i BIOETHANOL (BIO-FUEL) PRODUCTION FROM AGRO-WASTE USING YEAST ISOLATES FROM NIGERIA BY EBABHI, ABOSEDE MARGARET B.Sc., M.Sc., BOTANY (LAGOS) MATRIC No: 980803010 A THESIS SUBMITTED FOR THE DEGREE OF DOCTOR OF PHILOSOPHY (Ph. D) BOTANY DEPARTMENT OF BOTANY, UNIVERSITY OF LAGOS, NIGERIA NOVEMBER, 2012 ii ERTIFICATION This work titled “Bioethanol (Bio-fuel) production from agro-waste using yeast isolates from Nigeria” submitted to the school of Postgraduate studies, University of Lagos, Lagos, Nigeria for the award of Doctor of Philosophy in Botany is an original research carried out by EBABHI, Abosede Margaret in the Department of Botany, University of Lagos, under the supervision of Professor A. A. Adekunle, Dr. A. A. Osuntoki and Dr. W. O. Okunowo. This work has not been submitted previously, in whole or part, to qualify for any other academic award. PROFESSOR A. A. ADEKUNLE Department of Botany, University of Lagos, Akoka, Lagos, Nigeria. Dr. A. A. OSUNTOKI (Senior Lecturer) Department of Biochemistry, College of Medicine, University of Lagos, Idi-Araba, Lagos, Nigeria. Dr. W. O. OKUNOWO (Lecturer I) Department of Biochemistry, College of Medicine, University of Lagos, Idi-Araba, Lagos, Nigeria. EBABHI, ABOSEDE MARGARET (Candidate) iii DEDICATION This study is dedicated to the glory of GOD for his mercy and compassion that endureth forever. To Engr. Larry C. Ebabhi To Daniel and Marvellous To Bukola Ikumapayi. iv ACKNOWLEDGEMENTS Foremost, I thank the Almighty God for the grace and privileges released to me throughout this research work. I would like to thank my supervisor Professor Adedotun A. Adekunle for the support and mentoring not only during my Ph.D study, but also as initial inspiration and introduction into the field of Mycology and fungal biotechnology. Likewise I express my great appreciation for the contributions of my other supervisors Dr. Akinniyi A. Osuntoki and Dr. Wahab O. Okunowo who did not only supervised me but were always ready to assist in the research. During this Ph.D. study, a lot of my research was carried out at the Biochemistry Laboratory, College of Medicine, University of Lagos with the assistance of the laboratory technicians. It was a great experience and I highly appreciate the help and guidance I was given by Mr. Sunday Adenekan. I am grateful for the enthusiastic, encouraging, and joyful atmosphere provided by all my lecturers in the Department of Botany especially during the bench-work. They include: Prof. J. D. Olowokudejo, Prof. O. T. Ogundipe, Dr. A. Akinsoji, Dr. (Mrs) C. E. Umebese, Dr. (Mrs) O.O. Shonubi, Dr. V. Odjebga, Dr. (Mrs) O.E. Ade-Ademilua, Dr. (Mrs) T.A. Adesalu, Dr. (Mrs) O. H. Adekanbi, Dr. (Mrs). E.M. Adongbede, Dr. A.B. Kadiri and Dr. A. P. Adeonipekun. I appreciate your support. I would also like to express my gratitude to Prof. O. B. Familoni of the Department of Chemistry for the equipment provided during the course of this research. I also value the assistance of the technical staff of the Department of Botany: Mr. E. Adefusi, Mr. S. Afi and Mrs. V. Illo with their counterparts in the Department of Microbiology: Mr. Aderibigbe, Mr Yusuf and Mr. Fred. To all other lecturers who had at one stage or the other imparted knowledge on me throughout my studies in the University of Lagos, I say thank you. I can never forget the University of v Lagos for the Graduate Fellowship I was privileged to enjoy throughout the duration of my Ph.D study. I am grateful to the families of Amusa, A. A., Onifade, Chief (Mrs) M. A. Aiyebgusi, Femi Olumide, Toriola Adewale, Pastor A. Adeshina, Adekunle, A. A., Osuntoki, A. and James Ebabhi. It was also an honour to have Dr. A. A. Sanyaolu, Dr. (Mrs) U. Kanife, Dr and Dr. (Mrs) O.B. Samuel, Mr O. Adeogun, Dr. T. Adeyemi, Mrs. A. Bankole, Mrs. N. Bambgose, Mr. Mayomi, Mr. F. Orotope, Mr. M. Olaifa, Mr. O. Oduoye and Mr. P. Bankole around me during the course of my research. I thank my mother, Mrs B. A. Ikumapayi, my in-laws, uncles, aunts, pastors and friends who are just too numerous to mention for their enormous support during my Ph.D study and their interest in my work. You are a great inspiration. Love and thanks to all of you! Finally, my unreserved love and thanks goes to the love of my life, my darling husband Engr. Larry Ebabhi for the strong support, encouragement, and guidance throughout the whole period – thank you for your shoulder to lean on, your happy face to smile with, and especially your loving heart that cares deeply. I also bless the Almighty God for my joy and champion, Oluwaferanmi amd Jesutomisin. To my ever supportive sister and brothers, thank you for your understanding, love and patience. May the plans and purposes of God for your lives be fulfilled in Jesus name (Amen). I appreciate you all. Ebabhi, A. M. vi TABLE OF CONTENTS TITLE PAGE I CERTIFICATION II DEDICATION III ACKNOWLEDGEMENT IV TABLE OF CONTENTS VI LIST OF TABLES XII LIST OF FIGURES XIII LIST OF PLATES XIV LIST OF APPENDICES XV ABSTRACT XVI CHAPTER ONE 1.0 . INTRODUCTION 1.1. Background study 1 1.2. Statement of problem 3 1.3. Aims and objectives of study 7 1.4. Significance of the study 7 1.5. Research questions 8 1.6. Operational definition of terms 8 CHAPTER TWO 2.0 LITERATURE REVIEW 2.1 General description of bio-fuel 11 2.2. Classification of bio-fuel 12 2.2.1. First generation bio-fuels 12 2.2.2. Second generation bio-fuels 13 2.2.3. Third generation bio-fuels 14 vii 2.2.4. Fourth generation bio-fuels 15 2.3. History of bio-fuels 16 2.4. Types of bio-fuels 17 2.4.1. Bioethanol 17 2.4.1.1 Physical properties of bioethanol 18 2.4.1.2. Steps in bioethanol production 20 2.4.1.3. Source of raw material 22 2.4.2. Bio-butanol 23 2.4.3. Biodiesel 23 2.4.4. Biogas 24 2.4.5. Syngas 25 2.5. Fermentation 26 2.5.1. Process of fermentation 27 2.5.2. History of fermentation 29 2.5.3. Types of fermentation 30 2.5.3.1. Batch fermentation 30 2.5.3.2. Continuous fermentation 31 2.6. Yeast 32 2.6.1. Occurrence of yeast 33 2.6.2. Reproduction in yeast 33 2.6.3. Life cycle of yeast 34 2.7. Bioethanol production in Nigeria 36 2.8. Selected plants used for isolation of yeast and production of bio-fuel 38 2.8.1. Manihot esculenta Crantz 38 2.8.1.1. Bio-fuel from cassava 40 2.8.2. Elaeis guineensis Jacq. 40 2.8.3. Zea mays Linn. 42 2.8.3.1. Bio-fuel from maize 43 viii 2.8.4. Cola acuminata Schott and Endl. 44 2.8.5. Solanum tuberosum Linn. 46 2.8.6. Musa paradisiaca Linn. 47 2.8.7. Oryza Linn. 48 2.8.8. Ipomoea batatas (L.) Lam. 49 2.8.9. Saccharum officinarum Linn. 51 2.8.10. Pennisetum glaucum (Linn.) Moench 52 2.8.11. Sorghum bicolor (Linn.) Moench 53 2.8.12. Anacardium occidentale Linn. 54 2.9. What is waste? 56 2.9.1. Waste generation in Lagos state 58 2.9.2. Waste management 59 2.9.3. Treatment of processing wastes 60 CHAPTER THREE 3.0. MATERIALS AND METHODS 3.1. SAMPLE COLLECTION AND PROCESSING OF THE SUBSTRATE USED 62 3.1.1. Manihot esculenta (Cassava) 62 3.1.2. Elaeis guineensis sap (Palm wine) 62 3.1.3. Zea mays (Maize) 62 3.1.4. Solanum tuberosum (Irish potato) 63 3.1.5. Cola acuminata (Kolanut) 63 3.1.6. Musa paradisiaca (Plantain) 63 3.1.7. Oryza glaberrima (Ofada rice) 64 3.1.8. Ipomoea batatas (Sweet potato) 64 3.1.9. Saccharum officinarum (Sugarcane) 64 3.1.10 Pennisetum glaucum (Pearl Millet 64 3.1.11 Sorghum bicolor (Guinea corn) 65 http://en.wikipedia.org/wiki/Carolus_Linnaeus ix 3.2. Isolation of the organisms 65 3.3. Identification of isolates 66 3.4. Gram staining technique 66 3.5. Biochemical analysis 67 3.5.1. Germ tube test 67 3.5.2. Sugar fermentation 67 3.5.3. Urea hydrolysis test 68 3.5.4. Study on sensitivity to chloramphenicol 69 3.6. Determination of spore count of yeast isolates 69 3.7. Growth study of isolates at 37°C 69 3.8. Growth study of the isolates on different media 70 3.9. DNA extraction and analyses 71 3.9.1. Procedure 71 3.9.2. Preparation of 1% Agarose Gel 72 3.9.3. DNA sequencing 72 3.10. Extraction from Anacardium occidentale shell 73 3.11. Study on enzyme production from plant extract 73 3.11.1. β 1, 4-endoglucanase activity 74 3.11.2. β -1,4-exoglucanase activity 74 3.11.3. Xylanase activity 74 3.12. Hydrolysis of samples 75 3.12.1. Hydrolysis of samples with H2SO4 and HCl 75 3.12.2. Hydrolysis of samples with cashew nut shell extract 76 3.13. Preparation of broth 76 3.14. Fermentation 77 3.15. Fractional distillation 77 3.16. Determination of concentration of bioethanol produced 77 x 3.17. Proximate analysis and determination of cellulose content in the agro-waste 78 3.18. Determination of reducing sugar in the hydrolysates using 3-5-dinitrosalicyclic acid (DNS) principle 79 3.19. Identification of specific simple sugars in the plant extract hydrolysates 79 3.20. Statistical analysis 80 CHAPTER FOUR 4.0 RESULTS 81 4.1. Identification of yeast isolates 81 4.2. Biochemical test 90 4.2.1. Gram stains and sugar fermentation 90 4.2.2. Effect of chloramphenicol on yeast 90 4.2.3. Growth study at a specific temperature 90 4.2.4. Germ tube test 91 4.2.5. Urea test 91 4.3. Genotypic characterization of the yeast isolates 93 4.3.1. Sequence data of the studied yeasts genomic DNA 96 4.4. Chemical composition of the agro-waste used 100 4.4.1. Proximate composition 100 4.4.2. Cellulose content of the agro-waste 101 4.5. Reducing sugar concentration in the hydrolysates 101 4.6. Types and concentration of sugars present in the cashew nut shell extract (CNSE) hydrolysates 102 4.7. Bioethanol production 104 4.7.1. Bioethanol produced from HCl hydrolysates 104 4.7.2. Bioethanol produced from H2SO4 hydrolysates 108 4.7.3. Bioethanol produced from CNSE hydrolysates 111 4.8. Enzyme assay of the cashew nut shell extract 114 xi 4.9. Identification and concentration of alcohol produced from some agro-waste hydrolyzed with CNSE 115 4.10. Concentration of alcohols produced from some substrates hydrolyzed with mineral acid 115 4.11. Growth studies of the yeast isolates in eight media 118 CHAPTER FIVE 5.0. DISCUSSION 124 SUMMARY OF FINDINGS 137 CONTRIBUTIONS TO KNOWLEDGE 138 CONCLUSION 139 SUGGESTION FOR FURTHER WORK 141 REFERENCES 142 APPENDICES 162 xii LIST OF TABLES Table 1: Some crops use in bio-fuel production 2 Table 2: Types of waste generated in Lagos State 59 Table 3: Cultural characteristics of yeast isolates 82 Table 4: Morphological characteristic of the yeast isolates on potato dextrose agar (PDA) 83 Table 5: Phenotypic characterization of the isolates 92 Table 6: Molecular identity of yeast species isolated from some crop plants 95 Table 7: Proximate composition of agro-waste 101 Table 8: Concentration of different sugar components of the agricultural waste (plant-extract hydrolysates) 103 Table 9: Amount of bioethanol from substrate hydrolyzed with 50% HCl 106 Table 10: Amount of bioethanol from substrate hydrolyzed with 40% HCl 107 Table 11: Amount of bioethanol from substrate hydrolyzed with 50% H2SO4 109 Table 12: Amount of bioethanol from substrate hydrolyzed with 40% H2SO4 110 Table 13: Amount of bioethanol from substrate hydrolyzed with cashew nut shell extract 112 Table 14: Amount of bioethanol obtained from unhydrolyzed samples 113 Table 15: Concentration of bioethanol in some selected crop plant 117 xiii LIST OF FIGURES Figure 1: Sequence data of Schizosaccharomyces pombe, CU 329671.1 96 Figure 2: Sequence data of Kluyveromyces marxianus IMI398399 96 Figure 3: Sequence data of Pichia caribbica