Iron (Fe) and zinc (Zn) are key trace-elements essential for human health, and their dietary deficiency affects over a quarter of the world population, causing physiological disorders, diseases, and even death, thus constituting a primary global public health challenge [1
]. Iron is a crucial constituent of proteins such as hemoglobin and myoglobin devoted to oxygen transportation within the human body, and it is a constituent and activator of a variety of enzymes that are involved in electron transport, oxidation-reduction reactions, and other biological functions [5
]. According to the World Health Organization (WHO), iron deficiency anemia is the most ubiquitous health disorder on the global level, and it is closely associated with poor diet, prevalence of cereal-based food, and low consumption of meat, fish, and fruit and vegetables, diets typical of low-income regions in both developed and developing countries [6
]. Iron is particularly critical for women of childbearing age and young children, and its deficiency increases the risks of maternal mortality. Deficiency can cause stunted growth and irreversible effects on the psychomotor and cognitive function of young children [6
]. Following Fe in terms of quantitative body requirements, Zn is a structural constituent of proteins or a catalyzing cofactor involved in the activity of several vital enzymes such as RNA polymerase, superoxide dismutase, cellular signaling proteins, thus playing a key role in multiple biological functions [8
]. Like Fe, Zn deficiency is associated with poor, non-diversified diets characterized by high prevalence of cereal-based food products which have low Zn content and bioavailability [2
]. Inadequate intake of Zn during pregnancy and in pre-school children can permanently impair physical growth, immunity to disease, reproductive function, and neuro-behavioral development [2
]. Similarly to Fe deficiency, the risk of Zn deficiency is significantly higher and widespread in low-income communities in both industrialized and developing countries and primarily affects young children and women [2
Regions with higher risks of Fe and Zn deficiency are generally characterized by soils containing low levels of these nutrients or with properties that limit phytoavailability of trace elements [12
]. Moreover, today, the negative effects of climate change on the availability of nutritious food is exacerbating the risks of malnutrition, especially in developing countries [13
], and addressing Fe and Zn deficiency in a growing world population could become an emergency. Increasing awareness about the negative impact, especially on women and children, of such an extended and underlying public health issue raised the attention of the scientific community, and a number of studies have highlighted the importance of supplementing the diet of populations that are at risk of Fe and Zn deficiency with food enriched with these microelements [4
]. Besides diversifying the diet and trying to improve food selection habits, biofortification of staple crops has been proposed as a primary solution to address Fe and Zn deficiency, as well as for other essential micronutrients [4
]. A number of research projects have focused on crop breeding and genetic engineering, demonstrating that it is possible to fortify and enhance the bioavailability of Fe and Zn in staple crops mainly by: (i) increasing the expression of metal-binding proteins and enzymes; (ii) enhancing the movement and transportation of Fe and Zn from the root to the edible portions; (iii) reducing the content of antinutrients that inhibit Fe and Zn absorption such as phytates, tannins, and dietary fibers; or (iv) increasing the concentration of compounds and factors that promote the absorption of Fe and Zn such as vitamin C, organic and amino acids, and β-carotene [4
]. Nevertheless, this approach has several constraints, and despite the availability of modern breeding and biotechnology tools, the cost and time required to develop genetically biofortified crops make this solution sustainable only in the long run [12
]. Especially in the case of engineered crops, due to the high level of regulation and long process of evaluation required, the development and final introduction of a biofortified crop can take years, if not decades, as it requires the approval of plant scientists, policy makers, nutritionists, economists, and last but not least, the acceptance of the entire community [24
As an alternative, agronomic biofortification, which consists of increasing the accumulation of target nutrients in edible plant tissues through fertilization or other eliciting factors has been increasingly proposed in recent years as a simpler, short-term approach to develop functional staple crops and vegetables and address micronutrient deficiency [26
]. The exogenous application of cations in plants is not new and has been long used in crop cultivation to overcome problems such as the use of marginal irrigation water and high salinity through the regulation of nutrient uptake and the alleviation of nutrient imbalances caused in plants grown under stress conditions [33
]. Moreover, according to Chrysargyris et al. [35
], foliar application of minerals such as Si and Zn may have a positive effect on growth, development, and mineral content of Lavandula angustifolia
