Current Knowledge on the Fungal Degradation Abilities Profiled through Biodeteriorative Plate Essays

Fungi are known to contribute to the development of drastic biodeterioration of historical and valuable cultural heritage materials. Understandably, studies in this area are increasingly reliant on modern molecular biology techniques due to the enormous benefits they offer. However, classical culture dependent methodologies still offer the advantage of allowing fungal species biodeteriorative profiles to be studied in great detail. Both the essays available and the results concerning distinct fungal species biodeteriorative profiles obtained by amended plate essays, remain scattered and in need of a deep summarization. As such, the present work attempts to provide an overview of available options for this profiling, while also providing a summary of currently known fungal species putative biodeteriorative abilities solely obtained by the application of these methodologies. Consequently, this work also provides a series of checklists that can be helpful to microbiologists, restorers and conservation workers when attempting to safeguard cultural heritage materials worldwide from biodeterioration.


Introduction
The Fungal Kingdom comprises a highly diverse eukaryotic group able to inhabit every ecological niche available on the Planet [1]. The growth and biological activity of fungal species in cultural heritage materials is known to develop serious damages by means of biodeterioration (the undesirable modifications of a valuable material occurring by the action of living organisms) [2,3]. Fungi are highly versatile, ubiquitous, chemoheterotrophic microorganisms, being able to grow in a vast number of materials and contributing to the development of various biodeterioration phenomena [2,3]. Such modifications are a result from fungal species settling, development and exploitation of various organic and inorganic compounds present in historic art-pieces and monuments [2][3][4][5][6][7][8][9][10][11][12][13][14][15]. The fungal biodeterioration of books, paper, parchment, textiles, photographs, paintings, sculptures and wooden materials occurs due to the aesthetic modifications, mechanical pressure and exoenzymatic action [2]. Various components of these materials such as cellulose, collagen, linen, glues, inks, waxes and organic binders can be oxidized, hydrolyzed, dissolved, stained or structurally modified as a result of the action of fungal enzymes, pigments and organic acids [2,3,[7][8][9][10][11]. A typical and widely known example of these phenomena is known as "foxing", the development of red-brownish localized spots, hypothesized to be a result from fungal proliferation and metabolization of organic acids, oligosaccharides and proteic compounds that can stain and modify the constituent materials of many paper-based and photographic supports [3,8,13]. Another example of microorganism's attack of organic materials is related to the biodeterioration of human remains, mummies and funerary materials, where opportunistic, saprotrophic and highly cellulolytic and proteolytic taxa are able to thrive and trough their actions severely alter them [2,14,15]. Complementarily, historic relics mainly composed of inorganic components such as stone, frescoes, glass and ceramics can also suffer deep aesthetical, physical and chemical modifications resulting from fungal grow and action [2][3][4][5][6]12,16]. In these supports, deterioration is caused by hyphae penetration into the substrate, the production and release of extracellular destructive organic acids, enzymes and metabolites and by the the formation of distinct colored outlines as a result of fungi high pigment contents, contribution to biofilm development and chemical reactions with inorganic compounds [2][3][4][5][6].
Due to the known biodeterioration problems arising from their proliferation, the accurate species identification and a consequent deteriorative profiling of isolates are crucial steps towards the development and the establishment of proper protective measures for the diverse cultural heritage treasures around the world. With the recent development of innovative culture independent methodologies such as -omics technologies, molecular data is becoming increasingly more valuable for the identification of the microbes, the characterization of their metabolic functions and their deteriorative byproducts [17]. Methodologies such as metagenomics, transcriptomics, metabolomics and proteomics revolutionized the field and are increasingly allowing understanding of microbial diversity, but also species specific and holistic contributions to various materials biodeterioration phenomena [17]. These methods are particularly relevant considering that traditional cultivation dependent methodologies hold the disadvantage of being unable to correctly infer microorganism's abundance and only allow the study of active forms, failing to provide information regarding viable non-culturable and non-viable forms [17][18][19][20][21][22][23][24][25][26][27]. Nonetheless, classical culture dependent methodologies still offer an important advantage when compared with modern methodologies, especially when considering that the isolation of microbes allows their natural biodeteriorative profiles to be studied in great detail. Culture media plates modified to specify a positive biodeteriorative ability upon the microorganism development and deteriorative action (see Figure 1 for examples) can provide valuable data that allow the evaluation of the microorganism's putative risks to cultural heritage materials. Moreover, they also offer a highly informative, rapid and low-cost platform [28] that can help in a quick and focused decision-making process aiming to protect valuable artifacts. Currently, plate assays aiming to identify fungal deteriorative characteristics, such as calcium carbonate solubilization, mineralization and various enzymatic activities, have been proposed and somewhat widely used.