IMI 398400 97 Figure 4: Sequence data of Candida tropicalis strain A, IMI 398401 97 Figure 5: Sequence data of Candida tropicalis, strain B, IMI 398401 98 Figure 6: Sequence data of Saccharomyces cerevisiae strain A, GU931323.1 98 Figure7: Sequence data of Saccharomyces cerevisiae strain B, GU931323.1 99 Figure 8: Phylogenetic tree of yeast isolates ITS sequences 100 Figure 9: Amount of reducing sugar in hydrolyzed agricultural waste 102 Figure 10: Amount of enzyme activity of plant extract 114 Figure 11: Concentration of bioethanol in some agricultural waste 116 Figure 12: Growth curve of Schizosaccharomyces pombe in eight broth media at 530nm 120 Figure 13: Growth curve of Kluyveromyces marxianus in eight broth media at 530nm 120 Figure 14: Growth curve of Pichia caribbica in eight broth media at 530nm 121 Figure 15: Growth curve of Candida tropicalis strain A in eight broth media at 530nm 121 Figure 16: Growth curve of Candida tropicalis strain B in eight broth Media at 530nm 122 Figure 17: Growth curve of Saccharomyces cerevisiae Strain A in eight broth media at 530nm 122 Figure 18: Growth curve of Saccharomyces cerevisiae Strain B in eight broth media at 530nm 123 Figure 19: Growth curve of Candida krusei in eight broth media at 530nm 123 xiv LIST OF PLATES Plate 1 Photomicrograph of Schizosaccharomyces pombe (x40) 86 Plate 2 Photomicrograph of Kluyveromyces marxianus (x40) 86 Plate 3 Photomicrograph of Pichia caribbica (x40) 87 Plate 4 Photomicrograph of Candida tropicalis strain A (x40) 87 Plate 5 Photomicrograph of Candida tropicalis strain B (x40) 88 Plate 6 Photomicrograph of Saccharomyces cerevisiae strain A (x40) 88 Plate 7 Photomicrograph of Saccharomyces cerevisiae strain B (x40) 89 Plate 8 Photomicrograph of Candida krusei(x40) 89 Plate 9: Agarose gel electrophoresis of PCR products with 8 identifying yeast DNA bands viewed under the UV light 93 P s e u d y p h a xv LIST OF APPENDICES Appendix I Oneway analysis of varience 162 Appendix II Identification/ accession number of taxa used in the phylogenetic analysis 192 Appendix III Raw DNA sequence data for yeast isolates 212 Appendix IV Chromatogram of the concentration of sugar in the plant extract hydrolysates 214 Appendix V Chromatogram of some bioethanol produced 218 Appendix VI Constituent of the liquid media used in the growth Studies 222 Appendix VII Standard curve for reducing sugar assay 224 Appendix VIII Some agro-waste used in this study 225 Appendix IX The distillating equipment used in this study 226 xvi ABSTRACT Bio-fuels are fuels derived from biological materials or their by-products such as agricultural waste. A study was carried out to assess the production of bio-fuel (bioethanol) from some hydrolyzed agricultural wastes using yeast species isolated from Cola acuminata, Ipomoea batatas, Manihot esculenta, Pennisetum glaucum, Sorghum bicolor, Solanum tuberosum, Zea mays and palmwine (from Elaeis guineensis). Eight yeast species including Candida krusei, Candida tropicalis strain A, Candida tropicalis strain B, Kluyveromyces marxianus, Pichia caribbica, Saccharomyces cerevisiae strain A, Saccharomyces cerevisiae strain B and Schizosaccharomyces pombe were used. The agro- wastes and starchy substrates were pretreated through milling, saccharification with mineral acid (H2SO4/HCl) and Anacardium occidentale (cashew) Nut Shell Extract (CNSE). Substrates were distilled after 72 h of fermentation. The quantity of bioethanol produced varied with substrates and organisms used. The analysis of the CNSE revealed the presence of hydrolytic enzymes such as endoglucanase, exoglucanase and xylanase in varying concentrations. Biochemical analysis and DNA sequencing revealed that some of the fungal species are probably new strains. Using 100 g hydrolyzed substrates, the highest amount (33.34 ± 2.81 g/L) of bioethanol was obtained from sweet potato tuber hydrolyzed with 50 ml of 50% HCl and fermented with S. pombe. Plantain peel hydrolyzed with CNSE and fermented with S. pombe produced 28.12±1.61 g/L of bioethanol. The quantity of reducing sugar in the agro-waste hydrolyzed with CNSE was maximum of 491 mg/g in sugarcane chaff and minimum of 46 mg/g in rice husk. Sugar fermentation test of the yeasts showed that they can ferment sugars such as glucose, lactose, maltose, fructose and xylose. Lactose and xylose were fermented xvii atypically by S. cerevisiae strain B. All strains tested were resistant to 30 µg/l of chloramphenicol and they were able to grow at 37°C except Candida krusei. In the urease hydrolysis test, S. pombe, P. caribbica and S. cerevisiae strain B were positive. Growth study of the yeast strains on eight broth media showed that potato dextrose broth, malt peptone broth and millet dextrose broth are the best media for growth and reproduction. This is probably the first report of isolation and characterization of fermenting yeast from maize, kolanut and sweet potato in Nigeria. Gas chromatographic (GC) assay of the CNSE hydrolysates showed the presence of simple sugars in varying concentrations with cassava peel possessing the highest concentration of glucose (38.19 mg/L) while plantain peel yielded the lowest concentratiom of 5.44 mg/L. The GC analysis of some of the agro-waste distillates showed considerable concentrations of bioethanol. Highest concentration of 2.41 x 10 4 mg/L was obtained from sweet potato peel fermented with S. cerevisiae strain A whereas the lowest concentration of 2.75x10 3 was obtained from plantain peel fermented with S. pombe. The cashew nutshell extract was able to biodegrade the agro-wastes due to the synergy of the cellulases that are present in it. This study showed that agro-waste which are diverse and commonly pose significant disposal problems can be used for the production of bio-fuel and other organic compounds. Fossil fuel causes environmental pollution and replacing it with bio-fuel like bioethanol from agro-waste will be more environmental friendly and will also reduce pressure on crop plants. 1 CHAPTER ONE 1.0 INTRODUCTION 1.1. BACKGROUND STUDY Plants are very important to life because they support the existence of all living things directly or indirectly. The ability of plant to capture solar energy sustains almost all life forms on earth. Plant based organic matter is consumed by herbivores and forms the basis of every food chain (Marinelli, 2004). Plant materials serve other diverse purposes such as food for man and animal (Etejere and Bhat, 1985; Brand-miller and Holt, 1998; McGee, 2004; Abdulrahaman and Kolawole, 2006); medicine (Ainslie, 1937; Hutchinson and Dalziel, 1958; Oliver, 1960; Sofowora, 1982; Burkill, 1995, 2000; Adekunle, 2001; Adekunle and Ikumapayi, 2006); and raw materials in industries (Liggett and Koffler, 1948; Burkill, 1998). Today, plants are used in biotechnology for bio-monitoring (Martin, 1998; Mertens et al., 2005) and phytoremediation (Meagher, 2000; Mukred et al., 2008). The stored solar energy in plant is also used as fuel in wood, alcohol; methane and these are referred to as bio- fuels (Aiba et al., 1973, Ellington et al., 1993; Giampietro et al., 1997; Hammerschlag, 2006; Bowyer et al., 2007; Demirbas, 2009; Mathiyazhagan et al., 2011). Bio-fuel can be defined as any solid, liquid or gaseous fuel derived from biological materials (biomass) or their by-products (Giampietro et al., 1997; Yunqiao et al., 2007). Theoretically, it can be produced from any (biological) carbon source (Righelato, 1980); although, the most common sources are photosynthetic plants. Bio- fuel is also called non-conventional fuel (Birol and Davie, 2001) whereas fossil fuel, 2 petroleum, coal, propane, natural gases and nuclear materials like uranium are called conventional fuel. Today, the use of bio-fuel has expanded throughout the world. Some of the major producers and consumers are Brazil, China, Germany and United States of America (Goldemberg, 2007; Luciano et al., 2007). There are currently lots of researches on the use of biomass for bio-fuel production and many crop plants are specifically grown for this purpose. Examples of such crop plants and countries where they are used are found in Table 1 below: TABLE 1: SOME CROPS USED IN BIOFUEL PRODUCTION AGRICULTURAL PRODUCE COUNTRY Saccharum officinarum(Sugarcane) Brazil, Mozambique, Tanzania Manihot esculenta (Cassava) and Sorghum bicolor (Sorghum) China Beta vulgaris (Sugar beet) Germany Jatropha curcas (Barbados nut) India Triticum aestivum (Wheat) and Hurdeum vulgare (Barley) Spain Cereals Sweden Zea mays (Corn), Panicum virgatum (switch grass) and Glycine max (Soybean) United States of America Source: Kolachov and Nicholson, 1951; Borgelt et al., 1994; Luciano et al., 2007 Bio-fuel is used for purposes such as heating homes, alcohol, chemicals, for cooking stoves but the main use is in the transportation sector (Hill et al., 2006; Azih, 2007; Goldemberg, 2007; Luciano et al., 2007; Demirbas, 2009). They include fuels like biodiesel, bioethanol, non-fossil methane, biobutanol, oilgae (diesel from algae) and other biomass sources (Kurtzman, 1983; Shay 1993; Hill et al., 2006; Azih, 2007; Mathiyazhagan et al., 2011). 3 1.2. STATEMENT OF PROBLEM Bioethanol is the commonest and the most widely used liquid bio-fuel in the world (Righelato, 1980; Farrell et al., 2006). It is used particularly as an alternative fuel to petrol (gasoline). In Brazil, the ethanol-based bio-fuel sector has had a successful development since the 1980‟s and has become the envy of other countries that depend on importation of petroleum (Goldemberg et al., 1985). In the United States of America, China, Australia, Japan, Europe and parts of Africa, it is becoming the major source of fuel (Hill et al., 2006; Yunqiao et al., 2007; Klass, 2008). Nigeria has also followed suit (Azih, 2007). The country (Nigeria) is presently looking at the prospect of using Manihot esculenta (cassava), Saccharum officinarum (sugarcane) and Zea mays (maize) as sources of bio-fuel (Azih, 2007; Agba et al., 2010; Agboola et al., 2011). Bioethanol is the same as alcohol found in beverages. It is considered renewable because it is the main result of the conversion of the sun‟s energy into usable energy through photosynthesis, provided that all minerals required for growth are returned to land. Currently, interest in bio-fuel mainly lies in bioethanol production from starch or sugar substrate found in a wide variety of crops such as bagasse, sugar beet, sorghum, switch grass, barley, potatoes, sweet potatoes, corn, wheat, cassava, palm sap (Abouzied and Reddy, 1986; Shay, 1993; Mosier et al., 2005). There are controversies by farmers, politicians and environmentalists all over the world, over the production of bio-fuel from crop plants despite the production success that accompanies it. The effect of bioethanol production on other food crop prices is indirect. The use of maize for bioethanol production for example increases the 4 demand, and therefore increases price of maize. This in turn results in farm acreage being diverted from other food crops to maize production. Supply of the other food crops is reduced while the price of maize is increased. Clearly, substantial food price increase would occur if ethanol derived from crop plants replaces even a small percentage of petrol. The Food and Agriculture Organization‟s World Food Program (2008) reported that it