Nuclepore polycarbonate membrane

The lyophilized peptide was solubilized in DMSO up to 40 mg/mL, sonicated in a water bath for 5 min, and stored at −20 °C. Working peptide samples at defined final concentrations were solubilized from stock solutions in 10 mM HEPES 150 mM NaCl buffer, pH 7.4. The final DMSO content was maintained at 2% (v/v) in all experiments. Working peptide samples were sonicated in an ultrasonic bath for 2 to 5 min before use.
Small unilamellar vesicles (SUV) were prepared as previously described [81 (link)]. The lipid mixture was first solubilized in chloroform in a round-bottom flask. The solvent was evaporated under nitrogen flow until a thin lipid film was formed on the flask wall. The lipid film was further dried under vacuum overnight. A multilamellar vesicles (MLV) suspension was obtained after rehydration with the sample buffer and a series of 10 freeze–thaw cycles. The MLV suspension was extruded through a 50 nm-pore-size Nuclepore polycarbonate membrane purchased from Whatman/GE Healthcare (Maidstone, UK) using a LiposoFast-Basic plus Stabilizer setup from Avestin (Mannheim, Germany). This allowed the reorganization of MLVs into SUV. POPC, POPC:Chol (2:1), and POPC:Chol:SM (1:1:1) mixtures were prepared.

Phototrophic microorganisms cultures were contaminated at different Cr(NO₃)₃ concentrations, 1, 5, 10, 25, 50, 100, and 200 μM Cr(III), and incubated under the same conditions as mentioned above for a period of 9 days.
For SEM analysis, cultures were filtrated in Nuclepore polycarbonate membranes (Whatman, Ltd.) and then were fixed in 2.5% glutaraldehyde diluted in Millonig phosphate buffer (0.1 M pH 4) at 4°C for 2 hours and washed four times in the same buffer, dehydrated in increasing concentrations of ethanol (30%, 50%, 70%, 90%, and 100%), and dried by critical-point (CPD 030 Critical Point Drier, BAL-TEC GmbH, 58579 Schalksmühle). Finally, samples were mounted on aluminium metal stubs and coated with a 5 μm gold layer (K550 Sputter Coater, Emitech, Ashford, UK) for better image contrast. A Zeiss EVOMA 10 scanning electron microscope (Carl Zeiss NTS GmbH, Oberkochen, Germany) was used to view the images.
For EDX microanalysis, cells were homogenously distributed and filtered on polycarbonate membrane filters. These filters were fixed, dehydrated, and dried by critical-point drying and then coated with gold. An EDX spectrophotometer Link Isis-200 (Oxford Instruments, Bucks, England) coupled to the microscope operating at 20 kV was used. Finally, EDX-SEM spectra from individual cells were obtained.

Flow experiments
were conducted similarly
to the description in previous work,^{33 (link)} but
replacing the inflow of the desired enzymes with the desired volume
of PEBs, which remained compartmentalized in a Continuously Stirred
Tank Reactor (CSTR) during the experiment. The openings of the reactors
were sealed with Whatman Nuclepore polycarbonate membranes (5 μm
pore size) to prevent outflow of PEBs. Cetoni Low-Pressure High-Precision
Syringe Pumps neMESYS 290N were used to control the dispense of the
different solutions, prepared in Gastight Hamilton syringes (2500–10 000
μL), into the CSTR. The precise flow profile of the desired
flow rates was programmed using the Cetoni neMESYS software.
To detect and determine outflow concentrations from the CSTR, both
online and offline detection was employed. Online absorbance detection
was achieved with an Avantes AvaSpec2048 Fiber Optic spectrometer
and Avantes AvaLight 355 nm LED combined with a custom designed flow
cuvette provided to us by LabM8. Alternatively, offline measurement
could be achieved by means of connecting the outflow to a BioRad Model
2110 fraction collector. These fractions could subsequently be probed
for NADH absorbance using a Tecan Spark M10 platereader, or probed
for ATP, ADP, NAD+, and NADH using a Shidmadzu Nexera X3 HPLC.
Further details on the instrumentation and experimental protocols
can be found in the Supporting Information.