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CHNG 3013 -Chemical Engineering Research Project

Hydrolysis of Ligno-cellulosic Fibers


Production of Bioethanol from corn cobs and wood. Supervisor : Professor W. A. Mellows

Vinita Manna (808000299)


12/16/2011

Hydrolysis of Lignocellulosic Fibers


Production of Bioethanol from Corn Cobs and Pine wood Page i

ABSTRACT
The use of ethanol is forecasted to grow rapidly in the coming years, increasing the need for Bioethanol production from lignocellulosic material due to the Food vs. Fuel crisis. As a substrate for bioethanol production it has a barrier in its complex structure therefore treatments are necessary that release monomeric sugars, which can be converted to ethanol by microbial fermentation. For the purpose of this research project, the overall objective of this project was to investigate the Hydrolysis of Lignocellulosic Fibers. The substrates chosen were corn cobs and pine wood due to availability. The production of ethanol from these sources entailed running preliminary test runs to determine the feasibility of the method in terms of the acid used for hydrolysis and the fermentation time. Following this, the corn cobs and pine wood sawdust were run using these conditions to obtain ethanol. The method involved the mechanical degradation of the raw materials by milling followed by prehydrolysis using 10% Sulphuric Acid at 90C for 1 hour. Originally, the intent was to vary the pre-hydrolysis conditions using 10%, 13% and 15% sulphuric acid, however time and material shortages made this difficult, hence only the 10% acid was used. After this, the samples were subjected to the hydrolysis conditions of 20%, 30% and 40% under the 90C and 1 hour condition. The hydrolysates were then left to ferment for 12 days. Reducing sugars concentrations were determined by the Lane and Eynon Method and distillation was performed to find the percentage ethanol yield. In general, results showed that the optimum hydrolysis concentration for the sulphuric acid was 40 % for both the pinewood sawdust and the corn cobs since the reducing sugars percent found using the Lane and Eynon was the highest for this concentration. It was also deduced that the highest ethanol yield was from the 40% sulphuric acid hydrolysates for which both the sawdust and corn cobs yieled approximately the same percent of ethanol. Overall, the ethanol yields for the corn cobs were higher than that of the pinewood sawdust.

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ACKNOWLEDGEMENTS
Firstly, I would like to thank God for giving me the strength, guidance and determination necessary to the completion of this project. I would also like to thank the following people whom without their help, this project would not be completed in time: Professor Winston Mellows, for being a very supportive supervisor throughout the course of the project. His recommendations and guidance were appreciated when a change of method was necessary due to the lack of the necessary reagents. Wayne for being an extremely helpful technician as well as providing continuous guidance and advice throughout this project. Karen Camejo for her lab assistance with respect to the Fermentation process as well as the Lane and Eynon method. Giselle Ramtahal-Dhun for always being willing to supply me with the necessary reagents and instruments. Larry Bachansingh for always finding a solution when a problem arose. Marlon (woodwork shop) for supplying me with more than enough sawdust to perform the labwork. Last but not least, my family and friends for being a great support system throughout and helping in any way possible.

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TABLE OF CONTENTS

ABSTRACT .................................................................................................................................... ii ACKNOWLEDGEMENTS ........................................................................................................... iii TABLE OF CONTENTS ............................................................................................................... iv LIST OF FIGURES...................................................................................................................... viii LIST OF GRAPHS ........................................................................................................................ ix LIST OF CHARTS ........................................................................................................................ ix LIST OF TABLES .......................................................................................................................... x 1 2 INTRODUCTION .................................................................................................................. 1 LITERATURE REVIEW........................................................................................................ 3 2.1 Ethanol.............................................................................................................................. 3 Ethanol as a Fuel ....................................................................................................... 3

2.1.1 2.2 2.3

Ligno-Cellulosic Materials ............................................................................................... 4 Historical Development .................................................................................................... 7 Early sulphuric acid wood lignin methods (prior 1920) ............................................ 7 Wood Lignin Isolation Methods (1920-1940) ........................................................... 8 Wood Sugar Quantification Methods (1930-1970) ................................................... 8 Wood Lignin Methods applied to Herbaceous Feedstocks (1930-1955)................... 9

2.3.1 2.3.2 2.3.3 2.3.4

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2.3.5 2.3.6 2.3.7 2.4

Applications to Food, Feed and Dietary Fiber (1970-1990)...................................... 9 Early Applications to Biofuels Feedstocks (1980-1998) ......................................... 10 Contemporary Contributions to Biomass Methods (1995-2010) ............................ 11

Pretreatment Methods ..................................................................................................... 11 Physical Pretreatments ............................................................................................ 13 Chemical Pretreatments........................................................................................... 15 Physicochemical Pretreatments ............................................................................... 20 Biological Pretreatments ......................................................................................... 22

2.4.1 2.4.2 2.4.3 2.4.4 2.5

Hydrolysis Methods........................................................................................................ 22 Acid Hydrolysis ...................................................................................................... 22 Enzymatic Hydrolysis of Lignocellulosic Biomass ................................................. 23

2.5.1 2.5.2 2.6

Fermentation ................................................................................................................... 24 Separate Hydrolysis and Fermentation (SHF) ......................................................... 25 Direct Microbial Conversion (DMC) ...................................................................... 25 Simultaneous Saccharification and Fermentation (SSF) ......................................... 25

2.6.1 2.6.2 2.6.3 2.7

Reducing Sugars Test ..................................................................................................... 25 Lane and Eynon Method ......................................................................................... 26 High Performance Liquid Chromatography ............................................................ 26

2.7.1 2.7.2 2.8

Product Recovery Process .............................................................................................. 27

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METHODOLOGY................................................................................................................ 29 3.1 3.2 3.3 Apparatus and Materials ................................................................................................. 29 Reagents ......................................................................................................................... 29 Experimental Procedure ................................................................................................. 30 Mechanical Degradation ......................................................................................... 30 Pre-hydrolysis ......................................................................................................... 31 Hydrolysis ............................................................................................................... 32 Fermentation ........................................................................................................... 33 Determination of Reducing Sugars content: Lane and Eynon Method ................... 35

3.3.1 3.3.2 3.3.3 3.3.4 3.3.5 4 5

RESULTS ............................................................................................................................. 39 ANALYSIS OF RESULTS................................................................................................... 44 5.1 5.2 Lane and Eynon Calculations ......................................................................................... 45 Distillation Calculations ................................................................................................. 49

DISCUSSION ....................................................................................................................... 52 6.1 Limitations...................................................................................................................... 55

7 8 9 10

CONCLUSION ..................................................................................................................... 56 RECOMMENDATIONS ...................................................................................................... 57 REFERENCES...................................................................................................................... 58 APPENDIX ........................................................................................................................... 60

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LIST OF FIGURES
Figure 1 Representation of lignocellulose structure showing cellulose, hemicellulose and lignin fractions (Teixeira, 2010). ............................................................................................................... 4 Figure 2 Illustration of a Cellulose Chain ....................................................................................... 5 Figure 3 Lignin Structure (Spiller, 2001) ........................................................................................ 6 Figure 4 Schematic of pretreatment effect on lignocellulosic Biomass......................................... 12 Figure 5 Steps involved in column chromatography ..................................................................... 27 Figure 6: Dried Corn cobs ............................................................................................................. 30 Figure 7: A 45 gram dried corn cob sample .................................................................................. 30 Figure 8 : Suspension being filtered using the Buchner Funnel .................................................... 31 Figure 9: Filtrates before neutralisation......................................................................................... 32 Figure 10: The Pre-hydrolysates and Hydrolysates before mixing. .............................................. 33 Figure 11: Ethanol Recovery (Distillation Apparatus) .................................................................. 34 Figure 12: Pycnometer used to determine the specific gravity and hence Ethanol content. .......... 34 Figure 13: Standardisation of Fehlings Solution ........................................................................... 36 Figure 14: End point of the Titration ............................................................................................. 37

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LIST OF GRAPHS
Graph 1 Trends in Brix readings over a 12 day period for Sawdust Samples ............................... 44 Graph 2 Trends in Brix readings over a 12 day period for Corn cobs Samples ............................ 44 Graph 3 Trends in Brix readings over a 12 day period for the Blank ............................................ 45

LIST OF CHARTS
Chart 1 Reducing Sugars % for Sawdust Samples at 20%, 30% and 40% Acid hydrolysis conditions. ..................................................................................................................................... 47 Chart 2 Reducing Sugars % for Corn Cobs Samples at 20%, 30% and 40% Acid hydrolysis conditions ...................................................................................................................................... 47 Chart 3 Comparison of the Average Reducing Sugars % between the Sawdust and Corn Cobs . 48 Chart 4 Weight % and Volume % Ethanol Yield for Sawdust at each Acid Concentration ......... 50 Chart 5 Weight % and Volume % Ethanol Yield for Corn Cobs at each Acid Concentration ..... 50 Chart 6 Comparison of the Average Weight % Yield between Sawdust and Corn cobs for each Acid Concentration. ...................................................................................................................... 51 Chart 7 Comparison of the Average Volume % Yield between Sawdust and Corn cobs for each Acid Concentration. ...................................................................................................................... 51

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LIST OF TABLES
Table 1 Main components of Lignocellulosic Waste (Parveen Kumar, 2009) ................................ 7 Table 2 Pretreatment Methods (Ashok Pandey, 2008) .................................................................. 12 Table 3 Sulphuric Acid Concentrations for Pre-Hydrolysis and Hydrolysis of each sample. ....... 39 Table 4 Mass of Calcium Carbonate added to neutralise each sample after pre-hydrolysis and the resulting pH ................................................................................................................................... 39 Table 5 Mass of Calcium Carbonate added to neutralise each sample after hydrolysis and the resulting pH ................................................................................................................................... 40 Table 6 Brix readings before and after adding Molasses over a period of 12 days ....................... 41 Table 7 Results of the Lane and Eynon Method for determining Reducing Sugars ...................... 42 Table 8 Results of the Distillation for Ethanol Recovery .............................................................. 43 Table 9 Percent Sugars Found from the Lane and Eynon Method ................................................ 46 Table 10 Percentage by Weight and Volume Ethanol Yield ......................................................... 49 Table 11 Comparison of Cellulose, Hemicellulose and Lignin Compostion of Pinewood Sawdust and Corn Cobs ............................................................................................................................... 52 Table 12 Determination of the Alcohol Content : Specific Gravities of Alcohol Water Mixtures 60

