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Electronic Theses, Treatises and Dissertations
The Graduate School
2009
Enzymatic Hydrolysis of Cellulose in a
NMMP/H2O Solution
Rilwan Oyetunji
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FLORIDA STATE UNIVERSITY
COLLEGE OF ENGINEERING
ENZYMATIC HYDROLYSIS OF CELLULOSE IN A NMMO/H2O SOLUTION
By
RILWAN OYETUNJI
A Thesis submitted to the
Department of Chemical Engineering
in Partial Fulfillment of the
requirements for the degree of
Master of Science
Degree Awarded:
Spring Semester, 2009
The members of the Committee approve the Thesis of Rilwan Oyetunji defended on
April 3, 2009
__________________________
Subramanian Ramakrishnan
Professor Co9Directing Thesis
__________________________
John Collier
Professor Co9Directing Thesis
__________________________
Billie Collier
Outside Committee Member
Approved:
_________________________________________________________________
Bruce Locke, Chair, Chemical Engineering
_________________________________________________________________
Ching9Jen Chen, Dean, College of Engineering
The Graduate School has verified and approved the above named committee members.
ii
I dedicate this thesis to my Spouse Wumi for her overwhelming encouragement, my
family members for their unfailing support, all my friends and colleagues: who never
failed to tell me the truth even when I did not want to hear it, to God for His infinite
mercies and for making all these wonderful people a part of my life.
iii
ACK OWLEDGEME TS
First and foremost, I would like to acknowledge my Advisors: Dr Ramakrishnan and Dr
Collier, for their: ideas, trust, guidance and support throughout the program. This thesis
would not have been complete without their detailed comments, suggestions and
encouragements.
I would also like to acknowledge Dr Paravastu for letting me use his laboratory to
analyze the results of the experiment, and my colleagues: Brett, Brian, Colt, Daniel,
Elizabeth, and Rachel for their ideas and help in performing the experiments.
Finally I would like to acknowledge the Department of Chemical and Biomedical
Engineering for accepting me into the program, The PREM foundation and Sun Grant for
supporting the research, and members of the FAMU9FSU College of Engineering who
helped me in one way or the other in setting up the experiments.
iv
TABLE OF CO TE TS
ACKNOWLEDGEMENTS ........................................................................................................... iv
TABLE OF CONTENTS ................................................................................................................ v
LIST OF TABLES ....................................................................................................................... viii
LIST OF FIGURES ........................................................................................................................ ix
Abstract ........................................................................................................................................... x
Chapter 1 Introduction .................................................................................................................... 1
1.1 Problem Identification ........................................................................................................... 2
1.2 Project Objectives .................................................................................................................. 3
Chapter 2 Literature Review ........................................................................................................... 4
2.1 Introduction ........................................................................................................................... 4
2.2 Cellulose ................................................................................................................................ 4
2.3 Glucose .................................................................................................................................. 5
2.4 Cellulose Hydrolysis ............................................................................................................. 7
2.4.1 Acid Hydrolysis Method ................................................................................................. 8
2.4.2 Enzymatic Hydrolysis Method ....................................................................................... 9
2.5 Pretreatment Processes .......................................................................................................... 9
2.6 Enzymatic Hydrolysis of Cellulose ..................................................................................... 11
2.6.1 Understanding the Cellulase System ............................................................................ 11
2.6.2 Understanding the Lignocellulosic Substrate ............................................................... 12
2.6.3 Understanding the Enzyme9Substrate Interaction ........................................................ 13
2.6.4 Understanding the Inhibition or Deactivation of the Enzyme ...................................... 14
2.6.5 Description of the Cellulose Hydrolysis Kinetics ......................................................... 14
2.7 The Purpose of this Research .............................................................................................. 20
Chapter 3 Experimental Methodology .......................................................................................... 22
3.1 Introduction ......................................................................................................................... 22
3.2: Estimation of Reducing Sugars by DNS Method .............................................................. 22
3.2.1 – Theory Behind the DNS Method ............................................................................... 22
3.2.2 – Preparation of DNS Reagent ...................................................................................... 23
3.2.3 – Determination of Absorbance Vs Concentration Calibration Points ......................... 24
3.3: Estimation of Enzyme Activity by the Filter Paper Unit (FPU) Method .......................... 26
v
3.4: Preparation of NMMO.x H20 ............................................................................................ 27
3.5: Preparation of 1% Cellulose Solutions .............................................................................. 28
3.6: Protocol for enzyme reactions ........................................................................................... 28
Chapter 4 Results .......................................................................................................................... 30
4.1 Enzyme Activity /FPU Determination of Accelerase™1000 and Spezyme® Cellulase .... 30
4.2 Effect of NMMO on the Enzymatic Hydrolysis of Cellulose ............................................. 33
4.2.1 Yield of Reducing Sugars for Different Concentrations of NMMO mixed with
Cellulase................................................................................................................................. 33
4.2.2 Comparison of NMMO/Cellulase Solution with Acetate Buffered Solution of various
pH for 1 Hr Reaction with Genencor Spezyme® ................................................................... 35
4.2.3 Comparison of NMMO/Cellulase Solution with Acetate Buffered Solution of Various
pH for 3 Hr Reaction with Accelerase™1000 Cellulase ....................................................... 37
4.2.4 Effect of Cellulase on Cellulose Dissolved in NMMO and Filter paper in acetate buffer
or NMMO medium ................................................................................................................ 37
4.3 Factors That Affect The Enzymatic Hydrolysis .................................................................. 41
4.3.1 Effect of Temperature .................................................................................................. 45
4.3.2 Effect of Enzyme Loading ........................................................................................... 50
4.3.3 Effect of pH................................................................................................................... 54
4.4 Interaction Between Temperature and pH ........................................................................... 59
Chapter 5 Conclusions .................................................................................................................. 63
Chapter 6 Recommendations ........................................................................................................ 65
APPENDIX A Sugar Yields (mg/mL) as a function of Time ....................................................... 66
APPENDIX A.1 40C DATA ..................................................................................................... 66
APPENDIX A.2 50C DATA ..................................................................................................... 71
APPENDIX A.3 60C DATA ..................................................................................................... 76
APPENDIX B. Percentage Cellulose Conversion as a function of Time ..................................... 81
APPENDIX B.1 40C DATA ..................................................................................................... 81
APPENDIX B.2 50C DATA ..................................................................................................... 86
APPENDIX B.3 60C DATA ..................................................................................................... 91
APPENDIX C Dilution, Weight of 1% lyocell solution and Enzyme activity for each experiment
....................................................................................................................................................... 96
REFERENCES:............................................................................................................................. 98
vi
BIOGRAPHICAL SKETCH ...................................................................................................... 102
vii
LIST OF TABLES
Table 2. 19 Relationship Between Structural Feature and Enzyme Digestibility1 .......................... 5
Table 2. 29List of Assumptions from Authors that have modeled the Hydrolysis Process .......... 17
Table 3. 19Glucose Concentration versus Absorbance Data ......................................................... 25
Table 4. 19pH of NMMO Solution after Cellulase Addition versus Concentration of water in
NMMO solution ............................................................................................................................ 35
Table 4. 29 Percentage Conversion of Cellulose at 40C and 50C, pH 5.7 and 7.4, and Enzyme
Loading of 122 FPU/g ................................................................................................................... 44
Table 4. 39Percentage Conversion of Cellulose at Different Temperatures, Enzyme Loading and
pH after 24 Hours Enzymatic Reaction......................................................................................... 48
Table 4. 49Percentage Conversion of Cellulose at Different Temperature, Enzyme Loading and
pH after 3 Hours Enzymatic Reaction........................................................................................... 49
Table 4. 59Percentage Conversion of Cellulose at Different Temperatures and Enzyme Loading
at pH 7.4 after 24 Hours Enzymatic Reaction ............................................................................... 53
Table 4. 69Percentage Conversion of Cellulose at Different Temperatures and Enzyme Loading
at pH 5.7, after 24 Hours Enzymatic Reaction .............................................................................. 53
Table 4. 79 Percentage Conversion of Cellulose at Different Temperatures, pH and Enzyme
Loadings, after 24 Hours Enzymatic Reaction.............................................................................. 58
viii
LIST OF FIGURES
Figure 2. 19 Diagram of Glucose, Cellulose and Amylose ............................................................. 7
Figure 2. 29Lignocelluloses organization into elementary fibrils and microfibrils7 ..................... 13
Figure 2.39Comparison Between Two Processes of Hydrolyzing Cellulose ................................ 21
Figure 3. 19Reaction of Glucose with DNS .................................................................................. 23
Figure 3. 29Glucose Concentration versus Absorbance Calibration Curve.The equation of the
curve and the R2 value are on the plot. .......................................................................................... 26
Figure 4. 19FPU Determination for Accelerase 1000 ................................................................... 31
Figure 4. 29FPU Determination for Genencor Spezyme ............................................................... 32
Figure 4. 39 Effect of NMMO on Enzymatic Hydrolysis of Filter Paper ..................................... 34
Figure 4. 49Comparison between NMMO medium and Acetate Buffer Medium Using Genencor
Spezyme ........................................................................................................................................ 36
Figure 4. 59Comparison between NMMO medium and Acetate Buffer medium using Accelerase
1000 ............................................................................................................................................... 39
Figure 4. 69 Effect of Hydrolyzing Cellulose while it is coming out of Solution ......................... 41
Figure 4. 79 Yield of Reducing Sugars as a function of Time at Different Reaction conditions .. 43
Figure 4. 89 Effect of Temperature at pH 5.7 and 34.9 FPU/g Enzyme Loading ......................... 46
Figure 4. 99Effect if Temperature at pH 5.7 and 122 FPU/g Enzyme Loading ............................ 47
Figure 4. 109 Effect of Temperature at pH 5.7 and 1445.6 FPU/g Enzyme Loading ................... 48
Figure 4. 119Effect of Enzyme Loading at 40C and pH 7.4 ......................................................... 51
Figure 4. 129 Effect of Enzyme Loading at 40C and pH 5.7 ........................................................ 52
Figure 4. 139 Effect of pH at 40C and 122 FPU/g Enzyme Loading ............................................ 55
Figure 4. 149 Effect of pH at 50C and 122 FPU/g Enzyme Loading ............................................ 56
Figure 4. 159Effect of pH at 60C and 122 FPU/g Enzyme Loading ............................................. 57
Figure 4. 169Interaction Between Temperature and pH at 34.9 FPU/g Enzyme Loading ............ 60
Figure 4. 179Interaction Between Temperature and pH at 122 FPU/g Enzyme Loading ............. 61
Figure 4. 189Interaction Between Temperature and pH at 1445.6 FPU/g Enzyme Loading ........ 62
ix
ABSTRACT
This thesis is focused on the enzymatic hydrolysis of cellulose while it is in an N9
methylmorpholine9 N9Oxide (NMMO)/H20 solution. The reason for using NMMO/H20 solvent
is due to the solvent’s utilization in making lyocell fibers, and its ability to pretreat cellulose by
breaking down its crystalline structure. This pretreatment leads to an increase in the yield of
reducing sugars from the enzymatic hydrolysis. The enzymatic hydrolysis is done in
NMMO/H20, so that one step in the pretreatment process, the removal of the pretreated cellulose
prior to enzymatic hydrolysis, can be eliminated. This enzymatic hydrolysis was achieved by
first dissolving the cellulose in the near monohydrate form of NMMO solution before adding
water or 10 % (w/w) acetic acid; together with a diluted cellulase solution. By so doing, the
structure of the cellulose substrate is changed, and, the hydrolysis reaction medium is different
from the typical 50C and pH 4.8 reaction medium. This reaction medium was investigated at
temperatures of 40, 50 and 60C; enzyme loadings of 34.9, 122 and 1445.6 FPU/g; and pH
conditions of 5.7 and 7.4. The yield of reducing sugars was lowest at 60C, when compared to
other temperatures.
For experiments at 40C and 50C, there was an interaction between the effect of temperature
and pH. The 7.4 pH systems seemed to favor temperatures of 40C, while the 5.7 pH systems
favored temperatures of 50C. Increases in enzyme loading led to an increase in the yield of
reducing sugars; however it was observed that the increase in enzyme loading was not
proportional to the increase in reducing sugar yields. For this reason, increases in enzyme
loading led to a decrease in sugar yield per enzyme loading. The highest cellulose conversion,
92% conversion, was achieved at a temperature of 40C, enzyme loading of 1445.6 FPU/g and a
pH of 7.4.
x
CHAPTER 1 I TRODUCTIO
Saccharification of agricultural materials using cellulases is a process which has been studied by
many others
1, 5, 8
. The agricultural residues that show potential as biomass for energy are
composed of cellulose, hemicelluloses and lignin9. Of these, cellulose has been identified as the
principal polysaccharide in plants and the most abundant constituent of lignocelluloses. The
degradation of cellulose to soluble sugars requires the cooperative action of a number of
enzymes: endoglucanases, exoglucanases and β9glusidases; collectively known as cellulases. The
saccharification of lignocelluloses to sugars can be used for the production of organic solvents,
organic gases, or single cell protein; and can help solve a major problem facing mankind today:
that of energy.
The need for alternative energy sources concerns the international community because of an
overall increase in pollution rates, and also in the energy demands of developing countries. Fossil
fuels, which are presently the major source of energy, are known to produce combustion
byproducts that are harmful to the health of living things and the environment, and these fuels
are currently being depleted due to increased utilization and the fact that they are non renewable.
For these reasons, considerable research is constantly being done in order to find a new energy
source that is both reliable and economical. One of these alternatives is the degradation of
cellulose to simple sugars also known as saccharification. Saccharification of agro9industrial
materials using cellulases is a process which involves the use of enzymes to break down the
glycosidic bonds of lignocelluloses. The enzymatic degradation of cellulose requires the action
of cellulases and, in most cases, water as a reactant according to the following general
mechanism in which glucose is shown as a product of the reaction. Other simple sugars can also
result.
