Rice (Oryza sativa L.) Seed Sterilization and Germination

Jul 12, 2016 - We designed a system to produce atmospheric hybrid cold-discharge plasma (HCP) based on microcorona discharge on a single dielectric ...
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Rice (Oryza sativa L.) Seed Sterilization and Germination Enhancement via Atmospheric Hybrid Nonthermal Discharge Plasma Natthaporn Khamsen,† Damrongvudhi Onwimol,‡ Nithiphat Teerakawanich,† Sanchai Dechanupaprittha,† Weerawoot Kanokbannakorn,† Komsan Hongesombut,† and Siwapon Srisonphan*,† †

Department of Electrical Engineering, Faculty of Engineering, and ‡Department of Agronomy, Faculty of Agriculture, Kasetsart University, 50 Ngam Wong Wan Road, Ladyao, Chatuchak, Bangkok 10900, Thailand S Supporting Information *

ABSTRACT: We designed a system to produce atmospheric hybrid cold-discharge plasma (HCP) based on microcorona discharge on a single dielectric barrier and applied it to inactivate microorganisms that commonly attach the rice seed husk. The coldplasma treatment modified the surface of the rice seeds, resulting in accelerated germination and enhanced water imbibition. The treatment can operate under air-based ambient conditions without the need for a vacuum. The cold-plasma treatment completely inactivated pathogenic fungi and other microorganisms, enhancing the germination percentage and seedling quality. The final germination percentage of the treated rice seeds was ∼98%, whereas that of the nontreated seeds was ∼90%. Microcorona discharge on a single dielectric barrier provides a nonaggressive cold plasma that can be applied to organic materials without causing thermal and electrical damage. The hybrid nonthermal plasma is cost effective and consumes relatively little power, making it suitable for the surface sterilization and disinfection of organic and biological materials with large-scale compatibility. KEYWORDS: hybrid nonthermal plasma, electrical discharge, Oryza sativa, seed germination, disinfection

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INTRODUCTION Fungal infestation can deteriorate the biological quality and nutritive value of crops and agricultural products, producing undesirable odors and colors.1,2 Although not all microorganisms associated with seed are pathogens, several types of pathogenic fungi, such as Aspergillus spp. and Penicillium spp., can invade and damage stored seeds, legumes, and even young seedlings, lowering the germination yield and quality.1,3,4 The growth of fungi is one of the main factors responsible for the formation of mycotoxins such as aflatoxins, which are hazardous to human health. 3,5,6 Fungal contamination results in unacceptable and improper edible-product grade and is a serious economic problem for agriculturally supplied countries.1,4−6 Therefore, the elimination or minimization of contamination by mycotoxins and fungi is crucial for biological applications. Rice is a staple food for more than half of the world’s population more than 4 billion people depend on rice for at least 20% of their daily calorie requirements.7 The demand for rice seed, the starting point of rice production, is increasing steadily. Seed-borne pathogens of rice are one of the main problems affecting rice production, especially in tropical zones.4 Many types of research with an emphasis on seed treatment have been conducted to reduce the number of microorganisms or other pathogens infesting the surface and inner tissue of seeds before planting.8−11 Most traditional methods use © 2016 American Chemical Society

fungicides, which leave residual compounds and chemicals that can be toxic to the seed and the environment.8,11 Other methods (e.g., heat and ionizing radiation) require many carefully controlled parameters such as temperature, concentration, and energy dose, which significantly influence the nutrients available to the seeds and can consequently reduce seed germination rates.9,10 Ideally, seed sterilization should be harmless to the seeds, generate no toxic residues, and provide efficient processing times at nearly room temperature. Temperatures above ∼45 °C can quickly cause stress or deteriorate the heat-sensitive organics and biomaterials in seeds.12,13 Plasmas consisting of highly energized atoms, reactive species, charged particles, electric fields, and UV radiation are utilized in a broad range of applications.14−16 In recent decades, since the achievement of a plasma phase relatively close to room temperature at atmospheric pressure, nonthermal plasma has been extensively studied and utilized for biological, medical, food-processing, and agricultural applications.17−20 There are several types of nonthermal plasmas classified by their operational characteristics and parameters such as plasma temperature, power consumption, and electrode geometry. Cold plasma based on dielectric barrier discharge (DBD), Received: April 17, 2016 Accepted: July 12, 2016 Published: July 12, 2016 19268

