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

Thai Nguyen University of Agriculture and Forestry Degree Program Bachelor of Environmental Science and Management Thesis Title ―Bioethanol production from Sugarcane Bagasse by Separate

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 Bachelor of Environmental Science and Management

Thesis Title ―Bioethanol production from Sugarcane Bagasse by Separate

Hydrolysis and Fermentation using YEAST ISOLATED FROM DURIAN ((Durio zhibetinus) FRUIT‖

Keywords: Bioethanol production; Biofuel; Feedstock; Lignocellulosic

biomass; Pretreatment; Hydrolysis; Fermentation

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

TABLE OF CONTENT

List of Figure……… vii

List of Tables……….…viii

List of Abbreviations……… ………ix

PART I INTRODUCTION 1

1.1 Research rationale 1

1.2 Research‘s objectives 2

1.3 Research questions and hypotheses 3

1.4 Limitations 3

1.5 Definitions 3

PART II LITERATURE REVIEW 5

2.1 Energy guide 5

2.2 Renewable energy or alternative energy 7

2.3 Bioethanol 8

2.3.1 First generation 8

2.3.2 Second generation 9

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

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

LIST OF FIGURES

Figure 1 Classification of biofuels 9

Figure 2 Framework of Ethanol Fermentation Process……… 17

Figure 3 Preparation of sample 21

Figure 4 A laboratory being used for the fermentation culture medium… 23

Figure 5 Preparation of inoculum growth cell 24

Figure 6 Diagram of ethanol standard curve 27

Figure 7 Comparison ethanol analysis with different hydrolysis time 30

Figure 8 Standard curve (reducing glucose) 31

Figure 9 The curve of reducing sugar in three different hydrolysis time… 32

LIST OF TABLES

Table 1 The difference hydrolysis time 22 Table 2 Absorbance of glucose analysis by DNS 25 Table 3 The correlation between the concentration of glucose at various levels and the value Absorbance derived from glucose analysis by DNS 33 Table 4 Analysis of ethanol on different incubation time 32

SHF – Separate Hydrolysis and Fermentation

SSF- Simultaneous Saccharification and Fermentation

YPD or YEPD- Yeast extract peptone dextrose

PART I INTRODUCTION

1.1 Research rationale

Lao PDR is an agricultural based country and it is the most important economic sector

of Lao PDR There are a lot of wastes generated every year from agriculture and forestry production, municipal waste or organic industrial Laos has high potential of energy crops, which can be used as feedstock for biofuel production and energy used

In the country is mainly in form of traditional fuels

Fossil fuels play important roles in energy sectors and global economy (Ullah K, 2015) and basic energy used by humans comes from fossil fuels Fossil fuels not only dominate in field of energy but also involved in accumulation of greenhouse gases in atmosphere resulting in global warming due to which such fuels become widely organized as unsustainable (Bai F, 2014) Day by day the voice is raised universally against global warming due to change in the weather patterns, world‘s sea level increase enveloping lowland, deltas and islands and a tendency for global temperature increase Biologically produced fuels are looked upon with much interest and identified as potential alternative energy source (Parmar A, 2011) Renewable energy

or alternative energy is a term used for an energy source that is an alternative to using fossil fuels These alternatives are intended to address concerns about such fossil fuels has high carbon dioxide emissions, an important factor in global warming Fossil fuels are non-renewable They are limited in supply and will one day be depleted (Zehner, 2012)

One of the most renewable energy is from bioethanol because bioethanol can be produced from agricultural feed stocks It can be made from very common crops such

as hemp, sugarcane, potato, cassava and corn There has been considerable debate about how useful bioethanol is in replacing gasoline Concerns about its production and used relate to increase for food prices due to the large amount of arable land required for crops (The madness of biofuel, 2011), as well as the energy and pollution balance of the whole cycle of ethanol production

The main objective of this thesis is to successfully produce bioethanol from sugarcane bagasse Sugarcane bagasse is an agricultural waste which was obtained from SRIWIJAYA laboratory in Palambang, Indonesia The experiment used to produce bioethanol by using separate hydrolysis and can be made from glucose solution from fermentation process To get the result of ethanol concentration from sugarcane bagasse, the standardization of ethanol was used to determine the intensity of ethanol from each samples The amount of ethanol was showed in peak area

1.2 Research’s objectives

The objective of this research project is to:

1 Find out and describe ethanol production

2 Find out the use of ethanol production from waste products that are lignocellulosic

3 To determine the optimum conditions of fermentation process for the production of bioethanol from lignocellulosic biomass

4 Producing (experiment) of ethanol from lignocellulosic biomass

5 Compering the economics of the production and yield of ethanol using separate hydrolysis and fermentation

1.3 Research questions and hypotheses

Bioethanol has a number of advantages over conventional fuels It comes from renewable source or energy crops To produce ethanol by not impact food consumption and feedstock the product can be development into the used of waste to produce ethanol fuel There is ongoing research to answer the research questions

a Whether ethanol 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?

c How much energy goes into the ethanol production process and how much come out?

