Production of Bioethanol from Olive Solid Waste · Production of Bioethanol from Olive Solid Waste"...

Islamic University – Gaza

Department of graduate studies

Faculty of science

Biological science master program

Production of Bioethanol from Olive Solid Waste

" "

By

Usama Ahmed Mansour

Supervisors

Dr. Tarek Elbashiti Dr. Kmal ALkahlout

Assoc. Prof. of Biotechnology Assist. Prof. of Biotechnology

A Thesis Submitted to the Faculty of High Graduate Faculty in

Partial Fulfillment of the Requirements for the Master Degree of

Biological Science

1435هـ - 2014م

قل إن صلاتي ونسكي ومحياي ومماتي لله رب العالمني لا

شريك له وبذلك أمرت وأنا أول المسلمني

162} اآلية ماألنعا {

إقرار

:أنا الموقع أدناه مقدم الرسالة التي تحمل العنوان

Production of Bioethanol from Olive Solid Waste

" "

وردباستثناء ما تمت اإلشارة إليه حيثما ,أقر بأن ما اشتملت عليه هذه الرسالة إنما هي نتاج جهدي الخاص

أو أي جزء منها لم يقدم من قبل لنيل درجة أو لقب علمي أو بحثي لدى أية مؤسسة ,وإن هذه الرسالة ككل

.تعليمية أو بحثية أخرى

DECLARATION

The work provided in this thesis, unless otherwise referenced, is the researchers

own work, and has not been submitted elsewhere for any other degree or

qualification.

Student's name: Usama A. A. Mansour أسامة أحمد عوض منصور :الطالباسم

Signature: التوقيع :

Date: 2014/1/7 7/1/4114 التاريخ

I

Abstract

Renewable energy sources have received increased interest from the international

community with biomass being one of the oldest and the most promising ones. In the

concept of exploitation of agro-industrial residues, the present study investigates the

pre-treatment and ethanol fermentation potential of the olive pulp. Olive solid residue

(jeft) is the solid waste generated during olive oil production process in three-phase

olive mills. It consists of the remaining pulp of olive processing after the extraction of

oil, as well as the cracked seeds of the olive fruits. As a lignocellulosic material, the

hemicellulose, cellulose and lignin are the main components of olive stone.

In our study we have standardized the production of ethanol from olive solid wastes

using Saccharomyces cerevisiae strains which isolated from yogurt, grape and

sugarcane. These strains were identified according to morphological and biochemical

characterization tests characters in comparison with the commercial standard strain S.

cerevisiae hismaya. In alcohol tolerance test, S. cerevisiae tolerate up to 10% of

ethanol in the medium. Optimization of culture condition such as pH and temperature

of yeast strain for different yeast strains was done. 10g Jeft was subjected to

hydrolysis to sugar by acids such as 5% HCl and microwave assisted with 5% HCl at

90°C in shaker water path for 3 hours. The fermentation process was carried out in

jeft under optimum conditions such as pH4.5, temperature 30°C and incubated for 72

hrs. The maximum amount of ethanol production (9.3 g/l) was observed by using

Ethanol Assay Kit. Compared with convection mode of heating of dilute 5% HCl

hydrolysis, the microwave assisted 5% HCl process improved the yield of ethanol by

33.3% compared to the non-microwave process.

II

الملخص

"الجفت"إنتاج اإليثانول الحيوي من المخلفات الصلبة للزيتون

للبيئة ورخيصة ايضا وآمنةكونها نظيفة المجتمع الدولي اهتماما متزايدا من المتجددة مصادر الطاقة تلقى

تمت هذه الدراسة في . المهمة إحدى هذه المصادر والصناعيةالزراعية المخلفات استغالل مفهومويعتبر

عملية من اجل عصر ثمار الزيتون عملية من المتولدة المخلفات الصلبة هو عبارة عن الذيمعالجة الجفت

مكونات عدة أهمها المركبات الكربوهيدراتية عديدة يتكون الجفت من و . الحيوي االيثانول نتاجإلتخميره

.واللجنين ،هيميسيلولوز سليلوزال:مثل لتسكرا

من المخلفات الصلبة للزيتون باستخدام ساللة من الخميرة التي الحيوي انتاج االيثانول الىهدف هذا العمل ي

وقد تم التعرف على هذه الساللة من . وعصير العنبعصير قصب السكر ،اللبن : ادر عدة تم عزلها من مص

حيث تم عمل عدة فحوص ، بواسطة المجهر المركب والفحوصات الكيميائية ايضاخالل الفحوصات المظهرية

من حيث درجة الحرارة والرقم الهيدروجيني وذلك منها تحمل تراكيز مختلفة من االيثانول و ايضا عمل معايرة

من 5%جرام من الجفت من اجل تحليلها بواسطة 01عمليا تم وضع .التي تنمو فيهالمعرفة افضل الظروف

من حمض الهيدروكلوريك وبمساعدة الميكروويف في جهاز الحمام المائي 5%حمض الهيدروكلوريك و

معايرته باستخدام هيدروكسيد وبعد ذلك تمت. ساعات 3لمدة C°01الساخن الرجاج عند درجة حرارة

حقن الخميرة بعد ذلك جاهز لعملية التخمر حيث ت ويصبح 5.5الصوديوم حتى يصل الرقم الهيدروجيني الى

وقد وجدنا ان هذه .ساعة 27لمدة C°30لدرجة حرارة تبلغ االختبار المعزولة في المحلول و في حضانة

C ° 31من اإليثانول عند درجة حرارة 01%الساللة من الخميرة لها القدرة على النمو في وسط تركيزه

.لتر/جرام 0.3وبلغت كمية االيثانول المنتج . .5.5ورقم هيدروجيني

وبمساعدة 5%كلوريك عند تركيز هذا وقد أوضحت الدراسة أن المعالجة باستخدام حمض الهيدرو

أكثر من طريقة المعالجة الكيميائية بدون 33%الميكروويف قد حسن إنتاج االيثانول إلى ما نسبته

الميكروويف

III

Acknowledgement

I would like to thank the Almighty for his blessings towards achieving this goal in my life.

And I would like to express my sincere gratitude and appreciation to my supervisor, Assoc.

Prof. Dr. Tarek Elbashiti and Assist. Prof. Dr. Kmal ALkahlout, for very kind discussions

throughout the project. The work in this thesis could not have been finished without their

encouragement, instruction and supervision.

I would also like to thank Mr. Ashraf Shafea for all his help and valuable advice when I

needed it. I am also grateful to Mr. Ismail Quqa for helping me during the work in the

laboratory and for encouraging me to give my best at all times.

Additional thank is extended to my friend Mr. Ramzi Mansour for his excellent technical

assistance and letting me work in his laboratory.

Most of all, I would like to appreciate the enormous endurance and sacrifices of my wife,

Asmaa, my sons, Abdullah and Ahmad and my family for their encouragement and

unconditional support. Mother and father, I owe all my success to you. I would like to express

my gratitude towards my parents.

IV

Contents

Page

Abstract……………………………..........................................................I

Acknowledgement……………………....................................................III

Contents…………………........................................................................III

List of Tables…………………................................................................IX

List of Figures…………………...............................................................X

List of Abbreviation………………........................................................XII

Content

1 INTRODUCTION……….…………………………………………...

1.1 Overview………………………………………………………………………….1

1.2 Bioethanol………………………………………………………………………...2

1.3 Aim of the study………………………………………………………………….3

1.4 Specific Objectives…………………………………………………………….....3

1.5 Significance of the Study……………….……………………………………......3

2 LITERATURE REVIEW……………….………………………....

2.1 Raw materials for ethanol production……………………………………………..5

2.1.1 Lignocelluloses……………………………………………………..........5

2.1.1.1Cellulose………………………………………………………….6

2.2.1.2 Hemicelluloses…………………………………………………..6

2.1.1.3 Lignin………………………………………………………….7

V

2.2 Olive plant…............................................................................................................8

2.3 Uses of olive solid waste "jeft"…............................................................................9

2.4 Microorganisms…………………………………………….…………………….11

2.4.1 Saccharomyces cerevisiae…….………………………………………..11

2.5 Previous Studies………………………………………………………………….13

2.5.1 Substrate………………………………………………………………..14

2.5.2 Pretreatment methods………….….…….……………………………...17

2.5.3 Physical pretreatment………….……………………….…….………...18

2.5.4 Microwave………………………………………………………….......19

2.5.5 Chemical pretreatment………………………………….…….………..21

2.6 Fermentation……………………………………………………………………...24

3 MATERIALS AND METHODS………..………….……….………

3.1 Materials…………………………………………………………………….........29

3.1.1 Apparatus………………………………………………………………...29

3.1.2 Reagents………………………………………………………………….30

3.1.3 Culture media…………………………………………………………….30

3.1.4 Microorganisms …………………………………………………………31

3.1.5 Media…………………………………………………………………….31

3.2 Methods………………….……………………………………………………….31

3.2.1 Sample collection and isolation of S. cerevisiae ………………………...31

3.2.2 Inoculum preparation…………………………………………………….32

3.2.3 Characterization of the selected yeast isolates……………………….......32

3.2.3.1 Morphological characterization………………………………..32

VI

3.2.3.2 Physiological characterization….……………………………...32

1) Carbohydrate source assimilation test……………………………32

2) Tolerance to ethanol……………………………………………...33

3.4 Optimization of the environmental condition……………………………………33

3.4.1 Optimization of pH…………………………………………………….33

3.4.2 Optimization of temperature…………………………………………...33

3.5 Collection and preparation of olive solid waste (jeft) …………………………...34

3.6 Hydrolysis process……………………………………………………………….34

3.7 Microwave-acid pretreatment……………………………………………………34

3.8 Estimation of reducing sugars….………………………………………………...35

3.8.1 Method…………………………………………………………………35

3.8.2 Principle……………………………………………………………......35

3.8.3 Assay Procedure……………………………………………………......36

3.8.4 Calculation……………………………………………………………..36

3.9 Fermentation process ….........................................................................................36

3.10 Estimation of ethanol…………………………………………………………...36

3.10.1 Kit Contents…………………………………………………………...37

3.10.2 Reagent Preparation…………………………………………………..37

3.10.2.1 Ethanol Probe……………………………………………….37

3.10.2.2 Ethanol Enzyme Mix………………………………………..37

3.10.3 Ethanol Assay Protocol……………………………………….37

3.10.3.1 Standard Curve Preparations…….………………………….37

3.10.3.2 Sample Preparation…….………………………….…….………….38

VII

3.10.3.3 Reaction Mix Preparation…………………………………………...38

3.10.4 Data Analysis…………………………………………………………39

4 Results.......................….……………………………………………..

4.1 Isolation of different strain of S. cerevisiae……………………………………...40

4.2 Characterization and identification of S. cerevisiae strains……………………...40

4.2.1 Morphological characterization………………………………………...40

4.2.2 Biochemical characterization…………………………………………..41

4.2.2.1 Carbohydrate source assimilation test………………………..41

4.2.2.2 Ethanol Tolerance test………………………………………..44

4.3 Optimization of growth conditions………………………………………………47

4.3.1 Optimization of Temperature………………………………………..47

4.3.2 Optimization of pH………………………………………………….50

4.4 Pretreatment of olive solid waste………………………………………………...53

4.4.1 Hydrolysis process……………………………………………………………..53

4.4.1.1 Effect of different concentrations of HCl ……………………………..……..53

4.4.1.2 Effect of different concentrations of H2SO4...………………………………..53

4.4.1.3 Microwave assistant with 5% HCl pretreatments…………………………....53

4.4 Fermentation process…………………………………………………………….54

4.5 Ethanol yield……………………………………………………………………..56

5 Discussion……………………………………………………………

5.1 Isolation and selection of yeast isolates………………………………………….58

5.2 Characterization and identification of S. cerevisiae strains……………………...58

VIII

5.2.1 Morphological characterization………………………………………...58

5.2.2 Physiological characterization………………………………………….59

5.2.2.1 Carbohydrate source assimilation test………………………..59

5.2.2.2 Ethanol tolerance test………………………………………...60

5.3 Optimization of culture conditions…………………………………………….....62

5.3.1 Optimization of temperature…………………………………………...62

5.3.2 Optimization of pH……………………………………………………..63

5.4 Hydrolysis process……………………………………………………………….65

5.4.1 Strong acid hydrolysis…………………………….……………………65

5.4.2 Dilute acid hydrolysis…………………………………………………..66

5.4.3 Combined Microwave-Chemical Pretreatments……………………….66

5.5 Fermentation process……………………………………………………………..67

6 Conclusion and Recommendations………………………………....

6.1 Conclusion…………………………………………………………………..69

6.2 Recommendation……………………………………………………………71

7 Reference………………………..………………………………….73

IX

List of Tables

Table Page

Table 2.1: Cellulose, hemicellulose and lignin content in common agricultural

residues and wastes……………………………………………………………………5

Table 2.2 : Classification of Olea europaea, common name Olive tree….……...8

Table 2.3 : Annual production of olives and olive oil in Palestine………………....9

Table 2.4 : Classification of S. cerevisiae………………………………………….12

Table 3.1 : List of the apparatus used in this work………………………………...29

Table 3.2 : List of the reagents used in this work………………………………….30

Table 3.3 : Suppliers for culture media…………………………………………....30

Table 3.4 : Ethanol kit components…......................................................................37

Table 4.1: Effect of different concentrations of HCl, H2SO4 and microwave on

hydrolysis of olive solid waste at different time intervals……………………………54

Table 4.2 : Concentration of glucose utilization during fermentation process by the

different isolated yeast strains by using 10% inoculums size, pH 4.5 at 35 °C for 5

days, after using microwave assistant 5% HCl pretreatments on hydrolysis of olive

solid waste for 3 hours……………………………………………………………….55

Table 4.3 : Concentration of glucose utilization during fermentation process by the

different isolated yeast strain at 10% inoculums size, pH 4.5 at 35 °C for 5 days. After

using 5% HCl pretreatments on hydrolysis of olive solid waste for 3

hours…………………………………………………………….…………………....56

Table 4.4: Estimation of ethanol yield from pretreated olive solid waste by two

methods (5% HCl, and microwave assisted 5% HCl) ………………………………57

X

List of Figures

Figure page

Figure 1.1: Global Ethanol Production……………………………………………..2

Figure 2.1: Schematic illustration of the cellulose chain…………………………...6

Figure 2.2: Structures of monolignols………………………………………………7

Figure 2.3: Process of ethanol production…………………………………………18

Figure 3.1: Ethanol Standard Curve: Performed as described in these kit

instructions…………………………………………………………………………...39

Figure 4.1: S. cerevisiae on yeast extract peptone glucose agar plate……………..40

Figure 4.2: Microscopic Morphology of S. cerevisiae……………………………..41

Figure 4.3: Carbon assimilation test for S. cerevisiae isolated from Sugarcane…...42

Figure 4.4: Carbon assimilation test for S. cerevisiae isolated from Grape………..42

Figure 4.5: Carbon assimilation test for S. cerevisiae isolated from Yogurt………43

Figure 4.6: Carbon assimilation test for S. cerevisiae Control 1……………….......43

Figure 4.7: Carbon assimilation test for S. cerevisiae Control 2…...........................44

Figure 4.8: Growth of S. cerevisiae isolated from sugarcane at different Ethanol

concentration…………………………………………………………………………44

Figure 4.9: Growth of S. cerevisiae isolated from grape at different Ethanol

concentration…………………………………………………………………………45

Figure 4.10: Growth of S. cerevisiae isolated from yogurt at different Ethanol

concentration…………………………………………………………………………46

Figure 4.11: Growth of S. cerevisiae control 1 at different Ethanol concentration...46

XI

Figure page

Figure 4.12: Growth of S. cerevisiae control 2 at different Ethanol

concentration................................................................................................................47

Figure 4.13: Growth of S. cerevisiae isolated from yogurt at different

temperatures………………………………………………………………………….48

Figure 4.14: Growth of S. cerevisiae isolated from grape at different

temperatures………………………………………………………………………….48

Figure 4.15: Growth of S. cerevisiae isolated from sugarcane at different

temperatures………………………………………………………………………….49

Figure 4.16: Growth of S. cerevisiae control 1 at different temperatures…………...49

Figure 4.17: Growth of S. cerevisiae control 2 at different temperatures…………...50

Figure 4.18: Growth of S. cerevisiae isolated from yogurt at different

pH…………………………………………………………………………………….51

Figure 4.19: Growth of S. cerevisiae isolated from grape at different pH…………..51

Figure 4.20: Growth of S. cerevisiae isolated from sugarcane at different pH….......52

Figure 4.21: Growth of S. cerevisiae control 1 at different pH……………………...53

Figure 4.22: Growth of S. cerevisiae control 2 at different pH………………….......53

Figure 4.23: Ethanol yield g /l …………………………………………....................57

XII

List of Abbreviations

°C = degree Celsius.

2POMSW = second stage of olive mill solid waste.

DP = degree of polymerization.

FPV/g = flow propagation velocity per gram.

G = gram.

G/L = gram per liter.

GC = gas chromatography.

GOD = glucose oxidase enzyme.

h = hour.

HMF = 5-(hydroxymethyl) furfural

HPLC = high performance liquid chromatography.

IR = Infrared spectroscopy.

IV = Intrinsic Viscosity.

JEFT = olive solid waste.

KGy = Kilo gray.

L = liter.

LCM = Lignocellulosic materials.

LSW = Lignocellulosic wastes.

M = morality.

ML = micro liter.

Μm = micrometer.

XIII

MT = metric tons.

OD = optical density.

OMSR = Olive mill solid residue.

OMSW = olive mill solid waste.

OMWW = olive mill waste water.

OPH = olive pulp hydrolysate.

OSW = olive solid waste.

OSWC = olive solid waste compost.

OSWC = olive solid waste compost.

pH = power of hydrogen.

POD = peroxidase enzyme

SEM = Scanning Electron Microscope.

SSF = simultaneous saccharification and fermentation.

YPG = yeast peptone glucose.

YPGA = yeast-Pepton- Glucose Agar.

YRS = yield of reducing sugars.

1

Chapter 1

Introduction

1.1 Overview

Worldwide high demands for energy, uncertainty of petroleum resources, and concern

about global climatic changes have led to the resurgence in the development of

alternative liquid fuels. Ethanol has always been considered a better choice as it reduces

the dependence on crude oil and promises cleaner combustion leading to a healthier

environment. Developing ethanol as fuel beyond its current role of fuel oxygenate

would require lignocellulosic as a feedstock because of its renewable nature, abundance,

and low cost (Saha et al., 2005).

Most of the fuel ethanol produced in the world is currently sourced from starchy

biomass or sucrose (molasses or cane juice), but the technology for ethanol production

from non-food plant sources is being developed rapidly such that large-scale production

will be a reality in the coming years (Lin and Tanaka, 2006).

Ethanol is currently produced from sugars, starches and cellulosic materials. The first

two groups of raw materials are currently the main resources for ethanol production, but

concomitant growth in demand for human feed similar to energy could make them

potentially less competitive and perhaps expensive feedstock's in the near future,

leaving the cellulosic materials as the only potential feedstock for production of ethanol

(Taherzadeh and Karimi, 2007). Cellulosic materials obtained from wood and

agricultural residuals, municipal solid wastes and energy crops represent the most

abundant global source of biomass (Lin and Tanaka, 2006). These facts have

motivated extensive research toward making an efficient conversion of lignocelluloses

into sugar monomers for further fermentation to ethanol.

Olive oil production is an important industry in many countries. Olives and their oil

have major contributions in the Palestinian economy. The annual production of olive

fruits in Palestine is about 100,000 tons producing more than 40,000 tons of solid waste

and about 25,000 tons of olive oil (Majed and Mohamad, 2002; Albarran et al.,

2006).

2

1.2 Bioethanol

Ethanol, also known as ethyl alcohol with the chemical formula C2H5OH, is a

flammable, clear, colorless and slightly toxic chemical compound with acceptable

odour. It can be produced either from petrochemical feedstock's by the acid-catalyzed

hydration of ethene, or from biomass feedstock's through fermentation. On a global

scale, synthetic ethanol accounts for about 3-4% of total production while the rest is

produced from fermentation of biomass – mainly sugar crops, e.g. cane and beet, and of

grains (mainly corn) (Licht, 2006).

Ethanol is used for production of alcoholic beverages, for industrial purposes (as a

solvent, disinfectant, or chemical feedstock), and in recent years, as a blending agent

with gasoline to increase octane and reduce carbon monoxide and other smog-causing

emissions. Low-level ethanol blends such as E10 (10% ethanol and 90% gasoline) can

be used in conventional vehicles, while high-level blends, such as E85 (85% ethanol

and 15% gasoline) can only be used in specially designed vehicles, such as flexible fuel

vehicles (FFVs). Regarding the world ethanol scenario, a regular increase in the

production has been observed (Figure 1.1). The Americas are the largest producer

continent of ethanol. The United States of America is the largest producer country of

ethanol with production levels over 51 billion liters (13.5 billion U.S. gallons) in 2011

(Licht’s, 2011).

