A thesis submitted in fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering)

(1)

ENZYMATIC SYNTHESIS OF BIODIESEL FROM MORINGA OLEIFERA OIL VIA

TRANSESTERIFICATION

BY

YARA HUNUD ABIA KADOUF

A thesis submitted in fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering)

Kulliyyah of Engineering

International Islamic University Malaysia

OCTOBER 2018

(2)

ii

ABSTRACT

Biodiesel has become one of the leading options to replace fossil fuels as a source of energy and traditionally uses edible plants as they have high acid values, which in turn produce high quality oil. Current methods of producing biodiesel generate toxic waste as a result of chemical catalysts used to accelerate the process. In order to reduce costs and to ensure conservation, a more environmentally friendly approach is required.

Hence, this study explored the biodiesel potential of mature Moringa oleifera seeds, which are non-edible and therefore do not compete with food resources, via transesterification using immobilised Candida antarctica lipase as a catalyst. Moringa oil was extracted using Soxhlet extraction, with hexane as the solvent of choice. The crude oil was then characterised with respect to its physico-chemical properties.

Candida antarctica lipase was purified and immobilised on functionalised activated carbon (FAC) and effectiveness of different acids for functionalisation on immobilisation capacity was tested by reflux with hydrochloric acid, nitric acid and sulphuric acid, with HCl-FAC giving the highest immobilization capacity (6.022 U/g).

The immobilisation conditions were optimised by first screening parameters (time, temperature, pH, and agitation) using one-factor-at-a-time (OFAT) analysis and optimising according to time, temperature and pH using Face Centred Composite Design (FCCCD) in Design Expert. The optimum conditions were found to be 40°C, pH 6 and 24 hours. OFAT was used once more to determine if agitation, time, temperature, catalyst concentration and methanol to oil ratio showed significant influence on biodiesel production. Design Expert software was used in order to determine the optimum conditions for transesterification of Moringa oil. FCCCD using was selected, and the parameters chosen were: methanol to oil ratio, temperature, catalyst concentration and time. The optimum conditions were methanol to oil ratio 4:1, 40°C, 4% catalyst loading and 24 hours which gives a maximum yield of biodiesel of 94.01%. The kinetics of the transesterification reaction found that the activation energy was 43.126 kJ/mol and the frequency factor was 1.758 × 10⁸ min^-1 from a pseudo-first order reaction rate. Validation of both optimisations was then carried out as suggested by the software. The biodiesel was characterised with respect to iodine value, acid composition, kinematic viscosity, density, cloud point and pour point. This step determined the extent of conformity of the resulting biodiesel to the specified ASTM D 6751 and EN 14214 standards. High quality biodiesel product was found to be well within given standards and was the expected result of this study.

(3)

iii

ﺚﺤﺒﻟا ﺔﺻﻼﺧ

ﻪﺟﺎﺘﻧا ﰲ مﺪﺨﺘﺴﻳ نﺎﻛ ﻦﻜﻟو ﺔﻗﺎﻄﻠﻟ رﺪﺼﻤﻛ يرﻮﻔﺣﻷا دﻮﻗﻮﻠﻟ ﻲﺴﻴﺋﺮﻟا ﻞﻳﺪﺒﻟاﻮﻫ يﻮﻴﳊا دﻮﻗﻮﻟا وأ لﺰﻳدﻮﻴﺒﻟا ﺢﺒﺻا ىﺮخأ ﺔﻴﺣ ﻦمو .جﺘنﳌا دﻮﻗﻮﻟا ﰲ ىﻠعأ ةدﻮﺟ ﱃإ يدؤﻳ ﺎﳑ ﺎهﻴف ﺔﻴضﻤﳊا ةدﺎﳌا عﺎﻔترﻻ كﻟذو ﻞﻛﻸﻟ ﺔﳊﺎﺼﻟا ت ﺎﺒنﻟا ﺒﻟا جﺎﺘﻧا ﰲ ًﺎﻴﻟﺎﺣ ﺔمﺪﺨﺘﺴﳌا قﺮﻄﻟا نﺈف ةﺪعﺎﺴم ﺔﻴﺋﺎﻴﻤﻴﻛ ﻞماﻮع لﺎﻤعﺘسﻻ ﺔجﻴﺘﻧ ﺔمﺎﺴﻟا ت ﺎﻔنﻟا ﻦم ًاﲑثﻛ فﻠﲣ لﺰﻳدﻮﻴ

