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Oxygen and Air Nanobubble Water Solution Promote the
Growth of Plants, Fishes, and Mice
Kosuke Ebina
1
*, Kenrin Shi
1
, Makoto Hirao
2
, Jun Hashimoto
3
, Yoshitaka Kawato
1
, Shoichi Kaneshiro
1
,
Tokimitsu Morimoto
1
, Kota Koizumi
1
, Hideki Yoshikawa
1
1 Department of Orthopaedic Surgery, Graduate School of Medicine, Osaka University, Suita, Osaka, Japan, 2 Department of Orthopaedic Surgery, National Hospital
Organization, Osaka Minami Medical Center, Kawachinagano, Osaka, Japan, 3 Department of Immunology, National Hospital Organization, Osaka Minami Medical Center,
Kawachinagano, Osaka, Japan
Abstract
Nanobubbles (,200 nm in diameter) have several unique properties such as long lifetime in liquid owing to its negatively
charged surface, and its high gas solubility into the liquid owing to its high internal pressure. They are used in variety of
fields including diagnostic aids and drug delivery, while there are no reports assessing their effects on the growth of lives.
Nanobubbles of air or oxygen gas were generated using a nanobubble aerator (BUVITAS; Ligaric Company Limited, Osaka,
Japan). Brassica campestris were cultured hydroponically for 4 weeks within air-nanobubble water or within normal water.
Sweetfish (for 3 weeks) and rainbow trout (for 6 weeks) were kept either within air-nanobubble water or within normal
water. Finally, 5 week-old male DBA1/J mice were bred with normal free-chaw and free-drinking either of oxygen-
nanobubble water or of normal water for 12 weeks. Oxygen-nanobubble significantly increased the dissolved oxygen
concentration of water as well as concentration/size of nanobubbles which were relatively stable for 70 days. Air-
nanobubble water significantly promoted the height (19.1 vs. 16.7 cm; P,0.05), length of leaves (24.4 vs. 22.4 cm; P,0.01),
and aerial fresh weight (27.3 vs. 20.3 g; P,0.01) of Brassica campestris compared to normal water. Total weight of sweetfish
increased from 3.0 to 6.4 kg in normal water, whereas it increased from 3.0 to 10.2 kg in air-nanobubble water. In addition,
total weight of rainbow trout increased from 50.0 to 129.5 kg in normal water, whereas it increased from 50.0 to 148.0 kg in
air-nanobubble water. Free oral intake of oxygen-nanobubble water significantly promoted the weight (23.5 vs. 21.8 g;
P,0.01) and the length (17.0 vs. 16.1 cm; P,0.001) of mice compared to that of normal water. We have demonstrated for
the first time that oxygen and air-nanobubble water may be potentially effective tools for the growth of lives.
Citation: Ebina K, Shi K, Hirao M, Hashimoto J, Kawato Y, et al. (2013) Oxygen and Air Nanobubble Water Solution Promote the Growth of Plants, Fishes, and
Mice. PLoS ONE 8(6): e65339. doi:10.1371/journal.pone.0065339
Editor: Jose Luis Balcazar, Catalan Institute for Water Research (ICRA), Spain
Received November 7, 2012; Accepted April 24, 2013; Published June 5, 2013
Copyright: ß 2013 Ebina et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reprod uction in any medium, provided the original author and source are credited.
Funding: This research was funded by Ligaric Company Limited and West Nippon Expressway Company. The funders had no role in study design, decision to
publish, or preparation of the manuscript.
Competing Interests: This research was funded by Ligaric Company Limited and West Nippon Expressway Company. There are no patents, products in
development or marketed products to declare. This does no t alter the authors’ adherence to all the PLOS ONE policies on sharing data and materials.
