Deep subsurface microbial life in impact‐altered Late Paleozoic granitoid rocks from the Chicxulub impact crater

In 2016, IODP‐ICDP Expedition 364 recovered an 829‐meter‐long core within the peak ring of the Chicxulub impact crater (Yucatán, Mexico), allowing us to investigate the post‐impact recovery of the heat‐sterilized deep continental microbial biosphere at the impact site. We recently reported increased cell biomass in the impact suevite, which was deposited within the first few hours of the Cenozoic, and that the overall microbial communities differed significantly between the suevite and the other main core lithologies (i.e., the granitic basement and the overlying Early Eocene marine sediments; Cockell et al., 2021). However, only seven rock intervals were previously analyzed from the geologically heterogenic and impact‐deformed 587‐m‐long granitic core section below the suevite interval. Here, we used 16S rRNA gene profiling to study the microbial community composition in 45 intervals including (a) 31 impact‐shocked granites, (b) 7 non‐granitic rocks (i.e., consisting of suevite and impact melt rocks intercalated into the granites during crater formation and strongly serpentinized pre‐impact sub‐volcanic, ultramafic basanite/dolerite), and (c) 7 cross‐cut mineral veins of anhydride and silica. Most recovered microbial taxa resemble those found in hydrothermal systems. Spearman correlation analysis confirmed that the borehole temperature, which gradually increased from 47 to 69°C with core depth, significantly shaped a subset of the vertically stratified modern microbial community composition in the granitic basement rocks. However, bacterial communities differed significantly between the impoverished shattered granites and nutrient‐enriched non‐granite rocks, even though both lithologies were at similar depths and temperatures. Furthermore, Spearman analysis revealed a strong correlation between the microbial communities and bioavailable chemical compounds and suggests the presence of chemolithoautotrophs, which most likely still play an active role in metal and sulfur cycling. These results indicate that post‐impact microbial niche separation has also occurred in the granitic basement lithologies, as previously shown for the newly formed lithologies. Moreover, our data suggest that the impact‐induced geochemical boundaries continue to shape the modern‐day deep biosphere in the granitic basement underlying the Chicxulub crater.

There is a growing understanding of the effects of impacts on life at the Earth's surface, including aquatic and terrestrial microbial community changes caused by drastic environmental changes resulting from meteorite impact (Bralower, Cosmidis, Fantle, et al., 2020;Schaefer et al., 2020).However, despite the global participation of deep subsurface microbial communities in biogeochemical cycling (Amend & Teske, 2005;Colwell & Smith, 2004;Magnabosco et al., 2018), little is known about to what extent geological deformations resulting from large impacts have played or continue to play a role in shaping the community composition and functioning of the modern deep biosphere.
In 2016, the International Ocean Drilling Program (IODP) and the International Continental Scientific Drilling Program (ICDP) joint expedition 364 recovered an 829.03-m-long core (spanning 505.7-1334.73mbsf) at the impact crater site (M0077A; Yucatán Peninsula, Mexico).This core comprises a ~ 130-m-thick suevite interval (747.14-617.28mbsf) that separated the ~111-m-thick post-impact Cenozoic interval from the underlying felsic granites (1334.73-766mbsf) (Gulick et al., 2019).One of the overarching aims of this joint IODP-ICDP expedition was to reconstruct the post-impact recovery of the deep biosphere at the actual site of impact.
The energy released from the impact created a hydrothermal system with temperatures exceeding 300°C (Morgan et al., 2016), comprising mainly meteoric water with seawater interference (Abramov & Kring, 2007;Kring et al., 2020).Moreover, the impact caused target rock resurfacing by mixing near-surface rocks with deeper rocks at the impacted site (Kring et al., 2017;Morgan et al., 2016).Numerical modeling of the Chicxulub impact crater suggested that granitic rocks were uplifted 8-10 km from their origin (Morgan et al., 2016).Before impact, the in situ temperature of the uplifted granites was estimated to range between ~160 and 200°C, based on a geothermal gradient of ~20°C per km (Xu et al., 2011;Yuan et al., 2006).Since microbial life has not been reported at temperatures >121°C (Kashefi & Lovley, 2003), the recovered basement rocks were already sterile before impact.However, the Chicxulub impact event also created conditions that would have benefited the recolonization and evolution of deep subsurface microbial life.
For example, the impact generated a 60 GigaPascal (Gpa) shock wave (Morgan et al., 2016), which resulted in increased porosity and permeability of the shattered granitic basement rocks, creating opportunities for a potential post-impact recolonization and development of deep subsurface microbial life (Cockell et al., 2002(Cockell et al., , 2021)).
Hydrothermal fluid flow, in addition to increased porosity and permeability, has facilitated the vertical transport of nutrients, minerals, and redox changes (Bralower, Cosmidis, Heaney, et al., 2020;Kring et al., 2020;Simpson et al., 2020), a requirement for microbial life to recover and sustain in otherwise impoverished granitic rocks (Cockell et al., 2002(Cockell et al., , 2021)).The earliest recovery would have been possible after the crater cooled down to temperatures low enough to allow the survival of microbial life, roughly ~2 Ma after impact (Abramov & Kring, 2007).Thus, the deep biosphere at the impact site must have had less than 64 Ma to recover and evolve from ground zero.Calcite veins have been reported from the deepest part of the sampled granitic basement rocks (Kring et al., 2004(Kring et al., , 2017(Kring et al., , 2020)).These veins are estimated to have precipitated at 70°C (Demeny et al., 2010) and could have transported microbial life and microbial growth factors (e.g., redox nutrients) to the shattered granitic rocks.
The presence of δ 34 S-depleted pyrite framboids (−5 to −35‰) and δ 34 S values between pyrite and sulfate of 25 and 54‰ was considered evidence of microbial fractionation of sulfur in impact breccias overlying the granitic basement rocks (Kring et al., 2021).
We furthermore reported increased cell biomass at impact-induced geological interfaces within the impact-shocked granitic basement and the overlying suevite (Cockell et al., 2021), which was deposited within the first few hours of the Cenozoic (Gulick et al., 2019).
Moreover, 16S rRNA gene profiling revealed the presence of diverse microbial communities, which differed significantly in their compositions among granitic basement rocks, the suevite, and the postimpact Cenozoic marine sediments.This demonstrated that the impact produced new lithological horizons that offered long-term improvement in deep subsurface colonization potential (Cockell et al., 2021).
However, only seven intervals were previously analyzed from the impact-deformed 587-m-long granitic core section Cockell et al., 2021).Here, we performed a 16S rRNA gene survey at higher sampling resolution using stringent controls for contamination to study microbial communities associated with impact-shocked granites (n = 31 samples), non-granitic rocks (n = 7), and cross-cut mineral veins (n = 7).Non-granitic rocks comprised suevite and impact

