PRODUCTION OF NANOPARTICLES USING COCOA WASTE
BY
FATEMEH SOROODI
A dissertation submitted in fulfillment of the requirement for the degree of Master of Science in (Biotechnology
Engineering)
Kulliyyah of Engineering
International Islamic University Malaysia
JUNE 2017
ii
ABSTRACT
Currently, a large number of physical, chemical, and biological methods are available to synthesize different types of nanoparticles (NPs) such as metals, semiconductors, and magnetic materials. While most of the synthetic physicochemical methods reported to date are heavily dependent on the use of organic solvents and toxic reducing agents, green synthesis of NPs provides advancements over other methods as it is simple, cost-effective, and relatively reproducible and often results in more stable materials. Cocoa pod husk (CPH) is a by-product obtained after removal of cocoa beans from cocoa fruit which causes many environmental problems. The analysis of CPH has shown that this waste material contains phytochemicals such as polyphenols and theobromines as well as high amounts of protein and sugar which can act as reducing agent in the green synthesis of NPs. In this study, aqueous extract and broth of cocoa waste (cocoa pod husk and leaf) were screened for their potential reduction of five salt precursors including copper (II) chloride, cerium (III) nitrate, cadmium sulfate, silver nitrate and iron (III) chloride into their relative nanoparticles (NPs) in a novel, eco-friendly and one step protocol. Formation of NPs by CPH extract was followed by the observation of distinguished colour change of the related NPs in the reaction mixture. The synthesis of CPH extract mediated selenium nanoparticles (Se NPs) was identified by change in the colour of the reaction mixture into bright red colour, while the other metal salt solutions did not present any colour changes expected for their respective metal NPs formation. UV-vis spectroscopy further confirmed the formation of Se NPs. Since the required contact time for the synthesis of Se NPs was relatively long, initial studies were carried out to improve the reaction condition. The addition of 8 ml CPH extract (50% w/v) to 42 ml sodium selenite (1 mM) at 30 °C while vigorously mixing by magnetic stirrer exhibited the distinguished light red colour change of the reaction solution within 48 hours indicating formation of Se NPs. A systematic study of the significant factors involved in the production of Se NPs was performed by fractional factorial design at two levels.
Sodium selenite and CPH extract concentration as well as mixing time were identified as significant which were optimized by response surface method. It was concluded that maintaining the mixing time at 5.3 days and CPH extract concentration at 36.5%
w/v as well as the use of 30 mM sodium selenite maximized the Se NPs concentration.
Characterization of Se NPs by field emission scanning electron microscopy (FESEM) determined the spherical shape of the Se NPs with the size range from 25-45 nm. The Fourier transformation infra-red (FTIR) spectroscopy demonstrated two main peaks in both control sample (CPH extract) and the produced Se NPs corresponding to N-H and O-H groups. This was interpreted as the contribution of the proteins and polysaccharides available in the CPH extract in the reduction of the selenite ions (SeO32-).
iii
ثحبلا ةصلاخ
ً ایلاح
ً
ًكانه
ًرطلاًنمًیربکًددع ایمیکلاًق
ئ
ًة
ًو یازیفلا ئ
ًوًة یویلحا ةً
لما أاً فتاختً یکرکلً حاتا یزجًنمًعاون
ئ
ًتا
ًونانلا ( NPs )
ًً
داعلماک
ًوًن أ
ً صولماًفاصن
ًوًتلا یسیطانغلماًداولما ة
ً.
ًو
ًابم أ
ًن أ اسولاً فغ ئل
ً ایمیکویزیفلا ةئ
ً عبتالما ةً
لآا طبترمًن
ً ایرثک ةًً
ً
ًتابیذلمبا
یوضعلا
ًوًة ففقلماًلماوعلا ةً
فً،مستافل تا
ًبرتاع
ًةقیرط أاًعینصتالا فلًرضخض
یزج ئ
ًتا
ًونانلا مدقتام ةً
اسولاًنمًاهیرغًیفع
ً ارظنًل ئ
ً
ًوً اهتاطاسبل ففکتالاً ثیحً نمً اهتایفعاف
ةً
ً اهجئاتانوً اهتایجاتاناً نعو فه
رثكأً ایبسن ً ی
ً ارارقتاسا ً واکاکلاً فلاغً رشقً .
( دامًوهً) CPH ةً
یونثا ةً
تجنا ةً
یفمعًنع ةً
ًرذبًعزن ةً
ًرمثلاًنمًواکاکلا ةً
فکشمًدعبًیذلاو ةً
یبئ یة
ًرمثلاًتاففختًلیفتحً.
