Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 14-Day Trial for You or Your Team.

Learn More →

Cellulolytic potential and filter paper activity of fungi isolated from ancients manuscripts from the Medina of Fez

Cellulolytic potential and filter paper activity of fungi isolated from ancients manuscripts from... Ann Microbiol (2014) 64:815–822 DOI 10.1007/s13213-013-0718-6 ORIGINAL ARTICLE Cellulolytic potential and filter paper activity of fungi isolated from ancients manuscripts from the Medina of Fez Fatimazahra El Bergadi & Faouzi Laachari & Soumya Elabed & Iraqui Houssaini Mohammed & Saad Koraichi Ibnsouda Received: 1 May 2013 /Accepted: 17 September 2013 /Published online: 18 October 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract The content of libraries in Fez is an important Introduction historical testimony and a treasure of knowledge of several civilizations. Unfortunately, this cultural heritage has suffered Morocco, crossroads of several civilizations, has precious man- deterioration caused by many factors, including microbial uscripts. These documents play a major role in the field of deterioration specifically by cellulolytic fungi. The aim of scientific and historical research. One of the Moroccan cities our study was the identification and characterization of the most characterized by the wealth of manuscripts is the city of microflora that damage historical manuscripts books from an Fez. Historically, Fez has been known as a home to the oldest old library of the Medina of Fez. A total of 31 filamentous university in the world, "al qarawiyine university" (Saoudi fungi were isolated from deteriorated paper of ancient books. 2008). However, much of this valuable cultural heritage that Nine of these isolates were screened positive for the ability to has been accumulated over time is today in an advanced state of degrade carboxymethylcellulose (CMC). The ability of the deterioration due to poor storage conditions. Indeed, all of these nine isolates to produce filter paper activity (FPUase) in liquid manuscripts suffer from various destructive agents especially media as well as the effect of temperature and pH were also from microbial degradation that causes different kinds of dam- studied. According to the molecular identification, the most age depending on the organisms responsible for the attack. The frequent species were Penicillium chrysogenum, Aspergillus biodeterioration of library materials surpasses 110 years (Zyska niger, Aspergillus oryzae and Mucor racemosus, and other less 1993), but the first research, which discuses microbial alteration frequent, such as Hypocrea lixii, Aspergillus melleus and of papers appears in 1917 (See 1919). Schizophyllum commune were also present. Also, the effect of Fungi are considered as serious degrading agents of paper fungal contamination in paper artificially attacked for 18 months manuscripts, particularly cellulolytic fungi (Fabbri et al. 1997). at 25 °C was examined. The fungal strains were able to degrade Some filamentous fungi frequently colonize paper, and are able filter paper to varying degrees. to degrade cellulose fibers through the action of cellulolytic enzymes (Reese and Downing 1951; Nyuksha 1983;Ciferri et al. 2000), or to alter aesthetic and visual appeal by releasing . . . Keywords Fez Filter Paper Activity Biodeterioration weak acids or pigments; a phenomenon referred as foxing (Arai Cellulose Fungi 2000; Montemartini et al. 2003;Zotti et al. 2011). Cellulose from native wood is used for paper production. In addition to cellulose fibers, paper can contain hemicelluloses (wood polyoses), lignin, and additives such as fillers and pig- : : : : F. El Bergadi F. Laachari S. Elabed I. H. Mohammed ments (Fellers and Wegener 1991). Some filamentous fungi S. K. Ibnsouda (*) frequently associated with paper degradation are capable of Laboratoire de Biotechnologie Microbienne, Faculté des Sciences et dissolving cellulose fibres through the synergistic actions of Techniques de Fès, Université Sidi Mohamed Ben Abdellah, endoglucanase (EC 3.2.1.4), exoglucanase or cellobiohydrolase B.P. 2202 – Route d’Imouzzer, Fès-Maroc, Morocco e-mail: ibnsouda@hotmail.com (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21) (Lynd et al. 2002; Zhang and Lynd 2004), causing serious damage to paper S. Elabed S. K. Ibnsouda materials of cultural and historical importance (Reese and Centre Universitaire Régional d’Interface, Université Sidi Mohamed Downing 1951; Nyuksha 1983; Ciferri et al. 2000). In this case, Ben Abdellah, Fès-Maroc, Morocco 816 Ann Microbiol (2014) 64:815–822 Porck (2000) reported that the hydrolytic degradation of the Isolation, culturing and incubation cellulose molecules is the most frequent reaction, particularly when the presence of water in the storage environment plays an For sampling, three methods were used. (1) The samples essential role. In fact, microbial activity depends largely on the were swabbed from the historical books. (2) Other sam- water activity (aw) of the paper. It is a measure of free water ples were scraped with a scalpel, then all of them were which is available for the growth of the mould and is defined as pre-incubated in lysogeny broth (LB) supplemented by the ratio of the vapour pressure of the water in the substrate chloramphenicol (500 mg/l) (Rojas et al. 2009). (3) The and the vapour pressure of pure water at the same tempera- collected fragments of deteriorated paper were introduced ture and relative humidity (RH) (Arai 1987). According to directly in the same medium for 24 h at 25 °C. After, the standard reaction, the rate of the hydrolytic process is deter- supernatants from each sample were serially diluted in mined by the combined effects of aw, the temperature, the sterile distilled water up to 10–4 and cultivated using pH and the relative humidity. At a RH level higher than different media: MEA (4 % malt extract, 1.8 % agar), 65 %, and a temperature higher than 20 °C, moisture content LB agar (1 % peptone, 1 % NaCl, 0.5 % malt extract, of papercan reach8to10%,withconsequentwater activity 1.8 % agar), and YPG antibiotics agar (1 % yeast extract, (aw) higher than 0.65, microbial spores can germinate and 2 % glucose, 2 % peptone, 1.8 % agar, ampicillin 60 μg/ develop by using the paper as a substrate of growth, conse- ml and kanamycine 30 μg/ml). The dishes were then quently affecting the object of cultural value (Kowalik 1980). incubated at 25 °C for 24 hours. The strains were isolated Furthermore, Prieto and Silva (2005) reported that the inten- and purified after streaking several times. sity of the microbial contamination is influenced by the climatic conditions of the atmosphere. Screening of hydrolytic activity The fungal contamination of Moroccan manuscripts stored in archives is therefore problematic because of the imperfect Pure isolated cultures of fungal mycelia were screened for knowledge of the deteriorative agents. Also, many efforts their cellulase activity; for this, they were transformed have focused on the preservation and maintenance of this individually on CMC agar plates containing (NaNO - written cultural heritage. For adequate conservation of these 3.0 g/l, K HPO -1.0 g/l, MgSO ·7H O-0.5 g/l, KCl- 2 4 4 2 archive collections, it is important to identify and characterize 0.5 g/l, FeSO ·7H O-0.01 g/l, CMC-1.0 g/l, Agar- 20 g/ 4 2 the fungal species for their control. l, pH 5.0) and then incubated at 25 °C for 72 hours. After This investigation was done to (1) assess the role of incubation the plates were stained with 1 % Congo Red fungi in the degradation of manuscripts in librairies from solution for 15 min, and afterwards the stain was neutral- the city of Fez, (2) identify fungi isolated from the ized with 1 M NaCl solution. The appearance of a decayed paper by molecular methods, and (3) evaluate discoloured zone around the mycelia of the isolates con- their FPUase and cellulase activities in laboratory. Little firms the ability of cellulose utilization and cellulase ac- is known about deterioration of historical manuscripts in tivity of fungal culture. Additionally, a more quantitative old Fez city, and our results provide new information on assay method was used in order to determine cellulase the degradation caused by fungi, and therefore provide infor- activity in liquid medium for pre-selected fungi (Ariffin mation that is crucial to conservators for the preservation of et al. 2006). these manuscripts. Molecular identification For molecular identification, the genomic DNA was extracted Materials and methods using thermal shock. The rDNA ITS region 1, 5.8S, and ITS region 2 were amplified utilizing primers ITS1 (5′TCC GTA Paper samples GGT GAA CCT TGC GG 3′) and ITS4 (5’TCC TCC GCT TAT TGA TAT GC 3’) (White et al. 1990). The PCR reaction Two types of historical books of an old library of the Medina mix was performed in a total volume of 20 μlconsistingof 1 x of Fez at an advanced stage of biodeterioration were used in PCR reaction buffer, 1.5 mM MgCl , 0.2 mM deoxy- this study. The first one is the sacred book of Islam, Qur’an, nucleotides, 1 μMof each primer, U Taq DNA polymerase dated from 1827. The second is an un-identified book of (Promega, Madison, WI, USA). The thermocycling program koranic interpretation from the 19th century. The samples was asfollows:94°Cfor 5 min;35cyclesof94°Cfor 1 min, books were selected and samples that present a visible alter- 50 °C for 1 min, 72 °C for 1 min followed by a final extension ation, mainly from the margins of degraded pages, were step of 72 °C