Article
pubs.acs.org/Langmuir

Identiﬁcation of ROS Produced by Nanobubbles and Their Positive
and Negative Eﬀects on Vegetable Seed Germination
Shu Liu,*,† Seiichi Oshita,*,† Saneyuki Kawabata,† Yoshio Makino,† and Takahiko Yoshimoto††

Graduate School of Agricultural & Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, JapanS Supporting Information*

ABSTRACT: Exogenous reactive oxygen species (ROS) produced by nanobubble (NB) wateroﬀer 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 (O2·−), hydrogen peroxide (H2O2), hydroxyl radical (·OH), andsinglet oxygen (1O2)). ·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 seedgroups 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 were54%, 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.

■

using a sensitive ﬂuorescent probe, 3′-p-(aminophenyl)
ﬂuorescein (APF).17 In the same year, Minamikawa et al.
reported that using electron spin resonance (ESR) spectra, free
radicals were likely to be generated by the collapse of NBs.18
The generation of ROS due to NBs oﬀered a reasonable
explanation for the physiological promotion eﬀect of NBs.
However, ROS are not single entities but represent a broad
range of chemically distinct reactive species with diverse
biological reactivities.19 Therefore, to develop and exploit the
NB technology, further research must be performed to identify
the types of ROS produced by NBs. The main types of ROS
include the superoxide anion radical (O2·−), hydrogen peroxide(H2O2), hydroxyl radical (•OH), and singlet oxygen (1O2),
which are metabolic byproducts in both plants and animals.20
Pure water is composed of two elements, hydrogen and oxygen;therefore, these four types of ROS are the most likely species tobe produced in oxygen NB water. ESR has been widely used todetect the free radical generation by MNBs in the presence ofdynamic stimuli such as ozone, strong acid, and copper.21−23
However, the high DO concentration in oxygen NB water willaﬀect the ESR spectra of free radicals in solution. As oxygen isparamagnetic, oxygen-radical collisions contribute to themagnetic relaxation of free radicals and cause subsequent

INTRODUCTION
Reactive oxygen species (ROS) have historically been viewed as
purely harmful metabolic byproducts that cause oxidative
damage to lipids, proteins, and nucleic acid.1 Research in recent
decades, however, has highlighted new roles for ROS as
important physiological regulators of cellular signaling pathways. The concept of a “biological window” or an “oxidative
window” was raised in the ﬁelds of both plant biology andhealth and medicine.2,3 Too few ROS may result in decreased
antimicrobial defense, whereas too many ROS could destroy
cells and produce pathological eﬀects; however, a moderatelevel of ROS will play a positive role in growth.2,4 Bothendogenous and exogenous sources contribute to the formationof intracellular ROS.5 Endogenous sources of cellular ROS
include NADPH oxidase, mitochondrial oxidant, cytochrome coxidase, and xanthine oxidase.6 Exogenous sources include
irradiation (UV, X-, and γ-rays)5 and chemicals such as
exogenous H2O2.7−9
In recent decades, many papers have presented case studies
on the eﬀects of micro and nanobubbles (MNBs) on thephysiological activity of living organisms, such as promoting the
growth of plants,10,11 ﬁsh,12−14 and cell cultures.15,16 Althoughsome reports have attempted to describe the reason for which
physiological activity is promoted, most have simply
represented case studies without elaborating the mechanisms.
Recently, a new characteristic of NBs was discovered by our
research group, namely, that without any stimuli, NB water
could continually produce small amounts of ROS in the water© 2016 American Chemical Society

Special Issue: Nanobubbles
Received: April 28, 2016
Revised: June 2, 2016
Published: June 3, 2016
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Figure 1. Schematic of experimental design for ROS identiﬁcation (FI: Fluorescence intensity; ↑: increase; ↓: decrease; : no change).

