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Influences of Air, Oxygen, Nitrogen, and Carbon Dioxide Nanobubbles on Seed

Germination and Plant Growth

Article  in  Journal of Agricultural and Food Chemistry · May 2018

DOI: 10.1021/acs.jafc.8b00333


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7 authors, including:

Ahmed Khaled Abdella Ahmed Xiaonan Shi

New Jersey Institute of Technology New Jersey Institute of Technology


Weihua Qing Wen Zhang

The University of Hong Kong Anhui University


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Cite This: J. Agric. Food Chem. XXXX, XXX, XXX−XXX

Influences of Air, Oxygen, Nitrogen, and Carbon Dioxide

Nanobubbles on Seed Germination and Plant Growth
Ahmed Khaled Abdella Ahmed,†,‡,§ Xiaonan Shi,†,‡ Likun Hua,‡ Leidy Manzueta,‡ Weihua Qing,‡,∥
Taha Marhaba,‡ and Wen Zhang*,‡

John A. Reif, Jr. Department of Civil and Environmental Engineering, New Jersey Institute of Technology, Newark, New Jersey
07102, United States
Department of Civil Engineering, Sohag University, Sohag 82524, Egypt

Department of Civil Engineering, The University of Hong Kong, Pokfulam 999077, China
S Supporting Information

ABSTRACT: Nanobubbles (NBs) hold promise in green and sustainable engineering applications in diverse fields (e.g., water/
wastewater treatment, food processing, medical applications, and agriculture). This study investigated the effects of four types of
NBs on seed germination and plant growth. Air, oxygen, nitrogen, and carbon dioxide NBs were generated and dispersed in tap
water. Different plants, including lettuce, carrot, fava bean, and tomato, were used in germination and growth tests. The seeds in
water-containing NBs exhibited 6−25% higher germination rates. Especially, nitrogen NBs exhibited considerable effects in the
seed germination, whereas air and carbon dioxide NBs did not significantly promote germination. The growth of stem length and
diameter, leave number, and leave width were promoted by NBs (except air). Furthermore, the promotion effect was primarily
ascribed to the generation of exogenous reactive oxygen species by NBs and higher efficiency of nutrient fixation or utilization.
KEYWORDS: nanobubbles, seed germination, plant growth, sustainable agricultural watering

1. INTRODUCTION Applying NBs in water treatment could efficiently remove water

Microbubbles (MBs) are generally defined as gaseous bubbles contaminants (e.g., rhodamine B, p-nitrophenol, and ala-
with a diameter less than 100 μm and larger than 10 μm.1 chlor).1,15 For example, fenitrothion is an agricultural pesticide
Nanobubbles (NBs) are bubbles with a diameter of <1 μm that is harmful to fish, animals, and humans. Ozone MBs were
(also known as ultrafine bubbles).2 The ultrasmall sizes of the shown to remove and degrade fenitrothion in lettuce, cherry
micro- and nanobubbles (MNBs) elicit many intriguing tomatoes, and strawberries.16
properties.3 For example, NBs have long residence times in Recently, NBs have been introduced into agricultural
the solutions as a result of their low buoyancy4 and high applications (e.g., promoting plant growth and germination).
efficiency of gas mass transfer as a result of the reduced bubble For example, several studies demonstrated that the use of NBs
size (high surface area) and increased internal pressure.4,5 promotes fast germination time and growth of seeds.17−19
Furthermore, the increased specific surface area of NBs Germination rates of barley seeds submerged in water-
increases the contact area between liquid and gas,6 which containing NBs (bubbles formed from gas mixtures of nitrogen
facilitates mass transfer, sorption, and chemical reactions at the and pure air) were 15−25% greater than those of seeds
gas/liquid interface. Therefore, MNBs have rapidly transformed submerged in distilled water with the same concentration of
into innovative technologies with versatile applications in DO.20 Germination of seeds is one of the first and most
agriculture, aquaculture, food engineering, wastewater treat- fundamental life stages of a plant and largely determines plant
ment, and medical applications (e.g., drug delivery for growth and yield production.21 Germination begins with water
chemotherapy).6−8 The application of oxygen NBs enhanced uptake by the seed (imbibition) and ends with the start of
the oxygen concentration from 7.7 mg/L in normal distilled elongation by the embryonic axis. Germination is a complex
water to 31.7 mg/L after 30 min.9 Thus, NBs are also used in process during which the seed must quickly recover physically
aquaculture to improve the water quality and replenish from maturation drying, resume a sustained intensity of
dissolved oxygen (DO), which increases the productivity of metabolism, complete essential cellular events to allow for the
fish in limited space.10 embryo to emerge, and prepare for subsequent seedling
The collapse of NBs creates shock waves, which, in turn, growth.22 For germination to occur, seeds require moisture, a
promote the formation of hydroxyl radicals (•OH), a highly suitable temperature, and in most cases an aerobic
reactive oxygen species (ROS) that non-specifically reacts with atmosphere.22
and decomposes organic matter.11,12 MBs have also been
demonstrated to remove residual pesticides of vegetables and Received: January 18, 2018
improve the quality of produce.13 The collapse of NBs creates Revised: April 5, 2018
the shock waves and promotes the formation of hydroxyl Accepted: May 3, 2018
radicals, which are essential for pollutant degradation.11,14 Published: May 3, 2018