for 5 min. We sequenced both strands of all collected. Also, the powdery of the samples were observed amplified fragments. Comparative sequence analyses were with an Olympus optical microscope. performed by comparing sequences with those available in Ann Microbiol (2014) 64:815–822 817 the online databases provided by the National Centre for inoculated with 2 ml of single-strain suspension containing Biotechnology Information (NCBI) using the BLAST search 10 spores/ml previously counted by optical microscopy in a program (Altschul et al. 1997). Malassez Chamber. Others paper samples were not inoculated with fungi, to serve as a control. All the samples were incu- FPUase activity and stability as a function of PH bated at 25 °C for 18 months. Additional 1 ml of sterile water was monthly added to samples. After various periods of and temperature incubation, the texture and color of pigments surface was well defined: brown, black, green, white and pink. Also, the my- The enzymatic activity of cellulases was determined by the method of filter paper assay (FPA) (Ghose 1987). One unit of celium of fungi growing on the filter paper was removed this activity, termed as FPU, was defined as the amount of vigilantly and paper was dried and weighed. Then weight loss was determined by: enzyme that catalyzes the formation of 1 mM of reducing sugars that are liberated from the hydrolysis of filter paper, per minute of culture filtrate under assay conditions. It was Percentage Weight Loss ¼ Initial weight  Final weight assayed by measuring the release of reducing sugars in a 100=Initial weight: reaction mixture containing Whatman filter paper no.1 (1.0× 6.0 cm =50 mg) as a substrate in 50 mM sodium phosphate Results and discussions buffer (pH 4.8) after a 30 min of incubation at 50 °C. Reducing sugars were assayed by the dinitrosalicylic acid Macroscopic observations (DNS) method. This procedure was repeated thrice and the average value of three absorbances was determined. The In order to determine the levels of the degradation of these concentration of the sample was determined from the standard important manuscripts, we first observed our samples at a curve (prepared with glucose). To determine the optimum macroscopic level. All samples presented evidence of strong temperature, the cellulase was measured at a wide range of superficial alteration. Generally, the spots appear in the exter- temperatures (20–50 °C) in 0.1 M sodium phosphate buffer nal pages of books; however, the internal pages were better (pH 4.0). To determine the optimum pH of the activity of preserved. Indeed, this visual inspection reveals a number of cellulase, the enzymatic assays were carried out at pH values spots of different colors. ranging from 3.0 to 6.0, using sodium phosphate buffer sys- Furthermore, the results of the observation with the optical tem (0.1 M). microscope of powdery samples showed the presence of fungal spores and pigmented cellular structures including Growth on paper some hyphae fragments and mycelia. Pieces of filter paper (Whatman filter paper No 1, F 55 mm) were cleaned with acetone, then rinsed twice with distilled Fungi isolated, identified and screened water and dried. The dried paper samples were put in Petri dishes, wetted with 2 ml of distilled water and autoclaved for During this study, we isolated 31 fungal strains from the ancient 15 min at 120 °C. Each piece of sterilized filter paper was documents; 16 of them presented different morphology and Table 1 Fungi isolated from ancient documents: original document, accession numbers, genetic similarity with existing NCBI sequence and the total cellulase are presented. ID Orginal document Identified fungi Accession number Similarity (%) Class Total cellulase (ICMC) CPF1 Quoran Aspergillus oryzae EF661560.1 99 Eurotiomycetes 1.5±0.05 CPF4 Aspergillus niger* –– Ascomycetes 2.7±0.03 CPF6 Hypocrea lixii FJ517550.1 99 Ascomycetes 1.9±0.02 CPF9 Mucor racemosus HM641690.1 99 Zygomycetes 1.7±0.03 CPF11 Penicillium commune* –– Ascomycetes 2.5±0.02 CPF12 Schizophyllum commune FJ478109.1 100 Ascomycetes 1.9±0.03 CPF13 Penicillium chrysogenum GU985086.1 99 Ascomycetes 1.7±0.03 CPF14 Books Penicillium chrysogenum JF834167.1 98 Ascomycetes 0.9±0.01 CPF15 Mucor racemosus HM641690.1 100 Zygomycetes 2.9±0.02 CPF16 Aspergillus melleus FM986320.1 99 Ascomycetes 2,3±0.03 *Isolates identified using morphological examination 818 Ann Microbiol (2014) 64:815–822 FPUase activity (UI) colour. Among these fungi, only nine were identified and 0.06 screened for their ability to produce cellulase. Table 1 lists the molecular identification of fungal isolates from the deteriorated 0.05 manuscripts. This report presents the first results of the micro- 0.04 bial contamination study of the archives in Morocco. 0.03 The fungal contaminants detected in the paper samples 0.02 were Aspergillus niger van Tieghem 1867, Aspergillus oryzae 0.01 (Ahilburg) Cohn 1884, Aspergillus melleus Yukawa 1911, 1.4E-16 Mucor racemosus Fresenius 1850, Hypocrea lixii Patouillard 1891, Schizophyllum commune Fries 1821, -0.01 Penicillium commune Thom 1910, Penicillium chrysogenum Thom 1910. Some of the mentioned genera occur more fre- quently, such as Aspergillus and Penicillium. Overall, the Fig. 1 FPUase activity of the fungal isolates grown on filter paper as the high frequency of those genera is in agreement with other only source of carbon (pH = 7, T = 30 °C) works (Hyvarinen et al. 2002; Di Bonaventura et al. 2003; Lugauskas and Krikstaponis 2004; Silva et al. 2006; Michaelsen et al. 2006; Neves et al. 2009; Sohailetal. dangerous to books and documents due to their capacity to 2009). These are almost ubiquitous taxa, and can produce produce cellulolytic enzymes at high levels (Gopinath et al. numerous mitospores and conidia that are easily dispersed 2005). Moreover, Schizophyllum commune was isolated from by air. Moreover, they are usually found as contaminants or biodeteriorated paper and it has been found in a wide range of biodeterioration agents in many different habitats and mate- environments (soils, air, murals). In many cases the wood rials, including those considered as representative of historical presents a specific substrate for this species (Cojocariu and and cultural heritage (Das et al. 1997; Abrusci et al. 2005; Tanase 2010). Sohail et al. 2009). Shamsian et al. (2006) described a high Finally, the fungal strains belong to genera already isolated frequency of the genera Aspergillus, Penicillium, Mucor and from paper material, with the exception of Hypocrea lixii,which Trichoderma in the biodegraded books and manuscripts. was never isolated on biodeteriorated paper until now. This Andersen et al. (2011)describe that Penicillium chrysogenum microorganism is a teleomorph of Trichoderma harzianum was evenly associated with wallpaper compared to other (Chaverri and Samuels 2002; Eida et al. 2011) and produced a organic materials (wood, polywood, plaster, gypsum). green pigment in conidial spores as reported by Hölker et al. In fact, fungi are well known agents of decomposition of (2002) and was recently reported to produce cellulase on several organic matter in general and degradation of cellulosic sub- researches (Chandra et al. 2009; De Castro et al. 2010;Eida strate in particular, as reported by Lynd et al. (2002). These et al. 2011; Cabero et al. 2012). microorganisms synthesized cellulases during their growth on All of these microorganisms are usually present in the air; cellulosic materials (Lee and Koo 2001) and as known, in their presence in these manuscripts may activate the start of a time, paper suffers from deterioration processes because of the biodeterioration process if adequate environmental conditions oxidation and hydrolysis of the various functional groups are met—thus, the microbial contamination acts as a prelim- from the structure of cellulose. Several studies have reported inary precursor for the alteration of the manuscripts. that Aspegillus niger was isolated from paper materials (Silva et al. 2006;Michaelsen etal. 2006). In addition, it is well established that Aspergillus niger produces several of cellu- FPUase activity (UI) lolytic enzymes responsible for the degradation of cellulose Aspergillus oryzae 0.09 Aspergillus niger from paper fibers (Deacon 1997). Commercial cellulase prep- 0.08 Hypocrea lixii arations, derived from culture filtrates of the fungus, have 0.07 Mucor racemosus been fractionated by several workers (Van Whyk et al. 2000; 0.06 Schizophyllum commune Sukumaran et al. 2005; Khalid et al. 2006; Villena and 0.05 Penicillium chrysogenum Gutiérrez-Correa 2007; Sibtain et al. 2009;Eida etal. 2011). 0.04 Aspergillus melleus Besides, Aspergillus melleus is also known to produce cellu- 0.03 lase (Ezekiel et al. 2010). 0.02 Similarly, Mucor racemosus and Penicillium commune are 0.01 often present on biodeteriorated paper of historical documents 20°C 25°C 30°C 35°C 40°C 45°C 50°C Temperature (Gallo 1992; Florian and Manning 2000; Valentin et al. 2002; -0.01 Abrusci et al. 2005; Zyani et al. 2009). Likewise, Andersen Fig. 2 Effect of the temperature on the FPUase activity of the fungal isolates grown on filter paper as the only source of carbon (pH = 5) et al. (2011)found Mucor racemosus on wallpaper. These are Ann Microbiol (2014) 64:815–822 819 FPUase activity UI Effect of temperature and pH on the FPUase activity Aspergillus oryzae 0.07 Aspergillus niger 0.06 Hypocrea lixii The effect of temperature on the FPUase activity of the seven Mucor racemosus identified species was examined at various temperatures rang- 0.05 Schizophyllum commune Penicillium chrysogenum ing from20to50°Casshown in Fig. 2. The FPUase activity 0.04 Aspergillus melleus of the isolates was greatly affected by temperature changes. 