Faculty of Biotechnology

The Effect of Different Parameters on Fermentation of Neisseria

Meningitidis to Produce Polysaccharide "A" Vaccine

By:

Zaid Mohamed Wasfi Hisham El-Husseini

125457

Under supervision of:

Internal supervisor:

Dr. Ahmed M. K. Nada

Associate Prof. in MSA University

External supervisor:

Dr. Lamiaa Salah ElDin Shaker

Manager of Pilot Plant unit-VACSERA

Spring 2015

Table of Content:

Subject Page No.

Acknowledgement 7

List of figures 8

List of tables 9

List of Abbreviations 10

Abstract 14

Introduction 15

1. Historical background 15

2. Description of meningococcal disease 16

3. Neisseria meningitidis bacteria 18

4. Pathology of Neisseria meningitidis 20

5. Symptoms of meningococcal disease 21

6. Epidemiology of meningococcal disease 22

7. Diagnosis of meningococcal disease 24

7.1. Imaging 24

7.2. Lumbar puncture 24

7.3. Other laboratory tests 24

8. Transmission of meningococcal disease 28

9. Immunization of meningococcal disease 28

10. Meningococcal vaccines 30

10.1. Meningococcal Quadrivalent

Polysaccharide Vaccine 30

10.2. Quadrivalent Meningococcal

Conjugate Vaccines 31

10.2.1. MenACWY-D 31

10.2.2. MenACWY-CRM 31

10.3. Hib-MenCY-TT 32

11. Fermentation 32

11.1. Batch Cultures: 33

11.2. Continuous Cultures 33

11.3. Fed-Batch Cultures 34

12. Phases of bacterial growth 34

12.1. Lag phase 35

12.2. Log phase 35

12.3. Stationary phase 35

12.4. Death phase 36

Materials 37

1. Biological materials 37

1.1. Strain 37

1.2. Chemicals and Reagents 37

1.3. Buffers 39

1.4. Media 39

1.4.1. Preparation of solid media 39

A. GC agar base medium 39

B. Hemoglobin 40

1.4.2. preparation of liquid medium 40

A. Frantz media 40

B. Preparation of solution A 40

C. Preparation of solution B 41

D. Preparation of Yeast Extract 41

E. Preparation of the medium 41

1.5. Equipments 41

1.6. Disposable 42

Methods 44

1. Organism and growth 44

2. Seed Preparation 44

3. Inoculums Preparation 44

primary inoculum 44

secondary inoculum 45

4. Fermentation 45

A. Installation 45

A.1. Vessel Assembly 45

A.2. Impellers insertion 45

A.3. Retention rings installation 46

A.4. Sparger installation 46

A.5. Harvest tube installation 46

A.6. Thermowell insertion 46

A.7. Sampler installation 46

A.8. Head plate installation on vessel 46

A.9. pH probe installation 47

A.10. DO probe installation 47

A.11. Vessel installation 47

A.12. Probe preparation and calibration 47

1- DO probe 47

2- pH calibration 48

B. Sterilization 48

C. Vessel reinstallation 49

D. Fermentation Data Sheet 50

E. DO cascade system 51

F. Inoculation 51

G. Sampling 52

5. Harvesting 52

6. Estimation of cell mass 53

Results 54

1-Seed preparation 54

2- Inoculums preparation 55

Primary inoculums 55

Secondary inoculums 55

3-Fermentation 56

3.1. Estimation of bacterial growth 57

3.2. Estimation of pH 58

3.3. Determination of dissolved oxygen 59

3.4. Cell mass estimation 60

Discussion 61

References 66

Acknowledgement:

I would like to take the opportunity to extend my deepest appreciation to

everyone who has made this project possible. I would like to thank VACSERA and

my supervisor Dr. Lamiaa Salah El Din for her endless guidance,

I have sincerely benefited from her experience and have been truly educated

these past 3 months. I could have never completed this write up without the endless

support of my internal supervisor Prof. Dr.Ahmed Nada whose meticulous character

has enabled the completion of the dissertation.

I would like to thank the University, its owner Prof. Dr. Nawal El Degwi and

Dr. Khairy our university president. Our beloved Dean Prof. Dr. Ayman Diab and

Prof. Dr. Gehan Safwat our course coordinator who enabled the allocation into this

venue and has always organized this unique experience, Prof. Dr. Osama Saad who

has been a mentor to us all. And all the other staff members for their continuous

support and efforts they are partly responsible for the individual I am today.

Finally I would like to extend the deepest appreciation for my family specially

my father Eng.Mohammed Wasfi El-Husseini and my gorgeous mother Mrs. Rafah

Al-Rayyes who have both been my back bone and friends and in particular Mr. Alaa

Al-Nomrosi, Ms. Gihan Hammad, Ms. Nancy Karem and Ms Rama Azhari have

always been The people that keep me on the positive end especially when things have

seemed darker, I thank you all and I could not have done this without you.

List of figures:

Figure # Description Page number

Figure (1) Meningitis cases statistics 17

Figure (2) Meninges layers 17

Figure (3) Neisseria meningitidis cell

structure 19

Figure (4) The appendages and cell envelope

of Neisseria meningitidis 19

Figure (5) Illustration of the invasion of

Neisseria meningitidis 21

Figure (6) Epidemiology of Neisseria

meningitidis 22

Figure (7) Neisseria meningitis sero-groups

around the world. 23

Figure (8) Flow chart of a N. meningitidis identification

25

Figure (9) Oxidase test 27

Figure (10) Phases of bacterial growth 36

Figure (11) Gram staining of N. meningitidis 45

Figure (12) Inoculums preparation of N.

meningitidis 55

Figure (13) Optical density 57

Figure (14) pH value 58

Figure (15) Dissolved oxygen percentage 59

List of tables:

Table # Description Page

Table( 1) Fermentation Data

Sheet table

50

Table (2) experimental results

obtained with batch A

and B

56

List of abbreviations:

Acronym Definition

Ab Anti-bodies

BAP Blood Agar Plate

CAP Chocolate Agar Plate

CDC Centers for Disease Control

Conc. Concentration

CPS Capsular polysaccharide

CSF Cerebrospinal fluid

CT Computerized tomography

CTA Cystine Trypticase Agar

d.H2O Distilled Water

DIC Disseminated intravascular coagulation

DNA Deoxyribonucleic Acid

DO Dissolved oxygen

Acronym Definition

FDA Food and Drug Administration

g/L Gram per liter

GMT Geometric mean titres

H. influenzae Haemophilius influenzae

Hib Haemophilus influenzae type-b

IgG immunoglobulin G

IgM immunoglobulin M

Kg Kilogram

L Liter

LOS Lipooligosaccharide

LPS Lipopolysaccharide

M Molar

MC Meningococcus

MCPSA Meningococcal capsular polysaccharide sero-group A

Acronym Definition

MenACWY

CRM

Meningococcal Quadravalent ACWY sero-groups

conjugated with non-toxic mutant diphtheria

Mg Milligram

min. minutes

Ml Milliliter

MPSV4 Meningococcal Quadravalent polysaccharide vaccine

M. wt. molecular weight

Nm Nanometer

N.

meningitidis

Neisseria meningitidis

OD Optical Density

pH potential hydrogen

Rpm revolutions per minute

SASG Slide Agglutination Sero-Grouping

T cell Thymus cell

U/L Unit per liter

Acronym Definition

WFC Waterhouse-Friderichsen Syndrome

WHO World Health Organization

YE Yeast Extract

4vMenCV Quadrivalent meningococcal conjugate vaccines

µg Microgram

µl Microliter

µm Micrometer

°C degrees Celsius

% Percent

Abstract:

Meningococcal disease is rare but severe disease that causes meningitis,

septicemia or both of them, Egypt is one of the high risk regions of epidemic of this

disease which is caused by the bacteria Neisseria meningiditis that contains 13 sero-

groups. Neisseria meningitidis serogroup A constitutes the antigen for the vaccine

against meningitis disease. The goal of this work was to show the effect of some

different parameters as pH and dissolved oxygen on the growth curve and cell mass.

