Article Recycling and metabolic flexibility dictate life in the lower oceanic crust

https://doi.org/10.1038/s41586-020-2075-5 Jiangtao Li1,2,9, Paraskevi Mara2,9, Florence Schubotz3,4, Jason B. Sylvan5, Gaëtan Burgaud6, Frieder Klein7, David Beaudoin2, Shu Ying Wee5, Henry J. B. Dick2, Sarah Lott2, Rebecca Cox2, Received: 16 August 2019 Lara A. E. Meyer3,4, Maxence Quémener6, Donna K. Blackman8 & Virginia P. Edgcomb2,9 ✉ Accepted: 10 January 2020

Published online: xx xx xxxx The lithifed lower oceanic crust is one of Earth’s last biological frontiers as it is Check for updates difcult to access. It is challenging for microbiota that live in marine subsurface sediments or igneous basement to obtain sufcient carbon resources and energy to support growth1–3 or to meet basal power requirements4 during periods of resource scarcity. Here we show how limited and unpredictable sources of carbon and energy dictate survival strategies used by low-biomass microbial communities that live 10– 750 m below the seafoor at Atlantis Bank, Indian Ocean, where Earth’s lower crust is exposed at the seafoor. Assays of enzyme activities, lipid biomarkers, marker genes and microscopy indicate heterogeneously distributed and viable biomass with ultralow cell densities (fewer than 2,000 cells per cm3). Expression of genes involved in unexpected heterotrophic processes includes those with a role in the degradation of polyaromatic hydrocarbons, use of polyhydroxyalkanoates as carbon-storage molecules and recycling of amino acids to produce compounds that can participate in redox reactions and energy production. Our study provides insights into how microorganisms in the plutonic crust are able to survive within fractures or porous substrates by coupling sources of energy to organic and inorganic carbon resources that are probably delivered through the circulation of subseafoor fuids or seawater.

Diverse and abundant microorganisms are confirmed to be present openings) within predominantly olivine gabbro, oxide gabbro and in the basaltic upper crust5–7, yet limited information exists about the gabbro (Fig. 2) with around 0.1–5% porosity and 0.2–4 wt% water distinctly different gabbroic lower crust that accounts for two-thirds content. Borehole logging indicates temperatures of 15–18 °C. Total of the volume of the crust8,9. Expedition 360 of the International Ocean organic nitrogen (0.002–0.003 wt%) and total organic carbon (0.004– Discovery Program (IODP) drilled the Atlantis Bank oceanic core com- 0.018 wt%) contents were extremely low (Supplementary Table 1). plex on the southwest Indian Ridge, Indian Ocean, where the lower oceanic crust is exhumed by detachment faulting to around 700 m below sea level, providing convenient access to an otherwise largely Detection of biomass and biomarkers inaccessible realm (Fig. 1 and Supplementary Table 1). Low-density (131–1,660 cells per cm3), heterogeneously distributed Our approach combined cell counts, microscopy and shipboard microbial communities were detected (Supplementary Table 3b), quantification of ATP levels to assess the abundance and distribution of in the same range as recent studies of young, cool and oxic ridge- microbial communities, exoenzyme activity assays, carbon, hydrogen, flank subseafloor basalts in the Atlantic Ocean10 and samples from nitrogen and sulfur (CHNS) and thin-section analyses of host rock sam- 0–15 m below seafloor (mbsf) obtained from the 3.5-million-year-old ples, marker gene (iTAG) analyses of prokaryotic diversity, analyses of Atlantis Massif11. Detection of archaeal intact polar lipids (IPLs) with cultures to assess the viability and activities of selected taxa (including a dominance of archaeol-based lipids over glycerol dialkyl glycerol those that might be rare and may be missed by molecular approaches), tetraethers (GDGTs) indicates the existence of indigenous archaeal and examination of expressed genes and lipid biomarkers. Rigorous communities that are distinct from those that are observed in typical efforts were made at all stages to control for contamination, includ- deep-biosphere sedimentary settings12 (Fig. 3b, Extended Data Fig. 1 ing from drilling fluids (Supplementary Table 2) during shipboard and Supplementary Table 3a). Bacterial diether glycerol (DEG) lipids sampling, and from laboratory and molecular kits (further details are were detected at all depths, which require less cellular maintenance provided in the Methods and Supplementary Information). than fatty-acid-containing lipids (Fig. 3c) and may indicate an adap- Samples along the 809-m depth of hole U1473A showed evidence tation to the stresses of living in the low-energy plutonic rocks of the of carbonate and/or clay-altered felsic veins (variable 0.01–6 mm deep biosphere13.

1State Key Laboratory of Marine Geology, Tongji University, Shanghai, China. 2Department of Geology and Geophysics, Woods Hole Oceanographic Institution, Woods Hole, MA, USA. 3MARUM, University of Bremen, Bremen, Germany. 4Department of Geosciences, University of Bremen, Bremen, Germany. 5Department of Oceanography, Texas A&M University, College Station, TX, USA. 6Université de Brest, EA 3882, Laboratoire Universitaire de Biodiversité et Ecologie Microbienne, ESIAB, Technopôle Brest-Iroise, Plouzané, France. 7Department of Marine Chemistry and Geochemistry, Woods Hole Oceanographic Institution, Woods Hole, MA, USA. 8Scripps Institution of Oceanography, University of California San Diego, La Jolla, CA, USA. 9These authors contributed equally: Jiangtao Li, Paraskevi Mara, Virginia P. Edgcomb. ✉e-mail: [email protected]

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abWater Fault zones of unknown orientation 15º N

Atlantis 10º N Atlantis II Transform Bank JOIDES Resolution 5º N

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33° S, 57° E 5º S Atlantis Bank IODP Expedition 360 Footwall Hole 1473A

10º S ~700 m Hanging wall 15º S

20º S Basalt ~800 m Madagascar 25º S Dikes ~3 km Gabbro 30º S Mantle (lower crust) Site Gabbro 35º S

Partially molten gabbro 40º S 25º E 35º E 45º E 55º E

Fig. 1 | Study site of the exploration of the intrusive oceanic crust by IODP presence of fractures along the depth of hole U1473A. The inset map is adapted Expedition 360. a, Map of study site. b, Schematic of lower oceanic crust from a previous study31; the background map was made using GeoMapApp exhumed at the seafloor at Atlantis Bank. Dashed lines indicate the detected (http://www.geomapapp.org/).