0.03 Moreover, the enzyme showed a good activity at 25 °C and 30 °C, and inactive at 50 °C for all the strains. 0.02 The optimal temperature of the enzyme in Aspergillus was 0.01 around 30 °C; however for Hypocrea lixii, Mucor racemosus 0 and Schizophyllum commune and P. chrysogenum was of pH 3 pH4 pH5 pH6 pH7 pH 8 25 °C. In contrast, most filamentous fungi are mesophilic -0.01 requiring optimal temperatures between 25 and 35 °C (Reid Fig. 3 Effect of the pH on the FPUase activity of the fungal isolates 1998; Suresh et al. 1999). Philippidis (1994)report thatsome grown on filter paper as the only source of carbon (T = 25 °C) Aspergillus strains showed higher enzyme yield at 30 °C which is in agreement with the present study. Enzyme activity assay As a degradation factor, temperature acts indirectly by pro- moting fungal growth by accelerating the already initiated Production of FPUase activity for the identified isolates is chemical reactions, as well as the biological degradation prod- showninFig. 1. On the basis of the results, the production of ucts. Keeping the thermo-hygrometric parameters constant is this enzymatic activity was first detected after 48 h of cultivation essential for a suitable, long-term preservation of the cellulosic and increased during growth and reached maximum level on the materials. Moreover, Abrusci et al. (2005) noted that tempera- eighth day for all the isolates. Three of these isolates presented a ture is a very important factor affecting rate and extent of high FPUase production, and the others presented low FPUase. biodegradation. In the present work, there was a good correla- It was observed that the levels of FPUase of A. niger, A. oryzae tion between the optimal temperature for enzyme activity and and H. lixii were higher than the level of the same enzymatic the temperature previously evaluated in the ancient libraries of activity of A. melleus, M. racemosus, S. commune and P. the ancient Medina of Fez that not exceed 30 °C. This could chrysogenum. reflect the adaptation acquired of these fungi to grow and to Aspergillus niger and Hypocrea lixii enzyme activity was possess a maximum activity in the conditions of the novel higher (more than 0.05U) on the eighth day. The first species environment they inhabit. is known to degrade filter paper (Villena and Gutiérrez-Correa Similarly, the effect of pH on the FPUase activity was 2007; Jahangeer et al. 2005), also, Schizophyllum commune is examined at pHs ranging from 3.0 to 8.0 as shown in Fig. 3. reported to produce a similar quantity of filter paper enzyme All isolates were able to produce FPUase activity at different (Valencia et al. 2011). Furthermore, Ezekiel et al. (2010) pH, but at various degrees. The optimal pH for fungal cellu- found that Aspergillus melleus has a good FPUase activity. lases varies between species, although in most cases the This finding indicates that the cellulase produced by iso- optimum pH ranges from 3.0 to 9.0 (Coral et al. 2002; lates (FPUase) to hydrolyze cellulose; the principal content of Niranjane et al. 2007). paper; to extract the nutrients required for fungal growth. For Enzymes from all isolates presented a major activity peak this, the filter paper activity would be an interesting subject to at pH 5.0, with the exception of S. commune, which showed be assessed on degrading-paper microorganisms. higher activity at pH 6.0. Our findings are similar to those Table 2 Paper decomposition by fungal strains and colours of pigmented stains Fungal strains Aspergillus oryzae Aspergillus niger Hypocrea lixii Mucor racemosus Aspergillus melleus Visual colors of stains Green yellow Black Green Dark grey Green yellow Percentage weight loss 0(months) 0 0 0 0 0 2 4,17 2,67 1,17 7,83 0,83 4 6,17 4,67 1,83 8,83 2,50 8 11,33 7,67 4,50 10,33 6,17 12 11,67 12,67 5,33 12,17 8,50 18 13,83 15,17 12,83 17,83 10,17 820 Ann Microbiol (2014) 64:815–822 reported by several works with different period and tempera- Conclusion tures of incubation. The present study showed that optimum pH for many Aspergillus cellulases is near pH 5.0 as reported In the present study, it could be concluded that the historical by Vries and Visser (2001). Also, Sibtain et al. (2009)used pH paper documents are colonized by variety of fungi including of 5.5 for cellulases production from H. lixii. Similarly, Aspergillus niger, Aspergillus oryzae, Aspergillus melleus, Youssef (2011) previously described that maximum induction Penicillium commune, Penicillium chrysogenum, Mucor of FPUase activity of A. oryzae was achieved at pH 5. racemosus, Hypocrea lixxi and Schizophyllum commune. Furthermore, the pH requirement was determined at pH 5.0 Most of these isolates were known as contaminants of paper, for A. melleus as described by Ezekiel et al. (2010). but some of them never were observed on paper materials of libraries. Moreover, all of these fungal strains were found to possess cellulose degrading ability when grown on Artificial attack carboxymethyl-cellulose or on filter paper as a sole source of carbon. In parallel, the capacity of the isolates to change During the examination of the artificially contaminated sam- aesthetic aspect and cause the mass losses of artificially con- ples using pure fungal strains, unanticipated results were taminated paper was confirmed. The presence of these fungi obtained. All the isolated fungal strains demonstrated a high within the paper materials, and their ability to have a cellulo- capacity to grow on paper material after their reinoculation on lytic potential and degrade cellulose in vitro, demonstrated sterile Whatman paper. In addition, the margins were extreme- that these fungi could be responsible of the degradation and ly deteriorated, and in some areas, we observed the stains and the apparitionof foxingin historicalpaper. spots of different colors like those naturally affected by bio- This report may increase the knowledge about the micro- logical agents. In fact, the brownish spots that appeared on bial communities colonizing paper of ancient library mate- paper are the typical marks of foxing resulting from the effect rials, as well as the relation between decomposition of paper of biotic agents (Zotti et al. 2011), especially from the meta- and fungi cellulolytic enzyme activity. For that reason, it is bolic activity and pigments of the mycelia and spores. Table 2 useful to better study the other enzymatic activities of these shows that these cellulolytic strains were able to decompose fungi and others, like proteases and amylases that will enable filter paper cellulose at varying degrees. them to use glues, starch and other additives of paper as a Aspergillus niger shows black staining properties, charac- nutrient source for survival and maintenance. teristic of pigments (e.g., melanins) in the hypha of this fungus. Also, A. oryzae, A. melleus, M. racemosus and H. lixii produce respectively yellow, grey and green mycelia, could be responsible of intense stains. In most cases, the References colours of the stains are often characteristic of particular genus or species of fungi. It is common for most fungal species to Abrusci C, Martin-Gonzàlez A, Del Amo A, Catalina F, Collado J, Platas have aesthetical destructive effects by pigments and metabolic G (2005) Isolation and identification of bacteria and fungi from products. Such stains often badly disfigure the surface of cinematographic films. Int Biodeterior Biodegrad 56:58–68 paper, especially historical paper containing high levels of Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, deterioration products like acidic components. Sterflinger Lipman JD (1997) Gapped BLAST and PSI-BLAST: a new gener- ation of protein database search programs. Nucleic Acids Res 25: et al. (1999) described that fungal growth on objects of cul- 3389–3402 tural heritage often causes a serious aesthetical spoiling due to Andersen S, Frisvad JC, Sondergaard I, Rasmussen IS, Larsen LS (2011) colony formation and fungal pigments. Associations between fungal species and water-damaged building Besides, all the fungal strains were able to degrade filter materials. Appl Environ Microbiol 4180–4188 Arai H (1987) Microbial studies on the conservation of paper and related paper at varying degree. Table 2 shows that the maximum properties. Sci Conserv 26:43–26 decomposition was observed with M. racemosus (17.8 %), Arai H (2000) Foxing caused by fungi: twenty-five years of study. Int this species was capable of decomposing the component of Biodeterior Biodegrad 46:181–188 paper at two months at high levels, compared to the others. Ariffin H, Abdullah N, Umi Kalsom MS, Shirai Y, Hassan MA (2006) Production and characterization of cellulase by Bacillus pumilus This can be explained by the fact that the test conditions EB3. Int J Eng Technol 3:47–53 coincide with the optimum conditions of the growth or/and Cabero K, Pozzo T, Lidén G, Karlsson EN (2012) A cellulolytic of the cellulase production of this strains. However, the min- Hypocrea strain isolated from South American brave straw produces imum was found with A. melleus (10.2 %). The loss of a modular xylanase. Carbohydr Res 356:215–223 Chandra M, Kalra A, Sharma PK, Sangwan RS (2009) Cellulase produc- strength caused in the structure of paper is the result of tion by six Trichoderma spp. fermented on medicinal plant enzymatic decomposition of the components