Recent Advances in Bioenergy Research Volume I Edited by SACHIN KUMAR, ANIL K SARMA Sardar Swaran Singh National Institute of Renewable Energy, Kapurthala, India i ISBN 978-81-927097-0-3 © Sardar Swaran Singh National Institute of Renewable Energy, Kapurthala-2013 Electronic version published by SSS-NIRE ALL RIGHTS RESERVED ii CONTENTS Preface ix Contributors xi Part-I: Biomass Assessment and Management for Energy Purpose 1 Characteristics of Biomass A.K Jain 1.1 Introduction 1.2 Physical Properties 1.3 Thermal Characteristics 1.4 Chemical Analysis 1.5 Correlation Models 1.6 Conclusions References Global warming: A new paradigm for Bio-Energy Research S.K Sharma 2.1 Introduction 2.2 New Research opportunities in Bio Energy 2.3 Conclusions References 21 14 17 19 19 21 22 26 26 Biomass Assessment for Growth of Bioenergy: A Case Study in Assam, India D.C Baruah, Moonmoon Hiloidhari Abstract 3.1 Introduction 3.2 Materials and methods 3.3 Results and discussions 3.4 Conclusion References 28 Bio Mass Fuel Generation- An Ultimate Energy Resource Ajeet Kumar Upadhyay Abstract 4.1 Introduction 4.2 Bio fuels from biomass 4.3 Bio ethanol from biomass 4.4 Biodiesel from biomass 4.5 Methane generation from microbial action 4.6 Hydrogen from biomass 4.7 Conclusions References 44 iii 28 28 31 36 41 42 44 44 46 48 48 49 50 51 51 Part-II: Thermo-chemical Conversion 52 Modeling of Biomass Gasification Processes in Downdraft Gasifiers: A Review Anjireddy Bhavanam, R.C Sastry Abstract 5.1 Introduction 5.2 Downdraft Gasifiers 5.3 Gasification Models 5.4 Model Validation 5.5 Conclusions References 53 Prospect of Bioenergy Substitution in Tea Industries of North East India B.J Dutta, D Baruah, M Saikia, R Bhowmik, D.C Baruah Abstract 6.1 Introduction 6.2 Materials and Method 6.3 Results and Discussion 6.4 Conclusions References 67 53 53 55 57 63 63 65 67 67 69 73 76 77 Drying Of Biomass Fuel Used For Gasifier Using Waste Heat R Soni, A.K Jain, B.S Panesar, P.K Gupta 7.1 Introduction 7.2 Methodology 7.3 Results and Discussion 7.4 Conclusions References 79 Improved Woodstove – Tehtana Experience Usha Bajpai, Suresh C Bajpai Abstract 8.1 Introduction 8.2 Energy, Health and Global Warming 8.3 The Indian National Programme on Improved Chulhas 8.4 Improved Woodstove at Tehtana 8.5 Conclusions References 89 Development of a Briquetting Machine for Jatropha Seed Cake H Raheman, B Singh, T Alam, D Padhee Abstract 9.1 Introduction 9.2 Materials and Method 9.3 Results and Discussion 9.4 Conclusions 105 iv 79 80 82 86 87 89 90 90 96 97 102 103 105 105 107 108 113 References 113 10 Charcoal Activation at Low Temperature A.P Singh Chouhan, S.P Singh Abstract 10.1 Introduction 10.2 Materials and Method 10.3 Results and Discussion 10.4 Conclusions References 115 115 115 117 118 125 126 Part-III: Biogas & Biohydrogen 128 11 MNRE Policy on Biogas Programme M.L Bamboriya 11.1 Introduction 11.2 Biogas Programme 129 129 129 12 Biogas Plant a Check for Environment Pollution and Global Warming Sarbjit Singh Sooch Abstract 12.1 Introduction 12.2 Materials and Method 12.3 Results and Discussion 12.4 Conclusions References 143 143 143 144 147 149 149 13 Todays Waste Tomarrows Fuel: Hyderabad to Get 50MW from Garbage (MSW) K.K Jain, J Praveen Abstract 13.1 Introduction 13.2 RDF Fuel Conversion from MSW (Segregated high CV fraction of MSW) 13.3 Testing Results of RDF 13.4 Monitoring Report of 6.6 MW Plant 13.5 Emission Characteristics of RDF 13.6 Details of 6.6 MW Power Plant 13.7 RDF from processed MSW 13.8 Case Study of Biogas from Slaughter House Waste to Energy 13.9 Conclusions References 151 14 Municipal Solid Waste to Energy: Experimental Studies on Biogas Plant Usha Bajpai, Puja Singh Abstract 158 v 151 151 153 153 153 154 154 154 155 156 157 158 14.1 Introduction 14.2 Materials and Methods of Experimental Studies 14.3 Results and Discussion References 15 Poultry Litter as an Alternate Feed Stock to Cattle Dung for Biogas Production and Power Generation Sarabjit Singh Sooch, Urmila Gupta, Anand Gautam Abstract 15.1 Introduction 15.2 Methodology 15.3 Results and Discussion 15.4 Conclusion 16 CFD Modelling of an UASB Reactor for Biogas Production from Industrial Waste/Domestic Sewage Partha Kundu, I.M Mishra Abstract 16.1 Introduction 16.2 Methods 16.3 Results and Discussion 16.4 Conclusions References 159 164 168 169 170 170 170 172 172 173 176 176 176 179 184 190 191 17 Algal Bio-Hydrogen- Prospects and Challenges Shailendra Kumar Singh, M.K