Research Article
pubs.acs.org/journal/ascecg

Oxidative Capacity of Nanobubbles and Its Eﬀect on Seed
Germination
Shu Liu,*,† Seiichi Oshita,*,† Yoshio Makino,† Qunhui Wang,‡,§ Yoshinori Kawagoe,∥and Tsutomu Uchida⊥†

Graduate School of Agricultural & Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
Department of Environmental Engineering, School of Civil and Environmental Engineering, University of Science and Technology
Beijing, 30 Xueyuan Road, Haidian District, Beijing 10083, China§
Beijing Key Laboratory on Resource-Oriented Treatment of Industrial Pollutants, University of Science and Technology Beijing, 30
Xueyuan Road, Haidian District, Beijing 10083, China∥
College of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, Japan⊥
Division of Applied Physics, Faculty of Engineering, Hokkaido University, N13W8 Kita-ku, Sapporo, Hokkaido 060-8628, Japan‡S Supporting Information*

ABSTRACT: Nanobubbles (NBs) have been reported to be eﬀective at accelerating themetabolism 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 indicatedthat 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 concentrationof H2O2 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 H2O2 respectively. The seedgermination tests were performed in the NB water, distilled water and H2O2 solutions. The
germination rate at each observation times of seeds submerged in gas-mixture NB water and
0.3 mM H2O2 solutions were both higher than those submerged in distilled water. The
amounts of superoxide radicals in the seeds were detected using NBT staining. The resultsof absorbance data proved that the amounts of O2•− in seeds submerged in gas-mixture NB water and in 0.3 mM H2O2 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.
KEYWORDS: Reactive oxygen species, Bubble number density, Microscope spectrophotometer, Hydrogen peroxide,
Fluorescence intensity■of plants, shellﬁsh, and yeast.6−10 On the other hand, MNBs arewidely used to improve the oxidation eﬀect.11−16 For example,
ozone MNBs are eﬀectively used to remove residual pesticides
in vegetables,11,12 to inactivate microorganisms,13 and to reduce
the organic material in wastewater.14−16 MNBs’ oxidative
promotion eﬀects were not only due to the reason that MNBs
can enhance the mass transfer of ozone but also that theexogenous reactive oxygen species (ROS) are generated by
MNBs in the presence of dynamic stimuli such as ozone, strong
acid, copper, and short-wavelength UV irradiation.17−20
The physiological promotion and oxidation eﬀects of MNB
water described above may seem contradictory to each other.
However, the generation of reactive oxygen species (ROS)
caused by NBs may oﬀer a reasonable explanation for these two

INTRODUCTION
Microbubbles (MBs) are bubbles with a diameter ranging fromseveral micrometers to about 100 μm. Nanobubbles (NBs) arebubbles with a diameter of submicrometer order.1 Since the1990s, many types of commercial micro- and nanobubble(MNB) generators have been invented in Japan, such as swirl
ﬂow, ejector, cavitation, pore, mechanical agitation, andsonication.2 These MNB generators can produce the watercontaining high number concentration of MNBs ranging from
106 to 108 particles/mL. MNBs show various characteristics
such as the increased solubility of gases in liquids, reduced
friction, either negative or positive ζ-potentials and thegeneration of free radicals.3−5 Because of their uniqueproperties, the MNBs application technology has widely beenused in many ﬁelds.
Recently, MNB physiological promotion eﬀect and oxidationeﬀect have both been reported. A series of investigations havereported that water containing MNBs can accelerate the growth© 2015 American Chemical Society

