Chitin extraction and characterization from Daphnia magna resting eggs

International Journal of Biological Macromolecules 61 (2013) 459–464

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Chitin extraction and characterization from Daphnia magna resting eggs Murat Kaya a,∗ , Idris Sargin b , Kabil Özcan Tozak b , Talat Baran b , Sevil Erdogan c , Göksal Sezen d a

Department of Biotechnology and Molecular Biology, Faculty of Science and Letters, Aksaray University, 68100 Aksaray, Turkey Department of Chemistry, Faculty of Science and Letters, Aksaray University, Aksaray, Turkey c Trakya University, Kes¸an Vocational College, Fisheries Programme, 22800 Kes¸an, Edirne, Turkey d Department of Biology, Faculty of Science-Literature, Harran University, S¸anlıurfa, Turkey b

a r t i c l e

i n f o

Article history: Received 7 February 2013 Received in revised form 1 August 2013 Accepted 11 August 2013 Available online 22 August 2013 Keywords: Crustacean TGA FTIR XRD SEM

a b s t r a c t New application areas for chitin and its derivatives have been extensively investigated and there is a solid, growing demand for new chitin sources. In this present study, chitin content of Daphnia magna resting egg (18–21%) was determined for the first time. FTIR, elemental analysis, TGA, XRD and SEM studies revealed the structural and thermal properties of extracted ␣-chitin. This study suggests that D. magna resting eggs can be exploited as an attractive alternative chitin source. © 2013 Elsevier B.V. All rights reserved.

1. Introduction Chitin is the most abundant biopolymer after cellulose. Despite its extensive application in such areas as biotechnology, agriculture and medicine [1,2], a few sources (shrimp, crab and krill) have been cited as commercial chitin resources [3]. Recent studies reveal new application areas for chitin and its derivatives [4–7]. Considering wide application of chitin and its derivatives, there is an increasing demand for unconventional chitin sources. Chitin has three different crystalline polymorphic forms in nature: ␤-chitin, ␣-chitin and ␥-chitin [8]. Each of crystalline forms has different utilization areas [9]. Determination of thermal properties of extracted chitin is of great importance to further applications of chitin and its variants [9]. Some workers investigated the chitin content of Daphnia and reported that Daphnia contained chitin 3–7% of its total weight [10]. Chitin content of resting eggs has yet to be determined in Daphnia species. Daphnia (Cladocera, Crustacean) is a zooplankton genus in aquatic ecosystems and has a major role in the upward transfer of energy from primary producers to the higher trophic levels in food webs [11]. Resting egg production in Daphnia is initiated in response to deteriorating conditions for growth and parthenogenesis to ensure survival of the organism. Resting eggs (ephippia) hatch

∗ Corresponding author. Tel.: +90 382 288 2184; fax: +90 382 288 2125. E-mail address: [email protected] (M. Kaya). 0141-8130/$ – see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.ijbiomac.2013.08.016

into parthenogenetic female juvenile daphnids as environmental conditions improve. Resting eggs, encased in robust ephippium, can withstand dryness and freezing and stay dormant for decades [12]. The study presented here aims (1) to determine the chitin content of Daphnia magna resting egg and (2) to characterize the extracted chitin by employing FTIR, elemental analysis (EA), TGA, XRD, and SEM. 2. Material and methods 2.1. Samples collection and preparation Buoyant Daphnia resting eggs, accumulated on the surface of Mamasın Dam Lake (Aksaray, Turkey), harvested in November, 2012. At the laboratory, the samples were sieved through a 400 ␮ sieve to isolate D. magna resting eggs from the other Daphnia species resting eggs. Washed resting eggs were dried at room temperature, and then D. magna resting eggs were collected using needles under light to ensure species selectivity. SEM image of D. magna resting egg is presented in Fig. 1. 2.2. Chitin extraction from D. magna 1 g of resting eggs was pulverized in a mortar and then dried in an oven at 70 ◦ C for 6 h. Then, it was refluxed in 50 ml of 1 M HCI solution for 16 h at 65–75 ◦ C for demineralisation. The mixture

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3. Results and discussion 3.1. Chitin content of D. magna resting eggs

Fig. 1. Resting egg of Daphnia magna.

was filtered and washed with distilled water. Then, the deproteinization step was carried out in 250 ml of 1 M NaOH at 65 ◦ C for 20 h. The solution was filtered and subsequently the filtrate was washed with distilled water and then dried at 70 ◦ C. Then, the last step, the decolourization process, was carried out. The sample was treated with chloroform, methanol and water (v/v 1:2:4) mixture at room temperature for 1 h. Finally, washed and dried sample was weighted and kept in an air-tight container. The extraction procedure was repeated several times and in each procedure chitin yield was calculated on basis of dry weight of resting eggs.

