Unlocking the mystery of deep biosphere hosted by the Archaean granitic crust underneath the Deccan Traps, Koyna, India

Life that inhabits deep within the continental crust remained enigmatic, and scientifically less explored. Recent scientific drilling of 3000-meter-deep scientific borehole at the Koyna seismogenic zone of the Deccan Traps, India provided an unprecedented opportunity to investigate the nature of life that evolved and existed within the extreme environment of Archean granitic basement. Rock core samples recovered from deep (up to ~2910 meter below surface), progressively hot (up to 74 °C), high pressure, alkaline, oligotrophic biosphere were analyzed through amplicon sequencing, metagenomics, and cultivation-based approaches. 16S rRNA gene amplicon sequencing showed considerable bacterial diversity. A significant proportion (50-63 %) of the microbial community, represented by the members of Actinobacteria, Gammaproteobacteria, Alphaproteobacteria, Bacilli and Clostridia was identified as endemic or core microbiome of this extreme realm. Relative abundance of different taxa of the core microbiome varied with depth in response to prevailing lithology and geochemistry. Ecological modeling depicted a major role dispersal limitation in explaining community variability suggesting the greater impact of stochasticity on the community assembly. Co-occurrence network analysis elucidated close interactions among autotrophic and organotrophic bacteria. Shotgun metagenomics revealed a major role of autotrophic carbon fixation via the Wood-Ljungdahl pathway and Rnf complex for energy and carbon metabolism. Presence of other mechanisms of energy generation such as ATPase, H₂, CO, CH₄ or sulfur oxidation system and the possibility of coupling of electron donors to NO₃⁻/SO₄²⁻ reduction illustrated the metabolic versatility of these organisms in terms of utilizing diverse resources. Deeper analysis suggested the existence of an ‘acetate switch’, coordinating biosynthesis and cellular homeostasis. Reactivation of the rock-hosted microbiome using different nutrients (carbon source, e- donor and acceptor) and at elevated temperatures (up to 70-degree C) and anaerobic conditions showed patterns of differential taxa recruitment for utilizing different substrate regimes. Enrichment of strict anaerobic members of taxa Peptococcaceae, Clostridiaceae Family XIV, Clostridiaceae 2 and Moraxellaceae in H₂+CO₂ condition corroborated well with their ability for acetogenesis. Universal enrichment of Burkholderiaceae, Bacillaceae and Sphingomonadaceae suggested their abilities to use diverse substrates. Our data provided deeper insights into microbial life and its mechanisms of carbon and energy metabolism within the nutrient- and energy-limiting deep granitic crust.