1.0 INTRODUCTION 1.1 Theme behind Over the last few decades, the negative impacts of fossil fuel on the environment and consequent global warming, progressive demand for energy, inevitable depletion of the world s energy supply, and the unstable oil market have renewed the interest of society in searching for alternative fuels (Himmel et al., 2007; Solomon et al., 2007). The alternative fuel are expected to satisfy several requirements including substantial reduction of greenhouse gas emission, worldwide availability of raw materials, and capability of being produced from renewable feed stocks. Many alternative fuel sources have been explored. Two of the biofuels that have received most attention are biodiesel and bioethanol. Both of these fuel sources are made from renewable feedstock. Biodiesel is made from vegetable oils or animal fats (Ma and Hanna, 1999) and ethanol is made from plant sugar (Wheals et al., 1999). Corn, sugarcane and lignocellulosic biomass have been explored as the major substrate for later. Production of fuel ethanol from biomass seems to be an interesting alternative to traditional fossil fuel, which can be blended with petrol or used as neat alcohol in dedicated engines, taking advantage of higher octane number and higher heat of vaporization; furthermore it is an excellent fuel for future advanced flexi-fuel hybrid vehicles (Hahn-Hagerdal et al., 2006). Ethanol is currently produced from sugars, starches and cellulosic material. The first two groups of raw material are currently the main resources for ethanol production, but concomitant growth in demand for human feed similar to energy could make them potentially less competitive and perhaps expensive feed stocks in the near future, leaving the cellulosic material as the only potential feedstock for production of ethanol (Taherzadeh and Karimi, 2007). Cellulosic material obtained from wood and agricultural residues, municipal solid Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 1
wastes and energy crops represent the most abundant global source of biomass (Lin and Tanaka, 2006). All these facts have motivated extensive research toward making an efficient conversion of lignocelluloses in to sugar monomers for further fermentation to ethanol. 1.2 Ethanol and its physicochemical properties Ethanol (ethyl alcohol, grain alcohol) is a clear, colorless liquid with a characteristic, agreeable odor. In dilute aqueous solution, it has a somewhat sweet flavor, but in more concentrated solutions it has a burning taste. Ethanol (CH 3 CH 2 OH) is an alcohol, a group of chemical compound whose molecules contain hydroxyl group (-OH), bonded to a carbon atom, melts at -114.1 o C, boils at 78.5 o C, and has a density of 0.789 g/ml at 20 o C. The word alcohol derives from Arabic alkuhul, which denotes a fine powder of antimony used as an eye makeup. Alcohol originally referred to any powder, but medieval alchemists later applied the term to the refined products of distillation, and this led to the current usage. Ethanol is miscible in all proportions with water and with most organic solvent. It is useful as solvent for many substances and in making perfumes, paints, lacquer, and explosives. It can be oxidized to form first acetaldehyde and then acetic acid. It can be dehydrated to form ether. Alcoholic solutions of nonvolatile substances are called tinctures, if it is volatile; the solution is called spirit (Shakhashiri, 2009). 1.3 Historical background Ethanol has been made since ancient time by the fermentation of sugar. All beverage ethanol and more than half of industrial ethanol is still made by this process. Production of alcoholic beverages is in fact as old as human civilization while production of pure ethanol apparently begins in the 12-14 th century along with improvement of distillation. During the middle ages, ethanol was used mainly for Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 2
production of medical drugs and for the manufacture of painting pigments. The knowledge of using starchy materials for ethanol production was first employed in the 12 th century in typical beer making countries like Ireland. It was only in 19 th century that this trade became an industry with enormous production figures due to the economic improvements of the distilling process (Roehr et al., 2000). Alcohol beverages still represent a large portion of industrial alcohol, but other applications are becoming more important. Alcohol can be used for various purposes in the chemical industry and its role as a fuel is well known. Ethanol as a neat fuel or even in the blended form with gasoline has a long history as automotive fuel. In 1860, German inventor Nicholas Otto used ethanol as a fuel in an early prototype of an internal combustion engine because; it was widely available throughout Europe for use in spirit lamps. A few years later, Henry Ford built his first automobile with an engine