Osmotic energy conversion in serpentinite-hosted deep-sea hydrothermal vents

Domain: earth_science
Published: 2024-09-25
Editorial outcome: accept

Abstract
Cells harvest energy from ionic gradients by selective ion transport across membranes, and the same principle is recently being used for osmotic power generation from salinity gradients at ocean-river interfaces. Common to these ionic gradient conversions is that they require intricate nanoscale structures. Here, we show that natural submarine serpentinite-hosted hydrothermal vent (HV) precipitates are capable of converting ionic gradients into electrochemical energy by selective transport of Na+, K+, H+, and Cl-. Layered hydroxide nanocrystals are aligned radially outwards from the HV fluid channels, constituting confined nanopores that span millimeters in the HV wall. The nanopores change the surface charge depending on adsorbed ions, allowing the mineral to function as a cation- and anion-selective ion transport membrane. Our findings indicate that chemical disequilibria originating from flow and concentration gradients in geologic environments generate confined nanospaces which enable the spontaneous establishment of osmotic energy conversion.

Introduction
Gradients such as temperature, pressure, redox, and concentration are ubiquitous in nature. The conversion of these gradients into usable energy is a key process in both biology and industry. In nature, cells interconvert electrochemical, ion, and chemical gradients using respiratory protein complexes embedded in a membrane1,2. In synthetic chemistry, ionic gradients are recently also being harnessed as energy sources using two-dimensional materials and confined nanostructures3–9.

Submarine HVs are fascinating geologic systems surrounded by steep, long-lasting gradients and have been hypothesized to function as natural energy converters that could lead to the formation of the first cells10–23. HVs are formed by mineralization when the hot, reduced ion-rich vent fluid solutions are injected into cooler, oxidizing seawater, and have been discovered as arising from a diverse range of geologic activity24–for example, along mid-ocean ridges25,26 (on-axis) and away from spreading centers27,28 (off-axis). Off-axis HVs are mainly formed by the serpentinization process, in which the hydrolysis of olivine ((Mg,Fe)2SiO4) generates alkaline fluids with diverse ion compositions17,29,30. Subsequent mixing of alkaline fluids with seawater produces structurally highly complex, porous precipitates rich in layered metal oxides, hydroxides, and carbonates that act as a diffusion barrier to maintain steep gradients between the two solutions28.

Here, we report the first detailed insights into the nanoscale structure and energy conversion functions of off-axis, serpentinite-hosted, HV precipitates. A combined analysis of synchrotron X-ray diffraction, optical, and electrical measurements revealed that the HV precipitate contains interconnected nanopores with variable surface charges, allowing it to convert diverse ionic gradients into electrical energy – a process called osmotic energy harvesting. Selective ion transport is critical for osmotic energy conversion in cells1,2 and energy utilization in engineering fields3–9. The spontaneous establishment of nano- to millimeter-scale ion transport channels discovered in geologic environments suggests an evolutionary relevance between HVs and cells12–19 and showcases a geomimetic approach toward material self-organization.

Results
Structure of HVs with nanopores
The HV precipitates examined here (Fig. 1a) were collected from the Shinkai Seep Field (SSF), which is located in the southern Mariana Trench, Pacific Ocean, at a depth of 5743 m (11°39.38′ N, 143°2.73′ E)31. The geochemistry in the SSF is primarily related to serpentinization, a process that form serpentine from olivine. During the initial stages of HV formation in the SSF, a brucite-rich white chimney forms. In time, the mineral composition of the HV precipitates changes from predominantly brucite to carbonate-rich as the discharge fluid activity diminishes31. The detailed mineralogy of SSF HVs was described in our previous publication31. Briefly, the mineralogy varies with vent fluid flow and is classified into types I, II, and III. In this study, a Type I sample of approximately 84 cm in height was analyzed, which contains brucite as the major component (~80%), with calcium carbonate and magnesium carbonate as minor components (Supplementary Figs. 1, 2).

Cross-polarized optical microscopy (POM; Fig. 1b, c) revealed a complex internal structure comprised of porous columnar structures that served as channels for discharging the vent fluid. The fluid channels were separated by 200~400-µm thick inorganic walls that were mainly composed of brucite (Mg(OH)2), a layered hydroxide crystal, (Fig. 1c and Supplementary Figs. 1, 2) and exhibited a periodic lamellar structure with a wall thickness of ~10 μm (Fig. 1c (red arrows), d, e). The sublayers of this structure consisted of consolidated plate-like brucite nanocrystals with a diameter of ~100 nm that were perpendicularly aligned to the surface of the channel (Fig. 1f–h). In an enlarged high-angle annular dark-field scanning transmission electron microscopy (HAADF-STEM) image (Fig. 1i), individual crystals contained porous nanostructures (2~3 nm) that appeared as local density variations. The stacking of layered nanocrystals at multiple scales resulted in the formation of interconnected nanopores (Supplementary Figs. 3–5). Nitrogen adsorption-desorption isotherm analysis identified open-end slit-like nanopores (H3-type hysteresis) in the inorganic walls with a various pore size ranging from 2 to 100 nm (Supplementary Fig. 6).

Alignment of brucite across the macroscale
The alignment of the brucite nanocrystals within the HV precipitates was further analyzed using synchrotron radiation X-ray diffraction (wide-angle X-ray scattering (WAXS)) scanning. Multiple areas of the horizontal slice of HV samples were scanned using a 5-μm X-ray beam, and a scattering intensity map was constructed from the total intensity of the diffractograms (Fig. 2a). Similar to that seen in the POM and SEM images, a band-like structure was observed, due to the periodic density changes of the brucite crystals within the HV wall (Fig. 2a and Supplementary Fig. 7a, b). Pronounced anisotropy of brucite 001 diffraction was detected, indicating that the (001) plane of brucite orients preferentially inside the HV wall (Fig. 2b, c and Supplementary Fig. 7c, d). The directionality of the preferential orientation of brucite (001) planes varied depending on the locations (Fig. 2b, c). A schematic of the layered (001) plane of brucite with the corresponding c-axis was shown below each WAXS pattern (Fig. 2c, bottom).

We next extracted the diffraction intensities of the anisotropic rings and azimuthal angles of the highest intensity in the WAXS data and created a two-dimensional vector field map (Fig. 2d, e). The vector directions indicate the preferred orientation direction of the (001) plane along the [001] direction or c-axis, and the vector colors and lengths represent the relative amount of plane alignment. For example, the ring patterns of 3 and 4 (Fig. 2c) are represented by a red vector in the top right and a green vector in the bottom right, respectively. Notably, over the entire scanned area, the vector plot shows a continuous change in the direction of preferred orientation, suggesting that the brucite layers are arranged in a concentric manner radiating outwards from the fluid channel (Fig. 2e, f). The alignment of brucite nanocrystals along the channel continues throughout the entire sample, even when the wall structure bends, diverges, or converges (Fig. 2e and Supplementary Fig. 8a, b). The crystal alignment was also observed in the vertical HV slices (Supplementary Figs. 9, 10, and Supplementary Notes).

WAXS mapping using a larger, 100-μm X-ray beam size confirmed that the continuous alignment of the nanocrystals in the walls extended to millimeter-scale (2.5 × 6.0 mm; Fig. 3a, b). Such long-range alignment of the brucite nanocrystals was corroborated by POM analysis conducted with a retardation plate (first order, 530 nm; Fig. 3c). The yellow and blue interference colors alternating along the angular direction of the inorganic wall represent the radial configuration of crystals surrounding the channel and are consistent with the schematic illustration presented in Fig. 2f.

POM and WAXS analysis were also performed for the samples collected from multiple locations in the Type I sample. Thin sections were made from different parts of HV (the upper (35 sections), middle (10 sections), and lower (20 sections)). Essentially the same POM interference colors and anisotropic ring patterns were observed in all thin sections sampled from locations ranging from 26 cm to 84 cm apart (Supplementary Figs. 11, 12), confirming that the alignment observed above extends to almost entire parts of Type I HV. Small-angle X-ray scattering (SAXS) measurements of the HV wall showed further ellipsoidal SAXS patterns attributable to nanoscale periodicity (Supplementary Fig. 13). Material characterization based on SEM, TEM, nitrogen adsorption-desorption isotherm, and WAXS/SAXS demonstrates the anisotropic alignment and multilayer stacking of plate-like brucite nanocrystals collectively, forming interconnected nanopores referred to as nanochannel inside the HV walls (Fig. 2f and Supplementary Figs. 3–6).

Ion adsorption and surface charge of HV minerals
In addition to the continuous alignment of nanocrystals in HV minerals (Figs. 1–3), the pores show variable surface charges depending on adsorbed ions. Brucite crystals rich in carbonate ions were detected at several locations in the inorganic HV wall (Fig. 4b, red spectrum; Fig. 4c, blue Raman map). In addition, crystals with different refractive indices were also observed on the surface at these locations (Fig. 4d) and were identified by Raman and X-ray diffraction mapping as Mg-carbonate crystals (Fig. 4a–c and Supplementary Fig. 14). The existence of both brucite with carbonate ions and Mg-carbonate crystals indicates that the interconnected HV walls are exposed to various inorganic ions that are present in the hydrothermal fluid and seawater. As brucite is able to adsorb a wide variety of inorganic ions32,33, the surface charge of HV precipitates can significantly change depending on the composition of the surrounding fluids (Fig. 4e, f). Indeed, Raman analysis of natural HV samples confirmed that brucite, which is the main constituent mineral of HVs, adsorbs K+, Ca2+, CO3 2-, NO3 -, and SO4 2- (Fig. 4e). Furthermore, the zeta potential of the HV samples varied greatly depending on the type of adsorbed ion, from −30 mV (CO3 2-) to +25 mV (Ca2+), indicating that the electrostatic properties of the HV surface are markedly influenced by the interaction with inorganic ions present in the environment (Fig. 4f).

Osmotic energy conversion in HVs
Nanopores, structural alignment, and variable surface charges found in HVs are hallmarks of materials associated with osmotic energy conversion3–9. Materials with high surface charge density and confined nanopores have been designed for industrial power generation systems from salinity gradients between seawater and river water3–5. Motivated by the findings above, we tested the osmotic energy conversion in the HV.

A two-chamber electrochemical cell equipped with an Ag/AgCl electrode (Fig. 5a, b) was used to record the current-voltage (I–V) response of the HV samples. The HV samples were exposed to various concentrations of KCl, and the current response was plotted (Fig. 5c). At high KCl concentrations ranging from 1 M to 1 mM, the conductance of HVs is proportional with the salt concentration, indicating that electrolyte properties dictate ionic conductivity. However, the conductance remained constant even when the KCl concentration was decreased below 1 mM KCl, which is a significant deviation from the bulk characteristic of ion transport seen at higher concentrations. This plateau in the conductance is indicative of surface charge-governed ion transport34 and, therefore, suggests that ion transport behaviors are primarily regulated by electric fields generated at solid-electrolyte interfaces in intra- and inter-brucite crystal spaces (Fig. 5d, e)35.

The HV samples were next exposed to geologically plausible chemical gradient conditions by placing them at the interface of two KCl solutions (0.1 mM and 0.1 M). The HV samples were adsorbed with either CO3 2- or Ca2+, and the I–V response of the samples was recorded (Fig. 6a, b, N=15, Supplementary Fig. 15). When carbonate ions were adsorbed to the HV nanopore walls, a negative surface charge was generated. Further, when the HV nanopores were exposed to KCl, electrical double layers (EDLs) were created within the nanopores depending on the KCl concentration (Fig. 5e, Debye screening length = 0.96 and 30.42 nm for 0.1 M and 0.1 mM KCl, respectively). In the HV mineral wall, the dominant pore size, created by the multilayer stacking of brucite nanocrystals, is 60 nm (Supplementary Fig. 6b and Fig. 5d, e), indicating that a 30 nm EDL formed by 0.1 mM KCl is completely overlapped inside the HV nanopore. The overlap of EDL within the narrow nanopores effectively results in a screen barrier permeable only to ions with a specific charge. This allows selective ion transport based on surface charge-governed ion transport mechanism34. Furthermore, the continuous arrangement of crystals, together with the slit-like pores (Figs. 1, 2, 5d and Supplementary Figs. 3–6, 13), enables the HV walls to act as a cation- and anion-selective ion transport membrane and thus harvesting electrical energy from diverse ionic gradients present in the environments as will be demonstrated below.

In the presence of a KCl gradient, the CO3 2--adsorbed HV sample generated the open circuit voltage (Voc) of 229 mV (Supplementary Fig. 15a, blue circle). Subtracting the redox potential of the Ag/AgCl electrode (Vredox, 163 mV; Method, Supplementary Table) yielded a pure osmotic potential (Vos, diffusion potential) of 66 mV shown in Fig. 6b (blue circle). The Vos and diffusion current (Ios) increased proportionally with the ionic concentration difference (Supplementary Fig. 16a, b), confirming that the detected electrical energy originates from the ionic gradient. Notably, a negative Vos value of −46 mV was observed when Ca2+ ions were adsorbed (Fig. 6b, red circle). This change in the Vos indicates that the surface charge resulting from CO3 2- and Ca2+ adsorption plays a key role in the selective transport of cations and anions in HV nanopores. In other words, changing the sign of Vos indicates that the dominant charge carrier is shifted from K+ to Cl− upon switching the adsorbed ions from CO3 2- to Ca2+ due to the change in polarity of the EDLs. Furthermore, due to the cation- and anion-selective nature of HV walls, the series connection of positively and negatively charged HV sample pairs increased diffusion potentials, demonstrating that this configuration allows the HV precipitates to harness the chemical potential originating from both K+ and Cl− gradient and thus amplify the Vos (Fig. 6c). When the direction of the HV sample was rotated 90° from the natural HV configuration, Ios was lower than that of the HV’s natural direction, suggesting that the arrangement of brucite in HV precipitates facilitates selective ion transport (Supplementary Fig. 17 and Supplementary Notes).

Selective ion transport within HV precipitates was further verified by the direct measurement of Na+ and Cl− ions over time (Fig. 6a, d, e and Supplementary Fig. 16c). Cation- (CO3 2--adsorbed) and anion-selective (Ca2+-adsorbed) HV samples were fixed between two reservoirs filled with either low (0.1 mM) or high (0.1 M) salt concentrations and the concentration changes of the Na+ and Cl- ions were monitored with respect to time (Fig. 6d, e). In the case of Na+ ion transport through CO3 2--adsorbed HV nanopores, the concentration of Na+ ions in the low-concentration reservoir gradually increased over time. In contrast, there was minimal change in the Na+ ion concentration across the Ca2+-adsorbed HV sample. Instead, the Cl− increased steadily on this side of the reactor, providing compelling evidence for selective ion transfer within the HV precipitates.

One of the most characteristic features of serpentinite-hosted HV systems is the presence of a large pH gradient between the seawater (pH ~7) and vented fluids (pH 9~11). Experimental simulation of alkaline HV systems has been reported in the context of the origin of life on the ancient Earth, using reducing high pH fluid (pH 10~12) and oxidative low pH seawater (pH 4~6) simulant22,36,37. To examine the conversion of a pH gradient to electrochemical energy, the cation-selective (CO3 2--adsorbed) HV sample was placed between reservoirs containing pH 10 and 4 solutions, and the electrical response was measured (Fig. 6f, gray circle). Under these conditions, 55 mV of Vos and −2 nA of Ios were generated. The cation-selective HV sample was also subjected to a deuterium gradient (pD), and the I–V response was monitored (Fig. 6f, red circle). The Ios decreased by 2.3 fold compared to the pH gradient case, while the potential was similar, indicating that proton movement was the source of the generated current. In contrast, when the anion-selective HV sample was subjected to the same test, a reduced H/D kinetic isotope effect was observed, suggesting that hydroxide is involved in the power generation (Supplementary Fig. 18).

Methods
Chemicals
MgCl2·6H2O (97.0%), CaCl2·2H2O (99.9%), NaNO3 (99.0%), Na2CO3 (99.8%), KCl (99.5%), KOH (85.0%), HCl (35.0–37.0%), NaClO·5H2O (39.0% (as Cl)) were purchased from FUJIFILM Wako Pure Chemical Corporation. Na2SO4 (99%) and NaCl (99.5%) were obtained commercially from Junsei Chemicals. For aqueous solution preparation, high-purity deionized water (18.2 MΩ cm−1) was used. D2O (99.9 atom % D), KOD solution (40 wt.% in D2O, 98 atom % D), and DCl solution (35 wt.% in D2O, ≥99 atom % D) were purchased from Sigma-Aldrich. PDMS (SYLGARD 184 Silicone Elastomer Kit) and Ag wire (φ0.50 mm, 99.99%) were provided by Dow Corning and Nilaco, respectively.

Materials
The hydrothermal vent (HV) sample was collected using the Shinkai 6500 manipulators during dive #1404 on cruise YK14-13 in 2014 (ID; 12541-14001_6K1404-R05). The sample was sectioned onboard immediately after recovery and stored at 4°C. The SSF is located on the forearc slope of a subduction zone and differs in tectonic setting from the Lost City Hydrothermal Field, the most well-studied off-axis, alkaline HV site. However, both vent systems are associated with serpentinization, forming similar precipitates in texture and mineral composition. Therefore, this study used the SSF samples to represent alkaline HV precipitates.

Sample preparations
At the time of HV collection in the deep sea, the investigated HV had a height of approximately 84 cm, divided into three parts (upper: ~28 cm, middle: ~26 cm, lower: ~30 cm) for further analysis (Supplementary Fig. 1a). The HV parts were cut into multiple pieces for inspection using an MC-420 diamond cutting saw (Maruto Instrument Co., Ltd.) and a razor blade. The gentle pressure applied during cutting induced cracks in the sample, and the spontaneously cut fragments were also collected and observed. To observe HVs with a polarized light microscope, samples were embedded into epoxy resin (E205, Nichika). The samples were then cut and polished, and then mounted on glass slides and further polished to the desired thickness (tens of microns thick of thin section). Multiple thin sections were made using samples collected from various locations in each HV part, upper, middle, and lower. For the synchrotron X-ray scanning, slices of HV samples (~120 µm) were prepared by embedding the HVs in a UV-curable resin, which was then cut and polished to the desired thickness. Cross-sectional analysis of HV-polymer samples was observed after milling the samples using an Ar ion beam milling system (Leica EM TIC 3X).

Characterization of HV
Cross-polarized optical microscope (POM) images were obtained using an IX-70 inverted microscope (Olympus) equipped with additional linear polarizers and a first-order retardation plate (530 nm). In order to obtain a high-resolution optical image of a wide area, images observed by a digital microscope were connected without magnification and color adjustment. Naturally cut samples were observed using a Quattro S SEM (Thermo Fisher Scientific) without coating. For TEM observation, the samples were crushed, and the powdered samples were loaded on the grid. Images were taken with a JEM-2100F TEM (JEOL) coupled with a high-angle annular dark-field (HAADF) detector.

Powder X-ray diffraction patterns of bulk samples were measured using a SmartLab X E diffractometer (RIGAKU). The synchrotron X-ray scanning experiments were conducted using the RIKEN Targeted Proteins Beamline (BL32XU) and RIKEN Structural Biology I Beamline (BL38B1) in SPring-8 (Hyogo, Japan). Two beamlines with different beam sizes were used for the wide-angle X-ray scattering (WAXS) experiment. At the BL32XU beamline, the X-ray beam was 5 µm (Horizontal, H) × 5 µm (Vertical, V) with a 0.6300 Å wavelength. The sample-to-detector distance was 125.0 mm, and an EIGER X 9M (Dectris) detector was used. Sliced samples were mounted on a goniometer and raster scanned in 5-μm steps using an X-ray beam with an exposure time of 10 ms per point. Scanning was performed over several regions of the HV slices at room temperature. In the case of BL38B1, the X-ray beam was collimated to a size of 120 µm (H) × 60 µm (V) and a wavelength of 1.0 Å. WAXS patterns were acquired using a Pilatus 3X 2M (Dectris) detector69. The scattering vector (q = 4π sin θ/λ; 2θ, scattering angle; λ, a wavelength of the X-ray beam) and the position of the incident X-ray beam on the detectors were calibrated with FIT2D software70,71 based on silver behenate (d = 58.38 Å). The sample-to-detector distance was 312.689 mm. The sliced sample was mounted on a motorized stage and linear-scanned with 100-µm steps and 10 seconds of exposure time at each point. The scan was conducted over a large area (~7 × ~3 mm) at room temperature. In the case of small-angle X-ray scattering (SAXS), the same beamline, RIKEN Structural Biology I Beamline (BL38B1), was used for measurement. The sample-to-detector distance was 2.5450 m. The beam is collimated to the size of 100 µm (H) × 100 µm (V). The WAXS patterns were analyzed using DPDAK v. 1.5.0, open-source software72. Information on the intensity and direction of the diffraction peaks was extracted using a Python program, which will be made available at github after acceptance. For the ion adsorption test, 5~10 mg of powder HV was dispersed in 1 M of each electrolyte solution (2 mL) adjusted to pH 10, and the solution was then sonicated for 1 h and allowed to sit overnight at room temperature. For Raman spectral acquisition of slice and ion adsorption samples, a Senterra Raman Microscope (Bruker Optics) was used. A 785 nm laser line with 25 mW of power was applied, and the exposure time varied from 5 to 60 s depending on the target. To obtain micro-Raman map images, a RAMANtouch (Nanophoton) system was used with a 532-nm laser line, 2 s of exposure time, and 9.6 mW power. The Raman was calibrated using Si peaks and cosmic ray reduction was applied for all Raman measurements. The surface charge of HV particles was evaluated using an ELSZ-2PL zeta-potential analyzer (Otsuka Electronics). Gas adsorption-desorption isotherms were obtained using a BELSORP-mini X surface area analyzer (Microtrac) with nitrogen gas.

