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International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Comparison of chitin structures isolated from seven Orthoptera species
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3 4 5 6 7 8
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Murat Kaya a,b,∗ , Sevil Erdogan c , Abbas Mol b,d , Talat Baran b,e a
Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray University, 68100 Aksaray, Turkey Science and Technology Application and Research Center, Aksaray University, 68100 Aksaray, Turkey Fisheries Programme, Kes¸an Vocational College, Trakya University, 22800 Kes¸an, Edirne, Turkey d Guzelyurt Vocational School, Aksaray University, Guzelyurt, Aksaray, Turkey e Department of Chemistry, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey b c
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10 23
a r t i c l e
i n f o
a b s t r a c t
11 12 13 14 15 16 17
Article history: Received 7 May 2014 Received in revised form 13 September 2014 Accepted 27 September 2014 Available online xxx
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Keywords: Orthoptera Chitin Characterization
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1. Introduction
19 20 21
25 26 27 28 29 30 31 32 33 34 35 36
Differences in the physichochemical properties of the chitin structure of the exoskeleton of seven species from four genera were investigated in this study. The same method was used to isolate the chitin structure of the seven species. The physicochemical properties of the isolated chitins were revealed by ESEM, FTIR, TGA and XRD analyses. The FTIR, TGA and XRD results from the chitin samples were similar. The surface morphologies of the chitins were investigated by ESEM and interesting results were noted. While the surface morphologies of the chitins isolated from two species within the same genus were quite different, the surface morphologies of chitins isolated from species belonging to different genera showed similarity. It was determined that the dry weight chitin contents of the grasshopper species varied between 5.3% and 8.9%. The results of molecular analysis showed that the chitins from seven Orthoptera species (5.2–6.8 kDa) have low molecular weights. Considering that these invasive and harmful species are killed with insecticides and go to waste in large amounts, this study suggests that they should be collected and evaluated as an alternative chitin source. © 2014 Published by Elsevier B.V.
Orthoptera is an order containing more than 25,000 species worldwide, and it has two suborders, Caelifera (short-horned grasshoppers) and Ensifera (long-horned grasshoppers) [1]. Many studies have been performed in relation to the Orthoptera in the areas of taxonomy [2–5], faunistic [6,7], ecology [8], morphology [9], molecular [10], behavior [11,12], anatomy and physiology [13] and biogeography [14,15]. However, no previous studies have been performed related to the extraction and physicochemical characterization of the chitin exoskeleton in Orthopteran species. The damage caused by grasshoppers swarming induces great economic losses. Grasshoppers damage the leaves, flowers, buds of cultivated plants, bark and roots of tree-like shrubs [9,16,17]. Seven
∗ Corresponding author at: Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray University, 68100 Aksaray, Turkey. Tel.: +90 382 288 2184; fax: +90 382 288 2125. E-mail addresses: [email protected] (M. Kaya), [email protected] (S. Erdogan), [email protected] (A. Mol), [email protected] (T. Baran).
grasshopper species’ chitin contents were investigated in this study, and the species were chosen due to them being widespread and causing damage to agricultural land by occasionally overbreeding. It is estimated that the commercial production of chitin, which is a very common amino polysaccharide in nature, is equal to the annual production of cellulose. Chitin is found in the exoskeleton of arthropods, cell walls of fungi and cuticle of nematods [18]. Chitin and chitosan are natural products that are compatible with both plant and animal tissues, biologically functional, biodegradable, non-toxic and eco-friendly [19–21]. Due to these properties, chitin and its derivatives have many application areas including waste water treatment, pharmacy, medicine, cosmetics, weight loss, edible biofilm production and reduction of bad cholesterol levels in blood. Besides, antioxidant, antimicrobial and antitumor effects of chitin are also known [20,22–24]. The surface morphology, acetylation degree and molecular weight (Mw) are the three main criteria determining the industrial use of chitin and its derivatives [20,21]. The aim of this study was to isolate the chitin structures of seven different Orthoptera species, belonging to four genera, with the same method and to reveal differences among the species
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Table 1 XRD peaks and CrI values of chitins isolated from seven Orthoptera species. Species
Chitin content of dry weight (%)
Crystalline peaks
Crystalline index value % (CrI)
Ailopus simulatrix Ailopus strepens Duroniella fracta Duroniella laticornis Oedipoda miniata Oedipoda caerulescens Pyrgomorpha cognata
5.3 7.4 5.7 6.5 8.1 8.9 6.6
9.3, 12.7, 19.6, 21.1, 23.8, 26.6 9.5, 12.8, 19.6, 20.8, 23.8, 26.4 9.5, 12.6, 19.4, 20.9, 23.5, 26.8 9.5, 12.8, 19.3, 20.7, 23.2, 26.5 9.7, 12.9, 19.6, 21, 23.7, 26,8 9.3, 12.7, 19.3, 20.7, 23.1, 26.9 9.4, 13.3, 19.6, 20.9, 23.4, 26,9
76 75 72 71 74 74 63
62
by determining the physicochemical properties of the chitin structures. In addition, their availability as an alternative chitin source was investigated by determining the chitin contents of the grasshopper species.
63
2. Materials and methods
64
2.1. Sample collection
59 60 61
98
Pyrgomorpha cognata (Krauss, 1877) belonging to the Pyrgomorphidae is distributed widely in Africa, Southwest Asia and Anatolia [15,25]. Samples belonging to this species were collected from Aksaray University campus (33◦ 58 37.71 E and 38◦ 19 30.33 N, 945 m) on 03.06.2012. Oedipoda caerulescens (Linnaeus, 1758) belonging to the subfamily Oedipodinae of Acrididae is commonly found in Europe, Northern Africa and Western Asia [7]. It is sometimes a pest of cultivated plants [16]. Individuals of this species were collected from Amasya: Ezinepazarı, Abacı village (35◦ 35 44.37 E and 40◦ 24 23.42 N, 1000 m) on 19.07.2004. Oedipoda miniata (Pallas, 1771) is distributed from Southern Europe to Northern Africa and Palearctic Asia [26]. Specimens were collected from Tokat: Zile, Yıldıztepe (35◦ 53 14.92 E and 40◦ 11 12.46 N, 750 m) on 16.07.2004. Aiolopus strepens (Latreille, 1804) is another species belonging to the subfamily Oedipodinae that has a distribution in Central and Southern Europe, Central Asia, Northern and Western Africa and the Canary Islands [27]. It was reported as a pest of citrus plantings in Lenkoran and of different crops in Egypt [16]. Aiolopus simulatrix (Walker, 1870) is distributed in Anatolia, Southern Asia, Central and Northern Africa [26]. Samples belonging to these two species were collected from Kayseri: Develi, Bakırda˘g village (35◦ 28 60.00 E and 38◦ 22 60.00 N, 1270 m) on 23.04.2013. Lastly, Duroniella fracta (Krauss, 1890) belonging to the subfamily Gomphocerinae of Acrididae has a distribution in Central Asia and the Arabian Peninsula. Samples were collected from Aksaray University campus (33◦ 58 37.71 E and 38◦ 19 30.33 N, 945 m) on 03.06.2012. Duroniella laticornis (Krauss, 1909) is distributed in Turkey and Palestine [16]. Individuals of this species were collected from C¸orum: between Sungurlu-Kızılırmak, Tu˘gcu village (34◦ 19 24.89 E and 40◦ 11 2.31 N, 800 m) on 05.07.2013. All the samples were handpicked, labeled and transported to the laboratory.
