CYPRUS INTERNATIONAL UNIVERSITY

INSTITUTE OF GRADUATE STUDIES AND RESEARCH

Department of Bioengineering

PRODUCTION OF CRUDE CELLULASE ENZYME BY SOIL FUNGI AND

ITS APPLICATION IN BIOCONVERSION

MSc. Thesis

Mohamed Salem Abdualla ALNAIED

Nicosia - 2016
 CYPRUS INTERNATIONAL UNIVERSITY
INSTITUTE OF GRADUATE STUDIES AND RESEARCH
Bioengineering Department

PRODUCTION OF CRUDE CELLULASE ENZYME BY SOIL FUNGI AND

ITS APPLICATION IN BIOCONVERSION

MSc. Thesis

Mohamed Salem Abdualla ALNAIED

Supervisor
Assoc. Prof. Dr. Hatice A. ERKURT

Nicosia - 2016
 THESIS APPROVAL CERTIFICATE

The Thesis study of Bioengineering Department graduate student Mohamed Salem Abdualla
ALNAIED with student number 20141020 titled: Production of crude cellulase enzyme by
soil fungi and its application in bioconversion has been approved with unanimity/majority of
votes by the jury and has been accepted as a Master of Bioengineering Thesis.

Thesis Defense Date: 13/01/2016

Jury Members Signature

1) Thesis Supervisor
Assoc. Prof. Dr. Hatice ERKURT ……………...…..………..
(Cyprus International University)

Jury Member:
2) Asst. Prof. Dr. Emrah ERKURT …….………..…….……...
(Cyprus International University)

Jury Member:
3) Asst. Prof. Dr. Doğa KAVAZ …..…………..…………...
(Cyprus International University)

Prof. Dr. Hikmet SECIM

Director of the Institute

 DECLARATION

Name and Surname:

Mohamed Salem Abdualla ALNAIED

Title of Dissertation:

Production of crude cellulase enzyme by soil fungi and its application in bioconversion

Supervisor:

Assoc. Prof. Dr. Hatice A. ERKURT

Year:

2016

I hereby declare that all information in this document has been obtained and presented in
accordance with academic rules and ethical conduct. I also declare that, as required by these
rules and conduct, I have fully cited and referenced all material and results that are not original to
this work.

I hereby declare that the Cyprus International University, Institute of Graduate Studies and
Research is allowed to store and make available electronically the present Dissertation.

Date: 13/05/2016

Signature:
 ACKNOWLEDGEMENT

First I would like to thank Allah Almighty for his blessings towards achieving this goal
in my life and I would like to express my sincere gratitude and appreciation to my
master’s thesis supervisor and course advisor, Assoc. Prof. Dr. Hatice Erkurt for her
patience, guidance and positive feedback.

I would also like to thank the following people who have contributed earnestly to make
this project possible, starting with my parents and all my family members whose
constant encouragement and support helped me get through difficult situations.

I am also grateful to all the staff of Bioengineering department for all their guidance in
the laboratory, in the classroom, and life in general.

I also extended my gratitude to all my friends who in one way or the other contributed to
the success of this project. Lastly, special thanks to all CIU Bioengineering 2016 set.

i
PRODUCTION OF CRUDE CELLULASE ENZYME BY SOIL FUNGI AND ITS
APPLICATION IN BIOCONVERSION

ABSTRACT

Enzymes production is a growing field of biotechnology. Cellulase enzymes are one

among of the hydrolytic enzymes capable of hydrolyzing the most abundant organic
polymer i.e. "cellulosic material" to produce the smaller sugar components i.e. "glucose,
cellobiose and oligomers". The soil fungi have been the best effective microorganisms
for cellulase production under solid state fermentation (SSF) technology.

Three different species of soil fungi " Aspergilus.sp, Pencillium.sp and Aspergilus.niger
" were isolated , selected and used for crude cellulase production under SSF. The effect
of incubation time on enzyme production by different fungi was studied, and the
maximum cellulase production was 3.34 FPU/gds from A.niger at optimum fermentation
time of 4 days while the FPase of cellulase production by Aspergilus.sp and
Pencillium.sp were 2.315 FPU/gds at 4 days as optimum time and 1.016 FPU/gds at 5
days of incubation time respectively.

The higher enzyme activity produced by isolated fungi were applied for bioconversion
and saccharification of olive oil mill solid waste biomass (OOMSW) and the enzymatic
hydrolysis of pretreated OOMSW with 1.25% NaOH for fermentable sugar production
was performed and the optimum enzymatic conditions were determined as follows: pH
of 5.00, temperature of 50⁰C and substrate concentration was 50g/L. The maximum total
reducing sugars yield (TRS) 0.48g/g substrate and bioconversion/saccharification yield
43% were obtained by cellulase of A.niger by optimum enzyme loading of (26.72FPU)
at steady time of 24h while the enzymes of Aspergilus.sp and Pencillium.sp resulted
0.44g/g and 0.39g/g substrate of TRS yield respectively and 40%, 35% of
saccharification yield % by optimum enzyme loading of (23.15FPU) and (8.0FPU)
respectively from enzymatic hydrolysis of OOMSW .
ii
The experimental result fit well to Michaela’s Menten and Line Weaver Burk equations
for effect of substrate concentration on enzymatic hydrolysis and the kinetic constant Km
of three enzymes sources from isolated fungi notably: Aspergilus.sp , Pencillium.sp and
A.niger were 37.5 ,17.4 and 24.7 g/L while Vmax were 0.096 , 0.076 and 0.127 g/L/min
respectively.

Keywords: soil fungi, solid state fermentation, cellulase, incubation time, olive oil mill
solid waste, bioconversion, optimization, enzymatic hydrolysis, saccharification.

iii
 TOPRAK FUNGUSLARINDAN SELÜLAZ ENZİMİ ÜRETİMİ VE BİYOLOJİK DÖNÜŞÜM
ÇALIŞMALARINDA KULLANIMI

OZET

Enzim üretimi, biyoteknolojinin gelişen bir dalıdır. Selülaz enzimleri, doğada yaygın
olarak bulunan selülozik maddenin glukoz, sellobiyoz ve oligomerler gibi daha küçük
şeker birimlerine parçalanmasından sorumlu olan hidrolitik enzim grubundandır.
Toprak fungusları, katı faz fermantasyonu (KFF) ile selüloz üretiminde en etkin
organizmalardır.

Çalışmada, Aspergillus sp., Penicilium sp. Ve Aspergillus niger olmak üzere 3 farklı torak
fungusu izole edilmiş ve KFF yöntemi ile selülaz üretiminde kullanılmıştır. Farklı
funguslarla selüloz üretiminde inkübasyon süresinin etkisi incelenmiş ve maksimum
selülaz aktivitesi Aspergillus niger tarafından inkübasyonun 4. dününde 3.34 FPU/gds
olarak saptanmıştır. Aspergillus sp. ve Penicilllium sp. için maksimum selülaz aktiviteleri
sırası ile inkübasyonun 4. ve 5. günlerinde 2.315 FPU/gds ve 1.016 FPU/gds olarak
saptanmıştır.

Maksimum enzim aktivitesi elde edilen enzim özütü, zeytin yağı fabrikası katı atığında
biyolojik dönüşüm ve şekerleştirme çalışmalarında kullanılmıştır. 1.25% NaOH ile ön
işlem uygulanmış zeytin yağı fabrikası katı atığın enzimatik hidrolizi gerçekleştirilmiş ve
optimum koşullar pH=5.00, sıcaklık 50oC, substrat derişimi 50g/L olarak saptanmıştır.
Aspergillus niger optimum enzim yüklemesi ile (26.72 FPU/24 hours) maksimum toplam
indirgen şeker verimi, 0.48 g/g ve biyolojik dönüşüm (şekerleştirme) verimi ise 43%
olarak saptanmıştır. Aspergillus sp. 23.15 FPU optimum enzim aktivitesi ile ve
Penicilium sp. 8.0 FPU optimum enzim aktivitesi ile maksimum toplam indirgen şeker
verimi sırası ile 0.44 g/g ve 0.39 g/g; şekerleştirme verimi sırası ile 40% ve 35% olarak
saptanmıştır.

Deneysel veriler, hammadde derişiminin enzimatik hidroliz hızına etkisinin gösterildiği

Michaelis Menten ve Line Weaver Burk denklemleri ile uyum göstermiştir. Bu kinetik
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modeller kullanılarak Aspergillus sp., Penicillium sp. ve Aspergillus niger için elde edilen
Km değerleri sırası ile 37.5, 17.4 ve 24.7; Vmax değerleri ise sırası ile 0.096, 0.076 ve
0.127 olarak saptanmıştır.

Anahtar kelimeler: toprak mantarı, katı faz fermantasyonu, selülaz, inkübasyon süresi,
zaytin yağı fabrikası katı atığı, biyolojik dönüşüm, optimizasyon, enzimatik hidroliz,
şekerleştirme

v
 TABLE OF CONTENT

ACKNOWLEDGEMENT……………………………………………...…………….…i

ABSTRACT/OZET ......................................................................................................ii-v

TABLE OF CONTENTS…………………………………………..….……………vi-xi

ABBREVIATIONS ………………………………………………….……...………...xii

LIST OF TABLES …………………………………………………………...……... xiii

LIST OF FIGURES ……………………………………………………...………xiv-xvi

CHAPTER 1…………………………………………………………..…………………1

1.1.0 Introduction………………………………………………………..……………….1

1.1.1 Fermentation………………………………………………………..………………2

1.1.2 Cellulase Enzymes…………………………………………………...……………..2

1.1.3 Biomass…………………………………………………………………..………...3

1.1.4 Bioconversion……………………...…………………………………………….3-4

1.2 Aims and Objectives……………………………………………………………...…..5

CHAPTER 2…………………………………………………………………...………..6

2.0 Literature Review…………………………………………………………...………..6

2.1 Cellulase Enzyme………………………………………………………….…………6

2.1.1 Classification of Cellulase Enzymes……………………………………………..6-7

vi
2.2 Cellulolytic Microorganisms……………………………………………..………..7-8

2.2.1 Cellulase Producing Bacteria………………………………………………...…….8

2.2.2 Cellulase Producing Fungi……………………………………………………….8-9

2.3 Fermentation Production of Cellulolytic Enzymes……………………….………9-10

2.3.1 Submerged fermentation (SmF)……………………………………….………….10

2.3.2 Solid State Fermentation (SSF)……………………………………………......10-11

2.3.3 Advantages and Challenges of SSF………………………………...……………..11

2.4 Activity Assay of Cellulase…………………………………………...…………….12

2.5 Industrial Applications of Cellulase…………………………………..…………12-13

2.6 Lignocellulolytic Biomass…………………………………………...………….14-15

2.6.1 Olive oil mill solid waste biomass (OOMSW)……………………..…………15-17

2.6.2 Biotechnological applications of OOMSW biomass ……………………………..17

2.7 Bioconversion……………………………………………………….………………17

2.7.1 Enzymatic Saccharification of Lignocellulosic biomass (Hydrolysis of Cellulosic

material by cellulase)…………………………………………………………………...18

CHAPTER 3……………………………………………………………………………19

3.0 Instrumentation……………………………………………………………...………19

3.1 pH Meter…………………………………………………………………….………19

3.2 Water Bath……………………………………………………………..……………20

3.3 Drying Oven……………………………………………………………..………….21

vii
3.4 Thermostat …………………………...……………………………………………..22

3.5 Magnetic Stirrer …………………………………………………………………….23

3.6 Weighing Scale………………………………………………………..….…………24

3.7 Spectrophotometer………………………………………………………………24-25

3.8 Centrifuge………………………………………………………..………………25-26

3.9 Microscope………………………………………………………….…….……..26-27

3.10 Incubator…………………………………………………………………..…...27-28

3.11 Autoclave…………………………………………………………...………….28-29

3.12 Ashing/Muffle Furnace……………………………………………...…………29-30

CHAPTER 4……………………………………………………………………..……..31

4.0 Material and Methods…………………………………………………….…..……..31

4.1 Collection of Soil Samples…………………………..……………………….……..31

4.2 Isolation of Cellulase Producing Fungi……………………………………………..31

4.3 Culture Media and Sub-Culturing…………………………………………………..32

4.4 Identification of Fungi…………………………………………………...………….32

4.5 Production of Crude Cellulase Enzymes…………………………………….……...32

4.5.1 Fermentation Medium for Enzyme Production…………………………..……32-33

4.5.2 Solid State Fermentation (SSF) and Optimization of Parameters for Enzyme…...33
Production by Fungal Isolates

4.5.3 Inoculation of Medium Production ………………………………...…….………33

4.5.4 Time Course of Enzyme Production under the SSF by Fungal Isolates………….34

viii
4.5.5 Extraction of Crude Cellulase…………………………...………………………..35

4.6 Enzyme Assay…………………………………………………..………..…………35

4.6.1 Total Enzyme Activity………………………………………...………………35-36

4.6.1.1 Procedure of Fpase Assay…………………………………………...………35-37

4.7.0 Bioconversion of Biomass by Crude Cellulase Produced from Fungal Isolates

(Enzymatic conversion of Biomass)…………………………………………………….37

4.7.1 Enzymatic Saccharification of OOMSW Biomass (Enzymatic Hydrolysis)…37-38

4.7.1.1 Determination of moisture Content in OOMSW sample…………….…………38

4.7.1.2 Determination of cellulose content in OOMSW biomass………………………38

4.7.1.2.1 Procedure of determination of crude cellulose content in OOMSW by first

method ………………………………………………………………………………….39

4.7.1.2.2 Procedure of determination of crude cellulose content in OOMSW by second

method………………………………………………………………………………39-40

4.7.1.3 Pretreatment of OOMSW biomass…………………………………...…………40

4.7.2 Enzymatic hydrolysis experiment………………………………………………...41

4.7.2.1 Effect of PH on enzymatic hydrolysis of OOMSW…………………….………41

4.7.2.2 Effect of temperature on enzymatic hydrolysis of OOMSW…………….……..41

4.7.2.3 Effect of Concentration of substrate (OOMSW) on enzymatic hydrolysis of

OOMSW…………………………………………………………………………..…….41

4.7.2.4 Effect of incubation time and loading of enzymes on enzymatic hydrolysis of

OOMSW…………………………………………………………………….………42-43

4.8 Kinetic models of study……………………………………………………………..43

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4.8.1 Michaelis Menten kinetic model………………………………………..……..43-44

