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Chitin is the second most abundant biopolymer and functions as the main structural component in a variety of living organisms. In nature, chitin rarely occurs in a pure form, but rather as nanoorganized chitin-proteins, chitin-pigments, or chitin-mineral composite biomaterials. Although chitin has a long history of scientific studies, it is still extensively investigated for practical applications in medicine, biotechnology, and biomimetics. The complexity of chitin has required the development of highly sensitive analytical methods for its identification. These methods are crucial for furthering disease diagnostics as well as advancing modern chitin-related technologies. Here we provide a summary of chitin identification by spectroscopic (NEXAFS, FTIR, Raman, NMR, colorimetry), chromatographic (TLC, GC, HPLC), electrophoretic (HPCE), and diffraction methods (XRD, WAXS, SAXS, HRTEM-SAED). Biochemical and immunochemical (ELISA, immunostaining) methods are described with respect to their medical application. This review outlines the history as well as the current progress in the analytical methods for chitin identification., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Studies on the identification, properties and function of chitin in sponges (Porifera), which are recognized as the first multicellular organisms on Earth, continue to be of fundamental scientific interest. The occurrence of chitin has so far been reported in 21 marine sponge species and only in two inhabiting fresh water. In this study, we present the discovery of α-chitin in the endemic demosponge Ochridaspongia rotunda, found in Lake Ohrid, which dates from the Tertiary. The presence of chitin in this species was confirmed using special staining, a chitinase test, FTIR, Raman and NEXAFS spectroscopy, and electrospray ionization mass spectrometry (ESI-MS). In contrast to the case of marine sponges, chitin in O. rotunda has been found only within its holdfast, suggesting a role of chitin in the attachment of the sponge to the hard substratum. Isolated fibrous matter strongly resemble the shape and size of the sponge holdfast with membrane-like structure., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Chitin, as a fundamental polysaccharide in invertebrate skeletons, continues to be actively investigated, especially with respect to new sources and the development of effective methods for its extraction. Recent attention has been focused on marine crustaceans and sponges; however, the potential of spiders (order Araneae) as an alternative source of tubular chitin has been overlooked. In this work, we focused our attention on chitin from up to 12 cm-large Theraphosidae spiders, popularly known as tarantulas or bird-eating spiders. These organisms "lose" large quantities of cuticles during their molting cycle. Here, we present for the first time a highly effective method for the isolation of chitin from Caribena versicolor spider molt cuticle, as well as its identification and characterization using modern analytical methods. We suggest that the tube-like molt cuticle of this spider can serve as a naturally prefabricated and renewable source of tubular chitin with high potential for application in technology and biomedicine., Competing Interests: The authors declare no conflict of interest.

Naturally occurring three-dimensional (3D) biopolymer-based matrices that can be used in different biomedical applications are sustainable alternatives to various artificial 3D materials. For this purpose, chitin-based structures from marine sponges are very promising substitutes. Marine sponges from the order Verongiida (class Demospongiae) are typical examples of demosponges with well-developed chitinous skeletons. In particular, species belonging to the family Ianthellidae possess chitinous, flat, fan-like fibrous skeletons with a unique, microporous 3D architecture that makes them particularly interesting for applications. In this work, we focus our attention on the demosponge Ianthella flabelliformis (Linnaeus, 1759) for simultaneous extraction of both naturally occurring ("ready-to-use") chitin scaffolds, and biologically active bromotyrosines which are recognized as potential antibiotic, antitumor, and marine antifouling substances. We show that selected bromotyrosines are located within pigmental cells which, however, are localized within chitinous skeletal fibers of I. flabelliformis . A two-step reaction provides two products: treatment with methanol extracts the bromotyrosine compounds bastadin 25 and araplysillin-I N20 sulfamate, and a subsequent treatment with acetic acid and sodium hydroxide exposes the 3D chitinous scaffold. This scaffold is a mesh-like structure, which retains its capillary network, and its use as a potential drug delivery biomaterial was examined for the first time. The results demonstrate that sponge-derived chitin scaffolds, impregnated with decamethoxine, effectively inhibit growth of the human pathogen Staphylococcus aureus in an agar diffusion assay.

Sponges are a valuable source of natural compounds and biomaterials for many biotechnological applications. Marine sponges belonging to the order Verongiida are known to contain both chitin and biologically active bromotyrosines. Aplysina archeri (Aplysineidae: Verongiida) is well known to contain bromotyrosines with relevant bioactivity against human and animal diseases. The aim of this study was to develop an express method for the production of naturally prefabricated 3D chitin and bromotyrosine-containing extracts simultaneously. This new method is based on microwave irradiation (MWI) together with stepwise treatment using 1% sodium hydroxide, 20% acetic acid, and 30% hydrogen peroxide. This approach, which takes up to 1 h, made it possible to isolate chitin from the tube-like skeleton of A. archeri and to demonstrate the presence of this biopolymer in this sponge for the first time. Additionally, this procedure does not deacetylate chitin to chitosan and enables the recovery of ready-to-use 3D chitin scaffolds without destruction of the unique tube-like fibrous interconnected structure of the isolated biomaterial. Furthermore, these mechanically stressed fibers still have the capacity for saturation with water, methylene blue dye, crude oil, and blood, which is necessary for the application of such renewable 3D chitinous centimeter-sized scaffolds in diverse technological and biomedical fields.