DOI: 10.1021/acsami.6b04555 ACS Appl. Mater. Interfaces 2016, 8, 19268−19275

Research Article

ACS Applied Materials & Interfaces

diameter with an ∼ 200 μm spacing gap; Figure 1b), yielding an ultrahigh electric field localized at the tip edge (up to ∼5 × 104 V/cm). Consequently, the structure allows a microcorona discharge plasma glow in the entire area. An array of top metal tips is connected to a high-voltage sinusoidal source (Matsushita Electronic Components; ∼14 kVpp, at a frequency of ∼700 Hz; see the Experimental Section). The gap distance between the top electrodes and the dielectric layer is designed to be adjustable, typically in the range of ∼5 mm to ∼1 cm, for proper application and a selective gas ambient. Although, the HCP can be obtained in atmospheric-air ambient conditions, other gas mixtures such as air with argon (air/Ar) may be utilized to achieve a uniform, large volume of nonthermal plasma and minimize the damage and energy transferred to the seed, resulting in efficient surface activation. In this work, we injected Ar gas into atmospheric-air microcorona discharge plasma at rate of ∼2.5 l/min ∼8 cm from the plasma active area. The measured power of operation was ∼4.8 W with a gap distance of ∼7 mm. We placed the seed samples on the dielectric layer, exposing them to the cold plasma under airbased ambient conditions. By exploiting the benefits of a highly localized fringe field from the cathode edge that allows microcorona discharge plasma on a single dielectric barrier, we generated atmospherichybrid nonthermal plasma in the form of a faint glow expanding through the entire volume, covering the whole surface of the treated materials (Figures 2a and 3a). We employed Thermal Imager (Fluke-Ti100) and Thermopoint (AGEMA Infrared Systems) to measure the real-time temperature of the plasmatreated rice grains (inset of Figure 2a). We measured the temperature continually for ∼15 min. The average temperature was ∼26.8 °C (maximum ∼27.7 °C, minimum ∼26.4 °C), whereas the base ambient temperature was ∼25 °C, indicating that the hybrid microcorona discharge plasma was certainly a nonthermal plasma. In contrast, high-voltage pulse-induced plasma in a DBD structure can increase the temperature ∼6− 13 °C after ∼1 min treatment,24 while pulse DC-driven plasma jets provide a nonthermal plasma with a rising temperature up to ∼40−85 °C13. To ensure that the microcorona discharge cold plasma was a nonthermal plasma, we placed a piece of white paper on the dielectric and exposed it to the glow discharge for ∼10 min. There was no observable thermal damage, which would be indicated by burning and a dark spot on the paper surface (see Supporting Information S1 for temperature measurements). Therefore, the microcorona charge produced in our system provides a promising plasmatreatment method for highly temperature-sensitive applications and is also feasible for prolonged operation. Although the average and peak of the electric field were estimated to be ∼9 × 103 V/cm and ∼1 × 104 V/cm, respectively, which are far below the values for a typical breakdown electric field in air (∼3 × 104 V/cm), the microcorona discharge was made possible by the contribution of the localized electric field up to ∼5 × 104 V/cm at the tip edge, allowing the corona discharge to stream from the edge of the tips. We employed the finite-element commercial software COMSOL Multiphysics to simulate the distribution of the localized electric field around the tip edge (Figure 2b). We designed the structure used in the simulation to have the same dimensions as our experimental system (see Supporting Information S2, electric field distribution). The dielectric barrier covering the plane electrode prevents arc formation and stabilizes the cold-plasma process. As a result, the