These kinetic parameters were carried out in two batches three litters of Frantz media

in 5 L fermentor (New Brunswick) the first batch (A) was at 36 ºC, air flow 2 L/min,

agitation frequency 200 rpm, dissolved oxygen reached to 10% and adjustment pH.

Another batch(B) was carried out under different conditions the pH not adjusted, DO

cascade system was applied maximum set points of agitation was 400 rpm, air flow

4L/min and DO was arrived to 40%. The highest cell mass (0.85 g/L) was obtained in

batch A when the pH was adjustment and DO was 10% while, another batch was low

cell mass (0.46 g/L). An empirical relation is proposed to relate the specific cell mass

rate during stationary growth phase of the batch A. pH parameter was the main factor

that affect fermentation process, even it increase airflow and agitation.

Key words:

N. meningitidis, culture medium, batch cultivation, meningococcal vaccine sero-group

A.

Introduction:

1. Historical background:

Scientists and Physicians were very curiosity about the meningococcus and

meningococcal disease for more than 100 years.

Meningococcal disease was first described by Vieusseux in 1805. After his

report by two years, an epidemic of "spotted fever" was raged through New England,

and there was a large loss that sustained in the Prussian Army from the same disease

in the same year. After the characterization of these outbreaks, the disease has become

well-known in Europe, Asia, and America. Steiner's book on military diseases during

the American Civil War was referring to several reports whose characterization very

close to meningococcal disease. However, Weichselbaum was first one described the

meningococcus in detail that was not until 1887, and Councilman In 1898 found

meningococci in 31 of 34 cases of meningitis, firmly established that this organism as

the etiological agent of epidemic cerebrospinal meningitis. (DeVoe, 1982)

Although a lot has been written about this gram-negative pathogen and an

assortment of clinical manifestations of meningococcal disease, In fact not too much

is known about the direct relationship between the biochemistry and the physiology of

the microbe, on one hand, and the pathogenesis of disease on the other hand. (DeVoe,

1982)

2. Description of meningococcal disease:

Meningococcal disease was described by Vieusseux in 1805 is rare but severe

disease that causes meningitis and septicemia or both of them, this disease is caused

by the bacteria Neisseria meningiditis that have 13 sero-groups (A, B, C, D, H, I, K,

L, W135, X, Y, Z, and 29E (Z')) the most lethal are sero-group (A,B,C,Y and W135),

taking in consideration that 5-10 % survive after being infected and from 10-30%

suffer from acute side effect like deafness, skin damaging and neurological problems

(NCIRS, 2014). Luckily this disease has various vaccines according to the type of

microorganism and the age of the recipient.

Meningitis develops fast between new born (0-4) and young people (15-24).

The disease becomes more morbidity and may lead to death, as it claim its' peak in

1980s and then declined (Cohn, 2013) as it shown in figure (1).

Meningitis is one of the common manifestations that are caused by the

meningococcal disease that form an inflammation in the layers around the brain

(meninges) shown in figure (2) and spinal cord. Meningitis can be caused by bacteria,

viruses, parasites or fungi, the virus infection is the most common one but the bacteria

is more serious. The most common bacteria that cause meningitis are Neisseria

meningiditis, Haemophilus influenza and Streptococcus pneumonia (Liorens and

George, 2013).

Figure (1): In the left side Statistics shows increase and decline of the disease in the

past 40 years showing that it reached the highest number of cases in 1980 and in the

right side another statistics show that according to age cases between 15-24 years

have the greatest percentage.

adapted from: http://www.nbcnews.com/id/7994214/ns/health-childrens_health/t/new-meningitis-

shots-urged-kids/

Figure (2): diagram showing the layers of meninges protecting the brain and

spinal cord. Meninges consist of Dura mater, Arachnoid mater and Pia mater.

Adapted from: https://embryology.med.unsw.edu.au/embryology/images/f/f3/Meninges_cartoon.jpg

Septicemia is another manifestation caused by meningococcal disease that has

other names like bacteremia or blood poisoning. if septicemia remained untreated it

can cause sepsis that leads to inflammation all over the body that can result in organ

failure and even death. The main cause of Septicemia is a bacterial infection in lung,

urinary track or abdomen that can penetrate in to the blood stream. (O'Connell, 2012)

3. Neisseria meningitidis bacteria:

Higher order taxonomy:

Bacteria>Proteobacteria>Betaproteobacteria>Neisseriales>Neisseriaceae>Neisseria.

(NCBI taxonomy)

Neisseria meningitidis is a Gram negative bacteria that is found normally in 10

to 30 percent from the population considered as carriers, commensal in the

nasopharynx within the respiratory path of healthy persons, that have a spherical

shape range from (0.6-1µm) aerobic, non-motile found in pairs, and considered the

leading cause of meningitis in children, Neisseria meningitidis strains are categorized

in to 13 sero-groups varies in there outer membrane capsule of polysaccharide and the

most invasive are sero-groups A,B,C,Y and W125 varies in there distribution around

the world (Darryl et al, 2010). Neisseria meningitides as other capsulated bacteria

contains polysaccharide capsule, cell wall, cell membrane, cytoplasm, ribosomes and

the genetic information are held on its DNA as shown in figure (3) below.

Figure (3): Neisseria meningitidis cell structure.

adapted from: https://www.pinterest.com/meningitidis/neisseria-meningitidis/

As Hill, (2010) mentioned too Neisseria meningitidis considered to be an

immunogenic difficult not just because of the variant of the polysaccharide but there

is another virulence factors like lipopolysaccharide and other proteins that assist in the

adherent of the bacteria on a surface as can be seen in figure (4).

Figure (4): The appendages and cell envelope of Neisseria meningitidis.

Adapted from: http://www.chori.org/Principal_Investigators/Moe_Gregory_R/moe_research.html

4. Pathology of Neisseria meningitidis:

Neisseria meningitidis transfer throw respiratory system and humans are the

only host of it by sneeze, coughs, kissing and nearby breathing. The infection begins

with the adhesion of the bacteria on the muscosal membrane surface in the

nasopharynx aria using its pilli as you can see in figure (5). And then they make their

own way throw until they reach the blood stream and from here they find their way to

the meninges around the brain, three characteristics make this bacteria one of the most

dangerous pathogens (Lappann., 2006).