Figure 1.1 World Annual Ethanol Production since 2006 (Licht’s, 2012).

3

The major producers of ethanol are Brazil and the US, which account for about 62% of

world production. The major feedstock for ethanol in Brazil is sugar cane, while corn

grain is the main feedstock for ethanol in the US (Kim and Dale, 2003)

1.3 Aim of the study

The aim of this study is the production of ethanol from olive solid waste "JEFT"

1.4 Specific Objectives

The following specific objectives will be achieved by:

1 - Isolation of Saccharomyces cerevisiae from grape, sugarcane and yogurt.

2 - Identification, characterization and selection of best yeast strain for ethanol

production.

3 - Pretreatment of jeft by physical and chemical procedures.

4 - Fermentation of the hydrolyzed material by the isolated yeast (S. cerevisiae) to

produce ethanol.

1.5 Significance of the Study

Gaza strip is one of the most crowded areas in the world. It suffers from lack of natural

resources. In addition to the control of Israeli occupation on petrol and chemical

supplies. This situation attracted our attention to look for alternatives such as biofuels.

The importance of this thesis lies in twofold:

First attempt to exploit the jeft in the production of materials of economic importance

such as ethanol. Ethanol is considered as important material and has many applications

for: Industrial purposes (as a solvent, disinfectant, or chemical feedstock). It can be used

in Fuel, as a blending agent with gasoline to increase octane and reduce carbon

monoxide and other smog-causing emissions.

Every year there is about 35.0 thousand MT (metric tons) of Jeft produced in Palestine

alone, which contains about 20 million pounds of carbohydrates. This amount of

carbohydrate is more than enough to supply the existing number of factories and

research institutes in Palestine with their requirements of cellulose and fine chemicals

(Shalabia, 2011).

4

Second, environmental importance: The production process of olive oil, one of the main

agricultural products in the Mediterranean area, leads to the generation of large

quantities of liquid and solid wastes. As shown from previous studies, these wastes can

represent an environmental hazard when disposed directly to the environment, due to

their high organic load and toxic effect to microorganisms, plants and, as recently

shown, to marine organisms (Azbar et al., 2004, Danellakis et al., 2011).

This work contributes to the clean environment and also production of good materials,

such as ethanol, which serves the society and contribute to community building in

addition to the creation new job opportunities for young people.

5

Chapter 2

LITERATURE REVIEW

2.1 Raw materials for ethanol production:

2.1.1 Lignocelluloses

Lignocellulose presents as a building block of plant cell wall structure (Perez et al.,

2002). Lignocellulosic materials such as agricultural and forest residues, crops and

herbaceous materials in large quantities are available in many countries with various

climatic conditions, making them suitable and potentially cheap feedstock's for

sustainable production of fuel ethanol. The global production of plant biomass, with

over 90% lignocellulose content, is estimated to be about 200×109 tons/year, where

about 8-20×109 tons of primary biomass remain potentially accessible annually (Lin

and Tanaka, 2006).

Lignocelluloses are complex mixtures of carbohydrate polymers, namely cellulose,

hemicellulose, lignin, and a small amount of compounds known as extractives. The

compositional structure of common agricultural residues and wastes is shown in Table

2.1.

Table 2.1 Cellulose, hemicellulose and lignin content in common agricultural residues

and wastes.

Adapted from: McKendry (2002), Prasad et al. (2007), Sun and Cheng (2002).

6

2.1.1.1Cellulose

Cellulose is the main structural polymer in plant cell walls and is found in a very

organized fibrous structure. This linear polymer consists of D-glucose subunits linked to

each other by β-(1,4)-glycosidic bond (Fig. 2.1). Due to this linkage, cellobiose is

established as the repeat unit for cellulose chains. The long-chain polymers (called

elemental fibril) linked together by hydrogen and van der Waals bonds result in a

packed micro fibril. Hemicelluloses and lignin cover the micro fibril. The degree of

polymerization (DP) of native cellulose is in the range of 7,000-15,000. Fermentable D-

glucose can be produced from cellulose by breaking the β-(1, 4)-glycosidic linkages by

the action of acid or enzymes.

Figure 2.1. Schematic illustration of the cellulose chain.

2.2.1.2 Hemicelluloses

Hemicelluloses consist of different monosaccharide units such as pentoses (xylose,

rhamnose and arabinose), hexoses (glucose, mannose and galactose) and unonic acid

(e.g. 4-o-methyl-glucuronic, D-glucuronic and D-galactouronic acids). The backbone of

hemicellulose can be either a homo-polymer or heteropolymer with short branches at

e.g. β - (1, 4) and occasionally β-(1, 3)-glycosidic bonds. In addition, hemicelluloses

contain some degree of acetylation e.g. in heteroxylan. The principal component of

hardwood hemicellulose is glucuronoxylan whereas glucomannan is predominant in

softwood. In contrast to cellulose, hemicellulose an easy hydrolyzable polymer due to

its branched nature and does not forming aggregates even when they are co-crystallized

with cellulose chains.

7

2.1.1.3 Lignin

In contrast to cellulose and hemicellulose, structure of Lignin is a complex, cross-linked

polymer of phenolic compound monomers that form a large molecule structure. It is

present in the cellular cell wall, conferring structural support, impermeability and

resistance against microbial attack and oxidative stress (Perez et al., 2002). There are

three phenyl propionic alcohols as monomers of lignin: coniferyl alcohol (guaiacyl

propanol), coumaryl alcohol (p-hydroxyphenyl propanol) and sinapyl alcohol (syringyl

alcohols). Guaiacyl units are dominant in the softwood while syringyl units are

dominant in hardwood.

Lignin is one of the most complicated natural polymers with respect to its structure and

heterogeneity, which make it extremely resistant to chemical and biological degradation

(Lee, 1997).

Figure 2.2 Structures of monolignols

8

2.2 Olive plant:

Table 2.2 Classification of Olea europaea, common name Olive tree (Omer., 2012).

DOMAIN Eukaryota

KINGDOM Plantae

PHYLUM Anthophyta

CLASS Dicotyledones

ORDER Scrophulariales

FAMILY Oleaceae

GENUS Olea

SPECIES Olea europaea

Olive tree (Olea europae L.) orchards are dominant crops in Mediterranean countries.

Olives are one of main crops in terms of cultivation surfaces across the Mediterranean

basin (Niaounakis and Halvadakis, 2004). The Mediterranean area alone provides

98% of the total surface area for olive tree culture and total productive trees, and 97% of

the total olive production.

Middle-east is the primary diversification centre for olive (Olea europaea L.) in the

Mediterranean basin and Palestine represents one of the countries supposed to have a

very rich germplasm variability, Olive represents the most important cultivation in

Palestine, but unfortunately, and mainly because of the socio-political instability of the

area in the last decades, there are several cultivars grown in Palestine. Olive tree

cultivated areas account for more than 80% of the fruit trees area, namely about 93,000

hectares (PCBS, 2009). Olive trees are grown everywhere in Palestine, but the greatest

productive areas are located in the western and northern West Bank: About 90- 95

percent of the Palestinian olive harvest is used to produce olive oil: In the past decade

average oil production in good years has been around 20,000-25,000 tons. The quantity

of olive oil produced in 2010 reached 23,754 tons (PCBS, 2011). In addition,

Palestinian oil is considered to be of high quality among other olive oils in the world

9

(Omar, 2012). Statistics of production exhibit low yields and sharp periodic alternate-

bearing behaviours (‘on’ and ‘off’ years). This phenomenon, bringing about yield

fluctuations up to 90% between year to year (see Table 2.3).

Tab 2.3 annual productions of olives and olive oil in Palestine (PCBS and MoA, 2009).

Production

Year

Olive oil

(tons)

Olives

(tons)

4.7.22 4427422 4004

447421 2274.1 4001

447402 547.24 4002

27.50 4.7140 4004

127004 4457045 4002

878.0 127011 400.

487442 807425 4008

27.24 407248 4005

2.3 Uses of olive solid waste "jeft"

During the olive oil production large volumes of wastes are generated that vary in

composition depending on which of the three olive oil production systems is used. The

olive itself consists of pulp (75-85% weight), nut (13-23% weight) and seed (2-3%

weight) (Aragon, 2000). Approximately volume of olive solid waste is about 50 to

60% of the olive fruit after oil processing. It can be used as stock feed if it is dry and

destoned, it can be used as a mulch or separated olive stone can be utilised as a fuel

source (Anonymous, 2001).

The organic fraction of agro-wastes (e.g. olive oil wastes, sugar beet pulp, potato pulp,

and potato thick stillage or brewer´s grains) has been recognized as a valuable resource

that can be converted into useful products via microbially mediated transformations.

Organic waste can be treated in various ways, of which bio-processing strategies

resulting in the production of bioenergy (methane, hydrogen, and electricity) are

promising (Khalid et al., 2011).

11

Application of raw olive oil solid waste (OSW) increased soil aggregate stability.

Application of OSW at the rate of 8% has significantly increased soil total organic

nitrogen contents. There are several researches in Mediterranean countries that

evaluated the effects of OSW compost on plants. Alberuque et al (2006) reported

‘alperujo’’ compost had no phytotoxicity, had considerable greater organic matter and

lignin contents than the other two organic amendments tested.

Olive solid waste and OSW compost (OSWC) effects on tomatoes growth was

evaluated by Kavdir and Killi (2008). Application of OSWC increased tomatoes

growth, plant chlorophyll content in Sandy and Loamysoils. The best compost rate was

4% w/w. Compared to olive solid waste, OSWC increased plant length, dry and fresh

weight significantly.

In the Palestinian Territories, the only treatment that is done to the wastes of olive mills

is the partial reuse of the overall produced olive cake for the soap factories. The olive

solid waste is collected and then extracted by hydrocarbons to extract the remaining oil

to be used for producing soap. The olive oil soap is used in the region for bathing and

washing. The remaining olive solid waste (Jeft) is dried and used as burning material to

produce energy for the extraction process in the soap factories. The Jeft is also used

partly for combustion to heat houses during the winter season. Nowadays, the remaining

huge quantities of olive solid wastes (jeft) from the soap factories create environmental

problems. One way to deal with the Jeft is to increase its specific surface area and to use

it as carbon filters. This needs further investigation.

The extracted olive mill solid waste (OMSW) has 30%-45% stones, 15%-30% olive

skin and 30%-50% pulp (Cruz et al., 2006). They are used for the co-generation of heat

and electricity in combustion-turbine cycles or a gas-turbine cycle in the same way as

OMSW. The oil extraction factory usually uses this type of energy for its own drying

process before extraction.

Currently there are several experimental treatments for OMSW using it as a source of

pharmaceutical compounds. A new process based on the hydrothermal treatment of

OMSW led to a final solid enriched in minor components with functional activities

(Lama-Muñoz et al., 2011). Other studies have been carried out using the bacteria

11

like Penibacillus Jamila for the production of exo-polysaccharides with OMSW as

growth media (Ramos-Cormenzana & Monteoliva-Sánchez, 2000). There are two

patented products extracted from 2POMSW: oleanoic acid and maslinic acid. Maslinic

acid is being used for treatment against the human immunodeficiency virus (HIV-1)

(Parra et al., 2009).

OMSW have also been used as feeding for animals. There are several studies about the

digestibility of the protein content in OMSW used as sheep and goat feed (Martín et

al., 2003; Molina Alcaide et al., 2003). Maslinic acid obtained from OMSW added to

the diet of rainbow trout increased growth and protein-turnover rates (Fernández-

Navarro et al., 2008).

The application of OMSW as a fertilizer has also been considered. Although the

vegetation water gives a phytotoxic effect similar to olive mill waste water (OMWW), it

has been observed that the fertilizer effect prevails over the phytotoxic effect when the

dosage is not very high (Sierra et al., 2007). An extremely low quantity of OMSW is

used in these treatments, so none could be used as an integral treatment for this

problematic waste.

Further researches on preparation, enhancement and utilization of activated charcoal

from olive solid waste have been implemented at An-Najah University. The activated

charcoal produced from olive waste is used in water purification from organic and

inorganic contaminants (Shalabia, 2011).

2.4 Microorganisms

2.4.1 Saccharomyces cerevisiae

Scientific name and Authority: Saccharomyces cerevisiae

Common Name: Baker's yeast

Synonyms: Zygosaccharomyces paradoxus (Batschinskaya) Klocker (1824)

Candia robusta Diddens ET Lodder (1942)

Saccharomyces gaditensis Santa Maria (1970)

12

Classification

Table 2.4 Classification of S. cerevisiae (Kurtzman, 1995)

Domain Eukaryota

Kingdom Fungi

Phylum Ascomycota

Sub- Phyllum Saccharomycotina

Class Saccharomycetes

Order Saccharomycetales

Family Saccharomycetaceae

Genus Saccharomyces

Species Cerevisiae

Microorganisms play a significant role in production of ethanol from renewable

resources and thus, selection of suitable strain is essential for the individual process.

Ethanol production is much more challenging and difficult when lignocellulosic and/or

cellulosic materials are to be used as raw materials (van Zyl et al., 2007).

Fermentation of lignocellulosic hydrolyzates involves great challenges: having high

yield and tolerance to high ethanol concentration; the necessity of converting pentose as

well as hexose sugars to ethanol; and resistance to inhibitors present in hydrolysates.

Microorganism as a key factor in fermentation has an important role in meeting these

challenges. Many microorganisms such as yeast (e.g. Saccharomyces and Pichia

species), bacteria (e.g. Escherichia coli, Klebsiella and Zymomonas) and fungi (e.g.

Mucor, Rizhopus and Rhizomucor) have been employed and even genetically modified

to achieve these goals (Abbi et al., 1996; Olsson and Hahn-Hägerdal; 1996, Ingram

et al., 1999; Nigam, 2001, 2002; Millati et al., 2005). However, the larger sizes,

thicker cell walls, better growth at low pH, less stringent nutritional requirements, and

greater resistance to contamination give yeast advantages over bacteria for commercial

fermentation (Jeffries, 2006).

Baker’s yeast, S. cerevisiae, is widely used in ethanol production due to its high ethanol

yield and productivity, no oxygen requirement, and high ethanol tolerance (Olsson and

13

Hahn-Hägerdal, 1993). These unusual capabilities are the result of adaptation to

efficient ethanol production from hexose sugars during thousands of years (Olsson and

Hahn-Hägerdal, 1996). However, S. cerevisiae cannot transport and use xylose as a

substrate, whereas the isomers of xylose (xylulose and ribulose) can be fermented

(Jeffries, 2006). Cell metabolic engineering and modification have been extensively

carried out to give the ability of xylose assimilation to yeast due to the wide availability

of xylose resources. However, the development is still in progress and there is no known

recombinant yeast strain that is efficient enough to ferment glucose, xylose and other

minor sugars in hydrolysates to ethanol (Jeffries, 2006).

Nevertheless, native S. cerevisiae is probably still the best choice for softwood

hydrolysates, where glucose and mannose constituents are dominant among other

sugars. In addition, the native yeasts are inexpensive and widely available. S. cerevisiae

obtained from different sources were used in the current work. A commercial baker’s

yeast (hismaya, Volant, turkey) was used in this work, while different strain of S.

cerevisiae was isolated from different source like: sugarcane juice, grape juice, and

traditional yogurt. Although baker’s yeast was directly used, the strains were kept on

agar plates made of yeast extract 10 g/L, peptone 20 g/L, microbiological agar 20 g/L

and D-glucose 20 g/L as an additional carbon source (Purwadi, 2006).

2.5 Previous Studies

Ethanol production from cellulosic materials may offer a solution to some of the recent

environmental, economic and energy problems facing in the world. Presently,

agricultural residues are being used in many of the distilleries as its ideal sugar content

suitable for production of ethanol. The high amounts of reducing sugars are required for

production of ethanol. Similar sugars are also being observed in sweet sorghum, sugar

beet and rain tree pods etc. which can form alternate substrate for ethanol production.

The lignocelluloses and olive solid waste rich substrates are also found to be potential

substrates for ethanol production. The various biomass substrates having potential for

ethanol production are being reviewed here.

14

2.5.1 Substrate

Ethanol can be obtained from energy crops and lignocellulosic biomass. The complexity

of the production process depends on the feedstock, several reviews have been

published on the theme of fuel ethanol production especially from lignocellulosic

biomass.

Pineapple wastes (containing 11.7 % soluble sugars) was fermented for ethanol

production by Bankoffi and Han (1990) and obtained 0.8% in 48 hours.

Cofermentation of glucose and xylose with immobilized Pichia stipitis and S. cerevisiae

was studied by Grootjen et al. (1990) and glucose conversion was found to be 0.13 g g-

1 h

-1. Czarnecki and Grajek (1991) studied the influence of temperature and

incubation time of starch gelatinization in wheat, rye and maize grain and found that rye

starch was the most susceptible to enzymatic hydrolysis and produced highest alcohol

yield of 65 %.

Lynd et al. (1991) mentioned about obtaining of 400 billion litres of ethanol by

microbial conversion of the sugar residues present in waste paper and yard trash and

from US landfills. The waste paper was treated with steam in pressure vessel at 170-220

°C and then enzymatically saccharified and fermented for ethanol production by Capek

et al., (1992) and obtained 460 liters of ethanol per ton of waste paper which was an

increase of 29 % over control.

Smith and Buxton (1993) showed that the sweet sorghum had potential to produce

3,100-5,235 L per ha and grain maize has the capacity to produce about 2,340L per ha

Mamma et al. (1996) studied on simultaneous saccharification and fermentation of

sweet sorghum carbohydrates to ethanol by a mixed culture of Fusarium oxysporum

and S. cerevisiae. The optimum yield of ethanol was 5.2-8.4 %. Sheorain et al. (2002)

stated that sorghum can potentially give good yield of alcohol of about 380 to 390 liter

absolute alcohol from 1 ton of grains. Kim and Dale (2003) showed that wasted

sorghum grains alone had potential to produce 1.4 GL of bioethanol along with

sorghum straw it could produce 4.9 GL of bioethanol globally.

15

Hammond et al. (1996) conducted the laboratory experiment to assess the ethanol

production from waste banana as a whole fruit, pulp, peel and obtained ethanol yield of

0.91, 0.082 and 0.006 l/kg respectively. Duff and Murray (1996) carried out

bioconversion of forest products industry such as pulp and paper industry waste to fuel

ethanol. Miyamoto (1997) obtained 150 - 200 litres of alcohol from 720 kg of raw

material like sugarcane bagasse, rice straw and forest waste in pilot scale unit.

Biochemical studies on bioconversion of rice straw to ethanol was conducted by

Sandhu et al. (1998) and revealed a maximum ethanol yield of 0.34 g/g based on sugar

utilized after fermentation efficiency of 66.87 % was obtained. Ethanol yield of 2.90 %

(v/v) from damaged sorghum and 2.09 % (v/v) by damaged rice grain was obtained by

Suresh et al. (1999a) by simultaneous saccharification and fermentation. An

experiment was conducted by Ahring et al. (1999) for ethanol production from wet

oxidized wheat straw by Thermoanaerobacter mathranii and they obtained highest

ethanol yield using hydrolysates produced at conditions of 3 bar oxygen pressure and 2-

6.5 g/l sodium carbonate. Alcoholic fermentation of cheese whey permeate were

investigated by Dominguez et al. (2001) using a recombinant flocculating S. cerevisiae

expressing the LAC4 and LAC12 genes of Kluyveromyces marxianus enabling for

lactose metabolism. They obtained about 10 g/l/h ethanol.

Bvochora et al. (2000) investigated on ethanol production from mixture of sweet

sorghum juice and sorghum grains, fermentation was carried out 96 h, using malted and

Unmalted milled sorghum grains from sorghum cultivars DC-75 and SV-2. Maximum

ethanol level were about 16.8 % (v/v) and 11 % (v/v) for media containing malted and

unmalted milled sorghum grains.

Ramanathan (2000) obtained an ethanol yield of 42 l kg-1

of feed stock by

fermentation of root crops namely cassava, potato, yam and sweet potato. Sharma et al.