HV-PDMS membrane fabrication
HV samples were first soaked in 1 M Na2CO3 or CaCl2 (adjusted to pH 10) overnight to adsorb carbonate or calcium ions. The ion-adsorbed HV sample was placed in a partially polymerized PDMS precursor liquid. A vacuum pump was used to infiltrate the PDMS into the HV samples to fill the macropores inside the HVs. The resulting HV-PDMS samples were kept at 40°C to solidify the PDMS. HV-PDMS was inserted in the middle of the PDMS film to prevent the mixing of the electrolyte or two different solutions in a two-chamber electrochemical cell while allowing the transport of ions only through the HV samples. After the PDMS embedding process, the upper surface of the HV sample was cut to remove the polymer coating, and the left and right sides of the HV surface were exposed to the solution. Before each measurement, the HV-PDMS membranes were fully hydrated by immersion in a pH 10 solution for 2 h. Raman, WAXS, and cross-section SEM image analysis confirmed that the material properties of brucite remain intact during membrane fabrication processes (Supplementary Figs. 20–22).

Electrical measurements
For KCl ion transport, an HV pellet-PDMS membrane was used. The HV material was ground and pelletized and then inserted into a PDMS film containing a 1 cm hole in the center of the film. The HV pellet-PDMS membrane was clamped between two reservoirs (two-chambers electrochemical cell equipped with Ag/AgCl electrode at both reservoirs) containing the same concentration of KCl solution. Current-voltage (I–V) curves of the membrane were recorded at various salt concentrations.

For the power generation test, low and high concentrations of KCl, NaCl, or protons were filled in the left (working electrode placement) and right (counter electrode placement) reservoirs, respectively, and the I–V response of the HV-PDMS sample was then measured. To suppress the dissolution of the HV samples, various concentrations of salt solutions were adjusted to pH 10. For I–V measurement in a pH gradient, 0.1 mM KCl was added to both sides of the reservoir to prevent the formation of AgOH on the Ag/AgCl electrode. All electrical measurements were conducted using an HZ-7000 electrochemical workstation (Hokuto Denko) in a Faraday cage. A pair of Ag/AgCl electrodes was used for the measurement. Ag/AgCl electrode wires were prepared by chemical chlorination and rechlorination was performed after each measurement. The applied voltage scan range was from –0.3 V to +0.3 V at a scan rate of 50 mV/sec. The concentration of Na+ and Cl− ions were detected using Orion Versa Star Pro electrochemistry meters (Thermo Scientific) coupled with ROSS sodium ion and chloride ion selective electrodes. Before conducting the deuterium experiments, the dried HV samples were immersed overnight in a pD 10 solution titrated with KOD in D2O.

Calibration
The open circuit voltage (Voc) measured with Ag/AgCl wires is a result of the pure osmotic potential (Vos) of the HV and the redox potential (Vredox) of the electrode-solution interface upon exposure to a different concentration of salt and is represented by the equation: Voc = Vos + Vredox. Subtracting the redox potential from Voc gives the pure osmotic potential of the HVs. To measure the redox potential of the Ag/AgCl wire, a commercial reference electrode (filled with saturated KCl in a glass housing, Hokuto Denko) and the prepared wire were placed in a specific concentration of KCl, and the potential was recorded. The potential difference at the specific KCl concentrations was used to calculate the redox potential increase (see Supplementary Table). The measured current (Imeas) was adjusted by the following; Ios = (Vos/Voc) × Imeas

