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Recent Res. Devel. Microbiology, 10(2006): ISBN: 81-308-0022-5

Bacteria present in cinematographic films stored in spanish archives. Biodegradation of photographic gelatine

C. Abrusci1, D. Marquina1, A. Santos1, A. Del Amo2 and F.Catalina3 1Departamento de Microbiología III, Facultad de Biología, Universidad Complutense de Madrid, José Antonio Novais, 2, 28040-Madrid, Spain 2Filmoteca Española, Magdalena 10, 28012-Madrid, Spain 3Departamento de Fotoquímica de Polímeros, Instituto de Ciencia y Tecnología de Polímeros, C.S.I.C, Juan de la Cierva 3, 28006-Madrid, Spain

Abstract Bacteria present in black and white cinematographicfilm samples were isolated and identified from samples collected in Spanish archives of Madrid, Barcelona and Gran Canaria. Fourteen strains of bacteria were isolated, five species of genus Staphylococcus i.e. S. epidermidis, S. hominis, S. lentus, S. haemolyticus and S. lugdunensis and five species of Bacillus i.e.

Correspondence/Reprint request: Dr. D. Marquina, Departamento de Microbiología III, Facultad de Biología Universidad Complutense de Madrid, José Antonio Novais, 2, 28040-Madrid, Spain E-mail: dommarq@bio.ucm.es

C. Abrusci et al. 2

B. amyloliquefaciens, B. subtilis, B. megaterium, B. pichinotyi, B. pumilus were identified together with Sphingomonas paucimobilis, Kocuria kristinae and Pasteurella haemolytica. Also, the effectiveness of the isolated and identify bacteria in the biodegradation of photographic gelatine grade material has been studied by viscometry in aqueous solution (at 37º, 6.67%w/w). From viscosity data, different variables such us molecular weight and chain scission were calculated. In all the bioassay experiments 25x106cell/ml were used as initial concentration of bacteria. Through the viscosity decay profiles with bioassay-time, the relative quantitative gelatinase efficiency of bacteria has been evaluated. The influence of the temperature has also been determined on the biodegradation of gelatine by the studied bacteria. A synergistic effect in the biodegradation of gelatine was observed with the natural mixture of B. amyloliquefaciens and B. subtilis present in the cinematographic polymer sample. 1. Introduction A photographic film is composed of three generic components: a plastic support, an image-forming material (black and white images are formed by metallic silver particles and colour images are made of colour dyes) and a binder that is commonly based on gelatine. The last two components are the main materials of the photographic emulsion layer. Along the cinematographic history, several supports have been used to manufacture professional motion-picture films: cellulose nitrate (from 1889 to 1950), cellulose triacetate (from 1948 to 2000) and polyethylene tereftalate (from 1990´s to nowadays). Hence, cellulose triacetate represent the bulk of the film collections today. It is possible to estimate the existence of approximately 1.5 million roll-cans in Spain, including the total of private (laboratories and cinematographic industries) and public archives (national and regional). Filmoteca Española has the responsibility for the conservation and preservation of 430.000 roll-cans that constitute an important and irreplaceable historic and cultural heritage. The image stability depends on intrinsic (e.g. nature of the polymer base) and extrinsic (e.g. environmental conditions) factors. Archivists have faced the film deterioration problem caused by inappropriate storage conditions of temperature and moisture. Many efforts have focussed on the decrease of the physical damage caused by the “vinegar syndrome” in cellulose acetate-based films [1] and the fading of colour dyes. In contrast, there is a lack of knowledge on the contamination by bacteria and fungi in the cinematographic archives and its influence in the film stability. In the cinematographic films the organic components mentioned before can be considered as potential carbon source for the growth of microorganisms if the environmental conditions are adequate.