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2.2. Chitin extraction process
65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
100 101 102 103 104 105 106
The grasshopper species were cleaned by washing with distilled water and left to dry at room temperature. Fully dried grasshoppers were pulverized by crushing in a mortar. The chitin analysis was conducted on 2 g of the ground sample for each grasshopper species. First, samples were demineralized. Demineralization was performed by treating the ground samples with 100 ml 4 M HCL. This process took 1 h at 75 ◦ C. Then, samples were filtered
and rinsed several times with distilled water. In the second step, samples were subjected to deproteinization. In this process, to remove protein residues samples were treated with 50 ml 2 M NaOH at 175 ◦ C for 18 h. After that samples were filtered again and rinsed with distilled water. Following this impurities were removed from the samples and they were decolorized. For this process, samples were passed through a mixture of chloroform, methanol and distilled water in the ratio of 1:2:4, and rinsed with distilled water. Samples were left in an oven at 60 ◦ C for 24 h; and thus, the chitin extraction process was completed. This extraction method was taken from Kaya et al. [28] Shrimp chitin (Pcode: 1001416772) obtained from Sigma–Aldrich was used for comparisons with the Orthopteran chitins. 2.3. Thermogravimetric analysis (TGA) A thermogravimetric analyzer (EXSTAR S11 7300 system) was used to examine the thermal decomposition behavior of the grasshopper chitins. Samples were heated at a heating rate of 10 ◦ C/min from 25 to 650 ◦ C. Changes in the decomposition degree of the samples were calculated using the changes in an empty heating container as a reference. 2.4. Environmental scanning electron microscopy (ESEM) Chitins obtained from the seven grasshopper species were analyzed with an Environmental Scanning Electron Microscope (ESEM) to reveal their microstructures in detail. The surface morphologies of the chitins were examined with a Quanta 200 FEG ESEM at magnifications of 20,000× and 40,000×. Before examination, chitin samples were coated with gold by Gatan Precision Etching Coating System (PECS). 2.5. Fourier transform infrared spectroscopy (FTIR) To determine the presence of characteristic IR bands, which is indicative of chitin, 1 mg of grasshopper chitin was analyzed with a Perkin Elmer FTIR spectrometer. Absorbance values were evaluated between 4000 and 625 cm−1 . 2.6. X-ray diffraction (XRD) The crystallinity of the grasshopper chitins was determined by X-ray diffraction (XRD) analysis. XRD patterns were recorded with a Rigaku D Max 2000 system. Data were collected at 40 kV, 30 mA and 2 with a scan angle between 5◦ and 45◦ . The crystalline index value (CrI) was calculated according to the formula: CrI110 =
(I
110
− Iam )
I110
× 100
where I110 is the maximum intensity at 2 ∼ = 20◦ and Iam is the ◦ ∼ intensity of amorphous diffraction at 2 = 13 [29].
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107 108 109 110 111 112 113 114 115 116 117 118 119 120
121
122 123 124 125 126 127
128
129 130 131 132 133 134 135
136
137 138 139 140
141
142 143 144 145 146
147
148 149
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Fig. 1. X-ray diffraction patterns of ␣-chitins from seven grasshopper species (a. chitin from Ailopus simulatrix, b. chitin from A. strepens, c. chitin from Duroniella fracta, d. chitin from Duroniella laticornis, e. chitin from Oedipoda miniata, f. chitin from O. caerulescens, g. chitin from Pyrgomorpha cognata and h. commercial chitin).
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Ailopus strepens
Duroniella fracta
Duroniella laticornis
Oedipoda miniata
Oedipoda caerulescens
Pyrgomorpha cognata
Commercial chitin
O–H stretching N–H stretching CH3 sym. stretch and CH2 asym. stretch CH3 sym. stretch C O secondary amide stretch C O secondary amide stretch N–H bend, C–N stretch CH2 ending and CH3 deformation CH bend, CH3 sym. deformation CH2 wagging
– Aliphatic compounds
3437 3261–3103 2927
3430 3260–3100 2930
3436 3262–3101 2930
3430 3260–3100 2930
3437 3263–3103 2930
3433 3260–3101 2930
3436 3260–3105 2930
3437 3259–3101 2937
Aliphatic compound Amide I
2878 1656
2877 1653
2876 1656
2877 1653
2878 1657
2871 1653
2871 1654
2867 1654
Amide I
1621
1621
1622
1621
1621
1619
1621
1620
Amide II –
1553 1414
1553 1420
1552 1415
1553 1420
1554 1415
1554 1417
1554 1418
1553 1430
–
1376
1375
1376
1375
1376
1376
1374
1376
Amide III, components of protein
1308
1307
1308
1307
1308
1305
1307
1318
1153
1155
1155
1155
1154
1151
1154
1155
1113
1111
1112
1111
1111
1111
1111
1114
Saccharide rings
1066
1060
1066
1067
1063
1062
1064
1068
–
1008
1008
1009
1008
1009
1009
1011
1024
Along chain Saccharide rings
952 896
950 895
952 895
951 895
953 895
949 893
951 895
952 896
Asymmetric bridge oxygen stretching Asymmetric in-phase ring stretching mode C–O–C asym. stretch in phase ring C–O asym. stretch in phase ring CH3 wagging CH ring stretching
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Table 2 FTIR bands of chitins isolated from seven Orthoptera species and commercial chitin.
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Fig. 2. ESEM photographs of chitins from seven grasshopper species at 3000–6000× magnifications (a. chitin from Ailopus simulatrix, b. chitin from A. strepens, c. chitin from Duroniella fracta, d. chitin from Duroniella laticornis, e. chitin from Oedipoda miniata, f. chitin from O. caerulescens, g. chitin from Pyrgomorpha cognata and h. commercial chitin).
