EXPERIMENTAL INVESTIGATION OF BIOFUEL

PRODUCTION FROM RICE STRAW BY LIQUID-STATE

FERMENTATION

KOK POH SING

COLLEGE OF ENGINEERING
UNIVERSITI TENAGA NASIONAL
2014
EXPERIMENTAL INVESTIGATION OF BIOFUEL PRODUCTION FROM RICE
STRAW BY LIQUID-STATE FERMENTATION

By

KOK POH SING

Project Supervisor:
PROF.DR.T.M. INDRA MAHLIA

THESIS SUBMITTED IN PARTIAL FULFILMENT OF

THE REQUIREMENTS FOR THE DEGREE OF
BACHELOR OF MECHANICAL ENGINEERING

COLLEGE OF ENGINEERING
UNIVERSITI TENAGA NASIONAL

2014
 ii

DECLARATION

I hereby declare that this thesis, submitted to Universiti Tenaga Nasional as partial
fulfilment of the requirements for the degree of Bachelor of Mechanical Engineering,
has not been submitted to any other university for any degree. I also certify that the
work described herein is entirely my own, except for quotations and summaries
sources of which have been duly acknowledged.

This thesis may be made available within the university library and may be
photocopied or loaned to other libraries for the purposes of consultation.

10 February 2014 Kok Poh Sing

ME088618
 iii

ACKNOWLEDGEMENT

First of all, I would like to give my deepest gratitude to my project supervisor Prof.
Dr. T. M. Indra Mahlia because without guidance and knowledge from him, this
project would have never been carried out smoothly. Furthermore, Prof. Dr. T. M.
Indra Mahlia is also the person behind for financial support for this project to be
carried by outsourcing equipment usage from FTU.GMP @ BIOTECH and, also
Fakulti Bioteknologi Dan Sains Biomolekul UPM.

I would like to say thank you to the all the officers from FTU.GMP @
BIOTECH and Fakulti Bioteknologi Dan Sains Biomolekul UPM for their generosity
to allow us to use the equipment available with financial agreement and their aid and
guidance in carrying out the experiment. A special thank you to Mr. Rizal, Mdm
Liyana from FTU.GMP @ BIOTECH and, Mr. Rosli and Mdm Aluyah from Fakulti
Bioteknologi Dan Sains Biomolekul UPM. The officers have been a great help in
guiding us throughout the months of experiment.

Furthermore, I would like to express my gratitude to my friends especially

Kenny Koo Weng Kiet and Lee Lai Hoong that are undertaking the project together
for their help and sharing of knowledge in carrying out the experiment.

I would like to say thank you to the personnel in UNITEN for their years of
teaching and guidance. Lastly, I would like to say thank you to my family that have
been supporting me since the beginning in moral support and financial support.
 iv

ABSTRACT

Energy consumption is increasing day after another. Therefore, cleaner, safe and
sustainable energy sources must be found. Second generation bioethanol has posted a
great potential as an alternative energy source especially in transportation sector. Rice
straw has been studied as a potential conversion of lignocellulosic biomass into
bioethanol. In this study, rice straw has been first treated with mechanical
pretreatment using home blender and followed by acid pretreatment using 2.0 M
H2SO4 at 90℃ for 60 minutes. The glucose yield is found to be 0.324 g/g rice straw
basis or 32.4% g/g rice straw basis. Then, rice straw is hydrolyzed using 24 mg of
Cellulase from Tichoderma Ressei ATCC 26921 for duration of 72 hours. The glucose
yield from enzymatic hydrolysis is found to be 0.382 g/g rice straw basis. The total
glucose yield from the study is 0.706 g/g rice straw basis. The glucose obtained is then
fermented. Glucose is found to be fermented successfully with approximately 97.68%.
The ethanol conversion of fermented solution from acid pretreatment and fermented
solution from enzymatic hydrolysis is found to be 11.26% and 52.75% compared to
the theoretical ethanol yield. Rice straw has a great potential to be converted to
bioethanol as it is able to give high glucose yield but it would need to be further
studied for potential presence inhibition that may affect fermentation process.
 v

CONTENTS

Page
DECLARATION ii
ACKNOWLEDGEMENT iii
ABSTRACT iv
CONTENTS v
LIST OF TABLES ix
LIST OF FIGURES x
LIST OF ABBREVIATIONS xii

CHAPTER I INTRODUCTION
1.1 Introduction 1
1.2 Background 1
1.3 Problem Statements 3
1.4 Objectives 4
1.5 Research Scopes 4

CHAPTER II LITERATURE REVIEW

2.1 Introduction 5
2.2 Rice Straw 5
2.3 Rice Straw Composition 9
2.4 Rice Straw to Bioethanol 10
2.5 Pretreatment of Rice Straw Biomass 11
2.5.1 Physical Pretreatment 13
2.5.2 Acid Pretreatment 13
2.5.3 Alkaline Pretreatment 14
2.5.4 Comparison of Parameters for Physical, Acid and
Alkaline Pretreatment 14
2.6 Hydrolysis 15
2.6.1 Acid Hydrolysis 16
2.6.1.1 Dilute Acid Hydrolysis 16
2.6.1.2 Concentrated Acid Hydrolysis 18
2.6.2 Enzymatic Hydrolysis 18
2.6.3 Comparison of Different Types of Hydrolysis 20
2.7 Fermentation 22
 vi

2.7.1 Inhibitors of Fermentation 22

2.8 Recovery of Ethanol 23
2.9 Reducing Sugars Analysis and Ethanol Analysis Method 23

CHAPTER III METHODOLOGY

3.1 Introduction 24
3.2 General Methodology on Ethanol Production 24
3.3 List of Equipment 25
3.4 Calibration of Glucose Standard Curve of Absorbance 26
3.5 Glucose Content Determination 27
3.6 Fermentation Analysis 28
3.6.1 Preparation of Yeast Media 28
3.6.2 Yeast Inoculum 29
3.6.3 Analysis of fermentation samples 29
3.7 Preparation of Raw Material 30
3.8 Pretreatment of Rice Straw 30
3.8.1 Physical Pretreatment 30
3.8.2 Acid Pretreatment 31
3.8.2.1 Preparation of Sulfuric acid (H2SO4) 32
3.8.2.2 Preparation of Sodium Hydroxide (NaOH) 32
3.8.2.3 Analysis of Reducing Sugar Concentration
from Acid Pretreatment 32
3.9 Enzymatic Hydrolysis of Pretreated Rice Straw 33
3.9.1 Preparation of Hydrochloric acid (HCL) 34
3.9.2 Preparation of Sodium Acetate (C2H3NaO2) 34
3.9.3 Analysis of Reducing Sugars from Enzymatic Hydrolysis 34
3.10 Fermentation of Reducing Sugars 35
3.10.1 Fermentation of Liquid Phase from Acid Pretreatment 35
3.10.2 Fermentation of Liquid Phase from Enzymatic Hydrolysis 35
3.10.3 Analysis of Reducing Sugar Content and Yeast Growth
in Fermentation using DNS Testing Method 36
3.11 Analysis and Distillation of Fermented Solution for
Ethanol and Glucose Content 37
3.11.1 Analysis using High Performance Liquid Chromatography (HPLC) 37
3.11.2 Distillation of Fermented Solution 38
 vii

CHAPTER IV ANALYSIS AND RESULTS

4.1 Introduction 39
4.2 Calibration Curve of Absorbance for Glucose 39
4.3 Relationship of Glucose and Yeast during Fermentation 40
4.4 Glucose Yield from Acid Pretreatment on Rice Straw 43
4.5 Glucose Yield from Enzymatic Hydrolysis on Rice Straw 45
4.6 Total Yield of Glucose from Acid Pretreatment and Enzymatic Hydrolysis 47
4.7 Liquid State Fermentation 48
4.7.1. Initial Analysis of Glucose Concentration and
Yeast Growth using DNS Testing Method 48
4.7.1.1 Liquid State Fermentation on Acid Pretreatment Solution 48
4.7.1.2 Liquid State Fermentation on Enzymatic Hydrolysis Solution 50
4.7.2 Analysis of Glucose and Ethanol Concentration using HPLC 52

CHAPTER V DISCUSSIONS
5.1 Introduction 56
5.2 Calibration Curve of Absorbance 56
5.3 Effect of Acid Pretreatment on Sugar Yield 57
5.4 Effect of Enzymatic Hydrolysis on Sugars Yield 58
5.5 Distillation of Ethanol from Fermented Sample 59
5.6 Effect of Fermentation on Ethanol Yield 59

CHAPTER VI CONCLUSION AND RECOMMENDATION

6.1 Conclusion 61
6.2 Future Recommendation 62

REFERENCES 63

APPENDICES

A Results of Glucose Curve of Absorbance Calibration 66

B Results of Acid Pretreatment and Sample Glucose
Concentration Calculation 67
C Sample Glucose Concentration Calculation For Enzymatic
Hydrolysis Process 69
D HPLC Analysis Results on Calibration on Glucose and Ethanol 70
E HPLC Analysis Results on Fermented Samples of
Acid Pretreatment and Enzymatic Hydrolysis 80
 viii

F Sample Ethanol Concentration Calculation 85

G Results of Acid Pretreatment at 121℃ for 15 Minutes
Retention Time 86
H Properties of Ethanol at Different Pressure 87
I Pretreatment of Rice Straw Sample 88
J Enzymatic Hydrolysis of Acid Pretreated Rice Straw Sample 90
K Fermentation of Solution Obtained From Acid
Pretreatment and Enzymatic Hydrolysis 91
L Distillation and HPLC Analysis 93
 ix

LIST OF TABLES

Table No. Page

2.1 Carbohydrates present in rice straw 9

2.2 Carbohydrate composition and theoretical ethanol
yield of rice straw 10
2.3 Effect comparison of physical, acid and alkaline pretreatment 14
2.4 Comparison of dilute acid hydrolysis and concentrated
acid hydrolysis 20
2.5 Comparison of dilute acid hydrolysis and enzymatic hydrolysis 21
3.1 Equipment used and its functions 25
2
4.1 Experimental R values and linear equations for
calibration curve of absorbance using DNS method 39
4.2 Results for yeast growth during fermentation 41
4.3 Glucose concentration during fermentation process 42
4.4 Results for sugars yield in acid pretreatment 43
4.5 Results of enzymatic hydrolysis with 3g solid substrate
and 24 mg enzyme loading 45
4.6 Results of total glucose yield from acid pretreatment
and enzymatic hydrolysis 47
4.7 Average of Glucose Conversion during Fermentation Process 48
4.8 Results of Sugar Fermentation of Enzymatic Hydrolysis 51
4.9 Results of ethanol yield 52
 x

LIST OF FIGURES

Figure No. Page

2.1 Final energy use in transport sector 7

2.2 Final Energy Demand by Type of Fuels 7
2.3 Final Consumption of Petroleum Products 8
2.4 Retail Fuel Prices in Malaysia 8
2.5 Flowchart showing biochemical conversion processes of
lignocellulosic feedstock to high quality bioethanol 11
2.6 Schematic pretreatment of lignocellulosic material 12
2.7 Conversion processes of cellulose and hemicellulose to ethanol 15
2.8 Glucose yield using 0.5% dilute H2SO4 with temperature
range from 188 - 234℃ 17
2.9 Schematic flow of cellulose conversion to glucose by enzymes 19
3.1 General Bioethanol flowchart 24
3.2 Preparation of yeast media 28
3.3 High speed home blender used for physical pretreatment
of rice straw 30
3.4 Rice straw samples added with acid solution 31
3.5 HPLC equipment 37
3.6 Distillation Using Rotatory Evaporator Equipment 38
4.1 Selected experimental standard curve of absorbance for glucose 40
4.2 Relationship of yeast growth and duration of fermentation 41
4.3 Relationship between glucose concentration and
duration of fermentation 42
4.4 Sugar yield (g/L) with different retention time and acid
concentration 44
4.5 Sugar Yield in Percentage of g/g Rice Straw Basis 44
4.6 Absorbance values with respect to duration of
hydrolysis and type of pretreatment applied 46
4.7 Sugar yield (g/g rice straw basis) from enzymatic
hydrolysis with respect to time 46
4.8 Total glucose yield of acid pretreatment and
enzymatic hydrolysis 47
 xi

4.9 Glucose Content during Fermentation Process of

Sugar Yield from Acid Pretreatment 49
4.10 Growth of Saccharomyces Cerevisae during Fermentation
of Sugar Yield from Acid Pretreatment 49
4.11 Glucose Content during Fermentation Process of
Sugar Yield from Enzymatic Hydrolysis 50
4.12 Growth of Saccharomyces Cerevisae during Fermentation
of Sugar Yield from Enzymatic Hydrolysis 51
4.13 Calibrated Curve of Glucose for HPLC 53
4.14 Calibrated Curve of Ethanol Concentration for HPLC 53
4.15 Results of analysis of fermented solution from
acid pretreatment after distillation 54
4.16 Results of analysis of fermented solution from
enzymatic hydrolysis after distillation 55
5.1 Effect of temperature on enzyme reaction rate 59
5.2 Performing Works Involved in Fermentation using
Laminar Air Flow System to Minimize Bacterial Contamination 60
 xii

LIST OF ABBREVIATIONS

ha Hectares

cm Centimeter

nm Nanometer

ml Milliliter

g Gram

kg Kilogram

t Tonne

ktoe Kilotonne of oil equivalent

L Liter

M Molar

kWh Kilowatt hour

℃ Celsius

% Percentage

rpm Rotation per minute

μL Microliter

kPa Kilopascal

hr Hour

A Absorbance

CaCl2 Calcium chloride

HCL Hydrochloric acid

H2SO4 Sulfuric acid

NaOH Sodium hydroxide

C2H3NaO2 Sodium acetate

DNS Dinitrosalicylic acid

CO2 Carbon dioxide

 xiii

CaOH2 Calcium hydroxide

KOH Potassium hydroxide

KH2PO4 Monopotassium phosphate

NH4Cl Ammonia chloride

$ Dollar

MYR Malaysia Ringgit

E85 85% ethanol fuel blend

LPG Liquefied petroleum Gas

ATF Aviation turbine fuel

AV Gas Aviation Gasoline

β Beta

RO Reverse osmosis

% w/w Percentage weight per weight

% g/g Percentage gram per gram

g/g Gram per gram

LAB Lactic acid bacteria

HPLC High Performance Liquid Chromatography

HMF Hydroxymethylfurfural
 CHAPTER I

INTRODUCTION

1.1 Introduction

This chapter reviews the discussion on current overview of energy demand in

Malaysia. Furthermore, problems in meeting current and future energy demand would
be discussed as well. An alternative solution in reducing usage of fossil fuels is
proposed in this chapter.