Bacteria present in cinematographic films stored in spanish archives 3

A large number of studies have been carried out on the biodegradation of cellulose acetates (CA) by both, bacteria and fungi. Results showed that the hydroxyl substitution degree (acetylation) was a factor with a high influence on biodegradation, being those CA with higher degree of acetylation more resistant to microbial attack [2,3,4]. CA biodegradation is mediated by the cooperative action of esterase(s), lipase(s) and cellulase(s). These studies were carried out adding pure cultures of a determinate species or, by the contrary, using mixed populations such as those included in compost [5]. The cellulose triacetate used in the photographic industry has a substitution degree of approximately 2.7. Hence, its resistance to biodegradation should be much higher than the other components of the film. In fact, it has been reported [6] that microorganisms attack the organic part of the film emulsion especially at relative humidity above 60%. In the tropical climates fungal growth is a special problem, but also it is possible to detect these troublesome growths during the summer in temperate zones with high relative humidity. Gelatine, the binder of the photographic emulsion, is the most biodegradable substrate. It is composed of high molecular weight polypeptides derived from collagen, and the most widely used photographic gelatine is type-B, obtained by basic pre-treatment of the cattle bones [7]. Gelatine for cinematographic applications requires strict specifications of purity and high bloom values are required. The latter is one of the most important properties of the gelatine and is a measure of the gel strength resistance which is determined in a bloom gelometer [8]. Commercial gelatines vary from 50 to 300g Bloom (force in grams required to depress an standard plunger 4mm into the gel prepared from a 6.67% aqueous solution of gelatine and cooled at 10ºC). The higher bloom gelatines are the less degraded and colourless materials obtained in the manufacturing process and are first extracted at lower temperatures. The bloom value or gel strength is a measure of the gelatine quality, hence, in the degradation process the viscosity and the bloom values decrease. Many prokaryotic and eukaryotic microorganisms exhibit proteolytic capacity, so they are able to degrade gelatine. Stickley [9] from Kodak photographic industry, studied by viscosimetry the biodegradation of gelatine 5% aqueous solutions at 37º C using a strain of the genus Bacillus and one of Pseudomonas. In both cases, decay in the viscosity Bloom values was detected after 24h. Studies about microbial contamination of cinematographic films stored in archives are scant and have been reviewed by Abrusci et al [10]. The main work was made by Opela [11] and involved the analysis of cellular concentration and the identification of the principal fungal contaminants in the air, floors, cans and cinematographic films in two different archives of the Slovak Republic. Like in other studies of indoor habitats, the results indicated that certain environmental conditions (air flow, temperature, personnel

C. Abrusci et al. 4

movement, etc.) were crucial for the microbial concentration values found in the diverse places analyzed. Besides, fungal biodiversity was higher in the air than in the surface of the films. Fungal biodiversity was limited in archives, with Penicillium spp. and Aspergillus spp. as predominant in indoor air and buildings from cold and temperate climate regions [12, 13]. The presence of spores and/or vegetative cells of microorganisms on the surface of materials indicate the possibility of a biodegradation or biodeterioration process in future. However, colonization or microbial growth on a material involves almost always biodeterioration phenomena. There are different ways in which microorganisms can hamper the structure and function of polymeric materials [14]. For instance, they can produce pigmentation or a degradation of one or more compounds. In other cases, microbial growth can produce hydration or corrosion of materials. Finally, microorganisms can produce fouling or penetrate in the polymeric material. To produce any of the above mechanisms of biodeterioration, microorganisms must exhibit an active metabolism and growth. Although the environment contains an extremely rich variety of microorganisms, it is quite usual that only a few of the isolated microorganisms from a substrate are responsible for damaging it. Due to the potential risk of cinematographic film biodeterioration in the Spanish archives, we carried out research to isolate and identify bacteria and fungi from selected samples of black and white cinematographic films collected in different archives [15]. In this paper the obtained results in these research works are reviewed including analysis of the ability of these isolates to hydrolyse the gelatinous component of cinematographic films [16]. 2. Experimental part 2.1 Cinematographic film samples and gelatine Film samples were supplied by Filmoteca Española and consisted of collections performed at Barcelona, Madrid and Gran Canaria archives. Apparently, pictures of the supplied films were in good condition. Gelatine used in this work was supplied by Aldrich Chemicals and used as received. The commercial product of type B gelatine was characterized by a Bloom or gel strength value of 225g. 2.2. Isolation and characterization of bacteria from cinematographic films The microorganism isolation procedure was the same for all samples. Manipulations were made in a sterile environment using a laminar- flow chamber. Manual operations were carried out using aseptic gloves to avoid external microbial contaminations, in particular, with microorganisms of human skin microbiota. From each cinematographic sample, two or three

Bacteria present in cinematographic films stored in spanish archives 5

fragments were placed in a Petri dish containing an adequate general culture medium. The media used were TSA (trypticase-soya-agar). Petri dishes were incubated at 30ºC for one or two weeks. Cultures were observed daily under a stereoscopic microscope to check the presence of bacterial colonies. Pictures of the microbial growth are shown in figure 1.

