Abstract
Microgreens are gaining prominence for their high nutritional value, rapid growth cycle, and suitability for controlled-environment agriculture (CEA). Among key agronomic factors, seed density critically influences both yield and microbial safety, and it also impacts production cost. This study evaluated: (1) the effects of various seed densities on the yield and microbial load of cress (Lepidium sativum L.), rocket (Eruca sativa), and pea (Pisum sativum L.); and (2) the influence of supplemental UV-A radiation on the biomass, microbial load, and phytochemical profile of pea microgreens. The study found that fresh biomass increased with increasing seed density across all species up to a threshold, achieving maximum yields at 12 seeds/cm2 for cress and rocket and 2 seeds/cm2 for pea. However, higher seed densities were also associated with increased levels of total aerobic bacteria (TAB), Enterobacteriaceae, and fungi, which could pose an increased risk of microbial hazards concerning food safety, e.g., TAB in cress increasing from 7.04 ± 0.09 to 7.94 ± 0.17 log10CFU/g as seed density increases from 6 to 14 seeds/cm2. Initially white light supplemented with UV-A recorded a lower yield (11 g) compared to white light (13 g), but the final biomass was comparable under both lights, with microbial load remaining stable at ~3.8–4.2 log10 CFU/g. A temporary increase in carotenoids exhibited significantly higher levels (2.00± 0.29 µg/mg DW) under white light supplemented with UV-A radiation compared to white light alone (1.48 ± 0.23 µg/mg DW). However, these increases were not maintained throughout the growing period. These results indicate that optimising seed density in these species is vital for balancing productivity and food safety, and continuous UV-A exposure did not lead to sustained higher phytochemical levels or reduced microbial load compared with white light alone.
Keywords: microgreen, seed density, yield optimisation, phytochemicals, microbial safety, light spectra
1. Introduction
Global demand for functional and nutritional foods is steadily rising in response to increasing awareness of diet, health, and sustainability [1,2,3]. Microgreens have emerged as a promising category of young, tender seedlings harvested shortly after germination. They are widely appreciated for their high nutritional value, vibrant flavour, and potential as functional foods [3]. Their short growth cycles, minimal input, and high harvest index (~90%) make them well-suited for home cultivation and resource-limited environments, including space-based agriculture [4]. Microgreens can be cultivated using diverse methods and settings, such as polytunnels, greenhouses, using various growing media, and both artificial and natural lighting [5]. It is crucial to focus on the details of these factors and cultivation methods to tailor the desired crop to maximise health and commercial benefits. This includes selection of plant species, substrates, quantity and quality of light, and irrigation, fertilisation, and sowing density [6]. Seed density is one of the most important factors affecting microgreen output and quality, as it maximises biomass production, microbiological safety, and space utilisation [6], while minimising input costs for producers.
This seed density varies widely among species and is shaped by factors such as seed size, germination rates, and the distinct growth characteristics of each variety. Generally, larger seeds, like those of peas and sunflowers, should be sown at lower densities such as 1 seed/cm2 to provide ample space for shoot development. However smaller seeds, such as radish and mustard, require higher sowing densities, e.g., 3 seeds/cm2 [7]. The type of seed is a key determinant of the optimal seed density. For instance, the ideal seeding density for broccoli microgreens is different from that of pea microgreens, reflecting variations in seed size and growth habits [6,7]. While some detailed guides and tables provide specific recommendations for various microgreens, the core principle remains: adjusting seed density can significantly affect yield and quality.
Seed density and fresh biomass yield are directly correlated in microgreen production [8]. A study found that increasing the sowing density to between 3 and 5 seeds/cm2 for rapini, kale, and cress can boost yield by 19% at the highest density compared to the lowest [9]. Another study demonstrated that increasing seed densities to between 50 and 200 g/m2, along with enhancing the conductivity of the nutrient solution, led to an increase in shoot dry matter and soluble solids index in rocket microgreen [10]. ecílio Filho, et al. [11] demonstrated that increased sowing density for red cabbage can cause greater competition among seedlings, which increases hypocotyl length and negatively impacts the cotyledon area and shoot dry mass of the microgreens. An optimal seed density is suggested to avoid reducing individual shoot biomass and overall quality. In practice, growers rely on their visual estimates to determine sowing density, often without precise quantification. Industry standards tend to be ambiguous, offering broad ranges (e.g., 61–122 g/m2) without providing specific details tailored to individual crops [12]. There is some discrepancy among the published studies on seed density. Therefore, it is essential to assess seed density to optimise yield and quality-specific product traits, such as the timing of harvest and costs. This research offers a new comprehensive assessment of how seed density impacts both the yield and microbial safety of cress, rocket, and pea microgreens. While previous studies concentrated only on optimising growth or evaluating other agronomic benefits, this study connects microgreen productivity with food safety, presenting a comprehensive approach for cultivating microgreens safely and effectively.
