Fungal biodeterioration and preservation of cultural heritage, artwork, and historical artifacts: extremophily and adaptation

SUMMARY

Fungi are ubiquitous and important biosphere inhabitants, and their abilities to decompose, degrade, and otherwise transform a massive range of organic and inorganic substances, including plant organic matter, rocks, and minerals, underpin their major significance as biodeteriogens in the built environment and of cultural heritage. Fungi are often the most obvious agents of cultural heritage biodeterioration with effects ranging from discoloration, staining, and biofouling to destruction of building components, historical artifacts, and artwork. Sporulation, morphological adaptations, and the explorative penetrative lifestyle of filamentous fungi enable efficient dispersal and colonization of solid substrates, while many species are able to withstand environmental stress factors such as desiccation, ultra-violet radiation, salinity, and potentially toxic organic and inorganic substances. Many can grow under nutrient-limited conditions, and many produce resistant cell forms that can survive through long periods of adverse conditions. The fungal lifestyle and chemoorganotrophic metabolism therefore enable adaptation and success in the frequently encountered extremophilic conditions that are associated with indoor and outdoor cultural heritage. Apart from free-living fungi, lichens are a fungal growth form and ubiquitous pioneer colonizers and biodeteriogens of outdoor materials, especially stone- and mineral-based building components. This article surveys the roles and significance of fungi in the biodeterioration of cultural heritage, with reference to the mechanisms involved and in relation to the range of substances encountered, as well as the methods by which fungal biodeterioration can be assessed and combated, and how certain fungal processes may be utilized in bioprotection.

INTRODUCTION

Fungi are ubiquitous inhabitants of the biosphere performing a range of vital functions that influence, e.g., soil structure and development, cycling of the elements, plant productivity, and ecosystem health (1–6). While generally associated with and often predominant in aerobic terrestrial ecosystems such as soil, rocks, and minerals, and in their symbioses with phototrophs, lichens, and mycorrhizas, fungi are also important in aquatic ecosystems and in environments previously thought to be inimical to fungal success. Like prokaryotes, and despite possessing a limited metabolic versatility, fungi are found in all manner of so-called extreme environments such as those typified by extremes of temperature, desiccation, salinity, solar irradiation, and the presence of potentially toxic organic and inorganic substances (2, 3, 7–13). Furthermore, the currently known limits for fungal growth are now extended to the deep subsurface, and fungal populations exist in the igneous oceanic crust, deep sea sediments, hydrothermal vents, and similar environments, co-existing and interacting with prokaryotes (14–16). Whatever the habitat, and regardless whether free-living or symbiotic, fungi have major roles in chemical transformations of organic and inorganic substrates, and therefore, in the cycling of elements within and between environmental compartments and between organisms. Fungi are prime decomposers of organic substances, the only organisms that can degrade lignin, and key biotic components of carbon and nitrogen cycles, as well as those of all other elements constituting biomass (2, 4, 5, 17). As geoactive agents, fungi are also interactive agents with rocks and minerals mediating physical and biochemical biodeterioration and dissolution as well as the neoformation of new and secondary minerals which are also key processes in elemental cycles for metals and associated elements such as phosphorus and sulfur (2, 18–20). Since wood, metals, rocks, and mineral-based materials are fundamental components of human-made structures in the built environment and cultural heritage, the transformative effects of fungi are significant and obvious, with the potential to cause decay and destruction to buildings, historical artifacts, and artwork should conditions exist amenable to their proliferation (18, 19, 21–24). Such effects can be irreversible, costly to control or remedy, and result in permanent loss of cultural heritage, thereby affecting knowledge of the history and development of human societies worldwide.

This article specifically focuses on fungi as significant biodeteriogens of cultural and historical buildings and artifacts. It should be noted that while the nature and chemical composition of the material affected by fungi can play a crucial role, the process of fungal biodeterioration has not been equally well studied for all specific materials. However, little studied does not mean less important. In this review, we consider extensively studied problems in more detail, while less studied issues naturally receive less coverage. Although fungi are frequently the dominant causative agents of biodeterioration of a variety of materials, especially degradable organic materials, and of rock and mineral-based substrates, other microorganisms can be present in biodeteriorative communities and contribute to the undesirable effects of colonization. Where possible, we allude to other microbial groups where relevant, but detailed discussion is beyond the scope of this review. Other detailed articles are recommended and the references therein for further information on the range of organisms involved in biodeterioration of cultural heritage and their significance (9, 25–45).

BIODETERIORATION

Biodeterioration refers to “any undesirable changes in the properties of a material caused by the vital activities of organisms” (46, 47). As such, the definition is broad and provides no information on underlying mechanisms and potentially includes all living organisms. The concept is closely associated with human activities since a material is “a substance or matter of which anything is made or to be made” (47). Biodeterioration can range from changes in the appearance of a material, such as discoloration or staining (48), to its complete destruction through physical and chemical mechanisms mediated by the colonizing organisms. Biodegradation is also a term that is often used interchangeably in these contexts, although it is most appropriately used in the context of degradation of organic matter by the activities of microbial decomposers (49). Biofouling refers to the undesirable growth of organisms on surfaces and structures, which may or may not also be associated with biodeterioration or degradation. Biocorrosion generally refers to detrimental activities of living organisms on metals, and related materials like alloys, with microbially induced corrosion (MIC) specifically emphasizing the involvement of microorganisms (50–52). Although the human context is inherent in these types of definition, it should be emphasized that the biodeteriorative or biodegradative mechanisms found in human-made structures or materials occur in the natural environment with organic and inorganic substances such as wood, rocks, and minerals. For rocks and minerals, another widely used applicable term is bioweathering, which refers to the detrimental activities of living organisms on rock- and mineral-based substances (18, 19, 53–55). Although different kinds of organisms may be involved in the above contexts, including plants, animals, insects, macroalgae etc., it is the activities of microorganisms that are most interesting and most serious because of their direct and indirect abilities to irreversibly alter the substrate through physical and chemical mechanisms. Unless stated otherwise, in this article, we will use the term biodeterioration to imply the actions of fungi, or other microorganisms, on the various materials used in cultural heritage, historical artifacts, and artwork, with due reference to the physico-chemical mechanisms that underly interactions with specific materials.

ASSESSMENT OF FUNGAL BIODETERIORATION

Establishment that biodeterioration is occurring on a particular substrate is often obvious through visible growth or colony formation, color, and structural changes. It should be noted, however, that some deterioration of materials may be a consequence of purely chemical action without involvement of living organisms. This might be the case for metallic articles where the abiotic influence of water and/or humidity can lead to corrosion, the visible manifestation of which may resemble patterns of microbial colonization. Water and dampness can also lead to metal leaching from mineral-based structural materials, such as plasters, mortar, and brickwork, and the formation of secondary mineral efflorescence and structures that might also resemble consequences of biodeterioration (56, 57). Water is in fact a prime determinant of biodeterioration since it markedly influences all aspects of microbial activity. Biodeterioration is severely limited or even halted in dry conditions.

Apart from visible changes, biodeterioration is usually initially confirmed by microscopic observations (light and electron microscopy, advanced imaging techniques) and isolation methods, with the usual caveats applying to such techniques (23, 58). It is often difficult to differentiate biotic and abiotic structures, especially if samples are air dried or fixed chemically, or to distinguish biogenic/mycogenic biominerals or products from other substances. It is well known that classical microbial isolation techniques only capture a very small proportion of the organisms that may be present and are highly subject to the isolation medium and incubation conditions used. For fungi, rapidly growing species can obliterate slow-growing organisms, and there may be preferential isolation of profusely sporulating species, which may or may not be the prime causative agents of the observed biodeterioration. It is not surprising that many studies based on culture-dependent methods invariably feature easily grown high sporing genera such as Aspergillus, Penicillium, Cladosporium, and Alternaria (59, 60). To identify the organisms involved in the biodeterioration of materials, it is possible, in the case of fungi, to take samples of aerial structures with adhesive tape (61) and observe diagnostic structures, such as spores and conidia, directly under the microscope. However, it is not always possible to distinguish forms with diagnostic value, and in any case, conidial fungi often have variable dimensions, pigmentation, and morphologies that depend on the substrate they have developed on (62). For bacteria, it is impossible to arrive at a diagnosis on a morphological basis from light microscopy preparations obtained directly from the materials. Isolates are needed for bacterial identification on a biochemical and colony-morphology basis. In any case, microorganisms developing in and on materials often have special nutritional requirements. For example, they could be halophilic or require particular trace elements. Therefore, starting with a range of substrates that at least covers different pH values, water activities, and different nutritional requirements (e.g., substrates for cellulolytic or proteolytic activities) is advisable. Obtaining strains of microorganisms by isolation from the biodeteriorated materials is a significant opportunity that allows testing of the interaction mechanisms with the substrate, perhaps even reproducing the observed damage (63) and arriving at accurate identifications based on molecular analysis of pure cultures. It is worth pointing out that many groups of black-pigmented rock-inhabiting fungi (RIF) are extremely slow growing in laboratory media.

A variety of mineralogical techniques, e.g., X-ray diffraction, X-ray fluorescence spectroscopy, and Fourier-transform infrared spectroscopy, may be applied to understand mineralogical changes (64), and these can reveal the presence of secondary biogenic mineral products of biodeterioration. If available, a comparison with non-deteriorated parts of the substrate may provide information on the mineralogical changes mediated by colonizing organisms (65). The ease of application of the various sampling and examination techniques is highly dependent on the nature of the biodeteriorated substrate (66). Stone, for example, provides more challenges than easily manipulated paper or fabrics.

Biochemical assays may be used to confirm the presence of organisms, their enzymes and metabolites, and other biosignatures. Some assays might be similar to those traditionally employed in soil microbiology to differentiate microbial communities and indicate metabolic activity. Adenosine triphosphate (ATP) and phospholipid analyses can indicate activity and relative significance of bacterial and fungal populations, while assays for enzymes involved in carbon metabolism, or other key transformations in elemental cycles, e.g., nitrogen and sulfur, can provide information on the biochemical changes occurring. Stable isotope probing (SIP), such as ¹³C- and ¹⁵N-isotope labeling, can link identified biochemical reactions with their associated organisms (67, 68). DNA-SIP can identify active organisms in a community that utilize carbonaceous or nitrogenous substrates. Labeled DNA can be separated from non-labeled DNA and used in further analyses, including sequencing-based methods (69).

In recent years, the suite of modern molecular and genomic techniques is also routinely applied to identify and characterize isolates as well as the communities of organisms involved in biodeterioration or other environmental processes and to understand the influence of various environmental factors including climate and substrate composition (70). High-throughput DNA sequencing has provided a means of assessing the composition and potential functions of the microbial communities in a given environment, and these have all been applied to the problem of biodeterioration (20, 32, 39, 43, 48, 71–78). Metagenomics reveals the taxonomic profile of the entire microbial community (79, 80) and can provide some functional information, although this may be inferred or only speculated upon from existing knowledge of the species identified or other close taxa whose whole genome is annotated and deposited in public databases (62, 81, 82). Other omics-based techniques are applied to identify the functions of community members. Metatranscriptomics provides information about the genes that are expressed in a given environment, which enables identification of active species and their function (77). Metaproteomics confirms the functional activity of a community by identifying the translated functional proteins and their abundance. In contrast, metabolomics identifies and quantifies the abundance of metabolites produced by the organisms, which provides understanding about the biochemical processes occurring and, therefore, mechanisms of biodeterioration (78). All such technologies have various advantages and disadvantages but are becoming increasingly refined over the years and more widely available with favorable costings. Various drawbacks and lack of resolution in earlier approaches are constantly superseded in modern approaches, although of course this can cast doubt on the relevance of early studies. For example, third-generation sequencers, such as PacBIO (83) and Nanopore (84), which sequence longer DNA fragments compared to Illumina technology, are increasingly being used in heritage biodeterioration studies (85–87) and allow for more accurate fungal species identification and phylogenetic analysis (88). The advantage of having long sequences lies in the possibility of using more extended regions of the fungal genome for their identification based on similarity comparisons with public databases. The larger the target region of DNA, the greater the probability that two species or even two strains of the same species can be distinguished. Species delimitation based on long diagnostic DNA regions is more reliable as it allows the detection of widespread polymorphism within markers. For example, in fungi, the whole internal transcribed spacer (ITS) region has the highest probability of identification for the broadest range of taxa, with the most clearly defined barcode gap between inter- and intraspecific variation (89, 90). However, even the use of long diagnostic sequences often leads to few identifications or lists of species that are unlikely or unrealistic. This is because, as mentioned, there are still few correctly identified species, even on a morphological basis, for which reference sequences are available in databases (63, 85–87, 91).

At any rate, all techniques have their limitations and, in some cases, may not be able to be applied, e.g., where in situ examination is the only feasible approach allowed because obtaining pieces or fragments of the deteriorated substrate is not permitted or may be too damaging. Thus, non-invasive, or micro-invasive sampling techniques may be applied (61, 92–96), although these can be limited in the amount of material that can be obtained which may in turn provide difficulties in understanding the statistical significance of the resulting data (77). Sampling methods are basic and have included the use of swabs, adhesive tape, and obtaining fragments by scraping, cutting, boring, etc. using commonplace implements as well as some innovative simple non-invasive approaches, e.g., smooth vacuuming of selected sampling points on the object using sterilized Gilson filter tips (96, 97).

The combination of multiple diagnostic techniques can support not only knowledge of the diversity of species involved and the complexity of the communities in place but also the mechanisms of action of biodeteriogenic microorganisms. However, it remains very difficult to develop standardized experimental protocols for analyzing biodeterioration, precisely because materials are highly variable in their manufacture and use. Each community can manifest diverse physical and biochemical mechanisms on the substrate (98–100). For instance, using both metagenomic and cultivation techniques may reduce some of the limitations inherent in each approach (44, 48, 101, 102).

Many taxa of fungi and even entire communities of fungi and bacteria that attack artworks are still unknown. Little is also known from metagenomics and metabarcoding studies that usually return long lists of species that reasonably cannot be all involved in biodeterioration mechanisms. Nonetheless, molecular studies of biodeteriorated materials suggest that the fungi (and bacteria) that can be isolated in culture are only a small part of the community and not necessarily the part responsible for biodeterioration. This is the case with species such as Eurotium halophilicum (103), Parengyodontium album (104), or the fungal communities that typically succeed in parchment already attacked by the archaeon Halobacterium salinarum (105). These are all species that have long been “invisible” because they can hardly be cultivated and are difficult to study. In the case of paper materials (manuscripts and graphic works), some of the culture-independent molecular techniques previously listed have helped expand knowledge of the number and traits of species associated with their biodeterioration (59, 60, 106, 107). However, as mentioned, the environmental species of interest are often not represented in the main public databases, and thus, many fungi that proliferate in books and archives are not identifiable at the species level. However, delineating them at a reliable taxonomic level is sometimes possible with molecular techniques or at least to define what they are not. Karakasidou et al. (108), for example, discovered for the first time that the fungi Chalastospora gossypii and Trametes ochracea were responsible for the biodeterioration of Greek historical documents dating back to the 19th and 20th centuries. Okpalanozie et al. (109) analyzed four volumes of a Nigerian museum library with signs of biodeterioration and found species that had never before been detected on archival materials. Sato et al. (110) found cellulolytic and salt-tolerant fungal species after the library material had been submerged by a seawater flood (tsunami following the March 2011 East Japan Earthquake). Coronado-Ruiz et al. (111) found species new to science in an investigation into the biodeterioration of 19th-century French drawings and lithographs. These authors could not phylogenetically ascribe two isolates by their ITS and α-actin sequences, later describing them as two new species characterized by significant cellulolytic activity: Periconia epilithographicola and Coniochaeta cipronana spp. nov. Vieto et al. (112) described a new fungal species isolated from the canvas of a biodeteriorated 19th-century painting conserved at the National Theatre of Costa Rica. The authors used phylogenetic and morphological analyses and named a new species of Myxospora (Myxospora theatro sp. nov.) that showed strong cellulolytic activity.

Another significant problem in heritage biodeterioration studies is that data obtained from DNA or RNA sequencing of the microbial community are often correlated with various environmental factors including substrate composition, pH, temperature, rainfall, humidity, and solar irradiation (113). However, relevant information on such parameters is often incomplete or not obtained at the time of sampling, while interpretation of sequencing data is descriptive and relies on statistical conclusions in an absence of practical verification which is a significant challenge to be addressed in future research (113). It should also be appreciated that many sequencing studies do not attempt to clarify the entire microbial community, just focusing on prokaryotes (114, 115), meaning that potentially important eukaryotes such as fungi are often ignored. Further detailed accounts of sequencing techniques and their application in the context of fungal communities and cultural heritage are available elsewhere (76–78).

FUNGI IN BIODETERIORATION

Of the many kinds of prokaryotic and eukaryotic microorganisms that colonize and biodeteriorate materials, fungi are of particular prominence in the built environment and possess a long history regarding contamination and spoilage of human-made products and cultural heritage. They are regarded as the most important colonizers on stone, mortar, and plaster (1, 18, 19, 21, 23, 24, 55, 116). Fungal success in any environment is underpinned by their filamentous explorative growth habit and chemoorganotrophic metabolism, and human-made structures and materials can provide ideal conditions for colonization and a plethora of potential nutrient sources. The ability of fungi to utilize a range of carbon compounds and excrete a wide variety of extracellular degradative enzymes which act on natural and synthetic polymers means that almost every carbon-containing compound can be colonized and attacked with obvious deleterious consequences for artifacts of high organic content, e.g., wood, paper, leather, textiles, etc. In addition to extracellular enzymes, fungi also excrete a variety of other extracellular substances that change the geochemical properties of their microenvironment such as organic acids, siderophores, polyphenolics, and polysaccharides, and these can significantly affect metals, alloys, rock and mineral-based structures and artifacts, ceramics, plasters, etc. through physical and chemical action. It is also notable that many fungi can survive, grow, and explore in nutrient-poor oligotrophic conditions, proliferating when conditions change, while several other properties aid survival in apparently hostile environments such as those characterized by extremes of temperature, water activity, and irradiation (7–13, 42, 43, 117). Dispersal by spores and other vegetative fragments also ensures a regular inoculation of material surfaces in indoor and exterior environments.