| Sampling
Samples for this research were retrieved aseptically from available core catcher material immediately after coring, as discussed in detail elsewhere (Cockell et al., 2021).Briefly, the core segments were flame sterilized with ethanol (70%) and sprayed with RNase-Away (Thermo Fisher Scientific, Malaga, WA, Australia) to remove possible surface contamination with extracellular DNA.Then, the surface-sterilized granites (n = 31 samples) and intercalated non-granite intervals (i.e., suevite, impact melt, dacite, and dolerite rocks; n = 7) were wrapped in heat-sterilized (8 hours at 500°C) aluminum foil and crushed with a hammer.To further minimize cross-contamination, only larger pieces of rock (>2 cm in each dimension) were subsampled using flamesterilized tweezers from the center of the crushed core halves.The subsamples (Figures S1, S2) were wrapped in sterile aluminum foil, double-bagged in sterile Whirl-Pak™ bags, and immediately stored at −80°C until further preparation for DNA extraction.Seven of the 38 sampled rock intervals contained anhydrite and silica veins, considered a third sample category.These cross-cut mineral veins were subsampled using a Dremel 9906 with flame-sterilized tungsten-carbide carving bits.However, the cross-cut mineral veins were only 1-3 mm thick, which complicated the ability to obtain subsamples without completely avoiding cross-contamination with the surrounding rock material.In addition, drilling mud samples (~10-30 mL) were obtained in parallel, which served as controls for contamination during coring.
Attempts to collect pore water from the rocks for geochemical analysis were unsuccessful.None of the samples used in this study were previously analyzed by Cockell et al. (2021).
Geochemical analysis from additional core catcher samples located more than 5 cm away from XRF-analyzed core sections was performed commercially using the XRF facility at Intertek Genalysis.For this study, XRF analysis could only be performed on the 31 granitic and seven non-granitic rock samples since all material from the additional seven cross-cut mineral vein samples was needed for DNA extraction.

| DNA extraction
Between 4 and 10 grams of surface-sterilized frozen rock samples were ground using a heat-sterilized pestle and mortars (baked The drilling site M0077A location in the Chicxulub crater, Yucatán Peninsula, Mexico (modified after Gulick et al., 2013).The map in the top left corner shows the crater's location within the Yucatán Peninsula, Mexico, and the drilling site within the crater's peak ring is marked with a yellow star.
for 8 h at 500°C while wrapped in aluminum foil) within a HEPAfiltered horizontal laminar flow bench inside the clean lab facility at Western Australia-Organic and Isotope Geochemistry Centre (WA-OIGC), Curtin University.Standard precautions were used to minimize the contamination of samples and reagents with foreign DNA (Capo et al., 2021;Cockell et al., 2021).DNA was extracted from the pulverized material using the FastDNA™ 50 mL spin kit for soil (MP Biomedicals).The extracted environmental DNA samples were further concentrated to ~250 μL using Microsep Advance centrifugal devices (PALL Life Sciences), purified using the OneStep® PCR Inhibitor Removal Kit (Zymo Research), and concentrated to ~25 μL using Amicon ultra 0.5 mL centrifugal filters (Millipore-Merck).Extracted DNA was quantified spectro-fluorometrically using the Quant-iT™ PicoGreen (Thermo Fisher Scientific) DNA reagent and a NanoDrop™ 3300 Spectrofluorometer (Thermo Fisher Scientific).An extraction without sample material present was performed during each extraction series to monitor the contamination of the reagents and consumables used for DNA extraction.These extraction blanks were also processed for sequencing.
In addition, DNA was extracted from 10 mL of drilling mud (n = 7) as described above and served as a control for contamination during the coring activities.

