Eur. Phys. J. Appl. Phys. (2013) 61: 24309DOI: 10.1051/epjap/2012120405

THE EUROPEANPHYSICAL JOURNAL

APPLIED PHYSICS

Regular Article

Atmospheric pressure low-power microwave microplasma sourcefor deactivation of microorganisms �

Jerzy Mizeraczyk1,2, Miros�law Dors1, Mariusz Jasinski1, Bartosz Hrycak1, and Dariusz Czylkowski1,a

1 Centre for Plasma and Laser Engineering, The Szewalski Institute of Fluid-Flow Machinery,Polish Academy of Sciences, Fiszera 14, 80-952 Gdansk, Poland

2 Department of Marine Electronics, Gdynia Maritime University, Morska 81-87, 81-225 Gdynia, Poland

Received: 28 September 2012 / Received in final form: 31 October 2012 / Accepted: 5 December 2012Published online: 18 February 2013 – c© EDP Sciences 2013

Abstract. This work was aimed at experimental investigations of deactivation of different types of mi-croorganisms by using atmospheric pressure low-temperature microwave microplasma source (MmPS).The MmPS was operated at standard microwave frequency of 2.45 GHz. Its main advantages are simpleand cheap construction, portability and possibility of penetrating into small cavities. The microplasma de-activation concerned two types of bacteria (Escherichia coli, Bacillus subtilis) and one fungus (Aspergillusniger). The quality as well as quantity tests were performed. The influence of the microorganism type,oxygen concentration, absorbed microwave power, microplasma treatment time and MmPS distance fromthe treated sample on the microorganism deactivation efficiency was investigated. All experiments wereperformed for Ar microplasma and Ar/O2 microplasma with up to 3% of O2. Absorbed microwave powerwas up to 50 W. The Ar flow rate was up to 10 L/min. The sample treatment time was up to 10 s.

1 Introduction

Recently, there has been a growing interest in thelow-temperature microplasmas used in the biomedical ap-plications such as sterilization of medical instruments,high-precision surgery, cells treatment and deactivation ofbacteria and viruses [1–4]. Sterilization is a physical orchemical process that impairs or eliminates, especiallymicroorganisms like bacteria. In a plasma the steriliza-tion is achieved due to the reactive species in the plasma(electrons, ions, radicals, reactive molecules), UV light,etc. [5,6].

Due to many plasma sterilization systems like low pres-sure [7] or atmospheric pressure [8], DBD [9], RF [10], wedesigned, built and tested experimentally a smallatmospheric pressure low-temperature microwave micro-plasma source (MmPS). It has the structure of a coaxialline, formed by an inner conductor, made of a brass rodwith a tungsten rod top, and outer conductor in the formof a brass cylinder. The MmPS is operated at standard mi-crowave frequency of 2.45 GHz. Microplasma generatedby the MmPS has the form of a microflame or micro-jet above the inner conductor top. The main advantages

� Contribution to the Topical Issue “13th International Sym-posium on High Pressure Low Temperature Plasma Chemistry(Hakone XIII)”, Edited by Nicolas Gherardi, Henryca DanutaStryczewska and Yvan Segui.

a e-mail: dczylkowski@imp.gda.pl

of our MmPS are atmospheric pressure operation, lowmicroplasma temperature, simplicity and low cost. Theexperimental investigations concerning microorganismsdeactivation by atmospheric pressure low-temperaturemicroplasma were performed using this MmPS. The inves-tigations involved Ar and Ar/O2 microwave microplasmatreatment of two types of bacteria (Escherichia coli,Bacillus subtilis) and one fungus (Aspergillus niger) sam-ples. The Escherichia coli is gram-negative bacteria whichis a standard indicator of fecal contamination of drinkingwater. The gram-positive bacteria of Bacillus subtilis aredistributed in soil. It has the ability to form spores resis-tive to extreme environmental conditions. The fungus ofAspergillus niger is a common contaminant of food causinga disease called black mold on certain fruits and vegetables(grapes, onions and peanuts).

2 Microwave microplasma source (MmPS)

A number of various designs of the microplasmasources have been developed. Sources supplied througha waveguide [11,12], consisting of coaxial line [13,14] orstripline structures [15,16], are known. Waveguide-basedstructures, in contrast to coaxial line and stripline struc-tures, have much bigger size and allow to operate withhigher microwave powers. The sketch and photograph ofour MmPS are presented in Figure 1. It is based, as itwas previously described in [17–20], on a coaxial line,

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Fig. 1. Sketch and photograph of MmPS.

