FERMENTATION TECHNOLOGY ASSINGMENT

Of

FINDING AND DEVELOPING AN ANTIBIOTIC EFFECTIVE AGAINST A NEW

BACTERIAL PATHOGEN

NAME : Mohammed Ridzuwan Bin Abdul Rahaman

MATRIC NUMBER : 1107151018

SUBJECT : Fermentation Technology

SUBJECT CODE : SBB 3184

LECTURERS NAME : Dr. Rahayu binti Ahmad

SUBMISSION DATE : 10 July 2017

FERMENTATION TECHNOLOGY SBB 3184

TABLE OF CONTENT

Contents Page

INTRODUCTION 1

LITERATURE RIVIEW 2

CLASSICAL METHOD IN POTENTIAL ANTIBIOTIC FINDING 4

Primary Screening 4

Secondary Screening 5

Phytochemical Analysis 5

Shake Flask Fermentation 5

IMPROVEMENT OF ANTIBIOTIC PRODUCING STRAIN 6

COMMERCIAL PRODUCTION OF ANTIBIOTIC BY FERMENTATION 7

Starting the culture 7

Fermentation 8

Isolation & Purification 8

Modify Produced Antibody Chemically 8

Marketing & Financial 8

CONCLUSION 8

REFERENCES 9

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INTRODUCTION
Fermentation, a process traditionally known for the anaerobic conversion of sugar to
carbon dioxide and alcohol by yeast, now refers to an industrial process of manufacturing a
wide variety of metabolites and biomaterials by using microorganisms or mammalian cells in
a controlled culture environment. Fermentation can be performed in batch mode, continuous
mode or in a combinatory, fed-batch mode, depending on the product of interest.
Fermentation technology has long been known for the production of various medically
important products such as antibiotics, solvents such as ethanol, intermediary compounds
such as citric acid, probiotics such as yoghurt etc. New generation fermentation products
include anti-viral drugs, therapeutic recombinant proteins and DNA, and monoclonal
antibodies. Apart from the drugs, fermentation is also used for the commercial production of
materials required for the development of diagnostic kits, drug delivery vehicles and medical
devices. Fermentation technology remains at the heart of rapidly growing biopharmaceutical
industry today, which is expected to expand even more in the days ahead, in parallel with the
progress in novel, targeted drug discovery.
Antibiotics are antimicrobial compounds produced by living microorganisms, and are
used therapeutically and sometimes prophylactically in the control of infectious diseases.
Over 4000 antibiotics have been isolated but only about 50 have achieved wide usage. The
other antibiotic compounds failed to achieve commercial importance for reasons such as
toxicity to humans or animals, ineffectiveness or high production costs. Antibiotics have been
extensively used in medicine since about 1945 with the arrival of penicillin (Smith, 2009).
The production of antibiotics has undoubtedly been a highly profitable part of the
pharmaceutical industries in the industrialised world. The world market for antibiotics and
anti-fungal was worth over US$26 billion in 2001 and is the most valuable segment of the
total pharmaceutical market (US$200 billion) (Bains & Evans, 2001). Due to biotechnology
innovation, as world sales of antibiotics increase their production costs have decreased.
Microorganisms have developed multiple resistance to many of the antibiotics currently
in common use. This is due to several factors some inherent in the nature of microorganisms,
others relating to use (or misuse) of antibiotics by humans. Some of the factors pertaining to
the human use of antibiotics include the wide spread and sometimes unnecessary use of
antibiotics, the prophylactic use of antibiotics, and the use of low doses of antibiotics for
encouraging the growth of farm animals. In the case of bacteria, their sheer numbers means
that there are a potentially large number of genotypes waiting to be selected and their short
generation time fuels the rapidity of this selection. Finally their ability to horizontally transfer
genetic materials through plasmids, transposons, and by conjugation and transformation set
the stage for the (almost) inevitability of the development of resistance among
microorganisms, especially bacteria. So there are wide needs for a new antibiotic
development around the world.
Antibiotics are produced by fermentation. The process may take a few days to obtain an
extractable amount of product. Antibiotic production is done by the batch process. Oxygen
transport is the major concern; therefore sufficient polymeric sugar and protein with a trace
amount of elemental growth factors are used to enhance production. An anti-biogram test is
used to observe the amount of antimicrobial agent in the fermentation broth. A bioassay
determines the activity unit of the bactericides (Akcesme, n.d.). The potential antibiotic first
identified by classical method where primary screening, secondary screening, phytochemical
test, and shake flask fermentation for high yield production will do. Improvement of strain of
interest and certification for the antibiotic must be done before industrial scale fermentation.
The major steps in the commercial production of antibiotics are as follows; starting the
culture, ferment the culture, extraction and purification, and modify chemically the antibiotic.
Then the marketing as well as financing process is done to commercialise the antibiotics.

