In industrial production of enzymes using the filamentous fungus Aspergillus niger supply of sufficient oxygen is often a limitation, resulting in the formation of by-products such as polyols. In order to identify the mechanisms behind formation of the different by-products we studied the effect of low oxygen availability, at different carbon source concentrations and at different specific growth rates, on the metabolism of A. niger, using continuous cultures. The results show that there is an increase in the production of tricarboxylic acid (TCA) cycle intermediates at low oxygen concentrations. Indeed, at these conditions, a decrease in the mitochondrial respiratory chain activity leads to an accumulation of NADH and to a decreased ATP production which uncouples catabolism and anabolism, influences the intracellular pH and leads to production and excretion of organic acids. Moreover, mannitol is being produced in order to ensure reoxidation of NADH, and this is the main cellular response to balance the ratio NADH/NAD at low oxygen availability. Mannitol production is also coupled to low specific growth rate, which suggests a control of carbon catabolite repression on the mannitol pathway. The roles of two other polyols, erythritol and glycerol, were also investigated. Both compounds are known to accumulate intracellularly, at high osmotic pressure, in order to restore the osmotic balance, but we show that the efficiency of this system is affected by a leakage of polyols through the membrane.