# **The Role of Fungi in Biodeterioration of Cultural Heritage New Insights for Their Control**

Edited by Filomena De Leo and Daniela Isola Printed Edition of the Special Issue Published in *Applied Sciences*

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## **The Role of Fungi in Biodeterioration of Cultural Heritage: New Insights for Their Control**

## **The Role of Fungi in Biodeterioration of Cultural Heritage: New Insights for Their Control**

Editors

**Filomena De Leo Daniela Isola**

MDPI • Basel • Beijing • Wuhan • Barcelona • Belgrade • Manchester • Tokyo • Cluj • Tianjin

*Editors* Filomena De Leo University of Messina Italy

Daniela Isola University of Tuscia Italy

*Editorial Office* MDPI St. Alban-Anlage 66 4052 Basel, Switzerland

This is a reprint of articles from the Special Issue published online in the open access journal *Applied Sciences* (ISSN 2076-3417) (available at: https://www.mdpi.com/journal/applsci/special i ssues/Fungi Biodeterioration Control).

For citation purposes, cite each article independently as indicated on the article page online and as indicated below:

LastName, A.A.; LastName, B.B.; LastName, C.C. Article Title. *Journal Name* **Year**, *Volume Number*, Page Range.

**ISBN 978-3-0365-6495-1 (Hbk) ISBN 978-3-0365-6496-8 (PDF)**

Cover image courtesy of Daniela Isola

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## **Contents**

## **About the Editors**

#### **Filomena De Leo**

Prof. Filomena De Leo graduated in Biological Sciences, specialized in "Applied Microbiology" and obtained a Postgraduate Degree (PhD) in Microbial Biotechnologies. She is currently an Associate Professor of Microbiology at the Department of Chemical, Biological, Pharmaceutical and Environmental Sciences of the University of Messina, Italy; professor of the "General Microbiology", degree Course CdL in "Biotechnology", and professor of the "Nutrimicrobiomic" degree Course CdLM in "Biology of Health, Applied Technologies and Nutrition". She is a member of the Council of PhD in "Applied Biology and Experimental Medicine" of the University of Messina, Italy. She is a member of the Reference Scientific Community of the "Stazione Zoologica Anton Dohrn", Naples, Italy (http://www.szn.it/index.php/it/chi-siamo/comunita-scientifica-di-riferimento, accessed on 7 October 2022). She is a member of the University Research Center for the study of extreme environments and extremophiles (CUR AEE) and of the National Consortium for Marine Sciences (CoNISMa). Her main interests are the study of the biodiversity, ecology and genetic diversity of bacterial and fungal populations involved in the biodeterioration of cultural heritage (especially stone monuments), and more recently the diversity of halophilic micro-organisms in deep-sea environments. One of her particular focuses is the taxonomy of black fungi, which are among the most harmful micro-organisms causing the biodeterioration of stone monuments. She has identified two new fungal species and four new bacterial species isolated from monuments. She has participated in numerous national and international research projects both as the scientist responsible and as a member of the Research Unit. Prof Filomena De Leo has published 51 articles in journals, 16 monographs (or book chapters), 19 Proceedings of International Congress and 54 posters and oral communications. Her publications are visible on "Researchgate" at https://www.researchgate.net/profile/Filomena De Leo, accessed on 7 October 2022.

#### **Daniela Isola**

Prof. Daniela Isola is a contract Professor of Botany Applied to Cultural Heritage at the University of Tuscia. She graduated in Biological Sciences at the University of Cagliari and received postgraduate specializations in Neuropharmacology and Environmental Hygiene (University of Cagliari), and Bioinformatics (La Sapienza University of Rome). She obtained a PhD in Biological and Biochemical Evolution at the University of Tuscia. Since then, she has focused on fungi from extreme environments and "black fungi". Her main interests are the biodeterioration of cultural heritage and biodegradation of aromatic hydrocarbons. Particular attention has been paid to the identification, diversity, phylogeny, and metabolic skills of the involved organisms, and low-impact methods to control biodeterioration. She is the author of 4 genera and 11 fungal species, more than a thousand sequences deposited in GenBank (covering bacteria, fungi, viruses and higher plants) and about 40 scientific papers, with an H-index of 22.

## *Editorial* **The Role of Fungi in Biodeterioration of Cultural Heritage: New Insights for Their Control**

**Filomena De Leo 1 and Daniela Isola 2,\***

#### **Introduction**

Fungi are nature's major decomposers, and they play an essential role in biogeochemical cycles. Thanks to their wide enzyme repository, they can break down organic matter and hard-to-degrade biopolymers such as lignin, cellulose, and chitin. They also produce several inorganic and organic metabolites, organic acids, and chelating agents of fundamental importance in geological processes, leading to chemical transformations at local and global scales [1]. Though frequently overlooked, fungi are ubiquitous and can be found even in environments that are not so suitable for life due to their ability to metabolize xenobiotics [1]. Therefore, is not surprising that fungi are considered the most detrimental threat to both indoor and outdoor artefacts of historical and/or artistic value; their presence can lead to physical, chemical, and aesthetical damages [2,3].

Cultural heritage conservation refers to the measures, protocols, and methods taken to extend the life of artefacts, monuments, and sites. Biological deterioration of historical and artistic artefacts has gained significant attention in recent decades, evidencing how limited our knowledge of biodeteriogens is when we consider the wide range of heritage materials used and the environments in which they are kept. In conservation practices, precise and reliable identification of biodeteriogens is useful; however, further research is needed to assess their ecological requirements and metabolic profiles. Indeed, the environmental conditions favoring various taxonomic groups, and their limited lives, are essential in assessing the risk for artefacts and designing indirect (preventive) and direct (biocide treatments) control methods.

In this Special Issue, we present seven study cases and two reviews addressing the deterioration of different materials, such as stone, textiles, wood, and scientific instruments, exposed to outdoor, confined, or semi-confined environments.

From the UNESCO heritage sites of Cuma, Ercolano, Nola, Oplonti, and Pompei, Petraretti and colleagues [4] performed a deep sampling on frescoes, marble, mortars, plaster, and tuff to assess the biodiversity of the culturable fungal fraction. In addition to isolating fungal strains with detrimental potential, the authors suggest the importance of collecting detrimental fungi for future research on cultural heritage.

The study performed by Isola and colleagues focused on a selection of black meristematic fungal strains belonging to the Culture Collection of Fungi from Extreme Environments (CCFEE, Viterbo, Italy). All strains were isolated from marble monuments of the Bonaria Cemetery (Cagliari, Italy) and, using plate assays, the main ecological and metabolic traits and tolerance to traditional biocides were assessed [5]. The preservation of traditional Romanian clothes was the focus of the Ilies study group [6]. In detail, different essential oils were applied to different materials comprising coats (e.g., wool, cotton, leather) to control the detrimental fungal species. Other artefacts from museums include optical lenses

**Citation:** De Leo, F.; Isola, D. The Role of Fungi in Biodeterioration of Cultural Heritage: New Insights for Their Control. *Appl. Sci.* **2022**, *12*, 10490. https://doi.org/10.3390/ app122010490

Received: 8 October 2022 Accepted: 13 October 2022 Published: 18 October 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

**\*** Correspondence<EMAIL_ADDRESS>
studied by Ngo and co-workers [7]. This study evidenced the surface corrosion produced by fungi, here favored by the high humidity which is typical of North Vietnam's climate.

The study performed by Jurado and colleagues is of interest, instead, for the conservation of show caves [8]. In this study, the microclimatological and aerobiological monitoring gave insight into the seasonal dynamics of airborne fungi in the Nerja cave (Spain).

Barboux and colleagues [9] proposed an alternative instrumental method (FTIR) for detecting *Serpula lacrymans*; this method could be especially useful for protecting wooden structures from the dry rot fungi which are prevalent in indoor environments, especially in the holarctic regions. The study performed by Chlebicki and colleagues was devoted to testing the in vitro inhibitory effect exerted by bioactive ions exposed to galvanic systems with changing electrode distance [10].

This Special Issue also presents two reviews. The study performed by Zucconi and colleagues addressing wall paintings accounts for 60 years of fungal biodeterioration reports [11]. An exhaustive list of identified fungi was produced. However, the technical and methodological improvements that occurred within that period evidenced some limitations in the fungal identification (mainly morphological) and culturingprotocols, posing the basis for future improvements.

De Leo and colleague reviewed 109 papers published within the last 30 years to critically summarize the current knowledge on black fungi associated with the biodeterioration of stone monuments with a look at control methods and future perspectives [12].

This Special Issue offers an insight into the vast world of fungal deteriogens. More research is necessary in this field; however, with the incoming improvements in instrumental technologies, molecular diagnoses, and sustainable control methods, the research prospects in this field are exciting.

**Author Contributions:** Conceptualization, D.I. and F.D.L.; writing—original draft preparation, D.I.; writing—review and editing, D.I. and F.D.L. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Acknowledgments:** We wish to thank the editorial team of Applied Sciences for the support given during this editorial project.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

#### **Short Biography of Authors**

**Prof. Filomena De Leo** graduated in Biological Sciences, specialized in "Applied Microbiology" and Post graduated Degree (PhD) in Microbial Biotechnologies. Currently is Associate Professor of Microbiology at the Department of Chemical, Biological, Pharmaceutical and Environmental Sciences of the University of Messina, Italy: professor of "General Microbiology", degree Course CdL in "Biotechnology", and professor of "Nutrimicrobiomic" degree Course CdLM in "Biology of Health, Applied Technologies and Nutrition". She is a member of the Council of PhD in "Applied Biology and Experimental Medicine" of the University of Messina, Italy. She is member of the Reference Scientific Community of the "Stazione Zoologica Anton Dohrn" Naples, Italy (http://www.szn.it/index.php/it/chi-siamo/comunita-scientifica-di-riferimento, accessed on 7 October 2022). She is member of the University Research Center for the study of extreme environments and extremophiles (CUR AEE) and she is member of the National Consortium for Marine Sciences (CoNISMa). Her main interest is the study of biodiversity, ecology and genetic diversity of bacterial and fungal population involved in the biodeterioration of Cultural Heritage (especially stone monuments) and more recently in the diversity of halophilic microorganisms in deep sea environments. One special task is the taxonomy of black fungi that are among the most harmful microorganisms causing biodeterioration of stone monuments. She described two new fungal species and four new bacterial species isolated from monuments. She has participated in numerous National and International research projects both as Scientific Responsible and as a member of the Research Unit. Prof Filomena De Leo pubblished 51 articles in Journals, 16 monographs (or book chapter), 19 Proceedings of International Congress and 54 Poster and oral communication. The pubblications are visible on "Researchgate" link https://www.researchgate.net/profile/Filomena\_De\_Leo, accessed on 7 October 2022.

**Prof. Daniela Isola** is a contract Professor of Botany Applied to Cultural Heritage at the University of Tuscia. She graduated in Biological Sciences at the University of Cagliari and received postgraduate specializations in Neuropharmacology and Environmental Hygiene (University of Cagliari), and Bioinformatics (La Sapienza University of Rome). She obtained PhD in Biological and Biochemical Evolution at the University of Tuscia. Since then, she has focused on fungi from extreme environments and "black fungi". Her main interests are the biodeterioration of cultural heritage and biodegradation of aromatic hydrocarbons. Particular attention has been paid to the identification, diversity, phylogeny, metabolic skills of the involved organisms, and low-impact methods to control biodeterioration. She is the author of 4 genera and 11 fungal species, more than a thousand of sequences deposited in GenBank (covering bacteria, fungi, viruses and higher plants) and about 40 scientific papers with an H-index of 22.

## *Article* **Community Composition and Ex Situ Cultivation of Fungi Associated with UNESCO Heritage Monuments in the Bay of Naples**

**Mariagioia Petraretti 1,\*, Karl J. Duffy 1, Angelo Del Mondo 1,2, Antonino Pollio 1 and Antonino De Natale 1**

**Abstract:** The Bay of Naples, Italy, is renowned for its archaeological heritage. However, this heritage is threatened by the combination of weathering and the biological activity of microorganisms. Fungi are among the major agents of microbial deterioration of cultural heritage since they can cause cracks and lesions in monuments due to the penetrating force of their hyphae. Such biodeterioration may weaken the stone structures and threaten the longevity of these culturally important monuments. To address this, we collected, identified, and maintained in culture filamentous fungi that colonize the external surface of monuments at five important archaeological sites near Naples, namely Cuma, Ercolano, Nola, Oplonti, and Pompei. We isolated a total of 27 fungal taxa, all of which can be cultivated in the laboratory, and form a part of our reference collection. Many of the described fungal taxa we found belong to groups that are involved in stone biodeterioration and can thus be considered as model organisms for in vitro studies. These results emphasize the importance of identifying and cultivating fungal stock cultures for non-invasive studies on biodeterioration. Our newly developed reference collection represents a useful resource that is available to other researchers to rapidly identify potentially hazardous fungi on other monuments.