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1 INTRODUCTION
As the demand for energy is rising, there is a need to develop alternative fuel supplies for future energy demands. With bioenergy being a subject of increasing attention, interest in lignocellulosic biomass feed stocks for conversion into transportation fuels has grown. Ethanol-blended gasolines have the potential to contribute significantly to the reduction of greenhouse gases. Ethanol is an alternative fuel derived from biologically renewable resources and can be employed to replace octane enhancers such as methycyclopentadienyl manganese tricarbonyl (MMT) and aromatic hydrocarbons such as benzene or oxygenates such as methyl tertiary butyl ether (MTBE). A potential source for low-cost ethanol production is to utilize lignocellulosic materials feedstock from agricultural waste such as corn stover and cobs, wheat straw, woody residues, paper products and grasses such as switch grass. Lignocelluloses are the most abundant organic compounds in nature and represent an important resource for producing valuable products(Ketkorn, 2009). As a substrate for bioethanol production lignocellulosic material has a barrier in its complex structure which resists hydrolysis. For it to be amenable to fermentation, treatments are necessary that release monomeric sugars which can be converted to ethanol by microbial fermentation. Pretreatment is one of the most important steps in the process and high cost in the production of ethanol from lignocellulosic materials. The most common chemical pretreatment methods used for cellulosic feedstocks are dilute acid, alkaline, organic solvent, ammonia, sulphur dioxide of other chemicals to make the biomass more digestible by enzymes. Lignocellulosic materials sometimes require a particular combination of the pretreatment methods to optimize the yields of the feedstock, minimize the degradation of substrate, and maximize the sugar yield. For the purpose of this research project, the cellulosic materials used were corn cobs and pinewood sawdust based on the basis of availability. These are two of out the many available substrates found in Trinidad. Corn which is available during this time of the year (Septemberdecember) was used because it was easily sourced. The pinewood saw dust was used because Trinidad has a large production sector for wood products making this a possibility for a future

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industry. The production of ethanol from these sources entails mechanical degradation, pretreatment of substrates, acid hydrolysis followed by fermentation. The objectives researched were: To determine the effect of varying sulphuric acid concentrations and find the optimum concentration for the pre-hydrolysis of each substrate. To determine the effect of varying sulphuric acid concentrations and find the optimum concentration for the hydrolysis of each substrate. To investigate the fermentation of the monosaccharides produced by the acid hydrolysis and determine the relationship between percentage ethanol (%) and acid concentration. To compare the ethanol yield of the various substrates used. The following sections of this research paper include the detailed theory of the processes used, as well as the results and recommendations.

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2 LITERATURE REVIEW
2.1 Ethanol
Ethanol (C2H5OH) is one of the most significant synthetic oxygen-containing organic chemicals because of its unique combination of properties as a solvent, fuel, germicide, beverage, antifreeze, and especially because of its versatility as an intermediate to other chemicals. Ethanol is one of the largest-volume chemicals used in industrial and consumer products. The main uses for ethanol are as an intermediate in the production of other chemicals and as a solvent. As a solvent, ethanol is second only to water. Ethanol is a key raw material in the manufacture of plastics, lacquers, polishes, plasticizers, perfume, and cosmetics. The physical and chemical properties of ethanol are primarily dependent on the hydroxyl group, which imparts polarity to the molecule and also gives rise to intermediate hydrogen bonding. In the liquid state, hydrogen bonds are formed by the attraction of the hydroxyl hydrogen of one molecule and the hydroxyl oxygen of another. This makes liquid alcohol behave as though it were largely dimerized. Its association is confined to the liquid state; in the vapor state, its monomeric. (Sunggyu Lee, 2007) 2.1.1 Ethanol as a Fuel As a volatile chemical compound viewed as a gasoline substitute, pure ethanol has one major drawback. Internal combustion engines burn fuels; ethanol, in comparison combustion in oxygen generates less energy compared with either a pure hydrocarbon or a typical gasoline. This is not mitigated by the higher density of ethanol because liquid volumes are dispensed volumetrically and higher weights in fuel tanks represent higher loads in moving vehicles; a gallon of ethanol contains, therefore, only 70% of the energy capacity of a gallon of gasoline (Mousdale, 2008). A review of the relative merits of alternative fuels in 1996 pointed out that ethanol not only had a higher octane of combustion products (gases) per energy unit burned; these factors in optimized ethanol engines significantly eroded the differential advantages of gasoline. The high miscibility of ethanol and refined oil products allows a more conservative option, that is, the use of low-ethanol additions to standard gasoline (e.g., E10: 90% gasoline, 10% ethanol) and requires no modifications to standard gasoline-burning vehicles.

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2.2 Ligno-Cellulosic Materials


Lignocellulosic biomass comprising forestry, agricultural and agro-industrial wastes are abundant, renewable and inexpensive energy sources. Such wastes include a variety of materials such as sawdust, poplar trees, sugarcane bagasse, waste paper, brewers spent grains, switchgrass, and straws, stems, stalks, leaves, husks, shells and peels from cereals like rice, wheat, corn, sorghum and barley, among others. Lignocellulose wastes are accumulated every year in large quantities, causing environmental problems. However, due to their chemical composition based on sugars and other compounds of interest, they could be utilized for the production of a number of value added products, such as ethanol, food additives, organic acids, enzymes, and others. Therefore, besides the environmental problems caused by their accumulation in the nature, the non-use of these materials constitutes a loss of potentially valuable sources. (Teixeira, 2010) The major constituents of lignocellulose are cellulose, hemicellulose, and lignin, polymers that are closely associated with each other constituting the cellular complex of the vegetal biomass. Basically, cellulose forms a skeleton which is surrounded by hemicellulose and lignin as shown in Figure 1.

Figure 1: Representation of lignocellulose structure showing cellulose, hemicellulose and lignin fractions (Teixeira, 2010).

Cellulose is the main structural constituent in plant cell walls and is found in an organized fibrous structure. The structure of cellulose is shown in Figure 1. Cellulose is a high molecular weight linear homopolymer of repeated units of cellobiose (two anhydrous glucose rings joined

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via a -1,4 glycosidic linkage). The long-chain cellulose polymers are linked together by hydrogen and van der Walls bonds, which cause the cellulose to be packed into microfibrils [2]. By forming these hydrogen bounds, the chains tend to arrange in parallel and form a crystalline structure. Therefore, cellulose microfibrils have both highly crystalline regions (around 2/3 of the total cellulose) and less-ordered amorphous regions. Cellulose is more susceptible to enzymatic degradation in its amorphous form. More ordered or crystalline cellulose is less soluble and less degradable (Teixeira, 2010).

Figure 2: Illustration of a Cellulose Chain

Hemicellulose is a linear and branched heterogeneous polymer typically made up of five different sugars - Larabinose, D-galactose, D-glucose, D-mannose, and D-xylose - as well as other components such as acetic, glucuronic, and ferulic acids. The backbone of the chains of hemicelluloses can be a homopolymer (generally consisting of single sugar repeat unit) or a heteropolymer (mixture of different sugars). According to the main sugar residue in the backbone, hemicellulose has different classifications e.g., xylans, mannans, glucans, glucuronoxylans, arabinoxylans, glucomannans, galactomannans, galactoglucomannans, -glucans, and

xyloglucans (Teixeira, 2010). When compared to cellulose, hemicelluloses differ thus by composition of sugar units, by presence of shorter chains, by a branching of main chain molecules, and to be amorphous, which made its structure easier to hydrolyze than cellulose. The polymers do not aggregate, even when they cocrystallize with cellulose chains. (Parveen Kumar, 2009)

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Lignin is a very complex molecule constructed of phenylpropane units linked in a large threedimensional structure. Three phenyl propionic alcohols exist as monomers of lignin: p-coumaryl alcohol, coniferyl alcohol and sinapyl alcohol. Lignin is closely bound to cellulose and hemicellulose and its function is to provide rigidity and cohesion to the material cell wall, to confer water impermeability to xylem vessels, and to form a physicchemical barrier against microbial attack. Due to its molecular configuration, lignins are extremely resistant to chemical and enzymatic degradation. (Teixeira, 2010)

Figure 3: Lignin Structure (Spiller, 2001)

The amounts of carbohydrate polymers and lignin vary from one plant species to another. In addition, the ratios between various constituents in a single plant may also vary with age, stage of growth, and other conditions. However, cellulose is usually the dominant structural polysaccharide of plant cell walls (3550%), followed by hemicelluloses (2035%) and lignin (1025%). Average values of the main components in some lignocellulose wastes are shown in Table 1 Main components of Lignocellulosic Waste.

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Table 1 Main components of Lignocellulosic Waste (Parveen Kumar, 2009)

The corn cobs and pinewood are therefore expected to have similar yields.

2.3 Historical Development


2.3.1 Early sulphuric acid wood lignin methods (prior 1920)

The use of a two-stage sulphuric acid hydrolysis for the analysis of lignin dates to the turn of the 20th century, although the use of concentrated acid to release sugars from wood dates to the early 19th century . Klason, in 1906, is often credited as the first to use sulphuric acid to isolate lignin from wood. The method became named after Klason, and the insoluble residue from the test is known as Klason lignin. An English translation of a Klason paper, from this period, describes his attempt to determine the structure of spruce wood lignin. Klasons method originally used 72wt % sulphuric acid; he later reduced this to 66 wt% to gelatinize the wood. He filtered the solids and subjected them to a second hydrolysis in 0.5 wt % hydrochloric acid. (Justin B. Slutter, Raymond O. Rutz, Christopher J. Scarlata, Amie D. Slutter And David W. Templeton, 2010)

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2.3.2 Wood Lignin Isolation Methods (1920-1940) In 1922, Mahood and Cable chose a 72 wt% sulphuric acid lignin method and analyzed sawdust after extracting it in an alcohol/benzene solvent. They used a 16 h, room temperature, 72 wt % primary hydrolysis step followed by dilution with water to 3 wt% acid for a 2 h, reflux boiling secondary hydrolysis. (Justin B. Slutter, 2010) In 1932, Sherrard and Harris gave an extensive history of different lignin isolation methods plus added hot and cold water extractions to the sample preparation. They tested different primary hydrolysis conditions (70 wt % sulphuric acid, 10C) on isolated maple cellulose (not whole wood). Their conditions minimized carbohydrate charring into insoluble products that would contaminate large-scale lignin preparations. (Sherrard & Harris, 1932) Ritter et al., that same year, studied different hydrolysis temperatures and times and suggested modifications (2 h, 20C) to the primary hydrolysis. This was found to give lower lignin values (i.e., less interference) with a faster and more consistent primary hydrolysis. Ritter and Barbour, in 1935, suggested running a third 95% alcohol extraction, prior to the alcohol/benzene and water extractions, on some types of wood, such as redwood and white oak, to remove catechol tannins prior to lignin determination. (Ritter & Barbour, 1935) 2.3.3 Wood Sugar Quantification Methods (1930-1970) Ritter et al., in 1933, sought to determine the causes for the wide variability in reducing sugar yields (not lignin yields) seen by previous researchers. They tested different primary hydrolysis time and temperature conditions on isolated spruce cellulose (not whole wood) and found maximum yields at 6 h at 16C or 2 h at 35C. They tested 4 wt% sulphuric acid, rather than 3 wt %, for the secondary hydrolysis without explanation. Saeman et al., in 1945, suggested changes to the Ritter et al. methods in an effort to speed analysis times while measuring the effect on reducing sugar yield rather than on lignin yield. The authors adjusted both hydrolysis steps to speed the analysis. They tested a 45 min, 30C, primary hydrolysis step and a 1 h autoclave secondary hydrolysis, instead of a 4 h boiling water reflux step. That same year, Saeman et al. described three sugar detection techniques for analyzing wood

