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SIMULTANEOUS SACCHARIFICATION AND FERMENTATION OF CELLULOSIC WASTE FOR BIOETHANOL PRODUCTION USING Saccharomyces cerevisiae ATCC 24859 Lee Wai Kin (36647) TP Bachelor of Science with Honours 358 (Resource Biotechnology) 1A82 2015 2015

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SIMULTANEOUS SACCHARIFICATION AND FERMENTATION OF CELLULOSIC WASTE FOR BIOETHANOL PRODUCTION

USING Saccharomyces cerevisiae ATCC 24859

Lee Wai Kin (36647)

TP Bachelor of Science with Honours 358 (Resource Biotechnology)1A82 2015 2015

P I'\at Khirtmat MakJumat Akadem i! U ·r s LAYS, 4. SARAW~ I

Simultaneous Saccharification and Fermentation of Cellulosic Waste for Bioethanol Production Using Saccharomyces cerevisiae ATCC 24859

Lee Wai Kin (36647)

A thesis submitted in partial fulfillment of requirement for the degree of Bachelor of Science with Honours (Resource Biotechnology)

.'

Supervisor: Dr. Micky Vincent

Program of Resource Biotechnology Department of Molecular Biology

Faculty of Resource Science and Technology University Malaysia Sarawak

2015

ACKNOWLEDGEMENT

First and foremost, I would like express my gratitude to God for His blessings and

guidance towards the completion of this project. Secondly, I would like to specially thank

the supervisor for this research, Dr. Micky Vincent who have given me this opportunity to

be involved in this interesting project as well as for all the professional and constructive

advices in order to improvise the experimental works. I would also like to thank every

post-graduate student in laboratory, especially Mr. Ennry Esut and Miss Norizawati who

have effectively monitored and assisted in the conduct of my experiment.

I would also like to show my greatest appreciation for my parent and family members for

their financial and moral support as well as their belief in my vision for science. In

• addition, the accomplishment of this project is also facilitated by the accompaniment and

knowledge-sharing with my other laboratory counterparts and course mates. Last but not

least, I would like to reach my most sincere tlianks to all individuals who have helped me

either directly or indirectly in accomplishing this project.

..

II

DECLARATION

I hereby declare that no portion of work referred in this project entitled 'Simultaneous

Saccharification and Fermentation of Cellulosic Waste for Bioethanol Production

Using Saccharomyces cerevisiae ATCC 24859' has been submitted in support of an

application for another degree qualification of this or any other university and institution of

higher learning.

"

Lee Wai Kin Resource Biotechnology Department of Molecular Biology Faculty of Resource Science and Technology University Malaysia Sarawak

III

Pusat Khidmat ifalJumat Akad('mj~· U1 IV ,'RSIrI Mi S ARJ MlAJ.

