Efficient Production of L-(+)-Lactic Acid Using Mycelial Cotton-like Flocs of Rhizopus oryzae in an Air-Lift Bioreactor Enoch Y. Park,* Yuuko Kosakai, and Mitsuyasu Okabe Laboratory of Biotechnology, Department of Applied Biological Chemistry, Faculty of Agriculture, Shizuoka University, 836 Ohya, Shizuoka 422-8529, Japan L-(+)-Lactic acid production was enhanced in a culture of Rhizopus oryzae by induction of a mycelial flocs morphology. By conventional culture the morphology of R. oryzae is that of a pellet-like cake; however, when mineral support and poly(ethylene oxide) are added to the culture, the morphology of R. oryzae takes on a cotton-like appearance. The formation of these cotton-like mycelial flocs was induced by the addition of 5 ppm poly(ethylene oxide) into a 12-14 h culture containing 3 g/L of the mineral support before the formation of the conventional pellet morphology. The cotton-like flocs were also formed in cultures grown in an air-lift bioreactor. This morphology allowed effective mass transfer inside the flocs and effective fluidity of culture broth in an air-lift bioreactor. L-(+)-Lactic acid concentration produced by mycelial flocs in an air-lift bioreactor, with the support and poly(ethylene oxide), was 104.6 g/L with a yield of 0.87 using 120 g/L of glucose as the substrate; for this culture without both, the concentration was 43.2 g/L. These results demonstrate that cotton-like mycelial flocs are the optimal morphology for use in the air-lift bioreactor culture of R. oryzae. Introduction Although substantial advancements have been made in the study of mold molecular biology in recent years, progress in mold-culturing technique has proceeded at a slow pace, despite the importance of mold microorgan- isms in the production of many industrial products. In most of cases, these microorganisms are used as chemical stocks, pharmaceuticals, and enzymes, but relatively little is known about the ability of mold microorganisms to change mycelial morphology or the correlation between this morphology and productivity (Vicht-Lifshitz et al., 1990). Since molds show many morphological variations in submerged fermentation, it is difficult to control the morphology such that it optimizes the reactor perfor- mance. In bioreactors, the morphologies from discrete fila- ments of hyphae to pellets of highly entangled hyphal mass are found. The filamentous fungi in a typical growth result in the formation of pellets or in highly viscous mycelial suspension (Gerin et al., 1993). The pellets are composed of highly interwoven hyphae and are generally spherical in shape. Such pellets are subject to mass transfer limitations which produce solute gra- dients through the spheres. Mycelium at the center of these pellets becomes nutrient limited as the pellet increases in size; growth is eventually continued in a shell of limited thickness at the surface of the pellet (Pirt, 1967). On the other hand, in the case of filamentous morphology, mass transfer limitations are commonly encountered and growth of the mycelium on the impeller or on the electrodes hampers optimal control of the process. The phenomenon of flocculation may potentially be utilized to culture mycelia (Mill, 1964). The other method that has been proposed for this purpose is the disper- sion of mycelia in culture broth. In a study using the latter techniques, we previously reported that the min- eral support dispersed mycelial pellets in the culture broth and emulsified soybean oil used as a carbon source in a Streptomyces culture, resulting in an enhancement of cephamycin C production (Ichida et al., 1997). The mineral support consisted of a bundle of fibers, 30-50 µm in length and 0.2 mm in diameter, which had many rectangular tunnels of 10 × 3.6 Å. Silica dioxide and magnesium oxide constituted approximately 53% and 23%, respectively, of the total components. The average particle size was 16 meshes, and the porosity of the support was 70%. The support enhanced an emulsion of soybean oil-water in the culture broth and also enhanced the dispersion of mycelia pellets, which led to an improvement of both oil consumption and antibiotic production. The dispersed growth of Strepto- myces is particularly difficult to achieve by conventional culture methods using chemically defined or a complex media. Moreover, this support has the ability to suspend a large pellet-like mycelial cake as a cotton-like mycelial floc. The morphology of Rhizopus oryzae, a potential L-(+)-lactic acid producer, was previously characterized by the formation of a large pellet-like cake using con- ventional culture methods such as a shake flask or fermentor. However, it has been shown that when 3 g/L mineral support and 5 ppm poly(ethylene oxide) are added to the culture, cotton-like mycelial flocs are induced (Kosakai et al., 1997). We found that addition of the mineral support increased the electrostatic repul- sion between mycelia 3.5-fold compared to that of myce- lia, which allowed a dispersed growth of R. oryzae in the early growth phase. This morphology allowed effective mass transfer inside the flocs and effective fluidity of culture broth in the reactor, which led to a 1.6-fold * Corresponding author. Telephone: 81-54-238-4887. Fax: 81- 54-237-3028. E-mail: yspark@agr.shizuoka.ac.jp. 699 Biotechnol. Prog. 1998, 14, 699-704 S8756-7938(98)00064-2 CCC: $15.00 © 1998 American Chemical Society and American Institute of Chemical Engineers Published on Web 09/03/1998