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Potential for ethanol from urban cellulosic wastes Leung, Clara 2010

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 POTENTIAL FOR ETHANOL FROM URBAN CELLULOSIC WASTES  by  CLARA LEUNG B.A.Sc., The University of British Columbia, 2007     A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF  THE REQUIREMENTS FOR THE DEGREE OF  MASTER OF APPLIED SCIENCE  in  The Faculty of Graduate Studies  (Chemical and Biological Engineering)     THE UNIVERSITY OF BRITISH COLUMBIA (Vancouver)  APRIL 2010  © Clara Leung, 2010  ii  ABSTRACT  Ethanol fuel can be used as a substitute for gasoline. It can be produced from numerous sources. There are several environmental and economic benefits associated with this feedstock; its abundant availability worldwide, the reduction of landfill space, and the production of a valuable, clean liquid fuel that when utilized. The successful bioconversion of urban cellulosic waste into ethanol will provide industries with an inexpensive raw material for ethanol production and a more competitive ethanol price.  The objective of this project is to investigate whether it is technically- and economically-viable to use urban cellulosic wastes as a feedstock for a full-scale ethanol plant. The urban cellulosic wastes evaluated include: grass, cardboard, and pulp mill clarifier sludge with switchgrass being the control. In order to maximize the ethanol yield from the urban cellulosic wastes, conditions for pretreatment and enzymatic hydrolysis were optimized. After the optimal condition for each substrate was determined, the hydrolysate from each condition was subjected to fermentation with Saccharomyce cerevisiae (S. cerevisiae) K1 strain.  In order to evaluate the efficiencies of oxygen delignification, the pretreatment used in this study, and enzymatic hydrolysis, the compositions (lignin, pentose and hexose) of the substrates that were not pretreated and pretreated at different temperatures were determined by acid hydrolysis. By comparing the lignin contents of non-pretreated substrates from acid hydrolysis to those that were reported in literature, they are out of range. Therefore, all the lignin contents determined from acid hydrolysis will not be used for any calculation. However, these lignin values have indicated that oxygen delignification is effective on switchgrass, grass and pulp mill clarifier sludge since their lignin contents reduced when the substrates are pretreated. Although oxygen delignification is not as effective for cardboard, the non-pretreated cardboard that was hydrolyzed at 10 g dry substrate/L and 40 FPU/g dry substrate has the highest sugar yield (6.12 g sugar/10 g dry substrate) among all substrates tested in all pretreatment and hydrolysis conditions. After the hydrolysate obtained from the hydrolysis of cardboard was subjected to fermentation, it has an ethanol yield of 0.32 gram of ethanol per each gram of sugar.  iii  TABLE OF CONTENTS  ABSTRACT ............................................................................................................................................ ii TABLE OF CONTENTS........................................................................................................................ iii LIST OF TABLES .................................................................................................................................. v LIST OF FIGURES ................................................................................................................................ vi ACKNOWLEDGEMENTS .................................................................................................................... ix 1.0 INTRODUCTION ............................................................................................................................. 1 2.0 LITERATURE REVIEW .................................................................................................................. 3 2.1 Fossil fuel and its pollution ...................................................................................................... 3 2.2 Ethanol .................................................................................................................................... 6 2.3 Ethanol fuel.............................................................................................................................. 7 2.4 Conventional fermentation process for ethanol production ........................................................ 9 2.5 Alternative processes and substrates for ethanol production .................................................... 11 2.6 Lignocellulosic biomass ......................................................................................................... 14 2.7 Urban cellulosic substrates ........................................................................................................... 16 2.7.1 Switchgrass ........................................................................................................................... 17 2.7.2 Grass ..................................................................................................................................... 18 2.7.3 Cardboard ............................................................................................................................. 19 2.7.4 Paper mill clarifier sludge...................................................................................................... 20 2.8 Pretreatment of lignocellulosic materials ................................................................................ 21 2.9 Oxygen delignification ........................................................................................................... 22 2.10 Overview of hydrolysis process .............................................................................................. 26 2.11 Fermentation process.............................................................................................................. 27 2.11.1 Types of yeast ..................................................................................................................... 28 2.11.2 Glucose metabolism ............................................................................................................ 28 3.0 RESEARCH OBJECTIVES ............................................................................................................ 31 4.0 MATERIALS AND METHODS ..................................................................................................... 32  iv  4.1 Substrate sources and preparation................................................................................................. 32 4.2 Determination of cellulose, hemicellulose and lignin .................................................................... 35 4.3 Oxygen delignification ................................................................................................................. 37 4.4 Enzymatic hydrolysis ................................................................................................................... 39 4.4.1 Procedures for cellulase activity determination ...................................................................... 39 4.4.2 Procedures for -glucosidase assay........................................................................................ 44 4.4.3 Procedure for enzymatic hydrolysis ....................................................................................... 45 4.5 Fermentation of hydrolyzed samples ............................................................................................ 48 4.5.1 Yeast growth ......................................................................................................................... 48 4.5.2 Determination of yeast concentration..................................................................................... 49 4.6 Analytical .................................................................................................................................... 51 4.6.1 Analysis of monomer sugars by HPLC .................................................................................. 51 4.6.2 Analysis of ethanol by gas chromatography ........................................................................... 53 4.6.3 Determination of various concentrations by spectrophotometer ............................................. 55 4.6.4 Measurement of pH ............................................................................................................... 56 5.0 RESULTS AND DISCUSSION ....................................................................................................... 57 5.1 Analysis of substrate composition ................................................................................................ 57 5.2 Sugar loss during oxygen delignification ...................................................................................... 59 5.3 Kinetics and yield of sugar production from enzymatic hydrolysis ............................................... 60 5.4 Yeast growth in YPG medium...................................................................................................... 65 5.5 Ethanol yield for fermentation of hydrolysates ............................................................................. 67 5.6 Material balance .......................................................................................................................... 69 5.6.1 Mass balance of lab scale experiment .................................................................................... 69 5.6.2 Mass balance of scaled up ethanol plant ................................................................................ 72 6.0 CONCLUSIONS ............................................................................................................................. 75 7.0 FUTURE WORK ............................................................................................................................ 76 REFERENCES...................................................................................................................................... 77 APPENDIX ........................................................................................................................................... 84  v  LIST OF TABLES  Table 1     Net energy return values obtained from producing ethanol from corn (7) ................................. 1 Table 2     U.S. primary energy consumption by source 2007 (18) ............................................................ 3 Table 3     Energy inputs and outputs for the life-cycle analysis of cellulosic ethanol manufacturing technologies (values are in kJ/L and the crop processed is assumed to be hybrid poplar – 11 dry metric tonnes/ha/year)(11) .............................................................................................. 13 Table 4      Contents of cellulose, hemicellulose and lignin in common agriculture residues and wastes (59) ...................................................................................................................................... 18 Table 5     Sugar yield in enzymatic hydrolysis of steam exploded washed grasses (Celluclast 1.5L FP 10FPU/g, -glucosidase 10nkat/g*, 45ºC, pH 5, 72 h) (60) ................................................... 19 Table 6     Wet to dry weight ratio for different substrates ...................................................................... 34 Table 7     Enzyme dilution used in filter paper assay ............................................................................. 40 Table 8     Glucose dilution used for standard ......................................................................................... 40 Table 9     Test tubes setup for filter paper assay .................................................................................... 41 Table 10   Absorbance of controls, standards and samples measured at 540 nm ...................................... 43 Table 11   Enzyme dilution used in beta-glucosidase assay .................................................................... 45 Table 12   Hydrolysis experimental conditions (91) ............................................................................... 46 Table 13   Initial factorial design for oxygen delignification and enzymatic hydrolysis ........................... 47 Table 14   Dilutions used in constructing yeast calibration curve ............................................................ 50 Table 15   Operating conditions for HPLC analysis ................................................................................ 52 Table 16   Nanopure specifications for water used in HPLC ................................................................... 53 Table 17   Sugar standards used in HPLC .............................................................................................. 53 Table 18   Operating conditions for GC analysis .................................................................................... 54 Table 19   Dilution for GC calibration curves used for correlating yeast concentration to absorbance ..... 55 Table 20   Compositions of substrates (urban cellulosic wastes) used in ethanol production ................... 58 Table 21   Contents of lignin and total sugar for non-pretreated substrates used...................................... 59 Table 22   Sugar loss in oxygen delignification step ............................................................................... 60 Table 23   Maximum sugar yield and percent sugars hydrolyzed in hydrolysis for factorial design conditions ............................................................................................................................ 62 Table 24   Mass balance for processes used in this project ...................................................................... 70    vi  LIST OF FIGURES  Figure 1     World liquid fuel consumption for the Energy Information Administration (EIA) projects (21)  .............................................................................................................................................. 4 Figure 2     Chemical structure of ethanol ................................................................................................. 7 Figure 3     Ethanol production (in millions of litres) by types (34) ........................................................... 7 Figure 4     Block flow diagram for a corn dry mill (40) ......................................................................... 10 Figure 5     Block flow diagram for a corn wet mill complex (40)........................................................... 10 Figure 6     Structure of cellulose (45) .................................................................................................... 14 Figure 7     Structure of hemicellulose (46) ............................................................................................ 14 Figure 8     Main components of lignin (47) ........................................................................................... 15 Figure 9     Schematic of enzyme attack on cellulose (49) ...................................................................... 16 Figure 10   Photograph of corrugated fiberboard .................................................................................... 19 Figure 11   Oxygen delignification in pulping industry ........................................................................... 23 Figure 12   Initial reactions in oxygen delignification (70) ..................................................................... 24 Figure 13   Reactions of hydroperoxide intermediates (70) ..................................................................... 25 Figure 14   Flow diagram for producing ethanol from urban cellulosic wastes ........................................ 32 Figure 15   Cuisinart mini-prep food processor ...................................................................................... 33 Figure 16   Switchgrass before (left) and after size reduction ................................................................. 33 Figure 17   Grass before (left) and after size reduction ........................................................................... 34 Figure 18   VWR model #1350FM drying oven ..................................................................................... 36 Figure 19   Muffle furnace (Thermo Scientific, FB1300)........................................................................ 36 Figure 20   Oxygen delignification apparatus ......................................................................................... 37 Figure 21   Front, side and top views of movable head unit of Parr reactor (86) ...................................... 