4.8.2 Line Weaver Burk Equation………………………………….………………..44-46

CHAPTER 5……………………………………………………………………………47

5.0 RESULTS AND DISCUSSIONS……………………………….………………….47

5.1 Isolation and identification of cellulytic fungi for crude cellulase production.…47-49

5.2 productions of cellulase enzymes under SSF and Fpase assay…….……………49-53

5.2.1 Time course of cellulase production by three different isolated fungal strains.53-58

5.3.0 Bioconversion and Saccharification of OOMSW sample by produced crude

cellulase extract……………………………………………………………………...58-59

5.3.1 Determination of moisture content in OOMSW sample………………..…….......59

5.3.2 Determination of crude cellulase content in the OOMSW sample……….…...59-60

5.4.0 Enzymatic hydrolysis of OOMSW and optimization of parameters conditions….60

5.4.1 Enzyme sources for enzymatic hydrolysis of biomass……………………………60

5.4.2 Substrate of enzymatic hydrolysis……………………………………….……60-61

5.4.3 Effect of the temperature on enzymatic hydrolysis of the OOMSW biomass...61-62

5.4.4 Optimization of pH for enzymatic hydrolysis of OOMSW biomass………….62-64

5.4.5 Effect of concentration of substrate on the enzymatic hydrolysis of OOMSW

biomass……………………………………………………………….……………...64-65

5.4.6 Kinetic models of produced crude cellulase and enzymatic hydrolysis of OOMSW
biomass………………………………………………………………………………66-68

5.4.7 Effect of enzyme loading on reducing sugar production from OOMSW biomass
with time course………………………………………………………..……………69-77

x
CHAPTER 6………………………..………………………………………………….78

6.1 CONCLUSION………………………………………………………………….78-79

REFERENCES………………..……………………………………………………80-88

xi
 ACRONYMS AND ABBREVIATIONS

TRS Total Reducing Sugar

FPase Filter Paper Activity

OOMSW Olive Oil Mill Solid Waste

SSF Solid State Fermentation

SmF Submerged Fermentation

DNS 3.5-Dinitrsalicylic Acid

FPU Filter Paper Unit

U/gds Unit per Gram Dried Substrate

PDA Potato Dextrose Agar

xii
 TABLE LIST

Table 2.1 Industrial application of cellulose enzymes…………………………….……13

Table 2.2 Chemical composition of OOMSW from some previous studies……………16

Table 4.1 Dilution of glucose standard and construction of standard curve………..…..36

Table 5.1 probable ID of isolated fungi…………………………………………..…….48

Table 5.2 previous studies reporting use of wheat bran as substrate…………….….50-51

Table 5.3 Effect of incubation on enzyme production…………………………………54

Table 5.4 Optimum fermentation time of isolated fungi ………………………………56

Table 5.5 Comparison of cellulase production and fermentation time result with other
previous studies………………………………………………………………….…..57-58

Table 5.6 Crude cellulase content in OOMSW sample…………………………………60

Table 5.7 Effect of temperature on enzymatic hydrolysis of OOMSW…………...……62

Table 5.8 Effect of pH on enzymatic hydrolysis of OOMSW…………………...……..63

Table 5.9 Effect of concentration of substrate on enzymatic hydrolysis of OOMSW.....65

Table 5.10 Effect of enzyme loading with incubation time on the enzymatic hydrolysis
of OOMSW…………………………………………………………..…………………71

Table 5.11 TRS yield released in g/g substrate and saccharification yield % of
substrate………………………………………………………………...……………….74

Table 5.12 Cellulase conversion % of enzymatic hydrolysis of OOMSW……………..77

xiii
 FIGURE LIST

Figure 2.1 lignocellulosic biomass structure……………………………………………14

Figure 2.2 Application of lignocellulosic material…………………………...…………15

Figure 2.3 Mechanism of enzymatic hydrolysis of cellulosic material………….……...18

Figure 3.1 pH meter………………………………………………………………..……19

Figure 3.2 Water Bath………………………………………………………………..…20

Figure 3.3 Drying oven……………………………………………………………….....21

Figure 3.4 Thermostat………………………………………………………………......22

Figure 3.5 Magnetic stirrer…………………………………………………………..….23

Figure 3.6 Weighing Scale……………………………………………………………...24

Figure 3.7 Spectrophotometer…………………………………………………..………25

Figure 3.8 Centrifuge………………………………………………………….…..…….26

Figure 3.9 Light Microscope………………………………………………………..…..27

Figure 3.10 Incubator………………………………………………………………..….28

Figure 3.11 Autoclave………………………………………………………………..…29

Figure 3.12 Ashing furnace…………………………………………………………..…30

Figure 4.1 Fermentation medium for enzyme production………………………………33

Figure 4.2 Glucose standard curve……………………………………………...………36

xiv
Figure 4.3 Dried OOMSW biomass……………………………………………….……38

Figure 4.4Effect of pretreatment of biomass on enzymatic hydrolysis………………....40

Figure 4.5 Example of Michaeli’s menten model………………………...…………….44

Figure 4.6 Example of line weaver-Burk plot…………………………………………..45

Figure 5.1 Final isolated colonies after 5days of incubation……………………………47

Figure 5.2 Microscopic observation of fungi isolates ………………………………….49

Figure 5.3 Growth of isolated fungi after 3 days……………………………………….50

Figure 5.4 Effect of incubation time on enzyme production…………………………...54

Figure 5.5 Effect of temperature on the enzymatic hydrolysis process of OOMSW…...62

Figure 5.6 Effect of pH on the enzymatic hydrolysis of OOMSW……………………..64

Figure 5.7 Effect of substrate concentration on the enzymatic hydrolysis of OOMSW..65

Figure 5.8 Michaelis Menten curve for enzymatic process of OOMSW by cellulase from
Aspergilus .Sp…………………………………………………………………………...67

Figure 5. 9 Michaelis Menten curve for enzymatic process of OOMSW by cellulase

from Pencillium. Sp……………………………………………………………………..67

Figure 5.10 Michaelis Menten curve for enzymatic process of OOMSW by cellulase
from A. niger…………………………………………………………………..………..68

Figure 5.11 Lineweaver-Burk plot of enzyme from Aspergillus. Sp…………...………69

Figure 5.12 Lineweaver-Burk plot of pencillium cellulase enzyme……………………69

Figure 5.13 Lineweaver-Burk plot of enzyme from A. niger…………………………..70

xv
Figure 5.14 Effect of enzyme loading with time by cellulose of Aspergillus sp on the
hydrolysis process of OOMSW………………………………………………..………..72

5.15 Effect of enzyme loading with time by cellulose of Pencillium sp on the hydrolysis
process of OOMSW…………………………………………………………….………72

5.16 Effect of enzyme loading with time by cellulase of A. niger on the hydrolysis
process of OOMSW…………………………………………………………….………73

5.17 TRS yields released per gram substrate by optimum loading of different enzyme
sources………………………………………………………………………….….……75

5.18 Saccharification yield % of OOMSW by optimum enzyme dosage………………75

xvi
 CHAPTER 1

1.1.0 INTRODUCTION

Bio-products such as enzymes, proteins, antibiotics, vitamins and organic acids are more
growing fields of biotechnology. Enzymes production is one of the important products
of biotechnology due to its uses in provision of human needs through diverse areas
(Sharada, 2013., papinutti, 2007), Food industries, Pharmaceutical industries and
Environmental biotechnological applications utilize enzymes at many stages (Sharada,
2013., and Ramesh, 2011).Current developments in biotechnology have shown useful
areas in the application uses of enzymes are very beneficial (Chetua et al., 2015).

Enzymes are catalysts synthesized by living systems and are used both in synthetic as
well as in degenerative processes (Awun Sasi et al., 2012). Enzymes for commercial or
industrial application are obtained from three main sources namely: plant, animal and
microorganisms. In the past, plant and animals served as the main sources of synthesis
of enzymes but recently, microbial sources became more effective (Abubaker and
Oloyede, 2013)., for obvious reasons such as:

 The synthesis of enzymes from plant and animal sources require a large amount
of plant and animal material.
 And the enzymes produced are low in quantity as a result of difficulties in the
extraction of these enzymes (Abubaker et al., 2013, Banaker and Thipp, 2012).
 On the other hand, microbial enzymes are not subject to any of these problems
related with plant and animal sources of enzymes.

Microorganisms are currently the primary sources of industrial enzymes. Production of

enzymes from Fungi and Yeasts represent for 50%, Bacteria 35%, while the remaining
15% are from other microorganisms such as Algae (Banaker and Thippe, 2012).

1
Microbial cells produce a variety of enzymes which aid in its growth and other cellular
activities (Arun et al., 2012).

Soil is the major source for the isolation of industrially important enzyme producing
microorganisms most especially fungi. Soil type, vegetation, climatic conditions and
nutrient composition of the soil affect fungal growth in the soil. Indigenous fungi in the
soil have more potential to produce industrially important enzymes than other isolates
(Banakar and Thippe, 2012).

1.1.1 FERMENTATION

Most manufactures produce enzymes using Sub-merged fermentation (SmF) and solid-
state fermentation (SSF) techniques. SSF is rapidly achievement interest as a cost
effective process for the production of enzyme , higher yields of cellulase has been
reported under SSF and low cost scale when compared to the liquid culture (SmF)
(Chetha et al., 2015).

1.1.2 CELLULASE ENZYMES

Cellulases are multi hydrolytic enzymes consist of group of enzymes which are capacity
of depolymerizing cellulose to smaller molecules "Monomers" (Chetna et al., 2015).,
and this refers to the class of enzymes produced chiefly by fungi and other
microorganism such as bacteria and Protozoan. Fungi are the main cellulase producing
microorganisms which are known to hydrolyze both soluble and insoluble cellulolytic
materials (Abuet et al., 2013). Filamentous fungi have been reported as good producers
of Lignocellulosic enzymes due to extra cellular release of the enzyme and also higher
yield compared to yeast and bacteria (Mohammed, 2013., papinutti, 2016).

Crude cellulase is a group of enzyme consisting of three different of enzymes: endo-1, 4-

β-D glucanase , exo-1,4- β-D glucanase)and β- glucosidase. These enzymes have the
ability to degrade carboxyl methylated cellulose (Chouhan, 2014). Microbial cellulase
have become main biocatalysts due to their complex nature and wide spread industrial

2
applications (Ramesh et al., 2011). Cellulase enzymes can be effectively used for
industrial applications and play role in many industries such as food, animal feed ,
textiles, pulp and paper industries, grain ethanol fermentation, starch and pharmaceutical
processing (Aftab and Patrick, 2008).

1.1.3 BIOMASS

Lignocellulosic material "biomass" is the most abundant renewable biopolymer on

earth. It is the most abundant waste material which is generated from both natural and
human made activities (Reeta, et al., 2006). It is the major cell wall polysaccharides of
plant, which are composed of three types of polymers: Cellulose 40% - 60%, hemi
cellulose 15% - 30%, and lignin 10-15 % (Rubeena et al., 2013., Nabila et al., 2001).,
Lignocellulosic material are chemically bonded by non-covalent bonds and covalent
cross-linkages. The complex molecular polymeric structure of cellulosic biomass makes
lignocellulose is strongly joined and highly resistant to chemical pretreatment and
bioconversion procedures for break down the lignocellulosic structures (Karin et al.,
2012). Biomass is a potential raw material for the microbial production such as food,
fuel and some chemicals as a result of utilization of cellulose in these process by
microorganisms as their source of carbon. With the help of cellulase enzyme system,
cellulose material can be converted to glucose by bioconversion process (Cellulolysis).

1.1.4 BIOCONVERSION

Bioconversion of lignocellulosic materials (Cellulolysis) is basically biological process

of breaking down or hydrolysis of cellulose material into smaller polysaccharides or
completely into glucose units , this process is controlled and processed via cellulase
enzymes (Vipal et al., 2012., Gomashe, 2013).The three groups of cellulase are
responsible for bioconversion of cellulosic material : 1,4- β-D glucanase or endo
glucanase (CMCase) is responsible for random and attacks cleavage of β-1,4-glycosidic
bond along cellulose polymeric chain. 1, 4- β-D exoglucanase or exoglucanase
(cellobiohydroase) is second hydrolytic enzyme and it is necessary for cleavage of the

3
non-reducing end of cellulose chain while β- glucosidase (cellobiase) enzyme convert
cellobiose of biomass and water-soluble cellodextrin to glucose (Asma et al., 2012,
Gomashe, 2013), bioconversion of the lignocellulosic biomass such as agricultural,
industrial and municipal cellulolysic waste material is more attractive than physical and
chemical conversion in converting biomass to glucose or other mono-sugars because of
due to its advantages such as low investment and low or non-pollution of bioprocess
(Dongang et al., 2011). The bio Products obtained from bioconversion or hydrolysis of
lignocellulosic biomass via cellulase enzymes consists mainly of glucose, cellobiose and
cello-oligosaccharides. Among of them, glucose is the most desired product serve as
valuable feed stuck for a large variety of bio production such as organic acid , biofuel,
bioethanol, and single-cell protein (Liwan et al 2014).

The commercial use of cellulases is dependent on the high enzymatic activity and low
production cost (Arun et al., 2012). Solid-state fermentation SSF has been reported as an
attractive technology to produce economically cellulase due to cultivation of
microorganism on solid medium that is closed to natural environment and its low capital
investment, low operating expenses. The large scale production of cellulase for
commercial and industrial purposes requires the identification of Fungi that has high
cellulase yielding capability (Prabavathy and Valli, 2011). And using of cheap
Lignocellulosic material as substrate such as wheat straw and wheat bran rather than the
expensive commercial cellulose powder which can reduce the cost of cellulase
production (Caixia and Yebo.,2012). Economic bioconversion requires an appropriate
pretreated biomass and an effective cellulase system for make all biomass is converted
and production of the high mono-sugars (Gomashe et al., 2013).

This study involves isolation, identification of some soil fungi and production of crude
cellulose enzymes from them by using wheat bran as substrate under solid state
fermentation (SSF). The enzyme activity for the different isolated enzymes produced is
studied and also study the application of produced enzymes in bioconversion of
OOMSW biomass as substrate .

4
1.2 AIMS AND OBJECTIVES

The aim of this study is the production of crude cellulase enzymes by soil fungi and its
applications in bioconversion processes.

The objectives of the study include:

 Isolation of some soil Fungi.

 Identification of some soil cellulolytic fungi.
 Production of crude cellulase enzymes by isolated fungi using wheat bran as
substrate under SSF.
 Application of enzymes in bioconversion of OOMSW Lignocellulosic biomass
for production the fermentable sugars.

