Hydrolysis of Lignocellulosic Materials for Ethanol Production

Department of Chemical Engineering National Institute of Technology Rourkela 2008

HYDROLYSIS OF LIGNOCELLULOSIC MATERIALS FOR ETHANOL PRODUCTION2008-09

A thesis submitted in partial fulfillment of the requirements for the degree of Bachelor of Technology in Chemical Engineering By Nirbhay Gupta Roll no. 10500027 Under guidance of

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National Institute of Technology Rourkela

CERTIFICATE

This is to certify that the thesis entitled HYDROLYSIS OF LIGNOCELLULOSIC MATERIALS FOR ETHANOL PRODUCTION submitted by Mr. Nirbhay Gupta in partial fulfillments for the requirements for the award of Bachelor of Technology Degree in Chemical Engineering at National Institute of Technology, Rourkela is an authentic work carried out by him under my supervision and guidance. To the best of my knowledge the matter embodied in the thesis has not been submitted by any other University/Institute for the award of any Degree or Diploma.

Date:

Dr. A. KumarDepartment of Chemical Engineering National Institute of Technology Rourkela-769008

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ACKNOWLEDGEMENT

I would like to make my deepest appreciation and gratitude to Prof. A. Kumar for his invaluable guidance, constructive criticism and encouragement during the course of this project.

I would like to thank Prof. R.K.Singh for being a uniformly excellent advisor. He was always open minded, helpful and provided us with a strong broad idea.

Grateful acknowledgement is made to all the staff and faculty members of Chemical Engineering Department, National Institute of Technology, Rourkela for their consistent encouragement.

In spite of the numerous citations above the author accepts full responsibility for the contents that follow.

DATE:

Nirbhay Gupta Roll: 10500027

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CONTENTSContents List of figures List of Tables Abstract vi v iii v

Chapter 1

INTRODUCTION

1

Chapter 22.1 2.2

LITERATURE REVIEWEthanol from Cellulose Ethyl Alcohol: Overview

34 7

Chapter 33.1

PRODUCTION METHODS Pretreatment of lignocellulosic materials

12 13 14 14 17 18

3.2 Physical Pretreatment 3.3 Chemical Pretreatment 3.4 Biological Pretreatment 3.5 Hydrolysis Process

Chapter 4

MATERIALS & EQUIPMENT USED 4.1 Materials Used 4.2 Equipments Required

21 22 22

4

Chapter 5

PROCEDURE OF EXPERIMENT 5.1 Pretreatment of bagasse 5.2 Hydrolysis 5.3 pH adjustment 5.4 Fermentation

25 26 26 27 27

Chapter 6

RESULTS 6.1 Standard plot for ethanol 6.2 Analysis of ethanol for different days 6.3 Comparison of ethanol concentration with increasing no. of days.

29 30 33

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Chapter 7

DISCUSSION

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Chapter 8

REFERENCES

45

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LIST OF GRAPHS 6.1 6.2 6.3 6.4 6.5 6.6 6.7 6.8 6.9 6.10 6.11 Standard plot for ethanol Absorbance vs Molarity curve for day 1 Absorbance vs Molarity curve for day 2 Absorbance vs Molarity curve for day 3 Absorbance vs Molarity curve for day 7 Absorbance vs Molarity curve for day 8 Absorbance vs Molarity curve for day 9 Absorbance vs Molarity curve for day 10 Ethanol conc. Vs no. of days curve for 0.2 M Ethanol conc. Vs no. of days curve for 0.3 M Ethanol conc. Vs no. of days curve for 0.5 M 32 33 34 35 36 37 38 39 40 41 42

LIST OF TABLES

6.1 6.2 6.3-6.9 6.10 6.11 6.12

Standard data for ethanol concentration Standard for ethanol concentration (revised) Data for molarity vs absorbance for 10 days Ethanol conc. Vs no.of days table for 0.2 M Ethanol conc. Vs no. of days table for 0.3 M Ethanol conc. Vs no. of days table for 0.5 M

30 31 33-39 40 41 42

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ABSTRACT

PURPOSE OF THE PROJECTBiofuel is defined as solid, liquid or gaseous fuel derived from relatively recently dead biological material and is distinguished from fossil fuels, which are derived from long dead biological material. Theoretically, biofuels can be produced from any carbon source; although, the most common sources are photosynthetic plants. Various plants and plant-derived materials are used for biofuel manufacturing. Increasing the use of bio-fuels for energy generation purpose is of particular experiment nowadays because they allow mitigation of greenhouse gases, provide independence and even may offer new employment possibilities. Bio-fuels are being investigated as potential substitutes for current high pollutant fuels obtained from conventional sources. There are two common strategies of producing liquid and gaseous agrofuels. One is to grow crops high in sugar, and then use yeast fermentation to produce ethyl alcohol. The second is to grow plants that contain high amounts of vegetable oil, such as oil palm, soybean, and algae. When these oils are heated, their viscosity is reduced, and they can be burned directly in a diesel engine, or they can be chemically processed to produce fuels such as biodiesel.

