POZNAN UNIVERSITY OF TECHNOLOGY FACULTY OF CHEMICAL TECHNOLOGY INSTITUTE OF TECHNOLOGY AND CHEMICAL ENGINEERING

Ph.D. THESIS

DEVELOPMENT OF NOVEL INORGANIC-ORGANIC CHITIN-BASED MATERIALS OBTAINED UNDER EXTREME BIOMIMETIC CONDITIONS

presented by

MARCIN WYSOKOWSKI, M.Sc., Eng.

For obtaining the degree of Doctor of Philosophy

Specialty: Chemical Technology

Supervisor: Professor TEOFIL JESIONOWSKI (Poland)

Co-supervisor: Professor HERMANN EHRLICH (Germany)

Poznan, 2015

Projekt współfinansowany przez Unię Europejską w ramach Europejskiego Funduszu Społecznego

Załącznik nr 11 do Regulaminu Stypendialnego

OŚWIADCZENIE DOTYCZĄCE PROMOCJI PROJEKTU pt. „Wsparcie stypendialne dla doktorantów na kierunkach uznanych za strategiczne z punktu widzenia rozwoju Wielkopolski”, Poddziałanie 8.2.2 PO KL realizowanego w latach 2013-2014

I declare that I am a scholarship holder within the project “Scholarship support for

Ph.D. students specializing in majors strategic for Wielkopolska’s development”, Sub- measure 8.2.2 Human Capital Operational Programme, co-financed by European Union under the European Social Fund.

Thesis was partially prepared within the framework of

ETIUDA 2 research project,

supported by Polish National Science Center

no DEC-2014/12/T/ST8/00080

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Thesis was prepared partially within the framework of

II edition of Mobilność Plus research project,

supported by Ministry of Science and Higher Education

no 920/MOB/2012/0

Thesis was prepared partially within the framework of

Research Grant for Doctorac Candidates and Young Academics and Scientists,

supported by DAAD – German Academic Exchange Service

no 323-91528917-50015537

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Acknowledgements

I am heartily thankful to my supervisors,

Professor Hermann Ehrlich and Professor Teofil Jesionowski,

who encouraged, guided and supported me from the initial to the final stage of this thesis. They enabled me to develop an understanding of the subject and were

always open for my activity related to finding the internship programs to study

abroad.

I am also grateful to Dr Vasilii V. Bazhenov (TU Bergakademie Freiberg),

Dr Mykhailo Motylenko (TU Bergakademie Freiberg), Dr Łukasz Klapiszewski

(Poznan University of Technology), M.Sc. Iaroslav Petrenko (TU Bergakademie

Freiberg), M.Sc. Tomasz Szatkowski (Poznan University of Technology) for all

helpful scientific discussions we have shared.

Especially, I would like to thank to B.Sc. Andre Ehrlich (BromMarin) for his

valuable discussions during scientific expeditions,

but mostly I am thankful for his help and creation of familiar atmosphere

during my internships in foreign country.

Finally, I offer my regards to all of those, especially

my family, Ehrlich family and my friends, who supported me,

in both, good and bad moments during the completion of this thesis.

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TABLE OF CONTENTS

CHAPTER 1 GENERAL INTRODUCTION ...... 8 CHAPTER 2 LITERATURE REVIEW ...... 11 2.1 BIOINSPIRATION AND BIOMIMETICS ...... 12 2.1.1 BIOMIMETICS ...... 12 2.1.2 EXTREME BIOMIMETICS ...... 17 2.1.3. HYDROTHERMAL VENTS AND HOT SPRINGS AS AN INSPIRATION FOR EXTREME BIOMIMETICS ...... 18 2.2 CHITIN AS A SUITABLE TEMPLATE FOR EXTREME BIOMIMETICS ...... 24 2.2.1 CHITIN SOURCES ...... 24 2.2.2 CHITIN STRUCTURAL PROPERTIES ...... 26 2.2.3 MORPHOLOGICAL BENEFITS OF CHITIN OF PORIFERAN ORIGIN ...... 32 2.2.4 CHITIN AS TEMPLATE FOR BIOMIMETICS ...... 37 2.2.5 THERMAL STABILITY OF CHITIN AS A KEY TO EXTREME BIOMIMETICS ...... 42 2.3 HYDROTHERMAL TECHNOLOGY ON SERVICE OF EXTREME BIOMIMETICS ...... 45 2.3.1 HISTORICAL LANDMARKS OF HYDROTHERMAL TECHNOLOGY ...... 45 2.3.2 FUNDAMENTALS OF HYDROTHERMAL TECHNOLOGY ...... 47 CHAPTER 3 AIM OF THE WORK ...... 55 CHAPTER 4 MATERIALS AND METHODS...... 58 4.1. ISOLATION OF THE α-CHITIN FROM MARINE SPONGES ...... 59 4.2. HYDROTHERMAL SILICIFICATION OF SELECTED CHITINOUS SCAFFOLDS ...... 60 4.3. FORMATION OF ZIRCONIUM DIOXIDE NANOPHASE USING CHITINOUS SCAFFOLDS UNDER HYDROTHERMAL CONDITIONS ...... 62 4.4. HYDROTHERMAL SYNTHESIS OF β-CHITIN/ZnO NANOSTRUCTURED COMPOSITES ...... 62

4.5. HYDROTHERMAL SYNTHESIS OF α-CHITIN/Fe2O3 NANOSTRUCTURED COMPOSITES ...... 63

4.6. HYDROTHERMAL SYNTHESIS OF CHITIN/GeO2 NANOSTRUCTURED COMPOSITES...... 63 4.7. CHARACTERIZATION TECHNIQUES ...... 64 4.7.1. FOURIER-TRANSFORM INFRARED SPECTROSCOPY...... 64 4.7.2. RAMAN SPECTROSCOPY ...... 64 4.7.3. POWDER X-RAY DIFFRACTION (XRD) ...... 65 4.7.4. X-RAY PHOTOELECTRON SPECTROSCOPY ...... 65 4.7.5. TRANSMISSION ELECTRON MICROSCOPY ...... 66 4.7.6. SCANNING ELECTRON MICROSCOPY ...... 67 4.7.7. FLUORESCENCE AND LIGHT MICROSCOPY ...... 67 4.7.8. THERMAL ANALYSIS ...... 67

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4.7.9. NEAR EDGE X-RAY ABSORPTION FINE STRUCTURE ...... 67 4.7.10. ELECTROCHEMICAL MEASUREMENTS ...... 68 4.7.11. PHOTOLUMINESCENCE MEASUREMENTS ...... 68 4.7.12. ANTIBACTERIAL ACTIVITY ...... 69 CHAPTER 5 STUDIES OF THE PHYSICOCHEMICAL PROPERTIES OF THE EXTREME BIOMIMETICALLY PREPARED CHITIN-SILICA MATERIALS ...... 70 5.1. STRUCTURAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-SILICA MATERIALS ...... 72 5.2. PHYSICOCHEMICAL CHARACTERIZATION OF HYDROTHERMALLY PREPARED CHITIN-SILICA MATERIALS ...... 73 CHAPTER 6 CHITIN AS A VERSATILE TEMPLATE FOR EXTREME BIOMIMETIC SYNTHESIS OF CHITIN- ZIRCONIA MATERIALS ...... 80 6.1. STRUCTURAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-ZIRCONIA MATERIALS ...... 82 6.2. PHYSICOCHEMICAL CHARACTERIZATION OF HYDROTHERMALLY PREPARED CHITIN-ZIRCONIA MATERIALS ...... 87 6.3. PRELIMINARY RESEARCH ON APPLICATION OF CHITIN-ZIRCONIA COMPOSITE IN ADSORPTION OF HAZARDOUS METAL IONS ...... 89 CHAPTER 7 AN EXTREME BIOMIMETIC APPROACH FOR HYDROTHERMAL SYNTHESIS OF β-CHITIN- ZnO NANOSTRUCTURED COMPOSITES ...... 93 7.1. STRUCTURAL AND PHYSICOCHEMICAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-ZnO MATERIALS ...... 95 7.2. ANTIBACTERIAL PROPERTIES OF CHITIN-ZnO COMPOSITE...... 100 CHAPTER 8 SYNTHESIS OF NANOSTRUCTURED CHITIN–IRON OXIDE COMPOSITES UNDER EXTREME BIOMIMETIC CONDITIONS ...... 104 8.1. STRUCTURAL AND PHYSICOCHEMICAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-IRON OXIDE MATERIALS ...... 106

8.2. ELECTROCHEMICAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-Fe2O3 MATERIALS . 115

CHAPTER 9 EXTREME BIOMIMETIC APPROACH FOR DEVELOPMENT OF NOVEL CHITIN–GeO2 NANOCOMPOSITES WITH PHOTOLUMINESCENT PROPERTIES ...... 119 9.1. STRUCTURAL AND PHYSICOCHEMICAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN- GERMANIUM DIOXIDE MATERIALS...... 121

9.2. PHOTOLUMINESCENT PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-GeO2 MATERIALS128 GENERAL SUMMARY ...... 132

REFERENCES ...... 135 ABSTRACT ...... 158 STRESZCZENIE ...... 161 SCIENTIFIC ACTIVITY ...... 165

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CHAPTER 1 GENERAL INTRODUCTION

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Nowadays much attention is being paid to the development of advanced materials for specific applications. Sustainable development concept as well green chemistry forces scientists to create technologies of their synthesis in environmentally friendly methods with use of biopolymers from renewable sources. Consequently, new sophisticated methods are developed on a current basis and have become the driving forces to meet future challenges of biopolymer-based materials. Among them special attention, definitively, has to be paid to biomimetism and bioinspiration as tools for the design of innovative hybrid materials that combine organic and inorganic components on a nanoscale with innovative controlled textures.

Nature has nominated organisms that can create specialized inorganic material structures in nano- or microscale in process termed - biomineralization. These organisms are capable to organizing nanoscale building blocks into large-scale hierarchical structures to form hard tissues serving different functions, such as mechanical support, filtration, light harvesting, gravity sensing, and locomotion [1,2]. Scientists ware always amazed by the high degree of specific organization on several hierarchical levels within the biocomposites. Therefore, a lot of research has been dedicated to adaptation of mechanisms and principles from biomineralized tissues to create functional structures for advanced applications, which are usually referred to biomimetics. Most of these methods are carried out at ambient conditions, and biomineralization processes found in extreme natural environments are surprisingly ignored or omitted by large community of scientists working on bioinspired synthesis. This is quite surprising because number of examples of hard tissues found in organisms, which dwell in extreme (arctic or hydrothermal waters) conditions, may have great contribution to modern materials science [1].

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Thus, this thesis is oriented on utilization of unique natural thermostable polymer matrix – chitin, which is commonly found in skeletal systems of organisms clustered around hydrothermal vents, in solvothermal synthesis of hybrid inorganic-organic chitin- based materials. It is worth highlighting that all studies presented in this thesis are based on utilization of three-dimensional chitinous scaffolds isolated directly from marine sponges of the Verongida order or Sepia cuttlebone. This allows overcoming the technical limitations of chitin processing into fibrous structures, which arise from poor solubility.

Presented in this thesis, Extreme Biomimetic methods for development of chitin-based biomaterials, might be a new gate into the world of biomimetics and bioinspired materials chemistry. This thesis provides comprehensive review, with respect to the current literature, dedicated to chitin as a suitable biomacromolecule for Extreme Biomimetics.

The detailed methodology applied for synthesis as well as physicochemical characterization of chitin-based materials is described. Discussion of obtained results with respect to physicochemistry as well practical applications of prepared chitin-based inorganic-organic hybrid materials is the main part of this thesis. Finally, all results are summarized and general conclusions are highlighted.

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CHAPTER 2 LITERATURE REVIEW

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2.1 BIOINSPIRATION AND BIOMIMETICS

2.1.1 BIOMIMETICS

2.1.2 EXTREME BIOMIMETICS

2.1.3 HYDROTHERMAL VENTS AS AN INSPIRATION HABITATS FOR EXTREME BIOMIMETICS

2.1.1 BIOMIMETICS

Nature has developed number of unique materials that combine many inspiring properties such as sophistication, miniaturization, hierarchical organization, unique mechanical properties. They show various functions from macroscale to nanoscale and are designed to fulfill specific role in the tissues of various organisms. Nature develops biological objects by means of growth or biologically controlled self-assembly, adapting to the environmental circumstances and using the most commonly found materials. Thus, biological materials are developed and improved through evolution over 3.8 Gyr [1] by using ways directed by the genetic code. Consequently, biological materials and tissues are created by hierarchical structuring at all levels [2]. Analyzing myths of ancient Greece and well-known legend about Daedalus and Icarus who constructed wings from bird feathers and wax, it can be stated that nature has been source of inspiration for millenia. Imitation of the models, systems, and elements of nature for solving complex human problems is now one of the most studied scientific direction in modern science and it has been named biomimetics. Several other synonyms for biomimetics have been established including ‘biomimesis’, ‘biomimicry’,

‘bionics’, ‘biognosis’, ‘biologically inspired design’ and similar words and phrases implying copying or adaptation or derivation from biology [3]. A framework of biomimetics is to use the discovered natural structures and processes as inspiration to

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design innovative devices, materials and systems with unique structural and physicochemical properties [1,4]. The general concept of biomimetic has been presented in the Figure 1. It can be seen that the area of biomimetics is highly interdisciplinary and involves the understanding of biological functions, assemblies, and roles of various objects found in nature by architects, physicists, biologists, chemists, material scientists, mathematics, programmers and the computation, design and fabrication of various materials and devices of commercial interest by widely- understood engineering [2]. Thus, these methods are promising scientific and technological challenges, which regularly sheds new light on materials science and robotics.

Figure 1. Interdisciplinary of biomimetics.

Recently, special attention in biomimetics field has been paid to the biomineralization-inspired synthesis of functional organic/inorganic hybrid materials

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[5,6]. Biomineralization refers to a biological process responsible for the formation of complex materials, such as bones, nacre, shells, spicules, teeth etc. These materials are characterized by a remarkable level of molecular control of the particle size, structure, morphology, aggregation, and crystallographic orientation of their mineral phases composition [7]. Living organisms are well-recognized for creating a wide range of highly sophisticated inorganic-organic biocomposites with nano-, micro- and macroscale organization specially designed and adapted to their function. The diversity of mineralized structures found in nature, in terms of mineral composition, morphologies, properties, and functions is astonishing [2]. More than 60 different types of minerals are known to be used by organisms from all 5 kingdoms, where each organism has evolved its own strategies for creating biominerals that are tailored for their function [8–10]. Unique degree of complexity and resulting specific mechanical properties of biomineralized tissues and biominerals inspire scientists to get insights into these materials by understanding mechanisms of their formation and in following adaptation of these principles into modern materials chemistry [11]. At the nanoscale level, biomineralization process involves the molecular creation of discrete self- assembled organic supramolecular systems (like micelles, vesicles, etc.) that are used as preorganized environments for controlling the formation of superbly oriented mineral materials, with size ranging from 1 to 100 nanometers [12]. The fabrication of consolidated biominerals, such as bone, teeth, diatoms and sponge skeletal frameworks or nacre also involves the construction of preorganized organic frameworks but the size is greater and the matrix is polymeric (i.e. collagen [13], chitin

[14] etc.). The building of these organic architectures in biomineralization usually involves hierarchical arrangements (i.e. Bouligand structure of chitin-protein system

[15]), in which the molecular-based construction of organic assemblies plays role of

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the structural framework for the synthesis of organized biominerals, which in turn are exploited as prefabricated units in the production of complex micro- and macrostructures [12]. At this point it should be stated that formation of mineral phase on organic templates by living organisms could have been abiotic, where organic matter serves solely as a template for nucleation, or biogenic which means induced by biological activities, or combination of both. Therefore, biomimetic mineralization in vitro is based on selection and utilization of proper biomacromolecules, which play a crucial role in nucleation, thermodynamic and kinetic regulation of crystal growth; but also can be used as a soft templates for growth of the inorganic structures [16]. In particular, the sophisticated three dimensional, nanostructural biocomposite-based scaffolds are essential, from the viewpoint of widely understood materials science, for a broad variety of advanced applications [17,18]. In result, the adaptation of principles derived from biomineralization research to the synthesis of inorganic materials with controlled properties is nowadays one of the most extensively investigated topic in modern materials science related to biomimetics [8]. During the last decades, many inorganic crystals or hybrid inorganic-organic materials with special sizes, morphology, crystalline structure have been synthesized via biomineralization inspired methods with the assistance of various biomolecules; especially enzymes

[6,19], proteins [20–22], peptides [23–25] and polysaccharides [14,26,27]. It has been established that these biomacromolecules not only lower the surface free energy of developing nuclei, and thereby promote heterogeneous nucleation over crystallization in bulk solution, but they can also direct the crystallization of biominerals by the stabilization of specific planes of crystal lattice [16,28]. However, despite of intensive research made in the field of biomineralization, scientists still discover new facts and new principles, which are surprising for broad community associated around this

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topic. Recent sensation in this field was biomineralization-inspired synthesis of artificial flexible calcite-based spicules published by Filipe Natalio and co-workers in

Science Magazine [19]. In this study authors used silicatein-α (the protein responsible for the biosilicification in some demosponges) to promote the self-assembly of calcite

“spicules” analogous to the spicules of the calcareous sponge Sycon sp.. Transferring a protein from a biological silicification system to in vitro calcite precipitation led to the synthesis of the rod-like structures (similar to spicules) with extremely high flexibility that can be used as waveguides for visible light. Rarely, the bioinspired mineralization is successful in reproducing of few properties in one material. Here, it is worth noting that Natalio et al. [19] obtained material with several properties [29], which are even better than biological material which was chosen as an inspiration for this study.

Intriguingly, despite the above-mentioned broad diversity of biominerals in Animal kingdom, most of the research is paid on bioinspired formation of calcite and aragonite

(CaCO3), hydroxyapatite Ca5(PO4)3(OH), magnetite (Fe3O4) and opal-A (SiO2)- based biomaterials. Usually, approaches related to the in vitro biomineralization of organic templates under biomimetic conditions are carried out at temperatures between 20 and 40 °C and at near-neutral pH, which are considered the most appropriate from biomimetic point of view. It is assumed that biological materials, especially protein- based ones, are not stable at temperatures higher than 40 °C. Nevertheless, nature developed several strategies of biomineralization pathways at extreme cold and extreme hot environments. Unfortunately, these unique processes are particularly ignored by scientists related with biomimetics. As a result, the principles and rules, which underlie these specific processes of creation of specific materials are still poorly understood, but proves that Nature is inexhaustible source of inspiration for development of biomineral-based materials and technologies.

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2.1.2 EXTREME BIOMIMETICS

Extreme Biomimetics is a novel scientific direction of modern biomimetics proposed for the first time by Hermann Ehrlich in 2010 [2]. The aim of Extreme

Biomimetics is to explore the mechanisms and principles that underlie biomineralization processes in extremophiles that are adapted to biologically extreme environments in terms of temperature, pressure as well as the presence of harmful compounds [2]. These range between the freezing point of seawater (-1.9 °C to 4 °C) and the extreme heat of hydrothermal vents and hot springs (between 60 °C and

121 °C). Is almost certain that understanding of the principles of biomineralization that are underlying survival of organism in these extremes of nature will result in establishment of the fundamental principles of Extreme Biomimetics and will employ them in a vast array of new technologies and commercial applications. The primary aim of mimicking of the engineering features achieved by these organisms found in the extreme niches will open the gate for the development of new synthetic routes for the generation of biocomposites that possess specific temperature and chemical resistance in vitro. Today, this state-of-the-art concept is based on the utilization of the specific thermostable biomacromolecules (proteins, enzymes, polysaccharides etc.) isolated from extremophiles, as a bioinspired tools for thermodynamic control of inorganic phase growth. It forces the scientific community to rethink role of thermostable molecules (specifically chitin, highly stable collagen or silk as well as anti-freezing proteins) in biomineralization. Extreme Biomimetic approaches can additionally be an important source of information for finding answers on fundamental questions about the mechanisms and principles of the biomineralization processes in the extreme environments of early Earth. It should be stated that Extreme Biomimetic concept is a very interdisciplinary field, which unites together chemistry, biology, physics and

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materials science. Furthermore, the computation and intelligent numerical modelling of the biomolecule assisted hydrothermal reactions would give important insights into optimization of Extreme Biomimetics with respect to thermodynamics, which is crucial when comes to the economical aspects of transfer of scientific knowledge regarding

Extreme Biomimetics to industrial practices.

2.1.3. HYDROTHERMAL VENTS AND HOT SPRINGS AS AN INSPIRATION FOR EXTREME BIOMIMETICS

Extreme environments in definition are characterized by “conditions that are far outside the boundaries in which most organisms live comfortably. These conditions include: pH, air pressure, temperature, salinity, radiation, dryness (desiccation), and oxygen level” [30]. Submarine hydrothermal vents, geothermal waters and terrestrial hot springs undoubtedly fall under this definition with respect to the number of their specific attributes, including acidic or alkaline pH, high hydrostatic pressure, high temperature, the presence of toxic compounds, reducing atmosphere, the absence of light etc. In spite of all these harsh parameters, extreme from biological point of view, it has been well reported that number of various thermophilic and hyperthermophilic organisms are widely distributed around these specific environmental niches. Vent communities are dominated by bacteria, arthropods, mollusks, and annelid worms, while cnidarians, chordates and sponges have a notable presence [31]. On the other hand, bacteria and algae dominate terrestrial hot springs and geothermal waters.

These thermophilic organisms are interesting for the Extreme Biomimetics because they are able to carry out biomineralization processes at harsh conditions (pH 1-3, temperature 40 °C - 120 °C) and, in result, form biomaterials with exceptional properties. For instance, Inagaki et al. [32,33] proved that extremophilic bacteria within the Thermus and Hydrogenobacter genera are predominant components among

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the microbial community in siliceous deposits formed within the pipes and equipment of Japanese geothermal power plants. These bacteria strains influence the rapid formation of massive siliceous deposits. Additionally, in vitro examination suggested that Thermus cells induced the precipitation of supersaturated amorphous silica during the exponential growth phase, concomitant with the production of a specific cell envelope protein. On the other hand, the role of microorganisms in silica precipitation in terrestrial hot springs is still topic of discussion. It is mainly considered as a passive process resulting from rapid cooling to ambient temperatures of stream saturated with silicic acid. Silicification onto growing cells can also be affected by evaporation and/or steam loss, mixing and changes in pH of the hydrothermal waters after discharge.

However, several studies shown that functional groups (for instance hydroxyl groups) present on the microbal surface serve as favorable nucleation sites for silica precipitation [34] and are responsible for reducing the activation energy barriers of silica nucleation, or act as templates for heterogeneous nucleation [35]. After silica precipitation is initiated upon the bacterial surface, continued growth presumably occurs auto-catalytically due to the increased surface area generated by the small silica phases. Organic surfaces provided by microbes can also facilitate the binding of silica polymers and colloids from solution. It should be kept in mind that the colloidal silica surface has a residual negative charge; however, the bacterial surface also has a negative charge, except extremely low pH conditions. Thus, sorption reactions can occur by either (1) hydrogen bonding between dissolved silica and cell hydroxyl functional groups, (2) metal cation bridging between anionic cell functional groups and the silica, or (3) direct electrostatic interaction of silica with cell surface ammonium

(-NH3+) groups [36]. Surprisingly, it has been proven that extremophile bacteria have no influence on precipitation of CaCO3 in hot spring waters with temperature above

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90 °C [37]. Additionally it has been reported that postmortem calcification, resulting from supersaturation, and transformation of aragonite to calcite - destroys the morphology of calcified cyanobacteria, whereas silicification helps to preserve their morphological features. Therefore, it has been suggested that the dissolved silica in geothermal hot waters or terrestrial hot springs may be a significant component in the maintenance of position and survival of microorganisms within these extreme niches

[2]. This unique feature has been recently used for a bioinspired approach to generate thermostable virus by introducing an artificial hydrated silica exterior on individual virion [38]. Similar to thermophiles, silicified viruses can survive longer at high temperature than their wildtype relatives.