© XXXX American Chemical Society A DOI: 10.1021/acs.jafc.8b00333

J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

In addition, MBs have proven to facilitate the growth of used daily. The pH values for ANBs, ONBs, NNBs, and tap water were
plants, such as lettuce, in the nutrient solutions.23 Water between 6 and 7, and the pH value for CNBs was around 4.5. We
containing air MBs led to a 2.1 times greater fresh lettuce leaf monitored DO of the freshly and stored NB suspension with Orion
weight and 1.7 greater dry leaf weight than macrobubble- Star A329 multiparameter meters (Thermo Fisher Scientific, Waltham,
MA, U.S.A.).
treated plants.24 NBs were shown to enhance the growth of 2.2. Examination of ROS Production in Water Suspension of
plants by improving the oxygen supply as nutrient elements.25 NBs. The generation of hydroxyl radicals (•OH) by NBs was detected
Moreover, according to a previous study,26 rice growth did not by a PL technique with terephthalic acid as a probe molecule.30,31
differ between plants irrigated with NB water (water saturated Terephthalic acid readily reacts with •OH to produce a highly
by oxygen NBs) and those irrigated with control water (without fluorescent product, 2-hydroxyterephthalic acid. The intensity of the
NBs), but NB water significantly reduced cumulative CH4 PL peak of 2-hydroxyterephtalic acid is in proportion with the amount
emission during the rice-growing season by 21%. The amounts of •OH radicals produced in water. This method relies on the PL
of iron, manganese, and arsenic that leached into the drainage signal at 425 nm of the hydroxylation of terephthalic acid with •OH
generated by NBs.
water before full rice heading were also reduced by the NB
Compressed air, oxygen, nitrogen, and carbon dioxide were
water. However, most of these previous studies used MBs for separately purged into 300 mL of the 5 × 10−4 M terephthalic acid
plant growth promotion, and only a few of them employed solution with a concentration of 2 × 10−3 M NaOH in a 1 L beaker for
NBs. Oshita and co-workers showed accelerated seed 30 min at a constant temperature of 20 °C. To increase the collapse
germination rates in mixed nitrogen and air NB water rate of NBs, an ultrasonic wave (100 W, 42 kHz ± 6%) was applied to
compared to that in distilled water and indicated that NBs the four paralleled samples of control and NB suspensions for different
promoted physiological activity of plants because of the times (0, 0.5, 3, and 6 min). After ultrasonication, the PL spectra of
generation of exogenous ROS and increased the mobility of these liquid samples were measured on a Hitachi fluorescence
the water molecules in bulk.18,19 One of the key advantages of spectrophotometer to determine the generated 2-hydroxyterephthalic
NBs for agricultural applications is that potentially environ- acid.
2.3. Germination Tests. Lettuce, carrot, and fava bean seeds were
mentally harmful chemicals in fertilizer or insecticide used in germination tests. The seeds of lettuce (Lactuca sativa), carrot
production and utilization can be reduced or eliminated. NBs Scarlet Nantes type (Daucus carota ssp. sativus), and fava bean (Vicia
could be a more environmentally sustainable alternative to faba) were purchased from The Home Depot in July 2017, with details
improve the crop yield. shown on the package in Figure S2 of the Supporting Information.
Despite the intensive studies on agricultural applications of For each seed type, five paralleled groups were prepared to
NBs, the enhancement mechanisms for NBs (e.g., different investigate the effects of four different NBs on their germination rates,
types or compositions) on seed germination and plant growth which were calculated daily by the percentage of germinating seed
remain elusive. Moreover, the roles of ROS produced in the number to the total number on the Petri dish.32 Among the five
suspension of NBs in the germination and growth process are groups, there was a control group using tap water without NBs. The
other four groups were performed by watering the seeds with
not fully understood. In this study, we investigated the effects of
suspensions of different NBs, including ANBs, ONBs, NNBs, and