Sero-group (A,B,C,W135 and Y) from the 13 sero-groups how have a

polysaccharide capsular coat protecting them from phagocytes, secondly the coding

genes of the pelus are very flexible making it more pathogen and finally the highly

production of endotoxin lipopolysaccharide (LPS) throw the exponential phase

(DeVoe I. 1982). The metabolism of Neisseria meningitidis based on the reduction of

iron that can be found in the human heme iron with other nutrients in the blood make

it as a growth media (Lappann., 2006).

Figure (5): An illustration of the invasion of Neisseria meningitidis inside the blood

stream.

Adapted from: http://guillaumedumenillab.weebly.com/neisseria-meningitidis.html

5. Symptoms of meningococcal disease:

The symptoms of meningitis and septicemia caused by meningococcal disease

might not be typical but in general and as Dr.Ferguson says that it begins with normal

flu symptoms with fever, rash, vomiting, tiredness, hallucination. And may develop

over few hours in to purpura (which is a purple or red spots on surface of the skin)

disseminated intravascular coagulation (DIC), coma, shudder and then death as a

result of the Fulminant meningococcemia known as water-house-friderichsen

syndrome(WFS). (Frguson, 2002)

6. Epidemiology of meningococcal disease:

Many of individuals from 10-25% of the population are holding Neisseria

meningitides in their noses and throats, but those hosts do not develop the disease

because of their immune defenses that rejects the bacteria from interring the body, and

in some cases it can overcome the defense mechanisms and develop the disease

(WHO, 2015).

Epidemic meningococcal disease can be found in all countries as seen in

figure (6), as the most category infected are children younger than 5 years, this cases

can be estimated from 1 to 3 every 100,000 annually (WHO, 2015).

Figure (6): This map shows the meningitis belt in red, the high risk regions of

epidemic in brown and in gray is all places that has a sporadic cases only (2004).

Adapted from: http://www3.chu-rouen.fr/Internet/services/sante_voyages/pathologies/meningite/

Some developing countries are showing an epidemic of meningitis frequently,

the most groups found are A,B,C,W135 and Y. and as can be seen in figure (7), sero-

group B and C are most common in Europe and the Americas , also A and W135 can

be found mostly in Africa meningitis belt and Far East (WHO, 2015).

Figure (7): The distribution of Neisseria meningitis sero-groups around the

world.

Adapted from: http://www.phac-aspc.gc.ca/publicat/ccdr-rmtc/09vol35/acs-dcc-4/gfx/figure1-eng.jpg

7. Diagnosis of meningococcal disease:

Diagnosis begins with searching for medical history and then there are some physical

exams and certain diagnostic tests. Those tests include checking some parts of the

body like head, throat and skin alongside the spine (CLINIC, 2013).

7.1. Imaging:

Taking an X-rays or computerized tomography (CT) that will show if there is an

inflammation in the brain tissue, or may help in checking the rest of the organs that is

associated with meningitis (CLINIC, 2013).

7.2. Lumbar puncture:

This test has another name as spinal tap. This test is vital because it looks in the

cerebrospinal fluid (CSF) for the level of glucose, protein and white blood cells, the

meningitis patient low glucose but an increase in protein and white blood cells

(CLINIC, 2013).

7.3. Other laboratory tests:

A laboratory tests are performed to determine the existence of Neisseria Meningitides

and the type of sero-group. Those tests are inexpensive and save time compared with

others (CDC, 2012). Those test are preformed according to the chart in figure (8).

Figure (8): Flow chart for identification and characterization of a N. meningitidis

isolate.

Adapted from: http://www.cdc.gov/meningitis/lab-manual/chpt07-id-characterization-nm.html

The first step is by culturing the bacteria in a Blood Agar Plate (BAP) or

Chocolate Agar Plate (CAP) and its ideal conditions with a temperature about 35-37oc

and 5% CO2 or placed in a candle jar. After 18-24 hours the morphology of the culture

can be observed as smooth, rounded, glistening and moist. To make sure that these

bacteria cultured are Neisseria meningitides an oxidase test is done if it is positive, a

carbohydrate utilization test is taking place, and then to know which sero-group of

bacteria is dealt with, a Slide agglutination sero-grouping (SASG) test is performed

(CDC, 2012).

Kovac's oxidase test, is a test done to detect the presence of cytochrome

oxidase. If it is present the Kovac's oxidase reagent which is tetramethyl-p-

phenylenediamine dihydrochloride will turn in to purple, this reaction happen if the

microorganism contains a cytochrome c in there respiratory chain. The problem is that

Neisseria meningitides is not the only one that gives positive reaction result but

Neisseria genus and other bacteria may give false positive. This test maybe used as

detergents or filter paper method (CDC, 2012).

If the oxidase test is positive, carbohydrate utilization (CTA) test is done to

insure that the bacteria is Neisseria meningitides. This test is performed by adding 4

different carbohydrates which are (glucose, maltose, sucrose, and lactose) into 4 tubes

containing cystine trypticase agar and phenol red as a sensitive indicator that will give

a yellow color in the presence of acid in pH less than 6.8 as can be seen in figure (9).

Neisseria meningitidis is the only one that oxidase just maltose and glucose from

Neisseria species, and that could be the key to identify it (CDC, 2012).

Figure (9): cystine trypticase agar oxidase test showing the positive of Neisseria

meningitides in tube the first and second tube that contain maltose and dextrose

(glucose).

Adapted from: http://www.cdc.gov/meningitis/lab-manual/chpt07-id-characterization-nm.html

The last test is to identify which sero-group of Neisseria meningitides by

performing (SASG) test. There are 13 sero-groups for Neisseria meningitides and

there are commercial antisera for them. But not all antisera are tested practically, just

the ones that is more likely to be within that geographic region. For example, if it was

in Africa antisera A and W135 are used. In this test using saline as a control is essential

to detect nonspecific autoagglutination (CDC, 2012).

8. Transmission of meningococcal disease:

Neisseria meningitis transmitted through respiratory droplets or throat

secretions form person to person (human), knowing that it does not infect animals.

The transmission can be done by close contact like coughing, kissing or sneezing, or

being around the infected person or the carrier such living in the same dormancy, or

being in crowded places as in some religious ritual (alHajj) (WHO, 2015).

As this bacteria (Neisseria meningitis) infects humans only, it can be found in

the throat and for not fully understood reasons it can penetrate the defenses and infect

the blood stream continuing to the brain causing inflammation on its layers. And it is

believed that from 10 to 20 percent of the population are carriers, but this rate can be

higher in the epidemic regions (WHO, 2015).

9. Immunization of meningococcal disease:

The body is exposed to many external influences like harmful radiation,

mechanical and chemical factors causing burns and wounds, other than that it is

exposed to microorganisms like bacteria, viruses and many others that can cause

diseases, the body in return uses alternative defensive mechanisms to protect himself

against those external influences (NIAID, 2003) .

Many host defense mechanisms occurring during meningococcal infection are

driven by anti-bodies (Ab), being complement activation by bacterial Ab as the most

important in terms of protection (Sanchez et al., 2002). The efficiency of anti-

meningococcal serum in the reducing mortality during the pre-antibiotic era provided

evidence supporting the importance of humeral immunity in resistance to

meningococcal disease (Flexner, 1913). Humeral immunity to N. meningitidis sero-

groups occurs between 2-12 years of age. However, there is a progressive annual

increase of approximately 5% in the proportion of children with Ab against

meningococcal sero-groups. Moreover, marked decrease in the incidence of disease

occurred at 6 to 8 years of age and more than 90% of children have Ab to sero-group

C (Goldschneider et al., 1969).