Figure 1. One-week Petri dish culture in TSA at 30oC showing on left bacteria growth and on right Bacillus colonization on a fragment of film. These experiments were repeated at least five times per sample. As a result of this procedure 14 strains of heterotrophic bacteria were isolated from the film samples. Each isolate was cultured on TSA either at 30ºC during 48hours. Replicates were made and preserved at 4ºC or –80ºC using TSB liquid medium supplemented with glycerol. The code for strains of bacteria used in this paper consists of an initial letter that indicates the location from which the sample comes i.e. B-Barcelona, M-Madrid, GC-Gran Canaria. The strain codes and origins are compiled in Table 1. Gram and simple staining were performed using exponential cultures in TSA of each bacterial strain. Besides, both oxidase and catalase test were done. Acid production from glucose and saccharose on OF basal medium, growth in Simmons citrate agar and starch and gelatine hydrolysis were tested. In all Gram-positive and some Gram negative bacterial strains, the ability to form spores was tested using 24, 48, 72 hours and one week cultures on TSA as well as the Schaeffer-Fulton specific stain [17]. Morphological observations were made with and optical microscope, Zeiss Laboval 4 equipped with differential interference contrast (Nomarski) and phase contrast. After the preliminary morphological and physiological characterization previously used by other authors [18], bacterial strains were identified by commercial identification kits. Depending on the bacterial nature, different commercial assays were employed such us API 20 NE, API 20E, API 50 CHB and API STAPH. The previous preparation and the inoculation procedures were done following the recommendations of the manufacturer (BioMerieux

C. Abrusci et al. 6

España S.A.). The strains were incubated at 30ºC for 24 hours. The numeric profiles obtained were compared with a bacterial database using the commercial software APILAB from BioMerieux. Simultaneously, additional diagnostic tests were made with some bacterial strains, such as growth on McConkey or mannitol salt agar (composition or reference) and coagulase and lysostaphin tests. 2.3. Viscosity measurements Water solution viscosity, often determined at 6.67% (w/v) is a widely characteristic property of the gelatine used in the industry as an estimation of the relative molecular weight [19]. In this work we have measured the viscosity of the gelatines at 37ºC in unbuffered water solution because these conditions favor the microbial grow and are interesting in the application point of view in order to evaluate the biodegradation of gelatines. To measure the viscosity an Ubbelohde micro-viscometer (filling capacity of about 2.5ml) from Schott, having a 0.40 mm capillary diameter was used. The instrument constant of the employed viscometer was K= 0.01mm2/s2, being suitable for the measurements of viscosities ranging from 0.4 to 6cP. The kinematic viscosity (ν) of the gelatine solutions in centipoises (cP) can be calculated using the instrument constant by the equation: ( ) tKcP ⋅=ν [1]

were K is the instrument constant and t is the flow time in seconds which was corrected when necessary according to the manufacturer of the viscometer and following DIN 51 562. The viscometer was conveniently cleaned with suitable solvents, oven dried and treated in an autoclave (120ºC- 30 min.) after each run to avoid contaminations. Solution viscosity is usually measured by comparing the falling times (t -polymer solution, t0-solvent) in the viscometer. These efflux times are proportional to the viscosities of the polymer solution (η) and solvent (η0) respectively. With these values it is possible to obtain, the relative viscosity (ηrel = η/η0), the specific viscosity (ηsp) and reduced viscosity (ηred = ηsp/c), were the polymer concentration, c, is expressed in grams per deciliter, g/dL. To make viscosity independent of the concentration, the intrinsic viscosity is used and calculated by extrapolation to c = 0, but is a function of the solvent. The expressions of such viscosities are shown in the following equations.

10

0

0

0 −=−

=−

= relsp ttt

ηηηη

η [2]

Bacteria present in cinematographic films stored in spanish archives 7

[ ] ⎟⎟⎠

⎞⎜⎜⎝

⎛=

→ csp

c

ηη

0lim [3]

Generally, viscosity data as a function of the concentration are extrapolated to infinite dilution by the well known Huggins [20], equation [4].

[ ] [ ]( )ckc hsp ηηη += 1/ [4] In order to avoid the measure of viscosity at different concentrations to calculate the intrinsic viscosity various single point methods have been used [21]. These methods are extremely useful for the evaluation of the viscosity decay with degradation and in this work we have used the semi-empirical equation of Shulz y Blaschke [22] (S-B), equation [5].

