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Production of nitric oxide using a microwave plasma torch and its application to fungal cell differentiation
This content has been downloaded from IOPscience. Please scroll down to see the full text. 2015 J. Phys. D: Appl. Phys. 48 195401 (http://iopscience.iop.org/0022-3727/48/19/195401) View the table of contents for this issue, or go to the journal homepage for more
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Journal of Physics D: Applied Physics J. Phys. D: Appl. Phys. 48 (2015) 195401 (7pp)
doi:10.1088/0022-3727/48/19/195401
Production of nitric oxide using a microwave plasma torch and its application to fungal cell differentiation Young Ho Na1,3, Naresh Kumar2,3, Min-Ho Kang1, Guang Sup Cho1,2, Eun Ha Choi1,2, Gyungsoon Park1,2 and Han Sup Uhm1,2 1
Department of Electrical and Biological Physics, Kwangwoon University, Seoul 139-701, Korea Plasma Bioscience Research Center, Kwangwoon University, Seoul 139-701, Korea
2
E-mail:
[email protected] and
[email protected] Received 20 January 2015, revised 2 March 2015 Accepted for publication 13 March 2015 Published 8 April 2015 Abstract
The generation of nitric oxide by a microwave plasma torch is proposed for its application to cell differentiation. A microwave plasma torch was developed based on basic kinetic theory. The analytical theory indicates that nitric oxide density is nearly proportional to oxygen molecular density and that the high-temperature flame is an effective means of generating nitric oxide. Experimental data pertaining to nitric oxide production are presented in terms of the oxygen input in units of cubic centimeters per minute. The apparent length of the torch flame increases as the oxygen input increases. The various levels of nitric oxide are observed depending on the flow rate of nitrogen gas, the mole fraction of oxygen gas, and the microwave power. In order to evaluate the potential of nitric oxide as an activator of cell differentiation, we applied nitric oxide generated from the microwave plasma torch to a model microbial cell (Neurospora crassa: non-pathogenic fungus). Germination and hyphal differentiation of fungal cells were not dramatically changed but there was a significant increase in spore formation after treatment with nitric oxide. In addition, the expression level of a sporulation related gene acon-3 was significantly elevated after 24 h upon nitric oxide treatment. Increase in the level of nitric oxide, nitrite and nitrate in water after nitric oxide treatment seems to be responsible for activation of fungal sporulation. Our results suggest that nitric oxide generated by plasma can be used as a possible activator of cell differentiation and development. Keywords: nitric oxide, microwave plasma, fungal differentiation, biological application of plasma (Some figures may appear in colour only in the online journal)
1. Introduction
be also possible that nitric oxide can trigger other biological activations such as cell differentiation and metabolic stimulation. Differentiation and activation of stem cells and effective microorganisms has been one of the foremost issues in current regeneration medicine and industry [13, 14]. Although an increasing number of studies have focused on developing technologies for the activation of cell differentiation and microbial metabolisms, tremendous research is still needed. A nitric oxide generator based on an arc discharge has been developed and demonstrated that treatment with gaseous
Studies have shown that reactive nitrogen species (RNS) play critical roles in regulating disease resistance, growth, and stress tolerance in plants [1–7], and blood pressure and muscle relaxation in animals [8–12]. Since nitric oxide (a major reactive nitrogen species) is a well known activator for blood circulation, wound healing, and disease resistance, it may 3
Equally contributed to this work.
