Plasma-driven biocatalysis

Abstract

Methods using plasma-driven generation of H.sub.2O.sub.2 in an aqueous liquid may provide a substrate to enzymes, which are then capable to oxidize or hydroxylate organic compounds. A plasma device may produce an aqueous liquid comprising H.sub.2O.sub.2 for use in an enzymatic reaction.

Claims

1. A method for enzymatically oxidizing or hydroxylating an organic compound, wherein the method comprises: treating an aqueous liquid with a plasma device to obtain an aqueous liquid comprising H.sub.2O.sub.2 to form an enzymatically oxidized or hydroxylated organic compound; wherein the aqueous liquid comprises at least one enzyme, at least one organic compound, and optionally at least one solvent; wherein the at least one solvent is not water; wherein the at least one enzyme is immobilized on a solid support and positioned at a distance from the surface of the aqueous liquid in proximity to the plasma device where the distance ranges from 1 mm to 20 cm; and optionally extracting the aqueous liquid to isolate the oxidized or hydroxylated organic compound.

2. The method according to claim 1, wherein the plasma device is discontinuously run.

3. The method according to claim 1, wherein: the aqueous liquid is water or an aqueous buffer; or the aqueous liquid is an aqueous buffer having a pH value ranging from 4 to 8.

4. The method according to claim 1, wherein the H.sub.2O.sub.2 concentration of the obtained aqueous liquid ranges from 0.05 to 5 mM.

5. The method according to claim 1, wherein the aqueous liquid is treated with the plasma device for an amount of time ranging from 1 min to 24 hours.

6. The method according to claim 1, wherein the plasma device uses one or more of the following: a frequency ranging from 30 to 20,000 Hz; a voltage ranging from 0.2 to 25 kV peak-to-peak; a power ranging from 1 to 10,000 mW; or combinations thereof.

7. The method according to claim 1, wherein the organic compound is selected from unsubstituted or substituted alkanes, alkenes, alkines, cyclic or aromatic hydrocarbons, heterocyclic hydrocarbons, amino acids, proteins, alkaloids, steroids, and terpenes, or mixtures thereof.

8. The method according to claim 1, wherein the aqueous liquid and/or the obtained aqueous liquid further comprise at least one auxiliary substance selected from superoxide dismutase, mannitol, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid, ebselen, uric acid, molecular chaperones, peroxynitrite, HOC, nitric acid, and combinations thereof.

9. The method according to claim 8, wherein the at least one auxiliary substance is superoxide dismutase.

10. The method according to claim 1, wherein the at least one enzyme is selected from oxidases, monooxygenases, peroxidases, peroxygenases, or combinations thereof.

11. The method according to claim 1, wherein the plasma device is an atmospheric pressure plasma device.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The accompanying drawings serve to afford an understanding of various embodiments. The drawings illustrate embodiments and together with the description serve to elucidate same. Further embodiments and numerous advantages from among those intended are evident directly from the following detailed description. The elements and structures shown in the drawings are not necessarily illustrated in a manner true to scale with respect to one another.

(2) FIG. 1: General scheme of the reaction, which forms the basis of Example 1: The DBD treatment of an aqueous liquid produces H.sub.2O.sub.2 and superoxide, the latter of which can be converted into H.sub.2O.sub.2 by SOD. The peroxide is then used to fuel the UPO catalyzed hydroxylation of ethylbenzene to 1-(R)-phenylethanol.

(3) FIG. 2: Concentration of 1-phenylethanol after different cycles of DBD plasma treatment either with free or immobilized enzyme of UPO.

(4) FIG. 3: General scheme of the plasma-driven H.sub.2O.sub.2 generation and the subsequent biocatalysis, which forms the basis of Example 2. A dielectric barrier discharge (DBD) is generating non-thermal plasma, interacting with the liquid and thus forming reactive oxygen and nitrogen species, e.g. peroxynitrite (ONOO.sup.), superoxide (O.sup.2), or H.sub.2O.sub.2. Some of the species can recombine or dissociate to the other reactive particles. Some ROS or RNS can serve as reactants fueling ROS- or RNS-dependent biocatalyses.

(5) FIG. 4: H.sub.2O.sub.2 accumulation in DBD-treated water and KPi buffer (100 mM, pH 6).

