comparative studies on the extraction of chitin- chitosan from golden apple snail shells at the ...

122 Pages·2016·3.65 MB·English

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COMPARATIVE STUDIES ON THE EXTRACTION OF CHITIN-
CHITOSAN FROM GOLDEN APPLE SNAIL SHELLS
AT THE CONTROL FIELD

SAYAN KAEWBOONRUANG

A DISSERTATION SUBMITTED IN PARTIAL FULFILLMENT OF
THE REQUIREMENTS FOR THE DOCTOR DEGREE OF
PHILOSOPHY IN ENVIRONMENTAL SCIENCE
FACULTY OF SCIENCE
BURAPHA UNIVERSITY
JUNE 2016
COPYRIGHT OF BURAPHA UNIVERSITY

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ACKNOWLEDGEMENT

This Ph.D. dissertation was written based on my research work carried out at
Graduate School Program iIn Environmental Science, Faculty of Science, Burapha
University, I would like to express my sincere gratitude and deep appreciation to my
major advisor, Asso Prof. Dr. Nantaporn Phatrabuddha, and my co-advisor, Asst.
Prof. Dr. Pichan Sawangwong and Asst. Prof. Dr. Somsak Pitaksanurat, for all of their
guidance and valuable advice throughout this study.
I would also like to express my heartfelt gratitude to principal examiner
Prof. Thamrong Prempridi and Asso Prof. Dr. Nantaporn Phatrabuddha, Asst. Prof.
Dr. Pichan Sawangwong, Asst. Prof. Dr. Somsak Pitaksanurat and Dr. Karnjana
Hrimpeng as examining committees for giving me valuable guidance and expertise in
research and taking up the examination job.
I would like to acknowledge all faculty staffs and friends at Burapha
University. Great appreciation is also given to Sirindhorn College of Public Health,
Khon Kaen, my working organization, for giving me a chance to continue my study in
the Doctor Degree of Philosophy in Environmental Science at Burapha University.
For all of the lecturers, staffs, and friends in Graduate School Program in
Environmental Science, Burapha University, sincere thanks for your support,
attention, and motivation.
Finally, heartfelt thanks to my family, for persistent and endless support
thorough my life and the long years of study. Great respect to Asst. Prof. Dr. Pichan
Sawangwon and Asst. Prof. Dr. Arnon Chaisuriya for your loving, patience,
caretaking, attention, and support throughout my study in both of Burapha University.
I offer special thanks to all my friends for your giving kindness, helping, solidarity,
and togetherness.

Sayan Kaewboonruang
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53810001: MAJOR: ENVIRONMENTAL SCIENCE; Ph.D.
(ENVIRONMENTAL SCIENCE)
KEYWORDS: CHITIN-CHITOSAN/ EXTRACTION/
GOLDEN APPLE SNAIL
SAYAN KAEWBOONRUANG: COMPARATIVE STUDIES ON THE
EXTRACTION OF CHITIN-CHITOSAN FROM GOLDEN APPLE SNAIL
SHELLS AT THE CONTROL FIELD. ADVISORY COMMITTEE: NANTAPORN
PHATRABUDDHA, Ph.D., PICHAN SAWANGWONG, Ph.D., SOMSAK
PITAKSANURAT, Ph.D. 111 P. 2016.

Chitin and chitosan are the second most available biopolymers after
cellulose. Chitosan as a result of N-deactylation of chitin and has linear chain of
β-(1, 4)-linked 2-acetamino-2-deoxy-β-D-glucopyranose and 2-amino-2-deoxy-β-
Dglucopyranose. The chitosan extraction consists of common steps such as
deproteinisation, demineralisation, and deacetylation. This study on the extraction of
chitin and chitosan from the shells of with is golden apple snail shells collection in the
control field. the extraction methods, by Method 1 using a mechanical stirrer rotating
at all times, Method 2 to using heat to boil, the high temperatures and Method 3 is
soaking up for a long time in the static reactor, to compare the productivity of chitin
and chitosan extraction from a chemical process in each Method 1 Method 2 and
Method 3 to compare the specific properties of chitin and chitosan extracted in
different method. All the resulting products were then characterized and confirmed
using the preliminary solubility test, Scanning Electron Microscopy (SEM) and
Electron Dispersive Spectroscopy (EDS), X-ray Diffractometry (XRD), Fourier
Transform Infrared Spectroscopy (FT-IR) and Nuclear Magnetic Resonance
Spectroscopy (NMR). The resulted from % chitin from Method 1, 2 and 3 was 1.99%,
0.55% and 0.03%, respectively. The percent of chitosan from Method 1, 2 and 3 was
42.56%, 23.02% and 15.06%, respectively.
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CONTENTS