BREIF DESCRIPTION OF THE EXPERIMENTThe raw material used for the experiment is bagasse. First the bagasse is chipped and grinded (may be up to powder form). Then this is taken for acid hydrolysis. Dilute sulphuric acid of 0.2 M, 0.3 M, and 0.5 M concentration were used in this process. For the acid hydrolysis, 5 gms of bagasse was put in 100 ml of each of the concentrations and was allowed to soak for 1 day. Then it was autoclaved for half an hour and allowed to cool. The fermentation media was prepared. 5 gms of yeast was added to the above media and kept in incubator for 1 day. 10 ml of this media was added to each of the sample in asceptical (laminar flow hood) manner and placed in orbital shaking incubator. The pH was adjusted to 5 and the fermenting temperature was kept at 35 0C. Fermentation may take up to 10 days.

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Chapter 1

INTRODUCTION

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Energy consumption has increased steadily over the last century as the world population has grown and more countries have become industrialized. Crude oil has been the major resource to meet the increased energy demand. Scientists used several different techniques to estimate the current known crude oil reserves and the reserves as yet undiscovered and concluded that the decline in worldwide crude oil production will begin before 2010. They also predicted that annual global oil production would decline from the current 25 billion barrels to approximately 5 billion barrels in 2050. Because the economy in the US and many other nations depends on oil, the consequences of inadequate oil availability could be severe. Therefore, there is a great interest in exploring alternative energy sources Unlike fossil fuels, ethanol is a renewable energy source produced through fermentation of sugars. Ethanol is widely used as a partial gasoline replacement in the US. Fuel ethanol that is produced from corn has been used in gasohol or oxygenated fuels since the 1980s. These gasoline fuels contain up to 10% ethanol by volume. As a result, the US transportation sector now consumes about 4540 million liters of ethanol annually, about 1% of the total consumption of gasoline. Recently, US automobile manufacturers have announced plans to produce significant numbers of flexible-fueled vehicles that can use an ethanol blend E85 (85% ethanol and 15% gasoline by volume) alone or in combination with gasoline. Using ethanol-blended fuel for automobiles can significantly reduce petroleum use and exhaust greenhouse gas emission. However, the cost of ethanol as an energy source is relatively high compared to fossil fuels. A dramatic increase in ethanol production using the current cornstarch-based technology may not be practical because corn production for ethanol will compete for the limited agricultural land needed for food and feed production. A potential source for low-cost ethanol production is to utilize lignocellulosic materials such as crop residues, grasses, sawdust, wood chips, and solid animal waste.

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Chapter 2

LITERATURE REVIEW

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Cellulose molecules consist of long chains of glucose molecules as do starch molecules, but have a different structural configuration. These structural characteristics plus the encapsulation by lignin makes cellulosic materials more difficult to hydrolyze than starchy materials. Hemicellulose is also comprised of long chains of sugar molecules; but contains, in addition to glucose (a 6-carbon or hexose sugar), contains pentoses (5-carbon sugars). To complicate matters, the exact sugar composition of hemicellulose can vary depending on the type of plant. Since 5-carbon sugars comprise a high percentage of the available sugars, the ability to recover and ferment them into ethanol is important for the efficiency and economics of the process. Recently, special microorganisms have been genetically engineered which can ferment 5-carbon sugars into ethanol with relatively high efficiency 2.1 ETHANOL-FROM-CELLULOSE In times of fuel shortages, fermentation ethanol has been commercially manufactured in the US from cellulosic biomass feedstock using acid hydrolysis techniques. However, it is only recently that cost-effective technologies for producing ethanol-from-cellulose (EFC) in the US have started to emerge. There are three basic types of EFC processesacid hydrolysis, enzymatic hydrolysis, and thermo chemicalwith variations for each. The most common is acid hydrolysis. Virtually any acid can be used; however, sulfuric acid is most commonly used since it is usually the least expensive

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12

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2.2 ETHYL ALCOHOL: OVERVIEW Ethanol, also called ethyl alcohol, pure alcohol or drinking alcohol, is a volatile, flammable, colorless liquid. It is a psychoactive drug, best known as the type of alcohol found in alcoholic beverages and in modern thermometers. Ethanol is one of the oldest recreational drugs known to man. In common usage, it is often referred to simply as alcohol or spirits. Ethanol has widespread use as a solvent of substances intended for human contact or consumption, including scents, flavorings, colorings, and medicines. In chemistry, it is both an essential solvent and a feedstock for the synthesis of other products. It has a long history as a fuel for heat and light and also as a fuel for internal combustion engines.Properties Molecular formula Molar mass Appearance Density

C2H6O 46.07 g mol1 colorless clear liquid 0.789 g/cm3 114.3 C, 159 K, 174 F 78.4 C, 352 K, 173 F

Melting point

Boiling point Solubility in water Acidity (pKa)

Fully miscible

15.9 1.200 mPas (cP) at 20.0 C

Viscosity

Dipole moment

5.64 fCfm (1.69 D) (gas

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2.2.1 PHYSICAL PROPERTIES Ethanol is a volatile, colorless liquid that has a strong characteristic odor. It burns with a smokeless blue flame that is not always visible in normal light. It is also used in finger nail polish remover. The physical properties of ethanol stem primarily from the presence of its hydroxyl group and the shortness of its carbon chain. Ethanols hydroxyl group is able to participate in hydrogen bonding, rendering it more viscous and less volatile than less polar organic compounds of similar molecular weight. Ethanol is a versatile solvent, miscible with water and with many organic solvents

2.2.2 CHEMICAL PROPERTIES Ethanol is classified as a primary alcohol, meaning that the carbon to which its hydroxyl group is attached has at least two hydrogen atoms attached to it.

