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Introduction
Cultural Heritage, either movable or immovable, is subject to degradation induced by diverse living organisms. Fungi are among the most active microorganisms in these processes. The nature of the support will determine the type of degradation. The alteration mechanisms are different on organic supports (wood, leather, textiles, etc.) and on inorganic supports (stone, glass, metals, etc.) due to the heterotrophic nutrition of fungi. While fungi can use the organic support itself as nutrients, in the case of inorganic supports these are transformed by several metabolites which are excreted and that may react with the support in different ways. Fungi Biology Fungi are living organisms that constitute an entire kingdom, which shows the great dimension of their diversity. As expected, they have numerous common characteristics, the main one being the heterotrophic nutrition, which means that they need organic matter for their metabolism. Most fungi are saprophytes, thus they decompose the organic matter in order to absorb the substances that are formed by that process. Therefore, the assimilation of nutrients is made by absorption of the necessary substances. Fungi are composed of thallus which may be unicellular or pluricellular. The latter is filamentous, the filaments are called hyphae and may be aseptate or coenocytic (without septa). In addition, fungi are generally composed of a fruiting body where the spores are produced in a great variety of colours, forms and sizes. The cell walls of fungi generally contain chitin besides other components. Their reproduction is sexual or asexual. Beside the presence of organic matter, fungi need for their development suitable parameters of environmental conditions such as humidity and temperature. If these conditions are adverse to the fungi needs, they will operate as limiting parameters of their development. Organic Materials The organic materials from which works of art are normally constituted belong to two main groups: cellulosic and proteinaceous. Among the first group are wood, paper and some textiles while the second contains other textiles such as wool and silk, and also leather and parchment. Fungi deteriorate organic material in respect to the aesthetic and degradative aspect although these are not independent as the aesthetic degradation is sometimes due to the external manifestation of the chemical transformation process that the support undergoes. Some other times it is simply a matter of stains or other alterations easy to solve, although this occurs in just a few cases. Cellulosic Supports The action of fungi on cellulosic supports is common for cellulolytic fungi that perform external digestion through cellulase complex enzymes that degrade the cellulose in basic molecules of glucose which are then absorbed by the fungus. However, there are other groups beside the cellulolytic fungi that attack the other components of the support as well. Taking the wood for example, we know that besides cellulose it is constituted by lignin which is far more difficult to digest by the fungus but even then there are several species that can achieve it successfully. Lignin is an amorphous polymer formed by the random combination of various phenols and acids that leads to a three-dimensional structure. The effects of some fungi on cellulose and lignin are known as rot. Three types of rot can be highlighted according to the residual state of the wood fibre after having been attacked by certain fungi. Brown rot. Also known as prismatic and dry, it occurs when the fungus attacks the cellulose and the short chains of other polysaccharides, leaving traces of lignin. In case the wood loses between 10% and 20% of its weight, it may lose up to 95% of its mechanical resistance which for the wood used in buildings (beams, altarpieces, etc.) may be really dangerous. In the case of movable heritage the immediate consequence could be the loss of the work depending on the degree of degradation. The wood darkens and dries forming a typically cubic craquelure network, both in the longitudinal and transversal fibbers. The fungi that cause this kind of rot are especially the Basidiomycetes such as Serpula lacrymans or Merulius lacrymans. White rot. Also known as corrosive and cavernous, is caused by the fungi that attack both lignin and cellulose through a system of ligninase and cellulase enzymes, leaving behind a white residue and inducing gradually weight loss. They need very high moisture content (30-60%) appearing mostly in the wood near the ground, such as sarcophagus and materials in basements, and near ceilings and walls, such as wood coffered ceilings (artesonados) and altarpieces. The wood might even lose all its resistance, becoming spongy, filamentous or laminated, and usually with a stained and discoloured aspect when compared to healthy wood. This type of rot is especially produced by the species of the genera Pholiota sp., Coriolus versicolor, Fomes sp., etc. Soft rot. In this kind of rot the fungi attack preferentially the cellulose of the secondary wall leaving the wood with a consistency similar to fresh cheese. However, they can also attack the hemicellulose and in a much lower degree the lignin. This type of rot is especially common in soaked wood, in conditions of high humidity and in wood that is in contact with the ground, in areas of archaeological diggings and in underground, underwater or water-saturated environments. These are Ascomycetes and Deuteromycetes fungi from where the following genera may be highlighted: Chaetomium, Xylaria, Alternaria, Coniothyrium, etc. Most part of the cellular wall is destroyed forming a typical craquelure when the wood is dried after the rotting process. Besides rotting wood, fungi also produce many other alterations that may not have such dangerous consequences but that are equally undesirable when wood is the support of works of art. Among these are the colorformers, fungi that stain the wood either through several pigmentations that they synthesize or by dark colour hyphae. A particular case of these processes is the so called blue-stain of the wood in which the fungus attacks the reserve cells but not the xylem. Thus, the wood resistance is not compromised which is very important for structural wood but not for ornamental wood, once it can undergo loss of pigment or other alterations. Among the fungi that produce these processes are the species of the genera Chlorociboria, Aspergillus, Aureobasidium, Fusarium, Penicillium, Trichoderma and Chaetomium.