38 Figure 22   Determination of glucose concentration ............................................................................... 43 Figure 23   Filter paper unit determination for cellulase activity ............................................................. 44 Figure 24   General growth curve (92) ................................................................................................... 49 Figure 25   Correlation of yeast K1 concentration and optical density at 600 nm .................................... 50 Figure 26   Determination of yeast K1 concentration.............................................................................. 51 Figure 27   Hexose concentration obtained from non-pretreated cardboard ............................................. 61 Figure 28   Glucose yield over time at different solid loadings of olive tree biomass (98) ....................... 63 Figure 29   Cellulose conversion (ratio of grams of glucose produced to grams of cellulose supplied in enzymatic hydrolysis) at different -glucosidase and cellulase concentrations (99) ............... 63  vii  Figure 30   Comparison of hexose concentration of non-pretreated cardboard (Hydrolyzed at 10 g dry substrate/L and 40 FPU/g) .................................................................................................... 65 Figure 31   Concentration of yeast strain K1 over time (growth conditions: incubated at 30°C and 150 rpm in YPG medium with 2% w/w glucose) ................................................................................ 66 Figure 32   Determination of specific growth rate .................................................................................. 67 Figure 33   Yeast concentration during fermentation (growth conditions: incubated at 30°C and 150 rpm)  ............................................................................................................................................ 68 Figure 34   Process flow diagram for mass balance ................................................................................ 69 Figure 35   Schematic diagram for ethanol production ........................................................................... 72 Figure 36   Switchgrass – pentose concentration (no oxygen delignification).......................................... 84 Figure 37   Switchgrass - hexose concentration (no oxygen delignification) ........................................... 85 Figure 38   Switchgrass – pentose concentration (oxygen delignification at 100°C) ................................ 85 Figure 39   Switchgrass – hexose concentration (oxygen delignification at 100°C) ................................. 86 Figure 40   Switchgrass – pentose concentration (oxygen delignification at 130°C) ................................ 86 Figure 41   Switchgrass – hexose concentration (oxygen delignification at 130°C) ................................. 87 Figure 42   Switchgrass – pentose concentration (oxygen delignification at 150°C) ................................ 87 Figure 43   Switchgrass – hexose concentration (oxygen delignification at 130°C) ................................. 88 Figure 44   Grass – pentose concentration (no oxygen delignification) ................................................... 88 Figure 45   Grass – hexose concentration (no oxygen delignification) .................................................... 89 Figure 46   Grass – pentose concentration (oxygen delignification at 100°C) .......................................... 89 Figure 47   Grass – hexose concentration (oxygen delignification at 100°C ............................................ 90 Figure 48   Grass – pentose concentration (oxygen delignification at 130°C) .......................................... 90 Figure 49   Grass – hexose concentration (oxygen delignification at 130°C) ........................................... 91 Figure 50   Grass – pentose concentration (oxygen delignification at 150°C) .......................................... 91 Figure 51   Grass – hexose concentration (oxygen delignification at 150°C) ........................................... 92 Figure 52   Grass – pentose concentration (oxygen delignification at 180°C) .......................................... 92 Figure 53   Grass – hexose concentration (oxygen delignification at 180°C) ........................................... 93 Figure 54   Cardboard – pentose concentration (no oxygen delignification) ............................................ 93 Figure 55   Cardboard – hexose concentration (no oxygen delignification) ............................................. 94 Figure 56   Cardboard – pentose concentration (oxygen delignification at 100°C) .................................. 94 Figure 57   Cardboard – hexose concentration (oxygen delignification at 100°C) ................................... 95 Figure 58   Cardboard – pentose concentration (oxygen delignification at 130°C) .................................. 95 Figure 59   Cardboard – hexose concentration (oxygen delignification at 130°C) ................................... 96 Figure 60   Pulp mill clarifier sludge – pentose concentration (no oxygen delignification) ...................... 96  viii  Figure 61   Pulp mill clarifier sludge – hexose concentration (no oxygen delignification) ....................... 97 Figure 62   Pulp mill clarifier sludge – pentose concentration (oxygen delignification at 100°C) ............ 97 Figure 63   Pulp mill clarifier sludge – hexose concentration (oxygen delignification at 100°C) ............. 98 Figure 64   Pulp mill clarifier sludge – pentose concentration (oxygen delignification at 130°C) ............ 98 Figure 65   Pulp mill clarifier sludge – hexose concentration (oxygen delignification at 130°C) ............. 99 Figure 66   Ethanol yield for switchgrass (pretreated at 130°C) .............................................................. 99 Figure 67   Ethanol yield for grass (pretreated at 150°C) ...................................................................... 100 Figure 68   Ethanol yield for cardboard (non-pretreated) ...................................................................... 100 Figure 69   Ethanol yield for pulp mill clarifier sludge (pretreated at 100°C) ........................................ 101  ix  ACKNOWLEDGEMENTS  The research work done for this project was sponsored by Dr. Sheldon Duff of the Department of Chemical and Biological Engineering at the University of British Columbia. I would like to express my gratitude to Dr Duff for his patience, mentorship and contribution on my thesis. I would also like to thank all of my peers in the Biofuels Research Lab and all the staff members in this department who provide assistance whenever I needed. In addition, I would like to express thanks to my thesis committee members for their valuable feedback. Lastly, I would like to thank my family and Jacky for their love and support.  1  1.0 INTRODUCTION  Fossil fuels are non-renewable fuels that were formed hundreds of millions of years ago. As our consumption rate doubles every forty years, the concern over decreasing availability of fossil fuels has heightened (1). Global warming and the associated climate change have also contributed to a sense of urgency to reduce our dependency on fossil fuels and seek alternative energy sources. Ethanol is one of the most promising alternative liquid transport fuels. According to the Renewable Fuels Association (RFA), the annual U.S. fuel ethanol demand increased 39%, from 6,500 to 9,150 million gallons from 2007 to 2008 (2). The benefit from using ethanol as transportational fuel is that even 10% ethanol-enriched fuel reduces the greenhouse gas emission by 12 – 19% when compared to gasoline fuel (3). Most of the ethanol produced today is called first generation bioethanol. It can be produced from energy crops, such as corn, sugar cane and sugar beet. These crops contain sugars or materials that can be converted into sugar (4). While first generation bioethanol production is a relatively mature technology that is widely used around the world, there are some drawbacks to using this technology (5). Since most of these energy crops are also food sources for both human and animal, as bioethanol demand elevates, it is foreseeable that arable lands for food production will be occupied by energy crops. Consequently, food supply will decline and the food prices will increase (6). The net energy return from producing bioethanol from energy crops is also a controversial issue with both positive and negative net energy values reported (Table 1). Thus, alternative raw materials should be sought for bioethanol production in order to minimize the energy input for producing the energy crops and thereby ensure there is a positive net energy return.  Table 1   Net energy return values obtained from producing ethanol from corn (7) Positive Net Energy Value Negative Net Energy Value Authors Input : Output Authors Input : Output Marland and Turhollow (1991) 1:1.25 Pimentel and Patzek (2005) (8) 1.29:1 Morris and Ahmed (1992) 1:1.34 Pimentel (1991) 1.34:1 Shapouri et al. (1995) 1:1.20 Keeney and DeLuca (1992) 1.10:1 Morris and Lorenz (1995) 1:1.38 Ho (1989) 1.05:1  2  The purpose of this project is to investigate the potential of ethanol production using urban cellulosic waste as the alternative raw material. This waste was selected as the target for investigation because it is relatively abundant, close to major markets for transport fuel and readily available world-wide. Recent years have seen the reduction of available landfill space (9). In order to minimize wastes, many countries have been proposing or implementing recycling programs, which will prolong the life-span of these landfills. Some of the recycled materials such as paper, grasses, newsprint, construction wastes, corrugated cardboard, and yard trimmings, contain cellulose, which can be used as raw material for ethanol fuel production (10) (11) (12). This can reduce the need for landfill space and reduce landfill gas emissions. These landfill gases also contribute to the “greenhouse effect” because they absorb and emit infrared radiation, which lead to the heating of the earth’s surface (13). In addition, the cost and pollution associated with transporting urban cellulosic wastes to an ethanol plant are minimized since the wastes are available locally. As less energy is used in the bioethanol production process, using urban cellulosic wastes will definitely increase the net energy return. In addition, recycling these waste materials can increase their economic value. For instance, 100% recycled paper products is 3 – 10% more expensive when compared to paper made from virgin fiber because the processes involved are complicated and more expensive (14). Also, the collected yard trimmings are usually composted by local municipalities and the compost is then given away or sold very cheaply (15). However, the value of these materials may be increased if they are to be used to produce ethanol fuel, which can be used as an alternative to gasoline. When gasoline prices increase, ethanol fuel made from lignocellulosic materials will likely to have a higher demand and economic value.  In this project, switchgrass, cardboard, grass clippings, and paper mill clarifier sludge, were used to determine which substrate is the best raw material for bioethanol production, their optimum hydrolysis conditions and evaluate ethanol yields from each substrate at the corresponding optimum condition. The aim of this work is to determine which substrate and the corresponding pretreatment condition which yields the best hydrolysis. The hydrolysate will then undergo fermentation to determine the ethanol yield.  3  2.0 LITERATURE REVIEW  2.1 Fossil fuel and its pollution  Petroleum, which is known as crude oil, is a well-known fossil fuel. It was formed from the action of heat and pressure on the remains of plants and animals that existed millions of years ago (16) (17). After petroleum is extracted from the ground, it is used for gasoline production. According to the Annual Energy Review done by the Energy Information Administration (EIA), petroleum is the largest source of energy consumed in the U.S.. Despite the efforts to boost renewable energy through subsidises and mandates by the U.S. government, only 7.7 quadrillion (1 x 1015) kJ (about 7% of energy consumed) is renewable energy (table 2) (18).  Table 2   U.S. primary energy consumption by source 2007 (18)   Energy Source (Quadrillion kJ) Energy Source (%) Petroleum1 39.1 37.40 Natural Gas2 25.1 23.99 Coal3 23.7 22.68 Renewable Energy4 7.7 7.36 Nuclear Electric Power 9.0 8.57 1 Excludes 0.6 quadrillion kJ of ethanol, which is included in "Renewable Energy.” 2 Excludes supplemental gaseous fuels 3 Includes 0.1 quadrillion kJ of coal coke net imports 4 Conventional hydroelectric power, geothermal, solar/PV, wind, and biomass   Gasoline is a liquid mixture that contains mostly aliphatic hydrocarbons, which consists of saturated and unsaturated carbon in chains (19). It is often enhanced with iso-octane or aromatic hydrocarbons like benzene and toluene to increase octane rating, which is a measure of how well the fuel burns in a controlled manner (20).  4  In the present world, gasoline is a source of energy that we cannot live without. It is the most common fuel used in vehicles (19). Based on the short term energy outlook from EIA (figure 1), the annual growth in world oil consumption has dropped since 2004. However, it is clear that the total consumption is stabilizing at a high level (21).   Figure 1   World liquid fuel consumption for the Energy Information Administration (EIA) projects (21)  Although more alternative fuel vehicles are becoming available in the marketplace, most vehicles are still dependent on gasoline. Thus, the air pollution problems associated with these vehicles is still a great concern for the public. In order to prevent this pollution, it is necessary to understand how vehicles produce pollutants. All engines in vehicles are known as internal combustion engines because they burn gasoline inside the engines. The advantages of using internal combustion engines are that they are more fuel-efficient and are smaller in size when compared to external combustion engines (22). There are different kinds of internal combustion engines available, such as gas turbines, diesel engines, conventional gasoline four – stroke engines, rotary engines and two-stroke engines. Currently, almost all vehicles utilize a process -2 -1 0 1 2 3 4 5 0 10 20 30 40 50 60 70 80 90 100 2002 2003 2004 2005 2006 2007 2008 2009 A nn ua l G ro w th            (M ill io n ba rr el s pe r da y) To ta l C on su m pt io n          (M ill io n ba rr el s pe r da y) Year Total Consumption Annual Growth - China Annual Growth - United States Annual Growth - Other Countries  5  called the four – stroke combustion cycle (also known as Otto cycle) to combust gasoline and generate power. The cycle includes an intake stroke, a compression stroke, a combustion stroke and an exhaust stroke (22).  During the intake stroke, a small amount of fuel is allowed through the intake port along with air as the piston moves downward. After the intake stroke, the piston travels upwards to compress the gasoline-air mixture. As soon as the piston reaches the top of its travel, the spark plug ignites the mixture to create an explosion, which drives the piston downward. Lastly, the exhaust gas will exit through the exhaust valve to the tailpipe when the piston moves toward the top again. The linear movement of the piston rotates the crankshaft, which translates into a rotational movement of the wheels (22).  Gasoline, which is made up of carbon and hydrogen, can be combusted completely into carbon dioxide and water if enough oxygen is available. However, there is not enough reaction time for complete combustion in an automobile. Three major pollutants are generated with gasoline powered vehicles. They are carbon monoxide, nitrogen oxides and unburned hydrocarbons. Other impurities, such as sulfur, which can also form sulfur dioxides, may also present and contribute to the pollution (23). Among these pollutants, unburned hydrocarbon causes the least problems because it is usually washed away by rain from air into water (24).  