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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.

The cave microbiota is assumed to be shaped by indoor microclimate, biotic and abiotic factors, which are largely dependent from outside environmental conditions; however, this knowledge is available at local or regional scales only. To address this knowledge gap, we reanalyzed over 1050 bacterial and fungal communities of caves worldwide, and found that outdoor temperature and rainfall play a critical role in explaining differences in microbial diversity patterns of global caves, selecting specific dominant taxa across gradients of growing aridity conditions with arid climate leading to a reduction in total cave microbial diversity. Moreover, we found that fungal (from 186 to 1908 taxa) and bacterial (from 467 to 1619 taxa) diversity increased under temperate-tropical and temperate-continental climatic regions, respectively, highlighting an opposite preference for the two microbial compartments. We hypothesized that outdoor geographical, climatic variables and lithology are critical epistatic drivers in assembling microbial communities and their dominant taxa, whose ecological responses could be useful to predict the fate of these subterranean environments in the context of climate change. Our work elucidates the intimate connection between caves microbiota and surface ecosystems highlighting the sensitivity of cave microbial communities to climatic changes and environmental degradation. This work also provides a natural benchmark for the biogeographic information for caves globally and for protection strategies aiming at conservation of underground environments.

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.

A flood event affecting Pindal Cave, a UNESCO World Heritage site, introduced a substantial amount of external sediments and waste into the cave. This event led to the burial of preexisting sediments, altering the biogeochemical characteristics of the cave ecosystem by introducing heightened levels of organic matter, nitrogen compounds, phosphorus, and heavy metals. The sediments included particulate matter and waste from a cattle farm located within the water catchment area of the cavity, along with diverse microorganisms, reshaping the cave microbial community. This study addresses the ongoing influence of a cattle farm on the cave ecosystem and aims to understand the adaptive responses of the underground microbial community to the sudden influx of waste allochthonous material. Here, we show that the flood event had an immediate and profound effect on the cave microbial community, marked by a significant increase in methanogenic archaea, denitrifying bacteria, and other microorganisms commonly associated with mammalian intestinal tracts. Furthermore, our findings reveal that one year after the flood, microorganisms related to the flood decreased, while the increase in inorganic forms of ammonium and nitrate suggests potential nitrification, aligning with increased abundances of corresponding functional genes involved in nitrogen cycling. The results reveal that the impact of pollution was neither recent nor isolated, and it was decisive in stopping livestock activity near the cave. The influence of the cattle farm has persisted since its establishment over the impluvium area, and this influence endures even a year after the flood. Our study emphasizes the dynamic interplay between natural events, anthropogenic activities, and microbial communities, offering insights into the resilience of cave ecosystems. Understanding microbial adaptation in response to environmental disturbances, as demonstrated in this cave ecosystem, has implications for broader ecological stu

In the absence of sunlight, caves harbor a great diversity of microbial colonies to extensive biofilms with different sizes and colors visible to the naked eye. One of the most widespread and visible types of biofilm are those with yellow hues that can constitute a serious problem for the conservation of cultural heritage in many caves, such as Pindal Cave (Asturias, Spain). This cave, declared a World Heritage Site by UNESCO for its Paleolithic parietal art, shows a high degree of development of yellow biofilms that represents a real threat to the conservation of painted and engraved figures. This study aims to: 1) identify the microbial structures and the most characteristic taxa composing the yellow biofilms, 2) seek the linked microbiome reservoir primarily contributing to their growth; 3) seed light on the driving vectors that contribute to their formation and determine the subsequent proliferation and spatial distribution. To achieve this goal, we used amplicon-based massive sequencing, in combination with other techniques such as microscopy, in situ hybridization and environmental monitoring, to compare the microbial communities of yellow biofilms with those of drip waters, cave sediments and exterior soil. The results revealed microbial structures related to the phylum Actinomycetota and the most characteristic bacteria in yellow biofilms, represented by the genera wb1-P19, Crossiella, Nitrospira, and Arenimonas. Our findings suggest that sediments serve as potential reservoirs and colonization sites for these bacteria that can develop into biofilms under favorable environmental and substrate conditions, with a particular affinity for speleothems and rugged-surfaced rocks found in condensation-prone areas. This study presents an exhaustive study of microbial communities of yellow biofilms in a cave, which could be used as a procedure for the identification of similar biofilms in other caves and to design effective conservation strategies in caves with valuab