1.2 Background

Energy is the fundamental to sustain life forms. Current time, people’s lifestyle has
been improving from time to time; making the demand of energy to rise dramatically.
World energy consumption is rapidly increasing due to strong growth in industrial,
population and economic. Global energy demand is estimated to double by year 2050
with existing technologies and consumption pattern (Roy et al., 2012). Considering
negligible changes in laws and energy consumption governing policy, the global
energy consumption is predicted to increase by 56% from 524 to 820 quadrillion Btu
between year 2010 and 2040. As effect, hasting the usage of fossil fuels. The emission
of greenhouse gases is predicted to increase from 31 billion metric tons to 45 billion
metric tons between the same years (U. S. Energy Information Administration, 2013).
Due to factors such as fast depletion of world primary energy supply which is fossil
fuels and high emission of carbon dioxide (U. S. Energy Information Administration,
2013); sustainable, cleaner and safe alternative energy sources must be found. One of
the biomass production, biofuel or more specifically, bioethanol has posted a great
potential as an alternative energy source especially in transportation sector by
replacing or reducing petroleum dependent (Dwivedi et al., 2009). Biofuel is
considered as carbon dioxide neutral because it does not contribute to greenhouse
effect by contrasting it to traditional fuels such as petrol (Nutawan et al., 2010).
 2

Bioethanol has posted a great potential as an alternative fuel in various energy

sectors especially in transportation sector. Ethanol has a great potential market as huge
as oil market and it has the potential to replace the fuel market for gasoline entirely
(Taherzadeh and Karimi, 2007a). In year 2007, United States of America (USA) arise
as the world’s largest bioethanol producer with production capacity of fuel alcohol of
51.5 billion litres from 180 bio-refineries plantation (Walker, 2010). Bioethanol has
been widely used as blending fuel in gasoline known as E85 in countries such as
Brazil and USA (Window on State Government). According to the BP statistic review
of world energy, Malaysia has started to produce biofuels (bioethanol and biodiesel)
in year 2006 with production amount of 48 thousands toe and it has risen to 97
thousands toe in year 2011 (BP, 2012). Malaysia has a planning to become one of the
significant exporters of biofuels in the world (Cushion et al., 2010). In year 2012, the
company; Sime Darby Berhad announced its plans to invest MYR 2 billion for the
setting up of 616 bio-refinery module lines in the next five years in Malaysia and
Indonesia (Choong, 2012). Malaysia biofuel research community has been on
developing the processes that turn different sources of cellulosic biomass into
bioethanol as an alternative transportation fuel, replacing gasoline and natural gas.
One of the potential lignocellulosic biomass feedstock for bioethanol production that
is abundant and cheap is rice straw waste.

Lignocellulosic biomass feedstock are categories as renewable raw material,

largely unutilised, low cost, huge variety of resources availability such as crop residue
like rice straw, and abundantly available sources of raw materials for the production of
biofuel. By liberate these raw materials by hydrolysis process, sugars polymers in
form of cellulose and hemicellulose can be obtained and subsequently fermented to
ethanol by microorganisms (Taherzadeh and Karimi, 2007a).

Upon harvesting rice grain from rice plant, rice straw is left over as waste
materials on paddy field upon paddy harvesting. It is the stalk of the rice plant (Lim et
al., 2012). Rice straw is one of the cellulosic type biomass feedstock and it has great
potential to be commercialize as the feedstock for bioethanol production due to its
abundant and availability throughout the year. Therefore, this paper will study the
yield of ethanol from rice straw as feedstock.
 3

1.3 Problem Statement

Production of ethanol from biomass has become an increasingly well-known

alternative to petrol as one option to cut down dependency on oil and reducing the
contribution to global warming (Diep et al., 2012). Ethanol can easily blend with
gasoline. Until current years, majority of the production of first generation bioethanol
is produced from food-based crops such as sugarcane and maize. Therefore, it invokes
the debates on long term supply and sustainability of these food-based crops due to
competition with world’s food and animal feed supply (Wirawan et al., 2012). To
provide an alternative, second generation of bioethanol production has been
introduced by using lignocellulose waste materials as feedstock because it does not
compete with food crops (Roy et al., 2012).

Since centuries till current days, farmers in most of the Asia countries are still
practicing open field burning of rice straw after harvesting season (Lim et al., 2012).
Open burning of rice straw contributes not only to greenhouse effect, burning rice
straw in large scale will result in serious health hazards there are increasing amount of
respiratory and eye illness among the local residents (Norhalim, 2010). Therefore,
many countries have enforced strict regulations to bound open field burning activities
due to health and environmental concerns. One of the best alternatives for managing
the rice straw waste from rice grain harvesting is to convert the biomass into
commercializes products such as enzymes, ethanol, and nutritive feedstock (Oberoi et
al., 2010). Nevertheless, rice straw disposal methods have switched towards the global
“waste to resource” (Lim et al., 2012).
 4

1.4 Objectives

1. To study the yield of glucose by applying different parameters such as

temperature, retention time and concentration of acid used for pretreatment
process.
2. To evaluate yield of glucose from enzymatic hydrolysis with different
retention time on acid pretreated rice straw.
3. To study the yield of ethanol from rice straw fermentation using
saccharomyces cerevisiae yeast and evaluate the ethanol produced through
liquid state fermentation.

1.5 Research Scopes

The research will cover the following scopes in achieving the objectives of the project
by following the limitation of equipment and chemical availability. Acid will be used
in pretreatment process to break the composition of rice straw. For enzymatic
hydrolysis, cellulase from Tichoderma Ressei will be used to hydrolyse hemicellulose
and cellulose into reducing sugars. Saccharomyces cerevisiae yeast (Baker’s yeast)
will be used to ferment reducing sugars to ethanol. HPLC will be used to evaluate
ethanol concentration in the fermented samples.
 CHAPTER II

LITERATURE REVIEW

2.1 Introduction

This chapter discusses the background of rice straw availability in Malaysia, the
composition of rice straw and also the potential to be converted to reducing sugars by
undergoing processes such as pretreatment and hydrolysis. A basic flow of bioethanol
conversion from feedstock is shown. Moreover, common type of pretreatments such
as physical pretreatment, acid pretreatment and alkali pretreatment and type of
hydrolysis available such as acid hydrolysis and enzymatic hydrolysis will be
discussed. Factors affecting the yield of reducing sugars produced and content testing
methods are to be reviewed as well.

2.2 Rice Straw

Lignocellulosic feedstock can be easily obtained as agricultural waste and it is one of

the most abundant and low cost biomass on globe, and hence becoming one of the
most promising feedstock for mass and rapid production of bioethanol (Wirawan et
al., 2012). Rice straw is one of the lignocellulosic feedstock available for bioethanol
production. Rice is ranked at third as major agricultural farm in the earth (Chandra et
al., 2012). According to Jeng Shiun Lim et al. (Lim et al., 2012), “Rice demand is
expected to remain strong in the next few decades due to the economic and population
growths in many countries across Africa and Asia”. Therefore, rice straw as one of the
waste of harvested rice grain would be a promising feedstock for mass bioethanol
production in Asia. For every kilogram of harvested paddy, there will be rice straw
waste range between 0.396 to 0.41 kg. Unfortunately, the farmers in many Asia
countries are still practicing open field burning of rice straw after harvesting season.
Nevertheless, rice straw disposal methods have switched towards the global “waste to
resource” (Lim et al., 2012).
 6

In Malaysia, it is estimated that the amount of rice straw produced yearly is

approximately two million tonnes through harvesting paddy from 350,000 ha of paddy
field. The country; Malaysia is attempting to increase the yield of rice to 10 t/ha (M.
Sashikala and Ong, 2009). In year 2010, the total planted area of paddy at granary
areas was 387,160 ha. Therefore, the attempt of increasing rice yield would give a
good promising of huge and continuous supply of rice straw for bioethanol production
in Malaysia.

Energy production in Malaysia is mainly powered by fossil fuels primarily

coal, fuel oil and natural gas. The energy produced is used to power sectors such as
agriculture, non-energy use, residential and commercial, transport and industrial. The
final energy use by transport sector has been showing an increase from 12,071 ktoe to
16,119 ktoe in year 2001 to 2011. The graph is shown as in FIGURE 2.1. Petroleum
and other products that have been used as energy have shown an increase in trend for
the past years to year 2011 as shown in FIGURE 2.2. The demand of petroleum
products and others remains the highest compared to others. The high demand of
petroleum products has led to higher harvesting rate of crude oil to meet the demand.
As consequences, the market selling price of petroleum products has shown an
increase due to economy and political issues. FIGURE 2.3 shows final consumption of
petroleum products in Malaysia while FIGURE 2.4 shows the selling prices of several
petroleum products used in transport sector in Malaysia from year 2001 to year 2011.
The latest selling price for RON 97, RON 95 and diesel are MYR 2.85, MYR 2.10 and
MYR 2.00. The fuel price is expected to rise. Transport sector remains as the highest
consumption of petroleum products.
 7

18000

16000

14000

12000

10000
ktoe

8000

6000

4000

2000

0
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011
Year

FIGURE 2.1 Final energy use in transport sector (KeTTHA, 2011)

50000

45000

40000

35000

30000 Coal and Coke

Natural Gas
25000 Electricity
ktoe
20000 Petroleum
Products and
15000 Others

10000

5000

0
2001 2002 2003 2004 2005 2006 2007 2008 2009 2010 2011
Year

FIGURE 2.2 Final Energy Demand by Type of Fuels (KeTTHA, 2011)

 8

Kerosene ATF & AV Gas

LPG 0% 9%
6% Non-Energy
Fuel Oil 3%
8%

Kerosene
ATF & AV Gas
Non-Energy
Motor Petrol
Diesel
Motor Petrol
36% Fuel Oil
Diesel LPG
38%

FIGURE 2.3 Final Consumption of Petroleum Products (KeTTHA, 2011)

2.5

1.5 RON 97
RM/litre RON95
1 Diesel

0.5

Date

FIGURE 2.4 Retail Fuel Prices in Malaysia (KeTTHA, 2011)

 9

Nevertheless, it is time to search for other alternative energy sources that are
clearer and much sustainable than fossil fuel to ensure future generation could meet
their own energy demand. Bioethanol production from lignocellulosic feedstock has
posted a great potential as an alternative fuel in Malaysia for transportation sector.
Recently, rice straw has received much attention from numerous researchers around
the globe on bioethanol production potential using different techniques and processes.

2.3 Rice Straw Composition

Lignocellulosic materials mainly contain predominant of a mixture of carbohydrates

polymers which are cellulose and hemicellulose, lignin, extractives and ashes
(Taherzadeh and Karimi, 2007a). The predominantly composition of rice straw are
cellulose (32 – 47 %), hemicellulose (19 – 27%) and lignin (5 – 24%) (Karimi et al.,
2006). Cellulose is a polymer of repeating β-D-glucopyranose units and is a chief
constituent of the feedstock while hemicellulose; a polymer to C5 and C6 sugars is
similar to cellulose, is a polysaccharide but less complex and can be easily hydrolysed
by acids to monomer components such as xylose, mannose, glucose, galactose,
arabinose (Dwivedi et al., 2009). The carbohydrate of rice straw is presented in
TABLE 2.1. Lignin roles as a cementing material and binds all other constituents in
position (Dwivedi et al., 2009). Therefore, lignin network consisting of polysaccharide
layers actually prevents the enzymatic hydrolysis of cellulose and hemicellulose in
rice straw (Lim et al., 2012). The structure of lignin varies in every different feedstock
making lignin content in rice straw to be lower than other common feedstock such as
corn stover and wheat straw (Hsu et al., 2010).

TABLE 2.1 Carbohydrates present in rice straw (Nutawan et al., 2010)

Carbohydrate of rice straw Content (%)
Glucose 41 – 43.4

Xylose 14.8 – 20.2

Arabinose 2.7 – 4.5
Mannose 1.8
Galactose 0.4
 10

It is studied that only cellulose and hemicellulose have the potential to be

converted to sugar by chemically or enzymatically hydrolysis before fermentation
(Roslan et al., 2011) and lignin remains as a by-product (Taherzadeh and Karimi,
2007a). It is further explained by Puneet Dwivedi et al. by saying that glucose is the
dominant sugar present in the cellulose while hemicellulose is a mixture of different
types of sugars namely C5 (xylose, arabinose, and rhamnose) and C6 (glucose,
mannose and galactose) (Dwivedi et al., 2009). TABLE 2.2 shows an example of
composition of carbohydrates polymers (cellulose and hemicellulose) with the
theoretical ethanol yield of rice straw and assuming hemicellulose fractions are all
polymers of xylose (Binod et al., 2010).

TABLE 2.2 Carbohydrate composition and theoretical ethanol yield of rice straw
(Binod et al., 2010)
Cellulose content 38.6%

Hemicellulose content 19.7%

Theoretical ethanol yield (L/kg dry) 0.42

2.4 Rice Straw to Bioethanol

Lignocellulosic feedstock will need to undergo few processes before ending up with
the final product of the project which is bioethanol. At present, there are many
methods that have been studied to produce bioethanol from lignocellulosic feedstock
and all these methods can be categorized into two board categories namely
biochemical conversion and thermochemical conversion (Dwivedi et al., 2009). In this
present paper, biochemical conversion method would be focused on producing
bioethanol from rice straw. There are few main steps involving in biochemical
conversion of lignocellulosic feedstock into fuel grade ethanol. The steps are pre-
treatment, saccharification/hydrolysis, fermentation, distillation and dehydration if
blending with gasoline is desired (Taherzadeh and Karimi, 2007a). FIGURE 2.5
shows the flowchart of processes involved in biochemical conversion method.
 11

Mechanical Chemical Saccharification /

pretreatment pretreatment Hydrolysis

Fermentation Distillation Dehydration

FIGURE 2.5 Flowchart showing biochemical conversion processes of lignocellulosic

feedstock to high quality bioethanol

2.5 Pretreatment of Rice Straw Biomass

Generally, pretreatment; also known as first stage hydrolysis has been viewed as one
of the most expensive processing steps involved in ethanol production from
lignocellulosic feedstock (Dwivedi et al., 2009, Binod et al., 2010). It is estimated to
account for 33% of the total cost of bioethanol production (Ibrahim, 2012). Enzymes
is unable to convert lignocellulosic feedstock into fermentable sugars without proper
pretreatment (Binod et al., 2010).