T ABLE OF CONTENT

ACKNOWLEDGEMENT I

DECLARATION II

TABLE OF CONTENT III

LIST OF ABBREVIATIONS VIII

LIST OF FIGURES IX

LIST OF TABLES X

ABSTRACT XI

1.0 INTRODUCTION 1

2.0 LITERATURE REVIEW

2.1. Ethanol 4

2.1.1 History 4

2.1.2 Physical and chemical properties 4

2.2 Bioethanol 5

2.3 Cellulosic Biomass 6

2.4 Paper Wastes 7

2.5 Cellulose 8

2.6 Cellulases 9 . 2.7 Enzymatic Diversity of Cellulases 10

2.8 Saccharomyces cerevisiae

2.8.1 Morphology 10

2.8.2 Biochemistry 11

2.8.3 Physiological Conditions 11

IV

~ 2.9 Simultaneous Saccharification and Fennentation 12

2.10 Fed-Batch Fennentation 12

2.11 Dinitrosalicylic (DNS) Acid Reducing Sugar Assay 13

2.12 Phenol-Sulphuric Total Carbohydrate Assay 14

2.13 High Perfonnance Liquid Chromatography 14

3.0 MATERIALS AND METHODOLOGY

3.1 Materials 15

3.2 Overall Methodology 16

3.3 Setting of Experimental Parameters 16

3.4 Culture Preparation of Saccharomyces cerevisiae 17

3.5 Stock Culture Preparation ofS. cerevisiae 17

3.6 Substrate Preparation 18

3.7 Chemicals Preparation of Citrate Buffer 18

. 3.8 Inoculum Preparation of S. cerevisiae 19

3.9 Simple Staining of S. cerevisiae 19

3.10 Harvesting ofS. cerevisiae 20

3.11 Simultaneous Saccharification and Fennentation 20

3.12 Feeding of Substrate 21

3.13 Sample§ Collection 21

3.14 Final Dry Biomass Detennination 21

3.15 Chemical Preparation for Sample Analysis

3.15.1 3, 5-Dinitrosalicylic Acid Reagent 22

3.15.25% Phenol 22

v

3.16 Standard Curve Preparation

3.16.1 3, 5-Dinitrosalicylic Acid Test 23

3.16.2 Phenol-Sulphuric Test 23

3.17 Samples Analysis

3. 17.1 Reducing Sugar Assay 24

3.17.2 Total Carbohydrate Assay 24

3.17.3 High Perfonnance Liquid Chromatography 25

4.0 RESULTS AND DISCUSSION

4.1 Qualitative Observations

4.1.1 Fennentation at 2.5% Substrate Loading 26

4.1.2 Fennentation at 2.5% Substrate Loading 27

4.1.3 Reducing Sugar Assay 28

4.1.4 Total Carbohydrate Assay 29

4.2 Quantitative Analysis

4.2.1 Total Carbohydrate Assay 30

4.2.2 Reducing Sugar Assay 32

4.2.3 Time Course for Cellobiose Profile 34

4.2.4 Time Course for Glucose Profile 38

4.2.5 l)me Course for Ethanol Profile 41

4.2.6 Time Course for Lactic Acid Profile 45

4.2.7 Time Course for Acetic Acid Profile 47

4.3 Final Biomass Oetennination 50

4.4 Recommendations 51

u

VI

5.0 CONCLUSION 53

REFERENCES 54

APPENDICES 58

.'

VII

CBH

DNS

EMP

EPA

FPU

g

h

HPLC

IUPAC

MEA

ml

NAD

nm

PSTC

rpm

SqSF

SSF

U.K.

U.S.A.

YMB

~l

~m

LIST OF ABBREVIATIONS

Cellobiohydrolase

Carbon dioxide

Dinitrosalicylic

Embden-Meyerhoff-Parnas

Environmental Protection Agency

Filter paper unit

Grams

Hour

High perfonnance liquid chromatography

International Union Pure and Applied Chemistry

Malt extract agar

Milliliter

Nicotinamide adenine dinucleotide

Nanometer

Phenol-sulphuric total carbohydrate

Revolutions per minute

Sequential saccharification and fennentation

Solid state fennentation .. United Kingdom

Unites States of America

Yeast malt broth

Microliter

Micrometer l

Degree Celsius

VIII ·

LIST OF FIGURES

Figure 1. Figure 2.

Figure 3.

Figure 4. Figure 5. Figure 6. Figure 7.

Figure 8a.

Figure 8b.

Figure 9. Figure 10.

Figure 11. Figure 12.

Figure 13.

Figure 14. Figure 15. Figure 16. Figure 17. Figure 18. Figure 19. Figure 20. Figure 21.

Figure 22. Figure 23. Figure 24. Figure 25. Figure 26.

