UTILIZATION OF SWEET SORGHUM: PRODUCTION

OF BIO-ETHANOL FROM NON-EDIBLE PART AND

EVALUATION OF THE JUICE CRYSTALLIZATION
POTENTIAL

BONFACE GODWIN MUKABANE

MASTER OF SCIENCE
(Energy Technology)

JOMO KENYATTA UNIVERSITY OF AGRICULTURE

AND TECHNOLOGY
2015
Utilization of sweet sorghum: production of bio-ethanol from non-
edible part and evaluation of the juice crystallization potential

Bonface Godwin Mukabane

A thesis submitted in partial fulfillment for the degree of Master of

Science in Energy Technology in the Jomo Kenyatta University of
Agriculture and Technology

2015

ii
 DECLARATION
This thesis is my original work and has not been presented for a degree in
any other university.

Signature: .. Date..

Bonface Godwin Mukabane

This thesis has been submitted for examination with our approval as
University supervisors

Signature: .. Date..

Dr. Willis O. Owino

JKUAT, Kenya

Signature: ... Date

Prof. George Thiongo

JKUAT, Kenya

Signature: Date

Dr. Benson B. Gathitu

TU-K, Kenya

iii
 DEDICATION
To my lovely wife Deborah, beautiful daughter Immaculate and wonder boy Victor.
They make my life delightful.

iv
 ACKNOWLEDGEMENT
My sincere thanks to the Almighty God for making this work a success. With heartfelt
gratitude, I wish to acknowledge my supervisors: Dr. Willis O. Owino and Prof. George
Thiongo, both of Jomo Kenyatta University of Agriculture and Technology and Dr.
Benson B. Gathitu from Technical University of Kenya, for critiquing this works for
technical content and providing guidance, suggestions and encouragement throughout
the period of my research.

I acknowledge the financial support for this research work from Jomo Kenyatta
University Innovation Fund for Technology Adoption for Industrial Exploitation of
Sweet Sorghum awarded to Dr. W. Owino. I also appreciate all the support provided by
the technical staff in the Department of Food Science and Technology, Faculty of
Agriculture (JKUAT), more so, Mr. Paul Karanja, David Abuga, Jessica Oruka and
David Vodha.

I am forever indebted to my dear wife, Deborah and children, Obuyanzi and Omulembe,
for their support and understanding for the many hours I spend in my study room away
from them.

Finally, I wish to thank my friends and colleagues who worked with me in the Food
Biochemistry Laboratory for moral support and assistance.

v
 TABLE OF CONTENTS
DECLARATION ............................................................................................................ iii

DEDICATION .................................................................................................................iv

ACKNOWLEDGEMENT ............................................................................................... v

LIST OF TABLES ..........................................................................................................ix

LIST OF FIGURES ......................................................................................................... x

LIST OF APPENDICES .............................................................................................. xii

LIST OF ACRONYMS AND ABBREVIATIONS ................................................... xiii

ABSTRACT .................................................................................................................... xv

CHAPTER ONE .............................................................................................................. 1

1.0 INTRODUCTION .................................................................................................. 1

1.1 Background .............................................................................................................. 1

1.2 The Problem Statement ............................................................................................ 3

1.3 Justification .............................................................................................................. 4

1.4 Research Question .................................................................................................... 5

1.5 Hypotheses ............................................................................................................... 5

1.6 Objectives ................................................................................................................. 5

1.6.1General Objective.5

1.6.2Specific Objective.5

CHAPTER TWO ............................................................................................................. 6

2.0 LITERATURE REVIEW ...................................................................................... 6

2.1 Introduction .............................................................................................................. 6

2.2 Second-Generation Bio-fuels ................................................................................... 7

2.2.1Introduction...7

vi
 2.2.2 Lignocellulosic feedstocks..9

2.3 Sweet sorghum ....................................................................................................... 13

2.4 Crystallization ........................................................................................................ 16

2.4.1Principlesofcrystallization...17

2.4.2Techniques of crystallization..19

2.5 Biofuel Production Technologies ........................................................................... 20

2.5.1.Introduction......20

2.5.3 Enzymatichydrolysis....25

2.5.4 Fermentation process for ethanol production.......26

2.5.5 Enzymatic hydrolysis and fermentation strategies for production of

bioethanol from lignocellulose biomass....27

2.5.6 Ethanol Purification ............................................................................................. 36

2.5.6.2 Ethanol Dehydration...37

CHAPTER THREE ....................................................................................................... 39

3.0 MATERIALS AND METHODS ......................................................................... 39

3.1 Sweet Sorghum varieties ........................................................................................ 39

3.2 Plant materials, experimental design and juice extraction ..................................... 39

3.3 Analytical Procedures............................................................................................. 40

3.3.1Analysis of juice and syrup...40

3.4 Clarification and treatment of Rio juice ................................................................. 40

3.5 Concentration of RIO juice .................................................................................... 41

3.6 Crystallization of RIO juice ................................................................................... 41

3.7 Production of bio-ethanol from RIO Sweet sorghum bagasse ............................... 41

3.7.1Size reduction of RIObagasse41

vii
 3.7.2Determination of Composition of RIO bagasse......41

3.7.3Riobagasse pretreatments...43

3.7.4Enzymatic hydrolysis.43

3.7.5 Fermentation......44

CHAPTER FOUR .......................................................................................................... 45

4.0 RESULTS AND DISCUSSION ........................................................................... 46

4.1 The total sugar content, glucose, fructose and sucrose concentration .................... 46

4.2 Sucrose /Apparent purity (AP) ............................................................................... 47

4.3 Crystallization process for RIO juice ..................................................................... 48

4.3.1 Extracted Rio juice.....48

4.3.2Rio syrup......49

4.3.3Rio syrup crystallization......51

4.4 Production of bioethanol from Rio sweet sorghum bagasse .................................. 52

4.4.1Composition of Rio SSB..52

4.4.2 Pretreatment....53

4.4.3 Enzymatic hydrolysis (saccharification) .. .....54

4.4.4 Fermentation...55

CHAPTER FIVE ............................................................................................................ 57

5.1 Conclusion .............................................................................................................. 58

5.2 Recommendations .................................................................................................. 58

6. REFERENCES ........................................................................................................... 59

7. APPENDICES ............................................................................................................ 70

viii
 LIST OF TABLES
Table 4.1: Total sugar content in 0Brix, glucose,fructose and sucrose in g/l.....47

Table 4.2: Apparent/Sucrose purity in percentage...48

Table 4.3: Characteristics of extracted Rio juice from the sugarcane presser.49

Table 4.4: Characteristics of Rio syrup from the evaporator......49

Table 4.5: The composition of Rio sweet sorghum bagasse (weight percent of dry
matter)...53
Table 4.6: The carbohydrate content of pretreated Rio sweet sorghum bagasse in
different conditions...54

Table 4.7: Yield of enzymatic hydrolysis of untreated and different pretreated sweet
sorghum bagasse (as percentage of theoretical yield)..55

ix
 LIST OF FIGURES
Figure 2.1: Block flow diagram for conversion of sweet sorghum juice to
sugar.....16

Figure 2.2: Equilibrium solubility, supersaturation, and metastable limit. An

undersaturated solution can be induced to crystallize by solvent evaporation(path a),
temperature change (path b) or both....18

Figure 2.3: Structures of (A) lignocelluloses, (B) celluloses, and (C)

hemicelluloses..22

Figure 2.4: A pretreatment process for lignocellulosic materials...23

Figure 2.5: Enzymatic hydrolysis of cellulose to glucose.............26

Figure 2.6: Simplified process flow diagram for separate enzymatic hydrolysis and
fermentation..28
Figure 2.7: Simplified process flow diagram for simultaneous saccharification and
fermentation......31
Figure 2.8: Simplified process flow diagram for nonisothermal simultaneous
saccharification and fermentation process (NSSF)......33
Figure 2.9: Simplified process flow diagram for simultaneous saccharification and co-
fermentation (SSCF).....35
Figure 4.1: Specific sugar concentrations in Rio juice after extraction prior to
clarification..................................................................................................................50

Figure 4.2: Specific sugars present in RIO syrup before crystallization.....50

Figure 4.3: Apparent purity of clarified Rio juice and Rio syrup before
crystallization.......................................................................................................51

Figure 4.4: Effect of different pretreatments on hydrolysis of cellulose.....55

Figure 4.5: Maximum ethanol yield (% theoretical yield)..57

x
Figure 4.6:Maximum ethanol production (g/L.h)......57

xi
 LIST OF APPENDICES
Appendix 1: Fructose standard curve......70
Appendix 2: Glucose standard curve...70

Appendix 3: Sucrose standard curve...71

Appendix 4: Three months old Sweet Sorghum in the JKUAT experimental farm..71

Appendix 5: Juice extraction using Sugarcane presser...72

Appendix 6: Sample injection into the HPLC instrument for sugar analysis................72

Appendix 7: Concentration of clarified juice using a rotary vacuum evaporator..73

Appendix 8: Enzymatic hydrolysis of pretreated Rio bagasse....73

Appendix 9: Ethanol analysis using GC equipment....74

Appendix 10: The ANOVA results of sucrose, glucose, fructose, Apparent Purity and
0
Brix analysis75

xii
 LIST OF ACRONYMS AND ABBREVIATIONS
ANOVA -Analysis of Variance

ADP -Adenosine diphosphate

AP - Apparent Purity

ATP - Adenosine triphosphate

BR & Di -Biomass Research & Development Initiative

CBP -Consolidated Bioprocessing

CIS - Commonwealth of Independent States

CO2 - Carbon dioxide

DoE - Department of Energy

EIA - Energy Information Administration

EJ - Exajoule

FAO - Food and Agriculture Organization

FAPRI - Food and Agricultural Policy Research Institute

FPU - Filter Paper Units

GJ -Giga-joules

HPLC - High Performance Liquid Chromatography

H3PO4 -Phosphoric Acid

H2SO4 -Sulphuric Acid

ICRISAT - International Crops Research Institute for the Semi- Arid Tropics

xiii
IEA - International Energy Agency

JKUAT - Jomo Kenyatta University of Agriculture and Technology

LHV - Low Heating Value

MgSO47H2O - hydrated magnesium sulphate

Mtoe - Million tons of oil equivalents

NaOH - Sodium Hydroxide

(NH)2HPO4 - Ammonium hydrogen phosphate

NRC - National Research Council

NREL - National Renewable Energy Laboratory

NSSF -Nonisothermal Simultaneous Saccharification and Fermentation

OECD -Organization for Economic Cooperation and Development

REN21 - Renewable Energy Policy Network for the 21st Century

SHF - Separate Enzymatic Hydrolysis and Fermentation

SSCF - Simultaneous Saccharification and Co-fermentation

SSF - Simultaneous Saccharification and Fermentation

xiv
 ABSTRACT
The availability of cheaper sources of energy is a key driver of any economic
development, more so for a developing country like Kenya. The vision of the Kenya
Government Biofuel Policy is to increase access to energy through sustainable biofuel
production, and reduce dependence on fossil fuels by 25% in volume by the year 2030.
The objective of this research was to characterize sweet sorghum cultivars which could
be used to produce crystal sugar from its juice and bio-ethanol from the bagasse. The
bio-ethanol could then be blended with gasoline and used in the transport sector. This
blending will serve to reduce the import bill of Kenyas fossil fuel while at the same
time mitigating the environmental change. Furthermore, since the juice can be
crystallized to produce crystal sugar, this will meet Kenyas annual sugar deficit of
200,000 tons. Sixteen sweet sorghum varieties namely: Madhura, Dale, Wiley,
Brandes, Theis, Rema, Ramanda, Rio, CMSXS636, CMSXS633, CMSXS644,
SPV1411, IESV91018LT, IESV92008DL, IESV92038/2SH, IESV93042SH, were
planted at the JKUAT experimental farm. After 16 weeks, 4 stalks of each variety were
cut, seeds, pinnacle and leaves removed. The juice was expressed from the stalks using a
sugarcane presser. The Brix was measured for each variety and glucose, fructose and
sucrose content were analyzed using HPLC. Rio cultivar with the highest apparent purity
(AP) of 83.91 was then chosen for crystal sugar and bio-ethanol production. Rio variety
was then harvested and juice extracted from the stalks, treated, concentrated and then
crystallized by cooling. The Rio sweet sorghum bagasse was comminuted after drying in
the green house and subjected to alkaline peroxide and phosphoric acid pretreatments.
This was then subjected to enzymatic hydrolysis using cellulase produced by
Trichoderma reesei, after which the hydrolysate was fermented using the bakers
yeast.The 0Brix for the juices ranged from 15.1 21.5, Sucrose content, 6.05 - 72.77
g/L, Glucose content, 2.65 - 16.42 g/L, Fructose content, 2.66 12.53 g/L and AP, 33.89
83.91%. The Rio syrup did not crystallize into raw crystal sugar. The yield for
enzymatic hydrolysis was 50%, 78% and 88% for the untreated bagasse, H3PO4 acid
pretreated and alkaline peroxide pretreated bagasse, respectively. The bioethanol yield

xv
was approximately 60%, 40% and 15%, respectively, for acid pretreated, alkaline
pretreated and untreated bagasse. Due to the lower purity(ratio of the %wt. of sucrose to
the %wt. of soluble) of the sugar extracted from sweet sorghum (about 75 AP) compared
to that of sugar cane or sugar beet (80-85 AP), it may require further technological input
to produce white sugar from sweet sorghum. Thus, the more likely markets for sorghum
sugar can be syrup for local foodstuffs or as raw material for the food industry.
According to the study, the following sweet sorghum cultivars namely; Rio,
CMSXS636, IESV91018LT, IESV93042SH and SPV1411 could have the potential to
be used in raw sugar production. Bio-ethanol can also be obtained from the Sweet
Sorghum bagasse after pretreatment and saccharification. This work therefore provides a
complimentary source of sugary products and bio-fuel.

xvi
 CHAPTER ONE

1.0 INTRODUCTION

1.1 Background
Sweet sorghum [Sorghum bicolor (L) Moench] has been considered as a particularly
important energy plant, due to its high-yields, drought tolerance, relatively low input
requirements and ability to grow under a wide range of environmental conditions
(Gnansounou et al., 2008). There are numerous published reports on intensive
research efforts progress in various countries such as the USA, China, India, Indonesia,
Iran, Philippines and Kenya in assessing the agro-industrial potential of sweet sorghums
(Rnolaet al., 2007; Reddy...et al., 2008; Tsuchihashi & Goto, 2008; Bennett & Anex,
2009; Pillay & Da Silva, 2009; Zhang et al., 2010; Olwenyet al., 2013; Owino et
al., 2013). More than 125 sweet sorghum germplasm resources have been registered in
China (Lu, 1997). The Sweet sorghum (SS) crop offers great potential as a food and an
industrial crop. It is a multifunctional crop that can be cultivated for simultaneous
production of grain for food and utilization of juice from stalk in production of value-
added products like syrup and ethanol. The leaves and stalks can be used for fodder, and
bagasse for animal feed or fiber production (Gnansounouet al., 2005).

