Influence of relative gas humidity on the inactivation efficiency of a low temperature gas plasma

ORIGINAL ARTICLE

Influence of relative gas humidity on the inactivationefficiency of a low temperature gas plasmaP. Muranyi1, J. Wunderlich1 and M. Heise2

1 Department of Food Technology, Fraunhofer Institute for Process Engineering and Packaging IVV, Freising, Germany

2 Department of Plasma Technology, Fraunhofer Institute for Laser Technology ILT, Aachen, Germany

Introduction

Plasma technology is an innovative and versatile method

for the treatment and modification of surfaces and is suit-

able for a variety of industrial applications, for example

etching, coating, cleaning and sterilization. The latter is

currently an active field of research because gas plasma

allows fast and safe sterilization of packaging materials

such as bottles, lids and films – without adversely affect-

ing the main properties of the packaging materials. This

approach is very interesting for heat-sensitive materials

such as plastics (e.g. polyethylene terephthalate and poly-

ethylene) which are becoming increasingly important in

the food, pharmaceutical and medical industries. Cur-

rently, ionizing radiation, ethylene oxide (EtO) and

hydrogen peroxide are widely used in medical fields for

the nonthermal sterilization of heat-sensitive items. The

disadvantages of high toxic EtO are residues absorbed on

the plastics and long storage times for venting. Energetic

radiation (e.g. gamma radiation) is costly and can modify

materials such as polypropylene (Moreira et al. 2004). In

the area of food packaging, hydrogen peroxide and per-

acetic acid in combination with moderate heat are the

main established methods for the sterilization of heat-sen-

sitive materials (Ansari and Datta 2003). Both agents are

harmful chemicals and it is therefore necessary to comply

with threshold limit values or maximum allowable con-

centrations making their use technologically elaborate.

Furthermore, it is challenging to remove residual chemi-

cals from the packaging, handling is difficult and from a

technical point of view it is very tricky to apply the cor-

rect amount of chemical in a homogeneous pattern.

These are some of the reasons why both hydrogen perox-

ide and peracetic acid technologies are inconvenient and

why new sterilization methods are required. Low temper-

ature plasma has the potential to replace or complement

current decontamination or sterilization technologies in

the medical and food industries. In previous studies we

have shown that a cascaded dielectric barrier discharge

(CDBD) inactivates a variety of micro-organisms within

Keywords

Aspergillus niger, Bacillus subtilis, CDBD, gas

humidity, low temperature plasma, spores.

Correspondence

Peter Muranyi, Department of Food

Technology, Fraunhofer Institute for Process

Engineering and Packaging (IVV),

Giggenhauserstr. 35, D-85354 Freising,

Germany. E-mail: [email protected]

2007 ⁄ 1015: received 29 June 2007, revised 7

November 2007 and accepted 10 November

2007

doi:10.1111/j.1365-2672.2007.03691.x

Abstract

Aims: To investigate the effect of relative gas humidity on the inactivation effi-

ciency of a cascaded dielectric barrier discharge (CDBD) in air against Aspergil-

lus niger and Bacillus subtilis spores on PET foils.

Methods and Results: The inactivation kinetics as a function of treatment time

were determined using synthetic air with different relative humidity as the pro-

cess gas. Spores of A. niger and B. subtilis respectively were evenly sprayed on

PET foils for use as bioindicators. In the case of A. niger, increased spore mor-

tality was found at a high relative gas humidity of 70% (approx. 2 log10). This

effect was more evident at prolonged treatment times. In contrast, B. subtilis

showed slightly poorer inactivation at high gas humidity.

Conclusions: Water molecules in the process gas significantly affect the inacti-

vation efficiency of CDBD in air.

Significance and Impact of the Study: Modifying simple process parameters

such as the relative gas humidity can be used to optimize plasma treatment to

improve inactivation of resistant micro-organisms such as conidiospores of

A. niger.

Journal of Applied Microbiology ISSN 1364-5072

ª 2008 The Authors

Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 1659–1666 1659

short-treatment times (Muranyi et al. 2007). Even bacte-

rial spores such as endospores of Bacillus atrophaeus can

be reduced by about five orders of magnitude in just a

few seconds.

