Pbpk models for the prediction of in vivo performance of oral dosage forms
OGU RICHARD 09CF09371
1. COLLEGE OF SCIENCE AND TECHNOLOGY
SCHOOL OF ENGINEERING
DEPARTMENT OF CHEMICAL ENGINEERING,
COVENANT UNIVERSITY, OTA.
ALKALINE PRETREATMENT AND ENZYMATIC HYDROLYSIS OF
RICE HULLS
A FINAL YEAR RESEARCH PROJECT
BY
OGU RICHARD AFENOKO
09CF09371
APRIL 2014
2. ii
ALKALINE PRETREATMENT AND ENZYMATIC HYDROLYSIS OF
RICE HULLS
A FINAL YEAR RESEARCH PROJECT
Presented to
College of Science and Technology
School of Engineering
The Department of Chemical Engineering
By
OGU RICHARD AFENOKO
MATRICULATION NO.: 09CF09371
In Partial Fulfilment of the requirements for the Degree Bachelor of Engineering in
Chemical Engineering
APRIL, 2014
3. iii
CERTICIFICATION
I hereby declare that the contained report on “Alkaline pre-treatment and enzymatic
hydrolysis of rice hulls” was researched, the results thoroughly analysed, under the
supervision of my project supervisor and approved having satisfied the partial
requirements for the award of Bachelor of Engineering in Chemical Engineering
(B.Eng.), Covenant University, Ota.
___________________________ _____________________________
OGU RICHARD AFENOKO Date
__________________________ ___________________________
DR. A.O AYENI Date
Supervisor
___________________________ _____________________________
PROF. F.K. HYMORE Date
Head of Department
4. iv
DEDICATION
I dedicate this to report to God Almighty, the reason why I live and also to my parents Chief
& Mrs Ogu.
5. v
ACKNOWLEDGEMENT
I want to express my unending gratitude to God Almighty for His extravagant grace upon my
life. Without Him I would be nothing.
I also appreciate my parents greatly for giving me the opportunity to come to Covenant
University and build a career in Chemical Engineering.
I am thankful to my supervisor, Dr. A.O Ayeni for his guidance and meticulous supervision of
my project work.
I also express my gratitude to the academic and non-academic staff of the Chemical
Engineering Department for all the assistance given to me during the course of my research. I
am too grateful.
Furthermore, I would like to appreciate my friends and course mates who gave me their support
and encouragement throughout the research period. You all are the best.
Finally, to my siblings Onuche, Arikpi and Daniel, I say a big thank you for your prayers and
love.
6. vi
ABSRTACT
Alkaline pretreatment was performed before the conversion of rice hulls to reducing sugars
through enzymatic hydrolysis using Trichoderma ressei cellulase enzyme. The effects of time,
temperature and hydrogen peroxide concentration were studied. A statistical software,
MINITAB was used to determine the optimum pretreatment conditions which were validated
experimentally. The validated optimized conditions of 49.8ºC, 11.36 hours and 3.68% with a
biomass loading of 4% and 25FPU/g enzyme loading gave the highest reducing sugar yield of
192.89mg/g of dry biomass. When compared with the reducing sugar yield of the untreated
sample which gave 32.8mg/g of dry biomass, it was seen that alkaline pretreatment could be
used to pretreat rice hulls to a substantial level for better reducing sugar yields after enzymatic
hydrolysis.
7. vii
TABLE OF CONTENTS
CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND STUDY ...............................................................................................1
1.2 AIMS AND OBJECTIVES..............................................................................................2
1.3 SCOPE .............................................................................................................................2
1.4 JUSTIFICATION.............................................................................................................4
1.5 RELEVANCE OF STUDY..............................................................................................4
1.6 RESEARCH LIMITATIONS ..........................................................................................5
CHAPTER TWO
LITERATURE REVIEW
2.1 BIOFUEL.........................................................................................................................6
2.1.1 Classification Of Biofuels.............................................................................................6
2.1.2 Types Of Biofuels .........................................................................................................7
2.1.3 Biofuel Vs Fossil Fuel...................................................................................................7
2.1.4 Greenhouse Gas (GHG) Emissions And Global Warming...........................................9
2.1.5 Current Trends...............................................................................................................9
2.2 LIGNOCELLULOSIC BIOMASS ..................................................................................9
2.2.1 Structure Of Lignocellulosic Biomass ........................................................................10
2.2.2 Products Of Lignocellulosic Biomass.........................................................................12
2.2.3 Production Of Ethanol From Lignocellulosic Biomass ..............................................12
2.2.3.1 Acid Hydrolysis........................................................................................................14
2.2.3.2 Enzymatic Hydrolysis ..............................................................................................15
2.2.3.2.1 Cellulosic Capability Of Organisms: Difference In The Cellulose-Degrading
Strategy.................................................................................................................................16
2.2.3.2.2 Characteristics Of The Commercial Hydrolytic Enzymes....................................21
2.3 RICE HULLS.................................................................................................................24
2.4 ABSORBANCE.............................................................................................................26
2.4.1 Measuring The Absorbance Of A Sample Using A Spectrophotometer ....................26
8. viii
2.4.2 The Importance Of Concentration...............................................................................26
2.4.3 The Importance Of The Container Shape ...................................................................27
CHAPTER 3
METHODOLOGY
3.1 MATERIALS USED......................................................................................................28
3.1.1 Biomass.......................................................................................................................28
3.1.2 Chemicals Required ....................................................................................................28
3.2 EQUIPMENT USED .....................................................................................................28
3.3 BRIEF SUMMARY OF WORK DONE .......................................................................28
3.4 ALKALINE PRETREATMENT...................................................................................32
3.5 ENZYMATIC HYDROLYSIS......................................................................................34
3.6 OPTIMIZATION OF THE ALKALINE PRETREATMENT AND ENZYMATIC
HYDROLYSIS CONDITIONS FOR RICE HULLS. .........................................................34
3.7 GLUCOSE ANALYSIS.................................................................................................35
CHAPTER 4
RESULTS AND DISCUSSIONS
4.1 SIEVE ANALYSIS........................................................................................................36
4.2 LABORAORY ANALYSIS ..........................................................................................36
4.3 ALKALINE PRETREATMENT...................................................................................41
4.4 ENZYMATIC HYDROLYSIS......................................................................................41
4.5 OPTIMIZATION OF PRETREATMENT CONDITIONS...........................................48
4.6 GLUCOSE TEST...........................................................................................................51
CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS
5.1 CONCLUSION..............................................................................................................55
5.2 RECOMMENDATIONS ...............................................................................................55
10. x
LIST OF FIGURES
Figure 1.1: Lignocellulosic materials: composition of major compounds. ...............................3
Figure 2.1: Reductions of GHG emission by first generation (American corn and Brazilian
sugarcane) ethanol and second generation.................................................................................8
Figure 2.2: Illustration of a cellulose chain. ............................................................................11
Figure 2.3: Scheme of a lignocellulosic biorefinery................................................................13
Figure 2.4: Process for production ethanol from lignocellulosic biomass...............................13
Figure 2.5: Schematic of the role of pretreatment in the conversion of biomass to fuel.........17
Figure 2.5: Schematic representation of a cellulosoma. ..........................................................22
Figure 2.6: Mechanism of action of cellulose..........................................................................25
Figure 3.1: Equipment and Experimental set up for the study ................................................31
Figure 4.1: Frequency distribution chart for screened rice hulls .............................................38
Figure 4.2: Plot of weight fraction against average particle sizes ...........................................39
Figure 4.3: Graph showing the different contents of rice hulls in % (w/w) ............................40
Figure 4.4: Surface plot of Yield vs. Time &Temp.................................................................46
Figure 4.5: Surface plot of Yield vs. H2O2 & Temp. ...............................................................46
Figure 4.6: Surface plot of Yield vs. H2O2 & Time.................................................................47
Figure 4.7: Optimization Plot for Pretreatment Conditions.....................................................47
Figure 4.8: Graph showing the reducing sugar yields against biomass loading and enzyme
loading variations.....................................................................................................................50
Figure 4.9: Chart showing the concentration of glucose and other reducing sugars in the yield
..................................................................................................................................................54
11. xi
LIST OF TABLES
Table 2.1: Biofuel comparison with fossil fuel..........................................................................8
Table 2.1: Cellulose, hemicellulose, and lignin contents in common agricultural residues and
wastes.......................................................................................................................................11
Table 2.3: Methods for biomass lignocellulosic pretreatment.................................................18
Table 2.4: Commercial cellulases able to work at temperature ranging from 50 to 60ºC.......25
Table 2.5: Typical composition of rice hulls. ..........................................................................25
Table 3.1: Experimental range and uncoded levels of factors for pretreatment ......................33
Table 3.2: Experimental order for pretreatment ......................................................................33
Table 4.1a: Table showing the particle sizes and sample weights for each batch of sample
during the sieve analysis ..........................................................................................................37
Table 4.1b: Table showing average particle sizes and weight fractions of different batches of
the sieve analysis......................................................................................................................37
Table 4.2: Average weight fractions and average particle sizes..............................................38
Table 4.3: Contents of Rice hulls.............................................................................................39
Table 4.4: Pretreatment Data ...................................................................................................43
Table 4.5: Various Pretreatment conditions and total reducing sugars yield of rice hulls after
enzymatic hydrolysis ...............................................................................................................44
Table 4.6: Variation of hydrolysis biomass loading and enzyme loading at 45°c using samples
that were not soaked before pretreatment ................................................................................49
Table 4.7: Variation of hydrolysis biomass loading and enzyme loading at 45°c using soaked
samples.....................................................................................................................................49
Table 4.8: Glucose data for optimized samples.......................................................................53
12. 1
CHAPTER ONE
INTRODUCTION
1.1 BACKGROUND STUDY
The quick depletion of fossil fuels and the negative impacts such as greenhouse gas
emissions into the atmosphere through combustion of these fuels has driven the world to
utilize renewable-energy sources such as biofuel in order to reduce the total dependency on
non-renewable energy sources. The growing industrialization has derived in an increasing
demand of fuels attempting to satisfy both the industrial and domestic demands. Second
generation bioethanol is based on raw materials rich in complex carbohydrates, resulting
an interesting alternative to reduce competition with food industry. The process to obtain
second generation bioethanol involves four basic steps: feedstock pretreatment, enzymatic
or acid hydrolysis, sugars fermentation, and ethanol recovery (Gómez Sandra, Andrade
Rafael, Santander, Costa, & Maciel, 2010).
Lignocellulosic agricultural residues are promising raw materials for sugar-platform
biorefinery on a large scale. These residues or wastes do not compete with primary food
production. However, few biorefinery processes based on sugar-platform are cost-
competitive in current markets because of the low efficiency and high cost of enzymatic
conversion processes (Himmel M. , et al., 2007). Lignocellulose is a generic term for
describing the main constituents in most plants, namely cellulose, hemicelluloses, and
lignin. Lignocellulose is a complex matrix, comprising many different polysaccharides,
phenolic polymers and proteins. Cellulose, the major component of cell walls of land
plants, is a glucan polysaccharide containing large reservoirs of energy that provide real
potential for conversion into biofuels. Lignocellulosic biomass consists of a variety of
materials with distinctive physical and chemical characteristics. It is the non-starch based
fibrous part of plant material.