(2002b) used amylolytic yeast strain S. cerevisiae SJ 31 to hydrolyse potato starch and

they reported 48 % hydrolysis and 3.4 % alcohol production with fermentation

efficiency of 91 %. Shiva et al. (2001) conducted an experiment for fermentation of

agriculture waste such as jowar stalk and left over corn stalk to ethanol. Steam

explosion of potato for the efficient production of alcohol was experimentally studied

16

by Kobayashi et al. (2002). The amount of water soluble starch increased with the

increase in steam pressure. The exploded potato was later hydrolysed by amylolytic

microorganism (Aspergillus awamorii) and fermenting microbe (S. cerevisiae). The

maximum ethanol produced was 4.2 g l-1

.

Lang et al. (2001) used a recycle bioreactor for bioethanol production from raw wheat

starch particles. They found that 95% of the starch particles were converted to ethanol

with 24 hours and the ethanol yield was 0.48g ethanol / g glucose.

Alcoholic fermentation of an enzymatic hydrolysate of exploded rice straw were studied

experimentally by Nakamura et al. (2001) and they obtained ethanol yield of about 86

% (w/w) by using Pichia stipitis yeast strain. An experiment were conducted by

Sharma et al. (2002a) for enzymatic saccharification of pretreated sunflower stalks and

they obtained maximum enzymatic saccharification of 57.8 % by treating 5 % (w/w)

pretreated sunflower stalks. The experimental trials of the dilute acid hydrolysis of

bagasse hemicellulose to produce xylose, arabinose, glucose, acid soluble lignin and

furfural were conducted using a temperature controlled digester by Lavarack et al.

(2002). They obtained the xylose yield of about 220 mg/g of solid material.

Sharma et al. (2002b) isolated yeast from different sources and screened for growth,

ethanol production and gluco-amylose activity. The selected yeast strain SM-10 showed

maximum gluco-amylose activity of 80 unit/ml and ethanol production from starch.

House et al., (2000) calculated that 1.2 - 2.3 million metric tons sorghum was used for

ethanol production, 3.7–7.5 % of the grain for ethanol production was sorghum, and

0.13–0.25 billion gallon (0.49–0.95 billion liters) of ethanol originated from sorghum.

Kim et al. (2003) conducted an experiment on corn stover. Pretreatment with aqueous

ammonia in a flow-through column reactor was employed. They obtained 99 %

enzymatic digestibility of cellulose with 60 FPV/g of glucan enzyme loading, within 90

mm of the process. Schell et al. (2004) studied on pilot scale production of bioethanol

from corn fiber feed stock using dilute sulphuric acid hydrolysis. They obtained 48 %

glucose yield after the acid hydrolysis. The conversion of bark-rich biomass mixture

into fermentable sugar by two stage dilute acid catalyzed hydrolysis. They obtained

glucose yield of 13.6 g/100 g of original dry feed stock.

17

Today most fuel ethanol is produced by either the dry grind or wet mill process. Current

technologies allows for 2.5 gallons (wet milling process) to 2.8 gallons (dry milling

process) of ethanol per bushels of corn (Bothast and Sclicher, 2005).

Sebastian (2008) reported the conversion of olive stones to ethanol. Olive stones were

pre-treated using high-pressure hot water then enzymes that degrade plant matter and

generate sugars were added. The hydrolysate obtained from this process was then

fermented with yeasts to produce ethanol. Yields of 5.7 kg of ethanol per 100 kg of

olive stones have been obtained. The low cost of transporting and transforming olives

stones make them attractive for biofuels production.

Olive cake was evaluated as a feedstock for ethanol production. To this end, the

lignocellulosic component of the olive cake was dilute-acid pretreated at a 13.5 % olive-

cake loading with 1.75 % (w/v) sulfuric acid and heating at 160°C for 10 min. This was

followed by chemical elimination of fermentation inhibitors. Soluble sugars resulting

from the pretreatment process were fermented using E. coli FBR5, a strain engineered

to selectively produce ethanol. 8.1 g of ethanol/L was obtained from hydrolysates

containing 18.1 g of soluble sugars. Increasing the pretreatment temperature to 180°C

resulted in failed fermentations, presumably due to inhibitory by-products released

during pretreatment (El Asli and Qatibi, 2009).

2.5.2 Pretreatment Methods

Conventional production of ethanol from cellulose via fermentation involves a complex

process of pretreatment in attempt to recover a maximum amount of sugars from the

hydrolysis of cellulose and hemicellulose, and to ferment them into ethanol.

Pretreatment is required to alter the biomass macroscopic and microscopic size and

structure as well as its submicroscopic structural and chemical composition and to

facilitate rapid and efficient hydrolysis of carbohydrates to fermentable sugars. The

pretreatment aims to increase pore size and reduce cellulose crystallinity (Petrova &

Ivanova, 2010). The pretreatment methods employed are physical, chemical and

biological. The physical pretreatment like ball milling and compression milling

decreases the degree of crystallinity and also molecular weight of cellulose (Tassinari

http://scialert.net/fulltext/?doi=tasr.2011.410.425&org=10#32581_an

18

et al., 1980). The dilute acid hydrolysis of certain softwood species is found to be

suitable for obtaining maximum soluble sugars (Kim et al., 2003).

Figure 2.3 process of ethanol production (Dashtban M., Schraft H., and Qin W.,

2009).

2.5.3 Physical Pretreatment

Waste materials can be comminuted by a combination of chipping, grinding and milling

to reduce cellulose crystallinity. This reduction facilitates the access of cellulases to the

biomass surface increasing the conversion of cellulose. The energy requirements of

mechanical comminution of lignocellulosic materials depend on the final particle size

and biomass characteristics. Although mechanical pretreatment methods increase

cellulose reactivity towards enzymatic hydrolysis, they are unattractive due to their high

energy and capital costs (Ghosh and Ghose, 2003).

The adsorption of cellulase on cellulose and a lignaceous residue were examined by

using cellulase from Trichoderma reesei by Ooshima et al. (1990). They used

19

hardwood and pretreated with dilute sulphuric acid along with explosive

decompression. The data showed that the pretreatment at a higher temperature results in

the more enzymatic adsorption on cellulose fraction and less on lignaceous residue

fraction and the maximum sugar yield obtained was 68.4 % after enzymatic

pretreatment.

An attempt was made to enhance enzymatic hydrolysis of wheat straw by Vallander

and Eriksson (1991). They found that steam pretreatment of wheat straw solubilized

most of the xylan present and the yield of monomeric sugars was found to be 69 % after

enzymatic pretreatment

Effects of time, temperature and pH during the steam explosion of poplar wood were

studied by Excoffier (1991) with the aim of optimizing both pentoses recovery and

enzymatic hydrolysis efficiency. They obtained 70 % of xylose as monomers and 70 %

of glucose after acid followed by enzymatic hydrolysis. The effect of hammer milling or

ball milling of wheat straw on pyrolysis, at up to 300°C in an inert atmosphere was

studied by Koullas et al. (1992) using a modified tubular laboratory reactor. The

process yield was determined on a dry basis and proximate analysis (Volatile matter

content and calorific value). They found that solid yield was negatively affected by the

crystallinity reduction during ball milling which improved the properties of the solid

fuel produced by increasing its calorific value from 18 to 20 mg/kg and by drastically

reducing value from 98 to 67 % on prepyrolyzed basis.

2.5.4 Microwave

Irradiation can cause significant breakdown of the structure of lignocellulosic wastes

(LSW). Microwave irradiation at a power of up to 700 W at various exposure times

resulted to weight loss due to degradation of cellulose, hemicellulose and lignin, and the

degradation rates are significantly enhanced by the presence of alkali (Zhu et al.,

2005a, 2005b, 2006). In addition, gamma radiation has been shown by Yang et al.

(2008) to cause significant breakdown of the structure of powder of 140 mesh wheat

straw, leading to weight loss and glucose yield of 13.40% at 500 k Gy.

The earliest known study involving microwave pretreatment examined the effect of

microwave radiation on rice straw and bagasse immersed in water reported an

21

improvement in total reducing sugar production by a factor of 1.6 for rice straw and 3.2

for bagasse in comparison to untreated biomass (Ooshima et al., 1984). Microwave

pretreatment of sugarcane bagasse and rice hulls soaked in water followed by lignin

extraction was reported to yield 77-84% of total available reducing sugars (Azuma et

al., 1984). A similar study involving microwave pretreatment of rice straw and

sugarcane bagasse followed by lignin extraction reported a yield of 43-55% of total

available reducing sugars (Kitchaiya et al., 2003). Microwave-based pretreatment of

rice straw soaked in dilute alkali resulted in glucose yield and total carbohydrate

conversion of 65 % and 78 % respectively (Zhu et al., 2005a). Recently studies on

microwave-based alkali pretreatment of 86 switchgrass have also been reported and are

discussed later in this study (Hu and Wen, 2008; Keshwani et al., 2007).

Keshwani et al. (2007) have to examine the feasibility of microwave pretreatment to

enhance enzymatic hydrolysis of switchgrass and to determine the optimal pretreatment

conditions. Switchgrass samples immersed in water, dilute sulfuric acid and dilute

sodium hydroxide solutions were exposed to microwave radiation at varying levels of

radiation power and residence time. Pretreated solids were enzymatically hydrolyzed

and reducing sugars in the hydrolysate were analyzed. Microwave radiation of

switchgrass at lower power levels resulted in more efficient enzymatic hydrolysis. The

application of microwave radiation for 10 minutes at 250 watts to switchgrass immersed

in 3% sodium hydroxide solution (w/v) produced the highest yields of reducing sugar.

Results were comparable to conventional 60 minute sodium hydroxide pretreatment of

switchgrass. The findings suggest that combined microwave-alkali is a promising

pretreatment method to enhance enzymatic hydrolysis of switchgrass.

Ravoof et al. (2012) were studied the effect of microwave heating in dilute nitric acid

pretreatment, enzymatic hydrolysis experiments were performed using convectional

heat dilute nitric acid pretreated rice straw. Microwave assisted dilute nitric acid

pretreatment could enhance the enzymatic digestibility of rice straw by 14% when

compared to the convection heating mode. It also reduces the pretreatment time from 60

min to 7 min and also highest yield of reducing sugars were obtained at 2% of acid

concentration.

21

Chittibabu et al. (2011) were to optimize the microwave assisted alkali pretreatment

and enzymatic hydrolysis of banana pseudostem (BPS) for the production of bioethanol.

Pretreatment of BPS was performed at different alkali concentration, liquid-solid ratio,

temperature and microwave exposure time. Enzymatic hydrolysis of pretreated BPS was

done at constant cellulase enzyme loading and yield of reducing sugars (YRS) with

respect to time was observed. It was found that when BPS was pretreated by 10 %

NaOH with 4:1 liquid to solid ratio at 90°C for 8 min, the yield of reducing sugars

reached 84 % by enzymatic hydrolysis of 110 h with cellulase enzyme loading of 30

FPU/g of solid. Compared with convection mode of heating of alkali pretreatment,

microwave assisted alkali pretreatment and enzymatic hydrolysis was more effective for

BPS.

Li et al. (2012) were examined the feasibility of microwave assistant KOH

pretreatments to enhance enzymatic hydrolysis of bamboo. Pretreatment was carried out

by immersing the bamboo in KOH (12% and 8% w/w bamboo) solutions and exposing

the slurry to microwave radiation power of 400 W for 30min. Chemical composition of

the pretreated substrates and spent liquor was analyzed. Pretreated substrates were

enzymatic hydrolyzed, and glucose and xylose in the hydrolysate were analyzed. The

results showed that the pretreated substrate with microwave assisted KOH had

significantly higher sugar yield than the untreated samples. The fermentation inhibitors

formic acid, furfural, 5-(hydroxymethyl) furfural (HMF) and levulinic acid were much

lower than acid pretreatment reported.

2.5.5 Chemical pretreatment

Chemical pretreatments that have been studied to date have had the primary goal of

improving the biodegradability of cellulose by removing lignin and/or hemicellulose,

and to a lesser degree decreasing the degree of polymerization (DP) and crystallinity of

the cellulose component. Chemical pretreatment is the most studied pretreatment

technique among pretreatment categories. Chemical pretreatment techniques, including

catalyzed steam-explosion, acid, alkaline, ammonia fiber/freeze explosion, organosolv,

pH-controlled liquid hot water, and ionic liquids pretreatments (Zheng et al, 2009).

22

An alternative approach was tested by Teixeira et al. (1999a, b) which employ a silo

type system by introducing the feedstock (bagasse or hybrid poplar) in plastic bags to

which a peracetic acid solution was added. Cellulose conversion of pretreated material

reached 93.1% during 120 h using 21 wt% acid concentrations or during 24 h using 60

wt% acid concentrations.

Ballesteros, et al. in 2001 were assayed olive pulp and fragmented stones as substrate

for ethanol production by the simultaneous saccharification and fermentation (SSF)

process. Pretreatment of fragmented olive stones by sulfuric acid-catalyzed steam

explosion was the most effective treatment for increasing enzymatic digestibility;

however, a pretreatment step was not necessary to bioconvert the olive pulp into

ethanol. The olive pulp and fragmented olive stones were tested by the SSF process

using a fed-batch procedure. By adding the pulp three times at 24-h intervals, 76% of

the theoretical SSF yield was obtained. Experiments with fed-batch pretreated olive

stones provided SSF yields significantly lower than those obtained at standard SSF

procedure. The preferred SSF conditions to obtain ethanol from olives stones (61% of

theoretical yield) were 10% substrate and addition of cellulases at 15 filter paper units/g

of substrate.

The olive stones were delignified with sodium hydroxide at 50°C and simultaneous

velocity of 250 rpm. It has undergone enzymatic hydrolysis. Various quantities of

cellulase in different concentrations of achieved cellulose materials have been used. The

findings of the study indicated that, although using more enzymes (80 cc) yields more

sugar, the amount of sugar produced will be half of the quantity (40 cc) used, with

regard to high cost of enzymes. The velocity of reactions dropped dramatically after 8 h.

With an 8-h double cycle of hydrolysis and enzymatic recovery, sugar concentration of

about 20 g/l is achieved using 200 g of primary cellulose compounds (100 g per cycle)

and 40 cc of enzymes. Thus, with 0.55 cc of alcohol efficiency to 1 g of sugar, 11 cc of

alcohol is produced in fermentation (Ahmadi et al., 2010).

Mishra et al. 2011 deals with the bioconversion of cellulose from press cakes of

Jatropha oilseeds into ethanol by using the methods of acid pretreatment, hydrolysis and

fermentation by S. cerevisiae. The process includes the pretreatment method of the

finely ground cellulosic solid oilseed cake with dilute sulphuric acid and heating the

23

mixture at a high temperature to break the crystalline structure of the lignocellulose to

facilitate the hydrolysis of cellulosic component by dilute acids. The hydrolysis of the

cellulose content into sugars is done by dilute acid hydrolysis that involves incubation

period at a high temperature for 72 hours. Finally, the fermentation of the hydrolyzed

waste is done using S. cerevisiae under proper incubation conditions to produce ethanol.

About 80 % ethanol was recovered as a result of the process (Mishra et al, 2011).

Shalabia, (2011) was study Jeft and extracted cellulose was subjected to hydrolysis to

sugar by acids such as HCl and H2SO4. Highest yield of 64% sugar was obtained using

acid alone (concentrated HCl). The yield of sugar was enhanced to about 49.89% using

diluted HCl (10%) with Lewis acid ZnCl2 (20%). These results indicate that cellulose

obtained from Jeft has a microcrystalline structure. These results are consistent with the

results obtained from Intrinsic Viscosity (IV), Scanning Electron Microscope (SEM),

and Infrared spectroscopy (IR) studies which all showed that cellulose has a

microcrystalline structure. Medium rate of hydrolysis could be attributed to the high

crystallinity of cellulose which reduces the accessibility of hydrolyzing agent to

cellulose structure.

Influence of different pretreatment methods on sugar conversion and bioethanol

production were investigated by kikas et al., (2012). Different dilute acid and alkaline

pretreatment methods are compared to determine the best pretreatment method to give

the highest glucose and ethanol yields under the mild operating conditions. Dilute

sulfuric acid, hydrochloric acid, nitric acid and potassium hydroxide solutions are used

for pretreatment in combination with enzymatic hydrolysis. Results indicate that the

highest cellulose-to-glucose conversion rate of 316.7 g kg-1

of biomass is achieved by

the pretreatment with nitric acid. The lowest glucose concentration of 221.3 g kg-1

is

achieved by hydrochloric acid (kikas et al., 2012).

Senkevich et al., (2012) were to investigate the effect of thermochemical pre-treatment

of OMSR, on the final ethanol yield from the yeast Pachysolen tannophilus. Nine

different types of OMSR-based substrates were tested i.e. Raw OMSR, hydrolysates

generated from pretreated OMSR with NaOH (0.5 %, 1.5 % w/v) and H2SO4 (0.5 %, 1.5

% v/v), and pretreated OMSR with NaOH (0.5 %, 1.5 % w/v) and H2SO4 (0.5 %, 1.5 %

24

v/v) whole biomass. It was shown that in all cases pretreatment enhanced the

consumption of carbohydrates as well as ethanol final yields.

2.6 Fermentation

Fermentation is one of the oldest biochemical processes known. It is used to produce a

variety of products, including foods, flavorings, beverages, pharmaceuticals, and value-

added chemicals like ethanol. The future of the fermentation industry with respect to

bioethanol production depends on three major strategies. First, its ability to exploit a

variety of microorganisms that are capable of efficient ethanol production by

fermentation; second, to utilize various substrates such as sugars, starches or celluloses

derived from a variety of different sources; and third, since utilizing starches and

celluloses requires enzymes, to locate, develop and investigate relatively inexpensive

sources of enzymes.

Starch granules from cassava, corn, bagasse and potato have been used (Lang et al.,

2001). Ueda et al. (1981) used raw cassava root starch for fermentation at pH 3.5 and

30°C for 5 days to produce ethanol with yields between 82.3% and 99.6% of the

theoretical value. Mikuni et al. (1987) performed batch runs for simultaneous

saccharification and fermentation of corn starch granules using S. cerevisiae, at pH 5.0

and 30°C and achieved ethanol yields between 63.5% and 86.8% of the theoretical

value.

Lee et al. (1995) studied ethanol production by fermentation using tapioca starch. They

reported that liquefaction and saccharification of tapioca starch resulted in a glucose-

maltose mixture containing approximately 92 % glucose and 8 % maltose. They

proposed a model that accurately represents ethanol production from a mixture of

glucose and maltose as substrates.

Saccharification of raw flour starch by Bacillus subtilis and fermented by S. cerevisiae

were investigated by Suresh et al., (1999b). The damaged grain sample comprised 50

% damaged and 50 % sound grains, and the damaged portion included kernels that were

broken, cracked, attacked by insects or discolored. The high-quality sorghum flour was

obtained locally. It was found that using a level of 25 % (w/v) substrate yielded 3.5 %

25

(v/v) ethanol from the damaged grain sample. For comparison, the high-quality

sorghum flour yielded 5.0 % (v/v) ethanol (Suresh et al., 1999b).

Sree et al. (1999) reported ethanol production by simultaneous saccharification

fermentation (SSF) of wheat products using S. cerevisiae, which was 44.2 g ethanol l-1

when fine wheat flour was used as substrate, and 34.1 g l-1

using damaged wheat flour.

Lee et al., (1992) studied ethanol production using Zymomonas mobilis and slurries

containing 100 g l-1

of sago starch and found 40 g l-1

of ethanol. An experiment was

conducted for conversion corn starch to fuel ethanol which was 72.2 g l-1

ethanol

produced in 120 minute residence time (Krishnan et al., 1999).

A maximum alcohol yield in three days during fermentation of yam to ethanol by S.

cerevisiae was observed by Ramanathan (2000). Ethanol production on a pilot scale

for the conversion of high solid saccharification of corn mash to ethanol by continuous

fermentation and CO2 stripping were demonstrated by Taylor et al. (2000). Ethanol

production by co-culture were studied by Verma et al. (2000) using S. diastaticus and

S. cerevisiae strain-21 in raw unhydrolysed starch which yielded ethanol of 48 % higher

(24.8 g l-1

) than that obtained with monoculture of S. diastaticus (16.8g l-1

). Amutha

and Gunasekaran (2000) used mixed culture of an amylolytic yeast strain S.

diastaticus and Z. mobilis for improved ethanol production from cassava starch. The

ethanol yield in mixed culture was 36.5 g l-1

that is higher than that of monoculture

(24.1 g l-1

). Four strains of Z. mobilis were screened by Panesar et al. (2001) for their

ability to produce ethanol from molasses medium at pH 6.

Various authors have reported about this early glucose extinction during the SSF, using

soluble starch (Fujii et al., 2001) or raw cassava starch (Roble, 2003) as substrate and

immobilized yeast for fermentation. The nutrient starvation might play an important

role in the saccharification performance (Suresh et al., 1999b).