Among marine demosponges (Porifera: Demospongiae), only representatives of the order Verongiida have been recognized to synthetize both biologically active substances as well as scaffolds-like fibrous skeletons made of structural aminopolysaccharide chitin. The unique 3D architecture of such scaffolds open perspectives for their applications in waste treatment, biomimetics and tissue engineering. Here, we focus special attention to the demosponge Pseudoceratina purpurea collected in the coastal waters of Singapore. For the first time the detailed description of the isolation of chitin from the skeleton of this sponge and its identification using diverse bioanalytical tools were carried out. Calcofluor white staining, FTIR analysis, electrospray ionization mass spectrometry (ESI-MS), SEM, and fluorescence microscopy as well as a chitinase digestion assay were applied in order to confirm with strong evidence the finding of alpha-chitin in the skeleton of P. purpurea. We suggest that the discovery of chitin within representatives of Pseudoceratinidae family is a perspective step in evaluation of these verongiid sponges as novel renewable sources for both chitin and biologically active metabolites, which are of prospective use for marine oriented biomedicine and pharmacology, respectively., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Sponges (Porifera) are recognized as aquatic multicellular organisms which developed an effective biochemical pathway over millions of years of evolution to produce both biologically active secondary metabolites and biopolymer-based skeletal structures. Among marine demosponges, only representatives of the Verongiida order are known to synthetize biologically active substances as well as skeletons made of structural polysaccharide chitin. The unique three-dimensional (3D) architecture of such chitinous skeletons opens the widow for their recent applications as adsorbents, as well as scaffolds for tissue engineering and biomimetics. This study has the ambitious goal of monitoring other orders beyond Verongiida demosponges and finding alternative sources of naturally prestructured chitinous scaffolds; especially in those demosponge species which can be cultivated at large scales using marine farming conditions. Special attention has been paid to the demosponge Mycale euplectellioides (Heteroscleromorpha: Poecilosclerida: Mycalidae) collected in the Red Sea. For the first time, we present here a detailed study of the isolation of chitin from the skeleton of this sponge, as well as its identification using diverse bioanalytical tools. Calcofluor white staining, Fourier-transform Infrared Spcetcroscopy (FTIR), electrospray ionization mass spectrometry (ESI-MS), scanning electron microscopy (SEM), and fluorescence microscopy, as well as a chitinase digestion assay were applied in order to confirm with strong evidence the finding of a-chitin in the skeleton of M. euplectellioides . We suggest that the discovery of chitin within representatives of the Mycale genus is a promising step in their evaluation of these globally distributed sponges as new renewable sources for both biologically active metabolites and chitin, which are of prospective use for pharmacology and biomaterials oriented biomedicine, respectively., Competing Interests: The authors declare no conflict of interest.

Since the discovery of chitin in skeletal structures of sponges (Porifera) in 2007, studies on search of novel species which possess this structural aminopolysaccharide continue up today. The most potential source of chitin is suggested to be localized in the four families of sponges related to the order Verongida (Demospongiae) which nevertheless require further clarification. Here, we report for the first time the isolation and identification of α-chitin from the Suberea clavata demosponge (Aplysinidae: Verongida). Raman spectroscopy, Calcofluor White staining, chitinase test and ESI-MS techniques were used to identify chitin. We suggest that the presence of chitin within fibrous skeletons of diverse species of Verongida order, and, especially in all species of the Aplysinidae family, may be useful for the identification of novel, previously unidentified marine demosponges., (Copyright © 2017 Elsevier B.V. All rights reserved.)

The recent discovery of chitin within skeletons of numerous marine and freshwater sponges (Porifera) stimulates further experiments to identify this structural aminopolysaccharide in new species of these aquatical animals. Aplysina fistularis (Verongida: Demospongiae: Porifera) is well known to produce biologically active bromotyrosines. Here, we present a detailed study of the structural and physico-chemical properties of the three-dimensional skeletal scaffolds of this sponge. Calcofluor white staining, Raman and IR spectroscopy, ESI-MS as well as chitinase digestion test were applied in order to unequivocally prove the first discovery of α-chitin in skeleton of A. fistularis., (Copyright © 2013 Elsevier B.V. All rights reserved.)

This work demonstrates that chitin is an important structural component within the skeletal fibers of the freshwater sponge Spongilla lacustris. Using a variety of analytical techniques ((13)C solid state NMR, FT-IR, Raman, NEXAFS, ESI-MS, Morgan-Elson assay and Calcofluor White Staining); we show that this sponge chitin is much closer to α-chitin, known to be present in other animals, than to β-chitin. Genetic analysis confirmed the presence of chitin synthases, which are described for the first time in a sponge. The presence of chitin in both marine (demosponges and hexactinellids) and freshwater sponges indicates that this important structural biopolymer was already present in their common ancestor., (Copyright © 2013 Elsevier Inc. All rights reserved.)

