Optimization of Fermentation Conditions for Citric Acid Production by Aspergillusniger in ...

126 Pages·2012·5.88 MB·English

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Optimization of Fermentation Conditions for Citric Acid Production by
Aspergillusniger in Submerged Culture

By
ShazaGamal Mohammed Ali
B.Sc. (Hons.) Botany, University of Khartoum, 1998
M.Sc. Botany, University of Khartoum, 2004

A Thesis Submitted to the University of Khartoum for the Award of the degree of
Ph.D. in Botany

Supervisor: Dr. Adil Ali El-Hussein

Co-Supervisor: Dr. Mohammad Amin Siddig

Department of Botany,
Faculty of Science,
University of Khartoum

Jan
2012
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Dedication

To my kids with love

Shaza

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Acknowledgements

Most of all thank “Almighty Allah” with countless thanks and gratitude
for giving me this great opportunity to complete this work.
I would like to express my sincere gratitude to my unique, splendid and
outstanding supervisor, Dr. Adil Ali El-Hussein, for his valuable
guidance, support, encouragement. My thanks are also due to my co-
supervisor, Dr. Mohammed Amin Siddig for his advice.
Words are short to express my appreciation to Dr. Elsayed Elsayed Hafez
and Prof. Osama Gabra, at Mubarak City for Scientific Research and

Technology, for their hospitality, valuable guidance and advice.
Thanks and gratitude are also due to all staff members of Mubarak City
for Scientific Research and Technology for their technical support during
this work.
I wish to thank my colleagues at the Botany Department, University of
Khartoum for their moral support. Special thanks are due Dr. Manal
Elsayed and Dr. Marmar for their ceaseless help, support and
encouragement.
Iam also grateful to my treasured family and friends who were a sure
source of strength and encouragement throughout this work, especially
mother, Rawhia, Nuha and Thabat.
My thanks are extended to Ustaz Badr Eldin Abd Al.Gader, for his help
in the statistical analysis of the results throughout this work.
My thanks and appreciations are extended to my real friend Yasir Sharef.
Last, but not least, I wish to express my sincere gratitude to my beloved
husband Haidar who supported me during this work.
This work would have been impossible without the sponsoring of Third
World Organization for Women in Science (TWOWS).
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Abstract

The objective of this study was to optimize citric acid production by
Aspergillus niger in submerged culture. During this study, six
presumptive isolates of A. niger were recovered from different sources,
and were characterized by microscopic and cultural characteristics on
different growth media. Results of characterization indicated that the
presumptive isolates colonies were powdery in texture, with a black
surface and pale yellow underneath. Microscopic examination showed
that each presumptive isolate consisted of a substrate white mycelia from
which vertical hyphae grow and carry at its tip globose heads with
smooth hyaline stipes, spherical vesicles, packed metulae and short
phialides that carry globose to elliptical conidia. Idtinification and
characterization was confirmed by sequencing of the 18s ribosomal RNA
gene. Partial sequencing of 18s rRNA of each isolate was aligned to the
reference isolate ATCC 9763. Phylogenetic analysis showed that the
highest homology (99 %) was obtained by isolates KU1 and KU8, low
identity percentage (93%) was observed for isolate KU3. RAPD-PCR
divided the isolates into three groups. The band patterns obtained were
consistent and reproducible, their numbers varied between 4 to 14 and the
size of the fragments was in the range of 1000 – 1500 bp.The isolates
were screened for citric acid production using Czapek-Dox medium
under submerged conditions. The results indicated that all isolates were
able, although to variable efficiencies, to convert sucrose to citric acid.
Based on the results of this experiment, only one isolate (KU 2) was
selected for further studies. Optimization of media constituents and other
fermentation conditions were studied in submerged culture using Czapek-
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Dox medium. Results of optimization experiments indicated that
optimum citric acid concentration of (g/l) 26.7, 17.8, 23.7, 29.1, 28.9
were obtained at 10 % sucrose, pH 5, 30°C, 0.3% Na NO and 4%
3
ethanol, respectively. Optimum medium volume and optimum inoculum
level were 25 ml in 250 ml vessel and 2.5 %, respectively. Maximum
citric acid concentration of 51.3g/l was obtained when fermentation
period was kept for 144h. El-Genaid cane molasses was tested as a
substrate for citric acid fermentation. H SO treated molasses
2 4
supplemented with 4 % ethanol gave 56 g/l citric acid in shake flask.
Scale up of citric acid production was conducted in 5 L stirred fermentor
and Czapek Dox medium was used as a fermentation substrate
supplemented with 100g/l sucrose. Maximum amount of citric acid
(50.01 g/l) was produced after144hrs and dry mycelium weight was 26.86
g/l while sugar consumed was 61.52 g/l with 81.65 % yield.
Results reported validates that the process of citric acid production by
fermentation can be explored for commercialization.