2.2.2.1 Acid-base chemistryEthanol's hydroxyl group causes the molecule to be slightly basic. It is almost neutral like water. The pH of 100% ethanol is 7.33, compared to 7.00 for pure water. Ethanol can be quantitatively converted to its conjugate base, the ethoxide ion (CH3CH2O), by reaction with an alkali metal such as sodium: 2CH3CH2OH + 2Na 2CH3CH2ONa + H2, or a very strong base such as sodium hydride: CH3CH2OH + NaH CH3CH2ONa + H2. This reaction is not possible in an aqueous solution, as water is more acidic, so that hydroxide is preferred over ethoxide formation.

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2.2.2.2 HalogenationEthanol reacts with hydrogen halides to produce ethyl halides such as ethyl chloride and ethyl bromide: CH3CH2OH + HCl CH3CH2Cl + H2O HCl reaction requires a catalyst such as zinc chloride. Hydrogen chloride in the presence of their respective zinc chloride is known as Lucas reagent. CH3CH2OH + HBr CH3CH2Br + H2O HBr requires refluxing with a sulfuric acid catalyst. Ethyl halides can also be produced by reacting ethanol with more specialized halogenating agents, such as thionyl chloride for preparing ethyl chloride, or phosphorus tribromide for preparing ethyl bromide. CH3CH2OH + SOCl2 CH3CH2Cl + SO2 + HCl

2.2.2.3 Haloform reactionThe haloform reaction is a chemical reaction where a haloform (CHX3, where X is a halogen) is produced by the exhaustive halogenation of a methyl ketone (a molecule containing the R-COCH3 group) in the presence of a base.

2.2.2.4 Ester formationUnder acid-catalyzed conditions, ethanol reacts with carboxylic acids to produce ethyl esters and water: RCOOH + HOCH2CH3 RCOOCH2CH3 + H2O. For this reaction to produce useful yields it is necessary to remove water from the reaction mixture as it is formed.

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Ethanol can also form esters with inorganic acids. Diethyl sulfate and triethyl phosphate, prepared by reacting ethanol with sulfuric and phosphoric acid respectively, are both useful ethylating agents in organic synthesis. Ethyl nitrite, prepared from the reaction of ethanol with sodium nitrite and sulfuric acid, was formerly a widely-used diuretic.

2.2.2.5 DehydrationStrong acid desiccants, such as sulfuric acid, cause ethanol's dehydration to form either diethyl ether or ethylene: 2 CH3CH2OH CH3CH2OCH2CH3 + H2O CH3CH2OH H2C=CH2 + H2O Which product, diethyl ether or ethylene, predominates depends on the precise reaction conditions

2.2.2.6 OxidationEthanol can be oxidized to acetaldehyde, and further oxidized to acetic acid. In the human body, these oxidation reactions are catalyzed by enzymes. In the laboratory, aqueous solutions of strong oxidizing agents, such as chromic acid or potassium permanganate, oxidize ethanol to acetic acid, and it is difficult to stop the reaction at acetaldehyde at high yield. Ethanol can be oxidized to acetaldehyde, without over oxidation to acetic acid, by reacting it with pyridinium chromic chloride.[19] The direct oxidation of ethanol to acetic acid using chromic acid is given below. C2H5OH + 2[O] CH3COOH + H2O The oxidation product of ethanol, acetic acid, is spent as nutrient by the human body as acetyl CoA, where the acetyl group can be spent as energy or used for biosynthesis.

2.2.2.7 ChlorinationWhen exposed to chlorine, ethanol is both oxidized and its alpha carbon chlorinated to form the compound, chloral. 4Cl2 + C2H5OH CCl3CHO + 5HCl

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2.2.2.8 CombustionCombustion of ethanol forms carbon dioxide and water: C2H5OH(g) + 3 O2(g) 2 CO2(g) + 3 H2O(l); (Hr = 1409 kJ/mol[21])

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Chapter 3

PRODUCTION METHODS

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The basic five stages of this process are: 1. A pretreatment phase to make the raw material such as wood or straw amenable to hydrolysis. 2. Hydrolysis, to break down the molecules of cellulose into sugars. 3. Separation of the sugar solution from the residual materials, notably lignin. 4. Yeast fermentation of the sugar solution.

