Chemical Engineering Science 93 (2013) 250–256

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Chemical Engineering Sciencejournal homepage: www.elsevier.com/locate/ces

Effects of nanobubbles on the physicochemical properties of water:
The basis for peculiar properties of water containing nanobubbles
Shu Liu a, Yoshinori Kawagoe b, Yoshio Makino a, Seiichi Oshita a,nab

Graduate School of Agricultural & Life Sciences, The University of Tokyo, Yayoi 1-1-1, Bunkyo-ku, Tokyo 113-8657, Japan
College of Bioresource Sciences, Nihon University, Kameino 1866, Fujisawa, Kanagawa 252-0880, JapanH I G H L I G H T S

G R A P H I C A L

c Germination

rates of barley seeds
dipped in nanobubbles(NBs) water
increased.
c The number of NBs had a positive
correlation with the T2 value of thewater.
c NBs could increase the mobility of
the water molecules in bulk.

G: germinating seed, NG: non-germinating seed.

a r t i c l e i n f o

a b s t r a c t

Article history:
Received 27 June 2012
Received in revised form
17 January 2013
Accepted 4 February 2013
Available online 11 February 2013

The mechanism with which nanobubbles (NBs) promote physiological activity is investigated using
germination tests and nuclear magnetic resonance (NMR) relaxation-time measurements. The germination rates of barley seeds dipped in water containing NBs (bubbles formed from gas mixtures ofnitrogen and pure air) were 15–25 percentage points greater than those of the seed dipped in distilledwater with the same concentration of dissolved oxygen (DO). In addition, the proton NMR relaxation
time, T2, of water containing NBs (bubbles formed from nitrogen) was measured and compared with
the T2 of control water (water without bubbles or DO). After T2 measurements, both water containing
NBs and control water were degassed, and the T2 values were subsequently measured again to examine
its changes before and after degassing. Water containing NBs exhibited T2 values that were statisticallylonger than those of control water. After degassing, the T2 values of water containing NBs decreased,
which indicated that the decrease in the NB number density shortened T2. On the basis of these results,
we concluded that the number of NBs was positively correlated with the T2 value of the water. The
increase in T2 with the generation of NBs indicated that the mobility of the water molecules increased;consequently, a longer time was required to reach the equilibrium state through spin–spin relaxation.
These observations indicated that the NBs in water could inﬂuence the physical properties of water andthat it could also be used to verify the stability of NBs in the water.
& 2013 Elsevier Ltd. All rights reserved.

Keywords:
BubbleFood processing
Metabolism
Nanostructure
Proton relaxation time T2
Water mobility

A B S T R A C T

1. Introduction
Micro- and nanobubbles (MNBs) have been a subject of intensive research over the past decade. The characteristics of MNBsnCorresponding author. Tel.: þ81 3 5841 5362.
E-mail address: aoshita@mail.ecc.u-tokyo.ac.jp (S. Oshita).

0009-2509/$ - see front matter & 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.ces.2013.02.004

include the increased solubility of gases in liquids, reduced friction,either negative or positive zeta potentials and the generation offree radicals (Takahashi et al., 2003; Serizawa et al., 2003; Chu
et al., 2008), leading to numerous promising applications. A few ofthese applications include the sewage treatment of polluted waterby air ﬂotation (Liu et al., 2012b, 2010), reducing the friction of
ﬂowing liquids (Hara et al., 2011) and the detergent-free cleaningof adsorbed proteins (Liu et al., 2010; Wu et al., 2008).

