Integrated fermentation, downstream processing, and formulation shape the performance of Bacillus thuringiensis-based biocontrol agents

Abstract The performance of Bacillus thuringiensis (Bt)-based biopesticides depends not only on their insecticidal proteins but also on the manufacturing process, from fermentation through downstream formulation. However, these stages are often investigated independently, limiting understanding of how process design influences the properties and performance of the final product. This study develops and evaluates a spray-dried formulation of Bacillus thuringiensis subsp. kurstaki (Btk) strain Lip by characterizing the fermentation growth–sporulation trajectory using controlled physicochemical parameters and off-gas monitoring, relating these dynamics to spore and protein production, characterizing the resulting spray-dried formulation, and evaluating its laboratory bioefficacy, and stability .Btk Lip was produced by submerged fermentation on a wheat bran medium under controlled operating conditions and converted to a spray-dried formulation (Lip-F) designed to enhance application performance and resistance to sunlight degradation, and as a unformulated dried material (Lip-U) that will serve as a control and a baseline for following experiments. Across four fermentations (72 h/batch), off-gas and pO 2 profiles were reproducible and coincided with rapid sporulation (9.0–10.0 log 10 CFU/mL) and protein accumulation (1.4–1.5 mg/mL). Quality control showed preserved Cry profiles (130 and 60–65 kDa) and comparable Cry densitometry to Delfin ® (5.0 ± 1.0 vs. 4.1 ± 0.5 mg/g), with higher total protein in Lip-F (104 ± 15 vs. 61 ± 13 mg/g). Bioassays across pests showed efficacy comparable to Delfin ® , with higher potency against Ostrinia nubilalis (LC 50 ratio 2.3; p = 0.03). Sunlight exposure improved stability, Lip-F retained 30 ± 6% CFU vs. 1.85 ± 1.18% (Lip-U), preserved Cry integrity, and maintained Spodoptera frugiperda mortality (Odds Ratio 0.217 vs. 0.0386).