Exploring the diversity, distribution and metabolic potential of chemolithotrophic and chemoorganotrophic microorganisms reactivated from deep continental granitic crust beneath the Deccan Traps, India

Abstract

Scientific drilling within the deep continental crust provides the unique opportunity for characterizing subsurface microorganisms from deep crystalline rocks and is of major scientific interest owing to the crucial role of these microorganism in global carbon cycles. Scientific drilling in Koyna seismogenic zone enables characaterization of deep Earth crust microbiome and provides a new insight into their biogeochemical role. In this study, we investigated the microbial populations reactivated (enriched) from the deep [1679 - 2908 meters below surface (mbs)] granitic rocks underneath the Deccan Traps with inorganic [CO2 (+H2)/ HCO3- ] and organic carbon (CH4 / Organic carbon mix/ Polymeric carbon mix) sources under hot (50°C), anaerobic conditions (180 days). Estimation of total protein, ATP and 16S rRNA gene qPCR suggested considerable microbial growth in all the enrichment setups. 16S rRNA gene amplicon sequencing showed a depth-wide distribution of diverse chemolithotrophic (and a few fermentative) organisms in the inorganic enrichments, whereas a substrate specific response was observed under the heterotrophic conditions with organic carbon sources. Comamonas, Burkholderia-Caballeronia-Paraburkholderia, Ralstonia, Klebsiella, unclassified Burkholderiaceae and Enterobacteriaceae were reactivated as dominant organisms from the enrichments of the deeper rocks (2335 - 2908 mbs) with both CO2 and HCO3-. From shallower depths, organisms of varied taxa were enriched. Pseudomonas, Rhodanobacter, Methyloversatilis, and Thaumarchaeota were major CO2 (+H2) utilizers, while Nocardioides, Sphingomonas, Aeromonas responded towards HCO3-. Enrichments with CH4 reactivated Actinophytocola, Pseudomonas, Methylobacterium, Nocardiodes members, while with the organic carbons, Bacillus and Thermoactinomyces were the most abundant taxa. Gemella, Pseudomonas and Nocardiodes got enriched with the polymeric carbon mix. Interestingly, enrichment of Cyanobacteria were observed mostly with CO2 (+H2), CH4  and polymeric carbon mix, which could be attributed to their ecological versatility, potential for H2-based lithoautotrophic metabolism and their ability to switch to fermentative lifestyle. Statistical analyses depicted depth-wise and substrate-wise distinct cladding of chemolithotrophic and chemoorganotrophic microbial communities, respectively. Phylogenetic analyses of the operational taxonomic units, showed similarities with sequences retrieved from other extreme deep subsurface environments. Co-occurrence networks suggested close interaction between chemolithototrophic and chemoorganotrophic/fermentative organisms. Predictive metabolic profiling revealed the presence of various genes for carbon and energy metabolisms as well as for valuable chemicals. The subsurface microorganisms showed the potential for answering the fundamental questions of deep life and for their exploitation as resources of valuable chemicals.