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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 fuctuated 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

purifcation 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 felds in

recent years. Shrinkage of microbubbles (MBs) in water can be observed through an optical

microscope. However, it is not possible to visually confrm whether the bubbles fnally

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 confrmed the physiological activity promoting

effect and sterilizing effect of water containing NBs (Takahashi, 2006). Adding to this, the

present authors confrmed experimentally and with good precision the existence of NBs

in water by the particle size distribution, zeta potential and proton spin–lattice relaxation time (T 1 ) 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 fnding 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 defnition. 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 specifc surface area is larger than that of ordinary bubbles with the same volume, thus signifcantly 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 feld. (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 specifc frequency of vibration. (6) The repeated application of vibration results in the formation of a high-temperature, high-pressure feld

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 purifcation effect of MBs, Goto et al. (2006) described the purifcation 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 flm 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 foatation in which air MBs

were applied to a suspension of fne carbon particles (Terasaka and Shinpo, 2007). This foatation 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 specifc gravity is large. The foatation 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 fotation signifcantly

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 confrmed 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 fowering 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 fve-fold increase of the mass-transfer coeffcient (oxygen transfer capacity coeffcient) of the broth (culture solution) of a flamentous 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 (coeffcient 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 frst molecular layers of water at the gas-liquid interface. It is also described by Najaf 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

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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 felds. Recently, both spin–lattice relaxation time (T 1 ) and spin-spin relaxation time (T 2 ) of germinated seeds dipped in water containing NBs were reported to be signifcantly greater than those of seeds dipped in distilled water (Liu et al., 2012).

Moreover, Ishibashi et al. (2005) have reported that T 2 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 T 2 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 T 2 of barley seeds was composed of two components: T 2a (long) and T 2b (short). As shown in Fig. 4, two repetitive experiments showed that the T 2 values of germinated seeds dipped in water containing NBs were signifcantly 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 fuctuated 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 verifed 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 infuence the bioelectric feld 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 effcient 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 fnancial 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

Figures Fig. 1. Particle size distribution (average values, n=10) of oxygen MNB water (a) just after

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.

Figures Fig. 1. Particle size distribution (average values, n=10) of oxygen MNB water (a) just after

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

Figures Fig. 1. Particle size distribution (average values, n=10) of oxygen MNB water (a) just after

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. T of germinated seeds dipped in the water containing NBs and distilled water (

Fig. 4. T 2 of germinated seeds dipped in the water containing NBs and distilled water (T 2a and T 2b are the long and the short components of T 2 ; The bars showed the standard error of fve replicates).

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