Imaging of Alginate-encapsulated Azotobacter vinelandii Cells: Insights into Hydrogel–Cell Interactions Using LV-STEM

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Alginate hydrogels are a valuable biomaterial with a wide range of applications in biotechnology, medicine, as well as in agriculture. Their water-binding capacity and biocompatibility make them a suitable carrier for living microorganisms and biologically active substances. At the same time, their structural properties, such as high water content and a fine, porous network, make these materials difficult to image with electron microscopy. One of the microorganisms closely associated with alginate is Azotobacter vinelandii, a soil bacterium with significant plant growth-promoting activities (PGPR). This organism is known for its ability to produce the extracellular polysaccharide alginate, which forms a protective layer around the bacterial cells and helps them to survive in challenging conditions, such as during soil drying. In this study, we focused on how A. vinelandii can be encapsulated in an alginate hydrogel and subsequently studied the fine structural relationships between the bacterial cells and the polymer matrix. For imaging, we employed low-voltage scanning transmission electron microscopy (LV-STEM), which enables the observation of ultrathin sections of biological samples with high contrast, even without the use of conventional heavy metal stains. A key step in the methodology was to chemically fix the samples so that the ultrastructure of the bacterial cells and the structure of the hydrogel network were preserved simultaneously. For this purpose, we optimized the fixation, post-fixation and dehydration protocol, including the addition of calcium ions (Ca²⁺) to help stabilize the alginate network without osmotic damage to the cells. Using LV-STEM, we observed not only intracellular structures, for example, PHA granules and vesicular bodies, but also complex extracellular structures. The cell surroundings consist of a bilayered polysaccharide shell (intine and exine), which is further connected to a polymeric hydrogel network. The samples showed differences in the polymer matrix structure depending on the presence of calcium ions during preparation, with a more ordered and denser network of fibers around the cells observed in the stabilized samples. The obtained results confirm that the combination of a suitably chosen fixation protocol and LV-STEM allows the study of the detailed structures of hydrogel-encapsulated microorganisms. These findings may be of significant value in the design of efficient biotechnological applications such as carriers for plant inoculation or targeted release of metabolites in agricultural environments. They also open the way for a deeper understanding of how microorganisms interact with their immediate surroundings in different types of polymeric environments.

Alginate; Azotobacter vinelandii; Electron microscopy; Encapsulation

MRÁZOVÁ, K.; HAVLÍČKOVÁ, A.; ČERNAYOVÁ, D.; SEDLÁČEK, P.; KRZYŽÁNEK, V.

17. 9. 2025

Czech-Bioimaging

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