of paper by processings. J Ind Microbiol Biotechnol 36:605–609 fungi, and like final results, the paper become thinner and Chaverri P, Samuels GJ (2002) Hypocrea lixii, the teleomorph of weaker. Trichoderma harzianum. Mycol Prog 1:283–286 Ann Microbiol (2014) 64:815–822 821 Ciferri O, Tiano P, Mastromei G (2000) Of Microbes and Art. The role of Michaelsen A, Pinzari F, Ripka K, Lubitz W, Pinar G et al (2006) microbial communities in the degradation and protection of cultural Application of molecular techniques for identification of fungal heritage. Kluwer Academic, New York communities colonising paper material. Int Biodeterior Biodegrad Cojocariu A, Tanase C (2010) Macromycetes identified on the construc- 58:133–141 tion wood of historical monuments from Moldavia and causes of Montemartini CA, Ferroni A, Salvo VS (2003) Isolation of fungal species their development. J Plant Dev 17:63–68 from test samples and maps damaged by foxing, and correlation Coral G, Arikan B, Unaldi MN, Guvenments H (2002) Some properties between these species and the environment. Int Biodeterior of crude carboxymethyl cellulase of Aspergillus niger Zio. Wild Biodegrad 51:167–173 type strain. Turk J Biol 2:209–213 Neves ER, Schafer S, Phillips A, Canejo J, Filomena MM (2009) Das MKL, Prasad JS, Ahmed SK (1997) Endoglucanase production by Antifungal effect of different methyl and propyl paraben mixtures paper-degrading mycoflora. Lett Appl Microbiol 25:313–315 on the treatment of paper biodeterioration. Int Biodeterior Biodegrad De Castro AM, Pedro KC, Da Cruz JC, Ferreira MC (2010) Trichoderma 63:267–272 harzianum IOC-4038: a promising strain for the production of a Niranjane AP, Madhou P, Stevenson TW (2007) The effect of carbohy- cellulolytic complex with significant beta-glucosidase activity from drate carbon sources on the production of cellulase by Phlebia sugarcane bagasse cellulignin. Appl Biochem Biotechnol 162:2111– gigantean. Enzym Microbial Technol 40:1464–1468 2122 Nyuksha YP (1983) Some special cases of biological deterioration of Deacon JW (1997) Modern Mycology, 3rd edn. Blackwell Science, books. Restaurator 5:177–182 Oxford, p 97 Philippidis GP (1994) Cellulase production technology. In: Enzymatic Di Bonaventura MP, De Salle R, Bonacum J, Koestler RJ (2003) conversion of biomass for fuel production. (Eds): Himmel ME et al., Phylogenetic trees, fungal infections and the Tiffany’sdrawings. ACS symposium series 566 In: Saiz-Jimenez C (ed) Molecular Biology and Cultural Heritage. Porck HJ (2000) Rate of paper degradation: the predictive value of Balkema, Lisse, pp 131–135 artificial aging tests. The European Commission on Preservation Eida MF, Nagaoka T, Wasaki J, Kouno A (2011) Evaluation of cellulo- and Access lytic and hemicellulolytic abilities of fungi isolated from coffee Prieto B, Silva B (2005) Estimation of potential bioreceptivity of granitic residue and sawdust composts. Microbes Environ 26:220–227 rocks from their instrinsic properties. Int Biodeterior Biodegrad 56: Ezekiel CN, Odebode AC, Omenka RO, Adesioye FA (2010) Growth 206–215 response and comparative cellulase induction in soil fungi grown on Reese ET, Downing MH (1951) Activity of the Aspergilli on cellulose, different cellulose media. J Life Phys Sci 3:52–59 cellulose derivatives and wool. Mycologia 43:16–28 Fabbri AA, Ricelli A, Brasini S, Fanelli C (1997) Effect of different Reid ID (1998) Influence of incubation temperature on activity of antifungals on the control of paper biodeterioration caused by fungi. ligninolytic enzymes in sterile soil by Pleurotus sp.and Int Biodeterior Biodegrad 57:61–65 Dichomitus squalens. J Basic Microbiol 40:33–39 Fellers G, Wegener, Papparstekik (1991) Institutionen for pappar-stekik. Rojas JA, Cruz C, Mikan JF, Villalba LS, Cepero de Garcia MC, Restrepo Stockholm S (2009) Isoenzyme characterization of proteases and amylases and Florian MLE, Manning L (2000) SEM analysis of irregular fungal spots partial purification of proteases from filamentous fungi causing in an 1854 book: population dynamics and species identification. Int biodeterioration of industrial paper. Int Biotederior Biodegrad 63: Biodeterior Biodegrad 46:205–220 169–175. Gallo F (1992) Il biodeterioramento di libri e documenti. Centro di Studi Saoudi N (2008) La ville de Fès célèbre 1200 ans de rayonnement. Islam per la Conservazione della Carta. ICCROM, Roma Tour 37:54–60 Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem See P (1919) La florule du papier : Etude systématique et biologique de 59:257–268 champignons chromogènes du papier pique. (Nature, origine, agents Gopinath CBS, Anbu P, Hilda A (2005) Extracellular enzymatic activity et remèdes de 1’altération des papiers) Thesis Paris. France, Cited in profiles in fungi isolated from oil-rich environments. Mycoscience Zyska B (1997) Fungi isolated from library materials: A Rev of the 46:119–126 Literature Int Biodeterior Biodegrad 40: 43–51 Hölker U, Dohse J, Höfer M (2002) Extracellular laccases in ascomy- Shamsian A, Fata A, Mohajeri M, Ghazvini K (2006) Fungal contami- cetes Trichoderma atroviride and Trichoderma harzianum. Folia nations in historical manuscripts at Astan Quds museum library, Microbiol 47:423–427 Mashhad. Iran Int J Agric Biol 3:420–422 Hyvarinen A, Meklin T, Vepsa lainen A, Nevalainen A (2002) Fungi and Sibtain A, Ammara B, Huma S, Mubshara S, Amer J (2009) Production action bacterian moisture-damaged building materials – concentra- and purification of cellulose degrading enzymes from a filamentous tions and diversity. Int Biodeterior Biodegrad 40:27–37 fungus Trichoderma harzianum. Pak J Bot 41:1411–1419 Jahangeer A, Khan N, Jahangeer S, Sohail M, Shahzad S, Khan A (2005) Silva M, Moraes A, Nishikawa M, Gatti M, Vallim de Alencar M, Screening and characterization of fungal cellulases isolated from the Brandao L, Nobrega A (2006) Inactivation of fungi from deteriorat- native environmental source. Pak J Bot 37:739–748 ed paper materials by radiation. Int Biodeterior Biodegrad 57:163– Khalid M, Yang WJ, Kishwar N, Rajput ZI, Arijo AG (2006) Study of 167 cellulolytic soil fungi and two nova species and new medium. J Sohail M, Naseeb S, Sherwani SK, Sultana S, Aftab S, Shahzad S, Zhejiang Univ Sci B 7:459–466 Ahmad A, Khan SA (2009) Distribution of hydrolytic enzymes Kowalik R (1980) Microbiodeterioration of Library Materials. Restaurator among native fungi: Aspergillus the pre-dominant genus of hydro- 4:99–114 lase producer. Pak J Bot 41:2567–2582 Lee SM, Koo YM (2001) Pilot-scale production of cellulose using Sterflinger K, Krumbein WE, Rullkotter J (1999) Patination of marble Trichoderma reesei Rut C-30 in fed-batch mode. J Microbiol sandstone and granite by microbial communities. Z Dt Geol Ges Biotechnol 11:229–233 150:299–311 Lugauskas A, Krikstaponis A (2004) Microscopic fungi found in the Sukumaran RK, Singhania RR, Pandey A (2005) Microbial cellulases libraries of Vilnius and factors affecting their development. Indoor production, application and challenges. J Sci Ind Res 64:832– Built Environ 13:169–182 844 Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS (2002) Microbial Suresh BJ, Hari KS, Sekhar RKC (1999) Studies on the production ad cellulose utilization: fundamentals and biotechnology. Microbiol application of cellulase from Trictoderma reesei QM- 9414. Mol Biol Rev 66:506–577 Bioprocess Biosyst Eng 22:467–470 822 Ann Microbiol (2014) 64:815–822 Valencia JWA, Jiménez AV, Gonçalves de Siqueira F, Medina KD, Innis Gelfand MADH, Sninsky JJ, White TJ (eds) PCR protocols: A Restrepo Franco GM, Ferreira Filho EX, Siegfried BD, Grossi-de- guide to methods and applications. Academic Press, Inc., New York, Sa MF (2011) Holocellulase activity from Schizophyllum commune pp 315–322 grown on bamboo: a comparison with different substrate. Curr Youssef GA (2011) Physiological studies of cellulase complex enzymes Microbiol 63:581–587 of Aspergillus oryzae and characterization of carboxymethyl cellu- Valentin N, Garcia R, de Luis O, Maekawa S (2002) Air ventilation for lase. Afr J Microbiol Res 5:1311–1321 arresting microbial growth in archives. Quatrièmes Journées Zhang YH, Lynd LR (2004) Toward an aggregated understanding of Internationales d’Etudes de l’ARSAG 139–150. Paris enzymatic hydrolysis of cellulose: non complexed cellulase system. Van Whyk JPH, Mogale MA, Seseng TA (2000) Saccharification Biotechnol Bioeng 88:797–824 of used paper with different cellulase. Biotechnol Lett 22: Zotti M, Ferroni A, Calvini P (2011) Mycological and FTIR analysis of 491–494 biotic foxing on paper substrates. Int Biodeterior Biodegrad 65:569– Villena GK, Gutiérrez-Correa M (2007) Production of lignocellulolytic 578 enzymes by Aspergillus niger biofilms at variable water activities. Zyani M, Mortabit D, Mostakim M, Iraqui M, Haggoud A, Ettayebi M, Electron J Biotechnol 10:124–140 Ibnsouda KS (2009) Cellulolytic of fungi in wood degradation from Vries RP, Visser J (2001) Aspergillus enzymes involved in deg- an old house at the medina of Fez. Ann Microbiol 59:699–704 radation of plant cell wall polysaccharide. Microbiol Mol Zyska B (1993) Preservation of library materials, factor deteriorating Biol Rev 65:497–522 materials in library collections. Uniwersytet SIgski, Katowice, White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct Cited in Zyska B (1997) Fungi isolated from library materials: A sequencing of fungal ribosomal RNA genes for phylogenetics. In: review of the literature. Int Biodeter Biodegr 40: 43–51 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Annals of Microbiology Springer Journals

