Article

Cite This: J. Agric. Food Chem. 2019, 67, 10813−10822 pubs.acs.org/JAFC

Effect of Dielectric Barrier Discharge Cold Plasma on Pea Seed

Growth
Xiaoting Gao,†,‡ Ai Zhang,† Paul Heŕ oux,§ Wolfgang Sand,† Zhuyu Sun,† Jiaxun Zhan,† Cihao Wang,†
Siyu Hao,† Zhenyu Li,† Zhenying Li,† Ying Guo,∥ and Yanan Liu*,†,‡
†
College of Environmental Science and Engineering, Donghua University, 2999 North Renmin Road, Shanghai 201620, China
‡
Shanghai Institute of Pollution Control and Ecological Security, Shanghai 200092, China
§
Department of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal H3A 0G4, Canada
∥
Department of Applied Physics, College of Science, Donghua University, Shanghai 201620, China
See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

*
S Supporting Information
Downloaded via COLUMBIA UNIV on October 22, 2019 at 06:14:15 (UTC).

ABSTRACT: Traditional seed pretreatment methods cause secondary pollution for the application of various chemicals. This
study investigated the effect of dielectric barrier discharge (DBD) cold plasma on seedling growth. Effects of plasma-activated
tap water (PATW) and plasma-activated seeds (PAS) were compared for germination rates, seedling height, dry weight, and
chlorophyll content. Results show that compared with controls these growth parameters were all increased by more than 50%.
The yields and contributions of hydrogen peroxide, nitrate, nitrite, and ammonium were quantified. Hydrogen peroxide and
nitrate have an important role in seedling growth. By etching, the seed epidermis free radicals can reduce the apparent contact
angle and increase the water absorption of the seeds. In addition to the low cost of PATW and PAS compared with commercial
fertilizers, DBD does not involve any chemical addition. Thus, both PATW and PAS can be an alternative for improvement of
agricultural production.
KEYWORDS: DBD plasma, plasma-activated tap water (PATW), plasma-activated seeds (PAS), seedling growth,
improvement mechanism

1. INTRODUCTION
According to the United Nations Food and Agriculture
Organization, deteriorating environment, climate changes,
and urbanization caused by human activities have dramatically
increased the global food demand.1,2 Traditionally, there are
two ways to improve seedling growth. The first one involves
the use of a variety of dormancy-breaking methods, including
physical pathways (magnetic fields, ultraviolet, hot water
immersion)3−5 and chemical agents (disinfectants, fungicides,
hormones)5−8 as presowing seed processes to enhance seed
germination and growth rate. The second way involves the
addition of chemical fertilizers to supply nitrogen, phosphorus,
and potassium needed for plant growth.1,9,10 However, these
methods have their own drawbacks, such as they are time
consuming, labor intensive, and causing secondary pollu-
tion.11,12
In recent years, nonthermal plasma has become an efficient,
innovative, and green alternative to traditional seed culture.
Dielectric barrier discharge (DBD) is one of the forms of
nonthermal plasma that can conveniently generate ultraviolet
light, high-energy electrons, and active particles at atmospheric
pressure.9,13 DBD can be used to produce plasma-activated tap
water (PATW) to irrigate the seeds or to activate seeds by
direct discharge on the seed surface (plasma-activated seeds,
PAS). Reactive oxygen species (ROS) and reactive nitrogen
species (RNS) are produced during discharge in the
atmosphere of air. Among them, both long-lived species
(such as H2O2, NO2−, NO3−, etc.) and short-lived ones (such
as •OH, O2−•, 1O2, ONOOH, etc.) can optimize the biological
growth process.14−16 It has been found that tap water activated
by plasma has a significant effect on plant growth. This seems
to be correlated to RNS such as NO2− and NO3−.17 Similarly, a
direct exposure of seeds to plasma also increases seedling
growth. Seeds treated with radiofrequency plasma for 10 s
experience increase of the germination rate (GR) of almost
100%.15 This can be attributed to the fact that DBD etches the
seed surface and allows nitrate to enter the seed. This process
simulates seed immersion in nitrate nitrogen-rich water.18
However, there are few reports studying the effects of PATW
and PAS on agricultural output. Moreover, most studies only
explore the germination rate to evaluate the effect of plasma
but ignore other parameters like plant growth.19−21 Addition-
ally, the contributions of RNS and ROS to plant growth have
not been studied quantitatively.
In this study, the mechanisms of PATW and PAS on
seedling growth were investigated. Germination rate, height,
dry weight, and chlorophyll were measured to assess the
impacts of PATW and PAS. Furthermore, hydrogen peroxide,
nitrate, nitrite, and ammonium were quantified to clarify the
mechanisms of PATW on seedling growth. An ion-containing
medium (supplying the same concentration as the discharge)
was used to explore the contribution of each ion.
Simultaneously, the morphology and hydrophilicity of the
Received: May 17, 2019
Revised: August 25, 2019
Accepted: September 6, 2019
Published: September 6, 2019