5
 CHAPTER 2

2.0 LITERATURE REVIEW

2.1 CELLULASE ENZYME

Cellulase is an enzyme system that breaks down cellulose in cell wall of plant biomass
(Zahangir et al., 2009).This enzymatic system aids the hydrolysis (breakdown ) of plant
cellulose leading to production of important economic product such as bioethanol, single
cell proteins and other chemicals (Zahangir et al., 2009).Microbial
utilization/degradation of lignocellulolisic waste and the products of this degradation are
as a result of synergetic effort of different enzymes and the most prevalent is the
cellulase enzymes which are majorly secreted by cellulase producing microorganisms
(Rajeev et al., 2005). One of the problems associated with Lignocellulosic bioethanol is
shortage of cellulase supply which aids the enzymatic hydrolysis of the lignocellulosic
biomass for production of ethanol (Damato et al., 2012). Production of enzymes that can
utilize lignocellulolisic biomass is of high importance because it will lead to creation of
sustainable energy sources and other economical chemicals such as ethanol.
Lignocellulolytic enzymes are biocatalyst capable of degrading lignin and cellulosic
materials (Godliving, 2012).

2.1.1 Classification of Cellulase Enzymes

According to studies by Rabinovich et al, (2002), cellulase can be divided into three
major enzyme activity classes notably: endo-glucanases or endo-1,4 β-glucanase (EC
3.2.4), Cellobiohyrolase (EC 3.2.1.91) and β-glucosidase (EC3.2.2.1.1.21). It has been
proven that microorganisms have pure form of Exo and Endo glucanases (Shen et al.,
1995), same with hemicellulases of which majority of the cellulases are multi-domain
proteins. These cellulases are employed for different applications such as in food
industry, and various other industrial applications (Ramesh et al., 2011).

6
Endoglucanases hydrolyze glucosidic bonds at the amorphous regions of the cellulose
resulting in long chain oligomers (non-reducing ends) for the action of exoglucanases or
cellobiohydrolases, which cleave the long chain Oligosaccharides generated by the
action of endoglucanase into short chain oligosaccharides. There are notably two types
of exoglucanases acting uni-directionally on the long chain oligomers which could be
from the deducting ends of the (E.C3.2.1.91) or from the (E.C.3.21.176) liberating
cellobiase, which is further hydrolyzed to glucose by β- glucosidase (E.C.3.2.1.21).
(Veeresh and Jin, 2014).

Glycosidic hydrolases (GH) is a group of cellulase enzyme and according to

carbohydrate- active enzyme data base (CAZy), endo glucanases are implicated amoung
the families of GH: 5-8,12,16,44,45,48,51,64,71,74,81,87,124, and 128. According to
Veeresh and Jin, (2014), both the cellobiohydrolases and the Exoglucanases are among
the GH families of 5-7 and 48.. While the β-glucosidase is associated with GH families
of: 1,3,4,17,30,116.

The complete cellulase family act in consortia in the production of glucose from
cellulose through conversion processes. Both the Exo-cellobiohydrolases and the
endoglucanases act in accordance to breakdown cellulose to its monomers. The
oligosaccharides (consisting of cellobiase) are now converted to glucose through
hydrolysis processes by β- glucosidase (Rajeev et al., 2005).

2.2 CELLULOLYSIC MICROORGANISMS

Cellulolysic microorganisms are majorly carbohydrate degraders, they are able to use
fats or proteins as their source of energy (source of carbon) (Poulsen and Peterson,
1988). Bacteria and algae as well as fungi can make use of cellulose and carbohydrates.
Anaerobic cellulolytic microorganisms have limited range to carbohydrate metabolism
limited to cellulose or its hydrolytic products. Certain strains of fungi are preferable for
the production of high amount of cellulases as a result of their capability to produce
greater amount of extracellular protein. One of these fungi specie with these

7
characteristics and also the most extensively studied fungi is Trichoderma reesei. Other
commonly studied cellulolytic microorganisms include: fungal species- Streptomyces,
Actinomucor, Actinomycetes, Streptomyce, Cellulomonas, Pseudomonas, Bacteria-
Bacilli, Aspergillus, Penicillium, Humicola, and Trichoderma (Rejeev et al., 2005).

Few strains of Fungi have the ability to secrete a complex of Cellulase enzymes, which
can be applied in the enzymatic hydrolysis of cellulose (Rajeev et al., 2005), such as T.
reesei and other fungi such as Humicola, Penicillium and Aspergillus. These
aforementioned genuses are capable of yielding large amounts of cellulases.
Cellulomonas, cellovibrio and Cytophaga are aerobic bacteria with the ability of
cellulose degradation in pure cultures (Lynd et al., 2002). Microorganisms commercially
used for Cellulase preparation are restricted to T. ressei, H.insolens, A. niger,
Thermomonospora fusca, Bascillus spp. (Rajeev et al., 2005).

2.2.1 Cellulase Producing Bacteria

Production of cellulase in cultures is growth associated involving microbial interactions

which can be influenced by various factors that in turn affect cellulase production.
Cellulase producing bacteria especially those characterized in the genera:
Thermonospora, Cytophage, Ruminococcus and Cellulomonas can make use of cellulose
and other carbohydrates to produce cellulase enzymes (Rajeev et al., 2005).

2.2.2 Cellulase Producing Fungi

Fungi including the Filamentous Fungi and Yeast are employed for several industrial
processes ranging from production of enzymes to other products such as vitamins,
Polysaccharides, polyhydric alcohols, lipids, and pigment and glycol lipids. Lots of these
products are made for commercial purposes and others used for biotechnological
purposes (Jose and Aenold, 2003). According to Gustave et al, (2015), the phylum
“Ascomycete” has the ability to produce cellulase enzymes in large quantity.

8
Strains of Ascomycetes that are often employed for commercial purposes are mostly
isolated from ecological niches with lots of agricultural waste (example: wheat, rice,
maize and sugar cane). Most of the promising strains of Ascomycetes was isolated from
hay, straw or husks, from cereal plants, Agricultural soils and compost. For example,
A. nidulan strains were isolated from hay and also decaying Vegetation (Gustav et al.,
2015).

Various literature have shown that various strains of fungi capable of producing
cellulose degrading enzyme can be gotten from substrata of lignocelluloses, mostly
debris from cereal waste, and soils from arable Agricultural fields. This goes to show
that good cellulase producers can be sourced from habitats containing cellulolysic
substrates. (Peterson et al., 2012).

Production of cellulase enzymes for industrial purposes employ the use of genetically
engineered fungal strains capable of producing a single enzyme class or a particular type
of enzyme with enzyme activities (Gustav et al., 2015).

Trichoderma Koningii AS 3.4262 according to study by Jian and Jichu, (2007), is

capable of degrading waste from vinegar industry to produce cellulase enzyme under
Solid State fermentation (SSF).

Newly isolated Oleaginous fungi often referred to as Aspergillus tubingensis has been
implicated in production of cellulase enzymes with high enzyme activity from palm
pressed fibre (PPF) and palm empty fruit bunches (EFB) as substrates (Suleeporn and
Benjamas et al.,2014).

2.3 FERMENTATION PRODUCTION OF CELLULOLYTIC ENZYMES

Production of cellulase enzymes from cellulosic biomass through fermentation processes

involves metabolic actions/processes by specific microorganisms mostly Fungi and
bacteria on cellulosic biomass. These metabolic reactions are critically dependent on the

9
physical and chemical conditions that exist in the fermentation medium/environment
(Doran, 2004).

Two types of fermentation methods for the production of cellulase exist, they include:
submerged type of fermentation and solid state type of fermentation (SmF and SSF
respectively) (Gustav et al., 2015)

2.3.1 Submerged fermentation (SmF)

Submerged fermentation is liquid medium is used as fermentation medium for growing

microorganism. The process parameters for the SmF can be controlled unlike the SSF.
The technology has been so developed that the parameters such as pH, aeration,
foaming, agitation and temperature which is a very important factor because it has an
effect on the growth rate, rate of evaporation in the medium, oxygen tension and product
formation (Gustav et al., 2015).

2.3.2 Solid State Fermentation (SSF)

The use of Solid-state fermentation (SSF) in the production of cellulase enzyme is

consistently gaining momentum as an area of biotechnology with low capital investment
cost (Tajeev et al., 2005). SSF process can be described as a fermentation with low or no
free water (i.e. Substrate is moist enough to support growth of microorganism) (Gustav
et al., 2015). Hence, the best type of fermentation process that imitates the natural
environment of most filamentous fungi is the SSF type of fermentation unlike the SmF
(Te et al., 2002). The fungi growing in solid states are of the mycelia form; because of
the possibility of producing both the aerial and substrate penetrating. As of resent,
demand for industrially produced cellulase is been met by adopting SSF technology in
the cultivation of filamentous fungi (Karishna, 2005). According to most literature,
enzyme yield is more with the SSF than the SmF, hence attention is being focused on
adopting the use of SSF type for commercial production of Cellulase enzyme (Provot et
al., 2013). For solid state fermentation, the enzyme could be attached/stocked to the
substrate therefore it acts on the substrate on close range thereby reducing the glucose

10
repression effect (Gustavet al., 2015). Type of substrate and the control measures
required are the determining factors for choice of SSF reactor. There are different type
of bioreactor design for both small-scale and large-scale applications using SSF such as
the packed bed reactor, tray reactor and the Drum reactor (Gustav et al., 2015).

2.3.3 Advantages and Challenges of SSF

Production of enzymes using SSF is higher over submerged fermentation. The initial is
associated with larger biomass and lower production breakdown (Viniegra et al., 2003).
Cost associated with energy utilization is lower for SSF when compared to SmF because
of the less water requirement, no mechanical fixing coupled with less energy
requirement for the downstream processing (Krishme, 2005).

An exceptional advantage of SSF is that substrates that cannot be used in submerged

fermentation processes can be applied for the SSF, using substrate with rich carbon and
nitrogen content. Also the use of antifoaming agent is irrelevant in SSF setting (Hocker
and Lenz, 2005).

Product purification and determination of fungal biomass are among the challenges of
SSF setting. This operations is affected as a result of limitations in monitoring online
process parameters, mixing heat provision and mass transfer (Rani et al.,2009).
Decreased porosity and agglomerated solids can occur where the substrate decomposes
due to mass and heat transfer limitations. Temperature control is a major challenge in
SSF setting because heat produced is in proportion to the level of respiration rate
(Biesebeke et al., 2002). Also, there are challenges with maintaining a constant moisture
level, hence water ought to be regularly monitored and replaced either by adding water
directly or by saturating the air (Ali and Zukali, 2011). Contamination control for long
term fermentation can be difficult with the SSF. It can be difficult to determine presence
of contaminants due to the different make up and instability of the substrate, unlike the
SmF where simple microscopic analysis can determine it.

11
2.4 ACTIVITY ASSAY OF CELLULASE

In terms of measuring cellulolytic activity, there are quite a number of assays available
(Gustav et al., 2015). However, there are methods approved by the standardized
international union of pure and applied chemistry (IUPAC) for determination of
cellulase activity; they include: carboxymethyl cellulose (CMC) assay, paper assay (FP),
and cellobiase assay. Recreation of the CMC and FP assay is usually a challenged
especially when using of 3.5-dinitrasalicylic acid (DNS) in the determination of
reducing sugars (glucose) from cellulose substrates. The use of DNS requires heating at
certain temperature for full color development; this can cause partial degradation of the
reducing sugar. Another back draw with the FP assay is its high dependence on the level
of BGs in the enzyme blends that is being evaluated (Dashtban et al., 2010). Enzyme
activity is usually represented as u/g of protein, where U represents the enzymatic units
relating to the level (µ. mol) of substrate transformed per minute. In cases where the
amount of protein is unknown, the activity is usually represented as u/ml of added
enzyme solution or as u/g carbon source or substrate (u/gs) in the medium. In some
cases, enzyme activity can be represented as (u/L/h) which shows the general potential
for the process (Gustav et al., 2015).

2.5 INDUSTRIAL APPLICATIONS OF CELLULASE

Microbial cellulases have enormous industrial applications and are often used by various
industries such as: pulp and paper industries, textile, Agricultural, wine and brewery,
food processing, Animal feed and Detergent industries (Ramesh et al., 2011). They are
also used in olive oil extraction, carotenoid extraction and for waste management
purposes (Ramesh et al., 2011). Table 2.1 shows the various industrial applications of
cellulase enzyme.

12
Table 2.1 Industrial applications of cellulase enzymes.

Industry Applications

Agricultural Reduced dependence on mineral fertilizer, plant pathogen

and disease control; improved soil quality; improved seed
germination and better root system.
Can be used for polishing of textile fibers; improve fabrics
quality; improved cellulosic fabrics; and for removal of
Textile
excess dye from fabrics.

Enzymatic deinking; production of biodegradable cardboard;

Pulp and Paper co-additive in pulp bleaching.

Detergents Cellulase-based detergents; anti-redeposition of ink particles;

removal of rough protuberances in cotton fabrics.
Removal of antioxidants from fruit and vegetable pomace;
Food starch and protein extraction; pressing and color extraction
production from fruits and vegetables; it is used in bakery to improve
texture and quality of bakery products.
Production of ethanol, organic acids and other economic
solvents from cellulosic materials, production of single cell
Bioprocess proteins, Animal feed production
conversion For improved malting and mashing; improved filtration rate
and Stability of wine production; improved fermentation and
quality of beer; improved pressing and color extraction of
Fermentation grapes; to enhance aroma of wines.

13
2.6 LIGNOCELLULOLYTIC BIOMASS

Biomass is referred to as large mass of material/ mater of organic origin (Pothiraj et al.,
2006). Lignocellulosic biomass is now gaining momentum as a vital feed stock for
production of various biochemical products such as bioethanol, biofuel and enzymes
(Chundowat et al., 2011). Two effective steps are required for the production of bio
products from lignocellulosic biomass through bioconversion processes. They include:
breakdown of the polymers into their monomers/simple sugars by microorganisms and
fermentation of produced sugar into bio products (Suleeporn and Benjamas, 2014).

Plant cell wall is made up of lignin, hemicelluloses and cellulose, which all together
make up the lignocellulosic residue. Proteins, pectin and ash can also make up
lignocellulosic residue (Himmel et al., 2007, Sanchez, 2009).

Figure 2.1 Lignocellulosic biomass structures

14
Lignocellulosic waste is generated in large quantities mostly from industries such as
agricultural, pulp and paper, food industries in addition to both animal and domestic
solid waste (Mehdi et al., 2009). These materials were regarded as waste in the past;
however, significant efforts have been made to utilize these lignocellulosic residues as
substrate for generation of useful economic products such as chemicals, biofuels and
animal feeds (Howard et al., 2003).the figure 2.1 show the application of lignocellulosic
biomass .

Figure 2.2 The application of lignocellulosic material

2.6.1 Olive oil mill solid waste biomass (OOMSW)

Olive oil production is on of an important and economic industry in Mediterranean area

,there are three exaction processes of oil , traditional , three-phase and two-phase

15
processes .The two-phase system has economic and environmental befits due to reduce
the water consumption during process and thus reduce the generated waste water
(Inmaculada et al .,2009) .These systems of olive oil production produces the oil around
20% and two waste ,solid waste (OOMSW) about 30% and waste water
(OOMWW)about 50% (Sherian et al .,2015). dry matter of OOMSW biomass is very
rich in organic matter which is composed of hull, stone and seeds of grinded core,
crushed skin and pulp of olive fruit that it contains of main components of cellulose ,
hemicellulose , lignin which can be sources of lignocellulosic material, cellulose about
35% to 50% and followed by phenolic , proteins , fat compounds and mineral elements
(Malika et al .,2013). In table (2.0) is the chemical composition of OOMSW of some
previous studies.

