(473f) Acoustic Resonance Agitation Improves the Production of Bacterial Extracellular Vesicles

Bacterial extracellular vesicles (BEVs) are nanostructured spherical particles released by Gram-negative and Gram-positive bacteria. Its release is a survival mechanism mediating its inter- and intraspecific communication. The physical, biochemical, and functional characteristics of BEVs are very particular, and due to their functions, there is interest in producing them in the development of new therapies as administration systems, and they have been part of the development of vaccines. However, the release of BEVs could be higher, which limits its applicability [1]. To improve BEV production, here we describe a biotechnological strategy that includes the growth of an E. coli strain under conditions of acoustic agitation that causes hypervesiculation. BEVs present heterogeneous sizes ranging from 10 to 400 nm, and their composition changes depending on the parent bacteria [1,2]. Gram-negative bacteria typically release vesicles from their outer membrane, known as outer membrane vesicles (OMVs) [1]. BEVs comprise lipopolysaccharides (LPS), lipids, peptidoglycans, outer membrane proteins (OMPs), periplasmic and cytoplasmic proteins, and metabolites. BEVs have recently gained attention due to their composition and ability to transport signaling molecules to develop next-generation therapies and drug delivery systems.

The bacteria cultures developed in conventional flasks under aerobic conditions usually reach lower cell density than well-agitated bioreactors [3,4] and usually, are affected by oxygen limitations and changes in pH, among other parameters [3]. During orbital agitation (OA), a rotational centrifugal force is generated through the fluid [3]. The mass transfer variations impact Escherichia coli's growth because oxygen participates as a nutrient during aerobic growth [4]. The alternative to overcome the oxygen transfer limitations of shake flask-based cultures is acoustic resonance agitation (ARA). The motion result of low-frequency acoustic resonance provokes one-dimensional oscillation through the liquid in an axial motion, micromixing patterns, and the formation of drops and tiny bubbles [2]. Previously, our group reported increased biomass in E. coli shake flask cultures with ARA versus OA [4]. This work aims to characterize and determine the difference in BEV production by E. coli shake flask cultures using ARA and OA.

We use the non-pathogenic E. coli BL21 strain cultured into 250 mL conventional Erlenmeyer flasks with a 50 mL working volume to compare ARA and OA in BEV production. The flasks were incubated at 37 °C under OA at 200 rpm (control condition, with specific oxygen transfer coefficient -k_La- of 78.9 ± 2.0 h⁻¹), ARA 7.5 x g (k_La of 82.0 ± 10.4 h⁻¹), and ARA 18 x g (k_La of 295 ± 1.5 h^-1). Cell growth, glucose consumption, and organic acid production were characterized to determine the cultures' kinetic and stoichiometric parameters. Glucose consumption and lactic acid production were quantified using the A15 analyzer (BioSystems, Barcelona, Spain) and the other metabolites by high-performance liquid chromatography (Shimadzu, Kyoto, Japan) [4]. The production of BEVs was evaluated in the exponential (3 h), pre-stationary (6 h), and stationary (12 h) growth phases. The isolation of the vesicles consisted of a clarification process by centrifugation for 15 min and 4°C. Then, BEVs were recovered and concentrated by filtration. An indirect quantification of BEVs was performed by measuring total protein using the Bradford method. The BEVs' morphology and size were analyzed using transmission electron microscopy (TEM). The size distribution of the BEVs was determined by dynamic light scattering and zeta potentials.

ARA increased E. coli growth by more than two-fold and total protein production in the supernatants by up to two-fold, an indirect measure of the number of BEVs. The growth of E. coli in OA and ARA is different, particularly between OA and ARA at 18 x g; it reaches a maximum biomass concentration 3.5 times higher in ARA at 18 x g. Kinetic and stoichiometric parameters stand out, such as the specific growth rate (μ) and the specific acetate productivity, with non-significant differences between OA and ARA 7.5 x g, which may be related to the fact that both conditions have similar k_La. In ARA, a greater abundance of BMVs vs OA is observed. Under both types of agitation, BEVs are spherical nanostructures ranging between 50 and 400 nm. ARA is an effective strategy to increase the productivity of BMVs, which is associated with greater biomass production and not with an effect due to cell rupture. Importantly, BEVs' size and protein composition depend on the agitation system. ARA at 18 x g causes significant changes in the size of small vesicles. ARA effectively increases the production of BEVs, which, when recovered in the pre-stationary phase under the three stirring conditions, present bimodal distributions with diameters ranging between 13.5 nm and 78.8 nm and populations with hydrodynamic diameters. Interestingly, no significant differences were found between the agitation conditions. On the other hand, for all BEVs, zeta potential values between -7 and -16 were obtained. Regarding the size distributions observed by TEM, greater size heterogeneity was found for OA, with diameters between 11.5 nm and 206.5 nm (n = 270). For 7.5 x g, the diameters of BEVs estimated from micrographs range from 11.18 nm to 185 nm (n = 286), while under 18 x g, the sizes were between 9.52 nm and 165 nm (n = 236).

Our work improves the acquisition of BEVs using acoustic resonance. Compared to OA, ARA increases the release of BEVs up to two-fold. OA and ARA at 7.5 x g produce BEVs with similar hydrodynamic sizes. It should be noted that the micrographs indicate a greater abundance of BEVs produced under ARA.

References

1. Liu H, Li M, Zhang T, Liu X, Zhang H, Geng Z, Su J. (2022) Chem. Eng. J., 450, pp. 138309
2. Z. Ou, X. He, Q. Li, N. Cao, M. Gao, B. He, M. Zhang, F. Hu, W. Yao, Q. Wang, L. Zheng, B. Chem. Eng. J., 446 (2022), Article 136847,
3. Reynoso GI, García RI, Valdez-Cruz NA, Trujillo-Roldán MA. (2016) Biochem. Eng. J., 105, pp. 379–390.
4. Valdez-Cruz NA, Reynoso GI, Pérez S, Restrepo-Pineda S, González J, Olvera A, Alagón A, Trujillo-Roldán MA (2017) Microb. Cell Fact.,16, no. 1, pp. 1–12.

Acknowledgment

Consejo Nacional de Ciencia y Tecnología – CONACyT (LMME 1102921). CONAHCyT CF-2023-I-1549. PAPIIT de la UNAM: IN210822; IV201220. IIB‑UNAM, institutional program: La producción de biomoléculas de interés biomédico en bacterias y hongos”