Fonnation of fossil fuels which takes millions of years. 1 Global municipal waste in 2012: Total of 251 million tons (before recycling). 2 The regeneration of combusted bioethanol in the environment. 5 The composition of cellulosic biomass. 6 Landfilling in Sibu, Sarawak. 8 The composition of cellulosic biomass. 9 Principal flow in simultaneous saccharification and fennentation. 12 Flow chart for simultaneous fed-batch saccharification and fennentation of office waste at 2.5% substrate loading. 16 Flow chart for simultaneous fed-batch saccharification and fennentation of office waste at 5.0% substrate loading. 16 Shredded office paper used as substrate. 18 Structure of simple stained S. cerevisiae under microscope. 20 High perfonnance liquid chromatography in FRST. 25 Observation on the texture of the paper during fennentation for 2.5% substrate loading. 26 Observation on the texture of the paper during fennentation for 5.0% subst~ate loading. 27 Reducing sugar assay at 2.5% substrate loading. 28 Reducing sugar assay at 5.0% substrate loading. 28 Total carbohydrate assay at 2.5% substrate loading. 29 Total carbohydrate assay at 5.0% substrate loading. 29 Graph of kinetics for total carbohydrate. 30 Graph of kinetics for reducing sugar. 32 Graph of time course for cellobiose profile. 34 Chromatogram of 12 h fennentation with 2.5% substrate loading shows the possible presence of degraded oligomers of cellulose. Graph of time course for glucose profile. Graph of time course for ethanol profile. Graph of time course for lactic acid profile. Graph oftime course for acetic acid profile.

partially 36 38 41 45 47

Pelletization of remaining paper substrate after 5 days of fennentation; for 2.5% (left) and 5.0% (right) substrate loadings.

IX ·

51

LIST OF TABLES

Table 1. Composition of different types of paper waste. 7 Table 2. Properties of cellulases enzymes. 10 Table 3. Proportion of stock glucose and distilled water for

different glucose concentrations. 23 Table 4. Initial and final dry biomass for fermentation at 2.5%

substrate loading. 50 Table 5. Initial and final dry biomass for fermentation at 5.0%

substrate loading . 50

.'

x

Simultaneous Saccharification and Fermentation of Cellulosic Waste for Bioethanol Production Using Saccharomyces cerev;s;ae ATCC 24859

Lee Wai Kin (36647)

Resource Biotechnology Faculty of Resource Science and Technology

University Malaysia Sarawak

ABSTRACT

The utilization of energy has subsequently surged up by 16 fold as human population becomes 4 times larger in the 21st century. Thus, fossil fuels are foreseen to be vastly diminished by 2050. Therefore, to improve the sustainability of energy production, cellulosic office waste materials are proposed to be used as substrates for bioethanol production. This research aims to study the hydrolysis of cellulose to fermentable sugar as well as to analyze the bioethanol production via simultaneous fed-batch saccharification and fermentation performed in the same vessel, using cellulosic office waste as substrate. The methodology involved inoculum preparation of Saccharomyces cerevisiae using yeast malt broth (YMS) as the main medium; overnight (24 hours) for. On the following day, the S. cerevisiae was harvested to obtain the cells pellet. Then, the S. cerevisiae pellet was introduced into the mixture of autoc1aved office waste with nutrient medium (yeast extract and peptone) at substrate loadings of2.5% and 5.0% where the substrate were fed at three intervals; 0, 24 and 48 h. Meanwhile, 50 FPU/g cellulases were simultaneously introduced into the fermentation broth which was incubated for 5 days at 130 rpm. Lastly, ethanol yield and composition of other fermentation products were analyzed upon the completion of fermentation via High Performance Liquid Chromatography (HPLC), reducing sugar assay and total carbohydrate test. From this experiment, the more effective ethanol production is observed at 5.0% substrate loading (2.41 giL) with dry biomass reduction of substrate of 44.98%.

Keywords: Bioethanol, Simultaneous Saccharification and Fermentation, Cellulosic Waste, Saccharomyces cerevisiae, High Performance Liquid Chromatography (HPLC)