Page
ABSTRACT ............................................................................................................. iv
CONTENTS .............................................................................................................. v
LIST OF TABLES .................................................................................................. vii
LIST OF FIGURES ............................................................................................... viii
CHAPTER
1 INTRODUCTION ............................................................................................. 1
Statements and significance of the problems ............................................ 1
Objectives ................................................................................................. 3
Hypotheses and Justification..................................................................... 3
Conceptual Framework ............................................................................. 4
Scope of Research ..................................................................................... 4
Expected Results ....................................................................................... 4
Definition of terms .................................................................................... 5
2 LITERATURE REVIEWS ................................................................................ 8
Biology and species of golden apple snail ................................................ 8
General information about the production of chitin-chitosan .................. 15
The extraction of chitin-chitosan methods ............................................... 20
Instrument use for properties extraction of chitosan ................................ 27
3 RESEARCH METHODOLOGY ..................................................................... 35
Study design ............................................................................................. 35
Area preparation for controlled field ....................................................... 36
Sample preparation .................................................................................. 37
Experimental procedures ......................................................................... 39
Experimental equipment .......................................................................... 43
Materials and chemicals ........................................................................... 43
Physical and chemical testing for chitosan properties ............................. 44
Statistical Analyses .................................................................................. 52
vi

CONTENTS (CONTINUED)

Chapter Page
4 RESULTS ......................................................................................................... 51
Properties of golden apple snail shells ..................................................... 51
Chitin and chitosan yields & efficiency of Methods ............................... 51
Analysis of chitin extraction .................................................................... 53
Analysis of chitosan extracted from chitin .............................................. 55
Characterisation of chitin and chitosan .................................................... 57
5 DISCUSSION AND CONCLUSION .............................................................. 70
Discussions .............................................................................................. 70
Conclusions .............................................................................................. 71
Benefit Cost Ratio (B/C) .......................................................................... 73
Recommendations .................................................................................... 74
REFERENCES ........................................................................................................ 75
APPENDIX .............................................................................................................. 81
APPENDIX A ................................................................................................... 82
APPENDIX B ................................................................................................... 92
APPENDIX C ................................................................................................. 103
BIOGRAPHY ......................................................................................................... 111
vii
LIST OF TABLES

Table Page
2-1 Sources of chitin and chitosan ....................................................................... 18
2-2 Shows the amount of chitin in various organisms (%) .................................. 18
2-3 Physicochemical characteristics of extracted and commercial chitosan
(mean ± standard deviation of triplicate determinations) .............................. 27
2-4 Wavenumbers and chemical group of FT-IR absorption bands for
chitosan .......................................................................................................... 32
3-1 Sample name, chemical, time and parameters for the stages of each method 59
4-1 Experimental yield of each stage for extraction of chitin and chitosan
from golden apple snail shells ....................................................................... 52
4-2 Analysis properties of chitin extraction ........................................................ 53
4-3 Analysis properties of chitosan extracted ...................................................... 55
5-1 Benefit Cost analysis the amount of raw golden apple snail shell
in the extraction of chitin-chitosan ....................................................... 73
A-1 Analysis of the ash content of golden apple snail shells ............................... 83
A-2 Analysis of the percentage calcium of golden apple snail shells .................. 83
A-3 Determination of chitin by Method 1, Method 2, Method 3 ......................... 84
A-4 Analysis moisture of chitin by Method 1, Method 2, Method 3 ................... 85
A-5 Analysis of the ash content of chitin- by Method 1, Method 2, Method 3.... 86
A-6 Analysis of chitosan extracted from chitin by Method 1, Method 2,
Method 3 ....................................................................................................... 87
A-7 Analysis moisture of chitosan by Method 1, Method 2, Method 3 ............... 88
A-8 Analysis of the ash content of chitosan by Method 1, Method 2, Method 3 . 89
A-9 Analysis of the percent DD chitosan by Method 1, Method 2, Method 3 .... 90
A-10 Analysis of the percentage of solubility chitosan by Method 1, Method 2,
Method 3 .................................................................................................... 91
A-11 Analysis of pH chitin and chitosan ............................................................ 91
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LIST OF FIGURES

Figure Page
1-1 Conceptual framework of comparative studies on the extraction of
chitin-chitosan from golden apple snail shells ....................................................... 4
2-1 The shape and style of the golden apple snail ........................................................ 9
2-2 Life cycle of the golden apple snail ......................................................................... 10
2-3 Reproduction of golden apple snail ......................................................................... 11
2-4 Spawning....................................................................................................................... 11
2-5 Biological characteristics of the golden apple snail shell ................................... 12
2-6 The structure of the golden apple snail shell ......................................................... 12
2-7 Chemical structure of chitin ...................................................................................... 15
2-8 Chemical structure of chitosan ................................................................................. 16
2-9 Schematic interpretation of the structure of the organic matrix in
Mollusk shells (not drawn to scale) ......................................................................... 23
2-10 Chemical manufacturing processes for chitin, chitosan,
and chitosan oligomers ............................................................................................... 24
2-11 Biotechnological processes for chitin, chitosan, and
chitosan oligomers production ................................................................................. 25
2-12 Scanning Electron Microscopy (SEM) ................................................................... 28
2-13 Electron Dispersive Spectroscopy as SEM ............................................................ 29
2-14 X-ray Diffractometry (XRD) .................................................................................... 30
2-15 Michelson interferometer in the FTIR spectrometer ........................................... 31
2-16 The wavelength in the range of electromagnetic spectrum that can be
made the transition ...................................................................................................... 33
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LIST OF FIGURES (CONTINUED)