2.3. Fourier transform infrared spectroscopy (FTIR) of chitin The IR spectra of extracted chitin were recorded with a Perkin Elmer FTIR Spectrometer over the frequency range of 4000–625 cm−1 .

2.4. Elemental analysis (EA) Elemental analysis was performed using Thermo Flash 2000. The degree of acetylation (DA) of isolated chitin was determined from date of elemental analysis. Eq. (1) was employed to determine DA as following [13]: DA =

C/N − 5.14 × 100 1.72

(1)

where C/N is ratio of carbon to nitrogen (w/w).

2.5. Thermogravimetric analysis (TGA) TG and DTG curves were obtained at the thermal degradation of chitin at a heating rate of 10 ◦ C min−1 by using EXSTAR S11 7300.

2.6. X-ray diffraction (XRD) X-ray diffraction data were obtained at 40 kV, 30 mA and 2 with the scan angle from 5 to 45◦ using a Rigaku D max 2000 system in Harran University (HÜMEL).

2.7. Scanning electron microscopy (SEM) The surface morphology of D. magna resting egg and extracted chitin was analyzed by using EVO LS 10 ZEISS scanning electron microscope. The samples were coated with gold for SEM analysis by Sputter Coater (Cressingto Auto 108).

This study revealed that D. magna resting egg is composed of ␣chitin, which constitutes 18–21% of its dry weight. The chitin yield in each extraction process fluctuated in a range of 18 to 21%. When the chitin content of D. magna resting egg is compared to those of some commercial organisms (crab; 13–26%, shrimp; 14–42% and krill; 34–49%) and other organisms (Holotrichia parallela and Artemia urmiana cyst shells; 15% and 29.3–34.5%, respectively) D. magna resting egg stands out as a promising alternative chitin source [3,4,14]. Chitin is the major component of cuticles of insects, corals, fungal cell walls and has wide industrial and biomedical applications [15–18]. The major source of industrial chitin comes from wastes of processed marine food (mainly crustacean shells, e.g. shrimp and crab shells or krill) [3]. Physicochemical characteristics of purified chitin in chitin production are of great importance because the quality of the final product determines its further utilization. Extraction methods and conditions are largely determined by the chitin source and harsh treatments can cause detrimental effects on the polymer. Regarding D. magna resting egg structure, chitin production from such small crustacean cysts seems to overcome the above-mentioned issues. A previous study by Cauchie et al. [10] revealed the chitin content of Daphnia (ranging from 3 to 7% on seasonal basis) and we report that chitin content of its resting egg (18–21%) is at least three times higher. Therefore, D. magna and its resting egg can be exploited as a commercial alternative chitin source. There are a limited number of papers on chemical composition of Daphnia resting eggs. Kawasaki et al. [19] reported that resting eggs have crystalline calcium phosphate and magnetic material besides its chitin content. Another paper by Kawasaki et al. [20] revealed the elemental composition of resting eggs (phosphorus, sulphur, potassium, and calcium). In this present study, we investigated the chitin content of D. magna for the first time in detail. Daphnia are members of the order Cladocera, (Crustacean). Daphnia is a large genus comprising about 200 taxa worldwide [21]. Since D. magna is larger than the other Daphnia species, it is easily cultured in pools as a popular live food in fish keeping. D. magna occurs worldwide and have nearly cosmopolitan distribution. Daphnia produce ephippial (resting) eggs under harsh conditions, such as extreme cold or drought to survive. Daphnia responds to especially sudden and sharp fallings in temperature by producing resting eggs [22,23]. As an easily cultured organism, D. magna can be manipulated to switch to sexual reproduction and produce fertilized resting eggs by changing culture water temperatures for chitin production. In this work, resting eggs were collected from Mamasın Dam Lake (Central Anatolia, Turkey). A dramatic decrease in population of a toothless fish species largely feeding on Daphnia led to an unprecedented increase in Daphnia population and subsequently in resting production in the early winter. This shows that such lakes or ponds can also be regarded as resting eggs source for chitin production. 3.2. FTIR Chitin may be regarded as a derivative of cellulose. In chitin molecule, hydroxyl group at the position C-2 is replaced by an acetamido group. Intramolecular hydrogen bonds (C3 OH· · ·C5) and intermolecular hydrogen bonds (NH· · ·C O and C6 OH· · ·O C) give chitin polymer stability [24]. Reported studies show that chitin exists in three different polymorphic forms (␣, ␤ and ␥) [25]. In nature chitin mainly exists as alpha form. ␣-chitin is found in