As discussed earlier, lignin network consists of polysaccharide layers that

prevent enzymatic hydrolysis of cellulose and hemicellulose in rice straw (Binod et
al., 2010). Hemicellulose similar to lignin, provides a protective covering to cellulose
as well (Dwivedi et al., 2009). In order to make cellulose and hemicellulose more
accessible to enzymatic action, pretreatment of rice straw is necessary and important
in order to break down lignin present in rice straw. FIGURE 2.6 shows the effect of
pretreatment on the lignocellulosic structure.

The aims of pretreatment are to increase biomass surface area, decrease

crystallinity (degree of structural order in a solid), eliminate hemicellulose, and
remove lignin network. Pretreatment allows cellulose to be more accessible to
enzymes. Thus, increasing the yield and rate of fermentable sugars from conversion of
carbohydrate polymers (Binod et al., 2010).
 12

FIGURE 2.6 Schematic pretreatment of lignocellulosic material (Haghighi Mood et

al., 2013)

There are many difference types of known pretreatment techniques such as

physical pretreatment, chemically pretreatment and thermally pretreatment and their
combinations (Binod et al., 2010). For chemical pretreatment, hemicellulose will be
hydrolysed into its basic sugars while small amount of cellulose will be hydrolysed
into glucose (Dwivedi et al., 2009).

Yield of lignocellulose to monomeric sugars and by-products is influenced by

numerous factors, making lignocellulosic materials and hydrolyses processes to be
complicated. Factors such as feedstock size, ratio of liquid to solid, type and
concentration of chemical used, temperature, and reaction time are examples of
factors that influence the yield of end products of the conversion (Karimi et al., 2006).
Nonetheless, these variables vary in different type of lignocellulosic feedstock,
making difficulty in data interpreting for a type of feedstock to another.
 13

2.5.1 Physical Pretreatment

Physical pretreatment is the most common pretreatment prior to any other

pretreatment of lignocellulosic feedstock. Physical pretreatment aims to increase the
total accessible surface area and size of lignocellulosic pores. Furthermore, physical
pretreatment will also aid in decreasing the crystallinity and degrees of polymerization
of cellulose (Binod et al., 2010). Examples of well common used physical
pretreatment methods include milling and grinding, steaming, irradiation, temperature
and pressure. Physical pretreatment is still considered unattractive due to cost and
energy (Ghose and Ghosh, 2003).

2.5.2 Acid Pretreatment

Acid pretreatment will result in more soluble hemicellulose but has little impact on
degrading lignin. It allows cellulose to have better accessible to the enzymes action
during hydrolysis (Binod et al., 2010). Consequently, the remaining components of
acid pretreated lignocellulosic feedstock are cellulose, a minor amount of
hemicellulose, and a reduced amount of lignin (Wirawan et al., 2012).

Common mineral acids that are used to carry out acid pretreatment are
hydrochloric acid (HCl) and sulfuric acid (H 2SO4) (Binod et al., 2010). Acid
pretreatment can be carried out either under low concentration with high temperature
setting or under high concentration with low temperature setting. High concentration
acid pretreatment is reported to be economical but several major drawbacks such as
high corrosion, toxicity, degradation of monosaccharaide polymer such as glucose,
and production of inhibitors which may affect fermentation yield, are preventing this
application from being commercialized. In addition, acid pretreatment at high
temperature will produce inhibitors such as furfural which will degrade into unwanted
by-products such as formic and levulinic (Haghighi Mood et al., 2013).

Dilute acid pretreatment is the most preferable pretreatment for lignocellulosic

feedstock to hydrolyse hemicellulose as it produces less amount of fermentation
inhibitors (Haghighi Mood et al., 2013, Dwivedi et al., 2009). Before fermentation,
neutralization of acid or removal of acid is required (Dwivedi et al., 2009).
 14

2.5.3 Alkaline Pretreatment

Alkaline pretreatment is effective in degrading lignin and a part of hemicellulose,

making the cellulose to be much accessible by enzyme, improving the yield of
hydrolysis. Alkaline pretreatment has shown better results in breaking ester bonds
between lignin, hemicellulose and cellulose, and avoiding hemicellulose polymers
from fragmentation compared to acid or oxidative reagents (Binod et al., 2010). In
additional, this method is also known for causing chemical swelling of fibrous
cellulose which will increase the solubility of glucose to the enzyme. The remaining
components after alkaline pretreated are cellulose, hemicellulose and a small amount
of lignin (Wirawan et al., 2012). The degradation of lignin in rice straw is reported to
be 36% by using alkaline pretreatment (Binod et al., 2010). The common bases used
for alkaline pretreatment are sodium hydroxide (NaOH), potassium hydroxide (KOH),
calcium hydroxide (CaOH2) and ammonia (Haghighi Mood et al., 2013).

There are certain advantages of choosing alkaline pretreatment over acid

pretreatment as it does not require complex reactors or equipment and it can be
performed under low temperature while the major drawbacks are longer residence
time which may require hours to days depending on factors such as equipment
technology, alkali concentration and temperature, and the need to neutralize the
pretreated slurry formed (Haghighi Mood et al., 2013).

2.5.4 Comparison of Parameters for Physical, Acid and Alkaline Pretreatment

TABLE 2.3 shows summarise for the effect of different pretreatment methods on
different parameters discussed in earlier section.

TABLE 2.3 Effect comparison of physical, acid and alkaline pretreatment (Haghighi
Mood et al., 2013)
Incremen
t Hemicellulos
Cellulose
Pretreatmen of e Formation of
specific decrystallizatio removal and Lignin inhibitor
t surface n solubilization removal compounds
a
Physical ++ ++ - - -
Acid ++ - ++ + ++
Alkaline ++ - + ++ +/-
 15

++a : High effect; + : Moderate effect; +/- : Low effect; - : No effect

The major drawback of physical pretreatment is the high energy consumption

during the process (Haghighi Mood et al., 2013). As the operation runs in
commercialized scale, energy costs would be high but physical pretreatment has good
advantages in increasing the yield of glucose in the further processes. The major
drawbacks of acid pretreatment are high corrosion, degradation of produced sugars
and required neutralization of pretreated slurry (Haghighi Mood et al., 2013). Still,
dilute acid pretreatment is the most preferable pretreatment for lignocellulosic
feedstock to hydrolyse hemicellulose as it produces less amount of fermentation
inhibitors compared to concentrated acid pretreatment (Dwivedi et al., 2009, Haghighi
Mood et al., 2013). Alkaline pretreatment shows the best pretreatment compared to
acid pretreatment as alkaline pretreatment removes most of the lignin composition and
produces less fermentation inhibitors. Unfortunately, long retention time has made
alkaline pretreatment to be less favourable than acid pretreatment (Haghighi Mood et
al., 2013). In additional, acid pretreatment with enzymatic hydrolysis has shown a
higher sugar yield compared to alkali pretreatment with enzymatic hydrolysis
(Nutawan et al., 2010).

2.6 Hydrolysis

Hydrolysis is a process of biopolymers degradation into smaller polymers or

monomers by chemical process. Therefore, hydrolysis process is used to convert
cellulose and hemicellulose to fermentable sugars by using either acids or enzymes
hydrolysis after the feedstock has been pretreated (Binod et al., 2010). A complete
hydrolysis of cellulose and hemicellulose will results in glucose and several pentoses
and hexoses respectively (Taherzadeh and Karimi, 2007a). FIGURE 2.7 shows the
hydrolysis conversion of cellulose and hemicellulose to its simple sugar base.
 16

Cellulose Hemicellulose
Hydrolysis Hydrolysis
Glucose Pentoses and
Hexoses
Fermentation Fermentation
Ethanol Ethanol

FIGURE 2.7 Conversion processes of cellulose and hemicellulose to ethanol

At present, there are three common hydrolysis methods which are dilute acid
hydrolysis, concentrated acid hydrolysis and enzymatic hydrolysis. Enzymatic
hydrolysis is much preferable for lignocellulosic bioethanol production because it is
more environmental friendly and it gives higher sugars yield than dilute acid
hydrolysis and concentrated acid hydrolysis (Wirawan et al., 2012). Furthermore,
enzymatic hydrolysis is able to carry out at mild conditions of temperature and
pressure (Dwivedi et al., 2009).

2.6.1 Acid Hydrolysis

Acid hydrolysis method can only be used to hydrolyse feedstock that has gone
through dilute acid pretreatment. There are two options available to hydrolyse the acid
pretreated feedstock which are dilute acid hydrolysis and concentrated acid
hydrolysis. Acid concentration used and temperature setting will be higher than
previous acid pretreatment to hydrolyse cellulose to glucose (Dwivedi et al., 2009).
The two common mineral acids used in acid hydrolysis are HCl and H2SO4.

2.6.1.1 Dilute Acid Hydrolysis

To date, dilute acid hydrolysis is the common employed pretreatment technology for
numerous herbaceous materials such as rice straw (Hsu et al., 2010). Batch reactor is
the most widely used reactor for ethanol production from lignocellulosic feedstock
using dilute acid hydrolysis (Taherzadeh and Karimi, 2007a). For dilute acid
hydrolysis, higher temperature is used compared to acid pretreatment. The
temperature ranges from 180℃ - 230℃ whereas the concentration of acid is around 4
% (Taherzadeh and Karimi, 2007a, Dwivedi et al., 2009). FIGURE 2.8 shows about
35% glucose yield is obtained from 0.5 % dilute H 2SO4 with temperature about 228℃
for retention time of 7 minutes (Taherzadeh and Karimi, 2007a). In additional, it is
reported that up to 95% hemicellulose sugars can be obtained using dilute acid
 17

hydrolysis, depending on the type of feedstock, physical and chemical conditions

(Ranjan and Moholkar, 2013).

0.4

0.35

0.3
Glucose Yield (25% dry weight)

0.25

0.2

0.15

0.1

0.05

0
180 190 200 210 220 230 240
Temperature (℃)

FIGURE 2.8 Glucose yield using 0.5% dilute H2SO4 with temperature range from 188
- 234℃ (Taherzadeh and Karimi, 2007a)

The major drawbacks of dilute acid hydrolysis operating at high temperature

are the degradation of sugars and formation of by-products which will influence the
sugar yield and fermentation process (Taherzadeh and Karimi, 2007a). Dilute acid
hydrolysis might result in sugars and other unwanted by-products Hydrolysis of
cellulose will result in glucose, hydroxymethylfurfural (HMF), levulinic acid while
hydrolysis of hemicellulose will result in xylose, arabinose, glucose, mannose,
galactose. These reactions may advance further and hydrolyse pentoses to furfural and
hexoses to HMF (Karimi et al., 2006). Furfural and HMF are inhibition to
fermentation process. In order to minimise degradation of sugars and formation of
 18

unwanted by-products, two-stage dilute acid hydrolysis has been introduced

(Taherzadeh and Karimi, 2007a).

2.6.1.2 Concentrated Acid Hydrolysis

Concentrated acid hydrolysis is reported to have very high sugar yield which is
approximately 90% of the theoretical glucose yield (Dwivedi et al., 2009). The
operation temperature for concentrated acid hydrolysis is relatively low (40℃) and
the process is relatively rapid (10 – 12 hours) as well as only causes a small
degradation of sugars (Taherzadeh and Karimi, 2007a, Dwivedi et al., 2009). The acid
concentration used is relatively high (30% - 70%). As high acid concentration is used
to hydrolyse feedstock, dilution and heating of the high acid concentration is
extremely corrosive. Thus, expensive reactors made specially to resist extreme
corrosion are required, drawing back the potential of concentrated acid hydrolysis
despite its high sugar yield. Although there are major drawback on investment and
environmental aspects, there are two full scale lignocellulosic ethanol conversion
projects under development in North America (Taherzadeh and Karimi, 2007a).

2.6.2 Enzymatic Hydrolysis

Enzymatic hydrolysis is one of the potential conversion technologies that may

dominate the total production of ethanol from lignocellulosic feedstock (Dwivedi et
al., 2009). Commonly, enzymatic hydrolysis is carried out under mild conditions
(temperature and pressure) and long retention time. This gives a higher glucose yields
and lower chances of fermentation inhibitor compounds formation, environmental
impact reduction and less significant of equipment is required compared to acid
hydrolysis (Binod et al., 2010, Dwivedi et al., 2009). It is possible to hydrolyse
cellulose to nearly 100% using enzymatic hydrolysis (Taherzadeh and Karimi, 2007b).
 19

In enzymatic hydrolysis process, cellulases; a complex of enzymes that act

synergistically to attack native cellulose, are the primarily implicated enzymes used
for the hydrolysis process. A variety of microorganisms such as bacteria,
actinomycetes and fungi are required to produce cellulases (Sarkara and Aikata,
2012). Trichoderma reesei is one the best options for cellulases production. Common
commercialized cellulases are produced from Trichoderma spp (Taherzadeh and
Karimi, 2007b). There are at least three major cellulases involve in the process namely
endoglucanase (EC 3.2.1.4), exoglucanase (E EC 3.2.1.91.) and β-glucosidase (EC
3.2.1.21) (Dwivedi et al., 2009). It could further understood from FIGURE 2.9.

FIGURE 2.9 Schematic flow of cellulose conversion to glucose by enzymes

(Taherzadeh and Karimi, 2007b)

Despite the advantages over acid hydrolysis, there are still several drawbacks
for enzymatic hydrolysis. Comparing to acid hydrolysis, enzymatic hydrolysis may
 20

takes up to several days duration rather than just minutes to hours for acid hydrolysis,
and the cost of enzymes are higher than acid used in acid hydrolysis. Furthermore, the
released sugars for enzymatic hydrolysis will inhibit the hydrolysis yield.

2.6.3 Comparison of Different Types of Hydrolysis

TABLE 2.4 shows the comparison in term of advantages and disadvantages of dilute
acid hydrolysis and concentrated acid hydrolysis. As concentrated acid hydrolysis will
have large environmental aspect issues, dilute acid hydrolysis is much preferable for
its lower cost and lesser environmental impacts (Taherzadeh and Karimi, 2007a).
TABLE 2.5 shows the comparison of dilute acid hydrolysis and enzymatic hydrolysis.
Enzymatic hydrolysis is much preferable as it gives higher sugar yield, low
environmental effects and does not produce fermentation inhibitors (Taherzadeh and
Karimi, 2007b).