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acid hydrolysates. The authors reported an electrometric titration, a reducing sugar assay, or a yeast sorption (i.e., a standardized fermentation) method to measure the sugars released from lignin hydrolyses. Saeman et al., in 1954, published an updated version of their earlier method in the TAPPI journal. The authors added a paper chromatography sugar detection method and introduced the use of loss factors to correct the sugars for degradation during hydrolysis. Paper chromatography became the standard sugar separation and quantification method for nearly 20 years. (Justin B. Slutter, 2010) 2.3.4 Wood Lignin Methods applied to Herbaceous Feedstocks (1930-1955) Peterson et al., in 1932, tested different primary hydrolysis temperatures (with a constant 18 h duration) on lignin measurements when applied to corn stalks and wood. Also in the 1930s, Norman and Jenkins (26,27) and Norman (28) studied the causes of interferences with the lignin method applied to straws and some grasses. They found that certain carbohydrates, protein, or nitrogenous substances could coprecipitate with lignin. Dunning and Lathrop, in 1945, described a slightly modified hydrolysis method and applied it to an acid saccharification process on agricultural residues. Four years later, Dunning and Dallas described a faster hydrolysis method that included a 55C, 5 min primary hydrolysis and a 25 min autoclave secondary hydrolysis. This method was used for control analyses supporting a semiworks pilot plant (1000 pounds per run) hydrolyzing agricultural residues. The authors describe factors used to correct values to corresponding Association of Official Analytical Chemists (AOAC) methods for pentosans, which suggests the methods were precise and fast, but not necessarily accurate. (Justin B. Slutter, 2010) 2.3.5 Applications to Food, Feed and Dietary Fiber (1970-1990) Prior to the 1970s, the sulphuric acid methods had been applied mainly to the analysis of wood, now these methods would be applied to different substrates. In 1971, Sloneker , of the USDA Northern Regional Research Laboratory, tested a miniaturized version of the Saeman hydrolysis to the analysis of total aldoses in foods, feeds, and feedlot wastes. He added a new sugar detection method that used gas-liquid chromatography (GC) to quantify the hydrolyzed sugars, after derivatizing them to alditol acetates. (Justin B. Slutter, 2010). The 72 wt% sulphuric acid

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hydrolysis methods were adapted for measurement of dietary fiber by various researchers for dietary fiber analyses in human nutrition research, and these methods were later adapted for biomass analyses. (Justin B. Slutter, 2010) 2.3.6 Early Applications to Biofuels Feedstocks (1980-1998) In 1984, Grohmann et al. described the hydrolysis of wheat straw using less severe hydrolysis conditions (Grohmann, Himmel, Rivard, Tucker, & Baker, 1984). They found low glucose yields (50%) on Sigmacell 50 using the regular Moore and Johnson method, and they attributed the low yield to undetected sulphonated glucose (Justin B. Slutter, 2010). Pettersen et al. of FPL compared the analysis of wood hydrolysate sugars by HPLC with paper chromatography, based on Saemans method (Pettersen, Schwandt, & Effland, 1984). The authors found some statistically significant differences between the methods, but they were judged to be small enough to make the HPLC method useful. Pettersen, also in 1984, published wood composition data collected from 1927 to 1968 and briefly described the FPL analytical methods used to generate the data. In 1991, Pettersen and Schwandt used anion chromatography and pulsed amperometric detection (PAD) to analyze wood sugar solutions. This was followed in 1998 with a paper by Davis M. W. in 1998 describing an updated anion chromatography/PAD method with improved peak resolution and sample throughput. In 1985, Theander, from the Swedish University of Agricultural Sciences in Uppsala, applied the sulphuric acid dietary fiber methods to plant materials for use as biofuels feedstocks. In 1991, he added 80%ethanol to extract lignocellulosic materials prior to sulphuric acid hydrolysis (Theander, 1991). Kaar et al., in 1991, developed a rapid, reliable summative analysis method for the analysis of wood. The authors performed the autoclave step in sealed bombs, thus retaining the volatile acetic acid and 2-furaldehyde, and analyzed the hydrolysates by HPLC. The authors measured hydroxymethylfurfural (HMF), furfural, acetic acid, levulinic acid, and uronic acids in the hydrolysates and added these amounts into their glucan and xylan components, instead of measuring sugar loss factors.

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2.3.7 Contemporary Contributions to Biomass Methods (1995-2010) Recently proposed modifications to the sulphuric acid method include a 1997 paper from Thammasouk et al. describing the effects of water or ethanol extractions on the compositions of switchgrass, corn stover, and fescue. Generally, extracted biomass had lower lignin or glucan values compared to the native biomass. These extractions were assumed to have removed interferences and led to more accurate lignin or glucan values. A decade later, Foyle et al. compared different hydrolysis methods for the analysis of waste paper and wheat straw and settled on a modified version of the Grohmann et al.method. Also that same year,Moxley and Zhang reported a modified quantitative saccharification method. They suggested that a separate 1% acid hydrolysis corrected for loss with an associated 1% sugar recovery standard is more appropriate to correct the labile sugars, xylose and arabinose. They did not present complete summative component closure data thatwould help confirm this hypothesis. (Foyle, Jennings, & Mulcahy, 2007) In 2007,Chen et al. analyzed the components found in water extractives from corn stover. Hames wrote a 2009 book chapter describing compositional analysis methods applicable to biomass samples. Dien wrote a 2010 book chapter discussing sulphuric acid biomass compositional analytical methods and their use for yield calculations needed to compare pretreatment experiments (Dien, 2010). Chen et al., in 2010, analyzed the water-extractable components found in switchgrass (Chen, Mowery, Svecik, Scarlata, & Chambliss, 2010)

2.4 Pretreatment Methods


The structure of ligno-cellulosics in the cell wall resembles that of a reinforced concrete pillar with the cellulose fibers being the metal rods and lignin the matrix cement. The carbohydrate polymers are tightly bound to the lignin, mainly by hydrogen bonds but also by some covalent bonds. The biodegradation of the untreated ligno-cellulose is slow and the extent of the degradation is often low. Hence, treatment of the biomass is essential in order to increase the

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accessibility and enzymatic hydrolysis. (Ashok Pandey, 2008)

Figure 4: Schematic of pretreatment effect on lignocellulosic Biomass

A large number of pretreatments exist which can be broadly classified into physical, chemical, physicochemical, and biological shown in .Sometimes a combination of two or more pretreatments is employed. These pretreatments open the structure of the potential cellulose substrate. An efficient pretreatment method is one that increases accessibility to the cellulase and enhances the complete solubilization of the polymer to monomer sugars without formation of degradation products.
Table 2 Pretreatment Methods (Ashok Pandey, 2008)

Pretreatment Methods Physical Ball milling, two-roll milling, hammer milling, colloid milling, vibro energy milling, pyrolysis, -irradiation, microwave irradiation Chemicals AlkaliNaOH, NH3, ammonium sulfite AcidH2SO4, H3PO4, HCl GasesClO2, NO2, SO2 Oxidizing agentsH2O2, ozone Cellulose dissolving agentscadoxen, CMCS, phosphoric acid/acetone, ionic liquids Solvent extractionethanol-water, benzene-ethanol, butanol-water, ethylene glycol Physiochemical Steam explosion (SE), SO2-catalyzed SE, CO2 explosion, SC-CO2 explosion,

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Biological

ammonia freeze explosion (AFEX) Fungi

2.4.1 Physical Pretreatments The crystalline structure excludes water molecules and other large molecules such as enzymes. The smaller particles have a large surface-to-volume ratio. The surface area available for the enzyme-substrate interaction is influenced by the pore size and shielding effect by the hemicelluloses. Physical treatments such as grinding, milling, high temperature, freeze/thaw cycles, and radiation are aimed at size reduction and mechanical decrystallization. Mechanical methods such as ball milling, two roll milling, colloid milling, and non-mechanical methods such as -irradiation, high-pressure steaming, and pyrolysis have all been attempted to change one or more structural features of the cellulose and enhance the hydrolysis. Most of these methods are limited in their effectiveness and often expensive. 2.4.1.1 Milling Milling reduces the particle size and crystallinity and increases the surface area and the bulk density. This method can be used for a variety of substrates but is highly energy intensive. Ball milling and two-roll milling have been found to increase the susceptibility of the cellulose to enzyme action. Fitz milling results in size reduction without changing the crystallinity and wet milling results in fibrillation and delamination of the cellulose with no change in the chain length and crystallinity due to the plastisizing action of water. (Ashok Pandey, 2008) 2.4.1.1.1 Ball milling The shearing and compressive forces of ball milling cause reduction in the crystallinity, decrease in the degree of polymerization (DP), decrease in the particle size, increase in the bulk density, and increase in the external surface area. Increase in the bulk density allows use of high substrate concentrations, and reduces the reactor volume and the capital cost. Milling at elevated temperatures shows an increase in the rate of enzymatic hydrolysis compared to milling at room temperature. Ball-milled cellulose can be completely hydrolyzed to sugars. However, the effectiveness of the milling varies with the cellulosic source, and softwood shows the least

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response. Although this is an effective treatment, time and cost make it prohibitive for use on a large scale. (Ashok Pandey, 2008) 2.4.1.1.2 Two-roll milling This mill consists of two cast-iron tempered surface rolls placed horizontally, with roll clearance that can be adjusted by screws. The cellulose substrates are fed into the roll and masticated for a specific period of time. The pretreated material is then scraped off. A variety of substrates, including cotton, maple chips, white pine chips, newspaper, etc., have been subjected to two-roll milling, also called differential roll milling. This method reduces the crystallinity as well as the DP and increases the bulk density. (Ashok Pandey, 2008) 2.4.1.1.3 Hammer milling A hammer mill consists of a rotor with a set of attached hammers. As the rotor turns, the hammers impact the substrate against a breaker plate. The hammer milling of cellulose improves the digestibility of newsprint to a limited extent. Prolonged hammering of the substrate is not recommended as it reduces the susceptibility of the cellulose to enzymatic hydrolysis. (Ashok Pandey, 2008) 2.4.1.1.4 Colloid milling A colloid mill consists of two disks set close to each other, revolving in opposite directions, while the substrate slurry is passed between the disks. There is only marginal improvement in the susceptibility of the cellulose to enzymatic hydrolysis and the method is uneconomical owing to the high operating cost. (Ashok Pandey, 2008) 2.4.1.1.5 Simultaneous milling and saccharification This method combines milling with saccharification in a single step. Simultaneous ball milling and enzymatic hydrolysis could improve the rate of saccharification and/or reduce the enzyme loading required to attain total hydrolysis. The effectiveness of the method depends on the lignified matrix of the cellulose microfibrils, the grinding elements, and the oscillation frequency