four types of NBs on the germination and growth of plants. Air, CNBs. Each group was composed of 25 seeds, and each 5 seeds were
oxygen, nitrogen, and carbon dioxide NBs were generated in separately submerged in 10 mL of the tested NB suspension inside a
tap water and applied on the germination and growth tests for non-sterilized Petri dish (Figure S3 of the Supporting Information).
lettuce, carrot, tomato, and fava bean. Tap water was used as All Petri dishes were kept at the same room temperature (∼23 °C)
watering reference data (control group) for comparison. A and natural light conditions. During the germination experiments, the
photoluminescence (PL) technique was used to characterize tap water and the NB solutions were changed every 24 h from the
the ROS generation in different suspensions of NBs. We further stored bottles to avoid the depletion of oxygen or water evaporation
analyzed the potential mechanisms behind the promotion and also to replenish the dose of NBs.19 Germination tests for lettuce,
effects of NBs and the roles of reactive oxygen radicals and carrot, and bean seeds lasted for about 6−10 days. The sizes and
weights of the seeds were measured before and after the tests. The
nutrient delivery in germination and plant growth. hypocotyl lengths were measured daily to compare hypocotyl
elongation. Images were captured daily for all tested seeds and
2. MATERIALS AND METHODS processed by the ImageJ software to measure the hypocotyl length of
2.1. Production and Storage of NB Suspensions Containing the seeds.33 The images, hypocotyl elongations, and germination rates
Air, O2, CO2, and N2. Different kinds of NBs, including air of the tested seeds were compared among all different applied NBs.
nanobubbles (ANBs), oxygen nanobubbles (ONBs), carbon dioxide 2.4. Plant Growth Tests. For the growth study, fava bean (V.
nanobubbles (CNBs), and nitrogen nanobubbles (NNBs), were faba), carrot, and tomato San Marzano (Solanum lycopersicum) were
generated by direct injection of compressed air (Ultra Zero Grade grown in the garden soil (Miracle-Gro soil). The plants were grown in
Air, Airgas, Inc.), oxygen (purity of 99.999%, Airgas, Inc.), carbon rectangle tabletop planters with saucers, as shown in Figure S4 of the
dioxide (purity of 99.99%, Airgas, Inc.), and nitrogen (purity of Supporting Information. The height, width, and length of the planters
99.999%, Airgas, Inc.) through a tubular ceramic membrane [100 nm were 16.75, 20.02, and 60.33 cm, respectively. Five groups of each
pore size, wave aberration function (WAF) of 0.1, refraction, U.S.A.] plant type were cultivated. All groups were subjected to irrigation
into tap water as we reported previously.27 The gases were injected every 3 days by saturated water suspension of ANBs, ONBs, NNBs,
continuously for 90 min under a pressure of 414 kPa and a flow of 0.45 CNBs, and tap water only (control group). For each group, five seeds
L m−1 to reach a stable bubble size distribution and a saturation were inoculated in one planter with an apart distance of approximately
point.27 The hydrodynamic diameters of the produced NBs were 10 cm. For growth tests, the length or diameter (centimeters) of the
measured by dynamic light scattering (DLS) on a Zetasizer Nano ZS leave, stem, and root were measured depending upon the growth rate
instrument (Malvern Instruments) as reported previously.27,28 The with a caliper. The results were expressed as an average with standard
bubble size distribution is shown in Figure S1 of the Supporting deviation as the error bars.
Information. The used tap water was first left at ambient temperature 2.5. Statistical Analysis. All comparisons were made between the
for 24 h to allow free residual chlorine to exit water.29 A fresh NB control samples (irrigated by tap water only) and the test groups that
suspension was used immediately for the plant growth tests. However, underwent the treatments with different NB water in all of the tests.
for germination tests, all types of NB suspensions were generated The significance of difference for comparison was analyzed by one-way
every 3 days, stored separately in closed 1 gallon water bottles, and analysis of variance (ANOVA, t test, two sided, with a significance level

