What drives the cost of biowaste-to-lipase production? Linking titre, rate and yield to techno-economic performance in batch-to-continuous Pichia pastoris fermentation

Fruit and vegetable (FV) waste represents an underutilized organic stream in Europe, yet its soluble sugar fraction remains poorly exploited as a bioprocess feedstock. Recombinant enzyme production from biowaste is often bottlenecked by heterogeneous substrate composition, low fermentable sugar titres, and uncertain process economics. This study evaluates the techno-economic feasibility of recombinant Candida rugosa lipase type 1 (CRL1) production in Pichia pastoris (Komagataella phaffii) under the constitutive glyceraldehyde-3-phosphate dehydrogenase promoter pGAP across three carbon sources: commercial glucose (Scenario A), mechanically pressed fruit and vegetable juice (Scenario B), and an enzymatically hydrolysed, nanofiltered fruit and vegetablesugar stream (Scenario C). Bench-scale fermentation kinetics were integrated into 24 SuperPro Designer models across four scales (1 to 5000 L nominal vessel capacity; 0.75 to 3750 L working volume) under batch and continuous chemostat ( \:D=0.05 h⁻¹ ) modes. Interpreted through the Titre, Rate, and Yield (TRY) framework, continuous operation on glucose increased volumetric CRL1 productivity 12.6-fold over batch (0.084 vs. 0.06 U L−1 h−1. Conversely, continuous operation on the hydrolysed stream did not improve productivity over batch, indicating potential kinetic inhibition from enzymatic pretreatment products or suboptimal dilution rate. At the 5000 L vessel scale under continuous mode, unit production costs were $1186, $36,049, and $3416 $$\:{kg}⁻¹$$ for Scenarios A, B, and C, respectively, all exceeding the commodity lipase benchmark ($25 \:kg⁻¹ ). Facility-dependent costs dominated Scenarios A and B (73% and 78% of OPEX), whereas enzyme reagents dominated Scenario C (75%). Sensitivity analysis confirmed that secreted protein titre and downstream recovery efficiency are the primary economic drivers, demonstrating that feedstock acquisition cost is not the binding constraint. Early-stage TEA driven by bench-scale kinetic parameterization provides essential screening prior to pilot-scale investment in biowaste-to-enzyme biorefineries.