Innovative Methodologies for the Conservation of Cultural Heritage against Biodeterioration: A Review

Abstract

The use of traditional biocidal products in cultural heritage has suffered a slowdown due to the risks related to human health and the environment. Thus, many studies have been carried out with the aim of testing innovative and environmentally friendly alternatives. In this framework, this review attempts to provide an overview of some novel potential products with biocidal action, tested to counteract the process of degradation of paper and stone materials due to microbial activity, keeping in mind the sustainability criteria. In particular, we have focused our attention on the testing of nanotechnologies, essential oils, DES (deep eutectic solvents) with low toxicity, and colloidal substances for conservation purposes.

1. Introduction

The cultural heritage is largely affected by biodeterioration, a phenomenon manifested commonly by microbial growth on surfaces causing aesthetic, physical, and chemical damage to a cultural object [1]. The prevention and the control of the diffusion of biodeteriogens represent a global necessity in the conservation of the cultural heritage [2]. The intensity of the biodeterioration processes depends on several factors, such as the environmental conditions to which the materials are exposed and the biological species.

Biodeteriogens consist of microorganisms [3,4], such as bacteria and fungi, capable of using a substrate to support their growth and reproduction [5]; for this reason, environmental factors such as the presence of water, temperature, natural or artificial light radiation, and the characteristics of the material, such as porosity, represent crucial aspects to be considered. These biofilms can cause both mechanical and chemical transformations, respectively, due to the microdecohesions and metabolic processes of microorganisms, as well as their secretions, combined with air pollutants, which accumulate in biofilms and corrode the substrate [6]. These effects can occur both on the surfaces and on the inner areas of the material, producing its long-term decay and loss of strength and durability over time [7]. Every material can be affected more or less intensely; among these, stone and cellulosic are particularly sensitive. In museum, archive, or library environments, paper can be colonized by numerous microorganisms that can cause serious damage to valuable documents. Therefore, the preservation of historical paper-based artifacts against deterioration due to the presence of bacteria and fungi is important in order to protect the cultural heritage [8]. Similarly, the stones used in the monuments or in decorative elements represent an ideal environment for the colonization of a wide variety of microorganisms, and some physical properties, such as porosity and surface roughness, make the stone susceptible to biological colonization, which can lead to aesthetic and/or physical and chemical damage [9].

Therefore, restoration interventions are necessary in order to hinder the development of biodeteriogens. Mainly, the techniques include both physical and chemical methods [6], and the latter ones will be described and discussed in this review. Even though traditional procedures with biocidal products act on microorganisms efficiently, they are harmful to the environment and the operator and therefore do not represent a sustainable alternative [10]. In fact, most biocidal products containing compounds such as acetone, ethanol, toluene, ethyl acetate, quaternary ammonium salts, etc., are toxic substances or pollutants that do not degrade easily and can remain in the environment for a long time [11], hence the need to develop innovative, sustainable, and safe products [12,13].

When it comes to biocides, reference is made to many chemicals that can kill unwanted organisms [2]; these products inhibit the action of microorganisms by the presence of certain bioactive substances. The term biocidal product does not always have a negative connotation; its dangerousness depends on the circumstances of exposure and the levels of toxicity. However, the extensive use and repeated applications of biocides could lead to the evolution of other microorganisms and can create resistant and harmful species on cultural heritage objects [14]. In this regard, an interesting challenge that also represents a gap in this topic is the long-term monitoring of the evolution of the biofilms and the microorganisms at substrate/air interface after the application of biocidal products. In fact, sometimes, the interactions between organisms, the environment, and products can provide unpredictable effects such as the formation of new secondary biofilms, which is a fact worth investigating. In addition, it is crucial to not underestimate and to investigate biodeterioration due to the indirect action of compounds originating from the previous activity of the microorganisms.

Some biocides may produce undesirable effects that cause physical damage, such as color change, structure and permeability damage, or even chemical damage such as mineral solubilization and pH change [15,16]. Studies for the development of natural products aim to create alternatives that will, over time, replace conventional biocides, which are respectful of the environment and the material while, at the same time, being economical and effective [2,14].

Thus, this review highlights the complexity of the biodeterioration issue, account for the biodiversity, the different bioreceptivities, and adaptability of microorganisms to environmental changes. Moreover, it is worth noting the capacity of the microorganisms to develop resistance versus traditional biocidal materials; this requires the definition of ever more efficient strategies able to mitigate the effects of biodeterioration while respecting the environment, the health of the operators, and, obviously, the integrity of the artwork. For this reason, the review means to identify the status of progress in conservation practices applied to cellulosic and stone materials, comparing traditional techniques with the most recent products, clarifying the different performances, and identifying these new solutions as real, sustainable, and efficient alternatives.

Microbial agents on paper can cause damage due to chromatic changes but also due to structural alterations to both basic and additive components due to hydrolysis and oxidation phenomena, while, among the forms of degradation that affect stone materials, there are blemishes, patinas, pitting, crusts, biological patina, etc.

In what follows, alternative and ecological approaches will be presented, such as the use of nanotechnologies, essential oils, deep eutectic solvents (DES), and colloidal substances.

2. Cellulosic Materials: Paper

Paper consists mainly of cellulose, a naturally occurring polysaccharide. Cellulose is represented by its empirical formula, (C₆H₁₀O₅)_n, where “n” indicates the degree of polymerization, indicating the number of basic units (monomers) that make up its structure [17,18].

The degree of polymerization (DP) constitutes a primary parameter that reflects the number of glucose monomeric units joined together in the structure of cellulose. To determine the degree of polymerization of paper samples, viscosity measurements are employed. These measurements involve dissolving the samples in a 50% CED (cupryleethylenediamine) solution, following the international standard ASTM 04 243 from 1999 [19]. The calculation of the degree of polymerization is based on the empirical equation DP = K[η]^α, where η represents intrinsic viscosity, K is equal to 1.5, and α is equal to 1 [20]. This value can vary depending on the plant origin of cellulose (

Table 1. Degree of polymerization of the different materials from which cellulose is obtained [21].

Origin of the Paper	Degree of Polymerization (DP)
Fir	600
Pine	650
Straw	1600
Raw Cotton	2100
Jute	2200
Hemp	2250
Linen	2400

) and has a significant impact on the overall strength of the cellulose molecule. Non-uniformity in paper quality is observed due to discrepancies in DP values. For instance, spruce wood exhibits cellulose with an approximate DP of 600, which corresponds to a less resistant fiber compared to hemp or flax, which have a DP exceeding 2000. The DP value is frequently employed to define the characteristics of a paper sample and evaluate the preservation degree of ancient documents. In such contexts, the average DP is considered, representing the measure of the mean degree of polymerization of all cellulose molecules present in the analyzed sample. In general, high-quality paper tends to have an average DP around 1000, while extremely fragile or deteriorated paper may exhibit an average DP lower than 100. A significant decline in the average DP value denotes a series of ruptures in cellulose chains, indicating severe deterioration of the paper material [21].

Cellulose comprises numerous glucose molecules (Figure 1) that bond together to create macromolecules organized in a linear chain. These macromolecules then cluster via hydrogen bonds, giving rise to individual microfibrils. The assembly of multiple microfibrils leads to the formation of fibrils, and, in the end, these fibrils unite to constitute fibers [22].

Cellulose is predominantly sourced from various tree species, including poplar, eucalyptus, pine, red fir, beech, and others. For centuries, other materials, such as cotton rags, hemp, and flax, have also been traditional sources for cellulose pulp production. However, wood serves as the primary raw material for cellulose production, requiring treatment in order to separate cellulose fibers from other components, such as lignin, which is an intricate three-dimensional polymer composed of aromatic alcohols connected by various carbon–carbon bonds—the molecule is notably resilient, with only a select few fungi and bacteria possessing the capability to break it down—and hemicelluloses, encompassing pectic substances and diverse heteropolysaccharides, which are polysaccharides with limited solubility that are closely linked to cellulose. Exoenzymes known as hemicellulases, produced by most bacteria and fungi, facilitate their hydrolysis [24].

Aside from cellulose, lignin, and hemicellulose, paper includes additional elements tailored to bestow specific properties essential for applications. These supplementary components encompass binders, introduced to restrict ink absorption by the paper; mineral fillers, usually white, functioning as pore fillers to yield a smoother, whiter surface, while also acting as opacifiers; and dyes, incorporated to ensure even distribution of color throughout the paper’s thickness [25].

