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Exogenous reactive oxygen species (ROS) produced by nanobubble (NB) water offer a reasonable explanation for NBs' physiological promotion and oxidation effects. To develop and exploit the NB technology, we have performed further research to identify the specific ROS produced by NBs. Using a fluorescent reagent APF, a Fenton reaction, a dismutation reaction of superoxide dismutase and DMSO, we distinguished four types of ROS (superoxide anion radical (O·), hydrogen peroxide (HO), hydroxyl radical (·OH), and singlet oxygen (O)). ·OH was confirmed to be the specific ROS produced by NB water. The role of ·OH produced by NB water in physiological processes depends on its concentration. The amount of exogenous ·OH has a positive correlation with the NB number density in the water. Here, spinach and carrot seed germination tests were repeatedly performed with three seed groups submerged in distilled water, high-number density NB water, and low-number density NB water under similar dissolved oxygen concentrations. The final germination rates of spinach seeds in distilled water, low-number density NB water, and high-number density NB water were 54%, 65%, and 69%, respectively. NBs can also promote sprout growth. The sprout lengths of spinach seeds dipped in NB water were longer than those in the distilled water. For carrot seeds, the amount of exogenous ·OH in high-number density NB water was beyond their toxic threshold, and negative effects were shown on hypocotyl elongation and chlorophyll formation. The presented results allow us to obtain a deeper understanding of the physiological promotion effects of NBs.

Nanobubbles (NBs) have been reported to be effective at accelerating the metabolism of living organisms, but the mechanism is not yet well understood. In this study, the production of reactive oxygen species (ROS) by NBs and its effect on seed germinations were investigated. The fluorescence response of APF to NB water was measured. It changed depending on the NB number density which decreased with storage time. This indicated that NBs could produce ROS and the amount of ROS had positive correlation with the NB number density. The fluorescence intensity of APF increases linearly with the concentration of H 2 O 2 in the range of 0−1 mM. Just after the NB generation, the oxidative capacities represented by amount of ROS of oxygen NB water and gas-mixture (air + nitrogen) NB water were estimated to be equivalent to 0.5 and 0.3 mM H 2 O 2 respectively. The seed germination tests were performed in the NB water, distilled water and H 2 O 2 solutions. The germination rate at each observation times of seeds submerged in gas-mixture NB water and 0.3 mM H 2 O 2 solutions were both higher than those submerged in distilled water. The amounts of superoxide radicals in the seeds were detected using NBT staining. The results of absorbance data proved that the amounts of O 2•− in seeds submerged in gas-mixture NB water and in 0.3 mM H 2 O 2 solution were similar and significantly higher than those in the distilled water. These results indicated that moderate level of exogenous ROS produced by NB water played an important role in seed germination.

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