Ultrastructural Analysis of Azotobacter vinelandii Encapsulated in Alginate Hydrogel Using Cryogenic Preparation and Electron Microscopy Techniques

abstrakt

angličtina

One of the key aspects of sustainable agriculture is the development of novel biofertilizers, which often rely on plant growth-promoting microorganisms (PGPMs), including a subgroup of rhizobacteria (PGPRs). These rhizobacteria colonize plant roots and support growth through both direct and indirect mechanisms. One example of such a bacterium is Azotobacter vinelandii, a gram-negative bacterium capable of producing intracellular poly-3-hydroxybutyrate granules (PHB) and extracellular alginate, which helps retain water around plant roots. In its vegetative state, A. vinelandii has a rodshaped morphology, but under unfavourable conditions, encystation occurs. This process is characterized by the gradual rounding of central body and the formation of a protective capsule around it. This capsule consists of two layers: the outer layer is an electron-dense exine, beneath which lies an electron-transparent intine (using traditional contrast-enhancing techniques). In this research, alginate produced by the bacteria was crosslinked with selected agents (CaCl2, malic acid, glucono-δ-lactone (GDL) and GDL in a mixture with CaCO3), resulting in the encapsulation of the bacteria. The encapsulated bacteria in alginate were then prepared for cryogenic scanning electron microscopy (cryo-SEM) and low-voltage scanning transmission electron microscopy (LV-STEM). The preparation for cryo-SEM included high-pressure freezing (HPF), freeze fracturing, and freeze etching. The samples were examined using cryo-SEM, at −120 °C, with electron beam energy of 2 kV and probe current of 6.3 A. The preparation for LV-STEM also started with HPF, followed by freeze substitution, epoxy embedding, ultrathin sectioning, and contrasting of sections. The samples were observed in a SEM equipped with a STEM detector, using an electron beam energy of 20 kV and a probe current 6.3 A. Cryo-SEM enabled rapid imaging of the samples while preserving their near-native state, without the need for sputter-coating. In contrast, LV-STEM provided high-resolution ultrastructural analysis while avoiding structural alterations associated with chemical fixation. The ultrastructure of A. vinelandii bacteria was observed in the encystation phase and in the mature cyst state. Apart from shape, the key difference between these states was the formation of an inner layer of the capsule, called intine, which forms later in the encystment process than the exine. Furthermore, vesicles surrounded by a bilayer membrane were also observed outside the bacteria central body, where they were fusing. Regarding the surrounding hydrogel, changes in its structure were observed depending on the crosslinking agent used. Samples crosslinked with CaCl2 and malic acid showed similar structures, but the hydrogel was significantly denser in the samples with GDL, and even denser in the sample with GDL in a mixture with CaCO3. These findings demonstrate the potential of cryogenic preparation and electron microscopy techniques for ultrastructural analysis of biological samples, advancing methodologies for studying encapsulated bacteria and similar complex systems. Further detailed investigations are ongoing to expand our understanding of these structural features and their implications. This research is part of a broader study focused on the development of sustainable biological fertilizers.

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

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

7. 9. 2025

Slovenian Microscopy Society

Ljubljana

978-961-94264-4-9

17th Multinational Congress on Microscopy 17 MCM Book of Abstracts

1st

272

273

2

https://17mcm.si/wp-content/uploads/2025/09/Book-of-Abstracts_17MCM_final.pdf