Continuing with the cellulosic supports, it is important to have in attention the paper, which is part of a variety of movable heritage, such as documents, books, paintings, etc. Old papers are primarily made from cellulose although they can contain other compounds depending on the manufacturing process. It is not out of the ordinary for paper to contain certain quantities of lignin, hemicellulose, pectin, dyes, proteins, etc.
The fungi that affect paper may be cellulolytic, degrading thus the cellulose, or non-cellulolytic, degrading any of the other compounds. Some cellulosic and proteinic alterations can affect the mechanical resistance and the weight of the paper, while others affect the aesthetics of the work by pigmentation or discolouration as a consequence of both endo- and exopigments produced by fungi. All these processes, of course, are conditioned by the quantity of moisture that the support contains and the environmental conditions. Among the cellulolytic fungi some species of the genera Alternaria spp., Aspergillus spp., Fusarium spp., Humicola, Myrothecium, Penicillium spp., Stachybotrys, etc. may be found and among the noncellulolytic, several species of Chaetomium. These fungi have been frequently found in books, documents and prints. There are two very frequent alterations of paper: foxing and moisture-induced consolidation of paper. In both cases, fungi are among the main causes of these alterations along with other microorganisms. Textiles of vegetable origin, including cotton, linen, jute and sisal (hemp) are subject to a particular case of fungi action on cellulosic supports. These are composed of cellulose derivatives: linen of flax phloem fibbers, sisal of leave fibbers and cotton of seeds. Among the most frequent alterations produced by fungi on textiles are stains, discolouration and resistance loss. Susceptibility to fungi attack depends on both the cellulose content and on other non-cellulosic compounds. For example, the presence of lignin decreases the susceptibility of attack while pectin and pentose increase it. Cotton contains a 5% of non-cellulosic compounds and linen 15%. Textiles with high content of lignin are more resilient to microbial attack than those that contain less lignin. Protein Materials Protein and cellulosic materials undergo a similar degradation process, except for those that are specific to each of the support compounds. Fungi degrade proteins. Proteins are polymers composed of polypeptides, which are made of amino acids. For the decomposition of these, living organisms use two types of enzymes, peptidases and proteinases. The function of these proteolytic enzymes is to separate the proteins in peptides and then into amino acids for an easier use by the fungal cells. The most common protein supports are parchment and leather. The most frequent alterations produced by fungi in these materials are granulations, stains, loss of elasticity and stiffness. Parchment has its origin in the city of Pergamon from where its name derives. It was made from non-tanned skins of lamb, goat, pig and donkey. A particular case was the vellum, made from lamb and calf embryos. Parchment is composed mainly of collagen but also has other substances such as keratin and elastin, and smaller amounts of albumin and globulin. Fungi can cause proteolysis of collagen, but there are a number of factors that facilitate the process, such as the storage environmental conditions and some substances that reside in the original skin (other proteins, lipids, carbohydrates, mineral constituents and impurities) which can also be used by the fungi metabolism and facilitate their colonization. Among the types of fungi found in ancient scrolls are Cladosporium, Fusarium, Ophiostoma, Scopulariopsis, Aspergillus, Penicillium, Trichoderma, etc. Chemically the leather is very similar to parchment but it undergoes a skin tanning process. Its susceptibility to biodeterioration by fungi is also similar but varies according to the different tanning process and the type of dyes used, such as animal, vegetable, chrome tanning, etc. The latter has a fungistatic capacity which serves as protection to microbiologic attack. Sometimes, however, some species of the genera Penicillium and Paelomyces, which are tolerant to chromium-based dyes, have developed on tanned leather. In this case, the proteins are not directly affected by the fungi but the leather is attacked by the organic acids they produce. Besides the mentioned protein supports we should not forget other important and extensively used supports in cultural heritage that are textiles, in particular wool and silk used in clothing, flags, banners, etc. The fibres of these are composed of fibrous protein structure which confers them a high resistance to microbial attack. Under certain conditions, however, there are a number of bacteria and fungi capable of degrading them. Among the fungi, representatives of the genera Fusarium, Aspergillus and Trichoderma stand out. Inorganic Materials The biodeterioration of inorganic supports is radically different because as fungi are heterotrophic organisms they do not use the supports for nutrition but they do alter them deeply with synthesis products from their own metabolism, such as inorganic and organic acids. The latter can produce chelation and form complexes with metallic cations, which are obtained from the support. In the case of stone monuments, the development of fungal colonies appears over layers of organic matter of different origins. Species of fungi