Harikrishna et al. (2001) carried out simultaneous saccharification and fermentation

(SSF) to produce ethanol from lignocellulosic wastes (Sugarcane leaves) using

Trichoderma reesei cellulose and yeast cells. Kluyveromyces fragile NCIM 3358

performed better than S. cerevisiae NRRL – Y – 132 in SSF process and resulted in

26

high yield of ethanol 2.5 – 3.5 % (W/V). Increased ethanol yields were obtained when

the cellulose was supplemented with β – glucosidase.

Pretreatment of olive pulps was pretreated in a laboratory scale stirred autoclave at

different temperatures (150–250°C) for Olive pulp. Pretreatment was evaluated

regarding cellulose recovery, enzymatic hydrolysis effectiveness ethanol production by

a simultaneous saccharification and fermentation process (SSF), and phenols recovery

in the filtrate. The pretreatment of olive pulp using water at temperatures between

200°C and 250°C enhanced enzymatic hydrolysis. Maximum ethanol production (11.9

g/L) was obtained after pretreating pulp at 210°C in a SSF fed-batch procedure.

Maximum hydroxytyrosol recovery was obtained in the liquid fraction when pretreated

at 230°C (Ballesteros, et al. 2002).

Sharma et al. (2002a) used S. cerevisiae strain SJ-31 which was potential for ethanol

production from starch substrate because of its ability to produce amylase and gluco-

amylose .The ethanol produced was 3.4 % with fermentation efficiency of 91 %. Chen

et al. (2008) investigated that Surface-engineered yeast S. cerevisiae co displaying

Rhizopus oryzae gluco-amylose and Streptococcus bovis α -amylase on the cell surface

was used for direct production of ethanol from uncooked raw starch. The final ethanol

concentration could reach 53 g l-1

in 7 days.

A 100 % respiration-deficient nuclear petite amylolytic- S. cerevisiae NPB-G strain was

generated, and its employment for direct fermentation of starch into ethanol was

investigated. In a comparison of ethanol fermentation performances with the parental

respiration-sufficient WTPB-G strain, the NPB-G strain showed an increase of ca. 48%

in both ethanol yield and ethanol productivity (Oner et al., 2005).

Neves et al. (2006) studied that fermentation performance of low-grade wheat flour

(LG) and wheat bran (WB) and compared to wheat flour (WF) α- amylase or cellulase

was used for liquefaction, followed by simultaneous saccharification and fermentation

(SSF) by gluco-amylose and Z. mobilis. The final ethanol concentration, overall

productivity and yield obtained from LG (51 .4 g ethanol l-1

, 2.72 g ethanol l-1

h-1

and 0

.17 g ethanol g-1

flour, respectively) were considerably higher compared to WB (18.1 g

27

l-1

, 1.09 g l-1

h-1

and 0.02 g g-1

) . High low-grade fermentation rates, reaching the

highest ethanol productivity (4 .4 g l-1

h-1

) within 6 h of SSF.

Cot et al. in 2007 performed aerated fed-batch fermentation on 2% glucose with

S. cerevisiae strain CBS 8066. The production phase was not coupled to the growth

phase and they found that 20 % (v/v) ethanol was produced in 45 hours (Cot et al.,

2007). Hill et al. (1990) reported results of 14 batch runs performed on glucose at a

temperature of 30°C and a pH of 4.0, using S. cerevisiae strain NRRL Y132. They

modeled the data and found the best value of the Monod constant to be 2 g/L.

In industry, molasses, a by-product of the sugarcane industry, is the most widely used

sugar for ethanol fermentation. This molasses contains approximately, by weight, 35 –

40 % sucrose, 15 – 20 % invert sugars such as glucose and fructose and 28 – 35 % non-

sugar solids (Grylls homepage, 2012). Govindaswamy et al. (2007) performed

fermentation experiments on glucose and xylose alone as well as on combinations of

both glucose and xylose. They obtained maximum specific growth rates of 0.291 h-1

and

0.206 h-1

for experiments performed on 20 g/L glucose and 20 g/L xylose, respectively.

In medium containing combinations of glucose and xylose, they found that glucose was

exhausted first followed by xylose.

Fermentation of undiluted olive pulp hydrolysate (OPH) resulted in the maximum

ethanol produced (11.2 g/L) with productivity of 2.1 g/L/h. Ethanol yields were similar

for all tested OPH concentrations and were in the range of 0.49-0.51 g/g. Results

showed that yeast could effectively ferment OPH even without nutrient addition,

revealing the tolerance of yeast to olive pulp toxicity. Because of low xylan (12.4%) and

glucan (16%) content in olive pulp, this specific type of olive pulp is not a suitable

material for producing only ethanol and thus, bioethanol production should be

integrated with production of other value-added products (Georgieva and Ahring.,

2007).

The commercial enzyme treated hydrolysate of fresh and spoilt sorghum grains was

inoculated with fermentative organisms. The maximum ethanol yield was 28.13 g l-1

in

case of Saccharomyces diastatitcus NCIM-3392 followed by S. cerevisiae HAU strain

28

(21.09 g l-1

) with respect to fresh sorghum grains. In insect damaged sorghum grains,

S. diastaticus NCIM-3392 produced 23.47 g l-1

followed by S. cerevisiae HAU strain

19.04 g l-1

, respectively. Further optimization of parameters such as pH and nutrient

supplementation were carried out to enhance yields of ethanol (Nagesha, 2009).

29

Chapter 3

Materials and Methods

3.1. Materials

3.1.1 Apparatus

The Apparatus used in this study are listed in Table 3.1.

Table 3.1 List of the apparatus used in this work

Apparatus Manufacture Country

Shaking Incubator

N-Biotech

Korea

Shaker and incubator

Biological safety cabinet

Centrifuge combi 514r Hanil science industrial

Vertical pressure steam sterilization

Bouxun

China

Heat drying oven

Microwave oven JAC

Light microscope

Analytical balance (max 150g-d 0.005g) ae-ADAM equipment

U.S.A

Analytical balance (Max 120g- d 0.0001g)

pH/mV Meter Azzota

Vortex (Turbo mixer) LW scientific Georgia

Refrigerated cabinets “Medilow” 0 °C Up

To 15 °C. Jp selecta s.a Spain

Spectrophotometer(CT-2200) Chrom Teck Taiwan

Orbital Shaker Boeco Germany

31

3.1.2 Reagents

The reagents used in this study are listed in Table 3.2.

Table 3.2 List of the reagents used in this work

Reagents Manufactures Country

Hydrochloric acid Himedia

India Tartaric acid

Sulphanilic acid (white) Oxford laboratory reagent

Ethyl alcohol

Frutarom

"Occupied Palestine"

Glycerin

Sodium hydroxide

Chloramphenicol birzeit –Ltd company Palestine

Ethanol Assay Kit Biovision USA

Glucose assay kit Diasys Germany

3.1.3 Culture media

The culture media that were used are listed in Table 3.3.

Table 3.3 Suppliers for culture media

Media Manufactures Country

Glucose

Himedia

India

Maltose

Lactose

Galactose

Xylose

Sucrose

Yeast extract

Peptone

Agar agar

31

3.1.4 Microorganisms

Microorganisms have important role in ethanol production. In this work S. cerevisiae

was isolated from three different sources such as grape juice, sugarcane juice, and

yogurt. Commercial yeast species of S. cerevisiae (Hismaya, Turkey) and S. cerevisiae

(Volant , Turkey) were used as a control strain.

3.1.5 Media

The culture media used in this work was yeast pepton (YP) medium (10 g/L yeast

extract, 10 g/L peptone, 20 g/L agar) supplemented with different carbon sources at the

concentration of 20 g/L (glucose, sucrose, galactose, maltose, lactose, xylose) with or

without the addition of 80 mL/L ethanol. The medium YP supplemented with 20 g/L

glucose (YPG) were also supplemented with 6 g/L tartaric acid, 30 mg/mL

chloramphenicol for isolation strain.

3.2 Methods

3.2.1 Sample collection and isolation:

The yeasts (S. cerevisiae) were isolated from different sources of grapes juice,

sugarcane juice, and traditional yogurt. Each juice exposed to air for 24 h and by using

techniques such as serial dilution and spread plate method using yeast peptone glucose

agar medium (YPG). The samples were plated on YPG and incubated at 30°C for 72

hours. After incubation, the colonies were plated on YPG medium supplemented with

30 mg/mL chloramphenicol and incubated at the same conditions (Thais et al., 2006).

Pure culture of the colonies were prepared on YPG agar media by keeping the plate at

300C for at least 3 days and maintained on YPG agar slants at 4°C. Yeast cultures were

stored in 40% sterile glycerol at -80°C. The strains were sub cultured to YPG agar

media and incubated at 30°C for 3 days and then used to inoculate preculture broths.

The preculture broth was prepared by inoculating 10 ml YPG media broth in test tube

with a loop full of the cultured yeast and incubated at 30°C for 12 hours then we put 10

ml of inoculums to 40 ml of YPG media broth and incubated at 300C for 48 h. When the

density of the yeast cells in the liquid medium was adequate, suspension of S. cerevisiae

32

at OD 660 = 0.1 was used as the inoculum in the fermentation medium (Mishra et al.,

2011).

3.2.2 Inoculum preparation:

The isolated yeasts and the commercial strain were inoculated in YPG broth and

incubated at 30°C for 12 h with constant shaking at 110 rpm. After incubation, a

suspension was prepared and adjusted to an optical density of 0.1 at 660 nm. An aliquot

of 50 mL was plated on specific broth media used for carbon source assimilation,

temperature tolerance test, and ethanol tolerance test. The volume of suspension (10 ml

of inoculum in 40 ml of specific broth media) was used that able to provide an

absorbance between 0.1 and 0. 2 at OD 660 nm (Thais et al., 2006).

3.2.3 Characterization of the selected yeast isolates:

Yeast isolates were identified based on the morphological characters (Kreger-Van Rij,

1984; Mesa et al., 1999) and physiological characters.

3.2.3.1 Morphological characterization:

The isolated strains were cultured by inoculation of 10ml of preactivated culture in 100

mL of sterile YPG media broth in 250 mL conical flask, incubated at 30°C for 48 h and

examined for vegetative cells shape and budding pattern under phase contrast

microscope (Olympus, Japan).

3.2.3.2 Physiological characterization:

The isolated strains along with standard S. cerevisiae were screened for their growth on

different carbon sources such as described by Wickerham (1948). Tolerance to ethanol

was performed by using the standard protocols (Bowman and Ahearan, 1975).

1) Carbohydrate source assimilation test:

Yeast fermentation broth medium was used for identification of yeast based on the

utilization of various carbohydrates (Glucose, Galactose, Maltose, Xylose, Sucrose and

Lactose). In this test, 50 ml of YP media, each containing a specific carbohydrate was

33

inoculated with yeast isolate. Incubated at 30°C for 96 hours with constant shaking at

150 rpm. Samples were taken approximately after 24, 48, 72 and 96 hours. Yeast

growth was measured in these samples as turbidity by using of a spectrophotometer

(Chrom Teck, UV-1601, Taiwan) at a wavelength of 660 nm.

2) Tolerance to ethanol:

Tolerance of yeast stains to ethanol was tested in comparison with the standard strain S.

cerevisiae. 10 ml of 24 h old culture was inoculated in 100 ml YPG broth and subjected

to different concentrations of ethanol (5%, 8%, 10%, 13% and 15% ml/L) in the YPG

broth and incubated at 30°C for 4 days with constant shaking at 150 rpm. After

incubation, the population was estimated by a spectrophotometer (Chrom Teck, UV-

1601, Taiwan) at a wavelength of 660 nm at different time intervals. The growth curves

were constructed to find out the best growth at specific ethanol concentration (Khaing

et al., 2008).

3.4 Optimization of the environmental condition:

3.4.1 Optimization of pH

The different strains of organisms were further studied to know the optimum pH for

working and then for bioethanol production. The different isolated strains and

commercial strains were inoculated in 50 ml of liquid YPG media broth with different

pH (4, 4.5, 5 and 5.5) at 30°C for 72 hours with constant shaking at 150 rpm. A pH

meter was used to measure the pH of each solution during and after establishment

(Tahir et al., 2010b).

3.4.2 Optimization of temperature

The different isolated strains and commercial strains were inoculated in 50 ml of liquid

YPG media broth. The inoculums were incubated in flasks with different temperatures

(25°C, 30°C, 35°C and 40°C) for 72 h with constant shaking at 150 rpm. Samples were

taken approximately after 2, 4, 6 and 24 hours (Neelakandan and Usharani, 2009).

34

3.5 Collection and preparation of olive solid waste (jeft)

Fresh Jeft was obtained from an olive factory from the city of Rafah in Palestine in

October 2012. For pretreatment purpose, samples were soaked in water for overnight to

remove dirt and excess oil. The oil slowly left the solid waste to float on the water

surface, the solid waste were then separated from the dirty water using a sieve and dried

in air to make the weight constant before pretreatment. The biomass was dried at 50°C

by using oven until stabilization of weight (1day). The dried biomass was subjected to

mechanical treatment using stainless steel grinder mill. Stones with diameter above

1mm were removed by using sieve then stored at -20°C until their use. In order to be

used for, the powder jeft was dried at 50°C for 1 day in oven (Mishra et al., 2011).

3.6 Hydrolysis process

In this process, acids and microwave were used to catalyze conversion complex

polysaccharides in Jeft to simple sugar. The pretreated samples of the jeft were

subjected to hydrolysis by diluted H2SO4 or diluted HCl.

Around 10 g of powder Jeft was placed in a round bottom flask (100 mL) fitted with a

condenser and with a magnet stirrer bar for mixing. About 100 ml of diluted H2SO4 or

100 mL of diluted HCl was added to the contents. Hydrolysis was performed at various

concentration of H2SO4 ranging from 3%, 5%, and 7%. This process was proceeded at

90°C temperature in shaker water path for various periods of time (1 hour, 2 hours, 3

hours, 5 hours, and 7 hours). Then the reaction mixture was neutralized with 4M NaOH

solution (Shalabia, 2011).

3.7 Microwave-acid Treatment

Microwave treatment was carried out using a domestic microwave instrument (JAC,

China). The microwave instrument was operated at 2450 MHz and 450 W. For acid

pretreatment, 100 ml of 5% HCl was mixed with 10 g of jeft in 250 ml Erlenmeyer

flask for 5 min, and shaking well. The mixture then exposed to microwave for 5 min.

The resulted mixture was passed to shaking water path at 90°C temperature for 3 h.

After the period of time was over, the reaction mixture was neutralized with 4M NaOH

solution. All experiments were carried out in duplicate, and the given numbers are the

mean values (Binod et al., 2012).

35

3.8 Estimation of reducing sugars:

The amount of reducing sugars was estimated by using glucose assay kit (Diasys,

Germany).

3.8.1 Method

“GOD-PAP“: enzymatic photometric test.

3.8.2 Principle

Determination of glucose after enzymatic oxidation by glucose oxidase. The

colorimetric indicator is quinoneimine, which is generated from 4-aminoantipyrine and

phenol by hydrogen peroxide under the catalytic action of peroxidase (Trinder’s

reaction).

Glucose + O2 GOD Gluconic acid + H2O2

2 H2O2 + 4-Aminoantipyrine + Phenol POD Quinoneimine + 4 H2O

3.8.3 Assay Procedure

Wavelength 500 nm, Hg 546 nm

Optical path 1 cm

Temperature 20 – 25 °C/37 °C

Measurement Against reagent blank

36

3.8.4 Calculation

3.9 Fermentation process

Anaerobic batch fermentation in 250 ml Erlenmeyer flask of broth media consisting of

pretreated and hydrolyzed jeft was carried out in order to convert the released sugars

into ethanol. The conversion process being accomplished by the enzymes released by S.

cerevisiae. The pH of the solution was brought to pH 4.5 by adding required amount of

4 M NaOH to optimize yeast growth. The volume of the broth was brought to 115 ml.

The hydrolyzed material was completely sterilized by autoclaving (120°C, 15 psi

pressure and 30 min) before inoculating the yeast. After the substrate was prepared, 10

mL of inoculum was added to each flask (Mishra et al. 2011).

The fermentation was carried out in a closed conical flask at temperature of 30°C,

agitation rate at 150-rpm in shaker incubator. The flasks were tightly sealed to maintain

anaerobic condition and an outlet was provided to release CO2. The other end of the

outlet was dipped in lime water to confirm the release of CO2 as it turns lime water

milky. Duplicate fermentation broths of same composition were prepared and incubated

in the same conditions. The fermentation was continued for 3 days and samples were

taken from each of the two broths every day for analysis to get duplicate results. The

samples were frozen immediately after extraction in by the microcentrifuge tube until

use. All experiments including media preparation and sampling were carried out in lab

safe cabinet.

3.10 Estimation of ethanol:

The ethanol was estimated calorimetrically by using ethanol assay kit (Biovision, USA).

37

3.10.1 Kit Contents

Table 3.4 Ethanol kit components

Components Volume Cap color

Ethanol Assay

Buffer 25 ml WM

Ethanol Probe (in

DMSO,anhydrous)

200 µl

Red

Ethanol Enzyme

Mix

1 vial

Green

Ethanol Standard

(MW:46.07,

17.15N)

0.5 ml

Yellow

3.10.2 Reagent Preparation:

3.10.2.1 Ethanol Probe:

Ready to use as supplied. Warm to room temperature prior to use. Store at –20°C, avoid

contamination with water, protect from light. Use within two months.

3.10.2.2 Ethanol Enzyme Mix:

220 μl Ethanol Assay Buffer was added to the Ethanol Enzyme Mix and mix well. Store

at 4°C. Use within two months.

3.10.3 Ethanol Assay Protocol:

3.10.3.1 Standard Curve Preparations:

For the colorimetric assay, 50μl of pure ethanol standard was added to 808.7μl Ethanol

Assay Buffer, mix well. Then 10 μl of the dilution. Was taken into 990 μl assay buffer

to generate 10 nmol/μl of ethanol standard. 100 μl of the dilution was added to 900 μl

38

assay buffer to generate 1mM (1nmol/ μl). We add 0, 2, 4, 6, 8, 10 μl to a series of wells

in a 96 well plate and adjust the volume of each to 50 μl with Assay Buffer to generate

0, 2, 4, 6, 8, 10 nmol/well ethanol Standard.

3.10.3.2 Sample Preparation:

Samples can be diluted directly in Assay Buffer and tested. Biological samples such as

serum (containing ~ 0.01-0.16% w/v) should be diluted 1:10-1:100 and volumes in the

range of 10-306l used. For beverages which contain 100X more alcohol,

correspondingly greater dilutions should be used. We suggest making several dilutions

of your sample so that the sample reading is within the standard curve range.

Adjust the final volume to 50 μl using Assay Buffer.

3.10.3.3 Reaction Mix Preparation:

Mix enough reagents for the number of assays performed: For each well, prepare a total

50 μl Reaction Mix containing:

1. 46 μl Ethanol Assay Buffer

2. 2 μl Ethanol Probe

3. 2 μl Ethanol Enzyme Mix

4. Add 50μl of the Reaction Mix to all wells.

5. Incubate for 60 minutes at room temperature or 30 minutes at 37°C protected

from light.

6. Measure O.D. at 570 nm for colorimetric assay or Ex/Em = 535/590 nm.

7. Correct background by subtracting the background value derived from the 0

ethanol control from all samples (The background reading can be significant and

must be subtracted from sample readings). Calculate ethanol concentrations of

the test samples from the standard curve, multiplied by the dilution factor.

C = Sa/Sv nmol/Al or mM

Where: Sa is sample amount from the Standard Curve (nmol).

Sv is sample volume added into the sample well (6l).

Ethanol molecular weight: 46.07 g/mol.

39

Figure 3.1 Ethanol Standard Curve: Performed as described in the ethanol kit

instructions.

3.10.4 Data Analysis

Statistical analysis of experimental data was conducted using the PROC GLM

functionality in SAS, version 9.1.3 (SAS Institute Inc., Cary, NC).

41

Chapter 4

Results

4.1 Isolation of different strains of S. cerevisiae

Ethanol fermentation and recovery were not only depending on the substrate used, but

also depends mainly on the efficiency of yeast strain to convert the reducing sugar to

ethanol. In the present study; an attempt was made to evaluate olive solid wastes for

ethanol production by isolating elite yeast strains from three sources and optimizing

conditions for fermentation.

4.2 Characterization and identification of the isolated S. cerevisiae

strains

4.2.1 Morphological characterization:

Morphological characterization is an important tool for classic identification of yeasts.

In figure 4.1, we observed complete colonies that were 2–3 mm in diameter, slightly

convex, of a smooth, creamy consistency, white to cream in color and having a sweet

smell that is typical of yeast.

