*Article* **Continuous Production of DHA and EPA Ethyl Esters via Lipase-Catalyzed Transesterification in an Ultrasonic Packed-Bed Bioreactor**

**Chia-Hung Kuo 1,2,\* , Mei-Ling Tsai <sup>1</sup> , Hui-Min David Wang <sup>3</sup> , Yung-Chuan Liu <sup>4</sup> , Chienyan Hsieh <sup>5</sup> , Yung-Hsiang Tsai <sup>1</sup> , Cheng-Di Dong <sup>6</sup> , Chun-Yung Huang 1,\* and Chwen-Jen Shieh 7,\***


**Abstract:** Ethyl esters of omega-3 fatty acids are active pharmaceutical ingredients used for the reduction in triglycerides in the treatment of hyperlipidemia. Herein, an ultrasonic packed-bed bioreactor was developed for continuous production of docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) ethyl esters from DHA+EPA concentrate and ethyl acetate (EA) using an immobilized lipase, Novozym® 435, as a biocatalyst. A three-level–two-factor central composite design combined with a response surface methodology (RSM) was employed to evaluate the packed-bed bioreactor with or without ultrasonication on the conversion of DHA + EPA ethyl ester. The highest conversion of 99% was achieved with ultrasonication at the condition of 1 mL min−<sup>1</sup> flow rate and 100 mM DHA + EPA concentration. Our results also showed that the ultrasonic packed-bed bioreactor has a higher external mass transfer coefficient and a lower external substrate concentration on the surface of the immobilized enzyme. The effect of ultrasound was also demonstrated by a kinetic model in the batch reaction that the specificity constant (*V* 0*max*/*K*<sup>2</sup> ) in the ultrasonic bath was 8.9 times higher than that of the shaking bath, indicating the ultrasonication increased the affinity between enzymes and substrates and, therefore, increasing reaction rate. An experiment performed under the highest conversion conditions showed that the enzyme in the bioreactor remained stable at least for 5 days and maintained a 98% conversion.

**Keywords:** docosahexaenoic acid ethyl ester; eicosapentaenoic acid ethyl ester; lipase; packed-bed reactor; ultrasonication; kinetics; mass transfer; solvent-free; ethyl acetate