1.4 Limitations

The limitation of the production of ethanol from cellulosic include:

1 Pretreatment process to reduce lignin is needed

2 To apply all sugar content needs microbial agent which can ferment not only glucose but also xylose and arabinose

3 The need to find or genetically engineer organisms to efficiently ferment these sugars

4 Costs of collection and storage of low density biomass feedstock

1.5 Definitions

Bioethanol production process has three steps which are pretreatment, hydrolysis and fermentation process The scope of this study was to determine the yield of bioethanol

can be produced glucose from sugarcane bagasse in fermentation process The 150 ml

fermentation will conducted to investigate the effect of temperature and fermentation

time The optimum ethanol production of glucose 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 timed.

PART II LITERATURE REVIEW 2.1 Energy guide

The need to meet the ever-increasing demand for energy is probably the greatest challenge that society has to grapple with in this new millennium Virtually every aspect of life on planet Earth (heating, transportation, etc.) requires energy input in one form or another Population growth has always been and will remain one of the key drivers of energy demand, along with economic and social development Growing population consume more energy, placed on arable land, the increase in the consumption of crude oil, cost of energy fuel are rising (Bartlett, 1994)

Energy resources are the estimated maximum capacity for energy production given all available resources on Earth They can be divided by type into fossil fuel, nuclear fuel and renewable resources However, it has been recognised that global crude oil reserves are finite, and their depletion is occurring much faster than previously predicted (Grant, 2005; Möller, 2006; Bai et al., 2008) In addition, shortterm price volatility has heightened apprehension about the future of global energy security (Hahn-Hägerdal et al., 2006) In 2008, before the global economic recession began, crude oil sold for over USD135 per barrel in the market However, conventional petroleum is essentially non-renewable and intertwined with this practical impediment

is an apparent moral dilemma of environmental pollution arising from its very usage (Wackett, 2008) The combustion of these hydrocarbons makes significant contributions to greenhouse gases (GHG) in the atmosphere and inevitably contributes significantly to global warming (Wigley, 2005) The transport sector alone accounts for 60% of global oil consumption (International Energy Agency (IEA), 2008), 19%

of carbon dioxide and 70% of carbon monoxide emissions (Goldemberg, 2008) With the world human population projected by the United Nations to hit 9 billion and the number of cars 2 billion (World Business Council for Sustainable Development (WBCSD), 2004) by 2050, it is no longer sustainable to continue to combust fossil fuel without regard for the environment Consequently, the need for environmentally sustainable and renewable energy sources cannot be overemphasized, given the rapid rate of global industrial development (Zaldivar et al., 2001; Gray et al., 2006)

Bioethanol can be used in various blends with gasoline, such as 5% bioethanol (Demirbas, 2008), 10% & 20% (E10 & E20) (Gray et al., 2006), 22% (Wyman, 1994)

or even 85% (E85) (Balat et al., 2008) and because of its favourable physicochemical properties, ethanol is considered an excellent alternative transportation fuel to gasoline that can considerably improve the quality of the atmosphere (Philippidis, 1993) A major boost for the biofuels industry has come from automakers, GM, Chrysler and Ford, who have stated that half of all the cars they produce worldwide in 2010 will be

‗flex-fuel‘, or E85 (85% bioethanol)- compatible (Waltz, 2007) Other benefits come from using bioethanol as biofuel: it is totally biodegradable and sulphur free, and the products from its incomplete oxidation (acetic acid and acetaldehyde) are less toxic in comparison to other alcohols (Minteer, 2006)

