Development of xyloseutilizing and inhibitortolerant yeast strains for bioethanol production

iv LIST OF FIGURES Figure 2.1: General principle of bioethanol production Figure 2.2: Structures of cellulose and hexose components of hemicelluloses Figure 2.3: The required stages in

Vrije Universiteit Brussel Katholieke Universiteit Leuven Universiteit Antwerpen

Interuniversity Program Molecular Biology (IPMB)

Development of xylose-utilizing and inhibitor-tolerant

yeast strains for bioethanol production

Thesis submitted in partial fulfillment of the requirements for the Degree of

Master of Science in Molecular Biology

Tung Thanh Dinh

Promoter: Prof Johan Thevelein

Co-promoter: Dr Françoise Dumortier

Supervisor: Mekonnen M Demeke

Laboratory of Molecular Cell Biology Department of Molecular Microbiology

Faculty of Science K.U.Leuven

Academic year 2010-2011

CONFIDENTIAL DOCUMENT

This thesis is a piece of examination that has not been corrected after the defense The results of this thesis might be used for a patent application Therefore, all the data of this document should be considered as confidential They may by no means made public; and there should not be any reference to them To know the date of public release, the promoter of this thesis can be contacted

i

TABLE OF CONTENTS

TABLE OF CONTENTS i

LIST OF FIGURES iv

LIST OF TABLES vi

ACKNOWLEDGEMENT vii

ABSTRACT viii

1 INTRODUCTION 1

2 LITERATURE REVIEW 2

2.1 BIOETHANOL PRODUCTION – AN OVERVIEW 2

2.1.1 First generation bioethanol 2

2.1.2 Second generation bioethanol 3

2.2 INDUSTRIAL REQUIREMENTS, CURRENT STATUS AND CHALLENGES 4

2.2.1 Industrial requirements 4

2.2.2 Current status 5

2.2.3 Challenges 9

2.3 INHIBITORS IN HYDROLYSIS PRODUCTS OF LIGNOCELLULOSES 10

2.3.1 Origin 10

2.3.2 Effects and Mechanisms 11

2.3.2 Solutions for inhibitor 12

2.4 XYLOSE FERMENTATION 15

2.4.1 Xylose fermentation in yeast 15

2.4.2 Recombinant xylose-fermenting strain development 16

3 OBJECTIVES 20

4 MATERIAL AND METHODS 21

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4.1 CULTURING MEDIA 21

4.1.1 Inoculation media 21

4.1.1 Screening media 21

4.2 YEAST STRAINS 22

4.3 YEAST MANIPULATION 23

4.3.1 Sporulation 23

4.3.2 Tetrad analysis 23

4.3.3 Mating type determination 23

4.3.4 Spot test 23

4.3.5 OD600 measurement 24

4.3.6 Dry weight measurement 24

4.3.7 Fermentation 24

4.4 DNA MANIPULATION 25

4.4.1 DNA isolation 25

4.4.2 Polymerase chain reaction (PCR) 25

4.4.2 Agarose gel electrophoresis 25

4.5 SUGAR AND METABOLITE ANALYSIS 26

4.5.1 Rate of sugar consumption 26

4.5.2 Ethanol, Sugar and metabolite analysis 26

4.6 GENOME SHUFFLING 26

5 RESULTS AND DISCUSSION 28

5.1 EXPERIMENTS ON ETHANOL RED 28

5.1.1 Segregant preparation 28

5.1.2 High-throughput inoculating segregants with non-specified amounts of cells 28

5.1.3 High-throughput inoculating segregants with specified amounts of cells 29

5.1.4 Fermentation of Ethanol Red segregants 32

iii

5.2 FERMENTATION OF SACCHAROMYCES CEREVISIAE STRAINS 33

5.2.1 Effect of furan derivative on fermentation of ER, BY4742, and TMB3400 strains 33

5.2.2 Fermentation of ISO12 ISOB57 ER TMB3400 strains in 50%, 60%, and 70% Hydrolysate and containing YP and 13% glucose 35

5.3 GENETIC MAPPING BASED ON AMTEM 38

5.3.1 Genetic mapping strategy 38

5.3.2 AMTEM for inhibitor tolerance phenotype 38

5.4 GENOME SHUFFLING 40

6 CONCLUSION AND RECOMMENDATION 42

7 BIBLIOGRAPHY 44

iv

LIST OF FIGURES

Figure 2.1: General principle of bioethanol production

Figure 2.2: Structures of cellulose and hexose components of hemicelluloses

Figure 2.3: The required stages in case cellulose is the main substrate

Figure 2.4: Inhibitors in the lignocellulosic biomass

Figure 2.5: Schematic view of inhibition mechanisms of inhibitors

Figure 2.6: Three main fermentation modes used in fermentation technology

Figure 2.7: Xylose (a,b) and arabinose (c,d) utilization pathways in bacteria (a,c) and in fungi (b,d)

Figure 2.8: Metabolic pathways used by yeasts: the nonoxidative pentose phosphate pathways Figure 5.1: OD600 values of ER segregants on 65% liquid hydrolysate in 7 days

Figure 5.2: ER segregants from 1A to 44D on 60% solid hydrolysate in YPD pH=6 after 2 days

Figure 5.3: ER segregants from 1A to 44D on 70% solid hydrolysate in YPD pH=6 after 2 days

Figure 5.4: ER segregants from 1A to 44D on 80% solid hydrolysate in YPD pH=6 after 3 days

Figure 5.5: Spot test of ER segregants on 50% solid hydrolysate after 4 days

Figure 5.6: Spot test of ER segregants on 60% solid hydrolysate after 4 days

Figure 5.7: OD600 values of ER and its 12 selected segregants and in 60% and 70% liquid hydrolysate media supplemented with YP and 2% glucose after 48hrs

Figure 5.8: Sugar consumption of ISO12, ER and its 6 segregants in 60% spruce hydrolysate + YP + 13% glucose

Figure 5.9: Effect of furan derivatives on fermentation of BY4742

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Figure 5.10: Effect of furan derivatives on fermentation of ER

Figure 5.11: Effect of furan derivatives on fermentation of TMB3400

Figure 5.12: Sugar consumption of ISO12, ISOB57, ER, and TMB3400 in 50% hydrolysate

supplemented with YP and 13% glucose

Figure 5.13: Sugar consumption of ISO12, ISOB57, ER, and TMB3400 in 60% hydrolysate

supplemented with YP and 13% glucose

Figure 5.14: Sugar consumption of ISO12, ISOB57, ER, and TMB3400 in 70% hydrolysate

supplemented with YP and 13% glucose

Figure 5.15: Ethanol yields measured from the residual fermentation medium of ISO12,

ISOB57, ER, and TMB3400 in 50%, 60%, and 70% hydrolysate supplemented with YP and 13% glucose