On the other hand, carbon monoxide, nitrogen oxides (NOx) and sulfur dioxides are harmful to both humans and the environment. Carbon monoxide, which is the most abundant pollutant emitted from vehicles, can affect the oxygen transporting ability of hemoglobin, which may trigger symptoms such as headache and drowsiness (24). In addition carbon monoxide reacts with hydroxyl radicals (•OH) in the atmosphere and thereby reduces the concentration of hydroxyl radicals in the atmosphere. Since hydroxyl radicals can help shorten the lifetime of green house gases in the atmosphere by reacting with them, the presence of carbon monoxide will indirectly contribute to global warming (25). Nitrogen oxide is created when nitrogen and oxygen are reacted at high temperature. Photochemical smog is produced when nitrogen oxide reacts with volatile organic compounds (VOCs) under sunlight. This leads to the existence of particulate matter, which are  6  either man made or natural tiny solid or liquid pollutant particles that suspend in gas or liquid, and tropospheric ozone (26), which can induce lung and respiratory illnesses such as asthma and bronchitis (27). Although nitrogen oxide affects both global warming and our health, its breakdown can actually increase the amount of •OH radicals in the atmosphere, which can, in turn, reduce the lifetime of greenhouse gases (28). In addition, sulfur dioxides can create acid rain. Ironically, the presence of sulfur dioxides can reduce the effect of global warming. Studies have found that areas with low concentration of sulfur dioxides suffered regional warming because sulfate aerosols, which are formed by reacting the sulfur dioxide, water and oxygen in the air, can reflect sunlight back into space, which is the exact opposite of what happens with greenhouse gases (29). Due to these effects caused by the emissions from the vehicles, many researchers are striving to find an alternative renewable fuel that burns more cleanly. In this project, ethanol has been chosen for investigation.  2.2 Ethanol  Ethanol (CH3CH2OH), also known as ethyl alcohol, grain alcohol and EtOH, is a clear, colourless, volatile, flammable and slightly toxic chemical compound. Ethanol is one of a group of chemical compounds whose molecules contain a hydroxyl group, bonded to a short carbon chain (Figure 2). It can be used in alcoholic beverages, pharmaceuticals, cosmetics, solvents, chemicals and fuels (30). One of the most common uses of ethanol is ethanol fuel. The history of ethanol as a fuel can be dated back to the early days of the automobile. However, it was replaced by petroleum in the early 1900s because ethanol was more expensive. Recently, due to the rising price of oil and the depletion of fossil fuels, ethanol fuel has once again attracted the public interest (31). Moreover, global warming has become a major energy and environmental issue (32). It is widely believed that CO2 emissions from automobiles are a major contributor to global warming. Studies have shown that 9 kg of CO2 is created by 3.8 L of gasoline used (33). As a result, the use of a renewable and cleaner burning fuel is of particular interest.  7   Figure 2   Chemical structure of ethanol   2.3 Ethanol fuel  Ethanol fuel can be produced from any feedstock that contains sugars or materials that can be converted into simple sugars (4). Because of this, it has become one of the most promising alternative fuels. In 2003, it was estimated that ethanol for fuel accounted for 70% of world ethanol production (figure 3).   Figure 3   Ethanol production (in millions of litres) by types (34) 0 10000 20000 30000 40000 50000 60000 70000 80000 90000 1990 1995 2000 2005 Et ha no l p ro du ce d (m ill io n lit re s) Years Beverage Industrial Fuel  8   Due to the increasing interest in ethanol fuel, the overall production of ethanol has dramatically increased. The top three ethanol producing countries are the United States, China and Brazil. In 2006, their combined ethanol production accounted for approximately 75% of the world’s annual production (35). Ethanol can be produced in two ways: acid-catalyzed hydration of ethylene and fermentation of sugars with yeast. The chemical reaction of hydration of ethylene is: C2H4 (g) + H2O (g)  CH3CH2OH (l) This reaction is performed with excess high pressure steam at 300°C. The catalysts used in the reaction, such as phosphoric acid, are absorbed onto a porous support, such as charcoal (36). The ethanol produced by the hydration method is used by commodity chemical companies while the ethanol used in beverages and fuel is usually produced by the fermentation process. The fermentation process will be discussed further in section 2.11.  Oxygenated substances are added to traditional fuel in order to reduce the carbon monoxide formed during combustion (37). One of the most commonly-used oxygenates is methyl tert-butyl ether (MTBE). As a result of leaking fuel tanks and spills, MTBE is found in groundwater throughout the United States. MTBE is very soluble in water, thus it is very difficult to remove it from groundwater. As a result, the use of MTBE is being banned in most areas of the US. MTBE can be replaced by ethanol, which is a safer alternative. In addition, ethanol can be used as an octane booster, which enhances the gasoline-ethanol fuel performance, as well as being used as a standalone fuel (38). Another benefit of using pure ethanol fuel is that, ideally, it burns particulate-free and combusts cleanly with oxygen to produce carbon dioxide and water (3). In comparing the uses of ethanol fuel and gasoline, it is found that less carbon monoxide is emitted when using ethanol (39). Thus, ethanol fuel is an improved source of energy.    9  2.4 Conventional fermentation process for ethanol production  As previously mentioned, bioethanol production has grown dramatically over the past decade. The two most practiced and mature bioethanol production technologies are: the yeast-catalyzed production of ethanol from sucrose in sugarcane, and from corn.  Producing ethanol from sugarcane involves two major steps. First, the sugarcane stalks are squeezed to produce a juice that contains sucrose. The fiber that is left is called bagasse and is used as fuel for the process. The sucrose juice is then fermented into ethanol.  To produce ethanol from corn, two processes – dry and wet milling (figure 4 & 5), are employed. In the dry milling process, the corn is ground and cooked. Then, amylases are added to break starch into simple sugars, which are then fermented into a dilute beer. It is then distilled to produce pure ethanol and a high protein product. The high protein product is centrifuged and dried, and may be sold as an animal feed. Wet milling separates corn kernels by steeping, milling, and centrifuging. The resulting starch is converted into ethanol by the same method as in the dry milling process. Wet milling plants are generally bigger than dry milling, and are coal- powered. This imposes a negative impact on the environment, thus these plants are looking for alternative fuels, such as biogas from landfills, as their operational fuel to reduce this negative effect on the environment (11).   10   Figure 4   Block flow diagram for a corn dry mill (40)  Figure 5   Block flow diagram for a corn wet mill complex (40)  11  2.5 Alternative processes and substrates for ethanol production  Due to the increasing world population, it is foreseeable that the demand for food will continue to escalate. Recently, it has become an issue of public concern to use food sources, such as corn and sugarcane, as raw materials for ethanol production (41). Other materials have also been considered by researchers. Lignocellulosic material is the most promising alternative because it has high productivity, which means that there are lower production costs and less environmental consequences from fuel production (11). In addition, this process yields a positive net energy balance and can be nearly carbon neutral, which means there should not be any significant changes in the accumulation of carbon in the atmosphere during the production of bioethanol (11)(42). Two conversion processes, which are anticipated to be the main focus in converting lignocellulosic materials into ethanol, are thermo-chemical and biological conversion (11).  In thermo-chemical conversion, syngas is produced by biomass gasification with a metal catalyst. Syngas, which contains mostly CO and H2, is sent to the catalytic reactor to produce a mixture of alcohols. This mixture of alcohols includes methanol, which is recycled back into the catalytic reactor, ethanol, which is the target product, and higher alcohols, which can be used as solvents. Some studies have shown that the higher alcohols can increase the compatibility of ethanol fuel and gasoline. The National Renewable Energy Laboratory (NREL) claims that about 380 L of ethanol can be produced from every dry metric tonne of biomass by using this process (11).  Although thermo-chemical conversion can produce higher alcohols as a by-product for a more efficient ethanol fuel, extensive syngas cleaning is required to avoid catalyst poisoning. Alternatively, ethanol fermentation by Clostridium ljungdahlii can be used after the biomass gasification to replace the metal catalyst process. The advantages of using ethanol fermentation are high selectivity and less cleaning required. The yield for this process is 290 – 440 L of ethanol produced per dry metric tonne of biomass, which is comparable to the thermo-chemical conversion. In observing both gasification practices, they each have high theoretical yields.  12  However, gasifiers have drawbacks, such as tar formation and the need for gas cleaning. Thus, other methods are being considered for lignocellulosic material conversion (11).  Enzymatic hydrolysis of lignocellulosic material followed by ethanol fermentation has become one of the most popular methods for biological ethanol production, which is the focus of this MASc thesis research, because it has less environmental liabilities and the enzyme cost is expected to be lower as technology matures(43). This process is being practiced by Iogen Corp. (Ottawa, Canada) at a scale of 50 tons feedstock (corn stover) per day. This process involves a chemical or physical pretreatment, such as a dilute acid pretreatment, which removes lignin and hydrolyzes the hemicellulose that blocks the cellulose conversion. After the pretreatment, cellulase enzymes are used to break cellulose into simple sugars – pentoses and hexoses. The sugars then undergo fermentation. Recent research has focused on using genetically-modified organisms for converting both pentose and hexose into ethanol, since no natural organism has the capability to convert both sugars efficiently(11). The lignin residue is used to provide energy for the process. The resulting yield of this method is about 255 – 340 L of ethanol per dry metric tonne of cellulosic biomass (11). It has been discovered that the energy ratio of the life-cycle of cellulosic ethanol manufacturing is 10.97 (table 3), which means that the ethanol produces 997% more energy than the amount of fossil fuel energy it consumes. This energy ratio is evidence of the environmental benefits of this conversion technology (11).   13  13  Table 3   Energy inputs and outputs for the life-cycle analysis of cellulosic ethanol manufacturing technologies (values are in kJ/L and the crop processed is assumed to be hybrid poplar – 11 dry metric tonnes/ha/year)(11)  Biomass gasification / metal catalysis (BG/MC) Biomass gasification / ethOH fermentation (BG/EF) Enzymatic saccharification / ethOH fermentation (base) (ES/EF-B) Enzymatic saccharification / ethOH fermentation (advanced) (ES/EF-A) Enzymatic saccharification / acetic acid fermentation (AAF/CP) Cellulosic mixed- acid fermentation (MAF/CP) Energy source for conversion Incoming biomass Incoming biomass Undigested residue Undigested residue Undigested residue Undigested residue Fossil fuels input      Crop production 1,003 1,110 1,139 909 735 723      Crop transport 1,098 1,215 1,246 994 804 791      Other feedstocks 0 0 0 0 0 0      Alcohol conversion 0+ 0++ 0‡ 0‡ 0‡ 0‡‡      Alcohol distribution 443δ 443 443 443 443 443δ      Subtotal 2,543 2,768 2,828 2,346 1,982 1,956 Co-product credits      Process co-products 0 0 0 0 0 0      Other fuel or power export 0 1,915 2,186 2,341 0 0      Subtotal 0 1,915 2,186 2,341 0 0 Net energy value 20,858 22,548 22,759 23,396 21,419 21,445 Energy ratio 9.2 9.15 9.05 10.97 11.81 11.96 Values are in kJ/L of equivalent ethanol on an energy basis because the product is mixed alcohols. + NEV and ER are a little overestimated as chemicals and catalyst consumption is not included. ++ It is assumed that no fossil-fuel derived nutrients are needed. This is close, as only a small amount of nutrients is needed because cells are recycled. ‡ It is assumed that no fossil-fuel derived nutrients are needed. This could be true, or it could be small, if a natural and clean source of nutrients is used. ‡‡ No fossil-fuel derived nutrients are needed as cells are recycled and a nutrient-rich biomass (e.g. manure, sewage sludge) may be employed. δValue for distribution of mixed alcohols will actually be lower, as studies show that pipeline shipping might be feasible, even as gasoline blends.   14  2.6 Lignocellulosic biomass  As previously stated, urban sources of lignocellulosic biomass are the raw materials used in this ethanol conversion project. Therefore, it is important to understand the structure of the various forms of lignocellulosic biomass. Lignocellulosic biomass consists of celluloses (figure 6), hemicelluloses (figure 7) and lignin (figure 8). Hydrogen and covalent bonds bind celluloses and hemicelluloses to lignin, which limits the accessibility of the cellulose to enzymes. As a result, pretreatment is typically required to partially remove lignin and to render the cellulose amenable to hydrolysis (44).   Figure 6   Structure of cellulose (45)  Figure 7   Structure of hemicellulose (46)  15    Figure 8   Main components of lignin (47)   To liberate monomer, fermentable sugars from cellulose and hemicellulose, hydrolysis is required. In the process of enzymatic hydrolysis, -1,4-glucanase breaks the cellulose into cellobiose, which is then broken down by -glucosidase into glucose (figure 9). Studies have shown that the kinetics of the hydrolysis can be modeled by using the Michaelis-Menten rate expressions (48).  16   Figure 9   Schematic of enzyme attack on cellulose (49)  In addition to enzymatic hydrolysis, dilute acid can also be used to catalyze the hydrolysis of cellulose. In acid hydrolysis, the protons in acid start forming conjugate acids with the glycosidic oxygen, which links the two sugar units. Then, the separation of C-O bond and the breakdown of the conjugated acid take place. Water is added to the cyclic carbonium ion forming a monosaccharide and a proton (50). The disadvantage of acid hydrolysis is that chemicals, such as furfural, hydroxymethylfurfural, and acetate will be produced during the hydrolysis. They are inhibitory towards ethanol production. Therefore, enzymatic hydrolysis will be used in this project.  2.7 Urban cellulosic substrates  Previous works have shown urban cellulosic wastes are capable of producing bioethanol(51)(52). Li et al. (51) used five different municipal solid wastes – carrot peelings, potato peelings, grass, newspaper and scrap paper. Among all substrates, scrap paper has highest cellulose content, which is 67.07 wt%. Different pretreatment methods were investigated, which are dilute acid, steam treatment, microwave treatment or a combination of any of two of them. They were conducted prior to enzymatic hydrolysis that hydrolyzed pretreated substrate with cellulase (10 and 60 FPU/g substrate). After hydrolysis, glucose yield obtained from different pretreatment and hydrolysis conditions were compared. It was found that the highest glucose yields were achieved by pretreating the substrates using H2SO4 (4%), followed by steam treatment and grass has the highest glucose yield (62%) among all substrates that were pretreated at this  17  condition(51). This proves that many of the urban cellulosic wastes have potential for producing ethanol. The four substrates that were being evaluated in this project are switchgrass (standard), grass, cardboard and paper mill clarifier sludge.  2.7.1 Switchgrass  Switchgrass, also known as Panicum virgatum or tall panic grass, is an endogenous perennial grass that was once abundant before European settlers arrived in North America. As more lands were needed for homes and productive plants, such as corn and wheat, the switchgrass was replaced. However, switchgrass has recently attracted interest as a biomass source for ethanol production. This grass has the advantage of being drought-tolerant because it uses a mechanism called C4 fixation (53)(54). Unlike corn, which needs good soils, adequate water, and fertilizer, switchgrass can be grown in all different types of soil, including those that are inappropriate for growing other crops. Moreover, switchgrass does not need re-seeding after harvest and is able to survive for at least ten years, which makes it an ideal substrate for ethanol production (55). According to Samson et al. (56), each hectare of land can produce 10,000 kg of switchgrass or 0.167 million MJ of energy every year. It is concluded that the energy yield is 11 kcal of energy from ethanol production from every kcal of fossil fuel input for its production (8). In addition, Schmer et al. (57) has concluded that switchgrass is capable of producing 5.4 MJ of renewable energy for every MJ of non-renewable energy consumed for cellulosic ethanol production based on 10 farms that have different precipitation and temperature levels. Other research done by U.S. Department of Agriculture has confirmed that about 13.1 MJ of energy is produced from ethanol when one MJ of petroleum is used (52). Since research has proven that switchgrass is a successful substrate for ethanol production, ethanol production from switchgrass is being used as a control for the other three substrates.      