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1 Florida State University Libraries Electronic Theses, Treatises and Dissertations The Graduate School 2009 Enzymatic Hydrolysis of Cellulose in a NMMP/H2O Solution Rilwan Oyetunji Follow this and additional works at the FSU Digital Library. For more information, please contact lib-ir@fsu.edu

2 FLORIDA STATE UNIVERSITY COLLEGE OF ENGINEERING ENZYMATIC HYDROLYSIS OF CELLULOSE IN A NMMO/H 2 O 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

3 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

4 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

5 ACKOWLEDGEMETS 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

6 TABLE OF COTETS ACKNOWLEDGEMENTS... iv TABLE OF CONTENTS... v LIST OF TABLES... viii LIST OF FIGURES... ix Abstract... x Chapter 1 Introduction Problem Identification Project Objectives... 3 Chapter 2 Literature Review Introduction Cellulose Glucose Cellulose Hydrolysis Acid Hydrolysis Method Enzymatic Hydrolysis Method Pretreatment Processes Enzymatic Hydrolysis of Cellulose Understanding the Cellulase System Understanding the Lignocellulosic Substrate Understanding the Enzyme9Substrate Interaction Understanding the Inhibition or Deactivation of the Enzyme Description of the Cellulose Hydrolysis Kinetics The Purpose of this Research Chapter 3 Experimental Methodology Introduction : Estimation of Reducing Sugars by DNS Method Theory Behind the DNS Method Preparation of DNS Reagent Determination of Absorbance Vs Concentration Calibration Points : Estimation of Enzyme Activity by the Filter Paper Unit (FPU) Method v

7 3.4: Preparation of NMMO.x H : Preparation of 1% Cellulose Solutions : Protocol for enzyme reactions Chapter 4 Results Enzyme Activity /FPU Determination of Accelerase 1000 and Spezyme Cellulase Effect of NMMO on the Enzymatic Hydrolysis of Cellulose Yield of Reducing Sugars for Different Concentrations of NMMO mixed with Cellulase Comparison of NMMO/Cellulase Solution with Acetate Buffered Solution of various ph for 1 Hr Reaction with Genencor Spezyme Comparison of NMMO/Cellulase Solution with Acetate Buffered Solution of Various ph for 3 Hr Reaction with Accelerase 1000 Cellulase Effect of Cellulase on Cellulose Dissolved in NMMO and Filter paper in acetate buffer or NMMO medium Factors That Affect The Enzymatic Hydrolysis Effect of Temperature Effect of Enzyme Loading Effect of ph Interaction Between Temperature and ph Chapter 5 Conclusions Chapter 6 Recommendations APPENDIX A Sugar Yields (mg/ml) as a function of Time APPENDIX A.1 40C DATA APPENDIX A.2 50C DATA APPENDIX A.3 60C DATA APPENDIX B. Percentage Cellulose Conversion as a function of Time APPENDIX B.1 40C DATA APPENDIX B.2 50C DATA APPENDIX B.3 60C DATA APPENDIX C Dilution, Weight of 1% lyocell solution and Enzyme activity for each experiment REFERENCES: vi

8 BIOGRAPHICAL SKETCH vii

9 LIST OF TABLES Table Relationship Between Structural Feature and Enzyme Digestibility Table 2. 29List of Assumptions from Authors that have modeled the Hydrolysis Process Table 3. 19Glucose Concentration versus Absorbance Data Table 4. 19pH of NMMO Solution after Cellulase Addition versus Concentration of water in NMMO solution Table Percentage Conversion of Cellulose at 40C and 50C, ph 5.7 and 7.4, and Enzyme Loading of 122 FPU/g Table 4. 39Percentage Conversion of Cellulose at Different Temperatures, Enzyme Loading and ph after 24 Hours Enzymatic Reaction Table 4. 49Percentage Conversion of Cellulose at Different Temperature, Enzyme Loading and ph after 3 Hours Enzymatic Reaction Table 4. 59Percentage Conversion of Cellulose at Different Temperatures and Enzyme Loading at ph 7.4 after 24 Hours Enzymatic Reaction Table 4. 69Percentage Conversion of Cellulose at Different Temperatures and Enzyme Loading at ph 5.7, after 24 Hours Enzymatic Reaction Table Percentage Conversion of Cellulose at Different Temperatures, ph and Enzyme Loadings, after 24 Hours Enzymatic Reaction viii

10 LIST OF FIGURES Figure Diagram of Glucose, Cellulose and Amylose... 7 Figure 2. 29Lignocelluloses organization into elementary fibrils and microfibrils Figure 2.39Comparison Between Two Processes of Hydrolyzing Cellulose Figure 3. 19Reaction of Glucose with DNS Figure 3. 29Glucose Concentration versus Absorbance Calibration Curve.The equation of the curve and the R 2 value are on the plot Figure 4. 19FPU Determination for Accelerase Figure 4. 29FPU Determination for Genencor Spezyme Figure Effect of NMMO on Enzymatic Hydrolysis of Filter Paper Figure 4. 49Comparison between NMMO medium and Acetate Buffer Medium Using Genencor Spezyme Figure 4. 59Comparison between NMMO medium and Acetate Buffer medium using Accelerase Figure Effect of Hydrolyzing Cellulose while it is coming out of Solution Figure Yield of Reducing Sugars as a function of Time at Different Reaction conditions.. 43 Figure Effect of Temperature at ph 5.7 and 34.9 FPU/g Enzyme Loading Figure 4. 99Effect if Temperature at ph 5.7 and 122 FPU/g Enzyme Loading Figure Effect of Temperature at ph 5.7 and FPU/g Enzyme Loading Figure Effect of Enzyme Loading at 40C and ph Figure Effect of Enzyme Loading at 40C and ph Figure Effect of ph at 40C and 122 FPU/g Enzyme Loading Figure Effect of ph at 50C and 122 FPU/g Enzyme Loading Figure Effect of ph at 60C and 122 FPU/g Enzyme Loading Figure Interaction Between Temperature and ph at 34.9 FPU/g Enzyme Loading Figure Interaction Between Temperature and ph at 122 FPU/g Enzyme Loading Figure Interaction Between Temperature and ph at FPU/g Enzyme Loading ix