Cellulolytic potential and filter paper activity of fungi isolated from ancients manuscripts from the Medina of Fez

Download PDF
8 pages

Loading next page...
 
/lp/springer-journals/cellulolytic-potential-and-filter-paper-activity-of-fungi-isolated-1E6kwcvf00

References (58)

Publisher
Springer Journals
Copyright
Copyright © 2013 by Springer-Verlag Berlin Heidelberg and the University of Milan
Subject
Life Sciences; Microbiology; Microbial Genetics and Genomics; Microbial Ecology; Fungus Genetics; Medical Microbiology; Applied Microbiology
ISSN
1590-4261
eISSN
1869-2044
DOI
10.1007/s13213-013-0718-6
Publisher site
See Article on Publisher Site

Abstract

Ann Microbiol (2014) 64:815–822 DOI 10.1007/s13213-013-0718-6 ORIGINAL ARTICLE Cellulolytic potential and filter paper activity of fungi isolated from ancients manuscripts from the Medina of Fez Fatimazahra El Bergadi & Faouzi Laachari & Soumya Elabed & Iraqui Houssaini Mohammed & Saad Koraichi Ibnsouda Received: 1 May 2013 /Accepted: 17 September 2013 /Published online: 18 October 2013 Springer-Verlag Berlin Heidelberg and the University of Milan 2013 Abstract The content of libraries in Fez is an important Introduction historical testimony and a treasure of knowledge of several civilizations. Unfortunately, this cultural heritage has suffered Morocco, crossroads of several civilizations, has precious man- deterioration caused by many factors, including microbial uscripts. These documents play a major role in the field of deterioration specifically by cellulolytic fungi. The aim of scientific and historical research. One of the Moroccan cities our study was the identification and characterization of the most characterized by the wealth of manuscripts is the city of microflora that damage historical manuscripts books from an Fez. Historically, Fez has been known as a home to the oldest old library of the Medina of Fez. A total of 31 filamentous university in the world, "al qarawiyine university" (Saoudi fungi were isolated from deteriorated paper of ancient books. 2008). However, much of this valuable cultural heritage that Nine of these isolates were screened positive for the ability to has been accumulated over time is today in an advanced state of degrade carboxymethylcellulose (CMC). The ability of the deterioration due to poor storage conditions. Indeed, all of these nine isolates to produce filter paper activity (FPUase) in liquid manuscripts suffer from various destructive agents especially media as well as the effect of temperature and pH were also from microbial degradation that causes different kinds of dam- studied. According to the molecular identification, the most age depending on the organisms responsible for the attack. The frequent species were Penicillium chrysogenum, Aspergillus biodeterioration of library materials surpasses 110 years (Zyska niger, Aspergillus oryzae and Mucor racemosus, and other less 1993), but the first research, which discuses microbial alteration frequent, such as Hypocrea lixii, Aspergillus melleus and of papers appears in 1917 (See 1919). Schizophyllum commune were also present. Also, the effect of Fungi are considered as serious degrading agents of paper fungal contamination in paper artificially attacked for 18 months manuscripts, particularly cellulolytic fungi (Fabbri et al. 1997). at 25 °C was examined. The fungal strains were able to degrade Some filamentous fungi frequently colonize paper, and are able filter paper to varying degrees. to degrade cellulose fibers through the action of cellulolytic enzymes (Reese and Downing 1951; Nyuksha 1983;Ciferri et al. 2000), or to alter aesthetic and visual appeal by releasing . . . Keywords Fez Filter Paper Activity Biodeterioration weak acids or pigments; a phenomenon referred as foxing (Arai Cellulose Fungi 2000; Montemartini et al. 2003;Zotti et al. 2011). Cellulose from native wood is used for paper production. In addition to cellulose fibers, paper can contain hemicelluloses (wood polyoses), lignin, and additives such as fillers and pig- : : : : F. El Bergadi F. Laachari S. Elabed I. H. Mohammed ments (Fellers and Wegener 1991). Some filamentous fungi S. K. Ibnsouda (*) frequently associated with paper degradation are capable of Laboratoire de Biotechnologie Microbienne, Faculté des Sciences et dissolving cellulose fibres through the synergistic actions of Techniques de Fès, Université Sidi Mohamed Ben Abdellah, endoglucanase (EC 3.2.1.4), exoglucanase or cellobiohydrolase B.P. 2202 – Route d’Imouzzer, Fès-Maroc, Morocco e-mail: ibnsouda@hotmail.com (EC 3.2.1.91), and β-glucosidase (EC 3.2.1.21) (Lynd et al. 2002; Zhang and Lynd 2004), causing serious damage to paper S. Elabed S. K. Ibnsouda materials of cultural and historical importance (Reese and Centre Universitaire Régional d’Interface, Université Sidi Mohamed Downing 1951; Nyuksha 1983; Ciferri et al. 2000). In this case, Ben Abdellah, Fès-Maroc, Morocco 816 Ann Microbiol (2014) 64:815–822 Porck (2000) reported that the hydrolytic degradation of the Isolation, culturing and incubation cellulose molecules is the most frequent reaction, particularly when the presence of water in the storage environment plays an For sampling, three methods were used. (1) The samples essential role. In fact, microbial activity depends largely on the were swabbed from the historical books. (2) Other sam- water activity (aw) of the paper. It is a measure of free water ples were scraped with a scalpel, then all of them were which is available for the growth of the mould and is defined as pre-incubated in lysogeny broth (LB) supplemented by the ratio of the vapour pressure of the water in the substrate chloramphenicol (500 mg/l) (Rojas et al. 2009). (3) The and the vapour pressure of pure water at the same tempera- collected fragments of deteriorated paper were introduced ture and relative humidity (RH) (Arai 1987). According to directly in the same medium for 24 h at 25 °C. After, the standard reaction, the rate of the hydrolytic process is deter- supernatants from each sample were serially diluted in mined by the combined effects of aw, the temperature, the sterile distilled water up to 10–4 and cultivated using pH and the relative humidity. At a RH level higher than different media: MEA (4 % malt extract, 1.8 % agar), 65 %, and a temperature higher than 20 °C, moisture content LB agar (1 % peptone, 1 % NaCl, 0.5 % malt extract, of papercan reach8to10%,withconsequentwater activity 1.8 % agar), and YPG antibiotics agar (1 % yeast extract, (aw) higher than 0.65, microbial spores can germinate and 2 % glucose, 2 % peptone, 1.8 % agar, ampicillin 60 μg/ develop by using the paper as a substrate of growth, conse- ml and kanamycine 30 μg/ml). The dishes were then quently affecting the object of cultural value (Kowalik 1980). incubated at 25 °C for 24 hours. The strains were isolated Furthermore, Prieto and Silva (2005) reported that the inten- and purified after streaking several times. sity of the microbial contamination is influenced by the climatic conditions of the atmosphere. Screening of hydrolytic activity The fungal contamination of Moroccan manuscripts stored in archives is therefore problematic because of the imperfect Pure isolated cultures of fungal mycelia were screened for knowledge of the deteriorative agents. Also, many efforts their cellulase activity; for this, they were transformed have focused on the preservation and maintenance of this individually on CMC agar plates containing (NaNO - written cultural heritage. For adequate conservation of these 3.0 g/l, K HPO -1.0 g/l, MgSO ·7H O-0.5 g/l, KCl- 2 4 4 2 archive collections, it is important to identify and characterize 0.5 g/l, FeSO ·7H O-0.01 g/l, CMC-1.0 g/l, Agar- 20 g/ 4 2 the fungal species for their control. l, pH 5.0) and then incubated at 25 °C for 72 hours. After This investigation was done to (1) assess the role of incubation the plates were stained with 1 % Congo Red fungi in the degradation of manuscripts in librairies from solution for 15 min, and afterwards the stain was neutral- the city of Fez, (2) identify fungi isolated from the ized with 1 M NaCl solution. The appearance of a decayed paper by molecular methods, and (3) evaluate discoloured zone around the mycelia of the isolates con- their FPUase and cellulase activities in laboratory. Little firms the ability of cellulose utilization and cellulase ac- is known about deterioration of historical manuscripts in tivity of fungal culture. Additionally, a more quantitative old Fez city, and our results provide new information on assay method was used in order to determine cellulase the degradation caused by fungi, and therefore provide infor- activity in liquid medium for pre-selected fungi (Ariffin mation that is crucial to conservators for the preservation of et al. 2006). these manuscripts. Molecular identification For molecular identification, the genomic DNA was extracted Materials and methods using thermal shock. The rDNA ITS region 1, 5.8S, and ITS region 2 were amplified utilizing primers ITS1 (5′TCC GTA Paper samples GGT GAA CCT TGC GG 3′) and ITS4 (5’TCC TCC GCT TAT TGA TAT GC 3’) (White et al. 1990). The PCR reaction Two types of historical books of an old library of the Medina mix was performed in a total volume of 20 μlconsistingof 1 x of Fez at an advanced stage of biodeterioration were used in PCR reaction buffer, 1.5 mM MgCl , 0.2 mM deoxy- this study. The first one is the sacred book of Islam, Qur’an, nucleotides, 1 μMof each primer, U Taq DNA polymerase dated from 1827. The second is an un-identified book of (Promega, Madison, WI, USA). The thermocycling program koranic interpretation from the 19th century. The