foresees an urgent problem with world hunger resulting from rising food prices. In Nigeria, the land degradation problem in the Niger Delta, climate change and the pointer to the fact that oil reserves will be exhausted in the nearest future, 2050 (Agba et al., 2010) coupled with the need for economic development would necessitate a shift towards bio-fuel production. The basic steps for large scale production of bioethanol include hydrolysis, microbial (yeast) fermentation of sugars, distillation, dehydration and denaturing (Kolachov and Nicholson, 1951; Abouzied and Reddy, 1986; Buzas et al., 1989; Tyiagi et al., 1992; De Figueroa et al., 2000; Zafar and Owais, 2006). Prior to fermentation, some biomass type require saccharification or hydrolysis of the polysaccharides into simple sugar because of microbial fermentation (Rhee et al., 1984; Odunfa and Shasore, 1987; Ofuya and Nwajuiba, 1990; Amutha and Gunasekaran, 2001; Ueda et al., 2004). Two major components of plants, starch and cellulose, are both made up of sugars and can in principle be converted to sugar for fermentation. Currently, only the starch portions can be economically converted and this account for the bulk of energy constituents of crop plants while the cellulose linked with lignin (lignocelluloses) which make up the stalk, peels, husk, chaff of crop plants are difficult to break due to the complexity involved. Lignocellulose is the woody structural material of plants http://en.wikipedia.org/wiki/Lignocellulose 5 which gives them strength and rigidity. It is usually composed of three primary constituents including cellulose, hemicelluloses and lignin (Hayes, 1977; Reshamwala et al., 1995; Cheung and Anderson, 1997). These feedstocks are abundant, diverse, and in some cases pose a significant disposal problem. There is a lot of energy stored in lignocellulose (Mosier et al., 2005) and so, it is important to find ways of making it easier to get at this energy and extract it in the form of sugars that can be fermented to produce bioethanol and other products. This is because, the sugars suitable for fermentation processes and alcohol yield are only found in the cellulose and hemicellulose constituents. The cellulose is the most abundant organic biomolecule on earth and the major constituent of all plant materials (Murai et al., 1998; Narasimha et al., 2006). It is a linear biopolymer that contains hexose sugars (mainly glucose) and linked by a β1- 4 glycosidic bond (Gielkens et al., 1999). The hemicellulose which is the second most abundant component of lignocellulosic biomass contains pentose sugars (mainly xylose) and sugar acids (Zheng et al., 2009). The xylose in wood and straw represents about a third of the sugars that could potentially be used to make bioethanol, but it is not readily available. Releasing the energy from lignocellulose is an important challenge to tackle as it will allow the production of fuels from plants in a sustainable way that does not affect the food chain. In order to obtain the sugars needed for fermentation pre- treatments such as chemical pre-treatment or biological pre-treatment of lignocellulosic biomass is necessary (Mosier et al., 2005). The most widely used pre-treatment method is mineral acid hydrolysis (Zheng et al., 2009). Concentrated acid hydrolysis, which is one of the acid hydrolysis methods involves the use of concentrated acid (sulfuric acid - H2SO4 or hydrochloric acid - 6 HCl) under high temperature (190 - 215°C) to split the cellulose and hemicellulose into simple sugars (Zheng et al., 2009). However, the products of mineral acid hydrolysis (acid hydrolysate) become very acidic and may contain a variety of inhibitory compounds such as furfural or furfuraldehyde 5-hydroxymethyl-furfural (5- HMF), acetate and other phenolic compounds (Palmqvist and Hahn-Hagerdal 2000; Luo et al., 2002; Hayes et al., 2005). The use of these mineral acids in breaking down the lignocellulose makes the bioethanol produced corrosive on pipes and other containers. A lot of developing nations will have to depend on importation of the mineral acids and a lot of hazards are encountered in the transportation from place to place. Also there are problems of emission of poisonous gases in the disposal of the mineral acid containers. Finding alternative means of hydrolyzing cellulolytic waste will reduce the corrosive nature of ethanol produced and make the production of bioethanol more economical. It has become essential to produce bioethanol from residues of biomass which are materials derived from recently living organisms. These include plant and animal by- products, such as garden waste, crop residue, animal dung and waste plant materials. Also, biodegradable outputs from industries, agriculture, forestry and households can be used for bioethanol production. These cellulosic ethanol production using non-food crops or inedible waste products, does not divert food away from the animal or human food chain. Using waste feedstock to produce fuel can therefore contribute to waste management, fuel security, reduced pollution and reduction in global warming. However, there is little or no scientific investigation into the genetic constituents and biochemical properties of the yeast strains isolated in Nigeria and there is dearth of information on plant-based acids for the purpose of hydrolysis. http://en.wikipedia.org/wiki/Cellulosic_ethanol 7 1.3. AIM AND OBJECTIVES OF STUDY The aim of this work is therefore to isolate and characterize yeasts from various substrates. It is also aimed at using the isolated yeast to produce bio-ethanol from agro-waste hydrolyzed with cashew nut shell extract. The objectives are to: 1. isolate and characterize yeasts from tropical plant sources: cassava, sorghum, kola nut, sweet potato, millet, Irish potato, maize and palm wine. 2. identify the yeasts phenotypically based on standard conventional techniques and genotypically using molecular analysis. 3. Extract constituents from cashew nut shell (Anacardium occidentale) and examine its efficacy in hydrolyzing agro-waste. 4. inoculate pure culture of the yeast (individually) on the hydrolyzed agro-waste and determine the best yeast isolate for production of bioethanol from agro- waste. 5. produce bioethanol from the agro-waste hydrolyzed with the cashew nut shell extract and compare with bioethanol produced from mineral acid hydrolyzed waste. 1.4. SIGNIFICANCE OF THE STUDY This study shall provide information on yeast strains from tropical plants which can be useful in microbial fermentation in industries, plant base extract that can be used for hydrolysis and agro-waste that can be useful in the production of bio-fuel in Nigeria. 8 1.5. RESEARCH QUESTIONS 1. Can we use yeast isolates from substrates in Nigeria to produce bioethanol? 2. Which Nigerian plant can produce extract for hydrolysis of agro-waste? 3. Are there agro-waste that can be used for the production of bioethanol? 4. Which part of Lagos produces the various kinds of waste? 1.6 OPERATIONAL DEFINITION OF TERMS Arthrospores: Arthrospores are conidia that are produced very simply by the last cell on a hypha breaking off and dispersing as a propagule. Ascocarp: Fruiting body in the sub division ascomycotina. Batch fermentation: Batch fermentation process refers to the process that starts with the inoculation and end with the retrieval of the product. This happens inside a single fermenter with no intermediate steps. Biodegradation: The transformation of a substance into new compounds through biochemical reactions or by the actions of microorganisms such as fungi. Biodeterioration: The breakdown of biomass by microorganisms. Bioethanol: A colourless, volatile, flammable organic compounds that contain the hydroxyl group (OH) and that form esters with acids. With the chemical formula C2H5OH, synthesized or obtained by fermentation of sugars and starches. It is also called alcohol, ethyl alcohol, grain alcohol. http://www.mushroomthejournal.com/greatlakesdata/Terms/arthr620.html#conid162 http://www.mushroomthejournal.com/greatlakesdata/Terms/hypha136.html http://www.mushroomthejournal.com/greatlakesdata/Terms/propa165.html 9 Biofuel: Bio-fuel can be defined as any solid, liquid or gaseous fuel derived from relatively dead biological materials or their by-products. Biomass: The term biomass is actually an abbreviation of the term biological mass. It describes the total quantity or mass of organic material produced by living organisms in a particular area, at a given time. Bio-waste: Biodegradable waste is a type of waste that originates typically from plant and animal sources which can be broken down by other living organism. Cellulose: This is a polysaccharide that is made up of glucose units that constitute the main part of the cell wall of plants. Continuous fermentation: The process by which cells are maintained in culture in the exponential growth phase by the continuous addition of fresh medium that is exactly balanced by the removal of cell suspension from the bioreactor. Distillation: This is the process of separating mixtures based on differences in their volatilities in a boiling liquid mixture. It is a physical separation process. Feedstock: This refers to the raw material that is required for some industrial process. Fermentation: The process by which micro-organisms break down complex organic substances generally in the absence of oxygen to produce alcohol and carbon dioxide. Fossil fuels: A general term for buried combustible geologic deposits of organic materials, formed from decayed plants and animals that have been converted to crude oil, coal, natural gas, or heavy oils by exposure to heat and pressure in the Earth's crust over hundreds of millions of years. http://www.greenfacts.org/glossary/mno/organic.htm 10 Hemicelluloses: Hemicelluloses are plant cell wall polysaccharides that are not solubilized by water but are solubilized by aqueous alkali or mineral acid. Hydrolysate: This is the product of hydrolysis. Hydrolysis: The chemical process by which carbohydrates or starches are simplified into organic soluble, usually by facultative anaerobes. Lignocellulose: This is a matrix of cross-linked polysaccharide networks, glycosylated proteins, and lignin which consist of three main components: cellulose (38–50%), hemicellulose (17–32%), and lignin (15–30%). Non-conventional fuel: Also known as alternative fuels or advanced fuels, are any materials or substances that can be used as fuels, other than conventional fuels. Pseudohyphae: A chain of easily disrupted fungal cells that is intermediate between a chain of budding cells and a true hypha, marked by constrictions rather than septa at the junctions. Saccharification: Is the process of breaking a complex carbohydrate (starch or cellulose) into its monosaccharide components. Starch: Complex carbohydrate used by plants as a way to store glucose (sugar). It is found in grains, roots, tubers and other foods. Yeast: This is a microscopic, unicellular fungus (usually in sub-division Ascomycotina) consisting of single oval cells that reproduce vegetatively by budding or fission and are capable of converting sugar into ethanol and carbondioxide. http://en.wikipedia.org/wiki/Fuel http://en.wikipedia.org/wiki/Material http://en.wikipedia.org/wiki/Chemical_substance http://en.wikipedia.org/wiki/Fuel 11 CHAPTER TWO 