Masters Thesis

University Code: 10384
Student ID No.: 20420081153696

Optimization of medium components for natamycin production by Streptomyces Gilvosporeus
Na me : M AN I RAF A SH A E m ma n ue l
MASTERS DEGREE THESIS

Optimization of medium components for natamycin
production by Streptomyces Gilvosporeus
By
MANIRAFASHA Emmanuel
RWANDA
S up erv iso r: P ro fe sso r Lu Y ing h ua
Supervisor: Professor Lu Yinghua

Major: Chemical Engineering
Date of Graduation: July, 2010
A THESIS SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS FOR THE

AWARD OF THE DEGREE OF MASTER OF ENGINEERING
Xia me n Un iv e rs ity
DEPARTMENT OF CHEMICAL AND BIOCHEMICAL ENGINEERING
COLLEGE OF CHEMISTRY AND CHEMICAL ENGINEERING
XIAMEN UNIVERSITY
CERTIFICATION
I, Professor Lu Yinghua, hereby certify that I have read this manuscript and recommend for
acceptance by the Xiamen University a dissertation entitled Optimization of medium
components for natamycin production by Streptomyces Gilvosporeus in fulfillment of degree
of Master of Engineering at Xiamen University, Peoples Republic of China.
Signed
Supervisor
Date
Department of Chemical and Biochemical Engineering
College of Chemistry and Chemical Engineering
Xiamen University
Xiamen, Fujian Province
P.R. China
ORIGINAL STATEMENT
The research described in this thesis Masters of Engineering was conducted under the
supervision of Professor Lu Yinghua at the Department of Chemical and Biochemical
Engineering, Xiamen University. I hereby declare that the work submitted is my own and that
appropriate credit has been given where reference has been made to the work of others. I also
confirm that it has not been previously or concurrently submitted for any other degree, diploma
or any other qualifications at Xiamen University, P.R China or other institutions.
COPYRIGHT DECLARATION
All rights reserved. No part of this dissertation may be reproduced, stored in any retrieval system,
or transmitted in any form by any means: electronic, mechanical, photographing, recording or
otherwise without prior written permission of the author or Xiamen University.
MANIRAFASHA Emmanuel
Date
Acknowledgements
ACKNOWLEDGMENTS
It gives me great pleasure to acknowledge the many people who have assisted me throughout this
work. I would like to thank my supervisors Professor Lu Yinghua who has accepted, without
beating around the bush, to assist and guide me over the course of this project.
Im extremely grateful to the Government of Rwanda for its great support for my undergraduate
level where I gained the background which helps me to accomplish this masters level; I also
wish to extend my thanks to the Government of Peoples Republic of China for giving me the
masters program scholarship.
I would like to thank all my country mates who are in Xiamen University, classmates and lab
mates especially Mr. Zeng and Miss Wang Baobei (). I am very grateful for
meeting you, for warm environment and for our relationship. Your encouragement and
understanding is endless and I look forward to sharing many more accomplishments with you in
the future.
Finally, I want to thank my entire family especial BASHONGA family and my girl friend for her
affection, prayer and help. Without your help and patience, this would not have been possible. I
feel extraordinarily blessed to have such a network of wonderful people in my life. Thank you all
for believing in me and helping me reach my goal.
i
Abstract
ABSTRACT
Natamycin, also known as pimaricin, is a member of the class of antibiotics known as polyene
macrolide, which are toxic to yeast and fungi but not to bacteria. Natamycin is a naturally
occurring antifungal agent produced by submerged fermentation with some of the actinomycete
bacterium Streptomyces strains such as Streptomyces natalensis, Streptomyces chattanoogensis
and Streptomyces Gilvosporeus. It is mostly separated from the fermentation broth by extraction.
It is a very attractive agent. The importance of this antibiotic is due to the fact that Natamycin
broad-spectrum activity against yeasts and molds, with low toxicity against mammalian cells.
The Natamycin is one, among the few antibiotics being advised by FDA (Food and Drug
Administration) as a food additive and classified as a GRAS (Generally Regarded As Safe)
compound, it has been widely used as a natural preservative to prevent the mold contamination
in food industry. Beside its application in food industry, the Natamycin is also used to treat
fungal keratitis because it is especially affective against Aspergillus and Fusarium corneal
infections.
Due to the important commercial value of Natamycin, it is necessary to improve its productivity.
During this study, we tried to optimize the fermentation culture medium for Natamycin
production as one factor among factors which are influenced the yield of natamycin. The effects
different carbon sources (starch soluble and glucose) and nitrogen sources (soybean powder,
yeast extract and peptone) concentrations in the culture medium were investigated during this
study in order to design the new optimal culture medium for the Natamycin production by
Streptomyces Gilvosporeus ATCC13326.
The development of new optimal culture medium in this study, however, was optimized by one
at a time strategy: varying one culture medium component while keeping all others constant. The
culture conditions were the follows: seed age 38-44hours, temperature 28 , inoculum level
10%, and precursor: 0.6% sodium propionate fed at 24 hours of the culture and the total culture
time of 5 days.
ii
Abstract
By examining the effect of different starch soluble concentrations, the results showed that the
highest natamycin production was obtained in a cultivation medium containing 100 g/L starch
soluble and the maximal Natamycin production at that point was 5.85 g/L while in the controller
was 2.2 g/L (15 days spores were used to inoculate the seed medium at this time). We have
found that the fermentation is strongly controlled by carbon and nitrogen sources but strongly by
starch soluble.
When the all optimal factors were combined together in order to design the new optimal
natamycin production medium, the natamycin yield was 4.3 g/L which is less than the value
obtained by evaluating the effect of starch soluble. We decided to change starch soluble
concentration and maintain the other medium component at the same value as in original
medium unless the CaCO3 which was reduced from 15 g/L to 7.5 g/L. This optimization strategy
let to a natamycin yield of 5.85 g/L in shake flask, which was nearly 134% higher than that
obtained in the original medium (2.5 g/L).
Keywords: Natamycin, fermentation, Streptomyces Gilvosporeus, production medium,

optimization.
iii
Abstract

ATCC13326.
38-44 h28 10%24 h0.6%

120 h100 g/L
5.85 g/L2.2 g/L
4.3 g/L
7.5 g/L
5.85 g/L,
2.5 g/L2.34
iv
Table of contents
TABLE OF CONTENTS
ACKNOWLEDGMENTS ............................................................................................................... i
ABSTRACT .................................................................................................................................... ii
ABSTRACT (in Chinese) .............................................................................................................. iv
TABLE OF CONTENTS ................................................................................................................ v
LIST OF FIGURES ..................................................................................................................... viii
LIST OF TABLES .......................................................................................................................... x
CHAPTER 1: INTRODUCTION ................................................................................................... 1
1.1. What is Natamycin? ......................................................................................................... 1
1.1.1. Discovery and Origin..................................................................................................... 1
1.1.2. Physical and Chemical Properties ................................................................................. 2
1.2. Motivation and objectives ................................................................................................ 4
1.3. Overview of the thesis .......................................................................................................... 5
CHAPTER 2: LITERATURE REVIEW ........................................................................................ 6
2.1. Microorganism Culture Methods ......................................................................................... 6
2.1.1. Modes of culture................................................................................................................ 6
2.2. Fermentation Technology .................................................................................................... 9
2.2.1. Microbial Growth in Batch Fermentation ..................................................................... 9
2.2.2. Methods to determine the growth curve ...................................................................... 14
2.3. Fermentation media ............................................................................................................ 18
2.3.1. General review ............................................................................................................. 18
2.3.2. Medium components ................................................................................................... 18
2.3.3. Complex medium ........................................................................................................ 20
v
Table of contents
2.3.4. Defined medium .......................................................................................................... 22
2.3.5. Fermentation medium formulation .............................................................................. 23
2.4. Criteria for design and optimization of fermentation process ............................................ 24
2.4.1. The role of precursor during the fermentation............................................................. 25
2.5. Natamycin inoculum propagation and fermentation .......................................................... 25
2.6. Introduction to separation process of natamycin................................................................ 26
CHAPTER 3: MATERIAL AND METHODS............................................................................. 27
3.1. Materials, Equipments and Media.................................................................................. 27
3.1.1. Microorganisms ...................................................................................................... 27
3.1.2. Media and culture conditions .................................................................................. 27
3.1.2. Equipments ............................................................................................................. 30
3.2. Strategy to optimize the natamycin production medium ............................................... 30
3.2.1. Carbon sources ........................................................................................................ 31
3.2.2. Nitrogen sources ..................................................................................................... 31
3.3. Analytical method .......................................................................................................... 32
3.3.1. Measurement of cell growth ................................................................................... 32
3.3.2. Determination of Natamycin concentration ............................................................ 32
3.3.3. Chemical reagents and solutions ................................................................................ 35
3.4. Natamycin extraction ......................................................................................................... 36
CHAPTER 4: RESULTS AND DISCUSSION ............................................................................ 37
4.1. Fermentation in shake flask................................................................................................ 37
4.1.1. Effect of initial glucose concentration, in natamycin production medium, on

natamycin production by Streptomyces Gilvosporeus................................................... 37
4.1.2. Effect of different starch soluble concentrations on cell growth and natamycin
production by Streptomyces Gilvosporeus ............................................................................ 38
vi
Table of contents
4.1.3. Effect of soybean powder concentration on cell growth and Natamycin production by
Streptomyces Gilvosporeus.................................................................................................... 40
4.1.4. Effect of yeast extract on the cell growth and Natamycin production by Streptomycin
Gilvosporeus .......................................................................................................................... 41
4.1.5. Effect of peptone on the cell growth and Natamycin production by streptomycin
Gilvosporeus .......................................................................................................................... 42
4.2. Fermentation in the bioreactor ........................................................................................... 43
4.3. Natamycin extraction results .............................................................................................. 45
4.3.1. Natamycin extraction with different dilutions ............................................................. 45
4.3.2. Effect of pH on natamycin extraction.......................................................................... 46
CHAPTER 5: CONCLUSION AND RECOMMENDATIONS .................................................. 47
5.1. Conclusion.......................................................................................................................... 47
5.2. Recommendation ................................................................................................................ 48
REFERENCES ............................................................................................................................. 49
Appendices .................................................................................................................................... 54
vii
List of figures
LIST OF FIGURES
Figure1. 1. The structure of Natamycin [6] ...................................................................................... 2
Figure1. 2. The structure of Natamycin in 3 dimensions................................................................. 2
Figure2. 1 Microbial Growth in Batch Fermentation [23] ............................................................ 10
Figure2. 2Figure 2.1.b: Typical growth phases for micro-organisms in submerged culture [21]
.................................................................................................................................................... 10
Figure2. 3. The cell growth which is substrate limited [9]............................................................. 12
Figure2. 4. Spectrophotometer pictured above .............................................................................. 14
Figure2. 5. Block flow diagram of biotechnology fermentation process ...................................... 15
Figure2. 6. Shake flask...................................................................................................................... 15
Figure2. 7. The shake flasks in a rotary shaker ............................................................................. 16
Figure2. 8. A schematic of a fermentor [28] ................................................................................... 17
Figure3. 1. the morphology of Streptomyces Gilvosporeus at 38h cultivated in liquid medium at

28, Picture taken by author. ................................................................................................. 27
Figure3. 2.The pattern of Streptomyces Gilvosporeus on the slant medium, picture taken by
author ......................................................................................................................................... 28
Figure3. 3. Natamycin concentration standard curve ................................................................... 33
Figure3. 4. Chromatography chart of natamycin .......................................................................... 34
Figure4. 1. Effect of initial glucose concentrations, in natamycin production medium, on

natamycin production by Streptomyces Gilvosporeus .......................................................... 38
Figure4. 2. Effect of different starch soluble concentrations on cell growth and natamycin
production by Streptomyces Gilvosporeus ............................................................................... 40
Figure4. 3. Effect of different soybean powder concentrations on cell growth and natamycin
production by Streptomyces Gilvosporeus ............................................................................... 41
viii
List of figures
Figure4. 4. Effect of different yeast extract on cell growth and Natamycin production by
Streptomyces Gilvosporeus ........................................................................................................ 42
Figure4. 5. Effect of different peptone concentrations on cell growth and Natamycin

production by Streptomyces Gilvosporeus ............................................................................. 43
Figure4. 6. Curves of natamycin fermentation in 3.6 L bioreactor .............................................. 44
Figure4. 7. Natamycin extraction with different dilutions ............................................................ 45
Figure4. 8. Effect of different pH on natamycin extraction .......................................................... 46
ix
List of tables
LIST OF TABLES
Table2. 1.Complex fermentation media often applied in the fermentation industry[30] ........... 20
Table2. 2. Elemental Composition of Bacteria[34, 45] .................................................................. 23
Table3. 1. Equipments names and manufacturers ......................................................................... 30
Table3. 2. The chemical reagents ..................................................................................................... 35
Table4. 1. The effect of different starch soluble concentration on cell growth and Natamycin
production by Streptomyces Gilvosporeus ............................................................................. 39
Table4. 2. Data from fermentation in the 3.6 L bioreactor using the original natamycin
production medium ................................................................................................................... 44
x
Chapter 1: Introduction
CHAPTER 1: INTRODUCTION
1.1. What is Natamycin?