C6H11O69(C6H10O5)n9C6H11O6+ (n9x) H2O→ (n9x) C6H12O6+ C6H11O6 9 (C6H10O5)x9 C6H11O5
The simple sugars formed from this reaction can be further decomposed into alcohols and
gaseous organic compounds, including methane and hydrogen, which can be used to provide
electricity. However, the degradation step is critical because cellulose has a highly rigid structure
which is very difficult to decompose. In contrast, the research on the hydrogen production from
sugars has been carried out for many years.
1
The two approaches to degrading cellulose are: acid hydrolysis and enzymatic hydrolysis. Acid
hydrolysis has proved unsuccessful due to high operational cost, while interest in enzymatic
hydrolysis is growing due to recent advances in biochemistry, which has greatly reduced the
price of enzymes.
The three raw materials that are most used for the conversion of biomass to energy byproducts
utilizing enzymes are sugars, starch and cellulose. Of these three, cellulose materials represent
the most abundant source of biomass (global production is 200×109 tons per year3); however the
utilization of the lignocellulosic feedstock is currently impractical due to high costs of
transportation, scattered stations, and a slow conversion rate.
In order to improve the conversion rate, the pretreatment of the cellulose before enzyme
hydrolysis also needs to be taken into consideration. The chemical pretreatment of
Lignocellulose is used to improve the accessibility of enzymes to the cellulose molecules. There
are various types of chemical pretreatment that can be used to increase the digestibility of the
biomass material. For this reason, the most efficient pretreatment method would considerably
decrease the amount of time required for the hydrolysis of cellulose to glucose.
1.1 Problem Identification
In natural plant materials, the cellulose is bound with lignin and hemicelluloses, and forms a
rigid structure which is highly resistant to hydrolysis. In order to extract the cellulose from the
raw materials, pretreatment of the cellulose prior to enzymatic hydrolysis is required. This
pretreatment is used to decrease the crystallinity of the cellulose molecule, to increase the surface
area, and to remove or make the the lignin9hemicelluloses sheath that surrounds the cellulose
more penetrable. However, most pretreatment methods which decrease cellulose crystallinity
involve the use of extreme temperatures9 (e.g. sub and supercritical H2O), high pressures, 11 or
chemical pretreatment using specialized chemicals (ionic liquids12) in order to break down the
structure of the cellulose. These methods usually lead to high operating costs, because the
pretreated cellulose needs to be extracted before the enzyme hydrolysis, which usually occurs
under mild conditions. For example most enzyme reactions usually take place at 50C and in
moderately acidic environments (pH of 5).
2
1.2 Project Objectives
For the purpose of achieving high cellulose conversion rates and explaining the important factors
that affect enzyme hydrolysis, dissolving pulp cellulose, which has minute amounts of lignin and
hemicelluloses, was dissolved in N9methylmorpholine9N9Oxide (NMMO) before the addition of
known amounts of precipitating solvents, and enzyme dilutions. The precipitating solvents were
used to vary the pH of the system, and various enzyme dilutions were used to control the enzyme
loading in the system. NMMO was used because it is known to dissolve cellulose by interacting
with the hydrogen bonds within the cellulose molecule. Cellulose precipated from NMMO/H20
has a less crystalline structure and is, therefore, more susceptible to enzymatic hydrolysis. When
this method is used, the cellulose need not need be extracted from the system before enzyme
addition. The key factors that were varied in this study are: temperature, pH and enzyme
loadings. These factors were chosen because their effect on enzyme hydrolysis is well
documented, and also because NMMO is a highly basic solvent. That is, a low pH requirement
may not be economically feasible if it cannot be obtained from the precipitating solvent.
Through investigation of how these factors affect the enzymatic hydrolysis of the cellulose, the
optimum conditions for enzymatic hydrolysis, using NMMO as the reaction medium, can be
obtained.
3
CHAPTER 2 LITERATURE REVIEW
2.1 Introduction
For the purpose of decreasing the United States dependency on foreign oil, mandates have been
proposed to displace 30% of the nation’s gasoline use by 2030. This proposal would require
approximately a billion dry tons of biomass. The key benefits of the proposal is: 1) the reduction
of green house emissions, 2) a decreased dependency on foreign oil, which would lead to better
national security and 3) the growth of energy supplies and the creation of jobs38. A major barrier
to achieving these goals, by using biofuels to displace the gasoline, is the recalcitrance of the
cellulosic biomass to enzymatic degradation. This is because most biofuels are produced from
the fermentation of reducing sugars by bacteria or fungi. While cellulosic and starch containing
crops or materials can be used for the production of sugars, cellulosic materials are usually
slowly degraded by enzymatic reactions due their structure. This review discusses the factors that
need to be considered for the successful enzymatic degradation of cellulose to sugars.
2.2 Cellulose
Cellulose is the major component of cell walls of cellulosic fiber. It is a linear condensation
polymer consisting of D9anhydroglucopyranose units, joined together by β91, 49glycosidic
bonds. Coupling of adjacent cellulose polymeric units by hydrogen bonds and van der Waal’s
forces, results in a parallel alignment that leads to a highly crystalline structure which is resistant
to decomposition. That is, these hydrogen bonds inhibit the cellulose structure from being broken
by water, or mild chemicals, and make it resistant to enzymatic decomposition17.
The degree of polymerization (DP) of cellulose, refers to the number of anhydroglucopyranose
units (glucose units) linked together by the glycosidic bonds, and the DP differs according to the
type and origination of the cellulosic material. In native, cellulose up to 10000 β9anhydroglucose
residues can be linked to the long chain molecule 17. Native cellulose, which is also referred to as
cellulose I, has two distinct crystallite forms which can be converted to other forms of cellulose
by using various treatment methods.
Various pretreatment methods are used to break down the hydrogen bonds that give cellulose
its structural stability. Usually, all chemical reactions take place at the glucosidic linkage and
4
hydroxyl groups of the cellulose molecule. In order to increase the access of enzymes to these
places, the chemical or physical features of the cellulose molecule needs to be changed. The
physical features that affect the cellulose digestibility are surface area, crystallinity, degree of
polymerization, pore volume and particle size. Table 2.1 summarizes how these properties affect
the digestibility of the enzyme. A positive effect means an increase in that property leads to an
increase in enzyme digestibility while a negative effect means a decrease in that property leads to
an increase in enzyme digestibility. As can be seen from the table some properties have no
substantial effect on the digestibility of the cellulose by the enzyme (no correlation).
Table 2. 1+ Relationship Between Structural Feature and Enzyme Digestibility1
Structural Features
Relationship between structural feature and digestibility
Physical
Surface Area
Crystallinity
Degree of Polymerization
Pore volume
Particle size
Positive
Negative/No correlation
Negative/No correlation
Positive
No correlation
Lignin
Hemicellulose
Acetyl Group
Negative
Negative
Negative
Chemical
2.3 Glucose
The basic unit of cellulose, as well as starch and glycogen, is glucose. It is the most common
organic group in nature, and is one of the main energy sources for plants and animals. Other facts
of glucose are as follows:
it has the molecular formula C6H12O6
it is a monosaccharide, since it’s made up of one unit
two glucose units linked together is called a dissacharide; three glucose units linked
together is called a trisaccharide,
three to ten glucose units are sometimes referred to as an oligosaccharide
very large number of units can be linked to form a polysaccharide
its carbon atoms are easily oxidized to form carbon dioxide, and energy is released in the
process
5
it is highly soluble in water due to its numerous hydroxyl groups (hydrogen bonds are
easily formed with water molecules)
Of the 16 stereoisomers of glucose there are three most often considered structural
isomers: D9glucose from starch, D9galactose, a sugar in milk, and fructose, a sugar found
in honey
For these reasons many polysaccharides made up of glucose subunits are usually used for
sources of food or sources of energy. The differences between polysaccharides made up of
glucose subunits are the linkages which join the glucose molecule together and the cyclical form.
The linkages affect the ease at which glucose can be retrieved from the polysaccharide. For
example, the body of human beings has the ability to easily break down starch (carbohydrates)
but cannot break down Cellulose, which is easily broken down by sheep or other ruminants.
Below is a diagram of the glucose chain and various polysaccharides. Glucose units in cellulose
are linked together by β91, 4 linkages, while units in the natural starch (amylose) are linked
together by α91, 4 linkages. Amylopectin is another type of glucose polysaccharide which
consists of several amylose chains joined together by α91, 6 linkage
6
Ring form of Glucose
Cellulose
Amylose
Figure 2. 1+ Diagram of Glucose, Cellulose and Amylose
2.4 Cellulose Hydrolysis
Cellulose hydrolysis can be defined as the degradation of cellulose to glucose or other simple
sugars, in order to utilize the sugars produced, for the production of energy. Each cellulose
molecule is a straight chain consisting of 1000 to 1 million D9glucose units, linked by β91, 4
glycosidic bonds. It is a structural molecule for plants and this makes it resistant to degradation.
Cellulose from various biomass sources differ in crystalline structures and in their binding to
other components. The cellulose present in lignocelluloses is composed of two components:
crystalline and amorphous, and, the amorphous component degrades more easily than the
crystalline component. The lignin in lignocelluloses forms a barrier which inhibits cellulose from
degradation by either enzyme or acid. For this reason, pretreatment of the cellulose by the
removal of lignin leads to an increase in the accessibility of cellulose molecules and increases the
7
degradation rate. The two methods of hydrolyzing the cellulose are: the Acid Hydrolysis Method
and the Enzymatic Hydrolysis method.
2.4.1 Acid Hydrolysis Method
The hydrolysis of biomass by dilute acid was the first technology used for the conversion of
biomass to ethanol: with the first commercial process being built in Germany in 1898. However,
this process was considered to be uneconomical in the 1980’s after it was attempted by the U.S.
during the Second World War30.
The acid hydrolysis process is a process, which uses acid to change the molecular structure of
cellulose thereby reducing its degree of polymerization. The two major types of acid hydrolysis
process are: diluted and concentrated acid hydrolysis24.
The dilute acid process involves the contact of cellulosic materials with dilute acid. This leads to
the formation of hydrocellulose, then the formation of soluble polysaccharides before the
formation of simple sugars. This process is conducted at a high temperature and pressure; and is
usually completed within a few minutes. An advantage of the dilute acid process is its ability to
convert biomass to sugars within minutes and achieve a sugar recovery of approximately 80%,
while the disadvantages are: the high temperature and pressure required; the requirement of
expensive materials for the process and; the neutralization of the acid before the sugar
recovery31.
The concentrated acid process is a process that can utilize a low temperature and low pressure for
the degradation of cellulose materials to simple sugars. The process has a sugar recovery of 90%
for both hemicellulose and cellulose sugars, however, in comparison to the dilute acid system, at
low temperatures, it is a slow process and the cost of acid recovery systems is high. The acid
recovery system is important because neutralization of the acid would lead to large sugar
deposits, which can increase the cost of the process due to disposal requirements24.
The products that are produced by the acid hydrolysis of cellulose are: sugars, furfurals and
organic acids. The major sugars produced by the process are xylose, glucose and cellobiose; the
furfurals produced are fufuraldehyde and hydroxylmethyl furfural; while the organic acids
produced are levulinic acid, formic acid and acetic acid. In general the hydrolysis of natural
8
cellulose sources by acids leads to the formation of many by9products, which makes it difficult to
produce pure sugars and limits the viability of the acid hydrolysis process.
2.4.2 Enzymatic Hydrolysis Method
Since a cellulose molecule consists of a long chain of β91→4 glycosidic linkages, the enzyme
complex which is capable of breaking down the glycosidic linkages consists of three main
groups of enzymes. These are: endo9β9glucanase, exo9β9glucanase and β9glucosidase,
collectively known as cellulases. The ease at which the cellulases are able to break down the
cellulose depends on the cellulose structure or configuration. There are basically two types of
cellulose structures: crystalline and amorphous. The amorphous structure of cellulose has a more
random structure and is therefore more easily degradable by the cellulases. Because natural
cellulose sources (i.e. lignocelluloses) contains lignin compounds, which pose as an obstacle to
enzymatic hydrolysis of cellulose, and generally consist of the crystalline form of cellulose, the
susceptibility of the cellulose to cellulases needs to be improved before the enzymatic hydrolysis
of cellulose. For these reasons, pre9treatment processes that can remove lignin and
hemicelluloses, and change the crystalline structure of cellulose to an amorphous structure, are
often essential prior to the enzymatic hydrolysis of cellulose7, 18.
2.5 Pretreatment Processes
The type of process used to pre9treat biomass materials ultimately depends on the particular
biomass because different biomass materials have different compositions of lignin,
hemicelluloses and cellulose. In general the pre9treatment processes, that have been used, can be
classified into four main categories: physical, physico9chemical, chemical and biological pre9
treatments. One or more combinations of these pre9treatment options can be used to pre9treat a
particular type of biomass. The pre9treatment processes used, should: improve the formation of
sugars or the ability to form sugars; reduce or avoid the degradation of carbohydrate; prevent the
formation of byproducts during the hydrolysis process and; be economical and cost effective24, 26.
The main purpose of the physical pretreatment is to physically break down the lignocellulosic
biomass into particles that can be easily hydrolyzed by acidic or enzymatic hydrolysis. For this
reason most physical pretreatment methods are energy intensive. Examples of these are:
9
pyrolysis, milling of the biomass material, radiation, freezing, and mechanical comminution. The
crystallinity and the degree of polymerization of the cellulose are reduced after these processes24.
In Physico9chemical pre9treatment the biomass is treated with high pressure for a certain time
period, and then the pressure is reduced swiftly. With this treatment the biomass material
undergoes a rapid decompression which exposes the internal surface area to enzymatic
hydrolysis. The main types of physico9chemical pretreatment includes: steam explosion,
ammonia fiber explosion and CO2 explosion. Some of these pre9treatment require the biomass to
be first chipped into small sizes before it undergoes pre9treatment 24, 28.