Increased interest in bio-fuels is mainly driven by (1) the rising oil prices and
recognition of the fact that the global fossil fuel reserves are getting exhausted, (2) the
requirements of the Kyoto Protocol on carbon emissions, and (3) the provision of
alternative outlets for agricultural producers.

Global bio-fuel production has been increasing rapidly over the last decade, but the
expanding bio-fuel industry has recently raised pertinent concerns. In particular, the
sustainability of many first-generation bio-fuels, fuels made from edible sugars and
starch, has been increasingly questioned over concerns such as reported displacement of
food crops, effects on the environment and climate change. In general, there is growing
consensus that if significant emissions reductions in the transport sector are to be
achieved, bio-fuel technologies must become more efficient in terms of net lifecycle
greenhouse gas emission reduction while at the same time be socially and
environmentally sustainable. It is increasingly understood that most first-generation bio-
fuels, with the exception of sugarcane ethanol, will likely have a limited role in the
future transport fuel mix (IEA, 2008).

Cellulosic biomass- plant stalks, trunks, stems and leaves contain cellulose (44%),
hemicellulose (30%) and lignin (26%) (US Department of Energy [DOE], 2006). These
biomass materials can be broken down to release cellulose. The cellulose obtained can
be hydrolyzed to glucose which can then be fermented to produce ethanol. According to
the US DOE (2006) Workshop on Breaking the biological barriers to cellulosic
ethanol, about 4.1 billion gallons of ethanol were produced from corn in the USA in
2005. This was less than 2% of USAs transport energy demand. Therefore cellulosic
biomass will add to supply of materials needed to produce ethanol.

Insufficient energy supply is a threat to Kenyas realization of Vision 2030 strategic

plan, whereby Kenya wants to be a middle income nation by this period. Gasoline, hydro
and geothermal powers are unable to meet Kenyas energy demand. This coupled with
fossil fuel price hikes due to political situation in producing countries may reduce
Kenyas economic growth expectations. Nowadays, the tendency is to turn to bio-fuels
to fill the gap between energy demand and supply.

Bio-fuels are divided into three categories:

i. First-generation bio-fuels made largely from edible sugars and starches,

for instance sugar cane and corn.
ii. Second-generation bio-fuels made from non-edible plant materials, for
example maize stover, wheat straw, saw dust, sugar cane bagasse and
sweet sorghum bagasse.
iii. Third-generation bio-fuels made from algae and microbes (Larson, 2008).

Alternatively, bio-fuels can also be categorized as:

i. Simple sugar based like those from sugar cane and sweet sorghum
ii. Starch based maize, cassava and microalgae

2
 iii. Lignocellulose based- stalks, straws, grass and other agricultural wastes

Kenyas sugar demand is not met by the local factories that rely on sugarcane.
Furthermore cane requires humid conditions and a lot of water to grow and the varieties
grown in the Western part of the country take 22 to 26 months to mature. Therefore an
alternative crop has to be sought that uses less water, takes a shorter time to mature and
one that can grow in arid and semi-arid areas to fill this gap.

1.2 The Problem Statement

Kenyas demand for energy and sugar has increased to outstrip supply. This has
continued to push the prices of fossil fuels, hydroelectricity and sugar higher up. The
consequence of this is that it has led to the increase of prices of crucial goods and
services thus lowering the standard of living of most Kenyans. If this trend continues, it
will reduce economic and industrial growth hence jeopardizing the realization of Vision
2030. Sweet sorghum (SS) is C4 crop growing in geographical areas with a temperate
climate and requires limited fertilizer rates and water (Goshadrouet al., 2011). It also
has a shorter growing season than sugar cane, higher sugar yield per hectare than sugar
beet, and high resistant to drought and salinity (Barbantiet al., 2006; Dolciottiet al.,
1998; Zhao et al., 2009). The juice extracted from the fresh stems of this plant
contains approximately 16-18% fermentable sugars predominately sucrose, glucose and
fructose. Furthermore SS has a high yield of green biomass (20-30 t/ha), and huge
amount of lignocellulosic residue are produced as byproduct of SS (Maiorella, 1985;
Sipos et al., 2009; Teetor et al., in press; Wu et al., 2010). This lignocellulosic
residue, so-called bagasse, has only non-food applications e.g., cattle feed roughage and
soil fertilizer after composting (Sipos et al., 2009). This biomass can be used to
produce bio-ethanol to satisfy fully the energy demand in the motor industry. However,
this has not been realized because the method of production has remained expensive.
The current methods of breaking lignocelluloses for ethanol production involve
chemical and heat explosion. These methods are not environmentally friendly and
consume the much needed energy. The first-generation bio-fuels use feedstock that is
food for humans hence leading to increase in food prices. This is not socially and
economically acceptable, hence not sustainable. This therefore necessitates a thorough

3
research on alternative uses of SS in addressing energy and sugar challenges in the
country.

1.3 Justification
There has been increased interest in the production of sweet sorghum in Kenya and the
suitable areas for production in the Western, Central, Eastern and Coastal regions of
Kenya are estimated at 46.4% of the total Kenya surface area (Ndegwa et al., 2011).
As one of the value addition pathway to support the SS value chain in Kenya, it is
important to explore alternative uses of sweet sorghum in addressing some of the energy
and sugar challenges within the country.

One of the pillars of Kenyas Vision 2030 is economic development which cannot be
achieved without a secure, reliable and sustainable energy source. The vision of the
Kenya Government Bio-fuel Policy is to increase access to energy through sustainable
bio-fuel production, and reduce dependence on fossil fuels by 25% in volume by the
year 2030.

Lignocellulose is of great interest as a feedstock for second generation ethanol

production. In this case, the involved technologies are more complex and the ethanol
production costs are higher when compared to first generation feedstocks.
Lignocellulosic materials are byproducts of agricultural activities and industrial residues
and show great potential for the production of fuel ethanol at large scale in Kenya. It is
considered that lignocellulosic biomass will become the main feedstock for ethanol
production in the near future.

Therefore, exploring a method of converting lignocellulosic materials to bio-fuels cost-

effectively may lead to production of enough energy leading to the realization of Vision
2030. Furthermore, more energy will contain the ever rising price of fossil fuel and
lower energy cost. Venturing into bio-ethanol production will encourage
entrepreneurship ventures thus creating employment and spurring development of the
rural areas where these crops and factories will be established. Lignocellulose which is
ordinarily seen as an agricultural waste will be used as the energy raw material hence
lowering the cost of the final product.

4
Crystallization of the sweet sorghum juice could be a major breakthrough for the supply
of sugar to the local Kenyan market which normally has a shortfall of 200,000 metric
tons annually hence lowering the sugar prices which are currently very high (Anyanzwa,
2014).

1.4 Research Question

1. Is it possible to crystallize sweet sorghum juice to crystal sugar?

2. Can Sweet Sorghum Bagasse be used to produce bio-ethanol?

1.5 Hypotheses
1. There is no significant difference in sucrose content of selected sweet
soghum varieties
2. There is no significant difference in crystallization process between sweet
sorghum and sugarcane
3. Specific pretreatments of sweet sorghum bagasse has no significant
difference in fermentation of lignocellulose

1.6 Objectives

1.6.1 General Objective

To explore the potential of stalk juice from specific varieties of sweet sorghum to
crystallize and production of bio-ethanol from the bagasse.

1.6.2 Specific Objectives

The specific objectives were:

1. To determine sucrose content in selected sweet sorghum varieties;

2. To determine the crystallization potential of the extracted juice from specific

sweet sorghum variety;

3. To compare the yield of untreated, acid and alkaline pretreatments of enzymatic

hydrolysis and fermentation of lignocellulose of RIO sweet sorghum.

5
 CHAPTER TWO

2.0 LITERATURE REVIEW

2.1 Introduction
Sweet sorghum [Sorghum bicolor (L) Moench] has been regarded as one of the most
promising crops for ethanol production (Gnansounou et al., 2005). Because it is a C4
crop with higher photosynthetic efficiency and sugar and biomass yield. It also has a
wide adaptability to harsh growth conditions, such as higher drought, water logging,
salinity, and alkalinity tolerance (Corredor et al., 2009; Rubin, 2008). Based on these
characteristics, the marginal lands could be utilized to grow SS for ethanol and sugar
production, which might give a potential solution to the competition between foods and
fuels (Shen, 2011). Additionally, approximately 9.5% (wet basis) soluble carbohydrates
(glucose, fructose, and sucrose) and 10% (wet basis) insoluble carbohydrates (cellulose
and hemicellulose) exist in SS stalk (Sipos et al., 2009). These parts have both been
regarded as the important sources for bioethanol production (Yu et al., 2010).

Bio-fuels are made from organic matter. They are renewable, since their sources can be
re-grown (Cheng, 2010). Advanced bio-fuels offer environmental benefits such as lower
carbon emissions and lower sulphur compared with first-generation bio-fuels and
conventional fossil fuels. About 95% of bio-fuels produced in the world are derived
from agricultural products. According to a World Bank Paper on production of second-
generation bio-fuels (Miguel et al., 2010), global bio-fuel production has been
increasing rapidly over the last decade, but the expanding bio-fuel industry has recently
raised important concerns. In particular, the sustainability of many first-generation bio-
fuels has been increasingly questioned over concerns such as reported displacement of
food-crops, effects on the environment and climate change. In general, there is growing
consensus that if significant emission reductions in the transport sector are to be
achieved, bio-fuel technologies must become more efficient in terms of net lifecycle
greenhouse gas emission reductions while at the same time be socially and
environmentally sustainable. It is increasingly understood that most first-generation bio-
fuels, with exception of sugar cane ethanol, will likely have a limited role in the future
transport fuel mix. The increasing criticism of the sustainability of many first-generation
6
bio-fuels has raised attention to the potential of so-called second-generation bio-fuels.
Depending on the feedstock choice and cultivation technique, second generation bio-fuel
production has the potential to provide benefits such as consuming waste residues and
making use of abandoned land. In this way, the new fuels could offer considerable
potential to promote rural development and improve economic conditions in emerging
and developing regions (Carriquiry & Timilsina, 2010). However, while second-
generation bio-fuel crops and production techniques are more efficient, their production
could become unsustainable if they compete with food crops for available land. Thus,
their sustainability will depend on whether producers comply with criteria like minimum
lifecycle greenhouse gas reductions, including land use change, and social standards.

Technically, it would be possible to produce a large portion of transportation fuels using

advanced biofuel technologies, specifically those that can be grown using a small
portion of the worlds land area for example, microalgae or those grown on arable lands
without affecting food supply for instance agricultural residues (Cheng, 2010). However,
serious technical barriers limit the near-term commercial application of advanced bio-
fuels technologies. Key barriers include:

i. Low conversion efficiency from biomass to fuel,

ii. Limits on supply of key enzymes used in conversion,
iii. Relatively large energy requirements for operation and
iv. Dependence, in many cases, on commercially unproven technology (Cheng,
2010).

2.2 Second-Generation Bio-fuels

2.2.1 Introduction
Bio-fuels production and consumption has been rapidly growing in the last few years.
Led by Brazil and the United States, global production of fuel ethanol more than
doubled during the last four years, increasing from 31.3 billion litres in 2005 to over
72.8 billion litres in 2009 (Licht, 2009). Although being produced in smaller quantities
than ethanol, the relative growth experience by biodiesel is even stronger, surpassing
12.8 billion litres in 2008 up from 3.9 billion litres in 2005 (Licht, 2008). Currently, bio-

7
fuels provide for over 1.5 % (about 34 Mtoe) of energy used for transport (IEA, 2008).
The Energy Information Administration (EIA) of the U.S Department of Energy projects
the 2030 world energy consumption of liquid forms at 112.5 million barrel of oil
equivalent per day (about 238 EJ/yr), of this, 60% or 142.8 EJ/yr would be consumed by
the transport sector. On the other hand EIA (2009), projects that the total production of
bio-fuels will be between 10.1 and 15.1 EJ per year by 2030 depending on the
assumptions on oil prices.