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Acceptable with a revison.\nThis manuscript elucidates the phenomenon of ionic gradient conversions occurring within\nthe water-rock system proximate to hydrothermal vents. The authors substantiate the ion\nadsorption and surface charge characteristics of hydrothermal vent (HV) minerals and\nexpound upon the energy conversion processes within HVs utilizing a custom-fabricated\ndevice. This research is both intriguing and contributes valuable insights into the energy\nconversion mechanisms operating in the vicinity of deep-sea hydrothermal vents.\nRegarding this paper, the section on the Structure of HVs with nanopores may be more\nsuitably incorporated into the Supplementary Materials, as it’s not that significant. In\nconjunction with the osmotic energy conversion in HVs section, Supplementary Figures 10 to\n12 may be integrated into the main context of the Conclusion, and Figures 10 to 12 can be\namalgamated into a singular composite figure. In addition, the visual design of current\nSupplementary Figure 10 can be further refined. Furthermore, the intricate physical and\nchemical principles underlying ion migration should be synthesized in a more detailed and\nspecialized manner by combining with the integrated figure.\nWhile you propose that the conversion of ionic gradients near HVs may serve as a reference\npoint for the study of life's origins, this concept remains in a hypothetical stage based on the\ncurrent study's lack of specific and supportive data. Substantiating this conclusion presents a\nformidable challenge. The authors are in the process of establishing connections, but it is not\nyet robust enough to warrant a definitive conclusion. If the authors want to conclude this in\nthe Abstract, additional analyses and experiments are imperative to fortify this assertion.\nFig. 1 is difficult to identify the letters from a-i.\nRedundancy in Line 72.\nReferences: formatting issues (some list all names of five authors, while others being\npresented as Fujisawa, T. et al).", "reviewer_2": "This is a beautiful piece of work and I highly recommend its publication. The authors perform\nan extensive series of analyses of material from a deep-sea alkaline hydrothermal vent and\nfind excellent evidence for osmotic properties that may be of great importance for resolving\nsome of the questions surrounding the origin of life on Earth. I think the findings will be of\ngreat interest and importance to researchers in various fields including alkaline hydrothermal\nvents, osmotic processes in geology, and the origin of life.", "reviewer_3": "Lee Ms Nature 2023 444925-7903865 Russell September 6\nOverall assessment: Truly exceptional and I strongly recommend publication.\nI cannot rate this Ms too highly. It brings a much needed new footing to the long assumed\nrelationship between submarine alkaline vents and the emergence of life. The Ms derives\nand is evolved from Okumura (the second author to the present Ms) and colleagues’ 2016\nreport of the discovery of brucite-carbonate chimneys at a depth of nearly 6km in the\nMariana ocean trench right through to exquisite Synchroton operando analyses.\nCalling upon transdisciplinary research including, and since, Mitchell’s chemiosmosis\nepiphany, the Ms involves many of the current interests of the scientific community: free\nenergy conversion, electrochemistry, electrodialysis, diffusiophoresis, redox chemistries,\nbioenergetics, early metabolism and nanoconfinement, all tied into the serpentinization\nprocess and the energetics driving life today and at its origin. The research uses all the\navailable and appropriate techniques for the thorough analyses of the micro-to-\nnanostructures produced at the interface between alkaline, reduced seepage waters and\nrelatively oxidized ocean water.\nExquisite photographs and optical images of synchrotron X-ray diffraction analysis of the\nbrucite precipitates demonstrate multi-layering of the nanopore walls and, most importantly,\nthe preferred orientation of the (001) plane crystallographic cleavage planes which, as they\nshow, are aligned radially outwards from the fluid channels, constituting confined nanopores\nthat span millimeters in the hydrothermal vent precipitates.\nThe paper demonstrates that the effect of nano-to-millimetric diffusion of both cations and\nanions – the ionic disequilibria – is to produce potentials reaching ~300 millivolts, quite\nenough to engender an emergent metabolism given the complex hydrothermal environment\nthey allude to.\nI could find no flaws in this beautifully constructed Ms, written as it is by leaders in the\nrespective fields covered and impeccably and exhaustively referenced. The paper (and the\naccompanying SI) is state of the art and so original that I for one didn’t see it coming! It takes\nthe ambit of alkaline vent research and understanding to a new level and provides a strong\nbase for future theory and practice.\nQuestion to the authors\nYou reference Khan and O’Hare’s LDS intercalation paper (your [32]) and allude to Morello\net al.’s enzyme cascades ([59]) but elsewhere your note channels are nano to millimeter\nscale. Could you clarify whether the lack of dampening of the potassium ion flux shown in\nFigure 5c (except at very low concentration) implies that diffusion is entirely inter- and not\nintra-crystalline? The fact that flux force linearity is not observed would be at variance with\nenzymatic behaviours in biological membranes, perhaps worth a note ? (see:\nStucki, J. W. (1980). The thermodynamic buffer enzymes. Europ. J. Biochem. 109, 257-267.\nStucki, J.W. (1983). Compiani, M.; Caplan, S.R. Efficiency of energy conversion in model\nbiological pumps; Optimization by linear n‐onequilibrium thermodynamic relations. Biophys.\nChem. 18, 101-109.)\nMinor quibbles\n54 protein complexes embedded in a membrane1,2. In synthetic “chemistry”, ionic gradients\nare\n59 … HVs are formed by mineralization chemistry when the hot, “reduced” ion-rich vent fluid\nsolutions are injected into the cold, oxidizing seawater, and discovered in a\n65 precipitates rich in layered metal oxides, “hydroxides” and carbonates that act as a\ndiffusion barrier to maintain steep gradients “between” the two solutions\n71 electrical energy – a process [delete “so”] called osmotic energy harvesting. Selective ion\ntransport is critical for osmotic energy conversion in [delete 2nd “in”] cells\n82 serpentinization, a process that forms serpentine from olivine. During the initial stages of\nHV formation in the SSF, a brucite-rich white chimney forms. “In time” the mineral\ncomposition of the HV precipitates change from predominantly brucite to carbonate-rich as\nthe discharge fluid activity “diminishes”.\nthicknesses of\nangles of [delete “the”] the highest intensity in the WAXS\nimage of large HV wall sample area with overlaid vector plots [delete “, which were”]\ngenerated from a single scan direction and [delete “are”] plotted as white arrows.\n277 and anion-selective (Ca2+-adsorbed [delete “HV”) HV samples were fixed between two\n373 the HV system, the self-assembly of [delete “a”] brucite,\n375 and functions may offer [delete “s”] new synthetic", "reviewer_4": "Thank you for inviting me to review this article. The article contains a lot of interesting\ntechnical information and it was very nice to read it. The paper of Lee et al., contains three\nmain claims:\n1) The formation of a nanoporous channel texture in brucite crystals forming the walls of\nhydrothermal vents.\n2) These channels are vital in converting geochemical ionic gradients into electrochemical\npotentials\n3) Electrochemically coupled ion transport - a universal process in cell bioenergetics -\nemerged at the origin of life.\nIt is important to note that the sample studied by the authors is not representative of\nhydrothermal vents as the title and the term “hydrothermal vent (HV)” used along the text\nsuggest. It is a sample of white fumaroles, i.e. of hydrothermal vents linked to the\nserpentinization process. There are many other hydrothermal vents completely different from\nthe geochemical, hydrochemical, and mineralogical point of view.\nThe walls of most white hydrothermal vents are mostly formed by brucite but they are not\nmonophasic. Depending on the geological context there is also an important mineralogical\ncontribution of calcium carbonate (calcite and aragonite) and also of magnesium carbonate.\nActually, the powder X-ray diffraction pattern in Supplementary Figure 2 shows the presence\nof these minerals. If the authors did a quantitative study of the mineralogical content of\nrepresentative samples of white fumaroles, they would find is very high proportion of calcium\ncarbonate, both calcite and aragonite, as well as magnesium and calcium carbonate. The\nreason is that the fluids expelled by hydrothermal vents associated with serpentinization are\nvery alkaline, with pH values of around 11, that, a pH at which the CO2 dissolved in the\nocean, will be as carbonate anion.\nThe study of wide-angle X-ray scattering is very nice but I am afraid that it is not convincing.\nThe entire diffraction study only proves the formation of brucite concentric layers with\ncrystals oriented perpendicular to the layers at the local scale. No nanochannels or anything\nsimilar are observed. The only information reporting nano-structures is in Figure 3 of the\nsupplementary materials, and they are nano-pores, not nano-channels. The authors wrote\nthat \"Pronounced anisotropy of brucite 001 diffraction was detected, indicating that (001)\nplane of brucite orients preferentially along the [001] crystallographic direction\" but (001)\nplanes are always perpendicular to [001] crystallographic direction. The plots shown in\nFigure 2 might be convincing of a radial texture of the brucite crystallites at the local\n(micrometer) scale but they are not representative of the large scale. Actually, why such a\ntexture should appear? The concentric layers of brucite could be the results of episodic\ngrowth related to periodic fluid dynamics but there is no reason inducing the brucite\nmicrocrystals to be co-oriented to each other.\nThe experimental procedure to fabricate a “membrane” from the sample of the wall of the\nwhite fumaroles is not very “clean” and I suspect that would modify the properties of the\nmaterial. The experimental method to measure with these “membrane” the electrical\nchemical energy does not mimic the natural process. The solutions to use should be those of\nplaying in the natural vents, i.e. a reducing high pH fluid inside the wall and seawater outside\nthe wall. The experimental measurements -even if they are not very conclusive- do not\nmodel properly the natural scenario. The existence of the generation of an electrochemical\npotential in osmotic-driven structures has been demonstrated and measured (see Glaab et\nal., (Angew. Chem. Int. Ed. 2012, DOI: 10.1002/ange.201107754 ). The electrochemical\npotential was proposed to be a summation of three factors, of which a large pH difference is\na major contributor. Perhaps the authors could find ideas for experimental testing and\nexplanations in that article. And of course, a porous structure connecting the two sides of the\nmembranes is key for the formation of the electrochemical potential.\nThroughout the manuscript, the authors try to prove that the brucite crystallites have\nconfined nanochannels with charged surfaces, which are the requirements for the\nconversion to work. The idea is good and the implications -if the hypothesis is demonstrated-\nare interesting, but the results that count do not demonstrate, in the opinion of this referee,\nthe existence of osmotic energy conversion processes in hydrothermal vents."}, "author_response": "As to the comments of Reviewer #1\nNotice: All revisions based on this reviewer’s comments are highlighted in green in the\nmanuscript and Supplementary Materials.\nReviewer comment 1:\nReviewer #1 (Remarks to the Author): Acceptable with a revision.\nThis manuscript elucidates the phenomenon of ionic gradient conversions occurring within the\nwater-rock system proximate to hydrothermal vents. The authors substantiate the ion adsorption\nand surface charge characteristics of hydrothermal vent (HV) minerals and expound upon the\nenergy conversion processes within HVs utilizing a custom-fabricated device. This research is\nboth intriguing and contributes valuable insights into the energy conversion mechanisms\noperating in the vicinity of deep-sea hydrothermal vents.\nAuthor reply 1:\nWe thank the reviewer for their interest in our work and for the constructive comments,\nwhich\nhave greatly helped us improve the quality of our work.\nReviewer comment 2:\nRegarding this paper, the section on the Structure of HVs with nanopores may be more\nsuitably incorporated into the Supplementary Materials, as it’s not that significant. In\nconjunction with the osmotic energy conversion in HVs section, Supplementary Figures 10 to\n12 may be integrated into the main context of the Conclusion, and Figures 10 to 12 can be\namalgamated into a singular composite figure. In addition, the visual design of current\nSupplementary Figure 10 can be further refined. Furthermore, the intricate physical and\nchemical principles underlying ion migration should be synthesized in a more detailed and\nspecialized manner by combining with the integrated figure.\nAuthor reply 2:\nWe thank the reviewer for their invaluable suggestion about schematic presentation of ion\ntransport. According to the reviewer’s suggestion, we integrated Supplementary Figs. 10 to 12\nto create new figures, which were added as Figs. 5 and 6 in the revised manuscript. Also, we\nadded more detailed explanation about the ion transport mechanisms in the caption of Fig. 5.\nMeanwhile, we believe that Fig. 1 provides invaluable information about the hieratical structure\nthat submarine HV self-generates. This structural information is the basis for a state-of-the-art\nwide-angle X-ray scattering (WAXS) and small-angle X-ray scattering (SAXS) analysis of\nnanocrystal alignment in HVs, we decided to keep Fig. 1 as is. Also, according to the reviewer’s\ncomment (Reviewer comment 4), we revised the letter from a-i in Fig. 1.\nFig. R1 (Fig. 5) Ionic transport by hydrothermal vent (HV) precipitates. a, A photograph\nof the HV-PDMS membrane sample used for the measurement. b, Experimental setup for\nmeasuring current-voltage response of the HV samples. For conductance measurement, both\nsides were filled with KCl solution of the same concentration. c, Electrical conductance of HV\nsample as a function of KCl concentrations with pH 10. d, Schematics showing the continuous\narrangement in HV inorganic wall, comprising multilayers of the brucite (001) plane at the\nangstrom level, plate nanocrystal stacks at the nanometer level, and a radial alignment structure\nextending to the millimeter level. This configuration results in continuous channels, allowing\nthe HV walls to function as a selectively permeable membrane for ion transport. e, Schematics\nshowing osmotic power generation. Upon exposure to KCl, an electric double layer (EDL) is\nformed on the surface of brucite crystals, exhibiting varying Debye screening lengths (λ )\nD\ndepending on KCl concentration. Blue represents the EDL formed on the brucite surface by\nexposure to ion concentration gradient. The prevalent pore size in HV walls is measured to be\n60 nm, which means that the 30 nm EDL formed by 0.1 mM KCl completely overlaps inside\nthe HV nanopore. Effective overlap of EDLs within narrow nanopores establishes a screening\nbarrier that is permeable only to ions with specific charges. Acting as a selective membrane,\nthe HV wall allows selective movement of counterions from a high concentration to a low\nconcentration, generating an ionic current.\nFig. R2 (Fig. 6) Osmotic power generation by selective ion transport in hydrothermal vent\n(HV) precipitates. a, Schematic illustration of the experimental setup for the ion selectivity\nmeasurements. The anion- and cation-selective HV samples were placed between reservoirs,\nand the HVs were exposed to alternating salt concentrations (0.1 mM / 0.1 M / 0.1 mM). b,\nOsmotic energy conversion under a KCl gradient. Current-voltage curves were measured under\na 1000-fold salinity gradient (0.1 mM / 0.1 M KCl) for CO 2--adsorbed (blue dots) and Ca2+-\n3\nadsorbed (red dots) HV samples. The redox potential of the electrode was subtracted from\nmeasured potential and the diffusion potential (V ) and current (I ) are shown in the figure.\nos os\nThe opposite polarity is generated from the different charge selectivity of HV samples. c,\nDiffusion potential of a series of connected HVs as a function of time. A combination of anion-\nand cation-selective HV pairs was placed under NaCl gradient conditions (0.1 mM / 0.1 M), as\nillustrated in the schematic on the top. Multiple connections of HVs enhanced the diffusion\npotential. The electrolyte was not refreshed during the measurement. d, e, Concentration\nchanges of Na+ (d) and Cl- (e) ions over time at locations 1 and 2 were measured and plotted. f,\nOsmotic power generation in the presence of pH (pH 10/ pH 4) and pD (pD 10/ pD 4) gradients.\nThe cation-selective (CO 2--adsorbed) HV sample was used for the analysis.\nResults, Osmotic energy conversion in HVs part\ntherefore suggests that ion transport behaviors are primarily regulated by electric fields\ngenerated at solid-electrolyte interfaces in intra- and inter-brucite crystal spaces (Fig. 5d and\ne)35.\nFig. 5 caption\nSchematics showing osmotic power generation. Upon exposure to KCl, an electric double layer\n(EDL) is formed on the surface of brucite crystals, exhibiting varying Debye screening lengths\n(λ ) depending on KCl concentration. Blue represents the EDL formed on the brucite surface\nD\nby exposure to ion concentration gradient. The prevalent pore size in HV walls is measured to\nbe 60 nm, which means that the 30 nm EDL formed by 0.1 mM KCl completely overlaps inside\nthe HV nanopore. Effective overlap of EDLs within narrow nanopores establishes a screening\nbarrier that is permeable only to ions with specific charges. Acting as a selective membrane,\nHV wall allows selective movement of counterions from a high concentration to a low\nconcentration, generating an ionic current.\nAdded Figures:\nFig. 5 Ionic transport by hydrothermal vent (HV) precipitates.\nFig. 6 Osmotic power generation by selective ion transport in hydrothermal vent (HV)\nprecipitates.\nReviewer comment 3:\nWhile you propose that the conversion of ionic gradients near HVs may serve as a reference\npoint for the study of life's origins, this concept remains in a hypothetical stage based on the\ncurrent study's lack of specific and supportive data. Substantiating this conclusion presents a\nformidable challenge. The authors are in the process of establishing connections, but it is not\nyet robust enough to warrant a definitive conclusion. If the authors want to conclude this in the\nAbstract, additional analyses and experiments are imperative to fortify this assertion.\nAuthor reply 3:\nWe agree with the reviewer’s comment. Therefore, we revised the final sentence in the abstract\nas follows:\nOriginal:\nThe finding that inorganic precipitates in submarine HVs convert geochemical ionic gradients\ninto electrochemical potentials may provide a link to how electrochemically coupled ion\ntransport - a universal process in cell bioenergetics - emerged at the origin of life.\nRevised:\nThe finding that inorganic precipitates in submarine HVs convert geochemical ionic gradients\ninto electrochemical potentials - a vital process in cell bioenergetics - may have implications\ntowards the hydrothermal vent theory for the origin of life on Earth.\nReviewer comment 4:\nFig. 1 is difficult to identify the letters from a-i.\nAuthor reply 4:\nWe agree with the reviewer’s comment. Therefore, we enlarged the letter from a-i in Fig. 1.\nFig. R3 (Fig. 1) Structure of hydrothermal vent (HV) precipitates.\nReviewer comment 5:\nRedundancy in Line 72.\nAuthor reply 5:\nThank you very much for pointing out the typo. We deleted “in”.\nReviewer comment 6:\nReferences: formatting issues (some list all names of five authors, while others being presented\nas Fujisawa, T. et al).\nAuthor reply 6:\nThank you for reviewing the details of our manuscript. In terms of the references style, we\nfollowed the Nature Communications guideline for authors.\nThe guideline specifies that\n\"Nature Communications uses standard Nature referencing style. All authors should be\nincluded in reference lists unless there are six or more, in which case only the first author should\nbe given, followed by 'et al.'.\"\nIn cases with up to five authors, all authors are included in the reference.\nThis concludes our response to the reviewers’ comments. We are grateful for their comments\nwhich have helped us improve the rigor and quality of our manuscript.\nAs to the comments of Reviewer #2\nReviewer comment:\nReviewer #2 (Remarks to the Author):\nThis is a beautiful piece of work and I highly recommend its publication. The authors perform\nan extensive series of analyses of material from a deep-sea alkaline hydrothermal vent and find\nexcellent evidence for osmotic properties that may be of great importance for resolving some\nof the questions surrounding the origin of life on Earth. I think the findings will be of great\ninterest and importance to researchers in various fields including alkaline hydrothermal vents,\nosmotic processes in geology, and the origin of life.\nAuthor reply:\nWe are very grateful to the reviewer for their interest in our study and strong support for its\npublication to Nature Communications. We agree that our research will be of great interest to\nresearchers in the fields of alkaline hydrothermal vents, osmotic energy conversion and the\norigins of life.\nThis concludes our response to the reviewers’ comments. We are grateful for their comments\nwhich have helped us improve the rigor and quality of our manuscript.\nAs to the comments of Reviewer #3\nNotice: All revisions based on this reviewer’s comments are highlighted in yellow in the\nmanuscript and supporting information.\nReviewer comment 1:\nReviewer #3 (Remarks to the Author):\nTruly exceptional and I strongly recommend publication.\nI cannot rate this Ms too highly. It brings a much needed new footing to the long assumed\nrelationship between submarine alkaline vents and the emergence of life. The Ms derives and\nis evolved from Okumura (the second author to the present Ms) and colleagues’ 2016 report of\nthe discovery of brucite-carbonate chimneys at a depth of nearly 6km in the Mariana ocean\ntrench right through to exquisite Synchroton operando analyses. Calling upon transdisciplinary\nresearch including, and since, Mitchell’s chemiosmosis epiphany, the Ms involves many of the\ncurrent interests of the scientific community: free energy conversion, electrochemistry,\nelectrodialysis, diffusiophoresis, redox chemistries, bioenergetics, early metabolism and\nnanoconfinement, all tied into the serpentinization process and the energetics driving life today\nand at its origin. The research uses all the available and appropriate techniques for the thorough\nanalyses of the micro-to-nanostructures produced at the interface between alkaline, reduced\nseepage waters and relatively oxidized ocean water. Exquisite photographs and optical images\nof synchrotron X-ray diffraction analysis of the brucite precipitates demonstrate multi-layering\nof the nanopore walls and, most importantly, the preferred orientation of the (001) plane\ncrystallographic cleavage planes which, as they show, are aligned radially outwards from the\nfluid channels, constituting confined nanopores that span millimeters in the hydrothermal vent\nprecipitates. The paper demonstrates that the effect of nano-to-millimetric diffusion of both\ncations and anions – the ionic disequilibria – is to produce potentials reaching ~300 millivolts,\nquite enough to engender an emergent metabolism given the complex hydrothermal\nenvironment they allude to. I could find no flaws in this beautifully constructed Ms, written as\nit is by leaders in the respective fields covered and impeccably and exhaustively referenced.\nThe paper (and the accompanying SI) is state of the art and so original that I for one didn’t see\nit coming! It takes the ambit of alkaline vent research and understanding to a new level and\nprovides a strong base for future theory and practice.\nAuthor reply 1:\nWe are very grateful to the reviewer for their interest in our work and strong support for its\npublication to Nature Communications. We agree that our research has the potential to provide\nthe basis to bridge chemistry, catalysis and biology, all consolidated to the alkaline\nhydrothermal vent theory of origin of life.\nReviewer comment 2:\nYou reference Khan and O’Hare’s LDS intercalation paper (your [32]) and allude to Morello\net al.’s enzyme cascades ([59]) but elsewhere your note channels are nano to millimeter scale.\nCould you clarify whether the lack of dampening of the potassium ion flux shown in Figure 5c\n(except at very low concentration) implies that diffusion is entirely inter- and not intra-\ncrystalline? The fact that flux force linearity is not observed would be at variance with\nenzymatic behaviours in biological membranes, perhaps worth a note ? (see: Stucki, J. W.