Hyaluronic acid has a wide range of biomedical applications and its commercial value is highly dependent on its purity and molecular weight. This study highlights the utility of aqueous two-phase separation as a primary recovery step for hyaluronic acid and for removal of major protein impurities from fermentation broths. Metabolically engineered cultures of a lactate dehydrogenase mutant strain of Lactococcus lactis (L. lactis NZ9020) were used to produce high-molecular-weight hyaluronic acid. The cell-free fermentation broth was partially purified using a polyethylene glycol/potassium phosphate system, resulting in nearly 100% recovery of hyaluronic acid in the salt-rich bottom phase in all the aqueous two-phase separation experiments. These experiments were optimized for maximum removal of protein impurities in the polyethylene glycol rich top phase. The removal of protein impurities resulted in substantial reduction of membrane fouling in the subsequent diafiltration process, carried out with a 300 kDa polyether sulfone membrane. This step resulted in considerable purification of hyaluronic acid, without any loss in recovery and molecular weight. Diafiltration was followed by an adsorption step to remove minor impurities and achieve nearly 100% purity. The final hyaluronic acid product was characterized by Fourier-transform IR and NMR spectroscopy, confirming its purity.

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Supported liquid membrane (SLM) is the most effective technique to extract and recover the desired product from the biomass products in a single step. The study of the operation parameters in SLM system is very important to improve the yield of extraction and recovery of the product. In this study, different types of stripping agents such as sodium hydroxide (NaOH), sodium carbonate, hydrochloric acid, trimethylamine, and water were tested in the SLM system to extract levulinic acid (LA). By using 0.3 M trioctylamine in 2-ethyl-1-hexanol as liquid membrane phase, it was found that NaOH was the best stripping agent to extract LA. The concentration of the NaOH stripping agent was varied from 0.25 M to 1 M. The best stripping agent concentration was 0.5 M, which gave an LA extraction of 86% from a 10 g/L LA aqueous solution. The flow rate of the feed and stripping phase was investigated from 25 mL/min to 125 mL/min. SLM operated at 75 L/min was found to be adequate in reducing the boundary layer thickness at both sides of the SLM phases without any leakage of the liquid membrane and breakage of the matrix support. At the best stripping condition, 89% of the LA was extracted using 0.5 M NaOH that operated at flow rate of 75 mL/min.