Enhancement of Cellulosic Ethanol Production through Aspergillums Modification
ABSTRACT
Simultaneous Saccharification and Fermentation (SSF) were carried out to produce ethanol from maize stalk in 500ml conical flask. Aspergillus niger strains were isolated from four difference sources, maize stalk, soil, bambaranut, rotten wood.
Cellulose degrading ability was screened by zone of clearance carried out by simple agar method.. Apergillus strain from rotten wood (ANRW) produced the largest zone of clearance of 6.5mm; hence it was selected for further studies.
The effect of pH, temperature, substrate particle size, and substrate concentration were studies and optimized to be 5.0, 3o C, 300um, 3% respectively.Aspergillus niger was modified using UV irradiation technique by varying the exposure timings.
The strain expose at 30 minutes gave largest zone of clearance and hence was selected. The ethanol yield by simultaneous saccharification and fermentation of modified and unmodified strain of A.
TABLE OF CONTENTS
TITLE PAGES
TITLE PAGE- – – – – – – – – – i
DECLARATION- – – – – – – – – – ii
CERTIFICATION- – – – – – – – – – iii
DEDICATION- – – – – – – – – – iv
ACKNOWLEDGMENT- – – – – – – – – v
ABSTRACT- – – – – – – – – – – vi
TABLE OF CONTENTS- – – – – – – – – vii
CHAPTER ONE: Introduction – – – – – – 1
1.0 INTRODUCTION- – – – – – – – – 1
1.1 RESEARCH PROBLEM- – – – – – – – 3
1.2 AIM- – – – – – – – – – – 3
1.3 RESEARCH OBJECTIVES- – – – – – – 3
1.4 JUSTIFICATION- – – – – – – – – 4
1.5 SCOPE- – – – – – – – – – 4
1.6 LIMITATION – – – – – – – – – 4
CHAPTER TWO: Literature Review – – – – — 5
2.0 Literature Review.- – – – – – – — – 5
2.1 Ethanol – — – — – – – – – – 5
2.1.1 Uses of ethanol – — – – – – – – – 6
2.1.2 Ethanol as disinfectants – – – – – – – – 6
2.1.3 Ethanol as domestic lighting agent – – – – – – 6
2.1.4 Ethanol as transportation fuel- – – – – – – 7
2.1.5 Other uses of ethanol- – – – – – – – – 8
2.1.6 Performance of ethanol in Engines- – – – – – – 8
2.2 Chemistry of bioethanol – – – – – – – – 9
2.2.1 Properties of ethanol- – – – – – – – 10
2.2.2 Bioethanol production process – – – – – – – 10
2.2.3 Process description of simultaneaous saccharification
and fermentation (SSF).- – – – — – – 11
2.2.4 Fermentation- – — – – – – – – — 12
2.2.5 Distillation – – – – – – – – – — 13
2.2.6 Dehydration – — – – – – – – – – 13
2.3 Cellulose- – – – – – – – – – 14
2.3.1 Chemistry of Cellulose- – – – – – – – 15
2.3.2 Cellulases- – – – – – – – – 16
2.2.3 Cellulose conversion – – – – – – – – 16
2.4 Genus Aspergillus- – – – – – – – – 17
2.4.1 Aspergillus niger group – – – – – – – – 18
2.4.2 Saccharomyces cerevisiae- – – – – – – – 18
2.4.3 Macroscopic morphology of yeasts- – – – – – 20
2.4.4 Enzyme production by fungi – – – — – – 20
2.4.5 Cellulolytic enzymes from Fungi – – – – – – – 20
2.4.6 Kinetics of Ideal Enzyme Reactor Systems – – — – – 21
2.5 Biodegradation – – – – – – – – – 22
2.5.1 Bioconversion of agricultural residues (waste) to glucose – – – 23
2.5.2 Pretreatment of lignocelluloses materials- – – – – – 24
2.5.3 Factors affecting enzymatic hydrolysis of cellulose- – – – – 25
2.6.0 Optimization of culture condition- – – – – – – 26
2.6.1 Enzyme related factors affecting hydrolysis – — – — – 26
2.6.2 Effect of substrate particle size – – – – – — — 27
2.6.3 Optimal substrate concentration.- – – – – – – 28
2.6.4 Optimal fermentation pH – — – – – – 29
2.6.5 Optimal fermentation temperature.- – – – – – – 30
2.6.6 Substrates – – – – – – – – – – 32
2.6.7 Substrate inhibition – – – – – – – – – 32
2.6.8 Product inhibition- — – – – – – – 33
2.7 Strain improvement – – – – – – – – – 33
2.7.1 Mutation – – – – – – – – – – 34
2.7.2 Gene cloning – — – – – – – – – 35
2.7.3 Protoplast fusion – – – – – – – – – 35
2.7.4 Transformation – – – – — – – – 36