Figure 5.16: AMTEM strategy employed to identify the genes or mutation responsible for

inhibitor-tolerant and xylose-fermenting phenotype

Figure 5.17: OD600 values of ER and ISOB57 after 72hrs growth in hydrolysate supplemented with YP and 2% glucose

Figure 5.18: Spot test result of F1 hybrid [ISOB57 x AMS928α] segregants on 40% hydrolysate supplemented with YP and 2% glucose solid medium after 2 days

Figure 5.19: Spot test result of F1 hybrid [ISOB57 x AMS928α] segregants on 50%

hydrolysate supplemented with YP and 2% glucose solid medium after 4 days

Figure 5.20: OD600 values of initial mating products along with MDX1051, MDX1052, MDX1053, MDX1054, ER, and TMB3400 in YP + 2% xylose after 24hrs and 48hrs (with same initial OD600 of 1)

Figure 5.21: OD600 values of mating mix in 50% and 60% hydrolysate supplemented with YP and 2% xylose after 24hrs and 48hrs

Figure 5.22: Sugar consumption of the final mating mix in YP supplemented with 2% xylose

vi

LIST OF TABLES

Table 2.1: Annual world ethanol production by country

Table 2.2: The most commonly used strategies applied to S cerevisiae for pentose

fermentation

Table 4.1: Yeast strains used in experiments

Table 4.2: Composition of buffers and medium used in genome shuffling experiment

Table 5.1: Composition of the spruce hydrolysate supplied by SEKAB Company

vii

ACKNOWLEDGEMENT

First, I would like to thank Belgian government namely BTC for financing my Master study I have really enjoyed the time living in this beautiful country I also want to show my

most sincere gratitude to my promoters Professor Johan Thevelein and Dr Francoise

Dumortier for giving me the opportunity to do my thesis at Molecular Cell Biology

Laboratory (MCB), KULeuven I really appreciate my supervisor Mekonnen M Demeke for

his devoted support during my time at the lab

I want to devote the thesis to my parents who brought me up with love and care My family including my younger brother has been always behind me when I was in trouble and shared happiness with me in the time of success Without my family, I would not be what I

am today

I would like to thank all IPMB teachers because of precious knowledge I have learnt from them, as well as administrative staffs for their support during my study period My

deepest gratitude to IPMB coordinator Professor Eddy Van Driessche and Mr Rudi Willems

for their enthusiastic guidance and assistance I really enjoyed the time with my IPMB classmates and the memory of our class will always be in my heart

viii

ABSTRACT

Second generation bioethanol production has been considered as a solution for energy crisis in the future due to fossil fuel depletion Despite being promising, this technology confronts several challenges to reach its full potential One of them is the presence of inhibitors generated during the hydrolysis of lignocellulosic biomass Another which is not less important is the incapacity of the most commonly used microorganism in the industry, Saccharomyces cerevisiae, to ferment pentose sugar such as arabinose and xylose

The first objective of this study was to isolate a segregant of an inhibitor tolerant industrial yeast strain, Ethanol Red, and subsequently use this segregant for mapping genomic regions responsible for inhibitor tolerance By tetrad analysis, 188 segregants were isolated and applied to microbial techniques in order to find segregants which are at least as tolerant as ER They were then spotted on solid spruce hydrolysate media (60%, 70%, and 80%) without pre-determined amount of cells Segregants which grew nearly as good as ER were subsequently subjected to Spot test for further screening and 12 segregants with best growth were kept Afterwards, these 12 segregants were inoculated in liquid hydrolysate media and only 6 segregants which were able to grow on highly inhibiting 70% hydrolysate were chosen for fermentation evaluation As a result, we manage to get 1 segregant (8D) that ferments better than ER in 60% spruce hydrolysate containing YPD

With the aim of combining the inhibitor-tolerance and xylose-fermenting phenotypes capacity from strains possessing one of these two phenotypes, genome shuffling of Ethanol Red (inhibitor tolerant strain) along with four xylose utilizing yeast strains was performed After the spores of these 5 strains were isolated and crossed all together, the resulting diploids were able to grow in both xylose and inhibitor containing medium The mating products were proven to grow significantly faster in YPXylose than their xylose-fermenting parents but fermented xylose with very slow rate This indicates that good growth on xylose does not mean good fermentation to ethanol and further genome shuffling cycle and evolutionary adaptation in anaerobic condition should be done to obtain strains of better xylose fermenting capacity

1

1 INTRODUCTION

The fossil fuel all over the world has been on the way of exhaustion and to meet the ever increasing demand of energy, especially for transport purpose, human being has to search for alternative renewable sources One of the solutions is to convert biomass of the environment into biofuel For instance, biomass like starch or lignocellulosic material can be hydrolyzed into monomeric sugars which are then fermented by microorganism, usually yeast, into ethanol This kind of biofuel called bioethanol has become one of the most developed energy sources nowadays It not only alleviates our dependency on fossil fuel but also reduces green house gas emission (Farrell et al., 2006) Plenty of companies and laboratories have invested enormous amount of money and resources in research of bioethanol production

Lignocellulosic material is the most abundant renewable resource Approximately, plants on earth can supply 1.3x1010 tons of wood per year which is equivalent to 7x109 tons

of coal and sufficient to meet human’s energy requirement Every year, about 180 million tons of cellulosic feedstocks are produced (Demain et al., 2005) Moreover, if people can take advantage of inexpensive lignocelluloses from agriculture and forestry residues, it can help to reduce pollution and greenhouse effect problems

Although being promising, the second-generation bioethanol production has confronted several challenges One of them is the presence of inhibitor generated during the hydrolysis

of lignocellulosic biomass Another which is not less importance is the incapacity of the most

commonly used microorganism in the industry, Saccharomyces cerevisiae, in fermenting

pentose sugar such as arabinose and xylose (Träff, 2001) The aim of this study was to unravel the genes or mutations responsible for inhibitor tolerance and xylose-fermenting

capacity of Saccharomyces cerevisia strains Thereby, a strain which is inhibitor-tolerant and

at the same time xylose-fermenting could be developed

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2 LITERATURE REVIEW

2.1 BIOETHANOL PRODUCTION – AN OVERVIEW

2.1.1 First generation bioethanol

Bio-ethanol can be produce from a wide variety of carbohydrate sources: mono-, di-, or polysaccharides The biomass sources from which ethanol is produced in the industry can be sweet juice (e.g., sugar cane or molasses) and starch (e.g., corn, wheat, barley)