Received: October 26, 2015
Revised: December 3, 2015
Published: December 11, 2015
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concentration in the range from 0.04 to 12 mg L−1. During the MNB
production, the water had a milky appearance. This appearance wasobserved just a few seconds after we started the MNB generation andit was maintained during the whole bubble generation period. Whenthe MNB generation stopped, the milky appearance graduallyvanished. In about 10 min, the water in the 2 L Erlenmeyer ﬂask
became transparent. Most of the MBs can exist in the water for only
about 10 min, whereas NBs are relatively stable and can exist in thepure water for days.29,30 Therefore, the expression of “gas-mixture NB
water” is used in this paper. NB number densities were measured using
the nanoparticle tracking analysis method (NanoSight-LM10,
Quantum Design Inc., Japan). Using a laser-illuminated optical
microscope, we observed NBs as light-scattering centers moving
under Brownian motion. Bubble-size distributions were measured
using the nanoparticle tracking analysis method, a kind of dynamic
light scattering (NanoSight-LM10, Quantum Design Inc., Japan),which has been widely used to determine the sizes of nanoparticles inliquids.31
Production of ROS by NBs. APF (5 mM, Sekisui Medical Co.,
Ltd., Japan), a ﬂuorescent reagent that is highly resistant toautoxidation, was used for detecting generation of ROS (mainlyhydroxyl radicals, OH) by NBs.32 APF itself has almost no
ﬂuorescence intensity. After APF reacts with ROS, ﬂuorescein (withstrong ﬂuorescence intensity) is generated. Other chemicals werepurchased from Kanto Chemical Co., Inc. and were of the highest
obtainable purity. H2O2 solutions were prepared prior to each use. To
obtain ﬂuorescence excitation−emission matrixes (EEMs), excitation
wavelengths were incrementally increased from 450 to 550 nm at 1 nm
steps; for each excitation wavelength, the emission at longer
wavelengths was also detected at 1 nm steps using a ﬂuorescence
spectrophotometer (F-7000, Hitachi High-Tech Co. Ltd., Japan). The
ﬂuorescein caused by ROS has an EEM peak located at excitation
wavelength 490 nm and emission wavelength 515 nm. The slit width
was 5 nm for both excitation and emission. The photomultipliervoltage was set at 800 V. The ﬂuorescent intensity was given as arelative value corresponding to a combination of parameters’ setting
such as slit width, location of lamp, voltage, etc.
NBs were generated in 2 L of phosphate buﬀer (0.1 M, pH 7.4)
without APF for 60 min at a constant temperature of 20 °C. NBs wereformed with pure oxygen (purity 99.999 95%, Taiyo Nippon Sanso
Co. Ltd., Japan) or gas-mixture (described in Generation of NB
Water). Then the NBs solution was stored in the sealed biochemical
oxygen demand (BOD) bottles. After a certain NB water storage time,
10 mL of buﬀer containing NBs was quantitatively taken out for eachsample. Then 2 μL of APF was added for each sample, and the samples
were measured for ﬂuorescence intensities. The ﬁnal concentration of
APF was 1 μM. The control water is the phosphate buﬀer aerated withpure oxygen gas through a tube with an inner diameter of 4 mm. The
DO concentration of this control water was around 40 mg L−1, andfew bubbles were observed using a laser-scattering image system(Zeecom, Microtech Co. Ltd., Japan).
To increase the disappearing speed of NBs number in the sample, alow intensity ultrasonic wave (150 W, 38 kHz) was applied to thecontrol water and NBs water for 60 s. After the samples were taken out