CHAPTER THREE: Materials and Methods — — 37
3.0 Materials and methods. – – – – – – – 37
3.1 List of Equipment and Materials- – – – – – — 37
3.2.0 Sample collection- – – – – – – – 39
3.2.1 Sample preparation- – – – – – – – – 39
3.3.0 Media and reagent preparation- – – – – – – 39
3.3.1 Potato dextrose agar preparation- – – – – — 39
3.3.2 Preparation of reagents- – – — – – – – 40
3.4.0 Enrichment for mould growth- – – – – – 40
3.4.1 Isolation of Aspergillus niger – – – – – – – 40
3.4.2 Screening for cellulose degrading Aspergillus niger– – – – 41
3.4.3 Preparation of inoculums- — – – – – — – 41
3.4.3.1 Aspergillus niger inoculum – – – – – – – – 42
3.4.3.2 Saccharomyces cerevisiae inoculums- – – – – – 42
- 4.4.3 Substrate preparation – — – – – – – – 42
3.5.0 Experimental set – up and fermentation procedure- – – – – 43
3.5.1 Mutagenic treatment of isolated Aspergillus niger- – – – – 43
3.5.2 Fungal mycelia dry weight- – – – – – – 44
3.5.3 Determination of ethanol concentration. – – – – – – 44
3.5.4 Preparation of ethanol standard curve- – – — – – 46
3.5.5 Preparation of DNS reagent – – – – – – – 46
3.5.6 Determination of residual sugar in the fermentation medium.- – – 46
3.5.7 Calibration for the quanlitative determination of glucose concentration – 47
3.6.0 Optimization of the culture condition – – – – – – 48
3.6.1 Optimization of substrate concentration – – – – – – 48
3.6.2 Optimization of pH- – – – – – – – – 48
3.6.3 Optimization of temperature- – – – – – — – 48
CHAPTER FOUR: Results and Discussions – 49
4.0 Results and Discussions – – – – – – 49
4.1 Macroscopic observation- – – – – – – — 49
4.2 Microscopic observation- – – – – – — 49
4.3 Screening for cellulose degrading performance of Aspergillus niger- – 55
4.4 Effect of Substrate particle size on ethanol yield – – — – – 59
4.5 Effect of substrate particle size on reducing sugar – – – – – 59
4.6. Effect of substrate concentration on ethanol yield- – – – – 73
4.7 Effect of substrate concentration on reducing sugar- — – – – 74
4.8 Effect of substrate concentration on cell dry weight.- – – – 74
4.9. Effect of pH on ethanol yield – – – – – – – 75
4.10 The effect of pH on reducing sugar- – – – – – – 75
4.11 The effect of pH on cell dry weight- – – – – – – 76
4.12 Effect of temperature on ethanol yield – – – – – – 76
4.13 Effect of temperature on reducing sugar- – – – – – 77
4.14 Effect of temperature on cell dry wiegth – – – – -. 77
4.15 The effect of UV on ethanol yield- – – – – – – 77
4.16 The effect of UV on reducing sugar – – – – – – 78
4.17. The effect of UV on cell dry weight- – – – – – — 78
CHAPTER FIVE : Conclusions and Recommendations – 80
5.1 CONCLUSIONS – – – – – – – – – 80
5.2 RECOMMENDATIONS- – – – – – – – 81
REFERENCES- – – – – – – – – – 82
APPENDICES – – – –
INTRODUCTION
In view of continuously rising petroleum cost and dependence upon fossil fuel resources, considerable attention has been focused on alternative energy resources.
Production of ethanol or ethyl alcohol [CH3CH2CH2OH] from biomass is one way to reduce both the cost of consumption of crude oil and environmental pollution. Ethanol represents an important, renewable liquid fuel for motor vehicles (Lewis, 1996).
The use of bioethanol as an alternative motor fuel has been steadily increasing around the world for a number of reasons. Domestic production and use of ethanol for fuel can decrease dependence on foreign oil, reduce trade deficits, create jobs in rural area, reduce air pollution, and reduce global climate change due to carbon dioxide buildup.
Ethanol unlike gasoline is an oxygenated fuel that contains 35% oxygen, which reduces particulate and NO2 emission from combustion. When burned, ethanol derived from fermentation produces no net increase in carbon dioxide in the atmosphere.
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