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Contents lists available at ScienceDirect

Colloids and Surfaces A: Physicochemical andEngineering Aspects
journal homepage: www.elsevier.com/locate/colsurfa

Evidence of the existence and the stability of nano-bubbles in water
Fernanda Yumi Ushikubo a , Takuro Furukawa a , Ryou Nakagawa a , Masatoshi Enari a ,Yoshio Makino a , Yoshinori Kawagoe a , Takeo Shiina b , Seiichi Oshita a,∗ab

Faculty of Agriculture, The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku, Tokyo 113-8657, Japan
Distribution Engineering Laboratory, National Food Research Institute, 2-1-12 Kannondai,Tsukuba, Ibaraki 305-8642, Japana r t i c l e

i n f oArticle history:
Received 26 October 2009
Received in revised form 3 March 2010
Accepted 4 March 2010
Available online xxx
Keywords:
Micro-bubble
Nano-bubble
Nuclear magnetic resonanceParticle size distribution
Zeta potential
Bubble stabilitya b s t r a c t
Although micro- and nano-bubble technology has been attracting attention in many ﬁelds, the state ofwater after the introduction of those bubbles is still not clear. In this study, the existence and stabilizationof nano-bubbles after the generation of bubbles were investigated. The presence of nano-sized particles
was detected through dynamic light scattering for days, when pure oxygen was used to generate thebubbles, and for less than 1 h, in the case of air bubbles. NMR spin–lattice relaxation time increased
with the introduction of micro- and nano-bubbles in manganese ions solution, indicating the presenceof a gas–liquid interface which adsorbed the manganese ions. Furthermore, the zeta potential measured
in the water after the introduction of oxygen micro- and nano-bubbles was in the range from −45 mV
to −34 mV and from −20 mV to −17 mV in water bubbled with air, indicating the presence of stableelectrically charged particles. This study suggested a strong possibility of the existence of nano-bubbles
in water for a long time. The stability of nano-bubbles is supported by the electrically charged liquid–gas
interface, which creates repulsion forces that prevent the bubble coalescence, and by the high dissolved
gas concentration in the water, which keeps a small concentration gradient between the interface andthe bulk liquid.
© 2010 Elsevier B.V. All rights reserved.

1. Introduction
Micro- and nano-bubbles (in this study, referred to as “MNB”)
present characteristics that make them special in relation to theordinary bubbles (macro-bubbles) because of their reduced diameter size. Some of the advantages of the MNB are their high speciﬁcarea (surface area per volume) and the high stagnation in the liquid phase, which increase the gas dissolution. Moreover, it wasreported that when micro-bubbles collapse, free-radical generation occurs, due to the high density of ions in gas–liquid interface
just before the collapse [1].
There are applications of MNB in different ﬁelds, which include
the water treatment by ﬂotation [2,3], taking advantage of the highspeciﬁc area of MNB; the sterilization using ozone gas [4], which
has its dissolution increased with MNB generation; contrast agent
for ultrasonography [5] and the possible use in food industry forfoam products, carbonated drinks and as a nutritional supplement
carrier [6], in which bubble stability is desired.
Furthermore, the use of MNB in water was reported to be effective for the acceleration of metabolism in shellﬁshes and vegetables[7,8], as well as in aerobic cultivation of yeast [9], accelerating the∗ Corresponding author. Tel.: +81 35 841 5362; fax: +81 35 841 8174.
E-mail address: aoshita@mail.ecc.u-tokyo.ac.jp (S. Oshita).

growth and increasing the yield of the products. This effectiveness,
however, cannot be explained only by the increase of the dissolved
oxygen (DO) concentration [8], using MNB in the solution for thehydroponic cultivation of lettuce, observed acceleration in growthin comparison with lettuce grown in solution containing similar DO
concentration but without MNB. Therefore, the MNB should play animportant role in the physiological activity in cells.
However, there are still many questions concerning the nanobubbles that are not clear. Some of them are the stability and size ofnano-bubbles after the introduction of MNB. This basic information
is important to understand the properties of the water after the
introduction of bubbles.
For this purpose, in this study, the presence of bubbles in nanoscale was investigated using dynamic light scattering (DLS). DLS isgenerally used to measure the size of particles such as polymers,
micelles, emulsions, proteins and so on, by detecting the Brownian
movement of particles.
Moreover, in order to obtain information about the electricalcharge and stability of particles, -potential (zeta potential) was
measured in water after the introduction of MNB.
In addition, in order to examine the existence of bubbles inwater by a method based on a different physical principle than DLS,
nuclear magnetic resonance (NMR) spectroscopy was used. This
technique can detect weak molecular interactions such as hydrogen bonding, molecular mobility and steric effects [10], so it was