Appl. Sci. 2021, 11, x FOR PEER REVIEW 2 of 37 them [2,14,15]. Complementarily, historic relics mainly composed of inorganic components such as stone, frescoes, glass and ceramics can also suffer deep aesthetical, physical and chemical modifications resulting from fungal grow and action [2][3][4][5][6]12,16]. In these supports, deterioration is caused by hyphae penetration into the substrate, the production and release of extracellular destructive organic acids, enzymes and metabolites and by the the formation of distinct colored outlines as a result of fungi high pigment contents, contribution to biofilm development and chemical reactions with inorganic compounds [2][3][4][5][6].
Due to the known biodeterioration problems arising from their proliferation, the accurate species identification and a consequent deteriorative profiling of isolates are crucial steps towards the development and the establishment of proper protective measures for the diverse cultural heritage treasures around the world. With the recent development of innovative culture independent methodologies such as -omics technologies, molecular data is becoming increasingly more valuable for the identification of the microbes, the characterization of their metabolic functions and their deteriorative byproducts [17]. Methodologies such as metagenomics, transcriptomics, metabolomics and proteomics revolutionized the field and are increasingly allowing understanding of microbial diversity, but also species specific and holistic contributions to various materials biodeterioration phenomena [17]. These methods are particularly relevant considering that traditional cultivation dependent methodologies hold the disadvantage of being unable to correctly infer microorganism's abundance and only allow the study of active forms, failing to provide information regarding viable non-culturable and non-viable forms [17][18][19][20][21][22][23][24][25][26][27]. Nonetheless, classical culture dependent methodologies still offer an important advantage when compared with modern methodologies, especially when considering that the isolation of microbes allows their natural biodeteriorative profiles to be studied in great detail. Culture media plates modified to specify a positive biodeteriorative ability upon the microorganism development and deteriorative action (see Figure 1 for examples) can provide valuable data that allow the evaluation of the microorganism's putative risks to cultural heritage materials. Moreover, they also offer a highly informative, rapid and low-cost platform [28] that can help in a quick and focused decision-making process aiming to protect valuable artifacts. Currently, plate assays aiming to identify fungal deteriorative characteristics, such as calcium carbonate solubilization, mineralization and various enzymatic activities, have been proposed and somewhat widely used.  Although differences among distinct isolates, assays and incubation conditions are known and expected, the available literature concerning distinctive fungal species deteriorative profiles obtained using such methodologies remains pending a deep summa-rization. With this in mind, this work aims to provide an overview of available plate assays, as well as the fungal putative biodeteriorative profiles obtained solely through such tests so far. In addition, we also aimed at providing a series of quick and straight forward checklists that can be consulted by microbiologists, restorers and conservation staff, when working to safeguard important cultural heritage materials worldwide. These checklists were also annotated to contain currently accepted fungal names according to Mycobank (www.mycobank.org, last accessed on 26 April 2021) and Index Fungorum (www.indexfungorum.org, last accessed on 26 April 2021) in order to ensure an updated identification for fungi displaying such profiles, and to facilitate information sharing in the future.