Jha, Ajay Bansal, Apurba dey Abstract 17.1 Introduction 17.2 Physiology of H2 production in green algae 17.3 Challenges and prospects 17.4 Design and cost of photobioreactors 17.5 Conclusions References 194 Part-IV: Production Aspects of Biodiesel 207 18 Jatropha (Jatropha Curcas) L Plantations and Climate Change Avtar Singh Abstract 18.1 Introduction 18.2 Status of jatropha plantations in the world and future potential for expansion 18.3 Soil for Jatropha cultivation 18.4 Genetic improvement in Jatropha 18.5 Tissue culture in Jatropha curcas 18.6 Seedling production in nursery 18.7 Plantation establishment 18.8 Plant protection vi 208 194 195 196 197 202 203 204 208 209 209 209 210 210 211 212 215 18.9 Plant responses to climate change 18.10 Effect of Jatropha on climate change References 19 Biodiesel Production from Algal Species Grown on Dairy Wastewater Richa Kothari, Vinayak V Pathak, D.P Singh Abstract 19.1 Introduction 19.2 Materials and Methods 19.3 Results and Discussion 19.4 Conclusions References 215 217 218 221 221 221 222 224 227 227 20 Green Technology for Biodiesel Production using Waste Material Based Heterogeneous Catalyst Anil Kumar Sarma, Ashish P Singh Chouhan Abstract 20.1 Introduction 20.2 Materials 20.3 Results and Discussion 20.4 Conclusions References 230 21 Production and Studied of Fuel Properties of Sunflower Ethyl Ester and its Blends R Kumar, A.K Dixit, S K Singh, G.S Manes, R Khurana Abstract 21.1 Introduction 21.2 Materials and Methods 21.3 Results and Discussion 21.4 Conclusions References 241 Part-V: Lignocellulosic Ethanol Production 248 22 Thermophiles: smart bugs for ethanol production from agricultural residues Sachin Kumar, Pratibha Dheeran, Dilip K Adhikari Abstract 22.1 Introduction 22.2 Materials and methods 22.3 Results and Discussion 22.4 Conclusions References 249 23 Study of Bioethanol Production from Brewer’s Spent Grain using Fusarium oxysporum Abhay Dinker, Arvind Kumar, Madhu Agarwal 258 vii 230 231 232 234 238 238 241 241 242 244 246 246 249 249 251 252 255 256 Abstract 23.1 Introduction 23.2 Materials and Methods 23.3 Results 23.4 Conclusions References 258 258 260 262 262 263 viii Preface Sachin Kumar, A.K Sarma Bio-energy research has received tremendous attention all over the world due to steep hike in petroleum prices and environmental concerns At the current electricity generating capacity and other available energy sources, a huge gap exists between the demand and supply (above 15%) and the Conventional Energy resources of the country are meagre Agricultural crop residues production in the country is about 550 Mt/year and is likely to increase in the coming years Majority of the crop residues are either processed in uneconomic way or get destroyed as such Apart from the crop residues, other biomass such as animal excreta, forest wastes and agro-industrial wastes are also available in abundance and can play a major role in supplementing the energy resources of the country Waste biomass materials include various natural and derived materials, such as woody and herbaceous species, bagasse, agricultural waste, waste from paper, municipal solid waste, industrial waste, sawdust, grass, food processing waste, waste oil, non-edible oil or shell of oil-bearing seed, aquatic plants and algae, etc., which could be potentially used for production of useful fuels and chemicals The average majority of biomass energy is produced from wood and wood wastes (64%), followed by municipal solid waste (24%), agricultural waste (5%) and landfill gases (5%) Waste and degraded lands are generally used for energy plantation and biomass production There is no debate on the issue that renewable energy is the only sustainable energy in nature Biomass energy in particular is one of the cleanest form of energy gifted by nature This is also the ‘waste to wealth’ making weapons for the farmers Because, all forms of derived agricultural waste can be converted to useful energy that directly contribute to the income of farmers and nation as well Moreover, they are highly beneficial from the viewpoint of environmental pollution control and an asset for carbon credit Keeping in view the need and importance of bioenergy research in our country, we express pleasure to introduce the first edition of ‘Recent Advances in Bioenergy Research- Volume-I’ in the form of a book The book is divided in five parts viz Part-I: Biomass Assessment and Management for Energy Purpose; Part-II: Thermo-chemical Conversion; Part-III: Biogas & Biohydrogen; Part-IV: Production Aspects of Biodiesel; ix Part-V: Lignocellulosic Ethanol Production Each section includes respective chapters from Eminent Academician, Scientists and Researchers in the field We are really grateful for their commendable contribution for this book Emphasis is given such that current trends of research and investigation in the bioenergy sector can be easily worked out from the in-depth study of this book Our efforts will be successful if the readers dig up the expected gain out of these articles x in future At present India produces only 1.3 million liters of ethanol from molasses However, considering 10 % ethanol supplementation in petrol and diesel, its demand in the transportation sector is projected to be nearly 12 billion liters per year (Ghosh and Ghose, 2003) As future demand of ethanol as transportation fuel increases, it is quit unlikely that molasses could be the sole feed-stock for ethanol production Therefore, cellulosic and starchy biomass biomass are one of the most abundant, cheap and renewable resources for ethanol production The recent development in acid and enzymatic hydrolysis of cellulose and starch to fermentable sugars may possibly lead to commercially viable production of ethanol from the vast and renewable quantities of cellulose available on the earth Sugarcane bagasse is mostly utilized for producing steam and electricity required for the cane processing plant Because of its high carbohydrate content, relatively low lignin content and its availability as an industrial waste product, sugarcane bagasse is a particularly appropriate substrate for bioconversion to ethanol The microorganisms, which are able to ferment both glucose and xylose, are required for an efficient conversion of bagasse to ethanol (Martín et al., 2002) Other biomass feed-stocks rich in sugars include sugar beet, sweet sorghum, and various fruits (Badger, 2002) Whereas cassava, a high starch accumulating root (70-80 %) needs to be explored as a low cost feed-stock to meet the future demand for ethanol Cassava was cultivated around 6.7 million tons in 2004 in India The entire quantity of the crop has potential to produce around 12 billion liters of ethanol The potential applications of thermophilic microorganisms in industrial applications have been widely reported The important advantages at higher temperature include the possible use of continuous ethanol stripping as a method of ethanol recovery, significant restriction of contamination chances and reduction in the volume of distillery cooling-wastewater effluent (Kosaric, 1996; Lee, 1997; Nigam et al., 1997; Banat et al., 1998; Kumar et al., 2009a) In the present paper we report the production of bioethanol from sugarcane bagasse and cassava by thermophiles The hydrolysis of bagasse and cassava into fermentable sugars was carried out by acid hydrolysis and enzymatic hydrolysis, respectively These fermentable sugars were fermented into bioethanol by thermophilic strain Kluyveromyces sp IIPE453 at 50 ºC 250 22.2 Materials and methods 22.2.1 Sugarcane bagasse hydrolysis and detoxification Sugarcane bagasse was chopped into small pieces of 3-5 mm size 100 g of dried sugarcane bagasse was mixed with L of % (v/v) H2SO4 solution and was kept in an autoclave at 15 psi and 121 ºC for 90 The hydrolysate was separated from residual sugarcane bagasse by filtration Hydrolysate was neutralized with calcium oxide (CaO) The furfural in hydrolysate was removed by solvent extraction 30 ml of hydrolysate and 10 ml of ethyl acetate was taken into 250 ml conical flask, and extracted by agitation for 30 The mixture was transferred into a separating funnel The organic (ethyl acetate) and aqueous (hydrolysate) phases were separated Air stripping was conducted through extracted hydrolysate phase to remove volatile components (i.e ethyl acetate) 22.2.2 Cassava hydrolysis by thermostable amylase Thermostable amylase was isolated from Geobacillus sp IIPTN was used for the hydrolysis of cassava starch (Dheeran et al., 2010) The amylase was produced by IIPTN at 60 The hydrolysis of cassava was carried out by ultra filtered amylase at 80 ºC and pH 5.0 with constant shaking The unhydrolysed cassava was removed by centrifugation at 15,000 rpm for 10 The clear cassava hydrolysate was then used for ethanol production 22.2.3 Microorganisms and culture conditions The strain used for ethanol