0022-3727/15/195401+7$33.00
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© 2015 IOP Publishing Ltd Printed in the UK
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J. Phys. D: Appl. Phys. 48 (2015) 195401
nitric oxide has improved healing of complicated wounds in rats and humans [15–17]. These studies suggest that exogenous gas type nitric oxide can be used for therapeutic purposes with significant health benefits. However, the beneficial effects of gaseous nitric oxide have not been actively explored in other application areas. Since cell proliferation and differentiation are essential steps in wound healing, plasma generated nitric oxide can possibly stimulate these steps. In a previous study, we developed a microwave plasma torch system producing nitric oxide and demonstrated a theoretical basis for nitric oxide generation [18]. As a follow-up study, we present here experimental data on nitric oxide generation from a microwave plasma torch and effects of nitric oxide on cell differentiation using Neurospora crassa (bread mold), a saprophytic fungus (non-pathogenic) known as a model organism for eukaryotes. Our results exhibit that nitric oxide can elevate the formation of spores and expression of a sporulation related gene in N. crassa. Since N. crassa is a model system for eukaryotic cells, application of nitric oxide to N.crassa differentiation can provide useful information on differentiation of other eukaryotic cells such as mammalian and stem cells. 2. Experimental setup 2.1. System configuration of nitric oxide generating microwave plasma
The microwave plasma system for nitric oxide generation consists of a magnetron, waveguide components (WR-340 for 2.45 GHz) and a microwave plasma torch, as shown in figure 1(a). Nitrogen gas mixed with oxygen enters the discharge tube through feeders as a swirl gas that creates a vortex flow in the discharge tube, where a plasma torch with a temperature of 6000 K and a plasma density on the order of 1013 cm−3 is generated. The design and operation of the atmospheric microwave plasma torch are reported in detail in the literature [19]. The gas emitted from the torch flame has a very high temperature. Therefore, a microwave plasma-torch system is connected to a water cooling system for medical applications. The gas is monitored by a gas analyzer. Nitrogen torch flames at a microwave power of 400 W are presented in figure 1(b). The nitrogen working gas is mixed with oxygen with a small mole fraction. The flames in figure 1(b) are the result with 10 standard liters per minute (slm) of nitrogen gas with an oxygen mixture gas of 10 ~ 50 standard cubic centimeters per minute (sccm). The torch flame becomes longer as the electrical power increases. In addition, the overall torch flames increase as the oxygen gas increases. The flames in figure 1(b) heat the quartz tube, which in turn emits its own light, masking the torch flames in the photographs. The torch flames in figure 1(b) exhibit two distinctive regions, the first a bright region of the high-temperature zone for a typical torch based on plasma species and the second a blurry, dimmer region of a relatively low-temperature zone of nitrogen atoms burning in oxygen gas.
Figure 1. Microwave plasma torch system and analysis of plasma flame. (a) System configuration of the nitric oxide generator, consisting of a power supply, a magnetron, a circulator, a power monitor, a three-stub tuner, a tapered waveguide, a discharge tube made of fused quartz, and a water cooling device. (b) Nitrogen torch flames shown inside a quartz tube. The flames are for 10 slm of nitrogen gas with an oxygen gas input in the range of 10 ~ 50 sccm. (c) Plot of the apparent flame length versus the oxygen input in sccm for a nitrogen flow rate of 10 slm. The bright purplish region of the high-temperature zone is about 5 cm for a nitrogen gas flow of 10 slm.
2.2. Assessment for the effects of nitric oxide on fungal differentiation
Differentiation of the fungus N. crassa starts with germination of spores followed by formation of fungal mycelia (stage of vegetative growth). N. crassa then begins to form aerial hypha and produce multinucleate asexual spores, macroconidia at the end of aerial hypha (stage of sporulation) [20]. We examined if treatment of fungal spores with nitric oxide generated from a microwave plasma torch can accelerate any stage in fungal differentiation. Treatment of fungal spores with nitric oxide was carried out as follows: sterile water was added in a 2 week old culture flask and the flask was vigorously shaken. The suspension was filtered through 2 layers of miracloth (Calbiochem, Darmstadt, Germany) to remove fungal mycelia. The filtered suspension was centrifuged at 4000 rpm for 5 min to pellet down fungal spores. The spore 2
Y H Na et al
J. Phys. D: Appl. Phys. 48 (2015) 195401
pellets were resuspended in new sterile water to get the concentration of 106 spores per milliliter (ml). The spore suspension (5 ml) was placed in a petri dish (90 mm in diameter) and exposed to different levels of nitric oxide for 5 min. The level of nitric oxide was controlled by changing the flow rate of oxygen gas. A plasma torch was generated by using a microwave of 2.45 GHz and 400 W, N2 gas of 10 slm (standard liter per minute) and O2 gas of various flow rates. A spore suspension with no treatment was used as control. After exposure to nitric oxide or no treatment (control), 2 µl of suspension was placed in the center of VM (Vogel’s Minimal) agar plate and incubated at 30 °C for 24 h. After 24 h, fungal vegetative growth was assessed by measuring the diameter of the fungal mycelial mat formed on the VM agar plate. Sporulation of the fungus after nitric oxide treatment was quantified by counting the number of spores formed on aerial hypha. After the spore suspension was treated with different levels of nitric oxide, 2 µl of suspension was placed on VM agar media in a 100 ml Erlenmeyer flask and the flask was incubated at 30 °C for 5 d. After 5 d, 10 ml of water was added into the flask and the flask was vigorously shaken to suspend spores into water. The spore suspension was collected and the number of spores per ml suspension was counted. The processes involved in fungal sporulation are carried out by the action of many sporulation related genes. Thus, we examined if nitric oxide treatment can activate the expression of sporulation related genes, in other words, increase the level of mRNA for sporulation related genes. The level of mRNAs for 10 candidate sporulation related genes (NCUs 08769, 07846, 09235, 07325, 07324, 08726, 08457, 09873, 00478, 07617) was measured by real time RT (Reverse Transcriptase) PCR (Polymerase Chain Reaction). Real time RT PCR was performed as described previously [21].