from the ultrasonic device, APF was added into each sample. Then the
ﬂuorescence intensities of the samples were measured.
Seed Material. Seeds of barley (Hordeum vulgare L.), which were
harvested in 2012 and stored under controlled conditions (roomtemperature), were obtained from the University of Ehime, Japan.
These seeds were screened with a magnifying lens, and only large
seeds without visible defects were selected.
Germination Test. Germination tests were repeatedly performed
with ﬁve seed groups. Each of them was composed of 100 barley seeds.
Then every group was sealed in plastic net bags; one group was
submerged in a 1 L beaker ﬁlled with water containing MNBs (themixture of nitrogen and air was used to adjust the DO of MNB water
to be the same as the distilled water), one in a beaker ﬁlled with
distilled water and the others in beakers ﬁlled with diﬀerent
concentrations (0.1, 0.3, and 0.5 mM) of H2O2 solutions. During
the germination experiments, to avoid the lack of oxygen and toeﬀects because excess concentration of ROS has oxidationeﬀect and moderate concentration of ROS can show
physiological promotion eﬀect. As we know, ROS have long
been regarded as damaging compounds. ROS can cause
oxidative damage to macromolecules, thus leading to lipid
peroxidation, DNA damage, and DNA breaks.22,23 Owing tothese principles, ROS are widely used in sterilization andoxidation processes. However, as has reported in recent year,
ROS play a dual role in seed physiology behaving.21
Appropriate amount of ROS does not act as harmful
compounds but instead, it plays an important role as cell wallloosening factor and essential signaling molecules in thegrowing process of plant.24,25 It has been accepted thatexogenously supplied H2O2, a kind of ROS, can promote thegermination of cereal plants such as barley, wheat, rice, andZinnia elegans seeds.26−28
In our latest research on NBs, we reported that thegermination rates of barley seeds submerged in NB water
were 15−25% greater than those of seeds submerged in
distilled water with the same concentration of DO.29 According
to this result and the dual roles of ROS described above, wehypothesize that without dynamic stimuli, NBs can also supply
a small amount of exogenous ROS in the water. With this idea
in mind we have studied the production of ROS by NB waterusing a ﬂuorescent probes, 3′-p-(aminophenyl) ﬂuorescein(APF). As the ﬂuorescence response of APF to NB waterchanged with storage time, it is important to grasp therelationship between the amount of ROS and the NB number
density in water. The ﬂuorescence intensity of APF increases
linearly with the concentration of H2O2 in the range of 0−1
mM. Therefore, the oxidative capacity of NB water was
estimated using the correlation equation between the
ﬂuorescence intensities of APF and the concentrations of
H2O2 solutions.
A second aim of this work was to study the role of exogenous
ROS produced by NB water in the seed germination process.
The germination tests were performed among the barley seeds
submerged in the NB water, distilled water and diﬀerent
concentrations of H2O2 solutions under the similar DO
concentrations. The amounts of superoxide radicals in theseeds submerged in the NB water, distilled water, and diﬀerent
concentrations of H2O2 solutions were detected using nitro
blue tetrazolium (NBT) staining. We suppose that as signaling
molecules, the exogenous ROS produced by NBs can play animportant role in seed germination.