Fig. 2. Photographs of the microwave microplasma for various operating conditions.

formed by the inner conductor, made of a brass rod witha tungsten rod top, and outer conductor in the form ofa brass cylinder. The inner conductor is fixed inside theouter conductor tightly with a PTFE centering disk.The MmPS is supplied from the microwave generator atstandard frequency of 2.45 GHz through a 50 Ω coaxialline using an N-type connector. The operating gas is in-troduced through a duct between the inner and outer con-ductors. The microplasma was generated by the MmPS inthe form of a tiny candle-like flame (in Ar, Ar/O2 at lowabsorbed powers) or a plasma jet (in Ar/O2 at high ab-sorbed powers) above the inner conductor top. The lengthand diameter ranged from 5 to 25 mm and 0.5 to 2 mm,respectively, for Ar microplasma and from 2 to 30 mmand 2 to 16 mm, respectively, for Ar/O2, depending onthe operating parameters. The MmPS was operated withceramic tip made of MACOR. The tip formed a kind ofnozzle that increased the velocity of argon in plasma form-ing zone. It prevented breakdowns between the inner andouter conductors. The tip covered the hotter part of themicroplasma column, thus exposing only the lowest tem-perature microplasma (i.e., its tip). Figure 2 shows pho-tographs of the Ar and Ar/O2 microwave microplasma forvarious operating conditions. The operation of MmPS isdescribed in detail in [20].

In Figure 3 the measured emission spectra of the at-mospheric pressure Ar microplasma in the range of

Fig. 3. Measured emission spectrum of Ar microplasma. Arflow rate 2 L/min, absorbed microwave power 15 W. The ob-servation area within visible part of Ar microplasma column:base of the visible part of column. Lines identified with NISTatomic spectra database [21].

300–600 nm can be seen. The spectra were measured atAr flow rate of 2 L/min and absorbed microwave power of15 W. The lines identification was performed with NISTatomic spectra database [21]. The Hβ , OH, N2 and NHspectral lines were observed in the emission spectrum.

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Fig. 4. Comparison of the measured and simulated emissionspectra of OH (A-X) band. Ar flow rate of 10 L/min, absorbedmicrowave power of 10 W.

This is due to generation of the Ar microplasma in anambient atmosphere.

The comparison of the measured and simulated emis-sion spectra of OH (A-X) band in Ar plasma (for Arflow rate of and absorbed microwave power of 10 W) is

shown in Figure 4. For simulation of the optical spec-tra the Specair [22] program was used. The code of thisprogram allows to calculate the spectral radiation emittedby gases and plasmas of various compositions at given in-dividually vibrational and rotational temperatures. Basedon the above-described methods the rotational tempera-ture was determined to be about 500 K for OH radicals.The OH rotational temperature increases with increasingabsorbed microwave power and decreases with increasingargon flow rate. For absorbed microwave power and ar-gon flow rate range applied during the microplasma mi-croorganisms sample treatment, small changes of the OHrotational temperature could be observed.

The microplasma core and tip temperature for differ-ent working conditions measured using a thermocoupleare presented in Figures 5 and 6. Figure 5 shows the tem-perature of the core and tip of Ar (Fig. 5a) and Ar/O2

(Fig. 5b) microwave microplasma as a function of Ar flowrate in the range of 2–10 L/min. The absorbed microwavepower was 35 W.

From the presented results one can see that, for both,the Ar and Ar/O2 microplasmas the temperaturedecreases with increasing the gas flow rate. For given

Fig. 5. Temperature of the core and the tip of Ar microwave microplasma (a) and Ar/O2 microwave microplasma (b) as afunction of Ar flow rate.

Fig. 6. Temperature of the core and the tip of Ar/O2 microwave microplasma: (a) as a function of absorbed microwave power,(b) as a function of O2 concentration.

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Fig. 7. The diagram of the experimental setup for deactivationof microorganisms.

experimental conditions the microplasma temperaturedoes not exceed 300 ◦C.

Figure 6a presents the influence of absorbed microwavepower on microplasma core and tip temperature. The ab-sorbed microwave power was up to 80 W. The increaseof the microplasma core and tip length with increasingthe absorbed microwave power can be observed. For ab-sorbed microwave power of 80 W the microplasma coretemperature reaches 300 ◦C. The influence of O2 additivein the range of 0–3% on microplasma core and tip tem-perature is shown in Figure 6b. As seen in figure the mi-croplasma temperature increased with increasing O2 con-centration although the temperature of the core lookedlike saturating at 2–3% of O2 addition.

3 Experimental procedure

The experimental investigations concerning microorgan-isms deactivation by our MmPS were carried out in the mi-crobiological laboratory of Innovation Enterprise“IMPULS”, Pruszcz Gdanski, Poland. We performedquality as well as quantity tests. The influence of the

microorganism type, O2 concentration, microwave powerlevel, treatment time and microplasma distance from thetreated sample on the microorganisms deactivation effi-ciency was investigated. The microorganisms treated bythe microplasma were bacteria (Escherichia coli, Bacillussubtilis) and fungus (Aspergillus niger). The test was per-formed for Ar microplasma and Ar/O2 microplasma withup to 3% of O2. Absorbed microwave power was up to50 W. The Ar flow rate ranged from 2 to 10 L/min.The sample treatment time was up to 10 s.

