Nanobubbles are microbubbles smaller than 0.2 μm (Jung et al., 2016) and are applied to various fields such as surface washing, water purification, growth promotion, and fluid friction reduction with their unique physical properties (Liu et al., 2013; Song et al., 2013). Nanobubbles are affected by less buoyancy than macrobubbles (≥50 μm) or microbubbles (10–50 μm) due to nanosized bubble diameter and rise very slowly, which is why they remain in liquid for a long time (Jeong et al., 2017). Shock waves generated as nanobubbles break apart in nanobubble water promote formation of highly reactive hydroxyl radical (−OH) and effectively decompose organic matter, thereby enabling water purification (Ahmed et al., 2018).

In general, the amount of dissolved oxygen (DO) contained in water is 1–3 ppm for groundwater and 3–4 ppm for tap water or purified water. Compared to groundwater, tap water, and purified water, nanobubble water includes 45–60 times more DO (Song et al., 2013). Lower oxygen content of soil inhibits root respiration and growth (Greenway et al., 2006). Supplying sufficient oxygen to the root zone of plants promotes absorption of soil moisture and nutrients, thereby increasing crop yield (Du et al., 2018; Sang et al., 2018; Zhou et al., 2019). The germination rate of barley was 58% in 17 hours of the experiment after applying oxygen nanobubble water that has 15–25% higher DO than distilled water, thereby showing a 28% increase compared to applying distilled water (Liu et al., 2016). Increasing DO is effective in plant cultivation, but the extent varies depending on the crop. When micro-nanobubble is applied with DO 15 mg·L⁻¹ to tomatoes and cucumbers, soil enzyme content and the amount of beneficial soil microbes increased in root zone soil (Zhou et al., 2019). Moreover, leaf length, leaf width, number of leaves, and leaf area of lettuce increased in the plant applying DO 8.6 mg·L⁻¹ using microbubbles compared to the control plot (DO 6.15 mg·L⁻¹) (Abu-Shahba et al., 2021). Vitamin C and water-soluble sugar content were highest in the 15 mg·L⁻¹ treatment plot (Zhou et al., 2019). It is necessary to explore the most suitable oxygen nanobubble water concentration for germination and growth of each crop.

The propensity to consume fresh foods is constantly growing in terms of choosing and consuming plants based on increased living standard and income (Jun et al., 2012). Due to the change in dietary culture, vegetable sprouts and leafy vegetables for wraps are receiving attention. Sprouts refer to edible fresh shoots from seeds at an early stage of growth, and they are harvested quickly with the growth duration of 3–7 days (Lee et al., 2011). The market size for sprouts accounts for about 3% of the whole market, and starting with radish sprouts in the 1980s, various sprouts are being cultivated in the 2000s such as Leguminosae, Gramineae, and Cruciferae (Seo, 2013). Leafy vegetables have lower calories than other foods and include many important nutrients necessary for maintaining health, and thus the consumption propensity is increasing along with the change in food consumption patterns (Jun et al., 2012; Lee et al., 2016; Lee, 2017). Among leafy vegetables, cruciferous vegetables reduce the risk of diseases such as cancer and diabetes, have a powerful antioxidant effect, and are beneficial for health (Lee, 2017). Red mustard among cruciferous vegetables is a popular and typical red vegetable (Rural Development Administration [RDA], 2018), and pak choi is well received by consumers for its high content of antioxidant glucosinolates (Podsędek et al., 2007).

This experiment was conducted to investigate the effect on seed germination and seedling growth when applying oxygen nanobubble water in various DO concentrations in substrate cultivation of 3 types of sprouts and 2 types of leafy vegetables commonly consumed in Korea and determine an effective DO concentration.