[ ] ( )spSBsp

kcη

ηη

+=

1 [5]

In an earlier paper we demonstrated6 the good fit between the intrinsic viscosities obtained by extrapolation for commercial gelatines and those obtained by the equation of S-B using the value of kSB constant for a dilute solution 0.28. In the biodegradation experiments the viscosity changes will be also determined by this equation. Also under our experimental conditions, the equation [6] determined by Pourardier and Venet [23] can be applied to estimate the molecular weigh average number (Mn). In the pioneering works of these authors, molecular weight fractions from Mn = 46.000 to 207.000 of photographic gelatines (Kodak F-14) determined by osmotic pressure measurements were correlated with intrinsic viscosity of undegraded fraction of gelatines and degraded gelatine mixtures (at pH 4.75) using the same relationship [6].

[ ] [ ] 885.051066.1 nMx −=η [6] The application of the Pourardier and Venet relationship to the [η] values determined by the S-B equation at the gelatine concentration of 6.67% (w(w), gives molecular weigh average number (Mn) of 77.260 for the gelatine of Bloom 225 employed in this work. Another useful parameter frequently used to evaluate the degradation of a polymer is the well known number of main chain scission (S), which can be calculated by equation [7]. This variable can be useful to compare the biodegradation activities of the fungi.

C. Abrusci et al. 8

[ ][ ]

1

0 01 1αη

η⎛ ⎞

= − ≈ −⎜ ⎟⎜ ⎟⎝ ⎠

n

nt t

MS

M [7]

Where the sub-indexes, 0 and t, indicates the “initial” and “at time t” respectively of the Mn and [η]. The value of the exponent α is 0.885 from equation [6]. 2.4. Bioassay procedure In order to assess the rate and the extent of biodegradability of gelatine in solutions by bacteria, aerobic bioassays were conducted at 37ºC in water solution of gelatine 6.67 % (w/w). Hence, a 50ml graduate flask was filled with the corresponding gelatine weight (3.335g) and approx. 40 ml of bi-distilled and deionised water (MiliQ), the mixture was shacked in a thermostatised bath at 37ºC until complete solution of the gelatine (about 1hour). After that, 5ml of bacteria inoculums were added to the flask. After this addition, water at 37ºC was added until to make level in the flask. Under such conditions the gelatine solutions contain a concentration of 25x106cell/ml. The bacteria suspensions for inoculums were prepared previously fixing an absorbance of 0.2 at 550nm in saline buffer (0.9g/L). All of these solutions contained about 25x107cell/ml. The microbial populations were estimated using the standard plate-count technique. These values of cells per millilitre are in agreement with the average concentration of McFarland turbidimetric standard [24, 25] for this absorbance. The absorbances of the different bacteria suspensions prepared in this work for inoculums are shown in table 1. From this bioassay solution and at different intervals of time (0, 12, 24... hours), a volume of 2.5ml was taken each time and filled in the viscometer in order to determine de viscosity of the gelatine. This procedure enabled the change of gelatine viscosity with time to be monitored in a reproducible way since all the experiments were repeated and the results were coincident. In the case of the bioassays at lower temperatures, (30, 20 and 4ºC), gelatines are not dissolved and a swelling hydrogel filled the flask. Hence, for these experiments, identical solutions were used but placed in graduated flasks of 5ml. At the corresponding test-time, and previously to the measurement of the viscosity, the gelatine hydrogel was dissolved again shaking the flask for 5 minutes at 60ºC. Afterwards, the solutions were thermostated at 37ºC and the viscosity for each run was measured. 3. Results and discussion 3.1. Characterization and identification of the bacterial isolates Fourteen bacterial strains have been isolated from cinematographic films stored in three different archives of Filmoteca Española. Most of the isolates

Bacteria present in cinematographic films stored in spanish archives 9

were Gram-positive bacilli and cocci. The rod-like bacteria exhibited motility and were oxidase- and catalase-positive. All were exopolimer producer strains. Gram-positive cocci are represented by six different strains, which depending on the isolate present a cellular arrangement in chains (B2 and B4C), tetrads (M4), which produces a yellow pigment, or individual cells (B5, GC1B and M3). None of the spherical bacteria exhibit mobility and all were catalase-positive. Only two strains of Gram-negative rods have been isolated and they were oxidase-catalase-positive, and they were identified as P. haemolytica and S. paucimobilis Results from bacterial identification are shown in Table 1.

Table 1. Identification of bacterial strains present in cinematographic films.

