Polysaccharide-degrading archaea dominate acidic hot springs: genomic and cultivation insights into a novel <i>Thermoproteota</i> lineage

The expansion of sequencing technologies and bioinformatics has greatly advanced our understanding of microbial "dark matter," yet the isolation of pure cultures, especially among Archaea, remains rare and challenging. Cultivation is still essential for the reliable characterization of microbial metabolism, which cannot be fully replaced by metagenomics and other omics-based approaches. Here, we report the first cultivated representatives of a deep-branching archaeal lineage previously known as <i>Candidatus</i> Marsarchaeota. Our phylogenomic analyses place these isolates within the phylum <i>Thermoproteota</i> as a novel order, <i>Tardisphaerales</i>. Members of <i>Tardisphaerales</i> dominate the prokaryotic communities in acidic hot springs below 70°C, comprising up to 40% of the total microbial population, underscoring their ecological significance. Functional genomics and culture experiments reveal a thermoacidophilic, anaerobic lifestyle, with energy metabolism based on carbohydrate fermentation, particularly of polysaccharides. This metabolic capability is supported by numerous glycosidase-encoding genes and by unprecedented metabolic versatility among thermoacidophiles. The isolates possess complete glycolysis, Entner-Doudoroff, and pentose-phosphate pathways, allowing them to utilize different sugars. Specialization in polysaccharide hydrolysis presumably provides an adaptive advantage for these slow-growing archaea, as most other heterotrophic thermoacidophiles prefer peptides or simple sugars. Furthermore, robust defense mechanisms against reactive oxygen species and persistence in acidic conditions enable <i>Tardisphaerales</i> to outcompete other heterotrophs and maintain dominance in these extreme habitats. The discovery and cultivation of this new order expand prokaryotic taxonomy and reveal the key players in carbon cycling in acidic geothermal ecosystems.IMPORTANCEMost of the dominant prokaryotes in natural environments remain uncultivated, and their metabolic potential and ecological role can be inferred solely from metagenomics. However, cultivation is essential for comprehensive functional characterization and identification of novel traits. Here, we describe the first cultivated representatives of the new archaeal order <i>Tardisphaerales</i> within the novel class <i>Tardisphaeria</i> (phylum <i>Thermoproteota</i>), a lineage abundant in acidic hot springs. Through the whole-genome reconstruction and microbiological experiments in pure cultures, we demonstrate that these archaea are metabolically distinct from the known thermoacidophiles, making them the key degraders of the complex organic matter in hot, acidic environments. Their genomes encode a diverse set of glycosidases that allow efficient polysaccharide breakdown at high temperatures and low pH, a trait with promising biotechnological applications.