Journal of Environmental Biotechnology
Vol. 15, No. 1, 41–44, 2015

Technical Note

A Simple Method for Nanobubble Generation and Stability of the Bubbles
Masayuki Ohmori1,2*, Kasumi Haruta2, Shinji Kamimura1, Hiroyuki Koike1, Tsutomu Uchida3
and Haruko Takeyama2
Department of Biological Sciences, Chuo Univ., Kasuga 1–13–27, Bunkyo-ku, Tokyo 112–8551, Japan
Institute for Nanoscience and Nanotechnology, Waseda Univ., Wakamatsu-cho 2–2, Shinjuku-ku, Tokyo 162–8480, Japan3
Division of Applied Physics, Graduate School of Engineering, Hokkaido Univ., N13W8 Kita-ku, Sapporo 060–8628, Japan12* E-mail: ohmori@bio.chuo-u.ac.jp(Received; 29 June, 2015/Accepted; 10 August, 2015)
Key words: Nanobubble, Stability of nanobubble

1. Introduction
It has been reported that ultra-small gas bubbles inliquid with diameters of micron and submicron-order, so
called micro-nanobubbles, have some characteristic physicaland chemical properties. They show slow buoyancy, negative surface charges 19,22), free radical formation 12,20) andincreased water molecule mobility 14,22). These properties ofmicro-nanobubbles might cause miraculous effects on growthof plants 4,18) and fishes 4,17). Application of these tiny bubbles
for many fields of technology attracts great attention 1). In
the field of environmental biotechnology, miro-nanobubbles
is applied for cleaning solid surface 13,23) and for waste water
treatment, for example, degradation of noxious chemicals
such as phenol 11). Further, it is expecting to use micronanobubbles in medical field such as drug delivery 6) andtumor destruction 15). The use of ozone nanobubble water as
an adjunct to periodontal treatment is proposed 5).
However, basic study of micro-nanobubbles, particularly
nanobubbles, remains still in the beginning. It has long been
believed that nanobubbles cannot exist for a long period,
since a high internal gas pressure of the bubbles drivesgas diffusion across the interface resulting instantaneous
dissolution of bubbles 2). No fundamental research on themechanism of biological effect of nanobubbles has beenmade. To conduct a laboratory experiment, we need to get
nanobubbles that are stable in size and exist for long periods.
Generation of micro-nanobubbles in water are mainly
performed by two methods that are based on decompression
and gas-water circulation 1). Ultrasonic waves are also used togenerate microbubbles through the cavitation of water 9,15). A
small-porous-glass membrane has been reported as a usefuldevice for nanobubble formation 10). Anyhow, a sophisticate
device has been used to generate micro-nanobubbles, andbubble size and quantity were variable among each instrument.
In the present experiment, we have succeeded to generate
air-nanobubbles easily using a household hand mixer. The
size and stability of the air-nanobubbles were determinedusing a dark field microscope, an electron microscope, anda nanoparticle analyzer. It was concluded that most of thenanobubbles generated were smaller than 100 nm in diameterand existed for over one month. In the present report we use
the term, “gas nanoparticle (GNP)”, instead of nanobubble.

2. Materials and methods
Pure water was prepared by Direct Q-UV (Millipore S.A.S,Molsheim, France) and the conductivity of water was higher
than 18.5 MΩ ⋅ cm. The screw of the hand mixer (Braun
MR 400plus, Braun Co. Germany) was dipped into 100 mL
water in a 300 mL-beaker and the water was swirled for 5 to
30 min on ice. Since a swirling period of 30 min gave about
ten times higher concentration of GNPs than that of 5 min,we adopted 30 min swirling to generate GNPs regularly. To
prevent overheating of the motor, one swirling was shorter
than 5 min, so that 6 motors were used for 30 min swirling.
The GNPs were observed with a dark field microscope (IX71-TIR, Olympus Co. Japan) equipped with
epi-illumination device and high speed ECD camera. TheGNPs were also observed by electron microscopy as reported
previoously 21). A field-emission gun-type TEM (JEOL Ltd.,Tokyo, Japan, JEM-2010) was used to observe the replica
film at a 200-kV acceleration voltage.
Numbers of the GNPs were measured by direct counting at
800×magnification. The polystyrene beads with diameters of
50 nm, 100 nm and 200 nm (Polysciences Inc., PA, U.S.A.)
were used as standards for size and number. The size and zeta
potential were measured by a nanoparticle analyzer based ondynamic light scattering (Zetasizer Nano, Malvern/SysmexCo., England). Generally, this type of particle analyzer cannot
measure the quantity of particles but shows size distribution in percentage only. Thus, we added a known amountof internal standard to the sample and roughly estimatedthe amount of GNP in the sample. Thus obtained values
were well agreed with those using dark field microscopy andelectron microscopy.

Ohmori et al.