Antibody voracity has been used as an alternate marker of immunological

memory, because T cell-dependent antigens are normally change in affinity over time

after vaccination and there is rapid production of high-affinity responses after

increasing with such antigens (Borrow et al., 2002).

Children less than 2 years old and young infants are the major group that can

be infected with N. meningitidis knowing that polysaccharide vaccines are poorly

immunogenic in this group and this type of vaccines does not have the ability to

induce immunological memory that is because they stimulate the production of IgM

and IgG2 antibodies which are weak activators for complements more over they fail

induce antibody avidity maturation and isotype switching (Girard et al., 2006).

Changes in the ratio of IgG1/IgG2 after vaccination can indicate the activation

of cellular control mechanisms typical of a T cell-dependent response (Findlow et al.,

2006).

Polysaccharide vaccines have restricted effect on asymptomatic carriage and if

there is an effect on immunity in general it would not last long. Polysaccharide

vaccines can induce hypo-responsiveness such that repeated dosages of

polysaccharide vaccines do not achieve geometric mean titres (GMT) as high as those

achieved after the first dosage. That raises worries that the individuals who have been

vaccinated would not show the optimal immune response when they exposed to N.

meningitides increasing the possibility of the infection and susceptibility to disease

specially in children between 3 to 5 (Canada communicable disease report, 2007).

Immunogenicity studies can predict the short-term effectiveness as for the

long term effectiveness cannot be predictable always, and those studies do not predict

the effect of vaccination on carriage and herd immunity (Canada communicable

disease report, 2007).

10. Meningococcal vaccines:

There are two types of meningococcal vaccines either purified capsular

polysaccharide vaccines or conjugated vaccines, for each type there are deferent

vaccines with deferent age target but all of these vaccines are used for the prevention

from meningococcal disease sero-groups A, C, W and Y (CDC, 2012).

10.1. Meningococcal Quadrivalent Polysaccharide Vaccine (MPSV4):

MPSV4 is a purified polysaccharide vaccine that was licensed in 1981. This

vaccine is for sero-groups A, C, W135 and Y, and for individuals who are more than 2

years. This type of vaccine is taken as a single dose each dose consist of 50 µl for

each of the four sero-groups purified polysaccharide (CDC, 2012).

10.2. Quadrivalent Meningococcal Conjugate Vaccines (4vMenCV)

4vMenCV are type of vaccines with purified capsular polysaccharide that is

conjugated to a protein carrier contains T-lymphocyte epitopes that convert the

immune response from T-lymphocyte–independent to T-lymphocyte–dependent.

These types of conjugated vaccines have improved the immune primary response to

the antigen, mainly in children and tough immunogenic memory (CDC, 2012).

And there are two types of (4vMenCV).

10.2.1. MenACWY-D

MenACWY-D was licensed in January 2005 by FDA, this vaccine is taken as

a single dose for people between 2 and 55 years and 2 dose for infants between 9 and

23 months. This 0.5ml single dose contains 4 µg of each capsular polysaccharide from

the 4 sero-groups conjugated to nearly 48 µg of diphtheria toxoid. MenACWY-D is in

the market in single dose vessels and taken as an intramuscular injection(CDC, 2012).

10.2.2. MenACWY-CRM

MenACWY-CRM this vaccine is taken as a single dose for individuals

between 2 and 55 years. The 0.5 ml single dose consists of 10 µg of purified capsular

polysaccharide of sero-group A and 5 µg purified capsular polysaccharide of sero-

group C, Y and W that is conjugated to 33-64 µg of CRM197 which is a non-toxic

form of diphtheria toxin obtained from Corynebacterium diphtheriae. This vaccine is

prepared by reconstructing the lyophilized serogroup A conjugate with the liquid

serogroups C, W, and Y conjugate components (CDC, 2012).

10.3. Hib-MenCY-TT

Hib-MenCY-TT was licensed and approved in june 2012 by FDA. This

vacscine is taken as 4 dose series for infants between 6 weeks and 18 months. And it

is consist of a lyophilized, sterile powder reconstructed with saline diluent for

intramuscular injection. The components of 0.5 ml vaccine are 5 µg of polysaccharide

sero-group Y conjugated with 6.5 µg of tetanus toxoid, 5 µg of polysaccharide sero-

group C conjugated to 5 µg of tetanus toxoid and 2.5 µg capsular polysaccharide of

Haemophilus influenzae type b conjugated to 6.25 µg of tetanus toxoid (CDC, 2012).

11. Fermentation:

Fermentation starts under anaerobic conditions with no presence of light and

absence of electron acceptors. The bacteria goes under this situation are anaerobic or

facultative anaerobic that gain the ability to catabolize the organic compound by the

concept of oxidation-reduction reactions, by this concept the organic compound is

served as electron donor and acceptor, as the adenosine triphosphate is synthesized by

the process of phosphorylation. Taking in consideration in modern fermentation

processes aerobic conditions are taking place and maintained in a closed fermenter with

submerged cultures, the contents of the fermenter are agitated with an impeller and

aerated by forcing sterilized air (Muller, 2001).

There are three methods for fermentation according to the addition and

harvesting which are batch culture, fed-batch culture and continuous culture.

11.1. Batch Cultures:

The batch operation is about a closed system, means that the medium

components and row materials are added in the beginning of the fermentation and not

during the process, the same thing for the outcome product either it was extracellular

or intracellular it has to be harvested after the end of the process. For the parameters

which are temperature, pH and dissolved oxygen (DO), those parameters are held

constant throw the operation in the batch reactor. The only optimization parameters

are the initial medium composition. The initial medium composition has been the only

optimization parameters. While, the pH and temperature of the profile optimization

may cause an improvement in the performance over the operation that may carried out

at constant pH and temperature (Henry and Sung Shin, 2011).

11.2. Continuous Cultures

The fed in the continuous operation are continuously, in which the nutrients

can be feed more than one. On the other hand, the effluent stream removes the cells,

residuals, and products continuously. Also the steady state is done by preserving the

volumetric flow rate equally for the effluent streams and feeding. And thus at the

steady state values, nutrient concentration and volume will stay constant. Other

industries like chemical industry the continuous reactor is preferred, as the production

of certain beer, single cell protein, and waste treatment procedures. But the

continuous cultures are not commonly used as a major culture for the industries,

because of its complexity in the protection of attacking phages or mutation, and

maintaining sterile condition. Studies say that the dynamic operation has more yield

than the steady state operation, because of unknown reason (Henry and Sung Shin,

2011).

11.3. Fed-Batch Cultures

In fed batch culture or semi-batch operation the media that is necessary for the

cell growth and production is added continuously or in separated time to the culture in

two or more feed streams. But like the batch culture the product are harvested in the

end of the process as full harvest or it can be partially harvested. The fed batch culture

can be repeated a number of times if the bacteria were fully viable and can produce

more product. Sources of phosphates, nitrogen, carbon, nutrients or inducers are fed

either in continuous or discontinuous way into the culture by operating the feed

amounts during the run (Henry and Sung Shin, 2011).