**Keywords:** fungi; ex situ collection; biodeterioration; biodegradation; cultural heritage

#### **1. Introduction**

Fungi play an important role in the deterioration of buildings. Deterioration caused by fungal colonization involves both physical and chemical damage of stone surfaces, and in most cases, they take place simultaneously [1]. Physical damage is related to the ability of fungal hyphae to penetrate into the substratum, where pores and fissures provide a useful microhabitat for fungal growth. The pressure exerted by fungal growth leads to further damage due to cell turgor pressure and exopolysaccharide formation that, in addition to fungal adhesion on stone surfaces, increases mechanical pressure [2]. Moreover, chemical damage occurs due to byproducts of fungal metabolism that leads to corrosion and discoloration of stone surfaces. Fungi are able to excrete a large variety of organic acids that act as metal-chelators [3] and mediate the precipitation of secondary minerals produced through the reaction of anions from excreted acids with cations from the stone. The formation of secondary minerals, such as carbonates, oxalates, and phosphates, can cause blistering, scaling, granular disintegration, and flaking or "spalling" of outer layers, leading to stone decay [4]. There is a close relationship between material and colonizing organisms [5]; indeed, the degree of fungal colonization of a stone surface also depends on the structure, wetness, and chemical and mineralogical compositions of the substrata as well as environmental conditions [6,7]. Different lithotypes, e.g., brick, limestone, marble,

**Citation:** Petraretti, M.; Duffy, K.J.; Del Mondo, A.; Pollio, A.; De Natale, A. Community Composition and Ex Situ Cultivation of Fungi Associated with UNESCO Heritage Monuments in the Bay of Naples. *Appl. Sci.* **2021**, *11*, 4327. https://doi.org/10.3390/ app11104327

Academic Editors: Filomena De Leo and Daniela Isola

Received: 12 March 2021 Accepted: 6 May 2021 Published: 11 May 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

tuff, and porphyry, provide a diverse range of substrates that fungi may use to acquire nutrients and grow.

Despite the large number of studies in the literature in which damage to cultural heritage is directly associated with fungi colonization, the occurrence of fungi on cultural heritage monuments does not necessarily mean that these fungi cause the loss of chemical and physical properties of the substrate; indeed, filamentous fungi as well as lichens could protect colonized materials, especially against environmental parameters [8], or they could bear no influence on the material properties. The multifaceted role of fungi in biodeterioration can be effectively assessed on the basis of preliminary in vitro tests, particularly recommended in the issues of monument protection, that require ex situ conservation strategies for fungal strains isolated from monuments. Ex situ collections may significantly improve our knowledge of the role of fungi in stone cultural heritage biodeterioration, providing the basis for an appropriate and effective maintenance and restoration strategy. The importance of maintaining a broad range of taxa in collections for ex situ conservation accessible to researchers prompted us to perform a survey campaign along the archaeological remains of Campania, Italy. Campania hosts a large number of works of art and monuments made of different stone materials, spanning the last three thousand years. Despite this unique cultural heritage, a deep sampling aimed to assess the biodiversity of cultivable fungi in these historical areas has never been conducted.

Using a combination of microscopical, genetic, and culture techniques, here we describe the taxonomic diversity of fungi that occur in the UNESCO heritage sites of Cuma, Ercolano, Nola, Oplonti, and Pompei, with the overall aim of developing an ex situ collection of fungal strains from these archeological sites.

#### **2. Materials and Methods**

#### *2.1. Sampling*

The sampling campaign in this study was carried out in March 2018 at some of the most important cultural heritage sites in Campania, namely the Sibyl Caves in Cuma, the Suburban Baths in Ercolano, the Roman Amphitheater in Nola, the House of Poppea in Oplonti, and the House of Fauno and the House of Castricio in Pompei (Figure 1).

**Figure 1.** Location of the sampling site of UNESCO heritage monuments in the bay of Naples, Campania, Italy.

At every site, we measured ecological parameters, e.g., temperature and relative humidity, using a thermo-hygrometer (model HI 9564, Hanna® Instruments, Smithfield, RI, USA) and light intensity using a Climalux N light meter (Laboratori di Strumentazione

Industriale S.p.a., Milan, Italy). We measured pH on substrates at sampling points using a pH test paper strip. All the environmental parameters are shown in Table 1. The sampling points were chosen on the basis of the visibility of the fungal presence on the surface. Biofilm samples were taken by gently scraping the walls of the sampling sites with a sterile scalpel and adhesive tape strips were also used as a non-destructive sampling method [9]. The materials were deposited into sterile vials, until arrival at the laboratory.

**Table 1.** Values of environmental parameters (light, pH, relative humidity, and temperature) at each sampling site.

#### *2.2. Confocal Laser Scanning Microscope Analysis*

The recorded adhesive tape samples were cut into small sections (approximately 1 × 1 cm, [9]), placed on a glass slide, and observed on a Confocal Laser Scanning Microscope (CLSM), Zeiss LSM 700 (Carl Zeiss AG, Munich, Germany, using the software Zen 2011), by capturing images with a 63× water immersion objective. Images were acquired in three channels simultaneously: the red channel was used to discriminate phototrophs containing autofluorescence pigments (chlorophyll a and phycobilins), with excitation beams at 488 and 639 nm and emissions at 590–800 nm; the green channel was used to detect extrapolymeric matrix (EPS) using concanavalin-A with Alexa 488, with the excitation beams at 488 nm and emissions at 553–636 nm; and calcofluor-white was used to evidence the bacteria and hyphae with the excitation beams at 405 and 488 nm and emissions at 415–506 nm (blue channel) [10].

#### *2.3. Isolation of Fungal Strains*

After the sampling campaign, samples were inoculated on agar medium, such as Potato Dextrose Agar (PDA) prepared according to Samson et al. [11], Bold's Basal Medium (BBM) [12] added to sucrose (12 g/L) according to Jeger et al. [13], and Malt-Yeast Extract-Sucrose Agar (MEA, Difco™) prepared according to Skaar and Stenwig [14]. Incubation was carried out at 22 ± 2 ◦C for 30 days. At the end of the incubation period, enumeration of microorganisms as cfu/g of sample was carried out and the several mycelia obtained were isolated with the aid of a stereomicroscope. Afterwards, fungi were separately cultivated on PDA and finally observed with a stereomicroscope.

#### *2.4. Identification of Fungal Isolates*

Fungal strains were identified through a polyphasic approach that is an integrated approach of identification based on morphological and molecular features of microorganisms [15]. According to Barnett and Hunter [16] and Fassatiovà and Ellis [17], the morphological identification of fungi was based on the macroscopic features of colonies growing on agar plates and the micromorphology of the reproductive structure. The morphological analysis was then confirmed by molecular analysis. For each fungal isolate, the following procedure was applied: DNA was extracted with a modified DNA extraction

protocol [18] and used for a Polymerase Chain Reaction with primers targeting the internal transcribed spacer region (ITS) (primer forward, 5 -TCCGTAGGTGAACCTGCGG-3 ; primer reverse, 5 -TTCAAAGATTCGATGATTCAC-3 ). The ITS is the region spanning ITS1, 5.8S rRNA, and ITS2 was recently elected to be the universal barcode marker for fungi [19]. This DNA region has enough gaps between the intraspecific and interspecific variation across the kingdom Fungi and has been shown to have a high amplification success rate in various fungal taxa, e.g., it can discriminate the majority of species in Mucorales [20]. The barcode region together with a well-curated database of DNA sequences may constitute a reliable and fast tool for culture collection in the task of providing certification of fungal cultures. The amplification reaction was carried out in a reaction volume of 25 μL containing 2.5 μL of 10× reaction buffer, 1.5 μL of MgCl2, 2 μL of dNTP, 1.5 μL of each of the primers, and 0.2 μL of Taq polymerase (EconoTaq, Lucigen, Middleton, WI, USA). An amount of DNA, approximately 100 ng, was added to each reaction mixture in a PCR tube. The profile used was the same described by Del Mondo 2017 [21]. Amplification was run in an Applied Biosystem 2720 thermal cycler. The amplification product was then evaluated on 1.2% (*w/v*) agarose gel in an electrophoretic purified with a QIAquick® PCR Purification kit (Qiagen Inc, Valencia, CA, USA). The sequence reaction was obtained with the BigDye Terminator Cycle Sequencing technology (Applied Biosystems, Foster City, CA, USA), purified automatically using the Agencourt CleanSEQ Dye terminator removal Kit (Agencourt Bioscience Corporation, 500 Cummins Center, Suite 2450, Beverly, MA, USA) and a robotic station Biomek FX (Beckman Coulter, Fullerton, CA, USA). The product was analyzed on an Automated Capillary Electrophoresis Sequencer 3730 DNA Analyzer (Applied Biosystems). The amplification primers were used as the sequencing primers. The obtained sequence was searched for in BLAST version 2.0 (National Center for Biotechnology Information databases) and identified. The ITS sequences obtained in this study have been deposited in GenBank (the accession numbers are listed in Table 2).

**Table 2.** Identification of the fungal species complex level based on ITS sequences with the description of their sampling site and lithic substrate. ACUF Collection Codes and Gene Bank Accession numbers are given for each strain.

#### *2.5. Fungal Preservation for Ex Situ Conservation*

In according to the World Federation for Culture Collection Guidelines, more than one method was applied for each fungal strain for successful preservation. Our fungal strains are stored by different methods: (a) PDA on Petri dishes at a temperature range of between 22 ◦C and 25 ◦C in darkness; (b) in a corked glass tube with sterilized water at room temperature [22]; and (c) at −80 ◦C in glycerol (selected strains only). This method, namely cryopreservation together with freeze drying, is considered to be a long-term preservation method [23]. To maintain fungi in a viable state, to evaluate purity, and to avoid devitalization, monthly checking and refreshment of cultures were performed. All the fungal strains are maintained in the Algal Culture Collection (ACUF) at the Department of Biology, University of Naples Federico II, Italy. This collection, traditionally devoted to the maintenance of aero-terrestrial microalgae and cyanobacteria [24], has been enriched with a special section devoted to the maintenance of fungal strains isolated directly from archeological sites in Campania. Each strain is included in a private database with all the information regarding sampling sites, origin substrate, data on collection, ecological notes, cultivation and maintenance methods, phenotypic characteristics, and genomic analysis. In order to maintain the safety of the data associated with each of the strains preserved in the collection, all computer files are duplicated and kept in a separate area. Furthermore, we deposited our isolates at the Mycotheca Universitatis Taurinensis, Turin, Italy (MUT), a renowned collection specialized in fungal preservation.

#### **3. Results**

#### *3.1. Description of Damage and Substrate Change*

Biological growth on stone can result in changes in surface color and structure depending on the identity of the organism and their growth and behavior. At our sampling sites, the biological colonization on stone surfaces assumed the forms of epilithic formations with a patina aspect. In particular, these organisms formed a subaerial biofilm, which is a type of biofilm that occurs at the atmosphere–rock interface. This type of biofilm has been frequently reported in the literature on hypogean monuments, such as catacombs [25], and on walls, statues, and wetlands. These formations may have a colored patinas aspect, depending on the type of biocenosis and of the growth phase of the prevailing species. As

shown in Figure 2b,d,e, the stone surface appears with a green and greenish stain, probably due to the presence of organic pigments (e.g., chlorophylls, carotenoids, melanins) [26]. In Figure 2h,k,n, the stone surface appears with a black stain and this is related to the mixed association of different fungal groups.

**Figure 2.** Archeological sites: Sibyl Caves in Cuma (**a**), Suburban Baths in Ercolano (**d**), the Roman Amphitheater in Nola (**g**), the House of Poppea in Oplonti (**j**), and the House of Fauno (**m**) and the House of Castricio (**p**) in Pompei; the visible alteration at the same sites (**b**,**e**,**h**,**k**,**n**,**q**); the recorded adhesive tape samples observed on the CLSM (**c**,**f**,**i**,**l**,**o**,**r**; scale bar, 50 μm).

#### *3.2. Confocal Laser Microscopy*

All the samples analyzed by CLSM revealed that many cells contained chlorophyll and phycobilin (red auto-fluorescence), which were ascribed to algae and cyanobacteria and polysaccharide polymers (e.g., cellulose and chitin) in their cell walls (blue color), which were ascribed to fungi (Figure 2c,f,i,l,o,r).

#### *3.3. Molecular Identifications*

Table 2 shows the identification of the isolated fungal species retrieved from the sampled UNESCO monuments, together with sites and source sampled as well as the collection code linked to the fungal strains and the GenBank accession numbers of the obtained sequences.

A total of 18 fungal taxa, belonging to 3 different Classes, 5 different Orders, and 10 different Families, were obtained (Figure 3) and kept in culture.

**Figure 3.** The relative abundance of fungal isolated according to (**A**) classes, (**B**) orders, and (**C**) families.

Overall, the most common genera were *Aspergillus* in Cuma (60%), *Fusarium* in Ercolano (42.86%), *Penicillium* in Nola (41.67%), *Fusarium* in Oplonti (45.45%), *Penicillium* (60%) in Pompei, Fauno, and *Aspergillus* (50%) in Pompei, Castricio (Figure 4).

**Figure 4.** The relative prevalence of fungal genera found in: Sibyl Caves in Cuma (**A**), Suburban Baths in Ercolano (**B**), the Roman Amphitheater in Nola (**C**), the House of Poppea in Oplonti (**D**), and the House of Fauno (**E**) and the House of Castricio (**F**) in Pompei.