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of the shaker. While glass beads are effective for pure cellulose, stainless steel beads are more effective for lignocellulosics. (Ashok Pandey, 2008) 2.4.1.2 Effect of Temperature Freezing cellulosic materials in water suspension at -75C is reported to enhance chemical reactivity (as measured by dye absorption). The effect was more pronounced with repeated freezing and thawing cycles. The cryomilled cotton cellulose obtained by hammer milling in liquid nitrogen showed 36% more hydrolysis compared to untreated sample. Pyrolysis involves heating the biomass at 200C and is reported to increase hydrolysis. The type of gaseous atmosphere during pyrolysis affects the reaction. Pyrolysis in the presence of oxygen results in depolymerization, oxidation, and dehydration. In inert atmosphere, depolymerization is slow and by-product formation decreases. 2.4.1.3 Effect of -Irradiation High energy radiation was found to enhance in vitro digestibility as well as acid/enzymatic hydrolysis of the cellulose. The radiation treatments are effective in breaking the lignin-cellulose complex as evidenced by the increased presence of phenolics in the irradiated samples. The irradiation is reported to cause increase in the surface area, while its effect on the crystallinity of the cellulose is controversial. Irradiation in the presence of oxygen, milling, or the addition of nitrate salts, or treatment with acid or alkali prior to irradiation increased the digestibility of the treated sample. 2.4.2 Chemical Pretreatments There are two types of swelling of cellulose, intercrystalline and intracrystalline. Intercrystalline swelling can be affected by water and is a prerequisite for any microbial reaction to occur. Intracrystalline swelling requires a chemical agent that is capable of breaking the hydrogen bonds of the cellulose. Aqueous solutions of acid and alkali belong to this group of chemical agents. Chemical pretreatment approaches have gained significant attention to increase the accessibility to hydrolytic attack. A wide variety of chemicals as pretreatment agents have been reported in the literature, which include cellulose solvents, sodium hydroxide, aqueous ammonia, calcium

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hydroxide plus calcium carbonate, phosphoric acid, alkaline hydrogen peroxide, sulphur dioxide, carbon dioxide, inorganic salts with acidic properties, ammonium salts, Lewis acids and organic acid anhydrides, acetic acid, formic acid, sulphuric acid, organic solvents, n-butylamine, npropylamine, and alcohols such as methanol, ethanol, or butanol in the presence of an acid or alkaline catalyst. Chemical pretreatments are generally more effective in solubilizing a greater fraction of lignin while leaving behind much of the hemicelluloses in an insoluble polymeric form and opening up the crystalline cellulosic substrate. A few of the most commonly used pretreatment methods are discussed below. 2.4.2.1 Cellulose Dissolving Agents The cellulose dissolving agents fall into four groups: strong mineral acids such as H2SO4 and H3PO4, quaternary ammonium bases, transition metal complexes, and organic solvents. Strong mineral acids and transition metal complexes are commonly used as cellulose dissolving agents. Solvents such as cadoxen and CMCS are able to swell and transform solid cellulose into a soluble state. This ability to dissolve the cellulose has been exploited as a means of pretreatment. The crystalline structure of the native cellulose can be completely destroyed by dissolving in a solvent and on reprecipitation the cellulose is regenerated as a soft floc and is highly reactive. Enhancement in reactivity is observed both with acid and enzymatic hydrolysis and quantitative yields of sugar are obtained. Solvent pretreatment results in higher moisture regain values, larger pore size distribution, and lower crystallinity. The most common solvents are cadoxen, CMCS, H2SO4 and H3PO4. Only concentrated acids act as cellulose solvents. 2.4.2.2 Organic Solvents Delignification using organic solvents with mineral acids as catalysts has also been reported as a pretreatment method. This method breaks the internal lignin and hemicellulose bonds and separates the lignin and hemicellulose fractions that can be potentially converted to useful products. Methanol, ethanol, butanol, n-butylamine, acetone, ethylene glycol, etc., have been used in the organosolv process. Organic acids such as oxalic, acetylsalicylic, and salicylic acid can be used as catalysts. The hardwoods are readily delignified in acid-catalyzed systems, whereas

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softwoods require higher temperature. At high temperatures (above 185C), the addition of catalyst was unnecessary for satisfactory delignification. 2.4.2.3 Dilute Acids Those pretreatments that use dilute acid result in the hydrolysis of a significant amount of the hemicellulose fraction of biomass, leading to high yields of soluble sugars from the hemicellulose fraction. The hot-wash process, a variation of the dilute acid pretreatment, involves hightemperature separation and washing of the pretreated solids, which is thought to prevent reprecipitation of the lignin and/or xylan that may have been solubilized under the pretreatment conditions. The reprecipitation of the lignin can negatively affect the subsequent enzymatic hydrolysis. Complete removal of the hemicellulose from the lignocellulosic material during pretreatment is a necessary prerequisite for the successful enzymatic hydrolysis of the cellulosic fraction. Dilute acid pretreatment is effective in removing the hemicelluloses fraction from the lignocellulose. Hemicellulose removal increases the porosity of the native lignocellulosics and, thus, enzymatic accessibility to the cellulosic fraction. The amount of lignin and cellulose dissolved during this pretreatment method is usually minor. Dilute acid is an efficient pretreatment method suitable for all kinds of lignocellulosic substrates such as corn stover, newsprint, etc., to improve the enzymatic hydrolysis of substrates. 2.4.2.3.1 Sulphuric acid Acid catalyzed hydrolysis uses dilute sulphuric, hydrochloric, or nitric acids. Dilute sulphuric acid (0.51.5%) at temperatures above 160C was found to be most suitable for industrial application, because of its high sugar yields from the hemicellulose hydrolysis (xylose yields of 7590%). A dilute acid pretreatment method involving two steps was reported for hardwoods (Sunggyu Lee, 2007). In the first step, a temperature of 140C was used to hydrolyze the easily degradable fraction and in the second step, the temperature was slowly increased to 170C to hydrolyze the hemicellulose fractions that were more difficult to degrade. Treatment with 1 to 2%

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H2SO4 at less than 220C and retention times of a few minutes reduces the DP of the cellulose, while the crystallinity does not decrease. Enzymatic hydrolysis results in complete conversions within 24 h based on theoretical values. The acid has to be removed or neutralized before fermentation, yielding a large amount of gypsum. Although close to theoretical yields can be achieved, this process involves high capital investment, acid consumption, and acid recovery costs. 2.4.2.3.2 Peracetic acid Delignification of corn stalks and sawdust with 20% peracetic acid showed significant increase in the rate of enzymatic hydrolysis. Peracetic acid resulted in 76.2% delignification with concomitant increase in the surface area of wheat straw and drastic increase in digestibility. This method causes significant reduction in crystallinity due to structural swelling and dissolution of the crystalline cellulose. The oxidizing action of peracetic acid causes relatively low decrease in hemicellulose, leaving hemicellulose-rich material. 2.4.2.4 Alkali Pretreatment Among the various chemical pretreatments, alkali treatment with bases like sodium hydroxide is most widely used to enhance in vitro digestibility and enzymatic hydrolysis of the lignocellulose. All the lignin and part of the hemicellulose are removed and the reactivity of cellulose for hydrolysis is sufficiently increased. The success of this method depends on the amount of lignin in the biomass. 2.4.2.4.1 Sodium Hydroxide Sodium hydroxide is used as an intracrystalline swelling agent for both crystalline and amorphous cellulose. Dilute sodium hydroxide causes separation of the structural linkages between the lignin and carbohydrate and disruption in the lignin structure; swelling leads to increase in the internal surface area and pore size, as well as decrease in the DP and crystallinity. The mechanism is believed to be saponification of intermolecular ester bonds cross-linking the hemicelluloses and lignin. The porosity of the lignocellulosic materials increases with the removal of the cross links,

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leading to swelling, and enhances the accessibility to the enzymes. The extent of the hydrolysis increases with increase in NaOH concentration used for pretreatment. It was observed that following alkali treatment, digestibility of the softwoods increases slightly as compared to hardwoods. This difference in response appears to be related to the lignin content of the wood. 2.4.2.4.2 Ammonia Ammonia exerts a strong swelling action and brings about phase change in the cellulose crystal structure. The benefits of this method include breakage of glucuronic acid ester crosslinks, solubilization of lignin, and disruption of crystalline structure, swelling and Increase in the accessible surface area of cellulose. Pretreatment with liquid ammonia has been used mostly to increase in vitro digestibility of animal feed. Wheat straw treated with 50% ammonium hydroxide showed 20% delignification, with threefold increase in hydrolysis. 2.4.2.4.3 Alkaline Oxidation Pretreatment This method is a combination of the alkaline and oxidative pretreatments. Pretreatment with H2O2 in an alkaline environment or combining it with a preceding alkali treatment step is an effective pretreatment for lignocelluloses. In weak alkaline media, H2O2 only selectively acts on phenolic compounds originated from partial scission of lignin, causing its degradation without affecting the cellulosic fraction. Only the lignin and hemicellulose are solubilized. 2.4.2.5 Gases Pretreatment with gases has the advantage that gases can penetrate uniformly throughout the substrate. However, their recovery for reuse poses problems. Chlorine dioxide, nitrogen dioxide, sulphur dioxide, sodium hypochlorite, HCl gas, and ozone have been used as pretreatment agents to solubilize the lignin and increase in vitro digestibility. Chlorine dioxide is an active agent in chlorine pulping to solubilize the lignin. Sulphur dioxide disrupts the lignin-carbohydrate complex and depolymerizes lignin.

