Effect of dielectric barrier discharge cold plasma on pea seed

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Agricultural and Environmental Chemistry

Effect of dielectric barrier discharge cold plasma on pea seed growth Xiaoting Gao, Ai Zhang, paul Héroux, Wolfgang Sand, Zhuyu Sun, Jiaxun Zhan, Cihao Wang, Siyu Hao, Zhenyu Li, Zhenying Li, Ying Guo, and Yanan Liu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03099 • Publication Date (Web): 06 Sep 2019 Downloaded from pubs.acs.org on September 6, 2019

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Journal of Agricultural and Food Chemistry

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Effect of dielectric barrier discharge cold plasma on pea seed growth

2 3

Xiaoting Gaoa,b, Ai Zhanga, Paul Hérouxc, Wolfgang Sanda, Zhuyu Suna, Jiaxun

4

Zhana, Cihao Wang a, Siyu Hao a, Zhenyu Lia, Zhenying Lia, Ying Guod, Yanan

5

Liua,b*

6

(a College of Environmental Science and Engineering, Donghua University, 2999

7 8

North Renmin Road, Shanghai 201620, China. b Shanghai

9 10

200092, China. c Department

11 12 13

institute of pollution control and ecological security, Shanghai

of Epidemiology, Biostatistics and Occupational Health, McGill University, Montreal H3A 0G4, Canada

d

Department of Applied Physics, College of Science, Donghua University,Shanghai 201620, China

14

*Corresponding author: Tel/fax: 86-21-67792538

15

E-mail: [email protected] (Y. Liu)

16

ACS Paragon Plus Environment


18

Abstract

19

Traditional seed pretreatment methods cause secondary pollution for the application

20

of various chemicals. This study investigated the effect of dielectric barrier discharge

21

(DBD) cold plasma on seedling growth. Effects of plasma activated tap water (PATW)

22

and plasma activated seeds (PAS) were compared for germination rates, seedling

23

height, dry weight and chlorophyll content. Results show that compared with controls

24

these growth parameters were all increased by more than 50 %. The yields and

25

contributions of hydrogen peroxide, nitrate, nitrite, and ammonium were quantified.

26

Hydrogen peroxide and nitrate have an important role in seedling growth. By etching

27

the seed epidermis free radicals can reduce the apparent contact angle and increase the

28

water absorption of the seeds. In addition to the low cost of PATW and PAS

29

compared with commercial fertilizers, DBD does not involve any chemical addition.

30

Thus, both PATW and PAS can be an alternative for improvement of agricultural

31

production.

32

Key words ： DBD plasma; plasma activated tap water (PATW); plasma activated

33

seeds (PAS); seedling growth; improvement mechanism


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1. Introduction

36

According to the United Nations Food and Agriculture Organization, deteriorating

37

environment, climate changes, and urbanization caused by human activities have

38

dramatically increased the global food demand

39

to improve seedlings growth. The first one involves the use of a variety of

40

dormancy-breaking methods including physical pathways (magnetic fields, ultraviolet,

41

hot water immersion) 3-5 and chemical agents (disinfectants, fungicides, hormones) 5-8

42

as pre-sowing seed processes to enhance seed germination and growth rate. The

43

second way involves the addition of chemical fertilizers to supply nitrogen,

44

phosphorus and potassium needed for plant growth

45

have their own drawbacks, such as time consuming, labor intensive, and secondary

46

pollution11-12.

1-2.

Traditionally, there are two ways

1, 9-10.

However, these methods

47

In recent years, non-thermal plasma has become an efficient, innovative, and green

48

alternative to traditional seed culture. Dielectric barrier discharge (DBD) is one of the

49

forms of non-thermal plasma that can conveniently generate ultraviolet light,

50

high-energy electrons, and active particles at atmospheric pressure

51

used to produce plasma activated tap water (PATW) to irrigate the seeds, or to

52

activate seeds by direct discharge on the seeds surface (plasma activated seeds, PAS).

53

Reactive oxygen species (ROS) and reactive nitrogen species (RNS) are produced

54

during discharge in the atmosphere of air. Among them, both long-lived species (such

55

as H2O2, NO2−, NO3−, etc.) and short-lived ones (such as OH, O2-, 1O2, ONOOH, etc)


9, 13.

DBD can be


14-16.