Symbiotic fungal growth forms are also highly significant in biodeterioration. Lichens are probably the most important in this context, especially on exterior stone- and mineral-based structures and materials, but they can colonize almost every kind of surface, including metals and metallic substances, fabrics, and plastics. As pioneer colonizers of rock surfaces, they are regarded as pivotal in the early stages of mineral soil formation from rocks, and stone-based materials and artifacts are also subject to their influence, being significant biodeteriorative agents of stone monuments, buildings, cement, and mortar (36, 118–123) (Fig. 1). Progressive colonization of surfaces and concomitant biofilm development of prokaryotic, algal, and fungal communities accelerate destructive activities and may finally result in colonization and development of lower plants such as mosses, liverworts, and ferns and higher plant communities, all with their associated microbiomes that may also include mycorrhizal fungi.

Fig 1.

Lichen colonization of (A and B) ancient lodge dwelling at Montecute House, Montecute, Somerset, England, UK and gravestones located at (C and D) St. Leonard’s Church, Shipham, Somerset, England, UK; (E and F) St. Andrew’s Parish Church, Blagdon, Somerset, England, UK; and (G and H) Carmylie Parish Church, Carmylie, Angus, Scotland, UK (images by G.M. Gadd).

FUNGAL COLONIZATION, BIOFILMS, AND OTHER MICROORGANISMS

For a substance to be biodeteriorated, it must be infected and colonized by the responsible organisms. Colonization of a surface is often discussed in terms of biofilm development (41) because it is now accepted that the majority of microorganisms in natural, synthetic, or engineered habitats live in biofilm communities of varying complexity (45, 51, 124–126). The biofilm term is broad, and even single pure culture microbial colonies on agar plates are now referred to as a biofilm. The term is therefore apposite to surface colonization by fungal species, including lichens.

It is commonly accepted that biofilm formation confers survival advantages to the organisms involved not least through adherence and adhesion to the substrate but also protection from environmental factors such as desiccation, temperature fluctuations, solar irradiation, and toxic agents (41). The formation of a “conditioning layer,” arising from organic substances such as polysaccharides and proteins, on uncolonized surfaces is believed to be an initial step in successful biofilm formation, although much biofilm research has been carried out with surfaces in aquatic media and planktonic bacterial systems. It is debatable whether a conditioning layer is essential for fungal development when considering the usual mode of aerial deposition on surfaces, and the surface irregularities that exist on most substrates can enable spore entrapment. However, deposition of organic substances on surfaces from atmospheric, animal, and industrial sources would no doubt be beneficial to fungal colonization and subsequent growth. As biofilms develop, the production of extracellular polymeric substances (EPS) can enhance substrate adherence and protective effects, as well as further colonization by other microbial species (127). Where phototrophs such as cyanobacteria and microalgae are primary colonizers, EPS production can enable co-aggregation and colonization by other organisms such as bacteria and fungi, also capable of EPS production. The EPS comprise polysaccharides and proteins as well as other substances such as lipids and can contain humic acids, clay minerals, secondary minerals, and other substances derived from the atmosphere, organisms or substrate.

Pure cultures of microorganisms seldom occur in nature, and colonization of exterior cultural heritage is no exception. Phototrophs and heterotrophic microorganisms are often components of colonizing biofilms, with photosynthate and EPS production from phototrophs benefiting heterotrophs as well as stabilizing colonization (41). It is commonly stated that phototrophic lichens, algae, and cyanobacteria may be primary colonizers of, e.g., rock surfaces, and phototroph-driven microbial communities can be significant in bioweathering in the built environment. However, this appears not always to be a prerequisite for bioweathering of human-made structures, or indeed in the natural environment (7, 128, 129). Fungi may be late colonizers of phototrophic communities (130), but organic matter deposition may enable fungal development in the absence of phototrophs (129, 131). Fungi seem especially suited for phototroph-independent colonization through their morphological and physiological adaptation mechanisms to potentially extreme or stressful environments, and capacity for nutrient acquisition.

FUNGAL SURVIVAL, ADAPTATION, AND EXTREMOPHILY

Exterior heritage surfaces are usually considered low nutrient environments (41) as well as subject to varying extremes of environmental stress. Rocks or other subaerial surfaces are inhabited by poikilotrophic organisms (i.e., able to deal with varying extremes in micro-climatic conditions, e.g., light, salinity, pH, and water potential) that can thrive under these extreme conditions over considerable periods of time (34, 53, 117, 132), and fungi are ubiquitous components of such communities. Oligotrophic growth and survival of fungi is well known (13), while abundant nutrient sources can arise from, e.g., atmospheric deposition, particulate entrapment, anthropogenic activities, and waste products and detritus from animals and insects (53). Colonization of subsurface cracks, fissures, and cavities can also confer protection from environmental extremes (7). Rock- and mineral-inhabiting fungi can occur on surfaces (epiliths) and in cracks, fissures, and pores of the subsurface (endoliths). Chasmoendoliths colonize open fissures, cracks, and holes; cryptoendoliths occupy subsurface pores and cavities, while some fungi may actively tunnel into rock and mineral substrates (euendoliths) (1, 53, 133, 134). Extremophilic fungi, including lichens, can therefore survive in and exploit microhabitats on the surface and within mineral substrata (1, 53, 54, 135–137), and exhibit a range of morphological and physiological adaptations and stress responses (138).

The hyphal growth form underpins the ecological success of filamentous fungi, and many morphological and structural properties can determine colonization, growth, and survival in adverse habitats. The significance of sporulation for dispersal as well as dormancy is obvious, as is the development of other resistant cell forms, such as melanized cells, hyphae and chlamydospores, and structures such as sclerotia (139, 140). A highly important feature of hyphal growth is that it enables spatial exploration of the environment in order to locate and exploit new favorable substrates and nutrient sources (141–143). This is mediated by a range of sensory responses, known as tropisms, that determine the direction of hyphal growth. This could be toward a new carbon and energy source, away from a toxicant, or toward or away from light, for example (144, 145). Such responses may influence patterns of fungal colonization on all manner of solid substrates. Of particular relevance to rocks and minerals and other solid substrates is thigmotropism or contact guidance. This is a well-known property of fungi that grow on mineral surfaces and within solid substrates (146, 147). The direction of fungal growth can be influenced by grooves, ridges, and pores in the solid substrate and, therefore, may be more prevalent in weakened mineral surfaces.

Many fungi are oligotrophic and can maintain sparse growth in nutrient-limited habitats (13, 148, 149). It has been suggested that such organisms have properties that enable efficient utilization of low nutrient supplies which would confer an ecological advantage (149). Nutrients may also be utilized through cryptic growth, where hyphal growth is maintained through nutrients released from degradation or autolysis of older parts of the colony (150). Atmospheric gases and volatile compounds may also contribute as a source of fungal nutrition (149). It has also been shown that both exploitative highly branched mycelia as well as sparsely branched explorative hyphae may be produced by fungi in variable responses to external stress and nutritionally poor conditions (145).

Microcolonial fungi (MCF) include those black pigmented fungi that occur on rock and mineral surfaces as spherical clusters of tightly packed thick-pigmented-walled cells and/or as moniliform thick-walled hyphae (117, 135, 136, 151). Such fungal groups are often referred to as “rock-inhabiting fungi,” although many other free-living and symbiotic fungi can be found on and in rock substrata that do not exhibit this pattern of growth. Melanization is a common fungal trait in response to stress factors providing protection from, e.g., desiccation, temperature extremes, solar irradiation, and toxic substances, and also enables dormancy which can be broken once favorable external conditions ensue. Apart from the microcolonial forms, many species of melanic fungi can be associated with exterior cultural heritage such as rock and minerals, painted surfaces, etc.

The prevailing fungal genera commonly found in almost all kinds of biodeteriorated objects of cultural heritage, e.g., textiles, canvas paintings, wood, paper, parchment, stone, wall paintings, metals, and audio-visual materials, are Alternaria, Aspergillus, Cladosporium, Fusarium, Penicillium, and Trichoderma, and they manifest a marked predominance of the phylum Ascomycota (77, 106, 152–155). A worldwide meta-analysis of the diversity of fungi associated with biodeterioration of wall paintings of cultural and historical value revealed that the most diverse fungal genera were Aspergillus (with its teleomorphs Eurotium and Emericella) and Penicillium (with its teleomorph Talaromyces), including over 46 and 48 fully-identified species, respectively (156). Also, according to an overview summarizing data from the 21st century, Aspergillus and Penicillium were reported to be the most diverse genera on stained glass along with the Cladosporium genus (154). The representatives of these genera are ubiquitous fungi in soil and in outdoor and indoor air biota and indoor dust, and many possess an intrinsic ability to tolerate wide ranges of temperature and water activity (xerotolerance), as well as metal- and mineral-rich environments, which makes them common agents of biodeterioration. An unappreciated fungal biodeteriogen is Parengyodontium album which has been found in numerous examples of cultural heritage, including historic and religious buildings, monuments, museums, libraries, and wall paintings, and on all common substrates such as stone, plaster, brick, glass, paper, and wood (104). Causative effects range from discoloration and staining, salt efflorescence, and white, pink, or dark surface or patina development (104, 157, 158). It is mesophilic but appears capable of growth over a wide range of environmental conditions, particularly humid or salty environments, and utilizes a wide range of carbon substrates, including chitin, which may be linked to a frequent association with insects and arachnids and their remains (104).

For controlled indoor environments with very low humidity, e.g., museums, repositories, and archives, a particular aspect of fungal biodeterioration is associated with extremophiles, specifically obligately xerophilic aspergilli from the section Restricti (95, 159, 160). Among them, the most remarkable fungal agent of biodeterioration of a plethora of heritage materials (e.g., paper, leather, wood, film, and murals) worldwide is Aspergillus halophilicus (teleomorph Eurotium halophilicum) (156, 159, 161, 162). A. halophilicus is one of the most-extreme xerophiles in the Earth’s biosphere, able to grow at a water activity down to 0.651, at very high NaCl concentrations and at temperatures around 0°C (162–165). A. halophilicus is the only fungus from the section Restricti having a sexual reproductive state (Fig. 2), which gives this species an additional evolutionary advantage in its survival and adaptation compared to other xerophilic aspergilli (160). It also has increased biodeteriorative potential due to production of many more secondary metabolites (e.g., chaetoviridin A, deoxybrevianamid E, pseurotin A, pseurotin D, rugulusovin, stachybotryamide, and tryprostatin B) than anamorphic species of section Restricti, A. penicillioides and A. vitricola, which are often found as A. halophilicus associates on biodeteriorated materials (95, 160, 161, 166).

Fig 2.

Scanning electron microscopy images of Aspergillus halophilicus on restoration plaster from the walls of the medieval cathedral, St. Sophia, Kyiv, Ukraine, showing (left) numerous cleistothecia in the biodeteriorated area and (right) ascospores emerging from a broken-open cleistothecium (images by M. Fomina, unpublished).

Prior to the development of metagenomic taxonomic profiling of fungal communities involved in biodeterioration of cultural/historical heritage objects, A. halophilicus was largely underestimated because of difficulties of isolation and cultivation. This species does not grow on common media conventionally used for fungal isolation and is very slow growing on media selective for obligate xerophiles. It can be cultivated only at low water activity values which are prohibitive for many other fungi, e.g., on a medium containing at least 15% NaCl (162, 165). However, due to the presence of distinctive hairy structures covering the hyphae and conidiophores of A. halophilicus along with conidia of specific ornamentation, this fungus can be readily observed using high vacuum scanning electron microscopy (SEM), if an adequate sampling strategy is applied (162) (Fig. 3). Some SEM images that appeared in earlier publications might serve as possible evidence of the presence of this species, e.g., in biodeteriorated areas of 16th century murals in St. Martin’s church in Greene, Kreiensen, Germany (167) and in foxing spots in a 19th-century book in the Royal British Columbia Museum, Canada (168) where fungi with hair-like structures were observed but not fully identified.

Fig 3.

SEM images of Aspergillus halophilicus on the restoration plaster of the walls of the medieval cathedral St. Sophia, Kyiv, Ukraine, showing conidia and hair-like structures covering the hyphae (images by M. Fomina, unpublished).

A. halophilicus is thought to benefit from the covering of hair-like microfilaments that absorb water and maintain a layer of humid air adjacent to the hyphae as well as enabling salt deliquescence within the surrounding EPS to maintain conditions consistent with a suitable water activity for growth (162). The hair-like structures of A. halophilicus, therefore, play an important role in the substrate-air interface mechanisms of this tonophilic species under severe water stress and contribute to its remarkable ability to shift from extreme natural environments to artificial anthropogenic niches making it an invasive species for museums, libraries, and archives (162) (Fig. 4). However, the specific nature of the extreme stress tolerance mechanisms of A. halophilicus under dynamic changes in water regimes of the surface habitats on which it commonly occurs is still unclear.

Fig 4.

Parchment biodeterioration. (A) An illuminated ancient manuscript made of parchment spoiled by purple spots; (B) fungal hyphae in conjunction with a purple spot; (C) appearance of the parchment surface affected by purple spots. The arrowhead indicate the loss of material and the profound damage caused by microbial collagenolytic activity, resulting in the collagen fibers becoming broken and fissured. The arrowhead with an asterisk indicates biogenic crystals; (D) fungal-fruiting bodies (Chaetomium sp.) immersed in the parchment to form craters (arrowhead) containing spores of the fungus; (E) appearance of parchment after microbial attack, which even at low magnification shows deep gaps and structural brittleness; (F) the co-occurrence of bacteria (actinobacteria, arrowhead with asterisk) and fungi (arrow, an echinate fungal spore) on biodeteriorated parchment. Images B, C, and F were obtained using high-vacuum scanning electron microscopy on gold-coated samples (Zeiss SEM EVO 50), and images D and E were obtained using a variable-pressure scanning electron microscope (Zeiss SEM EVO 50) fitted with a backscattered electron detector (images by F. Pinzari, unpublished).

LICHENS

Lichens are a fungal growth form (169) and are a symbiotic partnership between a fungus and a phototrophic organism, either an alga or a cyanobacterium, but sometimes both. It is now known that some species also contain species of symbiotic yeast (169–172). Bacteria are also a significant component of the lichen microbiome and are thought to contribute to the overall successful functioning of the entire symbiosis (173, 174). Lichens can colonize almost every exterior natural and anthropogenic material but are particularly associated with rocks as pioneer colonizers and initiators of bioweathering biofilms (37, 123, 175–177). Conversely, the formation of lichen crusts, often biomineralized, may stabilize and protect certain rock surfaces from weathering (48, 55, 178). Unsurprisingly, they are significant inhabitants of rock- and mineral-based cultural heritage (36, 122) (Fig. 1). Lichens can also entrap airborne particulates and pollutants (179).

Rock-living lichens are called “saxicolous” lichens and include all the morphological lichen forms, i.e., crustose, foliose, and fruticose (123, 177, 179, 180). Crustose lichens form crusts on or beneath the rock surface with their lower surface becoming tightly attached and integrated within the stone substrate. Foliose (leaf-like) and fruticose (bush-like) lichens attach to rocks employing only a proportion of their underside (179). Significant geochemical transformations occur at the lichen-rock interface, and free-living cyanobacteria, bacteria, algae, and fungi can also be involved in this region (19, 27, 179). Some (especially crustose species) are epilithic (surface dwellers) and/or endolithic (interior dwellers), with cryptoendoliths occupying structural cavities, chasmoendoliths inhabiting fissures and cracks, and euendoliths capable of active rock penetration (181, 182). These terms are also applicable to other rock-inhabiting microorganisms, although there are overlaps for a given species and between different organisms. Endolithic colonization of rock- and mineral-based substrates may provide protection from environmental extremes, as with free-living organisms. Lichenized and non-lichenized fungal hyphae can occur in endolithic microhabitats (182). The mineralogical composition of the rock can influence lichen colonization and diversity, especially in the presence of different metals (183).

MECHANISMS OF FUNGAL BIODETERIORATION

Mechanisms of fungal biodeterioration depend on the nature of the substrate, although there is some commonality between all substrates with the filamentous branching explorative mode of growth and chemoorganotrophic metabolism underpinning all aspects of the lifestyle of mycelial fungi. Hyphal growth determines colonization and spread as well as penetrative growth within the substrate, with a variety of sensory mechanisms and tropisms facilitating the colonization of favorable habitats and nutrient acquisition. Metabolism alters the physico-chemical environment around the fungus through uptake of organic and inorganic nutrients, production of extracellular polymeric materials, and the excretion of reactive metabolites, including H⁺, CO₂, siderophores, and organic acids. Fungi can also excrete amino acids, and proteins, of which extracellular enzymes are highly significant in the degradation of organic materials such as wood, leather, and cellulosic materials.