of different genera such as Cladosporium herbarum, Aspergillus niger, Stachybotrys spp. and Alternaria have been found on these supports. Many of these fungi are responsible, along with other chemical and biological factors, for the formation of black crusts due to the melanin in their hyphae. The hyphae of the fungus can penetrate the limestone calcite crystals previously dissolved by enzymes. Some fungi are called endolithic because they penetrate into the substrate causing "pitting", a surface that appears to have many small holes. This alteration has been found on monuments such as the gate of the Cathedral of Huesca in Spain, shown in Figure 5. Due to the presence of organic acids produced and excreted by the fungi, the stone support suffers a decrease of pH. Acids may produce chelation, among which the oxalic acid that induces a large corrosion of primary minerals and the complete decomposition of iron-based components of clay. Organic acids also destroy the feldspar in granites and participate in the sandstone weathering. Control and eradication of biodeterioration produced by fungi in cultural heritage Once we understand the way fungi act in the biodeterioration of works of art, it is very important to know which methods of control and eradication are available for treatment. Of course, the intervention will be different for movable or immovable heritage and will depend on the organic or inorganic nature of the support. The first phase is the identification of the attack, which means we must confirm that there is truly a fungal attack. To this end, a sample should be taken in order to identify the species or the cause of the problem. By knowing which species we deal with, we know which damage can occur, according to its metabolic needs - if there is a species that causes an aesthetic damage or one inducing chemical degradation of the support, for example. The identification of fungal species may be performed in 2 ways: by traditional identification methods, using optical and scanning electron microscopy or by modern techniques of DNA identification using PCR and sequencing. In the first case the samples obtained directly from the object are grown in a culture media suitable for fungi. Later, using the techniques of cellular biology that include specific staining and microscopic observation, determinant characteristics such as shape and size of the spores’ fruiting bodies are detected. Subsequently, the classification is made with aid of dichotomous classification tables to obtain the identification of the species involved. In immovable works, through the cultivation methods calculations can be done to find out, for example, not only which pollutants but also how many contaminants are in the environmental samples, which is clearly very useful to estimate the contamination degree. Nowadays, the new molecular biology techniques are gradually adapting to the study of the biodeterioration of cultural heritage, such as the Polymerase Chain Reaction (PCR). Using this technique, a complex mixture of DNA can be taken to localize a single gene (rRNA 18S in fungi), to multiply it and to obtain a pure solution for study. Potential applications of PCR are virtually limitless. Roughly the protocol that is followed is: 1. Culture or environmental sampling; 2. The environmental samples are subject to freezethaw cycles (-20º C, +60º C) for DNA extraction. Once the DNA is obtained, a first PCR is performed for amplification if possible, using a series of reagents to determine the initial concentrations. Protocols are already established for other areas in biology. The DNA amplification is achieved by using a temperature ramp. 3. The results of the first PCR are then subjected to a first DGGE (Denaturing Gradient Gel Electrophoresis) and the results are checked with agarose gel. 4. The product of the first PCR is used as DNA template for making a second PCR whose results are subject to an environmental DGGE. 5. The results are checked with agarose gel and DNA is extracted from the bands in order to proceed with their sequencing. Sometimes a third PCR is required. The sequences obtained are compared with the NCBI database and only those sequences above 95% are considered. The process for the culture samples is shorter once we already started from isolated microorganism unlike with environmental samples where different DNA is mixed. Thus, in the second case a single PCR and a single DGEE may be enough. Despite the work load and high cost of this technique, it presents a series of advantages with respect to the traditional analysis, namely the accuracy in the species identification, the smaller quantity of the sample required for the identification, which is truly important in cultural heritage, and the retrieval of more real contaminant data. However, the efforts and the expenses should be taken into consideration, depending on the seriousness of the contamination, the extension of the problem and the nature of the support. In inorganic supports of immovable heritage these techniques are being increasingly used, not only for fungi but especially for bacteria. In works on wooden support which are placed inside museums and in controlled environmental conditions, where contaminants are known because they are more specific, conventional analysis methods are generally used. Once the fungus or fungi are identified, their removal should be addressed taking into account a series of factors: 1- Works located in a museum, archive, library, etc. 2- Works located in an exterior