S. Liu et al. / Chemical Engineering Science 93 (2013) 250–256

Recently, the application of MNB technology in biologicalprocesses has been focused upon considerably. Water that contains MNBs has been reported to accelerate the growth of plants
and shellﬁsh and has also been used in the aerobic cultivation ofyeast. The air micro-bubble supply resulted in a better cultivationof oysters (Heterocapsa circularisquama) in terms of size andtaste (Ohnari, 2001). Kurata et al. (2008), who applied oxygen
micro-bubbles in an osteoblast cell-culture system, reported
greater alkaline phosphatase activity, which was related toincreased osteoblastic cell activity. Park and Kurata (2009) foundthat fresh weights of micro-bubble treated lettuces were 2.1 times
greater than those of the macro-bubble treated lettuces, whengrown under a similar dissolved oxygen (DO) concentration.
Ushikubo et al. (2008) showed that when barley coleoptile cells
were ﬂoated in water after the generation of oxygen MNBs,
cytoplasmic streaming rates inside the cells were accelerated.
Moreover, nanobubbles (NBs) may provide a transport mechanism for gas delivery to a membrane or cell and thus affect transmembrane proteins or the membrane structure. Both effects
considerably alter the cell function (Dzubiella, 2010; Seddon
et al., 2012).
However, no explanations have been provided for these newscientiﬁc ﬁndings. Furthermore, according to classical thermodynamic theory, NBs do not exist (Ljunggren and Eriksson, 1997).
Although numerous researchers have reported their existence inwater on the basis of the results of experiments (Ushikubo et al.,2010; Uchida et al., 2011; Weijs et al., 2012), a few fundamental
aspects of the water containing NBs still remain unclear (Philip,2012). Thus, basic research on the physicochemical properties ofwater containing NBs is important.
In this study, we attempted to use a germination test toverify the ability of NBs to promote the metabolism of barleyseeds and provide reasonable and convincing explanations of theseeffects by basic research on the physical properties of watercontaining NBs.

2. Materials and methods
2.1. Seed germination
2.1.1. Seed material
Seeds of barley (Hordeum vulgare L.), which were harvested in2011 and stored under controlled conditions (room temperature),
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.
2.1.2. Germination tests
Germination tests were performed with a pair of seed groups
composed of 100 and/or 120 barley seeds each. Each group wassealed in plastic net bags; one group was dipped in a 1-L beaker
ﬁlled with distilled water containing NBs and the other in a
beaker ﬁlled with distilled water. The gas mixture, which contained nitrogen (purity 99.99995%, Taiyo Nippon Sanso Co. Ltd.,
Japan) and air (CO2 o1 ppm, THCo1 ppm, Taiyo Nippon Sanso
Co. Ltd., Japan), was introduced into distilled water through a
micro-bubble generator (OM4-GP-040, Aura Tec Co. Ltd., Japan)
for 1 h at a constant temperature of 20 1C to prepare ‘watercontaining NBs. The DO concentration of water containing NBs
was adjusted to be the same as that of distilled water through amixed-gas ﬂow regulator (Log MIX-D100A-0050 and Log MIXD100A-0052, FRONTO Co. Ltd., Japan). Germination temperature
was maintained at 25 1C with a water bath. After the seeds had
been dipped in the water for approximately 20 h, germination
rate was determined.