12. Phases of bacterial growth:

Bacteria goes throw 4 phases which are Lag phase, exponential (Log) phase,

stationary phase and death phase, as seen in figure (10), depends on the number of

bacteria and factors that affect the growth. They're

12.1. Lag phase:

The growth of the inoculated bacteria does not occur immediately but takes a little

while, during this time the bacteria are introduced and adapted to the medium and

break down the substances in the growth medium by the enzymes that the bacteria

have synthesized. The cells in this time are very active metabolically, but the number

of cells changes very slowly.(Todar, 2008)

12.2. Log phase:

The growth rate of the bacteria steadily increases and reproduces very quickly at the

same time the bacteria introduced and adapted to the new medium. The population

doubles every generation and there is a rapid cell growth. A plot of the log number of

cells is against the time gives a linear relationship; in this phase the cells are at their

greatest activity. The cells can be maintained active by transferring cultures to the

fresh medium at regular period of time. An active culture can quickly control any new

environment easily (Todar, 2008).

12.3. Stationary phase:

After certain time of log phase, the growth rate of bacteria slow down, due to

continuously reducing the nutrients concentration, or increasing the accumulation of

toxic waste products. At the same time, the cells are dying off: A state of equilibrium

is achieved between the formation of new cells and the death of the old cells. So that

the death rate equals the rate of reproduction, resulting the cells number remain

constant. This phase is called the stationary phase (Todar, 2008).

12.4. Death phase:

After the stationary phase, the formation of new cells ceases and the culture enters a

steady state. The biomass remains constant, except when definite accumulated

chemicals in the culture lyse the cells. So that the death rate equals the rate of

reproduction, due to the limiting factors in the environment. This is called the death

phase (Todar, 2008).

Figure (10): this graph is showing a classical growth curve of bacteria with its phases,

Adapted from:

http://upload.wikimedia.org/wikipedia/commons/thumb/c/c0/Bacterial_growth_en.svg/2000px-

Bacterial_growth_en.svg.png

Materials:

1. Biological materials

1.1. Strain:

Neisseria meningitidis strain number M1027 serotype A was kindly obtained

from American Type Culture Collection number 13077 (ATCC). The strain used is

recommended by the WHO, for polysaccharide production (Branham, 1958).

1.2. Chemicals and Reagents:

Reagents and

chemicals

Company

Ammonium chloride Riedel-de Haën, M wt 53.49

Antifoam 204 Sigma

Crystal violet Sigma, M wt 408

Dextrose Difco (USA)

Ethanol 95% Panreac, M wt 46.67

GC medium Difco (USA)

Glycerine Reidel-deHaën, M wt 92.09

Hemoglobin freeze

dried

Difco (USA)

Reagents and

chemicals

Company

Hexadecyl - trimethyl-

ammonium bromide

(Cetavlon)

Sigma, USA , M wt 364.46

Iodine Panreac

IsoVitalex Becton, Dickinson and Company (BD, BBL, USA)

L- Cysteine

hydrochloride

BDH, M wt 157.62

L-Glutamic acid Sigma, M wt 147.1

Magnesium sulphate

dehydrat

Reidel-deHaën, M wt 246.47

O2 electrolyte Mettler –Toledo GmbH

Potassium chloride BDH, M wt 74.55

Potassium iodide Adwic,- M wt 166

Safranin Sigma

Sodium chloride Riedel-de Haën, M wt 58.44

Sodium hydroxide Fisher scientific company M wt 40.0

Sodium phosphate

diabasic

Riedel-de Haën M wt 177.99

Yeast extract Difco (USA)

1.3. Buffers:

Buffer solution pH 7 Sigma, (USA)

Buffer solution pH 4 Sigma, (USA)

Buffer solution pH 10 Sigma, (USA)

1.4. Media:

1. Preparation of solid media

A. GC agar base medium

7.2 g of the powder was suspended in 100 ml distilled water (d. H2O), mix thoroughly

and heated with frequent agitation then boiled for 1 min to completely dissolve the

powder. The media was sterilized by autoclaving at 121ºC for 15 min. Cooled to 45 to

50 ºC. 100 ml was added aseptically to sterile hemoglobin solution and 2ml

supplement isovitalex mix well, and poured in plates to the thickness of

approximately 4mm.the plates were allowed to be completely cooled and dried. They

were stored in plastic bags at +4ºC. A sterility test was done for the media plates by

putting a plate in the incubator overnight.

B. Hemoglobin

10 g of hemoglobin was dissolved in 1 litter of medium. The hemoglobin was

sterilized by autoclaving at 121ºC for 15 min. while it still hot, the solution was

combined with GC base medium and mix thoroughly before dispensing into

appropriate containers.

2. preparation of liquid medium

A. Frantz media

The modified Frantz medium was supplemented with yeast extract and solution B.

The medium contains neither high molecular components, nor components that will

form a precipitate upon the addition of the cationic detergent hexadecyl –trimethyl-

ammonium bromide (Cetavlon) (Frantz, 1942).

B. Preparation of solution A:

1.3 g. of L-Glutamic acid, 0.02g of L-Cysteine-HCl, 2.5g of Na2PO4.H2O, 0.09g of

KCL , 6.0g of NaCl , 1.25g of NH4Cl needed for 1L , were dissolved in the

desirable amount of d. H2O, the pH was then adjusted to 8.2 using 5N NaOH, and

completed to 1L of the d. H2O. The media was sterilized by autoclaving at 121ºC for

20 min.

C. Preparation of solution B:

0.06g of MgSO4.7 H2O and 5.0g of dextrose were dissolved in 20ml of d. H2O, the

solution was filtered by 0.2 µm membrane filter, and kept at +4°C for two days at the

maximum.

D. Preparation of Yeast Extract:

80 g of Bacto-yeast extract was dissolved in 400ml of d.H2O. It was ultrafiltered by

utrafilteration device vacuum sterilized using 0.2 µm membrane filter.

E. Preparation of the medium:

970 ml of solution A was added just prior to inoculation, 20ml of solution B, and 10

ml YE was taken place and the pH was then adjusted to 7.6 using 5N NaOH.

1.5. Equipments:

Equipment Company

Fermentors BioFlo3000 (New Brunswick scientific USA)

5 Litters

Freeze dryer Ilshin- Korea

CO2 Incubator NAPCO, USA

Stirrer Thermolyne Nuova II ,USA

Equipment Company

Centrifuge Kendro , Sorvall super T21, USA

Spectrophotometer Perkin Elmer- EZ301, USA

pH meter Jenway -3510, UK

Light Microscope CETI- Belgium

Refrigerator Kriazi (No Frost),EGYPT

Balance Sartorius - CP2245, Germany

Biological Safety

Cabinet

NUAIRE - Class II, Japan

Oven Bourdon, USA

Autoclave Consolidated, USA

Shaker incubator New Brunswick, USA

Pippet aid Jencons, UK

1.6. Disposable:

Disposable Tools Company

Disposable serological

pipette (1-5-10-25 ml)

Corning incorporated Costar

sterile pasteur pipettes LP Italiana SPA

Petri dish ISO LAB GMBH

Surgical gloves Femto Trade CO.Egypt

Disposable Tools Company

Examination gloves Grinener-Germany

Face mask Winice

Syringe (3-5-10ml) BRiMO

Screw cap (15ml) Corning incorporated Costar

Sterile tubes (50ml) Corning incorporated Costar

Syringes (1ml) Euromed

Stericup&steritop 0.2

µm (250-500ml-1L)

Millipore

Cotton Teba company

Methods:

1. Organism and growth

Neisseria meningitidis is a pathogenic organism; appropriate precaution

should be taken to minimize the potential for laboratory acquired infection. Transfer

of cultures should be done in a laminar flow hood and care should be taken to

minimize the generation of aerosols (WHO, 1976).

