative humidity inside the greenhouse between 60 and 80%. For the ventilation fan system, two ventilation fans (370 W, 50 cm in diameter, DF5B-4, Guangdong Zhaoqing Deton Co., Ltd., Zhaoqing, China) were equipped at 2 m above floor level on opposite greenhouse sidewalls operated from 10:00 to 16:00 h for air ventilation inside the greenhouse only in the fan operated treatment. In addition to the shade system, a commercial black plastic shade (50% shading effect) was operated at 13:00–15:00 h in the shading-operated treatment. The environmental conditions, consisting of temperature, humidity, and light intensity, were constantly monitored with sensors inside and outside the greenhouse, which connected to the IoT system.

2.2. IoT System for Environmental Data Acquisition

An IoT system for data acquisition comprises temperature, humidity, and light intensity sensors, microcontrollers, and a database. SHT31 sensors were implemented in the system as the air temperature and humidity sensors. The light intensity is measured with the phototransistor sensors designed and fabricated at the Thai Microelectronics Center (TMEC, Amphur Muang, Chachoengsao, Thailand). The sensors were calibrated with RK200-02 Quantum PAR (photosynthetically active radiation) Sensor (Hunan Rika Electronic Technology Co., Ltd., Changsha, Hunan, China) from Rika for PAR detection. The sensors were installed at the level of plant pots so that they represent the microclimate at the plant level. Due to the analog signal output, Arduino NANO was used in the system for A/D conversion. The digitalized acquired data were transferred from Arduino NANO to ESP32 using the RS232 (serial) communication protocol. Finally, the received dataset in EPS32 was forwarded to the database every ten minutes for later analysis.

2.3. Growth Conditions

Green romaine lettuce was selected as the experimental plant. Lettuce seeds were sown in 104-cell polystyrene trays (1 seed/cell) using commercial peat moss as the planting medium. After fifteen days, uniformized seedlings at the four-leaf stage were subsequently transplanted into individual plastic pots (D: 20 cm × H: 15 cm) filled with commercial growing media (soil: composted rain tree leaves: filtrate cake: chicken manure: coconut coir dust: chopped coconut husk: rice husk ash at a ratio of 4: 1: 2: 1.5: 0.4: 0.3: 0.3: 4 by volume), one seedling was planted in each pot. The chemical properties of growing media used in this experiment were as follows: pH 6.39, EC 3.00 dS m⁻¹, 35.13% organic matter, 1.31% total N, 2.27% total P, 1.32% total K, 1.59% total Ca, 0.53% total Mg, and a C/N ratio of 15.58.

Trail pots were placed on greenhouse benches and irrigated equally with drip irrigation twice daily (8:00 and 16:00 h), as required to maintain vigorous growth until harvesting. Natural extracts were sprayed to eliminate diseases and insects when needed, and mechanical methods permanently eliminated weeds.

2.4. Plant Growth and Biomass Accumulation

For each treatment, five plant samples were chosen at random. The growth parameters, including plant height, plant width, leaf number, and leaf area, along with plant biomass (including both upper parts that are marketable and root fresh weight), were recorded every five days for 30 days after transplanting. The plant dry weights were measured after oven-drying at 80 °C for 72 h or until weights were constant.

2.5. Physiological Evaluations

Net photosynthetic rate, stomatal conductance, and transpiration rate were measured by using a LI-6800 portable photosynthesis system (Li-Cor Inc., Lincoln, NE, USA). Fully expanded leaves (fourth frond from the upper shoot) with non-senescent were selected for measurements that were operated in an artificial climatic chamber, with a photosynthetic photon flux density (PPFD) of 1200 μmol m⁻² s⁻¹, a CO₂ concentration of 1000 µmol mol⁻¹ and a constant airflow rate of 500 μmol s⁻¹, to avoid the effect of environmental fluctuations on gas-exchange measurements. The measurements were carried out between 8:00 and 12:00 h, covering the daily maximum plant physiological activity [27]. The data were simultaneously recorded and analyzed by Photosyn Assistant ‘software, version 2.1 [28].

2.6. Phytochemical Analysis

2.6.1. Chlorophyll Content

The chlorophyll content was assessed by adapting the technique described by Mackinney [29]. The lettuce leaf was pierced with a 6 mm cork borer into ten pieces (total area was 2.82 cm²). Then, these ten pieces were placed in a test tube containing 80% acetone with a volume of 10 mL. Chlorophyll was extracted for 72 h at 4 °C in the dark. After that, the absorbance of the extract was measured using a spectrophotometer at 645 and 663 nm. Then, the total chlorophyll concentration was calculated and expressed as the content per unit leaflet area (µg cm⁻²) using the following equation.

