First chitin extraction from Plumatella repens (Bryozoa) with comparison to chitins of insect and fungal origin

Accepted Manuscript Title: First chitin extraction from Plumatella repens (Bryozoa) with comparison to chitins of insect and fungal origin ˇ Author: Murat Kaya Vykintas Baublys Ingrida Satkauskien˙ e Bahar Akyuz Esra Bulut Vaida Tubelyt˙e PII: DOI: Reference:

S0141-8130(15)00304-9 http://dx.doi.org/doi:10.1016/j.ijbiomac.2015.04.066 BIOMAC 5072

To appear in:

International Journal of Biological Macromolecules

Received date: Revised date: Accepted date:

16-2-2015 20-4-2015 25-4-2015

ˇ Please cite this article as: M. Kaya, V. Baublys, I. Satkauskien˙ e, B. Akyuz, E. Bulut, V. Tubelyt˙e, First chitin extraction from Plumatella repens (Bryozoa) with comparison to chitins of insect and fungal origin, International Journal of Biological Macromolecules (2015), http://dx.doi.org/10.1016/j.ijbiomac.2015.04.066 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

First chitin extraction from Plumatella repens (Bryozoa) with comparison to chitins of insect and fungal origin Murat Kayaa, *, Vykintas Baublysb,c, Ingrida Šatkauskienėb, Bahar Akyuza, Esra Buluta,

Department of Biotechnology and Molecular Biology, Aksaray University, 68100,

cr

a

ip t

Vaida Tubelytėb

us

Aksaray, Turkey E-Mail: [email protected]

Institute of Biology and Plant Biotechnology, Faculty of Agronomy, Aleksandras Stulginskis

M

c

Department of Biology, Vytautas Magnus University, LT-44404 Kaunas, Lithuania

an

b

Ac ce p

Corresponding Author

te

d

University, LT-53361 Akademija, Kaunas region, Lithuania.

* Address: Aksaray University, Faculty of Science and Letters, Department of Biotechnology and Molecular Biology, 68100, Aksaray, Turkey. E-Mail: [email protected] Tel.: +90-382-288-2184 Fax: +90-382-288-2125

1

Page 1 of 24

Abstract Chitin immediately suggests the representatives of the kingdom Fungi, as well as such phyla as Annelida, Mollusca, Porifera, Cnidaria and, mostly, Arthropoda. Although Bryozoa also represents a chitin-containing phylum, no study has been developed yet on the isolation or characterization of the chitin from it. In this study, physiochemical properties of the chitin

ip t

isolated from Plumatella repens belonging to the phylum Bryozoa was determined for the first time. The chitin structure was also studied comparatively by isolating chitin from an

cr

insect species (Palomena prasina) of the phylum Arthropoda, and Fomes fomentarius belonging to the kingdom Fungi. It was observed that the bryozoan chitin was in the α form,

us

as in the arthropod and fungal chitins. The chitin contents in the dry weight of the bryozoan, fungal and insect species were observed to be 13.3%, 2.4%, and 10.8% respectively. The insect chitin exhibited the highest thermal stability followed by that of the bryozoan and then

an

the fungal chitins. Surface morphologies reveal that the insect and bryozoan chitins were composed of nano fibre and pore structures, whereas the fungal chitin had no pores or fibres.

M

The crystallinity of the insect chitin (CrI=84.9%) was higher than the bryozoan (CrI=60.1%) and fungal chitins (CrI=58.5%).

1. Introduction

te

d

Keywords: Bryozoa, Plumatella, insect, fungi, chitin, extraction

Ac ce p

Studies conducted on chitin isolation and characterization have focused primarily on

organisms from the phylum Arthropoda [1] and kingdom Fungi [2,3]. Recently, some research has also been conducted on the chitin structures of some specific species belonging to the phyla Porifera [4-6] and Cnidaria [7]. There is a limited amount of literature on the chitin structure of the phylum Bryozoa, and it suggests that such organisms have low or no chitin contents.

