Production Of Bioethanol From Pomelo (citrus Maxima) Peel (mandia) 2019.docx

PRODUCTION OF BIOETHANOL FROM POMELO (Citrus maxima) PEEL

A Research Presented to The Faculty of the Chemical Engineering Department College of Engineering, Eastern Visayas State University Tacloban City

In Partial Fulfillment of the Requirements for the course CHE 362 (Methods of Research II) Bachelor of Science in Chemical Engineering

by:

FIONA N. MANDIA

BSCHE-4A

2019

APPROVAL SHEET

In partial fulfillment of the requirements for the degree of Bachelor of Science in Chemical Engineering, this research entitled: “PRODUCTION OF BIOETHANOL FROM POMELO (Citrus maxima) PEEL” has been prepared and submitted by FIONA N. MANDIA. who is recommended for Oral Examination. ENGR. ELVIRA DOLORES M. URGEL Adviser

Approved by the Committee on Oral Examination with a rating of __________.

ENGR. EVELYN A. CARDOSO Chairperson ZENAIDA L. ANDRADE, Ph.D. Member

MARIA LINA A. DOLLETE, Ph.D. Member

ELVIRA DOLORES M. URGEL, Ph.D Member

PROF. RICHARD S. BRUN Member

Accepted in partial fulfillment of the requirements for the course, CHE 362 (Methods of Research II), Bachelor of Science in Chemical Engineering.

ENGR. EVELYN A. CARDOSO Instructor, CHE 362 (Methods of Research II) Eastern Visayas State University, Tacloban City

Date of Oral Examination February 22, 2019

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ACCEPTANCE SHEET

This research hereto attached entitled: “PRODUCTION OF BIOETHANOL FROM POMELO (Citrus maxima) PEEL” prepared and submitted by FIONA N. MANDIA. in partial fulfillment of the requirements for the course, CHE 362 (Methods of Research II), Bachelor of Science in Chemical Engineering, is hereby accepted.

ZENAIDA L. ANDRADE, Ph.D. Member, Oral Examination Committee

MARIA LINA A. DOLLETE, Ph.D. Member, Oral Examination Committee

PROF. RICHARD S. BRUN Member, Oral Examination Committee

ENGR. EVELYN A. CARDOSO Chairperson, Oral Examination Committee

Accepted in partial fulfillment for the requirements for the course, CHE 362 (Methods of Research II), Bachelor of Science in Chemical Engineering.

ENGR. EVELYN A. CARDOSO Instructor, CHE 362 (Methods of Research II)

MARIA LINA DOLLETE, Ph.D. Head, Chemical Engineering Department Eastern Visayas State University, Tacloban City

ANNABELLE B. PILAPIL, Ph.D. Dean, College of Engineering Eastern Visayas State University, Tacloban City

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ACKNOWLEDGEMENT First, the researcher would like to express her gratitude to the Almighty God for His guidance and love all the time, especially when hard situations occurred on this journey. The undying support of the researcher’s family is worthy to offer the success of this research and friends who uplift and cheer whenever problems come. The researcher also wants to give thanks to the mentors who played very important roles in school life; To Engr. Evelyn Cardoso, Engr. Nida B. Lacaba and Engr. Zenaida L. Andrade, and Engr. Elvira Dolores M. Urgel who helped and advised the researcher by sharing their knowledge and things they can offer. To Engr. Ma. Lina A. Dollete, who allowed me to conduct the experiments in the Chemistry Laboratory. To Mr. Pascualito M. Ilagan, MRD, who allowed me to perform the experiments and use the laboratory equipment in the Chemistry Laboratory. To Mrs. Fe T. Piedad, R.Ch., who shared her knowledge and expertise about the study. To Mr. Darwin C. Gomez, R.Ch., I would like to express my heartfelt gratitude for all the time, effort, sacrifice and for helping me all the way to make this study successful. The study would not be positive without your help for I feel so lost at times and do not know what to do anymore. Words are not enough to express how grateful I am for the encouragement throughout. To Blessel, Ada, Neil, Romeo, Jolie, Maura, Isip, Franz, Paul, and other 4 th year BSChemistry students, who helped me I would like to thank all of you, even for just a short time, I’m happy I made some friends there. To everyone who was part on creating the study, I recognized all of you. Thank You! – FIONA 4

Table of Contents TITLE PAGE

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APPROVAL SHEET

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ACCEPTANCE SHEET

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ACKNOWLEDGEMENT .

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TABLE OF CONTENTS

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LIST OF FIGURES, TABLES AND APPENDICES

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ABSTRACT .

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Background of the Study

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Objectives of the Study

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Null Hypothesis

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Conceptual Framework

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Significance of the Study

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Scope and Delimitation of the Study .

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Definition of Terms

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CHAPTER 1: INTRODUCTION

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CHAPTER II: RELATED LITERATURES AND STUDIES CHAPTER III: METHODOLOGY Research Design

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Research Subject and Sampling Procedure

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Research Instrument .

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Research Procedure

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Methods of Analysis .

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Statistical Treatment Tool

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CHAPTER IV: RESULTS AND DISCUSSION .

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CHAPTER V: CONCLUSION AND RECOMMENDATIONS .

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BIBLIOGRAPHY

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APPENDICES

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LIST OF TABLES, FIGURES AND APPENDICES Figures 1.1 Conceptual Framework of the Study

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3.1 Process flow of the experimental procedure of the study

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Tables 3.1 Chemical Composition of Raw Pomelo Peel

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3.2 Chemical Composition of Pomelo Albedo

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4.1 Summary of Experimental Conditions Using Hydrothermal Treatment 34 4.2 Summary of Experimental Conditions Using Hydrothermal Treatment 35 4.3 Results

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Appendices APPENDIX A: Raw Data

APPENDIX A2: Ethanol-Water Mixtures

APPENDIX B: Documentation on the Making of Rodenticide from Color .