Several reasons can be advanced as fuelling growth in bio-fuels. Salient drivers are
increased oil prices over the past decade, as well as oil price volatility. This has led to
increased public support for renewable fuels for instance, subsidies and mandated
consumption by many countries (REN21, 2009). The oil crisis of the 1970s prompted
some interest in bio-fuels. However, that impulse was short lived and faded in most
countries with the subsequent decline in the price of petroleum. Brazil is an exception to
this pattern, with ethanol production continuing to expand, with the help of blending
mandates, throughout the 1980s. Without blending and oxygenations mandates, and
other forms of intervention, the market for bio-fuels would be limited when the price of
crude oil is below US$60-70 per barrel (OECD-FAO, 2009).

The recent rapid growth of bio-fuel production has become controversial. The wide
support that bio-fuels enjoyed just three or four years ago has eroded more recently as
new studies began to emerge linking their production to rising food prices, questioning
their ability to displace fossil energy, and criticizing their potential contribution to
monoculture and deforestation (Fargione et al., 2008; Mitchell, 2008; Searchinger
et al., 2008). The combined impacts of these effects have stimulated greater interest, and
even some sense of urgency, for the development of bio-fuels produced from non-food
biomass, commonly referred to as second-generation bio-fuels. These are less land and
water intensive, and/or use residues from agriculture. Despite increased interest in
expanding second-generation bio-fuels and progress made in recent years, significant
hurdles still need to be overcome before second-generation bio-fuels can be produced at
commercial scale, even with the massive investments in Research and Development
observed in recent years (IEA, 2008).

8
The economic potential for second-generation bio-fuel production depends critically on
both the amount of land that would be used, relative to other land uses, the productivity
of biomass cultivation and the cost of converting various types of biomass to liquid
fuels. While second-generation bio-fuels could make significant contributions to global
energy supply, the economic potential market for these bio-fuels will likely be more
limited due to the amount of feedstocks that can be produced at affordable costs with
available land, as well as the costs of production relative to liquid fossil fuels (Miguel
et al., 2010).

The potential feedstocks for second-generation bio-fuels production are biomass from
crop residues, other non-food energy crops, wood/forestry residues, jatropha and algae.
Of major interest are potential feedstocks of lignocellulosic nature (Miguel et al.,
2010).

2.2.2 Lignocellulosic feedstocks

The major components of lignocellulosic feedstocks are cellulose and hemicelluloses
(over 67% of dry mass), which can be converted to sugars through a series of
thermochemical and biological processes and eventually fermented to bio-ethanol. In
general, lignocellulosic feedstocks are divided into three categories:

i. Agricultural residues for instance crop residues and sugarcane bagasse

ii. Forest residues
iii. Herbaceous and woody energy crops (Miguel, 2010).

2.2.2.1 Agricultural residues

Agricultural residues differ in their chemical composition, which leads to different bio-
fuel yields per unit of feedstock. For instance, percentage composition of carbohydrates
for barley straw, corn stover, rice straw, sorghum straw, wheat straw and sugar cane
bagasse is 70.0, 58.3, 49.3, 61.0, 54.0 and 67.2, respectively, and the corresponding
biofuel yield in litres/hectare is 367, 503, 392, 199, 410 and 3133, respectively (EIA,
2001; Kim & Dale, 2004; NRC, 1958; US DOE, 2008; US DOE, 2008a).

The availabilities of agricultural residues are estimated by extending the approach

followed by Lal (2005) to calculate the global amount of crop residue production. Since

9
data on the amount of crop residues produced is usually unavailable, these are
approximated using the ratio between residue and crop production and the commodities
production levels. A second step would involve determining how much of the crop
residues produced could be actually removed and used for bio-fuel production. Medium-
run (2015/2016) projected crop production levels for major producing countries were
obtained from FAPRI (2009). The amount of residues that can be sustainably collected
is still a contentious issue, and is affected by many factors, including topography,
nutrient management, crop yields, climate and tillage practices (Andrews, 2006; Blanco-
Canqui & Lal, 2009). Given the large geographic distribution of potential availability
and possible feed and other usage, using the work of Kim and Dale (2004), it was
assumed that 40% of total residues can be used for bio-fuel production for most crops.
The exceptions would be rice straw and bagasse where 100% of residues can be
removed.

A major advantage of using residues for bio-fuel production when compared to the grain
crops and dedicated energy crops is that no additional land is needed (Miguel et al.,
2010). By avoiding the competition for land, residue based bio-fuel production should
have minimal direct impact on food prices. Furthermore, greenhouse gas emissions
associated to direct and indirect land use changes are also avoided- hence improving
their carbon balance (Searchinger et al., 2008). Crop residue removal can also be
beneficial for some crops and situations as it may help control pests and diseases, and
increase soil temperature in the spring facilitating seed germination (Andrews, 2006).
On the other hand, crop residues are important to conserve soil properties, conserve
water, enhance soil productivity, and to sequester carbon in soils. Excessive removal
will have adverse impacts not only on soil properties and the environment,but also on
crop production (Blanco-Canqui & Lal, 2009).

2.2.2.2 Forest residues

Forest residues include logging residues produced from harvest operations, fuel-wood
extracted from forest lands, and primary and secondary wood processing mill residues
(Perlack et al., 2005). Forest residues feedstock namely black locust, hybrid poplar,
eucalyptus, pine and switchgrass contain cellulose ranging from 31.98 to 49.50 (% dry

10
mass), hemicelluloses, 13.07 to 21.90, carbohydrates, 57.15% to 66.45%, lignin, 18.13%
to 27.71% and their theoretical ethanol yield in litres/dry ton ranged from 377 to 436
(Hamelinck et al., 2005; US DOE, 2008b). The US National Renewable Energy
Laboratory (2007) assumed yields of 65 gallons (246 litres) per ton of feedstock; the
same source provides target yields of 90 and 94 gallons (341 and 356 litres) of ethanol
per ton of cellulosic feedstock by 2012 and 2020 respectively.

Several factors restrict the potential use of forest residues for bio-fuel production
(Perlack et al., 2005). The first factor is the economic costs of transportation where
limited accessibility largely increases the operation costs of logging/collection activities.
Another factor is the potential reduction of recoverability in harvest areas due to
environmental considerations (Richardson, 2008).

2.2.2.3 Herbaceous and woody energy crops

Biofuel crops can be broadly classified between grassy such as herbaceous or forage and
woody or treecrops (Miguel et al., 2010).

2.2.2.3.1 Herbaceous or Forage crops

Perennial forage crop species are a promising source of feedstock for second-generation
biofuels. Switchgrass (panicumvirgatum L.) is frequently mentioned because of its
relatively low water and nutrition input and requirement costs, positive environmental
impact, and adaptability to low quality land (Keshwani & Cheng, 2009). Other perennial
forage crops such as alfalfa (Medicago sativa L.), reed canarygrass
(Phalarisarundinacea L.), napiergrass (PennisetumpurpureumSchumach.), and
bermudagrass (Cynodon spp.) could also serve as potential bioenergy crops (Miguel et
al., 2010).

2.2.2.3.1.1 Switchgrass
Among 34 herbaceous species, switchgrass is identified as a leading candidate of
dedicated bioenergy crop by the Biofuels Feedstock Development Program at Oak Ridge
National Laboratory (ORNL, 2008). Switchgrass is widely and naturally distributed
from Central America to Southern Canada. Research indicates that while soil type does

11
not have much impact on switchgrass production, water-holding-capacity is important in
its growth (Miguel et al., 2010).

A wide range of yield expectations have been reported in the literature. Given ideal
establishment and growing conditions, Thompsonet al., (2005) reports dry mass
potential yields based on for upland populations as high as 18 to 20 Mg/ha, while yields
in lowland forms could reach 23-27 Mg/ha.

2.2.2.3.1.2 Other Species

Miscanthus is a grass native to Asia and a compelling herbaceous biomass feedstock for
Europe (Lewandowski et al., 2003), in part because of its cold tolerance and low
levels of nitrogen needed. A drawback to this species is that it takes 2-3years to start full
production as it must be established and propagated via rhizome cuttings. Other major
limitations identified are: (1) Limited availability of genotype, (2) important losses over
winter, and (3) high costs of establishments (Lewandowski et al., 2003).

Reed canarygrass is commonly used for hay and forage. It is well adapted to temperate
agro-economic regions and to weathered soils (Carlson et al., 1996). Reed
canarygrass can be slow to establish and become an invasive species in native wetland
(Merigliano & Lesica, 1998).

Alfalfa is a forage crop that can be used to both supply biomass feedstock and as a high
quality animal feed (Delong et al., 1995). Several other subtropical and tropical
grasses have been explored as potential biomass feedstocks in the US, including
bermudagrass (Boateng et al., 2007), napiergrass (Schank et al., 1993), eastern
gamagrass and prairie cordgrass (Springer & Dewald, 2004; Boe & Lee, 2007).

The yield of these perennial species in terms of biofuel per ton of feedstock (and thus by
hectare) will depend on their theoretical potential, and the conversion efficiency of the
process. For switchgrass, the theoretical potential is around 100 gallons (377 litres) per
dry ton (Hamelinck et al., 2005).

12
2.2.2.3.2 Woody energy crops
Broadly referred to as woody energy crops, some fast growing tree species have also
shown promise for biofuel production. Important attributes include the relatively high
yield potential, wide geographical distribution, and relatively low levels of input needed
when compared to annual crops (Smeets et al., 2007). Their versatility as a source of
solid and liquid energy is also a plus according to these authors. Poplar (Populus spp.),
willow (Salix spp.), and eucalyptus are among the species most frequently mentioned for
this end (Miguel et al., 2010).

Dedicated energy crops as feedstock for biofuel production have some advantages over
the feedstocks currently used to that end. These energy crops are in general less
demanding in terms of inputs, reduce erosion and improve soil properties, and provide
better wildlife habitat (Miguel et al., 2010). Additionally more energy per unit of land
can be obtained from these crops as a higher proportion of the biomass can be utilized.
On the other hand, while highly yielding, dedicated energy crops do not entirely escape
the food versus fuel debate as additional land is needed for their production (Miguel et
al., 2010). In order not to compete for land with food production, these crops (woody or
forages) should only be installed in lands where neither food crop production nor
grazing pastures are feasible activities, or that are not needed (Miguel et al., 2010).

As with the case of crop and forest residues, the logistics of feedstocks obtained from
dedicated energy crops is still a challenging issue to be resolved. These feedstocks are
simply bulky and difficulty to transport. For the case of switchgrass, Epplin et al.,
(2007) indicated that the corresponding infrastructure for harvest, storage,
transportation, and spot markets still do not exist. In addition, a narrow harvesting
window and yield viability may potentially increase feedstock cost and force bio-
refineries to maintain a feedstock buffer for continuous biofuel production (Miguel et
al., 2010).

2.3 Sweet sorghum

Sweet Sorghum is a C4 crop (open stomata during the day and close during the night,
hence maximizing on the sunlight) in the grass family belonging to the genus Sorghum
bicolor L. Moench which also includes grain and fiber sorghum and is characterized by a

13
high photosynthetic efficiency (Goshadrouet al., 2011). Sweet sorghum is often
considered to be one of the most drought resistant agricultural crops as it has the
capability of remaining dormant during the driest periods (Woods, 2000). Like other
sorghum types, sweet sorghum probably originated from East Africa and spread to other
African regions, Southern Asia, Europe, Australia and the United States. Although
native to the tropics, sweet sorghum is well adapted to temperate climates
(Goshadrouet al., 2011). The plant grows to a height of from 120 to above 400 cm,
depending on the varieties and growing conditions and can be an annual or short
perennial crop. More than 125 sweet sorghum germplasm resources have been registered
in China (Lu, 1997). Seed germination takes place within 24 h in warm and moist soils,
and the time to maturity lies between 90 and 120 days (Gnansounouet al., 2005).
Although the juice, grain and bagasse from sorghum provide opportunities for many
uses, most applications around the world are for syrup and forage. An average yield of
1900 L (500 gallons) of syrup per hectare can be achieved, although yields of 800-1200
L (200-300 gallons) per hectare can result if weather conditions are poor
(Gnansounouet al., 2005). In forage applications, chickens can be fed with seed heads
and ruminant livestock can use the grains, leaves and stalks. The organic by-product
from sweet sorghum syrup processing is often fed to livestock, left on the field or
composted (Gnansounou et al., 2005).

Because of the relatively high sugar content in sweet sorghum compared to sugar cane
and sugar beet (Cheng, 2010), sugar can be readily extracted from the plant and sold in
local and world markets. However, due to the lower purity (ratio of the %wt. of sucrose
to the %wt. of solubles) of the sugar extracted from sweet sorghum (about 75 apparent
purity, AP) compared to that of sugar cane or sugar beet (80-85 AP), it is more costly to
produce white sugar from sweet sorghum (Gnansounouet al., 2005).

The technology considered for juice extraction involves a series of tandem roller mills
with countercurrent juice flow to leach soluble content. On this basis, the sugar
extraction (i.e, the proportion of initial sugars present in the juice after extraction)
reaches 87%. Because of the relatively high fibre content in sweet sorghum, it is

14
unlikely that the yield will be as high as from sugarcane (Cundiff & Vanghan,1987;
Woods, 2000).