(6) FIG. 5: Conversion of colorimetric HRP substrates by DBD plasma treatment in the presence and absence of HRP. Bars in the right panel show the absorption after 5 minutes of plasma treatment in the presence (black) and absence (grey) of HRP.

(7) FIG. 6: Plasma-driven biocatalysis using HRP as biocatalyst (1 U mL.sup.1) and guaiacol (10 mM) as substrate, which are directly exposed to plasma in the KPi buffer system (100 mM, pH 6). Reaction volumes of 200 L were treated with DBD plasma for the indicated times. Immediately after plasma treatment, the absorbance at 470 nm was measured.

(8) FIG. 7: Plasma treatment of unknown peroxidase (40 L of 1 mg/mL protein solution, 2 mm distance, 13.5 kV, 300 Hz, enzyme activity against treatment time [s]).

(9) FIG. 8: Plasma treatment of unspecific peroxygenase from Agrocybe aegerita (UPO) (40 L of 1 mg/mL protein solution, 2 mm distance, 13.5 kV, 300 Hz, relative activity against treatment time [s]).

(10) FIG. 9: Plasma treatment of vanadium chloroperoxidase from Curvularia inaequalis (40 L of 1 mg/mL protein solution, 2 mm distance, 13.5 kV, 300 Hz, (9A) enzyme activity against treatment time [s], (9B) concentration against treatment time [s], (9C) fluorescence against treatment time [s]).

(11) FIG. 10: Plasma treatment of ScDYP1 (40 L of 1 mg/mL protein solution, 2 mm distance, 13.5 kV, 300 Hz, (10A) enzyme activity against treatment time [s], (10B) concentration against treatment time [s]).

(12) FIG. 11: Plasma treatment of ScDYP2 (40 L of 1 mg/mL protein solution, 2 mm distance, 13.5 kV, 300 Hz, (11A) enzyme activity against treatment time [s], (11B) concentration against treatment time [s]).

(13) FIG. 12: Plasma tuning of horseradish peroxidase (40 L of 10 U/mL protein solution, 2 mm distance, (12A) relative activity of HRP after 1 min of treatment at different applied voltages (kV), (12B) relative activity of HRP after 1 min of treatment at different frequencies (Hz)).

(14) FIG. 13: Relative activity of horseradish peroxidase after treatment of free and immobilized HRP with 5 min treatment time (40 L of 10 U/mL protein solution, 2 mm distance, 13.5 kV, 300 Hz).

(15) FIG. 14: Relative activity of differently concentrated HRP against treatment time [s] (40 L of protein solution, 2 mm distance, 13.5 kV, 300 Hz).

(16) FIG. 15: Plasma-driven biocatalysis using UPO: (15A) Product analysis, (15B) Relative conversion with H.sub.2O.sub.2 or plasma-treated buffer, (15C) Reusability of the immobilized enzyme with H.sub.2O.sub.2 or plasma-treated buffer, (15D) product formation using direct plasma treatment or treatment with plasma-treated buffer (conditions: 150 nM UPO, 1 mM H.sub.2O.sub.2, 100 mM KPi, pH 7.5, 5 L ETBE (150 L final volume), reaction time 30 min, extraction with 150 L ethyl acetate containing 2 mM 1-octanol as internal standard, analysis with Hydrodex -6TBDM column).

(17) FIG. 16: Activity of immobilized UPO against plasma treatment time [min] (40 L of 1 M immobilized protein solution; 2 mm distance, 13.5 kV, 300 Hz).

(18) FIG. 17: Specific enzyme activity [A405/s*M UPO] at varying experimental conditions after 5 min treatment time (40 L of 1 M protein solution; 2 mm distance, 13.5 kV, 300 Hz).

DETAILED DESCRIPTION

Examples

Example 1

(19) Production of Unspecific Peroxygenase (UPO)

(20) UPO from the fungus Agrocybe aegerita was produced as reported in Molina-Espeja et al., Enzyme Microb. Technol., 2015, 73, 29-33.

(21) The purity of the protein was tested by SDS-PAGE. Two bands were found in the gel, which correspond to the two isoforms of the protein as described in Pecyna et al., Appl. Microbiol. Biotechnol., 2009, 84, 885-897.

