© 2022 ASABE. All Rights Reserved.Fungal cellulases and xylanases are commonly used for biological pretreatment of lignocellulosic biomass into simple sugars, which can then be converted into many value-added products including bioethanol. However, these enzymes are costly to produce and need a feedstock that should be more economical and high in fiber and other nutrients. Distillers dried grains with solubles (DDGS) is the byproduct of corn ethanol production and can be considered an appropriate feedstock. Among various problems in adaptation of a submerged fungal fermentation process to produce these enzymes, the lower activities are one of the most prominent challenges. To overcome this issue, different levels of aeration, agitation, and nutrient concentrations should be studied to evaluate their effects on enzyme production. Therefore, in this study, the dilute acid hydrolyzed DDGS media with solid DDGS particles was used with different dilution rates to evaluate the effect of sugar concentration and enzyme productions in 2L bench-top bioreactors by Aspergillus niger (NRRL 330).The effect of aeration and agitation was evaluated for each dilution factor for both cellulase and xylanase productions. It was observed that at lower agitation and aeration rates, the enzyme production increased gradually from day 1 to day 9. On the other hand, a sharp increase in cellulase and xylanase enzymes was observed with higher agitation and aeration rates. The highest cellulase activity (0.76 IU/ml) was obtained on the day 7 of the fermentation with dilution factor of 2, aeration of 1 vvm and agitation of 500 rpm. Highest xylanase activity was 29.04 IU/ml for the same reactor conditions but on day 8 of fermentation. In conclusion, this research shows that dilution, aeration and agitation have positive effect on enzyme production in the early days of fermentation.