International Journal of Biological Macromolecules 75 (2015) 7–12
Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Description of a new surface morphology for chitin extracted from wings of cockroach (Periplaneta americana) Murat Kaya a,b,∗ , Talat Baran b,c a b c
Faculty of Science and Letters, Department of Biotechnology and Molecular Biology, Aksaray University, 68100 Aksaray, Turkey Science and Technology Application and Research Center, Aksaray University, 68100 Aksaray, Turkey Department of Chemistry, Faculty of Science and Letters, Aksaray University, 68100 Aksaray, Turkey
a r t i c l e
i n f o
Article history: Received 30 October 2014 Received in revised form 3 December 2014 Accepted 8 January 2015 Available online 15 January 2015 Keywords: Chitin New morphology Nanoporous ESEM Insect Thermal properties
a b s t r a c t In this study a new morphology of chitin, which could find wide applications in the fields of medicine, pharmacy, agriculture, food and textiles, has been described. The chitin was isolated from the wings of Periplaneta americana employing a conventional method. Considering chitin isolation studies conducted previously, chitin has three surface morphologies, which are (1) hard and rough surface without pores or nanofibers, (2) surface solely composed of nanofibers and (3) surfaces with both pores and nanofibers. In this study, the surface of the chitin, examined with environmental scanning electron microscopy (ESEM), only has oval nanopores (230–510 nm) without nanofibers, and this is different from the above mentioned surface morphologies. The nanopores are not distributed on the chitin surface randomly. Typically, there is a pore in the center that is surrounded by six or seven other pores in an ordered manner. Structures similar to cell walls exist between the pores. Chitin with the new surface morphology was characterized using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG), X-ray diffraction (XRD) and elemental analysis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The molecular weight, the degree of acetylation or deacetylation of the chitin and its derivatives together with the surface morphology are very important aspects of chitin when considering the effective use of the biomaterial [1–3]. Chitin and chitosan can be used in a lot of areas thanks to their non-toxicity, biocompability and biodegradable properties. These biopolymers have been widely used in controlled drug release capsule production and weight loss pills that have a high oil retention capacity [4,5]. They are also used in the food and textile industries due to their antimicrobial and antioxidant effects [5–7]; while, additionally they are used in agriculture, waste water treatment and veterinary and human medicine [6]. For more applications of chitin and its derivatives, there are a range of review studies [8–12]. Therefore, the
∗ Corresponding author at: Faculty of Science and Letters, Department of Biotechnology and Molecular Biology, Aksaray University, 68100 Aksaray, Turkey. Tel.: +90 382 288 2184; fax: +90 382 288 2125. E-mail addresses: [email protected] (M. Kaya), talatbaran [email protected] (T. Baran). http://dx.doi.org/10.1016/j.ijbiomac.2015.01.015 0141-8130/© 2015 Elsevier B.V. All rights reserved.
description of a new surface morphology of chitin that has a wide range of applications is closely related to many branches of science. Scientists have focused on chitin isolation and characterization from crayfish, crabs and shrimp because of their commercial production [13]. The use of chitin and its derivatives is increasing worldwide, both in overall consumption levels and areas of application. Due to this reason, new chitin sources are being sought. Recently, some organisms such as insects, anthozoan, fungi and sponges have been researched as alternative chitin sources [13–23]. In the present study, the wings and other body parts of Periplaneta americana were screened for their ability to be an alternative chitin source. Periplaneta americana is an insect species that is distributed worldwide. The adults have a body size of 35–40 mm, and they show excessive growth, especially when it is hot, humid and there is abundant food. People are familiar with this insect because they live in the trash, kitchens, bathrooms, near radiators, sewers and drainage channels. Therefore, they are described as a big problem in terms of human health; they cause asthma and intestinal diseases in humans [24,25]. In addition, the body of this insect has been found to carry pathogenic bacteria such as Klebsiella, Pseudomonas, Escherichia coli, Staphylococcus, Enterobacter, Streptococcus, Serratia, Bacillus and Proteus [25]. They can achieve huge numbers with
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extreme reproduction and development in moist and warm areas [26]. In this study, a new surface morphology of chitin, from the wings of an insect (P. americana), was recorded and defined. The surface morphologies of the chitins isolated from the wings of an insect and other body part were examined comparatively using ESEM. In addition, the new chitin morphology was characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG), X-ray diffraction (XRD) and elemental analysis.
2. Materials and methods 2.1. Sample collection Individuals belonging to P. americana were collected by handpicking in Kirikan (Hatay, Turkey) on October 14, 2013. After collection, the samples were taken to the laboratory in a 20% ethanol solution. The samples were washed with distilled water for the removal of possible residues, and then the wings were removed with the help of forceps. The separated wings and other parts of the insect were incubated for 2 days to dry in an oven at 60 ◦ C. The pictures of P. americana and its wings were shown in Fig. 1.