2.1.1.1 Sucrose-to-Ethanol

The most common disaccharide used for bioethanol production is sucrose which is

fermented by industrial yeast like Saccharomyces cerevisiae First, sucrose which is

composed of glucose and fructose is hydrolyzed by invertase (an enzyme produced by yeast) into these two monosaccharides Afterwards, zymase (an enzyme complex produced by yeast) ferments glucose and fructose into ethanol and carbon dioxide Theoretically, one ton

of sucrose can be converted into 511 kg ethanol, yet the practical efficiency is only about 92% of this theoretical value (Pandey, 2009)

In the industry, the main source of sucrose is sugarcane and sugar beet or in some cases sweet sorghum Brazil has been the main producer of fuel produced from sugarcane juice, giving 16 billion liter of ethanol in 2005, 2 billion of which were exported European Union (EU) mainly produces ethanol from sugar beet juice which plays a less important role than wheat previously However, usage of wheat has increased significantly thanks to incentive of

EU policy for energy crops In 2005, about 950 million liters of bio-ethanol were produced in the EU (F.O Licht 2006)

2.1.1.2 Starch-to-Ethanol

To convert starch to ethanol, the polymer is first broken down by the action of glucoamylase enzyme into dextrose or D-glucose This is followed by the fermentation and complementary processes to produce ethanol In the industry the starch feedstock are mainly grains like corn, wheat, or barley among which corn is the dominant source worldwide containing 60-70% starch (Pandey, 2009)

There are two methods of converting starch to ethanol, namely the dry and wet mills In the dry mill method, the grain which is ground to powder is hydrolyzed and the sugar present

in the hydrolysate is fermented to ethanol while the remaining is converted into distiller

3

grains This co-product of ethanol can be used as animal feed (figure 2.1) Carbon dioxide, a by-product, may also be taken for other usage such as in beverage industry This method is the most commonly used in industrial field The wet mill method is also applied in a large number of factories The grains are wet milled to separate various components like starch, protein, fiber and germ before being converted into different products (Pandey, 2009)

The United States is the leading producer of bioethanol from corn, producing 8 billion liters in 2002 and up to 15 billion liters in 2005 Production of fuel ethanol from corn is responsible for 93% of the whole 18.5 billion liters of U.S bioethanol yield in 2006 It has been shown that corn-to-ethanol production helps to increase energy gain but only insignificantly reduces total CO2 emission (Demirbas, 2009)

Figure 2.1: General principle of bioethanol production (Demirbas, 2009)

Ethanol production from plant substrates has been proven to increase the available source for gasoline blending and help to reduce greenhouse gas emission and other gaseous pollutants (Bergeron, 1996) However, it on the other hand is not cost-effective and influences the food supply and biodiversity (Mousdale, 2008)

2.1.2 Second generation bioethanol

To meet the ever increasing energy demand especially for transportation and to reduce green house gas emission, ethanol has been produced from other feedstock than saccharides and starch Lignocellulosic material appears to be a good candidate and in some countries inulin also has attracted attention Second-generation in which bioethanol is produced from lignocellulosic materials like agricultural and forestry by-products can help to decrease the use of fossil fuel However, a number of challenges have to be circumvented, for instance developing enzymes for lignocellulose hydrolysis, microorganism which can use a variety of substrates, and so on Second generation bioethanol production has been developed since 1920s by Scholler and was commercially marketed in former Soviet Union until the late 1980s (Keller, 1996)

Figure 2.2: Structures of cellulose and hexose components of hemicelluloses

As mentioned above, second-generation bioethanol is produced from lignocellulosic biomass containing cellulose, hemicelluloses, and lignin which is a cementing substance, making cross-linking and hindering the hydrolysis process Cellulose, a structural polymer in plants, is highly insoluble, mixed with other polysaccharides like hemicelluloses and

4

protected from enzymatic attack in woods by the presence of lignins Cellulose contents in plant vary from 38 to 57% (Mousdale, 2008) While cellulose is a linear polymer of glucose, hemicelluloses is a branched heteropolymer of a mixed group of polysaccharides with different structures of two or three types of sugars (sometimes O-methylated or O-acetylated) and a sugar acid (figure 2.2) The major components of hemicelluloses are xylose, arabinose (pentoses) and glucose, galactose and mannose (hexoses) To convert biomass into ethanol, first cellulose and hemicellulose have to be chemically or enzymatically hydrolyzed into monomeric sugars (figure 2.3) which are subsequently fermented into ethanol (Mousdale, 2008) If hemicellulosic sugars are also considered as substrates, either hemicellulases or other microorganisms are needed for simultaneous or subsequent fermentations

Figure 2.3: The required stages of conversion of biomass into ethanol using cellulose as the main

substrate

The promising future of second-generation bioethanol production has been statistically persuasive It is estimated that the total biomass energy crops potential for bioethanol production is around 1.3-2.3 billion tons, being capable of supplying 30-50% U.S gasoline consumption In contrast, even if all the cultivated areas were devoted for energy, the production of bioethanol from corn would only meet 12% of energy demand According to the figures from Canada, in 2004, 2025 million liters of ethanol was produced from wheat, barley, corn, and potatoes while the amount from nonfood crops like straw, wood residues, and forest residues already reached about 11500 liters (Mousdale, 2008) Recently, there have been some policies and initiatives to increase the ethanol production from cellulosic materials This has promoted the interest in bioengineering solutions to the challenges in lignocellulosic bioethanol, in the hope of creating a sophisticated biofuel program (Economic, Financial, Social Analysis and Public Policies for Fuel Ethanol Phase 1, Natural Resources Canada, Ottawa, November 2004)

2.2 INDUSTRIAL REQUIREMENTS, CURRENT STATUS AND CHALLENGES 2.2.1 Industrial requirements

Up to now, most bioethanol production is first generation and it is a tradition to use

yeast as fermenting microorganism to produce ethanol Among yeast, S cerevisiae is the

most commonly used one in the industry nowadays thanks to its capacity of fermenting fast

and efficiently Compared to other yeast and filamentous fungi, S cerevisiae possesses plenty

5

of traits which are favorable in the industrial conditions, especially its high tolerance toward ethanol and as a result high concentration of ethanol can be reached (Hahn-Hägerdal et al., 2007) It can grow under aerobic and anaerobic condition and ferment well in industrial context It can also tolerate a wide range of pH, working optimally at acidic pH, which

enables fermentation by S cerevisiae less susceptible to infection than bacterial fermentation

S cerevisiae can survive up to about 40oC and optimally between 30-35oC which is easily achievable in the industry (Skoog and Hahn-Hägerdal, 1988)

Because S cerevisiae has been used from a long time ago in human history for the

production of food, it is “generally regarded as safe” (GRAS Since, it has been also utilized

perpetually in the industry, S cerevisiae can tolerate high concentration of sugar and ethanol

(above 10%), which allows non-sterile process operation and relatively high osmotic

pressure Furthermore, S cerevisiae isolated from industry shows robustness against

inhibitory compounds in fermentation process (Hahn-Hägerdal et al., 2005; Lindén et al., 1992)