B DOI: 10.1021/acs.jafc.8b00333
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

α = 0.05). The results of the t tests are summarized in Table S1 of the

Supporting Information. The normality of replicate data on seed
germination and plant growth data was examined when necessary
upon Shapiro−Wilks W test by SPSS 11.5 (p > 0.05).

3.1. Effect of Different Types of NBs on the
Germination Rates of Vegetable Seeds. Figure 1 shows

Figure 2. Effect of different types of NBs on the hypocotyl length

during germination of (a) lettuce, (b) carrot, and (c) bean.

Figure 3 shows the hypocotyl growth process of lettuce under

immersion into different NB water and tap water. Clearly, the
promotion effects by NBs became evident on the fourth and
sixth days of incubation. Seeds exposed to NBs had a higher
germination rate and hypocotyl length than seeds treated with
Figure 1. Effect of different types of NBs on the germination rates of tap water.
(a) lettuce, (b) carrot, and (c) bean.

that four types of NBs consistently promoted the germination

rates of lettuce, carrot, and bean. After 6 days of submersion,
the lettuce germination rate reached 100% with NNBs (p <
0.05, as shown in Table S1 of the Supporting Information),
followed by CNBs, ONBs, ANBs, and tap water, which
corresponded to the germination rates of 85, 85, 82, and 80%,
respectively. The same results were achieved on carrot and
bean, for which the germination rates were highest under
irrigation by NNB water. For carrot and bean, ANBs and CNBs
did not significantly promote germination compared to tap
water (p > 0.05). Thus, NNBs had a considerable promotion
effect on the germination rate, probably because of the effective
delivery of nitrogen elements or other growth factors by NBs.4,5
3.2. Effect of NB Type on the Hypocotyl Length of
Vegetable Seeds. Figure 2 shows that the hypocotyl length
growth was fasten by exposure to NNBs or ONBs (p < 0.05).
For all three kinds of seeds, CNBs made no significant
difference to the hypocotyl length (p > 0.05). ANBs appeared
to slow down the growth rates of the hypocotyl length Figure 3. Photos of the hypocotyl growth process of lettuce seeds at
compared to tap water, which is observed in the last 2 days. different submersion days.

