The Loss of Structural Integrity of 3D Chitin Scaffolds from Aplysina aerophoba Marine Demosponge after Treatment with LiOH

Abstract

Aminopolysaccharide chitin is one of the main structural biopolymers in sponges that is responsible for the mechanical stability of their unique 3D-structured microfibrous and porous skeletons. Chitin in representatives of exclusively marine Verongiida demosponges exists in the form of biocomposite-based scaffolds chemically bounded with biominerals, lipids, proteins, and bromotyrosines. Treatment with alkalis remains one of the classical approaches to isolate pure chitin from the sponge skeleton. For the first time, we carried out extraction of multilayered, tube-like chitin from skeletons of cultivated Aplysina aerophoba demosponge using 1% LiOH solution at 65 °C following sonication. Surprisingly, this approach leads not only to the isolation of chitinous scaffolds but also to their dissolution and the formation of amorphous-like matter. Simultaneously, isofistularin-containing extracts have been obtained. Due to the absence of any changes between the chitin standard derived from arthropods and the sponge-derived chitin treated with LiOH under the same experimental conditions, we suggest that bromotyrosines in A. aerophoba sponge represent the target for lithium ion activity with respect to the formation of LiBr. This compound, however, is a well-recognized solubilizing reagent of diverse biopolymers including cellulose and chitosan. We propose a possible dissolution mechanism of this very special kind of sponge chitin.

Keywords: Aplysina aerophoba; bromotyrosines; chitin; dissolution; marine sponges.

Figure 11

Schematic view of the dissolution mechanism of chitin in A. aerophoba. The molecules of selected bromotyrosines have been adapted from [89].

Figure 1

Image of the dried fragment of the A. aerophoba sponge with finger-like bioarchitecture. The chitin-based skeletal microfibers became visible (arrows) during the drying of the sponge body.

Figure 2

The sample of A. aerophoba sponge before the experiment (a) (see also Figure 1) and after insertion into 1% LiOH solution when disintegrated chitin scaffolds have been obtained (b). SEM image (c) shows non-regular microfibrous matter without typical microarchitecture observed with chitin scaffolds.

Figure 3

Dissolved and next dialyzed and lyophilized chitin of A. aerophoba sponge (a) after LiOH treatment. Digital microscopy images represent the partially dissolved chitin scaffold (b), where a few residual, pigmented chitinous microfibers (arrows) are still visible, and the finally-formed film (c,d).

Figure 4

Digital microscopy imagery of isolated chitin scaffolds of A. aerophoba demosponge origin after 10% NaOH treatment (a,c,e) vs. chitin fibers partially dissolved in 1% LiOH (b,d,f).

Figure 5

FTIR spectra of chitin and chitosan standards and in LiOH-dissolved sponge chitin in the range (a) 400—4000 cm⁻¹ and (b) 400—1800 cm⁻¹.

Figure 6

Normalized XRD spectra of chitin and chitosan standards in comparison to dissolved A. aerophoba sponge chitin.

Figure 7

SEM images of the A. aerophoba chitin scaffold after NaOH treatment (a,c) and after dissolution in LiOH (b,d). The destruction of the structural integrity after insertion into LiOH solution on the microlevel is clearly visible.

Figure 7

SEM images of the A. aerophoba chitin scaffold after NaOH treatment (a,c) and after dissolution in LiOH (b,d). The destruction of the structural integrity after insertion into LiOH solution on the microlevel is clearly visible.

Figure 8

Comparison of different chitin samples before and after the LiOH-procedure. Only in the case of A.aerophoba sponge chitin was the milky suspension (arrow) obtained. The structural peculiarities of this phase are represented in the Figure 7d.

Figure 9

CFW-stained samples of (a,b) partially dissolved chitin fibers; (c,d) dried film of the obtained lyophylisate. Images (a,c) were obtained via the DAPI channel, images (b,d)—using bright field conditions. Light exposure time: (a) 1/6800 s; (c) 1/1100 s.

Figure 10

Bromotyrosine-containing extract (a) obtained during the chitin dissolution procedure based on LiOH treatment. (b) Digital microscopy image of the dialyzed and dried extract, (c) image in higher magnification. (d) Comparative FTIR spectra of the obtained dialyzed and dried extract and the Isofistularin-3 standard in the range 400–4000 cm⁻¹ and (e) in the range 400–1800 cm⁻¹.

Figure 12

Schematic presentation of the A. aerophoba sponge chitin dissolution procedure in LiOH.