Thermophilic bacteria are not the only example of biomineralization, which take place in hydrothermal vents, geothermal and hot waters. Diatoms occupy the most varied water biotopes, including warm and hot waters. These microalgae are sensitive to a variety of environmental parameters that have made them useful tools in environmental monitoring. The most common in the hot springs are the following species: Pinnularia microstauron, Rhopalodia gibberula, Navicula cincta,

Achnanthidium minutissimum, Gomphonema parvulum, Amphora coffeaeformis,

Nitzschia amphibian, and Achnanthidium exiguum [2]. All of them present unique ability for formation of biosilica-based skeletons at extreme conditions. Analysis of chemical composition of above-mentioned diatoms with respect to organic molecules, which are responsible for silicification, is now topic of great importance in perspective of advanced Extreme Biomimetics. The recent discovery of chitin in siliceous cells of diatom [39–41] species living at ambient conditions, may suggest that this polymer can play crucial role in hydrothermal silicification as well, especially when we take into account unique thermal stability of chitin [42].

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It is well reported that also specially adapted Metazoans not only tolerate extreme hot conditions but they often thrive in them [43]. The classical examples are two chimney-dwelling polychaetes: Alvinella pompejana and Riftia pachyptyla, which live on newly formed vent walls very near the super-hot vent fluid. Pompeii worms (A. pompejana) are capable of withstanding temperatures up to 105 °C [44]. The multilayered structural exoskeleton of A. pompejana, which incorporates minerals

(sulfates), have attracted the attention of materials scientists. Exoskeleton secreted by these worms is concentrically multilayered structure assemblage of biopolymers composed of about 50% of proteins. Unique assembly and composition of this skeleton results in high thermal (0-100 °C) and chemical stability (acid and alkaline pH) and high hydrophobicity [45]. The unique type of collagen present in A. pomejana skeletons is the second key to adaptation of this organism to extreme hot environments. The size of the triple helices of cuticular collagen isolated from these worms fluctuate between

2400 and 2600 nm [2] and thus belong to the longest collagen molecules known thus far. High thermal stability (up to 45 °C) of this collagen is a result of significantly larger content of proline in the Y position. Moreover, it is reported [46] that almost all of the proline residues in the Y position of the Gly-X-Y triplets are fully hydroxylated. This phenomenon promote hydrogen bonding of water molecules in crystals of the peptides and increase stability of collagen helix. Additionally, in situ electrochemical speciation and discrete sample measurements provided that the ability of A. pompejana to thrive in extreme microhabitats is caused by the unique interaction of Fe2+ and free S2- to form iron sulfide species at high temperatures, which lowers pH and detoxifies sulfide into biologically unavailable [47]. The fact, which is worth of noting is that the predominant mineral phase found at the outer tube of this organism is FeS2 marcasite [48], while the

(Zn,Fe)S (wurzite) is always predominant at the inner surfaces and within the matrix

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of the tubes. Marcasite precipitation beneath worm tubes and worm migration to newly formed areas of chimney, combined together, create a moving front of biomineralization that keeps pace with chimney growth [49].

A lot of research has been dedicated to another representative of hydrothermal vent fauna – R. pachyptila. This organism is also characterized by the presence of unusual cuticular collagen. In contrast to A. pompejana, the glycosylated threonine is located at the Y-position of the Gly-X-Y triplets of the amino acid sequence and presumably enhances the thermal stability of the triple helices of collagen as an adaption to hydrothermal environment in R. pachyptila [2]. However, the significant feature responsible for unique chemical and thermal stability is the presence of β-chitin in the skeletal structures of R. pachyptila. In skeleton of this organism the microfibrils and crystallites of β-chitin are embedded in a protein matrix, and together form flat liquid- crystal-like structures [50], which are different from the commonly known chiral nematic (cholesteric) or isotropic arrangement of classical chitin [51,52] and chitin- protein systems [53]. The size of the chitin crystallites is extraordinary high and equal

50 nm, whereas the most common size for β-chitin crystallites found for instance in squid pens are about 3-10 nm [54,55]. Several crisscrossing layers of these ribbons build up the tube wall. These huge crystallites are composed of up to 6000 β-chitin chains and are secreted by large cup-shaped microvilli-like pyriform glands (present in all the four main regions of the animal except obturaculum [56,57]). Chamoy et al.

[57] identified protein bonded to β-chitin in R. pachyptila skeletons with 21.3 kDa size, named Riftia chitin-binding protein (RCBP). This protein is closely related to type II chitin-binding domains that are restricted to the animal kingdom, however the unique property, showed by affinity assay and immunogold labeling, is that this protein binds specifically β-chitin and is unable to bind the most common α-form found in chitin

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secreting animals [57]. Riftia tube was shown to be very resistant to aggressive in vitro chemical and physical treatments [58]. It is well known feature of chitin, which has been reported to be stable even in 40 % HF [39]. While unique stability against enzymatic degradation of Ritifia tubes may be a result of the disulfide bonds of the specific protein-chitin complex [2].

Hot-vent gastropod with iron sulfide dermal sclerites has been reported by

Warén et al. [59] as an example of exceptional biomineralization phenomenon related with hydrothermal vents. The discovered gastropod lives at the base of black-smoker chimneys in the Kairei vent field in the Indian Ocean and have feet covered by sclerites composed of conchiolin, mineralized with pyrite (FeS2) and greigite (Fe3S4) [59,60].

The term “sclerite” refers to an scale- or spine-shaped element of a exoskeleton, in which the elements are held together to form a protective coating layer [61]. The sclerites have size up to 8 mm long and they cover the sides of the gastropod foot similarly to roof-tiles [59]. Sclerites are a three-layered structures (composed of sulfates, conchiolin and pulp [59]) with exceptional mechanical properties making this materials even harder and stiffer than human tooth enamel [61]. The hardness and stiffness gradually decrease from the iron sulfide layer to the conchiolin layer. Results published by Suzuki et al. [61] undeniably indicate that iron sulfide mineralization is directly mediated by the gastropod. Application of advanced methods for analysis of mechanical properties revealed that each layer of the shell is responsible for distinct and multifunctional role in mechanical protection against predators and to maximize survivability in harsh hydrothermal environments [62]. Mechanical phenomena of these sandwich-type three-layered “armors” could also potentially be employed for use in protective military and engineering applications [62,63]. It is worth noting that a new morphotype (solitaire) of ‘scaly-foot’ gastropod without iron-sulfide dermal

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sclerites has been also discovered. This result suggests that the two morphotypes of

‘scaly-foot’ gastropod have different mineralization capabilities. Surprisingly the solitaire morphotype showed greater mechanical strength of the whole structure than did that from the Kairei morphotype.

To summarize this chapter it seems that chitin-based (R. pachyptila), collagen- based (A. pompejana) and conchiolin-based structures found in organisms thriving at hydrothermal vents possess unique chemical and thermal stability owing to their extraordinary ability for controlled biomineralization. Therefore, these organic templates have intriguing potential with respect to classical biomimetics as well

Extreme Biomimetic concepts. The deep understanding of their role in extreme biomineralization has significant potential to expand current knowledge of the functional structures in biology, as well as to inspire development of the new technological processes and novel generation of biomaterials.

2.2 CHITIN AS A SUITABLE TEMPLATE FOR EXTREME BIOMIMETICS

2.2.1 CHITIN SOURCES

2.2.2 CHITIN STRUCTURAL PROPERTIES

2.2.3 MORPHOLOGICAL BENEFITS OF CHITIN OF PORIFERAN ORIGIN

2.2.4 CHITIN AS TEMPLATE FOR BIOMIMETICS

2.2.5 THERMAL STABILITY OF CHITIN AS A KEY TO EXTREME BIOMIMETICS

2.2.1 CHITIN SOURCES

It has been estimated that every year about 1011 tons of chitin are produced by organisms [64,65], thus after cellulose, chitin is the second most abundant polysaccharide on the Earth. The great natural abundance of chitin arises from the fact

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that it is structural component of the broad range of species. These include yeast and fungi [66,67]; diatoms [39,40]; coralline alga [68], protists [69,70], corals [71]; mollusk

[72,73], polychaetes [57]; spiders [74]; insects [75]; crustaceans including crabs [76], lobsters [15,77], shrimps [78], krill [79,80]. It has also been found within vertebrates

(fish and amphibia) [81,82]. By intensified research in this field, Ehrlich and co- workers [83] published report indicating that chitin is also an endogenous material within the siliceous skeletons of Farrea occa – representative of the glass sponges

(Hexactinellida: Porifera). Thanks to this discovery, it was hypothesized [83] that the chitin molecules might be part of a very old template system involved in biosilicification phenomena. In 2009, Brunner et al. [84] revealed that chitin-based scaffolds are an integral part of skeletons from the marine demosponge Ianthella basta of the Verongida order. This study showed that marine sponges developed mechanism to produce unique, highly organized two-dimensional networks composed of cross- linked chitinous nanofibers. These fibers consist of loosely packed chitin with rough, deeply fissured surfaces. In the native sponge skeleton, proteins and bromotyrosine- related compounds are intercalated into the chitinous scaffold and are cross-linked with chitin, thus preventing it from forming unperturbed crystalline chitin [84].

Systematic studies regarding isolation of chitin on numerous representatives of the

Aplysinidae family revealed the presence of a chitin-based scaffold closely resembling the shape and morphology of the original sponge in several specimens of the Aplysina genus from the Mexican Pacific [85], and in the Aplysina fistularis sponge skeleton [86].

Intriguingly, Ehrlich and co-workers published recently two papers describing the discovery of α-chitin as a part of the skeletal structures of fresh water sponges like

Lubomirskia baicalensis [87] and Spongilla lacustris [88]. These sensational discoveries of chitin within the skeletons of both marine and freshwater sponges can also yield

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information useful for classifying sponge species. What is more, Ehrlich et al. [89] reported that fragments of chitin fibres survived in the 505 million year old (Middle

Cambrian) Burgess Shale Vauxia gracilenta sponge fossils. Compared to previously reported chitin fossils [90,91], the chitin found in Vauxia sponge fossils is thus far the oldest known, which highlight exceptional chitin preservation. Besides, the oldest chitin found up to today is also of sponge origin.

Despite wide diversity of chitin sources, the isolation procedure is relatively easy and can be described in three basic steps: (i) treatment with 20% acetic acid (or diluted 3-5% hydrochloric acid) for decalcification, (ii) treatment with sodium hydroxide for deproteinization, desilicification, (iii) decoloration with use of hydrogen peroxide or sodium hypochlorite. It is important to add that deproteinization should be performed with use of NaOH concentration not exceeding 4M. Application of higher sodium hydroxide concentration can result in transformation of metastable β- and γ- chitin into α polymorph, and can result in uncontrolled deacetylaton of the product.

2.2.2 CHITIN STRUCTURAL PROPERTIES

From chemical point of view, chitin is a structural polysaccharide made up of many repeating units of a sugar termed N-acetyl glucosamine units bonded by β-1,4- glycoside bond [92]. Chemical structure of chitin repeating units has been presented in

Figure 2. It is worth noting that utilization of NaOH for chitin purification

(deproteinization) results in partial deacetylation. In consequence, obtained chitin is never fully acetylated and it is a copolymer of N-acetylglucosamine and glucosamine units [92–94].

In nature chitin exists in several crystalline polymorphs. This fascinates scientists since 1920, and up to today it was commonly accepted [14,95] that the α- and

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β-forms are the two main polymorphs of chitin. Both forms can be differentiated by infrared; 13C NMR [96] spectroscopy, and X-ray diffraction [97–100]. A third allomorph, γ-chitin, has been also described in literature [101].

α-Chitin has been the subject of intensive crystallographic research since 1920

[92]. A detailed model for the crystal structure of the fibrous polysaccharide α-chitin was proposed in 1957 [102]. In this fundamental work, Diego Calström agreed with the theoretical assumptions of Meyer and Pankow (made based upon that of the cellulose)

[92] that the orthorhombic unit cells containing chitobiose units run in opposite directions with the planes of the pyranose rings perpendicular to the a axis. However, from experimental data he obtained different dimensions of unit cells (a=4.76 Å, b=10.28 Å and c=18.85 Å) and also indicated the space group was P22121 or P212121

[102]. Measured distance of C(3)-OH---O(5) intramolecular hydrogen bond (equal to

2.68 Å), an acceptable glycosidic bond (107°) and a b repeating distance 10.28 Å proved that the chitin chain could not be considered as a straight line and he proposed a “bent chain” theory. Additionally, Calström considered the –NHC(O)CH3 groups and suggested planarity of the aminoacetyl groups, suggesting that due to the limited rotation of C(2)-N and measured distance of 2.69 Å for the C(2)-NH---O=C(7), the -NH group has to point approximately towards the carbonyl oxygen of an adjacent chain in the a axis direction [102]. The model proposed by Calström was without any doubt a breakthrough in determination of α-chitin structure. Nevertheless, he had to face a wave of criticism [92]. One of the mostly highlighted critical remark was that

Calström’s model suggested that –C(5)OH are not involved in formation of hydrogen bonds, and a second was that the splitting of I amide band cannot be arranged in the same environment. Thus, over years number of scientists have revised this model,

(please see for review [92]). Finally, Minke and Blackwell [103] demonstrated the

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formation of additional (not reported by Calström) intramolecular hydrogen bonds

C(6)–OH---O=C(7) in one chain as well as intermolecular hydrogen bonds C(6)–OH---

HO–C(6) between two chains. As a result, the proposed chitin structure, contained two types of amide groups, which differed in their hydrogen bonding environments; explaining the splitting of the I amide band in the IR spectra of α-chitin [103,104]. Up to now, it had been a well-accepted model but it underwent several slight modifications and improvements. For instance, Sikorski et al. [99] by application of high resolution

XRD analysis proved formation of special hydrogen bond networks, and Petrov et al.

[105] by ab initio studies gave evidence of intermolecular hydrogen bonds in the G-G+ conformations. The chemical structure of α-chitin is presented in Figure 2a.

In case of the β-chitin, the earliest model of its structure was proposed by

Dweltz [106] when he determined the P21 space group, and noted that the –NH groups are involved in the formation of intermolecular hydrogen bonds along the a axis; and proposed formation of intermolecular hydrogen bonding through water molecules in the c axis direction. In contrast to α-chitin the unit cell in β-chitin has monoclinic crystal system [107], and adjacent sheets along the c axis have the same direction. Worth of noting is the lack of hydrogen bonds along the b axis, which make β-chitin more susceptible for intra-crystalline water swelling [108–110]. The swelling phenomenon takes place without destruction of the initial microfibrillar morphology, which can be recovered after removal of the intra-crystalline guest molecules [110]. Sawada et al.

[111] by application of neutron fiber diffraction, proved the formation of O3---O5 hydrogen bonds in β-chitin, with a hydrogen to acceptor distance of 1.77 Å and a hydrogen bond angle of 171°. Additionally, Sawada et al. [112] reported that water can interact with β-chitin by formation of hydrogen bonds. Intriguingly, for β-chitin dihydrate complex, authors reported absence of an intramolecular H-bond between O3

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and ring oxygen O5, instead, O3 donates hydrogen to water molecule [112]. On the other hand, β-chitin is synthesized by living organisms under water and thus it is suggested that β-chitin in the native state is a hydrated complex.

Figure 2. Schematic view on antiparallel chain organization in α-chitin. b) High-resolution image of cross-sectioned α-chitin microfibril showing the lattice fringe corresponding to (0 2 0) plane. c) Model of cross-sectioned α-chitin microfibril adapted from [100]. d) Lattice images of a cross- sectioned microfibrils corresponding to the (0 1 0) plane of anhydrous b-chitin. e) The model of cross-sectioned β-chitin microfibrils adapted from [119].

Kobayashi et al. [113] studied the crystal transition between hydrated and anhydrous

β-chitin by using synchrotron X-ray fiber diffraction and confirmed hypothesis that

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β-chitin in natural environment occurs in form of dihydrates. However, these authors proved with strong evidence that it is not possible to return from anhydrate to the initial dihydrate form, even when the relative humidity is 100%. It should be mentioned that β-chitin is metastable polymorph of chitin and it can be transformed into α-chitin by treatment with NaOH, however the 30% as the minimal concentration of alkali is needed [114].

Rudall [115] reported the presence of a third, very rare polymorph of chitin whereas two chains are aligned parallel and one is antiparallel. Jang et al. [101] confirmed, by the XRD measurements that maximum peaks around 2Θ=9.6° and

2Θ=19.8° which means that γ -chitin, are close in its XRD patterns to α-chitin.

The diversity of the chitin polymorphs seems to be related to the multiplicity of functions in various tissues [92,98]. Thus, α-chitin is mainly isolated from organisms where extreme mechanical properties are required (hardness) and it is commonly associated with inorganic compounds [67]. The most representative example of this phenomenon is lobster cuticle. Raabe and co-workers [15,116–118] in a series of papers indicated that chitin chains are organized in an antiparallel fashion forming

α-chitin polymorph that in turn form nanofibrils assembled into honeycomb shaped arrays. These are stacked along their normal direction of about 180° and referred to as the plywood or Bouligand layer.

Application of the sychrotron wide angle X-ray diffraction method proved that samples taken from different parts of the lobster exoskeleton are characterized by a similar crystallographic pattern, which in result indicate that the α-chitin has a very similar orientation along the entire cuticle surface. This phenomenon, considered together with the “mechanical selection theory”, encouraged Raabe and co-workers

[116] to hypothesize that the α-chitin matrix relative to the local sample coordinates

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turns out to be mechanically favorable, providing maximum protection. Similar phenomenon of unique mechanical properties achieved thanks to mineralized α-chitin

Bouligand structures has been reported in the dactyl club of the Mantis shrimp –

Odontodactylus scyllarus [11,120,121]. This biomaterial has been recently a huge scientific sensation due to its unique mechanical properties, thanks to which this material is adapted for powerful close-range combat and is able to withstand multiple high-energy impacts [120]. The mineralized α-chitin fibers can dissipate the energy released by propagating microcracks; an oscillating elastic modulus that provides further shielding against catastrophic crack propagation; a modulus mismatch in the impact region that acts as a crack deflector near the impact surface [120]. Ehrlich et al.

[88] likewise showed that incorporation of α-chitin into proteinaceous skeletons of fresh water sponge (Spongilla lacustris) appears to be evolutionally favored because of the stiffening of the skeleton. Moreover, α-chitin presented within the holdfast of endemic freshwater sponge Lubomirska baicalensis plays a major role in the adhesion of sponge’s to rocky substrate [87].

In comparison, β-chitins are usually found in organisms where both flexibility and durability are required [92,98]; for example in squid pens [55] or tube worms

[50,56,57]. Interestingly, all three polymorphs has been indicated within different parts of the Loligo sp. squid pen (α-chitin in thin cuticle lining stomach, β-chitin in pen,

γ-chitin in thick cuticle lining stomach) and in some insect cuticles [98]. This phenomenon clearly indicates that different forms are related to the function which they play in various tissues, rather than to taxonomic grouping [92,98].

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2.2.3 MORPHOLOGICAL BENEFITS OF CHITIN OF PORIFERAN ORIGIN

Thanks to the unique biocompatibility of chitin and a possibility of degradation by human lysozyme to non-toxic products, recently a rapid “bloom” of research dedicated to the use of chitin-based materials in many biomedical applications is being observed. Especially the use of chitin-based biomaterials in the field of tissue engineering [122–124] and regenerative medicine [125,126] has been intensively growing over the past years [127]. The present generation of materials for structural supports in tissue engineering is based on seeding cells onto porous biodegradable polymer matrices [128]. Thus, in perspective of tissue engineering applications it is crucial to prepare scaffolds in porous sponge-like forms on a cellular spatial scale, offering microscale channels for the migration of host cells and nutrients as well growth of regenerated tissue into the matrix [127].

On the other hand, three-dimensional chitin materials have also exhibited other intriguing physicochemical properties, which make them highly effective for oil separations. This arises from the existence of the hydrophilic hydroxyls and acetyl amino group, combined with hydrophobic pyranose rings in the chitin molecules, which give the overall material amphilicity, and allows uptake of both polar and nonpolar liquids [129]. In result, artificially prepared three-dimensional chitinous sponges are able to collect organics both on the surface and bottom from the water and are highly effective removers of oil from water, showing potential application in the pollutant remediation field especially in perspective of management of oil-spills and leaks into the environment [129]. Unique adsorptive properties arising from the presence of functional groups in the chitin molecule make this biopolymer attractive for heavy metals [130] and fluorides removal [131]. Therefore, application of chitinous

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sponge-like structures in design of fixed bed columns for wastewater management

[131] seems a very promising area of research.

Unfortunately, there is a dearth of ‘prefabricated’ natural 3D chitin scaffolds. The chitin isomorphs isolated so far from sea-food waste (crabs, lobsters, shrimps, crayfish) occur in the form of granules, flakes or powder - not as 2D or 3D scaffolds.

Thus, recently the main challenge is to create chitinous two- or three-dimensional structures with open pores, controlled diameter of fibers, specially designed mechanical properties, which will pass rigorous demands imposed by special applications. Several attempts have been made to achieve this goal. The popular way is lyophilization of the chitin gel. By this method Okamoto et al. [132] created and applied polymeric chitinous sponge-like structures to veterinary practice for treatment of various types of trauma, abscess, surgical tissue defects and herniorrhaphy.

Correspondingly, Abe et al. [133] developed freeze-drying method for production of biodegradable and biocompatible β-chitin sponge and used it as a scaffold for three- dimensional culture of rabbit chondrocytes. The key to this method is preparation of chitinous-gel, which in turn can be lyophilized. Taking into consideration fact that chitin, due to strong hydrogen bonds, is particularly insoluble in most common solvents it seems to be a very challenging task. However several methods of chitin gel preparation have been developed. These include nanofibrilation by mechanical grinding [76,134,135] or ultrasound treatment [136,137], TEMPO-mediated oxidation

[138], acid hydrolysis [139], utilization of specific solvents (ionic liquids

[123,140,141], deep eutectic solvents [142] or hexafluoro-2-propanol [143]). These allow the chitin to dissolve, transform into gel and facilitate processing chitin into sponge-like structures, membranes or fibers. However, most of these procedures,

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which are usually followed by freeze-drying are unattractive from an economic standpoint because of high cost of the reagents or high-energy inputs.

As it was mentioned above, synthesis of two- as well three-dimensional chitinous structures is a major challenge, which needs sophisticated processess and unit operations. Therefore, recent discovery of naturally occuring, morphologically defined chitin-based skeletons of sponge origin is of great importance in perspective of preparation of advanced materials attractive for a wide range of practical applications.

Special attention in this field has been paid to chitinous scaffolds isolated from

Ianthella basta skeletons [84]. Scaffolds isolated from this sponge form two- dimensional networks composed of a highly organized chitin cross-linked nanofibers

(see Figure 3) with diameter of the chitin tubes fluctuating in range 40-100 nm

[84,104]. These naturally prefabricated chitinous structures isolated from Ianthella basta resemble the structure of comercially used bandages. High swelling ability, good compatibility with blood and possibility of drug loading characteristic for chitin, make them attractive for designing of wound dressing materials and treatment of skin wounds of various etiology. Additionally, it is worth to note that Rohde and Schupp

[54] demonstrated that I. basta is an organism capable for regeneration of the damaged chitin-containing tissue. It was found to have a reasonably rapid growth rate of 20 cm/year. Therefore, marine sponge I. basta is an renewable source of naturally prefabricated two-dimensional bandage-like chitin.