The chemical pretreatment method utilizes a solvent to dissolve the crystalline structure of the
cellulosic material. Various chemicals have been utilized for this pre9treatment. The overall
effect, of the pre9treatment on the biomass depends on the chemical that is used. Alkalis, acids,
ozone, hydrogen peroxide, mixtures of organic solvents with inorganic acid catalysts
(organosolv) and, more recently cellulose ionic solvents have been employed in the pre9
treatment of biomass. Ozone has been used to degrade lignin and hemicellulose in the biomass
material, and the pre9treatment process can be carried out at room temperature. However a large
amount of ozone is required for the process to be effective. The oxidative delignification or the
use of hydrogen peroxide is able also remove the lignin and hemicelluloses from the biomass
material
24, 26, 28
. However at low temperatures (~30C) longer times are required for the process
to be effective while at high temperatures (~170C) the pre9treatment can be completed within 59
10 minutes. The Alkaline treatment of the biomass causes structure swelling that is able to
increase the internal surface area, and decrease the degree of polymerization
25, 27
. This pre9
treatment is also able to disrupt the lignin structure and separate the linkages between lignins and
cellulose/hemicelluloses. Acids, such as dilute H2SO4 and HCl, serve as catalysts for the
hydrolysis of cellulose rather than as a reagent for pretreatment, however this pre9treatment
process requires reactors that are resistant to corrosion due to the corrosive nature of the solvents.
Cellulose solvents such as NMMO, 19Butyl939methylimidazolium Chloride, 19ethyl939
methylimidazolium acetate, Cadoxen (a complex compound of cadmium and ethylene diamine),
etc can swell and transform solid cellulose into a soluble state, thus enhancing hydrolysis process
although some of these solvents are toxic and expensive, a few of these solvents are not toxic,
have a low vapor pressure, and can be easily recovered prior to cellulose hydrolysis 12, 25, 29.
10
Biological pre9treatment uses micro9organisms, such as brown9, white9, and soft9rot fungi to
solubilize lignin and hemicelluloses, and soften cellulose material, which leads to easier access
for the cellulases. These processes require less energy, but also proceed very slowly26.
2.6 Enzymatic Hydrolysis of Cellulose
The enzymatic hydrolysis of cellulose consists of two main steps: the hydrolysis of cellulose into
cellobiose by endo9β9glucanases and exo9glucananases, and the conversion of cellobiose to
glucose by β9glucosidase. The most studied enzyme complex used for degrading cellulose is
derived from the Trichoderma Reesei fungi. This enzyme complex consists of the three main
groups of enzymes used to degrade cellulose: endo9β9glucanase, exo9β9glucanase and β9
glucosidase, collectively known as cellulases. These are all required to hydrolyze cellulose
efficiently.
The process occurs as follows: the cellulase binds to cellulose to form a thermodynamically
favorable complex, which stabilizes the protein on the cellulose and limits the enzyme’s
penetration into the fibrils of the cellulose; an increase in mechanical pressure due to the
presence of these enzyme molecules is exerted on the cavity walls of the cellulose molecule; the
cellulose structure swells; water gets accommodated within the cellulose molecule; this leads to
the breaking of the hydrogen bonds, the dissociation of the microfibrils and, the formation
glucose15.
Based on this process, reviews on cellulose hydrolysis have classified the factors that need to
be considered in order to understand the enzymatic hydrolysis of cellulose into five. These are:
1) understanding of the cellulase system, 2) understanding the lignocellulosic substrate, 3)
understanding the enzyme9substrate interaction 4) understanding the inhibition or deactivation of
the enzyme.
2.6.1 Understanding the Cellulase System
The most studied cellulase systems were secreted from
and
organisms due to the high levels of cellulase that are secreted from these organisms,
although, there are other organisms which are capable of secreting cellulases. The nature of these
cellulases determines the mode of action, the activity of the components, and the inhibitory
11
effects of products or intermediates. The
cellulase consists of: two
cellobiohydrolases, five endoglucanases, β9glucosidases and hemicellulases. For the hydrolysis
of cellulose, the cellobiohydrolases are known to remove glucose units from the non9reducing
end of the cellulose; endoglucanase hydrolyzes bonds of the cellulose in random action; while β9
glucosidase hydrolyzes the soluble sugars (cellobiose) that are released by the cellobiohydrolases
and endoglucanases. The efficiency of the hydrolysis depends on the presence of the cellulase
components and their corresponding ratios because these enzymes are known synergistically
hydrolyze the cellulose7, 10, 18.
During the modeling of cellulose hydrolysis, some investigators use a combined catalytic
activity to describe how the enzyme hydrolyzes the cellulose, however the more recent models,
which have been used to describe the process, separate the enzyme components into three:
endoglucanase, exoglucanase and β9glucosidaase.
2.6.2 Understanding the Lignocellulosic Substrate
Majority of the cellulose that exists can be found in lignocellulosic biomass. Examples of
lignocellulosic biomass materials are woods, grasses and cellulose wastes (like papers, leaves,
tree barks etc). These materials differ in property, but they all have a relatively similar
composition, which consists of cellulose, hemicelluloses and lignin and are usually termed
Lignocelluloses. The major component of these materials is usually cellulose and it accounts for
40950% of the material weight, hemicellulose constitutes of about 20940% of the material, and
the remaining component is lignin. Cellulose and hemicelluloses form the main structure, while
lignin acts as a joining material and binds the fibers together. Lignin and hemicelluloses also
play a role in structural stabilization of the lignocellulose material; they make cellulose resistant
to decay and insect attack, however, lignin also prevents cellulosic materials from enzymatic
hydrolysis of glucose and other simple sugars. Figure 2.2 describes how these materials are
arranged with respect to one another.
12
Figure 2. 2+Lignocelluloses organization into elementary fibrils and microfibrils7
2.6.3 Understanding the Enzyme+Substrate Interaction
The interaction of the enzymes and the substrate is dependent on an adsorption process and it is
very rapid in comparison to the time required for hydrolysis. For this reason the incorporation of
the adsorption into models, has been ignored by some researchers and Michelis9Menten model is
usually used to describe the enzymatic hydrolysis, although this approach may not be applicable
at high substrate concentrations. The cellulase adsorption and desorption on the cellulose surface
has been described as irreversible, semi9reversible and reversible. It has been suggested that
cellulase adsorbs onto surface of cellulose and performs catalytic actions while moving along
substrate, while it has also been suggested that enzyme adsorbs, desorbs and then readsorbs
depending on its catalytic action
10
. The Langmuir isotherm, derived on the basis that the
adsorption process can be described by one adsorption equilibrium constant, is usually used to
model the adsorption process. Due to the simplicity of this assumption, some researchers have
used dynamic models and empirical models, e.g. Freudlich 9 Langmuir isotherms, to also
describe the adsorption process5,
32
. It has been observed that the adsorption process is
independent of pH, but strongly dependent on temperature18.
Mass transfer resistances, which are usually ignored during the modeling of cellulose
hydrolysis, are also known to affect the adsorption process. The three key resistances are: 1) the
external mass transfer resistance through the stagnant film layer, 2) the rate of enzyme
adsorption on the solid substrate, and 3) the rate of cellulose catalysis. The continued hydrolysis
13
of the substrate depends on the enzyme’s penetration through the fibrils and the diffusion of the
enzymes10.
2.6.4 Understanding the Inhibition or Deactivation of the Enzyme
Enzyme activity is affected by product inhibition and deactivation of the enzyme either by
environmental conditions or mass transfer constraints. Cellulase enzymes are susceptible to
deactivation when exposed to fluid shear stress, due to turbulence, in the reaction zone.
Kastelyanos et al found that glucose inhibited cellobiase, while cellobiose inhibited endoglucase
and exoglucanase was not inhibited33. Severe inhibition was observed by cellobiose and mild
inhibition was observed by glucose. Cellulolytic enzymes are inhibited by cellobiose and
glucose. Some models assumed competitive inhibition while others assumed non9competitive
inhibition. Some researchers have argued competitive inhibition while others argued non9
competitive inhibition (some said both). It has also been reported that glucose inhibits hydrolysis
of cellulase by T. viride
10
. Degree of deactivation is more serious at air9liquid interface. Reese
and Ryu suggest that enzyme deactivation was caused by unfolding of protein molecules at gas9
liquid interface. The temperature and pH also deactivate the enzyme.
2.6.5 Description of the Cellulose Hydrolysis Kinetics
Inorder to describe the kinetics of cellulose hydrolysis, Michaelis9Menten types of rate
expressions with substrate or product inhibition terms have been used. The inhibition terms were
added because experimental studies20 have shown that the enzymatic hydrolysis is affected by
the cellobiose and glucose products. A combination of enzyme adsorption and kinetic rate
models have been proposed based on Michaelis9Menten kinetics. One of the more recent
hydrolysis models proposed by Q Gan et al10 used Michelis9Menten kinetics to estimate the
glucose yield. The model was used to understand the effect of active and inactive cellulose
particles and, the effect of shear deactivation on cellulose hydrolysis. The concentration of active
substrate, at the water9cellulose9enzyme reaction interface was modeled to change as the reaction
progressed. The key assumptions made were: the cellulases had a combined catalytic function;
the cellulosic material was composed of two fractions9 a region easily hydrolyzed by enzymes
and a region that was inert to hydrolysis; the substrate concentration was based on the surface
concentration of hydrolysable cellulose and not the total concentration of cellulose; new
14
cellulose and inert substrates emerged from the inner region of substrate solids after the
dissolution of the first layer; there was a gradual decrease in the quality of the reaction interface;
the sugars produced from the enzyme inhibit the enzymes activity and; the deactivation of the
enzyme is related to the shear field residence time.
The results showed that binding of the enzyme to inert regions plays an important role in the
continuous reduction of the reaction rate. It was observed that: the initial surface concentration
accessible to enzyme binding and catalysis was 2% of the total substrate mass stock inserted in
the system; a small proportion of soluble enzymes retained their original catalytic power and; the
substrate particle size had a strong influence on the initial rate of enzyme hydrolysis. However
the effect of shear field on the kinetic model and experimental results were virtually
insignificant. In comparison to older models that had been used to predict glucose yield, three
new parameters were added and evaluated. These were: the cellulose accessibility coefficient,
surface active cellulose concentration coefficient and the relative shear field residence time.
Converse et al21 used two models to elucidate the factors which affect the enzymatic
hydrolysis. Although both models were based on Michaelis9Menten kinetics, the assumptions
made in acquiring the models were very different. The first model was based on the inactivation
of the enzyme by products, while the second model was based on mass transfer resistances
within the cellulose fibril. The key assumptions made by the inactivation model were: 1) the
enzyme may be inactivated by inactive substrate,2) the rate of hydrolysis was proportional to
concentration of active adsorbed enzyme, 3) Product inhibition is assumed to be in equilibrium
to yield and 4) the desorption and adsorption process was pseudo9steady state. In the mass
transfer model, it was assumed that, 1) the rate of formation of adsorbed but inactive enzyme is
proportional to the enzyme in solution 2) the steric factors of the enzyme and cellulose, hindered
the synergy between the components of all the enzyme in the fibril. Therefore, enzyme in the
fibril is deactivated 3) there was equilibrium between the enzyme in solution, enzyme9product
complex and the product and 4) the enzyme had four components9 active enzyme adsorbed by
the cellulose, enzyme in solution within the cellulose fibril, enzyme9product complex within the
fibril, enzyme adsorbed on the inactive cellulose within the fibril. The key difference between
both models is that in the inactivation model, the rate of formation of deactivated enzyme
15
depends on concentration of adsorbed enzyme, while in second model it depends on
concentration of free enzyme in solution.
The model was applied to a 10 fold range of substrate concentrations and the result showed
that both models predicted the glucose yield successfully, however, the mass transfer model
provided a better representation of adsorbed enzyme activity. Although it was assumed that the
adsorption sites on the cellulose molecule is proportional to square of substrate concentration in
both models, from the results it seems that the deactivation of the enzymes is dependent on the
concentration of free enzyme in solution. Table 2.2 shows a list key assumptions and conclusions
from various authors that have modeled the cellulose hydrolysis process.
16
Table 2.2+List of Assumptions from Authors that have modeled the Hydrolysis Process
Conclusions
Author
Key Assumptions
Movargenejad et
Transfer of enzyme from solution
Mean absolute deviation
al19
bulk to cellulose surface is rapid in
of 6% was observed
comparison to rate of reaction
The limit of the reaction
Only adsorbed enzymes hydrolyze
was
the cellulose
model
well predicted by
occurs;
Initial rates of reaction
enzymes adsorb solely at the
was under estimated by
cellulose external surface
model
No
internal
Cellulose
diffusion
sites
available
for
Some discrepancies were
enzyme is proportional to surface
observed
between
area of cellulose particle
experimental results and
Activity of enzyme adsorbed at
those determined from
cellulose surface decreases with
model
time
Inhibition
by
products
occur
during the reaction
cellulose particles shrink during
reaction
Liao et al5
Enzyme first adsorbed by different
Model
components
predict adsorption and
(cellulose,
was
able
to
hemicellulose and lignin)
hydrolysis data at high
Two types of bonding sites on
substrate concentrations(
cellulose9 active and inactive
50g/L)
Enzymes on active sites produce
Use of enzyme activity
sugars
instead
Glucose was assumed to be the
concentration effectively
only inhibitor of hydrolysis.
predicted the adsorption
Enzymatic hydrolysis divided into
and hydrolysis of the
17
of
enzyme
Table 2. 2 continued
Author
Conclusions
Key Assumptions
two
parts:
adsorption
sample
and
Langmuir adsorption was
hydrolysis
Langmuir
isotherms
used
to
significantly
changed
describe the enzyme adsorption
during hydrolysis for 5
Competitive model of glucose
samples
inhibition was used to simulate
hydrolysis reactions
3 major enzymes in conversion of
An empirical equation
cellulose to glucose: exoglucanase,
was used to describe
endoglucanase and β9glucosidase
change
Competitive enzymatic hydrolysis
constant
of
adsorption
is first order reaction on cellulose
surface
Structure
of
fiber
matrix
is
uniform in terms of enzyme
adsorption
Lignin and hemicellulose only
influence enzyme adsorption
Effect
of
different
concentrations
substrate
and
structure
difference were negligible
Available cellulose is related to
ratio of remained cellulose to
initial total cellulose
Shen et al22
-
Cellulase enzyme is assumed to
-
parameter
model
have a single combined effect in
developed could describe
the
a wide range of sugar
hydrolysis
of
insoluble
substrate
-
Two
concentration with time
Surface and structure of insoluble
fiber are homogeneous and there is
18
-
Model was successfully
used to fit experimental
Table 2. 2 continued
Author
Conclusions
Key Assumptions
no distinction between amorphous
-
and crystalline regions
-
-
data of cotton gin waste
Highest yield of reducing
Enzyme deactivation is assumed to
sugars were obtained at
be a second order reaction
lowest
Adsorption of free enzyme in
concentrations
initial
enzyme
suspension is based on Langmuir
isotherm adsorption
-
Quasisteady state is assumed for
the formation of enzyme9substrate
complex
-
Enzyme deactivation by insoluble
substrate
is
independent
of
hydrolysis rate
-
Diffusivity of enzyme on insoluble
substrate
was
estimated
from
Fick’s second law
The major researches that have been done in the area of cellulose hydrolysis are generally
focused on two areas: improving the conversion rate of cellulose hydrolysis and understanding
the kinetics of the conversion process. In order to improve the conversion rate, the key aspects of
the hydrolysis process that have been used to optimize the rate are: 1) increasing the
susceptibility of the substrate to enzyme decomposition; 2) varying the composition of the
enzyme components; and 3) changing the system properties. In order to understand the kinetic
processes the intricate details that need to be considered are: 1) the nature of the enzyme, 2) the
physical structure of the cellulosic material, 3) the substrate enzyme interactions, and the 4)
enzyme inhibition and deactivation during the hydrolysis.