أ ایمیکلاًداولماًنمًدیدعلاًدوجوًرهظ ةئ
ً یتابنلا لاًلثمًة لاونیف
ًوًةدیدعلاًت کوًینموربویثلا
فش ت
ً بسنًدوجوًنع ةً
یلاع
ًنمًة
ًوًتانیتوبرلا ضفختًلماوعًدعتًیتالاًتیارکسلا
ةً
یفمعًیف ةً
أاًعینصتالا جًرضخض
یزئ تا
ً ونانلا
ً.
ف ساردلاًهذهًی ةًً
ًنمًلک
الماًصفختاسلما
ًوًیئ
ًرثمًتاففختًجیزم ةً
اوًفلاغلاًرشق(ًواکاکلا أ
صحفًتمً)قارو ه
ًردقً شکلًا ةً
ا
ًنمًسملخًالهازتاخض
ًوًحلامأاًتابكرم یه
ًً
دیروفك
ًا
ًساحنل ( II )
ًموییرسلاًتارکینً،
( III )
ً ضفلاًتارکنً،مویمداکلاًتاتایبرکً،
ًوًة
ًدیدلحا إ
ًیل
یزج ئ صالخاًونانلاًتا ةً
یبفلًقیدصًدیدجًلوکوتوربًیفًابه ةئ
ً فحرمًوذ ةً
دحاو ة یزجًلکشتً.
ئ
ًونانلاًتا ( NPs )
ً
ًنم
ًصفختاسم
ًواكاكلاًةرشق (
تاعباتامًتمً) CPH ةً
ظحلامًقیرطًنع ةً
یزجًنولًیرغت ئ
ًونانلاًتا ( NPs )
ً
ً.لعافتالاًطیفخضًیف
نعً شکلاًتم
ً عینصت
ً لًونانلاًتائیزج
ًموینیفیسف ( Se NPs )
ً
ًنم
ًصفختاسم واكاكلاًةرشق
ً(
ًنولًیرغتابً) CPH
ًلعافتالاًطیفخض إ
أاًنوفلاًیل
ً تًلمًینحًیفً،عقافلاًرحم
ً هظ أاًیقباًر یندعلماًحلام
ةً
أاًیفًعقوتالماًیفًیرغتالا انثاًناول
ءً
ًلکشت
یزج ئ ونانلاًتا .ً
ًدكأ لاًلیفحتالا
ًقوفًیفیط لا
ًیجسفنب أ
یزجًلیکشتًاضی ئ
ونانلاًتا .ً
ًو
ًابم أ
ًلیکشتالًبوفطلماًتقولاًن
یزج
ً لایوطًناکًونانلاًتا ئ
ً،
أًدقف
ًتاسردًتیرج أ
یلو ةً
ل
ً.لعافتالاًفورظًینسحتا
ًلثم فاضا ةً
8
ً
ًصفختاسمًنمًلم
ًةرشق
لا واكاك
ًإ
ًیل 42
ًم ل
ً تانیفیسًنم
ً مویدوصلا
ً(
1
ً رلاومًیفیم )ً
ًرارحًیفع ةً
30
ً جرد ةً
ًةیوئم
ًینحًیف
ًیوقلاًطفلخاًمتای
یسیطانغلماًطلالخبا
،ً
ًیرغتًضرع إ
أاًنوفلاًیل
ًللاخضًلعافتالاًجیزلمً یفلخاًرحم 48
ً عاس
ً ارشوم ةً
ً ذب ل ك
ً
ًلکشت
ًتائیزج
لا موینیفیس
ً ( Se NPs )
ًتتمً.
سارد ةً
یجهنم ةً
ًرثولماًلماوعفل ةً
ًلاًعینصتًیف Se NPs
ً میمصتًقیرطًنع
ً براجتالا
ً
لًنيورکكللإاًبوسالحبا اًلماوعف
ةرثؤلم
ً مًیفع ینیوتاس اهمً،
ً تانیفیسًزیکرت
ً مویدوصلا
ًًو
ًصفختاسم واكاكلاًةرشق
ً لإبا فاض ةً
إ قوًیل
ًوًاهتایفعافًنمًدکاتالاًتمًجزلماًت
ًجهنمًقیرطًنعًاهنیستحًتمًدق لاا
باجتاس
ً
ًتقولاًتیبثتًنأًانصفختاساً.ةیریدقتالا
ًةدلم 5.3
ً
ًوًموی
ًزیکرت 36.5
ً
% w/v
ً
ًصفختاسلم واكاكلاًةرشق
ً لإباو فاض ةًإ
ًمادختاساًیل 30
ً مًیفیم
ًنمًرلاو
ًتانیفیس
لا
ًققیحًمویدوص أ
زیکرتًیصق
ً ج ونانلاًتائیز
ً Se NPs
ًتمً.