In addition to seed density, both the quality and duration of light exposure play crucial roles in influencing plant growth and the accumulation of phytochemicals [13]. Light affects plant physiological processes through photoreceptors that are sensitive to various light spectra, including ultraviolet (UV), blue, red, and white light. In particular, UV radiation is known for inducing stress responses in plants, which can result in an increased production of secondary metabolites, such as phenolics and antioxidants [13]. These compounds enhance the nutritional value of microgreens and contribute to extended shelf life by inhibiting microbial growth on plant tissue [14]. Thus, white light, which encompasses a wide range of photosynthetically active radiation (PAR), is vital for optimal photosynthesis and overall plant growth. Ultraviolet A (UV-A) radiation has been proven to boost the nutritional and antioxidant properties of microgreens, thereby increasing their health benefits [15]. This improvement is facilitated by the activation of specific photoreceptors and the formation of reactive oxygen species (ROS), which drive the production of bioactive compounds [16]. hang, et al. [17] highlighted the enhanced quality of microgreens by increasing the levels of beneficial bioactive compounds. Specifically, when microgreens are subjected to 402 nm UV-A for a prolonged duration of 16 h, there is a notable rise in total phenolic content and α-tocopherol levels. Conversely, exposure to shorter wavelengths, such as 366 nm, results in higher concentrations of lutein/zeaxanthin and β-carotene. Furthermore, longer UV-A wavelengths played a positive role in the accumulation of essential mineral elements, thereby improving the nutritional profile of mustard microgreens without hindering their growth. Similar studies have shown that exposure to UV-A radiation, at wavelengths of 366 and 390 nm, significantly enhanced the phytochemical composition and biomass of microgreens like pak choi and mustard, while also positively affecting their antioxidant characteristics [15,18]. Additionally, the duration of light exposure, referred to as the photoperiod, can further modulate these effects by influencing biomass accumulation, enzyme activity, and metabolite synthesis [17].
The microbiological safety of microgreens is critically important because they are eaten raw, without a thermal ‘kill step’ to reduce pathogens. Unlike mature leafy greens, microgreens require high moisture and are densely sown, creating a humid environment that encourages the growth of spoilage fungi and harmful bacteria such as Salmonella enterica and Listeria monocytogenes [19]. Research demonstrates that industrial production is constrained by rapid quality deterioration, with high respiration rates and the “presence of microorganisms” responsible for spoilage playing a major role [20].
This research explores maximising yield and quality while ensuring microbial safety in cress, rocket, and pea microgreens. By integrating a study on supplemental UV-A radiation and extended harvesting time, this work connects agronomical productivity with consumer safety. Our findings offer a multidimensional framework for optimising indoor cultivation, ensuring that the drive for maximum yield does not compromise the functional or microbial integrity of microgreens. This highlights the critical need to optimise seed density to balance yield and microbial quality, providing valuable insights for indoor farming and urban agriculture systems.
2. Materials and Methods
2.1. Materials
Organic, microgreen seeds of cress (Lepidium sativum L.), rocket (Eruca sativa), and pea (Pisum sativum L.) were purchased from Seeds Ireland, 6 Old Schoolhouse, Patrickswell, Limerick V94 Y3C3, Ireland, for the purposes of optimising seed density, harvesting day, and yield (fresh and dry biomass). Seed germination percentage was calculated under the same conditions for all species [8] (Table 1). All three varieties were sown in four replicates at all seed densities, based on seed size, as shown in Table 1. Each experiment was repeated three times.
Table 1.
Species-specific seeding densities.