* E-mail: k-ebina@umin.ac.jp
Introduction
Nanobubbles are miniature gas bubbles in liquids with
,200 nm in diameter, and have several unique physical properties
[1]. They remain stable in water for a long time because of their
negatively charged surface (zeta potential), whereas macrobubbles
increase in size, rise rapidly and burst at the water surface [2,3]. In
addition, internal pressure of nanobubbles in liquids is much
higher than that of their environment, which accelerates
dissolution of the gas into the liquids [4,5]. This remarkable
property of nanobubbles, highly efficient gas solubility, was
previously reported in super-saturation of oxygen gas in water
[1,3]. The smaller the bubble size, the higher the oxygen pressure
(PO
2
) values in water [6], suggesting that nannobubbles increase
the PO
2
values in water to greater extent than that of
microbubbles (10–50 mm in diameter) [1,2]. It has been reported
that high oxygen gas solubility of microbubbles is beneficial for
oxygenation of hypoxic tissues [7,8,9], and their variable
applications for medicine are expected to be useful [10,11,12].
Very recent ex vivo study has demonstrated that oxygen-
nanobubble saline effectively improved hypoxic conditions of
swine blood [2].
Previous studies have demonstrated that hyperoxia promotes
the growth of plants [13] and animals [14], from which we
assumed that air and oxygen-nanobubbles may affect the growth
of life by changing oxygen condition. Indeed, a previous study
showed that air-microbubbles promoted the growth of leaf lettuce
compared to air-macrobubbles [15], but it was the study only on
plants, not on animals. Moreover, no study of the effect of fine
nanobubbles on the growth of lives have so far been reported.
In this paper, we studied whether air and oxygen-nanobubble
water can be safely used and can affect the growth of plants, fishes,
and mammals.
Materials and Methods
Ethics Statement
The animal experimental protocol was approved by the ethics
review committee for animal experimentation of Osaka University
School of Medicine.
PLOS ONE | www.plosone.org 1 June 2013 | Volume 8 | Issue 6 | e65339
Fine microbubbles of gas were generated first after brief
sonication. Then nanobubbles were generated using a nanobubble
aerator (BUVITAS; Ligaric Company Limited, Osaka, Japan),
gas-liquid mixing system with hydrodynamic function (Figure 1).
In this apparatus, gas was supplied at 0.1 MPa and 0.7 L/min into
microbubbles water for 30 min, which is introduced by a pump
(3600 r.p.m.), spirals along the inner wall and finally goes down to
the outlet. The high-speed centrifugal force caused by the
circulation separates the microbubbles into fine nanobubbles by
the strong shearing force in the dispersed water.
Dissolved oxygen concentration (DO) of oxygen-nanobubble
water during generation by BUVITAS was measured sequentially
by Winkler’s method [16]. To confirm the sequential change of
number and diameter of generated air-nanobubbles, water
containing air-nanobubbles was morphologically analyzed by
Multisizer 3 (Beckman Coulter, Inc., Miami, FL, USA) [17].
Brassica campestris (32 stumps in each group) were cultured
hydroponically for 4 weeks within air-nanobubble water or within
normal water. Water was kept circulated (38 L/min) in the tank
(125 L) and air-nanobubble solution was continuously generated
using the nanobubble aerator. The water level of the tank and
water temperature (from 23.2 to 24.3uC was maintained
throughout the culture. The height, length of leaves, and aerial
fresh weight of plants were monitored after 4 weeks. In addition,
DO (by Winkler’s method), pH, and other growth-affecting
elements (nitrogen, phosphorus, potassium, calcium, and magne-
sium) of cultured water were also monitored sequentially. To assess
the effect of air-nanobubbles on the growth of fishes, sweetfish (for
3 weeks) and rainbow trout (for 6 weeks) were kept either within
air-nanobubble water (nanobubble aerator was always kept
working to circulate 67 L air-nanobubble water/min) or within
normal water. Normal fishbait was provided by 13% of primary
total weight per day in each group. Finally, to assess the effect of
oxygen-nanobubbles on the growth of mice, 5 week-old male
DBA1/J mice were bred for 12 weeks with free-normal chaw
(Oriental Yeast, Tokyo, Japan) and free-drinking either of oxygen-
nanobubble distilled water or of normal distilled water. All distilled
water was purchased from Otsuka Pharmaceutical Factory, Inc.
