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Cave heritage is often threatened by tourism or even scientific activities, which can lead to irreversible deterioration. We present a preventive conservation monitoring protocol to protect caves with rock art, focusing on La Garma Cave (Spain), a World Heritage Site with valuable archaeological materials and Palaeolithic paintings. This study assessed the suitability of the cave for tourist use through continuous microclimate and airborne particles monitoring, biofilm analysis, aerobiological monitoring and experimental visits. Our findings indicate several factors that make it inadvisable to adapt the cave for tourist use. Human presence and transit within the cave cause cumulative effects on the temperature of environmentally very stable and fragile sectors and significant resuspension of particles from the cave sediments. These environmental perturbations represent severe impacts as they affect the natural aerodynamic control of airborne particles and determine bacterial dispersal throughout the cave. This monitoring protocol provides part of the evidence to design strategies for sustainable cave management.

Castañar is a cave with strict visitor control measures since it was open to public visits in 2003. However, in recent years, the cave suffered two fungal outbreaks, the first in 2008 and controlled by cleaning the contaminated sediments and subsequent closure of the cave until 2014. The cave was reopened but limited to a maximum of 450 visitors/year. Despite these restrictions on visit, the cave experienced a second outbreak in 2021, originating from the installation of a steel grating walkway, aiming at protecting the ground sediments from the visitors’ footsteps. Here, we conducted an analysis using Next-Generation Sequencing and culture-dependent techniques to investigate the fungal communities related to the second outbreak and compare with those present before the cave suffered the outbreak. The results show that the most abundant fungi involved in the 2021 outbreak were already detected in 2020, and even in 2008 and 2009, although the main species that originating both outbreaks were different, likely due to the different carbon sources introduced into the cave.

The Gypsum Karst of Sorbas, Almeria, southeast Spain, includes a few caves whose entrances are open and allow the entry and roosting of numerous bats. Caves are characterized by their diversity of gypsum speleothems, such as stalactites, coralloids, gypsum crusts, etc. Colored biofilms can be observed on the walls of most caves, among which the Covadura and C3 caves were studied. The objective was to determine the influence that bat mycobiomes may have on the fungal communities of biofilms. The results indicate that the fungi retrieved from white and yellow biofilms in Covadura Cave (Ascomycota, Mortierellomycota, Basidiomycota) showed a wide diversity, depending on their location, and were highly influenced by the bat population, the guano and the arthropods that thrive in the guano, while C3 Cave was more strongly influenced by soil- and arthropod-related fungi (Ascomycota, Mortierellomycota), due to the absence of roosting bats.

Cueva del Viento, located in the Canary Islands, Spain, is the Earth's sixth-longest lava tube, spanning 18,500 m, and was formed approximately 27,000 years ago. This complex volcanic cave system is characterized by a unique geomorphology, featuring an intricate network of galleries. Despite its geological significance, the geomicrobiology of Cueva del Viento remains largely unexplored. This study employed a combination of culture-dependent techniques and metabarcoding data analysis to gain a comprehensive understanding of the cave's microbial diversity. The 16S rRNA gene metabarcoding approach revealed that the coloured microbial mats (yellow, red and white) coating the cave walls are dominated by the phyla Actinomycetota, Pseudomonadota and Acidobacteriota. Of particular interest is the high relative abundance of the genus Crossiella, which is involved in urease-mediated biomineralization processes, along with the presence of genera associated with nitrogen cycling, such as Nitrospira. Culture-dependent techniques provided insights into the morphological characteristics of the isolated species and their potential metabolic activities, particularly for the strains Streptomyces spp., Paenarthrobacter sp. and Pseudomonas spp. Our findings underscore the potential of Cueva del Viento as an ideal environment for studying microbial diversity and for the isolation and characterization of novel bacterial species of biotechnological interest.

European caves contain some of the world’s greatest Paleolithic paintings, and their conservation is at risk due to the use of artificial lighting. Both lighting and high CO2 promotes the growth of phototrophic organisms on walls, speleothems and ground sediments. In addition, the combined effect of increases in CO2, vapor concentration and temperature variations induced by visitors can directly affect the development of corrosion processes on the cave rock surfaces. An early detection of the occurrence of phototrophic biofilms on Paleolithic paintings is of the utmost importance, as well as knowing the microorganisms involved in the colonization of rocks and walls. Knowledge of the colonizing species and their ecology will allow the adoption of control measures. However, this is not always possible due to the limited amount of biomass available for molecular analyses. Here, we present an alternative approach to study faint green biofilms of Chlorophyta in the initial stage of colonization on the Polychrome Panel in El Castillo Cave, Cantabria, Spain. The study of the biofilms collected on the rock art panel and in the ground sediments revealed that the lighting of the cave promoted the development of the green algae Jenufa and Coccomyxa, as well as of complex prokaryotic and eukaryotic communities, including amoebae, their endoparasites and associated bacteria and fungi. The enrichment method used is proposed as a tool to overcome technical constraints in characterizing biofilms in the early stages, allowing a preliminary characterization before deciding for direct or indirect interventions in the cave.

Antimicrobial resistance has become a global problem in recent decades. A gradual reduction in drug discoveries has led to the current antimicrobial resistance crisis. Caves and other subsurface environments are underexplored thus far, and they represent indispensable ecological niches that could offer new molecules of interest to medicine and biotechnology. We explored Spanish show caves to test the bioactivity of the bacteria dwelling in the walls and ceilings, as well as airborne bacteria. We reported the isolation of two strains of the genus Crossiella, likely representing a new species, isolated from Altamira Cave, Spain. In vitro and in silico analyses showed the inhibition of pathogenic Gram-positive and Gram-negative bacteria, and fungi, as well as the taxonomical distance of both strains from their closest relative, Crossiella cryophila. The presence of an exclusive combination of gene clusters involved in the synthesis of lanthipeptides, lasso peptides, nonribosomal peptides and polyketides indicates that species of this genus could represent a source of new compounds. Overall, there is promising evidence for antimicrobial discovery in subterranean environments, which increases the possibility of identifying new bioactive molecules. Crossiella; predicted gene clusters; Spanish show caves

