2.8. Determination of potassium and phosphorus leakage

This experiment was performed to estimate the secondary metabolism of the tested bacterial strains by computing the proportion of potassium, and phosphorus secreted into the medium. Potassium and phosphorus ion efflux was determined according to a previously described method [36],[37]. The concentration of free potassium, and phosphorus ions in the bacterial suspension of each bacterial strain was measured after the exposure of bacterial cells to nutrient broth for 20, 60, and 100 min. The mixture was incubated at 37 °C. Three replicates of each tube were conducted. At each pre-established interval, extracellular potassium (EasyRA Medica, Bedford, USA) and phosphorus (COBAS® INTEGRA 400 Plus Analyzer, Roche, Basel, Switzerland) concentrations were measured using photometric procedures. 300 µM of Cetyl Trimethyl Ammonium Bromide (CTAB) was used as positive control for giving 100% permeabilization (Sigma-Aldrich, Merck, Darmstadt, Germany). Results were expressed as the amount of extracellular free K⁺ and PO₃⁻ in the growth medium (µmol/mL). Each treatment was performed in triplicate.