ABSTRACT
The production of bioethanol from waste pineapple and watermelon, using dried active baker‘s yeast strain (Sacchromyces cerevisiae) was investigated using two methods of production (Direct Fermentation and Simultaneous Saccharification and Fermentation). The proximate composition, pH and the sugar content were determined. The mean value obtained for moisture, ash, crude protein, crude fiber, fat and carbohydrate was 91.52 ± 0.01 %, 0.22 ± 0.01 %, 0.45 ± 0.05 %, 0.19 ± 0.01 %, 0.18 ± 0.01 %, 7.18 ± 0.05 % respectively for watermelon and also the mean obtained for moisture, ash, crude protein, crude fiber, fat and carbohydrate was 82.20 ± 0.07 %, 1.23 ± 0.15 %, 0.45 ± 0.07 %, 0.70 ± 0.01 %, 0.47 ± 0.03 %, 4.06 ± 0.20 % respectively for pineapple waste. The reducing sugar assay of the pineapple waste gave 7.8 g/100 g and watermelon gave 4.5 g/100 g. Effect of yeast concentration, duration of fermentation, pH and temperature as they relate to the optimisation of the ethanol production were also investigated. The result showed that the fermented fruits waste produced ethanol: 5.88 ± 0.03 % and 1.91 ± 0.01 % (v/v) for pineapple and watermelon respectively using direct fermentation and 14.18 ± 0.03 %, 3.07 ± 0.01 % (v/v) for pineapple and watermelon respectively using simultaneous saccharification and fermentation, the result shows that the rate of ethanol production by fermentation of both waste fruits by baker‘s yeast (Sacchromyces cerevisiae) increases with fermentation period and maximum ethanol yield was at 72 h.
TABLE OF CONTENTS
Title page – – – – – – – – – – iii Declaration – – – – – – – – – – iv Certification – – – – – – – – – – v Dedication – – – – – – – – – – vi Acknowledgement – – – – – – – – – vii Abstract – – – – – – – – – – viii Table of Contents – – – – – – – – – ix List of Tables – – – – – – – – – xv List of Figures – – – – – – – – – xvi CHAPTER ONE – – – – – – – – – 1
1.0 Introduction – – – – – – – – – 1
1.1 Biofuel – – – – – – – – – 3 1.1.1 Types of Biofuel- – – – – – – – – 3 1.1.2 Bioethanol – – – – – – – – – 3 1.1.3 Biodiesel – – – – – – – – – 4 1.2 Alcohol – – – – – – – – – 4
1.2.1 Properties of Ethanol – – – – – – – – 5
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1.3 Crops with Biofuel Potential in Nigeria- – – – – – 6 1.3.1 Sorghum – – – – – – – – – 6 1.3.2 Cassava – — – – – – – – 7 1.3.3 Sugarcane – – – – – – – – – 8 1.3.4 Jatropha – – – – – – – – – 9 1.4 Fuel Ethanol – – – – – – – – – 12 1.4.1 Ethanol Production Technologies – – – – – – 12 1.4.2 Pretreatment – – – – – – – – – 13 1.4.3 Hydrolysis – – – – – – – – – 13 1.4.4 Acid Hydrolysis – – – – – – – – 13 1.4.5 Dilute Acid Hydrolysis – – – – – – – 14 1.4.6 First Stage Dilute Acid Hydrolysis- – – – – – – 15 1.4.7 Two Stage Dilute Acid Hydrolysis – – – – – – 16 1.4.8 Enzymatic Hydrolysis – – – – – – – – 16 1.5 Separate Hydrolysis and Fermentation – – – – – 17 1.6 Simultaneous Saccharification and Fermentation – – – – 17 1.7 Distillation – – – – – – – – – 19
1.8 Feedstock for Ethanol Production- – – – – – – 20
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1.9 Feedstock Containing Simple Sugar – – – – – 20 1.10 Feedstock Containing Starch – – – – – – 21 1.11 Lignocellulosic Biomass Feedstock – – – – – – 21 1.12 Microbial Fermentation – – – – – – – 22 1.13 Significance of the Study- – – – – – – – 23 1.14 The Statement of the Problem – – – – – 23 1.15 Aim – – – – – – – – – – 24 1.16 Objectives- – – – – – – – – – 24
CHAPTER TWO – – – – – – – – – 25
2.0 Literature Review – – – – – – – – 25
2.1 Ethanol Production – – – – – – – – 25
2.1.1 Continuous Fermentation – – – – – – – 25
2.1.2 Fermenting Organism – – – – – – – – 26 2.1.3 Thermophilic Ethanol Producing Bacteria – – – – – 27 2.1.4 Biomass Yield – – – – – – – – – 27 2.1.5 Substrate Range – – – – – – – – 27 2.1.6 Growth and Reaction Rate – – – – – – – 28
2.1.7 Continuous Product Removal – – – – – – – 28
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2.1.8 Condition of the Fermenting Broth- – – – – – – 28 2.1.9 Risk of Contamination – – – – – – – 28 2.2.0 By Product Formation – – – – – – – – 29 2.2.1 Growth Inhibition – – – – – – – – – 29 2.2.2 The Benefits of Fuel Ethanol – – – – – – – 29 2.2.3 Ethanol Production and Environmental Issue – – – – – 30
CHAPTER THREE – – – – – – – – – 41
3.0 Materials and Methods – – – – – – – – 41
3.1 Apparatus – – – – – – – – – 41
3.2 Reagent – – – – – – – – – 41
3.3 Sample Collection – – – – – – – – 41
3.4 Direct Fermentation of the Fruit (Whole) – – – – – 41
3.4.1 Sample Preparation – – – – – – – – 41
3.4.2 Determination of Moisture – – – – – – – 42
3.4.3 Determination of Ash – – – – – – – – 42
3.4.4 Determination of Crude Fibre – – – – – – – 42
3.4.5 Determination of Lipids – – – – – – – 43
3.4.6 Determination of Crude Protein – – – – – – 44
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3.4.7 Determination of Carbohydrate – – – – – – 45
3.4.8 Fermentation – – – – – – – – – 45
3.5 Simultaneous Saccharification and Fermentation – – – – 46 3.5.1 Sample Preparation – – – – – – – – 46 3.5.2 Acid Hydrolysis of Fruit – – – – – – – 46 3.5.3 pH Adjustment – – – – – – – – – 46 3.5.4 Fermentation – – – – – – – – – 47 3.5.5 Distillation – – – – – – – – – – 47 3.5.6 Alcoholic Content – – – – – – – – 47 3.6 Density Measurements – – – – – – – – 48 3.7 Data Analysis – – – – – – – – 48