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Fig. 1. Scheme of micro-bubble generator.

expected to ﬁnd some modiﬁcation of the intermolecular structurewith the addition of MNB.
Thus, the present study aimed to investigate the stabilizationof nano-bubbles in water by the measurement of particle size
distribution, -potential and proton NMR spin–lattice relaxationtime.
2. Materials and methods
2.1. Micro- and nano-bubbles generation
Ultrapure water was used to produce micro- and nano-bubbles.
This water was obtained using a water puriﬁcation system (DirectQ, Nihon Millipore Ltd., Japan), which is equipped with a reverse
osmosis cartridge and modules of ion-exchange resins and activated carbon. In the particle size distribution measurements, 2 l ofultrapure water was prepared. For the -potential measurements,
1 l of commercial ultrapure water (Kanto Chemical Co. Inc., Japan)
was used, since a very strict control of water purity was required.
In the case of the NMR measurements, manganese ions solution
was used as a control (see Section 2.5) and the volume of solution
was 1 l. The manganese ions solution was prepared using ultrapure
water prepared with the Direct-Q puriﬁcation system.
The main gas used to produce MNB was oxygen (O2 , purity
99.999%, Nissan Tanaka Co., Japan). For comparison reasons, air(setting a ﬁlter in the gas inlet) and xenon (Xe, purity 99.995%,
Iwatani Sangyo Co. Ltd., Japan) were also used to produce MNB
in some experiments.
A micro-bubble generator (OM4-GP-040, Aura Tec Co. Ltd.,
Japan) was used for the production of micro- and nano-bubbles(Fig. 1). In this system, the gas was introduced in the inlet part of amagnetic gear pump (MDG-R2RVA100, Iwaki Co. Ltd., Japan) while
the water was suctioned. Then, the mixture of water and gas was
subjected to a high pressure (0.25–0.27 MPa) in a pressurized tankto increase the dissolution of gas in the water. When the water
was released under atmospheric pressure, the depressurization of
the gas-supersaturated water led to the nucleation of the bubbles,
which were expelled and dispersed through a nozzle at the outlet.
The water was circulated in this system for 40 min at 20 ◦ C constant temperature, controlled by a water bath (NTT-1300, Tokyo
Rikakikai Co. Ltd., Japan). In this study, the water obtained after
this procedure is referred to as “MNB water”, indicating the waterin which micro- and nano-bubble were introduced.
According to [11], the magnetic gear pump is non-corrosive, has
no seals and is free of leakage. This pump is recommended for applications in which extreme clean conditions are necessary, such as
pure water processing machines and medical applications. There-

fore, the contamination of the water by the magnetic gear pump isunlikely to occur. Furthermore, in order to remove possible impurities from the equipment, three cycles of 10 min each, exchanging
the water between them, were performed before the production of
bubbles in the micro-bubble generator.
2.2. DO concentration and pH analyses
The DO (dissolved oxygen) concentration and the pH were both
determined at 20 ◦ C, using a dissolved oxygen meter (SG6, MettlerToledo GmbH, Switzerland) and a pH meter (D-55, Horiba Ltd.,
Japan), respectively.
2.3. Particle size distribution
The particle size distribution in nano-scale was measured using
a green badge (532 nm laser) Zetasizer Nano ZS particle size
analyzer (ZEN3500, Sysmex Co., Japan), which detects Brownian
motion of particles through dynamic light scattering (DLS). The
range of particle size which can be detected is from 0.6 nm to 6 ␮m.
The refractive index of the material of the “particles”, was set to
1.0, corresponding to the air. The measurements were performed
at 20 ◦ C in a glass cell with square aperture. For each sample, 5–10
replications were done.
In the particle size measurement, two different gases were used
to produce MNB water: oxygen and air. Oxygen MNB water was
produced at two different DO concentrations. This modiﬁcation was
done by the use of nozzles with different capacities to disperse thegas into the water in the outlet of the generator.
The ﬂask containing each type of MNB water was stored
at a 20 ◦ C constant temperature room, sealed with Paraﬁlm(Pechiney Plastic Packaging, USA). Samples were taken periodically for DLS analysis until the particle size distribution showed norepeatability.
The data was analyzed through the geometric mean diameter of
the average particle size distribution. The geometric mean diameter(dg , nm or ␮m) and the geometric standard deviation (Sg ) were
calculated according to the standard ASAE S319.4 [12], as describedin Eqs. (1) and (3), respectively:⎡ n⎤(Fi log di ) ⎥⎢⎢⎥−1 ⎢ i=1⎥
dg = log ⎢
n
⎥