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2.4.3 Physicochemical Pretreatments Several pretreatment processes combine physical and chemical methods. In this regard, high pressure steaming, with or without rapid decompression (explosion), has been claimed as one of the most successful options for fractionating wood into its three major components and enhancing the susceptibility of the cellulose to enzymatic attack. 2.4.3.1 Steam Treatment (Autohydrolysis) One of the most common physicochemical pretreatment methods is steam explosion or liquid hot water (LHW) treatment. This process involves treatment with steam under high pressure and temperature, followed by quick release of the pressure, causing the biomass to undergo an explosion and shatter the structure in a popcorn-like effect. The advantages of this method are the low energy input and negligible environmental impact. However, steam explosion does not always break down all the lignin, requires small particle size, and can produce compounds that may inhibit subsequent fermentation. Despite these drawbacks, steam explosion is currently the most popular method for separating the lignin and hemicellulose from the cellulose. 2.4.3.2 Acid-Catalyzed Steam Explosion The addition of dilute acid in the steam explosion can effectively improve enzymatic hydrolysis, decrease the production of inhibitory compounds, and lead to more complete removal of the hemicellulose. Limitations include destruction of a portion of the xylan fraction, incomplete disruption of the biomass structure, and the generation of inhibitory compounds. The necessary water wash decreases the overall sugar yields. 2.4.3.3 Ammonia and Steam Explosion The lignocellulosic materials can also be exploded using ammonia and involves liquid ammonia and steam explosion. This method is considered one of the leading biomass pretreatments. Ammonia freeze explosion (AFEX) treats the biomass with concentrated ammonia under pressure and at temperatures up to about 100C. After a few minutes under these conditions, the pressure is rapidly released (the explosion). The ammonia evaporates and is recovered. AFEX disrupts the

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lignocellulose and reduces the cellulase requirement but removes neither hemicelluloses nor lignin. The treated biomass is now much more easily converted by enzymes to sugars and then to ethanol. 2.4.3.4 CO2 -Catalyzed Steam Explosion CO2 explosion is similar to steam and ammonia explosion. The glucose yields in the later enzymatic hydrolysis are lower compared to steam and ammonia explosion. CO2 explosion is more cost effective than ammonia explosion and does not cause the formation of inhibitors as in steam explosion. 2.4.3.5 Supercritical Carbon Dioxide (SC -CO2) Supercritical carbon dioxide is carbon dioxide above its critical point of 31C and 73 atm. This pretreatment has many advantages, such as nontoxicity, low solvent cost, (CO2), low pretreatment temperatures, easy recovery of CO2, and high solids concentrations in the pretreated materials. 2.4.3.6 Advantages and Disadvantages of the Steam Explosion 2.4.3.6.1 Advantages Ability to separate the three components of wood: modifies lignocellulose to allow fractionation of hemicellulose in autohydrolysis steam, lignin in aqueous alcohol or alkali, and cellulose as insoluble biomass. Cellulose is highly susceptible to acid or enzymatic hydrolysis. Lignin in suitable form for conversion to chemicals. Hemicellulose is easily converted to liquid fuels. Inhibitors are easily extractable. 2.4.3.6.2 Disadvantages Produces substrates with low bulk density. Does not always break down the lignin completely. Requires small particle size.

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Some of the compounds produced can be inhibitory to the subsequent ethanol fermentation. A limitation of all the pretreatment processes is their capital intensive nature. 2.4.4 Biological Pretreatments In these pretreatments, the natural wood attacking microorganisms that can degrade lignin are allowed to grow on the biomass, resulting in lignin degradation. The main biological pretreatments include fungi and their enzymes. There is significant loss of the xylan and mannan components of the hemicellulose during the lignin hydrolysis. This is the most promising organism for biological pretreatment of lignocellulose. The various means to use these organisms are: use of naturally occurring white-rot fungi; use of cellulose-less mutants as efficient lignin degraders and/ or to repress the enzymes that degrade wood carbohydrates.

2.5 Hydrolysis Methods


The most commonly considered hydrolysis processes are the concentrated hydrochloric acid process, the two-step dilute acid hydrolysis, and enzymatic hydrolysis. During the hydrolysis of lignocellulosic materials a wide range of compounds are released which are inhibitory to microbial fermentation. The composition of the inhibitors differs depending on the type of lignocelluIosic hydrolysates. 2.5.1 Acid Hydrolysis 2.5.1.1 Dilute Acid Hydrolysis Dilute acid hydrolysis of biomass is the oldest technology for converting biomass to ethanol. The hydrolysis occurs in two stages to accommodate the differences between the hemicellulose and the cellulose and to maximize the sugar yields from the hemicellulose and cellulose fractions of the biomass. The first stage is operated under milder conditions to hydrolyze the hemicellulose, while the second stage is optimized to hydrolyze the more resistant cellulose fraction. The liquid hydrolysates are recovered from each stage, neutralized, and fermented to ethanol. (Sunggyu Lee, 2007)

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During the degradation of the lignocellulosic structure, not only fermentable sugars are released, but a broad range of compounds, some of which might inhibit the fermenting microorganism. In the prehydrolysis step, the hemicellulose is liquefied, resulting in a mixture of mono- and oligosaccharides. The hydrolysis of the cellulose is usually performed by weak acids or by enzymes. 2.5.1.2 Concentrated Acid Hydrolysis This process is based on concentrated acid decrystallization of the cellulose followed by dilute acid hydrolysis to sugars at near theoretical yields. The separation of acid from the sugars, acid recovery, and acid reconcentration are critical operations. The fermentation converts sugars to ethanol. Concentrated hydrochloric acid has also been utilized where the prehydrolysis and hydrolysis are carried out in one step. Generally, acid hydrolysis procedures give rise to a broad range of compounds in the resulting hydrolysate, some of which might negatively influence the subsequent steps in the process. A weak acid hydrolysis process is often combined with a weak acid prehydrolysis. 2.5.2 Enzymatic Hydrolysis of Lignocellulosic Biomass The enzymatic hydrolysis or saccharification of lignocellulosic biomass is preceded by a pretreatment process in which the lignin component is separated from the cellulose and hemicellulose to make it amenable to the enzymatic hydrolysis. The lignin interferes with hydrolysis by blocking the access of the cellulases to the cellulose and by irreversibly binding the hydrolytic enzymes. Therefore, the removal of the lignin can dramatically increase the hydrolysis rate. For the efficient enzymatic hydrolysis of lignocellulosic biomass a pretreatment step is necessary. 2.5.2.1 Factors Governing Enzymatic Hydrolysis There are different factors that affect the enzymatic hydrolysis of cellulose, namely, substrates, cellulase activity, and reaction conditions (temperature, pH, as well as other parameters). To

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improve the yield and rate of enzymatic hydrolysis, research has been focused on optimizing the hydrolysis process and enhancing the cellulose activity. The yield and initial rate of enzymatic hydrolysis of cellulose is affected mainly by the substrate concentration. At low substrate levels, an increase of substrate concentrations yields an increase in the reaction rate of the hydrolysis and the products. However, substrate inhibition is caused at high substrate concentration, which considerably lowers the rate of hydrolysis. The ratio of the enzyme to substrate in the hydrolysis reaction is crucial to establish the level of substrate inhibition. The hydrolysis of cellulosic substrates by the enzymes depend to a large extent on the structural features of the substrate, such as cellulose crystallinity, degree of cellulose polymerization, surface area, and content of lignin. The yield and rate of hydrolysis of the cellulosic substrate can be increased to a certain extent by increasing the dosage of the cellulases in the process, but that would significantly increase the cost of the process. The adsorption of the cellulase enzymes onto the surface of the cellulose, the biodegradation of cellulose to fermentable sugars, and desorption of the cellulase are three steps involved in enzymatic hydrolysis of the cellulose. The cellulase activity decreases during hydrolysis because of the irreversible adsorption of the cellulase on the cellulose. The cellulose surface property can be modified and the irreversible binding of the cellulase can be minimized by the addition of surfactants during the hydrolysis.

2.6 Fermentation
The hydrolysis of lignocellulosic biomass yields reducing sugars. Once the sugars are available, its fermentation to ethanol is not a difficult task as many technologies have been developed. Essentially, there are three different types of processes by which this can be achieved, namely, Separate hydrolysis and fermentation (SHF) Direct microbial conversion (DMC) Simultaneous saccharification and fermentation (SSF) SSF has been shown to be the most promising approach to biochemically convert cellulose to ethanol in an effective way.

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2.6.1 Separate Hydrolysis and Fermentation (SHF) This is a conventional two-step process where the lignocellulose is hydrolyzed using enzymes to form reducing sugars in the first step and the sugars thus formed are fermented to ethanol in the second step using Saccharomyces or Zymomonas. 2.6.2 Direct Microbial Conversion (DMC) This process involves three major steps, namely, enzyme production, hydrolysis of the lignocellulosic biomass, and the fermentation of the sugars, all occurring in one step .The relatively lower tolerance of the ethanol is the main disadvantage of this process. A lower tolerance limit of about 3.5% has been reported as compared to 10% of ethanologenic yeasts. Acetic acid and lactic acid are also formed as by-products in this process in which a significant amount of carbon is utilized . Neurospora crassa is known to produce ethanol directly from cellulose/hemicellulose, because it produces both cellulase and xylanase and also has the capacity to ferment the sugars to ethanol anaerobically . 2.6.3 Simultaneous Saccharification and Fermentation (SSF) The saccharification of lignocellulosic biomass by enzymes and the subsequent fermentation of the sugars to ethanol by yeast such as Saccharomyces or Zymomonas take place in the same vessel in this process . The compatibility of both saccharification and fermentation processes with respect to various conditions, such as pH, temperature, substrate concentration, etc., is one of the most important factors governing the success of the SSF process.

2.7 Reducing Sugars Test


Reducing sugar determination, although not saccharide specific, is nevertheless often carried out as this analysis most commonly indicates glucose and fructose concentrations in the sample. The two methods considered for the determination of the reducing sugars content were the Lane and Eynon method and the High Performance Liquid Chromatography (HPLC).

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2.7.1 Lane and Eynon Method The procedure for the Lane and Eynon titration method is based on the ability of reducing sugars carbonyl group to react with an alkaline cupric complex which is alkaline in nature and reduce it to cuprous oxide. The source of the alkaline copper solution comes from Fehlings solution which is made up of two reagents- CuSO4 solution (Fehling A) and sodium potassium tartrate and NaOH solutions (Fehling B) which is present in equal proportions. When the Fehlings solution is boiled with the reducing sugar, the blue colour due to the cupric tartrate is discharged and a red precipitate of Cu2O forms indicated by orange-red colour of the liquid. Time, temperature, and reactant concentrations must be controlled. (Scales, 2011) This method takes part in the following steps: A bulk of the solution containing the carbohydrate is added to the boiling Fehlings solution after which the remaining carbohydrate solution (usually less than 1 ml) is added drop by drop until the blue colour is almost discharged. The endpoint is difficult to detect and thus a redox indicator should be used. The indicator usually used is aqueous methylene blue solution (1%). Titration is continued until the colour of the indicator is completely discharged. 2.7.2 High Performance Liquid Chromatography High Performance Liquid Chromatography (HPLC) is one of the most common analytical techniques used today. It is a form of column chromatography in which analytes are under high pressure and separation of the analytes is based on the polarities and interaction with a stationary phase. The units comprising an HPLC system are an injector valve, column, pump, mobile phase (eluent) and detector. The mixture is separated into its components by the interaction of the solutes with the column stationary phase (immobile packing in the column) and the mobile phase (liquid phase). Figure 5 below shows manner in which different components (indicated by red and blue) of a sample are eluted.

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Figure 5: Steps involved in column chromatography

This HPLC method is the preferred method for the analysis, however the proper columns and standards were not available for use.

