Thesis for Doctor of Philosophy

Carla da Conceição Caramujo Rocha de Carvalho
“Bioconversion of (-)-carveol to (-)-carvone catalysed by whole cells of Rhodococcus erythropolis. Influence of reaction conditions on cell morphology and physiology.”
Ph. D. Biotechnology, IST, 2003
Supervisor: Prof. M. Manuela R. da Fonseca

Abstract

Cells of Rhodococcus erythropolis DCL14 have several carveol dehydrogenases which allow them to carry out the cofactor dependent stereoselective oxidation of (-)-trans-carveol to (‑)‑carvone. When a mixture of (-)-carveol is supplied, diastereomeric resolution is concomitantly achieved.

A 24 mL n-dodecane:mineral medium reaction system, with a 1:5 phase ratio, was effective in overcoming substrate and product low water solubility, while allowing the maintenance of cell viability (monitored off-line by fluorescence microscopy and image analysis) and high production rates.

At higher scale, maximum trans-carveol conversion (92%) and specific production rate (1.69 mg carvone/h.mg prot) were obtained in a 500 mL biphasic mechanically stirred reactor at, respectively, ambient temperature in phosphate buffer, and 28ºC in mineral medium. At both scales, carvone highly inhibited its own production at concentrations above 50 mM. This was overcome in an air-driven column reactor after adapting the cells to the presence of solvent, substrate and product. The maximum production of carvone, 1.56 g at 341h, as well as high carveol conversion rates were achieved in a 130 mL reactor with a G4 porous sparger, after adapting the cells for 136h. Still, the highest productivity (0.19 mg carvone/h.mL org phase), yield (0.96 g carvone/g carveol) and final concentration (1.03 M) were attained in a 65 mL reactor with a G2 glass sparger when cells adapted for 268h were used as biocatalyst.

Keywords: Rhodococcus erythropolis, carveol, carvone, fluorescence microscopy, image analysis, organic solvents.