2.2. Chitin extraction The dried wings and other parts were pulverized separately, in a mortar, into a powdered form. Then a demineralization step was performed in which 10 g of each material was weighted and refluxed in a 4 M HCl solution for 2 h at 75 ◦ C. Subsequently, the mixture was filtered with a 1 m filter paper and rinsed with distilled water to a neutral pH. To remove the protein, the filtrate was refluxed in a 4 M NaOH solution for 20 h at 150 ◦ C. Then the mixture was filtered and washed with distilled water to a neutral pH. Afterwards, the extract was mixed with a magnetic heating stirrer in a solution of water, methanol and chloroform (in the ratio of 4:2:1) for 4 h at 30 ◦ C. Then the extract was filtered and washed again with distilled water to a neutral pH. Finally, the material was dried in an oven at 60 ◦ C for 2 days. The chitin contents of the dried wings and other parts of the insects were calculated. 2.3. ESEM The dried wings and other parts of P. americana were coated with gold using a “Sputter Coater” (Cressingto Auto 108) at Selc¸uk University. Then the surfaces of the samples were examined employing a QUANTA- FEG 250 ESEM (at 15,000×, 40,000×, 80,000× and 100,000× magnifications) at the Science and Technology Application and Research Center (Aksaray University). 2.4. Fourier transform infrared analysis (FTIR) Analysis of chitin isolated from the wings of P. americana was conducted using a Perkin–Elmer FTIR at 4000–625 cm−1 . 2.5. Thermogravimetric analysis (TG) TGA analysis of extracted chitin from the wings of P. americana was done using an EXSTAR S11 7300 from 25 to 650 ◦ C at a heating rate of 10 ◦ C min−1 . 2.6. X-ray diffraction (XRD) XRD peaks of chitin extracted from the wings of P. americana were taken at 40 kV, 30 mA and 2 with a scan angle from 5◦ to 45◦ using a Rigaku D max 2000 system in Harran University (HUMEL). The crystalline index (CrI) values were calculated with the formula given below: CrI110 =
(I
110
− Iam )
I110
× 100
(1)
∼ 19.58◦ and Iam is where I110 is the maximum intensity at 2 = the intensity of amorphous diffraction at 2 ∼ = 12.88◦ . 2.7. Elemental analysis A Thermo Flash 2000 was employed to determine the N, H and C contents of the chitin structure isolated from the wings of P. americana. Then degree of acetylation (DA) of the chitin was calculated using the following formula [27]:
DA =
(C/N − 5.14) 1.72
× 100
(2)
3. Results and discussion 3.1. ESEM
Fig. 1. Periplaneta americana: (a) habitus, (b) wings.
ESEM analyses conducted on chitin have revealed that it has three main surface morphologies: (1) a hard and rough surface
M. Kaya, T. Baran / International Journal of Biological Macromolecules 75 (2015) 7–12
9
Fig. 2. ESEM pictures of chitin isolated from the wings of Periplaneta americana (magnifications: (a) 15,000×, (b) 40,000×, (c) 80,000× and (d) 100,000×).
without pores or nanofibers, with chitin isolated from certain fungi and Daphnia magna eggs having this type of surface morphology [28,29]. (2) A surface solely composed of nanofibers, with this type of morphology having been observed in samples isolated from fungi [30,31] as well as Antarctic krill, certain aquatic insect species and aquatic Asellus aquaticus (Cructacea) [23,32]. (3) A surface with both pores and nanofibers, which is the most common morphology [33–35]. In this study, a new morphology was defined: pores but no nanofibers. The shape of the pores is oval and their diameter ranges from 230 to 510 nm. Previous studies reported that the diameters of pores on chitin isolated from shrimp, Gammarus and silkworm chrysalides ranged from 150 to 250 nm [33–35]. The pores on the chitin that are defined in this study are larger than the ones reported in the literature. This new morphology has cell walllike structures between the pores. These pores are hollow and not very deep (Fig. 2). The distance between two pores is in the range of
125–340 nm. Fig. 2 shows this new chitin morphology at 15,000×, 40,000×, 80,000× and 100,000× magnifications. Another important point is that more than 50% of the chitin surface is composed of pores (Fig. 2). This highly porous surface could have significant biomaterial applications. The pores are regular and positioned on the surface orderly (Fig. 3). It is evident from Fig. 3 that there is a pore in the center that is surrounded by six (Fig. 3a) or seven (Fig. 3b) other pores in an ordered manner. Chitin with this magnificent design may find important applications in various fields. Fig. 4 depicts a comparison of the new type morphology with previously known types. Fig. 4a represents a hard and rough surface without pores or nanofibers [29]. Fig. 4b shows the surface morphology solely composed of nanofibers [23]. A surface with both pores and nanofibers can be seen Fig. 4c [35]. The surface of chitin extracted from other parts of P. americana (not the wings) has both pores and nanofibers (Fig. 5), which is an example of type 3.