\n(1980). The thermodynamic‐buffer enzymes. Europ. J. Biochem. 109, 257-267. Stucki, J.W.\n(1983). Compiani, M.; Caplan, S.R. Efficiency of energy conversion in model biological\npumps; Optimization by linear nonequilibrium thermodynamic relations. Biophys. Chem. 18,\n101-109.)\nAuthor reply 2:\nThank you for raising the question of whether ions are transported via intra- or inter spaces of\ntwo-dimensional brucite crystals. In the original manuscript, we demonstrated “surface charge-\ngoverned ion transport” as a main mechanism for selective ion transport (reference [34] in the\nmanuscript). Surface charge-governed ion transport is a process in which the mobile charged\nions are repelled or attracted to the nanoscale pore based on the electric field established at the\nsolid-electrolyte interface. Under our experimental conditions, the Debye screening length is\nbetween 0.96 nm (0.1 M KCl) and 30.42 nm (0.1 mM KCl). This means that nanopores with a\ndistance of up to 60 nm can act as a channel for selective ion transport. This value is consistent\nwith the dominant pore size of the HV material (60 nm, Supplementary Fig. 3b), indicating that\na 30 nm electrical double layer is completely overlapped inside the HV nanopore. Based on\nabove consideration, our reply to the reviewer's question is that both intra- and interstitial spaces\nof brucite crystals can function as selective ion transport channels. To clarify this point, we\nadded new sentences and figures in the revised manuscript as follows:\nFig. R1 (Fig. 5) Ionic transport by hydrothermal vent (HV) precipitates. a, A photograph\nof the HV-PDMS membrane sample used for the measurement. b, Experimental setup for\nmeasuring current-voltage response of the HV samples. For conductance measurement, both\nsides were filled with KCl solution of the same concentration. c, Electrical conductance of HV\nsample as a function of KCl concentrations with pH 10. d, Schematics showing the continuous\narrangement in HV inorganic wall, comprising multilayers of the brucite (001) plane at the\nangstrom level, plate nanocrystal stacks at the nanometer level, and a radial alignment structure\nextending to the millimeter level. This configuration results in continuous channels, allowing\nthe HV walls to function as a selectively permeable membrane for ion transport. e, Schematics\nshowing osmotic power generation. Upon exposure to KCl, an electric double layer (EDL) is\nformed on the surface of brucite crystals, exhibiting varying Debye screening lengths (λ )\nD\ndepending on KCl concentration. Blue represents the EDL formed on the brucite surface by\nexposure to ion concentration gradient. The prevalent pore size in HV walls is measured to be\n60 nm, which means that the 30 nm EDL formed by 0.1 mM KCl completely overlaps inside\nthe HV nanopore. Effective overlap of EDLs within narrow nanopores establishes a screening\nbarrier that is permeable only to ions with specific charges. Acting as a selective membrane, the\nHV wall allows selective movement of counterions from a high concentration to a low\nconcentration, generating an ionic current.\nFig. R2 (Supplementary Fig. 10) X-ray scattering patterns of hydrothermal vent (HV)\nsamples. a, Small angle X-ray scattering (SAXS) pattern of HV sample. b-d, Wide angle X-\nray scattering (WAXS) pattern of HV samples at different locations. WAXS patterns are also\ndisplaying the SAXS pattern at the center. Yellow dashed lines show highest intensity of WAXS\nplots indicating alignment direction of (001) plane of brucite. Green dashed lines indicate major\naxis of ellipsoid of SAXS region suggesting alignment direction of particles or voids. e,\nSchematic of the (001) brucite plane and particle alignment shown in (d). The alignment\n(periodicity) direction observed in WAXS and the alignment direction observed in SAXS are\nmatched in the same direction.\nAdded sentences:\nResults, Alignment of brucite across the macro scale part\nMaterial characterization based on SEM, TEM, nitrogen adsorption-desorption isotherm and\nWAXS/SAXS demonstrates collectively the anisotropic alignment and multilayer stacking of\nplate-like brucite nanocrystals, forming interconnected nanopores referred to as nanochannel\ninside the HV walls.\nResults, Osmotic energy conversion in HVs part\ntherefore suggests that ion transport behaviors are primarily regulated by electric fields\ngenerated at solid-electrolyte interfaces in intra- and inter-brucite crystal spaces (Fig. 5d and\ne)35.\nAdded Figures:\nFig. 5 Ionic transport by hydrothermal vent (HV) precipitates.\nSupplementary Fig. 10 X-ray scattering patterns of hydrothermal vent (HV) samples.\nThank you also for introducing the two papers regarding enzymology under flow conditions. In\nour experiments, ion flux across a chimney precipitate shows a linear relation with applied\nvoltage (please see Fig. 6b), meaning that electrochemical driving forces linearly correlate with\nion flux. This is a typical behavior for selective ion transport processes in electrochemical\ndevices (Macha, M. et al. (2019) Nat. Rev. Mater. 4, 588-605). Although the two papers\ndescribed the non-classical behavior of enzyme (ATP synthases), we find it difficult to\nincorporate these in the present format of the paper. We are grateful for the reviewer’s\nsuggestion, but we decided not to include these two papers in the revised manuscript.\nReviewer comment 3:\nMinor quibbles\n54 protein complexes embedded in a membrane1,2. In synthetic “chemistry”, ionic gradients\nare\nAuthor reply 3:\nThank you very much for your valuable suggestion\nWe revised “materials” to “chemistry”.\nReviewer comment 4:\n59 … HVs are formed by mineralization chemistry when the hot, “reduced” ion-rich vent fluid\nsolutions are injected into the cold, oxidizing seawater, and discovered in a\nAuthor reply 4:\nThank you very much for your valuable suggestion\nWe added “reduced” to the corresponding sentence.\nReviewer comment 5:\n65 precipitates rich in layered metal oxides, “hydroxides” and carbonates that act as a diffusion\nbarrier to maintain steep gradients “between” the two solutions\nAuthor reply 5:\nThank you very much for your valuable suggestion\nWe added “hydroxide” to the corresponding sentence.\nWe revised “in” to “between”.\nReviewer comment 6:\n71 electrical energy – a process [delete “so”] called osmotic energy harvesting. Selective ion\ntransport is critical for osmotic energy conversion in [delete 2nd “in”] cells\nAuthor reply 6:\nThank you very much for pointing out the typos.\nWe deleted “so” and “in”.\nReviewer comment 7:\n82 serpentinization, a process that forms serpentine from olivine. During the initial stages of\nHV formation in the SSF, a brucite-rich white chimney forms. “In time” the mineral\ncomposition of the HV precipitates change from predominantly brucite to carbonate-rich as\nthe discharge fluid activity “diminishes”.\nAuthor reply 7:\nThank you very much for your valuable suggestion\nWe revised sentence accordingly.\nReviewer comment 8:\nthicknesses of\nAuthor reply 8:\nThank you very much for your valuable suggestion\nWe deleted “a”.\nReviewer comment 9:\nof [delete “the”] the highest intensity in the WAXS\nAuthor reply 9:\nThank you very much for your valuable suggestion\nWe deleted “the” from the corresponding sentence.\nReviewer comment 10:\nimage of large HV wall sample area with overlaid vector plots [delete “, which were”] generated\nfrom a single scan direction and [delete “are”] plotted as white arrows.\nAuthor reply 10:\nThank you very much for your valuable suggestion\nWe deleted “which were” “are” from the corresponding sentence.\nReviewer comment 11:\n277 and anion-selective (Ca2+-adsorbed [delete “HV”) HV samples were fixed between two\nAuthor reply 11:\nThank you very much for your valuable suggestion\nWe deleted “HV” from the corresponding sentence.\nReviewer comment 12:\n373 the HV system, the self-assembly of [delete “a”] brucite,\nAuthor reply 12:\nThank you very much for your valuable suggestion\nWe deleted “a” from the corresponding sentence.\nReviewer comment 13:\n375 and functions may offer [delete “s”] new synthetic\nAuthor reply 13:\nThank you very much for your valuable suggestion\nWe deleted “s” from the corresponding sentence.\nThis concludes our response to the reviewers’ comments. We are grateful for their comments\nwhich have helped us improve the rigor and quality of our manuscript.\nAs to the comments of Reviewer #4\nNotice: We have split some of the comments (for example Comment 4A-E) to clarify our point-\nby-point response. All revisions based on this reviewer’s comments are highlighted in blue in\nthe manuscript and supporting information.\nReviewer comment 1:\nThank you for inviting me to review this article. The article contains a lot of interesting technical\ninformation and it was very nice to read it. The paper of Lee et al., contains three main claims:\n1) The formation of a nanoporous channel texture in brucite crystals forming the walls of\nhydrothermal vents. 2) These channels are vital in converting geochemical ionic gradients into\nelectrochemical potentials 3) Electrochemically coupled ion transport - a universal process in\ncell bioenergetics - emerged at the origin of life.\nAuthor reply 1:\nWe thank the reviewer for their interest in our work and for the constructive comments,\nwhich\nhave greatly helped us improve the quality of our work.\nReviewer comment 2:\nIt is important to note that the sample studied by the authors is not representative of\nhydrothermal vents as the title and the term “hydrothermal vent (HV)” used along the text\nsuggest. It is a sample of white fumaroles, i.e. of hydrothermal vents linked to the\nserpentinization process. There are many other hydrothermal vents completely different from\nthe geochemical, hydrochemical, and mineralogical point of view.\nAuthor reply 2:\nWe appreciate the reviewer’s invaluable comments regarding the types of hydrothermal vent\n(HV). To clarify the HV type studied in this work, we have added the term of “serpentinite-\nhosted” in the title, abstract, introduction, and discussion of the revised manuscript.\nRevised sentence:\nTitle\nOsmotic Energy Conversion in Serpentinite-Hosted Deep-Sea Hydrothermal Vents\nAbstract\nHere, we show that natural submarine serpentinite-hosted deep-sea hydrothermal vent (HV)\nprecipitates are capable of converting ionic gradients into electrochemical energy by selective\ntransport of Na+, K+, H+, and Cl-.\nIntroduction\nOff-axis HVs are mainly formed by the serpentinization process, in which the hydrolysis of\nolivine ((Mg,Fe) SiO ) generates alkaline fluids with diverse ions compositions17,29,30.\n2 4\nHere, we report the first detailed insights into the nanoscale structure and energy conversion\nfunctions of off-axis, serpentinite-hosted, HV precipitates.\nDiscussion\nNamely, in serpentine-hosted HV systems, a concentration gradient is established between the\nhot, reduced ion-rich vent fluid solutions and oxidizing seawater36,37,52,53, generating an electric\nfield perpendicular to the flow direction.\nBy performing a comprehensive material analysis of HVs, we have found the formation of\nnano- to millimeter-scale ion transport channels that provide new perspectives on the\nmultifaceted roles that serpentinite-hosted HVs may play.\nReviewer comment 3:\nThe walls of most white hydrothermal vents are mostly formed by brucite but they are not\nmonophasic. Depending on the geological context there is also an important mineralogical\ncontribution of calcium carbonate (calcite and aragonite) and also of magnesium carbonate.\nActually, the powder X-ray diffraction pattern in Supplementary Figure 2 shows the presence\nof these minerals. If the authors did a quantitative study of the mineralogical content of\nrepresentative samples of white fumaroles, they would find is very high proportion of calcium\ncarbonate, both calcite and aragonite, as well as magnesium and calcium carbonate. The reason\nis that the fluids expelled by hydrothermal vents associated with serpentinization are very\nalkaline, with pH values of around 11, that, a pH at which the CO2 dissolved in the ocean,\nwill be as carbonate anion.\nAuthor reply 3:\nWe are grateful to the reviewer’s insightful comments on mineralogy of serpentinite-hosted\nHVs. As the reviewer mentioned, our HV precipitate is not monophasic. Dr. Okumura, the\nsecond author of our work, has performed the intensive analysis of mineralogical, geological,\nand biological properties of Shinkai Seep Field (SSF) HV. The SSF is a location point of a\nserpentinite-hosted HV system in the southern Mariana forearc. A detailed quantitative analysis\nof the mineralogical distribution of HV was described in Dr. Okumura’s 2016 paper, Okumura,\nT., et al. (2016), Geochem. Geophys. Geosyst., 17, 3775-3796 (reference 31). As described in\nthe paper, the mineralogy of HV varies with vent fluid flux and is categorized into types I, II,\nIII (Fig. R1a). The ternary plot (Fig. R1b) illustrates the diverse mineral distribution among\nthree types. For our study, type I samples, characterized by the strongest fluid flux, were used\nfor analysis. Type I samples were further divided into three distinct parts (upper, middle, lower),\nand their compositions were plotted in the ternary plot as red dots. Brucite was the main\nconstituent of Type I (over 80%). The remaining 20% is composed of calcium carbonate and\nmagnesium carbonate, and these crystals are mainly localized in specific locations. In the case\nof calcium carbonate, it is primarily located on the outer surface of the HV, on the sea water\nside (Fig. R2). Magnesium carbonate, is observed on the surfaces of some channels (Fig. 4a-d\nand Supplementary Fig. 11).\nFig. R1 Previous study showing the mineralogical distribution of the Shinkai Seep Field\n(SSF) hydrothermal vents (HVs). a, A schematic of different types of HV depending on the\nvent fluid. b, Brucite-calcite-aragonite ternary plot showing mineralogy of the three types of\nSSF HVs.\n[Redacted]\nFig. R2 Powder X-ray diffraction patterns of HV samples. The difference of a and b is\nsampling position. Outside part of HV precipitate shows higher ratio of calcium carbonate\ncrystal than inside. The Synchrotron radiation powder X-ray diffraction patterns of were\nrecorded in the transmission mode (0.3 mm diameter capillary, λ = 0.4099 Å, 2θ range =\n2.986°–155.5309°, step width = 0.005°) on a diffractometer equipped with the OHGI system\nat RIKEN Materials Science Beamline (BL44B2) at SPring-8 (Kato, K. et al. (2019) J.\nSynchrotron Rad. 26, 762–773. Proposal No.: 20210064).\nWith regard to mineralogy of HV sample, we added the following sentences in the revised\nmanuscript to provide detailed information to the readers.\nAdded sentences:\nResults, Structure of HVs with nanopores part\nThe detailed mineralogy of SSF HVs was described in our previous publication31. Briefly, the\nmineralogy varies with vent fluid flow and is classified into types I, II, and III. In this study,\nType I sample of approximately 84 cm in height was analyzed, which contains brucite as the\nmajor component (~80%), with calcium carbonate and magnesium carbonate as minor\ncomponents.\nSupplementary Fig. 2 caption\nThe HV precipitate is mainly composed of brucite (constituting over 80%)1, with additional\nmixtures of calcium carbonate and magnesium carbonate crystals. These crystals exhibit\ndistinct localization within the HV structure: brucite is primarily found in the wall, calcite is\nlocated on the exterior and certain interior areas, and magnesium carbonate crystal is observed\non the surface of wall.\nReviewer comment 4A:\nThe study of wide-angle X-ray scattering is very nice but I am afraid that it is not convincing.\nThe entire diffraction study only proves the formation of brucite concentric layers with\ncrystals oriented perpendicular to the layers at the local scale. No nanochannels or anything\nsimilar are observed. The only information reporting nano-structures is in Figure 3 of the\nsupplementary materials, and they are nano-pores, not nano-channels. The authors wrote\nthat \"Pronounced anisotropy of brucite 001 diffraction was detected, indicating that (001)\nplane of brucite orients preferentially along the [001] crystallographic direction\" but (001)\nplanes are always perpendicular to [001] crystallographic direction. The plots shown in\nFigure 2 might be convincing of a radial texture of the brucite crystallites at the local\n(micrometer) scale but they are not representative of the large scale. Actually, why such a\ntexture should appear? The concentric layers of brucite could be the results of episodic\ngrowth related to periodic fluid dynamics but there is no reason inducing the brucite\nmicrocrystals to be co-oriented to each other.\nAuthor reply 4A:\nWe agree with the reviewer’s concern that the wide-angle X-ray scattering (WAXS) represents\nthe crystal orientation at the local scale, not in an entire part of HVs. To respond this comment,\nwe performed new experiments to clarify the location-dependent crystal alignment using\npolarized optical microscope (POM) and X-ray scattering measurements. As explained below,\nwe confirmed that the brucite crystals are aligned in a concentric manner radiating outwards\nfrom the fluid channel in nearly an entire part of HV material (approximately 84 cm height).\nAt the time of HV sampling from Shinkai Seep Field, the type-1 HV sample had a height of\napproximately 84 cm. This sample was divided into three parts (upper: ~28cm, middle: ~26cm,\nlower: ~30cm) for analysis in this study. We prepared 10-μm thickness thin sections (65\nsections) from the upper (35 sections), middle (10 sections), and lower (20 sections) parts of\nHV, and these thin sections are subjected to POM and WAXS analysis. Fig. R3 a-c represent\nPOM images of thin sections from the upper part, while Fig. R3 d and e are from the lower part.\nDue to its inherent birefringence, brucite displays distinctive interference colors (yellow or\nblue) when observed under cross-polarized conditions with the insertion of a 530 nm retardation\nplate. The appearance of a yellow subtractive interference color indicates perpendicular\nalignment of brucite c-axis relative to slow axis of 530 nm plate, while the presence of a blue\nadditive interference color suggests parallel alignment. Please note that essentially the same\nPOM interference colors were observed in all thin sections sampled from locations spanning\nfrom 26 cm to 84 cm apart (Fig. R3). This observation substantiates the presence of crystal\nalignment across the entire brucite wall of type I, SSF HV.\nFig. R3 (Supplementary Fig. 8) Polarized optical microscope (POM) images of the\nhydrothermal vent (HV) thin section samples. a-e, Thin section images analyzed by a cross-\npolarized condition with 530 nm retardation plate. The HV thin section samples were collected\nfrom different positions within the upper (a-c) and lower (d, e) part. A slow axis of retardation\nplate is shown as a double-headed arrow. The parallel direction of the slow axis of the\nretardation plate and the c-axis of brucite generates a blue interference color, whereas a\nperpendicular direction displays a yellow interference color. All scale bars 200 μm.\nFor the WAXS analysis samples, we newly collected the slice samples from different locations\nto those presented in our original manuscript (Figs. 2 and 3). Using a 100 μm X-ray beam, the\nslice samples were scanned, and the corresponding WAXS and small angle X-ray scattering\n(SAXS) patterns for each location are presented in the Fig. R4. Similar to Fig. 2, asymmetric\nring patterns were observed in samples from different locations, providing further support that\ncrystal alignment is present on HV walls across multiple locations.\nFig. R4 (Supplementary Fig. 9) Multiple location X-ray scattering analysis of\nhydrothermal vent (HV) walls. a,b, Optical images of HV slices with the wide-angle X-ray\nscattering (WAXS) scanning area indicated by a yellow rectangular box. c, Sequential plots of\nWAXS patterns scanned along lines A and B as shown in a. The line scan was conducted using\na 100 μm-sized beam with measurements taken at intervals of 100 μm. For line A, 8 points were\nmeasured (top plots in c), and for line B, 6 points were measured (bottom plots in c). Points 4\nand 5 in line A, corresponding to the resin part, were omitted. d, WAXS pattern results scanning\nalong line C. As shown in b, 18 points were measured, and the corresponding complete WAXS\npatterns are displayed. The yellow dashed line within the WAXS pattern indicates the direction\nof the strongest asymmetry. Asymmetric rings constantly appear in various parts of the HV wall,\nwith the direction continuously changing depending on the location. These findings align with\nFig. 2, illustrating a consistent alignment in HV walls across multiple locations.\nSmall-angle X-ray scattering (SAXS) pattern is also shown at the center of diffractogram.\nRed dashed lines indicate major axis of ellipsoid of SAXS region originated from the alignment\ndirection of particles or voids at nanoscale.\nWe added the new sentences to explain POM and WAXS/SAXS data in the revised manuscript\nas follows. We also added the corresponding POM and WAXS/SAXS data as Supplementary\nFig. 8 and Supplementary Fig. 9 in the revised Supplementary information.\nAdded sentences:\nResults, Alignment of brucite across the macro scale part\nPOM and WAXS analysis were also performed for the samples collected from multiple\nlocations in Type I sample. Thin sections were made from different parts of HV (the upper (35\nsections), middle (10 sections), and lower (20 sections)). Essentially the same POM interference\ncolors and anisotropic ring patterns were observed in all thin sections sampled from locations\nranging from 26 cm to 84 cm apart (Supplementary Figs. 8 and 9), confirming that the alignment\nobserved above extends to almost entire parts of Type I HV. Small angle X-ray scattering\n(SAXS) measurements of the HV wall showed ellipsoidal SAXS patterns attributable to\nnanoscale periodicity (Supplementary Fig. 10).\nMethod, Sample preparations part\nAt the time of HV collection in the deep-sea, the investigated HV had a height of approximately\n84 cm, divided into three parts (upper: ~28cm, middle: ~26cm, lower: ~30cm) for further\nanalysis.\nMultiple thin sections were made using samples collected from various locations in each HV\npart, upper, middle, and lower.\nAdded Figures:\nSupplementary Fig. 8 Polarized optical microscope (POM) images of the hydrothermal\nvent (HV) thin section samples.\nSupplementary Fig. 9 Multiple location X-ray scattering analysis of hydrothermal vent\n(HV) walls.\nReviewer comment 4B:\nNo nanochannels or anything similar are observed. The only information reporting nano-\nstructures is in Figure 3 of the supplementary materials, and they are nano-pores, not nano-\nchannels. The authors wrote that \"Pronounced anisotropy of brucite 001 diffraction was detected,\nindicating that (001) plane of brucite orients preferentially along the [001] crystallographic\ndirection\" but (001) planes are always perpendicular to [001] crystallographic direction. The\nplots shown in Figure 2 might be convincing of a radial texture of the brucite crystallites at the\nlocal (micrometer) scale but they are not representative of the large scale. Actually, why such a\ntexture should appear? The concentric layers of brucite could be the results of episodic\ngrowth related to periodic fluid dynamics but there is no reason inducing the brucite\nmicrocrystals to be co-oriented to each other.\nAuthor reply 4B:\nWe are grateful to the reviewer’s comment about nanochannels. According to the comments,\nwe have attempted to provide a more description of nano-channel structures and additional\nexperimental data obtained by SAXS measurements.\nThe morphology of pores can be analyzed by the hysteresis loop shape in gas adsorption\ndesorption isotherm data (K. S. W. Sing. et al. (1985) Pure & Appl. Chem., 57, 603-619).\nIUPAC provides a classification of hysteresis loops by relating hysteresis types (H1 to H4)\nto adsorbent texture (Fig. R5a) (M. Thommes, (2010) Chemie Ingenieur Technik, 82, 1059).\nAmong four types, H3 represents a slit-like pore which is characterized by the absence of\nlimiting adsorption, in contrast to the closed pore with saturating adsorption peak at high gas\npressure range (H2). The H3 hysteresis curve is often observed for clays and shale materials, in\nwhich the multilayer stacking of two-dimensional plate-like particles constitutes slit-like pores\n(Fig. R5 b, c). Please note that nitrogen adsorption-desorption isotherm analysis for our HV\nsamples also exhibits the H3-type hysteresis, demonstrating the presence of slit-like pores with\nno closed ends (Fig. R6). The nitrogen gas adsorption analysis indicates that the slit-like pores\nconstructed by multiple stacking of brucite nanoplate has an interconnected pore with a size\nranging from 2 to 100 nm (Fig. R6).\nConsistent with the H3-type hysteresis for the HV samples, SEM and TEM inspections also\nmanifested the multiple hierarchical stacking of plate-like structures (Fig. 1, g, h). Arrangement\nof the brucite nanocrystals results in slit-like spaces between the flat surfaces.\n[Redacted]\nFig. R5 Previous studies showing hysteresis loops of gas adsorption desorption isotherm.