ESR line broadening.24 Meanwhile, using the ESR method,some researchers have observed free radical generation by NB
water itself,18 whereas some did not.25 For the above reasons,an alternative higher sensitivity methodology is needed for
detection of the production small quantities of ROS by NB
water itself without any stimuli. The ﬂuorescence methodology,
associated with the use of suitable probes, is an excellent
approach to measure ROS because of its high sensitivity,
simplicity in data collection, and high spatial resolution in
microscopic imaging techniques.26 Thus, in this paper we try to
identify what type of ROS is produced by NB water using a
ﬂuorescent probe, APF. A strong oxidizing power is required
for the ipso-substitution reaction of APF. Therefore, due to thediﬀerent oxidizing powers of various ROS, the ﬂuorescence
responses of APF to them also diﬀer. Based on the above fact,
we can build a set of methods to distinguish the four types of
ROS using APF and identify the speciﬁc type of ROS producedby oxygen NB water.
ROS are a double-edged sword. A moderate increase in ROS
concentration may promote cell proliferation and survival.
However, when ROS concentration reaches the toxic threshold,it may overwhelm the cellular antioxidant capacity and trigger
the cell death process. Therefore, as signal molecules, diﬀerent
amounts of exogenous ROS produced by NBs will havediﬀerent eﬀects on the growth of living organisms.
Furthermore, the amount of exogenous ROS positively
correlated with the NB number density in the water.17 Here,
we study the eﬀects of NB number density on the germination
processes of two types of vegetable seeds (spinach and carrot).
The germination rates and the elongation of roots andhypocotyls are both compared among the seeds submergedin water with diﬀerent NB number densities. We intend toobtain a deeper understanding of the mechanism behind thephysiological promotion eﬀects of NBs, together with theidentiﬁcation of the ROS generated by them.

■(SOD) will lead to the formation of H2O2. Then, we will add ferrous
ions to the solution. The ferrous ions would react with H2O2 to
produce •OH, causing a dramatic increase in ﬂuorescence intensity. If
the ﬂuorescence intensity of samples does not dramatically increase,
the existence of O2•− will be excluded. Third, we will try to identify
whether the ROS produced by NB water is •OH or 1O2. DMSO is
known to quench •OH but not 1O2. Hence, after DMSO is added, if
the ﬂuorescence intensity of NB water decreases, the ROS will be•OH. Otherwise, the ROS will be 1O2. More detail is provided in the
Results and Discussion section.
Reagents. APF (5 mM, Sekisui Medical Co., Ltd., Japan), a
ﬂuorescent reagent that is highly resistant to autoxidation, was used to
identify the diﬀerent ROS. APF itself has almost no ﬂuorescenceintensity. After APF reacts with ROS, ﬂuorescein (with strong
ﬂuorescence intensity) is generated. SOD (Bovine Erythrocytes
5612U/mg) was obtained from Nacalai Tesque Inc. Japan. Ultrapure
water (1 mL) was added in 3000 units of SOD powder and thesolution was used within 2 days. H2O2 (30%, Kanto Chemical Co.,
Inc., Japan) is prepared prior to each use. Other chemicals werepurchased from Kanto Chemical Co., Inc., Japan and were of thehighest obtainable purity.
Fluorescence Spectrophotometry. To obtain ﬂuorescence
excitation−emission matrices, the excitation wavelength was increased
from 450 to 550 nm in 1 nm steps; for each excitation wavelength, theemission at longer wavelengths was also detected in 1 nm steps with aﬂuorescence spectrophotometer (F-7000, Hitachi High-Tech Co. Ltd.,
Japan). The slit width was 5 nm for both excitation and emission. The
photomultiplier voltage was set to 700 V. Because the baseline
ﬂuorescence varies from probe to probe and from experiment toexperiment, the ﬂuorescence intensity was given as a relative value
corresponding to a combination of parameters such as slit width,
location of lamp, and voltage.27 The ﬂuorescein caused by ROS had an
excitation−emission matrix peak located at an excitation wavelength of
490 nm and an emission wavelength of 515 nm.
Hydroxyl Radical Formation. Hydroxyl radical was generated by
the well-known Fenton reaction.28 Ferrous iron(II) is oxidized by
H2O2 to ferric iron(III), a hydroxyl radical, and a hydroxyl anion,
which is shown in eq 1. H2O2 of 0.1 mM was added to the sodium
phosphate buﬀer (0.1 M, pH 7.4) containing APF and then various
amounts of ferrous sulfate solutions were added. The ﬁnal theoreticalconcentrations of •OH were in the range 0−25 μM. Ferrous sulfate
solution was freshly prepared prior to each use.
Superoxide Anion Radical Formation. Reactions with potassium superoxide (KO2) were performed during conditions of very low
relative humidity. KO2 powder was carefully weighed in dry beakers
before use. Buﬀer solution containing APF was added with vigorous
mixing in the beaker containing KO2 powder. After reacting with KO2
for 2 min, the ﬂuorescence intensity was determined. The ﬁnal
theoretical concentrations of KO2 ranged from 0 to 1 mM.
Singlet Oxygen Formation. Singlet oxygen was formed by 3(1,4-epidioxy-4-methyl-1, 4-dihydro-1-naphthyl) propionic acid (EP).
EP powder was carefully weighed in dry beakers before use. Buﬀer
solution containing APF (buﬀer containing 10% ethanol) was added

