Sampling Methodology:

Sediment coring was performed by the IODP (described in Expedition 327 Scientists, 2011; Integrated Ocean Drilling Program). SSU rRNA genes were obtained as described in Jungbluth et al., 2013; Proceedings of the Integrated Ocean Drilling Program (see Supplementary Information Document(.DOC) for this publication).

CORK borehole fluids were sampled using a custom-built water sampler (described in Cowen et al., 2012). For a detailed description of the pump system see Lin, et al. 2012.

Pore water dissolved organic carbon (from Lin et al., 2015 Materials and Methods ).

Sedimentary pore water DOC concentrations were measured by high-temperature combustion using a Shimadzu TOC-VCSH analyzer. The combustion temperature was set at 720C to ensure complete oxidation of organic matter. Samples were acidified to pH <2 by the addition of 45 uL of 2 M HCl to 3 mL samples. No acid contamination was observed based on monitoring the DOC value of low-carbon deionized water. Samples were purged with nitrogen gas within the autosampler syringe for 2 min in order to remove inorganic carbon. An injection volume of 150 uL was used, with five or six injections per sample. The reproducibility between replicate injections was <1 uM. Analytical reference materials (ARM) supplied by Dr. Dennis Hansell (RSMAS, University of Miami) were measured before, between, and after analysis of environmental samples (Sharp et al., 2002; Dickson et al., 2007). At least one ARM was measured every five samples. The average measured concentration of the ARM was 42 plus or minus 2 uM (n = 44); the reported value was 41–43 uM. Our detection limit for DOC concentrations was ~2 uM.

Sediment organic carbon and nitrogen (relevant text extracted from Lin et al., 2015 Materials and Methods ).

Whole sediment samples were analyzed for concentration of total carbon, organic carbon, and total nitrogen using an elemental combustion system (Costech ECS 4010) connected inline to an isotope-ratio mass spectrometer (Thermo Finnigan Delta XP). The amount of powdered sediment used for the analyses was optimized to provide sufficient carbon and nitrogen for isotopic composition analysis and varied between 26 and 425 mg. A subset of samples was acidified by fuming with concentrated HCl (Hedges and Stern, 1984) in order to remove inorganic carbon and quantify the particulate organic carbon (POC) content. Acid fuming did not remove inorganic nitrogen, resulting in insignificant differences between whole and acid-fumed total particulate nitrogen (PN) concentrations.

Analytical methods for geochemistry

Text below extracted from Supplementary Information (PDF) Junbluth et al., 2016. See reference for full description.

Major ions (Ca2+, Mg2+, K+ , Na+, Cl- , SO4 2- and Br- ) were analyzed by ion chromatography on a Dionex ICS-1100s (Sunnyvale, CA, USA). In addition, magnesium and calcium concentrations were also analyzed by EDTA (colorimetric) and EGTA (electrometric) titration (Grasshoff et al., 1999), or inductively coupled plasma optical emission spectroscopy (ICP-OES) (Lin et al., 2012).

Silicate, nitrate, nitrite, phosphate, dissolved sulfide and dissolved manganese concentrations were measured by colorimetry (Brewer and Spencer, 1971; Phillips et al., 1997; Grasshoff et al., 1999).

Ammonium concentrations were measured by a flow injection-fluorometric method (Jones, 1991). The detection limit was ~2 µM for ammonium in basement fluids and the analytical uncertainty is 0.5 µM.

Ferrous iron was measured directly by a Ferrozine colorimetry method (Stookey, 1970; Gibbs, 1976).

For total iron analysis, samples were first reduced with ascorbic acid and analyzed as ferrous iron. The detection limit for both ferrous iron and total iron was 0.1 µM.

Dissolved organic carbon (DOC) was measured by high-temperature combustion using a TOC-VCSH analyzer (Sharp et al., 2002a; Dickson et al., 2007) (Shimadzu Corp., Kyoto, Japan).

Total dissolved nitrogen (TDN) was measured with a chemiluminescence detector in-line with a Shimadzu TOC-VCSH analyzer (Sharp et al., 2002b).

Alkalinity was determined by acid titration. Acid (0.1N HCl) was standardized with CO2 certified reference materials (CRMs) purchased from the office of Andrew Dickson at Scripps Institution of Oceanography.

An Orion 911600 Semi-micro pH electrode (ThermoFisher Scientific, Waltham, MA, USA) was used to measure the pH and electrode potential during the titration process. The Gran function plot method was used to evaluate titration end-points and calculate sample alkalinity (Dickson et al., 2007). The analytical reproducibility for alkalinity measurements was <0.02 mM.

SSU rRNA gene cloning and sequencing (from Supplementary Information Document(DOC) for Junbluth et al., 2013).

Small subunit ribosomal RNA (SSU rRNA) gene fragments were amplified via the polymerase chain reaction (PCR) using the universal oligonucleotide forward and reverse primers 519F (5’-CAGCMGCCGCGGTAATWC-3’) and 1406R (5’-ACGGGCGGTGTGTRC-3’), respectively. Each 20 ul PCR reaction contained 0.25 U of PicoMaxx high fidelity DNA polymerase (Stratagene, La Jolla, CA), 1x PicoMaxx reaction buffer, 200 uM of each of the four deoxynucleoside triphosphates (dNTPs), 200 nM of both forward and reverse primer, and ~3-4 ng of environmental DNA template. PCR cycling conditions consisted of an initial denaturation step at 95C for 4 minutes, followed by 35 to 38 cycles of 95C denaturation for 30 sec, 55C annealing for 1 min, 72C extension for 2 min, and a final extension step at 72C for 20 min. For the 2008 borehole fluid sample, a 3-cycle reconditioning PCR was performed in order to help eliminate heteroduplexes (Thompson et al., 2002). Amplification products of the anticipated length were excised from an agarose gel and subsequently purified using the QIAquick gel extraction kit (Qiagen, Valencia, CA). Products were cloned using either the pGEM-T Easy kit (Promega, Madison, WI) or the TOPO TA Cloning kit (Invitrogen, Carlsbad, CA) following the manufacturer’s instructions. Clones were sequenced unidirectionally on an ABI 3730XL DNA Analyzer (Applied Biosystems, Carlsbad, CA).

Fluorescence Microscopy: microbial cell counts (from Supplementary Information Document(DOC) for Junbluth et al., 2016).

Sample preparation for microscopy and fluorescence microscopy Fluid samples for microscopy collected in 2011 were prepared in similar fashion to those collected in sampling years 2008-2010 and described previously (Jungbluth et al., 2013). Briefly, 40 to 120 ml sub-samples were fixed with a final concentration of 3% of 0.2 um-filtered formaldehyde for 2 to 4 hours at 4C, and subsequently filtered through 0.2 um pore-sized polycarbonate membranes (Whatman, Maidstone, United Kingdom). After air-drying, membranes were stored desiccated at -80ºC until microscopic analysis. Filter sections were prepared for fluorescence microscopy using a mix of Citifluor/VectaShield/PBS/DAPI as described previously (Jungbluth et al., 2013a). Stained filter sections were inspected with a Leica DM5000B epifluorescence microscope (Leica Microsystems, Wetzlar, Germany) (samples: SSF1-2, SSF4, MIX1-4, SW1-5, SW9-11, SW14-15) or an Eclipse 90i (Nikon Corp., Tokyo, Japan) epifluorescence microscope (all other samples). Both microscopes were equipped with 100x objectives and filter sets appropriate for DAPI fluorescence.

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