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Figure 3. Fragment of marine sponge I. basta preserved in 70% ethanol (a), isolated fibrous 2D skeletal chitinous scaffolds (b), SEM image of the chitin structure indicating high level of organization of chitinous fibers (c) schematic view on chitinous tubular scaffolds isolated from I. basta.

Another source of naturally prefabricated chitin are demosponges from the Aplysina genus. Chitinous scaffolds isolated from these specimens are characterized by unique tubular, three dimensional, highly macroporous, fibrous morphology (Figure 4) with number of chambers, channels, and the high swelling ability [144]. The practical value of the sponge skeletons arises in their relatively high porosity and surface area estimated for isolated skeletons at 6-8 m2/g. Thanks to the intricate network of tubes and chambers, acting like capillars these skeleton can be characterized with high swelling ability. This property has been documented as favorable for cell

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adhesion [128]. As a result of open pores structure, the 3D chitinous scaffolds derived from marine sponges of Aplysina genus promote proliferation and differentiation into osteogenic and adipogenic directions of human adipose tissue-derived mesenchymal stromal cells, which could provide broad opportunities for creation of new biocompatible and functionally active bioengineered structures [128].

Figure 4. Dried fragments of marine sponge A. cauliformis with the fingerlike bodies (a), are a renewable source for isolation of the fibrous 3D skeletal chitinous scaffolds (b), adapted from Wysokowski et al. [145]. Scale bars 1 cm (a) and 5 mm (b).

To sum up, it is worth of highlighting that isolation of morphologically defined two-dimensional or three-dimensional chitin structures from sponges, overcomes several technical limitations associated with manufacturing chitin into above-

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mentioned structures. Therefore, application of chitin from sponge origin is highly cost-effective and efficient, and aligns closely with concepts of “green chemistry” and sustainable development. Thanks to ability to regeneration of damages chitin- containing tissue, it is quite feasible to grow or farm the amount of material needed for manufacturing from natural, renewable sources via marine ranching or primmorph cultivation of sponges. It is strongly believed that sponge-derived chitin is therefore poised to make deep inroads within bioinspired materials science [144].

2.2.4 CHITIN AS TEMPLATE FOR BIOMIMETICS

Polysaccharides play an exceptional role as a structural and crystal directing templates in biomineralization [14,146]. Chitin is a polysaccharide that is the most commonly associated with the biomineralized skeletons of various invertebrates including diatoms [39,40], corals [71], mollusk [73,147–153], coralline alga [68], crustaceans [154], polychaetes [57,108], freshwater [87,88] and marine sponges

[83,84,86,155–157]. Mineralized chitin offers a structural framework for mechanical support as well as providing strain energy storage. Crystalline chitin fiber features a high stiffness to weight ratio [118] and Young’s modulus even higher than 150 GPa

[158] and it is related to polymorph. Living organisms take advantage of the different stiffness of chitin polymorphs and during evolution developed strategies for creation of chitin-based biomaterials with sophisticated organization starting from molecular level through nano-, micro- and the macroscale [116]. This unique hierarchical relationship with intricate intimate architectures (for example Bouligand structure) serves many different functional needs for both soft and hard tissues of chitin- containing organisms [144]. Chitin in biomineralization phenomena acts as a structural substrate which is able to bind other macromolecules or ions and in turn

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induce their nucleation [14]. It has been reported that chitin in various invertebrates could be regarded as the substrate that binds other organic macromolecules (proteins) and form chitin-protein complex that in turn induce nucleation of the mineral phases

[14]. This phenomenon is very well described for mollusk shells especially for nacre, but β-chitin is the framework also for other macromolecular components that obviously promote the calcification process, even in the regime of crystal polymorphism [159–161]. The β-chitin fibrils and the protein polypeptide chains of the nucleating proteins are aligned respectively with the a and b crystallographic axes of the aragonite crystals [161]. The current model of mollusk shell nacre formation proposed by Levi-Kalisman et al. [160] is as follows: the matrix consists of sheets of

β-chitin that are surrounded by a silk-like protein gel, mixture of chitin–protein pre- fills the space to be mineralized, and chitin is the ordered structure that ultimately dictates the orientation of the mature crystals [14]. Biomineralized crustacean’s exoskeletons have evolved over millions years into a rigid, tough, and complex cuticles, that are served as structural support for protection of vital organs, and defense against predation [11], as well as preying tools [162]. Raabe et al. [116] indicated that in lobster cuticle chitin chains are arranged in an antiparallel fashion forming α-chitin, which in turn form nanofibrils assembled into honeycomb shaped arrays stacked along their normal direction of about 180° and referred to Bouligand layer. The three or four upper layers of this cuticle are biomineralized with CaCO3 precipitated into this

Bouligard structure [14]. Similar phenomenon has been found in dactyl club of mantis shrimp, however here the mineral phase is hydroxyapatite [120].

Chitin biomineralization phenomenon is not only restricted to calcium-based minerals. Löwenstam [148,149] proved that chiton teeth contain α-FeO(OH)

(goethite), Fe3O4 (magnetite) and γ-Fe2O3 (lepidocrocite). Recently published detailed

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research, dedicated to radula teeth, revealed that α-chitin is the main organic component of the material and is tightly associated with the iron-based mineral phase

[151,153]. These studies proved additionally that radula displays active mineralization process, whereas the degree of mineralization increasing from a completely non- mineralized structures consisting of α-chitin at the posterior end to a fully mineralized tooth at the anterior end, whereas dominant phases in radula teeth are α-chitin and magnetite (Fe3O4).

Discovery of chitin-based organic networks as integral part of cell wall biosilica in the diatom (Thalassiosira pseudonana) as well as a structural component in several glass sponges (Hexactinellida) [83,163] suggest that this biomacromolecule is a part of a very old template system involved in biosilicification phenomena. Systematic study on numerous representatives of the Aplysinidae family revealed that α-chitin is also involved in formation of multiphase, nanostructured silica-chitin-aragonite biocomposites [164]. This discovery prompted research on the phenomenon multiphase biomineralization in other organisms. Recently, Michels et al. [162] proved, for example, that Copepods teeth are multiphase composites of chitin, amorphous silica and Al or Zn containing silicates [165].

Utilization of chitin as a structural template in biomineralization-inspired experiments in vitro have been well studied. Manoli et al. [27] reported in vitro crystallization of calcite CaCO3 (calcite) on chitinous substrate. Performed investigations indicated that deposition of calcite on chitinous substrate, from supersaturated solutions is favored at pH 8.50 and the number of ions forming the critical nucleus was found to be n* = 3. By kinetic analysis, the growth order was found to be 2, typical for processes controlled surface diffusion [27]. The negative charge in

–N-C=O groups is shifted towards the oxygen atom, thus the nucleation and formation

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of calcite probably is initiated through the interaction of Ca2+ ions with the end of the

C=O bond and local supersaturation [27]. Falini and co-workers [166] studied preparation of biomimetic composites of β-chitin with CaCO3 polymorphs, by precipitation of the mineral into a chitin scaffold by use of a double diffusion system.

Authors proved that it is possible to obtain a composite material made of β-chitin and calcium carbonate, and that the chitin matrix governs the location and polymorphism of the formed mineral. Additionally, they proved that the presence Mg2+ ions affect formation of aragonite, without presence of magnesium ions formation of calcite occurs more likely. Correspondingly, Ma and Feng [167] have recently published study dedicated to the role of magnesium ions and β-chitin in formation of calcium carbonate.

Authors indicated that Mg2+ ions, as co-solutes of calcium, have higher hydration energy [168], and dehydration of the magnesium ions prior to incorporation in the calcite lattice creates a barrier to the growth of calcite nuclei. This explains why aragonite crystals can be obtained more easily in the presence of sufficient magnesium.

It is worth noting that magnesium ions induce the formation of aragonite and are not incorporated into crystalline lattice [168]. Munro and McGrath [169] developed biomimetic approach to form chitin-aragonite composites using Kitano method. Their study revealed that addition of the high charge density acid-rich additives, in this case poly(acrylic acid), to the mineralization solutions favorite formation of aragonite phase. Utilization of chitin as a structural template for mineral formation is not only limited to calcium carbonates.

Exploitation of chitin as a structural template for biomimetic mineral formation is not only limited to calcium carbonates. Wan et al. (1998) reported successful preparation of a chitin–apatite composite by in situ precipitation onto porous chitinous scaffolds. The mechanism of chitin mineralization with respect to calcium phosphate

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may be explained as follows: the reaction is based on binding ability of –OH groups present in chitin molecule, at C6 and C3 positions. Hydroxyls can interact with both

Ca2+ and PO43- in a loose fashion, increasing the local degree of supersaturation in the vicinity of the chitin surface and promote nucleation and crystal growth. Falini et al.

[170] prepared β-chitin composites with octacalcium phosphate in vitro by double membrane system. A strong relationship between the orientation of β-chitin chains and octacalcium phosphate crystals has been documented. Additionally, it was also noted that the crystal orientations strongly depend on the nucleation point inside the membrane. Moreover, the deposition of (001) blades of octacalcium phosphate crystals, inside the chitin matrix, induces a reorganization of the β-chitin chains to a more distinct layered structure. Oriented growth can be a result of both: (i) mechanical factors as well (ii) epitaxial crystal growth.

Retuert et al. [171] proposed a method for synthesis of chitin-based polymeric hybrid materials by mixing of prehydrolyzed tetraethyl orthosilicate with a partially deacetylated chitin. The resulting hybrid materials were homogeneous and transparent glassy. Basing on FTIR analysis authors suggested the possible mechanism of interactions based on hydrogen bonding between the functional groups of the partially deacetylated chitin and the silanol groups. Ogasawara et al. [172], chosen β- chitin scaffold isolated from Sepia officinalis cuttlebone, as a template for biomimetic silicification. In contrast to Retuert et al. [171], silicification of chitinous scaffold proposed by Ogasawara et al. [172] has been performed with use of the sodium silicate solutions as a source of silicic acid. This choice is easily explainable, sodium silicate correspond to the usual form of soluble silica found in nature [173]. Authors indicated that silicate ions and silica oligomers preferentially interact with glucopyranose rings exposed at the β-chitin surface, presumably through polar and H-bonding interactions.

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Correspondingly to Ogasawara et al. [172] report, recently, Spinde et al. [174] successfully silicified β-chitin, under biologically relevant conditions (pH 5.5, 20 °C), using sodium silicate as the precursor compound. Application of advanced instrumental methods (i.e. 13C and 29Si NMR, FTIR) confirmed previous assumptions that silica interacts with chitin via hydrogen bonds between silanols and –OH groups located at C6 carbon position in chitin molecules. Additionally, by application of the molybdenum blue test it has been confirmed that this weak interaction does not result in an acceleration of the silica polycondensation process.

To sum up chitin can be served as a superior scaffolding architecture for bioinspired mineralization studies in order to develop of new class of chitin-based materials. However, all above-mentioned methods have been performed at ambient conditions with temperature not exceeding 37 °C. This is because large community of scientist has been convinced that temperatures below 40 °C are the most proper from biological point of view. However, almost all representatives of hydrothermal vent fauna, where biomineralization processes has been detected, contain in their skeletal structures chitin or its monomer N-acetylglucosamine [2]. This strongly suggests that chitin can be used as a template for Extreme Biomimetic experiments.

2.2.5 THERMAL STABILITY OF CHITIN AS A KEY TO EXTREME BIOMIMETICS

The main factor, which decides about application of chitin in Extreme

Biomimetics, is thermal stability of this biopolymer. Analysis of the literature revealed a few basic methods that have been widely used for monitoring thermal degradation of chitin and its derivatives. First studies related to chitin thermal stability were performed by Köll et al. [175,176]. These Authors proved by thermogravimetry, that the degradation of chitin with an acetylation degree equal 62% started at 200 °C, and

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it is a two-step process. Application of on-line mass spectrometry by these authors revealed that formation of volatile acetamide compounds is the first step of chitin thermal degradation. In contrast, Wanjun and co-authors [177] by FTIR analysis of chitin before and after thermal degradation, concluded that decomposition of chitin starts from the scission of the glycosidic bond in the biopolymer backbone, and then a subsequent depolymerization to monomers is followed by dehydratation of the pyranose ring and deacetylation. Nam et al. [178] performed comparative study of the thermal decomposition of chitin and its main derivative – chitosan. They proved with strong evidence that thermal decomposition of the chitin nanofibers occurred in a single step reaction, whereas the chitosan nanofibers with a deacetylation degree higher than 50%, showed a two-step degradation mechanism. Additionally, this study showed that the maximal decomposition temperature of the chitin nanofibers is higher than that of the chitosan nanofibers. Higher thermal stability of chitin is a result of strong inter- and intramolecular hydrogen bonds within the chitin, related with crystalline structure and acetylation of nitrogen at C2 position. Stawski et al. [42] confirmed that thermal stability of chitin is associated with crystallinity. Additionally, these authors indicated that the monoclinic β-chitin obtained from squid is thermally much less stable than the orthorhombic α-chitins originating from krill, crab and shrimp. These differences are easily explained by the different crystal structures, and the different orientation of chains, which were described above (subchapter 2.2.2).

Strong intra- and intermolecular hydrogen bonds in chitin polymorphs have positive influence on stability of chitin in hydrothermal solutions. Sakanishi et al. [179] performed detailed studies of chitin stability in hydrothermal solution. The hydrothermal decomposition of chitin was carried out using a stainless steel tube reactor at 300-400 °C for 30-120 s under pressures of 15-30 MPa. Authors proved that

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cellulose undergoes complete (100%) degradation at 350 °C, whereas observed degradation of chitin at this temperature was only 32%. Moreover, hydrothermal degradation of chitin at 400 °C was just slightly higher and equal 45.8%. The mechanism of degradation is strongly associated with both: (i) changes of solid properties of chitin and (ii) changes of physicochemical properties of water in function of temperature. Structure of water changes significantly near the critical point because of the breakage of infinite network of hydrogen bonds, and water exists as separate clusters with a chain structure [180]. In fact, the dielectric constant of water decreases considerably near the critical point, which causes a change in the dynamic viscosity and also increases the self-diffusion coefficient of water. These changes of water near the supercritical conditions provide a completely different homogeneous reaction environment. Hot compressed water acts as both reactant and reaction medium. Thus hot compressed water [181] usually leads to decomposition reactions such as hydrolysis, deacetylation and dehydration. However, it has been also confirmed by

Nam et al. [181] that high crystallinity of chitin protect this biopolymer from degradation and dissolution in compressed hot water up to 200 °C. Recently, Deguchi et al. [182] performed in situ microscopic observations of chitin behaviours in hydrothermal water revealed that for a complete chitin dissolution temperature of

390 °C is needed [182]. It can be concluded that high crystallinity of chitin is one of, but not the only, beneficial property to graft principles of biomineralization processes found in hydrothermal vents [61,183,184], into modern materials science, according to Extreme Biomimetic concept [2].

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2.3 HYDROTHERMAL TECHNOLOGY ON SERVICE OF EXTREME BIOMIMETICS

2.3.1 HISTORICAL LANDMARKS OF HYDROTHERMAL TECHNOLOGY

2.3.2 FUNDAMENTALS OF HYDROTHERMAL TECHNOLOGY

2.3.1 HISTORICAL LANDMARKS OF HYDROTHERMAL TECHNOLOGY

There is number of examples that mimicking of the processes found in Nature fascinates broad community of scientists, and very often leads to discovery of the new materials and technologies. Sir Roderick Murchison (1792-1887) has made excellent example of this. British geologist in 1840 [185,186], studied the role of water at elevated temperature and pressure in the genesis of rocks and minerals in Earth crust, by mimicking of the natural conditions (high temperature and pressure) for crystallization of artificial minerals in the laboratory scale. Therefore, the term hydrothermal is of geological origin [186,187]. The first reactions following the principles of the hydrothermal technology were carried out in Germany. In 1845,

German chemist Schafthaul synthesized small quartz crystals using Papin’s digestor

[185]. In 1848, Robert Wilhelm Bunsen used aqueous reaction media for synthesis of barium carbonate and strontium carbonate in closed, thick-walled glass ampoules at temperatures above 200 °C and pressures higher than 100 bars [188]. However, the introduction of modern hydrothermal synthesis into the geological sciences is ascribed to H. De Senarmont (1851), which is known today as the founder of hydrothermal synthesis in geoscience [186,187,189]. De Senarmont developed hydrothermal method for synthesis of six-sided quartz prism by using sealed glass ampoules as reaction vessels placing them in autoclaves (to avoid explosion due to high pressure in the ampoule) and heated up to 200-300 °C. De Senarmont synthesized in this way a

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number of oxides, carbonates, fluorides, sulfates, and sulfide minerals [187]. This breakthrough encouraged geochemists and mineralogists, especially from Germany,

Italy, France, Japan and USA to synthesize various other minerals. It is worth noting that majority of the early hydrothermal studies, carried out during the 1840s to early

1900s, mainly dealt with the nanocrystalline products. However, these experiments were discarded as failures by scientist, mainly due to the lack of sophisticated electron microscopic techniques available during that time to observe materials of such small- size [186]. Major development of hydrothermal technology with regard to synthesis of minerals as well as construction of reactors was forced by World War II because quartz crystals with high purity were essential for construction of military radio communication devices [188]. The development of autoclaves and other novel lining materials capable of withstanding high pressure and temperature, combined with understanding of the physical chemistry of hot compressed water, made this technique a powerful tool for synthesis of advanced crystalline materials, which allowed for control and manipulation of crystal growth [190,191]. Outstanding progress in hydrothermal synthesis has been achieved and published by Brarrer, in series of more than 20 papers, who utilized this technique to controlled synthesis of various zeolites.

Together with R. M. Milton, Brarrer is called the founding father of hydrothermal synthesis of zeolites [192]. Nowadays, these technique have widely been applied in the synthesis of nanomaterials (including inorganic oxides [193], zeolites [192,194], hybrid materials [195]). Moreover, more and more evidence supports the hydrothermal origin of life [196]. Therefore, the novel challenge of hydrothermal technology is to apply previously gathered knowledge into non-enzymatic hydrothermal synthesis from inorganic simple compounds to complex organic biological species [195,197]. It will be helpful for better understanding of life evolution

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but also it will have interesting contribution into modern materials science and industry. According to previously described thermal stability of chitin, hydrothermal technology opens a new way for development of novel scientific direction called

Extreme Biomimetic for bioinspired synthesis of various biocomposites.

2.3.2 FUNDAMENTALS OF HYDROTHERMAL TECHNOLOGY

Hydrothermal processes are one of the most environmentally significant methods which allow for synthesis of crystalline inorganic materials without calcination step, which is beneficial from economical point of view as well it is in line with green chemistry [195]. Additionally, solvothermal reactions are considered to be advantageous due to several benefits including high reaction yields (mostly approaching 100%), not complicated experimental setup, low costs of reagents and precise control of synthesis with respect to morphology of obtained crystalline materials [187,188,198]. Definition of solvothermal synthesis underwent evolution but finally scientist agreed to defined it as: “any heterogeneous or homogeneous chemical reaction performed in the presence of a solution above room temperature and at pressure greater than 1 atm in a closed system” [198–201]. It should be stated that hydrothermal reaction is the specific, but very common case where water is used as a solvent; generally, this technique is referred to as a solvothermal reaction. General rule of hydrothermal technique is heating hermetically closed vessels using water as a solvent so that the autogenous pressure exceeds the ambient pressure and in consequence it allows to bring the solvents to temperatures above their boiling points

[202]. It is used to increase reaction velocities, dissolve and recrystallize substances.

From this point of view, it can be easily seen that properties of water, which depend on temperature, are the key factors in hydrothermal synthesis. Therefore, it is necessary

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to understand basic principles behind the thermodynamic properties of water, which are in strong relationship with pressure and temperature.

Pressure-volume-temperature relations of water and aqueous solutions as well as physicochemical properties of water in the temperatures and pressures required for hydrothermal synthesis are well studied and widely described in numerous articles

[203–205] and are presented on Figure 5 and 5 b, respectively. For experimental hydrothermal synthesis the PT diagram of water is very important (Figure 5 a). The pressure prevailing under working conditions is determined by the filling degree of autoclave with the reaction mixture. With a fill degree above 32% fluid-gas meniscus is curved upwards and the reaction vessel is completely filled with the fluid phase below the critical temperature of 374 °C. The higher percentage of filling, the lower temperature is needed to obtain autoclave completely filled with liquid. The filling degree is correlated with density as function of temperature (Figure 5 b, black dotted line). It can be easily explained with an example of autoclave with reaction volume of 1 dm3 filled with 0.8 kg of water. When reactor is heated up to 245 °C the density decrease to achieve 0.8 kg/dm3 that means that water sealed inside of the reactor will have volume of 1 dm3, thus reaction vessel will be completely filled with liquid phase.

Further heating will increase the pressure [188]. It should be kept in mind that Figure

5 specify the properties of pure water. Any additional components like precursors and mineralizer will have impact on solution and in consequence they will shift the temperatures needed to obtain autoclave completely filled with liquid [206].

The ionization constant of water (Kw) is a benchmark property in aqueous solution chemistry and Kw has been experimentally obtained over wide ranges of

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temperatures and pressures. The ionization constant of water is attributed to the following reaction:

+ − 2 퐻2푂 ↔ 퐻3푂 + 푂퐻 (1)

The ion product (Kw) for high-temperature liquid (close to supercritical) water is about

3 orders of magnitude higher than that for ambient liquid water [207]. Consequently, water at these conditions will have concentrations of H+ and OH- ions that are naturally higher than in ambient liquid water [207].

Figure 5. Pressure–temperature dependence of water for different filling degrees of the autoclave during hydrothermal synthesis (a). Density, dielectric constant and ionic product, Kw of water at 30 MPa as a function of temperature (b).

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The dielectric constant of water (Figure 5b, red line) drops drastically as water

is heated and approaches that of non-polar solvents around supercritical conditions

[206]. In consequence, the solubility of nonpolar species increases, whereas that of

ionic and polar compounds decreases [207].

As it has been already reported the great benefit of hydrothermal syntheses is

possibility to obtain crystalline inorganic structures at mild conditions up to 300 °C. In

Table 1 several examples of various inorganic products that can be obtained using

hydrothermal technology have been presented

Table 1 Examples of various inorganic oxides synthesized under hydrothermal conditions at temperatures not exceeding 300 °C.