CHAPTER FOUR – – – – – – – – – 49
4.0 RESULTS – – – – – – – – – – 49 4.1 Proximate Composition – – – – – – – 50 4.2 Optimisation of Ethanol Production with Direct Fermentation – – 51
CHAPTER FIVE – – – – – – – – – 61
5.0 DISCUSSION – – – – – – – 61
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5.1 Proximate Composition – – – – – – – 61
5.1.1 Moisture Content – – – – – – – – 61
5.1. 2 Ash Content – – – – – – – – – 61 5.1.3 Carbohydrate Content (CHO) – – – – – – – 61 5.1.4. Crude Protein – – – — – – – – – 61 5.1.5 Fat – – – – – – – – – – – 62 5.2. Optimization – – – – – – – – – 62 5.3. Statistical Analysis of the Experimental Results – – – – – 63 5.4. Effects of Experimental Variables on Hydrolysis – – – – 63 5.5. Stepwise Simultaneous Saccharification and Fermentation of Fruits – 65 CHAPTER SIX – – – – – – – – – 66 6.0 SUMMARY, CONCLUSION AND SUMMARY – – – – 66 6.1 Summary – – – – – – – – – 66 6.2 Conclusion – – – – – – – – – 68 6.3 Recommendation – – – – – – – – 68 REFERENCES – – – – – – – – – 69 APPENDICES – – – – – – – – – 89 Appendix I: A Variation of Yeast Concentration with % Volume of Ethanol Formed from Pineapple and Water Melon (Direct Fermentation) – – 89 Appendix II: Three Dimensional and Cube Plot of Varying Parameters for the Production of Ethanol from Pineapple – – – – 90
CHAPTER ONE
1.0 INTRODUCTION One of the oldest biological processes known to mankind is the fermentation of biomass into bioethanol. The process has been known since prehistoric time for the production of alcoholic drinks but has lately gained growing importance due to the interest in producing fuel ethanol. Industrial production of fuel ethanol started in Brazil during the oil crisis in the 1970s, and has ever since been widely important there. The know-how and achievements from Brazil have been used and further developed around the world. Today ethanol is produced from all sorts of biomass rich in sucrose, starch and cellulose. These are easily hydrolyzed by acids or enzymes to C6 sugars (mainly glucose), which are fermented into ethanol and CO2. The largest producers of bioethanol are Brazil and the USA using sugarcane and corn respectively as feedstock. Mainly wheat crops and sugar beet have so far been used in Europe. The world production of fuel ethanol in 2007 was around 50 billion liters (Renewable Fuel Association, 2008). The energy in this ethanol corresponds to less than 1.2 % of the total fuel consumption of 88.2 quadrillion Btu in 2005. In the USA (the largest producer of bioethanol) the production of bioethanol has more than doubled from 2002 to 2007 (Renewable Fuel Association, 2008).
The types of feedstock used today for production of bioethanol are primarily based on edible bio-resources, which can also find use as food for humans and animals (Klass, 1998). This has together with increasing food prices resulted in an almost religious debate about the ethics in using food for fuels (Renewable Fuel Association, 2008). Furthermore it has also increased the interest in the next generation of bioethanol. Second generation bioethanol or cellulosic ethanol as it is also called is made from biomass like agricultural waste, straw and wood. These consist of large amounts of cellulose, hemicelluloses and lignin (termed lignocellulose) and much less starch and sugar
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compared to the feeds for first generation bioethanol. Due to the complex structures in which glucose is bound in lignocellulosic biomass more energy is required to release the glucose for fermentation (Klass, 1998). In particular rice straw in Asia has a huge potential as lignocellulosic feed for second generation bioethanol production (Klinke et al., 2004). These new improved technologies for biomass conversion use less non-renewable energy and give both a higher CO2 reduction and a higher product yield than first-generation plants due to a higher utilization of the feedstock. Therefore, there is globally an intense research on developing this so-called second generation bioethanol. Another significant aspect is that abundant coal resources are the primary source of the non-renewable energy in conversion of biomass into ethanol. In that way, a premium liquid transportation fuel is produced from a less valuable energy resource, which further reduces the need for import of petroleum (Shapouri, 2002).The use of ethanol as an automobile fuel is not a new invention. Already in 1908, Ford‘s model (T) could be adjusted to run on either gasoline or alcohol (Saha et al., 2005). However, after World War II the interest in using ethanol as a fuel declined because cheap gasoline made from petroleum was available. In the 1970‘s, the interest in fuel ethanol was renewed due to the oil crisis (Aro et al., 2005). More recently, ethanol has become used as an additive in gasoline. MTBE (methyl tertiary butyl ether) is used as a gasoline additive to increase the oxygen content and the octane number. During the last few years, the use of MTBE has been banned in several states in the USA due to the risk of contamination of water. Many companies have replaced MTBE with ethanol to give the gasoline similar clean burning and octane boosting properties as MTBE-blended gasoline (Dien et al., 2006).