The high IPL-to-core-lipid ratios support a likely in situ source of known to have wide metabolic flexibility, including carbon fixation detected lipids. After cell death, the labile head groups of membrane through Rubisco, and an ability to oxidize alkanes, methane and/or lipids are enzymatically cleaved, leaving behind the core lipids that can sulfur to generate energy15,16—all potential metabolisms in the crustal be preserved in the rock record over millions of years14. Concentrations basement. The detected DEG lipids could also be in part derived from of core lipids were as low as for their IPL counterparts, ranging between these Deltaproteobacteria17. The putative sulfur and hydrogen oxidizers 4 and 3,700 pg g−1 of rock (Supplementary Table 3a). The resulting IPL- SAR406 Marinimicrobia were also detected in all samples18. to-core-lipid ratios are high, on average more than 70% for glycosidic Non-metric multidimensional scaling (Extended Data Fig. 4) indi- (G)-GDGTs/(G-GDGTs + core GDGTs) and 35% for glycosidic archaeols cates that the measured ATP concentrations, fracturing intensity and (G-ARs)/(G-ARs + core ARs), pointing to the minimal accumulation of graphic lithology all had the most-significant effect on observed depth fossil material. In comparison, IPLs typically comprise less than 10% of variance in diversity for the 11 samples (P ≤ 0.05). Locations of fractur- the sum of core lipids and IPLs in sedimentary settings12. ing and alteration are where seawater from above, or low-temperature Raman spectroscopy of a sample from 182 mbsf shows Raman bands fluids from below, can most readily permeate the rock. Unfortunately, that are consistent with amino acids and sugars. Scanning electron it was not possible to sample borehole fluids for important studies microscopy shows filamentous inclusions of organic compounds of in situ fluid chemistry during Expedition 360. Other studies of the associated with iron and manganese oxides, surrounded by, and subseafloor lithosphere show that such fluids can carry the required 19 inter-grown with, calcite (CaCO3), evidence of in situ origin (Fig. 3a). substrates to support microbial life . The viability of the cells is further supported by (1) the detection of methane production in long-term enrichments (average production −4 −2 −1 mRNA reveals diverse strategies from 9.62 × 10 –5.66 × 10 nmol CH4 g of rock per day) (Extended Data Fig. 2); (2) the cultivation of more than 100 unique fungal isolates Recovery of mRNA was lower for the shallowest samples from 11 and from samples along the entire depth of the hole; (3) ATP concentrations 168 mbsf, consistent with observations that the top of the drill hole above detection levels (1 pg cm−3) in 6 out of 11 rock samples (Fig. 2); was primarily dense unaltered gabbro with few fractures to support and (4) detectable alkaline phosphatase exoenzyme activity rates of microbial populations (Supplementary Table 4; further details for all 0.04–2.3 pmol cm−3 h−1 in all 5 samples for which measurements were analyses are provided in the Supplementary Discussion). Detection of attempted (Supplementary Table 3b). transcripts for various metabolic processes indicates that microorgan- Marker gene (iTAG) libraries reveal low diversity (Simpson scores isms have taken the prerequisite steps towards these processes. Addi- (1 − D) of 0.48–0.93) of putative heterotrophic and chemoautotrophic tional measurements of activities, for example, through stable-isotope taxa, many of which have been described from deep-sea and polyex- probing, in addition to those performed in this study are needed to treme habitats (Extended Data Fig. 3). These include Nitrosopumi- demonstrate the occurrence of these processes. Expression of genes laceae, SAR202 Chloroflexi, Nitrospinaceae, SAR406 Marinimicrobia, unique to peptidoglycan synthesis was detected in 5 out of 11 samples, SAR324 Deltaproteobacteria and Thioglobaceae, among other hetero- indicating that cells are engaging in cell division and/or maintenance geneously distributed taxa. Variation in the taxonomic composition of cell integrity (Supplementary Table 4). Transcripts involved in bio- among samples suggests that the availability of carbon and energy synthesis of different amino acids were detected in 10 out of 11 samples substrates is also heterogeneous. The SAR324 (marine group B) line- as well as transcripts implicated in vitamin E and B12 synthesis (for age of Deltaproteobacteria was detected in 8 out of 11 samples and is example, cbiC and cbiJ) in 9 samples (Supplementary Table 4). The cbiJ

2 | Nature | www.nature.com Diabase Diorite/felsic veins Oxide gabbros Oxide-bearing gabbros Disseminated oxide gabbros Oxide gabbronorite Gabbro Olivine gabbro

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800 800 800 800 800 800 800 800 800 800 0 100% 0 24650 0 5 0 5 0 550 102 104 01020 00.5 1.0 U1473A lithology ATP Carb veins Clay veins All veins Alteration Fracturing Average cells Volatile content Clay OH (recovered (pg cm–3) (rank) (rank) (rank, (intensity, (intensity, (per cm3) (wt%) (150–300 °C) 2-m average) 1-m average) 1-m average) 2-m average)

Fig. 2 | Summary of hole U1473A of IODP Expedition 360. Downhole light-grey horizontal zone around 500 mbsf indicates a zone that was drilled properties of the gabbroic section determined by shipboard macroscopic but not cored. ATP was measured once per sample. CCSF, core composite descriptions and measured microbiological indicators. Labels indicate the depth below seafloor. Replicate cell-count data are provided in Supplementary property shown in each panel. Carb, carbonate. A key to lithology is included; Table 3b. Source data are available at http://web.iodp.tamu.edu/DESCReport/ rank and intensity range between 0 and 5. Faults are indicated by pale-green (file name, 360_U1473A_macroscopic.xlsx). lines. Microbiological sample depths are highlighted by dashed blue lines. The sequence was annotated to Chroococcidiopsis sp., a cyanobacterial RNA gene sequences of know methanogens were recovered (Extended taxon that may contribute to anaerobic production of cobalamin or Data Fig. 3). However, the abundance of SAR324 Deltaproteobacteria pseudocobalamin in the lower crust using a hydrogen-based lithoau- in our iTAG libraries indicates potential methanotrophy15,16 coupled totrophic metabolism20. with methane production in our long-term enrichments (Extended Although we observed the production of methane in long-term Data Fig. 2). Some methane metabolism is probably present, but its enrichments (Extended Data Fig. 2), methanogens appear to be rare importance is unclear in these samples. Detection of glycosidic archaeol in our samples, explaining the low recovery of transcripts involved and acyclic GDGT (GDGT-0) may indicate the presence of methanogens; in methane metabolism (Supplementary Table 4), and no ribosomal however, these lipids may also derive from other archaeal sources.

Nature | www.nature.com | 3 Article a

Fe/Mn oxide Calcite Org. laments oup Intensity Fe-O-Fe gr Fe-O etch str 2 tc h etch etch deformation str and CH st re 2 amide III 2 3 2 1,182.43 osine r CH bend PO CH str CH Ty CH C-H Phenylalanine

500 1,000 1,500 2,000 2,500 3,000 3,500 Raman shift (cm–1)

b Archaeal IPLs (%) IPL (pg g–1) c Bacterial DEGs (%) DEG (pg g–1) 0420 0 60 80 100 0102 104 0420 0 60 80 100 0102 104 0 0 10.7 10.7

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228.7 200 228.7 200 274.6 274.6 300 300 382.7 382.7

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747.8 747.8 800 800 Diethers Diethers 1G-AR2G-AR C28–C30 C31–C33 Tetraethers C34–C36 C37–C39 1G-GDGT 2G-GDGT

Fig. 3 | Biological signatures at hole U1473A revealed by Raman archaeal IPLs within the 11 independent samples. c, Diversity (left) and spectroscopy and membrane lipid analyses. a, Raman spectral features of concentration (right) of bacterial DEGs within the 11 independent samples. The organic inclusions in sample 21R2 from 182 mbsf. Inset, back-scattered electron summed carbon chain lengths from C28 to C39 are shown. 1G, monoglycosidic; image of an organic inclusion. Scale bar, 100 μm. Spectral measurements were 2G, diglycosidic; AR, archaeol. Lipid data are from single measurements owing performed at two spots on this particular feature, 10 independent times per to sample constraints. Source Data are available online. spot, with similar results. b, Diversity (left) and concentration (right) of

