Background: Biofilm-immobilized continuous fermentation has the potential to enhance cellular environmental tolerance, maintain cell activity and improve production efficiency. Results: In this study, different biofilm-forming genes (FLO5, FLO8 and FLO10) were integrated into the genome of S. cerevisiae for overexpression, while FLO5 and FLO10 gave the best results. The biofilm formation of the engineered strains 1308-FLO5 and 1308-FLO10 was improved by 31.3% and 58.7% compared to that of the WT strain, respectively. The counts of cells adhering onto the biofilm carrier were increased. Compared to free-cell fermentation, the average ethanol production of 1308, 1308-FLO5 and 1308-FLO10 was increased by 17.4%, 20.8% and 19.1% in the biofilm-immobilized continuous fermentation, respectively. Due to good adhering ability, the fermentation broth turbidity of 1308-FLO5 and 1308-FLO10 was decreased by 22.3% and 59.1% in the biofilm-immobilized fermentation, respectively. Subsequently, for biofilm-immobilized fermentation coupled with membrane separation, the engineered strain significantly reduced the pollution of cells onto the membrane and the membrane separation flux was increased by 36.3%. Conclusions: In conclusion, enhanced biofilm-forming capability of S. cerevisiae could offer multiple benefits in ethanol fermentation. Graphical

Galactooligosaccharides (GOS) are one of the most important functional oligosaccharide prebiotics. The surface display of enzymes was considered one of the most excellent strategies to obtain these products. However, a rough industrial environment would affect the biocatalytic process. The catalytic process could be efficiently improved using biofilm-based fermentation with high resistance and activity. Therefore, the combination of the surface display of β-galactosidase and biofilm formation in Pichia pastoris was constructed. The results showed that the catalytic conversion rate of GOS was up to 50.3% with the maximum enzyme activity of 5125 U/g by screening the anchorin, and the number of the continuous catalysis batches was up to 23 times. Thus, surface display based on biofilm-immobilized fermentation integrated catalysis and growth was a co-culture system, such that a dynamic equilibrium in the consolidated integrative process was achieved. This study provides the basis for developing biofilm-based surface display methods in P. pastoris during biochemical production processes.

Biofilms enable bacterial cells to adhere and thrive on surfaces, with associated changes in growth and gene expression aiding their survival in challenging environments. While previous research has explored E. coli biofilm formation, there has been limited exploration of its application in industrial production. Prior studies have shown that immobilized fermentation can enhance L-threonine production. This study aims to augment biofilm formation and subsequently increase L-threonine production in E. coli by regulating the quorum sensing system, focusing on key AI-2-related genes, including luxS, lsrB, lsrK, and lsrR. In +pluxS and +plsrB strains, AI-2 levels were significantly altered, resulting in enhanced biofilm formation, increased curli expression, shorter free-cell fermentation periods, and improved production efficiency through immobilized continuous fermentation. In a single batch of free-cell fermentation with E. coli W1688, L-threonine production was 10.16 g/L. However, +pluxS and +plsrB strains achieved L-threonine yields of 15.27 g/L and 13.38 g/L, respectively, after seven fermentation batches. Additionally, the fermentation period was reduced from 36 h to 28 h and 30 h, respectively.