IV
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κϠΨΘδϣ

ήτϓΔτγ΍ϮΑϚϳήΘδϟ΍ξϣΎΣΝΎΘϧϻϰϠΜϤϟ΍ήϴϤΨΘϟ΍ϑϭήχΪϳΪΤΗϭςΒπϟΔγ΍έΪϟ΍ΖϓΪϫ
ΓέϮϤϐϤϟ΍ΔϋέΰϤϟ΍ήϴϤΨΗΔϴϨϘΗϡ΍ΪΨΘγΎΑAspergillus nigerΩϮγϷ΍ϦϔόΘϟ΍
Δγ΍έΩΖϤΗϭΔϔϠΘΨϣέΩΎμϣϦϣΩϮγϷ΍ϦϔόΘϟ΍ήτϓϦϣΕϻΰϋΔΘγϝΰϋΔγ΍έΪϟ΍ϩάϫϝϼΧϢΗ
Ξ΋ΎΘϨϟ΍ΕήϬχ΃ΚϴΣΔϳήϬΠϤϟ΍ι΍ϮΨϟ΍ϭΔϔϠΘΨϣΔϴ΋΍άϏρΎγϭ΃ϡ΍ΪΨΘγΎΑΔϴϋέΰϤϟ΍ι΍ϮΨϟ΍
Ξ΋ΎΘϧΎϣ΃ϰ΋΍άϐϟ΍ςγϮϠϟϲϠϔδϟ΍ΐϧΎΠϟ΍ϰϓήϔλ΃ϭϱϮϠόϟ΍΢τδϟ΍ϰϠϋΩϮγ΃ήτϔϟ΍ϥϮϟϥ΃
ϭΔϔΣ΍ίΕΎϔϳΎϫϭ˯ΎπϴΑΎϴϠδϴϣϰϠϋϯϮΘΤΗΕϻΰόϟ΍ϥ΃ΖΤοϭ΃ΪϘϓΔϳήϬΠϤϟ΍ι΍ϮΨϟ΍
ΩϮγ΃ϥϮϟΕ΍ΫϭΓήϴΒϛήτϔϠϟΔϳΪϴϧϮϜϟ΍α΅ϭήϟ΍ϥ΃ΎϤϛˬϯϭήϛα΃έΎϬϴϠϋϝϮϤΤϣΔϤ΋ΎϗϯήΧ΃
ϥϮϜΗϭΔϠλϮΤϟ΍΢τγϰϠϋΔϳΩϮϤϋΔΟήϔϨϣϞγϼγϰϟ·ϝϮΤΘΗΎϬϨϳϮϜΗΔϳ΍ΪΑϰϓΔϳϭήϛϭΪΒΗ
ΪϳΪΤΗΔτγ΍ϮΑΔϳΪϴϛ΄Θϟ΍Ε΍έΎΒΘΧϹ΍Ξ΋ΎΘϧΖΤοϭ΃ϭΔϳέϭέΎϘϟ΍ΐϴϛ΍ήΘϟ΍ϰϠϋΔϟϮϤΤϣΕ΍ΪϴϧϮϜϟ΍
A. niger ήτϓϦϋΓέΎΒϋΕϻΰόϟ΍ϩάϫϊϴϤΟϥ΃s rRNAϦϴΠϟϱΪϴΗϮϴϠϛϮϴϨϟ΍ϞδϠδΘϟ΍
ϱϭϮϨϟ΍ξϤΤϠϟϝΎϜηϷ΍ΩΪόΘϤϟ΍ϰ΋΍Ϯθόϟ΍έΎΜϛϹ΍ΔϘϳήσϡ΍ΪΨΘγΎΑΕϻΰόϟ΍ϩάϫΖμΨηϚϟάϛ
