ARTICLE Improved Reactor Performance and Operability in the Biotransformation of Carveol to Carvone Using a Solid–Liquid Two-Phase Partitioning Bioreactor Jenna L.E. Morrish, Andrew J. Daugulis Department of Chemical Engineering, Queen’s University, Kingston, Ontario, Canada K7L 3N6; telephone: 613-533-2784; fax: 613-533-6637; e-mail: daugulis@chee.queensu.ca Received 15 January 2008; revision received 14 April 2008; accepted 21 April 2008 Published online 2 May 2008 in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/bit.21957 ABSTRACT: In an effort to improve reactor performance and process operability, the microbial biotransformation of ()-trans-carveol to (R)-()-carvone by hydrophobic Rho- dococcus erythropolis DCL14 was carried out in a two phase partitioning bioreactor (TPPB) with solid polymer beads acting as the partitioning phase. Previous work had demon- strated that the substrate and product become inhibitory to the organism at elevated aqueous concentrations and the use of an immiscible second phase in the bioreactor was intended to provide a reservoir for substrates to be delivered to the aqueous phase based on the metabolic rate of the cells, while also acting as a sink to uptake the product as it is produced. The biotransformation was previously underta- ken in a two liquid phase TPPB with 1-dodecene and with silicone oil as the immiscible second phase and, although improvement in the reactor performance was obtained relative to a single phase system, the hydrophobic nature of the organism caused the formation of severe emulsions leading to significant operational challenges. In the present work, eight types of polymer beads were screened for their suitability for use in a solid–liquid TPPB for this biotrans- formation. The use of selected solid polymer beads as the second phase completely prevented emulsion formation and therefore improved overall operability of the reactor. Three modes of solid–liquid TPPB operation were considered: the use of a single polymer bead type (styrene/butadiene copo- lymer) in the reactor, the use of a mixture of polymer beads in the reactor (styrene/butadiene copolymer plus Hytrel 1 8206), and the use of one type of polymer beads in the reactor (styrene/butadiene copolymer), and another bead type (Hytrel 1 8206) in an external column through which fermentation medium was recirculated. This last configura- tion achieved the best reactor performance with 7 times more substrate being added throughout the biotransforma- tion relative to a single aqueous phase benchmark reactor and 2.7 times more substrate being added relative to the best two liquid TPPB case. Carvone was quantitatively recovered from the polymer beads via single stage extraction into methanol, allowing for bead re-use. Biotechnol. Bioeng. 2008;101: 946–956. ß 2008 Wiley Periodicals, Inc. KEYWORDS: carvone; solid–liquid two phase partitioning bioreactor; polymer beads; Rhodococcus erythropolis DCL14 Introduction The worldwide flavor and fragrance industry is commer- cially significant and was estimated to be valued at US$ 19,910,000,000 in 2007 (www.leffingwell.com). Many flavor and fragrance compounds come from natural sources such as the essential oils of plants and seeds; correspondingly, because of the limited supply, there are only about 400 aromas that are manufactured on a scale greater than 1 ton per year (Krings and Berger, 1998) and many of these can be very costly. There is significant consumer interest in the natural production of these valuable compounds due to increasing health- and nutrition-conscious lifestyles (Abra- ham et al., 1994), and the rising demand for natural products used in food applications has increased the necessity for the production of natural flavor compounds (Etschmann and Schrader, 2006). An alternative production method for flavor/aroma chemicals is via microorganisms (Gatfield, 1988) whose products are considered to be natural. The natural label is important for the profitability of microbiologically produced flavors/aromas (Krings and Berger, 1998). Carvone is a common flavor and fragrance compound that is produced by over 70 different plants (Burdock, 1995) and exists as two enantiomers, (R)-()-carvone which has a spearmint aroma and (S)-(þ)-carvone which has a caraway aroma. The essential oil containing carvone is produced primarily by caraway (Carum carvi), dill (Anethum grave- olens) and spearmint (Mentha spicata) plants (Ravid et al., Correspondence to: A.J. Daugulis Contract grant sponsor: Natural Science and Engineering Research Council of Canada 946 Biotechnology and Bioengineering, Vol. 101, No. 5, December 1, 2008 ß 2008 Wiley Periodicals, Inc.