\na, Four hysteresis loops depending on the particular features of the pore structure (Sing, K. S.\nW. et al. (1985) Pure & Appl. Chem., 57, 603–619). b, c, Nitrogen adsorption–desorption curves\nof (b) the Es s shale in the Dongying Depression (Yong, Y. et al. (2022) ACS Omega 7, 36748–\n4\n36761) and (c) natural palygorskite (black and red lines), a hydrated magnesium-rich silicate\nclay mineral (Wang, W. et al. (2016) Sci. Rep. 6, 39599). These natural shale and clay show H3\nhysteresis.\nFig. R6 (Supplementary Fig. 3). Nitrogen adsorption-desorption analysis of hydrothermal\nvent (HV) sample. a, Adsorption desorption isotherm. Based on the features of the hysteresis\nloop, H3 type, the shape of pore is predicted to be open-ended (slit or plate-like pores)2.\nIn an attempt to provide the additional support for interconnected nanopores, we have\nperformed SAXS measurements using 100 μm X-ray beam. In contrast to WAXS, which\nprovide angstrom-level of periodicity such as the interplanar distance of brucite, SAXS shows\nnanometer scale of periodicity in the material. Please note that a clear elliptical shape was\nobserved in the SAXS pattern of the brucite wall (Fig. R7). This ellipsoid is originated from the\nexistence of nanometer-scale periodic scattering of particles and/or voids inside the sample,\nindicating that the plate crystals are indeed orientated with their flat surfaces (Liebi, M. et al,\n(2015) Nature 527, 349–352, Polishchuk, I. et al., (2017) Science 358, 1294–1298, Grünewald,\nT. A., et al., (2020) Sci. Adv. 6, eaba4171). The orientation of the alignment of mineral particle\nwith long axis observed in SAXS (major axis of ellipsoid, green line) is co-aligns with the\norientation of c-axis of brucite in WAXS (strong asymmetry direction, yellow line). This shows\nthat nanoscale periodic alignment exists along the direction of brucite [001] vector, as illustrated\nin the Fig. R7 e, confirming nanoscale channel-like structures, inside the HV wall. This co-\nalignment of the brucite (001) plane and particles is observed throughout the inorganic wall of\nHV (Fig. R4). The periodic nanoscale voids (Fig. R7 e, right) can provide nano-distance\nchannel-like structures.\nFig. R7 (Supplementary Fig. 10) X-ray scattering patterns of hydrothermal vent (HV)\nsamples. a, Small angle X-ray scattering (SAXS) pattern of HV sample. b-d, Wide angle X-\nray scattering (WAXS) pattern of HV samples at different locations. WAXS patterns are also\ndisplaying the SAXS pattern at the center. Yellow dashed lines show highest intensity of WAXS\nplots indicating alignment direction of (001) plane of brucite. Green dashed lines indicate major\naxis of ellipsoid of SAXS region suggesting alignment direction of particles or voids. e,\nSchematic of the (001) brucite plane and particle alignment shown in (d). The alignment\n(periodicity) direction observed in WAXS and the alignment direction observed in SAXS are\nmatched in the same direction.\nBased on the nitrogen adsorption-desorption isotherm and WAXS/SAXS analysis, we have\nadded new schematics showing the alignment of plate-like brucite crystals with interconnected\nnanopores.\nFig. R8 (Fig. 5) Ionic transport by hydrothermal vent (HV) precipitates. a, A photograph\nof the HV-PDMS membrane sample used for the measurement. b, Experimental setup for\nmeasuring current-voltage response of the HV samples. For conductance measurement, both\nsides were filled with KCl solution of the same concentration. c, Electrical conductance of HV\nsample as a function of KCl concentrations with pH 10. d, Schematics showing the continuous\narrangement in HV inorganic wall, comprising multilayers of the brucite (001) plane at the\nangstrom level, plate nanocrystal stacks at the nanometer level, and a radial alignment structure\nextending to the millimeter level. This configuration results in continuous channels, allowing\nthe HV walls to function as a selectively permeable membrane for ion transport. e, Schematics\nshowing osmotic power generation. Upon exposure to KCl, an electric double layer (EDL) is\nformed on the surface of brucite crystals, exhibiting varying Debye screening lengths (λ )\nD\ndepending on KCl concentration. Blue represents the EDL formed on the brucite surface by\nexposure to ion concentration gradient. The prevalent pore size in HV walls is measured to be\n60 nm, which means that the 30 nm EDL formed by 0.1 mM KCl completely overlaps inside\nthe HV nanopore. Effective overlap of EDLs within narrow nanopores establishes a screening\nbarrier that is permeable only to ions with specific charges. Acting as a selective membrane, the\nHV wall allows selective movement of counterions from a high concentration to a low\nconcentration, generating an ionic current.\nTo reflect the above explanation and experimental data in the revised manuscript, we made the following revision:\n(1) To increase the accuracy of the explanation of pore morphology, we revised the term of\n“nanochannel” to “interconnected nanopores” in a whole part of manuscript.\n(2) To increase the accuracy of the explanation of pore morphology, we added the term of “slit-\nlike pores (H3 type)” in the relevant sentence in the revised manuscript.\n(3) We added the SAXS data and corresponding explanation in the Results, Fig. 5d caption, and\nSupplementary Fig. 10.\n(4) We created the new schematic to explain ion transport through the charged nanopores\nconstructed by multiple staking and alignment of brucite nanocrystal (Fig. 5).\nAdded sentences:\nResults, Alignment of brucite across the macro scale part\nMaterial characterization based on SEM, TEM, nitrogen adsorption-desorption isotherm and\nWAXS/SAXS demonstrates collectively the anisotropic alignment and multilayer stacking of\nplate-like brucite nanocrystals, forming interconnected nanopores referred to as nanochannel\ninside the HV walls (Fig. 2f).\nMethod, Characterization of HV\nIn the case of small-angle X-ray scattering (SAXS), the same beamline, RIKEN Structural\nBiology I Beamline (BL38B1), was used for measurement. The sample-to-detector distance\nwas 2.5450 m. The beam is collimated to the size of 100 µm(H) x 100 µm(V).\nRevised Figures:\nSupplementary Fig. 3. Nitrogen adsorption-desorption analysis of hydrothermal vent\n(HV) sample.\nAdded Figures:\nFig. 5 Ionic transport by hydrothermal vent (HV) precipitates.\nSupplementary Fig. 10 X-ray scattering patterns of hydrothermal vent (HV) samples.\nReviewer comment 4C:\nThe authors wrote that \"Pronounced anisotropy of brucite 001 diffraction was detected,\nindicating that (001) plane of brucite orients preferentially along the [001] crystallographic\ndirection\" but (001) planes are always perpendicular to [001] crystallographic direction.\nAuthor reply 4C:\nWe appreciate the reviewer’s comment. We noticed that our original sentence was misleading\nand therefore have revised it as follows.\nRevised sentence:\nPronounced anisotropy of brucite 001 diffraction was detected, indicating that (001) plane of\nbrucite orients preferentially inside the HV wall.\nReviewer comment 4D:\nThe plots shown in Figure 2 might be convincing of a radial texture of the brucite crystallites\nat the local (micrometer) scale but they are not representative of the large scale.\nAuthor reply 4D:\nAs described in Author reply 4A, to respond to the reviewer’s comments, we have performed\nthin section analysis using samples collected from multiple locations. When obtaining HV\nsamples from the deep-sea, we divided the HV structure (84 cm total height) into three parts\n(upper, middle, lower). Samples were collected from each part to create thin sections. One of\nthese thin sections was shown in Fig. 3c through POM analysis in original manuscript (collected\nfrom top part). As detailed in Author reply 4A, POM analysis provides an effective method for\nidentifying the orientation of brucite. Using POM with 530 nm retardation plate, we\nadditionally analyzed the thin section samples collected from upper and lower parts (Fig. R3).\nIn our original manuscript (Fig. 3), the distinctive alternating blue and yellow interference\ncolors observed in the horizontal slice indicates the radial alignment of the brucite inorganic\nwall. This feature is further evident in thin section images at different locations (Fig. R3 a, d).\nThe collection locations for each sample are spaced roughly 26 cm to 84 cm apart,\ndemonstrating a consistent alignment across the entire HV wall.\nReviewer comment 4E:\nActually, why such a texture should appear? The concentric layers of brucite could be the results\nof episodic growth related to periodic fluid dynamics but there is no reason inducing the brucite\nmicrocrystals to be co-oriented to each other.\nAuthor reply 4E:\nThank you very much for raising the question about the anisotropic crystal arrangement found\nin natural HV materials. In the discussion section of original manuscript, we proposed\ndiffusiophoresis generated by chemical gradients as a force to arrange the brucite nanocrystal.\nSentence copied from the original manuscript:\nIn addition to the periodic pattern, the anisotropic crystal arrangement observed in the HV\nsamples (Figs. 2, 3, Supplementary Figs. 4-7) may be attributable to diffusiophoresis, a\nphenomenon in which charged particles move along a concentration gradient of ions, such as\nNaCl, KCl, protons, and hydroxides42-44. Namely, during HV formation, a concentration\ngradient is established between charged brucite precipitates in the vent fluid and seawater45-47.\nThe resulting diffusiophoretic field provides a directional driving force to the charged\nprecipitates and promotes the concentric arrangement of particles around the channels\n(Supplementary Notes).\nPrevious studies have shown the alignment of anisotropic particles, such as two-dimensional\ngraphene oxide sheets, under flow conditions. Anisotropic particles typically align along the\nflow direction (shear flow-induced alignment) (Hayter, J. B. & Penfold, J. (1984) J. Phys. Chem.\n88, 4589-4593, Xin, G., et al. (2019) Nat. Nanotechnol. 14, 168-175, Zhao, C., et al. (2020)\nNature 580, 210-215). Contrary to the shear flow-induced orientation, the orientation of brucite\nin HVs deviates from the direction of vent fluid flow (Supplementary Fig. 7). Namely, the two-\ndimensional brucite sheets align perpendicularly to the flow direction. This in turn means that\nthey align with the direction of chemical gradients present between vent fluid and seawater.\nWe therefore hypothesized that diffusiophoresis, which generates an electric field perpendicular to\nthe flow direction, is the force for the brucite orientation.\nDiffusiophoresis is the phenomenon where particles move along a concentration gradient of\nsalt. In gradient condition, difference in cation and anion diffusion speed generates an electric\nfield and when a charged particle exposed to this field, the particle moves following the field\ndirection. Additionally, the creation of different electrical double layers (Debye screening\nlength) on particle surface due to the salt gradient results in a pressure gradient, inducing a\ntangential flow near the surface. This motion of a particle generated by chemical gradients can\nbe explained by the equation below. (Prieve, D. C. et al. (1984) J. Fluid Mech., 148, 247-269)\n𝜖 𝑘𝑇 𝑘𝑇 2 𝑍𝑒𝜁\n𝑈wh=ere ε, η, k[,𝛽 T𝜁, −Z,2 e, ζ,l nan(d1 −C,t aanreh, respe)c]ti∇vellny,𝐶 the electrical permittivity, fluid viscosity,\n4𝜋𝛽𝑍𝑒 𝑍𝑒 4𝑘𝑇\nBoltzmann constant, absolute temperature, valence, the charge of an electron, a zeta potential\nof particle, and the ionic concentration field. β is the diffusivity difference factor, where D and\n+\nD are, respectively, the diffusion constants of the cations and anions.\n-\n𝐷+ −𝐷−\n𝛽Th=erefore, the particle velocity, U, is influenced by local fluid properties (ε, η, ζ) and salt\n𝐷+ +𝐷−\ncharacteristics (β, Z, C).\nTo test the hypothesis of diffusiophoresis-induced orientation, we are currently conducting\nthe microfluidic experiments to replicate the chemical gradients and precipitation processes in\nserpentinite-hosted HV systems. As shown in Reviewer Only Material, we have confirmed that\nchemical gradients between two fluids can indeed induce crystal alignment. As expected from\nthe nature of diffusiophoresis explained above, the optimizing parameters, including local fluid\nproperties and salt characteristics, enhanced the degree of brucite alignment, resulting in\nessentially the same orientation with those found in natural HV samples. In this response letter,\nwe present preliminary data in a separated \"reviewer-only\" material to support this, which\nwill be addressed in another publication.\nAdded sentences:\nDiscussion\nIn addition to the periodic pattern, it is worth noting the anisotropic crystal arrangement\nobserved in nearly the entire part of the HV samples (Figs. 2, 3, Supplementary Figs. 4-10).\nPrevious studies have demonstrated the alignment of anisotropic particles, such as two-\ndimensional graphene oxide sheets, under flow conditions45,46. Anisotropic particles typically\nalign along the flow direction (shear flow-induced alignment)45-47. Contrary to the shear flow-\ninduced orientation, the two-dimensional brucite sheets align perpendicularly to the flow\ndirection (Supplementary Fig. 7). We speculate diffusiophoresis, a phenomenon in which\ncharged particles move along a concentration gradient of ions, such as NaCl, KCl, protons, and\nhydroxides48-51, acts as a driving force for the alignment. Namely, in serpentine-hosted HV\nsystems, a concentration gradient is established between the hot, reduced ion-rich vent fluid\nsolutions and oxidizing seawater36,37,52,53, generating an electric field perpendicular to the flow\ndirection. The resulting diffusiophoretic field provides a directional driving force to the charged\nprecipitates and promotes the concentric arrangement of nanocrystals around the channels\n(Supplementary Notes).\nReviewer comment 5A:\nThe experimental procedure to fabricate a “membrane” from the sample of the wall of the white\nfumaroles is not very “clean” and I suspect that would modify the properties of the material.\nThe experimental method to measure with these “membrane” the electrical chemical energy\ndoes not mimic the natural process.\nAuthor reply 5A:\nTo address the reviewer’s concern, we have analyzed the HV samples before and after\nmembrane fabrication using Raman spectroscopy (before: Fig. 4e, after: Fig. R9) and WAXS\n(before: Supplementary Fig. 2a, after: Supplementary Fig. 4c, d). Raman spectra of the HV\nsamples after membrane fabrication show the characteristic peaks of brucite at 280 cm-1 and\n444 cm-1. The spectrum was identical with that observed for the sample before membrane\nfabrication (no peak shifts at the spectral resolution of 4 cm-1). Consistent with the Raman\nanalysis, membrane fabrication shows negligible changes in the X-ray scattering (diffraction)\npeaks for brucite (Supplementary Fig. 4c, d). Therefore, we can conclude that the material\nproperties of brucite remain intact after the membrane fabrication process.\nFig. R9 (Supplementary Fig. 17) Raman measurements of the cross-section of the cut\nhydrothermal vent (HV)-PDMS membrane sample. a, Optical image displaying the cross-\nsection of the HV-PDMS membrane with marked Raman measurement points. b, Raman\nspectra at each position. Clear and unshifted peaks at 280 and 444 cm-1, corresponding to brucite,\nare distinctly visible (yellow rectangle).\nTo make these points clear, we added the following sentence in the revised manuscript.\nAdded sentences:\nMethod, HV-PDMS membrane fabrication part\nRaman and WAXS analysis confirmed that material properties of brucite remain intact during\nmembrane fabrication processes (Supplementary Fig. 17).\nAdded Figures:\nSupplementary Fig. 17 Raman measurements of the cross-section of the cut hydrothermal\nvent (HV)-PDMS membrane sample\nReviewer comment 5B:\nThe solutions to use should be those of playing in the natural vents, i.e. a reducing high pH fluid\ninside the wall and seawater outside the wall. The experimental measurements -even if they are\nnot very conclusive- do not model properly the natural scenario.\nAuthor reply 5B:\nThank you for pointing out the important issue. We agree with the reviewer's comments\nabout the importance of simulating the natural environment. In the original manuscript, we\nexperimentally demonstrated the selective ion transfer across the natural HV sample from an\nionic gradient of NaCl and KCl. This was explicitly verified by in-situ monitoring of the\nosmotic potential and the direct detection of cation (Na+) and anion (Cl-) using Na+ and Cl- ion-\nsensitive electrodes. In addition, we experimentally demonstrated the selective transport of H+\nunder simulated alkaline hydrothermal conditions. Namely, the natural HV sample was placed\nbetween a reducing high pH fluid and an oxidizing low pH solution.\nSentence copied from the original manuscript:\nOne of the most characteristic features of serpentinite-hosted HV systems is the presence of a\nlarge pH gradient between the seawater (pH ~ 7) and vented fluids (pH ~ 9-11). To examine\nconversion of a pH gradient to electrochemical energy, the cation-selective (CO 2--adsorbed)\nresponse was measured (Fig. 5g, gray circle).\nSpecifically, we have attempted to simulate alkaline hydrothermal conditions based on the\nrepresentative papers in the research field of simulated alkaline hydrothermal vent experiments\n(Hudson, R. et al. (2020) Proc. Natl. Acad. Sci. U.S.A., 117, 22873-22879, Barge, L. M. et al.\n(2012) Langmuir 28, 3714-3721, Moller, F. M. et al. (2017) Angew. Chem. Int. Ed. Engl. 56,\n2340-2344). To verify which ion carrier is responsible for the osmotic energy conversion,\nprotons or hydroxide ions, we also performed a deuterium gradient (pD) (pD 4 and pD 10). A\nhigh H/D kinetic isotope effect (2.3) manifests that under simulated alkaline HV conditions,\nprotons (not hydroxide ions) play a predominant role in converting pH gradient into electrical\nenergy. To clarify the above point, we have added the following sentences in the revised\nmanuscript.\nAdded sentences:\nResults, Osmotic energy conversion in HVs part\nExperimental simulation of alkaline HV systems has been reported in the context of origin of\nlife on the ancient Earth, using reducing high pH fluid (pH 10 ~ 12) and oxidative low pH\nseawater (pH 4 ~ 6) simulant22,36,37.\nIn our effort to further respond to the reviewer’s comments, we have conducted osmotic\npower generation experiments by replicating actual chemical compositions of alkaline vent fluids\nand sea water based on the geochemical dataset at Shinkai Seep Field (SSF). The simulant\nsolutions were prepared by referencing a paper that analyzed the solution of SSF HV\n(Onishi Y., et al. (2018) PLoS ONE, 13, e0199000).\nTable R1. Chemical compositions of Shinkai Seep Field (SSF) hydrothermal vent (HV)\nfluid and sea water.\nA mixed solution consisting of NaCl (500mM), MgCl (50mM), CaCl (10mM), and Na SO\n2 2 2 4\n(30mM), which were considered to be the major charged components, was prepared. The pH\nlevels were adjusted to 6.6 for the seawater simulant and 9.9 for the vent fluid simulant.\nHowever, please note that, in the presence of multiple ions, the selective ion transfer and\nsubsequent generation of electric potential cannot be monitored experimentally due to the lack\nof ion-selective sensor electrodes. Namely, in the research field of osmotic power generation,\nAg/AgCl is widely used as a sensor electrode. It can indeed monitor the concentration change\nof Na+, K+, H+, Cl- and OH- when the ionic gradient is derived from mono-ionic species.\nHowever, the electrochemical signal in principle becomes the summation of the open circuit\nvoltage derived from transport of mixed ion species. In fact, unlike the mono-ionic species (Fig.\n6), we were unable to detect the reliable electrochemical signals from the simulant of SSF HV\nconditions. This is an intrinsic technical problem raised by multi-ion component systems,\nleaving room for advancement of ion-sensitive sensor electrodes. We believe that this is beyond\nthe scope of our study and hope that our experimental data under simulated alkaline\nhydrothermal vent conditions based on the paper by Hudson, R. et al. (Proc. Natl. Acad. Sci.\nU.S.A., 2020, 117, 22873-22879) adequately respond to the reviewer's comments.\nReviewer comment 5C:\nThe existence of the generation of an electrochemical potential in osmotic-driven structures has\nbeen demonstrated and measured (see Glaab et al., (Angew. Chem. Int. Ed. 2012, DOI:\n10.1002/ange.201107754 ). The electrochemical potential was proposed to be a summation of\nthree factors, of which a large pH difference is a major contributor. Perhaps the authors could\nfind ideas for experimental testing and explanations in that article. And of course, a porous\nstructure connecting the two sides of the membranes is key for the formation of the\nelectrochemical potential.\nAuthor reply 5C:\nWe appreciate the reviewer to introduce a relevant paper to our work. In the paper by Glaab et\nal, a silicate-based tubular membrane structure was fabricated by immersing a tablet of CoCl\n2\nin silica solution (chemical garden system). By placing two Pt electrodes inside and outside the\ntubular membrane structure, they monitored the time course of overall cell potential (ΔE ). The\nh\nΔE , which is also refer to as electrochemical potential, was then divided by three components:\nh\npH (ΔE ), diffusion (ΔE ), and membrane (ΔE ) potential.\npH diff m\nΔE = ΔE + ΔE + ΔE\nh pH diff m\nIn our study, we performed essentially the same analysis as that reported by Glaab et al.\nexcept that we used an Ag/AgCl electrode instead of a Pt electrode. The use of an Ag/AgCl\nelectrode allows to increase the sensitivity to monitor the electrochemical potential (ΔE )\nh\ncaused not only by pH, but also by concentration change of cations (Na+ and K+) and anions\n(Cl-) (Macha, M. et al. (2019) Nat. Rev. Mater. 4, 588-605, Siria, A. et al. (2013) Nature 494,\n455-458, Feng, J. et al. (2016) Nature 536, 197-200). Therefore, in contrast to the work by\nGlaab et al, we can monitor the osmotic power generation caused by selective transport of Na+,\nK+, H+, and Cl-. Using an Ag/AgCl electrode, we monitored the open circuit voltage (V )\noc\nestablished across the HV membrane, which corresponds to ΔE . Then, the redox potential\nh\n(V ) of the electrode-solution interface was experimentally monitored by immersing two\nredox\nAg/AgCl electrodes to the solutions without HV membrane, which corresponds to ΔE .\npH\nFinally, subtracting the redox potential from V gives the pure osmotic potential of the HVs\noc\n(V = V + V ).\nos oc redox\nIn the revised manuscript, we have cited the paper by Glaab et al (Angew. Chem. Int. Ed.\n2012, DOI: 10.1002/ange.201107754) as reference 53.\nReviewer comment 6:\nThroughout the manuscript, the authors try to prove that the brucite crystallites have confined\nnanochannels with charged surfaces, which are the requirements for the conversion to work.\nThe idea is good and the implications -if the hypothesis is demonstrated- are interesting, but the\nresults that count do not demonstrate, in the opinion of this referee, the existence of osmotic\nenergy conversion processes in hydrothermal vents.\nAuthor reply 6:\nOnce again, we thank the reviewer for their interest in our work and for their constructive\ncomments, which have helped us greatly improve the quality of our work. As described above,\nwe have addressed all the comments raised by the reviewer, which we believe provides\nappropriate responses. Based on the newly conducted experiments (SAXS, POM, Raman and\nmicrofluidics) and the detailed discussion of mineral composition, nanopores morphology (slit-\nlike pores), crystal orientation mechanisms (diffusiophoresis), simulation of alkaline\nhydrothermal vent conditions (pH4~10), and measurement details of osmotic power generation,\nwe believe that our main finding of cation- and anion-selective ion transfer across natural HV\nmaterials is strengthened.\nThis concludes our response to the reviewers’ comments. We are grateful for their comments\nwhich have helped us improve the rigor and quality of our manuscript.\nREVIEWER COMMENTS\nReviewer #1 (Remarks to the Author):\nThere are a total of four reviewers who seem to come from different background to provide\nvaluable comments about this manuscript. Overall, the critical comments are from Reviewer\n#4, about which the authors mostly have addressed, with some relevant additional\nexperiments, data syntheses, and comment incorporation. This reviewer thinks that the\nrevised manuscript is acceptable.\nReviewer #2 (Remarks to the Author):\nI enjoyed reading the revised manuscript. In my opinion this is an important piece of work\nthat should be published.\nReviewer #3 (Remarks to the Author):\nAll my concerns have been addressed and to the best of my knowledge the manuscript is\nacceptable and now ready for publication.\nReviewer #4 (Remarks to the Author):\nsee attached.