Lakhasly

1.However, reports about the application of EOs in cultural heritage conservation, as a valid alternative to traditional biocides, are still very limited to in vitro applications and need an application "on-field" to test the effectiveness, the proper way of application, and a monitoring strategy to define the efficiency over time. In vitro analysis is limited as biofilms are known to have different biological properties on stone surfaces than in suspension [10]. In addition, other factors such as the surface of application, the environment, and the weather could influence the effectiveness of essential oils in the field. The aim of this study was to test a combination of treatments, especially focusing on evaluating the effectiveness of selected EOs according to their biocidal activity as defined by the literature, through the application in a real case study [1,38-40]. Pure EOs and mix of EOs were compared to a conventional product based on QACs and two commercially green products based on EOs, which were taken as references. The monitoring of their effectiveness was assessed when applied on a white and black mosaic in the XIX room of "Insula delle Muse" in the Archaeological Park of Ostia Antica (Rome).The biocidal effect of many EOs has already been tested, such as for: Origanum vulgare, Rosmarinus officinalis, Lavandula angustifolia, Thymus vulgaris, Allium sativum, Pimpinella anisum, Eugenia caryophyllata, Calamintha nepeta, Cinnamum zeylanicum, Carum copticum, S. aromaticum, Citrus sinensis, Melaleuca alternifola, Cuminum cyminum, Eucalyptus globulus etc.In addition, Tabata and colleagues [14] reported Pseudomonas species to be responsible for the degradation of chloride compounds, contained in QACs, since microor- ganism consortia can feed on carbon sources from organic residuals contained in QACs, thus encouraging recolonization.Traditionally, the biocides derive from chemical products including acids, pyridines, quaternary ammo- nium salts, and organometallic compounds, with benzalkonium chloride, permethrin, and sodium fluoride [1] that are dangerous for human health and for the environment [9].This was apparent in the reported case study, in the Cave of Lascaux (France), where the fungus Fusarium solani during repeated treatments gained resistance, hence favoring the spread of other microorganisms (e.g., Ochroconis lascauxensis, Ralstonia spp.Stupar and colleagues (2014) [37], applying the biocide activity of Origanum vulgare, Rosmarinus officinalis, and Lavandula angustifolia EOs, against fungal strains isolated from different artifacts, discovered that O. vulgare showed the strongest inhibitory response.Ketones are the main constituents of sage oil (thujone, camphor) and peppermint oils (menthone, carvone), while fennel, eucalyptus and rosemary oils are rich in ethers: an ethole and 1,8-cineole, respectively.According to the colonizing organisms and the characteristics of the inter- ested area (surfaces, cracks, or pores), the entity of the damage changes, thus varying from exterior damage to irreversible disintegration of the inner substrate [4].EO configuration depends on many factors including the genotype (species, cultivation and ecotype), the ecological factors (geographic origin, climate, and soil characteristics), and processing techniques [22].EO activity in affecting microbial growth takes place through different pathways: indeed, EOs inhibit growth by affecting cytoplasmic membrane integrity, influencing cellular metabolism, affecting enzymatic activity, and impacting protein synthesis [13,30-32].Veneranda et al. [1] analyzed 10 different EO constituents that ratified thymol (Thyme sp. EO), eugenol (Clove sp. EO) and cinnamaldehyde (Cinnamon sp. EO) as the best enduring inhibitors.Department of Biology, Ecology and Earth Science DIBEST, University of Calabria, Via Pietro Bucci, * Correspondence: [email protected] Methods Protoc.2022, 5, 37.[1,16,29,33-36].

1. Introduction
   Bio-colonization is one of the main problems affecting the cultural heritage sector [1]. This phenomenon takes place on structures exposed to an environment characterized by specific local conditions, such as high moisture, high salinity, and abundance of organic nutrients [2,3]. According to the colonizing organisms and the characteristics of the inter- ested area (surfaces, cracks, or pores), the entity of the damage changes, thus varying from exterior damage to irreversible disintegration of the inner substrate [4].
   Department of Biology, Ecology and Earth Science DIBEST, University of Calabria, Via Pietro Bucci, * Correspondence: [email protected]
   Methods Protoc. 2022, 5, 37. https://doi.org/10.3390/mps5030037 https://www.mdpi.com/journal/mps