EXPERIMENTAL SECTION

Experimental Design. Figure 1 shows a schematic of ROS
identiﬁcation. The ﬂuorescence response of APF to four diﬀerent typesof ROS will be studied. Due to the diﬀerent oxidative capacities of
ROS, which are related to electrode potential, the ﬂuorescence
responses should be diﬀerent. First, we will try to identify whether the
ROS produced by NBs is H2O2. The formation of •OH by a Fenton
reaction will cause a dramatic increase in the ﬂuorescence intensity.
Therefore, if this phenomenon is not observed after adding ferrous
ions to the H2O2 solution, the existence of H2O2 will be excluded.
Second, we will try to identify whether the ROS produced by NBs is
O2•−. The dismutation of O2•− catalytic with superoxide dismutase
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Langmuirwith vigorous mixing in the beaker containing the EP powder. The
mixtures were stirred a 37 °C for 15 min. The ﬁnal theoreticalconcentrations of singlet oxygen ranged from 0 to 250 μM.
Fluorescence Intensity of NB Water. Pure oxygen (purity
99.99995%, Taiyo Nippon Sanso Co. Ltd., Japan) was introduced into
a 2-L phosphate buﬀer (0.1 M, pH 7.4) and circulated through an
MNB generator (OM4-GP-040, Aura Tec Co. Ltd., Japan) for 1 h at aconstant temperature of 20 °C to obtain “oxygen NB water.” The DOconcentration of oxygen NB water was above 40 mg L−1. APF of 2 μL
was added into 10 mL of water containing NBs and the ﬂuorescenceintensities were measured. This was repeated more than ﬁve times.
The ﬁnal concentration of APF was 1 μM.
Seed Germination. Seeds of carrot (Daucus carota subsp. sativus)
and spinach (S. oleracea L.), which were harvested in 2015 and stored
under controlled conditions (room temperature), were obtained from
Takii & Co. Ltd.
Germination tests were repeatedly performed with three seedgroups. Each group was composed of 50 seeds. We used distilled water
as a control. The DO concentration of distilled water was about 8
ppm. For NB water, a mixture of nitrogen and air was used to adjust
the DO of MNB water to be the same as that of the distilled water. NB
water was prepared following the method described in our previous
paper.17 To study the eﬀect of NB number density on the germination
process, we diluted NB water with distilled water in order to adjust the
number density to be 20% of the original value. The original NB water
is termed “100% NB water.” The total NB number densities in control
water, 100% NB water, and 20% NB water were about 0, 1.22 × 108,
and 2.44 × 107 particles/mL, respectively (Supplemental data 1). NB
number densities were measured using the nanoparticle tracking
analysis method (NanoSight-LM10, Quantum Design Inc., Japan).
The nanoparticle tracking analysis cannot distinguish between NBs
and foreign matters. However, the decrease of total particle number
density with storage time proved that most of the particles that exist in
the water are actually NBs rather than solid nanoparticles.11,17
Spinach and carrot seeds were submerged in beakers ﬁlled with
distilled water, 20% NB water, and 100% NB water in a ratio of 10 mLof water per seed. During the germination experiments, to avoid thelack of oxygen and to maintain a certain amount of NBs in the water,
the 100% NB water, distilled water, and 20% NB water were changed
twice a day. Germination tests for spinach and carrot seeds lasted forabout 7 and 14 days, respectively.
Chlorophyll Content. Twenty carrot seeds were ground to ﬁne
powder with liquid nitrogen in a mortar and pestle and transferred to 2
mL tubes. Acetone solution (80%) of 500 μL was added to each tube
and the contents were mixed, homogenized, and incubated at 4 °C in
the dark for 2 h. Homogenates were clariﬁed by centrifugation at
15 000 rpm for 10 min at 4 °C. Clariﬁed extracts were used forchlorophyll content analyses. The absorption of the extracts wasmeasured with a spectrophotometer by considering the spectrum
between 500 and 700 nm. The absorption at 652 nm is used torepresent the total chlorophyll content.