production, Kluyveromyces sp IIPE453, was isolated from sugar industry waste (Kumar et al., 2009b) The strain was grown in salt medium containing 0.15 g/l di-sodium hydrogen ortho phosphate, 0.15 g/l potassium di-hydrogen ortho phosphate, 2.0 g/l ammonium sulphate, 1.0 g/l yeast extract and 10 g/l glucose with pH 5.0 at 50 ºC Fermentation was carried out in medium prepared in hydrolysate containing 0.15 g/l potassium di-hydrogen ortho phosphate, 1.0 g/l ammonium sulphate, 1.0 g/l yeast extract with pH 6.0 at 50 ºC 22.2.4 Fermentation conditions Fermentation of 450 ml sugarcane bagasse hydrolysate was performed in litre three neck flat bottom jacketed flask by free cells of Kluyveromyces sp IIPE453 in batch mode The dry cell mass concentration was kept 5.5 g/l Initial sugar conc in sugarcane bagasse hydrolysate was 25 g/l The temperature was maintained at 50 ºC and stirred by 251 magnetic stirrer at 50 rpm Fermentation of cassava hydrolysate was performed in 250 ml capped bottle by free cells of Kluyveromyces sp IIPE453 in batch mode The temperature was maintained at 50 ºC in shaker at 100 rpm 22.2.5 Analytical methods Dry cell weight in broth: ml sample was centrifuged in eppendorf tube by using Eppendorf Centrifuge 5415 C at 10000 rpm for and washed twice with distilled water and dried in vacuum oven at 70 ºC to a constant weight Reducing sugars in media and fermented broth were determined by dinitrosalicylic acid (DNS) method at 575 nm by Double Beam UV-VIS Spectrophotometer 2600 (Summer and Somers, 1944) DNS was prepared as: 1.87 g 3,5 di-nitrosalicylic acid and 3.48 g sodium chloride were mixed in 250 ml D/W and added 53.9 g potassium sodium tartrate, 1.34 ml phenol and 1.46 sodium metabisulphite Xylose in sugarcane bagasse hydrolysate was analysed by p-bromo aniline by at 520 nm by Double Beam UV-VIS Spectrophotometer 2600 (Pesez and Bartos, 1974) pbromo aniline was prepared as: 2g p-bromo aniline was mixed in 100 ml glacial acetic acid and saturated with thiourea Ethanol was also determined by colorimetry method in which chromic acid was used (Horwitz, 1980) Furfural was measured by Double Beam UV-VIS Spectrophotometer 2600 at 277 nm 22.3 Results and Discussion 22.3.1 Hydrolysis of sugarcane bagasse The total reducing sugar (glucose and xylose) in sugarcane bagasse hydrolysate was 28 g/l The furfural concentration was 560 mg/l The maximum lignocellulose was converted into fermentable sugar within 90 in acid hydrolysis The total fermentable sugar recovered from sugarcane bagasse was 328.5 g/kg of sugarcane bagasse whereas sugarcane bagasse contains 614 g sugar (glucose and xylose)/kg sugarcane bagasse fibers (Lee, 1997) Thus, 53.5 % sugars (glucose and xylose) of total sugars could be recovered in sugarcane bagasse Hernández-Salas et al (2009) reported 37.21 % reducing sugar yield from sugarcane depithed bagasse by using 1.2 % (v/v) HCl at 121 ºC for h 252 22.3.2 Detoxification of sugarcane bagasse hydrolysate The hydrolysate was neutralized by CaO and furfural from hydrolysate was removed by ethyl acetate Ethyl acetate was able to remove 75.67 % of furfural without any sugar loss in first stage extraction This process was repeated three times 93 % of furfural could be removed from hydrolysate in three stages of extraction process (Fig 22.1) Fig 22.1 % removal of furfural in bagasse hydrolysate in three stage extraction with ethyl acetate 22.3.3 Ethanol production from bagasse hydrolysate Fermentation was carried out with sugarcane bagasse hydrolysate in batch process by using Kluyveromyces sp IIPE453 (Fig 22.2) The total sugar in hydrolysate was consumed in h with overall yield of 52 % of theoretical yield and productivity 0.65 gl1 -1 h Boyle et al (1997) reported 0.2 gl-1h-1 ethanol productivity on pretreated barley straw in SSF by using Kluyveromyces marxianus IMB3 Ballesteros et al (2004) reported 16-19 g/l ethanol concentration in 72-82 h on defferent lignocellulosic biomass in SSF by Kluyveromyces marxianus CECT 10875 at 42 oC whereas by the same strain Tomás-Pejó et al (2009) reported maximum 32 g/l ethanol with productivity 0.44 gl-1h-1 on wheat straw in SSF batch process The dry cell mass was almost constant throughout the fermentation Fig shows that the rate of fermentation was very fast till h after that the sugar in the broth was very less so the rate was decreased The ethanol yield was 253 low on the sugarcane bagasse hydrolysate due to the low percentage of glucose present in hydrolysate