■

EXPERIMENTAL SECTION

Generation of NB Water. 2 L of distilled water was placed in an
Erlenmeyer ﬂask. The distilled water was produced in a distillation unit
(Autostill WA-53, Yamato Scientiﬁc Co., Ltd., Japan), which isequipped with a water storage tank of 40 L capacity. The gas wasintroduced into the distilled water and circulated through a micro andnanobubble generator (OM4-GP-040, Aura Tec Co. Ltd., Japan) for 1
h at a constant temperature of 20 °C to obtain “water containing
MNBs”. The DO concentration of distilled water was about 8 mg L−1.
The DO concentrations of oxygen MNB water, air MNB water, andnitrogen MNB water were about 40, 12, and 0.04 mg L−1, respectively.
To adjust the DO concentration of MNB water to be the same as thedistilled water, the gas-mixture of nitrogen (purity 99.999 95%, Taiyo
Nippon Sanso Co. Ltd., Japan) and air (CO2 < 1 ppm, THC < 1 ppm,
Taiyo Nippon Sanso Co. Ltd., Japan) was used. The ratio of nitrogenand air was adjusted through a mixed-gas ﬂow regulator (Log MIXD100A-0050 and Log MIX-D100A-0052, FRONTO Co. Ltd., Japan).
In this way, we can obtain the MNB water with a certain DO
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maintain a certain amount of NBs in the water, NB water, distilledwater, and H2O2 solutions were changed every 12 h. The MBs canonly exist in water for about 10 min and then will disappear, so ingermination experiments it is only NBs left in the water that areeﬀective. Germination tests were performed at 20 °C in the dark.
Germination rates obtained from three independent replicates areshown as mean ± standard deviation (SD).
Amount of Superoxide Radical in Seeds. Superoxide radicals(ROS, O2•−) were detected and quantiﬁed by staining withnitrobluetetrazolium (NBT, Tokyo Chemical Industry Co., Ltd.
Japan). After 17 and 37 h of submerging time, ﬁve germinated seeds
in each group were incubated in a mixture of 1 mM NBT and 10 mM
Tris−HCl buﬀer (pH 7.34) at room temperature for 30 min. Eachseed was horizontally placed in a stainless steel cube container (15 ×
15 × 15 mm). The sample containers were ﬁlled with super cryoembedding medium (SCEM-L1, Section-lab Co. Ltd., Japan). The
samples were rapidly frozen in an isopentane solution (Kanto chemical
Co. Inc., Japan) at −105 °C for approximately 3 min. The preparedcube samples were ﬁxed on the sample holder and stabilizedon thecryostat platform (Leica CM1950, Leica Biosystems Co. Ltd., Japan)
for about 10 min. After the frozen samples were stabilized, they wereattached to the cryostat stage one at a time. The working temperature
for the cutting process was −20 °C. The longitudinal axis of each seed
was vertical to the cutting knife direction (Surigipath DB80LS,
Section-lab Co. Ltd., Japan). The cutting process was as follows. A 15mm wide transparent adhesive ﬁlm (Cryoﬁlm type II C (9); Sectionlab Co. Ltd., Japan) was laid on the sample surface and was ﬁrmly
pressed to adhere to the cutting surface of the sample. The sample wasthen slowly cut into 100 μm slices continuously from the tip of the
sprout. The ﬁlm with the cut sample was placed on a glass slide withboth the ends of the ﬁlm section fastened with double sided adhesive
tape. The measurement processes for superoxide radicals in seeds areshown in Figure S1.
Subsequently, the samples were measured spectrophotometrically.
Superoxide radicals were visualized as deposits of dark blue insoluble
formazan using a microscope spectrophotometer (MSV-5000, JASCO
Co. Ltd., Japan). The microscope spectrophotometer uses UV−visible
spectra, and the spectral analyses were conducted on each preset circleof 30 μm in diameter. The measured absorbance value is the average ofthe absorbance in each cycle area. The distribution of ROS inside thebarley seed cells was determined using ﬁxed wavelength measurements(absorbance diﬀerence between 560 and 700 nm), with absorbance at
560 nm representing the amount of dark-blue colored formazan. The
diﬀerence in absorbance values at these wavelengths represents theamount of ROS at each location and also reduces the spectral
background caused by impurities or radiation scattering.
After the maximum concentration of ROS in the sample was locatedby ﬁxed wavelength measurements, spectrum measurements wereconducted at the locations with the maximum concentration of O2•−in the samples from 450 to 700 nm with intervals of 1 nm. For eachsample, we selected 5−12 measurement circles of 10 μm in diameter.
The spectrum measurements were conducted at 450−700 nm at
locations that showed a maximum amount of ROS.

Figure 1. Bubble size distribution in the gas mixture NBs with
diﬀerent generation times (black error bars indicate standard error of
the mean (n = 3)).

Production of Exogenous ROS by NBs. In the following
experiments, the ﬂuorescence response of APF to oxygen NB
water was further studied to verify whether NBs can produce
ROS in water. The ﬂuorescent intensity of APF iscorresponding to the amount of ROS in the water. As can beseen in Figure 2A, the ﬂuorescence intensities of water

Figure 2. (A) Fluorescence response of 1 μM APF to oxygen NB
water with water storage time. Fluorescence intensities were acquiredin 0.1 M phosphate buﬀer at pH 7.4 (λex 490 nm, λem 515 nm).
Phosphate buﬀer was aerated with pure oxygen as the control whose
DO concentration was above 40 mg L−1. To obtain suﬃcient bubble
number density, oxygen NBs generated for 1 h and then was stored inseveral biochemical oxygen demand (BOD) bottles. The error bars
show the standard errors of 8 parallel samples. (B) Time course
measurement for total bubble number concentrations and DO
concentrations of oxygen NB water. The error bars show the standard
errors of 3 measurements.