(Tokyo, Japan). Oxygen-nanobubbles distilled water was filtered
immediately after generation, and normal distilled water was
filtered immediately after opening the lid, with 0.22 um pore size
cellulose acetate membrane (Corning, Cambridge, MA) to avoid
contamination. After filtration, DO was measured by handheld
dissolved oxygen meter DO-24 P (DKK-TOA Corporation,
Tokyo, Japan). Size (detection range; 10–1000 nm) and concen-
tration of nanoparticles were measured by NanoSight LM10-
HSBT14 (NanoSight Ltd, Salisbury, UK). Water was kept in 4uC
for 12 weeks and sufficiently supplied in water bottles of mice twice
a week after restoring to room temperature. All mice were
purchased from CLEA Japan (Tokyo, Japan), and housed in a
room under controlled temperature (2361uC) and humidity (45–
65%).
Statistical Analysis
Data are expressed as mean 6 standard error (SE) in the growth
of Brassica campestris. Other data are expressed as mean 6
standard deviation (SD). Differences between groups were assessed
by Student’s t-test. Any P value,0.05 was considered statistically
significant.
Results
Oxygen concentration was 7.7 mg/L in original normal
distilled water, whereas it increased to 31.7 mg/L in oxygen-
nanobubble water immediately after running nanobubble aerator
with 100 L water for 30 min (Figure 2). Figure 3 shows the
Figure 1. Nanobubble aerator (BUVITAS; Ligaric Company Limited, Osaka, Japan). Fine microbubbles of gas were generated first after
brief sonication. Then nanobubbles were generated using this gas-liquid mixing system with hydrodynamic function. In this apparatus, gas was
supplied at 0.1 MPa and 0.7 L/min into microbubble water for 30 min. The high-speed centrifugal force caused by the circulation separates the
microbubbles into fine nanobubbles by the strong shearing force in the dispersed water.
doi:10.1371/journal.pone.0065339.g001
Figure 2. Sequential dissolved oxygen concentration (DO) in
oxygen-nanobubble mixed water. Oxygen-nanobubbl es were
generated by the same methods as Figure 1 with 100 L water. Oxygen
concentration was measured sequentially by Winkler’s method.
doi:10.1371/journal.pone.0065339.g002
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Figure 3. Sequential changes of number and diameter of generated air-nanobubbles. After generating air-nanobubble water by the same
methods as Figure 1, sequential changes of number and diameter of generated air-nanobubbles were analyzed by Multisizer 3 (Beckman Coulter, Inc.,
Miami, FL, USA).
doi:10.1371/journal.pone.0065339.g003
Figure 4. Growth of Brassica campestris cultured with either normal water or air-nanobubble water for 4 weeks. Brassica campestris
(32 stumps in each group) were hydroponically cultured for 4 weeks. Water was circulated (38 L/min) in a tank (125 L/tank) and air-nanobubble
solution was continuously generated using the nanobubble aerator. The water level of the tank and water temperature (from 23.2 to 24.3uC) was
maintained throughout the culture. Data are shown as mean 6 S.E. (n = 32 in each group). **P,0.01; ***P,0.001.
doi:10.1371/journal.pone.0065339.g004
Nanobubble Water Promotes the Growth of Lives
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chronological change and distribution of number and diameter of
air-bubbles in water after generation. Approximately 70% of the
generated air-bubbles were smaller than 2 mm in diameter
immediately after generation. Moreover, even 2.5 months after
generation, approximately 60% still remained smaller than 2 mm
in diameter.