| Library preparation and sequencing
The universal forward primer 519fM (5'-CAG CMG CCG CGG TAA-3') (Cockell et al., 2021) was used in combination with the universal reverse primer 803R (5'-GGA CTA CHV GGG TWT CTA AT-3') to amplify the V4 region of 16S rRNA genes (Caporaso et al., 2012).Both forward and reverse primers contained the Illumina flow cell adapters and pad regions.In addition, the reverse primer had a unique 12-base Golay barcode sequence to support the pooling of samples (Caporaso et al., 2012).A PCR mixture of 20 μL was prepared by adding DNA from rock samples and controls, 1x TB Green Premix Ex Taq (Tli RNase H Plus) (Takara Bio), and 0.2 μM final concentration of for-  et al., 2016): Breccia with impact melt fragments (lighter blue and green fragments) and underlying impact melt rock (pale green with pink fragments); pre-impact dikes (yellow); shocked granites (pink); impact melt rock dykes (darker green with pink fragments); and impact breccia dykes (darker blue/grey).Different shades of blue and green were used to indicate a possible different origin between the breccia underlying the felsic basement and the dykes within the felsic basement.The temperature anomaly (i.e., negative spike) at 920 mbsf was the result of a loss of mud circulation.
Picogreen dsDNA Assay Kit on a VersaFluor™ Fluorometer (BIO-RAD Laboratories).The barcoded amplicons were pooled in equimolar amounts and concentrated using an Amicon Ultra-0.5 Centrifugal Filter 30 kDa MWCO (MilliporeSigma).The concentrated library was subjected to agarose gel electrophoresis, and the expected fragment was excised and gel purified using the Monarch® Gel Extraction Kit (New England Biolabs).Roughly 400 ng of the pooled library was submitted to the Australian Genomic Research Facility (AGRF) for paired-end Illumina MiSeq sequencing (600 cycles).

| Bioinformatics and biostatistics
Quantitative Insights into Microbial Ecology 2 (QIIME2, version 2020.11) was used to process the paired-end sequence data (Bolyen et al., 2019).Paired-end reads were demultiplexed using q2-demux, and primer sequences and Illumina adapters were removed using q2-cutadapt (Martin, 2011).The Divisive Amplicon Denoising Algorithm (DADA2) was used for denoising and chimera removal (Callahan et al., 2016).The QIIME2 feature-classifier classify-sklearn (Pedregosa et al., 2011) was used for the taxonomic annotation of the amplicon sequence variants (ASVs) that passed the stringent quality control steps against the SILVA 138 database (Silva-138-99-515-806-nb-classifier.qza)(Quast et al., 2012).Contaminant ASVs that appeared in the samples and the controls were stringently removed and analyzed separately.The R package Venn diagram (Chen & Boutros, 2011) was used to visualize the number of shared microbial taxa at the ASV level among granites, mineral veins, and nongranite samples.Partial least square-discriminant analysis (PLS-DA) (Lê Cao et al., 2011) was performed in the R package MixOmics (Rohart et al., 2017) as a classification and ordination approach to examine the relationship between ASVs and the three sample categories (granites, non-granites, and mineral veins).Before PLS-DA analysis, zero entries in the ASV x Sample Abundance matrix were offset by 1 to allow a centered log-ratio transformation (CLR).Global and pairwise analysis of similarity (ANOSIM) was used to verify if microbial communities identified at the ASV level differed significantly between the three sample categories.This analysis was performed in PRIMER-e v7 (Clarke & Gorley, 2015) using Bray-Curtis dissimilarities of square root-transformed relative abundance data.The R package PerformanceAnalytics (Peterson et al., 2018) was used to demonstrate the Spearman co-correlation between XRF-inferred chemical compounds.CCA analysis (Legendre & Legendre, 2012) revealed the association between relative changes in the microbial community composition and the physicochemical parameters.The significance of these associations was confirmed using analysis of variance (ANOVA) (Fisher, 1992).Both analyses were performed in the R-package Vegan (Dixon, 2003).The R package Pheatmap (Kolde, 2019) was used to generate a heatmap of Spearman correlations between relative changes in the microbial community composition and the physicochemical parameters, which were analyzed in parallel.For the Spearman correlation analysis, the species abundance matrix was based on combining the ASVs assigned at the identical lowest reliable taxonomic ranks.Only taxa representing more than 5% of the community in one sample or more were included.