2. Seed Preparation:

An ampoule of lyophilized Neisseria meningitidis serotype A is reconstituted

and streaked on GC Agar. The plates were incubated overnight at 36°C in CO2

incubator (5% CO2 atmosphere), (Gotschlich et al., 1969), cell growth and purity

were checked by Gram stain and colony morphology.

3. Inoculums Preparation:

Primary inoculum

A loopful from the overnight growth was transferred to 150 ml liquid medium

and was incubated at 36°C with vigorous shaking (150 rpm) for 18 h, cell growth was

checked by measuring optical density (OD) of the culture at absorbance 650 nm and

purity checked by Gram stain.

Secondary inoculum

A 5% inoculum from this culture was added to 300ml of medium which was

incubated under the same condition for 4-6 h only. This young culture was used as the

inoculum (5% still) for cultivation in large volumes. Smears Gram-stained

(Cruicksbank et al., 1975) and cell growth was checked by measuring OD of the

culture at absorbance 650 nm and purity checked by Gram stain.

4.Fermentation

A. Installation

A.1. Vessel Assembly:

Baffle was installed insertion, the baffle was assemblied inside the glass vessel

by gently compressing the baffle ring at its ends and then sliding the assembly into the

vessels.

A.2. Impellers insertion

The impellers were slided onto the agitation drive shaft, the lower impeller

should be positioned about 1/4 inch above the bottom of the battle. The upper

impeller should be one to one- and one- half impeller diameters above the lower

impeller. The impeller was clamped down in place.

A.3. Retention rings installation

The head plate was lubricated the head plate o-ring and the lower retention ring

o-ring with a light coat of silicon grease. Clamping screw was tightening firmly.

A.4. Sparger installation

Sparger tube was inserted into the sparger port. The lock nut was finger tighten

on the sparger.

A.5. Harvest tube installation

The harvest tube was inserted and finger tighten the lock nut.

A.6. Thermowell insertion

Thermowell tube was inserted; thermocouple was inserted and connecting to

the temperature control port.

A.7. Sampler installation

Sampler was inserted assembly into the sample port.

A.8. Head plate installation on vessel

The head plate was installed on the vessel flange and was secured to the upper

retention ring. The clamping screws were tighten.

A.9. pH probe installation

pH probe was coated with glycerol. The probe was gently inserted into the

appropriate port.

A.10. DO probe installation

DO probe was coated with glycerol and was inserted gently into its adaptor.

A.11. Vessel installation

Motor assembly installation, the motor assembly was positioned on top of the

bearing housing. Motor cable was connected to its place. All cables were connected

from all probes to their respective sockets on the face of the console. The exhaust

condenser was connected to the exhaust filter using flexible tubing to the top of the

condenser, and was secured with tubing ties. The water lines were connected to the

heat exchanger in the vessel base and to the exhaust condenser using the quick

connects.

A.12. Probe preparation and calibration

1- DO probe

Set to zero: The DO cable was removed from the DO electrode. The DO

function was set to zero. The DO cable was reconnected to the DO electrode. Set the

span: The DO function was set to span. The display was set to read 100 by setting the

DO span column to 100.

2- pH calibration

pH probe was connected with cable. The pH probe was calibrated using two

external buffer solution of known pH usually 7.00 and 4.00. The pH calibration was

checked after autoclaving, immediately prior to inoculation. Sample was taken from

the vessel and was compared by an external pH meter.

B. Sterilization:

The motor was removed from the top of the vessel and was placed it on the

motor mount at the top of the console. The air line was disconnected from the inlet

filter. All probes were disconnected and the probe cables were removed, the sampler

rubber bulb was removed, cotton was inserted into sampler port, and sampler valve

was closed. Just before placing the vessel into the autoclave, the glass sample bottle

was loosen by 1/2 turn. The media for fed batch fermentation was inserted into the

vessel. The entire vessel assembly (consisting of glass jar, headplate and headplate

components) was inserted into an autoclave and sterilized for 25 min at 121°C.

During autoclaving, the glass vessel was vented at all times, electrolyte in pH probe

was not allowed to boil.

C. Vessel reinstallation

Vessel was returned to console, water lines to the heat exchanger and the

exhaust was connected. The motor was placed on the bearing house, on top of the

vessel assembly. DO and pH short cap were removed, pH cable and DO were

connected. Liquid addition system was installed a sterile (0.2 µm) filter in one of the

two penetration on the addition bottle cap was aseptically installed. The harvest tube

was connected aseptically land threaded the tubing through the desired peristaltic feed

pump. The tubing was connected and securing it with a plastic tie to the appropriate

addition port on the head plate clamp was removed. Temperature probe was installed,

glycerin was added to the thermowell and temperature probe was inserted, and

temperature cable was connected.

D- Fermentation Data Sheet table:

Item Culture A Culture B

Seed Volume 200 ml 200 ml

Agitation 200 rpm 200-400 rpm

Temperature 36◦C 36◦C

Air Flow 2 L /min 2 L /min

Dissolved Oxygen

(DO)

Air saturation by

automatic adjustment

of the impeller speed

and air flow.

Air saturation by

automatic adjustment

of range impeller

speed from 200-400

rpm.

pH 7.6 was adjusted by

using 5 N NaOH

Not adjusted

Antifoam 0.5 ml/l was added of

antifoam.

0.5 ml/l was added of

antifoam.

Duration 18 h 18 h

DO/Air Cascade No cascade Cascade with air flow

range 2-3 l/h

E- DO cascade system

Cascading brings several systems together to work jointly to achieve goal.

Cascade was designed to control DO through controlled agitation speed and oxygen

output. When the actual DO value rises above the DO set point the agitation speed

will automatically decrease until DO set point is reached. Conversely, when the actual

DO value drops below the set points, the cascade system acts to bring it back up. If

DO value rises above the set point, agitation and airflow rate will remain at their

minimum value. If DO drops below the set point, first agitation speed will gradually

increase to its maximum set point, then airflow rate will gradually increase to its

maximum set point.

F- Inoculation

The plug was removed from the inoculation port. The inoculum was removed

aseptically from its flask with the inoculation syringe. The inoculum was injected

through the septum in the inoculation port. The plug was reinstalled in the port.

When the whole lot of the liquid media is ready about three L for each

fermentors), i.e, solution A supplemented with solution B and YE and the pH adjusted

to 7.6 and add a 5% inoculum (purity testing of the inoculum is examined by gram

stain). The growth of the cultures takes place at 36°C with shaking incubator 200 rpm

for fermentor 1 and 400 for fermentor 2 for 18 hrs.

