Gypsum Cave Biofilm Communities are Strongly Influenced by Bat- And Arthropod-Related Fungi.

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.

Scenarios for the Altamira cave CO2 concentration from 1950 to 2100.

A data-driven approach insensitive to the initial conditions was developed to extract governing equations for the concentration of CO2 in the Altamira cave (Spain) and its two main drivers: the outside temperature and the soil moisture. This model was then reformulated in order to use satellite observations and meteorological predictions, as a forcing. The concentration of CO2 inside the cave was then investigated from 1950 to 2100 under various scenarios. It is found that extreme levels of CO2 were reached during the period 1950-1972 due to the massive affluence of visitors. It is demonstrated that it is possible to monitor the CO2 in the cave in real time using satellite information as an external forcing. For the future, it is shown that the maximum values of CO2 will exceed the levels reached during the 1980s and the 1990s when the CO2 introduced by the touristic visits, although intentionally reduced, still enhanced considerably the micro corrosion of walls and pigments.

A Second Fungal Outbreak in Castañar Cave, Spain, Discloses the Fragility of Subsurface Ecosystems.

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.

Adaptive response of prokaryotic communities to extreme pollution flooding in a Paleolithic rock art cave (Pindal Cave, northern Spain).

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 studies and underscores the importance of considering temporal dynamics in conservation efforts.

Microclimate, airborne particles, and microbiological monitoring protocol for conservation of rock-art caves: The case of the world-heritage site La Garma cave (Spain).

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.

Prokaryotic communities inhabiting a high-radon subterranean ecosystem (Castañar Cave, Spain): Environmental and substrate-driven controls.

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 methods used to study the negative impacts of tourism on cave ecosystems.

New insights into the structure, microbial diversity and ecology of yellow biofilms in a Paleolithic rock art cave (Pindal Cave, Asturias, Spain).

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 valuable cultural heritage.

Diversity of Microfungi in a High Radon Cave Ecosystem.

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.

Role of subterranean microbiota in the carbon cycle and greenhouse gas dynamics.

Subterranean ecosystems play an active role in the global carbon cycle, yet only a few studies using indirect methods have focused on the role of the cave microbiota in this critical cycle. Here we present pioneering research based on in situ real-time monitoring of CO2 and CH4 diffusive fluxes and concurrent δ13C geochemical tracing in caves, combined with 16S microbiome analysis. Our findings show that cave sediments are promoting continuous CH4 consumption from cave atmosphere, resulting in a significant removal of 65% to 90%. This research reveals the most effective taxa and metabolic pathways in consumption and uptake of greenhouse gases. Methanotrophic bacteria were the most effective group involved in CH4 consumption, namely within the families Methylomonaceae, Methylomirabilaceae and Methylacidiphilaceae. In addition, Crossiella and Nitrosococcaceae wb1-P19 could be one of the main responsible of CO2 uptake, which occurs via the Calvin-Benson-Bassham cycle and reversible hydration of CO2. Thus, syntrophic relationships exist between Crossiella and nitrifying bacteria that capture CO2, consume inorganic N produced by heterotrophic ammonification in the surface of sediments, and induce moonmilk formation. Moonmilk is found as the most evolved phase of the microbial processes in cave sediments that fixes CO2 as calcite and intensifies CH4 oxidation. From an ecological perspective, cave sediments act qualitatively as soils, providing fundamental ecosystem services (e.g. nutrient cycling and carbon sequestration) with direct influence on greenhouse gas emissions.

Dominance of Arcobacter in the white filaments from the thermal sulfidic spring of Fetida Cave (Apulia, southern Italy).

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.

Environment-driven control of fungi in subterranean ecosystems: the case of La Garma Cave (northern Spain).

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.

Prokaryotic communities from a lava tube cave in La Palma Island (Spain) are involved in the biogeochemical cycle of major elements.

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.

Biologically mediated release of endogenous N2O and NO2 gases in a hydrothermal, hypoxic subterranean environment.

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. This raises many questions on the metabolic behaviour of this and other archaeal phyla.

High radon levels in subterranean environments: monitoring and technical criteria to ensure human safety (case of Castañar cave, Spain).

Castañar cave contains the highest radon gas ((222)Rn) concentration in Spain with an annual average of 31.9 kBq m(-)(3). Seasonal variations with summer minimums and maximum values in fall were recorded. The reduction of air-filled porosity of soil and rock by condensation or rainfalls hides the radon exchange by gas diffusion, determining this seasonal stair-step pattern of the radon activity concentration in underground air. The effective total dose and the maximum hours permitted have been evaluated for the guides and public safety with a highly detailed radon measurement along 2011 and 2012. A network of 12 passive detectors (kodalphas) has been installed, as well as, two radon continuous monitoring in the most interesting geological sites of the subterranean environment. A follow up of the recommended time (max. 50 min) inside the underground environment has been analysed since the reopen to public visitors for not surpassing the legal maximum effective dose for tourists and guides. Results shown that public visitors would receive in fall a 12.1% of the total effective dose permitted per visit, whereas in summer it is reduced to 8.6%, while the cave guide received a total effective dose of 6.41 mSv in four months. The spatial radon maps allow defining the most suitable touristic paths according to the radon concentration distribution and therefore, appropriate fall and summer touristic paths are recommended.

Subterranean atmospheres may act as daily methane sinks.

