Enhancing Sustainability and Productivity in Komagataella phaffii Fermentation: A Techno-Economic Comparison of Fed-Batch and Continuous Cultivation with Mixed Induction Strategies

The increasing demand for recombinant proteins has driven innovation in bioprocessing strategies using Komagataella phaffii as a host organism. Conventional fed-batch cultivation with pure methanol induction remains widely used but presents challenges including high methanol consumption, extended downtime, and elevated operational costs. This study evaluates alternative strategies combining mixed induction (methanol/sorbitol) with continuous cultivation to enhance productivity, sustainability, and improved economic outcome. Using KEX2 protease as a model industrial recombinant protein, we compared four cultivation modes: fed-batch with methanol (benchmark), fed-batch with mixed induction, continuous with methanol, and continuous with mixed induction. Cell growth, volumetric yield, and specific productivity were evaluated at 5L scale and then modelled to simulate industrial scales (40 L and 400 L). Results demonstrate that continuous cultivation with mixed induction significantly improves yield up to 9-fold compared to conventional fed-batch and reduces methanol usage and oxygen demand. Techno-economic simulations reveal that a 40 L continuous process can match or exceed the output of two 400 L fed-batch runs, while lowering capital and operating costs and minimizing environmental footprint. This integrated strategy offers a scalable, low-cost, and safer method for recombinant protein production, supporting compact and sustainable manufacturing solutions.