Quaatro

Samples for dissolved organic carbon (DOC) and total dissolved nitrogen (TDN) were filtered onto combusted Whatman GF/F filters (∼0.8 μm pore size) and immediately stored at −20°C until analysis. DOC and TDN analyses were performed by a high-temperature catalytic method using a Shimadzu TOC-L analyzer equipped with a total nitrogen unit. Calibration for non-purgeable organic carbon (NPOC) was done with potassium phthalate, and potassium nitrate was used for TDN. Results were validated with Surface Seawater Reference (SSR) material for DOC and TDN (CRM Program, Hansell Lab). Reproducibility was no greater than 2% (Hansell and Carlson, 1998 (link)).
Dissolved inorganic nitrogen (NH₄⁺, NO₂^–, NO₃^–) and dissolved inorganic phosphorus (PO₄^3–) concentrations were determined spectrophotometrically using segmented flow analysis (QuAAtro, Seal Analytical) following standard methods (Hansen and Koroleff, 2007 (link)). Validation and accuracy of results were checked with reference material (KANSO Co., Ltd.) before and after sample analyses. Quality control is performed annually through participation in an intercalibration program (QUASIMEME Laboratory Performance Study).

Soil horizons of each soil core were divided into Oi and Oe horizons (organic materials), and OA and A horizons. The OA horizon was placed in between organic and mineral A layers, and most A horizons were found below 50 cm depth. We defined here Oi horizon as “top soil,” and Oe, OA, and A horizons as “sub-soil.” Soil texture was determined by the pipette method (Gee and Bauder, 1986 ). Electrical conductivity (EC) and soil pH were measured in a soil-water suspension (1:5 ratio, w/v) using a pH/EC meter (Orion Star A215, Thermo Scientific, Waltham, MA, USA). Water content (WC) was determined by measuring the weight change in soils after oven-drying at 105°C for 48 h. Total carbon (TC) and total nitrogen (TN) contents in soils were measured using an elemental analyzer (FlashEA 1,112 Thermo Electron corporation, Waltham, Massachusetts, USA). For NH₄⁺-N and NO₃⁻-N contents analysis, fresh soil was extracted using 2 M KCl solution and subsequently filtrates were analyzed on an auto-analyzer (QuAAtro; Seal Analytical, Norderstedt, Germany). Fresh soil was mixed with deionized water and then filtered through Whatman filter paper #42 firstly and then 0.45-μm filter to acquire water extractable carbon (WEC) and nitrogen (WEN).

Parallel cores were recovered for geochemical analyses and porewater was extracted from 23 depths from SK cores and 16 depths from BB cores directly after recovery by using Rhizon samplers (Rhizosphere Research Products B.V, Wageningen, The Netherlands) (Seeberg-Elverfeldt et al. 2005 (link)). Porewater samples for NO₃⁻ and NH₄⁺ were kept frozen until later analysis at the IOW (Leibniz Institute for Baltic Sea Research Warnemünde) and measured after the methods of Grasshoff et al. (1999 ) by using a continuous flow nutrient analyser (QuAAtro, Seal Analytical GmbH, Norderstedt, Germany). Additional geochemical measurements of these cores were made and are discussed in Reyes et al. (2016 (link)).

Dissolved inorganic macronutrient data were measured on-board, following Jeon et al. [59 (link)]. In short, dissolved inorganic phosphate and silicic acid concentrations were measured according to the Joint Global Ocean Flux Study (JGOFS) protocols [61 ], using a four-channel Auto-Analyzer (QuAAtro, Seal Analytical, Germany). Precisions for measurements were ±0.02 and ±0.28 µmol kg⁻¹ for phosphate and silicic acid, respectively. Al-though nitrate and ammonium data were not available, the overall average concentrations of dissolved phosphate and silicate suggest non-limiting conditions [59 (link)], i.e., 1.7 ± 0.3 µM and 80 ± 8 µM, respectively (Table 1). Dissolved iron (dFe) was measured as described by van Manen et al. [62 ]. Samples were UV-digested for 4 h and pre-concentrated. Dissolved trace metal concentrations were calibrated using a mixed stock solution. Dissolved iron (dFe) concentrations were relatively low in surface waters for all stations (average 0.19 ± 0.09 nM; Table 1).

Dissolved inorganic nitrogen compounds (NH₄⁺, NO₂⁻, and NO₃⁻) and dissolved inorganic phosphorus (PO₄³⁻) concentrations were determined spectrophotometrically by QuAAtro segmented flow analysis (Seal Analytical, Norderstedt, Germany) following standard methods (30 ). The validation and accuracy of the results were checked with reference material (Kanso Technos, Osaka, Japan) before and after sample analyses. The quality control is performed annually by participating in an intercalibration program (QUASIMEME Laboratory Performance Study).

The in situ temperature of vent fluids was determined by scuba divers using a thermocouple. Salinity values were measured onboard shortly after collection with an Autosal 8400B salinometer calibrated with IAPSO standard seawater. pH values were measured with a pH meter (Radiometer PHM-85, Denmark) at 25 °C, with a precision better than 0.003 pH unit. Dissolved oxygen was measured using a gas chromatographic method. Nitrate, nitrite, and ammonium nitrogen concentrations were colorimetrically analyzed with a continuous flow autoanalyzer (QuAAtro, SEAL Analytical Inc.). Dissolved sulfide concentrations were assayed by the methylene blue method [26 ] (the precision was 0.7% at 20 μM, n = 6), immediately after returning to the land-based laboratory. Dissolved CH₄ was measured by gas chromatography using the gas-stripping method [27 (link)]. A dissolved inorganic analyzer (AS-C3, Apollo SciTech, United States) was employed for the analysis of dissolved inorganic carbon values, with a precision of 0.1%. Chl a was extracted from the filters using the acetone extraction method and measured with a fluorometer according to the procedure summarized in [28 ]. Prokaryotic abundances were determined with an Accuri C6 flow cytometer (BD Biosciences).

A total of 9 geochemical properties of soil were assessed, including pH, water content, organic carbon, organic nitrogen, ammonium nitrogen (NH ${}_4^{+}$ -N), silicate (SiO ${}_4^{2-}$ -Si), nitrite nitrogen (NO ${}_2^{-}$ -N), phosphate phosphorus (PO ${}_4^{3-}$ -P) and nitrate nitrogen (NO ${}_3^{-}$ -N) (Table 1). Soil pH was measured by adding 10 ml of distilled water to 4 g of soil and recording pH using a pH electrode (PHS-3C, Shanghai REX Instrument Factory, Shanghai, China). Water content was determined as gravimetric weight loss after drying the soil at 105°C until constant weight. Analysis of organic carbon and organic nitrogen was performed using an Elemental Analyzer (EA3000, Euro Vector SpA, Milan, Italy). The other properties were analyzed using a High Performance Microflow Analyzer (QuAAtro, SEAL Analytical GmbH, Norderstedt, Germany).