Total chlorophyll (a + b) = [(20.20 × A645) + (8.02 × A663)] × (V/A)

(1)

where V = Final volume of chlorophyll-extracting solution; A = Frond area of plant samples taken for chlorophyll.

2.6.2. Total Phenolics Content

The total phenolic content was measured using the Folin-Ciocalteu assay, following the method of Chutimanukul et al. [30] with some modifications. For the sample extraction, 10 mg of dried sample was homogenized with 5 mL of methanol containing 1% hydrochloric acid at 25 ± 1 °C for 3 h and centrifuged at 12,000× g for 5 min. The supernatants were collected, and the total phenolic content was evaluated. Briefly, a 200 µL aliquot of the obtained sample was mixed with 200 µL of 1 N Folin-Ciocalteu reagent and 600 µL of 7.5% sodium carbonate solution. The mixture was incubated at 25 ± 1 °C for 1 h, and the absorbance was measured at 730 nm with an ultraviolet spectrophotometer. The total phenolic content was calculated from the calibration gallic acid curve, and the result was expressed as mg gallic acid equivalent (GAE) g⁻¹ dry weight.

2.6.3. DPPH Radical Scavenging Activity

The modified method from Chutimanukul et al. [30] was carried out to assay the 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical scavenging. Briefly, 100 µL of an extracted sample (10 mg of dried sample extracted with 5 mL of methanol containing 1% hydrochloric acid) was mixed with 900 µL of 0.1 mM DPPH working solution and then incubated in the dark for 30 min at 25 ± 1 °C. The absorbance of the mixed solution was measured at 515 nm. A calibration curve was determined using Trolox, and the result was expressed with the inhibition percentage of DPPH absorbance as following formulae:

DPPH radical scavenging (%) = [(Ac − As)/Ac] × 100

(2)

where Ac = control reaction absorbance; As = sample reaction absorbance.

2.7. Nutrient Accumulation in Plant Tissues

The dried shoot samples were ground and were then sieved through a 2 mm sieve to provide for the analysis of N, P, and K contents on a % dry weight basis. To determine the total N content, the Kjeldahl method was used, and for determining the P and K contents in the plant tissues, the standard protocol of the Association of Official Analytical Chemists (AOAC) was followed with some modifications. Each sample, weighing 1.0 g, was digested in 15 mL of nitric-perchloric acid (HNO₃:HClO₄ at a ratio of 2:1 v/v). After digestion, the samples were diluted with distilled water until they reached a volume of 50 mL and were then stored in plastic tubes at room temperature. The P content in the distilled samples was analyzed by using a spectrophotometer (UV-1280, Shimadzu, Kyoto, Japan) at 420 nm. In contrast, the K quantification was analyzed using an atomic absorption spectrometer (PinAAcle900F, Perkin-Elmer, Waltham, MA, USA).

2.8. Sensory Test

The quantitative descriptive analysis (QDA) method was carried out to evaluate the sensory qualities, consisting of sweetness (with and without dressing), bitterness (with and without dressing), crispness, and juiciness, by the six-member trained panelists from the Sensory Analysis Center, Food Innopolis Thammasat University. The sensory evaluation was performed by using the 15 cm line scale, with increments of 5, where 0 represented no intensity, and 15 represented the highest intensity. The scales were presented on printed ballots, and the scale length was independently evaluated on each sensory. In brief, room-temperature fresh-cut samples of green romaine lettuce from each treatment were served in plastic dishes and identified by a random three-digit code. The order in which samples were evaluated was randomized. During the testing, participants were given reverse osmosis drinking water and unsalted crackers to cleanse their palate between samples. The salad dressing used in this study was Japanese soy sauce sesame dressing.

2.9. Statistical Analysis

The experiment comprised a completely randomized design (CRD) with five replications. Data were analyzed through one-way analysis of variance (ANOVA). The means were evaluated by Duncan’s multiple range test (p ≤ 0.05).

3. Results

3.1. Environmental Conditions in the Greenhouse

Throughout the 1–3 harvesting periods, during the day, the average hourly temperature, relative humidity, and light intensity both inside and outside of the greenhouse with various temperature control systems were recorded (

Table 1. Micro-environmental conditions under different treatments among experimental periods.

