EVALUATION OF ELEPHANT GRASS (PENNISETUM PURPUREUM) AS SUBSTRATE FOR BIOETHANOL PRODUCTION USING CO-CULTURES OF ASPERGILLUS NIGER AND SACCHAROMYCES CEREVISIAE


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TABLE OF CONTENTS

Title page
Abstract
Table of Contents

CHAPTER ONE
1.0INTRODUCTION
1.1Statement of Research Problem
1.2Justification
1.3       Aim
1.4       Specific Objectives

CHAPTER TWO
2.0       LITERATURE REVIEW
2.1       Ethanol
2.1.1 History
2.1.2 Physical Properties
2.1.3 Production of Ethanol
2.1.4 Ethanol Purification
2.1.5 Grades of Ethanol
2.1.6 Uses of Ethanol
2.2       Lignocellulosic Ethanol Production
2.2.1 Structure of Lignocellulosic Biomass
2.2.2    Value-Added Products from Lignocellulosic Wastes (LCW)
2.2.3    Bioprocessing of Lignocellulosic Materials
2.2.4    Pretreatment Technologies for Lignocellulosic Wastes
2.3       The Genus Aspergillus
2.3.1    Aspergillus niger (Black mould)
2.4       The Genus Saccharomyces
2.4.1    Saccharomyces cerevisiae
2.5       Elephant Grass (Pennisetum purpureum)

CHAPTER THREE
3.0MATERIALS AND METHODS
3.1       Collection of Samples
3.1.1    Sample collection for Aspergillus niger
3.1.2    Sample Collection for Saccharomyces cerevisiae
3.1.3    Sample collection and Identification of Elephant Grass
3.2       Media for Isolation
3.3       Isolation of Test Organisms
3.3.1    Isolation of A. niger
3.3.2    Isolation of S. cerevisiae
3.4       Identification Of Test Organisms
3.4.1    Identification of A. niger Isolates
3.4.2    Identification of S. cerevisiae Isolates
3.5       Preparation of Standard Reagents
3.5.1    Preparation of 3, 5- Dinitro Salicylic Acid (DNS) reagent
3.5.2    Preparation of 0.05M sodium citrate buffer pH 4.8
3.6       Preparation of Standard Curves
3.6.1    Preparation of glucose curve for Total saccharifying cellulase (FPase) assay
3.6.2    Preparation of glucose curve for reducing sugars measurement
3.6.3    Preparation of standard ethanol density curve
3.6.4    Preparation of standard ethanol specific gravity curve
3.7       Screening of A. niger Isolates
3.7.1    Qualitative Cellulase Assay
3.7.2    Quantitative Enzyme Assay
3.8       Screening of Yeast Isolates
3.8.1    Growth at 50% (W/V) Glucose Concentration
3.8.2    Fermentation of 50% (W/V) Glucose
3.8.3    Ethanol Tolerance Test
3.9       Proximate Analyses of Elephant Grass Sample
3.9.1    Determination of Moisture Content
3.9.2    Determination of Ash Content
3.9.3    Determination of Crude Lipid Content
3.9.4    Determination of Crude Fibre Content
3.9.5    Determination of Nitrogen and Crude Protein Content
3.9.6    Determination of Soluble Carbohydrates
3.10   Determination of Lignocellulose Content
3.10.1Determination of Neutral Detergent Fibre (NDF)
3.10.2  Determination of Acid Detergent Fibre (ADF)
3.10.3  Determination of Acid Detergent lignin (ADL)
3.11   Fermentation of Elephant Grass
3.11.1Sample Preparation and Pretreatment
3.11.2Media Preparation for Fermentation
3.11.3  Inocula Preparation
3.12                 Analytical Procedure
3.12.1  Measurement of Cell Dry Weight
3.12.2  Determination of Reducing Sugar Concentration
3.12.3  Quantitative and Qualitative Determination of Ethanol Concentration
3.13                 Statistical Analyses