containing oxygen NBs were statistically higher than those ofcontrol water. With diﬀerent NB water storage time, theﬂuorescence response of APF to oxygen NB water wasmeasured. We found that the higher value of ﬂuorescentintensities of oxygen NB water remained within the 2 day
storage time, indicating that oxygen NBs can continuously
produce a small amount of ROS. Furthermore, we found thatthe total NB number densities in the water were decreasing
with storage time. After 2 day storage, the total NB number
density was only 1/5 of the value just after the NB generation(Figure 2B). The DO concentration of oxygen NB water
decreased from 40 to about 13 mg L−1 (Figure 2B). At thesame time, the statistical diﬀerences between the ﬂuorescenceintensities of oxygen NB water and the control water gradually
decreased from 60 to approximately 20. These results meantthat the amount of ROS produced by NB water has positive
correlation with the NB number density in the water. The
nanoparticle tracking analysis cannot distinguish between NBs■

RESULTSNB Number Density. As can be seen in Figure 1, bubblenumber densities increased with the MNB generation time.
The geometric mean bubble size observed was around 108,
112, and 105 nm after 10, 30, and 60 min bubble generation,
respectively. The total bubble-number densities of gas-mixture
NBs were 0.41, 0.75, and 0.83 × 108 particles/mL after 10, 30,
and 60 min bubble generation, respectively. These results
showed that the mean bubble sizes will not change with the
bubble generation time. But the bubble number density will
increase with bubble generation time, and then get stable 30
min later. The following experiments were conducted with the
60 min generation NB water.
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Table 1. Germination of Barley Seeds at 20 °C in the Darka

and foreign matters. However, the decrease of total particle
number density proved that most of the particles that exist in
the water are actually NBs rather than solid nanoparticles.
Compared to the oxygen NBs, the amount of ROS produced
from the gas-mixture NBs which was used in the germination
tests was relatively fewer. The ﬂuorescent increase caused bygas-mixture NB water was about half of the oxygen NB water(Figure S1).
Oxidative Capacity of NBs in the Water. H2O2 is a weak
ROS. Low concentration of H2O2 is mainly used as a cellsignaling molecule but not an oxidizing substance.33 The
physiological promotion eﬀects of NB water were similar tomoderate concentrations of H2O2 solutions. Thus, we estimate
the oxidative ability of NB water using H2O2 solutions. Under
the same measuring conditions, the ﬂuorescence response of
APF to NB water and diﬀerent concentrations of H2O2 werestudied. In this way, we estimated the oxidative ability of watercontaining NBs by using the correlation equation between the
ﬂuorescence intensities of APF and the concentrations of H2O2
solutions. Diﬀerent concentrations of H2O2 were added to thephosphate buﬀer solution, following which 1 μM APF was
added. Figure 3A−D shows the contours of EEM spectrum ofgermination rate (%)
water type

17 h

37 h

distilled water
0.1 mM H2O2
0.3 mM H2O2
0.5 mM H2O2
NBs

28 ± 0.9
48 ± 4.0
58 ± 4.0
54 ± 4.0
58 ± 3.8

98 ± 0.4
98 ± 0.4
100 ± 0100 ± 0100 ± 0a

The DO concentrations of NBs water were adjusted to be the sameas those of the distilled water. Data are means of three replicates ± SD.
P < 0.05.