© 2019 American Chemical Society 10813 DOI: 10.1021/acs.jafc.9b03099

J. Agric. Food Chem. 2019, 67, 10813−10822
Journal of Agricultural and Food Chemistry
Figure 1. Schematic diagram of the experimental system.
seed surface were investigated to clarify the mechanism of PAS
action on seedling growth. These findings can give options for
improvement of agricultural production in the future.
2. MATERIALS AND METHODS
2.1. Materials. Pea seeds were purchased from a local agricultural
market. The seeds were shaken before taking the samples, and each
300 seeds weighed between 150−180 g.
The reagents, including sodium hydroxide, hydrogen peroxide,
ethanol, acetone, titanium potassium oxalate, sodium nitrate, sulfuric
acid, sulfamic acid, phosphoric acid, p-aminobenzenesulfonamide,
ammonium chloride, sodium potassium tartrate, N-(1-naphthyl)-
ethylenediamine dihydrochloride terephthalic acid, and hydrochloric
acid were of analytical grade and purchased from Sinopharm
Chemical Reagent Co., Ltd (Shanghai, China). The solutions were
prepared using deionized water, while nitrate and hydrogen peroxide
solutions used for reference experiments were made with tap water.
2.2. Plasma Apparatus and Determination of Power. A
dielectric barrier discharge (DBD) plasma reactor (CTP-2000K,
Nanjing Suman Electronics, China) was used as the plasma source at
atmospheric pressure. The schematic diagram of the experimental
apparatus is shown in Figure 1. It consisted of three main parts: a high
voltage alternating current power source, a DBD reactor device (self-
designed), and an oscilloscope (TDS 2012B, Tektronix).
The DBD reactor is the core of the experimental system. The
reactor contains two parallel metal electrodes and a quartz container
for seeds and tap water. The electrode is 1 cm thick and 15 cm in
diameter. The diameter, internal height, and external height of the
quartz container are 15, 0.8, and 1.3 cm, respectively. Air can enter
through small holes on both sides of the quartz container. The
oscilloscope is fitted with two probes: Tektronix P6015A for the high
voltage and Tektronix TPP0101 for the capacitor voltage.
2.3. Experimental Procedure. Tap water (TW) was selected as
the water matrix for this study. Fifty milliliters of water was used for
each experiment. Discharge powers ranged from 60 to 164 W, with
reaction times between 5 and 20 min.
Seeds were divided into two 300-seed groups, which were soaked in
1 L of TW or PATW for 15 h. Then, the seeds were placed on a wet
paper filter in a Petri dish (285 × 205 × 40 mm3) filled with 50 mL of
water (TW or PATW). Two groups were irrigated with TW or
PATW, each for 12 h.
For the DBD plasma treatment, 300 seeds were placed in Petri
dish-like containers and exposed to plasma with a discharge power of
9−35 W. The reaction times ranged from 1−10 min. The treated
seeds were soaked in 1 L of TW for 15 h and processed as follows: the
seeds were put on a wet filter paper in a 285 × 205 × 40 mm3 Petri
dish with 50 mL of TW, which was refilled after every 12 h. For
comparison, the same number of seeds, but without plasma discharge,
were treated in the same way.
When the sprout of the plant reached 1 cm height, the plants were
exposed to a simulated sunlight (P = 10 W, λred: 620−660 nm, λblue:
10814
Article
450−470 nm, λred/λblue = 3:1) for 12 h per day. The entire growth
cycle took 15 days.
2.4. Analytical Methods. After 15 days, the plants were harvested
and the individual heights were measured. The average height was
calculated as follows
total height of all plants
AH (cm) =
total number of plants (1)
The numbers of germinated seeds were also counted. The
germination rate (GR) was calculated as follows
number of germinated seeds per dish
GR (%) = × 100
number of total seeds per dish (2)
The harvested plants were then dried in an oven (60 °C) for 3 days.
Afterward, the dry weight of each group and the chlorophyll
concentration of the leaves was measured (Supporting Information,
determination of chlorophyll). The chlorophyll concentrations C(mg/
g) were calculated as follows22
C (mg/g) = (8.02A 663 + 20.2A 645)V /1000W (3)
where V means the total volume of the sample solution (mL), W
means the weight of sample (g), A663 means the absorbance of the
solution at 663 nm, and A645 means the absorbance of the solution at
645 nm.
The concentrations of hydrogen peroxide (H2O2), ammonia
nitrogen (NH4+-N), nitrite nitrogen (NO2−-N), and nitrate nitrogen
(NO3−-N) in the PATW were determined colorimetrically by
spectrophotometric measurements.23,24 The hydroxyl radicals were
combined with terephthalic acid, which in the presence of •OH forms
2-hydroxyterephthalic acid (HTA). HTA absorbs light at 310 nm and
emits fluorescence at 425 nm.25
Plasma-treated and nontreated seeds were examined both under a
scanning electron microscope (SEM) (Hitachi S4800) to analyze the
surface structure. Surface structures of cross and longitudinal sections
of the plant root were also visualized by SEM. A detailed analytical
method is provided in the SI.
For the determination of water absorption, 300 dry seeds were first
weighed and recorded as m0. Subsequently, seeds were soaked in 1 L
of TW for 1 h up to 20 h. The seeds were then removed from the
water, the excess surface moisture wiped off with blotting paper, and
then weighed and recorded as mt. The water adsorption was
calculated using the following equation2
m t − m0
water adsorption (%) = × 100
m0 (4)
The apparent contact angle was measured using the sessile drop
technique (SL200KS, KINO Industry Co. Ltd). The test was
performed by dripping 2 μL of distilled water on the seed surface.
An image was taken under an optical microscope in conjunction with
computer-aided measurement. Plasma was diagnosed using optical
DOI: 10.1021/acs.jafc.9b03099
J. Agric. Food Chem. 2019, 67, 10813−10822
Journal of Agricultural and Food Chemistry
Figure 2. Seedling growth in PATW under different discharge powers and times. (a) TW treated with different powers for 10 min. (b) TW treated
at 87 W for different times.
Figure 3. SEM of the cross section through a seedling root (a) cultured with TW and (b) cultured with PATW treated at 87 W for 10 min.
emission spectra (OES) (2048TEC, Avaspec, Netherlands) in the
range of 198−947 nm.
To analyze the microbial community structure, untreated seeds and
seeds treated at 15 W for 3 min were used for DNA extraction and
metagenomic sequencing (Majorbio Bio-Pharm Technology Co.,
Ltd., Shanghai, China). Details of the analytical methods are provided
in the SI.
2.5. Statistical Analysis. All treatments were done in triplicates,
and experiments were repeated three times. The data are presented as
the mean ± standard deviation of the triplicates. Statistical analyses of
the data were performed using student’s T-test to establish
significance between data points, and significant differences were
based on p < 0.05 or 0.01.
3. RESULTS AND DISCUSSION
3.1. Seedling Growth after Irrigation with Plasma-
Activated Water. As illustrated in Figure 2, the seedling
growth of all treated samples was considerably higher than in
the control group. This means that seedling growth was
improved by irrigating with PATW. In Figure 2a, it is shown
that an applied power of 60 W is not sufficient to promote
seedling growth. However, if the applied power exceeds 60 W,
the seedling height was much higher than the control. On the
other hand, no significant differences were noted in the heights
of seeds cultivated with TW at discharge power values of 73,
87, and 122 W. An exemption was the result of the 164 W
discharge, where a slight decrease in height was recorded. The
low power values are obviously not conducive to the
production of hydrogen peroxide, nitrogenous compounds,
10815
Article
and other nutrients. In contrast, with high power plasma
treatment conditions, excess H2O2 was produced, which is
toxic to growth.17 The values for chlorophyll and dry weight
indicated low enhancement and high inhibition for increased
treatment power. An applied power value of 87 W was the
optimal value for the effect on seedling growth.
The effects of discharge time on seedling growth are shown
in Figure 2b. With treatment times of 5 min or more, the
height and chlorophyll concentrations of the seedlings were
notably higher than those for the control. However, there was
no significant difference observed between the treatment
periods of 5 and 20 min. After 10 min of treatment, dry weight
reached a maximum value and no further increase was
observed. The modification of the operational conditions
favored the generation of nitrogen by the discharge process.26
Short-term discharges produced sufficient nitrogen compounds
as nutrients for the cultivation of the seeds. Thus, considering
the growth enhancement and energy consumption, the
optimum discharge power and treatment time are 87 W and
10 min.
The root channel organization of the plant is responsible for
long-distance transport of water, inorganic salts, and nutrients
in the plant. It was therefore necessary to compare the root
cross sections of plants that had been cultivated either with
TW (Figure 3a) or with PATW (Figure 3b). Compared with
the roots of plants cultured in TW, the PATW-cultured plant
roots had thinner phloem walls and larger pore diameters.
DOI: 10.1021/acs.jafc.9b03099
J. Agric. Food Chem. 2019, 67, 10813−10822
Journal of Agricultural and Food Chemistry Article