Fig. 3. ESEM pictures of chitin extracted from the wings of Periplaneta americana ((a) one pore in the middle and six pores around and (b) one pore in the middle and seven pores around).
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Fig. 4. SEM pictures of chitin types isolated from various organisms: (a) hard and rough surface without pores or nanofibers (isolated from Daphnia magna resting eggs), (b) surface solely composed of nanofibers (isolated from Asellus aquaticus) and (c) surface with both pores and nanofibers (isolated from Gammarus).
3.2. FTIR Chitin has three crystalline forms: alpha, beta and gamma, but there is little information regarding the gamma form [36]. The most important feature of FTIR spectra is that they can be assessed to distinguish the alpha form from the beta via whether the Amide I band is split or not. In the alpha form, the Amide I band splits into two bands at about 1650 and 1620 cm−1 [32]. In the beta form, only one Amide I band is observed at 1656 cm−1 . Beta chitins are found in organisms such as squid pens [36]. It is known that alpha chitin is found in the Arthropoda [19,27]. In the FTIR spectra of the chitin extracted in this study, the Amide I band is split at 1654 and 1621 cm−1 (Fig. 6), which indicates that this chitin from P. americana is in the alpha form. The other FTIR spectrum bands were observed as the following; 3435 cm−1 (O–H stretching), 3259 cm−1 (asymmetric N–H stretching), 3108 cm−1 (symmetric N–H stretching), 2924 cm−1 (asymmetric C–H stretching), 2857 cm−1 (symmetric C–H stretching), 1420 cm−1 (CH2 ending and CH3 deformation), 1376 cm−1 (CH bend, CH3 symmetric deformation), 1308 cm−1 (CH2 wagging),
1154 cm−1 (C–O–C asymmetric stretching), 1114 cm−1 (asymmetric bridge oxygen stretching), 1114 cm−1 (asymmetric in-phase ring stretching mode), 1068 m−1 (C–O–C asymmetric stretch in phase ring), 1012 cm−1 (C–O asymmetric stretch in phase ring), 952 cm−1 (CH3 wagging), 895 cm−1 (CH ring stretching) [15]. The FTIR bands of commercial chitin were observed to be as follows; 3438, 3260, 3103, 2932, 2865, 1654, 1623, 1555, 1428, 1376, 1310, 1155, 1115, 1068, 1016, 952, 896 cm−1 . High similarity was recorded between the commercial and the chitin extracted from wings of P. americana. 3.3. TG In the literature, characterization studies for chitin extracted from crab, shrimp and insects using TG analysis have revealed that there are two mass loss steps: one at around 100 ◦ C and the other at 350–390 ◦ C [36]. In the present work we obtained similar results to the previous studies (Fig. 7). The first mass loss step represents the evaporation of absorbed water molecules; the second one corresponds with the decomposition of the chitin polymer. In the first
Fig. 5. ESEM pictures of chitin isolated from the insect’s (Periplaneta americana) body without wings (magnifications: (a) 30,000× and (b) 40,000×).
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Fig. 8. XRD peaks of chitin extracted from the wings of Periplaneta americana.
organisms can vary in the range of 54–90% [13,37]. This wide range can be attributed to differences in chitin source and purity of the isolated chitin. Fig. 6. FTIR bands of chitin isolated from the wings of Periplaneta americana.
