Electron Microscopy of Cryo-Fixed Azotobacter vinelandii Encapsulated in Alginate Hydrogel: Effects of Crosslinking Agents

Electron Microscopy of Cryo-Fixed Azotobacter vinelandii Encapsulated in Alginate Hydrogel: Effects of Crosslinking Agents

Abstrakt

Azotobacter vinelandii is a gram-negative plant growth-promoting rhizobacterium (PGPR) that can contribute to sustainable agriculture through nitrogen fixation, phytohormone production, and biopolymer synthesis. It produces intracellular poly-3-hydroxybutyrate (PHB) and extracellular alginate, which promotes soil moisture retention. Under stress, it undergoes encystation, forming a two-layered capsule: an outer electron-dense exine and an inner electron-transparent intine. In this study, alginate-producing A. vinelandii cells were encapsulated using three crosslinking agents: CaCl₂, malic acid, and glucono-δ-lactone (GDL) with CaCO₃. Samples were analysed by cryogenic scanning electron microscopy (cryo-SEM) and lowvoltage scanning transmission electron microscopy (LV-STEM). Both techniques sterted with high-pressure freezing. Cryo-SEM included freeze-fracturing and freeze-etching, and imaging at −120 °C. LV-STEM included freeze substitution, epoxy embedding, ultrathin sectioning, and post-contrasting, using the same SEM with a STEM detector. Cryo-SEM and LV-STEM revealed that hydrogel ultrastructure varied significantly depending on the crosslinking agent. CaCl₂ and malic acid produced a similar structure with relatively large pores, while GDL with CaCO₃ formed significantly denser structures. LV-STEM showed slightly denser crosslinking with malic acid than with CaCl₂, probably due to a heterogeneous distribution of alginate, as the polysaccharide was produced by A. vinelandii itself. Most of the bacterial cells were encysted or encysting, and thus showed rounding and capsule formation. The exine formed first, and then the intine, with vesicles present in both layers and in close proximity to the cell. Cryo-SEM enabled relatively rapid imaging without conductive coating, while LV-STEM preserved the ultrastructure without artefacts from chemical fixation or significant staining. These methods revealed differences in hydrogel architecture and bacterial morphology, highlighting their utility for studying complex biological samples.

Azotobacter vinelandii is a gram-negative plant growth-promoting rhizobacterium (PGPR) that can contribute to sustainable agriculture through nitrogen fixation, phytohormone production, and biopolymer synthesis. It produces intracellular poly-3-hydroxybutyrate (PHB) and extracellular alginate, which promotes soil moisture retention. Under stress, it undergoes encystation, forming a two-layered capsule: an outer electron-dense exine and an inner electron-transparent intine. In this study, alginate-producing A. vinelandii cells were encapsulated using three crosslinking agents: CaCl₂, malic acid, and glucono-δ-lactone (GDL) with CaCO₃. Samples were analysed by cryogenic scanning electron microscopy (cryo-SEM) and lowvoltage scanning transmission electron microscopy (LV-STEM). Both techniques sterted with high-pressure freezing. Cryo-SEM included freeze-fracturing and freeze-etching, and imaging at −120 °C. LV-STEM included freeze substitution, epoxy embedding, ultrathin sectioning, and post-contrasting, using the same SEM with a STEM detector. Cryo-SEM and LV-STEM revealed that hydrogel ultrastructure varied significantly depending on the crosslinking agent. CaCl₂ and malic acid produced a similar structure with relatively large pores, while GDL with CaCO₃ formed significantly denser structures. LV-STEM showed slightly denser crosslinking with malic acid than with CaCl₂, probably due to a heterogeneous distribution of alginate, as the polysaccharide was produced by A. vinelandii itself. Most of the bacterial cells were encysted or encysting, and thus showed rounding and capsule formation. The exine formed first, and then the intine, with vesicles present in both layers and in close proximity to the cell. Cryo-SEM enabled relatively rapid imaging without conductive coating, while LV-STEM preserved the ultrastructure without artefacts from chemical fixation or significant staining. These methods revealed differences in hydrogel architecture and bacterial morphology, highlighting their utility for studying complex biological samples.

cryo-SEM, low-voltage STEM, Azotobacter vinelandii, alginate

cryo-SEM, low-voltage STEM, Azotobacter vinelandii, alginate

HAVLÍČKOVÁ, A.; MRÁZOVÁ, K.; HRUBANOVÁ, K.; SÚKENÍK, M.; SEDLÁČEK, P.; KRZYŽÁNEK, V.

17.09.2025

Czech-BioImaging

Rozdrojovice

CZECH-BIOIMAGING CONFERENCE Imaging Principles of Life 2025 ABSTRACT BOOK

1st

66

67

2

https://www.czech-bioimaging.cz/files/2025/09/IPL-2025-abstract-book.pdf