251

2.1.3. NMR measurements
The proton spin–spin relaxation times (T2) were measured
using a pulsed spectrometer (JNM-MU25A, JEOL, Japan) operated at
25 MHz and at a constant temperature of 20 1C. The pulse sequence
used for T2 was the Carr–Purcell–Meiboom–Gill sequence. Fivereplicates of each sample were collected in NMR tubes with anouter diameter of 10 mm and were then sealed.
2.1.4. Samples for NMR measurements
Twelve seeds were collected from the beaker ﬁlled with 1-L
distilled water or from water containing NBs. The seeds were
wiped gently with a tissue paper to remove external water andwere immediately analysed using NMR. The volume height of theseed sample in the NMR tube was approximately 2 cm.
2.2. Properties of water with and without NBs
2.2.1. Ultrapure water
Ultrapure water was obtained using a water-puriﬁcation system (Direct-Q, Nihon Millipore Ltd., Japan), which was equippedwith a reverse-osmosis cartridge and ion-exchange and activatedcarbon modules. According to the results of nanoparticle-tracking
analysis (Z-NTA, Quantum Design Inc., Japan), ultrapure water did
not contain any particles or bubbles (data not shown), but it
naturally contained approximately 6–9-mg/L dissolved oxygen.
2.2.2. Control water
Ideally, control water should exhibit a low DO concentration
and be free of bubbles. To remove DO from ultrapure water,
bubbling method was used. Dissolved gasses were purged in 2-L
ultra-pure water via the direct introduction of nitrogen gas
through a tube with an inner diameter of 4 mm. The DOconcentration of this control water was less than 0.15 mg/L, andfew bubbles were observed using a laser-scattering image system(Zeecom, Microtech Co. Ltd., Japan).
2.2.3. Water containing NBs
Control water was placed in an Erlenmeyer ﬂask. The gas (N2,purity 99.99995%, Taiyo Nippon Sanso Co. Ltd., Japan) was
introduced into the water through a micro-bubble generator for
1 h at a constant temperature of 20 1C to obtain ‘water containing
NBs’. The difference between DO concentration in control waterand that in water containing NBs was less than 0.05 mg/L.
2.2.4. Samples for NMR measurement
For NMR measurements, the volume of water samples was
typically 0.4 mL. In this study, two methods were used toeliminate the paramagnetic effect of dissolved oxygen. In the ﬁrst
method, NMR tubes were ﬁlled with water and then sealed. In thesecond method, after 0.4-mL water was placed in each tube, and avacuum pump (5 L/min, 60 Hz and 0.02 MPa) was used to degas
the air in the headspace of the water sample for 15 s. Next, the air
was replaced by nitrogen from a gas bag through a three-way
value. All tubes were stored in an incubator at 20 1C. Fivereplicates of each sample were collected in pressure-tight tubes
with inner diameters of 8.5 mm and then sealed.
2.2.5. Bubble-size distribution
Bubble-size distributions 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 centres moving under Brownian motion. After NBs were generated, water containing NBs wasstored in 15 BOD bottles in an incubator at 20 1C. Bubble-size

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S. Liu et al. / Chemical Engineering Science 93 (2013) 250–256

distribution was subsequently measured as a function ofstorage time.

100

2.2.6. Degassing
A vacuum pump (5 L/min, 50 Hz and 0.02 MPa) was used todegas water samples. The pressure for degassing was approximately 0.02 MPa. After the water was degassed, the head space of
the NMR tubes was ﬁlled with nitrogen to maintain the water
sample under atmospheric pressure. Degassing was conductedwith the samples placed in a water bath at 30 1C.

80

Germination rate (%)

Distilled water

60

40

2.3. Zeta potential
Zeta-potential measurements were performed with a Zeta
Potential Analyzer (Zeecom, Microtech Co. Ltd., Japan). Thissystem detects the electrophoretic mobility of particles and isequipped with a microscope and CCD camera to observe themovement of particles in the range 20–100 mm. Small particles
can be observed by the scattering of laser light. Fifty particles
were tracked manually to determine their speed, and zeta
potential was calculated using the Smoluchowski equation.
2.4. DO concentration
DO concentration was measured in both control water andwater containing NBs at 20 1C using a DO metre (SG6, Mettler
Toledo GmbH, Switzerland).

NBs water

20

1

2

3
4
Test number

5

6

Fig. 1. Comparison of barley seed germination rate between the water containing
NBs and the distilled water under the same DO concentration (The average valuesof germination rates in the water containing NBs and the distilled water were 77.9
and 59.2%, respectively).

90

10Distilled water

NBs water

Distilled water

NBs water

9

80

8
70
7

2.5. Statistical analysis
Statistical tests were performed using the EXCEL 2007 software. The paired-sample t-test was used to compare the difference between control water and water containing NBs with asigniﬁcance level (p-value) of 0.0005–0.25.

