BACKGROUND: The profitable production of gluconic acid (GA), an organic acid extensively used in the food and beverages industries, relies on the use of cheap and available substrates with high sugar content. The high oxygen demand for the microbial conversion of glucose to GA is still a bottleneck of industrial production in conventional stirred-tank reactors. Therefore, in this study, for the first time, the effect of increased air pressure up to 4 bar on GA production and sugar consumption in batch and step-wise fed-batch cultures in sugarcane molasses (ScM) and grape must (GM) medium was studied.RESULTS: ScM and GM, by-products of the agro-food industry, proved to be adequate feedstocks to replace pure glucose in GA batch production by Aspergillus niger MUM 92.13. A threefold improvement in GA productivity was obtained in batch experiments carried out at 4 bar with ScM and GM compared to those at 1 bar. Furthermore, the addition of two pulses of ScM, under pressurized conditions, increased the GA production, reaching a final concentration of 140 g L −1 . CONCLUSION: This work proved that the use of total air pressure up to 4 bar is an effective approach to enhance GA productivity using ScM and GM as substrates in batch and step-wise fed-batch cultures. Also, this study provides a novel strategy for developing a biotechnological bioprocess for GA production by A. niger from low-cost and sugar-rich by-products performed in pressurized stirred tank reactors.