Kinetic models have shown that the rate and extent of cellulose hydrolysis by cellulase
enzymes is influenced by a variety of substrate and enzyme factors. The overall reaction rate is
19
affected by mass transfer resistances, which includes the film resistance around cellulose
particles, the bulk phase resistance, and the resistance through the capillary of cellulose particles.
Reaction conditions also influence the hydrolysis rate. Therefore conditions that have been
used to improve the conversion rates are: the concentration and susceptibility of the cellulose
particles, the enzyme concentration, the pH of the medium, the agitation intensity and the
temperature. The optimal conditions for the hydrolysis is dependent upon the origination of the
cellulose, the ratio of the enzyme groups in the enzyme complex, and where the enzyme was
extracted from. The lignin content also acts as a barrier to the enzymes. However it was observed
by Kaya et al that the addition of dissolved lignin improved the enzymatic reaction. The
reasoning given for this occurrence was that the enzymes which bind to the lignin in solution
were sustained by the lignin and were able to hydrolyze the adjacent cellulose easily.
2.7 The Purpose of this Research
In our research we are trying to improve the conversion rate, of cellulose hydrolysis to sugars,
by changing the structure of the substrate and the reaction medium. The structure of the
crystalline cellulose material is changed to an amorphous structure by first dissolving the
cellulose in the monohydrate form of N9methylmorpholine9N9Oxide (NMMO) and then
precipitating it from the solution by adding water to the reaction medium. Studies4, 41 have shown
that the hydrolysis of the amorphous cellulose structure obtained from this precipitation
increased the conversion rate by a factor of three when compared to the hydrolysis of the
crystalline cellulose structure. Another reason why NMMO/H20 is used as the reaction medium
is due to its commercial utilization by the lyocell process. The lyocell process uses
NMMO/H20/Cellulose mixture (lyocell solution) to manufacture cellulose fibers which are used
for manufacturing cloth materials. For this reason, the rheological properties of
NMMO/water/cellulose solutions is well documented42,43 and therefore the commercialization of
a cellulose hydrolysis process using NMMO as the reaction medium can be easily achieved .
Figure 2.3 describes how this approach differs from previous methods used to convert
biomass to sugars. Typically the cellulases used for the conversion of biomass to sugars require
a slightly acidic reaction medium (pH 5) and a temperature of 50C. For this reason, previous
methods of converting biomass to sugars require the pretreatment of the biomass using the
20
methods previously described, before separating and hydrolyzing the pretreated biomass to
sugars in a reaction medium suitable for the cellulases. Therefore by using NMMO/H2O reaction
medium, the need to separate the pretreated biomass would not be required, and a higher yield of
sugars can be easily achieved.
Pretreatment of Cellulose in
solvent
Pretreatment/dissolution of
cellulose in NMMO/H20
Regeneration of
cellulose and
separation of
solvent is required
Hydrolysis of
cellulose to simple
sugars at 50C and
pH 4.8
Adjustment of pH using acetic
acid or deionized water and
hydrolysis to sugars
Figure 2.3+Comparison Between Two Processes of Hydrolyzing Cellulose
Comparison between Previous methods of Hydrolyzing cellulose and the Hydrolysis of cellulose
in a NMMO/H2O reaction medium
21
CHAPTER 3 EXPERIME TAL METHODOLOGY
3.1 Introduction
For the conversion of biomass to energy by9products, the hydrolysis of cellulose to glucose,
or cellulose to reducing sugars, is the process that consumes the majority of the cost. The sugars
produced can be used to produce hydrogen using hydrogenase bacteria. In this research, we are
trying to improve the conversion rate of the hydrolysis by; changing the structure of the substrate
and the reaction medium.
3.2: Estimation of Reducing Sugars by D S Method
The concentration of reducing sugars in a sample was estimated by diluting 0.2 mL of the
sample’s supernatant ( the sample was first centrifuged) to 1mL using distilled water9 in a test
tube9 and then adding adding 3mL dinitrosalicylic
acid (DNS) reagent. The test tube was
inserted in a 100ºC water bath for 10 minutes, and then inserted into an ice water bath in order
for the reaction to stop, and to stabilize the color, which is temperature sensitive. Two mL
deionized water was added to the test tube and the absorbance of the sample at wavelenght
540nm was acquired using a spectrophotometer. The spectrophotometer used was Lambda 25
UV/Vis Spectrometer and the cuvettes used were Quartz spectrophotometer cell (Starna cells,
Catalog no. 19Q910).
3.2.1 – Theory Behind the D S Method
The DNS method for estimating the concentration of reducing sugars in a sample was
originally invented by G. Miller34 in 1959. Figure 3.1 shows an example of the chemical reaction
that occurs during the DNS assay, in this case, glucose is used as the reducing sugar. A reducing
sugar is one that in a basic solution forms an aldehyde or ketone. The aldehyde group of glucose
converts DNS to 39amino959nitrosalicylic acid,which is the reduced form of DNS). The amount
of 39amino959nitrosalicylic acid is proportional to the amount of glucose. Water is used up as a
reactant and oxygen gas is released during the reaction.
22
O
O
OH
O
OH
O
N
+
N
O
HO
+
OH
OH
OH
+
O
OH
+
O
N
HO
+
OH
+ H2O
HO
O
OH
OH
+
NH 2
O
O2
O
HO
HO
OH
Figure 3. 1+Reaction of Glucose with D S
The formation of 39amino959nitrosalicylic acid results in a change in the amount of light
absorbed, at wavelenght 540 nm. The absorbance measured using a spectrophotometer is directly
proportional to the amount of reducing sugar. For this reason, a calibration curve of absorbance
vs concentration can be developed by measuring the absorbance of known concentrations of
glucose, and the equation of the calibration curve can be used to calculate the unknown
concentrations corresponding to their absorbance. D9(+) Glucose obtained from Sigma Aldrich
was used for the calibration. A reducing sugar is any sugar that forms aldehyde or ketones in a
basic solution. Sugars containing acetal or ketal linkages are not reducing sugars, while all
monosacharides and some dissacharides e.g. cellobiose are reducing sugars.
3.2.2 – Preparation of D S Reagent
Five hundred mL of 2% DNS solution was prepared by adding 10.0g of DNS acid powder to
500mL deionized water, and then mixing the solution with 100mL of 10.67 w/v % sodium
hydroxide solution. The solution was inserted into a 45C water bath until the contents were fully
dissolved; 300g of potassium sodium tartarate salt was then added with continuos mixing.
Deionized water was used to bring the total volume of the solution to 1L. The solution was kept
in an amber bottle due to the light sensitivity of the DNS solution.
The 10.67w/v % sodium hydroxide solution was prepared by dissolving 16g NaOH pellets
(Obtained from Fisher Scientific chemicals 98.5% Purity, Lot no. 033972) to 150 mL distilled
23
water, and the 2% DNS solution was prepared by adding 10g of DNS ( obtained from Alfa Aesar
Chemicals, 97% Purity) to 500mL distilled water.
3.2.3 – Determination of Absorbance Vs Concentration Calibration Points
Twenty standard glucose solutions (as given in Table 1), with concentration of 0.1mg/mL , to
3.0mg/mL were made by diluting 3mg/mL solution with the appropriate amount of deionized
water. 1mL of each solution was placed in a test tube before the addition of 3mL of DNS reagent
into each test tube. The samples were inserted in a 100C water bath for 10 minutes. The test
tubes were then inserted into an ice water bath, 2mL deionized water was added to each test tube,
and the absorbance at 540nm was measured with a spectrophotometer. The absorbance value of
the 20 standard solutions was used to develop the calibration curve, and the equation of
absorbance vs. concentration was obtained. The values of the absorbance at each concentration
is given in Table 3.1. Figure 3.2 is a plot of absorbance vs concentration for the glucose solution
and the curve fit developed from this figure is used for calculating the unknown glucose
concentrations.
24
Table 3. 1+Glucose Concentration versus Absorbance Data
Glucose Concentration (mg/mL)
Absorbance
0.1
0.0913
0.2
0.2065
0.3
0.3244
0.4
0.4423
0.5
0.5688
0.6
0.6872
0.7
0.7970
0.8
0.9288
0.9
1.0470
1.0
1.1835
1.2
1.4334
1.4
1.7038
1.6
1.9605
1.8
2.2262
2.0
2.4791
2.2
2.7179
2.4
2.9654
2.6
3.1985
2.8
3.3785
3.0
3.5813
25
4
2
y = 0.0441 + 1.23786x R = 0.99919
3.5
Absorbance
3
2.5
2
1.5
1
0.5
0
0
0.5
1
1.5
2
2.5
3
3.5
Glucose Concentration (mg/ml)
Figure 3. 2+Glucose Concentration versus Absorbance Calibration Curve
The equation of the curve and the R2 value are on the plot.
3.3: Estimation of Enzyme Activity by the Filter Paper Unit (FPU) Method
In order to estimate the filter paper activity of the enzyme, the enzyme was first diluted with
acetate buffer; 1 mL of the diluted enzyme, together with 1mL acetate buffer was added to 0.05g
Whatman filter paper, which had already been inserted in different test tubes. The acetate buffer
was prepared by mixing 2.95mL glacial acetic acid with 900mL deionized water, and then
adding drops of 50% NaOH solution till the pH of the buffer was 4.8. The test tubes were
inserted in an incubator with the temperature set to 50ºC. A set of test tubes were also used as
control test tubes, that is, they contained the same quantities of acetate buffer and enzymes, but
26
had no filter paper. This was done in order to subtract any sugars that may have been dissolved
in the enzyme sample before estimating the concentration of sugars yielded by the enzyme.
At specific times, a test tube was removed and inserted into a boiling water bath for 5 minutes
in order to deactivate the enzyme. The amount of sugars released by the enzyme was estimated
using the DNS test. The FPU was then calculated using the following equation:
/
= ((
1ℎ
∗
)/ ( 0.18016 ∗ 60
)∗
)
The FPU is defined as the enzyme that releases 1 µmol of glucose equivalents per minute from a
Whatman No. 1 filter paper.
3.4: Preparation of MMO.x H20
A round bottom flask with a lid was filled with approximately 500g of 50% NMMO
solution. The NMMO solution was obtained from Sigma Aldrich Co. The flask was connected to
a rotovaporator that was capable of rotating the flask under vacuum, and the flask was inserted in
an 85C water bath. The sample was heated under vacuum, till the amount of water in the sample
was approximately 17%. This was determined gravimetrically since the melting point of
anhydrous NMMO is 184ºC and since it has a negligible vapor pressure; therefore it was
assumed that only the water in the sample evaporated. Propyl gallate, which is usually added to
NMMO to prevent cellulose from degrading, was not added in this case, in order to avoid any
complication it may have with the enzymatic hydrolysis. Due to the slightly dark color of the
83% NMMO, some decomposition of the NMMO may have occurred. The sample was then
separated into 2 jars: Jar A and Jar B. Cellulose was dissolved in Jar A as described in the
following section, while Jar B which contains 83% NMMO and 17% H2O was separated into 20
mL Vials. Each vial contained approximately 5g of the sample. The vials were closed with a
screw cap and stored in a desiccator till the start of the enzymatic hydrolysis. These vials were
used as enzyme blanks in order to ensure that the enzyme effect on the 83% NMMO was taken
into account before estimating the yield of sugars in the reaction sample.
27
3.5: Preparation of 1% Cellulose Solutions
Dissolving cellulose pulp obtained from Buckeye Technology was first ground, using a
Black and Decker coffee grinder, till the cellulose had a fluffy texture. The cellulose was added
to Jar A (described in the previous section). The jar was inserted into an 85C water bath and the
solution was stirred using a mixer (manufactured by Arrow Engineering Co., serial number
C0795134) for approximately 20 minutes or until a homogeneous lyocell solution was observed.
While the cellulose was dissolving into the NMMO solvent, it was essential to wrap the jar with
aluminum foil to avoid water from entering the NMMO since it is a hygroscopic solvent and can
easily absorb water. The amount of cellulose added was 1 w/w% of the total mixture. Five grams
of this solution (also known as 1% lyocell solution) was pipetted into 20 mL vials and stored in a
desiccator at room temperature till the start of the enzymatic hydrolysis.
3.6: Protocol for enzyme reactions
The contents of the enzymatic reaction vessel was prepared by adding 5.8mL deionized water
or 10% acetic acid solution (depending on desired pH), into 5g 1% lycocell solution stored in
20mL vials. For subsequent tests on the room temperature stored samples, the 5g lyocell solution
was first inserted in a 85ºC water bath for 10 minutes inorder to melt the solution (the solution is
solid at room temperature). The total solution pH when deionized water was used as the
buffering medium was 7.4, while the solution pH when 10% acetic acid was used as the
buffering medium was 5.7. Phase separation was observed after the buffers were added. Figure 2
shows a picture of the regenerated cellulose slowly coming out of the 1% lyocell solution. After
the addition of deionized water, the sample and the diluted enzyme solution was equilibrated at
the desired reaction temperature for approximately 10 minutes before starting the reaction,
6.44mL of enzyme solution was then added to the vial and the vial was shaken vigorously before
being inserted into a temperature controlled shaker bath( Precision temperature controlled shaker
bath Cat No. 51221076) . Enzyme concentrations of 34.9 FPU/g, 122 FPU/g and 1445.6FPU/g
were used inorder to see the effect of enzyme concentration on the rate of hydrolysis. The
temperatures used for the reaction were 40C, 50C and 60C. Unless otherwise mentioned, all
samples were run in duplicates or triplicates.