Table (2.2) The chemical composition of OOMSW from some previous studies .

chemical composition % of OOMSW

Dry matter Crude cellulose Hemicellulose lignin ash references
Crude matter 89.11 46.2 23.83 19.89 4.78 Abdi et al
(2000)
Treated matter 84.22 42.53 0.17 0.151 10.28

Crude matter 70.20 33.42 15.12 22.0 1.95 Malikaet al

(2013)

Crude matter 87-94 17.37-24.14 7.92-11 1-14.18 1.7-4 Dermeche

et al (2013)

Crude matter 46.8 49 - - - Senkevich

et al (2012)

Treated matter 84.22 42.53 0.17 0.151 10.28 Mameri et

at (2000)
Sumitra et
Crude matter 85.2 40 - - 4.2 al (2007)

The chemical composition of OOMSW depends on the some parameters and various
such as geographic natural of area , olive oil fruit varieties , cultivation environmental
conditions and oil extraction processes .(Elisa and Paris .,2016)

16
Disposal of olive oil mill solid waste biomass (OOMWW) and (OOMSW) has raised
environmental problem. They are characterized by low pH, high COD, a significant
suspended solids fraction with inhibiting compounds such as polyphenols; Phenolic
compounds have been proven to be the main determinants of the phytotoxic effect of
OOMSW (Dannibale et al., 2004).

2.6.2 Biotechnological applications of OOMSW biomass

Olive oil mil solid waste have been widely applied for production of bio products such
as fermentable sugars , industrial enzymes , vitamins , bio pesticides , antibiotics ,
organic acids , single –cell proteins ,bioethanol and other biochemical . It have been also
used as feed supplements ,as preparation of protein hydrolysate , as animal feed sources
, as energy sources and as bio control agents (Sumitra et al .,2007).

2.7 BIOCONVERSION

Bioconversion is defined as the appropriate bio treatment of lignocellulosic material by

converting polymer into mono sugars; Biomass conversion for production of useful
economic products is preferable less harmful and environmental friendly sources of
energy. When compared to other alternative sources of energy (Meri et al., 2009).

Cellulolytic Microorganisms mostly fungi and bacteria which capable of degrading and
convert it by complex of enzymes into simple compounds the bioconversion of
lignocellulosic residues. Trichoderma reesei and Aspergillus niger secrete larger amount
of extracellular cellulolytic enzymes when compared to bacteria (Mahdi et al., 2009).

Bioconversion of lignocellulosic residues to valuable materials require four steps of

processing, the first three are bio related while the fourth is basically a chemical
engineering process. They include: (i) pretreatment (ii) saccharification, (iii)
Bioconversion, (iv) Purification of the products (Hahn-Hagerdal et al., 2006).

17
2.7.1 Enzymatic Saccharification of Lignocellulosic biomass (Hydrolysis of
Cellulosic material by cellulase).

Cellulose and hemicelluloses content in the biomass can be hydrolyzed after

pretreatment process into soluble monomeric sugars notably: cellobiose , glucose ,
xylose , hexoses and pentoses, with the use of cellulase and hemicellulases respectively.
Low pH and High temperature tolerant enzymes are preferable in hydrolysis process
because current pretreatment processes mostly rely on acid and heat. Also, thermo-stable
enzymes have higher catalytic activity which will enhance the general hydrolytic
performance as proposed by Viikari et al, (2007). Fungal strains such as T. terrestris, T.
emoresonii, T. aurentiacus, S. thermophile, Chaetonium thermophilium and Corynascus
thermophilus can produce thermo-stable enzymes that are active and stable even at
temperatures greater than their optimum growth temperature (>60⁰C) (Bhat and
Maheshwari, 1987, Maheshaori et al., 2000, Gomez et al., 2000, Grassick et al., 2004,
Li et al., 2006). Thermophilic fungal enzymes are good potential enzymes for hydrolysis
of lignocellulosic residue with emphasis to industrial scale production as a result of their
acid tolerance and thermo-stability (Mehdi et al., 2009),

Figure 2.3 mechanism of enzymatic hydrolysis of lignocellulosic biomass

18
 CHAPTER 3

3.0 INSTRUMENTATION

The laboratory instruments which were used to for this research are described in this
chapter with emphasis on the working principles, specification, features and various
applications of these laboratory instruments

3.1 PH METER

A pH Meter is a laboratory device that measures the hydrogen-ion concentration "pH".

pH is the measure of the acidity or alkalinity of a substance or solution. pH range is from
0 to14, with 7 as neutral. pH less than 7 is acidity, whereas pH measurement greater than
7 is basicity. "InoLab PH 720, Germany ", is the type of pH meter that was used for the
lab work of this study. Its system features are:

 Reciprocating on/off switch.

 Redox and conductivity measurement.
 Performs pH measurements and provides the highest degree of operating .
 Comfort and reliability.
 Safe for all applications.

Figure 3.1 PH meter.

19
3.2 WATER BATH

A water bath is an instrument used in laboratories for the purpose of incubating samples
at a constant temperature in water, and to a larger extent, constant shaking/rotation. Used
For histology, pathology, chemistry, bacteriology and in clinical laboratories. ‘GFL
1083’ shaking water bath is the water bath model used for this study. System features
are:

 Temperature range: approx. 5⁰c above ambient to +99.9⁰c and Exact temperature
control, precision of ± 0.5 °C.
 Shaking motion: reciprocating, with on/off switch.
 Linear shaking movement with amplitude of 20 mm.
 Main bath body made from black anodized aluminum
 Housing (D) × (H) × (W): 45.5×33×71.5cm.
 Interior (D) × (H) × (W): 32.5×18.5×48cm.
 Technical specifications: 220 V, 50 Hz, 1.5 kW, 7 A.
 Good visibility of slides on black rim.

Figure 3.2 Water bath.

20
3.3 DRYING OVEN

Drying ovens are instruments with a compartment chamber for removing moisture in
samples/materials. The type of drying oven used for this lab work in this study is Model
“INB 200”. Its features include:

 Intuitive and easy-to-use operating menu.

 Electronic PID controller with permanent power matching auto-diagnostic
system for rapid fault finder.
 Temperature range up to +220°C
 Pre-heated fresh air prevents temperature fluctuations.
 Recessing push/turn control for simple operation of the oven
 Visual alarm indication.
 Monitor relay to switch off heating in case of fault.

Figure 3.3 Drying oven.

21
3.4 THERMOSTAT (TS 606/2-i WTW)

Thermostat is an instrument used to manage temperature tasks within 10⁰c to 40⁰c

temperature range. It is used for several analysis and experimental applications such as:

 BOD determination at 25⁰c

 Test for enzyme activity at 25⁰c
 Microbial Colony count at 37⁰c

The model of thermostat used for this study is “TS606/2i WTW”. Its features include:

 A LED display to show the internal temperature

 It is equipped with tangible blower for recirculation of internal temperature
 Inter temperature sensor and insulated cabinet which aids in controlling the
internal temperature.

Figure 3.4 (TS 606/2-I WTW)

22
3.5 MAGNETIC STIRRER

A magnetic stirrer is a laboratory device that uses rotating magnetic field to cause a stir
bar immersed in a liquid to spin very quickly, thus causing a stir. It is use for diversified
laboratory applications, biotechnological, Chemical, microbiological, pharmaceutical
applications, dissolving nutrients and solids and medical Growing microorganisms.
Stirring & Heating type of stirrer by "Velp" with aluminum hot plate stirrer is the
magnetic stirring instrument used for this study and its features include:

 Electronic speed control up to 1500 rpm

 Temperature regulation from room temperature to 370⁰c
 Stirring system with high power driving magnet type “PCM”.
 The highest safety standards available on the market.
 Accepts sample volumes up to 15 liter flasks .
 Excellent resistance to chemicals.

Figure 3.5 Magnetic stirrer.

23
3.6 WEIGHING SCALE

Weighing scales are important laboratory instrument used to measure weight of

substances and material or calculate the mass of a substance. Working with the
microbalance requires a steady hand and a smooth touch. “Saturius CP, model CPA
2250” is the weighting scale that was employed in this study. It is an electronic scale
with weighing capacity of 0.1mg-220g. Its pan size is Ø 80mm and it has a response
time of 3-6s.

Figure 3.6 Weighing scale.

3.7 SPECTROPHOTOMETER (DR 2800 LANGE)

A spectrophotometer is mostly employed to ascertain the transmission or reflection of

solution/materials as a function of wavelength. “DR 2800 Spectrophotometer is a
portable spectrophotometer that is known to be user friendly. Some of its features
include:

 It can be used for 240 analytical methods

 It is touch screen with small footprint
 High accuracy and accommodates multiple cell sizes.

24
 Figure 3.7 spectrophotometer (DR 2800 LANGE)

3.8 CENTRIFUGE

A centrifuge is an instrument that makes an object or substance rotate while still on a

fixed axis (Spinning it in a circle). Sedimentation principle is the working principle of
the centrifuge in which the centrifugal acceleration causes denser substances and
particles to move outward in the radial direction. Centrifuges are used in clinical
laboratories as well as in virology, bacteriology and genetic research.

The centrifuge model used for this study is the “MIKRO 220R” made by “Hettich”. Its
features include: (i) powerful compact bench top centrifuges for processing microlitre
tubes of 0.2mL to 2.0Ml and even tubes up to 50Ml volume. (ii) Short run-up and run-
down times even at high speed. (iii) Quiet and smooth running.

25
 Figure 3.8 MIKRO 220R centrifuge

3.9 MICROSOPE

A microscope is an instrument used for the magnification of an object, thereby making it

possible to be seen by the eye. It is mostly used in the clinical laboratories, as well as for
virology and bacteriology studies. The model of microscope used for this study is the
“LEICA DM 4000B”. Its features include:

 Brilliant images with stunning contrast

 Fluorescence intensity manager (FIM) that enables fast precise and reproducible
setting of fluorescent light intensity.
 Superior image quality with led illumination.

26
 Figure 3.9 LEICA DM 400B Microscope

3.10 INCUBATOR

Incubator is a device mostly used for laboratory work/study in microbiology, molecular

and cell biology. It is used to monitor microbiological (Bacteria, Fungi and eukaryote)
and cell cultures by maintaining optimal carbon dioxide level, oxygen level, optimal
temperature and humidity conditions. Incubator made by “Memmert” was use for this
study. Its features include:

 Visual alarm indicator

 Maximum temperature of 220 ⁰c
 Simple operation with recessing push/ turn control knob.

27
 Figure 3.10 incubator (MEMMERT)

3.11 AUTOCLAVE

Autoclave is equipment with a pressure chamber used both for industrial processes,
laboratory and medical applications for sterilization purpose. The material or substance
to be sterilized is subjected to high pressure saturated steam usually at 121⁰c (249⁰F) for
about 15 – 20 minutes depending on the material or substance to be sterilized. The type
of autoclave used for the study is the “Wiseclave” made by “Wisd”. Its features include:

 Excellent mobility
 Safety pressure mechanism
 Easy to use digital controller
 LCD display
 Solid or liquid mode options
 Strong screw handle door.

28
 Figure 3.11 Autoclave.

3.12 ASHING/MUFFLE FURNACE

Ashing/muffle finance is a heating instrument used in research facility/laboratories for

high-temperature application. It is used to determine the proportion of a sample that is
non-combustible and non-volatile (i.e., Ash). “PFL 110/6” made by ‘Brotherm
Furnaces’ is a chamber type of ashing furnace with maximum heating temperature of
1100⁰c.

System Features:

 Standard special design door

 It is equipped with DC1010 digital controller
 High-quality fiber material
 Standard door safety switch
 High level temperature uniformity

29
 System operation with solid-state-relays
 Bottom protection, alumina plates on the floor Rugged and durable design
 Intuitive controller user interface
 High quality heating elements
 Electrical protection
 Dual skin housing for low external temperatures and high stability

Figure 3.12 Ashing/Muffle furnace.

30
 CHAPTER 4

4.0 MATERIAL AND METHODS

4.1 Collection of Soil Samples

The potential natural environment for the isolation of cellulytic fungi is the soil
(Utharalakshmi and Kumar, 2014). The soil samples were collected from different sites
around Cyprus International University campus, Nicosia, North Cyprus. Five different
samples were collected in sterile plastic bags and were transported to the university
laboratory under sterile conditions for isolation of cellulytic fungi.

The five different samples were mixed to form a composite soil sample followed by
crushing using mortar- pestle. It was then sieved to remove the big particles and plant
waste (Chouhan, 2014).

4.2 Isolation of Cellulase producing Fungi.

Isolation of cellulase producing fungi from collected soil samples was done by using
serial dilution method.

One gram of soil sample was added into ten mL of sterile distilled water in test tubes.
Then the suspension was homogenizes by shaking and then 1mL of sample suspension
above was added into 9 ml of distilled water (Chetha et al., 2015).

The dilution were made up to 10-5 and then 1ml of each dilution “10-1, 10-2, 10-3, 10-4,
10-5” were gotten using the pipette and then spread on the culture media (Lekh et al.,
2014, Gomashe et al., 2013).

31
4.3 Culture Media and Sub-Culturing

For the purpose of this work, potato dextrose agar (PDA) was used as the culture media.
It was prepared by adding 39g of commercial PDA powder to 1L of distilled water and
mixing to dissolve it while heated.

PDA was autoclave at 121⁰c for 15 min and was plated out (Gomashe et al., 2013). After
solidification of media on to plates, 1mL of the serially diluted sample was spread on the
PDA plates. The plates were incubated at 30⁰c for 7 days. The cultures were monitored
daily and three different colonies of fungal growth were isolated on the basis of
morphological characteristics of colony.

These isolated fungi were sub-cultured on a fresh sterile PDA plate. The Sub-culturing
process was repeated until the pure fungi isolate were obtained. The stuck cultures were
maintained on PDA plates at 4⁰c which were used for production of crude cellulase
enzyme (Nazanin et al., 2012, and Chouchau, 2014).