ABSTRAK

Penggunaan tenaga semakin meningkat sebanyak 16 kali ganda sedangkan populasi manusia menjadi 4 kali ganda lebih besar daripada abad ke-21 ini. Oleh itu, bahan api Josil diramalkan akan jauh berkurangan menjelang 2050. Oleh itu, untuk meningkatkan kelestarian pengeluaran tenaga, bahan-bahan buangan pejabat sellulosik dicadangkan untuk digunakan sebagai substrat untuk penghasilan bioetano!' Kajian ini bertujuan untuk mengkaji hidrolisis daripada selulosa kepada gula simpleks dan juga untuk menganalisis penghasilan bioetanol melalui saccharifikasi dan Jermentasi serentak dengan penambahan substrat, dalam bekas yang sama, di mana bahan buangan pejabat sellulosik menjadi substrat. Metodologi pertama bagi kajian ini melibatkan penyediaan inoculum untuk Saccharomyces cerevisiae menggunakan 'yeast malt broth' (YMB) sebagai media utama selama 24 jam. Pada hari berikutnya, Kultur s.,. cerevisiae dituai untuk mendapatkan pelet sel-se!. Kemudian, pelet s.,. cerevisiae telah dimasukkan dalam tiga set sisa kertas yang dicampur dengan medium nutrien (ekstrak yis dan pepton) dan telah diautoklaf, pada dos substrat sebanyak 2.5% dan 5.0%, di mana subtrat tambahan dimasukkan pada 0, 24 dan 48 h. Sementara itu, 50 FPU I g cel/ulases dimasukkan serentak ke dalam kaldu Jermentasi dan dikultur selama 5 hari pada 130 rpm. Akhir sekali, hasilan etanol dan komposisi produk Jermentasi lain akan dianalisis setelah 5 hari melalui Kromatograji Cecair Berprestasi Tinggi (KCB7), dinitrosalicylic (DNS) asid assay dan penilaian jumlah karbohidrat. Daripada eksperimen ini, pengeluaran etanol yang lebih berkesan diperhatikan pada 5.0% dos substrat (2.41 giL) dengan pengurangan biojisim kering substrat sebanyak 44.98%.

Kata Kunci: Bioetanol, Saccharifikasi dan Fermentasi Serentak. Sisa Buangan Selulosik, Saccharomyces cerevisiae. Kromatograji Cecair Berprestasi Tinggi (KCB7)

XI

1.0 INTRODUCTION

This project revolves the emphasis of goal in introducing paper waste as a bio-resource for

bioethanol production and its high potential as a fuel enhancer or sole substitute in the

future to reduce fossil fuel dependency, elaboration of experimental data as well as further

future recommendations.

The key crisis in this present era of globalization, 90% of global energy consumption by

human is derived from the burning of fossil fuels, also known as dead carbon. According

to Vincent (2010), the utilization of energy has subsequently surged up by 16 fold as

human population grows 4 times larger in the current 21 st century. This leads to following

an estimated increment of energy consumption from the current 13TW to approximately

27-42 TW in the upcoming decades (Himmel et aI., 2007). According to the modified

formula of Klass model, the reserve depletion duration for petroleum and natural gas is

about 35 and 37 years respectively, thus foreseen to be depleted by 2050 (Shafiee & Topal,

2009).

Figure 1. Formation of fossil fuels which takes millions of years. (Retrieved from http://shanahanl.pbworks.com/w/page/16014137/Earth%20Science)

The excessive burning of fossil fuels mainly for economical purposes has also contributed

to the emission of approximately 82% greenhouse gases, equivalent to about 7.0 billons

tons of carbon annually (Lens et aI., 2005). Hence to reduce dependency on fossil fuels,

biomass has been introduced to produce biofuels such as bioethanol which are sustainable

1

and renewable bio-resource to be used as transportation fuels. In Malaysia, the domestic

demand for bioethanol has been promising, figuring up to 6, 677 ton/day (Goh et aI., 2010).

To improve the sustainability of bioethanol production without threating the food security,

lignocellulosic waste materials from the agrifood chain are proposed to be used as biofuel

substrates instead. However, the production of ethanol from researches is still currently

economically impractical due to overly expensive pretreatments, difficulty to obtain high

substrate loadings as well as the diversity of enzyme to achieve appropriate hydrolysis

(Elliston et aI., 2013).

Thus, this research introduces usage of cellulosic waste as substrate, which is paper waste.

Paper waste which is the most abundant among the municipal waste have undergone

pulping do not require energy-intense chemical or thermo-physical pretreatments for

enhanced enzymolysis as they are already de-lignified with reduced amount of

hemicellulose which ferments poorly (Elliston et aI., 2013).

