**Abstract**

Significant percent of world cultural heritage artifacts is threatened by fungal infestation. Fungi can deteriorate different substrates via various physical and chemical mechanisms. Hyphal growth and penetration into the substrate can cause symptoms like discoloration, biopitting, cracking, exfoliation and patina formation. On the other hand, chemical mechanisms include acid secretion, release of extracellular enzymes, pigment production, oxidation/reduction reactions and secondary mycogenic minerals formation. These processes can lead to serious, both esthetic and structural, alterations which may be irreversible and could permanently impair artworks. Proper isolation and identification of autochthonous isolates, as well as employment of different microscopic techniques and *in vitro* biodegradation tests are pivotal in understanding complex biodeterioration mechanisms caused by microorganisms, including fungal deteriogens. Biodeterioration and biodegradation studies require multidisciplinary approach and close collaboration of microbiologists, chemists, geologists and different personnel responsible for the safeguarding of cultural heritage monuments and artifacts, especially restorers and conservators.

**Keywords:** alterations, biodegradation, cultural heritage, fungi, multidisciplinary research

## **1. Introduction**

*Ars longa, vita brevis* – states the ancient Roman proverb, emphasizing that human need for artistic expression is as old as the civilization itself. Unfortunately, extant artworks are only a fragment of humanity's creations throughout history. Along with artistic creation, there is a need for protection of the artwork from external, frequently damaging influences. Since works of art are an essential part of the cultural heritage legacy of every nation, they ought to be protected for future generations. Biodeterioration is defined as any undesired alteration of the property of the material which is caused by living organisms and cultural heritage objects are frequently prone to this process [1]. Mentioned alterations can be induced by both macroorganisms (plants and animals) and microorganisms (bacteria, algae and fungi). Inadequate storage and irregular maintenance of artifacts in archives, museums and depots oftentimes favorize microbial, especially fungal, proliferation [2]. Since fungi are ubiquitous organisms, with pronounced metabolic activities, they

are capable of colonizing various types of microenvironments therefore constantly causing problems in cultural heritage collections around the world [3].

Fungal propagules - spores and mycelial fragments, are always present in the air, their concentrations being dependent on environmental factors [4, 5]. Namely, during their life cycle, fungi produce various types of sexual and asexual spores which are actively or passively released into the surrounding environment and dispersed by air currents to available substrates [3]. The successful colonization of available substrates requires propagules to be viable in addition to favorable growth conditions [4, 6]. It is known that due to their metabolic activities, numerous fungal species could cause both esthetic and physical damage to a variety of substrates, including stone, paint, paper, wood, textile and other materials of which cultural heritage artworks are made. Therefore, the application of adequate microscopic techniques, proper species identification and physiological characterization of autochthonous isolates are very important to appropriately assess potential threats to cultural heritage artworks, especially on those stored in inadequate conditions [3]. Consequently, biodeterioration and biodegradation studies require a multidisciplinary approach and a close collaboration of scientists (microbiologists, chemists, geologists etc.) and the specialists responsible for the safeguarding of cultural heritage objects, such as restorers and conservators. Therefore, this work addresses general mechanisms of biodeterioration caused by fungi and their role in the deterioration of different materials which constitute cultural heritage artworks.

### **2. Biodeterioration mechanisms**

Fungi present on artworks can affect them in two ways – mechanically and chemically. The aforementioned processes, more often than not, are taking place simultaneously. Depending on the substrate's nature, exogenic and endogenic factors, the effect of one process can prove more prominent than the other [7, 8]. Notably, depending on its location, fungal colonizers can affect the substrate in two ways – from the surface to its interior and *vice versa* [7].

#### **2.1 Physical processes**

Physical processes are taking place under the influence of hyphal apical growth or by the formation of fruiting bodies on the surface and/or the inner layers of the colonized material. If the fungal growth is superficial, it results in the formation of spreading mycelium which covers the substrate and changes the original appearance, hence the esthetic value of the artifact [7]. Inner fungal growth might lead to further damage of the artworks and, especially if paintings are concerned, to the detachment of painted layers (exfoliation). Melanized micromycetes are well known inducers of mechanical deterioration, especially of stone substrates, since melanin provides mechanical rigidness to fungal structures, enhances the turgor pressure and facilitates hyphal penetration [8, 9]. In order to study mechanical deterioration, the application of different microscopic techniques is pivotal, especially *in situ* optical microscopy and scanning electron microscopy (**Figure 1**). The multimicroscopic approach is essential to ensure detailed information, not only about the deterioration status, but also to elucidate alterations that affect works of art, and to detect potential biodeterioration "culprits" [10].

#### **2.2 Chemical processes**

Mechanisms of chemical biodeterioration are much more complex and prominent than physical ones. Fungi can chemically alter the substrate via assimilation

#### **Figure 1.**

*Scanning electron micrographs depicting deteriorated surfaces of cultural heritage objects: A, B. deteriorated icon silk fibers with cracks and gaps formed by Chaetomium globosum hyphal growth; C, D. profusion of Cladosporium sp. hyphal network with conidial mass on deteriorated wall painting; E, F. Mycobiont of lichenized fungi from the surface of deteriorated limestone monument; G, H. anamorphic state of Xylariaceae infesting wooden iconostasis.*

and dissimilation processes [11]. In case of the former, fungi utilize nutrients from the substrate by secreting various enzymes which catalyze the macromolecules' degradation. In contrast, dissimilation represents the production of various extracellular metabolites such as organic acids and pigments. These substances modify

or damage the colonized substrate. Since hyphae have high surface to volume ratio, these metabolites can quickly diffuse between the cells as well as from the cell into the substrate [7, 8]. Nowadays, various microbiological, biochemical and petrographical tests are employed to study chemical biodeterioration. *In vitro* tests provide rapid, cost effective estimation of fungal degradation capacity, which helps in evaluating a potential risk to cultural heritage artifacts [3].