Mechanisms of biodeterioration are often separately discussed as being physical or biochemical in nature, but it is doubtful that there are many instances where both physical and biochemical mechanisms do not operate together, not least because of effects of the growing mycelium. Physical effects also depend on metabolism in any case, and where they might not, the biodeteriorative agent is a product of metabolism. Biophysical effects include the effects of fungal colonization and penetration disrupting the structure of a substrate, which can be aided by the significant turgor pressure within the growing mycelium (151, 184–188). In rocks and minerals, fungi can penetrate fissures and cracks, colonize pores, and there is evidence for tunneling or boring in some materials, the latter aided by metabolic products such as siderophores and organic acids (189–191). Such effects may be pronounced in a weakened or porous substrate (190, 192). Tunnels may also result after fungal formation of a secondary mineral matrix of the same or differing mineralogical composition as the substrate, e.g., secondary CaCO₃ or an oxalate (193). This can result in “cementation” of fissures and cracks with such biominerals. After death and degradation of the fungal biomass, tunnels of similar dimensions to hyphae remain within the minerals. Weakening of a mineral lattice can also occur through wet and dry cycles and associated expansion or contraction of biomass. EPS can also influence rock and mineral biodeterioration through wet-dry cycles, complexation of metals, and also underpin biofilm growth and survival on surfaces, the mucilaginous matrix providing adhesion and protection as well as being an excellent matrix for geochemical reactions including the precipitation of secondary minerals (193, 194). The formation of secondary minerals such as calcites and oxalates can also contribute to physical disruption and/or cementation of the rock-mineral substrate (18, 19, 53, 134, 137, 195).

The main mechanisms of biodeterioration or bioweathering discussed under the biochemical heading are metabolism dependent and related to the excretion of degradative or geoactive substances (18, 19, 28, 31, 33, 34, 36–38, 41, 196). The fungal capability for extracellular enzyme excretion means that a wide variety of organic materials can be degraded and used as carbon and energy sources, providing there is adequate moisture and supply of other essential nutrients such as a nitrogen source. Excretion of H⁺, CO₂, siderophores, and organic acids can have significant effects on rocks, minerals, metals, and mineral-based structural materials causing pitting, etching, or dissolution (20, 151, 176, 197, 198). Some fungi are also capable of sulfur oxidation which can lead to acidic dissolution (199). Surface attachment is clearly important for the efficacy of such mechanisms (200). Siderophores remove Fe(III) from iron-containing minerals, while H⁺ and CO₂ can affect the pH of the fungal environment which in turn can mediate metal solubilization from minerals and precipitation of secondary minerals. The release of metals from rocks and minerals from weathering or fungal-mediated mechanisms can result in formation of secondary mineral precipitates that include carbonates, phosphates, oxides, and oxalates (197, 198, 201, 202). Such biomineral formations may contribute to physical disruption, staining and discoloration, and development of rock coatings (3, 7, 19, 193). Respiratory CO₂ production can also lead to carbonate precipitation. Biogenic organic acids are very effective at mineral dissolution which emphasizes the importance of fungi in biochemical weathering (3, 189, 202, 203). Organic acids, such as citric, oxalic, malic, gluconic, etc., can complex and solubilize metals from minerals and metals leading to dissolution, metal release, or corrosive effects. Oxalic acid can also precipitate metals as insoluble metal oxalates when produced in adequate amounts, and these can contribute to the formation of biomineralized patinas and crusts as well as substrate disruption through expansion in pores and fissures (18, 19, 53, 201, 202). Fungal biomineralization can also result from oxidation or reduction of a metal species (which leads to altered solubility of that species). Soluble Mn(II) can be oxidized by many fungal species which results in the formation of black Mn oxides (19, 204). Physical and biochemical effects of fungi on rocks are highly influenced by environmental factors, rock chemistry, and mineralogy (34, 70, 128, 200).

Other features of fungal colonization and biodeterioration include color changes on and/or in the substrate; the consequences of which may be aesthetically unpleasant but may also reflect the effects of biodeterioration mechanisms. Many fungi produce pigments that are located in and around cell walls or excreted extracellularly. As mentioned, many biodeteriorative fungal species are melanized which can result in blackening and spotting of surfaces reflecting patterns of colonization (205, 206). Some color changes can be a result of mineral dissolution, the formation of secondary minerals, or MIC on metallic substrates.

Lichens exert physico-chemical changes on a rock substrate, altering appearance and weathering rates, and interact with other rock-inhabiting microorganisms. As a result of lichen bioweathering, many rock-forming minerals exhibit extensive surface alteration, biodeterioration, and chemical transformations (36, 175, 176, 178, 180) (Fig. 1). Lichens can cause mechanical damage to rocks and minerals through penetration of their anchoring structures, e.g., rhizines in foliose lichens, between mineral grains and cleavage planes, and through expansion or contraction of the thallus in response to varying moisture levels or freeze/thaw cycles (41, 177, 180). Separated rock and mineral grains can be incorporated into the thallus as well as any produced secondary minerals such as oxalates (199). Such effects, as well as removal of lichen cover and associated substrate by animals, rain, and wind, can lead to visible damage in a few years (202). Oxalic acid is possibly the most significant bioweathering agent produced by lichens. In some extreme cases, up to 50% of certain lichen thalli may comprise metal oxalates, which are the main secondary biomineral products of lichen bioweathering (178, 183). Secondary mineral formation through reaction of lichen-excreted organic acid anions with cations from the rock can result in “efflorescence,” causing blistering, scaling, disintegration, and flaking (or “spalling”) of outer rock layers. This is often considered to be a major mechanism of rock decay (56). Lichens also produce a spectrum of other agents collectively called “lichen acids” (mainly polyphenolic compounds), which also cause damage (177, 180, 207).

FACTORS INFLUENCING FUNGAL BIODETERIORATION

A plethora of factors can influence fungal biodeterioration of cultural heritage, artwork, and historical artifacts. This has been described in numerous studies (22, 39, 41, 97, 153–155, 208) and is summarized in Fig. 5. The main three categories of factors relate to (i) the nature and chemical composition of the biodeteriorated material, (ii) macro- and microclimatic characteristics of the environment and exposure to exogenous and endogenous environmental influences, and (iii) the specifics of housekeeping management, including the quality of cleaning and ventilation (22). The initiation and progress of certain biodeterioration processes are determined by a particular combination of factors and conditions (41). Regarding the third category, the importance of simple cleaning and dusting is often underestimated since accumulation of dust and other substances on the surface of the objects means a concentration of fungal spores and bacteria as well as nutrients for colonizing microorganisms (22, 39, 155).

Fig 5.

The main factors that influence fungal biodeterioration of cultural heritage.

Some species of fungi have functional and structural capacities to adapt to man-made environments or materials in indoor environments, which are selective for extremophilic species that can withstand long periods of dormancy and then develop rapidly as soon as environmental conditions permit. Climate change and the unique environmental conditions that increasingly characterize cities are also factors that can favor new ecological niche “jumps” (209–211). In other words, the spread of certain species of fungi, usually considered rare in nature, in environments created by humans or on synthetic materials (e.g., plastics, mortar, concrete, xenobiotics, etc.) may change the biogeography of certain taxa. Without human interference, these species would remain bound to microhabitats and, in general, relegated to highly selective ecological niches, e.g., rare natural extreme conditions. These findings call for a revisiting of Baas-Becking’s theories on microbial biogeography: “Some things are everywhere and some things are not. Sometimes the environment selects and sometimes it doesn’t,” offering some evidence to the role of humans and species’ functional traits in the spread and dispersal of fungi around the world (211).

The most abundant organic compound on Earth is cellulose (30%–50% of the dry weight of plants), which in natural environments hosts complex successions of microorganisms for which cellulose is the main source of carbon and energy (212). By creating libraries and archives, humans have created new ecological niches for many species of microorganisms that have specialized over millions of years to degrade cellulose with great efficiency and under different, often competitive conditions (213). It is difficult to ascertain whether certain species of fungi, that are normally considered to be rare and have recently begun to be found as major biodeteriogens of certain materials, are more easily detected and identified because of new technologies, or whether they are really species that have “jumped” from one environment to another.

Fungi that colonize organic materials, such as paper, leather, and both plant- and animal-derived textiles such as silk and wool inside buildings, can have very different functional traits (107, 214–216). Such environments, although artificial, offer differentiated food niches and often host truly specialized communities (108, 217–220). For example, species that proliferate in archives and libraries not only feed on cellulose but can also degrade more complex chemical substances such as lignin, keratin, glues, and synthetic polymers (214–216) and easily disperse between environments and on materials through spores which can be transported by air, mites, or insect vectors (221, 222) (Fig. 4). Fungal infections in indoor environments often have a point of origin from which the amplification and dispersal begins such as a source of free water represented by the dripping condensation from an air conditioner or leakage from a water pipe.

In addition to the many different substances fungal species can use as substrates, environmental conditions can also strongly delimit their activity (223), making broad categorizations possible. If there is a lot of water available in the material or environment, as in the case of a sudden event such as a flood, the microorganisms that attack organic materials such as paper and parchment are “r” selected (224–226) and spread rapidly, produce colored spots, and emit many volatile substances, including strong odors (110, 226). Once the event of high-water availability has passed, different species of microorganisms follow one another during the drying of the material, exploiting the partially degraded substrate and the remains of the first colonizers. These species grow abundantly in the presence of free water, possess powerful cellulolytic and proteolytic enzymes, and thus cause structural damage through a targeted attack on the covalent chemical bonds of macromolecules (e.g., cellulose and collagen). They also produce abundant organic acids (e.g., oxalic, citric, malic), which, in turn, promote substrate oxidation mechanisms (218) and have considerable competitive capabilities, e.g., production of antimicrobial compounds to outcompete other species (227). For paper, fungi not only attack the cellulose with oxidative reactions and targeted enzymes but are substantially influenced by the contextual availability of starch, gelatine, glues, salts, and mineral compounds that can strongly influence the outcome of the attack and determine the type of mechanical damage and staining produced (205, 228) (Fig. 6). The most prevalent fungal pigments in materials attacked by fungi are melanins, which are particularly damaging to books and cultural heritage objects (205). Melanins are contained in the cell walls of fungi, which, by penetrating the structure of materials, cause dark stains that can remain deep in the body of the material (229). The exact chemical structure of fungal melanin is complex and highly variable. The color can be different even in the same species of fungus depending on growth conditions, nutrient availability, and substrate properties. Thus, the same species of fungus can manifest itself very differently on different materials, causing stains of varying color and consistency depending on different substrates (230). Paper attacked by fungi is also often disfigured by polyketide quinones, carotenoids, and other compounds where synthesis or color may depend on nitrogen availability, pH, and the presence of other limiting nutrients and enzyme cofactors such as metal cations, e.g., Fe, K, Ca, Mg, and Mn (205, 228) (Fig. 7).

Fig 6.

Toxic metals. Details of the illuminated parchment ‘‘Corazzata Sicilia’’ conserved in Rome at the Memorial Museum of the Flags showing biodeterioration and colonization by the rare xerophilic fungus Diploöspora rosea; (A) gold lamina and white lead pigments with attached fungal hyphae (arrows), image obtained using a variable-pressure scanning electron microscope (Zeiss SEM EVO 50) fitted with a backscattered electron detector; (B) illuminated parchment showing detachment of decorative layers; (C) a detached sample used for scanning electron microscopy (SEM) and energy dispersive X-ray analysis (EDS) (SEM-EDS) analyses; (D) results of energy dispersive X-ray analysis (EDS, Oxford INCA 250) of gold and “emerald green” layers of the pigments showing the presence of lead (Pb) and chromium (Cr) where the fungus grew despite the potential toxicity of these elements [images by F. Pinzari, unpublished, see also Tanney et al. (231)].

Fig 7.

Paper and glue. Fungi capable of rapid growth at high water activity can develop at the expense of both cellulose and glues and other compounds used in manufacture. (A) A book volume which was soaked with water after flooding and subsequently attacked by several fungal species. (B) Growing hyphae of a melanic fungus (Aureobasidium pullulans) firmly attached to cellulose fibers (arrowhead). Image obtained using a Leica optical microscope in bright field mode; (C) A. pullulans hyphae attached to cellulose fibers (variable-pressure scanning electron microscope, Zeiss SEM EVO 50 with backscattered electron detector); (D) A. pullulans cells firmly attached to paper fibers without apparently causing damage to the cellulose and presumably growing at the expense of glues or sizing compounds [image obtained using high-vacuum scanning electron microscopy (Zeiss SEM EVO 50) on gold-coated samples].

In ecology, r-K selection is a theoretical model that describes the dynamics by which a population of a given species grows and becomes established in an ecosystem: r-selected species have a high growth rate but low survivability, while the K-selected species have a low growth rate but high survivability. The theory, expressed in quantitative terms, is fundamentally based on the relationship between two variables: the biotic potential and the environment’s carrying capacity. The r-K selection theory defines for a defined environment the potential for success of a given species. When free water in materials is scarce, the fungal species that develop in indoor environments are very different from the “r”-type species and also have ecological traits of a selective “K” type (224) that bring them closer to those species living in extreme environments, from which they sometimes originate. These climatic situations occur frequently and typically in stable environmental conditions or particular environments, such as closed shelving (159, 166, 219). These species also produce enzymes, organic acids, and pigments but more slowly, so the appearance of damage on the material may not be immediate, with the effects often underestimated (Fig. 7).

A substrate’s physical and chemical properties play a primary role in the growth and survival of fungi. Water availability is one of the selective factors for spore germination and for fungal penetration and utilization of the substrate itself. Some fungal species are xerotolerant, while others require high water potentials to grow. A “pioneer” fungus, capable of germinating and growing on a substrate with little available water, can alter the substrate itself through its biochemical and mechanical actions, making it suitable for more demanding species, in an ecological succession. The remains of the pioneer fungus offer nitrogen and trace elements to the species that follow. The properties of paper to absorb and release water vary considerably in the presence of a living organism or its dead residues in the form of mycelial fragments and metabolic exudates (232).

The affinity for water of a heterogeneous organic material is often expressed by means of its water activity (a_w), which relates the vapor pressure of pure water (P_o) to that of the water contained in the matrix of the material itself (P). The water activity is, therefore, given by (233, 234):

$${\displaystyle a_w=P/P_o}$$

where P differs from P_o due to the effect that the presence of solutes has on the vapor pressure of the solution itself (vapor pressure is the force with which water molecules on the surface of the solution tend to move from the liquid phase to the vapor phase). The maximum (theoretical) value of a_w is 1, when the water contained in the material is also pure, while the minimum value of a_w is 0, when the water contained in the material has no vapor pressure (all of its molecules are strongly retained in the material itself).

Water activity, however, remains a measure of water concentration and, for the relationship between a material and a fungus, is only a theoretical indication and does not allow for consideration of all the forces that come into play in the regulation of water flow between the substrate and the organism. The measurement of the medium’s water potential is far more informative for this purpose since it is an expression of the energy state of the system and takes into account the forces at play in the process of water uptake by the fungus (235, 236).

For fungal cells to absorb water from the external environment, they must be able to regulate their internal osmotic potential to create a favorable inflow of water, enabling maintenance of the correct turgidity. Osmoregulation in fungi is a process that requires much metabolic energy, as it requires active transport of ions and non-electrolytes from the outside to the inside of the cell against the chemical gradient. The internal osmotic potential inside fungal cells must be lower than the external osmotic potential; otherwise, water leaves the hyphae instead of entering. The ability to maintain a favorable osmotic potential relative to the environment varies from species to species in fungi and affects the ability to grow on substrates with very low water potentials. Some fungal species under water stress, e.g., activate the synthesis of compounds capable of increasing the intracellular osmotic potential, such as glycerol and mannitol (237, 238).

The climate is changing globally, greatly reducing the predictability of weather forecasting models on a planetary and local scale (239, 240). The changes occurring at the ecosystem level have repercussions on certain microbial communities in both the natural and built environment, some of which are unpredictable. Fungi that are xerophilous have a wide distribution range and possess resilience to multiple stresses that can favor their colonization of man-made environments. One possible example is the fungus Eurotium halophilicum C.M. Chr., Papav. & C.R. Benj. This is an ascomycete first described by Christensen and initially associated with foods with low water activity values (241) but is now increasingly associated with monospecific infections within museums, archives, and libraries (103, 162) (Fig. 8). E. halophilicum is an obligate xerophile, and the astonishing ability of this fungus to cope with changing conditions could lead to its increasing spread and persistence in both natural and synthetic ecosystems.

Fig 8.

Paper and cellulose. Fungi attacking paper often release polyketide quinones, carotenoids, and other staining pigments; (A) purple stains of fungal origin and fungal-fruiting structures on a biodeteriorated archival document; (B) clean paper before the fungal attack [variable-pressure scanning electron microscope (VP-SEM), Zeiss SEM EVO 50, backscattered electron detector] showing scattered minerals corresponding to carbonates used as sizing materials in paper manufacture: the light appearance depends on the atomic number of calcium; (C) the same paper grade shown in (B) after the fungal attack. The fungi can produce organic acids and transform the sizing mineral, precipitating biogenic crystals of calcium oxalate (arrowhead), while using cellulose as a substrate (deteriorated cellulose fiber: arrowhead with asterisk); (D) fungal defacement of paper often depends on fungal melanins. Melanins are located in the cell walls, and, by fungal penetration of the material structure, cause dark stains that can remain deep in the body of the material. Epicoccum sp. is a fungus that attacks paper and causes dark stains (optical microscopy, bright field); (E) Epicoccum sp. spores attached to cellulose fibers that appear to be torn and fissured by fungal enzymatic action (VP-SEM, Zeiss SEM EVO 50, backscattered electron detector).