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Odor .

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APPENDIX C: Documentation on Producing Ethanol from Pomelo Peels in a Laboratory Scale . . . . . . . . . . 56

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ABSTRACT

PRODUCTION OF BIOETHANOL FROM POMELO (Citrus Maxima) PEEL

FIONA N. MANDIA Eastern Visayas State University Tacloban City

ENGR. ELVIRA DOLORES M. URGEL Adviser

Energy is vital for life. Many billion people left cold and hungry without it. Due to global energy consumption and dependence on non-renewable energy, the cost of transport and environmental problems has increased. There was an urgent need for a clean, renewable and economical energy source. This study presents an alternative raw material for bioethanol production to address energy source problems. The pomelo peels were processed to reduce size, treat hydrothermal and then fermented with alkaline peroxide. Commercial ethanol and sucrose setup were designed to serve as the experiment's control variable. The fermentation process lasted two days and was then distilled, the distillate was tested using gravimetric analysis from different pretreatments. The distillate density from commercial ethanol, hydrothermal and alkaline-peroxide treatment was 0.9926, 8

0.9915 and 0.9963. The hydrothermal treatment yielded 11.7 percent and 4.92 percent in the treatment of alkaline-peroxide. The distillate pH from commercial treatment with ethanol, hydrothermal and alkaline peroxide was 3.39, 3.41 and 3.40. The results show that the density and pH of the distillates with commercial ethanol differs slightly. There is a qualitative difference between distillate and commercial ethanol. Ethanol was therefore obtained from pomelo peels for this study.

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CHAPTER I INTRODUCTION Background of the Study Energy is vital for life. Many billion people left cold and hungry without it. The main supply of energy comes from fossil fuels, which is why most industrialized and developing countries are currently using fossil fuels such as oil, coal and gas. Burning fossil fuels is considered the most cost-effective way to generate electricity, but it can lead to serious environmental problems such as air pollution and is a limited and non-renewable energy source. Due to global energy consumption and dependence on non-renewable energy, the cost of transport and environmental problems has increased. There was an urgent need for a clean, renewable and economical energy source. Ethanol was a promising renewable source, according to Farrell et al. (2006). Bioethanol has gained more attention as a replacement for fossil fuels as a clean and renewable fuel (P. Wei et al., 2014). It can be produced from different types of raw materials classified into three categories of agricultural raw materials: feedstocks containing sucrose, starch and lignocellulosic materialsLignocellulosic materials are used for the production of second-generation biofuel as a cheap and abundant feedstock compared to sugar and starch-based materials. (Balat, 2011). Lignocellulose mainly consists of cellulose, hemicellulose and lignin, which can be converted into fermentable sugars. These sugars are used by fermenting microorganisms to produce ethanol as a metabolism by-product (Garrote, 2002). A large amount of citrus waste is generated with grown citrus production and consumption, which accounts for approximately 50 percent of the fruit weight. The waste includes fruit peel, membrane residues and other by-products traditionally disposed of 10

through a burning process that leads to severe environmental pollution and large waste. It is highly desirable to develop green and cost-effective ways of dealing with citrus waste and using it to produce staple or added value products. Citrus waste consists mainly of soluble mono-and disaccharides (e.g. glucose, fructose or sucrose), insoluble polysaccharides (e.g. cellulose, pectin), and lignin. This chemical composition makes waste a potential candidate for biofuels and other chemicals based on sugar (Huang et al., 2014). The aim of this study is to determine the viability of pomelo peel as a potential raw material source for the production of bioethanol. The researcher decided to carry out a study on one of the citrus fruits that was thought to produce ethanol. It contains abundant waste of pectin-rich biomass and can be used as a fuel source. Citrus maxima, also known as Pomelo, is the said fruit peel chosen by the researcher as the object of the study. Raw pomelo peel consists of cellulose, hemicellulose, pectin, lignin and soluble sugars, making it an interesting choice for appropriate organisms to produce metabolites such as ethanol. However, an individual or combination of mechanical, chemical and biological treatments is required to break down cellulose, hemicellulose and pectin polymers in the cell walls of pomelo peels and convert them into monomers of sugars then fermented into ethanol.

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Objectives of the Study This study aims to utilize pomelo peels as an alternative raw material for the production of bioethanol; specifically the study seeks to answer the following: 1. Determine the percent yield of ethanol obtained. 2. Determine the quality of ethanol from pomelo peels. a. Color b. Odor c. Density d. pH 3. Determine the significant difference between the quality of ethanol from pomelo peels and commercial ethanol. a. Color b. Odor c. Density d. pH

Null Hypothesis Pomelo peel is not a sustainable bioethanol raw material. In particular, the quality of ethanol from pomelo peels and the quality of commercially produced ethanol do not differ statistically significantly.

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Conceptual Framework The Republic Act No. 9367, known as the “Biofuels Act of 2006” is an act to direct the use of biofuels to reduce dependence on imported fuels as an alternative to fossil fuels (Republic Act No. 9367, 2007). The Republic Act No. 9513 known as the “Renewable Energy Act of 2008” passed and strengthened R.A. 9367. It declared the State’s policy to achieve energy security by reducing reliance on fossil fuels and minimizing exposure to price fluctuations in oil markets (Sta. Lucia, 2015). The U.S. Grains Council delegation visited ethanol plants and talked with ethanol producers in the Philippines about the country’s plans to increase the national ethanolblending mandate to E20 by 2020. The movement “E20 by 2020” is an upward transition that will result in increased domestic ethanol production as well as the potential for additional imports (Council Shares U.S. Ethanol Experience In The Philippines, South Korea, 2018).