| Recovery of genomic DNA
Between 250 and 1116 pg DNA was recovered per gram of granitic rock sampled between 774 and 1050 mbsf.The DNA content in the deeper granites (1050-1333 mbsf) ranged between 215 and 2136 picogram (g rock) −1 with a maximum at 1090 mbsf (Figure 2a).The DNA content within the granitic rock intervals showed a significant positive correlation (t-test, n = 31) with available borehole temperature data (49.3-66.5°C;r = .406,p = .021),but not with porosity (0.10%-0.33%; r = .138,p = .45)and TOC content (0.1%-0.8%; r = .261,p = .149).The DNA content in the mineral veins was generally higher than in the paired granite samples, with the highest difference observed at 1005.86 mbsf (i.e., 1669 vs. 1100 pg/g DNA in the mineral vein and paired granite sample, respectively).In contrast, the DNA content was comparable between the parallel non-granite rock and vein sample at 1306.86 mbsf (Figure 2a).

| Chemical composition
XRF analysis revealed the abundance (%) of the main bioavailable elements (S, Fe, P, Mn, and Si), as well as major-(Mg, Na, K) and transition (Ca, Ba, Cr, and Ti) elements in the 31 analyzed granite and 7 non-granite rock intervals (Table S2).All elements were measured as oxides, except for sulfur, which was quantified as weight % due to the low sulfur oxide content in the granite rock intervals.As mentioned above, XRF elemental analysis could not be performed on the thin cross-cut anhydride and silica veins since all available sample material was required for DNA extraction.Substantial differences in the downcore distribution of the identified chemical compounds were only observed for Fe 2 O 3 , MgO, CaO, MnO, P 2 O 5 , and elemental S (Table.S2) and the relative abundance of these compounds was highest in the non-granite rock intervals (Figure 3a).We observed significant moderate-to-strong cross-correlations (r > 0.48; p ≤ .01) between these compounds, except for S, which only showed moderately strong and marginally significant cross-correlations (r = ~0.5;p < .05)with Fe and Mg (Figure 3b).

| General statistics of the recovered ASVs and quality controls
After the removal of singletons, 2080 ASVs were recovered from all samples and controls combined.We stringently removed 825 ASVs that were considered contaminants, which were analyzed separately and mineral veins (n = 7), respectively (Figure S5).Grouping the ASVs assigned at the lowest possible taxonomic level yielded 340 unique taxa, of which 270 comprised more than 1% of the total community in at least one of the analyzed rock samples.These more abundant taxa could be assigned to 24 phyla, 47 classes, 107 orders, 161 families, 300 genera, and 18 species.The total number of taxa identified at the lowest reliable taxonomic levels varied most strongly in granites (14-66 taxa) and mineral veins (19-67 taxa).The highest number of taxa (67) was observed in a white mineral vein associated with granite from 1005.86 mbsf.Between 16 and 38, taxa were recovered and identified from the non-granites (Figure S6).

| Microbial community composition in granites, intercalated non-granites, and cross-cut mineral veins
Proteobacteria, Bacteroidota, Actinobacteriota, Firmicutes, and Deinococcota were relatively abundant in all three sample categories and, combined, they comprised more than 91% of the microbial community composition (Figures 4a, S6).Acidobacteriota, F I G U R E 3 Averaged relative abundance of biologically relevant minerals that differed significantly between granites and non-granites versus Spearman correlation coefficients between mineral pairs.(a) Bar graphs show the relative abundance of Fe, Mg, Ca, Mn, and P (as oxides) and S with standard deviations.Only minerals that differed significantly between both sample categories are shown.See Table S1 for a complete overview of the mineral compositions.Silica (Si) and Aluminum (Al) combined constituted ~ 70% of the elements identified (Table S1) but were randomly distributed and not further discussed in this study.Oceanospirillales, Xanthomonadales, and Legionellales comprised more than 1% of Gammaproteobacteria in all three sample categories (Figure 4c).Among those, Burkholderiales were the relatively most abundant Gammaproteobacteria (~28%-56%), followed by Pseudomonadales (3%-12%) and Oceanospirillales (~1%-9%) (Figure 4c).The following orders were detected in all three sample categories but only relatively abundant in one or two sample categories: Enterobacteriales (granites and non-granites), Nitrococcales (nongranites and minerals), and Pasteurellales (non-granites).In contrast, Nitrosococcales were relatively abundant Gammaproteobacteria in granites and not detected in the other sample categories (Figure 4c).