C DOI: 10.1021/acs.jafc.8b00333
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

3.3. Effect of NB Type on the Vegetable Growth.

Figure 4 presents the leaf number of tomato, carrot, and beans

Figure 4. Influence of the water type on the number of leaves of

tomato, carrot, and bean after 37 days. ∗ denotes significant differences
(p < 0.05) between the values of NB treatment groups and the control
group of the same kind of plants.

under exposure to different kinds of NBs. In comparison to tap

water, the number of leaves increased with exposure to most of
the tested NBs. NNBs led to a significant increase in tomato
leave number (p < 0.05), whereas ANBs did not increase but
instead reduced the number of tomato leaves. The t test
indicated that, in comparison to tap water, NNBs had a
significant promotion effect on all plants (p < 0.05), CNBs only
Figure 5. Effect of different types of NBs on stem length of (a) bean,
had a significant promotion effect on bean plants, and ONBs (b) carrot, and (c) tomato and stem diameter of (d) bean, (e) carrot,
promoted tomato. Conversely, ANBs had a negative effect on and (f) tomato.
tomato growth. Other groups did not show significant
differences from the control group. Figure S5 of the Supporting
Information shows that beans after 1 week of watering by four
different NBs grew quite differently. NB-treated beans grew
faster with apparent leaves sprouting out of their buds, whereas
the tap-water-treated beans had no leaf sprout during the same
initial growth period.
The effect of NBs on the stem length and diameter is
demonstrated in Figure 5 (p < 0.05). Similar to the results of
the leaf number, some of the results show that stem length and
diameter were both increased by NBs. However, ANBs
appeared to inhibit the growth of the stem length for bean
and tomato after 30 days compared to tap water. Stem length
exhibited distinct time-dependent growth (left column of
Figure 5). However, the stem diameter did not change with
exposure time, and also the effect of NBs varied on three types
of vegetables. For example, for the stem diameter of beans,
NNBs promoted its growth and the effects of CNBs/ONBs
were negligible, whereas ANBs had an inhibitory effect. For
tomato, the stem diameter was considerably increased by
ONBs, NNBs, and CNBs.
Panels a−c of Figure 6 indicate that there was no significant
difference for the leave length of beans and carrots watered by
NBs and tap water (except tomato). Panels d and e of Figure 6
also show the promotion effect of NNBs on the leaf width of
beans and carrots compared to the control group with tap water
(p < 0.05), whereas other NBs made no obvioius differences.
For tomato, its leaf length and width were both enhanced by
NNB water but inhibited by ANBs. Our results are inconsistent Figure 6. Effect of different types of NBs on leave length of (a) bean,
with a previous study that showed that ANBs significantly (b) carrot, and (c) tomato and leave width of (d) bean, (e) carrot, and
promoted the height and length of leaves and aerial fresh (f) tomato.
weight of Brassica campestris.9 Figure 7 shows that tomatoes
with submersion to CNBs, NNBs, and ONBs had bigger leaves
than those with tap water.
D DOI: 10.1021/acs.jafc.8b00333
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

Figure 7. Photos of tomato leaves after the same amount and frequency of watering by NB water and tap water.