Over the last 20 years, E. halophilicum has been found worldwide in several kinds of indoor environment. It has been observed on many different materials such as canvas covers of books, wood, leather, paintings, and frescoes (159, 162, 166). E. halophilicum is not considered the most halophilic or extremophilic fungus but can grow at a significantly lower water activity than saturated NaCl (162, 164) and can germinate at 0.651 water activity on media supplemented with glycerol, NaCl, and sucrose (165). A recent study highlighted how E. halophilicum utilizes the deliquescence of salts as part of its resistance strategy at low ambient water availability. The hypothesis proposed by Micheluz et al. (162) is that the kosmotropicity of certain extracellular polymeric substances produced by the fungus contributes to the concentration of ions and their precipitation in the form of salts (Fig. 8). This implies that the deliquescence of salts is influenced by the fungus, even though the deliquescence of salts itself is driven by physico-chemical abiotic factors. Therefore, through the secretion of abundant extracellular polymeric substances, the production of compatible solutes, the assimilation, transport and concentration of elements into the mycelium, and the effect of salt deliquescence, the fungus possesses a coordinated and effective strategy of water regulation. In addition, the hyphae of E. halophilicum are covered in non-cellular microfilaments with a diameter of several nanometers. Micheluz et al. (162) hypothesized that these microfilaments of E. halophilicum provide a large surface area capable of absorbing moisture. Clearly, this fungus evolved and differentiated long before the invention of libraries and the development of urbanized habitats where it now dominates in many indoor and artificial environments. This species’ new ecological niches and synanthropic distribution range could be an excellent model for studying the ecology of similar fungal species that “jump” from rare natural ecological niches to extremely widespread but artificial conditions. There are other examples besides E. halophilicum, such as the Cladosporium species associated with bathroom tile grout throughout the world (242, 243), or the fungal species growing in washing machines (244), which suggests ecological mechanisms of colonization and dispersal based more on a combination of environmental variables rather than on food materials and resources. Eggins wrote in 1968 (245) “Biodeterioration, by its very definition, is concerned with the interaction of materials with organisms. Thus substrate specificity, which may not coincide with habitat specificity, should be the overriding criterion of study in ecological considerations of biodeterioration.” In light of the most recent findings on the biogeography and ecology of certain species of fungi with an anthropogenic distribution, such a statement must necessarily be deepened and defined in terms of how certain materials come into equilibrium with environmental conditions.

FUNGAL BIODETERIORATION OF SPECIFIC MATERIALS

Stone

Being an important aspect of geomycology, fungal interactions with stone, rock, and minerals is an extensively researched area. Rock- and stone-based cultural heritage is readily colonized by microorganisms with fungi as an important component, sometimes dominant, of the surface and sub-surface microbial communities and biofilms (9, 18, 24, 26, 28, 31–33, 36, 37, 55, 116, 122, 123, 181, 246). Fungal colonization of stone can have multiple effects ranging from staining and discoloration to biodeterioration and crust formation (18, 48, 122, 204, 207, 247–250). Colonization can be affected by fluctuating environmental and climatic factors, such as wind, rain, relative humidity, particulate deposition, industrial emissions, and temperature, as well as the physical and chemical nature of the stone itself are subject to abiotic weathering and alteration (24, 35, 41, 44, 70, 130, 239, 251–257).

Bioreceptivity refers to the capacity of a stone to hold organisms and depends on the type of mineral, its chemical composition, petrographic features, porosity, roughness, and permeability of water (41, 42, 258). Unsurprisingly, increased porosity and surface roughness enhance bioreceptivity because of the better water holding capacity and more efficient trapping of airborne propagules and spores, particulates, and nutrients (41). Bioreceptivity is an important concept because it can aid the understanding of the colonization of stone as well as other materials (35, 36, 259–263), although it is difficult to define objectively (130). A bioreceptivity index has been devised for some granitic rock types (264, 265), but application to other substrates is limited, unsurprising due to the complex and fluctuating parameters that influence colonization (130, 253, 254). Bioreceptivity may be increased through the biodeteriorative actions of colonizing microorganisms, bioprotective or cleaning measures, and abiotic weathering, or reduced through formation of protective layers or crusts (41, 266). Different kinds of stone can show different patterns of colonization, and fungal and lichen communities can show different distributions depending on mineralogical composition (41). Sandstone is regarded as a stone of high bioreceptivity which in combination with exposure to water, as in the Buddhist cave temples Maijishan and Tiantishan Grottoes in China, can boost surface colonization and biofilm formation by an inter-connected and dynamic community of fungi and bacteria (39).

Mineral and elemental composition and nutritional needs of colonizing organisms markedly affect microbial colonization (200, 267, 268). In favorable conditions, lichen colonization of granitic buildings may be rapid (247). Other factors affecting stone colonization include substrate pH. Many fungi involved in biodeterioration of stone can grow under a very broad pH range, whereas alkaline rocks are generally reported to be more susceptible to fungal attack than acidic rocks. There are some species that prefer substrata such as granites and some sandstones of low acidity (41, 133, 269, 270). Orientation and aspect of cultural heritage to prevailing wind and rain can also have significant effects on microbial colonization, including fungi and lichens (271).

Hyphomycetes and black meristematic fungi are main fungal groups colonizing stone cultural heritage (123, 250, 272). These are often referred to as RIF, but this term is not specific and many fungi associated with rocks are also common inhabitants of other environments such as soil and decomposing organic matter (250). Hyphomycetes produce asexual spores (conidia) that are commonly aerially dispersed, meaning fungal biodiversity on surfaces may reflect the diversity of fungal airspora (22, 257, 273–276). Fungi isolated from a museum limestone false door included several Aspergillus and Alternaria genera, and Cladosporium herbarum which were found capable of modern limestone colonization (277). Genera commonly isolated from stone monuments included Alternaria, Aspergillus, Cladosporium, Curvularia, Dematium, Fusarium, Mucor, Penicillium, and Rhizopus (23, 257, 274–276, 278). Alternaria tenuissima could rapidly penetrate stone under suitable conditions of humidity and temperature, resulting in cryptoendolithic growth and further stone biodeterioration (131). Although common fungal species may dominate limestone communities, other rarer taxa are commonly isolated, and shifts in community composition occur with time, particularly in porous limestone possibly reflecting changes in the tolerance of certain species toward desiccation (279). Several fungal species are dematiaceous, which refers to their black or dark-colored appearance (250, 277, 280). Black fungi such as Aureobasidium pullulans have been shown to be pioneer colonizers of marble (281, 282). Many colonizing fungi are capable of organic acid production, and this can lead to dissolution, biopitting, secondary mineral formation, and formation of crusts and patinas on stone surfaces (23, 101, 202, 203, 249, 283). Organic acids are capable of significant mineral dissolution including silicates, phosphates, and carbonates (203, 284). Secretion of organic acids and other substances can cause deep alterations in rock subsurfaces (283, 285, 286). Organic acid mineral dissolution by ligand-mediated dissolution processes can be accompanied or followed by proton-mediated dissolution (200, 203). Penicillium, Fusarium, and Aspergillus genera are frequently isolated organic acid secreting genera (23, 101, 189, 274, 287–289). A range of organic acids are routinely found to be produced by rock isolates including oxalic, citric, fumaric, acetic, gluconic, malic, and succinic acid (189, 274, 287, 288), with oxalic acid particularly associated with dissolution and oxalate precipitation (23, 175, 203, 289–291). Calcium oxalate-containing patinas and crusts are often associated with fungal and lichen colonization of stone with the monohydrate (whewellite) and dihydrate (weddelite) both being recorded (23, 178, 257, 292). Such patinas may also contain calcite or vaterite, resulting from secondary precipitation of calcium after, e.g., carbonate dissolution, or phosphates (23, 195, 257, 289, 291).

Black meristematic fungi, e.g., Hortaea, Sarcinomyces, Exophiala, and Knufia spp., possess thick dark melanin-pigmented walls and form slow-growing colonies that grow by isodiametric enlargement of the cells (21, 22, 293, 294). Meristematic black fungi can be considered the true stone-inhabiting fungi (22, 250, 295, 296). These are also referred to as MCF because of their typical cauliform colonies (250). Advances in molecular analyses have led to the identification of many new genera and species of MCF (117, 251, 294, 297–302). Several species can also exhibit yeast-like growth and included among the so-called “black yeasts” (250, 282). Oligotrophy and the microcolonial-melanized growth form give clear advantages by conferring resistance to environmental extremes of temperature, salinity, desiccation, and solar irradiation, and enable dormancy in dry conditions (21, 43, 117, 123, 250, 282, 294, 295, 303–305). The involvement of biochemical and biomechanical mechanisms is proposed for stone biodeterioration by MCF, such as local release of EPS, organic acids, or siderophores, and subsequent biomineralization, and colonial expansion and rock penetration (187, 188, 194, 286, 306, 307). They may grow and disrupt mineral crystal structure in, e.g., marble, and also cause biopitting (308), although their slow growth may mean that their contribution to bioweathering is over the long term (309). On darkened white marble, Ascomycota was the dominant phylum detected by molecular analyses and also dominant for those fungal species that could be isolated (302, 310). Metataxonomic analysis showed the prevalent organisms in the darkened marble fungal community were meristematic fungi from well-known genera, including Cladophialophora, Devriesia, Knufia, Lithophila, and Vermiconia (296, 302, 307). Lichen biodeterioration of stone is a serious problem for cultural heritage with, as mentioned previously, a plethora of biomechanical and biochemical mechanisms being involved, mutualistic interactions with other microorganisms, including MCF, and variable effects between different lichen species and stone composition (9, 41, 122, 123, 175, 177, 181, 207, 290, 311).

Concrete, mortar, plaster, and buon frescoes

Another important group of inorganic substrata in cultural heritage includes concrete, mortar, and plaster. Most of these materials are cement based. Currently, the most used building material in the world, concrete, is usually made of cement (containing 75% calcium silicates), water, and aggregates (quartz sand and granite gravel) (312). Mortar, made from cement mixed with water and sand, is a bonding agent for masonry units, bricks, and tiles, whereas plasters, which can be also cementitious, or lime- and gypsum based, are used for rendering on the outside and inside of walls.

Traditionally, studies of biodeterioration or conservation treatment of built heritage, including architectural works and monumental sculptures, mainly focused on preserving natural stone used before the 20th century (313). At the turn of the 19th and 20th centuries, the great mechanical properties, versatility, and ease of handling of cementitious materials brought a revolution in building technologies, which in turn brought modern architecture with concrete/cement-based masterpieces such as the Park Güell and Sagrada Familia by Gaudi in Barcelona, Spain and the House with Chimaeras by Horodecki in Kyiv, Ukraine (313, 314). Nevertheless, concrete-based architecture was poorly recognized as an element of cultural heritage, and little studied in terms of its preservation (313). In an investigation of the state of conservation of concrete heritage buildings in Europe, it was found that around 70% of objects were unrestored (315). The percentage of biological growth-related damage of heritage objects made of or supported on concrete was found to be very similar for all studied environments: industrial, urban, rural, and maritime (12%–14%); and moisture-related processes were considered as the most important cause of biological growth-related damage comprising 80% of all hypothetical causes of this type of damage (315). It is known that, in abiotic terms, long-term concrete exposure to water may compromise concrete stability resulting in progressive leaching of the cement phases and calcium (312). Moisture also is one of the key factors promoting fungal and other microbial colonization on the surface of such building materials, where formation of hydrophilic slimes and biofilms contributes to water retention and expansion of clay minerals (316).

Another important factor defining concrete colonization is its alkalinity, which decreases with time from the initial values of pH 12–13 for fresh concrete due to a carbonation process with atmospheric carbon dioxide resulting in an increase of bioreceptivity of this material (317). However, the ability of many fungi to grow over wide ranges of pH is well known and also includes the highly alkaline pH values of cementitious materials, mortars, and frescoes (41, 270). As concrete is a composite material, bioreceptivity of its surface to fungi is heterogeneous, reflecting the nature of each component. It was demonstrated that the most susceptible component to fungal biodeterioration is cement paste in contrast to the coarse aggregate granite gravel (Fig. 9). The fine aggregate minerals, quartz and feldspar, are almost unreactive (312).

Fig 9.

SEM images of concrete biodeterioration by Aspergillus niger during a year-long experiment in microcosm showing differing bioreceptivity and susceptibility to fungal destruction of cement paste (with spalling, cracking, and massive biomineralization of calcium oxalate) and granite (with a relatively smooth surface) [images by M. Fomina, unpublished, see also Fomina et al. (312)].

Concrete heritage objects, like any other concrete, can be successfully colonized by fungi from genera, including Alternaria, Aspergillus, Aureobasidium, Cladosporium, Fusarium, Paecilomyces, and Penicillium, which can biodeteriorate this material through both biochemical (excretion of protons and ligands) and biomechanical mechanisms as described previously. In concrete, the cement paste components, Ca(OH)₂ and calcium-silicate hydrate, are the first to be subject to fungal biochemical attack, mobilizing calcium (Ca²⁺) and silicon (mostly as silicic acids [SiOn(OH)_4−2n]_x). Leached elements can be accumulated by the fungi and reprecipitated in the microenvironment, where secondary mycogenic minerals such as calcium oxalates (whewellite and weddellite) can be formed by oxalate-excreting fungi (312). Because mortar and cementitious plasters are of the same chemical composition as concrete, apart from coarse aggregates such as granite gravel, all these materials share the same biochemical and biomechanical mechanisms of biodeterioration leading to their structural damage.

Plaster has been often composed of gypsum and widely found in historic buildings and the built environment and susceptible to fungal biodeterioration (1, 18, 21, 55). Fibrous plaster has also long been used in decorative interiors and ceilings, and subject to degradation and biodeterioration that can have serious consequences should ceiling collapse occur (318, 319). Moisture and fungal biodeterioration are the synergistic biodeteriorative determinants of plaster failure. Moisture affects mechanical properties of the plaster and facilitates fungal growth. Fungal colonization is facilitated by porosity and cracks, and the fibrous organic component (e.g., hessian-containing cellulose, hemicellulose, and lignin) may be attacked and degraded by fungi (319). Organic acid excretion by fungi can solubilize gypsum and lead to extensive production of calcium oxalate (320), which can further lead to loss of structural integrity. Colonizing fungi are frequently well-known indoor and outdoor airborne species including Cladosporium, Penicillium, and Chaetomium genera (319, 321–324).

Gypsum plasters have been used since the time of Late Epipaleolithic (~10,000 BC). Another group of ancient plasters was lime plaster, made of lime, water, and sand, with the earliest recorded use around 7,200 BC (325, 326). Mixed gypsum-lime plasters and renders were also used in many parts of the world, where the addition of gypsum enabled the lime plaster to set faster. In the past, lime plaster was a common multi-purpose material used in buildings and artworks, including frescoes.

While a mural is any wall painting of great size, a fresco is a type of mural where water-soluble paint is applied on wet (buon fresco) or dry (secco fresco) lime plaster. Buon fresco is unique and the most durable technique of painting murals, derived from the Italian “buon” meaning “good or true” and “fresco” meaning “fresh” regarding the fresh plaster. In this technique, the mineral pigments are completely incorporated into the wet plaster becoming an integral part of the wall surface (65). In contrast, in the fresco secco (“dry fresco”) technique, the pigments, applied to dry finished walls, do not penetrate the mineral matrix of the lime plaster but create a film on the surface like most other painting techniques (apart from buon fresco). The secco technique is necessary for fine detailed painting and retouching of the fresco.

Due to natural abiotic deterioration and microbial biodeterioration, where fungi are particularly significant, of mural materials, increasing attention is given to conservation of ancient frescoes and other wall paintings. A comprehensive world-wide meta-analysis of the diversity of fungi deteriorating wall paintings of historical value has been published, as well as a review of the general factors and mechanisms that influence the deterioration of valuable murals (156, 327). However, there has been little research carried out on fungal attack of buon frescoes, and information on this problem is still limited.

The dark-spotted biodeterioration of medieval buon frescoes in Saint Sophia Cathedral in Kyiv, Ukraine, is a good illustration where a combination of environmental variables and the nature of the substrate define colonization and spread of specific fungi, which are the prime agents of this biodeterioration (65).

One of the key factors affecting the development of dark-spotted biodeterioration is the mineral-rich substrate of the walls (Fig. 10). The mineral composition of the 11th century mineral buon frescoes was found to be very similar to the restoration plaster applied in the 1950s to fill the gaps between the lost original frescoes. The main mineral in both materials was calcite. Generally, high alkalinity and the lack of organic content of such mineral-rich substrates make buon frescoes more durable and less susceptible to microbially induced decay. Nevertheless, such a hostile substrate can harbor many fungi able to tolerate and grow under such mineral-rich conditions. Another crucial factor is the very low humidity maintained in the cathedral, with relative humidity values around 37% (Fig. 10). Such conditions are unsuitable for the growth of many fungi except xerotolerant and xerophilic species. The temperature of the indoor air in the cathedral, maintained within the range 14–16°C in winter and 20–22°C during the summer (65), is favorable for growth of many fungi. Finally, a very important factor influencing fungal colonization and dispersal was related to the aerodynamic characteristics of the near-wall environment (Fig. 10). The air velocity in the general passageways of the cathedral, where people are walking, varied from 0.08 to 0.22 m per second, which met accepted standards and regulations. However, there was a heterogeneity in aerodynamic characteristics because of areas of slower air movement in the near-wall environment of the corners and small altars. The air velocity in the near-wall environment in the direction toward the ceiling was half of that for the passageways, while the air velocity in the downward direction to the floor was an order of magnitude lower compared to the passageways. This created a “toss-up” effect when the air flow at reduced speed goes up the wall and then significantly slows down while descending to the floor, thereby providing suitable conditions for attachment of dust and fungal spores to the surface of biodeteriorated walls. Only when all these factors coincided was the dark-spot biodeterioration observed (Fig. 10). Of these influencing factors, the combination of a mineral substrate and a very low humidity/water activity was key for the selection of obligate xerophilic aspergilli, which were able to grow and cause staining and biodeterioration under such harsh conditions. As a result of fungal colonization, growth, and biogeochemical activity, the mineral matrix of the original frescoes and restoration plaster lost integrity through mineral dissolution via heterotrophic leaching and the neoformation of the secondary mycogenic minerals: Ca-malate, hydrated Al-phosphate (AlPO₄⋅1.67H₂O), and strengite (FePO₄⋅2H₂O) (65). Unlike Ca-oxalate, Ca-malate biomineralization by fungi is rarely found in nature and biodeteriorated cultural heritage and could be triggered by the combination of abundant calcite with limited nitrogen availability and extremely low water activity (65, 328). Generally, the presence of malate along with oxalate and other low molecular weight organic acid salts has been suggested to be markers of prior fungal activity in wall paintings (328).