4.4 Identification of Fungi

The three different colonies of isolated fungi were identified according to their basic of
cultural morphological characteristics such as color of the colony, growth pattern studies
and according to the some microscopic characteristics of the colonies using light
microscope with lower power objective lens of (10×) and high power (40×) objective
lens (Chouhan , 2014; Hawjot and Deepti, 2015, Uttam et al., 2014).The cultural
morphological and microscopic characteristics of three different isolates were compared
with book (Tsuneo Watanabe ,. 2002) as reference

4.5 Production of Crude Cellulase Enzymes.

4.5.1 Fermentation Medium for Enzyme Production

The medium use for enzyme production under SSF was wheat bran. The wheat bran was
chosen as a substrate for production of crude cellulase enzyme in this study because it is

32
a cheap substrate and no pretreatment is required so as to get high yield of enzyme
production (Namita et al., 2012).

The wheat bran was washed using distilled water and dried in oven at 50⁰c for 24h.

The fermentation medium for Enzyme Production before incubation, it is shown in

Figure (4.1).

Figure 4.1 The fermentation medium for Enzyme Production

4.5.2 Solid state fermentation (SSF) and optimization of parameters for enzyme
production by fungal isolates.

The SSF of crude cellulase production medium was the non-pretreated wheat bran
(Washed and Dried). SSF was carried out in 500mL Erlenmeyer flasks which contained
40g of substrate. Medium was humidified with moistening agent which is 24ml of
sodium phosphate buffer 0.1M, pH 5.0 with ratio of 60% (v/w) (Ali et al., 20050; Reeta
et al., 2006). After optimization, the moisture content and pH buffer was sterilized using
an Autoclave at 121⁰c for 30min (Chetna et al., 2015; Soma and Rangasamy, 2014).

33
4.5.3 Inoculation of Medium Production

After the cooling of the sterilized medium flasks, each of them was inoculated by the 5
pieces ~ (1cm) of fungal stock culture which grow on PDA (Namita et al., 2012; Ali et
al., 2005).

The inoculation of fermentation medium was done by three different isolates of fungal
colonies. The content of flask were mixed well to distribute the fungal inoculum
throughout with medium, and then the inoculated flasks were covered via cotton and
incubated at different days. The incubation temperature was adjusted at 30⁰c for all
fermentation times and for all isolates,

4.5.4 Time course of Enzyme Production under the SSF by fungal isolates.

The time course for crude cellulase enzyme production was carried out by preparing
different incubation times of the inoculated flasks (3, 4, 5 and 6 days) which contains
40g of Wheat bran (Substrate) moistened with 24 mL 0.1M sodium phosphate buffer of
pH 5.0. These flasks were incubated at 30⁰c.

4.5.5 Extraction of Crude Cellulase.

After incubation of the inoculated fermentation medium at different fermentation time,

the cultures grown on SSF medium were dried in an incubator at 40⁰c for a day; then the
dehumidified culture media were grinded using a grinder for 2 min (Ali et al., 2005).
0.1M Sodium phosphate buffer with pH of 5.0 was added into the powdery fermented
culture media in a ratio of 1:10 (w/v) g/mL and the mixture was incubated at room
temperature in a shaking water bath of 150 rpm and then filtered using a metallic sieve
to remove the solid contents. The extract was centrifuged at 600rpm for 10 min (Namita
et al., 2012; Devendra et al., 2012; Chetna et al., 2015). The suspension of extract was
used as source of crude cellulase enzyme for further uses for determination of enzyme
activity and saccharification of OOMSW biomass. The crude enzyme was maintained at
4⁰c.

34
4.6 Enzyme Assay

4.6.1 Total Enzyme Activity

Cellulase enzyme activity was assayed by filter paper cellulase activity (Fpase)
according to Goshe Method (Ghoshe, 1987) with a little modification. Fpase for total
cellulase activity was determined by measuring total reducing sugars TRS using DNS
reagent (3, 5 –dinitrosalicylic acid) and glucose concentration standard curve (Asma et
al., 2012).In this assay, the Whatman filter paper was used as substrate; crude cellulase
of culture filtrate from each isolate of different incubation times was used as source of
enzyme and 0.1M sodium phosphate buffer of pH5.0 was used as the reaction buffer.

4.6.1.1 Procedure of Fpase Assay.

Whatman filter paper no 1 (1×6 cm) ~ 50g were placed into glass test tubes 25mL. 1mL
of buffer was added to each tube and the 0.5mL of produced crude cellulase enzymes
was added into the tubes. All the tubes containing the reaction mixture were incubated in
a shaking water bath at 50⁰c ± 1 for 1h at 150rpm.

After incubation for 1h, all tubes were removed from the water bath and the 3mL DNS
reagent [1416mLD water-106g DNS- 19.8g NaOH – 306g Na- K tartrate – 3.6g phenol
8.3g Na Sulfite] was added to all tubes to stop the enzyme substrate reaction.

The tubes were placed in boiling water bath for 5 min for red brown color development,
then all tubes were cooled in an ice water bath after which 20ml of distilled water were
added to all tubes and completely mixed by inverting the tubes several times and then
the tubes were left at room temperature for 20 min. the light absorbance was measure
using a spectrophotometer at 540nm and was compared with standard curve of glucose
to translate the color of samples to TRS equivalent (Chouhan, 2014; Namita, 2014;
Chetna et al., 2015).

The activity was calculated as measured reducing sugar of sample (mg of glucose) ×
0.185 which was expressed as the Fpase Unit (FPU) (Bhat and Ramesh, 1987; Ghose,

35
1987). One unit of filter paper activity (FPU) is the amount of enzyme required to form
1µ mol of glucose (TRS) from substrate (Filter paper) per minute under the assay
conditions and during the hydrolysis reaction. The enzyme activity of SSF has been
expressed as U/g dry substrate (U/gds) which was used in the production fermentation
media (dry mycelia wheat bran) (Soma and Rangasamy, 2011).

The glucose standard was done by prepared the glucose stock solution (1 g / 100ml) and
the dilution of glucose standard and construction of standard calibration curve are show
in the table (4.1) and figure (4.1.)

Table 4.1 Dilution and construction of glucose standard calibration curve.

Glucose stock (mL) buffer (mL) Dilution Concentration Abs. 540 nm

1.0 0.5 1:1.5 7.6 mg/ mL 0.611
1.0 1.0 1:2 5.0 mg/ mL 0.481
1.0 2.0 1:3 3.3 mg/mL 0.272
1.0 4.0 1:5 2.0 mg/mL 0.154

Figure 4.2 Construction of glucose standard calibration curve for TRS measurement.

36
The glucose standard curve was used to determination of unknown total reducing sugar
concentration for both, for essay the cellulase activity and estimate the Enzymatic
Saccharification of OOMSW Biomass by using below equation

(Y = 10.53x + 0.2032) ( 4.1)

When X = Abs of samples (OD)

4.7.0 Bioconversion of Biomass by Crude Cellulase Produced from Fungal Isolates

(Enzymatic conversion of Biomass).

The enzymatic conversion of biomass involves the substrate (biomass), enzyme source
and optimization conditions for hydrolysis of lignocellulosic material into fermentable
reducing sugars (Enzymatic Saccharification) (Lei et al., 2015). The application of
produced enzyme in bioconversion in this study was done by using olive oil mill solid
waste (OOMSW) biomass as the substrate and a high activity crude cellulase produced
from fungal isolates as the enzyme sources.

4.7.1 Enzymatic Saccharification of OOMSW Biomass (Enzymatic Hydrolysis).

The OOMSW sample was collected from olive oil mill in Nicosia , North Cyprus and
transported into CIU laboratory. The sample was washed with water to remove the
residue oil and it was then dried at 105⁰c for 1day. The dried sample was grinded and
sieved using a metallic sieve and then stored at room temperature condition until used
for enzymatic hydrolysis process.

37
Figure 4.3 Dried OOMSW biomass

4.7.1.1 Determination of moisture Content in OOMSW sample.

The moisture of OOMSW biomass was determined by drying 5g of biomass in an oven

at 105⁰c for 24h. The moisture content was calculated by using the below equation:

weight of initial sample − weight of dried sample

Moisture content % = x 100 (4.2)
weight of initial sample

4.7.1.2 Determination of cellulose content in OOMSW biomass

Two methods were used for estimate the crude cellulose content in the OOMSW
biomass, (1) Weendize methods as described previously by (Amir et al., 2011). (2) ISO –
AFNOR norm (NFV 0340, 1977) method as described previously by (Mameri et al .,
2000 ). Both of them based on corresponding to organic residue undissolved in both
H2SO4 and NaOH media.

38
4.7.1.2.1 Procedure of determination of crude cellulose content in OOMSW by first
method

1g of OOMSW sample which was dried and grinded was placed in the 200mL flask and
100mL of 1.25% (w/v %) sulfuric acid solution was added. Then flask was boiled for
30min on the heating magnetic stirrer and then the sample was filtered and washed
several times with hot distilled water to adjust natural PH. The solid residual of sample
was placed again in 200mL flask and 100ml of 1.25% (w/v %) sodium hydroxide
solution was added to it and it was boiled for 30mins. The samples was washed and
filtered again. The solid residual of sample was treated with ethyl alcohol 70 % to
remove dyes and raw ash complex and then the sample was washed with distilled water.
The residual of sample was dried at 105⁰c for 24h and weighed to calculate the content
of cellulose in the sample.

weight of residue of dried treated sample

Crude cellulose content % = x 100 (4.3)
weight of initial dried sample of biomass

4.7.1.2.2 Procedure of determination of crude cellulose content in OOMSW by

second method

3g of OOMSW was added into flask of 250ml and was mixed and treated with 200ml of
0.13M,sulfuric acid for 30 min at boiling temperature and then the hot water was used
for washed and filtered of solution and 200ml of 0.31M sodium hydroxide was added
into filtered residue of biomass for 30 min .The solution was filtered and washed again
with hot water and solid residue of biomass was heated in oven at 130⁰c for one h and
the product P1 was weighted and then product 1 was heated second time at 550⁰c for 1
h and mass of second remaining product P 2 was weighted again.

The percentage of crude cellulose in OOMSW was calculated by below equation

39
 Mass of P1 – Mass of P2
Crude cellulose content % = x 100 (4.4)
Mass of initial dried biomass material

4.7.1.3 Pretreatment of OOMSW biomass

The pretreatment step of hydrolysis process is required for efficient enzymatic

hydrolysis of biomass. The pretreatment process of OOMSW is required to reduce the
complex size and structure of lignocellulosic material as well as its chemical
composition to enhance the enzymatic hydrolysis process so that make the hydrolysis of
carbohydrate fraction of OOMSW into monomeric sugars is achieved rapidly and with
high greater yields of fermentable sugar (Caixia and Yebo, 2012). The OOMSW
biomass sample was treated by chemical pretreatment using sodium hydroxide NaOH
with 1.25% of NaOH at a ratio (1:10 w/v) 100g/L (Abdi et al., 2000).

50g of OOMSW sample was placed in 1liter flask; 500ml of 1.25% NaOH was added
into flask then heated at 100⁰c for 1h on the magnetic stirrer at 4 rpm. After mixing for
1h, the sample was cooled, filtered and washed with distilled water until neutral pH was
achieved and then the sample was dried at 105⁰c in oven for a day.

The pretreated OOMSW was maintained at 4⁰c for enzymatic hydrolysis (Abdi et al.,
2000; Maviuuthu et al., 2010).

Figure 4.4 Effect of pretreatment of biomass on enzymatic hydrolysis

40
4.7.2 Enzymatic hydrolysis experiment.

The hydrolysis processes of OOMSW biomass by crude cellulase as the enzyme source
from different three isolates at high activity were carried out in 200ml flask. 0.1M
sodium phosphate buffer was used as reaction buffer during all experiments and Alkali
pretreated OOMSW biomass as the substrate. The biomass material was autoclaved for
sterilization at 120⁰c for 30 min before enzymatic hydrolysis process .The reaction of
mixture was performed in the shaking water bath of 150rpm and the optimization
parameters of enzymatic hydrolysis of OOMSW were studied .

4.7.2.1 Effect of PH on enzymatic hydrolysis of OOMSW

The effect of PH on enzymatic process of treated biomass was done by using 4 different
parameter of PH "4.5 , 5.0 , 5.5 and 6.0" for each enzyme source .The temperature was
adjusted at 50⁰c,Concentration of substrate was 1g/50ml ( 20g/L ) , 1 mL of each
enzyme (4.63 FPU of Aspergilus sp), (2.0 FPU of Pencillium sp) and (6.68 FPU of
A.niger) were used , reaction mixture was incubated for 2h of time and finally Volume
of buffer was adjusted of 50ml.

4.7.2.2 Effect of temperature on enzymatic hydrolysis of OOMSW

The effect of temperature on the enzymatic hydrolysis of biomass was carried out by
using different temperature of reaction "40 , 50 and 60 ⁰c " 5.0 of PH , Concentration of
substrate was 1g/50ml ( 20g/L ) , 1 mL of each enzyme (4.63 FPU of Aspergilus sp),
(2.0 FPU of Pencillium sp) and (6.68 FPU of A.niger) were used , reaction mixture was
incubated for 2h and finally Volume of buffer was adjusted of 50ml.

4.7.2.3 Effect of Concentration of substrate (OOMSW) on enzymatic hydrolysis of

OOMSW

The effect of concentration of biomass "substrate" was investigated on enzymatic

process and amount of TRS by using different concentration of pretreated OOMSW "
20, 30 , 40 , 50 , and 60g/L " pH was 5.0 , temperature of reaction was 50 ⁰c and 1 mL

41
of each enzyme (4.63 FPU of Aspergilus sp), (2.0 FPU of Pencillium sp) and (6.68 FPU
of A.niger) were used . Reaction mixture was incubated for 2h and finally Volume of
buffer of reaction mixture was adjusted of 50ml.

4.7.2.4 Effect of incubation time and loading of enzymes on enzymatic hydrolysis of

OOMSW

The enzymatic hydrolysis was done by using 5 different dosage of enzyme which was
expresses as 5 different volume and FPU of each enzyme source i.e. "1ml (4.63 FPU),
2ml (9.26FPU), 3ml (13.89FPU), 4ml (18.52FPU) and 5ml (23.15FPU) " of Aspergilus
sp cellulase , " 1ml (2.0 FPU), 2ml (4.0FPU), 3ml (6.0FPU), 4ml (8.0FPU) and 5ml
(10.0FPU) " of Pencillium sp cellulase and " 1ml (6.68 FPU), 2ml (13.36FPU), 3ml
(20.0FPU), 4ml (26.72FPU) and 5ml (33.4FPU) " of A.niger cellulase with 2 , 4 , 8 , 16
, 20 and 24h of time course of reaction to study the effect of enzymes dosage on
enzymatic hydrolysis with time for determine the optimum loading of enzyme and the
steady state of reaction time. Concentration of pretreated OOMSW was 50g/L, pH was
5.0, temperature of reaction was 50 ⁰c and finally Volume of buffer of reaction mixture
was adjusted of 50ml.