Sphingomonadales, Rhizobiales, Rhodobacterales, and
Caulobacterales were relatively abundant Alphaproteobacteria in at least one of the three averaged sample categories (Figure 4c).
Rhizobiales contributed 6%-11% of Alphaproteobacteria in the three sample categories.Rhodobacterales comprised 2.5% of Alphaproteobacteria in the minerals veins and less than 1% in the rock sample categories.Acetobacterales represented 1.6% and 0.8% of Alphaproteobacteria in the granites and minerals, respectively, and were not detected in the non-granites (Figure 4c).

| Similarities and dissimilarities in the microbial community composition between sample categories
PLS-DA analysis (Lê Cao et al., 2011) yielded separate clusters for granites (n = 31 samples), non-granites (n = 7), and mineral veins (n = 7) based on sample-specific dissimilarities in ASV compositions (Figure 5).However, only a small percentage of variance was explained by both X-variates (Figure 5), and according to pairwise analysis of similarities (ANOSIM), bacterial communities only differed significantly (p = .016)between granites and non-granites (Table S3).

| Correlation between microbial taxa and environmental parameters
CCA revealed that Fe, Mg, and Mn contributed significantly (p < 0.003) to the differences in the overall community composition (identified at the lowest reliable taxonomic levels) in the granite and non-granite rock samples (Table S4).The Spearman rank correlation coefficient was used to examine the strength (r-values) of associations between relative changes in the microbial community composition and downcore changes in physical (borehole temperature and porosity) and chemical (TOC, S, and oxides of Fe, Mg, Ca, and P) parameters (Figure 6; Table S5).The Spearman correlation analysis Taxa associated with Cluster 3 correlated positively with most of the measured physicochemical parameters but only significantly with S, Fe, Mn, and Mg content.Cluster 4 includes taxa that showed a significant negative correlation with borehole temperature and a weak positive correlation with some chemical parameters (Figure 6; Table S5).
Significant positive Spearman correlations with borehole temperature were observed for Meiothermus, Achromobacter, env.
OPS_17 (Sphingobacteriales), and Sediminibacterium (Figure 6, Table S5).The latter two taxa also showed a significant positive correlation with porosity.A non-significant but positive Spearman correlation with borehole temperature was observed for Bacillus, Phreatobacter, Leptospira, and Empedobacter.In contrast, Azoarcus, Halomonas, Yersiniaceae, Aliidiomarina, and Neisseriaceae showed significant negative Spearman correlations with borehole temperature.A significant positive Spearman correlation with %S was observed for Effusibacillus, Empedobacter, and Hydrogenophilus (Figure 6; Table S5).Beggiatoaceae, Curtobacterium, unclassified Enterobacteriaceae, Cupravidus, Pseudomonas, and Methylonatrum represented notable taxa that furthermore correlated positively, albeit not significantly, with %S (Figure 6).Most taxa in Cluster (Cluster 4) correlated significantly with %Fe.A non-significant but positive correlation with %Fe was observed for Pasteurellaceae, Gemmata, and Prevotella in Cluster 4. At the same time, none of these taxa co-correlated positively with %Fe (Figure 6).Moreover, Gemmata in Cluster 4 was the only taxon that significantly correlated with %Ca and showed 99.2% sequence similarity with an environmental clone from freshwater lithifying microbiolites (Table S5).

F I G U R E 6
Heatmap showing Spearman correlation strength (r-values) between relative changes in the microbial community composition and concomitant changes in physical (borehole temperature and porosity) and geochemical properties (TOC, S, and oxides of Fe, Mg, Ca, and P).Spearman's r-values (between −0.5 and + 0.5) are shown in the color key for comparison.Significance levels (p): 0 "***" 0.001 "**" 0.01 "*" 0.05.Correlations with p < .05are considered significant and shown in the heatmap.The horizontal dendrogram shows separate clusters for chemical properties versus temperature, porosity, and TOC content.See the main text for a detailed explanation of the four horizontal clusters.Only taxa that comprised more than 5% of the community composition in at least one interval were included in the analysis.Detailed information about the Pearson r and p values can be found in Table S6.

| Granites and non-granitic rocks harbor different microbial communities
This study examined the composition of thermophilic deep subsurface microbial communities and their predicted roles in elemental cycling in the upper 587 m granitic basement below the Chicxulub impact crater.Hydrothermally altered shocked granites, nutritionally rich non-granite rocks (e.g., intercalated intervals of pre-impact subvolcanic, suevite, and impact melt rocks), and cross-cut mineral veins of anhydrite and silica were analyzed from this core section.All three sample categories were dominated by Proteobacteria (notably Gammaproteobacteria), followed by Bacteroidota, Firmicutes, and Actinobacteriota, which is consistent with the predominant microbial communities previously recorded from the main lithologies of the Chicxulub Impact crater (Cockell et al., 2021) and those associated with deep subsurface igneous rocks such as basalt, granite, and inactive hydrothermal vents (e.g., Dutta et al., 2018;Hou et al., 2020;Jørgensen & Zhao, 2016;Mandal et al., 2022;Zhang et al., 2016).
Despite a predominance of available granite samples and an unequal distribution of ASVs in favor of the granites, ANOSIM analysis showed that the overall microbial community composition differed significantly between the granite and non-granite rock intervals.CCA analyses revealed significant correlations between relative changes in the total microbial community composition and Fe, Mn, and Mg content.This suggests that microbial niche separation has occurred in the impact-deformed granite and intercalated non-granite rock intervals and that differences in the relative abundances of electron donors and acceptors between the two rock categories continue to shape the modern microbiome in the craters' granitic basement.