Figure 2. Fluorescence response of APF (0.1 M, pH 7.4) to diﬀerent
kinds of ROS. The ﬂuorescence intensity was determined at 515 nmwith excitation at 490 nm (a) Detection of •OH in the Fenton
reaction (In the horizontal axis, the concentration of Ferrous iron(II)is equal to that of hydroxyl radicals, H2O2 (0.1 mM) was added tobuﬀer solution of APF and then ferrous sulfate was added. The
ﬂuorescence increases were the ﬂuorescence intensities minus thebaseline signal obtained after adding 0.1 mM H2O2). (b) Fluorescenceincrease of APF in H2O2 system. (c) Fluorescence increase of APF in
KO2 system. (d) Fluorescence increase of APF in EP system. The
ﬂuorescence increases were the ﬂuorescence intensities minus thebaseline signal.

reaction. Ferrous sulfate was added at various concentrations(1−25 μM) into the buﬀer solution of APF containing an
excess of H2O2 (0.1 mM). According to the Fenton reaction
shown in eq 1, ferrous iron(II) is oxidized by H2O2 into ferriciron(III), a hydroxyl anion, and a hydroxyl radical. The
concentration of ferrous iron is equal to the concentration of
the hydroxyl radical:
Fe 2 + + H 2O2 → Fe3 + + OH− + •OH(1)

Figure 2b shows the relationship between the ﬂuorescenceincrease of APF and the concentration of H2O2. In the range oflow H2O2 concentrations (0−1 mM), APF can be used todetect the existence of H2O2 in terms of a dose-dependent
increase of ﬂuorescence. By comparing panels a and b in Figure
2, when similar ﬂuorescence intensities were produced in the
APF solution, the concentration of H2O2 was about 2000-foldgreater than that of •OH.
APF was incubated with various concentrations of KO2
solution to test its reactivity with O2•−, and the increase inﬂuorescence intensity was shown in Figure 2c. The ﬂuorescence
response of APF to •OH was 180-fold greater than that ofO2•−, similar to the 200-fold increase in the literature.29
APF was incubated with various concentrations of EP
solutions to test its reactivity with 1O2, and the increase inﬂuorescence intensity was shown in Figure 2d. We found that
APF response was about 120-fold greater to •OH than to 1O2.
These results coincide with the results in other papers.29,30
A strongly oxidizing species is required for the ipsosubstitution reaction of APF.29 Therefore, the diﬀerent
ﬂuorescence responses of APF to ROS in Figure 1 are due todiﬀerences in the oxidizing powers of ROS. Using the diﬀerent■

RESULTS AND DISCUSSION
Ground-state oxygen may be converted into ROS by energy- or
electron-transfer reactions.6 Energy transfer leads to theformation of 1O2, whereas the successive one-electron
reduction of molecular oxygen yields O2•−, H2O2, •OH, andwater. Each ROS has a characteristic oxidative capacity and a
speciﬁc role in living cells. Based on the above, we tried to
identify the certain type of ROS produced by NB water throughthe reactivity of APF with •OH, O2•−, H2O2, and 1O2.
Fluorescence Response of APF to Diﬀerent Types of
ROS. We compared the ﬂuorescence responses of APF to•OH, O2•−, H2O2, and 1O2. From Figure 2, we can see therelationship between the ﬂuorescence increases of APF with
diﬀerent concentrations of ROS. As shown in Figure 2a, theﬂuorescence increase of APF was proportional to theconcentration of •OH. The •OH was formed from the Fenton
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Langmuirexcess in the reaction solution. Hence, 20 μM ferrous ion willproduce 20 μM O2•−. According to the equation in Figure 2c,when 20 μM O2•− is produced, the ﬂuorescence increase
should be about 15. The value in Figure 3 coincided with our
calculated value from the correlation curve in Figure 2c.