To evaluate and validate the efficiency of sterilization

methods, selected test strains are used as biological indi-

cators. Characteristics of these micro-organisms are their

high resistance to the sterilizing agent and nonpathoge-

nicity. Examples of such test strains are Bacillus subtilis

for hydrogen peroxide, Geobacillus stearothermophilus for

superheated steam and Bacillus pumilus for gamma radia-

tion (Bernard et al. 1990). To find an appropriate test

germ for a newly developed sterilization method, it is

necessary to investigate a wide range of micro-organisms.

We have found that conidiospores of Aspergillus niger

have high resistance to CDBD plasma treatment in air.

Therefore, A. niger conidiospores could be used for vali-

dation and optimization of the efficiency of plasma

decontamination in common. Generally, the aim of the

optimization process is the improvement of A. niger inac-

tivation. In the case of plasma there is the possibility of

varying parameters such as the process gas, pressure or

applied power density. Previous studies with various pro-

cess gases have shown that air seems to be very efficient

for destroying A. niger conidiospores (Muranyi et al.

2007) and for this reason we have tried to improve this

microbicidal effect by changing the gas humidity.

In this study, we investigated the mortality of two dif-

ferent micro-organisms, spores of B. subtilis DSM 4181

and A. niger DSM 1957, as a result of plasma treatment

with the CDBD set-up and as a function of the humidity

of the synthetic air process gas.

Materials and methods

Experimental set-up

The set-up for decontamination experiments has been

previously described in detail (Muranyi et al. 2007). The

respective lab-scale device is equipped with a so-called

CDBD which is a special kind of atmospheric pressure

plasma. It is suitable for the decontamination of flat

materials because of its geometric constraints (mm to

cm). A drawback regarding decontamination might be

that UV emitting plasmas are often not very chemically

reactive and vice-versa. By spatially combining two differ-

ent plasmas in one single set-up this problem is overcome

in the CDBD. In the first discharge gap (a closed, flat

quartz vessel) UV emitting excimer gases are utilized. In

our case a gas mixture of Xe and Br was used, which

emits monochromatic light of 282 nm. In the second gap

air or other oxidizing gases are applied directly to the

sample. As the first gap is only separated from the second

gap by a UV-transparent dielectric, both kinds of plasma

act on the sample at the same time. As Falkenstein

(1997a, 1997b) has shown, it is possible to increase the

total amount of hydroxyl radicals generated in a standard

barrier discharge in humid air or argon mixtures by illu-

mination with UV radiation at 254 nm. Moreover, the

discharge, consisting of many single micro-discharges,

more uniformly covers the treated surface area and this

was also confirmed for CDBD (Heise et al. 2004). Com-

pared with standard barrier discharges, we therefore

expect the CDBD to produce higher radical concentra-

tions (OH, O) in dry or humid air and this could

improve the microbicidal effect.

Conditioning of gas humidity

The water content of synthetic air was adjusted with a

special gas conditioning set-up (Fig.1). The device con-

tains closed loops of dry gas and wet gas, which are con-

trolled by a mass flow controller (Mass-Flo, 2000 sccm;

MKS Instruments, Berlin, Germany). For creating wet

gas, a dry synthetic air flow is fed through a water tank.

The final gas humidity is achieved by blending the two

gases and the ratio is determined by a humidity sensor

(Therm 8736; Ahlborn, Holzkirchen, Germany). Com-

mercial dry synthetic air [composition 80% N2 : 20% O2

(v ⁄ v)] from a gas bottle was fed with an initial pressure

of 1 bar into the gas conditioning device, resulting in a

gas flow of 2000 sccm. Experiments were carried out at

relative gas humidities of between 0% and 80%, increas-

ing in steps of 10%, and at a gas temperature of about

20�C. Dry synthetic air with 0% moisture was directly fed

in the CDBD set-up controlled by a gas flowmeter (ABB,

Gottingen, Germany). The CDBD set-up was directly

connected to the gas conditioning device by flexible pres-

sure tubing.