⎣
⎦

(1)

Fi

i=1

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⎡ n
⎤1/2

2
Fi (log di − log dg ) ⎥
⎢
⎢ i=1
⎥
⎥
Slog = ⎢
n
⎢
⎥

⎣
⎦

(2)

Fi

i=1

Sg =

−1
1
dg [log−1 Slog − (log−1 Slog ) ]2(3)

where n is the number of intervals; Fi is the relative frequency of
1/2
the ith interval (%); di is calculated as (di × di+1 )(nm or ␮m), in
which di is the lower size in a size interval and di+1 is the upper sizein a size interval of the histogram; Slog is the geometric standard
deviation of log-normal distribution in ten-based logarithm.
The coefﬁcient of variation (CV), here expressed in percentage,
measures the dispersion of the particle size distribution:
Sg
CV =
× 100
dg

(4)

The particle size distribution was presented as relative frequency of particles expressed in intensity of scattered light.
2.4. -Potential measurement
-potential measurements of the oxygen and air MNB water
were performed using a Zeta Potential Analyzer (Zeecom,
Microtech Co. Ltd., Japan). This system detects the electrophoretic
mobility of particles and is equipped with a microscope and a CCD
camera to observe the movement of particles in the range of 20 nm
to 100 ␮m in a 1 mm ×10 mm glass cell. The small particles can beobserved by the scattering of a halogen light or laser. Fifty particles were tracked manually to determine their speed and the-potential was calculated using Smoluchowski equation.
2.5. Proton NMR spin–lattice relaxation time measurement
Five replications of each sample (control and MNB) were collected in tightly closed 10 mm diameter NMR tubes, up to a 1.5 cm
high liquid column. Proton spin–lattice relaxation time (T1 ) was
measured in a pulsed spectrometer (JNM-MU25A, JEOL, Japan) at
25 Hz frequency and at constant temperature of 20 ◦ C, using thesaturation recovery pulse sequence.
As shown in previous study [13], the presence of oxygen in thesamples was an interferer in the T1 measurements. The reason was
the paramagnetic characteristic of oxygen, that is, the magnetogyric ratio of the electron in this molecule is much higher thanthat of a proton, which leads to a very efﬁcient relaxation [14] and,
consequently, to a decrease in T1 .
The ideal condition to perform NMR analyses would be, therefore, the complete removal of oxygen gas from the samples.
However, the oxygen in the form of micro- and nano-bubble isexactly the target of this experiment. Furthermore, some difﬁculties
were found to degas the water to a very low DO concentration. Thus,a different approach was taken in order to mask the paramagnetic
effect of oxygen.
As manganese ions are paramagnetic, the addition of these ionsin the water before the generation of bubbles was expected to cause
an excess of paramagnetism and mask the interference of oxygenin the NMR analysis. Thus, solutions of 3, 5, 10, 15, 40 mM of manganese (II) chloride tetrahydrate (Mn2 Cl·4H2 O, Kanto Chemical Co.
Inc., Japan) were prepared (control sample), then oxygen MNB was
produced (“O2 MNB solution”). Besides, xenon MNB was generated
in 10 mM manganese solution (“Xe MNB solution”). Xenon was chosen to verify the properties of MNB made by a non-paramagnetic
gas. For this purpose, other gases could be used, but some factors

