Bioethanol production from sugarcane bagasse by separate hydrolysis and fermentation using YEAST ISOLATED FROM DURIAN ((durio zhibetinus) FRUIT

THAI NGUYEN UNIVERSITY UNIVERSITY OF AGRICULTURE AND FORESTRY SRIWIJAYA UNIVERSITY SANGVONE SOULIYA BACHELOR THESIS Study Mode : Full-time Major : Environmental Science and Management Faculty : International Programs Office Batch : K45 AEP (2013-2017) Thai Nguyen,22/10/ 2017 Thai Nguyen University of Agriculture and Forestry Degree Program Student Name Student ID Thesis Title Supervisor (s) Bachelor of Environmental Science and Management Sangvone Souliya DTN1454290077 ―Bioethanol production from Sugarcane Bagasse by Separate Hydrolysis and Fermentation using YEAST ISOLATED FROM DURIAN ((Durio zhibetinus) FRUIT‖ Hermansyah,Ph.D Nguyen Huu Tho,Ph.D Supervisor‘s signature (s) Abstract: Sugarcane bagasse is the most abundant agriculture waste in the world It is an attractive feedstock for large-scale biological production of bioethanol Therefore, production of ethanol (bioethanol) from lignocellulosic biomass is one way to reduce environment pollution and crude oil from fossil Bioethanol can produce biofuel by fuel in gasoline engine mixed with ethanol Bioethanol production from sugarcane bagasse can produce with three main steps by pretreatment, hydrolysis and fermentation In this study Pretreatment or delignification conducted physical method using steam autoclave for hour to remove lignin Followed by hydrolysis in addition of 5% in each samples to dilute or concentration acid hydrolysis Fermentation used Na-OH to measure ethanol concentration was obtained pH 4.5 The results showed that the highest of ethanol production was 0.55% or 0.041ml during 30 minutes hydrolysis timed and fermentation process by shaking incubator was days Keywords: Bioethanol production; Biofuel; Feedstock; Lignocellulosic biomass; Pretreatment; Hydrolysis; Fermentation ii Number of pages Date of Submission 44 October, 2017 iii ACKNOWLEDGMENT I gratefully acknowledge the support and guidance of faculty members of Chemistry Department of Mathematic and Natural Science Faculty in Sriwijaya University, Indonesia Most particularly I would like to express my deep thanks to my supervisors Hermansyah,Ph.D of Chemistry Department of Mathematic and Natural Science Faculty in Sriwijaya University, Indonesia and Nguyen Huu Tho,Ph.D of Department of Science Management and International Relation, Thai Nguyen University of Agriculture and Forestry (TUAF), Vietnam who so kindly participated in this research by giving their generously of their time Without their thoughtful encouragement and careful supervision This thesis would never have taken shape I am also deeply thankful to Mr Deddy and Prof Aldes from research laboratory and all of my friends from Indonesia for giving me very kindly and helpful during doing the experiment Finally, I would like to express my deepest thanks from my heart to my family for their very supportive in every way and all good friends that beside me for along iv TABLE OF CONTENT List of Figure………………………………………………………………… vii List of Tables……………………………………………………………….…viii List of Abbreviations………………………………………………… ………ix PART I INTRODUCTION 1.1 Research rationale 1.2 Research‘s objectives 1.3 Research questions and hypotheses 1.4 Limitations 1.5 Definitions PART II LITERATURE REVIEW 2.1 Energy guide 2.2 Renewable energy or alternative energy 2.3 Bioethanol 2.3.1 First generation 2.3.2 Second generation 2.3.3 Third generation 10 2.4 Lignocellulosic biomass 12 2.4.1 Hemicellulose 13 2.4.2 Cellulose 13 2.4.3 Lignin 14 2.5 Processing of lignocellulosic to ethanol 14 2.5.1 Pretreatment 15 2.5.2 Hydrolysis 16 2.5.3 Fermentation 16 2.5.4 Separate hydrolysis and fermentation 17 PART III METHODOLOGY 18 v 3.1 Materials 19 3.2 List of apparatus 20 3.3 List of reagents 21 3.4 Methods 23 3.4.1 Processing of lignocellulosics to bioethanol 23 3.4.2 Pretreatment 24 3.4.3 Hydrolysis 24 3.4.4 Fermentation 25 3.4.4.1 Preparation of fermentation medium 25 3.4.4.2 Preparation of YPD agar medium 25 3.4.4.3 Preparation of YPD broth 26 3.4.4.4 pH 27 PART IV RESULT AND DISCUSSION 30 4.1 Ethanol standard 30 4.2 Ethanol analysis 31 4.3 Ethanol analysis curve 33 PART V CONCLUSION 37 REFERENCES 38 APPENDICES 44 vi LIST OF FIGURES Figure Classification of biofuels Figure Framework of Ethanol Fermentation Process…………………… 17 Figure Preparation of sample 21 Figure A laboratory being used for the fermentation culture medium… 23 Figure Preparation of inoculum growth cell 24 Figure Diagram of ethanol standard curve 27 Figure Comparison ethanol analysis with different hydrolysis time 30 Figure Standard curve (reducing glucose) 31 Figure The curve of reducing sugar in three different hydrolysis time… 32 vii LIST OF TABLES Table The difference hydrolysis time 22 Table Absorbance of glucose analysis by DNS 25 Table The correlation between the concentration of glucose at various levels and the value Absorbance derived from glucose analysis by DNS 33 Table Analysis of ethanol on different incubation time 32 viii LIST OF ABBREVIATIONS C - carbon - carbon dioxide DNS - Di-Nitro Salicylic Acid g - gram g/L - gram per liter - methane H – hydrogen O - Oxygen Mg- Magnesium - minutes mL - milliliter mg - milligram Na – sodium OH - Hydroxide % - Percentage ix °C - degree celsius μL – microliter n – a