step the mass loss is 5% and in the second step it is 76%. The DTGmax where chitin monomers decompose has been determined to be 389 ◦ C. It has been reported in the literature that the DTGmax values for chitin from crab, shrimp and Antarctic krill were 365–380 ◦ C. Another chitin characterization study by Sajomsang and Gonil [19] reported that the DTGmax for chitin from cicada sloughs (insect) was 379 ◦ C. The DTGmax value that we determined in this work is very similar to the values reported in literature. 3.4. XRD X-ray diffraction analysis of chitin shows two strong peaks and four faint peaks [19,31,32,36]. We observed two strong peaks (9.14 and 19.58) and four faint peaks (12.88, 20.98, 23.12 and 26.8) (Fig. 8). The crystallinity index (CrI) has been calculated to be 86.7%. Liu et al. [13] reported that the CrI for chitin from Holotrichia parallela (insect) was 89%. The CrI values of chitin from different
3.5. Elemental analysis We determined the C, N and H contents of the chitin from the wings of P. americana to be 45.74%, 6.69% and 6.59% respectively. The N percentage value of completely acetylated chitin is known to be 6.89 [17]. The N percentage values for chitin from different organisms are as follows: shrimp, 4.85%; bumblebee, 5.92%; crab, 5.3%; Holotrichia parallela (beetle), 6.45%; another shrimp, 6.24%; and cicada sloughs, 5.92% [13,17,19,38]. It is known that the N content of chitin from fungi is low (2.96%) due to the glucan residues in chitin [31]. The N content determined in this study approached the value of completely acetylated chitin. This indicates that chitin obtained in this study has higher purity than that reported in the literature. The degree of acetylation of chitin cannot exceed 100% [19], and the degree of acetylation of the chitin we isolated was calculated employing the formula given in the materials and methods section; it was found to be 98.67%. Some workers reported that the degrees of acetylation of chitin from shrimp, insect and crap were 112–237%, 101–132% and 104% respectively [13,17,19,38]. These values are higher than 100%, which indicates that the isolated chitin contains some impurities, like protein and inorganic materials. 3.6. Chitin content of wings and insect body (without wings) It has been determined that the dry weight of the wings of the P. americana was 18% chitin, while the chitin content of the other parts of the organism was 13%. The dry weights in other organisms are Holotrichia parallela contains 15% chitin [13], Bombyx mori 15–20% [37] and Cicada sloughs 36% [19]. Our chitin content result is similar to those of H. parallela and B. mori. 4. Conclusion
Fig. 7. TG thermograms and DTG curves of chitin from the wings of Periplaneta americana.
Periplaneta americana is a cosmopolitan species that can be cultured easily. Twenty individuals of this species were obtained from Hacettepe University (Ankara) and cultured in our animal biodiversity laboratory. The temperature and humidity of the laboratory is conditioned to be 30 ◦ C. Many newborns and eggs have been generated in only 2 months; this species multiples in hot and humid conditions [26]. Since this species is an invader and can be easily
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cultured, this organism could be considered as an alternative chitin source to crab, shrimp, crayfish and prawn. Chitin is found in the body structures of more than one million invertebrate organisms including representatives of fungi, diatoms, protists, anthozoans, nematods, sponges and mostly arthropods. Considering the chitin isolation and characterization studies conducted so far, around 50 different species have been investigated. In this study, the isolation and characterization of chitin was conducted using only the wings of an organism and not the whole body for the first time, and a new surface morphology for chitin was defined. The study showed that the physical characteristics of chitin can be different depending on the body part of the same organism. The chitin isolation and characterization procedure that we described here can be applied to various organisms. Furthermore, it is possible that this chitin isolation and characterization process can be assessed regarding commercial chitin sources, such as crab, shrimp and crayfish. The number of pores on the chitin’s surface is an important parameter in metal sorption studies [9]. Chitin with this new morphology may be utilized to remove heavy metal ions. In addition, chitin with pores can be used in tissue engineering applications [39]. In the most common morphology (referred to as type 3) nanofibers and pores are randomly disturbed on the chitin surface [33–35]. In the new morphology that we defined here, the distribution pattern of the pores on the chitin has a magnificent design, and this design is depicted in this study for the first time. In conclusion, (1) chitin from the wing of P. americana was isolated and characterized using FTIR, TG, XRD and elemental analysis, (2) the isolated chitin is in the alpha form and (3) very similar to the commercial chitin from shrimp, crab and crayfish. The feature that distinguishes this chitin from the others is its surface morphology. We believe that chitin with this new morphology could be a promising material for further studies and a product that is desired in a wide range of applications. References [1] I. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, G. Galed, A. Heras, Curr. Chem. Biol. 3 (2009) 203–230. [2] H. Ehrlich, Int. Geol. Rev. 52 (2010) 661–699. [3] R.A.A. Muzzarelli, Mar. Drugs 9 (2011) 1510–1533. [4] P.A. Felse, T. Panda, Bioprocess Eng. 20 (1999) 505–512. [5] B.K. Park, M.M. Kim, Int. J. Mol. Sci. 11 (2010) 5152–5164. [6] P.K. Dutta, J. Dutta, V.S. Tripathi, J. Sci. Ind. Res. India. 63 (2004) 20–31. [7] A. Anitha, S. Sowmya, P.T. Sudheesh Kumar, S. Deepthi, K.P. Chennazhi, H. Ehrlich, M. Tsurkan, R. Jayakumar, Prog. Polym. Sci. 39 (2014) 1644–1667. [8] M.N.V. Ravi Kumar, React. Funct. Polym. 46 (2000) 1–27.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Description of a new surface morphology for chitin extracted from wings of cockroach (Periplaneta americana) Murat Kaya a,b,∗ , Talat Baran b,c a b c