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461

Transmittance (%)

2852 3262

2922 1308 1652

1556

1376

1622

11153

953

1060 1022

4000.0

3600

3200

2800

2400

2000

1800

1600

1400

1200

1013

800

1000

650.0

Wavenumbers Fig. 2. IR spectra of chitin extracted from resting eggs of Daphnia magna.

crab, lobster, shrimp and other arthropods [26]. In ␣ form, piles of chitin polymer chains are aligned in an antiparallel manner. In this orthorhombic form, the chains are organized very tightly [27]. In nature, physiological role and tissue characteristics determine the molecular arrangement of chitin. Unlike ␣-chitin, pure ␤-chitin is presented in fewer organisms [28]. In ␤ form, chitin polymer takes the monoclinic crystalline form and in this form, chains are packed in parallel sheets, in which they are tightly held by intrasheet hydrogen bonds [29]. In contrast to ␣-chitin, ␤-chitin has only intrasheet hydrogen bonding in its crystalline structure [29]. This makes ␤-chitin more susceptible to intra-crystalline swelling. It is, therefore, more soluble in for solvents and shows higher reactivity in modification reactions than ␣-chitin [28]. In the ␥ form, the polymer chains are aligned in one parallel and two antiparallel arrangements [30]. The ␥ form has reported to be a variant of ␣ family [31]. The structure analysis by Fourier transforms infra-red (FTIR) studies have indicated that in ␣ form, the absorption band of hydrogen bonding between the carbonyl groups ( C O) of amide I and amine groups ( NH ) of amide II is observed at 1660 cm−1 , and the absorption band of hydrogen bonding between the side chain ( CH2 OH) and the carbonyl group ( C O) appears at 1620 cm−1 . Splitting of the amide I band appears in the FTIR spectrum of ␣chitin, which is not the case for ␤-chitin. In ␤ crystalline form,

chains have only intrasheet hydrogen bonding and the absorption band of hydrogen bonding between the carbonyl groups ( C O) of amide I and amine groups ( NH ) of amide II appears at 1650 cm−1 [8]. The FTIR spectrum of ␣-chitin extracted from D. magna resting eggs is presented in Fig. 2. In the spectrum, three absorption bands were observed at 1652 cm−1 , 1622 cm−1 (characteristic of amide I stretching of C O and amid II of N H bend) and 1556 cm−1 (C N stretching). Characteristically, these three peaks indicate existence of ␣-chitin in D. magna resting eggs. Also in the spectrum, 1060 cm−1 corresponds to asymmetric bridge O stretching (C O C) and 1022 cm−1 is ascribed to asymmetric C O stretching. More details are presented in Table 1. Liu et al. [14] studied FTIR spectra of commercial ␣-chitin from shrimp and they observed 1654 cm−1 (amide I), 1627 cm−1 (amide II) and 1560 cm−1 (C N stretching) characteristic bands. These findings are consistent with FTIR spectra of ␣-chitin from D. magna resting eggs. 3.3. Elemental analysis (EA) Chitin isolated from D. magna resting eggs was studied with elemental analysis. Percent of N, C, H and C/N ratio and degree of

Table 1 IR spectrum data of the chitin. Functional group and vibration modes

Classification

O H and N H hydroxyl 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 C O C asym. stretch in phase ring C O asym. stretch in phase ring CH ring stretching

– Aliphatic compounds Aliphatic compound Amide I Amide I Amide II – – Amid III, components of protein Saccharide rings – Saccharide rings

Wavenumber (cm−1 ) frequency 3262 2922 2852 1652 1622 1556 1432 1376 1308 1060 1022 950

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Table 2 Results of elemental analysis (EA) and degree of acetylation (DA). Samples

N (%)

C (%)

H (%)

C/N

DA (%)

Daphnia magna resting egg Standard ␣-chitin (shrimp Shell) (Liu et al. [14])

6.66 6.24

44.2 43.75

6.30 6.40

6.63 7.07

87 109

acetylation (DA) values are given in Table 2. In chitin characterization studies by elemental analysis, theoretical nitrogen content (6.9%) is stated for completely acetylated chitin [14]. In this work, nitrogen content of chitin isolated from D. magna resting eggs was found to be 6.66%. DA is employed for determination of degree of purification of isolated chitin. Elemental analysis is one of the major techniques for determination of DA. In a study conducted by Liu et al. (2012) DA of ␣-chitin was reported to be 109% [14]. In our study DA value was determined to be 87%. According to Sajomsang and Gonil (2010), DA should not exceed 100% [34]. In cases where DA is over 100%, incomplete removal of some inorganic materials from the polymers structure can be mentioned [34]. Here, it can be stated that chitin isolated from D. magna resting eggs is purer than standard shrimp chitin investigated by Liu et al. [14]. 3.4. TGA The thermogram of ␣-chitin from D. magna resting eggs is presented in Fig. 3. In the thermogram, two decomposition steps were observed. In the first step, 5.78% mass loss at 37.2 ◦ C can be related