2.2.2 Current status

Nowadays, the United States and Brazil are the two world’s largest ethanol producers For Brazil, the biomass which has been used is sugar cane while for the United States, corn has been utilized Sugar cane consists of a large portion of sugar in which the content of sucrose can be nearly 20% (www.suedzucker.de, www.nedalco.com) Sucrose can be

hydrolyzed into glucose and fructose and then fermented by S cerevisiae to bioethanol For

the production of ethanol from sugar cane, the viscous residue left after sugar crystallization has high osmolarity, which makes it possible to be stored for a long period of time without microbial infection In Brazil, fed-batch model along with yeast recycling has been applied commonly Since the fermentation is performed with high cell density, the rate is relatively

fast in which fermentation is almost completed in 6-11hrs and S cerevisiae has been almost

exclusively used (Amorim et al., 2004)

Table 2.1: Annual world ethanol production by country (adapted from

www.ethanolrfa/industry/statistics)

(million gallons)

2009 (million gallons) USA

Brazil

9000.0 6472.2

10,600.00 6577.89

6

European Union China

Thailand Canada Other Colombia India Australia

733.6 501.9 89.8 237.7 128.4 79.29 66.0 26.4A

1039.52 541.55 435.20 290.59 247.27 83.21 91.67 56.80

2.2.2.1 Hydrolysis of lignocellulosic biomass

There are three widely used methods of hydrolysis in industry, namely, the two-step dilute acid hydrolysis, concentrated acid hydrolysis and enzymatic hydrolysis After these processes, a variety of inhibitors are generated and the composition of which depends on type

of lignocellulosic origin In the three methods, the biomass materials are usually mechanically pretreated to enhance the accessibility of the substrates Besides, each method has its own advantages and disadvantages Acid hydrolysis is fast and easy to operate but its drawbacks are the non-selectivity of the hydrolysis and inhibitors formation In contrast, enzymatic hydrolysis has higher substrate specificity and under milder condition of temperature and pH, problem of corrosion by acid can be avoided However, the use of expensive biocatalyst lowers the cost-effectiveness of the method (Demirbas, 2009)

2.2.2.1.1 Dilute Acid Hydrolysis

Hydrolysis by diluted acid is performed under high temperature and pressure with reaction times within seconds or minutes, which is favorable for continuous processing Most

of the processes can only reach sugar efficiency of about 50% The reason is sugar degradation and the fact that furfural and other degradation products are poisonous to the fermenting microorganisms The most striking advantage of dilute acid hydrolysis is its fast rate reaction, which facilitates continuous processing However, combination of acid and high temperature and pressure necessitates special reactor material, which can be very costly (Demirbas, 2009)

Dilute acid hydrolysis is the first technology which has been developed The hydrolysis

is performed in two stages due to the differences between the hemicelluloses and the cellulose (Harris et al 1985), maximizing the conversion yield Since 5-carbon sugars

7

degrade more rapidly than 6-carbon sugars, one way to decrease sugar degradation is to have

a two-stage process In the first step, which is called prehydrolysis step, a mild condition is applied and hemicellulose is broken down to obtain 5-carbon sugars, while in the second step

a more harsh condition is performed to hydrolyze the more resistant cellulose component producing 6-carbon sugars The two hydrolyzed streams can be fermented into ethanol either together or separately and in the latter case they are mixed and ethanol is then distilled The prehydrolysis can also be carried out physically (steam pretreatment, milling, freeze explosion), biologically (white rot fungi), by other acids (phosphoric acid, sulfuric acid, sulfur dioxide, by alkaline (sodium hydroxide, ammonia), or by organic solvents (ethylene glycol) (Vallander and Eriksson, 1990; Saddler et al., 1993) In this step, hemicelluose is liquefied and converted to mono- and oligosaccharides (Olsson and Hahn-Hägerdal, 1996) A process devised by National Renewable Energy Laboratory (NREL) gives the following steps: for stage 1: 0.7% sulfuric acid, 190oC and for stage 2: 0.4% sulfuric acid, 215oC condition The liquid hydrolysates are retrieved and converted into alcohol while the remaining cellulose and lignin in the solids are used as boiler fuel for electricity or steam production Yields of 89% for mannose, 82% for galactose, and 50% for glucose can be achieved In another process, by applying very low acid and temperature condition of autohydrolysis of sawdust, a yield of 70% glucose was obtained (Ojumu and Ogunkunle, 2005)

2.2.2.1.2 Concentrated Acid Hydrolysis

Concentrated acid processes use mild temperatures and pressures are only involved in pumping materials from vessel to vessel Reaction times are usually longer than that of diluted acid hydrolysis This method generally applies concentrated sulfuric acid before diluting with water to dissolve and hydrolyze the substrate into sugars The process gives a complete and quick conversion to sugars with little degradation The decisive factors required for the economic feasibility are the optimization of the recovery of sugars and the cost effectiveness of the acid recycling The biggest advantage of the concentrated acid method is its high sugar yield The acid and sugar can be separated by ion exchange followed by the re-concentration of acid via multiple evaporators The application of low temperature and pressure allows the use of low-cost materials such as fiberglass tanks and piping However, the process is slow and cost effective acid recovery systems have been difficult to develop Without acid recovery, large quantities of lime must be used to neutralize the acid present in

a striking feature is the usage of a membrane to separate acid from sugar in the product stream The efficiency of acid recovery was very high 80% (Wenzl, 1970) In another method developed by the Soviet Union, concentrated hydrochloric acid was utilized and in this case the prehydrolysis and hydrolysis were operated in one step, resulting in a variety of compounds, some of which interfere with the subsequent fermentation step In the time of World War II, scientists at the U.S Department of Agriculture’s Northern Regional Research Laboratory in Peoria, Illinois improved the process, working with concentrated sulfuric acid

on corn cobs and achieving a yield of 15-20% xylose and 10-20% glucose which was readily fermented to ethanol at 85-90% theoretical yield (Pandey, 2009) Another modification in the United States in 1992 is the addition of recycling of dilute acid from the hydrolysis step and the improvement of sulfuric acid recycle, which helps to reduce the use of it Moreover, recycling of acid increases the cost-effectiveness of the process According to U.S.b Patent 5,188,673, the use of 30-70% weight of sulfuric acid at temperature below 100oC acid results

in high conversion of biomass (80-90% cellulose and hemicelluloses are hydrolyzed) but low product yield caused by degradation and the recovery of acid The drawback of this model is clearly demonstrated by the fact that concentrated acids are toxic and corrosive, which necessitates reactors resistant to corrosion (Von Sivers and Zacchi, 1995)