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P

Ethanol from Pomelo Peels

Commercial Ethanol

A R A

Color

Color

Odor

Odor

Density

Density

M

E T E R S

pH

pH

Figure 1.1 Conceptual Framework

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Significance of the Study Together with the pollution and waste generation abundant in the environment due to the disposal of cellulosic wastes (e.g. pomelo peels), recycling these cellulosic wastes for the production of ethanol would lead to a more efficient and eco-friendly substitute type of energy source. To the Researcher who conducted the study. It is to improve the proficiency and skills in the field of the researcher, which can be useful in the line of work. The researcher aims to help everyone to minimize the effects of pollution. To the Chemical Engineering Field. This research may provide additional information and data that can be used for the future generation that is relevant to the study and production of energy from sustainable sources. To the Fuel Industries. This study may be of help to them since fossil fuel is rapidly depleting, to produce ethanol to produce ethanol from a cheap source that can also minimize the polluting effects of the usual energy sources. To the Community. This research may give insights and lead everyone not only to attract them to use the product but also to challenge them in joining our cause to help the environment. They can also make their own bioethanol at home for their vehicles to reduce expenses. There are many people producing bioethanol at home for their own use and many of them opt for a homemade “still” for the production process (makebiofuel, 2018) To the Environment. The aim of this research is to minimize the emission of greenhouse gases since ethanol burns clearly compared to pure gasoline. Under current 15

conditions, use of ethanol-blended fuels as E85 (85% ethanol and 15% gasoline) can reduce the net emissions of greenhouse gases by as much as 37.1%. Ethanol-blended fuel such as E10 (10% ethanol and 90% gasoline) reduces greenhouse gases by up to 3.9%. The environmental implications of feedstock production associated with the production of ethanol for fuel are biological renewability, sustainable agriculture, and energy balance (Environmental Benefits of Ethanol, 2000). To the Future Researchers. The data and information from this study can be utilized for further researchers and future applications. Scope and Delimitation of the Study This study is limited on the viability of pomelo peel as a possible source for the production of bioethanol. The study is also limited in terms of determining the percent yield of ethanol obtained, the quality of ethanol from pomelo peels, and determining if there is a significant difference between the quality of ethanol from pomelo peels and commercial ethanol. The parameters checked for the produced ethanol are color, odor, density, and pH and these characteristics are compared with the commercially produced ethanol. It is limited on alkaline-peroxide and hydrothermal treatment as the process used in the study. The study is restricted in gravimetric analysis as the technique used to determine the density of ethanol in the distillate. The study is limited in the survey of five respondents for their opinion on color and odor of the distillate and commercial ethanol. Pretreatment conditions used in the study were limited to mechanical, hydrothermal, and alkaline-peroxide techniques.

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This study is limited on determining the viability of pomelo peel as a source of bioethanol by using Saccharomyces cerevisiae as a catalyst in the fermentation process. The study will utilize the available facilities located within the Eastern Visayas State University-Main Campus, Tacloban City. The pomelo peels will be taken from the Tacloban City. Definition of Terms The following terms are defined for the better understanding of the reader with the study at hand: Alkaline-peroxide treatment. This form of pretreatment utilizes alkaline solutions such as NaOH, KOH, NH4OH, or Ca(OH)2 (Taherzadeh and Karimi, 2008). Sodium hydroxide is the most commonly studied pretreatment alkali and is seen as an alternative to sulfuric acid (Silverstein et al., 2007; Kumar et al., 2009). This is one of the methods applied to produce ethanol in the study. Bioethanol. It is ethanol that is produced from agricultural products (such as sugar cane or corn) and that is used as a fuel supplement (Merriam-Webster, 2018). Color. It is the aspect of the appearance of objects and light sources that may be described in terms of hue, lightness, and saturation (Merriam-Webster, 2018). Distillation. The process of purifying a liquid by successive evaporation and condensation (Merriam-Webster, 2018). It is the process used in the study to separate ethanol from water.

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Ethanol. A colorless volatile flammable liquid C2H5OH that is the intoxicating agent in liquors and used as a solvent and in fuel (Merriam-Webster, 2018). Fermentation. An enzymatically controlled anaerobic breakdown of an energyrich compound (such as a carbohydrate to carbon dioxide and alcohol or to an organic acid) (Merriam-Webster, 2018). In this study, the process takes place in which the glucose is broken down to ethanol by the action of enzymes in the yeast used. Gravimetric analysis. It is the process of producing and weighing a compound or element in as pure form as possible after some form of chemical treatment has been carried out on the substance to be examined (Gravimetric Analysis, 2019). The method used to determine the specific gravity of the distillate. Hydrothermal treatment. It refers to a thermochemical process for decomposing carbonaceous materials such as coal and biomass with water in a high temperature and high pressure condition (Yuliansyah & Hirajima, 2012). This is one of the methods applied in the study to produce ethanol. Lignocellulosic Biomass. It is a renewable and abundant resource suitable for the production of bio-based materials such as biofuels and chemicals and is mainly composed of cellulose, hemicellulose and lignin (Dutra, et al., 2017). Odor. A characteristic or predominant quality (Merriam-Webster, 2018). Percent Yield. The percent ratio of actual yield to the theoretical yield (Geankoplis, 1993). In this study, it refers to the percent ratio of the mass of ethanol to the mass of the pomelo peels.

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Saccharification. It is the process of breaking a complex carbohydrate such as starch or cellulose into its monosaccharide components (Saccharification, 2019). In this study, this is one of the process that the experiment underwent. Saccharomyces cerevisiae. It is a type of budding yeast that is able to ferment sugar into carbon dioxide and alcohol and is commonly used in the baking and brewing industries (Encyclopaedia Brittanica, 2018). It is the microorganism used in the fermentation process of the study.