Bryozoa is a phylum belonging to the Animal kingdom with more than 6000 marine and 65 freshwater species identified worldwide [8]. Almost all bryozoans are colonial, and composed of anywhere from a few to millions of individuals. The size of colonies ranges from 1 cm2 to 1 m2. They cling to the lower parts of aquatic ecosystems and feed on phytoplanktonic

organisms

[9].

This

phylum

has

three

classes:

Gymnolaemata,

Phylactolaemata, and Stenolaemata. Of them, the order Cheilostomata of the class 2

Page 2 of 24

Gymnolaemata has been reported to contain chitin in its cuticle structure [10,11]. Another report [12] indicated that the exoskeleton of bryozoans may be made of inorganic calcium carbonate or organic materials (polysaccharide, protein, or chitin). Chitin is found in statoblasts, which are a survival form produced by bryozoans against poor ambient conditions. Once normal ambient conditions are restored, the statoblast develops to enable the

ip t

production of new individuals [13]. There is a limited amount of studies reporting the presence of chitin in bryozoan. For this reason, the structure and physiochemical properties of

cr

chitin of bryozoan origin remain unclear in detail.

us

Three species, Plumatella repens (phylum Bryozoa), Fomes fomentarius (kingdom Fungi), Palomena prasina (phylum Arthropoda), were selected for chitin isolation in this study. The bryozoan species, P. repens, is a member of the genus Plumatella that only lives in

an

fresh water [14]. P. repens displays a cosmopolitan distribution and is abundantly present, particularly in European freshwater [15]. The fungal species, F. fomentarius, lives on sick and

M

weakened trees and is widely distributed throughout Europe, Asia, Africa, and North America. F. fomentarius is also known as tinder fungus, and is non-toxic, but due to its hard and woody structure it cannot be consumed as food [16]. An insect species, P. prasina (green

d

shield bug), was also selected for chitin isolation because of its wide distribution in Palearctic

te

areas (very common in Europe) [17]. In addition, this insect species harms plants such as hazelnut, cherry, and apple, and has overgrowth to form an invasion [17,18]. The fungus is

Ac ce p

not consumed as a foodstuff, the insect is classified as a pest during overgrowth in the population, and both are cosmopolitan species. These attributes meant they were our preference for use in this study. Furthermore, the fact that there were no previous studies on the chitin structures of the selected fungi and insect species is the other reason underlying our choice.

This is the first study on the physiochemical characteristics of chitin isolated from a

species (P. repens) belonging to phylum Bryozoa.

The present study also includes a

comparison of the bryozoan chitin to chitin from the class Insecta (Arthropoda) and kingdom Fungi.

2. Material and methods 2.1 Sample collection 3

Page 3 of 24

The locations are: Adult individuals (from colonies) of P. repens were collected from the Lazdijai region (Lake Snaigynas, Lithuania) in September 2014. F. fomentarius was collected from Bakirdagi, Kayseri (Turkey) in April 2013. P. prasina was collected from Reservation Plokštynė (Žemaitija National park, Lithuania) in May 2013. The studied species

ip t

for chitin extraction are showed in Fig. 1.

cr

Figure 1

Samples were rinsed with distilled water several times and cleaned to remove potential

us

particles that clung to the outer surface of the three species. Cleaned samples were dried in an oven for seven days at 50oC. Dried samples were then ground into a powder mechanically in a mortar, and the study was performed with initial amounts of 0.8g, 5g, and 1.1g of P. repens,

an

F. fomentarius, and P. prasina, respectively.

M

The same method was employed to isolate the chitin from the three species. Fourier transform infrared spectroscopy (FTIR), elemental analysis, thermogravimetric analysis (TGA), X-ray diffractometry (XRD), and scanning electron microscopy (SEM) analyses were

d

used in the characterization of the isolated chitins as in Kaya et al. [19]. In addition, the

te

acetylation degree (DA) and crystalline index (CrI) values of chitins were calculated

Ac ce p

according to Kaya et al. [19].