(22) Plasma-Driven Reactions

(23) In general, a solution of potassium phosphate buffer (110 L, 250 mM, pH 7.0) was treated with the DBD (dielectric barrier discharge) device for different amounts of time (typically 5 minutes). The used DBD device has one driven, cylindrical copper electrode covered with aluminum oxide with a total diameter of 20 mm. Plasma was generated by applying voltage pulses with a maximum of 13.5 kV, a trigger frequency of 300 Hz and a surface power density of 130 mW/cm.sup.2.

(24) Then the sample was let to rest for five minutes at room temperature in order to eliminate the short-lived species produced by the plasma. The plasma-activated buffer obtained (110 L) was combined with ethylbenzene (5 L for a total concentration of 80 mM) and a solution of unspecific peroxygenase (final enzyme concentration 100 M) to a total volume of 150 L and incubated for 10 minutes at 30 C. and 600 rpm. Plasma-activated buffer was added two times (100 L) and the reaction was incubated for 10 minutes. The final reaction volume (355 L) was extracted with ethyl acetate containing 1-octanol (2 mM) as injection standard and analyzed by gas chromatography. The organic phase was dried with MgSO.sub.4 and measured with a Shimadzu 2010 GC system containing a Hydrodex--6TBDM column (Macherey-Nagel, Germany) with a column temperature of 120 C.

(25) Effect of the Plasma Treatment Time in the Production of 1-(R)-Phenylethanol

(26) TABLE-US-00001 TABLE 1 Effect of the plasma treatment time in the production of 1-(R)-phenylethanol using a solution of purified UPO (100 M) in different conditions. Plasma treatment performed at room temperature. Hydroxylation reaction performed at 30 C. and 600 rpm. Final 1- phenylethanol Treatment concentration Entry time (min) Buffer (mM) pH (mM) TTN 1 0 KPi 250 mM 7 0 0 2 5 KPi 250 mM 7 0.46 0.01 4576 3 10 KPi 250 mM 7 0.97 0.00 9729 4 15 KPi 250 mM 7 1.28 0.05 12868 8 5 KPi 1M 7 0.53 0.04 5336 9 5 KPi 250 mM 7 0.62 0.02 6245 10 5 KPi 50 mM 7 0.55 0.00 5475 11 5 TRIS 50 mM 7 0.69 0.01 6864 12 5 HEPES 50 mM 7 0.89 0.06 8913 13 5 MES 50 mM 7 0.64 0.01 6368

(27) The product formation correlates with the plasma treatment time, showing the accumulation of 1-(R)-phenylethanol (Table 1, Entry 1 to 4). In all cases the product is optically pure (ee>99%), which suggests that the hydroxylation reaction is based on an enzymatic reaction. Calculated total turnover numbers confirm that this is comparable to previously reported TTNs of UPO. Negative controls without enzyme, plasma treatment or ethylbenzene did not result in any detectable product formation.

(28) In the experiments, buffered solutions were used. Variations of the pH from pH 4 to 8 and the buffer salt concentration of the potassium phosphate buffer had an influence on product formation. Different buffer salts and concentrations were tested (potassium phosphate, TRIS, HEPES, MES, Table 1, Entries 8-11). While TRIS-buffer and MES-buffer show a small improvement over potassium phosphate buffer, the use of HEPES proved to be the best performing buffer for the reaction yielding 62% more product than using KPi.

(29) Peroxide-Formation with the Aid of Additional Enzymes

(30) Reactive oxygen species can interact directly with the peroxygenase resulting in amino acid modifications or complete inactivation of the enzyme. This drawback can be overcome by different approaches, like the addition of other detoxifying enzymes, shielding the peroxygenases against the short-living reactive species, or adjustments in the plasma generation. For example, the superoxide dismutase A (SOD) from Escherichia coli can further increase the peroxide production by conversion of plasma-generated superoxide to H.sub.2O.sub.2.

(31) The addition of different amounts of SOD during the plasma treatment resulted in a slight increase of the formation of 1-phenylethanol.

