cone septum and crimped sealed. Vials were vortexed and then stored upside down at 4°C (Andrén et al., 2015).

* Remaining material from the interior of COMGAS1 was used for shipboard total carbon/inorganic carbon (CARB) analyses of the solid phases (see Solid-phase analysis) and for sedimentological investigations (see Lithostratigraphy).

* Remaining material from the interior of COMGAS2 was subsampled for shore-based characterization of organic matter (385VHTOC) and mineral-bound nitrogen characterization (YAMA).

At other sites, COMGAS1 and COMGAS2 were combined into a single 7 cm long COMGAS whole-round core. From this whole-round core, samples were taken for 385VHCO2, 385VHCH4, 385JACH4 in replicate, CARB, 385VHTOC, and YAMA approximately once per 9.5 m advance.

During hard rock coring, a small (5–15 cm long) whole-round sample of igneous rock was placed into a trilaminated foil barrier bag and immediately sealed. These samples were incubated for at least 24 h at 70°C to stimulate degassing. Bags were then sampled by gas-tight syringe through a polytetrafluoroethylene/silicone septum to quantitatively remove gas. The volume of gas was recorded along with the weight of the rock segment. This gas was then subsampled into crimped septa vials prefilled with saturated NaCl solution for shore-based stable isotope (385VHCH4) and clumped isotope (385JACH4) analyses. Additionally, 10 mL of this gas was immediately subsampled for shipboard hydrocarbon, H₂, and CO analyses.

When the Kuster FTS was deployed to sample borehole fluids, samples of enclosed gases and fluids were taken using an incremental depressurization approach: ~5 mL of gas was released into a stainless steel tube equipped with two Swagelok valves. One valve served to open and close the connection of the tube toward the Kuster FTS and the other valve served to open and close the tube toward a gas-tight syringe connected by means of a luer lock (see Figure F23). In this manner, the gas content of the Kuster FTS was quantitatively sampled and further treated like a void gas sample. In addition, the fluid was transferred into a 20 mL headspace and sealed with a polytetrafluoroethylene/silicone septum and a metal crimp cap. A 5 mL headspace of pure nitrogen was inserted into the vial, and dissolved gases were allowed to exsolve into this headspace for 20 min prior to analysis.

### 9.1.2. Hydrocarbon gases

For safety gas monitoring, the molar ratios of hydrocarbon gases, including methane (C₁ or CH₄), ethene + ethane (C₂), propene + propane (C₃), i-butane (i-C₄), n-butane (n-C₄), i-pentane (i-C₅), n-pentane (n-C₅), i-hexane (i-C₆), and n-hexane (n-C₆), were monitored following standard headspace gas sampling and analysis procedures (Kvenvolden and McDonald, 1986) and IODP pollution prevention and safety protocol (JOIDES Pollution Prevention and Safety Panel, 1992; Shipboard Scientific Party, 1994; Fritz, 1980). When present, unsaturated compounds were at low concentration. To derive molar concentrations of dissolved hydrocarbon gases, we followed the approaches of previous ocean drilling expeditions that had a strong biogeochemical focus (Shipboard Scientific Party, 2003a; Expedition 301 Scientists, 2005; Expedition 307 Scientists, 2006; Expedition 311 Scientists, 2006; Expedition 322 Scientists, 2010; Expedition 337 Scientists, 2013; Morono et al., 2017). In addition to these compounds, four additional hydrocarbons were also monitored and semiquantified: an unknown compound almost co-eluting with iso-pentane; 2,2-dimethylbutane; 2,3 dimethylbutane; and 3-methylpentane. The three latter compounds were previously described in headspace gas samples from Guaymas Basin sediments (Whelan and Hunt, 1982) and were identified based on their elution properties. Additional compounds were also observed in some samples but not monitored (Figure F24). From the volume concentration in parts per million of individual hydrocarbons, the C₁/C₂ and C₁/C₄ values were calculated. The latter ratio was calculated as follows:

$$C_1/C_4 = C_1/(C_2 + C_3 + i-C_4 + n-C_4 + i-C_5 + n-C_5 + i-C_6 + n-C_6). \quad (10)$$

For shipboard C₁–C₆ hydrocarbon gas analysis, 5 cm³ of headspace gas was extracted from the 21.5 mL headspace vials with a gas-tight syringe and manually injected onto an Agilent 7890A gas
<figure><img src="image_27.png" /></figure>

**Figure F23.** The Kuster Flow Through Sampler (Kuster FTS) is used to obtain downhole fluid samples from an open borehole. A, B. Fluid sampling procedure upon recovery of the Kuster FTS during Expedition 385. The sample chamber is connected to a (A) syringe and (B) glass container used for sampling via two valves connected in series to regulate the fluid (and potential volatile) flow. C. The instrument consists of a sample chamber with a spring-loaded valve on each end. A latching mechanism connects the valves and holds them open. Above the chamber, a clock is used to program the closing time and a ball-operated tripping mechanism to release the valves. The lower end has a removable bullnose with ports to allow fluid to enter. When the valves are open on each end, fluids pass freely through the chamber. At an interval programmed on the surface, the valves close, trapping the fluid. The sample contained in the chamber remains in the same state as it was in the well, with no change in pressure. The sample can be removed from the sampler and transferred to a container suitable for storage without changing the pressure of the sample or contaminating it. Image showing the different Kuster FTS tool parts taken from de Ronde et al. (2019).

<figure><img src="image_28.png" /></figure>

**Figure F24.** Example of hydrocarbon gas chromatogram (void gas Sample 385-U1547B-24F-1, 75 cm). Inset shows the expanded region of the chromatogram where additional compounds were observed. C₁ = methane, C₂ = ethane, C₃ = propane, iC₄ = *iso*-butane, nC₄ = linear butane, U = unknown, iC₅ = *iso*-pentane, nC₅ = linear pentane, 2,2D = 2,2-dimethylbutane, 2,3D = 2,3-dimethylbutane, iC₆ = *iso*-hexane, aC₆ = 3-methylpentane (*anteiso*-hexane), nC₆ = linear hexane. Stars = unknown compounds not monitored.
chromatograph (GC) equipped with a split/splitless injector and a flame ionization detector (FID). The chromatogram conditions were as follows:

* Injector temperature: 300°C.

* Split ratio: 30:1.

* GC column: DB-1 precolumn (5 m × 320 µm diameter × 0 µm film thickness) coupled to an Agilent J&W PLOT Alumina/S column (25 m × 320 µm diameter × 80 µm film thickness).

* Oven temperature: hold at 35°C for 5 min, increase from 35° to 200°C at 25°C/min, and a final hold at 200°C for 5 min.

Helium was used as the carrier gas at a flow rate of 3 mL/min. Total run time per injection was 15.6 min. Data were collected and analyzed using the Agilent OpenLab CDS ChemStation software (2001–2016). The FID response was calibrated against gas standards with variable quantities of low molecular weight hydrocarbons provided by Scott Specialty Gases (Air Liquide). For quantification of the additional hydrocarbons monitored, in the absence of reference compounds, the FID response factor of the unknown compound was considered identical to that of closely eluting isopentane. For the branched C₆ compounds, the response factors were considered identical to that of iso-hexane. Concentrations of hydrocarbon gases are reported in parts per million by volume.

Methane concentration in IW was derived from the headspace concentration using a mass balance approach (modified from Expedition 322 Scientists, 2010):

$$CH_4 = [\chi_M \times P_{\text{atm}} \times V_H]/[R \times T \times V_{\text{pw}}], \quad (11)$$

where

$\chi_M$ = molar fraction of methane in the headspace gas (obtained from GC-FID analysis),

$P_{\text{atm}}$ = pressure in the vial headspace (assumed to be the measured atmospheric pressure when the vials were sealed),

$V_H$ = volume of headspace in the sample vial,

R = universal gas constant (8.314 × 10⁻² L·bar/mol·K),

T = temperature of the vial headspace in Kelvin, and

$V_{\text{pw}}$ = volume of pore water in the sediment sample.

The volume of IW in the sediment sample ($V_{\text{pw}}$) was determined based on the mass of the bulk wet sample ($M_b$), the porosity of the sediment ($\phi$; taken from shipboard MAD measurements in adjacent samples), grain density ($\rho_g$), and the density of the pore water ($\rho_{\text{pw}}$):

$$V_{\text{pw}} = M_{\text{pw}}/\rho_{\text{pw}} = \phi \times M_b/[\phi \times \rho_{\text{pw}} + (1 - \phi) \rho_g], \quad (12)$$

where

$M_{\text{pw}}$ = mass of pore water in the sample,

$\rho_{\text{pw}}$ = 1.024 g/cm³, and

$\rho_s$ = 2.8 g/cm³.

The volume of headspace in the sample vial ($V_H$) was determined by the difference between the total volume of the vial ($V_{\text{vial}}$) and the volume of the bulk sediment sample ($V_b$):

$$V_H = V_{\text{vial}} - V_b = V_{\text{vial}} - M_b/\rho_b, \quad (13)$$

where

$V_{\text{vial}}$ = 21.5 cm³ and

$\rho_b$ = bulk density of the sediment (from MAD data).

After measurement of the headspace gases using GC-FID, the mass of the bulk wet sample ($M_b$) was determined to a precision of 0.01 g using two electronic balances and a computer averaging system that compensates for ship motion (Mettler Toledo XS204 dual analytical balance system).

The same mass balance approach was used to derive a first estimate for the molar concentrations of methane in igneous rocks that had been incubated in trilaminated foil barrier bags using the
molar fraction of methane in the gas phase, the total volume of the gas phase inside the bag used for incubation, the weight of the incubated rock, and an assumed average porosity of 14% and density of 2.7 g/cm³.

### 9.1.3. Dissolved hydrogen and carbon monoxide

The analysis of dissolved H₂ and CO follows the approach described by Lin et al. (2012) and has been applied and modified during recent expeditions (Expedition 322 Scientists, 2010; Expedition 329 Scientists, 2011; Expedition 337 Scientists, 2013; Morono et al., 2017). The method relies on the nearly complete extraction of dissolved H₂ and CO into a defined H₂/CO-free gas volume. To this end, samples were taken and prepared as described above. Prior to measurement, the vials were vortex mixed for 3 s to ensure that the dissolved H₂ and CO were concentrated in the headspace.

The molar fraction of H₂ and CO in the headspace gas was quantified using a Peak Performer 1 (PP1) GC (Peak Laboratories LLC, USA), a third-party tool provided by the University of Georgia. The PP1 was fitted with a 100 µL sample loop and calibrated on a daily basis using a 10 ppmv H₂ gas standard and a 10 ppmv CO gas standard balanced in nitrogen. A 5-point calibration curve was obtained by diluting the 10 ppmv H₂ and CO with nitrogen gas in a gas-tight syringe (0, 1, 2.5, 5, and 10 ppmv). To perform a measurement, 1 mL of headspace gas was extracted using a gas-tight syringe with a needle and injected into the PP1. The measured H₂ and CO concentrations of the headspace were then converted to molar IW H₂ and CO concentrations using the following equations:

$$n_{H_2} = X_{H_2}(V_{\text{headspace}}/V_{\text{molar}}) \text{ and} \quad (14)$$

$$n_{\text{CO}} = X_{\text{CO}}(V_{\text{headspace}}/V_{\text{molar}}), \quad (15)$$

where

$n$ = total number of moles (H₂ or CO) in the sample (mol),

$X$ = molar fraction of H₂ (or CO) in the headspace gas (ppm, obtained from GC analysis),

$V_{\text{molar}}$ = molar volume of headspace gas at laboratory temperature (L/mol), and

$V_{\text{headspace}}$ = volume of the headspace (L),

and

$$[H_2]_{\text{IW}} = n_{H_2}(V_{\text{sed}}/\phi) \text{ and} \quad (16)$$

$$[\text{CO}]_{\text{IW}} = n_{\text{CO}}(V_{\text{sed}}/\phi), \quad (17)$$

where

$[H_2]_{\text{IW}}$ = concentration of H₂ dissolved in the IW (M),

$[\text{CO}]_{\text{IW}}$ = concentration of CO dissolved in the IW (M),

$V_{\text{sed}}$ = sediment sample volume (L), and

$\phi$ = sediment porosity.

Each sample was analyzed in duplicate. Procedural blank samples, which were headspace vials solely filled with 18 MΩ water, were prepared for each whole-round core section sampled for gas analysis. The procedural blank associated with this method averaged 23 ± 3 nM for H₂ and 43 ± 15 nM for CO. The PP1 H₂ and CO blank-corrected concentration detection limit obtained using this protocol was 40 nM for H₂ and 150 nM for CO based on 6× and 7× the standard deviation of the blank, respectively. Laboratory air H₂ and CO concentrations were monitored regularly throughout the span of the measuring campaign.

## 9.2. Solid-phase analysis

### 9.2.1. Solid-phase sampling

Samples for shipboard solid-phase analysis were taken as regular subsamples from the COMGAS samples (see Gas sampling), as well as from intervals of sedimentological interest noticed in the
split cores. About 5 cm³ of sediment was transferred into sampling bags using a syringe or a ceramic knife. Samples were freeze-dried for ~12 h, ground and homogenized with an agate pestle and mortar, and then analyzed for TC, TIC, TS, and TN. In addition, solid-phase samples were taken from whole-round cores and preserved for shore-based analyses as follows (Table T13):

* Organic matter diagenesis: under nitrogen atmosphere in a glove bag, the central part (~30 cm³) of a 5 cm whole round was recovered with a spatula and transferred into a 60 mL glass jar (I-chem) with polytetrafluoroethylene-lined polypropylene caps. Samples were stored frozen at -20°C.

* Shore-based incubation experiments: 10–20 cm whole rounds were sampled as described in Microbiology.

## 9.2.2. Inorganic carbon content

Inorganic carbon (IC) concentrations were measured using a Coulometrics 5012 CO₂ coulometer. Approximately 10 mg of freeze-dried and powdered sediment was accurately weighed using two electronic balances and a computer averaging system that compensates for ship motion (Cahn C-31 automatic electrobalance). The sediment was then reacted with 6.5 mL of 2 M HCl in a glass vial. The liberated CO₂ was reacted with a solution containing excess monoethanolamine, forming the carbamate salt of monoethanolamine. This solution was titrated with electrochemically generated OH⁻ to a colorimetric end point, and the change in light transmittance was monitored with a photodetection cell. The weight percent of calcium carbonate was calculated from the IC content, assuming that all the evolved CO₂ was derived from the dissolution of calcium carbonate, using the following equation:

$$CaCO_3 (wt\%) = IC (wt\%) \times 100/12. \qquad (18)$$

No correction was made for the presence of other carbonate minerals. A standard reference material (>99.9% CaCO₃; Fisher Scientific) was used to monitor analytical precision and accuracy (standard deviation less than ±0.4 wt%).

## 9.2.3. Total carbon, nitrogen, and sulfur contents

For TC, TS, and TN contents, samples were freeze-dried and powdered, and ~15 mg (±5 mg) was accurately weighed into tin cups with the Cahn C-31 automatic electrobalance. Samples were mixed with an equivalent mass of V₂O₅ catalyst, and the tin cups were carefully folded closed. Analysis was conducted using a Thermo Finnigan FlashEA 1112 CHNS elemental analyzer, in which the sample was combusted (950°C) in a stream of O₂, nitrogen oxides were reduced to N₂, and the mixture of CO₂, N₂, and SO₂ gases was separated by packed column CHNS/CNS GC (Thermo Electron) and detected using a thermal conductivity detector (TCD). A CHNS calibration standard (2.5-bis[5-tert-butyl-benzoxazol-2-yl]thiophene, BBOT; C = 72.53 wt%, N = 6.517 wt%, and S = 7.44 wt%) and two soil reference standards (NIST 2704, Buffalo River Sediments; Soil CNS Reference Material, Säntis Analytical) run every 10 samples were used to ascertain analytical precision and accuracy (standard deviation was less than ±0.04% for N, ±0.43% for C, and ±0.09% for S).

Because of analytical problems during the final elemental analysis campaign offshore, elemental analysis of the samples from Hole U1548E was duplicated onshore at the LOG laboratory (Lille University, France), using a FlashEA 1112 CHNS elemental analyzer. The analysis was performed on 1.5–2 mg of sample weighed on a MX5 Mettler Toledo microbalance in addition to ~5 mg of V₂O₅. BBOT was used as a calibration standard and was run every 10 samples to ascertain analytical precision and accuracy (standard deviation was ±0.01% for N, ±0.30% for C, and ±0.02% for S). TOC and TN data were comparable to offshore data, so only S is presented here.

TOC contents were calculated by the subtraction of IC from TC contents as determined by elemental analysis. Finally, the TOC/TN atomic ratio was determined as a source indicator of organic matter (Meyers, 1994).

TS and TOC relationships in sediments are often compared to the so-called “normal marine” trend, which has a TOC/TS ratio of 2.8. This trend is observed in fine-grained, reducing, and organic-rich (TOC < 6 wt%) platform sediments deposited under an oxygenated water column
(Goldhaber and Kaplan, 1974; Berner, 1982). In such sediments, organoclastic sulfate reduction is dominant in the sediment and the quasi-absence of bioturbation leads to limited escape of generated H₂S. Reduced sulfur is thus fixed within the sediment, generally as iron sulfide, in proportion to remaining organic carbon (Goldhaber and Kaplan, 1974; Berner, 1982). TOC/TS < 2.8 can be indicative of anoxic depositional conditions (Leventhal, 1983) or of fluid circulation in the sediment (e.g., Passier et al., 1996).

### 9.2.4. Source rock analysis (organic matter characterization)

For selected intervals, the thermal maturity of the organic matter was evaluated by pyrolysis assay using the SRA (Weatherford Laboratories). This assay also yields quantification of the TOC and indicates the type of organic matter. The SRA parameters are the following:

* S1 peak (mg HC/g rock): free hydrocarbons (HC) already present in the samples. They were released during a first heating step at 300°C for 3 min under helium.

* S2 peak (mg HC/g rock): hydrocarbons generated by the pyrolysis of organic matter during the programmed temperature increase from 300° to 610°C at 25°C/min under helium.

* Tmax: nominal temperature at which the hydrocarbon yield is maximum during the pyrolysis step.

* S3 peak (mg CO₂/g rock): CO₂ released during pyrolysis between 340° and 390°C.

* S4 peak (mg C/g rock): CO₂ produced by oxidizing the pyrolysis residue at 580°C under air.

TOC<sub>SRA</sub> was calculated from S1, S2, and S4, assuming that S1 and S2 are 83% carbon, as follows:

$$TOC_{SRA}(\%) = [0.83 \times (S1 + S2) + S4]/10. \qquad (19)$$

The hydrogen index (HI; mg HC/g TOC) and the oxygen index (OI; mg CO₂/g TOC) were calculated as follows:

$$HI = (100 \times S2)/TOC_{SRA} \text{ and} \qquad (20)$$

$$OI = (100 \times S3)/TOC_{SRA}. \qquad (21)$$

Between 60 and 150 mg of freeze-dried, ground sediment was used. All measurements were preceded by a blank and then calibrated to a rock standard from Weatherford Laboratories (STD 533). The same standard was used for quality control every 10 samples.

Organic matter types are based on the chemical composition of the insoluble organic matter (i.e., kerogen) (Tissot and Welte, 1978). Type I kerogen is hydrogen rich and oxygen poor and prone to oil formation upon thermal maturation. It is usually derived from algal organic matter deposited in a lacustrine setting. Type II kerogen is moderately hydrogen rich and oxygen poor and is moderately prone to oil formation upon thermal maturation. It usually stems from algal organic matter deposited in anoxic marine basins. Type III kerogen is hydrogen poor and oxygen rich and prone to gas formation upon thermal maturation. It is mainly derived from terrestrial plant debris, in particular wood. It must be noted that because the chemical composition of the organic matter is susceptible to evolution during burial, the type (I, II, III, or a mixture of them) as determined from the chemical composition is not a strong indicator of the origin of the organic matter (Tissot and Welte, 1978).

