Highly accumulative production of l(+)-lactate from glucose by crystallization fermentation with immobilized Rhizopus oryzae

In order to produce microbiologically large amount of l(+)-lactic acid (LA) from glucose, batch and fed-batch (intermittent addition of sterilized glucose powder aseptically) cultures of Rhizopus oryzae NBRC 5384 (identical to NRRL 395 and ATCC 9363) whose mycelia were immobilized in situ within sponge-like cubic particles (3.5 mm edge long) were carried out at 37°C in a three baffled shake flask. Appropriately calculated amount of fine powdery calcium carbonate (CaCO3) was added initially or intermittently to control pH of the culture liquids. High accumulations of LA (145 g/L and 231 g/L, in reality 176 g/L and 280 g/L as anhydrous calcium lactate) were achieved by a batch (glucose concentration = 150 g/L) and a fed-batch cultures (the initial glucose concentration = 150 g/L and the intermittent addition of glucose equivalent to 100 g/L). In these cultures the yields and productivities of LA were, 95.0%, 1.42 g/L·h and 92.5%, 1.83 g/L·h, respectively. Existence of considerable amounts of calcium lactate (Ca(LA)2) as crystals in the culture broth was experimentally proved by two evidences: (i) heating up (70°C) followed by quick low centrifugal force to remove remaining CaCO3 solids from culture broth and then cooling down (37°C) followed by incubation of the culture supernatant at 37°C to observe recrystallization of Ca(LA)2, and (ii) the measurement of solubility of Ca(LA)2 in the culture media. It was conceptually discussed to be able to avoid the product inhibition by means of crystallization fermentation for the high accumulation of LA by R. oryzae.