Faculty of Science and Letters, Department of Biotechnology and Molecular Biology, Aksaray University, 68100 Aksaray, Turkey Science and Technology Application and Research Center, Aksaray University, 68100 Aksaray, Turkey Department of Chemistry, Faculty of Science and Letters, Aksaray University, 68100 Aksaray, Turkey
a r t i c l e
i n f o
Article history: Received 30 October 2014 Received in revised form 3 December 2014 Accepted 8 January 2015 Available online 15 January 2015 Keywords: Chitin New morphology Nanoporous ESEM Insect Thermal properties
a b s t r a c t In this study a new morphology of chitin, which could find wide applications in the fields of medicine, pharmacy, agriculture, food and textiles, has been described. The chitin was isolated from the wings of Periplaneta americana employing a conventional method. Considering chitin isolation studies conducted previously, chitin has three surface morphologies, which are (1) hard and rough surface without pores or nanofibers, (2) surface solely composed of nanofibers and (3) surfaces with both pores and nanofibers. In this study, the surface of the chitin, examined with environmental scanning electron microscopy (ESEM), only has oval nanopores (230–510 nm) without nanofibers, and this is different from the above mentioned surface morphologies. The nanopores are not distributed on the chitin surface randomly. Typically, there is a pore in the center that is surrounded by six or seven other pores in an ordered manner. Structures similar to cell walls exist between the pores. Chitin with the new surface morphology was characterized using Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG), X-ray diffraction (XRD) and elemental analysis. © 2015 Elsevier B.V. All rights reserved.
1. Introduction The molecular weight, the degree of acetylation or deacetylation of the chitin and its derivatives together with the surface morphology are very important aspects of chitin when considering the effective use of the biomaterial [1–3]. Chitin and chitosan can be used in a lot of areas thanks to their non-toxicity, biocompability and biodegradable properties. These biopolymers have been widely used in controlled drug release capsule production and weight loss pills that have a high oil retention capacity [4,5]. They are also used in the food and textile industries due to their antimicrobial and antioxidant effects [5–7]; while, additionally they are used in agriculture, waste water treatment and veterinary and human medicine [6]. For more applications of chitin and its derivatives, there are a range of review studies [8–12]. Therefore, the
∗ Corresponding author at: Faculty of Science and Letters, Department of Biotechnology and Molecular Biology, Aksaray University, 68100 Aksaray, Turkey. Tel.: +90 382 288 2184; fax: +90 382 288 2125. E-mail addresses: [email protected] (M. Kaya), talatbaran [email protected] (T. Baran). http://dx.doi.org/10.1016/j.ijbiomac.2015.01.015 0141-8130/© 2015 Elsevier B.V. All rights reserved.
description of a new surface morphology of chitin that has a wide range of applications is closely related to many branches of science. Scientists have focused on chitin isolation and characterization from crayfish, crabs and shrimp because of their commercial production [13]. The use of chitin and its derivatives is increasing worldwide, both in overall consumption levels and areas of application. Due to this reason, new chitin sources are being sought. Recently, some organisms such as insects, anthozoan, fungi and sponges have been researched as alternative chitin sources [13–23]. In the present study, the wings and other body parts of Periplaneta americana were screened for their ability to be an alternative chitin source. Periplaneta americana is an insect species that is distributed worldwide. The adults have a body size of 35–40 mm, and they show excessive growth, especially when it is hot, humid and there is abundant food. People are familiar with this insect because they live in the trash, kitchens, bathrooms, near radiators, sewers and drainage channels. Therefore, they are described as a big problem in terms of human health; they cause asthma and intestinal diseases in humans [24,25]. In addition, the body of this insect has been found to carry pathogenic bacteria such as Klebsiella, Pseudomonas, Escherichia coli, Staphylococcus, Enterobacter, Streptococcus, Serratia, Bacillus and Proteus [25]. They can achieve huge numbers with
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extreme reproduction and development in moist and warm areas [26]. In this study, a new surface morphology of chitin, from the wings of an insect (P. americana), was recorded and defined. The surface morphologies of the chitins isolated from the wings of an insect and other body part were examined comparatively using ESEM. In addition, the new chitin morphology was characterized by Fourier transform infrared spectroscopy (FTIR), thermogravimetric analysis (TG), X-ray diffraction (XRD) and elemental analysis.
2. Materials and methods 2.1. Sample collection Individuals belonging to P. americana were collected by handpicking in Kirikan (Hatay, Turkey) on October 14, 2013. After collection, the samples were taken to the laboratory in a 20% ethanol solution. The samples were washed with distilled water for the removal of possible residues, and then the wings were removed with the help of forceps. The separated wings and other parts of the insect were incubated for 2 days to dry in an oven at 60 ◦ C. The pictures of P. americana and its wings were shown in Fig. 1.