The largest potential feedstock for ethanol is lignocellulosic biomass. Lignocellulosic
biomass includes materials such as agricultural residues (corn stover, crop straws, rice hulls
and bagasse), herbaceous crops (alfalfa, switchgrass), short rotation woody crops, forestry
residues, waste paper and other wastes (municipal and industrial). Bioethanol production
from these feedstock could be an attractive alternative for disposal of these residues.
Importantly, lignocellulosic feedstock do not interfere with food security. Moreover,
bioethanol is very important for both rural and urban areas in terms of energy security
reason, environmental concern, employment opportunities, agricultural development,
foreign exchange saving, socioeconomic issues etc.
13. 2
1.2 AIMS AND OBJECTIVES
Rice hulls, which represent 20% dry weight of the harvested rice, can serve as a low cost
abundant feedstock for production of fuel (Saha B., 2007). They are considered waste
materials because of their low value as animal feed due to low digestibility, peculiar size
distribution, low bulk density, high ash/silica contents, and abrasive characteristics. They
can be easily collected from rice-processing sites and contain about 36% cellulose and 12%
hemicellulose, so they can be used after transformation for bioethanol production. For this
purpose, these polymers must be hydrolyzed to simple sugars, which are subsequently
fermented to ethanol. However, rice husks also contain high quantities of ash (20%) and
lignin (16%), which combined with hemicelluloses, results a complex structure around the
cellulose, being more difficult its use as a lignocellulosic feedstock for conversion to
ethanol. For this reason, pretreatments are generally applied in order to make these
polymers more accessible to the enzymes to be converted into fermentable sugars (Mosier,
et al., 2005).
The aim of this research is to study the capacity and functioning of rice hulls as feedstock
for ethanol production. Specific objectives of this research are as follows:
1. To study the effect of alkaline pretreatment of the rice hulls for effective enzymatic
hydrolysis.
2. To study the effects of hydrolysis of the pretreated rice hulls using cellulase enzyme.
3. To perform analysis using a 2 level, 3 factor central composite design, a form of response
surface design for the optimization of the pretreatment conditions. Time, temperature and
hydrogen peroxide concentration are the factors to be considered.
4. To validate the optimized pretreatment conditions
5. To investigate the influence of enzyme loading and biomass loading on enzymatic
hydrolysis yield.
1.3 SCOPE
In this work, milled rice hulls with a screen size of 1.18 mm was analyzed. This substrate
was used for comparison of reducing sugar production by commercially prepared
Trichoderma ressei cellulase. Factors considered in this work included; pretreatment
temperature, pretreatment time and alkaline hydrogen peroxide (H2O2) concentration. The
optimum conditions were evaluated using the Central Composite Design (CCD).
This research work was limited to alkaline pretreatment and enzymatic hydrolysis of rice
hulls.
14. 3
Figure 1.1: Lignocellulosic materials: composition of major compounds (Kumar, Barrett,
Delwiche, & Stroeve, 2009).
15. 4
1.4 JUSTIFICATION
The environmental impact from the production of fuels is an important factor in
determining its feasibility as an alternative to fossil fuels. Over the long run, small
differences in production cost, environmental ramifications, and energy output may have
large effects. It has been found that cellulosic ethanol can produce a positive net energy
output. The reduction in greenhouse gas (GHG) emissions from corn ethanol and cellulosic
ethanol compared with fossil fuels is drastic. Corn ethanol may reduce overall GHG
emissions by about 13%, while that figure is around 88% or greater for cellulosic ethanol.
As well, cellulosic ethanol can reduce carbon dioxide emissions to nearly zero.
Also, despite its lower energy content than gasoline, ethanol’s high octane rating reduces
engine knock thereby improving engine performance even in dilute ethanol–gasoline
blends (Bromberg L. et al., 2006).
Pretreatment is done because enzyme hydrolysis is greatly hindered by the crystallinity of
cellulose and the protective sheath of lignin and hemicellulose that wrap around cellulose
(Laureano-Perez, Teymouri, Alizadeh, & Dale, 2005). An effective pretreatment method
can weaken all these hindrances and exposes cellulose to cellulase enzymes for effective
hydrolysis. (Alizadeh, Teymouri, Gilbert, & Dale, 2005) Reported that only less than 20 %
glucose is released from lignocellulosic biomass without pretreatment while the yield can
be as high as 90 % with proper pretreatment.
The hydrolysis of cellulolytic materials with diluted acids is well known, but this process
generates toxic products of hydrolysis. Other negatives factors related to the acid hydrolysis
are the corrosion and the high amounts of salts resulting from the acid neutralization.
Enzymatic hydrolysis is preferred because of the higher conversion yields and less
corrosive, less toxic conditions compared to acid hydrolysis.( Ngamveng J. et al. 1990)
1.5 RELEVANCE OF STUDY
Long-term economic and environmental concerns have resulted in a great amount of
research in the past couple of decades on renewable sources of liquid fuels to replace fossil
fuels. Burning fossil fuels such as coal and oil releases CO2, which is a major cause of
global warming. With only 4.5% of the world’s population, the United States is responsible
for about 25% of global energy consumption and 25% of global CO2 emissions. The
average price of gasoline in 2005 was $2.56 per gallon, which was $0.67 higher than the
average price of gasoline in the previous year.
Yet, in June 2008, the average price of gasoline in the United States reached $4.10 per
gallon. Conversion of abundant lignocellulosic biomass to biofuels as transportation fuels
presents a viable option for improving energy security and reducing greenhouse emissions.
Unlike fossil fuels, which come from plants that grew millions of years ago, biofuels are
produced from plants grown today. They are cleaner-burning than fossil fuels, and the short
cycle of growing plants and burning fuel made from them does not add CO2 to the
atmosphere. It has been reported that cellulosic ethanol and ethanol produced from other
biomass resources have the potential to cut greenhouse gas emissions by 86%.
Lignocellulosic materials such as agricultural residues (e.g., wheat straw, sugarcane
bagasse, corn stover, rice hulls), forest products (hardwood and softwood), and dedicated
crops (switchgrass, salix) are renewable sources of energy. These raw materials are
sufficiently abundant and generate very low net greenhouse emissions
16. 5
1.6 RESEARCH LIMITATIONS
Lignocellulosic agricultural residues are promising raw materials for sugar platform
biorefinery on a large scale. However, few biorefinery processes based on sugar-platform
are cost-competitive in current markets because of the low efficiency and high cost of
enzymatic conversion processes (Himmel M. E., et al., 2007).
Rice hulls also contain high quantities of ash (20%) and lignin (16%), which combined
with hemicelluloses, results a complex structure around the cellulose, being more difficult
its use as a lignocellulosic feedstock for conversion to ethanol. For this reason,
pretreatments are generally applied in order to make these polymers more accessible to the
enzymes to be converted into fermentable sugars (Mosier, et al., 2005). Pretreatment
processes can be physical, chemical, biological or a combination of these methods (Ana,
Julie, Ana, Ignacio, & Ildefonso, 2013). Although many different types of pretreatments
were tested in different conditions over the past years, advances are still needed for overall
costs to become competitive.
Pre-treatment is considered to be the most expensive step to convert lignocellulosic
biomass into ethanol. Most pretreatment methods disrupt cell walls of the plant fibers to
expose the sugar polymers, but do not remove much lignin. But, alkaline and alkaline
peroxide pre-treatments which belong to chemical methods are effective processes for
pretreating lignocellulose material (Ana, Julie, Ana, Ignacio, & Ildefonso, 2013).
The use of enzymes in the hydrolysis of cellulose is more advantageous than use of
chemicals, because enzymes are highly specific and can work at mild process conditions.
Despite these advantages, the use of enzymes in industrial applications is still limited by
several factors: the costs of enzymes isolation and purification are high; the specific activity
of enzyme is low compared to the corresponding starch degrading enzymes. As
consequence, the process yields increase at raising the enzymatic proteins dosage and the
hydrolysis time (up to 4 days) while, on the contrary, decrease at raising the solids loadings
(Berlin, Maximenco, Gilkes, & Saddeler, 2007).
Despite the challenges of using lignocellulose, there is a vast supply of this biomass
available across many climates. Crops intended for lignocellulosic ethanol production are
sometimes cheaper to grow and harvest than sugar or starch-rich crops (Adetayo, 2013).
17. 6
CHAPTER TWO
LITERATURE REVIEW
2.1 BIOFUEL
Biofuel is a hydrocarbon fuel made by or from a living organism that we humans can use
to power something. This definition of a biofuel is rather formal. In practical consideration,
any hydrocarbon fuel that is produced from organic matter (living or once living material)
in a short period of time (days, weeks, or even months) is considered a biofuel. This
contrasts with fossil fuels, which take millions of years to form and with other types of fuel
which are not based on hydrocarbons (nuclear fission, for instance).
What makes biofuels tricky to understand is that they need not be made by a living
organism, though they can be. Biofuels can also be made through chemical reactions,
carried out in a laboratory or industrial setting, that use organic matter (called biomass) to
make fuel. The only real requirements for a biofuel are that the starting material must be
CO2 that was fixed (turned into another molecule) by a living organism and the final fuel
product must be produced quickly and not over millions of years. Biomass is simply organic
matter. In others words, it is dead material that was once living. Kernels of corn, mats of
algae, and stalks of sugar cane are all biomass. Before global warming related to burning
fossil fuels became a major factor in determining where energy came from, the major
concern was that fossil fuels, which are considered limited in supply, would run out over
the next century. It was thought that if we could produce hydrocarbons another way, and
quickly, then we could meet our energy demands without much problem. This leads to one
of the major separating factors between a biofuel and a fossil fuel - renewability.
Fossil fuel is not considered renewable because it takes millions of years to form and
humans really cannot wait that long. Biofuel, on the other hand, comes from biomass,
which can be produced year after year through sustainable farming practices. This means
biomass and biofuel are renewable (we can replace used biofuel over a very short period of
time).
It is important to note that 'renewable' energy is not the same thing as 'green' energy.
Renewable energy simply won’t run out any time soon, like biofuels, hydroelectric, wind,
and solar. A “green” energy is one that is also good for the planet because it does not harm
ecosystems, contribute to acid rain, or worsen global warming. Solar energy is a 'green'
energy. All 'green' energy is considered renewable, but not all renewable energy is green.
Biofuels are examples of renewable energy sources that aren’t always green because they
produce greenhouse gases (Biofuel Facts, 2010).
2.1.1 Classification of Biofuels
Biofuels are often broken into three generations.