2.8 Product Recovery Process


Distillation is an energy-intensive separation process used to recover water-soluble products from fermentation. Distillation separates two liquids by taking advantage of their difference in boiling point temperatures. A distillation still consists of a vessel to heat the combined liquids to boiling, a condenser to cool the vapors back into liquid, and receivers to collect the concentrated liquids. Distillation follows one of two main methods. In the first method, the heated mixture may consist of two liquids with significantly different boiling points. The vapor that is given off will be a majority of one or the other liquid, which after condensation and collection effects the separation. A second method, fractional distillation, is more effective at separating liquids with similar boiling points and therefore applies better for the expected ethanol-water mixture. This method relies upon a gradient of temperatures existing in the condenser stage of the equipment. Often in this technique, a vertical condenser, or column, is used. By extracting products that are liquid at different heights up the column, it is possible to extract liquids that have different boiling (and condensing) temperatures. Under ideal conditions, distillation can be used to effect a nearcomplete separation of one type of liquid from another. The fractions can be purified further by a second distillation.

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Distillation is not effective for recovering many fermentation products. Even where distillation may work, lower cost, lower energy separation techniques are being developed and used. In the ethanol industry, distillation accounts for about 40% of the total energy needed for corn-toethanol conversion (Vane, 2003) . Other recovery methods include precipitation and other chemical based techniques, and various types of membrane separation. Organic acid fermentation products may be recovered as salts of calcium and other metals. Recovery involves concentration of the salt, and conversion back into the acid. Other techniques use caustic chemicals to precipitate fermentation products. Recovery through these methods results in large volumes of waste salt materials such as gypsum. (Energy Center of Wisconsin, 2004) Membrane separation technology applies to the use of a membrane, an engineered barrier with special properties that restricts the transport of various chemicals in a selective manner. Transport through the barrier by selective chemicals may be driven by convection, diffusion, or electric charge (electrodialysis), or by pressure, temperature, or concentration differences. Membranes can provide substantial energy savings over distillation, and offer flexibility and modularity in design. A relatively new technology called pervaporation may provide considerable energy savings over traditional distillation technologies. (Energy Center of Wisconsin, 2004) Pervaporation is a membrane-based process used to separate and concentrate volatile compounds from a liquid mixture by selective permeation through a non-porous membrane into a vacuum permeate stream. The remaining liquid thus becomes depleted in the compound that permeates the membrane.

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3 METHODOLOGY
3.1 Apparatus and Materials
Autoclave Beakers Buchner Funnel Burettes Conical Flasks Digital Balance Dropper Fermentation vessels Fermentation Locks Filter Paper Fractional distillation equipment Funnels Glass rods Refractometer Large bucket Magnetic Stirrers Measuring Cylinders Oven pH meter Pycnometer Pipette Scale Stopwatch Vacuum Pump Volumetric Flasks Wash bottles Water bath Wiley mill

3.2 Reagents
Alkaline Tartrate Solution Calcium Carbonate Concentrated Hydrochloric Acid Copper Sulphate solution Distilled water Yeast Methylene Blue indicator Molasses Concentrated Sodium Hydroxide Sucrose Wine Yeast Substrates ( Corn Cobs & Pine Sawdust)

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3.3 Experimental Procedure


3.3.1 Mechanical Degradation 1. Firstly, 2kg of the raw materials, corn cobs and pine sawdust, were grinded using the Wiley mill to produce a finely shredded material. 2. The milled material was then subjected to a moderate temperature of 50 C for a period of 4hrs to drive off all the moisture.

Figure 6: Dried Corn cobs

3. The substrates were weighed into 45 gram samples and stored until use.

Figure 7: A 45 gram dried corn cob sample

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3.3.2 Pre-hydrolysis 4. A 3000 ml Sulphuric acid solution of concentration 10% was made up from dilution of a 96% solution. 5. 45 grams of the dried corn cobs was placed in a 500ml conical flask and 225ml of the 10% sulphuric acid added, after which it was sealed off and placed in a water bath at 90 degrees for 60 minutes. 6. The resulting suspension was the filtered using the 150 mesh screen keeping both the filtrate and residue.

Figure 8 : Suspension being filtered using the Buchner Funnel

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Figure 9: Filtrates before neutralisation

7. The filtrate was then neutralized using CaCO3 until the fizzing stopped and a pH between 5.5 and 7.5 was achieved. This was filtered using a Buchner funnel and the filtrate kept as the pre-hydrolysates. The residue was discarded. 8. The residue from the first filtration was used for the hydrolysis step. 3.3.3 Hydrolysis 9. Sulphuric acid solutions of concentrations of 20%, 30% and 40 vol. % were made up by dilution of a 96% solution. 10. The residue from the pre-hydrolysis in step 6 was placed in a 500ml conical flask and 225ml of the 20% sulphuric acid added, after which it was sealed off and placed in a water bath at 90 degrees for 60 minutes. 11. The resulting suspension was the filtered using the 150 mesh screen keeping only the filtrate. 12. The filtrate was then neutralized using CaCO3 until the fizzing stopped and a pH between 5.5 and 7.5 was achieved. This was filtered using a Buchner funnel and the filtrate kept as the hydrolysates. The residue was discarded. 13. The steps were repeated using the different concentrations of 20%, 30% and 40% and for each substrate. All were done in duplicates.

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3.3.4 Fermentation 14. The pre-hydrolysates and hydrolysates were mixed and the brix of the mixture recorded.

Figure 10: The Pre-hydrolysates and Hydrolysates before mixing.

15. All apparatus to be used for the fermentation process were autoclaved for 30 minutes. 16. 100 ml of molasses stock was added to each of the samples to bring the brix into the fermentable range. The resulting brix was also recorded. The samples were autoclaved for 30 minutes. 17. Approximately 0.2 g of wine yeast was added and the vessels sealed and left for 12days, during which the brix was recorded daily. 18. Fractional distillation was used to recover the ethanol.

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Figure 11: Ethanol Recovery (Distillation Apparatus)

19. The density of the bioethanol was then found using a pycnometer and the total weight and volume percent calculated.

Figure 12: Pycnometer used to determine the specific gravity and hence Ethanol content.

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3.3.5 Determination of Reducing Sugars content: Lane and Eynon Method 3.3.5.1 Inversion of Sample 1. A 10 ml aliquot of the hydrolysates sample was placed in a beaker using a pipette and 5 ml of concentrated Hydrochloric Acid added to invert sample. This was left overnight. 2. The solution was then neutralized using 20% NaOH and a few drops of phenolphthalein. 3. The sample was made up to 200 ml in volumetric flask. 4. A 10 ml aliquot was pipette into conical flask and this used for the titration. 3.3.5.2 Determination of Total Sugars (Reference: AOAC Pgs 533, 539 - 540) 3.3.5.2.1 Reagents Preparation 1. Copper Sulphate Solution (Fehlings 1) Dissolved 6.93 gm CuSO4 5H2O in H2O, diluted to 100 ml and filtered through filter paper. 2. Alkaline Tartrate Solution (Fehlings 11) Dissolved 34.69 gm KNa (tartrate), 4H2 O (Rochelle Salt) and 50 gm NaOH in H2O. Diluted to 100 ml. Left to stand for 2 days and then filtered. 3. Invert sugar standard Solution 1% To a solution of 9.5 gm of pure sucrose, 5 ml concentrated HCl was added and diluted with H2O to 100 ml. This was stored for 3 days at room temperature and then dilute to 1 liter. 4. Working solution 5 mg/ml 100 ml of the stock 1% Invert Sugar solution was placed into a 200 ml volumetric flask using a pipette. It was neutralized with 20% NaOH and checked with a few drops phenolphthalelin. It was diluted to 200 ml and mix well. This solution was prepared fresh daily. 5. 1% Methyl Blue Indicator.

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3.3.5.2.2 Standardization of Fehlings Solution 1. 50 ml burette was filled with working standard solution. 2. 10 ml of each Fehlings I and Fehlings II were pipetted into a conical flask.

Figure 13: Standardisation of Fehlings Solution

3. From the burette 19 ml of standard Working Solution to reduce the Cu. 4. The cold mixture was placed on the heater and regulated so that the boiling will begin in approximately 3 mins, and maintained at moderate boiling exactly 2 mins, reducing heat if necessary to prevent bumping. 5. Without removing flask from heater, 4 drops 1% aq. Methyl blue indicator was added and the titration completed within total boiling time of Ca 3 mins, by dropwise addition of standard working solution at intervals of Ca 10 seconds until boiling mixture resumes bright orange appearance which it had before indicator was added. Continuous emission of steam was maintained to prevent re-oxidation by air.

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Figure 14: End point of the Titration

6. Standardization was repeated several times. Factor F is the average volume (ml) standard sugar solution required to completely reduce 20 ml fehlings solution.

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3.3.5.2.3 Titration of Sample 1. 10 ml of each Fehlings I and Fehlings II were accurately pipetted into a 300 ml conical flask. 2. 10 ml of Inverted Sample solution was added. (This value was varied to ensure the amount of standard solution added was between 12ml and 19 ml). 3. The flask was placed on the heater, regulating heat so that boiling begins inapproximately 3 mins. 4. After liquid boils 10 15 secs, observe change in colour of solution. If blue colour persists working standard solution was added 0.5 1.0 ml at a time (pausing for a few seconds between additions) until it was safe to add more without risk of passing end point. 5. 3 drops methyl blue indicator solution was added and the adding of sugar solution continued at 1 ml at a time, at intervals of 10 seconds until indication is completely decolourized. Let total volume standard solution used be M. 3.3.5.2.4 Calculations for % Invert Sugar % Invert Sugar =
( )

Where F = Cu factor (Vol. std. sugar solution required to reduce 20 ml Fehlings solution). M = Vol. std. sugar solution used in titration sample. I = gm invert sugar in 1 ml of working std. sugar solution. W = gm sample in aliquot used.