18  2.7.2 Grass  The different types of grasses studied as substrate for production of ethanol include plants in the Gramineae (Poaceae), rust (Juncaceae) and sedge (Cyperaceae) families (58). Most species of grasses have about 25 – 40 wt% of cellulose and 35 – 50 wt% of hemicellulose (table 4). The sugar yield (62%) obtained in the Li et al. (51) study have shown contrast to another study that showed enzymatic hydrolysis of three different steam-exploded pure grass species (reed canary grass autumn, reed canary grass spring and barley straw) produces an average sugar yield of 76% (table 5). The differences between yields from these studies are probably due to the difference in the way that yield was calculated. The waste grass mixture study only quantified glucose yield over total carbohydrates, while the pure grass species study utilized total sugar yield over total carbohydrates.  Table 4    Contents of cellulose, hemicellulose and lignin in common agriculture residues and wastes (59) Lignocellulosic materials Cellulose (%) Hemicellulose (%) Lignin (%) Hardwoods stemas 40 - 55 24 - 40 18 - 25 Softwood stems 45 - 50 25 - 35 25 - 35 Nut shells 25 - 30 25 - 30 30 - 40 Corn cobs 45 35 15 Grasses 25 - 40 35 - 50 10 - 30 Paper 85 - 99 0 0 -15 Wheat straw 30 50 15 Sorted refuse 60 20 20 Leaves 15 - 20 80 - 85 0 Cotton seed hairs 80 - 95 5 - 20 0 Newspaper 40 - 55 25 - 40 18 - 30 Waste papers from chemical pulps 60 - 70 10 - 20 5 - 10 Primary wastewater solids 8 - 15 not available 24 - 29 Swine waste 6 28 not available Solid cattle manure 1.6 - 4.7 1.4 - 3.3 2.7 - 5.7 Coastal Bermuda grass 25 35.7 6.4 Switchgrass 45 31.4 12  19  Table 5   Sugar yield in enzymatic hydrolysis of steam exploded washed grasses (Celluclast 1.5L FP 10FPU/g, -glucosidase 10nkat/g*, 45ºC, pH 5, 72 h) (60)  Arabinose (%) Glucose (%) Xylose (%) Xylo-oligo saccharides (%) Total Yield (%) Reed canary grass (autumn) 0.12 65.7 8.7 0.15 75 Reed canary grass (spring) 0.1 71.3 5.9 0.15 77 Barley straw 0.21 64.4 10.9 0.1 76     Average 76 *nkat = nanokatal (amount of enzyme required to raise the reaction rate by 1 nmol/s under defined condition)  2.7.3 Cardboard  Corrugated cardboard boxes, one type of post-consumer paper, are made from corrugated fiberboard, which is usually made from kraft pulping of softwood such as pine. The composition is composed of 59.7±0.1 wt.% cellulose, 13.8±0.2 wt.% hemicellulose, 14.2±0.1 wt.% lignin and 12.3±0.4 wt.% other material (10). Corrugated fiberboards consist of a fluted corrugated sheet that is sandwiched between two flat paperboards, which are approximately 0.3 mm thick (figure 10).  Figure 10   Photograph of corrugated fiberboard  20   Corrugated cardboard boxes are used for packing, storing, and transporting goods to and from factories, offices, and homes. In the corrugated cardboard recycling process, the cardboard boxes are sorted and sent to a hydropulper, where the cardboard is cleaned and processed with water (61). Afterward, the pulp has to be cleaned to remove contaminants, such as wax coatings, plastics, food, garbage, metal fasteners, staples, and nails. This cleaning may also be important for using cardboard as a raw material for the bio-ethanol process since chemicals in the contaminants may reduce the conversion rate (10). One advantage of using cardboard boxes for ethanol conversion is that some lignin, which is one of the main constraints in the enzymatic attack on cellulose, has already been removed in the kraft cooking process when the corrugated cardboard is first made (62). Thus, the enzymatic digestibility is relatively high compared to other untreated raw materials. Alonso et al. (2004) have done a two step saccharification of randomly sampled corrugated cardboards.  They were pretreated with 3% w/w H2SO4 for 180 minutes. The liquor was analyzed and shown that it contained 10 grams of hemicellulosic sugar and 9.2 grams of glucose per liter. The solid phase from the acid pretreatment was then subjected to enzymatic hydrolysis at 48.5 °C, pH 4.85 with a liquor-to-solid ratio of 30 g/g. The resulting solution contained 17.9 g/L glucose, which corresponds to a conversion yield of 63.6% (10). These results are comparable to the yield from grass, as discussed in section 2.7.2. 2.7.4 Paper mill clarifier sludge  In British Columbia, approximately 50 million hectares of land are forest and 96% of these are softwood. Thus, lumber (softwood), pulp, paper and paperboard are the top three products that are being exported(63).  Because of this geographical advantage, many pulp and paper mills are located in British Columbia. Kruger Inc. is one of the major producers of publication papers, tissue, lumber and other wood products, and corrugated cartons from recycled fibers (64). In this project, a sample of paper mill clarifier sludge was collected from Kruger Inc., New Westminster, British Columbia. It is assumed that Kruger Inc. uses softwood as the feed and since the composition of the pulp mill clarifier sludge varies depending on the feed used in the pulp and paper mill, it is assumed that the composition of the pulp mill clarifier sludge is similar to the softwood. It contains 43 – 45% (w/w) cellulose, 20 – 23% (w/w) hemicelluloses and 28%  21  (w/w) lignin. According to studies done by Galbe and Zacchi, softwood is generally harder to hydrolyze than hardwood or other agriculture residues (65).  2.8 Pretreatment of lignocellulosic materials  There are different kinds of pretreatment processes being evaluated as a means of enhancing sugar yield in enzymatic hydrolysis. Some of the examples are dilute acid pretreatment (66), steam explosion (51)(59)(66), ammonia fiber explosion (59), organosolv (59) and oxygen delignification (62)(67)(68).  Dilute acid pretreatment uses acid, namely sulphuric acid (H2SO4), nitric acid (HNO3), and hydrochloric acid (HCl), as a pre-hydrolysis treatment. The conversion is performed at around 110 – 220°C with an acid concentration ranging from 0.3 – 1.1%. In this treatment, most of the hemicelluloses are removed from the raw materials. In addition, the dilute acid can increase the surface area of the materials for enzymatic hydrolysis. If the temperature for pretreatment is too high, furfurals may be produced, which can inhibit ethanol production (66).  Steam explosion, also known as steam treatment, has the simplest setup among all the evaluated pretreatment processes. It treats cellulosic substrates with steam at 160 – 260°C and 0.69 – 4.83 MPa for between 30 seconds to 20 minutes. The substrate is then taken out from the reactor  and exposed to atmospheric pressure afterward. The high temperature causes decomposition of both hemicellulose and lignin (59)(66). It can also be done with an autoclave at an initial temperature of 121°C for 15 minutes. The pressure is then rapidly reduced to atmospheric (51). The shortcoming of this pretreatment is the high energy requirement (51)(66). In addition, steam explosion has less effect on softwood than hardwood due to its rigid structure and higher lignin content (65).   22  Ammonia fiber explosion (AFEX) is a pretreatment similar to steam explosion, which also utilizes moderate temperature (90°C) and high pressure. The only difference is that lignocellulosic material is exposed to 1 – 2 kg of liquid ammonia per kilogram of dry biomass for 30 minutes. Afterward, the pressure is reduced immediately. When compared to dilute acid pretreatment, the solubility of hemicellulose with AFEX is low when raw material with high lignin content is treated, such as newspaper (18-30%) and aspen chips (25% lignin). Thus, the compositions of the sugars in the materials stay almost the same. In addition, the advantage of AFEX is that hemicellulose is not converted to inhibitors for the downstream fermentation process (59).  In the organosolv process, organic solvents such as ethanol, acetone, methanol and ethylene glycol are used to remove lignin. It is reported that organic solvent mixtures that contain acid catalysts such as HCl or H2SO4 can also be used to breakdown lignin, but acid catalysts are not essential at temperatures above 185°C. All of the solvents used in the organosolv process must be regenerated by distillation in order to improve the process economics and to minimize inhibition in the enzymatic hydrolysis and fermentation steps that comes after the pre-treatment (59). Although the pretreatments mentioned above are being widely studied, the pretreatment used in this project is oxygen delignification.  2.9 Oxygen delignification  Oxygen delignification, also known as oxygen bleaching or prebleaching, is a widely used process in the pulping industry (62). Although this pretreatment may not be the most effective, it is the only pretreatment technique that has been applied to lignocellulosic feedstocks on a full scale. Thus, this process has been chosen as the pretreatment for this project.   23  In the pulping industry, oxygen delignification is used as a pre-bleaching step and further delignifies the wood pulp (69). Figure 11 shows a simple schematic of the oxygen delignification process in the pulping industry.   Figure 11   Oxygen delignification in pulping industry  In the oxygen delignification process, high pressure oxygen (150 psi) is used under alkaline (5 – 6% caustic) conditions. Since it can be used as a pre-bleaching process, the use of downstream bleaching chemicals may be reduced. Therefore, chemical costs can be minimized in pulp mills (70).  During oxygen delignification, the raw materials being treated are placed in a pressurized vessel with oxygen in an alkaline environment. The temperature inside the vessel is increased and stabilized at around 150°C. A study regarding high selectivity oxygen delignification has shown  24  that this pretreatment can remove 35–50% of lignin from brownstock pulp. The reactions in oxygen delignification are shown in figure 12 and 13. Oxygen free radicals attack the electron rich substrate. These reactions increase the solubility of the lignin, which is the ultimate goal of this pretreatment. Details of the reactions are explained in the high selectivity oxygen delignification project technical report (70).  Figure 12   Initial reactions in oxygen delignification (70)  25   Figure 13   Reactions of hydroperoxide intermediates (70)  Oxygen delignification has attracted more interest than when it was first developed in the 1970’s due to increased environmental concerns. Since the liquor from the oxygen delignification operation can be recycled into the kraft chemical recovery system, the pulp mill pollution problem can be reduced (62). Although oxygen delignification has good selectivity toward  26  lignin, further delignification is not possible because degradation of cellulose will occur, which will affect the pulp strength (62).  Many studies have proven the potential for oxygen delignification as pretreatment prior to hydrolysis. In Martin et al. study, oxygen delignification has shown better hydrolysability of sugarcane bagasse (57.4%) than steam explosion (48.9%). This pretreatment method can also increase the cellulose convertibility four times for corn stover when compared to the untreated one. The hydrolysability of softwood can also be enhanced by oxygen delignification. By using oxygen delignification at 200°C for 10 minutes, 79% of original carbohydrates is hydrolyzed.  2.10 Overview of hydrolysis process  Hydrolysis is a reaction between a chemical and water. A hydrolysis reaction that is involved in hydrolysis is the hydrolyzation of the cellobiose (71): (C6H10O5)n + nH2O ↔ nC6H12O6 In this project, hydrolysis is used to break down polysaccharides, such as cellulose, into monosaccharides, such as glucose, in the presence of water. The monosaccharides will be used by yeast during subsequent fermentation.  There are two common hydrolysis methods: acid hydrolysis and enzymatic hydrolysis. Acid hydrolysis is a traditional method that converts the cellulose into glucose using mineral acids. It can be performed at two different acid concentrations: concentrated acid and dilute acid. Each of the concentrations is performed under different conditions. Concentrated acid is used to react with cellulose at low temperature (about 37.8°C) and atmospheric pressure, while dilute acid is reacted at higher temperature (about 215°C) and pressure (10 – 35 bar) (72) (73) (74). The drawbacks of acid hydrolysis are the toxic products that are formed, which can inhibit the  27  fermentation process. As well, the hydrolysate requires neutralization after the hydrolysis process to allow efficient downstream fermentation (74) (43). Due to the extra cost imposed for neutralization of the product, the separation and recycling of the acid, acid hydrolysis was not chosen for this project.  In this project, enzymatic hydrolysis is used for the conversion of cellulose for ethanol production. Enzymatic hydrolysis employs cellulase enzymes to catalyze the hydrolysis of polysaccharide (75). In the process, cellulase enzymes are used under mild conditions (50°C and pH 5). Thus, no byproducts will be generated to hinder the enzyme activity or fermentation process, which is an advantage over acid hydrolysis (74). Another advantage is that enzymatic hydrolysis requires less energy than acid hydrolysis because of the mild operating conditions. The only drawback of enzymatic hydrolysis is the relatively high cost of the enzymes. However, many enzyme companies have invested significant time and money to find ways to reduce enzyme cost through mass production (74). For instance, Novozymes and the National Renewable Energy Laboratoy (NREL) have reduced the cost of enzyme to $0.01–0.18/gal of ethanol, which is a 30-fold reduction between 2001 and 2005 (76). Thus, it is anticipated that the enzyme price will drop to a more competitive level in the near future. In this project, two commercial enzyme preparations are chosen. They are Novozyme and Celluclast, which were obtained from Novozymes A/S (Bagsvaerd, Denmark).  2.11 Fermentation process  Fermentation is one process by which microorganisms can obtain energy through the oxidation of carbohydrates (77). This process is commonly used in the food processing, wine making, and beer brewing industries (78). In all of these processes, yeast is used anaerobically to oxidize the sugars, such as glucose, fructose and sucrose, into ethanol (79) (80). The general chemical equation is (80): C6H12O6 → 2 C2H5OH + 2 CO2  28  2.11.1 Types of yeast  Yeasts are unicellular eukaryotic microorganisms that belong to the fungi kingdom, and 1500 species of yeast have been identified (81). They can inhabit diverse environments, from aerial to aquatic. Because of this diversity, the metabolisms of yeasts are complicated, especially in light of the presence of different carbon and energy sources (82). According to the type of metabolic processes the yeasts utilize, they can be categorized into non-, facultative- or obligate- fermentative yeasts. Non-fermentative yeasts exhibit only respiratory metabolism, which takes place in the presence of oxygen. Thus, they cannot produce alcohol anaerobically. On the other hand, obligate-fermentative yeasts only go through the fermentative metabolism that occurs in the absence of oxygen. Facultative-fermentative yeasts comprise most of the identified yeast population. Depending on the availability of sugars and oxygen, they are capable of both respiratory or fermentative or a mixture of both metabolisms (82). The yeast that is used in this experiment is S. cerevisiae. The yeast strain of S. cerevisiae that is being investigated is K1. Strain K1 is a robust industrial yeast (83). S. cerevisiae is known to be facultative-fermentative yeast (82). Thus, it can undergo respiratory and fermentative metabolisms.  2.11.2 Glucose metabolism  Glucose (or other carbohydrates) is the most important carbon and energy source for organisms. Glucose metabolism for organisms can be classified into two main pathways – aerobic respiration and anaerobic metabolism. Glucose catabolism is divided into the Embden- Meyerhof-Parnas (EMP) pathway and the Krebs or tricarboxylic acid (TCA) cycle (84).  