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ARTICLE IN PRESS C. Abrusci et al. / International Biodeterioration & Biodegradation 56 (2005) 58–68 65 Fig.3.Microsatellite primed PCR fingerprinting for A. alternata strains HB1 (lane 2) and HB41N (3); P. chrysogenum strains HLV1 (5), HMI (6), HGC3B (7), HGC1 (8), HB7 (9), HB6 (10) and HB41B (11); (4), (12) control, 100 bp ladder. oxygen availability.In a previous study (Abrusci et al., 2004c), it was stated that these gelatinase-positive bacterial isolates can degrade photographic grade gelatin (Bloom 225) in aqueous solution (37 1C, 6.67%, w/w) causing progressive viscosity decrease, with each species showing a characteristic viscosity decrease profile, which changed with temperature.At 4 1C, only B. amyloliquefaciens, B. subtilis, B. pumilus and B. megaterium were able to degrade gelatin aqueous solution.Nevertheless, in studies of several industrial gelatin production processes (De Clerck and De Vos, 2002) and semi-final gelatin extracts (De Clerck et al., 2004), the authors reported that under experimental conditions the majority of isolates exhibited gelatinase activity. All filamentous fungi isolated in this work were able to degrade gelatin in the tube test (Table 2), but the yeast did not, although other strains of this species have been reported as proteolytic (Wade et al., 2003).Unlike yeasts, filamentous fungi are very important microbial Table 2 Gelatinase activity of microbial strains (bacteria and fungi) isolated and identified from cinematographic films Strain Identification Gelatin hydrolysis been reported as soil and airborne fungi.The biodiversity in that research was therefore different from that in our investigation.It might be due to the higher climatic differences existing between the Spanish archives, especially if we consider that two of them are placed in coastal areas where environmental humidity is much higher than in Madrid. The only yeast isolated was identified as Cryptococcus albidus, which has been isolated from air, soils, and a range of habitats, as well as from man and other mammals (Barnett et al., 2000).Although in general terms yeasts cannot survive or develop in habitats with low water activity.However, it has been reported that C. albidus var. diffluens shows adaptation to extreme low humidity (water activity) (Aksenov et al., 1973). 3.3. Gelatin microbial biodegradation tests In cinematographic films, gelatin degradation is a highly relevant problem, because it causes the loss of the image.Since some microbial contaminants found in our film samples were able to grow actively and produce severe deterioration of the films, the ability of all the microorganisms isolated from cinematographic films to degrade gelatin was checked. Table 2.Seven of the 14 bacterial isolates degraded gelatin in both Petri dish and tube hydrolysis tests, six being Bacillus spp.In addition, S. lentus hydrolysed gelatin in the Petri dish assay, but not in gelatin test tube assay, perhaps owing to the lower B3BA Bacillus amyloliquefaciens + B7 Bacillus megaterium + GC2 Bacillus megaterium + M2 Bacillus pichinotyi + M5 Bacillus pumilus + B3BS Bacillus subtilis + M4 Kocuria kristinae GC1A Pasteurella haemolytica B4A Sphingomonas paucimobilis B2 Staphylococcus epidermidis M3 Staphylococcus haemolyticus B4C Staphylococcus hominis + B5 Staphylococcus lentus a GC1B Staphylococcus lugdunensis HB1 Alternaria alternata + HGC3 Aspergillus versicolor + HM3 Aspergillus nidulans var. nidulans + HB2 Aspergillus ustus + HGC2B Aspergillus ustus + HB3A Cladosporium cladosporioides + HM4 Mucor racemosus + HB41B Penicillium chrysogenum + HB41N Alternaria alternata + HB6 Penicillium chrysogenum + HB7 Penicillium chrysogenum + HMI Penicillium chrysogenum + HGC1 Penicillium chrysogenum + HGC3B Penicillium chrysogenum + HLV1 Penicillium chrysogenum + HGC3N Phoma glomerata + HGC2 Trichoderma longibrachiatum + LB6 Cryptococcus albidus + ¼ positive;— ¼ negative. a Negative in tube-test but positive in Petri-dish assay.
66 ARTICLE IN PRESS C. Abrusci et al. / International Biodeterioration & Biodegradation 56 (2005) 58–68 Fig.4.Individual film frames (on moist culture medium) showing extreme biodeterioration of the image caused by B. subtilis (left) and P. chrysogenum (right). agents of biodeterioration; they not only degrade many kinds of molecules and materials, but also contribute to the formation of biofilms.As well as various bacteria (mainly Bacillus, Pseudomonas, Sphingomonas), fungi such as Alternaria, Trichoderma, Aspergillus and Penicillium have been found to degrade, at least partially, certain natural proteinaceus materials, e.g. silk (Seves et al., 1998), keratin (Yamamura et al., 2002) and leather (Orlita, 2004). Of course, neither presence nor absence of gelatinase activity in the microbial contaminants isolated from Spanish cinematographic film is indicative of inability to degrade other components of film.Diverse species of bacteria can degrade cellulose triacetate (Buchanan et al., 1993; Nelson et al., 1993; Sakai et al., 1996; Ishigaki et al., 2000), either in conditions of aerobiosis or anaerobiosis and there are also fungal biodegraders (Itävaara et al., 1999; Gu et al., 1993a, b).In all these studies cellulose triacetate with a higher degree of substitution was more resistant to biodegradation (Buchanan et al., 1993). 3.4. Visual observation of cinematographic film biodeterioration and microscopic analysis Bacterial and fungal growth can usually be observed on the emulsion side of film as irregular dull spots.The spots increase in size and number when the material is left unattended.Eventually, microorganisms can degrade the emulsion to the point that it is useless.A degree of extreme growth of bacteria and fungi on the side of the emulsion is shown in Fig.4. Microbial growth can be observed on individual frames between loose coils at the beginning of rolls of film and also on the margins in the appressed coils.Damage to the image is not immediate. If the microbial growth is discovered in time it is possible to remove it and to prevent recurrence. Continued growth causes permanent damage and the destruction of gelatin (Fig.5).The cryogenic fracture of the film at 77 K under liquid nitrogen gives a transversal profile of the gelatin layer where there are hyphae of a Penicillium.The surface of the film material is also colonized. 4. Conclusions This investigation has provided evidence that black and white cinematographic films stored in the Spanish archives are contaminated by diverse species of bacteria and fungi, and as judged by the properties of many of the microbial isolates, particularly fungi, microbial colonization might result in severe biodeterioration, including aesthetic variation or chemical and mechanical degradation.Most microorganisms that were isolated can resist environmental conditions adverse to microbial life, e.g. desiccation or nutrient starvation, and they can adhere to the substrates by hyphae or exopolymers.This may give rise to biofilm development, increasing the corrosive action of microbial metabolic products.In addition, some of the microbial contaminants can degrade the gelatin and probably other components of the films.Therefore, it is crucial that cinematographic archives such as museums, historic libraries, etc.have a strategy for microbiological quality control of the indoor environment in order to prevent, or minimize, the potential risk of microbial colonization of the materials.
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