Nanobubble Characteristics and Its Application to Agriculture and Foods

S. Oshita and S. Liu

Graduate School of Agricultural and Life Sciences,

The University of Tokyo, 1-1-1, Yayoi, Bunkyo-ku,
Tokyo 113-8657, Japan

Abstract
After an overview of past researches on micro and nanobubbles (MNBs),
our research was introduced, in which the dynamic light scattering measurement
indicated the particles with a few hundred nanometers in diameter. A good repeatability
of particle size distributions corresponding to air and oxygen bubbles in water
was maintained for some periods, and then it fluctuated and disappeared. This
phenomenon suggested an evidence of nanobubbles (NBs) existence. The zeta potential
measurements showed that NBs were negatively charged in the given range of pH and
that the higher electrical charge was observed in oxygen NBs. As an application, the
germination tests of barley seeds were performed with a pair of seed groups composed
of 100 and/or 120 barley seeds each. Each group was dipped in the water containing
NBs and the distilled water, respectively, at 25°C in the dark for 20 h. The dissolved
oxygen concentration of the water containing NBs was adjusted to be the same as that
of distilled water. The germination rate of barley seeds dipped in the water containing
NBs was obviously higher than that in the distilled water.

Keywords: Nanobubble, seed germination, water absorption, zeta-potential, NMR relaxation

time

INTRODUCTION
There have been many reports on the effects and applications of MNBs, such as
purification of wastewater; water quality improvement, sterilization, decolorization and
cleaning of contaminated water and promotion of the physiological activities of living
organisms. As such, MNBs have attracted considerable attention from various fields in
recent years.
Shrinkage of microbubbles (MBs) in water can be observed through an optical
microscope. However, it is not possible to visually confirm whether the bubbles finally
disappear by dissolving in water or they remain in water as nanobubble (NBs) whose
diameters are smaller than the spatial resolution of the microscope. Therefore, whether NBs
can remain for extended periods of time has still been a point to be discussed. For example,
the lifetime of colloidal size air bubbles in water is very short. There is a calculation result
indicating that the lifetime of air bubbles with radii between 10 and 100 nm ranged from
about 1 to 100 μs (Ljunggren and Eriksson, 1997). Furthermore, there is a report that stable
NBs could exist only in liquid under highly tensile stress or large negative pressure according
to simulation results, and that NBs observed under atmospheric pressure are those in the
shrinking process or foreign substances that are mixed in (Matsumoto and Tanaka, 2008).
On the other hand, results of an experiment confirmed the physiological activity promoting
effect and sterilizing effect of water containing NBs (Takahashi, 2006). Adding to this, the
present authors confirmed experimentally and with good precision the existence of NBs
in water by the particle size distribution, zeta potential and proton spin–lattice relaxation
time (T1) measurements (Ushikubo et al., 2010). Direct observation of NBs were also
aimed by Uchida et al. (2011) and they observed oxygen NBs that formed in pure water
and compared the size distributions of NBs obtained by a transmission electron microscope

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images along with the freeze-fractured replica technique with those by the Dynamic Light
Scattering (DLS) measurements.
The above two opinions depending on theoretical and experimental observations
may seem contradictory to each other, but it is natural to think that physical properties of
water as a bulk material, such as surface tension, are affected by interactions between water
molecules and other components and foreign substances in water used in experiments,
thus differing from the physical properties of water models used in theoretical calculations
and simulations. Nonetheless, there has been no finding that can accurately explain the
discrepancy between the above two opinions. In view of this, it is important to clarify
the characteristics unique to water containing MNBs and verify the stability of NBs in
order to effectively utilize MNB technology, seek potential applications, and develop
commercialization technology.

