Embed Size (px)
Citation preview
OPTIMIZATION OF BIOETHANOL
PRODUCTION FROM OIL PALM TRUNK SAP
NORHAZIMAH BINTI ABDUL HALIM
DOCTOR OF PHILOSOPHY
(BIO-PROCESS ENGINEERING)
UNIVERSITI MALAYSIA PAHANG
UNIVERSITI MALAYSIA PAHANG
DECLARATION OF THESIS AND COPYRIGHT Author’s full name : NORHAZIMAH BINTI ABDUL HALIM
Date of birth : 1st
DECEMBER 1986
Title : OPTIMIZATION OF BIOETHANOL
PRODUCTION FROM OIL PALM TRUNK SAP
Academic Session : Semester II 2015/2016
I declare that this thesis is classified as:
CONFIDENTIAL (Contains confidential information under the
Official Secret Act 1972)
RESTRICTED (Contains restricted information as specified by the
organization where research was done)
OPEN ACCESS I agree that my thesis to be published as online open
access (Full text)
I acknowledge that Universiti Malaysia Pahang reserve the right as follows:
1. The Thesis is the Property of Universiti Malaysia Pahang.
2. The Library of Universiti Malaysia Pahang has the right to make copies for
the purpose of research only.
3. The Library has the right to make copies of the thesis for academic exchange.
Certified By:
(Student’s Signature) (Signature of Supervisor)
DR. CHE KU MOHAMMAD
861201-11-5168 FAIZAL BIN CHE KU YAHYA
New IC / Passport Number Name of Supervisor
Date : 17 June 2016 Date : 17 June 2016
SUPERVISOR’S DECLARATION
We hereby declare that we have checked this thesis and in our opinion this thesis is
adequate in terms of scope and quality for the award of the degree of Doctor of
Philosophy in Bio-process Engineering.
(Supervisor’s Signature)
Name of Supervisor : DR. CHE KU MOHAMMAD FAIZAL BIN CHE KU YAHYA
Position : ASSOCIATE PROFESSOR FACULTY OF ENGINEERING
TECHNOLOGY
Date : 17 JUNE 2016
(Co-Supervisor’s Signature)
Name of Co-Supervisor : DR. MIOR AHMAD KHUSHAIRI BIN MOHD ZAHARI
Position : SENIOR LECTURER FACULTY OF CHEMICAL AND
NATURAL RESOURCES ENGINEERING
Date : 17 JUNE 2016
STUDENT’S DECLARATION
I hereby declare that the work in this thesis is my own except for quotations and
summaries which have been duly acknowledged. The thesis has not been accepted for
any degree and is not concurrently submitted for award of other degree.
(Author’s Signature)
Name : NORHAZIMAH BINTI ABDUL HALIM
ID Number : PKB12002
Date : 17 JUNE 2016
vi
TABLE OF CONTENTS
Page
DECLARATION
TITLE PAGE i
DEDICATIONS ii
ACKNOWLEDGEMENTS iii
ABSTRACT iv
ABSTRAK v
TABLE OF CONTENTS vi
LIST OF TABLES ix
LIST OF FIGURES xi
LIST OF SYMBOLS xiv
LIST OF ABBREVIATIONS xv
CHAPTER 1 INTRODUCTION
1.1 Background of Study 1
1.2 Problem Statement 2
1.3 Objectives and Scope 5
CHAPTER 2 LITERATURE REVIEW
2.1 Bioethanol Production 7
2.2 Biomass for Bioethanol Production 14
2.3 Microorganism for Bioethanol Production 17
2.4 Oil Palm Trunk 19
2.5 Kinetic Modelling 22
2.6 Statistical Method Design of Experiment 27
2.6.1 Plackett-Burman Design 30
2.6.2 Steepest-ascent Method 30
2.6.3 Box-Behnken Design 30
2.6.4 Analysis of Statistical Data 31
2.6.5 Validation of Model 31
2.7 Conclusion 32
vii