TABLE 2.4 Comparison of dilute acid hydrolysis and concentrated acid hydrolysis
(Taherzadeh and Karimi, 2007a)
Type of Hydrolysis Advantages Disadvantages
Dilute acid  Low amount of  Operates at high
acid usage temperature
 Short retention  Low sugar yield
duration  Corrosion effect
 Formation of unwanted
inhibitor by-products
Concentrated acid  Operates at low  High amount of acid
temperature usage
 High sugar yield  Long retention time
 High corrosion effect
 21

 High energy usage for

acid recovery

TABLE 2.5 Comparison of dilute acid hydrolysis and enzymatic hydrolysis

(Taherzadeh and Karimi, 2007b)
Type of Hydrolysis Advantages Disadvantages
Dilute acid  No formation of  Operates at high
product inhibition temperature and
during hydrolysis pressure
 Low cost of acid  Low sugar yield
 Short retention  Formation of
duration inhibitors by-
products
 Corrosion effect
Enzymatic  Operates under  High cost of
mild conditions enzyme
(temperature and  Long retention
pressure) duration
 High yield of  Product inhibition
sugars during hydrolysis
 No formation of
inhibitor by-
products
 No corrosion effect
 22

2.7 Fermentation

Fermentation of hydrolysed lignocellulosic feedstock is commonly done by using

baker’s yeast named Saccharomyces cerevisiae. Saccharomyces cerevisiae is used to
ferment C6 sugars under anaerobic/aerobic conditions (Dwivedi et al., 2009).
However, the limitation of Saccharomyces cerevisiae is its incapability to ferment
xylose (Taherzadeh and Karimi, 2007a). Referring to TABLE 2.1, composition of
xylose in rice straw carbohydrate is 14.8 – 20.2%. Many alternatives have been
researched and introduced to ferment xylose as well. For an example, Escherichia coli;
an engineered microbes has been developed to ferment both C6 and C5 sugars. Other
than that, separation fermentation of glucose and xylose could be done to obtain
higher ethanol yield. A second yeast named Pachysolen tannophilu could be used to
ferment xylose after distillation of unconverted xylose from fermented glucose using
Saccharomyces cerevisiae (Dwivedi et al., 2009). Eq 2.1 shows the conversion of
glucose into by-products consist of ethanol and CO2. The theoretical conversion of
glucose to ethanol is found to be 51% on a weight basis (Lee et al., 2007).

C 2 H 12 O 6 =2C 2 H 5 OH +2 C O2 (2.1)

2.7.1 Inhibitors of Fermentation

Bioethanol production form lignocellulosic feedstock has been proven to be a

challenging process from pretreatment to fermentation. Inhibitors are the major
challenge for bioethanol production during fermentation where inhibition could
disrupt replication of cellular and metabolism of sugar and also disrupt integrity of
membrane (SUTTON, 2011). Fermentation inhibitors can be divided into at least four
major groups which are weak acids, furfural and hydroxymethyl furfural (furan
 23

derivatives), and phenolic compounds (Palmqvist and Hahn-Hägerdal, 2000,

SUTTON, 2011). It is suggested by Kate Brandon Sutton that optimization of
lignocellulosic feedstock pretreatment is the best way to minimize the formation of
inhibitors.

2.8 Recovery of Ethanol

Conventional distillation is the most reliable method of ethanol recovery that is

commonly used to purify ethanol produced from lignocellulosic material to a purity
range from 90 – 95% (Taherzadeh and Karimi, 2007a, Ghose and Ghosh, 2003).
Distillation aims to separate water present in the ethanol produced after fermentation
process (Ghose and Ghosh, 2003). To enable ethanol produced from lignocellulosic
materials to blend with gasoline; fuel grade ethanol, further dehydration of ethanol is
required (<1% water), leading the required purity of ethanol to above 99%. It is
known as dehydrated ethanol or anhydrous ethanol. This could be achieved by passing
desired ethanol through inorganic materials (molecular sieves technology) such as
calcium chloride (CaCl2) (Taherzadeh and Karimi, 2007a, Ghose and Ghosh, 2003).

2.9 Reducing Sugars Analysis and Ethanol Analysis Method

Dinitrosalicylic acid (DNS) reagent could be used to analyse the total concentration of
reducing sugar (monosaccharide) present in the pretreated, hydrolysed and fermented
lignocellulosic feedstock. Spectrophotometer with absorbance measured at 540 nm is
commonly used in DNS testing method to estimate total reducing sugars present
where glucose or xylose is used as standard (Sarkara and Aikata, 2012). A standard
graph of absorbance versus concentration of glucose can be plotted by obtaining data
from spectrophotometry analysing and the concentration of glucose can be determined
from the experimental obtained data.

Due to the presence of complex media in the treated feedstock, large errors
might occur by using DNS testing method. Another way to analyse the samples
thoroughly and much accurate is by using chromatography testing method
 24

(Taherzadeh and Karimi, 2007a). Ethanol concentration can be determined by using

high performance liquid chromatography (HPLC) or gas chromatography (Zhu et al.,
2005).
 CHAPTER III

METHODOLOGY

3.1 Introduction

In this chapter, the methods used throughout the study will be explained thoroughly.
Methodology on obtaining standard glucose curve, pretreatment of rice straw,
enzymatic hydrolysis of rice straw, fermentation, distillation and dehydration of
ethanol are presented on this chapter.

3.2 General Methodology on Ethanol Production

Ethanol can be produced by using different approaches with rice straw as feedstock.
FIGURE 3.1 shows a general flow for bioethanol production from collection of
feedstock to anhydrous ethanol (<1% water concentration).

Collection and transportation

Rice straw is collected and tranported for further processing

Feedstock Preparation
Rice straw is washed and milled to increase surface area

Pretreatment
Rice straw is pre-treated to breakdown lignin and hemicellulose

Hydrolysis / Saccharification
Pre-treated rice straw undergo hydrolysis to convert into fermentable sugar

Fermentation
Sugars are fermented to ethanol

Distillation
Ethanol is distilled to produce concentrated ethanol (90 - 95%)

Dehydration
Ethanol is dehydrated to <1% water concentration

FIGURE 3.1 General Bioethanol flowchart

 26

There are two processes that can be chosen to convert lignocellulosic

feedstock into energy products. The processes are namely bio-chemical process and
thermo-chemical process. In this project, bio-chemical process is studied and
understood.

3.3 List of Equipment

TABLE 3.1 Equipment used and its functions

Equipment Function(s)
To provide continuous even shaking experience and desired
Incubator shaker temperature to samples

pH meter To measure the pH value of samples

Weighing balance To measure the weight of samples

Magnetic stirrer To mix mixture of solution thoroughly

To heat samples according to desired temperature (max 100
Water bath ℃)

Laminar airflow To provide contaminant free working environment

To heat samples to high temperature. Mainly used for

Autoclave samples sterilization

HPLC To measure glucose and ethanol concentration

Spectrophotometer To measure glucose concentration in samples

Home blender To reduce samples surface area

Centrifugal To centrifuge liquid and solid phase in samples

Rotatory Evaporator To distill ethanol from samples

3.4 Calibration of Glucose Standard Curve of Absorbance

 27

A standard curve of absorbance had been plotted for absorbance value against known
concentration. The calibration curve is needed to determine the glucose concentration
later for the rice straw samples from pretreatment, hydrolysis and fermentation.
Absorbance is also known as optical density.

0.5 grams of dry solid monosaccharide glucose was weighed using high
sensitivity weighing balance and added into a volumetric flask. Then, 500 ml of
distilled water was added into the volumetric flask. By using pipette, 0.2 ml of diluted
glucose solution was transferred into each of 5 test tubes. It followed by adding 1.8 ml
of distilled water and 2.0 ml of DNS reagent into each of the test tubes.
Dinitrosalicylic acid (DNS) reagent was prepared by lab technician.

The procedures were repeated by increment of 0.2 ml of glucose and

decrement of 0.2 ml of distilled water respectively while amount of added DNS
solution remained constant. Then, the test tubes opening were covered with parafilm
to prevent water droplets from entering the test tubes during heating process. The test
tubes containing the mixture solution were inserted into water bath for 5 minutes at 90
℃.

After 5 minutes of heating, the test tubes were taken out and parafilm sheet
was removed. Then, the mixtures were left to cool down at room temperature. Then,
the mixture solution was tested using spectrophotometer at wavelength of 540 nm
(Saqib and Whitney, 2011). The data were taken down. A graph of absorbance (A)
versus glucose concentration was plotted and it will be used to determine the glucose
concentration in the later samples from rice straw.

3.5 Glucose Content Determination

 28

Glucose content in the samples was calculated based on the Eq 4.1 obtained from
standard curve of absorbance for glucose; FIGURE 4.1 and also based on the dilution
factor used. Below shows the simple formulas used in the study for glucose content
determination by utilizing the Eq 4.1 and dilution factor.

g Absorbance ( A )∗Dilution factor

Glucose content ()
L
=
5.076
(3.1)

The glucose content determine from Eq 3.1 is in g/L. In order to determine

how much sugar is actually successfully yield from a gram of acid pretreated rice
straw in the added 100 ml of solution, Eq 3.2 will be used. In term of percentage, Eq
3.2 is multiplied with 100%.

g Glucose content
Glucose yield ( g rice straw basis )
=
Substrate
mass∗1000 ml
(3.2)
acid volume

3.6 Fermentation Analysis

 29

Fermentation analysis was carried out to study the behaviour of yeast growth and
behaviour of glucose concentration level in the samples with respect to allowable
retention time. Preparation of yeast media, yeast inoculum techniques and analysis
techniques of samples collected were learned.

3.6.1 Preparation of Yeast Media

In order to cultivate yeast, a suitable medium is required. The required mediums for
yeast to be cultivated are yeast extract, glucose, ammonia chloride (NH 4Cl) and
Monopotassium phosphate (KH2PO4). The pH value should be ranging from 5.0 – 6.0.
400 ml of yeast extract was prepared.

40 g of dry solid monosaccharide glucose, 4 g of yeast extract powder, 1.6 g of

KH2PO4, and 0.8 g of NH 4Cl were weighed using electronic weighing balance. Then,
the measured materials were transferred into a 500 ml conical flask. 400 ml of reverse
osmosis (RO) water was measured and transferred into the conical flask. Then, the
medium was stirred using magnetic stirrer. The temperature setting was set to be 50
℃ to assist mixture process. 100 ml of the yeast medium prepared was measured and
transferred into the conical flasks respectively and enclosed loosely with conical flask
closure. The conical flasks containing the medium were sterilized using an autoclave
at 121 ℃ for 15 minutes. The conical flasks were left to cool down at room
temperature.

FIGURE 3.2 Preparation of yeast media

3.6.2 Yeast Inoculum
 30

After sterilization process of the conical flasks containing yeast medium, a small
amount of earlier cultivated yeast; Saccharomyces Cerevisiae was transferred into
three of the conical flasks containing yeast medium while one remained as control
experiment. Then, the samples were placed into an incubator shaker at 37℃ and at
100 rpm. 1 ml of each sample was taken at 6 hours, 8 hours, and 10 hours. The
samples taken were transferred into eppendorf tubes.

3.6.3 Analysis of fermentation samples

The samples contained in 1ml eppendorf tubes were centrifuged at 10,000 rpm for 5
minutes to separate liquid phase and solid phase. The liquid phase of each centrifuged
samples with approximately 1 ml was transferred into 50 ml centrifuge tubes
accordingly and 49 ml of reverse osmosis (RO) water was added into each centrifuge
tubes. Then, 1 ml of RO water was added into each eppendorf tubes to dilute the solid
phase (yeast cell) and was shook well to ensure the solid phase mixed completely.

0.1 ml from each sample in centrifuge tubes was transferred into different test
tubes accordingly and mixed with 1.9 ml of RO water. 2 ml of DNS reagent was
added into each test tubes each containing 0.1 ml of the sample. A control solution
was prepared by mixing 2 ml of RO water with 2 ml of DNS reagent. Parafilm was
used to cover all the test tubes opening. The test tubes were heated using water bath
for 5 minutes at 90 ℃. Spectrophotometer with wavelength setting of 540 nm was
used to obtain the data for level of absorbance.

The samples in each eppendorf tubes were analysed using spectrophotometer

with wavelength setting at 640 nm without any additional of DNS reagent. The data
obtained was taken.

3.7 Preparation of Raw Material

 31

Rice straw used throughout the study was obtained from Sekinchan, Selangor during
harvesting season on July. The rice straw obtained was cleaned with running tap water
to remove dust and any other unwanted impurities. Then, the washed rice straw was
air dried using hot air oven for 6 hours at 60℃. The composition of rice straw is
reported in earlier chapter where cellulose is 32 – 47%, hemicellulose is 19 – 27%,
and lignin is 5 – 24% (Karimi et al., 2006).

3.8 Pretreatment of Rice Straw

There were two pretreatment that had been carried out on the lignocellulosic
feedstock. The pretreatments carried out were physical pretreatment and acid
pretreatment. Physical pretreatment aims to increase the total accessible surface area
and size of lignocellulosic pores. Furthermore, physical pretreatment will also aid in
decreasing the crystallinity and degrees of polymerization of cellulose. Acid
pretreatment was carried out under dilute concentration condition. Acid pretreatment
allows cellulose to have better accessible to the enzymes action during hydrolysis and
have little effect on degrading lignin (Binod et al., 2010).

3.8.1 Physical Pretreatment

The cleaned rice straw was physically treated by using a high speed home blender.
The physical pretreated rice straw was stored inside a container at room temperature
for further use. FIGURE 3.3 below shows physical pretreatment using a home
blender.

FIGURE 3.3 High speed home blender used for physical pretreatment of rice straw
 32

3.8.2 Acid Pretreatment

6 sets of samples, each with 3 g of physical pretreated rice straw were weighed
using a weighing balance and transferred into respective conical flask. The samples
were labeled accordingly with 2 sets for retention time of 30 minutes, 2 sets for
retention time of 60 minutes and 2 sets for retention time of 90 minutes. 100 ml of
prepared 1 Molar (M) sulfuric acid was added into each of the conical flask. The
mixture was shook well to ensure the samples were completely covered by the acid
solution as shown in FIGURE 3.4. Parafilm was used to cover all the conical flasks
top. The samples were heated using water bath at 90℃ for retention time of 30
minutes, 60 minutes and 90 minutes. The samples were taken out after the retention
time and were left to cool down at room temperature. 100 ml of prepared 2 M sodium
hydroxide was added into each conical flask for neutralization purpose. This would
give a dilution factor of 2. The mixture was shook well to ensure even mixture
experience. The procedures were repeated with 1.5 M and 2.0 M of sulfuric acid and
3.0 M and 4.0 M sodium hydroxide respectively to obtain different samples. The
samples were kept for further analysis.