and lower ethanol yield on xylose, which can be further increased by increasing glucose concentration in hydrolysate 22.3.4 Cassava Hydrolysis 10 g cassava was taken in 200 ml acetate buffer of pH 5.0 50 ml amylase enzyme with total 326 mg protein was used to hydrolyze the cassava The sugar concentration in hydrolysate was 27 g/l The total sugar yield from cassava was 67.5 % 22.3.5 Ethanol production from cassava hydrolysate Fermentation was carried out with cassava hydrolysate in batch process by using Kluyveromyces sp IIPE453 (Fig 22.3) The total sugar in hydrolysate was consumed in 138 h with productivity 0.09 gl-1h-1 The ethanol yield was obtained 45 % on the basis of fermentable sugars and the overall ethanol yield on dry cassava basis was 33 % The dry cell mass was almost constant through out the fermentation Fig 22.2 Ethanol production from sugarcane bagasse hydrolysate in batch process 254 Fig 22.3 Ethanol production from cassava hydrolysate in batch process 22.4 Conclusions The process used for acid hydrolysis yielded 53.5 % of total fermentable sugar in sugarcane bagasse The hydrolysis process requires the improvement for high fermentable sugar yield The strain Kluyveromyces sp IIPE453 is capable to produce ethanol at faster rate as compared to other industrial ethanalogens The fermentation of hydrolysate performed very poor yield but the final broth showed complete conversion of sugar It may be due to conversion of xylose to other products So we need a strain or mixture of strains which can convert both glucose and xylose to ethanol The strain Kluyveromyces sp IIPE453 is able to convert cassava hydrolysate sugars to ethanol but at very slow rate Acknowledgements We thank Dr M O Garg, Director, IIP, Dehradun for his valuable suggestion and facilities provided to carry out the work Sachin Kumar and Pratibha Dheeran also gratefully acknowledge the Research Fellowship awarded by Council of Scientific and Industrial Research (CSIR), India 255 References Badger P.C (2002) Ethanol from cellulose: A general review In: Janick, J., Whipkey, A., (Eds.), Trends in new crops and new uses ASHS Press, Alexandria, VA, pp 17-25 Ballesteros M., Oliva J.M., Negro M.J., Manzanares P and Ballesteros I (2004) Ethanol from lignocellulosic materials by a simultaneous saccharification and fermentation process (SFS) with Kluyveromyces marxianus CECT 10875 Process Biochem., 39:1843-1848 Banat I.M., Nigam P., Singh D., Marchchant R and McHale A.P (1998) Review: Ethanol production at elevated temperatures and alcohol concentrations: Part I - Yeasts in general W J Microbiol Biotechnol., 14:809-821 Boyle M., Barron N and McHale A.P (1997) Simultaneous saccharification and fermentation of straw to ethanol using the thermotolerant yeast strain Kluyveromyces marxianus IMB3 Biotechnol Lett., 19:49-51 Dheeran P., Kumar S., Jaiswal Y.K and Adhikari D.K (2010) Characterization of hyperthermostable α-amylase from Geobacillus sp IIPTN Appl Microbiol Biotechnol., 86:1857-1866 Ghosh P and Ghose T K (2003) Bioethanol in India: Recent past and emerging future In: Scheper T (Ed.), Advanced Biochemical Engineering/Biotechnology, Biotechnology in India II Springer, New York, 20, pp 1-27 Hernández-Salas J.M., Villa-Ramírez M.S., Veloz-Rendόn J.S., RiveraHernández K.N., González-César R.A., Plascencia-Espinosa M.A and TrejoEstrada S.R (2009) Comparative hydrolysis and fermentation of sugarcane and agave bagasse Bioresour Technol., 100:1238-1245 Horwitz W (1980) Beverages: Distilled Liquors In: Official Methods of Analysis of the Association of Official Analytical Chemists, 13th ed, pp 147 Kosaric N (1996) Ethanol-potential source of energy and chemical products In: Biotechnology, Products of Primary Metabolism, Rehm H J & Reed G (eds), 2nd ed VCH Publishers Inc., New York, Vol 6, pp 135 256 10 Kumar S., Singh S.P., Mishra I.M and Adhikari D.K (2009a) Ethanol and xylitol production from glucose and xylose at high temperature by Kluyveromyces sp IIPE453 J Ind Microbiol Biotechnol., 36:1483-1489 11 Kumar S., Singh S.P., Mishra I.M and Adhikari D.K (2009b) Recent advances in production of bioethanol from lignocellulosic biomass Chem Eng Technol., 32:517-526 12 Lee J (1997) Biological conversion of lignocellulosic biomass to ethanol J Biotechnol., 56:1-24 13 Martín C., Galbe M., Wahlbom C.F., Hahn-Hägerdal B and Jönsson L.J (2002) Ethanol production from enzymatic hydrolysates of sugarcane bagasse using recombinant xylose-utilising