A microbial oasis in karstic caves: a metagenomic view of active moonmilk microbiomes

Karstic caves are extreme critical zone habitats that remain underexplored mostly by virtue of the logistical difficulties in accessing them. However, despite the absence of sunlight and nutrients, microbial life is prevalent in karstic caves and is posed to play significant roles in the dissolution and re-precipitation of CaCO3, therefore aiding and guiding the formation of primary speleothems such as stalactites, but also of secondary speleothems, which include moonmilk. Previous investigations using cultivation, prokaryotic marker gene and stable isotope approaches indicated that the microbiomes of this "cream-cheese-like" (Barton & Jurado, 2007) secondary speleothem have the potential to fix atmospheric greenhouse gases such as CO2 and CH4. Here, we present metagenomes generated for active and dried moonmilk, for the first time allowing inferences of microbial function in this microenvironment from high-quality MAGs. Active moonmilk harbored several archaeal MAGs of phyla Thermoproteota and Nanoarchaeota that were not detected in dried moonmilk. Other MAGs uniquely detected in active moonmilk presented metabolic pathways indicative of carbon fixation potential via the pentose phosphate pathway. Predicted antiviral systems in active moonmilk MAGs were diverse, but largely exclusive of CRISPR-Cas systems, suggesting viral infection in this microenvironment may be limited by the unique chemistry of its inner carbonate matrix. The complex microbial ecology of active moonmilk resulted in the naming of two bacterial genera: Viracochaeum gen. nov. (Viracochaeales ord. nov., of Zixibacteria class MSB-5A5) and Tunupaeum gen. nov. (Tunupaeaceae fam. nov., Paceibacteria, Patescibacteria). Moonmilk is a common encrusted feature of karstic cave walls for which the first MAGs hereby recovered have exhibited diverse microbial communities of relevance to Earth"s carbon cycling.