Natamycin, also known as pimaricin, is a member of the class of antibiotics known as polyene
macrolide, produced by submerged fermentation with some of the actinomycete bacterium
Streptomyces such as Streptomyces Natalensis, Streptomyces Chattanoogensis and Streptomyces
Gilvosporeus [1-3].
1.1.1. Discovery and Origin

The origin of Natamycin started from 1955s when the first microorganism used to produce
natamycin was taken from a soil sample near the town of Pietermaritzburg in Natal, a province
of South Africa by Dutch scientists; that microorganism was named Streptomyces natalensis. A
new, highly active antibiotic was isolated from culture filtrates of Streptomyces natalensis and
was named pimaricin. Gist-brocades B.V. (Delft, The Netherlands) filed two patents on
pimaricin: the patent application, Pimaricin and Process of Producing the Same, on March 13,
1956 in the Netherlands and the U.S. patent application for pimaricin on March 7, 1957[4, 5].
The antibiotic tennecetin was isolated from a soil sample in Chattanooga, TN from a
Streptomyces strain in 1959. The strain was named Streptomyces chattanoogensis. Around the
same time, another antibiotic, a 5283, was isolated from Streptomyces gilvosporeus. Tennecetin
and A 5283 were proved to have the identical chemical structure of pimaricin. The production of
pimaricin from Streptomyces gilvosporeus was applied for patent in the U.S. by the American
Cyanamid Company on July 23, 1956.
In the late 1960's the World Health Organization (WHO) established a regulation specifying that
antibiotics produced from Streptomyces must carry names ending in ...mycin. Therefore, the
antibiotic pimaricin changed its name to natamycin. A literature review shows that natamycin
and pimaricin are interchangeably used to describe this antibiotic.
The food industry and most of the British Commonwealth use the name natamycin to describe
this substance, while most of the rest of the world and medical community use the old name,
pimaricin, more prevalently[4].
1
1.1.2. Physical and Chemical Properties

Natamycin has the empirical formula C33H47NO13 and a molecular weight of 665.72518 g/mol
(figure1.1 and figure1.2). It has a crystalline form and belongs to the group of polyene macrolide
antifungals, or more specifically the tetraenes [6],[7].
H O OH
O OH
H3C O H OH O O
HO
O O CH3
HO OH
NH2
Figure1. 1. The structure of Natamycin [6]
Figure1. 2. The structure of Natamycin in 3 dimensions
The molecule is amphoteric with one acid and one basic group. Natamycin is poorly soluble in
water (30 100 ppm at room temperature) and almost insoluble in non-polar solvents. However,
it shows good solubility in strongly polar organic solvents, e.g. glycerol (15000 ppm).
The poor solubility in water is, most of the time, not a problem because of the relatively low
concentrations required for Natamycin to be effective. On the contrary, the low solubility can be
an advantage because the preservative remains effective on the surface of the food for longer
2
periods. When first applied, only 30 50 ppm will be present on the surface, the remainder will
be present in the more stable crystal formation[2]. Natamycin is most effective at pH-values
between 5 and 7. Below pH 4, 5 and above pH 9 its effectiveness may drop by as much as 30%.
Aqueous solutions/suspensions of Natamycin at neutral pH remains stable for 24 hours at 50
but longer periods of exposure to this temperature will cause a reduction in effectiveness due to
hydrolysis of the ring structure [7]. The products perform well between the pH range of 5 and 7
with only little reduction in activity, the suggested pH value is 4-7. They remains stable at
ambient temperature and are unaffected by short periods of exposure to temperatures as high as
100 but must be protected from exposure to direct sunlight. Natamycin is extremely stable
under dry condition. However, the stability can be greatly affected by PH value, temperature,
daylight, oxidant and heavy metal content. Natamycin has sensitive to oxidant and ultraviolet
radiation and melting point of 180 (decomposed) [2, 8].
Exposure to high temperatures 100 for shorter periods shows little reduction in activity.
Exposure to ultraviolet light for longer periods reduce the activity of Natamycin and contact with
oxidizing agents and heavy metals should also be avoided. Heavy metals reduce the stability of
dilute solutions and chemical oxidation leads to reduced effectiveness of the antimycotic.
Therefore, exposure to direct sunlight should be avoided and glass, plastic or stainless steel
containers should be used. Natamycin in solution has an ultraviolet absorption spectrum with
minima at 250, 295.5, and 311 nm, and maxima at 220, 290, 303, and 318 nm [2].
Natamycin is effective against moulds and yeasts but not against bacteria, viruses or other
microorganisms such as protozoa. This is because natamycin acts by combining with ergosterol
and other sterols, e.g. 24- and 28-dehydroergosterol and cholesterol, which are present in the cell
membranes of moulds and yeasts but not in bacteria, with few exceptions. The binding of
Natamycin to the sterols disrupts the cell membrane leading to increased permeability and
leakage of essential cellular material. This in turn leads to a rapid drop in intracellular pH and
possibly cell lysis. Natamycin also inhibits the glycolysis and respiration [2, 9]
Natamycin is white to yellow crystalline powder. Natamycin has a little or no odor or taste.
Because of these properties natamycin will no influence on the taste and the appearance when
applied on food or in drinks [10].
3
1.2. Motivation and objectives

Natamycin is predominantly a strong, broad-spectrum antifungal agent. It is a very attractive
agent; the importance of this antibiotic is due to the fact that Natamycin broad-spectrum activity
against yeasts and molds, with low toxicity against mammalian cells. Therefore, its production
attracts researchers. There are some researches which have already carried out on natamycin
production and its separation. Few reports were found to study the optimization of fermentation
culture medium to enhance the yield of natamycin, such as Natamycin production by
Streptomyces Gilvosporeus base on statistical optimization (the Natamycin yield was 2.45 g/L);
optimization of the culture medium for Natamycin production by Streptomyces natalensis (the
Natamycin yield was 1.5 g/L); Optimization of Natamycin fermentation culture medium and
conditions using Plackett-Burman design combined with the path of steepest ascent method and
responses surface methodol (the natamycin yield was 2.19 g/L); Optimizing fermentation
medium of high Natamycin producing strain (the Natamycin yield was 2.12 g/L); Optimization
of fermentation medium compositions and conditions of fusant Sr-1 of Streptomyces natalensis
and Streptomyces chattanovgensis for producing Natamycin (the Natamycin yield was 3.07 g/L)
[11-15].
Even though there are some researches which have already carried out on Natamycin production
but till now its productivity is still very low. Therefore, due to the important commercial value of
Natamycin, it is of great importance to enhance the research and development of production
process.
The rate of Natamycin formation and productivity are dependent on many factors such as strain,
fermentation medium, fermentation conditions, precursor as well as the separation method. The
natamycin production strain has been screened in our lab that is reason we chose to optimize the
natamycin production medium during this study.
As the media have the most important effect on cell growth and natamycin production, the
overall aims of this research were to optimize natamycin production medium components
concentration for their effects on Streptomyces Gilvosporeus growth and Natamycin production.
4
The data would be analyzed to identify the importance of each input variable and to identify
whether there were any interactions between medium components and their optimal
concentration range. The specific objectives were:
To investigate the effects of different Natamycin production medium components on

Natamycin production in the shake flasks;
Improve the culture conditions for Natamycin yield in shake flasks and fermenter;
To scale up the fermentation process in 3.6 L, 10L bioreactors by adjusting fermentation

conditions.
To improve the Natamycin extraction methods in order to get high quantity and quality of
Natamycin from the fermentation broth.
1.3. Overview of the thesis

Chapter two reviews Microorganism Culture Methods, fermentation technology, fermentation
media, criteria for design and optimization of fermentation process and natamycin inoculum
propagation and fermentation. Additionally, this chapter reviews medium optimization
techniques in fermentation medium development, the different types of nutrient sources, and
their mechanism of action. Finally, the chapter reviews introduction to separation process of
natamycin.
Materials, equipment and media, analytical methods, experimental conditions and strategy to
optimize the natamycin production medium used in this research are described in chapter three.
This chapter also contains the protocol of natamycin extraction used during this study. Chapter
four presents the experimental results and discussions. Data were used to investigate the effects
of the medium components and their optimal concentration range on cells growth and Natamycin
yield. Conclusions and recommendations for further work are summarized in chapter five.
5
Chapter 2: Literature review
CHAPTER 2: LITERATURE REVIEW
2.1. Microorganism Culture Methods
For any microbiological culture, there are some demands which must be taken into consideration:
firstly, it is necessary to have a contamination free seed culture, and secondly the equipment and
media that are used, should be fully sterilized[16]. Most of the industrially useful bacteria and fungi
are cultured on either a solid medium or a liquid medium with a support. In either case, the medium
should contain all essential ingredients needed for optimum growth of that particular microorganism.
Solid medium is generally prepared by melting agar at 100 (in water) and then solidifying it at
about 45 . The medium, which is still in a molten state, is poured into a suitable container and is
allowed to cool and solidify. It is then inoculated by dipping a needle into the suspension of bacteria
or fungi and drawing this needle right across the surface of the medium. After a few days of
inoculation, colonies (spores) of microorganism appear on the surface of the medium. In liquid
cultures, all essential nutrients are dissolved in water; Agar or any other solidifying agent is not
needed [17].
2.1.1. Modes of culture

Culture techniques can be classified into batch, fed-batch, and continuous operations[18, 19].
2.1.1.1. Batch culture
This is the most common method for cultivation of microorganisms cells. In a simple batch culture
system, a limited amount of complete culture medium and microorganisms inoculum are placed in
a culture vessel and incubated in a favorable environment for growth. Some form of agitation, such
as shaking or impeller mixing, is necessary to ensure nutrient and gaseous exchange at the cells.
The culture vessel can be a simple conical flask, Erlenmeyer flask or an environment controlled
fermenter.
Batch culture is widely used for commercial cultivation of mostly of microorganisms for its ease of
operation and simple culture system. Since the process is batch wise, there is low requirement for
complete sterilization. For large scale microorganisms culture production, a portion of the culture
could be retained as inoculum for the next batch culture.
6
Some advantages of using the batch culture are the followings: Simplicity of use. A batch culture
can be easily readied, and, depending on the microorganism used, can be finished in less than 24
hours; Fewer possibilities of contamination: all of the materials required for the bioprocess are
present in the vessel and sterilized before the run starts. The only material added (with the
exception of the inoculum at the beginning of the bioprocess) and removed during the course of
batch fermentation are the gas exchange, and if using a bioreactor, sterile antifoam and pH control
solutions if required; It is easy to assign a unique batch number to each run, generating high
confidence in the history of each batch of product. This is critically important in a highly regulated
environment[19].
The batch culture has some limitations such as culture ageing, and more importantly differentiation,
can be a specific problem, especially so with growth-related products; if using the organism from
one bioprocess to seed another culture, degeneration or differentiation may occur, which could
affect the bioprocess and product formation; the use of batch cultures in industrial systems can lead
to an increased nonproductive period due to down time required for cleaning, resterilisation, filling
and cooling of equipment[19].
The different phases, which may occur in a batch culture, reflect changes in the biomass and in its
environment were discussed in the section 2.2 [19, 20].
2.1.1.2. Fed-batch culture
In a fed-batch culture, the medium is added continuously or intermittently whereas the culture is
harvested periodically, thus the culture volume may not be constant and the rate of dilution varies
with the culture volume.
A quasi steady state is reached when the biomass concentration and other culture parameters vary in
a repeating pattern within a fed-batch cycle. Fed-batch culture is the most widely used industrial
continuous flow culture process, where concentrated culture medium (such as acetate) is fed
continuously or intermittently, and the culture is harvested at the end of the cultivation cycle [19,
20].
Some advantages of fed-batch culture are the following: Controlling the concentration of the
limiting substrate prevents the repressive effects of high substrate concentration and avoids
catabolite repression, Reduction of broth viscosity. This is particularly important in filamentous
fungal fermentations, or where the product is highly viscous, such as the polysaccharide products of
7
Sphingomonas elodea gellan gum; and Xanthomonas campestris xanthan gum. The addition of
fresh medium during the fermentation run, leads to the broth being diluted, and a brief viscosity
drop, allowing better aeration and agitation within the system[19]. Even though the fed-batch has
some advantages but it has also the disadvantages such as the process operator must be fully trained
and highly skilled [19].
2.1.1.3. Continuous cultures
Historically, continuous culture techniques have not been widely used in laboratory scale, but are
more common in industry where these techniques are used for such processes as vinegar production,
waste water treatment, ethanol production and single cell protein production. In the laboratory,
these techniques have been used increasingly to study the growth and physiology of
microorganisms. In particular, continuous systems have been used to study proteomics,
transcriptomics and flux analysis, showing increased reproducibility and accuracy of data when
compared with similar studies in batch cultures.
Continuous cultivation is a method of prolonging the exponential phase of an organism in batch