Spore preparation

Bacillus subtilis DSM 4181 and Aspergillus niger DSM

1957 were used as test strains and obtained from the Ger-

man National Resource Centre (DSMZ, Braunschweig,

Germany). Bacillus subtilis was selected because of the

highly resistant endospores and their function as a bioin-

dicator in hydrogen peroxide sterilization (Leaper 1984).

Bacillus subtilis DSM 4181 is recommended as test strain

against hydrogen peroxide by the German Association

VDMA (Industry Association for Food Processing

Machines and Packaging Machines), for validation of

aseptic filling machines in food industry (VDMA 2006).

Aspergillus niger was chosen because of its different mor-

phology and high UV resistance. Previous studies have

shown that both test strains have high resistance to

Influence of gas humidity on plasma inactivation efficiency P. Muranyi et al.

1660 Journal compilation ª 2008 The Society for Applied Microbiology, Journal of Applied Microbiology 104 (2008) 1659–1666

ª 2008 The Authors

CDBD plasma treatment, especially conidiospores of

A. niger (Muranyi et al. 2007).

Bacillus subtilis spores were obtained after inoculation

of tissue culture flasks containing 400 ml PCA medium

(Plate count agar; Merck, Darmstadt, Germany), 10 mg

manganese sulphate and 1 ml B. subtilis cells from an

overnight culture followed by incubation at 30�C for

10 days. The spores were harvested by scraping in sterile

ringer solution (Oxoid, Wesel, Germany), washed several

times by centrifugation (10 000 g for 10 min at 20�C)

and re-suspended in sterile ringer solution. After this pro-

cess the spore suspension was heated at 80�C for 32 min

to kill vegetative cells and then stored at 4�C.

Conidiospores of A. niger were formed on YGC plates

(Yeast extract glucose chloramphenicol; Merck) which

were inoculated with a suspension of cell material of A.

niger. After an incubation period of 10 days at 30�C the

spores were harvested by tapping the plates with sterile

sea sand. For deposition of the sand particles, the agglom-

erates of sand and spores were suspended in sterile ringer

solution treated with ultrasound for 1 min. The superna-

tant containing the conidiospores was removed and

finally stored at 4�C until usage.

Bioindicator preparation

Bioindicators carrying B. subtilis endospores or A. niger

conidiospores were prepared according to a previously

described inoculation method (Muranyi et al. 2007).

Planar squares with an area of 10 · 10 cm2 were cut

from polyethylene terephthalate blank foils (Mitsubishi

Polyester, Wiesbaden, Germany) and used as carriers for

the test strains. For disinfection and cleaning, the foils

were immersed in a bath of ethanol (70% v ⁄ v) and then

dried. Because of the very low initial microbial count of

the material this procedure was adequate.

Spores of the test strains were deposited on the surface

of the foils using a special spray device. An appropriate

dilution of an aqueous spore suspension was placed in a

holding tank and fed to a two-substance nozzle (Dusen

Schlick, Coburg, Germany) via a gas flow of nitrogen.

The air cap of the nozzle was adjusted to give a fine mist

of spore suspension to ensure a homogenous distribution

on the surface of the PET foils, which was verified by

microscopy. To prevent backside contamination only a

sub-area of 4 · 4 cm2 was spray inoculated by covering

the PET foil with a sterile metal template. The atomizing

pressure was 2Æ5 bar and the spray time was varied to

deposit 0Æ01 g suspension on the surface of the PET foil.

In this way, the microbial concentration on the samples

was controlled. Aspergillus niger had an initial inoculation

density of approx. 105 CFU ⁄ sample and B. subtilis of

106 CFU ⁄ sample. For avoiding an influence of different

cell densities on the inactivation efficiency (e.g. because of

agglomeration), the initial microbial load on the PET foil

was kept nearly constant for each test strain. The differ-

ence between the inoculation density of A. niger and B.

subtilis depends on the lower initial microbial count of

the spore suspension for A. niger. Finally the PET foils

were removed from the spraying set-up and allowed to

dry for 1 h under a laminar air flow cabinet.

