Cyclosporin A (CyA) is a neutral cyclic polypeptide as an immunosuppressive drug that usually was produced from the culture medium of Tolypocladium inflatum. In this study, screening of six environmental components (dairy sludge (%), molasses (%), glucose (g/mL), malt extract (%), TES (mL), and ammonium sulfate (%)) and five culture parameters (temperature (°C), pH, time (h), shaking (rpm), and inoculation (mL)) of CyA production based on Plackett–Burman experimental design was performed. The most important factors affecting fermentation including carbon source (dairy sludge, glucose), nitrogen source (malt extract, ammonium sulfate), and trace elements solution (TES) were optimized using central composite rotatable design (CCRD). Based on the results obtained from the statistical design, the culture medium with combinations of 46.47% of carbon source, 9.56% of nitrogen source, and 1.49 mL of mineral matter was selected as optimal conditions. The treatments of ultrasound (US), high hydrostatic pressure (HHP), and pulsed electric field (PEF) were applied to the culture medium on the 1st, 5th, and 10nth days of fermentation. Scanning electron microscopy (SEM) images related to treatment were prepared to evaluate the morphology of the fungus, and the bioassay method was used to compare the amount of CyA production in terms of clear zone creation of each plate. The amount of CyA in the optimal was measured by high performance liquid chromatography (212 ppm) and US stress on the 10th day of fermentation (334 ppm), respectively, which indicates a twofold increase in the production of CyA. The results show the optimized culture medium containing dairy sludge waste with US treatment can have a suitable efficiency for the production of CyA

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