UNESCO defines cultural heritage as “the legacy of physical artefacts and intangible attributes of a group or society that are inherited from past generations, maintained in the present and bestowed for the benefit of future generations”. The roots of each country and region are anchored in its own cultural heritage. Cultural heritage is an invaluable legacy and is integral to our future, but due to its fragile and finite nature we sought to identify the best, most sustainable means of preserving cultural heritage. This implies the conservation of movable (paintings, sculptures, artifacts) and immovable (monuments, archaeological sites, industrial archaeology) properties. Today, cultural heritage is exposed to air pollution, flooding, earthquakes, wrong management activities, etc., which threaten its integrity. To mitigate damages, research should focus on analyzing and alleviating deterioration and provide technological solutions for enhancing the conservation of cultural heritage. These goals can be achieved through interdisciplinary and transdisciplinary studies. This Special Issue on Interdisciplinary Researches for Cultural Heritage Conservation in Applied Sciences aims to bring together some of the latest researches in this field. A total of 10 papers focusing on different aspects of cultural heritage are categorized into the three topics detailed below and summarized.

Castañar Cave (Caceres, Spain) is a unique show cave known for its high natural radiation levels. This study presents a comprehensive analysis of its prokaryotic diversity, specifically focusing on investigating the influence of environmental conditions and substrate characteristics on the prokaryotic community structure in the cave sediments. Additionally, the research aims to evaluate the potential impact of human activities on the cave ecosystem. The identification of distinct bioclimatic zones within the cave was made possible through a combination of environmental and microbial monitoring (ATP assays). The results reveal sediment texture as a significant factor, notably affecting the structure, diversity, and phylogenetic variability of the microbial community, including both Bacteria and Archaea. The proportion of clay minerals in sediments plays a crucial role in regulating moisture levels and nutrient availability. These substrate properties collectively exert a significant selective pressure on the structure of prokaryotic communities within cave sediments. The molecular approach shows that heterotrophic bacteria, including those with chitinolytic enzymes, primarily inhabit the cave. Furthermore, chemoautotrophic nitrifiers such as the archaea Nitrososphaeria and the genus Nitrospira, as well as methanotrophic bacteria from the phyla Methylomirabilota, Pseudomonadota, and Verrucomicrobiota, are also present. Remarkably, despite being a show cave, the cave microbiota displays minimal impacts from human activities and the surface ecosystem. Prokaryotic populations exhibit stability in the innermost areas, while the tourist trail area experiences slightly higher biomass increases due to visitor traffic. This suggests that conservation efforts have successfully limited the entry of external nutrients into the innermost cave areas. Additionally, the results suggest that integrating biomarkers like ATP into environmental monitoring can significantly enhance the m

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.

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.

La Cueva del Pindal (Ribadedeva, Asturias) y su arte rupestre paleolítico forman parte de la Lista del Patrimonio Mundial de la UNESCO desde 2008. Actualmente está abierta al público con visitas limitadas y en ella se desarrolla un proyecto de investigación sobre actividad microbiana en ecosistemas naturales subterráneos y su aplicación al diseño de estrategias de conservación. El sistema kárstico del Pindal se desarrolla en un macizo calcáreo modelado en forma de terrazas marinas (rasas), situadas entre 30 y 68 metros s.n.m que constituyen el área de captación hídrica preferente y en las que se observan numerosas estructuras exokársticas tipo dolina y poljé. Sobre una de las dolinas, situada casi en la vertical de la cavidad, se instaló una explotación ganadera en 1995. El 23 de octubre de 2019 tras un episodio extremo de precipitaciones (209 l/m2) se produjo una fuerte acumulación de agua en la dolina y una entrada masiva de agua y sedimentos en la cueva. El 31 de octubre se realizó un muestreo específico para conocer los cambios provocados en el ecosistema subterráneo. La inundación provocó un fuerte incremento de materia orgánica, nitrógeno, fósforo y potasio disponibles en los sedimentos y modificaciones muy significativas en la composición de las comunidades microbianas presentes en los sedimentos: aparición de nuevos filos bacterianos (Firmicutes y Bacteroidetes), incluyendo grupos de bacterias patógenas oportunistas (Corynebacterium, Thauera, Clostridiales) y la casi total desaparición de otros (Rokubacteria y Nitrospirae). Los resultados confirman que el agua y sedimentos arrastrados al interior de la cueva en la inundación fueron acompañados de residuos procedentes de la explotación ganadera, demostrando el alto grado de vulnerabilidad de este tipo de ambientes. Tras el estudio e informe correspondiente, la actividad ganadera cesó en 2021 y continúa el estudio de las poblaciones microbianas para conocer su evolución a medio-largo plazo.