Plasma treatment and spore recovery from the samples

Bioindicators were placed in the CDBD plasma device on

the ground electrode. The plasma was ignited by applying

an electrical power of about 170 W. To prevent microbial

contamination, the CDBD set-up was arranged under a

Figure 1 Experimental set-up for

conditioning the gas humidity. In our study

commercial synthetic air was humidified in

the range between 0% and 80% relative

humidity (20�C). The initial pressure was

1 bar and the gas flow was adjusted to

2000 sccm (ml min)1).

P. Muranyi et al. Influence of gas humidity on plasma inactivation efficiency

ª 2008 The Authors


laminar air flow cabinet. The applied dose was varied by

changing the treatment time in the range of 1–7 s. Com-

mercial synthetic air adjusted to different relative humidi-

ties was used as the process gas. After plasma treatment,

the samples and the untreated references were stored in

sterile Stomacher� bags (Seward, Norfolk, UK) filled with

100 ml sterile ringer solution containing 0Æ1% (w ⁄ v)

Tween-80 (Merck). Spores were removed from the surface

of the foil by washing for one minute with a Stomacher�

blender. Determination of B. subtilis survivors was carried

out using Koch’s plate dilution technique and spiral plate

method (Eddy Jet; IUL Instruments, Konigswinter, Ger-

many) on PCA. In the case of very low bacterial counts

micro-filtration was used for detection. For recovery of A.

niger only the plate dilution technique with YGC agar

(Merck) was utilized. Finally, after incubation at 30�C for

3 days, colony forming units were counted and microbial

reduction was determined as follows: logarithmic count

reduction = log10 N0 ) log10 N.

N0 is the mean of the initial microbial count of the

untreated reference samples, N is the mean of the number

of survivors after plasma treatment. For each tested

parameter, three samples were examined and data were

presented as averages.

Results

Inactivation of B. subtilis endospores as a function of gas

moisture content

Figure 2 shows the inactivation of B. subtilis endospores

using the CDBD plasma set-up as a function of the treat-

ment time and relative gas moisture. For comparison of

the different kinetics, results were graphically displayed as

N ⁄ N0. This is feasible because of the very similar initial

bacterial count of all the reference samples N0 (approx.

1 · 106 CFU ⁄ sample). It can be observed that the inacti-

vation of B. subtilis worsens with increasing gas humidity.

This effect is very evident after a short-treatment time of

1 s. To determine the extent of the humidity effect, we

have compared the results for the gas moisture limit

values. Table 1 displays the microbial log reduction

(log10 N0)log10 N) at 0% and 80% relative humidity as a

function of the treatment time and Fig. 3 shows the cor-

responding graph. After 1 s a mean microbial reduction

of 4Æ7 log10 was observed at 0% humidity and only

2Æ6 log10 at 80% humidity. This indicates that the spore

mortality of B. subtilis decreases by up to approx. 2 log10

by increasing the moisture level. Furthermore, spore mor-

tality was not proportional to the treatment time, which

indicates a bi-phasic progression of the kinetic as

reported by others (for example Purevdorj et al. (2003)).

A reason could be that plasma decontamination is a

method with limited penetration depth and therefore

stacked spores or surface layers can gradually slow down

the inactivation process.

Inactivation of A. niger conidiospores as a function of

the gas moisture content

The inactivation of A. niger conidiospores under the same

parameters ⁄ conditions as mentioned for B. subtilis is

shown in Fig. 4. Unlike for B. subtilis, an improvement of

A. niger inactivation with increasing relative gas humidity

70 80 1e–7

1e–6

1e–5

1e–4

1e–3

N/N

0

1e–2

1e–1

1e+0

60 50 40 30 20 10 0 8 Trea

tmen

t tim

e (s

)

7 6

5 4 3

2 1 0

Rel. gas humidity (%)

Figure 2 Inactivation of Bacillus subtilis endospores as a function of

the treatment time and the relative gas humidity of synthetic air. The

experiments were carried out with the cascaded dielectric barrier dis-

charge plasma set-up, equipped with a 282-nm excimer flat lamp.