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CHAPTER II REVIEW OF RELATED LITERATURE This chapter presents the various literatures and studies reviewed by the researchers, which provide more understanding and substance to this study. Related Literature The production of bioethanol from lignocellulosic biomass mainly involves two processes: Hydrolysis of cellulose in lignocellulosic biomass to reduce sugar production and fermentation sugars into ethanol (Sun & Cheng, 2002). However, the sugars necessary for fermentation are trapped in the lignocellulose structure, so biomass pretreatment is always necessary to remove the surrounding lignin and hemicellulose matrix before the hydrolysis of polysaccharides (cellulose and hemicellulose) in the biomass. Pre-treatment methods can generally be classified into three categories, including physical, chemical and biological. After pretreatment, residues can be converted into ethanol by means of wellestablished fermentation technologies when hydrolyzed into their component sugars (Zheng, Pan, & Zhang, 2009). The sugar concentration is determined after the pretreatment and hydrolysis steps and the yield of ethanol after fermentation. Sugars are quantified using spectrophotometric methods or chromatographic techniques, while ethanol is determined by simple distillation followed by specific gravity analysis or liquid chromatography.

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Bioethanol It is one of the most promising and environmentally friendly alternatives to fossil fuels from renewable sources (Zabed, Sahu, Boyce, & Faruq, production of fuel ethanol from lignocellulosic biomass: An overview of feedstocks and technological approaches, 2016). Bioethanol is produced from biomass containing free fermentable sugars or complex carbohydrates, which can be converted to fermentable sugars. These feedstocks can be divided into three groups of sugars, starchy crops and lignocellulosic biomass (Zabed, Sahu, Boyce and Faruq, Production of fuel ethanol from lignocellulosic biomass: An overview of feedstocks and technological approaches, 2016). The fermentation of raw materials based on sugar is referred to as bioethanol of "first generation," while the use of raw materials from lignocellulose is commonly referred to as bioethanol of "second generation.". Algal bioethanol's "third generation" is at an early stage of investigation. The main reasons for the increased development of bioethanol are its use as a favorable and near-carbon-neutral renewable fuel, which reduces CO2 emissions and related climate change; its use as an octane enhancer in unleaded gasoline; and its use as an oxygenated fuel mix for cleaner gasoline combustion (Kang, Appels, Tan, & Dewil, 2014). Lignocellulosic Materials As a sustainable source of sugars and chemical platforms for conversion into renewable fuels, fine chemicals and materials, lignocellulosic biomass is of particular interest. It is also the only accessible non-fossil carbon source that can be processed into liquids that are easily incorporated into the existing fuel infrastructure for transport (Xu & Sun, 2016). It consists mainly of cellulose, hemicellulose and lignin, as well as small fractions and ash extraction. It requires a stage of pre-treatment to convert it into energy 21

products. The main purpose of pretreatment is to disrupt the recalcitrance of the lignocellulosic matrix and facilitate the separation of polysaccharides and lignin, which leads to increased accessibility to enzymatic hydrolysis (Dutra, et al., 2017). Hydrothermal Pretreatment Pretreatments that mainly use steam or liquid water at high temperatures can efficiently convert biomass into a form that enzymes can easily digest by facilitating autohydrolysis reactions in biomass. Hydrothermal pretreatment is known as hot water or steam processes as the primary chemical. Both forms of hydrothermal pre-treatment use steam (explosion of steam) and water (pre-treatmentof liquid hot water). The pretreatment of liquid hot water is the form of hydrothermal pretreatment used in the study. During the pre-treatment of liquid hot water, water is present as a liquid instead of gas (Waldron, 2010). Alkaline Hydrogen Peroxide Pretreatment It is an oxidative pretreatment process that acts in the delignment of lignocellulosic biomass, which allows for greater efficiency in the recovery of sugars in the liquid phase of enzyme hydrolysis, since the presence of lignin makes it difficult for enzymes to attack the substratum. This type of pretreatment is low-energy and does not produce inhibitors such as hydroxymethylfurfural and furfural (Dutra, et al., 2017). Hydroxides of sodium, potassium, calcium and ammonium are often used to treat alkaline reagents and can improve the efficiency of lignin removal. Sodium hydroxide (NaOH) pretreatment is one of the most common methods of bioconversion of lignocelluloses and has been extensively

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studied. Treatment with NaOH is very effective in increasing the digestibility of low lignin content hardwood and agricultural residues (Xu & Sun, 2016). Pomelo Peels Pomelo or Pummelo is a Philippine citrus fruit produced from Davao or popularly known as the "Philippine Fruit Basket." The greenish-yellow rind of the pomelo peel is thick and contains a thick layer of spongy pith. Instead of throwing the pomelo peel, use it for bioethanol production. Table 3.1 Chemical Composition of Raw Pomelo Peel Composition

Percentage

Cellulose

16.5%, based on dry matter

Hemicellulose

6.86%

Pectin

35.42%

Lignin

3.16%

Soluble sugars

12.62%

Ash

4.14%

Raw pomelo peel consists of cellulose (16.5 %), hemicellulose (6.86 %), pectin (35.42 %), lignin (3.16 %), soluble sugar (12.62 %), and ash (4.14 %) (Renliang Huang, 2014). Pomelo peel is one of the under-used waste materials that can produce bioethanol because it contains the above-mentioned bioethanol components and is a lignocellulosic material. In terms of energy security, environmental concerns, employment opportunities, agricultural development, foreign exchange savings, socio - economic issues, etc.,

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lignocellulosic feedstocks do not interfere with food security and are necessary for each rural and specific area (Zafar, Biofuels from Lignocellulosic Biomass, 2018). Table 3.2 Chemical composition of Pomelo Albedo Composition (% w/w, dry basis) Carbohydrate