Treatment	Greenhouse Temperature (°C)			Relative Humidity (%)			Photosynthetically Active Radiation (PAR, μmol m−2 s−1)	VPD (kPa)
	Average	Maximum	Minimum	Average	Maximum	Minimum
1st harvesting
T1	35.90	37.76	32.91	61.30	67.30	57.65	886.08	2.31
T2	33.13	34.64	31.43	68.21	73.81	62.95	592.17	1.62
T3	33.98	36.30	32.82	66.47	70.19	61.16	506.28	1.83
T4	33.58	35.69	31.96	67.28	70.00	63.38	499.11	1.71
Outside	32.56	34.15	30.19	68.57	76.95	62.97	1309.53	1.55
2nd harvesting
T1	35.15	36.45	33.31	57.68	61.58	54.60	636.76	2.41
T2	32.25	33.82	30.65	63.77	69.02	57.14	669.03	1.76
T3	32.78	34.34	30.99	64.40	67.44	59.36	472.28	1.77
T4	32.85	34.40	31.52	63.26	65.34	60.30	422.02	1.84
Outside	33.30	34.04	31.89	58.39	64.53	55.90	1429.80	2.13
3rd harvesting
T1	35.85	37.11	33.01	52.14	57.41	49.94	495.91	2.83
T2	31.33	32.27	30.20	62.21	66.70	59.35	529.90	1.73
T3	32.66	33.82	31.29	56.07	60.53	53.16	512.38	2.17
T4	32.32	33.90	31.62	58.09	60.67	56.10	443.04	2.15
Outside	33.33	34.40	30.95	49.29	53.97	45.42	1383.97	2.61

Data were collected and averaged from 10:00–15:00 h daily during each harvesting month. T1: control (no temperature control system), T2: fogging + ventilation fan, T3: fogging + shading, and T4: fogging + ventilation fan + shading.

, Figure 2). As the microclimate averaged during the daytime (10:00–15:00 h), presented in Table 1, the operation of all patterns of temperature control systems (fogging + ventilation fan, fogging + shading, and fogging + ventilation fan + shading) presented similar or lower values of the greenhouse‘s internal temperature in all three harvesting periods, compared to the outside air temperature. On the other hand, the condition of not utilizing any temperature control systems always showed higher average values of the greenhouse‘s internal temperature during the daytime (10:00–15:00 h), which were 3.34, 1.85, and 2.52 °C, rather than that of outside air temperature in 1–3 harvesting periods, respectively (Table 1). The relative humidity value under non-temperature control operation was lower than other treatments at all determined periods (Table 1). More specifically, the relative humidity inside the greenhouse was increased throughout the duration of the fogging operation in all treatments and was higher than outside conditions (Table 1, Figure 2). However, this result was not observed in the first harvesting periods. This could be due to rainfall, which is particularly high in September, when the first harvesting period was observed. Nonetheless, it seems that the operation of fogging with a ventilation fan, shading, or ventilation fan and shading was not influenced by the differences in relative humidity in the greenhouse, in which the average relative humidity ranged from 66.47–38.21%, 63.26–64.40% and 56.07–62.21% during the 1–3 harvesting periods, respectively (Table 1).

In terms of average light intensity during the daytime (10:00–15:00 h) in the first harvesting period, it was observed that the light intensity under the treatment of a non-operated temperature control system or operation of fogging and ventilation fan decreased by 32.34 and 54.78% compared to the outside condition. On the other hand, shading operation with fogging (T3) or fogging and ventilation fan (T4) recorded lower light intensity in the greenhouse that decreased by 61.34 and 61.89% compared to the outside conditions. In the 2nd and 3rd harvesting periods, however, it was discovered that the light intensity in the greenhouse of all treatments was comparable, with the values greatly reduced from the external light intensity, around 53.21–70.49% and 61.71–67.99%, respectively (Table 1).

The vapor pressure deficit (VPD) value, as shown in Figure 2, is a measurement related to the air temperature and relative humidity. It is one of the most important microclimate factors influencing plant growth and productivity by directly affecting transpiration and stomatal functions. The calculated VPD values in the current study showed that the mean values of VPD during hourly daytime periods were higher in the control site (T1: no operating system), whereas these values were reduced when the temperature control systems (T2: fogging + ventilation fan, T3: fogging + shading and T4: fogging + ventilation fan + shading) were operating. More specifically, the VPD values under the treatment of fogging and ventilation fan systems operating (T2) were lower than those of the other treatments, which were 1.62, 1.76, and 1.73 kPa in the 1–3 harvesting. In contrast, other operating patterns (T3: fogging + shading and T4: fogging + ventilation fan + shading) lead to higher VPD values in 1–3 harvesting periods ranging from 1.71–2.31, 1.77–2.41, and 2.15–2.83 kPa, respectively. Nevertheless, the trails without any temperature control systems had the highest VPD values of all harvesting periods, which were 2.31, 2.41, and 2.83 kPa, respectively (Table 1, Figure 2).