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can optimize the biological growth process

It has been found that tap water

57

activated by plasma has a significant effect on plant growth. This seems to be

58

correlated to RNS such as NO2− and NO3− 17. Similarly, a direct exposure of seeds to

59

plasma also increases seedling growth. Seeds treated with radiofrequency plasma for

60

10 s experience increases of the germination rate of almost 100 %

61

attributed to the fact that DBD etches the seed surface and allows nitrate to enter the

62

seed. This process simulates seed immersion in nitrate nitrogen rich water

63

However, there are few reports studying the effects of PATW and PAS on agricultural

64

output. Moreover, most studies only explore the germination rate to evaluate the

65

effect of plasma, but ignore other parameters like plant growth 19-21. Additionally, the

66

contributions of RNS and ROS to plant growth have not been studied quantitatively.

15.This

can be

18.

67

In this study, the mechanisms of PATW and PAS on seedling growth were

68

investigated. Germination rate, height, dry weight and chlorophyll were measured to

69

assess the impacts of PATW and PAS. Furthermore, hydrogen peroxide, nitrate,

70

nitrite, and ammonium were quantified to clarify the mechanisms of PATW on

71

seedling growth. An ion-containing medium (supplying the same concentration as the

72

discharge) was used to explore the contribution of each ion. Simultaneously, the

73

morphology and hydrophilicity of the seed surface were investigated to clarify the

74

mechanism of PAS action on seedling growth. These findings can give options for an

75

improvement of agricultural production in the future.

76

2. Materials and methods


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78 79

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 g -180 g.

80

The reagents, including sodium hydroxide, hydrogen peroxide, ethanol, acetone,

81

titanium potassium oxalate, sodium nitrate, sulfuric acid, sulfamic acid, phosphoric

82

acid, p-aminobenzenesulfonamide, ammonium chloride, sodium potassium tartrate,

83

N-(1-naphthyl)-ethylenediamine dihydrochloride terephthalic acid and hydrochloric

84

acid were of analytical grade and purchased from Sinopharm Chemical Reagent Co.,

85

Ltd (Shanghai, China). The solutions were prepared using deionized water, while

86

nitrate and hydrogen peroxide solutions used for reference experiments were made

87

with tap water.

88

2.2 Plasma apparatus and determination of power

89

A Dielectric Barrier Discharge (DBD) plasma reactor (CTP-2000K, Nanjing

90

Suman Electronics, China) was used as the plasma source at atmospheric pressure.

91

The schematic diagram of the experimental apparatus is shown in Fig. 1. It consisted

92

of three main parts: a high voltage alternating current power source, a DBD reactor

93

device (self-designed), and an oscilloscope (TDS 2012B, Tektronix, USA).

94

The DBD reactor is the core of the experimental system. The reactor contains

95

two parallel metal electrodes and a quartz container for seeds and tap water. The

96

electrode is 1cm thick and 15 cm in diameter. The diameter, internal height and



97

external height of the quartz container are 15 cm, 0.8 cm and 1.3 cm, respectively. Air

98

can enter through small holes on both sides of the quartz container. The oscilloscope

99

is fitted with two probes: a Tektronix P6015A for the high voltage and a Tektronix

100

TPP0101 for the capacitor voltage.

101 102

103

Fig. 1 Schematic diagram of the experimental system.

2.3 Experimental procedure

104

Tap water (TW) was selected as the water matrix for this study. 50 mL water was

105

used for each experiment. Discharge powers ranged from 60 W to 164 W, with

106

reaction times between 5 min and 20 min.

107

Seeds were divided into two 300-seed groups, which were soaked in 1 L of TW or

108

PATW for 15 hours. Then the seeds were placed on a wet paper filter in a petri dish

109

(285 mm×205 mm×40 mm) filled with 50 mL water (TW or PATW). Two groups

110

were irrigated with TW or PATW each 12 hours.

111

For the DBD plasma treatment, 300 seeds were placed in petri dish-like containers,


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112

and exposed to plasma with a discharge power of 9 W to 35 W. The reaction times

113

ranged from 1 min to 10 min. The treated seeds were soaked in 1 L of TW for 15

114

hours and processed as follows: the seeds were put on a wet filter paper in a

115

285×205×40 mm Petri dish with 50 mL TW, which was refilled each 12 hours. For

116

comparison, the same number of seeds, but without plasma discharge, were treated in

117

the same way.

118

When the sprout of the plant has reached 1 cm height, the plants were exposed to a

119

simulated sunlight (P=10W, λ red: 620-660nm, λ blue: 450-470nm, λ red:λ blue=3:1)

120

for 12 hours per day. The entire growth cycle took 15 days.

