The mixing zone of the Kuroshio and Oyashio Extension (KOE) is a critical carbon sink in the global ocean, significantly influencing global warming. However, the impact of ocean mixing on methane (CH4) biogeochemical cycling in the KOE remains poorly understood. In this study, we integrated station observations and isotope tracer incubations to examine the controls on methane methanotrophic activity in the dynamic KOE region (135.98°-151.99°N; 29.5-42.4°E) of the Northwest Pacific during cruises in 2021 (May-June) and 2022 (June-July). Samples were collected using 12 L Niskin bottles, equipped with an SBE 911 plus conductivity-temperature-depth (CTD) probe. CH4 samples were taken in 120 mL brown bottles following the standard dissolved gas sampling procedure, with 50 μL of saturated HgCl2 added to inhibit biological activity. Dissolved CH4 was measured using a cryogenic purge-and-trap system coupled to an Agilent GC-8890 gas chromatograph with a flame ionization detector (FID). Seawater samples for bacterial production (BP) and methane oxidation (MOx) rate measurements were gently introduced into 1 L polycarbonate bottles without bubbles or vortexing, and stored at 4°C in the dark before analysis. MOx rates were quantified using a radiotracer approach with 3H-labeled methane, and BP rates were measured with 3H-labeled leucine.

Seawater samples collected by 12 L Niskin bottles equipped with SBE 911 plus a conductivity-temperature-depth (CTD) probe. The methane sample was collected in 120 mL brown bottles and then 50 μL saturated HgCl2 was added to inhibit biological activity. Samples were sealed with aluminum caps containing Teflon septa and temporarily stored in the dark at 4°C.Dissolved methane was measured using a cryogenic purge-and-trap setup connected to an gas chromatograph (GC-8890, Agilent, USA) with a flame ionization detector (FID) according to the detailed methods given in (Mao et al., 2022; Li et al., 2023; Liu et al., 2024; Wang et al., 2024). The 3H radiotracer approach was used to measure the aerobic MOx rate (Mao et al., 2022; Liu et al., 2024). Seawater samples (triplicate samples and one killed control) were filled into 20 mL Hungute tubes with butyl rubber stoppers without headspace. ~50 μL 3H-CH4 gas was injected using airtight needles into each sample by displacing the same volume of seawater. Controls were killed with a final concentration of 3.7% formaldehyde prior to tracer additions. All samples were incubated in the dark at in situ sampled temperature for 48 hours. The concentration of CH4 added to the samples was less than ~2 nM. Once the incubation was finished, ~100 μL subsample was collected in 2 mL glass vials filled with scintillation liquid, which was used to measure total radioactivity (DPM-3H-H2O and DPM-3H-CH4) using a scintillation counter (Tri-Carb 3110TR, USA). The remaining sample was transferred to a 50 mL falcon tube containing 2 mL 37% formaldehyde and purged with N2 for 45 min to remove the unused 3H-CH4. Then 2 mL samples added 5 mL scintillation liquid was quantified for the measurement of DPM-3H2O. For the measurement of BP rates, the procedure of 3H-leucine incorporation was followed (Zhou et al. 2023). About ~25 μL leucine (final concentration of 0.012 nM) with a specific activity of >106 disintegrations per minute, DPM) was added to ~2 mL seawater sample. The bacterial activity in control samples was terminated bacterial activity by trichloroacetic acid (TCA). Incubations were carried out for about 48 hours at sampling temperature. After incubation, all samples were killed by TCA and centrifugated at 10,300 rpm for 15 minutes. Samples were processed by washing with 5% TCA and 80% Ethanol. Radioactivity was measured by the liquid scintillation counter (Tri-Carb 3110R, PerkinElmer, USA) with 2 mL scintillation cocktail (Ultima Gold, PerkinElmer, USA).