3.2. Plant Growth Characteristics

As shown in Figure 3 for the first harvesting period, green romaine lettuce grew normally even when temperature control systems were not operated. However, different operating temperature control systems markedly influenced the lettuce sizes, especially 15 days after transplanting. During the second and third harvesting periods, the green romaine lettuce grew the tallest when the fogging and ventilation fan systems were in use. There was no noticeable difference between using fogging and shading versus using fogging, ventilation fans, and shading together. In addition, the greatest lettuce width of the first and third harvesting periods was recorded in plots with the fogging and ventilation fan systems operating. Under all the operating patterns of temperature control systems, the higher leaf number and leaf area tended to occur when fogging and ventilation fan systems were operating. At the same time, they were not statically different from one another treatment in some harvesting periods. The highest marketable fresh weight of all harvesting periods was observed when fogging and ventilation fan systems were operating, and no significant differences existed among some treatments studied. On the other hand, the root fresh weight of green romaine lettuce was not affected by the different operating temperature control systems, which were in a range of 3.35–6.55 g plant⁻¹ in all harvesting periods (

Table 2. Growth parameters of green romaine lettuce under different greenhouse temperature control systems.

Treatment	1st Harvesting	2nd Harvesting	3rd Harvesting
Plant height (cm)
T1	24.70 ± 1.30	21.94 ± 1.61 b	23.32 ± 2.08 b
T2	25.00 ± 1.92	25.82 ± 0.78 a	26.60 ± 0.42 a
T3	24.00 ± 1.30	18.98 ± 1.05 c	25.36 ± 0.68 a
T4	25.00 ± 1.87	22.74 ± 0.70 b	26.20 ± 0.70 a
p-value	0.413	0.000	0.002
Plant width (cm)
T1	28.34 ± 1.86 bc	25.26 ± 1.32	25.18 ± 0.77 b
T2	30.80 ± 0.83 a	25.54 ± 0.38	27.88 ± 1.35 a
T3	26.80 ± 1.79 c	26.22 ± 2.42	24.30 ± 1.21 b
T4	29.60 ± 1.14 b	25.88 ± 1.67	24.88 ± 0.64 b
p-value	0.004	0.804	0.000
Leaf number
T1	16.60 ± 1.14 a	13.20 ± 0.44 c	15.20 ± 1.30 b
T2	17.40 ± 2.30 a	14.80 ± 0.84 ab	18.40 ± 1.14 a
T3	12.00 ± 1.00 b	14.60 ± 0.55 b	15.00 ± 1.00 b
T4	17.20 ± 1.48 a	15.60 ± 0.55 a	16.40 ± 0.55 b
p-value	0.000	0.000	0.000
Leaf area (cm2)
T1	795.15 ± 41.73 b	630.08 ± 23.21 c	665.72 ± 30.97 b
T2	984.76 ± 64.91 a	900.78 ± 58.07 a	731.53 ± 11.30 a
T3	556.65 ± 23.61 c	657.91 ± 71.94 c	539.79 ± 20.86 c
T4	766.55 ± 58.82 b	754.93 ± 43.30 b	720.13 ± 22.15 a
p-value	0.000	0.000	0.000
Marketable fresh weight (g plant−1)
T1	75.81 ± 9.51 a	38.31 ± 6.60 b	68.41 ± 6.36 b
T2	80.82 ± 8.42 a	65.24 ± 7.21 a	81.90 ± 3.13 a
T3	49.32 ± 2.54 b	40.86 ± 2.54 b	47.35 ± 5.48 c
T4	77.85 ± 8.06 a	59.67 ± 5.13 a	71.83 ± 2.20 b
p-value	0.000	0.000	0.000
Root fresh weight (g plant−1)
T1	5.29 ± 0.28 a	3.35 ± 0.63	6.33 ± 1.05 a
T2	4.41 ± 0.36 a	4.67 ± 0.50	5.59 ± 0.61 ab
T3	3.35 ± 0.81 b	4.26 ± 0.38	4.80 ± 0.52 b
T4	4.54 ± 0.98 a	6.55 ± 0.42	5.76 ± 0.67 ab
p-value	0.003	0.000	0.036

Mean with different letters in the same column indicates a significant difference according to Duncan’s multiple range test at p ≤ 0.05. T1: control (no temperature control system), T2: fogging + ventilation fan, T3: fogging + shading, and T4: fogging + ventilation fan + shading.

).