3.4. ROS Generation by NBs and Dissolved Oxygen compared the physiological promotion effects of ONB water
Measurement. The ultrasound could accelerate the aggrega- and H2O2 solutions,17 indicating that the oxidative capacity of
tion process of NBs in an aqueous solution and promote ONB water was equivalent to 0.5 mM H2O2 and the oxidative
collapse and ROS generation.34 We measured the •OH radicals capacity of gas mixture NB water was equivalent to 0.3 mM
in the water saturated with NBs under different sonication H 2 O 2 . ONB water and equivalent concentration H 2 O 2
times. Without sonication, the PL intensity of different NB solutions exhibited a similar effect in promoting the
solutions is similar to the control group, indicating that germination rate of barley seeds.17 Additionally, NB water
terephthalic acid could self-decompose slightly and there were was shown to induce the expression of genes related to cell
no significant or detectable amounts of ROS in NB water division and cell expansion.18
without sonication. Figure 8 reveals that •OH radicals were In the past, ROS production in seeds has been widely
regarded as a symptom of oxidative stress that potentially leads
to deleterious consequences, such as cell death and cell damage
(e.g., oxidative degradation of lipids, proteins, and nucleic
acids).35 Research in recent decades, however, has highlighted
new roles for ROS as important physiological regulators of
cellular signaling pathways.19 Additionally, both endogenous
and exogenous sources contribute to the formation of
intracellular ROS.36 Owusu-Ansah and Banerjee discovered
that a moderately high ROS level in the progenitor population
sensitizes them to differentiation and establishes a signaling role
for ROS in the regulation of hematopoietic cell fate.37,38 Bailly
et al. suggested a concept of the “oxidative window for
Figure 8. Fluorescence intensity of different NB water under
germination”,39 a critical range of the ROS level at which the
sonication (100 W). ∗ denotes significant differences (p < 0.05) occurrence of the cellular events associated with germination is
between the values of NB groups and the control group under the optimal. As reported previously,39 a low level of ROS during
same sonication time. imbibition is inhibitory to germination and may result in
decreased antimicrobial defense. A high level of ROS could
produced in the NB water and both ONBs and ANBs destroy cells and produce pathological effects.40,41 The amount
generated considerable amounts of •OH radicals. NNBs of ROS produced in the NB water could be tuned to match the
seemed to quench some radical formation, resulting from range of the “oxidative window”,17 depending upon specific
sonication, which produced some •OH radicals under types of NBs (pure oxygen or oxygen mixture with air).36
sonication. CNBs did not produce considerable levels of •OH Although ROS, such as •OH, have a short lifetime, NB water
radicals in the solution under sonication. may be able to yield a constant sub-micromolar level of •OH,17
Figure S6 of the Supporting Information shows the changes which substantially enhances germination and plant growth by
of DO levels in water that was saturated with different kinds of NBs.
NBs and tap water. ANB water had almost the same DO In our research, ONBs generated a considerable amount of
concentration (8.7 mg L−1 on average) as tap water. NNBs and ROS and ONBs did not significantly promote the germination
CNBs reduced DO levels below the level in tap water. The DO of carrot seeds. However, ONBs had a promotion effect on the
level in ONB suspension progressively reduced from 41.8 to growth of tomatoes, which indicates that the ROS effect could
15.0 mg L−1, whereas the level of DO increased in the water be plant-specific. A previous study revealed that the oxidative
suspension of NNBs and CNBs, probably as a result of oxygen window for carrot seeds is narrow and the amount of
gas transfer from ambient air. The water was respiked with NBs exogenous •OH produced by ONBs could be above the toxic
every 3 days, which replenished DO in ONB suspension from threshold of carrot seeds,19 which is why ONBs had no obvious
15.0 to 41.8 mg L−1. promotion on carrot seeds. Meanwhile, although NNBs only
generated a small amount of ROS, it significantly promoted
4. DISCUSSION seed germination for all three kinds of seeds, which could be
4.1. ROS Effects on Germination and Plant Growth. related to not only the proper ROS but also the possible
Because ROS is one of the activation agents involved in cell increased nitrogen accessibility to plants.
wall loosening and cell elongation,18 the continuous supply of 4.2. Effects of the Growth Elements Delivered by NBs.
ROS by NBs may sustain a long-lasting stimulation of living Nitrogen and carbon, as key elements of biomass growth, have
organisms and, thus, promote plant growth.17 Liu et al. the profound effect on the germination rate, plant growth, and
E DOI: 10.1021/acs.jafc.8b00333
J. Agric. Food Chem. XXXX, XXX, XXX−XXX
Journal of Agricultural and Food Chemistry Article