STARTING COMPOUND MINERALIZER CONDITIONS MORPHOLOGY REF COMPOUNDS

72 h; NaOH + Stöber silica 200 °C; [208] NaCl

SiO2 (quartz)

1.5 h; Fumed silica NaOH [209] 300 °C

Titanium 4 h; TiO2 HCl [210] isopropoxide 150 °C

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Titanium 4 h; HCl [210] isopropoxide 150 °C

TiO2

12 h; H/K titanate HNO3 [211] 180 °C

NaOH

2 h; FeOOH FeCl3·6H2O [23] 200 °C

NaOH + EDTA

HCOOH 6 h; 90 °C

Cu2O Cu(NO3)2 [212]

HCOOH + 6 h; 90 °C NH3OH

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6 h; Cu(CH3COO)2 Urea [213] 90 °C

CuO Urea 12 h; 150 °C

Cu(NO3)2 [214]

20 h; NaOH 120 °C

CeCl3 12 h; + - [215] acrylamide 140 °C

CeO2

12 h; Ce(NO3) Na3PO4 [216] 170 °C

GeO2 24 h; CuGeO3 - [217] + Cu(CH3COO)2 120 °C

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FeSO4

30 h; Fe2O3 NaOH [218] 180 °C

FeSO4 + CTAB

1.5 h; NaS2 [219] 135 °C

AgNO3 + Silver Ethylene glycol + PVP

2.5 h; FeCl3 [220] 160 °C

12 h; [221] 120 °C

ZnO Zn(CH3COO)2 NaOH

13 h; [222] 180 °C

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48 h; ZrO(NO3)2 [223] 200 °C

NaOH ZrO2 -

Zirconium 24 h; [224] n-propoxide 150 °C

Table 1 presents only small percent of various crystalline structures, which can be synthesized hydrothermally. But these examples clearly illustrate that hydrothermal technology offer an exceptional possibilities to control rate and uniformity of nucleation, growth and aging, which affects size, morphology and aggregation control of which can not be controlled so easily in other methods. Additional advantage of hydrothermal technology is that it can be hybridized with other processes like microwave, electrochemistry, ultrasound, mechano-chemistry, optical radiation and hot-pressing to gain advantages such as enhancement of reaction kinetics and increase ability to make new materials. All syntheses shown in Table 1 has been performed at temperatures whereas chitin is thermally stable. Thus it strongly syggest that application of chitin as a structural template in hydrothermal syntheses can pave the way for modern biomaterials science and biomimetics.

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CHAPTER 3 AIM OF THE WORK

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The main aim of the doctoral thesis was utilization of chitinous scaffolds of poriferan origin as structural templates in hydrothermal reactions in order to obtain novel chitin-based inorganic-organic materials.

The scope of the research included following stages:

1. Synthesis of chitin-silica materials by hydrothermal depositon of silica on

chitinous support. Detailed characterization of morphological, structural as well as

physicochemical properties of obtained materials with respect to determination of

possible mechanism of interactions between silica and chitin particles.

2. Synthesis of chitin-zirconia hybrid materials under hydrothermal conditions with

use of ammonium zirconium(IV) carbonate at 150 °C. Detailed characterization of

crystalline structure of inorganic phase deposited on chitinous substrate by use of

X-ray diffraction (XRD), high resolution transmision electron microscopy

(HRTEM) selected area electron diffraction (SAED) and fast Fourier

transformation (FFT) methods. Determination of interactions between chitin and

ZrO2 nanoparticles. Analysis of potential application of prepared materials in

waste water treatment processes.

3. Preparation of chitin-ZnO film like materials under hydrothermal conditions at

90 °C with comprehensive analysis of morphology as well as crystalline structure

of obtained materials, by using XRD, HRTEM, SAED and FFT analytical techniques.

Evaluation of antibacterial properties to determine the possibility of using this

materials as a potential wound dressing materials.

4. Hydrothermal deposition of iron oxide on chitinous supports at 90 °C. Evaluation

of influence of precursor concentration (iron(III) chloride) on morphological

properties, crystalline structure as well interactions between chitinous template

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and iron oxide nanoparticles. Analysis of electrochemical performance of

supercapacitors prepared with use of chitin-hematite as a filler of active carbon.

5. Hydrothermal synthesis of chitin-GeO2 nanomaterials at 185 °C with

comprehensive analysis of influence of chitin on morphology, crystal structure and

size of crystallites as well photoluminescent properties of growing germanium

dioxide nanophase.

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CHAPTER 4 MATERIALS AND METHODS

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4.1. ISOLATION OF THE α-CHITIN FROM MARINE SPONGES

4.2. HYDROTHERMAL SILICIFICATION OF SELECTED CHITINOUS SCAFFOLDS

4.3. FORMATION OF ZIRCONIUM DIOXIDE NANOPHASE USING CHITINOUS SCAFFOLDS UNDER HYDROTHERMAL CONDITIONS

4.4. HYDROTHERMAL SYNTHESIS OF β-CHITIN/ZnO NANOSTRUCTURED COMPOSITES

4.5. HYDROTHERMAL SYNTHESIS OF α-CHITIN/Fe2O3 NANOSTRUCTURED COMPOSITES

4.6. HYDROTHERMAL SYNTHESIS OF CHITIN/GeO2 NANOSTRUCTURED COMPOSITES

4.7. CHARACTERIZATION TECHNIQUES

4.1. ISOLATION OF THE α-CHITIN FROM MARINE SPONGES

The marine sponges Aplysina aerophoba (Aplysinidae: Verongida: Demospongiae:

Porifera) were collected in the Adriatic Sea (Kotor Bay, Montenegro) in August 2008 by

SCUBA diving. Due to the regeneration capacity of A. aerophoba, divers only cut the apical parts of the sponge body. The remained basic parts intact and allow the sponge to regenerate under natural conditions. Sponge samples were put in ziplock bags underwater, brought back to the laboratory at Institute of Marine Biology (Kotor,

Montenegro) and frozen less than 1 h after collection. The sponge fragments as collected were prepared, lyophilized and transported by the INTIB GmbH (Germany) to the

Laboratory of Biomineralogy & Extreme Biomimetics Group, TU Bergakademie Freiberg

(Germany). In brief, isolation was performed in three basic steps: (i) removal of water soluble salts and impurities by washing with distilled water; (ii) removal of proteins and residual pigments by treatment with 2.5M NaOH solution; (iii) removal of calcium and magnesium carbonates by treatment with 20% acetic acid. The isolation procedure was repeated few times to obtain colorless, tubular chitin scaffolds (Figure 6). These scaffolds were stored in glass bottles with ultra-pure water at 4 °C.

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Corresponding procedure of isolation of chitinous scaffolds has been performed for Ianthella basta, Aplysina cauliformis and Aplysina fistularis. Schematic view on chitin scaffold isolation is presented in Figure 6.

Figure 6. Step-by-step scheme of chitin-based scaffolds isolation procedure from the skeleton of marine demosponge I. basta [225].

4.2. HYDROTHERMAL SILICIFICATION OF SELECTED CHITINOUS SCAFFOLDS

Chitin–silica composite was synthesized in two main steps. At first stage, appropriate silica dispersions were synthesized independently by a modified Stöber method [226,227]. In brief, silica dispersions were synthesized by heating of appropriate volume of ethanol solution (anhydrous, 99.8% Sigma-Aldrich, Germany) to a desired

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temperature. To the heated solution tetraethyl orthosilicate (TEOS) (reagent grade, 98%

Sigma-Aldrich, Germany) and aqueous ammonia solution (reagent grade, 25% Chempur

SA, Poland) were quickly added upon vigorous stirring. Dispersion of micrometric silica was achieved at room temperature, by mixing ethanol, ammonia and TEOS at 1.7:0.1:0.03 molar ratio respectively, for 1 h. Nanosilica dispersion was prepared at 37 °C by mixing the same reagents at the molar ratio 5:0.095:0.048 for 20 h. To determine the size of the silica particles Zetasizer Nano-ZS (Malvern Instruments Ltd. USA) was used. Particles size distribution of applied micro- and nanosilica dispersions are presented in Figure 7a and b, respectively. In the second step, the earlier isolated I. basta chitin scaffolds were separately incubated in a dispersion of micro- or nanosized silica. This process was carried out in a closed glass ampoule (10 cm3, SCHOTT, Germany) that was incubated at

120 °C (during 24 h at Memmert Incubator, Germany). After that chitin–silica composite was isolated, washed with ethanol (analytical grade, 99.9% VWR, Germany) and then with distilled water and finally dried at 105 °C during 12 h.

Figure 7. Particle size distributions of prepared micrometric (a) and nanometric (b) silica dispersions.

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4.3. FORMATION OF ZIRCONIUM DIOXIDE NANOPHASE USING CHITINOUS SCAFFOLDS UNDER HYDROTHERMAL CONDITIONS

At first fragments of three-dimensional chitinous matrix, isolated from A. cauliformis and A. aerophoba were inserted into 3 cm3 of ammonium zirconium(IV) carbonate solution (Sigma-Aldrich, Germany) in a glass ampoule (10 cm3, Schott). The structure of ammonium zirconium(IV) carbonate is presented in Figure 8. The glass ampoule with the biopolymer matrix in solution was melted on the edge. For the next step, the ampoule was heated at 150 °C for 48 h in a thermostat (Nabertherm, Germany). White crystals precipitated during the hydrothermal reaction. Finally, the ampoule was carefully opened and the chitinous matrix, now covered by nano- and microcrystals, was isolated.

Isolated scaffolds were washed subsequently with ethanol and distilled water and dried at 105 °C for 24 h.

The same reaction was carried out using quartz wool fibers of 10 µm in diameter

(K991571738, Merck, Germany) as a reference substrate, as well as in glass ampoules without any kind of matrices.

Figure 8. Chemical formula of the ammonim zirconium(IV) carbonate.

4.4. HYDROTHERMAL SYNTHESIS OF β-CHITIN/ZnO NANOSTRUCTURED COMPOSITES

In a synthesis experiment, 20 cm3 of (0.0625M) Zn(CH3COO)2 (Sigma-Aldrich,

Germany) solution was added to 20 cm3 of NaOH (1M) to form a transparent solution.

Afterwards, the β-chitin membrane was added to the reaction mixture at pH 14.0. The total reaction system was transferred into the Teflon®-lined vessel of the hydrothermal

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reactor (Parr, USA) and heated to 70 °C for 5 h. After this time the chitin template covered with ZnO crystals was carefully isolated, washed with distilled water (up to pH 6.8) and dried at 70 °C over 48 h (Memmert incubator, Germany). As a control, ZnO particles were also prepared within the same reaction system without the presence of any chitin template.

4.5. HYDROTHERMAL SYNTHESIS OF α-CHITIN/Fe2O3 NANOSTRUCTURED COMPOSITES

In a typical experiment, hydrothermal deposition of Fe2O3 on a chitinous template was performed by the forced hydrolysis of iron(III) chloride (Sigma-Aldrich, Germany).

In brief, a measured amount of anhydrous FeCl3 was dissolved in 10 cm3 of ultra-pure water to achieve a desired concentration. Afterwards the solution was added to a mixture containing 90 cm3 of ultra-pure water and 0.75 cm3 of 1M HCl. In the next step, sponge chitin fragment (1.0 x 0.5 cm) was added to the solution and the whole volume was transferred into a Teflon-lined® vessel (200 cm3) of the hydrothermal reactor (Parr, USA), and heated to 90 °C for 48 h. After this time, the chitin template covered with Fe2O3 nanocrystals was carefully isolated, washed with distilled water in an ultrasound bath

(Elmasonic GmbH, Germany) for 20 min, brought up to pH 6.8 and dried at 90 °C for 48 h

(Memmert incubator, Germany). As a control, Fe2O3 particles were also prepared within the same reaction system without the presence of any chitin templates.

4.6. HYDROTHERMAL SYNTHESIS OF CHITIN/GeO2 NANOSTRUCTURED COMPOSITES

For the synthesis, a piece of chitinous scaffold (0.5 cm × 0.5 cm) and 1 cm3 tetraethoxygermanium(IV) – TEOG (Sigma-Aldrich, 99.95%) were added with rapid stirring to 10 cm3 of a water/ethanol solution (70% water) with an appropriate ammonium hydroxide concentration. Stirring was continued for 15 min, after which the

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solution was transferred to a Teflon-lined® hydrothermal reactor (Hydrion Scientific

Instruments, USA), closed, and heated up to 185 °C. After 24 h, the reactor was cooled down and opened; chitin–GeO2 composite was carefully isolated and washed three times with anhydrous ethanol. To remove all unbound particles of germanium oxide, the composite was washed with anhydrous ethanol in an ultrasound bath (Elmasonic GmbH,

Germany) for one hour. Subsequently, it was dried in a vacuum oven at 110 °C for 12 h, and cooled at room temperature in a desiccator. As a control, GeO2 crystals were also prepared within the same reaction system without the presence of any chitin templates.

4.7. CHARACTERIZATION TECHNIQUES

4.7.1. FOURIER-TRANSFORM INFRARED SPECTROSCOPY

Infrared spectra were recorded on a Nicolet 380 Fourier-transform infrared spectrometer (USA). Approximately 3 mg of the sample was mixed with 400 mg KBr and compressed. We recorded 100 scans at a spectral resolution of 2 cm–1. All spectra were baseline corrected with a two-point linear baseline (at 845 and 1890 cm–1).

4.7.2. RAMAN SPECTROSCOPY

Raman spectra were recorded using a Raman spectrometer (RamanRxn1™, Kaiser

Optical Systems Inc., Ann Arbor, USA) coupled to a light microscope (DM2500 P, Leica

Microsystems GmbH, Wetzlar, Germany). The excitation of Raman scattering was obtained with a diode laser emitting at a wavelength of 785 nm, propagated to the microscope with a 100 µm optical fibre and focused on the samples by means of a

50x/0.75 microscope objective, leading to a focal spot of about 20 µm. The Raman signal was collected in reflection configuration and sent to the f/1.8 holographic imaging spectrograph by using a 62.5 µm core optical fibre. The spectral resolution in the range

150-3250 cm-1 was 4 cm-1. Raman spectra were recorded using an integration time of 1 s

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and averaging 50 spectra in order to improve the signal-to-noise ratio. The laser power measured in the focal spot was 15 mW. The spectra were analyzed with MATLAB toolboxes (MathWorks Inc., Natick, USA). In order to eliminate the background due to the fluorescence, a variable baseline was calculated for each spectrum applying the function

“msbackadj” within multiple windows of 200 cm-1 width shifted with a 100 cm-1 step; a linear interpolation method was chosen. Four spectra were acquired in different positions of the samples to account for the small composition of inhomogenities; the averages were taken as representative spectra of each sample type.

4.7.3. POWDER X-RAY DIFFRACTION (XRD)

XRD measurements were performed using a Bruker D8 Advance diffractometer equipped with a Cu X-ray tube, Goebel mirror, and silicon strip detector. Samples were prepared on a glass plate without adding further substances. Measurements used a parallel beam with equal incident and emergent angles within the ranges indicated in the figures. Compositions and crystallite sizes were determined through Rietveld-refinement using the program Bruker Topas 4.2.

4.7.4. X-RAY PHOTOELECTRON SPECTROSCOPY

XPS analyses were performed using a ESCALAB 250Xi from Thermo Scientific

(USA), with a monochromatic Al Ka X-ray source (1486.6 eV). The X-ray source has a spot size of 650 mm and operates at a power of 14.8 kV and 19.2 mA. The spectra were taken with a pass energy of 20 eV and an energy step width of 0.1 eV. The base pressure was

2 X 10-10 mbar, but during the measurement the pressure increased to 3 X 10-7 mbar due to the ion gas flow from the flood gun, which was used for charge compensation.

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4.7.5. TRANSMISSION ELECTRON MICROSCOPY

For high-resolution micrographs, thicknesses even below 10 nm are necessary.

Preparation of samples for TEM analysis is crucial to obtain high-quality data.

Heterogeneous etching of different phases, surface contamination, radiation damage, and structural and chemical changes during the preparation process are often challenges requiring extended testing and adjustments for sample preparation [228]. Therefore, sophisticated method of samples preparation for TEM studies was developed. Fragments of prepared hybrid inorganic-organic chitin-based materials were disrupted mechanically using liquid nitrogen and an agate mortar to obtain nanosized powder, this powder was suspended in ethanol. Afterwards, drop of the as prepared suspension was deposited on the carbon film of an electron microscopy grid (Plano GmbH, Wetzlar, Germany), followed by drying in air.

The recording of a selected area for the electron diffraction pattern (SAED),

HRTEM investigations, and energy dispersive X-ray (EDX) analysis, were performed on a

TEM JEM-2200FS (JEOL, Japan) at an acceleration voltage of 200 kV by using an ultra- high-resolution objective lens, Cs corrected illumination system, and an in-column filter.

Application of the aberration corrector makes a revolutionizing the performance of HR-

TEM/STEM instruments, allowing one to achieve a spatial resolution better than 0.8 Å and an energy resolution better than 0.2 eV, thereby making the characterization of the local structure at sub-atomic scale available [229,230].

The SAED and HRTEM images were taken using a 2K × 2K CCD camera of Gatan Inc

(USA). The orientation of local sample regions and the phase composition were determined through the analysis of interplanar distances and angles by taking into account the diffraction spots on SAED or frequencies of local Fast Fourier Transformation

(FFT). For the evaluation of HRTEM images, the simulation of atomic arrangement inside

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of inorganic crystallites with successional simulation of HRTEM contrast was performed using the software packages in CrystalMaker and JEMS.

4.7.6. SCANNING ELECTRON MICROSCOPY

The scanning electron micrographs were observed using a FEI Helios NanoLab

600i DUALBeam FIB/SEM equipped with a Schottky field emission gun. Besides the usual

Everhart Thornley Detector (ETD) an in-lens “through-the-lens” detector (TLD) served mainly for the detection of the secondary electrons (SE) at a working distance (WD) of 4.0 mm. Before testing, the samples were fixed in a sample holder and covered with Au for 45 s using an Edwards S150B sputter coater.

4.7.7. FLUORESCENCE AND LIGHT MICROSCOPY

Samples of prepared pure reference samples and chitinous matrix prior to and after hydrothermal syntheses were observed using Keyence BZ-9000 (Japan) microscope in light as well in fluorescence microscopy modus.

4.7.8. THERMAL ANALYSIS

Thermogravimetric (TG) and Differential Thermal Analyses (DTA) of synthesized chitin-based materials were carried out with a Jupiter STA 449F3 (Netzsch, Germany) analyzer using an Al2O3 crucible. Measurements were performed in oxidative atmosphere at the heating rate of 10 °C min-1. The samples were heated to 1000 °C, starting from

25 °C.

4.7.9. NEAR EDGE X-RAY ABSORPTION FINE STRUCTURE

NEXAFS measurements were performed at the Helmholtz-Zentrum Berlin für

Materialien und Energie (HZB), the electron storage ring BESSY II, using the facilities of

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the Russian-German beamline [231]. NEXAFS spectra were recorded in a total-electron yield mode and normalized to the incident photon flux.

4.7.10. ELECTROCHEMICAL MEASUREMENTS

Electrochemical characterization was performed in a two electrode Swagelok® system. The composition of pellets was: 85 wt% of active materials, 10 wt% of polyvinylidene fluoride (PVDF Kynar Flex 2801) and 5 wt% of acetylene black. The active materials used were commercially available activated carbon Norit® DLC Supra 30 (S30) with a surface area of 1588 m2/g, synthesized chitin–Fe2O3 hybrid material (Ch–H) and a mixture of 80% activated carbon Norit® DLC Supra 30 with 20% of the chitin–Fe2O3 hybrid material (Ch–H (20%) + S30 (80%)). The masses of the electrodes were in the range of 7–9 mg and a geometric surface area of one electrode was 0.8 cm2. As an electrolyte, 6M KOH was used. The capacitance properties of the materials (expressed per mass of one electrode) were estimated by galvanostatic charge/discharge (100 mA/g –

1000 mA/g), cycling voltammetry (CV; 1–100 mV s-1) and electrochemical impedance spectroscopy (100 kHz to 1 mHz) using VSP Biologic, France.

4.7.11. PHOTOLUMINESCENCE MEASUREMENTS

Photoluminescence (PL) spectra were excited using a HeCd laser emitting at 325 nm

(3.815 eV). A power of 10 µW was focused to a spot of 130 µm diameter on the samples.

Room-temperature luminescence was detected by a liquid-nitrogen cooled CCD camera

(SPEC-10:100BR_eXcelon, Princeton Instruments, USA) coupled to an Acton SP2560i monochromator using an exposure time of 100 ms. All PL spectra have been corrected by the spectral response of the detection system.

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4.7.12. ANTIBACTERIAL ACTIVITY

Antibacterial activity testing was carried out using an agar diffusion method with two bacterial species: Gram-negative Escherichia coli (ATCC 25923) and Gram-positive, endospore-forming Bacillus subtilis B9 (Collection of Department of Biotechnology and

Food Microbiology, Poznan University of Life Sciences). Microorganisms were grown in nutrient broth (OXOID CM 0001), containing 1 g/dm3 meat extract, 2 g/dm3 yeast extract,

5 g/dm3 peptone, and 5 g/dm3 sodium chloride with 15 g/dm3 agar addition. The pH of the solution was adjusted to 7.4 ±0.2. Quadratic pieces (6 x 6 mm) of the tested materials were placed on plates, previously inoculated using indicator microorganisms in the range of 106 CFU cm3/dm3. Then the plates were incubated at 37 °C for 48 h. The diameter of inhibitory zone surrounding pieces of the tested materials was then measured in mm

(after 24 and 48 h) [232].

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CHAPTER 5 STUDIES OF THE PHYSICOCHEMICAL PROPERTIES OF THE EXTREME BIOMIMETICALLY PREPARED CHITIN-SILICA MATERIALS

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INTRODUCTION

Recently, researchers discovered a chitin–silica composites which build unique biocomposite-containing skeletal structures in diatoms [39,40], glass sponges [83] as well as silica–chitin–aragonite composites in demosponges of the order Verongida [164]. In addition it has been proved that the presence of inorganic minerals in combination with chitin matrix ensures proper chemical stability, flexibility and mechanical strength of skeletons of the organisms studied which are fascinating for modern materials scientists. Silica is known as the most popular reinforcing agent in polymer blends but offers also very interesting properties such as ability to be easily modified [233] and bioactivity associated with highly reactive silanols and large specific surface area [234]. Combination of biodegradability of a biopolymer with bioactivity and reinforcing properties of silica permit preparation of advanced bioinspired chitin–silica hybrid materials designated for wide range of applications including waste-water treatment [235], catalysis [236], chromatography [237] and biomedicine [238]. In result, nowadays scientists developed several strategies of biomimetic synthesis of chitin-silica composites. Alonso and Belamie [239] reported preparation of bioinspired chitin–silica nanocomposite by self-assembly of α-chitin nanorods and silica colloids (obtained by acid catalyzed sol–gel methods). To the best of our knowledge, preparation of chitin–silica composites via a base-catalyzed Stöber method has not been reported previously. Additionally, all proposed methods associated with preparation of bioinspired chitin-silica materials were carried out at room temperature (not exceeding 40 °C). It is quite confusing, especially in accordance to numerous reports regarding vast range of thermophilic microorganisms as well as thermotolerant diatoms, which are involved in the biosilicification under extreme environmental conditions with respect to high temperatures (50-96 °C). Therefore, the purpose of this study was silicification of 2D chitinous matrix isolated from I. basta marine sponge skeletons under combined sol– gel/hydrothermal technique because of both, the Extreme Biomimetic concept [2] and the examination of highly structured sponge chitin as scaffold useful for hydrothermal synthesis.

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5.1. STRUCTURAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-SILICA MATERIALS

To analyze the micromorphology of the surface of I. basta chitinous scaffolds prior to and chitin-silica materials obtained after hydrothermal silicification procedure based on Stöber method, SEM microscopy was used. The size of silica particles in the Stöber dispersions strongly influenced the morphology of the obtained composites. The use of the colloidal silica of micrometric size leads to the homogeneous distribution of spherical

SiO2 particles on the surface of sponge chitin fibers (Figure 9 b). When the colloidal silica with nanometric size particles is used, the chitinous scaffolds are completely covered with the siliceous layer composed of spherical nanoparticles with particle size bellow 220 nm

(Figure 9 c,d). In contrast, the reported chitin–silica materials obtained by acid catalyzed sol–gel synthesis [236,239] usually are characterized by monolithic morphology.

Observed the homogeneity of silica layer on the surface of chitinous scaffold is a probably a result of compatibility of silanols in silica with functional –OH groups presented in chitin molecule. Intriguingly, for materials obtained with use of nanosilica dispersions, it was also observed that nanosilica infiltrated into the nanoorganized fibers of chitinous scaffold; this is marked by an arrows and can be easily seen in Figure 9 d. This is likely caused by intrafibrillar infiltration of primary silica into hierarchical organization of chitin fibers and thus immobilization of nanoparticles between chitin nanofibers can be easily observed. Corresponding phenomenon has been previously observed for sol-gel silicification of collagen [240,241].