ﬂuorescence responses of APF to various ROS, we attempted to
identify the ROS produced by NBs.
Identiﬁcation of ROS Produced by Oxygen NB Water.
To Examine Whether the ROS Produced by NB Water Is
H2O2. H2O2 is cytotoxic but is considered a relatively weakoxidizing reagent (electrode potential: E (H2O2, H+/•OH,
H2O) = 0.39 V). H2O2 is stable and has a long lifetime fromhours to days in water. It can permeate into membranes andhas a long lifetime within the cell. Here, we tried to verify
whether H2O2 can be produced by NB water.
Figure 3 shows the eﬀects of ferrous ions on the ﬂuorescenceintensities of NB and control water (baseline ﬂuorescence by

Fe 2 + + O2 → Fe3 + + O•−
2(2)

To Examine Whether the ROS Produced by NB Water Is
O2•−. O2•− has two electrode potentials (E(O2/O2•−) = −0.35
V and E(O2·−, 2H+/H2O2) = 0.91 V), showing that O2•− canact both as an oxidant and as a mild reductant.31 Compared toother radicals, O2•− has a relatively long lifetime in the range of
milliseconds to seconds,32 which enables diﬀusion within thecell and thereby increases the number of the potential targets.
In this section, we tried to verify whether O2•− could be
produced by NB water.
From the results in Figure 2c and Figure 3, if the ROS
produced by NBs is O2•−, the NB water must contain about 50
μM of O2•−. According to eq 3, the dismutation of 1 mol ofO2•− catalytic with SOD will lead to the formation of 1/2 amole of H2O2.33 Thus, in the presence of 50 μM of O2•− in NB
water, we can obtain about 25 μM H2O2. According to theformulas in Figure 2b, 25 μM of H2O2 will not cause asigniﬁcant increase in the ﬂuorescence intensity of APF.

Figure 3. Eﬀect of ferrous ion (20 μM) on the ﬂuorescence intensitiesof the buﬀer solutions with and without NBs. Before ferrous ion wasadded, the ﬂuorescence intensities of oxygen NB water (hollow
column) were higher than control (probe, hollow column) by about
40. After ferrous ion was added, the ﬂuorescence intensities of oxygen
NB water (shaded column) and the probe (shaded column) increased
by about 15 and 70, respectively.

SOD

•−
O•−
2 + O2 =⇒ O2 + H 2O2(3)

Thus, after adding SOD to the NB water, the ﬂuorescenceintensity of NB water should be the same as that of the control
water. When we add ferrous ions to the NB water, they willreact with H2O2 to produce •OH according to eq 1. The
ﬂuorescence intensity of NB water will signiﬁcantly increase.
To verify our hypothesis, we applied the above procedure toboth the KO2 solution and NB water. First, we prepared 0.1 M
KO2 stock solution. Immediately after the preparation, we
added 30 μL of KO2 solution to the buﬀer solutions of APF
with and without 6 units of SOD. As the lifetime of O2•− wasvery short, the real concentration of O2•− was much lower than
the theoretical concentration (0.3 mM). As can be seen in
Figure 4a, after adding the KO2 solution, the ﬂuorescenceprobe). Before ferrous ions were added, the ﬂuorescenceintensities of oxygen NB water were statistically higher thanthose of control water. The ﬂuorescence increase of the APF
solution caused by oxygen NB water was approximately 40.
According to the formula in Figure 2b, the oxidative capacity ofoxygen NB water was equivalent to 0.5 mM H2O2. Similarly,
the oxidative capacity of oxygen NB water was equivalent to0.25 μM of •OH, 50 μM of O2•−, or 30 μM of 1O2 according to
the formulas in Figure 2c,d. Thus, if the ROS produced by NBs
is H2O2, the NB water should contain about 0.5 mM of H2O2.
According to the Fenton reaction shown in eq 1, in thepresence of excess amounts of H2O2, the concentration ofadded ferrous ions is equal to that of •OH. As can be seen in
Figure 2a, in the presence of 0.1 mM H2O2, the ﬂuorescenceintensity of APF increased substantially with the addition offerrous sulfate in the range 0−25 μM. We supposed that 0.5
mM H2O2 existed in the NB water. Thus, after 20 μM of
ferrous sulfate was added, 20 μM •OH should be produced.
Compared to the values before 20 μM ferrous sulfate wasadded, the ﬂuorescence intensities of NB water would increaseby more than 2000. However, in Figure 3, after 20 μM ferrous
sulfate was added, the ﬂuorescence intensity of NB water
increased only by about 70. Thus, we can conclude that the
ROS produced by NBs was not H2O2.
From Figure 3, the ﬂuorescence intensity of control watersigniﬁcantly increased by about 15 after 20 μM ferrous ions wasadded. The reason was considered to be as follows. According
to eq 2, ferrous ions will react with dissolved oxygen andproduce O2•−. The concentration of the added ferrous ions was
20 μM. The dissolved oxygen concentration in the control
water was about 8 mg/L (over 250 μM), which showed the