ﻩﺬﻫ نﺈف ﻪﻴﻠعو .ًﺎضﻳأ ﺔﻔﻠﻜت ﻞﻗأو ﺔﺌﻴﺒﻟا ىﻠع ﺔﻈفﺎﳏو ﺔمءﻼم ﺮثﻛا ىﺮخأ ﺔﻘﻳﺮﻃ دﺎﳚإ مزﻼﻟا ﻦم نﺎﻛ اﺬﳍ .ﺔﻴﻠﻤعﻟا ﻞﻴجعﺘﻟ وا ﺎجنﻳرﻮﳌا تﺎﺒﻧ بﻮﺒﺣ ﻦم لﺰﻳدﻮﻴﺒﻟا جﺎﺘﻧا تﺎﻴﻧﺎﻜمإ فﺸﻜﺘﺴت ﺔسارﺪﻟا اﲑﻔﻴﻟ

(Moringa oleifera) .ﺔجﺿﺎنﻟا

ﰎ ﺔسارﺪﻟا ﻩﺬﻫ ﰲ .نﺎﺴﻧﻺﻟ ﺔﻴﺋاﺬغﻟا داﻮﳌا سفﺎنت ﻻ ﱄﺎﺘﻟ ﻲهف ﻲﺋاﺬغﻟا كﻼهﺘسﻼﻟ ﺔﳊﺎﺻ ﲑغ بﻮﺒﳊا ﻩﺬﻫ نأ ﺎﲟو ةﱰسﻷا ﺔﻴﻠﻤع ﻖﻳﺮﻃ ﻦع بﻮﺒﳊا ﻩﺬﻫ ﻦم لﺰﻳدﻮﻴﺒﻟا جﺎﺘﻧا (transesterification)

زﺎﺒﻴﻟ ﱘﺰﻧا لﺎﻤعﺘس كﻟذو

(Candida antarctica) ﺔﻘﻳﺮﻄﺑ ﺎجنﻳرﻮﳌا ﺖﻳز صﻼﺨﺘسا ﰎ .ﺪعﺎﺴم ﻞمﺎعﻛ ﺖﺒثﳌا

Soxhlet ةدﺎم لﺎﻤعﺘس و

Hexane

زﺎﺒﻴﻟ ﺖﻴﺒثتو ﺔﻴﻘنت ﰒ ﺔﻴﺋﺎﻴﻤﻴﻛﻮﻳﺰﻴﻔﻟا ﻪﺼﺋﺎﺼﲞ مﺎﳋا ﺖﻳﺰﻟا ﺰﻴﻴﲤ ﰎ كﻟذ ﺪعﺑ .يرﺎﻴﺘخا ﺐﻳﺬﻤﻛ

(Candida antarctica) ﻂﺸنمو ﻞعﻔم نﻮﺑﺮﻛ ىﻠع

(FAC) ﲪﻻا ﺔﻴﻠعﺎف رﺎﺒﺘخا ﰎ ﰒ

ﺪنع ﺎهﺋادأو ﺔﻔﻠﺘﺨﳌا ضﺎ

نأ ﺮهظو .كﻴﺘﻳﱪﻜﻟاو كﻳﱰﻴنﻟاو كﻳرﻮﻠﻛورﺪﻴﳍا ضﺎﲪأ عم داﺪترﻻا ىﻠع ﺎ رﺪﻘم ىﺪﲟ كﻟذو ﺖﻴﺒثﺘﻟا HCI-FAC

ﺖﻴﺒثﺘﻟا ىﻠع ةرﺪﻘم ىﻠعﻷا ﻮﻫ (6.022U/g)

ﻞماﻮع ﻲﻫ ﺔنﻴعم ﲑﻳﺎعم ﺐﺴﺣ ﺖﻴﺒثﺘﻟا ﺔﻴﻠﻤع فوﺮظ ﲔﺴﲢ ﰎ ﰒ .

ﻦمﺰﻟا) – ةراﺮﳊا ﺔﺟرد -

pH – ﻻاو ﺖﻗو ﻞﻛ ﰲ ﺪﺣاو ﻞمﺎع ﻞﻴﻠﲢ ﺔﻘﻳﺮﻃ ماﺪﺨﺘس كﻟذو (جﺎﲡر (OFAT)

ﺔﻘﻳﺮﻃ ماﺪﺨﺘس كﻟذو ﺔﺿﻮﻤﳊا ﺔﺟردو ةراﺮﳊا ﺔﺟردو ﻦمﺰﻠﻟ ًﺎﻘفو ﲔﺴحﺘﻟا ﰒ ﻦمو (Face-centred central

composite design) (FCCCD) جم ﺮﺑ ﻦم

(Expert Design) ﻲﻫ فوﺮﻈﻟا ﻞثمأ نأ ﺪﺟو .