Strain Identification % Archive Base B3BA B. amyloliquefaciens 88,1 Barcelona CTA

B7 B. megaterium 99,9 Barcelona CN

GC2 B. megaterium 99,9 Gran Canaria CTA

M2 B. pichinotyi 99,0 Madrid CTA

M5 B. pumilus 99,4 Madrid CTA

B3BS B. subtilis 86,2 Barcelona CTA

M4 K. kristinae 99,1 Madrid CTA

GC1A P. haemolytica 86,5 Gran Canaria CTA

B4A S. paucimobilis 99,2 Barcelona CTA

B2 S. epidermidis 97,3 Barcelona CTA

M3 S. haemolyticus 90,1 Madrid CTA

B4C S. hominis 98,0 Barcelona CTA

B5 S. lentus 99,9 Barcelona CN

GC1B S. lugdunensis 98,0 Gran Canaria CTA CTA: Cellulose triacetate; CN: Cellulose Nitrate

The Gram-positive bacilli were identified as four different species of the genus Bacillus. In fact, they were in general sporulated bacteria and cells were disposed in the typical long chains of this genus. Special mention must be given to strain M2, isolated from the Madrid archive, which corresponds to B. pichinotyi (AF 519460). This species was first isolated from tropical rice soils [26]. However, our strain has at least two differential features with respect to other Bacillus spp. Thus, the results from Gram staining were always positive, and endospore formation was not observed, being the latest one of the most typical characteristics of the genus.

C. Abrusci et al. 10

Two strains of B. megaterium were characterized; one from Gran Canaria (Canary Islands) and one from Barcelona. Both isolates produce abundant exopolymers and poly-β-hydroxybutyrate granules. Cocci have been identified as members of the genus Staphylococcus, with the exception of the pigmented strain M4 which corresponds to the species Kocuria kristinae. Finally, Gram-negative bacilli were represented by two species with quite different features, P. haemolytica and S. paucimobilis. The analysis of the main habitats and the morphological/physiological characteristics of the bacterial isolates are relevant in order to explain the potential causes that determine the bacterial contamination of the cinematographic films. Members of the genus Staphylococcus are widespread in nature. Due to their ubiquity and adaptability, they are a major group of bacteria inhabiting the skin, skin glands and mucous membranes of humans, other mammals and birds [27]. These bacteria are especially resistant to desiccation. Besides, at least one of the cocci found in the cinematographic films produced slime [28]. S. epidermidis and S. hominis are two of the most prevalent and persistent bacteria infecting the human skin [29, 30]. S. haemolyticus shares the same habitats of S. hominis, but it is usually found in smaller populations. It has been occasionally associated with mastitis in cattle [31]. S. lentus has been isolated in large populations from domestic sheeps and goats [32] and occasionally from other animals in farms [33]. Finally, the main habitat of S. lugdunensis is the human nasal cavity where S. haemolyticus can be also found [34]. It is known that most environmental sources contain transient and small populations of staphylococci, many of them are probably contaminants disseminated by humans or animals. Mammalian skin is considered the primary habitat of K. kristinae and the related micrococci, but this species has been also isolated in food industries, specially from fermented sausages and Spanish dry-cured ham [35]. Regarding the Gram-negative bacilli, the most interesting species is S. paucimobilis. This microorganism is widespread in nature due to its physiological and metabolic versatility [36]. It can degrade diverse recalcitrant or xenobiotic aromatic hydrocarbons, such us, phenanthrene, anthracene and fluoronanthrene [[37]. Besides, most of strains (included the isolates of this work) produce an extracellular polysaccharide known as gellan gum, using diverse substrates as nutrient sources [38, 39]. The gellan gum production has a commercial interest in the biotechnological industry. Unfortunately, these exopolysaccharides are involved in biofilm formation, which has been associated with biodeterioration processes and nosocomial infections [40]. The other Gram-negative bacterium isolated from cinematographic films, P. haemolytica causes the bovine pneumonic mannheimiosis, a common and

