Scientific deep drilling at Koyna, western India provides a unique opportunity to explore microbial life within deep biosphere hosted by ~65 Myr old Deccan basalt and Archaean granitic basement. Characteristic low organic carbon content, mafic/felsic nature but distinct trend in sulfate and nitrate concentrations demarcates the basaltic and granitic zones as distinct ecological habitats. Quantitative PCR indicates a depth independent distribution of microorganisms predominated by bacteria. Abundance of dsrB and mcrA genes are relatively higher (at least one order of magnitude) in basalt compared to granite. Bacterial communities are dominated by Alpha-, Beta-, Gammaproteobacteria, Actinobacteria and Firmicutes, whereas Euryarchaeota is the major archaeal group. Strong correlation among the abundance of autotrophic and heterotrophic taxa is noted. Bacteria known for nitrite, sulfur and hydrogen oxidation represent the autotrophs. Fermentative, nitrate/sulfate reducing and methane metabolising microorganisms represent the heterotrophs. Lack of shared operational taxonomic units and distinct clustering of major taxa indicate possible community isolation. Shotgun metagenomics corroborate that chemolithoautotrophic assimilation of carbon coupled with fermentation and anaerobic respiration drive this deep biosphere. This first report on the geomicrobiology of the subsurface of Deccan traps provides an unprecedented opportunity to understand microbial composition and function in the terrestrial, igneous rock-hosted, deep biosphere. status released display-pdf yes is-olf no is-manuscript no is-preprint no is-journal-matter no is-scanned no is-retracted no Received 2018 Jan 8; Accepted 2018 Nov 5; Collection date 2018. Introduction Microbial life that resides within the deep continental subsurface represents one of the largest and most diverse biospheres on this planet 1 . Recent assessments confirm that in spite of considerable physical as well as chemical constraints, the oligotrophic, dark biosphere underneath the continental crustal system (including fluid-filled pores, and fractures of the igneous rocks) harbour 2–19% of Earth’s total biomass represented by bacteria, archaea and fungi 1 – 4 . Overall, a combination of metabolic processes encompassing both autotrophy (including chemolithoautotrophy) and heterotrophy is implicated in deep terrestrial ecosystems 18 , 20 , 21 . Endolithic microbial Exploration of deep biosphere of granitic-basaltic crustal system is highly imperative to answer the fundamental questions on microbial life within deep, dark, oligotrophic crust, their mode of interaction and role in Earth’s biogeochemical processes, origin of life on Earth and even on extra-planetary locations (e.g., Mars). Compared to a reasonable level of understanding of deep life within marine subsurface, geomicrobiology of subterranean igneous rocks at greater depths remains almost elusive. Results Geochemical characteristics of the samples Thirteen rock core samples covering BS, TZ and GR horizons are collected from three bore holes. Core samples from different depths portray geochemical characteristics of the subsurface system (Fig. 1 , Supplementary Tables S1 and S2 ). Based on the tested parameters we find distinct and characteristic geochemical nature of the granitic and basaltic horizons. The distribution pattern of carbon, nitrogen and phosphorous has been analysed with respect to depth in order to assess the general nutritional condition of the subsurface crustal system. Total organic carbon content shows a strong negative correlation with depth followed by total carbon and total inorganic carbon. Nitrogen (NO 3 − and NO 2 − ) is generally low whereas phosphate is below detection limit except for samples obtained from the deeper horizon (PV8, P1 and U11). Figure 1 Geochemical characteristics of the samples ( a ) Heatmap showing relative abundances of different measured geochemical parameters. Rock types are depicted in red for basalts, green for transition zone samples and blue for granites; Normalized depth stands for depth from mean sea level (msl) – positive values for samples above msl and negative values for samples below msl. * unit of alkalinity is mg/kg. The samples are arranged according to the increasing normalized depth. ( b ) Principal component analysis of measured geochemical parameters; Rock types are depicted in red for basalts, green for transition zone samples and blue for granites. P: Panchgani, U: Ukhalu and PV: Phansavale. Sequencing data Sequence information and diversity indices are summarized in Table 1 . 11.5 million reads are obtained for 13 samples. This particular observation presents a sharp contrast to the high degree of overlap in OTU distribution pattern in deeply buried oceanic crust and sedimentary habitat above 26 . Interestingly, the observed partitioning of BS and GR microbiomes (as observed in db-RDA) corroborates well with the previous reports of a basalt specific microbiome 85 , 86 . Based on these results, we propose that although taxonomically similar organisms have occupied different igneous rocks at varied depths, environment specific taxon recruitment based on local availability of electron donors, acceptors and carbon sources, and limited opportunities of migration led to the distinct taxonomic assemblages. Microbial life in oligotrophic deep terrestrial subsurface hosted by igneous rocks are highly constrained by metabolic resources. Due to the extremely low cell turnover rates (hundreds to thousands of years), it is difficult to describe microbial processes by direct measurements 20 . Nevertheless, whole genome metagenome based studies, supported by 16S rRNA gene based analysis allow us to gain a better understanding. The metagenomic inventory supports our observation that there is a syntrophic relationship between autotrophic and heterotrophic organisms which could exchange fixed carbons, CO 2 , H 2 , etc. for their sustenance.