ًتافصاومًدیدتح
ًونانلاًتائیزج ل
مویدوصلاًتانیفیس
ً
ًنع
ًتناورککللااًحسمًتثااعبناًقحًقیرط ( FESEM )
ً تانیفیسلاًتائیزجًیورکلاًلکشلاًاددمح
ً
ًسایقب 25 - 45
ً
ًروفًلیوتحًلامعتاساًقیرطًنعًیفیطلاًلیفحتالاً.رکموننا یر
ً لأل عش ةً
ارملحاًتتح ءً
( FTIR )
ًأ نیعًنمًلکًیفًینتامقًرهظ ةً
نراقلما
،ة
ًًو صفختاسم
ً واكاكلاًةرشق
ًًو لاًتانیفیس مویدوص
ً )ونانلا(ًةقیقدلا
ً عنصلما ةً
فباقلما ةً
ًیتاعوملمج N - H
ًو O - H
ً
ًاذه
تًتم ع فیف ةً
هماسمًیفع
ًوًتانیتوبرلاًة ددعتام
ةً
دیارکسلا تا
ً جاوتالما ةد
ً
ًلاًصفختاسمًیف
ً CPH
ًلازتاخضلا آ نوی ةً
یفیسلا
ًتان
2 3-
. SeO
ً
iv
APPROVAL PAGE
I certify that I have supervised and read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Science (Biotechnology
Engineering).
………..
Ibrahim Ali Noorbatcha Supervisor
………..
Parveen Jamal Co-Supervisor
I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a dissertation for the degree of Master of Science (Biotechnology Engineering).
………..
Hamzah Mohd Salleh Internal Examiner
………..
Ma'an Alkhatib Internal Examiner
This dissertation was submitted to the Department of Biotechnology Engineering and is accepted as a partial fulfillment of the requirements for the degree of Master of Biotechnology Engineering.
………..
Faridah Yusof Head, Department of
Biotechnology Engineering This dissertation was submitted to the Kulliyyah of Engineering and is accepted as a partial fulfillment of the requirements for the degree of Master of Biotechnology Engineering.
………..
Erry Yulian Triblas Adesta Dean, Kulliyyah of Engineering
v
DECLARATION
I hereby declare that this dissertation is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.
(Fatemeh Soroodi)
Signature ... Date 15.6.2017
vi
INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA
DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH
PRODUCTION OF NANOPARTICLES USING COCOA WASTE
I declare that the copyright holders of this dissertation are jointly owned by the student and IIUM.
Copyright © 2017 (Fatemeh Soroodi) and International Islamic University Malaysia. All rights reserved.
No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below
1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.
2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.
3. The IIUM library will have the right to make, store in a retrieved system and supply copies of this unpublished research if requested by other universities and research libraries.
By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialization policy.
Affirmed by (Fatemeh Soroodi)
……..……….. 15.6.2017 Signature Date
vii
ACKNOWLEDGEMENTS
My dissertation has not been finished without the help from many people. First and foremost, I would like to thank my supervisor Prof. Dr. Ibrahim Ali Noorbatcha for teaching me the rigorous nature of research, importance of details, thoroughness, and offering me effective advice in dissertation writing and time management. He reviewed and edited my manuscripts numerous times and gave me comments, gave me confidence, and strongly encouraged me to continue working on my thesis.
Moreover, I appreciate Prof. Dr. Parveen Jamal as my co-supervisor whose help was indeed a great contribution to my study.
Second, my special thanks go to my husband Sadraddin Eslami whose encouragement and motivation made me be hopeful and strong towards doing my dissertation.
Third, I thank my mother Hafseh Hazanzadeh and my father Ahmad Soroodi who always taught me the importance of learning and improving myself. I also thank my mother and father in law Sharifeh Qurashi and Mohammad Noor Eslami for encouraging me throughout my studies.
Fourth, I would like to thank my friends Soofia Khan Ahmadi, Marjan Karimi pour, Ben Belgacem Farah, Oualid Abdelkader Bellag, Br. Aziz Ahmad and Br. Deni Subara.