Figure 2. Global municipal waste in 2012: Total of251 million tons (before recycling). (Retrieved from http://www.epa.gov/epawaste/nonhaz/municipal/index.htm)

2 ' .

This research also analyzes the effects of different feedstock loadings (2.5% & 5.0%)

towards the simultaneous fed-batch fermentation process as the parameters of this research.

Based on the additional literature on effects of feedstock loading to fermentation, a

hypothesis is made for the possible outcome from the parameters tested:

Ho: The fermentation at 5.0% feedstock loading results in an EQUAL rate of ethanol

production compared to 2.5% substrate loading (J1s.0% = J12.S%).

HA: The fermentation at 5.0% feedstock loading results in a DIFFERENT rate of

ethanol production compared to 2.5% substrate loading (J1S0% -t J12.S0/o).

This study will also involve sequential saccharification and fermentation in the same vessel,

in which cellulose enzymolysis and ethanolic fermentation of hexose occur concurrently in

an integrated step. This significantly prevents the cellulase inhibition by end-products such

as glucose and cellobiose and reduces containment cost (Blaschek et aI., 2010; Olofsson et

aI., 2008; Zhang & Lynd, 2010).

The objectives for this research are:

1. To quantify the reducing sugar and total carbohydrate content during the fermentation

paper waste using Saccharomyces cerevisiae.

2. To investigate the hydrolysis of polymeric cellulosic substrate (paper waste) into

fermentable monomeric sugar using commercial cellulolytic enzymes via simultaneous

saccharification.

3. To analyze the production bioethanol and other co-products from fermentation of

paper waste of 2.5% and 5.0 % feedstock loadings, conducted in a fed-batch system.

3

2.0 LITERATURE REVIEW

2.1.0 Ethanol

2.1.1 History

In the late 19th century, ethanol was first used as a fuel extender for gasoline. This was

initiated by the efforts of Otto and Benz who developed the internal combustion engine

(Lens et aI., 2005). Following the First World War (1920-1930), ethanol production first

peaked throughout the world where several countries legislatively agreed the use of

ethanol-blended gasoline. Nevertheless, in the 1930s, the price of gasoline dropped

tremendously, causing fuel ethanol programs and corresponding legislations to be

abandoned prior to World War Two. Due to the fluctuation of oil prices since the 1970s as

well as realization to reduce fossil fuel dependency, the usage of ethanol was reintroduced

in the early 1980s, mainly via ProAlcool in Brazil and the US Fuel Ethanol Program (Lens

et aI., 2005).

2.1.2 Physical and chemical properties

Ethanol, also known as ethyl alcohol, is physically a colorless and volatile liquid organic

compound which has a strong odor (Speight, 2011). Composing chemically of carbon,

hydrogen and oxygen in a straight chain, a typical molecule of ethanol consist of an ethyl

group (CH3CH2-) and one hydroxyl group (-OH). Thus chemical formula of ethanol is .'

CH3CH20H and its molecular weight is 46.07 glmol (Vincent, 2010). The presence of the

hydroxyl group in ethanol enables hydrogen bonding, which explain its higher viscosity

but lower volatility compared to less polar organic compounds of similar size. The

hydrogen bonding also renders pure ethanol to be hygroscopic, where it absorbs water

readily from air (Speight, 2011). In addition, ethanol is a versatile solvent as it is miscible

with water as well as numerous organic solvents and low-boiling hydrocarbons.

4

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rn 1\ RA\\lAJ..:

2.2 Bioethanol

Bioethanol is a C02-neutral biomass-derived energy source. The carbon dioxide amount

emitted from the combustion ofbioethanol is equivalent to the atmospheric carbon dioxide

that is originally fixed by the biomass, simultaneously capturing part of the solar energy

(Lens et aI., 2005; Speight, 2011). Bioethanol is a promising substitute for transportation

fuel because it is biodegradable, oxygenated (contains 35% oxygen) as well as has high

octane number, flammability limits, flame speeds and heat of vaporization (Speight, 2011;

Zimmennann & Entian, 1997).