Table 1. Seedling Growth in PATW at Different pH

pH height/cm IR (%) germination/% IR (%) dry weight/g IR (%) chlorophyll content/(mg/L) IR (%)
3 15.37 ± 2.68 −2 75.33 ± 2.24 −20 11.32 ± 0.55 −13 4.90 ± 0.2 −15
5 11.62 ± 1.68 −26 96.67 ± 1.71 2 10.39 ± 0.61 −20 3.73 ± 0.03 −35
6 12.20 ± 1.85 −22 98.33 ± 1.54 4 9.99 ± 0.22 −23 3.77 ± 0.14 −35
7 15.64 ± 2.20 0 94.33 ± 1.77 0 13.00 ± 0.25 0 5.78 ± 0.26 0
8 15.01 ± 2.24 −4 92.67 ± 1.02 −2 12.02 ± 0.61 −8 4.87 ± 0.21 −17
9 11.97 ± 1.63 −23 93.67 ± 1.42 −1 10.19 ± 0.69 −22 3.59 ± 0.18 −38

Increased diameters for transport cells enhance nutrient
exchange.27 Therefore, a seedling treatment with PATW
created an improved growth by changing the physiological
structure of the plants.
3.1.1. Effect of PATW with Different pH Scales on Seedling
Growth. The treatment with DBD plasma resulted in a pH
decrease of PATW (Tables S1 and S2). The decrease can be
attributed to the generation of nitric acid, hydrogen peroxide,
and ozone by the discharge process.15,26 Sivachandiran et al.
proved that the acidity of plasma-activated water affected
seedling growth.1 To explore the effects of pH on plant growth,
PATW (87 W, 10 min) was adjusted to different pH. The
results are shown in Table 1. The initial pH of TW after
discharge for 10 min at 87 W was 2.86. The pH of PATW was
adjusted with 1 mM NaOH to pH of 3−9. The increase rate
(IR) was calculated using eq 5, where growth parameters
represent height, germination, dry weight, and chlorophyll
content
IR (%) = [(growth parameter value at different pH
− growth parameter value at pH 7)
/growth parameter value at pH 7] × 100
(5)
It was noted that pH above 8 or pH below 6 did not promote
plant growth (as shown in Table 1). A pH between 6 and 8 did
not affect the germination rate, but other growth parameters,
such as height, dry weight, and chlorophyll content were
reduced. In addition, the changes in pH had no effect on the
concentrations of H2O2 and NO3− during the plasma
activation process (shown in Figure 4f). Therefore, this
study points to neutralized PATW as the optimal culture
solution.
3.1.2. Contribution of H2O2 to Seedling Growth. Hydrogen
peroxide is considered as a stable product of PATW. Its half-
life period is 8 h-20 days. So it has an effect for quite a long
time.9,28 Hydrogen peroxide interferes with the levels of
abscisic acid, thus changing the dormancy period of seeds. In
this way, seed development and growth quality are improved.9
As shown in Figure 4a, with a discharge power below 87 W,
the hydrogen peroxide content increased slowly, while at
values exceeding 87 W, the content increased sharply.
According to previous studies, there are three possible ways
to generate H2O2: the dissolution of gaseous H2O2;29
electrolysis of water30,31 in which the electrolyzed O2 captures
electrons from the cathode to form O2− and further combines
with H+ to form H2O2 (eqs 6−8); the transfer of energy
between excited species and water molecules caused by high
electron density.26,32 The reaction mechanisms are shown in
eqs 9−10
O2 + e− → O2− (6)
O2− + H+ → HO2 (7)
10816
2HO2 → H 2O2 + O2 (8)
e− + H 2O → H + OH + e− (9)
OH + OH → H 2O2 (10)
Under optimal reaction conditions, the amount of H2O2
produced was 17.1 mg/L (Figure 4a). This is consistent with
the results of Zhou et al., who found that plasma discharge can
produce 17.4 mg/L H2O2 in 10 min.26 As shown in Figure 4g,
•
OH (maximum intensity at 309 nm in the emission spectrum)
was the major contributor to PATW reactivity. Besides, the
amount of •OH radical production under optimal conditions
was 2.29 mmol/L (Figure 4b), which can theoretically produce
38.93 mg/L H2O2. Therefore, the •OH radicals can partially be
converted to H2O2.33 With an increase of discharge time, the
H2O2 further reacted with some compounds in PATW to form
other active substances, such as peroxynitrite (ONOOH/
ONOO−) and nitrate.28,34
To further analyze the contribution of H2O2, the study
configured H2O2 solution to culture seeds (shown in Figure
4c). The concentration of H2O2 was the same as that produced
by the DBD discharge. The variation of seedling height and dry
weight during H2O2 treatment was in good agreement with the
data for the DBD treatment. However, the improving effect of
DBD treatment was slightly higher than that for the H2O2
treatment. This means that other synergistic effects (such as
NO3−, NH4+, •OH) also contributed to the growth promotion.
3.1.3. Contribution of Nitrogen Compounds to Seedling
Growth. Nitrogen is indispensable for plant growth. However,
the natural form of nitrogen (diatomic nitrogen, N2) has a
strong triple bond, making absorption by plants impossible.
Plasma synthesis is considered a green alternative to traditional
nitrogen fixation, as it involves no chemical agents and cause
no secondary pollution.9 During discharge, ionized nitrogen,
such as nitrite (NO2−) and nitrate (NO3−), is thought to be
generated.26 It is reasonable to suppose that a solution
containing a suitable nitrogen source will form an environment
conducive to the growth of pea seeds. In view of this, it was
necessary to analyze the nitrogen content and conversion in
PATW.35 As shown in Figure 4g, the nitrogen second positive
system (N2), the nitrogen ion (first negative system, N2+), and
the NH emission are visible in the optical emission spectra.
These groups are the precursors of nitrogen compounds useful
as nutrients.36,37
The amounts of nitrogenous substances in PATW appeared
to follow the order NO2− < NH4+ < NO3−. Figure 4e
demonstrates that both ammonium and nitrate concentrations
increased with an increase of discharge power and treatment
time. The concentration of ammonium is less than 2 mg/L,
which is much lower than that of nitrate at 250 mg/L. Nitrite
could only be detected under low power conditions, since it
would be converted to nitrate nitrogen, with high discharge
power and time.38,39 It can be speculated that nitrate nitrogen
DOI: 10.1021/acs.jafc.9b03099
J. Agric. Food Chem. 2019, 67, 10813−10822
Journal of Agricultural and Food Chemistry Article