G- Sampling

The sample bottle was checked to be slightly loose, not tight against

the gasket. The valve on the sampler tube was closed. The bulb was

squeezed and holding it compressed, the sample bottle was tightened

against the gasket. The valve was opened and the rubber bulb was let go

gradually to obtain the desired sample volume. When the desire volume

was obtained, the valve was closed. The sample bottle was unscrewed

from the sampler, the cap was taken from a new bottle and placed on the

sampler-filled bottle. The new bottle was installed in the sampler and

made sure that the sample bottle was sealed firmly against the sampler

gasket and sampling intervals was 2 h.

5. Harvesting

The culture had been harvested from late stationary phase culture grown for 18

h at 36°C, The culture was harvested after killing the organism by heating to 56 °C for

30 min. The cultures are harvested after purity testing of the inoculum is examined by

gram stain) from each container of two fermentors and centrifuged at 3000 for 45

minutes for clarification. The supernatant is collected. The cells, on the other hand,

are pooled and kept frozen (Gotschlich et al., 1969).

6. Estimation of cell mass:

Cell concentration was expressed as dry biomass weight per liter (g/l) after

centrifugation of 3 L culture, followed by pellet drying at 60 °C for 48 h (Barugue-

Ramos et al., 2005)

Results:

1-Seed preparation:

The Gram smear of N. meningitidis was utilized as indicator for the purity of

the culture and demonstrated the morphological characteristic of Gram negative,

diplococci bacteria whereas that figure (11)

Figure (11)

The Gram staining of N. meningitidis ATCC 13077, the bacteria were

identified as Gram negative, diplococci.

2- Inoculums preparation:

Primary inoculums

Three 250 ml flasks of primary inoculums were examined by Gram stain and

optical density. The highest optical density was 1.038 and pH was 6, this culture was

chosen for secondary inoculums and showed identical Gram negative diplococci.

Secondary inoculums

Three 500 ml flasks of secondary inoculums were examined by Gram stain

and optical density. The highest optical density was 0.9 and pH was 6.4, smear of

culture showed pure identical Gram negative diplococcic as shown in figure (12), this

culture was taken for seed two batches fermentation with different parameters.

Figure (12)

Inoculums preparation Gram negative diplococcic of N. meningitidis .

3-Fermentation:

Two batches of N. meningitidis were cultivated in BioFlo 3000 fermentor

sterilized and calibrated, fermentation of 3 L culture and 200 ml of bacterial seed at

36 ºC for 18 h, the summary of results obtained were described in table below.

Summary of experimental results obtained with batch A and B table

Culture

duration

(h)

Agitation pH Dissolved

oxygen

Temp Dry cell

mass

g/L

Supernatant

of culture

Batch

A

18 h 200 rpm 7.6 10% 36 ºC 0.85 g/L 2720 ml

Batch

B

18 h 400 rpm 5.4 40% 36 ºC 0.46 g/L 2800 ml

Batch A pH was adjusted using 24 ml N NaOH, agitation was fixed at 200

rpm, air flow was about 2 L/min at all culture time, dissolved oxygen was measured

automatically decreased by time and reached to min/ value 10%. Batch B pH was not

adjusted, agitation/ oxygen/ DO cascade system was applied maximum set points of

agitation was 400 rpm, air flow 4 L/min was arrived after 12 h . DO was at fix point

40%.

3.1. Estimation of bacterial growth:

Comparison of bacterial growth for batch A and B were estimated, 5 ml

samples were taken every two hours and measure optical density using

spectrophotometer at 650 nm, growth curve was drawn using optical density versus

time. Batch A showed higher optical density than batch B shown in figure (13).

Figure (13):

Growth curve comparison of N. meningitidis fermentation for Batch

A and B.

0

0.2

0.4

0.6

0.8

1

1.2

1.4

0 2 4 6 8 10 12 14 16 18 20 22

Op

tica

l De

nis

ty

Time/h

Batch A Batch B

3.2. Estimation of pH

pH value of batch A and B were estimated automatically and the curve for

comparison between two batches were drawn. Batch A was linear adjusted pH while

batch B, pH was decreased graduating by time will reached finally to 5.4 as shown in

figure (14).

Figure (14)

The effect of pH on the growth of N. meningitidis showing the growth

occurred from pH 7.6 to 5.4 during the exponential growth.

4

4.5

5

5.5

6

6.5

7

7.5

8

0 2 4 6 8 10 12 14 16 18 20 22

pH

val

ue

Time/ h

Batch A Batch B

3.3. Determination of dissolved oxygen:

Dissolved oxygen was determined automatically during culture, a curve

compared between batch A and batch B was drawn. Batch B was higher DO than

batch A as agitation /O₂/ DO cascade increase final DO to 40% as shown in figure

(15).

Figure (15)

The effect of agitation/ O₂/ DO cascade on DO% during and at the

end of batch A and B.

0

20

40

60

80

100

120

0 2 4 6 8 10 12 14 16 18 20 22

Dis

solv

ed

Oxy

gen

Pe

rce

nta

ge

Time/ h

Batch A Batch B

3.4. Cell mass estimation:

After 18h fermentation, the culture were harvested and centrifuged and pellets

of culture A and B were dried at 60 ºC for 48 h and dry cell mass of culture A was

0.85 g/L, supernatant of culture was 2720 ml, while cell mass of culture B was 0.46

g/L and supernatant was 2800 ml.

Discussion:

Meningococcal disease is rare but severe disease that causes meningitis,

septicemia or both of them, Egypt is one of the high risk regions of epidemic of this

disease which is caused by the bacteria Neisseria meningiditis that contains 13 sero-

groups. Neisseria meningitidis sero-group A constitutes the antigen for the vaccine

against meningitis disease. The goal of this work was to show the effect of some

different parameters as pH and dissolved oxygen on the growth curve and cell mass.

These kinetic parameters were carried out in two batches three litters of Frantz media

in 5 L fermentor (New Brunswick) the first batch (A) was at 36 ºC, air flow 2 L/min,

agitation frequency 200 rpm, dissolved oxygen reached to 10% and adjustment pH.

Another batch(B) was carried out under different conditions the pH not adjusted, DO

cascade system was applied maximum set points of agitation was 400 rpm, air flow

4L/min and DO was arrived to 40%. The highest cell mass (0.85 g/L) was obtained in

batch A when the pH was adjustment and DO was 10% while, another batch was low

cell mass (0.46 g/L). An empirical relation is proposed to relate the specific cell mass

rate during stationary growth phase of the batch A. pH parameter was the main factor

that affect fermentation process, even it increase airflow and agitation.

Many trials and experiments of extracting the polysaccharide capsule from the

bacteria Neisseria meningitidis are done all over the world in research labs to produce

an efficient and effective vaccine for meningitis disease, these experiments are

searching for the best way to get the heist yield of bacterial culture, further more they

are testing the effects of different parameters as pH, temperature, agitation and

dissolved oxygen, and nitrogen and glucose source found in the medium.