<~ .II~ Combustion

Ethanol Carbon o ox ide~

.

Figure 3. The regeneration of combusted bioethanol in the environment. (Retri ved from http://www.ecosmartfire.comlabout! about -bioethano 1)

Thus, this reduces emission of particulates, unburned hydrocarbons and hannful gases ­

nitrous oxides, carbon monoxides, sulfur dioxide from compression-ignition engines as

ethanol results in complete combustion (Lens et aI., 2005). Based on Speight (2011),

several types of eth~nol-blended fuel are the E85 (85% ethanol), gasohol (24% ethanol)

and EI0 (10% ethanol).

Recent strategy is to promote cellulosic ethanol production from non-edible lignocellulosic

waste, also known as second generation bioethanoI. These cellulosic wastes, mainly

composed of cellulose are generated from agricultural, forestry and urban sources as well

as landfilled municipal solid residues (Speight, 2011). According to Lens et aI. (2005), the

5

world's production of residual plant biomass is approximately 200 x 109 ton/year, where it

is estimated that 100,000 million gallons of ethanol can be potentially produced from this

biomass. Meanwhile, Malaysia is capable of producing 26,161 ton/day of second-

generation bioethanol as the biomass availability of the country is 47,402 dry kton/ year,

regionally reducing 19% of C02 emissions (Goh et aI., 2010).

2.3 Cellulosic Biomass

Cellulosic biomass is defined as components of 'recent biological origin' such as trees,

agricultural crops and agro waste (Speight, 2011). The estimated global annual production

of lignocellulose is approximately 2-5 x 1012 metric tons. On average, most trees contain

lignocellulose with composition of 45% cellulose, 30% hemicellulose and 25% lignin

(Glazer & Nikaido, 2007). The cell wall of higher plants vascular tissues are comprised of

cellulose fibrils embedded within an amorphous matric containing hemicellulose and lignin.

The three polymers are bounded to each other by covalent crosslinks and non-covalent

forces, forming a complex composite materials termed lignocellulose which represents

about 90% of the plant's dry weight (Glazer & Nikaido, 2007).

Plant Cell Wall

o~o o

HJCO~ );;;;; 0

Figure 4. The composition of cellulosic biomass. (Retrieved from http://biofuel. webgarden.coml sectionslbloglpictures-for-lignocellulose)

6

2.4 Paper Wastes

Paper waste is a type of cellulosic waste, which typically contains 85-99% cellulose, 0%

hemicellulose and 0-15% lignin as they have undergone pulping and delignification

(Howard et aI., 2004). Judging on paper consumption (kilograms per capita) based on The

Bureau of International Recycling (2000), the North Americans rank first (323 kg),

followed by Australasia (322 kg), Europe (125 kg), Latin America (36 kg), Asia (28 kg)

and Africa (6 kg), averaging the world's paper consumption to 53.8kg per capita.

Examples of paper wastes (secondary fibers) from mills are after-used corrugated

cardboard, copier paper and newspaper:

Table 1. Composition of different types ofpaper waste (Howard et aI., 2004).

Paper type Cellulose Hemicellulose Lignin (%) (%) (%)

Conventional paper 85-99 0 0-15 Newsprint 40-55 25-40 18-30

Chemical pulp wastes 60-70 10-20 5-10

A study by the Environmental Protection Agency (EPA) reported that paper mills produce

a total 2.5 million dry ton/year of paper sludge (Wang et aI., 2009). Since 1andfilling and

incinerating paper waste involves high cost, takes up huge spaces and causes air pollution

(Duff & Murray, 1996), these wastes are potential feedstock for bioethanol production.

Landfilling paper wastes releases methane which is 25 times more toxic than carbon

dioxide, according t9 the International Institute for Environment and Development (lIED).