2.2. Chitin extraction The dried wings and other parts were pulverized separately, in a mortar, into a powdered form. Then a demineralization step was performed in which 10 g of each material was weighted and refluxed in a 4 M HCl solution for 2 h at 75 ◦ C. Subsequently, the mixture was filtered with a 1 m filter paper and rinsed with distilled water to a neutral pH. To remove the protein, the filtrate was refluxed in a 4 M NaOH solution for 20 h at 150 ◦ C. Then the mixture was filtered and washed with distilled water to a neutral pH. Afterwards, the extract was mixed with a magnetic heating stirrer in a solution of water, methanol and chloroform (in the ratio of 4:2:1) for 4 h at 30 ◦ C. Then the extract was filtered and washed again with distilled water to a neutral pH. Finally, the material was dried in an oven at 60 ◦ C for 2 days. The chitin contents of the dried wings and other parts of the insects were calculated. 2.3. ESEM The dried wings and other parts of P. americana were coated with gold using a “Sputter Coater” (Cressingto Auto 108) at Selc¸uk University. Then the surfaces of the samples were examined employing a QUANTA- FEG 250 ESEM (at 15,000×, 40,000×, 80,000× and 100,000× magnifications) at the Science and Technology Application and Research Center (Aksaray University). 2.4. Fourier transform infrared analysis (FTIR) Analysis of chitin isolated from the wings of P. americana was conducted using a Perkin–Elmer FTIR at 4000–625 cm−1 . 2.5. Thermogravimetric analysis (TG) TGA analysis of extracted chitin from the wings of P. americana was done using an EXSTAR S11 7300 from 25 to 650 ◦ C at a heating rate of 10 ◦ C min−1 . 2.6. X-ray diffraction (XRD) XRD peaks of chitin extracted from the wings of P. americana were taken at 40 kV, 30 mA and 2 with a scan angle from 5◦ to 45◦ using a Rigaku D max 2000 system in Harran University (HUMEL). The crystalline index (CrI) values were calculated with the formula given below: CrI110 =
(I
110
− Iam )
I110
× 100
(1)
∼ 19.58◦ and Iam is where I110 is the maximum intensity at 2 = the intensity of amorphous diffraction at 2 ∼ = 12.88◦ . 2.7. Elemental analysis A Thermo Flash 2000 was employed to determine the N, H and C contents of the chitin structure isolated from the wings of P. americana. Then degree of acetylation (DA) of the chitin was calculated using the following formula [27]:
DA =
(C/N − 5.14) 1.72
× 100
(2)
3. Results and discussion 3.1. ESEM
Fig. 1. Periplaneta americana: (a) habitus, (b) wings.
ESEM analyses conducted on chitin have revealed that it has three main surface morphologies: (1) a hard and rough surface
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Fig. 2. ESEM pictures of chitin isolated from the wings of Periplaneta americana (magnifications: (a) 15,000×, (b) 40,000×, (c) 80,000× and (d) 100,000×).
without pores or nanofibers, with chitin isolated from certain fungi and Daphnia magna eggs having this type of surface morphology [28,29]. (2) A surface solely composed of nanofibers, with this type of morphology having been observed in samples isolated from fungi [30,31] as well as Antarctic krill, certain aquatic insect species and aquatic Asellus aquaticus (Cructacea) [23,32]. (3) A surface with both pores and nanofibers, which is the most common morphology [33–35]. In this study, a new morphology was defined: pores but no nanofibers. The shape of the pores is oval and their diameter ranges from 230 to 510 nm. Previous studies reported that the diameters of pores on chitin isolated from shrimp, Gammarus and silkworm chrysalides ranged from 150 to 250 nm [33–35]. The pores on the chitin that are defined in this study are larger than the ones reported in the literature. This new morphology has cell walllike structures between the pores. These pores are hollow and not very deep (Fig. 2). The distance between two pores is in the range of
125–340 nm. Fig. 2 shows this new chitin morphology at 15,000×, 40,000×, 80,000× and 100,000× magnifications. Another important point is that more than 50% of the chitin surface is composed of pores (Fig. 2). This highly porous surface could have significant biomaterial applications. The pores are regular and positioned on the surface orderly (Fig. 3). It is evident from Fig. 3 that there is a pore in the center that is surrounded by six (Fig. 3a) or seven (Fig. 3b) other pores in an ordered manner. Chitin with this magnificent design may find important applications in various fields. Fig. 4 depicts a comparison of the new type morphology with previously known types. Fig. 4a represents a hard and rough surface without pores or nanofibers [29]. Fig. 4b shows the surface morphology solely composed of nanofibers [23]. A surface with both pores and nanofibers can be seen Fig. 4c [35]. The surface of chitin extracted from other parts of P. americana (not the wings) has both pores and nanofibers (Fig. 5), which is an example of type 3.