2.2.2.1.3 Enzymatic Hydrolysis

The third basic method is enzymatic hydrolysis including two technological approaches: enzymatic and direct microbial conversion methods Prior to enzymatic hydrolysis, the chemical pretreatment of cellulosic biomass is necessary The hydrolysis is conducted by using cellulolytic enzymes Different type of cellulases can be used to break down cellulose and hemicelluloses, for example a mixture of endoglucanases, exoglucanases, β-glucosidases, and cellobiohydrolases (Ingram and Doran, 1995; Laymon et al., 1996) The endoglucanases randomly break the cellulose chains to create shorter polysaccharides, while exoglucanases attack the non-reducing ends of these shorter chains and remove cellobiose moieties β-glucosidases hydrolyze cellobiose and other oligosaccharides to glucose

9

(Philippidis and Smith, 1995) To work efficiently, enzymes need to be in good contact with their corresponding substrates This necessitates a pretreatment process to separate hemicelluloses and lignin as well as break down the crystal structure of cellulose and as a result cellulose and hemicellulose molecules can be exposed for access A new generation of enzymes is required for cost-effective hydrolysis of cellulose to glucose since the technical bottleneck of the enzymatic approach is the low specificity of currently used enzymes and enzyme production cost The lignin component is also unconvertible during hydrolysis and fermentation processes (Ching, 2008) Due to the fact that lignin impedes the hydrolysis by obstructing the access of cellulase to cellulose and by irreversible binding to the enzymes, it has to be removed out of the biomass Lignin can be separated from hemicellulose and cellulose in the pretreatment step This improves the hydrolysis efficiency significantly (McMillan, 1994)

Because of the low hydrolysis yield of the previously described dilute-acid hydrolysis (50-60%) (Jones and Semrau, 1984), enzymatic hydrolysis has been developed in which the hydrolysis yield of 80-90% can be achieved theoretically (Söderström et al., 2003; Bura et al., 2003) Nevertheless, no large-scale ethanol production process based on enzymatic hydrolysis has been operated To achieve efficient hydrolysis of cellulose, the lignocelluloses have to be first mechanically treated to interrupt the matrix of cellulose-hemicellulose-lignin (Galbe and Zacchi, 2002) Material is processed into small size particles which in addition enhances mass transfer The mild condition of enzymatic hydrolysis reduces saccharide degradation and inhibitor formation (Jones and Semrau, 1984)

2.2.3 Challenges

The first challenge is in hydrolysate production In the beginning, hydrolysates were generated by acid hydrolysis and concentrated acid was used at first which gives high sugar yield Nevertheless, due to its corrosive nature, the recovery of acid was very costly To reduce acid consumption, single-state dilute-acid (0.4% H2SO4) hydrolysis process at high temperature was then applied, however, saccharide degradation was observed (Jones and Semrau, 1984) This problem can be circumvented by using a two-stage procedure in which hemicelluloses is hydrolyzed in the first step (150-190oC) and cellulose is then hydrolyzed in the second step (190-230oC) (Nguyen et al., 1999; Wayman et al., 1984)

Another challenge is that on one hand S.cerevisiae can utilize sucrose, glucose,

fructose, galactose and mannose (Lindén et al., 1992; Nilsson et al., 2002) but on the other

10

hand it cannot ferment a number of monosaccharides, disaccharides (cellobiose and xylobiose), trisaccharides originated from starch cellulose and hemicelluose, neither can it utilize pentose sugar like xylose and arabinose (Lynd et al., 2002; Zhang and Lynd, 2005) Major disadvantages of current industrial ethanol fermentation are related to the

metabolism of S.cerevisiae Under anaerobic conditions a significant amount of glycerol is

generated resulting from the formation of the reduced co-factor NADH Furthermore, the hexose sugar galactose is only utilized upon the depletion of glucose, which makes the fermentation of galactose-rich materials slower (Oura, 1973)

2.3 INHIBITORS IN HYDROLYSIS PRODUCTS OF LIGNOCELLULOSES

2.3.1 Origin

Hydrolysis of pretreated lignocellulosic biomass results in D-glucose as main components as well as D-galactose, D-mannose and D-rhamnose (hexoses) and that of hemicelluloses gives rise to D-xylose and L-arabinose (pentoses derived from hemicelluloses) Also, uronic acids, for instance -glucuronic and 4-O-methylglucuronic acids are generated from the hydrolysis of hemicelluloses Further degradation of monomeric sugars and lignin could bring about 3 main groups of inhibitors: (1) furan derivatives; (2) low molecular weight fatty acids (mainly acetic acid, formic acid and levulinic acid); and (3) phenolic compounds (Almeida et al., 2007)

Furan derivatives: The furan compounds including 5-hydroxymethyl-2-furaldehyde

(HMF) and 2-furaldehyde (furfural) are generated from the dehydration of hexoses and pentoses, respectively (Dunlop, 1948) The amount of furans depends on the type of raw material and pretreatment methods For example, HMF content can vary from 2.0g/L to 5.9g/L depending on whether one-step or two-step dilute acid hydrolysis is used (Larsson et al., 1999; Nilvebrant et al., 2003) In contrast, HMF is not present in wheat straw treated by wet-oxidation method (Klinke et al., 2003) The concentration of furfural is usually lower than that of HMF, though it is still high enough (about 1g/L) to inhibit fermentation

Figure 2.4: Inhibitors in the lignocellulosic biomass (Almeida et al., 2007)

Low molecular weight fatty acids: Formic and levulinic acid are the major low

molecular weight fatty acids present in lignocellulosic hydrolysates They are formed by the breakdown of HMF, while acetic acid is generated by the deacetylation of hemicelluloses

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Moreover, additional quantity of formic acid can be produced from furfural under acidic condition at high temperature (Dunlop, 1948)

Phenolic compounds: consists of a variety of phenolics that are formed by lignin and

carbohydrate degradation during acid hydrolysis (Popoff and Theander, 1976) The amount and kind of phenolic compounds depends on the biomass nature

2.3.2 Effects and Mechanisms

The inhibitors generated during pretreatment and hydrolysis have been shown to inhibit either the microorganism growth or ethanol production (figure 2.5)

Furan derivatives: HMF and furfural inhibit growth, cause longer lag phase, and

therefore lower ethanol yield Several mechanisms have been proposed to explain the inhibition effects of furan on fermentation Through in-vitro measurement, furfural and HMF have been shown to inhibit alcohol dehydrogenase (ADH), pyruvate dehydrogenase (PDH) and aldehyde dehydrogenase (ALDH) (Modig et al., 2002) Crude-cell extracts from cultures which contained furfural demonstrated the decrease in activity of ADH, hexokinase, and glyceraldehyde-3-phosphate dehydrogenase in the glycolysis pathway (Banerjee et al., 1981) The reduction of furans by yeast cells could lead to NAD(P)H depletion This was proven by the fact that after furfural was added to the medium, levels of excreted acetaldehyde

increased (Palmqvist et al., 1999) Moreover, furfural in S.cerevisiae results in the

accumulation of reactive oxygen species which damages several organs such as vacuole and mitochondrial membranes, chromatin and actin (Almeida et al., 2007) In short, furan derivatives make yeast cells to devote their energy for fixing the damage and at the same time decrease the intracellular ATP and NAD(P)H levels by inhibiting enzymes or consuming cofactors