2.0. LITERATURE REVIEW 2.1. GENERAL DESCRIPTION OF BIO-FUEL Bio-fuels are fuels produced directly or indirectly from organic materials (biomass) including plant materials and animal waste. As stated by Warabi et al. (2004), the term covers solid biomass, liquid fuels and various biogases. They include a wide range of fuels which are in some way derived from biomass (Demirbas, 2009). Bio-fuels may be derived from agricultural crops, including conventional food plants such as corn, soybean, cassava or from special energy crops like switch grass (Birol and Davie, 2001; Yunqiao et al., 2007) also from oil plants such as canola, Barbados nut and sunflower. Using raw potato starch for example, Abouzied and Reddy (1986) were able to produce a type of bio-fuel. Bio-fuel may also be derived from forestry, agricultural or fishery products or municipal wastes, as well as from agro-industry, food industry, food service by-products and wastes (Subhadra and George, 2011). There are basically two common strategies of producing bio-fuels. One is to grow crops high in either sugar (sugar cane, sugar beet, and sweet sorghum) or starch (Borgelt et al., 1994; Hammerschlag, 2006; Luciano et al., 2007) and then use yeast fermentation to produce ethyl alcohol (ethanol). The second is to grow plants that contain high amounts of vegetable oil, such as oil palm, rape seed, cottonseed, soybean, algae (Subhadra and George, 2011), or Barbados nut (Agarwal and Agarwal, 2007). When these oils are heated, their viscosity is reduced, and they can be burned directly in a diesel engine, or the oils can be chemically processed to produce fuels such as biodiesel (Demirbas, 2008). Wood and its byproducts can also be converted into bio-fuels as reported by Bowyer et al. (2007), http://en.wikipedia.org/wiki/Biofuels#Solid_biofuels http://en.wikipedia.org/wiki/Liquid_fuels http://en.wikipedia.org/wiki/Biogas http://en.wikipedia.org/wiki/Biomass http://www.greenfacts.org/glossary/abc/bio-fuels.htm http://schools-wikipedia.org/wp/s/Sugarcane.htm http://schools-wikipedia.org/wp/s/Sugar_beet.htm http://schools-wikipedia.org/wp/y/Yeast.htm http://schools-wikipedia.org/wp/e/Ethanol.htm http://schools-wikipedia.org/wp/s/Soybean.htm http://schools-wikipedia.org/wp/a/Algae.htm http://schools-wikipedia.org/wp/b/Biodiesel.htm 12 with common examples like wood gas and methanol. It is also possible to make cellulosic ethanol from non-edible plant parts, but this can be difficult to accomplish economically. Bio-fuels are gaining increased public and scientific attention, driven by factors such as oil price, the need for increased energy security, economic stability and concern over greenhouse gas emissions from fossil fuels (Righelato, 1980). There are two forms of biofuel namely:  Primary bio-fuel  Secondary bio-fuel Primary bio-fuels are traditional unprocessed biomass such as fire wood, charcoal, wood chips and pellets, organic materials and animal dung (Bowyer et al., 2007; Laird, 2008). They represent the main source of energy for a large number of people in developing countries who use it mainly for cooking, heating or electricity production. Secondary bio- fuels result from processing of biomass and include liquid bio-fuels such as bioethanol and biodiesel that can be used in vehicles and industrial processes. Hill et al., (2006) reported the development of bioethanol based stoves in Malawi to reduce dependence on charcoal, firewood and paraffin. All bio-fuels are the results of varying production processes which can be categorized into four generations: first, second, third and fourth generation bio- fuels. 2.2 CLASSIFICATION OF BIO-FUEL 2.2.1. First generation bio-fuels First generation bio-fuels are sourced from natural compounds such as sugars, starches, vegetable oils and fats, which are then, processed using conventional technologies. As such, they encompass the fuel types of biodiesel, bioalcohols, syngas and vegetable oil. http://schools-wikipedia.org/wp/m/Methanol.htm http://en.wikipedia.org/wiki/Oil_price_increases_since_2003 http://en.wikipedia.org/wiki/Oil_price_increases_since_2003 http://en.wikipedia.org/wiki/Energy_security http://en.wikipedia.org/wiki/Greenhouse_gas http://en.wikipedia.org/wiki/Fossil_fuel http://www.greenfacts.org/glossary/abc/biomass.htm http://www.greenfacts.org/glossary/wxyz/wood-energy.htm http://www.greenfacts.org/glossary/abc/biomass.htm http://www.greenfacts.org/glossary/abc/bio-fuels.htm http://www.greenfacts.org/glossary/abc/bio-fuels.htm 13 However, while these sources of bio-fuels offer a great alternative to the traditional fossil fuels, they do present some economic difficulties of their own. One of the greatest controversies facing the first generation bio-fuels is that they require the use of major food crops in their production. Thus, with first generation bio-fuels, there is an incredible stress on the agricultural sector, resulting in food shortages, or the expansion of farmlands. This in itself has huge environmental implications such as deforestation, soil erosion, water shortages and on the larger scale, climate change. Many countries are expanding or contemplating on the expansion of their first generation bioethanol production with Brazil and the United States of America having by far the largest plants (Goldemberg et al., 1985; Luciano et al., 2007). Interest in first generation bio-fuel is also growing in South East Asia, Malaysia, Indonesia and Thailand where majority of the world‟s palm oil for food is grown (Schott, 2009). Looking at the researches of Borgelt et al. 1994, Hill et al. 2006, Goldemberg 2007, Luciano et al. 2007 and Demirbas 2009, with all these detrimental impacts, the excessive use of first generation bio-fuels may defeat the purpose of bio-fuels in the first place, which is to provide a clean and renewable source of energy. 2.2.2. Second generation bio-fuels Second generation bio-fuels comprise the answer to the issues presented by their first generation counterparts as they are manufactured from inedible plant matter or non-food crops as well as the waste biomass produced by the agricultural sector. This includes the left over stalks, stems and leaves from the processing of maize, cassava (Adesanya et al., 2009), sugar cane, wheat, millet (Oyeleke and Jibrin, 2009), soybeans and other food crops. Because of the vast and diverse array of inedible biomass types and sources, second generation bio-fuels largely surpass the limitations of the first generation as they do not threaten our food reserves, food production or biodiversity. They are also a far more 14 sustainable resource, environmentally friendly and completely cost-effective because we are making fuel from what would previously be thought of as useless waste materials. Second generation bio-fuel can further be defined based on the mode being used for converting the bio-waste into fuel which can be either biochemical or thermochemical means. Some second generation bio-fuel like bioethanol and biobutanol can be made through biochemical processes while most others like biodiesel can be processed thermochemically (Spath and Dayton, 2003). The biochemical process includes pre- treatment, saccharification, fermentation and distillation (Coelho, 2006). According to Coelho (2006), the second generation ethanol is often referred to as cellulosic ethanol because it is sourced from lignocelluloses bio-waste. 2.2.3. Third generation bio-fuels The bio-fuel obtained from algae also referred to as oilgae in the bio-fuel industry, is the third generation bio-fuel. Microalgae are photosynthetic microorganisms that can produce lipids, proteins and carbohydrates in large amounts over short periods of time. These products can be processed into both biofuels and useful chemicals (Demirbas, 2011). Recent research into the use of algae as a source of fuel has shown that it can produce as much as 30 times more energy per unit growing area than land crops (corn, soybeans, wheat, etc.), although this is yet to be commercially implemented. The advantage of third generation bio-fuels is that it is 100% environmentally friendly, biodegradable and easy to grow, although the oil extraction process is a bit complex (Mathiyazhagan et al., 2011). Algae also has the benefits of naturally producing ethanol as a byproduct, which can easily be extracted without disturbing the plants, as well as absorbing carbon dioxide in the process of photosynthesis. This sink of carbon dioxide is fundamental in an environment which is suffering under the strain of excessive greenhouse gas emissions. 15 2.2.4. Fourth generation bio-fuels This is an idea of capturing and storing carbon, the fourth generation technology looks at the possibility of combining genetically modified feedstocks which can capture enormous amount of carbon. In this, the production of bio-fuels is aimed at not only producing sustainable energy but also a way of capturing and storing CO2. Biomass materials, which have absorbed CO2 while growing, are converted into fuel using the same processes as second generation biofuels. This process however, differs from second and third generation production as at all stages of production the carbon dioxide is captured using processes such as oxy-fuel combustion (Dmitri and Ross, 2011). The carbon dioxide can then be geosequestered by storing it in old oil and gas fields or saline aquifers. This carbon capture makes fourth generation biofuel production carbon negative rather than simply carbon neutral, as it locks away more carbon than it produces. This system not only captures and stores carbon dioxide from the atmosphere but it also reduces CO2 emissions by replacing fossil fuels. Although, it is concluded that fourth generation biofuel production has introduced the cell factory concept and shifted the research paradigm. There are still several technical bottlenecks in this biofuel research and development, which can only be solved by the use of post-genome tools on these photosynthetic organisms (Jing et al., 2011). Bio-fuels can also be referred to as alternative fuels which can be called non-conventional or advanced fuels. Bio-fuels are also considered as renewable source, some form of renewable energy is used to create alternative fuels. Renewable energy is used mostly to generate electricity. http://en.wikipedia.org/wiki/Fuel 16 2.3. HISTORY OF BIO-FUELS Ancient people have used biomass fuels in the form of solid bio-fuels (wood) for heating and cooking since the discovery of fire. With the discovery of electricity, man discovered another way of utilizing the bio-fuel. This form of fuel was discovered even before the discovery of the fossil fuels, but with the exploration of the fossil fuel like gas, coal and oil the production and use of bio-fuel suffered a severe impact. With the advantages placed by the fossil fuels, they gained a lot of popularity especially in the developed countries. Interestingly, Dorado et al. (2004) stated the use of liquid bio-fuel in the automobile industry since its inception which dates back to 1853 when scientists E. Duffy and J. Patrick conducted the first trans-esterification of a vegetable oil many years before the first diesel engine became functional. This concept was later used by Rudolf Diesel the German inventor of the diesel engine (Diesel, 1913). He designed his diesel engine (a single 10 feet iron cylinder with a flywheel at its base) to run on peanut oil and later Henry Ford designed the Model T car which was produced from 1903 to 1926. This car was completely designed to use hemp derived bio-fuel as fuel. Diesel believed that the utilization of biomass fuel is the future of his engine, as he stated in his 1912 speech saying, “the use of vegetable oils for engine fuels may seem insignificant today, but such oils may become in the course of time, as important as petroleum and the coal-tar products of the present time” (Diesel, 1913). However, with the exploration of huge supplies of crude oil in some parts of the United States, petroleum became very cheap and thus led to the reduction of the use of bio-fuels. The manufacturers of diesel engines then decided to alter their engines to utilize the lower viscous fossil fuel best known as petro-diesel rather than the biomass vegetable oil fuel. Most of vehicles like trucks and cars began using this form of fuel which was much cheaper and efficient ignoring the fact that decades later it would bring high pollution costs. 