CHAPTER FOUR
4.0                   RESULTS
4.1                   Characterisation of Aspergillus niger Isolates
4.2                   Characterisation of Saccharomyces cerevesiae Isolates
4.3                   Screening of A. niger Isolates
4.3.1    Qualitative enzyme assay
4.3.2    Quantitative enzyme assay
4.4                   Screening of S. cerevisiae Isolates
4.5                   Proximate Analyses and Lignocellulose Content of Elephant Grass Sample
4.6                   Fermentation of Substrate for Ethanol Production
4.6.1    Effect of glucose concentrations on biomass yield
4.6.2    Effect of glucose concentrations on reducing sugar yield
4.6.3    Effect of glucose concentrations on ethanol yield
4.6.4    Effect of pretreated Elephant grass concentrations on biomass yield
4.6.5    Effect of pretreated Elephant grass concentrations on reducing sugar yield
4.6.6    Effect of pretreated Elephant grass concentrations on ethanol yield
4.7       Optimization of Culture Parameters for Ethanol Production from 6% Pretreated Substrate
4.7.1    Effect of pH on ethanol yield
4.7.2    Effect of temperature on ethanol yield
4.7.3    Effect of agitation rate on ethanol yield
4.7.4    Effect of length of time on ethanol yield

CHAPTER FIVE
5.0DISCUSSION

CHAPTER SIX
6.0 CONCLUSION AND RECOMMENDATIONS
REFERENCES
APPENDICES



ABSTRACT

Elephant grass (Pennisetum purpureum) was evaluated for its ethanol production potential using co-cultures of Aspergillus niger and Saccharomyces cerevisiaeisolated from local sources. Proximate and lignocellulose analysis carried out on the plant sample showed that it had crude fibre, lignin, hemicellulose and cellulose contents of 31.5%, 26.78%, 18.76% and 34.16% respectively. Aspergillus niger strains were isolated from soil and bread and were further screened for both qualitative and quantitative cellulase production. Qualitative cellulase assay revealed clear zones around colonies indicative of enzyme activity on solid agar medium containing 0.1% carboxymethyl cellulose (CMC) for all the isolates. Quantitative cellulase assay showed that A. niger isolate AN-15 from soil gave highest cellulase yield of (0.1792 IU/ml/min) and was therefore selected as a co-culture with

S. cerevisiae. Saccharomyces cerevisiae strains were isolated from palm wine and burukutu. Isolate PW-4 was selected for fermentation based on ethanol tolerance tests and assimilation of more sugars compared to other isolates. Fermentation of grass substrate was carried out at different concentrations ranging from 2-10% and highest ethanol yield of 1.68g/100ml was observed at an optimum substrate concentration of 6% though the yield was much less than that obtained from equal concentration of glucose (8.38g/100ml). Optimization of culture parameters for ethanol production showed maximum ethanol yield at pH 5, 35oC and agitation rate of 300 rpm. The results of the research also revealed that ethanol production by S. cerevisiae beyond the fourth day of fermentation is significantly reduced.




CHAPTER ONE

1.0                                                                     INTRODUCTION

Ethanol fuel, ethyl alcohol (CH3CH2OH), is the same type of alcohol found in alcoholic beverages. It is an oxygenated fuel with a high octane value like that of petroleum fuels known to run combustion engines at higher compression ratios and thus provides superior performance (Wheals et al., 1999). The blending of ethanol into petroleum-based automobile fuels can significantly decrease petroleum use and decrease the release of greenhouse gas emissions. Furthermore, ethanol can be a safer alternative to the common additive, methyl tertiary butyl ether (MTBE), in gasoline. Methyl tertiary butyl ether is toxic and is a known contaminant in ground water. Thus, ethanol can be a substitute to mitigate the problems associated with the rising energy demands across the world as well as a way to reduce greenhouse gas emission to as high as 85% (Perlack et al., 2005).

Ethanol may be produced either from petroleum products or from biomass substrate. Today, most of the ethanol produced comes from renewable resources (Bothast and Saha, 1997). Although, most of the ethanol currently produced from renewable resources come from sugarcane and starchy grains, significant efforts are being made to produce ethanol from lignocellulosic biomass (almost 50% of all biomass in the biosphere such as agricultural residues are lignocellulosic biomass). The technological advances in recent years are promising to produce ethanol at low cost from lignocellulosic biomass (Bothast and Saha, 1997).

Bioethanol production from sugarcane and starch-rich feed stocks such as corn, potato, is considered a first generation process because it has already been developed (Joshi et al., 2011). The long-term viability of this process is in question because it requires.....


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