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Growth control of leaf lettuce with exposure to underwater ultrasound and

dissolved oxygen supersaturation

Article  in  Ultrasonics Sonochemistry · October 2018

DOI: 10.1016/j.ultsonch.2018.10.005

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 Ultrasonics - Sonochemistry 51 (2019) 292–297

Contents lists available at ScienceDirect

Ultrasonics - Sonochemistry
journal homepage: www.elsevier.com/locate/ultson

Growth control of leaf lettuce with exposure to underwater ultrasound and T

dissolved oxygen supersaturation
Yuta Kurashinaa,b, Tatsuya Yamashitaa, Shuichi Kurabayashic, Kenjiro Takemuraa, Keita Andoa,
⁎

a
Graduate School of Science and Technology, Keio University, Yokohama, Kanagawa, Japan
b
School of Materials and Chemical Technology, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan
c
Graduate School of Media and Governance, Keio University, Fujisawa, Kanagawa, Japan

ARTICLE INFO ABSTRACT

Keywords: The growth rate of vegetables in plant factories can be regulated by environmental factors including light,
Plant growth control temperature, and chemicals, which might give rise to mutation in leaf health. Here, we aim to devise a new way
Underwater ultrasound that allows for controlling the growth rate of plants in hydroponics as well as maintaining the product quality;
Dissolved oxygen supersaturation we apply underwater ultrasound and dissolved oxygen supersaturation as external stimuli to plants. As an ex-
Leaf lettuce
ample, we examine the growth of leaf lettuce in hydroponics with exposure to 28-kHz ultrasound and dissolved
Plant factory
oxygen supersaturation up to 36 mg/L at 20 °C. Our results show that exposure to the ultrasound of peak-to-peak
pressure at 20 kPa or larger works as the growth inhibitor of the leaves and the roots, while the oxygen su-
persaturation as the growth promoter, without any degradation of chlorophyll in the leaves. This suggests that
these external stimuli can be used in the growth control system of plant factories.

1. Introduction the integrity of chloroplast proteins. While the lighting can promote the
growth rate, it may possibly have an impact on chlorophyll, which is an
Food waste has recently been a critical issue in developed countries indicator of leaf health [14]. Owen et al. [15] examined the effect of
and its significant reduction is thus favored for better food sustain- higher-intensity LEDs on the red pigmentation of leaf lettuce and found
ability. The annual food production in the world is currently about four that such a direct stimulus to the leaves gives rise to their transmutation
billion metric tons. According to the estimate of Food and Agriculture like leaf discoloration. The similar phenomenon is expected to occur in
Organization of the United Nations and Stockholm International Water leaf vegetables including cabbage, mizuna, and spinach, which are the
Institute [1,2], 30–50% of the food production (i.e., 1.2–2 billion metric main crops in plant factories [16], as well as in botanical fruits [17,18].
tons) is not consumed and thus wasted every year. Hence, in this study, we explore other methods that do not rely on the
Plant factories are one of cultural systems for optimal plant culti- electric lighting technique.
vation (mainly in hydroponics) to achieve a better quality of crops by In addition to electric lighting, there are a number of candidate
precisely controlling the environmental factors—electric light, tem- stimuli that may affect the growth of plants [19]. Plants in nature are
perature, and atmosphere [3]. While producing crops in open fields or subjected to a combination of stress factors such as, for example, gravity
plastic greenhouses usually relies on plant growth regulators to pro- [20,21], wind [22,23], electrostatic fields [24–26], sound in the air
mote the productivity, plant factories will be able to control the growth [27,28], and the atmosphere with varying temperature and moisture
rate of plants only through the external stimuli. However, how the [29,30]; these external stimuli can affect morphology, growth, and thus
external stimuli affect plants’ growth is still unclear [4]. Hence, there is cell cycles of plants. Collins et al. [31] reported that the maximal
a need to quantify the impact of the external stimuli on the growth rate growth rate of plants is achieved when they are exposed to sound whose
of plants, which is expected to contribute to reductions in food waste. wavelength is similar to the dimension of their leaves. Moreover, the
In the past decades, electric lighting techniques have been widely experiment of Tabaru et al. [32] in which Japanese radish sprouts’ roots
studied as a growth control method [5–9]; it has been reported that were exposed to ultrasound suggested a possibility of underwater ul-
growth control can be realized by the use of light emitting diodes trasound as a growth inhibitor; however, the effect of the underwater
(LEDs) [10–12]. Muneer et al. [13] reported that higher-intensity blue ultrasound intensity has not been studied very well, to our knowledge.
LEDs can promote the growth of lettuces by appropriately controlling On the other hand, an increase in the amount of dissolved oxygen (DO)