3

were considered, such as the gas solubility, which is greater thannitrogen’s, for example, and because xenon is an inert gas.
In order to verify if the dissolved oxygen in the samples could
affect T1 , a “O2 without MNB solution” was produced and the T1
measured in this sample was compared to the O2 MNB solution’svalue. The O2 without MNB solution was prepared by the introduction of pure oxygen into a 10 mM manganese solution througha gas diffuser. This manganese solution showed DO concentration
similar to the O2 MNB solution at 10 mM of manganese ions andwas considered to contain no MNB.
3. Results and discussion
3.1. Particle size measurements
3.1.1. Control water
The particle size distribution of the ultrapure water showed thepresence of particles of geometric mean of 612 nm (CV = 130.8%).
The concentration of this particles should be very low because of
the irregular shape and the presence of multiple peaks in the particle size distribution. In addition, the result of the DLS measurement
is given in relative frequency of particles, so the absolute concentration of particles is not provided by this method.
The detected particles were supposed to be a very few amount of
air bubbles which were incorporated when the water was poured
into the ﬂask. The reason for that can be explained by the fact that
Direct-Q system, which was used for the preparation of ultrapure
water, can reject more than 99% of the particles, microorganisms
and organic matter (molecular weight above 100 Da). Besides, nosurfactants neither salts were added. Therefore, the water was
expected to contain no solid particles.
After degassing the ultrapure water under vacuum for 20 min,the particle size distribution peaks were detected randomly
throughout all the diameter range. Because these size distribution
curves showed no repeatability, it was understood that the data
were at noise level and no particles were detected. Based on theseresults, the ultrapure water prepared by Direct-Q system and subsequently degassed for 20 min was considered suitable as control
water for the production of MNB water.
3.1.2. Oxygen MNB water
Differently from the results obtained for the control water, theparticle size distribution of the oxygen MNB water showed a regularshape of a mono-modal log-normal curve and the measurements
had good repeatability (Fig. 2(a)). Based on this difference, there isa high possibility that the particles detected represent the diameter
distribution of nano-bubbles formed by the introduction of gas.
The geometric mean of the particle size distribution was 137 nm(CV = 61.2%) just after stopping the generation of bubbles. Thisgeometric mean oscillated slightly on the next hours until the following day (Fig. 2(b)), reaching values up to 272 nm. After threedays of observation, the geometric mean diameter detected waslarger (380 nm) and the variation of particles size was higher(CV = 107.4%). The larger geometric mean does not necessarily indicate that the bubbles increased in size or coalesced. The reason isthat the DLS method is more sensitive to big particles than small
ones, so a relative higher concentration of larger particles could
have been detected. This can occur when the particle concentration is low. At a low concentration in all sizes, smaller particles
can not be detected, while larger particles are still sensed. The low
concentration of particles on the third day was estimated by thepoor repeatability of the results shown by the error bars in Fig. 2(c)in comparison to the measurements on the previous days (Fig. 2(a)and (b)). On the third day, it seems that the particle concentration isat the limit of the minimum detection level of the particle analyzer.

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Fig. 2. Particle size distribution (average values, n = 10) of oxygen MNB water (a) just after stopping the gas introduction (DO = 36.9 mg L−1 ), (b) 1 day, (c) 3 days and (d) 6days later (DO = 8.9 mg L−1 ). The vertical bars represent the standard deviation of the replication data.

Finally, on the sixth day after the bubble generation, the particle size distribution curves were no longer mono-modal, theshape became irregular and there was no repeatability of the data
(Fig. 2(d)). This lack of repeatability could be understood as an insufﬁcient concentration of particles to be detected by the instrument.
The decrease in concentration of particles with time is one more
supporting evidence that the detected particles by DLS could beoxygen nano-bubbles.
In the literature, Kikuchi et al. [15] reported the presence ofoxygen nano-bubbles after water electrolysis through the measurement of particle size by DLS. The mean particle size was 30 nm on
the ﬁrst day and increased to 180 nm and to 250 nm on the second and third day, respectively. After this time, the bubbles shrank
until the ﬁfth day, when no particles were observed. The range ofsize of the bubbles on the second and third days were similar to
the results obtained in this present study, as well as the durationof the bubble detection. In other report, 290 nm air nano-bubbles
were measured through DLS method after the nucleation of bubbles controlled by temperature reduction [16]. These reports in theliterature and the measurements done in this present study showthat the use of this technique can be reliable for measuring bubbles
at nano scale, although an indirect method such as DLS is normallyused for measuring solid particles.
Furthermore, in this study, the change in the particle size distribution curves with time was in accordance with the change inthe DO concentration, which was at supersaturated level just after
the bubble generation (36.9 mg L−1 ) and at saturation level after six
days (8.9 mg L−1 ). The supersaturated condition could have kept asmall gradient concentration of gas between the bubble interface
and the bulk liquid, maintaining the bubbles stable, at a number
density high enough to be detected by the laser scattering sensor.
With time, the oxygen in the bubble dissolved into the water and,ﬁnally, diffused to the atmosphere. Thus, the bubble concentration
decreased while the DO concentration reached the saturation level.
By the use of a nozzle with higher capacity of dispersing gas
into the water, oxygen MNB water containing higher DO concentration (44.5 mg L−1 ) was produced. The particle size distribution
was similar as the previous experiment at lower DO concen-