Two Streptomyces strains, labeled as MZ03-37(T) and MZ03-48, were isolated from two different samples, a mucolite-type speleothem and a microbial mat on the walls of a lava tube from La Palma Island (Canary Islands). Phylogenetic analysis based on concatenated sequences of six housekeeping genes indicated that both strains belonged to the same species. The closest relatives for both strains were Streptomyces palmae CMU-AB204(T) (98.71%), Streptomyces catenulae NRRL B-2342(T) (98.35%), and Streptomyces ramulosus NRRL B-2714(T) (98.35%). Multi-locus sequence analysis (MLSA), based on five house-keeping gene alleles (i.e., atpD, gyrB, recA, rpoB, and trpB), indicated that both isolated strains were closely related to S. catenulae NRRL B-2342(T). Whole-genome average nucleotide identity (ANI) scores of both strains were in the threshold value for species delineation with the closest species. Both strains presented a G+C content of 72.1 mol%. MZ03-37(T) was light brown in substrate and white in aerial mycelium, whereas MZ03-48 developed a black aerial and substrate mycelium. No pigment diffusion was observed in both strains. They grew at 10 degrees C-37 degrees C (optimum 28 degrees C-32 degrees C) and in the presence of up to 15% (w/v) NaCl. MZ03-37(T) grew at pH 5-10 (optimal 6-9), whereas MZ03-48 grew at pH 4-11 (optimal 5-10). LL-Diaminopimelic acid was the main diamino acid identified. The predominant fatty acids in both strains were iso-C-16:0, anteiso-C-15:0, C-16:0, and iso-C-14:0. The major isoprenoid quinones were MK-9(H6) and MK-9(H8), and the main polar lipids were aminolipid, phospholipid, and phosphoglycolipid. In silico analyses for functional annotation predicted the presence of gene clusters involved in resistome mechanisms and in the synthesis of described antimicrobials such as linocin-M18 and curamycin, as well as different genes likely involved in mechanisms for active compound synthesis, both already described and not discovered so far. On the basis

Castañar Cave is a clear example of an oligotrophic ecosystem with high hygrothermal stability both seasonal and interannual and the particularity of registering extraordinary levels of environmental radiation. These environmental conditions make the cave an ideal laboratory to evaluate both the responses of the subterranean environment to sudden changes in the matter and energy fluxes with the exterior and also any impact derived from its use as a tourist resource under a very restrictive access regime. In 2008, a fungal outbreak provoked by a vomit contaminated the sediments which were removed and subsequently treated with hydrogen peroxide. Fungal surveys were carried out in 2008 and 2009. The visits were resumed in 2014. Here, 12 years after the outbreak, we present an exhaustive study on the cave sediments in order to know the distribution of the different fungal taxa, as well as the prevalence and spatio-temporal evolution of the fungi caused by the vomit over the years under the conditions of relative isolation and high radiation that characterize this cave.

Contents About the Editors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . vii Filomena De Leo and Valme Jurado Editorial for the Special Issue “Microbial Communities in Cultural Heritage and Their Control” Reprinted from: Appl. Sci. 2021, 11, 11411, doi:10.3390/app112311411 .. . . . . . . . 1 Zuzana Kisov´a, Matej Plan´ y, Jelena Pavlovi´c, M´aria Buˇckov´a, Andrea Puˇsk´arov´a, Lucia Krakov´a, Magdal´ena Kapustov´a, Domenico Pangallo and Katar´ına ˇ Soltys Biodeteriogens Characterization and Molecular Analyses of Diverse Funeral Accessories from XVII Century Reprinted from: Appl. Sci. 2020, 10, 5451, doi:10.3390/app10165451 . . . . . 5 Yin Jia, Liuyu Yin, Fengyu Zhang, Mei Wang, Mingliang Sun, Cuiting Hu, Zijun Liu, Yue Chen, Jie Liu and Jiao Pan Fungal Community Analysis and Biodeterioration of Waterlogged Wooden Lacquerware from the Nanhai No. 1 Shipwreck Reprinted from: Appl. Sci. 2020, 10, 3797, doi:10.3390/app10113797 . . . . . . . . . . . . . . . . . 25 Fernando Bol´ıvar-Galiano, Clara Abad-Ruiz, Pedro S´anchez-Castillo, Maurizio Toscano and Julio Romero-Noguera Frequent Microalgae in the Fountains of the Alhambra and Generalife: Identification and Creation of a Culture Collection Reprinted from: Appl. Sci. 2020, 10, 6603, doi:10.3390/app10186603 . . . . . . . . . . . . . . . . . 39 Giulia Caneva, Daniela Isola, Hyun Ju Lee and Yong Jae Chung Biological Risk for Hypogea: Shared Data from Etruscan Tombs in Italy and Ancient Tombs of the Baekje Dynasty in Republic of Korea Reprinted from: Appl. Sci. 2020, 10, 6104, doi:10.3390/app10176104 . . . . . . . . . . . . . . . . . 57 Silvia Sparacello, Giuseppe Gallo, Teresa Faddetta, Bartolomeo Megna, Giovanna Nicotra, Beatrice Bruno, Belinda Giambra and Franco Palla Thymus vulgaris Essential Oil and Hydro-Alcoholic Solutions to Counteract Wooden Artwork Microbial Colonization Reprinted from: Appl. Sci. 2021, 11, 8704, doi:10.3390/app11188704 . . . . . . . . . . . . 83 Lucre

Show caves have different grades of colonization by phototrophic biofilms. They may receive a varied number of visits, from a few thousand to hundreds of thousands of visitors annually. Among them, Tesoro Cave, Rincon de la Victoria, Spain, showed severe anthropic alterations, including artificial lighting. The most noticeable effect of the lighting was the growth of a dense phototrophic community of cyanobacteria, algae and bryophytes on the speleothems, walls and ground. The biofilms were dominated by the cyanobacterium Phormidium sp., the chlorophyte Myrmecia israelensis, and the rhodophyte Cyanidium sp. In many cases, the biofilms also showed an abundance of the bryophyte Eucladium verticillatum. Other cyanobacteria observed in different biofilms along the cave were: Chroococcidiopsis sp., Synechocystis sp. and Nostoc cf. edaphicum, the green microalgae Pseudococcomyxa simplex, Chlorella sp. and the diatom Diadesmis contenta. Preliminary cleaning tests on selected areas showed the effectiveness of hydrogen peroxide and sodium hypochlorite. A physicochemical treatment involving the mechanical removal of the thickest layers of biofilms was followed by chemical treatments. In total, 94% of the surface was cleaned with hydrogen peroxide, with a subsequent treatment with sodium hypochlorite in only 1% of cases. The remaining 5% was cleaned with sodium hypochlorite in areas where the biofilms were entrapped into a calcite layer and in sandy surfaces with little physical compaction. The green biofilms from the entire cave were successfully cleaned.