Today there are several flexi-fuel automobile models (vehicles that can run on mixtures of ethanol and gasoline containing up to 85 % ethanol) available from various manufacturers (Larsson et al.,
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1999). About 99 % of the fuel ethanol is produced from cultivated crops. Brazil has for a long time been the leading ethanol producer of the world. However, during the last years USA has increased its production and today both countries have an annual production of about 16000 000m3 (Nigam, 2002). 1.1 Bio-fuels These are alcohols, ethers, esters and other chemicals made from cellulose-based biomass. This includes herbaceous and woody plant, agricultural and forestry residues and a large portion of municipal and industrial waste materials. Bio-fuels are renewable since they are produced from biomass—organic matter, such as plants. They generate about the same amount of carbon dioxide (a greenhouse gas) from the tailpipe as fossil fuels, but the plants that are grown to produce the bio-fuels actually remove carbon dioxide from the atmosphere. Therefore, the net emission of carbon dioxide will be close to zero (Rainer and Dominik, 2007). The bio-fuels industry has evolved from using first generation feedstock (typically food crops) to using second and third generation feedstocks, for both ethanol and biodiesel. While the term bio-fuels denote any fuel made from biological sources, for most practical uses, the term refers to either ethanol or biodiesel. The last few years have seen tremendous growth in biofuel (Rainer and Dominik, 2007) 1.1.1 Types of Biofuel 1.1.2. Biothanol
Biothanol, a distilled colorless liquid fuel obtained from numerous potential feedstock varieties such as sugar beet, wheat, corn, cassava, fruits, baggasse, barley, molasses, skim milk (whey), potatoes, sorghum, switch grass and cellulose biomass such as wood, paper, straw and other
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cellulose wastes such as grasses, others includes municipal solid wastes. These various waste streams for bioethanol production have their peculiar properties and generally differ. Feedstocks prices and price of natural gas are predominant influential factors that determine the cost of biothanol production. Biothanol as an alternative fuel, offers a Sustainable economy by reducing the use of imported petroleum, emitting neutral CO2 (g), boost economy providing value added market opportunities for the Agricultural sector (Shell and Codex, 2006). Ethanol with its high octane count has is currently positively used as an automobile fuels with policies to promote its production most especially in Brazil, United States, majority of the European Union Countries and South Africa. 1.1.3 Biodiesel This is an ester that can be made from several types of oils, such as vegetable oils and animal fats. Biodiesel is typically used as a blend 20 % biodiesel and 80 % petroleum diesel called B20. B20 can be used in a conventional diesel engine with essentially no engine modifications. There is also a growing interest in using biodiesel where workers are exposed to diesel exhaust, in aircraft to control local pollution near airports, and in locomotives that face restricted use unless emissions can be reduced (Rainer and Dominik, 2007). 1.2 Alcohol
The word alcohol derives from Arabic al-kuhul, which denotes a fine powder of antimony produced by distilling antimony and used as an eye makeup. Alcohol originally referred to any fine powder, but medieval alchemists later applied the term to the refined products of distillation, and this led to the current usage. Ethanol, the most widely used bio-fuel, is made in a process similar to brewing beer. The ethanol in the end is blended with gasoline to improve vehicle performance and reduce air pollution.
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Blending ethanol with gasoline produces a fuel with a higher octane rating which increases the engine‘s compression ratio and results in increased thermal efficiency. In high altitude sports, ethanol-gasoline blends are used as oxidizer and with the exhaust gases contributing little to atmospheric pollution. Bioethanol used as fuel is produced from fermentation of sugars derived mainly from corn (primary bioethanol source in the US), sugar beet, straw, sugar cane (primary bioethanol source in Brazil), wheat, molasses and any other rich source of carbohydrate (Demirbas, 2007). 1.2.1 Properties of ethanol Ethanol, which is also called ethyl alcohol, is a colourless, biodegradable, a high-octane, water-free alcohol. It is low in toxicity and causes little environmental pollution if spilt. Ethanol, being a straight-chain alcohol is often abbreviated as EtOH. It has a widespread usability in alcohol industry as alcoholic beverages, in chemical industry as a base chemical for other organic compounds, in medical as an antiseptic or as a treatment for poisoning by other alcohols (Demirbas, 2007). The physical properties of ethanol are dictated primarily by the presence of the hydroxyl group and its short carbon chain. The hydroxyl group forms hydrogen bonds which makes ethanol less volatile than other organic compound of comparable molecular weight. The properties of ethanol stem primarily from the presence of its hydroxyl group and the shortness of its carbon chain. Ethanol’s 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, like most short-chain alcohols, is flammable, colorless, has a strong odor, and is volatile. Ethanol-water mixtures that contain ethanol in excess of 50 % are flammable and ignite easily.