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2.7. Molecular weight (Mw)
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The intrinsic viscosity and molecular weight (Mw) were measured with an Ubbelohde type dilution viscometer. To determine the viscosity-average Mw of the chitins from seven Orthopteran species, at different concentrations, chitin solutions were prepared using the solvent system: 5% LiCl–DMAc (DMAc: N,N dimetilasetamid). The experiments were performed at 25 ◦ C. The Mw of the Orthopteran chitins were determined using the Mark–Houwink equation [30,31]: [] : 2.1 × 10−4 Mv 0.88where [] is the intrinsic viscosity of chitin; Mv: viscosity − average molecular weight of chitin.
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3. Results and discussion
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3.1. Chitin content of seven orthopteran species
151 152 153 154 155 156 157 158 159
those of the other species (Table 1). There were differences in chitin contents of the two species belonging to the genus Ailopus. Although these two species were collected from within an area of approximately 1 m2 in the same habitat and on the same date, their chitin contents were different. It is known that chitin contents of organisms, such as shrimp, crayfish, krill and crab that are used for producing commercial chitin vary between 20% and 31% [32–34]. The dry weight chitin contents of insects vary according to species, however it is between 10% and 36% [29,35–37]. The chitin contents of the seven grasshoppers examined were less than those of other insects. 3.2. XRD
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The dry weight chitin contents of the seven grasshopper species belonging to four genera varied between 5.3% and 8.9%. The chitin contents of the species belonging to Oedipoda were higher than
The XRD peaks of chitins isolated from the seven species were similar to each other, and these peaks are given in Table 1. Two sharp (9.3–9.7 and 19.3–19.6) and four weak (12.6–13.3; 20.7–21.1; 23.1–23.8 and 26.4–26.9) peaks were observed in all the samples (Fig. 1). Peaks observed in alpha chitins obtained from organisms such as insects, crustaceans, anthozoans and
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178 179 180 181 182 183
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fungi in other studies were similar to those recorded in this study [29,36,38,39]. The CrI values of the chitins from seven Orthopteran species varied between 63% and 76%. The CrI values of species belonging to the same genus were similar (Table 1). The CrI value of the chitin isolated from P. cognata was slightly lower than other species. It has been revealed in previous studies that CrI values of chitins vary between 47% and 91% according to species and the isolation method [29,37,38]. The CrI values recorded in this study were in the middle of the range of the values recorded in previous studies. The XRD results of commercial chitin procured from Sigma–Aldrich showed two sharp peaks at 9.4◦ and 19.3◦ and weak peaks at 12.64◦ , 23.16◦ , 26.16◦ and 28.53◦ as in the Orthopteran chitins (Fig. 1). The CrI value (78%) of the commercial chitin was a little higher than that of the Orthopteran chitins. The XRD results revealed that Orthopteran chitin has similar characteristics as the commercial chitin in terms of crystallinity properties.
Table 3 TG/DTG results of chitins isolated from seven Orthoptera species. Species
Percentage of the first mass loss (between 0 and 150 ◦ C)
Percentage of the second mass loss (between 150 and 600 ◦ C)
DTG max (◦ C)
Ailopus simulatrix A. strepens Duroniella fracta D. laticornis Oedipoda miniata O. caerulescens Pyrgomorpha cognata
6
82
383
5 6
78 74
382 381
5 3
72 76
382 385
5 4
77 74
384 384
3.3. SEM
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The surface morphologies of the chitins extracted from the seven species were examined and three different surface morphologies were seen (Fig. 2). The first had a surface consisting of nanopores with long and wide nanofibres, which adhered to each other (Fig. 2a, c, d). This was identified in chitins extracted from A. simultarix, D. fracta and D. laticornis. The second had a surface consisting of nanofibres and nanopores that had a narrow and short fracture appearance, and this gave it a spongy appearance. (Fig. 2b, e, g). This second type was identified in chitins extracted from A. strepens, O. miniata and P. cognata. The third type was observed in chitin isolated from O. caerulescens and simply consisted of nanofibres (Fig. 2f). Nanopores were not seen in this type. Surface morphology is one of the most important properties that determines the efficient use of chitin and its derivatives [21]. The best usage area for chitin can be determined according to its surface morphology. Synowiecki and Al-Khateeb [40] stated that the number of pores in the chitin surface increased the chitin’s ability to absorb metal ions, while the chitin that has a fibrillar surface morphology can be use in textiles. In addition, a porous structure means the chitin can be a useful agent for tissue engineering [21]. It can be seen from previous studies that the surface morphologies of chitin and its derivatives obtained from crab, krill, insects and fungi are quite different [33,34,41–43]. The SEM analysis results showed that the commercial shrimp chitin does not have pores on its surface, unlike Orthopteran chitins, and it consists of irregularly arranged thick nanofibres (Fig. 2h). In this respect, it shows a similarity to the chitin obtained from O. caerulescens. However, it has nanofibres that are thicker and that more firmly adhered to each other than that of O. caerulescens. Liu et al. [29] reported that commercial chitin from shrimp has a rough and thick surface distinctly arranged in a microfibrillar criystalline structure. Three different types of surface morphologies were observed in the chitins extracted from the seven species in this study. Quite significant differences were observed in the surface morphologies of chitins isolated from two species belonging to the same genus. Therefore, it is difficult to identify a standard surface morphology for the alpha chitin.
Table 4 Molecular weights of chitins obtained from seven Orthopteran species.
Fig. 3. IR spectra for ␣-chitins from seven grasshopper species (a. chitin from Ailopus simulatrix, b. chitin from A. strepens, c. chitin from Duroniella fracta, d. chitin from D. laticornis, e. chitin from Oedipoda miniata, f. chitin from O. caerulescens, g. chitin from Pyrgomorpha cognata and h. commercial chitin).
Species
Molecular weights of chitins (kDa)
Ailopus simulatrix A. strepens Duroniella fracta D. laticornis Oedipoda miniata O. caerulescens Pyrgomorpha cognata
5.3 5.2 5.9 5.6 6.8 6.2 5.5
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Fig. 4. TGA curves for chitins from seven grasshopper species (a. chitin from Ailopus simulatrix, b. chitin from A.strepens, c. chitin from Duroniella fracta, d. chitin from D. laticornis, e. chitin from Oedipoda miniata, f. chitin from O. caerulescens, g. chitin from Pyrgomorpha cognata and h. commercial chitin)
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It has been determined that chitins isolated from each of the seven species had different surface morphologies. Therefore, the areas of utilization of the chitins obtained from different species, which have different surface morphologies, will be quite different. It is possible that the various forms of alpha chitin can be standardized in future studies by considering the nanofibre and pore structures of chitin and its derivatives as well as their pattern on the chitin surface.