Bacteria present in cinematographic films stored in spanish archives 11

economically important disease of cattle, especially from North America [41, 42]. Finally, the species included in the genus Bacillus are ubiquitous, due to their wide range of physiological characteristics and the ability to degrade mostly all substrates [43]. Besides, the majority of them are endospore-forming, being these resting forms usually very resistant to environmental extreme conditions, antibiotics, disinfectants and other chemicals [44]. Furthermore, the spores can be easily disseminated by air, dust, pollen and diverse animals. In the photographic film manufacturing process, contamination by gelatine-degrading bacteria of the genera Bacillus and Pseudomonas has been reported by Stickley [9]. More recently, it has been demonstrated that many bacteria can colonize gelatine during its production process. This substrate is a protein with many industrial applications, including the manufacture of cinematographic films. Most of bacteria isolated in this study belong to the genus Bacillus and other related endospore-forming genera. Besides, non sporulated bacterial strains identified as diverse species into the genera Salmonella, Kluyvera, Burkholderia, Pseudomonas, Yersinia, Brevundimonas, Enterococcus, Staphylococcus and Streptococcus were found [45]. All of these isolates showed the ability to liquefy gelatine. Later, De Clerck et al. [46] evaluated the bacterial contamination of semi-final gelatine extracts from several production plants. In this case, almost all bacteria were included into the genus Bacillus and other related endospore-forming genera (Brevibacillus, Geobacillus and Anoxybacillus). However, a strain of Enterobacter sakazakii and four species members of the genus Staphylococcus; S. epidermidis, S.lugdunensis and S.hominis, had been also identified. Authors concluded that al least some spores or vegetative cells of these bacteria were able to resist the final UHT treatment used to prevent the potential microbial contamination during the industrial process of gelatine manufacture. Finally, other important aspect is the presence of bacteria in indoor environments, such as rooms, stores or archives. It is known that vegetative cells or spores of endospore-forming bacteria are common contaminants present in industrial processes and hospitals [47, 48]. Besides, they are frequent inhabitants of indoor environments. In these habitats, other genera of bacteria, such as Kocuria, Micrococcus, Staphylococcus, Aeromonas and those members of Pseudomonadaceae have been isolated most frequently, at least in Central and Eastern European countries [49]. From all the ecological / physiological considerations above, we can conclude that in the case of cinematographic films the bacterial contamination might have three potential origins. First, some cinematographic films could be contaminated with vegetative cells and especially with spores during their manufacture. Besides, the species Pasteurella haemolytica can infect cattle. Second, diverse species (Bacillus spp., Staphylococcus spp.) can survive the gelatine industrial fabrication process, so microorganisms appear in semi-final

C. Abrusci et al. 12

gelatine extracts. In this work, several species which are common components of the nasal cavity and skin microbiota of humans have been found. In this case, bacterial contamination might be consequence of manipulation during the elaboration of film copies or during the maintenance of cinematographic films in the archives. Finally, a rather probable way is through spores or vegetative cells suspended in the air present during the storage in the archives, since most of the bacterial species that have been found as contaminants of cinematographic films are commonly present in indoor environments. With the strains of bacteria isolated from cinematographic films, the biodegradation of gelatine in solution was studied. In the next sections the corresponding results are presented. 3.2. Biodegradation of gelatine in solution by bacteria The reduction of gelatine molecular weight of by hydrolysis is slow [50, 51] at neutral pH (pH values between 5 and 7). In our media, at 6.67% (w/w) solution of gelatine in the absence of buffer, the pH was found to be 5.65. In the time scale and conditions of the bioassay experiments carried out in this work, the viscosity remained constant in the absence of bacteria inoculums under sterilised conditions. In a recent study, the hydrolytic degradation of gelatines at elevated temperatures [52] has been studied and the influence of different variables in the degradation has been determined. In figure 2, the evolution of kinematic viscosities (ν in centipoises) with time of bacterial biodegradation is shown. In all the experiments, the initial cell-concentration was maintained constant at 25x106 cell /ml approximately as described before. From the fourteen strains samples isolated from the cinematographic films and studied in this work, that correspond to fourteen bacteria (table 1), only six of them were efficient in the biodegradation of gelatine in solution. These active bacteria in agreement with those that were positive-efficient in the gelatinase activity assay in were a tube-test containing gelatine-agar. The active bacteria exhibited different rates of biodegradation at 37ºC confirming their different physiological characteristics. In the initial 12-24 hours there was a low growth rate of the bacteria depending on the species and this is in accordance with the existence of a lag phase when inoculums are placed into a fresh medium under batch conditions. This lag phase is due to the physiological adaptation of the cell to the culture conditions. After this period of time, a second evolution of fast bacterial growth was observed in the batch system. Hence, a drastic decrease of viscosity was measured after 12-24 hours from the inoculums. This indicates that there is an exponential phase of growth taking place. The Bacteria studied in figure 1 at 37ºC differ in the time-length and viscosity-decay of both phases. The corresponding results of chain scission (S) calculated by using equation [7] presented in figure 3.