40

و ﺔﻳﻮﺌم ﺔﺟرد pH 6

ةﺪمو 24 ﺪﺣاﻮﻟا ﺖﻗﻮﻟا ﰲ ﺪﺣاو ﻞمﺎع ىﺮخأ ةﺮم ﺖﻠﻤعﺘسا ﰒ ﺔعﺎس (OFAT)

نا ﺪﻳﺪحﺘﻟ

.لﺰﻳدﻮﻴﺒﻟا جﺎﺘﻧا ىﻠع لﺎعف ﲑث ﺎﳍ ﺖﻳﺰﻟا ﱃا لﻮﻧﺎثﻴﳌا ﺔﺒﺴﻧو ﺪعﺎﺴﳌا ﻞمﺎعﻟا ﰲ ﺰﻴﻛﱰﻟا ﺔﺟردو ةراﺮﳊا ﺔﺟردو جﺎﲡرﻼﻟ نﺎﻛ جم ﺮﺑ ىﺮخأ ةﺮم مﺪﺨﺘساو Expert Design

ىﻠثﳌا فوﺮﻈﻟا ﺪﻳﺪحﺘﻟ ﻞﻤعﺘسا .ﺎجنﻳرﻮﳌا ﺖﻳز ﰲ ةﱰسﻸﻟ

جم ﺮﺑ FCCCD ﻦمﺰﻟاو ﺪعﺎﺴﳌا ﻞمﺎعﻟا ﺰﻴﻛﺮتو ةراﺮﳊا ﺔﺟردو ﺖﻳﺰﻟا ﱃإ لﻮﻧﺎثﻴﳌا ﺔﺒﺴﻧ : ﻲﻫ ﲑﻳﺎعم عم

– نا ﺮهﻈف

ﺖﻳﺰﻠﻟ لﻮﻧﺎثﻴﳌا ﺔﺒﺴﻧ ﻲﻫ ىﻠثﳌا فوﺮﻈﻟا 4:1

ةراﺮﳊا ﺔﺟردو 40

و ﺔﻳﻮﺌم ﺔﺟرد 4

% 5 ﻦمﺰﻟاو ﻞمﺎعﻟا ﺰﻴﻛﺮت 24

ﺔعﺎس

ىﻠع ﻞﺼﳓ ﻲﻜﻟ ﻲﻫو لﺰﻳدﻮﻴﺒﻠﻟ جﺎﺘﻧا لﺪعم ىﻠعأ

94.01%

.ﻮﻫ ﺔﻗﺎﻄﻟا ﻂﻴﺸنت نا ةﱰسﻼﻟ ﻞعﺎﻔﺘﻟا ﺔﻴﻛﺮﺣ تﺪﺟوو .

34.126 kJ/mol ﻮﻫ ﺮتاﻮﺘﻟا ﻞمﺎع نأو

1

min

-

1.758 x 10

8

لﺰﻳدﻮﻴﺒﻟا ﻢﻴﻴﻘت ﰎ .ﻞعﺎﻔت ﺔﺒﺴﻧ لوا ﻪﺒﺷ ﻦم

.بﺎﺒﺼﻧﻹا ﺔﻄﻘﻧو ﻢﻴغﺘﻟا ﺔﻄﻘﻧو ﺔﻛﺮﳊا ﺔﺟوﺰﻟو ﻲضﻤﳊا ﻦﻳﻮﻜﺘﻟاو دﻮﻴﻟا ﺔﻤﻴﻗ ﺐﺴﺣ ﺔﻘﺑﺎﻄم ىﺪم ةﻮﻄﳋا ﻩﺬﻫ تدﺪﺣ

ﲑﻳﺎعﳌ جتﺎنﻟا لﺰﻳدﻮﻴﺒﻟاا ASTM D 6751

و EN 1421 ةدﻮﳉا ﱄﺎع لﺰﻳدﻮﻴﺑ دﻮﻗو كﻟذ ﻦع جﺘﻧ .ةدﺪﶈا

.ﺎمﺎﲤ ﺔسارﺪﻟا ﻩﺬﻫ ﺖعﻗﻮت ﺎﻤﻛ ةﺎﻄعﳌا تﺎﻔﺻاﻮﳌ و

(4)

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 thesis for the degree of Master of Science (Biotechnology Engineering).

………..

Nassereldeen Ahmed Kabbashi Supervisor

………..