7 "

Figure 5. Landfilling in Sibu, Sarawak. (Retrieved from http: //www.thebomeopost.coml20 II / I O/06/waste-management-weighs­

heavily-on-council-shoulders )

2.5 Cellulose

Cellulose, the Earth's most abundant organic material (> I011 metric tons of cellulose per

year), is a homopolysaccaharide polymer with a linear, flat and ribbon-like chain

composed of thousands of ~-D-glucopyranose units primarily bonded by ~-I ,4-glycosidic

bond and internally stabilized by hydrogen !>ond (Glazer & Nikaido, 2007; Howard et aI.,

2004; Lens et aI., 2005; Speight, 20 II). The consecutive glucose units are arranged at

rotation of 1800 with respect to neighboring molecules along the chain axis (Glazer &

Nikaido, 2007).

The hydrogen bonds between adjacent cellulose chains allow them to interact strongly and

become arrayed wi!h other chains (Speight, 2011; Yang, 2008). This results in a long,

highly ordered and insoluble crystalline aggregate called microfibrils which further

combines to from larger fibrils and then organized in thin layers as lamellae, forming

crystalline and amorphous regions (Glazer & Nikaido, 2007; Howard et aI., 2004;

Mousdale, 2008).

8 "

Figure 6. The composition of cellulosic biomass. (Retrived from http://www.generalbiomass.comlcellethanoll .htm)

2.6 Cellulases

Based on the Enzyme Comission, cellulases is a shorthand tenn for three enzymatic

reactions for the complete hydrolysis of cellulose to glucose, which is divided into three

groups, namely endoglucanases, exoglucanase and ~-glucosidases (Lens et aI., 2005).

According to Swiss-Plot online database, approximately 120 endogucanses, 22

cellobioshydrolase and 27 ~-glucosidases are completely sequenced at amino acid level,

mostly from fungi and bacteria but also include higher plants and blue mussels.

Endoglucanases (1 ,4-~-D-glucan-4-glucanohydrolases) reduce the degree of

polymerization the cellulose macromolecule (-10k) by randomly hydrolyzing mUltiple

accessible internal bonds at amorphous region throughout the cellulose fibre, in order to

break the linear cellulose chain and open up regions to be targeted by subsequent enzymes

(Glazer & Nikaido, 2007; Mousdale, 2008).

Exoglucanase, also·known as ceUobiohydrolase (CBH), is a monomeric enzyme which

accounts for 40-70% of total cellulolytic proteins in the cellulases system of fungus

(Howard et aI., 2004). CBH attack the chain ends of the intennediate products from

endoglucanases activity, which liberates disaccharides cellobiose (Glazer & Nikaido, 2007;

Olofsson et aI., 2008). ~-glucosidases, a mixture of monomeric, dimeric and trimeric

enzymes, hydrolyze the soluble cellobiose and cellodextrins to glucose (Mousdale, 2008;

Vincent, 2010). Table 2 sho several properties of cellulases:

9 '.

Table 2. Properties of cellulases enzymes (Baldrian & Valaskova, 2008; Dashtban et aI., 2009).

Molecular Weight Optimum Optimum pH (kDa) Temperature (GC)

Endoglucanases 22-45 50-70 4-5 Cellobiohydrolyse 50-65 37-60 4-5

p-glucosidases 35-450 45-75 5-6

2.7 Enzymatic Diversity of Cellulases

Trichoderma spp. produces approximately 70% cellobiosehydrolases, 20% endoglucanses

but only <5% of ~-glucosidases (Mousdale, 2008). Synergy with Aspergillus strains to

supplement additional ~-glucosidases can enhance the hydrolysis of cellulose to glucose as

well as prevent inhibition by cellobiose accumulation (Mousdale, 2008; Olofsson et aI.,

2008). Based on Haigler and Weimer (1991), some demonstrated synergistic effect

involved endoglucanase, exoglucanase and ~-glucosidase between the coupling of

Penicillium funiculosuml Trichoderma viride; P. funiculosum I P. pinophilum and P.

foniculosum I T. emersonii which is attributed by the stereospecificity of the cellulase

system. Despite the synergistic effects of a combination of cellulases to degrade sugar, the

yeast in a sequential system can still utilize the degraded sugar and prevent the sporulation

and inhibition by the fungi via a catabolic repression removal mechanism (Smith et aI.,

1983).