2.1. Paper Biodeterioration

Museums constitute the main institutions providing knowledge and cultural, political, economic, scientific, and historical information within communities. In the context of a museum, library, or archival environment, the presence of pests can lead to significant damage to materials of invaluable and irreplaceable value. Paper, like other materials, undergoes a degradation process over time [26]. However, the rate of deterioration can be accelerated by various factors, both internal, such as acidity, the presence of metal ions, lignin, or degradation products, and external, such as heat, humidity, ultraviolet light, oxygen, pollutants, or biodeteriogens. Biodeteriogens are organisms characterized by their ability to use a substrate as a source of sustenance for their growth and reproduction [5], and biodeterioration is defined as any undesired form of alteration of a material caused by the vital activities of such organisms. This phenomenon occurs as the microclimatic conditions become favorable for the proliferation of microorganisms that act chemically, physically, or mechanically [27]. Such attacks pose a formidable obstacle to the preservation of Cultural Heritage. To prevent the progressive destruction of our Cultural Heritage, restorers, conservators, and researchers have developed various methodologies to combat chemical and biological enemies. In the case of a biological attack, it is possible to act in two ways: by avoiding contact between aggressive bioagents and the objects to be preserved, or by eliminating such bioagents [28].

2.1.1. Factors Leading to the Biodeterioration of Paper

Paper artifacts primarily consist of an organic matrix that provides a favorable environment for the proliferation of biological agents responsible for their degradation. In fact, the degradation of paper directly affects the structure of its fibers, weakening the cellulose chains. In particular, microorganisms such as fungi, which are the main biodeteriogens of paper, use cellulose as a nutrient source, transforming it into simpler and easily assimilable molecules [29]. Among the degradation agents, fungi represent the most common and widespread group, as many species demonstrate a high capacity for adaptation to different environmental conditions they encounter [25].

Over two thousand fungal species are known to cause damage to both wood and cellulose, primarily through the physical penetration of their hyphae and the production of primary and secondary metabolites [30,31,32]. These metabolites include fungal pigments and various extracellular hydrolytic enzymes such as cellulases, amylases, and proteases; these enzymes—in particular, the cellulases—make the depolymerization of the cellulose possible, and the microorganism will be able to use the glucose monomer as nutrient. [31]. The main microorganisms responsible for biodeterioration encompass fungi and bacteria. Common species encountered in this context include the following: Alternaria, Bacillus, Chaetomium globosum, Trichoderma viride, Penicillium chrysogenum, Cladosporium herbarum, Aspergillus niger, Stachybotrys atra, Trichoderma koningii, Chaetomium elatum Pseudomonas, Staphylococcus, Micromonospora, and Virgibacillus [32,33].

Once fungal colonization takes place, the degraded cellulose material provides an enriched substrate for bacterial growth [34]. It has been reported that various bacterial species colonize and damage paper, but bacteria of the genus Bacillus are considered to be among the main causes of paper deterioration [35]. However, it is important to note that these bacteria may be present on the surfaces of paper documents as contaminants from dust and may not play a significant role as causal agents of degradation [36].

These agents are primarily heterotrophic organisms that feed on organic substances containing carbon and nitrogen, and they mostly require oxygen for their aerobic metabolism. Therefore, such biological agents initiate the process of the biodeterioration of paper materials, which involves a combination of physical and chemical processes triggered by the growth of organisms that, mainly transported through the air, settle on the surfaces of art objects, causing their alteration [25].

In addition to microorganisms, insects also contribute to the process of the biodeterioration of paper. Regarding insects that pose a threat to paper conservation, it is important to mention some common families of beetles, such as silverfish and wood-boring beetles, as well as those that develop and live within paper materials, such as termites.

Annually, a significant number of volumes are subject to attacks by insects, microorganisms, and, to a lesser extent, rodents. These attacks exhibit variations in terms of characteristics and magnitudes across different geographical areas, in relation to the specific climatic conditions of each area, the present microflora, and the architectural peculiarities of the buildings housing library collections, which do not always adhere to the standards considered essential by the scientific community for conservation. Biological alterations are largely favored by inadequate environmental conditions and, when they occur, often involve entire sections of libraries; furthermore, they can be exacerbated by extraordinary events such as floods, earthquakes, and collapses, assuming even more significant proportions in such circumstances.

In addition to the presence of biological deposits on a substrate, biodeterioration phenomena are influenced by a range of complex factors, including environmental and climatic conditions that promote the growth of biological organisms. Within libraries, biodeterioration processes are favored by conditions such as high temperatures, elevated humidity levels, and inadequate ventilation.

To ensure the optimal preservation of paper, it is crucial to maintain specific parameters of temperature and relative humidity. The ideal range for paper conservation is generally considered to be between 16 °C and 20 °C for temperature and between 40% and 60% for relative humidity. These values aim to provide a stable and controlled environment that minimizes the risks of biodeterioration and effectively preserves the paper material.

Paper, being a hygroscopic material, is susceptible to the influence of moisture content, which can promote the development of microorganisms and insects. Like all hygroscopic substances, paper can absorb and release water in both liquid and vapor states. When the water content exceeds 10%, favorable conditions for spore germination can occur, especially in environments with temperatures around 20 °C and relative humidity of 80%. To prevent the proliferation of microorganisms, it is essential to maintain the temperature below 18 °C and the relative humidity below 65% (

Table 2. Components of the microclimate: the lower the thermal values, the higher the hygrometric values at which microorganisms develop [29].

Fungal Species	Temperature	Minimum Relative Humidity for Microorganism Development
Penicillium Chrysogenum	10 °C 15 °C 25 °C	83.5% 77% 72.5%
Aspergillus Flavus	12 °C 16 °C 30 °C	95% 90% 81%
Aspergillus Tamarii	15 °C 20 °C 30 °C	90% 85% 79%

). However, these values are not sufficient to prevent the survival of certain insect species, which can thrive even in environments characterized by low relative humidity (40%–60%) and very low temperatures (below 0 °C). Therefore, to effectively counter the risk of insect infestation, additional preventive measures need to be implemented, in addition to humidity and temperature control, in order to preserve the integrity of the paper material [29].

Bacteria can have deteriorating effects on paper; however, due to their relatively higher moisture requirements, it is more likely for fungi to proliferate in environments such as libraries, archives, and museums. The environmental conditions typically found in such settings are more conducive to fungal growth compared to bacterial growth [26]. The organic components of books and archival documents readily absorb water, making these materials susceptible to microbial attacks. Consequently, the biodeterioration of paper is primarily attributed to the action of microorganisms that exploit its constituent components, such as cellulose, hemicellulose, lignin, etc., and transform them into simpler and easily assimilable molecules as a source of nutrients.

Another significant factor is the pH value, which plays a crucial role in the chemical stability of paper supports. pH influences the hydrolysis processes that lead to the breaking of bonds within cellulose, thereby affecting the fragility of the paper. Acidic treatments can compromise the polymeric structure of cellulose, and the rate of such deterioration depends significantly on the pH value. Microorganisms can adapt to pH values ranging from 3 to 9, although the intensity of their metabolic activity is most pronounced within a narrow range that varies from species to species. For most fungi, this range falls between 5 and 7 [29].

2.1.2. Consequences of Paper Biodeterioration

Several environmental and biological factors have been identified as responsible for the degradation process affecting archival materials, manifesting through phenomena such as chromatic alterations, discoloration, and loss of paper structure [37,38].

As previously mentioned, fungal attack is a common occurrence, although the damage caused by microorganisms often combines different forms of deterioration. These damages, which can vary in terms of appearance and severity, include chromatic alterations (known as “foxing”) resulting from the formation of stains with various shapes and colors, structural alterations of the underlying components of the materials leading to material fragility and potential destruction, and alterations to essential additive components such as binders, pigments, and inks. Although these components are not the primary elements of the material, their deterioration leads to the loss of support characteristics.

The damage inflicted on paper by microbial agents can manifest in various forms and severity, primarily attributable to the following:

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Chromatic modifications resulting from pigments and exudates, associated with cellular structures such as fungal mycelium and spores, giving rise to the appearance of stains with a wide range of chromatic characteristics, shapes, and sizes.

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Structural modifications of the fundamental components of the materials, induced by specific enzymes such as cellulases, proteases, lipases, etc., are evident through the intrinsic fragility of the materials themselves, which can lead to their complete disintegration. This type of damage tends to occur in advanced stages of infection and is widely recognized as the most devastating.