72.62 ± 0.42

Moisture

16.13 ± 0.16

Protein

6.27 ± 0.23

Ash

3.41 ± 0.05

Fat

1.56 ± 0.07

Cellulose

21.29 ± 1.90

Carbohydrate (72 %) consists mainly of dried pomelo albedo This value was significantly higher than the content of carbohydrates found in lemon pulp (70 %) (Brillouet JM, 1988) and lemon albedo (59 %) (Ros JM, 1996). Pomelo albedo also found moisture and protein content at 16.13 percent and 6.27 percent. These values were obviously higher than those recorded in albedo lemon, orange peel and orange pulp (Sariçoban C, 2008) (Nassar AG, 2008). Small amounts of ash (3.41 %) and fat (1.56 %) were observed in the meantime. This is in accordance with Martin et al. (Marin FR, 2007), which described the amount of ash in the citrus peel ranging from 2.56 to 8.09 percent, while the amount of fat is 1.51 to 4.00 percent. Meanwhile, the content of cellulose in pomelo albedo was 21.29 percent significantly higher than 14.4 percent in orange peel (Bicu I, 2011). Pomelo albedo may be a better source of cellulose than other types of citrus fruit peel (Nor Fazelin Mat Zain, 24

preparation and characterization of cellulose and nanocellulose from Pomelo (Citrus Grandis) Albedo, 2014). Related Studies The following studies were conducted by the researchers – local and abroad, and somehow have bearing to the present studies: Abiertas (2018) conducted a study using carabao grass (Paspalum conjugatum). Its result revealed that the condition used in the study failed to produce significant amount of ethanol. It was determined that the conditions used were not suitable to produce ethanol by using a flammability test (Abiertas, 2018). In a study conducted by (Gatela Jr., 2018), peanut shell waste was used as a raw material to produce ethanol. The result revealed that most of the characteristics of the produced ethanol from peanut shells were quite similar to the commercial. Only the purity, odor, and color of the ethanol produced had a significant difference from the commercial. Huang (2014) utilized pomelo peel waste by integrated hydrothermal treatment, multi-enzyme formulation, and fed-batch operation. The results show that hydrothermal treatment (120 °C, 15 min) could significantly reduce the use of cellulase (from 7 to 3.8 FPU g−1) and pectinase (from 20 to 10 U g−1). A multienzyme complex, which consists of cellulase, pectinase, β-glucosidase, and xylanase, was also proven to be effective to improve the hydrolysis of pretreated pomelo peel, leading to higher concentrations of fermentative sugars (36 vs 14 g L−1) and galacturonic acid (23 vs 9 g L−1) than those with the use of a single enzyme. The results indicate

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that the use of the fed-batch mode could alleviate the decrease in ethanol yield at high solid loading, which is caused by significant mass transfer limitation and increased inhibition of toxic compounds in the SSF process (Huang, et al., 2014).

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CHAPTER III METHODOLOGY This chapter presents the discussion on the research methodology of the study, the research design, research subject and sampling procedure, research procedure, research instrument, and statistical analysis used for accurate data analysis and interpretation. Research Design This study used an experimental design, which followed an adopted method from various sources. Cellulosic ethanol was produced from pomelo peels. Two pretreatment methods were used to ferment the sample and the distillate recovered from each method was compared with the commercial ethanol. Three major processes will be conducted and specifically centered on the conversion of the cellulose substrate (pomelo peels) into fermentable sugar, converting the sugar to alcohol, and distillation of alcohol solution to yield a high percentage of ethanol. In the first part of the process, the pomelo is peeled and separated from its fruit. The pomelo peels will be reduced to fine sizes and be introduced to the processes of hydrothermal treatment and alkaline-peroxide treatment then autoclaved to obtain sugar (hexose compounds) from the pomelo peels. A filter will be utilized to separate the aqueous solution with the solid residue. The filtrate is the main ingredient in the microbial fermentation process in producing ethanol. Yeast is the microorganism used for the fermentation. The fermented solution will undergo distillation to acquire ethanol. The ethanol taken is introduced to tests and analysis afterwards.

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Pomelo Peel Size Reduction 10 g w/v solid Hydrothermal treatment Filtrate

Residue

Fermentati on

Alkaline-peroxide treatment

Distillation Filtrate

Residue

Ethanol Fermentati on Distillation Ethanol

Tests and Analysis

Figure 3.1 Process of the experimental procedure followed in the study

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The study will be conducted in the Chemistry Laboratory located on the third floor of the Science Building (SB) of Eastern Visayas State University, Tacloban City. The pomelo peels will be taken from Tacloban City, Leyte. Research Subject and Sampling Procedure Pomelo peels and yeast are the primary materials in making bioethanol. They will undergo pretreatment, hydrolysis, filtration, fermentation, and distillation to obtain ethanol. The bioethanol will then undergo tests and analysis to determine the quality of ethanol in terms of its color, odor, density, and pH level. The quality of ethanol taken from pomelo peels has been compared with the commercially produced ethanol to see the difference between them. Research Instruments This refers to the materials or equipment used in the course of the study. The main raw material in the study are pomelo peels. In the size reduction of pomelo peels, a knife and scissors will be used. Furthermore, several materials and apparatuses such as plastic bowls, beakers, reagent bottles, and Erlenmeyer flasks will be used for the pre-treatment and fermentation. In the ethanol production and tests, several raw ingredients such as hydrogen peroxide, sodium hydroxide, yeast and distilled water will be used. Moreover, an autoclave equipment for reducing the bacteria to minimal and an equipment composed of a container for the liquid, a heat source, and a condenser will be needed for the distillation in the course of the experiment.

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A thermometer, pH meter, burette and small Erlenmeyer flasks were used to test the temperature, pH content, and specific gravity of the materials respectively.