| Species | Family | Average Germination Percentage (%) | Seed Density (Seed/cm2) |
|---|---|---|---|
| Lepidium sativum L. (cress) | Brassicaceae | 94 | 6, 8, 10, 12, and 14 |
| Eruca sativa (L.) Cav. (rocket) | Brassicaceae | 93 | 8, 10, 12, 14, and 16 |
| Pisum sativum L. (pea) | Fabaceae | 86 | 1, 2, 3, and 4 |
2.2. Method
Seeds were germinated in seedling compost (Westland John Innes Seed Sowing Compost; peat-free and enriched with vermiculite) in small plastic pots (7 cm width × 7 cm length × 8 cm height) at calculated seed density for growing (Table 1) and placed in a dark room for three days for cress and rocket and four days for peas. Following germination, pots were transferred to a plant growth tent (Light House LITE Grow Tent) purchased from The Grow Shop, Ardsallagh, Navan, Meath C15 E52P, Ireland, and equipped with a steel rack (OMAR 1 shelf sec 92 width × 36 depth × 181 height cm). Two light strips of grow lights (Cosmorrow LED (The Grow Shop, Ardsallagh, Navan, Meath C15 E52P, Ireland) 40W Grow, 90 cm, White 6500 °K, Spectrogram Figure 1a) with PPFD ~205 μmol/s/m2 as per the manufacturer’s datasheet were placed at a distance of 25 cm from the LED lights at the top of the pots and remained in that position for the experiment’s duration to replicate commercial settings, where microgreens are produced under fully or partially controlled conditions utilising different growing media. Each shelf contained two trays containing 24 pots each, and the pots were moved daily except during the germination period, to minimise positional effects. A Tinytag Explorer 6.0 sensor was positioned on the middle shelf to record the relative humidity (RH) and temperature inside the growth tent every four hours (Figure 1b). To continuously expel excess heat and humidity, a Ram Inline Duct Fan (100 mm, 277 m3/h) was operational around the clock, linked via a 102 mm × 10 M Ram Aluduct Low Noise aluminium duct to the growth tent (Figure 1c). To ensure optimal seed germination, the roots and growing medium were adequately hydrated by applying a light mist to the microgreens once or twice daily. This was combined with sub-irrigation of the compost by adding water to each tray (41 cm × 29 cm) containing twelve pots, to allow plants to absorb water more efficiently. After 30 min, the trays were drained of excess water. This technique was used to prevent water from weighing down the delicate microgreens growing above the soil surface [21].
Figure 1.
(a) Spectrogram as per manufacturer’s datasheet, (b) humidity and temperature records for the growth tent, and (c) experimental setup with LED lights and growing microgreens.
2.2.1. Harvesting and Measurements
Different harvesting time points were tailored according to the growth rate of each species to optimise the harvest time for microgreens with the goal of maximising the nutritional value and yield. Cress, rocket, and pea microgreens were harvested on days 7, 9, and 12 post-sowing, respectively. This adjustment in the harvesting schedule accommodated the developmental timeline of selected microgreens, ensuring that the study captured their growth characteristics accurately. Pots were selected by simple random selection on each harvesting day, and microgreens were collected by cutting the stem 1 cm above the growth medium surface using scissors cleaned with 70% ethanol [22].
Total fresh biomass (FW) was immediately determined using an analytical weighing balance (Fisherbrand™ Analytical Balances 220 g capacity Model FAS224/E, Boulevard, Waltham, MA, USA). Subsequently, samples were dried in an oven (Heraeus drying oven) at 65 °C for 72 h to record dry biomass [23].
After drying, the moisture content was calculated using a specific equation:
| Moisture content (%) = (FW−DW/FW × 100) | (1) |
This formula defines FW (g) as the mass of freshly harvested microgreens and DW (g) as the mass of microgreens after drying.
2.2.2. Influence of White Light and White Light Supplemented with UV-A Radiation on Pigment and Colour in Pea Microgreens
To evaluate the effects of light in pea microgreen, this study was performed using two different light regimes: growing light (control) (Cosmorrow LED 40W Grow, 90 cm, White 6500 °K) and white light supplemented with ultraviolet radiation (treatment) (Cosmorrow Ultraviolet LED, 40W, 90 cm, comprising 33% near-ultraviolet with PF ~64 μmol/s (365 nm) and 66% cool white LED (6500 °K) with PPFD ~205 μmol/s/m2) as per the manufacture’s datasheet, with a photoperiod of 16 h daylength and 8 h darkness. Pea microgreens at 1 seed/cm2 were used and subjected to either light regime (control or treatment) while maintaining consistent photoperiod, temperature, and humidity conditions across all plants. Growth parameters (fresh biomass, dry biomass, and moisture content) were recorded at intervals of the harvest schedule (day 12, 17, 23, and 30). Freshly harvested pea microgreens (Pisum sativum L.) were dried in an oven (Heraeus drying oven) at 65 °C for 72 h and ground into powder using a mortar and pestle. Then, 200 mg of dried plant material was mixed with 10 mL of 99.9% HPLC-grade methanol using a digital Ultra-Turrax homogeniser (IKA T25, Staufen, Germany) operating at 10,000 revolutions per minute (RPM) for 5 min. The resulting mixture was centrifuged at 7000× g RPM for 15 min to eliminate insoluble residues. The supernatant was then filtered through Whatman No. 1 filter paper (Whatman Ltd., Kent, UK) to obtain clear extracts for pigment analysis. To determine photosynthetic pigments, the concentrations of chlorophyll a (Chl a), chlorophyll b (Chl b), and total carotenoids in the pea microgreen extracts were determined using a UV-Vis spectrophotometer. Subsequently, absorbance was measured at wavelengths of 470 nm, 652 nm, and 665 nm. The pigment concentrations were calculated using the equations provided by Lichtenthaler (1987) [24] and expressed as micrograms per milligram of dry biomass (µg·mg−1 DW).
| Chlorophyll a: Chl a (µg/mL) = 16.72 × A665 − 9.16 × A652 | (2) |
| Chlorophyll b: Chl b (µg/mL) = 34.09 × A652 − 15.28 × A665 | (3) |
| Total Carotenoids: Ca (µg/mL) = (1000 × A470 − 1.63 × Chl a − 104.96 × Chl b)/221 | (4) |
These pigment concentrations provide insights into the photosynthetic status and potential nutritional value of the pea microgreens at different stages of harvest.