121

2.4 Analytical Methods

122 123 124 125

After 15 days the plants were harvested and the individual heights were measured. The average height (AH) was calculated as follows：

AH (cm) 

Total height of all plants Total number of plants

(1)

The numbers of germinated seeds were also counted. The germination rate (GR)

126

was calculated as follows:

127

GR(%) 

Number of germinated seeds per dish  100 Number of total seeds per dish

(2)

128

The harvested plants were then dried in an oven (60 °C) for 3 days. Afterwards the

129

dry weight of each group and the chlorophyll concentration of the leaves were

130

measured (SI, Determination of chlorophyll). The chlorophyll concentrations

131

(C(mg/g)) were calculated as follows22.



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132

C(mg / g) = (8.02A 663 + 20.2A 645 )V / 1000 W

133

where V means total volume of sample solution (mL), W means the weight of sample

134

(g), A663 means the absorbance of the solution at 663 nm and A645 means the

135

absorbance of the solution at 645 nm.

(3)

136

The concentration of hydrogen peroxide (H2O2), ammonia nitrogen (NH4+-N),

137

nitrite nitrogen (NO2--N), and nitrate nitrogen (NO3--N) in the PATW were

138

determined

139

hydroxyl radicals were combined with terephthalic acid, which in the presence of ·OH

140

forms 2-hydroxyterephthalic acid (HTA). HTA absorbs light at 310 nm and emits

141

fluorescence at 425 nm 25.

colorimetrically by spectrophotometric measurements

23-24.

The

142

Plasma-treated and non-treated seeds were examined both under a scanning

143

electron microscope (SEM) (Hitachi S4800) to analyze the surface structure. Surface

144

structures of cross and longitudinal sections of the plant root were also visualized by

145

SEM. A detailed analytical method is provided in the Supplementary Information.

146

For the determination of water absorption 300 dry seeds were first weighed and

147

recorded as m0. Subsequently, seeds were soaked in 1 L of TW for 1 h up to 20 h. The

148

seeds were then removed from the water, the excess surface moisture was wiped off

149

with blotting paper, and then weighed and recorded as mt. The water adsorption was

150

calculated using the following equation 2:

151 152

Water adsorption (%) =

m t - m0 × 100 m0

(4)

The apparent contact angle was measured using the sessile drop technique


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153

(SL200KS, KINO Industry CO. Ltd, USA). The test was performed by dripping 2 μL

154

of distilled water on the seed surface. An image was taken under an optical

155

microscope in conjunction with a computer-aided measurement. Plasma was

156

diagnosed using Optical Emission Spectra (OES) (2048TEC, Avaspec, Netherlands)

157

in the range of 198 up to 947 nm.

158

To analyze the microbial community structure, untreated seeds and seeds treated at

159

15 W for 3 min were used for DNA extraction and metagenomic sequencing

160

(Majorbio Bio-Pharm Technology Co., Ltd., Shanghai, China). Details of the

161

analytical methods are provided in the Supporting Information.

162

2.5 Statistical analysis

163

All treatments were done in triplicates, and experiments were repeated three

164

times. The data are presented as the mean ± standard deviation of the triplicates.

165

Statistical analyses of the data were performed using student's T-test to establish

166

significance between data points, and significant differences were based on p < 0.05

167

or p < 0.01.

168

3. Results and discussion

169

3.1 Seedling growth after irrigation with plasma activated water

170

As illustrated in Fig. 2, the seedling growth of all treated samples was considerably

171

higher than in the control group. This means that seedling growth was improved by



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172

irrigating with PATW. In Fig. 2(a), it is shown that an applied power of 60 W is not

173

sufficient to promote seedling growth. However, if the applied power exceed 60 W,

174

the seedling height was much higher than the control. On the other hand, no

175

significant differences were noted in the heights of seeds cultivated with TW at

176

discharge power values of 73 W, 87 W and 122 W. An exemption was the result of

177

the 164 W discharge, where a slight decrease in height was recorded. The low power

178

values are obviously not conducive to the production of hydrogen peroxide,

179

nitrogenous compounds, and other nutrients. In contrast, with high power plasma

180

treatment conditions excess H2O2 was produced, which are toxic to growth

181

values for chlorophyll and dry weight indicated low enhancement and high inhibition

182

for increased treatment power. An applied power value of 87 W was the optimal value

183

for the effect on seedling growth.

17.

