Enhancing Hyaluronic Acid Production: Statistical Approaches to Sustainable Optimization of Fermentation Media Components

This study developed a two-step statistically integrated optimization framework to identify the effects of key fermentation medium components controlling hyaluronic acid (HA) biosynthesis by Streptococcus zooepidemicus. As an initial phase, the Plackett–Burman design was employed to identify the most influential components among yeast extract, casein, peptone, beef extract, MgSO4·7H2O, K2HPO4, KH2PO4, and (NH4)2SO4 by conducting 12 fermentation runs, and 30 g/L of glucose was used as the carbon source. Among the eight ingredients, yeast extract, MgSO4·7H2O, and KH2PO4 were identified as the most significant factors in enhancing HA production. The following steps were based on the selection of the best carbon and yeast extract sources. Sucrose was selected as the optimal carbon source among glucose and lactose, and Tastone 900-Baker’s yeast extract was selected as the optimal nitrogen source among various yeast extract sources. The final phase of the optimization procedure employed the Box–Behnken design to determine the optimal concentrations of three ingredients: yeast extract (10–30 g/L), MgSO4·7H2O (0.2–2.0 g/L), and KH2PO4 (1–4 g/L). The results depicted that the optimized media formulation, composed of 30.0 g/L of yeast extract, 1.16 g/L of MgSO4·7H2O, and 4.0 g/L of KH2PO4, enhanced HA production and biomass OD600 to 545.9 mg/L with 1250–1500 kDa and 2.53 OD600 in a 250 mL shake flask scale, which was around a 10-fold increase in HA production compared with run #10 of Plackett–Burman (57.42 mg/L). This study provided preliminary results for future process conditions optimization, scale-up studies, and techno-economic evaluation.