CHAPTER 3 MATERIALS AND METHODS
3.1 Introduction 34
3.2 Preparation and Characterization of Oil Palm Trunk Sap 35
3.3 Inoculum Preparation 38
3.4 Selection of Microorganism 39
3.5 Kinetic Modelling and Parameter Determination 42
3.6 Effects of Sterilization to Bioethanol Production 42
3.7 Identification of Important Parameter Using Plackett-Burman Design 43
3.8 Identification of Optimum Region by Path of Steepest-ascent Method 44
3.9 Identification of Optimum Condition by Box-Behnken Design 45
3.10 Validation and Fermentation in 20 liter Bioreactor 47
3.11 Repeated-Batch in 2 liter Bioreactor 48
3.12 Analysis Method 48
CHAPTER 4 RESULTS AND DISCUSSION
4.1 Characterization of OPT Sap and Fermentation Broth 51
4.2 Effects of Different Microorganism on Bioethanol Production from OPT
Sap at Different Temperature of Fermentation 52
4.2.1 Influence on Ethanol Yield 52
4.2.2 Influence on Cell Biomass 63
4.3 Comparison of Kinetic Parameter for Selected Microorganisms 70
4.3.1 Sugar Consumption Rate, Ethanol Consumption Rate and Biomass
Consumption Rate 76
4.3.2 Effects of Substrate Concentration on Bioethanol Fermentation 78
4.4 Effects of Sterilization to Bioethanol Production 81
4.5 Identification of Important Parameter Using Plackett-Burman Design 82
4.6 Identification of Optimum Region by Path of Steepest-ascent Method 85
4.7 Identification of Optimum Condition by Box-Benhken Design 87
4.8 Validation and Fermentation in 20 liter Bioreactor 97
4.9 Repeated-Batch in 2 liter Bioreactor 99
4.11 Kinetic Analysis in 20 liter Bioreactor 101
viii
CHAPTER 5 CONCLUSION AND RECOMMENDATION
5.1 Conclusion 105
5.2 Improvement and Recommendation 106
LIST OF PUBLICATION 107
REFERENCES 109
APPENDICES 116
A Oil Palm Trunk Sap 116
B Calibration Curve 117
C Experimental data 121
D Calculation 191
ix
LIST OF TABLES
Table No. Title Page
2.1 Characteristics and properties of ethanol
7
2.2 Identification of influential parameter for bioethanol production
9
2.3 Research on bioethanol from forest, food residue and agricultural waste
15
2.4 Selection of microorganism for bioethanol production
18
2.5 Selected fermentatable sugar in OPT sap
20
2.6 Amino acid and organics acid in OPT sap
21
2.7 Comparison of primary RSM experimental design
28
3.1 Type of microorganisms for fermentation for bioethanol production
39
3.2 Percentage of microorganisms in fermentation medium
40
3.3 Parameter and range selected for bioethanol fermentation from OPT
sap
44
3.4 Experimental design layout for PBD
44
3.5 Path of steepest-ascent design for nutrient addition using one-factor-at-
a-time method
45
3.6 Level of variables for BBD
45
3.7 The experimental design layout for BBD design
46
3.8 Summary of analysis parameter in UV-Vis spectrophotometer
49
3.9 Summary of analysis parameter in HPLC
49
3.10 Summary of analysis parameter in gas chromatography
50
4.1 Selected fermentation sugar composition in OPT sap at different part
of trunk
51
4.2 Ethanol yield by using Zymomonas mobilis at 96 h 53