tration: mono-modal log-normal curves with good repeatability.
The geometric mean diameter was also in similar range of size
(125 ∼ 172 nm) and the dispersion of the particle size was small(CV = 55.4% ∼ 72.6%). However, at a higher initial DO concentration,
particles were observed for a longer time, as bubbles were detecteduntil the ﬁfteenth day after the MNB water production. Only on
the eighteenth day, non-uniform and very scattered curves wereobtained. The DO concentration of the MNB water became near thesaturation condition on the ﬁfth day (10.3 mg L−1 ). On the eighteenth day, the DO concentration had reached to the saturation
equilibrium at 9.1 mg L−1 .
Based on the data obtained, it was observed that the higher
initial DO concentration could extend the particle stability. This
corroborates with the possibility of the existence of nano-bubbles,
since at higher DO concentration, the oxygen diffusion time would
take more time. However, only the high DO concentration cannot
explain the extension of the nano-bubble stability. From the ﬁfth
day after the bubble generation, although the DO concentration wasnear the saturation condition, the particle size distribution could
still indicate clearly the presence of nano-particles. Therefore, there
should be other factors which can contribute to the stabilization of
nano-bubbles besides the high gas concentration in the liquid.
3.1.3. Air MNB water
When air was introduced in water instead of oxygen, therepeatability of data was not as good as that obtained for oxygen
MNB water and the shapes of the distribution curves were irregular. The geometric mean of the particle size distribution ﬂuctuatedfrom 350 nm in the ﬁrst 5 min to 140 nm after 25 min after the bubble generation. The dispersion of data was larger (107.3% at 5 min
and 137.5% at 25 min) than that obtained in O2 MNB water. Someparticles could still be detected 45 min after the bubble generation at the size of 205 nm (CV = 120.7%), but at 70 min, the particles
could not be detected. The DO concentration just after the air bubble generation was 11.7 mg L−1 and was kept stable until the last
observation time.
The poor repeatability and the irregular distribution curves of
air MNB water indicate that the concentration of particles should be

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Fig. 3. Average values of -potential measurements in O2 MNB water and air MNB
water with time (n = 50). The vertical bars show the standard deviation of themeasurements.

under the minimum detection level to obtain more accurate data.
The results suggest the low stability of air MNB compared to theoxygen MNB, since the concentration of air bubbles should be much
lower than that of oxygen MNB.
The big difference between the periods in which bubbles could
be observed stable in the oxygen MNB water (3 or 15 days) and in
the air MNB water (less than 1 h) should be related to the lower dissolution of the air in water in relation to the dissolution of oxygen.
At a lower concentration of dissolved gas in the water, the concentration gradient across the gas–liquid interface of the bubble ishigher, which leads to a more efﬁcient gas transfer to the liquid
phase.
3.2. -Potential
The measurement of -potential in O2 MNB and air MNB waterwith time showed negative values (Fig. 3). Moreover, the averageof absolute values of O2 MNB water (34–45 mV) were higher thanthose of air MNB water (17–20 mV). The pH of the O2 MNB water
was between 6.2 and 6.4 and that of the air MNB water was in therange of 5.7 and 6.2.
In the literature, Graciaa et al. [17] reported a -potential of−65 mV in air bubbles formed in deionized water, while Takahashi[18] obtained −35 mV in distilled water (pH 5.8). According to Graciaa et al. [19], the disparity in results in the literature is due to thedifferences in the puriﬁcation methods of the water. However, inboth reports, as well in this present study, the value of -potential
was negative. The negative value is attributed to the predominance of hydroxide ions in the ﬁrst molecular layers of water at
the gas–liquid interface [20].
According to Spanos et al. [21], the absolute -potential value
normally taken as a minimum to observe a potential stability is
30 mV. At a high absolute -potential, the electrical charged particles tend to repel each other, avoiding aggregation of particles
in a colloidal dispersion. In the case of a bubble dispersion, thehigh -potential could create repulsion forces that would avoid thecoalescence of bubbles and contribute to the stabilization of the5