While, the water loss observed in the first step was 5.9%, the mass loss due to decomposition of chitin in the second step was found to be 76%. This value was in the middle of the range of percentage mass losses for the Orthopteran chitins observed in the second step. The DTG max value (386 ◦ C) of commercial chitin was very close to those of the Orthopteran chitins. In addition, the thermal stabilities of the Orthopteran chitins were very close to the thermal stability of the commercial chitin.
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3.4. FTIR
3.6. Molecular weight The viscosity-average Mw of the chitins from the seven Orthopteran species and commercial chitin were determined by dilute solution viscometry. The Mw of the Orthopteran chitins varied between 5.2 and 6.8 kDa (Table 4). The Mw of the commercial chitin was determined to be 85.3 kDa and it was higher than those of grasshoppers species. Aranaz et al. [21] stated that to determine the Mw of chitin is not easy because of its low solubility. Therefore, there are only a few studies that have calculated the Mw of chitin. Jang et al. [47] calculated the average Mw of ␣-chitin, -chitin and ␥-chitin using the relative viscosity method to be 701, 612, and 524 kDa, respectively. Salah et al. [49] also determined the Mw of chitin from shrimp shell as 338 kDa. They also recorded the Mw of low Mw chitin as 2.48 kDa. Considering these values, it is seen that the chitins obtained from the seven Orthopteran species have low Mw. The Mw is one of the important criteria determining the industrial use of chitin and its derivatives [20,21,24]. The chitins that have different Mw can be used in different application areas. Salah et al. [49] reported that low Mw chitin is an attractive target for selective anticancer drug development.
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FTIR spectroscopy can clearly determine which form (␣ or  crystal) of chitin molecule is present by referring to the different hydrogen bonds. Chitin in the ␣-crystal form has two intramolecular and two intermolecular hydrogen bonds [44–46]. The FTIR bands in these hydrogen bonds are around 1660 and 1620 cm−1 , respectively. Chitin in the  crystal form has only weak intramolecular hydrogen bonds. A single FTIR band for this hydrogen bond is observed around 1650 cm−1 [47]. In the FTIR spectra of the chitins extracted from the seven grasshopper species in this study, two bands at around 1660 and 1620 cm−1 were observed (Fig. 3). These bands indicate that the extracted chitins were in the ␣-form. Other bands observed from the extracted chitins are given in Table 2. Bands obtained by FTIR analysis showed similarity in all the chitin samples (Fig. 3). In addition, the FTIR bands of the chitins in this study were similar to the FTIR bands of alpha chitins isolated from different organisms in other studies [29,33,34,38,39]. These results show that the chitins isolated from these grasshoppers are in the alpha form. The FTIR spectra of commercial shrimp chitin bought from Sigma-Aldrich is also shown in Table 2. The FTIR bands of chitins obtained from the seven Orthopteran species in this study were very similar to those of commercial shrimp chitin.
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3.5. TGA
239 240 241 242 243 244 245
248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268
271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300
The results of the TGA analyses of chitins isolated from living organisms like crab, shrimp and insects in previous studies revealed that mass losses occur in two different steps [33,39,48]. The first of these mass losses is because of evaporation of the water in the chitin, and the second is because of decomposition of the chitin structure [34,41,47]. The mass losses in the chitin isolated from the seven species in this study occurred in two different steps similar to previous studies (Fig. 4). The water content of the chitins varied between 3% and 6% of the total mass depending on species (Table 3). In the second step, the observed mass loss varied between 72% and 82% depending on species (Table 3). The maximum mass loss (82%) occurring in the second step was observed in the chitin extracted from A. simulatrix, and the minimum mass loss (72%) was observed in the chitin extracted from D. laticornis. The DTGmax values (381–385 ◦ C), which are the maximum decomposition temperature, were similar for all the isolated chitins (Table 3). In previous studies, it has been seen that the DTGmax value of alpha chitin varied between 350 and 400 ◦ C [34,36,37]. However, it was reported in a study by Juárez-de La Rosa et al. [39] that the DTGmax values of chitins obtained from two black coral species were around 300 ◦ C. Whereas, beta chitin showed decomposition around 300 ◦ C [47]. The DTGmax values of the chitins isolated from seven grasshopper species in this study were similar to the DTGmax values of chitins isolated from other living organisms, such as insects, crab and shrimp, and all the chitins in this study were in the alpha form [33,34,36,37,48]. The TGA analysis results of commercial chitin also showed that mass loss occurred in two steps as in the Orthopteran chitins (Fig. 4).
4. Conclusions The FTIR bands, water content determined by TGA, percentage mass losses observed in the second step, DTGmax values and XRD peaks of the chitins extracted from seven grasshopper species were similar to each other. Considering the CrI values of the chitins, it was seen that all of them were close to each other except for that of P. cognata. We noted that the dry weight chitin contents of these grasshoppers varied between different genera as well as between two species belonging to the same genus. The results of molecular weight analyses points to the chitins of the seven grasshoppers species having low molecular weights. It was revealed in this study that the chitins isolated, using the same method, from two different species belonging to the same genus had different surface morphologies. Locust swarms are commonly observed across the globe. A locust swarm occured in Israel and Egypt in March 2013 that generated worldwide media attention. Web search results also show that there are many local locust swarms. In Turkey, locust swarms were observed in various provinces, for example, C¸ankırı, Kastamonu, Mu˘gla and S¸anlıurfa. Moreover, these invasive grasshoppers cause serious damage in agricultural fields. These harmful organisms, which have a potential to provide huge amounts of chitin, are killed with insecticides and they go to waste. In this study, we suggest that grasshoppers in the regions where these kind of locust swarms occur should be collected and used as an alternative chitin source to produce widely used chitin. References [1] D.C. Eades, D. Otte, M.M. Cigliano, H. Braun, Orthoptera species file Online, Orthoptera Species File (Version 5.0/5.0), 2013 (site visited December) http://orthoptera.speciesfile.org/HomePage/Orthoptera/HomePage.aspx [2] D.N. Jago, Proc. Acad. Nat. Sci. Phila. 123 (1971) 205–343. [3] M.J. Ritchie, Entomol. Ser. 42 (3) (1981) 83–183.