Transcripts involved in nitrogen metabolism were detected in genes were primarily annotated to Pseudomonas (Supplementary 9/11 samples, including genes associated with denitrification and Table 4). Recycled proteins are another important source of carbon. dissimilatory nitrite reduction to ammonium, the latter of which was Expression of oligo-, dd-endo-, amino- and carboxypeptidases was found in zones where fracturing and vein intensity were greatest (274.6, detected (Supplementary Table 4). Expression of anabolic and cat- 460.4 and 619.6 mbsf). Transcripts involved in sulfur metabolism were abolic genes implicated in the synthesis and degradation of amino detected in most samples (Supplementary Table 4). acids known to be involved in Stickland reactions were detected in Autotrophy might be expected in the gabbroic basement given the 10 of the 11 samples, accounting for up to 24% of normalized reads, likelihood of only ephemeral sources of organic carbon via circulat- indicating that these reactions may have a role in the nutrient-poor ing fluids and the potential for seawater reactions with olivine-rich lower ocean crust (Fig. 4). Stickland reactions involve the oxidative rocks that can produce molecular hydrogen (H2), methane (CH4) and deamination of one amino acid with a higher oxidation state, and the other short-chain hydrocarbons that can be used as reducing agents reductive deamination of another amino acid with a lower oxidation for metabolic energy production19. Indeed, fluid inclusions present state, leading to ATP synthesis by substrate-level phosphorylation23. within gabbro from the Atlantis Bank represent a source of abiotic CH4 Heterotrophic processes such as the degradation of amino acids may be and H2, which can be released upon dissolution or fracturing of the the dominant terminal steps of organic carbon mineralization to CO2 in mineral host21. Although the expression of genes that are potentially these samples; however, the incomplete coverage of expressed genes involved in 5 of the 6 known carbon-fixation pathways was observed in the recovered metatranscriptomes means that we cannot exclude (Supplementary Table 4), transcriptomes suggest that heterotrophy is contributions from additional pathways, including sulfate reduction, also active, and that in situ microorganisms efficiently recycle and store that may be expressed at low levels. the available organic compounds (Fig. 4). For example, the expression Many biochemical pathways produce acetyl-coenzyme A (acetyl- of transglycosylase genes associated with peptidoglycan degradation CoA), propionyl-CoA or acetoacetyl-CoA as intermediates that may annotated to Desulfobacterales and Thermus was detected at 460.4 and contribute to the production of polyhydroxyalkanoates (PHAs)—bacte- 643.9 mbsf. Furthermore, polycyclic aromatic hydrocarbons that can rial and archaeal biopolymers known to serve as compatible solutes in be formed through natural processes (for example, pyrolysis in hot- halophiles24. PHAs also serve as carbon-storage molecules, reducing ter, deeper layers22) may serve as carbon sources in the lower oceanic equivalents and energy sources under carbon, nutrient and oxygen crust. We detected the expression of genes involved in polyaromatic limitation25,26. The primary role of PHAs in our samples is likely to be as and aromatic hydrocarbon degradation in 10 out of 11 samples; these energy storage in an environment in which the abundance of nutrients

4 | Nature | www.nature.com D-Erythrose Quinate PAH 4-phosphate degradation Vitamin E biosynthesis Phenylalanine Shikimate Chorismate Prephenate pathway Benzoate degradation Tyrosine

Phenazines L-Tryptophan

PHA Polyamine β-Oxidation storage Cell communication biosynthesis molecules Catechol

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PHA storage -Ketoglutarate molecules Propionyl-CoA KREBS α cycle Succinyl-CoA

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Hydroxypyruvate Phosphoserine Serine Pyruvate L-Leucine

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UDP-GIcNAc Two-component systems

Fig. 4 | Schematic representation of metabolic processes inferred from Green boxes highlight biosynthetic processes, degradation pathways and two- observed transcripts in core samples from IODP Expedition 360. As the component systems supported by transcripts. Yellow boxes highlight cellular expression of all of the genes in the presented pathways was not detected in activities supported by transcripts. Dashed red arrows represent expected every sample, the schematic is presented as a working hypothesis only. PAH, (but unobserved) connections based on the data. The KREBS cycle (also known polycyclic aromatic hydrocarbons; UDP-MurNAc, uridine diphosphate N- as the tricarboxylic acid cycle) generates energy via the oxidation of acetyl- acetylmuramic acid; UDP-GlcNAc, uridine diphosphate N-acetylglucosamine. CoA. β-Oxidation is the process of degradation of fatty acids to generate Amino acids are highlighted in bold. Blue boxes highlight PHA storage acetyl-CoA. The two processes have been circled. molecules, the production of which is supported by the detected transcripts.

can vary considerably over time. PHAs such as poly-3-hydroxybutyrate Expression of phosphate transporters was detected in samples from esters can be synthesized through an operon of three genes (phaCAB). 274.6, 460.4, 558.5, 619.6 and 747.7 mbsf. Alkaline phosphatase is part of Expression of phaB or phaC was detected in samples from 10.7 and the pho regulon that encodes extracellular enzymes capable of obtain- 619.6 mbsf and both were detected in the sample from 714.9 mbsf; ing inorganic phosphorus. Genes of the pho regulon were expressed in these genes were primarily annotated to Pseudomonas. Propionyl- samples from 619.6 and 643.9 mbsf, consistent with the detected alka- CoA can be used for the biosynthesis of poly(3-hydroxybutyrate-co- line phosphatase activity (Supplementary Table 3b). We hypothesize 3-hydroxyvalerate), a type of PHA that is produced by bacteria and that recycled organic carbon may be used as a source of phosphorus archaea27. Formation of poly(3-hydroxybutyrate-co-3-hydroxyvaler- for cells in this environment (Supplementary Tables 3, 4). ate) requires the action of acetyl-CoA carboxylase and propionyl-CoA Our transcriptome data of life in the lower oceanic crust reveal het- carboxylase. Expression of both genes was detected in the sample erotrophic activities that reflect the competition for limited and spo- from 619.6 mbsf (Supplementary Table 4). β-Oxidation of fatty acids radically available resources, adaptations for withstanding long periods through 3-ketoacyl-CoA thiolase, 3-hydroxyacyl-CoA dehydrogenase of resource scarcity, and efficient recycling of pools of organic matter and acyl-CoA dehydrogenase can produce propionyl-CoA, as can the in this challenging environment (further discussion of the results is catabolism of amino acids using 2-oxoisovalerate dehydrogenase and provided in the Supplementary Discussion). methylmalonyl-CoA mutase. One or more of these genes was detected in all samples, including those obtained from the two deepest samples (Fig. 4 and Supplementary Table 4). Collectively, our data suggest that Conclusion PHAs have an important role in survival in the deep biosphere. Circulation of fluids through fault zones and fractures in the lower

Inorganic phosphorus starvation is hypothesized to occur in the oceanic crust can facilitate delivery of volatiles (for example, H2, 28 basaltic basement and is a possible condition in the lower oceanic CO2 and CH4), nutrients, and abiotic and biotic electron donors and crust. The levels of inorganic phosphorus are efficiently regulated in acceptors. Fluid pathways may represent advantageous habitats for bacteria by a two-component system29 that involves ABC transporters. microorganisms, yet the distribution of life in the deep crust remains

