Small-scale optimization of Bacillus subtilis sporulation: genomic knockouts and media buffering for increased yields

Abstract Background Bacillus subtilis spore surface display technology has been applied for more than a decade, offering advantages such as increased stability, simple purification, and low cost compared to other enzyme immobilization methods. However, systematic attempts to optimize this technology beyond individual parameters remain scarce and its full potential has yet to be realized. Results The sequential deletion of skfA , spo0E , epsA-O , srfAA-AD , and comK improved sporulation efficiency and kinetics in Bacillus subtilis . The most pronounced gains were observed upon deletion of skfA , encoding an extracellular killing factor that delays sporulation commitment by inducing lysis of non-sporulating cells, and spo0E , encoding a phosphatase that negatively regulates sporulation initiation. Supplementation of 2xSG medium with MOPS or HEPES buffer further enhanced biomass accumulation across all genetic backgrounds, while phosphate and Tris buffers proved inhibitory despite comparable or stronger pH stability, at a lower reagent cost. Together, the knockout strategy and MOPS buffering produced a small 1.2-fold increase in maximum biomass and improved pH stabilization compared to the unbuffered parental strain, suggesting that genetic and environmental optimization can be complementary. Surface-displayed sucrose phosphorylase activity was further improved 1.32-fold upon preliminary scale-up in a simple airlift reactor, without a corresponding difference in growth. Conclusions This study demonstrates that meaningful improvements in spore surface display efficiency are achieved through targeted interventions in both strain design and culture conditions. The results suggest that the two approaches are complementary, with the full benefit of the genetic modifications only realized under buffered conditions. The optimized strain and culture system presented here provides a practical foundation for further development of spore-based enzyme display platforms.