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.

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.

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.

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.

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.