AbstractEarth’s crust contains a substantial proportion of global biomass, hosting microbial life up to several kilometers depth. Yet, knowledge of the evolution and extent of life in this environment remains elusive and patchy. Here we present isotopic, molecular and morphological signatures for deep ancient life in vein mineral specimens from mines distributed across the Precambrian Fennoscandian shield. Stable carbon isotopic signatures of calcite indicate microbial methanogenesis. In addition, sulfur isotope variability in pyrite, supported by stable carbon isotopic signatures of methyl-branched fatty acids, suggest subsequent bacterial sulfate reduction. Carbonate geochronology constrains the timing of these processes to the Cenozoic. We suggest that signatures of an ancient deep biosphere and long-term microbial activity are present throughout this shield. We suggest that microbes may have been active in the continental igneous crust over geological timescales, and that subsurface investigations may be valuable in the search for extra-terrestrial life. Biosignatures of ancient microbial life are present across the igneous crust of the Fennoscandian shield | Communications Earth & Environment Skip to main content Thank you for visiting nature.com. You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript. Advertisement Biosignatures of ancient microbial life are present across the igneous crust of the Fennoscandian shield Download PDF Download PDF Abstract Earth’s crust contains a substantial proportion of global biomass, hosting microbial life up to several kilometers depth. Yet, knowledge of the evolution and extent of life in this environment remains elusive and patchy. Here we present isotopic, molecular and morphological signatures for deep ancient life in vein mineral specimens from mines distributed across the Precambrian Fennoscandian shield. Stable carbon isotopic signatures of calcite indicate microbial methanogenesis. In addition, sulfur isotope variability in pyrite, supported by stable carbon isotopic signatures of methyl-branched fatty acids, suggest subsequent bacterial sulfate reduction. Carbonate geochronology constrains the timing of these processes to the Cenozoic. We suggest that Precambrian crystalline rock makes up the largest volumes of the continental crust, accounting for >70% of the total continental surface area and >80% of crust at depths below 2 km 6 , 7 . Environmental conditions in this cryptic habitat shift rapidly to anoxic within the upper tens of meters 8 . The majority of microorganisms living in deep fracture water therefore maintain an anaerobic lifestyle in the absence of sunlight and scarcity of organic carbon sources 9 . Metabolic pathways include sulfate reduction, fermentation, acetogenesis, methanogenesis, and methanotrophy 10 , 11 , 12 , 13 , 14 . The deep biosphere is believed to host some of the most ancient evolutionary lineages 2 , 15 and has been proposed to have held the majority of Earth’s live biomass prior to plant colonization of land ~400 Ma ago 16 . This suggests that deep environments, including ocean floors, have dominated on Earth for most of life’s history. However, in similarity with live communities, knowledge of the extent and nature of ancient microbial activity in the deep continental crust is still scarce. This secondary biogenicity evidence is crucial, as there have been reports from the sedimentary record that superheavy pyrite may form also from TSR 65 . Morphological evidence for deep ancient life As most of the samples are not newly retrieved from the mines, focus is on completely mineralized morphological remains of microbial communities, interpreted as indigenous. The remnants of completely mineralized interconnected networks of filamentous structures with anastomosing and branching filaments occurring in spatial relation to bitumen in a sample from Lund (Fig. It has been hypothesized that microbial activity in the continental igneous crust has been widespread during Earth’s history 16 , 67 , but direct evidence for this omnipresence is scarce. Our approach using a comprehensive set of subsurface mineral samples collected from 33 mines across the Fennoscandian shield reveals omnipresent biosignatures of deep ancient life across this craton, particularly in the central part where most the samples in this study are from. There are also isotopic signatures for MSR in pyrite from the eastern part of the Fennoscandian shield (Finland) 60 . Eme, L., Spang, A., Lombard, J., Stairs, C. W. & Ettema, T. J. G. Archaea and the origin of eukaryotes. Nat. Rev. Microbiol. 15 , 711–723 (2017). Article CAS Google Scholar McMahon, S. & Parnell, J. The deep history of Earth’s biomass. J. Geol. Soc. 175 , 716 (2018). Article Google Scholar Pedersen, K. et al. Evidence of ancient life at 207 m depth in a granitic aquifer. Geology 25 , 827–830 (1997). Article CAS Google Scholar Tillberg, M. et al. Re-evaluating the age of deep biosphere fossils in the Lockne impact structure. Geosciences 9 , https://doi.org/10.3390/geosciences9050202 (2019). Ivarsson, M. et al. Fungal colonization of an Ordovician impact-induced hydrothermal system. Sci. Rep. Biosignatures of ancient microbial life are present across the igneous crust of the Fennoscandian shield. Commun Earth Environ 2 , 102 (2021). https://doi.org/10.1038/s43247-021-00170-2 Download citation Received : 20 January 2021 Accepted : 20 April 2021 Published : 03 June 2021 Version of record : 03 June 2021 DOI : https://doi.org/10.1038/s43247-021-00170-2 Share this article Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a shareable link is not currently available for this article.