⁎
Corresponding author.
E-mail address: kando@mech.keio.ac.jp (K. Ando).

https://doi.org/10.1016/j.ultsonch.2018.10.005
Received 15 August 2018; Received in revised form 2 October 2018; Accepted 4 October 2018
Available online 05 October 2018
1350-4177/ © 2018 Elsevier B.V. All rights reserved.
Y. Kurashina et al. Ultrasonics - Sonochemistry 51 (2019) 292–297

Fig. 1. The plant cultivation device with application

of underwater ultrasound and bubble aeration. (a)
Schematic illustration and (b) photograph of the
plant cultivation device. Leaf lettuces mounted on
foamed polyurethane forms are cultured on the ac-
rylic base. Water is circulated by a pump.
Underwater ultrasound is exposed in the cultivation
chamber, and bubble aeration is performed in the
water tank to control the DO level. In the schematic
(a), the acrylic box was intentionally depicted as
transparent.

Fig. 2. Measurement of the underwater acoustic pressure. (a) Five measurement positions (red cross) 53 mm above the transducer. (b) Peak-to-peak pressure at the
five measurement positions. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)

in hydroponics can promote plant growth [33]. Ebina et al. [34] de- 2. Materials and methods
monstrated that the growth accelerates as the DO increases. These
studies suggest that plant growth may possibly be controlled with ex- 2.1. Hydroponic system
posure to underwater ultrasound and DO supersaturation.
To obtain a fundamental insight into growth control in plant fac- Fig. 1 shows our hydroponic system of leaf lettuces (Green wave,
tories, we examine the growth of leaf lettuce with exposure to under- Takii Co. Ltd., Kyoto, Japan). A cylindrical Langevin transducer of
water ultrasound and DO supersaturation, as a model example. Here, 65 mm in diameter (0.028Z50I, Japan Probe, Kanagawa, Japan) was
we develop a plant cultivation system that allows for qualitatively fixed to the bottom of an acrylic cultivation chamber (whose inner di-
evaluating the growth rate under precise control of these external sti- mensions are 150 mm × 150 mm in lateral directions and 100 mm in
muli. In what follows, we will show, for the first time, that a combi- height). Tap water (Kanagawa, Japan) is filled in the chamber; the
nation of ultrasound and DO supersaturation enables one to control the water level was set at five-quarters of the ultrasonic wavelength oper-
growth rate. ated at 28 kHz (i.e., 66 mm above the transducer). The transducer was

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Y. Kurashina et al. Ultrasonics - Sonochemistry 51 (2019) 292–297