Figure 4.1 S. cerevisiae on yeast extract peptone glucose agar plate.

41

Microphotographs of different colonies from different samples have shown in (Figure

4.2). Strains were observed for Saccharomyces characteristic oval cell shape and

budding characters.

Figure 4.2 Microscopic Morphology of S. cerevisiae.

4.2.2 Biochemical characterization:

4.2.2.1 Carbohydrate source assimilation test:

Yeast isolated from sugarcane, grape and yogurt was able to utilize various sugars such

as glucose, maltose, sucrose, galactose but not lactose and xylose, and was compared to

standard S. cerevisiae (Figures 4.3- 4.7)

42

Figure 4.3 Carbon assimilation test for S. cerevisiae isolated from Sugarcane.

Figure 4.4 Carbon assimilation test for S. cerevisiae isolated from Grape.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 20 40 60 80 100 120

O.D

66

0

Time"hour"

Galactose Glucose Sucrose

Lactose Maltose Xylose

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 20 40 60 80 100 120

O.D

66

0

Time "hour"



43

Figure 4.5 Carbon assimilation test for S. cerevisiae isolated from Yogurt.

Figure 4.6 Carbon assimilation test for S. cerevisiae Control 1.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 20 40 60 80 100 120

O.D

66

0

Time "hour"



0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 20 40 60 80 100 120

O.D

66

0

Time"hour"



44

Figure 4.7 Carbon assimilation test for S. cerevisiae Control 2.

4.2.2.2 Ethanol Tolerance test:

The growth of yeast isolates in different ethanol concentrations is given in figure 4.8.

Yeast isolated from sugarcane showed that it can tolerate ethanol at 10% concentration

and gradually decreased at higher concentrations.

Figure 4.8 Growth of S. cerevisiae isolated from sugarcane at different Ethanol

concentration.

0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 20 40 60 80 100 120

O.D

66

0

Time"hour"



0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

0 20 40 60 80 100 120

O.D

66

0

Time "hour"

5% Ethanol 8% Ethanol %10 Ethanol

13% Ethanol 15% Ethanol

45

Yeast isolated from grape in figure 4.9 showed that it can tolerate ethanol at 8%

concentration and totally decreased at higher concentrations.

Figure 4.9 Growth of S. cerevisiae isolated from grape at different Ethanol

concentration.

Yeast isolated from yogurt in figure 4.10 showed that it can tolerate ethanol at 8% a

concentration and gradually decreased at higher concentrations.

Figure 4.10 Growth of S. cerevisiae isolated from yogurt at different Ethanol

concentration.

0

0.5

1

1.5

2

2.5

3

3.5

0 20 40 60 80 100 120

O.D

66

0

Time "hour"



0

0.5

1

1.5

2

2.5

3

3.5

4

4.5

5

0 20 40 60 80 100 120

O.D

66

0

Time "hour"



46

Standard S. cerevisiae in figure 4.11 and 4.12 showed that it can tolerate ethanol at 8%

concentration and totally decreased at higher concentrations.

Figure 4.11 Growth of S. cerevisiae control 1 at different Ethanol concentration

.

Figure 4.12 Growth of S. cerevisiae control 2 at different Ethanol concentration.

0

0.5

1

1.5

2

2.5

3

3.5

4

0 20 40 60 80 100 120

O.D

66

0

Time"hour"

5% Ethanol 8% Ethanol 10% Ethanol


0

0.5

1

1.5

2

2.5

3

3.5

4

0 20 40 60 80 100 120

O.D

66

0

Time "hour" control 2



47

4.3 Optimization of growth conditions

4.3.1 Optimization of Temperature

The isolated S. cerevisiae and control strains were tested for their growth at different

temperature. The optimum temperatures for most of the S. cerevisiae strains were

estimated in the temperature range from 30°C to 35°C.

The OD660 value increased as temperatures increased from 25°C to 35°C, and then

declined when the temperatures were above 35°C. A sharp decrease of OD660 values

was detected when the temperature increased from 35°C to 40°C. The OD660 value

almost reached zero at 40°C. Suggesting that 40°C might be a suppressor temperature

for all isolated and standard strain.

As shown in figure 4.13 – 4.17 the optimum growth temperature for all the isolated S.

cerevisiae strains were found at 30°C.

Figure 4.13 Growth of S. cerevisiae isolated from yogurt at different temperatures.

0

0.5

1

1.5

2

2.5

3

3.5

4

0 10 20 30 40 50 60

O.D

66

0

Time"hour"

Yogurt 25°C Yogurt 30°C Yogurt 35°C Yogurt 40°C

48

Figure 4.14 Growth of S. cerevisiae isolated from grape at different temperatures.

Figure 4.15 Growth of S. cerevisiae isolated from sugarcane at different temperatures

The standard S. cerevisiae control 1 and control 2 strains were also tested for their

growth at different temperature. The optimal temperature for growth of isolated yeast S.

cerevisiae from sugarcane was 30°C.

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20 30 40 50 60

O.D

66

0

Time"hour"

Grape 25°C Grape 30°C Grape 35°C Grape 40°C

0

0.5

1

1.5

2

2.5

3

3.5

4

0 10 20 30 40 50 60

O.D

66

0

Time"hour"

Sugercane 25°C Sugercane 30°C

Sugercane35°C Sugercane 40°C

49

Figure 4.16 Growth of S. cerevisiae control 1 at different temperatures

Figure 4.17 Growth of S. cerevisiae control 2 at different temperatures

0

0.5

1

1.5

2

2.5

3

3.5

4

0 10 20 30 40 50 60

O.D

66

0

Time" hour"

Control 1 25 °C Control1 30°C

Control 1 35°C Control 1 40° C

0

0.5

1

1.5

2

2.5

3

3.5

0 10 20 30 40 50 60

O.D

66

0

Time "hour"

Control 2 25 °C Control 2 30°C

Control 2 35°C Control 2 40°C

51


The growth of S. cerevisiae isolates at different pH is given in (figures 4.18 – 4.22). The

isolated strains from yogurt and grape recorded maximum population at pH 4.5 (figures

4.18 – 4.19), the isolate strain from sugarcane recorded maximum population at pH 4.

Above pH 5.0 yeast populations declined, the optimal pH for ethanol production was

around 4.5.

Figure 4.18 Growth of S. cerevisiae which isolated from yogurt at different pH.

The effects of pH on the S. cerevisiae which isolated from grape strain growth are given

in Figure 4.19. The OD660 value increased by a fraction when the pH ranged from 4.0 to

5.0, while 4.5 was obviously the optimal pH.

0

0.5

1

1.5

2

2.5

3

0 5 10 15 20 25 30 35

O.D

66

0

Time "hour"

Yogurt pH =4 Yogurt pH =4.5 Yogurt pH =5 Yogurt pH =5.5

http://www.scielo.br/img/revistas/bjm/v42n2/31f05.jpg

51

Figure 4.19 Growth of S. cerevisiae which isolated from grape at different pH.

The effects of pH on the S. cerevisiae which isolated from sugarcane strain growth are

given in Figure 4.20. The OD660 value increased by a fraction when the pH ranged from

4.0 to 5.0, while 4 was obviously the optimal pH.

Figure 4.20 Growth of S. cerevisiae isolated from sugarcane at different pH.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 5 10 15 20 25 30 35

O.D

66

0

Time "hour"

Grape pH=4 Grape pH=4.5 Grape pH=5 Grape pH=5.5

0

0.5

1

1.5

2

2.5

0 5 10 15 20 25 30 35

O.D

66

0

Time "hour"

Sugarcane pH=4 Sugarcane pH=4.5

Sugarcane pH=5 Sugarcane pH=5.5


52

The effects of pH on the standard S. cerevisiae growth are given in Figure 4.21 and

figure 4.22. The OD660 value increased by a fraction when the pH ranged from 4.0 to

5.0, while 4.5 was obviously the optimal pH for standard S. cerevisiae. In control 1and

both pH 4 and pH 4.5 were optimal pH for slandered S. cerevisiae control 2.

Figure 4.21 Growth of S. cerevisiae control 1 at different pH.

Figure 4.22 Growth of S. cerevisiae control 2 at different pH.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 5 10 15 20 25 30 35

O.D

66

0

Time"hour"

Control 1 pH =4 Control 1 pH =4.5

Control 1 pH =5 Control 1 pH =5.5

0

0.2

0.4

0.6

0.8

1

1.2

1.4

1.6

1.8

2

0 5 10 15 20 25 30 35

O.D

66

0

Time "hour"

Control 2 pH=4 Control 2 pH=4.5

Control 2 pH =5 Control 2 pH=5.5


53

The optimum culture conditions were obtained from the statistical analysis. The optimal

culture conditions for S. cerevisiae from yogurt were suggested to be at a temperature of

30°C, pH 4.5 and inoculums of 10 %. These conditions were later tested to ascertain the

reliability of these results. In conclusion, we have successfully isolated a S. cerevisiae,

which demonstrated tolerance to high ethanol concentrations and showed potential as an

ideal strain for bioethanol production.

4.4 Pretreatment of olive solid waste

4.4.4.1. Hydrolysis process

4.4.4.1 Effect of different concentrations of HCl:

Effect of different concentrations of HCl on hydrolysis of olive solid waste was tested

with different time intervals of 1 hour to 7 hours by estimating the concentration of

reducing sugar (Table 4.1). At 3 hour of hydrolysis process, the maximum amount of

reducing sugar [312 mg/dl] was observed at 5% HCl concentration (Table 4.1).

4.4.1.2 Effect of different concentrations of H2SO4:

Effect of different concentrations of H2SO4 on hydrolysis of olive solid waste was tested

at different time intervals of 1 hour to 7 hours by estimating the concentration of

reducing sugar percentage (Table 4.2). At 3 hour of hydrolysis process, the maximum

concentration of reducing sugar was 274 mg/dl in 7% H2SO4, concentration (Table 4.2).

4.4.1.3 Microwave assisted with 5% HCl pretreatments

The results showed that the pretreated substrate with microwave assisted 5% HCl at 3

hours had significantly produce higher concentration of reducing sugar yield [389

mg/dl] than the samples treated with Chemical pretreatment alone 5% HCl (Table 4.1).

The results indicated that hydrolysis with microwave assistant 5% HCl pretreatments

for 3 hours enhanced the hydrolysis of olive solid waste and produced significantly

higher concentration of reducing sugar yield of 389 mg/dl.

54

Table 4.1: Effect of different concentrations of HCl, H2SO4 and microwave assistant 5%

HCl on hydrolysis of olive solid waste at different time intervals (mg/dl).

Microw

ave

With

5%

HCl

Conc.

H2SO4

mg/dl

Conc.

HCl

mg/dl

7 %

H2SO4

mg/dl

5 %

H2SO4

mg/dl

3 %

H2SO4

mg/dl

7 %

HCl

mg/dl

5 %

HCl

mg/dl

3 %

HCl

mg/dl

311 23 43.2 110.6 93.2 149.4 145.4 168 117.4

1

hour

384 25 63.8 167.4 162.4 153.6 214 145.6 173.6

2 hours

389 41 67.2 274 246 155 288 312 169.4

3 hours

1.5 44 .0 221 170 126 224 192 138.2

5 hours

144 44 24 220 151 113.2 220 171 125.2

7 hours

4.5 Fermentation process:

During fermentation process various parameters such as pH, reducing sugar percentage,

and temperature were assayed (Table 4.2). The final pH of the olive solid waste was

about 4.5, and reduction in the reducing sugar content was recorded as it shown in the

table where the fermentation process slowed down after the first day.

Reduci

ng

sugar

mg/dl

Time

55

Table 4.2 Concentration of glucose utilization during fermentation process by the

different isolated yeast strains by using at 10% inoculums size, pH 4.5 at 30°C for 5

days, after using microwave assisted 5% HCl pretreatments on hydrolysis of olive solid

waste for 3 hours mg/dl.

During fermentation process various parameters such as pH, reducing sugar

concentration and temperature were assayed (Table 4.3). The final pH of the olive solid

waste was about 4.5, and reduction in the reducing sugar content was recorded at zero

time was 1016 mg/dl. Through the table 4.3 note that the fermentation process occurred

on the first day and then stopped.

Control

2.1

mg/dl

Control

2

mg/dl

Control

1.1

mg/dl

Control

1

mg/dl

Sugarcane

3.1

mg/dl

Sugarcane

3

mg/dl

Grape

2.1

mg/dl

Grape

2

mg/dl

Yogurt

1.1

mg/dl

Yogurt

1

mg/dl

Microwave

with 5%

HCl

1314 0 Time

29.6 28.5 6.5 7.2 17.8 17.2 5.7 6.1 6 5.5 1 Day

29.3 26.9 5.6 5.9 10.3 8.9 5.7 6.1 6 5.1 2 Day

11.9 10.4 5.4 5.6 6.3 6 5.3 5.9 5.6 4.8 3 Days

9.9 5.6 5.4 5.3 5.3 5.4 5.8 5.8 5.2 4.5 5 Days

56

Table 4.3 concentration of glucose utilization during fermentation process by the

different isolated strains yeast strains at 10% inoculums size, pH 4.5 at 350C for 5days.

After using 5% HCl pretreatments on hydrolysis of olive solid waste for 3 hours

(mg/dl).

4.6 Ethanol yield:

The results for ethanol production by yeast fermentation using the three types of

isolated strain S. cerevisiae and two types of control strains are shown in Table 4.4. The

concentration of ethanol in the reactor that contained pretreated (using microwave

assistant 5% HCl pretreatments) olive solid waste was much higher than in the

pretreated one with 5% HCl. The amount of ethanol detected in those cultures reached

up to 9.3 g/L after 3 days of yeast fermentation by using yogurt S. cerevisiae isolate.

Control

2.1

mg/dl

Control

2

mg/dl

Control

1.1

mg/dl

Control

1

mg/dl

Sugarcane

3.1

mg/dl

Sugarcane

3

mg/dl

Grape

2.1

mg/dl

Grape

2

mg/dl

Yogurt

1.1

mg/dl

Yogurt

1

mg/dl

5%HCl

1016 0 Time

29.4 28.7 6.8 11.7 11.7 13.4 6.3 6.8 5.8 5.9 1 Day

15.9 16.5 6.4 10.1 8.9 7.5 6.2 6 5.7 5.7 2 Day

6.2 7.3 6.1 6.6 6.3 6.1 5.5 5.7 5.7 5.6 3 Days

5.7 5.3 6 6.4 6.3 6.1 4.3 5.7 5.5 5.4 5 Days

57

Table 4.4 Estimation of ethanol yield from pretreated olive solid waste by two methods

(5% HCl, and microwave assisted 5% HCl) g/L.

Figure 4.23 Ethanol yield (g /l).

0

1

2

3

4

5

6

7

8

9

Yogurt Grape Sugarcane Control 1 Control 2

eth

ano

l yie

ld g

/l

5% HCL Microwave

Control

2.1

g/l

Control

2

g/l

Control

1.1

g/l

Control

1

g/l

Sugarcane

3.1

g/l

Sugarcane

3

g/l

Grape

2.1

g/l

Grape

2

g/l

Yogurt

1.1

g/l

Yogurt

1

g/l

6.2 6.3 5.9 6.2 7.5 7.3 6.5 6.4 8.4 8.2 5% HCl

7 6.8 6.4 6.6 7.4 7.6 6.3 6.9 9.3 8.6

Microwave

with

5%HCl

58

Chapter 5

Discussion

5.1 Isolation and selection of yeast isolates:

Ethanol fermentation and recovery were not only depending on the substrate used, but

also depends mainly on the efficiency of yeast strain to convert the reducing sugar to

ethanol. In the present study; an attempt was made to evaluate olive solid wastes for

ethanol production by isolating elite yeast strains from three sources and optimizing

conditions for hydrolysis and fermentation. The yeast strain isolated from yogurt was

more prominent than that of the strains isolated from sugarcane and grape. Hence the

yogurt isolated strain was selected for further studies. Monte et al. (2003) observed

earlier that S. cerevisiae isolated from molasses showed higher ethanol production rate

at different dilutions of sugar-cane molasses than the other strains of the yeast.

Similarly, Bowman and Ahearan (1975) have characterized an S. cerevisiae strain

with fast residual sugar fermenting ability. Recent studies have clearly suggests the

impact of S. cerevisiae isolated from molasses in the production of ethanol from the

sago wastes (subashini et al. 2011).

The results regarding the identification of yeast species have been described under

section 4.1. Three yeast strains were isolated, purified and identified from different food

sources. Different tests were applied, including morphological and physiological

characteristics, which facilitate the opportunity for identification of the yeasts. The sets

of these tests allow the information gathering for the studied objects and for

determination of their systematic status to species.

5.2 Characterization and identification of S. cerevisiae strains:

All yeast strains were identified as S. cerevisiae according to their morphological

characteristics, ability to assimilate carbon sources and ethanol tolerance test.

5.2.1 Morphological characterization:

The yeast isolate was identified as S. cerevisiae based on the morphology which clearly

observed on figure 4.1 and 4.2. We observed complete colonies that were 2–3 mm in

59

diameter, slightly convex, of a smooth, creamy consistency, white to cream in color and

having a sweet smell that is typical of yeast.

Similar results were reported by Tikka et al. (2013). Yeast strains produced different

types of colonies on YEPDA medium such as raised, creamy white color colonies.

Microphotographs of different colonies from different sample. Strains were observed

for Saccharomyces characteristic oval cell shape and budding characters. Out of fifteen

isolates, seven isolates showed oval cell shape with budding character (Figure 4.2).

Similar results were reported by Petrea, (2008) that the colonies morphology of

S. cerevisiae were like cream-colored colonies, convex profile with circular perimeter,

slippery and with 1.5-2 mm in diameter; when plated on YPGA (yeast-Pepton- Glucose

Agar ).

Colony morphology is influenced by several environmental parameters, for example.

Some of these factors, the concentration of agar, may influence colony morphology by

changing the physical and chemical properties of the substrate (e.g. surface tension,

surface hydrophobicity etc . . . ). Other parameters, such as carbon source, likely act, at

least in part, by changing the physiology of the yeast. Because of these multiple

parameters, Voordeckers was hypothesized that colony morphology is likely regulated

by several complex physiological processes involving many gene products

(Voordeckers et al, 2012).

5.2.2 Physiological characterization:

5.2.2.1 Carbohydrate source assimilation test:

Yeast isolated from sugarcane, grape and yogurts were able to utilize various sugars

such as glucose, maltose, sucrose, galactose but not lactose and xylose, and were

compared to standard S. cerevisiae (Figure 4.3- 4.7).

Similar results were reported by Walker et al. (2006). They reported that all the isolate

ferment at least one type of sugar. However a majority of these isolates which ferment

glucose, galactose, maltose, sucrose and raffinose, were strain belonged to the genus

S. cerevisiae.

61

Also similar results were reported by Kumar et al, (2011). They tested by the

fermentation patterns of carbon sugars, some of the sugars like glucose, galactose,

maltose, sucrose, raffinose, cellulose were positive in fermentation process which

indicated by color change from red to yellow due to acid production.

Martini, (1996) reported that indigenous yeast S. cerevisiae with very high ethanol

producing capabilities in the natural environment are though to be very rare. The most

commonly used microorganism for ethanol production is ordinary baker’s yeast S.

cerevisiae. In the pretreatment process some inhibitors are formed and S. cerevisiae one

of the most inhibitor tolerant microorganisms used for the conversion of hexoses such

as glucose and mannose not pentoses such as xylose, arabinose that are found in the

hemicellulose portion (Olsson and Hahn, 1993). Also proved that the S. cerevisiae t2

was not able of fermenting the maltose as compared to the wild type, S. cerevisiae strain

and to mutant strain S.cerevisiae t1.

Maltose is taken up via maltose permease and then hydrolyzed intracellular by maltase

into two units of glucose. Maltose permease is encoded by MALT. MAL genes cannot

be induced if maltose cannot be transported into the cell (Goffeau, 2000). It is possible

that the maltose transport to have been affected by irradiation, which can explain why

the strain, S. cerevisiae t2, obtained by mutagenesis under UV radiation, cannot ferment

maltose.

During the process of wort fermentation, glucose is consumed first, followed by

fructose and sucrose (Anghel et al., 1989). The disaccharide sucrose and trisacharide

raffinose are hydrolyzed outside the cell membrane into monosaccharide, which are

then taken up by the cell (Ostergaard et al., 2000).

5.2.2.2 Ethanol tolerance test:

Ethanol tolerance, sugar tolerance and invertase activities are some of the important

properties for use in industrial ethanol production (Jameonoz and Benitez, 1986). The

growth of yeast isolates in different ethanol concentrations is given in figures 4.8 – 4.12.