Germination rates in all sprouts did not show a difference between the control plot and NB treatment plots in 7 days after sowing (Table 2), but in leafy vegetables, germination rates of red mustard were 93.3%, 95.3%, 91.3%, and 96.7% in NB 0%, NB 20%, NB 33%, and NB 100% treatment plots in 5 days after sowing and the rate was high in NB 100% treatment plot compared to the control plot (NB 0%) (Table 1). As a result of increasing the DO to 8 mg·L⁻¹ using microbubbles on barley seeds, the germination rate increased significantly compared to the control plot (distilled water), showing the highest significance (p ≤ .001) especially around 18 hours of treatment during the experiment. Thus, NB showed a close correlation at the initial stage of germination of barley seeds (Hwang et al., 2016). When applying two types of oxygen NB (DO 8, 40 mg·L⁻¹) to spinach and carrot seeds, the germination rate increased by at least 9% when DO increased (Liu et al., 2016). It was predicted that the germination rate will increase along with DO; but comparing the germination rate of red mustard in treatment plots, it was found that the rate of NB 33% treatment plot decreased compared to NB 20% treatment plot, and the rate was highest in NB 100% treatment plot (Table 1). Excessively high DO concentration may destroy the cells or cause diseases in the body (Bailly et al., 2008). In canola, osmotic pressure and physical stress occurred with hydroxyl radical generated from microbubbles when DO concentration was supersaturated (9–11 mg·L⁻¹) (Ikeura et al., 2017). In NB 20% treatment plot of this study, the germination rate increased with DO concentration of approximately 15 mg·L⁻¹, but the germination rate may have decreased in NB 33% treatment plot with DO concentration of approximately 21 mg·L⁻¹ due to supersaturation of DO concentration that inhibited growth. Hwang et al. (2016) and Liu et al. (2016) applied NB by immersing the seeds in oxygen sponges and beakers. However, this study conducted the experiment by sowing the seeds in horticultural substrates and watering them with oxygen NB considering the actual cultivating conditions. Thus, there may be an effect of organic matter in horticultural substrates that may have increased the germination rate of red mustard significantly in NB 100% treatment plot. Accordingly, the adequate DO is presumed to be NB 20% to increase the germination rate of red mustard. For other leafy vegetables except red mustard and sprouts, there was no difference between the control plot and treatment plots in germination rates and mean germination time of all materials, indicating that oxygen NB did not have much effect on germination. However, considering the germination rate, red mustard is expected to see a stable increase in the germination rate within the DO concentration range of 15.40 mg·L⁻¹ (NB 20%).

For leafy vegetables, leaf length and leaf width of red mustard were higher in NB 33% treatment in 5 days after sowing of the experiment than the control plot, but tended to decrease from 35 to 45 days after sowing in NB 33% and NB 100 % treatment plots compared to the control plot and NB 20% treatment plot (Figs. 1A–1D). Number of leaves and leaf area of leafy vegetables did not show a difference in NB treatment plots regardless of plant type (Figs. 1E–1H). Number of leaves and leaf length of cruciferous Brassica campestris increased in the treatment plot where oxygen nanobubbles concentration was increased to 31.7 mg·L⁻¹ compared to the control plot with 7.7 mg·L⁻¹ (Ebina et al., 2013). Moreover, for lettuce, leaf length, leaf width, number of leaves, and leaf area increased with statistical significance (p ≤ .05) when applying microbubble water with DO concentration of 8.6 mg·L⁻¹ compared to the control plot (DO 6.15 mg·L⁻¹) (Abu-Shahba et al., 2021). On the other hand, leaf length and leaf width of watercress did not show a significant difference between the control plot and the treatment plot applying microbubbles (Bok et al., 2019). Ahmed et al. (2018) reported that, after applying oxygen nanobubbles to tomatoes, carrots, and horse beans cultivated in soil, leaf number of tomatoes increased significantly, but there was no effect on carrots and horse beans, and leaf length did not have significance in all plants. Thus, the treatment effect of oxygen nanobubbles varies depending on plant type.