Md. Zahangir Alam Co-Supervisor

………..

Mohamed El-Wathig Saeed Mirghani

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 thesis for the degree of Master of Science (Biotechnology Engineering).

………..

Mohammed Saedi Jami Internal Examiner

………..

Abdulrahman Hamid Nour External Examiner

This thesis was submitted to the Department of Biotechnology Engineering and is accepted as a fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering).

………..

Faridah Yusof Head, Department of Biotechnology Engineering This thesis was submitted to the Kulliyyah of Engineering and is accepted as a fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering).

………..

Erry Yulian Triblas Adesta Dean, Kulliyyah of Engineering

(5)

v

DECLARATION

I hereby declare that this thesis 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.

Yara Hunud Abia Kadouf.

Signature ... Date ...

(6)

vi

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

ENZYMATIC SYNTHESIS OF BIODIESEL FROM MORINGA OLEIFERA OIL VIA TRANSESTERIFICATION

I declare that the copyright holders of this thesis are jointly owned by the student and IIUM.

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 Yara Hunud Abia Kadouf

……..……….. ………..

Signature Date

(7)

vii

ACKNOWLEDGEMENTS

First and foremost, all gratitude and praise are dedicated to Allah swt who has kept me strong and upright throughout this seemingly insurmountable goal.

I would also like to extend my deepest gratitude to my supervisors, who were incredibly patient with me and guided me with utmost kindness to ensure that I submitted work that met their quality standard. My appreciation also goes to the members of the Bioenvironmental Research Group, Head of Department and all those who aided in allowing me to complete this research.

And lastly, I would like to show my deepest gratitude and dedication to my dear friends from the Biotechnology Engineering Department and my family, especially my parents, who have supported and believed in me unwaveringly through this journey.

To all those who picked me up when I stumbled, thank you from the bottom of my heart and soul. This truly would not have been possible without you.

If – by Rudyard Kipling

If you can keep your head when all about you Are losing theirs and blaming it on you, If you can trust yourself when all men doubt you,

But make allowance for their doubting too;

If you can wait and not be tired by waiting, Or being lied about, don’t deal in lies, Or being hated, don’t give way to hating, And yet don’t look too good, nor talk too wise:

If you can dream—and not make dreams your master;

If you can think—and not make thoughts your aim;

If you can meet with Triumph and Disaster And treat those two impostors just the same;

If you can bear to hear the truth you’ve spoken Twisted by knaves to make a trap for fools, Or watch the things you gave your life to, broken,

And stoop and build ’em up with worn-out tools:

If you can make one heap of all your winnings And risk it on one turn of pitch-and-toss, And lose, and start again at your beginnings

And never breathe a word about your loss;

If you can force your heart and nerve and sinew To serve your turn long after they are gone, And so hold on when there is nothing in you Except the Will which says to them: ‘Hold on!’

(8)

viii

If you can talk with crowds and keep your virtue, Or walk with Kings—nor lose the common touch,

If neither foes nor loving friends can hurt you, If all men count with you, but none too much;

If you can fill the unforgiving minute With sixty seconds’ worth of distance run, Yours is the Earth and everything that’s in it, And—which is more—you’ll be a Man, my son!

(9)

ix

LIST OF TABLES

Table 2.1 Technical properties of biodiesel ... 10

Table 2.2 Some standards for biodiesel according to ASTM and EN ... 15

Table 2.3 Comparison of properties of petro-diesel and biodiesel ... 16

Table 2.4 Oil species for biofuel production ... 29

Table 2.5 Comparison of physical properties: beef tallow and chicken fat ... 30

Table 2.6 Fatty acid composition of Moringa oleifera oil ... 33

Table 2.7 Physico-chemical properties of Moringa oleifera oil compared with palm oil ... 33

Table 2.8 Summary of studies using enzymes as catalysts for biodiesel production .. 37

Table 3.1 Centre values for conditions of optimisation of percentage immobilisation 58 Table 3.2 Centre values for optimisation of production of biodiesel... 61

Table 4.1 Physical properties of Moringa oleifera oil ... 66

Table 4.2 Chemical properties of Moringa oleifera oil ... 68

Table 4.3 Fatty acid composition of Moringa oleifera oil ... 72

Table 4.4 Comparison of lipase activity for activated carbon functionalised with various acids, weight of matrix = 1g ... 73

Table 4.5 Centre points for influencing parameters for immobilisation of lipase on FAC ... 89

Table 4.6 Experimental matrix for the immobilisation of lipase on functionalised activated carbon using FCCCD of Design Expert software ... 90

