Improved monoterpene biotransformation with Penicillium sp. by use of a closed gas loop bioreactor

A closed gas loop bioprocess was developed to improve fungal biotransformation of monoterpenes. By circulating monoterpene-saturated process gas, the evaporative loss of the volatile precursor from the medium during the biotransformation was avoided. Penicillium solitum, isolated from kiwi, turned out to be highly tolerant towards monoterpenes and to convert α-pinene to a range of products including verbenone, a valuable aroma compound. The gas loop was mandatory to reproduce the production of 35 mg L⁻¹ verbenone obtained in shake flasks and also in the bioreactor. Penicillium digitatum DSM 62840 regioselectively converted (+)-limonene to the aroma compound α-terpineol, but shake flask cultures revealed a pronounced growth inhibition when initial concentrations exceeded 1.9 mM. In the bioreactor, toxic effects on P. digitatum during biotransformation were alleviated by starting a sequential feeding of non-toxic limonene portions after a preceding growth phase. Closing the precursor-saturated gas loop during the biotransformation allowed for an additional replenishment of limonene via the gas phase. The gas loop system led to a maximum α-terpineol concentration of 1,009 mg L⁻¹ and an average productivity of 8-9 mg L⁻¹ h⁻¹ which represents a doubling of the respective values previously reported. Furthermore, a molar conversion yield of up to 63% was achieved.