5. Distillation to produce 99.5% pure alcohol. 3.1 Pretreatment of lignocellulosic materialsThe effect of pretreatment of lignocellulosic materials has been recognized for a long time. The purpose of the pretreatment is to remove lignin and hemicellulose, reduce cellulose crystallinity, and increase the porosity of the materials. Pretreatment must meet the following requirements: (1) improve the formation of sugars or the ability to subsequently form sugars by enzymatic hydrolysis; (2) avoid the degradation or loss of carbohydrate; (3) avoid the formation of byproducts inhibitory to the subsequent hydrolysis and fermentation processes; and (4) be cost-effective. Physical, physico-chemical, chemical, and biological processes have been used for pretreatment of lignocellulosic materials

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3.2. Physical pretreatment 3.2.1. Mechanical comminution

Waste materials can be comminuted by a combination of chipping, grinding and milling to reduce cellulose crystallinity. The size of the materials is usually 1030 mm after chipping and 0.22 mm after milling or grinding. Vibratory ball milling has been found to be more effective in breaking down the cellulose crystallinity of spruce and aspen chips and improving the digestibility of the biomass than ordinary ball milling. The power requirement of mechanical comminution of agricultural materials depends on the final particle size and the waste biomass characteristics.

3.2.2. PyrolysisPyrolysis has also been used for pretreatment of lignocellulosic materials. When the materials are treated at temperatures greater than 300 C, cellulose rapidly decomposes to produce gaseous products and residual char. The decomposition is much slower and less volatile products are formed at lower temperatures. Mild acid hydrolysis (1 N H2SO4, 97 C, 2.5 h) of the residues from pyrolysis pretreatment has resulted in 8085% conversion of cellulose to reducing sugars with more than 50% glucose .The process can be enhanced with the presence of oxygen When zinc chloride or sodium carbonate is added as a catalyst, the decomposition of pure cellulose can occur at a lower temperature

3.3 Chemical pretreatment 3.3.1 OzonolysisOzone can be used to degrade lignin and hemicellulose in many lignocellulosic materials such as wheat straw, bagasse, green hay, peanut, pine, cotton straw, and poplar sawdust. The degradation was essentially limited to lignin and hemicellulose was slightly attacked, but cellulose was hardly affected.The rate of enzymatic hydrolysis increased by a factor of 5 following 60%21

removal of the lignin from wheat straw in ozone pretreatment. Enzymatic hydrolysis yield increased from 0% to 57% as the percentage of lignin decreased from 29% to 8% after ozonolysis pretreatment of poplar sawdust. Ozonolysis pretreatment has the following advantages: (1) it effectively removes lignin; (2) it does not produce toxic residues for the downstream processes; and (3) the reactions are carried out at room temperature and pressure. However, a large amount of ozone is required, making the process expensive.

3.3.2. Acid hydrolysisConcentrated acids such as H2SO4 and HCl have been used to treat lignocellulosic materials. Although they are powerful agents for cellulose hydrolysis, concentrated acids are toxic, corrosive and hazardous and require reactors that are resistant to corrosion. In addition, the concentrated acid must be recovered after hydrolysis to make the process economically feasible. Dilute acid hydrolysis has been successfully developed for pretreatment of lignocellulosic materials. The dilute sulfuric acid pretreatment can achieve high reaction rates and significantly improve cellulose hydrolysis. At moderate temperature, direct saccharification suffered from low yields because of sugar decomposition. High temperature in dilute acid treatment is favorable for cellulose hydrolysis. There are primarily two types of dilute acid pretreatment processes: high temperature (T greater than 160 C), continuous-flow process for low solids loading (510% [weight of substrate/weight of reaction mixture]), and low temperature (T less than 160 C), batch process for high solids loading (1040%). Although dilute acid pretreatment can significantly improve the cellulose hydrolysis, its cost is usually higher than some physicochemical pretreatment processes. A neutralization of pH is necessary for the downstream enzymatic hydrolysis or fermentation processes.

3.3.3. Alkaline hydrolysisSome bases can also be used for pretreatment of lignocellulosic materials and the effect of alkaline pretreatment depends on the lignin content of the materials. The mechanism of alkaline hydrolysis is believed to be saponification of intermolecular ester bonds crosslinking xylan hemicelluloses and other components, for example, lignin and other hemicellulose. The porosity

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of the lignocellulosic materials increases with the removal of the crosslinks. Dilute NaOH treatment of lignocellulosic materials caused swelling, leading to an increase in internal surface area, a decrease in the degree of polymerization, a decrease in crystallinity, separation of structural linkages between lignin and carbohydrates, and disruption of the lignin structure ( Fan et al., 1987). The digestibility of NaOH-treated hardwood increased from 14% to 55% with the decrease of lignin content from 2455% to 20%. However, no effect of dilute NaOH pretreatment was observed for softwoods with lignin content greater than 26%. Dilute NaOH pretreatment was also effective for the hydrolysis of straws with relatively low lignin content of 1018% used the combination of irradiation and 2% NaOH for pretreatment of corn stalk, cassava bark and peanut husk. The glucose yield of corn stalk was 20% in untreated samples compared to 43% after treatment with electron beam irradiation at the dose of 500 kGy and 2% NaOH, but the glucose yields of cassava bark and peanut husk were only 3.5% and 2.5%, respectively. Ammonia was also used for the pretreatment to remove lignin. Iyer et al. (1996) described an ammonia recycled percolation process (temperature, 170 C; ammonia concentration, 2.520%; reaction time, 1 h) for the pretreatment of corn cobs/stover mixture and switchgrass. The efficiency of delignification was 6080% for corn cobs and 6585% for switchgrass.