Saccharomyces cerevisiae Enzyme Microb Technol., 31:274-282 14 Nigam P., Banat I.M., Singh D., Mchale A.P and Marchant R (1997) Continuous ethanol production by thermotolerant Kluyveromyces marxianus IMB3 immobilized on mineral kissiris at 45 °C W J Microbiol Biotechnol., 13:283-288 15 Pesez M and Bartos J (1974) Colorimetric and fluorimetric analysis of organic compounds and drugs Schwartz (ed.) Marcel Dekker Inc., New York 16 Summer J.B and Somers G.F (1944) Laboratory Experiments in Biological Chemistry, Academic Press, New York, 34 17 Tomás-Pejó E., Oliva J.M., González A., Ballesteros I and Ballesteros M (2009) Bioethanol production from wheat straw by the thermotolerant yeast Kluyveromyces marxianus CECT 10875 in a simultaneous saccharification and fermentation fed-batch process Fuel, 88:2142-2147 257 CHAPTER 23 STUDY OF BIOETHANOL PRODUCTION FROM BREWER’S SPENT GRAIN USING FUSARIUM OXYSPORUM Abhay Dinker, Arvind Kumar, Madhu Agarwal Abstract Brewer’s spent grain is the byproduct of brew industry and is a low cost feedstock In the present study this spent grain used as the substrate for the Fusarium oxysporum Fusarium contains the enzymatic machinery for the degradation of lignocellulosic material found in the spent grain and then conversion of degraded polysaccharide into the ethanol Experiments were performed with sugar mixtures simulating the carbohydrate content of BG in order to determine the utilization pattern that could be expected during the fermentation of cellulose and hemicelluloses The alkali pretreatment of spent grain was performed that helps in lignin degradation and efficient bioethanol production was achieved using the lignocellulytic enzyme machinery of Fusarium oxysporium The growth of Fungi and fermentation study both was carried out in the submerged stage suggest an efficient production of bioethanol from the spent grain The ability of Fusarium oxysporum to degrade the lignocellulosic material was also studied Keywords: Brewer’s spent grain, Submeged fermentation, Bioethanol, Titration 23.1 Introduction The production of alcohol fuel has been achieved on industrial scale from the wheat, corn, Beet, Molasses Lately the use of starch and sugar crops as raw materials in the biofuels industry has been for the subject of discussion More and more scientific efforts are being made towards an efficient technology for the biological conversion of lignocellulosic materials to second-generation biofuel (Huige, 1994) The enzymatic hydrolysis of cellulosic materials to produce fermentable sugars has an enormous potential in meeting global bioenergy demand through the biorefinery concept, since agri-food processes generate millions of tons of waste each year, such as spent grain 258 from brewing (BG) and corn cob (CC) Thus, several companies around the world are currently working toward developing technologies for producing cellulosic ethanol on a commercial scale Brewer's spent grain is a byproduct of beer brewing consisting of the residue of malt and grain, which remains, in the mash-kettle after the mashing and lautering process It is also called as Brewer’s grain or draff It consists primarily of grain husks, pericarp, and fragments of endosperm BG is the most abundant brewing by-product, corresponding to around 85% of total by-products generated and is mainly used as low-value cattle food (Santos et al., 2003) The chemical composition of BG varies according to barley variety, harvest time, malting and mashing conditions, and the quality and type of adjuncts added in the brewing process The BG used in this study, as described previously, contains mainly hemicellulose in the form of arabinoxylans from the barley grain and cellulose BG has the potential to serve as a low-cost feedstock for the production of ethanol since hemicelluloses and cellulose content corresponds to 52% w/w of dry BG (Santos et al., 2003) Presently conversion of lignocelluloses to ethanol requires the chemical and enzymatic methods for the conversion of fermentable sugars to the ethanol The large amounts of enzymes required for enzymatic conversion of hemicelluloses and cellulose to fermentable sugars impacts severely on the cost effectiveness of this technology (Xiros et al., 2008) The physical support and the energy required for a fungus to grow and produce the desired metabolite is primarily provided by a substrate Generally, the production of cellulases and hemicellulases has been shown to be inducible and