seen in Table 1, when the H2O2 concentrations were higher
than 0.3 mM, they had a similar promotional eﬀect on thegermination speed of barley seeds as that of gas-mixture NB
water. After 37 h of submerging time, although nearly 100% of
the seeds in ﬁve groups (NB water, distilled water, H2O2
solutions of 0.1, 0.3, 0.5 mM) had germinated, the sprouting
parts of barley seeds submerged in NB water were considerablylarger than those in the other four groups (data not shown).
Superoxide radical in seeds. Various forms of ROS (e.g.,superoxide radical, hydrogen peroxide, and hydroxyl radicals)
exist in the seeds.21 Among these, the production of superoxide
radicals (O 2 •− ) is an important component for seedgermination, seeding growth, and development.34 The amountof superoxide radicals generated inside barley seeds was studied
to explain the faster germination process caused by NBs andH2O2. The superoxide radical was detected and quantiﬁed using
nitro blue tetrazolium (NBT) chloride. O2•− was visualized in
the seeds using NBT, which produced formazan within the seedat the sites of O2•− formation.
Quantiﬁcation of O2•− inside the seeds was achieved with thespectra from microscopic samples. Superoxide radical O2•−oxidized the yellow dye (NBT) to a blue colored formazan,
which was measured spectrophotometrically at 560 nm.24 The
sprouts of barley seeds started to break the seed coat after 15−
16 h submerging in the water, whereas after 37 h submerging
time, nearly all the seeds germinated. Thus, we measured theO2•− inside barley seeds after 17 and 37 h submerging times,
respectively. The concentrations of O2•− in the sprouting partsof seeds in the ﬁve groups were compared.
Figure 4 shows the superoxide radical distribution in barleyseeds. According to the results of Figure 4, Figure 5 displays the
spectra of formazan produced at the locations of a maximum
amount of superoxide radical. The average level of endogenous
ROS in the sprouting part of barley seeds germinated in
diﬀerent solutions were also calculated (Figure S3). It can beclearly observed that the concentrations of endogenous ROS inseeds after 17 h submerging time were much higher than thoseafter 37 h, which means ROS were mainly produced just after
the sprouts broke the seed coat (Figure 4, Figure S4, and Figure5). The relative absorbance values at around 560 nm for theseeds which germinated in H2O2 solutions were signiﬁcantly
higher than those of distilled water after submerging for 17 and37 h (Figure 5). The results above showed that the exogenous
ROS (such as H2O2) in the water could stimulate theendogenous ROS production of barley seeds.
After 17 h of submerging, the concentrations of endogenous
ROS of seeds that germinated in NB water were higher than all
those that were submerged in the H2O2 solutions (Figure 5A).
After 37 h of submerging, the concentrations of O2•− of seeds
submerged in the gas-mixture NB water and H2O2 solutions

Figure 3. (A−D) Fluorescence response of 1 μM APF to H2O2
solution. EEM spectra were acquired in 0.1 M phosphate buﬀer at pH
7.4 (the ﬂuorescence intensities were determined at 515 nm withexcitation at 490 nm, slit width 5 nm). (E) Linear correlation between
ﬂuorescence increase and concentrations of H2O2 solution. The error
bars show the standard errors of 3 parallel samples.

APF with diﬀerent concentrations of H2O2 recorded from 450
to 550 nm in the emission wavelength and from 450 to 550 nm
in the excitation wavelength. As indicated in Figure 3E, theﬂuorescence intensity of APF (λex = 490 nm, λem = 515 nm)
increased gradually upon the increase of H2O2 concentration.
We observed a linear correlation between the ﬂuorescenceintensity and the concentration of H2O2. Just after NB
generation, the ﬂuorescence increase of APF solution causedby oxygen NB water was approximately 60. According to theformula, just after the NB generation, the oxidative capacity ofoxygen NBs water is equivalent to 0.5 mM H2O2. Similarly, theoxidative capacity of gas-mixture NBs water is equivalent to 0.3
mM H2O2 (Figure S1).
Germination Tests. Then, we studied the role of exogenous
ROS produced by NBs in the water in seed germination
process. Table 1 shows the germination rates of barley seeds in
response to diﬀerent treatments at 20 °C. After 17 h
submerging in the water, the average germination rate ofbarley seeds immersed in the gas-mixture NB water was 58%,
which was 2 times higher than that in the distilled water. These
results indicated that NBs eﬀectively promoted the germination
speed of barley seeds. In our experiments, low concentrationsof H2O2 solutions were used as positive controls. As can be
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Figure 4. Superoxide radicals (ROS) distribution in barley seeds after 17 h of submerging time: the sprouting region of representative seeds
germinated in three groups (distilled water, NB water, 0.3 mM H2O2) after the nitro blue tetrazolium staining process. (A) Microscopic images ofthe sectioned surfaces of seeds germinated in three diﬀerent kinds of water. Spectra were conducted on each preset circle. The diameter of each circle
is 30 μm. (B) Superoxide radical distribution in samples. The x- and y-axes show the locations of the measurement cycles. The measured values are
the diﬀerences between absorbance values at 560 and 700 nm in each cycle area.

were similar (Figure 5B). According to our results, we conclude
that gas-mixture NBs have similar or even higher eﬀects on theendogenous ROS production compared with the H 2O 2
solutions used in this experiment.

