Analysis of Alginate Hydrogel Structure as a Function of the Crosslinking Agent in Azotobacter vinelandii Encapsulation

abstrakt

angličtina

Alginate is a linear polysaccharide composed of β-D-mannuronic acid (M) and its C-5 epimer α-L-guluronic acid (G) [1]. Its crosslinking is most commonly achieved using divalent cations such as Ca2+. This process is known as the “egg-box” model, where the chains are linked by the cation between the G-blocks of the alginate chains. Perhaps the most frequently used crosslinking agent is CaCl2, however its application leads to the rapid formation of an inhomogeneous hydrogel due to the high solubility of this compound in water at neutral pH [2, 3]. To achieve a more homogeneous hydrogel, almost water insoluble CaCO3 can be used in combination with glucono-δ-lactone (GDL), which gradually lowers the pH, dissolves the CaCO3, and releases Ca2+ ions for crosslinking [3, 4]. Protonation (H+) can also induce crosslinking, resulting in the formation of hydrogen bonds between alginate chains [2]. This study investigates the bacterium Azotobacter vinelandii, a producer of extracellular alginate. To examine its encapsulation, selected crosslinking agents (2% (w/w) CaCl2, 0.5 M malic acid, 1 M GDL and a mixture of 1 M GDL with 0.5 M CaCO3) were added to cultured bacterial samples. The encapsulated A. vinelandii cells were analysed using cryogenic scanning electron microscopy (cryo-SEM) and low-voltage scanning transmission electron microscopy (LV-STEM). Cryo-SEM sample preparation consisted of high-pressure freezing (HPF), freeze fracture and freeze etching. LV-STEM sample preparation also included HPF, however, it was followed by freeze substitution, resin embedding, ultrathin sectioning, and contrasting with lead citrate [5]. Encystation of A. vinelandii under unfavourable conditions leads to rounding of the cell’s central body and the formation of an outer capsule. The outer capsule consists of two layers, the exine, which is composed of polysaccharides, proteins and lipids in roughly equal proportions, and the intine, which also contains these macromolecules, but polysaccharides and lipids as the predominant components, while proteins are present in smaller amounts. The main polysaccharide in the capsule is the alginate, with the M/G ratio differing between the exine and intine. G-blocks predominate in the exine, whereas M-blocks are more abundant in the intine. This is supported by our research, as the exine appeared well crosslinked and highly contrasted, while the intine was observed as an electron-transparent layer. In addition to the capsule, alginate is secreted into the surrounding environment. When CaCl2 or malic acid was added, a similar hydrogel structure was formed, with GDL, the hydrogel was denser and more precise. An even denser and precise structure was observed for the samples with addition of GDL and CaCO3. These findings demonstrate the potential of cryogenic preparation and electron microscopy techniques for the ultrastructural analysis of encapsulated bacteria in hydrogel. Further research aims to deepen our understanding of the effect of different crosslinking reagents on the alginate structure.

Alginate, Azotobacter vinelandii, cryo-SEM, LV-STEM

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

19. 5. 2025

Czechoslovak Microscopy Society

Brno

978-80-909216-1-0

Microscopy 2025. Book of abstracts.

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