Air-nanobubble water significantly promoted the height
(16.762.0 vs. 19.162.5 cm; P,0.001), length of leaves
(22.464.3 vs. 24.462.5 cm; P,0.01), and aerial fresh weight
(20.364.3 vs. 27.366.9 g; P,0.01) of Brassica campestris
compared to normal water after hydroponical culture for 4 weeks
(Figure 4). DO of cultured water was significantly higher
(P,0.001) in air-nanobubble water compared to normal water
from day 1 to day 28 (Figure 5A). On the other hand, there was no
significant difference in pH (Figure 5B), nitrogen, phosphorus,
potassium, calcium, and magnesium concentration within cultured
water throughout the cultured period between two groups (data
not shown).
After 3 weeks, total weight of sweetfish increased from 3.0 to
6.4 kg in normal water, whereas it increased from 3.0 to 10.2 kg in
air-nanobubble water (Table 1). In addition, total weight of
rainbow trout increased from 50.0 to 129.5 kg in normal water,
whereas it increased from 50.0 to 148.0 kg in air-nanobubble
water after 6 weeks (Table 1).
In our preliminary experiments, hyperoxidation as much as
about 31.7 mg/L of DO in oxygen-nanobubble water took place
immediately after generation (Figure 2), but it fell to a constant
level about 8.7 mg/L at 5 hr after generation and then maintained
from day 7 to 70 (Figure 6 A). Nanoparticles were not detected in
normal distilled water initially, but were clearly detected after
generation of oxygen-nanobubble. The concentration of nanopar-
ticles was maximum at day 7 and mostly maintained at day 70,
while the size of nanoparticles decreased gradually but maintained
as long as day 70 (Figure 6 B and C). DO of oxygen-nanobubble
distilled water did not change before and after filtration (data not
shown).
After 12 weeks, free oral intake of oxygen-nanobubble distilled
water significantly promoted the weight (21.860.3 vs. 23.560.3 g;
P,0.01) and the length (16.160.1 vs. 17.060.1 cm; P,0.001) of
mice as compared to free oral intake of normal water (Figure 7 A,
C, D). As for food consumption, mice drinking oxygen-
nanobubble water took higher dose of food compared to that of
normal water (Figure 7 B).
Discussion
To the best of our knowledge, we demonstrated for the first time
that air and oxygen-nanobubbles promote the growth of plants,
fishes, and mice. Yoshida et al. demonstrated that cucumber
plants at higher dissolved oxygen concentration showed increased
area and weight of leaves [13]. In addition, Park et al demon-
strated that air-microbubbles promote the growth of leaf lettuce
compared to air-macrobubbles, in which report DO of cultured
water at the beginning was very similar to that of us (about
8.5 mg/L) [15]. On the other hand, DO in cultured water on day
14 was relatively higher in our study (air-nanobubble water; about
9.2 mg/L) as compared to Park’s report (air-microbubble water;
about 8.2 mg/L). This difference is in accordance with the
previous report which speculated nannobubbles increase the
oxygen partial pressure (PO
2
) values in water to greater extent
than that of microbubbles [1,2]. In our study, nutrient solution
except for oxygen did not differ between two groups (data not
shown), but Park et al also speculated that larger specific surface
area of the microbubbles as well as negative electronic charges on
their surface may promote the growth of plants because
Figure 5. Sequential changes of dissolved oxygen concentration (DO) and pH of cultured water. DO (A) and pH (B) of Brassica campestris
cultured water were monitored sequentially. Data are shown as mean 6 S.D. (n = 5 in each group). ***P,0.001.
doi:10.1371/journal.pone.0065339.g005
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microbubbles can attract positively charged ions that are dissolved
in the nutrient solution. Regarding vertebrates, Owerkowicz et al.
demonstrated that the growth of American alligators was the
fastest when maintained at hyperoxia, while it was the slowest at
hypoxia [14]. They suggested that combination of elevated
metabolism and low demand of breathing in hyperoxic condition
allows a greater proportion of metabolized energy to be available
for growth. They also suggested that hyperoxia may induce
hypermetabolic state to maintain higher rate of food digestion and
absorption. These reports are in accordance with the results of our
study, suggesting that air and oxygen-nanobubble water solution
may contribute to elevated metabolism, higher food intake, and
promoted growth.
