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.This research was supported by the Spanish Ministry of Science and Innovation through project PID2019-110603RB-I00 and the collaboration of PID2020-114978GB-I00 project, MCIN/AEI/FEDER, UE/10.13039/501100011033. This is a contribution from CSIC Interdisciplinary Thematic Platform Open Heritage: Research and Society (PTI-PAIS)

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.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

Microbial Activity in Subterranean Ecosystems: Recent Advances

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

Crossiella, a Rare Actinomycetota Genus, Abundant in the Environment

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.This research was funded by the projects PID2020-114978GB-I00 and PID2019-110603RB-I00. The Malaga City Council financed data from the archaeological basement of the Thyssen Museum of Malaga through a conservation contract for this Roman site

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.This work was supported by the Spanish Ministry of Science and Innovation through project PID2019-110603RB-I00 and the collaboration of PID2020-114978GB-I00 project, MCIN/AEI/FEDER, UE/10.13039/501100011033. The microbiological analyses of the 2019 flood samples were financed by the “Consejería de Cultura, Política lingüística y Turismo del Principado de Asturias”

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.This research was supported by the Spanish Ministry of Science and Innovation through project PID2019-110603RB-I00 and the collaboration of projects RTI2018-099052-B-I00 and PID2020-114978GB-I00, MCIN/AEI/FEDER/UE/10.13039/501100011033. This is a contribution from CSIC Interdisciplinary Thematic Platform Open Heritage: Research and Society (PTI-PAIS)

Unraveling the Drivers Controlling the Transient and Seasonal CO2 Dynamic in a Shallow Temperate Cave

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

The Marine Bacterial Genus Euzebya Is Distributed Worldwide in Terrestrial Environments: A Review

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

Early Detection of Phototrophic Biofilms in the Polychrome Panel, El Castillo Cave, Spain

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

<i>Crossiella</i>, a Rare <i>Actinomycetota</i> Genus, Abundant in the Environment

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