The monoterpene limonene is a cheap and readily available starting material for bioconversions to higher value compounds, namely perillic acid, perillyl alcohol and perillaldehyde. These compounds are used as pharmaceuticals, in chemoprevention and cancer therapy [1], as antimicrobial and as flavourings. Limonene is the major constituent of orange peel and other plant essential oils. Solvent-resistant strains therefore are excellent candidates for performing limonene bioconversions, since they are expected to be more resistant to the apolar substrate and to accumulated toxic terpenoids. The microbial toxicity and multiplicity of limonene metabolites result in low product concentrations and high downstream processing costs [2].

In this work new solvent-resistant Pseudomonas strains were identified and characterised. These strains were able to grow on water soluble carbon sources such as glucose, but also in apolar substrates namely limonene and organic solvents (n-hexadecane, 1-octanol, cyclohexane, toluene). Growth was followed by spectrofotometry and with a McFarland nefelometer. High-molecular-weight DNA molecules from the strains, grown in the presence and absence of organic solvents, were resolved by pulse-field gel electrophoresis.

Bioconversion experiments of limonene, 0,5% (v/v_Total), to perillic acid were carried out in a minimal medium at 36ºC, pH 7. Limonene and perillic acid were analysed by HPLC. Furthermore these strains can be used in a multiphase bioreactor with limonene as a second phase. Optimisation studies are being carried out.

[1] Shulz, S., Reinhold, D., Schmidt, H., Ansorge, S., Hollt, V., Biochem. Biophys.Res. Commun., 241, 720-725, 1997.

[2] Van der Werf, M., de Bont, J.A.M., Leak, D., Adv. Biochem. Eng. Biotechnol., 55, 147-177, 1997.