Hyaluronic acid production by polyurethane-immobilized Streptococcus zooepidemicus: process optimization, repeated-batch performance, and characterization of the recovered polymer fraction

This study investigated hyaluronic acid (HA) production by Streptococcus zooepidemicus immobilized in flexible polyurethane (PU) foam and characterized the recovered HA-enriched polymer fraction. Biomass loads of 4–10 g and the effects of temperature, agitation, and aeration were evaluated using the CTAB turbidimetric response. Under the central-point conditions (8 g biomass, 35 °C, 500 rpm, and 2 vvm), the mean apparent HA concentration was 0.471 g/L after 28 h, with individual values ranging from 0.445 to 0.508 g/L. Total organic carbon and total nitrogen decreased by 28% and 43%, respectively. Apparent kinetic parameters, calculated from CTAB-derived concentrations, included Y P/S = 0.01484, mass productivity = 0.01486 g/L·h, and specific product formation rate = 0.00847 g/g·h. During repeated-batch operation, a measurable CTAB-reactive response persisted for up to 15 cycles; however, approximately 84% of the immobilized material was lost, and dense clump formation limited further analysis. Ethanol/NaCl precipitation yielded 56.65% apparent recovery of CTAB-reactive material. The recovered fraction showed no detectable cytotoxic effect on Vero cells up to 1000 μg/mL and measurable antimicrobial and ABTS responses, although these findings cannot be attributed exclusively to HA. FTIR showed functional groups consistent with an HA-containing fraction, and thermogravimetric analysis indicated a main degradation event near 280 °C. Because CTAB quantification was not fully validated in the fermentation matrix and molecular weight, rheological properties, and absolute purity were not determined, the results should be interpreted as preliminary. Overall, PU immobilization supported repeated-batch HA-associated production, but further analytical and process validation is required.