1st generation biofuels are also called conventional biofuels. They are made
from things like sugar, starch, or vegetable oil. Note that these are all food
products. Any biofuel made from a feedstock that can also be consumed as a
human food is considered a first generation biofuel.
2nd generation biofuels are produced from sustainable feedstock. The
sustainability of a feedstock is defined by its availability, its impact on
greenhouse gas emissions, its impact on land use, and by its potential to threaten
the food supply. No second generation biofuel is also a food crop, though certain
food products can become second generation fuels when they are no longer
18. 7
useful for consumption. Second generation biofuels are often called “advanced
biofuels.”
Though not a traditional category of biofuel, some people refer to 3rd generation
biofuels. In general, this term is applied to any biofuel derived from algae. These
biofuels are given their own separate class because of their unique production
mechanism and their potential to mitigate most of the drawbacks of 1st and 2nd
generation biofuels.
2.1.2 TYPES OF BIOFUELS
The chemical structure of biofuels can differ in the same way that the chemical structure
of fossil fuels can differ. For the most part, our interest is in liquid biofuels as they are
easy to transport. The table below compares various biofuels with their fossil fuel
counterparts.
In Table 1 only limited list of the biofuels are available, covering only the most popular
and widely used. It is worth nothing that ethanol is found in almost all gasoline
mixtures. In Brazil, gasoline contains at least 95% ethanol. In other countries, ethanol
usually makes up between 10 and 15% of gasoline.
2.1.3 BIOFUEL VS FOSSIL FUEL
Biofuels are not new. In fact, Henry Ford had originally designed his Model T to run
on ethanol. There are several factors that decide the balance between biofuel and fossil
fuel use around the world. Those factors are cost, availability, and food supply.
All three factors listed above are actually interrelated. To begin, the availability of fossil
fuels has been of concern almost from day one of their discovery. Pumping fuel from
the ground is a difficult and expensive process, which adds greatly to the cost of these
fuels. Additionally, fossil fuels are not renewable, which means they will run out at
some point. As our ability to pump fossil fuels from the ground diminishes, the
available supply will decrease, which will inevitably lead to an increase in price.
It was originally thought that biofuels could be produced in almost limitless quantity
because they are renewable. Unfortunately, our energy needs far out-pace our ability to
grown biomass to make biofuels for one simple reason, land area. There is only so much
land fit for farming in the world and growing biofuels necessarily detracts from the
process of growing food. As the population grows, our demands for both energy and
food grow. At this point, we do not have enough land to grow both enough biofuel and
enough food to meet both needs. The result of this limit has an impact on both the cost
of biofuel and the cost of food. For wealthier countries, the cost of food is less of an
issue. However, for poorer nations, the use of land for biofuels, which drives up the
cost of food, can have a tremendous impact.
The balance between food and biofuel is what keeps the relatively simple process of
growing and making biofuels from being substantially cheaper than fossil fuel. When
this factor is combined with an increased ability (thanks to advances in technology) to
extract oil from the ground, the price of fossil fuel is actually lower than that of biofuel
for the most part.
19. 8
Table 2.1: Biofuel comparison with fossil fuel.
Biofuel Fossil Fuel Differences
Ethanol Gasoline/Ethane Ethanol has about half the energy per mass of
gasoline, which means it takes twice as much
ethanol to get the same energy. Ethanol burns
cleaner than gasoline, however, producing less
carbon monoxide. However, ethanol produces
more ozone than gasoline and contributes
substantially to smog. Engines must be modified
to run on ethanol.
Biodiesel Diesel Has only slightly less energy than regular diesel.
It is more corrosive to engine parts than standard
diesel, which means engines have to be designed
to take biodiesel. It burns cleaner than diesel,
producing less particulate and fewer sulphur
compounds.
Methanol Methane Methanol has about one third to one half as much
energy as methane. Methanol is a liquid and easy
to transport whereas methane is a gas that must
be compressed for transportation.
Biobutanol Gasoline/Butane Biobutanol has slightly less energy than
gasoline, but can run in any car that uses gasoline
without the need for modification to engine
components.
Figure 2.1: Reductions of GHG emission by first generation (American corn and
Brazilian sugarcane) ethanol and second generation (cellulosic) ethanol (adapted from
(Wang, Wu, & Huo, 2007))
20. 9
2.1.4 GREENHOUSE GAS (GHG) EMISSIONS AND GLOBAL WARMING
With the exception of cultivating sugarcane in warm climates (like Brazil‘s),
production of first generation biofuels is far from an ideal closed carbon cycle, since
there is a significant petroleum usage during the whole process (to make fertilizers,
power farm equipment, transport feedstock’s), which make greenhouse gas
reductions in the order of 20% to 50%.
The appearance of second and third generation biofuels came as a possible solution to
avoid direct competition for commodities, while benefiting from increased GHG
reductions. Second generation biofuels are produced from non-food crops or waste
materials, such as food wastes, manure and agricultural residues. Third generation
biofuels use algae to produce carbohydrates and lipids, which can be used for
producing bio-ethanol and biodiesel, respectively. This technology is still not very
mature, but has potentially very high yields per terrain usage, while not displacing
terrain for food production.
2.1.5 CURRENT TRENDS
Most gasoline and diesel fuels in North America and Europe are blended with
biofuel.
Biodiesel accounts for about 3% of the German market and 0.15% of the U.S.
market.
About 1 billion gallons of biodiesel are produced annually.
Bioethanol is more popular in the Americas while biodiesel is more popular in
Europe.
The U.S. and Brazil produce 87% of the world's fuel ethanol.
More than 22 billion gallons of fuel ethanol are produced each year.
Ethanol is added to gasoline to improve octane and reduce emissions.
Biodiesel is added to petroleum-based diesel to reduce emissions and improve
engine life. (Biofuel Facts, 2010)
2.2 LIGNOCELLULOSIC BIOMASS
Lignocellulose refers to plant dry matter (biomass), so called lignocellulosic biomass. It is
the most abundantly available raw material on the Earth for the production of bio-fuels,
mainly bio-ethanol. It is composed of carbohydrate polymers (cellulose, hemicellulose),
and an aromatic polymer (lignin). These carbohydrate polymers contain different sugar
monomers (six and five carbon sugars) and they are tightly bound to lignin. Lignocellulosic
biomass can be broadly classified into virgin biomass, waste biomass and energy crops.
Virgin biomass includes all naturally occurring terrestrial plants such as trees, bushes and
grass. Waste biomass is produced as a low value byproduct of various industrial sectors
such as agricultural (corn stover, sugarcane bagasse, straw etc), forestry (saw mill and paper
mill discards). Energy crops are crops with high yield of lignocellulosic biomass produced
to serve as a raw material for production of second generation biofuel examples include
switch grass (Panicum virgatum) and Elephant grass.
21. 10
2.2.1 Structure of Lignocellulosic Biomass
Lignocellulose is the primary building block of plant cell walls. Plant biomass is mainly
composed of cellulose, hemicellulose, and lignin, along with smaller amounts of pectin,
protein, extractives (soluble nonstructural materials such as nonstructural sugars,
nitrogenous material, chlorophyll, and waxes), and ash. The composition of these
constituents can vary from one plant species to another. For example, hardwood has
greater amounts of cellulose, whereas wheat straw and leaves have more hemicellulose
(Table 1). In addition, the ratios between various constituents within a single plant vary
with age, stage of growth, and other conditions.
Cellulose is the main structural constituent in plant cell walls and is found in an
organized fibrous structure. The structure of cellulose is shown in Figure 2. This linear
polymer consists of D-glucose subunits linked to each other by β-(1, 4)-glycosidic
bonds. Cellobiose is the repeat unit established through this linkage, and it constitutes
cellulose chains. The long-chain cellulose polymers are linked together by hydrogen
and van der Waals bonds, which cause the cellulose to be packed into microfibrils.
Hemicelluloses and lignin cover the microfibrils. Fermentable D-glucose can be
produced from cellulose through the action of either acid or enzymes breaking the β-
(1, 4)-glycosidic linkages. Cellulose in biomass is present in both crystalline and
amorphous forms. Crystalline cellulose comprises the major proportion of cellulose,
whereas a small percentage of unorganized cellulose chains form amorphous cellulose.
Cellulose is more susceptible to enzymatic degradation in its amorphous form.
The main feature that differentiates hemicellulose from cellulose is that hemicellulose
has branches with short lateral chains consisting of different sugars. These
monosaccharides include pentoses (xylose, rhamnose, and arabinose), hexoses
(glucose, mannose, and galactose), and uronic acids (e.g., 4-o-methylglucuronic, D-
glucuronic, and D-galactouronic acids). The backbone of hemicellulose is either a
homopolymer or a heteropolymer with short branches linked by β-(1, 4)-glycosidic
bonds and occasionally β-(1, 3)-glycosidic bonds. Also, hemicelluloses can have some
degree of acetylation, for example, in heteroxylan. In contrast to cellulose, the polymers
present in hemicelluloses are easily hydrolyzable. These polymers do not aggregate,
even when they cocrystallize with cellulose chains.
Lignin is a complex, large molecular structure containing cross-linked polymers of
phenolic monomers. It is present in the primary cell wall, imparting structural support,
impermeability, and resistance against microbial attack. Three phenyl propionic
alcohols exist as monomers of lignin: coniferyl alcohol (guaiacyl propanol), coumaryl
alcohol (p-hydroxyphenyl propanol), and sinapyl alcohol (syringyl alcohol). Alkyl-
aryl, alkyl-alkyl, and aryl-aryl ether bonds link these phenolic monomers together. In
general, herbaceous plants such as grasses have the lowest contents of lignin, whereas
softwoods have the highest lignin contents (Table 2.1).
22. 11
Table 2.1: Cellulose, hemicellulose, and lignin contents in common agricultural
residues and wastes (Adapted from (Jorgensen, Kristensen, & Felby, 2007)
.
Figure 2.2: Illustration of a cellulose chain.
23. 12
2.2.2 Products of Lignocellulosic Biomass
Lignocellulosic biomass is a potential source of several bio-based products according
to the biorefinery approach. Currently, the products made from bioresources represent
only a minor fraction of the chemical industry production. However, the interest in the
bio-based products has increased because of the rapidly rising barrel costs and an
increasing concern about the depletion of the fossil resources in the near future (Hatti-
Kaul et al., 2007). The goal of the biorefinery approach is the generation of energy and
chemicals from different biomass feedstock, through the combination of different
technologies (FitzPatrick et al. 2010).
The biorefinery scheme involves a multi-step biomass processing. The first step
concerns the feedstock pretreatment through physical, biological, and chemical
methods. The outputs from this step are platform (macro) molecules or streams that can
be used for further processing (Cherubini & Ulgiati, 2010). Recently, a detailed report
has been published by DOE describing the value added chemicals that can be produced
from biomass (Werpy, 2004).