Finally, I am deeply indebted to the Faculty of Engineering at IIUM and the respected staff Dr. Raha Raus, Dr. Azura Amid, Dr. Fatihilah Ali, Hj. Sukiman Bin Sangat, Br.
Mohd Iznan, Br. Sanadi, Br. Faiz, Br. Mohd Hafizul, and Br. Asalam for taking on many of my administrative tasks so that I could concentrate on my dissertation.
viii
TABLE OF CONTENTS
Abstract ... ii
Abstract in Arabic ... iii
Approval Page ... iv
Declaration ... v
Copyright Page ... vi
Acknowledgements ... vii
Table of Contents ... viii
List of Tables ... xi
List of Figures ... xiii
List of Abbreviations ... xv
CHAPTER ONE: INTRODUCTION ... 1
1.1 Background of the Study ... 1
1.2 Statement of the Problem... 3
1.3 Research Objectives... 4
1.4 Scope of Research... 4
1.5 Significance of Research ... 5
1.6 Dissertation Organization ... 5
CHAPTER TWO: LITERATURE REVIEW ... 8
2.1 Introduction... 8
2.2 Nanotechnology ... 9
2.3 Nanoparticles ... 10
2.4 Synthesis of Nanoparticles ... 10
2.4.1 Physical Synthesis of Nanoparticles ... 12
2.4.2 Chemical Synthesis of Nanoparticles ... 14
2.4.3 Green Synthesis of Nanoparticles ... 15
2.4.4 Synthesis of Nanoparticles by Microorganisms... 16
2.4.5 Synthesis of Nanoparticles by Plant ... 27
2.5 Cocoa Plant ... 37
2.5.1 Cocoa Waste ... 39
2.6 Green synthesis of Metal and Metal Oxide Nanoparticles ... 43
2.6.1 Copper Oxide Nanoparticles ... 43
2.6.2 Cerium Oxide Nanoparticles ... 44
2.6.3 Cadmium Oxide Nanoparticles ... 44
2.6.4 Selenium Nanoparticles ... 45
2.6.5 Iron and Iron Oxide Nanoparticles ... 46
2.7 Factors Affecting Green Synthesis of Nanoparticles... 47
2.7.1 Temperature ... 47
2.7.2 Extract Concentration ... 48
2.7.3 Salt Solution Concentration ... 48
2.7.4 pH ... 49
2.8 Characterization of Nanoparticles ... 49
2.8.1 UV-visible Spectroscopy ... 49
2.8.2 Field Emission Scanning Electron Microscopy (FESEM) ... 50
ix
2.8.3 Fourier Transform Infrared Spectroscopy (FTIR) ... 51
2.9 Application of Software in Experimental Study ... 52
2.9.1 Design of Experiment ... 52
2.9.2 Fractional Factorial Design ... 53
2.9.3 Response Surface Methodology... 54
2.10 Overview of Phytochemical Synthesized Nanoparticles Applications ... 55
2.11 Summary ... 56
CHAPTER THREE: RESEARCH METHODOLOGY ... 58
3.1 Introduction... 58
3.2 Materials ... 60
3.3 Equipment and Instruments ... 60
3.4 Extract Preparation ... 60
3.5 Aqueous Metal Salt Preparation ... 61
3.6 Washing and Purification ... 62
3.7 Screening for NanoParticles Production ... 62
3.8 Optimization ... 67
3.8.1 Fractional Factorial Design ... 67
3.8.2 Response Surface Methodology Design ... 70
3.9 Characterization of Nanoparticles ... 71
3.9.1 UV-visible Spectroscopy ... 71
3.9.2 Field Emission Scanning Electron Microscope ... 72
3.9.3 Fourier Transform Infrared Spectroscopy... 73
3.10 Summary ... 73
CHAPTER FOUR: RESULTS AND DISCUSSION ... 75
4.1 Introduction... 75
4.2 Screening for NanoParticles Production ... 76
4.2.1 Screening for the Synthesis of Copper/Copper Oxide Nanoparticles Using Cocoa Waste ... 76
4.2.2 Screening for the Synthesis of Cerium Oxide Nanoparticles Using Cocoa Waste ... 78
4.2.3 Screening for the Synthesis of Cadmium Oxide Nanoparticles Using Cocoa Waste ... 79
4.2.4 Screening for the Synthesis of Selenium Nanoparticles Using Cocoa Waste ... 81
4.2.5 Screening for the Synthesis of Iron/Iron Oxide Nanoparticles Using Cocoa Waste ... 82