(32) TABLE-US-00002 TABLE 2 Effect of the addition of superoxide dismutase (SOD) in the production of 1-phenylethanol. SOD Formation of 2- concentration phenylethanol Entry (mg/mL) SOD addition (mM) 1 0 0.4 2 0.5 After treatment 0.4 3 0.5 During treatment 0.47 4 1 During treatment 0.5

(33) Comparison of External Addition of Plasma-Activated Buffer and In Situ H.sub.2O.sub.2 Generation

(34) Two different methods of H.sub.2O.sub.2 provision to the biocatalyst were performed in comparison: 1. The buffer was treated with plasma and then added to a vial containing the UPO and ethylbenzene. 2. A solution containing the enzyme and ethylbenzene was directly placed under the DBD and treated for an equivalent amount of time with plasma.

(35) With both methods, product was obtained. However, in the first method, 9 times more product was produced.

(36) Plasma-Driven Biocatalysis with Immobilized UPO

(37) To provide an improved product formation of method 2, immobilized UPO was used. The UPO immobilization was carried out in EC-HA Sepabeads generously provided by Viazym (Delft, Netherlands) using the protocol provided by the manufacturer. 10 mg of carrier were used to immobilize 0.35 mM of enzyme. This amount of carrier was then used in 500 L of reaction volume to a final enzyme concentration of 0.7 M. After the addition of 5 L of ethylbenzene (80 mM), 100 L of the reaction volume were extracted (without removing the immobilized protein) and treated for 5 minutes with the DBD. After treatment, the sample was placed back in the reaction vial and incubated for 10 minutes at 30 C. and 600 rpm. Then the treatment was repeated between 3 and 7 times. Due to the immobilization, this step could be performed while keeping the enzyme in the reaction vial. As positive control, the reaction was carried out using free enzyme in the same concentrations and with 3 cycles of treatment as described previously.

(38) This strategy obtained the same amount of product than with external addition of plasma-activated buffer without diluting the reaction solution (method 1), which is a decisive advantage for any synthetic application. The product obtained in both cases presented an ee of >99%, indicating that the plasma treatment does not interact or racemize the 1-(R)-phenylethanol. Up to seven treatment cycles were tested, producing an accumulation of 1-phenylethanol up to a concentration of 0.88 mM (FIG. 2).

Example 2

(39) Horseradish peroxidase extracted from horseradish roots was obtained from Sigma-Aldrich (P8375) as lyophilized powder.

(40) Dielectric Barrier Discharge (DBD)

(41) The used DBD device has one driven, cylindrical copper electrode covered with aluminum oxide with a total diameter of 20 mm. Plasma was generated by applying voltage pulses with a maximum of 13.5 kV, a trigger frequency of 300 Hz and a surface power density of 130 mW/cm.sup.2.

(42) H.sub.2O.sub.2-Accumulation in DBD-Treated Water and KPi Buffer

(43) H.sub.2O.sub.2-accumulation was determined in DBD-treated water and KPi buffer (100 mM, pH 6). Samples of 200 L were placed onto a glass slide and treated with DBD plasma for the indicated amount of time (FIG. 4). Immediately after treatment, the samples were analyzed with a commercially available hydrogen peroxide test kit, following the manufacturer's instructions (Merck Spectroquant Hydrogen Peroxide). The H.sub.2O.sub.2-concentration was determined using a calibration curve.

(44) The overall production rate was about 0.1 mM min.sup.1 for both deionized water and KPi buffer. After 5 minutes, more H.sub.2O.sub.2 was accumulated in KPi buffer (FIG. 4). This result is explainable by the pH stabilizing effect of KPi buffer in contrast to deionized water.

(45) Conversion of Colorimetric HRP Substrates by DBD Plasma Treatment

(46) In the experiment, colorimetric HRP substrates were converted by DBD plasma treatment in the presence and absence of horseradish peroxidase (HRP). Pyrogallol (PG) and guaiacol (GC) were prepared in KPi buffer at 100 mM and 10 mM, respectively. For each substrate 100 L were treated with the DBD device for the indicated amounts of time (FIG. 5). Directly after treatment, the samples were analyzed photometrically using the respective absorption maxima (.sub.Pyrogallol=420 nm, .sub.Guaiacol=470 nm). The enzymatic conversion was determined by adding 50 L of substrate (20 mM and 200 mM for GC and PG, respectively) into a microtiter plate well, mixing with 50 L of 2 mM H.sub.2O.sub.2, subsequent addition of 1 L of 10 U mL.sup.1HRP solution and immediate determination of the absorption.