#### **4. Discussion**

The protection of cultural heritage often involves the study of the bio-receptivity of building materials and the biodegradation potential of microorganisms involved in deterioration, which can be primarily assessed through laboratory studies. In this work, a non-destructive method was used to sample microorganisms at UNESCO cultural heritage sites in Campania, Italy. In particular, we sampled using adhesive tape sampling coupled with microscopical analysis to identify the constituent microorganisms in biofilms of these monuments, which allows us to examine the existing relationships between the surface and the colonizing microorganisms [9]. The observations made using confocal light microscopy demonstrated fungal colonization in all the adhesive tape samples analyzed as a network of filamentous structures. Furthermore, we observed the presence of cyanobacteria and algae closely connected with filamentous structures, demonstrating that fungi actively colonize the rock as essential compounds of the biofilm sampled and not as contaminants. The isolation of fungi through culture-dependent methods, followed by maintenance of strains in culture, was aimed at obtaining quality-controlled isolates for further studies on biodeterioration processes and to develop innovative strategies for their control. For each strain, we collected data related to the substrate and environmental conditions at the sampling location and this information can be used to develop future laboratory experiments simulating specific environmental conditions under which these fungi can grow. Indeed, future perspectives are directed towards using our fungi as models to perform in vitro experiments for understanding the patterns of microbial colonization of

stone materials [21]. The fungal isolates in this study are widespread, frequently associated with soil particles and plant material, which is probably due to their broad tolerance to different environmental conditions and allows them to colonize a large array of terrestrial habitats [27]. Molecular identification of sampled strains was performed using the internal transcribed spacer (ITS) rDNA area, which is the most widely used marker for fungi [19]. Unfortunately, for many Ascomycota genera, such as *Penicillium* and *Aspergillus*, the ITS is not variable enough to allow for species-level identification [28]. Because of the limitations associated with the chosen molecular marker, herein we considered the species-complex level for an overview of fungal diversity on stone monuments. We recognize the necessity of further identification using taxon-specific markers (e.g., SSRs) for identifying isolates to species level as the collection grows. The genera isolated in our sampling include *Alternaria, Aspergillus*, *Cladosporium*, *Clonostachys*, *Curvularia, Fusarium, Lecanicillium, Neofusicoccum, Penicillium, Purpureocillium, Talaromyces,* and *Trichoderma,* which are known to be ubiquitous filamentous fungi of soil and are often airborne. Some of them were already described as colonizer, or occasionally pioneer, taxa of deteriorated monuments [29]. For most of these species, there is no representative strain of the wide range of morphology and physiology expressed within that species and therefore it is necessary to maintain a number of representative strains. Some collections, such as the CABI Bioscience Genetic Resource Collection, retain on average five strains for each species, but in some cases this number is not sufficient. This is the case for host specificity in the plant pathogen, such as *Fusarium oxysporum*, which has a large number of genetic variants [30]. Thus, ex situ conservation of microorganisms collected from monuments can ensure that all strains with their unique properties are preserved and maintained over time. In accordance with other studies, we observed that the genera most frequently identified as colonizers of several stone substrates are *Aspergillus*, *Fusarium,* and *Penicillium* [31]. The action of these fungi, included in the orders Capnodiales and Pleosporales, could lead to aesthetic alteration and biopitting of stone materials [32]. Moreover, some of the fungal isolates belonging to *Alternaria* section *Alternata*, *Cladosporium sp., Fusarium solani* species complex, and *Penicillium* section *Aspergilloides* are known to contribute to acidification and the dissolution of stone by excreting organic acids. In particular, oxalic acid secreted by fungi can dissolve limestone calcium carbonate, producing calcium oxalates, one of the most severe biodeterioration processes affecting limestone monuments [33]. In addition, recently it has been shown that the genus *Purpureocillium* may have halotolerant characteristics [34], which may further broaden the impact these fungi have on stone structures.

#### **5. Conclusions**

The collection and molecular identification of fungal strains and their associated ecological data, describing their site of sampling, type of substrate, and morphological diagnostic characteristics, represent a key resource for the development of biotechnological approaches devoted to the conservation of cultural heritage. The ex situ conservation of fungi sampled from bio-deteriorated environments can ensure that isolates are preserved to maintain their integrity and long-term survival. This is essential for future research on the preservation of historical monuments, including the ecological differentiation of fungal communities according to sampling sites and the production of desirable end products applicable for bioremediation.

**Author Contributions:** Conceptualization, M.P. and A.D.M.; methodology, M.P.; software, M.P. and A.D.M.; validation, A.D.M., A.P. and A.D.N.; data curation, M.P.; writing—original draft preparation, M.P.; writing—review and editing, K.J.D., A.D.N. and A.P.; supervision, A.P. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research received no external funding.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** Data are contained within the article.

**Acknowledgments:** The authors thank the Herculaneum Archaeological Park, the Pompei Archaeological Park, the Phlegrean Fields Archaeological Park, Soprintendenza Archeologia Belle Arti e Paesaggio della città metropolitana di Napoli, and the Società dei Naturalisti in Napoli (Naples, Italy) for the assistance with research. The authors gratefully thank Serena Di Lecce and Alessandra Di Leva for their valuable technical support.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Black Fungi and Stone Heritage Conservation: Ecological and Metabolic Assays for Evaluating Colonization Potential and Responses to Traditional Biocides**

**Daniela Isola 1,2,\*, Flavia Bartoli 2, Paola Meloni 3,4, Giulia Caneva 2 and Laura Zucconi 1**

**Abstract:** Identifying species involved in biodeterioration processes is helpful, however further effort is needed to assess their ecological requirements and actual activity. Black fungi (BF) represent one of the most underestimated threats to stone cultural heritage in the Mediterranean basin; they are difficult to kill or remove due to their ability to grow inside the rock and cope with several stresses. Despite this, little is known about BF and factors favoring their growth on stone surfaces. Eighteen BF species were here investigated for temperature and salt tolerance, and metabolic traits by plate assays. The relation between some highly damaged monuments and their BF settlers was assessed using X-ray diffraction analysis, mercury intrusion porosimetry, and SEM. The sensitiveness to four commonly used traditional biocides was also tested. All strains were able to grow within the range of 5–25 ◦C and in the presence of 3.5% NaCl. Instrumental analyses were fundamental in discovering the relation between halophilic strains and weathered marble sculptures. The acid, cellulase, esterase, and protease production recorded proved BF's potential to produce a chemical action on carbonate stones and likely affect other materials/historical artefacts. Besides, the use of carboxymethylcellulose and Tween 20 should be evaluated in restoration practice to prevent tertiary bioreceptivity. Agar diffusion tests helped identify the most resistant species to biocides, opening the perspective of its use as reference organisms in material testing procedures.

**Keywords:** benzalkonium chloride resistance; biodeterioration control; *Exophiala*; halophilic fungi; fungal acid production; *Knufia*; monuments salt weathering; QAC biocides; *Salinomyces thailandicus*; marble XRD; MIP investigations

#### **1. Introduction**

All materials exposed to outdoor conditions are inevitably subject to deterioration processes leading to biotic and abiotic alterations. Surface colonization and the related biodeterioration phenomena greatly vary depending on the substrate's bioreceptivity, macro- and microclimatic conditions (e.g., water availability, sun exposure, shading, and orientation), and nutrients supply [1–4].

Among the stone biodeteriogens, rock black fungi are considered one of the most remarkable issues for cultural heritage conservation, particularly in arid and semi-arid environments, where they live at their ecological optimum [5–7]. Black fungi (BF), also known as rock-inhabiting fungi (RIF), black yeasts, or microcolonial fungi (MCF), are a polyphyletic poikilotolerant morpho-ecological group with remarkable adaptations allowing them to cope with changes in extreme temperatures, drought, starvation, osmotic stress, and high solar and UV-radiation as it occurs on rock surfaces [8–12].

**Citation:** Isola, D.; Bartoli, F.; Meloni, P.; Caneva, G.; Zucconi, L. Black Fungi and Stone Heritage Conservation: Ecological and Metabolic Assays for Evaluating Colonization Potential and Responses to Traditional Biocides. *Appl. Sci.* **2022**, *12*, 2038. https:// doi.org/10.3390/app12042038

Academic Editor: Gyungsoon Park

Received: 27 January 2022 Accepted: 11 February 2022 Published: 16 February 2022

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2022 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

When colonizing rocks, BF can induce a chemical deterioration by secreting siderophorelike compounds, however the most relevant damages are believed to be due to a mechanical action. Hyphal morphology and the strong mechanical turgor pressure (up to 12.39 bar in penetrating silicate and carbonate rocks) allow them to dig cavities on the heritage stone surfaces at depths ranging from a few hundred microns to several millimeters [7,13,14]. Furthermore melanin, the most important factor in BF stress resistance, is also responsible of the aesthetic alterations imparting a dark, blackish-brown appearance to the lithic surface [15–17].

Knowledge of the deteriogens' diversity is undoubtedly useful, however more efforts should be given to outline the ecological needs of the occurring species and their involvement in deterioration processes. The metabolic and ecological profiles, such as the environmental conditions, which favor the various taxonomic groups, are crucial in defining the risk for the artefacts and in designing both preventive/indirect and corrective/direct (e.g., biocide treatments) control measures [1,18]. In fact, the awareness on the tolerance range of the involved species for each environmental factor has a great practical relevance in preventing undesirable growth [1,19]. Such information is also relevant when considering biocide treatments, due to the need to customize the treatment selecting the most efficient product against specific microorganisms [20].

Corrective measures against fungal spreading are often required and such interventions are mainly carried out using biocidal products both to remove the biomass in the early stages of artwork treatment and later to prevent re-colonization [21–23]. In stone treatments, BF represent a challenge for restorers being able to survive and re-colonize artworks after restoration treatments with biocides or synthetic polymeric coatings [6,11,24–27]. To date, very little information is available on their sensitiveness to biocides and on their metabolic and eco-physiological profile, mainly limited to a few model fungi [28,29]. In this light, traditional culture dependent assays can be useful to deepen in detail their ecological traits and biodeteriorative potential [30].

Based on the multidisciplinary investigation surveys performed on the deteriogenic phenomena affecting the monumental Cemetery of Bonaria in Cagliari since 2010, we focused on a selection of 18 BF strains isolated from some of its marble monuments.

The goals of the present study were: (i) the selection and identification of the BF strains of interest; (ii) the assessment of the main fungal traits with respect to thermal and salt growth preferences; (iii) the possible relation between the recorded fungal traits and the substrate in selected monuments; (iv) the detrimental potential of the investigated species; and finally (v) their sensitiveness to a few traditional biocides. The achieved results will also give the opportunity of testing the best techniques of evaluation of metabolic evaluation and control. Data will also provide new insights to design more efficient preventive and control protocols, as well as on the leading forces driving the colonization processes.

#### **2. Materials and Methods**

#### *2.1. Black Fungal Strains Selection and Identification*

The ancient monumental cemetery of Bonaria (39◦12 36.96 N 9◦07 26.13 E) in Cagliari, Italy, has been object of multi-disciplinary studies aimed at favoring its conservation, rediscovery as urban historical heritage, and valorization [5,17,31–34]. The climatic conditions of the site are characterized by hot dry summers, very mild winters, and humidity may be high due the sea proximity. Over the period 2011–2014, we performed a few samplings from some Carrara marble funerary monuments (Figure 1), thanks to the collaboration of the Superintendence of Heritage Landscape, Historical, Artistic and Ethno-Anthropological Heritage for the metropolitan city of Cagliari and for the provinces of Oristano, Medio Campidano, and Municipality Cemeteries Direction of Cagliari.

Forty BF strains were isolated and most of them were identified through phylogenetic analysis, and new species described [31]. Three strains not considered previously, namely CCFEE 5778, 5945, and 6327, were identified by internal transcribed spacer (ITS) and LSU sequencing (in case of poor ITS identities) followed by BLASTn comparison. The primer set used for amplifications were ITS4-ITS5 and LR0R-LR7, respectively. In short, the reactions were performed in a total volume of 25 μL using 5 pmol of each primer, Bioline BioMix (Bioline Reagents, London, UK), and about 30 ng of genomic DNA [35]. The PCR protocols consist of an initial denaturation and final extension and 35 cycles of amplification with annealing at 55 ◦C (ITS) and 52 ◦C (LSU). After identification, representative strains of the whole BF diversity found in the site have been subject to selection in order to give the wider representativeness possible with precedence to type strains (when available).

**Figure 1.** The sampled Carrara marble funerary monuments of the ancient monumental cemetery of Bonaria—Cagliari entitled to (**A**) Avv. Giuseppe Todde; (**B**) Antonio Viganigo; (**C**) A. M. Frau Carta; (**D**) Francesca Warzee; (**E**) Rossino Bolla spouse; (**F**) Giuseppina Ara dei conti Ciarella; (**G**) Zelina Ferrà Gastaldi Millelire; (**H**) Ignazio Ruda Roych conte di San Lorenzo; (**I**) Unknown Burial.

All strains are part of the Culture Collection of Fungi from Extreme Environments (CCFEE, Viterbo, Italy) and new sequences were deposited in GenBank.

#### *2.2. Thermal Preferences, Salt Tolerance, and Metabolic Assays*

Thermal preferences and salt tolerance were assessed by inoculating small fragments of fungal mycelia onto malt agar plates (MA: 30 g/L malt extract, 15 g/L bacteriological agar; VWR) and incubating them for a month within the range 0–40 ◦C with 5 ◦C intervals. The growth at 37 ◦C was also assessed.

Similarly, salt tolerance was determined inoculating small fragments of mycelia on MEA 2% plates (malt extract 20 g/L, 15 g/L bacteriological agar) with increasing concentration of NaCl (0, 3.5, 7, 10, 12.5, 15, 20% *w*/*w*), incubated at 20 ◦C for a month.

The detrimental potentials of isolates were assayed through plate trials for acid (CaCO3 Agar, ACID), amylase (AMY), lipase (LIP), and protease (namely caseinase; Skim Milk Agar, SM) production as previously described [36]. The cellulase and pectinase activities were assessed using CMC agar (CMC) and pectinase screening agar medium (PSAM), respectively. CMC was prepared using (NH4)H2PO4 1 g; KCl 0.2 g; MgSO4· 7H2O 1 g; yeast extract 1 g; carboxymethylcellulose low-density 26 g, and bacteriological agar 3 g per liter of solution. PSAM was instead prepared using pectin 1 g; (NH4)2 HPO4 0.3 g; KH2PO4 0.2 g; K2HPO4 0.3 g; MgSO4 0.01 g; and bacteriological agar 2.5 g per 100 mL

of solution, pH 5.5. Plates were incubated at 20 ◦C for a month. To readability, for AMY, CMC, ACID, and PSAM plates were flooded with Lugols' iodine solution and read after 15 min incubation and washed with saline (NaCl 0.9%). The colonies' diameters were taken weekly and the halos, expressed as measure from the colony border, were taken at the end of the experiment. All measures were expressed as average of three replicas and standard deviation (SD).