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4 RESULTS
Table 3 Sulphuric Acid Concentrations for Pre-Hydrolysis and Hydrolysis of each sample. Sample # 1 2 3 4 5 6 7 8 9 10 11 12 Substrate Sawdust Sawdust Sawdust Sawdust Sawdust Sawdust Corn Cobs Corn Cobs Corn Cobs Corn Cobs Corn Cobs Corn Cobs Mass of Sample used/grams 44.90 45.03 45.00 44.98 44.89 45.03 44.90 45.03 45.00 45.03 45.00 44.99 Pre-Hydrolysis Acid Concentration 10% 10% 10% 10% 10% 10% 10% 10% 10% 10% 10% 10% Hydrolysis Concentration 20% 20% 30% 30% 40% 40% 20% 20% 30% 30% 40% 40%

Table 4 Mass of Calcium Carbonate added to neutralise each sample after pre-hydrolysis and the resulting pH Mass of CaCO3 (grams) added to neutralize Samples Mass Mass After Mass Added/ grams Before 40.20 14.96 25.24 30.80 1.21 29.59 28.81 1.78 27.03 37.61 3.80 33.81 33.30 1.08 32.22 42.76 10.30 32.46 107.18 68.83 38.35 103.00 68.84 34.16 68.83 38.29 30.54 68.82 36.32 32.50 101.51 67.30 34.21 94.06 60.78 33.28

Sample # 1 2 3 4 5 6 7 8 9 10 11 12

Pre-Hydrolysis Acid Concentration 10% 10% 10% 10% 10% 10% 10% 10% 10% 10% 10% 10%

pH 5.90 6.09 5.84 5.81 6.08 6.64 6.79 6.96 6.55 6.65 7.10 6.92

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Table 5 Mass of Calcium Carbonate added to neutralise each sample after hydrolysis and the resulting pH Mass of CaCO3 (grams) added to neutralize samples Mass Before Mass After Mass Added/ grams 109.63 118.00 156.66 131.90 138.60 155.90 178.23 124.47 186.87 249.85 131.39 133.24 15.81 21.35 54.98 29.77 22.00 35.84 83.87 32.38 78.79 137.14 11.02 7.93 93.82 96.65 101.68 102.13 116.60 120.06 94.36 92.09 108.08 112.71 120.37 125.31

Sample Hydrolysis Acid Concentration # 1 20% 2 20% 3 30% 4 30% 5 40% 6 40% 7 20% 8 20% 9 30% 10 30% 11 40% 12 40%

pH 6.93 6.12 6.42 6.63 6.78 6.12 6.71 7.05 6.55 6.54 7.10 6.92

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Table 6 Brix readings before and after adding Molasses over a period of 12 days DAY Sample # 1 2 3 4 5 6 7 8 9 10 11 12 BLANK Brix 1.2 1.4 1.6 1.9 2.1 2.0 1.6 1.9 2.0 2.0 2.0 2.0 0.0 Brix After adding 100ml Molasses 17.3 17.5 20.0 20.5 24.5 23.9 20.0 20.1 22.2 21.6 21.5 22.0 17.0 1 16.2 16.2 18.5 19.0 22.4 22.3 19.1 19.2 20.7 20.3 20.2 20.8 16.4 2 15.1 14.9 17.0 17.5 20.3 20.6 18.2 18.3 19.2 19.0 19.0 19.6 15.8 3 14.0 13.6 15.5 16.0 18.2 19.0 17.3 17.3 17.8 17.6 17.7 18.4 15.6 4 13.0 13.1 14.3 14.8 16.6 16.9 16.4 16.4 16.3 16.3 16.5 17.2 15.0 5 12.0 12.5 13.0 13.6 15.0 14.8 15.5 15.5 14.8 15.0 15.2 16.0 14.0 6 11.0 11.3 11.7 12.1 13.3 13.1 14.5 14.3 13.7 13.9 13.6 14.2 13.3 7 10.0 10.1 10.5 10.7 11.5 11.3 13.5 13.0 12.5 12.7 12.0 12.3 13.0 8 9.0 8.8 9.2 9.2 9.8 9.6 10.6 11.4 9.7 10.0 10.6 10.5 12.0 9 8.0 7.6 7.9 7.7 8.0 7.8 9.0 10.0 8.0 9.0 9.0 8.7 11.2 10 7.8 7.6 7.8 7.6 8.1 7.9 8.7 9.7 7.8 8.0 8.9 8.5 10.6 11 7.7 7.3 7.6 7.6 8.1 7.9 7.6 9.3 7.7 8.0 8.7 8.2 10.5 12 7.6 7.2 7.5 7.5 8.2 8.0 8.0 9.0 7.5 7.5 8.6 8.0 10.5

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Table 7 Results of the Lane and Eynon Method for determining Reducing Sugars Sample Aliquot Volume Run# Initial Burette Reading # (cm3) Standard 10 1 10 2 10 3 1 40 1 40 2 2 40 1 40 2 3 30 1 30 2 4 30 1 30 2 5 30 1 30 2 6 30 1 30 2 7 40 1 40 2 8 40 1 40 2 9 30 1 30 2 10 30 1 30 2 11 30 1 30 2 12 30 1 30 2 Final Burette Reading (cm3) 21 3 23.4 11 9.2 40.7 31.7 22.8 12.9 40.3 30.2 20.6 11.1 40.4 31.3 11.2 9.3 40.9 31.5 22.7 13.9 41.3 31.2 20.6 11.1 41.4 31.7 41 23.4 43.5 21.1 19.7 50 40.7 33.7 23.5 50 40.3 30.2 20.6 50 40.4 21.1 19.7 50 40.7 33.7 24.5 50 40.3 30.2 20.6 49.8 40.4

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Table 8 Results of the Distillation for Ethanol Recovery Sample # 1 2 3 4 5 6 7 8 9 10 11 12 BLANK Empty Pycnometer Mass (grams) 39.6063 39.5987 39.4998 39.5493 39.5245 39.5633 39.5439 39.5536 39.6289 39.5913 39.6101 39.6007 39.6054 Mass of Full Pyncometer (grams) 62.0528 62.1097 61.0035 61.0022 60.3392 60.5054 62.0573 62.0094 61.0045 60.5963 60.3423 60.5085 62.9956 Temperature / Degrees Celcius 20 20 20 20 20 20 20 20 20 20 20 20 20

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5 ANALYSIS OF RESULTS

Graph 1 Trends in Brix readings over a 12 day period for Sawdust Samples
Sample 1: 20% Sample 4 : 30% 25.0 20.0 Brix Reading 15.0 10.0 5.0 0.0 0 2 4 6 Day 8 10 12 14 Sample 2 : 20% Sample 5 :40% Sample 3: 30% Sample 6: 40%

Graph 2 Trends in Brix readings over a 12 day period for Corn cobs Samples
Sample 7: 20% Sample 10 : 30% 25.0 20.0 Brix Reading 15.0 10.0 5.0 0.0 0 2 4 6 Day 8 10 12 14 Sample 8 : 20% Sample 11 :40% Sample 9: 30% Sample 12: 40%

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Graph 3 Trends in Brix readings over a 12 day period for the Blank
18.0 16.0 14.0 Brix Reading 12.0 10.0 8.0 6.0 4.0 2.0 0.0 0 2 4 6 Day 8 10 12 14 Blank

5.1 Lane and Eynon Calculations


Using results from Table 7, for sample 1, run# 1: % Invert sugar =
( )

Where F = Cu factor (Volume standard sugar solution required to reduce 20ml Fehlings solution (ml) F= ml (Refer to Table 9 )

M = Volume Standard sugar solution used in titration of sample (ml) = 10.1ml I = grams Invert sugar in 1 ml of working standard sugar solution (ml) = 0.005g W = grams sample in aliquot used = % Invert sugar = (
)

= 2.52%

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Table 9 Percent Sugars Found from the Lane and Eynon Method Sample Aliquot # Volume Standard Run# 10 10 10 40 40 40 40 30 30 30 30 30 30 30 30 40 40 40 40 30 30 30 30 30 30 30 30 1 2 3 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 1 2 Initial Burette Final Burette Reading (cm3) Reading (cm3) 21 3 23.4 11 9.2 40.7 31.7 22.8 12.9 40.3 30.2 20.6 11.1 40.4 31.3 11.2 9.3 40.9 31.5 22.7 13.9 41.3 31.2 20.6 11.1 41.4 31.7 M, Volume Used (cm3) 41 23.4 43.5 21.1 19.7 50 40.7 33.7 23.5 50 40.3 30.2 20.6 50 40.4 21.1 19.7 50 40.7 33.7 24.5 50 40.3 30.2 20.6 49.8 40.4 20 20.4 20.1 10.1 10.5 9.3 9 10.9 10.6 9.7 10.1 9.6 9.5 9.6 9.1 9.9 10.4 9.1 9.2 11 10.6 8.7 9.1 9.6 9.5 8.4 8.7 W, Grams of Sample in Aliquot Percent Invert Sugar Averages

1 2 3 4 5 6 7 8 9 10 11 12

2 2 2 2 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5 2 2 2 2 1.5 1.5 1.5 1.5 1.5 1.5 1.5 1.5

2.52% 2.42% 2.72% 2.79% 3.09% 3.19% 3.49% 3.36% 3.52% 3.56% 3.52% 3.69% 2.57% 2.44% 2.77% 2.74% 3.06% 3.19% 3.82% 3.69% 3.52% 3.56% 3.92% 3.82%

2.47% 2.75% 3.14% 3.42% 3.54% 3.61% 2.50% 2.75% 3.12% 3.76% 3.54% 3.87%

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Chart 1 Reducing Sugars % for Sawdust Samples at 20%, 30% and 40% Acid hydrolysis conditions.

4 3.5 Reducing Sugars % 3 2.5 2 1.5 1 0.5 0 20% 30% Acid Concentration 40%

Chart 2 Reducing Sugars % for Corn Cobs Samples at 20%, 30% and 40% Acid hydrolysis conditions

4 3.5 3 Reducing Sugars % 2.5 2 1.5 1 0.5 0 20% 30% Acid Concentration 40%

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Chart 3 Comparison of the Average Reducing Sugars % between the Sawdust and Corn Cobs

3.5

2.5 Reeducing Sugars %

Sawdust Corn Cobs

1.5

0.5

0 20% 30% Acid Concentration 40%

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5.2 Distillation Calculations


The specific gravity of ethanol extract was determined with pycnometer and specific gravity was calculated using the following formula.
W W

Using the specific gravity, the percentage ethanol by weight and volume were found from Table 12. Following these calculations, the Ethanol yields are shown in Table 10.

Table 10 Percentage by Weight and Volume Ethanol Yield Sample # 1 2 3 4 5 6 7 8 9 10 11 12 BLANK Empty Mass of Full Temperature Weight of Specific Percent Percent Pycnometer Pyncometer C Distillate Gravity by by Mass (grams) (grams) weight Volume 39.6063 62.0528 20 22.4465 0.8979 57.75 65.58 39.5987 62.1097 20 22.5110 0.9004 56.66 64.52 39.4998 61.0035 20 21.5037 0.8601 73.79 80.26 39.5493 61.0022 20 21.4530 0.8581 74.61 80.97 39.5245 60.3392 20 20.8147 0.8326 84.93 89.43 39.5633 60.5054 20 20.9421 0.8377 82.92 87.83 39.5439 62.0573 20 22.5134 0.9005 56.62 64.47 39.5536 62.0094 20 22.4558 0.8982 57.62 65.45 39.6289 61.0045 20 21.3756 0.8550 75.89 82.06 39.5913 60.5963 20 21.0050 0.8402 81.91 87.03 39.6101 60.3423 20 20.7322 0.8293 86.22 90.43 39.6007 60.5085 20 20.9078 0.8363 83.47 88.28 39.6054 62.9956 20 23.3902 0.9356 40.61 48.05

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Chart 4 Weight % and Volume % Ethanol Yield for Sawdust at each Acid Concentration

90 80 70 Percentage % 60 50 40 30 20 10 0 20 20 30 30 40 40 Acid Concentration Weight % Volume %

Chart 5 Weight % and Volume % Ethanol Yield for Corn Cobs at each Acid Concentration

100 90 80 Percentage % 70 60 50 40 30 20 10 0 20 20 30 30 40 40 Acid Concentration Weight % Volume %

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Chart 6 Comparison of the Average Weight % Yield between Sawdust and Corn cobs for each Acid Concentration.