driven by a function generator (WF1974, NF corporation, Kanagawa,

Japan), through magnification by a power amplifier (HSA 4011, NF
corporation, Kanagawa, Japan), in order to generate underwater ul-
trasound. The underwater pressure was measured at one wavelength of
the incident ultrasound wave (i.e. 53 mm) above the transducer by a
needle hydrophone (HCT-0310, Onda Co. Ltd., Ibaraki, Japan) and
recorded with an oscilloscope (Wavesurfer 454, Teledyne LeCroy, NY,
US) in the absence of the leaf lettuces. Each seedling sample was con-
tinuously subjected to the ultrasound whose peak-to-peak pressure is
either 2 or 20 kPa above the center of the acrylic cultivation chamber as
shown in (i) of Fig. 2(a). We measured the five measurement points (red
cross) of peak-to-peak pressures at (i)–(v) in Fig. 2(a). Fig. 2(b) shows
the peak-to-peak pressures. Note that the orders of the average pres- Fig. 3. Definition of measurement ranges of leaf lettuce. (a) Length of the main
sures among the different measurement positions were 103 and 104 Pa root, (b) length of the longest leaf, (c) number of leaves, and (d) weight of the
when the peak-to-peak pressures above the center of the acrylic box fresh leaves.
were 2 and 20 kPa, respectively. In the experiment of Tabaru et al. [32],
the growth of Japanese radish sprouts was promoted for the case of Table 1
average underwater ultrasound pressure up to 103 Pa, while that was Five measurement characteristics of leaf lettuce.
suppressed for the case of the average pressure between 103 and 104 Pa.
Measurement characteristic Abbreviation Label in Figs. 3 to 6
According to their result, we chose the peak-to-peak pressure of the
underwater ultrasound at either 2 kPa or 20 kPa in the present experi- Length of the main root Lr (a)
ment. Length of the longest leaf Li (b)
The water was constantly aerated by supplying air or pure oxygen Number of leaves N1 (c)
bubbles through a commercially available airstone in the tank. Here, we Weight of fresh leaves W1 (d)

aim to maintain DO saturation (approximately at 9 mg/L at 20 °C) by

air bubble aeration, while we apply pure oxygen aeration to produce
applied analysis of variance (ANOVA) with Ryan’s multiple comparison
DO supersaturation [35]. Air and pure oxygen were supplied by an
test [38]; the value of *p < 0.05 or **p < 0.01 means statistically
aeration pump (Suishin SSPP-3S, Suisaku Corporation, Tokyo, Japan)
significant.
and an oxygen generator (FOX-4, SHOKO Co., Ltd., Tokyo, Japan),
To see whether underwater ultrasound and DO supersaturation have
respectively. Note that ultrasound exposure without aeration gives rise
an impact on plant health, we evaluated the photosynthetic activity.
to DO subsaturation; this is the so-called ultrasound degassing [36] The
For noninvasive measurement of Ac, we visualized the chlorophyll
water circulation was driven by a water pump at the volume flow rate
fluorescence using an inverted fluorescent microscope (Eclipse TE2000,
of 40 mL/min from the tank and by an overflow from the chamber
Nikon Instruments Inc., NY, US) equipped with a CCD camera and a UV
(based on the Siphon principle). The DO concentration in the cultiva-
excitation filter (UV-1A, Nikon Instruments Inc., NY, US). Since chlor-
tion chamber can be different, depending on the cultivation conditions.
ophyll consists of auto-fluorescent molecules (wavelength of 735 nm
Thus, we monitored the DO concentration using a DO meter of fluor-
[39]), we can measure the intensity of re-emitted fluorescence (red
escence type (SevenGo pro, Mettler Toledo, OH, US) in the first and the
color) and correlate it to the chlorophyll concentration in the leaves.
last days of the plant growth experiment.
We extracted red color from the captured image by threshold analysis in
ImageJ (National Institutes of Health, Bethesda, MD, USA).
2.2. Preparation of seedling samples of leaf lettuce