things". The finding is significant because no other multicellular organism had ever been detected farther than 2 km (1.2 mi) below the Earth's surface Halicephalobus mephisto is a species of nematode, among a number of other roundworms, discovered by geoscientists Gaetan Borgonie and Tullis Onstott in 2011. It was detected in ore recovered from deep rock fracture water in several gold mines in South Africa 0.9 km (0.56 mi), 1.3 km (0.81 mi), and 3.6 km (2.2 mi) under the surface of the Earth. Onstott said that "it scared the life out of me when Halicephalobus mephisto is a species of nematode, among a number of other roundworms, discovered by geoscientists Gaetan Borgonie and Tullis Onstott in 2011. It was detected in ore recovered from deep rock fracture water in several gold mines in South Africa 0.9 km (0.56 mi), 1.3 km (0.81 mi), and 3.6 km (2.2 mi) under the surface of the Earth. Onstott said that "it scared the life out of me when I first saw them moving", and explained that "they look like black little swirly things". The finding is significant because no other multicellular organism had ever been detected farther than 2 km (1.2… Halicephalobus mephisto is a species of nematode, among a number of other roundworms, discovered by geoscientists Gaetan Borgonie and Tullis Onstott in 2011. It was detected in ore recovered from deep rock fracture water in several gold mines in South Africa 0.9 km (0.56 mi), 1.3 km (0.81 mi), and 3.6 km (2.2 mi) under the surface of the Earth. Onstott said that "it scared the life out of me when I first saw them moving", and explained that "they look like black little swirly things". The finding is significant because no other multicellular organism had ever been detected farther than 2 km (1.2 mi) below the Earth's surface. It is named after Mephistopheles, the Lord of the Underworld in the Faust story, and alludes to the fact it is found so deep under the Earth's surface. Research has shown that Halicephalobus mephisto possesses special heat-shock and stress-response genes that allow it to survive over 3 kilometers below Earth's surface, in high temperatures and low oxygen conditions. Halicephalobus mephisto is resistant to a temperature as high as 37 °C (higher than most terrestrial nematodes can tolerate), it reproduces asexually, and feeds on subterranean bacteria. According to radiocarbon dating, these worms live in groundwater that is 3,000–12,000 years old. It is the deepest-living animal ever found, able to withstand heat and crushing pressure, and the first multicellular organism found at deep subsurface levels. A previously known species found at similar depths in the same study was Plectus aquatilis. Borgonie said that the worm was similar to the detritus-feeding species found on the surface, and probably descended from surface species. Such species are also able to survive extremes of temperature, and so, for Borgonie, the fact the first animal discovered at this depth was a worm was unsurprising. The team hypothesised that the species was descended from animals on the surface that were washed down the Earth's crust by rainwater. Halicephalobus mephisto worms measure from 0.5 to 0.56 mm (0.020 to 0.022 in) in length. Though species in the genus Halicephalobus have few distinguishing features, H. mephisto can be differentiated from other species within its genus by its comparatively long tail, which is between 110 and 130 micrometres in length. It is somewhat closely related to the mammalian pathogen Halicephalobus gingivalis, but is more closely related to certain unnamed species of the genus. In 2019, genome sequencing of the nematode indicated that there were expansions of the 70 kilodalton heat shock protein (Hsp70) and avrRpt2 -induced gene 1 (AIG1) proteins, both of which are transcriptionally induced under heat stress. De novo Illumina assembly with PacBio reads produced a 61.4 Mb assembly made of 880 scaffold and 313 kb N50. The water Halicephalobus mephisto was found in contained aerobic and anaerobic bacteria. Unlike other nematodes, it does not prefer Escherichia coli, and would rather feed on the sulphophile endolith and depth specialist Desulforudis audaxviator. The found specimen of H. mephisto propagated through parthenogenesis. == References ==