2.2 Renewable energy or alternative energy

Renewable or alternative energy is a term used for an energy source that is an alternative to using fossil fuels These alternatives are intended to address concerns about such fossil fuels, such as its high carbon dioxide emissions, an important factor

in global warming Marine energy, hydroelectric, wind, geothermal and solar power generally it indicates energies that are non-traditional and have low environmental impact (zehner, 2012) The term alternative is used to contrast with fossil fuels according to some sources By most definitions alternative energy doesn't harm the environment, a distinction which separates it from renewable energy which may or may not have significant environmental impact Bioethanol is a form of renewable energy that can be produced from agricultural feed stocks It can be made from very common crops such as hemp, sugarcane, potato, cassava and corn There has been considerable debate about how useful bioethanol is in replacing gasoline (Oilcrash, 2011) Concerns about its production and use relate to increased food prices due to the large amount of arable land required for crops, as well as the energy and pollution balance of the whole cycle of ethanol production (Youngquist, 2012) Lignocellulosic ethanol offers promise because cellulose fibers, a major and universal component in plant cells walls, can be used to produce ethanol Lignocellulosic ethanol could allow ethanol fuels to play a much bigger role in the future (Champagne, 2007)

2.3 Bioethanol

Concerns about shortage of fossil fuels, increasing crude oil price, energy security and accelerated global warming have led to growing worldwide interests in renewable energy sources such as biofuels An increasing number of developed and rapidly developing nations see biofuels as a key to reducing reliance on foreign oil, lowering emissions of greenhouse gases (GHG), mainly carbon dioxide ( ) and methane ( ), and meeting rural development goals Biofuels are referred to solid, liquid or gaseous fuels derived from organic matter (Koh LP, 2008) They are generally divided into primary and secondary biofuels (Figure2.1) While primary biofuels such as fuel wood are used in an unprocessed form primarily for heating, cooking or electricity production, secondary biofuels such as bioethanol and biodiesel are produced by processing biomass and are able to be used in vehicles and various industrial processes The secondary biofuels can be categorized into three generations: first, second and third generation biofuels on the basis of different parameters, such as the type of processing technology, type of feedstock or their level of development (Nigam

PS, 2010)

2.3.1 First generation

There has been an un-ending debate over bioethanol produced from food crops and future food security Although at the moment the bioethanol produced by a nation is dependent on the prevalent feedstock (for example, sugarcane for Brazil, corn for USA and cassava for Nigeria), it is increasingly understood that 1st-generation bioethanol produced primarily from food crops is limited in its ability to achieve

targets for oil-product substitution, climate change mitigation and economic growth (Sims et al., 2009) The sustainable production of these fuels is still currently under review, as is the possibility of creating undue competition for land and water used for food and fiber production A possible exception that appears to meet many of the acceptable criteria is ethanol produced from sugar cane (Sims et al., 2009) and cassava These concerns have accelerated interest in developing bioethanol produced from non-food biomass

First-generation biofuels are transport fuels produced using conventional technologies from feedstock like wheat, corn, sugar, palm oil and sunflower oil, largely agricultural products that are also used as feed and food Food crops with energy crops also result

in an increased in food price, increased stress on arable land currently used for food production can lead to severe food storages (report, 2007)

2.3.2 Second generation

Second generation biofuels, also known as advanced biofuels, are fuels that can be manufactured from various types of biomass Biomass is a wide-ranging term meaning any source of organic carbon that is renewed rapidly as part of the carbon cycle Biomass is derived from plant materials but can also include animal materials Second generation biofuels describes a wide range of fuel pathways that offer one or more advantages over first generation biofuels The distinguishing characteristics of second generation biofuels are: they use a non-food feedstock (so-called lignocellulosic biomass, such as field crops residues, forest products residues, or fast-growing dedicated energy crops), sources have high content, which include wood and

organic waste and the fuel is a ―drop-in‖ replacement for conventional based fuels, meaning there are no limits on blending, or they can be used as is (without blending) in existing vehicles (Evans, 2008)

petroleum-2.3.3 Third generation

Ethanol from plants that were modified for easier processing (e.g poplar with lower lignin content), biodiesel from algae and other microorganisms are the third generation biofuel (Oliver R Inderwildi, 2009) Algae are considered as the only alternate to food crops for renewable fuel production as they contain energy rich lipids and carbohydrates (Peterson, 2008) Algae are the fastest growing plant in the world and generally divides into macro algae and microalgae based upon morphology and do not use arable land

Figure 1 Classification of biofuels

(Source:https://www.researchgate.net/figure/230238121_fig2_figure-1-Classification-of-biofuels-adapted-from-Nigam-and-Singh1)

Second generation biofuel technologies have been developed because first generation biofuels manufacture has important limitations (Evans, 2008) First generation biofuel processes are useful but limited in most cases: there is a threshold above which they cannot produce enough biofuel without threatening food supplies and biodiversity (Ramirez, Brown, & Rainey, 2015) Many first generation biofuels depend on subsidies and are not cost competitive with existing fossil fuels such as oil, and some

of them produce only limited greenhouse gas emissions savings When taking emissions from production and transport into account, life-cycle assessment from first

Biofuels

Primary Secondary

1 𝑠𝑡 generation Bioethanol or butanol by fermentation of starch (from wheat, barley, corn, potato) or sugars

(from sugarcane, sugar beet, etc.)

