Optimization of hyaluronic acid production from Bacillus subtilis strain PV154141.1in submerged fermentation

Abstract

This study presents a novel approach to hyaluronic acid (HA) production from wild-type Bacillus subtilis strain PV154141.1, avoiding the need for genetic modifications commonly used in previous research. HA production was conducted under aseptic conditions using submerged fermentation in a medium containing glucose, lactose, yeast extract and tryptone. Critical fermentation parameters including media composition, incubation temperature, initial pH, and inoculum level, were optimized resulting in a significantly enhanced HA yield. Following optimization, HA was extracted using two distinct methods, the conventional ethanol method, which mainly relies on centrifugation and ethanol usage, and the CTAB-ethanol method, which involves greater volumes of cetyltrimethylammonium bromide (CTAB) and ethanol. Results indicated that the CTAB-ethanol method yielded significantly higher HA concentrations (472 µg/ml) compared to the conventional method (59.1 µg/ml), using the same experimental setup, because of the combined effect of CTAB, NaCl and ethanol for selective precipitation of HA, which is statistically significant (p ≤ 0.05). Fourier transform infrared (FTIR) spectroscopy further characterized the extracted HA, confirming its desired molecular structure and associated functional groups. Characteristic absorption peaks for HA were identified at 685.83, 834.92, 998.92, 1148.02, 1297.1, 1617.66, 2892.41, and 3257.69 cm^− 1. Each peak represents a specific biomolecule. The functional groups present in our sample included amide groups, hydroxyl groups, polyphenols, and proteoglycan sugar rings, confirming the presence of HA in the sample. The optimized fermentation process and a more efficient extraction technique contribute to advancing HA production methodologies. This research contributes a cost-effective and scalable approach to HA production, positioning wild-type B. subtilis as a promising non-GMO alternative for industrial applications in biotechnology, cosmetics and pharmaceuticals.