3.3. Physiological Responses

The different operations of temperature control systems influenced (p ≤ 0.05) the physiological responses of green romaine lettuce for all three harvesting periods. The highest average photosynthetic rate of green romaine lettuce occurred when fogging and ventilation fan systems were operating, while the lowest was recorded when fogging and shading were operating, these tendencies were observed in all three harvesting periods (

Table 3. Net photosynthetic rate, stomatal conductance, and transpiration rate of green romaine lettuce under different greenhouse temperature control systems.

Treatment	1st Harvesting	2nd Harvesting	3rd Harvesting
Net photosynthetic rate (μmol m−2 s−1)
T1	8.00 ± 1.60 b	10.45 ± 0.86 b	12.00 ± 0.71 bc
T2	11.31 ± 1.52 a	16.71 ± 1.65 a	16.58 ± 1.09 a
T3	4.32 ± 0.53 c	9.71 ± 1.66 b	11.25 ± 0.91 c
T4	8.90 ± 0.25 b	11.46 ± 0.60 b	13.43 ± 0.61 b
p-value	0.001	0.001	0.000
Stromatal conductance (mol m−2 s−1)
T1	0.22 ± 0.04 ab	0.38 ± 0.05 a	0.23 ± 0.01 c
T2	0.26 ± 0.05 a	0.42 ± 0.08 a	0.68 ± 0.16 a
T3	0.11 ± 0.02 c	0.23 ± 0.06 b	0.34 ± 0.07 b
T4	0.16 ± 0.03 bc	0.25 ± 0.03 b	0.25 ± 0.02 c
p-value	0.004	0.000	0.001
Transpiration rate (mmol m−2 s−1)
T1	7.69 ± 0.27 b	8.76 ± 1.34 b	7.24 ± 1.31 ab
T2	10.36 ± 0.91 a	11.91 ± 0.50 a	9.00 ± 0.37a
T3	6.96 ± 0.27 b	7.41 ± 1.87 b	4.31 ± 1.44 c
T4	7.81 ± 1.47 b	8.51 ± 0.53 b	5.88 ± 0.95 bc
p-value	0.007	0.010	0.005

Mean with different letters in the same column indicates a significant difference according to Duncan’s multiple range test at p ≤ 0.05. T1: control (no temperature control system), T2: fogging + ventilation fan, T3: fogging + shading, and T4: fogging + ventilation fan + shading.

). The significantly highest stomatal conductance was also measured when fogging and ventilation fan systems were operating, and no significant differences were observed among those and the control treatment in the first and second harvesting periods. Significantly higher rates of transpiration were also recorded in all harvesting periods when fogging and ventilation fan systems were operating, compared with the other operating patterns. However, they were not statistically different from the control treatment in the third harvesting period. (Table 3). This specifically indicates that the higher values of stomatal conductance and transpiration rate correspond with higher photosynthetic rates in all observation periods.

3.4. Phytochemical Contents

In this exploration, the results of total chlorophyll, total phenolic, and DPPH radical scavenging analysis of the third harvesting samples indicated that operating different temperature control systems significantly influenced these phytochemical compositions in green romaine lettuce, as shown in

Table 4. Phytochemical contents of green romaine lettuce at the third harvesting period under different greenhouse temperature control systems.

Treatment	Total Chlorophyll (μg cm−2)	Total Phenolic (mg GAE g−1 DW)	DPPH Radical Scavenging (%)
T1	86.06 ± 5.58 b	60.78 ± 0.27 b	61.07 ± 1.45 a
T2	95.10 ± 5.45 a	67.93 ± 2.66 a	60.53 ± 3.36 a
T3	55.21 ± 1.73 c	38.07 ± 1.21 c	23.36 ± 3.03 b
T4	56.47 ± 1.78 c	41.35 ± 4.25 c	61.78 ± 1.24 a
p-value	0.000	0.000	0.000

Mean with different letters in the same column indicates a significant difference according to Duncan’s multiple range test at p ≤ 0.05. T1: control (no temperature control system), T2: fogging + ventilation fan, T3: fogging + shading, and T4: fogging + ventilation fan + shading.

. The significantly highest total chlorophyll and phenolic contents were found in lettuce grown under the operation of the fogging and ventilation fan system (T2), which was around 11–12% higher than that under the control treatment (no temperature control system). In contrast, the total chlorophyll and phenolic in lettuce leaf tissues were reduced when the shading was operated with fogging (T3) or fogging and ventilation fan (T4) systems with values of 34–36% and 32–37%, respectively, compared to the control treatment. Nevertheless, the DPPH radicle scavenging was dramatically reduced by 62% when lettuce was grown under the operation of fogging and shading, compared to the control treatment (Table 4).

