The effects of addition of menadione (MD) to induce elevated intracellular superoxide levels (superoxide stress) and of elevated oxygen availability (oxygen stress) was investigated in batch and chemostat cultures of a recombinant Aspergillus niger (B1-D). Culture responses to the two stressors were clearly distinct in terms of the changes in recombinant protein production, overall protein synthesis, and morphology. MD addition resulted in a significant increase in intracellular O-2(circle-) levels, but gassing with O-2-enriched air (similar to25%) led to decreased intracellular O-2(circle-) levels. Under elevated O-2 availability, the activity of the alternative respiratory pathway was enhanced, reducing endogenous O-2(circle-) generation, but the consequent decreased energy availability in such cultures is proposed to be the main reason leading to the decreased intracellular protein content and foreign protein secretion in batch cultures and chemostat cultures at low dilution rates (D<0.1 h(-1)). The negative effects of O-2 enrichment on protein synthesis were less pronounced at high D due to the relative dominance of the main respiratory pathway at high growth rates. Under stressed cultivation conditions, the mean number of tips per clump correlated well with the secretion of recombinant protein. Morphological change with 02 enrichment was not directly caused by the increased O2(&LCIRC;)(-) generation as proposed in some previous studies, but possibly by the significantly altered culture metabolism. (C) 2003 Elsevier Inc. All rights reserved.