Financial support: Pakistan Science Foundation under the project No supported the work P-GC/BIO/RES-283.

Keywords: agitator bioreactor, Aspergillus niger, citric acid, MnSO₄, recycling fed-batch cultivation system, TCA cycle.

The present investigation deals with a novel technique for high and consistent yield of citric acid in a fed-batch bioreactor of 9-L working volume. Five different potent cultures of Aspergillus niger were evaluated for citrate production using clarified cane-molasses as basal substrate. Among the cultures, NG^GCB101 (developed by UV/chemical mutation in our labs) gave maximum production of citric acid i.e., 87.98 g/l after 6 days of mycelial inoculation. The values of kinetic parameters such as substrate consumption and product formation rates revealed the hyperproducibility of citric acid by selected Aspergillus niger NG^GCB101 (LSD = 0.456, HS). The addition of MnSO₄ to the culture medium promoted the formation of small rounded fluffy pellets (1.55 mm², dia.), which are desirable for maximal citric acid productivity. During recycling fed-batch cultivation, high and consistent yield of citric acid monohydrate (128.68 g/l) was obtained when the sampling vs. substrate feeding was maintained at 4-L (44.50% working volume). The incubation period was reduced from 6 to only 2 days, making the process highly economical for citric acid production. MnSO₄ at a level of 2.0 x 10^-6 M, added during inoculation time optimised the commercial exploitation of molasses in recycling fed-batch cultivation system. The technique developed in the current studies is a new addition in fermentation technology due to high yields of product, recycling of the fermentation medium and fungal mycelia, low manpower and energy consumption and the use of low cost raw substrate without any additional supplementation.