Calcium Carbonate Solubilization or Dissolution
One of the greatest fungal effects on stone monuments is credited to their secretion of inorganic and organic acids that can alter the material properties [2][3][4][29][30][31][32]. In fact, carbonate weathering has been consistently linked to the excretion and action of these metabolites [33][34][35]. Evaluation of fungal calcium carbonate solubilization abilities in cultural heritage scenarios has been helpful to study the biodeteriorative contribution of isolates retrieved from air, mural paintings, wooden art objects, frescoes, catacombs, bricks, concrete, buildings and various limestone and plaster monuments and museums [28,33,[35][36][37][38][39][40][41][42][43][44]. Fungal calcium carbonate solubilization ability screening is usually conducted with CaCO 3 glucose agar and adapted formulations [33]. Nonetheless, the application of Malt extract agar and Reasoner's 2A agar amended with CaCO 3 (CMEA and CR2A) has also been successfully achieved [35]. Moreover, Kiyuna and colleagues [37] also highlighted the utility of Glucose Yeast extract calcium carbonate agar (GYC) [45] for such evaluation. Positive CaCO 3 dissolution is usually evaluated by the visualization of a halo around the growing colony after a period of incubation. In addition, calcium carbonate solubilization screening can also be conducted coupled with the evaluation of the media pH modifications. For this purpose, Creatine Sucrose agar (CREA) [46] followed by the analysis of medium color changes around growing colonies, or liquid media according to the formulations provided by Borrego and colleagues [47] followed by pH analysis, can also be applied. A quick overview of the known fungal species able of CaCO 3 dissolution points that isolates from more than fifty species have been found to display this biodeteriorative profile, with the great majority of them being Aspergillus and Penicillium species (Table 1). Both genera are known important biodeteriogens, producing various acidic molecules and contributing to the deterioration of materials [28,48]. The detection of species from these genera (as well as others for example, from Pestalotiopsis and Talaromyces among others) might indicate a putative threat to acid susceptible resources, such is the case of stone structures, mural paintings and frescoes.

Mineralization or Crystallization Development
Calcium carbonate solubilization by the action of fungal acids can often occur coupled with the recrystallization of minerals in the substrate [2,3,5,6,[49][50][51]. Such mineralization singularities can lead to the development of various biodeterioration phenomena [52,53]. They occur from the reactions of secreted acids (especially oxalic acid) with stone cations [32] and often result in the formation of carbonates and/or calcium magnesium oxalates [5,6]. Characterization of fungal crystallization abilities in cultural heritage scenarios has been helpful to study the biodeteriorative contribution of isolates retrieved from air, limestone monuments, stone stela, wall and mural paintings [35,[41][42][43][53][54][55][56]. Fungal mineralization ability screening is usually conducted using B4 (with calcium acetate) or modified B4 (with calcium carbonate) media and adapted formulations [57]. Moreover, CaCO 3 modified Malt agar, Nutrient agar (NA) with CaCl 2 and the above mentioned CMEA and CR2A media have also been found useful for such purposes [35,42,43,58,59]. Positive mineralization development is usually evaluated by the microscopical visualization of neo-formed minerals around or in fungal hyphae after a period of incubation. Moreover, further characterization of these crystals can also be achieved by applying analytical methodologies such as X-ray powder diffraction (XRD) and/or energy dispersive X-ray spectroscopy (EDS) in conjunction with scanning electron microscopy (SEM) methodologies. So far, circa sixty species have been found to display mineralization abilities in plate essays and, as similarly found for fungal calcium carbonate dissolution, multiple Aspergillus and Penicillium species have also denoted this biodeteriorative profile ( Table 2). Such findings can be correlated with their long-known abilities to secrete oxalic acid, among various other acids [41]. Nonetheless, a relevant number of species from genera Alternaria, Cladosporium, Colletotrichum, Pestalotiopsis and Trichoderma putatively displaying these biodeteriorative abilities can also be verified. Typical minerals detected include calcium carbonate in the form of calcite and vaterite-calcite, weddellite, whewellite, hydroxyapatite, hydrocerussite, pyromorphite, phosphate and other still unidentified calcium oxalates and minerals. The detection of species from these genera might indicate a putative threat to materials highly susceptible to fungal acidolysis and biomineralization, such is the case of limestone monuments and murals [5,6,60].