3.3.4. Oxidative delignificationLignin biodegradation could be catalyzed by the peroxidase enzyme with the presence of H2O2. The pretreatment of cane bagasse with hydrogen peroxide greatly enhanced its susceptibility to enzymatic hydrolysis. About 50% lignin and most hemicellulose were solubilized by 2% H2O2 at 30 C within 8 h, and 95% efficiency of glucose production from cellulose was achieved in the subsequent saccharification by cellulase at 45 C for 24 h. used wet oxidation and alkaline hydrolysis of wheat straw (20 g straw/l, 170 C, 510 min), and achieved 85% conversion yield of cellulose to glucose.

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3.3.5. Organosolv processIn the organosolv process, an organic or aqueous organic solvent mixture with inorganic acid catalysts (HCl or H2SO4) is used to break the internal lignin and hemicellulose bonds. The organic solvents used in the process include methanol, ethanol, acetone, ethylene glycol, triethylene glycol and tetrahydrofurfuryl alcohol. Organic acids such as oxalic, acetylsalicylic and salicylic acid can also be used as catalysts in the organosolv process. At high temperatures (above 185 C), the addition of catalyst was unnecessary for satisfactory delignification. Usually, a high yield of xylose can be obtained with the addition of acid. Solvents used in the process need to be drained from the reactor, evaporated, condensed and recycled to reduce the cost. Removal of solvents from the system is necessary because the solvents may be inhibitory to the growth of organisms, enzymatic hydrolysis, and fermentation.

3.4. Biological pretreatmentIn biological pretreatment processes, microorganisms such as brown-, white- and soft-rot fungi are used to degrade lignin and hemicellulose in waste materials. Brown rots mainly attack cellulose, while white and soft rots attack both cellulose and lignin. White-rot fungi are the most effective basidiomycetes for biological pretreatment of lignocellulosic materials. Hatakka (1983) studied the pretreatment of wheat straw by 19 white-rot fungi and found that 35% of the straw was converted to reducing sugars by Pleurotus ostreatus in five weeks. Similar conversion was obtained in the pretreatment by Phanerochaete sordida 37 and Pycnoporus cinnabarinus 115 in four weeks. In order to prevent the loss of cellulose, a cellulase-less mutant of Sporotrichum pulverulentum was developed for the degradation of lignin in wood chips also reported the delignification of Bermuda grass by white-rot fungi. The biodegradation of Bermuda grass stems was improved by 2932% using Ceriporiopsis subvermispora and 6377% using Cyathus stercoreus after 6 weeks. The white-rot fungus P. chrysosporium produces lignin-degrading enzymes, lignin peroxidases and manganese-dependent peroxidases, during secondary metabolism in response to carbon or nitrogen limitation (Boominathan and Reddy, 1992). Both enzymes have been found in the extracellular filtrates of many white-rot fungi for the degradation of wood cell walls. Other24

enzymes including polyphenol oxidases, laccases, H2O2 producing enzymes and quinonereducing enzymes can also degrade lignin. The advantages of biological pretreatment include low energy requirement and mild environmental conditions. However, the rate of hydrolysis in most biological pretreatment processes is very low

3.5 Hydrolysis processesThe cellulose molecules are composed of long chains of glucose molecules. In the hydrolysis process, these chains are broken down to "free" the sugar, before it is fermented for alcohol production. There are two major hydrolysis processes: a chemical reaction using acids, or an enzymatic reaction.

3.5.1 Acid hydrolysisAcid hydrolysis has been investigated as a possible process for treating lignocellulosic materials such as wood chips, rice straw, sugar beet pulp and wheat straw. The mineral acids act simply and rapidly as reaction catalyzers of polysacharide fractions. Sugarcane bagasse can be hydrolyzed using dilute acid to obtain a mixture of sugars with xylose as the major component. However, in the hydrolyzate some by-products generated in the hydrolysis, such as acetic acid, furfural, phenolic compounds, or lignin degradation products, can be present. These are potential inhibitors of a microbiological utilization of this hydrolyzate. Processes such as two-stage acid hydrolysis can be employed to produce xylose and glucose. Treatment with dilute sulphuric acid at moderate temperatures (the first stage of acid hydrolysis) has proven to be an efficient means of producing xylose from hemicellulose. In the second stage more drastic reaction conditions are employed and glucose can be produced from cellulose hydrolysis. In general, acid treatment is effective in solubilizing the hemicellulosic component of biomass. Proper combinations of pH, temperature, and reaction time can result in high yields of sugars, primarily xylose from hemicelluloses. Sulphuric acid is a catalyst for this reaction and, in this work, was used to study the hydrolysis of sugarcane bagasse hemicellulose. The effects of temperature, acid concentration and reaction time were25

studied, and the effectiveness of the hydrolysis was evaluated in terms of hemicellulose solubilization.