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.

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.

The genus Crossiella contains two species, C. equi, causing nocardioform placentitis in horses, and C. cryophila, an environmental bacterium. Apart from C. equi, which is not discussed here, environmental Crossiella is rarely reported in the literature; thus, it has not been included among “rare actinobacteria”, whose isolation frequency is very low. After C. cryophila, only five reports cover the isolation of Crossiella strains. However, the frequency of published papers on environmental Crossiella has increased significantly in recent years due to the extensive use of next-generation sequencing (NGS) and a huge cascade of data that has improved our understanding of how bacteria occur in the environment. In the last five years, Crossiella has been found in different environments (caves, soils, plant rhizospheres, building stones, etc.). The high abundance of Crossiella in cave moonmilk indicates that this genus may have an active role in moonmilk formation, as evidenced by the precipitation of calcite, witherite, and struvite in different culture media. This review provides an overview of environmental Crossiella, particularly in caves, and discusses its role in biomineralization processes and bioactive compound production.

In the absence of sunlight, caves harbor a great diversity of microbial colonies to extensive biofilms with different sizes and colors visible to the naked eye. One of the most widespread and visible types of biofilm are those with yellow hues that can constitute a serious problem for the conservation of cultural heritage in many caves, such as Pindal Cave (Asturias, Spain). This cave, declared a World Heritage Site by UNESCO for its Paleolithic parietal art, shows a high degree of development of yellow biofilms that represents a real threat to the conservation of painted and engraved figures. This study aims to: 1) identify the microbial structures and the most characteristic taxa composing the yellow biofilms, 2) seek the linked microbiome reservoir primarily contributing to their growth; 3) seed light on the driving vectors that contribute to their formation and determine the subsequent proliferation and spatial distribution. To achieve this goal, we used amplicon-based massive sequencing, in combination with other techniques such as microscopy, in situ hybridization and environmental monitoring, to compare the microbial communities of yellow biofilms with those of drip waters, cave sediments and exterior soil. The results revealed microbial structures related to the phylum Actinomycetota and the most characteristic bacteria in yellow biofilms, represented by the genera wb1-P19, Crossiella, Nitrospira, and Arenimonas. Our findings suggest that sediments serve as potential reservoirs and colonization sites for these bacteria that can develop into biofilms under favorable environmental and substrate conditions, with a particular affinity for speleothems and rugged-surfaced rocks found in condensation-prone areas. This study presents an exhaustive study of microbial communities of yellow biofilms in a cave, which could be used as a procedure for the identification of similar biofilms in other caves and to design effective conservation strategies in caves with valuab

Castañar Cave (Caceres, Spain) is a unique show cave known for its high natural radiation levels. This study presents a comprehensive analysis of its prokaryotic diversity, specifically focusing on investigating the influence of environmental conditions and substrate characteristics on the prokaryotic community structure in the cave sediments. Additionally, the research aims to evaluate the potential impact of human activities on the cave ecosystem. The identification of distinct bioclimatic zones within the cave was made possible through a combination of environmental and microbial monitoring (ATP assays). The results reveal sediment texture as a significant factor, notably affecting the structure, diversity, and phylogenetic variability of the microbial community, including both Bacteria and Archaea. The proportion of clay minerals in sediments plays a crucial role in regulating moisture levels and nutrient availability. These substrate properties collectively exert a significant selective pressure on the structure of prokaryotic communities within cave sediments. The molecular approach shows that heterotrophic bacteria, including those with chitinolytic enzymes, primarily inhabit the cave. Furthermore, chemoautotrophic nitrifiers such as the archaea Nitrososphaeria and the genus Nitrospira, as well as methanotrophic bacteria from the phyla Methylomirabilota, Pseudomonadota, and Verrucomicrobiota, are also present. Remarkably, despite being a show cave, the cave microbiota displays minimal impacts from human activities and the surface ecosystem. Prokaryotic populations exhibit stability in the innermost areas, while the tourist trail area experiences slightly higher biomass increases due to visitor traffic. This suggests that conservation efforts have successfully limited the entry of external nutrients into the innermost cave areas. Additionally, the results suggest that integrating biomarkers like ATP into environmental monitoring can significantly enhance the m