samples was asfollows:94°Cfor 5 min;35cyclesof94°Cfor 1 min, books were selected and samples that present a visible alter- 50 °C for 1 min, 72 °C for 1 min followed by a final extension ation, mainly from the margins of degraded pages, were step of 72 °C for 5 min. We sequenced both strands of all collected. Also, the powdery of the samples were observed amplified fragments. Comparative sequence analyses were with an Olympus optical microscope. performed by comparing sequences with those available in Ann Microbiol (2014) 64:815–822 817 the online databases provided by the National Centre for inoculated with 2 ml of single-strain suspension containing Biotechnology Information (NCBI) using the BLAST search 10 spores/ml previously counted by optical microscopy in a program (Altschul et al. 1997). Malassez Chamber. Others paper samples were not inoculated with fungi, to serve as a control. All the samples were incu- FPUase activity and stability as a function of PH bated at 25 °C for 18 months. Additional 1 ml of sterile water was monthly added to samples. After various periods of and temperature incubation, the texture and color of pigments surface was well defined: brown, black, green, white and pink. Also, the my- The enzymatic activity of cellulases was determined by the method of filter paper assay (FPA) (Ghose 1987). One unit of celium of fungi growing on the filter paper was removed this activity, termed as FPU, was defined as the amount of vigilantly and paper was dried and weighed. Then weight loss was determined by: enzyme that catalyzes the formation of 1 mM of reducing sugars that are liberated from the hydrolysis of filter paper, per minute of culture filtrate under assay conditions. It was Percentage Weight Loss ¼ Initial weight  Final weight assayed by measuring the release of reducing sugars in a 100=Initial weight: reaction mixture containing Whatman filter paper no.1 (1.0× 6.0 cm =50 mg) as a substrate in 50 mM sodium phosphate Results and discussions buffer (pH 4.8) after a 30 min of incubation at 50 °C. Reducing sugars were assayed by the dinitrosalicylic acid Macroscopic observations (DNS) method. This procedure was repeated thrice and the average value of three absorbances was determined. The In order to determine the levels of the degradation of these concentration of the sample was determined from the standard important manuscripts, we first observed our samples at a curve (prepared with glucose). To determine the optimum macroscopic level. All samples presented evidence of strong temperature, the cellulase was measured at a wide range of superficial alteration. Generally, the spots appear in the exter- temperatures (20–50 °C) in 0.1 M sodium phosphate buffer nal pages of books; however, the internal pages were better (pH 4.0). To determine the optimum pH of the activity of preserved. Indeed, this visual inspection reveals a number of cellulase, the enzymatic assays were carried out at pH values spots of different colors. ranging from 3.0 to 6.0, using sodium phosphate buffer sys- Furthermore, the results of the observation with the optical tem (0.1 M). microscope of powdery samples showed the presence of fungal spores and pigmented cellular structures including Growth on paper some hyphae fragments and mycelia. Pieces of filter paper (Whatman filter paper No 1, F 55 mm) were cleaned with acetone, then rinsed twice with distilled Fungi isolated, identified and screened water and dried. The dried paper samples were put in Petri dishes, wetted with 2 ml of distilled water and autoclaved for During this study, we isolated 31 fungal strains from the ancient 15 min at 120 °C. Each piece of sterilized filter paper was documents; 16 of them presented different morphology and Table 1 Fungi isolated from ancient documents: original document, accession numbers, genetic similarity with existing NCBI sequence and the total cellulase are presented. ID Orginal document Identified fungi Accession number Similarity (%) Class Total cellulase (ICMC) CPF1 Quoran Aspergillus oryzae EF661560.1 99 Eurotiomycetes 1.5±0.05 CPF4 Aspergillus niger* –– Ascomycetes 2.7±0.03 CPF6 Hypocrea lixii FJ517550.1 99 Ascomycetes 1.9±0.02 CPF9 Mucor racemosus HM641690.1 99 Zygomycetes 1.7±0.03 CPF11 Penicillium commune* –– Ascomycetes 2.5±0.02 CPF12 Schizophyllum commune FJ478109.1 100 Ascomycetes 1.9±0.03 CPF13 Penicillium chrysogenum GU985086.1 99 Ascomycetes 1.7±0.03 CPF14 Books Penicillium chrysogenum JF834167.1 98 Ascomycetes 0.9±0.01 CPF15 Mucor racemosus HM641690.1 100 Zygomycetes 2.9±0.02 CPF16 Aspergillus melleus FM986320.1 99 Ascomycetes 2,3±0.03 *Isolates identified using morphological examination 818 Ann Microbiol (2014) 64:815–822 FPUase activity (UI) colour. Among these fungi, only nine were identified and 0.06 screened for their ability to produce cellulase. Table 1 lists the molecular identification of fungal isolates from the deteriorated 0.05 manuscripts. This report presents the first results of the micro- 0.04 bial contamination study of the archives in Morocco. 0.03 The fungal contaminants detected in the paper samples 0.02 were Aspergillus niger van Tieghem 1867, Aspergillus oryzae 0.01 (Ahilburg) Cohn 1884, Aspergillus melleus Yukawa 1911, 1.4E-16 Mucor racemosus Fresenius 1850, Hypocrea lixii Patouillard 1891, Schizophyllum commune Fries 1821, -0.01 Penicillium commune Thom 1910, Penicillium chrysogenum Thom 1910. Some of the mentioned genera occur more fre- quently, such as Aspergillus and Penicillium. Overall, the Fig. 1 FPUase activity of the fungal isolates grown on filter paper as the high frequency of those genera is in agreement with other only source of carbon (pH = 7, T = 30 °C) works (Hyvarinen et al. 2002; Di Bonaventura et al. 2003; Lugauskas and Krikstaponis 2004; Silva et al. 2006; Michaelsen et al. 2006; Neves et al. 2009; Sohailetal. dangerous to books and documents due to their capacity to 2009). These are almost ubiquitous taxa, and can produce produce cellulolytic enzymes at high levels (Gopinath et al. numerous mitospores and conidia that are easily dispersed 2005). Moreover, Schizophyllum commune was isolated from by air. Moreover, they are usually found as contaminants or biodeteriorated paper and it has been found in a wide range of biodeterioration agents in many different habitats and mate- environments (soils, air, murals). In many cases the wood rials, including those considered as representative of historical presents a specific substrate for this species (Cojocariu and and cultural heritage (Das et al. 1997; Abrusci et al. 2005; Tanase 2010). Sohail et al. 2009). Shamsian et al. (2006) described a high Finally, the fungal strains belong to genera already isolated frequency of the genera Aspergillus, Penicillium, Mucor and from paper material, with the exception of Hypocrea lixii,which Trichoderma in the biodegraded books and manuscripts. was never isolated on biodeteriorated paper until now. This Andersen et al. (2011)describe that Penicillium chrysogenum microorganism is a teleomorph of Trichoderma harzianum was evenly associated with wallpaper compared to other (Chaverri and Samuels 2002; Eida et al. 2011) and produced a organic materials (wood, polywood, plaster, gypsum). green pigment in conidial spores as reported by Hölker et al. In fact, fungi are well known agents of decomposition of (2002) and was recently reported to produce cellulase on several organic matter in general and degradation of cellulosic sub- researches (Chandra et al. 2009; De Castro et al. 2010;Eida strate in particular, as reported by Lynd et al. (2002). These et al. 2011; Cabero et al. 2012). microorganisms synthesized cellulases during their growth on All of these microorganisms are usually present in the air; cellulosic materials (Lee and Koo 2001) and as known, in their presence in these manuscripts may activate the start of a time, paper suffers from deterioration processes because of the biodeterioration process if adequate environmental conditions oxidation and hydrolysis of the various functional groups are met—thus, the microbial contamination acts as a prelim- from the structure of cellulose. Several studies have reported inary precursor for the alteration of the manuscripts. that Aspegillus niger was isolated from paper materials (Silva et al. 2006;Michaelsen etal. 2006). In addition, it is well established that Aspergillus niger produces several of cellu- FPUase activity (UI) lolytic enzymes responsible for the degradation of cellulose Aspergillus oryzae 0.09 Aspergillus niger from paper fibers (Deacon 1997). Commercial cellulase prep- 0.08 Hypocrea lixii arations, derived from culture filtrates of the fungus, have 0.07 Mucor racemosus been fractionated by several workers (Van Whyk et al. 2000; 0.06 Schizophyllum commune Sukumaran et al. 2005; Khalid et al. 2006; Villena and 0.05 Penicillium chrysogenum Gutiérrez-Correa 2007; Sibtain et al. 2009;Eida etal. 2011). 0.04 Aspergillus melleus Besides, Aspergillus melleus is also known to produce cellu- 0.03 lase (Ezekiel et al. 2010). 0.02 Similarly, Mucor racemosus and Penicillium commune are 0.01 often present on biodeteriorated paper of historical documents 20°C 25°C 30°C 35°C 40°C 45°C 50°C Temperature (Gallo 1992; Florian and Manning 2000; Valentin et al. 2002; -0.01 Abrusci et al. 2005; Zyani et al. 2009). Likewise, Andersen Fig. 2 Effect of the temperature on the FPUase activity of the fungal isolates grown on filter paper as the only source of carbon (pH = 5) et al. (2011)found Mucor racemosus on wallpaper. These are Ann Microbiol (2014) 64:815–822 819 FPUase activity UI Effect of temperature and pH on the FPUase activity Aspergillus oryzae 0.07 Aspergillus niger 0.06 Hypocrea lixii The effect of temperature on the FPUase activity of the seven Mucor racemosus identified species was examined at various temperatures rang- 0.05 Schizophyllum commune Penicillium chrysogenum ing from20to50°Casshown in Fig. 2. The FPUase activity 0.04 Aspergillus melleus of the isolates was greatly affected by temperature changes. 