culture, whilst maintaining an environment that has less fluctuation in nutrients, cell number or
biomass. This is known as steady state. The organisms are fed with fresh nutrients, and spent
medium and cells are removed from the system at the same rate. This ensures that several factors
remain constant throughout the fermentation, such as, culture volume, biomass or cell number,
product and substrate concentrations, as well as the physical parameters of the system such as pH,
temperature and dissolved oxygen.
In continuous flow cultures, fresh culture medium is supplied to the homogeneously mixed culture
and culture is removed continuously or intermittently. The approach is based on the observations
that substrates are depleted and products accumulate during growth. Eventually, culture growth
ceases due to depletion of the growth limiting substrate or accumulation of a growth-inhibiting
product. To sustain cell growth, the growth-limiting substrate needs to be replenished and the
growth inhibitory product needs to be removed or diluted by adding fresh culture medium[19, 20].
In theory, a continuous process can be operated indefinitely; however, because long periods of
operation can result in mechanical failure, the process must be stopped occasionally to allow for
system maintenance; During the continuous culture, productivity and growth rate can be optimized
by changing the feed flow rate during production;
8
The effects of environmental or physical factors are more easily analyzed in a continuous system,
where any changes in the constant steady state are observed and can be attributed solely to the
change in those factors[19]. Although the continuous culture has some advantages, it still has also
the disadvantages. The US and Drug Administration (FDA) do not accept continuous culture in the
production of therapeutic products as a Current Good Manufacturing Practice (cGMP). This is
because they require the manufacturer to segregate such products into batches for traceability
purposes, precluding continuous culture as a means of production; Contamination can be a major
problem in continuous cultivation, and can result in the wash out of the desired organism and
therefore a loss of product[19].
2.2. Fermentation Technology

Though there are different perceptions of the nature of the process, fermentation can be defined as
the breakdown or catabolism of organic compounds by micro-organisms under both aerobic and
anaerobic conditions. This breakdown yields end products.
Fungi and bacteria are commonly used in submerged fermentation to produce a wide range of
primary and secondary metabolites.
Many other plant, insect and mammalian cell lines are also used to produce secondary metabolites,
but these systems usually are not as effective as bacteria and fungi.
2.2.1. Microbial Growth in Batch Fermentation
Three distinct phases of a microorganisms growth can normally be distinguished in submerged

culture fermentations: the lag, the exponential (also known as log and linear) and stationary phases
(as shown on Figure 2.1 and Figure 2.2). The autolytic, or decline, or death phase is not usually
classed as a growth phase.
The length of the lag phase, where cells adapt to the new environment and begin to grow depends
on the microorganism, initial cell concentration, environmental conditions and growth medium.
In the linear phase (sometimes called the tropophase), there is rapid growth and the stationary phase
(sometimes called the idiophase), there is no further net growth. Secondary metabolites such as
antibiotics are usually produced in the stationary phase [21], [22].
9
Deceleration Death
Stationary phase or
Decline
Exponential phase
Biomass
Lag phase
Time
Figure2. 1 Microbial Growth in Batch Fermentation [23]
Figure2. 2Figure 2.1.b: Typical growth phases for micro-organisms in submerged culture [21]
Submerged cultures are widely used to produce many secondary metabolites because they allow
filamentous fungi and bacteria to produce freely suspended mycelia and pellets, essential for
secondary metabolite production[21]. Submerged culture fermentations require a stirred nutrient
medium and, in the case of aerobic microorganisms, a supply of oxygen.
10
The main focus of fermentation is to enhance the production of the desired product, whether it is a
primary metabolite, secondary metabolite and/or biomass. Therefore optimizing the fermentation is
critical for ensuring the appropriate growth or production phase is extended.
Microorganism morphology during the growth phase is influenced by strain, culture initiation
method, growth medium and the hydrodynamic regime[21], [24].
The morphology of filamentous bacteria and fungi in submerged cultures can vary from a network
of freely dispersed mycelia to tightly packed, discrete pellets. This morphology can affect product
yield. Most bacterial and fungal fermentations have a high oxygen demand for biomass production
and metabolite formation [24-26]. Media must contain at least a nitrogen and a carbon source to
support microbial growth and metabolite production [27]. Rapidly growing filamentous fungi
increase viscosity of submerged culture and concomitantly impeding secondary metabolites
production [24].
2.2.1.1. Lag phase
An initial lag phase where the specific growth rate is at sub-maximum level may often be observed.
The growth lag could be due to the presence of non-viable cells or spores in the inoculum. The
growth lag could also be the period of physiological adjustment due to changes in nutrient or
culture conditions. Lag phase may be abolished when cells at a later exponential growth phase are
used as inoculum [20].
The first major phase of microbial growth in a batch fermentation process,
A period of adaptation of the cells to their new environment,
Minimal increase in cell density,
May be absent in some fermentations.
2.2.1.2. Log phase or exponential phase
At the late lag phase, the cells have adjusted to the new environment and begin to grow and
multiply (accelerating growth phase), and eventually enter the exponential (or logarithmic) growth
phase. At the latter phase, cells grow and divide as an exponential function of time, as long as
mineral substrates and light energy are saturated[20].
The second major phase of microbial growth in batch fermentation process,
11
Cells have adjusted to their new environment,
The cells are dividing at a constant rate resulting in an exponential increase in the number of
cells present. This is known as the specific growth rate and it is represented mathematically
by first order kinetics as the following:
dX
( Kd ) X
dt
Where X is the concentration, is the cell growth rate, and K d is the cell death rate. The
term K d can be referred to as net . The cell death rate is sometimes neglected if it is
considerably smaller than the cell growth rate. [23]
The cell growth is often substrate limited, as depicted in the figure below (figure 2.3).
Figure2. 3. The cell growth which is substrate limited [9]
The growth curve is well represented by Monod batch kinetics, which is mathematically
depicted in following equation:
max S

KS S
Where is the specific growth rate, max is the maximum specific growth rate, S is the
growth limiting substrate concentration, and K S is the saturation constant which is equal to
12
the substrate concentration that produces a specific growth rate equal to half the maximum
specific growth rate. All specific growth rates account for the term K d and should be
considered to be net values [23].
There are other models used to determine the growth rate that depend upon inhibition
Substrate inhibition
Production inhibition
Toxic compounds inhibition
The type of inhibition causes mathematical changes in the previously presented Monod
equation for batch kinetics
The substrate inhibition: in batch fermentation, this can occur during the initial growth phases while
substrate concentrations are high; if this is a major problem, continuous or fed-batch fermentation
methods should be considered.
Production inhibition: in the batch fermentation, this can occur after induction of the recombinant
gene.
2.2.1.3. Stationary phase
The third major phase of microbial growth in a batch fermentation process
Occurs when the number of cells dividing and dying is in equilibrium and can be the result of
following: depletion of one or more essential growth nutrients, accumulation of toxic growth
associated by-products, stress associated with the induction of a recombinant gene
Primary metabolite, or growth associated, production stops
Secondary metabolite, or non-growth associated, production may continue
2.2.1.4. Death phase
Although the death phase is not usually classed as a growth phase, is the phase which
Ends the microbial growth in batch fermentation
13
Known as the decline phase or the autolytic phase
The rate of cells dying is greater than the rate of cells dividing
Similar to exponential phase, it is represented mathematically by first order as the following:
dX
Kd X
dt
2.2.2. Methods to determine the growth curve

There are two main methods primarily used to establish a growth curve.
Viable cell count
Determined by plating a sample from the culture
Optical density
Determined by taking an optical measurement using a spectrophotometer (figure 2.4)
The spectrophotometer picture is taken by the author in the chemical engineering laboratory, Xiamen University
Figure2. 4. Spectrophotometer pictured above
Measuring the optical density with a spectrophotometer is a quick and easy way to develop a
growth curve. One takes a sample of the fermentation broth and measures the absorbance at a
particular wavelength, in the spectrophotometer, which is often 600nm [23].
The measured value can be compared to previous measurements made in conjunction with cell
plating or cell counting.
14
The disadvantage of using the optical density is that both viable and non-viable cells absorb this
wavelength. As a result, the values taken are not representative of only viable cells.
After understanding microbial cells growth in a batch process, it is time to see how a general
biotechnology fermentation process works. An example, of fermentation process is represented in
the block flow diagram shown below (figure 2.5).
Inoculum Shaker flask

vial
2nd seed Production Harvesting/

Media 1st seed
preparation fermentor fermentor fermentor Purification
Figure2. 5. Block flow diagram of biotechnology fermentation process
First, a frozen vial containing a few milliliters of one microorganism strain is taken out of a freezer
and thawed. This vial is sometimes referred to as an inoculum vial and its content is known as
inoculum.
After thawing, the inoculum is transferred in the sterile manner to a shake flask containing growth
media. This process is known as inoculation. For Streptomyces species, the initial pH of the media
is typically around 7 and is controlled by using alkaline and/or acidic agent (depending on kind of
media is used) in the media . A picture of a shake flask is depicted below (figure 2.6).
Picture taken by author in chemical engineering laboratory, Xiamen University

Figure2. 6. Shake flask
15
The volume of media in the shake flask is usually on the order of magnitude of hundreds of
milliliters. After inoculation, the shake flask is placed in an incubator shaker so the cells can grow
and reproduce (figure 2.7). The shaker is operated at a constant temperature, which is around 28oC
for Streptomyces species. The shake flask holders in the shaker are attached to an orbital plate that
rotates horizontally at a programmable rate.
Figure2. 7. The shake flasks in a rotary shaker
This shaking motion has two purposes:

Keep the cells and the nutrients in the growth media homogeneous
Increase the rate of oxygen uptake by the media for the aerobic cells. The cells are growth to
a particular density near the end of their exponential phase and used to inoculate a small
fermenter known as a seed fermenter.
Once the cells are transferred to the seed fermenter, they are grown to a particular density near the
end of their exponential phase [9][28].
A schematic of a fermenter is shown in figure2.8; it is representative of both a seed and a productive

fermenter. The microbial cells are supplied with filtered oxygen through the sparger located at the
bottom of the fermenter. The agitator is used to keep the mixture of cells and growth media inside
the fermenter relatively homogeneous. It also increases oxygen mass transfer by decreasing the size
of the oxygen bubbles. The fermenter is operated at a constant growth temperature to achieve the
required growth rate. Since cells liberate heat during the growth, a constant temperature is
maintained using either cooling jackets surrounding the fermenter, coils inside the fermenter, or
combination of both. In addition, the cells secrete acids as they metabolize, which decrease the pH
level within the fermenter. As a result, a base is usually added to the fermenter whenever the pH
drops below its set point value.
16
After the cells reach the required optical density in the seed fermenter, the cells can either be used to
inoculate several increasingly larger seed fermenter until the required volume and density is reached,
or the cells can be transferred directly to the production fermenter to where they will eventually
synthesize the co-protein or any biological products depend on the target of fermentation. When the
cells reach their required volume and density; they are transferred to the production fermenter
where they are grown to a particular density. The density in which they are grown to depends upon
the desired product being growth or non-growth associated. For growth associated, the cells are
grown to their mid to late exponential phase.
At this point, a chemical is added that induces the cells to begin over-expressing the gene
responsible for the recombinant protein. The over-expression of the particular gene and the
depletion of nutrients eventually cause the cells to enter their stationary growth phase.
Figure2. 8. A schematic of a fermentor [28]
At this point, the cells are no longer capable of producing appreciable amounts of the desired
protein and the fermentation is ended. Now that fermentation process is over, the fermentation broth
containing the cells and the extracellular media is removed from the production fermenter. This is
called harvesting and that completes the upstream process of fermentation (the upstream
biotechnology process is known as fermentation process).
17
After the cells are harvested, the recombinant protein or other kind of biological product (depending
on fermentation process target) needs to be separated from the cells that produce them. This is
accomplished through the downstream process of purification. After successful fermentation or
enzyme reactions, desired products must be separated and purified. This final step is commonly
known as downstream processing or bioseparation, which can account for up to 60 percent of the
total production costs, excluding the cost of the purchased raw materials[29].
2.3. Fermentation media

An important aspect in design of a fermentation process is the choice of fermentation medium,
which represents the raw material for the process. [30]
2.3.1. General review