At specific times during the enzymatic hydrolysis, 0.5mL aliquots of the reaction content
was retrieved. The retrieved samples were inserted in a vial, which was subsequently inserted
28
into a boiling water bath for 5 minutes, in order to deactivate the enzymes. The samples were
then centrifuged for 3 minutes at 9500 rpm. Two tenth mL of the supernatant was transferred to a
test tube, and distilled water was added to increase the liquid level to 1mL, the concentration of
glucose in the solution was then determined using the DNS assay procedure.
29
CHAPTER 4 RESULTS
4.1 Enzyme Activity /FPU Determination of Accelerase™1000 and Spezyme® Cellulase
For the estimation of the filter paper activity of the cellulase, low concentrations of the
cellulase were used for the hydrolysis of filter paper. This was necessary in order to ensure that
the substrate is in excess in comparison to the enzyme, so that the rate at which the enzyme binds
is the rate limiting step. Figure 4.1 and 4.2 shows the yield of reducing sugars with time for the
2 cellulases used in this project: Genecor Spezyme® and Accelerase™1000.
The cellulases were diluted to approximately 200 and 400 parts by weight. Accelerase™1000
cellulase of mass 0.0528g was diluted with 20.14g acetate buffer of pH 4.8, to acquire a dilution
factor of 382, while 0.1085 g of the same cellulase was diluted with 20.224 g acetate buffer to
acquire a dilution factor of 187. Similarly, Genecor Spezyme® cellulase was diluted to 176 and
382 parts (all dilutions had a pH of ~4.8). After using equation 3.2 to estimate the filter paper
activity at each dilution factor, the filter paper activity for Accelerase™1000 was determined to
be 61 FPU/mL and 51FPU/mL for dilution factors of 382 and 187 respectively, while the filter
paper activity for Genecor Spezyme® was estimated to be 50 FPU/mL and 44 FPU/mL for
dilution factors of 357 and 176 respectively. The variation in FPU/mL for the various dilution
factors, suggests that the rate of hydrolysis of the filter paper is not directly proportional to the
enzyme loading; and therefore the average of these values, 56 FPU/mL and 47 FPU/mL was
used as the filter paper activities of Accelerase™1000 and Genencor spezyme®
respectively.
30
cellulase
2.5
2
Yield of Reducing Sugars (mg/mL)
y = 0.18264 + 0.02034x R = 0.94816
2
y = 0.0214 + 0.01228x R = 0.924
2
1.5
1
0.5
0
0
20
40
60
80
100
120
140
Time (minutes)
Figure 4. 1+FPU Determination for Accelerase 1000
Yield of reducing sugars as a function of time for Accelerase™1000. ■ represents experiments
that were performed with cellulase dilution ratio of 187 while the ♦ represent experiments that
were performed with cellulase dilution ratio of 382. The solid lines are linear curve fits to the
experimental data for the two different dilutions. The equations of the curve fits are also given in
the figure. The hydrolysis occurred in acetate buffer.
31
2.5
2
Yield of Reducing Sugars (mg/mL)
y = 0.21252 + 0.01846x R = 0.99193
2
y = 0.17275 + 0.00977x R = 0.9997
2
1.5
1
0.5
0
0
20
40
60
80
100
120
140
Time (minutes)
Figure 4. 2+FPU Determination for Genencor Spezyme
Yield of reducing sugars as a function of Time. ■ Symbol represents experiments that were
performed with cellulase dilution ratio of 176 while the ♦ symbols represent experiments that
were performed with cellulase dilution ratio of 357. The solid lines are linear curve fits to the
experimental data for the two different dilutions. The equations of the curve fits are also given in
the figure. The hydrolysis occurred in acetate buffer.
A reason why the reaction rate may not be proportional to the enzyme loading could be due
to the fact that the cellulases are a mixture of enzymes, which work synergistically with one
another; therefore, it is possible that at different dilution factors, the synergistic cooperation of
the enzymes may be different from one another
32
4.2 Effect of MMO on the Enzymatic Hydrolysis of Cellulose
4.2.1 Yield of Reducing Sugars for Different Concentrations of
Cellulase
MMO mixed with
A look at the effect NMMO has on the enzymatic hydrolysis of cellulose is shown in Figure
4.3. The data was obtained by conducting an experiment by which the near monohydrate form of
NMMO that contained 17% water was diluted with deionized water9 in 20mL vials9 so that the
concentration of water in the NMMO solutions ranged from 30% to 100%. One mL of buffered
Genencor Spezyme cellulase (buffered using pH 4.8 acetate buffer) was then mixed with 1 mL of
each NMMO solution, in distinct test tubes, before the addition of 50 mg filter paper to the test
tube. The hydrolysis of filter paper was allowed to occur for specific time periods, at 50C, in an
incubator. The test tubes that did not contain filter paper, were used as enzyme blanks in order to
subtract any contributions that the NMMO and cellulase may have, in the detection of reducing
sugars, by the DNS analysis. The overall concentration of the cellulose was 25 mg/mL and the
enzyme loading used was 157 FPU/g. The reaction was carried out using Genencor spezyme®.
The increase in NMMO concentration lowered the enzyme activity significantly for
suspended filter paper. After 96 hours of the reaction, 93% conversion had occurred in the test
tube that contained only acetate buffer, while only 4% conversion had occurred in the test tube
that contained 30% water. Moreover a significant increase in the sugar yield occurred in test
tubes which had lower concentrations of NMMO.
33
100% Water
90% Water in
80% Water in
70% Water in
60% Water in
50% Water in
40% Water in
30% Water in
Yield of Reducing sugars (mg/ml)
30
NMMO solution
NMMO solution
NMMO solution
NMMO solution
NMMO solution
NMMO solution
NMMO solution
25
20
15
10
5
0
0
20
40
60
80
100
Time
Figure 4. 3+ Effect of MMO on Enzymatic Hydrolysis of filter Paper
Effect of NMMO on enzymatic hydrolysis using different concentrations of water in
NMMO/H20 solution Different concentrations of NMMO were used as the reaction medium,
together with 1 mL of diluted cellulase solution. Each symbol represents the concentration of
water in the NMMO/H20 solution. The inset shows what H20 concentration each symbol
represents.
The low conversion of cellulose due to the increased concentration of NMMO could be
due to a pH effect because NMMO monohydrate has a pH of ~12, and, the enzymes used are
active in slightly acidic environments (pH of ~5). Or it could also be a mass transfer effect
34
because it was observed that solutions which contained higher concentrations of NMMO had
higher viscosities than solutions that had lower concentrations of NMMO, therefore, it is
possible that the enzyme may be limited in its ability to diffuse through the NMMO. Finally, the
low conversion could also be because the strong dipole interaction between nitrogen and oxygen
in the NMMO may be deactivating some of the enzymes in the cellulase. This interaction can
lead to the folding of the enzyme i.e. the overlapping of the hydrophobic and hydrophilic portion
of the enzymes, thereby leading to the cellulase being deactivated.
4.2.2 Comparison of MMO/Cellulase Solution with Acetate Buffered Solution of various
pH for 1 Hr Reaction with Genencor Spezyme®
In order to test if the low conversion was a pH effect or a solvent effect, acetate buffered
cellulase, which had the same pH of the various NMMO and cellulase in the test tubes
mentioned in the previous section, was prepared by diluting the enzyme with a pH 4.8 acetate
buffer and gradually adding drops of 50 w/w% NaOH till the desired pH was acquired. Table 4.1
shows the pH of the NMMO and cellulase solutions and the acetate buffered cellulase acquired.
The hydrolysis of filter paper in test tubes that contained 2mL of the acetate buffered cellulase
was allowed to occur for 1 hour. The enzyme loading used was 157 FPU/g, 50mg filter paper
was used and the total concentration of filter paper was 25 mg/mL. The maximum amount of
sugars that can be acquired is 27.8 mg/mL assuming 1 gram cellulose yields 1.11 gram sugars.
Table 4. 1+pH of MMO Solution after Cellulase Addition versus Concentration of water
in MMO solution
Corresponding H2O Concentration in NMMO pH of NMMO after cellulase addition
100%
4.74
90%
6.14
70%
6.92
50%
7.64
30%
7.94
The results shown in Figure 4.4, shows that for the one hour time period, the yield of sugars
in the acetate buffer was slightly higher than that of the NMMO solution of the same pH. This
occurred for solutions which contained 50% and 30% water in the NMMO/water solution;
however with solutions that contained 80% and 90% water in NMMO solution, the yield of
35
sugars was approximately similar to the acetate buffered enzyme solution. Genecor Spezyme®
cellulase was used for this reaction.
Yield of Reducing sugars (mg/mL)
3.5
3
2.5
2
1.5
1
0.5
0
4
5
6
7
8
9
pH
Figure 4. 4+Comparison between MMO medium and Acetate Buffer Medium Using
Genencor Spezyme
Comparison between NMMO medium and acetate buffer medium of similar pH for 1 hour
reaction of filter paper Yield of reducing sugars as a function of pH for a 1 hour reaction of filter
paper with diluted Spezyme® cellulase. ■ symbol represents hydrolysis reactions performed
using acetate buffered cellulase, while the ♦ symbol represents NMMO/H20 solution with
different H20 concentrations as depicted in Table 4.1.
This result shows that for high NMMO concentrations, the hydrolysis could be affected by
the pH effect and the viscous nature of the NMMO solution, however at lower concentrations of
NMMO; the effect seems to be more of a pH effect than a viscosity effect. Although previous
work has shown that the enzymes were weakened by high concentrations of ionic liquids29, the
36
reason that was given to this occurrence was that the strong polar bonds, by which these liquids
are able to dissolve cellulose, are deactivating the enzymes.
4.2.3 Comparison of MMO/Cellulase Solution with Acetate Buffered Solution of Various
pH for 3 Hr Reaction with Accelerase™1000 Cellulase
A further test of the effect NMMO has on the enzyme activity was conducted by using
Accelerase™1000 cellulase which contains higher β9glucosidase activity (according to the
Accelerase™1000 technical bulletin #1) than the previous cellulase used. The hydrolysis was
allowed to run for 3 hours in this case; and the experimental conditions were similar to the
previous experiment, that is, acetate buffer of similar pH was used to conduct the hydrolysis of
filter paper, together with NMM0/H20 solvent of different NMMO concentrations.
Figure 4.5 shows the results of this experiment. The acetate buffered hydrolysis produced higher
glucose yields when compared to the solutions of NMMO, except for the solution that contained
10% NMMO. In comparison to the results given in the previous experiment, this shows that the
effect NMMO has on the enzyme activity could be due to mass transfer limitations of the
enzyme and/or interactions between the enzyme and the NMMO solvent. The low conversion
rates of the filter paper in NMMO solutions are more pronounced for NMMO concentrations of
50% or lower. For example, the cellulose conversion to sugars for NMMO containing 50% water
is, 7% lower when compared to acetate buffer of similar pH or 25% lower in comparison to
acetate buffer of pH 4.8.
This shows that for the hydrolysis of suspended filter paper, acetate buffer is a much preferred
medium when compared with various concentrations of NMMO and deionized water. The
hydrolysis reaction occurred for 3 hours while the hydrolysis reaction with the Genecor
Spezyme® cellulase (previous section) occurred for 1 hour. The enzyme loading used in this
experiment was 187 FPU/g and the overall cellulose concentration was 25 mg/mL.
4.2.4 Effect of Cellulase on Cellulose Dissolved in MMO and Filter paper in acetate buffer
or MMO medium
Although the cellulose activity is reduced for suspended cellulose by the NMMO medium, it was
necessary to investigate this effect under a different experimental condition. The dissolution of
cellulose by NMMO is a well documented effect. That is, the monohydrate or near monohydrate
37
form of NMMO (NMMO containing 13.3% 9 17% Water) has the ability to dissolve cellulose by
breaking down the crystalline structure of the cellulose. This form of NMMO is a solid at room
temperature and therefore was heated to 85C before it can dissolve the cellulose.
For the purpose of studying the effect of the hydrolysis process on cellulose dissolved in
NMMO, two cellulose substrates were used: Dissolving pulp (DP 1160) and filter paper (DP
7507). These substrates were hydrolyzed under 3 different reaction environments, of similar pH,
however, the dissolving pulp had a higher degree of polymerization than filter paper.
The first reaction medium contained one percent dissolving pulp that was completely dissolved
in NMMO solvent: the NMMO solvent contained 17% Water. Five grams of the
cellulose/NMMO solution were mixed with 5.8mL of 10 w/w% acetic acid in order to precipitate
the cellulose, and 6.44mL diluted enzyme (diluted with deionized water) was added to the
solution. The hydrolysis was allowed to proceed without the removal of the cellulose from the
medium, that is, the cellulase was acting on the substrate as it came out of the NMMO solution.
38
Yield of Reducing sugars (mg/mL)
8
6
4
2
0
+2
4.5
5
5.5
6
6.5
7
7.5
8
8.5
pH
Figure 4. 5+Comparison between MMO medium and Acetate Buffer medium using
Accelerase 1000
Comparison between NMMO medium and acetate buffer medium, of similar pH, for 1 hour
reaction of filter paper using Accelerase™1000 Yield of reducing sugars as a function of pH for
a 3 hour reaction of filter paper with diluted Accelerase™1000 cellulase. The ▲ symbol
represent hydrolysis reactions performed using acetate buffered cellulase, while the ■ symbol
represent hydrolysis reactions in NMMO/H20 solution with different H20 concentrations as
depicted in Table 4.1.
