Multiscale oxygen transfer enhancement in high‑density Komagataella phaffii fermentation: dynamic pressure control and CFD‑guided industrial scale‑up

Oxygen transfer limitation is a key bottleneck in high-cell-density Komagataella phaffii fermentation for recombinant protein production. Conventional approaches based on increasing agitation and aeration may result in high shear stress, foaming, and increased energy demand. In this study, a multiscale oxygen-transfer enhancement strategy integrating dynamic headspace-pressure regulation with computational fluid dynamics (CFD)-guided bioreactor optimization was investigated. At the 5 L scale, response surface methodology demonstrated the combined effects of agitation, aeration, and headspace pressure on the volumetric oxygen-transfer coefficient ( $$ k_{L} a $$ ). A two-stage dynamic pressure (TSP) strategy was subsequently evaluated under fermentation conditions. Compared with the conventional-pressure condition, TSP increased the final dry cell weight (DCW) by 11.2% and the maximum xylanase activity by 19.8%, reaching 120.25 ± 1.04 g/L and 213.23 ± 2.10 KU/mL, respectively. The strategy was further implemented in a 1000 L bioreactor, where the peak oxygen uptake rate reached 185.23 mmol/L/h, with a final DCW of 124.08 ± 2.47 g/L and xylanase activity of 225.07 ± 3.64 KU/mL. At the 30 m3 scale, CFD analysis identified scale-dependent hydrodynamic limitations in the original agitation system and guided a combined redesign from 0.7 m Rushton turbines to a 0.8 m CD-6/3KSX hybrid impeller system. Under identical simulation conditions, the redesigned configuration increased the CFD-predicted average $$ k_{L} a $$ by 46.4% and reduced the predicted mixing time by 43.3%. Following implementation of the redesigned system, the final DCW and xylanase activity reached 134.01 ± 2.98 g/L and 242.02 ± 4.07 KU/mL, respectively. These results demonstrate a case-based multiscale approach combining process-level oxygen-transfer regulation with reactor-level hydrodynamic optimization for the investigated recombinant K. phaffii fermentation process.