The instability of the protein expression in Pichia pastoris strains has been an issue for various peptide productions. Some modifications to the traditional fermentation process could potentially solve the problem. Here, we consider a four-stage fermentation process to express the CAP2 (cell-penetrating antimicrobial peptide 2) candidate in P. pastoris KM71H, a slow methanol utilization strain. During the fermentation process, CAP2 productivity is limited (6.15 ± 0.21 mg/L·h) by the low overall methanol consumption (approximately 645 g), which is mainly the result of the slow methanol utilization of the P. pastoris KM71H. To overcome this limitation, we increased the cell concentration two-fold prior to the induction stage. A fed-batch process with exponential and dissolved oxygen tension (DOT) stat feeding strategies was deployed to control the glycerol feed, resulting in an increase in cell concentration and enhancement of the volumetric methanol consumption rate. The improved fermentation process increased the overall methanol consumption (approximately 1070 g) and the CAP2 productivity (13.59 ± 0.24 mg/L·h) by 1.66 and 2.21 times, respectively. In addition, the CAP3 (cell-penetrating antimicrobial peptide 3) candidate could also be produced using this improved fermentation process at a high yield of 3.96 ± 0.02 g/L without any further optimization. Note that there was no oxygen limitation during the improved fermentation process operating at high cell density. This could be due to the controlled substrate addition via the DOT stat system.

Seaweed bioactive extraction generates de-extracted residual solids that remain carbohydrate-rich but are often underutilized. This study developed an integrated valorization route for Gracilaria fisheri spent biomass to produce fermentable sugars for β-carotene production by Rhodotorula paludigena CM33. Reducing sugar production was optimized using response surface methodology (Box–Behnken design) by varying reaction time, sulfuric acid concentration, and biomass loading at 90 °C. The predicted optimum (47.39 min, 2.50% (w/v) H₂SO₄, and 7.13% (w/v) biomass) yielded 22.41 g/L reducing sugars and was validated experimentally at 22.22 ± 0.19 g/L, indicating that the model reliably predicted reducing sugar production. The optimized condition was scaled up in a 22 L bioreactor with sequential acid hydrolysis followed by enzyme-assisted hydrolysis, increasing reducing sugars from ~30 to ~40 g/L. FTIR and SEM analyses indicated progressive modification of the carbohydrate matrix across processing stages. Batch cultivation of R. paludigena on the hydrolysate showed that ammonium sulfate supplementation significantly increased biomass, whereas β-carotene titers were not significantly different. Repeated-batch operation on non-supplemented hydrolysate sustained production over four cycles with β-carotene titers of 13.75–17.27 mg/L, demonstrating the operational feasibility of the hydrolysate-based system. Overall, this work demonstrates a practical seaweed biorefinery approach to upgrade G. fisheri spent biomass into sugars and carotenoid-rich yeast biomass.