The overall pH of the system was 5.7. The second reaction medium contained Whatman 4 filter
paper which was inserted in a NMMO medium that contained the same amount of acetic acid
and enzyme; however the filter paper was not allowed to dissolve in the NMMO medium before
the beginning of the hydrolysis. The last reaction medium contained Whatman 4 filter paper
inserted in a solution of acetate buffered enzyme and no NMMO was present. The concentration
of the cellulose in all three systems was approximately 3.1 mg/mL, the enzyme activity was 122
39
FPU/g and the pH was 5.77. The reaction was allowed to proceed for 4 hours in a 50C heating
shaker bath and was done in duplicate.
Figure 4.6 shows that the hydrolysis of the cellulose while it is precipitating from the NMMO
solution leads to a higher yield of reducing sugars when compared to the remaining two reaction
mediums.
For the duration of the enzymatic hydrolysis a significant increase in reducing sugars was
observed for reaction medium one even though it had a higher degree of polymerization than the
filter paper. However, the conversion of suspended filter paper to sugars, in a similar NMMO
medium (reaction medium two) was very little. It is therefore evident that the NMMO
pretreatment of the cellulose, followed by precipitation/gelation of the cellulose, before the
enzymatic hydrolysis yields approximately 20% higher conversion when compared to suspended
filter paper, of lower degree of polymerization, in an acetate buffer medium.
40
Yield of Reducing sugars (mg/ml)
2
1.5
1
0.5
0
0.5
1
1.5
2
2.5
3
3.5
4
4.5
Time(Hr)
Figure 4. 6+ Effect of Hydrolyzing Cellulose while it is coming out of Solution
Effect of hydrolyzing cellulose while it is coming out of the NMMO solution .Yield of reducing
sugars as a function of time using Accelerase™1000 cellulase The ■ symbol represents
hydrolysis of cellulose in NMMO/cellulase reaction medium as cellulose is precipitating from
the NMMO solution, the ♦ symbol represents hydrolysis of filter paper performed in acetate
buffer medium of similar pH with NMMO/Celluase reaction medium, while the ▲ represents
hydrolysis of filter paper in buffered NMMO/cellulase solution, however the filter paper was not
dissolved in the NMMO/H20 solvent before hydrolysis.
4.3 Factors That Affect The Enzymatic Hydrolysis
Some of the factors that affect the rate of enzymatic hydrolysis are: the enzyme loading, the
temperature, the system pH and the pre9treatment of the cellulose material. The enzymatic
hydrolysis often proceeds at a slow rate and long times may be required in order to achieve the
desired conversion percentage because the reaction rate decreases due to the inhibition caused by
41
end products
1,20
. Since the hydrolysis is conducted with constant shaking of the reaction vessel
and at a temperature higher than room temperature, it is an energy consuming process and
therefore, it would not be economical to prolong the reaction beyond a certain time period. In our
research we are trying to improve the conversion rate (therefore reducing the reaction time) of
the hydrolysis by changing the structure of the substrate and the reaction medium. The structure
of the crystalline cellulose material is changed to an amorphous structure by first dissolving the
cellulose in the near monohydrate form of N9methylmorpholine9N9Oxide (NMMO) and then
precipitating it from the solution by adding water to the system. A previous study4 has shown
that the hydrolysis of the amorphous cellulose structure obtained from this precipitation
increased the conversion rate by a factor of three (when compared to the hydrolysis of the
crystalline cellulose structure). The enzyme loading, the pH and the temperature of the system
were varied. The system was analyzed at a pH of 5.7 and 7.4. The reason these pH conditions
were used was due to the highly basic nature of the NMMO solvent. A low pH requirement
would require a large amount of acetic acid to be added to the system, which would increase the
overall cost of the process.
The experiments for the enzymatic hydrolysis were performed multiple times to ensure
repeatability. The different experimental conditions are given in Appendix C. In summary, three
different temperatures (40, 50 and 60C), two different pH's (5.7 and 7.4) and three different
enzyme loadings (34.9, 122 and 1445.6 FPU/g) were investigated at a constant cellulose
concentration of 3.1 mg/mL A low enzyme loading would be desired due to the current high
cost of enzymes. All the reactions were allowed to proceed for 24 hours except for low enzyme
loadings (34.9 FPU/g), which had a total reaction time of 48 hours, and 1mL aliquot was
removed at certain times, to determine the concentration of reducing sugars by the DNS method.
The glucose yields were measured using the DNS assay procedure and the percentages of
conversion were calculated. The yield of reducing sugars for reactions at 40C and 50C, using
acetic acid and deionized water as precipitating solvents, and an enzyme loading of 122 FPU/g,
is shown in Figure 4.7, and the percent conversion of cellulose is shown in Table 4.3.
42
40C, pH 5.7
50C, pH 5.7
40C, pH 7.4
50C, pH 7.4
Yield of Reducing Sugars (mg/mL)
3.5
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
35
Time (Hours)
Figure 4. 7+ Yield of Reducing Sugars as a function of Time at Different Reaction
conditions
Yield of reducing sugars as a function of time for dissolving pulp precipitating from
NMMO/H20 solvent at temperatures of 40C and 50C reaction pH of 5.7 and 7.4 and enzyme
activity of 122 FPU/g.
43
Table 4. 2+ Percentage Conversion of Cellulose at 40C and 50C, pH 5.7 and 7.4, and
Enzyme Loading of 122 FPU/g
Time
0.25
1
3
12
24
40C, pH 5.7
12%
31%
48%
61%
65%
40C, pH 7.4
19%
24%
33%
43%
51%
50C, pH 5.7
21%
38%
50%
60%
59%
50C, pH 7.4
16%
31%
35%
44%
45%
It can be seen from Figure 4.7 and table 4.2 that although the sugar yields for the hydrolysis
reactions at 40C and 50C increased continually with time, there was tendency that the rate of
hydrolysis decreased with time. After 24 hours periods, the systems that utilized acetic acid as a
precipitating medium had higher conversion rates when compared to the system in which
deionized water was used. This shows that the cellulase are affected by the pH of the system.
Moreover, the temperature of the system has an effect on the initial rate of hydrolysis, as can be
seen from table 4.2, after 1 hour of the reaction at 40C the systems that utilized acetic acid and
deionized water as precipiating solvents showed conversion rates of 31% and 24% respectively,
while at 50C, 38% and 31% conversion was achieved by these systems. However at longer
times, it seems like the lower temperature of the system favored higher cellulose conversion, for
system of lower pH. For example after 24 hour reaction time the 40C system which utiliized
acetic acid as the precipitating solvent yielded a 65% conversion of cellulose while the system at
50C remained at 59% cellulose conversion. Although the effect of enzyme loading cannot be
seen in this table, for the majority of the reaction conditions utilized by this project, the higher
enzyme loading yielded higher cellulose conversion rates. However, the conversion rate of all
the reactions was not directly proportional to the enzyme loading. The plots of glucose yield with
time for all the hydrolysis systems conducted in this project is in Appendix A.
Acetic acid and deionized water were utilized as pH adjusting solvents, which precipitate the
cellulose from the cellulose/NMMO/H2O solution, however a higher yield of reducing sugars
was achieved when acetic acid was utilized as the pH adjusting solvent. This can be attributed to
the lower pH achieved by the acetic acid solvent. NMMO has a pH of ~12, and the optimum
cellulase pH, according to the cellulase manufacturers is 4.8. Therefore the use of acetic acid as a
precipitating solvent would bring the pH closer to the optimum pH of the cellulase when
44
compared to utilizing the deionized water. The pH of the 10% w/w acetic acid solution was
found to be 2.2, while the pH of deionized water was 5.7.
Figure 4.7 showed that the highest conversion, with an enzyme loading of 122 FPU/g, was
achieved when acetic acid was used as a precipition solvent and the temperature of the system
was 40C.
4.3.1 Effect of Temperature
Although, the preferred temperature for cellulase in hydrolysis of biomass material has been
accepted to be between 35950C
16
, some investigators have found that some enzymes can be
stabilized at high temperatures in ionic liquids39,40, however, higher operational temperatures
would lead to higher energy consumption which may not be suitable for the operational cost of
the process. To determine the effect of temperature on hydrolysis, experiments that had similar
enzyme loadings and precipitation agents were conducted on dissolving pulp. The trials were
carried out in 40C, 50C and 60C water baths. The yields of reducing sugars with time at 40C,
50C and 60C at pH 5.7 and different enzyme loadings is shown in Figures 4.8 and 4.9, and 4.10.
45
Yield of Reducing Sugars (mg/ml)
2
1.5
1
0.5
0
0
5
10
15
20
25
30
Time (Hours)
Figure 4. 8+ Effect of Temperature at pH 5.7 and 34.9 FPU/g Enzyme Loading
Effect of Temperature at pH 5.7 and Enzyme loading 34.9 FPU/g Yield of reducing sugars with
time at pH 5.7, enzyme loading 34.9 FPU/g and temperatures of: 40C, 50C and 60C. ■40 C,
♦50C, ▲60C
46
Yield of reducing sugars (mg/ml)
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
35
Time (Hours)
Figure 4. 9+Effect if Temperature at pH 5.7 and 122 FPU/g Enzyme Loading
Effect of Temperature at pH 5.7 and Enzyme loading 122 FPU/g. Yield of reducing sugars with
time at pH 5.7, enzyme loading 122 FPU/g and temperatures of: 40C, 50C and 60C. ■40 C,
♦50C, ▲60C
47
Yield of Reducing Sugars (mg/ml)
3.5
3
2.5
2
1.5
1
0
5
10
15
20
25
Time (Hour)
Figure 4. 10+ Effect of Temperature at pH 5.7 and 1445.6 FPU/g Enzyme Loading
Effect of Temperature at pH 5.7 and Enzyme loading 1445.6 FPU/g. Yield of reducing sugars
with time at pH 5.7, enzyme loading 1445.6 FPU/g and temperatures of: 40C, 50C and 60C. ■40
C, ♦50C, ▲60C
Table 4. 3+Percentage Conversion of Cellulose at Different Temperatures, Enzyme Loading
and pH after 24 Hours Enzymatic Reaction
Enzyme loading, pH
40C
50C
60C
34.9 FPU/g, pH 5.7
46%
54%
17%
34.9 FPU/g, pH 7.4
33%
27%
6%
122 FPU/g, pH 5.7
65%
59%
28%
122 FPU/g, pH 7.4
51%
45%
9%
1445.6 FPU/g, pH 5.7
77%
89%
56%
1445,6 FPU/g, pH 7.4
92%
66%
32%
48
Table 4. 4+Percentage Conversion of Cellulose at Different Temperature, Enzyme Loading
and pH after 3 Hours Enzymatic Reaction
Enzyme loading, pH
40C
50C
60C
34.9 FPU/g, pH 5.7
16%
38%
37%
34.9 FPU/g, pH 7.4
9%
13%
19%
122 FPU/g, pH 5.7
29%
48%
50%
122 FPU/g, pH 7.4
15%
33%
35%
1445.6 FPU/g, pH 5.7
49%
77%
72%
1445.6 FPU/g, pH 7.4
46%
76%
69%
Figures 4.8, 4.9 and 4.10 show the effects of temperature on the yield of reducing sugars
versus time curve, at a pH of 5.7, and enzyme loadings of 34.9, 122 and 1445.6 FPU/g
respectively. The effect of temperature on these curves are similar to the 7.4 pH systems ( curves
attached in appendix A), in that the enzymatic hydrolysis at 40C and 50C have similar
conversion rates for the first six hours of the reaction , however after this time period the glucose
yield was dependent on the temperature and system pH.
Tables 4.3 and 4.4 tabulates the yield of reducing sugars after 3 hours and 24 hours
respectively. This table together with figures 4.8, 4.9 and 4.10, show that the yield of reducing
sugars was lowest at 60C for all identical experimental conditions. However, after 24 hour
reaction time, it can be observed from the table that 5.7 pH systems at 50C, had higher cellulose
conversion when compared to 7.4 pH systems, except for the 122 FPU/g enzyme loading system.
At this enzyme loading there was no distinct differece between the two temperatures (with the
inclusion of error bars). However, it can be observed from table 4.3 that reactions at 40C favored
7.4 pH conditions and the highest conversion rate achieved after 24 hours was 92%, with an
enzyme loading of 1445.6 FPU/g, at 40C, and pH 7.4. It can also be observed that after 3 hour
hydrolysis, the 40C and 50C reaction systems yielded approximately the same amount of
reducing sugars, except for the systems that had an enzyme dilution of 34.9 FPU/g and a pH of
7.4. For these systems 50C was the more favorable temperature. These results suggest that, from
an economic stand point, the cellulase is active at approximately 40C for most reaction
conditions, however the optimum temperature would ultimately depend on the enzyme loading
and the pH of the system.
49
The reactions at 60C showed the lowest yield of sugars for all pH and enzyme loadings.
From figures 4.8,4.9 and 4.10 it can be observed that at 60C, the conversion of cellulose to
sugars stops within the first hour of the reaction for all enzyme loadings, however the yield of
sugars within this hour is dependent on the enzyme loading and pH. This shows that 60C is the
least favorable temperature for the conversion of cellulose to sugars for this enzymatic reaction
system. For all the reaction systems, approximately 0.5 mg/mL reducing sugar concentration was
produced within 15 minutes of the enzymatic reaction, which shows that the temperature, pH and
enzyme loadings may not affect the initial adsorption of the cellulase on the cellulose surface.
4.3.2 Effect of Enzyme Loading
The commercial cellulase enzyme used in this study had a filter paper activity of 56 FPU/mL.
The type of enzyme or cellulase used would have an effect on the amount of reducing sugars
released due to the variation of enzyme components in the cellulase or the activity of the
enzymes. In a typical system, higher enzyme loadings would generate higher cellulose
conversion rates due to the increase in the catalytic conversion of the cellulose material to sugars.
However, from an economic standpoint it is necessary to produce sugars at low enzyme loadings
because the cellulase enzyme is an expensive mixture that may end up being the main cost of the
entire process. Figure 4.11 and 4.12 presents the reducing sugars yield of the cellulose substrate
using two different precipitation solvents to precipitate the cellulose from the NMMO solvent :
deionized water and 10 w/w% acetic acid. Therefore the pH of hydrolysis system in figure 4.11
is 7.4 while the pH of the hydrolysis system in figure 4.12 is 5.7. The enzyme loadings used
were: 34.9, 122 and 1445.6 FPU/g substrate. Each reaction was carried out at 40C, 50C and 60C
and for a 249hour period.