Fig 10.

The combination of factors that contribute to the development of dark-spotted biodeterioration caused by xerophilic aspergilli on the walls of St. Sophia Cathedral, Kyiv, Ukraine. One of the dark spots is shown within the white circle (diameter ~100 µm) in the light microscopy image. Humidity, temperature, and aerodynamic data (n = 6) were recorded in August 2020 (M. Fomina, unpublished).

Glass

Glass is derived from quartz and can be considered as a silicate and, therefore, subject to all the abiotic and biotic interactions that can occur with silicates in the natural environment. Glass contains other chemical components such as various metal oxides and other metallic materials, the latter imparting color to stained glass, chemical modifiers, and stabilizers (41, 77, 329). Such components may have effects on microbial colonization and biodeterioration. While glass might be considered a difficult and inhospitable surface to colonize, microbial communities can still develop over time and cause significant biodeteriorative effects (124). As with other surfaces, nutrients can accrue through atmospheric deposition, microbial and animal exudates, and wastes, leading to the development of complex bacterial and fungal communities, the latter usually constituting ubiquitous airborne genera (329–331). Common fungal species colonizing glass are similar to those colonizing other exterior surfaces and include Aspergillus (and its teleomorph Eurotium), Cladosporium, Trichoderma, Penicillium, Chaetomium, Aureobasidium, Phoma, and Scopulariopsis genera, many of which are dark pigmented (330). Among the fungal genera, there are also extremophilic aspergilli, obligate tonophiles/xerophiles from section Restricti of the genus Aspergillus, with a strong capability for growing on cleaned glass without addition of any nutrients (160, 332). One species, Aspergillus vitricola, was isolated from valuable lenses and prisms and received its species name “vitricola” from “vitrum” meaning “glass” in Latin (332). This species is also one of the most abundantly detected fungi in house dust all over the world.

Colonization and deleterious effects may be rapid under suitable conditions (333) with acid-producing fungi being particularly significant (334). Colonization is influenced by the usual factors including relative humidity, temperature, nutrient availability, and the chemical composition of the glass (334, 335). Ancient stained glass can show all the well-known consequences of rock and mineral bioweathering such as etching, pitting, crack expansion, discoloration and alteration of transparency, hyphal “footprints” depletion or enrichment of elements, and crust or patina formation (154, 329, 330, 333, 336, 337). On stained glass, the most common biodeterioration forms reported in laboratory studies were micro-cracking, pitting, hyphal footprints, depletion or enrichment of elements, and deposition of crystalline compounds. In one study, over 20 different genera were found on biodeteriorated stained glass: Alternaria, Aspergillus, Aureobasidium, Capnobotryella, Chaetomium, Cladosporium, Coniosporum, Didymella, Engyodontium, Fusarium, Geomyces, Hortaea, Kirschsteiniothelia, Leptosphaeria, Myrothecium, Penicillium, Penidiella, Phoma, Rhodotorula, Sistotrema, Stanjemonium, Trichoderma, Ustilago, and Verticillium. Of these, the highest diversity was observed for the common airborne fungal genera Aspergillus, Cladosporium, and Penicillium (154).

Metal oxidation may also contribute to discoloration (336), while elements leached from glass may react with other chemical compounds forming crust-like deposits (335). Other materials used in window construction and placement such as metals, wood, cement, and mortar may also be subject to fungal biodeterioration.

Ceramic building materials

Clay-based ceramic materials are widely found in historic buildings and monuments as, e.g., bricks and roof, wall, or floor tiles (338) and are subject to the similar biodeteriorative effects as on stone and other rock-mineral-based materials described above. Ceramic building materials include unglazed and glazed ceramic tiles, the latter especially common and important in historic built heritage because of cultural and artistic imagery on the glazed outer surfaces (263, 338). The glazed vitreous surface coating, due to the presence of glass, is more resistant to abiotic and biotic weathering than the porous subsurface ceramic material, but ceramic tiles can be readily colonized and biodeteriorated by microorganisms. Porosity and surface roughness play a major role in bioreceptivity to colonization (262, 338). As with other rock- and mineral-based structural materials, fungal biodeterioration ranges from discoloration, staining, and overgrowth to biomechanical and biochemical effects on the substrate (262). Such activities at the ceramic-glaze interface may cause disintegration (338). Dematiaceous and meristematic fungi are frequent colonizers of ceramic tiles and commonly detected in community analyses (263, 339). Like other surfaces, many fungal colonizers are common airborne species arising from, e.g., soil or decomposing organic matter, such as Alternaria, Aspergillus, and Penicillium species (338). Nineteen fungal genera were identified in studies of biodeteriorated outdoor majolica glazed tiles in Portugal, including Aspergillus, Capnobotryella, Capnodiales, and Penicilium, with the most diverse genus being Devriesia with species D. imbrexigena, D. modesta, D. neodevriesiaceae, and D. xanthorrheae being recorded (154). Lichens are also significant colonizers and biodeteriorative agents of ceramic tiles (340, 341). Fungal biodeterioration can result in the formation of crystalline deposits and crusts due to calcium mobilization, such as calcite, gypsum, and calcium oxalate, the latter a direct indicator of fungal activity (202) and causing physical damage as well as alterations in surface gloss and reflectance (263, 341, 342).

Metal corrosion

The involvement of fungi in iron corrosion has so far been very little studied, and even less investigated has been metal corrosion inside buildings or when the air relative humidity is low, and the organisms involved do not have a regular supply of free water. Plany et al. (343) proposed a mechanism of corrosion by fungi in indoor environments involving the removal of protective iron oxides from the surface of iron objects (nails) by acid dissolution, dissimilatory respiration, and assimilative processes. Plany et al. (343) also first described the formation of compartmentalized iron rust shells containing fungal-fruiting bodies (Fig. 11). In the presence of 65% or more relative humidity and adequate nutrients, fungi can produce acidic by-products that trigger iron corrosion even in indoor environments (344). The relative air humidity limits for atmospheric corrosion of iron (60%–65%) are also the limits of ambient water that can support fungal growth on materials (163). The water activity (a_w) scale ranges from 0 (dry) to 1.0 (pure water). Fungi include the most desiccant-resistant microorganisms and can remain active at a_w = 0.6, while few bacteria remain active below a_w = 0.9. Filamentous fungi growing in drier environments often produce EPS, which protect from dehydration and maintain an osmotically favorable microenvironment for cellular processes.

Fig 11.

Dust and fungal spore dispersal. A commonly used microbiological marker of cellular viability in microscopy is fluorescein diacetate (FDA), an uncharged, non-fluorescent, lipid-soluble dye that, after uptake by the fungal cells, is hydrolyzed to fluorescein by non-specific intracellular esterases. Free fluorescein is a polar molecule and is retained by intact cells. The accumulation of fluorescein results in a measurable fluorescence visualized using a fluorescence microscope. The fluorescein ion diffuses out of damaged membranes: cells that do not fluoresce are therefore considered non-viable; (A and B) Aspergillus halophilicus stained with FDA: some conidia show strong fluorescence compared to others in the same image and can be considered viable (Leica DM5000 fluorescence optical microscope); (C) dust collected from indoor air in an archive and stained with FDA: some airborne spores are viable: the arrow indicates an Alternaria-like muriform viable conidium; (D) in addition to aerial dispersal, many fungi rely on zoonotic dispersal. It is common to find filamentous biodeteriorative fungi associated with mites and insect droppings: the arrow indicates a mite close to fungal structures sampled from a book cover; (E and F) dust mites covered with fungal spores (F image was obtained using variable-pressure scanning electron microscope, the arrowhead indcated the mite); (G) droppings of a liposcelid (book louse) containing fungal spores (arrowhead), indicating the specific diet of the insect (high-vacuum scanning electron microscopy, gold-coated sample).

Museum metal armor used to suspend a whale skeleton was found to be heavily corroded (343). A patina containing fungal conidiophores, chains of conidia, and the hyphae of a fungus belonging to the Microascaceae family was covered with iron-rich material to form tubercle-like structures (Fig. 11). The fungus, which was not identified at the species level, was attributed to the Scopulariopsis genus or the anamorph of a species of Microascus based on morphological characters (345). Some species of Scopulariopsis are known to bioaccumulate and biomethylate metalloid elements, resulting in changes in their speciation, mobility, toxicity, bioavailability, and volatility (346). Stranger-Johannessen (347) also demonstrated rapid and severe pitting corrosion of steel in the holds of a ship caused by Scopulariopsis brevicaulis. It has also been suggested that some fungi corrode iron to facilitate iron assimilation. Iron is a redox mediator in essential eukaryotic cellular processes and an essential element for fungi (348). Odoni et al. (349) demonstrated that Aspergillus niger increased citrate secretion under iron-limited conditions as a physiological response consistent with the role of citrate acting like a fungal siderophore. Lloyd and Manning (350) established that rusting rates of iron in the atmosphere are low in the absence of strong electrolytes containing aggressive anions. An electrolyte is a substance that produces an electrically conductive solution when dissolved in a polar solvent, such as water (351). The electrolytes associated with corrosion of iron nails in the museum whale skeleton contained calcium, phosphorus, sodium, chloride, and silicon, in addition to iron, that were released by fungal bioweathering of the bone where the nails were inserted (343). Many fungi can produce low molecular weight organic acids, including acetic, citric, and oxalic acids. The corrosive strength of an organic acid is decisive as the corrosion current of a 1% organic acid solution can vary from 0.27 to 0.03 mA/cm² (343). The dissolution of Fe oxides by oxalic acid is optimal at pH 2–3. The reaction of Fe₃O₄ with oxalic acid converts an insoluble iron oxide into highly soluble ferrioxalate ions that act to solubilize Fe₂O₃. The dissolution of iron oxide by oxalic acid results in the complexation of iron (Fe²⁺ or Fe³⁺, i.e., [Fe²⁺(C₂O₄)₂]²⁻ and [Fe³⁺(C₂O₄)₃]³⁻) by oxalate (Fig. 11). Other indoor iron corrosion mechanisms have been described. For example, Coniophora puteana possesses an enzyme (cellobiose dehydrogenase) capable of coupling the oxidation of cellodextrins to the conversion of Fe³⁺ to Fe²⁺ (352). Recently, it was demonstrated that corrosion of indoor metals is significantly more pronounced under microgravity conditions (353). The authors conducted fungal corrosion experiments using Aspergillus carbonarius growing on five metal plates (titanium alloy, aluminum alloy, iron, aluminum, and copper plates) under simulated microgravity conditions. The fungus was able to corrode all five types of metal, and some of the metal ions extracted from the plates (iron, aluminum, and copper) concentrated in the spores suggesting a form of accumulation. It was hypothesized that the clinostat rotation used to simulate microgravity stimulated increased oxalic acid production which caused a more intense corrosion per unit of time and surface (354).

Bone and ivory

Bones and ivory are biogenic hydroxyapatite minerals with a composition approaching [Ca₁₀(PO₄)6(OH)₂] (355). The ratio of calcium to phosphate in them varies between 1.3 and 2.0 (by weight), and they also contain carbonate and trace elements such as magnesium, sodium, and potassium. Although objects made of bone or ivory have a mineral composition, microorganisms can grow on them even when stored indoors (Fig. 12). Their porosity, high salt content, and hygroscopicity make them capable of hosting extremophilic fungi that form biofilms even at low relative humidity and temperature (356). Museum objects made of bone or ivory, as well as archaeological bone artifacts excavated from the ground or found in graves or containers of various types, can be biotically attacked by fungi that change their appearance, structure, and sometimes chemical composition (357). The biodeterioration of bones and ivory in indoor environments has been scarcely studied, whereas bone diagenesis in the soil is the subject of dedicated studies as it is important for taphonomy (358) and archaeological studies (359–363). Fungal activity has been particularly documented (364–366) and often distinguished from bacterial activity (367–369), or cyanobacterial and algal colonization (370–372) mainly in soil but also in seawater (355, 373). On human bone material from archaeological excavations, species of Aspergillus, Chaetomium, and Cladosporium were found by Pitr et al. (374), with Penicillium chrysogenum identified as one of the main participants in the formation of a biofilm characterized by bacteria and fungi mixed with calcite crystals. The authors considered the secretion of exopolymeric substances to be a main phase of biofilm formation on skeletal material. In vitro experiments by Pinzari et al. (191) showed that fungi alone could dissolve hydroxyapatite, the main component of bone and teeth (Fig. 12). Large bone skeletons are often iconic exhibits in natural history museums. Pinzari et al. (356) studied biodeterioration of the blue whale “Hope” skeleton at the Natural History Museum in London. This skeleton has been displayed in the Mammal Gallery since 1934 and was restored to be suspended in the museum’s Hintze Hall in 2017. The study’s results showed how the bone material provides a niche for specialized fungi in indoor environments and is affected by peculiar alteration mechanisms, in which extremophilic fungi are at the origin of hydroxyapatite dissolution and secondary mineral formation (356) (Fig. 12). In addition to Cladosporium herbarum, which is one of the most frequent cosmopolitan fungi in indoor environments and in dust (375, 376), several yeast species were found in the Hope skeleton bone, identified by culture-independent methods as belonging to the genera Naganishia, Cryptococcus, Erythrobasidium, and Rhodotorula. The fungus Dipodascus geotrichum Butler & Petersen was also documented with SEM images on bone fragments. This is a species that grows readily on hard surfaces such as industrial equipment, walls and floors, gutters and building materials, and can solubilize, translocate, and store metals by reduction and chelation mechanisms (377, 378). SEM-EDS has documented the fungal biofilm adhering to the surface of Hope’s bone, and this was characterized by a high iron concentration, with the iron coming from the nails that were used to assemble the skeleton. The presence of yeast cells in biodeteriorated areas of bone (e.g., affected by pitting and leaching mechanisms) was examined using SEM, and some cell structures correlated with the identification of clones belonging to the yeast Mrakia frigida (99.69% similarity). Mrakia frigida is a psychrophilic basidiomycete yeast (Basidiomycota, Tremellomycetes, and Cystofilobasidiaceae) (379) (Fig. 12). Psychrophilic fungi are found in permanently cold environments such as polar regions, marine environments, and deep waters (380). The survival and germination of these microorganisms in indoor environmental conditions can be partly explained by hygroscopic materials such as gypsum and plaster that create a microenvironment on bone in which free water may be available (381). Rhombohedral biogenic crystals, a typical crystalline habit of calcite, were also found on the bones (195, 382) and appeared immersed in an exopolymeric material of microbial origin. These secondary biogenic minerals have been repeatedly reported in biodeteriorated bones, especially from archaeological excavations (366, 369, 383). In addition to calcite, calcium oxalates and secondary apatite appearing as aggregate lamellar crystals, originating from microbial solubilization and recrystallization of calcium phosphates, were also present on the fungal-attacked bone. Microbial solubilization of apatite is linearly dependent on acidification of the environment (384), which in turn depends on the release of organic acids by the fungi and the formation of carbonic acid as a result of CO₂ production by respiration (53, 137). In addition to lowering pH, organic acids form strong complexes with cations, thereby increasing dissolution rates of phosphate minerals. The concentration of phosphate species (H₃PO₄, H₂PO₄⁻, HPO₄²⁻, and PO₄³⁻) at the interface between the bone and fungal mycelium is a function of local pH (385). Phosphates are more stable than carbonates at low pH (<5). The chemical composition of phosphate minerals is more variable than that of carbonate minerals, and crystalline chemical substitution of the PO₄³⁻ group by CO₃²⁻, OH⁻, F⁻, and Cl⁻ is common. Several metals ions such as Ca²⁺, Mg²⁺, Fe²⁺, and Na⁺ can also be incorporated into the structure of phosphate minerals, generating different secondary structures (385) by apatite dissolution and recrystallization and by heteroionic substitution. In the case of iron substitution in contact with the original bone, vivianite, [Fe₃(PO₄)₂·8H₂O] was present and in the case of calcium substitution, brushite (CaHPO₄) and calcite (CaCO₃). Fungal attack resulting in the transformation of apatite into calcite often leads to the detachment of fragments and destruction of the material, as the substituted CaCO₃ occupies more space in the mineral matrix than calcium phosphate (361). Fungi growing on bone and ivory can also cause disfiguring discoloration, deeply penetrating due to the material’s porous nature. Fungal hyphae have, for example, been directly associated with the brown discoloration of bones by Grupe and Dreses-Werringlöer (386). Many unwanted discolorations can be traced to the secondary biogenic minerals mentioned before. In fossil mammoth bones, dark-colored vivianite was derived from biogenic processes (387). Many other kinds of discoloration on ivory and bones are due to secondary phosphates whose formation depends on initial solubilization of apatite by acid dissolution, e.g., santabarbaraite mainly contributes to the blue and yellow-brown oxide layers of mammoth ivory (388).

Fig 12.

Eurotium halophilicum. Monospecific contamination of books by the fungus Eurotium halophilicum C.M. Chr., Papav. & C.R. Benj. (A) Fungal colonies on the cover of books; (B) conidiophores and conidia of Aspergillus halophilicus, the anamorphic state of E. halophilicum (bright field optical microscope, Leica DM5000); (C) high-vacuum scanning electron microscopy (HV-SEM, Zeiss SEM EVO 50) image of a conidiophore with conidia of Aspergillus halophilicus (gold-sputtered sample); (D) mature ascocarps of the perfect form E. halophilicum (HV-SEM); (E) E. halophilicum hyphae that are generally covered with hair-like microfilaments, especially when growing at low water activity, that are several nanometers in diameter; (F) mature ascospores of E. halophilicum (HV-SEM) [images by F. Pinzari, unpublished, see also Micheluz et al. (162)].