that could run on ethanol. In 1908, Ford unveiled his Model T engine equipped with carburetors that could be adjusted to use alcohol, gasoline or a mixture of both fuels (Solomon et al., 2007). Ethyl alcohol as the fuel of the future was presented by Ford for first time. In 1925, he told the New York Times: the fuel of future is going to come from fruit like that sumac out by the road, or from apples, weeds, sawdust almost anything there is fuel in every bit of vegetable matter that can be fermented. There s enough alcohol in one year s yield of an acre of potatoes to drive the machinery necessary to cultivate the fields for a hundred years. Instead of fossil fuels, which were predominantly used for automobile transportation throughout the last century, obviously due to their lower production cost; hydrous ethanol can be used as a substitute for gasoline in dedicated engines and anhydrous ethanol, on the other hand is an effective octane booster when mixed in blends of 5% to 30% with no engine modification requirement (Licht, 2006). Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 3
It was the beginning of the 20 th century that it had become known that alcohol might be used as fuel for various combustion engines, especially for automobiles. However, production of large amounts of alcohol require large amount of sugar, which tends to limit the production. Still, the 20 th century various process were developed, based on for instance sugar cane, beet molasses and industrial by products and now, the ethanol industry is usually in close connection with agricultural production (Roehr et al., 2000). 1.4 Comparative account of ethanol and gasoline Gasoline is a hydrocarbon produced from crude oil by fractional distillation. It is non-water miscible (which makes it hydrophobic) and has a flash point of approximately -45 o F, varying with octane rating. It has vapor density between 3 and 4. Therefore, as with all products with vapor density greater than 1.0 gasoline vapors will seek low levels or remain close to ground level. Gasoline has a specific gravity of 0.72-0.76 which indicates it will float on top of water since it is not-water miscible or insoluble. Its auto-ignition temperature is between 536 o F and 853 o F, and it has boiling point between 100 o F and 400 o F depending on fuel composition. Gasoline is not considered a poison but does have harmful effects after long-term and high-level exposure can lead to respiratory failure (Anonymous, 2012). In contrast to gasoline, ethanol is an oxygenated fuel that contains 35% oxygen, which reduces particulate and nitrogen oxides (NO X ) emission from combustion. Pure ethanol is polar solvent that is water soluble and has 55 o F flash point. Ethanol has a vapor density of 1.59, which indicates that it is heavier than air. Consequently, ethanol vapors do not rise, similar to vapors from gasoline, which seek lower altitudes. Ethanol s specific gravity is 0.79, which indicates it is lighter than water but since it is water-soluble (hydrophilic). It will thoroughly mix with water. Ethanol has Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 4
an auto-ignition temperature of 793 o F and boiling point of 173 o F. Ethanol is less toxic than gasoline or methanol and after blended with gasoline fuel for automobiles, it can significantly reduce petroleum use, increases the oxygen content of the fuel, improving the combustion of gasoline and reducing the exhaust greenhouse gases emission (Wang et al., 1999). It is most commonly blended with gasoline in concentration of 10% bioethanol to 90% gasoline, known as E10 and nicknamed gasohol. 1.5 Environmental impact of fuel ethanol Fuel ethanol is suggested as a sustainable fuel which can be produced from renewable resources and led to maintain or even reduced the level of greenhouse gasses. The net emission of CO 2 are reported to be close to zero, since the CO 2 released during ethanol production and combustion is recaptured as nutrient by the crop plants (during photosynthesis), which are the raw materials for ethanol production (Spatari et al, 2005). Ethanol is blend with gasoline, increases octane and provides oxygen to promote more complete combustion. Burning ethanol made from plants is estimated to reduce greenhouse gas emissions by 86% (Wang et al., 2007). In addition, ethanol, as petroleum gasoline additive, is safer than the methyl tertiary butyl ether (MTBE) which is currently used for cleaner combustion. Unlike MTBE, which is reported to be toxic and has potential to contaminate ground water, ethanol is found to be water soluble and biodegradable in nature (Sun and Chang, 2002). 1.6 Global Scenario The most widely used biofuel, ethanol as substitute for gasoline are attracting growing interest around the world, with some government announcing commitments to biofuel programs as a way to reduce both greenhouse gas emissions and dependence on petroleum-based fuels. The United States, Brazil, and several Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 5