3. Results and discussion
3.1. Seed-germination tests
Seed germination is a crucial process in the seed-plant life
cycle and is also important for plant establishment in natural andagricultural ecosystems (Karin et al., 2011). During germination,
the seeds rapidly recover physically from maturation drying,
resume a sustained intensity of metabolism, complete essential
cellular events to allow the embryo to emerge and prepare forsubsequent seeding growth (Nonogaki et al., 2010). Therefore,
germination test is an appropriate method to verify the ability ofwater containing NBs to promote physiological processes.
Comparison experiments were performed using distilled water
and water containing NBs produced from each batch of distilledwater. The DO concentration of distilled water was approximately
9 mg/L. NBs were formed from the mixture of nitrogen and air
and the DO concentration of water containing NBs was adjusted
to be the same as that of distilled water. Germination rate wascalculated on the basis of the following formula:Germination rate ¼(The number of germinated seeds)/(thetotal number of seeds)  100%
As shown in Fig. 1, six repetitive germination experiments
showed that the germination rates of barley seeds dipped inwater containing NBs were 15–25 percentage points greater thanthat of those dipped in distilled water; these results clearly verify
the physiological effect of NBs. The earlier germination might be
explained by greater activities of germination-related enzymes,
the early hydration of the membrane and greater molecular

60
50

6

1

2
Test number

5

1

2
Test number

Fig. 2. T2 of germinated seeds dipped in the water containing NBs and distilledwater (T2a and T2b are the long and the short components of T2; The bars showed
the standard error of ﬁve replicates).

mobility of the bulk and hydration water fractions (Vashisth
and Nagarajan (2010)).
The proton–nuclear magnetic resonance relaxation time canbe used to detect weak molecular interactions such as hydrogenbonding, molecular mobility and steric effects (Balci, 2005). Thus, it
is widely used to study the mobility and diffusion of the watermolecules in agriculture and food ﬁelds. Recently, both spin–lattice
relaxation time (T1) and spin–spin relaxation time (T2) of germinated seeds dipped in water containing NBs were reported to besigniﬁcantly greater than those of seeds dipped in distilled water(Liu et al., 2012a). Moreover, Ishibashi et al. (2005) have reported
that T2 is a suitable indicator for examining the degree of endosperm
degradation in germinating seeds of rice, which was, in turn, related
to the availability of T2 as a probe for metabolic activities.
Thus, in this research, the water status of seeds dipped in watercontaining NBs and in distilled water during germination wasdetermined via NMR spectroscopy. The T2 of barley seeds wascomposed of two components: T2a (long) and T2b (short). As evident
in Fig. 2, two repetitive experiments showed that the T2 values ofgerminated seeds dipped in water containing NBs were signiﬁcantly
greater than those dipped in distilled water (po0.05).
3.2. Change in T2 values of control water and water containing NBs
as a function of the water-storage time
Considering these results, spin–spin relaxation times (T2) inboth control water and water containing NBs were measured