bubbles. Therefore, in this study, the O2 MNB could be stabilized inwater by the electrically charged surfaces, while the air MNB would
be below the limit of the stabilization by repulsion forces.
Furthermore, it was observed a relation between -potential
and DO concentration. In the measurements performed in O2 MNB
water until the ﬁrst day after the bubble production, -potential
presented absolute values in the range from 40 mV to 45 mV. During
this period, the DO concentration was above the saturation level:
40.8 mg L−1 just after the bubble generation and 14.9 mg L−1 one
day later. From the second day after the bubble generation, when
the DO concentration reached 10.3 mg L−1 and tended to stabilize
near the saturation level (9.1 mg L−1 at 20 ◦ C), the -potential absolute value was lower and was practically constant in the range from
34 mV to 38 mV.
In air MNB water, -potential, as well as the DO concentration
did not vary much with time. The DO concentration was 10.7 mg L−1
just after the bubble production, decreasing to 10.0 mg L−1 in theﬁrst hour after stopping the bubble generation and stabilizing atsaturation level in the next hour.
One important point to observe is that, during the measurements of the particle mobility, although the velocity of the particles
did not seem to vary with time (resulting in practically constant potential), the amount of particles did. Therefore, while the chargeof the particles practically did not change, the concentration of particles or bubbles should be decreasing with time (qualitative data).
For example, in the case of air MNB water, the concentration of particles after 3 days of the bubble generation became so low that the-potential measurements could not be continued.
In summary, the -potential measurements showed that the
MNB water is negatively charged and that the O2 MNB water
presented higher electrical charge than the air MNB water. The
high value of -potential can be related to the stability of bubbles, explained by the repulsion forces generated by the electricallycharged surfaces of bubbles, which avoid the bubble coalescence.
3.3. Proton NMR spin–lattice relaxation time (T1 )After the introduction of oxygen as micro- and nano-bubbles
in the manganese ions solution, it was veriﬁed that T1 increased(p < 0.05) in all the manganese concentrations tested, as shown in
Table 1.
The pH value seemed to increase very slightly with the introduction of oxygen, probably because of the release of the naturally
dissolved carbon dioxide from the solution due to the gas bubbling.
One important point is that the increase in T1 was caused by the
production of MNB, but not by the increase in DO concentration. A
more detailed examination in the case of 10 mM Mn2+ solutions isshown in Fig. 4.
The O2 without MNB solution at 10 mM Mn2+ concentration has
similar DO concentration as the O2 MNB solution: 37.1 mg L−1 and38.8 mg L−1 , respectively. However, the T1 of O2 without MNB solution did not differ statistically from the control sample (p < 0.05).
Therefore, the generation of micro- and nano-bubbles is the only
reason for the T1 increase observed in O2 MNB solution. In other

Table 1
T1 , DO concentration and pH of Mn2+ solution at different concentrations, before (control) and after introduction of oxygen micro- and nano-bubbles (O2 MNB).
[Mn2+ ] (mM)Control
T1 (ms)

3
5
10
15
40

36.7±0.1a
20.8±0.1a
11.57±0.03a
7.77±0.03a
2.894±0.004a

O2 MNB−1

DO (mg L
7.7
8.0
8.6
9.4
8.5

)

pH

T1 (ms)DO (mg L−1 )

pH

5.97
5.80
5.59
5.78
5.88

37.60±0.06b
21.35±0.06b
12.48±0.06b
8.18±0.03b
3.01±0.02b

39.6
39.1
38.8
39.6
35.8

6.30
6.12
5.71
5.83
5.69

Different superscript letter means that the T1 of control and O2 MNB samples differ signiﬁcantly (p < 0.05).