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Figure 9. SEM microphotographs of the surface of isolated I. basta chitinous scaffold prior to in vitro silicification (a); 0.5 μm large silica particles (b) and Stöber silica nanophase tightly distributed on the surface of chitinous scaffold (c) clearly visible silicified nanofibrillar organization of the chitinous template (d)

5.2. PHYSICOCHEMICAL CHARACTERIZATION OF HYDROTHERMALLY PREPARED CHITIN- SILICA MATERIALS

It has been mentioned previously (chapter 2.2.5.) that thermal stability of the biopolymers is the crucial factor, specifying their potential in experiments following the

Extreme Biomimetics concept. Therefore, thermal behavior of chitin scaffolds isolated from I. basta, was determined by thermogravimetry. The TG curve (Figure 10) of pure

I. basta chitin (green line) indicates that total mass loss is 93.37% and the main degradation of polymer, associated with decomposition of acetylglucosamine units [80], occurs at temperature range 280–510 °C. Therefore, these scaffolds are suitable for proposed silicification experiments at 120 °C. Corresponding analysis of as-prepared chitin-silica composites revealed that silicification of chitin matrix increase its thermal stability. The slower degradation of obtained chitin–silica composites is associated with

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desorption of hydrogen-bound or coordinately bound water molecules and condensation of silanols as described previously for silica-based compounds [237,242,243]. Similar increase of thermal stability has been reported for silicified cellulose fibers [244].

Additionally, chitin–nanosilica composite was characterized by the lowest mass loss

(51.87%), which was a consequence of a higher degree of silicification related to a higher degree of I. basta chitin fiber covering with silica nanoparticles, as shown in the SEM microphotographs (Figure 9).

Figure 10. TG curves of pure I. basta chitin (green lines) in comparison with. I. basta chitin– microSiO2 (blue lines) and I. basta chitin–nanoSiO2 (red lines).

To evaluate the possible interactions between chitin and silica in formed materials analyses with use of FTIR, RAMAN and XPS spectroscopy were performed. Figure 11 represents the FTIR spectra of a chitin scaffold isolated from I. basta marine sponge and silicified chitinous scaffolds. Analysis of the spectrum of non-silicified sponge chitin

(brown line) indicates the presence of strong absorption peaks associated with the

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presence of typical for α-chitin splitting of amide band I corresponding to stretching vibrations of C=O bond (1660 cm−1, 1630 cm−1) characteristic of α-chitin. The splitting of amide band I stems from the occurrence of stretching vibrations of the C=O bond

(1660 cm−1) connected by a hydrogen bond with the N–H group of the neighboring chain and from the stretching vibrations of C=O group (1630 cm−1) bonded by hydrogen bonds with N–H and OH-6 groups in the same chitin chain [96,245,246]. The presence of absorption peak at 1554 cm−1 is attributed to the bending vibrations of amide II band (N–

H) and amide III bands between 1312–1200 cm−1. The vibrational absorption band at

1378 cm−1 was interpreted as the rocking of the methyl group. Glycoside linkage and pyranose rings are confirmed by the presence of 896 cm−1 and for ether bond in pyranose ring at 1159 cm−1. At both presented spectra of silicified samples, the strong silica bands suppress the chitin bands. Analysis of the FTIR spectra of silicified silica-chitin composite revealed the presence of two new absorption bands at 1103 cm−1 and 796 cm−1

(Figure 11). Observed intensive absorption bands in the spectra of silicified samples are assigned to asymmetric Si–O–Si bond stretching and symmetric Si–O–Si bond stretching vibrations. These bands are evidencing the presence of silica in obtained materials.

Additionally, the peak at 479 cm−1 corresponds to the deformation vibrations of Si-O-C bond that probably originates from partially unreacted tetraethoxysilane.

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Figure 11. FTIR spectra of isolated I. basta chitin scaffolds and chitin–silica composites obtained from different Stöber dispersions.

Similar observation has been made with use of the Raman spectroscopy

(Figure 12). The appearance of new bands in chitin-silica material has not been noted, however the intensity of the bands changed as a consequence of overlapping of the bands characteristic of chitin and silica between 1010 and 1040 cm−1. The results of both Raman and FTIR analyses confirm the effectiveness of the proposed two-stage method of silicification of chitin matrix.

Figure 12. Raman spectra of isolated I. basta chitin scaffolds and chitin–silica composites obtained from different Stöber dispersions.

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On the basis of the results and taking into account the mechanisms of silica interaction with α-chitin [163] as well as β-chitin proposed independently by Ogasawara et al. [172] and Spinde et al. [174], it can be concluded that silica preferentially interact by hydrogen bonding with glucopyranose rings as well as with carbonyl groups of chitin.

The X-ray photoelectron spectroscopy (XPS) of core levels was used to get additional insight into the chitin-silica interactions and characterize the state of silicon incorporated into chitin matrix. Si 2p photoelectron spectra of chitin/nano-SiO2 and chitin/micro-SiO2 materials taken at photon energy 200 eV are presented in Figure 13.

Figure 13. XPS spectra of prepared chitin–silica composites.

Both reveal similar structure with two well-separated features with only difference in relative intensities of these peaks. Feature at binding energy around 99.5 eV is spin-orbit doublet Si 2p3/2–Si 2p1/2, its energy position reflects the presence of zero-valent silicon

(Si0 form) [247]. In performed Si 2p PE spectra of chitin-silica composites the second feature is shifted to higher binding energies (107 eV) and reflects decreasing of electron

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density localized on Si atoms. However, chemical shift is much higher than the expected one for various silicon oxides. When going from Si2O to SiO2 species value of the shift rises from about 1 eV to 4 eV. Thus, these features may be concerned with presence of some unreacted precursor compound that quantity may vary from sample to sample. It seems probable that in electron transfer from Si atom could be higher in TEOS (consisting of four ethyl groups attached to (SiO4)4-) than that in pure SiO2. Therefore, it will lead to appearance of new components at higher binding energies. On the basis of the results presented in this work, the mechanism of interactions between α-chitin scaffold and silica micro- or nanoparticles within hydrothermally formed silica–chitin materials, is presented in Figure 14. The proposed model is based on hydrogen interactions between silica hydroxyls with OH, C=O and NH groups of chitin molecule, especially with those located at carbon C6.

Figure 14. Schematic view on possible mechanism of chitin–silica interactions, adapted from Wysokowski et al. [225].

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SUMMARY

Results presented above have proved that the thermostable, two-dimensional

α-chitin scaffolds isolated from skeletons of the marine sponge I. basta can be effectively silicified by the two-step method with the use of Stöber silica micro- and nanodispersions.

The control of silica particles growth at base-catalyzed hydrolysis is crucial to obtain composites with desired silicification rate in vitro. These preliminary results strongly confirm that chitin seems to be a biological material with unique physicochemical properties with respect to biomineralization in organisms, which occur in hot (diatoms from hot springs, annelids from vent communities) aquatic environments. This feature open the gate for attempts to develop novel composite-based materials under similar conditions in vitro using the principles of Extreme Biomimetics [23].

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CHAPTER 6 CHITIN AS A VERSATILE TEMPLATE FOR EXTREME BIOMIMETIC SYNTHESIS OF CHITIN-ZIRCONIA MATERIALS

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INTRODUCTION

The successful results of chitin silicification under hydrothermal conditions served as an encouragement to develop novel chitin based materials by deposition of ZrO2 nanoparticles on chitinous scaffolds. Zirconia or zirconium dioxide is a well-known polymorphic material that exhibits three phases: monoclinic (m-ZrO2), tetragonal (t-ZrO2) and cubic (c-ZrO2) [248]. The crystal structure has great impact on many unique properties of zirconia, and in turn on their potential applications. Zirconia, in all of the physical forms tested thus far, does not induce cytotoxicity in soft tissues and is considered as biocompatible material and is used in periodontal dentistry and dental implantology as well as materials for preparation of hip substitutes. Its exceptionally low thermal expansion coefficient, high thermal and chemical stability favor its utilization for the production of ceramic and insulating materials [249]. Thanks to its amphoteric character and redox properties, zirconium dioxide is used as a catalyst or a catalytic support in organic synthesis. Due to its ability to form complexes with amine and carboxylic groups present in the enzyme molecules, zirconia is used as a support for enzyme immobilization and production of biosensors

[250,251]. Recent studies conducted by independent research teams indicated ZrO2 as an important component of renewable electrodes used in electrochemical biosensing of DNA hybridization [252]. Moreover, the photoluminescent properties of monodisperse spherical particles of zirconia open the way to apply it in production of cheap, efficient and environmentally friendly luminescent materials [253]. The combination of all of the above-mentioned attractive properties of zirconia with biodegradability, non-toxicity as well exceptional morphology characteristic for chitin of poriferan origin was promising in perspective of development of novel material with unique properties attractive for practical uses in various branches of technology. It should be highlighted that combination of biopolymers from the renewable sources with inorganic materials is of huge importance in perspective of “green chemistry” and sustainable development. Utilization of Extreme Biomimetics, based on hydrothermal technology, fits directly into these two trends.

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6.1. STRUCTURAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-ZIRCONIA MATERIALS

Chitinous scaffolds isolated from sponges of Aplysina family as described previously, possess 3D arrangement of translucent, pipe-like fibers and characteristic hollow spaces between them (Figure 15 a). It is worth noting that these chitinous structures of poriferan origin are extremely sensitive to drying at room temperature, which results in collapsing of fibers and degradation of 3D morphology. Consequently, it has negative influence on their swelling behavior. Thus, to prevent degradation of 3D morphology, the chitinous scaffolds were squeezed between filter papers to remove excess of H2O. Afterwards, those samples were immediately soaked with ammonium zirconium(IV) carbonate (AZC) solution transferred into glass ampoule, and used for hydrothermal reaction at 150 °C. After 48 h, corresponding microcrystals were well visible on the surface of sponge chitin. Analysis of both light microscopy and SEM images of chitin scaffolds taken before (Figure 15 a, c and e) and after the hydrothermal reaction

(Figure 15 b, d and f) indicate that proposed hydrothermal method allows for efficient mineralization of three dimensional chitin isolated from A. cauliformis with respect to zirconium dioxide. The nanometric size (100–200 nm) of the zirconia particles deposited on the surface of the sponge chitin fibers is very well visible on the SEM images

(Figure 15 d and f).

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Figure 15. Light and SEM microscopy images of chitinous scaffolds of poriferan origin before (a, c, and e) and after hydrothermal mineralization (b, d, and f) [254].

Noteworthy is that the deposition of mineral phase occurs also within the chitin tubes, as can be seen on images taken with use of light microscopy (Figure 15 b). Intriguingly, the ultrasound-assisted washing procedure showed no influence on the location of the obtained spherical nanoparticles and confirms that growing inorganic nanophase is tightly bonded to the biopolymer surface.

The investigation of chitin–zirconia nanocomposites by use of diffraction methods of TEM — selected area electron diffraction (SAED) and HRTEM with FFT —showed that

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the chitin fibrils have an amorphous structure. Meanwhile, the grown inorganic nanoparticles consists of conglomerated crystallites with size about 100 nm (Figure 16).

Figure 16. TEM images of sponge chitin nanofibril with surface grown conglomerates of monoclinic zirconia nanocrystallites and corresponding indexed SAED pattern.

Detailed analysis of SAED pattern (figure 16, upper right corner) confirm the existence of monoclinic phase of zirconia by presence of characteristic reflections. To define the phase composition, the exact calibration of the diffraction pattern by using the diffraction pattern of Au standard deposited on the other half of the grid was performed. The obtained pattern undoubtedly confirms crystalline character of deposited zirconium dioxide and diffraction lines of monoclinic zirconia are strongly broadened because of the influence of crystallite size and strain. For the comprehensive description of zirconia nanocrystallites the evaluation of HRTEM images by the analysis of local FFTs that were calculated using the “DigitalMicrograph” software was performed. It was estimated that the differently oriented nanocrystallites (zone axes of individual crystallites shows

Figure 17 a) have a size in range of 4–8 nm. The whole HRTEM micrograph calculated from FFT (Figure 17 b) shows the blurred reflections, which form broken diffraction lines

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and originate from individual crystallites with low distribution statistics. The boundaries of the crystallite (CBs; extra marked in Figure 17 a) have a brighter HRTEM contrast than the internal lattice because of the presence of interface defects and accompanying strains.

Figure 17. Experimental HRTEM image of monoclinic zirconia consisting of nanocrystallites of different orientations (a) with corresponding FFT (b) and simulated HRTEM contrasts (c) of triple junction in zirconia using corresponding atomic model (d).

The HRTEM images are result of phase contrast and accordingly they describe the sample structure not directly. Thus, the simulation of HRTEM contrasts, especially close to interfaces is necessary. The software packages CrystalMaker and JEMS were employed for the simulation of the HRTEM contrast near triple junction in zirconium dioxide nanophase

(area A in Figure 17 a) that contains of three m-ZrO2 crystallites of 231, 211 and 112 — orientations of zone axes, were used (Figure 17 c). Atomic arrangement (AA) of super cell ((Figure 17 d) shows only cut out of AA) was generated by CrystalMaker and included approximately 70,000 atoms. By use of multi-slice method of JEMS the following parameters were used: defocus 43 nm, sample thickness 6 nm, Cs = 0.5, accelerating

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voltage 200 kV. These results confirm the bright contrast of CBs and their small thickness with interatomic distances fluctuating between 2 nm and 4 nm.

Obtained results are in line with the parallelly performed XRD measurements

(Figure 18), which indicate that deposited nanophase has crystalline character and the obtained spectra perfectly match the monoclinic-ZrO2 reference (according to 37-1484

JCPDS card). Application of Retvield refinement reveal that the size of crystallites is about

5.4 nm and it strongly corresponds with results obtained from TEM analysis.

Figure 18. XRD spectra of α-chitin and chitin–ZrO2 composites.

Results presented above are in line with the Kisi and Howard's theory [255] that hydrolysis and condensation of zirconium cations can also be stimulated under hydrothermal conditions, which means that zirconyl salt solution heated up to, or above,

100 °C will hydrolyze to crystalline monoclinic zirconia.

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6.2. PHYSICOCHEMICAL CHARACTERIZATION OF HYDROTHERMALLY PREPARED CHITIN- ZIRCONIA MATERIALS

To determine the interactions between chitin and zirconia the FTIR and Raman spectroscopy were applied. Major bands obtained with use of Raman spectroscopy

(Figure 19) strongly confirm that proposed hydrothermal synthesis method allows us to obtain chitinous composites characterized with the presence of crystalline monoclinic zirconia [256].

Figure 19. Raman spectra of monoclinic- and tetragonal-ZrO2 references as well as obtained chitin-

ZrO2 composite (a) and table with Raman wavenumbers of the bands in spectra of chitin-ZrO2 and references of m-ZrO2 as well t-ZrO2 samples (b).

In the FTIR spectra of obtained chitin-ZrO2 materials (Figure 20), the bands at positions: 729 and 512 cm–1, which are characteristic for monoclinic-ZrO2 can be distinguished and assigned to Zr–O–Zr, Zr–OH vibrations, respectively [257,258].

Additionally, the obtained spectra of chitin–ZrO2 materials reveals shifts of the stretching

NH band, from 3285 to 3288 cm–1; as well as deformations of I and II amide band, which perhaps are caused by formation of the hydrogen bonds between oxides and chitin.

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The shift of Zr–OH band from 508 to 512 cm–1 is probably also caused by hydrogen interactions between chitin and zirconia. Formation of any new bands including C-O-Zr

[259] has not been detected in the obtained spectra and this supports the theory of hydrogen bond interactions between chitin and zirconium dioxide.

Figure 20. FTIR spectra of chitin, m-ZrO2 standard and chitin–ZrO2 hybrid material: (a) overall view at range 4000–400 cm-1 and detailed view at range 200-400 cm-1

Ammonium zirconium(IV) carbonate can react with polymers containing hydroxyl groups by forming hydrogen bonds, such as starch and cellulose therefore it is a widely used compound in paper production [260] as a cross-linking agent or as an reinforcing additive in biopolymer films [261,262]. Taking into consideration this piece of information as well data from obtained measurements, the mechanism of reaction between chitin and AZC has been proposed. The reaction mechanism, depicted in

(Figure 21) suggests that AZC can react with hydroxyl groups of chitin by forming hydrogen bonds. However, the cross-linking reaction can also occur between AZC molecules. Similar reaction mechanisms have been previously suggested for cellulose and starch [263].

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Figure 21. Schematic view on the reaction mechanism of ammonium zirconium(IV) carbonate with chitin, adapted from [254].

6.3. PRELIMINARY RESEARCH ON APPLICATION OF CHITIN-ZIRCONIA COMPOSITE IN ADSORPTION OF HAZARDOUS METAL IONS

As it has been mentioned in the introduction to this chapter, zirconia-based materials have huge potential in technological applications. One of the remarking feature of this kind of materials are the adsorptive properties with regard to hazardous metal ions or organic pollutants. The benefits of zirconia as an adsorptive materials lie in the presence of functional (hydroxyl groups), high thermal and chemical stability as well relatively low price. On the other hand, similar properties have been described for chitin that is commonly known as a very efficient material for adsorption of various pollutants

[264–266]. Therefore, in this part of work it has been decided to preliminary analyze the potential of prepared chitin-zirconia materials in wastewater treatment applications.

To confirm utilization possibility of chitin-zirconia composites in removal of harmful metal ions at first stage of performed study the BET surface area was estimated

(Figure 22).

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Figure 22. Nitrogen adsorption/desorption isotherms and surface area for zirconia and the chitin-

ZrO2 material.

Polysaccharides are usually known as substances with small porosity, a detailed review regarding this topic has been presented by Crini [267]. Of course, over the last decade, several sophisticated methods of processing of polysaccharides to materials with well-developed porous structure has been described [268]. However, these methods need very sophisticated equipment and are still not attractive from economical point of view.

In this study combination of chitin with inorganic zirconium dioxide results in formation of material with well-developed mesoporous structure. The isotherms can be classified as type IV (IUPAC classification), indicating that there is multilayer adsorption on a mesoporous solid. Mesoporous character and presence of various functional groups on the surface (including –NH, C=O, –OH). This unique combination increases the chance for application of prepared material as a selective adsorbent for harmful metal ions.

The main stage of this study was determination of the sorption properties with regard to harmful metal ions (Cd2+). Adsorption process was optimized with regard to

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time as well concentration of the model solutions. Figure 23 a illustrates the dependence of adsorption efficiency on time for concentration of the model solutions 50 mg/dm3 and

100 mg/dm3. It can be observed that higher removal efficiency was obtained for solutions with concentration equal to 50 mg/dm3. The adsorption equilibrium has been reached after 30 min for both concentrations and it corresponds to the maximal efficiency of the adsorption process. Based on these results the quantities of metal ions adsorbed at equilibrium has been calculated and it has been estimated that 1 g of chitin-zirconia composite material is able to adsorb 20 mg of Cd2+.

Figure 23. Effect of contact time on cadmium(II) adsorption by means of removal efficiency (a) and adsorption capacity (b).

Benguella et al. [269] proved that the sorption capacity of pure chitin with regard to cadmium was equal to 8 mg/g and adsorption equilibrium was reached after 6-7 hours and these values are drastically lower than in the case of chitin-zirconia composites. The huge difference is caused by both increased surface area and porosity of the chitin- zirconia in comparison to chitin. Additionally, the tetravalent zirconium in its hydrated form can generate tetranuclear ions as well as octanuclear species with abundant

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hydroxyl ions and water molecules that can take part in ligand substitution reactions with metal ion [270]. Additionally, the hydroxyl ion and water molecule that may be present in the coordination sphere could also be contributing to the ligand-exchange reactions with cadmium ion [270]. Results of this experiment proved that this material is suitable for application in wastewater treatment and removal of harmful metal pollution.

SUMMARY

The results presented in this chapter clearly revealed that chitinous scaffolds of marine sponge origin can be used as a thermostable organic matrix in the hydrothermal synthesis of monoclinic nanostructured zirconium dioxide from ammonium zirconium(IV) carbonate. However, according to Kisi and Howard [255] theory, the hydrothermal deposition zirconia from other zyrconyl salts can probably be equally effective. Due to the unique tube-form structure of sponge chitin fibers, it was possible to obtain the ZrO2 crystalline phase both within and on the surface of the organic scaffold.

Obtained material have attractive properties in perspective of water pollution removal with respect to heavy metals. However, due to combination of unique properties of chitin with zirconia (such as biodegradability, biocompatibility and affinity to peptides) this new material can discover its exceptional potential in much broader range of practical usage including catalysis, enzyme immobilization, biosensors design. Thus, it is strongly believed that this study might represent the key to the development of new extreme biomimetically fabricated, chitin-zirconia hybrid materials for novel applications in materials science, biomedicine, and other modern technologies.

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CHAPTER 7 AN EXTREME BIOMIMETIC APPROACH FOR HYDROTHERMAL SYNTHESIS OF β-CHITIN-ZnO NANOSTRUCTURED COMPOSITES

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INTRODUCTION

Zinc oxide is an attractive inorganic material with many practical applications [191]. These are the result of exceptional properties of this oxide such as antibacterial activity [271–274], wide bandgap [275] and photocatalytic [275–278] properties. Among diverse paths of ZnO nanostructure synthesis, recently, new biomimetic methods, which borrow key principles from biomineralization (in particular the use of biomolecules to direct nucleation), are being intensively developed. It has been proved that viruses [279] and various macromolecules like DNA [280], polypeptides [24,281], bioengineered proteins [282–284], and also polysaccharides [285,286], can be used for controlling of nucleation and growth of ZnO crystals. Application of these biomolecules allows for the formation of ZnO with a controllable morphology and unique structural as well as physicochemical properties, under so-called mild conditions. Chen et al. [287] reported the preparation of ZnO replicas of butterfly wing scales. Proposed method is based on two stages: (i) soaking chitinous-wings in zinc salt solution (ZnNO3) and (ii) carbonization. Application of this method led to development of the biomorphic, porous structures with 3D morphology that maintain the microstructural features of the original butterfly-wing scale and membrane morphology down to the sub-micrometer level. There are only a few papers in literature about combination of chitin with ZnO [288,289]. Most of the attention is currently paid to chitosan [290,291] and other chitin derivatives. Kumar et al. [288] reported a method for synthesis of chitin–ZnO composites, based on simple mixing of a chitin hydrogel with ZnO nanoparticles followed by lyophilization. These materials are promising candidates for the development of advanced biodegradable antibacterial wound dressings [292]. In this study, it has been decided to develop a ZnO-containing composite material using β-chitin isolated from Sepia officinalis cuttlebones. Sepia is a genus of dibranchiate cephalopod that habituates in the coastal waters of Europe, Africa, Asia, and the South Pacific [293]. From a materials science point of view, the cuttlebone appears to be a highly efficient calcium carbonate–chitin containing biocomposite, using a minimum of materials to achieve the required strength [294]. Cuttlebone has been used for centuries as a mold-making material for casting jewellery-scale objects in metals but today, this naturally occurring scaffold is one of the key players in biomimetic synthesis of nanostructured composites developed from marine biomaterials. Recently, it has been

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reported that chitinous cuttlebones were used for in vitro formation of calcium phosphate [295,296], silica-based [172] as well as silver- and gold-based [297] hybrid materials. It is suggested that application of morphologically defined β-chitin as a template for biomimetic ZnO deposition is very attractive from a technological perspective as it eliminates challenges associated with processing chitin to membranes or scaffolds. For this purpose β-chitin membranes were isolated from the cuttlebone of S. officinalis and used as a thermostable organic template for hydrothermal zinc oxide growth.