Yeast isolated from sugarcane tolerance at 10% concentration and gradually decreased

at higher concentrations.

61

A similar result was reported by Osho, (2005). Seventeen wine yeasts isolated from

fermenting cashew apple juice were screened for ethanol and sugar tolerance. Two

species of Saccharomyces comprising of three strains of S. cerevisiae showed

measurable growth in medium containing 9% (v/v) ethanol.

Similar results were reported by Tikka et al. (2013), seven strains of S. cerevisiae

obtained from different fruit sources were screened for ethanol tolerance. The results

obtained in this study showed a range of tolerance levels between 7%-12% in all the

stains.

Khaing et al. (2008). reported that the S. cerevisiae (KY1&KY3) strains has tolerate up

to 15% of ethanol in the medium and S. cerevisiae (KY2) tolerate up to 20% of ethanol

has leads to maximum ethanol production over a long incubation period. Cassey, (1996)

reported that the yeast strains survive to any extent in palm wines must have some

degree of ethanol tolerance, which have some importance in choosing a yeast strain for

industrial ethanol fermentation process. Use of efficient yeast strains with higher

ethanol tolerance to improve ethanol yields in the fermented wash would reduce

distillation costs and hence the profitability of the overall process (Chandrasena et al.,

2006).

But ethanol is well known to inhibit yeast growth and viability, affecting various

transport systems, such as- the general amino acid permease and glucose uptake process

(Arroyo-Lopez et al., 2010). It can cause damage to mitochondrial DNA and degrades

biological membrane (Lopes and Mauro, 2001; Mobini-Dehkordi et al., 2007) in

yeast cells and inactivates some enzymes, such as hexokinase and dehydrogenase.

Moreover, it can dissolve the fatty acid constituents of the cell membrane, disrupt

cytoplasmic membrane rigidity (Salmon et al. 1993., Swiecilo et al., 2000 Osho,

2005.), stop proton motive force and finally cell death (Swiecilo et al., 2000.,

Furukava and Kitano, 2004).

62

5.3 Optimization of culture conditions

5.3.1 Optimization of temperature

Temperature has a marked influence on the production of ethanol. According to Rivera

et al., (2006) suitable temperature in fermentation process is the good condition for the

yeast to react. Too high temperature kills yeast, and low temperature slows down yeast

activity. Thus, it is requires to keep a specific range of temperature. Normally ethanol

fermentation is conducted at temperature ranges between 30-35°C which stated by

Shuler and Kargi, (2002) that the ethanol will be produced at highest concentration.

The optical density value increased as temperatures increased from 25°C to 35°C, and

then declined when the temperatures were above 35°C. A sharp decrease of OD660

values was detected when the temperature increased from 35°C to 40°C. The OD660

value almost reached zero at 40°C suggesting that 40°C might be a repressor

temperature for all isolated and standard strains (figure 4.13- 4.16). As shown in figure

4.13 – 4.15 the optimum growth temperature for all the isolated S. cerevisiae strain was

30°C.

The effect of temperature on ethanol production was also analyzed by Kumar et al.

2011. S. cerevisiae produced maximum yield of ethanol (18.13 ± 0.16 g/l) at 30°C.

Similar results were reported by Petrova and Ivanova, (2010). The fermentation of

olive tree pruning hydrolysate containing xylose by Pachysolan tannpphilus was

produced maximum ethanol 0.38 g/g at 30°C and pH 3.5. Mariam et al. (2009)

reported that the maximum amount of ethanol (7.5%) was obtained from fermentation

by S. cerevisiae at the optimum pH 3.5 and incubation temperature 30°C.

There are many explanations for the decrease of OD660 values that detected when the

temperature increased from 35°C to 45°C (Mager and Siderius, 2002, Schuller et al.,

2004).

Tolerance to high temperature is also an important factor for increasing efficiency of

industrial scale. The fermentation efficiency of S. cerevisiae at high temperature is very

low due to increased fluidity in membranes to which the yeast responds by changing its

fatty acid composition. Moreover, it is well known, that, ethanol production is itself an

63

exothermic process (Kumar et al., 1998, Mager and Siderius, 2002; Schuller et al.,

2004).

Also there is another explanation, Above 40°C, yeast does not grow and below 30°C its

growth slows down dramatically. The main reason is the ability of the enzymes

that catalyze all the biochemical reactions in the yeast cell to function at those

temperatures. Enzymes have an optimal temperature range. When temperature goes

below the optimal range, their ability to catalyze the intended reaction slows down. On

the other hand, when the temperature increases, enzymes begin to denature or unfold

and thus become inactive. Each enzyme will have a different temperature range

where it becomes inactive. Even if one essential enzyme stops working, the organism

fails to grow. Hence, whichever is the first essential enzyme gets deactivated it defines

the maximal temperature at which that organism can grow. At the lower end it gets

more complicated. Usually, the enzymes are not inactivated, but rather just slow down

(Kumar et al., 1998).


Hydrogen ion concentration has a significant influence on industrial fermentation due as

much to its importance in controlling bacterial contamination as its effect on yeast

growth, fermentation rates and by-product formation. The best ethanol yields are

generally obtained at pH 4.5-4.7. At higher pH, more glycerol and organic acids are

formed at the expense of ethanol (Wayman and Parekh, 1990).

Under fermentation conditions, the intracellular pH of S. cerevisiae is usually

maintained between 5.5 and 5.75 when the external pH is 3.0 or between 5.9 and 6.75

when the external pH is varied between 6.0 and 10.0. The gap between the extracellular

pH and the intracellular pH widens, greater stress is placed on the cells and more energy

is expended to maintain the intracellular pH within the range that permits growth and

survival of the yeast. A greater proportion of glucose is converted to ethanol if the pH is

adjusted to 4.5. This increased conversion is independent of the presence of nutrient

supplements in the medium (Thomas et al., 2002). If the pH is adjusted to 7 or above,

acetic acid is produced from acetaldehyde due to the increased activity of aldehyde

64

dehydrogenase due to glycerol production which inhibits ethanol fermentation (Wang

et al., 2001).

The growth of S. cerevisiae isolates at different pH is given in different figures (figures

4.18 – 4.22). The isolated strains from yogurt and grape recorded maximum growth at

pH 4.5 (figure 4.18 - 4.19), the isolated strain from sugarcane was recorded maximum

growth at pH 4. Above pH 5.0 yeast populations declined and the optimal pH for

ethanol production was around 4.5 (figure.4.20 - 4.22).

Similar results were reported by Periyasamy et al. (2009) they reported the influencing

parameters that affect the production of bio-ethanol from sugar molasses are optimized.

The optimal values of the parameters such as temperature, pH, substrate concentration,

enzyme concentration and fermentation period are found to be 35°C, pH4.0, 300 gm/l, 2

gm/l and 72 hours respectively.

Similar results were reported by Lin et al .2011 Increased substrate supply did not

improve the specific ethanol production rate when the pH value was not controlled.

They found that pH 4.0 - 5.0 was the optimal range for the ethanol production process.

There is another result reported by Tahir et al., (2010a) indicated that S. cerevisiae

Bio-07 gave maximum productivity (52.0g/L). Fermentation conditions were optimized

for maximum production of ethanol. Maximum yield of ethanol (76.8 g/L) was obtained

with 15% molasses concentration, 3% inoculum size, pH 4.5 and temperature 30ºC.

The optimal pH range for yeast growth can be varied from pH 3-6. The intracellular

enzymes of yeast work best at its optimal pH it leads to maximal conversion of sugar

into ethanol. Goksungur and Zorlu, (2001) reported that continuous production of

ethanol from beet molasses by Ca-immobilized S. cerevisiae at 30°C and pH 3 are

optimum for maximum ethanol production. The maximum amount of ethanol (7.5%)

was obtained from fermentation by S. cerevisiae at the optimum pH 3.5 and incubation

temperature 30°C. Manikandan et al. (2010) reported that S. cerevisiae yeast isolated

from toddy and maximum yield of ethanol (40 g/l) compared with baker’s yeast S.

cerevisiae in the optimum pH 3.0, temperature 30°C and initial sugar concentration

20%.

65

The most likely explanation for the observed reduction in ethanol production when the

initial medium pH was lowered from 4.0 to 2.0 and higher than 6.5. During growth, it is

important for the yeast to maintain a constant intracellular pH. There are many enzymes

functioning within the yeast cell during growth and its metabolism. Each enzyme works

best at its optimal pH, which is acidic because of the acidophilic nature of the yeast

itself. When the extracellular pH deviates from the optimal level, the yeast cell needs to

invest energy to either pump in or pump out hydrogen ions in order to maintain the

optimal intracellular pH (Narendranath et al., 2001 and Thomas et al., 2002). If the

extracellular pH deviates too much from the optimal range, it may become too difficult

for the cell to maintain constant intracellular pH, and the enzymes may not function

normally. If the enzymes are deactivated, the yeast cell will not be able to grow and

make ethanol efficiently (Narendranath and Power, 2005).

5.4 Hydrolysis process:

5.4.1 Strong acid hydrolysis

Different concentrations of H2SO4 and HCl on hydrolysis of olive solid waste was tested

as shown in table 4.1, there is no effect of concentration H2SO4 and HCl on hydrolysis

process. These results contradict with previous published studies (Sun and Cheng,

2002; Blue Fire Ethanol, 2010; Biosulfurol, 2010).

Concentrated strong acids such as H2SO4 and HCl have been widely used for treating

lignocellulosic materials because they are powerful agents for cellulose hydrolysis (Sun

and Cheng, 2002), and no enzymes are needed subsequent to the acid hydrolysis.

Advantages of concentrated acid hydrolysis are the flexibility in terms of feedstock

choice, high monomeric sugar yield as well as mild temperature conditions that are

needed. Drawbacks of using concentrated acids are corrosive nature of the reaction and

the need to recycle acids in order to lower cost. To date, several companies are in the

process of commercializing strong acid hydrolysis of lignocellulosic biomass for

microbial fermentation purposes (Blue Fire Ethanol, 2010; Biosulfurol, 2010).

66

5.4.2 Dilute acid hydrolysis:

Different concentrations of dilute H2SO4 and HCl on hydrolysis of olive solid waste

were tested as shown in table 4.1. At 3 hours of hydrolysis process, the maximum

concentration of reducing sugar was 274 mg/dl in 7% H2SO4, concentration respectively

and the amount of 312 mg/dl was observed at 5% HCl concentration (Table 4.1- 4.2).

Similar results were reported by Mishra et al, (2011). The hydrolysis of the cellulose

content into sugars is done by dilute acid hydrolysis that involves incubation period at a

high temperature for 72 hours. The fermentation of the hydrolyzed Jatropha waste is

done using S. cerevisiae under proper incubation conditions to produce ethanol. About

80 % ethanol was recovered as a result of the process.

Dilute acid hydrolysis has been successfully developed for pretreatment of

lignocellulosic materials. The dilute sulfuric acid pretreatment can achieve high reaction

rates and significantly improve cellulose hydrolysis (Esteghlalian et al., 1997). At

moderate temperature, direct saccharification suffered from low yields because of sugar

decomposition. High temperature in dilute acid treatment is favorable for cellulose

hydrolysis (McMillan, 1994).

5.4.3 Combined Microwave-Chemical Pretreatments

The reducing sugar yields from hydrolysis of microwave pretreatments combined with

varying levels of dilute H2SO4 and HCl are shown in table 4.2 respectively. The results

showed that the pretreated substrate with microwave assisted 5% HCl at 3 hours had

significantly produce higher concentration of reducing sugar yield of 389 mg/dl than the

samples treated with Chemical pretreatment alone (Table 4.2).

As clearly shown, combined microwave-5% HCl pretreatments yielded significantly

higher reducing sugars than chemical pretreatments alone (H2SO4 and dilute HCl). The

yields from microwave-HCl pretreatments were approximately three times that of

chemical pretreatments alone. The highest yields from combined microwave- HCl

pretreatments were obtained at a concentration of 5% HCl and a residence time of 3

hours.

67

A review of published studies agree with our results which indicated that microwave

pretreatment in the presence of chemical reagents would be more effective. Further,

Sridar (1998) stated that efficient and rapid heating by microwave radiation can

accelerate chemical reactions. Volumetric and selective heating of lignocelluloses by

microwave, facilitates the disruption of their recalcitrant structures more efficiently

(Hendriks and Zeeman, 2009).

5.5 Fermentation process:

Fermentation is the process where yeast convert sugars to alcohol. The most commonly

used yeast is S. cerevisiae (Pretorius, 2000).

Our results for ethanol production by yeast fermentation using the three types of

isolated strain S. cerevisiae and two control stains are shown in Table 4.4. The

pretreatment of olive pulp was done using water at temperatures 90°C in shaking hot

water path. The concentration of ethanol in the reactor that contained pretreated (using

microwave assistant 5% HCl pretreatments) olive solid waste was much higher than in

the pretreated with 5% HCl. The amount of ethanol detected in those cultures reached

up to 9.3 g/L after 3 days of yeast fermentation by using S. cerevisiae isolated from

yogurt as shown in Table 4.4.

Similar results were reported by Ballesteros, et al. (2002). The pretreatment of olive

pulp using water at temperatures between 200°C and 250°C enhanced enzymatic

hydrolysis. Maximum ethanol production (11.9 g/L) was obtained after pretreating pulp

at 210°C in a SSF fed-batch procedure. Maximum hydroxytyrosol recovery was

obtained in the liquid fraction when pretreated at 230°C.

Also similar results were reported by Georgieva and Ahring (2007). Fermentation of

undiluted olive pulp hydrolysate (OPH) resulted in the maximum ethanol produced

(11.2 g/L) with productivity of 2.1 g/L/h. Ethanol yields were similar for all tested OPH

concentrations and were in the range of 0.49 - 0.51 g/g.

68

Our result is considered relatively acceptable and very close to the published data.

There are many factors affect our results such as:

1. Kind of pretreatment, many pretreatment used high pressure about 30 psi and

high temperature about 200oC to 250

oC as shown in literature review.

2. All of our work occurred in hood, and the hydrolyzed material was completely

sterilized by autoclaving (120°C, 15 psi pressure and 30 min) before inoculating

the yeast, but all these precautions do not prevent contamination. Microbial

contamination causes reduced ethanol yield and ethanol plant productivity

(Barbour and Priest, 1988). The most common organisms associated with

microbial contamination are lactobacilli and wild yeasts. These microbes

compete with S. cerevisiae for nutrients (trace minerals, vitamins, glucose, and

free amino nitrogen) and produce inhibitory end-products such as acetic and/or

lactic acid. Dekkera/Brettanomyces wild yeasts have become a concern in fuel

alcohol production (Abbott and Ingledew, 2005). A reduction in lactic acid

bacterial contamination is currently achieved by using antibiotics in fuel ethanol

plants (Narendranath and Power, 2005).

3. The general low yield of ethanol from acid hydrolysed jeft could be due to

unfermentable sugars such as hydroxy methyl furfural and hydroxyl-methyl-

furans. Keim has reported that the use of traditional methods of acid hydrolysis

formed large amounts of unfermentable and thus leads to low ethanol yields

(Keim, 1983).

4. Agitation is important for adequate mixing, mass transfer and heat transfer. It

assists mass transfer between the different phases present in the culture, also

maintains homogeneous chemical and physical conditions in the culture by

continuous mixing. Agitation creates shear forces, which affect microorganisms,

causing morphological changes, variation in their growth and product formation

and also damaging the cell structure (Kongkiattikajorn et al., 2007).

69

Chapter 6

Conclusion and Recommendations

6.1 Conclusion

The present work focused on bioethanol production from olive solid waste. Ethanol

production was carried out by means of isolating a new ethanol-producing

microorganism, characterizing it, and analyzing its behavior under different cultivation

conditions. Furthermore, the isolates were grouped under the genus Saccharomyces

depending up on their morphological and physiological characteristics and were closely

related to S. cerevisiae. The morphological and physiological characters of the isolates

showed that the colonies of the isolates appeared butyrous, raised, smooth and glossy,

utilized various sugars namely glucose, sucrose, maltose, galactose while the isolates

failed to utilize lactose. Growth of yeast isolates at different concentration of ethanol,

temperature and pH revealed. The optimum temperature required for the isolates was

30°C at optimum pH 4.5. Generally the isolates strains where able to tolerate ethanol

concentration of 10 and 13 % (v/v) respectively.

Olive solid wastes are promising lignocellulosic feedstocks for bioethanol production.

To produce economically feasible cellulosic ethanol, ethanol yield and enzyme

efficiency have to be improved by optimizing all unit processes (pretreatments,

saccharification and ethanol fermentation). Most of the pretreatments focused on low

temperature pretreatment to avoid over degradation of hemicellulose and to improve the

pentose yield. Impregnation by various chemical reagents such as HCl, H2SO4 and

application of microwave assisted dilute HCl pretreatment have been tried to lower

pretreatment temperatures while maintaining high enzymatic digestibility

The major contributions resulting from this work can be seen as following:

(1) Waste olive has much higher cellulose content than common plants or biomass, but

they are difficult to be decomposed into reducing sugars by general hydrolysis methods.

At 90°C temperature, the hydrolysis efficiencies of olive waste were at 5% HCl or 7%

H2SO4 concentrations.

71

(2) Microwave assisted dilute HCl hydrolysis technology can greatly improve the

hydrolysis efficiency and rate of olive solid waste. Experimental results showed that the

highest hydrolysis efficiency of olive waste can achieve by microwave assisted with 5%

HCl.

(3) The optimal microwave temperature and time were 90°C for 5 min. As the

hydrolysates were further fermented with the yeast S.cerevisiae, the highest

concentration of ethanol was 9.2 g/l at the fermentation time of 72 h.

71

6.2 Recommendations:

The present work is part of extensive research devoted to meeting challenges and

requirements that human society currently encounters in sustainable development.

Producing fuel from renewable resources is becoming an important issue for the whole

world. Therefore, the work needs to be continued for further development of ethanol

production from biomass. The following suggestions could be of interest for future

studies:

1. First recommendation for future studies would be the use of cellulose from

olive solid waste as the substrate for bioethanol production. In addition to other

common cellulosic biomass resources are market residue, wood, municipal

solid wastes, wastes paper, agricultural residues and industrial residues.

2. Genetically engineered microorganisms could be designed or genetically

engineered to be more efficient in terms of enhanced capacity to fully ferment

C5 and C6 sugars at high temperatures. The development of new strains of S.

cerevisiae designed for pentose utilization, with high tolerance to inhibitors,

and with a better genomic stability has not been yet fully addressed despite the

recent advances in genetic engineering. Process wise, biorefineries should focus

on designing new bioreactors, flow-patterns, new cocktails of enzymes to

optimize hydrolysis, the utilization of immobilized microorganisms and the

development of new distillation and ethanol dehydration technologies that

favors the total energy balance.

3. Alternate processing methods to get higher release of reducing sugars could be

tried to make the technology more efficient and profitable. Future work will

focus on the scale up of microwave-based pretreatment of lignocellulosic

biomass. Radio frequency heating as the energy source for pretreatment is

recommending for studying as well. We also recommended applying the

microwave-base pretreatment techniques to other feedstocks, such as municipal

solid wastes and other agricultural residues.

4. Development of new and more environmental-friendly pretreatments that

include the use of fiber degrading enzymes and hot water and new strains of

72

yeast and bacteria are critical points for the economics of biomass

transformation. Although economical ethanol production from fiber-based

sources still requires a lot of research, with significant risks and uncertainties,

we need to continue to be optimistic about its future.

73

References

1. Abbi, M., Kuhad, R. C., Singh, A., (1996). "Bioconversion of pentose sugars

to ethanol by free and immobilized cells of Candida shehatae (NCL-3501):

fermentation behavior." Process Biochemistry, 31: 555-560.

2. Abbott, D. A., and Ingledew, W. M., (2005). "The importance of aeration

strategy in fuel alcohol fermentations contaminated with

Dekkera/Brettanomyces yeasts." Journal of Applied Biochemistry and

Biotechnology, 69:16-21.

3. Ahmadi, H., Akbarpour, V., Shojaeian, A., (2010). "Investigation of olive

stones as lignocellulose material for bioethanol production." American-

Eurasian Journal of Agricultural and Environmental Science, 8(5): 520-524.

4. Ahring, B. K., Licht, D., Schmidt, A. S., Sommer, P. and Thomsen, A. B.,

(1999). "Production of ethanol from wet oxidized wheat straw by

Thermonanaerobacter mathranii." Bioresource Technology., 68: 3-9.

5. Albarran, A., Celis, R., Hermosin, M. C., Pineiro, A. L., Calvo, J. J. O., and

Cornejo, J., (2006). "Effects of solid olive mill waste addition to soil

onsorption, degradation and leaching of the herbicide simazine". Soil Use

Manage, 19:150-156.