Figure 4. Eﬀect of SOD (6 units) and ferrous ion on the ﬂuorescenceintensities of superoxide radical solution and NBs solutions.

increases of the buﬀer solutions of APF with and without 6
units SOD were about 7 and 40, respectively. According to
Figure 2c, when the ﬂuorescence increase was 40, the O2•−
concentration in the APF solution should be 50 μM. Therefore,
the real concentration of O2•− added to the APF solution was
about 50 μM. Thus, 25 μM H2O2 was formed in the APF
solution with 6 units of SOD. Then, excess amount of ferrous
sulfate (30 μM) was added, and the ﬂuorescence intensity
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ﬂuorescence intensities of these NB waters were measured. As
can be seen in Figure 6, without 0.2% DMSO, the ﬂuorescenceincreased by about 3500, as shown in Figure 4a, which wasconsistent with the theoretical value. On the contrary, theﬂuorescence increases of NB water with and without 6 units
SOD were 60 and 40, respectively (Figure 4b). In the presenceof SOD, after adding ferrous sulfate (30 μM), the ﬂuorescenceintensity increased by only 120 (Figure 4b). These results
oﬀered clear evidence that the ROS produced by NBs was not
O2•−.
To Examine Whether the ROS Produced by NB Water Is•OH or 1O2. •OH is the strongest oxidant and has a muchhigher electrode potential (E(•OH, H+/H2O) = 2.31 V) thanthe other ROS.31 Due to its high reactivity and short lifetime,on the order of nanoseconds,34 •OH’s mobility range isextremely limited. It is not membrane permeable but can only
react with surrounding molecules. 1O2 has a short lifetime,about 2−4 μs,35 but it is capable of diﬀusion and is permeate
membranes. The electrode potential of 1O2 is 0.81 V (E (1O2/O2•−)). In this section, we tried to identity whether the ROS
produced by NB water is •OH or 1O2.
DMSO is known to quench •OH but not 1O2.30 Figure 5ashows the ﬂuorescence response of APF to 4 μM •OH in the

Figure 6. Eﬀect of DMSO on the ﬂuorescence intensities of oxygen
NB water.