Simultaneously, total phenolic content in pea microgreens was analysed using the Folin–Ciocalteu reagent (Sigma-Aldrich, St. Louis, MO, USA) through a spectrophotometric method, as outlined by Singleton and Rossi (1965) [25]. In a 10 mL volumetric flask, 0.2 mL of the extract, blank, or standard was mixed with 6 mL of distilled water, followed by the addition of 0.5 mL of the Folin–Ciocalteu reagent. After a period of one to eight minutes, 1.5 mL of a 20% sodium carbonate solution (Thermo Scientific™, Waltham, MA, USA) was added, and the solution was topped up with distilled water. The colour that developed after two hours was measured at 760 nm using a spectrophotometer (UV-1240, Shimadzu Corporation, Kyoto, Japan). A standard curve was established using gallic acid (Sigma-Aldrich), with concentrations ranging from 0 to 750 mg/L, achieving an R2 value ≥ 0.99. The total phenol content was expressed as milligrams of gallic acid equivalents per gram of dry biomass (mg GAE.g-1, DW).
The colour of dried pea microgreens was evaluated using a colourimeter (Chroma Meter CR-400, Konica Minolta, Tokyo, Japan). This instrument analyses the colour spectrum in a three-dimensional format (CIE standard illuminant D65 and standard observer 10), where the “L” axis indicates the transition from black to white; the “a” axis shows the shift from green to red; and the “b” axis illustrates the change from blue to yellow. The L-axis is scaled from 0 to 100, with values exceeding 50 signifying lighter samples, while those below 50 denote darker samples. Measurements were taken in four replicates for accuracy.
2.2.3. Microbiological Analysis
Microgreens harvested on the given days were analysed to ascertain the total aerobic bacteria (TAB). A sample of 10 g was weighed in four replicates and added to 90 mL of sterile maximum recovery diluent buffer (MRD, Scharlau, Barcelona, Spain). Serial dilutions were performed using the same diluent, and 0.1 mL of each dilution was inoculated onto tryptic soy agar (TSA) for the total aerobic bacteria (TAB), onto violet red bile glucose agar (VRBGA) for Enterobacteriaceae, and onto potato dextrose agar (PDA) for fungi. Plates were incubated at 30 °C for 24–48 h to enumerate the TAB. This was performed three times to check reproducibility. Colony-forming units (CFUs) were expressed as log CFU/g of fresh biomass [26].
2.2.4. Statistical Analysis
Statistical analysis was conducted to compare microbial loads across different microgreen types and treatment conditions to provide insights into yield and microbial safety throughout cultivation.
All statistical analyses were conducted using R (https://cran.r-project.org/, accessed on 13 November 2023). When assumptions were met, data were analysed using one-way ANOVA (stats package, v4.4.1), and pairwise comparisons were performed with Tukey’s HSD post hoc test (emmeans and multcomp packages) at a significance threshold of p ≤ 0.05. For datasets that did not follow normality assumptions, the non-parametric Kruskal–Wallis test was applied, followed by Dunn’s post hoc test with Bonferroni correction (FSA package, 0.10.0). The results of pairwise comparisons were presented as significance groupings denoted by letter codes on boxplots generated with ggplot2 (v3.5.1), indicating statistically homogeneous subsets.
3. Results
3.1. Effect of Seed Density on Yield (Fresh Biomass)
In all three species, the fresh biomass yield increased by increasing seeding density until reaching an optimal point, after which it slightly decreased or remained unchanged (Figure 2a–c).
Figure 2.
Fresh biomass (g) of microgreens at different seed densities: (a) cress (6–14 seeds/cm2); (b) rocket (8–16 seeds/cm2); (c) pea (1–4 seeds/cm2). Data represent mean ± SD from three independent experiments (n = 3), each comprising four biological replicates. Different letters indicate significant differences at p < 0.05 at different seed densities.