Czapek-Dox ϰ΋΍άϐϟ΍ςγϮϟ΍ϰϠϋϚϳήΘδϟ΍ξϤΣΝΎΘϧ·ϰϠϋΕϻΰόϟ΍ϩάϫΓέΪϘϣέΎΒΘΧ΍ϢΗ
ΝΎΘϧ·ϰϠϋΓέΪϘϤϟ΍ΎϬϟΕϻΰόϟ΍ϊϴϤΟϥ΃Ξ΋ΎΘϨϟ΍ΖΤοϭ΃ΓέϮϤϐϤϟ΍ΔϋέΰϤϟ΍ήϴϤΨΗΔϘϳήτΑ
(5.9g/l) ϚϳήΘδϟ΍ξϣΎΣϦϣΰϴϛήΗϰϠϋ΃Γ˯ΎϔϜϟ΍ΔΟέΩϰϓϑϼΘΧϻ΍ϊϣϚϳήΘδϟ΍ξϣΎΣ
ΎϬϋΎπΧϹΔϟΰόϟ΍ϩάϫέΎϴΘΧ΍ϢΗΔΑήΠΘϟ΍ϩάϫΞ΋ΎΘϧϰϠϋ΍ΩΎϤΘϋ·ˬKU2Δϟΰόϟ΍Δτγ΍ϮΑΞΘϧ΃
ϑϭήχϰϠϋΓήΛΆϤϟ΍Ϟϣ΍Ϯόϟ΍Δγ΍έΩϢΗϚϳήΘδϟ΍ξϤΣΝΎΘϧ·ΓΩΎϳΰϟΕ΍έΎΒΘΧϹ΍ϦϣΔϠδϠδϟ
ϢΠΣˬϰ΋΍άϐϟ΍ςγϮϟ΍ϢΠΣˬΓέ΍ήΤϟ΍ΔΟέΩˬϰπϤΤϟ΍Ϣϗήϟ΍ˬϰ΋΍άϐϟ΍ςγϮϟ΍ΕΎϧϮϜϣϞΜϣήϴϤΨΘϟ΍
Ξ΋ΎΘϧΖΤοϭ΃ϭϚϳήΘδϟ΍ξϤΣϦϣΰϴϛήΗϰϠϋ΃ϰϠϋϝϮμΤϠϟήϴϤΨΘϟ΍ΓΪϣϭϯήτϔϟ΍ϖϠόϤϟ΍
αϷ΍ˬϝϢΟ26.7ϥϮΑήϜϠϟέΪμϤϛίϭήϜγϰϫήϴϤΨΘϟ΍ϑϭήχϞΜϣ΃ϥ΃Δγ΍έΪϟ΍ϩάϫ
ϡϮϳΩϮμϟ΍Ε΍ήΘϧˬϝϢΟ23.7ΔϳϮΌϣΔΟέΩˬϝϢΟ17.8ϲϨϴΟϭέΪϴϬϟ΍
21.8ϕέϭΪϟ΍ϢΠΣϦϣˬϝϢΟ28.9ϝϮϧΎΛ·ˬϝϢΟ29.1ϦϴΟϭήΘϴϨϠϟέΪμϤϛ
ΔϋΎγήϴϤΨΗΓήΘϓϭϝϢΟ23.3ϯήτϔϟ΍ϖϠόϤϟ΍ϢΠΣϦϣˬϝϢΟ
ϝϢΟ
ϦϣϝϢΟϰϠτϋ΍ϭϥϮΑήϜϠϟκϴΧέέΪμϤϛϚϴΘϳήΒϜϟ΍ξϤΤΑΞϟΎόϤϟ΍αϻϮϤϟ΍ϡΪΨΘγ΍
ΔϤϴϗϰϠϋ΍ΖϧΎϜϓϚϳήΘδϟ΍ξϤΣΝΎΘϧ΍ΓΩΎϳΰϟήΘϟΔόγήϴϤΨΗίΎϬΟϡΪΨΘγ΍ϚϳήΘδϟ΍ξϤΣ
ϝϢΟ