The present study reports a sudden fungal outbreak that occurred in the corridor near the entrance of the Catacombs of SS. Marcellino and Pietro in Rome (Italy) observed after 1 year of a restoration treatment that interested the walls of the entrance of the Catacombs and some artifacts placed in situ. The colonization was observed on the vault at the entrance and in correspondence with the restored marble pieces displayed on the left side of the corridor. No growth was observed on the right side where similarly treated marble slabs were placed. Samples taken in correspondence with fungal biofilm were analyzed through the combined use of microscopical, cultural, and molecular tools and showed that the vault and the left side of the corridor entrance were colonized by a complex fungal biofilm consisting mainly of Coniophora sp. and other genera, such as Hypomyces, Purpureocillium, Acremonium, Penicillium, and Alternaria, many of which are well known as responsible of biodeterioration of stone surfaces. Regarding the brown-rot basidiomycete Coniophora, it was able to form very large colonies on the substrata with a diameter of up to 57 cm. Although the direct observation under a light microscope evidenced the presence of abundant brown fungal conidia, several attempts to cultivate the microorganism failed, therefore only through DNA sequencing analyses, it was possible to identify and characterize this fungus. There is very little literature on the genus Coniophora which is reported as one of the causes of wet-rot decay of wood in buildings. A connection with calcium-containing materials such as bricks and mortars was demonstrated, but no data were available about the possible role of this species in the biodeterioration of stones. This study features the first finding of a strain related to the basidiomycetous genus of Coniophora in the order Boletales in association with evident phenomena of biodeterioration.

Los tubos de lava se encuentran distribuidos por todo el mundo. Estas cuevas volcánicas, inicialmente formadas por el flujo de lava de un volcán en erupción, son habitadas por complejas comunidades microbianas una vez alcanzan la temperatura ambiente. En las paredes y techos del tubo de lava de la Cueva del Viento (Icod de los Vinos, Tenerife, España) se han encontrado colonizaciones microbianas que forman biopelículas de diferentes colores (blancas, amarillas y rojas). Esta cueva es uno de los tubos de lava más grande del mundo, y su nombre se debe a las importantes corrientes de aire que se producen en su interior. La Cueva del Viento presenta unas características geomorfológicas únicas y una gran diversidad de fauna subterránea. Sin embargo, la información disponible sobre la biodiversidad bacteriana presente en su interior es limitada. En este trabajo se llevaron a cabo técnicas de cultivo y análisis moleculares para la caracterización de las biopelículas microbianas y comprender su biodiversidad, así como el estado de conservación de esta cavidad volcánica. Se aislaron 42 cepas bacterianas, distribuidas principalmente en los filos Actinomycetota y Bacillota. El género más numeroso dentro de cada filo fue Streptomyces (ocho cepas), el cual es abundante en suelos, y Bacillus (nueve cepas), respectivamente. La especie Paenarthrobacter nicotinovorans, perteneciente al filo Actinomycetota y hallada en las muestras de colonias amarillas y blancas (cinco cepas), se caracteriza por degradar nicotina, atrazina y otros herbicidas nitrogenados, lo que evidenciaría una posible contaminación proveniente del suelo localizado sobre la cueva. La muestra más diversa correspondió a la biopelícula de color blanco, situada en una de las zonas más contaminadas de la cueva, donde se aislaron 14 cepas bacterianas. La caracterización de las comunidades microbianas presentes en la Cueva del Viento ayudará a comprender mejor su diversidad y a concienciar al público sobre la importancia

Las cuevas vienen siendo estudiadas desde diversos puntos de vistas: ambiental, geológico, geoquímico, microbiológico, turístico, etc. Sin embargo, las cuevas y otros ambientes subterráneos han sido poco explorados desde el punto de vista biotecnológico, aunque representan nichos ecológicos indispensables que podrían ofrecer nuevas moléculas de interés para la medicina y la farmacología. Se han investigado las bacterias de diversas cuevas de la Península Ibérica, así como de las Islas Canarias para comprobar su bioactividad. Entre ellas, han mostrado un interesante potencial antimicrobiano algunas de las bacterias aisladas de la Cueva de Altamira. Los análisis in vitro e in silico mostraron la inhibición de bacterias patógenas Gram- positivas, Gram-negativas y hongos. El interés de esta investigación se apoya en la conocida resistencia de las bacterias patógenas a los antimicrobianos, lo que se ha convertido en un problema mundial en las últimas décadas. Nuestros datos muestran la presencia de bacterias productoras de compuestos antimicrobianos en ambientes subterráneos; y el estudio de sus genomas indicarían la existencia de genes implicados en la síntesis de nuevos compuestos. Palabras clave: Cueva de Altamira, compuestos antimicrobianos, Crossiella, Pseudomonas, predicción de genes.