Research Procedure 1. Selection and preparation of Pomelo Peels Pomelo is bought in Tacloban City, Leyte and selected based on the greenish color as a suitable raw material for its ripeness. The fruit is separated with its peels using a knife and cut to pieces manually with a scissor. The small pieces of peels will now be the raw material for the experiment. 2. Size reduction, Pretreatment, and Filtration The chopped samples were oven-dried for 24 hours at 60˚C and pulverized using a blender. Pulverized samples were sieved into a beaker and weighed using a top loading balance. Four samples of 10 g pulverized pomelo peels were prepared. A buffer 2 solution was prepared dissolving 1 f of urea and 1 g of potassium dihydrogen orthophosphate in a 1 liter distilled water. A 100 mL of buffer 2 solution was added to the 10 g fine powder of pomelo peel in the beaker. The mixture is autoclaved at 121˚C, 15 psi for 1 hour and further cooled to room temperature. The hydrolysate was filtered with a cheesecloth to separate the juice and residue. The juice obtained from hydrothermal treatment was set aside in an Erlenmeyer flask to be fermented. 2 g NaOH dissolved in a 400 mL buffer 2 solution and mixed with a 13.3 mL H2O2 was poured to the residue collected. The mixture is subjected to alkaline-peroxide pretreatment and autoclaved at 121˚C, 15 psi for 1 hour. It was cooled

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to room temperature, filtered with a cheesecloth until the aqueous solutions are the only components remaining in the Erlenmeyer flask. The pH was maintained at 4.55. 3. Fermentation Process and Distillation Fermentation is the next step after filtering the hydrolyzed raw materials. Two fermentation setups were made: the juice collected from hydrothermal treatment and alkaline-peroxide treatment. The first setup was the juice obtained from hydrothermal treatment, it was added with 150 mL buffer 2 and 2.5 g yeast to be fermented. The second setup was the liquid obtained from the alkaline-peroxide treatment, it was added with 60 mL buffer 2 and 2.5 g yeast. Each setup consists of an Erlenmeyer flask where the aqueous solutions are contained and sealed with a plastic wrap and pierced with a needle then covered with a balloon. The balloon act as a pathway for the carbon dioxide product that will be given off from the chemical reaction. The two setups were kept in an incubator at a temperature of 33˚C. The optimum temperature range for yeast fermentation is between 32˚C to 35˚C (Temperature is key to fermentation success, 2013). The fermentation lasted for two days. After two days, ethanol in each fermentation product will be distilled using simple distillation method. A 250 mL of fermentation product in each setup will be placed in a distillation flask to begin the process. A 50 mL distillate will be collected in a volumetric flask in each setup as a receiver in the distillation process.

31

4. Tests and Checking Gravimetric analysis was used to determine the density of the distillate. It is concerned with the process of producing and weighing a compound or element in as pure form as possible after some form of chemical treatment has been carried out on the substance to be examined (Gravimetric Analysis, 2019). Methods of Analysis After distillation, the distillate from different pretreatments were put into different tests and analysis. A gravimetric analysis was performed in both pretreatments to determine the density of the distillate. A burette, small Erlenmeyer flasks, and analytical balance were used to perform the method. No contamination should be present as much as possible because it can affect the results. All the equipments used and distillates obtained were stabilized at 20˚C. The burette was rinsed off with a small amount of the distillate then the remaining were poured into the equipment. An analytical balance was used to get the initial mass of the small Erlenmeyer flasks. The initial and final volume of the distillate was noted on the burette. One Erlenmeyer flask is used as a receiver for the distillate from the burette and the final mass was noted. The change in mass and volume were calculated for each trial. The density was determined by the change in mass over the change in volume. The testing of the odor and color of the ethanol produced from the study is surveyed. Five respondents are approached to rate the odor and color of the ethanol solutions. The categories for odor in the survey are 1 – favorable, 2 – slightly favorable, 3 – can’t decide if favorable or unpleasant, 4 – unpleasant, and 5 – very unpleasant. The categories of the

32

color are 1 – colorless, 2 – slightly blurred white, 3 – slightly blurred yellow, 4 – light yellow, and 5 – yellow. Statistical Treatment Tool Mean was used in quantitative measurements for determining the average results from the data gathered in the experiments. For the comparative analysis of the odor and color between the distillates of two pretreatments and commercial ethanol, Analysis of Variance (ANOVA) of Two-Factor Without Replication was used.

33

CHAPTER IV RESULTS AND DISCUSSION This chapter tackles about the different laboratory data, reports and results which were obtained during the experiment. The gathered information throughout the course of the experiment were tabulated and recorded. Such data were relevant and pertains to the statement of the problem. Cellulosic Ethanol from Pomelo Peels Table 4.1 I and 4.2 presents the summary of experimental conditions used to produce ethanol from pomelo peels. Findings revealed that the conditions used in the study succeeded to produce significant amount of ethanol that can be detected by gravimetric analysis. This implies that the conditions used were suitable to produce ethanol. Table 4.1 Summary of Experimental Conditions Using Hydrothermal Treatment Conditions

Experiment

Pretreatment: size reduction

Powder

Pretreatment: hydrothermal

121˚C autoclaved for 1 hr

Fermentation: ph; time

4.5; 2 days

Ethanol content: gravimetric analysis

Detectable

34

Table 4.2 Summary of Experimental Conditions Using Alkaline-Peroxide Treatment Conditions

Experiment

Pretreatment: size reduction

Powder

Pretreatment: alkaline-peroxide

121˚C autoclaved for 1 hour

Fermentation: pH; time

4.5; 2 days

Ethanol content: gravimetric analysis

Detectable

Pretreatment conditions used in the study were limited to mechanical, hydrothermal, and alkaline-peroxide techniques. The pomelo peel samples were reduced in size to increase the surface area of the raw materials. It has been reasoned that higher surface area result in increased sugar yield due to more efficient sugar hydrolysis (Koullas et al., 1992). One of the most promising and environmentally friendly biomass pretreatment methods available is hydrothermal treatment since it makes the lignocellulosic biomass vulnerable to enzymatic breakdown (Cybulska, Brudecki, & Lei, 2013). Hydrothermal pretreatment greatly increases surface area of cellulose (by nonchemical swelling), which significantly enhances possible enzyme access (Chang et al. 1981; Sun and Cheng 2002).