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Contents lists available at ScienceDirect
International Journal of Biological Macromolecules journal homepage: www.elsevier.com/locate/ijbiomac
Comparison of chitin structures isolated from seven Orthoptera species
1
2
3 4 5 6 7 8
Q1
Murat Kaya a,b,∗ , Sevil Erdogan c , Abbas Mol b,d , Talat Baran b,e a
Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray University, 68100 Aksaray, Turkey Science and Technology Application and Research Center, Aksaray University, 68100 Aksaray, Turkey Fisheries Programme, Kes¸an Vocational College, Trakya University, 22800 Kes¸an, Edirne, Turkey d Guzelyurt Vocational School, Aksaray University, Guzelyurt, Aksaray, Turkey e Department of Chemistry, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey b c
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10 23
a r t i c l e
i n f o
a b s t r a c t
11 12 13 14 15 16 17
Article history: Received 7 May 2014 Received in revised form 13 September 2014 Accepted 27 September 2014 Available online xxx
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22
Keywords: Orthoptera Chitin Characterization
24
1. Introduction
19 20 21
25 26 27 28 29 30 31 32 33 34 35 36
Differences in the physichochemical properties of the chitin structure of the exoskeleton of seven species from four genera were investigated in this study. The same method was used to isolate the chitin structure of the seven species. The physicochemical properties of the isolated chitins were revealed by ESEM, FTIR, TGA and XRD analyses. The FTIR, TGA and XRD results from the chitin samples were similar. The surface morphologies of the chitins were investigated by ESEM and interesting results were noted. While the surface morphologies of the chitins isolated from two species within the same genus were quite different, the surface morphologies of chitins isolated from species belonging to different genera showed similarity. It was determined that the dry weight chitin contents of the grasshopper species varied between 5.3% and 8.9%. The results of molecular analysis showed that the chitins from seven Orthoptera species (5.2–6.8 kDa) have low molecular weights. Considering that these invasive and harmful species are killed with insecticides and go to waste in large amounts, this study suggests that they should be collected and evaluated as an alternative chitin source. © 2014 Published by Elsevier B.V.
Orthoptera is an order containing more than 25,000 species worldwide, and it has two suborders, Caelifera (short-horned grasshoppers) and Ensifera (long-horned grasshoppers) [1]. Many studies have been performed in relation to the Orthoptera in the areas of taxonomy [2–5], faunistic [6,7], ecology [8], morphology [9], molecular [10], behavior [11,12], anatomy and physiology [13] and biogeography [14,15]. However, no previous studies have been performed related to the extraction and physicochemical characterization of the chitin exoskeleton in Orthopteran species. The damage caused by grasshoppers swarming induces great economic losses. Grasshoppers damage the leaves, flowers, buds of cultivated plants, bark and roots of tree-like shrubs [9,16,17]. Seven
∗ Corresponding author at: Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray University, 68100 Aksaray, Turkey. Tel.: +90 382 288 2184; fax: +90 382 288 2125. E-mail addresses: [email protected] (M. Kaya), [email protected] (S. Erdogan), [email protected] (A. Mol), [email protected] (T. Baran).
grasshopper species’ chitin contents were investigated in this study, and the species were chosen due to them being widespread and causing damage to agricultural land by occasionally overbreeding. It is estimated that the commercial production of chitin, which is a very common amino polysaccharide in nature, is equal to the annual production of cellulose. Chitin is found in the exoskeleton of arthropods, cell walls of fungi and cuticle of nematods [18]. Chitin and chitosan are natural products that are compatible with both plant and animal tissues, biologically functional, biodegradable, non-toxic and eco-friendly [19–21]. Due to these properties, chitin and its derivatives have many application areas including waste water treatment, pharmacy, medicine, cosmetics, weight loss, edible biofilm production and reduction of bad cholesterol levels in blood. Besides, antioxidant, antimicrobial and antitumor effects of chitin are also known [20,22–24]. The surface morphology, acetylation degree and molecular weight (Mw) are the three main criteria determining the industrial use of chitin and its derivatives [20,21]. The aim of this study was to isolate the chitin structures of seven different Orthoptera species, belonging to four genera, with the same method and to reveal differences among the species
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Table 1 XRD peaks and CrI values of chitins isolated from seven Orthoptera species. Species
Chitin content of dry weight (%)
Crystalline peaks
Crystalline index value % (CrI)
Ailopus simulatrix Ailopus strepens Duroniella fracta Duroniella laticornis Oedipoda miniata Oedipoda caerulescens Pyrgomorpha cognata
5.3 7.4 5.7 6.5 8.1 8.9 6.6
9.3, 12.7, 19.6, 21.1, 23.8, 26.6 9.5, 12.8, 19.6, 20.8, 23.8, 26.4 9.5, 12.6, 19.4, 20.9, 23.5, 26.8 9.5, 12.8, 19.3, 20.7, 23.2, 26.5 9.7, 12.9, 19.6, 21, 23.7, 26,8 9.3, 12.7, 19.3, 20.7, 23.1, 26.9 9.4, 13.3, 19.6, 20.9, 23.4, 26,9
76 75 72 71 74 74 63
62
by determining the physicochemical properties of the chitin structures. In addition, their availability as an alternative chitin source was investigated by determining the chitin contents of the grasshopper species.
63
2. Materials and methods
64
2.1. Sample collection
59 60 61
98
Pyrgomorpha cognata (Krauss, 1877) belonging to the Pyrgomorphidae is distributed widely in Africa, Southwest Asia and Anatolia [15,25]. Samples belonging to this species were collected from Aksaray University campus (33◦ 58 37.71 E and 38◦ 19 30.33 N, 945 m) on 03.06.2012. Oedipoda caerulescens (Linnaeus, 1758) belonging to the subfamily Oedipodinae of Acrididae is commonly found in Europe, Northern Africa and Western Asia [7]. It is sometimes a pest of cultivated plants [16]. Individuals of this species were collected from Amasya: Ezinepazarı, Abacı village (35◦ 35 44.37 E and 40◦ 24 23.42 N, 1000 m) on 19.07.2004. Oedipoda miniata (Pallas, 1771) is distributed from Southern Europe to Northern Africa and Palearctic Asia [26]. Specimens were collected from Tokat: Zile, Yıldıztepe (35◦ 53 14.92 E and 40◦ 11 12.46 N, 750 m) on 16.07.2004. Aiolopus strepens (Latreille, 1804) is another species belonging to the subfamily Oedipodinae that has a distribution in Central and Southern Europe, Central Asia, Northern and Western Africa and the Canary Islands [27]. It was reported as a pest of citrus plantings in Lenkoran and of different crops in Egypt [16]. Aiolopus simulatrix (Walker, 1870) is distributed in Anatolia, Southern Asia, Central and Northern Africa [26]. Samples belonging to these two species were collected from Kayseri: Develi, Bakırda˘g village (35◦ 28 60.00 E and 38◦ 22 60.00 N, 1270 m) on 23.04.2013. Lastly, Duroniella fracta (Krauss, 1890) belonging to the subfamily Gomphocerinae of Acrididae has a distribution in Central Asia and the Arabian Peninsula. Samples were collected from Aksaray University campus (33◦ 58 37.71 E and 38◦ 19 30.33 N, 945 m) on 03.06.2012. Duroniella laticornis (Krauss, 1909) is distributed in Turkey and Palestine [16]. Individuals of this species were collected from C¸orum: between Sungurlu-Kızılırmak, Tu˘gcu village (34◦ 19 24.89 E and 40◦ 11 2.31 N, 800 m) on 05.07.2013. All the samples were handpicked, labeled and transported to the laboratory.