4.3 Ethanol yield by using Zymobacter palmae at 96 h
55
4.4 Ethanol yield by using Pichia stipitis at 96 h 56
x
4.5 Ethanol yield by using S. cerevisiae (local) at 96 h
57
4.6 Ethanol yield by using S. cerevisiae JCM 2220 at 96 h
58
4.7 Ethanol yield by using S. cerevisiae Kyokai no. 7 at 96 h
59
4.8 Ethanol yield by using S. cerevisiae Kyokai no. 7 and Pichia stipitis at
96 h
61
4.9 Ethanol yield by using S. cerevisiae (local) and Pichia stipitis at 96 h 61
4.10
Parameters of different microorganisms performance in OPT sap at
30.0°C
77
4.11 Comparison between ethanol production with different substrate
concentrations at 30°C
78
4.12 Summarized data for bioethanol production by S. cerevisiae Kyokai
no.7 in a different medium of OPT sap
80
4.13 Result obtained for Plackett-Burman experimental design
82
4.14 Percentage contribution based on Plackett-Burman experimental
design
82
4.15 Result for steepest-ascent 86
4.16 Experimental design and result for BBD 87
4.17 Lack of fit test 87
4.18 ANOVA for Response Surface Quadratic Model
88
4.19 Regression of coefficient and parameter
94
4.20 Diagnostics Case Statistics
95
4.21 Constrains set for optimization using numerical method
96
4.22 Solution suggested by the Design Expert software
96
4.23 Performance of S. cerevisiae Kyokai no.7 in optimized condition
during fermentation in 13 cycles
99
4.24 General assumption for bioethanol production from the fermentation
of OPT sap
102
xi
LIST OF FIGURES
Figures No. Title Page
2.1 Pilot-scale squeezing equipment for OPT sap
20
3.1 Summary for overall research framework
35
3.2 Preparation of OPT sap
36
3.3 Oil palm trunk from upper to lower part (A to F)
38
3.4 Procedure of fermentation 40
3.5 Design of experiment for selection of microbes
41
3.6 Experiment plan for kinetic modelling
42
3.8 Experiment plan for optimization
46
3.9 20 liter bioreactor (BiostatTM)
47
4.1 Bioethanol production from OPT sap by Zymomonas mobilis at
25.0°C, 27.5°C, 30.0°C static, 30.0°C shaking, 32.5°C and 35.0°C
53
4.2 Bioethanol production from OPT sap by Zymobacter palmae at
25.0°C, 27.5°C, 30.0°C static, 30.0°C shaking, 32.5°C and 35.0°C
54
4.3 Bioethanol production from OPT sap by Pichia stipitis at 25.0°C,
27.5°C, 30.0°C static, 30.0°C shaking, 32.5°C and 35.0°C
55
4.4 Bioethanol production from OPT sap by S. cerevisiae (local) a at
25.0°C, 27.5°C, 30.0°C static, 30.0°C shaking, 32.5°C and 35.0°C
56
4.5 Bioethanol production from OPT sap by S. cerevisiae JCM2220 at
25.0°C, 27.5°C, 30.0°C static, 30.0°C shaking, 32.5°C and 35.0°
57
4.6 Bioethanol production from OPT sap by S. cerevisiae Kyokai no.7 at
25.0°C, 27.5°C, 30.0°C static, 30.0°C shaking, 32.5°C and 35.0°C
58
4.7 Bioethanol production from OPT sap by mixed culture of S.
cerevisiae Kyokai no.7 and Pichia stipitis at at 25.0°C, 27.5°C,
30.0°C static, 30.0°C shaking, 32.5°C and 35.0°C
59
4.8 Bioethanol production from OPT sap by mixed culture of
S.cerevisiae (local) and Pichia stipitis at 25.0°C, 27.5°C, 30.0°C
static, 30.0°C shaking, 32.5°C and 35.0°C.