In the sprout experiment of this study, the seedling height did not show a difference between the control plot and NB treatment plots depending on DO (Table 3). The plant height of leafy vegetables was not significantly different between all NB treatment plots compared to the control plot at 15 and 20 days after sowing, but those in NB 20% increased than the control plot at 45 days after sowing in red mustard (Fig. 2A). In pak choi, the plant height increased constantly in 15, 20, and 35 days after sowing in some concentrations when applying NB (Fig. 2C). It tended to increase in all NB treatment plots compared to the control plot at 40 days after sowing, but there was no difference between treatment plots in 45 days after sowing. This study did not additionally apply nutrient solutions to examine the effect of oxygen NB on germination and growth of fresh foods such as sprouts and leafy vegetables in cultivation; rather, it supplied a low level of EC at 0.14 – 0.16 dS·m⁻¹ to determine only the effect of DO concentration (Table 1). Pak choi is a crop that has a high need for nutrients, and EC 1.8 and 2.4 dS·m⁻¹ are reported as the suitable nutrient concentrations for growth of pak choi. Low EC of 0.0 – 0.6 dS·m⁻¹ induces nutrient stress, which may inhibit growth and deteriorate the quality of pak choi (Ding et al., 2018). Thus, the low EC of oxygen NB may not show any effect on the height of sprout or leafy vegetables at 45 days after sowing, which is the final point of the experiment.

Generation methods of microbubbles like nanobubbles include pressurized dissolution type, gas circulating type, gas-liquid swirling flow type, etc. (Yoon et al., 2013), and there may be a difference in plant growth depending on the generation method of microbubbles. When applying microbubbles generated by pressurized dissolution type, the iron in the culture medium may be oxidized, possibly inhibiting plant growth such as plant length and dry weight (Ikeura et al., 2017). Nanobubbles in this experiment were generated using frictional force. Unlike previous studies, this study cultivated the plants using substrates instead of hydroponics, and thus did not use the hydroponics system or nutrient solutions in the experiment. However, physicochemical characteristics of bubbles vary depending on how microbubbles are generated, and there may be an effect on nutrient and water absorption of roots due to the energy accumulated in the bubbles as well as generated radicals (Bok et al., 2019). The generation method of nano-bubbles may also have affected plant growth in this study. Thus, it is necessary to conduct additional research analyzing the physicochemical characteristics of bubbles when applying oxygen nanobubbles using frictional force and the nutrient and water adsorption capacity of roots.

Barley sprouts and red mustard showed an increase in both fresh weight and dry weight of the shoot and root in all treatment plots and NB 100% treatment plot, respectively, compared to the control plot. In particular, fresh weight and dry weight of the root of red mustard increased by 55% and 47%, respectively, in NB 100% treatment plot compared to the control plot (Fig. 3). Weight is an important indicator that determines the growth condition or relative growth rate of crops (Kim et al., 2016). Moreover, dry weight is one of the key factors in expressing the relationship between crop growth and the environment and determining the biomass of crop harvest season (Both et al., 1994). Root length of radish sprouts and wheat sprouts increased in NB 33% treatment plot compared to the control plot (Table 3). The root length of red mustard did not show any difference between the control plot and NB treatment plots in 45 days after sowing, but it increased more in NB 100% treatment plots than the control plot in pak choi (Figs. 2B and 2D). The negative surface charge of microbubbles directly supplied oxygen to the root surface and positively stimulated growth (Takahashi, 2005), thereby increasing fresh weight of radish sprouts and wheat sprouts when applying oxygen nanobubbles compared to the control plot. For leafy vegetables, leaf length and leaf width of red mustard tended to decrease in NB 33% and NB 100% treatment plots (Figs. 1A–1D), but fresh weight and dry weight increased, which can be interpreted as growth as the plant size becomes dense. Not only color and shape of leafy vegetables but also how dense they are is a significant factor in evaluating the optimum harvest period. Leafy vegetables that are not yet mature or too mature for harvest tend not to be dense or have too hard mesophyll (Gil and Tudela, 2020). Thus, healthy leaf growth of sprouts and leafy vegetables can be made possible by applying NB 100% oxygen NB.

This study could not precisely control the DO for seed germination and leaf growth since horticultural substrates are used, and did not show a consistent seed germination response in sprouts and leafy vegetables compared to previous studies using hydroponics. However, for radish sprouts and wheat sprouts, root length became longer and fresh weight increased in DO concentration of 20.93 mg·L⁻¹. In leafy vegetables, fresh weight and dry weight increased in both leafy vegetables and sprouts excluding pak choi, showing that oxygen NB treatment enables growth stimulation of vegetables. There is a difference in the scope of DO concentration that enables stable growth of sprouts and leafy vegetables within the scope of oxygen NB treatment, and for leafy vegetables, the adequate scope of DO concentration is 39.29 mg·L⁻¹.