3.6 Sugar Fermentation in Yeast

Yeast are able to metabolize some foods, but not others. In order for an organism to make use of a potential source of food, it must be capable of transporting the food into its cells. It must also have the proper enzymes capable of breaking the foods chemical bonds in a useful way. Sugars are vital to all living organisms. Yeast are capable of using some, but not all sugars as a food source. Yeast can metabolize sugar in two ways, aerobically, with the aid of oxygen, or anaerobically, without oxygen.

In this lab, you will try to determine whether yeast are capable of metabolizing a variety of sugars. Although aerobic fermentation of sugar is much more efficient, in this experiment we will have yeast ferment sugars anaerobically. When the yeast respire aerobically, oxygen gas is consumed at the same rate that CO2 is producedthere would be no change in the gas pressure in the test tube. When yeast ferments the sugars anaerobically, however, CO2 production will cause a change in the pressure of a closed test tube, since no oxygen is being consumed. We can use this pressure change to monitor the respiration rate and metabolic activity of the organism. A gas pressure sensor will be used to monitor the fermentation of sugar.

The fermentation of glucose can be described by the following equation: C6Hl2O6 2 CH3CH2OH +2 CO2 + energy

Note that alcohol is a byproduct of this fermentation.

1. Yeast cannot utilize all of the sugars equally well. While glucose, sucrose, and fructose all can be metabolized by yeast, lactose is not utilized at all.

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2. Yeast may not have the proper enzymes to either transport lactose across its cell membrane, or it may not have the enzyme needed to convert it from a disaccharide to a monosaccharide.

3. The yeast need to be incubated so that the oxygen in the test tube will be completely consumed. If the yeast respire aerobically, no pressure change occurs, because much oxygen is consumed as CO2 is produced. It also takes a few minutes for the yeast to transport the sugar into the cell, to respire at a constant rate, and to reach the proper temperature.

4. Some yeast live on other organisms. If they are warm blooded, they may be near the optimal temperature for yeast respiration, 37C. Many yeast live in soils. The temperature of soils may easily be measured at different times of the year.

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Chapter 4

MATERIALS & EQUIPMENT USED

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4.1 MATERIALS USED 4.1.1 Chemicals Required i. ii. iii.iv.

Dilute Sulfuric acid Concentrated NaOH Phenol Sodium hydroxide

= 0.2 M, 0.3 M, 0.5 M = dropwise = = 2 gm 10 gm

4.2 EQUIPMENTS REQUIRED 4.2.1 Vertical autoclave This equipment is primarily used for sterilization purpose. It is an enclosed space where steam bath is given to any equipment placed inside it. Water filled in it is heated by electric coils present at bottom. It has a vent at top, from where the steam can be released to maintain the required pressure. For our case, sterilization is required after maintaining the pH and also for each filtration. Pressure around 2.02 kg/cm2 is used. Once the pressure reaches 2.02 kg/cm2, it is maintained for half an hour. The equipments are allowed to cool down before removing from the autoclave.

Figure 1 Vertical autoclave

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4.2.2 Laminar flow chamber A laminar flow cabinet or laminar flow closet or tissue culture hood is a carefully enclosed bench designed to prevent contamination of semiconductor wafers, biological samples, or any particle sensitive device. Air is drawn through a HEPA filter and blown in a very smooth, laminar flow towards the user. The cabinet is usually made of stainless steel with no gaps or joints where spores might collect. Such hoods exist in both horizontal and vertical configurations, and there are many different types of cabinets with a variety of airflow patterns and acceptable uses

4.2.3 Vaccum Filtration Vacuum filtration is a technique for separating a solid product from a solvent or liquid reaction mixture. The mixture of solid and liquid is poured through a filter paper in a Buchner funnel. The solid is trapped by the filter and the liquid is drawn through the funnel into the flask below, by a vacuum.

4.2.4 UV-Spectrophotometer The UV-Visible spectrophotometer uses two light sources, a deuterium (D2) lamp for ultraviolet light and a tungsten (W) lamp for visible light. After bouncing off a mirror (mirror 1), the light beam passes through a slit and hits a diffraction grating. The grating can be rotated allowing for a specific wavelength to be selected. At any specific orientation of the grating, only monochromatic (single wavelength) successfully passes through a slit. A filter is used to remove30

unwanted higher orders of diffraction. The light beam hits a second mirror before it gets split by a half mirror (half of the light is reflected, the other half passes through). One of the beams is allowed to pass through a reference cuvette (which contains the solvent only), the other passes through the sample cuvette. The intensities of the light beams are then measured at the end. 4.2.5 Shaking incubator Incubator Shaker is a large capacity shaking incubator which may be stacked up to 3 high, offering a large capacity in a minimum of floor space. The ingenious design of the Infors shakers features a front-loading pullout platform, stainless steel interior panels, panoramic front window and inspection light. The patented drive system provides uniform motion and quiet operation without vibration. 4.2.6 pH- meter A pH meter is an electronic instrument used to measure the pH (acidity or alkalinity) of a liquid. A typical pH meter consists of a special measuring probe (a glass electrode) connected to an electronic meter that measures and displays the pH reading

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Chapter 5

PROCEDURE OF EXPERIMENT

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5.1 Pretreatment of bagasseAbout 1 kg of post harvest sugarcane bagasse is taken and dried to remove all the moisture present in it. This dried sugarcane bagasse undergoes size reduction by the help of a grinder. Even the powdered form is used for hydrolysis.