In addition to sugars, the juice contains other compounds and impurities which have to
be eliminated before crystalline white sugar can be made. Furthermore, sweet sorghum
sugars consist of 85% (wt) sucrose, 9% glucose and 6% fructose on average, and only
sucrose may readily be converted to white sugar (Woods, 2000). The first stage in juice
purification is the addition of lime milk (liming) followed by saturation with carbonation
gas (mainly carbon dioxide) to precipitate the lime milk in a clarifier and capture the
impurities in the raw juice. The lime and carbonation gas are produced in a lime kiln
through the decomposition of limestone. The settled solids (mainly calcium carbonate
and non-sugars) from the clarifier are filtered in membrane presses and sent to the spent
lime storage area, while the clear portion is again saturated in a second carbonation
station. The purified juice obtained after the consequent filtration is called thin juice and
is thickened in a multi-effect evaporator into thick juice. The thin juice that has been
diluted with water during extraction and purification enters the evaporation station with
an average sugar content of 15% while the thick juice leaving the evaporators contain
approximately 70% sugars (Gnansounouet al., 2005).

White sugar in its crystalline form is eventually obtained from the thick juice by
crystallization in vacuum pans at reduced temperature and pressure. The mixture of
crystals (sucrose only) and the mother liquor (green syrup) are separated in centrifuges,
where the sugar is washed with hot water (Gnansounouet al., 2005). The wet sugar is
dried in a drum drier, screened and finally stored in silos after cooling, while the syrup
from the centrifuge is passed through an additional boiling stage to extract most of the
remaining sugars (i.e. glucose, fructose and some of the sucrose left). The syrup left over
is known as molasses. Although molasses is about 50% sugars, the concentration of non-
sugars is so high that no further crystallization is economically possible in a standard
processing facility, and molasses are stored in large tanks to be shipped for use by other
industries (Gnansounouet al., 2005). A simplified flow diagram of the overall process
is given in Figure 2.1.

15
 Lime station
Lime station

Sweet juice
Purification Evaporation

Crystalline
Crystallisation Centrifugation
sugar

Molasses

Figure 2.1: Block flow diagram for conversion of sweet sorghum juice to sugar

Source: (Gnansounouet al., 2005)

2.4 Crystallization
According to McCabe (2005), crystallization is the physical transformation (phase
transition) of a liquid, solution, or gas to a crystal, which is a solid with an ordered
internal arrangement of molecules, ions, or atoms. Crystalline substances of importance

16
to food sciences and nutrition include sugars, sugar alcohols, salts, fats, fatty acids, and
artificial sweeteners. Crystallization is a means to isolate chemical substances in the
solid form for long-term storage and downstream processing. As a purification
technique, crystallization relies on the stringent structural requirement for crystal
formation to exclude impurities. Crystallization performed under different conditions
can yield crystals of different sizes and morphologies, thus providing a way of
modifying particles to desired specifications (Myerson, 1993).

2.4.1 Principles of crystallization

Crystallization usually occurs through a nucleation and growth mechanism (Myerson,
1993). Nucleation is the formation of stable molecular aggregates (nuclei) capable of
growing into macroscopic crystals. Crystal growth is the actual development of the
nuclei into visible dimensions. Both nucleation and growth require a thermodynamic
driving force. In the case of solution crystallization, the thermodynamic driving force is
supersaturation. Supersaturation is generated when the concentration of a solute exceeds
its equilibrium solubility. Supersaturation can be expressed as the difference between the
concentration of a saturated solution Cssand the equilibrium solubility Ceq* or as the ratio
between the two, equation 2.1.

C ss
C Css Ceq * Or S = ........................................Equation 2.1
C eq *

Source: (Myerson, 1993)

The thermodynamic driving force for crystallization from a melt is generated by

lowering the temperature below the crystal melting point, or undercooling. Although any
nonzero supersaturation can in principle cause crystallization from a solution,
crystallization usually does not occur unless the supersaturation exceeds a certain
threshold, or metastable limit. A diagram illustrating this phenomenon is shown in
Figure 2.2. The solid line depicts the equilibrium solubility of a solute as a function of
temperature. The region below the solid line is undersaturated, where crystallization is

17
Concentration Supersaturated
C Labile zone

Metastable zone
a

b Undersaturated
Solubility curve

Temperature, T

Figure 2.2: Equilibrium solubility, supersaturation, and metastable limit. An

undersaturated solution can be induced to crystallize by solvent evaporation (path
a), temperature change (path b) or both.

Source: (Xiongwei & Anting, 2010)

thermodynamically impossible. The region above the solid line is supersaturated, where
crystallization is thermodynamically possible, and the solution is metastable. The
metastable limit is indicated by the dashed line, and the region between the solid and
dashed lines is called the metastable zone. A supersaturated solution free of crystal seeds
can remain in the metastable zone indefinitely. For a solute to crystallize spontaneously,
the solution concentration must be raised not only above the equilibrium solubility (solid
line), but also above the metastable limit (dashed line) into the so-called labile zone. The
width of the metastable zone depends on the nature of the solute and conditions of
crystallization (e.g. stirring, solvent, temperature, pressure, and the presence of

18
impurities and the surface characteristics of the crystallization vessel) (Xiongwei &
Anting, 2010). Crystal nucleation may be classified as primary or secondary. Primary
nucleation refers to the formation of crystal nuclei from a solution that contained no
preexisting crystals. Primary nucleation occurs through both homogeneous and
heterogeneous mechanisms. Homogeneous nucleation is the formation of nuclei within a
homogeneous fluid, and heterogeneous nucleation is initiated by contact with foreign
particles and surfaces. The effectiveness of surfaces or interfaces as templates for
nucleation frequently makes the heterogeneous mechanism the dominant mechanism
when particulate contaminants are present. Secondary nucleation refers to the generation
of crystal nuclei from preexisting crystals, such as those introduced through seeding
(Black et al., 1986).

2.4.2 Techniques of crystallization

Crystallization techniques differ in the way in which supersaturation is generated and
relieved, and nucleation is initiated (Myerson, 2000). The importance of selecting an
appropriate technique lies in the fact that the same crystallization performed under
different conditions can yield crystals of different properties (size, morphology,
chemical purity, polymorphic form, etc.). To induce crystallization from a solution, the
necessary supersaturation can be generated in several ways, including solvent
evaporation, temperature change, antisolvent addition, and chemical reaction. In Figure
2.2, solvent evaporation at a constant temperature corresponds to a vertical line from the
undersaturated to the supersaturated region. Solvent removal at controlled temperatures
can be performed either with or without vacuum. Slow evaporation of solvent is
frequently used to obtain large, high-quality single crystals. Crystallization induced by
temperature change (temperature gradient) takes advantage of the temperature
dependence of solubility. In Figure 2.2, the generation of supersaturation by
temperature change without solvent removal corresponds to a horizontal line from the
undersaturated to the supersaturated region. If solubility increases with temperature, a
high-temperature solution can be cooled to generate supersaturation. In general, slower
cooling rates lead to nucleation at higher temperatures, producing fewer and larger
crystals. To effect additional control, it is customary to seed the solution so that
crystallization is initiated at the desired temperatures. Once crystallization begins, the

19
suspension can be cooled to improve the product yield (assuming that solubility
increases with temperature). Crystallization by antisolvent addition (also called
drowning out) depends on the solvent solubility. With this technique, a solution to be
crystallized is mixed with an antisolvent, which is miscible with the initial solvent and in
which the solute is less soluble. The addition of antisolvent lowers the solubility and
generates supersaturation. The onset of crystallization is signaled by turbidity, after
which precipitation usually follows (Davey & Garside, 2000).

2.5 Biofuel Production Technologies

2.5.1. Introduction
Biofuels can be produced using various feedstocks and conversion technologies. Sassner
and Colleagues (2008) evaluated the techno-economic feasibilities of bioethanol
production from different lignocellulosic materials.Most of the recent research activities
in cellulosic bioethanol production have been focused on more cost-effective
pretreatment technologies for lignocellulosic materials, low-cost and high-efficiency
cellulose enzymes and co-fermentation of 6-carbon and 5-carbon sugars for ethanol
production (Cheng, 2010).
Lignocellulosic material can be used for bioethanol production because they have a high
content of cellulose and hemicellulose. However, the conversion of lingnocellulosic
materials to ethanol is much more difficult than that of sugar-rich for instance sugar cane
or starch-rich for example corn materials. This is because the cellulose and
hemicelluloses molecules are tightly tangled together and the structure is firmly wrapped
up by lignin. The conversion of lignocellulose to ethanol involves three steps: pre-
treatment, hydrolysis and fermentation (Carriquiry & Timilsina, 2010). Pre-treatment is
necessary because of the unique compact structure of lignocellulosic materials. The
main purpose of the pre-treatment is to remove lignin from the material and reduce the
crystallinity and increase the porosity of the material, so the cellulose and hemicellulose
are accessible for hydrolysis to produce fermentable sugars. The complex process for the
conversion of lignocellulosic materials to ethanol makes the conversion more expensive
than the conversion of either sugars or starch to ethanol. With the rapid increase in the
prices of sugars and starch feedstock and limitation of cropland for the feedstock

20
production, the abundant and inexpensive lignocellulosic materials have a great potential
in substantial expansion of fuel ethanol production (Cheng, 2010).
Lignocellulose is composed of mainly cellulose, hemicellulose and lignin (Figure 2.3A).
Cellulose is a long chain homogeneous polysaccharide of D-glucose units linked by -1,
4 glycosidic bonds and contains over 10,000 glucose units (Figure 2.3B). Hemicellulose
is a complex, heterogeneous polymer of sugars and sugar derivatives which form a
highly branched network and the monomers include: hexoses namely glucose, galactose
and mannose and pentoses like xylose and arabinose (Figure 2.3C). It consists of about
100-200 sugar units. Lignin is a very complex heterogeneous mixture of mainly phenolic
compounds and their derivatives. It is composed of mainly phenolic compounds and
their derivatives. It is the main component in plant cell walls. Lignin holds the cellulose
and hemicelluloses fibres together and provides support to the plants (Cheng, 2010).

21
A.
Lignin

Hemicellulose
Cellulose

B. H OH CH2OH H OH CH2OH
H
HO C C C C OH
OH H C O H C O H
OH H OH H
C C
H O C OH H C O C H C O C H
C
C O H H
H OH
CH2OH C C C O H OH
H C C
H OH
CH2OH H OH
Non-Reducing
End-Group -1,4 glycosidic bond Reducing
End-Group

C. X X X X X X X X X

2 3

1
4-O-Me--D-GA 1-L-A

X-X: -1,4-linked D-xylopyranose units

Me: methoxy group

GA: glucuronic acid

A : esterified--L-arabinofuranose side chain

Source: (Cheng, 2010)

Figure 2.3: Structures of (A) lignocelluloses, (B) cellulose, and (C) hemicelluloses

2.5.2 Pre-treatment
A diagram of pre-treatment process is shown in figure 2.4. Pre-treatment technologies
have been extensively investigated in the last three decades; these include physical,
chemical and biological processes for lignocellulosic materials (Sun & Cheng, 2002).
22
 Cellulose

Lignin

Pre-treatment

Hemicellulose

Figure 2.4: A pre-treatment process for lignocellulosic materials

Source: (Cheng, 2010)

2.5.2.1 Physical pre-treatment

Physical pre-treatment includes mechanical comminution, steam explosion, ammonia
fiber explosion and pyrolysis (Cheng, 2010). Mechanical comminution combines
chipping, grinding and milling to break the lignocellulosic materials down to 0.2 to 2
mm and reduce the crystallinity of the materials. Steam explosion applies high-
temperature (160- 260 oC) and high pressure saturated steam to steep the lignocellulose
and then rapidly release the steam pressure to atmospheric, causing explosive
decompression which separates lignin from the carbohydrates and degrades the
hemicellulose. Similar to steam explosion, ammonia fibre explosion uses liquid
ammonia to soak the lignocellulosic materials at high temperature (around 100 oC) for a
period of time and then the materials are rapidly flashed to a low pressure, breaking the
chemical bonds between cellulose and hemicellulose and substantially increasing the
porosity of the materials. In pyrolysis, the lignocellulosic materials are exposed to a high
temperature (over 220 oC). At that temperature, the hemicellulose and some lignin and
cellulose will be degraded to gaseous and tarry compounds and the tight structure of the
lignocellulose will be broken. Physical pre-treatment can effectively break the structure
of the lignocellulosic materials and substantially improve sugar yield in the following

23
enzymatic hydrolysis. However, physical pre-treatment usually involves high energy
input (Sun & Cheng, 2002).

2.5.2.2 Chemical pre-treatment

Commonly used chemical pre-treatment technologies include acid and alkaline
hydrolysis. In dilute sulphuric acid pre-treatment, high temperatures (140-190 oC) are
applied to the mixed slurry of the lignocellulose and the acid. The acid decomposes the
hemicellulose at that temperature, resulting in the disintegration of the lignocellulosic
structure. Dilute acid pre-treatment has been used in pilot-scale lignocellulosic ethanol
production because the technology is quite mature and breaks the lignocellulosic
materials very efficiently (cheng, 2010). After the pre-treatment, the lignocellulosic
materials can be easily separated into a liquid portion and a solid portion, while cellulose
remains in the solid state. The separated cellulose is then hydrolyzed in the following
enzymatic hydrolysis to produce fermentable sugars for ethanol production. The main
disadvantage of dilute acid pre-treatment is the formation of chemicals such as furfurals,
during degradation of hemicelluloses, which inhibit the subsequent enzymatic hydrolysis
and microbial fermentation.
Alkaline hydrolysis is another chemical pre-treatment method at high temperature (100-
170 oC). During the alkaline pre-treatment, there are saponification reactions of
intermolecular ester bonds cross linking hemicellulose and cellulose or lignin in the
lignocellulosic materials. Alkaline pre-treatment can also disrupt lignin structure,
decrease crystallinity of cellulose and degree of sugar polymerization (Sun & Cheng,
2002). Although alkaline pre-treatment could cut the bonds between lignin and cellulose
or hemicellulose, a significant portion of lignin still remains mixed with cellulose after
the pre-treatment. The existence of lignin may inhibit cellulase enzyme during the
following enzymatic hydrolysis.

