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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 the flood produced very significant changes in the microbial composition of sediments: the appearance of the bacterial phyla Bacillota and Bacteroidota, including groups of opportunistic bacterial pathogens (Corynebacterium, Thauera, Clostridiales) and the almost complete disappearance of Rokubacteriales and Nitrospirota. Bacillota and Bacteroidota are common in the intestinal tract of mammals and are dominant in liquid and solid samples of manure from dairy farms. Overall, the results conclude that the sediments dragged into the cave were accompanied by residues from livestock farming and indicate the high degree of vulnerability of this type of cave. Although livestock activity finally ceased in 2021, we continue analyzing environmental parameters, waters, sediments and microbial populations to evaluate their evolution in the medium-long term.Research funded by PID2019-110603RB-I00 – SUBSYST project and PID2020-114978GB-I

Water plays a key role in the colonization and ecology of microorganisms in natural subterranean environments since it controls their metabolic activity and the microbe-mineral interactions. In turn, microbial activity leaves its signature in groundwater. For instance, the aerial hyphae of Actinobacteria colonizing cave-rock surfaces and sediments act as vapour condensation nuclei and facilitate the retention of interstitial water in the porous system of rocks, sediments and speleothems.Here, we present the results of exhaustive monitoring of cave waters from Pindal Cave (northern Spain), a shallow and well-ventilated cave on the coastline, with extensive microbial colonization on the sediments and walls surfaces. This study sheds light on the detection of biosignatures based on stables isotopes of cave waters and controlled by underground-dwelling microorganisms. The isotopic composition (d18O and dD) of seepage waters (fast drips, soda straws and gours) and condensation droplets were determined by cavity ring-down spectroscopy (CRDS). An analytical set-up based on a CRDS analyser coupled to an Induction Module enabled the step-heating of moonmilk deposits and other substrates with distinct degrees of microbial colonization, and the selective release of their interstitial water for isotope analyses.The δ18O and δD values of the in-cave waters correlate linearly (δD=7.71·δ18O+12.28; R2=0.97), like the Local Meteoric Water Line (δD=7.00·δ18O+6.77; R2=0.92, based on the of isotopes composition of regional rain). The offset of d-excess between the cave waters and the LMWL would indicate a significant recharge by occult precipitation linked to local coastal fogs. The isotopic composition of dripping water, -5.7‰ [-6.7 to -4.8 ‰] for d18O and -31.3‰ [-39.0 to -22.7 ‰] for dD, agree with the water samples from the gours located far from the cave entrance, with some slight deviations indicating evaporation processes or sudden contributions of meteoric water due to flash-flood events.The δ18O and δD values of condensation water collected from non-colonized surfaces are -5.4‰ [-5.9 to -4.5 ‰] and –28.4‰ [-32.9 to -21.3 ‰], respectively. This isotope composition is similar to the contemporary infiltration water, which suggests that condensation mostly comes from autochthonous vapour generated from in-cave waters. The Actinobacteria mats quickly absorb the condensation water and it is then retained during long periods on the rock surface. Thus, in the case of microbial induced deposits like moonmilk, condensate water becomes a reservoir of interstitial water exposed to isotopes fractionation linked to the microbial metabolism processes. Indeed, the δ18O and δD values of this interstitial water correlate linearly with the rest of the in-cave waters but show a distinctive composition with higher δ18O and δD values compared to the infiltration water and condensation droplets; -3.6‰ [-4.4 to -2.0 ‰] and –18.9‰ [-27.2 to -10.8 ‰], respectively. These findings lead to exploring the potential use of water isotopes as a tool for indirectly assessing microbial activity and its role in the water balances in underground ecosystems.Research funded by PID2019-110603RB-I00 – SUBSYST and PID2020-114978GB-I00 projects

