Production of perillic acid from orange essential oil by Yarrowia lipolytica using a top-aerated bioreactor

Abstract

R-(+)-Perillic acid, a promising anticancer and immunomodulatory agent, is the major product from the biotransformation of R-(+)-limonene-rich orange essential oil by the yeast Yarrowia lipolytica. Due to the abundance and low cost of orange essential oil, which is a byproduct of the citrus industry, we attempted to improve the biotransformation process by optimizing yeast cell mass production. Then, the whole process was transposed and adapted to a 2-L instrumented bioreactor. Cell mass production was optimized in shaker flasks using a statistical experimental design. The optimized medium (g·L^-1: 22.9 glucose, 7.7 peptone, 4.1 yeast extract and 1.0 malt extract) resulted in a 13.0 g·L^-1 final cell concentration and 0.18 g cell·L^-1·h^-1 productivity. A further increase to 18.0 g·L^-1 was achieved in a 2-L bioreactor upon fed-batch culture. High-purity limonene bioconversion was performed in the same bioreactor utilizing top aeration to diminish terpene volatilization; as a result, 839.6 mg·L^-1 perillic acid accumulated after 48 h. Under the same conditions, industrial orange essential oil afforded 806.4 mg·L^-1 perillic acid. The yeast growth medium optimization resulted in a twofold increase in biomass accumulation and a reduction in growth medium nitrogen sources, which lowered the catalytic biomass production cost. Compared with conventional bottom aeration, the bioreactor top aeration strategy resulted in higher bioconversion rates. The conditions developed for high-purity limonene bioconversion were successfully applied to low-cost orange essential oil, showing the robustness of Y. lipolytica yeast.

Keywords: Biotransformation; Limonene; Orange essential oil; Perillic acid; Top-aerated bioreactor; Yarrowia lipolytica.

Fig. 1

Perillic acid accumulation by Y. lipolytica ATCC 18942 cells collected at different growth phases in YMB medium. The results represent the average of three experiments and the standard deviation

Fig. 2

Kinetics of cell mass accumulation (solid line) and glucose consumption (dashed line) during Yarrowia lipolytica ATCC 18942 fed-batch cultivation in the 2-L bioreactor using the optimized medium. The arrows indicate the pulse addition of 50 ml of tenfold concentrated medium to the culture. The bioreactor was run at 28 °C, 1 vvm aeration, 30% pO₂, and stirring speed between 250–700 rpm. The results represent the average of three experiments and the standard deviation

Fig. 3

Perillic acid accumulation from high-purity limonene in surface aerated bioreactor. Operational conditions: 20 g·L⁻¹ cell mass, pH 6.9, 1% oxygen saturation, pO₂ 1%, aeration rate 0.5 vvm. Arrows indicate limonene pulses at 0.16% (v/v) every 24 h. The numerical results are the average of duplicate experiments

Fig. 4

Perillic acid production from orange essential oil in surface-aerated 2-L bioreactor. Operational conditions: 20 g·L⁻¹ cell mass, pH 6.9, 1% oxygen saturation, pO₂ of 1%, and aeration rate of 0.5 vvm. The arrows indicate limonene pulses at 0.16% (v/v) every 24 h. The numerical results are the average of duplicate experiments