Research State on Micro and Nanobubbles

Some researchers hold the opinion that MBs are extremely small bubbles measuring
approximately 10 to 30 μm, 50 μm or less, or less than 100 μm in diameter (Chu et al.,
2008; Matsumoto, 2008; Ohnari, 2001; Tabei et al., 2007; Takahashi, 2005; Takahashi et
al., 2007), but there is no agreed definition. At the present time, it is reasonable to consider
MBs as bubbles with a diameter ranging from several microns to about 100 μm, and NBs as
bubbles with a diameter of sub-micron order. Diameters of MBs were reported in a number
of papers, such as those describing the evaluation of bubble images obtained with a high-
resolution camera (Fujikawa et al., 2003), measurement of diameter distribution based on
the change in light transmitted through rising MBs (Tabei et al., 2007), and evaluation of
the diameter distribution using a light scattering method (Kukizaki and Goto, 2006). The
remarkable features of MNBs are as follows (Serizawa et al., 2003; Ohnari, 2005; Takahashi
et al., 2003). (1) The specific surface area is larger than that of ordinary bubbles with the
same volume, thus significantly promoting chemical reaction, physical adsorption and
mass transport in the gas-liquid interface. (2) The rising velocity or slip velocity in liquid
is low, thus easily resulting in a homogeneous reaction field. (3) They have various superb
physiological activity promoting effects. (4) The collapse of MBs results in the generation of
a shock wave. (5) They have acoustic-dynamic characteristics such as ultrasonic scattering
characteristic and volume oscillation with a specific frequency of vibration. (6) The repeated
application of vibration results in the formation of a high-temperature, high-pressure field
and the generation of OH radicals. (7) The dynamic boundary condition at the gas-liquid
interface becomes heterogeneous because hydrophobic organic substances are adsorbed
toward the gas side and hydrophilic organic substances are adsorbed toward the water side.
(8) They are relatively uniform in size. (9) They promote dissolution of gases. (10) The
bubble surface has positive/negative potential.
With regard to the separation and purification effect of MBs, Goto et al. (2006)
described the purification of oil-contaminated soil using MBs in their reports. The purifying
effect of MBs is attributed to the entrainment levitation effect. When MBs adhere on the
surfaces of contaminated soil particles, they coalesce with other air bubbles and grow in
size, adsorbing oil film to their surfaces. When the coalesced air bubbles grow in size and
their buoyant force exceeds the adhesive force, air bubbles detach from the surfaces of
soil particles and rise upward. There is another report on the floatation in which air MBs
were applied to a suspension of fine carbon particles (Terasaka and Shinpo, 2007). This
floatation utilizes the surface characteristics of particles. If the surfaces of suspended
particles in the liquid are hydrophobic, the particles adhere to the surfaces of air bubbles
and rise upward. When they are hydrophilic, the particles remain under water, and then
settle to the bottom of the liquid if their specific gravity is large. The floatation of kaolin
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using air MBs was reported by Li and Tsuge (2006). This report pointed out that not only
the surface characteristics (zeta potential) of both bubbles and particles to be removed but
bubble concentration is also important parameters.
In connection with the use of MBs for a cleaning purpose, Li et al. (2009) reported
that the collapsing air MBs led to the decomposition of phenol which is a common pollutant
in wastewater. According to this report, the phenol decomposition is dependent on pH of the
solution and the type of gas within the MBs (air, oxygen, nitrogen). There is also a report
that ozone MBs are effective for decolorization, decomposition of organic materials and
removal of chemical oxygen demand (COD) (Chu et al., 2008) in the treatment of textile
wastewater. Liu et al. (2010) reported that coagulation micro-bubble flotation significantly
reduced coagulant doses and enhanced the pretreatment rate. Because the micro-bubble
generator produced fog-like micro-bubbles with diameters in tens of micrometer which
showed longer preservation time in the water along with a higher oxygen transfer rate.
In addition, the sterilizing effect of ozone MBs was reported by Tsuge et al.
(2009). These authors examined the sterilizing effect of ozone MBs against Bacillus subtilis
spores under various operational conditions, and confirmed that ozone would provide a
higher sterilizing effect when in the form of MBs. Tsuge et al. (2009) suggested that the
high sterilizing effect was attributed to hydroxyl free radicals generated by active self-
decomposition of ozone.
Many papers have presented case studies of the effect of MNBs on the physiological
activity of living organisms, such as promoting the growth of oysters and providing
sterilization, improving the pearl-layer-forming ability of pearl oysters (Ohnari, 2001), and
prolonging the flowering period of the Phalaenopsis orchid (Takahashi, 2006). In recent
years, the application of air MBs to nutriculture has been studied. In a lettuce nutriculture
experiment, the introduction of air MBs helped lettuce grow 2.1 times larger in fresh
weight and 1.7 times in dry matter weight (Park and Kurata, 2009). In a cell culture, the
introduction of air MBs resulted in a five-fold increase of the mass-transfer coefficient
(oxygen transfer capacity coefficient) of the broth (culture solution) of a filamentous fungus
(Trichoderma reesei, which is known as a high cellulase-producing strain), improving the cell
productivity by 1.8 times (Weber and Agblevor, 2005). Recently, the present authors reported
the promotive effect of NBs on germination rate of barley seeds (Liu et al., 2011; 2013).
Although some reports attempted to describe why physiological activity was promoted,
many have simply presented case studies without elaborating on the mechanisms. Thus,
there is a need to clarify these mechanisms.