Micro-organisms incorporate components from the fermentation medium into biomass and/or use
them for metabolite biosynthesis. When a medium is used only for biomass production, biomass
formation can be estimated by stoichiometry [31]. If, however, the microorganism uses the medium
components for producing metabolites and biomass, metabolite efficiency determines the
concentrations of biomass and metabolite. The nutrient supplied to the microorganism significantly
affects metabolite concentration, both directly through metabolism and indirectly by influencing the
specific metabolite production rate [31]. Secondary metabolite specific production rate can be
influenced by catabolite regulation. Rapidly growing cultures usually have an adverse effect on
secondary metabolite production, suggesting that the fermentation medium is critical for optimal
metabolite production[32].
2.3.2. Medium components

Fermentation medium should allow a large biomass to be produced within the shortest time, and
sustain the productive stationary phase for as long as possible. Medium composition is therefore the
most important (and the most complicated) factor influencing production yields, particularly with
high producing industrial strains[22].
In general, the fermentation medium should fulfill the following criteria:

It should contain a carbon, nitrogen and energy source;
It should contain all essential minerals required for growth;
18
It should contain all necessary growth factors to ensure rapid growth and high yield of the
desired product(for example, vitamins, hormones and the trace elements iron, zinc, etc.);
It should be of a consistent quality and be readily available throughout the year;
It causes a minimum of problems in the downstream processing;
It causes a minimum of problems in other aspects related to the fermentation process, i.e. it
has no negative effect on the gas-liquid mass transfer[30, 33, 34].
Carbon sources provide both energy and the basic cellular building blocks for microbial activity.
The carbon content of cells is high and carbon also plays a major role in energy production.
Therefore, the carbon/energy component of the substrate is usually the major medium component. It
is frequently the major cost of media.
The most common carbon substrates used for antibiotic production are starch, oils, and various
types of simple sugars, such as glucose [22, 33].
Nitrogen is a crucial component of proteins, nucleic acids, amino acids and enzymes for cell growth
and function. Sources for industrial fermentations includes proteins, ammonia and ammonium salts,
urea and nitrate salts [35]. When proteins are used, they must first be hydrolysed by extracellular
proteolytic enzymes before they can be assimilated [35]. Proteins can supplement the carbon energy
supply when other carbon sources are depleted. This releases ammonia, causing pH to become
alkaline. Excessive ammonia levels can inhibit antibiotic production [36].
Necessary growth factors to ensure rapid growth and high yield of the desired product must be well
controlled. For example, the sources of phosphorus include inorganic phosphate and/or complex
organic phosphates. Phosphate concentration is often critical, with only minor differences between
concentrations that give satisfactory growth rates and those that inhibit antibiotic production [22].
Phosphorous is mainly incorporated into nucleic acids, phospholipids and cell wall polymers and is
occasionally stored as polymeta-phosphate [33]. The phosphorus requirement differs widely
between cultures and fermentation conditions, excess phosphate can strongly inhibit or repress
antibiotic production [35]. For example, the highest natamycin production was obtained in a
cultivation medium containing 0.05 g/l of potassium dihydrogen phosphate. Further increase in
phosphate concentration resulted in a significant increase in biomass concomitant with lower
antibiotic production[37].
19
2.3.3. Complex medium
Fermentation medium is a critical component of industrial fermentation and can directly affects the
growth profile, metabolite production, productivity (g L-1h-1) and operations such as sterilization,
shear sensitivity of cells, nutrient requirements and broth viscosity [27]. The first stage of media
development uses components known to assist microbial growth and metabolite production.
Complex carbon and nitrogen sources, such as yeast extract and peptones are commonly used
because they are inexpensive and provide a wide range of nutrients [27]. Yeast extract is produced
by autolysing bakers or brewers yeast at approximately 50oC. It is a rich source of various amino
acids, peptides, water-soluble vitamins, trace elements and carbohydrates [27, 38]. Due to the
poorly controlled starting material and downstream processing, biomass and growth rates on yeast
extract can vary by as much as 50% between different batches[39] . It is recommended that a
representative sample of each raw material batch of a complex medium component be tested for
several pre-determined parameters to minimize variability [19, 27, 39].
Table2. 1.Complex fermentation media often applied in the fermentation industry[30]
medium contents origin Typical application
Corns teep Lactate, amino acids, Starch processing from antibiotics

minerals, vitamins corn
liquor
Corn starch Starch, glucose corn Ethanol,
industrial enzymes
Barley malt Starch, sucrose barley Beer, whiskey
molasses Sucrose, betain, Sugar cane and Bakers yeast,

raffinose, glucose,
fructose, sugar beer ethanol
pharmacedia Carbohydrates, Cotton seed antibiotics

minerals, fats,
Vitamins,
Amino acids,
serums Amino acids, serum Recombinants,
20
growth factors proteins
whey Lactose, proteins milk Lactic acid
Yeast extract Peptides, vitamins, yeast enzymes
amino acids
The advantages of applying complex media are that they often contain an organic nitrogen source,
essential minerals and different growth factors. The disadvantages of complex media are that:
1. There may be a seasonal variation in composition;
2. The composition changes with storage and;
3. There may be compounds present that are undesirable. With a requirement for increased
documentation and reproducibility in the fermentation industry there is trend towards
application of more defined media, and often minimal media with a carbon and energy
source (glucose, sucrose or starch), an inorganic nitrogen source, a mixture of minerals and a
perhaps a few vitamins may replace complex media to the benefit to the producer.
It is important to consider also the benefits of using a defined medium in the subsequent
downstream processing[30].
Also in the pharmaceutical sector there is a desire to use defined media and particularly the use of
serum free medium is today considered a standard requirement in connection with production of
heterologous proteins using mammalian cells cultures. In the future it is expected that lignocellulose
containing material may serve as cheap and efficient carbon and energy source. Lignocellulose
basically consists of three components: lignin, cellulose and hemicellulose (a complex pentose rich
polymer), and it is present in many different plant fibers like corn cob, bagasse, straw and wood.
The exploitation of lignocelluloses as raw material may allow many commodity products like fuels,
polymers, and chemicals to be produced through fermentation processes in a cost efficient manner
[30]. It is possible to improve secondary metabolite production and consistency by screening
common complex media components known to influence microorganism growth or metabolite
production [27, 39].
21
2.3.4. Defined medium

Chemically-defined media (also called synthetic media) are fully characterized mixtures of carbon,
nitrogen and trace metal components. They can enhance metabolite production through directed
biosynthesis, improved process control, fermentation consistency and enhanced product recovery
[27, 40, 41]. Other benefits of defined medium included reduced foaming and viscosity, which
improves ease and economics of product recovery[41].
Using defined medium for secondary metabolite production allows consistent microbial growth and
metabolite production between batches, an essential requirement in large-scale industrial antibiotic
production [27, 40, 41]. It has been successfully used for antibiotic production by a wide range of
Streptomyces species.
It is proposed that using specifically-defined carbon and nitrogen is critical as the source of
precursors and cofactors for synthesizing secondary metabolites. [42]. Stringent starvation
conditions promoted by selective nutrients can promote secondary metabolite production. It is also
reported that adding chloride ions in a synthetic medium for Streptomyces aureofaciens produced
chlortetracycline whereas having no chloride produced tetracycline[43].
Define carbon sources can give specific metabolic activity during growth and may exert complex
regulations on gene expression and enzyme activity during antibiotic synthesis[42].
Nitrogen in defined media may be a combination of organic (amino acid, amines, etc.) and/or
inorganic (nitrates, ammonia, nitrite, etc.) sources. For most filamentous fungi, ammonium nitrate,
sodium nitrate, and urea can be used for biomass formation and metabolite production [42].
Filamentous bacteria, however, utilize organic nitrogen sources more effectively than inorganic
nutrients. For example, Mohamed, A.FARID et al (2000) reported that the organic nitrogen sources
are better than inorganic nitrogen sources for supporting antibiotic production; some organic
nitrogen source support the cell growth and some others support the antibiotic production.
Therefore, it is recommended to use the mixed nitrogen source to support both cell growth and the
antibiotic production because nearly doubled Natamycin production, may be produced, compared
to the other culture containing the simple nitrogen source [12].
Basal trace metal solutions used for most chemically-defined media for Streptomyces species
contains magnesium sulphate, calcium chloride, cobalt chloride, ferric sulphate, Zinc sulphate,
copper sulphate and di-potassium orthophosphate [40-42, 44].
22
2.3.5. Fermentation medium formulation
It is important to be able to cultivate microbial cells under suitable conditions to study their
characteristics. To be able to do this, one must know what food material and physical conditions are
required [34].Designing a nutrient medium for growth and product formation is a key step in
experimental or production runs. Chemical constituents of the medium must meet all elemental
requirements for cell mass and products and must supply appropriate energy for synthesis and
maintenance. Specific vitamins and trace minerals requirements must also be met. Most microbial
cells that are actively growing are about 90 percent water. Any medium must contain, as a
minimum, the correct proportions of the typical elemental composition of a microbial cell (Table
2.2).
Table2. 2. Elemental Composition of Bacteria[34, 45]
Components Dry Weight %
Carbon 50
Oxygen 20
Nitrogen 7-14
Hydrogen 8
Phosphorus 1-3
Sulfur 0.5-1
Metals 4
Although many microorganisms can grow well on simple mineral salts media, some
microorganisms cannot synthesize all their own biochemical components and require one or more
specific biochemical compounds. The most frequent requirements are for vitamins and amino acids.
Yeasts, for instance, often require biotin, thiamine, and riboflavin[45].
Microorganisms can convert basic chemicals into complex molecules, but the second law of
thermodynamics cannot be violated.
The complex series of synthetic reactions performed in the cell requires energy, which usually
comes from controlled oxidation of organic compounds.
23
Thus, carbon compounds produce energy for biosynthesis as well as to meet the cells elemental
carbon requirement. Most heat is evolved during the terminal oxidation stages, where energy-
carrying cofactors are used to reduce oxygen to water. To satisfy the cells requirement for carbon
and energy, sufficient carbon for biosynthesis and energy generation must be supplied. Oxygen, the
remaining nutrient is a sparsely soluble gas. Supplying oxygen involves mass transfer. Oxygen
demand depends on the carbon source and utilization efficiency[45]. Fermentation medium
formulation has a large effect on fermentation processes.
2.4. Criteria for design and optimization of fermentation process

It is much rarer to come across the perfect medium for cultivating specific cell lines there are
many that are highly suitable but the reader requires being able to identify them. Consequently a
range of questions needs to be asked, for example:
(i) What is the elemental composition of the cell line to be used?
(ii) What energy source does the cell line require?
(iii) What is the purpose of the bioprocess under consideration? Biomass/product/both?
(iv)Will the formulation of the medium impact on the product? For instance, pH can have a
significant effect on products of a proteinaceous nature and substrates that can affect pH must be
considered carefully, even where pH control is utilized, as localized pH hotspots can potentially
denature the protein even at short exposure times. The correct carbon and nitrogen sources, add the
required macro- and micronutrients and growth factors, and the yield of cells should be very good.
The key point here is that ideally, though, understanding of cell physiology, and quantitative
approaches to the latter, are probably all that is needed to formulate a suitable medium for most
fermentations[19].
The criteria used for design and optimization of a fermentation processes depends on the product.
Thus, the criteria used for a high volume/ low value added product are normally completely
different than the criteria used for a low volume/ high value added product. For products belonging
to the first category (which includes most whole cell products, most primary metabolites, many
secondary metabolites, most industrial enzymes, and most polysaccharides) the three most
important design parameters are:
Yield of product on substrate (typical unit: g product per g substrate),

24
Productivity (typical unit: g product per L reactor volume per hour),
Final titer (typical unit: g product per L reactor volume),
Yield of product on the substrate is here very important since the raw materials often account for a
significant part of the total costs. Productivity is important since this ensures an efficient utilization
of the production capacity, i.e. the bioreactors. Especially in an increasing market it is important to
increase the productivity since this may prevent new capital investments. Final titer is of importance
for the further treatment of the fermentation medium, for example, purification of the product. Thus
if the product is present in a very low concentration at the end of fermentation it may be very
expensive even to extract it from the medium with a satisfactory yield[30].
2.4.1. The role of precursor during the fermentation

A precursor is a substance from which another substance is formed (especially by a metabolic
reaction), in the other words it is any chemical reactant which takes place at any stage in the
production by whatever method of a toxic chemical. In chemistry, the precursor is a compound that
participates in the chemical reaction that produces another compound.
In biochemistry, the term "precursor" is used more specifically to refer to a chemical compound
preceding another in a metabolic pathway.
During the production of macrolide antibiotics, the formation of lactone group is the limiting factor,
lactone group is known as the lowest fatty acid molecule (such as acetate, propionate, butyrate, etc);
as the precursor units composed of such small molecule group, they cannot be synthesized by some
bacteria, thus leading to poor production of antibiotics. If precursors are added at appropriate level
of concentration during the fermentation, they can significantly increase the antibiotics production;
but if the precursor concentration is too high in the fermentation broth, it will affect the strains and
concomitant to the decrease of the yield of antibiotics, also at low concentration, there is a negative
effect on formation of the antibiotics. Therefore, we have studied the effect of different precursors
on natamycin production[46, 47].
2.5. Natamycin inoculum propagation and fermentation