In the present study, we have the inoculum of N. meningitidis was prepared

according to Gotschlich (Gotschlich, 1969). The strain was streaked out on GC

medium and incubated at 36°C for 18- 20 h in a candle jar (5-10 % CO2). The cells

from each plate were resuspended in the liquid Frantz medium and then transferred to

conical flasks, each containing 100 ml of Frantz medium and incubated at 36 °C on a

rotary shaker 150 rpm. Microscopic examinations were carried out using the Gram

technique. These results were agreement with (Baruque, 2005) and (WHO, 1976).

In 2003 da-Paz did an experiment which is culturing Neisseria meningitides

sero-group C for the production of polysaccharide. This experiment was done by

using three different cultivation media and those are Frantz, modified Frantz and

Cartlin 6. Starting with the Frantz media which is the same media used in the present

study but in different concentration of compounds as L-Glutamic acid 1.6 g\L and

1.23 MgSO4.7H2O and in this experiment media the concentration for L-Glutamic

acid 1.3 g\L and 0.06 for MgSO4.7H2O, other components as L-Cystine-HCl, KCl,

NaCl, NH4Cl and glucose where the same as 0.02 g\L, 0.09 g\L, 6.0 g\L, 1.25 g\L and

5.0 g\L. in this culturing media Paz used deferent parameters than parameters used in

this study like temperature 35ºC while it was 36ºC in this study for both batch A and

B, agitation was 120 rpm unlike both of the present study batches (200-400 rpm), air

flow of 5 L\min while it was 2 L\min in both batches and the pH was not adjusted and

reached 5 as in batch B, adding that Paz used 80 L Brunswick bioreactor with 42 L of

culture media while in this study the bioreactors used were two 5 L Brunswick

fermentors with 3 L of culture media. These differences resulted that the dry biomass

produced was 1.11 g\L after 18 h which is higher than the present experiment results

0.85 g\L in batch A and 0.46 g\L in batch B (da-Paz, 2003).

In the same experiment, Paz used modified Frantz media with different

compounds as glycerol instead of glucose as a carbon source in the same

concentration 5.0 g\L and different concentration of compounds as L-Glutamic acid

1.6 g\L and 1.23 MgSO4.7H2O while in the current experiment the concentration L-

Glutamic acid 1.3 g\L and 0.06 for MgSO4.7H2O and with the same other compounds

concentrations L-Cystine-HCl, KCl, NaCl and NH4Cl 0.02 g\L, 0.09 g\L, 6.0 g\L and

1.25 g\L, with almost the same parameters as the first one; temperature which is 35ºC,

air flow 5 L\min and agitation 120 rpm, but the pH were adjusted at 7.5, and the result

of dry biomass which is 1.2 was higher than the current result. The last media Paz

used that is Cartlin 6 which was different than this study media (Frantz media), the

carbon scores of this media was glucose with a concentration of 10.0 g\L with 0.04

g\L Fe(III).Citrate with no MgSO4.7H2O nor KCl instead of that K2HPO4 and K2SO4

were added in a concentration of 4.0 g\L and 1.0 g\L, more than that a variations in

the concentration of L-Glutamic acid, L-Cystine-HCl ,NaCl and NH4Cl as 3.9 g\L, 0.1

g\L, 5.8 g\L and 1.0 g\L, as for the parameters the same temperature was used as the

two experiments before (35ºC) with the same agitation (120 rpm) and the same air

flow (5 L\min), the pH was not adjusted and the result was higher than all of them

which is 1.4 g\L. Pazs results demonstrate higher efficiency than the present study

because these experiments were handled in a large scale bioreactor and more than one

experiment on the same media taking the average of it, which gave an advantage in

the results, and in the Cartlin 6 media where the highest result observed, there were

many alterations but the most significant one was adding 10 g\L of glucose with 0.04

g\L Fe(III).Citrate (da-Paz, 2003).

Another experiment done by Henriques studding the process of capsular

polysaccharide production by Neisseria meningitidis sero-group C, the medium used

for cultivation was Frantz medium composed of 6.0 g/L of NaCl, 2.5 g/L of Na2HPO4,

1.25 g/L of NH4Cl, 0.09 g/L of KCl, 2.0 g/L of yeast extract and 5.0 g/L of glucose

the same concentration were used in this current experiment, but there is some

variances in L-Glutamic acid 1.6 g/L as the concentration used in the experiment was

1.3 g/L , MgSO4.7H2O and L-cysteine hydrochloride concentration are also different

0.02 g/L, 0.12 g/L while in the current experiment were 0.06 g\L and 0.02 g\L. some

of the parameters Henriques used for the bioreactor were also dissimilar to the current

experiment like 37ºC temperature, 7 pH, 0.8 L\min airflow and agitation of 130 rpm

while in the current experiment the parameters used in the fermentor 36ºC

temperature,7.6 pH in batch A and not adjusted pH in batch B, 2 L/min airflow and

200 rpm agitation in batch A and 200-400 agitation in batch B. adding that the

experiment was run in 1.0 L glass-vessel bioreactors (BIOSTAT Q,B. Braun Biotech

International Diessel GmbH,Germany) with 0.5 L of culture media. That result a dry

biomass of 0.84 g/L which is lower than the current experiment that is 0.85 g/L in

batch A, that different is insignificant but higher than batch B that is 0.46 g/L

(Henriques, 2005).

Baruque did an experiment in 2001 which aim to detect the nitrogen

consumption of Neisseria meningitidis sero-group C, in this experiment Baruque used

Frantz media which contains the same components as the Frantz media used in this

current research used but in different concentrations like 1.6 g/L L-Glutamic acid,

4.67 g/L Na2PO4 and 1.23 g/L MgSO4 while in the current experiment the

concentration of L-Glutamic acid was 1.3 g/L, Na2PO4 2.5 g/L and MgSO4 0.06.

However, there are some concentrations that match the concentrations used in the

recent experiment like 0.02 g/L of L-Cysteine-HCl, 0.09 g\L of KCL, 6.0 g\L of

NaCl, 1.25 g\L of NH4Cl and 5.0 g\L of glucose. After that the pH was corrected to

7.4 while in this experiment it was adjusted to 8.2 with 5N NaOH. In the parameters

Baruque used for the bioreactor - 35 ºC temperature, 120 rpm agitation, 5L\min

airflow and not adjusted pH starting with 7.4 – where completely different than the

parameters used for the current experiment which was 200 rpm agitation, 36 º C

temperature, 5 L/min airflow and 7.6 adjusted pH in batch A and not adjusted pH in

batch B starting with 7.6. other than that, the bioreactor Baruque used was New

Brunswick model MPP 80 that has a total capacity of 80 L and media volume used

was 40 L unlike the bioreactor used in the current experiment which is New

Brunswick BioFlo3000 with 10,5 capacity and 3 L of Frantz media (Baruque, 2001).

The result of Baruque was higher than the result achieved in the present

experiment, as Baruque produced 1.15 g/L dry cell mass after 18 hour with a

significant different than the current experiment obtained in batch B which was 0.46

g/L and last result was poorer might be due to the agitation used which was 200-400

rpm comparing to the agitation used by Baruque 120 rpm, noticing that Baruque used

a much larger bioreactor that help in the cultivation of bacteria. Comparing Baroques'

result with batch A the results was very close as batch A biomass result .85 and

Baruque has a result of 1.1 despite the differences in the culture media with was in

batch A 3L while Baruque was 40L. (Baruque, 2001).

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