In this study, there were no obvious abnormalities or harmful
effects in fishes and mice with oral intake of air and oxygen-
nanobubbles, respectively. No morphological irregularity was
recongnized in Brassica campestris cultured with air-nanobubble
water, either. It has also been suggested that nanobubbles could be
clinically safe in human; because the filter pore size of
cardiopulmonary bypass machines are usually in the range of
Figure 6. Sequential changes of dissolved oxygen concentration (DO) and concentration/size of oxygen-nanobubble distilled water
from day 0 to 70. DO (A) and concentration/size (B) of oxygen-nanobubble distilled water were monitored sequentially. Distribution of
concentration and size of nanoparticles are shown (C). Data are shown as mean 6 S.D. (n = 3 in each group). **P,0.01; ***P,0.001 compared to
normal water.
doi:10.1371/journal.pone.0065339.g006
Table 1. Change in total weight of fishes after keeping within normal water or air-nanobubble water.
Keeping period Normal Water group Air-nanobubble water group
Sweetfish 3 weeks Initial total weight, kg 3.0 3.0
Final total weight, kg 6.4 10.2
Rainbow trout 6 weeks Initial total weight, kg 50.0 50.0
Final total weight, kg 129.5 148.0
doi:10.1371/journal.pone.0065339.t001
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28–40 mm, nanobubbles of ,10 mm in diameter are therefore
negligibly small as a causal substance of gas embolism in blood
vessels [18]. Cavalli et al also reported that fluorescent labeled
nanobubbles showed good capacity of loading oxygen without
hemolytic activity or toxic effect in Vero cells [19]. Taken
together, it may be said that air and oxygen-nanobubbles could be
safe accelerator of growth in most of lives. In addition, DO as well
as concentration/size of nanoparticles in oxygen-nanobubble
distilled water were relatively stable in 4uC from day 0 to 70.
There are several limitations in this study. In fishes and mice
experiment, food intake was not controlled. The promoting effect
of air and oxygen-nanobubble water on growth may be partially
due to increase in oral uptake. In addition, although previous
study have demonstrated that oxygen-nanobubble saline effective-
ly improved hypoxic conditions of swine blood ex vivo [2], actual
Po
2
in blood of fishes and mice are not monitored. As we have not
used other gas-nonobubble water, whether promoting effect on
growth is due to nanobubbles themselves or elevated oxygen
concentration in the water is still unresolved. Further examination
using other gas nanobubbles is required to determine the effect of
nanobubbles themselves on growth of lives. Although there are
several limitations, air and oxygen-nanobubble water significantly
promoted the growth of plants, fishes, and mice, and this novel
finding may bring new insight in effective growth of lives in the
future.
Acknowledgments
All oxygen and air nanobubble suspension water was prepared by Ligaric
Company Limited. The feeding experiment of Brassica campestris was
performed by West Nippon Expressway Company Limited and Research
Institute of Environment, Agriculture and Fisheries, Osaka Prefecture. The
feeding experiment of the fish was performed by Ligaric Company
Limited.
Author Contributions
Conceived and designed the experiments: KE KS MH JH HY. Performed
the experiments: KE YK SK TM KK. Analyzed the data: KE YK SK TM
KK. Contributed reagents/materials/analysis tools: KE YK SK TM KK.
Wrote the paper: KE KS.