known as direct plasma, is currently one of the attractive methods utilized in biomedical applications.18,21 Although the dielectric-barrier approach is feasible for large-volume applications, the DBD involves numerous instantaneous filament streamers confined at the electrode, producing nonuniformity and requiring high power consumption. In contrast, the coldplasma jet known as indirect plasma can provide a plasma phase that is not confined by electrodes, which is suitable for a selectively localized surface. It has been a challenge, however, to obtain uniform, large-volume cold plasma that is not limited to a localized spot area and uses low operation power. The implementation of large-scale, air-diffuse plasmas has been difficult, and only a few systems have been successfully translated into practical industrial utilizations. Cold plasmas do not require extra chemicals and do not leave any toxic residues for the operators or environments. In addition, cold plasma has the potential to inactivate or kill some microorganisms and pathogens such as bacteria, fungi, and spores without causing thermal damage to nearby membranes.22,23 Therefore, cold plasma offers benefits not only to biomedical applications but also to agricultural applications. Here, we demonstrate a hybrid cold plasma (HCP) configuration for sterilizing and modifying the surface of rice seeds. The HCP can perform under atmospheric conditions at relatively low temperature (∼27 °C) without destroying the seeds, while that of the tradition cold plasma can increase the temperature above room temperature ∼10−20 °C after ∼1 min treatment.13,24 We investigated the modification of the seed surface and wetting properties and the effects on the germination percentage and seedling quality caused by the HCP application.

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RESULTS AND DISCUSSION The contact angle of the seed provides a good demonstration of the influence of the plasma on the seed wettability. Scanning electron microscope (SEM) images showed the successful reduction and inactivation of microorganisms and pathogenic fungi. Figure 1 illustrates a schematic diagram of the HCP configuration, which is based on the combination of corona-

Figure 1. The hybrid cold discharge-plasma system. (a) Schematic of the microcorona discharge on the single dielectric barrier (see Experimental Section for more details). (b) The twin tips as the metal top electrode configuration. Inset (left) scale bar ∼1 cm; (right) scale bar ∼3 mm.

discharge (indirect) plasma and dielectric-barrier (direct) plasma. The system includes a sterilization chamber comprising the top metal electrode and the bottom planar ground electrode, which is covered by glass ∼500 μm thick as a DBD (Figure 1a). The microcorona discharge is obtained by using twin tips with a rounded-cut sharp edge (∼250 μm 19269

DOI: 10.1021/acsami.6b04555 ACS Appl. Mater. Interfaces 2016, 8, 19268−19275

Research Article

ACS Applied Materials & Interfaces

Figure 2. Hybrid cold plasma-discharge mechanism. (a) Photograph of the large-scale plasma treatment of rice grains with corresponding temperature. The tips were wired together and held by epoxy holders, forming an array of 10 × 70 electrodes (∼1400 tips in total) covering ∼30 cm2. The inset shows the corresponding temperature under plasma operation for 10 min (bottom-left) and the localized electric-field distribution around the edge as simulated by a finite-element method (top-left). (b) Optical emission spectra (OES) of plasma generated under the mixture of Ar-air (red line) and air ambient (blue line). (c) A diagram of the plasma mixture comprising UV and related reactive species composition.

Figure 3. Cold plasma-mediated hydrophilic surface modification and enhancement of water imbibition. (a) Operation of hybrid cold-discharge plasma on rice seeds (scale bar ∼7 mm). (b, d, f) Water-droplet morphology and imbibition enhancement of nontreated, pure air-treated, and air/ Ar-treated seeds, respectively. (c, e, g) Apparent contact-angle measurements of nontreated (∼100°), pure air-treated (∼75°), and air/Ar-treated (∼0°) seeds, respectively (scale bar ∼4 mm).