4.3 Effect of Process Parameters ... 84
4.3.1 Investigation of Higher Concentration of Metal Salt and Plant Extract ... 84
4.3.2 Investigation of the Experimental Condition for Selenium Nanoparticles Synthesis ... 86
4.3.3 Investigation of Process Parameters for Faster Selenium nanoparticles synthesis ... 87
4.3.4 Evaluation of the New Conditions for the Synthesis of Other NPs... 89
4.4 Optimization ... 92
x
4.4.1 Fractional Factorial Design ... 92
4.4.2 Response Surface Methodology Design ... 98
4.5 Characterization of Nanoparticles ... 103
4.5.1 UV-visible Spectroscopy of Se NPs ... 103
4.5.2 Field Emission Scanning Electron Microscope ... 110
4.5.3 Fourier Transform Infrared Spectroscopy... 113
4.6 Summary ... 120
CHAPTER FIVE: CONCLUSION AND RECOMMENDATIONS ... 122
5.1 Conclusion ... 122
5.2 Recommendations... 124
REFERENCES ... 125
APPENDIX A: SE NPS STANDARD CURVE ... 146
APPENDIX B: UV–VIS SPECTRA FOR CONTROL SOLUTIONS ... 147
APPENDIX C UV-VIS SPECTROSCOPY RESULTS AND OBSERVATION OF COLOUR CHANGE ... 149
APPENDIX D: SCIENTIFIC PUBLICATIONS ... 151
xi
LIST OF TABLES
Table No. Page No.
2.1 Engineered NPs of different morphology biosynthesized by bacterial
species 18
2.2 Engineered NPs of different morphology biosynthesized by fungal
species 20
2.3 Engineered NPs of different morphology biosynthesized by
actinomycetes 24
2.4 Engineered NPs of different morphology biosynthesized by yeast 25 2.5 Engineered NPs of different morphology biosynthesized by algae 26 2.6 Use of different parts of plants in NPs green synthesis 30
2.7 Cocoa waste content 41
2.8 Cocoa leaves content 42
2.9 Characteristic infrared absorption frequencies 51
2.10 Diverse application of biosynthesized NPs 55
3.1 “Series C” study of various ratio of salt solution (0.011 M) to CPH
extarct (25% w/v) on formation of Se Nps 65
3.2 “Series D” trial experiment for Se NPs synthesis in different ratio of
salt solution (1 mM) to CPH extract (50% w/v) 66
3.3 Final screening of NPs with recomended process values 67
3.4 FFD process parameter 68
3.5 Experimental design for identifying important parameters using FFD 69 3.6 Experimental design for optimization of Se NPs yield using RSM 70 4.1 Initial screening result for synthesis of copper/copper oxide NPs
using cocoa waste 77
4.2 Initial screening result for synthesis of cerium oxide NPs using cocoa
waste 79
4.3 Initial screening result for synthesis of cadmium oxide NPs using
cocoa waste 80
xii
4.4 Initial screening result for synthesis of selenium NPs using cocoa
waste 82
4.5 Initial screening result for synthesis of iron/iron oxide NPs using
cocoa waste 83
4.6 “Series A” experimental results after 1 h stirring (left) and 48 h
contact time (right) of the reaction mixtures 85
4.7 "Series B” experimental results for Se NPs synthesis 87 4.8 “Series C” study of various salt solution and CPH ratios on
formation of Se NPs 88
4.9 “Series D” study of various salt solution and CPH ratios on
formation of Se NPs 89
4.10 FFD result for identifying significant process parameters 93
4.11 FFD ANOVA table 94
4.12 Optimum configuration of the Se NPs synthesis condition based on
FFD 98
4.13 RSM result for optimization of Se NPs yield 99
4.14 Analysis of variance of quadratic model (RSM) for Se NPs synthesis 100
xiii
LIST OF FIGURES
Figure No. Page No.