| The role of the deep biosphere in metal and sulfur cycling
Spearman analysis revealed significant correlations between relative changes in various bacterial community members and bio-accessible elements (notably Fe and elemental S) (Figure 6, Table S5).Redoxactive elements such as Fe, Mn, and S support microbial life in the deep subsurface by providing electron acceptors for heterotrophic respiration or electron donors for lithotrophic and autotrophic metabolisms.For example, our data showed that Enterobacteriaceae (Gammaproteobacteria; Enterobacteriales), Microscillaceae (Bacteroidia; Cytophagales), and Effusibacillus (Firmicutes; Bacilli; Alicyclobacillales; Alicyclobacilliaceae), correlated significantly with %Fe (Figure 6; Table S5).The presence of Enterobacteriaceae in environmental 16S rRNA sequence data is usually considered contamination of the sampling location with sewage material.However, slow-growing thermophilic chemolithoautotrophic Enterobacteriaceae were recently cultivated from deep continental granitic crust underneath the Deccan Traps (Koyna, India) using H 2 as electron donor and nitrate, Mn(IV), Fe(III), or sulfate as suitable electron acceptors (Mandal et al., 2022).The Enterobacteriaceae in our core could not be classified beyond the family level but showed 98.4% sequence similarity with the chemolithoautotrophic isolates from the deep Deccan trap granites (Table S6).This suggests that thermophilic Enterobacteriaceae play a role in iron cycling in carbondepleted deep subsurface rocks.Microscillaceae have not yet been reported from comparable high-temperature deep subsurface environments.However, members of this family have been isolated from the marine environment and were shown to possess the genomic potential to grow on heme-bound iron as a sole Fe source (Hopkinson et al., 2008).The unclassified Effusibacillus that showed equally strong, positive Spearman correlation strength with both %Fe and %S (Table S5) shared >99% sequence similarity to a thermophilic enrichment culture and an uncultured clone from basaltic rocks (Table S6).
All known members of this family share the ability to oxidize S, which is stored as intracellular S globules.This feature contributes to their conspicuous filamentous or chain-like morphology and the high refractory nature of their cells (Flood et al., 2021;Salman et al., 2011).
Members of this family are predominantly benthic and have been identified from a vast range of habitats, including hydrothermal sediments of the Guaymas Basin (Buckley et al., 2019) and geothermally active submarine caves (Mattison et al., 1998).However, to the best of our knowledge, Beggiatoaceae have not yet been reported from thermogenic deep subsurface granitic rocks.The ASVs in our core could not be identified beyond the family level (Table S5) and most likely represent novel taxa that have been able to colonize these deeper rocks via vertical fluid flow.
Hydrogenotrophic sulfur-transforming microorganisms could potentially be a source of H 2 S for the SOB community members in the granitic basement.For example, ASVs assigned to Hydrogenophilus, which also correlated significantly with %S (Figure 6), showed 100% sequence homology to H. icelandicus strain 16C (Table S6).This strain was isolated from a hot spring capable of growing at temperatures up to 60°C and appeared to be a hydrogenotroph using thiosulfate or elemental sulfur for chemolithoautotrophic growth under microaerophilic conditions (Kawai et al., 2022).
The presence of δ 34 S depleted pyrite framboids (−5 to −35‰) and δ 34 S values between pyrite and sulfate of 25 and 54 ‰ was previously reported to be indicative of a biological-instead of inorganic-fractionation of S in the granitic basement rocks (Kring et al., 2021).Hence, microbially produced and isotopically depleted sulfide, which does not participate in pyrite formation, may have been or is still available for the prevailing sulfur-oxidizing bacterial community members described above.