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Additionally, modifications occur in the crucial additive components of the materials, as in certain cases, microorganisms proliferate by utilizing specific substances that, although present in the material, do not constitute its main element (such as adhesives, plasticizers, antioxidants, etc.). This phenomenon leads to the loss of the substrate’s unique characteristics, sometimes rendering it unusable [25,29].

Therefore, it is of paramount importance to prioritize prevention and mitigation of degradation, as well as the risk of infections and infestations, using specifically designed biocidal products for paper conservation. These products are developed with the aim of controlling microbiological deterioration and countering the deposition of microorganisms on the surfaces of materials without causing lasting damage to the paper and paper supports. Furthermore, it is essential that such biocides are safe for the environment and for humans [39]. Moreover, it has to be taken into account that exposure to microorganisms, fungal spores, and their related metabolites can cause significant adverse effects on the health of people working in libraries, as well as that of visitors, particularly in terms of skin and respiratory system [40].

2.2. Innovative Methods for Paper Preservation

Various physical and chemical strategies have been employed for paper conservation, such as gamma radiation and the use of chemical compounds, such as orthophenylphenol, calcium propionate, ethanol, ethylene oxide, formaldehyde, etc. However, some methods have several limitations, including temporary effects, high costs, and the utilization of toxic chemicals. For example, ethylene oxide, which, in addition to causing an increase in the susceptibility of objects to future microbial attacks, is classified as carcinogenic; formaldehyde, which, at low relative humidity, undergoes polymerization and precipitates on materials, thus having a low penetration power (as well as is being carcinogenic); and pentachlorophenol, which is highly toxic and carcinogenic [26]. Consequently, there is an urgent need to develop an alternative sustainable and environmentally friendly strategy (

Table 3. Summary of innovative methods for paper conservation.

Innovative Method/Activity	Reference Work
Innovative deacidification material/procedure	[8] [42]
Supercritical carbon dioxide (SCCO2)	[43]
Microbial inhibition	[41] [44]
Bait for insects/parasites	[45,46]
Use of DES	[47,48,49,50,51]

) that achieves long-term efficiency, cost-effectiveness, and safety [41].

The objective of conservation is to mitigate degradation processes and extend the lifespan of the artifact. Numerous strategies are available for paper conservation, targeting various aspects of deterioration, and which are further tailored according to specific objectives and implementation constraints. Paper conservation activities are classified into distinct categories, which can be considered as approximate representations of the generic phases involved in a paper conservation treatment: intervention preparation, pest control and disinfection/sterilization, surface dry cleaning, wet washing, deacidification, paper repair, and consolidation/strengthening. Documentation of the treatment is concurrently carried out throughout all these stages [52].

2.2.1. Technologies against Fungal and Bacterial Attacks

The overwhelming majority of antifungal strategies employed to prevent and/or counteract fungal biodeterioration in the preservation of paper-based artifacts can range from restricting fungal access to water to the application of chemical agents in gaseous or liquid form, as well as the use of physical methods such as extreme temperatures, radiation, or currents [26]. A frequently employed physical method is represented by gamma irradiation. However, gamma irradiation is responsible for a reduction in the mechanical strength of paper, as well as its acidification and subsequent yellowing. Additionally, it is important to note that chemical agents can also cause damage to paper; for example, the use of a 70% (v/v) aqueous ethanol solution can result in loss of gloss and damage to book parts [43].

In general, physical intervention methods exhibit long-term characteristics as their antimicrobial action is immediate and leaves no residues. Conversely, most chemical compounds, including those in gaseous form such as ethanol, leave residues that can prolong the antimicrobial effect for a limited period. A simple and harmless approach to inhibit fungal growth involves restricting access to water, thereby reducing water activity on the substrate. An appropriate antifungal method for materials should possess a broad spectrum of activity, good chemical stability, low cost, be non-toxic to humans, and have no negative effects on the treated material [26].

Among biocidal treatment research, a first potential tested product is represented by supercritical carbon dioxide (SCCO₂) in combination with an ethanol additive used for disinfecting ancient paper artifacts affected by fungal contamination [43]. Supercritical carbon dioxide (SCCO₂) refers to the physicochemical state assumed by CO₂ when confined in an environment characterized by temperature and pressure conditions exceeding the critical point [53]. This solution offers advantages such as eco-compatibility, cost-effectiveness, and wide availability, as well as non-flammability, without compromising the mechanical strength of cellulose fibers. Among the numerous fungi detected in a set of 294 samples of ancient documents affected by fungal growth, Aspergillus niger exhibited the highest frequency (36%), followed by Aspergillus flavus (20.7%), Eurotium amstelodami (15.5%), Acremonium spp. (14.9%), Aspergillus versicolor (4.4%), Penicillium chrysogenum (4.2%), Cladosporium subuliforme (3.4%), Rhizopus spp. (0.6%), and Epicoccum nigrum (0.3%). The results of this study indicate that fungal contamination of the samples was reduced through the application of supercritical carbon dioxide (SCCO₂) containing concentrations of 4% and 8% ethanol. The comparative analysis did not reveal significant differences in efficacy between the use of the two ethanol concentrations (4% and 8%) in combination with SCCO₂ [43]. Therefore, it was stated by the authors that this procedure could represent a highly promising option for the preservation and control of fungi in ancient documentary artifacts in the future.

In recent years, significant progress has been made in the application of nanomaterials as deacidification agents for the preservation of paper artifacts. The presence of bacteria and fungi on paper materials plays a crucial role in the acidification process. The extracellular secretions of these microorganisms contribute to increased acidity within the cellulose structure, ultimately leading to paper degradation. Therefore, to indirectly reinforce cellulose, the removal of bacterial and fungal colonies from the artifact can also be anticipated, preventing acidification. Considering this, the use of antimicrobial agents can be an effective strategy to prevent microbial growth. Additionally, nanomaterials can be employed to enhance antimicrobial activity. In relation to the latter, a study [8] involved the preparation of calcium/chitosan nanoparticles (Ca/CS NPs) to explore their potential as a novel approach for the preservation of paper documents. The aim of this study was to improve the efficacy of Ca/CS NPs for paper preservation. Tests were conducted on specific fungal and bacterial strains, including Micrococcus luteus (ATCC 15307), Bacillus megaterium (ATCC 14581), Bacillus subtilis (ATCC 6051), Aspergillus niger (CRM-16404), and Aspergillus fumigatus (MYA-4916). The synthesis of chitosan nanoparticles (CS NPs) was performed for the first time via dropwise addition of sodium tripolyphosphate (TPP) to the chitosan solution. By mixing calcium hydroxide with empty CS NPs, Ca/CS NPs were also obtained. Subsequently, the Ca/CS NPs were applied to the paper samples using a spray method. The spraying process was conducted at room temperature and at 20 cm from the paper samples.

The deacidification effect of the nanoparticles was studied through periodic pH measurements. The initial pH level of the paper samples was measured as pH = 4.71, and after spraying the Ca/CS NPs onto the samples, the pH level increased to pH = 6.17. This pH level was maintained for nearly 10 days and then gradually decreased in the following days. The pH level increase and stability were attributed to the removal of microorganisms facilitated by the antimicrobial effect of the nanoparticles. It can be inferred that the incorporation of calcium with CS NPs enhances antifungal activity. SEM images of the Ca/CS NPs on the paper samples revealed their presence both on the surface and interfaces, forming a protective coating against microorganisms.

The Ca/CS NPs exhibit superior antimicrobial effects against specific strains of bacteria and fungi commonly found on paper documents compared to empty CS NPs. Therefore, these nanoparticles showed increased antifungal and antibacterial potency with the addition of calcium, resulting in a stronger inhibitory effect on specific gram-positive bacteria such as Bacillus megaterium and Bacillus subtilis. Furthermore, this antimicrobial activity contributed to the pH stability of paper-based artifacts. Based on the results, it can be deduced that these nanoparticles have the potential to be preferred as conservation materials for paper-based artifacts.

In the field of nanotechnology, an assessment was conducted on the preservative effect of paper models treated with biosynthesized zinc oxide (ZnO) and silver (Ag₂O) nanoparticles (NPs) against strains of Bacillus subtilis (B3) and Penicillium chrysogenum (F9) [41]. Isolation, identification, and characterization of microorganisms involved in the deterioration of archaeological manuscripts dating back to the 17th century (1677 A.D.) were performed, leading to the identification of 11 bacterial species and 15 fungal species. Among the obtained bacterial and fungal strains, B3 and F9 strains showed the highest cellulolytic activity, and their antimicrobial effect was evaluated using ZnO and Ag₂O NPs.