Figure 4. Production of chemical substances under different powers and times and their effects on plant growth. (a) H2O2 production and its
concentration change after pH adjustment; (b) •OH production; (c) effect of H2O2 (same concentration produced by DBD), and error bars are
unilateral; (d) effect of NO3− (same concentration produced by DBD), and error bars are unilateral; (e) NO2−, NH4+, and NO3− productions
under different powers and times; (f) NO3− production under different powers and times and its concentration change after pH adjustment; (g)
OES of PATW (87 W,10 min); (h) simulation of the seed growth process.

10817 DOI: 10.1021/acs.jafc.9b03099

J. Agric. Food Chem. 2019, 67, 10813−10822
Journal of Agricultural and Food Chemistry
dominated plant growth compared with other nitrogen-
containing compounds.
Ammonium was derived from the dissolution of ammonia
under acidic conditions, which was synthesized by excited
nitrogen and hydrogen in the plasma atmosphere.40 The low
amount of water vapors in air may explain the very low amount
of ammonia compared with nitrate.
N2 + 6H 2O → 2NH3(g) + 6OH
NH3(aq) + H+ → NH+4(aq)
The nitrate synthesis process is sophisticated, involving the
basic processes of eqs 13−16 (Table 2)36−38 and the collision
Table 2. Equation for Formation of Nitrate by Plasma
Discharge from Nitrogen in the Air
phase1 N2 + O → NO + N (13)
N + O2 → NO + N (14)
NO + O + M → NO2 + M (15)
O2 + O + M → O3 + M (16)
phase2 2NO2(g) + H 2O → NO2(aq) + NO3(aq) + 2H+(aq)
NO(g) + NO2(g) + H 2O → 2NO2(aq) + 2H+(aq) (18)
phase3 NO2 + O3 → NO3 + O2 (19)
NO2 + H 2O2 + H+ → NO3 + H 2O + H+ (20)
of activated oxygen and nitrogen leading to the rapid
conversion into nitrogen oxides (NOX). Some of the activated
oxygen atoms combined to the strongly oxidizing ozone
because of ionization of air by the high voltage. In the second
stage (eqs 17 and 18 in Table 2), nitrogen oxides dissolve in
water-produced nitrite and nitrate.32,39,41 However, due to the
strong oxidation of ozone and H2O2, nitrite is rapidly oxidized
to nitrate (eqs 19 and 20, Table 2).34,38,39 The nitrite−nitrate
conversion mechanism also explains why PATW was strongly
acidic, from the formation of hydronium ions.
In this experiment, the effect of nitrate in PATW on seedling
growth was tested using 40−230 mg/L NaNO3 as the culture
solution. The results are shown in Figure 4d. The different
nitrate concentrations did not increase the height significantly.
Figure 5. Seedling growth after treatment of seeds with DBD plasma: (a) treatment for 3 min with various power values, (b) treatment at 15 W for
different durations.
Article
This indicates that the lowest nitrate concentration (40 mg/L)
had already met the growth needs of the peas. The effect of
nitrate on the dry weight of plants is generally presented as a
low-promotion and high-repression influence, owing to excess
nitrogen produced as toxic nitrides that interfere with plant
growth.26,42 However, an appropriate amount of nitrate
nitrogen, as obtained in this study, can characterize PATW
as a green fertilizer. This study simulates the entire growth
(11)
process of the seed, which is shown in Figure 4h.
3.2. Seedling Growth of Plasma-Activated Seeds. In
(12)
addition to PATW, seeds treated directly by plasma also
showed enhanced growth. As shown in Figure 5, in comparison
with untreated seeds, almost all measured parameters were
improved significantly by DBD plasma treatment. As illustrated
in Figure 5a, the average height, chlorophyll, and dry weight
increased by 30−50, 20−45, and 30−40%, respectively,
compared with controls. There were no significant differences
measurable for discharge powers of 9 and 35 W. However, if
the power increase above 40 W, seed height and dry weight
declined by 15 up to 50% (Figure S1). Obviously, excessive
DBD plasma is detrimental to the seeds.15
With 3 min of discharge time, the best stimulation of
(17) seedling growth was achieved (shown in Figure 5b). The
average height, chlorophyll content, and dry weight increased
to 51, 46, and 34%, respectively. Although the dry weight
remained at a steady increase of 30%, the average height and
chlorophyll content of seedlings decreased with prolonged
treatment times. This means that seedlings are dose sensitive
and that attention needs to be paid to optimize DBD treatment
conditions.
3.2.1. Seed Characteristics and Water Absorption.
Correlations between seed growth and seed surface structure
changes after plasma treatment have been reported.15,17,19,20
To explain the enhancement of seed growth by plasma
treatment, the surface structure of pea seeds was recorded by
SEM. As shown in Figure 6, the surface network structure of
pea seeds became distorted and partially destroyed by DBD
plasma treatment. The ridges on the seed epidermis gradually
disappeared. This observation is similar to that of Lee et al.,
who described that plasma treatment caused surface
modification of spinach seeds.43 DBD plasma bombards
seeds with free radicals and ions. This leads to seed coat
10818 DOI: 10.1021/acs.jafc.9b03099
J. Agric. Food Chem. 2019, 67, 10813−10822
Journal of Agricultural and Food Chemistry Article