In the first step of glucose metabolism, the EMP pathway (also known as glycolysis), each glucose molecule is broken down into two pyruvate molecules. The reaction begins with a molecule of glucose, which is then phosphorylated into glucose-6-phosphate (G-6P) by  29  hexokinase and adenosine triphosphate (ATP). The G-6P is transformed into fructose-6- phosphate (F-6P) by phosphoglucose isomerise. F-6P is converted to fructose 1,6-diphosphate by phosphofructokinase with the help of ATP molecules. By using aldolase, fructose 1,6- diphosphate is converted into dihydroxyactone phosphate (DHAP) and glyceraldehyde-3- phosphate (GA-3P), which are in equilibrium. After this critical step, all subsequent reactions occur twice before glycolysis is ended. As DHAP and GA-3P are in equilibrium, GA-3P is utilized for producing 1,3-diphosphoglycerate (1,3-dP-GA) with an inorganic phosphate (Pi) and glyceraldehyde-3-phosphate dehydrogenase. Then 1,3-dP-GA becomes 3-phosphoglycerate (3P- GA). 1,3-dP-GA will release a phosphate group simultaneously, which is added to ADP to form ATP. 3P-GA, with the help of phosphoglyceromutase, will be converted into 2-phosphoglycerate (2P-GA), which will subsequently dehydrate into phosphenol pyruvate (PEP) by using enolase. Phosphenol pyruvate will finally be dephosphorylated into pyruvate (Pyr) by pyruvate kinase with the generation of ATP. The overall glycolysis reaction is: glucose + 2 ADP + 2 NAD+ + 2 Pi  2 pyruvate + 2 ATP + 2 (NADH + H+) with NAD+ is nicotinamide adenine dinucleotide and NADH is its reduced form.  Glycolysis occurs in both aerobic respiration and anaerobic metabolism. However, pyruvate will react differently under different conditions.  Under aerobic conditions, CO2 and NADH are produced from pyruvate after the TCA cycle, which provides electrons (NADH) for the electron transport chain and biosynthesis, supplies carbon skeletons for amino acid synthesis and generates energy. Prior to the TCA cycle, coenzyme-A is acylated by pyruvate. pyruvate + NAD+ + CoA-SH  acetyl CoA + CO2 + NADH + H+ With pyruvate dehydrogenase, acetyl CoA, CO2 and NADH are produced. Acetyl CoA is then used in the TCA cycle. The overall reaction of the TCA cycle is: acetyl-CoA + 3 NAD+ + FAD + GDP + Pi + 2 H2O  CoA + 3 (NADH + H+) + FADH2 + GTP + 2 CO2  30  Where FAD is flavin adenine dinucleotide, which is reduced to FADH2, and GDP is guanosine diphosphate, which is converted to Guanosine triphosphate by using pyruvate kinase and phosphoenolpyruvate. After TCA cycle, electron transport chain or oxidative phosphorylation takes place. The major role of the electron transport chain is to regenerate the NADs for glycolysis and ATP for biosynthesis.  On the other hand, pyruvate can be converted into lactic acid, ethanol or other products, such as acetone, butanol, and acetic acid when the yeast is grown under anaerobic conditions. Fermentation refers to energy generation without the electron transport chain under anaerobic conditions. The target product in this project is ethanol. After the glycolysis step that is shared by both aerobic and anaerobic conditions, pyruvate is converted into acetaldehyde and carbon dioxide by pyruvate decarboxylase. With the help of alcohol dehydrogenase, acetaldehyde is then transformed into our final product, ethanol (84).  31  3.0 RESEARCH OBJECTIVES  The objective of this project is to determine the potential of producing bioethanol using urban cellulosic wastes. In order to evaluate the potential, the processes include:  Obtain lignocellulosic wastes  Perform oxygen delignification and hydrolysis on substrates  Ferment hydrolysate with yeast strain, S. cerevisiae K1  Compare sugar and ethanol yields at different conditions  Evaluate the feasibility to use urban cellulosic wastes as feedstock for a full scale ethanol plant economically    32  4.0 MATERIALS AND METHODS  This project aimed to evaluate the ethanol yield which could be produced from different substrates that were readily available in the Metro Vancouver area.  As part of this overall objective, a number of steps were carried out, including; size reduction of the biomass samples, determination of moisture content and individual components, pretreatment of samples using a process called oxygen delignification, hydrolysis of the pretreated substrates, and fermentation of the resultant hydrolysates (Figure 14).  Figure 14   Flow diagram for producing ethanol from urban cellulosic wastes  4.1 Substrate sources and preparation  In this experiment, four substrates were evaluated: switchgrass (control), grass clippings, cardboard and paper mill clarifier sludge. The switchgrass variety was Cave Rock, which was grown and harvested in Manitoba 2007, and stored in a plastic container at a moisture content of 6 – 8%. It was then transferred to the laboratory and stored in Ziploc bags at room temperature. Grass clippings were harvested from a typical household backyard in Richmond BC during March 2008. The grass was harvested by cutting approximately 3 – 4 cm blades using a scissors. The grass was stored in plastic Ziploc bags at 4ºC.  Cardboard boxes were obtained from a dumpster and cut into 1 inch x 1 inch (2.5 cm × 2.5 cm) pieces. They were re-pulped in an agitated batch reactor at 2% consistency with hot tap water for approximately 15 minutes in order to ensure uniform moisture content and particle size throughout the entire old corrugated Substrate Preparation Oxygen Delignification Enzymatic Hydrolysis Fermentation  33  cardboard batch. The paper mill clarifier sludge was acquired from the primary clarifier in a liquid waste water treatment plant from Kruger paper mill. Samples of switchgrass and grass clippings underwent size reduction prior to oxygen delignification.  In both cases, they were processed at room temperature using a Cuisinart Mini- prep Food Processor (figure 15). The size of switchgrass fragments varied from 1 – 5 centimeters in length while grass samples range from two to four centimeters. After 5 minutes of grinding, switchgrass and grass had approximately uniform size of one and two centimeter(s) in length, respectively. The switchgrass and grass samples (before and after grinding) are shown in figure 16 and 17 respectively.   Figure 15   Cuisinart mini-prep food processor   Figure 16   Switchgrass before (left) and after size reduction  34    Figure 17   Grass before (left) and after size reduction  The dry weight of each substrate was determined and was used in subsequent calculations. In order to do this, wet substrate was placed in an aluminum dish. The aluminum dish and the wet substrate were weighed and the weight recorded. After drying in the oven at 105°C overnight, the weight of the dry substrate along with the aluminum dish was measured. By subtracting the dry substrate weight from wet substrate weight, the weight ratio between the wet and dry substrate could be determined and is shown in table 6. Table 6   Wet to dry weight ratio for different substrates Substrate Moisture Content (%) Wet to Dry Weight Ratio (g/g) Switchgrass 14.5 1.1695 Grass 57.7 2.3619 Cardboard 6.7 1.0723 Re-pulped Cardboard 79.9 4.9869 Paper Mill Clarifier Sludge 85.9 7.1153  35  4.2 Determination of cellulose, hemicellulose and lignin  The goal of analyzing the substrate composition was to accurately quantify the sugars and lignin. In this analysis, an acid hydrolysis procedure is used because it could achieve close to 100% sugar conversion in a shorter period of time than enzymatic hydrolysis. After the acid hydrolysis, the hydrolysate was analyzed with the HPLC to determine the sugar concentrations. It was assumed that that all pentose sugars were hydrolyzed from hemicellulose while hexose sugars were derived from cellulose (±10%) (73).  The composition of the substrates was determined using the NREL protocol that was published in 2008 (85). The substrate was initially dried to 10 wt% at 105°C in a drying oven (VWR, model #1350FM). Then, 150 mg dry weight of each substrate was dispensed into a 50 mL conical-bottom Pyrex glass test tube. Then, 1.5 mL of 72% sulfuric acid was pipetted into each tube. The contents of each tube was mixed with a glass stirring rod for one minute, then placed in a water bath for 60 minutes at 30°C. During this hour the contents of each tube were stirred every five to ten minutes. After 1 hour, 42 mL of deionized water was used to dilute the acid to 4%. The contents of the tubes were transferred into 150 mL serum vials which were sealed with butyl rubber stoppers and crimped aluminum caps. The serum vials were then placed in an oil bath that had been stabilized at 121°C inside a drying oven (figure 18). After one hour, the serum vials were cooled to room temperature in a fume hood, and the contents of each tube were vacuum filtered using a pre-weighed KIMAX 30 mL-30M crucible filter. The filtrate was collected and analyzed for sugars. The crucibles were weighed again and then placed in a 105°C oven overnight and reweighed to determine the acid insoluble lignin content. The crucibles were then placed in a muffle furnace at 575°C (Thermo Scientific, Type FB1300, figure 19) for 24 hours to determine the ash content.  36   Figure 18   VWR model #1350FM drying oven   Figure 19   Muffle furnace (Thermo Scientific, FB1300)     37  4.3 Oxygen delignification  Oxygen delignification was used in this project as a pretreatment prior to hydrolysis of lignocellulosic material. The effectiveness of the pretreatment was evaluated in three ways: (1) lignin removal, (2) increase in sugar yield in the hydrolysis and (3) yield of product. Each oxygen delignification run was carried out at 2 wt%. To prepare for the run, 10 grams dry weight of wet substrate was dispensed into a tared 500 mL Teflon cylinder liner. Approximately 300 mL of distilled water was then dispensed into the cylinder. The appropriate amount of 50% sodium hydroxide was added in order to achieve the desired caustic loading.  Finally, the weight of the contents was brought up to 500 g using distilled water.  The Teflon cylinder was placed inside the Parr high pressure reactor unit (Figure 20). The head was placed on the reactor and then secured using the 6 hexagonal machine screws, and the cylindrical safety ring. The removable head unit consists of a stirrer, gas release valve, gas inlet valve, pressure gauge and thermal well (Figure 21).   Figure 20   Oxygen delignification apparatus  38   Figure 21   Front, side and top views of movable head unit of Parr reactor (86)  The stirrer was set at 100 rpm using the Parr 4843 electronic controller in order to mix the contents of the reactor evenly during oxygen delignification. Once the reactor was assembled, the heater was started, and the thermocouple which was connected to the temperature control unit was placed in the thermowell on the head of the reactor. This allowed feedback control of temperature in the reactor.  To begin a run, the quick connect fitting on the gas inlet valve was connected to a nitrogen cylinder (Ultra High purity 5.0). Both the gas release valve and the inlet valve were opened. The needle valve on the outlet line from the nitrogen cylinder was slowly opened in order to allow a flow of nitrogen gas into the reactor. The reactor was sparged at 150 psi (1034 kPa) for 5 minutes in order to remove free oxygen from the reactor. At the end of the sparging period, the gas outlet valve and the gas inlet valve were closed with the nitrogen supply turned off. The reactor was then allowed to heat to the desired reaction temperature, at which point oxygen was introduced into the reactor. To do this, the oxygen cylinder was connected to the quick connect fitting on the gas inlet valve of the reactor. Care must be taken to ensure that the second stage  39  pressure from the oxygen regulator exceeded that in the reactor so that backflow did not occur. Once the oxygen cylinder was connected and the needle valve on the outlet line from the cylinder opened, the inlet gas valve to the reactor was opened. Then, the outlet gas valve from the reactor was slowly opened to allow oxygen to flow into the reactor.  The pressure of oxygen through the reactor was adjusted manually to approximately 150 psi. The reaction was allowed to proceed for 1 hour, after which the inlet and outlet valves were closed, the oxygen supply disconnected and the entire reactor was removed from the holder and placed in an ice bath. Once the reactor temperature cooled to below 70ºC, the outlet valve was slowly opened to allow the reactor pressure to equilibrate with the atmosphere. After the reactor cooled to approximately 40°C, and the pressure decreased to atmospheric pressure, the reactor was disassembled. A small sample (3 mL) of the liquor in the reactor was removed with a pipette to quantify sugar that had been lost into the supernatant and the solids were then captured by suction filtration in a Buchner funnel (water aspiration) using Whatman paper No.1, diameter 110 mm. The pretreated substrate was washed thoroughly with approximately 3 litres of distilled water and then removed from the pad, sampled for moisture content, and stored in a Ziploc bag at -20°C for subsequent enzymatic hydrolysis.  4.4 Enzymatic hydrolysis  The objective of enzymatic hydrolysis was to break down cellulose and hemicellulose into simple sugars by using enzymes. Commercial enzymes were used in the hydrolysis of the pretreated substrates. The enzymes were obtained from Novozymes A/S (Bagsvaerd, Denmark), and were supplied as liquids called Celluclast 1.5 L (primarily endocellulase and cellobiohydrolase) and Novozyme 188 (primarily beta-glucosidase).  4.4.1 Procedures for cellulase activity determination  To measure the ability of an enzyme mixture to degrade cellulose, an assay called the filter paper assay was used. This was a test protocol first developed by Mandels and Webber (87) and subsequently refined by the National Research Energy Laboratory (NREL) (88). Briefly, this test  40  measured the ability to hydrolyze cellulose in the form of a 50 mg strip of Whatman Number 1 filter paper, and thereby release reducing sugars. The reducing sugars were quantified through a reaction with dinitrosalicylic acid (DNS) to form a coloured solution. The intensity of the colour corresponded to the amount of sugar formed and was quantified at 540 µm using a spectrophotometer. One filter paper unit (FPU) was defined as the amount of enzyme that releases 2 mg of sugar from a 50 mg strip of filter paper under the assay conditions.  Before initiating the assay, the enzyme assay solutions, DNS reagent (89), 50 mM sodium acetate and citrate buffer were prepared. Then, five different enzyme dilutions and four different glucose standard solutions were prepared (Table 7, 8).  Table 7   Enzyme dilution used in filter paper assay  Citrate Buffer (L) 1:20 Enzyme (L) Concentration of Enzyme (L/L) Enzyme Dilution 1 1650 350 0.00875 Enzyme Dilution 2 1700 300 0.00750 Enzyme Dilution 3 1800 200 0.00500 Enzyme Dilution 4 1850 150 0.00375 Enzyme Dilution 5 1900 100 0.00250  Table 8   Glucose dilution used for standard   Citrate Buffer (mL) 10 mg/mL Glucose (mL) Glucose Concentration (mg/mL) Glucose Standard 1 0.5 1.0 6.7 Glucose Standard 2 1.0 1.0 5.0 Glucose Standard 3 2.0 1.0 3.3 Glucose Standard 4 4.0 1.0 2.0  After appropriate dilutions were made, enzyme assay test tubes are prepared as shown in table 9. For each enzyme dilution, three test tubes were prepared containing 1 mL of 50 mM acetate buffer with a pH of 4.8, and a 50 mg strip of Whatman No. 1 filter paper. The test tubes were then capped and placed in a water bath at 50oC for ten minutes to equilibrate to the reaction temperature. A 0.5 mL sample of the diluted Celluclast solution (Table 7) was then added to the  41  appropriate test tubes and the tubes were incubated for one hour at 50 oC.  Enzyme (tube 7, 10, 13, 16 and 19, table 9) and substrate blanks (tube 2, table 9) were also included in this assay. After one hour, 3 mL of Dinitrosalicylic Acid (DNS) reagent was added to each test tube and the tube contents were gently stirred to stop the reaction. All test tubes were then placed in a boiling water bath for exactly five minutes to allow the DNS to react with the reducing sugars. A 20 mL volume of deionized water was then added to each tube and the absorbance was measured at 540 nm to determine the concentration of reducing sugars present in the solution. The reducing sugar concentration was determined from a standard graph relating the mass of glucose in solution to the absorbance. The sugar released was then plotted against the enzyme concentration used in each assay tube, which was used to find the FPU.  