17 In the period of World War II bio-fuel again became valuable as a strategic alternative for imported fuel. In this period, Germany was one of the countries that underwent a serious shortage of fuel. It was during this period that various other inventions took place like the use of gasoline along with alcohol that was derived from potatoes. Britain was the second country to come up with the concept of grain alcohol mixed with petrol (Sheehan et al., 2004). The war frames were the periods when the various major technological changes took place but, during the period of peace, cheap oil from the gulf countries as well as the Middle East again eased off the pressure. With the increased supply the geopolitical and economic interest in bio-fuel faded away. According to Sheehan et al. (2004), a serious fuel crisis again hit various countries during the period of 1973 and 1979, because of geopolitical conflict. Thus, the Organization of Petroleum Exporting Countries (OPEC), made a heavy cut in exports especially to the non-OPEC nations. The constant shortage of fuel attracted the attention of the various academics and governments to the issues of energy crisis and the use of bio-fuels. The twentieth century came with the attention of the people towards the use of bio-fuels. Some of the main reasons for the people shifting their interest to bio-fuels were the rising prices of oil, emission of the greenhouse gases and interest like rural development (Bender, 1999). 2.4. TYPES OF BIO-FUELS There are different categories of bio-fuel depending on their chemical make-up or composition, but what they all have in common is that they are completely organic in origin. 2.4.1. Bioethanol The most commonly used and widely researched of the bioalcohols is bioethanol. Bio- ethanol is a type of alcohol that can be produced primarily through the fermentation of any 18 feedstock containing significant amounts of sugar or by the hydration of ethylene from petroleum and other sources (Myers and Myers, 2007). Ethanol can be blended with petrol or burned in nearly pure form in slightly modified spark-ignition engines. A litre of ethanol contains approximately two thirds of the energy provided by a litre of petrol. However, when mixed with petrol, it improves the combustion performance and lowers the emissions of carbon monoxide and sulphur oxide. Ethanol has widespread use as a solvent of substances intended for human contact or consumption (Chastain, 2006), including scents, flavourings, colourings, 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 more recently as a fuel for internal combustion engines (Reshamwala et al., 1995). Ethanol is the systematic name defined by the IUPAC nomenclature of organic chemistry for a molecule with two carbon atoms (prefix "eth-"), having a single bond between them (suffix "-ane"), and an attached -OH group (suffix "-ol") (Lide, 2000). 2.4.1.1 Physical properties Ethanol is a volatile, colourless liquid that has a slight odour (Anonymous, 2008). It burns with a smokeless blue flame that is not always visible in normal light. Ethanol is a versatile solvent, miscible with water and with many organic solvents, including acetic acid, acetone, benzene, carbon tetrachloride, chloroform, diethyl ether, ethylene glycol, glycerol, nitromethane, pyridine, and toluene (Windholz, 1976; Lide, 2000). It is also miscible with light aliphatic hydrocarbons, such as pentane and hexane, and with aliphatic chlorides such as trichloroethane and tetrachloroethylene (Windholz, 1976). According to Morrison and Boyd (1972), ethanol‟s miscibility with water decreases sharply as the number of carbons increases. The miscibility of ethanol with alkanes is limited to alkanes http://www.greenfacts.org/glossary/def/feedstock.htm http://www.greenfacts.org/glossary/abc/bio-fuels.htm http://en.wikipedia.org/wiki/Internal_combustion_engine http://en.wikipedia.org/wiki/Systematic_name http://en.wikipedia.org/wiki/IUPAC_nomenclature_of_organic_chemistry http://en.wikipedia.org/wiki/Miscible http://en.wikipedia.org/wiki/Acetic_acid http://en.wikipedia.org/wiki/Acetic_acid http://en.wikipedia.org/wiki/Acetone http://en.wikipedia.org/wiki/Benzene http://en.wikipedia.org/wiki/Carbon_tetrachloride http://en.wikipedia.org/wiki/Chloroform http://en.wikipedia.org/wiki/Diethyl_ether http://en.wikipedia.org/wiki/Ethylene_glycol http://en.wikipedia.org/wiki/Glycerol http://en.wikipedia.org/wiki/Nitromethane http://en.wikipedia.org/wiki/Pyridine http://en.wikipedia.org/wiki/Toluene http://en.wikipedia.org/wiki/Pentane http://en.wikipedia.org/wiki/Hexane http://en.wikipedia.org/wiki/1,1,1-Trichloroethane http://en.wikipedia.org/wiki/Tetrachloroethylene http://en.wikipedia.org/wiki/Alkane 19 up to undecane, mixtures with dodecane and higher alkanes show a miscibility gap below a certain temperature (about 13°C for dodecane) (Dahlmann and Schneider, 1989). The miscibility gap tends to get wider with higher alkanes and the temperature for complete miscibility increases. Ethanol-water mixtures have less volume than the sum of their individual components at the given fractions. The Mixture of equal volumes of ethanol and water results in only 1.92 volumes of mixture (Lide, 2000). Mixing ethanol and water is exothermic. At 298 K, up to 777 J/mol are set free (Costigan et al., 1980). Mixtures of ethanol and water form an azeotrope at about 96 volume percent ethanol and 4% water at normal pressure and temperature of 351 K. This azeotropic composition is strongly temperature and pressure dependent and vanishes at temperatures below 303 K (Pemberton and Mash, 1978). The addition of even a few percent of ethanol to water sharply reduces the surface tension of water. This property partially explains the “tears of wine” phenomenon. When wine is swirled in a glass, ethanol evaporates quickly from the thin film of wine on the wall of the glass. As the wine‟s ethanol content decreases, its surface tension increases and the thin film “beads up” and runs down the glass in channels rather than as a smooth sheet. Mixtures of ethanol and water that contain more than about 50% ethanol are flammable and easily ignited. Ethanol-water solutions that contain less than 50% ethanol may also be flammable if the solution is first heated. Ethanol is slightly more refractive than water, having a refractive index of 1.36242 (Lide, 2000) (at λ=589.3 nm and 18.35°C). The physical 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. Hydrogen bonding causes pure ethanol to be http://en.wikipedia.org/wiki/Undecane http://en.wikipedia.org/wiki/Dodecane http://en.wikipedia.org/wiki/Exothermic http://en.wikipedia.org/wiki/Azeotrope http://en.wikipedia.org/wiki/Surface_tension http://en.wikipedia.org/wiki/Tears_of_wine http://en.wikipedia.org/wiki/Flammable http://en.wikipedia.org/wiki/Refractive_index http://en.wikipedia.org/wiki/Hydroxyl 20 hygroscopic to the extent that it readily absorbs water from the air. The polar nature of the hydroxyl group causes ethanol to dissolve many ionic compounds, notably sodium and potassium hydroxides, magnesium chloride, calcium chloride, ammonium chloride, ammonium bromide, and sodium bromide while sodium and potassium chlorides are slightly soluble in ethanol (Windholz, 1976). Thus, ethanol cannot be transported in metallic pipes or vessels and because the ethanol molecule also has a non-polar end, it will also dissolve non-polar substances, including most essential oils and numerous flavouring, colouring and medicinal agents. 2.4.1.2. Steps in bioethanol production According to Kolachov and Nicholson (1951), after harvesting the feedstock such as sugarcane, corn, there are seven steps to making ethanol suitable to use as a petrol additive. These are: 1. Milling: The feedstock is ground into a fine powder called meal. 2. Liquefaction: The meal is mixed with water and alpha-amylase, passing through cookers where the starch is heated to 120-150°C and liquefied and then cooked further at 95°C. 3. Saccharification: The mash from the cookers is cooled and enzymes are added to convert the liquefied starch to fermentable sugars. 4. Fermentation: Yeast is added to ferment the sugars to ethanol and carbon dioxide. Using a continuous process, the fermenting mash is allowed to flow, or cascade, through several fermenters until the mash is fully fermented and then leaves for the final tank. 5. Distillation: The fermented mash, now called “beer,” contains about 10% alcohol, as well as all the non-fermentable solids from the meal and the yeast cells. The mash is pumped to the distillation system where the alcohol will be removed from the solids and http://en.wikipedia.org/wiki/Hygroscopic http://en.wikipedia.org/wiki/Sodium_hydroxide http://en.wikipedia.org/wiki/Potassium_hydroxide http://en.wikipedia.org/wiki/Magnesium_chloride http://en.wikipedia.org/wiki/Calcium_chloride http://en.wikipedia.org/wiki/Ammonium_chloride http://en.wikipedia.org/wiki/Ammonium_bromide http://en.wikipedia.org/wiki/Sodium_bromide http://en.wikipedia.org/wiki/Sodium_chloride http://en.wikipedia.org/wiki/Potassium_chloride http://en.wikipedia.org/wiki/Essential_oil http://en.wikipedia.org/wiki/Essential_oil 21 the water. The alcohol leaves the final column at about 96% strength, and the residue mash, called stillage, is transferred from the base of the column to the co-product processing area. 6. Dehydration: The alcohol then passes through a dehydration system where the remaining water will be removed. Most ethanol plants use a molecular sieve to capture the last bit of water in the ethanol. The alcohol product at this stage is called anhydrous (pure, without water) ethanol and is approximately 200 proof. 