As a seedling sample, we cultivated the seeds of leaf lettuce until 3. Results

their germination. The seeds were immobilized in foamed poly-
urethanes (23 mm × 23 mm × 28 mm, CP-613-S-1, TAKIGEN, Tokyo, 3.1. Parametric study
Japan), which consist of inert porous materials and are thus proper for
plant substrates [37]. The substrates were immersed in fertilizer-satu- To examine the effects of underwater ultrasound and DO super-
rated water (Hyponica liquid fertilizer—0.2% Fertilizer A and 0.2% saturation on the seedling growth, we make comparisons based on 8
Fertilizer B, Kyowa Co. Ltd., Osaka, Japan) and kept in a growth different conditions as listed in Table 2. In this table, we also reported
chamber (Biotron, Nippon Medical & Chemical Instruments Co. Ltd., on the DO concentrations. When the aeration was not applied (see
Osaka, Japan) with a 9-hour light and 15-hour dark cycle under the UlowA(−) and UhighA(−)), the DO concentration was lowered (and thus
atmosphere at 20 °C, while the water temperature was at 21 ± 2 °C, for subsaturated) by ultrasound degassing. As expected, the reduction in
2 weeks. Note that pH of fertilizer-saturated water was 6.3 ± 0.1. We the DO concentration was emphasized as the ultrasound intensity in-
selected seedlings of similar size to eliminate fluctuations in the growth creases. For the cases of air bubble aeration (see UlowAair, UhighAair, and
rate measurement due to poor germination of the seeds. U(−)Aair), on the contrary, we obtained the DO concentrations close to
We then examined the growth of the germinated seedlings with the saturation level, regardless of the ultrasound exposure. For the cases
varying parameters in the cultivation; see Section 3.1. Twenty five of oxygen bubble aeration (see UlowAoxy, UhighAoxy, and U(−)Aoxy), we
seedling samples were mounted by an acrylic plate in the cultivation obtained the DO superasaturation (approximately 4 times larger than
chamber of the hydroponic system with a 9-hour light and 15-hour dark the saturation level).
cycle under the atmosphere at 20 °C, while the water temperature was As summarized in the parametric study in Table 3, we aim to study
at 21 ± 2 °C, for a week. each condition in a step-by-step matter. In Section 3.2 (Case 1 and Case
2), we first examine the effect of the lower/higher-intensity ultrasound
2.3. Evaluation of plant growth exposure on the seedling growth from comparisons of UlowAair and
UhighAair to U(−)Aair. We also compare UhighAair and UhighA(−) to stress
To evaluate the seedling growth, we examined the five parameters the dominant role of sufficiently strong ultrasound in the plant growth
summarized in Fig. 3 and Table 1. When measuring the length, we (in comparison to that from the DO saturation level). In Section 3.3
made roots straight and leaves flat. In evaluating the plant growth, we (Case 3), we next examine the impact of DO supersaturation on the

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Y. Kurashina et al. Ultrasonics - Sonochemistry 51 (2019) 292–297

Table 2
Eight different conditions for comparison in the seedling growth.
Condition UlowAair UhighAair U(−)Aair UlowA(−) UhighA(−) UlowAoxy UhighAoxy U(−)Aoxy

Ultrasound low intensity high intensity not applied low intensity high intensity low intensity high intensity not applied (0 Pa)
(2 kPa) (20 kPa) (0 Pa) (2 kPa) (20 kPa) (2 kPa) (20 kPa)
Aeration air (DO 8.34 mg/L) air (DO 8.41 mg/L) air (DO not applied not applied oxygen oxygen oxygen
8.28 mg/L) (DO 6.76 mg/L) (DO 6.76 mg/L) (DO 35.7 mg/L) (DO 36.1 mg/L) (DO 35.7 mg/L)

Table 3
Parametric study between 4 different cases.
Comparison conditions

Case 1 UlowAair UlowA(−) U(−)Aair

Case 2 UhighAair UhighA(−) U(−)Aair
Case 3 UlowAair UlowAoxy U(−)Aair U(−)Aoxy
Case 4 UhighAair UhighAoxy U(−)Aoxy

seedling growth from comparisons between U(−)Aoxy and U(−)Aair (i.e.,

without the ultrasound exposure). In Section 3.4 (Case 3 and Case 4),
we finally compare results from UhighAair and UhighAoxy and from
U(−)Aoxy and UhighA(−) to show the combination effect of sufficiently
strong ultrasound and DO supersaturation on the seedling growth.