Figure 2.5: Schematic view of inhibition mechanisms of inhibitors (Almeida et al., 2007)

Low molecular weight fatty acids: The inhibition effect of these acids has been linked

with intracellular anion uncoupling and accumulation as well as a decrease in biomass formation (Russel, 1992) The undissociated form of these fatty acids can diffuse from the fermentation media through the plasma membrane, dissociate under high intracellular pH, and thus lower the pH in the cytosol (Verduyn et al., 1992) To counteract this phenomenon, the ATPase in the plasma membrane has to pump out protons at the expense of ATP hydrolysis As a result, there is less ATP available for biomass formation However, low

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levels of these acids enhance ethanol yield (Larsson et al., 1999) because it stimulates ATP production In contrast, when the concentrations are high the ATP demand becomes so high that the cell has to acidify the cytosol (Larsson et al., 1999) The anionic form of the acid inside the cell is captured and the undissociated acid will diffuse into the cell until equilibrium is reached Additionally, these weak acids have been known to lower the uptake

of aromatic amino acids in the medium due to strong inhibition of Tat2p amino acid permease (Bauer et al., 2003) and consequently inhibit yeast growth

Phenolic compounds: The inhibition mechanism of phenolics in S cerevisiae has not

been clarified still it is assumed that these compounds can partition into cell membrane and therefore disrupt membrane barrier integrity (Hage et al., 2001; Heipieper et al., 1994) Phenolic compounds with weak acidity may interfere with the electrochemical gradient due

to the fact that protons are transported back through the mitochondrial membranes (Terada, 1990)

S cerevisiae innate tolerance to furan and phenolics: Some strains of S cerevisiae

have shown furan tolerance owning to their ability to convert HMF and furfural to less inhibitory compounds Under either aerobic or anaerobic conditions, HMF is reduced to 2,5-bis-hydroxymethylfuran (HMF alcohol) while furfural is converted to furfuryl alcohol which

can be oxidized to formic acid under aerobic conditions (Palmqvist et al., 2000) S cerevisiae

also possesses the innate ability to metabolize phenolics present on the lignocellulosic hydrolysate (Klinke et al., 2003) This could be caused by the presence of phenylacrylic acid decarboxylase (PAD) which is a enzyme capable of metabolizing aromatic acid such as

cinnamic, p-coumaric and ferulic acids since these acids inhibit the ethanol production of S

cerevisiae (Goodey et al., 1982)

2.3.2 Solutions for inhibitor

To deal with the intrinsic inhibitors in the lignocellulosic hydrolysate, three strategies have been studied and implemented: Fermentation technology, Detoxification, and Strain development

2.3.2.1 Fermentation technology

In fermentation technology there are 3 main fermentation mode used, namely the batch, fed-batch and continuous modes For the second and the third modes, the rate of substrate addition can be controlled Since yeast is able to convert inhibitors in the hydrolysate into less

2.3.2.2.1 Biological Detoxification Methods

Biological detoxification exploits specific enzymes or microorganism which act on inhibitors present in hydrolysates and alter their composition Usage of enzymes like

peroxidases and laccase obtained from lignolytic fungus Trametes versicolor results in two to

three fold increases in maximal ethanol productivity from hemicelluloses hydrolysate of willow owning to their effects on acid and phenolic compounds (Jönsson et al 1998) The

fungus Trichoderma reesei has been used in inhibitor degradation of hemicelluloses

hydrolysate obtained after steam pretreatment of willow, which help to increase the maximal ethanol productivity by about three times and ethanol yields by four times (Palmqvist et al

1997) This results from the fact that T reesei removes acetic acid, furfural, and benzoic acid

derivatives The use of microorganism has also been proposed to selectively remove inhibitors from lignocellulosic hydrolysates

2.3.2.2.2 Physical Detoxification Methods

One of the methods of physical detoxification is by vacuum evaporation to reduce the concentration of volatile compounds like low molecular weight fatty acids, for example acetic acid, furfural, and vanillin present in the hydrolysate However, this approach increases fairly the concentration of nonvolatile inhibitors and consequently the level of fermentation inhibition

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2.3.2.2.3 Chemical Detoxification Methods

For chemical detoxification, inhibitors can be precipitated or ionized under particular

pH values in which the toxicity degree of inhibitors is altered (Mussatto, 2002) Toxic compounds can be alternatively adsorbed on activated charcoal (Dominguez, Gong, and Tsao, 1996; Mussatto and Roberto 2001), on diatomaceous earth (Ribeiro et al 2001) and on ion exchange resins (Larsson et al 1999; Nilvebrant et al 2001) Inhibitors in hydrolysate can also be eliminated through treatment of alkali, ozone, ion-exchange resins and enzymes (Alriksson et al., 2006; Jönsson et al., 1998; Nilvebrant et al., 2003; Nilvebrant et al., 2001; Santos et al., 2003) Nevertheless, this will raise the production cost and most detoxification procedures potentially reduce the saccharide component in the hydrolysate (Larsson et al., 1999b; Nilvebrant et al., 2003)

2.3.2.3 Strain development

2.3.2.3.1 Recombinant S cerevisiae

Improvement of yeast tolerance to inhibitors present in lignocellulosic hydrolysates can

be achieved by overexpressing homologous or heterologous genes encoding for enzymes which provide resistance to certain inhibitor(s) An example is the homologous alcohol dehydrogenase (ADH6p) gene encoding for a NADPH-dependent enzyme which can reduce HMF and furfural in yeast Overexpressing of this gene gives rise to a strain that can take in 4 times more of HMF in defined medium under aerobic or anaerobic conditions (Petersson et al., 2006) For tolerance to phenolics compounds, the PAD1 gene encoding phenylacrylic

acid decarboxylase was overexpressed in S cerevisiae cultured in ferulic and cinnamic acid

containing media The transformants were capable of converting ferulic acid and cinnamic acid 1.5 and 4 times faster, respectively than the control strain under aerobic conditions, which lead to 50 to 100% improvement in ethanol productivity (Larsson et al., 2001)