A holdfast is a root- or basal plate-like structure of principal importance that anchors aquatic sessile organisms, including sponges, to hard substrates. There is to date little information about the nature and origin of sponges' holdfasts in both marine and freshwater environments. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis. Using a variety of techniques (near-edge X-ray absorption fine structure, Raman, electrospray ionization mas spectrometry, Morgan-Elson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much closer to α-chitin than to β-chitin. Most of the three-dimensional fibrous skeleton of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly, the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This discovery of chitin within freshwater sponge holdfasts highlights the novel and specific functions of this biopolymer within these ancient sessile invertebrates.

Evolution and multi-functionality of the chitin system in sponges (Porifera) is currently a prime focus in modern experimental zoology including in phylogenetic studies of diverse poriferan taxa. Here, we present the first evidence of chitin in the skeleton of the demosponge originally defined as Dendrilla lacunosa (Porifera: Dendroceratida: Keratosa). The presence of chitin has been clearly confirmed using special Calcofluor white staining, Fourier-transform Infrared (FTIR) and Raman Spectroscopy, electrospray ionization mass spectrometry (ESI-MS), as well as a chitinase digestion assay. This feature is, however, unusual for Keratosa sponges with skeletons usually made of structural protein – spongin, and at the same time is typical for sponges of the Verongiida (subclass Verongimorpha). Consequently, obtained bioanalytical results, together with our findings about distinctive morphological and molecular characters of this Dendrilla -like sponge confirmed the necessity of renaming this organism as a novel species Ernstilla lacunosa in the new family Ernstillidae. This study is the first example of the impact of the reporting of chitin on the systematics of sponges. • We proved that, the skeleton of a representative of the new genus Ernstillidae: Ernstilla lacunosa possesses chitin, and the presence of chitin has been confirmed using various analytical methods. [ABSTRACT FROM AUTHOR]

The morphology, chemical, and mineralogical composition of subsurface filamentous fabrics (SFF) from the Deccan Volcanic Province (DVP) were investigated to determine the origin of these spectacular aggregates. SFF occur in a wide variety of morphologies ranging from pseudo-stalactites to irregular fabrics and are classified as SFFIr (irregular) or SFFMa (matted). The SFF samples exhibit a thread-like (or filament-like) center from which mineral precipitation starts to form the final macroscopic morphologies. Detailed investigations revealed organic material (fungal chitin) in the innermost filamentous core, which may have acted as an initial nucleus for the mineralization processes. The morphometric characteristics of certain filamentous fabrics are very similar to those of microbial filaments and the fabrics formed from them but are clearly distinct from similar types of non-biological precipitates (fibrous minerals, speleothems, and "chemical gardens"). These features indicate that the filamentous cores might be products of microbial communities that were active in the basaltic cavities. The SFF cross-sections display similar concentric layers of the mineral succession and reach thicknesses of several centimeters with spectacular lengths up to 100 cm and constant diameters. The typical mineralization sequence points to temporal variation in the chemical composition of the mineralizing fluids from Fe(Mg)-rich (Fe-oxides/-hydroxides, Fe-rich sheet silicates such as celadonite and di-/tri-smectite) to Ca-dominated (Ca-rich zeolites) and finally pure SiO2 (opal-CT, chalcedony, and macro-crystalline quartz). Assuming biological activity at least during the early mineralization processes, circumneutral pH conditions and maximum temperatures of 100–120 °C were supposed. The formation of filamentous cores including Fe-bearing phyllosilicates probably occurred near the surface after cooling of the lava, where the elements necessary for mineral formation (i.e., Si, Mg, Al, Fe) were released during alteration of the volcanic host rocks by percolating fluids. [ABSTRACT FROM AUTHOR]

A holdfast is a root- or basal plate-like structure of principal importance that anchors aquatic sessile organisms, including sponges, to hard substrates. There is to date little information about the nature and origin of sponges' holdfasts in both marine and freshwater environments. This work, to our knowledge, demonstrates for the first time that chitin is an important structural component within holdfasts of the endemic freshwater demosponge Lubomirskia baicalensis. Using a variety of techniques (near-edge X-ray absorption fine structure, Raman, electrospray ionization mas spectrometry, Morgan–Elson assay and Calcofluor White staining), we show that chitin from the sponge holdfast is much closer to α-chitin than to β-chitin. Most of the three-dimensional fibrous skeleton of this sponge consists of spicule-containing proteinaceous spongin. Intriguingly, the chitinous holdfast is not spongin-based, and is ontogenetically the oldest part of the sponge body. Sequencing revealed the presence of four previously undescribed genes encoding chitin synthases in the L. baicalensis sponge. This discovery of chitin within freshwater sponge holdfasts highlights the novel and specific functions of this biopolymer within these ancient sessile invertebrates. [ABSTRACT FROM AUTHOR]