The inoculum of the Streptomyces species (appropriate) is subsequently fermented which produces
high yields of natamycin when cultivated into the suitable natamycin fermentation medium.
25
The inoculum is typically exposed to a series of propagation steps wherein each step increases the
quantity of the natamycin production Streptomyces cells. After the quantity of the cells is adequate,
the Streptomyces is exposed to an environment and/or a medium which is designed to enhance
natamycin production when the Streptomyces species ferments.
By providing an improved environment for the Streptomyces species the yield of Natamycin may be
increased. Even though, there are so many factors (such temperature, pH, aeration, so forth) which
influence the production of natamycin in submerged fermentation culture; the inoculum propagation
and fermentation media play a very important role in increasing of yields of Natamycin in a
fermentation process. An organism capable of producing Natamycin is placed into a contact with a
predetermined medium to produce an inoculum and then into a predetermined fermentation
production medium that will support maximum metabolic activity of the organism during further
propagation and Natamycin producing fermentation[48].
2.6. Introduction to separation process of natamycin

High purity natamycin can be recovered from a fermentation broth containing natamycin by
extraction with methanol under controlled pH and temperature conditions. The fermentation broth
produced in the fermentation process comprises solid suspended natamycin, biomass and water. The
broth is used as the feed stream for the recovery process, either directly, or after concentration by
removal or some or nearly all the water. The methanol may be added to a natamycin feed stream to
form an extraction medium, and maintain the extraction medium at a temperature of about 0-35
but better at low temperature. After getting extraction medium, the pH must be first adjusted at low
value about 1-4.5; thereafter, it should be readjusted at high value of about 6-9 in order to
precipitate natamycin from extraction liquor[49].
26
Chapter 3: Material and methods
CHAPTER 3: MATERIAL AND METHODS

3.1. Materials, Equipments and Media
3.1.1. Microorganisms
These studies were carried out with the Streptomyces Gilvosporeus ATCC13326 (Figure3.1),
obtained from fermentation technology lab, Chemical and Biochemical Engineering, Xiamen
University, store where Stock cultures were stored in 1mL aliquots in 20% glycerol solutions (i.e.
0.5 mL of strain in 0.5 mL 20% glycerol solution) and held at -80 .
Figure3. 1. the morphology of Streptomyces Gilvosporeus at 38h cultivated in liquid medium at 28,
Picture taken by author.
3.1.2. Media and culture conditions

The three different main media were used during study; two of them were used as inoculum
propagation media (slant medium and seed medium) and the other one (Natamycin production
medium/fermentation culture medium) was used to producing Natamycin.
3.1.2.1. Slant medium (solid medium)
It was used for Streptomyces Gilvosporeus spore germination. This medium contained per liter of
distilled water starch soluble 20.0g; NaCl 0.5 g; KNO3 1 g; K2HPO4.3H2O 0.5 g; MgSO4.7H2O
0.5 g; FeSO4.7H2O 0.01 g and agar 20.0 g. The pH of this medium was pre-adjusted to 7.2-7.4
before sterilization by autoclaving at 121 for 20 minutes.
27
After inoculation of slant medium with 0.1 mL of the strain on the petridish (plate), the slant
culture was incubated at 30 ; the culture sporulated heavily within 5 to10 days depend on
suitable conditions (Figure3.2).
Figure3. 2.The pattern of Streptomyces Gilvosporeus on the slant medium, picture taken by author
3.1.2.2. Seed medium (liquid medium)
It was composed, per liter of distilled water, of starch soluble, 20.0 g; glucose 8.0 g; extract 8.0 g;
Soybean powder 10 g; peptone 5.0 g; CaCO3 2 g and pH was adjusted at 7.07.2 before
sterilization by autoclaving at 121 for 20 minutes. An arisen spore from the slant culture was
used to inoculating 30mL of seed medium into 300 mL Erlenmeyer flask and then the seed
culture was incubated on a rotary shaker at 200 rpm (revolutions per minute) for 38 to 44 hours
at 28 . Some portion of seed culture was used to inoculating the Natamycin production
medium and to keep for further use. The 0.5 mL of the seed culture was mixed with the same
quantity of 20% glycerol, and then 1.0 mL of the mixture (named stock strain solution) was put
into a 1.5mL microfuge tube and preserved at -20 and -80 for further use.
3.1.2.3. Natamycin production medium (liquid medium)
28
As the one of the target of this study is optimization of natamycin production medium
components, the different production media were used during this study but we have started with
original Natamycin production medium which was composed of starch soluble; 50.0 g/L glucose,
10 g/L; soybean powder 15.0 g/L; peptone, 5.0 g/L; yeast extract, 5.0 g/L; NaCl, 2.0 g/L; CaCO3,
15.0 g/L; and pH was adjusted at 7.2 with NaOH before sterilization. The fermentation was taken
place on the shake flasks and in the bioreactor (fermenter).
3.1.2.4. Natamycin fermentation conditions on the shaker
The cultivation conditions were as follows: seed culture age 38 to 44 hours, inoculum level 10 %
(i.e. a 3 mL portion of seed culture was used to inoculating 30 mL Natamycin production
medium into 300 mL Erlenmeyer flask), initial pH value was 7.0-7.2 before sterilization of the
culture medium. The production culture (fermentation on the shaker) was incubated on an orbital
shaking at 200 rpm for 120hours (i.e. 5 days). The 0.6% sodium propionate was fed into the
fermentation broth at 24 hour s of culture, as precursor [50-52].
3.1.1.5. Natamycin fermentation conditions in the bioreactor
Using the distilled water, the Natamycin production medium (discussed above) was prepared and
put fermenter vessel. Thereafter, the fermenter vessel containing Natamycin production medium
was sterilized into autoclave for 25 minutes at 115 . After incubation of seed culture at 28oC
for 38-44 hours on a rotary shaker at 200 rpm, 10% volume of seed culture was transferred into
Natamycin production medium as inoculum. The temperature was controlled at 28oC, the stirrer
was set at 400 rpm as set point but it was increasing as necessary during fermentation process in
order to maintain the dissolved oxygen (DO) at 30% and aeration rate of 2 v/v-min (volume per
volume medium per minute). At 24 hours of culture, the feeding of 0.6% sodium propionate as
precursor was started[51, 52].
Samples were taken at various intervals of time from 0 hour till the end of cultivation for
analysis to measure dry cell weight and Natamycin concentration.
29
3.1.2. Equipments
Table3. 1. Equipments names and manufacturers
Equipment name Manufacturer
3.6 L Bioreactor INFORSE. Switzerland
Microcentrifuge, Universal 320R Hettich Zentrifugen, Germany
Microscopy OLYMPUS CO.LTD. Japan
Freeze drier FD-1000 EYELA CO.LTD. Japan
Water-Circulation multifunction Vacuum Zheng Zhou Great Wall Scientific

pump Industry and trade CO.LTD
Microwave GALANZ CO.LTD. China
Incubator BOXUN CO.LTD. China
Rotary shaker incubator SUKUN TECH CO.LTD. China
Rotary evaporator SUKUN TECH CO.LTD. China
HPLC System AGILENT TECHNOLOGIES.

USA
Vacuum dryer JINHONG INSTRUMENT.

CO.LTD. China
Autoclave, HICLAVE HV-119 HIRAYAMA, Japan
Refrigerator HISENSE CO.LTD. China
Balance SARTORIUS, Germany
Bechtop AIRTECH CO.LTD. China
3.2. Strategy to optimize the natamycin production medium

During this study, we used the one at a time strategy: varying one culture medium component
while keeping all others constant, to optimize the new optimal natamycin production medium for
Natamycin production by Streptomyces Gilvosporeus AT CC13326; as one factor among factors
30
which influence the yield of natamycin. The effects different carbon sources (starch soluble and
glucose) and nitrogen sources (soybean powder, yeast extract and peptone) concentrations in the
culture medium were investigated.
3.2.1. Carbon sources

Starch soluble and glucose are the carbon sources used during this study to optimize the new
Natamycin production medium. The effect of starch soluble concentration in the medium, on
cell growth and natamycin production, was investigated by using the following different starch
soluble concentrations: 20, 40, 60, 80, 100, 120, 150 and 200 g/L. we did not mention the 0 g/L
concentration because we had used this concentration before and the yield of natamycin was
almost zero. The effect of glucose concentration in the medium, on natamycin production, was
investigated by using the following different glucose concentrations: 0, 5, 10, 20, 30 g/L.
3.2.2. Nitrogen sources
Complex nitrogen sources are generally recommended for large scale antibiotic production. Also,
Eisenschink, M.A., Millis, J.R. and Olson, P.T. (1997) reported that natamycin cultivation
medium should contain a mixture of non-yeast protein nitrogen and yeast protein nitrogen
compounds to increase the Natamycin production[48]. However, soybean powder was used as
non-yeast protein nitrogen and yeast extract and peptone were used as yeast protein nitrogen
compounds during this study.
The effect of soybean powder concentration in natamycin production medium, on cell growth
and natamycin production, was investigated by using the following different soybean
concentrations: 0, 5, 10, 15, 30 and 40 g/L.
The effect of yeast extract concentration in the natamycin production medium, on cell growth
and natamycin production, was investigated by using the following different yeast extract
concentrations: 0, 5, 10, 15, 20 and 30 g/L.
The effect of peptone concentration in the Natamycin production medium, on cell growth and
natamycin production, was investigated by using the following different peptone concentration: 0,
5, 10, 15 and 20 g/L.
31
3.3. Analytical method
3.3.1. Measurement of cell growth

The dry cell weight (DCW) of a sample from the production stage in shake flasks and in
bioreactor was determined by placing a 8 mL sample of fermentation broth into two pre-weighed
5mL microfuge tubes (4 mL in each tube) and centrifuging at 6000 rpm for 5 minutes. The of
supernatant was decanted; the pellet in each tube was then two times by mixing the pallet with
1mL of distilled water and centrifuging again at 6000 rpm for 5 minutes. After removing the
supernatant, the cells in the tubes were subsequently dried in an oven at 80 for 24 hours[53].
Dry mass was estimated after placing tubes in a desiccator for at least 1 hour before reweighing,
and the estimation was continued until a constant mass was obtained. By averaging the weight
from two tubes, results were expressed as gram dry weight per liter broth.
3.3.2. Determination of Natamycin concentration

3.3.2.1. Preparation of HPLC samples from Natamycin fermentation broth
The 0.5 mL of fermentation broth was mixed with 4.5ml of 100% methanol into 15mL test tubes,
the mixture was treated with ultrasonic batch for 30 minutes, and then the mixture solution was
centrifuged at 3800 rpm for 10 minutes to remove the mycelia. The supernatant from
centrifugation was diluted to a suitable concentration to be analyzed by HPLC.
For example, when the dilution of 50 times is needed, 0.5 mL of supernatant was mixed with 2
mL of 70% methanol. Finally, the samples were filtrated through 0.45 l filtration film in order
to get the samples which were ready to be analyzed by HPLC (High Performance Liquid
Chromatography).
3.3.2.2. HPLC natamycin standard samples and standard curve
A 50mg of pure Natamycin (99.1%, Shandong Lukang Pharmaceutical Co., Ltd, China) was
mixed 100% methanol into 50 mL volumetric flask till the total volume of 50 mL. The prepared
solution was 1 g/L concentration (called 1 g/L pure Natamycin solution), which has used to
prepare different low concentration samples.
1. A standard sample of 100 ppm was prepared by putting a 1 mL of 1 g/L pure Natamycin
solution into 10 mL volumetric flask and add 70% methanol till the gauge line (i.e. till the
total volume of 10 mL).
32
2. A standard sample of 80 ppm was prepared by putting a 0.8 mL of 1 g/L pure Natamycin
solution into 10 mL volumetric flask and add 70% methanol till 10 mL total volume of
solution.
solution.
solution.
solution.
The standard curve was generated by injecting a known amount of pure Natamycin (20 ppm, 40
ppm, 60 ppm, 80 ppm and 100 ppm) (figure3.3).
Figure3. 3. Natamycin concentration standard curve
3.3.2.3. HPLC conditions
33
Natamycin concentration in samples from shake flasks and bioreactor was analyzed using a
HPLC with an Agilent 1200 series DAD wavelength detector at 303 nm and at room temperature.
The HPLC system was equipped with a solvent degasser, Quaternary pump and DAD (Agilent
technologies, 1200series).The samples were eluted through an Agilent HC-C18 reversed phase
HPLC column (Agilent technologies). The Agilent HC-C18 column was 250 mm in length and
4.6 mm in diameter with a particle size of 5 m. Natamycin retention time was between 6.5 to
8.5 minutes.
The mobile phase composition was methanol, water and phosphoric acid (64:36:0.3 v/v/v) and
set at a low flow rate of 1 mL/min was used to elute the samples. The injection volume was 20
L [53-55].
From the standard sample
From fermentation broth sample