7.1. STRUCTURAL AND PHYSICOCHEMICAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-ZnO MATERIALS

Images obtained using stereomicroscopy (Figure 24 a) indicate that applied method of β-chitin isolation from S. officinalis cuttlebone permit to obtain translucent films. Worth noting is that these structures are extremely sensitive to drying. To preserve their unique structure, the films should be kept wet within hermetic containers with small amounts of water prior to hydrothermal synthesis. After the hydrothermal reaction with using zinc acetate solution, the chitinous scaffold turned the translucent structure to a milky color (Figure 24 b).

Figure 24. Images of β-chitin before (a) and after (b) hydrothermal ZnO deposition taken with use of stereomicroscopy.

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The formation of chitin-ZnO clusters can be characterized by the presence of flower- like microclusters of zinc oxide on the chitin surface with use of the scanning electron microscopy (Figure 25 c, d). These microclusters are made of crystalline nanorods of ZnO which can be observed using SEM after hydrothermal synthesis without use (reference sample) (Figure 25 a, b) and with use of the chitinous template (Figure 25 c, d), respectively.

Figure 25. Images of β-chitin before (a) and after (b) hydrothermal ZnO deposition taken with use of scanning electron microscopy. Arrows indicate that crystals are connected with chitin nanofibers.

It is well visible that using the β-chitin during hydrothermal formation of ZnO leads to the formation of uniform micro-sized rods with a sharp top that grow along the c-axis and are similar to structures, which grow without any template. However, it is clearly seen that

ZnO structures in chitin-ZnO composite originate from the surface of the chitin

(Figure 25 c) and are tightly bound to this substrate by nanofibrils (Figure 25 d), they

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resemble the structure of those obtained without the presence of organic matrix.

The nanomorphology of the obtained ZnO crystals (Figure 25) corresponds to that reported previously in the literature data [298–301]. The presence of single microrods with sharp top in the chitin-ZnO sample might be a consequence originating from interactions of chitin with ZnO nuclei. It has also been reported [302] that the mechanism of complex formation of metals with polysaccharides is manifold and probably dominated by different processes taking place simultaneously, such as physical adsorption, ion- exchange, and chelation, under different conditions [303]. Waltz et al. [286,304] proved that polysaccharides can specifically interact with the positively charged face (001) of

ZnO crystals and influence crystal growth in the c-axis direction. However, the complex of sodium with tetrahydroxozincate ion can react with the surface at different sites of a single ZnO nucleus. Thus, in short time, every site may act as new nucleation center for growth of the branching structure. Thus, the growth units [Zn(OH)4]2- begin to incorporate into ZnO along the c-axis at different sites resulting in formation of flowerlike crystals [298,301].

To evaluate the crystallographic properties of the ZnO structures templated by the chitinous matrix, the electron diffraction (HRTEM) as well as XRD techniques.

The brightfield image of the chitin-ZnO composite is presented on Figure 26 a and it clearly indicates the presence of ZnO crystallites of rectangular form with 0.3–0.5 µm width and 2–5 µm length. Comprehensive examination of the SAED pattern and FFT from various local regions of ZnO particles shows their hexagonal character. The size of single crystallites is about 100 nm. The evaluation of the typical SAED pattern and HRTEM image with FFT (Figure 26 b and c, respectively) that were recorded in the marked region of

ZnO particle (Figure 26 a, region A), shows that the crystallites have an [110]-orientation.

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Figure 26. Bright field image of chitin-ZnO composite (a) with marked investigated region A of the sample and corresponding results of SAED (b), HRTEM/FFT (c) and EDX analysis (d).

The XRD data obtained (Figure 27), additionally confirm the results of HRTEM investigations. A reference pattern for hexagonal ZnO has been overlaid

[ICSD no. 26170] [305]. From the ZnO data, the crystallite size of 82 ± 10 nm has been determined, and it corresponds with size estimated by SAED pattern and FFT.

Figure 27. XRD spectra of β-chitin and chitin–ZnO composite.

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Raman analyses were performed for chitin-ZnO composite as well for ZnO and

β-chitin as reference samples (Figure 28). The Raman spectrum of ZnO is characterized with the presence of a broad peak at 1100 cm-1, a strong sharp peak at 435 cm-1 and a few small peaks at 328 and 377 cm-1 assigned to optical phonon modes [306,307]. While the

Raman spectrum of chitin-ZnO reveals the presence of a small band at 438 cm-1 that confirms the presence of ZnO in the composites. However, in comparison to the ZnO spectrum we can observe that the E2high band is shifted from value 435 to 438 cm-1.

Additionally recorded band associated with glycosidic bonds in β-chitin standard is shifted in the spectrum of chitin-ZnO from 896 cm-1 to 903 cm-1 and the amide II band is not observed in the spectrum of chitin-ZnO composite. This phenomenon suggests that

ZnO interacts also with the –NH groups of chitin and these interactions are most likely dominated by different processes taking place simultaneously, including physical adsorption, ion-exchange, and chelation [302,308].

Figure 28. Raman spectra of ZnO, β-chitin and chitin-ZnO composite.

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7.2. ANTIBACTERIAL PROPERTIES OF CHITIN-ZnO COMPOSITE

Above discussed results show with strong evidence that chitinous scaffolds of

Sepia's cuttlebone origin can be chemically transformed under hydrothermal conditions into the chitin-ZnO composite material and resemble the initial film-like structure, where nanostructured ZnO is tightly bound to the surface of the organic template and cannot be removed by the ultrasound-assisted washing procedure. The number of literature reports suggest that chitin is a versatile biopolymer for design of specific biodegradable wound dressing materials which can be applied in the management of skin wounds of various etiology [309–311]. On the other hand, zinc oxide is a well-known inorganic oxide with confirmed antibacterial activity. An important aspect of the use of ZnO as an antibacterial agent is the requirement that the particles are not toxic to human cells and ZnO is indexed as a safe by the European Medicine Agency as well U.S. Food and Drug Administration

[312]. This prompted investigations into the antibacterial activity of chitin-ZnO films. The results of antibacterial activity testing for the analyzed materials are shown in Table 2.

Based on the long-established interpretative criteria for the agar diffusion method, an inhibition zone ~8 mm means weak antibacterial activity and ~14 mm – good antibacterial activity [232]. According to these criteria, chitin-ZnO composite displayed a very good level of antibacterial activity against Gram-positive Bacillus subtilis (after both

24 and 48 h of incubation). However, there is no effect of this complex observed in the case of Gram-negative Escherichia coli (Table 2). For comparison, chitin has no antibacterial activity against the two tested bacterial species (Figure 29).

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Table 2. Antibacterial activity of β-chitin and β-chitin/ZnO against tested microorganisms. Time of Diameter of inhibition Diameter of inhibition zone Bacteria incubation zone for β-chitin sample for chitin-ZnO sample (h) (mm) (mm) Escherichia 24 0 0 coli ATCC 25923 48 0 0

Bacillus 24 0 22.50±0.50 subtilis B9 48 0 26.40±0.36

This observation could also be indicative of higher Gram-negative strain resistance/tolerance against such nanomaterials over Gram-positive bacterial strains and this finding is in line with references [312,313], which reported that the ZnO nanoparticle effect is more pronounced against Gram-positive bacterial strains than Gram-negative bacterial strains. The Gram-positive and Gram-negative bacteria strains differ in the structure and composition of the cell wall. Gram-positive bacteria possess a thick cell wall containing many layers of peptidoglycan and teichoic acid. While, Gram-negative bacteria have rather a thin cell wall, composed of only a few layers of peptidoglycan surrounded by a second lipid membrane containing lipopolysaccharides and phospholipids, which face into the external environment [314,315]. One of the mechanisms of antibacterial action is providing damage to their membranes. This double outer membrane of Gram- negative bacteria can make it less susceptible to surface damage than the Gram-positive

B. subtilis (which has only a single membrane). The possible mechanism of action of ZnO on bacteria may be as follows. ZnO particles can be able to create reactive oxygen species

(ROS) in the presence of light (however, formation of ROS under dark conditions has been also reported for ZnO) like •OH, •O2 – radicals and H2O2 on the surface of chitin-ZnO composite films. These active free radicals cause oxidative stresses in the microorganisms cells and their diffusion inside the cell may result in the perturbation of cell membranes

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and damage of cellular proteins and DNA. Due to the presence of additional layer of negatively charged lipopolysaccharide, Gram-negative bacteria surface exhibited higher negative potential than Gram-positive bacteria [316] and chitin-ZnO zeta potential is negative.

Figure 29. Representative picture of inhibitory zones for Escherichia coli and Bacillus subtilis. The chitin-ZnO composite film are located in the upper part of each Petri dish, and the pure chitin films on the lower part.

According to hypothesis of Arakha et al. [316] that interfacial potential at the ZnO- bacteria interface which triggers possible reactions leading to bacterial non-viability is largely responsible for the antimicrobial propensity of ZnO. The observed differences might be a result of negative charge and repulsive forces that act between Gram-negative strain and chitin-ZnO composite.

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SUMMARY

Biomimetic synthesis is a driving force in engineering novel, advanced materials, which exhibit unique electronic, photonic and catalytic properties. Especially, morphologically defined biological architectures that combine biomacromolecules with inorganic crystals are of fundamental interest for applications in wide range of materials science. In this chapter it has been proved that β-chitin isolated from the S. officinalis cuttlebone can be used as a structural template for hydrothermal growth of hexagonal crystalline ZnO and in result the unique chitin-ZnO materials were obtained. Presented results indicate that synthesis under extreme biomimetic conditions is a new powerful tool for the development of β-chitin-based hybrid inorganic–organic nanostructured composites in the form of films with special antimicrobial properties against Gram- positive bacteria, which gives them good prospects in development of chitin based inorganic–organic wound dressing materials.

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CHAPTER 8 SYNTHESIS OF NANOSTRUCTURED CHITIN–IRON OXIDE COMPOSITES UNDER EXTREME BIOMIMETIC CONDITIONS

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INTRODUCTION

In natural environments, iron oxides occurs in several crystalline polymorphs including poorly-ordered minerals (i.e., green-rust and ferrihydrite) and more crystalline forms, such as hematite (α-Fe2O3), magnetite (Fe3O4), goethite (FeO(OH)), lepidocrocite (γ-FeO(OH)), [317]. Iron is the fourth most abundant element in the earth’s crust therefore it is not surprising that biogenic iron-oxides are one of the most wide spreaded biominerals in the Earth [318]. Biogenic iron-based minerals have been discovered in radula teeth of a chiton [148], limpets and sponge skeletons [2,319]. Extensive studies dedicated to the formation and occurrence of biogenic iron-rich minerals are related to bacteria intracellular and extracellular biomineralization [318]. It is proven [320] that exopolysaccharides from various bacteria strains (Gram-positive as well Gram-negative) are able to capture Fe3+ ions from solution and trigger precipitation of hematite or ferrihydrite outside of the microorganism cell. Representatives of fauna clustered around hydrothermal vents are one of the best examples for inspiring novel biomineralization routes [317]. For example, it is well known that hydrothermal vents are rich in iron. Iron is highly enriched in high temperature hydrothermal fluids as it is leached from host rocks during hydrothermal circulation of seawater [321]. It is also proved that vent-derived iron can rapidly oxidize and precipitate around vents in the form of various oxides and sulfides [322]. It has been proven that exomicrobiological iron oxide formation occurs also in hydrothermal vents in which bacteria are involved into stabilization of ferrihydride [323]. Therefore, hydrothermal vents are sources with number of examples of biomineralization phenomena, based on the formation of iron-based biominerals where unique organic matrices play a crucial role [61,324]. Mechanisms for this “extreme biomineralization” still remain unknown. However, several factors suggest that polysaccharides play an important role in the formation of biogenic iron-based minerals. Therefore, usage of polysaccharides as nucleating and structural templates in synthesis of iron oxide materials is reasonable and can by a “holy grail” [25] in bioinspired materials science. Therefore, in this chapter utilization of chitin from selected marine sponges as a structural template for the hydrothermal synthesis of iron-based minerals from a 0.1M as well from saturated solution of FeCl3 will be described. An experiment with the use of saturated solution was performed to mimic the biomineralization of microbial organisms

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in hydrothermal fluids which is often caused by supersaturation of the water with various minerals [36]. Moreover, tuning of saturation level of Fe source gives the possibility to control the morphology of obtained nanoparticles [325].

8.1. STRUCTURAL AND PHYSICOCHEMICAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-IRON OXIDE MATERIALS

Images taken with light microscopy clearly indicate that chitinous tubes are not only covered by iron oxide on their surface (Figure 30). It is well visible that deposition of inorganic phase occurs also inside chitinous tubes resulting in the formation of a visible iron oxide core with red color. This phenomenon has been observed for both composites prepared with 0.1M as well with saturated FeCl3. Similarly, like in previous studies application of ultrasound-based washing procedure has no influence on the location of the obtained hematite phase, confirming strong bonding of the iron oxide to chitin.

Figure 30. Light microscopy images of A. aerophoba chitinous scaffolds after hydrothermal synthesis showing the formation of iron oxide prepared wit use of saturated FeCl3 solution.

Application of the scanning electron microscopy indicates the differences in the micromorphology of obtained composites with respect to concentration of iron precursor used for their synthesis. The presented SEM images (Figure 31 a) indicate that using the

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chitin from the sponge A. aerophoba as a structural template, prior to hydrothermal reaction possess smooth surface and well organized morphology (Figure 31 b). While chitin after hydrothermal reaction with 0.1M iron(III) chloride is homogeneously covered, with uniform, spherical inorganic nanoparticles (Figure 31 c),

Figure 31. SEM images of the surface of the isolated A. aerophoba chitinous scaffold before (a,b) and after hydrothermal reaction with use of 0.1 M (c,d) and saturated (e,f) FeCl3 precursor of inorganic phase.

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As it was mentioned before, these nanoparticles are so tightly bonded to the chitinous substrate (Figure 31 d) that they cannot be removed from its surface, even after the ultrasound-assisted washing procedure. Intriguingly, inorganic structures obtained with use saturated FeCl3 solution are entirely different. In the Figure 31 e and f, it can be observed that growing structures are also spherical and homogenously distributed within polymer matrix. Especially Figure 31 e gives a strong impression that these structures are growing like from inside of the biopolymer and they are covered with biopolymer fibers. Further SEM observations confirmed this assumption (Figure 32 a). Presented image strongly confirms that precursor of inorganic phase or nuclei infiltrate the chitin hierarchical organization and start to nucleate and promote crystal growth on the chitin nanofibers.

Figure 32. SEM image of the chitin-iron oxide composite obtained with use of supersaturated solution of FeCl3 contains both chitin nanofibrils (arrows) and nanospheres of hematite (a). Schematic view of this structure (b).

Because crystals can grow within the chitin matrix, they are surrounded by chitin nanofibers, and are protected against aggregation and formation of large conglomerates.

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Similar phenomenon has been reported for collagen silicification [326], synthesis of collagen-zirconia [241] and calcification of chitin-chitosan hydrogels [327]. Similar influence of polysaccharides (cellulose) on morphology and crystallinity of in vitro hydrothermally prepared iron oxides has been previously reported by Ma et al. [328].

The morphology as well crystal structure of nanostructured chitin–iron oxide composites, which consists of chitin–nanofibers with oval nanoparticles of inorganic oxide, is also quite visible using TEM. The spherical nanoparticles are especially clearly visible in the dark field image (Figure 33 b). The size of the iron oxide particles ranges between 50 and 100 nm. The analysis of the region of interest (Figure 33 c) by the combination of scanning TEM (STEM) and EDX-measurements and evaluation of EDX- mapping (Figure 33 d) showed the high concentrations of iron and oxygen in the nanoparticles. The increase in concentration of the corresponding elements toward the center of the nanoparticle means that the particles have a spherical shape.

Figure 33. TEM investigations of composite obtained with use of 0.1M FeCl3. Bright-field (a) and dark-field (b) images of the chitin-Fe2O3 composite on graphitic template and the results of elemental analysis of ROI (c) by EDX-mapping (d) and local EDX-measurements (e).

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Figure 34. Detailed analysis of orientation of hydrothermal Fe2O3-particles on sponge chitin obtained with use of 0.1M FeCl3 (a) by evaluation of HRTEM micrograph (b) and SAED pattern (c).

Detailed local analysis of distinct Fe2O3 particles by HRTEM (FFT) and SAED has shown that these are mostly monocrystalline with diverse random orientations (Figure 34). The indexed FFT from the HRTEM micrograph (Figure 34 b) shows a particle with a [152] zone axis. The contrast of HRTEM images is caused by nearby spherical shape of the particle. Another two particles have an [7-4-1] and slightly tilted [2-1-0-1] orientations

(the selected area aperture overlap two particles), which are indexed with yellow and pink text respectively (Figure 34 c).

The XRD patterns of the chitin–Fe2O3 composites hydrothermally prepared with use of

0.1M FeCl3, α-chitin standard, and α-Fe2O3 references are shown in figure 35. The obtained pattern for chitin-Fe2O3 perfectly match the hematite reference (according to

33-664 JCPDS card). Rietveld refinement of these data reveals that the hematite reference sample, obtained without presence of chitin, exhibits crystallites with typical size of

74.7 ± 2.1 nm. The size of hematite crystallites on the chitin is 54.3 ± 5.2 nm, and thus our obtained data closely reflect the HRTEM results.

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Figure 35. XRD patterns of α-chitin from A. aerophoba, hematite and obtained chitin-Fe2O3 composite.

In case of chitin-iron oxide composite obtained with use of saturated FeCl3 the bright field

STEM image in the chitin the contrast from large and small microparticles are seen. The large particles have nearly spherical shape with a diameter of 0.5-0.7 microns, while the small particles have a lamellar form (Figure 36 a). In addition, a large number of small light grey nanoparticles, which are relatively frequently distributed is observed. The EDX analysis, which was carried out by elemental mapping, shows that both the large and small dark particles offer a relatively high concentration of iron and oxygen and a small amount of potassium and chlorine (Figure 36 b). This proves that the particles consist of an iron oxide with a small amount of ferric chloride.

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Figure 36. The bright-field STEM image of the area of the composite – chitin fibril with Fe2O3 particles obtained with saturated iron chloride solution (a,c) and the results of analysis of the corresponding sample areas by means of elements mapping (b) and HRTEM (d) with subsequent simulation of the diffraction pattern (f) by indexing of FFT determined orientation of the local area (e).

Detailed analysis of these particles by HRTEM and FFT shows (Figure 36 c-f) they are rather monocrystalline (in Figure 36 d) the particle has an [11-1]-orientation and have the structure of Fe2O3. The nanocrystalline particles with a high content of chlorine were mainly observed at the surface of these Fe2O3-particles. These are the remains of ferric chloride that can also be seen in the rest of the fibril as light grey particles.

To determine interactions between chitin and formed hematite phase the analyses with use of Raman spectroscopy (Figure 37) and XPS were performed (Figure 38).

According to reference data [329] the Raman shift of peaks characteristic for hematite strongly depends on particle size. The Raman spectrum of α-Fe2O3 reference sample show all the characteristic hematite bands which belong to the D3d6 space group: two A1g modes (224 cm-1and 494 cm-1) and five Eg modes (at 245 cm-1, 289 cm-1, shoulder at 298 cm-1, 407 cm-1, 608 cm-1). While, the presence of several characteristic bands at

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255, 323, 368, 395, 457, 501, 648, and 710 cm-1 that correspond to δ(CCC) ring deformation in the Raman spectrum of chitin reference sample [330] is observed.

Figure 37. Raman spectra of α-chitin reference sample, hematite and the obtained chitin–Fe2O3 composite.

Raman spectra of the obtained chitin–Fe2O3 composites show the presence of four very intense peaks characteristic of hematite (vertical lines). It is well known [331] that the position and width of the hematite nanoparticles peaks at 226 cm-1 and 412 cm-1 are influenced by the nanoparticles' dimensions. According to this assumption, from the obtained chitin–Fe2O3 spectra it was also possible to estimate a dimension of 80–100 nm for the particles that is in agreement with SEM and TEM results. Because of the clearly visible deformations of –NH and C=O bands in the Raman spectra of the chitin–hematite and bands characteristic for carbonyl groups in the Raman spectra of the chitin–Fe2O3 composite it has been suggested that these groups are interacting with Fe2O3.

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Good insights into the role of chitin and formation mechanism of chitin–hematite composites were obtained using the XPS analysis (Figure 38). The O 1s peak from chitin– hematite composites provides the best information about interactions between chitin and iron oxide. For example, the analysis of the results show subpeaks at 529.42 eV and

527.65 eV, which are ascribed to O atoms of chitin bound to Fe in α-Fe2O3 nanoparticles

[332]. Additionally, the peak at 531.70 eV is assigned to –OH groups of chitin. It is worth noting that the intensity of this peak decreases in spectra of the chitin–Fe2O3 composite.

Figure 38. XPS O 1s (a) Fe 2p (b) and N 1s (c) spectra for chitin reference sample, hematite reference sample and chitin-Fe2O3 composite.

The deconvolution Fe 2p peak of the chitin–hematite sample indicate the presence of two distinct subpeaks at 710.7 and 724 eV (Figure 38 b) and a satellite peak at

~718 eV [333]. The presence these peaks indicates that the iron is almost completely in the Fe3+ state. Additionally, the XPS N 1s peak (Figure 38c) was examined in detail to confirm the chemical state of nitrogen atoms present in the chitin and chitin–hematite materials.

On the basis of the results presented in this work and previously reported studies realized with the chitinous butterfly wings [334] and chitin derivatives [335], a mechanism of formation and interactions between α-chitin scaffold and hematite nanoparticles within chitin–Fe2O3 composite has been proposed (Figure 39). The model

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proposed is based on the formation of an –O–Fe bond between chitin and hematite that correspond to the case described by Zhang et al. [332,336] for PET–hematite composites.

However, the presence of hydrogen bonds and the chelating effect between C=O, NH and

OH groups of the chitin and iron oxide are also included [337].

Figure 39. A schematic view on the possible mechanism of chitin–hematite interactions under hydrothermal conditions.

8.2. ELECTROCHEMICAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-Fe2O3 MATERIALS

The question about the possible practical applications of the chitin–Fe2O3 composites obtained is of immense significance. Accordingly, the electrochemical properties of this novel composite material in two-electrode cells using an alkaline electrolyte were, to the best author knowledge, investigated for the first time.

Voltammometric characteristics at 10 mV/s are shown in Figure 40 a and differences in capacitances between the initial materials and composite are well visible. The capacitance

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of raw chitin–Fe2O3 hybrid material (Ch–H) is negligible. Active carbon (S30) with high surface area shows a regular rectangle shape of the CV curve and good charge propagation. Considerable increase of the capacitance is noticed for the composite of chitin-Fe2O3 mixed with active carbon. The increase can be the result of both redox reactions of the chitin–Fe2O3 hybrid material in an alkaline electrolyte, and the developed surface area of the active carbon component. Application of the active carbon/chitin-

Fe2O3 composite permits to improve the capacitances of the electrochemical capacitor.

This is due to the appearance of reduction and oxidation reactions on the electrodes.