423. Results and discussion
No nanoparticle was detected in pure water when analyzing
with a dark field microscope or nanoparticle analyzer,
suggesting that contamination of other particles from air isnegligible. When pure water was swirled for 30 min, GNP
was generated with many different diameters, namely, 40
to 500 nm. The pattern of size distribution was variable ineach experiment. Fig. 1A shows a typical pattern of the size
distribution of GNP in pure water. As for quantity of GNP,
several GNPs were observed by dark field microscopy at amagnification of 800. The numbers of GNPs were estimatedroughly to be less than 3×108 mL–1. The addition of glycerol
to pure water at 1% before swirling did not increase thenumbers of GNP.
When we added ethanol to pure water at 1% and swirled
for 30 min, the GNP numbers increased to several times
comparing those of pure water (Data not shown). The addition of ethanol at 2% slightly enhanced GNP generation
than that at 1% ethanol. However, the addition of ethanol
at 3% decreased the numbers of GNP to less than 70%
comparing with those at 1% ethanol. The addition of ethanol
to pure water at 10% did not enhance GNP generation.
Since we are attempting to apply GNP for bacterial growth
regulation, and 2% ethanol is toxic enough for the growth ofordinal microorganisms, we adopted ethanol concentrationof 1% to generate GNPs.
Fig. 1B shows the size distribution pattern of GNPs in 1%ethanol, in which a sharp peak appears at about 40 nm diam-Fig. 1. Size distribution of GNPs
GNPs were generated in pure water (A) or in 1% ethanol (B).
Size of GNPs was measured using Zetasizer Nano. Vertical in
the figure shows relative numbers of GNP (%) of each size tototal GNP numbers.

eter. On rare occasions, a very small gentle peak appears at
about 160 nm diameter. No GNP or contaminated particle,
larger than 500 nm diameter, was observed. In the presenceof 1% ethanol, the peaks in diameter were fixed around
40 nm for over one month (Data not shown). It is concluded
that the size of GNPs was stabilized in a diameter smaller
than 100 nm by the addition of ethanol at 1%. The lengthof swirling period did not affect stability of the size of GNPs,
namely, GNP concentration would not affect its stability.
Fig. 2 shows the change in numbers of GNP in the presenceof 1% ethanol when the GNP water was stored in refrigerator
for about 2 months. It is shown that the numbers of GNP (1to 2×109 per mL) were kept unchanged for over two months;
that is, once formed GNP in small sizes were present in water
stably so long periods as reported previously 16). It has beenreported that the life time of bubbles with mean bubble radiiof less than 1 μm becomes over one year when amphiphilicmolecules crystallize on the air-liquid interface 3).
Microbubbles have been reported to be negatively charged
with a zeta potential about –30 mV 19), and became morenegative with increasing pH 9). It has also been reported thatnanobubbles formed in α-cyclodextrin aqueous solution are
unstable in a higher ionic strength 7). We determined zeta
potential of the GNPs and the effect of NaCl on zeta potential.
The pH of the GNP water was about 6.5. In the absence ofNaCl, the average zeta potential was –32.9 ± 3.0 mV. When
NaCl was added at concentrations of 0.002% and 0.5%,
the zeta potential became –16.0 ± 1.6 and –5.8 ± 0.3 mV,
respectively. The addition of NaCl seemed to cancel out thenegative charge of GNP. It is speculated that Na+ might
be attracted to the negatively charged surface of GNP, andwhich might cancel out the negative charge of the GNP.
Fig. 3 shows the effect of NaCl on the size of GNP. The
addition of NaCl up to 0.3% did not significantly affect thesize of GNPs; that is, the diameters were kept smaller than
100 nm. However, the diameter increased to 190 nm in 1%NaCl, and to 600 nm in 5% NaCl. These results were wellagreed with the observation using a dark field microscope,where the GNPs disappeared by adding 5% NaCl. It isconsidered that 10 times increase in diameter results in 1,000Fig. 2. Durability of GNPs
GNPs in 1% ethanol were stored at 4°C. Numbers of GNPs
were measured using Zetasizer Nano.

Nanobubble generation

434. Conclusion
We could generate very small air-nanobubbles, having
diameters smaller than 100 nm, using a household handmixer. The bubbles were stable in water containing 1%
ethanol more than one month and show zeta potential of–30 to –40 mV. The addition of NaCl to nonobubble-water
caused changes in the diameter and the number of bubbles;
larger in size and lesser in numbers.

Acknowledgements
The authors thank Dr. K. Sou, Waseda University, for kindoffer to use Zetasizer Nano and his useful discussion. This
work was supported by a grant-in-aid for basic science (C)24620009 from the Ministry of Education, Culture, Sports,Science, and Technology to M.O.

References

Fig. 3. Effect of NaCl on the size of GNPs
(A): without the addition of NaCl. (B): NaCl was added at1%. (C): NaCl was added at 5%. Size of GNPs was measured
using Zetasizer Nano. Vertical in the figure shows relative
numbers of GNP(%) of each size to total GNP numbers.

times increase in the volume of GNPs; that means, 1,000
small GNPs disappears when one big GNP with ten times
larger diameter formed. Under the circumstances, we canunlikely detect GNPs in the field of microscope (800×). The
decrease in negative zeta potential might result in coalescenceof GNPs, which make the size of GNPs larger 8). Considering
these matters, to produce small GNPs in sea water will bevery difficult.
Why 40 nm size GNP was predominant in 1% ethanol, is avery difficult question. It has been proposed that the additionof a small amount of propanol affect the hydrogen-bonding
network at the gas-water interface 19). A certain thermodynamic situation may determine the size and quantity ofGNPs. Other physicochemical properties should be revealed
to know real nature of GNPs, and to use GNPs more effectively in biotechnology. Precise estimation of GNP numbers
is still a matter of problem. We need a reliable standard assay
technique for GNP counting. We are now going to elucidate
the mechanism of the effect of GNP on biological processes
using the water rich in GNPs.

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