Bio pigments produced by Monascus spp. have potential applications mainly in the food and medical industries. In the present study, a two-step statistical method was used to optimize the production of yellow, orange, and red pigments from Monascus purpureus. Eleven independent variables, including four carbon sources (wheat, barley, rice, and potato extracts), two nitrogen sources (ammonium nitrate and urea), nutrient elements (P, K, and microelements), and four bioreactor conditions (temperature, aeration, stirring, and pH) were optimized through Plackett-Burman design (PBD) and response surface method (RSM) methods. The model for each pigment was constructed and validated. With regard to carbon sources, the highest level of pigments was achieved at 2 g/l of rice and 9 g/l barley for yellow pigment, 2 g/l of rice and 18 g/l of barley for orange pigment, and 18 g/l of rice and 18 g/l wheat for red pigments. Temperature and barley extracts triggered the production of yellow pigments. The orange pigment was increased by pH and barley. Rice and wheat have a positive significant influence on red pigments. Aeration, pH, and stirring increased the production of the pigment. Citrinin is a biotoxin produced by Monascus during the fermentation process. The concentration of citrinin varied from 0.054 to 0.135 (μg/mL). The lowest amount of citrinin was achieved at 2 L/Min aeration or 6 L/Min stirring. This system is critical for the bioprocess, as it inhibits the citrinin product, and it could be a promising step in increasing pigment yield.

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