6. Alburquerque, J. A., Gonza´lvez, J., Garcı´a, D., Cegarra, J., (2006).

"Composting of a solid olive-mill byproduct ("alperujo") and the potential

of the resulting compost for cultivating pepper under commercial

conditions." Waste Management, 26: 620–626.

7. Amutha, R. and Gunasekaran, P., (2000), "Improved ethanol production by a

mixed culture of Saccharomyces diastaticus and Zymomonas mobilis from

liquefied cassava starch." Indian Journal of Microbiology, 40:103-107.

8. Anghel, I., Voica, C., Toma, N., Cojocaru, I. (1989) "Biologia si tehnologia

drojdiilor I, Editura Tehnica." Editura Tehnica, Bucuresti p: 308-312.

74

9. Anonymous, (2001). "Regional Australian Olive Oil Processing Plants".

RIRDC Project GGO 1A, Publication No 00/187.

10. Aragon, J. M., (2000). PROJECT IMPROLIVE – "Improvements of

Treatments and Validation of Liquid-Solid Waste from the Two-Phase

Olive Oil Extraction (FAIR CT96-1420)." Final report- Annex A2, p.79

11. Arroyo-Lopez, F. N., Salvado, Z., Tronchoni, J., Guillamon, J. M., Barrio, E.

and Querol, A., (2010). "Susceptibility and resistance to ethanol in

Saccharomyces strains isolated from wild and fermentative environments."

Yeast., 27(12): 1005-1015.

12. Azbar, N., Bayram, A., Filibeli, A., Muezzinoglu, A., Sengul, F., Ozer, A.,

(2004). "Areview of waste management options in olive oil production."

Critical Reviews in Environmental Science and Technology, 34: 209–247.

13. Azuma, J. I., Tanaka, F., Koshijima, T., (1984). "Enhancement of enzymatic

susceptibility of lignocellulosic wastes by microwave radiation." Journal of

Fermentation Technology, 62(4): 377-384.

14. Ballesteros, I., Oliva, J. M., Negro, M. J., Manzanares, P., and Ballesteros, M.,

(2002). "Ethanol production from olive oil extraction residue pretreated

with hot water." Applied Biochemistry and Biotechnology, 98-100(1-9):717-

732.

15. Ballesteros, I. Oliva, J. M., Saez, F., Ballesteros, M., (2001). "Ethanol

production from lignocellulosic byproducts of olive oil extraction." Applied

Biochemistry and Biotechnology., 91-93:237-52.

16. Bankoffi, L. and Han, Y. W., (1990). "Alcohol production from pineapple

waste". World Journal of Microbiology and Biotechnology, 6:281-284.

17. Barbour, E. A., and Priest, F. G., (1988). "Some effects of Lactobacillus

contamination in scotch whisky fermentations." Journal of the Institute of

Brewing., 94:89-92.

http://www.ncbi.nlm.nih.gov/pubmed?term=Ballesteros%20I%5BAuthor%5D&cauthor=true&cauthor_uid=11963854

http://www.ncbi.nlm.nih.gov/pubmed?term=Oliva%20JM%5BAuthor%5D&cauthor=true&cauthor_uid=11963854

http://www.ncbi.nlm.nih.gov/pubmed?term=Saez%20F%5BAuthor%5D&cauthor=true&cauthor_uid=11963854

http://www.ncbi.nlm.nih.gov/pubmed?term=Ballesteros%20M%5BAuthor%5D&cauthor=true&cauthor_uid=11963854

http://www.ncbi.nlm.nih.gov/pubmed/11963854


75

18. Binod, P., Satyanagalakshmi, K., Sindhu, R., Janu, K. U., Sukumaran, R. K.,

Pandey, A., (2012). "Short duration microwave assisted pretreatment

enhances the enzymatic saccharification and fermentable sugar yield from

sugarcane bagasse." Renew Energy, 37:109–116.

19. Biosulfurol (2010): http://biosulfurol-energy.com (accessed on April 5, 2013).

20. Blue Fire Ethanol (2010): http://bluefireethanol.com(accessed on April 5,

2013).

21. Bothast, R. J. and Schlicher, M. A., (2005). "Biotechnological processes for

conversion of corn into ethanol." Applied Microbiology and Biotechnology,

67:19-25.

22. Bowman, P. I. and Ahearan, D. G., (1975). "Evaluation of the Uni-Yeast-Tek

Kit for the identification of medically important yeasts." Journal of Clinical

Microbiology, 2: 354 -357.

23. Bvochora, J. M., Read, J. S. and Zvauya, R., (2000). "Application of very high

gravity technology to the co-fermentation of sweet stem sorghum juice and

sorghum grain." Industrial Crops and Products, 11: 11-17.

24. Capek, M. E., Follez, P., and Chornet, G., (1992). "Pretreatment of waste

paper for increased ethanol yields." Biotechnology Letters, 14:985-988.

25. Cassey, G., (1996)."Ethanol tolerance in Yeasts." critical reviews in

microbiology, 13:219-290.

26. Chandrasena, G., Keerthipala, A. P., Walker, G. M., (2006). "Isolation and

Characterization of Sri Lankan Yeast Germplasm and Its Evaluation for

Alcohol Production." Journal Institute Brewing, 112:302-307.

27. Chen, J. P., Wu, K. W. and Fakuda, H., (2008), "Bioethanol Production from

Uncooked Raw Starch by Immobilized Surface-engineered Yeast."

Applied biochemistry and biotechnology, 145: 59-67.

http://biosulfurol-energy.com/

http://bluefireethanol.com/

76

28. Chittibabu, S., Rajendran, K., Santhanmuthu, M., Saseetharan, M. K. (2011).

"Optimization of microwave assisted alkali pretreatment and enzymatic

hydrolysis of Banana pseudostem for bioethanol production." 2nd

International Conference on Environmental Science and Technology IPCBEE

vol.6.

29. Cot, M., Loret, M., François, J. and Benbadis, L., (2007). "Physiological

behavior of Saccharomyces cerevisiae in aerated fed-batch fermentation for

high level production of bioethanol." FEMS Yeast Research, 7, 22-32.

30. Cruz, P., Palomar, J. M. and Ortega, A., (2006). "Integral energy cycle for the

olive oil sector in the province of Jaén (Spain)." Grasas y Aceites., 57(2):219-

228.

31. Czarnecki, M. and Grajek, W., (1991)," Starch hydrolysis and its effect on the

product yield and microbial contamination in yeast ethanol fermentation."

World Journal of Microbiology and Biotechnology, 7: 467-469.

32. Danellakis, D., Ntaikou, I., Kornaros, M., Dailainis, S., (2011)."Olive oil mill

wastewater toxicity in themarine environment: alterations of stress indices

in tissues of mussel Mytilus galloprovincialis." Aquatic Toxicology, 101:358-

366.

33. Dashtban, M., Schraft, H. and Qin, W. (2009). "Fungal bioconversion of

lignocellulosic residues: opportunities & perspectives”. International Journal

of Biological Sciences, Vol. 5, pp. 578-595.

34. Domingues, L., Lima, N. and Teixeira, A. J., (2001), Alcohol production from

cheese whey permeate using genetically modified flocculent yeast cells."

Biotechnology and Bioengineering., 72: 507-514

35. Duff, B. J. S. and Murray, D. W., (1996)." Bioconversion of forest products

industry waste cellulosic's to fuel ethanol: A review." Bioresource

Technology., 55: 1-33.

77

36. El Asli. A., and Qatibi, A., (2009)." Ethanol Production from Olive Cake

Biomass Substrate Biotechnol." Biotechnology and Bioprocess Engineering,

14(1):118-122.

37. Esteghlalian, A., Hashimoto, A. G., Fenske, J. J., Penner, M. H., (1997).

"Modeling and optimization of the dilute-sulfuric-acid pretreatment of corn

stover, poplar and switchgrass." Bioresource Technology. 59:129–136.

38. Excoffier, G., Toussaint, B. and Viguon, R. M., (1991), "Saccharification of

steam exploded popular wood." Biotechnology and Bioengineering, 38: 1308-

1317.

39. F.O. Licht, 2006; World watch Institute, 2007.

40. Fernandez-Navarro, M., Peragon, J., Amores, V., Dela Higera, M. and Lupianez,

J. A. (2008). "Maslinic acid added to the diet increases growth and proteina-

turnover rates in the White muscle of rainbow trour (Oncorhynchus

mykiss)." Comparative Biochemistry and Physiology, Part C., 147: 158-167.

41. Fujii, N., Sakurai, A., Suye, S. and Sakakibara, M., (2001). "Ethanol

production from starch by immobilized A.awamorii and S. pastorianus

using cellulose carriers." Indian Journal of Microbiology and Biotechnology.,

27: 52-57

42. Furukava, K., Kitano, H., (2004)."Effect of cellular inositol content on ethanol

tolerance of Saccharomyces cerevisiae in sake brewing." Journal of bioscience

and bioengineering., 98: 107-113

43. Georgieva, T. I., Ahring, B. K. (2007). "Potential of agro industrial waste

from olive oil industry for fuel ethanol production." Biotechnology journal,

2(12):1547-55.

44. Ghosh, P., Ghose, T. K., (2003), "Bioethanol in India: recent past and

emerging future." Advances in Biochemical Engineering/Biotechnology., 85:

1–27.

45. Goffeau, A., (2000), "Four years of post-genomic life with 6,000 yeast genes."

Federation of European Biochemical Societies letters, 480:37-41.

http://www.ncbi.nlm.nih.gov/pubmed?term=Georgieva%20TI%5BAuthor%5D&cauthor=true&cauthor_uid=17979161

http://www.ncbi.nlm.nih.gov/pubmed?term=Ahring%20BK%5BAuthor%5D&cauthor=true&cauthor_uid=17979161


78

46. Goksungur, Y., Zorlu, N., (2001). "Production of ethanol from Ca-Alginate

immobilized yeast cells in a packed-bed reactor." Turkey Journal of Biology,

25:265-275.

47. Govindaswamy, S. and Vane, L. M., (2007). "Kinetics of growth and ethanol

production on different carbon substrates using genetically engineered

xylose-fermenting yeast." Bioresource Technology., 98: 677-685.

48. Grobben, N. G., Eggink, G., Cuperus, F. P. and Huizing, H. J.,(1993).

"Production of acetonebutonol and ethanol (ABE) from potato waste:

fermentation with integrated membrane extraction." Applied Microbiology

and Biotechnology, 39:494-498.

49. Grootgen, R. J., Meijlink, L. H. H. M., Vanderlans, R. G. J. M. and Luyben, K.

A. M., (1990). "Cofermentation of glucose and xylose with immobilized

Pichia stipitis and S7 cerevisiae." Enzyme and Microbial Technology, 12: 860-

864.

50. Grylls, F. S. M. and Harrison, J. S. Kang, J. Fermentation of Ethanol

http://www.andrew.cmu.edu/user/jitkangl/Fermentation%20of%20Ethanol

/Fermentation%20 of%20Ethanol.htm. (Accessed on June 10, 2012 ( .

51. Hammond, J. B., Egg, R., Diggins, D. and Coble, C. G., (1996)." Alcohol from

bananas". Bioresource Technology., 56: 125-130.

52. Harikrishna, S., Reddy, T. J. and Chowdary, G. V., (2001). "Simultaneous

saccharification and fermentation of lignocellulosic wastes to ethanol using

a thermotolerant yeast." Bioresource technology., 77: 193-196.

53. Hendriks, A. T. W. M., and Zeeman, G., (2008). "Pretreatments to enhance

the digestibility of lignocellulosic biomass." Bioresource Technology., 100(1):

10-18.

54. Hill, G. A. and Robinson, C. W. (1990). "A Modified Ghose Model for Batch

Cultures of Saccharomyces cerevisiae at High Ethanol Concentrations". The

Chemical Engineering Journal, 44(3): 69-80.

http://www.andrew.cmu.edu/user/jitkangl/Fermentation%20of%20Ethanol/Fermentation

http://www.andrew.cmu.edu/user/jitkangl/Fermentation%20of%20Ethanol/Fermentation

79

55. House, L. R., Gomez, M., Murty, D. S., Sun, Y. and Verma B. N., (2000).

"Development of some agricultural industries in several African and Asian

countries." In: C. W. Smith and R.A. Frederiksen, Editors, Sorghum: Origin,

History, and Technology, and Production, Wiley, New York, pp. 131–190.

56. Hu, Z. H., Wen, Z. Y., (2008). "Enhancing enzymatic digestibility of

switchgrass by microwave-assisted alkali pretreatment." Biochemical

Engineering Journal, 38 (3): 369–378.

57. Ingram, L. O., Aldrich, H. C. , Borges, A. C., Causey, T. B., Martinez, A.,

Morales, F., Saleh, A., Underwood, S. A., Yomano, L. P., York, S. W.,

Zaldivar, J., Zhou, S., (1999). "Enteric Bacterial Catalysts for Fuel Ethanol

Production." Biotechnology Progress, 15(5):855-866.

58. Jeffries, T. W., (2006). "Engineering yeasts for xylose metabolism". Current

Opinion in Biotechnology 17(3):320.

59. Jimenez, J., Benitez, T., (1986). "Characterization of wine yeasts for ethanol

production." Applied Microbiology and Biotechnology, 25: 150-154.

60. Kang, J. Fermentation of Ethanol.

http://www.andrew.cmu.edu/user/jitkangl/Fermentation%20of%20Ethanol/FerF

ermentat%20 of%20Ethanol.htm (accessed on April 13, 2012).

61. Kavdir, Y. and Killi, D., (2008). "Influence of olive oil solid waste

applications on soil pH, electrical conductivity, soil nitrogen

transformations, carbon content and aggregate stability." Bioresource

Technology., 99(7): 2326-2332.

62. Keim C. R. (1983). "Technology and economics of fermentation alcohol – an

update." Enzyme Microbial Technology, 5(2): 103–14.

63. Keshwani, D. R., Cheng, J. J., Burns, J. C., Li, L., and Chiang, V., (2007).

"Microwave Pretreatment of Switchgrass to Enhance Enzymatic

81

Hydrolysis." Conference Presentations and White Papers: Biological Systems

Engineering, Paper 35.

64. Khaing, T. W., Weine, N., Mya, M. O., (2008). "Isolation, Characterization

and Screening of Thermo tolerant, Ethanol Tolerant Indigenous Yeasts and

Study on the Effectiveness of Immobilized Cell for Ethanol Production."

Journal of Science and Technology, 1:12-14.

65. Khalid, A., Arshad, M., Anjum, M., Mahmood, T. and Dawson, L., (2011). "The

anaerobic digestion of solid organic waste." Waste Management, 31:1737-

1744.

66. Kikas, T., Tutt, M. and Olt, J., (2012). "Influence of different pretreatment

methods on bioethanol production from wheat straw." Agronomy Research

Biosystem Engineering., Special Issue 10(1): 269-276

67. Kim, S. and Dale, B. E., (2003). "Global potential bioethanol production

from wasted crops and crop residues." Biomass and Bioenergy, pp. 1-15.

68. Kim, T. H., Kim, J. S. and Sunwoo, Y. Y., (2003), "Pretreatment of corn

stover by aqueous ammonia." Journal of Applied Biochemistry and

Biotechnology, 90: 39-47.

69. Kitchaiya, P., Intanakul, P. and Krairiksh, M., (2003). "Enhancement of

Enzymatic Hydrolysis of Lignocellulosic Wastes by Microwave

Pretreatment under Atmospheric Pressure." Journal of Wood Chemistry and

Technology, 23(2): 217-225.

70. Kobayashi, M., Itoh, K., Suzuki, T., Osanai, H., Nishikawa, K., Katoh, Y.,

Takagi, Y., and Yamamoto, M., (2002) "Identification of the interactive

interface and phylogenic conservation of the Nrf2-Keap1 system." Genes

Cells., 7(8): 807-820.

71. Kongkiattikajorn, J., Rodmui, A., and Dandusitapun, Y., (2007)." Effect of

Agitation Rate on Batch Fermentation of Mixture Culture of Yeasts during

81

Ethanol Production from Mixed Glucose and Xylose." Journal of

Biotechnology., pp:1-4

72. Koullas, D. P., Christookopoulos, P. R., Kekos, D., Macris, B. J. and Koukios,

E. G., (1992). "Correlating the effect of pretreatments on the enzymatic

hydrolysis of straw." Biotechnology and Bioengineering, 39:113-116.

73. Kreger-Van Rij, N. J. W., (1984). "The Yeast a Taxonomic Study." Elsevier

Science Publisher BV, Amsterdam, pp: 1082.

74. Krishnan, M. S., Nghiem, N. P. and Davison, B. H., (1999). "Ethanol

production from corn starch in a fluidized bed bioreactor." Applied

Biochemistry and Biotechnology, 77:359-372.

75. Kumar, J. V., Shahbazi, A. and Mathew, R., (1998). "Bioconversion of solid

food wastes to ethanol." Analysts, 123:497-502.

76. Kumar, S. R., Shankar, T. and Anandapandian, K. T. K., (2011).

"Characterization of alcohol resistant yeast Saccharomyces cerevisiae

isolated from Toddy." International Research Journal of Microbiology (IRJM),

2(10): 399-405.

77. Kurtzman, C.P., (1995). "Relationships among the genera Ashbya,

Eremothecium, Holleya and Nematospora determined from rDNA sequence

divergence." Journal of industrial microbiology & biotechnology., 14(6):523-

530.

78. Lama-Munoz, A., Rodriguez-Gutiérrez, G., Rubio-Senent, F., Gnmez-Carretero,

A., Fernandez- Bolanos, J., (2011). "New hydrothermal treatment of alperujo

enhances the content of bioactive minor components in crude pomace olive

oil." Journal of Agricultural and Food Chemistry., 59:1115-1123.

79. Lang, X., Hill, G. A., and Macdonald, D. G., (2001). "Recycle Bioreactor for

Bioethanol Production from Wheat Starch I. Cold Enzyme Hydrolysis."

Energy Sources, 23: 417-425.

82

80. Lavarack, B. P., Griffin, J. G. and Rodman, D., (2002). "The acid hydrolysis of

sugarcane bagasse hemicellulose to produce xylose, arabinose, glucose and

other products." Biomass and Bioenergy, 23:367-380.

81. Lee, C. G., Kim, C. H. and Rhee, S. K., (1992). "A kinetic model and

simulation of starch saccharification and simultaneous ethanol

fermentation by amyloglucosidase and Zymomonas mobilis." Bioprocess

Engineering, 7 (8):335-341.

82. Lee, J., (1997). "Biological conversion of lignocellulosic biomass to ethanol."

Journal of Biotechnology, 56:1-24.

83. Lee, Y., Lee, W. G., Chang, Y. K. and Chang, H. N., (1995). "Modeling of

Ethanol Production by Saccharomyces cerevisiae from a Glucose and

Maltose Mixture." Biotechnology Letters, 17 (8):791-796.

84. Li, Z., Jiang, Z., Fei, B. Yu, Y. and Cai, Z., (2012)., "Effective of Microwave-

KOH Pretreatment on Enzymatic Hydrolysis of Bamboo." Journal of

Sustainable Bioenergy Systems., 2(4):104-107.

85. Licht, F.O., (2006). World Ethanol Market: The Outlook to 2015, Tunbridge

Wells, Agra Europe Special Report, UK.

86. Licht’s FO, F.O. Licht's World Ethanol & Biofuels Report. Available from

http://www.agra-net.com/portal2/showservice.jsp?servicename=as072 (accessed

21 June 2012).

87. Lin, Y., and Tanaka, S., (2006). "Ethanol fermentation from biomass

resources: current state and prospects." Applied Microbiology and

Biotechnology, 69:627-642.

88. Lin, Y., Zhang, W., Li, C., Sakakibara, K., Tanaka, S., Kong, H., (2011).

"Factors affecting ethanol fermentation using Saccharomyces cerevisiae

BY4742." Biomass and Bioenergy., Volume 47 Elsevier.

http://www.deepdyve.com/browse/journals/biomass-and-bioenergy

83

89. Lopes D. H. J. and Mauro S. P., (2001). "Urea increases tolerance of yeast

inorganic pyrophosphatase activity to ethanol". Archives of biochemistry and

biophysics. Supplement, 394: 61-66.

90. Lynd, L. R., Cushman, J. H., Nicholos, R. J. and Hyman, C. E., (1991)." Fuel

ethanol from cellulosic biomass". Science, 251:1318-1323.

91. Mager, W. H., Siderius, M., (2002). "Novel insights into osmotic stress

response of yeast." FEMS yeast research, 2:251-257.