Characterization of Distillate Gravimetric analysis was used to determine the density of the distillate. The analysis revealed that the ethanol content can be determined gravimetrically. Downstream analysis of distillate was performed in terms of its color, odor, density, and pH.

35

Table 4.3 Results Treatment

Densitya Percent Yieldb

pH

Color

Odor

Hydrothermal

0.9915

11.7

3.41

Colorless

Alkaline-Peroxide

0.9963

4.92

3.40

Slightly blurred white

Commercial Ethanol (5% v/v)

0.9926

-

3.39

Slightly blurred white

Can’t decide if favorable or unpleasant Can’t decide if favorable or unpleasant Favorable

a b

determined at 20 °C calculated as mass ethanol (g)/gram dry weight of pomelo peels x 100

Based on the table, the density of the distillate in a hydrothermal and alkaline peroxide treatment were 0.9915 g/mL and 0.9963 g/mL respectively. There is only a small difference between them and compared with commercial ethanol (5% v/v) density of 0.9907 g/mL. The percent yield in a hydrothermal and alkaline-peroxide treatment is 11.7% and 4.92%. It can be inferred in the data that the percent yield of a hydrothermal treatment is higher than the alkaline-peroxide treatment because it hydrolyzed the fermentable sugars in the sample. The hydrothermal treatment was the first method applied in the sample and the residue collected was used for alkaline-peroxide treatment. The remaining soluble sugars in the residue were hydrolyzed in the alkaline-peroxide treatment. The pH of the distillate from a hydrothermal and alkaline-peroxide treatment were 3.41 and 3.40, commercial ethanol has a pH of 3.39. There is only a 0.01 difference between the distillates and commercial ethanol. A survey was conducted with five respondents for the color and odor of the distillate. It can be inferred from the mean data that hydrothermal treatment is colorless, 36

alkaline pretreatment is slightly blurred white and commercial ethanol is slightly blurred white. In the odor of the distillate and commercial ethanol, the respondents cannot decide if it is favorable or unpleasant. In the mean data of the respondents, the odor of commercial ethanol is favorable.

37

CHAPTER V CONCLUSIONS AND RECOMMENDATIONS This chapter presents the conclusions from the light of the study and a number of recommendations to improve the study in different ways. CONCLUSION The main goal of the study was to analyze the potential of pomelo peels to produce ethanol. The density and pH of the produced ethanol and ethanol distillate from hydrothermal and alkaline treatment was almost similar. The color and odor of the ethanol produced from pomelo peels have a significant difference from the commercial. The following are the specific conclusions made from the study. 1. The percent yield of hydrothermal treatment is 11.7% and 4.92% in alkalineperoxide treatment. 2. The color of the product in a hydrothermal treatment is colorless and slightly blurred white in the alkaline peroxide treatment and commercial ethanol. 3. The respondents cannot decide if the odor of the product in a hydrothermal and alkaline-peroxide treatment are favorable or unpleasant. The odor of the commercial ethanol is favorable. 4. The densities have a uniform value for the first two decimal places. 5. The pH of the distillates and commercial ethanol were measured using a pH meter. There is a difference of 0.01 pH between the distillates and commercial ethanol. 6. There was a significant difference between the ethanol from hydrothermal treatment and alkaline pretreatment with the commercial ethanol in terms of its

38

color, odor, density, and pH. Therefore, it has been concluded that pomelo peel can be a substitute raw material for commercial ethanol. RECOMMENDATIONS The specific recommendations with regards to the study are listed below: 1. For future researchers, it is recommended to try enzymes in the hydrolysis of the biomass. Enzymatic hydrolysis process converts pre-treated substrate to monomeric sugars, which could be further converted to ethanol (Enzyme Hydrolysis, 2019). 2. It is recommended to try different solid loadings in an experiment. High solid loadings offers many advantages over lower-solid loadings, including sugar and ethanol concentration and decreased production and capital costs (AA & SE, 2012). 3. The researcher suggests to follow the procedure in the study but utilize the remaining residue from alkaline peroxide with enzymatic hydrolysis to get the soluble sugars the past two pretreatments failed to separate from cellulose, hemicellulose, and lignin.

39

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APPENDICES

47

APPENDIX A RAW DATA

Table 1 ETHANOL CONCENTRATION IN SUCROSE FERMENTATION Replicate No.

Mass of Solution (g)

Volume of Solution (mL)* 1 14.7879 14.97 2 14.7480 14.94 3 14.9037 15.10 *determined at 20.0 ± 1.0 °C

Specific Gravity* 0.98784 0.98715 0.98700

Mean Specific Gravity*

Ethanol Concentration

0.98733

9.296 g/L

Table 2 WATER Replicate Mass of Volume of Solution No. Solution (g) (mL)* 1 14.4327 14.47 2 14.9585 14.97 3 15.0952 15.08 *determined at 20.0 ± 1.0 °C

Specific Gravity* 0.99742 0.99923 1.00100

Mean Specific Gravity* 0.99922

Table 3 DISTILLED WATER Replicate Mass of Volume of Solution No. Solution (g) (mL)* 1 15.05419 15.07 2 14.6451 14.64 3 15.0116 15.02 *determined at 20.0 ± 1.0 °C

48

Specific Gravity* 0.99895 1.00034 0.99944

Mean Specific Gravity* 0.99958

Table 4 ETHANOL CONCENTRATION IN ALCOHOL STANDARD (5%) Replicate No.