#### *2.3. Marble Substrate Investigations*

To deepen the possible relation between the fungal traits recorded and the marble substrate, from which they were isolated, a selection of highly weathered monuments (e.g., Ruda Rojch and Viganigo) were analyzed by X-ray diffraction analysis (XRD), mercury intrusion porosimetry (MIP), light refraction optical microscopy and scanning electron microscopy (SEM).

In detail, detached and not replaceable scales were micronized in an agate jar, to obtain powders passing to 0.63 μm sieve and pressed in a glass holder. XRD spectra were acquired on powders using a diffractometer Rigaku Miniflex II, equipped with a graphite monochromator, Cukα wavelength in the following instrumental conditions: 15 kV, 30 mA, Ni filter, scan from 4 to 70◦2θ, step sampling 0.01◦θ. The identification of minerals was carried out using the search Crystal Impact MATCH! software v. 3.10, which uses, for comparison, both the JCPDS Database (Joint Committee on Powder Diffraction), and the COD Database (Crystallography Open Database) [37]. These results were compared to a quite unaffected old marble from a slab (Zelina Ferrà Gastaldi Millelire).

Total open porosity was measured by mercury intrusion porosimetry (MIP) on 1 cm3 (c.a.) degraded and control marble fragments oven-dried at 50 ± 5 ◦C until reaching constant mass. An AutoPore IV 9500 (Micromeritics Instrument Corporation, Norcross, GA, USA) operating at 2200 bar, and 10 s of equilibration time was used. Results were compared to unaffected-new Carrara white marble fragments (Statuarietto lithotype) used as control.

Specimens of about 0.5 × 0.5 cm, were preliminarily observed in light reflection mode using a MoticTM binocular microscope (Kowloon, Hong Kong) to evaluate the conservation conditions of samples and to select the portions of major interest for SEM analysis. These fragments were then glued in epoxy resin (Struers Ltd., Catcliffe, UK), cut in thin sections (30 μm thick) and finally polished. Other fragments were coated with a graphite layer (10 to 20 nm thick) and observed with a Zeiss EVO LS 15 environmental scanning electron microscope (Carl Zeiss SMT AG, Oberkochen, Germany), LaB6 cathode, EHD 5 kV, WD 13.5 mm.

#### *2.4. Selected Chemicals*

Among the available traditional biocidal formulations [20,21], we selected four products. Two of them contain quaternary ammonium compounds (QAC): Benzalkonium Chloride (CTS srl, Altavilla Vicentina, Italy; benzalkonium chloride 90%; BZC), and Preventol RI50 (Bresciani srl, Milano, Italy; benzalkonium chloride 50%; PREV). Otherwise, Lichenicida 264 (Bresciani srl, Milano, Italy; LICH) has *N*,*N*-dimethyl-*N* -phenyl-*N* -(fluorodichloromethyl)-sulphamide (also known as dichlofluanid) as active molecule, and Biotin R (CTS srl, Altavilla Vicentina, Italy; BioR) contains 3-iodo-2-propynyl butyl carbamate (IPBC), and 2-*n*-octyl-4-isothiazolin-3-one (OIT) dissolved in 2-(2-butoxyethoxy) ethanol. BZC and PREV were diluted in distillated water, LICH in alcohol [38], and BioR in white spirit.

#### *2.5. Sensitiveness to Biocides*

The sensitiveness of fungal isolates to biocides was measured using the agar diffusion tests (ADT) method. Due to their dimorphic clumped growth habit, the strong but variable melanization, and the absence of spores, the BF suspension was standardized as dry weight as follows: a MA slant culture was suspended scaping its surface in 3 mL of sterile saline solution (NaCl 0.9%), transferred into a 5 mL conical tube, homogenized by sterile pestle, and used to inoculate a 250-mL Erlenmeyer flask with malt extract broth (3% *w*/*v*; 30 mL). The incubation was performed at room temperature and 180 rpm on a rotary shaker for 1–3 days. ADTs were performed, in triplicate using 90 mm-Petri dishes. For each plate 200 μL of fungal suspension (8 ± 0.2 mg/mL dry weight) were added to 15 mL of MA by inclusion; a cellulose sterile disk (6 mm diam) was placed on the culture medium and soaked with 6 μL of biocide solution. Biocides were tested at the following concentrations: 0.5, 1, 1.5, 2, and 3% for BZC and PREV; 0.25, 0.5, and 1% for LICH, and 0.5, 1, 2, 3, 4, and 5% for BioR.

#### *2.6. Statistical Analyses*

PAST software (PAleontological STatistics, ver. 4.06b, [39]) was used to perform a Principal Component Analysis (PCA) assessing similarities/differences among the strains in study with respect of their salt tolerance, metabolic traits, and resistance to biocides.

Two-way ANOVA with Tukey post hoc test comparison (*p* < 0.05) was performed using GraphPad Prism version 9.3.1 (GraphPad Software, San Diego, CA, USA) to assess significance between biocides increasing doses.

#### **3. Results**

#### *3.1. Identification and Selection of BF Species*

Based on GenBank sequence match, the three strains were identified as follows. Strain CCFEE 5778 has been reported as *Saccotheciaceae* sp. due to the ITS (OM390240) best match with *Saccothecium rubi* MFLUCC 14-1171 (98.16%), and LSU (OM346745) match with *Selenophoma linicola* CBS 468.88 (98.90%) and *Aurobasidium pullulans* AFTOL-ID 912 (98.70%). Due to his very poor ITS match (OM568835) with *Diaporthe longispora* CBS 194.36 (coverage below 40%) and 97.21% LSU (OM346746) identity with *Phaeotrichum benjaminii* CBS 541.72, CCFEE 5945 was referred as "Unknown dothideomycete". Differently, CCFEE 6327 (OM390241) was identified as *Exophiala oligosperma* due to its 99.83% identity with the species type strain CBS 725.88.

The selected strains were in total 18, one for each species found, and among them were 4 *Knufia*, 3 *Neodevriesia*, and 2 *Exophiala* species (Table 1).

**Table 1.** The 18 strains in study, collection number (CCFEE), and monuments of isolation. (T) indicates type strain. In bold the new identified strains. All monuments were built in white Carrarese marble.

\* this new combination has been proposed by Czachura and colleagues as synonym of *Hortaea thailandica* [40].

#### *3.2. Main Ecological Traits Assessment: Thermal and Salt Tolerance*

All the tested strains were able to grow within the range of 5–25 ◦C (Figure 2, Table S1); the majority can grow at 0 ◦C (13/18), less than a half (7/18) at 35 ◦C; only *E. oligosperma* CCFEE 6327 at 37 ◦C, and none at 40 ◦C. Most of the strains grew best at 20 ◦C, except *K. petricola* CCFEEE 5776, *N. capensis* CCFEE 6200, and *E. oligosperma* CCFEE 6327 having an optimum at 25 ◦C.

**Figure 2.** Thermal preference chart. The bubble size is proportional to the average growth recorded at the end of the experiment (1 month). Table S1 reports the full average values and SD recorded.

Growth rates were extremely variable among the studied strains; for instance, after one month of incubation strains *A. pullulans* CCFEE 5736, *Saccotheciaceae* sp. 5778, *E. bonariae* CCFEE, 5792, and *E. oligosperma* CCFEE 6327 grew from about four (3.6) to seven times more than *V. calcicola* CCFEE 5770 and *C. uncinatum* CCFEE 5737.

Different degrees of salt tolerance were recorded (Figure 3, Table S2). *Verr. dirinae* CCFEE 5707, *N. bulbillosa* CCFEE 5704, *Sal. thailandicus* CCFEE 5723, and *Neoph. triangularis* CCFEE 5703 grow better in the presence of 3.5%. Five strains (namely the previous and *A. pullulans*) were able to grow at 20% NaCl, the highest concentration tested. *A. pullulans* CCFEE 5736, although able to grow in the presence of salt, drastically reduced its colony size up to 46% at 3.5% and 95.6% at 20% of NaCl. A high sensitivity to salt was instead found for the unknown dothideomycete CCFEE 5945 with a dramatic decrease in colony diameter of up to 74% in the presence of NaCl 3.5%, and no growth at 7%. Similarly, the slow-growing *C. uncinatum* CCFEE 5737 was unable to grow at 7%. The remaining strains showed instead an intermediate tolerance having *K. petricola* CCFEE 5776 as the most tolerant of the strains able to grow up to 10% NaCl.

**Figure 3.** Salt tolerance tests results. (**A**) The measures taken after one-month incubation at 20 ◦C are calculated as a percentage compared to the control plate (0% NaCl). (**B**) The PCA plot evidenced in the right side of the separated group of halophilic strains, and others (from right to left side) ordered according to their salt tolerance.

#### *3.3. Mineralogical and Physical Investigations*

Similar XRD diffraction patterns were recorded in the two deteriorated marbles affected by granular disintegration and crumbling (namely Antonio Viganigo, AV; and Ruda Roych, RR-data not shown). Significative differences were instead evidenced when the diffraction pattern of the AV deteriorated and soluble salt affected marble (Figure 4B) was compared to a gypsum film from an old compact marble monument (Ferrà Gastaldi Millelire; FGM, Figure 4A). Soluble salts and other decay phases were found in different amounts. In the grey film of FGM monument gypsum (CaSO4·2H2O) and calcite (CaCO3) prevailing on whewellite (CaC2O4·H2O), quartz (SiO2), halite (NaCl), sylvine (KCl) and titanite (CaTiSiO5). On a more weathered monument (AV), affected by granular disintegration and crumbling, the whitish crust shows mineralogical phases related to natural pollution (marine and soil particles) as halite, quartz, albitic plagioclase, as well gypsum and calcite.

**Figure 4.** XRD analysis results. XRD patterns of gypsum grey film on old compact marble (FGM) (**A**) and whitish crust on crumbling-granular disintegration marble (AV) (**B**).

MIP porograms (Figure 5A,B) evidenced high differences when comparing a new "Statuarietto" Carrara marble having a porosity of 0.7% ± 0.2 (black), and historical marble affected by granular decoesion 5% ± 0.2 (AV, gray) and high granular decoesion 8% ± 0.2 (RR, red). The porograms show macro- and micro-pores; the former are represented by

microcracks or confluent calcitic crystal dislocation. The secondary porosity, acquired by physical and chemical decay processes, represents a key indicator of biodeterioration/bioreceptivity.

**Figure 5.** MIP porosity results. MIP porosity of an unaffected-new "Statuarietto" marble (black), a decohesed (gray, AV), and highly decohesed marble (red, RR). (**A**) Cumulative Hg intrusion vs. pressure and (**B**) Log. differential intrusion vs. pore size distribution.

Light microscopy evidenced the presence of salts and other pollution materials in dark crusts (Figure 6B,D). SEM investigation confirmed the deep decohesion of calcitic crystals clasts, the presence of gypsum (desert rose twinning, Figure 6E), and halite crystals already evidenced in the previous analyses; these latter trials give an impacting measure of the phoenomena being marble crystals completely covered by salt (Figure 6H).

**Figure 6.** Granular disintegration marble: a salty niche. Particular to the monuments affected by granular disintegration into sand and powder devoted to (**A**) Ruda Roych (RR) and (**C**) Antonio Viganigo (AV). (**B**,**D**) Dark sulfation crust of AV monument by light reflection optical microscopy: (**C**) cross section, (**D**) surface and fracture on dark crust and salt crystals (NaCl) deposition. (**E**–**H**) RR sample SEM observation. (**E**) gypsum crystals; (**F**,**G**) granular disintegration, (**H**) salt crystals (NaCl) on marble surface. The bar corresponds to 10 μm in (**F**), 200 μm in (**G**); and 2 μm in (**H**).

#### *3.4. Detrimental Potential of the Investigated Species: The Metabolic Screenings*

As reported in Table 2, acid production was observed after iodine pouring in nine out eighteen investigated strains. No production was recorded for the two *Exophiala* species, while positive records were found for all the *Knufia* species. All tested strains were positive for the esterase activity (LIP) being able to use Tween 20 as a sole carbon source and precipitate calcium salt crystals. The majority of the tested strains showed amylase (14/18), and cellulase (15/18) activity, while caseinase and pectinase activities were recorded in half of them (9/18).

**Table 2.** Results of the metabolic assays performed on selected BF. The activities tested were for acid (ACID), lipase (LIP), amylase (AMY), protease (SM), pectinase (PSAM), and cellulase (CMC) production.

(−) negative; increasing positive responses expressed in mm measured from the colony border: (+) 0.1–5 mm, (++) 5.1–9.9 mm, (+++) 10–14.99 mm, (++++) 15–20 mm.

PCA analysis performed on halos occurred after metabolic assays evidenced in the right side the most responsive strains (Figure 7). All the halophilic strains take place within the most responsive strains. Interestingly *K. mediterranea* CCFEE 5710, due to the intense halos recorded, occupied quite a far position in respect to the other *Knufia* species in the study (namely *K. petricola* CCFEE 5776, *K. karalitana* CCFEE 6001, and *K. marmoricola* CCFEE 6204).

**Figure 7.** PCA analysis performed on halos recorded for each plate test.

Moreover, during metabolic screenings it was possible to collect additional information such as the loss of melanization by the dothidealean fungi (namely *A. pullulans* CCFEE 5736 and *Saccotheciaceae* sp. CCFEE 5778) when grown in SM (Figure 8L), the volcano-like growth showed by *K. mediterranea* CCFEE 5710 in presence of high concentration of glucose (Figure 8B), and the inhibitory action of *K. marmoricola* and *K. petricola* against a cladosporiacean plate contaminant (Figure 8M,N, respectively).