negative type, which is confined by the generation mechanisms to the near-electrode regions.25 Thus, the fringe-field enhanced corona discharge at the electrode edge was likely dominated by the positive-streamer corona. Ion neutralization occurred on the order of 10−100 ns near the dielectric surface, allowing relatively low-temperature plasma with ions and neutral temperatures on the order of a few 100 K.25,26 By exploiting the benefits of the sinusoidal source, we regularly alternated the positive and negative corona discharges (or cathode and anode), including the self-organized electric field direction, resulting in spatial homogeneity. Atmospheric gaseous plasma discharges can produce numerous charged particles, reactive oxygen species (ROS), and reactive nitrogen species (RNS).23 Because of their

microcorona discharge cold plasma streams from the edge of the tips and gradually decreases to the sample (Figures 2a and 3a). The electric field between the two tips is much weaker (Figure 2b) but is sufficient to break down the air-forming microcorona discharge. The simulation results showed the distorted field strength near the tip with a small curvature radius (∼100 μm) caused by the influence of the electric field and the redistribution of the impact-ionization rate induced by the space charge of the directed streamer cathode. The plasma source had a symmetrical, sinusoidal high voltage. Therefore, the HCP system combined the effects of positive and negative corona discharges. Typically, the positive streamer-corona discharge allows a larger volume of plasma creation by photoionization in the interelectrode gap compared with the 19270

DOI: 10.1021/acsami.6b04555 ACS Appl. Mater. Interfaces 2016, 8, 19268−19275

Research Article

ACS Applied Materials & Interfaces

Figure 4. Rice (Oryza sativa L.) seed sterilization and germination. (a, b) Day 14 rice seedlings that did not experience cold plasma before planting. All the seedlings show poor germination due to pathogenic fungi and seed-transmitted diseases (Scale bar ∼3.5 mm). (c, d) Day 14 rice seedlings that experienced cold plasma before planting. All the seedlings appear healthy and without pathogenic fungi. (e, f) Blotter paper used to incubate the seedlings after the day 14. The fungi from the nontreated rice seeds spread throughout the paper, especially (e) around the seedling area, whereas there was significantly less pathogenic fungi spreading from (f) the treated sample. (g) Statistical variation of germination rate and seed quality on day 14 of nontreated and treated samples.

oxidation−reduction (redox) biochemistry, the ROS and RNS play important therapeutic roles in aerobic and plant biology.27,28 Moreover, the plasma is expected to interact with cell membranes at the submicron scale. Hybrid nonthermal plasma is thus a potential approach for deactivating or eliminating fungal growth and mycotoxin formation on rice seeds and seedlings. It is therefore important to establish the impact of the cold-plasma treatment on the wettability and germination characteristics of the rice seeds. Figure 3a illustrates the atmospheric cold-plasma treatment of rice seeds under air-based ambient conditions with an injection of Ar at a flow rate of ∼2.5 l/min from a glass tube (∼5 mm diameter) located ∼8 cm from the treated sample. We measured the water uptake and contact angle of a ∼ 2 μL water droplet on nontreated and treated seeds immediately after plasma application to evaluate the cold plasma-mediated hydrophilic surface modification and the enhancement of water imbibition (see the Experimental Section). The nontreated rice seed is naturally hydrophobic, resulting in very slow water absorption (Figure 3b and 3c). The measured contact angle was ∼100° initially and gradually decreased due to absorption into the rice seed. The nonirradiated sample took ∼30 min to complete the water uptake process under ambient conditions. In contrast, the sample treated with air/Ar plasma showed no observable contact angle (∼0°), with the water droplet

instantaneously spreading over the surface. The duration of the water-uptake process was reduced to ∼1 min for the same amount of water (Figure 3e and 3f). The effective cold plasmatreatment time depends on the type of target material. In this case, 1 min plasma treatment under the mixture of air-Ar ambient was sufficient to hydrophilize the rice-seed surface, substantially enhancing the water imbibition (see Supporting Information S5, water imbibition experiment of rice seeds under different treatment times and ambient conditions). Composed of highly energized electrons, plasma gas can naturally enable the etching process on the seed surface, increasing the seed coat permeability and the rate of water uptake. To substantiate the hypothesis that the etching process on the seed surface was the primary mechanism of hydrophilization and enhancement of water imbibition, we gently scribed the rice seeds to remove the glume and any pericarp. The scarified seed surface was more hydrophilic than the nonscarified seed surface but was not comparable to that of the sample treated with the hybrid air/Ar cold plasma. The contact angle of the scribed sample was ∼40° and required ∼25 min to completely consume ∼2 μL of water (see Supporting Information S3, scarification test). Therefore, the hydrophilization process was determined not only by the scarified or etched surface but also by the nanoscale surface modification caused by the interaction between the reactive species in the 19271