2.5.2.3 Biological pre-treatment

Biological pre-treatment processes use microbes such as brown-, white-, and soft-rot
fungi to degrade lignin and hemicellulose in lignocellulosic materials (Schurz, 1978).
Brown rots mainly attack cellulose, while white and soft rots attack both cellulose and

24
lignin. White-rot fungi are the most effective basidiomycetes for biological treatment of
lignocellulosic materials (Fan et al., 1987). Biological pre-treatment is probably the
most economical pre-treatment technology for the lignocellulosic materials. However, it
is also a very time consuming process as the pre-treatment usually takes a few weeks.

2.5.3 Enzymatic hydrolysis

Enymatic hydrolysis of pretreated lignocellulosic materials involves enzymatic reactions
that convert cellulose into glucose and hemicelluloses into pentoses (xylose and
arabinose) and hexoses (glucose, galactose, and mannose). The conversion of cellulose
and hemicelluloses is catalyzed by cellulase and hemicellulase enzymes, respectively.
The enzymes are highly specific (Bguin & Aubert, 1994). The enzymatic hydrolysis is
usually carried out at mild conditions (pH 4.8 and temperature 45 to 50 0C).

Cellulases or -(1-4) glycoside hydrolases are a mixture of several enzymes and at least
three major groups of cellulases are involved in the hydrolysis of cellulose (Cheng,
2010): endoglucanase, exoglucanase, and -glucosidase. After the pre-treatment, most of
lignin is removed from the lignocellulosic materials, the crystallinity of the materials is
significantly reduced, and the porosity is substantially increased, which allows the
enzymes to penetrate into the materials and access the substrates. Endoglucanase
randomly attacks regions of low crystallinity in the cellulose fibre and hydrolyze the -
(1, 4) glycosidic bonds of cellulose to produce cello-oligosaccharides with free-chain
ends. Exoglucanase can hydrolyze the -(1, 4) glycosidic bonds from the non-reducing
ends of the cello-oligosaccharides to generate cellobiose which is further hydrolyzed by
-glucosidase enzymes to glucose (Cheng, 2010). The joint hydrolysis of the three
groups of enzymes completes the conversion of cellulose into glucose, as shown in
figure 2.5.

25
 Cellulose
Endoglucanase

Exoglucanase Cello-oligosaccharides

Cellobiose

-Glucosidase

Glucose

Figure 2.5: Enzymatic hydrolysis of cellulose to glucose

Source: (Cheng, 2010)

2.5.4 Fermentation process for ethanol production

During the enzymatic hydrolysis, the cellulose from the lignocellulosic materials is
converted to glucose which is then fermented by yeast to ethanol and carbon dioxide
according to equation 2.2.

Yeast
Glucose Ethanol + Carbon dioxide + Heatequation 2.2

Source: (Cheng, 2010)

In yeast fermentation, the glucose solution obtained from cellulose hydrolysis is mixed
with acclimated yeast culture under aseptic conditions. The optimum temperature for
yeast fermentation is around 32 0C (Cheng, 2010). Glucose in the solution penetrates
into yeast cells and is converted by a group of enzymes created by yeast cells through a
series of enzymatic reactions to eventually ethanol, carbon dioxide, and energy. Some of

26
the released energy and glucose are utilized by the yeast cells to support their growth
during the fermentation. The rest of the energy becomes heat to the fermentation broth
and may increase the temperature if not taken out of the system. Both ethanol and
carbon dioxide penetrate out of yeast cells. Carbon dioxide readily dissolves in water,
but can be easily saturated in fermentation broth. The excess carbon dioxide bubbles out
of the liquid and can be collected for food and soft drink preparation. Ethanol dissolves
in water at any ratio and the carbon dioxide bubbling helps the transportation of ethanol
from around the yeast cells to the bulk fermentation broth, avoiding the occurrence of
high ethanol concentration in local areas that may be toxic to yeast cells. The overall
biochemical reactions to convert glucose to ethanol and carbon dioxide in yeast
fermentation can be expressed as equation 2.3 (Cheng, 2010),

C6H12O6 + 2ADP 2C2H5OH + 2ATP + 10.6 kj.Equation 2.2

where ADP and ATP represent adenosine diphosphate and adenosine triphosphate,
respectively. The process involves a series of enzymatic reactions carried out by the
enzymes generated by yeast cells under anaerobic conditions (Cheng, 2010).

2.5.5 Enzymatic hydrolysis and fermentation strategies for production of

bioethanol from lignocellulose biomass

2.5.5.1 Separate Enzymatic Hydrolysis and Fermentation (SHF)

In this process, pre-treated lignocellulose are hydrolyzed to glucose and subsequently
fermented to ethanol in separate units (Figure 2.6). The major advantage of this method
is that it is possible to carry out the cellulose hydrolysis and fermentation at their own
optimum conditions. The optimum temperature for cellulaseis usually between 45 and
50 oC, depending on cellulase producing micro-organism (Taherzadeh & Karimi, 2007).
The optimum temperature for most of the ethanol-producing micro-organisms is
between 30 and 37 oC. Inhibition of cellulase activity by the released sugars, mainly
cellobiose and glucose, is the main drawback of SHF. For instance, at a cellobiose
concentration of as low as 6 g/L, the activity of cellulase is reduced by 60%. There is
also a problem of contaminations and the process is long, for instance it can take1 to 4
days.

27
 Hexose
fermenting Nutrient and
microorganism antifoam

Pre-treated
solid Hexose rich CO2 to gas
hydrolyzate scrubber
Cellulytic Hydrolysis
Enzymes Reactor

Condensate Cooling
Steam
water out
Cooling
water in Hexose fermentation
bioreactor

Solid residue

To ethanol
distillation

Centrifuge

Nutrient and
Pentose
antifoam
fermenting
microorganism
CO2 to gas
Pentose rich scrubber
hydrolyzate Pentose fermenting
bioreactor
Cooling Cooling
water in water out

Figure 2.6: Simplified process flow diagram for separate enzymatic hydrolysis and
fermentation
Source: (Taherzadeh & Karimi, 2007)

28
2.5.5.2 Simultaneous Saccharification and Fermentation (SSF)
One of the most successful methods for ethanol production from lignocellulosic
materials is combination of the enzymatic hydrolysis of pre-treated lignocellulose and
fermentation in one step (Figure 2.7). In this process,the glucose produced by the
hydrolyzing enzymes is consumed immediately by the fermenting micro-organism
present in the culture. The inhibition effects of cellobiose and glucose to the enzymes are
minimized by keeping a low concentration of these sugars in the media (Taherzadeh &
Karimi, 2007). SSF gives higher reported ethanol yields from cellulose than SHF and
requires lower amounts of enzyme. The risk of contamination in SSF is lower than in the
SHF process, since the presence of ethanol reduces the possibility of contamination. The
number of vessels required for SSF is reduced in comparison to SHF, resulting in lower
capital cost of the process (Taherzadeh & Karimi, 2007). An important strategy in SSF
is to have the optimum conditions for the enzymatic hydrolysis and fermentation as
close as possible, particularly with respect to temperature and pH. However, the
difference between optimum temperatures of the hydrolyzing enzymes and fermenting
micro-organisms is still a drawback of SSF. The optimum temperature for cellulases is
usually between 45 and 50 oC, whereas S. cerevisiae has an optimum temperature
between 30 and 35 oC and is practically inactive at more than 40 oC. The optimum
temperature for SSF by using Trichoderma reesei (cellulase) and Sacharomyse
cerevisiae (fermentation enzyme) was reported to be around 38 oC, which is a
compromise between the optimal temperatures for hydrolysis and fermentation
(Tengborg, 2000). Hydrolysis is usually the rate-limiting step in SSF (Philippidis &
Smith, 1995).
Several thermotolerant bacteria and yeasts such acidothermophilum and Kluyveromyces
marxianus have been proposed for use in SSF to raise the temperature close to the
optimal temperature of hydrolysis. Inhibition of cellulase by produced ethanol might be
a problem in SSF. It was reported that 30 g/L ethanol reduces the enzyme activity by 25
Wyman, 1996). Ethanol inhibition maybe a limiting factor in producing high ethanol
concentration. However, there has been less attention to ethanol inhibition of cellulase,
since practically it is not possible to work with very high substrate concentration in SSF
because of the problem with mechanical mixing and insufficient mass transfer. Despite

29
the mentioned problems, SSF is the preferred method in many laboratory studies and
pilot scale studies for ethanol production (Taherzadeh & Karimi, 2007).

30
Hexose fermenting Nutrient and antifoam
microorganism

Pre-treated
solid CO2 to gas Solid residue
scrubber (mainly lignin)
Cellulytic
enzymes

Cooling
Cooling
water Centrifuge
water out
SSF
Bioreactor

To ethanol
distillation

Nutrient and
Pentose fermenting antifoam
microorganism

CO2 to gas
scrubber
Pentose rich
hydrolyzate

Cooling Cooling
water in
water out

Pentose fermenting
Bioreactor

Figure 2.7: Simplified process flow diagram for simultaneous saccharification and
fermentation
Source: (Taherzadeh & Karimi, 2007)

31
2.5.5.3 Nonisothermal Simultaneous Saccharification and Fermentation (NSSF)
The enzymatic hydrolysis reaction in the SSF process is operated at a temperature lower
than the optimum level of enzymatic hydrolysis. This forces the enzyme activity to be
far below its potential, which results in raising the enzyme requirement. In order to
overcome this problem, a nonisothermal simultaneous saccharification and fermentation
process (NSSF) was suggested (Wu & Lee, 1998). In this process, saccharification and
fermentation occur simultaneously but in two separate reactors at different temperatures
(Figure 2.8). The lignocellulose is retained inside a hydrolysis reactor and hydrolysed at
the optimum temperature for the enzymatic reactions (for instance 50 oC). The effluent
from the reactor is recirculated through a fermenter, which runs at its optimum
temperature (for instance 30 oC). The cellulase activity is increased 2-3 times when the
hydrolysis temperature is raised from 30 to 50 oC. The NSSF process has improved the
kinetic enzymatic reaction compared to SSF, resulting in reduction of the overall
enzyme requirement by 30-40%. Higher ethanol yield and productivity have been
observed in the NSSF compared to SSF at an enzyme loading as low as 5 IFPU/g
glucan. The terminal yield, which has been obtained in 4 days with the SSF, was
obtained in 40hours with NSSF (Taherzadeh & Karimi, 2007).
Varga et al., (2004) reports another form of NSSF for production of ethanol from
pretreated corn stover. In the first step, small amounts of cellulase were added at 50 oC,
the optimal temperature of enzymes, in order to obtain better mixing conditions due to
some liquefaction. To maximize the solid concentration, the pre-hydrolysis step was
carried out in fed-batch manner to obtain better mixing conditions by some liquefaction
of the cellulase containing substrate. In the second step, more cellulases were added in
combination with the fermenting organism, S. cerevisiae, at 30 oC. This method made it
possible to carry out, the SSF at a higher dry matter content, and is referred to as
nonisothermal SSF.

32
 Pre-treated
lignocellulose

Cellulytic Hexose
enzymes fermenting
Nutrients and
microorganism
antifoam

Steam Warm
water out

CO2 to gas
Hydrolysis scrubber
reactor

Cooling
water out

Solid residue
(mainly lignin)

To ethanol
Cooling
distillation
water in

Centrifuge

Figure 2.8: Simplified Process flow diagram for Nonisothermal Simultaneous

Saccharification and Fermentation process (NSSF)
Source: (Taherzadeh & Karimi, 2007)

33
2.5.5.4 Simultaneous Saccharification and Co-fermentation (SSCF)
Co-fermentation refers to the fermentation of both 5-carbon and 6-carbon sugars to
ethanol. The hydrolyzed hemicellulose during pre-treatment and the solid cellulose are
not separated after pre-treatment, allowing the hemicellulose sugars to be converted to
ethanol together with SSF of the cellulose (Teixeira et al., 2000). The SSCF process is
considered to be an improvement to SSF (Hamelincket al., 2005) and is meanwhile
being tested at pilot scale by the United States Department of Energy. In SSF bioreactor,
only hexoses are converted to ethanol, and pentoses can be fermented in another
bioreactor with different micro-organism. Therefore, two bioreactors and two biomass
production setups are required in SSF (Taherzadeh & Karimi, 2007). In SSCF process
(Figure 2.9), it is suggested to ferment both hexoses and pentoses in a single bioreactor
with a single micro-organism. Therefore, only a single fermentation step is required to
process hydrolyzed and solid fractions of the pre-treated lignocellulose (McMillan,
1997). Lawford and Ronsseau (1998) used a genetically engineered strain Zymomonas
mobilis that can co-ferment glucose and xylose, developed at the National Renewable
Energy Laboratory (NREL) for ethanol production by SSCF from a synthetic hardwood
prehydrolyzate and glucose. McMillan and colleagues (1999) used an adapted variant of
the NREL xylose-fermenting Z. mobilis for ethanol production from dilute-acid-
pretreated yellow poplar by SSCF. The integrated system produced more than 30g/L
ethanol and achieved 54% conversion of all potentially available sugars in the biomass
(total sugars) entering SSCF. Kim and colleagues (2006b) used a recombinant E. coli in
the SSCF of corn stover, which was pre-treated by ammonia. Both the xylan and
glucanin the solid were effectively utilized, giving an overall ethanol yield of 109% of
theoretical maximum based on glucan, a clear indication that at least some of the
xylancontent was being converted into ethanol. Teixeira and colleagues (2000) used a
recombinant strain of Z. mobilis for ethanol production from hybrid poplar wood and
sugarcane bagasse. The biomasses were pre-treated by peracetic acid combined with an
alkaline pre-treatment. The SSCF with the recombinant strain resulted in ethanol yields

34
 of 92.8 and 91.9% of theoretical from pretreated hybrid poplar wood and sugarcane
bagasse respectively.