■

DISCUSSION
Reactive oxygen species are often blamed as damaging
compounds for the development of cancer and other diseases.
However, in recent years scientists found that a moderate levelof ROS may have beneﬁcial eﬀects. For example, in plantbiological ﬁeld, it is accepted that ROS plays a dual role in seedphysiology behaving. Bailly et al.21 raised a concept of the“oxidative window for germination”, which restricts theoccurrence of the cellular events associated with germination
to a critical range of ROS level, enclosed by lower and higher
limits. Below this window, the amount of ROS during
imbibitions is too low for allowing germination. Above thiswindow, ROS became deleterious and caused cellular oxidative
damage. Owusu-Ansah and Banerjee35,36 discovered that

Figure 5. Spectra of formazan produced as a result of superoxide
radical production in the sprouting region of germinated barley seeds
immersed in distilled water, NB water, and 0.3 mM hydrogen peroxide
solution. The measurements were conducted at locations with amaximum amount of ROS according to the ﬁxed wavelength
measurements described in Figure 4. For each group we measured
ﬁve seeds. Each curve represents the mean value of more than 120
microscopic sample dots (dot diameter = 10 μm). (A) 17 h ofsubmerging time. (B) 37 h of submerging time.

Figure 6. Eﬀect of ultrasonic wave (US) on the ﬂuorescent intensities of the buﬀer solutions with and without NBs ((A and B) EEM spectra of
buﬀer solution without NBs (control) before and after US was applied; (C and D) EEM spectra of buﬀer solution containing oxygen NBs before andafter US was applied; (E and F) EEM spectra of buﬀer solution containing oxygen NBs with 2 days storage before and after the application of US.
The values represent the ﬂuorescent intensities at 515 nm with excitation at 490 nm. (G) Error bars show the standard errors of 8 parallel samples.
DO concentrations of oxygen NBs water and the control water were above 41.17 and 40.03 mg/L, respectively. APF was added after the US wasapplied. APF concentration was 1 μM).
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intensity measurement can be a new method for the detectionof existence of NBs in the water.
The production of ROS by NBs is a continual process. The
stimulation on living organisms by NBs would last for a very
long time, as is preferable for the metabolism of livingorganisms. Additionally, the number of NBs can be controlled
to meet our needs through the operation of NB generator.
When large amount of ROS are needed, diﬀerent kinds of NBs
like air NBs and oxygen NBs can also be used. Finally, theapplications of NBs should not be limited to germination
promotion. Because of their ability to produce ROS, it isanticipated that the prospects of NBs will be immense;
however, much progress is still required. It is proposed that
our study will act as a basis not only for applying NB
technology to many areas of science and technology but also todeveloping new technology.