Besides ethanol, several other products can be obtained following the hydrolysis of the
carbohydrates in the lignocellulosic materials. For instance, xylan/xylose contained in
hemicelluloses can be thermally transformed into furans (2-furfuraldeyde,
hydroxymethil furfural), short chain organic acids (formic, acetic, and propionic acids),
and cheto compounds (hydroxy-1-propanone, hydroxy-1-butanone) (Güllü, 2010;
Bozell & Petersen, 2010). Furfural can be further processed to form some building
blocks of innovative polymeric materials (i.e. 2, 5-furandicarboxylic acid). In addition,
levulinic acid could be formed by the degradation of hydroxymethil furfural
(Demirabas, 2008). Another product prepared either by fermentation or by catalytic
hydrogenation of xylose is xylitol (Bozell & Petersen, 2010). Furthermore, through the
chemical reduction of glucose it is possible to obtain several products, such as sorbitol
(Bozell & Petersen, 2010). The residual lignin can be an intermediate product to be
used for the synthesis of phenol, benzene, toluene, xylene, and other aromatics.
Similarly to furfural, lignin could react to form some polymeric materials (i.e.
polyurethanes) (Demirabas, 2008).
2.2.3 PRODUCTION OF ETHANOL FROM LIGNOCELLULOSIC BIOMASS
Ethanol is the most common renewable fuel recognized as a potential alternative to
petroleum-derived transportation fuels. It can be produced from lignocellulosic
materials in various ways characterized by common steps: hydrolysis of cellulose and
hemicellulose to monomeric sugars, fermentation and product recovery. The main
differences lie in the hydrolysis phase, which can be performed by dilute acid,
concentrated acid or enzymatically (Galbe & Zacchi, 2002).
24. 13
Figure 2.3: Scheme of a lignocellulosic biorefinery. The shape of each step describes
the type of process used, chemical, biological, and physical (legend) (FitzPatrick,
Champagne, Cunningham, & Whitney, 2010)
Figure 2.4: Process for production ethanol from lignocellulosic biomass. The circle in
the scheme indicates two alternative process routes: simultaneous hydrolysis and
fermentation (SSF); separate hydrolysis and fermentation (SHF). (Adapted from
(Alessandra, Isabella, Emanuele, & Vincenza, 2012)
25. 14
2.2.3.1 Acid Hydrolysis
The main advantage of the acid hydrolysis is that acids can penetrate lignin without any
preliminary pretreatment of biomass, thus breaking down the cellulose and
hemicellulose polymers to form individual sugar molecules. Several types of acids,
concentrated or diluted, can be used, such as sulphurous, sulphuric, hydrocloric,
hydrofluoric, phosphoric, nitric and formic acid (Galbe & Zacchi, 2002). Sulphuric and
hydrochloric acids are the most commonly used catalysts for hydrolysis of
lignocellulosic biomass (Lenihan, et al., 2010). The acid concentration used in the
concentrated acid hydrolysis process is in the range of 10-30%. The process occurs at
low temperatures, producing high hydrolysis yields of cellulose (i.e. 90% of theoretical
glucose yield) (Iranmahboob, Nadim, & Monemi, 2002). However, this process
requires large amounts of acids causing corrosion problems to the equipment. The main
advantage of the dilute hydrolysis process is the low amount of acid required (2-5%).
However this process is carried out at high temperatures to achieve acceptable rates of
cellulose conversion. The high temperature increases the rates of hemicellulose sugars
decomposition thus causing the formation of toxic compounds such as furfural and 5-
hydroxymethyl-furfural (HMF).These compounds inhibit yeast cells and the
subsequent fermentation stage, causing a lower ethanol production rate (Alessandra,
Isabella, Emanuele, & Vincenza, 2012). In addition, these compounds lead to reduction
of fermentable sugars (Kootstra, Beeftink, Scott, & Sanders, 2009). In addition, high
temperatures increase the equipment corrosion (Jones & Semrau, 1984).
In 1999, the BC International (BCI) of United States has marketed a technology based
on two-step dilute acid hydrolysis: the first hydrolysis stage at mild conditions (170-
190°C) to hydrolyze hemicellulose; the second step at more severe conditions to
hydrolyze cellulose 200-230°C (Wyman, 1999). In 1991, the Swedish Ethanol
Development Foundation developed the CASH process. This is a two-stage dilute acid
process that provides the impregnation of biomass with sulphur dioxide followed by a
second step in which diluted hydrochloric acid is used. In 1995, this foundation has
focused researches on the conversion of softwoods using sulphuric acid (Galbe &
Zacchi, 2002).
26. 15
2.2.3.2 Enzymatic Hydrolysis
A pretreatment step is necessary for the enzymatic hydrolysis process. It is able to
remove the lignin layer and to decrystallize cellulose so that the hydrolytic enzymes
can easily access the biopolymers. The pretreatment is a critical step in the cellulosic
bioethanol technology because it affects the quality and the cost of the carbohydrates
containing streams (Balat., Balat, & Oz, 2008). Pretreatments methods can be classified
into different categories: physical, physiochemical, chemical, biological, electrical, or
a combination of these (Kumar, Barrett, Delwiche, & Stroeve, 2009), (Table 2.3).
On the whole, the final yield of the enzymatic process depends on the combination of
several factors: biomass composition, type of pretreatment, dosage and efficiency of
the hydrolytic enzymes (Alvira, Tomás-Pejó, Ballesteros, & Negro, 2010).
The use of enzymes in the hydrolysis of cellulose is more advantageous than use of
chemicals, because enzymes are highly specific and can work at mild process
conditions. Despite these advantages, the use of enzymes in industrial applications is
still limited by several factors: the costs of enzymes isolation and purification are high;
the specific activity of enzyme is low compared to the corresponding starch degrading
enzymes. As consequence, the process yields increase at raising the enzymatic proteins
dosage and the hydrolysis time (up to 4 days) while, on the contrary, decrease at raising
the solids loadings. One typical index used to evaluate the performances of the cellulase
preparations during the enzymatic hydrolysis is the conversion rate to say the obtained
glucose concentration per time required to achieve it (g glucose/L/h/).Some authors
reported conversion rates of softwoods substrates (5%w/v solids loading) in the range
0.3-1.2 g/L/h (Berlin, Maximenco, Gilkes, & Saddeler, 2007). In general, compromise
conditions are necessary between enzymes dosages and process time to contain the
process costs.
In 2001, the cost to produce cellulase enzymes was 3-5$ per gallon of ethanol (0.8-
1.32$/liter ethanol), (Novozymes and NREL). In order to reduce the cost of cellulases
for bioethanol production, in 2000 the National Renewable Laboratory (NREL) of USA
has started collaborations with Genencor Corporation and Novozymes. In particular, in
2004, Genencor has achieved an estimated cellulase cost in the range $0.10-0.20 per
gallon of ethanol (0.03-0.05$/liter ethanol) in NREL´s cost model (Genencor, 2004).
Similarly, collaboration between Novozymes and NREL has yielded a cost reduction
in the range $0.10-0.18 per gallon of ethanol (0.03-0.047$/liter ethanol), a 30-fold
reduction since 2001 (Mathew, Sukumaran, Singhania, & Pamdey, 2008).
27. 16
Unlike the acid hydrolysis, the enzymatic hydrolysis, still has not reached the industrial
scale. Only few plants are available worldwide to investigate the process (pretreatment
and bioconversion) at demo scale. More recently, the steam explosion pretreatment,
investigated for several years in Italy at the ENEA research Center of Trisaia (De Bari,
et al., 2002) (De Bari, Nanna, & Braccio, SO2-catalyzed steam fractionation of aspen
chips for bioethahnol production: Optimization of the catalyst impregnation, 2007), is
now going to be developed at industrial scale thanks to investments from the Italian
Mossi & Ghisolfi Group.
2.2.3.2.1 Cellulosic Capability of Organisms: Difference in the Cellulose-Degrading
Strategy
Different strategies for the cellulose degradation are used by the cellulase-
producing microorganisms: aerobic bacteria and fungi secrete soluble extracellular
enzymes known as non-complexed cellulase system; anaerobic cellulolytic
microorganisms produce complexed cellulase systems, called cellulosomes (Sun &
Cheng, 2002). A third strategy was proposed to explain the cellulose-degrading
action of two recently discovered bacteria: the aerobic Cytophaga hutchinsonii and
the anaerobic Fibrobacter succinogenes (Ilmén, Saloheimo, Onnel, & Pentillä,
1997).
Non-complexed cellulose system.
One of the most fully investigated non-complexed cellulase system is the
Trichoderma reesei model. T. reesei (teleomorph Hypocrea jecorina) is a
saprobic fungus, known as an efficient producer of extracellular enzymes
(Bayer, Chanzy, Lamed, & Shoham, 1998). Its non-complexed cellulase
system includes two cellobiohydrolases, at least seven endo-glucanases,
and several β-glucosidases. However, in T. reesei cellulases, the amount
of β-glucosidase is lower than that needed for the efficient hydrolysis of
cellulose into glucose. As a result, the major product of hydrolysis is
cellobiose. This is a dimer of glucose with strong inhibition toward endo-
and exo-glucanases so that the accumulation of cellobiose significantly
slows down the hydrolysis process (Gilkes, Henrissat, Kilburn, Miller, &
Warren, 1991). By adding β-glucosidase to cellulases from either external
sources, or by using co-culture systems, the inhibitory effect of cellobiose
can be significantly reduced (Ting & Makarov, 2009).
31. 20
It has been observed that the mechanism of cellulose enzymatic hydrolysis
by T.reesei involves three simultaneous processes (Ting & Makarov,
2009):
1. Chemical and physical changes in the cellulose solid phase. The
chemical stage includes changes in the degree of polymerization, while
the physical changes regard all the modifications in the accessible
surface area. The enzymes specific function involved in this step is the
endo-glucanase.
2. Primary hydrolysis. This process is slow and involves the release of
soluble intermediates from the cellulose surface. The activity involved
in this step is the cellobiohydrolase.
3. Secondary hydrolysis. This process involves the further hydrolysis of
the soluble fractions to lower molecular weight intermediates, and
ultimately to glucose. This step is much faster than the primary
hydrolysis and β-glucosidases play a role for the secondary hydrolysis.
Complexed cellulose system.
Cellulosomes are produced mainly by anaerobic bacteria, but their presence
have also been described in a few anaerobic fungi from species such as
Neocallimastix, Piromyces, and Orpinomyces (Alessandra, Isabella, Emanuele,
& Vincenza, 2012). In the domain Bacteria, organisms possessing cellulosomes
are only found in the phylum Firmicutes, class Clostridia, order Clostridiales
and in the Lachnospiraceae and Clostridiaceae families. In this latter family,
bacteria with cellulosomes are found in various clusters of the genus Clostridium
(McCarter & Whiters, 1994; Wilson, 2008). Cellulosomes are protuberances
produced on the cell wall of the cellulolytic bacteria grown on cellulosic
materials. These protuberances are stable enzyme complexes tightly bound to
the bacteria cell wall but flexible enough to bind strongly to cellulose (Lentig &
Warmoeskerken, 2001). A cellulosome contains two types of subunits: non-
catalytic subunits, called scaffoldins, and enzymatic subunits. The scaffoldin is
a functional unit of cellusome, which contain multiple copies of cohesins that
interact selectively with domains of the enzymatic subunits, CBD (cellulose
binding domains) and CBM (carbohydrates binding modules). These have
complementary cohesins, called dockerins, which are specific for each bacterial
species (Alessandra, Isabella, Emanuele, & Vincenza, 2012).