99
2.2. Chitin extraction process
65 66 67 68 69 70 71 72 73 74 75 76 77 78 79 80 81 82 83 84 85 86 87 88 89 90 91 92 93 94 95 96 97
100 101 102 103 104 105 106
The grasshopper species were cleaned by washing with distilled water and left to dry at room temperature. Fully dried grasshoppers were pulverized by crushing in a mortar. The chitin analysis was conducted on 2 g of the ground sample for each grasshopper species. First, samples were demineralized. Demineralization was performed by treating the ground samples with 100 ml 4 M HCL. This process took 1 h at 75 ◦ C. Then, samples were filtered
and rinsed several times with distilled water. In the second step, samples were subjected to deproteinization. In this process, to remove protein residues samples were treated with 50 ml 2 M NaOH at 175 ◦ C for 18 h. After that samples were filtered again and rinsed with distilled water. Following this impurities were removed from the samples and they were decolorized. For this process, samples were passed through a mixture of chloroform, methanol and distilled water in the ratio of 1:2:4, and rinsed with distilled water. Samples were left in an oven at 60 ◦ C for 24 h; and thus, the chitin extraction process was completed. This extraction method was taken from Kaya et al. [28] Shrimp chitin (Pcode: 1001416772) obtained from Sigma–Aldrich was used for comparisons with the Orthopteran chitins. 2.3. Thermogravimetric analysis (TGA) A thermogravimetric analyzer (EXSTAR S11 7300 system) was used to examine the thermal decomposition behavior of the grasshopper chitins. Samples were heated at a heating rate of 10 ◦ C/min from 25 to 650 ◦ C. Changes in the decomposition degree of the samples were calculated using the changes in an empty heating container as a reference. 2.4. Environmental scanning electron microscopy (ESEM) Chitins obtained from the seven grasshopper species were analyzed with an Environmental Scanning Electron Microscope (ESEM) to reveal their microstructures in detail. The surface morphologies of the chitins were examined with a Quanta 200 FEG ESEM at magnifications of 20,000× and 40,000×. Before examination, chitin samples were coated with gold by Gatan Precision Etching Coating System (PECS). 2.5. Fourier transform infrared spectroscopy (FTIR) To determine the presence of characteristic IR bands, which is indicative of chitin, 1 mg of grasshopper chitin was analyzed with a Perkin Elmer FTIR spectrometer. Absorbance values were evaluated between 4000 and 625 cm−1 . 2.6. X-ray diffraction (XRD) The crystallinity of the grasshopper chitins was determined by X-ray diffraction (XRD) analysis. XRD patterns were recorded with a Rigaku D Max 2000 system. Data were collected at 40 kV, 30 mA and 2 with a scan angle between 5◦ and 45◦ . The crystalline index value (CrI) was calculated according to the formula: CrI110 =
(I
110
− Iam )
I110
× 100
where I110 is the maximum intensity at 2 ∼ = 20◦ and Iam is the ◦ ∼ intensity of amorphous diffraction at 2 = 13 [29].
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107 108 109 110 111 112 113 114 115 116 117 118 119 120
121
122 123 124 125 126 127
128
129 130 131 132 133 134 135
136
137 138 139 140
141
142 143 144 145 146
147
148 149
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Fig. 1. X-ray diffraction patterns of ␣-chitins from seven grasshopper species (a. chitin from Ailopus simulatrix, b. chitin from A. strepens, c. chitin from Duroniella fracta, d. chitin from Duroniella laticornis, e. chitin from Oedipoda miniata, f. chitin from O. caerulescens, g. chitin from Pyrgomorpha cognata and h. commercial chitin).
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Ailopus strepens
Duroniella fracta
Duroniella laticornis
Oedipoda miniata
Oedipoda caerulescens
Pyrgomorpha cognata
Commercial chitin
O–H stretching N–H stretching CH3 sym. stretch and CH2 asym. stretch CH3 sym. stretch C O secondary amide stretch C O secondary amide stretch N–H bend, C–N stretch CH2 ending and CH3 deformation CH bend, CH3 sym. deformation CH2 wagging
– Aliphatic compounds
3437 3261–3103 2927
3430 3260–3100 2930
3436 3262–3101 2930
3430 3260–3100 2930
3437 3263–3103 2930
3433 3260–3101 2930
3436 3260–3105 2930
3437 3259–3101 2937
Aliphatic compound Amide I
2878 1656
2877 1653
2876 1656
2877 1653
2878 1657
2871 1653
2871 1654
2867 1654
Amide I
1621
1621
1622
1621
1621
1619
1621
1620
Amide II –
1553 1414
1553 1420
1552 1415
1553 1420
1554 1415
1554 1417
1554 1418
1553 1430
–
1376
1375
1376
1375
1376
1376
1374
1376
Amide III, components of protein
1308
1307
1308
1307
1308
1305
1307
1318
1153
1155
1155
1155
1154
1151
1154
1155
1113
1111
1112
1111
1111
1111
1111
1114
Saccharide rings
1066
1060
1066
1067
1063
1062
1064
1068
–
1008
1008
1009
1008
1009
1009
1011
1024
Along chain Saccharide rings
952 896
950 895
952 895
951 895
953 895
949 893
951 895
952 896
Asymmetric bridge oxygen stretching Asymmetric in-phase ring stretching mode C–O–C asym. stretch in phase ring C–O asym. stretch in phase ring CH3 wagging CH ring stretching
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Table 2 FTIR bands of chitins isolated from seven Orthoptera species and commercial chitin.
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Fig. 2. ESEM photographs of chitins from seven grasshopper species at 3000–6000× magnifications (a. chitin from Ailopus simulatrix, b. chitin from A. strepens, c. chitin from Duroniella fracta, d. chitin from Duroniella laticornis, e. chitin from Oedipoda miniata, f. chitin from O. caerulescens, g. chitin from Pyrgomorpha cognata and h. commercial chitin).