The experimental setup for deactivation of microor-ganisms consisted of a 2.45 GHz magnetron generator,microwave power supplying and measuring system (bi-directional coupler, dual-channel power meter), microwavemicroplasma source (MmPS), gas supply and flow controlsystem. The diagram of this setup is shown in Figure 7.The bi-directional coupler was used to sample the incidentPI and reflected PR microwave power at the plasma gener-ator input. Thus, the system enables direct measurementsof an incident PI and reflected PR microwave power. Anabsorbed power PA was obtained from the subtraction ofPI − PR.

Figure 8a illustrates the microorganism sample scan-ning procedure. Note that, because of small dimensionsof Ar/O2 plasma flame, the flame had to scan the sam-ple fourfold to cover its surface (10 × 10 mm). Single mi-croplasma exposure time was about 0.25 s over an area of2.5 mm × 2.5 mm. The arrows in figure show the treat-ment path. In Figure 8b, a photograph of the microwavemicroplasma processing of the microorganism sample isshown.

The MmPS placed perpendicularly to the treated sam-ple can be seen. The microorganism sample in the form ofdroplet was placed on the rectangular sterile laboratoryslides. The sample droplet volume and the initial samplenumber of colony-forming units were chosen individuallyfor each kind of test. The distance between the MmPS andthe sample during the treatment microplasma was fixed.The slides were manually moved below the microplasmato scan the sample full area. Depending on the treatmenttime the different number of scans with constant speedwas performed. Concerning microorganisms deactivation

Fig. 8. (a) Scanning procedure of the microorganism sample by microwave microplasma. Usually several scans in one treatment.Single microplasma exposure time ∼0.25 s over an area of 2.5 mm × 2.5 mm. (b) Microorganism treatment by microwavemicroplasma.

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(a) (b) (c)Fig. 9. The photographs of the Escherichia coli reflecting the influence of the treatment time on the deactivation effi-ciency: (a) sample not treated by microplasma, (b) sample treated time 2.4 s (12 scans), (c) sample treated time 3.6 s(18 scans). Ar flow rate – 10 L/min, the admixture of O2 – 0.5%, the absorbed microwave power – 25 W.

by our MmPS we performed quality as well as quantitytests.

4 Results

The visual effect of the influence of the microwave mi-croplasma treatment time on the Escherichia coli deac-tivation efficiency is showed in photographs of Figure 9.Figure 9a shows Escherichia coli sample not treated bymicroplasma. Figures 9b and 9c show samples treatedfor 2.4 s and 3.6 s, respectively. The time of 2.4 s cor-responds to 12 scans and time of 3.6 s to 18 scans. The Arflow rate was set at 10 L/min, the admixture of O2 was0.5%. The absorbed microwave power was 25 W. From the

Fig. 10. The influence of the treatment time on theEscherichia coli and Bacillus subtilis deactivation. Ar flow rateof 10 L/min (in the case of Escherichia coli treatment), and4 L/min (in the case of Bacillus subtilis treatment). Admix-ture of O2 0.5%. The absorbed microwave power 25 W. Thenumbers in brackets signify the number of scans.

photographs one can see the lower number of Escherichiacoli colony-forming units in the case of microplasma-treated samples (Figs. 9b and 9c) comparing to not treatedsample shown in Figure 9a. The photographs qualitativelyprove the increase of the Escherichia coli deactivation withincreasing the sample treatment time.

The quantity results of the influence of the treatmenttime on the Escherichia coli and Bacillus subtilis deactiva-tion are presented in Figure 10. The presented results cor-respond to Ar flow rate of 10 L/min (for Escherichia colitreatment), and 4 L/min (for Bacillus subtilis treatment)with admixture of 0.5% of O2. The absorbed microwavepower was 25 W. Numbers in brackets shown in the figuresignify the number of scans. The results show that the in-creasing of the microplasma treatment time resulted in the

Fig. 11. The influence of the O2 admixture on the Escherichiacoli deactivation. Ar flow rate of 10 L/min. The initial numberof colony-forming units 4 × 107. Treatment time 1.8 s (ninescans). The absorbed microwave power 25 W.

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Table 1. The influence of the microwave power level and thedistance of the microplasma source from the treated sampleon the Escherichia coli (the initial sample number of colony-forming units was 2.8 × 107 per sample) inactivation. Ar flowrate of 4 L/min, the admixture of O2 0.5%. Treatment time1.4 s (7 scans).

Microorganism Absorbed Distance Number oftype power (W) (mm) survived CFU

Escherichia coli 22 8 4.5 × 103

17 8 5.4 × 103

18 4 3.7 × 103

22 4 7.3 × 102

higher deactivation efficiency. For microplasma treatmenttime higher than 10 s the number of both Escherichia coliand Bacillus subtilis was reduced by more than three or-ders of magnitude.