2.8 Saccharomyces cerevisiae

.' 2.8.1 Morphology

Saccharomyces cerevisiae also known as the baker's yeast, is a yellow-green globular

shaped (5-10 J.lm) unicellular eukaryotic yeast categorized in the fungi kingdom (Senawi,

2013). S. cerevisiae have cell wall that is composed of chitin and ester bond-linked lipids,

with absence of peptidoglycan (Senawi, 2013).

10 '

2.8.2 Biochemistry

S. cerevisiae is known to be a facultative fennenting microbe, whereby it completely

hydrolyzes sugar in the glycolytic/ EMP (Embden-Meyerhoff-Pamas) pathway to carbon

dioxide and water in micro-aerobic environment, producing ethanol as a co-product

(Mousdale, 2008). According Lens et al. (2005), co-fennentation of S. cerevisiae with

Candida shehatae produces a yield of 4.8 g-ethanollg-sugar, showing that S. cerevisiae is

potentially a high yielding microorganism. A study by Yang (2008) also reports that S.

cerevisiae produces high ethanol yield of 0.45 g g-I at optimal conditions and high specific

rate of 1.3 g g-I cell mass h- I.

In the EMP pathway, the C02 molecule in pyruvate, an intennediate metabolite is reduced

to produce acetaldehyde, which is in tum reduced to ethanol via NADH-NAD+ redox

reaction (yang, 2008). All wild-type S. cerevisiae are able to fennent D-glucose, 0­

fiuctofuranose and D-mannose to ethanol and carbon dioxide, but unable to fennent

cellulose, hemicellulose, most pentoses as well as oligosaccharides such as cellobiose and

cellodextrin (Zimmennann & Entian, 1997). The glucose-to-ethanol fennentation by yeast

is based on the Gay-Lussac equation in 1810 (Glazer & Nikaido, 2007):

.' 2.8.3 Physiological Conditions

Based on Arroyo-Lopez et al. (2009), the optimum condition for yeast metabolism is

34.1 °C and pH 4.76. The metabolism rate is gradually lower in the range of 35-43 °c and

decreases drastically above 43°C (Glazer & Nikaido, 2007). Changes of pH in the range of

3.5-7.5 minimally affects the growth rate of S. cerevisiae, however the growth rate is

decelerated below pH 3.5 and stopped below pH 2 or above pH 8 (Zimmennann & Entian,

1997). S. cerevisiae also has a considerably high ethanol tolerance, up to 100 giL (Yang,

2008).

2.9 Simultaneous Saccharification and Fermentation

Simultaneous saccharification and fermentation (SSF) is a modified technique that

involves the occurrence of the substrate hydrolysis and fermentation in the same vessel to

prevent end product inhibition (glucose and cellobiose), avoid by-products inhibition

(furfurals and acetic acids) and reduce contamination (Blaschek et aI., 2010; Moo-Young

et aI., 1986; Mousdale, 2008). Respiratory inhibition of S. cerevisiae by the high

concentration of the exogenous D-glucose is known as Crabtree effect (Mousdale, 2008;

Zimmermann & Entian, 1997).

Cellulases & S. cerevisiae Introduction

Substrate Saccharification

Same Vessel

Liquid State Fermentation

Figure 7. Principal flow in simultaReous saccharification and fermentation.

SSF also includes additional criteria of incorporating prior cellulases production by solid

state fermentation in the same vessel which is then continued sequentially with SSF for

preservation of high cellulolytic enzyme yield, to ensure complete hydrolysis, thus greater

availability of fermentable sugar for ethanol production (Grohmann & Bothast, 1997). The

preferred temperat~re for SSF is 37°C, between that of cellulases (55°C) and S. cerevisiae. (34.1 °C) (Olofsson et aI., 2008).

2.10 Fed-Batch Fermentation

Fed-batch fermentation is the fermentation process performed by introducing substrate of a

particular concentration at either at fixed and differing rates, with or without replacement

of fermentation medium (Szymanowska & Grajek, 2009). The fed-batch mechanism is a

complicated, time-variant and non-linear system (Cheng et aI., 2009). In bioprocess