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Table 1.0: Physical properties of ethanol
Property
Value
Molecular Formula Molecular Mass Density Melting Point Boiling Point Viscosity
C2H6O, CH3CH2OH 46.07 g/mol 0.789 g/mol3 -114.3 0C 78.4 0C 1.20 Cp at 20 0C
(i) (ii) Figure 1.0 Chemical structure of Ethanol and its Functional group 1.3 Crops with Biofuel Potential in Nigeria 1.3.1 Sorghum Sorghum is one of the high drought resistance crops cultivated in about 50 % of the Nigeria agricultural land, mostly in the Northern region (8 0N to 14 0N latitude), accounting for 6.86 million hectares of land. Annual production has been estimated to rise by 45 % from the total production of 4.8 million tonnes in 1978 (Ogbonna, 2002). This Figure gives Nigeria the opportunity to be the highest producer of sorghum in Sub Saharan Africa, accounting for about 70 % of the total production in the region. The commonly grown varieties are the Farfara, Guinea and Kaura, which are all resistance to different killer weeds, and scientifically classified.
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Sorghum is currently use in Nigeria for two main categories of purpose classified here as local and industrial. Traditionally, the crop is mostly cultivated by poor farmers to meet their local demands; they mainly use their harvest for food, beverages, and variety of drinks. Non-food uses include roofing and fencing of compounds in local communities. The local application accounts for about 73 % of annual sorghum usage in the country. Industrially, the crop is used in malting. In 1984 and 1985 the demand for industrial sorghum malt in Nigeria was computed as 134170 and 161043 kg, accounting for 64 and 74 million Naira market value respectively (Ilori, 1991). This Figure had since risen by about 45 %, considering the large scale demand of sorghum both locally and industrially, diversion of the crop for fuel ethanol production could have severe consequences. First, the peasant farmers would definitely shift from cultivating other subsistence crops to sorghum, creating an imbalance in the food circle. Secondly, the objective of the biofuel policy would be defeated by sudden rise in food price and inappropriate use of agricultural land. Thirdly, most of the agricultural land would be exposed to degradation due to continuous mono-cropping, and this can severely add to the already existing problems of soil erosion and desertification in the Northern parts (Galadima, et al., 2007). 1.3.2 Cassava
Cassava is another crop grown on both local and commercial scales in some major parts of Nigeria, especially the rainforest, and the savannah areas of North West and North Central, due to availability of well-drained deep loamy soils. The spread of cassava production in the country could be traced to the period between 1930 and 1946, when yam production was considered unprofitable due to considerable damage by pests. Over sixty different varieties are currently cultivated. Initially, sweet varieties that could be eaten by the local people without further processing were the dominants. However, these were subsequently matched with other improved varieties such as TMS
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30572, 4(2)1425, 92/0326 and NR 8082. The annual production was estimated to have increased by about 66 % from 382,000 hectare per year from 1946 (Nweke, 2004). Like the sorghum, cassava is used at both local and industrial scales. Peasant farmers employ the tubers for production of food in form of garri, fufu and fermented flour (Ugwu and Nweke, 1996). Industrially, the crop is used as raw material for starch, chips, pellets, unfermented flour and more importantly in beer manufacture. Cassava has been given a great emphasis for fuel ethanol production under the current biofuel implementation plan than sorghum. In areas where its production remain the only source of food and household incomes for the local farmers, an imbalance could be created, although may not be very severe if the existing pre-exploited land is used in preference. Careful planning is therefore necessary to ensure that, large scale cassava production is carried out screening out food-to-fuel diversion issues. 1.3.3 Sugarcane Since its introduction into the country through the eastern and western coasts by the European Sailors in fifteen century, sugarcane has become an important crop grown in many parts of Nigeria. Traditionally, sugarcane is grown on small holdings (usually 0.2 to 1.0 ha) for chewing as juice and preparing livestock feed. However, with the increased demand for sugar in the country, the crop is grown on large scale as raw material for sugar industry.
Around 1997, the major sugar companies operating; Bacita, Lafiagi, Numan and Sunti utilized about 12,000 ha out of the total 30, 000 ha for sugar-based sugarcane production (Agboire, 2002). In the year 2007/2008 an estimate of 100, 000 tonnes were produced compared to 80, 000 tonnes in 2006/2007, however, due to the persistent increased in sugar demand to 1.50 billion tonnes, making Nigeria the second largest in Africa, the local sugarcane production is insufficient to meet the
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demand. With the current shift to biofuel ethanol production by the government, more companies were invited to participate in sugarcane production across the country. In the last few years, a US-based company (Lemna International) proposed to establish the first ethanol production plant in Taraba State. The project analysed to cost 50 million US$ would require a land covering 30,000 to 50,000 ha for local raw material cultivation. The NNPC have clearly identifies sugarcane and cassava as the major raw materials for the bioethanol production program. Currently, investors have already invested over $3.86 billion for the construction of 19 ethanol bio- refineries, 10,000 units of mini-refineries and feedstock plantations for the production of over 2.66 billion litres of fuel grade ethanol per annum from sugarcane and cassava, leading to land requirement of 859,561 ha (Ohimain, 2010). Sugarcane-based fuel ethanol production would have very little threat to the local people, as the crop is not used for daily food like sorghum or cassava. However, sudden rise in prices of sugar and sugar products would be a great challenge. To address this, importation and selling of sugar to peasants at a subsidized rate is necessary. Similarly, an unbiased food price versus food-fuel feasibility research should be executed simultaneously, such that proper policy modification is carried out in line with real situation. 1.3.4 Jatropha