Biodiesel by transesterification of oil crops (rapeseed, soybeans, sunflower, palm, coconut, used cooking oil, animal fats, etc.)

2 𝑛𝑑 generation Bioethanol and biodiesel produced from

conventional technologies but based on novel starch, oil and sugar crops such

as Jatropha, cassava or Miscanthus;

Bioethanol, biobutanol, syndiesel produced from lignocellulosic materials (e.g straw,bagasse, wood, and grass)

3 𝑟𝑑 generation Biodiesel from microalgae Bioethanol from microalgae and seaweeds Hydrogen from green

microalgae and microbes

generation biofuels frequently approach those of traditional fossil fuels (Ademe, 2015) Second generation biofuels can help solve these problems and can supply a larger proportion of global fuel supply sustainably, affordably, and with greater environmental benefits

2.4 Lignocellulosic biomass

Lignocellulosic material constitutes the world‘s largest bioethanol renewable resource Aside from being an environmentally friendly process, agricultural residues help to avoid reliance on forest woody biomass and thus reduce deforestation (non-sustainable cutting plants) Unlike trees, crop residues are characterized by a short-harvest rotation that renders them more consistently available to bioethanol production (Perlack, 2005) Lignocellulose refers to plant dry matter (biomass), so called lignocellulosic biomass It is the most abundantly available raw material on the Earth for the production of biofuels, mainly bio-ethanol It is composed of carbohydrate polymers (cellulose, hemicellulose), and an aromatic polymer (lignin) (Knauf M, Lignocellulosic biomass processing, 2004)

Lignocellulosic material can generally be divided into three main components: cellulose (25-45%), hemicellulose (28-32%) and lignin (15-25%) of sugarcane bagasse (Edye, 2008) Cellulose and hemicelluloses make up approximately 70% of the entire biomass and are tightly linked to the lignin component through covalent and hydro genic bonds that make the structure highly robust and resistant to any treatment (Pettersen, 1984)

2.4.1 Hemicellulose

Hemicellulose, also known as polyose, is a matrix of polysaccharides, such as arabinoxylans, that exist along with cellulose in almost all the plant cell walls It is a polysaccharide that is present in the biomass of most plant, about 20%-30% dry weight of plants (Saha, 2003) Hemicellulose, combined with cellulose, provides physical and structural strength to the cell wall In addition to glucose, the other structural components in hemicelluloses are xylose, galactose, mannose, rhamnose, and arabinose Hemicellulose has shorter chains of 500 and 3000 sugar units with a branched structure (Abey, 2016)

2.4.2 Cellulose

Cellulose is an organic polysaccharide molecule with the molecular formula It has a linear chain of several hundred to thousands of D-glucose units Cellulose is a natural polymeric compound found in many natural materials; for instance, it is the structural component of the primary cell wall in green plants It can be also found in many forms of algae species (Klemm, Heublein, Fink,

& Bohn, 2005) Cellulose is the commonest organic polymer on Earth Many natural compounds are rich in cellulose; for example, the cellulose content of wood, cotton fiber, and dried hemp are 40–50%, 90%, and 57% respectively (A, energy source 2005)

2.4.3 Lignin

Lignin is a very complex molecule constructed of phenyl propane units linked in a three-dimensional structure which is particularly difficult to biodegrade (Gruyter, 2012) Lignin is the most recalcitrant component of the plant cell wall, and the higher the proportion of lignin, the higher the resistance to chemical and enzymatic degradation Generally, softwoods contain more lignin than hardwoods and most of the agriculture residues There are chemical bonds between lignin and hemicellulose and even cellulose Lignin is one of the drawbacks of using lignocellulosic materials

in fermentation, as it makes lignocellulose resistant to chemical and biological degradation