3.5. Nutrient Concentrations in Plant Tissues

The lettuce tissue’s total nitrogen content of the third harvesting sample was similar among the different operations of temperature control systems. The range in the amount of total nitrogen in lettuce tissues was between 3.06 and 3.20. On the other hand, the significantly higher contents of total phosphorus of the third harvesting samples were measured when lettuce was grown under the operation of fogging and ventilation fan systems (T2) and fogging, ventilation fan, and shading (T4). In addition, the higher potassium contents in lettuce tissues of the third harvesting samples were also found in those operating systems (T2 and T4), which were significantly greater than the other operating systems. In contrast, the lowest total potassium content was observed in lettuce grown under fogging and shading. The nutrient accumulations in this study indicated that the proportion of total nitrogen and potassium contents was 2–3 times the total phosphorus content in lettuce tissues (

Table 5. Nutrient concentration in green romaine lettuce under different greenhouse temperature control systems at the third harvesting period.

Treatment	N (%)	P (%)	K (%)
T1	3.06 ± 0.13	1.16 ± 0.05 b	3.82 ± 0.10 b
T2	3.19 ± 0.04	1.70 ± 0.07 a	5.39 ± 0.14 a
T3	3.08 ± 0.03	1.23 ± 0.08 b	2.64 ± 0.39 c
T4	3.20 ± 0.10	1.69 ± 0.18 a	5.32 ± 0.04 a
p-value	0.191	0.000	0.000

Mean with different letters in the same column indicates a significant difference according to Duncan’s multiple range test at p ≤ 0.05. T1: control (no temperature control system), T2: fogging + ventilation fan, T3: fogging + shading, and T4: fogging + ventilation fan + shading.

).

3.6. Sensory Test Results

Different criteria, such as bitterness (with or without dressing), sweetness (with or without dressing), crispness, and juiciness, were selected for the sensory evaluation based on what the customer thought captured all the marketable lettuce. The sensory profiles of the green romaine lettuce samples of the third harvesting period obtained by quantitative descriptive analysis are shown in the radar chart (Figure 4). The significantly highest crispness and juiciness ratings were observed in green romaine lettuce produced under the fogging and ventilation fan systems. At the same time, crispness and juiciness were the lowest for lettuce cultivated under the control treatment (no temperature control system). The sweetness levels did not significantly differ between lettuce with or without dressing, and the bitterness levels remained consistent across all treatments. However, the lettuce cultivated under the control treatment exhibited the highest bitterness compared to the lettuce grown under the operation of fogging and ventilation fan systems, which had the lowest level of bitterness at 72%. There was a clear difference between the treatments of different temperature control systems in bitterness rather than sweetness (Figure 4).

4. Discussion

Greenhouse technology is becoming more popular as a viable option for commercial vegetable production. While it may be more costly than traditional outdoor farming due to specialized growing technologies and electrical control systems, the benefits are numerous. These include higher yields per unit area, predictable plant growth and development, and the ability to continue production even in inclement weather. In this experiment, researchers from the National Science and Technology Development Agency (NSTDA) in Thailand used a newly designed greenhouse. The microclimate in the current data collection showed that the indoor temperature, even under the control treatment (no temperature control system), was only 2–3 °C higher than that of the outside conditions in all harvesting periods, in contrast to what has been reported for the traditional greenhouse in Thailand, where the inside temperature was observed to be 3–5 °C higher compared to the outside conditions [31]. Numerous studies show that the optimal air temperature for lettuce plants, classified as cool-season vegetables, in a plant factory or temperate zone is between 22 and 26 °C [9,32]. However, the ideal temperature for lettuce grown in tropical environments was reported to be between 22 and 30 °C [33], or even 33 °C for other leafy vegetables like mustard greens [34]. As shown in the current study, the indoor temperature under the control treatment with non-operating temperature control systems was higher than 35 °C and some obvious tipburn and bolting occurrences were observed. Chen et al. [35] demonstrated that bolting (producing a flowering stem prematurely) tends to accelerate under high-temperature conditions. Additionally, bolting has been widely reported to relate to stimulating changes in plant hormone levels, accounting for bitter-tasting leaves and reducing the yield marketability [35,36,37]. Furthermore, the tipburn occurrence is usually prevalent in newly developed leaves of nearly mature lettuce. Choi et al. [19] reported a direct correlation between high temperature and tipburn severity of lettuce grown under constant illumination. Indeed, tipburn occurrence is caused by calcium deficiency commonly due to water stress and heat formation, resulting in reduced calcium translocation from the root via reduced transpiration under the hot climate. In the current investigation, mature lettuce produced under the control treatment (no temperature control system) with the high air temperature (>35 °C) circumstances in all harvesting times was the only lettuce that showed some obvious tipburn and bolting occurrences (Figure 5). Therefore, achieving year-round higher crop production, especially during hot climatic seasons, requires proper interior temperature regulation.