0.03 Moreover, the enzyme showed a good activity at 25 °C and 30 °C, and inactive at 50 °C for all the strains. 0.02 The optimal temperature of the enzyme in Aspergillus was 0.01 around 30 °C; however for Hypocrea lixii, Mucor racemosus 0 and Schizophyllum commune and P. chrysogenum was of pH 3 pH4 pH5 pH6 pH7 pH 8 25 °C. In contrast, most filamentous fungi are mesophilic -0.01 requiring optimal temperatures between 25 and 35 °C (Reid Fig. 3 Effect of the pH on the FPUase activity of the fungal isolates 1998; Suresh et al. 1999). Philippidis (1994)report thatsome grown on filter paper as the only source of carbon (T = 25 °C) Aspergillus strains showed higher enzyme yield at 30 °C which is in agreement with the present study. Enzyme activity assay As a degradation factor, temperature acts indirectly by pro- moting fungal growth by accelerating the already initiated Production of FPUase activity for the identified isolates is chemical reactions, as well as the biological degradation prod- showninFig. 1. On the basis of the results, the production of ucts. Keeping the thermo-hygrometric parameters constant is this enzymatic activity was first detected after 48 h of cultivation essential for a suitable, long-term preservation of the cellulosic and increased during growth and reached maximum level on the materials. Moreover, Abrusci et al. (2005) noted that tempera- eighth day for all the isolates. Three of these isolates presented a ture is a very important factor affecting rate and extent of high FPUase production, and the others presented low FPUase. biodegradation. In the present work, there was a good correla- It was observed that the levels of FPUase of A. niger, A. oryzae tion between the optimal temperature for enzyme activity and and H. lixii were higher than the level of the same enzymatic the temperature previously evaluated in the ancient libraries of activity of A. melleus, M. racemosus, S. commune and P. the ancient Medina of Fez that not exceed 30 °C. This could chrysogenum. reflect the adaptation acquired of these fungi to grow and to Aspergillus niger and Hypocrea lixii enzyme activity was possess a maximum activity in the conditions of the novel higher (more than 0.05U) on the eighth day. The first species environment they inhabit. is known to degrade filter paper (Villena and Gutiérrez-Correa Similarly, the effect of pH on the FPUase activity was 2007; Jahangeer et al. 2005), also, Schizophyllum commune is examined at pHs ranging from 3.0 to 8.0 as shown in Fig. 3. reported to produce a similar quantity of filter paper enzyme All isolates were able to produce FPUase activity at different (Valencia et al. 2011). Furthermore, Ezekiel et al. (2010) pH, but at various degrees. The optimal pH for fungal cellu- found that Aspergillus melleus has a good FPUase activity. lases varies between species, although in most cases the This finding indicates that the cellulase produced by iso- optimum pH ranges from 3.0 to 9.0 (Coral et al. 2002; lates (FPUase) to hydrolyze cellulose; the principal content of Niranjane et al. 2007). paper; to extract the nutrients required for fungal growth. For Enzymes from all isolates presented a major activity peak this, the filter paper activity would be an interesting subject to at pH 5.0, with the exception of S. commune, which showed be assessed on degrading-paper microorganisms. higher activity at pH 6.0. Our findings are similar to those Table 2 Paper decomposition by fungal strains and colours of pigmented stains Fungal strains Aspergillus oryzae Aspergillus niger Hypocrea lixii Mucor racemosus Aspergillus melleus Visual colors of stains Green yellow Black Green Dark grey Green yellow Percentage weight loss 0(months) 0 0 0 0 0 2 4,17 2,67 1,17 7,83 0,83 4 6,17 4,67 1,83 8,83 2,50 8 11,33 7,67 4,50 10,33 6,17 12 11,67 12,67 5,33 12,17 8,50 18 13,83 15,17 12,83 17,83 10,17 820 Ann Microbiol (2014) 64:815–822 reported by several works with different period and tempera- Conclusion tures of incubation. The present study showed that optimum pH for many Aspergillus cellulases is near pH 5.0 as reported In the present study, it could be concluded that the historical by Vries and Visser (2001). Also, Sibtain et al. (2009)used pH paper documents are colonized by variety of fungi including of 5.5 for cellulases production from H. lixii. Similarly, Aspergillus niger, Aspergillus oryzae, Aspergillus melleus, Youssef (2011) previously described that maximum induction Penicillium commune, Penicillium chrysogenum, Mucor of FPUase activity of A. oryzae was achieved at pH 5. racemosus, Hypocrea lixxi and Schizophyllum commune. Furthermore, the pH requirement was determined at pH 5.0 Most of these isolates were known as contaminants of paper, for A. melleus as described by Ezekiel et al. (2010). but some of them never were observed on paper materials of libraries. Moreover, all of these fungal strains were found to possess cellulose degrading ability when grown on Artificial attack carboxymethyl-cellulose or on filter paper as a sole source of carbon. In parallel, the capacity of the isolates to change During the examination of the artificially contaminated sam- aesthetic aspect and cause the mass losses of artificially con- ples using pure fungal strains, unanticipated results were taminated paper was confirmed. The presence of these fungi obtained. All the isolated fungal strains demonstrated a high within the paper materials, and their ability to have a cellulo- capacity to grow on paper material after their reinoculation on lytic potential and degrade cellulose in vitro, demonstrated sterile Whatman paper. In addition, the margins were extreme- that these fungi could be responsible of the degradation and ly deteriorated, and in some areas, we observed the stains and the apparitionof foxingin historicalpaper. spots of different colors like those naturally affected by bio- This report may increase the knowledge about the micro- logical agents. In fact, the brownish spots that appeared on bial communities colonizing paper of ancient library mate- paper are the typical marks of foxing resulting from the effect rials, as well as the relation between decomposition of paper of biotic agents (Zotti et al. 2011), especially from the meta- and fungi cellulolytic enzyme activity. For that reason, it is bolic activity and pigments of the mycelia and spores. Table 2 useful to better study the other enzymatic activities of these shows that these cellulolytic strains were able to decompose fungi and others, like proteases and amylases that will enable filter paper cellulose at varying degrees. them to use glues, starch and other additives of paper as a Aspergillus niger shows black staining properties, charac- nutrient source for survival and maintenance. teristic of pigments (e.g., melanins) in the hypha of this fungus. Also, A. oryzae, A. melleus, M. racemosus and H. lixii produce respectively yellow, grey and green mycelia, could be responsible of intense stains. In most cases, the References colours of the stains are often characteristic of particular genus or species of fungi. It is common for most fungal species to Abrusci C, Martin-Gonzàlez A, Del Amo A, Catalina F, Collado J, Platas have aesthetical destructive effects by pigments and metabolic G (2005) Isolation and identification of bacteria and fungi from products. Such stains often badly disfigure the surface of cinematographic films. Int Biodeterior Biodegrad 56:58–68 paper, especially historical paper containing high levels of Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, deterioration products like acidic components. Sterflinger Lipman JD (1997) Gapped BLAST and PSI-BLAST: a new gener- ation of protein database search programs. Nucleic Acids Res 25: et al. (1999) described that fungal growth on objects of cul- 3389–3402 tural heritage often causes a serious aesthetical spoiling due to Andersen S, Frisvad JC, Sondergaard I, Rasmussen IS, Larsen LS (2011) colony formation and fungal pigments. Associations between fungal species and water-damaged building Besides, all the fungal strains were able to degrade filter materials. Appl Environ Microbiol 4180–4188 Arai H (1987) Microbial studies on the conservation of paper and related paper at varying degree. Table 2 shows that the maximum properties. Sci Conserv 26:43–26 decomposition was observed with M. racemosus (17.8 %), Arai H (2000) Foxing caused by fungi: twenty-five years of study. Int this species was capable of decomposing the component of Biodeterior Biodegrad 46:181–188 paper at two months at high levels, compared to the others. Ariffin H, Abdullah N, Umi Kalsom MS, Shirai Y, Hassan MA (2006) Production and characterization of cellulase by Bacillus pumilus This can be explained by the fact that the test conditions EB3. Int J Eng Technol 3:47–53 coincide with the optimum conditions of the growth or/and Cabero K, Pozzo T, Lidén G, Karlsson EN (2012) A cellulolytic of the cellulase production of this strains. However, the min- Hypocrea strain isolated from South American brave straw produces imum was found with A. melleus (10.2 %). The loss of a modular xylanase. Carbohydr Res 356:215–223 Chandra M, Kalra A, Sharma PK, Sangwan RS (2009) Cellulase produc- strength caused in the structure of paper is the result of tion by six Trichoderma spp. fermented