**Figure 8.** Plate assays outcomes. (**A**) *C. uncinatum* CCFEE 5737 AMY test; (**B**) *K. mediterranea* CCFEE 5710 on CaCO3 agar: the colonies raise in the center and detach from plate; (**C**) *K. petricola* CCFEE 5776 esterase activity test (LIP); (**D**) *E. oligosperma* CCFEE 6327 esterase activity test (LIP); (**E**) *N. bulbillosa* CCFEE 5704 culture media darkening in presence of salt: from left to right side NaCl 0%, 3.5% and NaCl 7%; (**F**) *K. mediterranea* CCFEE 5710 PSAM test; (**G**) *Neoph. triangularis* CCFEE 5703 amylase test (AMY); (**H**) *Neoph. triangularis* CCFEE 5703 SM test; (**I**) *Neoph. triangularis* CCFEE 5703 colonies color shift in presence of salt from left to the right side NaCl 0% and 3.5%; (**J**) *Verr. dirinae* CCFEE 5707 PSAM test; (**K**) *V. calcicola* CCFEE 5770 AMY test; (**L**) *A. pullulans* CCFEE 5736 SM test; (**M**) Contaminated PSAM plate with *K. marmoricola* CCFEE 6204 creating inhibition halos (white arrow). (**N**) Contaminated PSAM plate with *K. petricola* CCFEE 5776 creating inhibition halos (white arrows); (**O**) *K. petricola* CCFEE 5776 acid production plate; (**P**) *N. bulbillosa* CCFEE 5704 cellulase test plate (CMC); (**Q**) *K. mediterranea* CCFEE 5710 cellulase test plate; (**R**) *Verr. dirinae* CCFEE 5707 negative acid production test; (**S**–**U**) *K. karalitana* CCFEE 6001, *K. mediterranea* CCFEE 5710, *K. marmoricola* CCFEEE 6204 positive acid production plate test; (**V**,**X**) *E. oligosperma* CCFEE 6327 colony morphology shifts from flat (**V**) grown at 25 ◦C to meristematic (**X**) grown at 35 ◦C; (**Y**) *N. capensis* CCFEE 6200 AMY test.

#### *3.5. Sensitiveness to Traditional Biocides*

The tested black fungal strains showed different degrees of sensitiveness to biocides (Table 3) and no linear response to incremental doses (Table S3). Two successive increasing biocide doses frequently did not significantly increase inhibitory halos (Table S4).

**Table 3.** Inhibition halos recorded after the application of the highest dose suggested by suppliers. The different colors represent the different sensitiveness to biocides, as indicated in the legend below, where *h* is the halo recorded in millimeters. The complete results are shown in Table S3.

The majority of strains evidenced a low sensitiveness to both BZC and PREV, and others, such *E. bonariae* CCFEE 5792, proved quite tolerant to all the biocides tested.

PCA analysis performed considering all responses to the different doses of biocides applied (Table S3), evidenced a sort of gradient where *N. bulbillosa* CCFEE 5704, *V. calcicola* CCFEE 5770, and *S. tyrrhenica* CCFEE 5935 resulted the most sensitive species to the tested biocides (Figure 9A).

**Figure 9.** Results of sensitiveness to traditional biocides. (**A**) PCA plot based on the ADT halos recorded using four different biocides at all the tested doses; from the left to the right there is an increasing sensitiveness, in the opposite sense an increasing tolerance to biocides. (**B**,**C**) ADT performed on *E. oligosperma* CCFEE 6327: (**B**) sharp inhibition halo obtained using BZC 3%, (**C**) double halo obtained when BioR 4% was used. There are notable zones of total (next to the disc), and partial inhibition (external) where growth is slowed.

Interestingly BZC always produced sharp halos (Figure 9B) while the other biocides (PREV, LICH, and BioR), especially at low doses, produced thin total inhibition halos surrounded by halos of partial inhibition (Figure 9C) as the growth was mainly slowed down.

#### **4. Discussion**

Biodeterioration processes are rarely caused by only one group of microorganisms, and more frequently are due to polymicrobic associations coexisting at the same time, in the same place, on a given artifact. To study them, two main approaches are available: the high throughput sequencing and the traditional culture-based approach. The biomolecular approach has become the dominant method of studying the microbial diversity in the last decade [41]. In fact, the main part of environmental microorganisms are unculturable and the behavior of a single species can frequently be explained in a community context [41]. However, beyond the exponentially increasing information, there is not an equally rapid increase in knowledge regarding the mechanisms and the main actors of the deterioration phenomena [42]. Moreover, the identification and characterization of monument settlers from a physiological and ecological point of view is of the utmost importance in order to design preventive control methods and biocide treatments [21,36,43–45]. From this perspective, an increasing number of papers propose traditional metabolic plate assays as a useful, easy and cost-effective tool to unveil the potential risk tied to some cultural heritage settlers [30,36,46–50]. Such a proposal is confirmed in this work.

#### *4.1. Selection and Identification of the BF Strains of Interest*

Even if they were all isolated from a single site, the eighteen strains considered in this study could represent other BF lithic communities even at different latitudes. *Knufia*, *Neodevriesia*, *Exophiala, Coniosporium*, and *Vermiconidia* are indeed well known genera due to their frequent finding and broad distribution on stone monuments in urbanized areas and natural crops.Their presence has been recorded in the Mediterranean countries as well as in Russia, Ukraine, Austria, Germany, Poland, China and, not least, Antarctica [51–53]. Information on their environmental preferences and metabolic traits could be helpful in the future to better understand their geographical distribution.

#### *4.2. Temperature and Osmotic Tolerance as Indicators of Environmental Preferences*

The thermal trials yielded important information on the optimum temperature of the studied strains, their growth range, and also on their growth rate. The majority of the strains grew within the range 5–30 ◦C, 39% of them instead within 0–35 ◦C, and only one, *E. oligosperma* CCFEE 6327, at 37 ◦C. With respect to their growth rate, the studied strains can be clustered into three groups: fast-; mid-fast-; and slow-growing black fungi. The majority of the slow-growing fungi (e.g., *V. calcicola* CCFEE 5770, *C. uncinatum* CCFEE 5737, and *Knufia* species) have been recorded from stone surfaces only; the others, namely the fast-growing (*A. pullulans* CCFEE 5736, and *Saccotheciaceae* sp. CCFEE 5778) and the mid-fast- growing species (e.g., *E. bonariae* and *E. oligosperma*, Unknown CCFEE 5945, *Verr. dirinae* CCFEE 5707, and *N. capensis* CCFEE 6200), can be considered at least in part to be occasional. *A. pullulans* is considered, indeed, a generalist that can inhabit different habitats without substantial specialization at the genomic level [54], and *E. oligosperma* is a well-known opportunist isolated from a number of household environments such as tap water and diesel car tanks [41,55,56]. This confirms the two survival strategies in fungi from extreme environments: they can be either widespread generalists, thriving in both extreme and moderate conditions, or specialists more or less confined to extremes [57–59].

Growth rate can even be drastically affected when salt is addicted to culture medium. All the tested strains were able to grow at 3.5% of NaCl. This trait increases their fitness for the site, being close to the sea with saltiness periodically deposited and washed away from surfaces with rains. Anyway, different degrees of tolerance were recorded, and strains tested grouped with respect to their response to increasing salt concentration and to their limits. The lowest tolerance has been recorded for *C. uncinatum* CCFEE 5737

and the unknown CCFEE 5945. The majority of the strains have their limits between 7 (6/18) and 10% (5/18), respectively. Five, instead, can grow up to 20%. While *A. pullulans* showed a broad-range tolerance to salt, *Verr. dirinae* CCFEE 5707, *N. bulbillosa* CCFEE 5704, *Sal. thailandicus* CCFEE 5723, and *Neoph. triangularis* CCFEE 5703 proved to be halophilic. The halophilic trait for a part of them was already reported [60,61], however, it was not for *N. bulbillosa* CCFEE 5704 and *Verr. dirinae* CCFEE 5707, which ability was suggested by indirect evidences only. For example, *N. bulbillosa* was isolated from limestone formations in Cala Saint VincenG in Mallorca Island, so possibly subjected to some extent to saltiness [62]. Otherwise, *Verr. dirinae* is worth a mention as even if described as a parasite of the lichen *Dirina massiliensis* [63], it has been isolated from other substrates/salted niches such as wood and marine macroalgae [64,65]. Moreover, the presence of some BF has been suggested to be in relation with the biodeterioration degree of tombstones [66].

#### *4.3. Marble Deterioration Degree Selects the Resident Fungal Community*

The instrumental stone investigations performed (namely XRD, light reflection microscopy, and SEM) on the two most deteriorated monuments evidenced the presence of gypsum and chloride crystals (mainly NaCl), the latter of which was found in high amounts. Their presence on marble stone is strictly related to the microclimatic condition, salt solubility and surface physical conditions (e.g., porosity, roughness). The severe alteration of the decayed marble has been shown by SEM and by MIP pore size distribution, where yields were up to 10 times higher than the new unaffected control specimen.  Previous studies showed that the most damaged marble pieces in the cemetery of Cagliari were all characterized by low values of solar radiation, high humidity and a reduced thermoforesis, favoring sulfatation and weathering along with the surfaces' colonization [32,33,67,68]. Indeed, the nearby and dense vegetation, in addition to the low exposition to the dominant winds (Libeccio and Scirocco from south and Mistral from north-west), favored the stagnation of marine air and its penetration into damaged stones. Sea spray acts as a marine inoculum migrating between grains by capillarity; here the salt wet-dry cycles gradually and increasingly alter the materials' structure [69–72]. Each crevice and intergrain space can become a salty micro-niche available for specialized microorganisms; along with the salt physical action, the selected community can contribute to stone structural destabilization [73–76]. The exclusive isolation of the halophilic fungi from sand disaggregated monuments only, highlighted as salt-attacked monuments constitute a suitable habitat for selected osmophilic/osmotolerant microorganisms [75,77,78]. However, this phenomenon seems to be progressive since *Neoph. triangularis*, *N. bulbillosa*, and *Verr. dirinae* were isolated from the RR monument (8% MIP porosity) only, while *S. thailandicus* and *K. petricola* from the AV monument (5% MIP porosity). On this basis, further studies are needed to investigate the possible use of the halophilic and the most halotolerant black fungal strains (e.g., *K. petricola* CCFEE 5776) as possible indicators of stone degradation in the early steps of salt-mediated decoesion or, by contrast, the use of low salt tolerant BF (e.g., *C. uncinatum*) as indicators of stone integrity in coastal environments.

#### *4.4. Metabolic Assay, Ecological Traits, and Detrimental Potential of the Investigated Species*

Knowledge on the relationship between microorganisms and colonized substrate drives the identification of the major conservation threats and may address the control strategies designed to face biodeterioration [79]. Plate assays were useful to outline some important metabolic traits in BF. Acid production has been recorded in half of the studied strains. Even if not leading to a complete dissolution of carbonates in the culture media, BF showed a detrimental potential well beyond their assessed biomechanical action [7]. A local decrease in the pH has been previously reported for *K. petricola* A95 and found here, for example, in all the *Knufia* species and in two of the three *Neodevriesia* [80]. Further studies are needed to asses if this trait is limitedly present in some selected genera or not. In any case more methods and/or protocol adjustments are needed to detect even weak responses.

The amylases, pectinases, esterases and cellulases activities recorded showed that these fungi can feed on vegetal sources and debris. According to the conceptual model for MCF proposed by Chertov and colleagues [81], the availability of organic nutrients is the dominant factor limiting their development on stone surfaces in European temperate and Mediterranean climates, while growth becomes very intensive in the presence of water. The metabolic features highlighted here support the well-known yet rarely applied practice of removing the organic matter from monuments to prevent fungal growth [82]. The production of esterases and caseinases activities suggests as these strains may also feed on wall paintings where oils and proteinaceous binders were used as already reported [83–86]. Furthermore, the inhibition halo (competition for space) produced by two *Knufia* species (namely *K. petricola* and *K. mediterranea*) against a cladosporiacean plate contaminant, provided evidence that some BF species in peculiar conditions (PSAM test) can compete, more than expected [87–89], against high sporulating fungi.

From a conservative point of view, the ability to metabolize carboxymethylcellulose (CMC) and Tween 20 (LIP) should be careful evaluated in restoration practice for their possible drawbacks. These chemicals, indeed, are commonly used as thickening agents and surfactants, respectively [90,91], and their residues on the treated artifact could lead to a tertiary bioreceptivity if not thoroughly removed.

#### *4.5. Tolerance to Biocides*

Despite the significant impact of the green revolution in the biocides market [92], traditional biocidal products are still widely used [93]. This is due to the consolidated protocols used on different materials, the wide knowledge on the pros and cons of their use, and the non-negligible economic aspects.

In situ trials are often used in restoration practice to customize treatments as they confidently reproduce, in a short time, the interaction of biocides with biota and materials, and provide a more reliable indication of the in situ persistence of the biocide [94–97]. However, several factors can negatively affect the efficiency of any restoration protocol. The metabolic state of the organism is important for their responsiveness to biocides treatments. Metabolically active colonies are, indeed, more sensitive to chemical and physical stresses [98]. BF, being poikilohydric organisms, withstand the unfavorable conditions in a dormant state mainly induced by desiccation (anhydrobiosis) [12,99]. In addition, fungi growing in stone cracks and fissures may be not easily reached by biocides applied on a monument surface during restoration treatments. Even the typical clump-like BF colonies, ensuring the optimal surface-volume ratio, minimizes the direct exposure to external stressors, including biocides; moreover, external cells in the colony shield the inner layers from external injuries (shadow effect) [100]. Conversely ADT plate assays have an undeniable advantage as they reproduce the best conditions to record the sensitivity to biocides of single metabolically active cells in a medium that cannot seize the biocide as stones frequently do. For these reasons, the tolerance/resistance recorded in the most responsive condition should warn about their possible survival after treatments, as well as their spread.