Figure3. 4. Chromatography chart of natamycin
34
3.3.3. Chemical reagents and solutions

Sodium propionate
Sodium propionate was prepared by dissolving 18 g of anhydrous sodium propionate in 400 mL
of distilled water and sterilized by autoclaving at 121 for 20 minutes.
75% Methanol
75% methanol was prepared by mixing 400 mL of 100% methanol and 100 mL distilled water
into 500 mL graduated cylinder.
Table3. 2. The chemical reagents
Name of reagents Grade Manufacturer
Ethanol AR Sinopharm Chemical

Reagent Co.,Ltd. (SCR)
Methanol AR, HPLC SCR
Sodium propionate CR Beijing solarbio science&

Technology Co., Ltd.
Starch soluble AR SCR)
glucose AR SCR
Soybean powder Made by ourself
Yeast extract BR Oxoid ltd., basingstoke,

hampshire, england.
peptone BR Guadong huankai micrdobial

sci&tech, co., ltd.
Sodium chloride AR SCR
Calcium carbonate AR SCR
Sodium hydroxide AR SCR
Natamycin MR Shandong Lunkang

Pharmaceutical CO.LTD,
China
Potassium phosphate AR SCR
35
diabasic trihydrate
Magnesium sulfate AR SCR

heptahydrate
Iron (II) sulfate AR SCR

heptahydrate
Potassium nitrate AR SCR
Agar powder AR Regal Biotechnology

Company
3.4. Natamycin extraction

We have used the following procedure to extract natamycin from dry feed stream (powder from
the FUJIAN HUITIAN BIOLOGICAL PHARMACY CO.LTD.):
1. Mixing the dry feed stream with 75% methanol (with different dilutions in different
bottles) to form the extraction medium;
2. Adjusting the pH at different values (1, 2, 3, 4, 10,11,12 and 13) in order to investigate
the effect of pH on natamycin extraction;
3. Shaking the extraction medium at 200 rpm, controlling the temperature at 28 and take
sample at different interval of time (0.5, 1, 1.5, 2, 2.5, 3, 3.5, 4, 4.5, 5, 6,7, and 8hours) in
order to check effect of time on natamycin extraction;
4. removing solids from the extraction medium to form an extraction liquor by centrifuging
the extraction medium at 10000 rpm for 5 minutes and then adjust the pH of the
extraction liquor at about 7.0;
5. Evaporating the extraction liquor from step 5 using a rotary evaporator at 50-60 till the
solution will turn almost yellow;
6. Put the solution from step 6 into fridge at -4 for overnight;
7. Centrifuge the solution from step 7 to separate the natamycin from extraction liquor and
collect the pallet and wash it with little distilled water ;
8. put pallet into the fridge at -20 for overnight;
9. Take the pallet from the fridge into the freeze dryer till it absolutely get dry;
10. Finally, weighting and check the quality of obtained natamycin powder.
36
Chapter 4: Results and discussion
CHAPTER 4: RESULTS AND DISCUSSION

4.1. Fermentation in shake flask
4.1.1. Effect of initial glucose concentration, in natamycin production medium, on

natamycin production by Streptomyces Gilvosporeus
Earlier experiment has shown that the glucose is among the best carbon source for natamycin
production [12, 53]; therefore, the glucose concentrations in the cultivation medium must be
optimized. For this purpose glucose was applied in different concentrations varied from 0 to
40g/L. The other carbon source and other medium components in original natamycin production
medium were kept constant (starch soluble was 50 g/L) at this time. The results in figure 4.1
showed that natamycin production reached a maximal value at glucose concentration of 5 g/L.
Below and above this concentration, the natamycin production decreased. Therefore, this
concentration was used in the subsequent experiment, to optimize the natamycin production
medium using different medium components, as initial glucose concentration. These results are
in agreement with Martin, J.F. and L.E. Mc Daniel(1977) who reported that In most polyene
antibiotic fermentation higher initial glucose concentration retarded cell growth and resulted a
significant decrease in antibiotic production[56, 57]. It is advisable to start with low glucose
concentration and feed it during the fermentation process in order to keep it at optimum value in
the culture medium instead of start with the whole amount needed during the process[58].
37
4.0
3.5
natamycin concentration (g/L)

3.0
2.5
2.0
1.5
1.0
0.5
0.0
-5 0 5 10 15 20 25 30 35
glucose concentration (g/L)
Figure4. 1. Effect of initial glucose concentrations, in natamycin production medium, on natamycin

4.1.2. Effect of different starch soluble concentrations on cell growth and natamycin
Natamycin production by Streptomyces Gilvosporeus is highly dependent on the composition of
cultivation medium; that is reason during our research, we put more enhance on evaluation of
natamycin production medium components. In order to evaluate the effect of starch soluble on
the production of natamycin by Streptomycin Gilvosporeus, cultivations were carried out with
different starch soluble concentrations in Natamycin production medium. Different starch
soluble concentrations varied from 20 to 200 g/L. when we used 200 g/L starch soluble, the
medium turned almost solid that is reason we did not show DCW at that point in the presented
results (table 4.1). Some spores form the slant culture (after 15 days) was used to inoculate 50mL
of the seed medium into 300 mL Erlenmeyer flask and then the seed culture was carried out on a
rotary shaker at 200 rpm for 41 hours at 28 . Finally, the 3 mL portion of seed culture was
used to inoculate 30 mL of the Natamycin production medium with different starch soluble
38
concentrations in different 250 mL Erlenmeyer flasks and then the fermentation was taken place
in the same conditions as the seed culture for 5 days.
The maximal Natamycin production of about 5.85 g/L was obtained by using 100 g/L starch
soluble concentration in Natamycin production medium while the maximal cell growth was
obtained by using 120 g/L starch soluble concentration (figure 4.2). The Natamycin production
medium contained 110 g/L and 130 g/L carbon source when 100 g/L and 120 g/L starch soluble
were used respectively (together with 10 g/L glucose) in the medium; These results are in
agreement with Eisenschink, MA et al (1997) who reported that the suitable Natamycin
production medium must contain at least 80 to 250 g/L carbon source.
Table4. 1. The effect of different starch soluble concentration on cell growth and Natamycin production
by Streptomyces Gilvosporeus
Starch soluble concentration Natamycin concentration (g/L) DCW (g/L)
20 0.40 26.22
40 0.64 29.01
60 2.22 35.12
80 4.75 35.45
100 5.85 41.96
120 4.66 45.94
150 4.32 40.91
200 0.27 -
39
50 10

45 8
7
40
6
DCW (g/L)
5
35
4
3
30
2
25 1
0
20 40 60 80 100 120 140 160 180 200
starch soluble concentration (g/L)
Figure4. 2. Effect of different starch soluble concentrations on cell growth and natamycin production by
Streptomyces Gilvosporeus
4.1.3. Effect of soybean powder concentration on cell growth and Natamycin production by
The soybean powder is one non-yeast protein nitrogen sources examined during this study. We
evaluated the effect of soybean powder by varying its concentrations from 0 to 40 g/L and
keeping other medium components constant in original Natamycin production medium. The
results were tested after 120 hours of whole fermentation process; the high yield of Natamycin
and cell growth were obtained by using 30 g/L and 40 g/L respectively ( as shown in Figure 4.3).
When there was no soybean powder in the culture medium, the natamycin yield decreased from
the high value of 3.5 g/L to 0.5 g/L. From the above results we find that the soybean powder has
a big effect on cell growth and natamycin production.
40
50 5
45
Natamycin concentration (g/L)

4
40
DCW (g/L)
3
35
30
2
25
1
20
15 0
-5 0 5 10 15 20 25 30 35 40 45
soybean powder concentration (g/L)
Figure4. 3. Effect of different soybean powder concentrations on cell growth and natamycin production
by Streptomyces Gilvosporeus
4.1.4. Effect of yeast extract on the cell growth and Natamycin production by Streptomycin
Gilvosporeus
The yeast extract is among the best complex nitrogen source which are generally supported the
Natamycin production and antibiotic in general. The effect of yeast extract (one of the yeast
protein nitrogen sources components) on cell growth and Natamycin production was studied by
varying its concentration in the Natamycin production from 0 to 30 g/L as shown in figure 4.4.
The high Natamycin yield and high cell growth were obtained when 10 g/L and 30 g/L yeast
extract in the medium were respectively used. The increase of the yeast extract concentration in
the culture medium from 15 g/L to 30 g/L caused the inhibition of Natamycin yield apparently
from 3.11 g/L to 0.17 g/L while the cell growth was somehow increased. From the above results,
the yeast extract concentration in the Natamycin production medium for Natamycin production
should be set at about 10 g/L because it could be suitable for cell growth and Natamycin yield.
41
40 4
38

3
DCW (g/L) 36
34
2
32
30
1
28
26 0
0 5 10 15 20 25 30
Yeast extract concentration (g/L)
Figure4. 4. Effect of different yeast extract on cell growth and Natamycin production by Streptomyces
Gilvosporeus
4.1.5. Effect of peptone on the cell growth and Natamycin production by streptomycin
Gilvosporeus
The peptone is the second yeast protein nitrogen sources component used to optimize the
Natamycin production medium. The effect of peptone was examined by varying its concentration
in the culture medium from 0 to 20 g/L while all others medium components were keep constant.
The results showed that the high cell growth and Natamycin production were obtained at value
of 5 g/L peptone in the culture medium (figure 4.5). These results were the same as those
obtained in the original Natamycin medium (i.e. this value was obtained in the fermentation
broth where the controller medium was used). That concentration was used to develop the new
optimal Natamycin medium.
42
5.0
34 4.5

4.0
32
DCW (g/L) 3.5
3.0
30
2.5
2.0
28
1.5
26 1.0
0.5
24 0.0
0 5 10 15 20
peptone concentration (g/L)
Figure4. 5. Effect of different peptone concentrations on cell growth and Natamycin production by
4.2. Fermentation in the bioreactor

The figure 4.6 shows time course of dry cell weight, dissolved oxygen, pH variation, and
natamycin production of S. Gilvosporeus obtained during fermentation process in 3.6L
bioreactor using original culture medium. The growth curve was not exact and clear well because
we have fed the precursor manual (see appendix 7), which caused some changes in the stationary
phase. After a 24 hours lag phase, the cells started to grow rapidly, the cell dry weight reached a
maximum of 46.6 g/L at 60 hours. The pH value in the medium decreased from 7.27 to near 6.91
in the initial 40 hours, where rapid cell growth was observed. From 40 to 48 hours, the culture
arrived at late log phase and the pH of the medium ranged from 6.96 to 7.42. During this period,
the accumulation of natamycin was increased gradually (Figure 4.6). Natamycin production
achieved a maximum of 5.45 g/L at 120 hours.
43
Table4. 2. Data from fermentation in the 3.6 L bioreactor using the original natamycin production
medium
Time (hour) pH DO (%) DCW (g/L) Natamycin concentration (g/L)

0 7.27 96.6 25.38 -
12 7.34 59.5 30.25 0.554
24 7.05 35.2 40.13 0.885
36 6.91 48.6 45.25 1.316
48 7.01 41.2 44.25 2.137
60 6.96 40.4 46.25 2.933
72 6.99 29.9 44.38 3.394
84 7.17 23.2 38.75 3.924
96 7.42 32.2 37.88 3.9785
108 7.16 34.7 36.88 4.303
120 7.16 34.5 36.13 5.401
14
10 100 60
9 55
12 90
50
8
80 45
10
7
(g/L))
70 40
DO (%)
DCW (g/L)
6 8 35
(Natamycin
60
pH
5 30
6
4 50 25
4 20
3 40
15
2
2 30 10
1 5
20
0
0 0
0 20 40 60 80 100 120
time (hours)
Figure4. 6. Curves of natamycin fermentation in 3.6 L bioreactor
44
4.3. Natamycin extraction results
4.3.1. Natamycin extraction with different dilutions