Bacteria present in cinematographic films stored in spanish archives 13

Figure 2. Viscosity decay (in centipoises) of photographic gelatine with biodegradation time at 37ºC by the gelatinase-active bacteria isolated form cinematographic films.

Figure 3. Chain scission evolution with the biodegradation time of photographic gelatine solution at 37ºC. The evolution of main chain scission shows, with increasing values, the previously observed lag and exponential phases. The increase rate of of cells in the culture is proportional to the chain scission number calculated from the viscosity data. It is interesting to remark on the good reproducibility obtained in the viscosity measurements. This can be illustrated by the case of Bacillus megaterium, which was isolated and identified in two separate cinematographic samples (form Barcelona, B7 and Gran Canaria GC2). Both strains exhibited identical behaviour in the viscosity decay of gelatine.

C. Abrusci et al. 14

From the obtained viscosity data different parameters can be used to compare the biodegradation rates with the bacteria. Hence the rates of biodegradation at different bioassay times at 37ºC are compiled in table 2. Table 2. Biodegradation rates of photographic gelatine by bacteria (37ºC) at different bioassay times.

Strain code Bacterium

ts=0.24 (h)

R48/72h (S·h-1)

R72h (S/h-1)

B3BA B. amyloliquefaciens 33 0.023 0.026

B3BS B. subtilis 22 0.043 0.053

B4C S. hominis 52 0.011 0.020

B7 B. megaterium 33 0.016 0.015

M2 B. pichinotyi 48 0.026 0.047

M5 B. pumilus 63 0.004 0.005

GC2 B. megaterium 33 0.017 0.019

Trypsin S=7.90 (2.5 hours) (a)

Pepsin Enzymatic degradation in optimal

conditions. S= 0.16 (19 hours) (a) (a) Calculated values from the bibliographic data, A.Courts, Biochem.J., 58 (1955) 74 The order in the biodegradation efficiency at 37ºC after 72hours of bioassay was as follow: Bacteria (R72h in S·h-1): B. subtilis (0.053) > B. pichinotyi (0.047> B. amyloliquefaciens (0.026) > S. hominis (0.015-20) ≈ B. megaterium (0.019) > B. pumilus (0.005) The rates of the biodegradation are physiologically dependent on the nature of the bacteria. In all cases the biodegradation rates were much lower than that found with the enzymatic biodegradation under optimum conditions. With photographic gelatine type-B at 37ºC trypsin was one of the most effective proteolytic enzymes and pepsin was much less efficient. The gelatinase active bacteria isolated in this work were much less efficient than the enzymatic biodegradation with these enzymes as the calculated values of chain scission, shown in table 3, put in evidence. The behaviour of the bacteria is also different in the latency phase as confirmed by the biodegradation rate in the first 24 hours. 3.3. Temperature effect in the biodegradation of photographic gelatine From the point of view of the gelatine bio-stability the effect of temperature is an important matter. Numerous publications1, some of them

Bacteria present in cinematographic films stored in spanish archives 15

promoted by the International Film Archives Federation (FIAF), recommend different conservation temperatures for de rolls of films. The realistic interval for cellulose supports ranges from 4 to 21ºC. Hence, in this work we have studied the biodegradation of gelatine by bacteria in this interval of film conservation. At temperatures lower than 35ºC the gelatine solution becomes a swelled network and the bioassay takes place in a hydrogel state. The influence of the temperature has been studied carring out the bioassay experiments at 30, 20 and 4ºC. The viscosity decays in the biodegradation experiments at 30 and 20ºC are shown in figures 4 and 5.

Figure 4. Viscosity decay of photographic gelatine with biodegradation time at 30ºC.

Figure 5. Viscosity decay of photographic gelatine with biodegradation time at 40ºC.

C. Abrusci et al. 16

From the figures it is possible, for almost all the bacteria studied in this paper, to observe a significant change in the biodegradation of gelatine with temperature. For comparison, the rates of biodegradation in the interval of assay-time 48-72 hours at 37, 30 and 20ºC are summarised in table 3. Table 3. Rates of biodegradation of photographic gelatine at 37, 30 and 20ºC in the bioassay-time interval 48-72hours.