FIGURE 3.4 Rice straw samples added with acid solution

 33

3.8.2.1 Preparation of Sulfuric acid (H2SO4)

An amount of 96 % or equivalently to 18.01 molar (M) of H 2SO4 was diluted to

prepare different molarity of H2SO4. To prepare 1 M of H2SO4 with total volume of 1
litre (L), it was calculated that 55.5 millilitre (ml) of 96 % H 2SO4 is required. 55.5 ml
of H2SO4 was added slowly into 944.5 ml of distilled water into a 1 L media storage
bottle. Then, the mixture was stirred slowly to ensure complete mixture. The
procedures were repeated to prepare 1.5 M and 2.0 M of H 2SO4 with 83.25 ml and 111
ml of H2SO4 respectively.

3.8.2.2 Preparation of Sodium Hydroxide (NaOH)

40 gram (g) of NaOH is equivalent to 1 molar (M) when it is mixed with 1 liter (L) of
distilled water. To prepare 2 M of NaOH, 80 g of NaOH pellets were weighed using
an electronic weighing balance. Then, the pellets were added into a 1 L media storage
bottle with 1 L of distilled water and was stirred using magnetic stirrer until all the
pellets dissolved completely in the solution. The procedures were repeated to prepare
3.0 M and 4.0 M of NaOH with 120 g and 160 g of NaOH respectively.

3.8.2.3 Analysis of Reducing Sugar Concentration from Acid Pretreatment

40 ml of the neutralized solution from each sample was transferred into centrifugal
bottle accordingly. Then, the samples were centrifuged to separate liquid and solid
phase in the samples. The centrifuge was set at temperature of 4℃ and rotation speed
of 1000 rpm for 10 minutes.

Analysis of reducing sugar level present in the neutralized samples was done
with further dilution. Dilution factor of 50 is selected due to spectrophotometer
limitation where the spectrophotometer is unable to give reading more than 2.500
Absorbance (A). Firstly, 0.1 ml of stock solution was taken from each sample and
mixed with 4.9 ml of distilled water. Then, 2 ml of the diluted stock solution was
transferred into new test tubes with 2 ml of DNS solution added into it. The test tubes
opening were then covered with parafilm to prevent water droplet during heating
process. Next, the test tubes were heated using water bath at 90℃ for 5 minutes. The
parafilm was removed and the mixtures were left to cool down before reducing sugar
concentration was tested using spectrophotometer at wavelength of 540 nm.
 34

3.9 Enzymatic Hydrolysis of Pretreated Rice Straw

The conditions for highest yield of glucose from acid pretreatment were carried
forward for enzymatic hydrolysis. Six samples from acid pretreatment (2 M with 60
minutes retention time); each initially containing 3 g of pretreated rice straw were
separated into solid and liquid phase by pressing through conventional cheesecloth.
The solid phase was washed with running tap water to remove reducing sugars left by
acid pretreatment and neutralise the pH value. The pretreated rice straw was then
weighed and dried in an oven for 8 hours at 60℃. The liquid phase was brought
forward for fermentation.

The dried samples were adjusted to 3 g each for enzymatic hydrolysis.

Cellulase from Tichoderma Ressei ATCC 26921 purchased from Sigma-Aldrich was
used as enzyme in this study.

4 samples; 3 samples with pretreated rice straw (Acid pretreatment and

mechanical pretreatment) and 1 sample with only mechanical pretreated rice straw
were prepared by having 3 g of rice straw in each flask. 100 ml of Sodium Acetate
was added into each of the flask. Then, 0.8% w/w (24 mg for 3 g of substrate) of
cellulase enzyme was weighed and added into each of the flask. The samples were
shook well to ensure the enzyme mixed evenly with the solution. Then, the pH value
for the samples was adjusted to 5.0 using hydrochloric acid (HCL) or Sodium
hydroxide (NaOH); depending on the initial pH value of the samples. The samples
were then incubated in a shaking incubator for up to 96 hours. The samples were
divided into batches where the samples were taken out after specific duration. One
sample was taken out at duration of 24 hours and the second sample was taken out at
duration of 48 hours. For the other 2 samples (1 sample with acid pretreated and 1
sample without acid pretreated), 1 ml was taken from each sample and the samples
were placed back onto the shaking incubator for another 24 hours to study the yield.
The samples were heated at 90℃ for 15 minutes and stored at 4℃ for further
analysis.
 35

3.9.1 Preparation of Hydrochloric acid (HCL)

An amount of 37 % or equivalently to 12.18 molar (M) of HCL was diluted to prepare

1 M of HCL. 200 ml of 1 M of HCL was prepared by mixing 16.5 ml of 37% HCL
with 183.5 ml of distilled water. The solution was shook well to ensure complete
mixing.

3.9.2 Preparation of Sodium Acetate (C2H3NaO2)

400 ml of sodium acetate was prepared by weighing 2.72 g of sodium acetate powder
and mixed with 400 ml of RO water.

3.9.3 Analysis of Reducing Sugars from Enzymatic Hydrolysis

1 ml was taken from the samples and transferred into eppendorf tubes and labelled
accordingly. The eppendorf tubes were then centrifuged at 10,000 rpm for 5 minutes
to separate the liquid and solid phases. Using dilution factor of 50 times, 0.1 ml from
each sample was transferred into test tubes and 4.9 ml of distilled water was added
into each test tubes. Then, 2 ml of each diluted sample was transferred into new test
tubes and 2 ml of DNS reagent was added into each test tubes. It followed by heating
the samples using a water bath at 90℃ for 5 minutes. The samples were let to cool
down at room temperature before taking the reading using spectrophotometer at
wavelength setting of 540 nm. The solid phase of the samples was ignored in this
study.
 36

3.10 Fermentation of Reducing Sugars

The fermentation was carried out by liquid state fermentation process. The yeast used
for the study was Saccharomyces Cerevisiae. The liquid phase from acid pretreatment
and enzymatic hydrolysis was fermented and studied separately for better
understanding.

3.10.1 Fermentation of Liquid Phase from Acid Pretreatment

The samples from acid pretreatment were separated into solid and liquid phase using
conventional cheesecloth. The liquid phase was then filtered by using filter paper and
a funnel. There were three samples studied for fermentation where one of the samples
acts as control experiment. Average values were taken in the analysis. The required
yeast media except glucose was introduced into the solution by ratio to the standard
amount studied earlier according to the total volume of the solution. In this study, 100
ml of solution from acid pretreatment will be fermented in each sample of
fermentation. The pH value was then tested. If the solution is in acidic range, NaOH
will be added while if the solution is in alkali range, H 2SO4 will be added. The pH
value was adjusted to 5.5. The samples were then autoclaved at 121℃ for 15 minutes.

After sterilization using autoclave, a small amount of Saccharomyces

Cerevisiae was introduced into the samples except the one which acted as control
experiment. Then, the samples were incubated using an incubator shaker for 48 hours
at setting of 100 rpm and 37℃. 1 ml was taken from each sample at 0 hr, 6 hrs, 8 hrs,
10 hrs, 12 hrs, and 24 hrs and 48 hrs. The samples taken were stored in eppendorf
tubes for later analysis.

3.10.2 Fermentation of Liquid Phase from Enzymatic Hydrolysis

The procedures were similar to chapter 3.10.1. After separation of solid and liquid
phase using conventional cheesecloth, the liquid phase was filtered by using filter
paper and a funnel. There were two samples prepared where the first sample was from
liquid phase from enzymatic hydrolysis and one was yeast media acting as control
experiment. First sample contained 100 ml of solution from enzymatic hydrolysis
while another sample as control experiment was prepared by following procedures
discussed in section 3.6.1.
 37

The required yeast media except glucose was introduced into the solution from
enzymatic hydrolysis by ratio to the standard amount studied earlier according to the
total volume of the solution while glucose was introduced into the control experiment.
The pH value was then tested. If the solution is in acidic range, NaOH will be added
while if the solution is in alkali range, H 2SO4 will be added. The pH value was
adjusted to 5.5. The samples were then autoclaved at 121℃ for 15 minutes.

After sterilization using autoclave, a small amount of Saccharomyces

Cerevisiae was introduced into the sample except the one which acted as control
experiment. Then, the samples were incubated using an incubator shaker for 72 hrs at
setting of 100 rpm and 37℃. 1 mm was taken from each sample at 0 hr, 6 hrs, 8 hrs,
10 hrs, 12 hrs, and 24 hrs, 48 hrs and 72 hrs. The samples taken were stored in
eppendorf tubes for later analysis.

3.10.3 Analysis of Reducing Sugar Content and Yeast Growth in Fermentation using
DNS Testing Method

The samples contained in 1ml eppendorf tubes were centrifuged at 10,000 rpm for 5
minutes to separate liquid phase and solid phase. The liquid phase of each centrifuged
samples with approximately 1 ml was transferred into 50 ml centrifuge tubes
accordingly and 49 ml of distilled water was added into each centrifuge tubes. Then, 1
ml of distilled water was added into each eppendorf tubes to dilute the solid phase
(yeast cell) and was shook well to ensure the solid phase mixed completely.

2 ml from each sample in centrifuge tubes was transferred into different test
tubes accordingly and 2 ml of DNS reagent was added into each of the test tubes. A
control solution was prepared by mixing 2 ml of RO water with 2 ml of DNS reagent.
Parafilm was used to cover all the test tubes opening. The test tubes were heated using
water bath for 5 minutes at 90 ℃. Spectrophotometer with wavelength setting of 540
nm was used to obtain the data for level of absorbance. The dissolved solid phase of
each sample in were analysed using spectrophotometer with wavelength setting at 640
nm without any additional of DNS reagent. Distilled water was used as reference
reading. The data obtained was taken.
 38

3.11 Analysis and Distillation of Fermented Solution for Ethanol and Glucose
Content

The fermented solution from acid pretreatment and enzymatic hydrolysis was then
distilled using a rotary evaporator where the distillation process was carried out in
vacuum condition. The analysis and distillation of fermented solution from acid
pretreatment and enzymatic hydrolysis was carried out separately where each sample
contained about 100 ml.

3.11.1 Analysis using High Performance Liquid Chromatography (HPLC)

Glucose content and ethanol content were analysed using HPLC by following the
standard guidance given. The content of glucose and ethanol was measured before and
after distillation.

1 ml of the fermented solution was taken out from each sample (acid
pretreatment and enzymatic hydrolysis) and filtered using filter attached syringe and
stored into eppendorf tubes. Then, 30μL of fermented solution of acid pretreatment
was injected into HPLC injection inlet and HPLC analysis was started. The running
time of HPLC was set to be 20 minutes. The procedures were repeated by testing
samples from enzymatic hydrolysis fermentation and also distilled fermented solution
from acid pretreatment and enzymatic hydrolysis. The data obtained was saved.

FIGURE 3.5 HPLC equipment

 39

3.11.2 Distillation of Fermented Solution

The temperature of the heating was set to be 73℃ and the distillation was left to run
for 30 minutes under vacuum condition. The pressure achieved was 80 kPa. The
boiling temperature of ethanol with respect to pressure could be found in APPENDIX
H. The amount of distilled solution was measured and taken for content analysis using
high performance liquid chromatography.

FIGURE 3.6 Distillation Using Rotatory Evaporator Equipment

Eq 3.3 and Eq 3.4 were used to calculate the ethanol yield in this present study.

EthanolContent Measured ∈the sample ( g g−1 )

Ethanol yield= (3.3)
Theoretical Ethanol(g g−1 )

Theoretical Ethanol ( g g−1 )=Initial glucose measured ( g ) ×0.5(3.4 )

 CHAPTER IV

ANALYSIS AND RESULTS

4.1 Introduction

In this chapter, data obtained from the study will be analysed. The experimental
results consist of glucose standard curve of absorbance, acid pretreatment of rice
straw, enzymatic hydrolysis of rice straw, fermentation of hydrolysed rice straw and
distillation of ethanol produced. Graphs are plotted to represent the relationship
between parameters that manipulating the results of each experiment.

4.2 Calibration Curve of Absorbance for Glucose

Referring to TABLE 4.1, the highest coefficient of determination, R 2 value is 0.9847

or 98.47% with linear equation as shown in Eq 4.1. R 2 is the measure of how much x
variable can attribute y variation in term of percentage or whole number (Michael,
2013). FIGURE 4.1 shows the accuracy of the experiment data obtained by plotting a
linear regression line on the graph. Eq 4.1 is the equation selected from the curve of
absorbance and it is to be used to determine level of glucose concentration in samples
throughout the study. The detailed results could be found in APPENDIX A.

Y =5.076 x
(4.1)

TABLE 4.1 Experimental R2 values and linear equations for calibration curve of
absorbance using DNS method
Test No. R2 Linear equation

1 0.7124 y = 6.4133x

2 0.9847 y = 5.076x
3 0.9368 y = 5.286x
 42

2.5
f(x) = 5.08 x
R² = 1

Absorbance (A) 1.5

0.5

0
0 0.1 0.2 0.3 0.4 0.5 0.6
glucose concentration (g/L)

FIGURE 4.1 Selected experimental standard curve of absorbance for glucose

4.3 Relationship of Glucose and Yeast during Fermentation

Samples were taken from the yeast media during fermentation process to study the
relationship between yeast growth and glucose concentration. TABLE 4.2 shows the
results obtained by analysing the samples under spectrophotometer with wavelength
setting at 640 nm. The absorbance values show an increasing pattern; directly
proportional to duration. In other word, yeast was cultivated during the fermentation
process. The relationship can be seen much detailed in FIGURE 4.2 where the
relationship is clearly shown.