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6 | Nature | www.nature.com Methods Contamination controls Data reporting Rotary coring contaminates the exteriors of core samples due to the No power analyses were done to pre-estimate sample size. The sample circulation of drilling fluids (a mixture of Sepiolite and surface seawater) size was determined by sample availability. The molecular control around the drilling bits. Extreme care was taken to remove or minimize samples and actual samples were identified by different numbers and this contamination and to not introduce new contaminations during were treated blindly until the final stages during which these labels were sample handling and analysis. During IODP Expedition 360, a new less- replaced with the actual depth-associated identifiers. volatile tracer, perfluoromethyldecalin, was successfully tested and calibrated and thus used to quantify the intrusion of drilling fluids into Study site the interior of samples. As a further control for laboratory contamina- The Atlantis Bank was uplifted from beneath sheeted dikes and pillow tion, open Petri dishes containing microbiological medium used to lavas in the local rift valley32 at the palaeo ridge-transform intersection. culture fungi were placed inside the laminar flow hood during sample On the crest of the bank, there is a 25-km2 wave-cut platform that is processing and these plates then were stored at room temperature for the location of an 809-m hole (U1473A) drilled during IODP Expedi- the duration of the cruise. In addition, extraction blanks, procedural tion 360 (32° 42.3402′ S, 57° 16.6910′ E) along a plate-spreading flow controls and samples (of drilling fluids) were analysed for lipid and line at the centre of the gabbro massif. The eroded platform exposes nucleic acid analyses (see the ‘Lipid extraction and UHPLC–MS’, ‘DNA massive foliated gabbro and oxide gabbro mylonites with enclaves extraction and small subunit ribosomal-RNA marker-gene analysis’ and of un-deformed olivine gabbro31. Oolitic sands and deep subaerial ‘RNA extraction and metatranscriptome analysis’ sections). weathering occur on the flanks of the Bank down to 2,500 m depth, indicating the massif once rose to as much as 1.8-km above sea level, Cell counts before subsiding to its present 700 m depth31, where it lies more than Paraformaldehyde (4 ml; 4% solution in 100 mM phosphate-buffered 3 km above the surrounding seafloor of the same age. Flexural uplift at saline) was added to autoclaved 7-ml plastic tubes. Then, 1 ml of pow- the inside corner high can only account for around 1 km of this uplift, dered rock material was added to each tube, bringing the volume to the rest is associated with later normal faulting accompanying isostatic 5 ml total. Two replicate tubes were prepared for cell counts for each uplift that occurred around 12 million years ago33,34. A notable feature sample and—where possible—vein material alone was aliquoted into one of the gabbro massif is that the boundary between crust and mantle sample and whole-rock powder was aliquoted into the other. Preserved rock is exposed along the transform valley wall for some 30 km. This samples were stored at 4 °C for onshore analysis. For cell counts, 1 ml structural geometry suggests that the overlying gabbro massif is in of the fixed sample slurry was used in the quantification procedure35, direct contact with partially serpentinized peridotite at a depth of which was modified to use 40 cycles of sonication instead of 20 to around 4,500 m below site U1473A33. better release cells attached to the powdered rock. Cells were enumer- ated on filters stained with a 1:40 dilution of the stock SYBR Green I in Sampling TE buffer by counting either around 400 fields of view if fewer than a Hole U1473A was drilled with a rotary coring bit, recovering cored rock total of 40 cells were detected, or at least 40–50 cells in fewer fields in 10-m increments within core barrels that were brought on deck of when possible. The limit of quantification was defined as 3× the s.d. the drill ship JOIDES Resolution and laid out on trays before splitting of the mean of the negative-control counts. One negative control was the cored rock samples into approximately 10-20-cm-long whole round processed and analysed for every 11 experimental samples. All samples core sections that were immediately selected and removed from the were counted in duplicate. core liner for microbiological analysis by scientists who wore gloves and masks; each rock sample was placed into a sterile plastic bag for transit ATP and exoenzyme assays to the microbiology laboratory. Prioritized core samples exhibited ATP concentration was assessed for all samples on board the JOIDES evidence of low-temperature alterations by aqueous fluids, including Resolution using luminescence methods36 with the ATP Biolumines- carbonate veins and clay-altered felsic veins. We collected 68 samples cence Assay Kit (Sigma-Aldrich) and a Turner Designs BioSystems 20/20 for microbiological analysis; 11 of these—spanning the depth of hole luminometer (Promega). The presence of ATP is indicative of microbial U1473A—were selected for analyses from among those that showed biomass. A standard curve of 0 (sterile MilliQ water), 1 and 100 ng l−1 the highest concentration of altered felsic and/or carbonate veins. ATP standard was run with each analysed set of samples. ATP in core In the microbiology laboratory on-board the ship, each core sec- samples was measured by placing 500 μl of ATP Assay Mix into a clean, tion was rinsed three times with sterile distilled and deionized water, sterile microcentrifuge tube after which the tube was incubated at changing the bag each time, and then sprayed with 200-proof ethanol. room temperature for 3 min to allow hydrolysis of endogenous ATP, At this point, after around 5 min, the core section was transferred to a thus decreasing the background signal. Then around 0.5 cm3 of pow- custom laminar flow hood equipped with a HEPA filter and air supply dered sample was added (all tubes were weighed) to the same tube and that maintained positive air pressure. Within the hood, samples were the solution was immediately transferred to a clean 1.9-ml screw-top photographed and the exterior approximately 1–2 cm of each whole glass vial (acid washed) with the cap off, using a 1-ml pipette tip with round core was removed using a sterile chisel and rock hammer within a the tip cut off. The vial was placed into the luminometer and results sterilized custom-made 0.3-m × 0.2-m × 0.1-m stainless-steel rock box. were read immediately. From the beginning of the expedition until After removing core exteriors, core interiors were divided for different drilling reached 218 mbsf, samples for ATP assays used leftover pow- analyses. Three times during the cruise (during the first, second and dered rock in the steel rock box after separation of sample material for third week of drilling), 1-litre samples of drilling fluid were filtered other assays. After observing largely undetectable ATP concentration, onto 45-mm 0.2-μm-pore-size Millipore Express Plus polycarbonate a second approach was used because microbial cells—if present—are filters and frozen at −80 °C for DNA and RNA analysis. At the same time, likely to be more concentrated along cracks, within veins and vugs. 50-ml samples of drilling mud (Sepiolite) were also collected and fro- Thus, starting at 218 mbsf, material from these specific features were zen. Materials for DNA, RNA and lipid analyses were stored in sterile included for these analyses. 