Table 4.7 Model data ... 91

Table 4.8 Analysis of variance (ANOVA) for response surface quadratic model [partial sum of squares] for immobilisation ... 93

Table 4.9 Validation of experimental model for immobilisation ... 97

(13)

xiii

Table 4.10 Centre points influencing parameters for immobilisation of lipase on FAC ...107 Table 4.11 Experimental matrix for the production of biodiesel via transesterification using FCCCD of Design Expert software ... 107 Table 4.12 Model data ... 109 Table 4.13 Analysis of variance (ANOVA) for response surface quadratic model [partial sum of squares] for transesterification reaction ... 112 Table 4.14 Validation of experimental model for transesterification 116 Table 4.15 Comparison of Moringa biodiesel produced with MPOB, ASTM and EN

standards 119

(14)

xiv

LIST OF FIGURES

Figure 2.1 Types of fatty acids - saturated, unsaturated, polyunsaturated (Rennison &

Van Wagoner, 2009) ... 14 Figure 2.2 Intermediate steps in conversion of triglycerides into methyl ester and glycerol by transesterification (Borges & Díaz, 2012) ... 25 Figure 2.3 Chemical reaction for biodiesel production via transesterification (Wen, Bantz, Bachmann, Brodrick, & Schweitzer, 2009) ... 26 Figure 2.4 Moringa plant seeds pods and leaves ... 32 Figure 2.5 Catalytic mechanism of CALB for hydrolysis or transesterification (Li, Tan,

Zhang, & Feng, 2010) ... 35 Figure 2.6 Block diagram of heterogeneous biodiesel production (Konwar, Boro, &

Deka, 2014) ... 38 Figure 2.7 Surface oxygen containing groups (Shafeeyan et al., 2010) ... 42 Figure 4.1 FT-IR peaks of the functionalised activated carbon (a) with water (b) with

sulphuric acid (c) with hydrochloric acid and (d) with nitric acid ... 76 Figure 4.2 SEM images (x4000) of functionalised activated carbon (a) with water (b) with sulphuric acid (c) with hydrochloric acid and (d) with nitric acid .... 79 Figure 4.3 Effect of time in %immobilisation (reaction parameters were 150 rpm, 40C, 8mg FAC/ml lipase, pH 6) ... 82 Figure 4.4 Effect of temperature in %immobilisation (reaction parameters were 150 rpm, 24 hours, pH 6, 8mg FAC/ml lipase) ... 84 Figure 4.5 Effect of agitation on %immobilisation (reaction parameters were 150rpm, 40C, pH 6, 24 hours, 8mg FAC/ml lipase) ... 86 Figure 4.6 Effect of pH (reaction parameters were 40C, 24 hours, 150rpm, 8mg FAC/ml lipase) ... 87 Figure 4.7 Regression coefficients determined using data from FCCCD for

optimisation (Rashid, Anwar, Ashraf, et al., 2011) ... 89 Figure 4.8 Normal probability plot ... 94 Figure 4.9 Actual vs. Predicted response values ... 94

(15)

xv

Figure 4.10 3D contour plots showing the interactions of operating parameters. (a) Interaction between pH and temperature. (b) Interaction between pH and time. (c) Interaction between time and temperature. ... 96 Figure 4.11 Reusability test of immobilised enzyme (reaction parameters were 10g of Moringa oil, 8mg FAC/ml lipase, 4:1 methanol to oil ratio, 40°C and 24 hours reaction time) ... 98 Figure 4.12 Effect of temperature on biodiesel yield (reaction parameters were 4:1 methanol to oil ratio, 4% catalyst loading, 250 rpm agitation rate, 24 hours) ... 100 Figure 4.13 Effect of time on biodiesel yield (reaction parameters were 4:1 methanol to oil ratio, 4% catalyst loading, 250 rpm agitation rate, 40°C) ... 102 Figure 4.14 Effect of agitation on biodiesel yield (reaction parameters were 4:1 methanol to oil ratio, 4% catalyst loading, 24 hours, 40°C) ... 104 Figure 4.15 Effect of methanol to oil ratio on biodiesel yield (reaction parameters were 40°C, 4% catalyst loading, 24 hours, 250 rpm agitation) ... 105 Figure 4.16 Effect of catalyst loading on biodiesel yield (reaction parameters were 40°C, 4:1 methanol to oil, 24 hours, 250 rpm agitation) ... 106 Figure 4.17 Normal probability plot ... 110 Figure 4.18 Predicted vs actual model responses ... 111 Figure 4.19(a), (b), (c): 3D contour plots showing the interactions of operating parameters. (a) Interaction between catalyst loading and temperature. (b) Interaction between catalyst loading and time. (c) Interaction between methanol to oil and catalyst loading (d) Interaction between methanol to oil and temperature (e) Interaction between methanol to oil and time (f) Interaction between temperature and time. ... 115 Figure 4.20 Plot of -ln(1-C) against time for varying temperatures ... 117 Figure 4.21Arrhenius plot of ln k against 1/T*10^3 for transesterification of Moringa feedstock oil ... 118