The policy identifies Jatropha oil as the main pilot raw material for the biodiesel industry. Jatropha is non-edible plant and therefore has not been on the large scale production by either the Nigerian food or commercial farmers. Some few research plantations were established in the recent years, as pilot studies for checking soil desertification. However, with the current biofuels plan some northern states namely Kebbi, Sokoto, Zamfara, Katsina, Kano, Jigawa, Bauchi, Yobe, Borno,
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Adamawa and Gombe are selected for large scale production. A number of literature studies have indicated Jatropha to be a very good source of oil for biodiesel production; yielding nearly 100 % of the fuel in short transesterification time under both homogeneous and heterogeneous conditions (Lu, 2009; Sahoo and Das, 2009; Vyas, 2009). From the economic perspective studies indicated successes in large scale Jatropha plantations in different tropical countries. Studies by Prueksakorn (2010) in Thailand showed that, both 20 years perennial system and annual cultivation method, involving harvesting the trees for wood and the seed for biodiesel could produce up to 4720 and 9860 GJ of net energy per ha. In India, production and use of Jatropha biodiesel have reported to triggers 82 % decrease in fossil diesel demand and 52 % decrease in global warming potential (Achten, 2010). Therefore, selection of Jatropha in Nigeria would be a multipurpose opportunity. In addition to the sources of energy, soil degradation, desertification, and deforestation problems could be addressed. If only 10 % of the available agricultural land (60,000,000 ha) in the selected states could be utilized, additional revenue of $3 billion, which is more than the annual allocation to these states, could be generated. However, the poor farmers may shift from food crops to Jatropha cultivation due to foreseeable market value, deforming the food circle. Similarly, continuous plantation is associated with soil acidification and eutrophication (Achten, 2010). 1.4 Fuel Ethanol
The production of ethanol from food such as corn, cassava etc. is the most predominate way of producing ethanol. This will definitely lead to a shortage in food, in-balance in food chain, increased food price and indirect land use. In 1925, Henry Ford had quoted ethyl alcohol, ethanol, as “the fuel of the future.‖ He furthermore stated, “The fuel of the future is going to come from apples, sugar
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cane, pawpaw, weeds and sawdust – almost anything. There is fuel in every bit of vegetable matter that can be fermented.” Today Henry Ford‘s futuristic vision significance can be easily understood. In the current time, the importance of alternative energy source has become even more necessary not only due to the continuous depletion of limited fossil fuel stock but also for the safe and better environment, with an inevitable depletion of the world‘s energy supply, there has been an increasing worldwide interest in alternative sources of energy (Wyman, 1999; Lynd, 2004; Herrera, 2004; Lin and Tanaka, 2006). Keeping in view all the above said advantages, biomass based fuel development technologies should rapidly gain momentum and the barriers imposed earlier should be removed for successfully attempting the production of bioethanol at the commercial level. It is welcome to understand that the use of bioethanol as a source of energy would be more than just complementing for solar, wind and other intermittent renewable energy sources in the long run (Lin and Tanaka, 2006). During the last two decades, advances in technology for ethanol production from biomass have been developed to the point that large-scale production will be a reality in next few years (Moiser, 2005). The ethanol yields processes and economics along with the technical maturity and environmental benefits of using ethanol blend fuel are the key parameters that determine the feasibility of bioethanol production (Nguyen and Saddler, 1991). The burning fossil fuel at the current rate is likely to create an environmental crisis globally.
Ethanol production process only uses energy from renewable energy sources, and as such no net carbon dioxide is added to the atmosphere, making ethanol an environmentally beneficial energy source (Bull et al., 1992; Kheshgi et al., 2000). Furthermore, fuel ethanol from waste fruits may also open new employment opportunities in rural areas, and thus make a positive socio-economic impact (Wyman, 2003). Producing ethanol as fuel, beyond its current role as fuel oxygenates will require
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developing waste fruit biomass as a raw material because of its abundantly available and low cost (Bevan and Franssen, 2006). The world ethanol production in 2004 was estimated to be 40 giga litres (GL) (Berg, 2004; Kim and Dale, 2004). Brazil and the US are the world leaders, which together accounted for about 60 % of the world ethanol production exploiting sugarcane and corn respectively. In India, lignocellulosic biomass (crop residues, forestry, fruit, vegetable waste and weeds) is available in quantity. The important issue that we wish to address affirmatively here is that the bioethanol production, without doubt, needs an economical approach to address the global fuel needs. Research efforts are needed to design and improve the process, which would produce sustainable and economically feasible transportation fuel. The new designed cellulase enzyme cocktail are important factors to consider in establishing a cost effective technology, besides the low cost of feedstock (Mojovic, 2006; Gray et al., 2006). 1.4.1 Ethanol Production Technologies Bioconversion of fruit waste to ethanol consists of four major unit operations: pretreatment, hydrolysis, fermentation and product separation/ distillation. 1.4.1.1 Pretreatment
Plant biomass is made up of lignocellulosic materials which make up the majority of plant cell walls (Pandey et al., 2000). This lignocellulose is difficult for microorganisms to break down through direct fermentation (Kamm et al., 2006). Pretreatment is required to alter the biomass macroscopic and microscopic size and structure as well as its submicroscopic chemical composition and structure so that hydrolysis of carbohydrate fraction to monomeric sugars can be achieved more rapidly and with greater yields (Sun and Cheng, 2002; Moiser et al., 2005).