DOI: 10.1021/acsami.6b04555 ACS Appl. Mater. Interfaces 2016, 8, 19268−19275

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ACS Applied Materials & Interfaces

Seed-borne pathogens of rice commonly invade and contaminate the seeds during harvesting and storage, especially in tropical zones.4,8−11 Figure 4b, d shows typical day 14 seedlings of nontreated and treated rice seeds, respectively. The fungi grew on almost the entire area of the natural seeds, particularly around the mesocotyl, seed coat surface, and seedling, whereas the treated seeds demonstrated a very clean surface with no fungi (see Supporting Information S4, representative data of seedling after day 14 of germination). The fungi also spread throughout the blotter, especially beneath the nontreated seedlings, as shown in Figure 4e. In contrast, we observed only an infinitesimal amount of fungi in the paper of the treated sample illustrated in Figure 4f. The four replicated experiments of each treated and nontreated sample showed the same results, which confirms the substantial reduction or deactivation of pathogens on the rice seeds. That result implies that the coldplasma treatment not only modified the surface from a hydrophobic state to a hydrophilic state but also completely inhibited the fungi, resulting in a higher growth rate and germination percentage. The plasma irradiation covered a range from the UV to the VIS because of the excitation and relaxation of excited Ar molecules (600−900 nm wavelength) and N2 molecules (300− 400 nm wavelength).20,22 Plasma generation in air mixed with Ar can produce several ROS and RNS such as ozone, (O3), hydroxyl (•OH), nitric oxide (NO), and peroxynitrite (ONOO−).20,22,28 Although UV is known to have the ability to inhibit bacterial replication,23 plasma-radiated UV is not a key mechanism in the process of killing or deactivating microorganisms because of its ultrathin penetration depth.19,30 On the other hand, the plasma-induced active species play an important role in modifying the seed surface and killing and deactivating microorganisms and pathogens. For example, some of the ROS such as hydroxyl radicals and singlet oxygen (1O2) cannot be produced in pure-air ambient conditions.20 Therefore, the hydroxyl radicals (•OH) and singlet oxygen (1O2) generated in the air/Ar plasma were responsible for the fast surface modification, enabling the hydrophilic surface. The experiments that directly monitor the reactive species levels of HCP system is fundamentally important to understand the underlying physic of seed surface−plasma interaction processes. Therefore, we utilized the optical emission spectroscopy (OES) for determination of the relative reactive species level. The OES was performed by using CCD spectrometers (THORLABS CCS200, wavelength 200−1000 nm) to analyze the reactive species generated under pure-air and the mixture of air-Air plasma treatment (see Experimental Section). Figure 2b demonstrate the optical emission spectra of HCP system under the mixture of Ar-air (red line) and pure air (blue line). Two regions of peaks were clearly observed under Ar-air ambient between 600 and 900 nm and 300−400 nm corresponding to Ar excited and N2 excited species, respectively.20,22 In contrast, there was no observable peaks of pure-air cold plasma. The amount of ROS and RNS in the system are directly related to exited Ar and N2 species generation.20,22,28 Therefore, the overall results suggest that ROS and RNS were significantly amplified in HCP system. This result is in good agreement with the results of water imbibition experiment that seeds treated by ∼1 min air-Ar HCP requires significantly less time to absorb the 2 μL water droplet compared to the seed treated by pure-air HCP and nontreated sample (see Supporting Information S5, water imbibition experiment of rice seeds under different treatment times and