7. Denaturing: Ethanol that will be used for fuel is then denatured with a small amount (2-5%) of some product, like gasoline, to make it unfit for human consumption. However, the simplified process for the production of bioethanol can be given as follows: Through the process of photosynthesis, plant produces sugar in the form of glucose: 6CO2 + 6H2O + light → C6H12O6 + 6O2 During fermentation performed primarily by yeast, glucose is decomposed into ethanol and carbondioxide: C6H12O6→ 2C2H6O + 2CO2+ heat During combustion, ethanol is heated with oxygen to produce carbondioxide and water 2C2H6O + 3O2→ 2CO2 + H2O + heat The net reaction for the overall production and consumption of ethanol is simply light plus heat (Uno et al., 2001; Agarwal and Agarwal, 2007). Ethanol and methanol fuel are typically primary sources of energy; they are convenient fuels for storing and transporting energy. These alcohols can be used in "internal combustion engines as alternative fuels". Methanol can be produced from a wide variety of sources including fossil fuels, 22 agricultural products, municipal waste, wood and varied biomass. More importantly, it can also be made from chemical recycling of carbondioxide (such as from the CO2 rich fuel gases of fossil fuel, burning power plant or exhaust cement and other factories). Recent developments with cellulosic ethanol production and commercialization may allay some of the concerns on use of large arable farm land for growing energy crops. 2.4.1.3. Source of raw materials The raw materials used in the manufacture of ethanol through fermentation vary and are conveniently classified under three types of agricultural raw materials: sugar, starch and cellulose materials. Sugars (from sugar cane, sugar beet, molasses and fruits) can be converted to ethanol directly. Starch (from grains, potatoes, root crops) must first be hydrolyzed to fermentable sugars using mineral acids or by the actions of enzymes from malt or molds (Righelato, 1980). Cellulose (from wood, agricultural residues, waste sulfite, liquor from pulp and paper mills) must also be converted to sugars generally by the actions of mineral acids. Once simple sugars are formed, enzymes from yeast can readily ferment to ethanol. All alcoholic beverages: wine and brandy from natural sugars present in fruits; beer and whiskey from grain starches and vodka, rum from cane sugar are produced through the process of fermentation (Cook, 1958; Aiba, 1973; Kurtzman, 1983). In all cases, the fermentation must take place in a vessel that allows carbon dioxide to escape, but prevents outside air from coming in, as exposure to oxygen would prevent the formation of ethanol. Similarly, yeast fermentation of various carbohydrate products is used to produce much of the ethanol used for fuel (Righelato, 1980; Rhee et al., 1984). http://en.wikipedia.org/wiki/Cane_sugar 23 2.4.2. Biobutanol Butanol is a 4-carbon alcohol (butyl alcohol). Biobutanol is butanol produced from biomass feedstocks (Atsumi et al., 2008) such as sugar beets, sugar cane, corn grain, wheat and cassava; non-food energy crops such as switch grass, as well as agricultural by-products such as straw and corn stalks. The difference from ethanol production is primarily in the fermentation of the feedstock and minor changes in distillation. Dürre (2007) reported the use of Clostridium acetobutylicum specifically for the fermentation process. These are introduced to the sugars produced from the biomass; the sugars are broken down into various alcohols, which include ethanol and butanol. Unfortunately, a rise in alcohol concentration causes the butanol to be toxic to the microorganisms, killing them off after a period of time. This makes the fermentation process expensive and unrealistic when compared to the petroleum costs of the late 50‟s. However, new technological advances and the discovery of new microbes have improved the efficiency and cost of the fermentation process tremendously. Through genetic engineering, researchers have been able to modify the most efficient microbes to be able to withstand high alcohol concentrations (Huang et al., 2010). New modifications are constantly being researched, including the modification to enzymes and genes involved in butanol formation from biomass fermentation. 2.4.3 Biodiesel Biodiesel refers to a non-petroleum-based diesel fuel consisting of short chain alky1 (methyl or ethyl) esters, made by trans-esterification of vegetable oil or animal fat (tallow), which can be used (alone, or blended with conventional petrol or diesel) in unmodified diesel-engine vehicles (Demirbas, 2008). Trans-esterification is the process of using an alcohol (ethanol or methanol) in the presence of a catalyst like sodium hydroxide (NaOH) http://en.wikipedia.org/wiki/Sugar_beet http://en.wikipedia.org/wiki/Sugar_cane http://en.wikipedia.org/wiki/Maize http://en.wikipedia.org/wiki/Grain http://en.wikipedia.org/wiki/Wheat http://en.wikipedia.org/wiki/Cassava http://en.wikipedia.org/wiki/Panicum_virgatum http://en.wikipedia.org/wiki/Agricultural_byproduct http://en.wikipedia.org/wiki/Agricultural_byproduct http://en.wikipedia.org/wiki/Straw http://en.wikipedia.org/wiki/Maize http://en.wikipedia.org/wiki/Stalk_(botany) http://en.wikipedia.org/wiki/Distillation 24 or potassium hydroxide (KOH) to chemically breakdown the molecule of raw renewable oil into methyl or ethyl esters of the renewable oil with glycerol as a by-product (Dorado et al., 2004). Biodiesel is distinguished from the straight vegetable oil (SVO) (sometimes referred to as waste vegetable oil, used vegetable oil, pure plant oil) used (alone or blended) as fuels in some converted diesel vehicles. It is standardized as mono-alky1 ester and other kinds of diesel-grade fuels of biological origin are not included. Biodiesel can be derived from a wide range of oils, including rapeseed, soybean, palm, coconut or Jatropha curcas oils (Agarwal and Agarwal, 2007) and therefore the resulting fuels can display a greater variety of physical properties than ethanol. Bio-oil or triglyceride that is, completely organically sourced oil obtained from plants such as Jatropha, canola flowers, soy-bean, rapeseed, sunflowers, palms (Shay, 1993), algae and used vegetable oil are all considered to be great sources of biodiesel (Dorado et al., 2004). The bio-oil is usually heated to reduce its viscosity, after which it can be used as an additive in diesel fuel. Alternatively, it can be further treated to produce biodiesel, which can be used to power absolutely any technology that subsists off of diesel fuel. It can be blended with traditional diesel fuel or burned in its pure form in compression ignition engines. Its energy content is somewhat less than that of diesel (88 to 95%). Diesel engines can also run on vegetable oils and animal fats, for instance used cooking oils from restaurants and fat from meat processing industries. The production processes for both bioethanol and biodiesel yield additional by-products such as animal feed. 2.4.4. Biogas Biogas is the result of anaerobic transformation of organic materials (such as animal dung) with the help of anaerobic organisms to produce a mixture of gases (containing up to 60 percent methane and CO2). Through specially designed collectors and closed systems, the http://www.greenfacts.org/glossary/abc/bio-fuels.htm http://www.greenfacts.org/glossary/abc/bio-fuels.htm 25 gas can be captured off landfills or waste deposit sites and used for a number of applications such as the source of electricity and to heat buildings and water. As a fuel, its primary use is in engine with internal combustion. It is about 20% lighter than air and has an ignition temperature in the range of 650 to 750°C. It is an odourless and colourless gas that burns with a clear blue flame similar to liquefied petroleum gas (LPG). The solid by- product of bacterial digestion can also be used as a potent fertilizer, ideal for agricultural use. 2.4.5. Syngas Syngas is essentially a mixture of carbon monoxide (a poisonous and potent greenhouse gas) and hydrogen (a harmless and naturally found atmospheric gas). This is produced through the partial combustion of biomass in an oxygen-starved environment that yields a more potent and efficient form of fuel, which can be used to drive vehicle engines as well as the generation of electricity by turbines. It production begins with gasification or pyrolysis (Devil et al., 2003). The gasification is based on the process by which various biofuel can be produced including Fisher Tropsch Liquid (FTL), dimethyl ether (DME) and various alcohols. During gasification, bio-waste is heated for easy conversion. The mixture of combustible and non-combustible gases contaminant in the gas is removed. This is followed by adjusting the composition of the gas to prepare it for further downstream process. The major components of non-clean and concentrated syngas are carbon monoxide (CO) and hydrogen (H2) with a little amount of methane (CH4). The carbon monoxide and hydrogen react when passed over a catalyst to produce liquid fuel. The design of the catalyst determines what fuel is produced. In most plant designs, not all the syngas passed over the catalyst is converted to liquid fuel. 26 2.5. FERMENTATION Fermentation is the process of deriving energy from the oxidation of organic compounds, such as carbohydrates without the use of oxygen as an electron acceptor (Klein et al., 2004). It is also a process by which complex organic compounds, such as glucose, are broken down by the action of enzymes into simpler compounds without the use of oxygen. Fermentation results in the production of energy in the form of two ATP molecules, and produces less energy than the aerobic process of cellular respiration. The other end products of fermentation differ depending on the organism. In many bacteria, fungi, protists, and animals cells (notably muscle cells in the body), fermentation produces lactic acid and lactate, carbon dioxide, and water. In yeast and most plant cells, fermentation produces ethyl alcohol, carbon dioxide, and water. During fermentation, pyruvate is metabolized to various different compounds. In contrast, respiration is where electrons are donated to an exogenous electron acceptor, such as oxygen, through an electron transport chain. Sugars are the most common substrate of fermentation and typical examples of fermentation products are ethanol, lactic acid, and hydrogen. However, other compounds such as butyric acid and acetone can be produced by the process of fermentation. Typically, fermentation in food processing industries is the conversion of carbohydrates to alcohols and carbon dioxide or organic acids using yeasts, bacteria, or a combination thereof, under anaerobic conditions. Yeast carries out fermentation in the production of ethanol in beers, wines and other alcoholic drinks, along with the production of large quantities of carbondioxide. Voet and Voet (1995), had also stated the process of fermentation in mammalian muscle during periods of intense exercise where oxygen supply becomes limited, resulting in the production of lactic acid. mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Redox mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Substrate_(biochemistry) mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Ethanol mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Lactic_acid mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Hydrogen mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Butyric_acid mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Acetone http://en.wikipedia.org/wiki/Food_processing http://en.wikipedia.org/wiki/Carbohydrate http://en.wikipedia.org/wiki/Alcohol http://en.wikipedia.org/wiki/Yeast http://en.wikipedia.org/wiki/Bacteria http://en.wiktionary.org/wiki/anaerobic mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Yeast mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Fermentation_(food) mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Ethanol mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Beer mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Wine mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Carbon_dioxide mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Mammal mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Muscle mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Lactic_acid 27 2.5.1. Process of fermentation Fermentation is a process of energy yielding metabolism that involves a sequence of oxidation-reduction reaction in which both the substrate (electron donor) and the terminal electron acceptor(s) are organic compounds. In aerobic respiration, molecular oxygen is the final electron acceptor while in anaerobic respiration or fermentation molecular energy is not required. Before fermentation takes place, one glucose molecule is broken down into two pyruvate molecules. This is known as glycolysis (Klein et al., 2004). Glycolysis is summarized by the chemical equation: C6H12O6 + 2ADP + 2Pi + 2NAD + → 2CH3COCOO − + 2ATP + 2NADH + 2H2O + 2H + The chemical formula of pyruvate is CH3COCOO − . Pi stands for the inorganic phosphate. As shown by the reaction equation, glycolysis causes the reduction of two molecules of NAD + to NADH. Two ADP molecules are also converted to two ATP and two water molecules via substrate-level phosphorylation. The chemical equation below summarizes the fermentation of glucose, whose chemical formula is C6H12O6 (Purves et al., 2004). One glucose molecule is converted into two ethanol molecules and two carbon dioxide molecules: C6H12O6 + Zymase → 2C2H5OH + 2CO2 In some organisms, the pyruvate goes through ethanol fermentation also referred to as alcoholic fermentation. This is a biological process in which sugars such as glucose, fructose, and sucrose are converted into cellular energy and thereby produce ethanol and carbon dioxide as metabolic waste products. Because yeasts perform this conversion in the absence of oxygen, ethanol fermentation is classified as anaerobic. Usually only one of the products is desired; in bread-making, the alcohol is baked out and in alcohol production, http://en.wikipedia.org/wiki/Glucose http://en.wikipedia.org/wiki/Pyruvate http://en.wikipedia.org/wiki/Glycolysis http://en.wikipedia.org/wiki/Glycolysis http://en.wikipedia.org/wiki/Chemical_equation http://en.wikipedia.org/wiki/Chemical_formula http://en.wikipedia.org/wiki/Pyruvate http://en.wikipedia.org/wiki/Phosphate http://en.wikipedia.org/wiki/Glycolysis http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide http://en.wikipedia.org/wiki/Nicotinamide_adenine_dinucleotide http://en.wikipedia.org/wiki/Adenosine_diphosphate http://en.wikipedia.org/wiki/Adenosine_triphosphate http://en.wikipedia.org/wiki/Substrate-level_phosphorylation mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Chemical_equation mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Glucose mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Chemical_formula mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Ethanol mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Carbon_dioxide mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Ethanol_fermentation http://en.wikipedia.org/wiki/Glucose http://en.wikipedia.org/wiki/Fructose http://en.wikipedia.org/wiki/Sucrose http://en.wikipedia.org/wiki/Ethanol http://en.wikipedia.org/wiki/Carbon_dioxide http://en.wikipedia.org/wiki/Yeast http://en.wikipedia.org/wiki/Oxygen http://en.wikipedia.org/wiki/Anaerobic_respiration 28 the carbon dioxide is released into the atmosphere or used for carbonating the beverage. When the ferment (enzyme) has a high concentration of pectin, minute quantities of methanol can be produced. In animals and some bacteria, the pyruvate can go through lactic acid fermentation. Lactic acid fermentation is the simplest type of fermentation. Essentially, it is a redox reaction. In anaerobic conditions, the cell‟s primary mechanism of ATP production is glycolysis. Glycolysis reduces (transfers) electrons to NAD + , forming NADH. For glycolysis to continue, NADH must be oxidized (have electrons taken away) to regenerate the NAD + . This is usually done through an electron transport chain in a process called oxidative phosphorylation; however, this mechanism is not available without oxygen (Uno et al., 2001). Instead, the NADH donates its extra electrons to the pyruvate molecules formed during glycolysis. Since the NADH has lost electrons, NAD + regenerates and is again available for glycolysis. Lactic acid, for which this process is named, is formed by the reduction of pyruvate (Uno et al., 2001). Lactic acid fermentation can be heterolactic or homolactic. In aerobic respiration, the pyruvate produced by glycolysis is further oxidized completely, generating additional ATP and NADH in the citric acid cycle and by oxidative phosphorylation. However, this can only occur in the presence of oxygen. Oxygen is toxic to organisms which are obligate anaerobes and are not required by facultative anaerobic organisms. If oxygen is present, some species of yeast (Kluyveromyces lactis, Kluyveromyces lipolytica) oxidize pyruvate completely to carbon dioxide and water (respiration). Thus these yeasts produce ethanol only in an anaerobic environment. However, many types of yeast such as the commonly used baker's yeast Saccharomyces cerevisiae and Schizosaccharomyces pombe prefer fermentation to respiration. These yeasts will produce ethanol even under aerobic conditions given the right sources of mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Pectin mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Methanol mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Redox mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Electrons mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Electron_transport_chain mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Oxidative_phosphorylation mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Oxidative_phosphorylation mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Lactic_acid mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Aerobic_respiration mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Citric_acid_cycle mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Oxidative_phosphorylation mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Oxidative_phosphorylation mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Obligate_anaerobe mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Facultative_anaerobic_organism mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Facultative_anaerobic_organism http://en.wikipedia.org/wiki/Kluyveromyces_lactis http://en.wikipedia.org/w/index.php?title=Kluyveromyces_lipolytica&action=edit&redlink=1 29 nutrition. In the absence of oxygen, one of the fermentation pathways occurs in order to regenerate NAD + ; lactic acid fermentation is one of these pathways. Hydrogen gas can also be produced in many types of fermentation (mixed acid fermentation, butyric acid fermentation, caproate fermentation, butanol fermentation, glyoxylate fermentation), as a way to regenerate NAD + from NADH. Electrons are transferred to ferredoxin, which in turn is oxidized by hydrogenase, producing H2. 2.5.2. History of fermentation Ancient man considered fermentation as some mystery not knowing that he was dealing with the activities of microorganisms. Man had knowledge of fermentation a long time ago when he observed that meat left to stand for a few days was tastier than meat eaten after killing and that intoxicating drinks could be made from grains and fruits (Aiba et al., 1973). This concept has been known to man for many years and humans have been controlling the fermentation process. The earliest evidence of winemaking dates from eight thousand years ago, in Georgia, in the Caucasus area (Cavalieri et al., 2003). There is strong evidence that people were fermenting beverages in Babylon around 5000 BC (FAO, 2007), ancient Egypt around 3150 BC (Cavalieri et al., 2003), pre-Hispanic Mexico around 2000 BC and Sudan around1500 BC. There is also evidence of leavened bread in ancient Egypt around 1500 BC (Dirar, 1993; FAO, 2007) and of milk fermentation in Babylon around 3000 BC. According to Dubos (1951), the French chemist, Louis Pasteur was the first known scientist to connect yeast to fermentation in 1856. Pasteur originally defined fermentation as "respiration without air". He later proved that alcoholic fermentation was brought about by yeast, when studying the fermentation of sugar to alcohol by yeast. He concluded that the fermentation was catalyzed by a vital force (ferments) within the yeast cells. The ferments were thought to function only within living organisms. He concluded that, mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/NAD+ mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Mixed_acid_fermentation mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Butyric_acid mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Caproate mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Butanol mhtml:file://C:/Users/Ebabhi/Desktop/fermen/Fermentation%20(biochemistry)%20-%20Wikipedia,%20the%20free%20encyclopedia.mht!/wiki/Glyoxylate http://en.wikipedia.org/wiki/Georgia_(country) http://en.wikipedia.org/wiki/Caucasus http://en.wikipedia.org/wiki/Babylon http://en.wikipedia.org/wiki/5000_BC http://en.wikipedia.org/wiki/Ancient_Egypt http://en.wikipedia.org/wiki/3150_BC http://en.wikipedia.org/wiki/2000_BC http://en.wikipedia.org/wiki/Sudan http://en.wikipedia.org/wiki/1500_BC http://en.wikipedia.org/wiki/Ancient_Egypt http://en.wikipedia.org/wiki/1500_BC http://en.wikipedia.org/wiki/3000_BC http://en.wikipedia.org/wiki/France http://en.wikipedia.org/wiki/Louis_Pasteur http://en.wikipedia.org/wiki/Alcohol http://en.wikipedia.org/wiki/Yeast http://en.wikipedia.org/wiki/Vitalism 30 alcoholic fermentation is an act correlated with the life and organization of the yeast cells and not with the death or putrefaction of the cells (Dubos, 1951). Nevertheless, it was known that yeast extracts ferment sugar even in the absence of living yeast cells. Many scientists, including Pasteur, had attempted unsuccessfully to extract the fermentation enzyme from yeast (Lagerkvist, 2005). Success came in 1897 when the German chemist Eduard Buechner ground up yeast, extracted a juice from them, then found to his amazement that this "dead" liquid would ferment a sugar solution, forming carbon dioxide and alcohol much like living yeasts. The "unorganized ferments" behaved just like the organized ones. From that time on the term “enzyme” came to be applied to all ferments. It was then understood that fermentation is caused by enzymes which are produced by microorganisms (Lagerkvist, 2005). He termed the ferment secretion zymase (Harden and Young, 1906). It is on record that it took scientists/researchers one hundred years to uncover the enzymatic process of degradation of carbohydrate by yeast into alcohol and carbondioxide (Cook, 1958; Aiba et al., 1973). Fermentation usually implies that the action of microorganisms is desirable and the process is used to produce alcoholic beverages such as wine, beer, and cider. It is also employed in the leavening of bread and for preservation techniques to create lactic acid in sour foods such as yogurt or vinegar (acetic acid) for use in pickling foods. 