Hexoses and Pentoses

fermenting producing
Nutrients and
microorganism
antifoam
Cellulytic
enzymes

Hemicellulose
hydrolyzate and CO2 to gas
pre-treated scrubber
lignocellulose
SSCF
Bioreactor
Cooling
Cooling
water in
water out

Solid (mainly lignin +

microorganism biomass)

Liquid effluent to
ethanol distillation

Centrifuge

Figure 2.9: Simplified process flow diagram for simultaneous

saccharification and co-fermentation (SSCF)
Source: (Taherzadeh & Karimi, 2007)

35
2.5.5.5 Consolidated Bioprocessing (CBP)
In all of the processes considered thus far, a separate enzyme production unit operation
is required or the enzymes should be provided externally. In Consolidated bioprocessing
(CBP), ethanol together with all of the required enzymes is produced in a single
bioreactor by a single micro-organisms community (Taherzadeh & Karimi, 2007). This
process is also known as direct microbial conversion (DMC). It is based on utilization of
mono- or co-cultures of micro-organisms which ferment cellulose to ethanol. CBP
seems to be an alternative approach with outstanding potential and the logical endpoint
in the evolution of ethanol production from lignocellulosic materials. Application of
CBP entails no operation costs or capital investment for purchasing enzyme or its
production (Hamelinck et al., 2005; Lynd et al., 2005).

2.5.6 Ethanol Purification

When the fermentation process is completed, ethanol concentration in the fermentation
broth is usually 10-15% (w/w) (Cheng, 2010). To have a fuel-grade ethanol, ethanol
needs to be purified to over 99% (Cheng, 2010). The purification process normally
occurs in two steps: fractional distillation and dehydration.

2.5.6.1 Fractional distillation

This is a thermal physical process, based on phase equilibrium of the ethanol-water
mixture. The process is performed in a fractionation column with plates or packing
materials. Ethanol can be mixed with water to form a solution at any ratio. The boiling
point of pure ethanol and water at atmospheric pressure is 78.4 and 100 oC, respectively.
However, the ethanol-water solution is a non-ideal solution. At certain ethanol
concentration between 0 and 93% (w/w), the solution has a higher dew point than a
boiling point (Cheng, 2010). In the fractionation column, the ethanol-water mixture is at
the boiling status, which generates an up-flow vapor with higher ethanol content and a
down-flow liquid with lower ethanol content. Thus, the effluent collected from the top of
the column has much higher ethanol content than that from the bottom of the column.
Theoretically, if there are enough plates in the fractionation column, the effluent
collected from the top of the column can be close to 93% (w/w) ethanol and the ethanol
content in the bottom effluent can be close to 0% (Cheng, 2010). Ethanol concentration

36
of 93% (w/w) is the theoretical maximum for fractional distillation because of the
azeotropic characteristics of ethanol solution at 93 to 100% (w/w). Normally, ethanol
concentration can be increased through fractional distillation to around 90% (w/w) from
the fermentation broth (Cheng, 2010).
To further remove the rest of the water, dehydration is necessary to increase ethanol
content to over 99% for use as automobile fuel. There are several methods used to
further purify ethanol beyond 90%.

2.5.6.2 Ethanol Dehydration

The following methods may be used for ethanol dehydration

Molecular Sieves
A commonly used technology for the dehydration of ethanol-water mixture is molecular
sieve adsorption. The fundamental principle is based on the different sizes of water and
ethanol molecules. The diameters of water and ethanol molecules are approximately
0.28 and 0.40 nm, respectively (Cheng, 2010). The molecular sieves used in the
dehydration to produce anhydrous ethanol usually have pores with a diameter of 0.3 to
0.35 nm which adsorb water molecules but not ethanol, so ethanol can be separate from
water (Cheng, 2010).

Drying using lime or salt

After distillation ethanol can further be purified by drying it using lime (CaO) or using
a hygroscopic material such as rocksalt. When lime is mixed with the water in ethanol,
calcium hydroxide forms. The calcium hydroxide can then be separated from the
ethanol. Similarly, a hygroscopic material will dissolve some of the water content of the
ethanol as it passes through leaving a purer alcohol (Mathewson, 1980).

Addition of an Entrainer
The ethanol-water azeotrope (mixture containing equal composition of ethanol and
water) can be broken by the addition of a small quantity of benzene or cyclohexane.
Benzene, ethanol and water form a ternary azeotrope with a boiling point of 64.9 oC.
Since this azeotrope is more volatile than the ethanol-water azeotrope, it can be

37
fractionally distilled out of the ethanol-water mixture, extracting essentially all of the
water in the process. The bottoms from such a distillation is anhydrous ethanol, with
several parts per million residual benzene. Benzene is toxic to humans and cyclohexane
has largely supplanted benzene in its role as the entrainer in this process. However, this
purification method leaves chemical residues which render the alcohol unfit for human
consumption (Mathewson, 1980).

Membrane separation
Membranes can also be used to separate ethanol and water. The membrane can break the
water-ethanol azeotrope because separation is not based on vapour-liquid equilibria.
Membranes are often used in the so-called hybrid membrane distillation process. This
process uses a pre-concentration distillation column as first separation step. The further
separation is then accomplished with a membrane operated either in vapour permeation
or pervaporation mode. Vapour permeation uses a vapour membrane feed and
pervaporation uses a liquid membrane feed (Mathewson, 1980).

Pressure Reduction
At pressures less than atmospheric pressure, the composition of the ethanol-water
azeotrope shifts to more ethanol-rich mixtures and at pressures less than 9.333 kPa, there
is no azeotrope and it is possible to distill absolute ethanol from an ethanol-water
mixture (Mathewson, 1980). While vacuum distillation of ethanol is not presently
economical, pressure-swing distillation is a topic of current research. In this technique, a
reduced-pressure distillation first yields an ethanol-water mixture of more than 95.6%
azeotrope, leaving anhydrous ethanol at the bottom.

38
 CHAPTER THREE

3.0 MATERIALS AND METHODS

3.1 Sweet Sorghum varieties

The following sweet sorghum varieties were sourced from the indicated sources.
Madhura hybrid seeds were sourced from Nimbkar Agricultural Research Institute
(NARI) Maharashta, India; Dale, Wiley, Brandes, Theis, Rema, Ramanda, RIO,
CMSXS636, CMSXS633 and CMSXS644 varieties were sourced from Brazilian
Enterprise for Agricultural Research (EMBRAPA), Brazil; while SPV1411,
IESV91018LT, IESV92OO8DL, IESV92038/2SH, IESV93042SH, were sourced from
International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Nairobi
Office-Kenya.

3.2 Plant materials, experimental design and juice extraction

The selected 16 sweet sorghum varieties were evaluated during the short rainy season of
September December 2012 and the long rains of April- July 2013 at the JKUAT
experimental farm. The type of soils at JKUAT area is rhodicferralsols with pH of 6.2
with an annual rainfall of 856 mm and a mean temperature of 25-27 C. The
experimental design consisted of a randomized complete block design (RCBD) with
three replications and each variety was sown in a plot size of 4 rows, 5 m long and 3 m
wide (15 m), the spacing was 75 cm by 30 cm and cultural practices such as weeding
and disease control were done to obtain optimum stalk and sugar yields.

At specific periods, harvesting was done manually, where sweet sorghum plants were
selected randomly from the middle rows, their leaves, heads, and pinnacles were
stripped and weighed individually. The SS juice was extracted using electrical stalk juice
crushers, filtered, clarified and held at -20 C in a freezer until further analyses and
thebagasse stored in the greenhouse to dry.

39
3.3Analytical Procedures

3.3.1 Analysis of juice and syrup

Total Sugars in terms of Brix was measured using a digital refractometer (Model PAL-
1, Atago Co. Ltd., Tokyo, Japan).

The specific sugars: The SS juice was filtered through filter paper number 1 and further
microfiltered using a 0.45 m membrane. The sugars were analyzed using a high
performance liquid chromatography (HPLC) (Model LC-10AS, Shimadzu Corp., Kyoto,
Japan) fitted with aminopropylsilyl column and a refractive index (RI) detector. The
mobile phase was acetonitrile / water at ratio of 80:20 (v/v) at a flow rate of 1 ml/min,
and the column and detector temperature was maintained at 351C. The standard stock
solution of glucose, fructose and sucrose were prepared with suitable concentrations of 5
mg/ml, 10 mg/ml and 15 mg/ml and standard curves drawn that were used to quantify
the reducing sugars of the samples. (See appendix 1, 2 and 3).

The data was analyzed statistically using Microsoft excel and analysis of variance at 5%
level of significance using the statistical software Genstat Version 14.1, and the means
were separated using Duncans Multiple Range Test (DMRT) to determine whether
there was significance difference in total and specific sugars, and apparent purity among
the varieties.

3.4 Clarification and treatment of Rio juice

This was carried out according the method of Klug (1993), with modifications.
Approximately 2 L of SS juice was heated to 50 0C and lime milk added till pH was 11
and carbon dioxide bubbled to drop the pH to 10. It was allowed to settle for 1 hr then
filtered under vacuum to give a clear juice. The juice was reheated to 50 0C and carbon
dioxide bubbled through once more to lower the pH to 7.1. The temperature was then
raised to 90 0C for 5 min and allowed to cool to 60 0C and 0.20 g (0.0097% weight of
feed) of calcium chloride and 0.26 g (0.0122% weight of feed) of amylase were added
and allowed to react for 1 hr and then vacuum filtered to get a clear thin juice. Calcium
chloride was added to remove aconitic acid whereas amylase enzyme hydrolyzed starch
to glucose.

40
3.5 Concentration of RIO juice
Approximately 1.3 lts of clarified juice was concentrated into syrup using a rotary
vacuum evaporator (Bibby Sterilin Ltd, RE 100B, UK). The temperature of the water
was set at 90 0C.

3.6 Crystallization of RIO juice

Refined sucrose was ground using a top bench grinder and passed through a 93 m sieve
to be used for seeding. Approximately 75 ml (95 g) of syrup in a 100 ml beaker with a
magnetic stirrer was cooled using ice cold water to 18 0C and the seed in ethanol as a
carrier was introduced into the syrup at this temperature.

3.7 Production of bio-ethanol from RIO Sweet sorghum bagasse

3.7.1 Size reduction of RIO bagasse

After 2 months of storage in the greenhouse, the bagasse was ground using a diesel
driven disintegrator, chopper and grinder (DPM-4, Brazil) to less than 0.84 mm.This
was further ground by a laboratory bench top mill to a size less than 0.42 mm. The
ground bagasse was then sealed in a black plastic bag.

3.7.2 Determination of Composition of RIO bagasse

3.7.2.1 Moisture determination

Moisture dishes which had been dried in a hot air oven for 1 hour and cooled in
desiccatorswere weighed using a 4 decimal analytical balance and approximately 2.00 g
of sample put in them (AOAC, 1995). Then the weight of dish plus sample was taken.
This was placed in a hot air oven and the temperature set at 105 0C and dried for 2hrs.
The samples were removed and put in a desiccator to cool to room temperature and then
reweighed. They were then put back in the hot air oven for 30 minutes and removed and
put in a desiccator for 15 minutes to cool to room temperature and then reweighed. This
was stopped when a constant weight was observed. The formula used was equation 3.1,

W1 W2
%Moisture (Wet basis) = *100 .Equation 3.1
W1 W0

Where W1-Weight of sample plus dish before drying

41
 W2- Weight of sample plus dish after drying
W0-Weight of empty dish

3.7.2.2 Determination of total ash content

Using a marker pen, 3 silica crucibles were marked and approximately 3 g of RIO
bagasse was weighed into the numbered crucibles in triplicate. The sample was then
charred on a hot plate in the fume cupboard till no visible smoke was present (AOAC,
1995). Once charred; the crucibles were placed on a heat resistant mat and transferred to
a muffle furnace heated to 550 0C and heated in the furnace for 7 hrs. Since the sample
was not yet white or grayish white, it was moistened with distilled water and reheated on
a hot plate in the fume cupboard, then transferred to the muffle furnace for further 9 hrs,
total time taken was16hrs. Samples were removed using tongs and cooled in a desiccator
and weighed to determine the mass of ash (Equation 3.2).

Average weight of ash

% Ash( w / w) = 100 Equation 3.2
Weight of sample

3.7.2.3 Determination of extractives by soxhlet extraction

Approximately 3 g of RIO sweet sorghum bagasse was put in a thimble in triplicate and
numbered and the thimble top closed with cotton wool. The thimble was then inserted in
the soxhlet apparatus and extracted with petroleum ether for 12 hrs contained in pre-
weighed round bottom flasks (AOAC, 1995). After extraction, the solvent was distilled
off from the round bottom flask using the rotary evaporator. The flasks and extractives
(thimbles) were then dried on a hot plate in the fume cupboard and then weighed using
the analytical balance and percentage extractives calculated.