Mines, like other subterranean environments, have ecological conditions which allow the thriving of microorganisms. Prokaryotes and fungi are common inhabitants of mines, developing a metabolism suitable for growing in such inhospitable environments. The mine of Lousal, Portugal, is an interesting site for the study of microorganisms present in their galleries. Aerobiological studies resulted in the isolation of a Pseudomonas sp., strain L5B5, closely related to the opportunistic fish pathogen P. piscis MC042T, and to the soil bacteria P. protegens CHA0T, P. protegens Cab57, and P. protegens Pf-5. Strain L5B5 was able to inhibit the growth of the pathogenic bacteria Bacillus cereus, Staphylococcus aureus, and Acinetobacter baumanii, as well as the cave fungi Aspergillus versicolor, Penicillium chrysogenum, Cladosporium cladosporioides, Fusarium solani, and Ochroconis lascauxensis. In silico analyses based on de novo genome hybrid assembly and RNA-Seq, performing seven conditions based on culture and phases of growth resulted in the prediction and detection of genetic mechanisms involved in secondary metabolites, with the presence of a possible new gene cluster transcribed under the tested conditions, as well as feasible virulence factors and antimicrobial resistance mechanisms

El patrimonio natural y cultural de las cuevas se ve a menudo amenazado por el turismo y por otras actividades de carácter científico que las expone a riesgos que pueden provocar la alteración irreversible de sus condiciones ambientales de carácter irreversible. Las cuevas con arte rupestre y yacimientos arqueológicos suponen un caso especial y complejo. Cualquier modificación en sus condiciones climáticas conlleva un cambio en el ecosistema subterráneo que, a menudo, favorece el desarrollo y proliferación de poblaciones microbianas que llegan a colonizar los diferentes sustratos presentes en la cueva. La cueva de La Garma constituye un ejemplo destacado de cavidad con una larga frecuentación humana desde el Paleolítico antiguo hasta la Edad Media. Desde su descubrimiento en noviembre de 1995, la cueva sólo es accesible para la investigación arqueológica y la monitorización de sus condiciones ambientales. En este trabajo se presentan los resultados del estudio detallado de sus condiciones microclimáticas, y su influencia en la dispersión de microorganismos en el aire subterráneo y en el desarrollo de biofilms sobre la superficie de las rocas y los sedimentos. La dispersión bacteriana en el aire de la cueva está directamente influenciada por la circulación del aire, con fuertes incrementos durante los pulsos de ventilación vinculados a los vientos de componente sur en el exterior. Las conclusiones del estudio desaconsejan la adecuación de la cueva para su uso turístico y el control exhaustivo de cualquier trabajo arqueológico que implique perforaciones, zanjas o movimientos de tierras- sedimentos, ya que se favorecería la proliferación inmediata de colonias microbianas y con ello el desarrollo de procesos irreversibles de biodeterioro.

Background: Marine actinobacteria are a potential resource for natural products; their secondary bioactive metabolites have shown several biological activities. Most of the isolated and identified actinobacteria in Algeria were usually explored from caves, Saharan soil or palm groves. The marine ecosystem is poorly explored and documented. Methods: Five Streptomyces strains producing bioactive compounds were isolated from Rachgoun Island located in Western Algeria and characterised phenotypically and genotypically using microbiological and 16S rRNA sequencing methods, respectively. The crude extract of the most representative strain ¿Streptomyces sp. strain SM2.4¿ and its seven active fractions were characterised by GC/MS analysis. Results: Streptomyces sp. strain SM2.4 revealed the strongest activity against the Gram-positive bacteria Staphylococcus aureus and Bacillus subtilis, the fungus Aspergillus niger and was inactive against Gram-negative bacteria. GC/MS analysis of the methylated crude extract of Streptomyces sp. strain SM2.4 revealed the presence of 11 major compounds including fatty acids methyl ester (12-methyltridecanoic acid methyl ester, 9-hexadecenoic acid methyl ester, hexadecanoic acid methyl ester, 14-methylhexadecanoic acid methyl ester and 16-methylheptadecanoic acid methyl ester), 2,4-di-tert-butylphenol, (4S,4aS,8aR)-4,8a-dimethyloctahydro-4a(2H)-naphthalenol (geosmin), 2,4-dimethylbenzaldehyde, 3,4-difluorobenzaldehyde, dimethylfuran-2,4-dicarboxylate and pyrrolo(1,2-a)pyrazine-1,4-dione, hexahydro-3-(2-methylpropyl)-. Partial purification of the crude extract by Thin-layer chromatography provided seven active fractions which were tested by radial diffusion assay. GC/MS analysis of the active TLC-fractions revealed the presence of a mixture of active compounds from which 2-(bromomethyl)-2-(2-methylphenyl)-1,3-dioxolane was found to be a new 1,3 dioxolane derivative. Furthermore, 3,4-dimethylbenzamide and pyrido[2,3-d] pyridazine-1,4-dione, he

Nerja Cave, Southern Spain, was revealed as an important biodiversity reservoir from which several novel species of Aspergillus were described. We carried out an aerobiological study in Nerja Cave to assess the origin of airborne fungi. This study quantified the fungi present in the air of ten representative halls covering the three sectors comprising the cave: Touristic Galleries, High Galleries, and New Galleries. Microclimatological monitoring allowed us to understand the dynamic of airborne fungi in two seasons of the year (winter and summer), corresponding to the strongest and the lowest cave ventilation, and to validate the influence that the transport of airborne fungi from outside may have on the cave itself. The data show that cold air enters in winter, as confirmed by the abundant presence of Aspergillus and Penicillium spores inside and outside the cave. In summer, the abundance of some fungi in the air of Nerja Cave, which are not detected outside, indicates a stagnation or low ventilation, and therefore, the concentration of fungal spores is maxima. The high occurrence of Cladosporium outside the cave and the scarce abundance inside support the cave stagnation in this season.

The aerobiology of caves in Southern Spain possesses special characteristics, different from caves located in Northern Spain. Previous studies demonstrated the influence of outdoor air on caves in the north and the existence of two different patterns, depending on the season. In summer there is an abundance of Ascomycota, whereas in winter Basidiomycota predominates, which are related to the periods of stagnation and ventilation, respectively. In caves in Southern Spain the presence of airborne Basidiomycota is scarce and Ascomycota represents the main group of fungi widely distributed across the caves in all seasons. The most characteristic features were the abundant presence of entomopathogenic fungi (Beauveria bassiana, Parengyodontium album, Pochonia chlamydosporia, Leptobacillium symbioticum, Leptobacillium leptobactrum) and Cladosporium cladosporioides in Cueva del Tesoro, Cueva de Ardales and Gruta de las Maravillas. However, the presence of yeasts of the genera Cutaneotrichosporon, Trichosporon, Cryptococcus, Naganishia, Cystobasidium, Microstroma and Phragmotaenium was exclusive to Gruta de las Maravillas. Fungal hazard in the three show caves were determined using an ecological indicator based on the concentration of spores in cave air. © 2021 by the authors. Licensee MDPI, Basel, Switzerland.