European Union member states have the largest programs promoting bio-fuels in the world. The different feedstock used in different countries including corn in US, sugarcane in Brazil and other biofuels elsewhere, have been produced to meet the increasing global demand (Ralph et al., 2010). Biofuel policies tend to vary according to available feedstock for fuel production and national agricultural policies. With all of the new government programs in America, Asia and Europe in place, total global fuel ethanol demand could grow to exceed 125 billion liters by 2020. Brazil and United States are world leader which exploit sugarcane and corn respectively and together account for about 70% of world bioethanol production. The United States is the world s largest producer of bioethanol, accounting for nearly 47% of bioethanol production while Brazil is the world s largest exporter of bioethanol and second largest producer after the United States (Balat et al., 2008; Demirbas, 2007a; Balat and Balat, 2009). Brazil has a long history of biofuel production dating to 1975 when National Alcohol Fuel Program (ProAlcool) was initiated. The program aimed to increase production of bioethanol as a substitute for expensive and extremely scarce gasoline. With substantial governmental interventions to increase alcohol demand and supply, Brazil created assets and developed institutional and technological capabilities for using renewable energy on a large-scale. Presently it is the only country which has more than 80% of its vehicle running with bioethanol and even small aero plane engine are now being developed (Soccol et al., 2010). Next to Brazil is United States which has large corn based ethanol industries with capacity of over 15 billion liters per year and production capacity is anticipated to continue rising to about 28 billion liter per year by 2012, furthermore US administration proposed mandate of 35 billion gallons of ethanol by 2017. Though Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 6
practical limit of corn based ethanol is 15 billion gallon per year, the differences of 20 billion gallons are expected to be from cellulosic ethanol. In addition, since the United States has the potential to produce over 1 billion tons of biomass each year, US department of agriculture and department of energy have targeted replacing 30% of the liquid petroleum transportation fuel (about 300 billion liter) with biofuels by 2025 (Perlack et al., 2005). In European countries, beet molasses are the most utilized sucrose containing feed stocks for bioethanol production (Cardona and Sanchez, 2007). Initially European countries blend 5% of ethanol under the EU quality standard EN 228. This blend requires no engine modification and is covered by vehicle warranties while with engine modification, bioethanol can be used at higher levels, for example, E85 (85% bioethanol) (Demirbas, 2008). On 23 rd January 2008, the European commission (European Commission, 2008) proposed a blending minimum target of 10% for the share of biofuels in transport in the context of the EU directive on the promotion of the use of energy from renewable sources that envisages a 20% share of all renewable energy sources in total energy consumption by 2020. France established an ambitious biofuel plan, with goals of 7% by 2010, and 10% by 2015 while Belgium was set a target of 5.75% (Pfuderer and Castillo, 2008; Thamsiriroj and Murphy, 2009; Balat and Balat, 2009). The largest producer of ethanol in Asia is China with world s largest producing plant; Jilin Tianhe with an initial capacity of 600,000 tons/year (2.5 million liters/day) is located in it. (Talebnia, 2008). Moreover, the China government has pledged that 10% of the nation s energy will come from renewable energy sources by 2020 (World Watch Institute, 2006). Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 7
India is the fourth largest producer of ethanol in world after Brazil, United States of America (USA) and China and second largest producer of ethanol in Asia after China, producing approximately 2000 million liters of ethanol mainly by fermentation of sugarcane molasses (Ray et al., 2011). 