5.2 HydrolysisThe cellulose molecules are composed of long chains are broken down to free the sugar, before it is fermented for alcohol production. Though hydrolysis is of many types, dilute acid hydrolysis is an easy and productive process. Also the amount of alcohol produced in case of acid hydrolysis is more than that of alkaline hydrolysis. Concentrated acid hydrolysis is not used as it is a hazardous and corrosive process and also acid has to be separated out after hydrolysis for the experiment has to be feasible.

5.2.1 Steps involved in dilute acid hydrolysis are1. 4 nos. of 250 ml of conical flasks were taken and to each of them 5 gm of grinded sugarcane bagasse is added carefully. 2. Another flask of 500 ml size is taken and to it 5 gm of grinded sugarcane bagasse is added carefully. 3. 100 ml of dilute sulfuric acid of concentration 0.2 M is added to the 500 ml flask. 4. 100 ml of dilute sulfuric acid of concentration 0.3 M is added to two of them 250 ml flasks. 5. 100 ml of dilute sulfuric acid of concentration 0.5 M is added to the remaining two 250 ml flasks. 6. All this samples are left to be soaked to dilute sulfuric acid for 24 hrs.

7. The bottles were then capped with the help of cotton plugs.

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5.3 pH adjustmentBefore addition of any micro-organism to the above prepared samples, pH of these samples has to be adjusted. Otherwise the micro-organism will die in hyper acidic or basic state. A pH of around 5-5.5 is maintained.

5.3.1 Steps involved in pH adjustment are1. The 500 ml flask containing 0.2 M dilute sulfuric acid hydrolyzed bagasse is taken and its pH is checked with the help of a pH meter. 2. As samples are acid hydrolyzed, a highly basic solution is added to bring the pH in the range of 5-5.5. 3. For this purpose, a highly concentrated NaOH solution is prepared by mixing weater with Na pellets. 4. This NaOH solution is added drop wise to the 0.2 M 500 ml flask with constant stirring until the pH reaches to a range of 5-5.5 5. If suppose the pH goes beyond 5-5.5, concentrated HCl acid is added drop wise to maintain the pH in the range. 6. The above steps are repeated for the 0.3 M and 0.5 M dilute sulfuric acid hydrolyzed bagasse. After maintaining the pH, the samples are kept in a Vertical autoclave for hr at 1200C (around 2.02 kg/cm2 pressure) and allowed to cool.

5.4 Fermentation 5.4.1 Media preparationFor preparing 100 ml media, we add Sugar (Dextrose) Yeast extract Urea Make up water = 10 gm = 0.2 gm = 1 gm = 100 ml

34

5.4.2 Steps involved in fermentation are1. To the above 100 ml media, 0.5 gm of yeast ( Saccharomyces cerevisiae) is added in a 250 ml conical flask. 2. This conical flask is then placed in a shaking incubator for 24 hrs. 3. 10 ml of this medium is then added to each of the 5 autoclaved samples aseptically in a Laminar flow chamber. 4. These flasks are properly covered with cotton plug. 5. These flasks are then placed in the shaking incubator at a temperature of 35oC and 120 rpm. 6. After 24 hrs, the flasks are covered with aluminium foils over the cotton plug.

35

Chapter 6

RESULTS

36

6.1 Standard plot for ethanol

16 ml of absolute ethanol was mixed with 24 ml of water and an absorbance of 0.897 nm was found in the UV spectrophotometer. This was assumed to be 100 % ethanol and the standard plot was drawn. SL.NO. Amount of ethanol (in ml) 1 2 3 4 5 6 7 8 9 10 11 0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4 2.7 3.0 Amount of water (in ml) 3.0 2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 0 0 10 20 30 40 50 60 70 80 90 100 0 0.185 0.223 0.291 0.376 0.419 0.611 0.665 0.734 0.8 0.897 Conc. Of ethanol Absorbance

Table 6.1

37

The 100 percent of this ethanol is actually the 40 percent concentration of absolute alcohol. Therefore the actual standard plate is given in the next table.

SL.NO.

Amount of ethanol ( in ml)

Amount of water ( in ml) 3.0 2.7 2.4 2.1 1.8 1.5 1.2 0.9 0.6 0.3 0

Conc. Of ethanol

Absorbance

1 2 3 4 5 6 7 8 9 10 11

0 0.3 0.6 0.9 1.2 1.5 1.8 2.1 2.4 2.7 3.0

0 40 180 120 160 200 240 280 320 360 400

0 0.185 0.223 0.291 0.376 0.419 0.611 0.665 0.734 0.8 0.897

Table 6.2

38

1 0.9 0.8 0.7 Absorbance 0.6 0.5 0.4 0.3 0.2 0.1 0 0 100 200 Ethanol conc. 300 400 500