to the evaporation of water molecules that had already adsorbed to the polymer chains. In the second step, at 351.6 ◦ C, 62.64% mass loss was observed. The degradation of the polysaccharide structure of the molecule and decomposition of the acetylated and deacetylated units of chitin polymer can account for this mass loss [32]. In a study by Juárez-de La Rosa et al. [35] it was observed that standard ␣-chitin (from crab shell) had main decomposition temperature (DTGmax ) at 350 ◦ C. In this study, we also report nearly same temperature (DTGmax ; 351.6 ◦ C) for main decomposition of chitin from D. magna resting eggs. These findings indicate that chitin from D. magna resting eggs is in ␣ form. 3.5. XRD XRD result of chitin extracted from D. magna resting eggs shows two strong peaks at 9.02, 19.68◦ and one faint peak at 26.48◦ . According to literature [8], ␣-chitin has classic sharp peaks at 9 and 19◦ . Some other small peaks can be seen at 12, 21, 23 and 26◦ [14,33–35]. Here, the chitin extracted from D. magna resting eggs is found to have alpha character (Fig. 4).

351,6 oC TG DTG 90.0

80.0

TG 70.0

60.0

40.0

37,2oC 30.0

100.0

200.0

300.0

400.0

500.0

o

Temperature ( C) Fig. 3. TG-DTG curves of chitin extracted from resting eggs of Daphnia magna.

600.0

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Fig. 4. X-ray diffractometry (XRD) of chitin extracted from D. magna resting eggs. Table 3 XRD peaks of ␣-chitin (◦ ) in the literature. Organisms

XRD peaks of ␣-chitin (◦ )

Resting egg of D. magna Standard ␣-chitin Commercial shrimp chitin Commercial crab chitin

9 9 9 9

12 12 12 12

References 19 19 19 19

21 21 21 21

23 23 23 23

Fig. 5. SEM pictures of extracted chitin from resting eggs of Daphnia magna.

26 26 26 26

Present study [35] [9,14] [9]

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Table 3 shows the comparison of XRD peaks of chitin from D. magna resting eggs with those of other standard ␣-chitin studied in previous papers. It can be seen from the table that XRD peaks of chitin from D. magna resting eggs are exactly same as those of standard ␣-chitin [35]. 3.6. SEM Surface characteristics of the ␣-chitin extracted from D. magna resting eggs are shown in SEM pictures taken in different magnifications in Fig. 5. The SEM images exhibit rough and thick surface morphology of the polymer surface. 4. Conclusion This paper is the first to determine chitin content of D. magna resting egg (18–21%) and it also confirmed the presence of ␣chitin. Detailed physicochemical characterization of ␣-chitin was carried out by FTIR, elemental analysis, TGA, XRD and SEM studies. D. magna is a widely cultured Daphnia species and can easily be manipulated for resting egg production. This study shows that resting egg of D. magna can be exploited for chitin production as a new source. References [1] M. Rinaudo, Prog. Polym. Sci. 31 (2006) 603–632. [2] B.K. Park, M.M. Kim, Int. J. Mol. Sci. 11/12 (2010) 5152–5164. [3] H. Tajik, M. Moradi, S.M.R. Rohani, A.M. Erfani, F.S.S. Jalali, Molecules 13 (2008) 1263–1274. [4] J. Synowiecki, N.A. Al-Khateeb, Crit. Rev. Food Sci. Nutr. 43 (2003) 145–171. [5] C. Chang, N. Peng, M. He, Y. Teramoto, Y. Nishio, L. Zhang, Carbohydr. Polym. 91/1 (2013) 7–13. [6] M. Sabitha, N.S. Rejinold, A. Nair, V.K. Lakshmanan, S.V. Nair, R. Jayakumar, Carbohydr. Polym. 91/1 (2013) 48–57. [7] R. Salah, P. Michaud, F. Mati, Z. Harrat, H. Lounici, N. Abdi, N. Drouiche, N. Mameri, Int. J. Biol. Macromol. 52 (2013) 333–339. [8] M.K. Jang, B.G. Kong, Y.I. Jeong, C.H. Lee, J.W. Nah, J. Polym. Sci. Part A: Polym. Chem. 42 (2004) 3423–3432.

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