Another candidate is the heterologous Trametes versicolor laccase gene, the enzyme of which

can reduce oxygen molecule into water and radicals that in turn can metabolize and hence eliminate phenolics (Larsson et al., 1999) Laccase-expressing strain cultivated in hydrolysate

in the presence of coniferyl aldehyde at the concentration of 1.25mmol/L showed growth

while the control strain did not Moreover, this recombinant S cerevisiae strain converted

aldehyde faster and gave higher ethanol productivity in the hydrolysate supplemented with

coniferyl aldehyde (Larsson et al., 2001) It can be concluded that recombinant S cerevisiae

strains acquiring an increased tolerance for inhibitors have performed better in the

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hydrolysates Determining of gene(s) or enzyme(s) involved in inhibitor conversion has been

a promising strategy to obtain more robust S cerevisiae strains

2.3.2.3.2 Improved S cerevisiae strains via evolutionary engineering

Inhibitor-tolerant S cervisiase strains have been developed by strain adaptation

exploiting its ability of adapting to inhibitory hydrolysates Significant improvement in fermentation efficiency can be achieved by short pre-cultivation on hydrolysate (Alkasrawi et al., 2006) Another approach is by continuous transferring of yeast to increased concentration

of hydrolysate An example is the case of sequential adding of increasing concentration of

HMF and furfural to synthetic media After more than 100 times of adding, the two S

cerevisiae strains namely 307-12H60 and 307-12H120 demonstrated better reduction

capacity of HMF at high concentration of as 30 and 60 mmol/L, respectively Furthermore, they both grew and fermented glucose faster than the control strain Y-12632 (Liu et al., 2005)

2.4 XYLOSE FERMENTATION

2.4.1 Xylose fermentation in yeast

S cerevisiae which is a facultatively fermentative microorganism can convert sugars

completely to CO2 and H2O in either aerobic or microaerobic condition, producing large

amounts of ethanol In anaerobic condition, S cerevisiae does not grow because essential

substances such as unsaturated fatty acids and sterols cannot be synthesized due to the absence of O2 (Lagunas, 1986) However, S cerevisiae uses a narrow range of fermentable

substrates Glucose, fructose, sucrose, galactose, mannose, and maltose are easily metabolized In contrast, cellobiose, lactose, xylose, rhamnose, sorbose, and maltotetraose cannot be used

Figure 2.7: Xylose and arabinose utilization pathways in bacteria (a,c) and in fungi (b,d)

(Satyanarayana, 2009)

S cerevisiae which is used in bioethanol industry traditionally cannot ferment

D-xylose However, it can slowly metabolize D-xylulose which is an isomer of D-xylose The

wild-type S cerevisiae genome possesses genes for both xylose reductase and xylitol

dehydrogenase Therefore, it can convert xylose into xylulose (figure 2.7) and the resulting xylulose after being phosphorylated by the action of xylulokinase can enter the pentose phosphate pathway (PPP) (Toivari et al., 2004) This is a biochemical pathway for xylose

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metabolism and has been found in almost all cellular organisms, the roles of which is to supply D-ribose for nucleic acid biosynthesis, D-erythrose 4-phosphate for aromatic amino acids synthesis and NADPH for anabolic reactions The PPP has two phases The oxidative phase converts the hexose, D-glucose 6P into the pentose, D-ribulose 5P, CO2 and NADPH The non-oxidative phase converts D-ribulose 5P into D-ribose 5P, D-xylulose 5P, D-sedoheptulose 7P, D-erythrose 4P, D-glyceraldehyde 3P, and D-fructose 6P which participates in glycolysis (figure 2.8) In yeasts, this process goes through a reduction and an oxidation step, which are mediated by xylose reductase (XYL1, Xyl1p) and xylitol dehydrogenase (XYL2, Xyl2p), respectively (Jeffries, 2006) The cofactor requirements of these two reactions determine cellular demands for oxygen Even when glucose fermentation occurs, the fermentation of xylose still requires O2 Xylose is metabolized to xylitol thanks to NADPH-dependent xylose reductase If O2 is absent, fermentation stops due to the fact that the presence of NAD-dependent xylitol dehydrogenase causes a disturbed redox balance of reduced and oxidized cofactors since NADH cannot be then reoxidized (Bruinenberg et al., 1983)

Figure 2.8: Metabolic pathways used by yeasts: the non-oxidative pentose phosphate pathways 2.4.2 Recombinant xylose-fermenting strain development

Xylose-utilizing strains of S.cervisiae have been obtained according to recent literatures

by introducing heterologous genes encoding enzymes involved in xylose metabolism like xylose reductase (XR), xylitol dehydrogenase (XDH), and xylulokinase (XK) (Ho et al., 1998; Wahlbom et al., 2003) Another method is expressing the xylose isomerase (XI) gene

in S.cervisiae and the first case of which is from the bacterium Thermus thermophilus,

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however, the enzymatic activity was very low (Walfridsson et al., 1996) A more recent case

in which a eukaryotic gene extracted from an anaerobic fungus Piromyces in spite of higher

activity (Kuyper et al., 2003) also faces the same problem of too low specific activity (Karhumaa et al., 2007b)

Increasing of XK activity resulted in an increase in consumption of xylose as the sole carbon source converted into ethanol The intracellular concentration of xylose-derived metabolites like xylulose 5-phosphate and ribulose 5-phosphate went up significantly However, the disadvantage is that the less O2 available, the more xylitol produced exceeding ethanol production (Toivari et al., 2001) In another case, by combining the XR and XDH enzymes into a fusion protein separated by short peptide linkers, the expression of the fusion

gene in S cerevisiae led to lower xylitol and glycerol concentration but higher ethanol

productivity although the activities of XR and XDH in the novel bifunctional enzyme did not

change (Anderlund et al., 2001) Introduction of a gene from Aspergillus acleatus for

displaying β-glucosidase on the cell surface along with high activities of XR, XDH, and XK

gave rise to a S cerevisiae strain capable of using xylose- and cellulose-derived

oligosaccharides from an acid hydrolysate of wood chips and producing 30g/L of ethanol from a total 73g/L of of hexose and pentose sugars in 36 hours (Katahira et al., 2006)

Besides, the expression of xylA gene in S cerevisiae enhanced XI activity but could not

induce ethanol production with xylose as the carbon source (Kuyper et al 2003) The

engineered bacterial XI gene from T thermophilus into S cerevisiae enabled it to grow

aerobically using xylose as sole carbon source, producing ethanol at 30ºC although XI activity was low (Karhumaa et al., 2005)

Another approach is disrupting the oxidative pentose phosphate pathway genes involved in the oxidation of 6-phosphate or 6-phosphogluconate, leading to improvement in ethanol yield and decrease in xylitol production from xylose by significantly reducing or even removing the main supply route for NADPH This, however, enhances the generation of side products like acetic acid and glycerol and a more negative consequence is the striking drop in xylose consumption rate due to low intracellular NADPH as a coenzyme in the XR reaction (Jeppsson et al., 2002) A strain converting xylose mainly to xylitol and CO2 can be obtained

by deleting the gene for glucose 6-phosphate dehydrogenase and introducing one for dependent D-glyceraldehydes 3-phosphate dehydrogenase (Verho et al., 2003)