150
2.7. Molecular weight (Mw)
160
The intrinsic viscosity and molecular weight (Mw) were measured with an Ubbelohde type dilution viscometer. To determine the viscosity-average Mw of the chitins from seven Orthopteran species, at different concentrations, chitin solutions were prepared using the solvent system: 5% LiCl–DMAc (DMAc: N,N dimetilasetamid). The experiments were performed at 25 ◦ C. The Mw of the Orthopteran chitins were determined using the Mark–Houwink equation [30,31]: [] : 2.1 × 10−4 Mv 0.88where [] is the intrinsic viscosity of chitin; Mv: viscosity − average molecular weight of chitin.
161
3. Results and discussion
162
3.1. Chitin content of seven orthopteran species
151 152 153 154 155 156 157 158 159
those of the other species (Table 1). There were differences in chitin contents of the two species belonging to the genus Ailopus. Although these two species were collected from within an area of approximately 1 m2 in the same habitat and on the same date, their chitin contents were different. It is known that chitin contents of organisms, such as shrimp, crayfish, krill and crab that are used for producing commercial chitin vary between 20% and 31% [32–34]. The dry weight chitin contents of insects vary according to species, however it is between 10% and 36% [29,35–37]. The chitin contents of the seven grasshoppers examined were less than those of other insects. 3.2. XRD
163 164 165
The dry weight chitin contents of the seven grasshopper species belonging to four genera varied between 5.3% and 8.9%. The chitin contents of the species belonging to Oedipoda were higher than
The XRD peaks of chitins isolated from the seven species were similar to each other, and these peaks are given in Table 1. Two sharp (9.3–9.7 and 19.3–19.6) and four weak (12.6–13.3; 20.7–21.1; 23.1–23.8 and 26.4–26.9) peaks were observed in all the samples (Fig. 1). Peaks observed in alpha chitins obtained from organisms such as insects, crustaceans, anthozoans and
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177
178 179 180 181 182 183
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fungi in other studies were similar to those recorded in this study [29,36,38,39]. The CrI values of the chitins from seven Orthopteran species varied between 63% and 76%. The CrI values of species belonging to the same genus were similar (Table 1). The CrI value of the chitin isolated from P. cognata was slightly lower than other species. It has been revealed in previous studies that CrI values of chitins vary between 47% and 91% according to species and the isolation method [29,37,38]. The CrI values recorded in this study were in the middle of the range of the values recorded in previous studies. The XRD results of commercial chitin procured from Sigma–Aldrich showed two sharp peaks at 9.4◦ and 19.3◦ and weak peaks at 12.64◦ , 23.16◦ , 26.16◦ and 28.53◦ as in the Orthopteran chitins (Fig. 1). The CrI value (78%) of the commercial chitin was a little higher than that of the Orthopteran chitins. The XRD results revealed that Orthopteran chitin has similar characteristics as the commercial chitin in terms of crystallinity properties.
Table 3 TG/DTG results of chitins isolated from seven Orthoptera species. Species
Percentage of the first mass loss (between 0 and 150 ◦ C)
Percentage of the second mass loss (between 150 and 600 ◦ C)
DTG max (◦ C)
Ailopus simulatrix A. strepens Duroniella fracta D. laticornis Oedipoda miniata O. caerulescens Pyrgomorpha cognata
6
82
383
5 6
78 74
382 381
5 3
72 76
382 385
5 4
77 74
384 384
3.3. SEM
202
The surface morphologies of the chitins extracted from the seven species were examined and three different surface morphologies were seen (Fig. 2). The first had a surface consisting of nanopores with long and wide nanofibres, which adhered to each other (Fig. 2a, c, d). This was identified in chitins extracted from A. simultarix, D. fracta and D. laticornis. The second had a surface consisting of nanofibres and nanopores that had a narrow and short fracture appearance, and this gave it a spongy appearance. (Fig. 2b, e, g). This second type was identified in chitins extracted from A. strepens, O. miniata and P. cognata. The third type was observed in chitin isolated from O. caerulescens and simply consisted of nanofibres (Fig. 2f). Nanopores were not seen in this type. Surface morphology is one of the most important properties that determines the efficient use of chitin and its derivatives [21]. The best usage area for chitin can be determined according to its surface morphology. Synowiecki and Al-Khateeb [40] stated that the number of pores in the chitin surface increased the chitin’s ability to absorb metal ions, while the chitin that has a fibrillar surface morphology can be use in textiles. In addition, a porous structure means the chitin can be a useful agent for tissue engineering [21]. It can be seen from previous studies that the surface morphologies of chitin and its derivatives obtained from crab, krill, insects and fungi are quite different [33,34,41–43]. The SEM analysis results showed that the commercial shrimp chitin does not have pores on its surface, unlike Orthopteran chitins, and it consists of irregularly arranged thick nanofibres (Fig. 2h). In this respect, it shows a similarity to the chitin obtained from O. caerulescens. However, it has nanofibres that are thicker and that more firmly adhered to each other than that of O. caerulescens. Liu et al. [29] reported that commercial chitin from shrimp has a rough and thick surface distinctly arranged in a microfibrillar criystalline structure. Three different types of surface morphologies were observed in the chitins extracted from the seven species in this study. Quite significant differences were observed in the surface morphologies of chitins isolated from two species belonging to the same genus. Therefore, it is difficult to identify a standard surface morphology for the alpha chitin.
Table 4 Molecular weights of chitins obtained from seven Orthopteran species.
Fig. 3. IR spectra for ␣-chitins from seven grasshopper species (a. chitin from Ailopus simulatrix, b. chitin from A. strepens, c. chitin from Duroniella fracta, d. chitin from D. laticornis, e. chitin from Oedipoda miniata, f. chitin from O. caerulescens, g. chitin from Pyrgomorpha cognata and h. commercial chitin).
Species
Molecular weights of chitins (kDa)
Ailopus simulatrix A. strepens Duroniella fracta D. laticornis Oedipoda miniata O. caerulescens Pyrgomorpha cognata
5.3 5.2 5.9 5.6 6.8 6.2 5.5
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Fig. 4. TGA curves for chitins from seven grasshopper species (a. chitin from Ailopus simulatrix, b. chitin from A.strepens, c. chitin from Duroniella fracta, d. chitin from D. laticornis, e. chitin from Oedipoda miniata, f. chitin from O. caerulescens, g. chitin from Pyrgomorpha cognata and h. commercial chitin)
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It has been determined that chitins isolated from each of the seven species had different surface morphologies. Therefore, the areas of utilization of the chitins obtained from different species, which have different surface morphologies, will be quite different. It is possible that the various forms of alpha chitin can be standardized in future studies by considering the nanofibre and pore structures of chitin and its derivatives as well as their pattern on the chitin surface.
