HIGH-FREQUENCY COLD PLASMA – AN ECOLOGICAL TREATMENT FOR THE MICROBIAL DECONTAMINATION OF ORGANIC MATERIALS

materials of organic nature has shown that the microbiological investigations confirm the biocide action of the HF plasma in fighting bacteria (after 45 minutes in the samples of icon wood and natural silk, 60 minutes in the cotton samples, 90 minutes in the archaeological wood and leather, and 100 minutes in flax and paper samples). The results were attained using a rigorously designed and employed experimental system, which included microbiological, physical, and chemical determinations prior to and after the treatment of the samples examined. The research tests performed corroborate the conclusion that the treatment with high- frequency cold plasma, when conducted in non-destructive conditions, does not degrade the collagen fibres, thus providing an ecological and more efficient alternative for biological decontamination treatments of cultural items on organic supports. The researches contribute to collection of certain literature data on fighting microorganisms specific to cultural items on organic support ecologically. Key words: organic material, microorganism, decontamination, high-frequency cold plasma INTRODUCTION The action of biological agents on organic supports has as a result the occurrence of biodeterioration, generated primarily by two mechanisms: the nutritive one, by which the material is used in the nutrition of the degradation agent, and the metabolic mechanism exerted by the products of metabolism of the degradation agent [2]. Patrimony pieces on organic supports such as leather, textile materials, wood or paper are the most susceptible to the attacks of fungi or microorganisms [8], humidity exceeding 60 % concurrent with temperature higher than 260 C, which stimulate their rapid growth [1,4]. The regular procedures of decontamination involve the use of non-selective chemical substances, extremely aggressive gamma radiations whose persistence and deteriorating effects on the supports have not been sufficiently examined; that is the reason why the current research studies focus on finding ecological techniques, which are less harmful [3]. The treatment with high-frequency cold plasma, for instance, provides an effective alternative with a high application potential, less used nowadays in the field of conservation. The decontamination treatments involve the use solely of HF cold plasmas with a temperature not exceeding 500 C [5]. The high-frequency cold plasma used, rich in accelerated electrons, molecules, free radicals and electromagnetic radiations, and highly reactive determines the efficacy of such medium in fighting biodegradation [7]. A medium rich in oxygen, hydrogen peroxide, nitrogen, argon, gives the plasma oxidizing, reduction and biocide properties, its effectiveness becoming thus higher. The objectives of this paper were as follows: identification of the contaminating microbiota which impacts on the supports examined (wood, leather, silk, wool, cotton, flax, and paper); decontamination treatment with high-frequency plasma of the samples; microbiological evaluation of the efficacy of the high-frequency plasma decontamination. MATERIALS AND METHODS Small size samples were collected from museum items or archaeological material, according to the strict specifications of the appropriate conservation regulations. The wood subjected to examination fell into two categories: archeological wood dating back to the 18th century and wood collected from an icon dating back to the 19th century. In what concerns the leather, two samples of archeological leather tanned with vegetable agents were selected: one treated with polyethylene glycol (PEG), discovered at Baia and dating back from the 15th century, and the other with no previous treatments, more recent (the 19th century). The textile materials examined consisted in old textile supports and modern fabrics. From the former category, the study included a sample of cotton fabric collected from an icon on wood; in terms of the latter, the study included samples of natural silk, wool, flax, and cotton fabrics. The piece of cotton cloth was collected from the lining of a military uniform from the 19th century, while the flax fabric was taken from a 16th century painting consolidated with the flax piece at the end of the 19th century. Another sample subjected to examination consisted in paper material taken from an 18th century psalm book. The samples were identified by means of the following conventional notation: I. Archaeological wood; II. Icon wood; III. Archaeological leather treated with PEG; IV. Archaeological leather untreated with PEG; V. Textile support –cotton (the icon); VI. Natural silk fabric (modern material); VII. Wool fabric (modern material); VIII. Flax fabric; IX. Cotton fabric; X. Paper material. For the microbiological examination of the organic supports, a simple growth medium was used: gelose. The medium was inoculated with a sample of the appropriate support specimen using the imprint method. The microbial growth was examined after incubation at