3.7.2.4 Determination of lignin concentration and neutral sugars

Extracted bagasse samples were weighed in quantities of 300 mg in triplicate and mixed
with 3 ml of 72% H2SO4. The mixture was put in a water bath (MODEL: SHA-C, SN:
10706002) at 30 0C for 1 hr for the hydrolysis reaction to take place. The acid was then
diluted by adding 73 ml of distilled water to a final concentration of 3%. The sample
was then placed in an autoclave (Model Autoclave SS-325, Tomy Seiko Co., Ltd,

42
Tokyo, Japan) at 125 0C for 1 hr, cooled and filtered through filter paper number 1. The
solids were dried to constant weight at 80 0C using a hot air oven and the resulting mass
was taken as acid insoluble lignin. The filtrate was analyzed for neutral sugars namely
glucan, galactan, mannan, and xylan using HPLC (Model LC-10AS, Shimadzu Corp.,
Kyoto, Japan) under similar conditions as those for soluble sugar analysis.

3.7.3 Rio bagasse pretreatments

3.7.3.1 Phosphoric acid acetone pretreatment

Approximately 6 g of bagasse were weighed using an analytical balance and put in 50
ml plastic centrifuge tubes and 60 ml (85%) H3PO4 added. The mixture was then put in a
water bath (MODEL: SHA-C, SN: 10706002) at 50 0C at 90 rpm for 30 minutes. The
treated slurry was washed with 20 ml cold acetone and centrifuged (H-200 000,
Kokusan Corporation, Japan) at 4000 rpm for 20 minutes and at a temperature of 27 0C.
The bagasse was then washed three times with 40 ml acetone, followed by three times
with 40 ml distilled water. The residual acetone from washing stages was removed from
the supernatant by simple evaporation in a fumehood.The treated bagasse was washed
using hot distilled water several times till clear liquid was produced at pH 7 (Jeihanipour
& Taherzader, 2009; Zhang et al., 2007). It was then dried in a hot air oven at 401
0
C for 2 days and kept in a refrigerator at -20 0C.

3.7.3.2 Alkaline hydrogen peroxide pretreatment

Approximately 50 ml of hydrogen peroxide was added to 700 ml of distilled water and
the pH adjusted to 12.70 using sodium hydroxide. Four grams of bagasse were treated
with 100 ml of the pretreatment agent in 250 ml flasks in a water bath (MODEL: SHA-
C, SN:10706002) at 2.5 Hz for 4 hrs. It was then washed with warm distilled water till
pH was neutral and then dried at 45 0C in a hot air oven.

3.7.4 Enzymatic hydrolysis

Six, 150 ml conical flasks were labeled and approximately 1 g of untreated, phosphoric
acid pretreated and alkaline peroxide pretreated bagasse, in duplicate was put in the
labeled conical flasks. The pH of citrate buffer was adjusted to 4.8 using 0.5 M H2SO4
and 100 ml of citrate buffer was added to each conical flask. This was then autoclaved

43
(Model Autoclave SS-325, Tomy Seiko Co., Ltd, Tokyo, Japan) at 121 0C for 1 hr and
allowed to cool in the autoclave to 50 0C. Approximately 3.3 mg (20FPU) of cellulase
produced by Trichoderma reesei was then added to each flask and incubated in a
shaking water bath at 50 0C for 72 hrs. Samples were drawn after 21 hrs, 30 hrs, 45 hrs,
54 hrs and 72 hrs for sugar analysis and placed in a boiling water bath for 15 minutes to
deactivate the cellulase and then stored in a deep freezer at -20 0C. The resulting
hydrolysate was then deactivated in a boiling water bath for 15 minutes and then stored
in a deep freezer at -20 0C awaiting fermentation.

The yield of enzymatic hydrolysis was calculated as a ratio of theoretical glucose

production yield using equation 3.3:

Yield of enzymatic hydrolysis (%)

Pr oduced glu cos e ( g / l ) 100

= ..Equation 3.3
1.111 Substrate concentrat ion ( g / l ) F

(Source: Goshadrou et al., 2011)

Where F in the denominator is the biomass glucan fraction, and is presented in Table 4.5
for untreated and Table 4.6 for different pretreated bagasse. The conversion factor of
1.111 was applied to consider the conversion of glucan to glucose.

3.7.5 Fermentation
The pH of the hydrolysates was adjusted to 6.00.1 using 0.5 M NaOH or 0.5 M H2SO4.
A mass of 1 gm of glucose each was put into two more conical flasks for use as the
reference. These were autoclaved (Model Autoclave SS-325, Tomy Seiko Co., Ltd,
Tokyo, Japan) at 121 0C for 1 hr and then cooled to 30 0C. Approximately 0.3 g/100ml
of the yeast strain sacharomyce cerevisiae was added into each of the conical flasks that
had glucose and the hydrolysates and supplemented with 0.005 g/100ml of (NH)2HP04
and 0.001 g/100ml MgSO4.7H2O as nutrients. The samples were then placed in a water
bath (MODEL: SHA-C, SN:10706002) at 30 0C shaking at 150 rpm and fermented for
48 hrs. Approximately 5 ml samples were collected from each flask and centrifuged at
13000 rpm for 5 minutes and the supernatants was then stored in a deep freezer at -20 0C

44
for GC analysis. The fermentation broth was placed in a boiling water bath for 15
minutes to deactivate the enzyme and then stored in a deep freezer at -20 0C. Equation
3.4 was used for calculation of ethanol production yield:

Yield of ethanol production (%)

Pr oduced ethanol ( g / l ) 100

= ... Equation 3.4
0.511.111 Substrate Concentrat ion ( g / l ) F

(Source: Goshadrou et al., 2011)

3.7.5.1 Ethanol analysis

The ethanol concentration was determined by a gas chromatography using a GC-9A
Shimadzu, equipped with a packed column and a flame ionization detector. The
temperature of the injector was 220 0C and that of the detector 240 0C. For the column, a
gradient of 50-150 0C was used and nitrogen was used as a carrier gas while hydrogen
and air were used as the combustion gases. The ethanol, chromatographic grade and
butanol (0.5 g/L) were used for the standard curve and internal standard substance
separately. Samples of 1 l were directly injected into the column. All quantifications
were done by means of standard curves, and the final results were the average of two
repetitions.

45
 CHAPTER FOUR

4.0 RESULTS AND DISCUSSION

4.1 The total sugar content, glucose, fructose and sucrose concentration
Data is presented in Table 4.1 of various SS varieties. Madhura had the lowest 0Brix of
15.05 while Dale had the highest 0Brix of 21.50 after 16 weeks of planting in the field.
The results are similar to what was observed by Reddy et al., (2005) of 16- 23 0Brix
and slightly higher than that observed by Woods (2000) of 11.0-18.5 0Brix. This
variation could be attributed to stalk variety, different soils and climatic conditions. All
the varieties except Madhura, Wiley, Brandes and IESV93042SH had a 0Brix higher
than that of sugarcane of 16.8 (Woods, 2000). Madhura had the lowest sucrose
concentration of 6.05 g/L whereas variety IESV91018LT had the highest sucrose
concentration of 72.77 g/L. These values far exceed those observed by Reddy et al.,
(2005). These differences could also be attributed to different soils and climatic
conditions. Those observed by Woods (2000) ranged from 6.3 12.8 g/L and for
sugarcane the sucrose concentration was 14.1 g/L. Variety CMSXS636 had the lowest
glucose and fructose concentration of 2.65 g/L and 2.66 g/L, respectively, whereas
Wiley had the highest glucose and fructose concentration of 16.42 g/L and 17.14 g/L,
respectively. To the best of authors knowledge, there is no literature quantifying
glucose and fructose concentrations in sweet sorghum varieties, as they are lumped
together as reducing sugars, hence there is no basis for comparison. According to the
different positions of sugar contained in the stalks, it can be divided into saccharin-type
SS and syrup-type SS (Li Dajue, 1995). Saccharin-type SS, which mainly contains
sucrose, can be used for refining crystal sugar. Syrup-type SS, which mainly contains
glucose, can be used for producing syrup. Also, syrup-type SS is a material of quality for
making drinking wine and alcohol.

46
Table 4.1: Total sugar content in 0Brix, glucose, fructose and sucrose in g/L

0
Variety Brix Glucose, Fructose, Sucrose, g/L
g/L g/L
z
Madhura 15.050.92a 3.020.05b 3.130.16b 6.050.08a

Wiley 16.201.41ab 16.410.15l 17.140.13k 17.200.14d

Brandes 16.400.85ab 8.030.07h 7.870.09f 15.990.19c

IESV93042SH 16.750.49abc 3.110.15b 3.170.09b 22.330.32g

CMSXS633 17.000.00bc 3.120.05b 3.140.19b 19.200.28e

Rema 17.001.41bc 3.790.01c 7.300.16e 25.110.16h

Ramanda 17.700.42bcd 6.910.04g 10.000.18g 38.070.33k

Theis 17.900.14bcd 10.300.24j 11.400.24h 38.190.13k

IESV91018LT 18.000.14bcd 8.390.24i 12.530.43j 72.770.15m

CMSXS636 18.500.71cd 2.650.14a 2.660.15a 26.100.15i

IESV92008DL 19.000.28de 7.010.16g 16.950.08k 38.370.50k

IESV92038/2SH 19.000.71de 5.620.02f 5.900.02d 20.670.68f

SPV1411 19.000.71de 4.980.14e 3.160.06b 27.210.14j

CMSXS644 20.500.71ef 3.760.14c 4.110.17c 9.620.87b

Rio 20.700.14ef 4.620.06d 3.210.13b 40.860.11l

Dale 21.501.41f 12.470.09k 11.990.14i 21.240.34f

Values are presented as Mean SD, n=2z means within columns followed by the same
letter(s) were not significantly different (P0.05)

4.2 Sucrose /Apparent purity (AP)

In Table 4.2 are tabulated values for sucrose purity for the sixteen sweet sorghum
cultivars which were calculated according to the equation 4.1. Wiley had the minimum
value of 33.89% whereas 6 varieties namely, CMSXS633, SPV1411, IESV91018LT,
IESV93042SH, CMSXS636 and Rio had values above 75% of, 75.42%, 76.99%,

47
 77.68%, 78.08%, 83.08% and 83.91%, respectively. A similar report was given earlier
by Woods (2000) where the AP for the sweet sorghum varieties considered varied from
48.2% - 69.7% whereas that of sugarcane juice was 83.6%. These cultivars have the
potential of crystal sugar production as they have an AP greater than 75%(Woods,
2001).

Pol
Sucrose/Apparent purity = ( 100 )..Equation 4.1
Brix

Table 4.2: Apparent / Sucrose purity in percentage

Variety Wiley Dale Madhura Brandes CMSXS644 IESV92008DL Theis IESV92038/2SH

AP, % 33.890.20az 46.480.17b 49.600.52c 50.130.54c 54.971.27d 61.550.08e 63.790.43f 64.210.68f

Ramanda Rema CMSXS633 SPV1411 IESV91018LT IESV93042SH CMSXS636 RIO

69.240.09g 69.350.47g 75.420.45h 76.990.54i 77.680.52i 78.080.42i 83.080.69j 83.910.07j

Values are presented as Mean SD, n=2

z
means within columns followed by the same letter(s) were not significantly different
( P0.05)

4.3 Crystallization process for RIO juice

After characterization of the sweet sorghum cultivars, the one with the highest apparent
purity of 83.91%, Rio, (Table 4.2) was chosen for crystal sugar production. Sucrose
purity is used to calculate the ease with which sucrose can be extracted and crystallized
and 75% is required as the minimum (Woods, 2001).

4.3.1 Extracted Rio juice

From Table 4.3, it can be deduced that 1000 kg (1 metric ton) of Rio stalk after 17
weeks in the farm will yield 312 L of thin unclarified juice of 18.8 0Brix and 642 kg of
bagasse. The 0Brix of the juice is similar to that recorded by Gnansounou et al.,

48
(2005) of 17.5. The minimum 0Brix required for crystal sugar production is 12 (Woods,
2001) hence the juice qualifies to be used for crystal sugar production. From Figure 4.1,
the extracted juice had approximately 132 g/L of sucrose, 6 g/L of glucose and 7 g/L of
fructose and an apparent purity of 91% (Figure 4.3). This is quite appropriate for
saccharine-type juice for crystal sugar production as it should have a higher sucrose
concentration relative to the reducing sugars.

Table 4.3: Characteristics of extracted Rio juice from the sugarcane presser

0
Variety Age in Weight of Volume of Weight of bagasse Brix of juice pH of
days stalks (kg) expressed produced (kg) on juice
juice (lts) wet basis

Rio 122 12.50.71 3.90.14 8.0250.25 18.80.28 4.750.07

Values are presented as MeanSD, n=2

4.3.2 Rio syrup

The Rio syrup from the evaporator had a 0Brix of 68.05 (Table 4.4). From the results, it
can be deduced that 1000 lts of clarified juice fed to the vacuum rotary evaporator will
produce approximately 58.8l L of syrup. From Figure 4.2, the syrup had 603 g/L of
sucrose, 19 g/L of glucose, 14 g/L of fructose and an apparent purity of 95%
(Figure 4.3).