The genus Crossiella contains two species, C. equi, causing nocardioform placentitis in horses, and C. cryophila, an environmental bacterium. Apart from C. equi, which is not discussed here, environmental Crossiella is rarely reported in the literature; thus, it has not been included among "rare actinobacteria", whose isolation frequency is very low. After C. cryophila, only five reports cover the isolation of Crossiella strains. However, the frequency of published papers on environmental Crossiella has increased significantly in recent years due to the extensive use of next-generation sequencing (NGS) and a huge cascade of data that has improved our understanding of how bacteria occur in the environment. In the last five years, Crossiella has been found in different environments (caves, soils, plant rhizospheres, building stones, etc.). The high abundance of Crossiella in cave moonmilk indicates that this genus may have an active role in moonmilk formation, as evidenced by the precipitation of calcite, witherite, and struvite in different culture media. This review provides an overview of environmental Crossiella, particularly in caves, and discusses its role in biomineralization processes and bioactive compound production.

“Rare actinobacteria” are non-Streptomyces actinobacteria whose isolation frequency is much lower than Streptomyces strains, commonly isolated by conventional methods [1][2]. Tiwari and Gupta [3][4] reported 120 new genera of “rare actinobacteria” in the first decade of the 21th century. A total of 40 out of 120 genera were isolated from soils, with comparatively lower percentages from other environments: marine and freshwater sediments, marine animals, plants, buildings, etc. A few reports included the rare genera Actinomadura, Nonomuraea, Micromonospora, Streptosporangium, Nocardiopsis, and Pseudonocardia as the most frequent in diverse environments [5][6][7][8][9]. It is noteworthy an abundance of “rare actinobacteria” in extreme environments, as exemplified in Atacama [7] and other deserts [10][11][12][13]. However, Crossiella has not been included among “rare actinobacteria” thus far. Researchers have found that Crossiella is an abundant genus in most studied Spanish caves, whether they are gypsum, karstic or volcanic [14][15][16][17][18][19][20][21], and in other terrestrial and aquatic environments., Texto completo disponible en: https://encyclopedia.pub/entry/45605

Pindal Cave (Asturias, Spain) and its Paleolithic art have been part of the UNESCO World Heritage List since 2008. The importance of this Paleolithic art led to a research project that deals with deciphering the relationships between environmental conditions and microbial activity in natural underground ecosystems and its application to the design of conservation strategies. The Pindal karstic system develops in a calcareous massif (Carboniferous) modeled in the form of an erosional marine terrace (rasa) by coastal morphogenetic processes. This marine terrace level is located at an elevation of 30-68 meters above current sea level and constitutes the preferred catchment area for runoff water from another higher level (140-170 m) developed on quartzite layers with very low permeability (Ordovician). The cave is the main endokarstic feature of the system. On the surface of the 30-68 m rasa there are numerous exokarstic structures of sinkhole and polje type. On one of the sinkholes, located almost vertically to the cave, a cattle farm was installed in 1995. Between October 19 and 23, 2019, an extreme episode of rainfall occurred in the area with a cumulative total of 209 l/m2. This event caused a strong accumulation of water in the aforementioned sinkhole that finally collapsed, flooding the cave for several days. Immediately after the cave had been drained, environmental measurements and sediment samplings were carried out at various points in order to determine the changes caused in the underground ecosystem. In the most affected area by the flood, changes in humidity and temperature of air and sediments were recorded for several months. Biogeochemical data indicated that the sediments deposited as a result of the flooding presented high values of available organic matter, nitrogen, phosphorus and potassium, much higher than those of the innermost areas did not directly affect by the flooding. The comparative microbiological study of sediment samples revealed that th

The Cross of the Inquisition, sculpted in 1903 and raised on a column with a fluted shaft and ornamented with vegetable garlands, is located in a corner of the Plateresque façade of the Seville City Hall. The Cross was vandalized in September 2019 and the restoration concluded in September 2021. A geological and microbiological study was carried out in a few small fragments. The data are consistent with the exposure of the Cross of the Inquisition to an urban environment for more than 100 years. During that time, a lichen community colonized the Cross and the nearby City Hall façades. The lichens, bryophytes and fungi colonizing the limestone surface composed an urban community, regenerated from the remains of the original communities, after superficial cleaning of the limestone between 2008 and 2010. This biological activity was detrimental to the integrity of the limestone, as showed by the pitting and channels, which evidence the lytic activity of organisms on the stone surface. Stone consolidation was achieved with Estel 1000. Preventol RI80, a biocide able to penetrate the porous limestone and active against bacteria, fungi, lichens, and bryophytes, was applied in the restoration.