gas-discharge plasma and the seed surface. Each experiment was performed on more than 50 seeds to confirm the reliability of the results. Overall, the wettability of the rice seed was improved significantly by the plasma treatment, leading to increased water absorption. To differentiate the importance of the interactions between the reactive species generated in the pure-air and air/Ar cold plasmas, respectively, and the seed morphology, we conducted an additional contact-angle experiment and water-absorption test under pure-air plasma conditions with the same 1 min exposure time. The sample with the pure-air plasma had a contact angle of ∼76° and needed ∼20 min to completely consume the 2 μL water droplet. That result suggests that the ROS generated by the Armediated cold plasma play important roles in the surface modification and wettability enhancement (more discussion appears in the germination test). To substantiate the hypothesis that HCP can provide benefit to rice germination, we irradiated 100 rice seeds with hybrid plasma for 1 min using the same protocol described for the hydrophilic treatment conditions. We placed 100 treated seeds and another 100 nontreated seeds on moist seed-germination paper kept in transparent acrylic seed-germination trays. Each tray contained ∼25 rice seeds (total ∼8 trays). The experiments were repeated four times under the same setup conditions to confirm the reproducibility of processes. We used seeds of Oryza sativa var. Indica cv. KDML105 that were kept in a clean polyethylene box under ∼10% relative humidity. During the plasma treatment, we randomly chose ∼20% of the total treated seeds and performed contact-angle measurements to ensure the successful hydrophilic enhancement of the seeds and also the uniformity and reproducibility of the plasma-treatment process. All of the samples were incubated in Plant Growth Chambers (Daihan Labtech Model LGC-5201) at 20 °C in the dark for 16 h and 30 °C in cool light for 8 h. The controlled relative humidity was ∼85% (see the Experimental Section). We examined the seeds every 24 h and considered them germinated when the radicle emergence was ∼0.5 cm. We calculated the maximum germination percentage by counting the seedlings on days 7 and 14.29 Figure 4 shows the overall results for the treated and nontreated seedlings after day 14. During the first 7 days, the average radical emergence percentage was ∼98% for treated and ∼90% for nontreated seeds. However, there was no significant difference of the seedling length between the nontreated and treated rice seeds. The average seedling length of both condition was ∼4.3 cm. After 10−14 days, the treated samples clearly demonstrated higher seedling quality with an average final germination percentage of ∼98%, while that of the nontreated samples was ∼90% (Figure 4g). Moreover, ∼ 90% of the germinated (plasma treated) seeds had a seedling length longer than 12 cm, while only ∼66% of the nontreated seedlings had a length longer than 12 cm. Only the seedlings that grew were counted to calculate the seedling length. Overall, the average height of the treated and nontreated seedlings was ∼13 and ∼11 cm, respectively. That result implies that the plasma treatment did not decrease or influence the essential nutrients required for rice seedling growth. On the other hand, the plasma significantly enhanced the growth rate. We attributed the acceleration and enhancement of germination to the reduction or deactivation of microorganisms such as fungi that commonly contaminate natural rice seeds. The treated samples (Figure 4c) were much healthier (with no contamination by photogenic fungi) compared with the nontreated samples (Figure 4a). 19272

DOI: 10.1021/acsami.6b04555 ACS Appl. Mater. Interfaces 2016, 8, 19268−19275

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ACS Applied Materials & Interfaces

Figure 5. SEM micrographs of rice seeds, including the seed coat−lemma and palea−in untreated control and cold plasma-treated KDML105 rice. (a−c) Nontreated rice seeds with scale bars ∼500, 200, and 50 μm, respectively. (d−f) Treated rice seeds with scale bars ∼500, 200, and 50 μm, respectively. The blue dashed line windows demonstrated (b, c) the microorganism living along the trench and (e, f) fragments of microorganisms.