The tests of the influence of the O2 additive on the bio-cidal effect of microplasma were performed withEscherichia coli for Ar, and Ar/O2 microplasma. The

sample treatment time was 1.8 s and the absorbed mi-crowave power was 25 W. The Ar flow rate was 10 L/min.The initial number of colony-forming units was 4 × 107.Figure 11 presents results of tests under above-describedconditions. The microplasma temperatures during thesamples treatment are indicated in the figure. As it canbe seen, in any case, even in the case or Ar plasma with-out additive O2, the result indicated reduction of bacteriapopulation. No effect of O2 admixture was observed, whichcan be explained by much higher concentration of O2 inthe ambient air.

Table 1 presents results of experiments on the influenceof the microwave power level and distance of the MmPsfrom the treated sample on the Escherichia coli bacte-ria deactivation. The Ar flow rate was 4 L/min and theadmixture of oxygen was 0.5%. The treatment time wasset to 1.4 s. The initial sample number of colony-formingunits was 3 × 107 per sample. The presented results provethe dependence of the bacteria deactivation efficiency onboth of above-mentioned parameters. In particular, theinfluence of microwave power can be clearly seen.

(a) (b) (c) (d)

Fig. 12. The photographs of the fungal showing the influence of the treatment time on the Aspergillus niger deactivation:(a) sample not treated by microplasma (number of CFU > 150), (b) sample treated time 0.6 s (3 scans), (c) sample treated time1.8 s (9 scans) and (d) sample treated time 2.4 s (12 scans). Ar flow rate – 10 L/min, the admixture of O2 – 0.5%, absorbedmicrowave power – 25 W.

Fig. 13. The photographs of untreated (a and c) and microwave microplasma treated (b and d) PTFE sample.

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The visualization of the influence of the microwave mi-croplasma treatment time on the Aspergillus niger fungaldeactivation can be seen in Figure 12. The photographswere taken 7 days after microplasma sample treatment.The microplasma operating conditions were as follows:Ar flow rate of 10 L/min, O2 concentration 0.5% andabsorbed microwave power 25 W. Figure 12a shows theAspergillus niger fungal on the sample not treated bymicroplasma. In that case almost 150 fungal centers canbe seen. The fungus on the sample treated by microplasmafor 0.6 s, 1.8 s and 2.4 s is shown in Figures 12b–12d, re-spectively. In the case of Figure 12b there was five fungalcenters while in the case of Figures 12c and 12d, four fun-gal centers were observed.

Thus, the presented photographs incline the influenceof the microplasma treatment time on Aspergillus nigerdeactivation.

To fulfill requirements for bio-sterilization, tests on theheat effect of microwave microplasma on polytetrafluo-roethylene (PTFE) surface were done. The 1 mm-thickPTFE sample was treated by microplasma of parameterstypical for our investigations: Ar flow rate of 10 L/min, theO2 admixture of 0.5%, absorbed microwave power 25 W.The sample treatment time was 180 s. Figure 13 showsphotographs of untreated (Figs. 13a and 13c) and mi-crowave microplasma-treated (Figs. 13b and 13d) PTFEsamples. As it can be seen, owing to a relatively low mi-croplasma temperature (90 ◦C) no heat effect on heat-sensitive medical material (PTFE) was observed.

5 Conclusions

Using the low-temperature Ar and Ar/O2 microplasmagenerated by coaxial microwave microplasma source(MmPS) the deactivation of the microorganisms(Escherichia coli, Bacillus subtilis, Aspergillus niger) wasstudied experimentally. The unquestioned merits of such amicroplasma are small size (from µm to several mm) andportability of the source which allows to penetrate intosmall cavities. Other benefits are connected with easy-to-use, low investment and operation costs.

The influence of the O2 concentration, absorbed mi-crowave power, microplasma treatment time and micro-plasma distance from the treated microorganism sampleon the deactivation efficiency was investigated. Our exper-iments showed the possibility of microorganism colony re-duction by a few orders of magnitude of the initial numberof colony-forming units in several seconds. The increasingof the treatment time resulted in the higher deactivationefficiency but no effect of O2 admixture on the deactiva-tion efficiency was observed. Our investigations show noheat effect on heat-sensitive medical material (PTFE) ow-ing to a relatively low microplasma temperature.

The presented in this paper experimental results al-low us to conclude that our MmPS is a promising devicefor the biomedical applications. Further tests are underpreparation.

This research was supported by The Szewalski Institute ofFluid-Flow Machinery, Polish Academy of Sciences under theprogram IMP PAN O3Z1T1 and by Innovation Enterprise“IMPULS”, ul. Jelenia 2, 80-336 Gdansk.

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