Enhancement of the Microbial Biotransformation of (-)-trans-carveol to (R)-(-)-carvone by Rhodococcus erythropolis DCL14 in Various Two Phase Partitioning Bioreactor Configurations by Jenna Lee Ellen Morrish A thesis submitted to the Department of Chemical Engineering in conformity with the requirements for the degree of Master of Science (Engineering) Queen’s University Kingston, Ontario, Canada January, 2008 Copyright © Jenna Lee Ellen Morrish, 2008 Abstract Carvone is a flavor and fragrance compound that is prominent in nature and is found in the essential oils of many plants. Carvone exists as two enantiomers, (R)-(-)- carvone which has a spearmint aroma and (S)-(+)-carvone which has a caraway aroma and can be used in a variety of applications: as a common food additive, as an antimicrobial/antifungal agent and as a potato sprout inhibitor. Carvone is currently produced by the extraction of essential oils from plants where the yield and quality of the extracted oil depends largely on successful agricultural production of dill, spearmint and caraway plants. Biotechnological production can offer a constant supply of carvone that is independent of several agricultural limitations. In this study, it was confirmed that the substrate and product of the microbial biotransformation of trans-carveol to (R)-(-)-carvone by Rhodococcus erythropolis DCL14 can be inhibitory to the cells at high concentrations. As such, a two phase partitioning bioreactor was employed where the function of the second phase (immiscible organic solvent or solid polymer beads) was to partition the inhibitory substrate into the aqueous phase at a rate governed by the metabolic demand of the cells and uptake the inhibitory product as it accumulated in the aqueous phase. Rational selection strategies were employed when determining the appropriate organic solvent and solid polymer to be used as the second phase. The performance of the reactor was evaluated based on volumetric productivity, length of biotransformation and total volume of substrate added to the reactor. The most successful reactor configuration was one in which styrene/butadiene copolymer beads were used as a second phase in the reactor and the fermentation medium was continuously circulated through an external extraction column i packed with Hytrel® 8206 polymer beads. The volumetric productivity, length of biotransformation and total volume of substrate added to this reactor were 99 mg/L.h, 48.75 h and 35 mL, respectively whereas in the single phase benchmark reactor the performance indicators were only 31 mg/L.h, 15.25 h and 5 mL, respectively. These results clearly show the advantage of employing a partitioning bioreactor configuration for the biotechnological production of high value chemical species that exhibit cytotoxicity. ii Co-authorship Ms. Emily Brennan and Ms. Helen Dry provided experimental assistance to portions of the material presented in Sections 3.4.2, 3.8.2 and 4.4. iii Acknowledgements I would especially like to thank my family for their constant support throughout my education. I would like to thank my mother and father (Stan and Sherrill Smith) for encouraging me to take on a second degree because they believed that I could do it. I would also like to thank my sister (Angela Smith) for first introducing me to Chemical Engineering at Queen’s as I’ve truly enjoyed my time here. I would like to thank my husband, Tim for his ever present support, and for learning to enjoy Kingston during our time here. He has been a great friend throughout my studies who helped me to laugh off the insignificant stresses and celebrate any small victories. I would like to thank my supervisor, Dr. Andrew Daugulis for his ever positive attitude, his continual interest and enthusiasm towards my work and his motivational support. I never considered completing graduate work for anyone else and have truly enjoyed getting to know him over past couple of years. Apart from Andrew’s support, there are three people to which I owe a big thank you for helping me throughout my degree. These individuals include Steve Hodgson who was always there to answer questions and fix my equipment crises and Emily Brennan and Helen Dry for their contributions as part of their fourth year thesis project. Last, but certainly not least, I would like to thank the lab mates that I have had the privilege of working with throughout my studies. I have to thank Dave Nielsen for introducing me to the lab and patiently supervising our lab work. I also have to thank George Prpich, Lars Rehmann, Jen Littlejohns, Matt McMahon, Fang Gao and Pedro Isaza for the fun times in the lab, their welcome advice and their continued support. iv Table of Contents Abstract............................................................................................................................... i Co-authorship...................................................................................................................iii Acknowledgements .......................................................................................................... iv List of Tables ..................................................................................................................viii List of Figures................................................................................................................... ix Chapter 1.0 Introduction.................................................................................................. 1 Chapter 2.0 Literature Review ........................................................................................ 4 2.1 Flavor and Fragrance Industries........................................................................... 4 2.2 Terpenes................................................................................................................... 5 2.3 Carvone.................................................................................................................... 7 2.3.1 Applications and Commercial Value ........................................................... 10 2.3.2 Current Extraction Methods for Carvone................................................... 12 2.3.3 Microbial Biotransformation........................................................................ 15 2.4 Overview of Microbial Biotransformations........................................................ 17 2.4.1 Isolated Enzyme Versus Whole Cell Biotransformations .......................... 19 2.5 Rhodococcus erythropolis...................................................................................... 21 2.5.1 DCL14 ............................................................................................................. 24 2.6 Partitioning Bioreactor Configurations.............................................................. 26 2.6.1 Adsorption ...................................................................................................... 28 2.6.2 Membrane Reactors....................................................................................... 29 2.6.3 Two Phase Partitioning Bioreactors............................................................. 31 2.6.3.1 Two Phase: Solvent................................................................................. 32 2.6.3.1.1 Solvent Selection............................................................................... 33 2.6.3.2 Two Phase: Polymer Beads.................................................................... 34 2.6.3.2.1 Polymer Selection............................................................................. 34 2.7 Discussion of Previous Work ............................................................................... 35 2.8 Summary and Scope of Current Work............................................................... 42 Chapter 3.0 Materials and Methods.............................................................................. 43 v 3.1 Chemicals and Polymers ...................................................................................... 43 3.2 Microorganism and Medium Composition ........................................................ 44 3.2.1 Microorganism ............................................................................................... 44 3.2.2 Medium Composition .................................................................................... 44 3.2.3 Inoculum Preparation ................................................................................... 45 3.3 Analytics................................................................................................................. 45 3.3.1 Gas Chromatography .................................................................................... 45 3.4 Quantification of Biomass .................................................................................... 46 3.4.1 Biomass Estimation: Absence of Organic Solvent, Substrate or Product 47 3.4.2 Biomass Estimation: Organic Solvent Present............................................ 48 3.4.3 Biomass Estimation: Inhibitory Substrate and/or Product Present ......... 49 3.5 Substrate and Product Toxicity........................................................................... 49 3.6 Solvent Selection.................................................................................................... 50 3.6.1 Biocompatibility: Determination of Critical log P...................................... 50 3.6.2 Bioavailability................................................................................................