on medicinal plant enzymatic decomposition of the components of paper by processings. J Ind Microbiol Biotechnol 36:605–609 fungi, and like final results, the paper become thinner and Chaverri P, Samuels GJ (2002) Hypocrea lixii, the teleomorph of weaker. Trichoderma harzianum. Mycol Prog 1:283–286 Ann Microbiol (2014) 64:815–822 821 Ciferri O, Tiano P, Mastromei G (2000) Of Microbes and Art. The role of Michaelsen A, Pinzari F, Ripka K, Lubitz W, Pinar G et al (2006) microbial communities in the degradation and protection of cultural Application of molecular techniques for identification of fungal heritage. Kluwer Academic, New York communities colonising paper material. Int Biodeterior Biodegrad Cojocariu A, Tanase C (2010) Macromycetes identified on the construc- 58:133–141 tion wood of historical monuments from Moldavia and causes of Montemartini CA, Ferroni A, Salvo VS (2003) Isolation of fungal species their development. J Plant Dev 17:63–68 from test samples and maps damaged by foxing, and correlation Coral G, Arikan B, Unaldi MN, Guvenments H (2002) Some properties between these species and the environment. Int Biodeterior of crude carboxymethyl cellulase of Aspergillus niger Zio. Wild Biodegrad 51:167–173 type strain. Turk J Biol 2:209–213 Neves ER, Schafer S, Phillips A, Canejo J, Filomena MM (2009) Das MKL, Prasad JS, Ahmed SK (1997) Endoglucanase production by Antifungal effect of different methyl and propyl paraben mixtures paper-degrading mycoflora. Lett Appl Microbiol 25:313–315 on the treatment of paper biodeterioration. Int Biodeterior Biodegrad De Castro AM, Pedro KC, Da Cruz JC, Ferreira MC (2010) Trichoderma 63:267–272 harzianum IOC-4038: a promising strain for the production of a Niranjane AP, Madhou P, Stevenson TW (2007) The effect of carbohy- cellulolytic complex with significant beta-glucosidase activity from drate carbon sources on the production of cellulase by Phlebia sugarcane bagasse cellulignin. Appl Biochem Biotechnol 162:2111– gigantean. Enzym Microbial Technol 40:1464–1468 2122 Nyuksha YP (1983) Some special cases of biological deterioration of Deacon JW (1997) Modern Mycology, 3rd edn. Blackwell Science, books. Restaurator 5:177–182 Oxford, p 97 Philippidis GP (1994) Cellulase production technology. In: Enzymatic Di Bonaventura MP, De Salle R, Bonacum J, Koestler RJ (2003) conversion of biomass for fuel production. (Eds): Himmel ME et al., Phylogenetic trees, fungal infections and the Tiffany’sdrawings. ACS symposium series 566 In: Saiz-Jimenez C (ed) Molecular Biology and Cultural Heritage. Porck HJ (2000) Rate of paper degradation: the predictive value of Balkema, Lisse, pp 131–135 artificial aging tests. The European Commission on Preservation Eida MF, Nagaoka T, Wasaki J, Kouno A (2011) Evaluation of cellulo- and Access lytic and hemicellulolytic abilities of fungi isolated from coffee Prieto B, Silva B (2005) Estimation of potential bioreceptivity of granitic residue and sawdust composts. Microbes Environ 26:220–227 rocks from their instrinsic properties. Int Biodeterior Biodegrad 56: Ezekiel CN, Odebode AC, Omenka RO, Adesioye FA (2010) Growth 206–215 response and comparative cellulase induction in soil fungi grown on Reese ET, Downing MH (1951) Activity of the Aspergilli on cellulose, different cellulose media. J Life Phys Sci 3:52–59 cellulose derivatives and wool. Mycologia 43:16–28 Fabbri AA, Ricelli A, Brasini S, Fanelli C (1997) Effect of different Reid ID (1998) Influence of incubation temperature on activity of antifungals on the control of paper biodeterioration caused by fungi. ligninolytic enzymes in sterile soil by Pleurotus sp.and Int Biodeterior Biodegrad 57:61–65 Dichomitus squalens. J Basic Microbiol 40:33–39 Fellers G, Wegener, Papparstekik (1991) Institutionen for pappar-stekik. Rojas JA, Cruz C, Mikan JF, Villalba LS, Cepero de Garcia MC, Restrepo Stockholm S (2009) Isoenzyme characterization of proteases and amylases and Florian MLE, Manning L (2000) SEM analysis of irregular fungal spots partial purification of proteases from filamentous fungi causing in an 1854 book: population dynamics and species identification. Int biodeterioration of industrial paper. Int Biotederior Biodegrad 63: Biodeterior Biodegrad 46:205–220 169–175. Gallo F (1992) Il biodeterioramento di libri e documenti. Centro di Studi Saoudi N (2008) La ville de Fès célèbre 1200 ans de rayonnement. Islam per la Conservazione della Carta. ICCROM, Roma Tour 37:54–60 Ghose TK (1987) Measurement of cellulase activities. Pure Appl Chem See P (1919) La florule du papier : Etude systématique et biologique de 59:257–268 champignons chromogènes du papier pique. (Nature, origine, agents Gopinath CBS, Anbu P, Hilda A (2005) Extracellular enzymatic activity et remèdes de 1’altération des papiers) Thesis Paris. France, Cited in profiles in fungi isolated from oil-rich environments. Mycoscience Zyska B (1997) Fungi isolated from library materials: A Rev of the 46:119–126 Literature Int Biodeterior Biodegrad 40: 43–51 Hölker U, Dohse J, Höfer M (2002) Extracellular laccases in ascomy- Shamsian A, Fata A, Mohajeri M, Ghazvini K (2006) Fungal contami- cetes Trichoderma atroviride and Trichoderma harzianum. Folia nations in historical manuscripts at Astan Quds museum library, Microbiol 47:423–427 Mashhad. Iran Int J Agric Biol 3:420–422 Hyvarinen A, Meklin T, Vepsa lainen A, Nevalainen A (2002) Fungi and Sibtain A, Ammara B, Huma S, Mubshara S, Amer J (2009) Production action bacterian moisture-damaged building materials – concentra- and purification of cellulose degrading enzymes from a filamentous tions and diversity. Int Biodeterior Biodegrad 40:27–37 fungus Trichoderma harzianum. Pak J Bot 41:1411–1419 Jahangeer A, Khan N, Jahangeer S, Sohail M, Shahzad S, Khan A (2005) Silva M, Moraes A, Nishikawa M, Gatti M, Vallim de Alencar M, Screening and characterization of fungal cellulases isolated from the Brandao L, Nobrega A (2006) Inactivation of fungi from deteriorat- native environmental source. Pak J Bot 37:739–748 ed paper materials by radiation. Int Biodeterior Biodegrad 57:163– Khalid M, Yang WJ, Kishwar N, Rajput ZI, Arijo AG (2006) Study of 167 cellulolytic soil fungi and two nova species and new medium. J Sohail M, Naseeb S, Sherwani SK, Sultana S, Aftab S, Shahzad S, Zhejiang Univ Sci B 7:459–466 Ahmad A, Khan SA (2009) Distribution of hydrolytic enzymes Kowalik R (1980) Microbiodeterioration of Library Materials. Restaurator among native fungi: Aspergillus the pre-dominant genus of hydro- 4:99–114 lase producer. Pak J Bot 41:2567–2582 Lee SM, Koo YM (2001) Pilot-scale production of cellulose using Sterflinger K, Krumbein WE, Rullkotter J (1999) Patination of marble Trichoderma reesei Rut C-30 in fed-batch mode. J Microbiol sandstone and granite by microbial communities. Z Dt Geol Ges Biotechnol 11:229–233 150:299–311 Lugauskas A, Krikstaponis A (2004) Microscopic fungi found in the Sukumaran RK, Singhania RR, Pandey A (2005) Microbial cellulases libraries of Vilnius and factors affecting their development. Indoor production, application and challenges. J Sci Ind Res 64:832– Built Environ 13:169–182 844 Lynd LR, Weimer PJ, Van Zyl WH, Pretorius IS (2002) Microbial Suresh BJ, Hari KS, Sekhar RKC (1999) Studies on the production ad cellulose utilization: fundamentals and biotechnology. Microbiol application of cellulase from Trictoderma reesei QM- 9414. Mol Biol Rev 66:506–577 Bioprocess Biosyst Eng 22:467–470 822 Ann Microbiol (2014) 64:815–822 Valencia JWA, Jiménez AV, Gonçalves de Siqueira F, Medina KD, Innis Gelfand MADH, Sninsky JJ, White TJ (eds) PCR protocols: A Restrepo Franco GM, Ferreira Filho EX, Siegfried BD, Grossi-de- guide to methods and applications. Academic Press, Inc., New York, Sa MF (2011) Holocellulase activity from Schizophyllum commune pp 315–322 grown on bamboo: a comparison with different substrate. Curr Youssef GA (2011) Physiological studies of cellulase complex enzymes Microbiol 63:581–587 of Aspergillus oryzae and characterization of carboxymethyl cellu- Valentin N, Garcia R, de Luis O, Maekawa S (2002) Air ventilation for lase. Afr J Microbiol Res 5:1311–1321 arresting microbial growth in archives. Quatrièmes Journées Zhang YH, Lynd LR (2004) Toward an aggregated understanding of Internationales d’Etudes de l’ARSAG 139–150. Paris enzymatic hydrolysis of cellulose: non complexed cellulase system. Van Whyk JPH, Mogale MA, Seseng TA (2000) Saccharification Biotechnol Bioeng 88:797–824 of used paper with different cellulase. Biotechnol Lett 22: Zotti M, Ferroni A, Calvini P (2011) Mycological and FTIR analysis of 491–494 biotic foxing on paper substrates. Int Biodeterior Biodegrad 65:569– Villena GK, Gutiérrez-Correa M (2007) Production of lignocellulolytic 578 enzymes by Aspergillus niger biofilms at variable water activities. Zyani M, Mortabit D, Mostakim M, Iraqui M, Haggoud A, Ettayebi M, Electron J Biotechnol 10:124–140 Ibnsouda KS (2009) Cellulolytic of fungi in wood degradation from Vries RP, Visser J (2001) Aspergillus enzymes involved in deg- an old house at the medina of Fez. Ann Microbiol 59:699–704 radation of plant cell wall polysaccharide. Microbiol Mol Zyska B (1993) Preservation of library materials, factor deteriorating Biol Rev 65:497–522 materials in library collections. Uniwersytet SIgski, Katowice, White TJ, Bruns T, Lee S, Taylor JW (1990) Amplification and direct Cited in Zyska B (1997) Fungi isolated from library materials: A sequencing of fungal ribosomal RNA genes for phylogenetics. In: review of the literature. Int Biodeter Biodegr 40: 43–51

Journal

Annals of Microbiology – Springer Journals

Published: Oct 18, 2013

There are no references for this article.