Evidence of NBs Through Size Distribution and Zeta Potential

The present authors generated bubbles by introducing air and oxygen gas into
ultrapure water by using a MB generator (OM4-GP-040, Aura Tec Co. Ltd., Japan), then
observed the approximate size of the bubbles displayed as laser light scattered images.
The zeta potential of bubbles was also measured as well as the residence time (Ushikubo
et al., 2010).
The size distribution of oxygen bubbles generated, which were measured by
using the DLS technique (Zetasizer Nano ZS particle size analyzer: ZEN3500, Sysmex
Co., Japan), showed stable distribution of NBs immediately after the generation of bubbles
as shown in Fig. 1a. The measurements were performed at 20°C. For each sample, 5-10
replications were done. The geometric mean of the bubble diameter was 137 nm (coefficient
of variation (CV) = 61.2%). This geometric mean diameter oscillated slightly in the
next few hours until the following day (Fig. 1b), reaching values up to 272 nm. Three
days after the generation of NBs, the geometric mean of the bubble diameter of NB was
380 nm (CV = 107.4%) but there was a decrease in reproducibility (Fig. 1c), which was
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suspected to have resulted from a low bubble density. The larger geometric mean diameter
does not necessarily indicate that the bubbles increased in size or coalesced. The reason
is that the DLS method is more sensitive to big particles than small ones, so a relatively
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 cannot
be detected, while larger particles are still sensed. The low concentration of particles on the
third day was estimated by the poor repeatability of the results shown by the error bars in
Fig. 1c in comparison to the measurements on the previous days (Fig. 1a and 1b). Six days
later, the distribution became irregular and reproducibility was lost (Fig. 1d). The decreased
bubble density is believed to be one of the causes of the loss of reproducibility of bubble
size distribution, and this in itself suggests that the bubbles captured by the dynamic light
scattering method were oxygen NBs.
The concentration of dissolved oxygen is also connected with the bubble size
distribution. The dissolved oxygen (DO) concentration (36.9 mg·L-1) immediately after the
generation of bubbles indicated an extremely high level of super saturation, but it decreased
with time. Six days later, the DO concentration decreased to a saturation level (8.9 mg·L-1).
Since the state of super saturation indicates a small concentration gradient at the interfacial
boundary of bubbles and water, it is believed to contribute to the stability of NBs.
As to air NB, the repeatability of data was not as good as that obtained for oxygen
NBs water and the shapes of the distribution curves were irregular. The poor repeatability
and the irregular distribution curves of air NBs water indicated that the concentration
of particles should be under the minimum detection level to obtain more accurate data.
The results suggested the low stability of air NBs compared to the oxygen NBs, since
the concentration of air NBs should be much lower than that of oxygen NBs. The big
difference was observed for the residence time, in which bubbles could be stable, between
the oxygen NBs (3 days) and the air NBs (less than 1 hour). This should be related to the
lower dissolution of the air in water comparing to the dissolution of oxygen.
The zeta potential, on the other hand, was in the range from -17 to -20 mV
(pH = 5.7 to 6.2) in the case of air NBs, and in the range from -34 to -45 mV (pH = 6.2
to 6.4) in the case of oxygen NBs. As such, the zeta potential varied depending on the
kind of gas introduced (see Fig. 2). The zeta potential of both air and oxygen NBs was
negative in the above pH range. The negative value is explained by Kelsall et al. (1996) as
attributed to the predominance of hydroxide ions in the first molecular layers of water at the
gas-liquid interface. It is also described by Najafi et al. (2007) that the negative charge on the
bubble surface is believed to be due to preferential adsorption of hydroxyl ions (OH-). It is
also described that as the enthalpy of hydration of hydrogen ion (H+) and OH- is -1104 and
-446.8 kJ·mol-1, respectively, H+ preferentially remain in the bulk aqueous phase, leaving
space at the gas-water interface for OH-. Similar understanding is that an increase in OH-
concentration near the bubble surface suppresses the dissolution of gas from bubbles into
water and serves as “shells” for the bubbles, thus improving stability (Takahashi, 2005).
Apart from the zeta potential, an explanation for the NBs stability is reported as the interface
of NBs consists of hard hydrogen bonds that are similar to the hydrogen bonds found in
ice and gas hydrates (Ohgaki et al., 2010).
According to Spanos et al. (2002), the absolute zeta potential value normally taken
as a minimum to observe a potential stability is 30 mV. At a high absolute zeta potential, the
electrical charged particles tend to repel each other, avoiding aggregation of particles in a
colloidal dispersion. In the case of NB dispersion, the high absolute values of zeta potential
could create repulsion forces that would avoid the coalescence of NBs and contribute to
the stabilization of the NBs. Therefore, in our study, the oxygen NBs could be stabilized in
water by the electrical charged surfaces, while the air NBs would be below the limit of the
26 Proceedings of AFHW 2013
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August 5-8, 2013, Golden Tulip Sovereign Hotel, Bangkok, Thailand
stabilization by repulsion forces leading to a short residence time. Although the absolute
values were different between NBs of air and oxygen, each of those did not vary so much
irrespective to the stabilization limit of zeta potential until when NBs could not be observed
due to reaching the lower bubble concentration than particle detection level (Fig. 2). This
experimental observation supported also the NB dispersion indicating each NBs’ existence
independent from others.
In conclusion, the higher initial concentration of dissolved gas in water could
explain the extension of the NB stability because a higher dissolved gas concentration is
expected to suppress the dissolution of gas from NB into water. However, only this cannot
explain the extension of the NB stability. One of other factors could be found in zeta
potential indicating the negative charge on the bubble surface within the observed range
of pH because of preferential adsorption of OH- on bubble surface. It is not still clear if the
increase in OH- could serve to form “shells” suppressing the dissolution of gas into water
or hard hydrogen bonds could be formed at the gas-water interface, further understanding
of zeta potential can contribute to clarify the stabilization of NBs besides the high gas
concentration in water.