2.2 Demineralization

Dry samples in powder form were added to a 2 M HCl solution and refluxed at 100oC

for two hours using a heating magnetic stirrer to remove contained minerals. After refluxing, the samples were placed in blotting paper with a pore diameter of 1 µm and rinsed with distilled water until a neutral pH value was obtained.

2.3 Deproteinization

Demineralized samples were added to a 2 M NaOH solution and refluxed at 140oC for 20 hours to remove the proteins. Then, the same procedure of rinsing the samples with distilled water in blotting paper with a pore diameter of 1 µm until a neutral pH value was obtained was repeated.

4

Page 4 of 24

2.4 Decolourization To remove oil and pigments from the samples after demineralization and deproteinization a solution of distilled water, methanol, and chloroform at a mixing ratio of 4:2:1 respectively was prepared and then refluxed at room temperature for two hours. The derived chitins were filtered with distilled water and then rinsed again until a neutral pH value

ip t

was obtained. The chitins isolated from the species were allowed to rest for three days in the oven at 50oC to complete the drying process. The dry weight of the chitins obtained from the

cr

three different species was measured on a precision scale to calculate the percentage chitin

3. Results and discussion 3.1 Chitin content of Bryozoa, Fungi, and Insecta

us

content in the initial amounts.

an

The percentage chitin content of the dry weight of the whole body structures of the species P. repens, F. fomentarius and P. prasina were found to be 13.3, 2.4 and 10.8

M

respectively (Table 1). Bryozoa was observed to contain more chitin than expected. The dry weight of the general body structures of insects are known to contain chitin in a range of 1020% [20-23]. On the other hand, crustacean shells were found to have a chitin content varying

d

between 7% and 40% by species [24]. As seen, the chitin content of the bryozoan species was

te

the same as that of some insects and crustacean shells. Which suggests that Bryozoa may be an alternative resource for chitin as it exits widely in aqueous ecosystems and allows easy

Ac ce p

chitin extraction.

3.2 Elemental Analysis

The elemental analysis of the chitin from Bryozoa, Fungi, and Insecta, including

the carbon (C), nitrogen (N), and hydrogen (H) contents as well as the corresponding degree of acetylation (DA) are shown in Table 1. The N content (5.74%) of the bryozoan chitin was recorded as slightly higher than the insect chitin (5.56%) but it was found to be lower in the fungal chitin (2.92%). While the C content of the bryozoan and insect chitins were very close to each other, it was found to be higher in the fungal chitin. For the H values, the insect chitin was at the top of the list followed by fungal and bryozoan chitins (Table 1). The DA (%) values calculated from the results of the elemental analysis reveal a DA value of 622.8% for the fungal chitin, 100.5% for the bryozoan chitin, and 83.7% for the insect chitin (Table 1). 5

Page 5 of 24

The N % value of pure chitin was known to be 6.89 [22] and the DA value was assumed as 100% [25]. In this study, the fungal chitin had a very low N content but quite high DA value. Ifuku et al. [3] observed very low N content in fungal chitins and a high DA value (much higher than 100%). These results imply the existence of a high amount of glucan

ip t

residues in fungal chitins [3]. Taking into account the N content and DA value of the fungal chitin in the present study, glucan residues are obvious. The N % and DA values of the

cr

bryozoan chitin were much closer to pure chitin than the insect chitin.

us

Theoretically DA value of the pure chitin is known 100%. In earlier studies, DA values of chitins extracted from different arthropod species were found between 79.8 and 237.2% [22,25-27]. In the present study, DA values of the chitins isolated from the bryozoan

an

and insect were found very close (100.5% for the bryozoan chitin, and 83.7% for the insect chitin) to theoretically described DA value for chitin. Also, According to Hajji et al. [1], DA

M

of chitin affects the solubility, chemical reactivity and biodegradability in many industrial

3.3 FTIR

d

processing.

te

Chitin is known to have three crystal forms, α, β, and γ [28]. FTIR spectroscopy is one of the most popular methods to identify the α, β, or γ form of chitin [29]. Existing studies

