SUPPLEMENTARY MATERIAL
Characterization of α-chitin extracted from a lichenised fungus species Xanthoria
parietina
Murat Kayaa,b*1, Mehmet Gökhan Halıcıc, Fatih Dumanc, Sevil Erdoğand, Talat Barane
a
Department of Biotechnology and Molecular Biology, Faculty of Science and Letters
Aksaray University, 68100, Aksaray, Turkey.
b
Science and Technology Application and Research Center, Aksaray University, 68100,
Aksaray, Turkey.
c
Department of Biology, Faculty of Science, Erciyes University, 38039, Kayseri, Turkey.
d
Fisheries Programme, Keşan Vocational College, Trakya University, 22800 Keşan, Edirne,
Turkey.
e
Department of Chemistry, Faculty of Science and Letters, Aksaray University, 68100,
Aksaray, Turkey.
1
*Corresponding author: Murat Kaya
E-Mail: muratkaya3806@yahoo.com
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Abstract
Lichens are symbiotic associations formed mainly by ascomycete fungi and green
algae or cyanobacteria. The presence of chitin in the fungal cell wall has been revealed by
previous studies. Considering the presence of fungi in the lichens, this work will determine
the presence of chitin in a cosmopolitan lichen species Xanthoria parietina. In this study,
chitin was derived from a lichen species for the first time and its physicochemical properties
were determined by Fourier transform infrared spectroscopy, Thermogravimetric analysis, Xray diffraction, Scanning Electron Microscopy and Elemental Analysis. The dry weight chitin
content of X. parietina was 4.23%, and this chitin was in the α-form. The crystalline index
(CrI) value of the lichen chitin was calculated as 70.1%. The chitin from X. parietina had a
smooth surface.
Key words: Lichen; chitin; isolation; characterization; biopolymer
3. Experimental
3.1. Collection of lichen samples
Lichen samples belong to the species X. parietina were collected for the extraction of
their chitin from an oak tree in Edirne (Saçlımüsellim, N 41°25.998', E 26°37.877', 62 m) on
21.04.2013 (Figure S5). The samples were rinsed with distilled water to remove all the
residues on lichen surface. After this the samples were put in an oven to dry at 50 °C for 5
days.
Twenty-five specimens of this lichen were dried and stored with a code number
(TUKMYOBOT 2013-01) in Keşan Vocational College, Trakya University, Edirne, Turkey.
3.2. Extraction of chitin from X. parietina
Dry lichen samples, 4 g in weight, were prepared for analysis by crushing them into a
powder. The chitin extraction process was carried out using the chemical method in three
steps.
Demineralization: Minerals in the lichen sample were removed by treating it with 4M
100 ml HCl. The lichen sample was kept in the acidic solution at 50-75 °C for 12 hours and
stirred. Then the sample was filtered and washed with distilled water.
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Deproteinization: The filtrates were added into another volumetric flask, and 50 ml of
2M NaOH was added in the flask. The filtrate was kept at 150-175 °C with stirring for 24
hours. The protein free substance was filtered and washed with distilled water.
Decolourization: To obtain a clean chitin, the substance was treated with a mixture
(total volume of 50 ml) of chloroform, methanol and distilled water (in the ratio of 1: 2: 4).
The mixture was stirred at 1000 rpm for 1 hour without heating.
The final product was filtered and washed with distilled water to eliminate chemicals.
It was dried in an oven at 60 °C for 48 hours.
In the present study, shrimp chitin provided from SIGMA-ALDRICH company was
used to make a comparison with lichen chitin. Code number of this commercial chitin was
1001416772.
3.3. Fourier transform infrared spectroscopy (FTIR) analysis
The chemical structure of the lichen chitin was investigated by IR analysis. Attenuated
Total Reflection (ATR) technique was performed for FTIR analysis. A Perkin Elmer FTIR
Spectrometer was used to measure the infrared spectra. The spectra were obtained in a
wavenumber range of 4000-625 cm-1.
3.4. Thermogravimetric (TGA) analysis
The thermal behavior of the chitin from the lichen was investigated with an EXSTAR
S11 7300 at a heating rate of 10oC per min. The mass loss and thermal decomposition of the
lichen chitin were measured as a function of temperature and its thermal stability was
determined.
3.5. X-ray diffraction (XRD) analysis
A Rigaku D max 2000 X-ray diffractometer was used to characterize the crystallinity
of the lichen chitin. To determine the XRD patterns of the lichen chitin, the X-ray
diffractometer was operated at 40 kV, 30 mA and 2θ with the scan angle from 5º to 45º. The
crystalline index value (CrI) was calculated according to the equation (1):
Equation (1): CrI110 = [(I110− Iam)/I110] × 100.
I110 = the maximum intensity at 2θ 20°
Iam = the intensity of amorphous diffraction at 2θ 16° (Liu et al. 2012).
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3.6. Scanning Electron Microscopy (SEM)
The surface morphology of the lichen chitin was observed on a Quanta 200 FEG
Environmental Scanning Electron Microscope. The surface of the sample was coated with
gold before the SEM analysis using a Gatan Precision Etching Coating System (PECS).
3.7. Elemental Analysis
Elemental analysis of the lichen chitin was performed using the method and the
devices reported in Kaya et al. (2013).
References
Kaya M, Sargin I, Tozak KÖ, Baran T, Erdogan S, Sezen G. 2013. Chitin extraction and
characterization from Daphnia magna resting eggs. Int J Biol Macromol. 61: 459–
464.
Table S1. FTIR bands of the chitins extracted from the lichen (Xantoria parietina) and
commercial chitin.
Functional group and vibration modes
Classification
O–H stretching
-
N-H stretching
Wavenumber (cm-1) frequency
X. parietina
Commercial
Chitin
3430
3431
3101-3268
3102-3268
CH3 sym. stretch and CH2 asym. stretch
Aliphatic compounds
2919
2920
CH3 sym. stretch
Aliphatic compound
2853
2854
C=O secondary amide stretch
Amide I
1654
1655
C=O secondary amide stretch
Amide I
1624
1622
N–H bend, C–N stretch
Amide II
1560
1558
Amida III,
components of protein
1422
1375
1310
1421
1376
1312
1153
1110
1060
1153
1111
1060
CH2 ending and CH3 deformation
CH bend, CH3 sym. deformation
CH2 wagging
Asymmetric bridge oxygen stretching
Asymmetric in-phase ring stretching mode
C–O–C asym. stretch in phase ring
Saccharide rings
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C–O asym. stretch in phase ring
CH3 wagging
CH ring stretching
-
1012
1011
along chain
954
955
Saccharide rings
891
893
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Figure S1. IR spectra for the α-chitins. (a. chitin from Xanthoria parietina, b. commercial
chitin).
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Figure S2. TGA curve of the chitins (a. chitin from Xanthoria parietina, b. commercial
chitin)
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Figure S3. X-ray diffraction pattern of the α-chitins (a. chitin from Xanthoria parietina, b.
commercial chitin)
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Figure S4. SEM pictures of the chitin obtained from Xanthoria parietina
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Figure S5. General view of the lichen Xanthoria parietina
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