Figure 7. Sequential changes of food intake, body weight, and body length of mice. 5 weeks old male DBA1/J mice were bred with normal
free-chaw and free oral intake either of oxygen-nanobubble distilled water or of normal distilled water for 12 weeks (A). Total food intake (B), body
weight (C), and body length (D) were monitored in each mouse. Data are shown as mean 6 S.D. (n = 4 in each group). **P,0.01; ***P,0.001.
doi:10.1371/journal.pone.0065339.g007
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References
1. Agarwal A, Ng WJ, Liu Y (2011) Principle and applications of microbubble and
nanobubble technology for water treatment. Chemosphere 84: 1175–1180.
2. Matsuki N, Ichiba S, Ishikawa T, Nagano O, Takeda M, et al. (2012) Blood
oxygenation using microbubble suspensions. Eur Biophys J 41: 571–578.
3. Takahashi M, Chiba K, Li P (2007) Free-radical generation from collapsing
microbubbles in the absence of a dynamic stimulus. J Phys Chem B 111: 1343–
1347.
4. Eriksson JC, Ljunggren S (1999) On the mechanically unstable free energy
minimum of a gas bubble which is submerged in water and adheres to a
hydrophobic wall. Colloids and Surfaces a-Physicochemical and Engineering
Aspects 159: 159–163.
5. Ljunggren S, Eriksson JC (1997) The lifetime of a colloid-sized gas bubble in
water and the cause of the hydrophobic attraction. Colloids and Surfaces a-
Physicochemical and Engineering Aspects 130: 151–155.
6. Li P, Takahashi M, Chiba K (2009) Enhanced free-radical generation by
shrinking microbubbles using a copper catalyst. Chemosphere 77: 1157–1160.
7. Bitterman H (2009) Bench-to-bedside review: oxygen as a drug. Crit Care 13:
205.
8. Abdelsalam M, Cheifetz IM (2010) Goal-directed therapy for severely hypoxic
patients with acute respiratory distress syndrome: permissive hypoxemia. Respir
Care 55: 1483–1490.
9. Guo S, Dipietro LA (2010) Factors affecting wound healing. J Dent Res 89: 219–
229.
10. Barbosa FT, Juca MJ, Castro AA, Duarte JL, Barbosa LT (2009) Artificial
oxygen carriers as a possible alternative to red cells in clinical practice. Sao Paulo
Medical Journal 127: 97–100.
11. Betit P, Craig N (2009) Extracorporeal Membrane Oxygenation for Neonatal
Respiratory Failure. Respiratory Care 54: 1244–1251.
12. Kulikovsky M, Gil T, Mettanes I, Karmeli R, Har-Shai Y (2009) Hyperbaric
Oxygen Therapy for Non-Heating Wounds. Israel Medical Association Journal
11: 480–485.
13. Yoshida S, Kitano M, Eguchi H (1996) Water uptake and growth of cucumber
plants (Cucumis sativus L.) under control of dissolved O2 concentration in
hydroponics. Acta Hortic 440: 199–204.
14. Owerkowicz T, Elsey RM, Hicks JW (2009) Atmospheric oxygen level affects
growth trajectory, cardiopulmonary allometry and metabolic rate in the
American alligator (Alligator mississippiensis). J Exp Biol 212: 1237–1247 .
15. Park JS, Kurata K (2009) Application of Microbubbles to Hydroponics Solution
Promotes Lettuce Growth. Horttechnology 19: 212–215.
16. Numako C, Nakai I (1995) Xafs Studies of Some Precipitation and Coloration
Reaction Used in Analytical-Chemistry. Physica B 208: 387–388.
17. Zhao G, He LQ, Zhang HF, Ding WP, Liu Z, et al. (2004) Trapped water of
human erythrocytes and its application in cryopreservation. Biophysical
Chemistry 107: 189–195.
18. Barak M, Katz Y (2005) Microbubbles: pathophysiology and clinical
implications. Chest 128: 2918–2932.
19. Cavalli R, Bisazza A, Giustetto P, Civra A, Lembo D, et al. (2009) Preparation
and characterization of dextran nanobubbles for oxygen delivery. Int J Pharm
381: 160–165.
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