The used biocides have different formulations, mechanisms of action, and thus different outcomes. BZC (CAS No 8001-54-5) is a quaternary ammonium compound (QAC) routinely used for chemical antisepsis and disinfection and widely applied in cultural heritage preservation treatments. According to the concentration used, BZC acts as a highly active detergent causing cell membrane permeability alterations, irreversible damage to the barrier function and proteins denaturation [101]. Benzalkonium chloride is also the only active compound in the PREV patent, albeit in a lower concentration (50%) than in BZC (90%). Despite the lower concentration of QAC salt in PREV, the inhibition halos produced were frequently larger than BZC, such as for *Sal. thailandicus* CCFEE 5723, *K. petricola* CCFEE 5776, and *S. tyrrhenica* CCFEE 5935. This apparent incongruence, already evidenced in

previous studies [36], has been explained with the possible different composition in terms of n-alkyl groups length and/or degree of C-C saturation in the two chemicals [102], and by additives, not specified in the composition of biocides but included in the patent of the commercial formulas [21,103]. The other two biocides tested have instead intracellular targets. As a matter of fact, *N*,*N*-dimethyl-*N* -phenyl-*N* -(fluoro-dichloromethylthio)-sulphamide (CAS No 1085-98-9), the active principle of LICH, is supposed to affect a number of enzymes by reacting with –SH bonds [104]. While the fungicidal and antimicrobial action played by IPBC (3-iodo-2-propynyl butyl carbamate, CAS No 55406-53-6) and OIT (2-*n*-octyl-4 isothiazolin-3-one; CAS No 26530-20-1), the active principles of BioR, are probably due to iodine toxicity, and inhibition of cell respiration/ATP production, respectively [105,106].

The achieved results evidenced an increasing biocidal power, in terms of inhibition halos and number of strains completely inhibited, as follows: BZC-PREV, LICH, and BioR. The thick multilayered and strongly melanized cell wall typical of these fungi serve as an efficient barrier for toxic substances with positive influence on resistance. Moreover, the presence of halos of total and partial inhibition recorded frequently when PREV, LICH, and BioR were applied (Figure 6C), could be explained by a certain ability to repair sub-lethal damages as resistance mechanism to biocides. Several strains, such as *E. bonariae*, *A. pullulans*, *E. oligosperma*, *Verr. dirinae*, and secondly *K. petricola*, and *C. uncinatum*, exhibited low responsiveness even to the most powerful biocide. This fact should warn against the use of extremely powerful biocides as a quick remedy in controlling whatever biological patina as the drastic decrease in competitors favors the spreading of the ones who survived. In the meanwhile, a stronger biocidal action is often backed to a higher toxicity for the operator and the environment. Conversely, the widespread use of ammonium quaternary salts (e.g., benzalkonium chloride), should be reduced to oppose the rapid recolonizations by serving as nutrients for micro- and macro-organisms [107]. The wide use of sub-inhibitory concentrations of benzalkonium chloride (and QACs), it has been suggested, may be responsible for a significant decrease in microorganisms' sensitivity to biocides, driving the evolution of polyextremotolerant fungi towards the enhancement of their stress tolerance [108]. For this reason, the rotation of disinfectants is often recommended in practice, in order to kill the resistant biota [109]. In this context plate assays could be useful to test multiple chemicals. The ability to cope with different biocidal products shown by these strains also make them promising candidates as new reference organisms in material testing procedures [110].

#### **5. Conclusions**

This research gave an assessment of the main BF traits with respect to thermal and salt growth preferences, such as on the possible relation between the recorded fungal traits and the substrate. Plate assays resulted a fast, easy, cost-effective, and useful tool for such aims. To deepen the BF detrimental/colonizing potential, more plate tests should be designed or improved, and physical analyses were fundamental in proving the relation between halophilic strains and highly deteriorated marble surfaces. We also assessed the detrimental potential of the investigated species through metabolic assays, which were also useful to determine the conditions favoring their growth and substances that should be avoided to prevent tertiary bioreceptivity. We also evaluated the sensitivity to a few traditional biocides through ADT tests, which were useful to identify the most recalcitrant BF species.

**Supplementary Materials:** The following supporting information can be downloaded at: https: //www.mdpi.com/article/10.3390/app12042038/s1, Table S1: Thermal preferences recorded within the range 0–40 ◦C, Table S2: Salt tolerance recorded within the range 0–20% NaCl, Table S3: ADT results, Table S4: ADT two-way ANOVA Tuckey test row analysis.

**Author Contributions:** Conceptualization, D.I.; methodology, D.I., F.B. and P.M.; data curation, D.I., P.M. and F.B.; writing—original draft preparation, D.I.; writing—review and editing, D.I., L.Z., P.M., F.B. and G.C.; All authors have read and agreed to the published version of the manuscript.

**Funding:** The Ordine Nazionale dei Biologi for funding a scholarship for the project "Fungal deteriogens and stone monuments".

**Institutional Review Board Statement:** No humans or animals are involved in this study.

**Informed Consent Statement:** No humans are involved in this study.

**Data Availability Statement:** All data resulting from this research are fully reported here or as Supplementary Material.

**Acknowledgments:** The authors wish to thank the Superintendence of Heritage Landscape, Historical, Artistic and Ethno-Anthropological Heritage for the metropolitan city of Cagliari and for the provinces of Oristano, Medio Campidano and, Municipality Cemeteries Direction of Cagliari for collaboration and sampling permission. Gianfranco Carcangiu is acknowledged for support in elaboration of XRD analysis.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Investigations of the Surface of Heritage Objects and Green Bioremediation: Case Study of Artefacts from Maramure¸s, Romania**

**Dorina Camelia Ilies, 1, Nicolaie Hodor 2, Liliana Indrie 3, Paula Dejeu 4, Alexandru Ilies, 1, Adina Albu 3, Tudor Caciora 1,\*, Marin Ilies, 5, Lucian Barbu-Tudoran 6,7 and Vasile Grama 1**

**Abstract:** Old textiles are important elements of thecultural heritage. As a result of their composition mostly of natural elements old textiles are extremely prone to physical and chemical degradation due to fungal action. The treatments usually applied for the cleaning of heritage textiles target the use of synthetic fungicides, which are potentially harmful to both human health and the environment. Numerous studies highlight as an alternative to the use of conventional antifungals, the employment of essential oils and plant extracts, which are environmentally friendly and which have no adverse effects on human health. Against this background the present study aims to test six essential oils (*Lavandula angustifolia, Citrus limon, Mentha piperita, Marjoram, Melaleuca alternifolia, Origanum vulgare*) to establish their inhibitory effects against fungi identified on an old piece of traditional Romanian clothing from Maramure¸s. For the study, the types of fungi present on the objects was determined primarily through the open plates technique and microscopic identification. After identification, the essential oils were applied to the delimited surfaces, and their effects observed up to 32 days after application. The results show that these essential oils have a strong inhibitory effect on such fungal genera as *Penicillinum* sp., *Cladosporium* sp., *Aspergillus* spp., *Candida guillermondii, Botrys* sp., *Mucor* sp., having no observable side-effects on the physical properties of the materials concerned. The antimicrobial effects that essential oils and plant extracts have in the short term must be tested in future to ensure the enhanced preservation of heritage textiles and the health integrity of the restorers and visitors who view them in museums, collections or exhibitions.

**Keywords:** cultural heritage; materials; fungi; essential oils; antifungal; inhibitory effects

#### **1. Introduction**

The traditional folk costume from Maramure¸s is an important component of the Romanian cultural heritage. Certain elements of the peasant's traditional garments are two thousand years old, as evidenced by the scenes on Trajan's Column in Rome, Italy

**Citation:** Ilies, , D.C.; Hodor, N.; Indrie, L.; Dejeu, P.; Ilies, , A.; Albu, A.; Caciora, T.; Ilies, , M.; Barbu-Tudoran, L.; Grama, V. Investigations of the Surface of Heritage Objects and Green Bioremediation: Case Study of Artefacts from Maramure¸s, Romania. *Appl. Sci.* **2021**, *11*, 6643. https:// doi.org/10.3390/app11146643

Academic Editors: Filomena De Leo and Daniela Isola

Received: 2 June 2021 Accepted: 14 July 2021 Published: 20 July 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

(113 AC), those on the Monument to Adamclisi, *Tropaeum Traiani* (108–109 AC), from Dobruja, Romania and some other important archaeological discoveries from Romania. The preservation and conservation of popular traditional cloths is of great importance, especially now, when the modernisation of society and globalisation generate a growing standardization and lack of differentiation in the appearance of outfits [1,2], most especially those for men. While Maramure¸s celebration clothes still retain their former appearance, recently some materials and colours have come to be produced by relatively advanced and modernised techniques. The traditional objects are those most subject to degradation, due to the numerous insects, moths, fungi, bacteria and mechanical forces, among others, to which they are subjected over the years. At the same time traditional methods of preservation and conservation can be lost. Therefore, the preservation, in good condition, of the old components of traditional garments has acquired increasing importance, for the use, the identity and the pride of the future inhabitants of the region/country. Arguably, Maramures, has become an identity brand, symbolising the art and the creative power, as well as the youth, vigour and elegance of the Romanian inhabitants of Maramures, .

Under investigation is an aged garment object which is mainly made up of natural sheep's wool; for the popular motifs that adorn it materials as sheepskin, cotton yarn, pearly beads and hemp yarn also are used. All of these materials are organic, undergoing chemical processing to give them a long life, stability and elasticity, if they are stored and used properly. Historically, the causes of damage to natural materials vary over time. Biological damage is a factor that makes the leather and furs particularly vulnerable. Due to the acidity of the skin under conditions of high humidity, the development potential for colonies of microorganisms is very high, with them adhering to the substrate along with dust particles and other environmental substances. Furs and wool can be attacked by insects such as moth larvae, beetles and others, and specific microclimatic conditions, characterised by conditions of high humidity, low intensity of light and others, favour their multiplication. Bio-damage can lead, in time, to the complete destruction of the object. Physical and chemical damage is caused by natural factors, with chemical damage being faster than physical damage, with the former's effects being profound and irreversible [3]. Deterioration can be caused by natural factors [4]. Temperature levels can pose problems that are difficult to solve in relation to the conservation of collections if favouring the development of moulds (along with high humidity), along with insects and rodents, and accelerating chemical damage to materials. The amount of damage incurred is increased when the materials are present in a relative humidity below 50%, under which conditions they tend to become dry and brittle, and their physical strength decreases. At high humidity or on contact with aqueous solutions, the collagen present is destroyed, with negative effects occurring in terms of destruction of the structure and an increase in the hydrophilicity of the fibres involved. Light, due to photochemical effects, can also cause significant degradation, in terms of the weakening of the resistance of the material substrate, friability, chromatic changes, rupture of collagen fibres and others. Ultraviolet light should be removed without affecting the lighting concerned. The presence of dust promotes biological attacks on the skin and furs. Damage to the objects due to anthropogenic causes can also occur, as a result of improper storage and exposure (e.g., materials being placed directly on top of one another, wrinkled, placed in contact with metal objects, fixed with needles and/or nails, the improper affixing of labels among others) [5,6].

Under certain environmental conditions the colonization of microorganisms is possible with the combination of factors including: temperature, humidity fluctuation, natural or artificial lighting (favorable premises for the installation and evolution of microorganisms are improper exposure to sunlight and weather [7–9], dust content and carbon dioxide high values or when nutrients are favorable); these can induce very frequent biodeterioration, alteration processes [10–12]. Wool fibers are most susceptible to attack by bacteria and fungi, especially in conditions where moisture can be accentuated due to the hygroscopic properties of wool. The microorganisms most frequently [13] mentioned in degradation of wool and other protein fibers are bacteria which belongs to the genus:

#### *Bacillus*, *Proteus*, *Actinomycetes (Streptomyces* sp.*)* and micro-fungi: *Aspergillus*, *Fusarium and Trichoderma*, *Penicillium*.

The biodeteriogens represented by fungi can lead to the deterioration of the cultural heritage organic composition materials, with their hyphae penetrating the substrate material favouring the germination of spores. The possible physical damage is likely to be colouring/discoloration, the smell of mould, fissures, fragment detachments, fragility and the variation of dyeing properties, among others [13–17]. Possible chemical damage and changes can be wrought by fungal-derived carboxylic acids, including oxalic, citric, succinic, formic, malic, acetic, fumaric and others. In addition, chemical changes can also be reflected in aesthetical damages to the cultural materials concerned, such as in the form of the discoloration and deterioration of the surfaces involved, leading to the appearance of stains that may alter the original colour of the garment [18–21]. The presence of organic residues (e.g., glue, dirt, dust) may accelerate the processes of degradation, with the aesthetical changes and chromatic alteration taking place. At the same time, they can lead to the loss of strength and elongation specific to the material, oxidation, discoloration or coloration, due to the pigments involved and the modification of the molecular structures present [22].

Remediation can be realised using toxic materials (e.g., ethylene oxide [EtO], gamma rays, etc.), but the use of such materials can significantly affect the biodiversity and ecological systems concerned. Therefore, specialists in the field try to apply environmentally and ecologically friendly biocides instead. The use of antifungal natural extracts (i.e., essential oils [EOs]) has always been a viable alternative to the use of harmful chemicals, with, for example, plant extracts (*Allium ursinum* and *Ocimum basilicum*) having been tested as successful antifungals [23].