| The role of physical parameters in shaping the granitic microbiome
Spearman correlation analysis showed that temperature played a substantial role in shaping the granite-associated microbial communities.
This finding agrees with a recent study that reported temperature as the leading environmental contributor in determining the presence of the granitic microbiome (Cockell et al., 2021), and the latter study suggested that thermophilic members in the granitic basement originate from impact-generated hydrothermal systems that prevailed for over 2 Ma (Abramov & Kring, 2007;Kring et al., 2020).In this study, 16S rRNA gene profiling of the granitic basement rocks at higher sampling resolution revealed that the thermophilic communities were vertically stratified along the temperature gradient, which naturally increased from 49 to 67°C with depth (~774-1334 mbsf) (Figures 2 and S7).For example, Meiothermus (Deinococci; Thermales), which showed the most significant and strongest positive Spearman correlation with temperature (Figure 6, Table S5), was recovered between 1018 and 1333 mbsf in the granitic basement and was closely related to isolates from hot springs in central France with optimum growth temperatures of ~55 to 60°C (Albuquerque et al., 2009), comparable to the measured borehole temperature at the shallowest section of the core where Meiothermus started to increase in relative abundance.In addition, env.OPS_17 group (Sphingobacteriales) with ~99% sequence homology to environmental sequences from hot springs in Yellowstone Park, USA, revealed a significant positive Spearman correlation with temperature (Figure 6, Table S5), but started to increase in relative abundance at a shallower depth of 838 mbsf and a lower in situ temperature of ~49°C (Figures 2 and S7).
In contrast, some taxa in the Spearman correlation heatmap negatively correlated with temperature.For example, Azoarcus (Burkholderiales) showed 100% sequence homology to a strain of Azoarcus taiwanensis (Table S5), which was isolated from a hot spring in Yang-Ming Mountain (Taiwan) and incapable of growing at temperatures above 55°C (Lee et al., 2014).In the granitoid basement, ASVs assigned to Azoarcus were recovered from rock intervals down to 1000 mbsf, where comparable maximum borehole temperatures were measured (i.e., 47-57°C) (Figure S7).Most taxa in Figure 6 also showed a moderate-to-weak positive correlation with porosity, which is 10 times higher in the shattered granitic basement rocks than typically observed in non-impacted granites (Christeson et al., 2018).The resulting increase in permeability could have facilitated vertical fluid flow, bringing microbial growth factors such as nutrients and energy sources to the granitic basement, which created opportunities for post-impact recolonization (Cockell et al., 2002(Cockell et al., , 2021; and references therein).

| Chicxulub's deep biosphere and significance for planetary science
The recovery of microbes in the Chicxulub granitic basement has significant implications for the habitability of the subsurface of tiple oxidation states with the potential to yield energy for microbial metabolism (Grotzinger et al., 2014;Vaniman et al., 2014).The probability of habitable conditions from the Phoenix landing site in the northern polar region of Mars has also been suggested (Stoker et al., 2010).However, petrologic interpretations of thermal emission spectra from Mars indicated that the Martian surface has basaltic or partly altered basalt compositions that are comparatively different from the Chicxulub lithology (Wyatt et al., 2004).
Nevertheless, our results show how impacts alter the geology and geochemical characteristics of the deep subsurface, thus influencing the metabolisms that can be supported.Given the large number of preserved craters on Mars, the subsurface habitability of that planet can only be understood with reference to the observed effects of impacts on microbial communities as described herein.
Thus, impact craters on Mars represent high-priority targets to explore the planet's habitability, past and present.