(47) Pyrogallol (PG) was found to be converted to the violet product purpurogallin to a significant extend by plasma treatment alone, even without HRP being present. After 5 minutes of plasma treatment of PG, the absorption was higher than for the enzymatic conversion with HRP and liquid H.sub.2O.sub.2. Guaiacol (GC) proved to be sufficiently plasma-stable. GC was transformed by HRP plasma-dependently (FIG. 5).

(48) HRP Activity Under Direct Plasma Treatment

(49) In this experiment, HRP and the substrate guaiacol were directly exposed to plasma. HRP was diluted to 1 U mL.sup.1 in KPi buffer (100 mM, pH 6) and GC was added to a final concentration of 10 mM. Reaction volumes of 100 L were treated with DBD plasma for the indicated times (FIG. 6). Immediately after plasma treatment, the absorbance at 470 nm was measured.

(50) With increased plasma treatment time, absorption at 470 nm increases, indicating conversion of GC to tetraguaiacol (tGC). HRP thus is able to utilize plasma-generated H.sub.2O.sub.2 in the reaction. Additionally, the enzyme was not inactivated within 5 min of DBD plasma exposure (FIG. 6).

Example 3

(51) In the following, enzymes were characterized in more detail with respect to direct plasma treatment time [s]:

(52) Unknown peroxidase (FIG. 7), unspecific peroxygenase from Agrocybe aegerita (UPO) (FIG. 8), vanadium chloroperoxidase from Curvularia inaequalis (FIG. 9A, 9B, 9C), DyP-type peroxidase from Streptomyces chartreusis, variant 1 (ScDYP1) (FIGS. 10A and 10B) and DyP-type peroxidase from Streptomyces chartreusis, variant 2 (ScDYP2) (FIGS. 11A and 11B) were treated with plasma with a distance between the surface of the aqueous liquid and the electrode of the plasma device of 2 mm with a voltage of 13.5 kV and a frequency of 300 Hz over 600 seconds. A total concentration of 40 L of protein solution (1 mg/mL) was used in each case. 100% is based on the untreated enzyme.

(53) Activity Assay Parameters:

(54) Unknown peroxidase: 5 L of treated sample in 2.5 mM ABTS in 50 mM sodium acetate buffer, pH 5.5; 1 mM H.sub.2O.sub.2; absorption measurement at 405 nm. Activity was calculated from linear slope in the absorption measurement.

(55) UPO: 5 nM UPO; 2.5 mM ABTS in 50 mM sodium acetate buffer, pH 5.5; 1 mM H.sub.2O.sub.2. Activity was calculated from linear slope in the absorption measurement.

(56) Vanadium chloroperoxidase: 10 L of treated sample in 200 M phenol red in 50 mM Tris-SO.sub.4; 5 mM KBr and 5 mM H.sub.2O.sub.2. Absorption measurement at 582 nm. Activity was calculated from linear slope in the absorption measurement.

(57) ScDYP1: 10 L of a 1:20 dilution of the treated sample was used for activity measurements. Final concentrations: 2.5 mM ABTS in 50 mM Britton-Robinson buffer, pH 4; 200 M H.sub.2O.sub.2.

(58) ScDYP2: 10 L of treated sample; 2.5 mM ABTS in 50 mM Britton-Robinson buffer, pH 8; 25 mM H.sub.2O.sub.2.

(59) Protein concentration was determined using the Bradford method with a commercially available test kit (Roti NanoQuant), following the manufacturer's instructions.

(60) Fluorescence was measured with a 1:5 dilution of the treated protein, with excitation wavelength 280 nm and emission wavelength 350 nm.

(61) In samples which were directly plasma-treated, the substrate conversion was determined by adding commercially available H.sub.2O.sub.2 to the sample after direct plasma treatment. The substrate used for the respective assay was added after direct plasma treatment.

Example 4

(62) Relative activity of horseradish peroxidase (HRP) was determined after 1 min of direct plasma treatment at different applied voltages and trigger frequencies. The plasma treatment was carried out with a distance of 2 mm with 40 L of a protein solution (10 U/mL) (FIGS. 12A and 12B).

(63) Activity assay parameters: 2 L of treated sample; 5 mM guaiacol (GC) in 100 mM potassium phosphate (KPi) buffer, pH 7; 0.5 mM H.sub.2O.sub.2.

