Industrial-scale microbial fermentation processes often face limitations in mixing and mass transfer, leading to the formation of environmental gradients within the bioreactor. These gradients expose microbes to heterogeneous conditions over time and space. In this study, we evaluated the effects of combined substrate and dissolved oxygen (DO) gradients on the metabolic response of Penicillium chrysogenum at an industrial scale. Three representative heterogeneous environments were simulated in scale-down systems: (1) feed inlet (high glucose, low oxygen (HGLO): CS > 20 mM, DO < 0.012 mM), (2) aeration inlet (high oxygen, low glucose (HOLG): CS < 0.8 mM, DO > 0.2 mM), and (3) global environment (periodic 360 s fluctuation cycle with 45 s of HGLO and 75 s of HOLG conditions). Results showed that prolonged exposure to feed inlet conditions led to a complete loss of penicillin production capacity, accompanied by significant excretion of intracellular metabolites, and this effect was largely irreversible. While, cells randomly walking under the top impeller zone did not lose production capacity but showed signs of premature degeneration due to increased energy demand. When exposed to the global environment, cells finely tuned their metabolism in a periodical manner, with nearly a 50% loss of penicillin productivity. In summary, substrate gradients alone did not cause irreversible effects, but large substrate gradients contributed to reduced productivity. Oxygen gradients, however, not only reduced production but also caused irreversible cellular damage. These findings provide valuable insights for developing scale-up criteria and strain engineering strategies aimed at improving large-scale culture performance.@en