While, the water loss observed in the first step was 5.9%, the mass loss due to decomposition of chitin in the second step was found to be 76%. This value was in the middle of the range of percentage mass losses for the Orthopteran chitins observed in the second step. The DTG max value (386 ◦ C) of commercial chitin was very close to those of the Orthopteran chitins. In addition, the thermal stabilities of the Orthopteran chitins were very close to the thermal stability of the commercial chitin.
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3.4. FTIR
3.6. Molecular weight The viscosity-average Mw of the chitins from the seven Orthopteran species and commercial chitin were determined by dilute solution viscometry. The Mw of the Orthopteran chitins varied between 5.2 and 6.8 kDa (Table 4). The Mw of the commercial chitin was determined to be 85.3 kDa and it was higher than those of grasshoppers species. Aranaz et al. [21] stated that to determine the Mw of chitin is not easy because of its low solubility. Therefore, there are only a few studies that have calculated the Mw of chitin. Jang et al. [47] calculated the average Mw of ␣-chitin, -chitin and ␥-chitin using the relative viscosity method to be 701, 612, and 524 kDa, respectively. Salah et al. [49] also determined the Mw of chitin from shrimp shell as 338 kDa. They also recorded the Mw of low Mw chitin as 2.48 kDa. Considering these values, it is seen that the chitins obtained from the seven Orthopteran species have low Mw. The Mw is one of the important criteria determining the industrial use of chitin and its derivatives [20,21,24]. The chitins that have different Mw can be used in different application areas. Salah et al. [49] reported that low Mw chitin is an attractive target for selective anticancer drug development.
269
FTIR spectroscopy can clearly determine which form (␣ or  crystal) of chitin molecule is present by referring to the different hydrogen bonds. Chitin in the ␣-crystal form has two intramolecular and two intermolecular hydrogen bonds [44–46]. The FTIR bands in these hydrogen bonds are around 1660 and 1620 cm−1 , respectively. Chitin in the  crystal form has only weak intramolecular hydrogen bonds. A single FTIR band for this hydrogen bond is observed around 1650 cm−1 [47]. In the FTIR spectra of the chitins extracted from the seven grasshopper species in this study, two bands at around 1660 and 1620 cm−1 were observed (Fig. 3). These bands indicate that the extracted chitins were in the ␣-form. Other bands observed from the extracted chitins are given in Table 2. Bands obtained by FTIR analysis showed similarity in all the chitin samples (Fig. 3). In addition, the FTIR bands of the chitins in this study were similar to the FTIR bands of alpha chitins isolated from different organisms in other studies [29,33,34,38,39]. These results show that the chitins isolated from these grasshoppers are in the alpha form. The FTIR spectra of commercial shrimp chitin bought from Sigma-Aldrich is also shown in Table 2. The FTIR bands of chitins obtained from the seven Orthopteran species in this study were very similar to those of commercial shrimp chitin.
270
3.5. TGA
239 240 241 242 243 244 245
248 249 250 251 252 253 254 255 256 257 258 259 260 261 262 263 264 265 266 267 268
271 272 273 274 275 276 277 278 279 280 281 282 283 284 285 286 287 288 289 290 291 292 293 294 295 296 297 298 299 300
The results of the TGA analyses of chitins isolated from living organisms like crab, shrimp and insects in previous studies revealed that mass losses occur in two different steps [33,39,48]. The first of these mass losses is because of evaporation of the water in the chitin, and the second is because of decomposition of the chitin structure [34,41,47]. The mass losses in the chitin isolated from the seven species in this study occurred in two different steps similar to previous studies (Fig. 4). The water content of the chitins varied between 3% and 6% of the total mass depending on species (Table 3). In the second step, the observed mass loss varied between 72% and 82% depending on species (Table 3). The maximum mass loss (82%) occurring in the second step was observed in the chitin extracted from A. simulatrix, and the minimum mass loss (72%) was observed in the chitin extracted from D. laticornis. The DTGmax values (381–385 ◦ C), which are the maximum decomposition temperature, were similar for all the isolated chitins (Table 3). In previous studies, it has been seen that the DTGmax value of alpha chitin varied between 350 and 400 ◦ C [34,36,37]. However, it was reported in a study by Juárez-de La Rosa et al. [39] that the DTGmax values of chitins obtained from two black coral species were around 300 ◦ C. Whereas, beta chitin showed decomposition around 300 ◦ C [47]. The DTGmax values of the chitins isolated from seven grasshopper species in this study were similar to the DTGmax values of chitins isolated from other living organisms, such as insects, crab and shrimp, and all the chitins in this study were in the alpha form [33,34,36,37,48]. The TGA analysis results of commercial chitin also showed that mass loss occurred in two steps as in the Orthopteran chitins (Fig. 4).
4. Conclusions The FTIR bands, water content determined by TGA, percentage mass losses observed in the second step, DTGmax values and XRD peaks of the chitins extracted from seven grasshopper species were similar to each other. Considering the CrI values of the chitins, it was seen that all of them were close to each other except for that of P. cognata. We noted that the dry weight chitin contents of these grasshoppers varied between different genera as well as between two species belonging to the same genus. The results of molecular weight analyses points to the chitins of the seven grasshoppers species having low molecular weights. It was revealed in this study that the chitins isolated, using the same method, from two different species belonging to the same genus had different surface morphologies. Locust swarms are commonly observed across the globe. A locust swarm occured in Israel and Egypt in March 2013 that generated worldwide media attention. Web search results also show that there are many local locust swarms. In Turkey, locust swarms were observed in various provinces, for example, C¸ankırı, Kastamonu, Mu˘gla and S¸anlıurfa. Moreover, these invasive grasshoppers cause serious damage in agricultural fields. These harmful organisms, which have a potential to provide huge amounts of chitin, are killed with insecticides and they go to waste. In this study, we suggest that grasshoppers in the regions where these kind of locust swarms occur should be collected and used as an alternative chitin source to produce widely used chitin. References [1] D.C. Eades, D. Otte, M.M. Cigliano, H. Braun, Orthoptera species file Online, Orthoptera Species File (Version 5.0/5.0), 2013 (site visited December) http://orthoptera.speciesfile.org/HomePage/Orthoptera/HomePage.aspx [2] D.N. Jago, Proc. Acad. Nat. Sci. Phila. 123 (1971) 205–343. [3] M.J. Ritchie, Entomol. Ser. 42 (3) (1981) 83–183.
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