The sample that was incubated for more than 1h was added to it (0.001% NaN3) (w/v) to
prevent contamination (Bin et al., 2006).The volume of reaction buffer was adjusted
at50ml with different volumes of enzymes.

At required times, the flasks of reaction mixture were taken out from water bath and
then filtered using metallic sieve and centrifuged for 10min at 6,000rpm.

The concentration of total reducing sugar TRS in supernatant which was produced
during hydrolysis process was determined according to DNS method and glucose
standard curve (Ghose, 1987, Chetna , et al 2015) the TRS data were corrected by
enzyme blanks .

42
1.5mL of supernatant of each sample was placed in the test tube and 1.5mL of DNS
reagent was added into test tube and then the mixture was boiled in a water bath for
5min to perform color reaction. The tubes of samples were cooled in ice water bath and
left at room temperature. The absorbance reading of sample was taken at 540nm.

The concentration of TRS (g/L) which was obtained from the hydrolysis process was
used for determination of bioconversion (saccharification yield) % and TRS yield g/g
substrate of OOMSW using the equation as described by (Faten et al., 2016) .

g
Concentration of TRC ( )x0.9
L
Saccharification yield % = × 100 (4.5)
concentration of substrate used in hydrolyusis (g/L)

Concentration of TRC (g/L)

TRS yield g/g = (4.6)
concentration of substrate used in hydrolyusis (g/L)

The cellulose conversion % was calculated using equation as described by (Ahmed et

al., 2012)

g
Concentration of TRC ( )x0.9
L
Cellulose conversion % = × 100 (5.7)
concentration of cellulose used in hydrolyusis (g/L)

4.8 Kinetic models of study

The kinetic models of this work was investigated by using Michaelis Menten kinetic and
line Weaver-Burk plot to estimate the kinetic models of enzymatic hydrolysis process of
OOMSW biomass .

43
4.8.1 Michaelis Menten kinetic model

The Michaelis-Menten model is the one of the known and suitable approaches to estimate
enzyme kinetics. It is an equation form relating reaction velocity to substrate concentration when
the enzyme (E) interacts with the substrate (S) by binding to its active site to form the enzyme-
substrate complex ( ES ) which then reacts irreversibly to generate a new p roduct ( P ) and the
free enzyme (E ) when reaction is followed by the decomposition of (ES). This system
can be represented schematically as follows:

The Michaelis-Menten equation for this system is:

(4.8)

Figure 4.5 An example of Michaelis Menten kinetic model

Where V = rate of reaction velocity mg/l/min.

V max = the maximum velocity achieved by the system.

44
 KM = the Michaelis constant mg/ l, is the substrate concentration at which the
reaction velocity is 50% of the Vmax.
[S] = concentration of the substrate.
The rate of reaction (V) can be obtained from equation:

d (𝑃) d (𝑆)
V= = (4.9)
dt dt
When P = concentration of product mg/L

t = time of hydrolysis (min)

The velocity of reaction (V) can be plotted against the substrate concentration (S) to
estimate the V max and KM of enzyme substrate reaction .

4.8.2 Line Weaver Burk Equation

Lineweaver-Burk analysis is method of linearizing substrate-reaction rate data that is

used to estimate the kinetic constants Km and Vmax. One creates a secondary, reciprocal
plot: 1/velocity, 1/[substrate], the Lineweaver-Burk plot is a straight line with :
y = m.x+b (4.10)

Figure 4.6 An example of a Lineweaver-Burke plot

45
 1 𝐾𝑚 + [S]
= (4.11)
𝑣 𝑉 max [S]

1 𝐾𝑚 1 1
= ( 𝑉 max) . ( [S]) + (4.12)
𝑣 𝑉 max

Apply this to equation for a straight line y= m.x+b

1
y= 𝑣

1
x = [S]

𝐾𝑚
m = slope = 𝑉 max

1
b = y intercept = 𝑉 max

The velocity of reaction 1/ v is plotted versus the substrate concentration 1/ [S] .The
slope of line is k m/ V max, x is -1/ K m and y is 1/ V max.

46
 CHAPTER 5

5.0 RESULTS AND DISCUSSIONS

5.1 Isolation and identification of cellulytic fungi for crude cellulase production.

The soil samples were obtained from some sites around CIU campus and the isolation
and identification of cellulase producing fungi were done.

Three strains of filamentous fungal colonies (Isolate 1, 2 and 3) were selected and
isolated by serial dilution method from soil samples for production of crude cellulase
enzyme.

isolate (1) isolate (2) isolate (3)

Figure 5.1 colonies of fungal isolates after 5 days of incubation

The isolated colonies of fungi were identified according to their morphological

characteristics which are the color and growth behavior studies of colony and also to
their microscopic characteristics which were examined under the light microscope
(Charitha and Sunilkumar, 2012).

47
In this study, the identification of isolated fungi was compared with book (Tsuneo
Watanable., 2002).

Table (5.1) and (figure 5.2) show the three probable identities of fungal isolates.

Isolate (1) is seen to be similar in structure to Aspergilus sp. Isolate (2) is seen to be
similar to Pencillium sp and isolate (3) is seen to be similar to Aspergilus niger.

Table 5.1 Probable ID of isolated Fungi

Isolate Colony Microscopic Probable ID of

morphological observation Fungi
observation
1 Dark green colored Septate hyphae,
growth non-septate Aspergilus Sp.
conidiophores,
columnar head

2 Dark brown color Long chains of

conidia with Penicillium sp.
smooth, brush
shaped
Conidiophores.

3 Dark black colony Septate hyphae ,

non-septate Aspergilus niger
conidiophores,
columanar head
black conidiospores

48
 Aspergilus sp Pencillium sp Aspergilus niger

Figure 5.2 Microscopic observations of fungal isolates

5.2 Productions of cellulase enzymes under SSF and Fpase assay

The use of available and cost-effective material as substrate for production of crude
cellulase to achieve high yield that can reduce the overall cost of cellulase production
(Namita et al 2012). The production of cellulase under SSF is fast gaining interest as a
cost effective technology with an almost tenfold, predicted production in costs and much
higher yield as well as it has some advantages including simple technique, less water
output, low energy requirement and better products when compared with submerged
SmF technology. (Dongyang et al., 2011). Wheat bran was applied in this study as
substrate because it has been reported in several publication studies. It has high protein
content more than 17% and around 19% of starch content in dry matter and it has more
than 39% of cellulose and hemicelluloses content. This thus makes it an excellent
fermentation culture medium to provide carbon sources for cellular growth of fungi and
an excellent support for production of crude cellulase enzyme based on the high content
of cellulose (Kilikian et al., 2014).

49
 (1) Aspergilus Sp (2) Pencillium sp(3) Aspergillus niger

Figure 5.3 Growth of isolated fungi after 3 days.

Presented in table (5.2) are some of the previous studies which reported that wheat bran
can be used as substrate for cellulase production.

Table 5.2 Some of previous studies.

Reference Fermentation Substrate Fungi Strain Fpase

technology
Namita et al SSF Wheat bran + Aspergillus 16.8 U/g
(2012) mineral salt niger
solution

Badhan et al SSF Wheat bran + Myceliophthora 0.7 U/g

(2007) mineral salt Sp.
solution

Deepa et al SSF Wheat bran + Fomitopsis Sp 3.49 U/g

(2011) mineral
solution

Devendra et al SSF Wheat bran + Trichoderma 2.4 U/g

(2012) mineral reesei.
solution

50
Devendra et al SSF Wheat bran + Trichoderma 1.4 U/g
(2012) mineral resei
solution

Khushal et al SSF Wheat bran + Aspergillus 10.7 U/g

(2010) Soybean hulls niger +
+ Mandels Trichoderma
solution resei

The cost-effective technology and substrate are needed for enzymes production with
high yield. SSF is a suitable technology for economical cellulase production by using
cheap material as substrate.

Three different strains of isolated Fungi in this study i.e. (1) Aspergilus Sp, (2)
Pencillium sp and (3) Aspergillus niger were used for production of crude cellulase. The
production fermentation process was carried out in 500mL of flasks under SSF
technology, wheat bran was used as substrate and the enzyme was assayed by measuring
the total enzyme activity FPase.

The temperature of fermentation process for enzyme production in this study was
adjusted at 30⁰C for all isolated strains that according to previous studies which reported
the optimum temperature for production of the high cellulase yield by various fungi is in
range of 25⁰C to 30⁰C (Namita et al., 2012).

In a study by Namita et al (2012), the optimum temperature of cellulase production by

A.niger was reported at 30⁰C in SSF. The highest yield of cellulase and optimum
temperature in a range of 28⁰C to 30⁰C for cellulose production by Trichoderma
harzianum, has been reported by Rubeena et al (2013). In the study by Deepa et al
(2011), 30⁰C was optimized for the best growth and cellulase production by brown rot
fungus Fomitopsis.sp. Also in the study by Khushal et al (2010) studied that 30⁰C is

51
optimum temperature for production of cellulase in mixed-culture of Trichoderma reesei
and Aspergillus Oryzae.

It indicates that the crude cellulase enzyme has high sensitivity to temperature and thus
an increase in temperature of fermentation process lead to decrease in the growth of
fungi and then decrease of enzyme activity, this may be due to the fact that higher
temperature can reduce the metabolic activity of fungus and also can denature the
enzyme (Rubeena et al,. 2013).

pH moistening agent for enzyme production (Phosphate buffer) was adjusted at 5.0 in all
processes for cellulase production and enzyme extraction processes. Most previous
studies has reported the optimization of pH at the range 4.5 to 6.0.

The optimum pH in SSF consisting of wheat bran and mineral salt solution is 4.8 by
Trichoderma reesei., this optimization of pH was shown and reported the maximum of
cellulase activity as explained by Devendra et al (2012). In a study by Chetha et al
(2015) determined the optimal pH for crude cellulase production by some selected
fungal strains in SSF which showed higher cellulase yields was 4.5 -5.0.The optimum
pH was reported to be 5.0 for cellulase enzyme production by soil fungus Pencillium sp.
(You Ree et al., 2015). The moisture content of fermentation medium by moisture
agents such as (Distilled water, phosphate buffer and mineral salt solution) is essential
for fungi metabolism and can effect on the physiology of the fungi.

In solid state fermentation, SSF technology both high and low moisture level of culture
medium by moisture agents can affect the activity and productivity in case of the high
moisture content reduces porosity and media that leads to changes in substrate particle
structure and lowers O2 transfer, whereas the lowering of moisture level decreases in the
solubility of nutrient of substrate and thus a lower level of swelling.

The optimum moisture content of growth medium in SSF depends on the nature of
substrate, microorganism and type of products (Deepa et al., 2011 and Namita et al.,
2012).

52
In general, the optimum moisture content of cellulase production by fungi is in a range
of 50-70%. In present study, the moisture level of wheat bran is SSF with phosphate
buffer was adjusted at 60% (v/w). This level of substrate moisture has been reported in
many previous studies as the optimum moisture content of substrate for cellulase
production.

Khushal et al (2010) reported that 70% of moisture content was the optimum level for
cellulose production by mixed culture of T.reesai and A.oryza under SSF and using
Wheat bran and Soybean hulls as substrate. Namita et al (2012) studied the substrate-
moisture ratio (60%) was the best suitable for high yield of cellulose production by
A.niger and wheat bran as substrate. Study by Reeta et al (2006) reported that 66.4% is
the optimum moisture content of substrate for cellulose production by T. reesei.

5.2.1 Time course of cellulase production by three different isolated fungal strains.

The optimization and evaluation and estimate of time course is first importance for crude
cellulase production by fungi (Rubeena et al., 2013). The incubation time is directly
related with biosynthesis of cellulase enzyme (Asma et al., 2012).

In this work, the investigation of time course for crude cellulase production was studied
under SSF process in 500mL flasks and wheat bran was used as substrate.

The incubated flasks of three different isolated fungus strains (Aspergilus sp, Pencillium
sp and A. niger) were harvested after every desired fermentation time (3, 4, 5, and 6
days) and it were analyzed for enzyme activity by Fpase. Table (5.3) and figure (5.4)
represent and show the results of effective time course (incubation time) on the yield and
activity of crude cellulase production by different selected fungal strains according to
determination of total cellulase activity by filter paper activity assay Fpase.

53
Table 5.3 Effect of incubation time on enzymes production

cellulase activity FPU/gds

Incubation time Isolated fungal strains
(Days) Aspergillus Sp Pencillium Sp Aspergillus niger

3 0.562 0.548 0.370

4 2.315 0.763 3.340
5 0.695 1.016 0.769
6 0.254 0.936 0.428

Effect of incubation time on enzymes production

cellulase activity FPU/gds

3,5
3
2,5
2 A.Sp
1,5
P.Sp
1
0,5 A. niger
0
3 4 5 6
Incubation time (Days)

Figure 5.4 Effect of incubation time on enzymes production.

The results from above table and figure indicates that produced cellulase yield which is
expressed as enzyme activity Fpase by isolate (3) Aspergilus niger reached its maximum
enzyme production after 4 days and Fpase was (3.340 FPU/gds). The enzyme yields
gradually decreased at 5 days and 6 days of incubation period (0.769 FPU/gds and 0.428
FPU/gds respectively). The results show that it is possible for A.niger to produce crude
cellulase at a large scale by using wheat bran as substrate.

54
The isolate (1) Aspergilus Sp. Produced the cellulase enzyme and showed increase in
enzyme activity with time and the maximum production of enzyme was obtained at 4
days of fermentation time by SSF yielding (2.315 FPU/gds) after that the enzyme
activity and yield decreased as depicted in table (5.3)

The result also indicates the production of enzyme by isolate (2) pencillium Sp. Which
gradually increased with time and the maximum yield of enzyme was obtained at 5 days
when expressed as cellulase activity (1.016 FPU/gds). And the enzyme activity declined
at the end of fermentation time of 6 days. so the effect of optimization of time course on
the cellulase production by three isolated fungal strains under SSF was investigated and
should note that any further increase in incubation time will not favor an increase in the
yield of cellulase production by isolated fungi. The increase in enzyme production with
increase in incubation period until reached the maximum point due to rapid hydrolysis of
cellulose content in fermentation medium (Marimuthu etal., 2010).

The case of increase of enzyme production and reach its high level at specific time to
achieve maximum cellulase activity and after that, the yield can be decreased at the end
of incubation period; Which is probably due to the nutrients is reduced with time in the
fermentation medium which stressed the fungal physiology resulting in the inactivation
of secretary machinery of enzyme (Asma et al., 2012). It also might be due to the release
and the drop in pH of the production medium. (Namita et al., 2012); and it may be due to
decrease in moisture content with increase in time which causes reduction in solubility
and thus can affect the production of enzyme with increase in time of incubation.
However, the optimum fermentation time for cellulase production by isolated fungal
strains in this study is represented in table (5.4).