NADP-18

In a nutshell, the disadvantage of recombinant xylose-utilizing S.cervisiae is that the

rate of pentose fermentation is much slower than that of hexose fermentation (Hahn-Hägerdal

et al., 2007) Moreover, fermentation of xylose brings about significant amount of xylitol which can cause the depletion of reduced co-factors (Bruinenberg et al., 1984) However, simultaneous pentose and glucose consumption at low glucose concentration can enhance the pentose fermenting efficiency (Jeffries et al., 1985; Karhumaa et al., 2007a; Meinander et al., 1999; Öhgren et al., 2006)

Once the pentose utilization gene is settled down, the strains can be further improved

by modifying cellular functions including sugar transport (Hamacher et al., 2002; Leandro et al., 2006) or by optimizing initial pentose metabolism (Eliasson et al., 2001; Karhumaa et al., 2007a, b), the pentose phosphate pathway (PPP) (Johansson and Hahn-Hägerdal, 2002a, b),

or inhibitor tolerance (Almeida et al., 2007) Evolutionary engineering (Sauer, 2001) along with adaptation and selection techniques also have been applied to obtain xylose-fermenting strains (Ho et al., 1998; Karhumaa et al., 2005; Sonderegger and Sauer, 2003; Wahlbom et al., 2003)

The final goal of developing pentose-utilizing S cerevisiae strains is to apply them in

large-scale ethanol production from lignocellulosic raw materials This necessitates strains which are robust and tolerant against inhibitors formed in the industrial processes (Almeida et al., 2007) It is desirable to transfer the metabolic engineering strategies from the laboratory yeast strains into industrial ones This strategy is commonplace however the deletion of genes

in industrial strains is difficult arising from the multiple chromosomes and unknown sequences

Table 2.2: The most commonly used strategies applied to S cerevisiae for pentose fermentation

Kötter and Ciriacy (1993) Tantirungkij et al (1993) Walfridsson et al (1996) Kuyper et al (2003) Deng and Ho (1990) Richard et al (2003)

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Additional modifications

Fungal arabinose pathway Fungal xylose and bacterial arabinose pathway Aldose reductase Gre3 deletion Oxidative PPP disruption Overexpression of the non-oxidative PPP

Through the literature review, it can be clearly seen that despite being promising, the second generation bioethanol industry has to deal with several problems One of these is the generation of inhibitors during the hydrolysis of lignocellulosic biomass which considerably decreases the ethanol productivity Non-genetic solutions have been proven to be inefficient

or not cost-effective Another problem is the incapability of the most commonly used

microorganism in the industry recently, S cervisiae, in fermenting pentose sugars As a

result, a significant amount of xylose and arabinose is wasted If these two problems could be solved, the second generation production would reach its full potential and commercial success

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3 OBJECTIVES

As can be seen in the literature review, substantial efforts have been made to improve the bioethanol production from lignocellulosic material by developing genetically engineered

Saccharomyces cerevisiae Nevertheless, the productivity of bioethanol has not reached its

full potential in the industry (Hahn-Hägerdal et al., 2007a) due to the presence of inhibitor in

the hydrolysate and the inability of Saccharomyces cerevisiae in fermenting pentose sugars

including xylose and arabinose

Therefore, the first objective of this study was to identify the genes or mutations responsible for the inhibitor tolerance phenotype of Ethanol Red (an industrial strain) and its derivative strains ISO12, ISOB57 (Lund University, Sweden) by linkage analysis method The second objective was to develop inhibitor tolerant and xylose utilizing yeast strain using genome shuffling technique by bulk crossing of spores from naturally xylose-fermenting strains with those of the robust non-xylose-fermenting strain Ethanol Red

sugar-Synthetic media (SC) consisted of 1.7g/L DifcoTM yeast nitrogen base without amino acids ammonium sulfate (Becton, Dickinson and Company, USA), 5g/L Ammonium Sulfate (SIGMA-ALDRICH, France), 740mg/L CSM-Tryptophan (MP Biomedicals, LLC, USA), and 100mg/L L-Tryptophan (MP Biomedicals, LLC, USA) in Milli-Q water The mixture was then dissolved and adjusted to pH 5.5 using 4M KOH solution

4.1.2 Screening media

To evaluate and select strains/segregants based on their inhibitor tolerance phenotype, the media containing spruce hydrolysate from 30% to 80% were prepared either in liquid or solid form which was also used in Spot test experiments

4.1.2.1 Liquid media: The hydrolysate (SEKAB) was centrifuged and the supernatant was

then filter-sterilized After being adjusted to pH 5 by 4M KOH, YP and additional sugar were added to achieve the desired content of hydrolysate (30-80%)

4.1.2.2 Solid media: Solid media were prepared in rectangular Petri dishes for screening

The hydrolysate (SEKAB) from the company was centrifuge and the supernatant was then sterile-filtered and adjusted to pH 6 by 4M KOH The proper amount of sterile YPAgar mixture which was kept at 60ºC was mixed with the calculated amount of liquid hydrolysate

to obtain 70ml with the desired content of hydrolysate (30-80%) The mixture was

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homogenized carefully and poured into rectangular Petri dish and allowed to be solidified by itself In some experiments, instead of using the supernatant, the crude hydrolysate which is also adjusted to pH 6 was utilized

4.2 YEAST STRAINS

Yeast strains which were used were listed in table 4 below Industrial strains used were Ethanol Red (ER) and TMB3400 Experimental yeast strains were ISO12 (diploid), ISOB57 (haploid) Artificial marker AMS921α was used for linkage analysis MV1000 and MV1001 were tester strains which were utilized for mating type determination BY4741 and BY4742 were used as control strains in mating type determination and fermentation MDX1051, MDX1052, MDX1053, MDX1054 were used for genome shuffling experiments

Table 4.1: Yeast strains used in experiments

TMB3400 HIS3 :: (ADH1p-XYL1-ADH2t,

PGK1p-XYL2-PGK1t, PGK1p-XKS1-PGK1t) Plus

random mutation

Barbel Hahn-Hagerdal Dept of Applied Microbiology Lund University, Sweden ISO12 ER background, evolution engineering Lund University, Sweden

BY4741 MAT a, leu2∆0, met15∆0, ura3∆0, his3∆1 Research Genetics

BY4742 MAT α, his3∆1, leu2∆0, lys2∆0, ura3∆0 Research Genetics

AMS928α BY4742 background, contains 189 artificial

markers

MCB, KULeuven