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Fig. 4. T1 value of 10 mM Mn2+ solutions: control, O2 without MNB solution, O2
MNB solution and Xe MNB solution. Different letters means that the samples differ
signiﬁcantly between each other (p < 0.05).

words, the difference between the T1 values of O2 without MNB andO2 MNB solutions indicates that the paramagnetic effect of oxygen
was successfully masked by the manganese ions.
Moreover, it was observed that the Xe MNB solution indicatedthe same tendency of increasing T1 as that observed in the O2 MNB
solution (Fig. 4). Similarly, in this case, the T1 values were independent of the DO concentration, which were 38.8 mg L−1 in O2 MNB
solution and 0.5 mg L−1 in Xe MNB solution. Furthermore, it seems
that the presence of MNB itself is more decisive for the dynamic
state of the solution than the type of gas that composes the bubbles.
In a previous study, Oshita et al. [22] reported the shortening of
T1 when xenon gas was applied to distilled water under high pressure. The authors related this result to the increase in the viscosityof water after it was subjected to the xenon gas. The change in viscosity was caused by the formation of “structured water” as a resultof the application of xenon under high pressure.
On the other hand, the opposite tendency was observed in thispresent study, with the T1 increase after Xe MNB production. This
opposite result could be explained by the difference of structures
formed when xenon gas is applied under high pressure and whenintroduced as micro- and nano-bubbles. The “structured water”
was not detected in this case.
One major possible reason for the relaxation time change in Xe
and O2 MNB solutions could be the change in apparent concentration of manganese ions. As bubble surfaces are negatively charged(see Section 3.2), some amount of manganese ions should adsorb
on the bubble surface, resulting in a lower apparent Mn2+ concentration in the solution. Since the paramagnetic material decreases
T1 only when it directly contacts with a water molecule, the lower
apparent concentration of Mn2+ of the solution induced a lower
paramagnetic effect. As a consequence, a longer T1 was observed insolutions containing MNB.
In short, it was veriﬁed that the increase in T1 with the introduction of MNB was caused by the presence of bubbles. From a different
approach, the existence of nano-bubbles in water was suggested
though NMR spectroscopy.
3.4. Discussion about the stabilization of nano-bubbles
In micro-scale, bubbles tend to shrink under the water until theycollapse, as the inner pressure increases [23]. The Young–Laplace
equation describes the relation between the pressure difference
and the radius of the bubble:
P = Pl + 2


r

(5)

in which P is the gas pressure, Pl is the liquid pressure,  is thesurface tension and r is the bubble radius.

According to Eq. (5), nano-bubbles should not exist at stablecondition under atmospheric pressure, because of the very highinternal pressure. A bubble of 100 nm radius, for example, shouldhave an internal pressure of approximately 0.15 MPa (using  =0.072785 N m−1 , [24]). At this high pressure, nano-bubbles shouldcollapse in a very short time. Ljunggren and Eriksson [25], throughcalculations based on the mass transfer of the gas at the bubble
interface to the bulk liquid, and considering the Young–Laplace
equation, found that the lifetime of a 100 nm radius gas bubbles
would be 100 ␮s.
Moreover, Matsumoto and Tanaka [26], when verifying thevalidity of Eq. (5) for nano-bubbles using molecular dynamics simulation, found out that the surface tension and the vapor pressure
were practically constant, independently of the bubble size. They
concluded that the condition for the nano-bubble to exist obeying
Eq. (5) is that the liquid phase should be under a large vacuum orhigh tensile stress. Therefore, according to this study, the nanobubbles should not be in equilibrium state under atmosphericpressure.
However, the validity of Eq. (5) for nano-bubbles is not a consensus in the literature. According to Hemmingsen [27], it may not beapplicable considering a constant surface tension of the liquid, since
the surface tension is greatly affected by the interface curvature andthe internal gas pressure at nano scale. In addition, Tolman [28]calculated theoretically that the surface tension in drops should
decrease signiﬁcantly at small sizes. That is, the surface tension
changes with the curvature at the gas–liquid interface. A lower surface tension at the nano-bubble interface would result in a lower
internal pressure, which could contribute to the stabilization of the
bubbles.
In agreement with the hypothesis of a lower internal pressure ofnano-bubbles, [29], based on molecular simulation data, concluded
that there are too few vapor atoms inside nano-bubbles, so the interior gas pressure would not be high enough to support the force
balance of a nano-bubble. The authors stated that the internal pressure should be much lower than predicted by Eq. (5), so it should not
be valid for nano-bubbles. Instead, the liquid–gas interface shouldplay an important role in the stabilization of nano-bubbles.
In this study, some evidence of the existence of nano-bubbles
was found experimentally. The particle size distribution curves
were very clear after the production of bubbles and remained stable on the following measurements. After a period, the distribution
curve became ﬂuctuated, which would indicate the disappearanceof bubbles. If those particles were not bubbles but some solid particles, the particle size distribution should not ﬂuctuate with time.
Moreover, the increase in T1 could be an indication of the presenceof bubbles, which led to the adsorption of manganese ions on theirsurface.
Therefore, there should be some mechanism that explain thestabilization of the bubbles at nano scale. One factor observed in thisstudy was the gas supersaturation condition of the water, which can
reduce the gas transfer rate from the bubble to the liquid. However, this factor should be not the only one that can explain the
nano-bubble stability, since nano-bubbles should exist even nearor after reaching the saturation equilibrium. Other contribution to
the nano-bubble should be related to the electrical charge at the
bubble surface, as indicated by the -potential measurements. The
high absolute value of -potential could avoid the bubble coalescence by the creation of repulsion forces, as discussed in Section
3.2.
The existence of nano-bubbles in saturated liquids was also
reported by Bunkin et al. [30,31], who justify the stability of the
bubbles by the adsorption of ions on their surface, which makes
Coulomb repulsion forces compensate surface tension forces. In thelater report, the authors concluded that 70–90 nm radii air bubbles
are gathered in micro-scale clusters in pure water.