increase of APF solution caused by oxygen NB water wasapproximately 40. In the presence of 0.2% DMSO, theﬂuorescence intensities of NB water and control water(probe) showed no signiﬁcant diﬀerence, meaning thatDMSO quenched APF ﬂuorescence by oxygen NB water.
These results clearly showed that the ROS produced by NB
water is •OH. According to the formulas in Figure 2a, oxygen
NB water could produce about 0.25 μM of •OH.
In a MNB generator, hydrodynamic cavitation may occur. It
is widely known that hydrodynamic cavitation can generate•OH and also H2O2, whose concentrations are in the micro
molar level.36,37 Although micro molar level •OH may form
during the MNB generation, it will immediately disappear due
to the very short lifetime. The NB water we used in this paper
was taken after we stopped the MNB generator. Thus, the •OH
we observed was produced due to NBs themselves rather than
MNB generator during the MNB generation process. Micromolar level of H2O2 can exist for a relatively long time. In
Figure 3, after 20 μM ferrous sulfate was added, theﬂuorescence intensity of oxygen NB water increased by about
70. Based on the calculation from the formula in Figure 2a, it ispossible that the ﬂuorescence increase was due to the Fenton
reaction between the existence of submicro molar level H2O2
and excess amount of ferrous sulfate. Although the micromolar
H2O2 has almost no eﬀect on the physiological activity of livingorganisms, the H2O2 may form the strongest oxidant •OH
associated with the disappearance of NBs in the water. The
research for •OH formation by NB water is worth doing in thenear future.
Eﬀect of NB Number Density on the Germination of
Vegetable Seeds. Most of the previous studies have focusedon analyzing the eﬀect of exogenously added H2O2 on the seedphysiology. Exogenous H2O2 stimulates the germination of
dormant seeds of barley,7 Arabidopsis,38 rice,9 and Zinnia
elegans.39 Other ROS such as •OH have been largely ignored
because of their short lifetimes. As NB water can continually
produce submicromolar levels of •OH17 (Figures 2−6), it ispossible to study the role of exogenously added •OH in theseed germination process.
We studied the role of exogenous •OH produced by NBs in
the water in the seed-germination process. As the amount of•OH is positively correlated with the NB number density inwater,17 waters of two diﬀerent NB number densities (100%
NB water and 20% NB water) were prepared for germination
tests, as described in the Experimental Section.
Figure 7a displays the germination rates of spinach seeds in
response to diﬀerent treatments at 20 °C. For spinach seed,

Figure 5. Eﬀect of DMSO on the ﬂuorescence intensities of hydroxyl
radical solution and singlet oxygen solutions (a) The ﬂuorescence
response of APF to 4 μM •OH in the presence of diﬀerent amount of
DMSO. (b) The ﬂuorescence response of APF to 40 μM 1O2 in thepresence of 0.2% DMSO. The •OH was formed by the Fenton
reaction; the 1O2 was formed by the EP powder dissolved in buﬀer
solution containing 10% ethanol.

presence of diﬀerent amounts of DMSO. Without DMSO, theﬂuorescence increase of APF caused by 4 μM •OH was about
530. After DMSO was added, the ﬂuorescence intensities
substantially decreased, as shown in Figure 5a. The ﬂuorescence
reaction of •OH with APF was quenched about 80% by 0.1%
DMSO and about 95% by 0.2% DMSO. The quenching of•OH with APF by 1% DMSO was similar to that by 0.2%
DMSO, which meant that DMSO above 0.2% did not havefurther eﬀects on quenching. Since DMSO is toxic to cells, forsafety concerns, a 0.2% concentration of DMSO was used in
the following experiments. Figure 5b shows the ﬂuorescence
response of APF to 40 μM 1O2 in the presence of 0.2% DMSO.
1
O2 was produced from EP powder which was dissolved in a
buﬀer containing 10% ethanol. As shown in Figure 5b, 0.2%
DMSO showed no quenching of APF ﬂuorescence by 1O2. The
above results showed that 0.2% DMSO could suppress about
95% of APF ﬂuorescence derived from •OH without aﬀecting
the signal derived from 1O2.
Then, we studied the eﬀect of DMSO on the ﬂuorescence
response of APF to oxygen NB water. APF was added to the
NB water with and without 0.2% DMSO, and then the
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Langmuirgreen. On the ﬁnal day of the germination tests, the chlorophyll
contents of carrot seeds submerged in three groups werecompared, as shown in Figure 8g. The absorbance value at 652
nm can be used to represent the chlorophyll content.40 It isclear that the chlorophyll contents of carrot seeds submerged in
100% NB water were obviously lower than those in distilledwater and 20% NB water. The low value of chlorophyll content
can be thought as the negative eﬀect caused by high-number
density NB water.
The amount, duration, and localization are very importantfactors when we consider the role of ROS in physiologicalprocesses. At the nanomolar and submicromolar levels, ROS
function as signaling molecules and are involved in regulation
development and pathogen-defense responses.41,42 At the
micromolar level, ROS can promote adaptation and possiblyorganismal ﬁtness. They are also involved in signal transduction
pathways in association with ROS-induced apoptosis andnecrosis.1,43 At the millimolar or higher, ROS will cause
irreversible damage to cellular compounds.
In NB water, the amount of •OH is at the submicromolar
level, and is beneﬁcial to cell growth and viability. However, theoxidative window for carrot seeds we used in our experiments isvery narrow. The amount of exogenous •OH produced by
100% NB water was beyond the toxic threshold of carrot seeds,
thus showing inhibition of the elongation of the hypocotyl.