Conferencia presentada en Goldschmidt 2022, Honolulu, USA and online 10-14 julio 2022, Moonmilk deposits is a well-known and widespread example of calcium carbonate biomineralization in subterranean environments. Moonmilk structure is spongy and wet during an emerging and growing active phase, while becoming crumbly, powdery and, finally, crusting during an evolved phase. The microbiota of moonmilk deposits varies throughout these development stages [1]. The RNA/DNA ratios also indicate an inactivation of microorganisms from incipient moonmilk toward consolidated deposits of calcium carbonate [2]. Therefore, a variety of biochemical mechanisms would be involved at each stage of moonmilk formation that could play a significant role in the carbon geochemical cycle. We quantify the CO2 and CH4 fluxes from microbial communities associated to moonmilk from Pindal cave (northern Spain), using a field-deployed chamber-based gas exchange system coupled with a cavity ring-down spectrometer for a simultaneous and real-time δ13C geochemical tracing. Moonmilk deposits were characterized by metagenomics analysis of 16S rRNA gene amplicons in terms of composition and ecological functionality, particularly those related with the cycle of both gases. Gas flux measurements show a continuous one-way CO2 efflux from moonmilk as well as a continuous one-way CH4 consumption from the cave air. These fluxes are intensified in the emerging and growing active phase of moonmilk. We found the key functional microbial groups in the moonmilk formation (Crossiella and the lineage wb1-P19) concluding that syntrophic relationships are established between bacteria, including ammonification (production of NH3 and CO2) → nitrification → CO2 capture and NH3 consumption triggering the fixation of the excess CO2 as calcite. Methylomonaceae is the main methanotrophic family responsible for the intense CH4 oxidation, up to 90% the atmospheric CH4 entering the cave environment. This research reveals moonmilk as a remarkable effective deposit in carbon sequestration. [1] Park et al (2020), Frontiers in Microbiology 11: 613. [2] Sanchez-Moral et al (2012), Geomorphology 139-140,285-292., This study was supported the Spanish Ministry of Science, Innovation through project PID2019-110603RB-I00, MCIN/AEI/FEDER, UE/10.13039/501100011033

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., This work was supported by the Spanish Ministry of Science, Innovation through project PID2019-110603RB-I00, MCIN/AEI/FEDER UE/10.13039/501100011033 and with collaboration of projects RTI2018-099052-B-I00 and PID2020-114978GB-I00. This research has also received funding from the European Union's Horizon 2020 research and innovation programme under the Marie Skłodowska-Curie grant agreement No 844535 — MIFLUKE.

24 páginas.- 8 figuras.- 110 referencias.- Supplementary Materials: The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/applbiosci1010003/s1, 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., This research was funded by the Consejeria de Universidades, Igualdad, Cultura y Deporte del Gobierno de Cantabria, and the Spanish Ministry of Science, Innovation through projects PID2020-114978GB-I00 and PID2019-110603RB-I00, MCIN/AEI/FEDER, UE/10.13039/501100011033.

18 pág.- 3 figuras.- 3 tablas.- 96 referencias, 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., This research was funded by Nerja Cave Foundation. This work is part of an Interdisciplinary Research Project for the conservation of Nerja Cave, authorized by the Ministry of Culture of the Andalusian Government.

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., This work was supported by the Spanish Ministry of Economy and Competitiveness through project CGL2016-75590-P with ERDF funds and PID2019-110603RB-I00/AEI/10.13039/501100011033. We acknowledge support of the publication fee by the CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

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., This work was supported by the Spanish Ministry of Economy and Competitiveness through projects CGL2013-41674-P, CGL2016-75590-P, PID2019-110603RB-I00, AEI/FEDER, UE and CSIC Open Access Publication Support Initiative through its Unit of Information Resources for Research (URICI).

19 páginas.- 9 figuras.- 2 tablas.- 91 referencias.- Supplementary Information The online version contains supplementary material available at https://doi.org/10.1007/s10123-021-00193-x, 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., Open Access funding provided thanks to the CRUE-CSIC agreement with Springer Nature. This research was supported by the Consejería de Universidades, Igualdad, Cultura y Deporte del Gobierno de Cantabria and the Spanish Ministry of Innovation and Science through projects CGL2016-75590-P with ERDF funds and PID2019-110603RB-I00/AEI/10.13039/501100011033.