Table 9   Test tubes setup for filter paper assay Test Tube Acetate Buffer Filter Paper Corresponding Enzyme Dilution Corresponding Glucose Standard 1 Reagent Blank 1.5 mL 2 Substrate Control 1.5 mL  3 Glucose Standard 1 1.0 mL     0.5 mL 4 Glucose Standard 2 1.0 mL     0.5 mL 5 Glucose Standard 3 1.0 mL     0.5 mL 6 Glucose Standard 4 1.0 mL     0.5 mL 7 [E1] Control 1.0 mL   0.5 mL 8 [E1] Assay 1 1.0 mL  0.5 mL 9 [E1] Assay 2 1.0 mL  0.5 mL 10 [E2] Control 1.0 mL   0.5 mL 11 [E2] Assay 1 1.0 mL  0.5 mL 12 [E2] Assay 2 1.0 mL  0.5 mL 13 [E3] Control 1.0 mL   0.5 mL 14 [E3] Assay 1 1.0 mL  0.5 mL 15 [E3] Assay 2 1.0 mL  0.5 mL 16 [E4] Control 1.0 mL   0.5 mL 17 [E4] Assay 1 1.0 mL  0.5 mL 18 [E4] Assay 2 1.0 mL  0.5 mL 19 [E5] Control 1.0 mL   0.5 mL 20 [E5] Assay 1 1.0 mL  0.5 mL 21 [E5] Assay 2 1.0 mL  0.5 mL   42   The absorbance of the substrate control, glucose standard and enzyme assay tubes were measured at 540 nm, which were shown in table 10.  From this table, a glucose concentration versus absorbance could be constructed, which was shown as figure 22. By using this concentration figure, the average glucose released from each enzyme sample could be calculated and plotted in figure 23. Since FPU unit was represented by the value of 2.0 mg of reducing sugar as glucose from 50 mg of filter paper in 60 minutes, a line was drawn between the two points that were closest. The equation y = 0.0023x – 0.0003 was obtained. Thus, the enzyme dilution that yields 2.0 mg per 0.5 mL of original enzyme was 0.0043. Since  and filter paper unit was defined as:  The filter paper activity for Celluclast was 86.05 FPU/mL.            43  Table 10   Absorbance of controls, standards and samples measured at 540 nm   OD1 OD2 OD3 Average Substrate Control 0.0267 0.0274 0.0274 0.0272 Glucose Standard 1 0.7536 0.6963 0.6972 0.7157 Glucose Standard 2 0.5323 0.5447 0.5449 0.5406 Glucose Standard 3 0.3554 0.3683 0.3682 0.3640 Glucose Standard 4 0.2133 0.2151 0.2155 0.2146 [E1] Control 0.0010 0.0009 0.0009 0.0009 [E1] Assay 1 0.6227 0.6508 0.6508 0.6414 [E1] Assay 2 0.6607 0.6785 0.6785 0.6726 [E2] Control 0.0038 0.0020 0.0020 0.0026 [E2] Assay 1 0.5777 0.5901 0.5860 0.5846 [E2] Assay 2 0.5735 0.5715 0.5707 0.5719 [E3] Control 0.0001 0.0009 0.0020 0.0010 [E3] Assay 1 0.5798 0.5413 0.5390 0.5534 [E3] Assay 2 0.4983 0.4904 0.5055 0.4981 [E4] Control 0.0031 0.0016 0.0016 0.0021 [E4] Assay 1 0.4355 0.4372 0.4385 0.4371 [E4] Assay 2 0.3807 0.3877 0.3868 0.3851 [E5] Control 0.0012 0.0037 0.0008 0.0019 [E5] Assay 1 0.2658 0.2645 0.2640 0.2648 [E5] Assay 2 0.2734 0.2591 0.2595 0.2640    Figure 22   Determination of glucose concentration y = 4.707x - 0.048 R² = 0.999 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0.0 0.2 0.4 0.6 0.8 C on ce nt ra tio n (m g/ 0. 5m L ) Absorbance at 540nm  44   Figure 23   Filter paper unit determination for cellulase activity  4.4.2 Procedures for -glucosidase assay  Beta-glucosidase is an enzyme that cleaves the -glucoside bond in cellobiose to form two glucose units. Celluclast, like most commercial celluloses, had some -glucosidase activity, however supplemental -glucosidase was required to achieve optimum activity. In order to assay  -glucosidase activity, the ability of the enzyme to cleave the -glucoside bond in a substrate analog, p-Nitrophenyl--D-glucoside, was determined under standardized conditions. Prior to conducting the assay, 50 mM citrate buffer (pH 4.8), 50 mM acetate buffer (pH 4.8) and 0.4 M glycine buffer (pH 10.8) were prepared. Typically, four different enzyme dilutions were used to evaluate the -glucosidase activity (Table 11).   y = 0.002x - 0.000 0.000 0.001 0.002 0.003 0.004 0.005 0.006 0.007 0.008 0.009 0.010 0.0 1.0 2.0 3.0 4.0 En zy m e D ilu tio n Average Glucose Released (mg/0.5mL) Determine for C=2mg/0.5mL Linear (Determine for C=2mg/0.5mL)  45  Table 11   Enzyme dilution used in beta-glucosidase assay   Citrate Buffer (L) 1:20 Enzyme (L) Dilution Factor Enzyme Dilution 1 4980 20 5000 Enzyme Dilution 2 4990 10 10000 Enzyme Dilution 3 4995 5 20000 Enzyme Dilution 4 9995 5 40000  To start the assay, test tubes that contain 1.8 mL of 50 mM acetate buffer (pH 4.8) and 1 mL of 5 mM of p-Nitrophenyl--D-glucoside in 50 mM acetate buffer were immersed in a water bath at 50°C for 10 minutes. Then, 200 L of 4 different dilution of the enzyme preparation (table 11) were added to the appropriate test tubes. Enzyme and substrate blanks, which had no substrate and enzyme in the mixture, respectively, were also included in the assay. The reaction started once the enzymes were in contact with the p-Nitrophenol solution. The reaction was stopped after 30 minutes by the addition of 4 mL of glycine buffer to each test tube. The addition of the glycine buffer caused the solution to change color because the yellow-coloured p-nitrophenyl was released (90). A sample was drawn from each test tube and the absorbance was measured in a spectrophotometer at 430 nm. The concentrations were then determined by a standard graph that correlates the absorbance to micromoles of p-Nitrophenol in solution. Cellobiose unit (CBU) was then determined as the amount of enzyme required to release 1 mol p-Nitrophenol in one minute.  4.4.3 Procedure for enzymatic hydrolysis  For this study, hydrolysis was carried out under a standardized set of conditions. The hydrolysis was performed using a 50 mL suspension of substrate in pH 4.8 acetate buffer in 250 mL Erlenmeyer flasks. The temperature was held at 50°C, shaking speed was set at 150 rpm, and the substrate concentration was either 10 g/L or 50 g/L (Table 12). Prior to the hydrolysis, the flasks containing the substrates, buffer and water were soaked overnight at room temperature. Before  46  the enzymes were added the flasks, the flasks were incubated for 15 minutes at a speed of 150 rpm and 50°C. After the 15 minute equilibration period, appropriate volumes of Celluclast and Novozyme were added to the substrate suspension using a pipettor, in order to achieve the desired enzyme loading (Table 12). The addition of the enzymes marked the commencement of the hydrolysis. The total incubation time was 48 hours. During enzymatic hydrolysis, 1.5 mL samples were drawn at 1, 2, 4, 8, 10, 24, 48 hours. Samples were centrifuged at 9447 × g for 5 minutes, and the supernatant was frozen for subsequent sugar analysis, which was performed with the HPLC.  Table 12   Hydrolysis experimental conditions (91) Condition Dry Substrate Concentration (g/L) Celluclast Activity (FPU/g) 1 10 20 2 10 40 3 50 16  To determine the effect of pretreatment temperature, substrate concentration and enzyme loading on the yield of sugars from hydrolysis of each substrate, a full factorial design was used (table 13). Response (or dependent) parameters included solid yield after oxygen delignification and sugar concentrations (arabinose, galactose, glucose, xylose, and mannose) after hydrolysis.    47  Table 13   Initial factorial design for oxygen delignification and enzymatic hydrolysis  Pretreatment Conditions Run Substrate N/A 100°C 130°C 10 g/L dry substrate, 20 FPU/g 10 g/L dry substrate, 40 FPU/g 50 g/L dry substrate, 16 FPU/g 1 Switchgrass      2     3    4       5      6     7      8      9     10 Grass      11     12    13       14      15     16      17      18     19 Cardboard      20     21    22       23      24     25      26      27     28 Pulp Mill Clarifier Sludge      29     30    31       32      33     34      35      36      48   4.5 Fermentation of hydrolyzed samples  Fermentation trials were conducted in this project to verify the fermentability of the hydrolysates. After the hydrolysates were obtained, they were frozen to be used for fermentation in the future. Two days prior to fermentation, yeast was transferred from petri dish to 50 mL YPG (1% w/w yeast extract, 2% w/w peptone, and 2% w/w glucose) nutrient broth for growth. The culture was incubated at 30°C and 150 rpm. To increase the inoculum size, yeast was transferred from the nutrient broth to another 50 mL fresh YPG media after 24 hours of growth, and allowed to grow for another 24 hours. Prior to fermentation, hydrolysates (30 – 45 mL) were thawed and transferred to 150 mL serum vials. At the same time, yeast broths were centrifuged at 5000 rpm and supernatants were discarded while retaining the yeast pellets. The yeast pellets were washed twice with and re-suspended in 10 mL of sterilized distilled water. Two mL of concentrated yeast suspension was inoculated into each hydrolysate sample. The serum vials were sealed tightly with rubber septa and crimped aluminum seals. Each septum was pierced with two hypodermic syringes. One of the syringes was for liquid sampling. The other syringe had its plunger replaced by foam packing. This syringe allowed CO2 to exit and minimize exposure to oxygen. After inoculation of the serum vials, they were incubated at 30°C and 150 rpm for 48 hours. Two samples (1 mL) of the remaining re-suspended yeast were taken and their dry cell weight and absorbance were measured. This provided the average cell concentration at time zero. Samples were taken at various times (1, 2, 4, 8, 10, 24, 48 hours) during the fermentation process, and yeast growth was monitored using absorbance at 600 nm. The samples were centrifuged at 13000 xg and frozen for subsequent analysis of ethanol.  4.5.1 Yeast growth  It was important to construct growth curves for yeast strains that were used in the experiment to test the viability of the yeast. To obtain a growth curve, yeast was first transferred from the petri dish into 50 mL YPG nutrient broth. It was allowed to grow for one day. After 24 hours of growth, the yeast would be spun down and the supernatant would be drawn out and discarded.  49  The yeast pellets would be re-suspended with 10 mL of sterilized distilled water using a vortex. Two mL of yeast would be added to a new yeast media for growth observation. Samples were drawn at 2, 4, 8, 10, 24, 48 hours and their absorbance would be measured by spectrophotometer (Mandel Scientific Inc., Pharmaspec UV-1700). A general growth curve was shown in figure 24. It included lag phase, log or exponential growth phase, stationary phase and death phase. Growth rate of the yeast strains can be calculated and will be discussed in section 5.4.   Figure 24   General growth curve (92)  4.5.2 Determination of yeast concentration  A yeast calibration curve was used to determine the yeast concentration of the samples that were taken during fermentation by correlating the concentration of yeast (g dry weight/L) with the absorbance. To construct the calibration curve, yeast was grown for one day and transferred into new medium to grow for another day. The yeast broths collected after 48 hours were spun down at 5000 xg and yeast pellets were collected. Each pellet was washed twice, with and re- suspended in 10 mL of sterilized distilled water and became the yeast stock. The dilutions of the stock were shown in table 14. The absorbance of yeast strain K1 dilutions were measured at 600 0 5 10 Lo g of  n um be rs  o f b ac te ri a -- > Time (hr.) Lag Phase Log or exponential growth phase Stationary phase Death or logarithmic decline phase  50  nm and plotted against their concentration (figure 25), which were obtained by measuring the dry cell weight of the yeast K1 stock.  Table 14   Dilutions used in constructing yeast calibration curve Dilution Factor Concentration (g/L) Dilution Factor Concentration (g/L) 1.0 48.34 500.0 0.0967 1.3 36.25 1000.0 0.0483 2.0 24.17 2000.0 0.0242 4.0 12.08 4000.0 0.0121 10.0 4.83 8000.0 0.0060 20.0 2.42 10000.0 0.0048 100.0 0.48 20000.0 0.0024 200.0 0.24   Figure 25   Correlation of yeast K1 concentration and optical density at 600 nm  A simple correlation between absorbance and yeast concentration could be obtained by using only the linear portion of the graph (figure 26), which was found to be: ܱܦ଺଴଴ = 2.2206 × [ݕ݁ܽݏݐ] + 0.0545  0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 0.0 10.0 20.0 30.0 40.0 50.0 60.0 O D 60 0 Concentration of Yeast (g/L)  51   Figure 26   Determination of yeast K1 concentration  4.6 Analytical  4.6.1 Analysis of monomer sugars by HPLC  High performance liquid chromatography (HPLC) is an improved form of column chromatography. Instead of utilizing gravity for separating the components from the mixture, it uses high pressure to force the mixture through the HPLC column (93). Stationary material was packed within the column, which would interact with mobile phase as it passes through (94). The separation was obtained due to the different chemical properties of the components in the mixture (95).  In this project, the HPLC system used was a DX 600 from Dionex Corp. It used an anion exchange column Carbopac PA-1 for identifying monosaccharides and disaccharides in aqueous samples collected during oxygen delignification and enzymatic hydrolysis. The instrument was controlled using software called the Chromeleon Chromatography Management System. This program used a pre-set carbohydrate method to analyze each sample and output a chromatogram and the corresponding sugar concentrations. The operating conditions for the carbohydrate y = 2.220x + 0.054 R² = 0.994 0.0 0.2 0.4 0.6 0.8 1.0 1.2 0.0 0.1 0.2 0.3 0.4 0.5 0.6 O D 60 0 Concentration of Yeast (g/L)  52  method were summarized in Table 15. The control panel of the Chromeleon program showed the real-time status of each component in the unit, such as the temperature, pressure and pH in the system.  Table 15   Operating conditions for HPLC analysis Temperature 30°C Pressure 200 - 2000 psi pH 10 - 13 Injection Volume 25 L Analysis Duration 60 minutes Eluent (flow rate) NaOH (1 mL/min) Diluent  Nanopure water  Prior to analysis with the HPLC, samples were removed from the freezer and thawed. Each sample was then diluted 50 times with nanopure water. A 5 mg/mL fucose solution was used as an internal standard. The diluted samples were then added to 1.5 mL HPLC screw-top vials and sealed with septa-lined lids. An autosampler, Dionex AS50, was used to withdraw a 20 µL sample from the HPLC vials and the sugars (fucose, arabinose, galactose, glucose, xylose, and mannose) were separated using a Dionex CarboPac PA1 column. Deionised water than had also be degassed was used as the mobile phase, with a flow rate 1.0 mL/min.  Nanopure water was used throughout the HPLC system due to the sensitivity of the column and ensures no impurity appears in the system. The specifications of the nanopure water used in this project were given in table 16. Four calibration standards were made (table 17) for generating a calibration curve.     53  Table 16   Nanopure specifications for water used in HPLC   Specifications Resistivity at 25 °C 18.2 MΩ cm2/cm Total Organic Carbon (TOC) < 1 ppb Bacteria < 1 CFU/mL pH at 25 °C 7 Particles < 0.2 m  Table 17   Sugar standards used in HPLC Standard 5 g/L Fucose (mL) 1 g/L Sugar (mL) Nanopure water (mL) [Sugar] (g/L) 1 0.10 2.00 2.90 0.40 2 0.10 0.50 4.40 0.10 3 0.10 0.25 4.65 0.05 4 0.10 0.10 4.80 0.02  To setup the HPLC, a sample list was input into the Chromeleon program. The program suggested the necessary nanopure water and dilute NaOH (0.75M) needed for the analysis of all samples. After they were placed in the bottles, the GP-50 gradient pump and TTL pump were primed to get rid of any bubbles in the system, because bubbles can affect the separation in the column. Then, the pumps were run for 5 minutes to ensure that there was no leakage anywhere in the system. Data acquisition was started to allow the signal to be stabilized before the analysis method was initiated in the control panel of the Chromeleon program.  4.6.2 Analysis of ethanol by gas chromatography  Gas chromatography (GC) was a chromatography technique that analyzes compounds that can be vaporized. During analysis, the gaseous compounds interact with different stationary phases that were coated on the wall of the column, which lead to different retention time of each compound. By looking at the retention time, each compound could be identified (96).  In this project, the GC system used was a CP-3800 manufactured by Varian Inc. The unit was used to quantify the concentration of ethanol by analyzing the samples collected during  54  fermentation. The GC system was connected to the computer and used an analytical program called Star. This program used a pre-set ethanol method to analyze each sample and output a chromatogram and peak area for the compounds of interest – ethanol and 1-butanol (standard). The operating conditions were shown in table 18.  Table 18   Operating conditions for GC analysis Column Chrompack Capillary Column           Length 60 m           Inside Diameter 0.25 mm           Film Thickness 0.25 m Oven Temperature 50°C Detector Temperature 200°C Injector Temperature 200°C Injection Volume 1 L Gas           Carrier Gas Hydrogen (40 psi and 30 mL/min)           Detector Gas Air (60 psi and 300 mL/min)           Make up Gas Helium (80 psi and 25 mL/min)   To analyze the samples from fermentation, they were first thawed and 200 L of the samples were mixed with 200 L of internal standard (5 g/L 1-butanol) and 600 L of deionized water. Each of the samples was prepared in 2 mL vial that was specially designed for the GC system. The internal standard was used to identify if there were any inconsistencies of the output signals. Standard curves were constructed by using 5 calibration standards, which were shown in table 19.    