A 5 g of Natamycin dry feed stream was mixed with 32.5, 45 and 57.5 mL (i.e. 6.5, 9, and 11.5
times respectively) of 75% methanol (pre-adjusted the pH at 3 before mixing) into 300 mL
Erlenmeyer flask i.e. six flasks were at that time because we duplicated each case. The samples
were taken at different interval of time in order to check the natamycin concentration in the
liquid phase. The samples were centrifuged at 6000 rpm for 5 minutes to remove the pallet and
thereafter the supernatant was diluted, using 75% methanol, to a suitable concentration to be
analyzed by HPLC. The results showed that the dilution of 11.5 times was the best one because
the natamycin is stable in the liquid phase (figure 4.7). The natamycin quantity in the liquid
phase increases with increase of the methanol quantity in the extraction medium and concomitant
with the increase of natamycin quantity from the dry feed stream. For example, 0.150, 0.259 and
0.310 g of natamycin were obtained from 5 g of Natamycin dry feed stream when 6.5, 9 and 11.5
times of 75 % methanol were respectively used to extract the natamycin from dry feed stream.
10
6.5times 75% MeOH
9times 75% MeOH
11.5times 75% MeOH
0 60 120 180 240 300 360 420 480

Time (min)
Figure4. 7. Natamycin extraction with different dilutions
45
4.3.2. Effect of pH on natamycin extraction

The pH was adjusted at different values (1, 2, 3, 4, 10, 11, 12, and 13) in the second step of
extraction procedure (see section 3.3); the samples were taken at different interval of time in
order to check the natamycin concentration in the liquid phase. The samples were centrifuged at
6000 rpm for 5 minutes to remove the pallet and thereafter the supernatant was diluted, using
75% methanol, to a suitable concentration to be analyzed by HPLC. The results showed that the
natamycin concentration was high in the liquid phase when the pH is adjusted at 13 but it
decreased as the time increases means there is no stability of natamycin in the liquid phase. It is
advisable to adjust the pH at about 13 if the extraction time is not over 0.5 hour. It is better to
adjust the pH at 2 in step 2 of this extraction process because there is stability of natamycin in
the liquid phase (figure 4.8). Those results were in agreement with Olson, Phillip Terry; Millis,
James R.; Reimer, Michael Henry (1995) who reported that the pH should be first adjusted to 1.0
to 4.5 to dissolve natamycin[59].
10
pH(1)
pH(2)
pH(3)
concentration of natamycin(g/L)
8 pH(4)
pH(10)
pH(11)
pH(12)
6 pH(13)
0
0 60 120 180 240 300 360 420 480 540
Time (min)
Figure4. 8. Effect of different pH on natamycin extraction
46
Chapter 5: Conclusion and recommendations
CHAPTER 5: CONCLUSION AND

RECOMMENDATIONS
5.1. Conclusion
The purpose of this study was to investigate the effects of different culture medium components
concentrations in the culture medium in order to optimize the fermentation culture medium for
natamycin production by Streptomyces Gilvosporeus ATCC 13326. The Natamycin production
medium was optimized by varying one factor while keeping all others constant. The medium
components investigated are the follows: starch soluble, glucose, soybean powder, yeast extract,
peptone and calcium carbonate. The starch soluble, glucose and soybean powder are the most
important medium components which showed a great effect on the growth and natamycin
formation. The results indicated that among the investigated components, the starch soluble
concentration had a significant effect for Natamycin yield.
The optimal starch soluble concentration was 100 g/L form 50 g/L in original natamycin
production medium. By using the optimal starch soluble concentration in natamycin production
medium, the natamycin yield was increased from 2.20 g/L to 5.85 g/L, which is an increase of
2.34 times as compared to that obtained using the starch soluble concentration in the original
natamycin production medium (50 g/L); the results obtained on the behalf of evaluating the
effect of starch soluble in this study have clearly demonstrated that the starch soluble is a critical
component in the natamycin production medium.
Concerning the effect of initial glucose concentration, in the fermentation culture medium, on the
natamycin production, it is better to start with the low glucose concentration in the medium (5
g/L) and feed it during the fermentation process. Toward the end of the fermentation process and
after the major fermentation period, the glucose addition must be discontinued so that little or no
glucose is left at the end of the fermentation cycle (e.g., the quantity of carbon source
substantially equates to the particular quantity of carbon source within the fermentation medium
which is necessary to complete the fermentation process).
47
Chapter 5: Conclusion and recommendations
When the all optimal factors were combined together in order to design the new optimal
natamycin production medium, the natamycin yield was 4.3 g/L which is less than the value
obtained by evaluating the effect of starch soluble. Because of limit time of our research, we did
not get opportunity to evaluate all interaction among factors, we decided to change starch soluble
concentration and maintain the other medium components concentrations at the same value as in
original medium unless the CaCO3 which is reduced from 15 g/L to 7.5 g/L. This optimization
strategy let to a natamycin yield of 5.85 g/L in shake flask, which was nearly 134% higher than
that obtained in the original medium (2.5 g/L).
Concerning Natamycin extraction, the data from extraction showed that the quantity of
Natamycin from fermentation broth could be achieved by using high quantity of methanol to
form the extraction medium. The pH of 75% methanol should be adjusted at 3 before mixing it
with dry feed stream or natamycin fermentation broth.
5.2. Recommendation
The one-at-time strategy is simple and easy, but it has a disadvantage of ignoring some
interactions among the factors. We suggest the further researchers to use statistical method to
investigate the interactions among factors since the culture medium components concentrations
range are known. We also suggest that the further research should clarify the effect of feeding
glucose, into this optimized medium, and optimize the fermentation culture conditions, such as
culture time, controlling pH, inoculum level, culture volume, so forth, during the fermentation
process.
48
References
REFERENCES
[1] Liu, N.; Jin, Z.; Luo, J.; Cen, P., Effects of precursor on natamycin biosynthesis. J Chin
antibiot 2005, 30:328-331.
[2] Thomas, L. V.; Delves-Broughton; Innovation, J. D.; Beaminster; Dorset, Natamycin
(Technical Paper, TP 21-1e). Elsevier Science Ltd: UK, 2005.
[3] Liang, J. G.; Xu, Z. N.; Liu, T. F.; Lin, H. P.; Cen, P. L., Effects of cultivation conditions
on the production of natamycin with Streptomyces gilvosporeus LK-196. Enzyme Microb
Tech 2008, 42(2):145-150.
[4] Suloff, E. C. Comparative study of semisynthetic derivative of natamycin and the parent
antibiotic on the spoilage ofshredded cheddar cheese. Virginia Polytechnic Institute and
State University, Blacksburg, Virginia, 1999.
[5] Suloff, E. C.; Marcy, J. E.; Hackney, C. R.; Sumner, S. S.; Bishop, J. R., Comparative
study of a semisynthetic derivative of natamycin and the parent antibiotic on the spoilage
of shredded cheddar cheese. J Food Protect 2003, 66(8):1499-1502.
[6] http://en.wikipedia.org/wiki/File:Natamycin.svg
[7] Introduction to Natamax Natural Antimicrobial (Technical Memorandum, TM 35-6e).
Danisco A/S
[8] http://www.penglaichem.com/OLDPAGE/Natamycin.htm
[9] Welscher, Y. M. T.; Ten Napel, H. H.; Balague, M. M.; Souza, C. M.; Riezman, H.; De
Kruijff, B.; BreukinkO, E., Natamycin blocks fungal growth by binding specifically to
ergosterol without permeabilizing the membrane. J Biol Chem 2008, 283, (10) :6393-6401.
[10] N.J, R.; Gould. G.W, Food preservatives, second edition. New York, 2003
[11] Guan-Qun, C.; Lu, F. P.; Du, L. X., Natamycin production by Streptomyces gilvosporeus
based on statistical optimization. J Agr Food Chem 2008, 56 (13):5057-5061.
[12] Mohamed, A. F.; A.EL-ENSHASY, H.; I.EL-DIWANY, A.; El-SAYED, A. E. S.,
Optimazation of the cultivation medium for natamycin production by Stretomyces
natalensis. J. Basic Microbiol 2000, 40:157-166.
[13] Xu, G. L., Jianmei; Yang, Deshan; Wang, Min, Optimization of natamycin fermentation
culture medium and conditions. Weishengwuxue Zazhi 2007, 27 (4):73-78.
49
References
[14] Wei, B. W., Xiaochen; Lin, Yushu, Optimizing fermentation medium of high natamycin
producing strain. Beijing Zhongniang Zazhishe 2009, 6:95-98.
[15] Meng, X. W., Lingxia; Zheng, Fenge; Wei, Baodong; Zhang, Qi. , Optimization of
fermentation medium compositions and conditions of fusant Sr-1 of Streptomyces
natalensis and Streptomyces chattanovgensis for producing natamycin. Zhongguo Shipin
Zazhishe 2009, 30(7):172-176.
[16] http://www.molecular-plant-biotechnology.info/use-of-microbes-in-industry-and-
agriculture/microorganism-sterilization-and-disinfection-methods.htm
[17] http://www.molecular-plant-biotechnology.info/use-of-microbes-in-industry-and-
agriculture/microorganism-culture-methods.htm
[18] Vogel, H. C.; Todaro, C. L., FERMENTATION AND BIOCHEMICAL E NG I N E E RI N
G HANDBOOK Principles, Process Design, and Equipment, Second Edition. Noyes
Publications: Westwood, USA, 1997.
[19] Brian, M.; Harvey, L. M., Practical Fermentation Technology. John Wiley & Sons, Ltd:
West Sussex PO19 8SQ, UK, 2008.
[20] Richmond, A., handbook of microalgal culture: biotechnology and applied phycology
Blackwell Science Ltd: Oxford , UK, 2004.
[21] Pelczar, M.; Reid, R., Microbiology. McGraw-Hill: Sydney, 1972.
[22] Giancarlo, L.; Rolando, L., Biotechnology of Antibiotics and Other Bioactive Microbial
Metabolites. Plenum Press: NY, USA, 1993.
[23] Shuler, M. L.; Kargi, F., Bioprocess Engineering Basic concepts, 2nd edition. Prentice Hall:
Upper Saddle River, NJ, 2002.
[24] Casas, L. J.; Sanchez, P. J.; Fernandez, S. J.; Rodriguez, P. E.; Chisti, Y., Pellet
morphology, culture rheology and lovastatin production in cultures of Aspergillus terreus. J
Biotechnol 2005, 116:61-77.
[25] Kalakoutskii, L.; Agre, N., Comparitive aspects of development and differentiation in
actionmycetes. Bacteriological Reviews 1976, 40(2):469-524.
[26] Monnet, D.; Vidal, D.; Creach, O.; , Influence of metabolic and physical factors on
production of diacetoxyscripenol by Fusarium sambucinum kuckel. Applied and
Environmental Microbiology 1988, 54(9) : 2167-2169.
50
References
[27] Zhang, J.; Reddy, J.; Buckland, B.; Greasham, R., Towards consistent and productive
complex medium for industrial fermentations: Studies on yeast extract for a recombinant
yeast fermentation process. Biotechnology and Bioengineering 2003, 82 (6):640-652.
[28] Stanbury Stanbury, P. F.; A. Whitaker, a. S. J. H.; P. F., A. W.; Hal, S. J., Principles of
Fermentation Technology Fermentation Technology,2nd ed. Butterworth Heinemann:
Oxford, 2000.
[29] Cliffe, K., Downstream Processing," in Biotechnology for Engineers, ed. Ellis Horwood
Ltd: England, 1988, 302-321.
[30] Nielsen, J.; John, V.; Liden, G., bioreaction engineering principles, 2nd edition. Kluwer
Academic Plenum New York, Boston, Dordrecht, London and Moscow, 2003.
[31] Atkinson, B.; Mavituna, F., Biochemical Engineering and Biotechnology Handbook.
Macmillan: Surrey, 1983.
[32] Aharonowitz, Y.; Demain, A., Carbon catabolite regulation of cephalosporin production in
Streptomyces clavuligerus. Antimicrob Agents Ch 1978, 14 (2):159-164.
[33] Pirt, S. J., Principles of Microbe and Cell Cultivation. Blackwell Scientific: Oxford, 1975.
[34] Dutta, R., Fundamentals of Biochemical Engineering. Ane Books India: New Delhi -
110002, India, 2008.
[35] Bader, F. G., Chapter 8 Physiology and Fermentation Development. . The Bacteria 1988,
IX, 282-321.
[36] Masuma, R.; Tunaka, Y.; Omura, S., Ammonium ion-depressed fermentation of tylosin by
the use of a natural zeolite and its significance in the study of biosynthetic regulation of the
antibiotic. Journal of Fermentation of Technology 1983, 61:607-614.
[37] Farid, M. A.; El-Enshasy, H. A.; El-Diwany, A. I.; El-Sayed, E. S. A., Optimization of the
cultivation medium for natamycin production by Streptomyces natalensis. J Basic Microb
2000, 40(3):157-166.
[38] Zakriskie, D.; Armiger, W.; Philips, D.; Albano, P., Traders guide to fermentation medium
formulation. Traders Protein 2300 East 50th Avenue: Lubbock, TX 79404 USA, 1980.
[39] Potvin, J.; Fonchy, E.; Conway, J.; Champagne, C. P., An automatic aurbidimetric method
to screen yeast extracts as fermentation nutrient ingredients. J Microbiol Meth 1997, 29:
153-160.
51
References
[40] Basak, K.; Majumdar, S. K., Ultilization of carbon and nitrogen sources by Streptomyces
kanamycesticus for kanamycin production. Antimicrob Agents Ch 1973, 4(1):6-10.
[41] Zhang, J.; Marcin, C.; Shifflet, M.; Salmon, P.; Brix, T.; Greasham, R.; Buckland, B.;
Chartrain, M., Development of a defined medium fermentation process for physostigmine
production by Streptomyces griseofuscus. Applied Microbiology and Biotechnology 1996,
44:568-575.
[42] Hajjaj, H.; Niederberger, P.; Douboc, P., Lovastatin biosynthesis by Asperillus tereus in a
chemically defined medium. Applied and Environmental Microbiology 2001, 67(6):2596-
2602.
[43] Darken, M.; Berenson, H.; Shirk, R.; Sjolander, N., Production of tetracycline by
Streptomyces aureofaciens in synthetic medium. Applied Microbiology 1960, 8, (1), 46-51.
[44] Delves-Broughton, J.; Thomas, L. V.; Williams, G., Natamycin as an antimycotic
preservative on cheese and fermented sausages. Food Aust 2006, 58 (1-2) :19-21.
[45] Wang, D. I. C.; Cooney, C. L.; A.L., D.; Dunnill, P.; Humphrey, A. E.; Lilly; M.D.,
Fermentation and Enzyme Technology. John Wiley: Toronto, 1979.
[46] http://www.thefreedictionary.com/precursor.
[47] http://en.wikipedia.org/wiki/Precursor_(chemistry), In.
[48] Eisenschink, M.; Millis, J.; Olson, P. Fermentation process for producing natamycin with
additional carbon and nitrogen. US patent 5,686,273, NOV. 11, 1997.
[49] Olson, P. T. U.; Millis, J. R. U.; Reimer, M. H. U. natamycin recovery. 09/16/1994
07/03/1996
[50] Luo, J.; Jin, Z.; Cen, P.; Wang, M., The study of high natamycin producing strain
breeding and fermentation condition optimization. Abstracts of Papers, 231st ACS National
Meeting. In Atlanta, GA, United States, 2006.
[51] Liu, N.; Jin, Z.; Jianmei, L.; Peilin, C., Effect of precursor on natamycin biosynthesis.
Zhongguo Kangshengsu Zazhi 2005, 30 (6):328-331.
[52] Luo, J.-m.; Jin, Z.-h.; Cen, P.-l., Effects of cultivation conditions on natamycin production
by Streptomyces gilvosporeus. Zhejiang Daxue Xuebao, Gongxueban 2005, 39 (2):296-300.
[53] Chen, G. Q.; Lu, F. P.; Du, L. X., Natamycin production by Streptomyces gilvosporeus
based on statistical optimization. J Agr Food Chem 2008, 56 (13):5057-5061.
52
References
[54] Wu, J.-Y.; Huang, J.-W.; Shih, H.-D.; Lin, W.-C.; Liu, Y.-C., Optimization of cultivation
conditions for fungichromin production from Streptomyces Pasanus PMS-702. Journal of
the chinese Institute of Chemical Engineers 2008, 39:67-73.
[55] Rybinska, K.; Postupolski, J.; Szczesna, M., Determination of natamycin residues in
ripening cheeses by high-performance liquid chromatography. Roczniki Panstwowego
Zakladu Higieny 1997, 48 (2):173-178.
[56] Martin, J. F.; Mc Daniel, L. E., Production of polyene macrolide antibiotics. Advances in
Appl. Microbiol 1977, 21:1-52.
[57] JIN, Z.; Xiu, C. E.; Peilin, C., Effects of Glucose and Phosphate on Spinosad Fermentation
by Saccharopolyspora spinosa. Chinese J. Chem. Eng. 2006, 14 (4):542-546.
[58] He, Y.; Wu, J.; Lu, F.; Du, L., Fed-batch fermentation to improve the yield of natamycin.
Yaowu Shengwu Jishu 2002, 9(4):224-226.
[59] Olson, P. T.; Millis, J. R.; Reimer, M. H. Natamycin recovery. WO/1995/007998, 23.03.
1995.
53
Appendices
Appendices
Appendix 1: Data for figure 4.1
Natamycin concentration
Glucose concentration (g/L)
(g/L)
0 0.839
5 3.216
10 2.48
20 1.986
30 1.912
Appendix 2: Data for figure 4.2
Soybean powder Natamycin