Ac ce p

have revealed that chitin with the α-crystallinity displays bands around 1650, 1620, and 1550 cm-1 [30-32]. In this study, these bands were at 1650 cm-1 (C=O secondary amide stretch, Amide I), 1625 cm-1 (C=O secondary amide stretch), and 1551 cm-1 (N–H bend, C–N stretch, Amide II) for bryozoan chitin, 1654, 1623, and 1552cm-1 for fungal chitin, and 1654, 1619, and 1550cm-1 for insect chitin (Fig. 2). The existence of these bands suggested that the extracted chitins were in α- form. In addition, there were no bands at 1540cm-1 in the spectral evaluation, which suggests no protein residues in the chitins [33]. This shows that the deproteinization process during chitin isolation was sufficient. Other major bands in FTIR spectra were as follows: 3434-3423cm-1 (O–H stretching), 3267-3262cm-1 (asymmetric N–H stretching), 3105-3097cm-1 (symmetric N–H stretching), 2923-2922cm-1 (asymmetric C–H stretching), 2878-2845cm-1 (symmetric C–H stretching), 1376-1375cm-1 (CH bend, CH3 symmetric deformation), 1154-1153cm-1 (C–O–C asymmetric stretching), and 1115-1110cm-1 6

Page 6 of 24

and 1022-1009cm-1 (C–O–C symmetric stretching ) [25,32,34]. The ß-glycosidic bond which is known for standard α-chitin was recorded as 897-896 cm-1 [35].

ip t

Figure 2

3.4 SEM

cr

The SEM results revealed that bryozoan and insect chitins had the same surface detected in surface morphology of fungal chitin (Fig. 3).

us

morphologies which are consisting of nanofibers and rare pores but no fibers or pores were Generally, chitin can be grouped in four distinct forms by surface morphology [19]. The first form is a smooth surface morphology without any nano fibres and pores. The second

an

form consists of nano fibres but no pores. The third form has nano fibres and pores together. The fourth form displays two types of pores in different sizes (one is big and the other is

M

small) in combination with nano fibres. In this study, the bryozoan and insect chitins were categorized into the third form characterized by pores and fibres on the surface. On the other

Ac ce p

3.5 TGA

te

Figure 3

d

hand, the chitin isolated from the fungus was the first form that contains no pores and fibres.

The chitins isolated from the three species displayed weight loss in two steps (Table

2). The insect chitin revealed the highest loss in total mass (80%), this was followed by the bryozoan chitin (71%) and fungal chitin (66%). The weight loss observed in the first step results from the evaporation of water in the chitin, and the water loss observed in the second step was due to the deterioration of polysaccharide molecules [25]. The temperature at which the peak maximum deterioration occurred (DTGmax) was observed to be 386ᵒC for the insect chitin, 355ᵒC for the bryozoan chitin, and 334ᵒC for the fungal chitin (Fig. 4). This result suggests that the insect chitin has the greatest thermal stability followed by the bryozoan chitin and fungal chitin.

Figure 4

7

Page 7 of 24

Previous studies also revealed weight losses in α chitins isolated from organisms such as crab, krill, shrimp, crayfish, and insect in two major steps in the range of 30-650ᵒC, which is consistent with this study [21,25,36,37]. Again, previous studies show that the DTGmax values of α chitins ranged between 350-390ᵒC [7,25,29,37]. The DTGmax value of beta chitin is around 300oC [7,29]. The DTGmax values for the bryozoan, fungal and insect chitins

ip t

suggest that these chitins are in α form. The DTGmax value of the fungal chitin was observed to be slightly lower (334ᵒC) than the bryozoan chitin. The low DTGmax value of the fungal

cr

chitin was attributed to the underlying glucan content that could not be fully removed. polymers have application in extreme biomimetics [38].