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Pretreatment affects the structure of waste by solubilizing hemicellulose, reducing crystallinity and increase the available surface area and pore volume of the substrate. Pretreatment has been considered as one of the most expensive processing step in biomass to fermentable sugar conversion with cost as high as 30 cents/gallon ethanol produced (Moiser et al., 2005). To assess the cost and performance of pretreatment methods, techno-economic analysis have been made recently (Eggerman and Elander, 2005). There is huge scope in lowering the cost of pretreatment process through extensive R and D approaches. Pretreatment of cellulosic biomass in cost effective manner is a major challenge of cellulose to ethanol technology research and development. Aqueous or steam pretreatment method is often acid or base catalyzed, the main objective of this method is to process the feedstock biomass with hot water or steam applications at high temperatures and pressures for short periods. In this process the use of acids and other chemicals are minimized with high sugar recovery tendencies and minimal fermentation inhibition. 1.4.1.2 Hydrolysis
After pretreatment there are two types of processes to hydrolyze the feed stocks into monomeric sugar constituents required for fermentation into ethanol. The hydrolysis methods most commonly used are acid (dilute and concentrated) and enzymatic. To improve the enzymatic hydrolytic efficiency, the lignin-hemicellulose network has to be loosened for the better amenability of cellulase to residual carbohydrate fraction for sugar recovery. Dilute acid treatment is employed for the degradation of hemicellulose leaving lignin and cellulose network in the substrate. Other treatments are alkaline hydrolysis or microbial pretreatment with white-rot fungi (Phaenerochate chrysosporium, Cyathus stercoreus, Cythus bulleri and Pycnoporous cinnabarinus etc.) preferably
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act upon lignin leaving cellulose and hemicellulose network in the residual portion. However during both treatment processes a considerable amount of carbohydrates are also degraded, hence the carbohydrate recovery is not satisfactory for ethanol production (Chandel et al., 2007). 1.4.1.3 Acid hydrolysis There are two types of acid hydrolysis process commonly used – dilute and concentrated acid hydrolysis. The dilute acid process is conducted under high temperature and pressure and has reaction time in the range of seconds or min. The concentrated acid process uses relatively mild temperatures, but at high concentration of sulfuric acid and a minimum pressure involved, which only creates by pumping the materials from vessel to vessel. Reaction times are typically much longer than for dilute acid 1.4.1.4 Dilute Acid Hydrolysis In dilute acid hydrolysis, the hemicellulose fraction is depolymerized at lower temperature than the cellulosic fraction. Dilute sulfuric acid is mixed with biomass to hydrolyse hemicellulose to xylose and other sugars. Dilute acid is interacted with the biomass and the slurry is held at temperature ranging from 120 – 220°C for a short period of time. Thus hemicellulosic fraction of plant cell wall is depolymerised and will lead to the enhancement of cellulose digestibility in the residual solids (Sun and Cheng, 2002).
Dilute acid hydrolysis has some limitations, if higher temperatures (or longer residence time) are applied, the hemicelluosic derived monosaccharides will degrade and give rise to fermentation inhibitors like furan compounds, weak carboxylic acids and phenolic compounds (Olsson and Hahn- Hagerdal, 1996). These fermentation inhibitors are known to affect the ethanol production performance of fermenting microorganisms (Chandel et al., 2006).
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In order to remove the inhibitors and increase the hydrolysate fermentability, several chemicals and biological methods have been used. These methods include over liming (Martinez et al., 2000), charcoal adsorption (Chandel et al., 2006), ion exchange (Nilvebrant, 2001), detoxification with lactase (Chandel et al., 2006), and biological detoxification (Lopez et al., 2004). The detoxification of acid hydrolysates has been shown to improve their fermentability; however, the cost is often higher than the benefits achieved (Palmqvist and Hahn- Hagerdal, 2000). Dilute acid hydrolysis is carried out in two stages- First-stage and two-stage. 1.4.1.5 First-stage Dilute Acid Hydrolysis The lignocellulosic material is first contacted with dilute sulfuric acid (0.75 %) and heated to approximately 50°C followed by transferring to the first stage acid impregnator where the temperature is raised to 190°C. Approximately, 80 % of the hemicellulose and 29 % of cellulose are hydrolyzed in the first reactor. The hydrolysate is further incubated at a lower temperature for a residence time of 2 h to hydrolyse most of the oligosaccharides into monosaccharides followed by the separation of solid and liquid fractions. The solid material again washed with plentiful of water to maximize sugar recovery. The separated solid material is sent to second stage acid hydrolysis reactor (Chandel, et al., 2007). 1.4.1.6 Two-stage Dilute Acid Hydrolysis
In two-stage dilute acid hydrolysis process, first, biomass is treated with dilute acid at relatively mild conditions during which the hemicelluose fraction is hydrolyzed and the second stage is normally carried out at higher temperature for depolymerization of cellulose into glucose. The liquid phase, containing the monomeric sugars is removed between the treatments, thereby avoiding degradation of monosaccharides formed. It is very important to avoid monosaccharide degradation
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products for improving the ethanol yield. Sanchez et al., (2004) carried out the two-stage dilute acid hydrolysis using Bolivian straw material, Paja brava. In first stage, P. brava material was pretreated with steam followed by dilute sulfuric acid (0.50 or 1.0 % by wt) hydrolysis at temperatures between 170 and 230°C for a residence time between 3 and 10 min. The highest yield of hemicellulose derived sugars were found at a temperature of 190°C, and a reaction time of 5 – 10 min, whereas in second stage hydrolysis considerably higher temperature (230°C) was found for hydrolysis of remaining fraction of cellulose. 1.4.1.7 Enzymatic Hydrolysis
The acid, alkaline or fungal pretreated lignocellulosic can be saccharified enzymatically to get fermentable sugars (Ghose and Bisaria, 1997; Kedah et al., 1997; Itoh et al., 2003). Bacteria and fungi are the good sources of cellulases, hemicellulases that could be used for the hydrolysis of pretreated lignocellulosic. The enzymatic cocktails are usually mixtures of several hydrolytic enzymes comprising of cellulases, xylanases, hemicellulases and mannanases. In the last decade, new cellulases and hemicellulases from bacterial and fungal sources have continued been isolated and regular efforts have been made for the improved production of enzymatic titers (Foreman et al., 2003). However, the cellulases were produced at a concentration too low to be useful. There is a group of microorganisms (Clostridium, Cellulomonas, Tricho-derma, Penicillium, Neurospora, Fusarium, Aspergillus etc.) showing a high cellulolytic and hemicellulolytic activity, which are also highly capable of fermenting monosaccharides. Genetic engineering is used to produce super strains, which are capable of hydrolysing cellulose and xylan along with fermentation of glucose and xylose to ethanol (Lin and Tanaka, 2006). The utilization of cellulose by microorganisms involves a substantial set of fundamental phenomena beyond those associated with enzymatic hydrolysis of cellulose (Lynd et al., 2002).