55  Table 19   Dilution for GC calibration curves used for correlating yeast concentration to absorbance Standard 5 g/L 1-butanol (L) 10 g/L Ethanol (L) Deionized water (L) [Ethanol] (g/L) 1 200 25 775 0.5 2 200 100 700 2.0 3 200 250 550 5.0 4 200 500 300 10.0 5 200 750 50 15.0  4.6.3 Determination of various concentrations by spectrophotometer  A spectrophotometer is a photometer that quantifies test samples based on their light absorption level (97). In this project, a double beam spectrophotometer (Mandel Scientific Inc., Pharmaspec UV-1700) was used. It compared the light intensity between the test sample and a reference sample (blank) at a specific wavelength (97), which yielded an optical density value (also called absorbance).  To measure the absorbance of a test sample, 3 mL of sample and 3 mL of deionized water or blank were prepared in 4 mL cuvettes. They were placed in two different slots for comparison. To correlate the concentration and the absorbance obtained from spectrophotometer at a specific wavelength, a calibration curve was needed. An example of a calibration curve was shown in Figure 26. If the absorbance exceeded the linear range, a correlation cannot be established and the sample needs to be diluted. The concentration that was corresponding to the absorbance obtained from diluted sample was multiplied by the dilution factor, which yields the actual concentration of the sample.  The spectrophotometer was used in determining the amount of sugar released in filter paper assay, concentration of p-Nitrophenol in beta-glucosidase assay and yeast concentration in fermentation.    56  4.6.4 Measurement of pH  A pH meter was an instrument that measures the pH of a liquid. The pH meter used in the laboratory was manufactured by Thermo Orion (model 710 pH meter). After the instrument had been idle for long term periods, calibration of the pH electrode should be performed with buffer solutions of pH 4, 7, and 10. During measurement, it was recommended that the liquid be continuously stirred to ensure adequate mixing for uniform measurement. Between measurements, a thorough washing of the pH electrode is required to avoid contamination.  The usage of pH meter included measuring and adjusting the glycine buffer to pH 10.8 in - glucosidase assay, the citrate buffer to pH 4.8 during both filter paper and -glucosidase assay and the sodium acetate buffer to pH 4.8 for both enzyme assay and enzymatic hydrolysis.   57  5.0 RESULTS AND DISCUSSION  5.1 Analysis of substrate composition  Acid hydrolysis was conducted to determine the compositions (cellulose, hemicellulose and lignin) of the non-pretreated substrates and substrates that were pretreated at different temperatures with oxygen delignification. This provided the amount of sugars (cellulose and hemicellulose) that were available for enzymatic hydrolysis. After the acid hydrolysis, monomer sugars, pentose and hexose, that were hydrolyzed from hemicellulose and cellulose were analyzed by the HPLC. They were reported as percentage monomer sugars (pentose and hexose) produced based on dry substrate used in the acid hydrolysis, which is shown in table 20.  As seen from table 20, the lignin contents in all substrates are reduced as pretreatment temperature rose. This proves that the pretreatment process, oxygen delignification, is effective in removing the lignin in switchgrass, grass and pulp mill clarifier sludge. However, it has a negligible effect on cardboard since the lignin content stayed about the same. Sugar yield, on the other hand, did not exhibit this trend. It rose to a maximum value at certain temperature and then reduced as the temperature continued to rise. For instance, switchgrass has the highest sugar yield at 130°C. However, the sugar yield reduced from 78.3% to 74.0% when the temperature rose to 150°C. This was probably caused by the degradation of hemicellulose or cellulose at high temperature during oxygen delignification, which was lost in the liquor. More detail is discussed in section 5.2.   58  Table 20   Compositions of substrates (urban cellulosic wastes) used in ethanol production  Pentose Hexose Total Sugars Lignin % Yield Standard Deviation % Yield Standard Deviation % Yield Standard Deviation % Yield Standard Deviation Switchgrass (non-pretreated) 28.4 0.2 37.6 0.1 66.0 2.8 23.8 2.2 Switchgrass (100C) 30.0 0.2 41.8 1.9 71.8 0.1 19.3 1.8 Switchgrass (130C) 32.4 0.4 45.9 2.5 78.3 1.9 14.9 1.8 Switchgrass (150C) 28.2 1.8 45.8 2.8 74.0 2.5 14.6 1.5 Grass (non- pretreated) 7.9 0.0 26.5 0.4 34.4 0.4 50.7 0.6 Grass (100C) 3.9 0.6 38.0 1.7 41.9 4.1 45.3 1.4 Grass (130C) 14.0 0.9 28.7 1.9 42.7 4.6 36.9 2.7 Grass (150C) 16.9 1.1 44.5 4.1 61.4 1.9 35.2 3.1 Grass (180C) 13.9 3.3 35.3 4.6 49.3 1.7 19.1 3.9 Cardboard (non- pretreated) 11.4 0.1 73.7 0.1 85.1 0.1 15.0 0.9 Cardboard (100C) 12.2 0.4 68.6 0.3 80.8 0.3 14.5 0.9 Cardboard (130C) 12.6 0.5 66.3 0.9 78.9 0.9 13.9 2.9 PMCS (non- pretreated) 9.0 0.1 41.6 3.0 50.6 4.1 46.6 3.6 PMCS (100C) 8.7 0.4 57.7 4.1 66.3 4.3 32.7 3.5 PMCS (130C) 8.7 0.9 50.6 4.3 59.3 3.0 28.7 4.6  The typical compositions for substrates are mentioned in section 2.7 and summarized in table 21. The composition of softwood is used as the composition for pulp mill clarifier sludge since the sludge is a waste product from softwood pulp mill. When the compositions of non-pretreated substrate that were obtained from this project are compared to the typical values, all of the experimental lignin content values are significantly higher than the typical values reported. The percentage differences in lignin for switchgrass, grass, cardboard and pulp mill clarifier sludge (PMCS) are 98.3%, 69.0 – 407%, 5.6% and 66.4% respectively. As we can see, the lignin and sugar values found for the substrates, except cardboard lignin value, are out of range. Therefore,  59  the lignin values will not be used in any subsequent calculations. Sugar values, on the other hand, will be used to determine the amount of sugar available that were unhydrolyzed.  Table 21   Contents of lignin and total sugar for non-pretreated substrates used  Typical Values Experimental Values % sugar % lignin % pentose % hexose % total sugars % lignin Switchgrass (59) 76.4 12 28.4 37.6 66.0 23.8 Grass (59) 60 – 90 10 – 30 7.9 26.5 34.4 50.7 Cardboard (10) 73.5 14.2 11.4 73.7 85.1 15 PMCS (65) 53 – 68 28 9.0 41.6 50.6 46.6   5.2 Sugar loss during oxygen delignification  Oxygen delignification is aimed at removing the lignin from a substrate for easier enzymatic attack on cellulose and hemicellulose. However, high temperature and pressure during oxygen delignification induces a risk of sugar loss. Thus, a liquid sample is drawn from the oxygen delignification liquor for each substrate to determine if sugar loss is a significant issue. By analyzing the sugar samples by HPLC, the amounts of sugar loss were tabulated in table 22. Substrates were partially dissolved during oxygen delignification. After they were pretreated at different temperatures, they were dried, weighed, and reported as the solid yields in table 22. On the other hand, the total amount of sugars in each substrate available for hydrolysis was calculated by multiplying the total sugar composition given in table 20 and the solid yield from oxygen delignification, which is shown in the sample calculation in the appendix. Based on these values, it was found that no significant sugar is lost during oxygen delignification step. The highest sugar loss is only 2.55 w/w%. However, it is observable that there is a higher sugar loss as the temperature increases. It was probably because more sugars are free from the lignin since there is higher lignin removal at more rigorous conditions. Thus, oxygen delignification is a good pretreatment that can remove the lignin while retaining the sugar content.  60   Table 22   Sugar loss in oxygen delignification step  Solid Yield (g dry substrate) Standard Deviation in Solid Yield Total Available Sugars (g) Sugar Loss (mg) Weight Percent of Sugar Loss (w/w%) Switchgrass – 100°C 8.88 0.15 6.37 0.1 0.002 Switchgrass – 130°C 8.39 0.38 6.57 0.3 0.005 Switchgrass – 150°C 6.64 0.79 4.92 3.0 0.061 Grass Clipping – 100°C 8.44 0.86 3.54 6.3 0.178 Grass Clipping – 130°C 4.85 0.23 2.07 16.3 0.787 Grass Clipping – 150°C 3.11 0.47 1.91 30.0 1.571 Grass Clipping – 180°C 2.78 0.61 1.37 35.0 2.554 Cardboard – 100°C 9.86 0.14 7.97 1.5 0.019 Cardboard – 130°C 9.72 0.13 7.67 0.3 0.004 Paper Mill Clarifier Sludge – 100°C 8.22 0.06 5.45 0.0 0.000 Paper Mill Clarifier Sludge – 130°C 7.60 0.01 4.51 0.4 0.009  5.3 Kinetics and yield of sugar production from enzymatic hydrolysis  The goal of enzymatic hydrolysis is to hydrolyze complex carbohydrates, such as hemicellulose and cellulose, in substrates into simple sugars, like pentose and hexose. The simple sugars are then used subsequently in the fermentation process. The factors that may influence sugar yield are pretreatment temperature, initial enzyme loading and substrate concentration (dry based).  The results for each substrate are categorized into pentose yield and hexose yield, which are expressed in grams of sugar per each gram of dry substrate used in the hydrolysis. Below is a sugar yield curves for non-pretreated cardboard, which shows the hexose sugar concentrations at three different conditions over time (figure 27).  61    Figure 27   Hexose concentration obtained from non-pretreated cardboard  Since switchgrass and grass have shown the maximum pretreatment temperature (130°C) would produce maximum sugar yield, they are subjected to higher temperature in the pretreatment step in order to investigate if higher pretreatment temperature will lead to higher sugar yield for grass and switchgrass. This ensured that the optimum condition has been reached. Both switchgrass and grass are pretreated at 150°C and hydrolyzed. As a result, switchgrass has exhibited a lower yield as temperature rises. On the other hand, grass has shown an increase in the yield, which is 0.56 gram total sugar per each gram of dry substrate used for hydrolysis. Therefore, grass is pretreated at 180°C and the sugar yield is reduced to 0.45 g total sugar/g dry substrate (table 23).   0 5 10 15 20 25 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data    62   Table 23   Maximum sugar yield and percent sugars hydrolyzed in hydrolysis for factorial design conditions   Pretreatment Temperature Maximum Sugar Yield (g sugar produced) Maximum Sugar Yield (%) % of Total Available Sugar Hydrolyzed Pentose Standard Deviation Hexose Standard Deviation Total Standard Deviation Total Standard Deviation SG N/A 0.0050 0.0004 0.0267 0.0005 0.0316 0.0009 6.3Δ 0.1 9.6 100°C 0.1670 0.0014 0.3671 0.0002 0.5341 0.0012 21.4° 0.2 29.8 130°C 0.3460 0.0127 0.6769 0.0518 1.0229 0.0645 40.9° 1.1 52.3 150°C 0.3574 0.0009 0.5518 0.0272 0.9091 0.0281 36.4° 2.6 49.1 G N/A 0.0064 0.0002 0.0525 0.0078 0.0589 0.0076 11.8 Δ 0.7 34.2 100°C 0.0260 0.0028 0.1059 0.0062 0.1319 0.0090 26.4 Δ 1.5 63.0 130°C 0.2080 0.0078 0.7900 0.0583 0.9980 0.0661 39.9° 1.4 93.5 150°C 0.0496 0.0002 0.2294 0.0034 0.2790 0.0035 55.8 Δ 1.8 90.9 180°C 0.0206 0.0005 0.2036 0.0076 0.2243 0.0071 44.9 Δ 2.6 90.1 CB N/A 0.0539 0.0104 0.2519 0.0055 0.3058 0.0159 61.2 Δ 3.1 71.8 100°C 0.0389 0.0009 0.2516 0.0168 0.2905 0.0159 58.1 Δ 3.3 71.9 130°C 0.2366 0.0157 1.2073 0.0923 1.4439 0.1080 57.8° 4.3 73.2 P N/A 0.0799 0.0065 0.4941 0.0080 0.5740 0.0145 23.0° 0.6 45.5 100°C 0.3723 0.0286 0.7431 0.0479 1.1154 0.0765 44.6° 1.8 67.3 130°C 0.0398 0.0039 0.1704 0.0051 0.2101 0.0090 42.0 Δ 3.1 70.9 SG = switchgrass; G = grass; CB = cardboard; P = pulp mill clarifier sludge Δ The enzymatic hydrolysis condition for the sugar yield is 10 g dry substrate/L and 40 FPU/g dry substrate ° The enzymatic hydrolysis condition for the sugar yield is 50 g dry substrate/L and 16 FPU/g dry substrate    63  From figure 36 – 65, it was observed that the sugar yields from the condition with low enzyme loading and solid loading (10 g dry substrate/L and 20 FPU/g) were lower than the yields obtained from the other two conditions most of the time. This trend is predictable in this experiment since it is well known that glucose yield increases as the solid loading (98) and enzyme loading (99) increases (figure 28, 29).   Figure 28   Glucose yield over time at different solid loadings of olive tree biomass (98)  Figure 29   Cellulose conversion (ratio of grams of glucose produced to grams of cellulose supplied in enzymatic hydrolysis) at different -glucosidase and cellulase concentrations (99) 0 10 20 30 40 50 60 70 0 10 20 30 40 50 60 70 80 G lu co se  (g /L ) Time (hr) 2% 5% 10% 20% 50% 0 10 20 30 40 50 0 50 100 150 200 250 C el lu lo se  c on ve rs io n (% ) b-glucosidase (IU/g cellulose) 12 FPU/g cellulose 24 FPU/g cellulose 48 FPU/g cellulose  64  The maximum amount of sugars that were obtained from different hydrolysis condition was tabulated in table 23. As seen from the table, all of the maximum sugar yields from enzymatic hydrolysis, which are expressed in percentage, are lower than that of acid hydrolysis (table 20). Since the hydrolysis condition that the maximum sugar concentration yielded from each substrate was different, the amount of sugars were calculated and tabulated in table 23. The fraction of sugars that are hydrolyzed during enzymatic hydrolysis based on the assumption that acid hydrolysis has converted all available cellulose and hemicellulose to sugars is also shown in the table. It can be concluded that non-pretreated switchgrass is the least susceptible substrate to enzymatic hydrolysis. On the other hand, grass clippings that are treated at high temperature (130°C – 180°C) have the greatest proportion of sugar hydrolyzed among the substrate tested. This proves that the pre-treatment with oxygen delignification is most effective for grass clippings at high temperature. Although oxygen delignification has a beneficial effect for some substrates, it has no effect on cardboard. Non-pretreated cardboard yields the highest sugar concentration after enzymatic hydrolysis of all substrates.  It has about 30% unhydrolyzed sugar left, which shows that it is possible to further enhance the sugar yield.  After determining the pentose and hexose sugar yields for each substrate at different conditions, an empirical model for enzymatic hydrolysis is developed and compared to the experimental data obtained. The objective of developing empirical model was to determine hexose concentration at different times. The nth-order model was used in order to taken all n reaction into account. The rate law equation for the breakdown of cellulose into glucose is: − ௗ஼ ௗ௧ = ݇ ቀ ஼ ஼೚ ቁ ௡                         (1) After integration from time zero to time t, equation (2) is developed (91). ܵ = ܥ௢ ቎1 − ቆ ଵ ଵା ೖ೟(೙షభ) ಴೚ ቇ భ ೙షభ ቏ × ଵ.ଵଵ ௚ ீ௟௨௖௢௦௘ ଵ ௚ ஼௘௟௟௨௟௢௦௘                     (2)  Where  S = hexose concentration at time t (g/L)             Co = cellulose concentration at time zero (g/L)              t = time (hr)           k, n = empirical constants  65   Figure 30   Comparison of hexose concentration of non-pretreated cardboard (Hydrolyzed at 10 g dry substrate/L and 40 FPU/g)  In order to estimate the empirical values n and k, excel was used to minimize the difference between the experimental and the calculated hexose concentration by changing the n and k values. For non-pretreated cardboard that was hydrolyzed at 10 g dry substrate/L and 40 FPU/g dry substrate, the n and k values are found to be 3.82 and 1.65 respectively by using excel solver and the empirical formula is simplified to: ܵ = 8.18 ቈ1 − ൬ 11 − 0.63ݐ൰଴.ଷହହ቉ The comparison between the data and model was shown in figure 30. More hydrolysis results are shown in appendix figure 36 – 65.  5.4 Yeast growth in YPG medium  As mentioned in section 4.5.1, the growth curve is used to determine the growth rate of the yeast used in this experiment. S. cerevisiae strain K1 was used to produce the growth curve. The biomass concentration over time curve is shown in figure 31. 0 1 2 3 4 5 6 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Data for condition 2 (10 g dry substrate/L, 40 FPU/g dry substrate) Model for condition 2 (10 g dry substrate/L, 40 FPU/g dry substrate)  66   Figure 31   Concentration of yeast strain K1 over time (growth conditions: incubated at 30°C and 150 rpm in YPG medium with 2% w/w glucose)  As observed from figure 31, there was no lag phase for yeast K1. The growth phase occurred immediately to 4 hours. Since cell growth is exponential over time, the cell growth rate is calculated as follow: ܺ = ܺ଴݁ఓ௧    (3) ݈݊ ௑ ௑బ = ߤݐ    (4) Therefore, specific growth rate (µ) can be determined by plotting ln(X/X0) versus time.  0 5 10 15 20 25 0 10 20 30 40 50 B io m as s C on ce nt ra tio n (g /L ) Time (hr) Biomass Concentration Exponential Growth Rate Model  67   Figure 32   Determination of specific growth rate  Thus, the specific growth rate is 0.35 hr-1, which is the slope in figure 32.  