Understanding the dynamics and spatial distribution of gases in the subterranean atmospheres is essential to increase the reliability of carbon balances in karst ecosystems or the paleoclimate reconstructions based on cave deposits. This scientific information is also very valuable for cave managers to ensure the safety of visitors and the conservation of the subterranean heritage. Through a comprehensive monitoring of the main air parameters in a shallow temperate cave, we decipher the physical drivers and mechanisms involved in the CO2 and radon exchange between the cave and the outer atmosphere, and how this process is triggered by the changes of local weather. Our results reveal that the biphasic infiltration (water plus air) in the network of penetrative structures from the overlying soil and host rock exercise remarkable control over the cave environment, delaying the thermal response of the cave air to the outer climate-driven changes and also the gaseous transfer between the cave atmosphere and the exterior. The cave location concerning the karstified outcrop determines that this subterranean site acts as a gas emitter during summer, which is contrary to what happens in many other caves. Prominent gas entrapment at a micro-local level is also registered in some upper galleries.

Understanding the dynamics and spatial distribution of gases in the subterranean atmospheres is essential to increase the reliability of carbon balances in karst ecosystems or the paleoclimate reconstructions based on cave deposits. This scientific information is also very valuable for cave managers to ensure the safety of visitors and the conservation of the subterranean heritage. Through a comprehensive monitoring of the main air parameters in a shallow temperate cave, we decipher the physical drivers and mechanisms involved in the CO2 and radon exchange between the cave and the outer atmosphere, and how this process is triggered by the changes of local weather. Our results reveal that the biphasic infiltration (water plus air) in the network of penetrative structures from the overlying soil and host rock exercise remarkable control over the cave environment, delaying the thermal response of the cave air to the outer climate-driven changes and also the gaseous transfer between the cave atmosphere and the exterior. The cave location concerning the karstified outcrop determines that this subterranean site acts as a gas emitter during summer, which is contrary to what happens in many other caves. Prominent gas entrapment at a micro-local level is also registered in some upper galleries.

The Cross of the Inquisition, sculpted in 1903 and raised on a column with a fluted shaft and ornamented with vegetable garlands, is located in a corner of the Plateresque façade of the Seville City Hall. The Cross was vandalized in September 2019 and the restoration concluded in September 2021. A geological and microbiological study was carried out in a few small fragments. The data are consistent with the exposure of the Cross of the Inquisition to an urban environment for more than 100 years. During that time, a lichen community colonized the Cross and the nearby City Hall façades. The lichens, bryophytes and fungi colonizing the limestone surface composed an urban community, regenerated from the remains of the original communities, after superficial cleaning of the limestone between 2008 and 2010. This biological activity was detrimental to the integrity of the limestone, as showed by the pitting and channels, which evidence the lytic activity of organisms on the stone surface. Stone consolidation was achieved with Estel 1000. Preventol RI80, a biocide able to penetrate the porous limestone and active against bacteria, fungi, lichens, and bryophytes, was applied in the restoration.

Research on cave microorganisms has mainly focused on the microbial communities thriving on speleothems, rocks and sediments; however, drip water bacteria and calcite precipitation has received less attention. In this study, microbial communities of carbonate precipitates from drip waters in Nerja, a show cave close to the sea in southeastern Spain, were investigated. We observed a pronounced difference in the bacterial composition of the precipitates, depending on the galleries and halls. The most abundant phylum in the precipitates of the halls close to the cave entrance was Proteobacteria, due to the low depth of this sector, the direct influence of a garden on the top soil and the infiltration of waters into the cave, as well as the abundance of members of the order Hyphomicrobiales, dispersing from plant roots, and other Betaproteobacteria and Gammaproteobacteria, common soil inhabitants. The influence of marine aerosols explained the presence of Marinobacter, Idiomarina, Thalassobaculum, Altererythrobacter and other bacteria due to the short distance from the cave to the sea. Nineteen out of forty six genera identified in the cave have been reported to precipitate carbonate and likely have a role in mineral deposition.

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.