1.Introducion

The use of micro and nano-bubbles (MNB) in water was reported to be effective for the acceleration of metabolism in shellfishes and vegetables [1, 2], accelerating the growth and increasing the yield of the products. This effectiveness, however, cannot be explained only by the increase of the dissolved oxygen (DO) concentration. Park and Kurata [2], using MNB in the solution for the hydroponic cultivation of lettuce, observed acceleration in growth in comparison with lettuce grown in solution containing similar DO concentration but without MNB. Therefore, the MNB should play an important role in the physiological activity of living organisms.

There are still many questions concerning the nano-bubbles that are not clear. Some of them are the stability and size of nano-bubbles after the introduction of MNB in water.

The present study aimed to investigate the state of water during and after the MNB generation and, more specifically, to study the existence and stability of bubbles in nano-scale in water after the introduction of MNB.

2.Micro and Nano-bubble Generation

Micro and nano-bubbles were produced in 2 liters of ultrapure water, obtained by a water purification system (Direct-Q, Nihon Millipore Ltd., Japan). The gas used to produce micro and nano-bubbles was pure oxygen.

A Micro-bubble Generator (OM4-GP-040, Aura Tec Co. Ltd., Japan) was used for the production of micro and nano-bubbles. The water and the oxygen were introduced in the inlet part of a magnetic gear pump (MDG-R2RVA100, Iwaki Co. Ltd., Japan) and subjected to a high pressure (0.25 to 0.27 MPa) in a pressurized tank. Flowing through the ejector nozzle in the outlet, the gas-supersaturated water was depressurized, leading to the nucleation of the bubbles, which were dispersed in the water. The water was circulated in this system for 40 minutes at 20°C. In this study, the water obtained after this procedure is referred to as "MNB water".

3.ζ-Potential

3.1.Materials and Methods

ζ-potential measurements of the oxygen MNB water was performed using a Zeta Potential Analyzer (Zeecom, Microtech Co. Ltd., Japan). This system detects the electrophoretic mobility of particles in the range of 20nm to 100 μm. The ζ-potential was calculated using the Smoluchowski equation.

3.2.Results

The ζ-potential for the oxygen MNB water just after the bubble generation was in the range from -44 to -40 mV. With time, the values decreased in absolute value (-38~-33 mV). The pH was practically constant at pH 6.2~6.4. This result was similar to the data obtained by Takahashi [3], which were around -35 mV in distilled water with air micro-bubbles at pH 5.8.

The negative value of ζ-potential indicates the polarization of water molecules in the water, as no ions were added in this system. The high values of ζ-potential indicate the stability of particles in the liquid phase, as they will repel each other. This could be a hint to understand the possible stability of nano-bubbles.

4.Particle Size Distribution

4.1.Materials and Methods

Zetasizer Nano ZS particle size analyzer (ZEN3500, Sysmex Co., Japan), green badge (532nm laser), was used to detect particles in MNB water after the bubble generation. This analyzer detects Brownian motion of particles from 0.6nm to 6 μm through Dynamic Light Scattering method. The measurements were performed at 20°C and 5 to 10 replications were done.

4.2.Results

The particle size distribution of the oxygen MNB water just after the bubble generation showed a geometric mean diameter of 137nm and a coefficient of variation of 61.2% (Fig 1(a)). The stability of the particle size distribution was shown by the regular shape of a mono-modal lognormal curve and good repeatability of the measurements. After three days of observation, larger particles with geometric mean diameter of 380nm were detected and the dispersion of the distribution size was higher, with a coefficient of variation of 107.4%.

Finally, on the sixth day after the bubble generation, the particle size distribution curves were no longer mono-modal, with no repeatability and the dispersion of the distribution became very high (181.2%) as shown in Fig 1(b). This result indicated an insufficient concentration of particles to be detected by the instrument. The decrease in the particles concentration suggests the disappearance of nano-bubbles due to the dissolution of oxygen into the water.

5.Proton NMR Spin-lattice Relaxation Time

5.1.Materials and Methods

Five replications of each sample (control and MNB) were collected in tightly closed 10mm diameter NMR tubes. Proton spin-lattice relaxation time (T1) was measured in a pulsed spectrometer (JNM-MU25A, JEOL, Japan) at 25 MHz frequency and at constant temperature of 20°C, using the saturation recovery pulse sequence.

The experiment consisted in using manganese solution as control. Solutions of 3, 5, 10, 15, 40mM of manganese (II) chloride tetrahydrate (Mn2Cl・4H2O, Kanto Chemical Co. Inc., Japan) were prepared (control sample), then oxygen MNB were generated in this solution (O2 MNB solution). A more detailed analysis was done at 10mM manganese solution, diffusing oxygen through a gas diffuser, which did not contain MNB (O2 without MNB solution).

5.2.Results

It was verified that T1 increased (p<0.05) after the introduction of oxygen as MNB in all the manganese concentrations tested. The DO concentration increased from about 7.7~9.4mg・L-1 in control sample to 35.8~39.6mg・L-1 in oxygen MNB solution.