Figure 7. Eﬀect of NB number density on the germination process ofspinach seeds and carrot seeds. (a) Spinach seeds. (b) Carrot seeds.
The error bars show the standard errors of three parallel samples.

visible radicle emergence was initiated within 1−2 days aftersubmerging in 100% NB water and 20% NB water. However,for the seeds submerged in distilled water, the radicle
emergence was delayed by 1 day. The ﬁnal germination ratesof spinach seeds in distilled water, 20% NB water, and 100%
NB water were 54%, 65%, and 69%, respectively. Besides, 100%
NB water can promote the growth of the sprout system. The
sprout lengths of spinach seeds submerged in 100% NB water
were longer than those submerged in the distilled water asshown in Figure 8a−c.

■

SUMMARY AND CONCLUSIONS
Using a sensitive ﬂuorescent probe APF, we established a set of
methods to distinguish the four major types of ROS and
identiﬁed the speciﬁc type of ROS using oxygen NB water. We
found clear evidence that NB water can produce submicromolar •OH in water. Together with the identiﬁcation of the ROS
generated by NBs, we have found that NB water with diﬀerent
bubble-number densities had diﬀerent eﬀects on the germination processes of spinach and carrot seeds. The amount ofexogenous •OH has a positive correlation with the NB number
density in the water. High-number density NB water (i.e.,
above 1 × 108 particles/mL) led to the higher ﬁnal germination
rates and faster germination processes for spinach seeds.
However, for carrot seeds, the same high-number density NB
water was beyond the toxic threshold and inhibited theelongation of the hypocotyl and the chlorophyll formation.
When the NB water was diluted, the relatively low number
density of NB water (2 × 107 particles/mL) did not show any
negative eﬀects while promoting the germination of carrot
seeds.
As the toxic thresholds for each type of seeds are diﬀerent,
the usage of NB water should be judged individually for eachtarget. Our study should inspire further work on the many roles
of NB water in both sterilization processes and growthpromotion for living organisms. To gain a deeper understanding of NB water’s impact on living organisms, molecularbiological and genomic research should also be done in the
future.

Figure 8. Eﬀect of NB number density on the hypocotyl elongationand chlorophyll content of carrot seeds and spinach seeds. Fifty
Spinach seeds are all shown in panels a−c. Ten carrot seeds in d−f arerepresentatives of 50 seeds. (g) The absorbance of chlorophyll content
of carrot seeds submerged in three kinds of waters. The error bars
show the standard errors of three parallel samples.

■

Figure 7b shows the germination rates of carrot seeds in
response to diﬀerent treatments at 20 °C. The ﬁnal germination
rates of carrot seeds in distilled water, 20% NB water and 100%
NB water were 38%, 62%, and 59%, respectively. However, forcarrot seeds, the 100% NB water prohibited the elongation ofradicles as seen in Figure 8d−f. The sprouts of carrot seeds
dipped in 100% NB water were smaller than those in 20% NB
water and distilled water. Moreover, the radicle did not turn

ASSOCIATED CONTENT* Supporting InformationS

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.langmuir.6b01621.
Bubble size distribution in the 100 % NBs water and 20%
NB water (PDF)
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AUTHOR INFORMATION

Corresponding Authors*Seiichi Oshita, E-mail: aoshita@mail.ecc.u-tokyo.ac.jp; TEL:
03-5841-5362; FAX: 03-5841-8174.
*Shu Liu, E-mail: liushu0313055@gmail.com.
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
A part of this research has been ﬁnancially supported by
Challenging Exploratory Research (25660202); a Grant in Aid
for Scientiﬁc Research (2402408) by JSPS; the Council for
Science, Technology and Innovation (CSTI); the Crossministerial Strategic Innovation Promotion Program (SIP);“Technologies for creating next-generation agriculture, forestry
and ﬁsheries” (funding agency: Bio-oriented Technology
Research Advancement Institution, NARO), Japan; and the
Mayekawa Houonkai Foundation in 2015.

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