The applied power was about 170 W. The initial bacterial count was

approx. 106 CFU foil)1.

Table 1 Mortality of Bacillus subtilis endospores after CDBD treat-

ment using synthetic air of different relative humidity

Treatment time (s)

Inactivation degree log10 (N0 ⁄ N)

Relative gas humidity (%)

0 80

1 4Æ7 ± 0Æ3 2Æ6 ± 0Æ3

3 5Æ1 ± 0Æ1 4Æ2 ± 0Æ7

5 5Æ0 ± 0Æ3 4Æ4 ± 0Æ1

7 4Æ9 ± 0Æ2 4Æ4 ± 0Æ2

CDBD, cascaded dielectric barrier discharge.

N0, initial microbial count of untreated reference samples.

N, final microbial count after CDBD treatment at 170 W and varying

times.

The spore mortality was calculated as the difference between the

untreated reference and the number of survivors after CDBD treat-

ment (log10 N0 ) log10 N).



ª 2008 The Authors

is observed. This effect becomes more evident at longer

treatment times. If we consider a treatment time of 7 s,

we observe that the microbicidal effect is most evident on

changing from 0% to 10% relative humidity and chang-

ing from 60% to 70% relative humidity. The highest inac-

tivation of 3Æ3 log10 was found for 70% relative gas

humidity and 7 s treatment time. This compared with

only 1Æ4 log10 at 0% relative humidity and the same treat-

ment time (Table 2, Fig. 5). This indicates an improve-

ment of approx. 2 log10 because of conditioning of the

relative gas humidity. Remarkable is that the inactivation

becomes slightly worse at the highest moisture level of

80% (see Table 2). To verify this result the experiment

was repeated with relative gas humidities of 65%, 70%

and 75% (Table 3, Fig. 6). Unfortunately the trial was

done with a new spore suspension of A. niger, meaning

that strict direct comparison of the two experiments is

not possible. The kinetics in this case also showed the

positive effect of high relative gas humidity on the inacti-

vation of A. niger spores, an effect which becomes more

evident at prolonged treatment times. The difference is

about 2 log10 at 70% relative gas humidity and 7 s treat-

ment time compared with dry air. This corresponds with

the results of the first experiment. A decrease in inactiva-

tion, as observed at 80% relative gas humidity, was not

observable at 75%.

Treatment time (s)

Sur

vivo

rs (

log 10

mea

n C

FU

foil–1

)

876543210

7

6

5

4

3

2

1

0

Figure 3 Spore mortality of Bacillus subtilis as a function of treat-

ment time and relative humidity. Inactivation tends to decrease with

increasing gas moisture. This phenomenon is more pronounced

at short-treatment times e.g. 1 s and for the highest relative humidity

(80%). Relative humidity: d, 80%; ,, 0%.

70 80 1e–4

1e–3

N/N

0

1e–2

1e–1

1e+0

60 50 40 30 20 10 0 8 Trea

tmen

t tim

e (s

)

7 6

5 4 3

2 1 0

Rel. gas humidity (%)

Figure 4 Inactivation kinetics of Aspergillus niger as a function of the

treatment time and the humidity of synthetic air. The plasma treat-

ment was carried out with the cascaded dielectric barrier discharge

set-up. The initial microbial count was in the range of approx.

105 CFU foil)1.

Table 2 Mortality of Aspergillus niger conidiospores after CDBD

treatment using synthetic air of different humidity

Treatment time (s)



0 70 80

1 0Æ7 ± 0Æ2 1Æ2 ± 0Æ2 0Æ8 ± 0Æ0

3 1Æ0 ± 0Æ1 1Æ8 ± 0Æ2 1Æ5 ± 0Æ1

5 1Æ2 ± 0Æ3 2Æ4 ± 0Æ4 2Æ2 ± 0Æ4

7 1Æ4 ± 0Æ3 3Æ3 ± 0Æ4 2Æ6 ± 0Æ4




times.