Fig. 3. ESEM pictures of chitin extracted from the wings of Periplaneta americana ((a) one pore in the middle and six pores around and (b) one pore in the middle and seven pores around).
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Fig. 4. SEM pictures of chitin types isolated from various organisms: (a) hard and rough surface without pores or nanofibers (isolated from Daphnia magna resting eggs), (b) surface solely composed of nanofibers (isolated from Asellus aquaticus) and (c) surface with both pores and nanofibers (isolated from Gammarus).
3.2. FTIR Chitin has three crystalline forms: alpha, beta and gamma, but there is little information regarding the gamma form [36]. The most important feature of FTIR spectra is that they can be assessed to distinguish the alpha form from the beta via whether the Amide I band is split or not. In the alpha form, the Amide I band splits into two bands at about 1650 and 1620 cm−1 [32]. In the beta form, only one Amide I band is observed at 1656 cm−1 . Beta chitins are found in organisms such as squid pens [36]. It is known that alpha chitin is found in the Arthropoda [19,27]. In the FTIR spectra of the chitin extracted in this study, the Amide I band is split at 1654 and 1621 cm−1 (Fig. 6), which indicates that this chitin from P. americana is in the alpha form. The other FTIR spectrum bands were observed as the following; 3435 cm−1 (O–H stretching), 3259 cm−1 (asymmetric N–H stretching), 3108 cm−1 (symmetric N–H stretching), 2924 cm−1 (asymmetric C–H stretching), 2857 cm−1 (symmetric C–H stretching), 1420 cm−1 (CH2 ending and CH3 deformation), 1376 cm−1 (CH bend, CH3 symmetric deformation), 1308 cm−1 (CH2 wagging),
1154 cm−1 (C–O–C asymmetric stretching), 1114 cm−1 (asymmetric bridge oxygen stretching), 1114 cm−1 (asymmetric in-phase ring stretching mode), 1068 m−1 (C–O–C asymmetric stretch in phase ring), 1012 cm−1 (C–O asymmetric stretch in phase ring), 952 cm−1 (CH3 wagging), 895 cm−1 (CH ring stretching) [15]. The FTIR bands of commercial chitin were observed to be as follows; 3438, 3260, 3103, 2932, 2865, 1654, 1623, 1555, 1428, 1376, 1310, 1155, 1115, 1068, 1016, 952, 896 cm−1 . High similarity was recorded between the commercial and the chitin extracted from wings of P. americana. 3.3. TG In the literature, characterization studies for chitin extracted from crab, shrimp and insects using TG analysis have revealed that there are two mass loss steps: one at around 100 ◦ C and the other at 350–390 ◦ C [36]. In the present work we obtained similar results to the previous studies (Fig. 7). The first mass loss step represents the evaporation of absorbed water molecules; the second one corresponds with the decomposition of the chitin polymer. In the first
Fig. 5. ESEM pictures of chitin isolated from the insect’s (Periplaneta americana) body without wings (magnifications: (a) 30,000× and (b) 40,000×).
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Fig. 8. XRD peaks of chitin extracted from the wings of Periplaneta americana.
organisms can vary in the range of 54–90% [13,37]. This wide range can be attributed to differences in chitin source and purity of the isolated chitin. Fig. 6. FTIR bands of chitin isolated from the wings of Periplaneta americana.