V
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List of Plates
Plate 1 (a-b): Microscopical and cultural characteristics of A. niger isolate

Plate 2 (a-f): Banding patterns shown by different isolates using different primers
Plate 3 (a-b): Pellets form of A. niger isolates under submerged culture
Plate (4): Pellets form of KU 2 isolate under submerged culture in stirred
fermentor.


VI
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List of Tables
Table (1): A . niger presumptive isolates and their source of isolation
Table (2a): Microscopical characteristics of A. niger isolates

Table (2b): Cultural characteristics of the A. niger isolates on different growth
media

Table (3): Sequences identity percentages of A. niger isolates
Table (4a): Degree of polymorphism between A. niger isolates using RAPD
primers

Table (4b):
Data matrix for association coefficients between each pair of
operating taxonomic units of A. niger isolates

: Screening for citric acid production by A. niger isolates in Czapek-
Table (5 )
Dox medium.

Table (6): Effect of different sugars on citric acid productivity by KU2.
Table (7): Effect of different sucrose concentrations on citric acid productivity
by KU 2.

Table (8): Effect of different nitrogen sources on citric acid productivity.
Table (9): Effect of different Na NO concentrations on citric acid production.
3

Table (10a): Effect of different stimulators on citric acid production.
Table (10b): Effect of stimulators on citric acid production kinetic parameters.

Table (11): Effect of different medium volumes on citric acid production.
Table(12): Effect of different inoculum volumes on citric acid production.
Table (13): Effect of different fermentation periods on citric acid production
Table (14): Effect of treated and untreated molasses on citric acid production.
.
Table (15a): Citric acid production by A. niger KU2 using Czapek dox medium
in stirred fermentor.
Table (15b): Citric acid production Kinetic parameters in stirred fermentor.

VII
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List of Figures
Fig. (1): Sequences of the 18s rRNA gene from different isolates.
Fig. (2): Dendrogram using Average Linkage (Between Groups)
Rescaled Distance Cluster Combine.

Fig. (3): Effect of different initial pH values on citric acid production, sugar
consumption, dry mass production and yield.
Fig. (4): Effect of different incubation temperature on citric acid production, consumed
sugar, dry cell mass, and yield.
Fig. (5a): : Effect of different incubation periods on citric acid formation (volumetric
rates) parameters.
Fig. (5b): Effect of different incubation periods on citric acid formation (yield
coefficient) parameters.
VIII
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Contents
Page

Dedication I
Acknowledgements II
Abstract III
Arabic Abstract V
List of Plates VI
List of Tables VII
List of Figures VIII
CONTENTS
1. CHAPTER ONE: INTRODUCTION & 1
LITERATURE REVIEW
1.1. General Introduction 1
1.2. Literature Review 5
1.2.1. Identification and Taxonomy of A. niger 5
1.2.2. Biochemistry of citric acid synthesis 7
1.2.3. Citric acid production techniques 8
1.2.3.1. Surface fermentation 8
1.2.3.2. Submerged fermentation 8
1.2.3.3. Solid state fermentation 10
1.2.4. Optimization of citric acid condition parameters 11
1.2.4.1. Initial pH 11
1.2.4.2. Fermentation temperature 13
1.2.4.3. Medium composition 14
1.2.4.3.1. Carbon source 14
1.2.4.3.2. Nitrogen sources 16
1.2.4.3.3. Phosphate 16
1.2.4.3.4. Micro-elements 17
1.2.4.3.5. Stimulating agents 18
1.2.4.4. Volume of culture media 19
1.2.4.5. Inoculum density 20
IX