For both cress and rocket, the maximum fresh biomass was observed at a seeding density of 12 seeds/cm2, whereas pea achieved its highest yield at 2 seeds/cm2. Specifically, cress biomass increased from 10.10 ± 1.65 g at 6 seeds/cm2 to 17.50 ± 1.64 g at 12 seeds/cm2, before experiencing a minor drop to 16.76 ± 2.64 g at 14 seeds/cm2 (Figure 2a, Table 2). Rocket exhibited a similar pattern, with the yield increasing from 12.13 ± 1.57 g at 8 seeds/cm2 to 18.45 ± 2.39 g at 16 seeds/cm2. For pea microgreens, fresh biomass increased from 15.31 ± 1.23 g at 1 seed/cm2 to 26.43 ± 4.86 g at 3 seeds/cm2; it then decreased to 19.79 ± 5.08 g at 4 seeds/cm2, likely due to overcrowding and competition among plants. The moisture content remained stable across all seed densities and species, ranging from 90.8% to 95.6%, which is typical for microgreens with high water content. Dry biomass values followed the trends observed in fresh biomass.
Table 2.
Yield parameters of microgreens at different seeding densities.
| Variety | Seeds/cm2 | Fresh Biomass (g) ± SD | Dry Biomass (g) ± SD | Moisture Content (%) ± SD |
|---|---|---|---|---|
| Cress | 6S | 10.10 ± 1.65 a | 0.54 ± 0.05 a | 94.56 ± 0.73 a |
| 8S | 13.50 ± 1.79 b | 0.65 ± 0.07 b | 95.10 ± 0.67 a | |
| 10S | 15.02 ± 2.60 bc | 0.75 ± 0.08 c | 94.88 ± 0.65 a | |
| 12S | 17.50 ± 1.64 d | 0.88 ± 0.11 d | 94.92 ± 0.36 a | |
| 14S | 16.76 ± 2.64 cd | 0.90 ± 0.08 d | 94.49 ± 1.11 a | |
| Rocket | 8S | 12.13 ± 1.57 a | 0.64 ± 0.07 b | 94.68 ± 0.54 b |
| 10S | 14.47 ± 1.38 b | 0.68 ± 0.04 ab | 95.26 ± 0.43 ab | |
| 12S | 17.78 ± 1.89 c | 0.82 ± 0.14 a | 95.36 ± 0.46 ab | |
| 14S | 17.50 ± 2.12 c | 0.77 ± 0.19 ab | 95.59 ± 0.95 a | |
| 16S | 18.45 ± 2.39 c | 0.83 ± 0.13 a | 95.47 ± 0.50 a | |
| Pea | 1S | 15.31 ± 1.23 a | 1.05 ± 0.05 a | 91.99 ± 0.44 c |
| 2S | 22.94 ± 1.08 b | 1.88 ± 0.03 b | 91.40 ± 0.38 b | |
| 3S | 26.43 ± 4.86 b | 2.23 ± 0.41 b | 92.06 ± 0.59 bc | |
| 4S | 19.79 ± 5.08 ab | 2.00 ± 0.45 b | 90.77 ± 0.40 a |
Various seed densities selected for each variety and their respective fresh weights, dry weights, and moisture contents. Different lowercase letters indicate significant differences (p < 0.05).
3.2. Effect of Seed Density on Microbial Load
Increasing seed density led to a significant increase in microbial load, total aerobic bacteria (TAB), Enterobacteriaceae, and fungi populations (Figure 3).
Figure 3.
The effect of seed densities on microbial load (log10 CFU/g fresh biomass) for (a) cress, (b) rocket, and (c) pea microgreens. Each panel shows the effect on total aerobic bacteria (TAB), Enterobacteriaceae, and fungi. Data represent mean ± SD from three independent experiments (n = 3), each comprising four biological replicates. Different letters indicate significant differences at p < 0.05 at different seed densities.
In cress microgreens, the total aerobic bacteria count rose significantly from 7.04 ± 0.09 log10 CFU/g to 7.94 ± 0.17 log10 CFU/g as the seed density increased from 6 to 10 seeds/cm2. Beyond this density, no further rise in microbial load was detected. Conversely, the counts for Enterobacteriaceae (~7 log10 CFU/g) and fungi (~7 log10 CFU/g) remained constant across all seed densities. A similar pattern was noted in rocket microgreens, where the total aerobic bacteria count significantly increased from 7.03 ± 0.05 log10 CFU/g to 7.88 ± 0.07 log10 CFU/g at 10 seeds/cm2. Enterobacteriaceae and fungal counts also showed an increasing trend. Previous studies have reported similarly high levels of bacterial count, typically ranging from ~8 to 9 log10 CFU/g, in cress and rocket microgreens [27]. In pea microgreens, which have larger seeds and plants, the total aerobic count was lower in comparison to cress and rocket, and it exhibited a significant increase with seed density, rising from 3.54 ± 0.07 log10 CFU/g to 5.58 ± 0.37 log10 CFU/g.
3.3. Effect of White Light and White Light Supplemented with UV-A Radiation on the Extended Growing Period on Pea Microgreens
Pea microgreens were selected to grow under white light and white light supplemented with UV-A for 30 days, to measure yield (biomass), microbial load, and phytochemicals (Figure 4a,b).