(64) Additionally, the relative activity of free and immobilized HRP after direct plasma treatment with a treatment time of 5 min (2 mm distance, 13.5 kV, 300 Hz) was determined in comparison to untreated HRP using the activity assay parameters as described above (FIG. 13).

(65) HRP was immobilized with Relizyme HA403 M beads (Resindion, Binasco, Italy). 10 mg of beads were activated by incubating in 100 mM KPi buffer (pH 7) with 0.4% glutaraldehyde for 1 h. After washing twice with deionized water, up to 5 mg of enzyme were added in 1 mL buffer. Immobilization was carried out over night at room temperature with constant shaking. Binding efficiency was checked by measuring the protein concentration of the supernatant after incubation and was found to be >80% in all cases.

(66) In addition, the effect of the protein concentration (1 kU/mL, 100 U/mL, 10 U/mL) on the relative activity of HRP over the treatment time [s] was measured (40 L of protein solution, 2 mm distance, 13.5 kV and 300 Hz using the activity assay parameters as described above (FIG. 14)).

(67) In samples which were directly plasma-treated, the substrate conversion was determined by adding commercially available H.sub.2O.sub.2 to the sample after direct plasma treatment. The substrate used for the respective assay was added after direct plasma treatment.

Example 5

(68) In the following, the products formed by plasma-driven biocatalysis using UPO were analysed (assay conditions: 150 nM UPO, 1 mM H.sub.2O.sub.2, 100 mM KPi, pH 7.5, 5 L ETBE (150 L final volume), reaction time 30 min, extraction with 150 L ethyl acetate containing 2 mM 1-octanol as internal standard, analysis with Hydrodex -6TBDM column).

(69) A chromatogram illustrating the racemic product reference (1-phenylethanol), the product formation of (R)-1-phenylethanol using plasma-treated buffer, and samples without plasma treatment, without substrate and without enzyme measured by gas chromatography is shown in FIG. 15A.

(70) A comparison of relative conversion of UPO when using commercially available H.sub.2O.sub.2 or plasma-treated buffer is shown in FIG. 15B.

(71) Additionally, it was determined how many cycles can be performed with the same enzyme, wherein one sample comprised plasma-treated buffer and immobilized UPO and the second sample comprised commercially available H.sub.2O.sub.2 and immobilized UPO. The data are illustrated in FIG. 15C.

(72) Then it was determined, whether plasma treatment can be coupled with running the reaction. For this, the 1-phenylethanol concentration formed based on direct plasma treatment was compared to the 1-PhOl concentration formed based on plasma-treated buffer (FIG. 15D). Assay conditions (15A-15D): 150 nM UPO, 1 mM H.sub.2O.sub.2, 100 mM KPi, pH 7.5, 5 L ETBE (150 L final volume), reaction time 30 min, extraction with 150 L ethyl acetate containing 2 mM 1-octanol as internal standard, analysis with Hydrodex -6TBDM column (GC).

(73) In samples which were directly plasma-treated, the substrate conversion was determined by adding commercially available H.sub.2O.sub.2 to the sample after direct plasma treatment. The substrate used for the respective assay was added after direct plasma treatment.

Example 6

(74) The effect of direct plasma treatment time [min] with a distance of 2 mm on the activity of immobilized UPO (40 L of 1 M immobilized protein solution, over 60 min) is demonstrated in FIG. 16. Activity assay parameters: 10 nM UPO; 2.5 mM ABTS in 50 mM sodium acetate buffer, pH 5.5; 1 mM H.sub.2O.sub.2 while constant shaking at 1400 rpm.

(75) The effect of enzyme immobilization and/or direct plasma treatment in comparison to free enzyme and/or samples without direct plasma treatment on the specific enzyme activity of UPO is illustrated in FIG. 17. (40 L of 1 M protein solution; 2 mm distance, 5 min treatment time).

(76) Activity assay parameters: 5 nM UPO; 2.5 mM ABTS in 50 mM sodium acetate buffer, pH 5.5; 1 mM H.sub.2O.sub.2. Activity was calculated from linear slope in the absorption measurement.

(77) In samples which were directly plasma-treated, the substrate conversion was determined by adding commercially available H.sub.2O.sub.2 to the sample after direct plasma treatment. The substrate used for the respective assay was added after direct plasma treatment.