3.6 XRD

us

Thermostability of chitin is one of the most important properties because thermostable

an

The XRD peaks of chitins isolated from the three different groups were quite similar (Table 3). In particular, two sharp peaks were observed around 9ᵒ and 19ᵒ, and four weak

M

peaks were observed around 13ᵒ, 21ᵒ, 23ᵒ, and 27ᵒ (Fig. 5). According to Jang et al. [29], these observed peaks suggested that the chitins were in the α form. The CrI values of the chitins were calculated to be 60.1%, 58.5%, and 84.9% for bryozoan, fungus, and insect respectively

d

(Table 3). The peak observed for the fungal chitin was 26.9ᵒ, which was sharper than the

te

bryozoan and insect chitins (Fig. 5). This sharper peak at 26.9ᵒ may be because the glucan

Ac ce p

could not be removed from the fungal chitin.

Figure 5

Previous studies reported that the CrI values of coral chitins were fairly low (25-29%)

[7], while chitins isolated from insects, shrimps, and crab shells had quite high CrI values (about 90%) [22,25]. On the other hand, Abdou et al. [36] measured the CrI values for chitins extracted from brown shrimp, pink shrimp, crab, and crayfish as 64%, 66.6%, 59.86%, and 56.94% respectively. These results revealed that the CrI values of chitins vary by the group of organism and the isolation method employed. In this study, the CrI value of the insect chitin was similar to that of the insects involved in other studies. In addition, the low CrI values of bryozoan and fungal chitins were similar to those of crab, shrimp, and crayfish chitins reported by Abdou et al. [36], which is advantageous in sorption studies. Aranaz et al. [39]

8

Page 8 of 24

reported that chitin with low crystallinity is desirable in heavy metal removal by providing low diffusion resistance in adsorption applications.

4. Conclusion The physiochemical properties of chitin from the phylum Bryozoa were revealed for

ip t

the first time with this study, and these properties were compared to those of insect and fungal chitins. The comparison indicated that there was a high similarity between the bryozoan and

cr

insect chitins in terms of chitin content, surface morphology, N content, and DA value. The thermal stability of the bryozoan chitin was lower than the insect chitin and higher than the

us

fungal chitin. On the other hand, the CrI values of the bryozoan and fungal chitins were determined to be similar and much lower than the insect chitin. The low crystalline of the bryozoan chitin may be efficiently employed in adsorption studies. The freshwater bryozoan

an

species examined in this study may be an alternative resource for chitin considering its cosmopolitan nature and high chitin content. Further studies on marine Bryozoa species

References

d

M

would help to better identify the chitin composition of this phylum.

te

[1] S. Hajji, I. Younes, O. Ghorbel-Bellaaj, R. Hajji, M. Rinaudo, M. Nasri, K. Jellouli,

Ac ce p

Structural differences between chitin and chitosan extracted from three different marine sources, Int. J. Biol. Macromol. 65 (2014) 298-306.

[2] M.T. Yen, J.L. Mau, Preparation of fungal chitin and chitosan from shiitake stipes, Fungal Sci. 21 (2006) 1-11.

[3] S. Ifuku, R. Nomura, M. Morimoto, H. Saimoto, Preparation of chitin nanofibers from mushrooms, Materials. 4 (2011) 1417-1425.

9

Page 9 of 24

[4]. H. Ehrlich, M. Maldonado, K-D. Spindler, C. Eckert, T. Hanke, R. Born, C. Goebel, P. Simon, S. Heinemann, H. Worch, First evidence of chitin as a component of the skeletal fibers of marine sponges. Part I. Verongidae (Demospongia: Porifera), J. Exp. Zool. B Mol.

ip t

Dev. Evol. 308B (2007) 347-356.

cr

[5]. H. Ehrlich, J.K. Rigby, J.P. Botting, M. Tsurkan, C. Werner, P. Schwille, Z. Petrasek, A. Pisera, P. Simon, V. Sivkov, D.V. Vyalikh, S.L. Molodtsov, D. Kurek, M. Kammer, S.

us

Hunoldt, R. Born, D. Stawski, A. Steinhof, W. Bazhenov, T. Geisler, Discovery of 505-

an

million-year old chitin in the basal demosponge Vauxia gracilenta, Sci. Rep. 3 (2013) 3497.