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 CO

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 δ13C 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 δ13C-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 release of large volumes of deep endogenous CO2, Vapor cave constitutes an effective sink of methane (CH4). The deep-sourced CH4 is continuously depleted and 13C-enriched along the vertical migration pathway into the cave (CH413C close to −30‰). Some anomalous concentrations of deep endogenous methane have been already registered in the cave air, e.g. during march 2016, with CH4 ranging 2.3 to 3.4 ppm and δ13C-CH4 lighter than that found in the local background atmosphere. These anomalous CH4 data could be related to the occurrence of contemporary earthquakes, characterized by a total amount of seismic energy released of 4.9 x 109 J and epicenter locations southwest of the cave and within a radius of 20 km.The continuous depletion of CH4 in the cave air constitutes itself a very valuable property in terms of using as potential earthquake precursor in combination with other geochemical indicators. Hence, any anomalous concentration and isotopic deviation of this gas in the cave atmosphere with reference to the background level in the cave atmosphere could denote a more intense migration of endogenous fluids through the upper vadose zone, which could be related with an increase of the regional seismotectonic activity.

On-line, del 4 al 8 de mayo de 2020, 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-chamber air samplings. We also characterized the soil microbial communities related to the carbon cycle by meta-barcoding analyses of bacterial 16S rRNA genes and Shotgun Metagenomics. Preliminary results show net CO2 emissions from cave-soil on a daily scale, resulting from respiration by chemotrophic microorganisms. We detect significant magnitude variations along the day, reaching occasionally values close to zero. This is remarkable in such thermo-hygrometric stable environment and absence of light. Changes in the cave ventilation regime seems to be the determining factor just in some cases. Intrinsic microbial processes appear to be decisive in others. The results also reveal net CH4 uptake from cave-soil on a daily scale, with no significant magnitude variations along the day. It seems to be linked to the metabolism of Nitrate-dependent methanotrophs belonging to the phylum Rokubacteria. Additionally, we detected significant variations in magnitude and different flow patterns in the cave-soils colonized by biofilms, most prominent in the case of moonmilk deposits. These preliminary results confirm that cave-soil is playing an outstanding role in the processes of production, consumption and storage of CO2 and CH4 and may be partially determining the strong variations of these major GHGs in natural subterranean ecosystems.

18 páginas._ 3 figuras.- 1 tabla.- 102 referencias, 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, Financial support was obtained through project 0483_PROBIOMA_5_E, co-financed by the European Regional Development Fund within the framework of the Interreg V-A Spain-Portugal program (POCTEP) 2014–2020. This work was also supported by the Spanish Ministry of Economy and Competitiveness through projects CGL2016-75590-P and PID2019-110603RB-I00, AEI/FEDER, UE.

On-line, del 4 al 8 de mayo de 2020, 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 Bacillus, Acinetobacter and Cupriavidus) from this hydrothermal and hypoxic ecosystem. On the other hand, CH4 concentrations and 13CH4 vary along the cave (with the deep), in deepest cave locations CH4 values are higher with lighter 13C values in comparison with the more superficial areas, which indicates a deep endogenous origin of methane. However, in areas near the entrance we observe lower concentrations of methane and heavier 13C values (CH4

24 páginas.- 14 figuras.- 5 tablas.- 136 referencias.- Supplementary data to this article can be found online athttps://doi.org/10.1016/j.scitotenv.2020.141218, 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. © 2020 Elsevier B.V., This work was supported by the Spanish Ministry of Economy and Competitiveness through projects PID2019-110603RB-I00 , CGL2016-78318-C2-1R and CGL2016-78318-C2-2R AEI/FEDER/UE, and with the scientific and technical contribution of the following projects: CGL2016-75590-P with ERDF funds, CGL2017-83931-C3-2-P and CGL2017-87216-C4-1-R , AEI / FEDER , UE. S. Cuezva was funded by the European Union's Horizon 2020 - Research and Innovation Framework Programme under the Marie Skłodowska-Curie grant agreement No 844535 — MIFLUKE. N. Otero was funded by the Serra Húnter Fellowship ( Generalitat de Catalunya , Spain). We thank to GERA-group of the Fire-fighters Service of Madrid Autonomous Region for the essential work on installing the system of tubes for air sampling and the Council of “Alhama de Murcia” for their technical assistance during fieldwork.