S. Liu et al. / Chemical Engineering Science 93 (2013) 250–256

using a pulsed spectrometer (JNM-MU25A, JEOL, Japan) at 25 MHz
to determine the effect of NBs on the physicochemical properties ofwater in bulk.
Under normal conditions, a signiﬁcance level greater than 0.05
is not meaningful. To compare the difference between control
water and water containing NBs as a function of storage time, testlevels with p values greater than 0.05 (0.1 and 0.25) were also
used. Water containing NBs displayed statistically longer T2values compared to those of control water, as shown in Fig. 3a.
Sample temperature was 20 1C. The pH values for control water
and water containing NBs were 6.31 and 7.06, respectively. The T2of the proton is independent of the pH value. The T2 values of bothcontrol water and water containing NBs decreased after 2 days,
and the difference in the T2 values observed on the day of thegeneration of NBs became unclear with time. T2 values and thedifference in T2 values decreased because oxygen (a paramagnetic
molecule) in the head space of the NMR tube dissolved in thewater, which resulted in elevated DO in sample water andconsequently, a reduced T2 value. Bubble-size distributions were
simultaneously measured using the nanoparticle tracking analysis
method, a kind of Dynamic Light Scattering (NanoSight-LM10,
Quantum Design Inc., Japan), which has been widely used todetermine the sizes of nanoparticles in liquids (Bunkin et al.,2012). Bubble-number density decreased with longer storage
times, which was supported by results shown in Fig. 4, in which
both the total number of bubbles and size of the main bubbles
decreased with longer storage time. The main bubble size
observed was classiﬁed into two sizes, 182 and 249 nm, on the
day of the generation of NBs; two days later, the bubble size was
186 nm, and seven days later it was 168 nm. The tendency of the
bubble sizes to shrink was clearly observed.
The total bubble-number density of water containing NBs was
1.97  108 particles/mL on the day of the generation of NBs. For
control water, the total bubble-number density was approximately 0.09  108 particles/mL, which was less than 1/20th thedensity of water containing NBs (Fig. 4). The measurement wasdone at room temperature, and the temperatures for the watercontaining NBs and the control water were 29.8 and 28.7 1C,
respectively. Therefore, the effect of NBs on T2 was partly masked
by the paramagnetic effect of oxygen during measurements
performed after 2 or more days.
The presence of oxygen would shorten the relaxation time. To
eliminate the paramagnetic effect of oxygen in the head space, we
ﬁlled pressure-tight tubes with both control water and watercontaining NBs to ensure that they contained no gas. As evident in

253

Fig. 3b, water containing NBs exhibited statistically longer T2values than those of control water. The sample temperature was
20 1C. The pH values for control water and water containing NBs
were 7.91 and 8.01, respectively. The T2 values of both control
water and water containing NBs did not substantially changeeven after 1 week, and the difference in their T2 values was still
obvious. Apart from the elimination of the paramagnetic effect ofoxygen, the closed environment, which precluded the presenceof gas above the water in the tube, might also account for the
prolonged existence of NBs.
3.3. Change in T2 values of control water and water containing NBs
after degassing
The results from the previously discussed experiments showedthat the introduction of NBs increased the T2 value of water. In
order to clarify the relationship between the T2 value of the waterand that of water containing NBs, degassing was used to remove
NBs from the water sample. If NBs were the only factor thatcaused the increase in the T2 of the water; then, the T2 of degassed
water sample should decrease to the original T2 value.
After T2 measurements, both water containing NBs and control
water were degassed under a pressure of 0.02 MPa. The T2 values
were subsequently measured again to examine the effects of thedisappearance of NBs due to degassing. As evident in Fig. 5a, theT2 value of one among ﬁve samples of degassed water containing
NBs decreased from 2.03 to 1.92 s. The T2 values did not change
until the samples were degassed for 5 min. After 30 min ofdegassing at room temperature, the T2 value decreased only
slightly. NBs were apparently so stable that under a degassing
pressure of 0.02 MPa, they could not be easily removed. Therefore,
we increased the degassing temperature to 30 1C; after another30 min of degassing at this temperature, the T2 value decreased to
1.96, and, 1 h later, the T2 value decreased to 1.92.
After the tendency of the T2 value to diminish with longer
degassing time was conﬁrmed, the other nine water samples (ﬁvesamples of control water and four samples of water containing
NBs) were also degassed for 45 min at 30 1C. As shown in Fig. 5b,
the T2 values of water containing NBs obviously decreased after
degassing, and the values after degassing were still statisticallylonger than that of control water.
For water containing NBs, after degassing, a few large bubbles
were formed from the coalescence of several NBs. Henry’s law
suggests that exposing a water droplet to an external pressure
lower than atmospheric pressure can reduce the solubility of air

Fig. 3. Time-dependent change of T2 values of the control water and the water containing NBs at 20 1C ((a) water sample of 0.4 mL in the NMR tube under air atmosphere;(b) water samples ﬁlled in pressure-tight tubes; The bars showed the standard error of ﬁve replicates; tubes containing 0.4-mL water sample).