Scanning electron microscopy (SEM) analysis revealed that a reduced concentration of nanoparticles, used in the context of this study, exhibits a protective effect on the cellulose fibers against bacterial deterioration, through its inhibitory influence was evident on the growth pattern of the B3 strain but not on the F9 strain. On the other hand, higher concentrations of NPs could provide protection against fungal deterioration of the cellulose fibers. Additionally, observation through SEM confirmed the presence of fillers, added to enhance paper properties such as opacity, whiteness, brightness, and printability. The constituent elements and chemical characterization of the filler material in the investigated archaeological manuscript were analyzed using energy-dispersive X-ray spectroscopy (EDX). The highest percentage was found for carbon, followed by oxygen. Other trace elements included silicon and aluminum. The analyses confirmed the use of kaolinite as a filler material, providing a soft characteristic to the paper. Furthermore, it was observed that calcium carbonate was utilized as a filler material to increase the opacity of the manuscript.

The application of Ag₂O nanoparticles at concentrations of 1.0 or 2.0 mM demonstrated a remarkable preservation effect on the paper models, achieving complete microbial inhibition against bacteria and fungi, respectively. Additionally, the NP-treated paper models exhibited slight chromatic changes. Therefore, the deterioration of manuscripts caused by bacterial action can be effectively treated with a low concentration (1.0 mM) of NPs, while for fungal inhibition and the long-term protection of archaeological documents from microbial attack, the use of a high concentration of NPs (2.0 mM) is recommended.

The newly developed functionalized polyamidoamines (PAAs) have been introduced for the sustainable protection of wood and paper [42]. These eco-friendly polymers exhibit a broad-spectrum protective action and have biostatic/biocide properties against various organisms responsible for biotic degradation. PAAs are synthetic linear polymers containing amide and tertiary amine groups that are evenly distributed along the polymer chain. They can be easily incorporated into aqueous media within lignocellulosic artifacts, where they primarily exert a deacidifying effect against bacteria, fungi, molds, and insects.

The objectives of treating paper with PAAs include deacidification, followed by protection against biotic degradation and consolidation. Preliminary studies have been conducted by immersing paper samples in PAAOH, which contains alcoholic groups, and it is soluble in aqueous environments. The deacidification process has proven to be highly effective; raising the pH from 5.4 to 7.4. PAAOH demonstrates promising biostatic activity against some molds responsible for paper degradation.

The collected data unequivocally demonstrate that treatments with PAAs effectively improve both the dynamic–mechanical properties and strength. It can thus be stated that such a treatment emerges as a valid approach to safeguarding the paper support of aged manuscripts from weakening caused by chemical and physical aging phenomena.

Essential oils have also been employed as antifungal agents against the growth of certain fungi. Their antifungal potency has been observed with three natural oils extracted from the peels of Citrus reticulata, the leaves of C. aurantifolia, and Linum usitatissimum (flaxseed), which were used as antifungal agents against the growth of Aspergillus flavus and Penicillium chrysogenum [44].

To assess the effect of the oil on paper, a sample of historical manuscript paper was considered. The samples were placed in Petri dishes containing cotton saturated with these oils, with no direct contact between the oil and the paper. Therefore, the process occurred through oil sublimation without impregnation.

The oils exhibited varying levels of antifungal activity against the studied fungi (Aspergillus flavus and Penicillium chrysogenum). Generally, the inhibitory effect of the oils increased proportionally with concentration, with maximum inhibition achieved at a final concentration of 2000 μL/ml. Thermogravimetric analysis revealed that the paper samples exhibited an initial weight loss of 2.86% at approximately 105 °C, primarily attributed to the evaporation of any absorbed moisture.

The oils played a crucial role in reducing color fading values in the historical paper, with Linum usitatissimum (flaxseed oil) exhibiting the most effective protection against color fading. Thermogravimetric analysis further demonstrated that flaxseed oil outperformed the other two oils, exhibiting superior inhibitory activity on historical documents and producing positive effects on them. Additionally, flaxseed oil provided the best protection against color change.

2.2.2. Strategies against Insect Infestation

The preservation of collections in museums, libraries, and archives, which comprise organic artifacts such as wood, linen, wool, and others, faces a significant risk from a diverse array of harmful insects jeopardizing their long-term conservation [45]. Non-chemical methods offer effective elimination of insect pests in museum, library, and historic building settings, prioritizing object preservation, comprehensive eradication of insects at all stages, and ensuring the well-being of both the environment and museum staff [54]. Physical treatments, such as freezing [55,56], controlled heating [57,58], microwave radiation exposure, and gamma irradiation, are employed. However, it is crucial to highlight that not all materials can be suitably treated using these methodologies. For delicate objects, anoxic treatments [59,60] are preferred, creating a low-oxygen atmosphere through nitrogen or argon-based approaches.

There is no universally perfect treatment method, and the selection of the most appropriate approach should consider factors such as time constraints, financial resources, available means, and the specific type of pests and materials requiring treatment [48].

An investigation was conducted to explore and identify the biodiversity of harmful insects infesting the Manuscript Library of the Coptic Museum (Egypt) [45]. Over a period of one year, from October 2018 to September 2019, monthly sampling was carried out using adhesive traps with a non-toxic sticky substance. The adhesive traps were strategically placed on the floor surface (at the corners of the library), on window and door edges, and behind fabrics and manuscripts to capture insects walking on them.

In total, 1047 specimens belonging to nine species were identified and categorized into five orders (Hymenoptera, Coleoptera, Thysanura, Psocoptera, Blattodea) and six families (Formicidae, Ptinidae, Dermestidae, Lepismatidae, Liposcelidae, Blattidae) within the studied library context [45]. The most abundant and consistently present species throughout all months was Monomorium pharaonis. It was found that the presence of insects in the library was higher during the summer months, with a range of 108 to 222 individuals, compared to the winter months, when 4 to 23 individuals were trapped. This seasonal difference could be attributed to the hot and humid climate, which favors the proliferation of pests.

The parasites infesting paper materials include both pests associated with molds and high humidity, such as Liposcelis bostrychophila (Booklice), which feed on microscopic molds that develop on paper and starch-based glue used in binding, often indicating the presence of harmful moisture and mold issues, as well as generic pests such as Thermobia domestica (Silverfish) that can damage paper, bindings, starch-based glue, and other cellulose materials. Additionally, common pests such as cockroaches, ants, and other insects can attack and cause problems within museums.

In conclusion, the baitless adhesive traps used proved to be more effective in collecting a wide range of harmful insects within museums compared to baited ones. To prevent the presence of moisture-related pests, measures need to be taken to adjust climatic conditions and reduce the presence of dust, microscopic fungi, and other organic substances. It is crucial to promptly address infested artifacts present in the library to prevent further spreading and damage.

The Ctenolepisma longicaudatum, also known as the “long-tailed silverfish”, represents an intradomestic parasitic agent of significant importance for libraries, archives, and museums. In a study [46], the impact of using an insecticidal gel bait containing indoxacarb as the active ingredient on populations of C. longicaudatum in three libraries, seven archives, and seven museums located in Norway and Austria was examined. To assess the effectiveness of bait application, the activity of the parasites was monitored using adhesive traps. This is a long-acting control strategy, which demonstrated a 90% reduction within a period of 3–4 months, requiring a minimal amount of bait to achieve complete eradication of the individuals. Significant decreases in parasite populations were observed in all sites where a few drops of bait were applied inside the buildings. In the context of this study, the maximum amount of bait used in a single location was 0.02 g per m².

Paper is universally recognized as a source of carbohydrates from which insects derive nourishment and can survive under abiotic conditions. However, to ensure their survival, C. longicaudatum also requires the intake of proteins, which are not present in the paper itself but can be obtained from dead insects, including those of the same species. It is important to note that these insects die because of ingesting baits containing sugars as stimulant agents, leading to primary poisoning. Once deceased, they become poisoned protein sources for other insects, thereby causing secondary poisoning.

Therefore, the use of bait has proven effective in preventing infestations within museums, representing an advantageous and low-risk approach that can be adopted as a conservation strategy in libraries, archives, and museums. The treatment cost, evaluated based on the amount of bait used and the hours of labor involved, has been found to be reasonable. The use of gel bait can provide a cost-effective alternative to reduce the presence of the parasite community; furthermore, the method has a limited impact on the health of operators and visitors in the treated areas, as well as on the objects present.