Enzymatic Action
Fungal ligninolytic action is often considered a threat to wooden structures [61][62][63][64][65][66]. Cultural heritage materials constructed with these materials can be affected by fungal hyphae penetration but also by the action of various exoenzymes [67]. Moreover, brown and white rot fungi are known to contribute to these substrates' deterioration and degradation in various contexts [68]. So far, fungal ligninolytic ability characterization in cultural heritage scenarios has been helpful to study the biodeteriorative contribution of isolates retrieved from air, wooden materials and art objects [61,69,70]. Ligninolytic ability screening can be conducted using media with Azure B (lignin peroxidase), Phenol Red (Mn peroxidase), Remazol Brilliant Blue R (laccase) [71][72][73][74] or, alternatively, by applying Potato Dextrose agar supplemented with guaiacol (PDA-guaiacol) [61,70]. Positive ligninolytic ability is usually evaluated by the clearance of the media specific color (Azure B, Phenol Red and Remazol Brilliant Blue R) or by the development of reddish-brown zones (PDA-guaiacol) after a period of incubation. As pointed by Pangallo and colleagues [36,69], data regarding ligninolytic abilities of filamentous fungi in biodeterioration contexts is still somewhat scarce. Nonetheless, as evidenced by Table 3, almost thirty species have been found to display these biodeteriorative abilities. Moreover, mainly species of genera Aspergillus, Chaetomium, Cladosporium and Penicillium represent the bulk of the currently studied lignin deteriorating fungi. As such, the detection of species from these genera might indicate a putative threat to lignin materials, such as the case of some types of paper and wood art pieces and objects. Fungi can also have an important role in the attack of animal-based objects, adhesives and additives. Textile materials such as silk and wool can suffer microbial mediated biodeterioration processes by the action of deteriorating enzymes [75,76]. In particular, the silks fibroin and sericin can both be the target of microbial attack [77]. Moreover, wool keratins can also be the target of attack by microbes [77]. Evaluation of fibroinolytic and keratinolytic action in cultural heritage scenarios has been helpful to study mummies, funeral clothes and accessories biodeterioration [78][79][80]. Moreover, fungal chitinolytic and pectinolytic action has also been pinpointed as a threat to Ancient Yemeni mummies preserved with diverse organic compounds [81]. Additionally, esterease action profiling has also been helpful to study isolates retrieved from wax seals, air, textiles and human remains [82][83][84]. Fibroinolytic screening can be conducted using fibroin agar, with the fibroinolytic action being evaluated by the isolates ability to grow in the culture-amended plates [85]. Moreover, keratinolytic action can be evaluated using feather broth and keratin medium and positive ability can be verified by media turbidity changes [79,85]. On the other hand, chitinolytic activity can be evaluated using powdered chitin agar [86] and pectinolytic activities can be evaluated with media containing pectin [87]. Both these deteriorative activities can be estimated and quantified [81]. Additionally, esterease action can be studied using Tributyrin agar and Tween 80 agar [83,84,88]. Their action can be detected by the development of clear zones (Tributyrin agar) or by the precipitation of insoluble salts and compounds (Tween 80) around colonies. As occurring with ligninolytic action, data regarding filamentous fungi fibroinase and keratinolytic action is still infrequent. Twenty-three species were found to be able of fibroinolytic activity, while more than twenty-five were found to have keratinolytic action. Again, Aspergillus and Penicillium species also dominate these biodeteriorative profiles (Tables 4 and 5). Moreover, various Alternaria species also displayed putative keratinolytic abilities. On the other hand, chitinolytic abilities have been identified for Aspergillus niger and Penicillium sp., while pectinolytic action has been identified in a slightly more diversified range of fungal genera and species (Aspergillus candidus, Mucor circinelloides, Penicillium echinulatum, Scopulariopsis koningii, Stachybotrys chartarum and Trichoderma hamatum) [81]. In parallel, fifty species have been identified as displaying estereolytic action, with a great dominance of Aspergillus and Penicillium species (Table 6). Understandably, the detection of these fungal species on crypt environments, human remains, buried materials, mummies, wax seals, textiles and clothes denotes a putative threat to these materials [3].    