## 10. Microbiology

During Expedition 385, microbiology sampling explored the evolutionary and functional diversity of microbes that inhabit the Guaymas Basin subsurface sediments and ultimately the limits of life in hydrothermal sediments where seismic data indicate potential magmatic intrusions of sills at various depths (Teske et al., 2014). Initial sequencing surveys of samples from shallow gravity piston coring to 5 mbsf obtained prior to Expedition 385 showed strong regional differences in bacterial and archaeal community composition and richness that reflect different settings such as organic-rich diatom ooze, strongly reducing methane-rich sediments, and hydrothermally influenced sediments above cooling sills (Teske et al., 2019; Ramírez et al., 2020). However, little is known about how tectonic processes shape subsurface habitats, transform organic matter, or
impact the structure and activities of local microbial communities. Expedition 385 provides an unprecedented opportunity to explore the limits of life in these deeply buried sediments, the past and present geologic processes that occur along divergent plate margin segments at sites in the Gulf of California, and the feedbacks between microbiology, diagenesis, hydrothermal alteration, and local geology.

## 10.1. Sampling

Sediment samples for microbiological studies were obtained using the APC, HLAPC, and XCB coring systems. APC drilling to refusal for microbial and biogeochemical samples was extended by HLAPC coring when sediments became increasingly indurated/heavily compacted (Teske et al., 2018) (see *Drilling and logging operations*); these drilling techniques produce the highest quality (cleanest and least disturbed) sediments (House et al., 2003; Lever et al., 2006). The RCB and XCB coring systems were used when sills and indurated sediments were encountered. For microbiological studies, sampling occurred immediately using syringes or by taking samples as whole-round cores to avoid any alteration or contamination introduced by sample handling before and during the core splitting process. Time-sensitive samples that required rapid processing were taken as soon as possible after a core was recovered on the core receiving platform. Samples for cell counting, 3-D structural imaging, and RNA analyses were collected using sterile 2 cm³ × 3 cm³ and 15 cm³ syringe tubes from near the center of the freshly cut end of core sections (cut using sterilized tools). Sediments for cell counts and 3-D structural analysis were transferred to a 15 mL centrifuge tube or 300 mL wide-mouth jar containing fixing solution and stored in the cold room of the microbiology laboratory (~4°C). Samples for mRNA analyses were transferred to 50 mL Falcon tubes and immediately frozen by dipping them in liquid nitrogen prior to placement in a –80°C freezer. Additionally, a 10 cm whole-round core sample was collected from the middle of each sediment core recovered in boreholes drilled for microbiology sampling for immediate shipboard frozen (–80°C) storage and eventual onshore archiving at –86°C in the IODP Gulf Coast Repository at Texas A&M University (USA). Further microbiology samples were not collected on the core receiving platform but were obtained after fast-track analysis of core sections for physical properties (see *Petrophysics*). From these core sections, whole-round samples listed in Table T14 were collected with ethanol-wiped spatulas, capped with ethanol-wiped end caps, and immediately transferred to the microbiology laboratory. There, the samples were placed in trifoil gas-tight

<table><caption>**Table T14.** Modified DSMZ media 141. All components were dissolved in distilled water except L-cysteine·HCl, Na₂S·9H₂O, vitamin supplement (ATCC MV-DS), acetate, formate, and trimethylamine. When required, pH was adjusted to 7.0. The medium was degassed by sparging with N₂ gas, moved to an anaerobic chamber, and allowed to sit for 30 min. L-cysteine·HCl was added to the media and, once the liquid had become clear, Na₂S·9H₂O was added. The media was aliquoted into sterile Balch tubes (8 mL per tube), which were then tightly capped with blue butyl stoppers and the stoppers crimped in place. The media filled tubes were autoclaved at 121°C for 30 min and then allowed to equilibrate to room temperature. Vitamin supplement (1% v/v) was added to all the tubes using sterile 23G needles. Substrate additions were added to the tubes as either 10 mM acetate, formate, trimethylamine, or no substrate (these tubes will have H₂/CO₂ gas serve as the only substrate). The headspace of all the tubes was then replaced with H₂/CO₂ (80/20) gas mix and pressurized to 2 bar. **Download table in CSV format.**</caption><thead><tr><td>Component</td><td>Concentration</td></tr></thead><tbody><tr><td>MgSO₄·7H₂O</td><td>28.75 mM</td></tr><tr><td>KH₂PO₄</td><td>1.03 mM</td></tr><tr><td>NH₄Cl</td><td>4.67 mM</td></tr><tr><td>NaCl</td><td>410 mM</td></tr><tr><td>MgCl₂·6H₂O</td><td>19.7 mM</td></tr><tr><td>KCl</td><td>4.56 mM</td></tr><tr><td>CaCl₂·2H₂O</td><td>0.95 mM</td></tr><tr><td>Fe(SO₄)₃·7H₂O</td><td>7 µM</td></tr><tr><td>NaHCO₃</td><td>30 mM</td></tr><tr><td>Yeast extract</td><td>0.2% w/v</td></tr><tr><td>Na₂S·9H₂O</td><td>1 mM</td></tr><tr><td>L-cysteine·HCl</td><td>3.2 mM</td></tr><tr><td>Na-resazurin solution (0.1% w/v)</td><td>0.05% v/v</td></tr><tr><td>Trace elements SL-10</td><td>1% v/v</td></tr><tr><td>Vitamin supplement (ATCC MV-DS)</td><td>1% v/v</td></tr><tr><td>Acetate, formate, trimethylamine, or none</td><td>10 mM</td></tr></tbody></table>
laminated bags, flushed with nitrogen, heat sealed, and then stored temporarily in the cold room at ~4°C until further processing, as outlined below. All further sampling procedures of whole-round samples were conducted either inside an anaerobic chamber (Coy Laboratory Products, USA) or on the bench with open clean zone system KOACH (KOKEN, Ltd.) on sterilized foil to maintain as sterile conditions as possible. Gloves were worn, and all utensils were sterilized with 70% ethanol between samples.

In addition to sediment cores, microbiological samples were also obtained from seawater injected into the drill pipe, drilling mud, and core liner fluid (when available). Drilling fluid analyses were primarily performed to monitor both the concentration of chemical tracer and any changes in microbial population size and structure.

**10.2. Contamination testing**

As part of the drilling process, surface seawater was injected into the borehole; injected volumes depended on the coring method. This is the major source of microbiological contamination of the cores collected. To evaluate the extent to which contaminating cells may have penetrated a sample, perfluorocarbon tracer (PFT) was continuously injected into the drilling fluid. Contamination will be further assessed postexpedition by DNA-based comparisons of microbial communities within the sediment samples and the drilling fluid at the time of coring.

Previous shipboard PFT studies have shown that the drilling and coring approach has a major impact on the degree of seawater contamination. Contamination tends to be much greater in RCB and XCB cores than in APC cores (Smith et al., 2000; House et al., 2003; Lever et al., 2006), which is related to the hydraulically actuated piston corer advancement for the latter versus advancing the hole through rotation of the drill string for RCB/XCB coring. Therefore, microbial and biogeochemical samples were recovered preferentially by APC coring to refusal, followed by using the HLAPC system when sediments became increasingly indurated or heavily compacted (Teske et al., 2018) (see **Drilling and logging operations**). The RCB and XCB systems were used when sills and indurated sediments were encountered (see **Drilling and logging operations**). Shipboard PFT studies have also consistently shown that the interiors of cores are much less contaminated (by factors of 3–300) than the core peripheries (Smith et al., 2000; House et al., 2003) and that the uppermost 1.5 m section of APC cores tends to be more contaminated than deeper sections (Lever et al., 2006). However, in all core categories, potential contamination varies considerably from sample to sample. Consequently, to avoid contamination-based distortions of microbiological results, contamination tests were conducted on each sediment core section or each sedimentary and igneous rock sample that is used for a microbiological experiment.

**10.2.1. Perfluorocarbon tracer**

Previous expeditions have used perfluoromethylcyclohexane (PFMC) or, more recently, perfluoro-methyldecalin (PFMD) injected into drilling fluid (seawater) to quantify the percolation of drilling fluid into the interior of samples (Smith et al., 2000; Lever et al., 2006; Inagaki et al., 2015; Dick et al., 2017; Fryer et al., 2018). The less volatile PFMD tracer was used while drilling holes for microbiology until it ran out, and we switched to PFMC starting at Site U1551. As a group, PFTs are nontoxic, inert, insoluble in water, and they can be detected with sensitivity using a GC with an electron capture detector (ECD). PFTs were introduced into the drilling fluids with a high-pressure liquid chromatography pump (rate of injection = 9.77 × 10⁻³ × [stroke of mud pump, 19.54 L/stroke]; 0.55 mL/min at 50 mud pump strokes/min) to provide a final concentration of ~0.5 mg/L. PFTs are dissolved in the drilling fluid and their presence on/in sampled core material is a direct indicator of contamination by drilling fluid. Because microbial cells are larger than these molecules, they may not penetrate a sediment or rock sample in proportion to the drilling fluid, and therefore the tracer should be regarded as providing a qualitative estimate of contamination potential.

**10.2.2. Contamination tracer measurements of sediment samples**

Samples were collected on the core receiving platform from the ends of freshly cut core sections immediately after core recovery to assess the possible degree of contamination within sediment core material using GC measurements of PFT concentration. Contamination was assessed along
the length of a core and across its diameter. Syringe samples ($V = 3 \text{ cm}^3$) were collected at the top of a core, where the highest contamination is expected, and from the central section and bottom of the core. To measure the contamination gradient within a core at each of these three locations, one ~3 $\text{cm}^3$ syringe sample was collected perpendicular to the z-axis of the core at the interface of the sediment core and core liner, a second sample was collected at the center of the sediment core, and a third was collected in between these two. All sediment samples were placed in 20 mL GC headspace vials, closed with screw caps and fluoropolymer seals, and stored at ~4°C for later analysis.

For rock samples collected for microbiology studies, pieces of the rock interior (after removing the outside ~1 cm surface layer using sterilized metal chisels) were placed in 20 mL headspace glass vials with metal caps and fluoropolymer seals; the same was done for the exterior chips of rock that were removed from the samples.

Headspace samples were heated to volatilize any tracer present, and the gas was injected into an Agilent 6890N GC with ECD after preparing and running calibration standards using $10^{-4}$, $10^{-6}$, $10^{-8}$, and $10^{-10}$ dilutions of each tracer. Any tracer detected on the interior of a whole-round sample is interpreted to imply drilling fluid was able to penetrate into the sample, and thus it is likely contaminated. The PFT molecules are smaller than microbial cells, and therefore PFTs indicate the potential but not the certain presence of microbial contamination.

We interpret the measurement of PFMC tracer concentrations with more caution than the measurement of the PFMD tracer because PFMC is extremely volatile (Dick et al., 2017). Once a few samples have entered the laboratory, it can generally be detected in most samples because it remains in the air in the laboratory. The GC was calibrated for each tracer by the shipboard technicians according to their standard protocols.

### 10.2.3. Contamination tracer measurements of rock samples

Only the interiors of sedimentary and igneous rock samples were sampled for microbiology to avoid the contaminated surfaces that are exposed to drilling fluid. The exterior ~1 cm of sedimentary rock cores was removed with a sterile spatula or ceramic knife prior to sampling the interior. The exteriors of whole-round igneous rock samples were washed briefly with sterile distilled and deionized water inside an anaerobic chamber (Coy Laboratory Products, USA) and then dried. At least 1 cm of the contamination-exposed surface of each igneous rock sample was then removed by careful chiseling using a sterilized stainless steel plate placed inside a large sterile sample bag to contain small rock pieces. All chisels and hammers used for this process were repeatedly sterilized with 70% ethanol or RNase AWAY (Thermo Fisher Scientific). The surface rock pieces were collected separately from the interior pieces and saved for analyses in which contamination is not a critical factor, for example, high-temperature enrichments that cold-adapted seawater microbes would not survive. The interior pieces were saved for the most critical analyses where the absence of contaminations is essential.

### 10.2.4. DNA assessments

In addition to monitoring the presence of tracer, contamination can be assessed by analyzing the microbial composition of the drilling fluid (primarily composed of surface ocean water) post-expedition using DNA sequencing approaches. Therefore, a sample of the drilling fluid was collected at the beginning of drilling in every hole established for microbiology sampling. The drilling fluids were collected directly from the injection pipe on the rig floor in sterile bottles with screw caps and handled using sterile equipment. Replicate 250 mL aliquots were filtered onto 45 mm, 0.20 µm pore-size Millipore Durapore filters and frozen at -80°C for shore-based DNA extraction and analysis of marker genes. Aliquots were preserved for cell counts, and 50 mL volumes were frozen at -80°C for lipid biomarker and other potential analyses in home laboratories. Additionally, drilling mud, or sepiolite, which is occasionally used to flush cuttings out of the hole while drilling, was collected in two 50 mL centrifuge tubes prior to injection into the drill pipe and frozen at -80°C for shore-based DNA extraction. Results will be compared with the composition of marker genes in our samples to look for overlap. Signatures of organisms found to be present in both the drilling fluid and rock samples will be an indication of potential contamination and will not be considered subsurface residents. Any samples in which all of the organisms detected during postexpedition analyses are also detected in the drilling fluid will be excluded from further analy-
sis. Although some species may be present both in surface seawater and the subsurface biosphere, the different physical, geochemical, and thermal conditions of the subsurface are more likely to select for different organisms, and any conclusions should be based conservatively on microorganisms known to be present in subsurface samples only.

## 10.3. Microbial cell detection and enumeration

### 10.3.1. Prokaryotic cell counts

Samples for cell detection and enumeration were taken using tip-cut plastic syringe (sediment) or hammer and chisel (rock). A 1 cm³ sediment or crushed rock sample was transferred into a sterile 15 mL centrifuge tube containing 4 mL of 3× phosphate-buffered saline (PBS) (pH = 7.5; Invitrogen 70013) with 2% (V/V) neutralized formalin as a fixative and then thoroughly mixed by vortexing to form a homogeneous suspension. The slurry sample was then subjected to either simple filtration (non-cell-extraction method) or the cell detachment and separation procedure.

The simple filtration procedure was completed as follows:

1. An aliquot of 50 µL, 1/5 diluted formalin-fixed sediment slurry was put into a 1.5 mL tube with 750 µL of 0.6 M NaCl solution. Then, 100 µL of detergent mix (100 mM ethylenediamine tetraacetic acid [EDTA], 100 mM sodium pyrophosphate, and 1% [V/V] Tween 80 [polyethylene glycol sorbitan monooleate solution]) and 100 µL of pure methanol were added.

2. The slurry samples were sonicated at 20 W for 60 s.

3. A total of 20 µL of the slurry was passed through a 0.22 µm polycarbonate membrane filter. Cells on the membrane filter were stained with SYBR Green I staining solution (1/40 [V/V] of SYBR Green I in TE [tris-EDTA] buffer) for epifluorescence microscopy. A total of 10 photomicrographs were taken for counting the number of SYBR Green I-stained cells with ImageJ software as described in Morono et al. (2009).

The cell detachment and separation procedure was completed as follows:

1. An aliquot of as much as 500 µL, 1/5 diluted formalin-fixed sediment slurry was put into a 15 mL tube. Then, 1.9 mL of 0.6 M NaCl solution, 300 µL of detergent mix (as above), and 300 µL of pure methanol were added.

2. The samples were shaken using the vortex at maximum speed for 30 min.

3. The samples were layered onto a density layer composed of 30% Nycodenz (1.15 g/cm³), 50% Nycodenz (1.25 g/cm³), 80% Nycodenz (1.42 g/cm³), and 66% sodium polytungstate (2.00 g/cm³), which was prepared by successively overlaying the higher density solutions with the lower density solutions.

4. Cells and sediment particles were then separated by centrifugation at 4000 × g for 30 min at 25°C with swinging rotors.

5. The light density layer that might contain microbial cells was carefully collected using a 20 gauge needle syringe.

6. 50% of the supernatant was passed through a 0.22 µm polycarbonate membrane filter. Cells on the membrane filter were stained with SYBR Green I staining solution (as above). The number of SYBR Green I-stained cells was enumerated by eye on an epifluorescence microscope as described in Morono et al. (2009).

All cell separation and filtration procedures for cell detection and enumeration were performed under ultraclean air conditions created by a KOACH system (KOKEN Ltd.) and an ionizer (Winstat, Shinshido Electrostatic, Ltd.) to neutralize static electricity charged on plastic wares and floating dust.

## 10.4. CARD-FISH cell counts of fungi

Catalyzed reporter deposition-fluorescence in situ hybridization (CARD-FISH) cell counts of fungi were prepared as follows:

1. 10 cm³ of sediment was placed in a 50 mL Falcon tube and diluted 50:50 with sterile PBS to a sample volume of 20 mL.
2. A total of 4 mL of 20% paraformaldehyde (PFA; final concentration = 4%) was added to each sample. Two replicate tubes were prepared for cell counts from each sample.

3. Samples were fixed for 2–12 h at 4°C.

4. The fixative was exchanged twice with PBS by adding ~25 mL 1× PBS each time and centrifuging for 5 min at 10,000 × g to pellet the sediment/cells.

5. The sediment was subsequently stored in a 50:50 (V/V) PBS/ethanol mixture at –20°C.

**10.5. Molecular biology**

**10.5.1. DNA-based molecular analysis**

For DNA analyses of bacteria and Archaea, we collected one or two whole-round core samples per selected core and stored the samples at –80°C for postexpedition analyses of marker genes. In addition, we collected two 50 mL samples in Falcon tubes for analysis of 18S rRNA genes and intergenic spacer sequences for eukaryotes. Generally, individual analyses by different shipboard scientists will differ by sample and primer choice. Because the portfolio of potential sequence-based analyses far exceeds the capabilities of any individual laboratory, mutual postexpedition prioritization and coordination between different laboratories will be required, and this section is therefore limited to methodological outlines and callouts of the most important alternate primer choices for gene amplification.

For analyses of marker genes of methanogenesis/methane oxidation, extracted DNA will be amplified using polymerase chain reaction (PCR) and customized primer pairs for the methyl coenzyme M reductase/alpha subunit of euryarchaeotal methanogens and ANME-1 Archaea (Lever and Teske, 2015). This multiprimer approach is superior to older primer versions that recover a lower diversity of methanogens and are generally biased against ANME-1 Archaea (Dhillon et al., 2005).

Partial 16S rRNA genes (the v4v5 region) will be amplified using domain-specific primers for bacteria and Archaea, as established by the Josephine Bay Paul Sequencing Center at the Marine Biological Laboratory under the sponsorship of the Deep Carbon Observatory (https://vamps2.mbl.edu/resources/primers).

According to these guidelines, which are widely accepted in the subsurface research community, bacterial 16S rRNA gene amplification is carried out using forward primer 518F (5'-CCAGCAG-CYGCGGTAAN-3') in combination with three versions of reverse primer 926R (Huse et al., 2014):

* 5'-CCGTCAATTCNTTTRAGT-3',

* 5'-CCGTCAATTTCTTTGAGT-3', and

* 5'-CCGTCTATTCCTTTGANT-3'.

The archaeal 16S rRNA gene is targeted using a combination of five variants of forward primer 517F:

* 5'-GCCTAAAGCATCCGTAGC-3',

* 5'-GCCTAAARCGTYCGTAGC-3',

* 5'-GTCTAAAGGGTCYGTAGC-3',

* 5'-GCTTAAAGNGTYCGTAGC-3',

* 5'-GTCTAAARCGYYCGTAGC-3',

and a single reverse primer 958R (5'-CCGGCGTTGANTCCAATT-3') (Topçuoğlu et al., 2016).