Figure 4.
Impact of white light and white light supplemented with UV-A radiation on (a) fresh biomass and (b) microbial load (total aerobic bacteria and Enterobacteriaceae). Values are mean ± SD (n = 4). Different lowercase letters indicate significant differences (p < 0.05) at different harvesting days under white light and white light supplemented with UV-A radiation, and uppercase letters show differences from 12 to 30 days under white light and white light supplemented with UV-A radiation.
3.3.1. Biomass and Microbial Load
On day 12, microgreens under white light had a notably higher fresh biomass (~13 g) than those under white light supplemented with UV-A radiation (~11 g; p < 0.05). However, by day 17, the microgreens exposed to white light supplemented with UV-A radiation had surpassed those under white light, reaching a fresh biomass of approximately 21 g, whereas the microgreens grown under white light only reached 16 g (p < 0.05). On day 23, the fresh biomasses of microgreens under both light conditions became similar, with no significant differences detected (p > 0.05), as yields stabilised between 24 and 30 g of FW per pot. The shift over time indicates that initial UV-A exposure caused mild photomorphogenic stress, leading to a temporary decrease in early biomass accumulation due to short-term inhibition of hypocotyl elongation and cell expansion [28,29]. By day 17, however, the plants appeared to have acclimated, potentially through the activation of antioxidant mechanisms and an increase in photosynthetic pigments, which enhanced light utilisation efficiency. Similar patterns of delayed growth recovery under UV-A have been documented in basil and mustard microgreens, where low doses of UV-A initiated photoprotective responses without affecting the final yield [30]. In the later stages of development (days 23–30), the absence of yield differences between white light and UV-A treatments suggests that exposure to commercially available white light supplemented with UV-A radiation does not significantly impact overall biomass production in pea microgreens. Total aerobic count and Enterobacteriaceae counts remained stable within each light treatment, ranging from ~3.8–4.2 log10 CFU/g. For the TAB, the only significant difference was observed on day 17, which was a slightly lower value under white light; this difference did not sustain throughout the growing period. The Enterobacteriaceae count varied significantly over time under both light conditions, but counts did not differ significantly between the white light and white light supplemented with UV-A groups. A previous study on beet microgreens grown under different light conditions showed a similar aerobic count of 3.67–5.07 log CFU/g and Enterobacteriaceae count of 3.94–5.54 log CFU/g [31]. Overall, white light supplemented with UV-A did not exert a sustained impact on microbial loads.
3.3.2. Phytochemical and Colour Analysis
The total phenolic content showed a notable increase from day 12 to day 23 (Table 3), followed by a decline by day 30 (p < 0.05) under both light treatments. There were no significant differences observed between the white light and supplemental UV-A treatments at any point. Brazaityte, Virsile, Jankauskiene, Sakalauskiene, Samuoliene, Sirtautas, Novickovas, Dabasinskas, Miliauskiene and Vastakaite [15] reported similar results in basil, beet, and red pak choi and concluded that changes could be species-specific under UV-A. Another study showed increased antioxidant capacity under UV-A for Brassica species [32].
Table 3.
Effects of white light and white light supplemented with UV-A radiation on total phenols, carotenoids, and chl in the extended growing period of pea microgreens.
| Day | Compounds | White± SD | UV-A± SD |
|---|---|---|---|
| 12 | Total phenols | 2.65 ± 0.19 aA | 2.63 ± 0.18 aA |
| 12 | Carotenoids | 1.48 ± 0.23 aB | 2.00± 0.29 bB |
| 12 | Total Chl A + B | 14.98 ± 0.61 aA | 15.57 ± 1.20 aA |
| 17 | Total phenols | 3.4 ± 0.27 aB | 3.48 ± 0.38 aB |
| 17 | Carotenoids | 1.06 ± 0.08 bB | 0.55 ± 0.07 aB |
| 17 | Total Chl A + B | 14.21 ± 0.64 aA | 15.93 ± 0.04 bA |
| 23 | Total phenols | 4.13 ± 0.37 aC | 4.09 ± 0.23 aC |
| 23 | Carotenoids | 1.14 ± 0.21 aB | 0.72 ± 0.35 aA |
| 23 | Total Chl A + B | 14.80 ± 0.70 aA | 16.40 ± 0.44 bA |
| 30 | Total phenols | 2.78 ± 0.19 aA | 3.01 ± 0.2 aAB |
| 30 | Carotenoids | 0.65 ± 0.13 aA | 0.54 ± 0.09 aA |
| 30 | Total Chl A + B | 18.35 ± 1.53 aB | 19.66 ± 1.21 aB |
Phytochemical content of pea microgreens under white light and white light supplemented with UV-A radiation at days 12, 17, 23, and 30. Values are mean ± SD (n = 4). Different lowercase letters indicate significant differences (p < 0.05) at different harvesting days under white light and white light supplemented with UV-A radiation, and uppercase letters show differences from 12 to 30 days under white light and white light supplemented with UV-A radiation.