letter that represents an unknown number - water EtOH – ethanol – Sulfate radical – ammonium GHG- Greenhouse gases SHF – Separate Hydrolysis and Fermentation SSF- Simultaneous Saccharification and Fermentation YPD or YEPD- Yeast extract peptone dextrose x CHAPTURE IV RESULTS AND DISCUSSION 4.1 Ethanol standard The standard was prepared at different concentrations such as 1%, 2%,3%,5% and 10% of ethanol The equation of ethanol standard was determined to be y=1E+06x ( = 0.9047) where y is the sample area of ethanol and x is % EtOH to calculate the percentage of ethanol the formula will be change to X=Y/1E Figure Diagram of ethanol standard curve 30 4.2 Ethanol analysis To know the optimum yield of ethanol in research used gas chromatography to separate ―volatile‖ compounds (those with a high-vapor pressure or a relatively low boiling point) so that they may be detected individually in complex mixtures The term chromatography applies to the separation of chemical constituents in a sample so they can be either detected or utilized individually Analysis of the factorial experiment for different incubation time during day until days The table showed significance for a quadratic response The significant factors be the two main effects with percentage of ethanol and volume 31 Table Analysis of ethanol on different incubation time Sample Hydrolysis Incubation Area %EtOH Volume Time(minutes) time (Day) E1 30 8423 0.008423 0.000632 E2 45 6108 0.006108 0.000458 E3 60 9373 0.009373 0.000703 A1 30 35257 0.035257 0.002644 A2 45 254527 0.254527 0.01909 A3 60 144100 0.1441 0.010808 B1 30 176784 0.176784 0.013259 B2 45 385668 0.385668 0.028925 B3 60 99155 0.099155 0.007437 C1 30 214897 0.214897 0.016117 C2 45 163025 0.163025 0.012227 C3 60 83864 0.083864 0.00629 D1 30 557612 0.557612 0.041821 D2 45 373135 0.373135 0.027985 D3 60 86584 0.086584 0.006494 (ml) 32 4.3 Ethanol analysis curve The result of experiment showed the different and comparison curves in different hydrolysis time The experiment resulted in higher analysis was showed on the blue curve during days for 30 minutes hydrolysis time Ethanol production during fermentation 0-8 days in from substrate resulted from various of hydrolysis time Figure Comparison ethanol analysis with different hydrolysis time 33 In this experiment, a standard curve for analysis of glucose will be constructed In order to this, a number of solutions of known concentration (standards) will be used in the reaction and the absorbance of each will be measured Figure Standard curve (Reducing glucose) 34 Standard curve for reducing sugar were reaction by graph below Glucose graph commonly start by high concentration to low concentration but in this study the result was opposite it means that the result was not optimum As we can see the graph start from low to high concentration Glucose concentration during fermentation process of sugarcane bagasse Glucose was initially resulted from hydrolysis of sugarcane bagasse using 5% sulfuric acid and auto clave at 121 Figure The curve of reducing sugar in three different hydrolysis times 35 Bioethanol contest was still low since not all glucose contest and other fermentable sugars in sugarcane bagasse such as xylose and arabinose was converted into ethanol In term of time during fermentation was too short It was effective parameter in pretreatment of lignocelluloses and their effects on improvement ethanol Furthermore, experimental condition in pretreatment, hydrolysis and fermentation should be optimized to obtain higher ethanol content 36 CHAPTURE V CONCLUSION An efficient dilution sulfuric acid is great importance in the SHF process and the focus in the thesis was therefore to optimize this step with respect to conversion to glucose Dilution and auto clave hydrolysis the temperature showed significant effects on the glucose concentration, while pH had no significant effect on the glucose concentration in the tested interval of pH 4.5 To increase the final glucose concentration since the maximum outcome the optimum concentration of sulfuric acid of the hydrolysis process was in 30 minutes and from by shaking incubation time of fermentation process it was on days until days The optimum yield of ethanol was 0.55% or 0.041ml on days in 30 minutes hydrolysis time SHF as a process alternative in an industrial bioethanol plant has both potential and limitations The main advantage is the possibility to separately optimize the process steps, especially to be able to run the enzymatic hydrolysis at an optimal temperature Although, even if the enzymatic hydrolysis is the key process in SHF, it is important to include all the process steps in the optimization work An enzymatic hydrolysis 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Sugarcane Bagasse by Separate Hydrolysis and Fermentation using YEAST ISOLATED FROM DURIAN ((Durio zhibetinus) FRUIT Hermansyah,Ph.D Nguyen Huu Tho,Ph.D Supervisor‘s signature (s) Abstract: Sugarcane. .. can be produced from sugarcane bagasse as raw material and yeast isolated from durian fruit as biological agent? b How much bioethanol can be produced using Separately hydrolysis fermentation? ... from sugarcane bagasse using yeast isolated from durian fruit was aimed Others parameter such as pH and the results will be compared the optimum ethanol from different hydrolysis and fermentation