92. Majed, A .Z. and Mohamad, W., (2002). "Influence of olive mills solid waste

on soil hydraulic properties." Communications in soil science and plant

analysis, 33.

93. Mamma, D., Koullas, D., Fountoukidis, G., Kekos, D., Macris, B.J. and

Koukios, E., (1996). "Bioethanol from sweet sorghum: simultaneous

saccharification and fermentation of carbohydrates by a mixed microbial

culture." Process Biochemistry, 31(4):377-381.

94. Manikandan, T., Umamaheswari, M., Jayakumari, M., Maheswari, K.,

Subashree, M., Mala, P., Sevanthi, T., (2010). "Bioethanol Production from

Cellulosic Materials." Asian Journal of Science and Technology, 1:5-11.

95. Mariam, I., Manzoor, K., Sikander, A., (2009). "Enhanced production of

ethanol from free and immobilized Saccharomyces cerevisiae under

stationary culture." Pakistan Journal of Botany, 41(2): 21-833.

96. Martín, A. I., Moumen, A., Yánez, D. R. and Molina, E. (2003). "Chemical

composition and nutrients availability for goat as and sheep of two-stage

olive cake and olive leaves." Animal Feed Science and Technology, 107:61-74.

97. Martini, A., (1996). "Origins and domestication of the wine yeast

Saccharomyces cerevisiae." Journal of Wine Research, 4:165-176.

98. McKendry, P., (2002). "Energy production from biomass (part 1): overview

of biomass." Bioresource Technology., 83: 37-46.

84

99. McMillan, J. D., (1994). "Pretreatment of lignocellulosic biomass, in

Enzymatic Conversion of Biomass for Fuels Production." (Eds M. E.

Himmel, J. O. Baker, and R. P. Overend), American Chemical Society,

Washington, DC, p. 292–324.

100. Mesa, J. J., Infante, J. J., Rebordinos, L., and Cantoral, J. M., (1999).

"Characterization of yeasts involved in the biological ageing of sherry

wines." LWT - Food Science and Technology, 32 (2): 114-120.

101. Mikuni, K., Monma, M. and Kainuma, K., (1987). "Alcohol fermentation of

corn starch digested by Chalara paradoxa amylase without cooking."

Biotechnology and bioengineering. 29:729-732.

102. Millati, R., Edebo, L., and Taherzadeh, M. J., (2005)." Performance of

Rhizopus, Rhizomucor and Mucor in Ethanol Production from Glucose,

Xylose and Wood Hydrolyzates." Enzyme and Microbial Technology., 36 (2-

3): 294-300

103. Mishra, S. M., Chandrashekhar, B., Tanushree, C., and Kanwal, S., (2011).

"Production of bioethanol from Jatropha oilseed cakes via dilute acid

hydrolysis and fermentation by Saccharomyces cerevisiae." International

Journal of Biotechnology Applications, 3(1):41-47.

104. Miyamoto, K., (1997). "Production of fuel alcohol from cellulosic

biomass." FAO Agricultural Services Bulletin, 128: 19-52.

105. Mobini- Dehkordi, M., Nahvi, I., Ghaedi, K. and Tavassoli, M., (2007).

“Isolation of high ethanol resistant strains of Saccharomyces cerevisiae."

Res. Pharm. Sc., 2: 85-91.

106. Monte, A. R., Rigo, M. and Joekes, I., (2003). "Ethanol fermentation

of a diluted molasses medium by Saccharomyces cerevisiae immobilized on

chryolite." Brazilian Archives of Biology and Technology, 46: 751-757.

85

107. Nagesha, L., (2009). "Bioethanol production from spoilt sorghum grain."

M. Sc (Department of Agricultural Microbiology) thesis, University of

Agriculture Sciences, Dharwad (India).

108. Nakamura, Y., Sawada, T. and Inoue, E., (2001). "Enhanced ethanol

production from enzymatically treated steams exploded rice straw using

extractive fermentation." Journal of Chemical Technology and Biotechnology,

76:879-884.

109. Narendranath, N. V., and Power, R., (2005). "Relationship between pH and

medium dissolved solids in terms of growth and metabolism of Lactobacilli

and Saccharomyces cerevisiae during ethanol production." Applied and

Environmental Microbiology, 71: 2239-2243.

110. Narendranath, N. V., Thomas, K. C., Ingledew, W. M., (2001). "Effects of

acetic acid and lactic acid on the growth of Saccharomyces cerevisiae in a

minimal medium." Journal of Industrial Microbiology and Biotechnology, 26

(3):171–177.

111. Neelakandan, K., Usharani, H., (2009). "Optimization and production

of bioethanol from cashew apple juice using immobilized yeast cells by

Saccharomyces cerevisiae." European Journal of Scientific Research 4 (2):85-

88.

112. Neves, M. A., Kimura, T., Shimizu, N. and Shiiba, K., (2006). "Production

of alcohol by simultaneous saccharification and fermentation of low-grade

wheat flour." Brazilian Archives of Biology and Technology, 49:481-490.

113. Niaounakis, M., and Halvadakis, C. P., (2004). "Olive-Mill Waste

Management: Literature review and Patent Survey." Typothito – George

Dardanos Publications, Athens, Greece.

114. Nigam, J. N., (2001),"Ethanol production from wheat straw hemicellulose

hydrolysate by Pichia stipitis." Journal of Biotechnology, 87(1):17-27.

86

115. Olsson, L. and Hahn-Hägerdal, B., (1996). "Fermentation of lignocellulosic

hydrolysates for ethanol production." Enzyme and Microbial Technology,

18(5):312.

116. Olsson, L., Hahn, B., (1993). "Fermentative performance of bacteria and

yeasts in lignocellulose hydrolysates." Process Biochemistry, 28:249-57.

117. Omer. R., (2012). "Morphological and Genetic Characterization of the

main Palestinian olive (Olea europaea L.) Cultivars". M. Sc. thesis,

department of plant, An-Najah National University, Palestine.

118. Oner, E. T., Oliver, S. G. and Kirdar, B., (2005). "Production of ethanol

from starch by respiration deficient recombinant Saccharomyces

cerevisiae." Applied and Environmental Microbiology, 71:6443-6445.

119. Ooshima, H., Aso, K., Harano, Y., and Yamamoto, T., (1984).

"Microwave Treatment of Cellulosic Materials for Their Enzymatic

Hydrolysis." Biotechnology Letters, 6(5):289-294.

120. Ooshima, H., Burns, S. D. and Converse, O. A., (1990). "Adsorption of

cellulase from Trichoderma reesei on cellulose and ligneous residue in wood

pretreated by dilute sulphuric acid with explosive decompression."

Biotechnology and Bioengineering, 36: 446-452.

121. Osho, A., (2005). "Ethanol and sugar tolerance of wine yeasts isolated

from fermenting cashew apple juice." African Journal of Biotechnology, 4:

660-662.

122. Ostergaard, S., Olsson, L. and Nielsen, J., (2000). "Metabolic Engineering

of Saccharomyces cerevisiae." Microbiology and Molecular Biology Review, p.

34-50.

123. Panesar, P. S., Marwaha, S. S., Gill, S. S. and Rai, R., (2001)."Screening of

Zymomonas mobilis strains for ethanol production from molasses." Indian

Journal of Microbiology, 41:187-189.

87

124. Parra, A., Rivas, F., Lopez, P. E., Garcia-Granados, A., Martinez, A.,

Albericio, F., Marquez, N. and Muñoz, E., (2009). "Solution- and solid-phase

synthesis and anti-HIV activity of maslinic acid derivatives containing

amino acids and peptides." Bioorganic and Medicinal Chemistry, 17:1139-

1145.

125. PCBS-Palestinian Central Bureau of Statistics and Ministry of Agriculture-

MoA., (2009). Palestine in Figures 2008 – May 2009.

126. Perez, J., Munoz-Dorado, J., De-la-Rubia, T., and Martinez, J., (2002),

"Biodegradation and biological treatment of cellulose, hemicellulose and

lignin: an overview." International Microbiology, 5:53-63.

127. Periyasamy, S., Venkatachalam, S., Ramasamy, S., Srinivasan, V. (2009),

"Production of Bio-ethanol from Sugar Molasses Using Saccharomyces

cerevisiae." Modern Applied Science., 3(8): 1913-1844

128. Petrea, L. (2008), "Characterization of two Saccharomyces cerevisiae

strains obtained by uv mutagenesis." Innovative Romanian Food

Biotechnology, 2:40–47.

129. Petrova, P., Ivanova, V. (2010), "Perspectives for the production of

bioethanol from lignocellulosic materials." Biotechnology and

Biotechnological Equipment, 24(2): 529-546.

130. Prasad, S., Singh, A., Joshi, H. C., (2007). "Ethanol as an alternative fuel

from agricultural, industrial and urban residues." Resources Conservation

and Recycling, 50: 1-39.

131. Pretorius, I.S., (2000). "Tailoring wine yeast for the new millennium:

novel approaches to the ancient art of winemaking." Yeast, 16:675-729.

132. Purwadi, R., 2006. "Continuous Ethanol Production from Dilute-Acid

Hydrolyzates: Detoxification and Fermentation Strategy." Chalmers

University of Technology, Sweden, pp: 1-72.

88

133. Ramanathan, M., (2000), "Biochemical conversion ethanol production

from root crops." In: Biomass Conversion Technology for Agriculture and

Allied Industries. Short Course Manual Organized by Department of Bioenergy,

Tamil Nadu University, Coimbatore, 4-13:155-162.

134. Ramos-Cormenzana, A. and Monteoliva-Sanchez, M., (2000). "Potencial

biofarmacéutico de los residuos de la industria oleicola." Ars Pharmaceutica,

41:129-136.

135. Ravoof, S. A., Pratheepa. K., Supassri. T., Chittibabu, S. (2012).

"Enhancing enzymatic hydrolysis of rice straw using microwave assisted

nitric acid pretreatment." International Journal of Medicine and Biosciences,

1(3):13 – 17.

136. Rivera, E. C., Costa, A. C., Attala, D. I. P., Margery, F., Mariel, M. R. W. and

Mariel Filch, R. (2006). "Evaluation of optimization techniques for

parameter estimation: Application to ethanol fermentation considering the

effect of temperature." Process Biochemistry, 41: 1682-1687.

137. Roble, N., (2003). "Development of production systems for raw cassava

starch bioconversion in novel bioreactors with cells immobilized in loofa

(Luffa cylindrica) sponge." Ph. D. Thesis, University of Tsukuba, Japan.

138. Saha, B. C., Iten, L. B., Cotta, M. A., Wu, Y. V., (2005). "Dilute acid

pretreatment, enzymatic saccharification and fermentation of wheat straw

to ethanol Pro." Biochemistry, 40 (12):3693–3700.

139. Salmon, J. M., Vincent, O., Mauricio, J. C., Bely, M. and Barre, P., (1993).

"Sugar transport inhibition & apparent loss of activity in Saccharomyces

cerevisiae as a major limiting factor of enological fermentation." American

Journal of Enology and Viticulture, 228:56-64.

140. Sandhu, H., Bajaj, K. L. and Arneja, J. S., (1998). "Biochemical studies on

bioconversion of rice straw to ethanol." Indian Journal of Ecology, 25:62-65.

141. Schell, J. D., Riley, J. C., Dowe, N., Farmer, J., Ibsen, N. K., Ruth, F. M.,

Toon, T. S. and Lumpkin, E. R., (2004). "A bioethanol process development

89

units: initial operating experiences and results with a corn fiber feed stock."

Bioresource Technology., 91:179-188

142. Schuller, D., Valero, E., Dequin, S., Casal, M,. (2004). "Survey of

molecular methods for the typing of wine yeast strains." FEMS

Microbiology Letters, 231: 19-26.

143. Sebastian, S., (2008)."Turning waste olive stones into bioethanol

Granada." Spain. Renewable Energy World.

144. Senkevich, S., Ntaikou, I., Lyberatos, G., (2012). "Bioethanol production

from thermochemical pretreated olive mill solid residues using the

Pachysolen tannophilus." Global NEST Journal, 14(2):118-124.

145. Shalabia, A., (2011). "Production of Bioethanol from Olive Pulp." M. Sc.

thesis, department of chemistry, An-Najah National University, Palestine.

146. Sharma, K. S., Kalra, K. L. and Grewal, H. S., (2002a), "Enzymatic

saccharification of pre-treated sunflower stalk." Biomass and Bioenergy,

23:237-243.

147. Sharma, S., Jain, M. and Dalel, S., (2002b). "Production of ethanol from

potato flour by an amylolytic Saccharomyces cerevisiae SJ-31." Annual

Conference of Association of Microbiologist of India. Department of

Microbiology, CCS Haryana Agriculture University Hisar, 11-13.

148. Sheorain, V., Banka, R. and Chavan, M., (2002). "Ethanol production

from sorghum." www.ethanolfromsorghumarticleicrisat.htm. pp. 1-11.

149. Shiva, C. A., Hamdapurkar, S. K. and Walkte, P.S., (2001). "A study on

biochemical conversion of agriculture starchy waste into glucose syrup." In:

Proceeding of the 42 Annual Conference of association of Microbiologist of

India held at Gulbarga University, November 9-11:72.

150. Shuler, M. L and Kargi, F., (2002). "Bioprocess Engineering: basic

concept." Second Edition, Prentice Hall, New Jersey.

91

151. Sierra, J.; Marti, E., Garau, M. A. and Cruenas, R., (2007). "Effects of the

agronomic use of olive oil mill wastewater: Field experiment." Science of the

Total Environment, 378:90-94.

152. Smith, G. A. and D. R. Buxton. (1993). "Temperate zone sweet

sorghum ethanol production potential." Bioresource Technology., 43:71–75.

153. Sree, N. K., Sridhar, M., Suresh, K. and Rao, L. V., (1999). "High ethanol

production by solid substrate fermentation from starchy substrates using

thermo tolerant Saccharomyces cerevisiae." Bioprocess engineering, 20:561-

563.

154. Subashini, D., Ejilane, J., Radha, A., Jayasri, M. A. And Suthindhiran,

K., (2011). "Ethanol Production from Sago Waste Using Saccharomyces

cerevisiae Vits-M1." Current Research Journal of Biological Sciences, 3(1):42-

51.

155. Sun, Y. and Cheng, J., (2002). "Hydrolysis of lignocellulosic materials

for ethanol production: A review." Bioresource Technology., 83(1): 1-11.

156. Suresh, K., Kiransree, N. and Rao, V. L., (1999a). "Utilization of damaged

sorghum and rice grains for ethanol production by simultaneous

saccharification and fermentation." Bioresource Technology., 68:301-304.

157. Suresh, K., Kiransree, N. and Venkateshwar Rao, L., (1999b). "Production

of ethanol by raw starch hydrolysis and fermentation of damaged grains of

wheat and sorghum." Bioprocess Engineering, 21:165–168.

158. Swiecilo, A., krawiec, Z., Wawryn, J. and Bartosz, G., (2000), "Effect of

stress on the life span of the yeast Saccharomyces cerevisiae." Acta

biochimica Polonica, 47:355-364.

159. Taherzadeh, M. J., and Karimi, K., (2007), "Acid-Based Hydrolysis

Processes for Ethanol from Lignocellulosic Materials: A Review."

Bioresource, 2: 472-499.

91

160. Tahir, A., Aftab, M. and farasat, T., (2010a)." Effect of Cultural Conditions

on Ethanol Production by Locally Isolated Saccharomyces cerevisiae Bio-

07." Journal of Applied Pharmaceutical Science, 3(2):72-78.

161. Tahir N, Bhatnagar R, Viswanathan L (2010). "Development of

resistance in Saccharomyces cerevisiae against inhibitory effects of

browning reaction products." Enzyme and Microbial Technology, 3:24–28.

162. Tassinari, T., Macy, C. and Spano, L., (1980)." Energy requirement and

process design consideration in compression milling pretreatment of

cellulosic wastes for enzymatic hydrolysis." Biotechnology and

Bioengineering, 22:1689-1705.

163. Taylor, F., Kurantz, M. J., Goldberg, N., Mcaloon, A. J. and Craig, J. C.,

(2000). "A dry grind processes for fuel ethanol fermentation and stripping."

Biotechnology progress, 64:103-111.

164. Teixeira, L. C., Linden, J. C., Schroeder, H. A., (1999a). "Alkaline and

peracetic acid pretreatments of biomass for ethanol production." Applied

Biochemistry and Biotechnology, 77–79:19–34.

165. Teixeira, L. C., Linden, J. C., Schroeder, H. A., (1999b). "Optimizing

peracetic acid pretreatment conditions for improved simultaneous

saccharification and co-fermentation (SSCF) of sugar cane bagasse to

ethanol fuel". Renewable Energy. 16:1070–1073.

166. Thais, M. G., Danilo, G. M., Iara, P. M., Cyntia, M. T., Fadel, P., Tania,

M. B. B., (2006). "Isolation and characterization of Saccharomyces cerevisiae

strains of winery interest." Brazilian Journal of Pharmaceutical and Sciences;

42 (1):119-126.

167. Thomas, K. C., Hynes, S. H., Ingledew, W. M., (2002). "Influence of

medium buffering capacity on inhibition of Saccharomyces cerevisiae

growth by acetic and lactic acids." Applied and environmental microbiology.

68:1616-1623.

92

168. Tikka, C., Osuru, H. P., Atluri, N., Raghavulu, P. C. V., yellapu, N. K.,

Mannur, I. S., Prasad, U. V., Aluru, S., Varma, N., and Bhaskar, M., (2013)."

Isolation and characterization of ethanol tolerant yeast strains."

Bioinformation, 9(8): 421-425.

169. Ueda, S., Zenin, C. T., Monteiro, D. A. and Park, Y. K., (1981). "Production

of ethanol from raw cassava starch by a nonconventional fermentation

method." Biotechnology and Bioengineering., 23: 291-199

170. Vallander, R. L. and Eriksson, L. K. E., (1991). "Enzymatic hydrolysis

of lignocellulosic materials: II Experimental investigations of theoretical

hydrolysis – process models for an increased enzyme recovery."

Biotechnology and Bioengineering, 38: 139-144.

171. Van Zyl, W., Lynd, L. R., den Haan, R., McBride, J. E., (2007).

"Consolidated Bioprocessing for Bioethanol Production Using

Saccharomyces cerevisiae." Biofuels, p 205-235.

172. Verma, G., Nigam, P., Singh, D. and Chaudhary, K., (2000). "Bioconversion

of starch to ethanol in a single step process by co-culture of amylolytic

yeasts and Saccharomyces cerevisiae." Bioresource Technology., 72:261-266.

173. Voordeckers, K., De Maeyer, D., van der Zande, E., Vinces, M. D., Meert,

W., Cloots, L., Ryan, O., Marchal, K., Verstrepen, K. J., (2012). "Identification

of a complex genetic network underlying Saccharomyces cerevisiae colony

morphology." Molecular Microbiology, 86(1): 225–239.

174. Walker, L. P., Hii, H., Wilson, D. B., (2006). "Enzymatic hydrolysis of

cellulose: An Overview." Bioresource Technology., 36:3-14.

175. Wang, Z. X., Zhuge, J., Fang, H., and Prior, B.A., (2001). "Glycerol

production by microbial fermentation: A review." Biotechnology Advances,

19: 201-223.

93

176. Wayman, M., Parekh, S. R., (1990). "Biotechnology of Biomass

Conversion; Fuel and Chemicals from Renewable Resources." Open

University Press, Milton Keynes., pp. 181-232.

177. Yang, C., Shen, Z., Yu, G., Wang, J., (2008). "Effect and after effect of

_radiation pretreatment on enzymatic hydrolysis of wheat straw."

Bioresource Technology., 99(14): 6240-6245.

178. Zheng, Y., Pan, Z., Zhang, R., (2009). "Overview of biomass pretreatment

for cellulosic ethanol production." International Journal of Agricultural and

Biological Engineering, 2(3): 51－68.

179. Zhu, S., Wu, Y., Yu, Z., Zhang, X., Li, H., Gao, M., (2006). "The effect of

microwave irradiation on enzymatic hydrolysis of rice straw." Bioresource

Technology. 97(15): 1964-1968.

180. Zhu, S., Wu, Y., Ziniu, Z., Liao, J., Zhang, Y., (2005b). "Pretreatment by

microwave/alkali of rice straw and its enzyme hydrolysis." Process

Biochemistry, 40(9):3082-3086.

181. Zhu, S., Yu, Z., Wu, Y., Zhang, X., Li, H., Gao, M., (2005a). "Enhancing

enzymatic hydrolysis of rice straw by microwave pretreatment." Chemical

Engineering Communications, 192(12):1559-1566.

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