The

184

The effects of discharge time on seedling growth are shown in Fig. 2(b). With

185

treatment times of 5 min or more minutes the height and chlorophyll concentrations of

186

the seedlings were notably higher than those for the control. However, there was no

187

significant difference observed between the treatment periods of 5 min and 20 min.

188

After 10 min of treatment, dry weight reached a maximum value, and no further

189

increase was observed. The modification of the operational conditions favored the

190

generation of nitrogen by the discharge process26. Short-term discharges produced

191

sufficient nitrogen compounds as nutrients for the cultivation of the seeds. Thus,

192

considering the growth enhancement and energy consumption, the optimum discharge

193

power and treatment time are 87 W for 10 min.


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(a)

(b) （b）

194 195 196

Fig.2 Seedling growth in PATW under different discharge power and time. (a), TW treated with different power for 10 min. (b), TW treated at 87 W for different times.

197

The root channel organization of the plant is responsible for long-distance transport

198

of water, inorganic salts and nutrients in the plant. It was therefore necessary to

199

compare the root cross-sections of plants that had been cultivated either with TW (Fig.

200

3a), or with PATW (Fig. 3b). Compared with the roots of plants cultured in TW, the

201

PATW cultured plant roots had thinner phloem walls and larger pore diameters.

202

Increased diameters for transport cells enhance nutrient exchange27. Therefore, a

203

seedling treatment with PATW created an improved growth by changing the

204

physiological structure of the plants.

205 206 207

Fig. 3 SEM of the cross section through a seedling root. (a), cultured with TW; (b), cultured with PATW treated at 87 W for 10 min.



208

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3.1.1 Effect of PATW with different pH scale on seedling growth

209

The treatment with DBD plasma resulted in a pH decrease of PATW (Tables S1

210

and S2). The decrease can be attributed to the generation of nitric acid, hydrogen

211

peroxide and ozone by the discharge process

212

the acidity of plasma activated water affected seedling growth 1. In order to explore

213

the effects of pH on plant growth, PATW (87 W, 10 min) was adjusted to different pH.

214

The results are shown in Table 1. The initial pH of TW after discharge for 10 min at

215

87 W was 2.86. The pH of PATW was adjusted with 1 mM NaOH to pH of 3-9. The

216

increase rate (IR) was calculated using Eqaution (5), where growth parameters

217

represent height, germination, dry weight, and chlorophyll content.

218

IR (%) =

15, 26.

Sivachandiran et al. proved that

Growth parameter value at different pH - Growth parameter value at pH 7 × 100 Growth parameter value at pH 7

(5)

219

It was noted that pH above 8 or pH below 6 did not promote plant growth (as

220

shown in Table 1). A pH between 6 and 8 did not affect the germination rate, but

221

other growth parameters such as height, dry weight and chlorophyll content were

222

reduced. In addition, the changes in pH had no effect on the concentrations of H2O2

223

and NO3- during the plasma activation process (shown in Fig. 4(a) and 4(f). Therefore

224

this study points to a neutralized PATW as the optimal culture solution.

225

Table 1 Seedling growth in PATW at different pH Germination

IR

Chlorophyll

Height/cm

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 %

/%

IR

Dry

pH

weight/g


content/（mg/L）

IR

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9

11.97±1.63

-23 %

93.67±1.42

-1 %

10.19±0.69

-22 %


3.59±0.18

-38 %


(a)

(c)

(b)

(d)

(e)

(f)

(g)

(h)

226


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227 228 229 230 231 232 233

growth. (a) H2O2 production and its concentration change after pH adjustment; (b) OH production; (c) The effect of H2O2 (same concentration produced by DBD), and error bars are unilateral; (d) The 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 seed growth process.

234

3.1.2 Contribution of H2O2 to seedling growth

Fig. 4 Production of chemical substances under different powers and times, and its effects on plant

235

Hydrogen peroxide is considered as a stable product of PATW. Its half-life period

236

is 8 h-20 d. So it has an effect for quite a long time 9, 28. Hydrogen peroxide interferes

237

with the levels of abscisic acid, thus to changing the dormancy period of seeds. In this

238

way seed development and growth quality are improved9.

239

As shown in Fig. 4(a), with a discharge power of below 87 W the hydrogen

240

peroxide content increased slowly, while at values exceeding 87 W the content

241

increased sharply. According to previous studies, there are three possible ways to

242

generate H2O2: the dissolution of gaseous H2O2

243

which the electrolyzed O2 captures electrons from the cathode to form O2- and further

244

combines with H+ to form H2O2 (Equation (6)-(8)); the last approach involves the

245

transfer of energy between excited species and water molecules caused by high

246

electron density 26, 32. The reaction mechanisms are shown in equations (9)- (10).