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1.5 Separate Hydrolysis and Fermentation (SHF) Enzymatic hydrolysis performed separately from fermentation step is known as separate hydrolysis and fermentation (SHF) (Sreenath et al., 2001).The separation of hydrolysis and fermentation offers various processing advantage and opportunities. It enables enzymes to operate at higher temperature for increased performance and fermentation organisms to operate at moderate temperatures, optimizing the utilization of sugars. 1.6 Simultaneous Saccharification and Fermentation (SSF) The most important process improvement made for the enzymatic hydrolysis of biomass is the introduction of simultaneous saccharification and fermentation (SSF), which has been improved to include the co-fermentation of multiple sugar substrates (Sreenath et al., 2001; Wingren et al., 2003). This approach combined the cellulase enzymes and fermenting microbes in one vessel. This enabled a one-step process of sugar production and fermentation into ethanol. Simultaneous saccharification of both carbon polymer, cellulose to glucose; and hemicellulose to xylose and arabinose; and, fermentation will be carried out by recombinant yeast or the organism which has the ability to utilize both C5 and C6 sugars. According to Alkasrawi et al., (2006) the mode of preparation of yeast must be carefully considered in SSF designing.
A more robust strain will give substantial process advantages in terms of higher solid loading and possibility to recirculate the process stream, which results in increased energy demand and reduced fresh water utilization demand in process. Adaptation of yeast to the inhibitors present in the medium is an important factor for consideration in the design of SSF process. More recently, Kroumov et al., (2006) demonstrated an unstructured model of SSF of starch to ethanol by genetically modified strain Saccharomyces cerevisiae, using two hierarchic levels of concept. In
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first concept, a mechanism of enzymatic hydrolysis of starch to glucose by combined action of two enzymes (alpha amylase and glucoamylase) secreted by recombinant yeast and the second concept was the enzymatic degradation of starch to glucose and simultaneous utilization of glucose to ethanol by microorganisms. SSF combines enzymatic hydrolysis with ethanol fermentation to keep the concentration of glucose low. The accumulation of ethanol in the fermenter does not inhibit cellulase action as much as high concentration of glucose; so, SSF is good strategy for increasing the overall rate of cellulose to ethanol conversion (Lin and Tanaka, 2006). SSF gives higher ethanol yield while requiring lower amounts of enzyme because end-product inhibition from cellobiose and glucose formed during enzymatic hydrolysis is relieved by the yeast fermentation (Banat et al., 1998). Simultaneous Saccharification and Fermentation (SSF) Bioethanol
Biomass Bioethanol
L Lignin
Residue-to-power Production Figure 1.1 A Schematic Design of Bioethanol Production 1.7 Distillation
Distillation is one of the steps of the purifications. Distillation is the method used to separate two liquid based on their different boiling points. However, to achieve high purification, several
Pre-treatment (Solubilisation of hemicellulose)
Enzymatic hydrolysis (Conversion of cellulose to sugar)
Fermentation (Conversion of sugar to bioethanol)
Distillation
and
Evaporation
Filter wash
Waste
Management
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distillations are required. This is because all materials have intermolecular interactions with each other, and two materials will co-distill during distillation. This means that proportion between two materials, in this case ethanol and water can be changed, and still, there are two materials in layers, the liquid and the vapor layers (Onuki, 2005). Whatever method of preparation is used, the ethanol is initially obtained in a mixture with water. The ethanol is then extracted from this solution by fractional distillation. Although the boiling point of ethanol, 78.3 0C, is significantly lower than the boiling point of water, 100 0C, these materials cannot be separated completely by distillation. Instead, an azeotrope mixture (i.e. a mixture of 95 % ethanol and 5 % water) is obtained, and the boiling point of the azeotrope is 78.15 0C. In a distillation, the most volatile material (i.e. the material that has the lowest boiling point) is the first material to distill from the distillation flask, and this material is the azeotrope of 95 % ethanol which has the lowest boiling point. If an efficient fractionating column is used, 95 % alcohol could be obtained first and then a small intermediate fraction of lower concentration, and then water. But no matter how efficient the fractionating column used, 95 % alcohol cannot be further concentrated by distillation because the vapor has exactly the same composition as the liquid; towards distillation, then, 95 % alcohol behaves exactly like a pure compound. 1.8 Feedstock for Ethanol Production In designing production processes for ethanol fuel, it is important to assess the various sources of feedstock and their utilization. Bioethanol can be produced from a variety of feedstock containing simple fermentable sugars. Bioethanol may also be produced from some complex polysaccharides that can be hydrolyzed to release the fermentable sugars. In the following sections, various feedstock for bioethanol production are discussed.