Treatment time (s)

876543210

Sur

vivo

rs (

log 10

mea

n C

FU

foil–1

)

6

5

4

3

2

1

0

Figure 5 Inactivation of Aspergillus niger conidiospores as a function

of treatment time and relative humidity. It can be observed that spore

mortality increases with increasing humidity. This effect becomes

more pronounced at prolonged treatment times. Relative humidity: d,

0%; ,, 70%; n, 80%.


ª 2008 The Authors


Discussion

The aim of this study was to optimize the CDBD inacti-

vation efficiency in air against A. niger conidiospores by

modifying the relative gas humidity. The mould A. niger

was chosen because of its high resistance to CDBD

plasma treatment as described previously (Muranyi et al.

2007). To examine the influence of gas humidity on bac-

teria simultaneously, endospores of B. subtilis were

selected. Generally, the properties of gas plasmas can be

modified and optimized by varying the main parameters

such as pressure, power and process gas. Each alteration

of one of these parameters changes the whole plasma

chemistry and influences, for example, the electron den-

sity, concentrations of charged or reactive particles and

the amount of emitted UV radiation. In the case of the

CDBD set-up, only the power, process gas composition

and the type of excimer flat lamp can be changed. To

achieve the highest inactivation rates, trials were carried

out with a maximum power of approx. 170 W (for this

set-up). Previous studies with different process gases had

shown that laboratory air is very effective for inactivation

of micro-organisms (Muranyi et al. 2007). There is also

the opportunity to change the wavelength of the excimer

flat lamp via selection of the excimer gas mixture, but

this has been studied elsewhere. In this work, we have

tried to improve the decontamination efficiency by taking

synthetic air having a gas composition of 80% nitrogen

and 20% oxygen and humidifying it to simulate atmo-

spheric conditions. Compared with normal air, minor

components such as argon and carbon dioxide are not

present in synthetic air. Previous experiments with

A. niger DSM 1957 and B. atrophaeus DSM 2277 have

shown that their effect can however be neglected (Fig. 7).

The studies have shown that the inactivation of A. niger

conidiospores can be improved by about 2 log10 by

increasing the relative gas humidity. The highest spore

mortality of approx. 3Æ3 log10 was found at 70% relative

humidity and a treatment time of 7 s. As shown by the

inactivation kinetics, the effect was enhanced at prolonged

plasma exposure which indicates a time-dependent reac-

tion. Remarkable is the slightly worse mortality at 80%

relative humidity. This could indicate that there is an

optimum relative humidity in the range of 70% for inac-

tivation of A. niger conidiospores.

Table 3 Repeat experiment with a new suspension of Aspergillus

niger conidiospores and synthetic air of different humidity

Treatment

time (s)



0 65 70 75

1 1Æ0 ± 0Æ0 1Æ5 ± 0Æ4 2Æ1 ± 0Æ1 1Æ9 ± 0Æ2

3 1Æ4 ± 0Æ1 2Æ1 ± 0Æ2 2Æ3 ± 0Æ2 2Æ4 ± 0Æ0

5 2Æ0 ± 0Æ0 2Æ0 ± 0Æ1 3Æ1 ± 0Æ2 4Æ0 ± 0Æ4

7 2Æ3 ± 0Æ2 3Æ1 ± 0Æ2 4Æ0 ± 0Æ4 4Æ0 ± 0Æ5




times.

Sur

vivo

rs (

log 10

mea

n C

FU

foil–1

)

Treatment time (s)

876543210

1e+6

1e+5

1e+4

1e+3

1e+2

1e+1

1e+0

Figure 6 Inactivation kinetics of Aspergillus niger conidiospores on

being subjected to cascaded dielectric barrier discharge treatment as a

function of time and at relative humidities of 0%, 65%, 70% and

75%. The process gas was synthetic air and the applied power was

about 170 W. Relative humidity: d, 0% s, 65%; ., 70%; n, 75%.

