Determination of the Reaction Yield during Biotransformation of the Volatile and Chemically Unstable Compound -Ionone by Aspergillus niger Franck Grivel, Christian Larroche,* and Jean Bernard Gros Laboratoire de Ge ´nie Chimique Biologique, Universite ´ Blaise Pascal, F-63177 Aubie `re Cedex, France Biotransformation of -ionone by Aspergillus niger IFO 8541 was a complex system, due to the low precursor solubility in water (0.88 mol/m 3 ) which gave a two-phase liquid system, its high volatility (vapor pressure 7.2 Pa), and its poor chemical stability. A dynamic model, considering transfers between organic, aqueous, and gas layers, was established. It also took into account the decrease in organic layer area exchange during a process, due to progressive precursor disappearance. Its solving needed separate determination of two parameters. The first-order kinetic constant for chemical degradation, K 1 , was estimated from side experiments involving a model system at thermodynamic equilibrium; its value was found to be close to 4.17 × 10 -6 s -1 . The volumetric transfer coefficient between gas and aqueous phase was estimated by analogy with results obtained for oxygen transfer. Results obtained demonstrated high liquid-gas transfer rates, while liquid-liquid transfers were severely limited. These data were then used to estimate the true yield of a biotransformation experiment, which was found to be close to unity. Introduction A large number of compounds with a trimethylcyclo- hexane ring have been isolated from tobacco during the last few decades. The compounds of this group have characteristic and low threshold odors (1), and they are thought to be key flavor components in the essential oils of air-cured tobaccos (2). Several investigators have therefore attempted to obtain tobacco flavorings by microbial conversion of either carotenoids or ionones. These last compounds are being considered to be key intermediates of carotenoids degradation (3). Krasnoba- jew and Helminger (4) described the microbial transfor- mation of -ionone by fungi, including Aspergillus, Bot- ryosphera, and Lasiodiplodia. All three genera were able to hydroxylate the ionone ring at positions 4 and 2 (5). They reported that, with Lasiodiplodia theobromae ATCC 28570, up to 10 g of -ionone/L of medium was converted with a yield of 90%, giving -cyclohomogeraniol as the main product. Mikami et al. (6) have studied the production of tobacco flavorings and have focused on the microbial conversion approach for this purpose. They found that the biotrans- formation of -ionone by Aspergillus niger JTS191 gave a mixture of more than 13 derivatives. This complex was very effective for tobacco flavoring at the parts-per- million level (1-5 ppm) (6, 7). The recovering yield of -ionone after this reaction time was close to 70%. Larroche et al. (8) have also published a similar process, which involved fed-batch biotransformation of -ionone by the commercial strain Aspergillus niger IFO 8541 entrapped in calcium alginate beads operated in an aerated, stirred bioreactor. The overall yield at the end of the experiments was here near 50%. It appeared, thus, that the apparent reaction yield was always less than 100%. Although some mineralization of compounds of the terpene family has been reported (9), one had to consider that no convincing argument was provided to explain this behavior. In this paper, we present a tool allowing investigation of the way in which the -ionone fraction not converted into metabolites disappeared from the aqueous medium, to determine the actual amount of precursor metabolized by the biocatalyst and, thus, the true reaction yield. The work is based on a modeling approach because the carrying out of blank experiments in parallel is difficult when the processes are performed using bioreactors. Materials and Methods Chemicals. -Ionone was a gift from SKW Biosystems (Grasse, France). Its purity was determined as 95% by gas chromatography, and it was used without further purification. All other chemicals were of reagent grade and were obtained from commercial sources. Microorganism. The strain A. niger IFO 8541 was used throughout this study. It was cultivated on potato/ dextrose/agar (Difco) in Petri dishes at 27 °C and was conserved by periodic replications on this medium. After 15 days of cultivation, the resulting Petri dishes were stored at -20 °C without any treatment. Conidia recovery was achieved by pouring the content of two dishes, thawed for 2 h at room temperature, into a 250 mL conical flask filled with 100 mL of distilled water. This suspension was then agitated on a rotary shaker operating at 4 s -1 for 15 min, and the upper layer was recovered. Its spore content was determined using a haematocytometer (Malassez cell). Culture Medium. It consisted of three solutions, A-C, which were prepared and autoclaved separately. Solution A contained 37.5 g/L sucrose, 6.88 g/L (0.05 mol/ * Corresponding author. Fax: 33 4 73 40 78 29. E-mail: larroche@gecbio.univ-bpclermont.fr. 697 Biotechnol. Prog. 1999, 15, 697-705 10.1021/bp9900312 CCC: $18.00 © 1999 American Chemical Society and American Institute of Chemical Engineers Published on Web 04/22/1999