Figure 6. Surface structure, apparent contact angle, and water absorption. (a−c) Untreated seeds; (d−f) seeds treated with DBD plasma for 3 min;
(g) seeds treated with different powers for 3 min; (h) seeds treated with 15 W for 0−10 min.

erosion.21,26,43,44 The altered seed coat increases the hydro- 3.2.2. Contribution of Plasma-Generated Reactive Species
philicity of the seed and in this way increases water absorption. and Other Synergistic Effects. The free radicals and ions
The wettability of the seeds is characterized by the apparent generated by plasma irradiation obviously play a major role in
contact angle, which takes into account both the chemical promoting the germination and growth of plants. Data in
structure and the roughness of the surface. The wetting of Figure 7 for the optical emission spectra allow to identify the
rough heterogeneous surfaces can be accounted for by the
Cassie and Wenzel models.19,45 The apparent contact angle of
the control seeds was 100°, while after 3 min of DBD plasma
irradiation, the angle decreased to 69° (Figure 6c,f). The
current findings are consistent with those of Zhou et al., who
found that plasma treatment significantly decreased the
apparent contact angle of Mung Bean seed.26
The change in wettability corresponds with a change in
water absorption, as presented in Figure 6g,h. There was rapid
water uptake in the first 5 h, followed by a slow one with 15 h.
Afterward, the seed mass remained approximately constant.
Consequently, the irradiated seeds absorbed more water than
the unirradiated seeds. Also, the water absorption rate was
initially increased. The most suitable DBD treatment was 3
min. Data in the literature indicate that legumes have a lipid
outer layer, which blocks water absorption. The results of da
Silva et al.20 indicate that the free radicals generated by the
Figure 7. OES of plasma-activated seeds (15 W, 3 min).
plasma oxidize this lipid layer and in this way chemically
modify the seed epidermis. This causes the increased water following radicals: N2(C−B), N2(B−A), N2+, NO, O2+, OH,
absorption of irradiated seeds. On the other hand, a prolonged
H(β), CO, H2, H(α), and N. In the study of Lee et al.,44 NO
treatment time and treatment power reduce the water
absorption. Different crops have different sensitivities and produced by plasma improved the quantity and speed of
tolerances to plasma, and excessive treatment may reduce germination and the development of alfalfa seedlings. Volin et
positive effects.46 This means that one needs to find the al.47 noted that deposition of nitrogen on the surface of seeds
optimum value for the treatment time and plasma intensity. had a positive effect on their germination, with N2 and N2+
10819 DOI: 10.1021/acs.jafc.9b03099
J. Agric. Food Chem. 2019, 67, 10813−10822
Journal of Agricultural and Food Chemistry Article

accounting for most of the peaks in the emission spectrum. range 300−400 nm at 293.88 mW/cm 2 for 3 min
The existence of CO and O2+ signals confirmed that chemical (corresponding to the optimal treatment time of this
etching of the seed surface by plasma played an important role experiment). Data in Figure S2 indicate that UV slightly
in stimulation of seed germination.48 increased the water absorption of the seeds, however far less
In previous studies, we have reported that DBD plasma also than the effect measured for the plasma application. This
has a sterilizing effect.49 Free radicals generated by the plasma means that the UV light contributed only by a small part to the
have an oxidizing effect, which partially killed the fungi on the plasma effect.
seed epidermis.50 3.3. Comparison between PATW and PAS. Although
The rarefaction curve constructed from the α diversity index both PATW and PAS promote seedling growth, they have
of each sample at different sequencing depths can be used to different application areas. As shown in Table 4, PATW has a
illustrate the integrity of the sample sequencing data.51 The
sobs index curve in Figure S3 tends to be flat, indicating that Table 4. Increase of Seedling Growth Parameters for PATW
the amount of sequencing data is reasonable. Application and PAS under Optimal Conditions
α diversity indexes can be used to quantitatively analyze the
increase (%)
richness and the diversity of microbial communities, in which
sobs, chao, and ace indices emphasize community richness. growth
parameters PATW/control (%) PAS/control (%) PATW/PAS (%)
Shannon emphasizes community evenness, and coverage
height 59 51 5.8
reflects the coverage of the community.51 According to
chlorophyll 48 46 1.6
Table 3, both community richness and evenness decrease
dry weight 68 34 25
after plasma treatment.

Table 3. Richness and Diversity Indices of Fungal Microbial

Communities on Untreated and Treated Seed Epidermis better incentive effect compared with PAS. Especially, the dry
sample/estimators ace chao sobs shannon coverage weight significantly increases. In addition, PATW is rich in
untreated 180.1841 180 180 3.4263 0.999985 nitrogen compounds required for plant growth. Therefore,
treated 141.8065 142 140 3.3412 0.999945 PATW can not only be an effective substitute for fungicides
but is also suitable for plants on poor soil or plants with poor
The distributions of the fungal community at phylum and growth.
genus level are depicted in Figure 8. All values for fungi of the It is worth mentioning that the production costs of PATW
and PAS are RMB 175 per ton and RMB 2.7 per ton,
respectively. These values are far below those for chemical
fertilizers (RMB 2500 per ton). PAS minimizes production
costs and has improved growth. This is a result of improved
germination because of facilitated epidermis breaking. These
findings provide options for increasing agricultural production.

