J2 21

After incubation, the culture was centrifuged at 5000 rpm at 4 °C for 20 min in a Beckman J2-21 with swinging rotor to separate the cell pellet from the cell-free supernatant. Nanoparticles were purified from both the supernatant and pellet. To recover CuNPs present in the cell-free supernatant, the solution was centrifuged at 17,000× g for 15 min in the same centrifuge with a fixed rotor. The CuNPs containing the pellet was then resuspended in double-distilled water (ddH₂O) and washed twice by repeated centrifugation steps.
To recover CuNPs from the cell pellet, ultrasonic wave shocks of short durations (15 s) were given to the ddH₂O-suspended pellet to rupture the microbial cell wall. After sonication, the sample was centrifuged 5000 rpm for 20 min (Beckman J2–21, Fullerton, California) and the NPs were recovered from the supernatant. This step was repeated three times to completely remove the cell debris from the supernatant. To recover CuNPs present in the cell-free supernatant, the solution was centrifuged at 17,000× g for 15 min in the same centrifuge with a fixed rotor. After being washed twice with deionized water and dried at 80 °C in an oven, the CuNPs were used for further characterization and experiments.

To obtain the peptide extracts, the original procedure was performed to remove all high-molecular weight components (preliminary proteins and carbohydrates) and secondary metabolites. Taking 10 g of the raw plant, over ground material, was ground, soaked in 500 mL of 1 M acetic acid (Merck, Darmstadt, Germany), and left for 1 h at room temperature. During the exposition, the suspension was obtained using ultrasonication (five times through 10 min). Subsequently, the suspension was heated at 100 °C for 30 min, cooled, and centrifuged (75,000× g, 40 min, using a J2-21 (Beckman, Krefeld, Germany). Acetone was added to collect the supernatant at a ratio of 2:5 and precipitated at +4 °C overnight. Then, a pellet was separated by centrifugation (40,000× g, 40 min, J2-21 (Beckman, Germany). To improve the dissolving of a peptide extract, the pellet was dissolved in 100 mM acetic acid and repeatedly centrifuged (twice, 40,000× g, 20 min). To remove residual acidic quantities, the pellet was lyophilized twice by dissolving in 50 mL of MQ water.

Liquid cultures were harvested using a high-speed centrifuge (Beckman J2–21, Baltimore, USA) at 4000 x g for 10 minutes. The pellets were stored at −80 °C and lyophilized for 24 h at −40 °C under freeze-dried machine.
FAME production followed the procedure provided by.^{47 (link)} FAMEs were analyzed using a Agilent’s Gas Chromatography (GC) system with discharge ionization detection equipped with a capillary column (Stabilwax-DA, 30 m 0.25 mm ID, film thickness 0.25 mm). GC inlet was set at 250 °C and the injections were in a volume of 1 μL. The temperature program started at 50 °C and then increased to 170 °C at a rate of 20 °C min⁻¹, with a plateau for 1 min. After this plateau, the temperature increased from 170 °C to 220 °C at a rate of 4 °C min⁻¹ and then kept constant for 14 minutes. The total analysis time was 35 minutes. Helium was used as carrier gas.