50
Yield of Reducing Sugars (mg/mL)
3.5
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
35
Time (Hours)
Figure 4. 11+Effect of Enzyme Loading at 40C and pH 7.4
Effect of Enzyme Loading at 40C and pH 7.4 Yield of reducing sugars with time at 40C, pH 7.4
and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g, ■122 FPU/g, ▲34.9
FPU/g
51
Yield of Reducing Sugars (mg/mL)
3.5
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
35
Time (Hours)
Figure 4. 12+ Effect of Enzyme Loading at 40C and pH 5.7
Effect of Enzyme Loading at 40C and pH 5.7 Yield of reducing sugars with time at 40C, pH 5.7
and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g, ■122 FPU/g, ▲34.9
FPU/g
From figures 4.11 and 4.12, which show the effect of enzyme loading at 40C and two
different pH conditions: 5.7 and 7.4. The experiments showed that increasing the enzyme loading
leads to an increase in cellulose conversion, however, the percentage increase in cellulose
conversion, by the increase in the enzyme loading, is dependent on the utilized enzyme loading:
the system pH, and the temperature. At all similar system conditions, except at 40C and a pH 5.7,
an enzyme loading of 1445.6 FPU/g produced a significant increase in the cellulose conversion
in comparison to enzyme loadings of 34.9 and 122 FPU/g. At 40C and a system pH of 5.7 the
overall cellulose conversion was not so significant utilizing an enzyme loading of 1445.6 FPU/g
52
in comparison to other enzyme loadings. This may be because 40C reaction systems favored
higher pH conditions as noted in section 4.3.1. The effect of cellulose conversion when the
enzyme loading is increased from 34.9 to 122 FPU/g is dependent on the pH and temperature of
the system.
Table 4. 5+Percentage Conversion of Cellulose at Different Temperatures and Enzyme
Loading at pH 7.4 after 24 Hours Enzymatic Reaction
40C
50C
60C
34.9 FPU/g, pH 7.4
33%
27%
122 FPU/g, pH 7.4
51%
45%
1445.6 FPU/g, pH 7.4
92%
66%
6%
9%
32%
Table 4. 6+Percentage Conversion of Cellulose at Different Temperatures and Enzyme
Loading at pH 5.7, after 24 Hours Enzymatic Reaction
40C
50C
60C
34.9 FPU/g, pH 5.7
46%
54%
122 FPU/g, pH 5.7 1445.6 FPU/g, pH 5.7
65%
77%
59%
89%
17%
28%
56%
From tables 4.5 and 4.6, which show the percentage conversion after 24 hours, it can be observed
that increasing the enzyme loading from 34.9 FPU/g to 122 FPU/g, while utilizing acetic acid as
the precipitaing agent, increased the conversion of the cellulose substrate from 54% to 59% at a
temperature of 50C. At 40C the same increase in the enzyme loading increased the conversion
rate from 46% to 64%. When deionized water was utilized as the precipitating agent (therefore
increasing the pH of the system) the corresponding conversion rate was increased from 27% to
45% at 50C, while at 40C an increase from 33% to 51% was observed. At 60C, this increase in
the cellulase loading was quite effective for a system pH of 5.7 ( 17% to 28%) but not really
effective for a system pH of 7.4 (6% to 9%).
A reoccuring trend that can be acquired is that the increase in enzyme loading is not proportional
to increase in cellulose substrate concentration. This may be due to limited accesible substrate
sites, that is, the amount sites available to the enzyme for the hydrolysis of the cellulose molecule
was limited by the substrate concentration. However, the effect of increasing the enzyme loading
depends on the pH and temperature of the system. At higher system pH the increase in enzyme
loading is effective, except at 60C; but when a lower pH was utilized, increases in enzyme
53
loadings were effective for systems at a temperature of 40C and 60C but not at 50C. According
to the manufacturers of the Trichoderma cellulase system, the optimum pH and temperature of
the cellulase are 4.8 and 50C respectively. Therefore variations in the enzyme loading at 50C and
a pH of 5.7, which is closer to the optimum cellulase pH of 4.8 when compared to a pH of 7.4,
may not have an effect on the sugars released by the system.
Calculation of the sugar yield per cellulase loading at different cellulase loadings would show
that the optimum sugar yield per enzyme loading is highest when the cellulase loading is 34.9
FPU/g. Therefore the optimum enzyme loading should depend on the desired conversion rate
and the time of hydroysis.
4.3.3 Effect of pH
There are various methods of pre9treating cellulose. Some of these methods involve the physical
or chemical modification of the cellulosic material. In this project the dissolving pulp was first
dissolved in a NMMO/H20 solvent, in order to break down the hydrogen bonds that give
cellulose its rigid structure. Due to the basic nature of this solvent (pH ~ 12), and the fact that the
solvent is a solid at room temperature (has a melting point of 78C), this medium would not be
suitable for the enzymatic hydrolysis of cellulose. The addition of a precipitating solvent that can
precipitate the cellulose before the enzymatic hydrolysis, reduce the pH of the medium and
change the chemical properties of the NMMO solvent would therefore increase the susceptibility
of the medium to cellulose hydrolysis. In this project, deionized water and Acetic acid (10 w/w
%) were used as precipitation solvents to serve this purpose. By using deionized water as the
precipitation solvent, the overall pH of the hydrolysis system was 7.4, while by utilizing acetic
acid as the precipitation solvent the overall pH was 5.7. By utilizing both precipitation solvents
the effect of pH system was analyzed. Figures 4.13, 4.14 and 4.15: show the sugar yields in
mg/mL resulting from these precipitating agents for enzyme loadings of 34.9, 122, 1445.6 FPU/g
respectively. Table 4.7 lists the conversion rates of the enzymatic hydrolysis.
54
Yield of Reducing Sugars (mg/mL)
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
35
Time
Figure 4. 13+ Effect of pH at 40C and 122 FPU/g Enzyme Loading
Effect of pH at 40C and enzyme loading 122 FPU/g Yield of reducing sugars with time at 40C
and an enzyme loading of 122 FPU/ at different pH 5.7 and 7.4 ■ 5.7 pH system, ♦7.4 pH system
55
Yield of Reducing Sugars (mg/mL)
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
Time (Hours)
Figure 4. 14+ Effect of pH at 50C and 122 FPU/g Enzyme Loading
Effect of pH at 50C and enzyme loading 122 FPU/g. Yield of reducing sugars with time at 50C
and an enzyme loading of 122 FPU/ at different pH 5.7 and 7.4 ■ 5.7 pH system, ♦7.4 pH system
56
Yield of Reducing Sugars (mg/mL)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
5
10
15
20
25
Time (Hours)
Figure 4. 15+Effect of pH at 60C and 122 FPU/g Enzyme Loading
Effect of pH at 60C and enzyme loading 122 FPU/g Yield of reducing sugars with time at 60C
and an enzyme loading of 122 FPU/ at different pH 5.7 and 7.4 ■ pH 5.7 systems, ♦ pH 7.4
systems
57
Table 4. 7+ Percentage Conversion of Cellulose at Different Temperatures, pH and Enzyme
Loadings, after 24 Hours Enzymatic Reaction
40C, 122 FPU/g
50C, 122 FPU/g
60C, 122 FPU/g
pH 5.7
65%
59%
28%
pH 7.4
51%
45%
9%
pH 5.7
pH 7.4
40C, 1445.6 FPU/g
77%
92%
pH 5.7
pH 7.4
40C, 34.9 FPU/g
46%
33%
50C, 1445.6 FPU/g 60C, 1445.6 FPU/g
89%
56%
66%
32%
50C, 34.9 FPU/g
54%
27%
60C, 34.9 FPU/g
17%
6%
Figures 4.13, 4.14 and 4.15 show that the lower the pH of the system the higher the cellulose
conversion rate for all temperatures and enzyme loadings, except at a temperature of 40C and an
enzyme loading of 1445.6 FPU/g (this figure is in appendix A), where it was observed that the
higher pH of the system yielded a higher conversion rate. A previous study by Hang and
Woodams16 has shown that at a pH of 5, sugar yields from corn husk were higher than that of pH
4 and pH 7. It was also observed that at 40C, 50C and 60C, the total sugar yields were slightly
different. The results of this study show a similar pH effect however, the total sugar yields is also
influenced by the temperature as can be seen in table 4.7. The higher conversion rate observed
using a higher pH, enzyme loading of 1445.6 FPU/g and temperature of 40C could be because
the large enzyme loading may be stabilized at a temperature of 40C when compared to 50C.
However the results indicate that in general, a lower system pH would be favorable.
At 60C and a pH of 7.4, even though the conversion of cellulose to sugars stopped within the
first hour, there was a gradual decrease in the amount of detectable soluble sugars by the DNS
method. According Adorjan et al35, monosaccharide sugars, under lyocell conditions, undergo
isomerization reactions similar to aqueous alkaline conditions and this leads to the formation of
aldopentoses and aldohexoses from glucose, thereby reducing the concentration of detectable
sugars in the system. This might be what is occurring in the system at 60C, another explanation
for this gradual decrease in reducing sugars may be the formation humins, which are sugars that
58
are linked by additional components thereby forming linkages that are resistant to enzymatic
hydrolysis. This explanation was explained by Sievers et al36, when a similar occurrence was
observed while hydrolyzing pine wood in an ionic liquid phase at high temperatures under acidic
conditions.
4.4 Interaction Between Temperature and pH
A factorial analysis on the interaction between pH and temperature was conducted in order to
determine if there was an interaction between the temperature and the pH. By using the reducing
sugar yields after 24 hours to conduct the analysis, Figures 4.16, 4.17 and 4.18 were obtained.
These figures show the effect of temperature on pH for enzyme loadings of 34.9, 122 and 1445.6
FPU/g. From these figures it was observed that the interaction between temperature and pH
occurred only at an enzyme loading of 1446.5 FPU/g (Figure 4.18). At this enzyme loading, a
temperature of 40C produced more sugar yields at a system pH of 7.4, while a temperature of
50C produced more sugar yields at a system pH of 5.7.
This shows that at this particular enzyme loading, a lower temperature (40C) favors a higher
system pH (7.4), while a higher temperature (50C) favors a lower system pH (pH 5.7).
59
Yield of Reducing Sugars (mg/ml)
2.00
1.80
1.60
1.40
1.20
1.00
pH 5.7
pH 7.4
0.80
0.60
0.40
0.20
0.00
30
40
50
60
70
Temperature C
tween Temperature and pH at 34.9 FPU/g Enzyme
nzyme Loading
Figure 4. 16+Interaction Between
60
Yield of Reducing Sugars (mg/ml)
2.50
2.00
1.50
5.7
7.4
1.00
0.50
0.00
30
40
50
60
70
Temperature C
tween Temperature and pH at 122 FPU/g Enzyme
zyme Loading
Figure 4. 17+Interaction Between
61
Yield of Reducing Sugars (mg/ml)
3.50
3.00
2.50
2.00
pH 5.7
1.50
pH 7.4
1.00
0.50
0.00
30
40
50
60
70
Temperature C
tween Temperature and pH at 1445.6 FPU/g Enzyme
Enzy
Loading
Figure 4. 18+Interaction Between
62
CHAPTER 5 CO CLUSIO S
The enzymatic reaction of cellulose was studied under different reaction conditions. At these
reaction conditions the glucose yield increased gradually with time however, the times at which
the enzyme was inactivated, or the time rate of the cellulose conversion ended, differed with the
reaction medium being used.
For all reaction mediums, it was observed that the rate of
hydrolysis was highest at the beginning, but gradually decreased with reaction time due to the
accumulation of end products or the gradual inactivation of the cellulase by the medium.
Through the comparison of different reaction mediums, it was seen that the reaction medium that
yielded the highest amount of reducing sugars was the medium in which the cellulose substrate
was originally dissolved in the near monohydrate form of NMMO, and the hydrolysis took place
while the cellulose was coming out of the solution. By using different precipitating solvents to
precipitate the cellulose from the NMMO solvent, the pH of the system was varied, and it was
determined that a lower system pH yielded a higher cellulose conversion rate. It was also
observed that an increase in the enzyme loading resulted in higher conversion rates, however the
sugar yield per unit enzyme decreased as the enzyme loading increased. The optimum enzyme
loading is dependent on the temperature of the system, the desired sugar yield and the available
hydrolysis time. It was observed that for the different pH and enzyme loading reaction systems
the favorable hydrolysis temperature was 40C. Increasing the temperature to 50C led to faster
enzyme inactivation, while increasing it to 60C resulted in lower sugar yields.
The enzymatic hydrolysis of dissolving pulp, originally dissolved in the near monohydrate
form of NMMO had a similar conversion rate at both 40C and 50C after 3 hours enzymatic
hydrolysis. However, under the same experimental conditions, it seems the decrease in
conversion rate at 50C is higher than at 40C, for example, after 24 hours enzymatic hydrolysis
65% conversion was observed at 40C and 122FPU/g enzyme loading while 59% conversion was
observed at 50C and the same enzyme loading. This was similar for all the enzyme loadings,
except at 34.9FPU/g and 1445.6 FPU/g.
The pre9treatment process used in this study included grinding of the dissolving pulp and
dissolving it in the near monohydrate form of NMMO before the enzymatic hydrolysis.
Dissolving the cellulose affected the enzymatic hydrolysis rate.
The cellulose conversion
observed when the hydrolysis occurred as cellulose was coming out of solution, with
63
NMMO/H2O, was higher than when the cellulose was suspended in an acetate buffer medium or
a similar NMMO/H2O medium. This suggests that the NMMO pretreatment of the cellulose is
effective.
The hydrolysis rate was improved by using 10 % (w/w) acetic acid as the precipitation
solvent rather than using deionized water, suggesting that the enzyme activity was improved at a
higher pH.
The higher the enzyme loading, the higher the yield of reducing sugars however, the increase
in reducing sugars was not proportional to the increase in enzyme loading.