Please cite this article in press as: F.Y. Ushikubo, et al., Evidence of the existence and the stability of nano-bubbles in water, Colloids Surf. A:
Physicochem. Eng. Aspects (2010), doi:10.1016/j.colsurfa.2010.03.005

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Furthermore, the negative -potential was related to the hydrogen bond network at the gas–liquid interface that has a different
structure from that of the bulk water [18]. The change in the hydrogen bond is also described by Ohgaki et al. [32], in which a “hardhydrogen bond” at the gas–liquid interface, detected by infrared
spectroscopy is reported. The change in the water structure at
the interface could be a hint to explain the stabilization of nanobubbles. This hypothesis can be related to the study of Nagayama
et al. [29] about the bubble interface, which should have some rolein keeping the nano-bubbles stable at a low internal pressure, as
discussed above.
4. Conclusion
This study conﬁrmed experimentally and with good precision
the existence of nano-bubbles in water by the particle size distribution, -potential and proton spin–lattice relaxation time (T1 )measurements. The particle size distribution measured by DLS
method indicated the presence of particles with a few hundreds
nanometers in diameter. These particles showed a good repeatability during a period after the bubble production. This period varied
from less than 1 h, in the case of air bubbles, to some days, in thecase of oxygen bubbles. The particle size distribution ﬂuctuatedafter this period and lost the repeatability. This fact is thought to be
one evidence of nano-bubbles existence and their subsequent disappearance. The stability of nano-sized bubbles was explained byboth the high dissolved gas concentration in water and the electrically charged interface of the bubbles. The latter reason was
supported by the -potential measurements, in which the highabsolute -potential obtained in O2 MNB water could have avoided
the coalescence of bubbles, while in air MNB water the absolute potential was not high enough. The presence of electrical charges
could be related to some special water structure at the interface,which promotes the stabilization of the nano-bubbles. Moreover,the change in T1 of Mn2+ solution after the introduction of oxygen
MNB could be one more evidence of the existence of electricallycharged bubbles, suggested by the adsorption of Mn2+ on the bubble surface.
Acknowledgments
The authors express their appreciation for the technical assistance given by Sysmex Co. (Japan) and for the ﬁnancial support
by the Food Nanotechnology Project of the Ministry of Agriculture,
Forestry and Fisheries of Japan and by Grant-in-Aid for Exploratory
Research (19658091). They are also grateful to the former professor Hisashi Uedaira at Hokkaido University, External Director
Toshio Ishii at Kyowa Interface Science Co., Ltd., and Prof. Kenichi
Yoshikawa at Kyoto University for the stimulating discussion about
the interpretation of the data of proton NMR relaxation time of
Mn2+ solution with and without bubbles.
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Please cite this article in press as: F.Y. Ushikubo, et al., Evidence of the existence and the stability of nano-bubbles in water, Colloids Surf. A:
Physicochem. Eng. Aspects (2010), doi:10.1016/j.colsurfa.2010.03.005