3.2. Seedling growth with underwater ultrasound of different intensity

To understand the role of underwater ultrasound exposure in the Fig. 5. Case 2: comparison between UhighAair, UhighA(−) and U(−)Aair to see
seedling growth, we consider the cases with ultrasound of different effects of the higher-intensity (20 kPa) ultrasound exposure on the seedling
intensity (and with the DO kept almost at saturation via air bubble under the different DO concentrations. (a) Length of the main root, Lr, (b)
length of the longest leaf, Ll, (c) number of the leaves longer than 5 mm, Nl, (d)
aeration). In Fig. 4, we see the effect of the lower-intensity (2 kPa) ul-
weight of the fresh leaves, Wl (mean ± SD, n = 25, *: p < 0.05, **: p < 0.01).
trasound on the seedling growth from comparison between UlowAair and
U(−)Aair. It follows that length of the main root Lr becomes only sta-
tistically higher (p < 0.05, ANOVA) by the ultrasound, while the other the seedling growth is insensitive to whether the water is saturated or
values (length of the longest leaf Ll, number of the leaves longer than subsaturated, provided the ultrasound intensity is sufficiently high.
5 mm Nl and weight of the fresh leaves Wl) are almost unaffected. In
Fig. 5, we see the effect of the higher-intensity (20 kPa) ultrasound on 3.3. Seedling growth with different DO supersaturation but without
the seedling growth from comparison between UhighAair and U(−)Aair. In ultrasound
this case, the ultrasound effect becomes clearer; Lr, Ll, Nl and Wl become
statistically lower (p < 0.01, p < 0.05, ANOVA) by the ultrasound. In Fig. 6, we see the effect of DO supersaturation on the seedling
Next, we examine the case of the higher-intensity ultrasound from growth from comparison between U(−)Aoxy and U(−)Aair. We again note
comparison between UhighAair and UhighA(−). It is important to again that the oxygen bubble aeration leads to DO supersaturation (ap-
note that the DO level was lowered by the ultrasound exposure (see proximately 4 times larger than the saturation level; see U(−)Aoxy in
UhighA(−) in Table 2). The comparison is made in Fig. 5, showing that Table 2). It follows that Ll, Nl, and Wl become statistically higher

Fig. 4. Case 1: comparison between UlowAair, UlowA(−) and U(−)Aair to see ef- Fig. 6. Case 3: comparison between UlowAair, UlowAoxy, U(−)Aair and U(−)Aoxy to
fects of the lower-intensity (2 kPa) ultrasound exposure on the seedling under see effects of the lower-intensity (2 kPa) ultrasound exposure and the DO su-
the different DO concentrations. (a) Length of the main root, Lr, (b) length of persaturation on the seedling growth. (a) Length of the main root, Lr, (b) length
the longest leaf, Ll, (c) number of the leaves longer than 5 mm, Nl, (d) weight of of the longest leaf, Ll, (c) number of the leaves longer than 5 mm, Nl, (d) weight
the fresh leaves, Wl (mean ± SD, n = 25, *: p < 0.05, **: p < 0.01). of the fresh leaves, Wl (mean ± SD, n = 25, *: p < 0.05, **: p < 0.01).

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Y. Kurashina et al. Ultrasonics - Sonochemistry 51 (2019) 292–297

chlorophyll. This indicates that underwater ultrasound and DO super-

saturation have no harmful impact on the photosynthetic activity. We
may say that our proposed method with these external stimuli will be
capable of controlling plant growth without any mutation in leaf health.