Figure 6.1

39

6.2 ANALYSIS OF ETHANOL FOR DIFFERENT DAYS

Day 1 Sl. No. 1 2 3 4 5 Sample 0.2 0.3 (I) 0.3(II) 0.5(I) 0.5(II)

Table 6.3 Absorbance (210 nm) 0.62 0.54 0.581 0.523 0.568

0.63 0.62 0.61 0.6 0.59 0.58 0.57 0.56 0.55 0.54 0 0.2 0.4 0.6

Figure 6.2

40



0.68 0.66 0.64 0.62 0.6 0.58 0.56 0.54 0.52 0.5 0 0.2 0.4 0.6

Figure 6.3

41



0.69 0.68 0.67 0.66 0.65 0.64 0.63 0.62 0.61 0.6 0.59 0 0.2 0.4 0.6

Figure 6.4

42



0.72 0.71 0.7 0.69 0.68 0.67 0.66 0.65 0.64 0.63 0.62 0 0.2 0.4 0.6

Figure 6.5

43



0.73 0.72 0.71 0.7 0.69 0.68 0.67 0.66 0.65 0.64 0 0.2 0.4 0.6

Figure 6.6

44



0.74

0.73

0.72

0.71

0.7

0.69

0.68 0 0.2 0.4 0.6

Figure 6.7

45



0.755 0.75 0.745 0.74 0.735 0.73 0.725 0.72 0.715 0 0.2 0.4 0.6

Figure 6.8

46

6.3 COMPARISON OF NUMBER OF DAYS For 0.2 M acid concentration

ETHANOL

CONCENTRATION

WITH

INCREASING

Table 6.10 SL. NO. 1 2 3 4 5 6 7 8 9 10 NO. OF DAYS 1 2 3 4 5 6 7 8 9 10 ETHANOL CONC. 276.47 297.43 302.34

317.50 322.40 327.76 335.78

The graph of the above table is as follows360 340 320 Ethanol conc. 300 280 260 240 220 200 0 2 4 6 no. of days 8 10 12

Figure 6.9

47

For 0.3 M acid concentration SL. NO. 1 2 3 4 5 6 7 8 9 10 NO. OF DAYS 1 2 3 4 5 6 7 8 9 10 Table 6.11 ETHANOL CONC. 249.94 275.39 287.85

299.44 305.24 321.74 326.19

The graph of the above table is as follows340 320 300 Ethanol conc. 280 260 240 220 200 0 2 4 6 No. of days 8 10 12

Figure 6.10

48

For 0.5 M acid concentration SL. NO. 1 2 3 4 5 6 7 8 9 10 NO. OF DAYS 1 2 3 4 5 6 7 8 9 10 Table 6.12 The graph of the above table is as follows340 320 300 Ethanol conc. 280 260 240 220 200 0 2 4 6 No. of days 8 10 12

ETHANOL CONC. ( ml/lt) 243.25 251.36 267.55

288.74 295.96 306.13 328.73

Figure 6.11

49

Chapter 7

DISCUSSION

50

1. The ethanol concentration in ml/lt of acid hydrolyzed bagasse increases with the increasing number of days. 2. This shows that the sugar is being fermented by the help of micro organism yeast for the production of ethanol. 3. The maximum concentration of ethanol is 328.73 ml/lt of acid hydrolyzed bagasse which was found on the 10th day of fermentation. 4. In absorbance vs molarity curve , after each progressive data the curve tends to come in a straight line. 5. For 0.3 M and 0.5 M we have taken two identical samples for each for better accuracy. 6. For drawing graphs, in case of 0.3 M, 0.5 M samples average is taken. 7. Still some deflection in the graphs can be considered due to precision error, long duration of experiment, and human errors.

51

Chapter 8

REFERENCES

52

1. Biosource technology, Volume 83 Issue 1, May 2002. 2. Biosource Technology, Volume 77, Issue 2, April 2001. 3. Cellulosic Ehanol- Wikipedia, The free encyclopedia. 4. Ethanol From Cellulose: A General Review Trends in new crops and new uses. 2002. J. Janick and A. Whipkey (eds.). ASHS Press, Alexandria, VA. 5. Ethanol production via enzymatic hydrolysis of sugar edition 1996 Elba P.S. Bon 6. www.osti.gov/bridge/servlets/purl/755492CufN/webviewable/755492 7. www.ethtec.com.au/downloads/Latest_News/ETHANOL_FROM_BAG ASSE_MEETINGS 8. Renewable and Sustainable Energy Reviews, Volume 13, Issues 6-7, August-September 2009, Pages 1418-1427 9. www.cellulosicroundtable.com/greenfieldethanol.htm 10. www.bioresourcesjournal.com/index.php/BioRes11. sciencestage.com/d/29329/lime-pretreatment-of-sugarcane-bagasse-

for-bioethanol-production12. www.academicjournals.org/ajb/PDF/pdf2008/18Jul/Damisa%20et%20

al.13. aiche.confex.com/aiche/2006/preliminaryprogram/abstract_58322.ht

m14.

www.rurdev.usda.gov/rbs/pub/sep06/ethanol.htm53

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55

Dr. Arvind Kumar

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Hydrolysis of Lignocellulosic Materials for Ethanol Production

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