step the mass loss is 5% and in the second step it is 76%. The DTGmax where chitin monomers decompose has been determined to be 389 ◦ C. It has been reported in the literature that the DTGmax values for chitin from crab, shrimp and Antarctic krill were 365–380 ◦ C. Another chitin characterization study by Sajomsang and Gonil [19] reported that the DTGmax for chitin from cicada sloughs (insect) was 379 ◦ C. The DTGmax value that we determined in this work is very similar to the values reported in literature. 3.4. XRD X-ray diffraction analysis of chitin shows two strong peaks and four faint peaks [19,31,32,36]. We observed two strong peaks (9.14 and 19.58) and four faint peaks (12.88, 20.98, 23.12 and 26.8) (Fig. 8). The crystallinity index (CrI) has been calculated to be 86.7%. Liu et al. [13] reported that the CrI for chitin from Holotrichia parallela (insect) was 89%. The CrI values of chitin from different
3.5. Elemental analysis We determined the C, N and H contents of the chitin from the wings of P. americana to be 45.74%, 6.69% and 6.59% respectively. The N percentage value of completely acetylated chitin is known to be 6.89 [17]. The N percentage values for chitin from different organisms are as follows: shrimp, 4.85%; bumblebee, 5.92%; crab, 5.3%; Holotrichia parallela (beetle), 6.45%; another shrimp, 6.24%; and cicada sloughs, 5.92% [13,17,19,38]. It is known that the N content of chitin from fungi is low (2.96%) due to the glucan residues in chitin [31]. The N content determined in this study approached the value of completely acetylated chitin. This indicates that chitin obtained in this study has higher purity than that reported in the literature. The degree of acetylation of chitin cannot exceed 100% [19], and the degree of acetylation of the chitin we isolated was calculated employing the formula given in the materials and methods section; it was found to be 98.67%. Some workers reported that the degrees of acetylation of chitin from shrimp, insect and crap were 112–237%, 101–132% and 104% respectively [13,17,19,38]. These values are higher than 100%, which indicates that the isolated chitin contains some impurities, like protein and inorganic materials. 3.6. Chitin content of wings and insect body (without wings) It has been determined that the dry weight of the wings of the P. americana was 18% chitin, while the chitin content of the other parts of the organism was 13%. The dry weights in other organisms are Holotrichia parallela contains 15% chitin [13], Bombyx mori 15–20% [37] and Cicada sloughs 36% [19]. Our chitin content result is similar to those of H. parallela and B. mori. 4. Conclusion
Fig. 7. TG thermograms and DTG curves of chitin from the wings of Periplaneta americana.
Periplaneta americana is a cosmopolitan species that can be cultured easily. Twenty individuals of this species were obtained from Hacettepe University (Ankara) and cultured in our animal biodiversity laboratory. The temperature and humidity of the laboratory is conditioned to be 30 ◦ C. Many newborns and eggs have been generated in only 2 months; this species multiples in hot and humid conditions [26]. Since this species is an invader and can be easily
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cultured, this organism could be considered as an alternative chitin source to crab, shrimp, crayfish and prawn. Chitin is found in the body structures of more than one million invertebrate organisms including representatives of fungi, diatoms, protists, anthozoans, nematods, sponges and mostly arthropods. Considering the chitin isolation and characterization studies conducted so far, around 50 different species have been investigated. In this study, the isolation and characterization of chitin was conducted using only the wings of an organism and not the whole body for the first time, and a new surface morphology for chitin was defined. The study showed that the physical characteristics of chitin can be different depending on the body part of the same organism. The chitin isolation and characterization procedure that we described here can be applied to various organisms. Furthermore, it is possible that this chitin isolation and characterization process can be assessed regarding commercial chitin sources, such as crab, shrimp and crayfish. The number of pores on the chitin’s surface is an important parameter in metal sorption studies [9]. Chitin with this new morphology may be utilized to remove heavy metal ions. In addition, chitin with pores can be used in tissue engineering applications [39]. In the most common morphology (referred to as type 3) nanofibers and pores are randomly disturbed on the chitin surface [33–35]. In the new morphology that we defined here, the distribution pattern of the pores on the chitin has a magnificent design, and this design is depicted in this study for the first time. In conclusion, (1) chitin from the wing of P. americana was isolated and characterized using FTIR, TG, XRD and elemental analysis, (2) the isolated chitin is in the alpha form and (3) very similar to the commercial chitin from shrimp, crab and crayfish. The feature that distinguishes this chitin from the others is its surface morphology. We believe that chitin with this new morphology could be a promising material for further studies and a product that is desired in a wide range of applications. References [1] I. Aranaz, M. Mengibar, R. Harris, I. Panos, B. Miralles, N. Acosta, G. Galed, A. Heras, Curr. Chem. Biol. 3 (2009) 203–230. [2] H. Ehrlich, Int. Geol. Rev. 52 (2010) 661–699. [3] R.A.A. Muzzarelli, Mar. Drugs 9 (2011) 1510–1533. [4] P.A. Felse, T. Panda, Bioprocess Eng. 20 (1999) 505–512. [5] B.K. Park, M.M. Kim, Int. J. Mol. Sci. 11 (2010) 5152–5164. [6] P.K. Dutta, J. Dutta, V.S. Tripathi, J. Sci. Ind. Res. India. 63 (2004) 20–31. [7] A. Anitha, S. Sowmya, P.T. Sudheesh Kumar, S. Deepthi, K.P. Chennazhi, H. Ehrlich, M. Tsurkan, R. Jayakumar, Prog. Polym. Sci. 39 (2014) 1644–1667. [8] M.N.V. Ravi Kumar, React. Funct. Polym. 46 (2000) 1–27.
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