One important point is that the increase in T1 was not caused by the increase in DO concentration. The O2 without MNB solution at 10mM Mn2+ concentration had similar DO concentration as the O2 MNB solution: 37.1mg・L-1 and 38.8mg・L-1, respectively. However, the T1 of the 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.

The major reason for the relaxation time change in oxygen MNB solutions was the change in apparent concentration of manganese ions. As bubble surfaces are negatively charged, as indicated by the ζ-potential measurements, 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 in direct contact with the water molecule, the lower apparent concentration of Mn2+ of the solution induced a lower paramagnetic effect. As a consequence, a longer T1 was observed in solutions containing MNB. This fact confirms the stability of nano-bubbles in the solution after the generation of oxygen MNB.

6.Conclusion

Using different approaches, the existence of stable nano-bubbles in water was strongly suggested. The particle size distribution indicated the presence of particles with a few hundreds nanometers in diameter during some days. Moreover, the change in T1 of Mn2+ solution after the introduction of oxygen MNB suggested the presence of nano-bubbles by the adsorption of Mn2+ on the bubble surface. The stability of nano-sized bubbles could be explained by both the highly dissolved gas concentration in water and the electrically charged interface of the bubbles, supported by the ζ-potential measurements.

Fig 1 Average particle size distribution (n=10) of the oxygen MNB water (a) just after stop the bubble production and (b) after 6 days. The vertical bars represent the standard deviation at each measurement point.

マイクロ・ナノバブル含有水は、廃水の浄化、湖沼などの閉鎖性水域の水質汚染の改善、殺菌、脱色、洗浄などの応用や生物の生理活性の促進効果などの事例が報告され、近年になって、様々な分野において注目を集めている。しかし、マイクロ・ナノバブル生成後の水中におけるナノバブルの安定性やサイズなど、多くの不明な点が残されたままである。そこで、本論文では、マイクロ・ナノバブルおよびそれら含有水の特性、中でもナノスケールのバブルの存在と安定性の解明を目的とした。

第1章では、マイクロバブル・ナノバブルの定義と特性および応用事例について概観し、問題点を指摘すると共にナノバブルの存在や水中での安定性を検証することの意味を指摘した。

第2章ではマイクロ・ナノバブル発生装置と共に水および温度条件を明確にし、第3章では、酸素マイクロ・ナノバブル含有水の特性を顕微鏡観察、溶存酸素(DO)濃度、pH、ゼータ電位を測定することにより明らかにした。顕微鏡観察では、直径がおよそ100μm程度のバブルは数時間の単位でバブルの状態を維持するが突然崩壊するバブルもあること、50μm以下のマイクロバブルは収縮して視認できなくなる過程を捉えた。また、DO濃度は、35mg・L-1から45mg・L-1という過飽和の状態まで上昇し、pHは6.0~6.5の範囲で安定していることを示した。一方、バブル生成直後のpH 6.2~6.4の水中のナノレベルの酸素バブルのゼータ電位は-44~-40mVであり、時間の経過により-38~-33 mVに変化することが示された。この結果、バブルが負のゼータ電位を有すことからバブル粒子は互いに反発し合い、ゼータ電位の絶対値が高いことが、水中におけるバブル粒子の安定性に寄与していることを指摘した。

第4章では、動的光散乱法によるバブル粒子のサイズ分布について検討した。バブル生成直後の酸素バブルの粒子サイズ分布は、幾何平均のピークを137nmに有する典型的な単峰性の対数正規曲線であり、このことから、粒子サイズ分布が安定していることが示された。生成から3日後にも粒子が観測されたが、その幾何平均は380nm以上に増大した。6日後には、粒子サイズ分布は単峰性を示さず、各粒子径における分散が増大した。これは、バブルから水へ酸素が溶解することによるナノバブルの消失を意味すると考えられた。

第5章では、NMRを用いたプロトン縦緩和時間(T1)よりバブル含有水の特性について検討した。10mMの塩化マンガン(II)四水和物(Mn2Cl・4H2O, Kanto Chemical Co. Inc., Japan)溶液に酸素マイクロ・ナノバブルを生成させた結果、マイクロ・ナノバブル生成によりT1が増大することが確認された(p<0.05)。これは、第3章で示したように、負のゼータ電位を持つバブル表面にマンガンイオンが吸着し、溶液中の見かけのマンガンイオン濃度が低下したためであると推察された。この事実は、酸素マイクロ・ナノバブル生成後も、水溶液中にナノバブルが安定して存在できる理由の1つであると考えられる。

以上、本論文は、様々な手法を用いて異なる視点から計測を行うことにより、マイクロ・ナノバブルおよびマイクロ・ナノバブル含有水の特性を明らかにする中で、ナノバブルが水中で安定に存在することを示したものであり、学術上・応用上貢献することが少なくないと考えられる。よって審査委員一同は、本論文が博士(農学)の学位論文として価値あるものと認めた。