(Mill.) plants, especially when grown under salinity stress conditions. However, simply increasing the concentration of micronutrients in edible plant tissues is not enough. Crop biofortification, either genetic or agronomic, is particularly challenging in the case of Fe and Zn, especially in the case of cereals, legumes, and oilseeds, which are characterized by limited micronutrient uptake and high levels of anti-nutrients such as phytates which significantly limit the absorption and bioavailability of Fe and Zn [36
A solution to this problem could be the biofortification of sprouted seeds instead of the regular grain crop as has been already proposed for soybean [38
], pea [39
], brown rice [29
], alfalfa, broccoli, radish, and mung bean [40
]. For several crops, it has been demonstrated in fact that during the germination process, as the content of phytate in the seed decreases, the bioavailability of Fe and Zn increases [29
]. Yet, agronomic biofortification conducted on a large scale also has some risks related to the variability of micronutrient uptake, which cannot be easily standardized due to the variable interactions between genotypes and environments, as well as to the interactions among nutrients during uptake. Repeated supplemental fertilization of the soil with metals like Fe and Zn can pose environmental and health risks due to their potential leaching into the ground water or the accumulation in the soil or in the plant tissues at excessive levels that can be toxic for the plants and other living organisms, as well as for the consumers [11
]. From this perspective, soilless growing systems combined with customized nutrient solutions of known concentrations could allow a standardization of the process of biofortification and fine control of the product quality, while avoiding or minimizing some of the risks associated with agronomic biofortification of soil grown crops [45
]. A number of studies have in fact proven the potential benefits of soilless growing systems in assuring efficient production of high quality and nutritious food crops, especially of leafy vegetables, even under conditions that are limiting or prohibitive for typical soil cultivation, such as in the case of contaminated soils, lack of growing space or availability of non-conventional growing space, scarce and/or low quality water resources, or other limiting conditions that are easily encountered in developing countries and marginal rural or urban areas [49
]. In selecting the method of biofortification and the crops that are most suitable for Fe and Zn biofortification, besides considering the potential physiological limits for the enrichment of a crop with specific micronutrients, it is important to consider the presence of factors that can inhibit or promote micronutrient availability, the presence of antinutritional factors or other risks, the potential for consumer acceptability of the biofortified product, and ideally, it would be good to consider the possibility of developing a method of biofortification simple enough to be transferred to the community in need, allowing them to self-produce biofortified food.
In recent years, microgreens have become increasingly popular as a rich source of vitamins, bioactive compounds, and minerals and have rapidly gained the appellative of “super food” or “functional food” [52
]. Like sprouts, microgreens are a promising crop category for Fe and Zn biofortification [55
]. In fact, using a variety of species, including wild edible plants, even with minimum availability of seeds, microgreens can be easily produced domestically in a relatively short time (7–21 days) within the framework of urban horticulture [54
]. The multitude of species suitable for the production of microgreens could guarantee a very rich and complete diet, providing a variety of essential nutrients, while the process of germination with the reduction of phytate as observed in sprouts, and the high content of Fe- and Zn-absorption promoters such as ascorbic acid and β-carotene that characterize microgreens, could assure a high bioavailability of both these trace elements. Although very limited research data is available on microgreen concentrations of Fe and Zn, data reported by Di Gioia et al. [54
] and Xiao et al. [56
] suggest that while there is significant variability among different genotypes, Brassicaceae species could be considered as a good source of both Fe and Zn, and their actual concentration in young plant tissues is highly influenced by the availability of nutrients during the growth period [55
]. Moreover, Brassicaceae microgreens are very popular, relatively inexpensive, easy to germinate and grow, and have great potential health-benefits thanks to their high content of glucosinolates, vitamins, and polyphenols [46
]. With such considerations, it is worth exploring the efficacy and potential implications of producing Fe and Zn agronomically biofortified Brassicaceae microgreens. To this purpose, a study was conducted to investigate the effects of increasing levels of Zn and Fe supplied through the nutrient solution on yield components and the mineral profiles of three popular species of Brassicaceae microgreens, namely arugula, red cabbage, and red mustard.