(16)

xvi

LIST OF ABBREVIATIONS

AC Activated Carbon

AER Annual Energy Review

ANOVA Analysis of Variance

AOAC Association of Official Analytical Chemists APEC Asia-Pacific Economic Cooperation

ASTM American standard test method

AV Acid Value

B100 Pure unblended Biodiesel

CFPP Cold Filter Plugging Point

CIE Compression ignition engine

CO Carbon monoxide

CO2 Carbon dioxide

DG Diacylglycerides

DoE Design of Experiment

EI Electron Impact

EIA Environmental Impact Assessment

EIC Electron ion chromatograms

EMA Engine Manufacturers Association

EN 14214 European standard of biodiesel fuel

EPA Environmental Protection Agency

EREC European Renewable Energy Council

FAAE Fatty acid alkyl ester

FAC Functionalized active carbon

FAO Food and Agricultural Organization, U.N.

FAME Fatty acid methyl ester

FCCCD Face centered central composite design

FFA Free fatty acids

FTIR Fourier Transformed Infrared Spectroscopic

GC-MS Gas Chromatography Mass Spectroscopy

GC-MSD Gas Chromatography Mass Spectroscopy Detector

GRAS Generally regarded as safe

H2SO4 Sulfuric acid

HC Hydrocarbon

IEA International Energy Agency

IU International Unit of Enzyme Activity

IV Iodine value

KOH Potassium Hydroxide

MG Monoglycerides

MPOB Malaysian Palm Oil Board

NaOH Sodium Hydroxide

NBB National Biodiesel Board

NG Natural Gas

NIST National Institute of Standards of Technology

(17)

xvii

NOX Nitrogen oxides

OECD Organization for economic co-operation and development

OFAT One-factor-at-time

OSI Oxidative stability index

PAHS Polycyclic Aromatic Hydrocarbon

PKC Palm kernel cake

PM Particulate Matter

pNPP P-nitrophenylpalmitate

PV Peroxide value

RES Renewable Energy sources

RME Rapeseed methyl esters

SME Scattering Electron Microscopy

SV Saponification value

SZA Sulfated Zirconia Alumina

TG Triacylglycerol/Triacylglycerides/Triesters

TIC Total ion chromatograms

Tscf Trillion standard cubic feet

UNDP United Nations Development Programme

USA United States of America

USDE US Department of Energy

(18)

xviii

LIST OF SYMBOLS

€ Euros

A Frequency Factor

Ao Initial lipase activity

At Lipase activity after immobilization

Avt Initial acid value

C Final acid value

ε Molar extraction coefficient

Ea Activation energy

GJ Giga joule

K Arrhenius Equation

k Mean change in conventional mass per volume of the fat sample due to the temperature change

Kwth kilowatt

M Mass of immobilized matrix

m1 mass in grams of empty pycnometer with stopper m2 Mass in grams of pycnometer and water and stopper m3 Mass in grams of pycnometer with oil sample with stopper MW Molecular weight of the alkaline solution

Mwth Megawatt

N Normality of the alkaline used

R² Coefficient of determination

U Immobilized Capacity

U Enzyme Activity

V Amount of alkaline used in the titration

vb Titre value for blank

vc Volume in milliliters of pycnometer at 25 ̊ C

vs Titre value for sample

W Mass of breaker

w Weight of sample in grams

Wb Mass of breaker and wet sample

Wd Mass of breaker and sample after drying

θc Calibration temperature

ρ Conventional mass per volume of water of calibration ρθ Conventional mass per volume of the oil sample at 25

(19)

1

CHAPTER ONE INTRODUCTION

1.1 BACKGROUND

Environmental concerns over pollution and the effect of greenhouse gases on the global climate is rapidly increasing, therefore the urgency to research and discover renewable energy sources is becoming more apparent. One of the main reasons for these phenomena is the consumption of fossil fuels, mostly used to meet the demanding requirements of the transport, agriculture, and engineering industries. Moreover, these non-renewable and therefore limited resources are being used now more than ever, due to the sudden world population boom as well as technological advancement worldwide (Rahman et al., 2014). Therefore, it is imperative that other forms of energy be investigated, such as wind or solar energy, in order to slowly but surely ease the heavy reliance most industries have on fossil fuels.