11 ABSTRACT This thesis is focused on the enzymatic hydrolysis of cellulose while it is in an N9 methylmorpholine9 N9Oxide (NMMO)/H 2 0 solution. The reason for using NMMO/H 2 0 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/H 2 0, 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 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 FPU/g and a ph of 7.4. x

12 CHAPTER 1 ITRODUCTIO 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 lignin 9. 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. C 6 H 11 O 6 9(C 6 H 10 O 5 ) n 9C 6 H 11 O 6 + (n9x) H 2 O (n9x) C 6 H 12 O 6 + C 6 H 11 O 6 9 (C 6 H 10 O 5 ) x 9 C 6 H 11 O 5 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

13 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 tons per year 3 ); 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 temperatures 9 (e.g. sub and supercritical H 2 O), high pressures, 11 or chemical pretreatment using specialized chemicals (ionic liquids 12 ) 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

14 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/H 2 0 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

15 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 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 jobs 38. 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 decomposition 17. 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 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

16 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 Relationship Between Structural Feature and Enzyme Digestibility 1 Structural Features Relationship between structural feature and digestibility Physical Surface Area Positive Crystallinity Negative/No correlation Degree of Polymerization Negative/No correlation Pore volume Positive Particle size No correlation Chemical Lignin Negative Hemicellulose Negative Acetyl Group Negative 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 C 6 H 12 O 6 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

17 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

18 Ring form of Glucose Cellulose Amylose Figure 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

19 degradation rate. The two methods of hydrolyzing the cellulose are: the Acid Hydrolysis Method and the Enzymatic Hydrolysis method 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 However, this process was considered to be uneconomical in the 1980 s after it was attempted by the U.S. during the Second World War 30. 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 hydrolysis 24. 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 recovery 31. 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 requirements 24. 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

20 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 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 cellulose 7, 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 effective 24, 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

21 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 processes 24. 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 CO 2 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 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 H 2 SO 4 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,

22 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 slowly 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 glucose 15. 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 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

23 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 cellulose 7, 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 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

24 Figure 2. 2+Lignocelluloses organization into elementary fibrils and microfibrils 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 process 5, 32. It has been observed that the adsorption process is independent of ph, but strongly dependent on temperature 18. 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

25 of the substrate depends on the enzyme s penetration through the fibrils and the diffusion of the enzymes 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 inhibited 33. 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 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 studies 20 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 al 10 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

26 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 al 21 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

27 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

28 Table 2.2+List of Assumptions from Authors that have modeled the Hydrolysis Process Author Key Assumptions Conclusions Movargenejad et al 19 Transfer of enzyme from solution bulk to cellulose surface is rapid in comparison to rate of reaction Only adsorbed enzymes hydrolyze the cellulose No internal diffusion occurs; enzymes adsorb solely at the cellulose external surface Cellulose sites available for enzyme is proportional to surface area of cellulose particle Activity of enzyme adsorbed at cellulose surface decreases with time Inhibition by products occur during the reaction cellulose particles shrink during reaction Mean absolute deviation of 6% was observed The limit of the reaction was well predicted by model Initial rates of reaction was under estimated by model Some discrepancies were observed between experimental results and those determined from model Liao et al 5 Enzyme first adsorbed by different components (cellulose, hemicellulose and lignin) Two types of bonding sites on cellulose9 active and inactive Enzymes on active sites produce sugars Glucose was assumed to be the only inhibitor of hydrolysis. Enzymatic hydrolysis divided into Model was able to predict adsorption and hydrolysis data at high substrate concentrations( 50g/L) Use of enzyme activity instead of enzyme concentration effectively predicted the adsorption and hydrolysis of the 17

29 Table 2. 2 continued Author Key Assumptions Conclusions Shen et al 22 two parts: adsorption and hydrolysis Langmuir isotherms used to describe the enzyme adsorption Competitive model of glucose inhibition was used to simulate hydrolysis reactions 3 major enzymes in conversion of cellulose to glucose: exoglucanase, endoglucanase and β9glucosidase Competitive enzymatic hydrolysis 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 substrate concentrations and structure difference were negligible Available cellulose is related to ratio of remained cellulose to initial total cellulose - Cellulase enzyme is assumed to have a single combined effect in the hydrolysis of insoluble substrate - Surface and structure of insoluble fiber are homogeneous and there is 18 sample Langmuir adsorption was significantly changed during hydrolysis for 5 samples An empirical equation was used to describe change of adsorption constant - Two parameter model developed could describe a wide range of sugar concentration with time - Model was successfully used to fit experimental

30 Table 2. 2 continued Author Key Assumptions Conclusions no distinction between amorphous and crystalline regions - Enzyme deactivation is assumed to be a second order reaction - Adsorption of free enzyme in 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 data of cotton gin waste - Highest yield of reducing sugars were obtained at lowest initial enzyme concentrations 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

31 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. Studies 4, 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/H 2 0 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 documented 42,43 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

32 methods previously described, before separating and hydrolyzing the pretreated biomass to sugars in a reaction medium suitable for the cellulases. Therefore by using NMMO/H 2 O 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/H 2 0 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 deionizedwater 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

33 CHAPTER 3 EXPERIMETAL 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 DS 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) Theory Behind the DS Method The DNS method for estimating the concentration of reducing sugars in a sample was originally invented by G. Miller 34 in 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