2. Materials and Methods
2.1. Experimental Site, Treatments, and Growing System
Two experiments were conducted during the spring of 2016, in a high tunnel covered with polyethylene film and lateral openings for natural air circulation, at the University of Florida (UF) Institute of Food and Agriculture Science (IFAS) Southwest Florida Research and Education Center (SWFREC) located in Immokalee, FL.
Three Brassicaceae species commonly used to produce microgreens were selected for both experiments: arugula (Eruca sativa (Mill.) Thell.) cv. ‘Astro’, red cabbage (Brassica oleracea L. var. capitata) cv. ‘Red Acre’ and red mustard (Brassica juncea (L.) Czern.) cv. ‘Purple Osaka’. Seeds of all three species were provided by Seedway LLC (Lakeland, FL, USA) and tested for germinability prior to seeding. Seeds were of high quality with a germination rate of 98%, 95%, and 97% at constant 20 °C, while the number of seeds per g was 520 ± 6.4, 180 ± 2.1, and 417 ± 10.8 (mean ± SD, n = 5) for arugula, red cabbage, and red mustard, respectively.
In both experiments, microgreens were grown in a soilless system using grow channels (Cropking Inc., Lodi, OH, USA) 0.25 m wide by 2.8 m long placed on adjustable benches with a slope of approximately 5% to enhance nutrient solution movement. Each channel hosted the three species grown on BioStrate-Felt (Cropking Inc., Lodi, OH, USA) growing mats (23 × 60 cm = 1380 cm2). Plants were fertigated with a half-strength modified Hoagland nutrient solution containing selected levels of zinc (0, 5, 10, and 20 mg L−1) in the first experiment (Exp-1), and selected levels of iron (0, 10, 20, and 40 mg L−1) in the second experiment (Exp-2). The standard nutrient solution was prepared using deionized water containing (mg L−1) 105.1 nitrogen, 15.5 phosphorus, 117.4 potassium, 26.0 magnesium, 92.5 calcium, 34.6 sulfur, 1.20 iron, 0.60 manganese, 0.15 zinc, 0.30 boron, 0.08 copper, and 0.03 molybdenum. Both macro and micronutrients were added to the nutrient solution using simple fertilizer grade salts such as calcium nitrate, potassium nitrate, ammonium nitrate, monopotassium phosphate, potassium sulphate, magnesium sulfate, copper EDTA, manganese sulfate, sodium borate, and sodium molybdate (Helena, Immokalee, FL, USA). In Exp-1, using laboratory grade zinc sulfate heptahydrate (ZnSO4 + 7H2O, Sigma Aldrich, St. Louis, MO, USA), zinc level was adjusted based on the predefined treatment rates (0, 5, 10, and 20 mg L−1), while iron concentration was the same for all Zn enrichment treatments (1.20 mg L−1). In Exp-2, using laboratory grade iron sulfate heptahydrate (FeSO4 + 7H2O, Sigma Aldrich, St. Louis, MO, USA), iron levels were adjusted based on the predefined treatment rates (0, 10, 20, and 40 mg L−1), while zinc concentration was the same for all Fe enrichment treatments (0.15 mg L−1). The final nutrient solution had an average electrical conductivity (EC) of 1.25 dSm−1 and pH 6.2. The nutrient solutions, stored in separate containers for each level of Zn and Fe in Exp-1 and Exp-2, respectively, were pumped to the growing benches and delivered at the upper end of each channel through an irrigation line with five pressure-compensated drippers (each with a delivery rate of 4.0 L h−1) per channel. Particular attention was paid to leveling the growing channels and defining the length of fertigation events to assure adequate and uniform distribution of the nutrient solution along the width and length of each channel at each fertigation event. The nutrient solution delivery was managed with an open cycle system and a 20% minimum drainage fraction in order to ensure that the entire growing mat area was completely wet at every fertigation event, while avoiding nutrient accumulation in the growing mats. The frequency of fertigation events of one or two minutes was adjusted through a timer that activated the individual pumps on each nutrient solution tank. The excess nutrient solution was collected in buckets at the end of each channel to measure the drainage volume and then was discarded. In a real production scenario, a closed cycle system with recirculating nutrient solution would be used for producing Zn and Fe enriched microgreens in order to optimize the use of water and nutrients; however, for the purpose of this research, an open management cycle was used to maintain a constant Zn and Fe concentration during the entire growing cycle in order to record the actual effect of Zn and Fe addition in the nutrient solution on plant nutrient content. Treatments were replicated three times and were arranged according to a split plot design, in which the single channels constituted the main plots and the growing mats of each species constituted the sub-plots.