Lava caves differ from karstic caves in their genesis and mineral composition. Subsurface microbiology of lava tube caves in Canary Islands, a volcanic archipelago in the Atlantic Ocean, is largely unknown. We have focused the investigation in a representative lava tube cave, Fuente de la Canaria Cave, in La Palma Island, Spain, which presents different types of speleothems and colored microbial mats. Four samples collected in this cave were studied using DNA next-generation sequencing and field emission scanning electron microscopy for bacterial identification, functional profiling, and morphological characterization. The data showed an almost exclusive dominance of Bacteria over Archaea. The distribution in phyla revealed a majority abundance of Proteobacteria (37,89%), followed by Actinobacteria, Acidobacteria and Candidatus Rokubacteria. These four phyla comprised a total relative abundance of 72,96%. The main ecological functions in the microbial communities were chemoheterotrophy, methanotrophy, sulfur and nitrogen metabolisms, and CO2 fixation; although other ecological functions were outlined. Genome annotations of the especially representative taxon Ga0077536 (about 71% of abundance in moonmilk) predicted the presence of genes involved in CO2 fixation, formaldehyde consumption, sulfur and nitrogen metabolisms, and microbially-induced carbonate precipitation. The detection of several putative lineages associated with C, N, S, Fe and Mn indicates that Fuente de la Canaria Cave basalts are colonized by metabolically diverse prokaryotic communities involved in the biogeochemical cycling of major elements.

The microbiota associated with vermiculations from karst caves is largely unknown. Vermiculations are enigmatic deposits forming worm-like patterns on cave walls all over the world. They represent a precious focus for geomicrobiological studies aimed at exploring both the microbial life of these ecosystems and the vermiculation genesis. This study comprises the first approach on the microbial communities thriving in Pertosa-Auletta Cave (southern Italy) vermiculations by next-generation sequencing. The most abundant phylum in vermiculations was Proteobacteria, followed by Acidobacteria > Actinobacteria > Nitrospirae > Firmicutes > Planctomycetes > Chloroflexi > Gemmatimonadetes > Bacteroidetes > Latescibacteria. Numerous less-represented taxonomic groups (< 1%), as well as unclassified ones, were also detected. From an ecological point of view, all the groups co-participate in the biogeochemical cycles in these underground environments, mediating oxidation-reduction reactions, promoting host rock dissolution and secondary mineral precipitation, and enriching the matrix in organic matter. Confocal laser scanning microscopy and field emission scanning electron microscopy brought evidence of a strong interaction between the biotic community and the abiotic matrix, supporting the role of microbial communities in the formation process of vermiculations.

Pseudomonas sp. strain L5B5 is an antimicrobial-producing bacterium isolated from an air sample collected in a pyrite mine in Lousal, Portugal. Genomic analyses predicted genes involved in virulence factors. Here, we report the complete genome sequence of this bacterium, which consists of a circular chromosome with a length of 6,811,662 bp. Copyright © 2021 Gonzalez-Pimentel et al.

This editorial focuses on the studies published within the present Special Issue presenting advances in the field of biodeterioration of cultural heritage caused by microbial communities with a particular focus on new methods for their elimination and control.

The Salón de Reinos, a remnant of the 17th century Palacio del Buen Retiro, was built as a recreational residence under the reign of Felipe IV between 1632 and 1640 and was the main room for the monarch’s receptions. This Salón owes its name to the fact that the coats of arms (shields) of the 24 kingdoms that formed Spain in Felipe IV’s time were painted on the vault, above the windows. In addition, the ceiling shows an original decorative composition. The painted ceiling and window vaults showed deterioration evidenced by fissures, water filtration, detachments of the paint layer, and black stains denoting fungal colonization related to humidity. Ten strains of bacteria and 14 strains of fungi were isolated from the deteriorated paintings. Their biodeteriorative profiles were detected through plate assays. The most frequent metabolic functions were proteolytic and lipolytic activities. Other activities, such as the solubilization of gypsum and calcite and the production of acids, were infrequent among the isolates.

Airborne microorganisms can cause important conservation problems in caves with Paleolithic art and therefore the knowledge of cave aerodynamic is essential. La Garma Cave (Cantabria, Spain), an exceptional archaeological site with several levels of galleries interconnected and two entrances, presents a complex atmospheric dynamics. An approach including aerobiological sampling together with microclimate monitoring was applied to assess the factors controlling the origin of airborne fungi. Here we show that winter ventilation is critical for the increasing of Basidiomycota spores in the cave air and the highest concentrations were found in the most ventilated areas. On the contrary, Ascomycota spores prevailed in absence of ventilation. Besides, most Ascomycota were linked to insects and bats that visit or inhabit the cave. The combination of aerobiological and microclimate data constitutes a good approach to evaluate the influence of external climatic conditions and design the most suitable strategies for the conservation of cultural heritage in the cave environment.

Morgana Cave is located in Val di Scerscen, Central Italian Alps. The cave opens at an altitude of 2,600 m a.s.l. close to the retreating glacier Vedretto di Scerscen, and its entrance was discovered 30 years ago hidden underneath the glacier. A characteristic of this cave is the occurrence of vermiculation deposits on the walls and ceiling. In general, the composition of the microbial communities in cave vermiculations is relatively unknown and rarely investigated. Here we present the data of a geomicrobiological study of vermiculations from an Alpine cave subjected to extreme climate conditions. The microbial communities were dominated by 13 main phyla of Bacteria, and contained a negligible percentage (<1%) of Archaea. The two major bacterial classes were Gammaproteobacteria and Betaproteobacteria, whose metabolic traits were mainly associated with the nitrogen cycle. In addition, psychrophilic and methanotrophic bacterial groups were identified. The occurrence of a large number of uncultured members, at the lowest taxonomic ranks, indicated the presence of still unexplored microbial taxa in the vermiculations.

Two Streptomyces strains were isolated from a lava tube in La Palma, Canary Islands. Genomic analyses suggest that the two strains could belong to the same species. Here, we report the draft genomes for these bacterial strains.