60
xii
4.9 Summary of bioethanol yield by different microorganism and mixed
culture at 30.0 °C
62
4.10 Biomass production by Zymomonas mobilis at at 25.0°C, 27.5°C,
30.0°C static, 30.0°C shaking, 32.5°C and 35.0°C
63
4.11 Biomass production by Zymobacter palmae at at 25.0°C, 27.5°C,
30.0°C static, 30.0°C shaking, 32.5°C and 35.0°C
64
4.12 Biomass production by Pichia stipitis at at 25.0°C, 27.5°C, 30.0°C
static, 30.0°C shaking, 32.5°C and 35.0°C
65
4.13 Biomass production by Saccharomyces cerevisiae (local) at 25.0°C,
27.5°C, 30.0°C static, 30.0°C shaking, 32.5°C and 35.0°C
66
4.14 Biomass production by Saccharomyces cerevisiae JCM2220 at
25.0°C, 27.5°C, 30.0°C static, 30.0°C shaking, 32.5°C and 35.0°C
66
4.15 Biomass production by Saccharomyces cerevisiae Kyokai no.7 at
25.0°C, 27.5°C, 30.0°C static, 30.0°C shaking, 32.5°C and 35.0°C
67
4.16 Biomass production by mixed culture of Saccharomyces cerevisiae
Kyokai no.7 and Pichia stipitis at 25.0°C, 27.5°C, 30.0°C static,
30.0°C shaking, 32.5°C and 35.0°C.
68
4.17 Biomass production by mixed culture of Saccharomyces cerevisiae
(local) and Pichia stipitis at 25.0°C, 27.5°C, 30.0°C static, 30.0°C
shaking, 32.5°C and 35.0°C
68
4.18 Summary of biomass production by different microorganism at 30 °C
with agitation
69
4.19 The fermentation curve of microbes in 72 h of fermentation
71
4.20 The natural log of the biomass growth versus time
73
4.21 Comparison between actual and predicted data of bioethanol obtained
using the Hinshelwood model
74
4.22 Sugar consumption rate for S. cerevisiae Kyokai no.7, mixed culture
and S. cerevisiae JCM2220 at 0 to 12 h of fermentation
76
4.23 Bioethanol productivity rate for S. cerevisiae Kyokai no.7, mixed
culture and S. cerevisiae JCM2220 at 0 to 12 h of fermentation
76
4.24 Glucose consumption for different substrate concentration
79
4.25 Ethanol production for different substrate concentration
79
xiii
4.26 Pareto chart
84
4.27 Result for steepest-ascent for CSL
85
4.28 Result for steepest-ascent for peptone
85
4.29 AB Interaction on ethanol yield by contour plot graph
89
4.30 AB Interaction on ethanol yield by response surface graph
89
4.31 AC Interaction on ethanol yield by contour plot graph
90
4.32 AC Interaction on ethanol yield by response surface graph
90
4.33 BC Interaction on ethanol yield by response surface graph
91
4.34 BC Interaction on ethanol yield by response surface graph
92
4.35 Perturbation graph at initial pH 6.20, CSL 7.0 and peptone 10 g/l.
93
4.36 Cube graph representing the effects of three factors at a time.
94
4.37 Desirability graph
97
4.38 Performance of S. cerevisiae Kyokai no.7 during fermentation in 13
cycles (bioethanol)
98
4.39 Sugar consumption of S. cerevisiae Kyokai no.7 during
fermentation in 13 cycles (sugar consumption)
99
4.40 Fermentation of bioethanol in 20 liter fermenter using enhanced
medium
101
4.41 Relationship between bioethanol production and cell biomass
growth
101
4.42 Process flow diagram for operation of bioethanol plant using OPT
sap as raw material
104
xiv
LIST OF SYMBOLS
°C degree celcius
g.g-1 gram per gram
g/l gram per liter
g/l.h gram per liter per hour
ha hectare
h hour
l liter
min minute
nm nanometer
rpm revolution per minute
v/v volume per volume
w/v weight per volume
xv
LIST OF ABBREVIATIONS
ANOVA Analysis of variance
ATCC American Type Culture Collection
BBD Box-Behnken Design
CDW Cell dry weight
DM Dried matter
EFB Empty fruit bunch
FFD Full factorial design
GC-FID Gas chromatography flame ionized detector
HPLC High performance liquid chromatography
JCM Japan Collection of Microorganism
KOS Saccharomyces cerevisiae Kyokai no.7 and Pichia stipitis
MAS Saccharomyces cerevisiae (local) and Pichia stipitis
OD Optical density
OFAT One-factor-at-a-time
OPT Oil palm trunk
PBD Plackett-Burman Design
RSM Response surface methodology
TRS Total reducing sugar