properties.42,43 Injecting the solution of NNBs could provide Hydrodynamic diameter of ANBs, ONBs, NNBs, and
more accessibility to N2 and enhance the efficiency of molecular CNBs (Figure S1), image of seeds: (a) carrot, (b)
nitrogen fixation by diazotrophs or nitrogen-fixing organisms,44 tomato, (c) lettuce, and (d) fava bean (Figure S2), setup
which convert N2 to ammonia (NH3) by nitrogenase and, thus, of seed germination tests (Figure S3), real photo of the
promote seed germination or plant growth.45,46 Thus, NNBs or setup of the plant growth tests (Figure S4), p values for
NB technologies may hold the potential to promote the the t tests between control groups (irrigated by tap water
element absorption and utilization efficiency of plants without only) and the test groups that underwent the treatments
secondary pollution. with different NB water on seed germination and plant
Oxygen is extremely important for nutrient absorption in growth data (Table S1), growth of beans taken after the
plants because oxygen is responsible for transporting nutrients first week of the test (Figure S5), and changes of DO
across the cell wall and into the roots of the plant. As more levels in water suspension of different NBs (Figure S6)
oxygen is absorbed into the root, nutrient absorption will be (PDF)

improved. The root of the crop needs enough oxygen to sustain
its own metabolism and the whole plant growth.47 Root AUTHOR INFORMATION
hypoxia can lead to the weakening of the root respiration,
which may shift from aerobic to anaerobic and decrease the Corresponding Author
root growth, ion transport, and root fluid flow. Therefore, *Telephone: 973-596-5520. E-mail:
consistent with the previous research,24 we found that ONBs ORCID
increased lettuce and carrot germination, probably because Xiaonan Shi: 0000-0002-1599-703X
ONBs support the breathing of the plant root.48 Likun Hua: 0000-0002-1228-6128
Overall, NNBs showed a considerable promotion in both Wen Zhang: 0000-0001-8413-0598
seed germination and plant growth for all species in our
experiments, similar to ONBs. Especially, NNBs enhanced the Author Contributions

germination rate of lettuce seeds by 25% higher than tap water Ahmed Khaled Abdella Ahmed and Xiaonan Shi contributed
alone. ANBs and CNBs did not show an obvious enhancement equally to this work.
on seed germination, but CNBs did show a mild enhancement Funding
on plant growth, especially with bean and tomato. According to The authors thank the Egyptian Ministry of Higher Education
PL results, four NBs had different ROS generation abilities. (Cultural Affairs and Missions Sector) for the scholarship
ONBs generated the highest concentration of ROS, followed by support and the Undergraduate Research Innovation Phase I
ANBs, CNBs, and NNBs, which provide a partial explanation and Phase II grants from New Jersey Institute of Technology
for the promotion on seed germination. Additionally, NBs may (NJIT), the Department of Civil and Environmental Engineer-
more effectively deliver nutrients (e.g., nitrogen or oxygen) to ing of NJIT, and Otto H. York Center for Environmental
plants or to nitrogen-fixing organisms in the root environment Engineering and Science for their financial and instrumental
as a result of the high surface area and mass diffusion rates. support.
Also, it is interesting that ANBs have a lower promotion effect Notes
than both ONBs and NNBs. Clearly, we speculate that nitrogen The authors declare no competing financial interest.

or oxygen exerts different mechanisms of plant growth
enhancement. For instance, enhanced nitrogen delivery might ABBREVIATIONS USED
be the governing factor for NNBs, while ONBs may play a
different role, such as enhancing the activity of aerobic root MBs, microbubbles; NBs, nanobubbles; MNBs, micro- and
microorganisms and indirectly promoting plant growth. In nanobubbles; ROS, reactive oxygen species; •OH, hydroxyl
contrast, ANBs have substantially different properties from radicals; ANBs, air nanobubbles; ONBs, oxygen nanobubbles;
NNBs or ONBs, which could explain the resulting different CNBs, carbon dioxide nanobubbles; NNBs, nitrogen nano-
phytoplankton effects. For example, ANBs has a different bubbles; PL, photoluminescence; DO, dissolved oxygen; NH3,

ionization energy than NNBs as well as different surface charges
and ζ potentials in aqueous solutions. For example, the
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