The search for substitutes to petroleum derivatives resulted in the invention of an alternative fuel known as biodiesel. First introduced in 1911 by Rudolph Diesel, the diesel engine was based on a compression-ignition system that could be fed by vegetable oils (Pinto et al., 2005).

Biodiesel is produced from vegetable oils and animal fat, which are examples of renewable organic sources. It is a highly available, renewable, as well as non-toxic resource, which makes it nearly ideal (Demirbas, 2009b). In addition, the burning of biodiesel produces low pollutant emissions and high biodegradability. Conventional means of producing biodiesels include the use of a reaction between animal fat or

(20)

2

vegetable oil with a monohydric alcohol such as methanol or ethanol in the presence of a chemical catalyst to produce mono-alkyl esters and glycerine. This process is known as transesterification (Abreu, Lima, Hamú, Wolf, & Suarez, 2004; Koh & Ghazi, 2011;

Noureddini, Gao, & Philkana, 2005).

Currently, the most common method to produce biodiesel form plant sources includes the use of edible oils such as canola, coconut, peanut of sunflower oils.

However, the concern of using oils that are important food sources means that non- edible plant materials, such as those derived from Moringa oleifera seeds and palm trees, are more suitable for the industrial, large scale application of production of biodiesels.

Additionally, the conventional method to produce biodiesel involves the use of harmful chemical ingredients and catalysts that often generates waste that causes wide- spread and undesirable environmental pollution. The use of biocatalysts have been explored, but not to any great extent as costs for these catalysts are often very high (Das, Thakur, & Deka, 2014; Kafuku & Mbarawa, 2010; Saifuddin, Raziah, & Farah, 2009).

However, use of cheaper biocatalysts such as lipase may reduce cost of production, as well as eliminate most of the harmful by-products that are the result of use of chemical catalysts (Saifuddin et al., 2009; Shimada et al., 1999), also reducing the cost of downstream processing, making biodiesel more profitable overall (Guldhe, Singh, Mutanda, Permaul, & Bux, 2015).

1.2 PROBLEM STATEMENT

As more nations continue to develop, the demand for a sustainable energy source is becoming increasingly obvious. The urgency for this type of energy comes from the need to ease reliance on non-renewable fossil fuels and to introduce an environmentally

(21)

3

friendly option to combat climate change (Wirawan & Tambunan, 2006). Biodiesel from crops is currently one of the top financially viable solutions for this problem because its production has a low impact on environmental waste. However, current production methods can still be significantly improved by using non-edible plant sources with high free fatty acid content (FFA), and use of biocatalysts such as lipase (Rashid, Anwar, Ashraf, Saleem, & Yusup, 2011; Saifuddin et al., 2009; Shimada et al., 1999).

Moringa oleifera is a well-known and an extremely efficient plant as nearly all

of the plant is edible, except for the seeds, which contain 40% of oil by weight (Kibazohi

& Sangwan, 2011). Current applications of Moringa is its use in fighting malnutrition in children as it contains 7 times more Vitamin C than oranges, 10 times more Vitamin A than carrots, 17 times more calcium than milk, 15 times more potassium than bananas and 25 times more iron than spinach. Additionally, Moringa is used in fighting both Type 1 and Type 2 diabetes as well has various forms of cancer. While the medicinal benefits of the plant have been heavily researched, the prospect for its use in biodiesel production has hardly been explored. Other applications include use as an anti- coagulant and as a cooking oil substitute for olive oil (Gopalakrishnan, Doriya, &

Kumar, 2016). Therefore, it is the aim of this research to explore the potential of Moringa oleifera seeds as a source of biodiesel by characterising it to determine its FFA

content, and then to compare the biodiesel produced to current industrial requirements for biofuels.

1.3 RESEARCH OBJECTIVES

The general objective of this research is to produce biodiesel from Moringa oleifera oil using lipase sourced from Candida antarctica immobilised on functionalised activated

(22)

4

carbon (FAC) via transesterification and the subsequent characterisation of the alkyl ester produced.

The specific objectives devised to achieve the main objective are:

 To optimise the immobilisation of Candida antarctica lipase on acid functionalised activated carbon.

 To optimise the production of biodiesel from Moringa oleifera oil using immobilised lipase as a catalyst via transesterification.