29;

electrolysis of water

30-31

in

247

O 2 + e- → O 2 -

(6)

248

O 2 - + H + → HO 2

(7)

249

2HO 2 → H 2O 2 + O 2

(8)

250

e- + H 2O → H + OH + e-


(9)


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251

OH + OH → H 2O 2

252

Under optimal reaction conditions the amount of H2O2 produced was 17.1 mg/L

253

(Fig. 4(a)). This is consistent with the results of Zhou et al., who found that plasma

254

discharge can produce 17.4 mg/L H2O2 in 10 min

255

(maximum intensity at 309 nm in the emission spectrum) was the major contributors

256

to PATW reactivity. Besides, the amount of OH radicals production under optimal

257

conditions was 2.29 mmol/L (Fig. 4(b)), which can theoretically produce 38.93 mg/L

258

of H2O2. Therefore, the OH radicals can partially be converted to H2O2 33. With an

259

increase of discharge time, the H2O2 further reacted with some compounds in PATW

260

to form other active substances, such as peroxynitrite (ONOOH / ONOO-) and nitrate

261

28, 34.

26.

(10)

As shown in Fig. 4(g), OH

262

In order to further analyze the contribution of H2O2, the study configured H2O2

263

solution to culture seeds (shown in Fig. 4(c)). The concentration of H2O2 was the

264

same as that produced by the DBD discharge. The variation of seedling height and dry

265

weight during H2O2 treatment was in good agreement with the data for the DBD

266

treatment. However, the improving effect of DBD treatment was slightly higher than

267

that for the H2O2 treatment. This means that other synergistic effects (such as NO3-,

268

NH4+, OH) also contributed to the growth promotion.

269

3.1.3 Contribution of nitrogen compounds to seedling growth

270

Nitrogen is indispensable for plant growth. However, the natural form of nitrogen

271

(diatomic nitrogen, N2) has a strong triple bond, making absorption by plants


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272

impossible. Plasma synthesis is considered a green alternative to traditional nitrogen

273

fixation, as it involves no chemical agents and cause no secondary pollution 9. During

274

discharge, ionized nitrogen, such as nitrite (NO2-) and nitrate (NO3-) are thought to be

275

generated 26. It is reasonable to suppose that a solution containing a suitable nitrogen

276

source will form an environment conducive to the growth of pea seeds. In view of this,

277

it was necessary to analyze the nitrogen content and conversion in PATW

278

shown in Fig. 4(g), the nitrogen second positive system (N2), the nitrogen ion (first

279

negative system, N2+) and the NH emission are visible in the optical emission spectra.

280

These groups are the precursors of nitrogen compounds useful as nutrients 36-37.

35.

As

281

The amounts of nitrogenous substances in PATW appeared to follow the order

282

NO2- < NH4+ < NO3-. Fig. 4(e) is demonstrating that both ammonium and nitrate

283

concentrations increased with an increase of discharge power and treatment time. The

284

concentration of ammonium is less than 2 mg/L, which is much lower than that of

285

nitrate with 250 mg/L. Nitrite could only be detected under low power conditions,

286

since it would be converted to nitrate nitrogen, with high discharge power and time

287

38-39.

288

other nitrogen containing compounds.

It can be speculated that nitrate nitrogen dominated plant growth compared to

289

Ammonium was derived from the dissolution of ammonia under acidic conditions,

290

which was synthesized by excited nitrogen and hydrogen in the plasma atmosphere 40.

291

The low amount of water vapors in air may explain the very low amount of ammonia

292

compared to nitrate.

293

N 2 + 6H 2O → 2 NH（ 3 g）+ 6 • OH


(11)


Page 18 of 34

294

NH3( aq ) + H + → NH +4 ( aq）

295

The nitrate synthesis process is sophisticated involving the basic processes of

296

Equatious(13) to (16) (Table 2) 36-38 and the collision of activated oxygen and nitrogen

297

leading to the rapid conversion into nitrogen oxides (NOX). Some of the activated

298

oxygen atoms combined to the strongly oxidizing ozone because of the ionization of

299

the air by the high voltage. In the second stage (Equations (17) and (18) in Table 2),

300

nitrogen oxides dissolve in water produced nitrite and nitrate32, 39, 41. However, due to

301

the strong oxidation of ozone and H2O2, nitrite is rapidly oxidized to nitrate

302

(Equations (19) and (20), Table 2)

303

explains why the PATW was strongly acidic, from the formation of hydronium ions.