Alternate primer combinations in wide use are the Earth Microbiome primer pair, 515F and 806R, which covers the majority of both bacteria and Archaea (Parada et al., 2016; Caporaso et al., 2011). It is important to keep in mind that, regardless of advertisement to the contrary, no single primer combination covers every microbial lineage, and many published primer combinations have “blind spots” that discriminate against certain subsurface lineages (for archaeal subsurface examples, see Teske and Sørensen, 2008). Therefore, it may prove useful to test multiple primer combinations to obtain more than one window into sequence diversity of the deep biosphere. For researchers who are particularly interested in archaeal community structure, the less frequently used archaeal primer combination A751F (5'-CGACGGTGAGRGRYGAA-3') and A1204R (5'-TTMGGGGCA-TRCNKACCT-3') (Baker et al., 2003) provides a “second opinion” that has proven useful in Guay-
mas Basin subsurface analyses (Ramírez et al., 2020). Finally, attempts will be made to isolate sufficient DNA for metagenomic analyses (Biddle et al., 2008), as done previously for surficial Guaymas Basin sediments collected from hydrothermally active spots covered with microbial mats (Dombrowski et al., 2017, 2018). Because of increasingly limited DNA yield downcore, DNA may have to be pooled and purified further after initial extraction from sediments.

**10.6. Eukaryotic RNA analyses**

Samples of core interiors were placed in sterile 50 mL Falcon tubes, labeled, and immediately frozen in liquid nitrogen on the core receiving platform and then stored at –80°C. For RNA analyses, using a 15–45 mL cutoff syringe, we collected two tubes of ~15–30 mL of sample (increasing volumes with core depth below seafloor) from the end of one dedicated whole-round sample immediately after the core had been cut on the core receiving platform. These samples will be analyzed during postexpedition research.

**10.6.1. Fungal lipids**

For analyses of fungal lipid biomarkers that complement the fungal intergenic spacer sequence diversity survey, ~50 mL samples were placed in sterile 50 mL Falcon tubes and frozen at –80°C for postexpedition analysis.

**10.7. Microbial activity measurements**

**10.7.1. Single-cell stable isotope analysis using nano(SIMS)**

After temporary storage (hours) under anaerobic conditions, whole-round samples were transferred to an anaerobic chamber for further processing in preparation for nanoscale secondary ion mass spectrometry (nanoSIMS) analysis to determine substrate and nutrient uptake on a cell-specific basis. The surfaces of whole-round core samples that faced the core liner were trimmed (~1 mm) using ceramic knives to remove drilling fluid contamination in cored sediments. One part sediment was mixed with two parts sterile anaerobic ammonium- and sulfate-free seawater media (Widdel and Bak, 1992). The final concentration of the media (after dilution with ²H₂O) contained 0.1× vitamin, 0.1× trace element, and 0.1× selenite-tungsten solution. A total of 35 mL of slurry was dispensed into 60 mL serum vials and sealed using black bromobutyl stoppers (Glasgerätebau Ochs, Bovenden, Germany). Serum vial headspace was flushed with argon, and the slurry was diluted with 5.8 mL of 70 atom% ²H₂O to a final isotope ratio of 10 atom% ²H (DLM-2259-70-1L). Incubations were additionally amended with Na¹⁵NH₄ (99 atom% ¹⁵N; NLM-467-1), Na₂³⁴SO₄ (90 atom% ³⁴S; Sigma Aldrich, USA), NaH¹³CO₃ (99 atom% ¹³C; CLM-441-1), Na¹³C₂H₃O₂, (99 atom% ¹³C; CLM-440-1), and/or ¹³CH₄ (99 atom% ¹³C; CLM-429-1-BS) to a final concentration of 3.3 mM, 2.0 mM, 6.1 mM, 1.7 mM, and 18.7% (V/V), respectively (all isotope compounds by Cambridge Isotope Laboratories, USA). Control stable isotope incubations using drilling fluid, drilling mud, and anaerobic artificial seawater medium were used to monitor possible contamination during coring operations and sample processing. In addition, parallel killed sediment controls were amended with zinc chloride (m/V). Incubations were overpressured to 20 psi (equivalent to 137.9 kPa) using argon, subsampled (see below), and incubated at their in situ temperatures. Incubations were subsampled on board after 0, 1, 2, and 4 weeks. At all time points, 2 × 1 mL of slurry was added to 2 × 1 mL of 4% (V/V) PFA in PBS (Electron Microscopy Sciences, USA) and incubated overnight at 4°C. Fixed cells were washed with PFA and ethanol, as previously described by Dekas and Orphan (2011), and will be analyzed onshore by nanoSIMS. Furthermore, at 0 and 4 weeks, geochemical subsamples were obtained to track activity over the course of the experiment and to choose the ideal time point for nanoSIMS. Pore water was obtained by centrifuging 2 × 2 mL of slurry for 30 s at 12,000 relative centrifugal force (rcf). Approximately 1 mL of pore water was added to N₂-filled exetainers containing 1 mL of 85% phosphoric acid (V/V). The mixture was stored at room temperature for onshore ¹³C DIC analysis. For the Cline assay, 200 µL of pore water was added to 200 µL of 0.5 M zinc acetate and stored at 4°C (Cline, 1969). Finally, for ion chromatography, 0.5–1.5 mL of pore water was frozen at –20°C.
**10.7.2. Stable and radioisotope probing of carbon substrate utilization experiments**

To elucidate the impact of temperature on carbon flow in the deep biosphere, whole-round core samples 10–20 cm in length were taken from selected horizons for shore-based stable isotope probing (SIP) and radio isotope probing (RIP) experiments to track microbial utilization of various substrates (DIC, acetate, glucose, and complex substrates), their transformation into metabolites and products, and their uptake into microbial membrane lipids (total lipid pool, intact polar lipids versus core lipids, and individual building blocks of lipids) as a function of temperature. Selected whole-round samples were closed with ethanol-wiped end caps, temporarily stored at ~4°C in N₂-flushed bags, and processed in an anaerobic chamber. For each sample, the core liner was removed and the outer 0.5 cm of the whole-round core was trimmed off with a sterile ceramic knife to remove drilling-induced contamination. The remaining sediment was transferred into a precom-busted glass bottle (250 or 500 mL; Schott) using a fresh set of sterile spoons and knives. The bottle was closed with an autoclaved butyl rubber stopper, and the sample was stored in the dark at 4°C.

**10.7.3. Stable isotope probing bioassays of hydrocarbon degradation**

¹³C-labeled phenanthrene and naphthalene were used to measure incorporation rates of these hydrocarbons by heterotrophic microbes, including fungi, using RNA-SIP. Four samples (two near-surface samples and two deeper samples) from the same cores will be assessed post-expedition for their fraction of active fungi using ~90 mL of sediment to examine whether and how fungi may respond to the addition of these available carbon sources in the subsurface in time-course SIP. These two hydrocarbons were chosen based on profiles of hydrocarbon composition in sediment push cores collected in the fall of 2018 using deep-sea submersible *Alvin* and the R/V *Atlantis* (Cruise AT42-05). For each sample, 90 mL of sediment core was collected in an anaerobic chamber and mixed with 810 mL anaerobic artificial seawater or filtered bottom water in a 1 L bottle. A 50 mL aliquot of this slurry was dispensed to sixteen 100 mL Hungate bottles. Labeled and unlabeled naphthalene or phenanthrene were added to bottles to a final concentration at 20x in situ average concentration. One bottle was incubated with no addition. Volumes of 0.76 mL labeled naphthalene (76 µg) or 1.14 mL (114 µg) labeled phenanthrene (both at 0.1 mg/mL stock) were added to four bottles each. The labeled substrates were labeled on all carbon atoms. The procedure was repeated for unlabeled substrates. Bottles were sealed using butyl rubber stoppers and crimp caps and bubbled for 5 min with biogas (a mixture of 70% N₂, 25% CO₂, and 5% H₂ (V/V)). Stock solutions contained 0.1 mg/mL naphthalene in nonane or phenanthrene in ethanol. Bottles were incubated at close to in situ temperature for 24 days. At each time point (*T*₀, *T*6 days, *T*12 days, and *T*24 days), half the volume of each replicate was frozen at -80°C for lipids and half for RNA extraction.

**10.7.4. Extremophile activity**

The subsurface of Guaymas Basin potentially harbors novel evolutionary lineages and metabolic types of microbial life that are distinct from those found in the nonhydrothermal subsurface. To explore this unknown microbial life and the thermal and energetic limits that shape the extent and persistence of microbial life in Guaymas Basin, microbial incubation experiments are planned in high-temperature and high-pressure conditions. For this purpose, whole-round core samples 30 cm in length were stored shipboard in N₂-flushed plastic bags at ~4°C and subsampled using 20 cm³ tip-cut syringes that were flushed with N₂ in oxygen-impermeable, trifoil laminated bags and stored in pressurized (20 MPa) and unpressurized (atmospheric pressure) conditions at ~4°C. Onshore laboratories, those samples will be separately incubated at various temperatures (~80°-130°C) under pressured conditions with stable isotope-labeled substrates.

**10.7.5. Rates of methane production and anaerobic methane oxidation**

We used radioactive tracer (or radiotracer) approaches to measure the potential rates of methane production and anaerobic methane oxidation. Whole-round samples (~30 cm long) were collected and temporarily stored in glove bags filled with nitrogen. In an anaerobic chamber (95:5 [V/V] N₂:H₂), ~5 mL of soft sediment from the innermost part of the core was transferred into cut-end Hungate tubes with cut-end syringes, and a modified butyl rubber stopper was added from the cut end to move the sediment to the threaded end of the tube. The septum was then placed onto the
sediment without headspace and tightened with the screw cap. All vials, stoppers, and septa were autoclaved before use.

At each sampling depth, triplicate live samples and one killed control (killed with 4 mL of 2 M NaOH before tracer addition) were collected for quantification of methane oxidation rates with ¹⁴C-labeled methane and methane production rates using five different ¹⁴C substrates (¹⁴C-labeled bicarbonate, formate, acetate, methanol, and methylamine). In the radiation van (aft of the microbiology laboratory), 100 µL of ¹⁴C-labeled methane (~15 kBq), bicarbonate (~850 kBq), ¹⁴C-formate (~380 kBq), ²⁻¹⁴C-acetate (~330 kBq), ¹⁴C-labeled methanol (~350 kBq), or ¹⁴C-labeled methylamine (~170 kBq) were injected into triplicate and control samples. Prior to radiotracer addition, killed control samples were injected with 4 mL of 2 M NaOH and homogenized (Zhuang et al., 2019). Samples were incubated for 2–3 weeks at different temperatures, and their microbial activities were terminated by injecting and mixing 4 mL of 2 M NaOH into each sample. Fixed samples were stored at room temperature and shipped to the University of Georgia (USA) after the expedition for further processing.

### 10.7.6. Sulfate reduction activity

We used radiotracer approaches to track the rate of sulfate reduction in sediment samples. Whole-round samples collected on the core receiving platform were transferred to trifoil laminated gas-tight bags which were flushed with N₂ gas, heat sealed, and stored at ~4°C until further processing. Samples were prepared inside an anaerobic chamber (95:5 [V/V] N₂:H₂). All vials, stoppers, and ceramic knives were autoclaved prior to use. The outermost ~7 mm of the whole-round core and both ends were cut off with a sterile ceramic knife to remove potentially contaminated material. The uncontaminated inner part of the whole-round core was chopped into small chips with a sterile ceramic knife. Approximately 10 g of chopped sample material was put into a 20 mL crimp vial with 5 mL of sulfate-reducing (5 mM SO₄²⁻, 5 mM HCO₃⁻) medium. The medium used for sulfate reduction rate measurements was based on the medium used during IODP Expedition 370 (Morono et al., 2017; see paragraph “Preparation of WRC samples for potential metabolic activity determination with radiotracers”) and contained the following in grams per liter of deionized water:

* KH₂PO₄, 0.2;

* NH₄Cl, 0.25;

* NaCl, 25.0;

* MgCl₂·6H₂O, 0.5;

* KCl, 0.5;

* CaCl₂·2H₂O, 0.15; and

* Na₂SO₄, 1.42.

The following additions were included:

* NaHCO₃, 84 g/L, 10 mL;

* Resazurin, 0.1 mass (m)/V solution, 300 µL; and

* Na₂S·9H₂O, 12 g/L, 3 mL.

Resazurin was added to the inoculation medium before autoclaving. The media and additions were autoclaved and purged with N₂ gas. During the N₂ gas purge of the media, NaHCO₃ was added through a sterile 0.2 µm (pore size) filter. Na₂S·9H₂O was added through a sterile 0.2 µm filter after autoclaving and gassing. After adding the sampling material and sulfate reducer medium, media vials were crimp sealed with nontoxic blue chlorobutyl stoppers and aluminum crimp caps. Triplicates were prepared for each sample. For approximately every other sample, an extra kill control was prepared by stopping microbial activity of the sample with 5 mL 20% [m/V] zinc acetate immediately after injection of the radiotracer. The headspace of the vials was flushed with N₂ gas to remove surplus hydrogen. To ensure a hydrogen concentration of ~400 nM, 40 µL of N₂:H₂ (95:5 [V/V]) was injected into each vial. In addition, 100 µL of radiolabeled ³⁵SNa₂SO₄ (5 MBq) was added to the medium/sample mix.

Samples were incubated at temperatures within their approximate in situ range at 20°, 30°, 40°, 50°, or 70°C. Because of time constraints, an incubation time of 10 days was chosen, forming a compromise between the maximum length of incubation and the duration of the expedition.
After 10 days of incubation, 5 mL of 20% (m/V) zinc acetate solution was injected into each vial and shaken to trap the produced H₂S gas. The slurry was quantitatively transferred to 50 mL Falcon tubes and the 20 mL headspace vial were rinsed two times with 5 mL 20% (m/V) zinc acetate solution and then shaken to distribute the zinc acetate and frozen immediately at -20°C for 2 days to terminate any microbial activity. The Falcon tubes were stored at 4°C afterward. Before cold chromium distillation, the inactive sediment (sediment with zinc acetate) in the 50 mL Falcon tubes was centrifuged at ~4000 × g for 10 min. Per sample, 50 µL of supernatant was pipetted into a small scintillation vial with the addition of 4 mL UltimaGold solution (Perkin Elmer) for analysis of the total injected activity. Additionally, per sample, 1 mL of supernatant was stored in a 1.5 mL cryovial in case replicate counts were needed. The rest of the supernatant was discarded. Postexpedition sulfate reduction rate measurements will be carried out according to the protocol of Kallmeyer et al. (2004).

## 10.8. BONCAT analyses

We determined the identity of fungi, archaea, and bacteria active under close to in situ conditions using shipboard incubations and a recently developed approach of bioorthogonal noncanonical amino acid tagging (BONCAT) (Hatzenpichler et al., 2014, 2015, 2016; Pasulka et al., 2018; Samo et al., 2014; Leizeaga et al., 2017). In BONCAT experiments, samples are incubated in the presence of a synthetic amino acid analog, which is taken up by cells and incorporated into newly made proteins of biosynthetically active cells due to the substrate promiscuity of the translational machinery (Kiick et al., 2002). We used the synthetic amino acid L-homopropargylglycine (HPG), a surrogate of L-methionine (Kiick et al., 2002). After incubation with HPG, active cells can be fluorescently detected using azide-alkyne click chemistry (Hatzenpichler et al., 2014, 2015, 2016).

Because cell activity is determined by fluorescence staining, active cells can be separated from inactive ones using fluorescence activated cell sorting (BONCAT-FACS). After sorting, identities of active cells can be determined by taxonomic marker gene sequencing. Alternatively, sorted cells that have been glycerol stored can be genome amplified (Hatzenpichler et al., 2016), or activity can be directly linked with taxonomic identity using rRNA FISH.

A 50 mM stock solution of HPG was prepared in 1× PBS and aliquoted to 1 mL cryovials that were stored at 4°C. For each group of four samples analyzed by SIP (as described in **Stable isotope probing bioassays of hydrocarbon degradation**), 130 mL sediment of the shallow core sample or 390 mL of the deeper sample were diluted with three volumes of sterile bottom water. A total of 40 mL of each slurry was removed to a 100 mL Hungate bottle as a no-addition control for the BONCAT assay because the addition of the labeled amino acid analog that is used for molecular tagging of active microbial cells could potentially alter microbial community structure and activity, particularly in substrate-depleted subsurface sediments. To the remaining slurry, the synthetic amino acid HPG was added to a final concentration of 0.05 mM. Then, the shallow and deeper slurries were divided after mixing: the shallow slurries were divided into 12 volumes of 40 mL each and paced into 100 mL Hungate bottles, and the deeper slurries were divided into 12 volumes of 120 mL each and placed into 250 mL bottles. This scheme provided sufficient slurry volumes for triplicate samples at all four points of time considered: immediately ($T_0$) and after 2 days, 1 week, and 2 weeks. By performing incubations at higher volumes for deeper samples, we improved the chances that enough active cells will be available for cell sorting later on. Depending on in situ temperature, the timescales for incubation might be varied because higher temperatures necessitate shorter incubation times. By taking samples after short and long incubation times, highly active cells can be detected already after short incubation times and differentiated from less active cells that are revealed by longer incubation. Incubation bottles were placed at in situ temperatures after gassing with Biomix, a mixture of 70% N₂, 25% CO₂, and 5% H₂ (V/V). At the time of sacrifice, slurry volumes were transferred to and spun in 50 mL Falcon tubes briefly (15 min at 4500 × g), the supernatant was decanted, and the sediment was washed in PBS to remove unincorporated HPG.

For near-surface sediment slurries, time-point samples of 40 mL were divided as follows:

* 3 mL of slurry was stored in 3 separate 1 mL cryotubes at -80°C for DNA analyses.

* 27 mL of slurry for FACS cell counts was stored in glycerol-TE (GlyTE) buffer following published protocols (Rinke et al., 2014). GlyTE buffer was prepared by mixing 20 mL of 100× TE
(pH 8), 60 mL Milli-Q water, and 100 mL molecular grade glycerol; the buffer was filtered through a 0.2 µm syringe filter and stored at -20°C during the expedition. GlyTE was added to slurry samples at a 9:1 (V/V) ratio of sample to GlyTE.

* 10 mL of slurry was stored for BONCAT-FISH by fixing it with 8 mL of 8% formaldehyde (in PBS; 3.6% final concentration) for 2–24 h. After spinning 5 min at max speed = 16,000 × g, the formaldehyde solution was exchanged twice with PBS and sediments were resuspended in 50:50 (V/V) PBS:ethanol and stored at -20°C.

For deeper sediment slurries, time-point samples of 120 mL were divided into 84 mL for FACS cell counts, 30 mL for BONCAT-FISH, and 6 mL for DNA extraction and preserved as above, scaling volumes accordingly.

## 10.9. Cultivation experiments

Cultivation-dependent studies will provide particularly useful information on physiology, potential biogeochemical function, diversity, and habitat range of subseafloor microorganisms. During Expedition 385, shipboard microbiologists initiated cultivations with a wide range of culture media, targeting various physiological types of microbes, as shown below. Generally, the inoculum slurries used for onboard cultivation experiments were individually prepared by individual shipboard scientists using subsamples taken from whole-round cores with cutoff syringes of various sizes. In addition, some sediment samples or inoculum slurries, as well as the sample-inoculated test tube cultures, were stored at 4°C or appropriate conditions for shore-based cultivation experiments.

### 10.9.1. Mesophilic, thermophilic, and hyperthermophilic methanogenic subsurface microorganisms

Sediments with in situ temperatures of 20°–80°C were selected to enrich mesophilic, thermophilic, and hyperthermophilic microorganisms. Thus, incubation temperatures of 50°, 60°, 70°, and 80°C were used to cover these different populations.

* For enrichment cultures, sediments from 30 cm long whole-round cores were stored in plastic bags flushed with nitrogen at 4°C or immediately transferred to an anaerobic chamber.

* Uncontaminated sediments (V = 100 cm³ per sample) from the innermost part of the whole-round core sample were collected anaerobically with cut-end syringes or sterile spatula for harder sediments, transferred to 500 or 250 mL glass bottles containing sterile, carbonate-buffered anoxic artificial seawater (of a similar volume to the sediment), and crimp sealed with butyl rubber septum stoppers and caps (Ochs Glasgerätebau, Bovenden, Germany). The carbonate-buffered artificial seawater was modified from the methanogen medium 141 of the German Collection of Microorganisms and Cell Cultures (DSMZ) (https://dsmz.de/microorganisms/medium/pdf/DSMZ_Medium141.pdf) (Vigneron et al., 2015) with the addition of 0.3 g/L of yeast extract.