55
Table 5.4 Optimum fermentation time of isolated fungi

Isolated fungal strain Optimum fermentation time

Isolate (1) Aspergilus Sp. 4
Isolate (2) Penicillium Sp. 5
Isolate (3) Aspergilus niger. 4

Uttam et al (2014) explained the reason of increase in enzyme production at specific

time and then become decreased may be due to increase in concentration of some toxic
wastes and nutrient in growth medium is depletion which leads to decline fungal growth
and enzyme production and also the fungal cells undergo a stationary phase condition
and then the enzyme production can be increased or decreased with specific time of
incubation times.

However, the table (5.5) shows the comparison of cellulase production and fermentation
time result in present study by isolated fungal strain with other in previous studies.

Table 5.5 The comparison of cellulase production and fermentation time result in
present study with other in previous studies

Fungi Strain Substrate Optimum FPase Reference

incubation time
(day)
Fomitopsis Sp Wheat bran + 11 3.492 FPU/gds Deepa et al
mineral salt (2011)
solution (SSF)

Trichoderma Wheat bran + 6 2.63 FPU/gds Devendra et al

reesei mineral salt (2012)
solution (SSF)

Trichoderma Wheat bran + 6 1.1 FPU/gds Namita et al

reesei mineral salt (2012).
solution (SSF)

Aspergilus niger Wheat bran + 4 16.8 FPU/gds Namita et al

Ns-2 mineral salt (2012).

56
 solution (SSF)

Aspergilus niger Wheat Straw + 4 2.8 FPU/gds Namita et al

mineral Solution (2012).
(SSF)
Aspergilus niger
KK2 Wheat Straw + 4 19.5 FPU/gds Kange et al
mineral (2004)
Solution(SSF)
Aspergilus niger
MTCC 7956 Wheat Straw + 4 4.55 FPU/gds Rajeev et al
mineral (2009)
Aspergilus Solution(SSF)
oryzae +
Trichoderma Wheat bran + 4 10.7 FPU/gds Khushal et al
reesei soy bean hulls + (2010)
mandel solution
Aspergilus niger (SSF)

Wheat Straw + 3-4 4.59 FPU/gds Chetha et al

Aspergilus niger mineral (2015)
Solution(SSF)

Penicillium Bagasse pith + 3-4 1.04 FPU/gds Chetha et al

oxalicum mineral salt (2015)
solution(SSF)

Penicillium Wheat bran 8 1.2 FPU/mL Reetu et al

echinulatum mandel media (2015)
(SmF)

Wheat bran + 5 8.3FPU/mL Laisa et al

Aspergilus niger cellulose + (2013)
mineral solution
(SmF)

Wheat bran + 4 3.34 FPU/gds Present study

Aspergilus Sp Phosphate
buffer(SSF)

Wheat bran + 4 2.315 FPU/gds Present study

Phosphate
Pencillium.sp buffer(SSF)

Wheat bran + 5 2.315 FPU/gds Present study

Aspergilus niger Phosphate
buffer(SSF)

57
Since the comparison of the results obtained in this study with those reported by others
studies as in table (5.5). In general, the results of the optimum fermentation time of the
fungal isolates in present study, is in agreement with observations from previous studies.

There is a variable in the result of Fpase between this work and other. The Fpase of
previous studies are higher than that in present study. Probably due to the substrate and
fermentation media which was used in enzyme production process.

In this study, the fermentation medium was wheat bran with phosphate buffer whereas in
the previous studies, the fermentation media was wheat bran or other substrate with
either Mendel's salt solution or mineral salt solution.

This fact is in agreement with study by Devendra et al (2012) which studied the
optimization of SSF conditions for cellulase production by Trichoderma.reesei and
reported that using mineral salt solution enhances the production of cellulase more than
using phosphate buffer solution or distilled water. However, using wheat bran without
any treatment with just phosphate buffer can be cost –effective in cellulase production.

The study results indicates that is possible to produce economic industrial cellulase at
large scale by isolated fungal strains under SSF which can be used in bioconversion
application of lignocellulosic material.

In conclusion with emphasis to the study results, production of cellulase at small scale
cost and large scale yield by using wheat bran as substrate with buffer solution and
without any treatment was successful.

The next step in this work was using the produced enzyme from isolated fungal strains
for bioconversion and saccharification of OOMSW into fermentable sugar which can be
used for other applications.

58
5.3.0 Bioconversion and Saccharification of OOMSW sample by produced crude
cellulase extract.

Olive oil mill solid waste (OOMSW) is the solid waste generated during olive oil
production processes in two and three-phase olive mills. It consists of remaining oil,
pulp, cracked seeds, peels and stones of the olive fruits. The OOMSW is rich in the
organic content Biomass mainly lignocellulosic material (Frank et al., 2009).

5.3.1 Determination of moisture content in OOMSW sample.

5g of sample was dried in an oven at 105 ⁰C for 24h and its moisture content was
calculated by (4.2) equation and represented as 33.6 % as the total content of sample and
its solid content was 66.4 % of the total content of OOMSW sample.

5.3.2 Determination of crude cellulase content in the OOMSW sample.

The two methods were used for determination of crude cellulose in biomass sample.

(1) Weendize methods as described by (Amir et al., 2011).

(2) ISO-AFNOR norm (NFV 0340, 1977) method as described by (Mameri et al., 2000).

The percentage of crude cellulase content was calculated by (4.3) (4.4) equations. Each
experiment was replicated twice and reported results indicate an average and standard
division at replicated experiments. Table (5.5) shows the crude cellulose content in
OOMSW sample and the concentration of crude cellulose in the sample was expressed
as percentage of dry matter.

59
Table 5.6 The crude cellulose content in OOMSW sample

Crude cellulose content %

Method Crude Sample NaOH treated sample

(1) 43 ± 1.26 41 ± 0.76

(2) 43.5 ± 2.3 42 ± 1.53

Most cellulosic materials consist of crystalline and amorphous domains, the crystalline
cellulose can be transformed to an amorphous state with chemical treatment and
heating.in this study the results indicate that the cellulose content of biomass sample was
decreased after alkali treatment that due to several conditions that can be effected on
transformation of crystalline to amorphous domains such as interaction of cellulose with
alkali solution , temperatures , NaOH solution concentration and time. Some of previous
studies show increase in the cellulose content of biomass after chemical treatment and
some of other show decrease in cellulose content. Abdi et al, (2000) reported the crude
cellulose content of OOMSW was 46.2% of crude sample and 42.53% of alkali treated
sample. In the study by Senkevich et al, (2012) reported the total carbohydrate in the
crude OOMSW biomass was 49% and after alkali pretreatment was 63.46%. Ravi et al
(2012) resulted the cellulose content of crude wheat straw sample was 25.2 % while in
the alkali treated sample was 49.0% .

However, the chemical composition of OOMSW depends on the olive oil extraction
process and also may be on the natural biological variability of olives and variations in
plant biosynthesis which is depending on the geographic location and climatic and soil
conditions (Maria et al., 2016).

5.4.0 Enzymatic hydrolysis of OOMSW and optimization of parameters conditions.

The crude extract enzymes which were obtained during the production process in SSF
were directly studied in terms of its capacity of enzymatic hydrolysis of OOMSW

60
biomass for production of fermentable sugars which can be used for further applications
such as bioethanol production, Bioenergy production and other biochemical products.

5.4.1 Enzyme sources for enzymatic hydrolysis of biomass.

Since the previous experiments and its obtained results, the maximum yields and activity
of produced crude cellulase from different isolated fungal strains were used as the source
of enzyme in the hydrolysis process of OOMSW.

1. Crude cellulase from Aspergilus.sp (2.315 FPU/gds)

2. Crude cellulase from Pencillium.sp (1.016 FPU/gds)
3. Crude cellulase from Aspergilus.niger (3.34 FPU/gds)

5.4.2 Substrate of enzymatic hydrolysis

Alkali pretreated OOMSW biomass was used as substrate in the hydrolysis processes.
The chemical pretreatment with NaOH was performed before the enzymatic hydrolysis
processes to enhance the Saccharification process of OOMSW.

Many previous studies reported that the alkali pretreatment of biomass before enzymatic
process for fermentable sugar production can generate high amount of sugar than acid
treatment or no treatment.

Abdi et al, (2000) studied and reported that alkali treatment of OOMSW enhanced the
enzymatic saccharification process. The enzymatic saccharification of alkali pretreated
wheat straw with 1% - 1.5% (w/v) of NaOH synthesized the higher amount of TRS more
than acid pretreatment (Khushal et al., 2010).

In this study, the effect of the dry matter concentration of OOMSW biomass, pH and
temperature of hydrolysis processes were studied and assayed. Furthermore the enzymes
loading which were expressed as the volumes of enzyme with different incubation times
of enzymatic hydrolysis were studied and tested in this work.

61
The total reducing sugar TRS was determined according to DNS methods which were
expressed as the bioconversion/ saccharification yield % in the enzymatic hydrolysis of
OOMSW and was calculated using equation (4.5).

5.4.3 Effect of the temperature on enzymatic hydrolysis of the OOMSW biomass.

The optimization of temperature of hydrolysis process was done with different

temperature i.e. 40, 50, 60 ⁰C for the three enzyme sources at 2h of incubation time. The
results are shown in table (5.6). The pH was 5.0 and substrate loading was 20g/L.

Table 5.7 The effect of temperature on hydrolysis process of the OOMSW biomass.

TRS g/L
Temperature ⁰C
Enzyme source 40 50 60

No enzyme (control) 2.52 2.51 2.54

Aspergilus.sp 4.9 7.47 4.22
Pencillium.sp 6.40 6.57 5.48
A.niger 8.42 9.38 3.94

Effect of temperature on enzymatic hydrolysis of OOMSW

10

8
TRS g/L

6 Enyme sources
Aspergilus.sp
4 pencilium.sp
2 A.niger
No Enzyme (control)
0
40 50 60
temperature c

Figure 5.5 effect of temperature of enzymatic hydrolysis process of OOMSW biomass.

pH = 5.0.substrate concentration =20g/L. enzyme loading =1ml. incubation time = 2h.

62
Since the results from the figure (5.5) the optimum temperature for enzymatic hydrolysis
of OOMSW sample was 50 ⁰C by all enzyme sources and the maximum level of TRS
yields 7.47g/L , 6.57 g/L and 9.38 g/L were obtained by Aspergilus.sp, Pencillium.sp
and A.niger respectively at 50⁰C.

This is in agreement with study by Abdi et al (2000) and Mameri et al (2000) which
reported that the optimum temperature for cellulasic hydrolysis of OOMSW was 50⁰C.

In the study by Ravi et al., (2012) which studied the production of fermentable sugars
from some lignocellulosic material by T.reesei, reported the optimum temperature for
the enzymatic hydrolysis was 50⁰C.

5.4.4 Optimization of pH for enzymatic hydrolysis of OOMSW biomass

The results of effect of pH on the enzymatic hydrolysis processes study with (4) levels
of pH i.e. 4.5, 5.0, 5.5, and 6.0 at 2h of incubation time. The temperature was 50⁰C and
substrate concentration was 20g/L are shown in table (5.8) and figure (5.6).

Table 5.8 Effect of pH on the enzymatic hydrolysis of OOMSW

TRS g/L

pH
Enzyme source 4.5 5.0 5.5 6.0

No enzyme (control ) 2.51 2.51 2.50 2.52

Aspergilus.sp 4.97 7.47 7.37 3.65
pencillium.sp 6.30 6.57 4.25 4.32
A.niger 4.39 9.38 8.73 3.59

63
Figure 5.6 Effect of pH on the enzymatic hydrolysis processes at 2h of incubation time.
The temperature = 50⁰C. Substrate concentration = 20g/L and volume of enzyme = 1 ml

The maximum levels of TRS as the results in figure (5.6) were 7.47 by enzyme of
Aspergilus.sp, 6.57 g/L by enzyme of Pencillium.sp and 9.38g/L by enzyme of A.niger
at pH 5.0 that hence the optimum pH of enzymatic hydrolysis of OOMSW biomass by
three produced enzyme sources was 5.0.

The result of this study agree with study by Abdi et al, (2000) which reported the
optimum pH of enzymatic hydrolysis of OOMSW by cellulase from T.reesei was 5.0
and also the results are in accordance with those by Ravi et al, (2012) who reported the
optimum pH for saccharification of cellulose powder by cellulase from T.reesei was 5.0

5.4.5 Effect of concentration of substrate on the enzymatic hydrolysis of OOMSW

biomass

The effect of substrate loading on the reducing sugar production (fermentable sugars)
was investigated by using different concentration of substrate (OOMSW sample) i.e.
20g/L, 30g/L, 40g/L, 50g/L and 60g/L. Based on the obtained results in previous studies
the optimum pH was adjusted at 5.0 and optimum temperature was 50⁰C the volume of

64
enzyme was 1mL of each enzyme source (4.63 FPU of Aspergilus sp), (2.0 FPU of
Pencillium sp) and (6.68 FPU of A.niger) and the incubation time was 2h.

Table (5.9) and figure (5.7) show the results of effect of concentration of substrate on the
hydrolysis process of biomass.

Table 5.9 Effect of substrate concentration on the enzymatic hydrolysis of biomass

TRS g/L

Substrate concentration g/L

Enzyme sources 20 30 40 50 60

No enzyme (control) 2.51 2.94 3.25 3.57 4.09

Aspergilus.sp 7.47 8.57 9.62 10.53 10.02
Pencillium.sp 6.57 8.11 9.49 9.98 9.25
A.niger 9.38 10.21 12.86 13.77 10.68

Figure 5.7 The effect of substrate concentration on the enzymatic hydrolysis of biomass. pH=
5.0. Temperature = 50⁰C. Volume of enzyme = 1mL of each enzyme source and the incubation
time = 2h.

65
Abdi et al, (2000) studied the enzymatic saccharification of OOMSW by cellulase of
T.reesei and reported that 40gdm-3 was the optimum concentration of substrate for
enzymatic hydrolysis. The low fermentable sugars yield at high concentration of
substrate may be due to two reasons: the lower enzyme volume to substrate ratio and
inhibition of end product feedback caused by the high concentration of TRS which is
produced during the enzymatic hydrolysis process. This fact is explained by Abdi et al,
(2000). The hydrolysis products of OOMSW can be contained glucose, Xylose,
Arabinose and particularly cellobiose which is considered to be strong enzymatic
inhibitor and thus may influence the enzymatic reaction.

In study by Nooshin (2014) reported the optimum loading of substrate for enzymatic
hydrolysis of rice straw by T.harianum was 50-70g/L.

5.4.6 Kinetic models of produced crude cellulase and enzymatic hydrolysis of

OOMSW biomass.