is affected by the nature of the substrate used in fermentation Therefore, the choice of an appropriate inducing co-substrate is of importance CC, the central wooden core of maize (Zea mays ssp mays L.), has been used as an excellent carbon source for enzyme production by fungi and especially Fusarium oxysporum Nowadays, CC, a chip agricultural by-product, is an important source of the furfural, an aromatic aldehyde used in a wide variety of industrial processes (Christkopoulus et al., 1989).The ethanol production by the mesophilic fungus F oxysporum by coupling solid state and submerged bioreactor fermentation was previously investigated The simultaneous production of cellulolytic and xylanolytic enzymes in solid-state culture, the increase of BG's saccharification and the consequent enhancement of ethanol production upon alkali pretreatment of BG was also investigated (Xiros et al., 2008) The submerged 259 culture technique was widely used for biotechnological applications as it is intrinsically less problematic (heat and oxygen mass transfer are much better, and culture homogeneity is usually superior) making it more reliable and reproducible, easier to monitor and to control key operational parameters, and it is more flexible In this study consolidated bioprocessing was implemented, taking advantage of the above-mentioned exceptional abilities of F oxysporum with regard to the bioconversion of BG to ethanol Both growth and production stages were carried out under submerged cultivation conditions The effect of BG on the simultaneous production of cellulolytic and xylanolytic enzymes in submerged culture was investigated Sugar mixtures simulating the composition of BG were used for the study of the fermentative performance of F oxysporum The fermentation of BG was also studied The increase of BG saccharification upon alkali pre-treatment was evaluated with regard to the enhancement of ethanol production under submerged conditions 23.2 Materials and method Brewer’s Grain was obtained from the S.A Brewery, Neemrana The material was frozen immediately after collected and stored at -60ºC in ultra freezer Before use it was dried at 60ºC for 48 hours in oven and then chopped in the hammer mill to particle size smaller than mm MTCC (Microbial Type Culture collection) of F Oxysporum (MTCC No 1755) was obtained from the IMTECH, Chandigarh All cultures was obtained in frozen dried state was revived on PDA (Potato Dextrose Agar) medium and then the cultures from the second generation of the revived culture were used for the fermentation studies (Beldmen et al., 2010) Inoculum was prepared by pouring 30 ml of de-ionized sterile water contains 200 µl Tween 80 solution on the prepared plates of the cultures and 10 ml of mixture from the plates then transfer to the 400 ml of mineral medium have (in g L-1) 1.0 KH2PO4, 0.30 CaCl2.2H2O, 0.30 MgSO4.7H2O, 10 (NH4)2HPO4, 6.94 NaH2PO4.2H2O, 9.52 Na2HPO42H2O, 20 Dextrose, 10 BG and then Incubate the flask at 30º C for days (Beldmen et al., 2010) Here we used Dextrose as of carbon source 23.2.1 Fermentation in bioreactor This prepared inoculums was transferred to a 2L thermo regulated double jacketed agitated bioreactor (Lark, India).with initial working volume of 35 g/L of BG was used Temperature was set to be 30 ºC in all cases and the agitation speed was 180 rpm The 260 bioreactor was sterilized at 121ºC for 20 before the transfer of Spent Grain and the Inoculum The NaOH was added along with the spent grain and water to the bioreactor, NaOH was added in the ratio of 8:1(w/w) The sterilization and pretreatment was carried out in the single step at 121ºC for 30 minutes After sterilization the pH was adjusted to by 1M sulphuric acid and then the 400 ml of fungal culture grown for 72 hours was then transferred to the Bioreactor The aeration rate (0.5v/v) and the pH were kept stable during the fermentation (Xiros et al., 2008) 23.2.2 Analysis of ethanol Qualitative analysis of ethanol was obtained by the reacting it with iodine in the presence of NaOH that gives crystals After qualitative analysis the quantitative estimation was carried out by the redox titration In this method the ethanol is oxidized into the ethanoic acid by reacting it with excess of potassium dichromate in the excess of potassium dichromate in acid The amount of unreacted dichromate was then determined by adding potassium iodide solution, which was also oxidized by the potassium dichromate forming