Almost all experimental conditions showed that at 50C a pH of 5.7 was preferred while at 40C a
pH of 7.4 was preferred.
64
CHAPTER 6 RECOMME DATIO S
The recommendations and suggestions on this study are as follows:
The detailed characterization of products of the enzymatic hydrolysis by HPLC would
help characterize how the cellulose is degraded to sugars. This would lead to information
about how the enzymes are affected by the reaction system.
The use of different cellulosic substrates and cellulose concentrations would help
determine the sensitivity of the system to various raw materials.
Further research on enzymes that can degrade the cellulose at high pH would reduce the
use of water or acetic acid that may be needed by the process.
Examination into the recycle of the enzymes, and the effect of recycling. This would help
determine how much fresh enzyme would be needed if there exists some deactivation of
recycled enzymes.
An integrated process system for the hydrolysis of cellulose may lead to an efficient and
economical process.
65
APPE DIX A SUGAR YIELDS (MG/ML) AS A FU CTIO OF TIME
APPE DIX A.1 40C DATA
Yield of Reducing Sugars (mg/mL)
3.5
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
35
Time (Hour)
Figure A.1: Effect of Enzyme Loading at 40C and pH 7.4. Yield of reducing sugars with time at
40C, pH 5.7 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g, ■122 FPU/g,
▲34.9 FPU/g
66
Yield of Reducing Sugars (mg/mL)
3.5
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
35
Time (Hour)
Figure A.2: Effect of Enzyme Loading at 40C and pH 5.7. Yield of reducing sugars with time at
40C, pH 5.7 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g, ■122 FPU/g,
▲34.9 FPU/g
67
Yield of Reducing Sugars (mg/mL)
3.5
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
Time (Hour)
Figure A.3: Effect of pH at 40C and enzyme loading 1445.6 FPU/g Yield of reducing sugars
with time at 40C and an enzyme loading of 1445.6 FPU/g at different pH 5.7 and 7.4. ■ 5.7 pH
system, ♦7.4 pH system
68
Yield of Reducing Sugars (mg/mL)
3.5
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
35
Time (Hour)
Figure A.4: Effect of pH at 40C and enzyme loading 122 FPU/g. Yield of reducing sugars with
time at 40C and an enzyme loading of 122 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH system, ♦7.4 pH
system
69
Yield of Reducing Sugars (mg/mL)
2
1.5
1
0.5
0
0
10
20
30
40
50
Time (Hour)
Figure A.5: Effect of pH at 40C and enzyme loading 34.9 FPU/g Yield of reducing sugars with
time at 40C and an enzyme loading of 34.9 FPU/g at different pH 5.7 and 7.4. ■ 5.7 pH system,
♦7.4 pH system
70
APPE DIX A.2 50C DATA
Yield of Reducing Sugars (mg/mL)
3.5
3
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
30
Time (Hour)
Figure A.6: Effect of Enzyme Loading at 50C and pH 5.7. Yield of reducing sugars with time at
50C, pH 5.7 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g, ■122 FPU/g,
▲34.9 FPU/g
71
Yield of Reducing Sugars (mg/mL)
3
2.5
2
1.5
1
0.5
0
0
10
20
30
40
50
Time (Hour)
Figure A.7: Effect of Enzyme Loading at 50C and pH 7.4. Yield of reducing sugars with time at
50C, pH 7.4 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g, ■122 FPU/g,
▲34.9 FPU/g
72
Yield of Reducing Sugars (mg/mL)
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
Time (Hour)
Figure A.8: Effect of pH at 50C and enzyme loading 122 FPU/g Yield of reducing sugars with
time at 50C and an enzyme loading of 122 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH system, ♦7.4 pH
system
73
Yield of Reducing Sugars (mg/mL)
3.5
3
2.5
2
1.5
1
0.5
0
5
10
15
20
25
30
Time (Hour)
Figure A.9: Effect of pH at 50C and enzyme loading 1445.6 FPU/g. Yield of reducing sugars
with time at 50C and an enzyme loading of 1445.6 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH system,
♦7.4 pH system
74
Yield of Reducing Sugars (mg/mL)
2
1.5
1
0.5
0
0
10
20
30
40
50
Time (Hour)
Figure A.10: Effect of pH at 50C and enzyme loading 34.9 FPU/g. Yield of reducing sugars
with time at 50C and an enzyme loading of 34.9 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH system, ♦7.4
pH system
75
APPE DIX A.3 60C DATA
Yield of reducing Sugars (mg/ml)
2
1.5
1
0.5
0
0
5
10
15
20
25
Time (Hour)
Figure A.11: Effect of Enzyme Loading at 60C and pH 7.4. Yield of reducing sugars with time
at 60C, pH 7.4 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g, ■122
FPU/g, ▲34.9 FPU/g
76
Yield of Reducing Sugars (mg/ml)
Time (Hour)
Figure A.12: Effect of Enzyme Loading at 60C and pH 5.7. Yield of reducing sugars with time
at 60C, pH 5.7 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g, ■122
FPU/g, ▲34.9 FPU/g
77
Yield of Reducing Sugars (mg/mL)
1.4
1.2
1
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
Time (Hour)
Figure A.13: Effect of pH at 60C and enzyme loading 122 FPU/g Yield of reducing sugars with
time at 60C and an enzyme loading of 122 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH system, ♦7.4 pH
system
78
Yield of Reducing Sugars (mg/mL)
2.5
2
1.5
1
0.5
0
0
5
10
15
20
25
Time (Hour)
Figure A.14: Effect of pH at 60C and enzyme loading 1445.6 FPU/g. Yield of reducing sugars
with time at 60C and an enzyme loading of 1445.6 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH system,
♦7.4 pH system
79
Yield of Reducing Sugars (mg/mL)
0.8
0.6
0.4
0.2
0
0
5
10
15
20
25
Time (Hour)
Figure A.15: Effect of pH at 60C and enzyme loading 34.9 FPU/g Yield of reducing sugars with
time at 60C and an enzyme loading of 34.9 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH system, ♦7.4 pH
system
80
APPE DIX B. PERCE TAGE CELLULOSE CO VERSIO AS A FU CTIO OF
TIME
APPE DIX B.1 40C DATA
100
% Conversion
80
60
40
20
0
0
5
10
15
20
25
30
35
Time (Hour)
Figure B.1: Effect of Enzyme Loading at 40C and pH 7.4. Percentage Conversion of cellulose
with time at 40C, pH 5.7 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g,
■122 FPU/g, ▲34.9 FPU/g
81
100
% Conversion
80
60
40
20
0
0
5
10
15
20
25
30
Time (Hour)
Figure B.2: Effect of Enzyme Loading at 40C and pH 5.7. Percentage Conversion of cellulose
with time at 40C, pH 5.7 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g,
■122 FPU/g, ▲34.9 FPU/g
82
100
% Conversion
80
60
40
20
0
0
5
10
15
20
25
Time (Hour)
Figure B.3: Effect of pH at 40C and enzyme loading 1445.6 FPU/g. Percentage Conversion of
cellulose with time at 40C and an enzyme loading of 1445.6 FPU/g at different pH 5.7 and 7.4. ■
5.7 pH system, ♦7.4 pH system
83
80
70
% Conversion
60
50
40
30
20
10
0
0
5
10
15
20
25
30
35
Time (Hour)
Figure B.4: Effect of pH at 40C and enzyme loading 122 FPU/g. Percentage Conversion of
cellulose with time at 40C and an enzyme loading of 122 FPU/ at pH 5.7 and 7.4. ■ 5.7 pH
system, ♦7.4 pH system
84
60
% Conversion
50
40
30
20
10
0
0
10
20
30
40
50
Time (Hour)
Figure B.5: Effect of pH at 40C and enzyme loading 34.9 FPU/g. Percentage Conversion of
cellulose with time at 40C and an enzyme loading of 34.9 FPU/g at different pH 5.7 and 7.4. ■
5.7 pH system, ♦7.4 pH system
85
APPE DIX B.2 50C DATA
100
% Conversion
80
60
40
20
0
0
5
10
15
20
25
30
Time (Hour)
Figure B.6: Effect of Enzyme Loading at 50C and pH 5.7. Percentage Conversion of cellulose
with time at 50C, pH 5.7 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g,
■122 FPU/g, ▲34.9 FPU/g
86
100
% Conversion
80
60
40
20
0
0
10
20
30
40
50
Time (Hour)
Figure B.7: Effect of Enzyme Loading at 50C and pH 7.4. Percentage Conversion of cellulose
with time at 50C, pH 7.4 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g,
■122 FPU/g, ▲34.9 FPU/g
87
70
60
% Conversion
50
40
30
20
10
0
0
5
10
15
20
25
Time (Hour)
Figure B.8: Effect of pH at 50C and enzyme loading 122 FPU/g. Percentage Conversion of
cellulose with time at 50C and an enzyme loading of 122 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH
system, ♦7.4 pH system
88
100
% Conversion
80
60
40
20
0
0
5
10
15
20
25
30
Time (Hour)
Figure B.9: Effect of pH at 50C and enzyme loading 1445.6 FPU/g. Percentage Conversion of
cellulose with time at 50C and an enzyme loading of 1445.6 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH
system, ♦7.4 pH system
89
60
% Conversion
50
40
30
20
10
0
0
10
20
30
40
50
Time (Hour)
Figure B.10: Effect of pH at 50C and enzyme loading 34.9 FPU/g. Percentage Conversion of
cellulose with time at 50C and an enzyme loading of 34.9 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH
system, ♦7.4 pH system
90
APPE DIX B.3 60C DATA
60
% Conversion
50
40
30
20
10
0
0
5
10
15
20
25
Time (Hour)
Figure B.11: Effect of Enzyme Loading at 60C and pH 7.4. Percentage Conversion of cellulose
with time at 60C, pH 7.4 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g,
■122 FPU/g, ▲34.9 FPU/g
91
60
% Conversion
50
40
30
20
10
0
0
5
10
15
20
25
Time (Hour)
Figure B.12: Effect of Enzyme Loading at 60C and pH 5.7. Percentage Conversion of cellulose
with time at 60C, pH 5.7 and enzyme loadings of: 34.9, 122 and 1445.6 FPU/g. ♦1445.6 FPU/g,
■122 FPU/g, ▲34.9 FPU/g
92
40
35
% Conversion
30
25
20
15
10
5
0
0
5
10
15
20
25
Time (Hour)
Figure B.13: Effect of pH at 60C and enzyme loading 122 FPU/g. Percentage Conversion of
cellulose with time at 60C and an enzyme loading of 122 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH
system, ♦7.4 pH system
93
% Conversion
0
5
10
15
20
25
Time (Hour)
Figure B.14: Effect of pH at 60C and enzyme loading 1445.6 FPU/g. Percentage Conversion of
cellulose with time at 60C and an enzyme loading of 1445.6 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH
system, ♦7.4 pH system
94
20
% Conversion
15
10
5
0
0
5
10
15
20
25
Time (Hour)
Figure B.15: Effect of pH at 60C and enzyme loading 34.9 FPU/g. Percentage Conversion of
cellulose with time at 60C and an enzyme loading of 34.9 FPU/g at pH 5.7 and 7.4. ■ 5.7 pH
system, ♦7.4 pH system
95
APPE DIX C DILUTIO , WEIGHT OF 1% LYOCELL SOLUTIO A D E ZYME
ACTIVITY FOR EACH EXPERIME T
1+58_pH 5.7
Dilution
58.9
58.5
59.7
59.7
59.7
1+58_pH 5.7
1% solution
5.0
5.1
5.0
5.0
5.0
FPU/g
122.2
120.6
121.5
122.0
120.3
5.1
5.1
5.0
118.0
117.6
121.4
4.9
4.8
5.2
5.1
1425.3
1465.0
4.9
5.0
5.0
5.0
5.0
5.0
5.1
5.1
1481.0
1433.5
1421.5
1412.7
203.1
5.0
35.5
203.1
5.0
35.4
203.1
5.0
35.6
5.0
5.1
5.1
35.7
34.9
35.1
Dilution
62.5
58.0
56.0
1+58_7.4
59.8
59.8
59.8
FPU/g
101.8
122.7
125.1
5.1
5.1
5.1
5.1
117.1
121.1
120.6
121.2
4.8
5.0
5.0
5.0
5.0
5.0
5.0
5.0
1478.9
1434.0
1430.7
1418.1
5.0
4.9
4.9
4.9
5.0
5.0
5.0
5.1
1440.8
1458.5
1459.3
1444.9
200.1
5.0
36.4
200.1
5.1
35.4
200.1
5.1
35.2
5.1
5.0
5.0
33.9
34.5
34.2
1+58_7.4
60.1
58.3
58.3
58.3
1+4_pH 5.7
1+4_pH 5.7
1437.2
1+201_5.7
1+201_5.7
35.5
1+201_7.4
201.3
201.3
201.3
1% solution
5.7
5.1
5.1
1+201_7.4
208.8
208.8
208.8
96
1+58_pH 5.7
Dilution
59.5
59.5
59.5
1% solution
5.0
5.1
5.0
FPU/g
120.3
119.6
121.4
5.0
5.1
5.0
5.0
5.0
5.0
121.9
121.2
122.9
121.6
120.9
121.7
5.0
5.0
5.0
5.0
5.0
5.0
1453.4
1434.4
1439.4
5.01
5.01
5.01
4.99
5.05
5.08
1444.21
1424.78
1418.18
5.07
5.00
5.08
35.58
36.04
35.51
5.0
5.0
5.1
35.1
35.2
34.8
1+58_7.4
58.8
58.8
58.8
59.8
59.8
59.8
1+4_pH 5.7
1+201_5.7
200.01
200.01
200.01
1+201_7.4
204.3
204.3
204.3
97
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101
BIOGRAPHICAL SKETCH
Mr Rilwan Oyetunji was born and raised in Lagos, Nigeria. He moved to the United States in
May, 2002 and obtained his B.Sc in Chemical Engineering from the University of Mississippi.
Prior to attending the University of Mississippi, he attended the Houston Community College,
Houston TX for a year.
He is currently married and he enjoys spending time with his spouse, Wumi, playing soccer and
going to the gym.
102