M

[6]. H. Ehrlich, O. Kaluzhnaya, E. Brunner, M.V. Tsurkan, A. Ereskovsky, M. Ilan, K.R. Tabachnick, V.V. Bazhenov, S. Paasch, M. Kammer, R. Born, A. Stelling, R. Galli, S.

d

Belikov, O.V. Petrova, V. Sivkov, D. Vyalikh, S. Hunoldt, G. Wörheide, Identification and

te

first insights into the structure and biosynthesis of chitin from the freshwater sponge Spongilla

Ac ce p

lacustris, J. Struct. Biol. 183 (3) (2013) 474-483.

[7] B.A. Juárez-de la Rosa, P. Quintana, P.L. Ardisson, J.M. Yáñez-Limón, J.J. Alvarado-Gil, Effects of thermal treatments on the structure of two black coral species chitinous exoskeleton, J. Mater. Sci. 47 (2012) 990–998.

[8] M. Hirose, S.F. Mawatari, Freshwater Bryozoa of tonle sap, Cambodia, Zool. Sci. 24 (2007) 630-641.

10

Page 10 of 24

[9] P.E. Bock, D.P. Gordon, Phylum Bryozoa Ehrenberg, 1831. In Zhang Z-Q (Ed) Animal biodiversity: an outline of higher-level classification and survey of taxonomic richness

ip t

(Addenda 2013), Zootaxa. 3703 (2013) 67–74.

cr

[10] W.C. Banta, The body wall of Cheilostome Bryozoa, J. Morphol. 125 (1969) 497-508.

[11] N.B. Lopanik, N.M. Targett, N. Lindquist, Ontogeny of a symbiont-produced chemical

us

defense in Bugula neritina (Bryozoa), Mar. Ecol. Prog. Ser. 327 (2006) 183-191.

an

[12] E.E. Ruppert, R.S. Fox, R.D. Barnes, Invertebrate zoology, a functional evolutionary

M

approach, seventh ed., Brooks Cole Thomson, Belmont, CA, 2004.

d

[13] R. Mitra, S. Crawford, A.F. Barton, S. Briggs, J.D. Orbell, A benign approach to the

te

preparation of freshwater bryozoan statoblasts for scanning electron microscopy (SEM)

Ac ce p

imaging, New. Zeal. J. Zool. 40 (2013) 154-159.

[14] T.S. Wood, The pipeline menace of freshwater bryozoans, Denisia. 16 (2005) 203-208.

[15] K.A. Økland, J. Økland, G. Geimer, J.A. Massard, Freshwater bryozoans (Bryozoa) of Norway IV: distribution and ecology of four species of Plumatella with notes on Hyalinella punctate, Hydrobiol. 501 (2003) 179–198.

[16] A. Afyon, M. Konuk, D. Yağız, S. Helfer, A study of wood decaying macro fungi of the western black sea region, Turkey, Mycotaxon. 93 (2005) 319-322.

11

Page 11 of 24

[17] T.R.E. Southwood, D. Leston, Land and water bugs of the British Isles the Wayside and Woodland Series, London, 1959.

ip t

[18] I. Saruhan, C. Tuncer, I. Akca, Development of green shield bug (Palomena prasina L,

cr

Heteroptera: Pentatomidae) in different temperatures, Zemdirbyste. 97 (2010) 55-60.

[19] M. Kaya, O. Seyyar, T. Baran, S. Erdoǧan, M. Kar, A physicochemical characterization

an

morphology, Int. J. Biol. Macromol. 65 (2014) 553–558.

us

of fully acetylated chitin structure isolated from two spider species: With new surface

M

[20] M. Zhang, A. Haga, H. Sekigushi, S. Hirano, Structure of insect chitin isolated from beetle larva cuticle and silkworm (Bombyx mori) pupa exuvia, Int. J. Biol. Macromol. 27

te

d

(2000) 99–105.

[21] A.T. Paulino, J.I. Simionato, J.C. Garcia, J. Nozaki, Characterization of chitosan and

Ac ce p

chitin produced from silk worm chrysalides, Carbohyd. Polym. 64 (2006) 98–103.