C for 48 hours. For the macromorphological description of the grown colonies, the following criteria were considered: colony type, shape, edge, elevation, consistency, transparency/opacity and color [6]. The examination of the micromorphological characters of the microorganisms isolated from the materials tested consisted in the preparation of smeared glass slides, slide staining using Gram’s method and observation by immersion microscopy (x 1000).The quantitative assessment was carried out using plate cultures and colony count (the number of colony forming units - CFU) [9]. The high-frequency cold plasma treatment of the samples was conducted in a dedicated plant which included a high-frequency generator, vacuum pump, vacuum and temperature measurement system, and a Pyrex glass vessel with cylindrical aluminium electrodes arranged concentrically inside.The samples subjected to the treatment were set on a stainless steel grind in-between the electrodes, which allowed the concurrent treatment of both sides of the material. A nitrogen cold plasma was used at the following working parameters: temperature inside the vacuum enclosure: not more than 500 C; pressure: 2-3 torrs; frequency: 1.2 MHz; intensity of the field: 150-300V/cm; power: 200 W. The samples were treated in the plasma with the above-mentioned characteristics for 45, and respectively 90 minutes. In addition, after changing several parameters of the plant (i.e. temperature range: 35 ? 400 C; frequency: 13.5 MHz; discharge power: 100 W; intensity of the electric field: 50 ? 100V/cm; pressure: 2.1 x 10-1 mbars) three other time intervals were tested in the flax, cotton, and paper sample: 30, 60 and 100 minutes. In the same samples, the CFU/ml was also calculated. The effectiveness of the HF plasma treatment was assessed based on the growth (lack of growth) of the microorganisms around the samples subjected to the treatment, after their inoculation on gelose and incubation at 370 C for 48 hours. The microbiological investigations were followed by scanning electronic microscopy test and thermogravimetric analysis of a leather sample collected from the examined supports. RESULTS AND DISCUSIONS Most untreated organic supports were found to exhibit bacterial contamination, except the textile material (i.e. wool) and the archaeological leather treated with PEG, in which the contamination was less obvious. The poorest bacterial growth was noticed in the sample of natural silk. The HF cold plasma decontamination treatment resulted in the following findings: while after 45 minutes, the bacteria multiplication rate reduced, after 90 minutes the reduction was obvious and significant in all the samples. The comparative analysis of the two sets of samples decontaminated for 45, and respectively 90 minutes, and the control samples (not treated with plasma) show that in the samples of archeological wood (I), textile support –cotton (from the icon) (V) and 19th century archaeological leather (IV), which exhibited high bacterial contamination at the beginning (Photo 1, respectively Photo

the growth was not extremely obvious after the 45 minute treatment (Photo 2, respectively, Photo 8), and was extremely poor after the 90 minute treatment (Photo 3). In the icon material (II) (Photo 4, respectively Photo 9) and natural silk fiber (VI) –(Photo

, the plasma treatment resulted in the inhibition of the microorganism growth even after

minutes (Photo 5, respectively Photo 11), the effect being easily perceived after 90 minutes (Photo 6, respectively Photo 12). The cold plasma treatment of the wool fabric (VII) only inhibited microorganism growth after 90 minutes (Photo 15), the growth being still obvious after 45 minutes (Photo 14) as in the case of the untreated sample (Photo 13). The macromorphological examination showed that all the organic supports subjected to study exhibited, in general, similar colony characters (Table I), with the predominance of rough, R-type, non-pigmented, loosely adherent to the substrate colonies. They differ solely in terms of the size of the colonies, which are much higher in the untreated samples, smaller in the samples treated with plasma for 45 minutes, and substantially reduced after 90 minutes. The microscopic examination (Table I) showed almost exclusively the presence of the Gram-positive bacilli, with differences in their arrangement and type of spores they generate, the only exception being the natural silk specimen in which Gram-positive cocci were found. The comparative analysis of the two samples of archaeological leather, one treated with PEG and the other untreated (Photo 7) shows in the latter the presence of the diffusible pigment (reddish brown pigment specific to the tannin extracted from oak), which migrates in depth of the culture medium coloring it, and its absence in the PEG treated fragments. The polymer probably inhibits pigment diffusion outside the fragment. The HF cold plasma treatment conducted for 30, 60, and 100 minutes revealed a number of differences among the supports subjected to examination, both in terms of quality and of the number of microorganisms. In terms of quantity (Table II), substantial decrease in the number of microorganisms was noticed in the paper material sample (X) following both the 30 minute treatment (120 CFU/ml) –Photo 23 and the 60 minute one (30 CFU/ml) –Photo 24 as compared to the control sample (5080 CFU/ml) –Photo 22. In the cotton fabric sample (VIII), the 30 minute treatment resulted in a decrease of the CFU/ml to 40 (Photo 20) as compared to the control sample (CFU/ml = 7920) –Photo 19. A similar effect as in the case of the paper sample (X) was noticed in the flax fabric sample (IX), being counted 4840 CFU/ml at 30 minutes (Photo 17) and 30 CFU/ml at 60 minutes (Photo 18), while the control sample exhibited 8240 CFU/ml –Photo 13. The overall analysis of the research results was able to show the efficacy of the plasma tretament after the specified time intervals and a considerable decrese in the number of microorganisms at 30 and respectively 60 minutes in the paper and flax samples as compared to the control ones. The bactericidal effect of plasma was evident in the cotton sample after a 60-minute treatment, and in all samples under investigation after a 100- minute treatment. The examination of the control samples collected from the leather item by scanning electronic microscopy using a TESLA BS 301 type microscope at an accelerating voltage of 15KV identified a compact appearance with micro-size pores and fine collagen fibers in parallel to the surface, specific to the papillary layer, incorporated in a granular polyethylene glycol mass. The sample treated with plasma did not exhibit changes of the dermal surface, thus confirming that the treatment does not degrade the collagen fibers. The thermogravimetric (TG) analysis of the samples using a Derivatograph type Q –