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1.9 Feedstock Containing Simple Sugars These are feedstock that contains sugar in its simple form. In this category is sugarcane either in the form of cane juice or cane molasses. This form is the most important feedstock utilized in tropical and sub-tropical countries for producing ethanol. In European countries, beet molasses are the most utilized sucrose-containing feedstock (Cardona and Sánchez, 2007). Because of the simple form in which these sugars exist, it is easy to convert them to ethanol during microbial fermentation. The major challenge faced by this source of feedstock for ethanol production is the competition created by the use of this same feedstock as food by the human population. There are indications that continued use of this source can affect food prices in the long run. The availability and cost of procuring raw materials play a vital role in terms of design of ethanol production processes from these kinds of feedstock. Maiorella et al., (1984) performed a detailed economic evaluation of alternative processes for ethanol production from molasses. They observed that the cost of raw materials (molasses) accounted for up to 70 % of the final ethanol sale price. 1.10 Feedstock Containing Starch Unlike simple sugars, bioconversion of starch to ethanol cannot be accomplished in a single step. This is because starch is a complex polysaccharide which requires hydrolysis (typically enzymatic) to release fermentable sugars which can then be converted to ethanol by a suitable microorganism. This type of feedstock is most utilized in North America and Europe (Cardona and Sánchez 2007). The economic evaluation of different starch containing feedstock is of prime importance with respect to the design of the ethanol production process. 1.11 Lignocellulosic Biomass Feedstock
For countries where the cultivation of crops for biofuels production is not sustainable, lignocellulosic materials are a viable alternative to conventional field crops. The main challenge
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facing this prospect comes from the nature of the feedstock. Due to the complex nature of the lignocellulose, there is a need to introduce a pretreatment step for its degradation, removal of lignin, hydrolysis of the hemicellulose and a reduction in the fraction of crystalline cellulose. During this pretreatment step, the cellulose undergoes hydrolysis to release the sugar which is then converted to ethanol during fermentation. Lignocellulosic materials are cheap and readily available because many of them are by-products of agricultural activities, domestic and industrial wastes and this offers considerable possibilities for the production of ethanol fuel on a large scale as well as its global consumption as a renewable fuel (Cardona and Sánchez, 2007). Currently, a considerable amount of research is being carried out on the utilization of lignocellulosic biomass as feedstock for ethanol fuel production. Hamelinck et al., (2005) evaluated the production of ethanol from cellulosic biomass for three stages of technological development (short-term, medium-term and long-term). It was observed that the production process contributes approximately 50 % of the capital investment for the short- and long-term. In another work, McBride et al., (2005) carried out a study to determine the cost of producing ethanol from corn starch and lignocellulosic feedstock. 1.12 Microbial Fermentation
The fermentation of sugars by microorganisms is the most common method of converting sugars present in biomass to liquid fuels such as ethanol. This process occurs by bioconversion or biocatalysis which is the use of microorganisms or enzymes to convert one material to another. Ethanol fermentation, also referred to as alcoholic fermentation, is a biological process by which sugars such as glucose, fructose and sucrose are converted to ethanol and carbon dioxide. The process is accompanied by the release of metabolic energy. Ethanol fermentation is facilitated by microorganisms the most common one being yeast. The microorganisms use glucose as carbon
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substrate to produce ethanol, carbon dioxide and more cells. In actual fact, the fermentation process proceeds through a network of intracellular reactions. 1.13 Significance of the Study All energy sources have an impact on the environment. Concerns about the greenhouse effect and global warming, air pollution, and energy security have led to increasing interest and more development in renewable energy sources such as bio-fuel, solar, wind, geothermal, and hydrogen. The fluctuating oil price has increased the interest of finding other possible ways to produce fuel. And the production of bio-ethanol has grown steadily during the last 25 years. 1.14 The Statement of the Problem
Although bioethanol fermentation from edible, cellulosic feedstock‘s using yeast cells has been carried out with success, very little research has been done on producing bioethanol from waste fruit. Inadequate municipal and industrial solid waste collection and disposal creates a range of environmental problems in Nigeria. A considerable amount of waste ends up in open dumps or drainage system, threatening both surface water and ground water quality and causing flooding, which provides a breeding ground for diseases-carrying pests. Open air burning of waste, spontaneous combustion in landfills and incinerating plants that lack effective treatment for gas emissions is a major cause of air pollution. The situation is exacerbated in slums where households cannot make use of garbage collection containers. Lack of the most basic solid waste services in crowded, low-income neighbors are a major contributor to the high morbidity and mortality among the urban poor. The adverse effects of inadequate solid waste service on productivity and economic development of the city are significant. Solid waste such as fruit peels and damaged whole fruits largely obtained as a by-product from fruits markets and also from its transportation to hotels,
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restaurants and juice processing houses in our country. These wastes can entail serious environmental problems unless they are changed or converted into some useful products or disposed properly. 1.15 Aims The aim of this work is to transform waste fruits by direct fermentation and simultaneous saccharification and fermentation to bioethanol using the fungus Saccharomyces cerevisiae as ethanol producing organism. 1.16 Objective The overall objectives of this work are to:
Produce bioethanol from fruit wastes.
Determine the proximate composition of both fruits
Compare the quality (pH, concentration and specific gravity) of the bioethanol produced by two unique methods (i) direct fermentation and (ii) simultaneous saccharification and fermentation.
Verify the chemical composition and determine the purity of the bioethanol.
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