* Sediment slurries were dispensed into 50 or 100 mL anaerobic glass vials. The vials were sealed with butyl rubber septum stoppers and aluminum crimp tops and then flushed with N₂ and stored at 4°C under a 200 kPa N₂/CO₂ (80/20 V/V) gas atmosphere. Resazurin (1 mg/L stock solution) was used as a oxidation-reduction (redox) indicator.

* Master sediment slurries were used to inoculate substrate-specific methanogen enrichments at 10%–20% (volumes of inoculum versus medium) for subsequent incubation.

All media and incubations were anaerobic, unless otherwise stated. To prepare separate enrichment media for CO₂-reducing, acetoclastic, methylotrophic, and alkanotrophic methanogens, media were usually supplemented individually with one of the following carbon substrates: bicarbonate, acetate (10 mM), trimethylamine (20 mM), dodecane, hexane, and hexadecane (100 µL) under an H₂/CO₂ (80/20 V/V; 200 kPa) and N₂/CO₂ (80/20 V/V; 200 kPa) gas atmosphere. Uninoculated media were used as negative controls. All cultures were statically incubated in the dark at near in situ temperature of sediment. The time-course enrichments were monitored through methane production by headspace using a GC and microscopic observations.
Toward the end of the expedition (Sites U1550–U1552), samples were prepared for time-course enrichments in home laboratories. Aliquots of sediment slurry were dispensed into 100 mL serum glass vials. The bottles and vials were flushed with argon, crimp sealed with butyl rubber septum stoppers and crimp top caps, and stored at ~4°C under a ~200 kPa argon gas atmosphere.

As a starting point for enrichment and incubation monitoring, subsamples of ~50 cm³ were frozen at –80°C within a few hours of core recovery for shore-based metagenomic and metabarcoding analyses.

### 10.9.2. Time-course stable isotope probing monitoring

For selected sediment samples, triplicates of 50 mL anaerobic sediments slurries were spiked with labeled (¹³C) and unlabeled (¹²C) substrates. Specifically, sediment slurries were amended independently with Na¹³C-acetate (1,2-¹³C₂, 99 atom%), NaH¹³C-CO₃ (99 atom%), trimethylamine (1,2,3-¹³C₃, 99 atom%), and ¹³C-methanol (99 atom%) to a final concentration of 10 mM (all compounds from Sigma Aldrich, USA). Vials were then statically incubated at near in situ temperatures in the dark. Controls were amended with the same volume of unlabeled ¹²C substrates. Sediment slurry samples of ~5 mL from microcosms were collected anaerobically at several time points for complex organic compound degradation, intermediates, and gas characterization and stored at –20°C for postexpedition analyses. Sediment slurry samples of 20 mL were collected anaerobically with cut-end syringes and stored in a glass vial crimp sealed at –20°C for postexpedition analyses of hydrocarbons.

Enrichment cultures were monitored during the incubation by subsampling 10 mL volumes in 50 mL centrifuge tubes using sterile 10 mL tip-cut syringes (Falcon) and freezing at –80°C for postexpedition metagenomic and metabarcoding analyses.

### 10.9.3. Methanogenic and sulfate-reducing microbial enrichments and DNA preservation for metagenomic analyses

Independently of previously described cultivations, further enrichments were performed with the intention of linking these with postexpedition metagenomic analyses. In order to observe the influence of local geochemistry on methanogenic and sulfate-reducing microbial communities, anaerobic methanogenic and sulfate-reducing microbial enrichments were started shipboard from the geochemically distinct Holes U1545B, U1546B, and U1547B at two in situ temperatures (20° and 50°C). Sediment was collected from the middle of a 20 cm long whole-round core using cut-end syringes. Sediment slurry was then prepared by adding 6 cm³ of sample sediment to 40 mL of preprepared anaerobic media for methanogens (Table T14) and sulfate reducers (Table T15). This slurry was aliquoted in a volume ratio of 5% into methanogenic media, in triplicate, that contained either 10 mM methanol, acetic acid, formic acid, or (80/20) H₂/CO₂ as substrate and was incubated at near in situ temperature. In the same volume ratio, the slurry was aliquoted in triplicate into sulfate reducing bacteria (SRB) media that contained a substrate mixture of acetic acid, pyruvate, and formic acid and incubated at near in situ temperature. A time-point 0 sample was taken from all enrichments for cell counts, from sulfate-reducing microorganism enrichments for sulfide concentration, and from methanogen enrichments for methane concentration.

Subsamples for shore-based DNA extraction and metagenomic analyses were taken from all whole-round cores collected from Holes U1545–U1545C, U1546B, U1546D, U1547B, U1550B, and U1551B. Subsamples were obtained from 20 cm long whole-round cores collected in Holes U1545A, U1545B, U1546B, and U1547B by cutting a 7 cm sample from the full 20 cm whole-round core, trimming the top 1 cm of the 7 cm sample, taking four 10 cm³ syringes of sediment from the remaining 6 cm, and freezing the syringe samples at –80°C in sterile Whirl-pak bags. The remainder of the 7 cm sample was stored in a sterile sample bag at ~4°C for mineralogic analysis. The remaining 13 cm of the whole-round core was sealed anaerobically in a gas-tight, trilaminated, foil barrier bag and stored at ~4°C for further shore-based enrichment experiments. From Holes U1545C, U1546D, U1550B, and U1551B, 10 cm long whole-round cores were collected and either frozen at –80°C in sterile Whirl-pak bags or stored at ~4°C in mylar bags for shore-based enrichments or DNA extractions, respectively.
Enrichments were also set up in duplicate with basaltic samples obtained from Hole U1547D for sulfate-reducing microorganisms and methanogenic Archaea. About 100 mL of chiseled/ground basaltic rock was mixed into 100 mL of medium (Parkes et al., 2010) to create a master slurry. This master slurry was then aliquoted as 10 mL into 30 mL serum bottles. The serum bottles were capped with blue butyl stoppers, crimped with aluminum caps, and pressurized to 2 bar (200 kPa) with either N₂/CO₂ (80/20 V/V) or H₂/CO₂ (80/20 V/V) gas. For methanogenic enrichments, either 10 mM acetate, 10 mM methanol, 10 mM formate, or H₂/CO₂ (80/20 V/V) served as a substrate. For sulfate-reducing microorganism enrichments, either a mixture of 10 mM formate, acetate, and pyruvate or H₂/CO₂ (80/20 V/V) served as a substrate. All enrichments were incubated at 80°C.

### 10.9.4. Cultivation experiments for fungi

Based on prior work on Guaymas Basin sediments, the functional groups of eukaryotic microbes include filamentous fungi and yeasts. For fungal enrichments, ~3 mL sediment was mixed 1:5 (V/V) with sterile bottom water in a 30 mL glass Hungate bottle with a rubber septum and topped up with sterile bottom water, leaving approximately a third of the bottle volume headspace that was gassed with Biomix gas (5% H₂, 75% N₂, 20% CO₂). Bottles were stored at 4°C for continuation of shore-based fungal culturing using high-throughput culturomics on diverse fungal media.

## 10.10. Structural imaging

### 10.10.1. 3-D spatial arrangement imaging

At the same frequency as cell count sampling, 5 cm³ of sediment was taken on the core receiving platform, sealed with parafilm, and temporarily stored at 4°C until further processing after several days. The syringe plug samples were capped with low-melting point agarose and fixed with 2% formaldehyde and 1% glutaraldehyde in 3x PBS. The fixed samples were shipped to a shore-based laboratory to be further stained with osmium and embedded in resin for postexpedition high-spatial resolution X-ray computed tomography analysis.

### 10.10.2. 3-D imaging of fungi

For the same cores and sections targeted for fungal SIP and BONCAT studies, syringe cores were collected for mapping fungal mycelia using micro-computed tomography (CT) scanning of filamentous fungal growth in those cores. We collected three 5–15 cm³ syringe plug samples from the

**Table T15.** Sulfate reducer media. All components were dissolved in distilled water except L-cysteine·HCl, Na₂S·9H₂O, vitamin supplement (ATCC MV-DS), acetate, formate, and pyruvate. When required, pH was adjusted to 6.8. The medium was degassed by sparging with N₂ gas, moved to an anaerobic chamber, and allowed to sit for 30 min. L-cysteine·HCl was added to the media and, once the liquid had become clear, Na₂S·9H₂O was added. The media was aliquoted into sterile Balch tubes (8 mL per tube), which were tightly capped with blue butyl stoppers and the stoppers crimped in place. The media-filled tubes were autoclaved at 121°C for 30 min and then allowed to equilibrate to room temperature. Vitamin supplement (1% v/v) was added to all the tubes using sterile 23G needles. A substrate mixture of acetate, formate, and pyruvate was added (5 mM each). The headspace of all the tubes was then replaced with N₂/CO₂ (80/20) gas mix and pressurized to 2 bar. **Download table in CSV format.**

<table><thead><tr><td>Component</td><td>Concentration</td></tr></thead><tbody><tr><td>Na₂SO₄</td><td>21 mM</td></tr><tr><td>KH₂PO₄</td><td>1.47 mM</td></tr><tr><td>NH₄Cl</td><td>5.6 mM</td></tr><tr><td>NaCl</td><td>410 mM</td></tr><tr><td>MgCl₂·6H₂O</td><td>14.76 mM</td></tr><tr><td>KCl</td><td>6.71 mM</td></tr><tr><td>CaCl₂·2H₂O</td><td>1.03 mM</td></tr><tr><td>NaHCO₃</td><td>30 mM</td></tr><tr><td>Yeast extract</td><td>0.2% w/v</td></tr><tr><td>Na₂S·9H₂O</td><td>1 mM</td></tr><tr><td>L-cysteine·HCl</td><td>3.2 mM</td></tr><tr><td>Na-resazurin solution (0.1% w/v)</td><td>0.05% v/v</td></tr><tr><td>Trace elements SL-10</td><td>1% v/v</td></tr><tr><td>Vitamin supplement (ATCC MV-DS)</td><td>1% v/v</td></tr><tr><td>Acetate</td><td>5 mM</td></tr><tr><td>Formate</td><td>5 mM</td></tr><tr><td>Pyruvate</td><td>5 mM</td></tr></tbody></table>
center of each designated whole-round core section using sterile cut-off syringes. The syringe
plunger was removed, and the bottom of each syringe was plugged with sterilized cellulose sponge
at its top and bottom. The syringe sample was then placed into a polypropylene Covidien 20 mL
Monoject syringe casing filled with 5–15 mL of 8% formalin (4% final concentration) and closed by
tightening its cap. Syringe core plugs were stored in a fume hood at room temperature and perco-
lated for 3 days. Formaldehyde was then exchanged with sterile 1× PBS twice over 4 days each, and
then syringe plugs were stored in 50:50 (V/V) PBS:ethanol at 4°C in the sealed casings at –20°C for
further processing and visualization in a shore-based laboratory. On shore, chitin-specific primary
antibodies labeled with gold will be incubated inside the core for 1–2 days. Subsequently, most of
the unbound antibodies will be flushed from the plug by incubating with sterile PBS prior to label-
ing with secondary antibodies. Labeled sediment cores will be visualized using micro-CT scan-
ning.

**10.11. Hydrocarbon analyses**

For a detailed profile of saturated hydrocarbons, polyaromatic hydrocarbons (PAHs), and
alkylated PAHs (~200 compounds) in samples targeted for studies of fungi (generally with in situ
temperature of 80°C or below), 20 mL of pore water was squeezed from ~100 mL of each sample
using a Carver titanium hydraulic squeezer (pressure < 10 MPa), filtered through a 0.22 µm
syringe filter, and stored at 4°C in 20 mL gas-tight vials for shore-based postexpedition analysis at
Alpha Analytical (USA). Sediment cakes (2 × 50 mL stored in Falcon tubes) were also analyzed.
Methane concentrations were measured by other shipboard scientists (see **Organic geochemistry**).
Selected samples will be analyzed by comprehensive 2-D GC (GC×GC) at the Woods Hole
Oceanographic Institution (USA).

**10.12. Nutrient analyses**

To quantify nutrients in 15 mL of pore water squeezed from each sample studied for fungi, pore water samples were set aside for postexpedition analysis at the Louisiana State University (LSU) College of the Coast and Environment Laboratory. These analyses include $NO_2^-$ + $NO_3^-$, $NH_4^+$, $PO_4^{3-}$ (5 mL pore water required), carbon (total dissolved organic and inorganic, DIC, dissolved organic carbon), and dissolved organic nitrogen (10 mL required for C and N analyses). Samples were stored at -20°C in 2 or 5 mL aliquots in screw-cap Eppendorf tubes without headspace. TOC, TC, and TN were measured in the sediment cakes remaining for each sample and stored in two 50 mL Falcon tubes (see Organic geochemistry).

**10.13. Summary of shipboard general objectives**

The general sampling objectives and codes for shipboard microbiology participants are outlined in Table T16 to supplement the list of samples collected by all participants presented in LIMS.
<table><caption>**Table T16.** MBIO sampling codes and research plans. Types of samples: 1 = frozen –80°C; 2 = frozen –20°C; 3 = live material +4°C; 4 = fixed material; 5 = enrichments. HC = hydrocarbons. **Download table in CSV format.**</caption><thead><tr><th>Scientist</th><th>Sample code</th><th>Sites sampled</th><th>Types of samples</th><th>Scientific objectives</th></tr></thead><tbody><tr><td>Andreas Teske</td><td>TESK1</td><td>All</td><td>WRND 1</td><td>DNA mcrA genes</td></tr><tr><td>Andreas Teske/ Michael Seidel (shore)</td><td>TESK2</td><td>All</td><td>WRND 1</td><td>High molecular weight compound composition of organic matter</td></tr><tr><td>Andreas Teske</td><td>TESK3</td><td>All</td><td>WRND 3</td><td>Heterotrophic enrichments</td></tr><tr><td>Diana Bojanova</td><td>BOJA1</td><td>All except U1549, U1551</td><td>WRND</td><td>Metagenomic sequencing for metabolic diversity</td></tr><tr><td>Diana Bojanova</td><td>BOJA2</td><td>All except U1549, U1551</td><td>WRND</td><td>Enrichments</td></tr><tr><td>Diana Bojanova</td><td>BOJA</td><td>All except U1549, U1551</td><td>GAS</td><td>Clumped isotope on CH<sub>4</sub></td></tr><tr><td>Nicolette Meyer</td><td>MEYE1</td><td>All</td><td>WRND</td><td>DNA</td></tr><tr><td>Nicolette Meyer</td><td>MEYE2</td><td>All</td><td>WRND</td><td>Stable isotope incubations for cell-specific anabolic activity</td></tr><tr><td>Nicolette Meyer</td><td>MEYE</td><td>All</td><td>WRND</td><td>Stable isotope incubations for cell-specific anabolic activity</td></tr><tr><td>Yuki Morono</td><td>MORO1</td><td>All</td><td>SYR 4</td><td>Microbial cell abundance, 3-D structural imaging</td></tr><tr><td>Yuki Morono</td><td>MORO2-1</td><td>All</td><td>WRND 1</td><td>Non-incubation control frozen sediment</td></tr><tr><td>Yuki Morono</td><td>MORO2-2</td><td>All</td><td>WRND 3</td><td>High-pressure and -temperature incubation</td></tr><tr><td>Florian Schubert</td><td>SCHU</td><td>All</td><td>WRND</td><td>Sulfate reduction rate</td></tr><tr><td>Virginia Edgcomb</td><td>EDGC1</td><td>All</td><td>WRND 1</td><td>DNA, lipid biomarkers</td></tr><tr><td>Virginia Edgcomb</td><td>EDGC2</td><td>All</td><td>WRND 3,4</td><td>CARD-FISH, fungal enrichments, live samples for HC degradation experiments, HC analyses</td></tr><tr><td>Virginia Edgcomb</td><td>EDGC</td><td>All</td><td>CYL 1</td><td>mRNA</td></tr><tr><td>Virginia Edgcomb</td><td>EDGC</td><td>All</td><td>CYL 4</td><td>3-D fungal imaging</td></tr><tr><td>Virginia Edgcomb</td><td>EDGC</td><td>All</td><td>IW 2,3</td><td>HC and nutrients</td></tr><tr><td>Laurent Toffin</td><td>TOFF1</td><td>All</td><td>WRND 1</td><td>Metagenomic</td></tr><tr><td>Laurent Toffin</td><td>TOFF2</td><td>All</td><td>3,5</td><td>Enrichments and <sup>13</sup>C incubations (SIP)</td></tr><tr><td>Guangchao Zhuang</td><td>ZHUA1</td><td>All</td><td>WRND 3</td><td>Methanogen activity rates</td></tr><tr><td>Guangchao Zhuang</td><td>ZHUA2</td><td>All</td><td>CYL 4</td><td>Hydrogen</td></tr><tr><td>Guangchao Zhuang</td><td>ZHUA</td><td>All</td><td>LIQ 1</td><td>Methanol and ethanol</td></tr><tr><td>Jayne Rattray (shore)</td><td>RATT</td><td>U1545–U1549</td><td>WRND 3</td><td>Spore-former cultivations and counts</td></tr><tr><td>Nagissa Mahmoudi (shore)</td><td>MAHM</td><td>U1545–U1547, U1550</td><td>WRND 1</td><td>Heterotrophic C degradation experiments with pure cultures</td></tr></tbody></table>

# 11. Petrophysics

High-resolution physical properties measurements were taken during Expedition 385 to aid litho-stratigraphic characterization and to link core observations with downhole measurements and seismic reflection profiles. In particular, physical properties data were used in hole-to-hole and site-to-site stratigraphic correlation, detection of lithologic discontinuities and heterogeneities, identification of changes in sediment composition and texture, and identification of major seismic reflectors. These physical properties include density (ρ), MS/MSP, P-wave velocity, NGR (i.e., measurements providing insights into elemental bulk composition of the cores), thermal conductivity, rheology (i.e., compressive and shear strength), and porosity. Physical properties of cores were characterized using a variety of techniques and methods on recovered whole-round core sections, section halves, and discrete samples. Finally, a variety of downhole logging tools have been used to characterize the following physical properties in situ: density, NGR, compressional (P-wave) and shear (S-wave) seismic velocities, electrical resistivity, and MS.

## 11.1. Core laboratory measurements

### 11.1.1. Standard core flow

A standard set of physical properties measurements were made on sediment cores in the core laboratory, following a standard sequence. We describe this standard sediment core flow here. Recovered whole-round sections were first stored in a rack for ~4 h for equilibration to room temperature (~20°C) and atmospheric pressure (~101 kPa). After thermal equilibration, a sequence of measurements was made on whole-round core sections. Whole-round sections were first run through the WRMSL, which measures density using the GRA system, MS, and compressional wave velocity ($V_p$) deploying the PWL. NGR emissions from the sections were then measured using the NGRL. The final whole-round measurement was thermal conductivity, which was made on one whole-round section per sediment core using a needle probe inserted into the section through a small hole drilled through the plastic core liner close to the middle of the section. When the sediment cores were too hard to insert the needle probe, a contact probe was used instead to measure the thermal conductivity on the section half after core splitting.
Core sections were split laterally after whole-round measurements were completed. One split-section half was designated as the archive half and the other as the working half for sampling and analysis. The archive half of the core was passed through section-half measurements starting with the SHMSL to measure MSP and color reflectance. The Section Half Measurement Gantry (SHMG) includes bidirectional P-wave velocity measurements (x- and z-axis directions) and two rheological measurements to estimate the compressive and shear strengths of the material. Finally, discrete samples were collected from the working halves (every other section) to measure wet bulk density, dry bulk density, water content, porosity, and grain density following the MAD procedure described below.

