Gttp isopore membrane filters

The supernatant from chemostat samples was acidified by adding 35 μl of 10% sulfuric acid (wt/wt) to 700 μl of supernatant. After centrifugation, it was filtered (0.22-μm nylon) and stored at 4°C until analysis. Ethanol, acetate, formate, lactate, pyruvate, malate, glucose, cellobiose, and fructose in the supernatant were quantified by HPLC (Waters, Milford, MA) using an Aminex HPX-87H column (Bio-Rad, Hercules, CA) equipped with a refractive index detector and UV detector. The column was run with 0.5 ml min⁻¹ of 2.5 mM H₂SO₄ at 60°C. Cell dry weight samples were measured in triplicate by filtering 5-ml samples through preweighed 0.2-μm GTTP Isopore membrane filters (Merck Millipore, Ltd.), washing with an equal volume of ultrapure water, and drying at 100°C for 24 h before weighing. For analysis of pellet total nitrogen (TN) and total organic carbon (TOC), frozen cell pellets were thawed and washed twice with ultrapure water. The cell TN and TOC were analyzed in a Shimadzu TOC-Vcph TOC analyzer with a TN unit and ASI-V autosampler added (Shimadzu Scientific Instruments, Columbia, MD), using an acidified glycine standard as described previously by Holwerda et al. (45 (link)). The offline OD was measured at 600 nm in triplicate in a Thermo Scientific Genesys 335901 visible spectrophotometer.

Samples for bulk XANES spectroscopy were collected after 3 days of experiment. For each sample, 10 ml of experimental medium was passed through a polycarbonate filter (GTTP Isopore membrane filters, Merck Millipore, pore size 0.2 μm). The materials collected on the filters were then rinsed three times in distilled water. The filters were then immediately placed at −20 °C and kept frozen until analyses. XANES spectra were collected at the S K-edge on beamline 4-3 at Stanford Synchrotron Radiation Lightsource. A liquid He cryostat was used to keep the samples frozen during XANES analyses. Between 2 and 3 spectra were accumulated for each sample to improve the signal-to-noise ratio. Energy calibration was performed at several intervals between samples using the S K-edge spectrum of a sodium thiosulfate standard, setting the position of the maximum of the first pre-edge feature to an energy of 2,472 eV (ref. 52 ). Two to four spectra were acquired and averaged per sample. A linear background determined in the pre-edge region (2,400–2,470 eV) was subtracted from the averaged data, and the spectra were then normalized at 2,510 eV. S K-edge XANES spectra of a polycarbonate filter and of a standard S⁰ compound (precipitated sulfur, Fisher Scientific) on sulfur-free polypropylene tape were also acquired and similarly processed.

Purified E. cloacae OMVs were fixed in 2% paraformaldehyde in PBS and filtered through 1:10 poly-L-lysine (Sigma-Aldrich) treated 0.2mm GTTP isopore membrane filters (Merck Millipore). Then the filtrates were fixed again with Trump’s fixative (EMS) and washed with 0.1 M sodium cacodylate (pH=7.24). The samples were post-fixed with 2% OsO4, washed with water, and then dehydrated in 25-100% graded ethanol series with 25% increments. Dehydrated samples were critical point dried by Tousimis Autosamdri-815 (Tousimis) and mounted onto a 12 mm carbon conductive adhesive tab and aluminium stub (EMS) having sputter gold/palladium coating. The grids were imaged using Hitachi SU-5000 FE-SEM (Hitachi High Technologies) and visualized using EM Wizard software.