[22] S. Liu, J. Sun, L. Yu, C. Zhang, J. Bi, F. Zhu, M. Qu, C. Jiang, Q. Yang, Extraction and characterization of chitin from the beetle Holotrichia parallela motschulsky, Molecules. 17 (2012) 4604–4611.

[23] M. Kaya, T. Baran, A. Mentes, M. Asaroglu, G. Sezen, K.Ö. Tozak, Extraction and characterization of α-chitin and chitosan from six different aquatic invertebrates, Food Biophys. 9 (2014) 145–57.

12

Page 12 of 24

[24] A. Tolaimate, J. Desbrieres, M. Rhazi, A. Alagui, Contribution to the preparation of chitins and chitosans with controlled physico-chemical properties, Polymer. 44 (2003) 7939–

ip t

7952.

[25] P. Gonil, W. Sajomsang, Applications of magnetic resonance spectroscopy to chitin from

cr

insect cuticles, Int. J. Biol. Macromol. 51(4) (2012) 514-522.

us

[26] J. Majtan, K. Bilikova, O. Markovic, J. Grof, G. Kogan, J. Simuth, Isolation and characterization of chitin from bumblebee (Bombus terrestris), Int. J. Biol. Macromol.

M

an

40 (2007) 237-241.

[27] M. Kaya, N. Bağrıaçık, O. Seyyar, T. Baran, Comparison of chitin structures derived

d

from three common wasp species (Vespa crabro Linnaeus, 1758, Vespa orientalis Linnaeus,

Ac ce p

press).

te

1771 and Vespula germanica (Fabricius, 1793)), Arch. Insect Biochem. Physiol. (2015) (in

[28] J.C. Jiang, M.Q. Xu, Kinetics of heterogeneous deacetylation of β-chitin, Chem. Eng. Technol. 29 (2006) 511-516.

[29] M.K. Jang, B.G. Kong, Y.I. Jeong, C.H. Lee, J.W. Nah, Physicochemical characterization of α-chitin, β-chitin, and γ-chitin separated from natural resources, J. Polym. Sci. Part. A: Polym. Chem. 42 (2004) 3423–3432.

[30] R.L. Lavall, O.B.G. Assis, S.P. Campana-Filho, Beta-chitin from the pens of Loligo sp: Extraction and characterization, Bioresour. Technol. 98 (2007) 2465–72. 13

Page 13 of 24

[31] M. Rinaudo, Chitin and chitosan: properties and applications, Prog. Polym. Sci. 3 (2006)

ip t

603–632.

[32] J. Brugnerotto, J. Lizardi, F.M. Goycoolea, W. Argüelles-Monal, J. Desbrieres, M.

cr

Rinaudo, An infrared investigation in relation with chitin and chitosan characterization,

us

Polymer. 42 (2001) 3569–3580.

[33] A. Morin, A. Dufresne, Nanocomposites of chitin whiskers from Riftia tubes and poly

M

an

(caprolactone), Macromolecules. 35 (2002) 2190–2199.

[34] K. Van de Velde, P. Kiekens, Structure analysis and degree of substitution of chitin, C NMR, Carbohyd.

te

Polym. 58 (2004) 409.

13

d

chitosan and dibutyryl chitin by FT-IR spectroscopy and solid state

Ac ce p

[35] M. Wysokowski, V.V. Bazhenov, M.V. Tsurkan, R. Galli, A.L. Stelling, H. Stöcker, S. Kaiser, E. Niederschlag, G. Gärtner, T. Behm, M Ilan, A.Y. Petrenko,T. Jesionowski, H. Ehrlich, Isolation and identification of chitin in three-dimensional skeleton ofAplysina fistularis marine sponge, Int. J. Biol. Macromol. 62 (2013) 94-100.

[36] E.S. Abdou, K.S.A. Nagy, M.Z. Elsabee, Extraction and characterization of chitin and chitosan from local sources, Bioresour. Technol. 99 (2008) 1359–1367.