2.1 Conventional production methods of nanoparticles 11
2.2 Intracellular synthesis of NPs 19
2.3 Extracellular synthesis of Ag NPs by Fusarium oxysporum 22 2.4 Probable phytochemicals of plant extract involved in the
bioreduction of metal ions 28
2.5 Mechanism involved in the formation of Ag NPs using
Terminalia chebula fruit extract . 29
2.6 Slender branches cocoa tree with yellow and red pods 38
2.7 Accumulated CPH after removing the bean 40
2.8 Molecular structure of theobromine 40
2.9 Phytochemicals present in CPH 42
3.1 General metodology of the study 59
3.2 Initial reaction conditions for NPs screening 63
3.3 “Series A” modified experimental conditions using CPH 64 3.4 “Series B” modified experimental conditions using Na2SeO3 65 4.1 Comparison of the UV-vis absorbance spectra of selenium
Nanoparticles 89
4.2 FFD Pareto chart 95
4.3 FFD Half-normal probability plot 95
4.4 FFD main effect plot 96
4.5 FFD interaction plot 97
4.6 The contour plot of the combined effects of Na2SeO3 concentration,
CPH concentration, and mixing time on Se NPs synthesis 102 4.7 The 3D response surface curves for the predicted optimum synthesis
of Se NPs 103
xiv
4.8 UV-vis absorption spectra of Na2SeO3 and CPH mixtures used in
FFD 105
4.9 Comparison of the red colour intensity after purification in the
reaction mixtures used in FFD 106
4.10 Comparison of the red colour intensity after purification in the
reaction mixtures used in RSM optimization 107
4.11 UV-vis absorption spectrum of Na2SeO3 and CPH mixtures used in
RSM optimization 108
4.12 FESEM images of Se NPs before optimization 111 4.13 FESEM images of the synthesized Se NPs by CPH extract in
fractional factorial design 112
4.14 FTIR spectra of Se NPs 115
4.16 Proposed mechanisms of green synthesis of NPs using plant extarct 119 4.16 Proposed mechanism of green synthesis of Se NPs using CPH
extract 120
xv
LIST OF ABBREVIATIONS
ANOVA Analysis of Variance
CCFD Central Composite Face-centred Design
CL Cocoa Leaf
CPH Cocoa Pod Husk
DW Deionized Water
FFD Fractional Factorial Design
FESEM Field Emission Scanning Electron Microscopy FTIR Fourier Transform Infrared Spectroscopy g gram
NPs Nanoparticles
RSM Response Surface Methodology rpm Revolutions per minute
mM milimolar
ml millilitre nm nanometre UV Ultraviolet Vis Visible µg Microgram λ max lambda maximum
1
CHAPTER ONE INTRODUCTION
1.1 BACKGROUND OF THE STUDY
Nanotechnology is a rapidly growing area of science which has attracted the interest of many scientists around the globe leading to a nano revolution. Nanoparticles (NPs) such as metals, semiconductors and magnetic materials are considerably notified for a wide variety of applications. The major applications of NPs include water purification, sunscreens, fuel-cell catalysts, UV protection, high density data storage, Nanoparticle Organic Memory Field-Effect Transistor, and many more. NPs are particles less than 100 nm in diameter that exhibit new and enhanced size-dependent properties compared to their bulk material (Baker et al., 2013) due to the increased surface area of NPs. These unique physicochemical characteristics including catalytic activity, optical properties, electronic properties, anti-bacterial properties, and magnetic properties have attracted the interest of scientist for the novel methods of NPs synthesis (Mittal et al., 2013).
Currently, a large number of physical, chemical, biological, and hybrid methods are available to synthesize different types of NPs (Salam et al., 2012).
However, most of the synthetic physicochemical methods reported to date is heavily dependent on the use of organic solvents and toxic reducing agents like thiophenol, mercapto acetate, sodium borohydride, etc. Most of these reagents are highly reactive and pose potential environmental and biological risks (Singh et al., 2011). By contrast, greener synthesis of NPs employs a biological system or its components for the formation of NPs, where the main reaction is reduction of raw material into NPs
2
(Baker et al., 2013). This approach provides advancements over other methods as it is simple, cost-effective, and relatively reproducible and often results in more stable materials (Kharissova et al., 2013). Owing to the rich biodiversity, plant mediated NPs synthesis has recently become a subject of interest with different plant species being rapidly explored and evaluated for the synthesis of NPs (Baker et al., 2013). The mechanism of production of NPs by plants is not completely yet known. It is possible that the chemicals present in the plants (phytochemicals) not only convert the metal salt solutions to metals or metal oxides, but also stabilize the NPs which are produced (Baker et al., 2013).
Theobroma cocoa L. (Sterculiaceae) is an economically important crop and
Malaysia is considered as one of the cocoa producers in the world. The cocoa beans are used primarily in chocolate manufacturing and a large quantity of by-product known as cocoa pod husk (CPH) is usually left to decompose in the plantation area.
This waste generates environmental issues such as unpleasant odour and botanical disease subsequently. On the other hand, researches have shown that the cocoa waste contains phytochemicals and protein. These components present in cocoa waste such as CPH and leaf can be expected to play a role in green production of NPs.