■
*
ASSOCIATED CONTENT
S Supporting Information
The Supporting Information is available free of charge on the
ACS Publications website at DOI: 10.1021/acs.jafc.9b03099.
Materials used and details of analytical methods; tables
showing pH values of solutions under different treat-
ment conditions (PDF)

■
Figure 8. Phylum- and genus-level distribution of fungi communities
from untreated and treated seeds.
AUTHOR INFORMATION
Corresponding Author
genus level indicate a reduction of abundance: Mycosphaerella *E-mail: liuyanan@dhu.edu.cn. Tel/Fax: 86-21-67792538.
(untreated 18.53%, treated 17.45%), Pleosporaceae (untreated ORCID
7.25%, treated 6.77%), Pithya (untreated 5.19%, treated
4.34%), Davidiellaceae (untreated 4.19%, treated 2.92%),
Wolfgang Sand: 0000-0002-6724-0960
Mortierella (untreated 1.28%, treated 1.13%), and Gibberella Yanan Liu: 0000-0002-2582-7501
(untreated 1.02%, treated 0.86%) They all belong to Notes
The authors declare no competing financial interest.

Ascomycota. Mycosphaerella, a dominant fungus, is the largest
genus of plant pathogens. The phyla, which are significantly
reduced in abundance, are parasites feeding on plant decay,
specifically Zygomycota, Chytridiomycota, and other fungi. Since
these plant pathogens were killed by the free radicals of the
plasma, the health of the plant was improved.
The plasma discharge is accompanied by ultraviolet light,
which is known to be able to change the wettability of
synthetic polymers.52 To quantify the contribution of UV in
DBD treatment, the seeds were exposed to UV light in the
10820
■
ACKNOWLEDGMENTS
This work was completed with the financial support of the
Fundamental Research Funds for Central Universities
(2232019A3-10), International Cooperative Projects of
Shanghai Municipal Committee of Science and Technology
(18230722800), and the National Natural Science Foundation
of China (Nos. 51578122, 51708096, 11475043). All financial
supports are gratefully acknowledged.
DOI: 10.1021/acs.jafc.9b03099
J. Agric. Food Chem. 2019, 67, 10813−10822
Journal of Agricultural and Food Chemistry
■ ABBREVIATION
DBD, dielectric barrier discharge
■ REFERENCES
(1) Sivachandiran, L.; Khacef, A. Enhanced seed germination and
plant growth by atmospheric pressure cold air plasma: combined
effect of seed and water treatment. RSC Adv. 2017, 7, 1822−1832.
(2) Meng, Y. R.; Qu, G. Z.; Wang, T. C.; Sun, Q. H.; Liang, D. L.;
Hu, S. B. Enhancement of Germination and Seedling Growth of
Wheat Seed Using Dielectric Barrier Discharge Plasma with Various
Gas Sources. Plasma Chem. Plasma Process. 2017, 37, 1105−1119.
(3) Soltani, A.; Gholipoor, A.; Zeinali, E. Seed reserve utilization and
seedling growth of wheat as affected by drought and salinity. Environ.
Exp. Bot. 2006, 55, 195−200.
(4) Poinapen, D.; Brown, D. C. W.; Beeharry, G. K. Seed orientation
and magnetic field strength have more influence on tomato seed
performance than relative humidity and duration of exposure to non-
uniform static magnetic fields. J. Plant Physiol. 2013, 170, 1251−1258.
(5) Błaszczak, W.; Doblado, R.; Frias, J.; Vidal-Valverde, C.;
Sadowska, J.; Fornal, J. Microstructural and biochemical changes in
raw and germinated cowpea seeds upon high-pressure treatment. Food
Res. Int. 2007, 40, 415−423.
(6) Graeber, K.; Nakabayashi, K.; Miatton, E.; Leubner-Metzger, G.;
Soppe, W. J. Molecular mechanisms of seed dormancy. Plant, Cell
Environ. 2012, 35, 1769−1786.
(7) Yang, Y. S.; Meier, F.; Lo, J. A.; Yuan, W. Q.; Sze, V. L. P.;
Chung, H. J.; Yuk, H. G. Overview of Recent Events in the
Microbiological Safety of Sprouts and New Intervention Technolo-
gies. Compr. Rev. Food Sci. Food Saf. 2013, 12, 265−280.
(8) Yao, Y.; Li, Y.; Yang, Y.; Li, C. Effect of seed pretreatment by
magnetic field on the sensitivity of cucumber ( Cucumis sativus)
seedlings to ultraviolet-B radiation. Environ. Exp. Bot. 2005, 54, 286−
294.
(9) Thirumdas, R.; Kothakota, A.; Annapure, U.; Siliveru, K.;
Blundell, R.; Gatt, R.; Valdramidis, V. P. Plasma activated water
(PAW): Chemistry, physico-chemical properties, applications in food
and agriculture. Trends Food Sci. Technol. 2018, 77, 21−31.
(10) Hou, J.; Fan, L.; Zhu, M.; Zhu, X.; Li, X.; Guan, J.; Gao, G.; Fu,
F. Combined Electrokinetic and Flushing Remediation of Multiple
Heavy Metals Co-contaminated Soil Enhanced with Acid Treatment.
Eng. Sci. Energy Environ. 2018, 2, 82−89.
(11) Martínez-Piernas, A. B.; Plaza-Bolanos, P.; Fernandez-Ibanez,
P.; Aguera, A. Organic Microcontaminants in Tomato Crops Irrigated
with Reclaimed Water Grown under Field Conditions: Occurrence,
Uptake, and Health Risk Assessment. J. Agric. Food Chem. 2019, 67,
6930−6939.
(12) Zhang, W.; Mace, W. J.; Matthew, C.; Card, S. D. The Impact
of Endophyte Infection, Seed Aging, and Imbibition on Selected
Sugar Metabolite Concentrations in Seed. J. Agric. Food Chem. 2019,
67, 6921−6929.
(13) Bogaerts, A.; Neyts, E.; Gijbels, R.; Mullen, J. V. D. Gas
discharge plasmas and their applications. Spectrochim. Acta, Part B
2002, 57, 609−658.