5.5 Ethanol yield for fermentation of hydrolysates  After the pretreatment and hydrolysis conditions for each substrate that yield the maximum sugar concentration are determined, the hydrolysates from these substrates are subsequently used for fermentation.  As seen from figure 33, there was minimal yeast growth during the fermentation. Conditions were chosen (high inoculums and microaerophilic condition) to minimize growth and maximize ethanol yield. y = 0.35x R² = 0.9386 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 0 1 2 3 4 5 ln  (X /X o) Time (hr)  68   Figure 33   Yeast concentration during fermentation (growth conditions: incubated at 30°C and 150 rpm)  Ethanol samples that were taken at different time points during fermentation were analyzed by GC and output as ethanol concentrations. The concentrations were divided by the hexose concentrations of the hydrolysates. A curve was generated for each substrate to show how much ethanol was produced from each gram of hexose sugar available. They were shown in figure 66 – 69. The maximum ethanol yields from switchgrass, grass, cardboard and pulp mill clarifier sludge were 0.21, 0.30, 0.32 and 0.30 gram of ethanol per gram of hexose sugar, respectively. Cardboard, which has the maximum sugar yield during hydrolysis, has the highest ethanol yield (0.32 g ethanol / g sugar) as well. It is 62.7% of theoretical ethanol yield (0.51 g ethanol / g hexose)(100)(101). As observed, switchgrass has the lowest ethanol yield among all the substrates tested. The other three substrates have relatively close ethanol yield when compared to each other and all of them have better ethanol yields than the control, switchgrass. This proves that all three substrates have potential to be the feed for ethanol production.   0 5 10 15 20 25 30 35 0 5 10 15 20 25 30 35 40 45 50 Y ea st  C on ce nt ra tio n (g /L ) Time (hr) Switchgrass (pretreated at 130°C) Grass (pretreated at 150°C) Cardboard (non-pretreated) Pulp Mill Clarifier Sludge (pretreated at 100°C)  69   5.6 Material balance  5.6.1 Mass balance of lab scale experiment  From the results shown in this section, it is technologically feasible to produce bioethanol from urban cellulosic wastes examined. However, it is important to know if there is any loss of materials during the ethanol production. The processes used in this project are shown in figure 34.      Figure 34   Process flow diagram for mass balance  The letters displayed in the diagram corresponds to different mass input and output. Table 24 summarizes these values and provides the overview of the processes.    (a) Substrate Pretreatment Hydrolysis Fermentation (b) (d) (c) (e) (f)  70 Table 24   Mass balance for processes used in this project     (a) (b) (c) (d) (e) Hydrolysates (f)   Conditions Substrate Used Sugar Loss Solid Yield Solid Filtered Vol. Max. Sugar Yield Max. Hexose Yield Ethanol Yield   g (dry weight) mg g (dry weight) g (wet weight) mL g/L g/L g ethanol /g hexose Switchgrass (non-pretreated) 1       2.73 43.5 0.4775 0.4200 2       3.98 43.0 0.6325 0.5325 3       20.40 26.0 1.4650 1.0625 Switchgrass (100°C) 1 10 0.1 8.88 2.55 41.5 1.7300 1.7275 2 3.77 39.5 2.0925 1.4200 3 17.86 29.0 10.6825 7.3425 Switchgrass (130°C) 1 10 0.3 8.39 2.38 43.5 3.4025 2.2075 2 3.15 41.5 3.7000 2.6125 3 14.85 30.0 20.4575 13.5375 0.2074 Switchgrass (150°C) 1 10 3.0 6.64 2.05 44.0 3.2650 1.9250 2 2.99 44.0 3.3025 1.9075 3 13.87 28.0 18.1825 11.0350 Grass (non-pretreated) 1       4.47 40.0 0.5825 0.5225 2       4.16 39.0 1.1775 1.0500 3       15.33 31.5 1.7425 1.3300 Grass (100°C) 1 10 6.3 8.44 5.32 39.0 1.8575 1.5150 2 5.09 38.5 2.6375 2.1175 3 19.28 27.5 10.0450 7.9600 Grass (130°C) 1 10 16.3 4.85 4.60 39.5 3.7525 2.9925 2 4.37 40.5 3.5025 2.6025 3 14.74 30.0 19.9600 15.8000 Grass (150°C) 1 10 30.0 3.11 6.19 41.5 5.1400 4.3675 2 5.86 40.5 5.5800 4.5875 0.3000 3 12.34 32.0 27.3950 22.9400  71     (a) (b) (c) (d) (e) Hydrolysates (f)   Conditions Substrate Used Sugar Loss Solid Yield Solid Filtered Vol. Max. Sugar Yield Max. Hexose Yield Ethanol Yield   g (dry weight) mg g (dry weight) g (wet weight) mL g/L g/L g ethanol /g hexose Grass (180°C) 1 10 35.0 2.78 10.67 34.0 3.9450 3.5900 2 8.18 36.0 4.4850 4.0725 3 19.9 25.5 20.5175 18.6450 Cardboard (non-pretreated) 1       2.98 42.0 5.2700 4.2150 2       2.79 42.8 6.1150 5.0375 0.3152 3       5.9 38.5 24.6850 19.3875 Cardboard (100°C) 1 10 1.5 9.86 2.07 43.0 3.4350 2.9225 2 1.69 42.5 5.8100 5.0325 3 4.56 38.0 27.1225 22.3825 Cardboard (130°C) 1 10 0.3 9.72 1.88 41.5 4.1575 3.5450 2 1.41 40.8 5.4300 4.6650 3 4.65 39.0 28.8775 24.1450 Pulp Mill Clarifier Sludge (non-pretreated) 1       1.59 41.5 1.5300 1.3800 2       2.83 40.8 2.1025 1.8750 3       7.87 39.0 11.4800 9.8825 Pulp Mill Clarifier Sludge (100°C) 1 10 0.0 8.22 1.96 41.8 3.5775 2.4175 2 0.90 43.0 4.0625 2.8675 3 5.95 39.0 22.3075 14.8625 0.2962 Pulp Mill Clarifier Sludge (130°C) 1 10 0.4 7.60 1.59 42.5 2.7525 2.2500 2 1.30 42.5 4.2025 3.4075 3 6.00 40.3 19.5075 15.0400  72  5.6.2 Mass balance of scaled up ethanol plant   After the mass balance for the lab scale experiment was performed, a scale up material balance was calculated, which can be used in the future for equipment sizing and cost estimation. When an economic analysis is done in the future, it can be determined if it is profitable to build a bioethanol plant that uses such wastes. Re-pulp cardboard was determined to yield the highest sugar concentration among the four substrates that were tested. It is assumed that the economic analysis performed in the future will estimate the cost of and profit from a full scale (130 million L / year) ethanol plant using re-pulped cardboard as a feedstock. To complete this economic analysis, the material balance for this full scale plant is performed based on the assumption that linear scale up factors can be applied. A schematic diagram is shown below to show the entire ethanol production process.   Figure 35   Schematic diagram for ethanol production    73  The chosen production capacity is 130 million litres per year (MMly). 130 × 10଺ ܮ ݕ݁ܽݎ × 0.7893݇݃ ݁ݐℎܽ݊݋݈ ܮ = 1.03 × 10଼ ݇݃ ݁ݐℎܽ݊݋݈ ݕ݁ܽݎ  The sugar needed for 130 MMly is: 1.03 × 10଼ ݇݃ ݁ݐℎܽ݊݋݈ ݕ݁ܽݎ × ݇݃ ℎ݁ݔ݋ݏ݁0.32 ݇݃ ݁ݐℎܽ݊݋݈ = 3.22 × 10଼ ݇݃ ℎ݁ݔ݋ݏ݁ݕ݁ܽݎ  Assuming no sugar is lost during filtration, the monomer sugar produced from hydrolysis is 3.22 x 108 kg of hexose/ year. The hexose produced from hydrolysis for non-pretreated cardboard (10 g dry substrate/L and 40 FPU/g dry substrate) is 0.2519 g hexose (table 23, section 4.3). 0.2519 ݃ ℎ݁ݔ݋ݏ݁50 ݉ܮ × 1000 ݉ܮܮ × ܮ10 ݃ ݀ݎݕ ݏݑܾݏݐݎܽݐ݁ = 0.504 ݃ ℎ݁ݔ݋ݏ݁݃ ݀ݎݕ ݏݑܾݏݐݎܽݐ݁ Thus, the amount dry substrates needed is: 3.22 × 10଼ ݇݃ ℎ݁ݔ݋ݏ݁ ݕ݁ܽݎ × ݇݃ ݀ݎݕ ݏݑܾݏݐݎܽݐ݁0.504 ݇݃ ݏݑ݃ܽݎ = 6.39 × 10଼ ݇݃ ݀ݎݕ ݏݑܾݏݐݎܽݐ݁ݕ݁ܽݎ which corresponds to: 6.39 × 10଼ ݇݃ ݀ݎݕ ݏݑܾݏݐݎܽݐ݁ ݕ݁ܽݎ × 5.335 ݇݃ ݓ݁ݐ ݏݑܾݏݐݎܽݐ݁1 ݇݃ ݀ݎݕ ݏݑܾݏݐݎܽݐ݁ = 3.41 × 10ଽ ݇݃ ݓ݁ݐ ݏݑܾݏݐݎܽݐ݁ݕ݁ܽݎ  Assuming that the dry weight of cardboard does not change during the re-pulp process, 6.39 x 108 kg of dry un-pulped cardboard is re-pulped. It is known that every kg of dry un-pulp cardboard corresponds to 1.0718 kg of wet un-pulped cardboard. The wet un-pulped cardboard needed is:  74  6.39 × 10଼ ݇݃ ݀ݎݕ ݑ݊ − ݌ݑ݈݌ ܿܽݎܾ݀݋ܽݎ݀ ݕ݁ܽݎ × 1.0718 ݇݃ ݓ݁ݐ ݑ݊ − ݌ݑ݈݌ ܿܽݎܾ݀݋ܽݎ݀1 ݇݃ ݀ݎݕ ݑ݊ − ݌ݑ݈݌ ܿܽݎܾ݀݋ܽݎ݀= 6.85 × 10଼ ݇݃ ݓ݁ݐ ݑ݊ − ݌ݑ݈݌ ܿܽݎܾ݀݋ܽݎ݀ ݕ݁ܽݎ  In this experiment, the consistency for re-pulping is 2%. Therefore, hot water needed for re- pulping is: 6.39 × 10଼ ݇݃ ݀ݎݕ ݑ݊ − ݌ݑ݈݌ ܿܽݎܾ݀݋ܽݎ݀ ݕ݁ܽݎ × 1000 ݃1 ݇݃ × 10 ܮ ݓܽݐ݁ݎ200 ݃ ݑ݊ − ݌ݑ݈݌ ܿܽݎܾ݀݋ܽݎ݀ × 1 ݇݃1 ܮ = 3.20 × 10ଵ଴ ݇݃ ݓܽݐ݁ݎ ݕ݁ܽݎ      75  6.0 CONCLUSIONS  Oxygen delignification is the pretreatment method that was used in this study. It is important to test if oxygen delignification is an effective pretreatment method. By quantifying lignin content in substrates that were not pretreated and pretreated at different temperatures, oxygen delignification contributed to lignin reduction in grass, switchgrass and pulp mill clarifier sludge. However, the lignin content stayed about the same for cardboard. The reduction in lignin is beneficial for enzymatic hydrolysis. Even though the oxygen delignification did not work for cardboard, non-pretreated cardboard has the highest sugar yield.  Each substrate has gone through acid hydrolysis to identify its composition of sugar and lignin. Non-pretreated cardboard has found to have highest amount of sugars available for hydrolysis. However, the enzymatic hydrolysis can only hydrolyze 71.8±0.1% of the sugar available. This indicates that there is potential for this substrate to yield more sugar during hydrolysis. This might be achieved by using a different pretreatment or hydrolysis method, which can be investigated further in the future.  Enzymatic hydrolysis was carried out for 48 hours and samples were taken at 1, 2, 4, 8, 10, 24 and 48 hours. An empirical model was developed through the rate law equation to compare the experimental results. The empirical model was found to be ܵ = 8.18 ቈ1 − ൬ 11 − 0.63ݐ൰଴.ଷହହ቉ With this equation, hexose concentration (S) can be determined at any given time (t).  The hydrolysates that were obtained after 48 hours of hydrolysis were subjected to fermentation. It was found that the yeast strain K1 used in this project has a specific growth rate of 0.35 hr-1. By transferring grown yeast to another nutrient solution after a day, the yeast was able to stay in stationary phase without nutrient limitation. Since the yeast did not require the nutrient to maintain itself, it can utilize the entire nutrient supply to maximize the production of the by- product, which is ethanol, in this project.  Non-pretreated cardboard, which has the highest sugar yield, has the highest ethanol yield of 0.32 g ethanol / g hexose.  76  7.0 FUTURE WORK  Oxygen delignification was found to be effective on switchgrass, grass, and pulp mill clarifier sludge. However, testing of other pretreatment methods, such as steam explosion and organosolv, on these substrates can provide a more detailed comparison and allow the researchers to determine if oxygen delignification is the best pretreatment for these substrates. Each method can also be evaluated based on other advantages or disadvantages, such as inhibitors or energy consumption, beside the effectiveness of the lignin removal.  It was observed that there is close to 30% unhydrolyzed sugar within the non-pretreated cardboard. It would be beneficial to test different enzymes for potential higher sugar yield. In addition, only three different hydrolysis conditions were tested in this study due to time constrain. If more conditions could be tested, the optimum condition for highest sugar yield would be more precise.  One of the objectives was to test the fermentability of the two yeast strains of S. cerevisiae – K1 and 259ST. 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[Online] 2009.    84  APPENDIX  The conditions listed in figure 36 – 65 are referred as follow: Condition 1 10 g dry substrate/L and 20 FPU/g dry substrate Condition 2 10 g dry substrate/L and 40 FPU/g dry substrate Condition 3 50 g dry substrate/L and 16 FPU/g dry substrate    Figure 36   Switchgrass – pentose concentration (no oxygen delignification) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  85   Figure 37   Switchgrass - hexose concentration (no oxygen delignification)  Figure 38   Switchgrass – pentose concentration (oxygen delignification at 100°C) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  86   Figure 39   Switchgrass – hexose concentration (oxygen delignification at 100°C)  Figure 40   Switchgrass – pentose concentration (oxygen delignification at 130°C) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  87   Figure 41   Switchgrass – hexose concentration (oxygen delignification at 130°C)  Figure 42   Switchgrass – pentose concentration (oxygen delignification at 150°C) 0 2 4 6 8 10 12 14 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  88   Figure 43   Switchgrass – hexose concentration (oxygen delignification at 130°C)   Figure 44   Grass – pentose concentration (no oxygen delignification) 0 2 4 6 8 10 12 14 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0.40 0.45 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  89   Figure 45   Grass – hexose concentration (no oxygen delignification)  Figure 46   Grass – pentose concentration (oxygen delignification at 100°C) 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0.0 0.5 1.0 1.5 2.0 2.5 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  90   Figure 47   Grass – hexose concentration (oxygen delignification at 100°C  Figure 48   Grass – pentose concentration (oxygen delignification at 130°C) 0 1 2 3 4 5 6 7 8 9 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  91   Figure 49   Grass – hexose concentration (oxygen delignification at 130°C)  Figure 50   Grass – pentose concentration (oxygen delignification at 150°C) 0 2 4 6 8 10 12 14 16 18 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  92   Figure 51   Grass – hexose concentration (oxygen delignification at 150°C)  Figure 52   Grass – pentose concentration (oxygen delignification at 180°C) 0 5 10 15 20 25 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0.0 0.5 1.0 1.5 2.0 2.5 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  93   Figure 53   Grass – hexose concentration (oxygen delignification at 180°C)  Figure 54   Cardboard – pentose concentration (no oxygen delignification) 0 2 4 6 8 10 12 14 16 18 20 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0 1 2 3 4 5 6 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  94   Figure 55   Cardboard – hexose concentration (no oxygen delignification)  Figure 56   Cardboard – pentose concentration (oxygen delignification at 100°C) 0 5 10 15 20 25 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  95   Figure 57   Cardboard – hexose concentration (oxygen delignification at 100°C)  Figure 58   Cardboard – pentose concentration (oxygen delignification at 130°C) 0 5 10 15 20 25 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  96   Figure 59   Cardboard – hexose concentration (oxygen delignification at 130°C)   Figure 60   Pulp mill clarifier sludge – pentose concentration (no oxygen delignification) 0 5 10 15 20 25 30 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 2.0 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  97   Figure 61   Pulp mill clarifier sludge – hexose concentration (no oxygen delignification)  Figure 62   Pulp mill clarifier sludge – pentose concentration (oxygen delignification at 100°C) 0 2 4 6 8 10 12 14 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0 1 2 3 4 5 6 7 8 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  98   Figure 63   Pulp mill clarifier sludge – hexose concentration (oxygen delignification at 100°C)  Figure 64   Pulp mill clarifier sludge – pentose concentration (oxygen delignification at 130°C) 0 2 4 6 8 10 12 14 16 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0.0 0.5 1.0 1.5 2.0 2.5 3.0 3.5 4.0 4.5 5.0 0 10 20 30 40 50 Pe nt os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model  99   Figure 65   Pulp mill clarifier sludge – hexose concentration (oxygen delignification at 130°C)   Figure 66   Ethanol yield for switchgrass (pretreated at 130°C) 0 2 4 6 8 10 12 14 16 18 0 10 20 30 40 50 60 H ex os e C on ce nt ra tio n (g /L ) Time (hr) Condition 1 Data Condition 2 Data Condition 3 Data Condition 1 Model Condition 2 Model Condition 3 Model 0.00 0.05 0.10 0.15 0.20 0.25 0 10 20 30 40 50 Et ha no l Y ie ld  (g  e th an ol /g  h ex os e) Time (hr)  100   Figure 67   Ethanol yield for grass (pretreated at 150°C)    Figure 68   Ethanol yield for cardboard (non-pretreated) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0 10 20 30 40 50 60 Et ha no l Y ie ld  (g  e th an ol /g  h ex os e) Time (hr) 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0 10 20 30 40 50 60 Et ha no l Y ie ld  (g  e th an ol /g  h ex os e) Time (hr)  101   Figure 69   Ethanol yield for pulp mill clarifier sludge (pretreated at 100°C)  Sample Calculation: Switchgrass (pretreated at 100°C) Cellulose and hemicellulose available in substrate = 71.8 w/w% Solid yield after oxygen delignification = 8.88 g dry substrate Amount of cellulose and hemicellulose ready for hydrolysis = 6.38 g (dry weight) Sugar loss = 0.1 mg Weight percent of sugar loss = (0.1 mg /6.38 g) * 100% = 0.002% 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 0 10 20 30 40 50 60 Et ha no l Y ie ld  (g  e th an ol /g  h ex os e) Time (hr)

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