2.5.3. Types of fermentation There are basically two types of fermentation namely: 1. Batch fermentation process: This is a process whereby a fermenter tank is filled with the prepared mash of raw materials to be fermented. The temperature and pH for microbial fermentation is properly http://en.wikipedia.org/wiki/Zymase http://en.wikipedia.org/wiki/Microorganisms http://en.wikipedia.org/wiki/Wine http://en.wikipedia.org/wiki/Beer http://en.wikipedia.org/wiki/Cider http://en.wikipedia.org/wiki/Leavening http://en.wikipedia.org/wiki/Bread http://en.wikipedia.org/wiki/Lactic_acid http://en.wikipedia.org/wiki/Yoghurt http://en.wikipedia.org/wiki/Vinegar http://en.wikipedia.org/wiki/Acetic_acid http://en.wikipedia.org/wiki/Pickling 31 adjusted and occasionally nutritive supplements are added to the prepared mash. The mash is steam sterilized in a pure culture process. The inoculum of a pure culture is added to the fermenter, from a separate pure culture vessel. Then the fermentation proceeds and after the proper time the contents of the fermenter are taken out for further processing. The fermenter is cleaned and the process is repeated. Thus, each fermentation process is a discontinuous process divided into batches. 2. Continuous fermentation process: In continuous fermentation, an open system is set up. Sterile nutrient solution is added to the bioreactor continuously and an equivalent amount of converted nutrient solution with microorganisms is simultaneously removed from the system (Maxon, 1960). Two basic types of continuous fermentations can be distinguished: Homogeneously Mixed Bioreactor: This is run as either a chemostat or a turbidostat. In the chemostat in the steady state, cell growth is controlled by adjusting the concentration of one substrate. Any required substrate (carbohydrates, nitrogen compounds, salts, O2) can be used as a limiting factor. In the turbidostat, cell growth is kept constant by using turbidity to monitor the biomass concentration and the rate of feed of nutrient solution is appropriately adjusted. Plug Flow Reactor: In this type of continuous fermentation, the culture solution flows through a tubular reactor without back mixing. The composition of the nutrient solution, the number of cells, mass transfer, and productivity vary at different locations within the system. At the entrance to the reactor, cells must be continuously added along with the nutrient solution 32 2.6. YEAST Yeasts are chemoorganotrophs as they use organic compounds as a source of energy and do not require sunlight to grow. The main source of carbon is obtained by hexose sugars such as glucose and fructose, or disaccharides such as sucrose and maltose (Kurtzman, 2006). Some species can metabolize pentose sugars, alcohols, and organic acids (Barnett, 1975). Yeast species either require oxygen for aerobic cellular respiration (obligate aerobes), or are anaerobic but also have aerobic methods of energy production (facultative anaerobes). Unlike bacteria, there are no known yeast species that grow only anaerobically (obligate anaerobes). According to Alexopoulos et al. (1996) yeasts are referred to as ascomycetes which possess a predominately unicellular thallus which reproduce asexually by budding or transverse division or both and produce ascospores in naked ascus. While Kurtzman and Fell, (2006) described yeasts as a growth form of eukaryotic microorganisms classified in the kingdom Fungi, with about 1,500 species described. It is estimated that only 1% of all yeast species have been described. Some organisms which are not known to produce ascospores but which possess all other characteristics above, and are not related to other group of fungi are listed as yeast because many have lost the ability to form ascospores or form ascospores under stringent conditions. The shape and size of the individual cell of some species vary slightly but in other species the cell morphology is extremely heterogenous. Cook (1958) gave the shape of yeast cells to be spherical, globose, ellipsoidal, elongate to cylindrical with rounded ends, more or less rectangular. This was also correlated by Alexopoulos et al. (1996), who added pear shaped, apiculate or lemon shaped, ogival or pointed at one end or tetrahedral to the shapes of yeast cells. The sizes of yeast vary in unicellular forms between 2 µm-10 µm in length (Cook, 1958). The length of cylindrical cells is often 20-30 µm and in some cases even greater. 33 2.6.1. Occurrence of yeast Yeast is ubiquitous in nature, existing on plants, animals, in water, sediments, soil, in terrestrial, aquatic and marine habitats (Cook, 1958; Alexopoulos et al., 1996; Sláviková and Vadkertiová, 2003); in the guts (Odiete and Akpata, 1981) and in decomposing organic matter (Alexander, 1977). Many species have high specific habitats whereas others are found on a variety of substrates in nature. Yeasts occur in an environment with temperature range of 25ºC – 30ºC and relative humidity of 80-90ºC. Optimum temperature for cultivation varies from species but those isolated from soil, air or water usually grow best at 28ºC (Arthur and Watson, 1976; Alexander, 1977). Yeast can grow both in aerobic and anaerobic conditions. All these factors are conducive for the cultivation and storage of yeast. Common media used for the cultivation of yeasts include; potato dextrose agar (PDA) or potato dextrose broth, Wallerstien Laboratories Nutrient agar (WLN), yeast peptone dextrose agar (YPD), and yeast mould agar or broth (YM). The antibiotic cycloheximide is sometimes added to yeast growth media to inhibit the growth of Saccharomyces yeasts and select for wild/indigenous yeast species (Seeliger, 1956). Methylene Blue is used to test for the presence of live yeast cells (Lee et al., 1981). 2.6.2. Reproduction in yeast Yeast basically undergoes asexual and sexual reproduction. Vegetative (asexual) reproduction is characterized by budding or fission. In the asexual reproduction where multiplication of yeast occur by budding process, the protoplasm pushes out of the cell wall in the form of a bud and forms a daughter cell that enlarges until it is separated from the mother cell by constriction at the base (Alexopoulos et al., 1996). Yeast buds are sometimes called blastospores or blastoconidia. When yeast reproduces by fission, the parent cell first elongates, the nucleus divides and a transverse wall is then formed to 34 separate the mother cell into two uninucleate daughter cells. The product of this mechanism is termed arthrospores or arthroconidia (Henrici, 1941). Sexual reproduction in yeast is differentiated from that of other fungi by sexual states that are not enclosed in a fruiting body. Yeasts are categorized into two groups based on their methods of sexual reproduction; the ascomycetous and basidiomycetous yeast. The sexual spores of the ascomycetous yeast are termed ascospores which are formed in simple structure often a vegetative cell. Such asci are called naked asci because of the absence of an ascocarp, which is a more complex fruiting body found in the higher ascomycotina. Two ascospores may assume the function of copulating gametangia which unite to form a zygote cell. Subsequently, an ascus is formed. This contains ascospores, the number of which depends on the proceeding development of the nuclei. Four to eight ascospores per ascus are usually formed but other numbers may also occur (Alexopoulos et al., 1996). If the vegetative cells are diploid, a cell may transform directly into an ascus after the 2N nucleus undergoes reduction or meiotic division. Some yeasts are heterothallic, that is, sporulation occurs when strains of opposite mating type are mixed on sporulation media. However, some strains may be homothallic (self-fertile) with reduction division and karyogamy (fussion of two haploid nuclei) taking place during the formation of sexual spores (Henrici, 1941). 2.6.3. Life cycle of yeast Yeast exhibits any of the three forms of life cycles below: a. The first form is a process when the diploid stage is short and confined to the zygote cell which undergoes meiosis immediately after karyogamy and develops ascospores. Example is found in Schizosacharomyces octosporus 35 b. The second form possesses a long diploid phase and very short haploid phase. This is the ascospores that sporulate in the yeast which undergoes the method. Example is found in Saccharomyces ludwigii c. In the third type of life cycle, both the haploid and diploid phases are perpetuated by budding so that both phases are of equal importance which may constitute a form of alternation of generation. Copulation takes place between haploid cells to form diploid cell by the process of plasmogamy and karyogamy (Henrici, 1941: Alexopoulos et al., 1996). Thus mature yeast cells are diploid containing double (2N) number of chromosomes. Basidiospores and teliospores are the sexual spores that are produced in the three classes of basidiomycetous yeast. Sexual reproduction and life cycle in these yeasts are typical in that it include both unifactorial (bipolar) and bifactorial (tetra polar) mating systems. The useful physiological properties of yeast have led to their use in the field of biotechnology. Fermentation of sugars by yeast is the oldest and largest application of this technology. Some yeasts have the ability to carry out an alcoholic fermentation while others lack this property. Fermentative yeasts have a fermentative type of metabolism whereas non-fermentative yeasts have only a respiratory or oxidative metabolism (Henrici, 1941). Both reactions produce energy, the energy from respiration is used for synthetic reactions such as assimilation and growth while part is lost as heat. When a fermenting (anaerobic) yeast culture is aerated, fermentation is suppressed and respiration increases. This is the Pasteur‟s phenomenon (Barnett, 2003). Many types of yeasts are used for making many foods: Baker's yeast in bread production, brewer's yeast in beer fermentation, yeast in wine fermentation and for xylitol (Prior et al., 1989). Instances are bound where yeasts like Kluyveromyces marxianus teleomorph of http://en.wikipedia.org/wiki/Teleomorph 36 Candida kefyr is used commercially to produce lactase enzyme similar to the use of other fungi such as those in the genus Aspergillus (Seyis and Aksoz, 2004). Saccharomyces cerevisiae and K. marxianus were yeasts used by Tyiagi et al. (1992) and Zafar and Owais, (2006) respectively in the production of ethanol. Some are able to grow in foods with a low pH, (5.0 or lower) and in the presence of sugars, organic acids and other easily metabolized carbon sources (Kurtzman, 2006). During their growth, yeasts metabolize some food components and produce metabolic end products. This causes the physical, chemical, and sensory properties of a food to change and the food is spoiled. Yeast also play other vital roles in industrial fermentation processes such as the production of industrial enzymes, chemicals, food products, malt, beverages and wine; genetic engineering and medical mycology (Boundy-Mills, 2012). 2.7. BIOETHANOL PRODUCTION IN NIGERIA In Nigeria the production of ethanol (alcohol) via local methods is as old as the people. Researches are currently on-going for large scale p