(14) Jeong, J.; Kim, J. Y.; Yoon, J. The role of reactive oxygen species
in the electrochemical inactivation of microorganisms. Environ. Sci.
Technol. 2006, 40, 6117−6122.
(15) Gómez-Ramírez, A.; Lópezsantos, C.; Cantos, M.; García, J. L.;
Molina, R.; Cotrino, J.; Espinós, J. P.; Gonzálezelipe, A. R. Surface
chemistry and germination improvement of Quinoa seeds subjected
to plasma activation. Sci. Rep. 2017, 7, No. 5924.
(16) Zhang, A.; Yin, X.; Shen, X.; Liu, Y. Removal of Fluticasone
Propionate and Clobetasol Propionate by Calcium Peroxide:
Synergistic Effects of Oxidation, Adsorption, and Base Catalysis.
Eng. Sci. Energy Environ. 2018, 1, 89−98.
(17) Bafoil, M.; Jemmat, A.; Martinez, Y.; Merbahi, N.; Eichwald,
O.; Dunand, C.; Yousfi, M. Effects of low temperature plasmas and
plasma activated waters on Arabidopsis thaliana germination and
growth. Plos One 2018, 13, No. e0195512.
10821
Article
(18) Š írová, J.; Sedlárǒ vá, M.; Piterková, J.; Luhová, L.; Petřivalský,
M. The role of nitric oxide in the germination of plant seeds and
pollen. Plant Sci. 2011, 181, 560−572.
(19) Bormashenko, E.; Grynyov, R.; Bormashenko, Y.; Drori, E.
Cold Radiofrequency Plasma Treatment Modifies Wettability and
Germination Speed of Plant Seeds. Sci. Rep. 2012, 2, No. 741.
(20) da Silva, A. R. M.; Farias, M. L.; Da, S. D.; Vitoriano, J. O.; de
Sousa, R. C.; Alves-Junior, C. Using atmospheric plasma to increase
wettability, imbibition and germination of physically dormant seeds of
Mimosa caesalpiniafolia. Colloids Surf., B 2017, 157, 280−285.
(21) Khamsen, N.; Onwimol, D.; Teerakawanich, N.;
Dechanupaprittha, S.; Kanokbannakorn, W.; Hongesombut, K.;
Srisonphan, S. Rice (Oryza sativa L.) seed sterilization and
germination enhancement via atmospheric hybrid non-thermal
discharge plasma. ACS Appl. Mater. Interfaces 2016, 8, 19268−19275.
(22) Qiao-yu, T.; Yong-yao, W.; Da-zhai, Z. Effects of selenium on
the Growth of Soybean in Seeding Stage. J. Hubei Inst. Natl., Nat. Sci.
Ed. 2005, 23, 124−126.
(23) Cheng-chun, J.; Su-yan, P.; Jun, M.; Wei-ping, X.; Yuan, Z.
Spectrophotometric Determination of Hydrogen Peroxide in Fenton
Reaction with Titanium Oxalate. China Water Wastewater 2006, 22,
88−91.
(24) Eaton, A. D. Standard Methods for the Examination of Water and
Wastewater, 21st ed.; APHA-AWWA-WEF: Washington, D.C., 2005.
(25) Tochikubo, F.; Shimokawa, Y.; Shirai, N.; Uchida, S. Chemical
reactions in liquid induced by atmospheric-pressure dc glow discharge
in contact with liquid. Jap. J. Appl. Phys. 2014, 53, No. 126201.
(26) Zhou, R.; Zhou, R.; Zhang, X.; Zhuang, J.; Yang, S.; Kateryna,
B.; Kostya, K. O. Effects of Atmospheric-Pressure N2, He, Air, and
O2 Microplasmas on Mung Bean Seed Germination and Seedling
Growth. Sci. Rep. 2016, 6, No. 32603.
(27) Forde, B.; Lorenzo, H. The nutritional control of root
development. Plant Soil 2001, 232, 51−68.
(28) Chen, Z. Y.; Liu, D. X.; Chen, C.; Xu, D. H.; Liu, Z. J.; Xia, W.
J.; Rong, M. Z.; Kong, M. G. Analysis of the production mechanism of
H2O2 in water treated by helium DC plasma jets. J. Phys. D: Appl.
Phys. 2018, 51, No. 9.
(29) Winter, J.; Wende, K.; Masur, K.; Iseni, S.; Dünnbier, M.;
Hammer, M. U.; Tresp, H.; Weltmann, K. D.; Reuter, S. Feed gas
humidity: a vital parameter affecting a cold atmospheric-pressure
plasma jet and plasma-treated human skin cells. J. Phys. D: Appl. Phys.
2013, 46, 453−460.
(30) Rumbach, P.; Witzke, M.; Sankaran, R. M.; Go, D. B.
Decoupling Interfacial Reactions between Plasmas and Liquids:
Charge Transfer vs Plasma Neutral Reactions. J. Am. Chem. Soc.
2013, 135, 16264−16267.
(31) Yamanaka, I.; Murayama, T. Neutral H2O2 synthesis by
electrolysis of water and O-2. Angew. Chem., Int. Ed. 2008, 47, 1900−
1902.
(32) Chandana, L.; Reddy, P. M. K.; Subrahmanyam, C.
Atmospheric pressure non-thermal plasma jet for the degradation of
methylene blue in aqueous medium. Chem. Eng. J. 2015, 282, 116−
122.
(33) Kovačević, V. V.; Dojčinović, B. P.; Jović, M. S.; Roglić, G. M.;
Obradović, B. M.; Kuraica, M. M. Measurement of reactive species
generated by dielectric barrier discharge in direct contact with water
in different atmospheres. J. Phys. D: Appl. Phys. 2017, 50, No. 19.
(34) Lukes, P.; Dolezalova, E.; Sisrova, I.; Clupek, M. Aqueous-
phase chemistry and bactericidal effects from an air discharge plasma
in contact with water: evidence for the formation of peroxynitrite
through a pseudo-second-order post-discharge reaction of H2O2 and
HNO2. Plasma Sour. Sci. Technol. 2014, 23, No. 015019.
(35) Lindsay, A. D.; Graves, D. B.; Shannon, S. C. Fully coupled
simulation of the plasma liquid interface and interfacial coefficient
effects. J. Phys. D: Appl. Phys. 2016, 49, No. 9.
(36) Judée, F.; Simon, S.; Bailly, C.; Dufour, T. Plasma-activation of
tap water using DBD for agronomy applications: Identification and
quantification of long lifetime chemical species and production/
consumption mechanisms. Water Res. 2018, 133, 47−59.
DOI: 10.1021/acs.jafc.9b03099
J. Agric. Food Chem. 2019, 67, 10813−10822
Journal of Agricultural and Food Chemistry Article