**Acid production and acidolysis** are the most studied biodeterioration mechanisms, particularly on inorganic materials [8, 12]. Due to their metabolic activities, fungi produce organic acids such as gluconic, citric, oxalic, malic, succinic, itaconic etc. [13]. Once the spore germination occurs, organic acids are produced by respiration in mitochondria as intermendiary products of the citric acid cycle. If the fungi grow on nutrient enriched substrates, these acids are formed in excess and excreted as secondary metabolites [6]. The secreted acids then react with different substances via cation solubilization and chelation reactions. The reaction of acids with different metals (i.e. K, Fe and Mn) results in the formation of organic salts and complex compounds [7]. It should be mentioned that many organic acids, especially the oxalic, are able to chelate different metals in the process called complexolysis. The oxalic acid is able to form complexes with diverse metals (Ca, Mg, Fe, Cu and others), consequently leading to secondary mycogenic minerals formation, calcium oxalate being the most well-known [14]. The aforementioned crystals are present in patinas on stone, frescoes, oil paintings, glass, wood and other materials [8]. It is ascertained that most of the fungi have the ability, in greater or lesser extent, to produce oxalic acid, and subsequently precipitate oxalates [3]. Furthermore, CO2, as a product of respiration, in the conditions of increased humidity is transformed into carbonic acid, which then solubilizes calcium carbonate and magnesium carbonate present in limestone, mortar and gypsum. As a result, water soluble bicarbonates are formed. Additionally, increased H+ concentration favorizes the colonization of acidofilic fungi, which further facilitates the biodeterioration process [7, 8].

**Enzymes.** Fungi are able to digest organic matter, altering and weakening those materials, by the action of extracellular hydrolytic enzymes, such as lignocellulases, proteases, lipases, pectinases, chitinases, etc. [15, 16]. Enzymes that convert large, complex and often water-insoluble compounds (cellulose, hemicellulose, lignin, proteins and lipids) into low-molecular-weight soluble compounds, play an important role in the biodeterioration and biodegradation processes [7]. Although filamentous fungi primarily use simple sugars as a carbon source, they can be producers of lignocellulolytic enzymes to depolymerize wood or cellulose material for nutritional purposes [17]. Cellulolytic enzyme complex, which is responsible for degradation of cellulose to glucose monomers, comprises of: endoglucanase (hydrolyzes β-1,4-glycosidic bonds within cellulose fibers), exoglucanase (hydrolyzes β-glycosidic bonds and remove cellobiose units from the free ends of chains) and β**-**glucosidase (hydrolyzes cellobiose and cellodextrin to glucose) [18, 19]. Hemicellulases hydrolyze hemicellulose, which is made up of hexoses (mannose, glucose, galactose) and pentoses (xylose, arabinose) to monomeric sugars and acetic acid. The complex of enzymes that hydrolyze hemicellulose consists of at least eight enzymes: endo-1,4-β-D xylanase, exo-1,4-β-D xylocuronidase, α-L arabinofuranosidase, endo-1,4-β- D mananase, β-mannosidase, acetyl-acid esterase, α-glucuronidase and α-galactosidase [19]. Lignin degradation, characteristic for white-rot fungi, is catalyzed by nonspecific polyphenol oxidases: manganese oxidizing peroxidases, lignin peroxidases, and laccase. This process involves breaking inter-monomer bonds, demethylation, hydroxylation, side chain modification, and aromatic ring cleavage [20, 21]. Some fungal species inhabit art objects that are substrates rich in fibrillar proteins (wool, parchment, leather, silk, etc.). Proteolytic enzymes (proteases) degrade various protein fibers such as collagen (wool), fibroin

#### *Fungal Deterioration of Cultural Heritage Objects DOI: http://dx.doi.org/10.5772/intechopen.98620*

(silk) and keratin (parchment) [11]. Lipases catalyze the hydrolysis of triacylglycerols to glycerol and fatty acids. These enzymes might take part in the degradation of widely used painting constituents, linseed and shellac, derived primarily from unsaturated oleic, linoleic and linolenic acids [22].

**Pigment production**. Micromycetes produce pigments which vary in chemical composition and color and are species specific [7, 8]. They are present in hyphae, conidia, or are secreted into the substrate whilst their production is determined by the availability of nutrients and minerals, UV radiation, pH, temperature and other environmental factors [6]. Pigment secretion on/into the substrate leads to the appearance of different, frequently irreversible, colorations leading to the observable changes on cultural heritage objects. This diminishes the aesthetic value of the artwork and accelerates biodeterioration process [7, 11, 23]. Many fungi produce dark colored pigments – melanins, which are responsible for characteristic brownish color of mycelia and reproductive structures. These water insoluble, very stable and resistant, molecules are formed by oxidative polymerization of phenolic compounds (ortho-dihydroxy phenols) [24]. Dark colored melanins are characteristic for dematiaceous fungi (representatives of the former family Deamtiaceae) and are present in cell walls in both granular of amorphic form, while their amount increases with aging [6, 25]. On the other hand, the secretion of various water soluble exopigments into the substrate can also cause different aesthetic damage to artworks, especially on those made from organic materials. A vast number of different pigments are identified and grouped in three main families: the derivatives of toluquinone, of naphthoquinone and of beta-methyl-quinone [8].