\nReviewer #4 attached file\nThe authors have made an extraordinary effort to answer the questions and criticisms of\nthe referees. I am very impressed by their careful response, and I wish to congratulate\nthem. In some cases, their answers are clear and satisfactory; however, to my\nunderstanding, other ones are not so convincing. Below, you can find my comments (in\nred) on specific points:\nReviewer comment 2:\nIt is important to note that the sample studied by the authors is not representative of\nhydrothermal vents as the title and the term “hydrothermal vent (HV)” used along the\ntext suggest. It is a sample of white fumaroles, i.e. of hydrothermal vents linked to the\nserpentinization process. There are many other hydrothermal vents completely different\nfrom the geochemical, hydrochemical, and mineralogical point of view.\nAuthor reply 2:\nWe appreciate the reviewer’s invaluable comments regarding the types of hydrothermal\nvent (HV). To clarify the HV type studied in this work, we have added the term of\n“serpentinite-hosted” in the title, abstract, introduction, and discussion of the revised\nmanuscript.\nThank you. I think this is an important clarification\nReviewer comment 3:\nThe walls of most white hydrothermal vents are mostly formed by brucite but they are\nnot monophasic. Depending on the geological context there is also an important\nmineralogical contribution of calcium carbonate (calcite and aragonite) and also of\nmagnesium carbonate. Actually, the powder X-ray diffraction pattern in Supplementary\nFigure 2 shows the presence of these minerals. If the authors did a quantitative study of\nthe mineralogical content of representative samples of white fumaroles, they would find\nis very high proportion of calcium carbonate, both calcite and aragonite, as well as\nmagnesium and calcium carbonate. The reason is that the fluids expelled by\nhydrothermal vents associated with serpentinization are very alkaline, with pH values of\naround 11, that, a pH at which the CO2 dissolved in the ocean, will be as carbonate\nanion.\nAuthor reply 3:\nWe are grateful to the reviewer’s insightful comments on mineralogy of serpentinite-\nhosted HVs. As the reviewer mentioned, our HV precipitate is not monophasic. Dr.\nOkumura, the second author of our work, has performed the intensive analysis of\nmineralogical, geological, and biological properties of Shinkai Seep Field (SSF) HV.\nThe SSF is a location point of a serpentinite-hosted HV system in the southern Mariana\nforearc. A detailed quantitative analysis of the mineralogical distribution of HV was\ndescribed in Dr. Okumura’s 2016 paper, Okumura, T., et al. (2016), Geochem.\nGeophys. Geosyst., 17, 3775-3796 (reference 31). As described in the paper, the\nmineralogy of HV varies with vent fluid flux and is categorized into types I, II, III (Fig.\nR1a). The ternary plot (Fig. R1b) illustrates the diverse mineral distribution among\nthree types. For our study, type I samples, characterized by the strongest fluid flux, were\nused for analysis. Type I samples were further divided into three distinct parts (upper,\nmiddle, lower), and their compositions were plotted in the ternary plot as red dots.\nBrucite was the main constituent of Type I (over 80%). The remaining 20% is\ncomposed of calcium carbonate and magnesium carbonate, and these crystals are\nmainly localized in specific locations. In the case of calcium carbonate, it is primarily\nlocated on the outer surface of the HV, on the sea water side (Fig. R2). Magnesium\ncarbonate, is observed on the surfaces of some channels (Fig. 4a-d and Supplementary\nFig. 11).\nFig. R1 Previous study showing the mineralogical distribution of the Shinkai Seep\nField (SSF) hydrothermal vents (HVs). a, A schematic of different types of HV\ndepending on the vent fluid. b, Brucite-calcite-aragonite ternary plot showing\nmineralogy of the three types of SSF HVs.\n[Redacted]\nFig. R2 Powder X-ray diffraction patterns of HV samples. The difference of a and b\nis sampling position. Outside part of HV precipitate shows higher ratio of calcium\ncarbonate crystal than inside. The Synchrotron radiation powder X-ray diffraction\npatterns of were recorded in the transmission mode (0.3 mm diameter capillary, λ =\n0.4099 Å, 2θ range = 2.986°–155.5309°, step width = 0.005°) on a diffractometer\nequipped with the OHGI system at RIKEN Materials Science Beamline (BL44B2) at\nSPring-8 (Kato, K. et al. (2019) J. Synchrotron Rad. 26, 762–773. Proposal No.:\n20210064).\nWith regard to mineralogy of HV sample, we added the following sentences in the\nrevised manuscript to provide detailed information to the readers.\nAdded sentences:\nResults, Structure of HVs with nanopores part\nThe detailed mineralogy of SSF HVs was described in our previous publication31.\nBriefly, the mineralogy varies with vent fluid flow and is classified into types I, II, and\nIII. In this study, Type I sample of approximately 84 cm in height was analyzed, which\ncontains brucite as the major component (~80%), with calcium carbonate and\nmagnesium carbonate as minor components.\nSupplementary Fig. 2 caption\nThe HV precipitate is mainly composed of brucite (constituting over 80%)1, with\nadditional mixtures of calcium carbonate and magnesium carbonate crystals. These\ncrystals exhibit distinct localization within the HV structure: brucite is primarily found\nin the wall, calcite is located on the exterior and certain interior areas, and magnesium\ncarbonate crystal is observed on the surface of wall.\nThat makes sense; calcite is mainly in the outer part because it is in contact with the\ncarbonate-rich seawater. Then, I think it is important to explain to the readers that your\napproach works for type-I hydrothermal vents. It is not clear to me what the difference\nis between type I upper, type I Middle, and type I lower. In any case, it seems that you\nalways have in type-I HV calcite in the outer part. Then, how the ionic flow occurs\nthrough the calcite outer layer?\nReviewer comment 4A:\nThe study of wide-angle X-ray scattering is very nice but I am afraid that it is not\nconvincing. The entire diffraction study only proves the formation of brucite concentric\nlayers with crystals oriented perpendicular to the layers at the local scale.\nAuthor reply 4A:\nWe agree with the reviewer’s concern that the wide-angle X-ray scattering (WAXS)\nrepresents the crystal orientation at the local scale, not in an entire part of HVs. To\nrespond this comment, we performed new experiments to clarify the location-dependent\ncrystal alignment using polarized optical microscope (POM) and X-ray scattering\nmeasurements. As explained below, we confirmed that the brucite crystals are aligned\nin a concentric manner radiating outwards from the fluid channel in nearly an entire part\nof HV material (approximately 84 cm height).\nAt the time of HV sampling from Shinkai Seep Field, the type-1 HV sample had a\nheight of approximately 84 cm. This sample was divided into three parts (upper: ~28cm,\nmiddle: ~26cm, lower: ~30cm) for analysis in this study. We prepared 10-μm thickness\nthin sections (65 sections) from the upper (35 sections), middle (10 sections), and lower\n(20 sections) parts of HV, and these thin sections are subjected to POM and WAXS\nanalysis. Fig. R3 a-c represent POM images of thin sections from the upper part, while\nFig. R3 d and e are from the lower part. Due to its inherent birefringence, brucite\ndisplays distinctive interference colors (yellow or blue) when observed under cross-\npolarized conditions with the insertion of a 530 nm retardation plate. The appearance of\na yellow subtractive interference color indicates perpendicular alignment of brucite c-\naxis relative to slow axis of 530 nm plate, while the presence of a blue additive\ninterference color suggests parallel alignment. Please note that essentially the same\nPOM interference colors were observed in all thin sections sampled from locations\nspanning from 26 cm to 84 cm apart (Fig. R3). This observation substantiates the\npresence of crystal alignment across the entire brucite wall of type I, SSF HV.\nFig. R3 (Supplementary Fig. 8) Polarized optical microscope (POM) images of the\nhydrothermal vent (HV) thin section samples. a-e, Thin section images analyzed by\na cross-polarized condition with 530 nm retardation plate. The HV thin section samples\nwere collected from different positions within the upper (a-c) and lower (d, e) part. A\nslow axis of retardation plate is shown as a double-headed arrow. The parallel direction\nof the slow axis of the retardation plate and the c-axis of brucite generates a blue\ninterference color, whereas a perpendicular direction displays a yellow interference\ncolor. All scale bars 200 μm.\nFor the WAXS analysis samples, we newly collected the slice samples from different\nlocations to those presented in our original manuscript (Figs. 2 and 3). Using a 100 μm\nX-ray beam, the slice samples were scanned, and the corresponding WAXS and small\nangle X-ray scattering (SAXS) patterns for each location are presented in the Fig. R4.\nSimilar to Fig. 2, asymmetric ring patterns were observed in samples from different\nlocations, providing further support that crystal alignment is present on HV walls across\nmultiple locations.\nFig. R4 (Supplementary Fig. 9) Multiple location X-ray scattering analysis of\nhydrothermal vent (HV) walls. a,b, Optical images of HV slices with the wide-angle\nthe wide-angle X-ray\nscattering (WAXS) scanning area indicated by a yellow rectangular box. c,\nSequential plots of WAXS patterns scanned along lines A and B as shown in a. The line\nscan was conducted using a 100 μm-sized beam with measurements taken at intervals of\n100 μm. For line A, 8 points were measured (top plots in c), and for line B, 6 points\nwere measured (bottom plots in c). Points 4 and 5 in line A, corresponding to the resin\npart, were omitted. d, WAXS pattern results scanning along line C. As shown in b, 18\npoints were measured, and the corresponding complete WAXS patterns are displayed.\nThe yellow dashed line within the WAXS pattern indicates the direction of the strongest\nasymmetry. Asymmetric rings constantly appear in various parts of the HV wall, with\nthe direction continuously changing depending on the location. These findings align\nwith Fig. 2, illustrating a consistent alignment in HV walls across multiple locations.\nSmall-angle X-ray scattering (SAXS) pattern is also shown at the center of\ndiffractogram. Red dashed lines indicate major axis of ellipsoid of SAXS region\noriginated from the alignment direction of particles or voids at nanoscale.\nWe added the new sentences to explain POM and WAXS/SAXS data in the revised\nmanuscript as follows. We also added the corresponding POM and WAXS/SAXS data\nas Supplementary Fig. 8 and Supplementary Fig. 9 in the revised Supplementary\ninformation.\nAdded sentences:\nResults, Alignment of brucite across the macro scale part\nPOM and WAXS analysis were also performed for the samples collected from multiple\nlocations in Type I sample. Thin sections were made from different parts of HV (the\nupper (35 sections), middle (10 sections), and lower (20 sections)). Essentially the same\nPOM interference colors and anisotropic ring patterns were observed in all thin sections\nsampled from locations ranging from 26 cm to 84 cm apart (Supplementary Figs. 8 and\n9), confirming that the alignment observed above extends to almost entire parts of Type\nI HV. Small angle X-ray scattering (SAXS) measurements of the HV wall showed\nellipsoidal SAXS patterns attributable to nanoscale periodicity (Supplementary Fig. 10).\nMethod, Sample preparations part\nAt the time of HV collection in the deep-sea, the investigated HV had a height of\napproximately 84 cm, divided into three parts (upper: ~28cm, middle: ~26cm, lower:\n~30cm) for further analysis.\nMultiple thin sections were made using samples collected from various locations in\neach HV part, upper, middle, and lower.\nAdded Figures:\nSupplementary Fig. 8 Polarized optical microscope (POM) images of the\nhydrothermal vent (HV) thin section samples.\nSupplementary Fig. 9 Multiple location X-ray scattering analysis of hydrothermal\nvent (HV) walls.\nReviewer comment 4B:\nNo nanochannels or anything similar are observed. The only information reporting\nnano-structures is in Figure 3 of the supplementary materials, and they are nano-pores,\nnot nano-channels. The authors wrote that \"Pronounced anisotropy of brucite 001 diffraction was detected,\nindicating that (001) plane of brucite orients preferentially along the [001]\ncrystallographic direction\" but (001) planes are always perpendicular to [001]\ncrystallographic direction. The plots shown in Figure 2 might be convincing of a radial texture of the brucite crystallites at the local (micrometer) scale but they are not representative of the large scale. Actually, why such a texture should appear? The concentric layers of brucite could be the results of episodic growth related to periodic fluid dynamics but there is no reason inducing the brucite microcrystals to be co-oriented to each other.\nAuthor reply 4B:\nWe are grateful to the reviewer’s comment about nanochannels. According to the\ncomments, we have attempted to provide a more description of nano-channel structures\nand additional experimental data obtained by SAXS measurements.\nThe morphology of pores can be analyzed by the hysteresis loop shape in gas\nadsorption desorption isotherm data (K. S. W. Sing. et al. (1985) Pure & Appl. Chem.,\n57, 603-619). IUPAC provides a classification of hysteresis loops by relating hysteresis\ntypes (H1 to H4) to adsorbent texture (Fig. R5a) (M. Thommes, (2010) Chemie\nIngenieur Technik, 82, 1059). Among four types, H3 represents a slit-like pore which is\ncharacterized by the absence of limiting adsorption, in contrast to the closed pore with\nsaturating adsorption peak at high gas pressure range (H2). The H3 hysteresis curve is\noften observed for clays and shale materials, in which the multilayer stacking of two-\ndimensional plate-like particles constitutes slit-like pores (Fig. R5 b, c). Please note that\nnitrogen adsorption-desorption isotherm analysis for our HV samples also exhibits the\nH3-type hysteresis, demonstrating the presence of slit-like pores with no closed ends\n(Fig. R6). The nitrogen gas adsorption analysis indicates that the slit-like pores\nconstructed by multiple stacking of brucite nanoplate has an interconnected pore with a\nsize ranging from 2 to 100 nm (Fig. R6).\nConsistent with the H3-type hysteresis for the HV samples, SEM and TEM\ninspections also manifested the multiple hierarchical stacking of plate-like structures\n(Fig. 1, g, h). Arrangement of the brucite nanocrystals results in slit-like spaces between\nthe flat surfaces.\n[Redacted]\nFig. R5 Previous studies showing hysteresis loops of gas adsorption desorption\nisotherm. a, Four hysteresis loops depending on the particular features of the pore\nstructure (Sing, K. S. W. et al. (1985) Pure & Appl. Chem., 57, 603–619). b, c, Nitrogen\nadsorption–desorption curves of (b) the Es s shale in the Dongying Depression (Yong,\nY. et al. (2022) ACS Omega 7, 36748–36761) and (c) natural palygorskite (black and\nred lines), a hydrated magnesium-rich silicate clay mineral (Wang, W. et al. (2016) Sci.\nRep. 6, 39599). These natural shale and clay show H3 hysteresis.\nFig. R6 (Supplementary Fig. 3). Nitrogen adsorption-desorption analysis of\nhydrothermal vent (HV) sample. a, Adsorption desorption isotherm. Based on the\nfeatures of the hysteresis loop, H3 type, the shape of pore is predicted to be open-ended\n(slit or plate-like pores)2.\nIn an attempt to provide the additional support for interconnected nanopores, we have\nperformed SAXS measurements using 100 μm X-ray beam. In contrast to WAXS,\nwhich provide angstrom-level of periodicity such as the interplanar distance of brucite,\nSAXS shows nanometer scale of periodicity in the material. Please note that a clear\nelliptical shape was observed in the SAXS pattern of the brucite wall (Fig. R7). This\nellipsoid is originated from the existence of nanometer-scale periodic scattering of\nparticles and/or voids inside the sample, indicating that the plate crystals are indeed\norientated with their flat surfaces (Liebi, M. et al, (2015) Nature 527, 349–352,\nPolishchuk, I. et al., (2017) Science 358, 1294–1298, Grünewald, T. A., et al., (2020)\nSci. Adv. 6, eaba4171). The orientation of the alignment of mineral particle with long\naxis observed in SAXS (major axis of ellipsoid, green line) is co-aligns with the\norientation of c-axis of brucite in WAXS (strong asymmetry direction, yellow line).\nThis shows that nanoscale periodic alignment exists along the direction of brucite [001]\nvector, as illustrated in the Fig. R7 e, confirming nanoscale channel-like structures,\ninside the HV wall. This co-alignment of the brucite (001) plane and particles is\nobserved throughout the inorganic wall of HV (Fig. R4). The periodic nanoscale voids\n(Fig. R7 e, right) can provide nano-distance channel-like structures.\nFig. R7 (Supplementary Fig. 10) X-ray scattering patterns of hydrothermal vent\n(HV) samples. a, Small angle X-ray scattering (SAXS) pattern of HV sample. b-d,\nWide angle X-ray scattering (WAXS) pattern of HV samples at different locations.\nWAXS patterns are also displaying the SAXS pattern at the center. Yellow dashed lines\nshow highest intensity of WAXS plots indicating alignment direction of (001) plane of\nbrucite. Green dashed lines indicate major axis of ellipsoid of SAXS region suggesting\nalignment direction of particles or voids. e, Schematic of the (001) brucite plane and\nparticle alignment shown in (d). The alignment (periodicity) direction observed in\nWAXS and the alignment direction observed in SAXS are matched in the same\ndirection.\nBased on the nitrogen adsorption-desorption isotherm and WAXS/SAXS analysis, we\nhave added new schematics showing the alignment of plate-like brucite crystals with\ninterconnected nanopores.\nFig. R8 (Fig. 5) Ionic transport by hydrothermal vent (HV) precipitates. a, A\nphotograph of the HV-PDMS membrane sample used for the measurement. b,\nExperimental setup for measuring current-voltage response of the HV samples. For\nconductance measurement, both sides were filled with KCl solution of the same\nconcentration. c, Electrical conductance of HV sample as a function of KCl\nconcentrations with pH 10. d, Schematics showing the continuous arrangement in HV\ninorganic wall, comprising multilayers of the brucite (001) plane at the angstrom level,\nplate nanocrystal stacks at the nanometer level, and a radial alignment structure\nextending to the millimeter level. This configuration results in continuous channels,\nallowing the HV walls to function as a selectively permeable membrane for ion\ntransport. e, Schematics showing osmotic power generation. Upon exposure to KCl, an\nelectric double layer (EDL) is formed on the surface of brucite crystals, exhibiting\nvarying Debye screening lengths (λ ) depending on KCl concentration. Blue represents\nD\nthe EDL formed on the brucite surface by exposure to ion concentration gradient. The\nprevalent pore size in HV walls is measured to be 60 nm, which means that the 30 nm\nEDL formed by 0.1 mM KCl completely overlaps inside the HV nanopore. Effective\noverlap of EDLs within narrow nanopores establishes a screening barrier that is\npermeable only to ions with specific charges. Acting as a selective membrane, the HV\nwall allows selective movement of counterions from a high concentration to a low\nconcentration, generating an ionic current.\nTo reflect the above explanation and experimental data in the revised manuscript, we\nmade the following revision:\n(1) To increase the accuracy of the explanation of pore morphology, we revised the term\nof “nanochannel” to “interconnected nanopores” in a whole part of manuscript.\n(2) To increase the accuracy of the explanation of pore morphology, we added the term\nof “slit-like pores (H3 type)” in the relevant sentence in the revised manuscript.\n(3) We added the SAXS data and corresponding explanation in the Results, Fig. 5d\ncaption, and Supplementary Fig. 10.\n(4) We created the new schematic to explain ion transport through the charged nanopores\nconstructed by multiple staking and alignment of brucite nanocrystal (Fig. 5).\nAdded sentences:\nResults, Alignment of brucite across the macro scale part\nMaterial characterization based on SEM, TEM, nitrogen adsorption-desorption isotherm\nand WAXS/SAXS demonstrates collectively the anisotropic alignment and multilayer\nstacking of plate-like brucite nanocrystals, forming interconnected nanopores referred to\nas nanochannel inside the HV walls (Fig. 2f).\nMethod, Characterization of HV\nIn the case of small-angle X-ray scattering (SAXS), the same beamline, RIKEN\nStructural Biology I Beamline (BL38B1), was used for measurement. The sample-to-\ndetector distance was 2.5450 m. The beam is collimated to the size of 100 µm(H) x 100\nµm(V).\nRevised Figures:\nSupplementary Fig. 3. Nitrogen adsorption-desorption analysis of hydrothermal\nvent (HV) sample.\nAdded Figures:\nFig. 5 Ionic transport by hydrothermal vent (HV) precipitates.\nSupplementary Fig. 10 X-ray scattering patterns of hydrothermal vent (HV)\nsamples.\nResponse to Reviewer comment 4A and 4B\nThank you very much for the detailed answer to my question. The POM and X-Ray\nanalysis inform very much about the texture and the gas-absiortion analysis the porosit;\nbut I cannot see the connectivity of the porous structure, perhaps my fault.\nReviewer comment 4C:\nThe authors wrote that \"Pronounced anisotropy of brucite 001 diffraction was detected,\nindicating that (001) plane of brucite orients preferentially along the [001]\ncrystallographic direction\" but (001) planes are always perpendicular to [001]\ncrystallographic direction.\nAuthor reply 4C:\nWe appreciate the reviewer’s comment. We noticed that our original sentence was\nmisleading and therefore have revised it as follows.\nRevised sentence:\nPronounced anisotropy of brucite 001 diffraction was detected, indicating that (001)\nplane of brucite orients preferentially inside the HV wall.\nReviewer comment 4D:\nThe plots shown in Figure 2 might be convincing of a radial texture of the brucite\ncrystallites at the local (micrometer) scale but they are not representative of the large\nscale.\nAuthor reply 4D:\nAs described in Author reply 4A, to respond to the reviewer’s comments, we have\nperformed thin section analysis using samples collected from multiple locations. When\nobtaining HV samples from the deep-sea, we divided the HV structure (84 cm total\nheight) into three parts (upper, middle, lower). Samples were collected from each part to\ncreate thin sections. One of these thin sections was shown in Fig. 3c through POM\nanalysis in original manuscript (collected from top part). As detailed in Author reply\n4A, POM analysis provides an effective method for identifying the orientation of\nbrucite. Using POM with 530 nm retardation plate, we additionally analyzed the thin\nsection samples collected from upper and lower parts (Fig. R3).\nIn our original manuscript (Fig. 3), the distinctive alternating blue and yellow\ninterference colors observed in the horizontal slice indicates the radial alignment of the\nbrucite inorganic wall. This feature is further evident in thin section images at different\nlocations (Fig. R3 a, d). The collection locations for each sample are spaced roughly 26\ncm to 84 cm apart, demonstrating a consistent alignment across the entire HV wall.\nReviewer comment 4E:\nActually, why such a texture should appear? The concentric layers of brucite could be\nthe results of episodic growth related to periodic fluid dynamics but there is no reason\ninducing the brucite microcrystals to be co-oriented to each other.\nAuthor reply 4E:\nThank you very much for raising the question about the anisotropic crystal arrangement\nfound in natural HV materials. In the discussion section of original manuscript, we\nproposed diffusiophoresis generated by chemical gradients as a force to arrange the\nbrucite nanocrystal.\nSentence copied from the original manuscript:\nIn addition to the periodic pattern, the anisotropic crystal arrangement observed in the\nHV samples (Figs. 2, 3, Supplementary Figs. 4-7) may be attributable to\ndiffusiophoresis, a phenomenon in which charged particles move along a concentration\ngradient of ions, such as NaCl, KCl, protons, and hydroxides42-44. Namely, during HV\nformation, a concentration gradient is established between charged brucite precipitates\nin the vent fluid and seawater45-47. The resulting diffusiophoretic field provides a\ndirectional driving force to the charged precipitates and promotes the concentric\narrangement of particles around the channels (Supplementary Notes).\nPrevious studies have shown the alignment of anisotropic particles, such as two-\ndimensional graphene oxide sheets, under flow conditions. Anisotropic particles\ntypically align along the flow direction (shear flow-induced alignment) (Hayter, J. B. &\nPenfold, J. (1984) J. Phys. Chem. 88, 4589-4593, Xin, G., et al. (2019) Nat.\nNanotechnol. 14, 168-175, Zhao, C., et al. (2020) Nature 580, 210-215). Contrary to the\nshear flow-induced orientation, the orientation of brucite in HVs deviates from the\ndirection of vent fluid flow (Supplementary Fig. 7). Namely, the two-dimensional\nbrucite sheets align perpendicularly to the flow direction. This in turn means that they\nalign with the direction of chemical gradients present between vent fluid and seawater.\nWe therefore hypothesized that diffusiophoresis, which generates an electric field\nperpendicular to the flow direction, is the force for the brucite orientation.