1.7 Nation s potential Indian economy is agricultural based fast growing economy and has 0.5% oil and gas resources of the world, but 16% of the world s population with the result that country depends heavily on oil import to meet the domestic demand (Sukumarn and Pandey, 2009). With the huge population and limited land resources, the nation is looking towards an alternative energy sources which must be renewable and environmental friendly. More than 70% needs of the country are met from import of crude oil and natural gas. The demand for motor gasoline has been growing at an average with an annual rate of 7% during the last decades and current consumption of petrol for transportation needs (motor gasoline) is estimated at 15.23 billion liters annually (MPNG 2009). Bioethanol might be one of the most potent solutions to overcome the demand for liquid transportation fuel. As mention earlier, India is the fourth largest producer of ethanol in the world after Brazil, USA and China, producing approximately 2000 million liters of ethanol. However it is consumed by three ways; of the total available ethanol the maximum, about 45% is used to produced potable liquor, about 40 per cent is used in the alcohol based chemical industry (as a solvent in synthesis of other organic chemicals) and the rest is used for blending with petrol and other purposes. The demand for ethanol has been continually increases on account of the growth of user industries and use to ethanol as a fuel in the country. However, the production and availability of ethanol has largely lagged behind ((Bharadwaj et al., 2007; Ray et al., 2011). Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 8
In India, there are approximately 400 sugar cane factories and 270 distilleries spread all over the country. Most of the distilleries are relatively small with typical production capacity of around 300,000 liters per year of primarily beverage and industrial grade ethanol (Virtus Global Partners, 2008). The average efficiency in production of molasses based ethanol is 85% and amount of fermentable sugar in molasses is about 42% with yield of about 222 liters of ethanol per tones of molasses. On the other hand, total annual demand for alcohol in the country excluding fuel application was about 1.3 billion liters which is 40% of total installed capacity. However the actual production of ethanol was only 1.69 billion liters which is not sufficient to meet the fuel ethanol demand if the entire gasoline in the country had to be doped at 5% level. If all the molasses had been converted to ethanol, the average annual yield would be only around 2.91 billion liters. Out of this the estimated surplus amount would be far less than the projected current demand of about 1.5 billion liters for the purpose of 10% blending (AIDA, 2006; Sukumaran et al., 2010). Due to very low sharing of biofuel production in consumption of transportation fuel, the ministry of petroleum and natural gas has mandatory to 5% blending of ethanol in gasoline in the 10 states including Andhra Pradesh, Goa, Gujarat, Haryana, Karnataka, Maharashtra, Punjab, Tamil Nadu, Uttar Pradesh and Uttaranchal and in 3 Union territories of Damn and Diu, Dadra and Nagar Haveli and Chanidigarh. The blending target for ethanol and biodiesel in gasoline and petroleum diesel were proposed as 10% and 20% respectively by 2012 (Planning Commission, 2003). In May 2009, the planning commission advised the government to consider providing incentives to encourage companies to acquire sugarcane plantations abroad, especially in countries such as Brazil. India s leading sugar refiner and ethanol Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 9
producer, Shree Renuka Sugars, has a long standing presence in Brazil and is now counted among the top five sugar producers there (Ray et al., 2011). The new biofuel policy was approved by the Union Cabinet in December 2009. In view of the multiplicity of departments and agencies, it was felt imperative to provide a higher level co-ordination and policy guidance/review of biofuel development, promotion and utilization. For this purpose, the policy proposed to set up a National Biofuel Coordination Committee (NBCC), headed by the prime minister and ministers from the concerned ministries were proposed to be members of the committee. The role and active participation of the states was considered crucial in the planning and implementation of the biofuel programme. The main objective of the policy has to encourage domestic production of ethanol and further the ethanol blending programme (EBP) in the country. As for as starchy biomass is concern (which is majorly available in the form of food crop), it is not possible to country like India with second largest population to feed, and with more than 238 million people living below poverty line (NSS, 2007), to spare food crop for ethanol production. Various laboratories across the country are engaged in R&D on different aspect of lignocellulosic ethanol including Biochemical Engineering Research Centre, IIT, Delhi and National Institute for Interdisciplinary Science and Technology (NIIST), Trivandrum, Kerala. As for as industrial sector is concern, PRAJ industries has 70% market share of the ethanol producing plants in India while US based Khosla Ventures recently purchased 10% stake in this company. Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 10