On day 12, carotenoid exhibited significantly higher levels (2.00 ± 0.29 µg/mg DW) under white light supplemented with UV-A radiation compared to only white light (1.48 ± 0.23 µg/mg DW). However, this trend had reversed by day 17, where the white light group maintained higher levels (1.06 ± 0.08 µg/mg DW) compared to the white light supplemented with UV-A radiation (0.55 ± 0.07 µg/mg DW) group. By days 23 and 30, both light treatments showed no statistically significant difference, similar to a previous study demonstrating that brief UV exposure enhances carotenoid biosynthesis as part of an abiotic stress response in microgreens [16]. Chlorophyll content was higher on day 30 regardless of light treatment; it increased significantly from day 12 to day 30, reaching 18.35 ± 1.53 µg/mg DW under white light and 19.66 ± 1.21 µg/mg DW under white light supplemented with UV-A radiation.
Pigments and phenolic levels either declined or remained the same regardless of the light treatments, supporting prior reports of pigment degradation and metabolic plateauing during extended growth of microgreens [16,33]. Collectively, these results underscore that white light supplemented with UV-A radiation acts as an abiotic elicitor to stimulate pigment biosynthesis transiently, but the effects on bioactive compounds are strongly modulated by the developmental stage and duration of exposure, as demonstrated in several published microgreen studies. Colorimetric analysis revealed subtle variations in colour between the two light treatments (Table 4).
Table 4.
Effects of white light and white light supplemented with UV-A radiation on the colour of pea microgreens.
| DOH | Light | L* | a* | b* |
|---|---|---|---|---|
| 12 | W | 42.51 ± 4.29 aAB | 0.53 ± 0.16 aA | 14.20 ± 2.48 aA |
| 12 | UV-A | 38.44 ± 0.83 aAB | 0.50 ± 0.04 aAB | 18.59 ± 0.53 bAB |
| 17 | W | 47.69 ± 1.47 bBC | 0.40 ± 0.06 aA | 15.23 ± 0.67 aA |
| 17 | UV-A | 42.05 ± 2.79 aA | 0.38 ± 0.10 aAB | 16.66 ± 1.19 aA |
| 23 | W | 40.60 ± 0.73 aA | 0.7 ± 0.06 aAB | 18.88 ± 0.33 aB |
| 23 | UV-A | 42.14 ± 2.51 aA | 0.65 ± 0.11 aAB | 19.17 ± 1.05 aB |
| 30 | W | 51.30 ± 1.44 bC | 0.99 ± 0.31 aB | 16.89 ± 1.52 aAB |
| 30 | UV-A | 47.31 ± 2.49 aB | 0.70 ± 0.23 aA | 16.31 ± 1.59 aB |
Colour parameters (L*, a*, b*) of pea microgreens grown under white light and white light supplemented with UV-A radiation across harvest days. Values are mean ± SD (n = 4). Different lowercase letters indicate significant differences (p < 0.05) at different harvesting days under white light and white light supplemented with UV-A radiation, and uppercase letters show differences from 12 to 30 days under white light and white light supplemented with UV-A radiation.
The L* coordinate, representing lightness (0 = black, 100 = white), exhibited an inverse relationship with the total chlorophyll content reported in Section 3.3.2.
These values show brightness, ranging from ~38 to 51, with no consistent trend between the two treatments. The green–red (a*) values remained close to zero, confirming the characteristic green hue of pea microgreens and exhibiting negligible treatment effects. By contrast, the yellow–blue axis (b*) revealed a transient increase in yellowness under UV, particularly at day 12 (18.59 ± 0.53), compared with white light (14.20 ± 2.48). This significant increase in b* at day 12 provides external validation for the phytochemical results (Table 3), where pea microgreens exhibited significantly higher carotenoid content (2.00± 0.29 µg/mg DW) than the control (1.48 ± 0.23 µg/mg DW). Because carotenoids absorb light primarily in the blue region (400–500 nm) and reflect it in the yellow–orange region (550–650 nm), the photoprotective response to UV-A stress resulted in the elevated b* value [34]. Overall, these findings suggest that while white light supplemented with UV-A radiation induces some limited and temporary changes in the phytochemical profiles and colour characteristics of pea microgreens, it does not significantly enhance overall yield or reduce microbial load when compared with white light.