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Therefore, research on the conservation of cultural heritage, as one of the main ob- jectives, has to explore novel solutions aimed at removing and controlling the so-called biodeteriogens [5–8].
Currently, the strategy to contrast biodeterioration is based on the application of synthetic biocidal products, followed by a cleaning procedure to mechanically remove the remnants of the biofilms to prevent and/or hinder biodeterioration. Traditionally, the biocides derive from chemical products including acids, pyridines, quaternary ammo- nium salts, and organometallic compounds, with benzalkonium chloride, permethrin, and sodium fluoride [1] that are dangerous for human health and for the environment [9].
Modern commercial products were developed from quaternary ammonium com- pounds (QACs): Algophase and Preventol®RI50. Despite all of them possessing moderate toxicity, they show short-time effectiveness. Furthermore, QACs may be harmful not only for the operator but even for the environment [10–12]. Moreover, several compounds are not biodegradable and may cause uncontrollable contamination near the area of ap- plication [13]. In addition, Tabata and colleagues [14] reported Pseudomonas species to be responsible for the degradation of chloride compounds, contained in QACs, since microor- ganism consortia can feed on carbon sources from organic residuals contained in QACs, thus encouraging recolonization. Another issue occurs in repetitive QAC treatments, the onset of resistance of treated biocolonizers. This was apparent in the reported case study, in the Cave of Lascaux (France), where the fungus Fusarium solani during repeated treatments gained resistance, hence favoring the spread of other microorganisms (e.g., Ochroconis lascauxensis, Ralstonia spp. and Pseudomonas spp.) [15].
As a consequence, some of the most effective biocidal products have been recently banned due to their toxicity, with the result that a biocide that is effective and completely safe has not yet been developed. During the last decades the need to find environmental and human eco-friendly products has increased [16–20] with the focus of recent studies on the promising features of natural biocides, such as plant extracts or essential oils (EOs).
EOs are a complex mixture of organic volatile compounds that can be obtained from plants by hydro- or steam distillation [16]. Mainly characterized by terpenes, EOs include hydrocarbons, alcohols, ethers, aldehydes, ketones, and esters [21]. EO configuration depends on many factors including the genotype (species, cultivation and ecotype), the ecological factors (geographic origin, climate, and soil characteristics), and processing techniques [22]. These factors are responsible for the flavor and fragrance of aromatic plants and the antimicrobial activity [23]. Due to their composition, EOs possess a wide and diversified range of properties and are exploited in numerous fields including traditional medicine (used as an alternative to the conventional antibiotics), food (to protect and control rancidity), cosmetics (having antioxidant effects), and pharmaceuticals [22–29]. They pro- duce a wide range of secondary metabolites with antibacterial, antioxidant, antimicrobial, antiviral, and anti-inflammatory properties.
Several researches have demonstrated that EOs efficiently contrast the growth of diverse strains of fungi and bacteria. EO activity in affecting microbial growth takes place through different pathways: indeed, EOs inhibit growth by affecting cytoplasmic membrane integrity, influencing cellular metabolism, affecting enzymatic activity, and impacting protein synthesis [13,30–32].
The biocidal effect of many EOs has already been tested, such as for: Origanum vulgare, Rosmarinus officinalis, Lavandula angustifolia, Thymus vulgaris, Allium sativum, Pimpinella anisum, Eugenia caryophyllata, Calamintha nepeta, Cinnamum zeylanicum, Carum copticum, S. aromaticum, Citrus sinensis, Melaleuca alternifola, Cuminum cyminum, Eucalyptus globulus etc. [1,16,29,33–36].
EOs oils obtained fromthyme and oregano plants have been reported to possess the most effective biocidal activity. Indeed, these EOs are mainly characterized by phenolic com- pounds, respectively thymol and carvacrol [30]. Thymol and carvacrol are monoterpenoid phenols that exhibit strong antimicrobial, anti-inflammatory, and antioxidant properties; another phenolic compound that shows these properties is eugenol, present in clove oil. On the contrary, EOs characterized by alcoholic compounds, such as tea tree oil (terpinen-4-ol

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and α-terpineol) express a slightly less effective action against microorganisms, showing antiseptic, cytotoxic, antifungal, antiviral and anti-inflammatory action and mainly used in the pharmaceutical and cosmetic industry [16,22]. Geranium oil (geraniol and citronellol), peppermint oil (menthol), and lavender oil (linalool and linalooll acetate) are members of this group. Rakotonirainy et al. [35], demonstrated the high antifungal activity of linalool and linalool acetate, present in Lavandula angustifolia against fungal strains isolated from library and archive storage areas. Ketones are the main constituents of sage oil (thujone, camphor) and peppermint oils (menthone, carvone), while fennel, eucalyptus and rosemary oils are rich in ethers: an ethole and 1,8-cineole, respectively. Rosemary extract, moreover, contains carnosic acid, as carnosol and rosmarinic which present antimicrobial, antioxidant, and anti-inflammatory properties.
Stupar and colleagues (2014) [37], applying the biocide activity of Origanum vulgare, Rosmarinus officinalis, and Lavandula angustifolia EOs, against fungal strains isolated from different artifacts, discovered that O. vulgare showed the strongest inhibitory response. Veneranda et al. [1] analyzed 10 different EO constituents that ratified thymol (Thyme sp. EO), eugenol (Clove sp. EO) and cinnamaldehyde (Cinnamon sp. EO) as the best enduring inhibitors.
However, reports about the application of EOs in cultural heritage conservation, as a valid alternative to traditional biocides, are still very limited to in vitro applications and need an application “on-field” to test the effectiveness, the proper way of application, and a monitoring strategy to define the efficiency over time. In vitro analysis is limited as biofilms are known to have different biological properties on stone surfaces than in suspension [10]. In addition, other factors such as the surface of application, the environment, and the weather could influence the effectiveness of essential oils in the field.
The aim of this study was to test a combination of treatments, especially focusing on evaluating the effectiveness of selected EOs according to their biocidal activity as defined by the literature, through the application in a real case study [1,38–40].
Pure EOs and mix of EOs were compared to a conventional product based on QACs and two commercially green products based on EOs, which were taken as references. The monitoring of their effectiveness was assessed when applied on a white and black mosaic in the XIX room of “Insula delle Muse” in the Archaeological Park of Ostia Antica (Rome).