The relationship between hyphal vacuolation, fragmentation and citric acid production by Aspergillus niger, was investigated in batch and fed-batch culture. Quantitative information on morphology and vacuolation was obtained by image analysis. Time profiles of the morphology parameters - mean perimeters of clumps, P, lengths of filaments, L - and vacuoles, together with, specific growth and production rates, were used to establish a link between vacuolation, fragmentation and product formation under various agitation conditions and glucose levels. Under intensive agitation conditions and during the early fermentation stages, the characteristics observed were the increased specific growth rates and hyphal branching, along with low vacuolation levels. These were followed by fragmentation of the highly vacuolated parts of filaments and regrowth at later stages, a process that maintained that the mycelium grew old and highly vacuolated, with limited renewal, conditions that did not favour increased biosynthetic activity. Increased vacuolation and low specific production rates were observed at low glucose levels in fed-batch culture. The results indicate that vacuolation weakened the hyphae and low glucose levels created the conditions that favoured fragmentation and made the mycelium more susceptible to it when exposed to increased agitation.