moderately high ROS level in the progenitor population
sensitizes them to diﬀerentiation, and establishes a signalingrole for ROS in the regulation of hematopoietic cell fate. In our
research, the amount of ROS existed in the NB water was in therange of “oxidative window”. Further investigation for ROS
existence in the NB water can help us deeply understand themechanism of NBs’ physiological promotion eﬀects.
Our results indicated that a small amount of ROS could beconstantly detected in NB water with storage time. ROS havevery short lifetimes, such as 1 × 10−9 s for ·OH,37 and in therange from milliseconds to seconds at neutral pH values forO2•−.38 Thus, we concluded that ROS can be continuously
produced in NB water. We also observed that both the DOconcentration and the bubble number density of NB water
decreases with storage time (Figure 2). These results indicatethat NBs gradually disappear with storage. We hypothesize thatthe disappearance of NBs causes the constant production of
ROS in water.
This hypothesis could be supported by the experimental
results as following. As we know, the ultrasound could
accelerate the aggregation process of the bubbles in an aqueous
solution,39 we tried to increase the disappearing number of NBs
per unit time through the application of ultrasonic wave. Firstof all, we made it sure that the ultrasonic wave used in thisstudy could only decrease but not increase the amount of NBs
in water. We applied the ultrasonic wave to ultrapure water for
60 s and no bubbles could be detected through the nanoparticle
tracking analysis method. For NB water, after the ultrasonicwave was applied for 60 s, the bubble number density
signiﬁcantly decreased (Figure S5). As can be seen in Figure
6, after the ultrasonic wave was applied to oxygen NBs waterfor 60 s, the ﬂuorescence intensities of the NB solutions
increased by 400, compared to those without ultrasonic wave
pretreatment. The ultrasonic wave pretreatment did not aﬀect
the ﬂuorescence intensity of control water. The above results
showed that ROS was produced during NBs’ disappearing
process.
We also found that after ultrasonic wave application, the stateof fast speed disappearance of NBs can last for a while. Having
taken out the samples from the ultrasonic device, we waited for
30 min and then added APF in the samples. And theﬂuorescent increase of NB solution was about 100 (Figure S6).
From Figure 6, after 2 days storage time, the statistical
diﬀerences between the ﬂuorescence intensities of oxygen NB
water and the control water decreased from 40 toapproximately 20. We applied the ultrasonic wave to the buﬀer
containing oxygen NBs after 2 days storage time. The
ﬂuorescence intensities of the NB solutions increased byapproximately 90, which is only one-ﬁfth of the value we
obtained just after generation of the oxygen NBs (Figure 6).
From our experiments, we can conclude that the amount of
ROS in NB water has positive correlation with the number of
NBs and the decreasing speed of NBs number.
In recent years, many researchers have conﬁrmed NBs’
existence in solution on the basis of the results of theirexperiments, such as degassing,40 relaxation time measurements,1,29 transmission electron microscope observation,41
measurement by dynamic light scattering method,42 etc.
Apart from the explanation of physiological promotion eﬀect
of NBs on seed germination, our results could also be used toprove the existence of NBs in the water. As disappearance of
NBs is correlated to the free radical generation, the ﬂuorescent■

ASSOCIATED CONTENT

S Supporting Information*

The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acssuschemeng.5b01368.
Schematic diagram for the measurement of superoxide
radical in seeds, ﬂuorescence responses of APF to gas
mixture NB water, average relative absorbance values ofendogenous ROS generation in barley seeds, distributionof endogenous ROS generation in barley seeds after 37 hof submerging time, eﬀect of ultrasonic wave on the total
bubble number concentration in gas mixture NB water,and eﬀect of ultrasonic wave on the ﬂuorescentintensities of the buﬀer containing oxygen NBs (PDF).

■

AUTHOR INFORMATION

Corresponding Authors*Seiichi Oshita, E-mail: aoshita@mail.ecc.u-tokyo.ac.jp.
*Shu Liu. E-mail: liushu0313055@gmail.com. Tel.: 03-58415362. Fax: 03-5841-8174.
Notes

The authors declare no competing ﬁnancial interest.

■

ACKNOWLEDGMENTS
A part of this research has been ﬁnancially supported by
Challenging Exploratory Research (25660202); Grant in Aid
for Scientiﬁc Research (2402408) by JSPS; Council for Science,
Technology and Innovation (CSTI); cross-ministerial Strategic
Innovation Promotion Program (SIP); “Technologies forcreating next-generation agriculture, forestry and ﬁsheries”(funding agency: Bio-oriented Technology Research Advancement Institution, NARO), Japan; and Mayekawa Houonkai
Foundation in 2013.

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DOI: 10.1021/acssuschemeng.5b01368
ACS Sustainable Chem. Eng. 2016, 4, 1347−1353