304

34, 38-39.

(12)

Nitrite-nitrate conversion mechanism also

Table 2 Equation for formation of nitrate by plasma discharge from nitrogen in the air Phase1

Phase2

Phase3

N 2 + O → NO + N

(13)

N + O 2 → NO + O

(14)

NO + O +M → NO 2 + M

(15)

O 2 + O + M → O3 + M

(16)

2 NO2（g）+ H 2O → NO2 ( aq）+ NO3( aq）+ 2H +（aq）

(17)

NO（g）+ NO 2（g）+ H 2O → 2 NO 2 ( aq）+ 2H +（aq）

(18)

NO 2 + O3 → NO3 + O 2

(19)

NO 2 + H 2O 2 + H + → NO3 + H 2O + H +

(20)

305 306

In this experiment, the effect of nitrate in PATW on seedling growth was tested

307

using 40 - 230 mg/L NaNO3 as culture solution. The results are shown in Fig. 4(d).

308

The different nitrate concentrations did not increase the height significantly. This


Page 19 of 34


309

indicates that the lowest nitrate concentration (40 mg/L) had already met the growth

310

needs of the peas. The effect of nitrate on the dry weight of plants is generally

311

presented as a low-promotion and high-repression influence, owing to an excess

312

nitrogen produced as toxic nitrides that interfere with plant growth 26, 42. However, an

313

appropriate amount of nitrate nitrogen, as obtained in this study, can characterize

314

PATW as a green fertilizer. This study simulates the entire growth process of the seed,

315

which shown in Fig. 4(h).

316

3.2 Seedling growth of plasma activated seeds

317

In addition to PATW, seeds treated directly by plasma also showed enhanced

318

growth. As shown in Fig. 5, in comparison with untreated seeds, almost all measured

319

parameters were improved significantly by DBD plasma treatment. As illustrated in

320

Fig. 5(a), the average height, chlorophyll and dry weight increased by 30 % - 50 %,

321

20 % - 45 % and 30 % - 40 %, respectively, compared to controls. There were no

322

significant differences measurable for discharge powers of 9 W and 35 W. However,

323

if the power increase above 40 W, seed height and dry weight declined by 15 % up to

324

50 % (Fig. S1). Obviously, excessive DBD plasma is detrimental to the seeds 15.

325

With 3 min of discharge time the best stimulation of seedling growth was achieved

326

(shown in Fig. 5(b)). The average height, chlorophyll content and dry weight

327

increased to 51 %, 46 % and 34 %, respectively. Although the dry weight remained at

328

a steady increase of 30 %, average height and chlorophyll content of seedlings

329

decreased with prolonged treatment times. This means that seedlings are dose



330

sensitive and that attention needs to be paid to optimize DBD treatment conditions. （a）

（b）

331 332 333

Fig 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.

334

3.2.1 Seed characteristics and water absorption

335

Correlations between seed growth and seed surface structure changes after plasma 15, 17, 19-20.

336

treatment have been reported

To explain the enhancement of seed growth

337

by plasma treatment, the surface structure of pea seeds was visualized by SEM. As

338

shown in Fig. 6, the surface network structure of pea seeds became distorted and

339

partially destroyed by DBD plasma treatment. The ridges on the seed epidermis

340

gradually disappeared. This observation is similar to that of Sang et al., who described

341

that plasma treatment caused a surface modification of spinach seeds 43. DBD plasma

342

bombards seeds with free radicals and ions. These lead to seed coat erosion 21, 26, 43-44.

343

The altered seed coat increases the hydrophilicity of the seed, and in this way

344

increases water absorption.

345

The wettability of the seeds is characterized by the apparent contact angle, which

346

takes into account both the chemical structure and the roughness of the surface. The


Page 20 of 34

Page 21 of 34


347

wetting of rough heterogeneous surfaces can be accounted for by the Cassie and

348

Wenzel models 19, 45. The apparent contact angle of the control seeds was 100°, while

349

after 3 min of DBD plasma irradiation the angle decreased to 69° (Fig. 6c+f). The

350

current findings are consistent with those of Zhou et al., who found that plasma

351

treatment significantly decreased the apparent contact angle of Mung Bean seed 26.

352

The change in wettability corresponds with a change in the water absorption, as

353

presented in Fig. 6g+h. There was a rapid water uptake in the first 5 hours, followed

354

by a slow one with 15 h. Afterwards the seed mass remained approximately constant.