90 80 70 Weight % Yield 60 50 40 30 20 10 0 20 30 Acid Concentration 40 Sawdust Corncobs

Chart 7 Comparison of the Average Volume % Yield between Sawdust and Corn cobs for each Acid Concentration.

90 80 70 Volume % Yield 60 50 40 30 20 10 0 20 30 Acid Concentration 40 Sawdust Corncobs

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6 DISCUSSION
The chemical composition of Pinewood sawdust and Corn Cobs taken from Table 1 is compared in Table 11 shown below.
Table 11 Comparison of Cellulose, Hemicellulose and Lignin Compostion of Pinewood Sawdust and Corn Cobs

Lignocellulosic Material

Cellulose %

Hemicellulose %

Lignin %

Pinewood Sawdust

45-50

25-35

25-35

Corn Cobs

45

35

15

After pretreatment at the 10% sulphuric acid concentration for a period of 1hr at 90C, the prehydrolysate was collected. The same conditions were used for the hydrolysis step whilst varying the concentration of the acid in concentrations of 20%, 30% and 40%. The hydrolysates were collected and mixed with the prehydrolysate collected for that particular sample. The total amount of reducing sugars released from samples is shown in Table 9. Analysis of the effect of acid concentration used for the hydrolysis step on the pinewood sawdust showed that as the concentration increased, the reducing sugars released also increased as shown in Error! Reference source not found.. The prehydrolysis step was expected to release the same mount of reducing sugars for each of the pinewood sawdust samples since the conditions for this were identical, hence it is assumed that the differences in reducing sugars is due to the different hydrolysis acid concentrations. Similar trends can be seen in the Corn cobs analysis shown in Chart 2. As the concentration of acid used increased, so did the amount of reducing sugars present. Comparison of the amount of reducing sugars present in the pinewood sawdust compared to that of the corn cobs is shown in Chart 3. As it can be seen, under the condition of 20% acid for the hydrolysis step, both substrates released approximately the same amount of sugars, however, as the concentration was

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increased, the corn cobs produced more sugars than the corncobs. This can be due to the fact that the corn cobs contained more cellulose than the sawdust. Hence since the concentrated acid is used to release the sugars from the cellulose part of the structure, more was released from the corncobs since more was available. This therefore coincides with the literature that more reducing sugars are expected from the lignocellulosic material with higher cellulose %. Also, as the concentration of acid increased with each substrate, the definite trend of an increase in reducing sugars released is observed. The mixture of the hydrolysates were all analysed using a refractometer to find the brix of the solutions. The brix values stated in Table 6 shows that the brix of the pinewood sawdust samples (1-6) had initial brix values of 1.2, 1.4, 1.6, 1.9, 2.1, 2.0. This is expected since the brix is a measure if the amount if sugars present and from the Lane and Eynon results, this is the trend. Both measures show an increase in the sugars released as the concentration of acid used is increased. The brix of the corn cobs samples (7-12) also show the same trend and show that an increase in acid concentration used, results in an increased amount of sugars released. Following this, 100 ml of molasses was added to each of the samples to ensure the brix was in the fermentable range as shown in Table 6. A blank was also made by using 100 ml of the molasses and 400 ml of water since that was the average volume of the samples. Yeast was then added to each sample and fermentation began. The brix of the samples were recorded over a period of 12 days after which each of the samples brix settled down to a value of approximately 7 to 8. The decreasing brix readings were seen as an indication of the fermentation taking place since the sugars were being used to form ethanol. The pinewood sawdust and corncobs brix readings are plotted in Graph 1and Graph 2. The blank showed different readings from all the samples such that its brix only went down to a value of 10.5 as shown in Graph 3. The results of the fermentation were analysed using the distillation. The specific gravity was found using a pycnometer and the values read off from Table 12 to find the weight and volume percent of ethanol in the mixtures. Firstly, comparison of the ethanol % both by volume and weight of the blank to the samples confirmed the presence of fermentable sugars derived from the

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lignocellulosic material. The samples all contained atleast 15% more ethanol than the blank. Analysis of ethanol yields in terms of weight and volume shown in Chart 4 for the pinewood sawdust shows that the yield increased as the acid concentration used increased. This also confirms that the amount of sugars was the most in the hydrolysates from the higher acid concentration hydrolysis. The 20% had the lowest ethanol yield with an average weight and volume percent of 57% and 65%. The highest yield was achieved by the 40% with an average weight and volume percent of 83% and 88%. Chart 5 shows the same trend as the pinewood sawdust such that the highest yield is the 40% with a weight and volume percent of 84% and 89%. Comparison of the average weight % yield and volume % yield are shown in Chart 6and Chart 7. The yields for the 20% for both the pinewood sawdust and the corn cobs were the same however, for the 30%, the corn cobs yielded more ethanol. The trend expected was that the 40% would have yielded more ethanol since analysis by the Lane and Eynon method stated that it contained more sugars, however the yield was the same for the pinewood sawdust as well as the corn cobs. This may have been due to experimental error since during distillation, only the first 30 ml was collected but sometimes this value was exceeded leading to possible errors. In general during acid hydrolysis, an increase of acid concentration improves reducing sugars production hence increasing the ethanol yield. The maximum sugar content occurred at 40 % of sulphuric acid with the corncobs, with a total reducing sugar of approximately 3.6% resulting 89% ethanol yield by volume.

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6.1 Limitations
The following limitations may have impacted on the results of the experiment: Faults within equipment such as the analytical balance may have affected readings in the weights of the samples such as that of the pycnometer. Due to availability of equipment, the various steps outlined in the method were not carried out immediately after the previous one. Time delays for 2 weeks occurred during which the samples were refrigerated. It is not certain if this led to any errors in the results. Detecting the end point during the titration in the Lane and Eynon method was very difficult and sensitive to any minute changes in reagents added. It was therefore difficult to have consistency among the titration runs carried out. The Lane and Eynon method is very susceptible to errors due to the many reagents that are to be prepared and standardized with a great degree of accuracy. During the titration, the conical flask containing the Fehlings and sample should be steaming out for the true end point to be detected. The titration has to be done within 3 minutes however some titrations took longer than this to achieve the end point.

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7 CONCLUSION

In conclusion, it was observed that The optimum hydrolysis concentration for the sulphuric acid was 40 % for both the pinewood sawdust and the corn cobs since the reducing sugars percent found using the Lane and Eynon was the highest for this concentration. The hydrolysates were fermented and distilled after which they were analysed to find the specific gravity. It was deduced that the highest ethanol yield was from the 40% sulphuric acid hydrolysates. Both the sawdust and corn cobs yieled approximately the same percent of ethanol. Overall, the ethanol yields for the corn cobs were higher than that of the pinewood sawdust.

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8 RECOMMENDATIONS

HPLC should be used to determine the sugars since it is the most accurate method based on the literature. HPLC grade sugars should also be order if HPLC analysis is to be used. The appropriate column and reagents for HPLC analysis should be ordered at least a year in advance to ensure that they arrive on time. Lane and Eynon method should be used as a last resort due to its tedious, time- consuming and lengthy procedure as well as its difficulty in determining the end point which is crucial in determining the sugar concentration.

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9 REFERENCES

Ashok Pandey. (2008). Handbook of Plant-Based Biofuels. CRC Press 2008. Chen, S.-F., Mowery, R. A., Svecik, R. S., Scarlata, C. J., & Chambliss. (2010). Compositional analysis of water-soluble materials in switchgrass. J. Agric. Food Chem. , 32513258. Dien, B. S. (2010). Mass balances and analytical methods for biomass pretreatment experiments. In A. A. Vertes, Biomass to Biofuels. New York: Wiley. Energy Center of Wisconsin. (2004). www.wisbiorefine.org. Retrieved 12 07, 2011, from Wiscosin Biorefining Development Initiative: http://www.biorefine.org/proc/fermlig.pdf Foyle, T., Jennings, L., & Mulcahy, P. (2007). Compositional analysis of lignocellulosic materials: evaluation of methods used for sugar analysis of waste paper and straw. . Bioresour. Technol. , 3026-3036. Grohmann, K., Himmel, M., Rivard, C., Tucker, M., & Baker, J. (1984). Chemical-mechanical methods for the enhanced utilization of straw. Biotechnol. Bioeng. , 137157. I. del Campo, I. A. (2006). Diluted acid hydrolysis pretreatment of agri-food wastes for bioethanol production. Industrial Crops and Products , 214-221. Justin B. Slutter, R. O. (2010). Compositional Analysis of Lignocellulosic Feedstocks. Review and Description of Methods. Journal of Agricultural and Food Chemistry , 9043-9053. Justin B. Slutter, Raymond O. Rutz, Christopher J. Scarlata, Amie D. Slutter And David W. Templeton. (2010). Compositional Analysis of Lignocellulosic Feedstocks. Review and Description of Methods. Journal of Agricultural and Food Chemistry , 9043-9053. Ketkorn, S. D. (2009). Comparison of Hydrolysis Conditions to Recover Reducing Sugars from Various Ligncellulosic Materials. Chaing Mai J. Sci , 384-394.

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M. Galbe, G. Z. (2002). A review of the production of ethanol from Softwood. Appl Microbiol Biotechnol , 618-628. Mousdale, D. M. (2008). Biofuels. Biotechnology, Chemistry, and Sustainable Development. CRC Press. Parveen Kumar, |. D. (2009). Methods for Pretreatment of Lignocellulosic Biomass for Efficient Hydrolysis and Biofuel Production. Ind. Eng. Chem. Res , 37133729. Pettersen, R. C., Schwandt, V. H., & Effland, M. J. (1984). An analysis of the wood sugar assay using HPLC - a comparison with paper-chromatography. J. Chromatogr. Sci. Ritter, G. J., & Barbour, J. H. (1935). Effect of pretreatments of wood on the lignin determination. Ind. Eng. Chem. Anal. , 238-240. Scales, F. M. (2011). The Determination of Reducing Sugars. The journal of Biological Science , 81-87. Sherrard, E. C., & Harris, E. E. (1932). Factors influencing properties of isolated wood lignin. . Ind. Eng. Chem. , 103106. Sunggyu Lee, J. G. (2007). Ethanol from lignocellulosics. Teixeira, S. I. (2010). Lignocellulose as raw material in fermentation processes. IBB Institute for Biotechnology and Bioengineering, Centre of Biological Engineering, University of Minho, Campus de . Theander, O. (1991). Chemical-analysis of lignocellulose materials. Anim. Feed Sci. Technol. , 35-44. Vane, L. (2003). Prevaporation - Membrane Process for Bioethanol Recovey, Solvent Dehydration, and Contaminant Removal. Washington DC: Environmental Protection Agency.

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10 APPENDIX

Table 12 Determination of the Alcohol Content : Specific Gravities of Alcohol Water Mixtures

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