The flow and processing of hard rock cores was slightly different from that of sediment cores. Drilling through and coring cohesive sedimentary and igneous rocks was done using the RCB coring system. RCB cores have a smaller diameter than cores recovered by the APC and XCB systems. RCB cores commonly consist of numerous discrete pieces, though a number of long, continuous hard rock cores were recovered during Expedition 385. Here we describe only the differing core flow process for sedimentary and igneous rock cores relative to the standard sediment core flow described above. The discrete pieces of a given recovered hard rock section were shaken into a sterile split liner in the core-splitting room for examination by a petrologist and/or a structural geologist, who determined the splitting line for obtaining working and archive halves from each whole-round piece (see *Structural geology*). The pieces were then put back into a full core liner and run through the WRMSL and NGRL. P-wave velocity was not measured because the air-filled space between the core liner and the hard rock pieces would make these measurements meaningless. After physical properties measurements on whole-round cores were complete, the core sections were split into working and archive halves and all rock pieces were labeled.

The archive halves of hard rock cores were passed through the SHMSL to measure MSP and color reflectance. Thermal conductivity of igneous rock pieces was measured once per core (i.e., on a single section-half rock piece chosen from the core) using a contact probe in a seawater bath after saturation of the piece in seawater for 4 h. Paleomagnetic, MAD, and P-wave velocity measurements were made on samples taken from the working half of the core at ~1 m intervals depending on lithologic variability. All three measurements were made on each sample in most cases. The P-wave velocity measurements for these samples were made in the x-, y-, and z-directions.

A full discussion of all methodologies and calculations used aboard *JOIDES Resolution* in the physical properties laboratory is available in Blum (1997). Details and procedures for each physical properties measurement are described below.

### 11.1.2. Whole-round measurements

GRA bulk density, MS, and P-wave velocity were measured nondestructively on the WRMSL (Figure F25); NGR was measured on the NGRL (Figure F26); and thermal conductivity was measured using a TeKa TK-04 thermal conductivity meter (Figure F27).

To optimize the measurement process, the same sampling intervals and measurement times were used for all sensors on each of the WRMSL instruments. These sampling intervals are common denominators of the distances between the sensors installed on the WRMSL (30–50 cm), which enables sequential and simultaneous measurements. For the WRMSL, the sampling interval was 2.0 cm with an integration time of 3 s for each measurement. A QC measurement was performed after every core by passing a single core liner filled with deionized water through the WRMSL. If deviations from expected values were observed, a recalibration procedure was performed.

#### 11.1.2.1. Gamma ray attenuation bulk density

The bulk density of a sample can be used to estimate the pore volume of the sample and to evaluate the consolidation state of sedimentary material. GRA provides a means to estimate material bulk density based on the attenuation of a gamma ray beam. The beam is produced by a ¹³⁷Cs gamma ray source at a radiation level of 370 MBq within a lead shield with a 5 mm collimator that is vertically directed through the whole-round core (Figure F25). The gamma ray receiver consists of a 75 mm³ sodium iodine (NaI) detector located on the upper side of the core, opposite to the source, that records the gamma radiation that passes through the core. The input gamma ray peak has a principal energy of 0.662 MeV and is attenuated as it passes through the core.
The attenuation of gamma rays occurs primarily by Compton scattering, in which gamma rays are scattered by electrons in the formation, and the degree of scattering is related to the material bulk density. Therefore, for a known thickness of sample, density (ρ) is proportional to the measured intensity of the attenuated gamma rays (Harms and Choquette, 1965):

$$ \rho = \ln(I_0/I)/(\mu d), \qquad (22) $$

where

I = the measured intensity of gamma rays passing through the sample,

I₀ = gamma ray source intensity,

μ = Compton attenuation coefficient, and

d = sample diameter.

Note: μ and I₀ are treated as constants so ρ can be calculated from I.

In general, WRMSL measurements are most accurate when taken on a completely filled core liner with minimal drilling disturbance; otherwise, measurements tend to underestimate true values. By default, the instrument reports measurements using the internal diameter of the core liner (66 mm) as the assumed sample diameter. This assumption is suitable for most sediment cores obtained using the APC system. However, for sediment and/or hard rock cored using the XCB or RCB system, core diameter is usually about 58 mm or smaller. Following Jarrard and Kerneklian (2007), the density measurements of cores obtained using the XCB or RCB systems may be corrected by multiplying the density values by 66/58 = 1.138 to account for this bias. We chose not to correct the raw values, but the offset is mentioned in the description of the measurements.

The spatial resolution of the GRA densitometer is <1 cm. The gamma ray detector is calibrated with a sealed calibration core consisting of one standard core liner filled with distilled water and a telescoped aluminum bar machined into discrete diameter steps of various diameters located at the top of the core. To establish the calibration curves, gamma ray counts were taken through each of the discrete diameter steps for 60 s. Recalibration was performed whenever the deionized water QC standard deviated significantly (1.00 ± 0.02 g/cm³).

<figure><img src="image_29.png" /><figcaption>**Figure F25.** The Whole-Round Multisensor Logger (WRMSL) measures gamma ray attenuation (GRA) bulk density, magnetic susceptibility, and P-wave velocity. Track direction indicated.</figcaption></figure>
<figure><img src="image_30.png" /><figcaption>**Figure F26.** Natural Gamma Radiation Logger (NGRL). Track direction indicated.</figcaption></figure>

<figure><img src="image_31.png" /><figcaption>**Figure F27.** Shipboard thermal conductivity facility. A. Insulated closed box for whole-round and section half sediment measurements. B. Insulated closed box for hard rock measurements. C. Thermal sensors for whole-round measurement in soft sediment (i.e., needle probe) and for measurements on section-half and half-space hard rock (i.e., puck).</figcaption></figure>
### 11.1.2.2. Magnetic susceptibility

MS is measured on whole-round core sections using a pass-through loop on the WRMSL (Figure F25). MS (χ) is a dimensionless measure of the degree to which a material can be magnetized by an external magnetic field:

$$ \chi = M/H, \qquad (23) $$

where M is the magnetization induced in the material by an external field of strength H.

MS measurements were made using a Bartington MS2C loop sensor with an 88 mm diameter (Bartington Instruments, 2011). An oscillator circuit in the sensor operating at a frequency of 0.565 kHz produces a low-intensity (250 μT), nonsaturating alternating magnetic field within the loop that has a strength proportional to the magnetic permeability within the loop. Sediment or hard rock core sections passing through the loop represent a change in permeability and thus cause a change in the induced field that can be measured as a change in oscillator frequency. Frequency information returned in pulse form to the susceptometer provides a direct estimate of the core material's magnetic permeability, which is converted into MS. The loop sensor has a spatial resolution of 20 mm and is accurate to within 5%. MS is reported in instrument units. To convert results into dimensionless units, it is necessary to multiply the data by a correction factor that is a function of the probe type, core diameter, and loop size. The sizes of small rock pieces in incomplete cores vary, so application of a single correction factor is not justified; therefore no correction is applied to the WRMSL MS data. This conversion factor does not change the order of magnitude of the measured susceptibility values, so the results are comparable with the susceptibility measured by the paleomagnetism group with the same physical units (SI).

The along-core response curve of the MS2C coil has a full width at half maximum of ~4 cm (Blum, 1997) and is consistent with the decay in magnetic intensity with distance from a dipole. Therefore, measurements of susceptibility from core pieces <8 cm long will significantly (>10%) underestimate MS. MS data recorded on the WRMSL must therefore be interpreted with reference to breaks and variable diameters of fragmented cores; this information can be obtained from the 360° photo imaging record.

MS varies with the type and concentration of magnetic grains, such as the ferromagnetic minerals (e.g., magnetite and maghemite), making it useful for identifying compositional variations. MS responds to variations in the magnetic composition of cored materials that are commonly related to variations in mineralogic composition (e.g., terrigenous versus biogenic materials) and diagenetic overprinting. Paramagnetic materials such as clay generally have MS that is several orders of magnitude lower than ferromagnetic minerals, which are common constituents of igneous material. Diamagnetic minerals such as calcite, aragonite, and opal, as well as water and plastics (such as the core liner), have a slightly negative MS. Thus, calcareous and siliceous biogenic deposits with very small amounts of clay and iron-bearing minerals have values approaching the detection limit of the MS meters.

### 11.1.2.3. Sonic velocity

P-wave velocity data can be used to evaluate small-strain elastic moduli, porosity, compaction, and cementation. P-wave velocity is the rate at which a compressional seismic wave travels through a medium, expressed in meters per second. P-wave velocity is dependent on the composition, porosity, bulk density, fabric, and temperature of the material, which in turn are functions of consolidation and lithification, state of stress, and degree of fracturing. P-wave velocity ($V_p$) of a homogeneous, isotropic material can be defined by the time required for a compressional wave to travel a specific distance:

$$ V_p = d/t_{core} \qquad (24) $$

where, for the case of a drill core, d is the path length of the wave across the core and $t_{core}$ is the traveltime through the core.

The PWL on the WRMSL transmits a 500 kHz P-wave pulse across the core liner at a specified repetition rate (Figure F25). The pulse transmitter and receiver are mounted on a caliper-type device and are aligned to make wave propagation perpendicular to the section's long axis. A linear
variable differential transducer measures the P-wave travel distance between the pulse source and the receiver. Good coupling between transducers and the core liner is facilitated with water dripping onto the contact from a peristaltic water pump system. Signal-processing software picks the first arrival of the wave at the receiver, and the processing routine corrects for the thickness of the liner. The total observed traveltime ($t_o$) is composed of

* $t_{delay}$ = time delay related to transducer faces and electronic circuitry,

* $t_{pulse}$ = delay related to the peak detection procedure,

* $t_{liner}$ = transit time through the core liner, and

* $t_{core}$ = traveltime through the cored material,

with the traveltime through the core expressed as

$$t_{core} = t_o - t_{pulse} - t_{delay} - 2t_{liner}. \qquad (25)$$

The system is calibrated using a core liner filled with distilled water, which provides control for $t_{delay}$, $t_{pulse}$, and $t_{liner}$. From these calibrations, $V_p$ can be calculated for the whole-round core in liners as

$$V_p = (d_{cl} - 2d_{liner})/t_{core}, \qquad (26)$$

where

$d_{cl}$ = measured diameter of core and liner,

$d_{liner}$ = liner wall thickness.

This equation assumes that the core completely fills the core liner. Measurements of $V_p$ made with the PWL when the core liner is not completely filled, and thus contains air-filled gaps, are not useful. Most cores recovered with the XCB and RCB systems do not fill the core liner and are thus not considered useful.

A series of acrylic cylinders of varying thicknesses are used to calibrate the PWL system. The regression of traveltime versus travel distance yields the P-wave velocity of the standard material, which should be 2750 ± 20 m/s. The thickness of the calibration samples, corrected for liner thickness, are divided by the traveltime to calculate P-wave velocity in meters per second. The calibration is verified by measuring a core liner filled with pure water at room temperature, and the calibration passes if the velocity is within ± 20 m/s of the expected P-wave velocity of water at room temperature and 1 atm (1485 m/s).

### 11.1.2.4. Natural gamma radiation

Gamma radiation is emitted from the decay series of mineral-hosted 238-uranium ($^{238}$U), 232-thorium ($^{232}$Th), and 40-potassium ($^{40}$K). The system in use (Figure F26) was designed and built at the JOIDES Resolution Science Operator (JRSO) at Texas A&M University (USA) (Vasiliev et al., 2011). When $^{238}$U, $^{232}$Th, and $^{40}$K radioisotopes decay, they and their daughter isotope products emit gamma radiation at specific energy levels unique to each isotope. NGR spectroscopy measures a wide energy spectrum that can be used to estimate the abundance of each isotope based on the strength of the signal at characteristic energies. Spectral data were collected and can be used for postexpedition processing to obtain U, Th, and K abundances, but they were not processed aboard the vessel. Total counts were used on board, and high counts usually identified fine-grained deposits containing K-rich clay minerals and their absorbed U and Th isotopes. NGR data thus reveal stratigraphic details that aid in core-to-core correlations. For example, in hard rocks, NGR measurements can yield information about the alteration and heat production of rocks (Dunlea et al., 2013) that is useful when correlating core measurements with downhole measurements of NGR (e.g., Révillon et al., 2002).

The main NGR detector unit consists of 8 NaI scintillator detectors arranged along the core-measurement axis at 20 cm intervals surrounding the section. The section is measured once and then moves 10 cm and is measured again every 10 cm to provide a final measurement spacing of 10 cm. The detector array has passive (layers of lead) and active (plastic scintillators) shielding to reduce background environmental and cosmic radiation. The overlying plastic scintillators detect
incoming high-energy gamma and muon cosmic radiation and cancel this signal from the total counted by the NaI detectors. The NGRL was calibrated using ¹³⁷Cs and ⁶⁰Co sources to identify peaks at 662 and 1330 keV, respectively.

Total counts are routinely summed over the range of 100–3000 keV. The quality of the measured energy spectrum depends on the concentration of radionuclides in the sample and on the counting time, with longer counting times providing better counting statistics. A live counting time of 5 min was set in each position for a total live count time of about 10 min per section. Sections were scanned only once. Sections less than 50 cm long were not measured.

### 11.1.2.5. Thermal conductivity

Thermal conductivity (k) is the coefficient of proportionality relating conductive heat flow to a thermal gradient. It reflects the rate at which heat is conducted through a material and is dependent on saturating fluid types, porosity, bulk density, water content, temperature, composition and texture of the mineral phases, and so on. At steady state, thermal conductivity is the coefficient of heat transfer (q) across a steady-state temperature difference (ΔT) over a distance (Δx):

$$q = k(\Delta T/\Delta x). \qquad (27)$$

Thermal conductivity measurements were attempted on the third section, or on a neighboring section if the third was unavailable. Measurements were performed on every core recovered from each drill site using the TeKa Berlin TK04 system described by Blum (1997) (Figure F27). For whole-round soft-recovery cores, a needle probe (number V10702) (Figure F27A, F27C) in full-space configuration (Von Herzen and Maxwell, 1959) was used, and for sedimentary and igneous rock split cores, a contact probe method in half-space configuration was used (Figure F27B–F27C). The probes contain a heater wire and calibrated thermistor.

For soft-sediment cores, the full-space needle probe (VLQ) was inserted into a 2 mm diameter hole drilled along one of the lines that later guided core splitting. The location was at around 60–70 cm from the top of the section or in an interval with low disturbance based on visual inspection. To avoid interference from air flow in the laboratory, the core was placed in an enclosed box insulated with foam (Figure F27A). For sedimentary and igneous rock cores, half-space determinations of thermal conductivity were made using standard half-space needle probe (HLQ) pucks (number H11017) and mini-HLQ pucks (number H51027). These pucks are recommended to be used on planar surfaces and consist of a Plexiglas block (k = 0.184 W/(m·K)) with a half-space needle probe embedded in its base. Heat is assumed to be transferred through the sample, and the TK04 system documentation indicates that heat flow through the Plexiglas block itself is only significant for samples with thermal conductivities <1 W/(m·K). The measurement was conducted in a polystyrene box that insulated the sample from the ambient air flow (Figure F27B).

All measurements were made at room temperature and pressure with no correction for in situ conditions. This system measures thermal conductivity by transient heating of the sample with a known heating power and geometry. Changes in temperature with time during heating are recorded and used to calculate thermal conductivity. Heating power can be adjusted for each sample depending on the type of probe used; heating power can be varied from 1 to 1.6 W/m for a standard puck and from 0.5 to 1.2 W/m for a mini-puck. For most samples, a heating power of 1.2 W/m for a standard puck and 0.8 W/m for a mini-puck was used. For the full-space needle probe, a heating power of 0.5–2.0 W/m was typically used in soft-sediment cores.

The solution to the heat-conduction equation for a line source of heat was fitted to the temperature measurements to obtain the thermal conductivity. Because the probe is much more conductive than sedimentary or igneous rock, the probe is assumed to be a perfect conductor. Under this assumption, the temperature of the probe has a linear relationship with the natural logarithm of the time after the initiation of the heat. The TK04 device uses a special approximation method to calculate conductivity and to assess the fit of the heating curve. This method fits discrete windows to the theoretical function of temperature (T) with time (t) for a continuously heated line source (Kristiansen, 1982):

$$T(t) = A1 + A2 \ln(t) + A3[\ln(t)/t] + (A4/t), \qquad (28)$$
where A1–A4 are constants that are calculated by linear regression. A1, A3, and A4 are related to the sample geometry and the material properties surrounding the needle probe, and A2 is related to its heating power and thermal conductivity. Having determined these constants (and how well they fit the data), the apparent conductivity (ka) for the fitted curve is time dependent:

$$ka(t) = Q/4\pi+A1+A3[1 - \ln(t)/t] - A4/t, \quad (29)$$

where Q is the input heat flux. The maximum value of ka and the time ($t_{max}$) at which it occurs on the fitted curve are used to assess the validity of that time window for calculating the thermal conductivity. The best solutions are those where $t_{max}$ is greatest, and these solutions are selected for use. Data are considered good if ka has a maximum value, $t_{max}$ is large, and the standard deviation of the least-squares fit is low.

Three measuring cycles separated by a cooling interval of 10 min were automatically performed to calculate average conductivity. A self-test, which included a drift study, was conducted at the beginning of each measurement cycle. Once the probe temperature stabilized, the heater circuit was closed and the temperature rise in the probe was recorded. The measurement apparatus was evaluated frequently by measuring the thermal conductivity of a certified ceramic standard ($k = 1.626 W/[m·K]$). For measurement of the ceramic standard, the half-space standard puck or minipuck was coated in silicone thermal contact gel and then placed on the standard; if the measured thermal conductivity was within 2% of the known value, then the puck was used for sample measurements. After the standard and prior to sample measurement, the silicone gel was thoroughly removed from the puck to avoid potential geochemical contamination of the core material. Conductivity measurements are reported in watts per meter degree Kelvin ($W/[m·K]$).

### 11.1.3. Section half logging and discrete measurements

Here we describe measurements made on split sections and discrete samples, including color reflectance spectrophotometry, MSP, rheology, seismic velocity, and MAD.

Color reflectance spectrophotometry and MSP of the archive section halves were measured with the SHMSL (Figure F28). MSP measurements were made using a Bartington Instruments MS2E point sensor (high-resolution surface-scanning sensor) (see Paleomagnetism). Measurement resolution is 1.0 SI, and each measurement integrated a volume of 10.5 mm × 3.8 mm × 4 mm, where 10.5 mm is the length perpendicular to the core axis, 3.8 mm is the width along the core axis, and 4 mm is the depth into the core. Three measurements were made at each measurement position.

Thermal conductivity was measured once per core on hard rock pieces using a contact probe on a piece of section half in a bath of seawater for igneous rock pieces (Figure F27B–F27C). Samples

<figure><img src="image_32.png" /><figcaption>Figure F28. Section Half Multisensor Logger (SHMSL).</figcaption></figure>
from igneous rock material were shared for both paleomagnetic and MAD measurements; P-wave velocity measurements were also made on these discrete samples.

For core-section halves from Holes A and B at each site, a MAD sample was collected from the bottom of the section where a headspace analysis of hydrocarbon gases was taken previously with a syringe in the splitting room. One section half per core was then selected according to its sedimentological characteristics to perform the discrete P-wave velocity, shear strength (automated vane shear [AVS]), and compressive strength (pocket penetrometer) measurements, and other sections were used for discrete paleomagnetic measurements. Finally, all sections were processed using the SHMSL, paleomagnetism (logging), and X-ray imaging upon request from lithostratigraphy and structural groups.

#### 11.1.3.1. Color reflectance spectrometry

Reflectance of visible light from the archive halves of sediment cores was measured using an Ocean Optics USB4000 spectrophotometer mounted on the automated SHMSL (Figure F28). Because the SHMSL demands flush contact between the MS2E point sensor and the split core, measurements were made on the archive halves of split cores, which were covered with clear plastic wrap after all visual core description, smear slide sampling, and X-ray image analyses were completed (see Lithostratigraphy). Measurements were taken at 2.5 cm spacing to provide a high-resolution stratigraphic record of color variation for visible wavelengths. Empty intervals, voids, and cracks were skipped to avoid spurious measurements. Intervals where the top of the flat surface was below the level of the core liner were lifted using foam or putty to ensure proper measurements. Each measurement was recorded in 2 nm wide spectral bands from 400 to 700 nm. Data are converted to L\*, a\*, and b\* and X, Y, and Z color reflectance parameters for efficient archi- val and display. Both zero and white calibrations were conducted before measurement on each core. Additional detailed information about measurement and interpretation of spectral data can be found in Balsam et al. (1997, 1998) and Balsam and Damuth (2000). In addition to a digital color image captured by the SHIL, the VCDs include a description of sediment color and the corre- sponding hue, value, and chroma data as determined qualitatively using Munsell Soil Color Charts for each major and minor lithology (Munsell Color Company, 2000, 2009). Further details are described in Lithostratigraphy.