355

Consequently the irradiated seeds absorbed more water than the unirradiated seeds.

356

Also the water absorption rate was initially increased. The most suitable DBD

357

treatment was 3 min. Data in the literature indicate that legumes have an lipid outer

358

layer, which blocks water absorption. The results of Da et al. 20

359

radicals generated by the plasma oxidize this lipid layer and in this way chemically

360

modify the seed epidermis. These causes the increased water absorption of irradiated

361

seeds. On the other hand, a prolonged treatment time and treatment power reduce the

362

water absorption. Different crops have different sensitivities and tolerances to plasma,

363

and excessive treatment may reduce positive effects46. This means that one needs to

364

find the optimum for the treatment time and plasma intensity.


indicate that the free


(a)

(b)

(c)

(d)

(e)

(f)

(g)

(h)

365 366 367 368

Fig. 6 Surface structure, apparent contact angle and water absorption. (a), (b), (c), untreated seeds; (d), (e), (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 min -10 min.

369

3.2.2 Contribution of plasma-generated reactive species and other synergistic

370

effects

371

The free radicals and ions generated by plasma irradiation obviously play a major

372

role in promoting the germination and growth of plants. Data in Fig. 7 for the Optical

373

Emission Spectra allow to identify the following radicals ： N2(C-B), N2(B-A), N2+,

374

NO, O2+, OH, H(), CO, H2, H(), and N. In the study of Sang et al. 44 NO produced

375

by plasma improved the quantity and speed of germination and the development of


Page 22 of 34

Page 23 of 34


47

376

alfalfa seedlings. Volinet et al.

noted that deposition of nitrogen on the surface of

377

seeds had a positive effect on their germination, with N2 and N2+ accounting for most

378

of the peaks in the emission spectrum. The existence of CO and O2+ signals confirmed

379

that chemical etching of the seed surface by plasma played an important role in

380

stimulation of seed germination 48.

381 382

Fig. 7 OES of plasma activated seeds (15 W, 3 min).

383 384

In previous studies we have reported that DBD plasma also has a sterilizing effect

385

49.

386

killed the fungi on the seed epidermis50.

387

Free radicals generated by the plasma have an oxidizing effect, which partially

The rarefaction curve constructed from the Alpha diversity index of each sample at



Page 24 of 34

388

different sequencing depths can be used to illustrate the integrity of the sample

389

sequencing data 51. The sobs index curve in Fig. S3 tends to be flat, indicating that the

390

amount of sequencing data is reasonable.

391

Alpha diversity indexes can be used to quantitatively analyze the richness and the

392

diversity of microbial communities, in which sobs, chao and ace indices emphasize

393

community richness. Shannon emphasizes community evenness, and coverage reflects

394

the coverage of the community

395

evenness have both decreased after plasma treatment.

396 397 398

51.

According to Table 3 community richness and

Table 3 Richness and diversity indices of fungal microbial communities on untreated and treated seed epidermis. Sample\Estimators

ace

chao

sobs

shannon

coverage

Untreated

180.1841

180

180

3.4263

0.999985

Treated

141.8065

142

140

3.3412

0.999945

399 400

The distributions of the fungal community at phylum and genus level are depicted

401

in Fig. 8. All values for fungi of the genus level indicate a reduction of abundance:

402

Mycosphaerella (untreated 18.53 %, treated 17.45 %), Pleosporaceae (untreated 7.25

403

%, treated 6.77 %), Pithya (untreated 5.19 %, treated 4.34 %), Davidiellaceae

404

(untreated 4.19 %, treated 2.92 %), Mortierella (untreated 1.28 %, treated 1.13 %) and

405

Gibberella (untreated 1.02 %, treated 0.86 %) They all belong to the Ascomycota.

406

Mycosphaerella, a dominant fungus, is the largest genus of plant pathogens. The

407

phyla, which are significantly reduced in abundance, are parasites feeding on plant

408

decay, specifically Zygomycota, Chytridiomycota and other fungi. Since these plant

409

pathogens were killed by the free radicals of the plasma, the health of the plant was


Page 25 of 34


410

improved.

411 412

Fig. 8 Phylum and genus level distribution of fungi communities from untreated and treated seeds.

413 414

The plasma discharge is accompanied by ultraviolet light, which is known to be 52.