\nDiffusiophoresis is the phenomenon where particles move along a concentration\ngradient of salt. In gradient condition, difference in cation and anion diffusion speed\ngenerates an electric field and when a charged particle exposed to this field, the particle\nmoves following the field direction. Additionally, the creation of different electrical\ndouble layers (Debye screening length) on particle surface due to the salt gradient\nresults in a pressure gradient, inducing a tangential flow near the surface. This motion\nof a particle generated by chemical gradients can be explained by the equation below.\n(Prieve, D. C. et al. (1984) J. Fluid Mech., 148, 247-269)\n𝜖 𝑘𝑇 𝑘𝑇 𝑍𝑒𝜁\n𝑈 = +𝛽𝜁 −2 ln21−tanh! 78∇ln𝐶\n4𝜋𝛽𝑍𝑒 𝑍𝑒 4𝑘𝑇\nwhere ε, η, k, T, Z, e, ζ, and C, are, respectively, the electrical permittivity, fluid\nviscosity, Boltzmann constant, absolute temperature, valence, the charge of an electron,\na zeta potential of particle, and the ionic concentration field. β is the diffusivity\ndifference factor, where D and D are, respectively, the diffusion constants of the\n+ -\ncations and anions.\n𝐷 −𝐷\n\" #\n𝛽 = \n𝐷 +𝐷\n\" #\nTherefore, the particle velocity, U, is influenced by local fluid properties (ε, η, ζ) and\nsalt characteristics (β, Z, C).\nTo test the hypothesis of diffusiophoresis-induced orientation, we are currently\nconducting the microfluidic experiments to replicate the chemical gradients and\nprecipitation processes in serpentinite-hosted HV systems. As shown in Reviewer Only\nMaterial, we have confirmed that chemical gradients between two fluids can indeed\ninduce crystal alignment. As expected from the nature of diffusiophoresis explained\nabove, the optimizing parameters, including local fluid properties and salt\ncharacteristics, enhanced the degree of brucite alignment, resulting in essentially the\nsame orientation with those found in natural HV samples. In this response letter, we\npresent preliminary data in a separated \"reviewer-only\" material to support this, which\nwill be addressed in another publication.\nAdded sentences:\nDiscussion\nIn addition to the periodic pattern, it is worth noting the anisotropic crystal arrangement\nobserved in nearly the entire part of the HV samples (Figs. 2, 3, Supplementary Figs. 4-\n10). Previous studies have demonstrated the alignment of anisotropic particles, such as\ntwo-dimensional graphene oxide sheets, under flow conditions45,46. Anisotropic\nparticles typically align along the flow direction (shear flow-induced alignment)45-47.\nContrary to the shear flow-induced orientation, the two-dimensional brucite sheets align\nperpendicularly to the flow direction (Supplementary Fig. 7). We speculate\ndiffusiophoresis, a phenomenon in which charged particles move along a concentration\ngradient of ions, such as NaCl, KCl, protons, and hydroxides48-51, acts as a driving force\nfor the alignment. Namely, in serpentine-hosted HV systems, a concentration gradient is\nestablished between the hot, reduced ion-rich vent fluid solutions and oxidizing\nseawater36,37,52,53, generating an electric field perpendicular to the flow direction. The\nresulting diffusiophoretic field provides a directional driving force to the charged\nprecipitates and promotes the concentric arrangement of nanocrystals around the\nchannels (Supplementary Notes).\nI have seen in laboratory experiments the crystalline co-orientation of microparticles in\nsolutions triggered by the Marangoni effect; I believe the shear-flow-induced alignment\nof nanoparticles reported in the literature. But in this particular case, the\ndiffusiophoretic explanation is speculative without further experimental evidence, as the\nauthors themselves realized. However, I admit that it is beyond the scope of this article.\nHowever, I wonder why in the laboratory experiments to synthetize chemical gardens, a\nlaboratory model some colleagues used for hydrothermal vents, no one so far has\nobserved the formation of oriented crystalline textures on the walls of the thus-forming\nosmotic chimney-like tubular structures.\nReviewer comment 5A:\nThe experimental procedure to fabricate a “membrane” from the sample of the wall of\nthe white fumaroles is not very “clean” and I suspect that would modify the properties\nof the material. The experimental method to measure with these “membrane” the\nelectrical chemical energy does not mimic the natural process.\nAuthor reply 5A:\nTo address the reviewer’s concern, we have analyzed the HV samples before and after\nmembrane fabrication using Raman spectroscopy (before: Fig. 4e, after: Fig. R9) and\nWAXS (before: Supplementary Fig. 2a, after: Supplementary Fig. 4c, d). Raman spectra\nof the HV samples after membrane fabrication show the characteristic peaks of brucite\nat 280 cm-1 and 444 cm-1. The spectrum was identical with that observed for the sample\nbefore membrane fabrication (no peak shifts at the spectral resolution of 4 cm-1).\nConsistent with the Raman analysis, membrane fabrication shows negligible changes in\nthe X-ray scattering (diffraction) peaks for brucite (Supplementary Fig. 4c, d).\nTherefore, we can conclude that the material properties of brucite remain intact after the\nmembrane fabrication process.\nFig. R9 (Supplementary Fig. 17) Raman measurements of the cross-section of the\ncut hydrothermal vent (HV)-PDMS membrane sample. a, Optical image displaying\nthe cross-section of the HV-PDMS membrane with marked Raman measurement points.\nb, Raman spectra at each position. Clear and unshifted peaks at 280 and 444 cm-1,\ncorresponding to brucite, are distinctly visible (yellow rectangle).\nTo make these points clear, we added the following sentence in the revised manuscript.\nAdded sentences:\nMethod, HV-PDMS membrane fabrication part\nRaman and WAXS analysis confirmed that material properties of brucite remain intact\nduring membrane fabrication processes (Supplementary Fig. 17).\nAdded Figures:\nSupplementary Fig. 17 Raman measurements of the cross-section of the cut\nhydrothermal vent (HV)-PDMS membrane sample\nWhen I suspected that the membrane fabrication would modify the properties of the\nmaterial, I was thinking on its properties such as porosity and texture. The comparison\nof the mineralogy by Raman and X-ray diffraction before and after fabrication informs\nabout the mineralogy -which is expected to be maintained- but not about modification\nof the properties linked to transport.\nReviewer comment 5B:\nThe solutions to use should be those of playing in the natural vents, i.e. a reducing high\npH fluid inside the wall and seawater outside the wall. The experimental measurements\n-even if they are not very conclusive- do not model properly the natural scenario.\nAuthor reply 5B:\nThank you for pointing out the important issue. We agree with the reviewer's comments\nabout the importance of simulating the natural environment. In the original manuscript,\nwe experimentally demonstrated the selective ion transfer across the natural HV sample\nfrom an ionic gradient of NaCl and KCl. This was explicitly verified by in-situ\nmonitoring of the osmotic potential and the direct detection of cation (Na+) and anion\n(Cl-) using Na+ and Cl- ion-sensitive electrodes. In addition, we experimentally\ndemonstrated the selective transport of H+ under simulated alkaline hydrothermal\nconditions. Namely, the natural HV sample was placed between a reducing high pH\nfluid and an oxidizing low pH solution.\nSentence copied from the original manuscript:\nOne of the most characteristic features of serpentinite-hosted HV systems is the\npresence of a large pH gradient between the seawater (pH ~ 7) and vented fluids (pH ~\n9-11). To examine conversion of a pH gradient to electrochemical energy, the cation-\nselective (CO 2--adsorbed) HV sample was placed between reservoirs containing pH 10\n3\nand 4 solutions and the electrical response was measured (Fig. 5g, gray circle).\nSpecifically, we have attempted to simulate alkaline hydrothermal conditions based on\nthe representative papers in the research field of simulated alkaline hydrothermal vent\nexperiments (Hudson, R. et al. (2020) Proc. Natl. Acad. Sci. U.S.A., 117, 22873-22879,\nBarge, L. M. et al. (2012) Langmuir 28, 3714-3721, Moller, F. M. et al. (2017) Angew.\nChem. Int. Ed. Engl. 56, 2340-2344). To verify which ion carrier is responsible for the\nosmotic energy conversion, protons or hydroxide ions, we also performed a deuterium\ngradient (pD) (pD 4 and pD 10). A high H/D kinetic isotope effect (2.3) manifests that\nunder simulated alkaline HV conditions, protons (not hydroxide ions) play a\npredominant role in converting pH gradient into electrical energy. To clarify the above\npoint, we have added the following sentences in the revised manuscript.\nAdded sentences:\nResults, Osmotic energy conversion in HVs part\nExperimental simulation of alkaline HV systems has been reported in the context of\norigin of life on the ancient Earth, using reducing high pH fluid (pH 10 ~ 12) and\noxidative low pH seawater (pH 4 ~ 6) simulant22,36,37.\nIn our effort to further respond to the reviewer’s comments, we have conducted osmotic\npower generation experiments by replicating actual chemical compositions of alkaline\nvent fluids and sea water based on the geochemical dataset at Shinkai Seep Field (SSF).\nThe simulant solutions were prepared by referencing a paper that analyzed the solution\nof SSF HV (Onishi Y., et al. (2018) PLoS ONE, 13, e0199000).\nTable R1. Chemical compositions of Shinkai Seep Field (SSF) hydrothermal vent (HV)\nfluid and sea water.\nA mixed solution consisting of NaCl (500mM), MgCl (50mM), CaCl (10mM), and\n2 2\nNa SO (30mM), which were considered to be the major charged components, was\n2 4\nprepared. The pH levels were adjusted to 6.6 for the seawater simulant and 9.9 for the\nvent fluid simulant. However, please note that, in the presence of multiple ions, the\nselective ion transfer and subsequent generation of electric potential cannot be\nmonitored experimentally due to the lack of ion-selective sensor electrodes. Namely, in\nthe research field of osmotic power generation, Ag/AgCl is widely used as a sensor\nelectrode. It can indeed monitor the concentration change of Na+, K+, H+, Cl- and OH-\nwhen the ionic gradient is derived from mono-ionic species. However, the\nelectrochemical signal in principle becomes the summation of the open circuit voltage\nderived from transport of mixed ion species. In fact, unlike the mono-ionic species (Fig.\n6), we were unable to detect the reliable electrochemical signals from the simulant of\nSSF HV conditions. This is an intrinsic technical problem raised by multi-ion\ncomponent systems, leaving room for advancement of ion-sensitive sensor electrodes.\nWe believe that this is beyond the scope of our study and hope that our experimental\ndata under simulated alkaline hydrothermal vent conditions based on the paper by\nHudson, R. et al. (Proc. Natl. Acad. Sci. U.S.A., 2020, 117, 22873-22879) adequately\nrespond to the reviewer's comments.\nYes, I remember that in the original paper, you demonstrated the selective ion transfer\nacross the natural HV mineral sample from an ionic gradient of NaCl and KCl. You also\ntry to mimic natural conditions of serpentinite-hosted HV systems i.e., pH gradient\nbetween the seawater (pH ~ 7) and vented reducing fluids (pH ~ 9-11), using pH 10 and\npH 4 solutions. As you realized, these fluids, these are far from being representative of\nnatural conditions. If I understood you properly, you are now using a mixed solution\nwith pH adjusted to 6.6 for the seawater simulant and 9.9 for the vent fluid simulant,\nand it was not possible to detect the existence of an electrochemical voltage. I agree\nwith you that this could be explained by the use of a solution mixture. But it also could\nbe due to the small pH gradient (three pH units lower). Perhaps you can make a test\nexperiment using the same fluid mixture but adjusting the pH to 10 and 4. This is\nunrealistic, but if you can detect an electrochemical voltage, it is actually the larger\ndifference in pH that matters.\nReviewer comment 5C:\nThe existence of the generation of an electrochemical potential in osmotic-driven\nstructures has been demonstrated and measured (see Glaab et al., (Angew. Chem. Int.\nEd. 2012, DOI: 10.1002/ange.201107754 ). The electrochemical potential was proposed\nto be a summation of three factors, of which a large pH difference is a major\ncontributor. Perhaps the authors could find ideas for experimental testing and\nexplanations in that article. And, of course, a porous structure connecting the two sides\nof the membranes is key for the formation of the electrochemical potential.\nAuthor reply 5C:\nWe appreciate the reviewer to introduce a relevant paper to our work. In the paper by\nGlaab et al, a silicate-based tubular membrane structure was fabricated by immersing a\ntablet of CoCl in silica solution (chemical garden system). By placing two Pt electrodes\n2\ninside and outside the tubular membrane structure, they monitored the time course of\noverall cell potential (ΔE ). The ΔE , which is also refer to as electrochemical potential,\nh h\nwas then divided by three components: pH (ΔE ), diffusion (ΔE ), and membrane\npH diff\n(ΔE ) potential.\nm\nΔE = ΔE + ΔE + ΔE\nh pH diff m\nIn our study, we performed essentially the same analysis as that reported by Glaab et\nal. except that we used an Ag/AgCl electrode instead of a Pt electrode. The use of an\nAg/AgCl electrode allows to increase the sensitivity to monitor the electrochemical\npotential (ΔE ) caused not only by pH, but also by concentration change of cations (Na+\nh\nand K+) and anions (Cl-) (Macha, M. et al. (2019) Nat. Rev. Mater. 4, 588-605, Siria, A.\net al. (2013) Nature 494, 455-458, Feng, J. et al. (2016) Nature 536, 197-200).\nTherefore, in contrast to the work by Glaab et al, we can monitor the osmotic power\ngeneration caused by selective transport of Na+, K+, H+, and Cl-. Using an Ag/AgCl\nelectrode, we monitored the open circuit voltage (V ) established across the HV\noc\nmembrane, which corresponds to ΔE . Then, the redox potential (V ) of the\nh redox\nelectrode-solution interface was experimentally monitored by immersing two Ag/AgCl\nelectrodes to the solutions without HV membrane, which corresponds to ΔE .\npH\nFinally, subtracting the redox potential from V gives the pure osmotic potential of the HVs (V\noc oc\n= V + V ).\nos redox\nIn the revised manuscript, we have cited the paper by Glaab et al (Angew. Chem. Int.\nEd. 2012, DOI: 10.1002/ange.201107754) as reference 53.\nActually, Glaab et al. proposed a combination of three contributions of the\nelectrochemical potential, diffusion (ΔEdiff) and membrane (ΔEm) potentials, as well as a\nthird pH-induced contribution (ΔEpH).\nReviewer comment 6:\nThroughout the manuscript, the authors try to prove that the brucite crystallites have\nconfined nanochannels with charged surfaces, which are the requirements for the\nconversion to work. The idea is good and the implications -if the hypothesis is\ndemonstrated- are interesting, but the results that count do not demonstrate, in the\nopinion of this referee, the existence of osmotic energy conversion processes in\nhydrothermal vents.\nAuthor reply 6:\nOnce again, we thank the reviewer for their interest in our work and for their\nconstructive comments, which have helped us greatly improve the quality of our work.\nAs described above, we have addressed all the comments raised by the reviewer, which\nwe believe provides appropriate responses. Based on the newly conducted experiments\n(SAXS, POM, Raman and microfluidics) and the detailed discussion of mineral\ncomposition, nanopores morphology (slit-like pores), crystal orientation mechanisms\n(diffusiophoresis), simulation of alkaline hydrothermal vent conditions (pH4~10), and\nmeasurement details of osmotic power generation, we believe that our main finding of\ncaton- and anion-selective ion transfer across natural HV materials is strengthened.\nThis concludes our response to the reviewers’ comments. We are grateful for their\ncomments which have helped us improve the rigor and quality of our manuscript.\nI am very sympathetic to this article. However, it seems to me that what the authors\nclaim is much more than what is demonstrated. It would be a pity if the article were not\npublished because it contains a lot of experimental information that can help to better\nunderstand the problem. The existence of these electrostatic potentials is important to\nunderstand how hydrothermal vents and the associated ecosystem function today. However,\nthe connection with the origin of life or the origin of metabolism is weak. As referee number 2 points out, claiming in the abstract that this study supports the hypothesis of the origin of life in hydrothermal vents is not consistent with the content of the study. Although the authors accepted the criticism of Referee 2, the old and new sentences in the original and revised manuscript are virtually identical. In my opinion, even without that life origin connection, the manuscript must be revised to make clear the actual finding and its geochemical implications.\nAs to the comments of Reviewer #1– second rebuttal\nReviewer comment:\nThere are a total of four reviewers who seem to come from different background to provide\nvaluable comments about this manuscript. Overall, the critical comments are from Reviewer\n#4, about which the authors mostly have addressed, with some relevant additional\nexperiments, data syntheses, and comment incorporation. This reviewer thinks that the\nrevised manuscript is acceptable.\nAuthor reply:\nWe appreciate Reviewer #1's thorough assessment of our manuscript and their critical\ncomments, which we have taken into careful consideration during the revision process. We are\npleased to note that our efforts to address Reviewer #1's concerns have resulted in an improved\nmanuscript. We are pleased that Reviewer #1 finds the revised version acceptable. Thank you\nfor your thoughtful evaluation.\nAs to the comments of Reviewer #2– second rebuttal\nReviewer comment:\nI enjoyed reading the revised manuscript. In my opinion this is an important piece of work that\nshould be published.\nAuthor reply:\nWe are deeply grateful to Reviewer #2 for their interest in our work and their strong support\nfor its publication in Nature Communications. We agree that our study has important\nrelevance to researchers in the fields of alkaline hydrothermal vents, osmotic energy\nconversion and the origin of life. Furthermore, we are grateful that Reviewer #2 recognized\nthe potential of our work to serve as a foundational link between chemistry, geology, and\nbiology to further enrich the alkaline hydrothermal vent theory of the origin of life.\nAs to the comments of Reviewer #3– second rebuttal\nReviewer comment:\nAll my concerns have been addressed and to the best of my knowledge the manuscript is\nacceptable and now ready for publication.\nAuthor reply:\nWe sincerely appreciate Reviewer #3's comprehensive assessment and constructive feedback\non our manuscript. Their critical comments have been carefully considered, and we have made\nconcerted efforts to address them. We are pleased to note that Reviewer #3 now finds the\nrevised manuscript acceptable, indicating that our efforts have been successful in meeting their\nexpectations. We thank Reviewer #3 for their valuable contributions to this process.\nAs to the comments of Reviewer #4 – second rebuttal\nNotice: All revisions based on this reviewer’s comments are highlighted in pink in the\nmanuscript and Supplementary Materials.\nReviewer comment 1:\nThe authors have made an extraordinary effort to answer the questions and criticisms of the\nreferees. I am very impressed by their careful response, and I wish to congratulate them. In\nsome cases, their answers are clear and satisfactory; however, to my understanding, other ones\nare not so convincing. Below, you can find my comments (in red) on specific points:\nAuthor reply 1:\nWe express our gratitude to the reviewer for their interest in our study and for providing\nvaluable feedback. Their constructive comments have significantly contributed to enhancing\nthe quality of our research. In response to the reviewer's comments, we have conducted\nadditional experiments and addressed each comment in a point-by-point manner, as shown\nbelow.\nReviewer comment 2:\nThank you. I think this is an important clarification\nAuthor reply 2:\nWe appreciate the reviewer’s invaluable comments regarding the types of hydrothermal vent\n(HV).\nReviewer comment 3:\nThat makes sense; calcite is mainly in the outer part because it is in contact with the carbonate-\nrich seawater. Then, I think it is important to explain to the readers that your approach works\nfor type-I hydrothermal vents. It is not clear to me what the difference is between type I upper,\ntype I Middle, and type I lower. In any case, it seems that you always have in type-I HV calcite\nin the outer part. Then, how the ionic flow occurs through the calcite outer layer?\nAuthor reply 3:\nWe are grateful to the reviewer’s comment about serpentinite-hosted HVs environment.\nTo enhance clarity regarding sample preparation, we have newly included an image of the HV\nsample at the collection point (depth 5743 m, 11°39.38′N, 143°2.73′E) and how it was\ndivided into three parts (Fig. R1a).\nFig. R1 (Supplementary Fig. 1) Hydrothermal vent (HV) structure at various locations.\na, A photograph of the HV sample collected at the Shinkai Seep Field (SSF) at a depth of 5743\nmeters (11°39.38′N, 143°2.73′E). The HV sample was divided into three parts as indicated in\nthe photograph. b, Photographs of a HV sample cut (upper part) in the vertical direction\nshowing the internal structure, which was divided into Zones 1 to 5. c, SEM images of HV\nsamples collected from the different zones shown in b. Most of the zones appeared to have a\nsimilar wall-type structural composition; however, aggregated structures were observed in the\noutermost layer (zone 1), fibrous structure was detected in zone 3.\nAdded Figures :\nSupplementary Fig. 1 Hydrothermal vent (HV) structure at various locations.\nRegarding the role of calcite, we find it difficult for the calcite crystal region to function as a\nselective ion transport due to the lack of nanopores and the inability to establish concentration\ngradients between inorganic walls. Osmotic power generation relies on layered materials,\nwhich can have distances at the angstrom scale at the unit cell level and distances at the\nnanometer scale through the stacking of layered structures (Xiao et al. (2019) Joule 3, 2364–\n2380; Macha et al. (2019) Nature Reviews Materials 4, 588–605; Zhang et al. (2021) Nature\nReviews Materials 6, 622–639). However, calcite crystals have a different crystallographic unit\ncell and morphology than the layered structures. Consequently, calcite crystals cannot provide\nslit-like nanopores as seen in the layered structures. Indeed, this difference in morphology can\nbe observed in Supplementary Fig. 2b and c of the original manuscript. Unlike brucite, which\nhas a layered crystal structure, calcium carbonate crystals exhibit large, discrete polygonal\ncrystal shapes. Therefore, in regions where calcite crystals are prevalent in Type I, random\nmixing of solutions may occur instead of directional selective ion transport. Furthermore, in\nType I, calcite is primarily located on the exterior. Unlike brucite walls, it would be difficult to\ncreate steep salt or pH gradients between vent fluid and seawater with calcite walls in between.\nFor these reasons, selective ion transport observed in the brucite region may be difficult to\nachieve in the calcite region.\nReviewer comment 4:\nThank you very much for the detailed answer to my question. The POM and X-Ray analysis\ninform very much about the texture and the gas-absiortion analysis the porosit; but I cannot see\nthe connectivity of the porous structure, perhaps my fault.\nAuthor reply 4:\nWe appreciate the reviewer's comment regarding the connectivity of the porous structure. To\naddress this concern, we have endeavored to enhance the clarity by providing more detailed\nimages of the nanostructures inside HV walls.\nIn HV walls primarily composed of brucite crystals, particles with sizes in the range of\nhundreds of nanometers, form an array, constructing a stratified wall structure (Fig. R2 a and\nb). The directional alignment of these particles naturally gives rise to predominantly uniaxial\ntextures in the fractured wall sections (Fig. R2 b and c). These distinctive textures observed\nconsistently throughout the HV wall, indicating a uniform alignment of particles along a single\naxis.\nFig. R2 (Supplementary Fig. 3) Array of particles inside hydrothermal vent (HV) wall.a,\nb, Enlarged SEM images shown in Fig. 1g. Yellow arrows show uniaxial texture inside HV\nwall. c, Schematic diagram showing pores that can be created through an array of particles.\nThe plate-like layered structure of brucite inherently leads to the formation of cavities oriented\nin a singular direction between particles, due to the directional alignment along one axis (Fig\nR2 c). To illustrate the arrangement and interconnectedness of these pores, the surface of the\nHV sample was milled, and the resulting pores were observed in the cross-section. An Ar ion\nbeam milling system (Leica EM TIC 3X) was used for cross-sectioning of the HV sample, and\n1~10 keV energy was used in cryo-stage condition using liquid nitrogen to minimize damage\nto the sample due to heat that may occur during the milling process. The milled brucite wall\nreveals particles arranged in nanometer scales, illustrating how the arrangement of these\nparticles creates pores between them (Fig. R3).