inactivate pathogenic fungi, consequently stopping mycotoxin formation. The energized charge particles originating in an atmospheric discharge via electron-impact excitation interact with the surface as a result of etching or rupture of the seed coat membrane.33 The cold plasma can thus enhance the wateruptake process and accelerate germination. It is still uncertain, however, whether the pathogens were temporarily or permanently deactivated, which will be a subject of further research. In addition, the generated RNS such as nitric oxide (NO) and Nitrate radical (NO3) can probably provide an important macronutrient to the seed and can proliferate on the seed to improve nitrogen fixation, resulting in accelerated germination compared with that of nontreated seeds.26,34

ambient conditions). That result is in agreement with the hypothesis that active species were responsible for the kinetics and mechanism of the inactivation process. During the plasma operation, the reactive species chemically interact with and infiltrate microorganisms, consequently impairing the contaminating microbes at the outer cell membrane. The reaction process can occur on a microsecond time scale.31 However, to substantiate with N2 excited species in pure air HCP, the OES was performed with longer integration time (∼20 s) under airHCP operation (see Supporting Information S6, optical emission spectrum of discharge plasma). The observed peak in region of 300−400 nm is in good agreement with the one that observed under air-Ar, confirming that only RNS species is generated under air ambient. Therefore, overall result suggest that the ROS generated by excited Ar species is an important for surface modification and water imbibition enhancement. Moreover, the strong electromagnetic field can contribute a large electric force that alters the electrostatic polarization inside the microorganism, resulting in changes in the cellular membrane and internal molecular structures or cell death. Hence, the cell membrane becomes dysfunctional.32 The electric field can enhance the permeability of microorganisms within a short time, which is sufficient for the chemical reaction to take place. Therefore, the bulk seed remains safe and healthy. For a detailed analysis of the modification of the resident microorganisms and morphology of the rice-seed surface and the consequent enhancement of the growth rate, we performed SEM on treated and nontreated rice seeds after the hybrid nonthermal plasma operation. The micrographs showed no significant difference between the nontreated (Figure 5a) and treated (Figure 5d) samples. There was no burning mark or thermal damage, confirming that the treatment was a coldplasma treatment. The microorganisms were living along the surface trenches, and there was a thin wax layer covering the seed surface (Figure 5b and 5c-blue dashed line windows). In contrast, the treated seeds (Figure 5e and 5f) had a cleaner surface with broken wax and fragments of microorganisms and pathogens such as fungi, indicating nanoscale modification of the surface morphology. The wax layer covering the seed surface is naturally thin for rice seed. The (blue dashed line) windows on Figure 5e and 5f demonstrate the broken of microorganism and the clearly opened area of wax on the rice seed surface. By incorporating Ar with air, hybrid cold plasma can produce ROS and RNS, which can decontaminate and

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CONCLUSION The hybrid cold-discharge plasma has the potential to modify rice-seed characteristics including surface properties. Not only does it increase the imbibition rate, but cold plasma also enhances the germination process, which could be advantageous for slow-to-germinate seeds. Furthermore, nonthermal plasma can operate at atmospheric pressure, which is suitable for some organic materials that cannot be sustained in a high vacuum ambient. Cold plasma is environmentally friendly and does not leave any contaminated aqueous waste. The coldplasma technology should be considered as the necessity approaches for the future use of seed-treatment technologies. Although the microorganisms on rice seeds were successfully inactivated, there are other fundamental issues that will require in-depth investigation. Understanding the underlying physics of plasma−biological interaction could lead to new applications in biological sterilization and antipathogenic therapy.