4. Discussion
Previous studies have predominantly focused on the combined effect of seed density and factors like light spectrum, growing media, and fertilisation to maximise yield and improve phytochemical content [35,36,37]. While these studies contribute valuable agronomic knowledge, they often overlook the essential aspect of food safety. Our research, however, not only assesses the influence of seed density on biomass yield but also addresses the growing risk of food safety due to microbial contamination in minimally processed microgreens.
The strong positive relationship between seed density and yield, particularly up to 12 seeds/cm2 for cress and rocket, mirrors previous findings that show a linear increase in fresh biomass with higher sowing rates (4, 8, and 12 seeds per cavity), where the highest yield was observed at 12 seeds [36]. However, beyond these optimal densities, as observed at 14 seeds/cm2 in cress and rocket, the yield either plateaus or slightly declines. This is likely due to factors such as overcrowding, reduced airflow, and intraspecies competition, which are known to impede individual plant growth and heighten the risk of microbial contamination. The elevated microbial counts at higher seed densities further underscore the trade-off between maximising yield and ensuring food safety. However, a study has observed the same or even higher levels of microbial load on over 20 microgreen varieties, including cress and rocket [27]. The significant differences in microbial load observed between white light and UV-A treatments were limited to specific sampling points and were not sustained throughout the growing period.
Furthermore, our findings showed that significant differences in microbial load (TAB) and Enterobacteriaceae load of pea microgreens grown under white light and white light supplemented with UV-A radiation were limited to specific sampling points and were not maintained throughout the growing period. The Enterobacteriaceae population exhibited significant proliferation over time, increasing from day 12 and reaching a higher level by day 30, while the TAB remained similar under white light and under UV-A radiation. However, comparing light conditions by day 30, the TAB was significantly lower under white light with UV-A radiation than under white light alone, suggesting that supplemental UV-A effectively limits late-stage bacterial overgrowth.
Exposure to white light with UV-A radiation resulted in short-term increases in pigments, with carotenoids reaching their highest levels on day 12 and chlorophyll on day 17. However, these increases were not maintained over the period of 30 days. UV-A radiation’s regular application for the purpose of enhancing phytochemicals under these conditions is not recommended without further refinement in terms of timing, dosage, and wavelength.
Fresh biomass exhibited minor increases with extended growth periods, independent of light quality. These results suggest that the commercially available white light supplemented with UV-A radiation used in this study, comprising 33% near-ultraviolet radiation (365 nm) and 66% cool white LED (6500 K), is not more effective than white light alone in enhancing phytochemical accumulation or reducing microbial load in pea microgreens under the given conditions. This highlights the need to critically evaluate the spectral composition and biological efficacy of supplemental lighting strategies before implementation in commercial microgreen production systems. Our research findings also highlight the need for further investigation into the consistently elevated microbial levels associated with Brassicaceae microgreens. Future research should focus on exploring microbial communities, particularly in regard to identifying organisms that may be pathogenic or responsible for spoilage. Simultaneously, it is crucial to develop effective methods for both pre-harvest and post-harvest phases to reduce microbial contamination and ensure the safety of these nutrient-rich crops.
Given the increasing importance of controlled-environment agriculture, particularly in urban and resource-constrained settings, this study offers practical advice to microgreen growers to maximise yield without sacrificing quality or safety.
5. Conclusions
Increasing seed density enhances yield (fresh biomass) up to an optimal threshold, beyond which yield either remains unchanged or declines.
Across all examined microgreen species, higher seed densities were consistently linked to higher microbial loads, including Enterobacteriaceae.
Prolonged exposure to white light supplemented with UV-A radiation did not maintain increased phytochemical levels or lower the microbial load.
Optimisation of seed density is critical for each microgreen species to maximise yield and minimise input cost, thereby maximising economic returns.
Surpassing the optimal seed density threshold may compromise both food safety and yield per unit area, highlighting the importance of seed density management in controlled-environment agriculture.
Acknowledgments
The authors would like to acknowledge Jean-Christophe Jacquier and Belay Dereje Olika for their valuable academic support and insightful discussions throughout this study. Special thanks are extended to Joey Henchy, the technical officer, for his essential assistance with the experimental setup.
Author Contributions
S.D.: performance of experiments, data accumulation, writing—original draft, and methodology; C.E.-K.: conceptualisation, data validation, revising the manuscript, and supervision; N.H.: conceptualisation linking extended growing period to the functional properties, and funding acquisition; D.H.: conceptualisation regarding the seed density and microbial load, supervision, and feedback on the original draft; A.R.: feedback on the original draft and visualisation. All authors have read and agreed to the published version of the manuscript.
Data Availability Statement
Data are contained within the article.
Conflicts of Interest
The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.
Funding Statement
The authors acknowledge funding support from the Government of Ireland Department of Agriculture, Food, and the Marine from research grant number 2021R523.
Footnotes
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Data Availability Statement
Data are contained within the article.