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S. Liu et al. / Chemical Engineering Science 93 (2013) 250–256

Fig. 4. Bubble size distributions with time of control water and the water containing NBs. (a) shows the comparison of bubble size distribution of control water and thewater containing NBs. (b) shows the change of bubble size distributions in the water containing NBs with different storage time.

Fig. 5. T2 of the control water and the water containing NBs before and after degassing measured at 20 1C ((a) change in T2 of one of 5 water samples as the watercontaining NBs during degassing; (b) change in mean value of T2 of the control water and the water containing NBs before and after degassing; The degassing pressure is
0.02 MPa; RT: room temperature (about 20 1C); the bars showed the standard error of ﬁve replicates; tubes completely ﬁlled with water).

and hence result in the local super saturation of the dissolved gas
in the droplet. As the bubbles increase in size, they may engulf
other nearby NBs, even if they most probably have the same
surface charge. After they are sufﬁciently large, the bubbles may
detach from the bulk water because of buoyancy. After degassing,a few large bubbles were actually observable with the naked eye.
Each large bubble was formed from a large number of NBs. For
control water, the T2 values also decreased after degassing.
However, the rate of decrease was slower than that of watercontaining NBs. Therefore, we conclude that control water may
also contain a small number of NBs although substantially fewer
than water containing NBs. On the basis of these results, weconcluded that the number of NBs had an apparent positive
correlation with the T2 value of the water. Moreover, apparently,
the stability of NBs was not disrupted by degassing under apressure of 0.02 MPa. The physical stability of NBs means that, as
Zimmerman et al. (2011) recently stated, ‘NBs, once formed, arehighly persistent’.
The device we used requires tubes with an outer diameter of
10 mm, and the volume of water samples is typically 0.4 mL.
Therefore, ﬁve NMR tubes containing 0.4-mL ultrapure water andﬁve tubes completely ﬁlled with ultrapure water (approximately
4 mL) were compared to test the reproducibility of T2 measurements using different water volumes. The results showed that thedeviation of T2 measurements for the water sample ﬁlled in the
tubes was less than that for the tubes containing 0.4-mL water
sample, which means for the sample preparation method, that is,
tubes completely ﬁlled with water, does not affect the reproducibility of the experiment.
Moreover, another sample-preparation method was used, i.e.,
0.4-mL water was placed into tubes, and the air in each tube was
replaced with nitrogen using a vacuum pump. As evident in Fig. 6,
the mean T2 values of water containing NBs were longer thanthose of control water. After degassing, the T2 values of watercontaining NBs obviously decreased. On the contrary, the T2 valueof control water showed no obvious changes. Therefore, therelationship between the existence of NBs and the T2 values ofwater was conﬁrmed using different sample-preparation methods.
3.4. Discussion about the inﬂuence of NBs on the physicochemical
properties of water
In addition to T2 measurements, the zeta-potential values ofwater containing NBs were also measured. NBs were formed byintroducing nitrogen. The zeta potentials of water containing
NBs, when they were formed with nitrogen, were 32.26
and  38.84 mV at pH 7.28 and 7.55, respectively. The charging
mechanism has been explained by the excess of hydroxyl ions

S. Liu et al. / Chemical Engineering Science 93 (2013) 250–256

255

network of bulk water; therefore, the mobility of the watermolecules increases. Therefore, the presence of ions could modify
the hydrogen-bonding network in water.
Katou and Okamoto (1970) found that the production ofhydrogen ions via the elongation of tissue and the transport ofhydrogen ions associated with the counter ﬂow of other cations
and water could generate a bioelectric ﬁeld within the elongatingzone itself. The bioelectric ﬁeld was closely related to theelongation growth of the plant (Okamoto et al., 1984). The orderof magnitude of the zeta-potential values of NBs was similar tothat of the membrane potential of plants. Another explanation isthat negatively charged NBs may inﬂuence the bioelectric ﬁeld ofplant tissue and thus enhance the metabolic activity.