2.2.3. DES (Deep Eutectic Solvent)

In the field of cultural heritage, degradation processes are widespread, and in the context of restoration and preservation of such materials, there is a gradual shift towards a “green” chemistry approach that focuses on the use of non-toxic solvents and reagents. In this perspective, deep eutectic solvents (DES) have been proposed as an alternative to Ionic Liquids (IL) [61]. DES can be described as a mixture of acids and bases or as a mixture of hydrogen bond donors and acceptors (HBD and HBA, respectively). When employed in the treatment of lignocellulosic biomass, DES can also serve as a pre-treatment medium to enhance the fiber structure.

In order to facilitate the production of micro fibrillated cellulose, a study was conducted to investigate the effects of DES on induced fibrillation processes [47]. Cellulose derived from eucalyptus and cotton pulp was utilized to evaluate these effects. The study examined the influence of three different DES systems (acidic, neutral, and basic) on the chemical and physical characteristics, including internal and external fibrillation, of cellulose fibers obtained from the aforementioned materials. The primary objective of the research was to assess the capacity of these solvents, when used as pre-treatment, to induce fiber fibrillation and achieve micro fibrillation without introducing significant modifications to the cellulose structure.

The obtained results highlight that the treatment with acid and neutral DES has modified the chemical composition of eucalyptus fibers. This treatment induced both internal and external fibrillation without significantly altering the chemical composition, crystallinity, and polymerization degree of the fibers. Furthermore, a notable improvement in the mechanical properties of the produced papers was observed, attributed to an increased degree of adhesion and preservation of fiber length. Therefore, the DES treatment can be regarded as a gentle and environmentally friendly “chemical” refinement process that could be employed as a pre-treatment method for cellulose micro-fibrillation.

In a previous study, the utilization of microwave-assisted deep eutectic solvent (MV-DES) treatment, combined with ultrasound treatment, was employed for the preparation of lignin-containing cellulose nanofibers (LCNF) [48]. Subsequently, the effectiveness of LCNF was evaluated through the application of reinforcement agents and UV-absorbing substances on polyanionic cellulose (PAC) films.

The resulting LCNF exhibited an intricate matrix, which can be attributed to the agglomerating role played by lignin among the cellulose nanofibers. The incorporation of LCNF led to a significant improvement in the stability of the film-forming suspension of polyanionic cellulose (PAC). Therefore, this study provides an environmentally friendly methodology to produce biomass derived LCNF. The addition of 5% LCNF to PAC films demonstrated a high capacity for UV protection, suggesting the broad applicability of the MV-DES method in the preparation of such nanofibers. The stability of the LCNF/PAC composite film suspensions exhibited excellent mechanical properties and an adjustable UV protection capacity based on the quantity and type of LCNF used.

Another study presents a novel approach for the direct modification of cellulose using deep eutectic solvents, providing a sustainable strategy for the modification of other materials [49]. The surface of cellulose particles was subjected to carboxylation in a deep eutectic solvent containing carboxylic acid (oxalic acid), acting as a hydrogen bond donor (HBD), while choline chloride was employed as an acceptor (HBA). The effects of key factors on cellulose carboxylation were investigated based on the carboxyl content.

The results of the analysis revealed that the type of carboxylic acid employed, the size of cellulose particles, and the pre-treatment methods significantly impact the efficiency of cellulose carboxylation process. The carboxylation reaction did not induce any alteration in the internal crystalline structure of cellulose; however, it led to the destruction of the surface crystalline structure, resulting in a remarkable enhancement in the dispersibility and hydrophilic properties of the product. In order to assess the potential practical applications of carboxymethyl cellulose modified using DES, a systematic study was conducted to investigate its adsorption capacity using methylene blue. Cellulose with a highly fine particle size exhibits a high susceptibility to degradation and carbonization when heated in DES containing oxalic acid, resulting in a significant reduction in the yield of carboxymethyl cellulose. This study revealed that cellulose carboxylation is more efficient in the presence of carboxylic acids characterized by high acidity in the DES, as well as a lower molecular size.

Another study presented a novel approach for investigating solvents for cellulose [50], focusing on the application of three DES for cellulose dissolution. Three new solvents were synthesized from combinations of urea/caprolactam, caprolactam/acetamide, and urea/acetamide, which are organic solid compounds with respective molar ratios of 1:3, 1:1, and 1:2. Their physical characteristics, including melting point, conductivity, and solubility, were studied.

The melting point represents a fundamental parameter for evaluating the behavior of a deep eutectic solvent, and in this study, it was observed that this parameter depended on the molar ratio of the two compounds. Specifically, it was found that the solvent composed of urea/caprolactam with a molar ratio of 1:3 had a melting point of 30 °C, the caprolactam/acetamide solvent with a molar ratio of 1:1 had a melting point of 18 °C, while the urea/acetamide solvent with a molar ratio of 1:2 exhibited a melting point of 48 °C.

Furthermore, taking the caprolactam/urea deep eutectic solvent as an example, it was observed that during the heating of the mixing system, the intramolecular hydrogen bonding of each precursor compound was disrupted, leading to the formation of new intermolecular hydrogen bonds between one urea molecule and multiple caprolactam molecules. This reaction resulted in a decrease in various physical factors, such as hydrogen bond energy, molecular arrangement degree, and electrostatic attraction between hydrogen and the organic molecule.

These weak ionic interactions contributed to the reduction of the crystalline lattice energy of the salts, resulting in salts with a low melting point. The conductivity of the deep eutectic solvent synthesized with the optimal molar ratio was approximately in the range of 10⁻⁵–10⁻⁴ S/m. Currently, one of the distinguishing properties of deep eutectic solvents compared to ionic liquids is their low conductivity, particularly for the deep eutectic solvent synthesized from urea and caprolactam, which was only approximately 3.2 × 10⁻⁵ S/m at 38 °C. At the same temperature, the conductivity of the three systems followed the following order: urea/caprolactam < caprolactam/acetamide < urea/acetamide. With increasing temperature, the conductivity increases.

It has been observed that the three solvents exhibit the ability to dissolve cellulose in the following order: urea/caprolactam > caprolactam/acetamide > urea/acetamide. The solubility of the deep eutectic solvent for cellulose is influenced by the quantity and intensity of hydrogen bonds formed with cellulose. In other words, a greater formation of hydrogen bonds with cellulose corresponds to a higher solubility of cellulose.

In conclusion, the three deep eutectic solvents exhibit a low melting point and low conductivity. Additionally, it has been found that the solubility of cellulose is limited. The studies have examined the application of DES on cellulose materials. However, studies have also been conducted on the use of these new molecules as preservation agents for stone materials as an alternative to traditional biocides that are toxic to the environment and operators.

A study was conducted on a mosaic located in the Archaeological Park of Ostia Antica to evaluate the effectiveness of five DES with different compositions containing choline chloride, ethylene glycol, malonic acid, glycerol, oxalic acid, and urea. These solvents were compared to the traditional biocide Preventol RL50 [51].

The DES employed in this research for the removal of biological patina are of pure nature and synthesized from natural precursors. Therefore, they are considered safe for both the environment and the operator, and they do not interact with the stone layer but exert solely antibacterial activity on the biofilm. It has been observed that DES 2 and 4, composed of malonic acid and oxalic acid, respectively, exhibit a lower pH and a higher biocidal effect, thereby inhibiting bacterial growth.

DES 5 (ChCl/U) has demonstrated significant efficacy at a slightly higher pH (7.2 ± 0.5). Subsequently, the effectiveness of DES was compared with that of Preventol RI50, and despite achieving positive performance, these new solvents do not reach the standards achieved by the traditional biocide. However, there is a substantial difference between the two products; in fact, DES exhibits stability, non-volatility, eco-compatibility, and does not require dilution with solvents as they can be used in their pure form as slightly viscous liquids at room temperature. The results obtained from this study highlight how the use of DES can represent a new environmentally friendly strategy for cleaning and preserving materials in the context of cultural heritage.

3. Stone Materials

Stone materials assume a predominant significance within the extensive panorama of our historical and artistic heritage, embodying a category of works that undergoes thorough and comprehensive investigation within the academic context of conservation and restoration.