Of the several critical challenges present in environmental microbiology today, one is the assessment of the contribution of microorganisms in the carbon cycle in the Earth-climate system. Karstic subterranean ecosystems have been overlooked until recently. Covering up to 25% of the land surface and acting as a rapid CH4 sink and alternately as a CO2 source or sink, karstic subterranean ecosystems play a decisive role in the carbon cycle in terms of their contribution to the global balance of greenhouse gases. Recent data indicate that microbiota must play a significant ecological role in the biogeochemical processes that control the composition of the subterranean atmosphere, as well as in the availability of nutrients for the ecosystem. Nevertheless, there are still essential gaps in our knowledge concerning the budgets of greenhouse gases at the ecosystem scale and the possible feedback mechanisms between environmental-microclimatic conditions and the rates and type of activity of microbial communities in subterranean ecosystems. Another challenge is searching for bioactive compounds (antibiotics) used for treating human diseases. At present, there is a global health emergency and a strong need for novel biomolecules. In recent decades, great research efforts have been made to extract antibiotics from marine organisms. More recently, caves have been receiving considerable attention in search of novel antibiotics. Cave methanotrophic and heterotrophic bacteria are producers of bioactive compounds and may be potential sources of metabolites with antibacterial, antifungal or anticancer activities of interest in pharmacological and medical research, as well as enzymes with a further biotechnological use. Here we also show that bacteria isolated from mines, a still unexplored niche for scientists in search of novel compounds, can be a source of novel secondary metabolites

The migration of geogenic gases in continental areas with geothermal activity and active faults is an important process releasing greenhouse gases (GHG) to the lower troposphere. In this respect, caves in hypogenic environments are natural laboratories to study the compositional evolution of deep-endogenous fluids through the Critical Zone. Vapour Cave (Alhama, Murcia, Spain) is a hypogenic cave formed by the upwelling of hydrothermal CO2-rich fluids. Anomalous concentrations of N2O and NO2 were registered in the cave's subterranean atmosphere, averaging ten and five times the typical atmospheric backgrounds, respectively. We characterised the thermal conditions, gaseous compositions, sediments, and microbial communities at different depths in the cave. We did so to understand the relation between N-cycling microbial groups and the production and transformation of nitrogenous gases, as well as their coupled evolution with CO2 and CH4 during their migration through the Critical Zone to the lower troposphere. Our results showed an evident vertical stratification of selected microbial groups (Archaea and Bacteria) depending on the environmental parameters, including O2, temperature, and GHG concentration. Both the N2O isotope ratios and the predicted ecological functions of bacterial and archaeal communities suggest that N2O and NO2 emissions mainly depend on the nitrification by ammonia-oxidising microorganisms. Denitrification and abiotic reactions of the reactive intermediates NH2OH, NO, and NO2− are also plausible according to the results of the phylogenetic analyses of the microbial communities. Nitrite-dependent anaerobic methane oxidation by denitrifying methanotrophs of the NC10 phylum was also identified as a post-genetic process during migration of this gas to the surface. To the best of our knowledge, our report provides, for the first time, evidence of a niche densely populated by Micrarchaeia, which represents more than 50% of the total archaeal abundance.

Cyanobacteria and microalgae are usually found in speleothems, rocks and walls of show caves exposed to artificial lighting. These microorganisms develop as biofilms coating the mineral surfaces and producing aesthetic, physical and chemical deterioration. A wide number of physical, chemical and environmental-friendly methods have been used for controlling the biofilms with different results. Natural biological control has been suggested by some authors as a theoretical approach but without direct evidence or application. Here we report the finding of a natural biological control of phototrophic biofilms on the speleothems of Nerja Cave, Malaga, Spain. The formation of plaques or spots where the phototrophic microorganisms disappeared can be assumed on the basis of processes of predation of bacteria, amoebas and some other organisms on the phototrophic biofilms. This study aims at investigating the potentialities of the biological control of phototrophic biofilms in caves, but the originality of these data should be confirmed in future studies with a larger number of biofilm samples in different ecological scenarios.

Streptomyces parvus strain C05 was isolated from the walls of a Roman tomb located in Carmona, Seville, Spain. Subsequent studies determined the capability of this strain for producing granaticins. Here, we report the 8.7-Mbp draft genome sequence for this bacterium.

Fetida Cave is an active sulfuric acid cave influenced by seawater, showing abundant microbial communities that organize themselves under three main different morphologies: water filaments, vermiculations and moonmilk deposits. These biofilms/deposits have different cave distribution, pH, macro- and microelement and mineralogical composition, carbon and nitrogen content. In particular, water filaments and vermiculations had circumneutral and slightly acidic pH, respectively, both had abundant organic carbon and high microbial diversity. They were rich in macro- and microelements, deriving from mineral dissolution, and, in the case of water filaments, from seawater composition. Vermiculations had different color, partly associated with their mineralogy, and unusual minerals probably due to trapping capacities. Moonmilk was composed of gypsum, poor in organic matter, had an extremely low pH (0–1) and low microbial diversity. Based on 16S rRNA gene analysis, the microbial composition of the biofilms/deposits included autotrophic taxa associated with sulfur and nitrogen cycles and biomineralization processes. In particular, water filaments communities were characterized by bacterial taxa involved in sulfur oxidation and reduction in aquatic, aphotic, microaerophilic/anoxic environments (Campylobacterales, Thiotrichales, Arenicellales, Desulfobacterales, Desulforomonadales) and in chemolithotrophy in marine habitats (Oceanospirillales, Chromatiales). Their biodiversity was linked to the morphology of the water filaments and their collection site. Microbial communities within vermiculations were partly related to their color and showed high abundance of unclassified Betaproteobacteria and sulfur-oxidizing Hydrogenophilales (including Sulfuriferula), and Acidiferrobacterales (including Sulfurifustis), sulfur-reducing Desulfurellales, and ammonia-oxidizing Planctomycetes and Nitrospirae. The microbial community associated with gypsum moonmilk showed the strong dominance (>6

