Depth wide distribution and metabolic potential of chemolithoautotrophic microorganisms reactivated from deep continental granitic crust underneath the Deccan Traps at Koyna, India

Abstract

Characterization of inorganic carbon (C) utilizing microorganisms from deep crystalline rocks is of major scientific interest owing to their crucial role in global carbon and other elemental cycles. In this study we investigate the microbial populations from the deep [up to 2,908 meters below surface (mbs)] granitic rocks within the Koyna seismogenic zone, reactivated (enriched) under anaerobic, high temperature (50°C), chemolithoautotrophic conditions. Subsurface rock samples from six different depths (1,679-2,908 mbs) are incubated (180 days) with CO₂ (+H₂) or HCO₃ ^- as the sole C source. Estimation of total protein, ATP, utilization of NO₃ ^- and SO₄ ^2- and 16S rRNA gene qPCR suggests considerable microbial growth within the chemolithotrophic conditions. We note a better response of rock hosted community towards CO₂ (+H₂) over HCO₃ ^-. 16S rRNA gene amplicon sequencing shows a depth-wide distribution of diverse chemolithotrophic (and a few fermentative) Bacteria and Archaea. Comamonas, Burkholderia-Caballeronia-Paraburkholderia, Ralstonia, Klebsiella, unclassified Burkholderiaceae and Enterobacteriaceae are reactivated as dominant organisms from the enrichments of the deeper rocks (2335-2,908 mbs) with both CO₂ and HCO₃ ^-. For the rock samples from shallower depths, organisms of varied taxa are enriched under CO₂ (+H₂) and HCO₃ ^-. Pseudomonas, Rhodanobacter, Methyloversatilis, and Thaumarchaeota are major CO₂ (+H₂) utilizers, while Nocardioides, Sphingomonas, Aeromonas, respond towards HCO₃ ^-. H₂ oxidizing Cupriavidus, Hydrogenophilus, Hydrogenophaga, CO₂ fixing Cyanobacteria Rhodobacter, Clostridium, Desulfovibrio and methanogenic archaea are also enriched. Enriched chemolithoautotrophic members show good correlation with CO₂, CH₄ and H₂ concentrations of the native rock environments, while the organisms from upper horizons correlate more to NO₃ ^-, SO₄ ^2- _, Fe and TIC levels of the rocks. Co-occurrence networks suggest close interaction between chemolithoautotrophic and chemoorganotrophic/fermentative organisms. Carbon fixing 3-HP and DC/HB cycles, hydrogen, sulfur oxidation, CH₄ and acetate metabolisms are predicted in the enriched communities. Our study elucidates the presence of live, C and H₂ utilizing Bacteria and Archaea in deep subsurface granitic rocks, which are enriched successfully. Significant impact of depth and geochemical controls on relative distribution of various chemolithotrophic species enriched and their C and H₂ metabolism are highlighted. These endolithic microorganisms show great potential for answering the fundamental questions of deep life and their exploitation in CO₂ capture and conversion to useful products.

Keywords: Koyna seismogenic zone; chemolithotrophic microorganisms; deep biosphere; inorganic carbon metabolism; terrestrial subsurface granitic rock.

Figure 1

Assessment of (A) cell biomass in terms of total protein, (B) ATP, (C) utilization of sulfate; provided as TEA, (D) utilization of nitrate; as TEA, (E) Bacterial 16S rRNA gene copy number per mL of enrichment in HC and BC sets. The black center line denotes the median value (50th percentile), the empty square denotes the mean value and the pink (HC) and green (BC) boxes contain the 25–75th percentiles of the data sets. The values beyond these upper and lower boundaries are considered as outliers, marked with black filled diamond shapes.

Figure 2

Principal Component Analysis for (A)HC/(B)BC enrichments based on the growth parameters.

Figure 3

Heatmap displaying the relative abundance of major taxa (abundance ≥1%) detected in (A) HC and (B) BC enrichments. Burkholderia-Caballeronia-Paraburkholderia is designated as B-C-P-burkholderia.

Figure 4

Rank abundance (based on relative percentage abundance) of top 50 OTUs detected in (A) HC and (B) BC enrichments. Burkholderia-Caballeronia-Paraburkholderia is designated as B-C-P-burkholderia.

Figure 5

Non-metric Multidimensional scaling of microbial community (at the taxa level) for microbial taxa belonging HC and BC enrichment based on their association with depth.

Figure 6

Canonical correspondence analysis for microbial taxa belonging (A) HC and (B) BC enrichment based on their association with depth and relevant geochemical parameters.

Figure 7

(A) Co-occurrence network of OTUs enriched from at least 4 samples (at the lowest taxa level) in HC enrichment. Node colour are proportional to the number of degrees. Nodes with higher degree are orange in colour than those with lower connections (blue in colour). Colour of the edges represents the strength of the Spearman’s correlations between the nodes. Edges with stronger correlation are orange in colour. For the Spearman correlation values |r| > 0.9 are only considered. (B) Co-occurrence network of OTUs enriched from at least 4 samples (at the lowest taxa level) in BC enrichment. Node colour are proportional to the number of degrees. Nodes with higher degree are orange in colour than those with lower connections (blue in colour). Colour of the edges represents the strength of the Spearman’s correlations between the nodes. Edges with stronger correlation are orange in colour. For the Spearman correlation values |r| > 0.9 are only considered.

Figure 8

Relative abundance of predicted genes related to CO₂ fixation pathway, lithotrophy related/energy metabolism and genes related to other important metabolism (amongst metabolic genes) as predicted by PICRUSt v2.