Fungal lipolytic action can have an important impact on the biodeterioration of parchment and leather related materials [91]. Fungi can attack lipids and take advantage of fatty materials as a mean to obtain carbon (while also contributing to the material deterioration) [92]. Fungal lipolytic ability characterization in cultural heritage scenarios has been helpful to study the biodeteriorative contribution of isolates retrieved from air, textiles, human remains, wax seals, albumen photographical materials, statues, wooden organs and pipes [83,84,89,93,94]. Lipolytic ability screening can be mainly conducted using Spirit Blue agar and Nile blue, and the positive action can be identified by the development of a halo around the colonies, after a period of incubation [89]. Circa sixty species were found to be able of lipolytic action ( Table 7). As similarly verified in other deteriorative analyses, Aspergillus and Penicillium species are still predominant in these profiles. The detection of these fungal species on materials rich in fatty compounds, such as wax seals and photographic materials should be considered putatively hazardous.

Conclusions
As pointed and reviewed by Pyzik and colleagues [107] the application of highthroughput Next-Generation sequencing technologies has highlighted that cultural heritage materials are inhabited by various unknown microorganisms still pending taxonomic description and their biodeteriorative profiling. The material biodeterioration is known to sometimes be caused by a predominant or specific microbial group, while more often the complex biodeterioration processes are a result of the synergistic action of a group of organisms resulting from various colonization events influenced by the impacts of multiple external factors throughout a time frame [77]. Cultivation methodologies often face limitations in what regards the ability for distinct organisms to be effectively cultivated and their original biodeteriorative characteristics replicated under laboratory conditions [108]. Understandably the application of more modern molecular techniques in cultural heritage biodeterioration studies has been increasingly being used and updated for the last two decades [27,108]. Although with their own set of limitations, culture-dependent methodologies still offer three main advantages: (1) The isolation of microbes for further differential analysis; (2) the possibility to isolate, characterize and describe previously unknown taxa; and (3) the development and improvement of biological and genetic databases. These aspects are especially important when considering that even the biodeteriorative role (but also their taxonomic classification) of long known species might also need to be constantly revised, updated and reevaluated [107,109]. For instance, the inclusion of the Fusarium solani Species Complex in the genus Neocosmospora was recently reevaluated and continues to be the focus of additional studies [110].
Fungi are constantly regarded as one if not the most important microorganism groups causing cultural heritage materials biodeterioration [2,3,30]. This review highlighted that, so far, isolates from more than two-hundred fungal species have been showed to exhibit biodeteriorative abilities when studied by specific plate essays. Based on the available studies performed so far, it is possible to verify that Aspergillus and Penicillium species dominate the biodeteriorative abilities usually screened in biodeterioration contexts. With this in mind, it should be reinforced that the detection of these species in various cultural heritage materials can, under specific conditions, result in severe biodeterioration of the substrate. Nonetheless, a careful analysis of these checklists, as well as, the biodeteriorative screening of obtained isolates, wherever possible, is strongly advised. Not all isolates might display deteriorative action or display similar degradative rates and thus a proper and specific analysis in each case and/or the implementation of additional tests (e.g., molecular identification of genes involved in biodeterioration (see for example [111])) is also recommended. In conjunction with molecular approaches not relying in cultivation, they can provide a holistic evaluation of a specific biodeterioration phenomena. As pointed by Sterflinger and colleagues [112], understanding deterioration mechanisms and the main microbial perpetrators is still one of the major challenges in historic and cultural materials biodeterioration research. As such, the information summarized in this work provides a contribution that can help microbiologists, restorers, conservators and curators in their attempt to preserve cultural heritage materials for future generations.