4. Discussion

4.1. Effects of underwater ultrasound on the seedling growth

From the data in Figs. 4 and 5 where the DO concentration was kept
at the saturation level by the air bubble aeration, we found that the
lower-intensity ultrasound exposure promotes the root growth but does
not affect the leaf growth, while the higher-intensity ultrasound ex-
posure suppresses the root and leaf growth. That is, we will be able to
control plant growth by underwater ultrasound with varying its in-
tensity. Note that the water was supplied to the tank constantly for the
Fig. 7. Case 4: comparison between UhighAair, UhighAoxy and U(−)Aair to see distance between the water surface and the transducer to be constant,
effects of the higher-intensity (20 kPa) ultrasound exposure and the DO su- which contributes to stabilization of the acoustic field. We speculate
persaturation on the seedling growth. (a) Length of the main root, Lr, (b) length that the higher-intensity ultrasound retards the absorption of water and
of the longest leaf, Ll, (c) number of the leaves longer than 5 mm, Nl, (d) weight nutrition molecules from the roots, as will be explained below.
of the fresh leaves, Wl (mean ± SD, n = 25, *: p < 0.05, **: p < 0.01). It is likely that the key to explain the difference in the seedling
growth by the ultrasound is the mechanical action of bubbles. For the
(p < 0.01, p < 0.05, ANOVA) by the DO supersaturation, while the case of the lower-intensity ultrasound, the probability of having cavi-
other values are almost unaffected. Namely, the growth promotion by tation bubbles in the water is expected to be lower. The number of small
the DO supersaturation appears in the leaves, not in the roots. As in the gas bubble nuclei in water tends to be augmented by the aeration, but
cases with ultrasound exposure, the leaf health is almost unaffected by their volume oscillation caused by such weak ultrasound forcing is
the DO level of our concern. expected to be mild. As a result of the mild oscillation of bubbles near
the seedling roots, there will appear microstreaming [40,41] and shear
3.4. Seedling growth with a combination of ultrasound and DO stress acting on the root surface [42]. These mechanical actions are
supersaturation believed to contribute to an increase of mass transfer across cell
membranes, thereby resulting in faster metabolic processes [43]. The
In Case 1 to Case 4, we studied the individual effect of ultrasound or similar trend can be confirmed in Fig. 4 that compares UlowAair and
a DO saturation level, separately, on the seedling growth. At the end, UlowA(−); the seedling growth is a bit slower unless the air bubble
we aim to evaluate their combination effect. In Fig. 7, we see the aeration is applied. This is perhaps due to the fact that the number of
combination effect of the higher-intensity ultrasound and the DO su- gas bubble nuclei will decrease by the ultrasound degassing effect.
persaturation from comparison between UhighAair and UhighAoxy. It is On the other hand, the growth suppression was observed in the case
seen that leaf length Ll becomes only statistically higher (p < 0.05, of the higher-intensity ultrasound. In this case, the amplitude of bubble
Student’s t-test) by the DO supersaturation, while the other values are oscillation is augmented and the resulting mechanical actions will thus
almost unaffected. In Fig. 7, we also compare UhighAoxy and U(−)Aoxy; be stronger, which might give rise to damage of the seedling roots (i.e.
the similar DO supersaturation was obtained in the two conditions (see cavitation erosion) [43,44]. As shown in Fig. 5, we again note that the
Table 2). It is seen that root length Lr becomes only statistically lower seedling growth under the higher-intensity ultrasound irradiation was
(p < 0.01, ANOVA) by the ultrasound, while the other values are al- insensitive to the DO supersaturation level. This might be explained as
most unaffected. From these figures, the combination effect of the ul- follows: the ultrasound intensity was strong enough to activate rectified
trasound and the DO supersaturation on the seedling growth is found to diffusion [45,46], which stands for bubble growth through net mass
be different from their individual effects, which will be discussed in influx resulting from nonlinear bubble oscillation, even under DO
Section 4.3. subsaturation [47]. As a result of the rectified diffusion, sufficiently
Finally, we draw a conclusion regarding the leaf health. Fig. 8 shows strong ultrasound can produce a number of oscillating bubbles, even
chlorophyll amount of representative smaples—U(−)Aair and UhighAoxy. when water is subsaturated with DO, thereby giving rise to cavitation
We did not observe any significant change in the amount of the leaf erosion, regardless of DO saturation levels.

4.2. Effects of DO on the supersaturation seedling growth

From Fig. 6 where no ultrasound was applied and the DO con-

centration was set either at supersaturation (oxygen bubble aeration) or
at saturation (air bubble aeration), we found that the DO super-
saturation can promote the leaf growth only. The similar observation
can be obtained even when the lower-intensity ultrasound was applied;
see Fig. 6 that compares UlowAair and UlowAoxy.
We can summarize the findings from Sections 4.1 and 4.2 where we
discussed the ultrasound effect and the DO effect separately: sufficiently
strong underwater ultrasound and DO supersaturation work, respec-
Fig. 8. Chlorophyll amount of representative samples. (a) Fluorescent micro- tively, as a growth inhibitor and promoter.
scope images of U(−)Aair and UhighAoxy (scale bar, 500 µm). (b) Amount of leaf
chlorophyll, Ac (mean ± SD, n = 10).

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