1.8 Lignocellulosic biomass as potential feedstock Ethanol can be produced from a variety of lignocellulosic biomass (Wheals et al., 1999). Lignocellulosic biomass is a plentiful and economical resource that can serve as a source for ethanol production on a large scale (Dien et al., 1999). Lignocellulosic biomass sources include: agriculture waste (wheat straw, corn stovers, soybean residues, sugarcane bagasse, and cotton stalk etc.), industrial waste (pulp and paper industry), forestry residue and municipal solid waste (Zaldivar et al., 2001). Cotton stalk is an example of a lignocellulosic agricultural waste. There are about 32.0 million hectares of cotton cultivable area across the world and about 10.0 million hectares in the country. India accounts for about 32% of the global cotton area and contributes to 21% of the global cotton produce, currently ranking second after China (Kranthi et al., 2011). Since cotton stalk is a byproduct of cotton crop (Guhagarkar, 1997); India has an abundance of this lignocellulosic biomass sources. These cotton stalks that are left behind after the cotton harvest, often becomes infested with pests such as boll weevils. Therefore farmers are in need of a way to get rid of the cotton stalk. However, cotton stalk contains approximate 45.5% of holocelluloses, which can be broken down into glucose for use in ethanol production (Williams, 2006). Using this byproduct for ethanol production would help to address the issue of disposing waste residue and turning it into something high value. The technology for bioethanol production from simple sugars and starch are well defined, however, production of ethanol from lignocellulosic biomass still requires extensive research to develop a feasible production method. For this reason, this study is focused on technology to convert lignocellulosic biomass to ethanol. Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 11
1.9 Research objectives The present research work is carried out to optimize the overall process of ethanol production from cotton stalk by exploring Saccharomyces cerevisiae and Pachysolen tannophilus. To complete the overall aim; the following specific objectives focus on several aspects of process. Determine the chemical composition of cotton stalk; Study the effect of different pretreatment process on native substrate in order to easily access the reagent to carbohydrate polymers; Investigate and optimize the effect of acid and enzyme during hydrolysis for the purpose to break down the cellulose and hemicellulose in to monomers; Investigate the inhibitory obstacles in hydrolyzate and study to overcome by various detoxification methods; Process optimization of some important parameter including temperature, ph, aeration, agitation, inoculum concentration, and time require for efficient fermentation process in order to evaluate the ethanol production potential from hydrolyzate of cotton stalk; Comparative study of co-culture fermentation by Saccharomyces cerevisiae and Pachysolen tannophilus with mono culture fermentation of each; Study the various aspects of immobilization and comparative analysis of free and immobilized cells of Saccharomyces cerevisiae and Pachysolen tannophilus; 1.10 Outline of thesis The structure was designed to focus different aspects of review and research, raised in objectives and is framed in six chapters, containing introduction, review of Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 12
literature, material and methods, results and discussion, concluding remarks and bibliography, which are presented as follows. The first two chapters focus on introduction and review of this research including energy demand, description of ethanol as a fuel, historical background, international and national scenario as well as environmental impact caused by the use of ethanol as a fuel. Principle steps required to achieve the fuel ethanol are given in chapter second. Bioconversion technologies, includes pretreatment, hydrolysis and fermentation of lignocellulosic biomass along with physiological phase of microorganisms is also reviewed in this chapter. Chapter third describes the materials and methods used in the work including collection of cotton stalk, compositional analysis, pretreatment hydrolysis and detoxification and fermentation as well as immobilization. The fourth chapter summarizes the results obtained from activities taken in order to achieve the proposed objectives of theses work (presented in 27 figures and 28 tables) and is discussed in light of previously done research and reviews. Chapter fifth summarizes the concluding remarks on results reported in previous chapter. Considering the conclusion, the recommendations are to be continuing the research regarding obtaining the bioethanol from cotton stalks and the future line of work to make it feasible for pilot level. The sixth chapter comprises the references, required in the documentation and implementations of this thesis, consulted 295 bibliographic references. Ph. D. Thesis, Mirza Zaheer Baig, 2014, Dr.BAMU, Aurangabad Page 13