EOs (e.g., lemon, spearmint, rosemary, fennel, marjoram, pin, eucalyptus, etc.) can successfully serve as part of a green bioremediation procedure to be applied in the sustainable conservation of artworks. Having been tested against microbial colonies isolated from different substrates using green potential strategies, they have been found to act as a sound alternative to traditional procedures in terms of their selective action, human safety and impacts on artworks [20,24–41]. The antifungal activity of *Origanum vulgare*, *Rosmarinus officinalis* and *Lavandula angustifolia* EOs has been tested, with positive results having been achieved against fungi isolated on stone (*Bipolaris* sp.) and against *Aspergillus* sp., *Penicillium* sp., and *Trichoderma* sp. on different objects by Stupar et al. [42], Savkovi´c et al. [43] and Bayramo ˘glu [44], among others. Some relevant studies in the field [45–50] confirm that thymus EO has been found to have good antimicrobial activity against *Bacillus, Staphylococcus*, *Fusarium* and *Aspergillus* spp., in comparison to the effects of EO with commercial biocide. Radwan et al. [49] point out the inhibitory effect of thyme, clove and cinnamon EOs on *Candida albicans* and different finds of mould. The EOs of eucalyptus and lavender tested with good results as natural preservatives for leather [51], with oregano EO being successfully used as a bactericidal agent in the leather industry by Bayramo ˘glu et al. [26,44]. Combinations of the EOs [52], *Thymus vulgaris* and *Pimpinella anisum* and methanol extracts, have shown outstanding antibacterial properties against the pathogenic bacteria, *Staphylococcus aureus, Bacillus cereus, Escherichia coli, Proteus vulgaris, Proteus mirabilis, Salmonella typhi, Salmonella typhimurium, Klebsiella pneumoniae* and *Pseudomonas aeruginosa*. EOs obtained from *Matricaria chamomilla* have been tested [53] as environmentally friendly in cultural heritage environments, against *Aspergillus* spp.; similarly, Mahilrajan et al. [54] pinpoint in the same study, the intense antifungal activity of camphor oil, in comparison with other tested oils. Pepa et al. [55] point out that EOs from southern Italy, derived from *Origanum*, tested with the minimal inhibitory concentrations, *in vitro*, with evident antibacterial and antifungal activity. EOs like *Origanum vulgare* and *Thymus vulgaris* have also been applied by Palla et al. [56] to emphasise the green remediation in the biodeterioration of cultural heritage artworks, which is induced by fungal colonisation (e.g., by that of *Aspergillus flavus*). The above-mentioned authors concluded that valid green conservation strategies

(with no negative effects for human health and no environmental pollution) could possibly replace traditional biocides.

In Romania, recently, an EO isolated from thyme *(Thymus vulgaris)* was assayed for antifungal activity against *Candida albicans* and *Aspergillus niger* on sheepskins, with it being shown to have outstanding antifungal proprieties [57–59]. Moreover, the studies by Niculescu et al. [60,61] and Marcu et al. [62] have emphasised the possibility of using antifungal ecological materials to treat cultural heritage objects made of natural leather.

Following the above, this research aimed to investigate the microbial load of an old heritage object, made of natural animal sheepskin, which metabolic activity could generate, thus accelerating the biodegradation process, and the loading of the aerosol with biological particles, like spores, toxins and allergens, as well as other harmful substances. Such action can cause problems regarding the health of workers and users and the application of natural biodegraders. Consequently, the antifungal properties of some EOs of *Lavandula angustifolia*, *Citrus limon*, *Mentha piperita*, *Marjoram*, *Melaleuca alternifolia*, *Origanum vulgare* were tested, with the results of their applications being recorded at certain time intervals.

#### **2. Materials and Methods**

#### *2.1. Investigated Object*

The object investigated was a handmade, short coat for young men, lined with sheepskin from the Mara Valley, Maramures, , Romania (Figure 1) [63–66], about 80 to 100 years old. The men's garment was created to cover the torso, with it being worn most often on important holidays or on Sundays.

**Figure 1.** Traditional Romanian men's coat; (**a**)—front view; (**b**)—back view.

The coat was made of hemp thread (also called '*pânză de tort*' in Romanian). The coat's lining consisted of sewn tanned lambskin, of curly wool. On all its edges and at the seams, an intensely dark-hued strip ('*cipcă*') or hem of red leather was applied, conveying a sense of perfect regularity and symmetry. On the inside, there was a large pocket on the left side, facilitating access by the right hand. The coat was decorated by hand-sewing (similar to embroidery), with coloured thread, and by applying decorative elements made of tanned leather, over the whole bridge of the coat, consisting of the tree of life, rose flowers called '*ruji*', stitched flowers encircling mirrors and pseudo zig-zag patterning. In the middle

of some rose flowers without mirrors, special metal buttons ('*bumbi*') had been placed, rounded and extended, with some fasteners facing inwards, which helped to affix them to the fabric structure, by means of their being bent. Near the free edge of the wings and on the back of the coat, close to the shoulders are several ornamental tassels (bell-shaped tassels) arranged symmetrically, in red, green and blue colours.

In the last half century, other examples of such fur coats (from Mara) have begun to be worn in other parts of Maramures, , becoming an identity brand [67–70], symbolising the art and creative power of the youth, as well as the vigour and elegance of the Romanians in Maramures, .

#### *2.2. Analytical Methods*

The procedure followed in the current study required the following materials: a delimiting frame; sterile swabs on a wooden rod; and six EOs with 100% purity from the Young Living Essential Oils, used for their antifungal properties [71–79]. The types of EOs employed were: *Mentha piperita, Lavandula angustifolia, Citrus limon, Melaleuca alternifolia, Marjoram* and *Origanum vulgare*. The products used consisted of: *Mentha piperita*, batch 103519, expiry date 7 August 2021; *Lavandula angustifolia*, batch 87491, expiry date 7 August 2021; *Citrus limon*, batch 103517, expiry date 7 August 2021; *Tea Tree*, batch 89075, expiry date October 2021; *Marjoram,* batch 86942, expiry date July 2022; and *Oregano*, batch 89094, expiry date September 2022. The material resources used consisted of: Sabouraud sterile culture medium for the yeast and mould isolation; an incubator ICT 18/FALC with a temperature range between 5 and 80 ◦C; a microbiological hood; glass slides and slides for the microscopic identification; microbiological handles; an optical microscope Micros Austria with binocular head series BIM-105B; an API yeast identification kit ®® 20C AUX; and a densitometer.

Six different areas on the face of the traditional coat were examined, as shown in Figure 2. The working areas were delimited by means of a metal dial with a size of 25 cm2. Small fragments (millimetres) of fur and sheepskin were removed with tweezers previously sterilized by the incandescent method for testing of the fungi concerned; so as to observe the effects that they might have had on the individual strands of material. The tests were conducted both before and after applying the EOs to each delimited area, although the primary samples were considered as blank references.

After delimiting the surfaces on which the in-situ procedures were to be performed, the samples of the surface were taken. The samples were extracted using sterile swabs from all six areas of the sheepskin coat, both before and after the application of the Eos (see Table 1). Ten drops (the equivalent to 300 μL) of the corresponding EO were applied directly to the tested material surface in the centre of each delimited area [75]. After the application of the EOs, three samples were taken for each of the six different work areas delimited on the area of sheepskin to which the EOs were applied. The three samples were taken at pre-set intervals, with the first sample being taken 30 min after the administration of the EOs, the second test being administered after 24 h and the third after 48 h. Table 1 also shows the type of material examined and the type of EO applied to each of the delimited areas. The EO used for each type of material concerned was selected in accordance with the recommendations made in the literature, especially that of Palla et al. [56], which state that certain EOs tend to be more effective than others when applied to certain types of material, such as those which constitute heritage objects.

It must be noted that the air in the room in which the sheepskin coat was stored and examined was not circulated, and neither was the room ventilated for 48 h during the entire examination, so as to avoid contamination with other types of spores from the atmosphere that could, otherwise, have been introduced by currents of air moving through the room. The temperature was maintained between 22 ◦C and 24 ◦C, while the humidity was kept between 52% and 55%.

**Figure 2.** Delimitation of the six work areas on the front of the coat, indicating the type of essential oil applied to each (**A**)—*Lavandula angustifolia*; (**B**)—*Cistrus lemon*; (**C**)—*Mentha piperita*; (**D**)—*Melaleuca alternifolia*; (**E**)—*Marjoram*; (**F**)— *Origanum vulgare*.

**Table 1.** Areas of the sheepskin coat examined, and to which essential oils applied.

With the EOs having been applied directly to the examined surfaces, all the samples were seeded on sterile Sabouraud culture media, with the plates used being incubated for 30 days at a temperature of 28 ◦C. The third stage of the procedure involved isolating and identifying the fungi developed on culture media. The plates were evaluated daily to observe their evolution in terms of the changes in the appearance of the colonies concerned, with the evaluation undertaken sequentially being that of shape, texture, consistency, diameter, colour and contour.

The moulds present were identified after evaluating the macroscopic and microscopic characteristics involved, and the yeast species was determined, using the API ®® 20 ◦C AUX6 identification kit, after examination of the 19 biochemical assimilation reactions that took place. The technique of working on the latter involved preparing the yeast suspension solution, calibrated at 2 McFarland, using a densitometer, and thereafter pipetting 100 μL into each microdwell of the gallery. After incubating the API gallery at 29 ◦C ± 2 ◦C for 72 h, the results were read, with a final result being obtained after comparing each well with the negative control. The final result was a 7-digit numerical profile, which was decoded using ApiwebTM computer software, so as to identify the corresponding yeast species.

#### **3. Results**

Fungal colonies were developed rapidly on Sabouraud media, from samples taken before the application of EOs. At 72 h of incubation, the first fungal colonies could be macroscopically visualised. After 10 days of incubation, the plates were completely invaded. Figure 3 shows the plates on the seventh day of incubation; each image indicates the examined area of the sheepskin coat. The high degree of fungal contamination of the coat can easily be observed.

**Figure 3.** Plates seeded with primary samples (before application of essential oils). Reading taken after 7 days of incubation (**A**)—*Lavandula angustifolia*; (**B**)—*Cistrus lemon*; (**C**)—*Mentha piperita*; (**D**)—*Melaleuca alternifolia*; (**E**)—*Marjoram*; (**F**)—*Origanum vulgare*.

In the above-mentioned way, seven different types of colonies were identified [80,81], with six being from the mould class (*Alternaria* sp., *Aspergillus* sp., *Botrytis* sp., *Cladosporium* sp., *Mucor* sp. and *Penicillium* sp.) and one from the yeast class (*Candida guilliermondii*) (Table 2).

As previously stated the number of samples taken after applying the EOs was 3 sets of 6, taken 30 min, 24 h and 48 h after applying the corresponding EO. The plates were monitored for 30 days in order to evaluate the inhibitory effects of the EOs. Figure 4 shows the plates, as read on day 14 of the incubation. The inhibitory effects of the EOs are evident. The only plate on which a fungal colony developed is the one corresponding to the inner area of the sheepskin coat, present in the sample taken 48 h after applying the EO of *Melaleuca alternifolia* (tea tree). The isolated fungal colony consisted of *Cladosporium* sp., which became macroscopically visible on day 5 of incubation. It was observed that this type of mould was not isolated from the primary sample, as is detailed in the discussion section below.

#### **Table 2.** Identified fungi and the effect of essential oils.

**Figure 4.** The plates read on day 14 of incubation: the samples at (**a**) 30 min, (**b**) 24 h and (**c**) 48 h.

Figure 5a–c show the plates, as read on day 30 of the incubation. The developed colonies became macroscopically visible after between 20 days to 25 days of incubation.

**Figure 5.** The plates read on day 30 of incubation: samples at (**a**) 30 min, (**b**) 24 h and (**c**) 48 h.

The results, obtained after the samples from the examined areas were examinedat 30 min, 24 h and 48 h following the application of the EOs, are presented in Table 3. The existing difference was due to the duration of the effect that they had on certain types of fungi as shown on Table 4. It is evident that some EOs (e.g., *Lavandula angustifolia, Melaleuca alternifolia, Marjoram, Origanum vulgare*) inhibited the action of some fungal genera up to a period of 22 days, whereas on others the effect lasted for a period of at least 30 to 32 days (Table 4).

**Table 3.** Interpretation of the seeded plates, with samples taken 30 min, 24 h and 48 h after applying the essential oil.

**Table 4.** Fungal species inhibited by tested essential oils and the tested duration of their inhibitory effect.

#### **4. Discussion**

After the tests were performed on the delimited surfaces of the 80- to 100-year-old clothing object, the conclusion was drawn that all the EOs had a strong inhibitory effect on the fungi present, even if they were applied on different materials (cotton yard, leather, wool, pearly beads). This is due to the fungicidal effect of EOs, which persists in some cases over 30 days after application. Similar results were obtained by different authors [82–85] following the application of these extracts on various materials. Most EOs (except for *Citrus limon* and *Mentha piperita*) were observed to have relatively little effect on *Botrytis* sp., which might indicate some resistance of this type of fungus to the action of the substances concerned.

It was revealed that the genus *Cladosporium* sp. developed at 5 days of incubation on the culture medium shown with the sample taken 48 h after the application of the EO, without the species involved having been isolated from the primary sample (taken before the application of the EO). The sample concerned was taken from the wool threads of the inner region of the coat. Woollen garments seem to be easily contaminated with fungal spores, with the degree of fungal contamination being most likely to differ from layer to

layer—superficial, intermediate and basal. The spores that are present in the lower layers may rise to the surface over time, and, if the EO is applied superficially or in insufficient quantity, its inhibitory effect is diminished. Moreover, in the case of studies like the present, the environmental conditions present in the room (temperature, humidity, air currents) must be taken into account, as well as the sampling procedure itself. All such factors form a set of features that can interfere with the final results.