415

able to change the wettability of synthetic polymers

416

contribution of UV in DBD treatment, the seeds were exposed to UV light in the

417

range 300 to 400 nm at 293.88 mW/cm2 for 3 min (corresponding to the optimal

418

treatment time of this experiment). Data in Fig. S2 indicate that UV slightly increased

419

the water absorption of the seeds, however far less than the effect measured for the

420

plasma application. This means that the UV light contributed only for a small part to

421

the plasma effect.

422

3.3 Comparison between PATW and PAS

423

In order to quantify the

Although both, PATW and PAS, promote seedling growth, they have different



Page 26 of 34

424

application areas. As shown in Table 4, PATW has a better incentive effect compared

425

to PAS. Especially the dry weight significantly increases. In addition, PATW is rich

426

in nitrogen compounds required for plant growth. Therefore, PATW can not only be

427

an effective substitute for fungicides, but also suitable for plants on poor soil or plants

428

with poor growth.

429 430

Table 4 Increase of seedling growth parameters for PATW application and PAS under optimal conditions Increase (%)

PATW/Control

PAS/Control

PATW/PAS

Height

59 %

51 %

5.8 %

Chlorophyll

48 %

46 %

1.6 %

Dry weight

68 %

34 %

25 %

Growth parameters

431 432

It is worth mentioning that the production costs of PATW and PAS are RMB 175

433

per ton and RMB 2.7 per ton, respectively. These values are far below those for

434

chemical fertilizers (RMB 2,500 per ton). PAS minimize production costs and have an

435

improved growth. This is a result of improved germination because of facilitated

436

epidermis breaking. These findings provide options for increasing agricultural

437

production.

438

ABBREVIATIONS USED

439

DBD, dielectric barrier discharge; PATW, plasma activated tap water; PAS,

440

plasma activated seeds; ROS, reactive oxygen species; RNS, reactive nitrogen species;

441

TW, tap water; AH, average height; GR, germination rate; SEM, scanning electron

442

microscope; OES, optical emission spectra; IR, increase rate.

443

Appendix A. Supplementary material


Page 27 of 34

444


Supplementary data associated with this article can be found in the supporting

445

information.

446

Acknowledgements

447

This work was completed with the financial support of the Fundamental Research

448

Funds for Central Universities (2232019A3-10), International Cooperative Projects of

449

Shanghai Municipal Committee of Science and Technology (18230722800), the

450

National Natural Science Foundation of China (No. 51578122, 51708096, 11475043).

451

All financial supports are gratefully acknowledged.



453 454 455 456 457 458 459 460 461 462 463 464 465 466 467 468 469 470 471 472 473 474 475 476 477 478 479 480 481 482 483 484 485 486 487 488 489 490 491 492 493 494

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605

Figure caption

606

Fig. 1 Schematic diagram of the experimental system.

607

Fig.2 Seedling growth in PATW under different discharge power and time. (a), TW

608

treated with different power for 10 min. (b), TW treated at 87 W for different times.

609

Fig. 3 SEM of the cross section through a seedling root. (a), cultured with TW; (b),

610

cultured with PATW treated at 87 W for 10 min.

611

Fig. 4 Production of chemical substances under different powers and times, and its

612

effects on plant growth. (a) H2O2 production and its concentration change after pH

613

adjustment; (b) OH production; (c) The effect of H2O2 (same concentration produced

614

by DBD), and error bars are unilateral; (d) The effect of NO3- (same concentration

615

produced by DBD), and error bars are unilateral; (e) NO2-, NH4+ and NO3-

616

productions under different powers and times;

617

powers and times and its concentration change after pH adjustment; (g) OES of

618

PATW (87 W,10 min); (h) Simulation of seed growth process.

619

Fig 5 Seedling growth after treatment of seeds with DBD plasma. (a), treatment for 3

620

min with various power values.(b), treatment at 15 W for different durations.

621

Fig. 6 Surface structure, apparent contact angle and water absorption. (a), (b), (c),

622

untreated seeds; (d), (e), (f), seeds treated with DBD plasma for 3 min; (g) seeds

623

treated with different powers for 3 min; (h) seeds treated with 15 W for 0 min -10

624

min.

625

Fig. 7 OES of plasma activated seeds (15 W, 3 min).

(f) NO3- production under different

626


Page 32 of 34

Page 33 of 34


627

Fig. 8 Phylum and genus level distribution of fungi communities from untreated and

628

treated seeds.



630

631 632

For Table of Contents Only


Page 34 of 34

Effect of dielectric barrier discharge cold plasma on pea seed

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