The term “stone materials” refers both to natural substrates, encompassing any variety of rocks utilized in the architectural field, and to artificial ones, which are materials resulting from the manipulation and transformation of naturally sourced raw materials such as mortars or ceramics. Within the natural stone materials, the possibility of classification based on their geological origin is highlighted: sedimentary rocks, such as limestone and sandstone, metamorphic rocks, including marble, and igneous rocks, such as granite, are distinguished. In each of the aforementioned categories, there exist further subdivisions or classifications that can be associated with chemical composition, color, porosity, utilization, and other related attributes.

Regarding artificial stone materials such as mortars and ceramics, they can be defined as a combination of inorganic or organic binders, predominantly fine-grained aggregates, water, and possible additions of organic and/or inorganic additives (or a mixture consisting solely of binder and water), in proportions necessary to confer the mixture with a suitable fresh state for processing, as well as appropriate physical properties in the hardened state, such as porosity, water permeability, and so forth, in addition to mechanical characteristics including compressive strength, tensile strength, flexural strength, adhesion, aesthetic appearance, and durability [62].

3.1. Degradation of Stone Material

Stone materials undergo a range of natural and non-natural processes that inevitably lead to a progressive alteration of their initial characteristics. In many cases, these processes occur without causing significant harm to the preservation of the material itself, while in others, they can manifest as genuine forms of degradation. These processes that can compromise the very structure of the asset can be linked to both natural factors (e.g., rainfall, temperature fluctuations, etc.) and anthropogenic factors such as pollution. It is worth pointing out that the mineropetrographic, physical, and chemical features of the stone substrate represent the first discriminant influencing the intensity and the typology of biodeterioration affecting the material.

Every kind of natural and artificial stone should be treated deeply and separately based on the different response to biodeterioration. However, the authors mean to provide a general overview on the progress of the strategies against this phenomenon, especially valorizing the “green” and most sustainable ones.

The in-depth analysis and understanding of the factors responsible for degradation, therefore emerge as a crucial element to implement preventive measures or, at the very least, delay the deteriorative processes, especially when dealing with materials exposed to external environmental conditions [63]. The implementation of strategies aimed at modifying moisture levels or mitigating the impact of pollutant agents proves to be inherently complex and requires meticulous application and management.

The modifications affecting the material are manifested through more or less significant variations in the stone matrices, giving rise to phenomena of alteration (material alteration that does not necessarily imply a deterioration of its chemical and physical properties from a conservation perspective) and/or degradation (alteration that always entails a deterioration of the original material’s chemical and physical characteristics) [64].

The magnitude of these effects will vary depending on the specific characteristics of the material itself, such as composition, origin, structure, and texture, as well as the combination of external agents, including environmental, physical, chemical, and biological factors.

Physical degradation occurs when external forces act upon the stone material, causing surface decohesion and a reduction in its mechanical strength, which can lead to the loss of material fragments in extreme cases [63]. The main factors responsible for physical degradation include water, salt crystallization, wind action, and thermal stress [65]. The result of these physical phenomena that affect the stone are the phenomena of alveolization [66,67], efflorescence [68], etc. Chemical degradation assumes a prominent role in stone materials due to its ability to induce chemical alterations in the original material, resulting in the formation of byproducts characterized by high solubility in many cases. The factors contributing to chemical degradation can stem from both natural and anthropogenic causes. Natural factors may be associated with marine aerosol, volcanic eruptions, and similar circumstances. As for anthropogenic chemical degradation factors, it is pertinent to mention the presence of atmospheric gases and particulate matter, including carbon compounds (CO_x), sulfur compounds (SO_x), and nitrogen compounds (NO_x). Acid precipitation, along with atmospheric particulate deposition, represent primary degradation factors that significantly impact the preservation of stone materials exposed to the outdoor environment [11]. Among the forms of physical degradation are the formation of black crusts [69,70], chromatic alteration [71], etc. Biological degradation is also observed, which is associated with the activity of microorganisms and organisms that attack the surface of stone materials. These agents include bacteria, algae, and higher organisms (i.e., plants) and often induce damages both aesthetically and chemical–physical damage that manifest itself through forms of degradation such as biological deposits [72] and biological patina [73].

It is essential, therefore, to identify the main causes of degradation and assess the feasibility of interventions aimed at mitigating or resolving them.

3.1.1. Stone Material Biodeterioration

Rocks, whether in natural geological formations or in stone monuments, serve as favorable environments that harbor a diverse array of microorganisms. The pervasive presence and metabolic activities of microorganisms contribute significantly to the degradation process experienced by stone structures [4,74,75,76].

Biodeterioration is a phenomenon characterized by the modification of an artifact’s original state, resulting from the metabolic activity of communities of organisms and microorganisms. These communities, comprising algae, bacteria, fungi, and lichens, collectively referred to as biodeteriogens, contribute to the process of alteration.

Biodeterioration is a subsequent phase that ensues after previous forms of alteration, establishing conducive conditions for subsequent colonization by living organisms. Stone surfaces undergo a sequence of transformative processes following their initial installation, involving diminished luster, heightened roughness, crack formation, organic matter accumulation, and increased moisture levels. These phenomena facilitate the colonization and proliferation of biodeteriogens.

Biodeterioration refers to a phenomenon in which the presence of microorganisms or organisms triggers physical and chemical processes that can utilize the substrate for both nutritional purposes (heterotrophs) and structural support (autotrophs).

Within the specific context of stone artifacts, which predominantly consist of inorganic materials, the initial colonization is mainly driven by autotrophic or photoautotrophic organisms, including a diverse array of bacteria, cyanobacteria, algae, and lichens. This is followed by the subsequent diffusion of heterotrophic organisms, encompassing various bacteria and fungi.

The adherence of microorganisms to the stone substrate occurs through the production of extracellular polymeric substances (EPS), which form a complex layer known as a biofilm [25]. These microbial aggregates form communities in which cells are enclosed within a self-generated gelatinous matrix composed of EPS [77]. This matrix, primarily consisting of water, encompasses diverse constituents, such as polysaccharides, proteins, and extracellular DNA, playing a vital role in substrate adhesion and the sustenance of the microbial community [3,78].

After the initial attachment, there is the establishment of a microbial community comprising diverse bacterial strains, progressing through subsequent stages of growth and maturation (Figure 2).

Within this progression, novel microbial species, including heterotrophic microorganisms, integrate into the community, utilizing organic matter in the form of deceased cells and secondary products as a source of nutritional sustenance.

The occurrence of biodeteriogens on surfaces is contingent upon multiple factors, encompassing the inherent composition of the structures, the degree of light exposure, the moisture levels, and the surrounding microclimate. These factors require meticulous preliminary analysis before making any decisions regarding the preservation of the object being examined.

The impact of these exogenous degradation agents is intensified by the bio-receptivity phenomenon, which pertains to the distinct response exhibited by the artifact, and it is influenced by a range of endogenous factors. These encompass the rock type, its origin, and the processing techniques employed, as well as its mineralogical and structural properties, including porosity, permeability, capillarity, and surface roughness. Furthermore, distinct microclimatic conditions associated with moisture, solar irradiation, wind, pollution, and, particularly, the presence of water, have a further impact on the bio-receptivity of the material.

Specifically, the hygroscopic nature of the material, intimately associated with its porosity, assumes a crucial role in the biodeterioration process. The amplification in relative humidity percentage within the external environment, coupled with an augmented water absorption capacity of the material, engenders more propitious conditions conducive to the proliferation of surface microflora.

Biofilms can also act as reservoirs for atmospheric pollutants, thereby exacerbating the processes of chemical substrate corrosion [6].

The process of biocorrosion, as an additional factor impacting biodeterioration, is initiated by the microbial secretion of both inorganic acids (such as nitric acid, sulfuric acid, carbonic acid, and nitrous acid) and organic acids. This secretion ultimately results in the solubilization and weakening of the stone matrix.

3.1.2. Consequences of Stone Biodeterioration

Microbial activity in biodegradation processes can lead to diverse forms of damage to stone monuments, encompassing aesthetic manifestations such as the development of surface crusts and patinas, mechanical repercussions resulting in significant fractures, and chemical modifications induced by metabolite production.

There exists a multitude of biological alterations that can manifest in various forms, such as patina, crust, discoloration, stains, and pitting, among others (

Table 4. Summary of degradation patterns induced by biological growths ([64] and modified from [79]).

