Polycarbonate membrane filter

All liposomes were made using
a 57:38:5 molar ratio of distearoylphosphatidylcholine (DSPC) (Avanti
Polar Lipids), cholesterol (Sigma-Aldrich), and polyethylene glycol-2000-distearoyl
phosphoethanolamine (PEG–DSPE) (Avanti Polar Lipids). Ligands
conjugated to PEG–DSPE were included in the 5% PEG–DSPE.
To assemble the liposomes, chloroform solutions of DSPC, cholesterol
and PEG–DSPE were mixed together. Excess chloroform was evaporated
using N₂ (g) and DMSO solutions of ligand–PEG–DSPE
were added to the dried liposome mixture. The liposomes were lyophilized
overnight then hydrated in PBS, pH 7.4 (Corning) to achieve a final
liposome concentration of 1–2 mM. To get the liposome mixtures
into solution, the mixtures were sonicated in a water bath 3×
for 30 s. Liposomes were extruded at room temperature using a miniextruder
(Avanti Polar Lipids) through polycarbonate membrane filters with
400 and 100 nm pore sizes (Millipore) 20 times each. Liposomes for
functional assays were purified over a Sepharose CL-4B column (Sigma-Aldrich)
upon extrusion.

Aliquots of 10 mg/ml stock lipid solutions in chloroform were mixed and chloroform was removed under nitrogen stream; the mixtures were held thereafter in a vacuum desiccator overnight. The dried lipid mixtures were re-hydrated with 150 mM KCl, 5 mM HEPES, pH 7.4 to a final concentration of 1 mM lipid for 60 min on ice with gentle agitation every 15 minutes. The lipid-buffer solutions were then vortexed for 30 seconds to fully homogenize the sample and passed through lipid extruder (Avanti Polar Lipids, Inc.) using polycarbonate membrane filters (Millipore) with the pore sizes of 200 and 100 nm, sequentially. Liposomes’ size and polydispersity were determined for each lipid composition and preparation by light scattering using Zetasizer Nano-ZS90 (Malvern). Homogeneous populations of Large Unilamellar Vesicles (LUVs) of 100 ± 26 nm diameter with a polydispersity <0.2 were used in FCS measurements. The liposome diameter measured for each lipid sample was used in analyzing the FCS data from that sample.

Samples for metagenome analyses were collected from St. 301 (5 m deep) and St. 307 (5, 30, 150, and 750 m deep) in October 2019 (coordinates 43.155517 N 28.005383 E and 43.1696 N and 29.001283 E, respectively). Up to 6.9 L of seawater from each sampling depth were filtered through a series of 20 μm Nylon Net filters (Millipore), 5 μm polycarbonate membrane filters (Millipore), and 0.22 μm SterivexTM Filter Units (Merck). DNA was then extracted only from these Sterivex filters using standard phenol-chloroform protocol [43 (link)]. In short, Sterivex filters were treated with CTAB lysis buffer and then treated with 1 mg ml^− 1 lysozyme and 0.2 mg ml^− 1 proteinase K (final concentrations). Then nucleic acids were purified with phenol/chloroform/isoamyl alcohol and chloroform/isoamyl alcohol.

Dark respiration and net photosynthetic oxygen evolution were measured in the middle of the light period within 3 h using a Clarke-type electrode (Hansatech, United Kingdom). Cells were harvested by filtering onto polycarbonate membrane filters (5 μm, Millipore, Germany) under gentle vacuum pressure (<0.01 MPa). These cells were then re-suspended in seawater buffered with 20 mM Tris–HCl with a final Chl a concentration of approximately 0.5 μg mL⁻¹. The pH levels of the Tris buffered-medium were pre-adjusted by adding hydrochloric acid or sodium hydroxide to the same levels of the cultures (pH 7.83 for HC and 8.13 for AC), and the O₂ levels were achieved by flushing the medium with pure N₂. The resuspended cells were injected into an oxygen electrode vessel with a magnetic stirrer held in a water-jacked chamber (temperature controlled at 27°C) under the same level of light intensity. The respiration rate was estimated in darkness by covering the reaction chamber with aluminum foil. Photosynthetic O₂ evolution was determined under growth O₂ levels.

On day 0 and day 10, a 500-ml seawater sample was collected from each bucket with or without the olivine addition. The collected seawater was firstly filtrated through a 20-μm nylon sieve and then sequentially filtrated through 3- and 0.2-μm pore size polycarbonate membrane filters (47-mm diameter; Millipore) for microbe collection at a negative pressure of <0.01 MPa. The bacteria that settled on the membrane filter of 3-μm pore size were defined as the particle-attached fraction (PA, 3–20 μm), while those that settled on the filter of 0.2-μm pore size were defined as the free-living fraction (FL, 0.2–3 μm). The DNA of bacteria from both the 3- and 0.2-μm filters were extracted using a DNeasy PowerSoil Kit (Qiagen, Germany) according to the manufacturer’s protocol.
Amplicon sequencing of the microbial community in the DNA extraction was performed by Tianjin Novogene Bioinformatic Technology Co., Ltd. (Tianjin, China). The PCR products of one replicate of the olivine-added group in surface seawater are not enough for sequencing, so this part of the data is excluded from the data processing. The V3–V4 region of the bacterial 16S rDNA gene was amplified using the primer pair 341F (5′-CCTAYGGGRBGCASCAG-3′) and 806R (5′-GGACTACNNGGGTATCTAAT-3′) (Zhang et al., 2016 (link)), and sequencing was performed on an Illumina Hiseq 2500 pe250.

The eDNA was sampled using two different methods. The first method used a 47‐mm‐diameter glass microfiber filter GF/F (nominal pore size 0.7 μm; GE Healthcare Life Science, Little Chalfont, UK) to estimate eDNA shedding and decay rates, and the second method used a series of 47‐mm‐diameter polycarbonate membrane filters (pore size 10, 3, 0.8, and 0.4 or 0.2 μm; MILLIPORE, US) to estimate eDNA size distribution. Disposable gloves were worn when collecting water samples, and the outside of the sampling bottles was washed with tap water after the samples were collected. This was to prevent contamination during water sampling and filtration. The filtering devices (i.e., filter funnels [Magnetic Filter Funnel, 500 ml capacity; Pall Corporation, Westborough, MA, USA], plastic holders [ADVANTEC, Japan], nipple joints [ADVANTEC, Japan], hoses [TOYOX, Japan], 1‐L beakers, tweezers, and sampling bottles used for water sampling) were bleached after every use in 0.1% sodium hypochlorite solution for at least 5 min.