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Forty-one fungal species belonging to 15 fungal genera isolated from Egyptian soil and sugar cane waste samples were tested for their capacity of producing acidity and gluconic acid. For the tests, the fungi were grown on glucose substrate and culture filtrates were examined using paper chromatography analysis. Most of the tested fungi have a relative wide potentiality for total acid production in their filtrates. Nearly 51% of them showed their ability of producing gluconic acid. Aspergillus niger was distinguishable from other species by its capacity to produce substantial amounts of gluconic acid when it was cultivated on a selective medium. The optimized cultural conditions for gluconic acid yields were using submerged culture at 30oC at initial pH 6.0 for 7 days of incubation. Among the various concentrations of substrate used, glucose (14%, w/v) was found to be the most suitable carbon source for maximal gluconic acid during fermentation. Maximum values of fungal biomass (10.02 g/l) and gluconic acid (58.46 g/l) were obtained when the fungus was grown with 1% peptone as sole nitrogen source. Influence of the concentration of some inorganic salts as well as the rate of aeration on the gluconic acid and biomass production is also described.