Wood

Wood is a widely used material in works of art, either as support (e.g., in canvases for paintings) or structural elements (furniture) or as a component of more complex objects, such as wooden covers of manuscripts (389). Technically, wood is xylem tissue originating from the cambium (the inner part of the bark of trees). The main components of wood are of a polysaccharide nature (cellulosic and hemicellulosic) and of a phenolic nature (polymers of phenylpropane:lignin), which, however, vary qualitatively and quantitatively depending on origin (e.g., coniferous and deciduous wood), age, and developmental conditions (390). The most important wood-deteriorating agents are the fungi that attack wood on the plant (parasitic fungi) and after harvesting, processing, and storage. Among the most dangerous fungi that cause significant damage are ligninolytic fungi that produce extensive lesions (caries, also known as rot) and chromogenic fungi that produce color changes. In the field of cultural heritage, if it is true that extensive lesions on wooden structures or artifacts represent very serious damage, but color changes can also be a serious problem to be prevented and treated. There are several studies on the biodeterioration of submerged wood, whether in fresh or salt water or buried in soils and sediments, as in the case of ancient shipwrecks. Under such conditions, aerobic and anaerobic bacteria also play a role (391, 392). However, only the biodeterioration of wood in indoor environments will be discussed here, a phenomenon that may mainly affect museum objects and furniture.

The fungal extracellular enzyme systems that can directly attack wood constituents (390) belong to the group of phenol oxidases, e.g., lignin peroxidase, manganese-dependent peroxidase (MnP), laccase, and dioxygenase (390). Other enzymes, such as superoxide dismutase and glyoxal oxidase, operate with phenol oxidases but never attack wood constituents independently. Other enzymes, such as glucose 1-oxidase, aryl alcohol oxidase, pyranose 2-oxidase, cellobiose:quinone oxidoreductase, and cellobiose dehydrogenase, play key roles in various forms of wood biodeterioration (393). Depending on the preferentially degraded wood component, wood decay is divided into white, brown, and soft rot. White rot occurs when degrading fungi preferentially attack lignin (394–396). Brown caries refer to rots caused by fungi that only degrade cellulose. Soft rots refer to caries caused by fungi that produce lytic enzymes that predominantly break down cellulose and hemicellulose. In general, damage caused to the wood of cultural heritage interest is caused by fungi that cause soft rot (saprophytic fungi). Fungi associated with soft rot are often ascomycetes. They can usually modify lignin without completely degrading it, decompose cellulose and hemicellulose, and create characteristic cavities due to degradation of the wood cell walls (397, 398). Laccase activities have been reported in saprophytic fungi belonging to the genera Penicillium and Aspergillus, which are also abundant in the indoor environments of museums and libraries (399). Laccases are multi-copper blue oxidases, frequently occurring as multiple isoenzymes in ascomycetes, and anamorphic fungi (399). Soft rot, however, affects wood when it is exposed to high humidity conditions, but the penetrative capacity of these fungi into the wood structure is generally very low so damage is generally limited to the outer layers of objects. Attacked wood in these cases presents itself with a dark, soft surface, often eroded by a reticular structure due to vertical cracks.

Much of the work on fungi that attack wood in indoor environments does not deal with xerophilous species of ascomycetes that proliferate on surfaces without penetrating deep into the wood (Fig. 13). There is more interest in species capable of causing extensive structural damage, such as the ligninolytic species growing at high water activity that cause rot by attacking wood in buildings when soaked in water (400). For example, Slimen et al. (401) identified fungi causing brown and white rot in 18th century French timber buildings, particularly Serpula lacrymans and Coprinellus aff. radians, but also listed numerous species of fungi belonging to the Ascomycota. Alfredsen et al. (402) listed 31 indoor wood rot fungal species in southern Norway. Schmidt (403) published a list of 74 species and genera of fungi isolated from biodeteriorated wood in buildings in Germany, a list later supplemented by Schmidt and Huckfeldt (404), who described 117 species and genera of wood decay fungi in German interiors. Fraiture (405) reported 101 species and genera of wood decay fungi in indoor environments in Belgium. However, there is also extensive evidence of fungal wood biodeterioration in conditions of low water activity. For example, Ortiz et al. (406) pointed out that wood deterioration can still occur in extreme environments, such as dry or salty desert sites. The authors analyzed biodeteriorated historical wooden structures in the Atacama Desert (northern Chile) belonging to the 1872 Humberstone and Santa Laura Saltpeter Works. The fungal species proliferating on wood in this highly saline environment were mostly halophilic or halotolerant species like Penicillium chrysogenum, Engyodontium album, Eupenicillium tropicum, Penicillium digitatum, Pseudotaeniolina globosa, Cladosporium phaenocomae, Aureobasidium pullulans, Penicillium virgatum, Coprinopsis sp., and Phanerochaete sordida. These fungi caused the defibration of the wood, with a particular degradation of the middle lamella between cells.

Fig 13.

Photographic materials biodeterioration. (A) A negative gelatine-silver film dating back to 1938–1940 and conserved at the Archivio Ente EUR-Archivio Centrale dello Stato, Rome, Italy; (B) detail of biodeterioration of the film showing fungal colonies on both sides (stereomicroscope, Leica MZ16); (C) variable-pressure scanning electron microscope (VP-SEM) image of an Aspergillus sp. that has produced conidiophores and degraded gelatine from the edges to the inner areas of the film; (D) biodeteriorated surface of the film showing a peculiar alteration with fungal hyphae covered with silver (light material indicated by the arrow) that the organism has accumulated from the lower layer of the photographic emulsion (VP-SEM, BSD detector); (E) alteration of the superficial layer of gelatine visibly eroded by fungal activity (VP-SEM, backscattered electron detector); (F) conidiophores and conidia of Zygosporium sp., a dematiaceous hyphomycete possessing dark-pigmented, curved vesicular cells (arrows) that give rise to two to four conidiogenic ampulliform structures: this fungus was found associated with photographic films and the cardboard supporting them [images by F. Pinzari, unpublished, see also Bučková et al. (85); Sclocchi et al. (407, 408)].

In indoor environments, it is rare for material to be soaked, and the most frequently observed changes to wooden objects are the presence of abnormal coloring and surface efflorescence, which can be observed even when the environmental conditions are suitable for conservation (Fig. 13).

Wood is a porous material that, at the microstructural level, can vary depending on the plant species of origin. Water vapor absorption from the air by a wooden object stored indoors encompasses several phases (409, 410). Water molecules can be adsorbed by capillarity or remain in vapor form and fill the empty spaces represented by the xylem vessels. For wood, a water activity of 0.80 corresponds to a minimum moisture content of 16%. Xerotolerant fungi can grow at moisture levels below 16%. Several filamentous ascomycetes common in indoor environments (e.g., species of the genera Penicillium, Aspergillus, Scopulariopsis, and Cladosporium) can maintain growth at a water activity of 0.70, which equates to a wood moisture content closer to 15%. Many authors have described alterations on wooden objects and furniture caused by xerophilic or xerotolerant fungi. Hallsworth (411) isolated a strain of Aspergillus penicillioides, a xerophilic but also environmentally ubiquitous generalist fungus (164), from the surface of an owl-shaped wooden handicraft object, showing hyphal growth at water activity values of 0.640 (411, 412). Hallsworth (411) also identified Eurotium repens from wooden objects stored inside a house near the Northern Ireland coast, Aspergillus vitricola from an antique mahogany tabletop, and Penicillium glabrum from an Indian wooden carving purchased in Japan. Such xerophilous fungi can survive prolonged periods of inactivity when dehydrated, and it can be assumed that their survival in indoor environments may also depend on their tolerance to periods of inactivity when water is less available. Choidis et al. (413) investigated the impact of climate change on the biological, mechanical, and chemical degradation of a wooden cabinet and a storage trunk from a historic timber building in Vestfold, Norway, showing how changes in local climate impact wooden materials and their susceptibility to biodeterioration, but there was a poor impact of moisture fluctuations on the abundance of biodeteriogenic fungi. A decisive role in the ability of xerophilic fungi to colonize the surfaces of wooden objects in indoor environments seems to be played by the substances used to treat or paint or assemble the wooden structure. Kavkler et al. (414) investigated the microbial contamination of the deteriorated 17th-century Celje Ceiling, a tempera painting in a wooden frame. They discovered that a coating layer made of protein binders and pigments, such as goethite, ultramarine and kaolinite, had an important role in mold development, with most of the damage caused by an unidentified xerophilic Aspergillus species. Štafura et al. (415) investigated fungal attack on 19th-century wooden pipe organs and isolated several species of filamentous fungi, including Alternaria mali, Eurotium cristatum, Aspergillus amstelodami, Penicillium crustosum, Aspergillus sydowii, Talaromyces rugulosus, Paecilomyces formosus, Cladosporium cladosporioides, Aspergillus versicolor, and Epicoccum nigrum. The authors used some of these strains to test the resistance to biodeterioration of new pipes treated with different glues and varnishes. It was found that at a relative humidity of 70%–75%, the mock pipes collapsed due to biodegradation of the glued joints. Sterflinger et al. (416) found that halotolerant aspergilli and penicillia with low optimal temperatures (below 20°C) were the most frequent invaders of pipe organs in churches. The authors showed that these fungi were not strictly halophilic but were all characterized by halotolerant behavior. These fungi were visible as white mycelia on the organic components of the organs, with preferential growth on the paint of the wooden pipes and parts containing organic glue. Kosel et al. (389) have documented how wood used in polychrome art or otherwise painted with different types of substances becomes differentially susceptible to attack by xerophilic fungi. Glues, varnishes, and pigments can substantially alter how the wood absorbs and yields water to biodeteriogenic microorganisms, while various chemicals can also interfere with use of the material as a food source.

Parchment

Parchment is a material prepared from animal skins (417, 418) that became widespread as a writing medium in Europe from the sixth century AD. The skins undergo a series of processes that lead to a product consisting of only the dermal layer, whose main component is collagen, an organic polymeric protein whose structural unit is a triple helix (419). Alkaline salts such as calcium hydroxide (420) and sodium chloride are used for manufacturing, and the finished parchments contain various substances derived from the skins (lipids, waxes, and other peptides) and manufacturing processes (salts and elements such as Al, Si, Mg, K, S, etc.) (93, 94, 220, 421). Salting (mainly with sea salt) was commonly used in the initial stages of parchment manufacture to prevent the skins from rotting. Parchments can be attacked by proteolytic bacteria and fungi that possess collagenase, the enzyme capable of cleaving the triple helix of collagen. However, many fungal species isolated from parchment are not collagenolytic but use the oils and waxes added during manufacturing processes as carbon sources. Parchment is a salt-rich environment, and the fungi that grow there are often halotolerant. Among the changes of biological origin that afflict parchment, worth mentioning is a form of “red heat” that also affects hides that have undergone salting (Fig. 14). Many ancient parchments of very different origins and ages share a distinctive form of biodeterioration that takes on the appearance of purple stains associated with the destruction of collagen fibers. Gallo and Strzelczyk (422) isolated several fungi capable of producing violet pigments from such alterations, such as Aspergillus versicolor (Vuill.) Tiraboschi, Penicillium notatum Westling, and Epicoccum sp. However, an attempt to reproduce these stains in vitro was unsuccessful, and it proved impossible to generate such violet-pigmented spots by inoculating just a single microorganism (422). Using culture-independent molecular methods, Pinar et al. (93, 94) attributed the damage to halophilic and halotolerant proteolytic bacteria that could develop due to the saline environment provided by the parchments (423). In particular, members of Actinobacteria such as Saccharopolyspora spp., and Aspergillus species, were detected in all the cases examined. Aspergillus versicolor appeared strongly associated with the deteriorated parchment samples, suggesting a form of co-occurrence between this fungus and the halophilic bacteria (Fig. 14). Recent articles have confirmed the presence of Saccharopolyspora spp. on many documents using massive DNA sequencing techniques (424, 425). Perini et al. (426) formulated a microbial attack model on the parchment in which halophilic bacteria present in the sea salt used in salting colonize the skins first by penetrating between the collagen fibers. However, the formation of the nucleated purple spots, according to this model, is due to the lysis of the halobacterial cells and the release of the bacteriorhodopsin pigment following the colonization of the materials by other halotolerant organisms from the Gammaproteobacteria and Firmicutes. The fungi predominantly attack parchments already affected by violet staining and bacterial damage in general. This role of fungi as secondary colonizers has emerged from several studies. For example, Pinzari et al. (220) quantified both ATP and fungal β(1-4)-N-acetyl-D-glucosaminidase activity in damaged and undamaged samples, concluding that viable fungal mycelium was present on the parchments analyzed but was not closely associated with purple staining. Pinar et al. (93) quantified the fungal β-actin gene by quantitative real-time polymerase chain reaction analysis in the Archimedes Palimpsest, also affected by purple staining, revealing a higher fungal abundance in degraded areas than in healthy areas.

Fig 14.

Bone and ivory biodeterioration. (A) A musealized bone fragment [high-vacuum scanning electron microscopy (HV-SEM)] showing clear surface alterations and cracks in the surface biofilm (arrows); (B) whale bones from a skeleton dismantled for restoration at the Natural History Museum, London; (C) yeast cells adhering to a bone fragment from a biodeteriorated museum skeleton, characterized by partial dissolution of hydroxyapatite and formation of other secondary minerals such as calcite [variable-pressure scanning electron microscope (VP-SEM)]; (D) biogenic calcite associated with the biofilm on deteriorated bones in the indoor environment (HV-SEM); (E) a fragment of ivory used in vitro to demonstrate the ability of Aspergillus niger to dissolve hydroxyapatite through the excretion of organic acids (stereomicroscopy, Leica MZ16); (F) a patina of secondary hydroxyapatite and calcium oxalate crystals formed at the interface between the ivory and fungal mycelium (VP SEM) [images by F. Pinzari, unpublished, see also Pinzari et al. (356)].

Mineral components in paper and parchment

Fungal attack of paper and parchment materials does not always affect the organic components (the cellulose of paper, the collagen of parchment, or the glues) but can also cause substantial changes in the mineral substances typically added during the manufacture or the use of these materials. Fungi can leach the minerals, transform them, reprecipitate in other forms, translocate certain elements, or selectively sequester them (198). Paper and parchment both contain various inorganic compounds in varying quantities. Various salts may be deposited in the materials as a result of the manufacturing processes, such as filler minerals or impurities in water in the case of paper, or metals from iron- and copper-based inks, and mineral compounds related to the use of pigments, especially in miniatures (the latter may contain orpiment, cinnabar, lapis lazuli, gold, silver, white lead, etc.) or seals (427). Calcium oxalates are frequently found in biodegraded papers and parchments, as well as other artworks (292, 342). The production of oxalates is due to the metal-precipitating capacity of oxalic acid generated by the fungi. The action of these acids combines with that of fungal respiratory CO₂, which generates carbonic acid in the growth environment. It can be hypothesized that the combined action of these two groups of acids produces the dissolution patterns observed on calcareous materials and that subsequent spontaneous recrystallization of fungal oxalic acid with Ca²⁺ on chitin nucleation sites leads to the formation of new crystals near fungal structures or directly on cellulose or collagen fibers (195, 428).

Tanney et al. (231) described the case of a rare halophilic fungus (Diploöspora rosea Grove) grown on the preparatory layer, composed of gypsum and calcium carbonate, of an illuminated parchment. The fungus had grown on the gold and green pigments used to illuminate the documents. The green layer contained arsenic, chromium, and iron, while the gold layer showed the presence of Pb, presumably from the underlying white preparation of basic lead carbonate (PbCO₃) (Fig. 15). The fungus, which developed in the presence of potentially toxic As, Cr, and Pb, caused large portions of the decorative gold illustrations to be exfoliated (Fig. 15). Pinzari et al. (220) documented that the biodeteriorated areas in an ancient parchment showed a decrease in the levels of Al and Si and an increase in those of S and P. Based on SEM-EDS analyses, the authors hypothesized that fungal and microbial species exerted a leaching effect on Al- and Si-containing minerals and were simultaneously able to accumulate and concentrate other elements from salts such as sulfates and phosphates. Observation of papers and parchments attacked by fungi through variable pressure electron microscopy with a backscattered electron detector (BSD) allowed these phenomena of translocation and concentration of both metallic and non-metallic elements in the hyphae to be documented very well. The hyphae are often clearly visible against the background consisting of organic material precisely because they concentrate high atomic weight elements, which, with the BSD, take on a clear chemical contrast.

Fig 15.

Wood biodeterioration. (A) Fungal colonies that developed inside a wooden cabinet in a library; (B) fungal hyphae on a fragment of oak wood attacked by filamentous fungi. The porous structure of the wood facilitates colonization and adhesion of the hyphae (variable-pressure scanning electron microscopy); (C) colonies of fungi that develop on wood in indoor environments often comprise several species together, the result of ecological successions or forms of commensalism between the airborne organisms. In this image, obtained with a bright-field light microscope, chains of conidia belonging to at least two different fungal species can be observed; (D) chains of conidia from two co-occurring fungi grown on wood indoors (high-vacuum scanning electron microscopy) (images by F. Pinzari, unpublished).