32. 21
For the bacterial cell, the biosynthesis of a cellulosome enables a specific
adhesion to the substrate of interest without competition with other
microorganisms. The cellulosome allows several advantages: (1) synergism of
the cellulases; (2) absence of unspecific adsorption (Alessandra, Isabella,
Emanuele, & Vincenza, 2012). Thanks to its intrinsic Lego-like architecture,
cellulosomes may provide great potential in the biofuel industry. The concept of
cellulosome was firstly discovered in the thermophilic cellulolytic and anaerobic
bacterium, Clostridium thermocellum (Wyman, Handbook on bioethanol:
production and utilization, 1996). It consists of a large number of proteins,
including several cellulases and hemicellulases. Other enzymes that can be
included in the cellulosome are lichenases.
Third cellulose-degrading strategy
The third strategy was recently proposed to explain the cellulose-degrading
behavior of two recently sequenced bacteria: Cytophaga hutchinsonii and
Fibrobacter succinogenes (Ilmén, Saloheimo, Onnel, & Pentillä, 1997). C.
hutchinsonii is an abundant aerobic cellulolytic soil bacterium (Fägerstam &
Pettesson, 1984), while F. succinogenes is an anaerobic rumen bacterium which
was isolated by the Rockville, (Maryland), and San Diego (California) Institute
of Genomic Research (TIGR) (Mansfieldet al., 1998). In the aerobic C.
hutchinsonii no genes were found to code for CBM and in the anaerobic F.
succinogenes no genes were identified to encode dockerin and scaffoldin. Thus,
a third cellulose degrading mechanism was proposed. It includes the binding of
individual cellulose molecules by outer membrane proteins of the
microorganisms followed by the transport into the periplasmic space where they
are degraded by endoglucanases (Ilmén, Saloheimo, Onnel, & Pentillä, 1997).
2.2.3.2.2 Characteristics of the Commercial Hydrolytic Enzymes
Most cellulase enzymes are relatively unstable at high temperatures. The maximum
activity for most fungal cellulases and β-glucosidase occurs at 50±5°C and a pH
4.5- 5 (Taherzadeh, 2007) (Galbe & Zacchi, 2002). Usually, they lose about 60%
of their activity in the temperature range 50–60 °C and almost completely lose
activity at 80°C (Gautam et. al.2010). However, the enzymes activity depends on
the hydrolysis duration and on the source of the enzymes (Tengborg, Galbe, &
33. 22
Zacchi, 2001). In general, cellulases are quite difficult to use for prolonged
operations. As mentioned before, the enzyme production costs mainly depend on
the productivity of the enzymes-producing microbial strain. Filamentous fungi are
the major source of cellulases and mutant strains of Trichoderma (T. viride, T.
reesei, T. longibrachiatum) have long been considered to be the most productive
(Gusakov, et al., 2005) (Galbe & Zacchi, 2002).
Figure 2.5: Schematic representation of a cellulosoma. Adapted from
(Alessandra, Isabella, Emanuele, & Vincenza, 2012)
34. 23
Preparations of cellulases from a single organism may not be highly efficient for
the hydrolysis of different feedstock. For example, Thrichoderma reesei produces
endo-glucanases and exo-glucanases in large quantities, but its β-glucosidase
activity is low, resulting in an inefficient biomass hydrolysis. For this reason, the
goal of the enzymes producing companies has been to form cellulases cocktails by
enzymes assembly (multienzyme mixtures) or to construct engineered
microrganisms to express the desired mixtures (Mathew, Sukumaran, Singhania, &
Pamdey, 2008). Enzyme mixtures often derive from the co-fermentation of several
micro-organisms (Ahamed, 2008) (Berlin, Maximenco, Gilkes, & Saddeler, 2007)
(Table 2.4). All the commercial cellulases listed in table 4 have an optimal condition
at 50°C and pH of 4.0-5.0. More recently, some enzymes producers have marked
new mixtures able to work in a higher temperature ranging from 50 to 60°C (Table
2.4).
In 2010, new enzymes were produced by two leading companies, Novozymes and
Genencor, supported by the USA Department of Energy (DOE). Genencor has
launched four new blends: Accelerase®1500, Accelerase®XP, Accelerase®XC
and Accelerase®BG. Accelerase®1500 is a cellulases complex (exo-glucanase,
endo-glucanase, hemi-cellulase and β-glucosidase) produced from a genetically
modified strain of T. reesei. All the other Accelerase are accessory enzymes
complexes: Accelerase®XP enhances both xylan and glucan conversion;
Accelerase®XC contains hemicellulose and cellulase activities; Accelerase® BG
is a β-glucosidase enzyme. In February 2010, Genencor has developed an enzyme
complex known as Accellerase®Duet which is produced with a genetically
modified strain of T. reesei and that contains not only exo-glucanase, endo-
glucanase, β-glucosidase, but includes also xylanase. This product is capable of
hydrolyzing lignocellulosic biomass into fermentable monosaccharides such as
glucose and xylose (Genencor, 2010). Similarly, Novozymes has produced and
commercialized two new enzymatic mixtures: cellic Ctec, and cellic Htec. Cellic
CTecis used in combination with Cellic HTec and this mixture is capable to work
with a wide variety of pretreated feedstock, such as sugarcane bagasse, corn cob,
corn fiber, and wood pulp, for the conversion of the carbohydrates in these materials
into simple sugars (Novozyme, 2010).
In order to meet the future challenges, innovative bioprocesses for the production
of new generation of enzymes are needed. As already described, conventional
35. 24
cellulases work within a range of temperature around 50°C and they are typically
inactivated at temperatures above 60-70 °C due to disorganization of their three
dimensional structures followed by an irreversible denaturation (Viikari,
Alapuranen, Puranen, Vehmaanperä, & Siika-aho, 2010). Some opportunities of
process improvement derive from the use of thermostable enzymes.
a) One FPU (filter paper unit) is the amount of enzyme that forms 1 µmol of
reducing sugars/min during the hydrolysis reaction of filter paper Whatman
No.1
b) One CBU (cellobiase unit) corresponds to the amount of enzyme which forms
2 µmol of glucose/min from cellobiose
2.3 RICE HULLS
Rice hulls, a byproduct generated during dehulling of rough rice (Oryza sativa), are
important lignocellulosic materials that could be considered for production of fuels and
chemicals. According to the world production of rice (FAO Food Outlook, 2009) and
based on the 20% yield of hulls of the harvested rice (Kim & Dale, 2004), the global
potential of rice hulls is around 139 million tonnes year.
Although there are several potential applications (Govindarao, 1980), rice hulls are
generally landfilled or burnt (Koopmans & Koppejan, 1997). The availability and
quality of rice hulls depend on the type and size of the rice mills.
Large rice mills generate high amounts of rather uniform hulls, whereas small village-
type (‘‘artisan’’) mills produce lower amount of rather heterogeneous hulls. Small
homemade mills, which are common for example in rural areas in Cuba, often lack a
good control on the milling, thus a high degree of grain breakage occurs during the
process and the hulls contain grain fragments and bran. Rice hulls are promising for
economical ethanol production as their carbohydrate content is high and they are readily
available from large production units without causing high transportation costs
(Moniruzzaman & Ingram, 1998); (Saha, Iten, Cotta, & Wu, 2005) (Martin, Lopez,
Plasencia, & Hernandez, 2006); (Martin, Alriksson, Sjode, Nilvebrant, & Jonsson,
2007a).
However, rice husks also contain high quantities of ash (20%) and lignin (16%), which
combined with hemicelluloses, results a complex structure around the cellulose, being
more difficult its use as a lignocellulosic feedstock for conversion to ethanol. For this
36. 25
Table 2.4: Commercial cellulases able to work at temperature ranging from 50 to 60ºC
Figure 2.6: Mechanism of action of cellulose. (Alessandra, Isabella, Emanuele, & Vincenza,
2012)
Table 2.5: Typical composition of rice hulls. (Ang, et al., 2011)
37. 26
2.4 ABSORBANCE
2.4.1 Measuring the Absorbance of a Sample Using a Spectrophotometer
For each wavelength of light passing through the spectrometer, the intensity of the light
passing through the reference cell is measured. This is usually referred to as I0 - that's I
for Intensity.
The intensity of the light passing through the sample cell is also measured for that
wavelength - given the symbol, I.
If I is less than I0, then obviously the sample has absorbed some of the light. A simple
bit of mathematics is then done in the computer to convert this into something called
the absorbance of the sample - given the symbol, A.
For reasons to do with the form of the Beer-Lambert Law (below), the relationship
between A (the absorbance) and the two intensities is given by:
A= log 10(I0/I)
On most of the diagrams you will come across, the absorbance ranges from 0 to 1, but
it can go higher than that.
An absorbance of 0 at some wavelength means that no light of that particular
wavelength has been absorbed. The intensities of the sample and reference beam are
both the same, so the ratio I0/I is 1. Log10 of 1 is zero.
An absorbance of 1 happens when 90% of the light at that wavelength has been
absorbed - which means that the intensity is 10% of what it would otherwise be.
In that case, I0/I is 100/10 (=10) and log10 of 10 is 1.
2.4.2 The Importance of Concentration
The proportion of the light absorbed will depend on how many molecules it interacts
with. Suppose you have got a strongly coloured organic dye. If it is in a reasonably
concentrated solution, it will have a very high absorbance because there are lots of
molecules to interact with the light.
However, in an incredibly dilute solution, it may be very difficult to see that it is colored
at all. The absorbance is going to be very low.
Suppose then that you wanted to compare this dye with a different compound. Unless
you took care to make allowance for the concentration, you couldn't make any sensible
comparisons about which one absorbed the most light.
38. 27
2.4.3 THE IMPORTANCE OF THE CONTAINER SHAPE
Suppose this time that you had a very dilute solution of the dye in a cube-shaped
container so that the light travelled 1 cm through it. The absorbance isn't likely to be
very high. On the other hand, suppose you passed the light through a tube 100 cm long
containing the same solution. More light would be absorbed because it interacts with
more molecules.
Again, if you want to draw sensible comparisons between solutions, you have to allow
for the length of the solution the light is passing through. (Clark, 2007)
39. 28
CHAPTER 3
METHODOLOGY
3.1 MATERIALS USED
3.1.1 Biomass
Rice hulls were used during this work. It was sourced from Ifo Local Government
Area in Ogun State. The hulls were sun dried and milled and kept in covered drums
at room temperature. The hulls were used in the entire process in this work.
