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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.

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

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.

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.

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.

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.

Los ambientes kársticos son los ecosistemas subterráneos más representativos a escala global ya que ocupan aproximadamente el 20 % de la superficie terrestre, albergan cavidades próximas a la superficie y forman parte de lo que se conoce como Zona Crítica de la Tierra. A pesar de que podrían jugar un papel importante en la regulación de la composición de la atmósfera externa, los ecosistemas subterráneos aún no han sido incluidos en las estimaciones globales de los balances de los principales GEI, pero son cada vez más numerosos los trabajos de investigación en este campo..., Karstic environments are the most representative underground ecosystems on a global scale since they cover approximately 20% of the land surface of the Earth and contain shallow caves that belong to the Critical Zone of the Earth. Although they could play an important role in regulation of the external atmosphere composition, the underground ecosystems have not yet been included in the annual carbon balances of the main GHGs. However, the research interest in this field has recently increased...

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

The advent of molecular tools, and particularly next-generation sequencing, has dramatically changed our knowledge of the diversity of microbial life on Earth. In recent decades, many studies on different terrestrial environments have described the intriguing diversity and abundance of Euzebyales/Euzebyaceae/Euzebya, yet its role in the geochemical cycle of elements is unknown. In addition, as far as we know, no Euzebya isolates have been obtained from terrestrial niches. In this review, it is shown that Euzebya and other haloalkaliphilic bacteria can thrive under harsh conditions, such as high concentrations of sodium and/or calcium, high electric conductivity and alkaline pH, highly variable temperatures, and water fluctuations. These conditions are quasi-extreme in the studied terrestrial environments. However, the culture media used so far for isolation have failed to reproduce the original conditions of these terrestrial ecosystems, and this is likely the reason why strains of Euzebya and other bacteria that inhabit the same niche could not be isolated. It is expected that culture media reproducing the environmental conditions outlined in this review could cope with the isolation of terrestrial Euzebya and other haloalkaliphilic genera.

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

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.

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.

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.

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.

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.

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.

A medida que se investiga y aprende sobre el funcionamiento de los ambientes subterráneos, lo que se conoce coloquialmente como las cuevas, se adquiere una mayor conciencia de la importancia de estos ecosistemas en el funcionamiento global del planeta: lo que sucede en superficie tiene influencia directa en el desarrollo y las condiciones de la vida en el subsuelo, así como también hay una retroalimentación entre los cambios biogeoquímicos del subsuelo, tanto estacionales como a largo plazo (ligados al cambio climático), y las condiciones de vida en la superficie. Es cada vez más numerosa la literatura científica que aborda diferentes estudios sobre la Zona Crítica de la Tierra, entendida esta como la capa más superficial de la corteza terrestre donde se producen e interactúan todos los procesos físicos, químicos y biológicos fundamentales para el mantenimiento de la vida (Moravec & Chorover, 2020). Los ecosistemas subterráneos forman una parte esencial de la Zona Crítica, en torno al 20% de la superficie continental libre de hielo se caracteriza por la presencia de rocas carbonáticas karstificables y que, por tanto, potencialmente pueden albergar cavidades próximas a la superficie. Son hábitats especiales para numerosas especies y contienen biomasa microbiana abundante y activa que vive tanto en el agua subterránea como en el sistema poroso de sedimentos y rocas. El hecho de que sean accesibles los convierte en laboratorios naturales de un enorme valor para numerosas disciplinas científicas.

The thermal spring of Fetida Cave, a still active sulfuric acid cave opening at sea level and located in Santa Cesarea Terme, southeastern Salento (Apulia region, Southern Italy) hosts abundant floating white filaments. The white filaments were mainly composed of sulfur crystals surrounded by microbial mass of the phyla Epsilonbacteraeota, Proteobacteria, Bacteroidetes, and Patescibacteria. The most abundant genus in the white filaments collected from the waters in the innermost part of the cave dominated by sulfidic exhalations was Arcobacter. This abundance can be related to the higher concentration of sulfide dissolved in water, and low oxygen and pH values. Conversely, lower Arcobacter abundances were obtained in the filaments collected in the entrance and middle part of the cave, where sulfidic water mixes with seawater, as the cave is subjected to tides and the mixing of fresh (continental) with marine water. The geochemical analysis of water and atmospheric gases confirmed these environmental constraints. In fact, the highest concentrations of H2S in the air and water were recorded closest to the spring upwelling in the innermost part of the cave, and the lowest ones near the cave entrance. The metabolic versatility of Arcobacter might provide a competitive advantage in the colonization of water bodies characterized by high sulfide, low oxygen, and dynamic fluid movement.