Photographic materials

As observed for parchment, other substrates, such as photographic materials, can also support entire communities of biodeteriogenic microorganisms, which typically are composite and present diversified trophic niches for generalists and specialized decomposers. In the field of biodeterioration, the co-occurrence of several species makes disinfection and conservation treatments more problematic (Fig. 16). If they are only effective against certain elements of the microbial community, biocidal chemicals risk triggering new forms of competition and ecological succession with emerging species that are more resistant and aggressive toward the materials to be preserved (429). The biological activity of fungi, regardless of the material attacked, be it the emulsion or the substrate, adversely affects the images contained in films, or other photographic materials, accelerates the aging process, and poses a health risk to people working with the contaminated materials. Habitats in which fungi and bacteria coexist can resemble ecological battlefields where microorganisms fight to dominate and/or annihilate each other (430). It can be assumed that on poly-material artifacts, there is a division of available space between deteriorating species according to functional and metabolic capabilities (Fig. 16). Bučková et al. (85) used as a model for a study on the ecology of biodeterioration a “preservation unit” of photographic material consisting of a cellulose nitrate negative film, a silver gelatine positive print, a cardboard frame, and a Pergamon paper envelope all kept together under poor preservation conditions. Positive and negative photographic media consist of at least three components: a rigid support (usually plastic or paper and sometimes glass and other materials), an image-forming material (black and white images are formed by particles of metallic silver), and a binder, which in 20th century documents is mainly gelatine based (407) (Fig. 16). Gelatine is a mixture of high molecular mass polypeptides produced from collagenous animal tissue that was used for all silver halide-based photographic materials (431). Biological damage on film collections has been particularly studied by Abrusci et al. (432) who isolated several fungal species from films, mainly in the genera Aspergillus (e.g., A. ustus, A. nidulans, and A. versicolor) and Penicillium. Szulc et al. (433) also analyzed photographic materials finding complex communities where the dominant fungi belonged to the genera Aspergillus, Alternaria, Penicillium, Chaetomium, Talaromyces, and Fusarium. The main limiting factor determining fungal development on photographic materials is recognized to be water, although many xerophilic/halophilic fungal genera and actinobacteria have been found in these materials. Sclocchi et al. (408) demonstrated the accumulation of silver nanoparticles on the surface of the fungal cell wall during biodeterioration of the silver halide photographic layer (Fig. 16). One or more of the fungal strains that caused the damage were able to reduce silver ions and form silver nanoparticles on the cell wall, presumably capped on structural proteins (408). Bučková et al. (85) documented the co-occurrence of bacteria and fungi on photographic materials, with species associated more with one material or the other, forming part of the photographic conservation unit. Galactomyces geotrichum is among the most abundant species on both positive and negative media and also one of the main contaminants of gelatine-coated photographic media due to its proteolytic activity (434). A Geotrichum sp. (the anamorph of the genus Galactomyces) was also found to be resistant to silver and capable of capturing Ag nanoparticles (435).

Fig 16.

Iron biodeterioration. (A) Iron nails used to mount and fix various museum specimens can be attacked by fungi that can develop at low water activity and by exploiting cyclic or otherwise variable thermo-hygrometric conditions. In the image, the arrows show fungal patinas covering iron nails that were inserted into the bones of a disassembled skeleton; (B) iron oxides formed around nails embedded within exopolymeric materials produced by filamentous fungi and bacteria [high-vacuum scanning electron microscopy (HV-SEM)]; (C) rust formations formed around the nails and fungal structures encapsulating them in a ferrous oxide shell (arrow with asterisk). Conidia of a Scopulariopsis-like fungus are visible (arrow) (HV-SEM); (D) fungal mycelium is often contained within these rust shells, indicating some role in metal corrosion phenomena (variable-pressure scanning electron microscopy, backscattered electron detector): the shells are made of iron oxide and inside there is fungal biomass in the form of conidia and a biofilm composed of condensed hyphae; (E) these secondary structures are the result of combined mechanisms with formations of a purely chemical nature, such as needle-shaped crystals of iron hydroxides and oxalates (arrow) and also forms directly shaped by the fungal structures themselves (arrow with asterisk) (HV-SEM) [images by F. Pinzari, unpublished, see also Planý et al. (343)].

In the case of photographic materials, as already observed for other types of cultural property, unusual fungi whose distribution range and ecological niche do not include indoor environments can become major biodeteriogens of anthropogenic materials. On photographic materials, Bučková et al. (85) documented the presence of uncommon fungal species such as a Zygosporium sp. identified not by barcoding (it was found to be an uncultured clone of Pezizomycotina) but by its very distinctive appearance. Zygosporium is a genus characterized as hyphomycetes (dematiaceous anamorphs) possessing dark-pigmented, curved vesicular cells that give rise to two to four conidiogenous ampulliform structures (Fig. 16). The vesicles may be pedunculate or sessile, and arise laterally to a setiform conidiophore or directly from the mycelium (436). Most genera and species of Zygosporium are found on dead plant material and leaf litter. Interestingly, Manimohan and Mannethody (437) associated a species of Zygosporium (Z. gibbum), morphologically very similar to the Zygosporium sp. observed by Bučková et al. (85), with a hyperparasitic lifestyle. The species is cosmopolitan and has been reported from tropical, subtropical, and temperate regions (438). It was found that another Zygosporium species was lethal as a contaminant of a bacterial culture (439). Its lytic action was due to exocellular α−1,3- and β−1,3-glucanases that degraded the bacterial cell wall. Other authors reported that some species of this genus are capable of producing an antibiotic compound (zygosporin A) with antimicrobial properties (440). These are all traits that point to a species capable of competing and surviving in complex assemblages of both bacterial and fungal species.

Oil paintings, tempera, and canvas

Whether ancient or modern, oil paintings on canvas are complex objects, usually consisting of several materials and a series of mixed or layered organic and inorganic substances. In antique oil-on-canvas paintings, there are textile fibers for the support (hemp, but also other fibers), layers of adhesives such as gelatine, and then natural organic substances such as egg yolk, flour starch, vegetable gums, oils, and resins, generally superimposed to form a series of layers that are more or less recognizable with spectroscopic techniques (441, 442). The pictorial layers containing pigments may also be rich in mineral substances. There may also be protective layers that generally represent the surface in direct contact with the air or, in some cases, with the wooden frames or exhibition glass (443, 444). Modern paintings are characterized by a very different manufacture, often containing acrylic pigments and also a wide variety of other materials, including synthetic polymers, that can be a substantial part of the work (414, 443, 445). From a microbiological perspective, paintings are substrates that can provide all the substances necessary for colonization by fungi and bacteria (organic substrates but also nitrogen, macro-, and micronutrients). Once again, water availability is the main limiting factor for the development of biodeteriogens. Some paintings may also contain salts that make them selective environments for halophilic fungal and bacterial species, or toxic metals, typically present in certain pigments, that make paintings even more restrictive environments for biotic attack (446). The fungal taxa most frequently isolated from oil paintings are representatives of genera that usually proliferate in indoor environments (e.g., Alternaria, Aspergillus, Cladosporium, and Penicillium). However, and as with other materials, culture-independent techniques have revealed the presence of many more species than can be isolated in vitro (96, 97). Interestingly, the few available studies entirely devoted to the microbiota attacking paintings on canvas have been able to ascertain that many of the species present on paintings as biodeteriogens possess many specialized enzymes to break down, e.g., lipids and proteins, and functional traits that make them particularly well equipped to decompose one or more types of such substances (447–450). Studies on bacteria attacking paintings are more numerous than those published on fungi (447, 448, 451). Văcar et al. (452) isolated 21 fungal strains from 4 oil paintings from the 18th and 20th centuries. Of these taxa, the genus Aureobasidium showed the greatest biodeterioration potential, followed by Cladosporium, Penicillium, Trichoderma, and Aspergillus, with evident secretion of organic acids capable of dissolving pigments of a mineral nature. Kavkler et al. (414) analyzed a biodeteriorated 17th-century tempera painting in a wooden frame, covering 143 m² of a ceiling using an interdisciplinary approach and subjecting 535 painting samples to machine learning analysis methods. The authors found that fungal colonization was strongly influenced by the position of the painting in the room and related microclimatic conditions. Interestingly, Kavkler et al. (414) discovered that the presence of a coating layer and the distribution of protein binders and pigments, such as goethite, ultramarine, and kaolinite, had an essential role in the pattern of colonization by xerophilic Aspergillus species. Vieto et al. (112) studied the colonization of oil paintings from the 19th century conserved at the National Theatre of Costa Rica. In particular, a large format painting on canvas (La Danza, size 9.83 × 5.13 m, painted in 1896 by Italian artist Vespasiano Bignami) with a supporting canvas made of hemp prepared with beeswax and binders containing linseed oil and white lead was attacked by species of Pestalotiopsis, Ustilago, and Penicillium and a newly discovered species of Myxospora.

Piñar et al. (96) analyzed the microbiome of two oil paintings on canvas from the 18th and 19th centuries applying metagenomic analyses through genome amplification based on the Oxford Nanopore sequencing technology (ONT). The authors discovered that fungi, belonging to the genus Aspergillus, and bacteria of the order Burkholderiales were responsible for biodeterioration of the paintings. Okpalanozie et al. (453) analyzed a contemporary oil painting on canvas from Nigeria and demonstrated that the fungal attack arose from the canvas, with fungi from the Ascotricha genus capable of decomposing cellulose that was the main focus of the biodeterioration. Ilieș et al. (454) isolated and identified the fungi present in the dust deposited on the surface of oil paintings. The authors sampled different materials from paintings (cotton, canvas, wood) and listed species belonging to several fungal genera including Arthrographis sp., Beauveria sp., Aspergillus sp., Penicillium sp., Alternaria sp., and Cladosporium sp., therefore demonstrating how deposited dust is a primary source of fungal colonization of paintings. López-Miras et al. (448) investigated the microbial community (bacteria and fungi) colonizing a biodeteriorated oil painting on canvas by applying a strategy comprising culture-dependent and -independent techniques. Interestingly the authors found contrasting results from the two approaches. The isolated fungal strains belonged to different genera of the order Eurotiales, such as Penicillium and Eurotium, while the non-cultivable genera belonged to species of the order Pleosporales and Saccharomycetales. The authors also demonstrated a co-occurrence between fungi and bacteria and a biodeterioration mechanism in which a Penicillium species and a bacterium of the Arthrobacter genus both contributed to spoilage of the painting.

PRESERVATION OF CULTURAL HERITAGE FROM FUNGAL BIODETERIORATION

To combat microbial attack on cultural and historical heritage, there are some common principles and approaches. Among these approaches, there are widely used traditional restoration methods preferred by most restorers (455), such as, e.g., use of paint brushes to apply required chemical substances to the damaged surfaces. There are also various modern physical, chemical, and biological approaches, some already successfully tested in heritage restoration. However, along with success stories, there are examples of poor outcomes of modern treatments (22, 327, 456). The most discussed methodical approaches and problems in heritage preservation, summarized in Fig. 17, are described in several other works on this topic (22, 34, 36, 42, 66, 153, 266, 313, 327, 455–469).

Fig 17.

Essential steps and methodical approaches for preservation of cultural heritage from fungal biodeterioration.

Before choosing the type of treatment for preservation, initial considerations are the chemical and physical properties of the material, the nature of the microbial agents of biodeterioration, and the mechanisms involved, as well as the variety of factors that may affect this process. It is also essential to critically analyze previous experience and outcomes with similar materials in order to learn from successes, mistakes, or problems. The common action strategies in heritage preservation of cleaning, disinfection, restoration, and protection are defined by the nature of the heritage material. All the steps in heritage preservation should be complemented by preventative “housekeeping” routines, including frequent simple dusting and surface cleaning procedures, adequate ventilation and regular microenvironmental control where applicable, and constant monitoring combined with instrumental analysis of the effects and long-term consequences of conservation treatments (22).

Common problems associated with the use of mechanical and physical methods for cleaning and disinfection are generally associated with structural and aesthetic damage of the material, e.g., accelerated aging effects on cellulosic materials in ɣ-radiation disinfection (22, 455). It should also be mentioned that disinfection of heritage objects by using irradiation can also result in enhanced color changes and aesthetic damage because of successions of radio-resistant fungi which possess pigments and other biomolecules that protect cells from irradiation (e.g., solar-, UV-, and ɣ-radiation), including melanins and mycosporines (470). For example, Cladosporium spp. accumulate melanin in the cell wall up to 30% of the total biomass, contributing to the remarkable tolerance and radiotropic responses of these fungi to severe abiotic stresses including ɣ-radiation (471).

Because of abundant surface contamination, the cleaning process is critically important, especially for preservation of extensive areas like murals (327, 472). An example of a simple mechanical approach was cleaning of visible fungal mycelium on wall paintings at the Maijishan Grottoes using a soft “make-up” brush combined with a vacuum pump cleaner (42). Another common problem in cleaning and disinfection is that the use of organic solvents and toxic chemicals can (i) potentially pose a health hazard for the restorers, (ii) have a negative effect on the materials, (iii) put selection pressure on the fungal communities leading to succession of organisms more resistant to treatment and which may be more aggressive agents of biodeterioration, and (iv) interfere with analyses of biomolecules (e.g., fumigation with ethylene oxide leads to intercalation with DNA/RNA) jeopardizing adequate assessment of the course of biodeterioration (22, 455). Other examples of highly toxic chemicals used in disinfection include organochlorine compounds (e.g., lindane or pentachlorophenol) used for preservation of wood and textiles, and organotins (tributyltin oxide) used for treatment of a mono-specific infection of a medieval wooden ceiling by Aspergillus glaucus, although organotin use is now largely prohibited worldwide due to its environmental toxicity (22). The Lascaux Caves, France, and the Cave of St. Paul in Ephesus, Turkey, are famous examples of the disastrous consequences of biocidal treatment of unique ancient wall paintings which led to severe aesthetic damage due to selection of more resistant and aggressive biodeteriorative fungal strains (22, 42, 473, 474). It might be concluded that fungal biodeterioration caused by a mono-specific infection or restricted fungal community would be easier to treat than that caused by highly diverse communities. However, treatment of a mono-specific biodeterioration process can result in attack by another species more adapted to the changing conditions. This occurred in restoration of the wall painting Marriage at Cana by Luca Longhi, Ravenna, Italy, where successful inhibition of the obligate xerophile Aspergillus halophilicus was achieved by applying only deionized water. However, the water alone facilitated attack by more hydrophilic fungi, including melanized Cladosporium sp. (466). Clearly, such treatments require thorough monitoring and documentation of the long-term consequences.

If affected by microbial colonization, book materials such as paper and parchment are among the most difficult to treat as they are more fragile and absorbent. Drying, wet and dry surface cleaning, disinfection, and consolidation or reinforcement are procedures and techniques developed and designed in various forms by conservators worldwide (63). These treatments have evolved together with scientific developments and the invention of new technologies. The use of hydrogels, biosensors, nanoparticles, photocatalysts, ionic liquids, plasma techniques, or laser cleaning is an example of recent applications to the conservation of library heritage. Unfortunately, the conservation of cultural heritage is an economically circumscribed market and therefore limited by little or no investment in research. Most substances and technologies used during conservation treatments are technological transfers of those used in other fields. It should also be added that, more recently, there has been a general trend toward minimally invasive treatments that are less toxic to restorers and less invasive to library materials (63, 475). In particular, in addition to the potential to cause chemical or physical degradation of materials, many conservation treatments also present the risk of erasing the objects’ inherent biological information content, especially regarding bioarchaeology and biocodicology. Indeed, because of the ability to study the environmental DNA present in books, and therefore apply this to metagenomic and forensic techniques, it is now possible to identify not only the microorganisms present in documents but also the plant species of the pollen accumulated through dust deposition over the centuries, the animal or plant species used in the manufacture of the writing media, and also other traces associated with the handling of documents (91, 424). These advances open and will increasingly open new possibilities in historical studies. Many treatments, however, can deteriorate DNA, thus preventing this type of analysis (63, 106).

One of the most promising approaches in heritage preservation is the use of gels as a reliable delivery system for cleaning biodeteriorated objects (e.g., stone, wall and easel paintings, paper, and textiles) contaminated with aged animal glue and other organic residues from past restoration, or showing stains, efflorescence (e.g., nitrate or sulfate salts), and metal corrosion products (22, 42, 153, 327, 455, 476). The gel cleaning process can be purely chemical (with hydrogels and organogels) and biological (gels containing microbial exoenzymes, metabolites, or viable cells) (Fig. 17). Some gels are biologically derived, e.g., xanthan or gellan gum, agarose, and chitosan (455). One of the problems associated with the application of gels, e.g., agar-agar, is that the surface of the biodeteriorated material should be smooth for adequate contact. To improve gel handling and manipulation, systems using agar-gauze or agar-Japanese paper have been tested (476, 477). For cleaning of medieval murals in St. Michael’s Chapel in Barcelona, Spain, the use of destructured agar on Japanese paper significantly improved contact with the not ideally smooth surface, and incorporation of tri-ammonium citrate or ethylenediaminetetraacetic acid in the agar gel successfully removed the animal glue and fungal stains on the biodeteriorated paintings (477).

Biogels are commonly bacteria based, where Pseudomonas stutzeri is the most used bacterium for removal of nitrogen salts and organic matter, and Desulfovibrio vulgaris is used for the removal of sulfates (153). However, some microbial enzymes may not be completely removed from the biogel-treated surface resulting in aesthetic damage over time, and there is a risk of further microbial attack because of the carbon source available in gels (42, 327). In the successful cleaning of murals using viable cells of P. stutzeri immobilized within an agar-gauze biogel, silver nanoparticles were subsequently applied to sterilize the treated murals at the final stage of restoration (476). The application of nanoparticles and nanomaterials (e.g., gold nanoparticles for enzyme immobilization, TiO₂ nanomaterials, and nanocomposite PAAG@Ca(OH)₂) in the protection of cultural and historical heritage is now attracting increasing attention (153, 327, 478). However, nanoparticles are unsuitable for some materials and often unsuccessful in the long term. Another approach currently attracting attention for preservation of cultural heritage, further discussed in the following section, is related to biorestoration or bioprotection through application of microbial biomineralization processes to create a protective coating using bacteria and fungi (153, 327).