 To determine the transesterification reaction kinetics as well as to characterise the Moringa oleifera biodiesel product.

1.4 RESEARCH METHODOLOGY OUTLINE

This study was conducted in shake flasks, in line with the scope which was limited to lab study. The research began by characterising the feedstock oil, Moringa oleifera, and the values found were used to determine the molarity of the oil.

Immobilisation was carried out to enhance the properties of the catalyst in terms of temperature stability so as to maximise reusability and reduce downstream processing costs. The immobilising matrix used in this study was activated carbon powder. The immobilisation capacity of the powder was sought to be increased so as to adsorb more lipase to its surface by way of refluxing it in three acids (sulphuric acid, nitric acid and hydrochloric acid) and was referred throughout the study as functionalised activated carbon (FAC). In order to further maximise the amount of lipase that was immobilised onto the immobilising matrix, in this case functionalised activated carbon powder, optimisation of pH, time and temperature was carried out using Face Centred Central Composite Design (FCCCD) of Design Expert Software DoE 6.0.8.

(23)

5

Optimisation of the production of biodiesel, also known as fatty acid methyl esters (FAME), was conducted using FCCCD and the parameters selected were time, catalyst loading, temperature, methanol to oil ratio and agitation.

The biodiesel produced was then purified and characterised in accordance to the official method for fuel tests. The results obtained were compared with the international standards for biodiesel fuel and the kinetics of the transesterification reaction was determined.

1.5 RESEARCH SCOPE

The development of potential feedstock options for the commercial scale of biodiesel which are cheap and available is important to ensure prolonged conservation of the environment. This research contributed not only to the expansion of non-edible oil feedstock options for biodiesel production, but it also seeks to improve the efficiency by which biodiesel is produced from Moringa oleifera oil by observing the optimum conditions at which maximum yield of biodiesel can be produced. The influence of temperature, time, agitation, catalyst loading, and methanol to oil ratio using Design Expert software was quantified and used for the optimisation of the biodiesel production process. Each experiment was conducted in triplicate in order to ensure an accurate and consistent response.

Additionally, the improvement of the processes relating to the immobilisation of lipase catalyst on functionalised activated carbon (FAC) helps in reducing downstream processing and increasing reusability of the catalyst. Parameters selected were time, temperature, agitation and pH. Reducing reliance on chemical methods of the production of biodiesel means that the waste products from the process will no longer have long term negative effects on the environment.

(24)

6

Finally, the results derived from both the optimisation procedures were validated and the biodiesel composition was analysed to determine the quality of the product and its suitability for use in diesel engines.

1.6 THESIS ORGANISATION

CHAPTER ONE introduces the background of the research work as well as the objectives devised in order to achieve the research methodology. Additionally, the scope of research as well as the reasons for carrying out the work was established.

CHAPTER TWO discusses the relevant findings relating to the topic introduced in this research work, namely various resources relating to manufacturing of biodiesel using varying feedstock oils including Moringa oleifera as well as findings informing on the variables which affect the product yield. Moreover, it identifies the gap in research that this work aimed to fill by adopting novel environmentally-friendly approaches. In CHAPTER THREE, the methods employed in the study to achieve the specific objectives were discussed in detail including the materials used and the procedure for analysis of the results. CHAPTER FOUR presents the analysis of the results obtained from the experimentation, including the statistical approach used to achieve the objectives outlines. Finally, CHAPTER FIVE concludes the study by presenting the results and analysis thereof in a concise manner, after which recommendations for further study into this research were made.

A thesis submitted in fulfilment of the requirement for the degree of Master of Science (Biotechnology Engineering)

ENZYMATIC SYNTHESIS OF BIODIESEL FROM MORINGA OLEIFERA OIL VIA

Soxhlet ةدﺎم لﺎﻤعﺘس و

DECLARATION

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

CHAPTER ONE: INTRODUCTION ... 1

CHAPTER THREE: METHODOLOGY ... 46

CHAPTER FOUR: RESULTS AND DISCUSSION ... 65

CHAPTER FIVE: CONCLUSION AND RECOMMENDATION ... 122

LIST OF TABLES

LIST OF FIGURES

LIST OF ABBREVIATIONS

LIST OF SYMBOLS

CHAPTER ONE INTRODUCTION

1.1 BACKGROUND

1.2 PROBLEM STATEMENT

1.3 RESEARCH OBJECTIVES

1.4 RESEARCH METHODOLOGY OUTLINE

1.5 RESEARCH SCOPE

1.6 THESIS ORGANISATION