environment: movable and immovable. To remove the biodeterioration produced by fungi, in the first place we ought to eliminate the conditions that foster their development, such as environmental conditions (humidity and temperature), nutrients, light, etc. If we take the example of museums, we can consider on one side the museum spaces and on the other the collections. It ought to provide the building with a suitable climate, to eliminate all the humidity sources and to keep it under stable conditions, never below 50% or above 62%. Concerning the temperature, this should never be higher than 20º C. These conditions are ideal for museums, as we already know, but to control the biodeterioration not only the needs of fungi are to be kept in mind. The problem should be tackled by evaluating the overall, including all the contaminants and, of course, the supports, since important variations of the parameters may affect them considerably. Concerning the living organisms, fungi may develop at relatively low temperatures, but although they have an ideal range (25 - 28º C) we all have experienced their development in refrigerators at temperatures of 5º C. Instead, they are very demanding with humidity which should be relatively high, although the spores may endure lower humidity levels. Although beneficial for many photosynthetic organisms and microorganisms, too much light is harmful for fungi, which always look for the darkest places. The nutrients in the inorganic supports are more controllable because fungi need organic matter. Thus, it ought to be careful in conservation so that no dust particles could provide organic matter that might be used as nutrients or other microbial contaminants. In case of collections, it is much easier to control the environmental conditions but the nature of the support is determinant because in most cases it is made of mixed materials and if it is organic, it can be a nutrient itself. If a work is contaminated, the best way to proceed is to isolate it from others and to treat it. The treatment will always depend of the extension and severity of the attack and the degree of damage of the support. Sometimes a simple mechanical removal may be effective although in most cases the use of a biocide is necessary. The objects located in the exterior present a completely different problematic for their conservation as it is not possible to control the climatic and environmental conditions. In any case the proliferation of water leaks and the ascension of water by capillarity should be prevented, as this favours not only the development of fungi but also of other microbial contaminants and the development of mosses, lichens and vascular plants. In such cases, the use of a broad-spectrum biocide is necessary in order to remove the contaminants. Acknowledgments I would like to thank the Laboratory of Biology from the Gabinete de Conservación y Restauración de la Oficina del Historiador de la Ciudad de La Habana, Cuba, for the use of the photos in Figures 3 and 4B and to Dr. Amelia Fernandez from La Habana for the photo in Figure 2B. The other photos belong to the present author and to Instituto del Patrimonio Cultural de España (IPCE). Bibliography [1] C. Ascaso, “Structural aspects of lichens invading their substrata", in Surface Physiology of Lichens, C. Vicente, D.H. Brown and M.E Legaz (eds.), Universidad Complutense de Madrid, Madrid, Spain, 1985, pp. 87-113 [2] R.M. Atlas, N.A. Chowdhury and K.L. Gauri, “Microbial calcification of gypsum-rock and sulfated marble”, Studies in Conservation 33, 1988, pp. 149-153 [3] E. Bryant, Climate process & change, Cambridge University Press, Cambridge, 1997 [4] G. Caneva, M.P. 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Jaton, “Facteurs biologiques de l'altération des pierres”, Biodeterioration of Materials, Elsevier, London, 1968, pp. 358-268 [21] O. Salvadori and L. Lazarini, “Lichens deterioration on stones of Aquileian monuments”, Botanika Chronika, 1989, in press [22] M.I. Sarró, A.M. García, V.M. Rivalta, D.A. Moreno and I. Arroyo, “Biodeterioration of the Lions Fountain at the Alhambra Palace, Granada (Spain)”, Building and Environment 41, 2006, pp. 1811-1820 [23] M.I. Sarró and I. Arroyo, “Microbiología y Biología molecular aplicada al patrimonio en el IPHE”, Bienes Culturales: Revista del Instituto del Patrimonio Histórico Español 8, 2008, pp. 197-210 [24] L. Tronchoni, “Patologías de materiales pétreos”, in Generalitat Valenciana, Conselleria de Cultura, Educació i Ciencia, Direcció General de Patrimonio Artísic (ed.), XII Congreso de Conservación y Restauración de Bienes Culturales, Valencia, 1998, pp. 341–352 About the author Irene Arroyo Conservation-scientist Contact: irene.arroyo@mcu.es Irene Arroyo Marcos, PhD, is specialised in Biology and its applications to cultural heritage conservation. She works since 1988 at the Scientific Department of the Institute of Cultural Heritage of Spain, Ministry of Culture. Previously she was lecturer of Biology at the University College Cardenal Cisneros and she worked at the Royal Botanic Garden of CSIC, the High Council of Scientific Research. As part of CSIC she has participated in conservation projects such as the conservation of the Romanic cloister of the cathedral of Pamplona and the conservation of the dome Regina Martyrum of the Basilica of Pilar from Zaragoza, Spain. Aside her research activity, she has taught as invited lecturer in masters and short courses in Spain and abroad.
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