3.1.2 Chemicals Required
a) Sodium hydroxide pallets – 40g
b) Hydrogen Peroxide – 600ml
c) 3-5, Dinitro Salicylic Acid – 25g
d) Crystalline Phenol – 500g
e) Sodium Metabisulphite – 10g
f) Sodium Potassium tartrate – 100g
g) Citric Acid – 40g
h) Sodium Citrate – 40g
i) Cellulase Enzyme
j) Acetone
k) Sulpuric Acid
l) Distilled Water
3.2 EQUIPMENT USED
a) Sieve Shaker
b) Conventional Oven
c) Soxhlet Extractor
d) Water Bath
e) Autoclave
f) Furnace
g) Magnetic Hotplate with Stirrer
h) Micropipette
i) Incubator
j) UV-Spectrophotometer
3.3 BRIEF SUMMARY OF WORK DONE
The milled rice hulls samples were sieved in order to get the right particle sizes for analysis.
The sieved samples were then analyzed in the laboratory to determine the extractives,
40. 29
moisture, lignin, ash, hemicellulose and cellulose contents. After this, the samples were
pretreated at the various conditions generated by the design of experiment, then enzymatic
hydrolysis was done on the pretreated samples.
The reducing sugar yields after enzymatic hydrolysis were analyzed and the pretreatment
conditions were optimized using the MINITAB software and a target yield value was
obtained and further pretreatments and hydrolysis were carried out in order to validate the
pretreatment conditions. Tests were also carried out to find the glucose concentration in the
reducing sugars.
42. 31
(g) (h)
(i) (j)
Figure 3.1: Equipment and Experimental set up for the study. (a) Oven, (b) Vacuum
Filtration Setup, (c) UV-Spectrophotometer, (d) pH Meter, (e) Water Bath, (f) Weighing
Balance, (g) Incubator, (h) Magnetic Hotplate , (i) Furnace, (j) Soxhlet Extractor
Figure 3.2: 0.15mm rice hulls (far left); unscreened rice hulls (2nd from left);
1.18mm rice hulls (2nd from right); 0.075mm rice hulls (far right)
43. 32
3.4 ALKALINE PRETREATMENT
The pretreatment was carried out in beakers. 5g of dry biomass was soaked in 100ml
mixture of distilled water and 1-3% of 30% H2O2. The pH of the mixture was raised to 11.5
with sodium hydroxide pellets. The temperature range for the pretreatment was 60-90ºc,
the time range was between 6-10hours.
The design of experiment was done using the statistical software MINITAB 16. The
response surface design method was used for the experimental design. A 2 level, 3 factor
central composite design was selected under the response surface design and 1 block was
selected in order to account for effects on the experiments due to the surroundings. The
levels of parameters for experimental design are shown in Table 3.1 and the total number
of experimental runs with the three variables that was designed according to the Central
composite design (CCD) is shown in Table 3.2.
45. 34
After the pretreatment, the dry weight analysis of each sample was done by putting 2g of
each sample in the oven for 3hours, cooling, weighing and putting them back into the oven
for another hour. The samples were dried till constant weight and the dry weight of the
pretreated samples was recorded.
The wet samples were stored in sample bottles and kept in the refrigerator before the
enzymatic hydrolysis.
3.5 ENZYMATIC HYDROLYSIS
The pretreated samples were hydrolyzed by the cellulase enzyme in order to check for the
efficiency of the alkaline pretreatment. The initial dry substance: liquid ratio was
maintained at 20gL-1
i.e. solid dry fraction of 2% (w/v). The solids were loaded into 100ml
sample bottles. 5ml of the 0.1M citrate buffer was added to the loaded biomass in order to
maintain the pH of the reaction at 4.8. The Trichoderma ressei cellulase enzymes were
prepared commercially. The activity of the enzymes was 57.8 FPU/ml and was added at a
loading of 25 FPU/g. The total volume was made to reach 20ml by adding an appropriate
amount of distilled water. The samples were then put into the incubator at 50ºc and
intermittent shaking was done. The experimental period was 96 hours.
3.6 OPTIMIZATION OF THE ALKALINE PRETREATMENT AND ENZYMATIC
HYDROLYSIS CONDITIONS FOR RICE HULLS.
After the yield of reducing sugars was obtain from calculations, optimization was done in
order to get the optimal process parameters for pretreatment. This was done using the
response optimizer of MINITAB 16.
After the yield of reducing sugars were obtained, the response surface design was analyzed.
The yield data was selected as the response of the pretreatment factors and model was set
up in order to examine the effects of the factors on the yield. Surface diagrams were drawn
to determine the individual and interactive effects of the factors on reducing sugar yield
and the optimal value of each factor to optimize the process response was generated using
the response optimizer.
The temperature during the optimized enzymatic hydrolysis was also changed to 45ºc, the
biomass loading was varied 2%, 3%, 4% & 5% biomass loading, the enzyme loading was
also varied between using 15FPU/g, 20FPU/g, 25FPU/g, 30FPU/g & 35FPU/g and some
46. 35
samples were soaked for 3 days while others were not. All these were the variations used
in the optimization process.
The untreated sample was also analyzed at 2% biomass loading and 25FPU/ (g biomass
loading) and the yields were compared.
3.7 GLUCOSE ANALYSIS
This test was performed on the optimized samples that were soaked. The test was done in
order to know the glucose content in the reducing sugars. The randox glucose test kit was
used in determination of the glucose concentration. The test kit had a buffer constituting of
phosphate buffer and phenol. The buffer had a pH value of 7.0. The kit also had a glucose
oxidase reagent. 4-aminophenazone, glucose oxidase and peroxibase where the
constituents of the glucose oxidase reagent. The final constituent of the kit was standard
glucose. The glucose was determined after enzymatic oxidation in the presence of glucose
oxidase. The hydrogen peroxide formed reacted under catalysis of peroxidase, with phenol
and 4-aminophenazone to form a red-violet quinoneimine dye as indicator. The reaction
principle is stated below;
Glucose + O2 + H2O GOD
→ gluconic acid + H2O2
2H2O2 + 4-aminophenazone + Phenol POD
→ quinoneimine + 4H2O
The procedure for the analysis is stated in appendix A7.
47. 36
CHAPTER 4
RESULTS AND DISCUSSIONS
4.1 SIEVE ANALYSIS
The milled rice hulls were subjected to particle size distribution. The arrangement for
the distribution was in the order (from bottom): Collection pan, 0.075mm, 0.15mm,
1.18mm and 2.36mm. Tables 4.1 (a) and 4.1 (b) show the obtained results.
After the sieve analysis, the 1.18mm particle sizes of rice hulls were kept and used for
the experiments. This was done because 1.18mm was the desired size amongst the other
sizes and there was considerable yield of this size after the sieve analysis.
4.2 LABORAORY ANALYSIS
The 1.18mm particle sizes of the rice hulls went through different tests in the laboratory
in order to obtain the various amounts lignocellulosic contents. Tests for moisture,
lignin, ash, hemicellulose and extractives contents were performed and the cellulose
content was obtained by;
Cellulose content % (w/w) = 100% - (Extractives + Hemicellulose + Lignin +Ash
+ Moisture) content % (w/w)
48. 37
Table 4.1a: Table showing the particle sizes and sample weights for each batch of
sample during the sieve analysis
Table 4.1b: Table showing average particle sizes and weight fractions of different
batches of the sieve analysis
BATCH 1 BATCH 2 BATCH 3 BATCH 4 BATCH 5
Particle
Size
(mm)
Sample
Weight
(g)
Particle
Size
(mm)
Sample
Weight
(g)
Particle
Size
(mm)
Sample
Weight
(g)
Particle
Size
(mm)
Sample
Weight
(g)
Particle
Size
(mm)
Sample
Weight
(g)
2.36 0 2.36 0 2.36 0 2.36 0 2.36 0
1.18 10 1.18 14 1.18 14 1.18 10 1.18 8
0.15 36 0.15 36 0.15 36 0.15 36 0.15 40
0.075 2 0.075 2 0.075 0 0.075 4 0.075 2
Pan 0 Pan 0 Pan 0 Pan 0 Pan 0
Total=
48
Total=
52
Total=
50
Total=
50
Total=
50
BATCH 1 BATCH 2 BATCH 3 BATCH 4 BATCH 5
Average
Particle
Size
(mm)
Wt.
Fraction
Average
Particle
Size
(mm)
Wt.
Fraction
Average
Particle
Size
(mm)
Wt.
Fraction
Average
Particle
Size
(mm)
Wt.
Fraction
Average
Particle
Size
(mm)
Wt.
Fraction
1.77 0 1.77 0 1.77 0 1.77 0 1.77 0
0.665 0.2083 0.665 0.2692 0.665 0.28 0.665 0.2 0.665 0.16
0.1125 0.75 0.1125 0.6923 0.1125 0.72 0.1125 0.72 0.1125 0.8
0.0375 0.0417 0.0375 0.0385 0.0375 0 0.0375 0.08 0.0375 0.04
0 0 0 0 0 0 0 0 0 0
Total 1.0 1.0 1.0 1.0 1.0
50. 39
Figure 4.2: Plot of weight fraction against average particle sizes
Table 4.3: Contents of Rice hulls
CONTENT %(w/w)
Cellulose
Content
Extractives
Content
Hemicellulose
Content
Lignin Ash
Content
Moisture
ContentInsoluble
Lignin
Soluble
Lignin
36.71 4.97 12.93 17.7 0.521 13.867 13.3
-0.1
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 1.8 2
Averagewt.fraction
Average particle sizes (mm)
Average weight fraction vs Average particle
sizes (mm)
51. 40
Figure 4.3: Graph showing the different contents of rice hulls in % (w/w)
0
5
10
15
20
25
30
35
40
Cellulose Hemicellul
ose
Lignin Ash Moisture Extractives
Series1 36.71 12.93 18.221 13.867 13.3 4.97
Content%(w/w)
Contents of Rice Hulls %(w/w)
52. 41
From the Figure 4.3, it is seen that the percentage of cellulose present in the rice hulls is
36.71% which is the highest among the other contents. This proves rice hulls to be a very
good lignocellulosic material due to its high concentration of cellulose. However, the
percentage of lignin and ash which combine with hemicellulose to form a complex
structure around the rice hulls is seen to be high also. This makes it difficult to use rice
hulls as an appropriate biomass for ethanol production. For this reason, pretreatment was
employed in order to break the complex chain of the lignin, ash and hemicellulose. Thus
making the cellulose easily accessible for hydrolysis and further fermentation for ethanol
production.
