Methyl-ketone production by Ca-alginate/Eudragit RL entrapped spores of Penicillium roqueforti Christian Larroche, Muhammad Arpah and Jean-Bernard Gros Laboratoire de G~nie Chimique Biologique, Universitd Blaise Pascal, F-63177 Aubiere Cedex, France (Received 8 July 1987; revised 22 April 1988) The synthesis of methyl-ketone from octanoic acid, sodium salt by Ca-alginate entrapped spores was studied. AIginate gel beads enclosed by an acrylate-methacrylate copolymer (Eudragit RL) were used due to their enhanced mechanical stability. The measurement of the carbon dioxide evolved showed that octanoic acid quantitatively yields 2-heptanone as the sole methyl-ketone. The reaction could be performed in a reactor with a nonbuffered medium by maintaining the pH at a constant value, 5.5. The determination of the volume of the hydrochloric acid solution added during the course of the biotransformation process allowed a continuous monitoring of the reaction progress performed in an aerated, stirred-tank reactor. Keywords: Penicillium roquefi~rti; methyl-ketone; 2-heptanone; immobilizedspores Introduction Fungal spores are generally stable and can be stored or transported as a concentrated biochemical catalyst which is readily available when required. Further- more, they give high, reproducible conversion of substrate and minimal amounts of undesirable prod- ucts.I Product recovery is also facilitated due to the lack of mycelium proliferation. Immobilization of fungal spores inside gel matrices has been far less investigated than immobilization of bacteria and yeasts. 2 However, some attempts have been made with nongrowing fungal spores used as biocatalysts. Aspergillus and Penicillium species have been entrapped on ion-exchange resins and used for sucrose inversion; 3 Aspergillus ochraceus spores have been immobilized on diatomaceous particles (Celite) and used in the 11 a-hydroxylation of progesterone. 4 Transformation of penicillin V to 6-aminopenicillanic acid has been performed using entrapped Fusarium sp. spores. 1 Penicillium roqueforti spores have been shown to carry out the conversion of fatty acids to methyl- ketones. 5'6 This reaction is of great practical impor- tance, since it exhibits a predominant role in flavor synthesis during the ripening of blue cheeses. 7-9 It could allow a decrease in the traditional blue cheese ripening time or the manufacture of new cheeses presenting a "blue taste" without the presence of any fungus. Methyl-ketones may also be used in the com- position of salad dressings, soups, crackers, and cakes. 7.10 In this paper, we report the behavior of Penicillium roqueforti spores entrapped in alginate beads during the course of 2-heptanone synthesis from octanoic acid and used in an aerated stirred reactor. Materials and methods Microorganism Penicillium roqueforti ATCC 64383,11 which was origi- nally isolated from French blue cheese by Lactolabo (Dange St-Romain, France), was conserved by repli- cating on Czapek agarJ z Spore production, storage, and recovery Spores were produced on buckwheat seeds using solid state fermentation conditions 13-15in one-liter bottles or in fixed bed fermentors. The medium harvested after the cultivation was stored at -20°C without further treatment. The spore-supporting medium stored as above was thawed for 2 h and the external spores were recovered by vortexing whole buckwheat grains in a sterile 0.05% Tween 80 solution. This spore suspension was 106 Enzyme Microb. Technol., 1989, vol. 11, February © 1989 Butterworth Publishers