4. Conclusions
Fig. 6. Change of T2 values of the control water and the water containing NBs after
the degassing (Degassing on the day of NB generation. pH of the water containing
NBs was 7.72 and pH of the control water was 8.19. DO of the water containing
NBs was 0.02 mg/L and DO of the control water was 0.15 mg/L. NS: No signiﬁcantdifference; 0.4-mL water was placed into tubes, and the air in each tube was
replaced with nitrogen using a vacuum pump).

relative to hydrogen ions at the gas–water interface. Two explanations for this result were proposed. The ﬁrst explanation is thatthe hydration energies of hydrogen and hydroxyl ions are different( 1104 and  446.8 kJ/mol for hydrogen and hydroxyl ions,
respectively), and hence hydrogen ions are more probable toremain in the bulk water phase than hydroxyl ions (Yoon and
Yordan, 1986; Najaf et al., 2007). The second explanation is that anelectric double layer is formed because of the orientation of water
dipoles at the interface, with hydrogen and oxygen atoms pointing
toward water and gas phases, respectively; this arrangement
causes the attraction of anions to the interface (Paluch, 2000;
Takahashi, 2005).
According to Ohgaki et al. (2010), the water molecules may
form shells of hard hydrogen-bonded ice-like structures around
MNB, and the greater surface tension (twice the normal value)that arises from the presence of the hard interface helps maintaina kinetic balance against high internal pressure. On the contrary,
Himuro (2007) found that the shrinkage of microbubbles considerably decreased the number of hydrogen-bonded water molecules,
thereby causing less surface tension. MNBs contributed to weakening the hydrogen-bonding network in the bulk water. We cannot
state which opinion is correct; however, our results from multiple
parallel experiments showed that the introduction of NBs increases
the T2 value of water.
Negatively charged bubbles adsorbed hydrogen ions onto theirsurface, resulting in Coulomb-repulsion forces that could compensate surface-tension forces (Chaplin, 2007; Bunkin et al., 2007,2008). The presence of ions, therefore, can modify the hydrogenbonding network in water. The hydrogen bonding around the
bubble surface might enhance the viscosity of water near theinterface of bubbles and decrease the mobility of a limited
number of the water molecules. However, for the rest of thelarger quantity of water, NBs contributed to the weakening of thehydrogen-bonding network of the water. The formation, shrinkage and disappearance of NBs would also affect the totalhydrogen-bonding network and structure of water. The disturbance of hydrogen bonding would accelerate the mobility of thewater molecules.
The dormancy release of seeds has been associated with lessbound water and increased molecular mobility within theembryonic axes (Bazin et al., 2011). Thus, one of our hypothesesis that the introduction of NBs can disturb the hydrogen-bonding

In summary, the germination rates of barley seeds dipped inwater containing NBs were 15–25 percentage points greater thanthose of seeds dipped in distilled water, which veriﬁed the clear
effect of NBs on the physiological activity. According to NMR
results, the number of NBs had a positive correlation with the T2value of the water, which indicated that NBs could increase themobility of the water molecules in bulk. These results suggested
that NBs in water could inﬂuence its physical properties, which
provides an explanation for the effect of NB promotion on thephysiological activity of living organisms. The other explanation isthat negatively charged NBs may inﬂuence the bioelectric ﬁeld ofplants, which is strongly related to their elongation growth. Aftercompletely understanding NBs’ ability to promote plant growth isachieved, the manipulation of NBs will provide an efﬁcient andcost-effective approach for the cultivation of hydroponic vegetables and allow the development of a new technology in agriculture applications.

Author contribution
Shu Liu and Seiichi Oshita planned and performed all of theexperiments. Shu Liu wrote the paper. All authors discussed theresults and commented on the manuscript.

Acknowledgements
A part of this research was ﬁnancially supported by the Food
Nanotechnology Project, MAFF of Japan. The authors also express
gratitude for the support offered by the Chinese ScholarshipCouncil. The nanoparticle tracking analysis data was technicallysupported by Ms. Ayako Irie at Quantum Design Inc., Japan.
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