A process to optimize L-lactic acid production from glucose by Rhizopus oryzae, based on sustaining floc morphology throughout the fermentation process, is herein performed. During the fermentation, supplementary ammonium sulfate was added intermittently to maintain the ammonia level of the culture medium always higher than 0.1 g/L. With replenish of nitrogen source, mycelia flocs did not aggregate, and the lactic acid production was optimized upon the fermentation being controlled at pH 4.3-4.5 by adding calcium carbonate slurry. In contrast, without supplementary addition of nitrogen source, mycelial clumps formed, resulting in a poor production of lactic acid. In the initial batch fermentation process, the final concentration of lactic acid produced was 109 g/L, with a yield (g lactic acid/g glucose consumed) of 0.87 and a productivity of 2.73 g/L h, using 125 g/L of glucose as substrate. For the first four cycles of repeated-batch fermentation, the average final concentration, the productivity and the yield of lactic acid were 113 g/L, 4.03 g/L h and 0.90, respectively. (C) 2007 Published by Elsevier Ltd on behalf of Taiwan Institute of Chemical Engineers.