Biodeteriogenic Agent	Description
Bacteria	Black crusts, black patinas, exfoliation, pulverization, colour change, stains
Fungi	Staining, exfoliation, pitting
Algae	Patinas and films of various colors and consistency
Lichens	Scaling, mottling, pitting
Superior plants	Grass, shrubs, and woody species induce fractures, collapses, detachment of material

). Biological patinas exhibit variations in color, thickness, and microstructure. These formations have the potential to comprise biofilms or evolve into authentic crusts, irrespective of the material type and environmental conditions. These “biopatinas” have the potential to generate mechanical actions resulting in the microdecohesion of substrates, as well as to initiate metabolic processes capable of inducing chemical transformations in materials.

The presence of biofilm formations can lead to the accumulation of atmospheric pollutants, thus intensifying the processes of chemical corrosion on substrates. Additionally, the occurrence of stains causes superficial chromatic alterations to the artwork, with microorganisms being implicated in certain instances. The occurrence of pitting signifies a manifestation of degradation resulting from the activity of microorganisms with the capacity to penetrate the microcavities found in the stone structure. This infiltration process leads to the generation of numerous invisible openings characterized by their proximity and high density.

3.2. New Technologies for Stone Restoration

Currently, there are multiple strategies available for the consolidation and disinfection of such artifacts; however, numerous issues arise, primarily related to compatibility with the original materials and the durability of restoration interventions.

Every restoration intervention on stone artifacts requires an initial phase of diagnosis of their conservation status, which includes the analysis of composition and deterioration. Subsequently, a cleaning phase is carried out to remove any superficial deposits such as dirt and alteration patinas. Finally, a consolidation phase is implemented to restore the structural characteristics of the artwork.

The widely adopted restoration techniques for the removal of biological patinas from stone surfaces involve the use of both physical and chemical approaches. Among the physical approaches, mechanical brushes are employed to remove the biofilm; however, it is important to consider that they can potentially damage the material itself, thereby increasing its susceptibility to biodegradation [80]. Traditional chemical methods, on the other hand, involve the use of biocides such as benzalkonium chloride, which can reduce the presence of the microbial community. However, this biocide is known to be potentially toxic to operators and harmful to the environment, especially when used in confined spaces.

Consequently, there emerges a necessity to conceive pioneering, ecologically sustainable, and secure solutions [12,13] to mitigate these impacts. As a result, research efforts have recently intensified towards the exploration of alternative eco-compatible tools [9]. In

Table 5. Summary of the innovative methods for stone reviewed.

Innovative Method	Activity	Reference Work
Sodium alginate hydrogel and hypochlorite ions	Biofilm removal	[11] Gabriele et al., 2021
Solvent gel of dimethyl sulfoxide		[81] Toreno et al., 2018
Lavandula angustifolia and Thymus vulgaris essential oil encapsulated in alginate hydrogel	Bacterial inhibition	[6] Ranaldi et al., 2022
Nanocapsules with essential oil of Origanum vulgare and Thymus capitatus		[82] Romano et al., 2020
Glyco-alkaloids extracted from Solanum nigrum, filtered without cells of the fungus Trichoderma harzianum and the bacterium Burkholderia gladioli		[9] Sasso et al., 2016
Silver nanoparticles		[10] Carillo-Gonzàlez et al., 2016

, we have summarized the innovative methods proposed against biodeterioration. They are based on a chemical action, since all the active components have a biocidal and/or bacterial inhibition feature. The main difference between biocidal and bacterial inhibition effect is that the first term refers to the ability to kill the microorganism, while the second one describes the ability to avoid the biological growth. The reviewed methods are divided in two classes: those methods related to the removal of the biofilm and those mainly focused on bacterial inhibition. Biofilm removal consists of the killing of the microorganisms and their removal from the surface; these methods cover all the conservation phases related to the cleaning of the stone. A bacterial inhibition method should be adopted after a cleaning phase since it is aimed to protect the surface against further colonization over time.

An alternative approach to the use of hypochlorite as a biocidal agent on stone substrates has been identified in order to mitigate the negative impact on such materials and preserve the health of the operators [11].

A hydrogel formulated with sodium alginate as an inert matrix and hypochlorite ions as biocidal agents has been tested to remove biological patinas from samples of Lecce limestone. It is one of the predominant materials used in the construction of historical buildings in the Baroque style located in southern Italy. This material generates considerable interest in the field of stone conservation studies, as it presents significant degradation issues both aesthetically and structurally, primarily associated with biofouling and chemical pollution.

Three samples of Lecce stone were selected as the control group, kept under conditions of intact preservation, while the other samples were subjected to induced biodeterioration or different treatments. Subsequently, an analysis was conducted to identify the phototrophic microorganisms constituting the present biofilms. It was found that these biofilms were predominantly composed of filamentous cyanobacteria and green microalgae. Optimizations were carried out on two different formulations of biocidal hydrogels to determine the best combinations of sodium alginate and calcium hypochlorite quantities to achieve a hydrogel with suitable consistency and maximum effectiveness.

The samples of Lecce limestone were subjected to specific biodeterioration processes, varying the duration to obtain three different levels of biodeterioration. Additional samples were subjected to a cleaning process using an aqueous solution containing 2.5% calcium hypochlorite. It was observed that in order to achieve complete removal of microorganisms from the stone, it would be necessary to use an oxidizing agent quantity approximately 10 times higher than the concentration present in the biocidal hydrogel with a higher amount of calcium hypochlorite. The presence of the biocide incorporated within the cross-linked structure of the hydrogel allows for the use of a reduced concentration of oxidizing agent.

The obtained results highlighted the effectiveness of the two optimized hydrogels in removing microorganisms from the stone surface, also demonstrating how the quantity of oxidizing agent to be incorporated in the hydrogel depends on the extent of the biocolonization process. This sanitization approach has made it possible to use hypochlorite as a biocidal agent, as potential side effects and health risks associated with operators are mitigated by its retention within the gel matrix. Thanks to the gel’s adherence to the stone surface, which ensures prolonged exposure times, an extremely low concentration of oxidizing agent could be used to eliminate the biofilm.

Another environmentally friendly method proposed for the removal of biofilms and lichens from surfaces is represented by a solvent gel containing dimethyl sulfoxide (DMSO) applied on marble artifacts affected by biological patinas [81].

The gel was applied to the surfaces of the marble blocks and subsequently removed after a 24-h period, followed by cleaning with distilled water. This protocol was repeated from two to six times. Surprisingly, excellent results were achieved after only two treatments with the gel.

Preliminary tests conducted on a non-colonized marble sample have demonstrated that treatment with the DMSO gel solvent does not significantly alter the physical parameters of the substrate. In fact, measurements taken using a profilometer and colorimeter before and after six applications did not show significant variations. Therefore, the DMSO gel treatment did not alter the natural color of the marble, and no significant variations in the surface profile of the stone were observed after all the applied treatments. There were no significant variations in terms of porosity, pore radius, saturation, and adsorption parameters, indicating that the marble substrate remains substantially unchanged after the cleaning treatments with the gel.

Before the gel treatments, it is possible to observe the widespread presence of a biofilm. However, after the first application of the gel, the biofilm appears partially detached from the substrate. At the end of the treatments, the rocky surfaces are completely clean and free of residues.

The comparative study conducted between the DMSO-based solvent gel and the currently used biocides revealed that, after the same number of treatments, the gel proved to be the most efficient. The number of gel applications required may be related to the thickness of the biofilm and its penetration into the substrate. In fact, the best results were achieved through repeated gel applications. Subsequently, a culture test conducted one year later did not detect the presence of colonies, confirming the effectiveness of the treatment.

In the context of cultural heritage restoration, the observation regarding the use of essential oils (EO) as natural biocidal agents has emerged as an effective alternative to conventional chemical compounds. Evidence supporting this observation has been provided by a scientific study [6] demonstrating the efficacy of Lavandula angustifolia Mill. and Thymus vulgaris L. essential oils at a concentration of 5% in combating cyanobacterial biofilms. During the comparative analysis, it was observed that the 5% concentration of Thymus vulgaris essential oil (EO) exhibited greater inhibitory efficacy against the photosynthetic activity of cyanobacterial biofilms over an extended period. Samples treated with thyme essential oil displayed significant discoloration without evidence of microbial growth, whereas only one of the samples treated with lavender essential oil exhibited such discoloration.

To develop new non-invasive methodologies for the conservation of stone monuments, various concentrations of thyme essential oil (EO) were experimented with. These concentrations were encapsulated within an alginate hydrogel (EO-HG) and applied for different time periods on cyanobacterial biofilms.