In recent years, methane (CH4) has received increasing scientific attention because it is the most abundant non-CO2 atmospheric greenhouse gas (GHG) and controls numerous chemical reactions in the troposphere and stratosphere. However, there is much that is unknown about CH4 sources and sinks and their evolution over time. Here we show that near-surface cavities in the uppermost vadose zone are now actively removing atmospheric CH4. Through seasonal geochemical tracing of air in the atmosphere, soil and underground at diverse geographic and climatic locations in Spain, our results show that complete consumption of CH4 is favoured in the subsurface atmosphere under near vapour-saturation conditions and without significant intervention of methanotrophic bacteria. Overall, our results indicate that subterranean atmospheres may be acting as sinks for atmospheric CH4 on a daily scale. However, this terrestrial sink has not yet been considered in CH4 budget balances.

The deterioration of Circular Mausoleum, Roman Necropolis of Carmona, Spain.

The Circular Mausoleum tomb in the Roman Necropolis of Carmona was carved on a calcarenite sequence in an ancient quarry located in the town of Carmona, Southern Spain. This rock-cut tomb, representative of Roman burial practices, currently suffers from serious deterioration. A detailed survey over several years permitted the identification of the main tomb's pathologies and damaging processes, which include loss of material (scaling, flaking, granular disintegration), surface modifications (efflorescences, crusts and deposits) and extensive biological colonization. The results obtained in this study indicated that anthropogenic changes were largely responsible and enhanced the main alteration mechanisms observed in the Circular Mausoleum. Based on the deterioration diagnosis, effective corrective actions were proposed. This study shows that any conservative intervention in the interior of the tomb should be preceded by accurate in situ measurements and laboratory analyses to ascribe the source of the deterioration damages and thus designing effective treatments.

Composition and spectra of copper-carotenoid sediments from a pyrite mine stream in Spain.

Mine drainages of La Poderosa (El Campillo, Huelva, Spain), located in the Rio Tinto Basin (Iberian Pyrite Belt) generate carotenoid complexes mixed with copper sulfates presenting good natural models for the production of carotenoids from microorganisms. The environmental conditions of Rio Tinto Basin include important environmental stresses to force the microorganisms to accumulate carotenoids. Here we show as carotenoid compounds in sediments can be analyzed directly in the solid state by Raman and Luminescence spectroscopy techniques to identify solid carotenoid, avoiding dissolution and pre-concentration treatments, since the hydrous copper-salted paragenesis do not mask the Raman emission of carotenoids. Raman spectra recorded from one of these specimens' exhibit major features at approximately 1006, 1154, and 1520 cm(-1). The bands at 1520 cm(-1) and 1154 cm(-1) can be assigned to in-phase C=C (γ(-1)) and C-C stretching (γ(-2)) vibrations of the polyene chain in carotenoids. The in-plane rocking deformations of CH3 groups linked to this chain coupled with C-C bonds are observed in the 1006 cm(-1) region. X-irradiation pretreatments enhance the cathodoluminescence spectra emission of carotenoids enough to distinguish organic compounds including hydroxyl and carboxyl groups. Carotenoids in copper-sulfates could be used as biomarkers and useful proxies for understanding remote mineral formations as well as for terrestrial environmental investigations related to mine drainage contamination including biological activity and photo-oxidation processes.

Deterioration of an Etruscan tomb by bacteria from the order Rhizobiales.

The Etruscan civilisation originated in the Villanovan Iron Age in the ninth century BC and was absorbed by Rome in the first century BC. Etruscan tombs, many of which are subterranean, are one of the best representations of this culture. The principal importance of these tombs, however, lies in the wall paintings and in the tradition of rich burial, which was unique in the Mediterranean Basin, with the exception of Egypt. Relatively little information is available concerning the biodeterioration of Etruscan tombs, which is caused by a colonisation that covers the paintings with white, circular to irregular aggregates of bacteria or biofilms that tend to connect each other. Thus, these colonisations sometimes cover extensive surfaces. Here we show that the colonisation of paintings in Tomba del Colle is primarily due to bacteria of the order Rhizobiales (Alphaproteobacteria), which were likely influenced by the neighbouring rhizosphere community and the availability of nutrients from root exudates.

The actinobacterial colonization of Etruscan paintings.

The paintings from Tomba della Scimmia, in Tuscany, are representative of the heavy bacterial colonization experienced in most Etruscan necropolises. The tomb remained open until the late 70's when it was closed because of severe deterioration of the walls, ceiling and paintings after decades of visits. The deterioration is the result of environmental changes and impacts suffered since its discovery in 1846. We show scanning electron microscopy and molecular studies that reveal the extent and nature of the biodeterioration. Actinobacteria, mainly Nocardia and Pseudonocardia colonize and grow on the tomb walls and this process is linked to the availability of organic matter, phyllosilicates (e.g. clay minerals) and iron oxides. Nocardia is found metabolically active in the paintings. The data confirm the specialization of the genera Nocardia and Pseudonocardia in the colonization of subterranean niches.

The biogeochemical role of Actinobacteria in Altamira Cave, Spain.

The walls and ceiling of Altamira Cave, northern Spain, are coated with different coloured spots (yellow, white and grey). Electron microscopy revealed that the grey spots are composed of bacteria and bioinduced CaCO(3) crystals. The morphology of the spots revealed a dense network of microorganisms organized in well-defined radial and dendritic divergent branches from the central area towards the exterior of the spot, which is coated with overlying spheroidal elements of CaCO(3) and CaCO(3) nest-like aggregates. Molecular analysis indicated that the grey spots were mainly formed by an unrecognized species of the genus Actinobacteria. CO(2) efflux measurements in rocks heavily covered by grey spots confirmed that bacteria-forming spots promoted uptake of the gas, which is abundant in the cave. The bacteria can use the captured CO(2) to dissolve the rock and subsequently generate crystals of CaCO(3) in periods of lower humidity and/or CO(2). A tentative model for the formation of these grey spots, supported by scanning electron microscopy and transmission electron microscopy data, is proposed.