was measured as described previously [22]. The level of nitric oxide within the cell was measured by DAF-FM diacetate (Life Technologies, Washington DC, USA) as described previously [21]. Intracellular nitric oxide was confirmed by using nitric oxide scavenger cPTIO (2-(4-Carboxyphenyl)-4,4,5,5tetramethylimidazoline-1-oxyl-3-oxide; Life Technologies, Washington DC, USA). After exposure to nitric oxide, fungal spores were treated with 2 mM cPTIO for 5 min and then intracellular nitric oxide was detected by DAF-FM diacetate as described above. 3. Results and discussion 3.1. Nitric oxide production
Production of nitric oxide was investigated using nitrogen plasma operated at a microwave power of 400 W. Typical flames from the microwave plasma torch are shown in figure 1(b) for a nitrogen flow rate of 10 slm. The length of the overall flames increases as the oxygen input increases. Particularly, the blurry, dimmer region of the relatively lowtemperature zone increases with an increase in the oxygen. This region is dimmer orange in color according to naked-eye observations. Establishing a reference point at the same level of brightness near the end of the dimmer region, we roughly measured the length of the flame from the base to the reference point. Shown in figure 1(c) is a plot of the apparent flame length versus the oxygen input in sccm for a nitrogen flow rate of 10 slm. The bright purplish region of the high-temperature zone is about 5 cm for 10 slm of nitrogen gas, where oxygen and nitrogen molecules dissociate preferentially in this region due to high density of plasma species. Nitrogen molecules are also excited in this high-temperature zone by high density electrons. The fluid element with a high concentration of nitrogen atoms flows through the relatively low-temperature zone, abundantly producing nitrogen monoxide. To identify the various excited plasma species generated by the microwave plasma torch, optical emission spectroscopy was applied in a wide wavelength range of 200–900 nm. Figure 2(a) shows the optical emissions of the torch flame of the microwave plasma when 10 slm nitrogen gas with an oxygen input of 50 sccm was injected. The emission spectrum was mainly dominated by the presence of excited nitrogen species, containing the first positive system of N2 due to the molecular excitation of nitrogen. In addition, the highly reactive radicals like nitric oxide (NO) at 250 nm are shown in figure 2(a). Atomic oxygen lines at 616 and 777.1 nm are buried by the strong emissions of the N2 first positive system, which is related to the excited metastable state of nitrogen molecules due to the small mole fraction of the oxygen gas. Atomic nitrogen lines at 747, 822 and 870 nm are also buried by the N2 emission. The hot nitrogen gas from the torch flame cools down while going through the cooling system, as shown in figure 1(a). The gas discharged through the cooling system was sampled and analyzed. Figure 2(b) shows the experimental data for the nitric oxide concentration in units of particulate per minute (ppm) in the gas discharged from the microwave plasma torch operated
2.3. Measurement of temperature, pH, and the level of intracellular and extracellular reactive species