Effect of Nanobubbles on Seed Germination

Seed germination is a crucial process in the seed-plant life cycle and is also
important for plant establishment in natural and agricultural ecosystems (Weitbrecht
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 for subsequent seeding growth (Nonogaki
et al., 2010). Therefore, germination test is an appropriate method to verify the ability
of water containing NBs to promote physiological processes. Comparison experiments
were performed using distilled water and water containing NBs produced from each
batch of distilled water (Liu et al., 2012). The DO concentration of distilled water was
approximately 9 mg·L-1. 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 was calculated on the basis of the following formula:

Germination rate = (The number of germinated seeds)/(the total number of seeds)×100%

As shown in Fig. 3, six repetitive germination experiments showed that the

germination rates of barley seeds dipped in water containing NBs were 15-25 percentage
points greater than that 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
mobility of the bulk and hydration water fractions (Vashisth and Nagarajan, 2010). The
proton-nuclear magnetic resonance relaxation time can be used to detect weak molecular
interactions such as hydrogen bonding, molecular mobility and steric effects (Balci,
2005). Thus, it is widely used to study the mobility and diffusion of the water molecules 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 be significantly greater than those of seeds dipped in distilled water (Liu et al., 2012).
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, the authors of this study
showed that the water status of seeds dipped in water containing NBs and in distilled water

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during germination was determined via NMR spectroscopy. The T2 of barley seeds was
composed of two components: T2a (long) and T2b (short). As shown in Fig. 4, two repetitive
experiments showed that the T2 values of germinated seeds dipped in water containing NBs
were significantly greater than those dipped in distilled water (p<0.05).

CONCLUSIONS
This article presented an overview of past research on MNBs, and then introduced
our research conducted mainly for the purpose of verifying the existence of NBs and
applicability to promotion of metabolic activity. The particle size distribution measured by
DLS method indicated the presence of particles with a few hundred nanometers in diameter.
It showed a good repeatability during a period after the bubble generation. The particle size
distribution fluctuated after a certain period and lost the repeatability. This fact is thought
to be an evidence of NBs existence and their subsequent disappearance. The zeta potential
measurements showed that the NBs water is negatively charged in the observed range of
pH and that the oxygen NBs presented higher electrical charge than the air NBs.
The germination rates of barley seed dipped in the water containing NBs were
15-25 percentage points higher than those in the distilled water which verified the clear
effect of NBs on physiological activity. One possible reason is that NBs could increase the
mobility of the water molecules in bulk. The other explanation is that the negative charged
NBs might influence the bioelectric field of plants, which had intimate relationship with
the elongation growth of plants.
Once a full understanding NBs’ effect on promotion of plant growth is achieved, the
manipulation of NBs will provide an efficient and cost-effective approach for the cultivation
of hydroponics vegetables and develop a new technology in agricultural applications.

ACKNOWLEDGEMENTS
The authors express their appreciation for the technical assistance given by both
Sysmex Co. (Japan) and Ms. A. Irie (Quantum Design Japan, Inc.), and the financial support
provided partly by Food Nanotechnology Project, the Ministry of Agriculture, Forestry
and Fisheries of Japan (19658091) and by the Japan Society for the Promotion of Science
(JSPS) Grant No. 25660202.

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Figures

Fig. 1. 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) 6
days later (DO=8.9 mg�L−1). The vertical bars represent the standard deviation of
the replication data.

Fig. 2. Average values of zeta potential measurements in O2 MNB water and air MNB water
with time (n=50). The vertical bars show the standard deviation of the measurements.

Fig. 3. Comparison of barley seed germination rate between the water containing NBs
and the distilled water under the same DO concentration (The average values of
germination rates in the water containing NBs and the distilled water were 77.9 and
59.2%, respectively).

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Fig. 4. T2 of germinated seeds dipped in the water containing NBs and distilled water (T2a
and T2b are the long and the short components of T2; The bars showed the standard
error of five replicates).

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