Among various types of NPs, metal and metal oxide NPs exhibit several unique properties due to which they play an extremely important role in the areas of electronics, chemistry, material sciences, drug–gene delivery and biosensor, etc. In this study, various NPs such as Se, CeO2, CuO, Fe2O3, Fe3O4 and CdO were screened for their ability to be produced by cocoa waste.
In order to produce NPs, the most important aspect that needs to be considered is the characterization of the synthetized NPs. The features of NPs are important to verify the chemical and physical properties of NPs that were obtained from the
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experiment. In the current study, UV-visible spectrophotometer was used to identify the formation and yield of the NPs. Field emission scanning electron microscopy was used to determine the size and the shape, and Fourier transform infrared spectroscopy determineded chemical characterization and composition.
Out of the successfully screened NPs, one NPs system was chosen for further studies to determine the optimum process conditions for the production of NPs using Design of Experiment software, 7th edition. Fractional factorial design (minimum run resolution IV) technique was used in order to find the effective factors involve in the high yield of the synthesized NPs and the central composite face-centred design i.e.
CCFD (a Respond Surface Method) was selected for the optimization study.
1.2 STATEMENT OF THE PROBLEM
For the time being, a large number of chemical and physical approaches are available to synthesize diverse types of NPs. But there are several problems arising when NPs are synthesized by these methods including usage of toxic chemicals, generation of dangerous by-products, long time for production and difficulty in purification. Thus, the use of such methods limits the usability of the NPs. Studies showed that promoting biosynthesis of NPs can influence the commercial applications of these NPs in the field of pharmaceuticals and other medical sciences which are limited factors for NPs synthesized via conventional methods. In addition, synthesis of metal NPs through green route is an ecologically friendly, cost effective without use of chemicals (Sankar et al., 2014).
Moreover, cocoa pod husk represent a disposal problem, since the growth of cocoa beans processing leads to increasing wastes which constitute more than half of world production estimated at 3.53 million tons (World Cocoa Foundation, 2010 and
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Marcel et al., 2011) and this is a good reason to come up with a useful outlet for this by-product (Mollea et al., 2008). The presence of biomolecules such as protein, polysaccharides and phytochemicals in cocoa waste can be used as reducing agents in the production of NPs and could be considered as a significant strategy to manage its disposal problem. Furthermore, this process economically lowers the cost of producing NPs compared to other methods as free available wastes were used for this process.
1.3 RESEARCH OBJECTIVES
This study concentrates on the following objectives:
1- To identify which metal or metal oxide nanoparticles could be produced by extract of cocoa waste, i.e. cocoa pod husk and leaf.
2- To optimize the process conditions for nanoparticles production.
3- To characterize the size and shape of nanoparticles using UV-Vis spectrophotometer, FESEM, and FTIR.
1.4 SCOPE OF RESEARCH
This research is carried out to find out which type of metal or metal oxide NPs can be produced using cocoa waste which is easily available. One of the successfully screened NPs was characterized using UV-visible spectrophotometer, field emission scanning electron microscopy, and Fourier transform infrared spectroscopy techniques. Optimum conditions for the production of selected NPs were investigated.
Concentration of the plant extract, type of the extract, salt solution, incubation time, and temperature are some of the factors which were studied in the optimization of the NPs produced.
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Along with the increasing industrialization of engineered NPs, the risks to human health and environment have also been a major concern (Salam et al., 2012).
For example, inhaled NPs may evade phagocytosis, cross cell membranes, and redistribute to other sites of the body, causing systemic health effects (Gwinn and Vallyathan, 2006). However, the current project does not address these issues.
1.5 SIGNIFICANCE OF RESEARCH
Plant mediated synthesis of NPs has great potential to reduce the environmental pollutions related to the chemical or high energy physical methods currently used for the NPs production. This method also reduces the cost of the production by utilizing the existing natural reducing agents in the plant extract such as proteins, polysaccharides, etc. Considering the importance of NPs toxicity in medical and diagnostic field, this study can provide a safe use of these biosynthesized NPs.
In addition to providing valuable green technology approach with all the above mentioned benefits for the production of NPs, it also adds value to the cocoa industry by expanding the optimum output and removing environmentally harmful waste at the same time to enhance the nanotechnology applications and research in Malaysia.
1.6 DISSERTATION ORGANIZATION
This dissertation consists of five chapters; Chapter one commences on a brief background about the importance of the nanotechnology and choosing cocoa pod husk for its high potential in the green synthesis of nanoparticles. In addition, the problem statement, research objectives, scope and the significance of the study are described in this chapter.