(37) Zhang, H.; Xu, Z.; Shen, J.; Li, X.; Ding, L.; Ma, J.; Lan, Y.; Xia,
W.; Cheng, C.; Sun, Q. Effects and Mechanism of Atmospheric-
Pressure Dielectric Barrier Discharge Cold Plasma on Lactate
Dehydrogenase (LDH) Enzyme. Sci. Rep. 2015, 5, No. 10031.
(38) Van Gaens, W.; Bogaerts, A. Kinetic modelling for an
atmospheric pressure argon plasma jet in humid air. J. Phys. D:
Appl. Phys. 2013, 46, 142−144.
(39) Liu, D. X.; Liu, Z. C.; Chen, C.; Yang, A. J.; Li, D.; Rong, M. Z.;
Chen, H. L.; Kong, M. G. Aqueous reactive species induced by a
surface air discharge: Heterogeneous mass transfer and liquid
chemistry pathways. Sci. Rep. 2016, 6, No. 23737.
(40) Maheux, S.; Duday, D.; Belmonte, T.; Penny, C.; Cauchie, H.
M.; Clément, F.; Choquet, P. Formation of ammonium in saline
solution treated by nanosecond pulsed cold atmospheric micro-
plasma: a route to fast inactivation of E. coli bacteria. RSC Adv. 2015,
5, 42135−42140.
(41) Oehmigen, K.; Hähnel, M.; Brandenburg, R.; Wilke, C.;
Weltmann, K. D.; Woedtke, T. V. The Role of Acidification for
Antimicrobial Activity of Atmospheric Pressure Plasma in Liquids.
Plasma Processes Polym. 2010, 7, 250−257.
(42) Wu, Z.; Luo, J.; Han, Y.; Hua, Y.; Guan, C.; Zhang, Z. Low
Nitrogen Enhances Nitrogen Use Efficiency by Triggering NO3−
Uptake and Its Long-Distance Translocation. J. Agric. Food Chem.
2019, 67, 6736−6747.
(43) Ji, S.-H.; Choi, K.-H.; Pengkit, A.; Im, J. S.; Kim, J. S.; Kim, Y.
H.; Park, Y.; Hong, E. J.; Jung, S.; Choi, E.-H.; Park, G. Effects of high
voltage nanosecond pulsed plasma and micro DBD plasma on seed
germination, growth development and physiological activities in
spinach. Arch. Biochem. Biophys. 2016, 605, 117−128.
(44) Ji, S. H.; Kim, T.; Panngom, K.; Hong, Y. J.; Pengkit, A.; Park,
D. H.; Kang, M. H.; Lee, S. H.; Im, J. S.; Kim, J. S.; Uhm, H. S.; Choi,
E. H.; Park, G. Assessment of the Effects of Nitrogen Plasma and
Plasma-Generated Nitric Oxide on Early Development of Coriandum
sativum. Plasma Process. Polym. 2015, 12, 1164−1173.
(45) Wenzel, R. N. Resistance of Solid Surfaces to Wetting by
Water. Ind. Eng. Chem. 1936, 28, 988−994.
(46) Meiqiang, Y.; Mingjing, H.; Buzhou, M.; Tengcai, M.
Stimulating Effects of Seed Treatment by Magnetized Plasma on
Tomato Growth and Yield. Plasma Sci. Technol. 2005, 7, 3143−3147.
(47) Volin, J. C.; Denes, F. S.; Young, R. A.; Park, S. M. T.
Modification of seed germination performance through cold plasma
chemistry technology. Crop Sci. 2000, 40, 1706−1718.
(48) Filatova, I.; Azharonok, V.; Kadyrov, M.; Beljavsky, V.;
Gvozdov, A.; Shik, A.; Antonuk, A. The effect of plasma treatment
of seeds of some grain and legumes on their sowing quality and
productivity. Rom. J. Phys. 2011, 56, 139−143.
(49) Yao, J. W.; Liu, Y. A.; Zhan, J. X.; Li, X.; Zhang, A.; Zhang, K.;
Yan, Z. H.; Cai, S. J.; Yang, C.; Sand, W.; Dai, Y.; Yang, H. H. Study
on Inactivation of Escherichia coli by Double Dielectric Barrier
Discharge. IEEE Trans. Plasma Sci. 2018, 46, 2026−2033.
(50) Selcuk, M.; Oksuz, L.; Basaran, P. Decontamination of grains
and legumes infected with Aspergillus spp. and Penicillum spp. by cold
plasma treatment. Bioresour. Technol. 2008, 99, 5104−5109.
(51) Ping, Q.; Lu, X.; Zheng, M.; Li, Y. Effect of CaO2 addition on
anaerobic digestion of waste activated sludge at different temperatures
and the promotion of valuable carbon source production under
ambient condition. Bioresou. Technol. 2018, 265, 247−256.
(52) Bormashenko, E.; Pogreb, R.; Whyman, G.; Bormashenko, Y.;
Jager, R.; Stein, T.; Schechter, A.; Aurbach, D. The reversible giant
change in the contact angle on the polysulfone and polyethersulfone
films exposed to UV irradiation. Langmuir 2008, 24, 5977−5980.

10822 DOI: 10.1021/acs.jafc.9b03099

J. Agric. Food Chem. 2019, 67, 10813−10822