| CON CLUS IONS
The microbial data of the granitic basement recovered within the Chicxulub impact crater site have provided insights into how an asteroid impact introduced features, such as hydrothermal systems, porosity, and nutrient availability, have shaped the modernday deep biosphere at the uplifted granite rocks (~747.14-1334.73mbsf) which were at sterilized conditions even prior to the asteroid impact.We observed that most recovered microbial communities resemble those found in hydrothermal systems.Spearman correlation analysis confirmed that the borehole temperature, which gradually increased from 47 to 69°C with core depth, significantly shaped a subset of the vertically stratified modern microbial community composition in the granitic basement rocks.
However, ANOSIM showed that the bacterial communities differed significantly between the impoverished shattered granites and nutrient-enriched non-granite rocks, even though both were at similar depths and temperatures.Furthermore, Spearman analysis revealed a strong correlation between the microbial communities and bioavailable chemical compounds and suggests the presence of chemolithoautotrophs, which most likely still play an active role in iron, manganese, and sulfur cycling.These results indicate that post-impact microbial niche separation has also occurred in the granitic basement lithologies, as previously shown for the newly formed lithologies.
Moreover, our data suggest that the impact-induced geochemical boundaries continue to shape the modern-day deep biosphere in the granitic basement underlying the Chicxulub crater.Our observations that impact-induced geological and geochemical defor- Chicxulub impact crater, continental deep biosphere, impact-altered granitoid basement, niche separation melt rocks intercalated into the granites during crater formation and strongly serpentinized pre-impact sub-volcanic, ultramafic basanite/ dolerite.Partial least square-discriminative analysis (PLS-DA), constraint correspondence analysis (CCA), and Spearman correlation analysis between relative changes in the downcore microbial community composition and the biologically relevant elements sulfur (S), iron (Fe), manganese (Mn), magnesium (Mg), calcium (Ca), phosphorus (P), and organic carbon (C) revealed insights into their biogeochemical cycling potential within this extreme environment.Moreover, our study investigated to what extent borehole temperature and porosity played a role in shaping the modern deep biosphere in the granitic basement below the impact crater.
ward and reverse primers.The newly formed SYBR®Green I-stained double-stranded DNA was monitored using a CFX Connect™ Real-Time System (Bio-Rad Laboratories, NSW, Australia).The following parameters were used for the quantitative PCR runs.Initial melting at 95°C for 60 s, melting at 95°C for 5 s, annealing at 60°C for 30 s, and primer extension plus imaging of newly formed double-stranded DNA at 72°C for 60 s.The reactions were stopped within the exponential phase (31-33 cycles) to prevent overamplification and the formation of artifacts.The amount of DNA in each barcoded amplicon was measured fluorometrically using the Quant-iT™ F I G U R E 2 Downcore variability in (a) DNA content in the analyzed shocked granites, intercalated non-granites, and cross-cut mineral veins in the felsic basement (nanogram x g −1 rock) (this study); (b) porosity (%); (c) TOC content (%); and (d) borehole temperature (°C) (data from Gulick et al., 2017).The lithologies of the analyzed core section are shown to the right of the figure (modified after Morgan ): 501 of the contaminant ASVs were uniquely present in the various controls (mud samples, extraction, and background blanks), whereas the remaining 324 contaminant ASVs were also present in the samples.The remaining 754 ASVs were considered indigenous to the various rock samples, with 425, 92, and 116 ASVs being unique for the granites (n = 31 intervals), non-granites (n = 7), (b)  Matrix showing the correlation strength between mineral pairs that differed significantly between granites and non-granites.Significance levels (p): 0 "***" 0.001 '**' 0.01 "*" 0.05 "." 0.1.p values lower than .05were not considered significantabundant phyla but comprised at least 1% of the community composition in one or two sample categories (Figures4, S6).Proteobacteria was the most abundant phylum, and at the class level, Gammaproteobacteria predominated over Alphaproteobacteria in relative abundance and the number of orders (Figure4b,c).At the order level, Burkholderiales, Pseudomonadales,

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Bar graphs show the average relative abundances of (a) phyla and (b) classes in all samples from the same categories combined.Only phyla and classes are shown that comprised >1% of the entire community.Panel C shows the relative abundance of the most abundant orders within the Gamma-and Alphaproteobacteria.
heatmap of Figure6revealed four separate clusters with the top 53 most relevant taxa: Cluster 1 includes taxa that negatively correlated with most physicochemical parameters.Cluster 2a comprises taxa that positively correlated with borehole temperature and porosity, whereas the taxa belonging to Cluster 2b lacked significant correlations with any of the analyzed physicochemical parameters.

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of the Spearman correlation heatmap also co-correlated positively with %Fe, %Mn, and %Mg.Within this cluster, Effusibacillus and unclassified Enterobacteriaceae correlated significantly with %Fe, while Leptospira and Curtobacterium correlated significantly with %Mn and %Mg, respectively.Furthermore, Microscillaceae F I G U R E 5 PLS-DA shows the spatial distribution of ASVs in the separation of samples based on differences in the ASV composition in the shocked granites (blue circles; n = 31), non-granites (green circles; n = 7), and mineral veins (red circles; n = 7).Despite the low variance explained by X-variates 1 and 2, pairwise ANOSIM analysis, denoted on the right y-axis of the PLS-DA plot, revealed that the overall ASV composition differed significantly (ANOSIM; p < .05) between granites and non-granites.
that the unclassified Effusibacillus in the granitic basement represent chemolithoautotroph(s), gaining energy by oxidizing inorganic compounds such as ferrous iron and elemental sulfur.Beggiatoaceae are another example of putative sulfur-oxidizing bacteria (SOB) other planets.Many large impact craters on Mars have been well preserved since its early history because of the lack of plate tectonics (McEwen et al., 2005; Michalski & Niles, 2010; Robbins & Hynek, 2012; Squyres et al., 2012).The National Aeronautics and Space Administration (NASA) Mars science laboratory investigated the Gale Crater and suggested that the ancient Martian lake had neutral pH and low salinity and contained iron and sulfur in mul- mations have played or continue to play a significant role in shaping the deep subsurface microbial biosphere and the metabolisms that can be supported, such as the biogeochemical cycling of metals and sulfur, have substantial implications for the habitability of the subsurface of other planets.Notably, the many well-preserved craters on Mars represent high-priority targets to explore the planet's past and present habitability, such as Gale Crater.Like the Chicxulub crater, this ancient Martian lake is thought to have had neutral pH and low salinity and contained iron and sulfur in multiple oxidation states with the potential to yield energy for microbial metabolism.