The temperature and pH of water treated with nitric oxide were measured by using an alcohol thermometer and a portable pH meter (Eutech Instruments, Singapore), respectively. In order to find the effect of water acidity on fungal differentiation, fungal spores were incubated in an HNO3 solution with different pH for indicated time. After incubation, 2 µl of suspension was placed on VM agar media and incubated at 30 °C. Hyphal growth, sporulation, and level of acon-3 expression were monitored as described above. The level of reactive oxygen species (OH radical) and reactive nitrogen species (nitric oxide) were measured in water and spore cells after treatment with nitric oxide. For measurement of OH radicals, terephthalic acid (TA; Sigma, St. Louis, MO, USA) was added in water (20 mM) and water was exposed to nitric oxide for 5 min. Then, the level of hydroxyterephthalic acid (oxidized TA by hydroxyl radical) was measured as described previously [22]. In order to measure the level of nitric oxide in water, DAF-FM (4-amino-5-methylamino-2′, 7′-difluorofluorescein; Life Technologies, Washington DC, USA) was added in 5 ml of water (final concentration 10 μM) right after being treated with nitric oxide and then fluorescence 3
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J. Phys. D: Appl. Phys. 48 (2015) 195401
The oxygen density at room temperature can be calculated through multiplying 2.6 × 1019 cm−3 by the ratio of the oxygen input to the nitrogen flow rate (a fixed flow rate). The nitrogen monoxide density nNO is modeled to be [18],
⎛ 2460 ⎞ T ⎟n O [1 − exp(−βn O )], exp⎜− 2 2 ⎝ Tr T ⎠ (1) nNO(n O2 ) = 6.29 × 10−2
Here, nNO and nO2 are the densities of the nitrogen monoxide, and molecular oxygen, respectively. T is the torch temperature and Tr = 298 K denotes the room temperature. The temperature T in equation (1) is the average flame temperature, which may be difficult to determine. It is therefore determined from the experimental data by a proper fitting. The parameter β must be determined from the gas flow rate and the experimental conditions. Equation (1) estimates the nitric oxide density in terms of the molecular oxygen density. Figure 2(c) shows the relationship between nitric oxide density and the oxygen molecular density for four different flow rates of nitrogen gas. The measured density of the nitric oxide in units of ppm shown in figure 2(b) is well matched with the line for the nitrogen flow rate of 20 slm in figure 2(c). The blue curve is obtained from equation (1) when T = 2950 K and β = 2.2 × 10−18 cm−3 for a 20 slm flow rate. Here, T = 2950 K and β = 2.2 × 10−18 cm−3 are least-square-fitted to the data. The flame temperature T and parameter β are also given for other flow rates as T = 2950 K and β = 1.4 × 10−17 cm−3 at 10 slm and T = 2950 K and β = 1.2 × 10−18 cm−3 at 30 slm. However, the flame temperature T and parameter β for a nitrogen flow rate of 5 slm are T = 4330 K and β = 5 × 10−17 cm−3, respectively, which are least-square-fitted to the four experimental data points in figure 2(b), resulting in the green curve in figure 2(c), which is obtained from equation (1). It is remarkable to observe that the flame temperatures T for the nitrogen flow rates of 10, 20 and 30 slm are identical. Deposition of microwave power (400 W) into a gas element with different flow rates may occur through the discharge tube. It may be possible that the gas element at a low flow rate may receive more microwave energy than that at a high flow rate, leading to an increase in flame temperature. However, the same average flame temperature was observed in the flow rate range of 10 ~ 30 slm although the visual flame length may shrink as the flow rate increases. Meanwhile, the average flame temperature (T = 4330 K) for the nitrogen flow rate of 5 slm is much higher than that of the other three nitrogen flow rates. The nitrogen gas enters the discharge tube as a swirl gas, creating a vortex flow. The flow rate in the range of 10 ~ 30 slm generates a stable vortex flow for steady torch flames, whereas a flow rate of 5 slm does not provide a stable vortex due to insufficient gas flow. As a result, the flame somehow wobbles and the discharge-tube part becomes hot, resulting in a higher flame temperature.