\nFig. R3 (Supplementary Fig. 4) A cross-section of a milled hydrothermal vent sample,\nrevealing pores formed by the particle array. From left to right, progressively higher\nmagnification images show inter-particle pores are created through the arrangement of particles\nof about 100 nm.\nThe High-Angle Annular Dark Field Scanning TEM (HADDF-STEM) images provide insights\ninto crystal stacking at a finer nanoscale, indicating the presence of inter-crystal pores (Fig.\nR4). Low-magnification STEM images exhibit overlaid layers, showing the stacking of\nparticles, similar to SEM observations (Fig. R4 left and Fig. R2 right). Upon closer examination,\nnumerous stacked nanodomains (nanocrystals) are evident within the 100 nm particles. This\nshows that inter-crystal pores are generated through nanocrystal stacking (Fig. R4 right).\nFig. R4 (Supplementary Fig. 5) High-Angle Annular Dark Field Scanning TEM\n(HAADF-STEM) images of the brucite crystals. The inset in the right corner shows the\nstructure that nanodomains may have.\nThe analysis of these images demonstrates stacking of crystals occurring at various levels,\ninevitably leading to the creation of pores. Moreover, the directional alignment of layered\nstructures results in the formation of connected pores oriented in one direction.\nThe previously presented nitrogen adsorption-desorption isotherm (Supplementary Fig. 6) and\nSAXS analysis (Supplementary Fig. 13) together with these image analyses collectively\ndemonstrate that the nanopore extends beyond the analyzed image area to the entire inorganic\nwall of the HV.\nWe have added new figures and sentences showing the interconnected nanopores.\nAdded Figures :\nSupplementary Fig. 3 Array of particles inside hydrothermal vent (HV) wall.\nSupplementary Fig. 4 A cross-section of a milled hydrothermal vent sample, revealing\npores formed by the particle array.\nSupplementary Fig. 5 High-Angle Annular Dark Field Scanning TEM (HAADF-STEM)\nimages of the brucite crystals.\nAdded sentences:\nStacking of layered nanocrystals at multiple scales resulted in the formation of interconnected\nnanopores (Supplementary Figs. 3-5).\nMethod, Sample preparations Characterization of HV\nCross-sectional analysis of HV-polymer samples was observed after milling the samples using\nan Ar ion beam milling system (Leica EM TIC 3X).\nReviewer comment 5:\nI have seen in laboratory experiments the crystalline co-orientation of microparticles in\nsolutions triggered by the Marangoni effect; I believe the shear-flow-induced alignment of\nnanoparticles reported in the literature. But in this particular case, the diffusiophoretic\nexplanation is speculative without further experimental evidence, as the authors themselves\nrealized. However, I admit that it is beyond the scope of this article. However, I wonder why\nin the laboratory experiments to synthetize chemical gardens, a laboratory model some\ncolleagues used for hydrothermal vents, no one so far has observed the formation of oriented\ncrystalline textures on the walls of the thus-forming osmotic chimney-like tubular structures.\nAuthor reply 5:\nThank you for raising the question about the anisotropic crystal arrangement found in natural\nHV materials and artificial HV laboratory experiments.\nWe are currently conducting more detailed mechanistic studies to provide a comprehensive\nexplanation for the observed anisotropic arrangement of crystals in HVs, leveraging insights\ngained from this current original manuscript experiments. We plan to address the phenomenon\nof the anisotropic arrangement of crystals in HV based on the artificial HV experiments in our\nupcoming paper.\nTo demonstrate the oriented crystalline textures resembling those found in HVs within\nlaboratory environments, we would like to highlight several other papers on artificial HV\nexperiments. Chemical gardens and similar HV settings experiments have reported the creation\nof inorganic walls or tube structures through the interaction of two differently composed\nsolutions. While the alignment of crystals wasn't a primary focus of these studies, the reported\nexperiments have revealed the directional growth of crystals along the walls of these tubes\n(Glaab et al (2012) Angew. Chem. Int. Ed.51, 4317–4321; Cartwright et al (2011) Langmuir\n27, 7, 3286–3293; Ibsen et al (2014) Chem. Eur. J. 20, 16112 – 16120; Barge et al (2015)\nAngew.Chem. Int. Ed. 54, 8184–8187).\nIn a study published in Chem. Eur. J. 2014, needle-shaped nanocrystals were observed, with\ntheir significant preferred orientation identified through XRD. The alignment of these needle\ntextures perpendicular to the tube axis corresponds closely with the crystal orientation observed\nin HVs. Similarly, in Angew. Chem. Int. Ed. 2015 paper, by interfacing of two different\nsolutions (acidic, iron-containing solution and alkaline, sometimes sulfidic solution) the\nauthors led to the creation of inorganic membranes with directionally aligned crystal textures.\nFurthermore, they observed membrane potential through this inorganic membrane. Although\nthe exact mechanisms underlying these phenomena were not explicitly explained in the paper,\nit is conceivable that such crystal arrangements might have influenced the generation of\ndiffusion potential.\nThese examples show that aligned crystalline textures readily emerge in HV conditions and\nenvironments where chemical gradients exist.\nReviewer comment 6:\nWhen I suspected that the membrane fabrication would modify the properties of the material,\nI was thinking on its properties such as porosity and texture. The comparison of the mineralogy\nby Raman and X-ray diffraction before and after fabrication informs about the mineralogy -\nwhich is expected to be maintained- but not about modification of the properties linked to\ntransport.\nAuthor reply 6:\nWe appreciate the reviewer’s valuable comments. In response to their concern, we have\nconducted an analysis of the cross-section of HV samples after the polymer embedding process.\nIn order to examine porosity changes that may occur during the membrane fabrication process,\nwe embedded HV samples in resin and observed the cross-section of the samples. For cross-\nsectioning, we utilized an Ar ion beam milling system (Leica EM TIC 3X) and conducted ion\nmilling using energy ranging from 1 to 10 keV under cryo-stage conditions with liquid nitrogen\nto minimize sample damage due to heat generated during the milling process. Fig. R5 shows\nSEM images of the cross-sectioned sample surface obtained through Ar ion milling. We\nobserved the interface between the polymer and HV wall (highlighted in the red box). The\nobserved HV wall is divided into brucite and magnesium carbonate crystal parts, and we\nobserved the effects of polymer at each location. The brucite and magnesium carbonate crystals\nare structured through the arrangement of multiple layered crystals, and pore formation is\nobserved within the crystals depending on the shape of the crystal arrangement. Fig. R5 c and\nd respectively show cross-section images of the crystal parts, demonstrating that the pores\nexisting between the crystals are empty spaces without polymer. This indicates that the polymer\nembedding process does not significantly affect the pores existing within the HV.\nFig. R5 (Supplementary Fig. 21) Cross-sectional images of hydrothermal vent (HV)\nsample processed by Ar ion milling after polymer insertion.a, b, Low-magnification SEM\nimages of cross-sectional samples. The inset in (a) is a photograph of the observed sample. The\nred dashed box corresponds to the location of (a). The location of (b) is shown in the red box\nin (a). c, d, High-magnification SEM images of magnesium carbonate (c) and brucite (d)\ncrystals.\nThe HV-PDMS membrane sample was also sliced by a blade and the cross-section was\nobserved using SEM (Fig. R6). Even at the interface of the polymer-inorganic wall, textures\nthrough the arrangement of crystals can still be observed.\nFig. R6 (Supplementary Fig. 22) Cross-section images of the HV-PDMS membrane. The\ninterface between the polymer and the inorganic HV wall was observed using SEM.\nThe porosity and texture observed on the inorganic wall remain even after the polymer\nembedding process and HV-PDMS membrane fabrication. Therefore, we concluded that there\nwas no significant effect of the polymer under our experimental conditions.\nTo make these points clear, figures and sentences have been added to the revised manuscript.\nAdded Figures:\nSupplementary Fig. 21 Cross-sectional images of hydrothermal vent (HV) sample\nprocessed by Ar ion milling after polymer insertion.\nSupplementary Fig. 22 Cross-section images of the HV-PDMS membrane.\nAdded sentences:\nMethod, Sample preparations Characterization of HV\nCross-sectional analysis of HV-polymer samples was observed after milling the samples using\nan Ar ion beam milling system (Leica EM TIC 3X).\nMethod, HV-PDMS membrane fabrication\nRaman, WAXS, and cross-section SEM image analysis confirmed that material properties of\nbrucite remain intact during membrane fabrication processes (Supplementary Figs. 20-22).\nReviewer comment 7:\nYes, I remember that in the original paper, you demonstrated the selective ion transfer across\nthe natural HV mineral sample from an ionic gradient of NaCl and KCl. You also try to mimic\nnatural conditions of serpentinite-hosted HV systems i.e., pH gradient between the seawater\n(pH ~ 7) and vented reducing fluids (pH ~ 9-11), using pH 10 and pH 4 solutions. As you\nrealized, these fluids, these are far from being representative of natural conditions. If I\nunderstood you properly, you are now using a mixed solution with pH adjusted to 6.6 for the\nseawater simulant and 9.9 for the vent fluid simulant, and it was not possible to detect the\nexistence of an electrochemical voltage. I agree with you that this could be explained by the\nuse of a solution mixture. But it also could be due to the small pH gradient (three pH units\nlower). Perhaps you can make a test experiment using the same fluid mixture but adjusting the\npH to 10 and 4. This is unrealistic, but if you can detect an electrochemical voltage, it is actually\nthe larger difference in pH that matters.\nAuthor reply 7:\nWe extend our gratitude for reviewer's suggestion. To investigate the possibility of\nundetectable signals due to a small pH gradient, we increased the pH gradient in both seawater\nand vent fluid simulant solutions to pH 4 and pH 10, respectively, and re-measured the potential.\nWe prepared the solutions based on the previously reported natural conditions of vent fluid and\nseawater in Shinkai Seep Field (Onishi Y., et al. (2018) PLoS ONE, 13, e0199000). The\nsolution, representing a natural scenario, includes various salt concentrations: NaCl (500 mM),\nMgCl (50 mM), CaCl (10 mM), and Na SO (30 mM). Additionally, in response to the\n2 2 2 4\nreviewer's comment, the pH level was adjusted to pH 4 for seawater and pH 10 for vent fluid.\nBelow are the open circuit voltage (V ) values of the HV samples measured in pH 4 and pH\noc\n10 gradients, encompassing various salts (Fig. R7 a). For comparison, we present the results\nfrom pH 6.6 and pH 9.9, as measured in the previous first rebuttal (Fig. R7 b). A very small\nlevel of potential was measured. We also plot the values measured through Ag/AgCl electrodes\nwithout HV membranes (Fig. R7 c). There was minimal variation in potential with changes in\npH conditions. When only pH gradient was present, V was detected as 250.45 mV at pH 10\noc\nand 228.47 mV at pH 4, providing 21.98 mV of V (Supplementary table in original\nredox\nmanuscript).\nFig. R7 Open circuit voltage measured in a salt mixture environment.a, b, Measured open\ncircuit potential (V ) of hydrothermal vent samples adsorbed with Ca2+ (red squares) and CO 2-\noc 3\n(blue squares) ions. The samples were placed under a pH gradient containing a salt mixture, L:\npH 10 and R: pH 4 (a) and L: pH 9.9 and R: pH 6.6 (b). c, Measured V of Ag/AgCl wires in\noc\ndifferent pH conditions (red-pH 4, sky-blue-pH 6.6, blue-pH 9.9) with a salt mixture.\nOnce again, these results reveal the challenge of measuring selective ion transport and electric\npotential in our Ag/AgCl electrode system when multiple ions are present. While these\nelectrodes excel at tracking individual ion concentrations in single-species gradients, they\nstruggle with combined electrochemical signals in scenarios with multiple ionic species. This\nis an intrinsic technical problem raised by multi-ion component systems.\nReviewer comment 8:\nActually, Glaab et al. proposed a combination of three contributions of the electrochemical\npotential, diffusion (ΔEdiff) and membrane (ΔEm) potentials, as well as a third pH-induced\ncontribution (ΔEpH).\nAuthor reply 8:\nWe are grateful to the reviewer for providing additional insights to improve our manuscript.\nThe suggested paper (Glaab et al (2012) Angew. Chem. Int. Ed.51, 4317–4321) measured all\npotentials formed during the growth of the silica garden. The three terms presented in that paper\ncorrespond to different states of the chemical garden: the potential before the formation of the\nsemi-permeable membrane (ΔE ), the potential with the semi-permeable membrane (ΔE ),\ndiff m\nand the redox potential arising from pH difference (ΔE ). We have concluded that the first\npH\nterm may not be applicable in our case because the HV precipitates already possesses a\nmembrane capable of selective ion transport.\nBelow is a more detailed explanation of each term (ΔE and ΔE ) :\ndiff m\nThe authors mention that in the initial step, the wall of the silica garden is permeable to all\nion species. Therefore, they calculated the diffusion potential (ΔE ) using the Henderson\ndiff\nequation. This equation accounts for the movement of all ions and the resulting liquid junction\npotential.\nIn the next section, the authors assume that as precipitation progresses, the diffusion of\nspecific ions becomes blocked, and it is no longer a membrane where all ions are transported\n(semi-permeable membrane). They then consider the membrane potential term (ΔE )\nm\ncalculated by the Goldman-Hodgkin-Katz (GHK) equation. In the GHK voltage equation, the\nterm for the relative permeability of ions is newly incorporated. The membrane potential\nobtained from the GHK equation is the potential generated in a membrane considering selective\nion transport.\nThe differences in these equations account for the potential variations observed in the silica\ngarden. Initially, as all ions move, a diffusion potential (ΔE ) emerges, which gradually\ndiff\ndiminishes as a semi-permeable membrane forms, leading to a continuous contribution of\nmembrane potential (ΔE ) to the overall potential. This feature is clearly reflected in the\nm\ncalculated overall potential (Figure S5 in the Glaab paper).\nFor HVs, where a semi-permeable wall is already present, it's plausible to consider that the\nmembrane potential, calculated using the GHK equation as discussed in the Glaab paper,\ncontributes to the pure osmotic (V ) potential.\nos\nReviewer comment 9:\nI am very sympathetic to this article. However, it seems to me that what the authors claim is\nmuch more than what is demonstrated. It would be a pity if the article were not published\nbecause it contains a lot of experimental information that can help to better understand the\nproblem. The existence of these electrostatic potentials is important to understand how\nhydrothermal vents and the associated ecosystem function today. However, the connection with\nthe origin of life or the origin of metabolism is weak. As referee number 2 points out, claiming\nin the abstract that this study supports the hypothesis of the origin of life in hydrothermal vents\nis not consistent with the content of the study. Although the authors accepted the criticism of\nReferee 2, the old and new sentences in the original and revised manuscript are virtually\nidentical. In my opinion, even without that life origin connection, the manuscript must be\nrevised to make clear the actual finding and its geochemical implications.\nAuthor reply 9:\nThank you for your valuable opinions. We have omitted a sentence related to the origin of life\nfrom the abstract. However, we wish to maintain the connection and implications of the origin\nof life in the discussion. We would like to point out that such implications are also mentioned\nin geological perspective paper reporting the discovery of alkaline HVs (Kelly et al. Nature\n(2001) 412, 145–149)\nThe correlation between HV and the origin of life has a long history. The discovery of black\nsmoker (on-axis HV) in 1979 illuminated previously unexplored realms of chemistry and\nsparked consideration of HV as a potential chemical environment for prebiotic synthesis. The\nunderstanding of origin of life, previously represented by a prebiotic organic soup theory, has\nbeen redefined with the discovery of HV (Martin et al, (2008) Nature Reviews Microbiology\n6, 805–814; Lane et al, (2024) Nature 626, 948-951). Over the past two decades, numerous\nlaboratory experiments have been conducted in the conditions present in black smoker HV,\nincluding high temperatures, pressure, sulfide minerals, and rich redox chemistry. These\nstudies have experimentally demonstrated essential carbon and nitrogen nonenzymatic\nmetabolic reactions crucial for life based on HV minerals (Huber et al, (1997) Science 276,\n245–247; McCollom et al, (2007) Chem.Rev. 107, 382−401; Varma et al, (2018) Nature\nEcology & Evolution 2, 1019–1024; Barge et al, (2019) PNAS 116 4828-4833; Preiner et al,\n(2020) Nature Ecology & Evolution 4, 534–542). The discovery of alkaline HVs (off-axis HV)\nin 2000, distinguished by their relatively moderate temperatures, high alkalinity, and\nhydroxide-based minerals, in contrast to black smokers, has unlocked fresh avenues in\nprebiotic chemistry. The intricate structure of vent fluid channels within alkaline HVs,\ncombined with thermal gradients, has been demonstrated experimentally to induce\nthermophoresis, facilitating the concentration increases of biomolecules or salts and\nintroducing new chemico-physical pathways for driving life processes (Baaske et al, (2007)\nPNAS, 104, 9346–9351; Niether et al, (2016) PNAS, 113, 4272–4277; Matreux et al, (2021)\nNature Chemistry 13,1038–1045).\nIn this context, our discoveries regarding HVs, including their aligned nanopores, selective\nion transport mechanisms, and osmotic energy conversion, hold promise for shedding light on\nthe origin of life through fundamental physical and chemical principles. Currently in the origin\nof life field, there is no clear explanations or evidence about the maintenance of pH gradients\nthrough membranes and their utilization (Vasiliadou et al, (2019) InterfaceFocus 9, 20190073).\nAn exploration of how selective ion transport membrane form and contribute to energy\nacquisition, based on our understanding of physicochemical principles, could offer direct\ninsights into these debates. Furthermore, highlighting the multifaceted possibilities that our\ndiscoveries offer, such as confinement-controlled chemical reactors and self-organized\nstructures in flow and gradient regime, has the potential to catalyze new directions in the study\nof life's origins. This could, in turn, spark interdisciplinary collaborations across diverse fields\nlike organic chemistry, geology, materials science, and microfluidics, fostering innovative\napproaches and collaborative insights.\nThe origin of life remains one of the most significant puzzles in the scientific community,\nwith no consensus yet reached. The field requires constructive disagreement and must welcome\ndiverse, well-founded frameworks for understanding the origin of life (Lane et al, (2024)\nNature 626, 948-951). Through this approach, the field may gradually approach a solution.\nThis concludes our response to the reviewers’ comments. We are grateful for their comments\nwhich have helped us improve the rigor and quality of our manuscript.\nREVIEWERS' COMMENTS\nReviewer #2 (Remarks to the Author):\nIn response to being asked for an additional opinion on how the authors have answered the\nconcerns of Reviewer #4:\nIndeed, this referee is getting into a great deal of technical detail on what might and can\ncause osmotic and electrostatic gradients. It seems to me the the authors have tried to fit as\nmuch as possible into the article, but those questions cannot be resolved within this piece of\nwork, and it’s really not a question here of whether a given orientation is proven. That ought\nto be future work in several fields, some of it inspired by this piece if and when it’s published.\nThese authors provide a hypothesis for the effects they observe and measure. Some of the\ndetails might turn out to need further work to clarify them, but that doesn’t invalidate the\npresent work. I don’t see that holding up the publication of this piece by asking the authors\nmore and more questions on some of those details is helpful, as indeed the referee seems to\nrealize with the comment \"It would be a pity if the article were not published because it\ncontains a lot of experimental information that can help to better understand the problem.\nThe existence of these electrostatic potentials is important to understand how hydrothermal\nvents and the associated ecosystem function today.” All of us working in the field know that\nthere are still huge gaps in our understanding of the path to the origin of life. We don’t expect\nany one piece of work to provide all the answers. I’ve just reread the abstract of the latest\nversion and I don’t see that the authors are making excessive claims.\nAuthor’s reply:\nWe would like to express our gratitude to Reviewer #2 for their fair and constructive evaluation of\nour responses to Reviewer #4.\nReviewer #4 (Remarks to the Author):\nAuthor’s reply:\nWe would like to express our gratitude once again to Reviewer #4 for their comprehensive review of\nour paper. As Reviewer #1 addressed, the detailed mechanistic study of the orientation and osmotic\nenergy conversion is beyond the scope of this single paper. In other words, our findings, derived\nfrom the first nanoscale analysis of submarine HVs, highlight the new research directions in material\nself-organization and the conversion of ionic energy. The detailed mechanistic studies are currently\nbeing conducted in our laboratory using microfluidic devices and computational fluid dynamics.\nReviewer comment 1:\nThank you for the answer. Unfortunately, it is not a clarification; rather, you confirm that the\nexternal calcite sheath that covers the hydrothermal vents you have studied prevents the selective\nosmotic gradient and the osmotic power generation, if I understood your response well.\nAuthor reply 1:\nThank you for your comment. Osmotic energy conversion is expected to mostly occur in the\npresence of magnesium hydroxide, which acts as a membrane-like structure to separate two solutions\nwith chemical gradients. The adsorption of Ca2+ ions onto the magnesium hydroxide generates a\npositive surface charge, which in turn facilitates anion-selective ion transport and osmotic energy\nconversion.\nReviewer comment 2:\nI know some of these papers very well, and to the best of my knowledge, they have no\ndemonstrated orientation of nano or microcrystals in the tubular walls of chemical gardens. Most of\nthe articles in which has been measured an electrostatic potential between the walls of the tubular\nstructures used iron or cobalt silica gardens whose walls are made of silica gel and nanocrystals of\niron oxyhydroxides. No orientation has been shown in these tubes. What really matters to trigger the\nformation of an electrostatic potential is porosity rather than orientation. See, for instance,\nKotopoulou et al., Nanoscale Anatomy of Iron-Silica Self-Organized Membranes: Implications for\nPrebiotic Chemistry. Angew Chem Int Ed Engl 2021, 60 (3), 1396-1402.\nAuthor reply 2:\nThe alignment observed in systems mimicking natural HV samples will be addressed in our\nupcoming paper. In our first response letter, we provided preliminary data in a separate \"reviewer-\nonly\" section to support this. We observed that flowing two solutions that replicate the HV\nenvironment resulted in precipitates with alignment, similar to the conditions seen in chemical\ngarden experiments. We hope to discuss the detailed mechanism and geochemical driven alignment\nin the forthcoming paper.\nReviewer comment 3:\nThen, it seems that the pH difference is what matters, right?\nAuthor reply 3:\nYes. The presence of a pH difference is crucial. We conducted experiments in a pH gradient\nenvironment that could have existed on early Earth, using simulants of reducing high pH fluids (pH\n10-12) and oxidative low pH seawater (pH 4-6). These data are detailed in the main text (Fig 6f, gray\ncircle).\nThis concludes our response to the reviewers’ comments. We are grateful for their comments which\nhave helped us improve the rigor and quality of our manuscript."}]

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