Response Surface-Based Optimization of Single-Cell Protein Production in Phaffia rhodozyma

The yeast Phaffia rhodozyma is widely used as a natural source of astaxanthin for aquaculture, but its protein-rich biomass has been less studied as single-cell protein (SCP). This study explored the potential of P. rhodozyma as a dual-purpose production system by optimizing a low-cost medium for a protein-enriched mutant (AEC3/9), while also monitoring astaxanthin formation. A central composite design and response surface methodology (RSM) were used to evaluate the effects of glycerol, urea, ammonium sulphate, and initial pH. Shakeflask cultivations performed in triplicate (250 mL baffled flasks, 20 mL working volume, 22 °C, 250 rpm, 7 days) showed that protein productivity reached its maximum on day 4, which was selected as the reference point for optimization. Astaxanthin was included in the model but was not used as the main optimization target. The optimization results suggested that moderate glycerol concentrations (70–90 g/L), low urea (<0.2 g/L), and slightly acidic pH (5.9) were the most suitable conditions. Under the selected conditions (70.54 g/L glycerol, 0.134 g/L urea, 4.98 g/L (NH₄)₂SO₄, pH 5.86), biomass reached 26.11 g/L with a protein content of 29.79 % (7.78 g/L). Scale-up in a 5 L stirred-tank bioreactor confirmed the feasibility of the process, reaching 33.50 g/L biomass and 6.82 g/L protein at 88 h. The results indicate that protein accumulation occurs before the phase associated with increased astaxanthin production. Optimizing nitrogen source and pH allows stable production of P. rhodozyma biomass on glycerol, supporting its use as an SCP co-product alongside astaxanthin.