Treatment time (s)

876543210

Sur

vivo

rs (

log 10

mea

n C

FU

foil–1

)

7

6

5

4

3

2

1

0

Figure 7 Inactivation kinetics of Aspergillus niger DSM 1957 coni-

diospores and Bacillus atrophaeus DSM 2277 endospores as a func-

tion of time for normal air and synthetic air. Relative humidity (20�C)

was kept constant for both gases. The treatment was done with the

cascaded dielectric barrier discharge at an applied power of about

180 W. The kinetics shows that there is no significant effect of minor

gases in normal air on the inactivation efficiency. (d) A. niger ⁄ room

air 47Æ5% RH; (s) A. niger ⁄ synthetic air 47Æ5% RH; (.) B. atro-

phaeus ⁄ room air 38% RH; (,) B. atrophaeus ⁄ synthetic air 38% RH.



ª 2008 The Authors

In contrast, the kinetics for B. subtilis endospores indi-

cated a slight worsening of inactivation at higher gas

humidity. Compared with dry air, the negative effect of

water vapour was most apparent (2 log10) for a treatment

time of 1 s and at 80% relative humidity.

We assume that the positive effect on inactivation

because of the increased gas humidity in the plasma

mainly arises from the decomposition of additional water

molecules into hydroxyl radicals as a result of impact by

other gas molecules or charged particles. The dissociation

of the O-H bond in a water molecule requires an energy

of approx. 5Æ1 eV. Such energy values are easily reached

by the accelerated electrons in the plasma. The photon

energy of the 282 excimer flat lamp (4Æ4 eV) is not suffi-

cient to dissociate the water molecules directly. However,

one main path of hydroxyl radical generation in a DBD

discharge is photodissociation of ozone into atomic sin-

glet oxygen and subsequent reaction of the atomic oxygen

with water molecules (Falkenstein 1997a). The maximum

absorption of ozone occurs at 255 nm, within the Hartley

absorption band (244–278 nm), so radiation emitted by

the 282 nm excimer surely contributes to the formation

of hydroxyl radicals.

The hydroxyl radicals are very electronegative and are

therefore strong oxidizing agents which can damage

microbial cells. The main targets of oxidative processes

include unsaturated fatty acids of the membrane lipid

bilayer and diverse enzymes and proteins of the cell ⁄ spore

(Laroussi 2005). The standard electrode potential of

hydroxyl radicals (+2Æ88 V) is much higher than other

species such as atomic oxygen (+2Æ42 V) or ozone

(+2Æ07 V). Furthermore, two hydroxyl radicals can react

to form hydrogen peroxide, which is also known to have

microbicidal effects. In this case, the effect is based on the

released atomic oxygen, which damages essential parts of

the cells: 2(ÆOH) fi H2O2 fi H2O + O. This mechanism

was also reported by Purevdorj et al. 2003.

Wallhaußer (1995) pointed out generally that in the

case of dry air (<32% relative humidity) a twofold dose

of UV radiation is necessary for sterilization compared

with air of 72% relative humidity. This agrees with our

results with A. niger conidiospores which showed

improved inactivation at 70% relative humidity. Further-

more, Wallhaußer (1995) reported that increased

amounts of water vapour (>80% relative humidity)

reduce the sterilization effect because of the poorer trans-

missibility of UV radiation in such air and protective

water films around the micro-organism cells. We also

observed reduced inactivation of A. niger conidiospores at

80% relative humidity. However, regarding the reduced

inactivation of A. niger spores, and especially B. subtilis

spores, at elevated gas humidities, we do not believe that

limited transmissibility or protective water films play a

major role, because Ansari and Datta (2003) and Hale

and Querry (1973) reported a UV light penetration depth

in water of up to 30 cm. The adjusted gap of our plasma

set-up has only a width of 1 mm. Therefore these aspects

must be further investigated.