#### 11.1.3.2. Point magnetic susceptibility

MSP is measured on the SHMSL using a Bartington MS2E contact probe with a flat 15 mm diameter sensor operating at a frequency of 0.580 kHz. The instrument takes and averages three measurements made at 1 s intervals to an accuracy of 5%. The area of response of the MS2E sensor is 3.8 mm × 10.5 mm, with a depth response of 50% at 1 mm and 10% at 3.5 mm (Bartington Instruments, Ltd., 2011). The MSP measurements made on section halves using the SHMSL therefore have a higher spatial resolution than MS measured by WRMSL on whole-round cores. The probe is zeroed in air before each measurement is made, and a background magnetic field is measured and removed from the data. Measurements are output in instrument units but reported in SI (see Magnetic susceptibility). MSP measurements were conducted at a resolution of 2.5 cm for sediment sections and 1 cm for igneous sections. Note that bulk MS data were also collected for discrete cubes taken from working section halves during analyses of anisotropic magnetic susceptibility (see Paleomagnetism).

#### 11.1.3.3. Moisture and density measurements

MAD analyses on working halves were conducted on unconsolidated materials, ~10 cm³ samples taken with plastic syringes or spatulas for analyses. Preweighed and numbered 16 mL Wheaton glass vials were used to process and store the samples. When a lithologic variation could be observed within a single core, two MAD samples were collected. However, in most cases one sample per core for Holes A and B was collected near the sampling location for headspace analyses of hydrocarbon gases (bottom or top of a given section, see Organic geochemistry). For sedimentary and igneous rock material, the discrete cubes initially used for paleomagnetism and P-wave velocity (P-wave caliper) measurements were also used for MAD analyses whenever possible.

Mass and volume measurements were made to determine wet and dry bulk density, grain density, water content, and porosity. After measurement of wet mass, samples were dried in a convection
oven for at least 24 h at 105° ± 5°C. Dried samples were then cooled in a desiccator for at least 1 h
before the dry mass and volume were measured.

The procedure for igneous rocks included saturation with seawater for 12 h before the wet mass
weight measurement was carried out.

**Dual balance system.** The weights of wet- and dry-sample masses were determined to a precision of 0.005 g using two Mettler Toledo (XS204) electronic balances and a computer averaging system to compensate for the ship's motion (Figure F29). A standard weight, with mass comparable to the sample's (maximum = >10% of the sample mass), was placed on a reference balance to increase accuracy. The default setting of the balances is 300 measurements. After wet mass determinations and P-wave measurements (for cube samples), samples were placed in the drying oven (24 h) and then allowed to cool in the desiccator (1 h) prior to the determination of dry masses.

**Hexapycnometer volume measurement.** Dry sample volume was determined using the hexapycnometer system of a six-celled, custom-configured Micromeritics AccuPyc 1330TC helium-displacement pycnometer (Figure F30). The system measures dry sample volume using pressurized He-filled chambers; the precision of each cell is within 1% of the full-scale volume when properly calibrated and operated. Volume measurements were preceded by three purges of the sample chamber with helium. For each measurement, five unknown cells and one cell that contained two stainless steel calibration spheres (3 and 7 cm³) with a total volume of ~10 cm³ were run. Calibration spheres were cycled through the cells to identify any systematic error and/or instrument drift. Spheres are assumed known to within 1% of their total volume. The volumes occupied by the numbered Wheaton vials were calculated before the expedition by multiplying each vial's weight against the average density of the vial glass. The procedures for the determination of these physical properties comply with the American Society for Testing and Materials (ASTM International, 1990).

**Moisture and density calculations.** The calculations given below are used as an IODP standard in accordance with Blum (1997). MAD properties reported and plotted in the Physical properties section of each site chapter were calculated with the MADMax shipboard program using the method applicable to saturated, fine-grained sediments called “Method C.” Method C is based on the measurement of wet mass (M<sub>wet</sub>), dry mass (M<sub>dry</sub>), and dry volume (V<sub>dry</sub>). The ratio of mass (rm) is a computational constant of 0.965 (i.e., 0.965 g of freshwater per 1 g of seawater). Salt precipitated in pores in the samples during the drying process is included in the M<sub>dry</sub> and V<sub>dry</sub> values. The mass of the evaporated water (M<sub>water</sub>) and salt (M<sub>salt</sub>) in the sample are given by

<figure><img src="image_33.png" /><figcaption>**Figure F29.** Dual balance system for wet and dry mass measurements.</figcaption></figure>
$$M_{\text{water}} = M_{\text{wet}} - M_{\text{dry}} \text{ and} \qquad (30)$$

$$M_{\text{salt}} = M_{\text{water}} [s/(1 - s)], \qquad (31)$$

where s is the assumed saltwater salinity (0.035) corresponding to pore water density ($ρ_{pw}$) of 1.024 g/cm³ and a salt density ($ρ_{salt}$) of 2.22 g/cm³. The corrected mass of pore water ($M_{pw}$), volume of pore water ($V_{pw}$), mass of solids excluding salt ($M_{solid}$), volume of salt ($V_{salt}$), volume of solids excluding salt ($V_{solid}$), and wet volume ($V_{wet}$) are

$$M_{\text{pw}} = (M_{\text{wet}} - M_{\text{dry}})/\text{rm}, \qquad (32)$$

$$V_{\text{pw}} = M_{\text{pw}}/ρ_{\text{pw}}, \qquad (33)$$

$$M_{\text{solid}} = M_{\text{wet}} - M_{\text{pw}}, \qquad (34)$$

$$M_{\text{salt}} = M_{\text{pw}} - (M_{\text{wet}} - M_{\text{dry}}), \qquad (35)$$

$$V_{\text{salt}} = M_{\text{salt}}/ρ_{\text{salt}}, \qquad (36)$$

$$V_{\text{wet}} = V_{\text{dry}} - V_{\text{salt}} + V_{\text{pw}}, \text{ and} \qquad (37)$$

$$V_{\text{solid}} = V_{\text{wet}} - V_{\text{pw}}. \qquad (38)$$

For all samples, water content (w) is expressed as the ratio of mass of pore water to wet sample (total) mass:

$$w = M_{\text{pw}}/M_{\text{wet}}. \qquad (39)$$

Wet bulk density ($ρ_{wet}$), dry bulk density ($ρ_{dry}$), sample grain density ($ρ_{solid}$), porosity ($φ$), and void ratio ($V_R$) are calculated as

$$ρ_{\text{wet}} = M_{\text{wet}}/V_{\text{wet}}, \qquad (40)$$

$$ρ_{\text{dry}} = M_{\text{solid}}/V_{\text{wet}}, \qquad (41)$$

$$ρ_{\text{solid}} = M_{\text{solid}}/V_{\text{solid}}, \qquad (42)$$

$$φ = V_{\text{pw}}/V_{\text{wet}} \text{ and} \qquad (43)$$

$$V_R = V_{\text{pw}}/V_{\text{solid}}. \qquad (44)$$

<figure><img src="image_34.png" /><figcaption>Figure F30. Hexapycnometer system for volume measurements.</figcaption></figure>
Precision and accuracy of MAD measurements are ~0.5%, although accuracy can vary with sediment type. The largest sources of potential error are the loss of material or change in moisture during the ~30–48 h procedure for each sample.

### 11.1.3.4. Rheology

Rheology measurements provide constraints on the finite-strain deformation of material under stress. The mode of deformation (e.g., viscous, plastic, or brittle) can vary considerably in sediments. The shear strength of relatively soft sedimentary materials can be characterized by two separate complementary measurements. The pocket penetrometer (PEN) is an unconfined compression strength test, and the vane shear strength (AVS) test is an undrained shear strength test. Both measurements quantify the effective shear strength of the material by applying known compressive and shear stress, respectively. The undrained condition specific to the AVS test implies that the quantified shear strength is the effective shear strength, which corresponds to the applied stress minus the intergranular pore fluid pressure:

$$ \sigma'_{m} = \sigma_{m} - P_{f}, \qquad (45) $$

where

$$ \sigma_{m} = (\sigma_{1} - \sigma_{3})/2. \qquad (46) $$

However, in practice, we observe that both tests in laboratory conditions aboard JOIDES Resolution do not really prevent the pore fluid from draining during the experiments. This is confirmed by the fact that both measurements consistently overlap whenever the measurements are reliable (see detail below). We therefore consider that both tests actually provide a measure of the shear strength ($\sigma_m$).

Note that the unconfined condition for the PEN measurement implies that loosely compacted sediments do not provide reliable values for the first drilled 50 m of sediment. However, the depth limit of the measurement capability for the PEN test is slightly greater than the AVS. AVS test measurements are not possible whenever the compressive strength is above ~250 kPa. Depending on the site, this translates to a depth of 150–300 mbsf. PEN measurements typically extend this maximum depth an additional 50 m.

**Pocket penetrometer.** Compressive strength was measured on every core with a pocket penetrometer (Figure F31). The yield envelope of a material can be defined as the maximum principal stress difference prior to failure (or maximum differential stress). This value is the same as the unconfined compressive strength of the material, that is, $\Delta\sigma_f = (\sigma_1 - \sigma_3)$. At prescribed stress, the shear strength of a material or yield ($\tau_f$) is related to the compressive strength ($\Delta\sigma_f$) by

<figure><img src="image_35.png" /><figcaption>Figure F31. Pocket penetrometers with and without foot adaptor.</figcaption></figure>
$$ \tau_f = \Delta\sigma_f/2. \qquad (47) $$

To obtain the shear strength, values read directly from the pocket penetrometer are divided by 2. The penetrometer is a flat-footed, spring-operated device (Figure F31) used to measure compressive strength by pushing a 6.4 mm (0.25 inch) diameter probe 6.4 mm (0.25 inch) deep (to the red marker) below the split-core surface. The selected probe location must be placed where surfaces are smooth. The mechanical scale is in kilograms per square centimeter, and data uploaded to the LIMS database are in these units. Conversion from kg/cm² to kPa is as follows:

$$ 2\tau_f (\text{kPa}) = 98.1 \times 2\tau_f (\text{kg/cm}^2). \qquad (48) $$

The maximum shear strength value measurable by the pocket penetrometer is 4.5 kg/cm².

A 2.54 cm (1 inch) adapter foot was available for measuring very soft sediments, providing a 16x increase in piston area. An allen wrench was used to adjust the set screw that holds the foot to the piston. When used, the reading obtained with the added foot adapter must be multiplied by 1/16 (0.0625).

**Vane shear strength.** The shear strength of a material describes the point at which the material yields (i.e., permanently deforms) in response to an applied shear stress. In contrast to the PEN experiment, the AVS theoretically provides a direct measurement of the effective shear stress at yield or shear strength ($\sigma'_m$) of the recovered sediment. The Giesa system is suited for measuring the shear strength of very soft to relatively stiff marine sediments (Figure F32). This geotechnical test is useful for determining the undrained strength of undisturbed clay- or silt-rich samples.

The Giesa system consists of a controller and a gantry for shear vane insertion. A four-bladed miniature vane was pushed carefully into the sediment of the working halves until the top of the vane was level with the core surface. The vane was then rotated at a constant rate of 60°/min to determine the torque required to cause the core material to shear (failure torque). All vane shear strength measurements were obtained using a 12.7 mm vane. Failure torque was determined by measuring the rotation of a torsional spring using a spring-specific relation between rotation angle and torque.

<figure><img src="image_36.png" /><figcaption>**Figure F32.** The Section Half Measurement Gantry (SHMG) measures (A) shear strength (AVS) and (B) P-wave velocity on caliper and bayonet axis. Track direction indicated.</figcaption></figure>
Vane shear strength (Su[V]) can be determined as follows:

$$Su(V) = T/Kv = (\Delta/B)/Kv, \quad (49)$$

where

T = torque required to induce material failure (N·m),

Kv = constant, depending on vane dimensions (m³),

Δ = maximum torque angle (°) at failure, and

B = spring constant that relates the deflection angle to the torque (°/N·m) (Blum, 1997).

We performed one measurement per core section until the recovered material became too stiff for vane insertion. Measurements were conducted in regions considered to be the most representative of the core lithology.

### 11.1.3.5. Discrete P-wave velocity

Discrete P-wave velocity was measured on every core using Panametrics-NDT Microscan delay line transducers transmitting at 0.5 MHz (Figure F32). The signal received through the section half or discrete sample was recorded by the Velocity Gantry 2.0.5.0 IODP software, where the peak of the first P-wave arrival is automatically or manually chosen. In case of a weak signal, manual picking of the first arrival was performed.

The distance between transducers was measured with a built-in linear voltage displacement transformer (LVDT). Calibration was performed daily with a series of acrylic cylinders of differing thicknesses and a known P-wave velocity of 2750 ± 20 m/s. The system time delay determined from calibration was subtracted from the picked arrival time to give a traveltime of the P-wave through the sample. The thickness of the sample (calculated by the LVDT, in meters) was divided by the traveltime (in seconds) to calculate P-wave velocity in meters per second. A clean first P-wave arrival can be difficult to pick depending on the material. Measurement attempts were conducted once per core.

For soft sediment cores, P-wave velocity measurements were performed on the working half before any sampling. Following standard IODP conventions, P-wave velocity measurements used the x-, y-, and z-axis of the SHMG and completed one analysis per core. Different positions with respect to lithology were chosen to generate viable data. Measurements of rock cube samples were conducted after orienting the sample (Blum, 1997) and placing it on a gantry that measures P-wave velocity in three orthogonal directions (x-, y-, and z-directions). P-wave anisotropy ratios were calculated from these multidirectional analyses.

### 11.1.4. Data cleaning procedure

Data acquired using automatic systems such as the WRMSL, NGR, and SHMSL (e.g., MSP) typically contained outliers that had to be removed. Most of these outliers are due to cracks or voids in core material that formed in response to volume expansion due to the change in confining pressure for recovered cores. Void formation was most dramatic when dissolved gases were exsolved due to this pressure change, which was common. Void gas volume and composition were measured at each site during this expedition and are documented in the organic geochemistry site reports. The measured value of a given physical property for any gas is typically orders of magnitude different from that property measured for any solid or fluid-saturated sediment. Cracks and voids formed through volume expansion are, by definition, not representative of in situ conditions, but aberrant measured physical property values can easily be recognized and removed by setting an expected value range for that property consistent with gas-free core material.

All the measurements conducted on instruments with automated core or section scanning may return aberrant values at the beginning and end of each section due to the proximity of the section end (i.e., void or plastic end caps of the core liners). Thus, our data-cleaning procedure removes the first two points near the beginning and end of each core section. Figure F33 shows an example of the overall application of the cleaning procedure on the measured GRA density from the WRMSL.
### 11.1.5. Core laboratory measurement summary

The WRMSL was used to measure GRA bulk density, MS, and *P*-wave velocity at 2 cm spacing. NGR was measured on whole-round core sections with the NGRL at 10 cm spacing (Table T17). Thermal conductivity was measured every 10 m when possible. Depending on the nature of the

<figure><img src="image_37.png" /><figcaption>**Figure F33.** Illustration of the data cleaning procedure based on GRA bulk density data.</figcaption></figure>

<table><caption>**Table T17.** Expedition 385 core laboratory measurement summary. Measurement spacing specified in brackets for all core scanning instruments (i.e., WRMSL, NGR, SHMSL, MSP, X-Ray Imager). The intended resolution for the discrete measurements were in general one every 10 m for the MAD, PEN, AVS, and discrete *P*-wave. Resolution may vary in practice due to the limit of the instrument and the recovered material. For example, AVS measurements are only possible in soft sediments. Whenever the dedicated hole to microbiology was in close distance to the Hole A we often lowered the resolution of the discrete measurements to one every 20 m, unless the first measurements on Hole A were unsatisfactory in terms of resolution. — = no samples analyzed. **Download table in CSV format.**</caption><thead><tr><th rowspan="2">Core lab<br>measurement</th><th rowspan="2"></th><th rowspan="2">Site:</th><th colspan="3">U1545</th><th colspan="3">U1546</th><th colspan="3">U1547</th><th colspan="3">U1548</th><th colspan="3">U1549</th><th colspan="3">U1550</th><th colspan="3">U1551</th><th colspan="3">U1552</th></tr><tr><th>Hole:</th><th>A</th><th>B</th><th>C</th><th>A</th><th>B</th><th>C</th><th>D</th><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th></tr></thead><tbody><tr><td rowspan="5">Whole round</td><td>WRMSL<br>(2 cm)</td><td>GRA</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td></tr><tr><td>MS</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td></tr><tr><td><i>P</i>-wave</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td></tr><tr><td>NGR (10 cm)</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td></tr><tr><td>TK04 (Every 10 m)</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>—</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td></tr><tr><td rowspan="7">Section half</td><td>SHMSL (2.5 cm)</td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td><td></td></tr><tr><td>MSP (2.5 cm sediments; 1<br>cm igneous sections)</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td></tr><tr><td>MAD (10 m)</td><td>V</td><td>V</td><td>—</td><td>V</td><td>V</td><td>V</td><td>—</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td></tr><tr><td>PEN (10 m)</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>—</td><td>V</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td></tr><tr><td>AVS (10 m)</td><td>V</td><td>V</td><td>—</td><td>V</td><td>V</td><td>—</td><td>V</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td></tr><tr><td>Discrete <i>P</i>-wave (10 m)</td><td>V</td><td>V</td><td>—</td><td>V</td><td>V</td><td>—</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td></tr><tr><td>X-Ray imaging</td><td>V</td><td>—</td><td>—</td><td>V</td><td>—</td><td>V</td><td>—</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td></tr></tbody></table>
recovered material, we measured thermal conductivity on whole-round sections for soft sediment and on working-half sections for indurated sediment and igneous rock. MSP measurements were made every 2.5 cm for sediment sections and 1 cm for igneous sections.

MAD measurements were conducted on samples collected every 20 m on standard holes (usually Hole A) and at every 10 m in coordination with headspace samples for hydrocarbon gas analysis (usually Hole B) (see **Organic geochemistry**). These MAD measurements were used to calculate wet bulk density, dry bulk density, sediment grain density, porosity, and void ratio. Rheological measurements (i.e., shear and compressive strength) were measured at discrete offsets on section halves to characterize the average rheological properties of the material. These measurements were only possible on soft to moderately indurated sediments at an intended spacing of 10 m. Discrete P-wave velocity was measured with the SHMG using the caliper for measurements through the split core and bayonets inserted into the sediment for measurements along and across the core. Rock cube samples collected on igneous material, in collaboration with the Paleomagnetism group, were measured through a specific discrete measurement procedure in three orthogonal directions. These cubes went through a special MAD procedure (see **Moisture and density measurements**) before being passed to the Paleomagnetism group for further analyses (Table T17).

## 11.2. Downhole measurements

A range of in situ physical properties and state measurements, collectively referred to as downhole measurements, were made using tools either attached to the BHA during drilling or lowered into the borehole on a conducting wireline after drilling. In situ downhole temperature measurements were acquired using the APCT-3 (Figure F34) and SET2 tools (Figure F35). These temperature measurements were conducted at all sites (see **Temperature measurements**; Table T18).