50-ml Falcon tubes, whereas microscopy and thin-section samples were Alkaline phosphatase activity was measured using fluorogenic placed inside sterilized aluminium foil. These samples were carefully substrate 4-methylumbelliferyl phosphate (Sigma-Aldrich) and its labelled, placed within plastic bags and immediately frozen at −80 °C. reference standard, methylumbelliferone. Fluorescence was meas- All equipment was rinsed and flame-sterilized between samples. ured using black, flat-bottom, 96-well microplates in a Spark 10M Article Multimode Microplate Reader (Tecan). Fluorescence of methylumbel- on a Hitachi TM3000 scanning electron microscope equipped with a liferone is greatest at pH 10, therefore 25 μl of 0.4 M NaOH was added Bruker energy-dispersive spectroscopy system for imaging and semi- to the wells (final concentration 40 mM) to be read. Then, 25 μl of 1 M quantitative element analysis. Promising samples with possible organic EDTA was added to the wells (100 mM final concentration) to prevent inclusions were analysed using a computer-controlled, high-resolution precipitation of carbonate from sampled veins37. Fluorescence was confocal Raman system (Horiba LabRam HR) equipped with three measured with an excitation wavelength of 380 nm and emission of lasers (633 nm, 532 nm and 473 nm), a motorized stage and a SWIFT 454 nm for all substrates and standards. We mixed 1 cm3 powdered fast-mapping option. Confocal Raman spectroscopy enables the non- rock with 5 cm3 of sterile artificial seawater (ASW) in a 8-ml serum vial destructive analysis and recognition of living and fossil (once-living) with 90:5:5 N2:CO2:H2 headspace. Then, 700 μl of each slurry was trans- microorganisms in altered igneous rocks. The achievable lateral and ferred with a sterile syringe into a 1.5-ml Eppendorf tube after set-up spectral resolution of this instrument is better than 1 μm and 2 cm−1, but before sealing the vial; this sample served as T0, which comprised respectively. Spectra were analysed using the BioRad Knowitall soft- triplicate 200 μl technical replicates. The 700-μl samples were briefly ware and spectral databases to identify organic compounds. centrifuged (60 s at 2,500 rpm) and the supernatant was used for the T0 analyses. Two additional samples were taken using the same methods as Lipid extraction and UHPLC–MS lipid biomarker analysis for T0 after at least 2 weeks and then again after 4–6 weeks to generate Crushed core samples stored in Falcon tubes at −80 °C were first milled a slope of activity. Incubations were kept at 10 °C, the inferred in situ for 10 min to a fine powder and subsequently extracted with a modified temperature, for the duration of each assay. Bligh and Dyer method according to a previous study41. Before mill- Autoclaved, powdered rock from each of the samples was tested ing and extraction of each sample, a procedure blank was performed. to determine the amount of fluorophore adsorbance to rock powder. First, a milling blank was performed using combusted sea sand (fired Adsorbance was found to behave in a systematic way, resulting in a at 450 °C for 5 h) to clean the mill and to limit cross-contamination of straight line when comparing fluorescence standards in ASW alone samples. Subsequently, this sea sand was transferred to geo-cleaned with fluorescence standards plus rock powder in ASW, although this (rinsed three times with a mixture of methanol and dichloromethane) relationship was found to be different when measured after 4 h versus Teflon containers used for the extraction of the samples and solvent- days later. Therefore, a correction factor for adsorbance was applied extracted in the same manner as the samples. For this, 100 ng of an inter- to the enzyme data for the initial measurement (T0, y = 1.90x − 676), nal standard (C46 glycerol trialkyl glycerol tetraether) and around 50 ml which was taken less than 2 h after experiment initiation, versus the of a solvent mixture of dichloromethane:methanol:buffer (2:1:0.8, v/v) second and third measurements (T1 and T2, y = 4.64x − 303), which were was added to the sample in the Teflon container and ultrasonicated taken days to weeks later. Negative controls consisting of the same ASW for 10 min using a geo-cleaned ultrasonic stick. After ultrasonication, used for the sample incubations plus substrate, but no sample, were the samples were centrifuged (1,750 rpm at 10 min) and the superna- consistently below the limit of detection. The limit of quantification tant was transferred to a fired separatory funnel. The samples were for the alkaline phosphatase assay, defined as 3× the s.d. of the blank, extracted in four steps, for the first two steps a phosphate buffer −3 −1 was 0.0242 pmol cm of rock h based on the analysis of 8 blanks. (K2HPO4, 50 mM at pH 7.4) was used, in the third step the phosphate buffer was replaced by 5% trichloroacetic acid (50 g l−1 at pH 2) and in Carbon and nitrogen analysis the last step only dichloromethane:methanol (9:1, v/v) was used. Equal Powdered rock material from each sample (produced in the laminar amounts of dichloromethane and deionized MilliQ water were added to flow hood on the JOIDES Resolution using a sterile mortar and pestle) the extract collected in the separatory funnel, the mixture was shaken, was immediately transferred to sterile, muffled glass containers and and the organic phase was collected as the total lipid extract and blown stored in a desiccator until analysis of carbon and nitrogen according to to dryness under a gentle stream of nitrogen. established methods38,39. In brief, samples were weighed into methanol- An aliquot of the total lipid extract was analysed using ultrahigh-pres- rinsed silver boats (4 mm × 6 mm, Costech). Then, 96-well glass plates sure liquid chromatography (UHPLC) coupled to mass spectrometry (combusted for 4 h at 450 °C) holding these samples were placed in a (MS) on a Dionex Ultimate 3000RS UHPLC connected to an ABSciEX vacuum desiccator that also contained an open dish with about 50 ml QTRAP4500 Triple Quadrupole/Ion Trap MS (UHPLC-triple quad-MS) fresh, concentrated (12 N) HCl. An inverted crystallization dish was using a Turbolon electrospray ion (ESI) source. Separation of com- placed over the samples to protect them from water condensation. The pounds was achieved on a Waters Acquity BEH C18 column (1.7 μm, desiccator was closed and pumped out with an air-driven aspirator, to a 2.1 mm × 150 mm) equipped with a guard column of the same material reading of about 0.5 atm and the desiccator was placed in an oven kept following a previously published protocol42. Compounds of interest between 60 and 65 °C. Acidification was allowed to run for 60 to 72 h, as were screened with multiple-reaction monitoring and selected-ion described previously40. When acidification was complete, the samples monitoring techniques as described previously42. Concentrations of were removed and set in the oven to dry (60–65 °C). Subsequently, the lipids were determined relative to the internal C46 glycerol trialkyl glyc- samples were placed in a vacuum desiccator charged with indicating erol tetraether standard and were corrected for individual response fac- silica gel (Fisher S162-500, activated by heating above 220 °C for sev- tors using commercially available standards (diC16-DEG, archaeol) and eral hours) and pumped down again and dried for about 24 h. Samples isolated standards from cultures (GDGT-0, 1G-AR, 2G-AR, 1G-GDGT-0 were then analysed on a Costech 4010 Elemental Analyzer connected and 2G-GDGT-0). Sciex Analyst 1.6.3 and Sciex MultiQuant 3.0.3 (AB via a Finningan-MAT Conflo-II interface to a DeltaPlus isotope ratio Sciex) were used for triple-quadrupole MS data acquisition and data mass spectrometer. processing. The presence of crenarchaeol was confirmed by core GDGT analysis Thin-section preparation, scanning electron microscopy and according to a previously published study43. In brief, an aliquot of the Raman analyses total lipid extract was analysed using a Dionex Ultimate 3000RS UHPLC Thin-section billets were cut from dedicated subsamples of the 11 sam- connected to a Bruker maXis ultrahigh-resolution quadrupole time-of- ples examined in this study. Thin sections were prepared by High Mesa flight MS, equipped with an atmospheric pressure chemical ionization Petrographics. Mosaic images were taken of all thin sections in transmit- (APCI) II source. Compounds were separated using two aquity BEH ted and reflected light and were used to guide the scanning electron HILIC amide columns (1.7 μm, 2.1 mm × 300 mm) in tandem maintained microscopy (SEM) and Raman analyses. Uncoated thin sections were at 50 °C, and n-hexane as eluent A and n-hexane:isopropanol (90:10, screened with SEM in low-vacuum mode to search for and visualize v/v) as eluent B (a detailed protocol has been published previously43). carbon-rich inclusions of possible organic origin. SEM was performed Drilling mud and extraction blank contamination controls were also run for lipid biomarker analyses (Extended Data Figs. 5, 6). Bruker Com- pass 1.9 and Bruker data analysis v.4.4 (Bruker Daltonics) were used for RNA extraction and metatranscriptome analysis quadrupole time-of-flight data acquisition and processing. Frozen rock material was crushed as above and then ground quickly into a fine powder using a precooled sterilized mortar and pestle, after DNA extraction and small subunit ribosomal-RNA marker-gene which RNA extraction started immediately. The jaw crusher was cleaned analysis and rinsed with 70% ethanol and RNaseZap RNase Decontamination Rock material was crushed while still frozen in a Progressive Exploration Solution (Invitrogen) between samples. About 40 g of material was Jaw Crusher (Model 150), the surfaces of which were sterilized with 70% extracted for each sample using the RNeasy PowerSoil Total RNA Iso- ethanol and RNase AWAY (Thermo Fisher Scientific) inside a laminar lation Kit (Qiagen) according to the manufacturer’s protocol with the flow hood. Powdered rock material was returned to the −80 °C freezer following modifications. Each sample was evenly divided into 8 bead until extraction. DNA was extracted from 20, 30 or 40 g of powdered tubes (Qiagen) and then 2.5 ml of Bead solution was added into the bead rock material, depending on the quantity of rock available. A DNeasy tube followed by 0.25 ml of solution SR1 and 0.8 ml of solution SR2. PowerMax Soil Kit (Qiagen) was used according to the manufacturer’s Bead tubes were frozen in liquid nitrogen and then thawed at 65 °C in protocol modified to included three freeze–thaw treatments before a water bath three times. RNA was purified using the MEGAclear Tran- the addition of Soil Kit solution C1. Each treatment consisted of 1 min scription Clean-up Kit (Ambion) and concentrated with an overnight in liquid nitrogen followed by 5 min at 65 °C. DNA extracts were con- isopropanol precipitation at 4 °C. Trace amounts of contaminating DNA centrated by isopropanol precipitation overnight at 4 °C. The low bio- were removed from the RNA extracts using TURBO DNA-free (Invitro- mass in our samples required whole-genome amplification before gen, USA) as described by the manufacturer. To ensure that DNA was PCR amplification of marker genes. Genomic DNA was amplified by removed thoroughly, each RNA extract was treated twice with TURBO multiple displacement amplification using the REPLI-g Single Cell Kit DNase (Invitrogen). A nested PCR reaction (2× 35 cycles) using bacterial (Qiagen) as described. Multiple displacement amplification bias was primers48 was used to confirm the absence of DNA in our RNA solutions. minimized by splitting each whole-genome amplification sample into RNA was converted to cDNA using the Ovation RNA-Seq System V2 triplicate 16 μl reactions after 1 h of amplification and then resuming kit (NuGEN) according to the manufacturer’s protocol to preferentially amplification for the manufacturer-specified 7 h (8 h total). DNA was prime non-rRNA sequences. The cDNA was purified with the MinElute also recovered from samples of drilling mud and drilling fluid (surface Reaction Cleanup Kit (Qiagen) and eluted into 20 μl elution buffer. water collected during the coring process) for negative controls, as well Extracts were quantified using a Qubit Fluorometer (Life Technolo- as two ‘kit control’ samples, in which no sample was added, to account gies) and cDNAs were stored at −80 °C until sequencing using 150-bp for any contaminants originating from either the DNeasy PowerMax paired-end reads on an Illumina NextSeq 550. Soil Kit or the REPLI-g Single Cell Kit. Bacterial small subunit ribosomal To control for potential contaminants introduced during drilling, RNA (rRNA) gene fragments were PCR amplified from multiple displace- sample handling and laboratory kit reagents, we sequenced a number ment amplification samples and sequenced at the Georgia Genomics of control samples as described above. This included two samples and Bioinformatics Core (University of Georgia). The primers used that controlled for potential nucleic acid contamination, a ‘method’ were: Bac515-Y and Bac926R44. Dual-indexed libraries were prepared control to monitor possible contamination from our laboratory with (HT) iTruS (Kappa Biosystems) chemistry and sequencing was extractions, which included around 40 g sterilized glass beads pro- performed on an Illumina MiSeq 2× 300-bp system with all samples cessed through the entire protocol in place of rock, and a ‘kit’ control combined equally on a single flow cell. to account for any signal coming from trace contaminants in kit rea- Raw sequence reads were processed using Trim Galore (http://www. gents, which received no addition. In addition, three field controls were bioinformatics.babraham.ac.uk/projects/trim_galore/), FLASH (http:// extracted: a sample of the drilling mud (Sepiolite), and two drilling ccb.jhu.edu/software/FLASH/) and FASTX Toolkit (http://hannonlab. seawater samples collected during the first and third weeks of drilling. cshl.edu/fastx_toolkit/) for trimming and removal of low-quality and/ cDNA obtained from these controls was sequenced together with the or short reads. Quality filtering included requiring a minimum average rock samples and co-assembled. quality of 25 and rejection of paired reads that were less than 250 nucle- Trimmomatic (v.0.32)49 was used to trim adaptor sequences (lead- otides. Operational taxonomic unit (OTU) clusters were constructed ing = 20, trailing = 20, sliding window = 04:24, minlen = 50). Paired reads at 99% similarity with the script pick_otus.py within the Quantitative were further quality checked and trimmed using FastQC (v.0.11.7) and Insights Into Microbial Ecology (QIIME) v.1.9.1 software45 and uclust. FASTX toolkit (v.0.014). Downstream analyses used paired reads. After Any OTU that matched an OTU in one of our control samples (drilling co-assembling reads with Trinity (v.2.4.0) from all controls (minimum fluids, drilling mud, extraction and whole-genome amplification con- length, 150 bp), Bowtie2 (v.2.3.4.1)50 was used (with the parameter ‘un- trols) was removed (using filter_otus_from_otu_table.py) along with any conc’) to align all sample reads to this co-assembly. Reads that mapped sequences of land plants and human pathogens that may have survived to our control co-assembly allowing 1 mismatch were removed from the control filtering due to clustering at 99% (filter_taxa_from_otu_table. further analysis (23.5–68.5% of sequences remained in sample data- py). As an additional quality-control measure, genera that are com- sets; Supplementary Table 4). Trinity (v.2.4.0) was used for de novo monly identified as PCR contaminants were removed46,47. assembly of the remaining reads in sample datasets (minimum length, Unclassified OTUs were queried using BLAST against the GenBank 150 bp). Bowtie aligner was used to align reads to assembled contigs, nr database and further information about these OTUs is provided in RSEM51 was used to estimate the expression level of these reads, and the Supplementary Discussion. OTUs that could not be assigned to TMM was used to perform cross-sample normalization and to gener- Bacteria or Archaea were removed from further analysis. For down- ate a TMM-normalized expression matrix. Within the Trinotate suite, stream analyses, any OTUs not representing more than 0.01% of the TransDecoder (v.3.0.1) was used to identify coding regions within con- relative abundance of sequences overall were removed as those are tigs and functional and taxonomic annotation was made by BLASTx unlikely to contribute considerably to in situ communities. The OTU and BLASTp against UniProt, Swissprot (release 2018_02) and RefSeq data table was transformed to a presence or absence table and the non-redundant (nr) protein sequence databases (e-value threshold Jaccard method was used to generate a distance matrix using the dist. of 1 × 10−5). BLASTp was used to look for sequence homologies with binary() function in the R package ade4. A hierarchical clustering den- the same e values. HMMER (v.3.1b2) was used to identify conserved drogram was created using hclust() and the stability of the clusters domains by searching against the Pfam (v 31.0) database52. SignalP was evaluated using the clusterboot() function in the fpc package in (v.4.1)53 and TMHMM (2.0c)54 were used to predict signal peptides and R with 500 iterations. transmembrane domains. RNAMMER (v.1.2)55 was used to identify Article rRNA homologies of archaea, bacteria and eukaryotes. Because the serum vials. The control for the methane measurements was a vial Swissprot database does not have extensive representation of protein of 27 ml of ASW with no rocks added. The vials were sealed with sequences from environmental samples, particularly deep-sea and butyl stoppers and gassed with a mixture of 90% N2, 5% H2 and 5% deep-biosphere samples, annotations of contigs used for analyses CO2, and incubated at 10 °C. Methane in the headspace of the vials of selected processes that we report were manually cross checked by was measured at 25 weeks and 60 weeks after incubation. The gas BLASTx against the GenBank nr database. in the headspace of the vials was extracted and measured using gas Aside from removing any reads that mapped well to our