All the examined concentrations of Thymus vulgaris essential oil encapsulated in an alginate hydrogel (T. vulgaris EO HG) demonstrated the ability to completely inhibit the photosynthetic activity of the biofilms.

As a result, the utilization of T. vulgaris EO-HG, even at very low concentrations, demonstrated a potent inhibitory power against microorganisms. Consequently, the utilization of Thymus vulgaris essential oil in an alginate hydrogel is considered a recommended formulation as it allows for the use of extremely limited amounts of biocide while maintaining high antimicrobial efficacy against cyanobacteria and reducing the volatility of terpenic components.

In the study [82], the use of nanocapsules (NC) containing essential oils of Origanum vulgare (Or-EO) and Thymus capitatus (Th-EO) is described to counteract the bacterial proliferation of two microorganisms (Escherichia coli and Kokuria rhizophila) on marble surfaces.

Plant essences, in the form of essential oils (EO), could represent an effective alternative to conventional chemical biocides. However, characteristics such as color, high volatility, insolubility in water, and sensitivity to oxygen, light, and heat may pose challenges in the use of these essential oils for the preservation of cultural heritage.

The process of nanoencapsulation of essential oils can be an effective strategy for creating new non-toxic and eco-friendly nanomaterials to be used in the field of cultural heritage. This technique allows for the protection of compounds, such as EO, from adverse environmental conditions, ensuring color masking, improved solubility, and reduced volatility. Furthermore, it enables controlled release of EO even at different temperatures.

No significant changes in the color, structure, or chemical composition of the stone were observed after the application of nano-encapsulated essential oil (EO-NC). After 6 h of contact, both Or-NC and Th-NC were able to reduce the bacterial concentration by three to four orders of magnitude for both microorganisms. After 24 h of contact, no viable bacterial cells were observed.

The nanostructured systems (EO-NC) have shown the ability to inhibit bacterial growth on the stone previously treated with a bacterial inoculum. It has been found that the nanocapsules loaded with thyme essential oil are more efficient compared to the nanocapsules loaded with oregano essential oil.

In the field of research, glycoalkaloids (GA) are also being studied as possible natural biocides, which are produced by plants of the Solanaceae family. In [9], they were tested against a phototrophic culture that develops on a substrate of limestone rock, filtered without cells of the fungus Trichoderma harzianum and the bacterium Burkholderia gladioli. To promote the development of a photosynthetic biofilm on the surface of a limestone rock, a phototrophic culture composed of different species was employed. The main components of the inoculum consisted of the genera Chlorella and Stichococcus. Sixteen blocks of white bioclastic limestone were used as the lithotype, onto which 200 mL of a multispecies phototrophic culture were applied on their respective upper surfaces.

The cell-free filtrates of T. harzianum and B. gladioli, along with the glycoalkaloids, exhibit limited biocidal activity that is primarily focused on filamentous phototrophic microorganisms in general. The treatment was not successful in effectively inhibiting the growth of photosynthetic biomass on stone surfaces. The glycoalkaloids appeared to reduce microbial growth during the initial month of incubation, but after 30 days of incubation, they promoted biofilm expansion. This also demonstrates the ineffectiveness of glycoalkaloids as biocides, especially against unicellular phototrophs, as these natural compounds can act as nutrient sources for microbial growth instead of controlling biological colonization. Among the three tested natural biocides, only the cell-free filtrate of the fungus Trichoderma proved to be effective against the photosynthetic community developed on limestone blocks during a 45-day incubation period. Additionally, the application of the treatments caused chromatic variations, manifesting as darkening and the yellowing of the blocks.

In summary, the application of cell-free filters and plant extracts on stone samples has been shown to suppress the growth of filamentous phototrophic microorganisms, but at the same time, it promoted the growth of unicellular phototrophic microorganisms. Therefore, the use of this biocide has proven to be ineffective.

Nanotechnology, which involves the production of nanomaterials using biological or natural components (green synthesis), is among the emerging alternatives in this context. It offers numerous advantages over chemical methods and has a low environmental impact. Silver nanoparticles (AgNP) have been synthesized using a green synthesis method and have been evaluated as potential antimicrobial inhibitors [10].

Two plant species, Foeniculum vulgare and Tecoma stans, were utilized in the synthesis of nanoparticles using aqueous leaf extracts and two extraction procedures. The potential of AgNP was evaluated as preventive/corrective treatments to safeguard materials (stucco, basalt, and limestone) from biodeterioration. AgNP exhibit unique physicochemical properties compared to individual particles, thereby conferring increased reactivity and high efficacy against a wide range of microorganisms.

A total of twenty-three bacterial species and fourteen fungal species were isolated from colored stains, patinas, and biofilms present on surfaces. AgNP produced by Foeniculum vulgare exhibited high efficacy in suppressing bacterial growth, surpassing that of nanoparticles produced by Tecoma stans. Studies have reported a correlation between bacterial growth and the increase in AgNP concentration. In particular, it was observed that a concentration of 200 μL of AgNP produced by both Foeniculum vulgare and Tecoma stans did not result in any significant reduction of bacterial growth. However, it was found that dosages of 300 μL consistently yielded high percentages of bacterial inhibition (85% and 77%, respectively). Furthermore, higher dosages (500 μL) demonstrated effective reduction of bacterial growth (71% and 64%).

A significant enhancement of the antibacterial effect was observed when the size of the nanoparticles (NP) was reduced from 100 to 20 nm. Additionally, it was observed that bacteria exhibit lower sensitivity to AgNP compared to fungi. The three types of materials show a similar degree of susceptibility to microbial colonization; however, limestone exhibits a higher tendency for alteration compared to other stone varieties due to processes such as material dissolution, salt crystallization, and biological activity. However, the use of AgNP contributes to enhancing the protection of the stone material. It is worth highlighting that during and after the treatments with AgNP, no surface alterations were observed in the three tested materials. The employment of AgNP as a preventive or corrective treatment to reduce microbial colonization in three different stone varieties used in historical masonry structures has shown remarkable success.

4. Conclusions and New Perspectives

From the examined literature it has been seen that the effectiveness of the compound or method used against biodeterioration varies greatly in the function of various factors, such as the mechanism of action, the concentration used, and the period of action.

Nowadays, the international scientific community dedicates considerable attention to researching and experimenting with innovative natural biocides that are both long-lasting, non-invasive, and eco-friendly.

Among the innovative methods concerning the biodeterioration of paper, functionalized nanoparticles and essential oils are the most effective methods of inhibiting microbial growth. Essential oils also have the benefit of being ecofriendly; however, some issue related to their color changing over time can arise. DES, in addition to having an inhibitory effect when combined with Preventol RL50, has been found to be effective in improving the mechanical properties of paper, which also affect biodeterioration. This is an example of the many possibilities of intervention that can be formulated and combined in a suitable way to test, case-by-case, different compounds of various origins.

This review conducted a critical assessment of the evidence regarding the biocidal properties of natural substances in the context of stone material preservation. In some cases, encouraging results have been observed when testing several natural substances, such as various essential oils and other non-harmful compounds, as biocidal agents on stone materials. Moreover, for stone materials, essential oils seem to be promising, although long-term observations should be carried out in order to properly understand the behavior. The performance of nanoparticles used as bacterial inhibitors has to be balanced with the recently emerging issues related to the dispersion of nanoparticles in the environment.

The issue is widely open considering the great heterogeneity of the artworks needing to be preserved, as well as the complexity of the degradation phenomena and the factors influencing them. Moreover, it is worth underlining the great variety of biodeteriogens, their strong adaptation skills with respect to the surrounding environment, and, consequently, their increasing resistance to intervention techniques over time. All this context means that the research around biodeterioration, restoration efforts, and the conservation of cultural heritage is constantly evolving.

In the future, it would be reasonable to improve the classification of the microorganisms present on paper and stone objects, investigating their metabolic functions and their close connection with the environment. Moreover, knowledge of the processes regulating biofilm composition, growth, and activities could contribute to identifying or formulating new biocontrol agents able to be, at once, efficient against biodeteriogens and preservative of the safety of other organisms, “from the micro to the macro”, such as the operators and end-users of cultural assets. Finally, it is worth noting that an experimental approach, with a case-to-case evaluation of the materials involved (whether stone or paper), the surrounding environmental conditions, and probable past restorative interventions, represents a most effective and always-current strategy.