The kinetics of isolated fungal enzymes was applied according to the Michaelis Menten
Kinetics (Michaelis and Menten ,1913).The concentration of OOMSW biomass was
20,30,40,50 and 60 g/L and the time of reaction was 2h ,the velocity of reaction was
calculated by using equation (4.9) .The velocity of reaction of different enzyme sources
increases with increase in substrate concentration.

The Michaelis Menten curve of reaction rate against OOMSW concentration were
obtained as show in figures (5.8), (5.9) and (5.10) .

66
Figure 5.8 The Michaelis Menten curve of enzymatic hydrolysis of OOMSW by
cellulase of Aspergilus.sp

Figure 5.9 The Michaelis Menten curve of enzymatic hydrolysis of OOMSW by

cellulase of pencillium.sp

67
Figure 5.10 The Michaels Menten curve of enzymatic hydrolysis of OOMSW by
cellulase of Aspergilus.sp

From Michaels Menten curve in figures above, the Vmax of reaction by enzymes of
Aspergilus.sp, Pencillium.sp and A.niger were approximately 0.0580 ,0.0534 and 0.0860
g/l/min and Km were 13 , 17 and 15 g/l respectively.

The kinetics of isolated fungal enzymes was applied also according to the line Weaver-
Burk plot (lineWeaver and Burk ,1934) .The reciprocal of the reaction rate (1/v) was
plotted against the reciprocal of OOMSW concentrations (1/[s]) and the linear
regression analysis of reaction by enzymes of Aspergilus.sp , Pencillium.sp and A.niger
resulted a slop Km/Vmax of 388.3 , 228.19 and 194.1 and the intercept 1/Vmax of
10.33 , 13.10 and 7.85 respectively.

68
Figure 5.11 Lineweaver –Burk plot of enzyme of Aspergilus.sp

Figure 5.12 Lineweaver –Burk plot of enzyme of pencillium.sp

69
Figure 5.13 Lineweaver –Burk plot of enzyme of A.niger

According to equations in the figures the calculated Km of produced enzymes which

were obtained from enzymatic hydrolysis via the different enzyme sources i.e.
Aspergilus.sp , Pencillium.sp and A.niger were 37.5 ,17.4 and 24.7 g/L while Vmax were
0.096 , 0.076 and 0.127 g/L/min respectively .

5.4.7 Effect of enzyme loading on reducing sugar production from OOMSW

biomass with time course

The enzyme dosage is an important parameter condition in enzymatic hydrolysis

affecting on the TRS yield. 5 different dosage of enzyme which was expresses as 5
different volume and FPU of each enzyme source i.e. " 1ml (4.63 FPU), 2ml (9.26FPU),
3ml (13.89FPU), 4ml (18.52FPU) and 5ml (23.15FPU) " of Aspergilus sp cellulase ,
" 1ml (2.0 FPU), 2ml (4.0FPU), 3ml (6.0FPU), 4ml (8.0FPU) and 5ml (10.0FPU)" of
Pencillium sp cellulase and "1ml (6.68 FPU), 2ml (13.36FPU), 3ml (20.0FPU), 4ml
(26.72FPU) and 5ml (33.4FPU) " of A.niger cellulase were investigated for hydrolysis
of OOMSW with the time course i.e. (2, 4, 8, 16, 20, and 24 h). Based on the results
obtained in previous experiments of optimum conditions in this experiment, the pH was
adjusted to 5.0 and temperature was 50⁰c and substrate concentration was 50g/L.

70
Table (5.9) shows the TRS of enzymatic hydrolysis by 3 enzyme sources.

Table (5.10) Effect of enzyme loading with incubation time on TRS of enzymatic
hydrolysis of OOMSW by three enzyme sources

TRS g/L

Incubation time (h)

Enzyme source 0 2 4 8 16 20 24
and loading

Aspergilus.sp
0 3.57 3.55 3.57 3.50 3.56 3.57 3.57
1ml(4.63 FPU) 3.57 10.52 10.62 10.94 11.89 11.80 11.80
2ml(9.26FPU) 3.57 11.47 12.94 13.47 13.50 13.48 13.48
3ml(13.89FPU) 3.57 11.94 14.88 16.22 18.71 19.68 19.71
4ml(18.52FPU) 3.57 13.51 17.26 19.05 21.15 21.16 21.15
5ml(23.15FPU) 3.57 16.84 19.31 24.11 24.95 26.00 26.03
Pencillium.sp
0 3.55 3.55 3.57 3.50 3.56 3.57 3.57
1ml(2.0 FPU) 3.55 9.47 9.89 10.31 11.68 11.70 11.68
2ml(4.0FPU) 3.55 10.20 11.99 13.58 14.00 14.52 14.61
3ml(6.0FPU) 3.55 10.62 13.04 13.80 16.52 16.84 16.85
4ml(8.0FPU) 3.55 11.68 16.52 20.84 22.84 23.26 23.30
5ml(10.0FPU) 3.55 11.70 17.50 20.83 22.31 22.17 22.15
A.niger
0 3.47 3.50 3.53 3.55 3.52 3.49 3.51
1ml(6.68 FPU) 3.47 13.68 14.52 14.63 14.63 14.64 14.63
2ml(13.36FPU) 3.47 14.10 15.79 18.95 18.95 19.37 19.39
3ml(20.01FPU) 3.47 14.11 16.42 16.84 16.84 22.52 22.57
4ml(26.72FPU) 3.47 15.47 19.73 21.53 21.53 27.63 27.60
5ml(33.42FPU) 3.47 15.58 20.74 21.52 21.52 27.31 27.57

In the figure (5.14) , (5.15) and (5.16) show the TRS yield and effect of enzyme loading
with time on enzymatic hydrolysis of OOMSW.

71
Figure 5.14 Effect of enzyme loading on TRS yield with time course by cellulase of
Aspergilus.sp , temperature =50c , PH= 5.0 , substrate concentration = 50g/L.

Figure 5.15 Effect of enzyme loading on TRS yield with time course by cellulase of
pencillium.sp , temperature =50c , PH= 5.0 , substrate concentration = 50g/L.

72
Figure 5.16 Effect of enzyme loading on TRS yield with time course by cellulase of
A.niger , temperature =50c , PH= 5.0 , substrate concentration = 50g/L.

Since the results in the table (5.10), in the general the enzymatic hydrolysis of OOMSW
by using various enzyme sources was achieved and the results indicated that the
fermentable sugars yield gradually increased with increase in enzyme loading and the
time for all the enzyme sources until the steady state rise was reached. The maximum
yield of TRS 24.13 g/L was reached at steady state of 24h by 4ml(26.72FPU) of
cellulase from A.niger. The enzymatic hydrolysis of OOMSW by crude cellulase from
Aspergilus.sp reached 22.5 g/L at the steady rise with 5mL(23.15FPU) of enzyme after
20h of incubation time. The crude cellulase from the Pencillium.sp resulted the low
amount of TRS in the hydrolysis process compared with another enzyme sources and
enzymatic hydrolysis by it was reached 19.89g/L of TRS at 24h as steady state by
4mL(8.0FPU) of enzyme loading . In the fact more active sites of enzyme would be
involved in conversion of substrate into fermentable sugars via enzymatic hydrolysis
when the enzyme loading is increased .The TRS yield decreased in some of the
hydrolysis process at the end of time that may be due to the end product inhibition in
enzymatic hydrolysis.

73
Based on the above result obtained in the study of enzymatic hydrolysis of OOMSW,
The optimum enzyme loading of the three enzyme sources for hydrolysis of OOMSW
were: 23.15 FPU of Aspergilus.sp cellulase, 8.0 FPU of Pencillium.sp cellulase and
26.72 FPU of A.niger cellulase. In table (5.11) show the result of TRS released per 1g of
substrate and the saccharification yield% of substrate which are obtained by the
optimum enzyme loading of all enzyme sources which was calculated according to (4.5)
and (4.6) equations.

Therefore any further increases in enzyme dosage in enzymatic hydrolysis of OOMSW

by the produced cellulase enzyme in this study do not result in the increase of the
hydrolysis yield.

Table 5.11 TRS yields released in g per g of substrate and the saccharification yield% of
substrate.

Enzyme sources
Aspergilus.sp Pencilium.sp A.niger
Time (h) g/g % g/g % g/g %

2 0.26 23 0.16 14 0.24 21

4 0.31 28 0.25 23 0.32 29
8 0.41 36 0.34 31 0.36 32
16 0.42 38 0.38 4 0.46 41
20 0.44 40 0.39 35 0.48 43
24 0.44 40 0.39 35 0.48 43
*g/g TRS released g per g of substrate (OOMSW)

*% Saccharification Yield %

The figure (5.14) and (5.15) show the results of TRS yield released per g substrate and
saccharification yield % of enzymatic hydrolysis of OOMSW with optimum conditions.

74
Figure 5.17 The TRS yield released in g per g of substrate by optimum loading of
different enzymes source.

Figure 5.18 Saccharification yield % of OOMSW by optimum loading of different

enzymes source.

75
Based on the results of enzymatic hydrolysis in the table (5.11) and the figures (5.17)
and (5.18) the saccharification yield % of hydrolysis process was increased with increase
in the enzyme loading and the time and the maximum hydrolysis yield of 43% was
achieved by the crude cellulase of A.niger and produced 0.48g/g substrate of sugars.
While use of crude cellulose by Aspergilus.sp for enzymatic hydrolysis of OOMSW
resulted in production of 0.44g/g substrate of TRS and hydrolysis yield of 40% was
reached. Where the crude cellulase from Pencillium.sp for hydrolysis resulted the low
level in the production of TRS 0.39g/g substrate and its hydrolysis yield was 35%.

These results when compared with previous studies show similarities in some aspects
and differences in other aspects. In general, the result of this work is in good agreement
with other studies. The result of this study is in accordance with those done by Abdi et
al, (2000) in the steady state of hydrolysis time, which was 24h incubation time. And it
is in agreement with Mameri et al (2000) in hydrolysis yield which was 45% at steady
state after 24h of batch reactor.

But the results are quite similar with other previous study in saccharification yield of
OOMSW by cellulase hydrolysis. In the study by Abdi et al (2000) studied the
enzymatic hydrolysis of OOMSW in batch reactor by cellulase from T.reesei and
reported the maximum hydrolysis yield was 50% and the TRS yield was 20g/L with
40g/L of substrate concentration. The results show also the reducing sugar yields which
obtained in this study by the different enzyme source where higher than that obtained by
study done by Maria et al (2016) which resulted 0.46g/g of solid substrates (extracted
olive pomace) at 15% (w/v) 15g /L of substrate concentration.

It is worthy to note that there are no more previous studies of Saccharification of

OOMSW that thus leads to comparison of this work with other studies which studied the
capacity of crude cellulase from fungi on the hydrolysis of some other biomass.

Nooshin et al, (2014) studied the Saccharification of the alkali treated rice straw by
crude cellulase from T.harzianum and resulted in production of 0.69g/g substrate at 72h.

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In the study by Ravi et al, (2012) studied the fermentable sugar production from Acid
treated wheat straw by T. reesei and reported the maximum TRS yield of hydrolysis was
0.205 g/g substrate.

The study done by Rajeev et al (2009) studied the production of cellulase by T. reesei
and its application in the lignocelluloses saccharification and resulted the maximum TRS
yield from rice straw and sugar cane bagasse were 26.3 g/L and 17.79g/L respectively
with 20g/L as substrate concentration.

In table (5.12) the result of cellulose conversion % of enzymatic hydrolysis of alkali

treated OOMSW by the produced crude cellulase enzymes at optimum experimental
condition were shown.

Table 5.12 Cellulose conversion % of enzymatic hydrolysis of alkali treated OOMSW

Cellulose conversion %
Time of incubation(h)
Enzyme sources 2 4 8 16 20 24

(23.15FPU)Aspergilus.sp 56 67 88 91 96 96
(8.0FPU)Pencillium.sp 34 55 74 82 84 84
(26.72FPU)A.niger 31 65 77 98 103 103

Since the results in the table (5.12) indicate the cellulose conversion percentage
increased with rise in the incubation time by all used enzyme sources . the enzyme of
A.niger resulted the highest conversion percentage of cellulose content in OOMSW
biomass during the hydrolysis processes which corresponds to 103%. The result
indicates that most of the cellulose content in the biomass was converted and it may be
noted that the conversion % of biomass have rather higher values than 100%. This might
be due to the possibility of other enzymes like Hemicellulases being found with the

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produce cellulase and this would have resulted in the observed higher conversion yield
due to the hydrolysis of hemicelluloses along with cellulase.

The enzyme of Aspergilus.sp resulted 96% of conversion yield and the enzyme of
Pencillium.sp resulted low hydrolysis of cellulose content which was 84%. However the
cost of cellulase enzyme contributes to the total cost of fermentable sugar production
processes. Hence it is suggested to minimize enzyme loading as much as possible.

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 CHAPTER 6

6.1 CONCLUSION

Cellulase is an enzyme that has cellulolytic capacity and produce mono sugars. The
crude cellulase enzymes consist of 3 main enzymes: endoglucanase (CM case),
exoglucanase (cellobiohydrolase) and β-glucosidase which act synergistically in the
hydrolysis process of cellulose material. The investigation of soil fungi to find their
ability for cellulase production and activity is necessary for industrial enzymes which
can be done by many filamentous fungi. In the current study, three different fungal
species for production of cellulase were selected and isolated from soil and it were
identified and the production of crude cellulase by them in SSF was done successfully
and the results observed the ability of isolated fungal genus to produce the enzymes and
have great potential to be used as fungal strains for industrial cellulase production.

The results of this study observed that the maximum cellulase production was achieved
by Aspegilus.sp , Aspergilus.niger and Pencillium.sp at optimal fermentation time .

Cost effective technologies are needed for enzyme production, the results of the study
indicate that the use of SSF technology show good cellulase production by isolated fungi
and it was possible to obtain large scale of crude cellulase complex from the isolated
strains using wheat bran as a very low cost substrate under SSF, with the view of
developing a low cost production system.

The study is novel because the substrate had no nutrient supplement in the fermentation
solid medium which consists of wheat bran simply moistened with phosphate buffer.

The test of the produced cellulase in this study for its ability in the bioconversion of
OOMSW and the production of fermentable sugars was investigated.

The Saccharification of OOMSW will not only be for production of fermentable sugars,
but also to decrease the pollution-load generated from OOMSW.

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The enzymatic hydrolysis of OOMSW by different enzyme sources was done
successfully and the optimum enzymatic conditions were determined.

These results of study show that in order to obtain maximum fermentable sugar yields
from OOMSW biomass, two steps in the hydrolysis process of OOMSW biomass is
required, pretreatment of biomass with 1.25% NaOH and enzymatic process . So the
produced crude cellulase enzymes can be used for bioconversion and saccharification of
biomass for achieving high fermentable sugars.

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