FUNGAL BIOPROTECTION OF CULTURAL HERITAGE

Bioprotection is identified as an Earth surface process and generally refers to a protective effect on the substratum mediated by growth of living organisms and/or their remains and products (479). It has been particularly considered in the context of bioprotection of rocks, and the concept has been extended into the built environment and cultural heritage objects (36, 480, 481). In this context, bioprotection can include consolidation, cleaning, and/or protection of stone affected by abiotic weathering and biodeterioration and can include cleaning and cementation by biomineralization processes (480, 482). Lichens are the organisms that have received the most attention in this area, although the bulk of published information concentrates on their biodeterioration activities (208, 479). Nevertheless, lichen bioprotection has frequently been observed where non-lichenized stone surfaces show increased abiotic weathering compared to colonized areas (483, 484). The biodeteriorative actions of the lichens on the stone were still occurring but were at a slower rate than the results of abiotic factors (479). Furthermore, lichen coverage may reduce effects of rain, water evaporation, and water entry into rock material, protect a surface from wind, abrasion, and temperature fluctuations, while biomineralized thalli, dead material, and crusts may further stabilize and protect rock surfaces (178, 201, 291, 335, 479, 485–487). However, this may not be true in all cases where the substrate and climatic conditions may greatly influence the outcome. For ancient temple sandstone in a tropical climate, the beneficial effect of lichen coverage in reducing deleterious effects of water evaporation did not outweigh the deteriorative effects of deep rock penetration by their hyphae and rhizines (29). It may be assumed that epilithic lichens may be more obvious bioprotective agents than biodeteriorating endolithic species (488), although biomineralizing activities of the latter may also “cement” surface layers (201, 260, 489). In addition, endolithic colonization by the lichens Verrucaria nigrescens and Caloplaca aurantia filled a porous rock substrate with a dense network of lichenized fungal hyphae which imbued waterproofing properties (489). Similar endolithic organic layers arising from ancient lichen growth have also been found on historical monuments and believed to contribute to preservation (489). Bioprotection by lichen coverage against rain and atmospheric deposition is referred to as an “umbrella effect” (479). Surface biomineralization by lichens or other microbial communities can also lead to the formation of robust varnish-like coatings (18, 490). Accelerated deterioration of surfaces may occur if outer layers of historic buildings and monuments are removed by physical and chemical cleaning methods (56, 128, 260, 491).

The relative significance of biodeteriorative or bioprotective mechanisms is a recurring topic regarding stone-based cultural heritage (29, 35, 36, 64, 201, 483). As described above, a multiplicity of mechanisms may be involved in microbial stone colonization and all influenced by the nature of the substrate and environmental conditions (35, 64), emphasizing the importance of site specificity and the difficulty of making generalized hypotheses. It has been proposed that bioprotection can be assessed and differentiated from biodeterioration and abiotic weathering by use of a “relative bioprotection ratio” (64). This was defined as a ratio between the sum of natural weathering and the sum of biodeterioration so that a relative bioprotection ratio >1 indicates that microbial colonization is bioprotective, whereas low values <1 indicate biodeterioration is more significant (64). Such a definition relies on the ability to disentangle natural weathering from microbe-mediated activities and produce quantitative data which are clearly a complex undertaking and possibly unattainable in many contexts. Characterization of the microbial communities involved can inform on the possible metabolic activities of the microbiota and their deteriorative or bioprotective relevance, although this is markedly influenced by environmental factors (64), and similar functions can have multiple effects. For example, organic acid excretion by fungi may cause mineral dissolution but also biomineralization, e.g., oxalate or secondary calcite formation, which may form protective crusts but may also disrupt mineral structure through crystal expansion as described earlier. Some coatings that incorporate oxalate are very stable and may provide protection from atmospheric weathering (180, 485, 492).

Fungal-mediated bioprotection through oxalate biomineralization has also been recorded for certain metal artifacts. Copper(II) oxalate [Cu(C₂O₄)·nH₂O (n <1)] has been found in patinas on copper metal (492). In fact, a fungal-derived copper oxalate coating was successfully developed for bioprotection of copper artifacts (493, 494). Corroded copper surfaces were covered with a nutrient gel containing Beauveria bassiana with the resulting copper oxalate patina being uniformly distributed and completed over a depth of several microns (495). Production of mycogenic biomineral coatings by B. bassiana has also been investigated for preservation of archaeological iron artifacts (348).

Numerous approaches have been used to prevent, control, or remediate microbial colonization and deterioration of stone heritage, with fungi offering considerable challenge to such attempts. Treatments may attempt to prevent colonization, remove biomass, and prevent re-colonization, and many natural and synthetic compounds, biocides, chemical substances, nanomaterials, gels, and coatings have been investigated as well as direct physical methods such as mechanical abrasion, ionizing, and laser irradiation (24, 25, 457, 462, 478, 480, 496–515). Such treatments are seldom completely effective and may result in mechanical damage and environmental contamination with potentially toxic chemicals (266, 482, 516–518). An extensive review of natural biocides showed that their efficacy was highly variable, with some showing adverse effects on the substrate, while the variability in experimental protocols inhibited convincing assessment (511). The abilities of fungi to survive biocidal treatments, degrade synthetic coatings, and re-colonize after restorative treatments are well known, with black-pigmented microcolonial fungi being particularly resistant to curative approaches (266, 519). One study showed that recolonization of marble, sandstone, and plaster after conservation treatment mainly depended on their bioreceptivity and climatic conditions. Although the treatments with water repellents and consolidants with biocides and copper nanoparticles reduced recolonization, they did not play a crucial role in preventing subsequent growth of biofilms and lichens (519).

Biocleaning has been explored for heritage surfaces where microbial systems may mediate effective removal of organic matter or pollutants (506, 518, 520). Biocleaning agents, including enzymes, have been applied for removal of organic and inorganic substances on paintings, paper, ceramics, and concrete as well as stone, with often favorable comparison to physical and chemical treatments (480, 495). Most attention has been on stone and bacterial systems for removal of, e.g., nitrates and sulfates (521–525), although one method involved “dry biocleaning” with viable dry yeast (518). Here, stonework was inoculated with dehydrated Saccharomyces cerevisiae: rehydration and subsequent metabolic activity resulted in better removal of salts and pollutants than cell-free controls (518). Alkaliphilic fungi have been investigated for biocleaning of corroded iron because they can remove the outer powdery chlorinated corrosion layers without damaging the underlying metal surface (348, 495).

There is growing information which suggests that fungal systems are of considerable potential when considering bioprotection approaches that employ natural or engineered colonization and their metabolic properties (481). A bioprotective effect may be achieved by physical protection and biomineralization. As mentioned previously, lichens, fungal biofilms, and coatings may physically inhibit weathering of surfaces through shielding and binding of the rock surface (479) (Fig. 1). Biomineralization can result from oxidation or reduction of a metal species, or the formation of secondary mineral precipitates as a result of fungal metabolism and metabolite excretion, such as carbonates, phosphates, oxides, and oxalates, which can contribute to the development of rock coatings (3, 7, 19, 193, 198, 202). One biologically initiated rock crust on sandstone formed by various fungi, and including lichens and green algae, was markedly more erosion-resistant and water impermeable than the underlying sandstone (526). Stable calcium oxalates (whewellite and weddellite) occur widely in patinas on stone buildings, monuments, plasters, cave and wall paintings, and sculptures, and these also contain calcium carbonate as well as organic residues that can contribute to longevity (202, 485). Several fungal species can oxidize Mn(II) to form black Mn(IV) oxides, which are a significant component of rock varnish (7). Precipitation of secondary calcite after fungal dissolution of limestone can result in cementation of the original limestone substrate (527). In fact, microbially induced calcium carbonate precipitation (MICP) has been widely explored in bioprotection with most research being carried out with bacteria (482, 516, 528–530) and rather less on fungal systems (531–533). There are several mechanisms by which carbonate cementation can be achieved, such as application of a suitable culture medium to a decayed porous substrate, e.g., limestone, that encourages development of carbonate-producing microorganisms, thereby ensuing cementation (35, 482, 516, 528, 529, 534). Such a process can also be termed biodeposition (528, 535). MICP has been widely investigated in the construction industry, especially regarding self-healing concrete for crack repair (531–533, 536–540). A frequent MICP approach involves microbial urea hydrolysis which leads to increased alkalinity and precipitation of calcium or other metal carbonates (541). Urease-based MICP has been applied to enhance the durability of structures by reducing water permeation and corrosion (537, 542), for crack cementation (543, 544), and the restoration of historic monuments (545). Several fungal species show high urease activity as well as alkalinity tolerance and have been shown to be effective in mediating calcite precipitation, as well as other metal carbonates, providing a system for bioprotection of porous cementitious materials as well as metal immobilization (546). The urease-positive fungus, Neurospora crassa, produced a biomineralized protective coating on porous building materials such as mortar and cement. The dense hydrophobic mycelial network provided a physical barrier to water infiltration, while the precipitated calcium carbonate acted as a biocement to clog pores and cracks, thus demonstrating that a fungal biomineralization process could be an effective biotreatment for porous mineral-based building materials (547). Possible application of lichen colonization for coverage and bioprotection offers an environmentally friendly approach in the built environment, although there are many practical difficulties arising from the unpredictable nature of lichen growth and rate of colonization, including methods of inoculation and nutrient supply, as well as environmental and species- and substrate-specific influence on the consequences of lichen action (481).

A distinct bioprotection focus is the use of entomopathogenic fungi to control insect and other arthropod infestations in museums, libraries, and archives. The protective potential of parasitic fungi against post-harvest insects and mites is well known (548). There are marketed products based on entomopathogenic fungi that are effective against the larvae or adults of various species that attack stored grains and dry food. The desired effect is often to reduce their populations by natural mechanisms, reducing infestation without using toxic substances. Entomopathogenic fungal products are tested to be harmless to humans. However, their applications in indoor environments and in the presence of organic substances such as those composing paintings, books, and perhaps even natural history collections give rise to some other problems that require a better knowledge of the functional traits of these organisms. In fact, since most of these fungi can live both as parasites and saprophytes, it must be verified that they cannot attack the materials one wishes to protect. Pinzari et al. (549) tested, e.g., a strain of Metarhizium anisopliae (Deuteromycotina: Hyphomycetes), already verified as a biocontrol agent of various insect species and marketed in different formulations, against species of the family Anobiidae both to assess its infectivity against the paper pathogens and its ability to degrade paper as a saprophyte. According to Pinzari et al. (549), the metabolic profile of the entomopathogenic fungus, analyzed with phenotypic microarrays and enzyme activity tests, is compatible with its use in libraries and archives. Such results could open new fungal bioprotection applications in the cultural heritage sector.

CONCLUSIONS

Fungi are ubiquitous and significant organisms in the biodeterioration of outdoor and indoor cultural heritage. They can be the most visible causative organism on a plethora of natural and manufactured organic and inorganic materials and also a significant component of mixed biodeteriorative microbial communities. It is likely they are present in all biodeteriorative contexts, although many “microbial” studies concentrate solely on the prokaryotic communities, sometimes understandable for convenience but also an indication of the prokaryotic-eukaryotic divide that occurs in many ecological and geomicrobial studies (550). The lichen symbiosis is a unique fungal entity of profound significance, particularly in outdoor colonization of stone- and mineral-based structures and artifacts. While fungi do not possess the metabolic diversity of prokaryotes, their chemoorganotrophic metabolism and growth mode underpin their success and adaptability as biodeteriogens. Free-living filamentous fungi, yeasts, microcolonial forms, and lichens can all be important. Hyphal growth, morphogenesis, and substrate colonization can respond to differing environmental conditions, including development of resistant forms, often pigmented, that withstand periods of desiccation or exposure to other environmental stresses. Many disperse widely through aerial spores, many have the capacity for oligotrophic growth, and all have various mechanisms for extraction of nutrients from insoluble organic and inorganic substrates. Even common ubiquitous fungi, such as inhabitants of soil or decomposing organic matter, that are frequent airborne contaminants of cultural heritage, possess advantageous survival traits. It seems that human-made structures provide an endless source of new environmental niches for fungal colonization.

Many fungi that grow at the expense of works of art or cultural heritage are extremophilic species, i.e., organisms adapted to extreme environments. Extremophilicity often concerns water availability and is evolutionarily defined in fungi through various mechanisms of resistance or tolerance. The production of osmolytes, the precipitation of brines or minerals capable of conserving water, and cellular structures that conserve or capture moisture are all documented examples found in several species of fungi that proliferate on cultural heritage. The presence of potentially toxic substances like xenobiotics, metals, preservatives, and biocides may also contribute to selection of extremophilic species in the “cultural heritage environment.” However, the reasons why cultural heritage may select extreme species of fungi are unclear but may be speculated upon in ecological terms. Manufactured materials and environments are selective and may represent completely new niches that are able to be colonized by certain species from other environments already able to take advantage of these conditions or to adapt to them. It can be hypothesized that the species that proliferate on cultural heritage or in museums, religious buildings, and other kinds of built environment, including private homes, are being selected because of their anthropogenic ranges and traits. For fungi growing on outdoor stone- or mineral-based substrates, analogies clearly exist for fungal communities growing on naturally occurring rocks and minerals. Again, although a wide diversity of fungi can be encountered on such materials, including ubiquitous airborne taxa, all possess significant survival mechanisms, with some groups, such as the microcolonial rock-inhabiting fungi and lichens, being characteristically associated with such an environment. The construction of historic and religious buildings, monuments, statues, and other items clearly provides many surfaces of different topographies and microenvironmetal conditions that might favor extensive colonization.

Island biogeography is a field of biogeography that examines the factors that influence the diversification of isolated natural communities and could be applicable in the context of fungal biodeterioration of cultural heritage. This theory was originally developed to explain the species-area relationship pattern in oceanic islands, but now the concept of insularity is used in reference to any isolated ecosystem in the sense of being “surrounded by different ecosystems” and has been extended to different environmental contexts, including natural habitats isolated from human land development. The first scientists to deal with it were the ecologists Robert H. MacArthur and E. O. Wilson in the 1960s (551, 552), who coined the term insular biogeography in their inaugural contribution to Princeton’s Monograph in Population Biology series, which sought to predict the number of species that would exist on a newly formed volcanic island. It seems that certain works of art, monuments, and interiors used as museums or archives and libraries could be considered as islands, i.e., ecosystems or isolated niches that are available for fungi to colonize, adapt, and evolve. As “islands,” these heritage ecosystems are of course not completely isolated from the surrounding environment, exchanging dust and aerosols, as well as inevitable contamination with organic matter and various microorganisms introduced through aerial routes and human activity. However, the most important distinguishing features for such “islands” are the chemical and physical nature of the materials that are colonized and biodeteriorated in combination with the microclimatic conditions that can be markedly different from the surrounding environment. Ironically, such anthropogenically created environments, designed to be ideal for heritage preservation, do not provide full protection because there are groups of extremophilic fungi with their requirements exactly matching the created microclimatic conditions. This microenvironment becomes less suitable for common ubiquitous fungi, the more amenable it becomes for extremophiles (e.g., A. halophilicus), which readily adapt to various materials and have now been found in museums throughout the world (103, 159, 161, 466). It should also be noted that fungi generally have a more damaging effect on cultural heritage than bacteria (153). Such challenges will be magnified by the current and future effects of climate change on colonization and subsequent effects of microbiota on the built environment and cultural heritage (18, 239). This means that fungal biodeterioration of cultural and historic heritage is an ever-present and increasing problem and a significant challenge for humankind to combat, preserve, restore, and save the heritage for generations to come.

Biographies

Geoffrey Michael Gadd has >45 years research experience of metal-mineral-microbe interactions and their applications in metal biorecovery, bioremediation, and mineral transformations. He has made significant contributions relating to fungal metal-mineral transformations, and the establishment of “geomycology” as a recognised research area. He holds the Boyd Baxter Chair of Biology and heads the Geomicrobiology Group in the School of Life Sciences, University of Dundee. He has published ~350 refereed papers, co-authored two books, and contributed to 6 patents as well as many co-edited books, invited chapters and reviews, and has delivered >125 invited lectures in >20 countries. He has received several national and international research awards and is a member of the European Academy of Microbiology, and elected Fellow of the Royal Society for Biology, the Linnean Society, American Academy of Microbiology, International Union of Pure and Applied Chemistry, the Learned Society of Wales and the Royal Society of Edinburgh.

Marina Fomina is a leading researcher at Zabolotny Institute of Microbiology and Virology of National Academy of Sciences of Ukraine (Kyiv, Ukraine). The focus of her research has been on microbial ecology, biotechnology, and geomicrobiology. Her PhD degree and later DSc degree were both related to fungal biology and geomycology. She has experience of many years of collaboration, including various research fellowships and projects, with the Geomicrobiology Group at the University of Dundee, Dundee, Scotland, UK, headed by Geoffrey Michael Gadd. Since 2016, she has shown special scientific interest in the field of fungal biodeterioration of cultural and historical heritage such as the medieval frescoes of the 11th century Saint Sophia Cathedral in Kyiv and has been working as a consultant for the National Reserve “Sophia of Kyiv” (Kyiv, Ukraine).

Flavia Pinzari is a senior researcher at the National Council for Research (CNR, Rome, Italy) and a scientific associate at the Natural History Museum of London. Her publications cover geomicrobiology, fungal physiology and community ecology in both natural environments and manufactured materials (cultural heritage). She obtained her Ph.D. with a thesis in mycology at the University "Sapienza" of Rome. She has been a Researcher at the Governmental Council for Agricultural Research (CREA) Rome, head of the Laboratory of Biology at the Italian Ministry of Cultural Heritage, and Deputy Director of the Italian University School of Restoration of Cultural Heritage. She is an expert in paper and parchment biodeterioration and has been in charge of analysing unique art objects, like Leonardo da Vinci's self-portrait and Archimedes' Palimpsest.