D MOM before (Fig. 1) and after the high-frequency plasma decontamination treatment (Fig. 2) detected a 2.5% loss in mass. The rate of the curves TG, TDG, TDA plotted up to 700°C for the same samples showed their similarity, thus indicating that HF plasma treatment did not result in thermal oxidizing degradations of the collagen fibers. Table I. Macro- and micro-morphological examination of bacterial colonies grown on the supports examined SAMPLE MACROMORPHOLOGICAL EXAMINATION MICROMORPHOLOGICAL EXAMINATION I. 18th century ARCHAEOLOGICAL WOOD

Beige, R-type colony with slightly lobate edges, and raised Gram + bacillus, arranged singly, sporulated, with central, non-distorting spores II. 19th century ICON WOOD Beige, R-type colony with undulate edges, and flat Large Gram + bacillus, arranged singly, curved, sporulated with fully formed spores III. PEG-TREATED ARCHAEOLOGICAL LEATHER Light beige, R-type colonies with lobate edges, rough surface, and flat Gram + bacillusarranged in chains, sporulated, with central, distorting spores IV. ARCHAEOLOGICAL LEATHER NOT TREATED WITH PEG Dark beige, R-type colony with undulate edges, ridged surface, and raised; exhibits diffusible pigment Gram + bacillus, arranged singly, completely sporulated, with fully formed spores V. TEXTILE SUPPORT (cotton) Light beige, R-type colony with irregular edges, and flat Large Gram + bacillusarranged in long chains, sporulated, with fully formed spores VI. NATURAL SILK FABRIC

Beige, S-type colony with irregular edges, and flat Gram + cocci, grouped in sarcinas VII. WOOL FABRIC Light beige, R-type colony with markedly lobate and slightly raised edges, and flat Gram + bacillus, arranged singly and grouped in pairs, sporulated, with central, non-distorting spores VIII. 19th century COTTON FABRIC

White, R-type colony with lobate edges, and raised Gram + bacillus, arranged in chains, not sporulated IX. 19th century FLAX FABRIC

White, R-type colony with undulate edges, flat Gram + bacillus, arranged in chains, sporulated, with central, non-distorting spores X. 18th century PAPER MATERIAL

White, R-type colony with fimbriate edges, slightly raised Gram + bacillus, arranged singly sporulated, with fully formed, central, non-distorting spores Table II. Quantitative analysis of microbiota in the samples examined CFU/ml Examined sample Not treated 30 min. treatment 60 min. treatment 100 min. treatment Paper material 5080 120 30 0 Cottonfabric 7920 40 0 0 Flax fabric 8240 4840 30 0 Fig. 1 Thermogram before plasma treatment Fig. 2 Thermogram after plasma treatment CONCLUSIONS

A number of 10 microorganism strains of bacterial type were isolated in pure cultures from the samples subjected to examination (i.e. wood, leather, cotton, wool, flax, and paper).

The examination of the macro- and micromorphological characters allowed the classification of the bacterial strains isolated into the Gram-positive sporulated bacilli group.

The 45 minute treatment with high frequency cold plasma has a bactericidal action in the icon wood and natural silk samples; the same bactericidal effect is found in the cotton samples after the 60 minute treatment.

The investigations proved the decontaminating effect of the high frequency cold plasma in most organic supports examined, following a 90 or 100 minute treatment, effect revealed by the inhibition of microorganism growth.

The research tests performed corroborate the conclusion that the treatment with high- frequency cold plasma, when conducted in the above-specified conditions, does not deteriorate the collagen fibres and the aesthetic appearance of the item, thus providing an ecological and more efficient biological decontamination alternative.

The results of this study recommend the use of cold plasma as a decontamination medium for a wide range of small and medium size patrimony items. SELECTIVE BIBLIOGRAPHY

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River, New Jersey. Organic supports decontamination with HF cold plasma Not treated 45 minute treatment 90 minute treatment I. 18th century ARCHAEOLOGICAL WOOD Photo 1 Photo 2 Photo 3 II. 19th century ICON WOOD Photo 4 Photo 5 Photo 6 IV. ARCHAEOLOGICAL LEATHER NOT TREATED WITH PEG Photo 7 Photo 8 Photo 9 VI. NATURAL SILK FABRIC Photo 10 Photo 11 Photo 12 VII. WOOL FABRIC Photo 13 Photo 14 Photo 15 Not treated 30 minute treatment 60 minute treatment (quantitative analysis) VIII. FLAX FABRIC Photo 16 Photo 17 Photo 18 IX. COTTON FABRIC Photo 19 Photo 20 Photo 21 X. PAPER MATERIAL Photo 22 Photo 23 Photo 24 ACKNOWLEDGEMENT The research presented in this paper was carried out under Programe CEEX - no.54/6948/2006 „Integrated multifunctional technology for the conservation of the national patrimony” (MITECH). The autors would like to express their gratitude to MATNANTECH PROGRAME for the financial support and for their valuable contribution to this research.