control chromatography–flame ionization detection (GC–FID), following co-assembly (1 mismatch), as an extra precaution, any sequence that protocols similar to previously described methods59. Calibrations exhibited ≥95% sequence identity over ≥80% of the sequence length were performed for the measurements using methane tanks at 0 ppm, to suspected contaminants (for example, human pathogens, plants or 1.0 ppm and 6.41 ppm, and the GC–FID loop was flushed for 1 min taxa known to be common molecular kit reagent contaminants, and not before each calibration measurement. The syringe used to inject the described from the marine environment) as described previously46,56 gas samples into the GC–FID loop was flushed three times with the were removed. This conservative approach potentially removed envi- 90% N2, 5% H2 and 5% CO2 gas mixture before each sample extraction ronmentally relevant data that were annotated to suspected contami- and injection. nants due to poor taxonomic representation from environmental taxa in public databases; however, it affords the highest possible confidence Reporting summary about any transcripts discussed. Additional functional annotations Further information on research design is available in the Nature of contigs were obtained by BLAST against the KEGG, COG, SEED and Research Reporting Summary linked to this paper. MetaCyc databases using MetaPathways (v.2.0) to gain insights into particular cellular processes, and to provide overviews of metabolic functions across samples based on comparisons of fragments per Data availability kilobase per million (FPKM)-normalized data. All annotations were iTAG data for the 11 samples as well as 13 different negative controls integrated into a SQLite database for further analysis. (drilling muds and fluids, seawater and kit controls) are deposited in the NCBI BioProject under accession number PRJNA497074. Results Statistics are presented at approximately phylum level; however, taxonomic Ocean-drilling legs with diverse sampling requirements can provide assignments at finer resolution are available from the correspond- only a limited volume of rock material for microbiology from each ing author upon request. Raw reads for transcriptome data have also depth horizon. Although still providing tremendously valuable insights been deposited in BioProject under accession number PRJNA497074; into the deep lithified biosphere, the absence of replicate samples limits iTAGs have been deposited in the NCBI SRA under accession numbers the types of statistical analyses that we can perform. A clustering analy- SRR8136794–SRR8136814 and transcript raw reads can be found in the sis of curated transcripts within the functional categories presented in SRA under accession numbers SRR8141073–SRR8141077. Assemblies this paper was performed using log + 1-transformed FPKM values and for curated portions of the data presented are available upon request the Ward method, and the distance matrix was constructed using the to the corresponding author. All relevant data are available from the Manhattan method and pvclust in R. Non-metric multidimensional corresponding author or are included with the manuscript as Sup- scaling (NMDS) analysis was performed on the Jaccard distance matrix plementary Information, and the Source Data for Fig. 2 are available at of prokaryotic OTU presence or absence data for 11 samples but not on http://web.iodp.tamu.edu/DESCReport/ (file name, 360_U1473A_mac- transcript data because we report here only a carefully curated subset roscopic.xlsx). 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M. & Fuhrman, J. A. Every base matters: assessing small Roughly 5 cm of rock from depths of 247.71, 279.55, 621.09, 724.68 subunit rRNA primers for marine microbiomes with mock communities, time series and and 747.78 mbsf were added to sterile ASW to 27 ml total in 30-ml global field samples. Environ. Microbiol. 18, 1403–1414 (2016). 45. Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing Acknowledgements We thank the captain, crew and all who sailed on JOIDES Resolution for data. Nat. Methods 7, 335–336 (2010). IODP Expedition 360, whose support was essential; T. Sehein and M. Torres-Beltran for 46. Salter, S. J. et al. Reagent and laboratory contamination can critically impact sequence- assistance with statistical analyses; Q. Ma, E. S. Taylor (WHOI Creative) and Andrew Newman based microbiome analyses. BMC Biol. 12, 87 (2014). Design for assistance with figures; S. Yvon-Lewis for providing materials and assistance with 47. Sheik, C. S. et al. Identification and removal of contaminant sequences from ribosomal methane measurements; and M. Sogin, J. Huber, B. Orcutt, K. Lloyd, J. Biddle and S. D’Hondt gene databases: lessons from the Census of Deep Life. Front. Microbiol. 9, 840 (2018). for helpful discussions on the relative merits of different molecular data-handling options for 48. Cole, J. R. et al. The Ribosomal Database Project: improved alignments and new tools for contamination controls. This study was funded by National Science Foundation grants OCE- rRNA analysis. Nucleic Acids Res. 37, D141–D145 (2009). 1658031 to V.P.E. and F.K., OCE-1658118 to J.B.S., and OCE-1450528 and OCE-1637130 to 49. Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina H.J.B.D. F.S. acknowledges funding from the DFG under Germany’s Excellence Strategy (no. sequence data. Bioinformatics 30, 2114–2120 (2014). EXC-2077-390741603) and the Gottfried Wilhelm Leibniz Program (HI 616-14-1). Support to J.L. 50. Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods was provided by the National Science Foundation of China (no. 41772358) and the Ministry of 9, 357–359 (2012). Science and Technology of China (no. 2012CB417302). 51. Li, B. & Dewey, C. N. RSEM: accurate transcript quantification from RNA-seq data with or without a reference genome. BMC Bioinformatics 12, 323 (2011). Author contributions V.P.E., J.B.S., F.K., F.S. and J.L. acquired funding. V.P.E. and J.B.S. 52. Finn, R. D. et al. The Pfam protein families database: towards a more sustainable future. collected samples, performed shipboard assays and established enrichment cultures. J.L. Nucleic Acids Res. 44, D279–D285 (2016). extracted RNA. P.M., J.L. and V.P.E. performed mRNA analyses. D.B., S.L. and R.C. extracted 53. Petersen, T. N., Brunak, S., von Heijne, G. & Nielsen, H. SignalP 4.0: discriminating signal DNA, and D.B. and S.Y.W. analysed iTAG data. F.S. and L.A.E.M. performed lipid biomarker peptides from transmembrane regions. Nat. Methods 8, 785–786 (2011). analyses. G.B. and M.Q. performed fungal isolations. F.K. performed Raman spectroscopy 54. Krogh, A., Larsson, B., von Heijne, G. & Sonnhammer, E. L. L. Predicting transmembrane experiments and J.L. carried out SEM with F.K. J.B.S. and S.Y.W. performed cell counts and protein topology with a hidden Markov model: application to complete genomes. J. Mol. exoenzyme assays, and analysed methane-generation experiments. H.J.B.D. and F.K. provided Biol. 305, 567–580 (2001). the geological context, and D.K.B. compiled downhole-core description data. P.M. conceived 55. Lagesen, K. et al. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. the working hypotheses in Fig. 4. V.P.E. wrote the manuscript draft and all authors contributed Nucleic Acids Res. 35, 3100–3108 (2007). to review and editing. 56. Glassing, A., Dowd, S. E., Galandiuk, S., Davis, B. & Chiodini, R. J. Inherent bacterial DNA contamination of extraction and sequencing reagents may affect interpretation of Competing interests The authors declare no competing interests. microbiota in low bacterial biomass samples. Gut Pathog. 8, 24 (2016). 57. Le Calvez, T., Burgaud, G., Mahé, S., Barbier, G. & Vandenkoornhuyse, P. Fungal diversity in Additional information deep-sea hydrothermal ecosystems. Appl. Environ. Microbiol. 75, 6415–6421 (2009). Supplementary information is available for this paper at https://doi.org/10.1038/s41586-020- 58. Burgaud, G., Arzur, D., Durand, L., Cambon-Bonavita, M.-A. & Barbier, G. Marine culturable 2075-5. yeasts in deep-sea hydrothermal vents: species richness and association with fauna. Correspondence and requests for materials should be addressed to V.P.E. FEMS Microbiol. Ecol. 73, 121–133 (2010). Peer review information Nature thanks Bo Barker Jørgensen, Jennifer Biddle and Steven 59. Valentine, D. L. et al. Propane respiration jump-starts microbial response to a deep oil D’Hondt for their contribution to the peer review of this work. spill. Science 330, 208–211 (2010). Reprints and permissions information is available at http://www.nature.com/reprints. Article