Recently, various cooling techniques and systems, including fan pad evaporative cooling, shade, misting or fogging, natural ventilation (through structure or employing exhaust fan), or combination systems, have become available for plant production in greenhouses [5]. Many studies have evaluated the appropriate systems for controlling microclimate in the greenhouse, especially the effect of fogging, shading, and ventilation fans on greenhouse performance for vegetable production in hot climates [6,38,39,40,41,42]. Several studies investigated whether mist/fogging systems could lower interior air temperature more than open field production [38] or under the greenhouse using only natural ventilation via roof opening [39,40,41] during summer periods. The fogging system was used for all the temperature control treatments. The result confirms that the operation of the fogging system with the other systems, including shading or ventilation fan or shading and ventilation fan, could reduce the interior temperature at all harvesting periods compared to the control treatment (no temperature control system). These results are consistent with those of Chansakoo et al. [43], who studied in the same Thailand regions and reported that the fogging system could reduce the interior temperature from 41 °C to 27.5 °C and increase relative humidity from 45% to 95% from the turmeric production in the greenhouse.

Additionally, to reduce the negative effects of high radiation and temperatures on crop production, shading is frequently used to limit and avoid intense solar radiation entering the greenhouse. This process is accelerated by high indoor air temperature caused by the accumulation of heat waves [44,45]. Ahemd et al. [41] and Laur et al. [42] also demonstrated that combining shading with evaporative cooling via a fogging system can lower indoor air temperature and impact the appropriate microclimate for lettuce growth. Furthermore, natural ventilation is still widely used for greenhouse operations, requiring little or no external energy [5]. However, it is greatly influenced by external environmental factors, thereby increasing the risk of under-ventilating in hot weather. Mechanical ventilation using fans has been widely reported to obtain the optimal air distribution inside and control temperature, humidity, and carbon dioxide concentration in the greenhouse, which are linked to crop growth well-being [24,46]. For instance, Xue and Zhao [47] observed that the operation of greenhouse ventilation from 11:50 to 16:50 h in the summer was necessary to reduce the temperature inside the greenhouse. Although our results did not show such a dramatic difference in inside temperature among the different operations of temperature control systems, it is interesting that fogging with a ventilation fan presented a lower indoor temperature than fogging with shading.

Based on the current study, it was observed that the growth rate and quality of lettuce varies depending on the microclimate created by different temperature control systems in the greenhouse. The use of fogging and ventilation fan systems resulted in a higher content of total chlorophyll, indirectly improving the photosynthetic capacity of the lettuce leaf and ultimately increasing the yield. This was found to be more effective compared to the control group or other operating patterns. Total chlorophyll, mainly including chlorophyll a and b, is the primary pigment of plant photosynthesis, which can absorb light energy, transfer electrons to the reaction center, and convert light energy into chemical energy of organic molecules in the form of sugars [48]. Our results are consistent with this general statement, which is the higher photosynthetic rate of lettuce plants exposed to the operation of fogging and ventilation fan systems. In addition to indoor air temperature, light intensity is another important factor in the photosynthetic performance of the lettuce grown at different temperature conditions. Low light intensity may reduce carbon photosynthetic assimilation enzyme activity and limit plant carbon assimilation [17].

As shown in the present study, the lettuce growth and yield grown under the operation of shading with fogging (T3) or fogging and ventilation fan (T4) was lower than that grown under the non-shading treatment (T2: fogging + ventilation fan), even though the average indoor temperature during the daytime periods was similar. Interestingly, the lower phenolic content was also observed when the shading was operated (T3, T4), compared to the non-shading treatment (T1, T2). These results agree with Galieni et al. [49], who reported that the limitation of photosynthetically active radiation (PAR) availability reduced both soluble and bound phenolic compounds, probably due to the induction of phenylalanine ammonia-lyase (PAL), an important enzyme in the biosynthesis of phenolic acids [50]. Although several studies reported that elevated antioxidant bioactive compound content improves the antioxidant capacity [16,18], there were no specific relationships between total phenolic content and DPPH radical scavenging capacity in this study, particularly when comparing T3 and T4 with the same shading operation. This suggested that other environmental factors (not only light intensity) or other phytochemical compounds may play a role in the DPPH radical scavenging activity. Nevertheless, it is interesting to observe that lettuce grown under the operation of fogging, shading, and ventilation fan (T4) tended to exhibit a higher yield than that under the operation of fogging and shading. These results are consistent with those of Perone et al. [51], who suggested that the right balance of air circulation can improve optimal gas and water diffusion in the leaf boundary layer, which enhances plant transpiration and photosynthesis and, as a result, fosters plant growth and development. In addition to the current investigation, even under the control treatment (no temperature control systems), the marketable fresh weight of lettuce seems to be higher in some harvesting periods. Although high temperature had a more pronounced influence on photosynthesis and yield in lettuce, Zhou et al. [17] revealed that the appropriate levels of light intensity could improve its potential photosynthetic capacity and result in maintaining the yield under improper temperature conditions.

