Primary recovery of hyaluronic acid produced in Streptococcus equi subsp. zooepidemicus using PEG-citrate aqueous two-phase systems

Given its biocompatibility, rheological, and physiological properties, hyaluronic acid (HA) has become a biomaterial of increasing interest with multiple applications in medicine and cosmetics. In recent decades, microbial fermentations have become an important source for the industrial production of HA. However, due to its final applications, microbial HA must undergo critical and long purification processes to ensure clinical and cosmetic grade purity. Aqueous two-phase systems (ATPS) have proven to be an efficient technique for the primary recovery of high-value biomolecules. Nevertheless, their implementation in HA downstream processing has been practically unexplored. In this work, polyethylene glycol (PEG)-citrate ATPS were used for the first time for the primary recovery of HA produced with an engineered strain of Streptococcus equi subsp. zooepidemicus. The effects of PEG molecular weight (MW), tie-line length (TLL), volume ratio (V-R), and sample load on HA recovery and purity were studied with a clarified fermentation broth as feed material. HA was recovered in the salt-rich bottom phase, and its recovery increased when a PEG MW of 8000 g mol(-1) was used. Lower V-R values (0.38) favoured HA recovery, whereas purity was enhanced by a high V-R (3.50). Meanwhile, sample load had a negative impact on both recovery and purity. The ATPS with the best performance was PEG 8000 g mol(-1), TLL 43% (w/w), and V-R 3.50, showing 79.4% HA recovery and 74.5% purity. This study demonstrated for the first time the potential of PEG-citrate ATPS as an effective primary recovery strategy for the downstream process of microbial HA.