2.5 Processing of lignocellulosic to bioethanol

The bioconversion of cellulose and hemicellulose to monomeric sugars for example carbohydrates with 5 and 6 carbons is harder to accomplish than the conversion of starch, presently used for bioethanol production (Bengtsson, 2006) One of the advantages of bioconversion with lignocellulosics is the opportunity to create a bio refinery, producing value-added co-products plus fuel bioethanol For instance, sugars may be subjected to bacterial fermentation under aerobic and anaerobic conditions, producing a variety of other products including lactic acid, which in turn may be processed into plastics and other products The noncarbohydrate components of lignin also have potential for use in value-added applications (Jensen, 2006) Processing of lignocellulosics to bioethanol consists of four major unit operations: pre-treatment, hydrolysis, fermentation and product separation/distillation

2.5.1 Pretreatment

The first step in bioconversion of lignocellosics to bioethanol is size reduction and pre-treatment (Graf, 2000) The goal of any pre-treatment technology is to alter or remove structural and compositional impediments to hydrolysis in order to improve the rate of enzyme hydrolysis and increase yields of fermentable sugars from cellulose

or hemicellulose (Mosier, 2005) Pre-treatment is an important tool for practical cellulose conversion processes Pre-treatment is required to alter the structure of cellulosic biomass to make more accessible to the enzymes that convert the carbohydrate polymers into fermentable sugars and to cellulose producing microorganisms (Patel, 2007) A successful pre-treatment must meet the following requirements (Silverstein, Comparison of chemical pretreatment methods, 2014): (i) improve formation of sugars or the ability to subsequently form sugars by hydrolysis, (ii) avoid degradation or loss of carbohydrate, (iii) avoid formation of by product inhibitory to subsequent hydrolysis and fermentation processes, (iv) separate raw materials and pretreatment by physical method (autoclave) and (v) be cost effective

Pre-treatment can be carried out in different ways such as mechanical pre-treatment (Rivers DB, 1987), steam explosion (Brownell HH, 1987), ammonia fiber explosion (Alizadeh H, 2005) (Teymouri F, 2004), supercritical treatment (Kim KH, 2001), alkali or acid pre-treatment (Silverstein RA, 2007), ozone pre-treatment (Indacoechea

I, 2006), and biological pre-treatment (Patel SJ, 2007)

A number of processes for hydrolyzing cellulose into glucose have been developed over the years The vast majority of processing schemes utilizes either cellulolytic enzymes or sulfuric acid of varying concentrations Historically, enzymes have been too expensive for economical production of fuel ethanol from biomass Sulfuric acid, itself, is less expensive than cellulolytic enzymes, although disposal costs associated with the use of sulfuric acid significantly increase its cost However, the single largest drawback to using sulfuric acid is that it also readily degrades glucose at the high temperatures required for cellulose hydrolysis (Mosier, 2002)

2.5.3 Fermentation

Lignocellulose is often hydrolyzed by acid treatment, the hydrolysate obtained is then used for bioethanol fermentation by microorganisms such as yeast Because such lignocellulose hydrolysate contains not only glucose, but also various monosaccharides, such as xylose, mannose, galactose, arabinose, and

oligosaccharides, microorganisms should be required to efficiently ferment these sugars for the successful industrial production of bioethanol (Katahira, 2006)

2.5.4 Separate hydrolysis and fermentation

Separate hydrolysis and fermentation (SHF) is a technology in which the fermentation

of glucose is decoupled from the enzymatic hydrolysis of the biopolymer starch or cellulose The enzymatic hydrolysis step is often in close collaboration with the following fermentation step in the ethanol production The layout of this process can

be designed in several ways, either by having separate hydrolysis and fermentation step (separate hydrolysis and fermentation, SHF) or by combining these two in one step (simultaneous saccharification and fermentation, SSF) Each process having its own pros and cons (Zacchi, 2002) This thesis will be focus on SHF, the process concept SHF involves a separation of the hydrolysis and fermentation by running the reactions in separate units Pretreated lignocellulosic material is in a first unit regarded

to monomeric sugars by cellulases and fermented to ethanol in a second, separate unit

The main advantage of this method is that the two processes (hydrolysis and fermentation) can be performed at their own individually optimal conditions This process is possibility to run the fermentation process in a continuous mode with cell recycling and this is possible because lignin residue removal can occur before fermentation (Zacchi, 2002) There is also have disadvantage with SHF is the risk of contamination Due to the relatively long residence time for the hydrolysis process, zero to eight days, there is risk of microbial contamination of sugar solution is not optimum