According to the relationship between the VPD value and plant productivity, high VPD (>2 kPa) generally has a negative impact on photosynthetic efficiency and reduces stomatal conductance and transpiration rate because plants close their stomata to prevent dehydration [1,52]. According to Bakker [53], the ideal VPD values for most plants were between 0.5 and 0.8 kPa. For instance, Amitrano et al. [1] found that Salanova lettuce plants grown at low VPD (0.69 kPa) had higher plant biomass than those cultivated at high VPD (1.76 kPa). Nevertheless, it was also discovered by Inoue et al. [52] that romaine lettuce grown at a VPD value of 1.32 kPa enhanced its leaf area and shoot dry weight in comparison to a 1.63 kPa VPD value. As shown in the present study, the mean values of VPD during the daytime (10:00–15:00 h) period were 2.31–2.83 kPa in the control site. However, they decreased to 1.62–2.15 kPa in all the operating patterns of temperature control systems, respectively. In addition, the relative humidity of the air rose by 8–19% compared to the control treatment when temperature control devices were in operation (fogging combined with a ventilation fan, shade, or ventilation fan with shading). This expressly suggests that the VPD value may be decreased or successfully managed by temperature control systems, particularly the fogging system, consistent with the findings in tomato [54] and the same romaine lettuce reported by Inoue et al. [52]. However, those findings were carried out at 24 °C throughout the experiment, roughly 10 °C lower than the air temperature recorded in the current investigation. According to numerous VPD charts for crop optimization, the ideal VPD values should not exceed 1.8–2.2 kPa and will rarely increase depending on how air temperature and relative humidity are managed. This was supported by Hoffman [55], who reported that the increase in VPD from 1.0 to 1.8 kPa causes major reductions in plant growth for several crops. The current study shows that the VPD values under the operation of fogging and ventilation fans for controlling indoor temperature were lower than the recommended marginal border (1.8 kPa). They showed a range of 1.62–1.76 kPa in all harvesting periods. In addition, this operating pattern of temperature control systems gave the highest yield and presented higher scores of crispness and lower bitterness since several pieces of literature indicate both crispness and bitterness as quality indicators directly concerning consumer expectations towards lettuce [56,57]. Thus, it would be feasible to use the VPD value as the indicator for controlling the optimal microclimate conditions in the greenhouse to achieve year-round higher yield production towards lettuce grown under the greenhouse. However, Inoue et al. [52] suggested that VPD in the greenhouse should be controlled not by intermittent regulation but by continuous regulation since fluctuating VPD consequently retarded plant growth. More studies would be required to improve the continuous management of VPD for plant production in the greenhouse while considering the mean VPD and the fluctuation range.

5. Conclusions

The present study clearly showed that the operation of temperature control systems is needed to maintain the indoor microclimate, especially indoor air temperature, for lettuce production in the greenhouse since it can reach severe levels during the daytime (10:00–15:00 h) and directly influence lettuce quantity and quality. Fogging and ventilation fan systems are cooling methods that are appropriate for improving the microclimate conditions, especially the VPD values at 1.62–1.76 kPa, considered the suitable growing conditions for the green romaine lettuce in the current greenhouse. In addition, our findings also demonstrated that fogging systems could improve the VPD values by enhancing the relative humidity, and the ventilation fan seems to obtain the optimal air distribution inside the greenhouse, resulting in improved plant productivity. Shading could lead to metabolic impairment, including a decreased photosynthetic rate, which accounted for the reduced biomass production in lettuce. However, from a prospective standpoint, it would be beneficial to concentrate on the frequency of fogging operations, especially during the summer months when temperatures are more intense. This will provide a more thorough understanding of the ideal temperature control systems and enable the greenhouse’s climate to be automatically controlled using “smart” algorithms.