Downhole wireline logging data were collected at Sites U1545–U1547. Two Schlumberger logging tool strings were used during Expedition 385: the triple combo tool string and the FMS-sonic tool string (Figure F36). Individual logging runs with each of these tool strings were done in succession after completion of coring operations. These downhole logging instruments collect continuous measurements of in situ physical, chemical, and structural properties along the length of the penetrated formation. These continuous data provide physical properties information over depth intervals where core recovery is potentially incomplete or disturbed, and they are particularly useful for stratigraphic and lithologic correlation between sites regionally. Downhole logging measurements can be directly tied to seismic profiles, which commonly have regional extent, by the creation of synthetic seismograms based on the logging data and, where appropriate, by the time-to-depth conversion of seismic profiles. The quality of logging data compared to core laboratory measurements depends on the physical property considered and other factors, including the spatial scale of interest. Downhole logging tools measure most formation properties averaged over spatial scales on the order of a meter, whereas laboratory measurements typically have a scale length of centimeters and geophysical surveys are sensitive to properties at the scale of tens of meters.

### 11.2.1. Temperature measurements

In situ temperature measurements were made using both the APCT-3 tool and the SET2 tool, with the latter being designed to measure and record formation temperature in semiconsolidated sediments that are too stiff for the APCT-3 tool to be deployed. Temperature measurements are useful

<figure><img src="image_38.png" /><figcaption>**Figure F34.** APCT-3 tool shoe (left) has a pocket to accept electronics, memory board, and battery (right) for temperature measurements while taking an APC core.</figcaption></figure>
for many reasons, but they were particularly useful for Expedition 385 in helping to determine
sampling plans for microbiological studies aimed at understanding the temperature dependence
of microbial populations and processes.

**11.2.1.1. Advanced piston corer temperature tool**

In situ temperature measurements were made with the APCT-3 tool (Figure F34) when the APC system was deployed. The APCT-3 thermistor tool fits directly into the coring shoe of the APC coring system and can therefore be used to measure formation temperatures during regular piston coring. The tool consists of a battery pack, a data logger, and a platinum thermistor probe calibrated over a temperature range from -5° to 55°C with an accuracy of ±0.02°C. Before entering the borehole, the tool is first held at the mudline for 5 min to thermally equilibrate and to estimate the bottom water temperature. However, the lowest temperature recorded during the run was occasionally used as an estimate of the bottom water temperature instead of the average temperature at the mudline because (1) it was more repeatable and (2) the bottom water is expected to have the lowest temperature in the profile. When the APCT-3 tool is fired into the formation, there is an instantaneous temperature rise from frictional heating. This heat gradually dissipates into the surrounding sediment as the temperature within the APCT-3 tool and adjacent sediment equilibrate to the temperature of the surrounding sediment volume. After the APCT-3 tool penetrates the sediment, it is held in place for 10 min while it records the temperature of the cutting shoe every second.

**11.2.1.2. Sediment temperature 2 tool**

Formation temperatures are measured using a thermistor in the SET2 tool (Figure F35) that
records actual in situ temperature and is located ~1 cm from the probe tip. The thermistor is cali-

<figure><img src="image_39.png" /><figcaption>**Figure F35.** SET2 tool schematic.</figcaption></figure>
brated for a temperature range of -2° to 50°C with an accuracy of ±0.002°C. Once the tool is retrieved, the data are downloaded and processed on the ship. The probe tip contains both the data logger and the thermistor string and is designed to enter the sediment with minimal disturbance. Features of the probe tip design include the following:

* High-strength stainless steel construction survives penetration into highly consolidated sediment and contact with igneous rock.

* The 8 mm spherical probe tip tapers upward at an included angle of 5° to minimize mechanical disturbance of the soil and the tendency to crack the formation upon penetration by the tool.

The SET2 tool is deployed by the motion-decoupled hydraulic delivery system (MDHDS). The SET2 tool may also be deployed by wireline using the collected delivery system (CDS) (see SET2 manual for information on the CDS tool, available at https://iodp.tamu.edu/tools/logging/index.html).

We used two devices each of the APCT-3 and SET2 tools. One of the devices of each tool setup was modified by technicians to operate at up to 90°C (upper limit of the electronics inside each device), and the counterpart was used at the standard calibrated temperature range of up to 55°C (APCT-3) and 50°C (SET2). High-temperature calibration for the APCT-3 and SET2 tools was achieved by using hot and cold baths in the laboratory to generate a known range of temperatures measured by thermometer while recording changing resistance values in the APCT-3 and SET2 tools with varying bath water temperatures and fitting a linear temperature regression line to those resistance values. Using this approach, it was possible to convert resistance measured by the tool into temperature value with an accuracy of ±2°–3°C for temperatures above the standard calibration range. A total of 93 in situ temperature measurements were conducted during the expedition using all four units of equipment of the two tool setups (Table T19). For holes in unusually warm sediments, the tool temperature may continue to rise after penetration. It is not possible to

<table><caption>**Table T18.** Expedition 385 downhole measurement summary. Overview of sites and holes where downhole measurements were conducted. * = instrument was present on both logging tool strings deployed. V = holes where the indicated test was performed. † = instrument removed in Holes U1546C and U1547B to enable a greater depth coverage for the other logging tools. ** = tests were also performed for these holes. Holes U1545A, U1546C, and U1547B were logged. NGR = natural gamma radiation, MS = magnetic susceptibility. Tools are defined in Tables T20 and T21. Download table in CSV format.</caption><thead><tr><th rowspan="2">Downhole measurement</th><th rowspan="2">Physical property</th><th rowspan="2">IODP site Tool/Hole</th><th colspan="3">U1545</th><th colspan="3">U1546</th><th colspan="3">U1547</th></tr><tr><th>A</th><th>B</th><th>C</th><th>A</th><th>B</th><th>C</th><th>D</th><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th></tr></thead><tbody><tr><td rowspan="2">Temperature measurements</td><td rowspan="2">Temperature</td><td>APCT-3 tool</td><td>V</td><td>V</td><td>—</td><td>V</td><td>—</td><td>—</td><td>V</td><td>V</td><td>—</td><td>—</td><td>—</td></tr><tr><td>SET2 tool</td><td>V</td><td>V</td><td>—</td><td>V</td><td>—</td><td>—</td><td>V</td><td>V</td><td>—</td><td>—</td><td>—</td></tr><tr><td rowspan="10">Formation wireline logging</td><td rowspan="2">Stress field</td><td>Caliper</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td></tr><tr><td>HNGS</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td></tr><tr><td>NGR*</td><td>HLDS</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Density*</td><td>HRLA</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Resistivity</td><td>DSI</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Acoustic velocity</td><td>FMS</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Formation image</td><td>MSS</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td></tr><tr><td>MS<sup>†</sup></td><td>GPIT</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td></tr><tr><td>FMS-sonic tool</td><td>Geochemistry</td><td>Kuster FTS</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Borehole fluid sampling</td><td>Geochemistry</td><td>Kuster FTS</td><td>V</td><td>—</td><td>—</td><td>—</td><td>V</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td></tr></tbody></table>

<table><thead><tr><th rowspan="2">Downhole measurement</th><th rowspan="2">Physical property</th><th rowspan="2">IODP Site Tool/Hole</th><th colspan="5">U1548</th><th colspan="3">U1549</th><th colspan="3">U1550</th><th colspan="3">U1551</th><th colspan="3">U1552</th></tr><tr><th>A</th><th>B</th><th>C</th><th>D</th><th>E</th><th>A</th><th>B</th><th>A</th><th>B</th><th>A</th><th>B</th><th>A</th><th>B</th><th>A</th><th>B</th><th>C</th></tr></thead><tbody><tr><td rowspan="2">Temperature measurements</td><td rowspan="2">Temperature</td><td>APCT-3 tool</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>V</td><td>—</td><td>V</td><td>—</td><td>V</td><td>—</td><td>V</td><td>—</td><td>V</td><td>—</td><td>V</td></tr><tr><td>SET2 tool</td><td>—**</td><td>V</td><td>V</td><td>—**</td><td>—**</td><td>—**</td><td>—**</td><td>—</td><td>V</td><td>—</td><td>—**</td><td>—</td><td>—**</td><td>—</td><td>—</td><td>—</td></tr><tr><td rowspan="10">Formation wireline logging</td><td rowspan="2">Stress field</td><td>Caliper</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>HNGS</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>NGR*</td><td>HLDS</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Density*</td><td>HRLA</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Resistivity</td><td>DSI</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Acoustic velocity</td><td>FMS</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Formation image</td><td>MSS</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>MS<sup>†</sup></td><td>GPIT</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>FMS-sonic tool</td><td>Geochemistry</td><td>Kuster FTS</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr><tr><td>Borehole fluid sampling</td><td>Geochemistry</td><td>Kuster FTS</td><td>—</td><td>V</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td><td>—</td></tr></tbody></table>
leave the tool in the seafloor long enough to achieve complete equilibration. This would require 40–60 min or more of stationary time. Instead, partial equilibration is achieved under such conditions. Processing of APCT-3 and SET2 tool data to infer the in situ formation temperature requires extrapolation of a short record of thermal equilibration.

### 11.2.1.3. Elevated Temperature Borehole Sensor

The ETBS is a slim-hole probe run on the coring line (Figure F37) that was modified and upgraded from the Ultra High Temperature Multisensor Memory Tool (UHT-MSM) prior to IODP Expedition 376 which successfully tested this new tool (de Ronde et al., 2019). The UHT-MSM was used during ODP Legs 169 and 193 (Shipboard Scientific Party, 1998, 2002a). The tool was originally developed for the University of Miami (USA) (Geophysical Research Corporation, 1994a, 1994b, 1996). The ETBS contains internal and ultra-high external temperature measuring devices, a pres-

<figure><img src="image_40.png" /></figure>

**Figure F36.** Wireline logging tool strings used during Expedition 385. LEH-MT = logging equipment head-mud temperature, EDTC = Enhanced Digital Telemetry Cartridge, HNGS = Hostile Environment Natural Gamma Ray Sonde, APS = Accelerator Porosity Sonde, HLDS = Hostile Environment Litho-Density Sonde, HRLA = High-Resolution Laterolog Array, MSS = Magnetic Susceptibility Sonde, DSI = Dipole Sonic Imager, GPIT = General Purpose Inclinometry Tool. Triple combo tool string takes downhole measurements of hole diameter (caliper), natural gamma radiation (HNGS), bulk density (HLDS), electrical resistivity (HRLA), and magnetic susceptibility (MSS). FMS-sonic tool string takes borehole resistivity images and measures natural gamma radiation and P- and S-wave velocities. See Table T18.
sure gauge, a multisensor memory unit, and a Dewar flask that acts as an insulator to maintain a stable temperature and cool-down rate for the tool (Figure F37). The heat shield is aircraft-grade aluminum bound at both ends by brass heat sinks. The Dewar flask can maintain an internal temperature suitable for tool operation for 4–5 h at an external temperature of 400°C. Operations are possible for as long as 10 h if the average temperature does not exceed 232°C.

The ETBS tool was connected to the core line via a new adapter designed on board to be compatible with both the ETBS and the Kuster FTS. This attachment enables deployment through the throat of the RCB coring bit. Such an approach minimizes the time that the drill string spends without circulation of seawater in the hole, which is when risk of borehole collapse is highest. The ETBS was fitted with Chemraz seals and Kalrez O-rings.

### 11.2.1.4. Thermal data reduction

The equilibrium temperature of the sediment is estimated by applying a mathematical heat-conduction model to the temperature-decay record (Horai and Von Herzen, 1985).

The theoretical thermal-decay curve for the APCT-3 tool is a function of the geometry and thermal properties of the probe and the sediment (Bullard, 1954; Horai and Von Herzen, 1985). The characteristic thermal time constant of the cutting shoe assembly into which the APCT-3 tool is inserted is ~2–3 min. Where the APC does not achieve a full stroke or where ship heave pulls the APC up from full penetration, the temperature equilibration curve is disturbed and temperature determination is less accurate. The nominal accuracy of APCT-3 temperature measurements is ±0.002°C.

Data reduction for the APCT-3 tool was performed using the program TP-Fit (Heeseman et al., 2006). Generally, it is not possible to obtain a perfect match between the analytical curves and the data because (1) the probe does not reach thermal equilibrium during the penetration period; (2) contrary to ideal theory, the frictional pulse upon insertion is not instantaneous; and (3) temperature data are sampled at discrete intervals, so the exact time of penetration is uncertain. Therefore, both the effective penetration time and equilibrium temperature must be estimated by applying a fitting procedure, which involves shifting the synthetic curves in time to obtain a match with the recorded data. Heeseman et al. (2006) suggests that the best fit is obtained from the final third of the data set. A single, laboratory-measured thermal conductivity was used for calculating heat flow and also input into the TP-Fit calculation for estimating in situ temperatures. Additional

<table><caption>**Table T19.** Summary of temperature measurement runs, reporting core and associated depth for each run during Expedition 385. Runs with high-temperature modified devices in bold. * = All SET2 tools were deployed into the top of the core they were attached to except for Core U1545A-74X, as that run was put down into the bottom of the hole below that core, after coring was finished in Hole U1545A. Download table in CSV format.</caption><thead><tr><th>Hole</th><th>APCT-3: Core (depth; mbsf)</th><th>Number of APCT-3 measurements</th><th>SET2: Core (depth; mbsf)*</th><th>Number of SET2 measurements</th></tr></thead><tbody><tr><td>U1545A</td><td>4H (33.0), 7H (61.5), 10H (90), 13H (118.5), 18F (143.2), 24F (172.9), 30F (193.5), 36F (231.2), <strong>47F (277.3)</strong></td><td>9</td><td><strong>62X (391.5), 67X (441.2), 71X (480.1), 74X (498.6)</strong></td><td>4</td></tr><tr><td>U1545B</td><td>4H (32.3), 7H (60.8), 10H (89.3), 13H (117.8), 16H (138.1), 20F (161.4), 27F (189.4), 33F (217.6), <strong>39F (245.8), 49F (287.9)</strong></td><td>10</td><td><strong>28F (190.4), 34F (218.6), 40F (246.8), 46F (275.0), 50F (288.9), 61X (329.5), 64X (358.6)</strong></td><td>7</td></tr><tr><td>U1546A</td><td>4H (32.3), 7H (60.8), 10H (89.3), 13H (117.8), 16H (146.3), 19H (174.8), 22F (198.5), 28F (218.4)</td><td>8</td><td><strong>36F (239.9), 43F (259.4), 52F (288.7), 59X (323.0)</strong></td><td>4</td></tr><tr><td>U1547A</td><td>4H (33.8), 6H (52.8), 8H (71.8), 10H (90.8), 10H (90.8)</td><td>4</td><td><strong>17F (101.9), 24F (124.2)</strong></td><td>2</td></tr><tr><td>U1547B</td><td>4H (34.7), 6H (53.7), 8H (72.7), <strong>10H (91.7), 12F (96.4)</strong></td><td>5</td><td><strong>21F (115.6), 27F (140.5)</strong></td><td>2</td></tr><tr><td>U1548A</td><td>4H (30.4), 6H (49.4), <strong>8H (68.4), 10H (87.4)</strong></td><td>4</td><td></td><td>—</td></tr><tr><td>U1548B</td><td>4H (34.4), 6H (53.4)</td><td>2</td><td><strong>8H (63.9), 9H (73.4)</strong></td><td>2</td></tr><tr><td>U1548C</td><td>4H (36.1)</td><td>1</td><td><strong>6H (46.6), 7H (56.1), 8H (65.6)</strong></td><td>3</td></tr><tr><td>U1548D</td><td>4H (34.8), 8H (72.8), 12H (105.8)</td><td>3</td><td></td><td>—</td></tr><tr><td>U1548E</td><td>4H (34.2), 7H (62.7), 10H (91.2)</td><td>3</td><td></td><td>—</td></tr><tr><td>U1549A</td><td>4H (35.0), 7H (63.5), 10H (92), 13H (120.5)</td><td>4</td><td></td><td>—</td></tr><tr><td>U1550A</td><td>4H (34.3), 6H (53.3), 8H (72.3), 12H (110.4)</td><td>4</td><td>16F (125.4)</td><td>1</td></tr><tr><td>U1551A</td><td>4H (31.1), 7H (45.0), 10H (68.7), 13H (99.3)</td><td>4</td><td></td><td>—</td></tr><tr><td>U1552A</td><td>4H (31.5), 7H (60.0), 10H (88.5), 12H (107.5)</td><td>4</td><td></td><td>—</td></tr><tr><td>U1552C</td><td>4H (32.8), 7H (61.3), 10H (89.8)</td><td>3</td><td></td><td>—</td></tr></tbody><tfoot><tr><td>Total</td><td></td><td>68</td><td></td><td>25</td></tr></tfoot></table>
laboratory measurements of core-sample thermal conductivity were not used to account for in situ conditions when fitting the APCT-3 data because the correction relative to a single, constant thermal conductivity would be small at the shallow depths over which the APCT-3 was used, owing to the high thermal gradient.

If heat transfer is by conduction and heat flow is constant, the thermal gradient will be inversely proportional to thermal conductivity, according to Fourier's law. This relationship can be linearized by plotting temperature as a function of cumulative thermal resistance (Bullard and Schonland, 1939):

$$T(z) = T_0 + q \sum_{i=0}^{N} \left( \frac{\Delta z_i}{k(z)_i} \right), \quad (50)$$

where

$T$ = temperature,

$z$ = depth,

$T_0$ = temperature at the seafloor,

$q$ = heat flow,

$\Sigma$ = thermal resistance,

$k$ = thermal conductivity, and

$N$ = number of thermal conductivity measurements.

In practice, $q$ and $T_0$ are estimated by plotting $T(z)$ against cumulative thermal resistance (Bullard plot) from the slope and intercept with $z = 0$, respectively. The conditions for the linearity of the

<figure><img src="image_41.png" /><figcaption>**Figure F37.** Elevated Temperature Borehole Sensor (ETBS) is a slim-hole probe run on the coring line and contains internal and ultrahigh external temperature measuring devices, a pressure gauge, a multisensor memory unit, and a Dewar flask that acts as an insulator to maintain a stable temperature and cooldown rate for the tool.</figcaption></figure>
plot are affected by conductive conditions, steady state, and absence of internal heat source. For
Expedition 385, three of these conditions may not be met: advective heat transport could be im-
portant and steady state is a poor assumption because of high sedimentation rate and the possibil-
ity of still-cooling sills, which would also be an internal heat source. Geothermal gradients were
quite linear (indicative of steady-state conditions) at all sites except Ringvent (Site U1547 and
Holes U1548A–U1548C).

11.2.2. Wireline logging

Downhole wireline logging was conducted using two Schlumberger tool strings: the triple combo tool string, which measures borehole (fluid) temperature, NGR, density, electrical resistivity, and MS (Figure F36), and the FMS-sonic tool string, which measures NGR and sonic velocity and captures FMS resistivity images of the borehole wall (Figure F36). Both tool strings also contain a telemetry cartridge for communicating through the wireline to the Schlumberger data acquisition system (multi-tasking acquisition and imaging system [MAXIS]) on the ship.

In preparation for logging, the boreholes were flushed of debris by circulating high-viscosity sepi-
olite mud. The BHA was pulled up to about 80 m WSF to cover the unstable upper part of the hole.
The tool strings were then lowered downhole on a seven-conductor wireline cable before being
pulled up at a constant speed of ~275 m/h to provide continuous measurements of several proper-
ties simultaneously. A wireline heave compensator (WHC) was used to minimize the effect of the
ship's heave on the tool position in the borehole, except when ship heave was minimal. During
each logging run, incoming data were recorded and monitored in real time on the Schlumberger
Minimum Configuration MAXIS (MCM) logging computer.

11.2.2.1. Log data quality

The main factor affecting logging data quality is the condition of the borehole wall. Where the borehole diameter varies over short intervals because of washouts (wide borehole) or ledges made of layers of harder material, the logs from tools that require good contact with the borehole wall (i.e., the FMS and density tools) may be degraded. Deep investigation measurements such as NGR, resistivity, and sonic velocity, which do not require contact with the borehole wall, are generally less sensitive to borehole conditions. Very narrow (“bridged”) sections also cause irregular log results. The quality of the borehole is improved by minimizing the circulation of seawater (drilling fluid) while drilling, flushing the borehole to remove debris prior to logging, and logging soon after drilling and hole conditioning are completed. During the expedition, circulation of drilling fluid, necessary to cool the bit and clear the hole of cuttings, washed out (widened) the corresponding borehole in places, particularly at locations with large contrasts in formation consolidation.