Table 4.4: Characteristics of Rio syrup from the evaporator

0
Sorghum Volume of Volume of Mass of syrup, BRIX
variety clarified juice syrup, ml grams

(feed), lts

Rio 1.250.07 73.52.12 92.151.63 68.050.21

Values are presented as MeanSD, n=2

49
 160
140

Sugar concentration, g/l

120
100
80
60
40
20
0
Sucrose Fructose Glucose
Specific sugar

Figure 4.1: Specific sugar concentrations in Rio juice after extraction prior to
clarification

700
Sugar concentration, g/l

600

500

400

300

200

100

0
Sucrose Fructose Glucose
Specific sugar

Figure 4.2: Specific sugars present in RIO syrup before crystallization

50
 96
95

Apparent Purity, %
94
93
92
91
90
89
88
Juice Syrup

Figure 4.3: Apparent purity of clarified Rio juice and Rio syrup before
crystallization

4.3.3 Rio syrup crystallization

The Rio syrup did not crystallize into crystal raw sugar as turbidity was not observed.
The sweet sorghum juice is not commonly used for crystallized sugar production
because of the presence of significant amounts of inverted sugars (glucose and fructose)
and antiquality compounds such as aconitic acid that makes crystallization difficult and
expensive (Wang, 2014). The sweet sorghum cultivar chosen for crystal sugar
production must have a high content of sucrose in the stalks and low levels of starch and
aconitic acid which impede sorghum sugar crystallization (Kulp, 2000). During
clarification, amylase enzyme was used to minimize the concentration of starch in the
juice, but this enzyme converts starch to glucose thus increasing the concentration of
glucose in the juice which is counterproductive. The concentration of glucose in the Rio
syrup was 19 g/L compared to 7 g/L in the unclarified Rio thin juice. On the other hand,
fructose concentration in the syrup and juice was 13.8 g/L and 6 g/L respectively.
Therefore the residual concentration of invert sugars in the Rio syrup was relatively high
and could have hindered crystallization. Calcium chloride was used during clarification
to eliminate aconitic acid but the concentration of the aconitic acid was never
determined to find out if it was completely removed from the sample. The other

51
probable reason for not getting crystal sugar is because of the presence and
concentration of dextran(Abdel-Rahman,2007). The presence of dextran in the sugar
factories leads to a falsely high polarization, increased viscosity, slowing of filtration,
lower evaporation rates, elongated crystals (needle grain), longer wash and separation
cycles in centrifuges and increase of sugar loss to molasses (Imrie & Tilbury, 1972;
McGinnis, 1982; Jolly & Prakash, 1987; Singletone et al., 2001; Singleton, 2002; Kim
& Day, 2004). However, the most damaging effects of elevated dextran concentrations
in a technical sucrose solution are foreseen in the crystallization process. Dextrans slow
down the crystallization rate or even inhibit crystallization (i.e., they have a high
melassigenic effect). It is estimated that for every 300 ppm dextran in syrup there is a
1% increase in molasses purity (the % ratio of sucrose) in total solids in a sugar solution
(Atkins & McCowage, 1984; Godshall et al., 1994; Clarkeet al., 1997; Cerutti de
Guglielmone et.al., 2000).

4.4 Production of bioethanol from Rio sweet sorghum bagasse

4.4.1 Composition of Rio SSB

The sweet sorghum bagasse used in this study contained glucan (38.26%), xylan
(17.22%), Acid insoluble lignin (21.07%) and ash (3.94%) (Table 4.5). Galactan,
mannan and arabinan were not detected. This does not mean that they were absent
because other researchers like Shen (2011) was able to detect them though their values
were less than 2%. The glucan content was higher than that of 35.1% got by Shen (2011)
but similar to that got by Wu (2011) of 38.7%. Approximately 60% of the total dry
matter was polysaccharide, in which the largest fraction was glucan occupying 38.26%
in total carbohydrate content. Thus, such high polysaccharide content could be
potentially made available for hydrolysis and subsequent ethanol fermentation.
Moreover, the ash content accounted for 3.94% of the total dry matter of SSB, and it was
lower than that obtained by Ballesteros (2004) for SSB of 4.8% but higher than that of
sugarcane bagasse of 1.6% obtained by Rabello (2011). Lower ash content in
lignocellulosic material was proven to be potentially beneficial for enzymatic hydrolysis
(Yu & Chen, 2010). The extractives consisted of 18.34% of the total dry matter.

52
Table 4.5: The composition of Rio sweet sorghum bagasse (weight percent of dry
matter)

Carbohydrates Extractives Acid Ash

Insoluble (%wt/wt)
(%wt/wt) (%wt/wt)
lignin
(%wt/wt)

Glucan Xylan Galactan Mannan

38.260.35 17.220.28 n.d.a n.d. 16.340.25 21.070.43 3.940.22

Values are presented as Mean SD, n=3

a
n.d. means not detected.

4.4.2 Pretreatment
The compositions of the bagasse after the pretreatments were analyzed and the results
are presented in Table 4.6. Glucan was the dominant component in the range of 49.12%
to 63.4% and xylan was second in the range of not detected to 14.7%. There was a
significant increase in glucan fraction after the pretreatments. The glucan fraction
increased by 65.7% and 28.4% after the pretreatment by sodium hydroxide and
phosphoric acid, respectively. This was so because the pretreatment eliminated lignin
thus exposing the cellulose to the pretreatment agent. Contrary to glucan, xylan
decreased by 14.6% when sodium hydroxide was used and it was not detected when
phosphoric acid was used. This implies that the pretreatment agents reacted with the
hemicellulose thus lowering the percentage xylan detected. According to Goshadrou
(2011), 58.66% glucan was observed when only sodium hydroxide solution was used as
the pretreatment agent (41.9% increase in glucan fraction) and 52.25% glucan (26.42%
increase in glucan fraction) when phosphoric acid was used as the pretreatment agent.
On the other hand xylan dropped by 29.2% and 33.85% when sodium hydroxide and
phosphoric acid were respectively used. The interest in the use of phosphoric acid is that
after neutralization of hydrolysates with NaOH, the salt formed is sodium phosphate
(Gmez et al., 2006). This salt can remain in the hydrolysates because it is used as

53
nutrient by micro-organisms (Cardona, 2010). Therefore, a filtration operation of it is
not needed with the subsequent advantages: improvement of process profitability
(avoiding salts removal and decreasing the amount of nutrients needed for fermentation)
and positive impact to the environment (the salt formed is not a waste).

Table 4.6: The carbohydrate content of pretreated Rio sweet sorghum bagasse in
different conditions

Pretreatment Glucan(%) Xylan(%) Mannan(%) Galactan(%)

method

NaOH 63.400.76 14.700.60 n.d. n.d.

H3PO4 49.122.31 n.d.a n.d. n.d.

Values are presented as Mean SD, n = 2

a
n.d. means not detected

4.4.3 Enzymatic hydrolysis (saccharification)

Different preparations of Rio sweet sorghum bagasse were subjected to 72 h enzymatic
hydrolysis by addition of cellulase. The most important hydrolysis results are presented
as percentages of theoretical sugar yield in Table 4.7 and glucose concentration in
Figure 4.4. The yield of hydrolysis of native bagasse was effectively improved after
sodium hydroxide and concentrated phosphoric acid pretreatments. Hydrolysis of the
untreated bagasse resulted in 15% and 50% conversion after 30 h and 72 h, respectively.
The hydrolysis yield increased from 50% to 78% after pretreatment by concentrated
phosphoric acid. The best results of enzymatic hydrolysis were obtained in the
hydrolysis of pretreated bagasse by NaOH solution, where more than 88% of the
theoretical glucose yield was obtained within 72 h. According to Figure 4.4, the rate of
glucose generation was low initially for all the pretreatments, and then increased steadily
and later slowed down except for the untreated SSB scenario. This could be due to the
fact that initially, the enzyme cellulase was undergoing acclimatization after which the
rate of glucose generation increased. After 54 h, the decline in glucose concentration is
due to the fact that the activity of cellulase enzyme is inhibited by the released sugars,

54
mainly cellobiose and glucose. For instance, at a cellobiose concentration of as low as
6g/L, the activity of cellulase is reduced by 60% (Taherzadeh, 2007).

Table 4.7: Yield of enzymatic hydrolysis of untreated and different pretreated

sweet sorghum bagasse (as percentage of theoretical yield)

Pretreatment method Yield of enzymatic hydrolysis(% theoretical sugar yield)

Hydrolysis time 30 h 54 h 72 h

Untreated 15% 29% 50%

NaOH 32% 84% 88%

Phosphoric acid 22% 76% 78%

14

12
Glucose concentration, g/l

10

8 NaOH
Phosphoric acid
6
untreated
4

0
0 30 54 72
Hydrolysis time, h

Figure 4.4:Effect of different pretreatments on hydrolysis of cellulose

4.4.4 Fermentation
Figures 4.5 and 4.6 show the results of fermentation. Pure glucose was selected as a
reference in fermentation and a mass ethanol yield and productivity of 0.51 g/g and
0.106 g/L.h, respectively were observed. The results showed significant improvements

55
in ethanol production from 15.33% of the theoretical yield for untreated bagasse to
40.45%-59.44% for the different pretreated materials depending on the pretreatment
method. According to Figure 4.5, phosphoric acid pretreatment improved the ethanol
production yield to 59.44% (44.11% increment), which was the better result for ethanol
production between the two applied pretreatment techniques. The results showed that
pretreatment with sodium hydroxide increased ethanol yield by 25.12%.

The rate of ethanol production for untreated and all pretreated materials is presented in
Figure 4.6. The rate of ethanol production for untreated bagasse hydrolyzate was 0.001
g/L.h, which was improved after the pretreatments. This was so because after the
pretreatments, the amount of cellulose available for hydrolysis to glucose and later
fermentation to ethanol increased (Table 4.6). The materials pretreated with sodium
hydroxide showed the highest productivity (0.019 g/L.h) whereas the phosphoric acid
pretreated bagasse had the productivity of 0.016 g/L.h. Ethanol yield for the
hydrolysates was lower than that obtained when glucose was used as substrate.
According to Cheng (2010), when the fermentation process of any lignocellulosic
material is completed, ethanol concentration in the fermentation broth is usually 10-15%
(w/w) or 13-19% (v/v). Therefore the results observed of 0.009-0.646% (v/v) of ethanol
in the fermentation broth are far much lower than those observed by Cheng (2010) of
13-19% (v/v). This difference could be due to the amount and type of enzyme used and
the conditions of fermentation and even the presence of fermentation inhibitors. For
instance, the bakers yeast used in this research can only ferment hexoses and not
pentoses thus affecting the ethanol yield observed.

56
 70

Maximum ethanol
60
50

yield,(%)
40
30
20
10
0
UNTREATED NaOH PHOSPHORIC
PRETREATED ACID
PRETREATED

Figure 4.5: Maximum ethanol yield (% theoretical yield)

0.12

0.1
Maximum volumetric ethanol
production(g/l.h)

0.08

0.06

0.04

0.02

0
Pure glucose NaOH Phosphoric acid Untreated

Figure 4.6: Rate of ethanol production (g/L.h)

57
 CHAPTER FIVE
5.0 CONCLUSION AND RECOMMENDATIONS

5.1 Conclusion
Due to the lower purity (ratio of the %wt. of sucrose to the %wt. of soluble) of the sugar
extracted from sweet sorghum (about 75 AP) compared to that of sugar cane or sugar
beet (80-85 AP), it may require further technological input to produce white sugar from
sweet sorghum. Thus, the more likely markets for sorghum sugar can be syrup for local
foodstuffs or as raw material for the food industry.

According to the study, the following sweet sorghum cultivars namely; Rio,
CMSXS636, IESV91018LT, IESV93042SH and SPV1411 could have the potential to
be used in raw sugar production. If starch, dextran and aconitic acid can be removed,
crystal sugar can be obtained from the sweet sorghum juice, especially from the variety
RIO. Bioethanol can also be obtained from the Sweet Sorghum bagasse after
pretreatment and saccharification. This work therefore provides a complimentary source
of sugary products and biofuel.

5.2 Recommendations
Further work is needed to increase the yield of bio-ethanol from Rio sweet sorghum
bagasse and production of crystal sugar from Rio syrup. The juice of IESV91018LT
cultivar should be subjected to the process of crystallization as it had the highest sucrose
concentration of 72.77 g/L (Rio had 40.86g/L) of all the varieties characterized and an
AP of 77.68%. Once this is done, the experiments should be scaled up from laboratory
scale to pilot plant scale before commercialization of the process of raw white sugar and
bio-ethanol production.

58
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 7. APPENDICES

Appendix 1: Fructose standard curve

Fructose
800000

600000 y = 48355x
R = 0.9994
400000

200000

0
0 5 10 15 20

Appendix 2: Glucose standard curve

1500000 Glucose
1000000 y = 72635x
R = 0.9923
500000

0
0 5 10 15 20

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Appendix 3: Sucrose standard curve

Sucrose
1200000
1000000
800000 y = 69279x
600000 R = 0.991
400000
200000
0
0 5 10 15 20

Appendix 4: Three months old Sweet Sorghum in the JKUAT experimental farm

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Appendix 5: Juice extraction using Sugarcane presser

Appendix 6: Sample injection into the HPLC instrument for sugar analysis

72
Appendix 7: Concentration of clarified juice using a rotary vacuum evaporator

Appendix 8: Enzymatic hydrolysis of pretreated Rio bagasse

73
Appendix 9: Ethanol analysis using GC equipment

74
Appendix 10: The ANOVA results of sucrose, glucose, fructose, Apparent Purity
and 0Brix analysis

Source ss df ms P value L.s.d S.e.d

Sucrose 7559.1849 15 503.9457 0.001 0.7613 0.3591

0
Brix 93.8050 15 6.2537 0.001 1.687 0.796

Fructose 760.4056 15 50.6937 0.001 0.3740 0.1764

Glucose 451.7111 15 30.1141 0.001 0.2738 0.1292

Apparent 6378.6328 15 425.2422 0.001 1.1319 0.5339

purity

75