As yield of yeast is increasing during fermentation process, glucose yield

shows a decrease pattern with respect to time. The relationship can be seen clearly in
FIGURE 4.3 where glucose concentration is inversely proportional to time during
fermentation process.
 43

TABLE 4.2 Results for yeast growth during fermentation

Spectrophotometer wavelength: 640 nm
Duration
(Hours) Sample Absorbance (A) Average Absorbance (A)
1 0.106
2 0.227
6 3 0.160 0.164
1 0.346
2 0.556
8 3 0.542 0.481
1 0.560
2 0.911
10 3 0.940 0.804

0.9

0.8
f(x) = 0.16 x − 0.8
0.7 R² = 1

0.6

0.5
Absorbance (A)

0.4

0.3

0.2

0.1

0
4 5 6 7 8 9 10 11
Time (Hours)

FIGURE 4.2 Relationship of yeast growth and duration of fermentation

TABLE 4.3 Glucose concentration during fermentation process

 44

Spectrophotometer wavelength: 540 nm

Duration
(Hours) Sample Absorbance (A) Average Absorbance (A)
1 0.505
2 0.499
0 3 0.503 0.502
1 0.449
6 2 0.421
3 0.453 0.441
1 0.319
8 2 0.303
3 0.322 0.315
1 0.221
10 2 0.238
  3 0.229 0.229

0.6

0.5

0.4
Absorbance (A)

0.3

0.2

0.1

0
0 2 4 6 8 10 12
Time (Hours)

FIGURE 4.3 Relationship between glucose concentration and duration of fermentation

4.4 Glucose Yield from Acid Pretreatment on Rice Straw

In the study, rice straw samples had been treated by varying two different parameters
which were acid concentration and retention time of pretreatment. The total reducing
sugar yield from each of the parameters is as shown in TABLE 4.4. From TABLE 4.4,
 45

the highest sugar yield is by using 2.0 M at retention time of 60 minutes. Further
retention time after 60 minutes at 2.0 M shows no significant increase in sugar yield.
The highest sugar yield obtained from the experiment is 9.71 g/L or 0.323 g/g on rice
straw basis. Therefore, the sugar yield in percentage is 32.3 % g/g. The standard
deviation of each data obtained shows a consistent relation. The highest deviation is
0.0903 while the lowest deviation is 0.0297. FIGURE 4.4 shows the relation of sugar
yield of each concentration with different retention time. From the graph, it shows
acid pretreatment with 2.0 M is able to give higher sugar yield with shorter treatment
time compared to 1.0 M and 1.5 M. The detailed results of acid pretreatment on rice
straw could be found at APPENDIX B.

TABLE 4.4 Results for sugars yield in acid pretreatment

Spectrophotometer wavelength: 540 nm
Sugar
Retention Average
time Absorbance Sugar Yield Yield (g/g
Concentration (minutes) (A) (g/L) %)
30 0.160 3.15 10.51
60 0.366 7.21 24.03
1.0 M 90 0.362 7.13 23.77
30 0.319 6.28 20.95
60 0.409 8.06 26.86
1.5 M 90 0.489 9.63 32.11
30 0.424 8.35 27.84
60 0.493 9.71 32.37
2.0 M 90 0.470 9.26 30.86
 46

12

10

8
Glucose Yield (g/L)

6
1.0 M
1.5 M
4 2.0 M

0
20 30 40 50 60 70 80 90 100
Retention Time (minute)

FIGURE 4.4 Sugar yield (g/L) with different retention time and acid concentration

35.00

30.00

25.00
Glucose Yield %(g/g rice strawbasis)

20.00
1.0 M
15.00 1.5 M
2.0 M
10.00

5.00

0.00
30 60 90
Duration (Minutes)

FIGURE 4.5 Sugar Yield in Percentage of g/g Rice Straw Basis

 47

4.5 Glucose Yield from Enzymatic Hydrolysis on Rice Straw

The acid pretreated rice straw under the best conditions (2M of H2SO4 with
retention time of 60 minutes) was carried forward for enzymatic hydrolysis process
with enzyme loading of 0.8% w/w of dry rice straw basis. The main varying parameter
is time factor. Furthermore, a sample without acid pretreatment versus samples with
acid pretreatment was also tested to study the effect of acid pretreatment on enzymatic
activity.

FIGURE 4.6 shows the results obtained by using samples with different
pretreatment process. Rice straw without acid pretreatment shows almost negligible
sugar yield even after undergoing enzymatic hydrolysis for 96 hours while rice straw
with acid pretreatment shows a good yield from enzymatic hydrolysis. The sugar yield
shows only small increment from duration of 72 hours to 96 hours. Total sugar yield
from enzymatic hydrolysis could be seen from FIGURE 4.7. The highest sugar yield
is 0.382 g/g dry rice straw basis or equivalent to 38.2% g/g dry rice straw basis for
enzymatic duration of 72 hours. Sugar yield of sample without acid pretreatment is not
further studied.

TABLE 4.5 Results of enzymatic hydrolysis with 3 g solid substrate and 24 mg

enzyme loading
Average Sugar Sugar Sugar
Duration Absorbance yield yield yield
Sample (hour) (A) (g/L) (g/g) (% g/g)

Acid + 0 0 0.000 0.000 0.00

Mechanical 24 0.803 7.910 0.264 26.37

Pretreated 48 1.078 10.619 0.354 35.40

Rice Straw 72 1.164 11.466 0.382 38.22

96 1.158 11.407 0.380 38.02

Mechanical 0 0 0.000 0.000 0.00

Pretreated 72 0.010 0.099 0.003 0.33

Rice Straw 96 0.005 0.049 0.002 0.16

 48

1.4

1.2

0.8
Absorbance (A)

0.6 Mechanical
Acid + Mechanical
0.4

0.2

0
0 20 40 60 80 100 120
Duration (Hour)

FIGURE 4.6 Absorbance values with respect to duration of hydrolysis and type of
pretreatment applied

0.450

0.400

0.350
Sugar Yield (g/g dry rice straw basis)

0.300

0.250

0.200

0.150

0.100

0.050

0.000
0 20 40 60 80 100 120
Duration (Hours)

FIGURE 4.7 Sugar yield (g/g rice straw basis) from enzymatic hydrolysis with respect
to time
 49

4.6 Total Yield of Glucose from Acid Pretreatment and Enzymatic Hydrolysis

The total yield of highest yield of glucose from acid pretreatment is 0.324 g/g rice
straw basis while highest glucose yield of enzymatic hydrolysis is 0.382 g/g rice straw
basis. Therefore, the total yield of these two processes have successfully yielded 0.706
g/g rice straw basis (70.6% g/g rice straw basis) in this present study.

TABLE 4.6 Results of total glucose yield from acid pretreatment and enzymatic
hydrolysis

Glucose Total
Yield glucose
(g/g rice yield
Method Parameters straw basis) (g/g rice straw basis)
Acid 2.0 M H2SO4 at retention time
Pretreatment 60 minutes 0.324
Enzymatic 24 mg Cellulase enzyme at
hydrolysis retention time 72 hours 0.382 0.706
Combination of treatment methods

0.32 0.38

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8

g/g rice straw basis

FIGURE 4.8 Total glucose yield of acid pretreatment and enzymatic hydrolysis
 50

4.7 Liquid State Fermentation

The liquid state fermentation of solution from acid pretreatment and enzymatic
pretreatment was carried out separately to study ethanol yield from sugars present in
the solution.

4.7.1. Initial Analysis of Glucose Concentration and Yeast Growth using DNS Testing
Method

DNS testing method was used to study the growth of yeast and reduction of glucose in
the samples before further analysis using HPLC.

4.7.1.1 Liquid State Fermentation on Acid Pretreatment Solution

The glucose content in the solution from acid pretreatment dropped relatively slower
during fermentation compare to fermentation of sugar yield from enzymatic
hydrolysis. The comparison can be clearly seen from FIGURE 4.9 and FIGURE 4.11
where FIGURE 4.11 shows a much steeper graph plotting.

TABLE 4.7 Average of Glucose Conversion during Fermentation Process

Duration Glucose Glucose Cellulose
Sample (Hours) (g/L) (g/g) Conversion (%)
0 7.664 0.255 0.00
6 7.250 0.242 5.40
8 6.777 0.226 11.57
10 6.481 0.216 15.43
12 6.186 0.206 19.29

A+B 24 5.733 0.191 25.20

48 4.531 0.151 40.88
72 3.684 0.123 51.93
96 1.005 0.033 86.89
120 0.926 0.031 87.92
 51

GLucose Content (g/L) 7

0
0 20 40 60 80 100 120 140
Duration (Hours)

FIGURE 4.9 Glucose Content during Fermentation Process of Sugar Yield from Acid
Pretreatment

0.9

0.8

0.7

0.6
Absorbance (A)

0.5

0.4

0.3

0.2

0.1

0
0 20 40 60 80 100 120 140
Duration (Hours)

FIGURE 4.10 Growth of Saccharomyces Cerevisae during Fermentation of Sugar

Yield from Acid Pretreatment
 52

4.7.1.2 Liquid State Fermentation on Enzymatic Hydrolysis Solution

Initial glucose content of solution from enzymatic hydrolysis was 11.47 g/L or 1.147
g/g rice straw basis and the final glucose content of the solution after fermentation is
0.266 g/L or 0.009 g/g rice straw basis as shown in TABLE 4.8. Thus, approximately
97.68% of the glucose in the solution was managed to be converted into ethanol. The
glucose content is observed to be dropping at a slow rate at the beginning and dropped
drastically after 8 hours while Saccharomyces Cerevisae yeast is observed to grow at a
consistently slow rate and showed minimum growth after 24 hours.

14

12

10
Glucose Content (g/L)

0
0 10 20 30 40 50 60 70 80
Duration (Hours)

FIGURE 4.11 Glucose Content during Fermentation Process of Sugar Yield from
Enzymatic Hydrolysis
 53

TABLE 4.8 Results of Sugar Fermentation of Enzymatic Hydrolysis

Cellulose
Sample Duration (Hours) Glucose (g/L) Glucose (g/g) Conversion (%)
0 11.466 0.382 0
2 10.855 0.362 5.33
4 10.619 0.354 7.39
6 9.614 0.320 16.15
A 8 8.757 0.292 23.63
12 5.506 0.184 51.98
24 0.699 0.023 93.90
48 0.552 0.018 95.19
72 0.266 0.009 97.68

1.4

1.2

0.8
Absorbance (A)

0.6

0.4

0.2

0
0 10 20 30 40 50 60 70 80
Duration (Hours)

FIGURE 4.12 Growth of Saccharomyces Cerevisae during Fermentation of Sugar

Yield from Enzymatic Hydrolysis
 54

4.7.2 Analysis of Glucose and Ethanol Concentration using HPLC

Calibrated standard curve of glucose and ethanol for HPLC can be found in Appendix
D. The standard curve for glucose and ethanol obtained by known concentration is
shown in FIGURE 4.13 and FIGURE 4.14. Eq 4.2 is the equation used to determine
glucose concentration by percentage in the samples while Eq 4.3 is the equation used
to determine ethanol concentration by percentage in the samples.

y=2 E 106 X (4.2)

y=749494 X ( 4.3)

As observed from the standard calibration curves data which can be found in
APPENDIX D, reading of glucose and ethanol concentration will be shown at
intervals between 7 – 8 minute and 16 – 17 minute respectively. For acid pretreatment
fermentation, the glucose concentration is 0.02 % while the ethanol concentration is
0.013%. For enzymatic hydrolysis fermentation, the glucose concentration is 0.24%
while the ethanol concentration is 0.1503%. Through conversion based on the
standard curves, glucose concentration and ethanol concentration for fermented acid
pretreatment solution in g/L are 0.2 g/L and 1.3 g/L respectively while glucose
concentration and ethanol concentration for fermented enzymatic hydrolysis solution
in g/L are 2.4 g/L and 1.503 g/L respectively. By using Eq 3.3 and Eq 3.4, it is found
that ethanol yield of fermented acid pretreatment is only 11.26% of the theoretical
ethanol yield while for ethanol yield of fermented enzymatic hydrolysis is found to be
as high as 52.75%. Detailed results of HPLC analysis on standard curve of glucose
and ethanol, and fermentation analysis on acid pretreatment and enzymatic hydrolysis
substrate can be found at APPENDIX E while sample calculation could be found at
APPENDIX F.

TABLE 4.9 Results of ethanol yield

Theoretical ethanol
Initial glucose yield Experimental
Method (g/g rice straw basis) (g/g rice straw basis) ethanol yield
Acid
Pretreatment 0.324 0.162 11.26%
Enzymatic
Hydrolysis 0.380 0.190 52.75%
 55

2500000
f(x) = 2489952.53 x
R² = 0.98

2000000

1500000
Response (Peak Area)

1000000

500000

0
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
Concentration (%)

FIGURE 4.13 Calibrated Curve of Glucose for HPLC

800000

700000 f(x) = 749494.47 x

R² = 0.97
600000

500000
Response (Peak Area)

400000

300000

200000

100000

0
0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9 1 1.1
Concentration (%)

FIGURE 4.14 Calibrated Curve of Ethanol Concentration for HPLC

 56

FIGURE 4.15 Results of analysis of fermented solution from acid pretreatment after distillation
 57

FIGURE 4.16 Results of analysis of fermented solution from enzymatic hydrolysis after distillation
 CHAPTER V

DISCUSSIONS

5.1 Introduction

In this chapter, the results obtained from the experiment will be discussed and
compared with other resources of similar techniques used for bioethanol production
using rice straw as lignocellulosic feedstock. Sugar yield from acid pretreatment and
enzymatic hydrolysis will be discussed as well as ethanol yield from fermentation and
concentration of ethanol produced.

5.2 Calibration Curve of Absorbance

For the curve of absorbance to be accepted as accurate, the value of coefficient of

determination, R2 has to be exceeding 0.90 or 90%. The experiment procedures were
repeated for three times to obtain different sets of results. The detailed results obtained
for the experiment can be found in Appendix A. The highest value of R 2 obtained
from the experiment was 0.9847 or 98.47 %. Therefore, error of glucose concentration
determination using the linear equation obtained Eq 4.1; as shown in FIGURE 4.1,
would have very low error from the actual value. It is possible to obtain a higher value
of R2 by minimizing errors during experiment. The possible errors that could have
occurred in the experiment were low sensitivity weighing balance used, inaccurate
amount of distilled water added to dilute glucose, and inaccurate pipette used for
solution transferring.
 59

5.3 Effect of Acid Pretreatment on Sugar Yield

As shown in TABLE 4.4, the highest sugar yield was obtained by using 2.0 M of
H2SO4 with retention time of 60 minutes at 90℃. Further retention time using 2.0 M
of H2SO4 shows no significant increase of sugar yield. This may be due to the
limitation of acid pretreatment using conventional water bath at 90℃ where reducing
sugar yield has reached the maximum possible yield. The highest sugar yield obtained
was 0.323 g/g or 32.3 % g/g. A study carried out by Nutawan Yoswathana et al. by
treating rice straw with 1% or 0.1875 M of H2SO4 at 121℃ gave 21.45% g/g of
reducing sugar (Nutawan et al., 2010). The amount of sugar yield in present study is
higher than Nutawan Yoswathana et al. reported. This may be due to mechanical
pretreatment on rice straw. In the study, rice straw was blended into fine state while
Nutawan Yoswathana et. al. chopped the rice straw to about length ≈ 2 cm.