14

Page 14 of 24

[37] Y. Wang, Y. Chang, L. Yu, C. Zhang, X. Xu, Y. Xue, Z. Li, C. Hue, Crystalline structure and thermal property characterization of chitin from Antarctic krill (Euphausia

ip t

superba), Carbohyd. Polym. 92 (2013) 90–97.

[38] M. Wysokowski, I. Petrenko, A.L. Stelling, D. Stawski, T. Jesionowski, H. Ehrlich,

cr

Poriferan Chitin as a Versatile Template for Extreme Biomimetics, Polymers. 7 (2015) 235-

us

265.

[39] I. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, G. Galed, A. Heras,

Ac ce p

te

d

M

an

Functional characterization of chitin and chitosan, Curr. Chem. Biol. 3 (2009) 203-230.

15

Page 15 of 24

Figure captions Figure 1: The studied species for chitin extraction: a) Bryozoa (Plumatella repens), b) Fungi (Fomes fomentarius), and c) Insecta (Palomena prasina).

Fungi (Fomes fomentarius), and c) Insecta (Palomena prasina).

ip t

Figure 2: FTIR spectrum data of chitins extracted from a) Bryozoa (Plumatella repens), b)

cr

Figure 3: Scanning electron microscopy (SEM) pictures of chitins isolated from a, b)

prasina).

us

Bryozoa (Plumatella repens), c, d) Fungi (Fomes fomentarius), and e, f) Insecta (Palomena

Figure 4: TGA of chitins extracted from a) Bryozoa (Plumatella repens), b) Fungi (Fomes

an

fomentarius), and c) Insecta (Palomena prasina).

M

Figure 5: X-ray diffractograms of chitins extracted from a) Bryozoa (Plumatella repens), b)

Ac ce p

te

d

Fungi (Fomes fomentarius), and c) Insecta (Palomena prasina).

16

Page 16 of 24

Tables Table 1. Elemental analysis result of chitin from Bryozoa, Fungi, and Insecta with the degree of acetylation-DA (%) and chitin content. Chitin sources

N (%)

C (%)

H (%)

DA (%)

Content

of

Bryozoa (Plumatella

5.74

39.4

6.30

100.5

2.92

46.29

7.26

622.8

5.56

36.5

8.41

Fungi (Fomes Insecta (Palomena

us

fomentarius)

83.7

2.4

10.8

Ac ce p

te

d

M

an

prasina)

13.3

cr

repens)

ip t

chitin (%)

17

Page 17 of 24

Table 2. Thermogravimetric analysis (TGA) results of chitin extracted from Bryozoa, Fungi, and Insecta. mass Total mass loss DTG max (ᵒC)

(%)

loss (%)

(%)

Bryozoa

6

65

71

3

63

66

5

75

80

(Plumatella repens) Fungi (Fomes

Insecta

334

us

fomentarius)

355

ip t

isolated from

cr

Chitin samples First mass loss Second

an

(Palomena

386

Ac ce p

te

d

M

prasina)

18

Page 18 of 24

Table 3. X-ray diffraction data of chitins isolated from Bryozoa, Fungi, and Insecta. Chitin

from XRD Peaks at 2θ

CrI (%)

organisms Bryozoa

60.1

9.2, 12.7, 19.8, 21.1, 23.8 and 27ᵒ

ip t

(Plumatella repens)

58.5

9.7, 13.5, 19.6, 21.1, 23.2 and 26.9ᵒ

cr

Fungi

fomentarius) Insecta

9.4, 13.4, 19.5, 21.1, 23.18 and 26.8ᵒ

84.9

an

(Palomena

us

(Fomes

Ac ce p

te

d

M

prasina)

19

Page 19 of 24

Ac

ce

pt

ed

M

an

us

cr

i

Figure 1

Page 20 of 24

Ac ce p

te

d

M

an

us

cr

ip t

Figure 2

Page 21 of 24

Ac ce p

te

d

M

an

us

cr

ip t

Figure 3

Page 22 of 24

Ac

ce

pt

ed

M

an

us

cr

i

Figure 4

Page 23 of 24

Ac ce p

te

d

M

an

us

cr

ip t

Figure 5

Page 24 of 24