Different results for Bacillus species were obtained in

the study of Purevdorj et al. (2003). In the case of B.

pumilus spores and a low pressure plasma set-up, they

observed increased spore mortality by more than two

orders of magnitude by moistening the air. The reasons

for this variance could be the high resistance of B. subtilis

endospores to oxidative agents, especially the tested strain

DSM 4181 (hydrogen peroxide bioindicator), wherefore

the positive effect of additional reactive species is not

obvious in our case. However, this is just an assumption

because plasma chemistry in low pressure systems is

rather different.

Black pigmented A. niger conidiospores have especially

high UV resistance and require dosage levels 20–100 times

higher (Ansari and Datta 2003), but they are also sensitive

to oxidation. The results of our study indicate that an

accumulation of reactive species from decomposed water

molecules could be responsible for the improved inactiva-

tion of A. niger at prolonged treatment times.

Also interesting are the results of Maeda et al. (2003)

regarding the inactivation of E. coli using atmospheric gas

plasma with a humidified working gas in the 0–70% rela-

tive humidity range. They reported survival rates as a

function of humidity which corresponds to a standardized

normal distribution. This means no inactivation at 0%

and 70% relative humidity, and maximum inactivation at

43% which corresponds to normal room conditions. It

therefore seems that the effect of moisture in air on the

microbiological targets depends not only on the amount

of water vapour but also on the microbiological species.

To achieve the highest positive inactivation effect of a gas

plasma, it hence makes sense to validate the influence of

gas humidity for every microbiological application.

Falkenstein (1997a) has investigated the micro-dis-

charge behaviour of dielectric barrier discharges in water–

air mixtures under UV illumination, which is similar to

our CDBD set-up. It was shown on the one hand that

increasing water concentration in air results in hydroxyl

radical production which increases oxidation rates. On

the other hand, the water vapour reduces the number of

micro-discharges. The reason for this effect is the reduc-

tion of the surface resistance of the dielectric caused by

the adsorption of water molecules (Falkenstein 1997a;

Falkenstein and Coogan 1997). The additional UV radia-

tion of the excimer flat lamp leads again to more and

smaller micro-discharges with lower residual OH-radical

concentration; but as these are nonlinear effects, an

increased total concentration of OH is ultimately


ª 2008 The Authors


observed. Nevertheless, it could be that an increased

amount of water vapour finally reduces the plasma

homogeneity of the CDBD (Falkenstein 1997b). These

assumptions do however have to be confirmed. Measure-

ments carried out with a normal dielectric barrier dis-

charge have shown that increasing gas humidity (80%,

24�C) reduces the discharge homogeneity (Franken et al.

2003). This could be a reason for the observed decrease

in B. subtilis endospore inactivation at high relative gas

humidity. Which of these effects dominate in the process

depends, among other things, on the properties of the

chosen micro-organism.

In summary, we assume in the case of our CDBD set-

up and using synthetic air as the process gas those high

gas humidities of, for example 70% could be optimal for

the inactivation of oxidation-sensitive micro-organisms

such as A. niger. By the use of germs such as B. subtilis,

where the inactivation pathway is based mainly on UV

radiation, the process will be slowed down by high water

vapour content. We believe that a possible effect of air

transmissibility or water films on UV penetration can be

neglected in this set-up. The influence of gas humidity on

plasma homogeneity has to be evaluated. The solution for

practical applications could be an arrangement of two

CDBD units in series, each with different process gases

(dry air and humidified air) to extend the target decon-

tamination spectrum. In all cases, it is important to adjust

the relative humidity of the plasma gas to a value which

results in optimum inactivation conditions for as many

strains as possible.

Acknowledgement

This work was partially funded by the European Commis-

sion under contract number G1RD-2002-00747 (COLA-

PE). The authors would also like to express their

gratitude to Mrs Berger and Mrs Steinbuchl for technical

support and to Dr Rieblinger for useful discussions.

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Influence of relative gas humidity on the inactivation efficiency of a low temperature gas plasma