4.3 ALKALINE PRETREATMENT
The alkaline pretreatment was done with the aim of fractionating the rice hull biomass into
a solid fraction containing as much cellulose and less lignin as possible and the liquid
fraction of the pretreatment containing solubilized hemicellulose. The order and variables
of the pretreatment is shown in table 3.1 and 3.2. The dry weight analysis was done in
order to estimate how much lignin, ash and hemicellulose was removed from the rice hull
sample. It was also done in order to calculate the mass of the biomass to use for enzymatic
hydrolysis. The weight of dry biomass in the solid fraction after drying 2g of the pretreated
sample ranged from 0.575g to 0.7556g.
The yield gotten after the enzymatic hydrolysis of each pretreated samples was studied
and used to optimize the alkaline pretreatment conditions using hydrogen peroxide as the
oxidant.
4.4 ENZYMATIC HYDROLYSIS
The results of the experiments after the enzymatic hydrolysis were analyzed by
considering the total reducing sugar yield of the pretreated rice hull samples as the
response variable. This analysis was done using the MINITAB software. The total
reducing sugar yield was expressed as milligram per gram dry biomass. Table 4.5 shows
the experimental design and the response column showing the yield of total reducing
sugars. From Table 4.5, it is seen that standard order 3 produced the maximum yield of
reducing sugars, followed by standard order 7. This shows that the enzymatic
hydrolysis of rice hulls for standard order 3 and 7 was affected by factors such as
53. 42
cellulose swelling, decrease of polymerization degree and crystallinity, increase in
internal surface area, disruption of the lignin structure and separation of structural
linkages between lignin and carbohydrates.
56. 45
From Table 4.5, one could also assume that other factors might affect enzymatic
hydrolysis yield. A potential factor could be the conversion of alkali into irrecoverable
salts and the incorporation of salts into the biomass during the pretreatment reactions.
Also, the alkaline reagents can also remove acetyl and various acid substitutions on
hemicellulose, thus reducing the accessibility of hemicellulose and cellulose to
enzymes.
From scientific literature, we see that alkaline pretreatment is more effective on
agricultural residues with low lignin content than on softwood with high lignin content.
(Bjerre, Olesen, & Fernqvist, 1996)
57. 46
Figure 4.4: Surface plot of Yield vs. Time, Temperature
Figure 4.5: Surface plot of Yield vs. H2O2, Temperature
50
100
50
75
150
6
4
100
10
8
(mg/g)
Time(hr)
Temp(°C)
h2o2 2
Hold Values
Surface plot of Yield vs Time, Temperature
0
40
80
50
75
80
120
1
0
100
3
2
(mg/g)
H2O2(%)
Temp(°C)
time 8
Hold Values
Surface plot of Yield vs H2O2, Temperature
58. 47
Figure 4.6: Surface plot of Yield vs. H2O2, Time
Figure 4.7: Optimization plot for pretreatment conditions
40
60
4
6
8
10
80
1
010
3
2
(mg/g)
H2O2(%)
Time(hr)
temp 75
Hold Values
Surface plot of Yield vs H2O2, Time
Cur
High
Low0.96074
D
Optimal
d = 0.96074
Maximum
Yield
y = 193.4294
0.96074
Desirability
Composite
0.3182
3.6818
4.6364
11.3636
49.7731
100.2269
time h2o2temp
[49.7731] [11.3636] [3.6818]
59. 48
Figures 4.4-4.6 show the surface plots of the interactive effect of pretreatment
temperature, time and percentage H2O2 on reducing sugar yield. The response
optimization was done and the graph is shown in figure 4.7. From the response
optimization, pretreatments at 49.8°C, time of 11.36 hours and 3.68% of H2O2 were the
optimum variables in order to attain a maximum reducing sugar yield of 193.43 mg/g
with a composite desirability of 0.96074.
4.5 OPTIMIZATION OF PRETREATMENT CONDITIONS
The response optimizer was used to obtain the optimum pretreatment conditions in
order to get a solid fraction with high cellulose content, low lignin and hemicellulose,
and a liquid fraction with low concentration of reducing sugars. The optimized
conditions were for pretreatments to occur at 49.77ºC, Time of 11.36 hours and
Hydrogen peroxide concentration of 3.68%. Additional experiments were carried out
in order to validate the optimized conditions. The experimental response gave a
maximum yield of 192.89 mg/g dry biomass with a predicted response of 193.43 mg/g
dry biomass, thus confirming the optimization process. This was gotten at 4% biomass
loading and 25 FPU/g enzyme loading.
The results of the different variations used in the optimization process and the
comparisons of their yields is shown below;
61. 50
Figure 4.8: Graph showing the reducing sugar yields against biomass loading and enzyme
loading variations
Untreate
d
Sample
2% B.
Loading
3% B.
Loading
4% B.
Loading
5% B.
Loading
15FPU/g 20FPU/g 25FPU/g 30FPU/g 35FPU/g
Unsoaked Pretreaments 32.8 70.04 79.89 184.66 110.36 54.67 61 61.71 57.68 60.24
Soaked Pretreatments 32.8 68.07 75.07 192.89 147.61 55.18 61.42 62.72 43.27 47.68
0
50
100
150
200
250ReducingSugarYield
(mg/gdrysolid)
Variations
Reducing Sugar Yields from different Variations at 45°C
Unsoaked Pretreaments Soaked Pretreatments
62. 51
From the results obtained, Figure 4.8 shows that increase in the biomass loading favored
higher reducing sugar yields. The highest yields of reducing sugars were obtained from
4% biomass loading at 25 FPU/g enzyme loading. These conditions gave the best
productivity at 45°C.
For the enzyme loading, the highest yields at constant biomass loading were obtained
between 20 FPU/g biomass and 25 FPU/g biomass. Thus the optimum enzyme loading
for rice hulls should be between 20–25 FPU/g biomass. This condition is very important
as overloading the biomass with enzymes can cause saturation of the substrate which
does not improve the yield, also insufficient loading could cause low enzyme
concentration and thus reduce the yield of reducing sugars.
Looking at the soaking, the soaked pretreated samples gave a better yield at higher
biomass loading than the pretreated samples that were not soaked. It is assumed that
due to the length of soaking, there was enough time to break the lignin complex and
dissolve more hemicellulose of the rice hulls more efficiently, thus enabling better
hydrolysis. Considering the yield of the untreated sample and comparing with the other
treated samples, it is clear that pretreatment greatly affects enzymatic hydrolysis yield.
The lowest yield on the graph was that of the untreated sample. This justifies the
pretreatment done on the samples.
The optimization of the pretreatment conditions helped in the achievement of a yield of
193.43 mg/g while the validated value was 192.89 mg/g
4.6 GLUCOSE TEST
The results of the glucose test are shown in Table 4.8
From Figure 4.9, it is seen that the glucose concentration in the reducing sugars was
very high in the optimized sample. This means that more cellulose was hydrolyzed in
the enzymatic hydrolysis, which is another indicator of effective pretreatment of the
biomass. The other reducing sugars in the yield were formed as a result of hydrolysis
of the hemicellulose. These other reducing sugars include uronic acids, pentoses,
hexoses and cellubiose. The highest yield of glucose was obtained at 4% biomass
loading and 25 FPU/g enzyme loading. Looking at the untreated sample, it is seen that
the glucose concentration after hydrolysis is far less than that of the reducing sugars.
63. 52
This validates the effect of pretreatment when comparing the glucose concentration of
the untreated sample to the yields of the other pretreated samples. The concentration of
the other reducing sugars is higher because more hemicellulose is hydrolyzed in the
untreated sample and the complex lignin structure around the cellulose prevents
efficient hydrolysis of the cellulose.
Possibly, the concentration of glucose in the reducing sugars could be higher if another
enzyme was used in the enzymatic hydrolysis. From (Gilkes, Henrissat, Kilburn, Miller,
& Warren, 1991), it is seen that in the Trichoderma ressei cellulase enzyme, the amount
of β-glucosidase is lower than the amount needed for efficient hydrolysis of cellulose
to glucose. As a result, a major product of the hydrolysis is cellubiose which is a dimer
of glucose.
64. 53
Table 4.8: Glucose data for optimized samples
Variation
Reducing
Sugar
Conc.
(mg/ml)
Absorbance
(500nm)
Glucose
conc.
(mg/ml)
% of glucose
in reducing
Sugar
% of other
reducing
sugars
Untreated Sample
0.0991 0.003 0.027 27.27 72.73
2% Biomass
Loading 0.86 0.046 0.41 48.05 51.95
3% Biomass
Loading 1.43 0.057 0.51 36 64
4% Biomass
Loading 1.51 0.083 0.75 49.62 50.38
5% Biomass
Loading 1.71 0.069 0.62 36.41 63.59
15FPU/g
0.67 0.034 0.31 45.83 54.17
20FPU/g
0.74 0.035 0.32 42.38 57.62
25FPU/g
0.76 0.039 0.35 46.24 53.76
30FPU/g
0.44 0.019 0.17 39.02 60.98
35FPU/g
0.48 0.021 0.19 39.14 60.86
The absorbance of the standard solution was 0.111.
65. 54
Figure 4.9: Chart showing the concentration of glucose and other reducing sugars in the
yield
0 10 20 30 40 50 60 70 80
2% Loading
3% Loading
4% Loading
5% Loading
15FPU/g
20FPU/g
25FPU/g
30FPU/g
35FPU/g
Untreated
48.05
36
49.62
36.41
45.83
42.38
46.24
39.02
39.14
27.27
51.95
64
50.38
63.59
54.17
57.62
53.76
60.98
60.86
72.73
% Concentration of glucose and other reducing sugars in
the yield
% Other reducing sugars % Glucose
66. 55
CHAPTER 5
CONCLUSIONS AND RECOMMENDATIONS
5.1 CONCLUSION
The investigation showed that alkaline pretreatment could be used to pretreat rice
hulls in order to give a substantial yield of reducing sugars after enzymatic
hydrolysis. Validated optimized conditions of 49.8°c, time of 11.36 hours and
3.68% of H2O2 with biomass loading of 4% and 25FPU/g enzyme loading gave a
reducing sugar yield of 192.89 mg/g dry biomass.
From the variations used during the enzymatic hydrolysis, it was seen that soaking
the samples in the pretreatment solution for 3 days before the actual pretreatment
helped in improving the reducing sugar yield. It was also noticed that enzyme
loading between 20FPU/g-25FPU/g gave higher yields than the other enzyme
loadings at 45°C. Also, increasing the biomass loading improved the reducing sugar
yield. The optimum biomass loading for best yield of reducing sugars was 4% at
45ºC.
The glucose concentration in the reducing sugars also helped validate the
pretreatment. The glucose concentration in the reducing sugars was higher in the
pretreated sample than the untreated sample.
5.2 RECOMMENDATIONS
1. More variations should be included in the optimization of the enzymatic
hydrolysis in order to get even better yields. Factors like hydrolysis temperature
can be varied in order to see how the hydrolysis responds to temperature
changes.
2. An incubator which stirs samples automatically during hydrolysis is
recommended, as the human factor in stirring or shaking samples is not
efficient.
3. A further evaluation is needed to determine the reducing sugar content in the
liquid fraction after pretreatment.