In this type of supercapacitor, the energy is stored in the electric double layer (active carbon) and by redox reactions (chitin-Fe2O3 composite). Adsorption/desorption processes of ions derived from the electrolyte occur in the pores of carbonaceous material, while in the pores of the nanostructured hematite occurs the deintercalation/intercalation processes. Moreover, during the process of oxidation and reduction on the electrode, consisting of active carbon/chitin-Fe2O3 composite, a reversible reaction [338] likely occurs:

− − 퐹푒2푂3 + 푂퐻 ↔ 퐹푒2푂3푂퐻 + 푒

The analysis of electrochemical impedance spectroscopy is presented in figure 38b. The results exhibit the improvement of capacitance and charge propagation using the composite as electrode materials for an electrochemical capacitor. Moreover, as shown in figure 38c, materials show the considerable stability during cycling measurements. It can be easily observed that after 5000 cycles of galvanostatic charge/discharge with the current regime of 1 A/g the capacitance of active carbon reduced negligibly – less than one percent. In the case of chitin–Fe2O3 hybrid material/active carbon composite, the rise of capacitance after cyclability measurements has been observed.

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Figure 40. Cyclic voltammograms of active carbon, carbon/chitin-Fe2O3 and chitin-Fe2O3 at 10 mV/s (a); capacitance–frequency dependence for active carbon and composite (b); cycling stability of active carbon and composite with current density 1 A/g (c).

Probably, a redox bioactive film is built on the surface of the composite and an increase of

(pseudo)capacitance is observed. Stability during cycling measurements is a very important parameter for practical applications of materials for electrochemical capacitors.

SUMMARY

The results presented and discussed in this chapter have shown that the hydrothermal route for development of novel chitin–hematite composites is facile and simple manipulating with concentration of the inorganic phase precursor can modulate its deposition. Composites of sponge chitin–Fe2O3 with active carbon could be successfully applied as electrode materials for electrochemical capacitors. Using suitable composite components with different mechanisms of energy storage can improve the electrochemical properties of electrode materials. Additionally, this facile method can be used for combining of other thermostable as well biocompatible and biodegradable polymers with iron oxide and opens new possibilities on the development of iron oxide-

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based materials for applications in supercapacitors [338,339], sensors[340] catalysts

[335] and drug carriers for anticancer therapy. Therefore, it is strongly believed that study presented in this chapter will influence wide range of research associated with hematite-based biomaterials.

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CHAPTER 9 EXTREME BIOMIMETIC APPROACH FOR DEVELOPMENT OF NOVEL CHITIN–GeO2 NANOCOMPOSITES WITH PHOTOLUMINESCENT PROPERTIES

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INTRODUCTION

Germanium dioxide nanoparticles have attracted recently huge attention in nanotechnology and especially in field of materials science, as GeO2 possesses unique physicochemical properties attractive for sophisticated applications. It exhibits a high dielectric constant, a high refractive index, high thermal stability, and high mechanical strength [341]. GeO2 is also known to display photoluminescence [341–343] and piezoelectric [344] properties. Owing to a combination of these intriguing features,

GeO2 possesses a special place among various semiconductor nanomaterials and is widely used in optical waveguides for integrated optical devices and systems [345]. Additionally, germanium oxide has applications in the preparation of catalysts for methanol steam reforming [346] and anodes for Li-ion batteries [346]. However, it is recognized that this material exhibits several polymorphs [347–349], and all above- mentioned, fascinating properties are strongly dependent on the crystalline structure of germanium oxide. Therefore, it is important to develop synthesis methods that will allow control over the morphology as well crystallinity of GeO2. Thus several different technologies have been developed for the synthesis of germanium oxide with desired properties, including sol-gel reactions [343], thermal evaporation [350], the reverse micelle method [351], direct precipitation followed by calcination [352], as well as thermal oxidation [353]. Template-directed approaches including hard templates using (for example carbon nanotubes [36]) or soft-templates with surfactants [354], 2-methyl pentamethylene diamine [355], have been investigated for their ability to synthesize specific crystalline phases of germanium oxide. Above-mentioned methods allow the preparation of germanium oxide with designed crystal phases as well as with desired morphology from nanowires, through nanospheres and nanocubes. Mostly these methods require harsh environments including high temperatures and extreme pH. Additionally, much attention is paid to the synthesis of germanium oxide organized into three-dimensional networks formed from nanofibers [356].

Intriguingly, in reference only few studies on the use of biological templates for forming the desired phases of germanium oxide can be found. These only relate to the formation of germania nanoparticles, and principally, no studies seem to exist on the development of germania-based biocomposites on the micro- and macroscale. The first biomimetic preparation of GeO2 was published by Patwardhan and Clarson [357], who

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proposed the utilization of two macromolecules: poly(allylaminehydrochloride) and poly-L-lysine to facilitate the synthesis of germania particles and control over product morphologies. This study strongly proved that these macromolecules can act as a catalyst/scaffold/template for particulate germania formation. On the other hand, Davis et al. [358] proposed the synthesis of germanium oxide using the sol-gel technique controlled by amino acid (lysine) introduced into reaction system. These researchers speculated that lysine might stabilize germanium species through complexation. Such stabilization may decrease rate of the crystal nucleation and growth, requiring additional (unstable) germania species (i.e., higher germania concentrations) before crystal nucleation can be initiated. Very recently, multicrystalline GeO2 was prepared from germanium tetraethoxide (TEOG) in the presence of different silk-based peptides [359]. It was reported that these organic materials incorporated into the mineral did not appear to affect the unit-cell dimensions. Moreover, the germania-binding peptide alone did not have any significant effect on reaction rate, yield, or the material’s properties in comparison with the control reference [45].

In this chapter utilization of chitin as a template for hydrothermal synthesis of germania based composites with sponge-like structure will be described and discussed.

9.1. STRUCTURAL AND PHYSICOCHEMICAL PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-GERMANIUM DIOXIDE MATERIALS

The presented SEM images (Figure 41 a-c) show that using α-chitin isolated from the A. cauliformis sponge as a structural template during the hydrothermal formation of germanium oxide from the TEOG precursor leads to the formation of chitin–GeO2 composites with the surface (Figure 41 c) as well inner space

(Figure 41 b) homogeneously covered with uniform germanium dioxide nanoparticles. Additionally, the presented SEM image (Figure 41 d) indicate that GeO2 nanoparticles are tightly bonded to the nanoporous chitin surface and similarly like in

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previous studies could not be removed even after ultrasound-assisted washing procedure.

Figure 41. SEM images indicating that he initially smooth surface of chitin fibers (a) became covered with nanocrystals of germanium dioxide on different locations (b,c) after insertion in hydrothermal reactor at 185 °C. SEM image obtained after 1 h of ultrasound treatment of the chitin–GeO2 nanocomposite, showing the nanoporous surface morphology of the prepared material (d).

Additionally, SEM images of mechanically fractured fragments of the composite

(Figure 42) show that nanofibrils of chitin (about 17 nm in diameter) represent the

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nucleation sites for the growth and formation of GeO2 nanocrystals that are up to 300 nm in size.

Figure 42. SEM of the fracture region within the chitin-GeO2 composite (a), clearly indicating that Germania nanocrystals are formed around nanofibrils of the sponge chitin (b).

It is well recognized that germanium dioxide has different crystalline polymorphs; the most widely known polymorphs are tetragonal and trigonal/hexagonal. To get insight into the crystalline nature of the inorganic phase growing on the chitinous matrices studied, the different highly sensitive methods including TEM and NEXAFS.

NEXAFS is proven to be a powerful tool to probe chemical bonding and molecular structures. Figure 43 shows the Ge L3-edge NEXAFS spectra of the chitin- based composite as well as the reference bulk hexagonal GeO2 sample. It should be noted that the spectral pattern of the reference sample is consistent with that obtained for full-density hexagonal GeO2 [360]. It is well visible that both spectra depicted in

Figure 43 are almost identical, this imply that newly synthesized chitin-based material contains hexagonal GeO2.

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Figure 43. Ge L3-edge NEXAFS spectra of chitin–GeO2 composite and the reference bulk hexagonal GeO2 sample.

This data has been confirmed using transmission electron microscopy. The TEM investigations of the prepared sample indicated that the chitin–GeO2 composites are observed in the form of GeO2 particles that adhere to the chitin fibers. The TEM bright field micrographs (Figure 44 a) present a chitin lamella with the dimensions of 0.4 µm x 2 μm with attached crystallites of GeO2 that have size fluctuating between 150 and

300 nm. The GeO2 nanoparticles, which have a hexagonal structure P3121 (fitted to the data used ICSD 637457) that is apparent from the SAED analysis (Figure 44 b). A small amount of GeO2 in the form of thin nanolamellae (width approximately 10 nm) has been also detected. The SAEDP of these sample areas represents the ring diagrams involving only a small number of reflections, or rather small randomly oriented nanocrystallites (Figure 44 c). Here, the crystallites also have a hexagonal structure; therefore, it is possible to say that deposited germania is monocrystalline.

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For a comprehensive analysis the bright-field TEM and HR-TEM micrographs were taken from areas with nanolamellae (Figure 44 d, e). The visual analysis and the analysis of local areas of HR-TEM images with FFT shows that the nanocrystallites of

GeO2 have a different orientation: an example (Figure 44 f) shows that region 1 has the [13-1]-orientation of hexagonal GeO2.

Figure 43. Bright-field TEM image of the chitin–GeO2 composite with SAEDP from the region of spherical particles (b) and lamellae (c) as well the TEM (d) and HR-TEM micrograph (e) with corresponding FFT from the local region of the lamellae (f) completed with the results of elemental analysis using of EDX point measurements (g) and EDS mapping of the entire region of the composite (h)–(i).

The results of the EDX analysis show the presence of Ge and O signals in both thick crystallites (EDX1, Figure 44 g) and nanolamellae (EDX2, figure 44 g). The surface analysis using the EDS mapping of regions of the chitin–GeO2 composites presents high

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concentrations of Ge as well O in the crystallites (Figures 44 h,i), which are grown on the chitin lamellae.

The Raman spectrum of GeO2 nanoparticles reference sample, prepared without chitin template, show strong peaks characteristic to α-quartz-like GeO2 hexagonal cells, including three symmetric phonon modes of A1 symmetry (at 259, 439, and 877 cm–1) and four modes of E symmetry (at 514, 589, 855, and 957 cm–1) that are divided into transverse (TO) and longitudinal optic (LO) modes [349,353]. The Raman spectra of the obtained chitin–GeO2 composites indicate the presence of five intense peaks typical for germanium oxide (vertical lines) this is an additional evidence that the nanoparticles interacting with the chitin surface have hexagonal crystalline structure.

Figure 45. Raman spectra of the α-chitin standard GeO2 reference, and chitin–GeO2 nanocomposite

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Figure 46 illustrates the FTIR spectrum of the chitin used for hydrothermal synthesis, GeO2 reference, and hydrothermally prepared chitin–GeO2 nanocomposite.

The triplet band, characteristic of the GeO2 hexagonal phase, is clearly visible between

588 and 520 cm–1 and is assigned to the Ge–O–Ge υ4 vibrational mode [348,358,361].

The low-frequency bands around 555 cm–1 are equivalent with the LO mode observed in the Raman spectrum at ~589 cm–1 (Figure 46). Furthermore, peaks assigned to Ge–

O–Ge (the antisymmetric stretching mode of hexagonal GeO2) were indicated at 892 and 960 cm–1[362] (Figure 46); these peaks are equivalent to TO and LO-split asymmetric stretching of the bridging oxygen, respectively [349]. In the FTIR spectrum of the chitin–GeO2 nanocomposite, observable are characteristic peaks for hexagonal

GeO2 however, some significant changes are also well visible. The main difference is that β-1,4-glycosidic bond peaks characteristic for α-chitin at 897 cm–1 [155] is shadowed by the strong GeO2 mode at 892 cm–1. The general view suggests that the lack of bands at ~680 and ~612 cm–1 indicates that formation of Ge–O–C and Ge–C bands and can be excluded [55]. Thus, it can be hypothesized that chitin interacts with germanium-oxide nanoparticles only through the formation of hydrogen bonds.

Compared with the chitin template, the absorption line of the O–H vibration is shifted from 3430 to 3439 cm–1 in the chitin–GeO2 composite because of hydrogen bonding.

Moreover, the shift in the infrared spectra from 1066 cm–1, that corresponds to the

C(3)–OH stretch in chitin [55], to 1071 cm–1 in the composite is well visible. Qin et al.

[363] reported that the formation of Ge–O–C bonds between germanium oxide and graphene oxide can be also indicated by the presence of a band at 1380 cm–1. However, in the case of the chitin-based composite, the absorbance at 1378 cm–1 is more-likely assigned to CH bending and asymmetric deformation of CH3 from chitin chain.

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Figure 46. FTIR spectra of the α-chitin standard GeO2 reference, and chitin–GeO2 nanocomposite.

9.2. PHOTOLUMINESCENT PROPERTIES OF HYDROTHERMALLY PREPARED CHITIN-

GeO2 MATERIALS

During microscopic observations with use of the fluorescence microscope, it has been noted that the chitin–GeO2 composite shows stronger autofluorescence

(Figure 47) in comparison with untreated chitin and the crystalline GeO2 sample obtained under similar hydrothermal conditions without the presence of any organic templates. The observed phenomenon paves the way to practical applications of 3D

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scaffolds based on chitin–GeO2 composites in the fields of medicine, sensors, and electronic devices.

Measurements performed with using photoluminescence spectroscopy

(Figure 47b) shows the PL spectra for the chitin–GeO2 composite together with those for the α-chitin and GeO2 reference samples. A strong enhancement in PL efficiency for the nanocomposite is readily visible. For quantification, all recorded spectra were fitted successfully by two Gaussian components; one centered around 2.6 eV and the other one just below 3 eV. Both components (dotted lines) and the resulting cumulative fit (thin continuous line) are shown in (Figure 47 b) for the chitin–GeO2 nanocomposite. For the other PL spectra, a similar cumulative fit quality (R2 > 0.997) was achieved by minor changes in the peak-component position and FWHM.

The integrated PL of the nanocomposite is found to be enhanced by 12 times as compared to that of the GeO2 reference and 4.3 times higher in comparison to chitin reference. The obvious differences in PL spectral shape between the different samples are reflected in the differing ratio between the two peak components. For the GeO2 reference, the ratio of the area of the 2.6 eV peak to the area of the 3 eV peak is equal to 3.2, whereas for the α-chitin reference, this ratio is only 0.6. The chitin–GeO2 nanocomposite demonstrated an intermediate ratio of 2.2. The observed chitin luminescence spectrum is in agreement with published data [364]. The reported luminescence spectra of GeO2 nanoparticles differ widely, probably because of the wide variety in preparation procedures, matrices, and shapes. Generally, luminescence in the 2–3 eV range has been attributed to either surface/interface defects or defects related to oxygen deficiency in the GeO2 crystals. These defects probably originate form incorporation of chitin nanofibers into the crystalline structure.

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Figure 47. Fluorescent microscopy images (light exposure time: 1/4 s) (a). Room-temperature

PL spectra of chitin–GeO2 composite (red line) show a 12-fold increase in PL intensity as compared to the GeO2 reference sample (blue line) (b). For comparison, pure chitin (grey line) shows intermediate PL intensity with a different PL band shape. All PL bands can be fitted using two Gaussian peak components centered around 2.6 eV and close to 3 eV. Here, the two components shown as dotted-green and dark-blue lines demonstrate the fitting of the chitin–

GeO2 nanocomposite PL band, with the cumulative fit given as a thin solid line (R² > 0.997).

SUMMARY

The results presented and discussed in this chapter have revealed a new hydrothermal route for developing novel chitin-GeO2 composites. The specific thermal stability of chitin open a gate to synthesize a crystalline phase of crystalline hexagonal

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GeO2 from the precursor germanium ethoxide at 185 °C. It has been proven with strong evidences that nanocrystals of GeO2 grew exclusively within and on the surface of the tube-like chitinous matrix, which signifies the importance of the typical morphology of the sponge skeleton. From this perspective, a solid chitin-GeO2 composite with specific sponge-like three-dimensional structure with functionalized surfaces that shows interesting fluorescence and photoluminescence properties attractive for sophisticated application, has been developed.

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GENERAL SUMMARY

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The main aim of this thesis was to develop new method of synthesis of advanced hybrid inorganic-organic chitin–based materials with respect to Extreme Biomimetic concept. On the basis of analysis of the reference data as well thermal analysis it has been proven that both α-chitin isolated from poriferan origin as well β-chitin from cuttlebones, thanks to exceptionally high thermal stability, can be effectively used as a biotemplate for hydrothermal deposition of various inorganic oxides including SiO2,

ZrO2, ZnO, Fe2O3 as well GeO2.

The intrafibrillar infiltration of inorganic precursors or primary nanoparticles likely occurred under applied conditions. In result, the nucleation of the inorganic crystals was observed on the nanolevel of chitin fibers organization. Interestingly, thanks to this phenomenon the deposition of inorganic core within the chitinous tubes was observed.

Application of sophisticated analytical methods proved that chitin interacts with formed inorganic oxides mainly by the formation of hydrogen bonds. However, it has been also proved that in case of hematite formation of complexes can occur as well.

All materials show exceptional stability with respect to the ultrasound-assisted treatment procedure, which confirms that inorganic nanoparticles are tightly bonded to the chitinous substrate.

Depending on the deposited inorganic phase, obtained hybrid materials are characterized by the unique properties that make them attractive for various sophisticated applications. For example, deposition of zirconia on chitinous substrate result in escalation of surface area, which in result greatly increase the adsorption capacity of obtained materials with respect to Cd2+ ions. This is an attractive feature for application of this material in wastewater treatment and removal of harmful

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pollutants. Moreover, chitin-ZnO nanomaterials with membrane-like morphology are characterized by antimicrobial properties against Gram-positive bacteria, which make them attractive in perspective of development of biodegradable wound dressing materials. Analysis of application potential of chitin-Fe2O3 materials, surprisingly revealed that these can be used as a fillers of active carbon and in result increase performance of the supercapacitors. Finally, the incorporation of chitin nanofibers into crystal lattice of germanium dioxide results in increase of the photoluminescence of the composite, that was 12 times higher than that of pure germania prepared without use of organic template. This make open the way to use them in preparation of sponge- like optical devices.

It should be highlighted that proposed methods are not only limited to morphologically defined chitin isolated from sponges. These can be applied for chitin nanocrystals, nanospheres, gels, nanofibers as well. Additionally, other chemically as well thermally stable biopolymers can be used in similar way as a templates for hydrothermal crystal growth of various biominerals. Therefore, it is strongly believed that research done within framework of this thesis will pave the way for the development of diverse biomaterials under hydrothermal synthesis within the

Extreme Biomimetic concept.

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ABSTRACT

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Presented thesis is focused on devpelopment of novel methods for synthesis of new generation of inorganic-organic chitin based biomaterials. The novelity of this thesis is based on non-linear approach to biomimetics and carrying synthesis under hydrothermal conditions (that are similar to these occurring in hydrothermal vents) in line with Extreme Biomimetic concept. Additionally, it is worth highlighting that research was realized with use of morphologically defined two-dimensional or three- dimensional chitin structures isolated from marine sponges or Sepia cuttlebones. This step allowd to overcome several technical limitations associated with manufacturing chitin into desired structures. Therefore, proposed methods are highly efficient, cost- effective, and align closely with “green chemistry” concept. It has been indicated that these chitininous templates are stable up to 280 °C and this property opens the key way to use chitinous matrices of poriferan origin as highly structured biological templates in a broad variety of hydrothermal synthesis in vitro. During next steps the novel approach for the design chitin-SiO2, chitin-ZrO2, chitin-ZnO, chitin-Fe2O3 and chitin-GeO2 has been developed. By detailed characterization of these materials using a variety of advanced analytical techniques it has been concluded that Extreme Biomimetic is on the rise as a powerful approach for the design of biomaterials with wide spectrum of practical applications

Chapter 1 is a general introduction into frames of presented thesis. Chapter 2 is a review of current state of art with respect to role of biomimetics and bioinspired chemistry in modern materials science. Within this chapter, the therm of Extreme Biomimetics has been defined wih regard to main inspirations and tasks. Addtionally, this chapter is dedicated to physicochemical properties of chitin as a structural template for Extreme Biomimetics. Moreover, the role of hydrothermal technology in biomimetics has been also presented. Chapter 3 covers the main objectives of presented thesis. Chapter 4 is dedicated to description of all materials and methods utilizeded within frames of this thesis. Chapter 5 describes hydrothermal silicification of two-dimensional chitin templates isolated from I. basta marine sponge with respect to morphology and physicochemical properties of obtained chitin-silicon dioxide materials.

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Chapter 6 deals with hydrothermal synthesis of chitin-ZrO2 composites in vitro. The reaction mechanism between chitin and ammonium zirconium(IV) carbonate under hydrothermal conditions has been proposed and described. Obviously, the morphology as well crystalline structure of obtained materials have been characterized in detail. Additionally, preliminary studies of practical application of chitin-zirconium dioxide as a novel effective adsorbent in wastewater treatment has been analyzed. Chapter 7 describes hydrothermal synthesis of chitin-ZnO biomaterials. The morphology of obtained materials as well crystalline structure have been described in detail. Additionally, the antibacterial properties have been described to evaluate the potential utilization of this properties in desing of modern wound dressing materials. Chapter 8 is dedicated to hydrothermal synthesis of chitin-iron oxide, with use of saturated and 0.1M FeCl3. This chapter covers insight into morphological and structural properties. The crystalline character is also characterized in detail. Additonally by applicatition of X-ray photoelectron spectroscopy, Raman spectroscopy and FTIR the interactions between chitin and hematite have been determined. Chapter 9 describes synthesis of unique photolumminescent three-dimensional chitin-GeO2 composites with sponge-like morphology. Analysis of crystalline structure and nanomorphology revealed that exceptional photoluminescent properties are effect of crystalline defects resulting from incoprotation of chitin nanofibers into growing

GeO2 hexagonal crystals.

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STRESZCZENIE

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Prezentowana praca zorientowana jest na rozwój nowych metod preparatyki nieorganiczno-organiczncych biomateriałów chitynowych. Interdyscyplinarny charakter pracy polega na zastosowaniu nieliniowego podejścia do zagadnień biomimetyki i prowadzenia syntezy w warunkach hydrotermalnych (zbliżonych do warunków panujących w źródłach hydrotermalnych), co jest zgodne z założeniami nowego kierunku badawczego jakim jest Ekstremalna Biomimetyka.

O wyjątkowości przedsięwziętej tematyki badawczej stanowi fakt, że we wszystkich prowadzonych badaniach wykorzystano dwu- i trójwymiarowe porowate, chitynowe szkielety gąbek morskich lub szkielety Sepia officinalis, co pozwaliło wyeliminować niepożądane procesy i operacje jednostkowe związane z przetworzeniem biopolimeru do właściwej formy morfologicznej. W ramach realizowanych prac stwierdzono, że wybrane matryce chitynowe są stabilne aż do 280 °C. Właściwość ta predysponuje do wykorzystania wybranych szkieletów chitynowych jako strukturanlnych matryc organicznych do nukleacji i wzrostu nieorganicznych kryształów w szerokiej gamie reakcji hydrotermalnych. Dowiedziono, że ta nowa metoda pozwala na preparatykę układów chityna-SiO2, chityna-ZrO2, chityna-ZnO, chityna-Fe2O3 oraz chityna-GeO2.

Dokonano szczegółowej analizy właściwości fizykochemicznych i strukturalnych, z wykorzystaniem zaawansowanych technik badawczych (HRTEM, SAED, XRD, SEM,

XPS, NEXAFS, etc.). Na podstwie uzyskanych rezultatów określono strukturę krystaliczną i wyznaczono rozmiar krystalitów uzyskanych materiałów. Ponadto zdefiniowano wpływ biopolimeru na morfologię wzrastających kryształów, a także ustalono charakter odddziaływań pomiędzy biopolimerem i wzrastającą fazą nieorganiczną. Finalnie, ocenie poddano potencjał aplikacyjny uzyskanych biomateriałów. Dowiedziono, że w zależności od wprowadzonego tlenku do matrycy

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chitynowej uzyskane materiały mogą znaleźć zastosowanie w adsoprcji szkodliwych metali, preparatyce materiałów opatrunkowych, superkondensatorów, a także układów optycznych.