2.2. Planting Date, Seeding Density, Treatment Differentiation, and Harvest
All three species were sown on 15 February 2016 and on 29 February 2016 for Exp-1 and Exp-2, respectively. Seeding density was defined after preliminary experiments, considering the number of seeds per gram and the germinability of the batch of seeds of each species. The equivalent of 55, 125, and 67 g m−2 of seeds were used in order to obtain a density of 2.9, 2.2, and 2.8 seeds cm−2 for arugula, red cabbage, and red mustard, respectively. After sowing, deionized water was sprayed on top of the seeds using a misting nozzle and growing benches were covered for two days with a white on black polyethylene film allowing seed germination in the dark in both experiments. After complete germination, the third day after sowing (on 18 February 2016 and 3 March 2016, in Exp-1 and Exp-2, respectively), the growth benches were uncovered and fertigation treatments with different levels of Zn or Fe enrichment were applied at each fertigation event until harvest. Microgreens of all three species were harvested 11 days after sowing (DAS) on 26 February 2016 and 11 March 2016 in Exp-1 and Exp-2, respectively. At harvest, all three species had fully expanded and turgid cotyledons with the appearance of the tips of the first true leaves. Harvest was conducted by cutting the shoots a few millimeters above the growing pad surface using clean cutter blades. To avoid border effects, a 2.5 cm strip on each end of the growing mat was excluded from the sampling area. Harvested microgreens were weighed to determine the fresh yield (g m−2) and a pre-marked 10 × 10 cm area in the middle section of each growing area was used for counting the number of germinated shoots to determine the shoot population density (shoots m−2) and the mean shoot fresh weight (mg shoot−1). Dry matter content (g 100 g−1 FW) was determined on fresh samples of approximately 300 g dried until constant weight at 65 °C in a forced-air oven. Dried plant tissue samples were ground using a mill and passed through a 1.0 mm sieve and were used to determine the concentration of P, K, Ca, Mg, Cu, Fe, and Zn.
2.3. Mineral Analysis
Dry and finely ground microgreen tissue samples collected at harvest were analyzed to determine the concentration of P, K, Ca, Mg, Cu, Fe, and Zn using the dry ash combustion digestion method [63
]. Analyses were performed at the University of Florida in the soil and plant analysis laboratory at the Southwest Research and Education Center, using an inductively coupled plasma atomic emission spectrometry (ICP-AES) system (OES Optima 7000 DV, PerkinElmer, Santa Clara, CA, USA). Dry plant tissue samples of 1.5 g were weighed and dry ashed at 500 °C for 16 h [65
]. The ash was equilibrated with 15 mL of 0.5 M HCl at room temperature for 0.5 h. The solution was decanted into 15 mL plastic disposable tubes and placed in a refrigerator at <4 °C [66
] until analyses using ICP-AES were performed using calibration and quality assurance procedures as described by Munter et al. [67
]. A three-point calibration was performed with standards within the expected range of concentrations. A quality control sample representing a blank, standard, or replicate was included after every 10 unknows in each sample set. Macronutrients and trace-element concentrations determined on dry weight (DW) basis (mg kg−1
of DW) were then transformed and presented on a fresh weight (FW) basis (mg 100 g−1
of FW) using the dry matter percentage previously determined on the same plant tissue samples (see Section 2.2
2.4. Statistical Analysis
Linear and quadratic regression analyses were used to estimate the yield and nutrient component response of each species to Zn and Fe concentration in the nutrient solution using the regression procedure (PROC REG) of the Statistical Analysis System software (SAS Institute, Cary, NC, USA). Collected data were subject to analysis of variance using the General Linear Models procedure of the Statistical Analysis System software (SAS Institute, Cary, NC, USA). Significant differences between treatments were analyzed using the Student–Newman–Keuls post-hoc multiple comparison procedure at p = 0.05.