Figure 2. Optical emission spectroscopy and nitric oxide concentration. (a) Optical emissions of the torch flame of the microwave plasma when 10 slm nitrogen gas with an oxygen input of 20 sccm was injected. The emission spectrum was mainly dominated by the presence of excited nitrogen species. (b). Plots of experimental data of nitric oxide concentration in units of particles per minute (ppm) versus the oxygen input in units of sccm for several different nitrogen flow rates. (c) Plots of the nitric oxide density versus the oxygen molecular density for four different flow rates of nitrogen gas. The curves are obtained from equation (1) for (T, β) = (4330 K, 5 × 10−17 cm−3) for 5 slm of nitrogen gas, (T, β) = (2950 K, 1.4 × 10−17 cm−3) for 10 slm of nitrogen gas, (T, β) = (2950 K, 2.2 × 10−18 cm−3) for 20 slm of nitrogen gas, and (T, β) = (2950 K, 1.2 × 10−18 cm−3) and for 30 slm of nitrogen gas.
at 400 W. The nitric oxide concentration in figure 2(b) is presented in terms of the oxygen input in units of sccm for several different values of the nitrogen flow rate. Each data point is the average of five measured values. The typical error bar is shown at an oxygen input of 120 sccm at a nitrogen flow rate of 20 slm, which is about 7%, including possible errors associated with the measurement device. The nitric oxide concentration increases as the oxygen input increases, as expected. The increase in the nitric oxide concentration with the oxygen input is more drastic as the nitrogen flow rate decreases. A nitric oxide concentration in a broad range is needed for the healing effects. A nitric oxide concentration in this range can easily be achieved for a broad range of physical parameters by making use of the microwave plasma torch.
3.2. Application of nitric oxide to fungal differentiation
In order to examine the effect of nitric oxide on germination, vegetative growth and sporulation of N. crassa, fungal spores suspended in water (106 ml−1) were exposed to different levels 4
Y H Na et al
J. Phys. D: Appl. Phys. 48 (2015) 195401
Figure 4. Expression of a gene related to asexual sporulation (acon-3) after nitric oxide treatment. The level of mRNA for acon-3 was monitored during incubation after fungal spores were exposed to nitric oxide generated under different levels of oxygen flow. Each value is an average of two replicate measurements from one experiment and the experiment was repeated three times.
Septa are formed between constricted bead like structures leading to spore formation [20]. These processes in sporulation are carried out by the action of many sporulation related proteins and therefore the expression (mRNA) level of genes coding for these proteins may be changed during sporulation. In N. crassa, about 25% of the predicted genes in the genome are differentially expressed during asexual development such as sporulation [23]. We examined changes in expression (mRNA) level of 10 candidate genes coding for sporulation related proteins (NCUs 08769, 07846, 09235, 07325, 07324, 08726, 08457, 09873, 00478, 07617) by quantitative PCR analysis after nitric oxide treatment. Figure 4 shows that among 10 genes, the mRNA level of a gene named as acon-3 (NCU07617) was increased after nitric oxide treatment under two oxygen flow conditions (200 and 400 sccm). Upon nitric oxide treatment, the level of acon-3 mRNA was significantly increased after 24 h and then decreased slightly after 48 h. Since the sporulation process in N. crassa starts after being grown on VM agar media for at least 24 h, and acon-3 is known to be expressed at early stage of conidiation and regulate early conidiophore development (a structure harboring spores) [23], our results indicate that nitric oxide treatment can accelerate the expression of acon-3 after 24 h and this may lead to the elevated sporulation. In order to identify factors responsible for the effects of nitric oxide on fungal sporulation, the temperature and pH of water after nitric oxide treatment were measured. As demonstrated in figure 5, the temperature of water was maintained at around 20 °C during nitric oxide treatment and the pH of water was decreased after nitric oxide treatment with a greater decrease in pH under higher oxygen flow. This indicates that slight acidification of water by nitric oxide treatment may play an important role in activating fungal sporulation. To test this hypothesis, fungal spores were incubated in water of similar pH adjusted by using nitric acid for 5 min and then growth and sporulation were examined on VM agar media. Figure 5(c) shows no significant changes in hyphal extension, spore numbers, and expression of acon-3 after treatment with nitric acid water. This suggests that reduction in the pH of water after treatment with nitric oxide may not be the significant reason for the accelerated fungal sporulation.
Figure 3. Hyphal growth and sporulation of N. crassa treated with nitric oxide. (a) Extension of fungal basal hypha measured as diameter of a mycelial mat formed on a VM agar plate and spore formation rate indicated as the number of spores harvested in ml water after nitric oxide treatment. Each value represents the average of 3 replicate measurements and the Student’s t test was performed; *p