Etruscan and Roman necropolises are cemeteries with different types of burial practices, which are remarkable for their magnificent mural paintings. The conservation of these paintings is difficult, mainly derived from environmental factors and the opening of the tombs to public visits. One of the most complex phenomena observed on these sites is biodeterioration. The Circular Mausoleum tomb (Roman Necropolis of Carmona, Seville, Spain) is characterized by a heavy colonization of phototrophic microorganisms on the walls and ceiling. In addition, some areas near the ceiling exhibited an important number of violet stains of unknown origin. Previously, Agarossi (1994) found similar violet stains, attributed to streptomycetes, in two Etruscan tombs. However, no data on the species involved or the chemical structure of the pigments were reported. Here we show that the violet stains observed in the Circular Mausoleum are produced by a strain of the actinobacterium Streptomyces isolated from the mortar walls, and able to synthesize the same violet pigment in the laboratory. Streptomyces parvus MC05 was identified after whole genome study by means of Next Generation Sequencing methods. Pairwise comparisons carried out for genomes of the type strain of S. parvus DSM40348T and the MC05 strain were performed using Jspecies service. While results observed for ANIb, ANIm and Tetra indexes indicated that both strains belong to the same species, the S. parvus MC05 strain differs from the DSM20348T type strain in genome size and secondary metabolites production. AntiSMASH analysis implemented for both strains showed that the S. parvus MC05, isolated from the tomb, presents the capacity of synthesize bioactive compounds which were absents in the type strain. HPLC-MS of the culture extracts from S. parvus MC05 showed the production of three main granaticin derivatives (dihydrogranaticin A, granaticin A and granaticin B) in addition to minor products of other granaticin analogues. Gra, References - Agarossi, G. 1994. In Studi e Ricerche sulla Conservazione delle Opere d’Arte Dedicati alla Memoria di Marcello - Paribeni. F. Guidobaldi (ed.), CNRS, pp. 1−11. - Dominguez-Moñino, I. et al. 2017. Science of the Total Environment 598, 889-899.

Subsurface granitic environments are scarce and poorly investigated. A multi‐disciplinary approach was used to characterize the abundant moonmilk deposits and associated microbial communities coating the granite walls of the 16th Century Paranhos spring water tunnel in Porto city (north‐west Portugal). It is possible that this study is the first record of moonmilk in an urban subsurface granitic environment. The morphology and texture, mineralogical composition, stable isotope composition and microbial diversity of moonmilk deposits have been studied to infer the processes of moonmilk formation. These whitish secondary mineral deposits are composed of very fine needle‐fibre calcite crystals with different morphologies and density. Calcified filaments of fungal hyphae or bacteria were distinguished by field emission scanning electron microscopy. Stable isotope analysis revealed a meteoric origin of the needle‐fibre calcite, with an important contribution of atmospheric CO2, soil respiration and urn:x-wiley:00370746:media:sed12431:sed12431-math-0001 from weathering of Ca‐bearing minerals. The DNA‐based analyses revealed the presence of micro‐organisms related to urban contamination, including Actinobacteria, mainly represented by Pseudonocardia hispaniensis, Thaumarchaeota and Ascomycota, dominated by Cladosporium. This microbial composition is consistent with groundwater pollution and contamination sources of the overlying urban area, including garages, petrol stations and wastewater pipeline leakage, showing that the Paranhos tunnel is greatly perturbed by anthropogenic activities. Whether the identified micro‐organisms are involved in the formation of the needle‐fibre calcite or not is difficult to demonstrate, but this study evidenced both abiotic and biogenic genesis for the calcite moonmilk in this subsurface granitic environment.

The Roman Necropolis of Carmona (Sevilla, Spain) has experienced a severe deterioration since the accidental discovery in 1868 and subsequent archaeological excavations starting in 1882. To this deterioration contributed the location in a soft calcarenite rock quarry, the adverse environmental conditions and the numerous and extensive wrong archaeological and managing interventions along more than a century. The cultural, artistic and religious importance of this Necropolis converts the protection and conservation of this archaeological site in a major issue. This work present the data obtained in a multidisciplinary research were geology, geomorphology, micro-environmental and climatic monitoring, rock petrophysical characterization, description of weathering forms and biological colonization were considered in order to propose corrective measures to minimize deterioration.

Two Gram-positive, catalase-positive, oxidase-negative, motile, endospore-forming, rod-shaped bacteria, designated as 0911MAR22V3T and 0911TES10J4, were isolated from air samples collected in two show caves, located in Andalusia, Southern Spain. Phylogenetic analysis based on 16S rRNA gene sequences indicated that both strains were indistinguishable and they were most closely related to Bacillus humi DSM 16318T (98%). DNA-DNA hybridization values of the strain 0911MAR22V3T with respect to strain 0911TES10J4 and B. humi DSM 16318T were 76.8% (73.9%, reciprocal) and 56.9% (63.3%, reciprocal analysis), respectively. Whole genome average nucleotide identity (ANI) values of both strains were in the threshold value for species delineation and less than 85% with B. humi. Strains 0911MAR22V3T and 0911TES10J4 grew at 10-47. °C (optimum 37. °C), at pH 6-9.5 and with 0-8% (w/v) NaCl (optimum 1%). In both strains the dominant isoprenoid quinone was MK-7, the major cellular polar lipids were diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine, and two more phospholipids, the predominant fatty acids were iso-C15:0 and anteiso-C15:0 and the DNA G + C content was 38. mol%. On the basis of their phylogenetic relatedness and their phenotypic and genotypic features, the strains 0911MAR22V3T and 0911TES10J4 should be attributed to a novel species within the genus Bacillus, for which the name Bacillus onubensis sp. nov. is proposed. The type strain is 0911MAR22V3T (=LMG 27963T = CECT 8479T); and strain 0911TES10J4 (CECT 8478) is a reference strain.