iodine The iodine was then titrated with a standard solution of sodium thiosulfate and the titration results were used to calculate the ethanol content of the original solution (Huige, 1994) 23.2.3 Titration of sample 5ml of sample was kept in a 10 mL beaker hanged in the flask tightly closed with the rubber stopper The water and ethanol slowly evaporate and as the ethanol comes in contact with the acid dichromate solution (0.01mol/L) at the bottom it first dissolves, and was then oxidized More ethanol evaporates until eventually all the ethanol from the beverage has left the sample and reacted with the dichromate Since this transfer takes time, it was necessary to leave the flask with the suspended sample in a warm place overnight Next morning the flask was allowed to come to room temperature, then the stopper was removed carefully and the sample holder was discarded The walls of the flask then rinsed with 100 mL of distilled water and mL of potassium iodide solution (1.2 mol/L) was added and mixed properly There blank titrations were also prepared by addition of 10 mL of acid dichromate solution to a conical flask, with addition of 100 mL of water and mL of potassium iodide solution and swirled well to mix The burette was filled with sodium thiosulfate solution and each flask was titrated with sodium thiosulfate (0.03 mol/L).When the brown iodine color fades to yellow, mL of starch solution was added and titration was continued until the blue color disappeared The 261 blank flasks was titrated first, and then repeated until concordant results were obtained (titres agreeing to within 0.1 mL) After that the sample of alcohol was titrated The average volume of sodium thiosulfate used for sample was then determined 23.2.4 Calculation of moles of ethanol Subtract the volume of the sodium thiosulfate solution used for the sample titration from the volume used for the blank titration This volume of the sodium thiosulfate solution was now used to determine the alcohol concentration The number of moles of sodium thiosulfate in this volume was calculated Using the equations, the relationship between the moles of sodium thiosulfate and the moles of ethanol was determined as: mol of S2O32- is equivalent to mol of Cr2O72 - and mol of Cr2O72- is equivalent to mol of C2H5OH - then mol of S2O32- is equivalent to 0.25 mol of C2H5OH This ratio was used to calculate the number of moles in the alcohol was then determined 23.3 Result As per the titration method stated above the amount of ethanol was obtained from the spent grain shown as in Table 23.1 On titrating the Acid dichromate solution against the sodium thiosulphate the moles of ethanol was determined by the method stated above and the using standard density of ethanol the amount of ethanol is calculated per Kg of spent grain The result was obtained for different samples and an average value of g ethanol /Kg of spent grain was obtained Table 23.1 Volume of Ethanol obtained from different Samples Sample /Batch No Titrating Volume (Titrated Difference in Blank and Sampled Acid Dichromate) 4.4 4.0 4.2 Ethanol (in g/Kg of BG) 5.3 4.9 5.0 Hence it was found that from Fusarium oxysporum on an average g /Kg of ethanol can be recovered 23.4 Conclusion In conclusion F Oxysporium was efficient for the production of Bioethanol from the Spent Grain The lignocellulytic enzyme machinery found in this microorganism can proves to be the most economic way for the production of Biofuel from the 262 lignocellulosic compounds.The spent grain is the good substrate for the enzymes present in the F Oxysporum it not only favors the degradation of the lignocelluloses and hemicelluloses present in the spentgrain but also efficiently converts these polysaccharide into the ethanol It was possible to control simultaneous production of cellulolytic and hemicellulolytic enzymes from F oxysporum and generate a multienzymatic system capable of hydrolyzing lignocellulosic substrates using a growth medium consisting of BG, and a mineral source under submerged conditions The fermentation study using sugar mixtures as carbon source simulating BG's carbohydrate content led to useful conclusions concerning the bioconversion of BG to ethanol Hydrolysis seems to be the bottleneck of the process while the fermentative performance of F oxysporum was satisfactory The high pentose content of BG and the ability of F oxysporum to ferment xylose make 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