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Chapter two overviews the previous studies conducted in the field of nanoparticles synthesis, different synthesis methods, characterizations and applications. A brief introduction is explained considering the role of nanotechnology its application in the industry. A review is also made on the main available methods and the need of clean method is the focus of the chapter. Hence, the different green synthesis methods of nanoparticles are well explained and the reasons for the selection of phytochemical synthesis i.e. use of plant extract are demonstrated. To justify potential bio-reduction ability, cocoa waste content as the medium for the nanoparticles synthesis are generally and specifically reviewed. The history of the selected nanoparticles synthesized by plant extract and the influence of the process parameters reported by the previous studies are discussed. Chapter Two also reviews different characterization methods and the analysis software used (Design of experiments) including fractional factorial design (FFD) and response surface method (RSM) to determine the significant factors based on the concentartion of nanoparticles. The last part of chapter two covers the summary of the application of biosynthesised nanoparticles from various sources.
Chapter Three explains in detail the materials, chemicals, equipment, apparatus used in this study. It also describes the procedure followed in this research in step-by-step starting from extraction, screening of nanoparticles, optimization of the selected nanoparticles and finally characterization, not excluding the charts and figures used for further explanation of the methodology.
The results and discussion of this research are described in Chapter four, beginning with the screening of the synthesis of nanoparticles from CPH and leaf extract and determining selenium nanoparticles (Se NPs) produced by the CPH extract as the objective of the optimization process. Laboratory scale optimization was carried
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out and the results were analysed using analysis of variance (ANOVA) following the design validation. UV-vis spectroscopy, FESEM and FTIR were further used to confirm the formation and characterization of Se NPs.
Chapter Five is the conclusion coupled with some recommendations to improve this study for future work.
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CHAPTER TWO LITERATURE REVIEW
2.1 INTRODUCTION
Richard Feynman first introduced the concept of nanotechnology in a lecture entitled
“There’s plenty of room at the bottom” in 1959 (Hulkoti and Taranath, 2014).
Nanotechnology is an outstanding field of modern science with potential implementation in different areas such as biotechnology, medicine, chemistry and physics. It has also provided the opportunities for researchers all over the world to initiate and conduct studies in developing technologies involved in the design and application of the novel materials with enhanced properties (Dhand et al., 2016).
A great concern of nanotechnology is to deal with the synthesis of nanoparticles (NPs) with different size ranging approximately from 1-100 nm which significantly affects the biochemical properties of the particles from their bulk materials (Nayak et al., 2016). Despite of existing conventional methods i.e. chemical and physical route of synthesis, in this study an inexpensive phytochemical approach was selected to synthesize NPs. The phytochemicals present in the plant extract accomplish the reduction process to form zero valent nanoparticles from its ions.
However, different parts of plant can be examined for their possible capability in the synthesis of NPs. Cocoa wastes including cocoa pod husk (CPH) and cocoa leaves (CL) were chosen as substrate component for the current work.
In this chapter, various methods for the synthesis of NPs are briefly discussed following by a detailed explanation regarding green synthesis of NPs. This comprises of a review on phytochemical production of NPs by different parts of plants and
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factors influencing the formation of NPs are argued. Furthermore, the application of the biosynthesized NPs in diverse field and industries is reviewed.
2.2 NANOTECHNOLOGY
Nanotechnology is one of the growing areas of science and technology in the past two decades because of the availability of new approaches and tools for the synthesis, characterization, and manipulation of nanomaterials in the form of nanoparticles, nanocrystals, nanopowder or nanoclusters. These nanomaterials offer multiple specialized improvements in miniaturizing the industry and have a great contribution to the use of the materials that are sparing from the nature (Nayak et al., 2016).
Despite the fact that nanotechnology is a new area of the science, its history traces back to the 9th century when gold and silver nanoparticles were applied in pottery for their glittering effects by the artisans of Mesopotamia. Experimental relations of gold (and other metals) to light by Michael Faraday in 1857 represent the first scientific documents describing the properties of nanoparticles (Singh et al., 2011). In 1980’s, the concept of nano-revolution was scientifically introduced by Eric Drexler who published the first article on nanotechnology in 1981 entitling “an approach to the development of general capacities for molecular manipulation”
(Drexler, 1981). Afterwards, the invention of characterization techniques such as transmission electron microscope (TEM), X-ray diffraction (XRD), atomic force microscope (AFM), etc. led the development of nanotechnology in the forefront of a wide range of disciplines including the borderline of chemistry, materials, medicine, electronics, optics, sensors, information storage, communication, energy conversion, environmental protection, aerospace, etc.