Extended Data Fig. 1 | Archaeal core lipid analyses. a, b, Downhole changes in respectively) and crenarchaeol are shown. c, Structures of the most abundant core lipid composition of archaeal IPLs (a) and archaeal core lipids (b). The archaeal lipids. 1G, monoglycosidic; 2G, diglycosidic; AR, archaeol; GDGT, relative abundances of the diether lipid archaeol and tetraether lipids with glycerol dialkyl glycerol tetraether. Lipid data are from single measurements zero, one, two and three rings (GDGT-0, GDGT-1, GDGT-2 and GDGT-3, owing to sample constraints. Source Data are available online. ASW Only )

ID 228.7 (26R2) (Sample

274.6 (31R1) seafloor below 619.6 (68R4) meters in

714.9 (81R2) Depth Average Rate for60Weeks (nmolCH4/g rocks∙day)

Average Rate for25Weeks (nmolCH4/g rocks∙day) 747.9 (84R6)

0.000 0.0050.010 0.015 0.0200.025 0.030 0.035 0.040 0.045 0.050 0.0550.060

Average Rate of Methane Production(nmol CH4/grocks∙day)

Extended Data Fig. 2 | Rate of methane production observed in long-term measured at 60 weeks and methane measured at 25 weeks divided by the time enrichment experiments. The rate at 25 weeks was derived from the value elapsed since the 25-week measurement. Data are from single measurements measured at 25 weeks minus the initial value (0) divided by the time elapsed in owing to limited sample availability, except for the ASW blank (n = 3), for which days. The rate at 60 weeks was derived from the difference between methane the mean is displayed. Source Data are available online. Article

Extended Data Fig. 3 | 16S rRNA iTAG composition and metabolic processes is given below the iTAG composition for each sample. The presence (black bars) detected in metatranscriptomes. Top, taxonomic composition at family level or absence (white bars) indicates the detection or non-detection of genes or deeper (where possible) of 16S rRNA iTAG sequences for taxa present as associated with the processes and activities listed in that sample. A detailed more than 1% of the total abundance in at least one sample. Taxa present at an discussion of metabolic pathways is provided in the Supplementary abundance of less than 1% in every sample were grouped into the ‘Others’ Information. Annotations at higher taxonomic resolution are available upon category. Bottom, an overview of categories of expressed genes in each sample request from the corresponding author. Extended Data Fig. 4 | See next page for caption. Article

Extended Data Fig. 4 | Non-metric multidimensional scaling and clustering distance matrix of the presence or absence data for 99% OTUs from 11 analyses of detected prokaryotic OTUs and transcripts. a, Non-metric IODP Expedition 360 samples. Jaccard similarity values of >0.8, calculated with multidimensional scaling analysis performed on the Jaccard distance matrix the clusterboot function in R, suggest a stable cluster. Shading highlights (Ward clustering using hclust; see Methods) of prokaryotic OTU presence or several samples that share certain OTUs. From left to right (1–4): 1, samples absence data for 11 biologically independent samples spanning the depth of share SAR11 clade II and SAR406; 2, samples share Nitrosopumilaceae; 3, hole U1473A showing only environmental vectors supported by P < 0.05. samples share SAR11 clade II; and 4, samples both contain the lowest OTU P values were generated as P = N + 1/n + 1 after a goodness-of-fit statistic, which counts. c, Clustering analysis of curated transcripts for 11 samples within the is the squared correlation coefficient, and was calculated on 999 random functional categories presented. Clustering analysis was based on permutations of the data using the vegan package of R. b, Clustering analysis of log + 1-transformed FPKM values and the Ward method, and a distance matrix presence or absence prokaryotic OTU data for 11 samples spanning the depth was constructed using the Manhattan method and the pvclust function in R. of hole U1473A. Hierarchical clustering dendrogram based on a Jaccard Extended Data Fig. 5 | Lipid sample blanks. a, Representative UHPLC–APCI- HPLC–ESI-MS chromatograms for selected bacterial DEG lipids with summed MS chromatogram, showing the detection of core GDGTs in sample 84R6 and chain lengths from C30 to C36 in sample 26R2 and the sample extraction blank. the sample extraction blank. b, Representative multiple-reaction-monitoring Lipid data are from single measurements owing to sample constraints. Article

Extended Data Fig. 6 | Drilling mud contamination control during lipid analysed in this study. e, Extracted ion chromatograms of targeted DEG lipids analysis. a, Depiction of outer, middle and inner sections subsampled from from C28 to C39 summed carbon chain lengths in the drilling mud and whole round core (WRC) sample 29R3 (259.03 mbsf) in order to test the subsampled WRC sample 29R3, showing the differences in relative abundance influence of drilling mud (DM) on the lipid biomarker composition. and isomer composition among these samples. Lipid data are from single b–d, Composition of archaeal core lipids (b), IPLs (c) and bacterial DEGs (d) in measurements owing to sample constraints. 2R1, 10.7 mbsf; 31R1, 274.6 mbsf; the drilling mud, subsampled WRC sample 29R3 and selected rock samples 68R2, 619.6 mbsf; 81R2, 714.9 mbsf. Source Data are available online.      

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