Strain code Bacterium

R48/72h 37ºC (S·h-1)

R48/72h 30ºC (S·h-1)

R48/72h 20ºC (S·h-1)

B3BA B. amyloliquefaciens 0.023 0.038 0.023 B3BS B. subtilis 0.043 0.043 0.043 B4C S. hominis 0.011 0.0003 - B7 B. megaterium 0.016 0.016 0.013 M2 B. pichinotyi 0.026 0.033 0.008 M5 B. pumilus 0.042 0.001 0.0004 GC2 B. megaterium 0.017 0.015 0.012

Except for B. subtilis and B. megaterium that exhibited a constant biodegradation rate in the studied interval of temperatures, the rest of bacteria shown optimum biodegradation rate at temperatures of 37ºC (S. hominis, B. pumilus) and 30ºC (B. amyloliquefaciens, B. pichinotyi). At 20ºC these bacteria decrease their biodegradation rates. The obtained results, viscosity decay and calculated chain scission with bioassay time at 4ºC are plotted in figures 6 and 7.

Figure 6. Viscosity decay of photographic gelatine with biodegradation time at 4ºC.

Bacteria present in cinematographic films stored in spanish archives 17

Figure 7. Chain scission of photographic gelatine with the biodegradation time at 4ºC. At this low temperature, only four bacteria were effective in the biodegradation of 225 Bloom gelatine, all of them from the genus Bacillus. The bioassay-time scale was three months in this case (days) since a drastic decrease in activity has been observed at this temperature. Except for B. subtilis, an important inhibition of the biodegradation can be observed confirming a large lag phase in the growth of these bacteria. These results are in agreement with the well known fact that the some species from the genus Bacillus can growth at temperatures as high as 75ºC or as low as -5ºC. Also, they can flourish at extremes of acidity and alkalinity, ranging from pH 2 to 10. Their resistance to the extreme conditions makes them extremely dangerous for the preservation of gelatine materials for photographic films. 3.4. Synergistic effect of Bacillus in the biodegradation of photographic gelatine From the photographic films, a natural mixture of bacteria was found in the sample B3. Initially this bacterial contamination was considered as a single bacterium, Bacillus sp., but after the morphological and biochemical analysis two different bacteria were identified: B. amyloliquefaciens (B3BA) and B. subtilis (B3BS). The separate evaluation of their biodegradation efficiencies and the activity of a mixture allows a study on the synergistic effect of these microorganisms. In Figure 8 and 9, the viscosity decays of gelatine with time at 37ºC and 4ºC respectively are shown.

C. Abrusci et al. 18

Figure 8. Synergistic effect on the viscosity decay of photographic gelatine with biodegradation time at 37ºC by the mixture of B. amyloliquefaciens and B. subtilis.

Figure 9. Synergistic effect on the viscosity decay of photographic gelatine with biodegradation time at 4ºC by the mixture of B. amylolicuefaciens and B. subtilis The mixture of these bacteria was the most active in the biodegradation of gelatine. The biodegradation activity of the mixture was greater than would be expected from the additivity law of the separate viscosity decay efficiencies. This synergistic effect supports the theory that the combination of their enzymes gives a better result in the overall gelatinase activity. The synergism effect was observed at 37 and 4ºC confirming that this behaviour takes place in

Bacteria present in cinematographic films stored in spanish archives 19

the overall interval of temperatures. At 4ºC (figure 9), the synergistic effect is more pronounced than that observed at 37ºC (figure 8) even though the relative biodegradation rates are very different and, in absolute values, much faster at 37ºC. Their complementary metabolisms could be the reason for these Bacillus species to share the habitat in the photographic films. 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. Most microorganisms that we isolated can resist environmental conditions adverse to microbial life, e.g. desiccation or nutrient starvation, and they can adhere to the substrates. The viscosimetry of gelatine solutions inoculated with bacteria and its evolution with time can be used as a quantitative method to evaluate their gelatinase activity and the biodegradation of the material. Also, the bioassay-temperature dependence allows one to establish the influence of this important variable in the biodegradation and subsequently to gain information on the interval of metabolic efficiency of the bacteria and, hence, different temperatures have been employed. Four species of Bacillus are still active at 4ºC, the biodegradation takes place in a one month time-scale. Also, a natural mixture of Bacillus (B.amyloliquefaciens and B.subtilis) present in the photographic films showed a synergistic effect in the biodegradation of gelatines. The obtained results put in evidence the risk of biodeterioration, even at low temperatures i.e. 4ºC) if the contaminated cinematographic films are stored in inadequate conditions or are exposed to elevated environmental relative humidity.

Acknowledgement The authors thank the Spanish Ministerio de Educación y Ciencia (MAT 2003-0119), Filmoteca Española and Fotofilm (inside the Consortium for Scientific Collaboration: CSIC-UCM-FE-FOTOFILM) for their financial support.

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