The fungal action identified on the garment investigated could be pathological for humans, especially for the personnel handling the garment. Although the fungi of the *Aspergillus* spp. family are generally harmless, they can cause various pathologies in those with compromised immune systems, underlying lung disease or asthma, who inhale the relevant fungal spores. The pathologies resulting from infections with such a fungus, which usually affect the respiratory system, have greatly varying signs and severity. In some (such as asthmatics), the spores trigger an allergic reaction, whereas others tend to develop mild to severe lung infections. The most serious form of aspergillosis, invasive aspergillosis, occurs when the infection spreads through the blood vessels, with the signs and symptoms of the infection varying, depending on the type of pathology that the patients concerned develop, and with it not being contagious. Daily exposure to *Aspergillus* is rarely a problem for people with healthy immune systems [86–88]. Fungi from the *Candida guilliermondii* family appear to be one of the most common pathogens in localised infections of the nails (onychomycosis) and/or of the skin on the toes. In patients with low immunity, infections with such a fungus can be a potential cause of fungal infections in the blood, namely fungemia, especially in patients with haematological malignancies, organ transplants and central venous catheters. This type of fungus is one of the most common opportunistic agents in severely immunocompromised patients. In such cases, depending on the associated pathologies, fungemia can prove fatal [89]. Fungal infections of the *Cladosporium* spp. family either aggravate the symptoms of an existing asthma, or are associated with atopy located mainly in the upper respiratory tract (allergic rhinitis/sinusitis) and less often in the skin (superficial or deep skin lesions). The impact of patients' symptoms (dyspnoea, sneezing, stuffy nose, pruritus, nasal/ocular secretions) was significantly correlated with spore concentrations. In patients with weakened immune systems, infections with this type of fungus can even cause disseminated infections, called fungemia [90–92]. Fungi of the genus *Penicillium* spp., which are occasionally the cause of infection in humans, can be isolated from patients with keratitis, endophthalmitis, otomycosis, necrotising esophagitis, pneumonia, endocarditis, peritonitis and urinary tract infections. Most *Penicillium* infections occur in immunosuppressed hosts, and the mechanism of infection with this type of fungus is either by means of inhalation (most commonly resulting in fungemia) or post-traumatic [93,94].

#### **5. Conclusions**

The clothing objects that make up the cultural heritage are prone to deterioration, due to fungal action, especially if they are stored or exposed in environments that lack careful adjustment of the internal microclimate parameters. In addition to the harmful effects that fungi can have on heritage objects, people (restorers, museographers, collectors, visitors) who come in contact with such infested objects can develop specific conditions. Thus, treating historical textiles in the most responsible way possible becomes a dual purpose issue; on the one hand, it is necessary to preserve the materials under the best possible conditions, and, on the other hand, it is necessary to ensure the integrity of the health of those who are interested in them. The current study demonstrates that EOs are one of the most viable solutions to help ensure responsible treatment given that their antifungal properties can exceed 30 days of use, and with the products being both ecofriendly and accessible in terms of cost. The six EOs (*Lavandula angustifolia, Citrus limon, Mentha piperita, Melaleuca alternifolia, Marjoram, Origanum vulgare*) applied on different types of materials served to inhibit the presence of six different fungal colonies (*Alternaria* sp., *Aspergillus* sp., *Botrytis* sp., *Cladosporium* sp., *Mucor* sp. and *Penicillium* sp.) and a class of yeast (*Candida*

*guilliermondii*) identified as being on the traditional coat. Simultaneously, the EOs had a strong antifungal effect on several materials (cotton, leather, wool) that formed part of the object investigated. Thus, all of the products studied were proven to be effective on certain surfaces and against certain types of fungi, in terms of both their cleaning and their protection effects. Prospects for the future must include the use of natural antifungals for the treatment of heritage objects; but at the same time, the tests must also aim at determining the possible negative effects that they may have on fragile objects.

**Author Contributions:** Conceptualisation: D.C.I. and N.H.; Methodology: P.D. and L.I.; Software: L.B.-T. and A.A.; Validation: P.D. and A.A.; Formal analysis: P.D., L.I. and L.B.-T.; Investigation: P.D., A.I. and M.I.; Resources: L.I. and A.A.; Data curation: P.D., D.C.I., N.H. and T.C.; Writing—original draft preparation: D.C.I., P.D., M.I. and T.C.; Writing—review and editing: D.C.I., T.C., A.I., M.I. and V.G. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research was funded by grant PN-III-P1-1.2-PCCDI-2017-0686.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** The data presented in this study may be obtained on request from the corresponding author.

**Acknowledgments:** The research undertaken was made possible by the equal scientific involvement of all the authors concerned. The authors wish to thank the anonymous reviewers for their thoughtful suggestions and comments made, and to acknowledge the support of the grant PN-III-P1-1.2-PCCDI-2017-0686. The investigations and analyses involved were done at the Medical Laboratory Service M.D., Bethany Medical Clinic Oradea, University of Medicine and Pharmacy of University of Oradea and at the National Institute of Research-Development for Isotopic and Molecular Technologies, Cluj-Napoca.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**

## *Article* **Identification of Fungal Community Associated with Deterioration of Optical Observation Instruments of Museums in Northern Vietnam**

**Cao Cuong Ngo 1,2,†, Quang Huy Nguyen 3,†, Thu Hoai Nguyen 2, Ngoc Tung Quach 1,4, Pravin Dudhagara 5, Thi Hanh Nguyen Vu 1,4, Thi Thanh Xuan Le 1, Thi Thu Hang Le 3, Thi Thu Hong Do 2, Van Duc Nguyen 6, Nam Trung Nguyen 1,4 and Quyet-Tien Phi 1,4,\***

**Abstract:** Fungi are the most harmful microorganisms responsible for the deterioration of nonmetallic materials such as glass, polymers, and composites. To date, biological aspects of glass deterioration have been poorly investigated. The present study aimed to evaluate the diversity of the fungal community colonizing eyepieces of binoculars collected from museums of the northern provinces of Vietnam and the biodeterioration effects on accurate glass reproductions. A total of 40 isolates belonging to 14 genera were identified based on internal transcribed spacer (ITS) sequencing, morphological features, and maximum likelihood analysis. The most abundant fungal genera included *Aspergillus* (43.8%) and *Penicillium* (31.3%). Among those detected, *Byssochlamys*, *Curvularia*, *Phomopsis*, *Coprinellus*, *Perenniporia*, *Talaromyces*, *Pithomyces*, *Neopestalotiopsis*, *Trichoderma*, *Pleospora,* and *Humicola* were found for the first time. Of the 40 strains tested, 8 strains showed great organic acid production, and the extent of mycelium covered from 33.6 to 46.24%. Specifically, the highest extracellular polymeric substance production was observed in *Byssochlamys spectabilis* BXMA1-2 (14.96 g/L), *Aspergillus niger* BXMA5-2 (12.17 g/L), and *Aspergillus ochraceopetaliformis* BMLC1-2 (9.89 g/L). Glass biodeterioration experiments revealed that the light transmission through the fungal-treated glasses was decreased by 30–42.2% as compared to the nontreated glass. In addition, the main alterations resulted from hyphal fingerprints and spots, leading to apparent damage and biocorrosion.

**Keywords:** glass corrosion; biodeterioration; optical instruments; fungal community; *Aspergillus*; *Penicillium*; light transmission

#### **1. Introduction**

For many years, the deterioration and corrosion of glass due to physicochemical processes have been documented [1]. Moreover, the role of microorganisms in glass deterioration has been investigated in recent decades [2–5]. Many studies have proved that along with the physicochemical attack, the deterioration process is strongly stimulated by microbial contamination. Microorganisms can grow on glass materials and cause damage

**Citation:** Ngo, C.C.; Nguyen, Q.H.; Nguyen, T.H.; Quach, N.T.; Dudhagara, P.; Vu, T.H.N.; Le, T.T.X.; Le, T.T.H.; Do, T.T.H.; Nguyen, V.D.; et al. Identification of Fungal Community Associated with Deterioration of Optical Observation Instruments of Museums in Northern Vietnam. *Appl. Sci.* **2021**, *11*, 5351. https://doi.org/10.3390/ app11125351

Academic Editor: Gyungsoon Park

Received: 5 May 2021 Accepted: 5 June 2021 Published: 9 June 2021

**Publisher's Note:** MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

**Copyright:** © 2021 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https:// creativecommons.org/licenses/by/ 4.0/).

in situ, such as crack formation, pitting, etching, chipping, leaching, and discoloration [6]. Consequently, the glass will lose its transparency and quality as well as light transmission. Many studies have been conducted mainly focusing on the deterioration of stained-glass samples at historic churches in Germany [7], Spain [8,9], and Brazil [10], but little is known about the biodeterioration of modern glass materials of optical instruments.

Among microorganisms, fungi are very widely dispersed and grow in almost every environment on the planet. Fungi are the most common biological invaders responsible for the biodegradation and biocorrosion of glass materials [3,6,10]. Fungi are well-known producers of organic acids and therefore contribute to the biodegradation processes [4,11]. The presence of a tiny amount of substrates from water vapor in the air is enough to initiate the life of fungi on the surface of the glass. The inorganic composition and physical properties of the glass support fungal growth, and fungi can acquire the elements needed for growth from the glass itself [12].

Moreover, fungi can form spores highly resistant to adverse conditions and easily travel into the air, binding to all materials [13]. The biodegradation of glass caused by fungi often occurs slowly but continuously [6]. *Aspergillus, Cladosporium, Trichoderma, Penicillium, Chaetomium, Aureobasidium, Eurotium, Phoma, Scopulariopsis*, and *Rhizopus* are commonly reported on the surface of glass materials [2,6,10]. Experimental evidence showed that significant chemical and morphological changes in the surface layer were observed in a short period after inoculation with fungi [3,4].

The tropical climate, characterized by temperatures above 25 ◦C and humidity of 80–100%, imposes challenging conditions for preserving materials. Such environmental conditions are perfect for the growth of fungi and, therefore, facilitate the colonization of inorganic materials, including glass surfaces [2,7]. Especially in the rainy season, many of the nutrients come continuously from water vapor in the air, promoting biodeterioration and corrosion activities of fungi [10]. Fungus-mediated alteration of the glass is a slow and multistep process starting with the deposition of the organic material on the glass surface [14]. In suitable environmental conditions, the glass surface facilitates the growth of airborne fungal species. Colonization of fungi initiates biofilm formation, and slowly the mineralization process occurs on the glass surface, leading to glass decay. In many African, South-East Asian, and Latin American regions, high temperature and relative humidity provide perfect growth conditions for fungi attacking glass materials, especially optical instruments [15]. However, the risk and severity of damage to instruments vary widely within these regions. Moreover, a recent study showed that the biodeterioration of optical glass is induced by lubricant used in the operation of optical instruments [16]. So, timely cleaning of the glass surface is essential for the durability and service life of glass and optical instruments.

In this context, the present study aimed to evaluate the co-occurrence and diversity of culturable fungi isolated from eyepieces of binoculars in the northern provinces of Vietnam. Another objective of this work was to assess the biodeterioration of optical glass reproductions by isolated fungi under laboratory conditions. Understanding the fungal community growing on eyepieces of binoculars plays an important role in developing modern glasses resistant to harmful fungi. In addition, this finding will also provide crucial information for effective conservation solutions and prolong the use of optical devices in studied areas.

#### **2. Materials and Methods**

#### *2.1. Materials*

The binoculars (model 6nu5 8 × 30 M) used frequently for nature and bird watching were collected between June and September 2019 from a total of 3 three different museums located in northern Vietnam, including Muong Cultural Space Museum, Hanoi city; Museum of Biology, Phu Tho province; Thu Museum, Vinh Phuc province. In each museum, five binoculars used for around 4 years were collected. During 4 years of use, the exterior of binoculars was cleaned with ethanol solution every month. After that, five eyepieces

contaminated with fungi were removed from five binoculars, placed in sterile plastic bags, and directly transported to the laboratory for fungal isolation.

#### *2.2. Methods*

#### 2.2.1. Microscopical Investigations

The surface of fungus-contaminated eyepieces was primarily observed using an OP-TIKA stereo microscope at a magnification of 60×. The figures were taken by using a digital camera via OPTIKA Vision Pro software. ImageJ v.1.51 software was used to analyze the figures to assess the level of fungal mycelium covering the glass surface. The colonization of fungi on the surface of eyepieces was then examined under a JEOL 5410 scanning electron microscope (SEM) (Japan). After that, the surface of eyepieces was cleaned twice with ethanol 70% to remove biomass and fungal byproducts (biofilms, biogenic crystals) entirely, and the biodeterioration patterns on the glass surface were analyzed.

#### 2.2.2. Fungal Isolation

For each sample, the surface of eyepieces was swabbed using a sterile tube previously wetted in sterilized water and submerged into 1 mL sterile water containing 0.05% Tween 80, shaking at 200 rpm/min for 30 min. About 100 μL of the supernatant was then spread on a Czapek–Dox agar medium (30 g sucrose, 7.5 g peptone, 1 g K2HPO4, 0.5 g MgSO4·7H2O, 0.5 g KCl, 0.01 g FeSO4, pH 7.0) and incubated at 30 ◦C for 4 to 5 days. Fungal colonies were streaked to obtain pure strains for the analysis of morphological features. Fungal spore and spore chain were observed under a light microscope.

#### 2.2.3. Identification and Analysis of Fungal Community