[ES]: La geomorfología del medio subterráneo depende de su historia geológica, y de los fenómenos que lo modelan, como la erosión y otros agentes externos. Además, sobre los sedimentos, las paredes o los espeleotemas de cuevas se desarrollan poblaciones de microorganismos que modifican la superficie mineral. Así, la colonización de este medio estará en función de las condiciones ambientales, del grupo de microorganismos en cuestión y de la biorreceptividad de los sustratos. Las cuevas se consideran espacios especializados que se caracterizan por una temperatura uniforme, elevada humedad y escasos recursos nutricionales y energéticos. Las cuevas turísticas presentan mayor disponibilidad de energía, generada por la luz artificial y la entrada de materia orgánica, y favorecen la presencia de un mayor número de organismos fotosintéticos y heterótrofos. El desarrollo de microorganismos sobre las paredes y los espeleotemas de las cuevas pueden producir procesos de biodeterioro, es decir, cambios indeseables en el sustrato. En este trabajo se presenta la información obtenida sobre la microbiota que coloniza espeleotemas iluminados de dos cuevas turísticas, la Cueva de Nerja y la Cueva del Tesoro (Málaga), mediante el empleo combinado de la microscopía y del análisis molecular. Los resultados ponen de manifiesto que los microorganismos se organizan en comunidades estructurales complejas denominadas biofilms. El empleo de la microscopía ha permitido conocer la composición del sustrato y su relación con el biofilm, así como observar procesos de biodeterioro relacionados con cambios físicos y químicos del sustrato tales como decoloración, pérdida de consistencia, ruptura y disolución. En relación con la composición de los biofilms, se ha observado una biodiversidad dominada por cianobacterias y microalgas, junto con actinobacterias, proteobacterias, arqueas, hongos e incluso protozoos y pequeños artrópodos. El análisis molecular revela la presencia de nuevas especies de microo, [EN]: The geomorphology of the subterranean environment depends on its geological history, and phenomena such as erosion and other external agents. Moreover, in the caves the development of microorganisms can change the surface of sediments, walls and speleothems. Therefore, the colonization of a cave will depend on the environmental conditions, the type of microorganisms involved and the substrata bioreceptivity. Caves are considered specialized areas characterized by a uniform temperature, high humidity and low nutritional and energy resources. Visited caves have more available energy, produced by artificial lighting and the input of organic matter, and these conditions allow the growth of photosynthetic and heterotrophic organisms. The development of microorganisms on the cave walls and speleothems may produce biodeterioration, i.e., undesirable changes in the substratum. This work provides information on the microbiota that colonizes illuminated speleothems in two tourist caves, Nerja and Tesoro (Málaga), through the combined use of optical and electron microscopy, together with molecular analysis. The results showed that, in both caves, microorganisms are organized into complex structural communities called biofilms. The microscopic approach provides data on the substratum composition and its relationship with the biofilm. Biodeterioration processes related to physical and chemical changes of the substratum such as discoloration, loss of consistency, breaking and dissolution, were also observed. The biofilms were dominated by cyanobacteria and microalgae together with actinobacteria, proteobacteria, archaea, protozoa, fungi and even small arthropods. Moreover, molecular analysis revealed the presence of new species of microorganisms in both caves. Knowledge about these communities and their mechanisms of biodeterioration allows the design of preventive and corrective actions, according with a proper conservation of natural and cultural heritage of both caves.

[ES]: La zona arqueológica de la Garma (Cantabria) forma parte de un gran complejo kárstico activo con varios niveles de galerías de desarrollo horizontal interconectados por conductos verticales. La Garma alberga un conjunto arqueológico excepcional que incluye un magnífico conjunto de arte parietal y suelos paleolíticos en un asombroso estado de conservación, así como valiosísimas evidencias del comportamiento simbólico de los cazadores-recolectores del Pleistoceno. Sus valores patrimoniales le han hecho merecedora de ser incluida en la Lista del Patrimonio Mundial de la UNESCO en el año 2008. La conservación de este enclave es en todo momento prioritaria y recientemente se ha puesto en marcha un programa de conservación preventiva que tiene como objetivo fundamental el conocimiento del ecosistema subterráneo y del impacto de la presencia humana en el medio subterráneo, a los efectos tanto de la propia gestión e investigación de la cavidad como ante una eventual apertura al público en condiciones de máxima seguridad. El funcionamiento de un sistema kárstico subterráneo como el de La Garma, con varios niveles de desarrollo y conectado directamente con el nivel freático activo, implica un complejo movimiento de materia (agua, masas de aire y aerosoles) y energía. Para alcanzar un adecuado conocimiento de su dinámica ambiental, desde febrero de 2015 se está llevando a cabo un estudio multidisciplinar mediante el empleo de metodologías específicas para cada interfase involucrada: microclima (cavidad en tres niveles internos del karst y atmósfera externa), hidroquímica (agua de infiltración y condensación), geoquímica isotópica (aire, suelo) y contaminación microbiológica (aire, suelo, roca). Los primeros datos muestran cambios estacionales muy marcados en el patrón de ventilación general de la cavidad, con mayor tasa de renovación de aire en la época invernal, y una estabilidad ambiental muy diferenciada entre zonas, con algunos sectores aislados del régimen general d, [EN]: The archaeological site of the Garma (Cantabria) is part of an active karst system with several levels of galleries interconnected by vertical shafts. La Garma houses an exceptional archaeological heritage that includes magnificent examples of Palaeolithic cave art and settlement floors in an amazing state of preservation, as well as valuable evidence of symbolic behavior of Pleistocene hunter-gatherers. Its heritage values have made it worthy of their inclusion on the World Heritage List of UNESCO in 2008. The conservation of this site is a priority and it has been recently launched a program of preventive conservation. The main objective of this project is to achieve a better knowledge of the underground ecosystem and the impact of human presence in the underground microenvironment, both for purposes of sound management and research of the cavity as well as for a hypothetical opening to the public in the future. The underground karst system of La Garma, with various levels of galleries and directly connected with active water table, involves a complex movement of matter (water, air masses and aerosols) and energy. To achieve an adequate knowledge of environmental dynamics, our research group is conducting a multidisciplinary study since February 2015 by employing specific methodologies for each interface involved: microclimate (cavity in three internal levels of karst and external atmosphere), hydrochemical (water infiltration and condensation), isotope geochemistry (air, soil) and microbiological contamination (air, soil, rock). Early data show very marked seasonal changes in the general ventilation pattern of the cavity (air renewal in winter) and areas with different microenvironmental stability, including sectors with a greater degree of isolation from the regime of general circulation of air in the cavity.