To further study the potential sugar yield from rice straw during acid
pretreatment, similar experimental procedures to Nutawan Yoswathana et al. were
carried out. Rice straw samples were pretreated with 1%, 3%, 5%, 7%, and 9% of
H2SO4 using autoclave machine at 121℃. The results obtained show that up to 0.506
g/g dry rice straw basis or equivalent to 50.6% g/g could be obtained by using 5% of
H2SO4. The results show different optimum concentration and higher sugar yield than
Nutawan Yoswathana et al. reported. This may again due to the result of mechanical
pretreatment used (blending compare to chopping to approximate 2 cm). The detailed
results and graph could be found in Appendix G.

Due to costing factor, conventional water bath is much preferable that

autoclave machine especially on large scale. According to the specifications of the
autoclave machine and water bath machine, a total of 1.5 kWh is required for
autoclave during pretreatment while only 1.2 kWh is required for water bath
pretreatment. Furthermore, a small scale autoclave would cost $12,500 while small
scale water bath costs about $4200 (Inc, 2014). In additional, a lower yield of
reducing sugars may not be considered as a concerning issue due the price of
lignocellulosic feedstock such as rice straw is relatively low (Taherzadeh and Karimi,
2007a).
 60

5.4 Effect of Enzymatic Hydrolysis on Sugars Yield

The pretreated rice straw from acid pretreatment was washed with running tap water
in order to study the sugar yield from enzymatic hydrolysis and acid pretreatment
separately as proposed by E. Yu. Vlasenko et. al. (Vlasenko et al., 1997).

Enzyme loading was not studied in this present paper as numerous similar
enzyme loading was used by other researchers which is approximately 0.8% w/w of
rice straw basis (Nutawan et al., 2010, Diep et al., 2012). The results of enzymatic
hydrolysis show no further increase in sugar yield after retention duration of 72 hours
with yield of 0.382 g/g rice straw basis. This shows the maximum possible yield of
glucose from cellulose by Cellulase enzyme on acid pretreated rice straw in this study.

As shown in FIGURE 4.6, sugar yield from non-acid pretreated rice straw
shows negligible yield with reaction time up to 96 hours. This shows that acid
pretreatment is essential in degrading lignin layers in order for enzyme to act on
cellulose and hemicellulose contain in the rice straw. Pretreatment is carried out to
increase the surface area and pore volume of solid phase of rice straw which will
greatly increase the glucose yield from enzymatic hydrolysis (Zhu et al., 2008).
Without acid pretreatment, cellulose and hemicellulose components are well protected
under lignin component, causing ineffective enzymatic hydrolysis process.

In order to stop the enzyme activity in enzymatic hydrolysis, the samples were
heated to high temperature (90℃) for a period of time. FIGURE 5.1 shows the
enzyme activity with respect to temperature. As temperature increases beyond
optimum temperature of the enzyme, the activity of the enzyme will start to degrade.
At high temperature, the structure of the enzyme will start to destabilise and even
collapse resulting in total denaturation (Corporation, 2014). Furthermore, samples
collected for analysis were stored at low temperature (4℃) to ensure immobilisation
of enzyme activity.
 61

FIGURE 5.1 Effect of temperature on enzyme reaction rate (Corporation, 2014)

5.5 Distillation of Ethanol from Fermented Sample

The results for ethanol distillation from fermented samples were inaccurate due to
faulty rotatory evaporator. Therefore, the collected solution from distillation was not
measured due to presence of other components in the solution.

5.6 Effect of Fermentation on Ethanol Yield

Contamination during fermentation would generally reduce the ethanol yield as sugars
are consumed by the bacteria. The major contaminants during ethanol fermentation
are Lactobacillus plantarum, L. paracasei and L. fermentum. These contaminants are
much commonly known as Lactic acid bacteria (LAB) (Katakura et al., 2011).
Therefore, to minimize the chances of bacterial contamination, all the work involved
in fermentation such as yeast inoculum and samples taking were conducted in a
bacteria free environment. The works were performed inside a laminar flow system as
shown in FIGURE 5.2.
 62

FIGURE 5.2 Performing Works Involved in Fermentation using Laminar Air Flow
System to Minimize Bacterial Contamination

For fermentation of acid pretreatment solution, the percentage yield of glucose

into ethanol was only 87.92%. This may be due to sugar inhibitions that formed
during acid pretreatment that limited the activity of Saccharomyces Cerevisiae during
fermentation process. It is further observed that the percentage of glucose successfully
converted into ethanol during fermentation of enzymatic is as high as 97.68%. It is
studied that enzymatic hydrolysis would not produce inhibitory that may affect the
performance of Saccharomyces Cerevisiae (Taherzadeh and Karimi, 2007b).

Analysis carried out by using HPLC showing low yield of ethanol in the
samples especially on the fermented acid pretreatment solution. This may be due to
improper precaution taken during the operation of HPLC. Furthermore, by studying
FIGURE 4.15, there is high possibility that the samples of acid pretreatment are
contaminated by bacterial; resulting in low ethanol concentration. Another possibility
is the formation of high salt concentration during neutralization and pH adjusting that
had been carried out prior to enzymatic hydrolysis and fermentation. High salt
concentration is also an inhibitor to yeast during fermentation (SUTTON, 2011).
Furthermore, fermented solution was not filtered before distillation; leaving much
impurities in the solution.
 CHAPTER VI

CONCLUSION AND RECOMMENDATION

6.2 Conclusion

Conversion of rice straw to bioethanol has been studied in this study. Rice straw
biomass was obtained from Sekinchan, Selangor during harvesting season on May
2013. Rice straw samples were treated with mechanical pretreatment prior to acid
pretreatment. Mechanical pretreatment has shown to be important in the study for
enzymatic hydrolysis as it allows enzyme to be much accessible during enzymatic
hydrolysis.

Mechanical pretreated rice straw was then treated using H2SO4 at 90℃. The
conditions for highest yield of glucose from acid pretreatment is found by using 2.0 M
of H2SO4 with retention time of 60 minutes. Highest glucose yield from acid
pretreatment on rice straw is found to be 0.324 g/g rice straw basis. Furthermore, acid
pretreatment has also shown to be an important treatment prior to enzymatic
hydrolysis. It is studied that glucose yield from rice straw samples during enzymatic
hydrolysis without acid pretreatment is almost negligible. Enzymatic hydrolysis was
then carried out by using enzyme purchased from Sigma. Cellulase from Tichoderma
Ressei ATCC 26921 was used in this study. Glucose yield from enzymatic hydrolysis
is found to be 0.382 g/g after retention time of 72 hours. Further retention duration
shows no increase in glucose yield. The total glucose yield from enzymatic hydrolysis
and acid pretreatment of rice straw is 0.706 g/g rice straw basis.

Fermentation was then carried out and glucose concentration is found to have
dropped from 11.47 g/L to 0.266 g/L. However, ethanol yield from fermented solution
of acid pretreatment and hydrolysis enzymatic is found to be 11.26% and 52.75%
compared to theoretical ethanol yield.
 64

In conclusion, the objectives of the study have been successfully studied.

Conversion of rice straw biomass into bioethanol is worthful to be researched as it
gives high glucose yield from the process and high potential ethanol yield. More
understanding on inhibitions produced during acid pretreatment and enzymatic
hydrolysis would need to be understood. It is believed that bioethanol would be able
to play a major contribution to the society especially in transportation sector.

6.1 Future Recommendation

For mechanical pretreatment, it is suggested that the rice straw samples are further
reduce in sizing for better accessible of enzyme. Only 3 g of rice straw loading had
been used throughout this study due to space limitation caused by high space
consumption of rice straw samples. Therefore, by further reducing the size of rice
straw, higher samples loading could be used to study the yield of potential ethanol
from rice straw.

Acid pretreatment that had been carried out in this study has shown a high
yield of reducing sugars by using low temperature with short retention time but the
fermentation of the acid pretreatment solution has shown an unpleasant results.
Therefore, it is recommended that further study would be carried out to study the
behaviour of such acid pretreatment method on rice straw.

For fermentation process, it is recommended that more precautions could be

practised to prevent contamination during the process. A stirrer tank reactor could be
used for fermentation as well. Furthermore, the fermented solution should be filtered
using filter paper before distillation to improve the results for HPLC analysis.

Time management is very significant in this study because of dependency of

equipment, materials and chemical from outsource facilities. Therefore, planning on
schedule, planning on type of equipment required, and chemicals required are
important to prevent faulty equipment during usage, running out of chemicals or
unavailable type of chemicals.
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APPENDICES
 APPENDIX A

RESULTS OF GLUCOSE CURVE OF ABSORBANCE CALIBRATION

TABLE A.1 Results of first trial glucose absorbance curve calibration

Glucose
concentration Distilled water DNS solution Absorbance
(g/L) Stock (ml) (ml) (ml) (A) R2
0 0 2 2 0
0.1 0.2 1.8 2 1.038
0.2 0.4 1.6 2 2.073
0.3 0.6 1.4 2 2.471
0.4 0.8 1.2 2 2.509
0.5 1 1 2 2.528 0.7124

TABLE A.2 Results of second trial glucose absorbance curve calibration

Glucose
concentration Distilled water DNS solution Absorbance
(g/L) Stock (ml) (ml) (ml) (A) R2
0 0 2 2 0
0.1 0.2 1.8 2 0.419
0.2 0.4 1.6 2 1.043
0.3 0.6 1.4 2 1.666
0.4 0.8 1.2 2 2.14
0.5 1 1 2 2.371 0.9847

TABLE A.3 Results of second trial glucose absorbance curve calibration

Glucose
concentration Distilled water DNS solution Absorbance
(g/L) Stock (ml) (ml) (ml) (A) R2
0 0 2 2 0
0.1 0.2 1.8 2 0.723
0.2 0.4 1.6 2 1.342
0.3 0.6 1.4 2 1.812
0.4 0.8 1.2 2 2.115
0.5 1 1 2 2.354 0.9368
 70

APPENDIX B

RESULTS OF ACID PRETREATMENT AND SAMPLE GLUCOSE

CONCENTRATION CALCULATION

Concentration: 1M
Dilution factor: 50

TABLE B.1 Results of acid pretreatment at 1.0 M H2SO4 for 30, 60, and 90 minutes
Absorbance Value (A) at 540 nm
Sample Retention time (min) DNS test 1 DNS test 2 Average A
A 30 0.129 0.141 0.160
B 30 0.171 0.2
C 60 0.425 0.473 0.366
D 60 0.281 0.293
E 90 0.354 0.379 0.362
F 90 0.343 0.392

Concentration: 1.5M
Dilution factor: 50

TABLE B.2 Results of acid pretreatment at 1.5 M H2SO4 for 30, 60, and 90 minutes
Retention Absorbance Value (A) at 540 nm
time DNS DNS test DNS test DNS test
Sample (min) test 1 2 3 4 Average A
A 30 0.265 0.273 0.268 0.333 0.319
B 30 0.32 0.322 0.322 0.365
C 60 0.312 0.412 0.435 0.4 0.409
D 60 0.445 0.403 0.614 0.38
E 90 0.444 0.518 0.444 0.556 0.489
F 90 0.436 0.471 0.569 0.529
 71

Concentration: 2 M
Dilution factor: 50

TABLE B.3 Results of acid pretreatment at 2.0 M H2SO4 for 30, 60, and 90 minutes
Retention time Absorbance (A) at 540 nm
Sample (min) DNS test 1 DNS test 2 DNS test 3 Average A
A 30 0.521 0.367 0.434 0.424
B 30 0.46 0.475 0.478
C 60 0.638 0.49 0.442 0.493
D 60 0.626 0.541 0.532
E 90 0.6 0.489 0.494 0.470
F 90 0.732 0.803 0.489

TABLE B.4 Results of glucose yield from different molarity of H2SO4

Retentio
n Glucos
time Averag e Glucose Yield Glucose
Concentratio (minutes e Yield (g/g rice straw Yield
n ) A (g/L) basis) (%)
30 0.160 3.152 0.1051 10.51
60 0.366 7.210 0.2403 24.03
1.0 M 90 0.362 7.132 0.2377 23.77
30 0.319 6.284 0.2095 20.95
60 0.409 8.058 0.2686 26.86
1.5 M 90 0.489 9.634 0.3211 32.11
30 0.424 8.353 0.2784 27.84
60 0.493 9.712 0.3237 32.37
2.0 M 90 0.470 9.259 0.3086 30.86

Sample calculation of glucose yield in g/g rice straw basis for 1.0 M at 30 minutes
retention time with 3 g loading substrate is as shown below.

0.160=5.076∗Glucose Concentration( g L−1 )

Glucose concentration( g L−1 )=0.03152∗100 ( Dilution factor )=3.152 g/ L

Glucose yield ( g g−1 rice straw basis ) =3.152 g L−1 ÷30 g samples L−1=0.1051 g g−1 rice straw basis
APPENDIX C

SAMPLE GLUCOSE CONCENTRATION CALCULATION FOR

ENZYMATIC HYDROLYSIS PROCESS

Sample calculation of glucose yield in g/g rice straw basis for retention time of 24
hours with 3 g loading substrate is as shown below. The loading of enzyme was 24
mg.