Figure Page
3-1 Experimental design for the extraction of chitin-chitosan .................................... 35
3-2 Control field ................................................................................................................. 36
3-3 Golden apple snail from control field ..................................................................... 38
3-4 Dried with air oven at 100+10oC for 1 hour .......................................................... 39
3-5 Golden apple snail sample size ....................................................................... 39
3-6 Scanning Electron Microscopy (SEM) and Energy dispersive
Spectroscopy (EDS) .................................................................................................... 48
3-7 X-ray Diffractometry (XRD) .................................................................................... 49
3-8 Fourier Transform Infrared Spectroscopy (FT-IR) .............................................. 49
3-9 Proton Nuclear Magnetic Resonance (1H NMR) ................................................. 50
4-1 SEM Micrograph of golden apple snail shells powder ....................................... 57
4-2 SEM Micrographs of chitin Method 1 .................................................................... 58
4-3 SEM Micrographs of chitin Method 2 .................................................................... 58
4-4 SEM Micrographs of chitin Method 3 .................................................................... 58
4-5 SEM Micrographs of chitosan Method 1 ............................................................... 59
4-6 SEM Micrographs of chitosan Method 2 ............................................................... 59
4-7 SEM Micrographs of chitosan Method 3 ............................................................... 60
4-8 EDS Spectrum and distribution of element for chitosan Method 1 .................. 61
4-9 EDS Spectrum and distribution of element for chitosan Method 2 .................. 61
4-10 EDS Spectrum and distribution of element for chitosan Method 3 .................. 62
4-11 XRD Diffractogram for chitosan Method 1 (M1), chitosan Method 2 (M2),
chitosan Method 3 (M3) and chitosan standard .................................................... 63
4-12 FT-IR Spectrum of chitosan Method 1 (M1) at Transmittance ......................... 64
4-13 FT-IR Spectrum of chitosan Method 2 (M2) at Transmittance ......................... 65
4-14 2 FT-IR Spectrum of chitosan Method 3 (M3) at Transmittance...................... 65
4-15 1H NMR Spectrum of commercial chitosan ......................................................... 66
4-16 1H NMR Spectrum of chitosan extracted Method 1............................................ 67
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LIST OF FIGURES (CONTINUED)

Figure Page
4-17 1H NMR Spectrum of chitosan extracted Method 2............................................ 67
4-18 1H NMR Spectrum of chitosan extracted Method 3 ........................................... 68
4-19 1H NMR Spectrum of chitosan extracted Method 1, chitosan extracted
Method 2, chitosan extracted Method 3 and chitosan standard ......................... 68
B-1 Peaks Data on XRD Diffractogram for chitosan method 1 (M1)...................... 93
B-2 Peaks Data on XRD Diffractogram for chitosan method 2 (M2)...................... 94
B-3 Peaks Data on XRD Diffractogram for chitosan method 3 (M3) ..................... 95
B-4 Bands Data of FT-IR Spectrum for chitosan method 1 (M1) ............................ 96
B-5 Bands Data of FT-IR Spectrum for chitosan method 2 (M2) ............................ 97
B-6 ..................................... 98
Bands Data of FT-IR Spectrum for chitosan method 3 (M3)
B-7 1H NMR Spectrum of commercial chitosan ......................................................... 99
B-8 1H NMR Spectrum of chitosan extracted method 1 (chitosan 01) ................. 100
B-9 1H NMR Spectrum of chitosan extracted method 2 (chitosan 02) ................. 101
B-10 1H NMR Spectrum of chitosan extracted method 3 (chitosan 03) ................. 102
C-1 Deproteinisation method 1, 2 M NaOH 1g:20ml at room temperature,
stirrer 2 hrs .................................................................................................................. 104
C-2 Rinse with clean water several times and filtered until a neutral pH ............. 104
C-3 Demineralisation method 1, 2 M HCl 1 g: 20ml at room temperature,
stirrer 2 hrs .................................................................................................................. 105
C-4 Deacetylation method 1, 50% NaOH 1g:20 ml 140+10oC, stirrer 2 hrs........ 105
C-5 Deproteinisation method 2, 2 M NaOH 1g:20ml 120°C, boil 4 hrs ............... 106
C-6 Demineralisation method 2, 2 M HCl 1 g: 20ml 100°C boil 24 hrs ............... 106
C-7 Deacetylation method 2, 50% NaOH 1g:20 ml 90-120oC, boil 4 hrs ............ 107
C-8 Deproteinisation method 3, 2 M NaOH 1g:20ml at room temperature,
soak it up 48 hrs ......................................................................................................... 107
C-9 Demineralisation method 3, 2 M HCl 1 g: 20ml at room temperature,
soak it up 72 hrs ......................................................................................................... 108