Fluid migration along faults can be highly complex and spatially variable, with channelled flow along karstified structures of the vadose zone. One such example is Vapor cave, near the urban area of Alhama de Murcia, situated along a tectonically active, NE-SW trending master fault as results of the convergence between Africa and the microplate of Iberia. Vapor cave represents an outstanding gases-blowout site from the upper vadose zone, developed in a favourably fissured carbonate-cemented conglomerate host rock under hypogene speleogenesis by the upwelling of hydrothermal (>33°C, and 100% relative humidity) and CO2-rich air, in or from the zone of fluid-geodynamic influence.In this study, we investigate the gaseous composition and, specifically, the geochemical fingerprint of deep-origin greenhouse gases (CO2, CH4) of both cave and soil air at Vapor cave. Detailed surveys were conducted to monitor the deep-origin gases exhaled by the cave, by using high precision field-deployable CRDS and FTIR spectrometers to in situ and real time measure the concentration and 13C of both carbon-GHGs. Inert gases like radon were also measured in parallel by a pulse-counting ionization chamber (alpha spectroscopy). The collected data provide new insights into the control exerted by active fault segments on deep-seated gas migration toward the surface.The C species of the deep-origin fluids are dominated by CO2 (concentration higher than 1% and 13C-CO2 ranging from 4.5 to 7.5) with the abundance of CH4 below the atmospheric background. It is estimated that the exhaled air represents between 1 to 3% of this pure¿theoretical CO2 added from the deep endogenous source feeding the cave atmosphere and linked to the fault activity. Anomalous radon concentrations recorded at this site also confirm the contribution of this geogenic gas in the cave atmosphere (222Rn ranges 40-60 kBq/m3 at -30 m depth) and its accumulation in the overlying soil (exceeding 10K kBq/m3).In contrast to the releas

The hydrothermal caves linked to active faulting have subterranean atmospheres with a distinctive gaseous composition containing deep endogenous gases, such as carbon dioxide, methane and nitrogen oxides (NOx). Ascending fluids through associated near-surface hydrothermal processes can mobilize endogenous gases into the Critical Zone and, ultimately, to the lower troposphere. Nitrogen oxides are polluting gases and can have adverse effects on human health, especially inhaled NO2. They also catalyse ozone (O3) production in the lower layers of the atmosphere and the greenhouse effect, when they react with volatile organic compounds. The largest source of NOx emissions is anthropogenic. The rest is produced naturally by microbial processes in soil and water, by lightning, volcanic activity, storms, etc. Production of N2O and NO2 is associated with soil and other active-geothermal ecosystems, far less is known about the sources and sinks of these gases within subterranean locations. Here, we report high N2O and NO2 concentrations detected along a hypogenic system associated with an active faulting (Vapour Cave, southern Spain), which enables direct gas exchange with the low-atmosphere. These anomalous concentrations of N2O and, NO2 are about ten and five times higher than the typical atmospheric background, respectively. Gaseous composition analyses of subterranean atmosphere were conducted by high precision field-deployable CRDS and FTIR spectrometers for measuring in situ the target tracer gases (NO2, N2O, CH4, CO2) and ¿13C of both carbon-GHGs. DNA extraction, sequencing and phylogenetic analyses were conducted to characterize the microbial community of cave sediments. The results showed that N2O and NO2 emission depends on the activity of nitrification by ammonia oxidizing microorganisms (such as members of the family Nitrosomonadaceae and phylum Thaumarchaeota) and/or as a result of incomplete denitrification by heterotrophic denitrifying bacteria (such as Bacillu

Karsts cover up to 25 % of the land surface and contain significant sedimentary deposits that become active cave-soils. Subterranean karst ecosystems play an active role in the global carbon cycle in terms of their contribution to the global GHG balance. They act alternately as a source or sink of CO2 and as a rapid sink of CH4. The most recent results indicate that microbiota must play a significant ecological role in the biogeochemical processes that control the subterranean atmosphere composition. Soils forming underneath the surface must host a large part of the subterranean microbiota. But to date, their behaviour concerning the production of gases and exchange with the ¿confined troposphere¿ has not been evaluated. Systematic direct estimates of CO2 and CH4 fluxes from cave-soils do not exist in literature. And they are needed before global generalizations can be made about the carbon budgets (emissions and sinks) of karstic ecosystems.Here we present pioneering research to evaluate the carbon fluxes from the cave soils directly exchanged with the cave atmosphere. This preliminary study is the first approach to systematically characterize the role of cave-soils in the production and transport of CO2 and CH4 in the subterranean environment. We carried out automatic in situ and real-time monitoring of CO2 and CH4 diffusive fluxes from a sedimentary alluvial soil in Pindal cave for one year (north Spain). We developed seasonal campaigns for CH4 and CO2 fluxes daily continuous monitoring by a LICOR closed chamber-based gas exchange system, in conjunction with a compatible Gasmet FTIR gas analyser. Moreover, autonomous equipment monitored the main micro-environmental parameters of the local subsurface-soil-atmosphere system. To interpret gas exchange processes and rates, and to understand the underlying mechanisms in soils, we also carried out seasonal ¿13C geochemical tracing by using Picarro cavity ring-down spectroscopy, through simultaneous cave atmosphere-soil