The quality of the wireline depth determination depends on several factors. The depth of the logging measurements is determined from the length of the cable payed out from the winch on the ship. The seafloor is identified on the NGR log by the abrupt reduction in gamma ray count at the water/seabed boundary (mudline). Discrepancies between the drilling depth and the wireline log depth occur. In the case of drilling depth, discrepancies are caused by core expansion, incomplete core recovery, or incomplete heave compensation. In the case of log depth, discrepancies between successive runs occur because of incomplete heave compensation, incomplete correction for cable stretch, and cable slip. Tidal changes in sea level affect both drilling and logging depths.

11.2.2.2. Wireline heave compensator

The WHC system is designed to compensate for the vertical motion of the ship and maintain a steady motion for the logging tools. The system uses vertical acceleration measurements made by a motion reference unit (MRU) located under the rig floor near the center of gravity of the ship to calculate the vertical motion of the ship. It then adjusts the length of the wireline by varying the distance between two sets of pulleys through which the cable passes. Real-time measurements of uphole (surface) and downhole acceleration are made simultaneously by the MRU and the Enhanced Digital Telemetry Cartridge (EDTC), respectively.

11.2.2.3. Logging data flow and log depth scales

Data for each wireline logging run were monitored in real time and recorded using the Schlumberger MAXIS 500 system. The initial logging data were referenced to the rig floor (WRF scale).
After logging was completed, the data were shifted to a seafloor reference (WSF scale) based on the step in gamma radiation at the water/seabed interface. The data were transferred to the Lamont-Doherty Earth Observatory Borehole Research Group (LDEO-BRG; Columbia University, USA), where standardized data reduction took place. The main correction was depth matching to remove depth offsets between data from different logging runs, producing a new depth scale (WMSF). The second uplog became the wireline depth reference, and its WSF scale is the same as its WMSF scale. The logs are documented with an assessment of log quality, and the data were converted to ASCII. Schlumberger Geo-Quest's Techlog software package is used for most of the wireline log data processing. Data were transferred back to the ship within a few days of logging completion, and this processed data set was made available to the science party in ASCII format.

### 11.2.3. Logged formation properties and tool measurement principles

The logged properties and the principles used to measure them are briefly described in this section. The logging tools are listed in Tables T20 and T21. More detailed information on individual tools and their geological applications may be found in Rider (1996), Goldberg (1997), and Ellis and Singer (2007). A complete online list of acronyms for Schlumberger tools and measurement curves is available at the Schlumberger Curve Mnemonic Dictionary (http://www.apps.slb.com/cmd/, accessed 30 September 2020).

#### 11.2.3.1. Borehole diameter (caliper)

The borehole diameter measured by the caliper provides an indication of formation strength and its response to the artificial erosion associated with drilling operations (Figure F36). Additionally, caliper measurements can provide information on the present-day stress field inferred from the ellipticity of borehole deformation induced by downhole operations.

The method for estimating stress-field orientation uses the orthogonal borehole diameter measured by the calipers of the FMS-sonic tool string (called C1 and C2, respectively) and the caliper orientation provided by the general purpose inclination tool (GPIT) (see below). Displaying this information along depth can provide evidence of breakouts or borehole ellipticity that can be interpreted in terms of maximum and minimum plane stresses. A breakout signal typically shows an increase in diameter in one of the orthogonal calipers, whereas the other caliper remains close to the bit size. Theoretically, the greater diameter highlights the minimum plane stress.

#### 11.2.3.2. Natural gamma radiation

The Hostile Environment Natural Gamma Ray Sonde (HNGS) was used on both the triple combo and FMS-sonic tool strings to measure and classify NGR in the formation (Figure F36). Each HNGS instrument has two bismuth germanate scintillation detectors that measure concentrations of K (in weight percent) as well as U and Th (both in parts per million) that are determined from the characteristic gamma ray energy spectra of isotopes in the ⁴⁰K, ²³²Th, and ²³⁸U radioactive decay series. An additional NGR sensor, housed in the EDTC, is run on both tool strings. Its sodium iodide scintillation detector measures the total NGR emission of the formation with no spectral information. The inclusion of an NGR sonde in every tool string improves core-log integration by enabling the use of NGR data for depth correlation between logging strings and

<table><caption>**Table T20.** Logging instrument assemblages of the Triple Combo and FMS-sonic tool strings for Expedition 385. **Download table in CSV format.**</caption><thead><tr><th>Tool string</th><th>Tool</th><th>Measurement</th><th>Sampling interval (cm)</th><th>Approximate vertical resolution (cm)</th></tr></thead><tbody><tr><td rowspan="6">Triple combo</td><td>EDTC</td><td>Total gamma ray</td><td>5 and 15</td><td>30</td></tr><tr><td>HNGS</td><td>Spectral gamma ray</td><td>15</td><td>20–30</td></tr><tr><td>APS</td><td>Porosity</td><td>15</td><td>38</td></tr><tr><td>HLDS and caliper</td><td>Bulk density</td><td>2.5 and 15</td><td>38</td></tr><tr><td>HRLA</td><td>Resistivity</td><td>15</td><td>30</td></tr><tr><td>MSS</td><td>Magnetic susceptibility</td><td>4</td><td>40</td></tr><tr><td rowspan="4">FMS-sonic</td><td>EDTC</td><td>Total gamma ray</td><td>5 and 15</td><td>30</td></tr><tr><td>GPIT</td><td>Tool orientation and acceleration</td><td>3.8</td><td>15</td></tr><tr><td>DSI</td><td>Acoustic velocity</td><td>15</td><td>107</td></tr><tr><td>FMS</td><td>Microresistivity and caliper</td><td>0.25</td><td>1</td></tr></tbody></table>
logging-run passes. This in turn enables more confident direct comparison of downhole and core laboratory physical properties measurements.

### 11.2.3.3. Density and photoelectric factor

Formation density was measured with the Hostile Environment Litho-Density Sonde (HLDS) (Figure F36). The sonde contains a radioactive cesium (¹³⁷Cs) gamma ray source (622 keV) and far and near gamma ray detectors mounted on a shielded skid that is pressed against the borehole wall by a hydraulically activated decentralizing arm. Gamma radiation emitted by the source undergoes Compton scattering, in which gamma rays are scattered by electrons in the formation. The number of scattered gamma rays that reach the detectors is proportional to the density of electrons in the formation, which is in turn related to bulk density. Porosity may also be derived from this bulk density if the matrix (grain) density is known.

The HLDS also measures the photoelectric effect factor (PEF), which is a measure of the photoelectric absorption of low-energy gamma radiation. Photoelectric absorption occurs when the energy of the gamma rays falls below 150 keV as a result of being repeatedly scattered by electrons in the formation. PEF is determined by comparing the counts from the far detector in the high-energy region, where only Compton scattering occurs, to those in the low-energy region, where count rates depend on both reactions. Because PEF depends on the atomic number of the ele-

<table><caption>**Table T21.** Acronyms and units used for downhole wireline tools and measurements, Expeditions 385. For the complete list of acronyms used in IODP and for additional information about tool physics, consult https://iodp.tamu.edu/tools/logging/index.html. Download table in CSV format.</caption><thead><tr><th>Tool</th><th>Output</th><th>Description</th><th>Unit</th></tr></thead><tbody><tr><td>ETBS</td><td></td><td>Elevated Temperature Borehole Sensor tool</td><td>Degree (°) C</td></tr><tr><td></td><td>Text, Tint</td><td>Temperature (external borehole and internal tool)</td><td>Degree (°) C</td></tr><tr><td></td><td>P</td><td>Pressure</td><td></td></tr><tr><td>EDTC</td><td></td><td>Enhanced Digital Telemetry Cartridge</td><td></td></tr><tr><td></td><td>GR</td><td>Total gamma ray</td><td>gAPI</td></tr><tr><td></td><td>ECGR</td><td>Environmentally-corrected gamma ray</td><td>gAPI</td></tr><tr><td></td><td>EHGR</td><td>High-resolution environmentally corrected gamma ray</td><td>gAPI</td></tr><tr><td>HNGS</td><td></td><td>Hostile Environment Gamma Ray Sonde</td><td></td></tr><tr><td></td><td>HSGR</td><td>Standard (total) gamma ray</td><td>gAPI</td></tr><tr><td></td><td>HCGR</td><td>Computed gamma ray (HSGR minus uranium contribution)</td><td>gAPI</td></tr><tr><td></td><td>HFK</td><td>Potassium</td><td>wt%</td></tr><tr><td></td><td>HTHO</td><td>Thorium</td><td>ppm</td></tr><tr><td></td><td>HURA</td><td>Uranium</td><td>ppm</td></tr><tr><td>APS</td><td></td><td>Accelerator Porosity Sonde</td><td></td></tr><tr><td></td><td>APLC</td><td>Near/array limestone corrected porosity</td><td>Dec. fraction</td></tr><tr><td></td><td>STOF</td><td>Computed standoff</td><td>Inch</td></tr><tr><td></td><td>SIGF</td><td>Formation capture cross section</td><td>Capture unit</td></tr><tr><td>HLDS</td><td></td><td>Hostile Environment Litho-Density Sonde</td><td></td></tr><tr><td></td><td>ROHM</td><td>Bulk density</td><td>g/cm³</td></tr><tr><td></td><td>PEFL</td><td>Photoelectric effect</td><td>barn/e⁻</td></tr><tr><td></td><td>LCAL</td><td>Caliper (measure of borehole diameter)</td><td>Inches</td></tr><tr><td></td><td>DRH</td><td>Bulk density correction</td><td>g/cm³</td></tr><tr><td>HRLA</td><td></td><td>High Resolution Laterolog Array Tool</td><td></td></tr><tr><td></td><td>RLA1-5</td><td>Apparent Resistivity from Computed Focusing Mode 1-5</td><td>Ωm</td></tr><tr><td></td><td>RT</td><td>True resistivity</td><td>Ωm</td></tr><tr><td></td><td>MRES</td><td>Borehole fluid resistivity</td><td>Ωm</td></tr><tr><td>MSS</td><td></td><td>Magnetic Susceptibility Sonde</td><td></td></tr><tr><td></td><td>LSUS</td><td>Magnetic susceptibility, deep reading</td><td>Uncalibrated unit</td></tr><tr><td>FMS</td><td></td><td>Formation MicroScanner</td><td></td></tr><tr><td></td><td>C1, C2</td><td>Orthogonal hole diameter</td><td>Inch</td></tr><tr><td></td><td>P1AZ</td><td>Pad 1 azimuth - Spatially oriented resistivity images of borehole wall</td><td>Degree (°)</td></tr><tr><td>GPIT</td><td></td><td>General Purpose Inclinometer Tool</td><td></td></tr><tr><td></td><td>DEVI</td><td>Hole deviation</td><td>Degree (°)</td></tr><tr><td></td><td>HAZI</td><td>Hole azimuth</td><td>Degree (°)</td></tr><tr><td></td><td>F<sub>x</sub>, F<sub>y</sub>, F<sub>z</sub></td><td>Earth's magnetic field (three orthogonal components)</td><td>Degree (°)</td></tr><tr><td></td><td>A<sub>x</sub>, A<sub>y</sub>, A<sub>z</sub></td><td>Acceleration (three orthogonal components)</td><td>m/s²</td></tr><tr><td>DSI</td><td></td><td>Dipole Sonic Imager</td><td></td></tr><tr><td></td><td>DTCO</td><td>Compressional wave slowness</td><td>μ/ft</td></tr><tr><td></td><td>DTSM</td><td>Shear wave slowness</td><td>μ/ft</td></tr><tr><td></td><td>DT1</td><td>Shear wave slowness, lower dipole</td><td>μ/ft</td></tr><tr><td></td><td>DT2</td><td>Shear wave slowness, upper dipole</td><td>μ/ft</td></tr></tbody></table>
ments in the formation (heavier elements have a higher PEF), it also varies according to the chemical composition of the minerals present and can be used for the identification of the overall mineral makeup of the formation. For example, the PEF of calcite is 5.08 barn/e⁻, illite is 3.03 barn/e⁻, quartz is 1.81 barn/e⁻, and hematite is 21 barn/e⁻. Good contact between the tool and the borehole wall is essential for good HLDS logs; poor contact results in underestimation of density values. Both the density correction and caliper measurement of the hole are used to check the contact quality. In the deeper parts of the hole, the PEF log should be used with caution, especially in washouts, because barium in the logging mud swamps the signal despite correction for mud effect.

### 11.2.3.4. Electrical resistivity

The High-Resolution Laterolog Array (HRLA) provides six resistivity measurements with different depths of investigation, including borehole mud resistivity and five measurements of formation resistivity with increasing penetration into the formation: 0.254, 0.508, 0.762, 1.524, and 2.286 m (Figure F36). The tool sends a focused current into the formation and measures the intensity necessary to maintain a constant drop in voltage across a fixed interval, providing direct resistivity measurements. Two curves are calculated from these values: true resistivity (RT) of the hole, which is always the closest value to the deepest measurements, and resistivity of the borehole fluids (RM). Because it is least affected by the borehole fluids infiltrating the formation, the deepest depth of investigation, RLT5, is consistently used to describe variations of resistivity with depth.

The tool has one central (source) electrode and six electrodes above and below it that serve alternately as focusing and returning current electrodes. By rapidly changing the role of these electrodes, simultaneous resistivity measurement at six penetration depths is achieved. The tool is designed to ensure that all signals are measured at the same time and tool position and to reduce the sensitivity to “shoulder bed” effects when crossing beds that are thinner than the electrode spacing. HRLA needs to be run centralized in the borehole for optimal results, so bowspring centralizers were used to keep the HRLA in the center of the borehole and knuckle joints allowed the density tool to be centralized and maintain good contact with the borehole wall (Figure F36). Calcite, silica, and hydrocarbons are electrical insulators, whereas ionic solutions like IW are conductors. Electrical resistivity can therefore be used to evaluate porosity for a given salinity and resistivity of IW. Clay surface conduction also impacts formation resistivity, but at high porosities this effect is relatively minor.

### 11.2.3.5. Magnetic susceptibility

The Magnetic Susceptibility Sonde (MSS) is a nonstandard wireline tool designed by LDEO (Figure F36). It measures the ease with which formations are magnetized when subjected to a magnetic field. The ease of magnetization is ultimately related to the concentration and composition (size, shape, and mineralogy) of magnetic minerals (principally magnetite) in the formation. These measurements provide one of the best methods for investigating stratigraphic changes in mineralogy and lithology because the measurement is quick, repeatable, and nondestructive and because different lithologies often have strongly contrasting magnetic susceptibilities.

The MSS dual-coil sensor provides ~40 cm resolution measurements with ~20 cm depth of horizontal investigation. The MSS was run as the lowermost tool in the triple combo tool string, and a data translation cartridge was used to enable the MSS to be run in combination with the Schlumberger tools. MS data from the MSS are plotted as noncalibrated units and are affected by temperature and borehole size (higher temperatures lead to higher susceptibility measurements). For quality control and environmental correction, the MSS also measures internal tool temperature, z-axis acceleration, and low-resolution borehole conductivity.

### 11.2.3.6. Acoustic velocity

The Dipole Shear Sonic Imager (DSI) measures the transit times between sonic transmitters and an array of eight receivers (Figure F36). It combines replicate measurements (i.e., upward and downward propagation), thus providing a direct measurement of sound velocity through formations that is relatively free from the effects of formation damage and borehole shape (Schlumberger, 1989). Along with the monopole transmitters found on most sonic tools, it also has two crossed-dipole transmitters that allow measurement of shear wave velocity in addition to com-
pressional wave velocity. Dipole measurements are necessary to measure formation shear velocities that are less than the compressional velocity of the borehole fluid. Such “slow formations” are commonly encountered in deep-ocean drilling.

### 11.2.3.7. Formation MicroScanner

The FMS provides high-resolution electrical resistivity-based images of borehole walls (Figure F36). The tool has four orthogonal arms and pads, each containing 16 button electrodes that are pressed against the borehole wall during logging. The electrodes are arranged in two diagonally offset rows of eight electrodes each. A focused current is emitted from the button electrodes into the formation, and there is a return electrode near the top of the tool. Resistivity of the formation at the button electrodes is derived from the intensity of the current passing through the button electrodes. Processing transforms the resistivity measurements into oriented high-resolution images that reveal geologic structures of the borehole wall. Features such as bedding, stratification, fracturing, slump folding, and bioturbation can be resolved (Luthi, 1990; Salimullah and Stow, 1992; Lovell et al., 1998). Because the images are oriented to magnetic north, further analysis can provide measurement of the dip and direction (azimuth) of planar features in the formation. In addition, when the corresponding planar features can be identified in the recovered core samples, individual core pieces can be reoriented with respect to true north.

Approximately 30% of a borehole with a diameter of 25 cm (9⅝ inches) is imaged during a single pass. Standard procedure is to make two full upward passes with the FMS to maximize the chance of getting full borehole coverage with the pads. The maximum extension of the caliper arms is 40.6 cm. In holes with a diameter greater than this maximum, the pad contact at the end of the caliper arms is inconsistent, and the FMS images may appear out of focus and too conductive. Irregular (rough) borehole walls will also adversely affect the images if contact with the wall is poor.

### 11.2.3.8. Acceleration and inclinometry

The GPIT was included in the FMS-sonic tool string to calculate tool acceleration and orientation during logging (Figure F36). Tool orientation is defined by three parameters: tool deviation, tool azimuth, and relative bearing. The GPIT utilizes a three-axis inclinometer and a three-axis fluxgate magnetometer to record the orientation of the FMS as the magnetometer records the magnetic field components ($F_x$, $F_y$, and $F_z$). Thus, the FMS images can be corrected for irregular tool motion, and the dip and direction (azimuth) of features in the FMS image can be determined.

### 11.2.4. Downhole fluid sampling

Borehole fluid sampling was accomplished using a Kuster FTS (Figure F23). The Kuster FTS is rated to 690 bar (10,000 psi) and 232.2°C, although it has been successfully run as high as 260°C (de Ronde et al., 2019). The Kuster FTS tool was fitted with a 1000 mL sample chamber. The tool was run directly after temperature logging with the ETBS. Procedures for downhole survey operation were followed in accordance with manufacturer guidelines (available from the JOIDES Resolution laboratory manuals, guides, and resources, https://wiki.iodp.tamu.edu/display/LMUG/Downhole+Logging#DownholeLogging-KusterFTS, accessed 30 September 2020).

The tool consists of a 600 mL capacity fluid sample chamber that can be connected to an additional 400 mL capacity tube with a spring-loaded valve on each end of the sample chamber. The lower end has a removable bullnose with ports to allow the fluid to enter. At the top is a rope socket for attaching the wireline. A latching mechanism connects the valves together and holds them open during deployment. A programmable mechanical clock is attached above the chamber, with either a 1 or 3 h clock for trigger times <50 and >50 min, respectively. This clock is preset at the surface to define the closing time, at which point a ball-operated tripping mechanism releases the valves, trapping the fluid in the sampler and maintaining the pressure of the sampling depth until the tool is recovered and the fluids are retrieved through a transfer head assembly (see Organic geochemistry). Operationally, 10 min was allowed to elapse following the estimated valve closure time before the Kuster FTS was recovered at an ascent rate of 70 m/min.

Between samples, the Kuster FTS was cleaned with ACS reagent grade acetone, rinsed with deionized water, and serviced following manufacturer guidelines. Likewise, it was ensured that the transfer head assembly was free of contaminants by washing with acetone and distilled water and
testing that the valves would easily open or shut. After recovery of the fluid sampler tool, all valves, connections, and O-rings were checked for mechanical wear and embrittlement and replaced as necessary prior to the next run.

### 11.2.5. Downhole measurements summary

A combined total of 93 downhole temperature measurements with both the APCT-3 and SET2 tools were carried out during the expedition (Tables T18, T19). Logging formation tool strings were deployed at Sites U1545–U1548 (Table T18), and downhole sampling with the Kuster FTS was conducted at Sites U1545, U1547, and U1548 (Table T18).

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