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EXPERIMENTAL SECTION

Seed Materials. Seeds of Oryza sativa var. Indica cv. KDML105 were obtained from the Rice Department, Bangkok, Thailand. The seeds had been stored at 5 ± 1 °C and 50% relative humidity for 9 months subsequent to harvesting in June 2015 from the Ubon Ratchathani Rice Research Center at Ubon Ratchathani, Thailand (15° 19′ 55.15″N, 104° 41′ 27.28″E). Prior to the experiment, the seeds were graded, and only seeds of 15−30 mg with confirmed uniformity were used in the experiments. High Voltage Generation in the HCP System. An array of top metal tips was connected to a high-voltage sinusoidal source (Matsushita Electronic Components, ∼ 14 kVpp at a frequency of ∼700 Hz) driven by a DC power supply (OMRON DC 24 V, 2.1 A). 19273

DOI: 10.1021/acsami.6b04555 ACS Appl. Mater. Interfaces 2016, 8, 19268−19275

Research Article

ACS Applied Materials & Interfaces

(Grant E5068-5814501294). S.S. acknowledges the financial support by the Thailand Research Fund and the Kasetsart University Research and Development Institute (Grant TRG5880237). We are very grateful for Dr. Varakorn Kasemsuwan for his helpful discussions.

The high voltage was measured using a High Voltage Probe (Tektronix P6015A) in conjunction with a DSO1012A-100 MHz oscilloscope (Agilent Technologies). An array of the tips was designed with 10 × 10 twin-tip electrodes covering an area of ∼4 cm2. Treatment Conditions and Seed Materials. Rice seeds were selected and exposed to hybrid cold-discharge plasma under air-based ambient conditions. Ar gas was supplied with a flow rate ∼2.5 l/min at a distance of ∼8 cm from the plasma-active area. Water Droplet, Apparent Contact Angle Measurement, Water Imbibition Enhancement. A water droplet was applied using a well-controlled 2 μL pipet (Lab-Mate Pro 20 LMP20 2−20 μL). The clear contact angle and water imbibition of the 2 μL water droplet were measured under an optical microscope in conjunction with computer-aided measurement. Germination Test. Germination testing was carried out immediately after the plasma treatments in accordance with ISTA,34 except that only 100 seeds were used per treatment (four replicates of 25 seeds each). The seeds were placed on top of moistened blotter papers in transparent 15 × 8 × 7 cm polyethylene boxes. Each box contained one replicate (25 seeds) of a single treatment. The boxes were placed in a plant-growth chamber (Daihan Labtech, Model LGC5201) with conditions of 20 °C in the dark for 16 h and 30 °C in cool light for 8 h at ∼85% relative humidity. Germination, normal seedling, and seedling growth were determined at 24 h intervals for 14 days. Statistical Analysis. Descriptive statistics including the mean, maximum and minimum values of four replicates were expressed to represent the statistical variation of germination rate and seed quality on day 14 of nontreated and treated samples. Optical Emission and Absorption Spectroscopy. Optical emission spectroscopy (OES) was performed using a CCD spectrometer (THORLABS CCS200, Germany) to analyze reactive species generation during plasma treatment. Detector optical fibers were placed near the plasma area and the emission spectrum monitored at wavelengths between 200−1100 nm with ∼10 s integration time. Scanning Electron Microscopy. Intact rice seeds, treated and nontreated, were equilibrated in 10−15% relative humidity using hermetic containers and zeolite drying beads for controlling the relative humidity. Dry seeds with a moisture content of 5−8% were stubbed for SEM at 25 ± 3 °C. Pictures were produced using a Hitachi SU 1500 (Japan).

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ASSOCIATED CONTENT

* Supporting Information S

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acsami.6b04555. Temperature measurement under atmospheric hybrid cold plasma, the calculation of electric field distribution between the two electrodes under plasma operation via finite element method (FEM), surface modification and water imbibition test on scarification seeds, representative data of seedling after day 14 of germination, water imbibition experiment of rice seed under different treatment times and ambient conditions, optical emission spectrum of the discharge dominated by excited N2 in an atmospheric air-HCP system (PDF)

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REFERENCES

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work is supported in part by the Graduate Program Scholarship from the Graduate School, Kasetsart University 19274

DOI: 10.1021/acsami.6b04555 ACS Appl. Mater. Interfaces 2016, 8, 19268−19275

Research Article

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DOI: 10.1021/acsami.6b04555 ACS Appl. Mater. Interfaces 2016, 8, 19268−19275