Rozdział 1 stanowi generalne wprowadzenie w zakres pracy doktorskiej.

Rozdział 2 dotyczy przeglądu aktualnego stanu wiedzy w odniesieniu do roli biomimetyki w nowoczesnej inżynierii materiałowej. W tym rozdziale zdefiniowano zagadnienie ekstremalnej biomimetyki w aspekcie głównych źródeł inspiracji, założeń i zadań. Ponadto w ramach tego rozdziału dokonano wnikliwej charakterystyki właściwosci chityny jako strukturalnej matrycy do zastosowań w Ekstremalnej

Biomimeryce. Znaczenie syntezy hydroteralnej w odniesieniu do ww. kierunku badawczego zostało również przedstawione w niniejszym rozdziale.

Rozdział 3 opisuje główne cele przedstawionej pracy doktorskiej.

Rozdział 4 jest dedykowany do opisu wszystkich materiałów i metod wykorzystanych w celu osiągnięcia założonych celów badawczych w ramach niniejszej pracy doktorskiej.

Rozdział 5 poświęcony jest hydrotermalnej silifikacji chitynowych matryc wyizolowanych z gąbek I. basta w odniesieniu do właściwości morfologicznych i fizykochemicznych uzyskanych biomateriałów chityna-krzemionka.

Rozdział 6 dotyczy hydrotermalnej syntezy chityna-ZrO2. W ramach tego rozdziału dokonano oceny właściwości morfologicznych, a także zbadano charakter krystaliczny uzyskanych materiałów. Na podstawie wyników spektroskopii Ramana oraz FTIR zaproponowano mechanizm reakcji między biopolimerem a prekursoem cyrkonu, w tym przypadku amonowęglanem. Ponadto dokonano wstępnej oceny praktycznego

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zastosowania układów chityna-ZrO2 jako nowych adsorbentów jonów metali w aspekcie oczyszczania modelowych roztworów ścieków.

Rozdział 7 opisuje hydrotermalną syntezę materiałów chityna-ZnO z wykorzystaniem chitynowych szkieletów Sepia officinalis w roli matrycy organicznej. Właściwości morfologiczne uzyskanych biomateriałów oraz ich struktura krystaliczna zostały szczegółowo opisane. Ponadto, zaprezentowano wyniki badań antybakteryjnych w odniesieniu do wykorzystania przygotowanych materiałów w roli materiałów opatrunkowych.

Rozdział 8 poświęcony jest hydrotermalnej syntezie układów chityna-tlenek żelaza.

W ramach tego rozdziału dokonano oceny wpływu stężenia zastosowanego prekursora na charakter morfologiczny finalnych produktów. Ponadto przedstawiono wnikliwą charakterystykę struktury krystalicznej materiałów chityna-Fe2O3.

Zastosowanie zaawansowanych technik badawczych takich jak spektroskopia fotoelektronów wzbudzonych promieniowaniem Rentgenowskim, spektroskopia

Ramana oraz FTIR, pozwoliły na ocenę charakteru oddziaływań pomiędzy chityną a wzrastającą fazą tlenku żelaza.

Rozdział 9 opisuje hydrotermalną syntezę unikatowych fotoluminescencyjnych, trójwymiarowych materiałów chityna-GeO2. Analiza struktury krystalicznej i nanomorfologii dowodzi, że wyjątkowe właściwości fotoluminescencyjne są efektem defektów w strukturze krystalicznej omawianych materiałów, które kolejno są następstwem wzrostu inkorporacją nanowłókien biopolimeru do sieci krystalicznej

GeO2

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SCIENTIFIC ACTIVITY

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PUBLICATIONS

CUMULATIVE IMPACT FACTOR: 67.909

1. Szatkowski T., Wysokowski M., Lota G., Pęziak D., Bazhenov V.V., Nowaczyk G., Walter J., Molodtsov S.L., Stöcker H., Himcinschi C., Petrenko I., Stelling A.L., Jurga S., Jesionowski T., Ehrlich H., Novel nanostructured hematite-spongin composite developed under an extreme biomimetic approach. RSC Advances (2015) 5, 79031- 79040. IF=3.840

2. Bartczak P., Norman M., Klapiszewski Ł., Karwańska N., Kawalec M., Baczyńska M., Wysokowski M., Zdarta J., Ciesielczyk F., Jesionowski T. Removal of nickel (II) and lead (II) ions from aqueous solution using peat as a low-cost adsorbent: A kinetic and equilibrium study. Arabian Journal of Chemistry – in press IF=3.725 doi:10.1016/j.arabjc.2015.07.018

3. Szatkowski T., Kołodziejczak-Radzimska A., Zdarta J., Szwarc-Rzepka K., Paukszta D., Wysokowski M., Ehrlich H., Jesionowski T., Synthesis and characterization of hydroxyapatite/chitosan composites. Physicochemical Problems of Mineral Processing (2015) 51(2), 575−585. IF=0.926

4. Zdarta J., Klapiszewski Ł., Wysokowski M., Norman M., Kołodziejczak-Radzimska A., Moszyński D., Ehrlich H., Maciejewski H., Stelling A.L., Jesionowski T., Chitin- lignin material as a novel matrix for enzyme immobilization. Marine Drugs (2015) 13, 2424-2446. IF=2.853

5. Wysokowski M., Materna K., Walter J., Petrenko I., Stelling A.L., Bazhenov V.V., Klapiszewski Ł., Szatkowski T., Lewandowska O., Stawski D., Molodtsov S.L., Maciejewski H., Ehrlich H., Jesionowski T., Solvothermal synthesis of hydrophobic chitin-polyhedral oligomeric silsequioxane (POSS) nanocomposites. International Journal of Biological Macromolecules (2015) 76, 224-229. IF=2.858

6. Bazhenov V.V., Wysokowski M., Petrenko I., Stawski D., Sapozhnikov P., Born R., Stelling A.L., Kaiser S., Jesionowski T., Preparation of monolithic silica-chitin composite under extreme biomimetic conditions. International Journal of Biological Macromolecules (2015) 76, 33-38. IF=2.858

7. Wysokowski M., Petrenko I., Stelling A.L, Stawski D., Jesionowski T., Ehrlich H., Poriferan chitin as a versatile template for extreme biomimetics. Polymers (2015) 7, 235-265. IF=3.681

8. Wysokowski M., Motylenko M., Beyer J., Makarova A., Stöcker H., Walter J., Galli R., Kaiser S., Vyalikh D., Bazhenov V.V., Petrenko I., Stelling A.L., Molodtsov S., Stawski D., Kurzydłowski K.J., Langer E., Tsurkan M.V., Jesionowski T., Heitmann J., Meyer D.C., Ehrlich H., Extreme biomimetic approach for development of novel chitin-GeO2

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nanocomposites with photoluminescent properties. Nano Research (2015) 8, 2288-2301. IF=7.010

9. Wysokowski M., Petrenko I., Motylenko M., Langer E., Bazhenov V.V., Galli R., Stelling A.L., Kljajić Z., Szatkowski T., Kutsova V.Z., Stawski D., Jesionowski T., Renewable chitin from marine sponge as a thermostable biological template for hydrothermal synthesis of hematite nanospheres using principles of extreme biomimetics. Bioinspired Materials (2015) 1, 12-22.

10. Klapiszewski Ł., Bartczak P., Wysokowski M., Jankowska M., Kabat K., Jesionowski T., Silica conjugated with kraft lignin and its use as a novel ‘green’ sorbent for hazardous metal ions removal. Chemical Engineering Journal (2015) 260, 684-693. IF=4.321

11. Wysokowski M., Motylenko M., Walter J., Lota G., Wojciechowski J., Stöcker H., Galli R., Stelling A., Himcinschi C., Niederschlag E., Langer E., Bazhenov V.V., Szatkowski T., Zdarta J., Pertenko I., Kljajić Z., Lesiegang T., Molodtsov S.L., Meyer D.C., Jesionowski T., Ehrlich T., Synthesis of nanostructured chitin-hematite composites under extreme biomimetic conditions. RSC Advances (2014) 4, 61743-61752. IF=3.840

12. Milczarek G., Motylenko M., Modrzejewska-Sikorska A., Klapiszewski Ł, Wysokowski M., Bazhenov V.V., Piasecki A., Konowal E., Ehrlich H., Jesionowski T., Deposition of silver nanoparticles on organically-modified silica in the presence of lignosulfonate. RSC Advances (2014) 4, 52476-52484. IF=3.840

13. Wysokowski M., Zatoń M., Bazhenov V.V., Behm T., Ehrlich A., Stelling A.L. Hog M., Ehrlich H., Identification of chitin in 200-million-year-old gastropod egg capsules. Paleobiology (2014) 40(4), 529-540. IF=2.658

14. Wysokowski M., Klapiszewski Ł., Moszyński D., Bartczak P., Szatkowski T., Majchrzak I., Siwińska-Stefańska K., Bazhenov V.V., Jesionowski T., Modification of Chitin with Kraft Lignin and Development of New Biosorbents for Removal of Cadmium(II) and Nickel(II) Ions. Marine Drugs (2014) 12, 2245-2268. IF=2.853

15. Klapiszewski Ł., Zdarta J., Szatkowski T., Wysokowski M., Nowacka M., Szwarc- Rzepka K., Bartczak P., Siwińska-Stefańska K., Ehrlich H., Jesionowski T., Silica/lignosulfonate hybrid materials: preparation and characterization. Open Chemistry (2014) 12 (6), 719-735. IF=1.329

16. Klapiszewski Ł., Wysokowski M., Majchrzak I., Szatkowski T., Nowacka M., Siwińska-Stefańska K., Szwarc-Rzepka K., Bartczak P., Ehrlich H., Jesionowski T., Preparation and characterization of multifunctional chitin/lignin materials. Journal of Nanomaterials Article ID 425726 (2013) IF=1.644

17. Wysokowski M., Motylenko M., Stöcker H., Bazhenov V.V., Langer E., Dobrowolska A., Czaczyk K., Galli R., Stelling A.L., Behm T., Klapiszewski Ł., Ambrożewicz D.,

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Nowacka M., Molodtsov S.L., Abendroth B., Meyer D.C., Kurzydłowski K.J., Jesionowski T., Ehrlich H., Extreme biomimetic approach: Hydrothermal synthesis of β-chitin/ZnO nanostructured composites. Journal of Materials Chemistry B (2013) 1, 6469-6476. IF=4.726

18. Wysokowski M., Bazhenov V.V., Tsurkan M.V., Galli R., Stelling A.L., Stocker H., Kaiser S., Niederschalg E., Gartner G., Behm T., Ilan M., Petrentko A.Y., Jesionowski T., Ehrlich H., Isolation and identification of chitin in three dimensional skeleton of Aplysina fistularis marine sponge. International Journal of Biological Macromolecules (2013) 62, 94-100. IF=2.858

19. Ehrlich H., Simon P., Motylenko M., Wysokowski M., Bazhenov V.V., Galli R., Stelling A.L., Stawski D., Ilan M., Stocker H., Abendroth B., Born R., Jesionowski T., Kurzydłowski K.J., Meyer D.C., Extreme Biomimetics: formation of zirconium dioxide nanophase using chitinous scaffolds under hydrothermal conditions. Journal of Materials Chemistry B (2013) 1, 5092-5099. IF=4.726

20. Wysokowski M., Motylenko M., Bazhenov V.V., Stawski D., Petrenko I., Ehrlich A., Behm T., Kljajic Z., Stelling A.L., Jesionowski T., Ehrlich H., Poriferan chitin as a template for hydrothermal zirconia deposition. Frontiers of Materials Science (2013) 7, 248-260. IF=1.000

21. Wysokowski M., Behm T., Born R., Bazhenov V., Meißner H., Richter G., Makarova A., Vyalikh D., Schupp P., Jesionowski T., Ehrlich H. Preparation of chitin-silica composites by in vitro silicification of two-dimensional Ianthella basta demosponge chitinous scaffolds under modified Stöber conditions. Materials Science and Engineering C (2013) 33, 3935-3941. IF=3.088

22. Pilarska A., Wysokowski M., Markiewicz E., Jesionowski T., Synthesis magnesium hydroxide and its calcinates by precipitation method with the use of magnesium sulphate and poli(ethylene glycols), Powder Technology (2013) 235, 148-157 IF=2.349

23. Wysokowski M., Piasecki A., Bazhenov V. V., Paukszta D., Born R., Schupp P., Jesionowski T., Poriferan chitin as the scaffold for nanosilica deposition under hydrothermal synthesis conditions. Journal of Chitin and Chitosan Science (2013) 1, 26-33.

24. Pilarska A., Linda I., Wysokowski M., Paukszta D., Jesionowski T., Synthesis Mg(OH)2 from magnesium salts with use NH4OH and direct functionalization by poly(ethylene glycols), Physicochemical Problems of Mineral Processing (2012) 48(2), 631−643. IF=0.926

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PATENT APPLICATIONS

1. Jesionowski T., Klapiszewski Ł., Ehrlich H., Wysokowski M., Majchrzak I., Multifunctional chitin-lignosulfonate materials and method of their synthesis – P.404661

2. Jesionowski T., Klapiszewski Ł., Ehrlich H., Wysokowski M., Majchrzak I., Multifunctional chitin-lignin materials and method of their synthesis – P.404660

3. Pilarska A., Wysokowski M., Linda I., Jesionowski T., Synthesis Mg(OH)2 from magnesium

salts with use NH4OH and direct functionalization by poly(ethylene glycols) – P.397985

4. Pilarska A., Wysokowski M., Jesionowski T., Synthesis of active form of magnesium hydroxide by precipitation metod with application of poly(ethylene glycols) – P.395643

CONFERENCES

1. Wysokowski M., Petrenko I., Bazhenov V.V., Kaiser S., Stelling A.L., Stawski D., Jesionowski T., Ehrlich H., Hydrothermal technology as a tool for development of novel chitin-based materials with respect to extreme biomimetic approach. XXI Conference of Polish Chitin Society – New Aspects in chemistry and applications of chitin and its derivatives (16– 18.09.2015, Szczecin, Poland) Oral presentation

2. Petrenko I., Bazhenov V.V., Wysokowski M., Gali R., Stelling A.L., Niederschlag E., Stöcker H., Walter J., Molodtsov S.L., Jesionowski T., Kutsova V.Z., Ehrlich H., Novel method for mineralization of 3D chitinous scaffolds of poriferan origin in vitro. XXI Conference of Polish Chitin Society – New Aspects in chemistry and applications of chitin and its derivatives (16–18.09.2015, Szczecin, Poland) Oral presentation

3. Bazhenov V.V., Petrenko I., Wysokowski M., Galli R., Stelling A.L., Niederschlag E., Stöcker

H., Walter J., Molodtsov S.L., Jesionowski T., Ehrlich H., Electrodeposition of Cu/Cu2O on 3D chitinous scaffolds of poriferan origin. 12th International Conference of the European Chitin Society/ 13th International Conference on Chitin and Chitosan (30.08-02.09.2015, Münster, Germany) Poster

4. Kaiser S., Wysokowski M., Motylenko M., Petrenko I., Bazhenov V.V., Stelling A.L., Ehrlich H., Hydrothermal synthesis of crystalline germnium oxide on sponge chitin matrices. 12th International Conference of the European Chitin Society/ 13th International Conference on Chitin and Chitosan (30.08-02.09.2015, Münster, Germany) Poster

5. Wysokowski M., Petrenko I., Bazhenov V., Kaiser S., Stelling A.L., Stawski D., Jesionowski D., Ehrlich H. Chitin as novel template for Extreme Biomimetics. 12th International Conference of the European Chitin Society/ 13th International Conference on Chitin and Chitosan (30.08-02.09.2015,Münster, Germany) Poster + Oral presentation

6. Wysokowski M., Materna K., Petrenko I., Klapiszewski Ł., Ehrlich H., Jesionowki T. Solvothermal synthesis of hydrophobic chitin-POSS nanocomposite. European Polymer Federation Congress 2015 (21-26.07.2015, Dresden, Germany) Poster

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7. Jesionowski T., Wysokowski M., Lewandowska O., Materna K., Ehrlich H. Synthesis of advanced chitin-POSS materials prepared via solvothermal method. Eurofillers Polymer Blends 2015, (26-30.06.2015 Montpellier, France) Oral presentation

8. Wysokowski M., Petrenko I., Szatkowski T.,Kaiser S., Bazhenov V.V., Kutsova V.Z., Jesionowski T., Ehrlich H. Extreme biomimetic strategies on duty for biomaterials science. Jahrestagung der Deutschen Gesellschaft für Biomaterialien, (06–08.11.2014, Dresden, Germany) Poster

9. Wysokowski M., Szatkowski T., Petrenko I., Bazhenov V.V., Kaiser S., Jesionowski T., Ehrlich H. Extreme biomimetic approach for development of chitin based hybrid materials. XX Conference of Polish Chitin Society – New Aspects in chemistry and applications of chitin and its derivatives (24.–26.09.2014, Lódz, Poland) Oral presentation – 1st AWARD for the best oral presentation

10. Szatkowski T., Wysokowski M., Petrenko I., Bazhenov V.V., Jesionowski T., Ehrlich H., Extreme biomimetics: Development of 3D spongin-based biocomposites via hydrothermal route Jahrestagung der Deutschen Gesellschaft für Biomaterialien, (06–08.11.2014, Dresden, Germany) Poster

11. Norman M., Pawełko A., Połczyńska D., Król W., Pisarek A., Bartczak P., Wysokowski M., Szatkowski T., Jesionowski T., Ehrlich H., Ocena skuteczności adsorpcji barwników naturalnych na powierzchni komercyjnej chityny przy użyciu metod spektroskopowych. VII Ogólnopolskiego Sympozjum Nauka i Przemysł - metody spektroskopowe w praktyce, nowe wyzwania i możliwości (10.06-12.06.2014 Lublin, Poland) Poster

12. Wysokowski M., Szatkowski T., Bazhenov V.V., Motylenko M., Petrenko I., Jesionowski T., Ehrlich H., Extreme biomimetic approach for synthesis of advanced organic-inorganic chitin-based composites. Euro Bio-Inspired Materials Conference 2014 (18-21.03.2014 Potsdam, Germany )Oral presentation

13. Milczarek G., Modrzejewska-Sikorska A., Klapiszewski Ł., Wysokowski M., Ehrlich H., Jesionowski T. Lignosulfonate-Mediated Synthesis of Silica/Nanosilver Composites. BaltSilica 2014 (01-03.06.2014 Poznań, Poland) Poster

14. Szatkowski T., Bazhenov V.V., Wysokowski M., Motylenko M., Jesionowski T., Ehrlich H., Novel nanostructured hematite-spongin biocomposites obtained using fibrous skeleton of Hippospongia communis marine sponge via hydrothermal route. Euro Bio-Inspired Materials Conference 2014 (18-21.03.2014 Potsdam, Germany) Oral presentation

15. Wysokowski M., Bazhenov V.V., Ehrlich H., Jesionowski T., Extreme biomimetic mineralization of poriferan chitin with use of polyhedral oligomeric silsesquioxane (POSS). 12th International Symposium on Biomineralization – Biomin 12 (27-30.08.2013 Freiberg, Germany) Poster

16. Jesionowski T., Wysokowski M., Ehrlich A., Kljajić Z., Szatkowski T., Stawski D., Ehrlich H., In vitro extreme biomimetic silicification of 3D chitinous scaffolds isolated from Aplysina aerophoba marine sponge skeleton. 12th International Symposium on Biomineralization – Biomin 12 (27-30.08.2013 Freiberg, Germany) Poster

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17. Zgłobicka I., Wysokowski M., Kaiser S., Bazhenov V. V., Makarova A., Vyalikh D., Zdunek J., Płociński T., Motylenko M., Sundareshwar P. V., Witkowski A., Kurzydłowski K. J., Ehrlich H., First investigations into biominerals formation within stalks of fouling diatom Didymosphenia geminata. 12th International Symposium on Biomineralization – Biomin 12 (27-30.08.2013 Freiberg, Germany) Poster

18. Szatkowski T., Wysokowski M., Bazhenov V.V., Ehrlich H., Jesionowski T. Preparation of silica-spongin biocomposite under hydrothermal conditions. 12th International Symposium on Biomineralization – Biomin 12 (27-30.08.2013 Freiberg, Germany) Poster

19. Pisera A., Kurek D., Simon P., Sivkov V., Tsurkan M., Wysokowski M., Ehrlich H., Demineralization of Cambrian fossil demosponge Vauxia gracilenta: Isolation and identification of skeletal chitin 12th International Symposium on Biomineralization – Biomin 12 (27-30.08.2013 Freiberg, Germany) Poster

20. Wysokowski M., Ehrlich H., Jesionowski T. Otrzymywanie biokompozytów chityna-SiO2 z wykorzystaniem chitynowych szkieletów gąbek morskich (P118) „Misja chemo-, bio- i nanotechnologii w Wielkopolskim Centrum Zaawansowanych Technologii – Materiały i Biomateriały” (Poznań, Poland 28-29.11 2011) Poster

21. Wysokowski M., Ehrlich H., Jesionowski T., Extreme biomimetic synthesis of chitin/silica composites (P526) International Conference on Biobased Polymers and Composites, (27- 31.05.2012 Lake Balaton, Siofok, Hungary,) Poster

22. Wysokowski M., Ehrlich H., Jesionowski T., Synthesis and characterisation of novel α- chitin/nanosilica composites, (P527) International Conference on Biobased Polymers and Composites, (27-31.05.2012 Lake Balaton, Siofok, Hungary,) Oral presentation

23. Wysokowski M., Ehrlich H., Jesionowski T., Synthesis and characterisation of novel

chitin/ZrO2 composites (P1751) 15th European Conference on Composite Materials – ECCM, (24-28.06.2012 Venice, Italy) Oral presentation

24. Wysokowski M., Ehrlich H., Jesionowski T., Chitin/nanosilica composites as promising two-dimensional materials for biomedicine. 2nd Summer Symposium on Nanomaterials and their application to Biology and Medicine (21-24.06.2012 Poznań, Poland) Poster

RESEARCH PROJECTS

1. NCN ETIUDA – Project investigator – Development of novel inorganic-organic chitin-based composites obtained under Extreme Biomimetic conditions. 2014/12/T/ST8/00080

2. NCN Preludium – Project investigator – Advanced biomaterials chitin-POSS: synthesis and characterization. 2012/07/N/ST8/03904

3. Mobilność Plus – Project investigator – Development of novel inorganic-organic chitin- based composites obtained under Extreme Biomimetic conditions. 920/MOB/2012/0 – Results of the project have been rewarded by an Interdisciplinary Scientific Team - appointed by the Polish Minister of Education

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SCHOLARSHIPS

1. NCN ETIUDA – Research Grants for Doctoral Candidates 01.01.2015 – 30.06.2015 TU Bergakademie Freiberg ▪ Institut für Experimental Physik

2. DAAD – Research Grants for Doctoral Candidates and Young Academics and Scientists up to 6 months 01.06.2014 – 30.11.2014 TU Bergakademie Freiberg ▪ Institut für Experimental Physik

3. Mobilność Plus – support forinternational mobility of scientists 02.01.2013 – 30.12.2013 TU Bergakademie Freiberg ▪ Institut für Experimental Physik -

4. Era Inżyniera – Scholarships for PhD students 01.06.2014 – 30.11.2014 TU Bergakademie Freiberg ▪ Institut für Experimental Physik

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