DCW (g/L)
concentration concentration (g/L)
0 18.7 0.524
5 27.1 1.977
10 30.5 2.022
15 32.4 2.117
30 42.6 3.529
40 46.7 3.004
Appendix 3: data for figure 4.4
54
Appendices
Yeast extract Natamycin

DCW (g/L)
concentration (g/L) concentration (g/L)
0 26.993 1.45
5 31.318 2.23
10 34.218 3.11
15 34.95 2
20 33.5375 0.22
peptone Natamycin
DCW (g/L)
concentration (g/L) concentration (g/L)
0 24.775 1.65
5 33.368 2.45
10 31.537 2.01
15 30.993 1.98
20 29.418 1.96
55
Appendices
Natamycin Natamycin Natamycin

concentration (g/L) concentration (g/L) concentration (g/L)
Time (min)
6.5times 75% 9times 75% MeOH 11.5times 75%
MeOH MeOH
5 8.70984 6.88565 4.98934
10 9.5629 7.89095 5.55225
20 9.72765 8.34167 6.23657
30 9.36649 7.50414 5.87633
45 8.71567 7.99041 6.12367
60 8.84799 7.00002 5.49054
90 7.67046 6.84714 6.33877
120 7.19358 6.57175 4.91048
150 5.89716 4.86512 4.85110
180 5.72024 5.72109 5.26286
210 5.57692 5.70197 5.18132
240 3.71794 5.0234 4.95213
300 3.73009 5.15054 4.73418
360 3.37283 4.88744 4.98424
420 2.8962 4.36383 4.84715
480 3.48578 4.38177 4.93418
56
Appendices
T (min) Natamycin concentration (g/L)
pH=1 pH=2 pH=3 pH=4 pH=10 pH=11 pH=12 pH=13
0 0 0 0 0 0 0 0 0
5 0.67257 2.5143 0.97949 1.17327 1.10189 0.75984 1.14324 0.59244
10 3.24069 4.3269 2.12165 1.97988 2.16756 1.88424 1.6189 6.52061
20 2.35349 4.39729 2.25745 1.97868 2.06444 2.05569 1.74325 6.84837
30 1.99506 4.57397 2.19238 2.19611 2.16415 2.00249 1.94396 8.69325
40 1.62362 4.39398 2.17957 2.14384 2.05776 2.05541 1.72813 7.39883
50 1.25487 4.25088 2.16336 2.03582 1.9476 1.98278 1.81403 6.96437
60 1.16458 4.19495 2.68946 1.91924 1.99918 1.97916 2.01741 7.17492
90 1.00847 4.38845 2.00106 2.21853 2.28277 1.98057 1.88664 7.07843
120 1.43761 4.10182 2.739 2.0999 2.14526 2.38253 2.10031 7.20887
150 0.8664 4.13466 2.3594 2.069 2.20247 1.99458 1.94097 6.46277
180 0.70765 3.95478 2.43258 2.02049 2.10902 1.86753 2.12128 6.10286
210 0.65475 4.45346 2.34472 2.14571 2.1614 2.0342 2.00312 6.18299
240 0 3.92217 2.30285 2.14549 2.18121 2.00697 2.02032 6.13393
300 0 4.05533 2.39215 2.10027 2.14257 2.13995 1.93208 5.86446
360 0 2.45816 2.11933 6.01887
420 0 4.36922 2.36692 2.29086 2.32805 2.03799 2.14121 5.36494
480 0 4.43336 2.32035 2.11645 2.17523 2.14553 2.02543 5.15571
57
Appendices
Appendix 7: Feeding time of the precursor (3.6L bioreactor)
Fermentation time (hours) Precursor quantity (mL)
24 10.36
30 14.8
43 7.4
48 7.4
54 10.36
67 7.4
72 14.8
80 17.76
90 10.36
58

Masters Thesis

Uploaded by

Document Information

Copyright

Available Formats

Share this document

Share or Embed Document

Sharing Options

Did you find this document useful?

Is this content inappropriate?

Copyright:

Available Formats

Masters Thesis

Uploaded by

Copyright:

Available Formats

University Code: 10384

Student ID No.: 20420081153696

MASTERS DEGREE THESIS

Supervisor: Professor Lu Yinghua

A THESIS SUBMITTED IN FULFILLMENT OF THE REQUIREMENTS FOR THE

DEPARTMENT OF CHEMICAL AND BIOCHEMICAL ENGINEERING

COLLEGE OF CHEMISTRY AND CHEMICAL ENGINEERING

Department of Chemical and Biochemical Engineering

College of Chemistry and Chemical Engineering

Xiamen, Fujian Province

Keywords: Natamycin, fermentation, Streptomyces Gilvosporeus, production medium,

38-44 h28 10%24 h0.6%

ABSTRACT (in Chinese) .............................................................................................................. iv

TABLE OF CONTENTS ................................................................................................................ v

LIST OF FIGURES ..................................................................................................................... viii

LIST OF TABLES .......................................................................................................................... x

CHAPTER 1: INTRODUCTION ................................................................................................... 1

1.1. What is Natamycin? ......................................................................................................... 1

1.1.1. Discovery and Origin..................................................................................................... 1

1.1.2. Physical and Chemical Properties ................................................................................. 2

1.2. Motivation and objectives ................................................................................................ 4

1.3. Overview of the thesis .......................................................................................................... 5

CHAPTER 2: LITERATURE REVIEW ........................................................................................ 6

2.1. Microorganism Culture Methods ......................................................................................... 6

2.1.1. Modes of culture................................................................................................................ 6

2.2. Fermentation Technology .................................................................................................... 9

2.2.1. Microbial Growth in Batch Fermentation ..................................................................... 9

2.2.2. Methods to determine the growth curve ...................................................................... 14

2.3. Fermentation media ............................................................................................................ 18

2.3.1. General review ............................................................................................................. 18

2.3.2. Medium components ................................................................................................... 18

2.3.3. Complex medium ........................................................................................................ 20

2.3.4. Defined medium .......................................................................................................... 22

2.3.5. Fermentation medium formulation .............................................................................. 23

2.4. Criteria for design and optimization of fermentation process ............................................ 24

2.4.1. The role of precursor during the fermentation............................................................. 25

2.5. Natamycin inoculum propagation and fermentation .......................................................... 25

2.6. Introduction to separation process of natamycin................................................................ 26

CHAPTER 3: MATERIAL AND METHODS............................................................................. 27

3.1. Materials, Equipments and Media.................................................................................. 27

3.1.1. Microorganisms ...................................................................................................... 27

3.1.2. Media and culture conditions .................................................................................. 27

3.1.2. Equipments ............................................................................................................. 30

3.2. Strategy to optimize the natamycin production medium ............................................... 30

3.2.1. Carbon sources ........................................................................................................ 31

3.2.2. Nitrogen sources ..................................................................................................... 31

3.3. Analytical method .......................................................................................................... 32

3.3.1. Measurement of cell growth ................................................................................... 32

3.3.2. Determination of Natamycin concentration ............................................................ 32

3.3.3. Chemical reagents and solutions ................................................................................ 35

3.4. Natamycin extraction ......................................................................................................... 36

CHAPTER 4: RESULTS AND DISCUSSION ............................................................................ 37

4.1. Fermentation in shake flask................................................................................................ 37

4.1.1. Effect of initial glucose concentration, in natamycin production medium, on

4.2. Fermentation in the bioreactor ........................................................................................... 43

4.3. Natamycin extraction results .............................................................................................. 45

4.3.1. Natamycin extraction with different dilutions ............................................................. 45

4.3.2. Effect of pH on natamycin extraction.......................................................................... 46

CHAPTER 5: CONCLUSION AND RECOMMENDATIONS .................................................. 47

5.2. Recommendation ................................................................................................................ 48

Figure1. 2. The structure of Natamycin in 3 dimensions................................................................. 2

Figure2. 1 Microbial Growth in Batch Fermentation [23] ............................................................ 10

Figure2. 3. The cell growth which is substrate limited [9]............................................................. 12

Figure2. 4. Spectrophotometer pictured above .............................................................................. 14