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Figure 2. Examples of factors that can influence various characteristics of BEVs (This figure was created using Figdraw). This figure illustrates seven potential factors that affect the consistency of BEV production. Effect of purification method: The figure demonstrates the disruption of BEV structure caused by filtration through a membrane, emphasizing how the choice of purification method influences the integrity of the vesicles. Culture time: It illustrates that extracellular proteases secreted during the stationary phase break down certain proteins t sensitive to these enzymes. Variations in the length of the stationary phase lead to inconsistencies in the degradation levels of these proteins, leading to differences in the protein profiles of isolated BEVs. Temperature influence: The figure illustrates the BEVs obtained from bacteria cultured at 15, 28, and 37 °C. SDS-PAGE analysis reveals differences in the protein composition and content of BEVs under these conditions, highlighting that temperature affects BEV consistency. Effect of pH: It shows that acidic pH and/or high counterion concentrations induce aggregation or fusion of BEVs, reflecting the effect of pH on BEVs. Role of culture medium: The figure depicts that BEVs produced in MC.6M medium contain more specific proteins such as TdfH, OpcA, OMP NMB0088, etc., compared to BEVs from bacteria cultured in Frantz medium. This underscores the influence of the culture medium on BEV consistency. Iron regulation: This figure illustrates how iron activates the PmrAB system. Iron first activates PmrB, which then phosphorylates PmrA. This activation regulates the addition of ethanolamine phosphate to lipid A and the LPS core by EptA and CptA, demonstrating iron’s effect on BEV lipid composition. Oxidative stress and vesicle production: The figure shows that hydrogen peroxide-induced BEV generation requires the presence of ΔwbpM S, but not Δrmd. This reflects that oxidative stress can be strategically used to produce vesicles. BEVs: Bacterial extracellular vesicles; TdfH, OpcA, OMP NMB0088: proteins expressed in BEVs; ΔwbpM: A B-band LPS deletion mutant; Δrmd: an A-band LPS deletion mutant; LPS: lipopolysaccharides.