4. Other enzymes can be investigated in order the check for their various effects
on glucose yields after hydrolysis.
67. 56
REFERENCES
Adetayo, A. (2013). Sodium hydroxide and hydrogen peroxide oxidative pretreatment and
enzymatic hydrolysis of siam weed for ethanol production. In A. Adetayo, Sodium
hydroxide and hydrogen peroxide oxidative pretreatment and enzymatic hydrolysis of
siam weed for ethanol production. Ota: Chemical Engineering Department, College of
science and technology, Covenant University.
Ahamed, A. &. (2008). Enhanced enzyme production from mixed cultures of Trichoderma
reesei RUT-C30 and Aspergillus niger LMA grown as fed batch in a stirred tank
bioreactor. In A. &. Ahamed, Enhanced enzyme production from mixed cultures of
Trichoderma reesei RUT-C30 and Aspergillus niger LMA grown as fed batch in a
stirred tank bioreactor (pp. 41-46). Biochemical Engineering Journal ,Vol.42, No.1
(October, 2008).
Alessandra, V., Isabella, D. B., Emanuele, R., & Vincenza, C. (2012). Hydrolysis of
Lignocellulosic Biomass: Current Status of Processes and Technologies and Future
Perspectives. In V. Alessandra, D. B. Isabella, R. Emanuele, & C. Vincenza,
Hydrolysis of Lignocellulosic Biomass: Current Status of Processes and Technologies
and Future Perspectives. Bioethanol, Prof. Marco Aurelio Pinheiro Lima (Ed.),
ISBN: 978-953-51-0008-9.
Alizadeh, H., Teymouri, F., Gilbert, T., & Dale, B. (2005). Pretreatment of switchgrass by
ammonia fiber explosion (AFEX). In T. F. Alizadeh H, Pretreatment of switchgrass
by ammonia fiber explosion (AFEX) (pp. 1133–1141). Appl Biochem Biotechnol
124.
Alvira, P., Tomás-Pejó, E., Ballesteros, M., & Negro, M. (2010). Pretreatment technologies
for an efficient bioethanol production process based on enzymatic hydrolysis: A
review. In P. Alvira, E. Tomás-Pejó, M. Ballesteros, & M. Negro, Pretreatment
technologies for an efficient bioethanol production process based on enzymatic
hydrolysis: A review (pp. 4851-4861). Bioresource Technology, Vol.101, No.13,
(July, 2009).
Ana, D., Julie, L. T., Ana, B., Ignacio, d. O., & Ildefonso, C. (2013). Pretreatment of Rice
Hulls with Alkaline Peroxide to Enhance Enzyme Hydrolysis for Ethanol Production.
In D. Ana, L. T. Julie, B. Ana, d. O. Ignacio, & C. Ildefonso, Ana, Diaz; Julie, Le
Toullec; Ana, Blandino; Ignacio, de Ory; Ildefonso, Caro. Chemical Engineering
Transactions.
Ang, T., Yoon, L., K.M., L., Ngoh, G., Chua, A., & Lee, M. (2011). Efficiency of ionic
liquids in the dissolution of rice husk. In T. Ang, L. Yoon, L. K.M., G. Ngoh, A.
Chua, & M. Lee, Efficiency of ionic liquids in the dissolution of rice husk (pp. 4790-
4800). Bioresources. 6(4).
Balat., M., Balat, H., & Oz, C. (2008). Progress in bioethanol processing. In M. Balat., H.
Balat, & C. Oz, Progress in bioethanol processing (pp. 551-573). Progress and
Energy Combustion science, Vol. 34, No.5, (October 2008).
Bayer, E., Chanzy, H., Lamed, R., & Shoham, Y. (1998). Cellulose, cellulases and
cellulosomes. In E. Bayer, H. Chanzy, R. Lamed, & Y. Shoham, Cellulose, cellulases
68. 57
and cellulosomes (pp. 548-557). Current Opinion in structural Biology, Vol.8, No.5
(October, 1998).
Berlin, A., Maximenco, V., Gilkes, N., & Saddeler, J. (2007). Optimazion of enzyme
complexes for lignocelluloses hydrolysis. In A. Berlin, V. Maximenco, N. Gilkes, &
J. Saddeler, Optimazion of enzyme complexes for lignocelluloses hydrolysis (pp.
pp.287-296). Biotechnology and Bioengineering, Vol.97, No.2 .
Biofuel Facts. (2010). Retrieved from Biofuel.org.uk: Biofuel.org.uk/Biofuels%20-
%20Biofuel%20Facts.htm
Bjerre, A., Olesen, A., & Fernqvist, T. (1996). Pretreatment of wheat straw using combined
wet oxidation and alkaline hydrolysis resulting in convertible cellulose and
hemicellulose. In B. A.B., O. A.B., & F. T., Pretreatment of wheat straw using
combined wet oxidation and alkaline hydrolysis resulting in convertible cellulose and
hemicellulose (pp. 568-577). Biotechnol Bioeng, 49.
Bozell, J., & Petersen, G. (2010). Technology development for the production of biobased
products from biorefinery carbohydrates-The US Department of Energy’s “Top10“
revisited. In J. Bozell, & G. Petersen, Technology development for the production of
biobased products from biorefinery carbohydrates-The US Department of Energy’s
“Top10“ revisited (pp. pp.525-728). Green Chemistry, Vol. 12., No.4, (Jun, 2010).
Clark, J. (2007). Chemguide. Retrieved from www.chemguide.co.uk:
www.chemguide.co.uk/analysis/uvvisible/beerlambert.html
De Bari, I., Nanna, F., & Braccio, G. (2007). SO2-catalyzed steam fractionation of aspen
chips for bioethahnol production: Optimization of the catalyst impregnation. In I. De
Bari, F. Nanna, & G. Braccio, SO2-catalyzed steam fractionation of aspen chips for
bioethahnol production: Optimization of the catalyst impregnation (pp. 7711-7720).
Industrial & Engineering Chemistry Research, Vol.46, No.23 (October, 2007).
De Bari, I., Viola, E., Barisano, D., Cardinale, M., Nanna, F., Zimbardi, F., . . . Braccio, G.
(2002). Ethanol Production atFlask and Pilot Scale from Concentrated Slurries of
Steam-Exploded Aspen. In I. De Bari, E. Viola, D. Barisano, M. Cardinale, F. Nanna,
F. Zimbardi, . . . G. Braccio, Ethanol Production atFlask and Pilot Scale from
Concentrated Slurries of Steam-Exploded Aspen (pp. 1745-1753). Industrial &
Engineering Chemistry Research, Vol. 41, No.7 (March, 2002).
Demirabas, A. (2008). Products from Lignocellulosic Materials via Degradation Processs. In
A. Demirabas, Products from Lignocellulosic Materials via Degradation Processs
(pp. 27-37). Energy Sources Part A, Vol.30, No.1,(January, 2008).
Fägerstam, L., & Pettesson, L. (1984). The primary structure of a 1,4–β-glucan
cellobiohydrolases of Trichoderma reesei QM9414. In L. Fägerstam, & L. Pettesson,
The primary structure of a 1,4–β-glucan cellobiohydrolases of Trichoderma reesei
QM9414 (pp. 97–101). FEBS Letters, Vol.119, No.2 (February, 1984).
FAO Food Outlook. (2009, June). Retrieved from www.fao.org: http://www.fao.org/giews/
FitzPatrick, M., Champagne, P., Cunningham, M., & Whitney, R. (2010). A biorefinery
processing perspective : Treatment of lignocellulosic materials for the production of
69. 58
value-addued products. In M. FitzPatrick, P. Champagne, M. Cunningham, & R.
Whitney, A biorefinery processing perspective : Treatment of lignocellulosic
materials for the production of value-addued products (pp. pp. 8915-8922).
Bioresource Technology, Vol.101, No.23, (December, 2010).
Galbe, M., & Zacchi, G. (2002). A review of the production of ethanol from softwood. In M.
Galbe, & G. Zacchi, A review of the production of ethanol from softwood (pp. pp.
618-628.). Applied Microbiology and BiotechnologyVol.59, No.6, (July, 2002).
Genencor. (2004, October 21). Retrieved from www.genencor.com:
http:/genencor.com/cms/connect/genencor/media_relations/news/archive/2004/gen_2
11004_en.htm
Genencor. (2010, January 14). Retrieved from www.genencor.com:
http://www.genencor.com/ wps/ wcm/connect/ genencor/ genencor/ products and
services/ business development/ biorefineries/products/ accellerase product line
en.htm
Gilkes, N., Henrissat, B., Kilburn, D., Miller, R. J., & Warren, R. (1991). Domains in
microbial β-1,4-glycanases: sequence conservation, function, and enzyme families. In
N. Gilkes, B. Henrissat, D. Kilburn, R. J. Miller, & R. Warren, Domains in microbial
β-1,4-glycanases: sequence conservation, function, and enzyme families (pp. 303–
315). Microbiological Reviews, Vol. 55, No.2.
Gómez Sandra, M., Andrade Rafael, R., Santander, C. G., Costa, A. C., & Maciel, F. R.
(2010). Pretreatment of sugar cane bagasse with phosphoric and sulfuric diluted acid
for fermentable sugars production by enzymatic hydrolysis. Chemical Engineering
Transactions.
Govindarao, V. (1980). Utilization of rice husk – a preliminary analysis. In V. Govindarao,
Utilization of rice husk – a preliminary analysis (pp. 395–415). J. Sci. Ind. Res. 39.
Gusakov, A., Sinitsyn, A., Salanovich, T., Bukhtojarov, F., Markov, A., Ustinov, B. B., . . .
Burlingame, R. (2005). Purification, cloning and characterisation of two forms of
thermostable and highly active cellobiohydrolase I (Cel7A) produced by the industrial
strain of Chrysosporium lucknowense. In A. Gusakov, A. Sinitsyn, T. Salanovich, F.
Bukhtojarov, A. Markov, B. B. Ustinov, . . . R. Burlingame, Purification, cloning and
characterisation of two forms of thermostable and highly active cellobiohydrolase I
(Cel7A) produced by the industrial strain of Chrysosporium lucknowense (pp. 57–69).
Enzyme and Microbial Technology, Vol.36, No.1, (January, 2005).
Himmel, M. E., Ding, S., Johnson, D., Adney, W., Nimlos, M., Brady, J., & Foust, T. (2007).
Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production. In
M. E. Himmel, S. Ding, D. Johnson, W. Adney, M. Nimlos, J. Brady, & T. Foust,
Biomass Recalcitrance: Engineering Plants and Enzymes for Biofuels Production.
Science 315, 804.
Himmel, M., Ding, S., Johnson, D., Adney, W., Nimlos, M., Brady, J., & Foust, T. (2007).
Biomass recalcitrance: engineering plants and enzymes for biofuels production. In D.
S. Himmel M.E., Biomass recalcitrance: engineering plants and enzymes for biofuels
production (pp. 804-807). Science.