Mass of Solution (g)

Volume of Solution (mL)* 1 14.969 15.09 2 14.8911 15.02 3 15.0212 15.12 *determined at 20.0 ± 1.0 °C

Specific Gravity* 0.99198 0.99142 0.99347

Mean Specific Gravity*

Ethanol Concentration

0.99229

4.124 g/L

Table 5 ETHANOL CONCENTRATION IN POMELO PEEL FERMENTATION USING ALKALINE-PEROXIDE PRETREATMENT Replicate No.

Mass of Solution (g)

Volume of Solution (mL)* 1 13.9161 13.95 2 14.0006 14.03 3 13.9402 14.03 *determined at 20.0 ± 1.0 °C

Specific Gravity* 0.99757 0.99790 0.99359

Mean Specific Gravity*

Ethanol Concentration

0.99636

2.006 g/L

Table 6 ETHANOL CONCENTRATION IN POMELO PEEL FERMENTATION USING HYDROTHERMAL PRETREATMENT Replicate No.

Mass of Solution (g)

Volume of Solution (mL)* 1 15.0455 15.14 2 14.8719 14.98 3 14.8332 15.01 *determined at 20.0 ± 1.0 °C

Specific Gravity* 0.99376 0.99278 0.98822

49

Mean Specific Gravity*

Ethanol Concentration

0.99158

4.788 g/L

ETHANOL ODOR SURVEY Respondent 1

Hydrothermal treatment Odor rating 3

Alkaline-peroxide treatment Odor rating 2

2

5

4

3

2

4

4

4

3

5

2

4

Sum

16

17

Mean average

3.2

3.4 16.5

Overall average

COMMERCIAL ETHANOL ODOR SURVEY Commercial Ethanol Respondent Odor rating 1

1

2

1

3

1

4

1

5

1

Sum

5

Mean average

1

50

ETHANOL COLOR SURVEY Respondent

Hydrothermal treatment

Alkaline peroxide treatment

Color rating

Color rating

1

1

1

2

1

1

3

1

2

4

1

3

5

1

1

Sum

5

8

Mean average

1

1.6

Overall average

1.3

51

COMMERCIAL ETHANOL COLOR SURVEY Respondent

Commercial Ethanol Color rating

1

2

2

2

3

2

4

2

5

2

Sum

10

Mean average

2

52

APPENDIX A2 ETHANOL-WATER MIXTURES Density 20˚C (g/cm3)

% by weight ethanol

% by volume ethanol

Density 20˚C (g/cm3)

% by weight ethanol

% by volume ethanol

1,00000*

0

0

0,91097

52

59,8

0,99813

1

1,3

0,90872

53

60,8

0,99629

2

2,5

0,90645

54

61,8

0,99451

3

3,8

0,90418

55

62,8

0,99279

4

5,0

0,90191

56

63,8

0,99113

5

6,2

0,89962

57

64,8

0,98955

6

7,5

0,89733

58

65,8

0,98802

7

8,7

0,89502

59

66,8

0,98653

8

10,0

0,89271

60

67,7

0,98505

9

11,2

0,89040

61

68,6

0,98361

10

12,4

0,88807

62

69,6

0,98221

11

13,6

0,88574

63

70,5

0,98084

12

14,8

0,88339

64

71,5

0,97948

13

16,1

0,88104

65

72,4

0,97816

14

17,3

0,87869

66

73,3

0,97687

15

18,5

0,87632

67

74,2

0,97560

16

19,7

0,87396

68

75,1

0,97431

17

20,9

0,87158

69

76,0

53

0,97301

18

22,1

0,86920

70

76,9

0,97169

19

23,3

0,86680

71

77,8

APPENDIX B COLOR

Anova: Two-Factor Without Replication SUMMARY

Count 1 2 3 4 5

A B C

3 3 3 3 3

Sum 4 4 5 6 4

Average 1.333333 1.333333 1.666667 2 1.333333

Variance 0.333333 0.333333 0.333333 1 0.333333

5 5 5

5 8 10

1 1.6 2

0 0.8 0

ANOVA Source of Variation Rows Columns Error

SS 1.066667 2.533333 2.133333

df MS 4 0.266667 2 1.266667 8 0.266667

Total

5.733333

14

54

F

P-value F crit 1 0.460905 3.837853 4.75 0.043672 4.45897

ODOR

Anova: Two-Factor Without Replication SUMMARY 1 2 3 4 5 A B C

Count Sum Average Variance 3 6 2 1 3 10 3.33333 4.33333 3 7 2.33333 2.33333 3 8 2.66667 2.33333 3 7 2.33333 2.33333 5 5 5

16 17 5

3.2 3.4 1

ANOVA Source of Variation Rows Columns Error

SS 3.06667 17.7333 6.93333

df MS 4 0.76667 2 8.86667 8 0.86667

Total

27.7333

14

55

1.7 0.8 0

F P-value F crit 0.88462 0.51455 3.83785 10.2308 0.00624 4.45897

APPENDIX C Documentation on Producing Ethanol from Pomelo Peels in a Laboratory Scale

Drying of pomelo peels in the oven at 60 ˚C for 24 hours

The oven dried pomelo peels were pulverized using a blender

Samples that went through a hydrothermal treatment (autoclaved at 121˚C, 1 hr)

A buffer 2 solution was prepared as a nutrient for the yeast

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The residue (left) and filtrate (right) obtained from hydrothermal treatment

Samples that went through an alkaline-peroxide treatment (autoclaved at 121˚C, 1 hr)

The residue (left) and filtrate (right) obtained from alkaline-peroxide treatment

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Distillation setup

The fermentation setup of two pretreatments Hydrothermal treatment (left) Alkaline-peroxide treatment (right)

Samples Autoclaved at 121˚ for 1 hour

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Survey conducted for the odor and color of each product

Fermentation setup in the incubator Hydrothermal treatment (left) Alkaline-peroxide treatment (right)

The distillates used for survey A- hydrothermal treatment B- alkaline-peroxide treatment C- commercial ethanol

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