0.803=5.076∗Glucose Concentration(g L−1 )

Glucose concentration( g L−1 )=0.0158∗50 ( Dilution factor )=7.910 g /L

Glucose yield ( g g−1 rice straw basis ) =7.910 g L−1 ÷ 30 g samples L−1=0.264 g g−1 rice straw basis
 APPENDIX D

HPLC ANALYSIS RESULTS ON CALIBRATION ON GLUCOSE AND

ETHANOL

File name : aluyah754.CH2

Info : sample: glu+eth 0.25%
flow rate: 1.ml/min
Mobile phase: 0.1% H2Po4

Vial # = 1 Rack # = 1
Injection Date :15-Jan-2014 12:11:28
Curr. Date : 21-Jan-2014 14:03:56
User : DEFAULT
Group : DATA
Control Method :

TABLE D.1 Results of calibration for ethanol and glucose at 0.25%

Area[RIU.Sec
# Name RT Quantity
]
1 0.108 115.916 0
2 0.417 1461.602 0
3 1.083 67.688 0
4 1.2 49.271 0
5 1.333 67.437 0
6 1.5 42.25 0
7 1.642 41.561 0
8 1.742 41.549 0
9 2.075 165.89 0
10 2.392 61.695 0
11 2.492 76.657 0
12 2.767 189.502 0
13 2.883 133.62 0
14 3.05 274.782 0
15 3.425 101.255 0
16 3.733 174.129 0
17 3.975 115.864 0
18 4.067 149.383 0
19 4.492 186.314 0
20 4.65 224.255 0
 74

21 5.067 521.839 0
22 5.192 303.613 0
23 5.383 185.639 0
24 5.842 297.531 0
25 6.367 71.158 0
26 6.825 1239.333 0
27 7.767 291574.249 0
28 9.625 1982.087 0
29 9.792 1345.85 0
30 9.975 1282.464 0
31 10.108 2170.96 0
32 10.433 3926.614 0
33 11.133 776.415 0
34 11.333 618.894 0
35 11.417 550.269 0
36 11.85 378.655 0
37 12.017 522.476 0
38 12.133 262.38 0
39 12.417 430.851 0
40 12.6 244.223 0
41 12.717 427.919 0
42 13.025 365.041 0
43 13.192 163.029 0
44 13.35 207.143 0
45 13.517 190.722 0
46 13.658 165.631 0
47 13.875 245.419 0
48 14.008 126.322 0
49 14.2 218.04 0
50 14.333 190.663 0
51 14.5 246.512 0
52 16.183 72947.442 0
53 17.817 581.286 0
54 18.175 363.734 0
55 18.433 772.837 0
56 18.617 486.798 0
57 18.992 248.33 0
58 19.083 371.559 0
59 19.775 77.348 0
60 19.975 17.529 0

Total Area of Peak = 390839.424 [RIU.Sec]

 75

FIGURE D.1 Results of 0.25% of glucose and ethanol on HPLC analysis

File name : aluyah753.CH2

Info :
 76

sample: glu+eth 0.50%

flow rate: 1.ml/min
Mobile phase: 0.1% H2Po4
Vial # = 1 Rack # = 1
Injection Date :15-Jan-2014 11:49:24
Curr. Date : 21-Jan-2014 14:03:20
User : DEFAULT
Group : DATA
Control Method :

TABLE D.2 Results of calibration for ethanol and glucose at 0.50%

Area[RIU.Sec
# Name RT
] Quantity
1   0.475 1986.25 0
2   1.167 170.231 0
3   2.658 722.305 0
4   2.808 251.994 0
5   3.325 619.548 0
6   3.567 567.054 0
7   3.992 921.548 0
8   4.292 829.153 0
9   4.558 344.41 0
1
5.167 1170.254 0
0  
1
5.483 765.893 0
1  
1
5.792 955.582 0
2  
1
6.008 354.822 0
3  
1
6.825 55093.571 0
4  
1
7.775 1266562.417 0
5  
1
10.192 34708.071 0
6  
1
11.525 19030.576 0
7  
1
11.95 32710.539 0
8  
1
12.35 24690.596 0
9  
2
12.667 16263.046 0
0  
 77

2
12.825 43342.327 0
1  
2
13.033 39048.822 0
2  
2
13.258 20372.566 0
3  
2
13.35 22631.12 0
4  
2
13.525 16984.349 0
5  
2
13.658 29356.542 0
6  
2
13.825 18649.488 0
7  
2
14.05 41995.041 0
8  
2
14.225 23082.567 0
9  
3
14.408 16885.57 0
0  
3
14.5 11053.628 0
1  
3
14.967 11115.237 0
2  
3
16.192 423942.596 0
3  
3
18.675 9790.27 0
4  
3
18.95 3353.271 0
5  
3
6   19.483 1763.639 0

Total Area of Peak = 2192084.893 [RIU.Sec]

 78

FIGURE D.2 Results of 0.50% of glucose and ethanol on HPLC analysis

File name : aluyah756.CH2

Info :
sample: glu+eth 0.75%
flow rate: 1.ml/min
Mobile phase: 0.1% H2Po4
Vial # = 1 Rack # = 1
Injection Date :15-Jan-2014 13:12:34
Curr. Date : 21-Jan-2014 14:04:46
User : DEFAULT
Group : DATA
 79

Control Method :

TABLE D.3 Results of calibration for ethanol and glucose at 0.75%

Area[RIU.Se
# Name RT Quantit
c]
y
1   0.508 1975.75 0
2   1.042 18.5 0
3   1.183 113.25 0
4   1.675 10.429 0
5   1.808 22.684 0
6   1.992 50.48 0
7   2.125 99 0
8   2.608 34.764 0
9   2.967 19.84 0
10   3.267 30.435 0
11   3.558 78.984 0
12   3.917 29.372 0
13   4.65 15.5 0
14   4.9 272.429 0
15   5.125 270.265 0
16   5.408 136.102 0
17   5.967 196.712 0
18   6.2 427.564 0
19   6.717 84121 0
20   7.833 2197415 0
21   13.625 28504.2 0
22   13.817 39933.5 0
23   14.333 26795.6 0
24   16.267 660851 0
25   19.792 520.788 0
Total Area of Peak = 3041942.885 [RIU.Sec]
 80

FIGURE D.3 Results of 0.75% of glucose and ethanol on HPLC analysis

Info :
 81

sample: glu+eth 1.0%

flow rate: 1.ml/min
Mobile phase: 0.1% H2Po4
Vial # = 1 Rack # = 1
Injection Date :15-Jan-2014 12:37:38
Curr. Date : 21-Jan-2014 14:04:24
User : DEFAULT
Group : DATA
Control Method :

TABLE D.4 Results of calibration for ethanol and glucose at 1.0%

Area[RIU.Sec
# Name RT
] Quantity
1   0.467 2375.05 0
2   2.158 19.02 0
3   2.475 58.526 0
4   2.633 50.178 0
5   2.958 46.682 0
6   3.933 191.607 0
7   4.4 100.272 0
8   4.492 52.656 0
9   4.633 34.75 0
10   5.233 109.5 0
11   5.458 55 0
12   6.683 86476.7 0
13   7.858 2314425 0
14   13.75 15300.2 0
15   14.517 26379.9 0
16   14.725 20026.9 0
17   14.9 46862 0
18   16.258 679456 0

Total Area of Peak = 3192019.894 [RIU.Sec]

FIGURE D.4 Results of 1.0% of glucose and ethanol on HPLC analysis

82
 APPENDIX E

HPLC ANALYSIS RESULTS ON FERMENTED SAMPLES OF ACID

PRETREATMENT AND ENZYMATIC HYDROLYSIS

File name : Ps Ten808.CH2

Info :
sample: APF distilled 5 days
flow rate: 1.ml/min
Mobile phase: 0.1% H2PO4
Vial # = 1 Rack # = 1
Injection Date :21-Jan-2014 13:44:42
Curr. Date : 21-Jan-2014 14:10:18
User : DEFAULT
Group : DATA
Control Method :

TABLE E.1 Results for fermented samples of acid pretreatment

Area[RIU.Sec
# Name RT
] Quantity
1   2.283 38.333 0
2   2.992 50.144 0
3   3.125 126.8 0
4   3.3 103.422 0
5   3.5 101.202 0
6   3.608 222.405 0
7   3.883 179.643 0
8   4.308 48.912 0
9   4.542 49.838 0
10   4.7 45.532 0
11   4.842 28.125 0
12   4.983 34.277 0
13   5.125 35.555 0
14   5.267 4.614 0
15   5.617 78.055 0
16   5.825 5.616 0
17   5.925 0 0
 84

18   6.017 8.036 0
19   7.692 11276.929 0
20   7.85 2741.34 0
21   8.008 2634.585 0
22   8.458 4212.771 0
23   8.7 1644.039 0
24   8.817 1572.041 0
25   8.983 1911.882 0
26   9.308 4074.953 0
27   9.392 1764.381 0
28   9.592 3798.12 0
29   9.842 760.032 0
30   9.992 1493.206 0
31   10.133 1997.195 0
32   10.342 2214.36 0
33   10.458 839.821 0
34   10.567 1347.234 0
35   10.733 2946.854 0
36   11.15 1688.417 0
37   11.45 2867.091 0
38   11.933 1025.239 0
39   12.05 1045.138 0
40   12.2 2314.153 0
41   13.033 54.332 0
42   13.217 98.862 0
43   13.475 68.038 0
44   13.625 114.365 0
45   13.808 249.09 0
46   13.975 218.734 0
47   14.183 451.997 0
48   14.575 202.301 0
49   14.717 704.408 0
50   14.867 999.669 0
51   15.175 696.702 0
52   15.292 542.161 0
53   15.483 726.394 0
54   16.283 10255.255 0
55   17.05 72.061 0
56   17.158 65.061 0
57   17.292 20.151 0
58   17.408 8.322 0
59   17.6 9.423 0
60   17.775 5.618 0
 85

61   17.958 49.944 0
62   18.183 130.101 0
63   18.575 107.417 0
64   18.892 318.687 0
65   19.208 136.809 0
66   19.517 311.452 0
67   19.65 250.231 0
68   19.858 117.214 0

Total Area of Peak = 74315.095 [RIU.Sec]

FIGURE E.1 Results for analysis of acid pretreatment fermented samples

File name : PS TEN787.CH2

 86

Info :
sample: EHF 48hrs
flow rate: 1.ml/min
Mobile phase: 0.1% H2Po4
Vial # = 1 Rack # = 1
Injection Date :16-Jan-2014 16:36:10
Curr. Date : 21-Jan-2014 14:43:48
User : DEFAULT
Group : DATA
Control Method :

TABLE E.2 Results for fermented samples of hydrolysis enzymatic

Area[RIU.Sec
# Name RT Quantity
]
1   0.367 1476.797 0
2   1.15 74 0
3   1.45 63.25 0
4   1.717 190.75 0
5   2.25 186.133 0
6   2.567 231.3 0
7   2.717 65.7 0
8   2.933 128.5 0
9   3.192 36.711 0
10   3.425 42.911 0
11   3.75 218.567 0
12   4.017 281.133 0
13   4.242 249.47 0
14   5.817 82521.989 0
15   6.2 84743.627 0
16   7.983 474885.968 0
17   8.308 444948.986 0
18   9.083 725597.14 0
19   13.308 157991.445 0
20   15.192 2615.855 0
21   15.325 3634.431 0
22   16.733 112671.554 0

Total Area of Peak = 2092856.217 [RIU.Sec]

 87

FIGURE E.2 Results for analysis of hydrolysis enzymatic fermented samples

 APPENDIX F

SAMPLE ETHANOL CONCENTRATION CALCULATION

By using Eq 3.3 and highest initial results from acid pretreatment on glucose yield,

Theoretical ethanol ( g g−1) =Initial glucose measured ( g g−1 ) × 0.5

Theoretical ethanol ( g g−1) =0.324 g g−1∗0.5=0.162 g g−1

The ethanol concentration measured is as followed. To simplify the calculation, it is

taken that 1% of ethanol is equivalent to 1 g in 100 ml of solution.

Ethanol concentration ( % )=10255.255 ÷ 749494=0.013 %

Ethanol concentration ( % )=0.1343 %∗4 ( Dilution Factor ) =0.055 %

0.055 g
Experimental ethanol concentration ( g g−1 )= =0.018 g g−1
3 g loading

Therefore, ethanol yield in percentage is as followed,

0.018 g g−1
Ethanol yield ( % )= ∗100 %=11.26 %
0.162 ¿−1

By using Eq 3.3 and highest initial results from enzymatic hydrolysis on glucose
yield,
−1 −1
Theoretical ethanol ( g g ) =Initial glucose measured ( g g ) × 0.5

Theoretical ethanol ( g g−1) =0.38 g g−1∗0.5=0.19 g g−1

The ethanol concentration measured is as followed. To simplify the calculation, it is

taken that 1% of ethanol is equivalent to 1 g in 100 ml of solution.

Ethanol concentration ( % )=112671.554 ÷ 749494=0.1503%

Ethanol concentration ( % )=0.1343 %∗2 ( Dilution Factor ) =0.301 %

0.301 g
Experimental ethanol concentration ( g g−1 )= =0.1002 g g−1
3 g loading

Therefore, ethanol yield in percentage is as followed,

0.1002 g g−1
Ethanol yield ( % )=
0.19 0≫¿−1∗100 %=52.75 % ¿
 APPENDIX G

RESULTS OF ACID PRETREATMENT AT 121℃ FOR 15 MINUTES

RETENTION TIME

TABLE G.1 Results of acid pretreatment using autoclave equipment

Absorbance Value @ 540 nm Average Glucose Glucose
Concentratio Absorbance Yield Yield
n DNS Test 1 DNS Test 2 (A) (g/L) (%)
1% 0.596 0.483 0.540 10.628 35.43
3% 0.649 0.583 0.616 12.136 40.45
5% 0.780 0.761 0.771 15.179 50.60
7% 0.588 0.564 0.576 11.348 37.83
9% 0.538 0.545 0.542 10.668 35.56
10.67% (2 M) 0.494 0.456 0.475 9.358 31.19

16
14
12
Glucose Yield (g/L)

10
8
6
4
2
0
0% 2% 4% 6% 8% 10% 12%
Acid Concentration

Figure G.1 Plotted results of acid pretreatment using autoclave

 90

APPENDIX H

PROPERTIES OF ETHANOL AT DIFFERENT PRESSURE

FIGURE H.1 Properties of ethanol at different pressure (Desormeaux, 2006)

 APPENDIX I

PRETREATMENT OF RICE STRAW SAMPLE

FIGURE I.1 Before and after mechanical pretreatment on rice straw sample

FIGURE I.2 Neutralised solution of acid pretreatment

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FIGURE I.3 Filtered solution of acid pretreatment using filter paper

FIGURE I.4 Dried acid pretreated rice straw

 APPENDIX J

ENZYMATIC HYDROLYSIS OF ACID PRETREATED RICE STRAW

SAMPLE

FIGURE J.1 Enzyme used for the study

FIGURE J.2 Prepared enzymatic solution for hydrolysis

 APPENDIX K

FERMENTATION OF SOLUTION OBTAINED FROM ACID

PRETREATMENT AND ENZYMATIC HYDROLYSIS

FIGURE K.1 Yeast media measurement

FIGURE K.1 Addition of yeast media into the fermented solution

 95

FIGURE K.2 Adjusting of pH value of prepared solution for fermentation

FIGURE K.3 Fermentation of samples

 APPENDIX L

DISTILLATION AND HPLC ANALYSIS

FIGURE L.1 Distilled fermented solution

FIGURE L.2 Analysis using HPLC for glucose and ethanol content