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LITERATURE REVIEW
The very first recorded observation on microbial antibiosis dates back to 1877.
Pasteur and Joubert described slower growth of Clostridium sp., in the presence of other
bacteria. In 1893, BartolomeoGosio, an Italian physician, discovered a compound in the
culture filtrate of Penicillium brevicompactum, which inhibited growth of Bacillus anthracis.
The accidental discovery of penicillin by Alexander Fleming in 1929 in England began the
golden era of antibiotics. He noted that some of his plates containing Staphylococcus aureus
were contaminated with the mould, P. notatum. Strangely, he observed that none of the
bacterial colonies could grow in the vicinity of the mould and concluded that the mould was
producing some inhibitory agent. He also noted that filtrates of the mould lyzed the
staphylococci and was nontoxic in animals. Attempts to isolate penicillin began in 1939 by
Howard W. Florey, Ernst B. Chain, Norman G. Heatley, Edward Abraham, and their
colleagues (Arnold, Demain, Vandamme, Collins, & Buchholz, 2017). s. Development of
submerged culture techniques has enhanced the cultivation of the mould in large-scale
operations using sterile air supply. The commercial production of penicillin and other
antibiotics is the most dramatic example of industrial microbiology. The annual production of
bulk penicillin is about 33 million pounds, with annual sales market of more than US$344
million (Pelczar, Chan, & Krieg, 1993). Penicillin production can be studied as an example
for the antibiotic world because it was the first antibiotic produced on a large scale, and also
the techniques used for the industrial production of penicillin have served as a model for the
industrial production of other antibiotics or secondary metabolites.
Antibiotics are products of secondary metabolism that can be produced commercially
by microbial fermentation. Commercially useful antibiotics are produced mainly by
filamentous fungi and by bacteria of the actinomycete group. As secondary metabolites, each
antibiotic is produced by a relatively limited number of species and is encoded by sets of
dispensable genes. These compounds are synthesized at the end of the exponential growth
phase and during the stationary phase, and their formation is highly influenced by the growth
conditions, especially by the composition of the culture medium (Chater & Bibb, 1997).
Antibiotics that affect a wide range of microorganisms are termed broad spectrum, for
example, choramphenicol and the tetracyclines, which can control such unrelated organisms
as Rickettsia, Chlamydia and Mycoplasma species. In contrast, streptomycin and penicillin
are examples of narrow spectrum antibiotics, being effective against only a few bacterial
species (Golub, 1994). The production of antibiotics has undoubtedly been a highly profitable
part of the pharmaceutical industries in the industrialised world. The world market for
antibiotics and anti-fungal was worth over US$26 billion in 2001 and is the most valuable
segment of the total pharmaceutical market (US$200 billion).Due to biotechnology
innovation, as world sales of antibiotics increase their production costs have decreased (Bains
& Evans, 2001).
New techniques such as protoplast fusion and gene transfer technologies are leading to
the development of new strains with higher productivity, improved stability and possible new
products. These improvements have all resulted in continued decreases in overall costs of
production. At present all antibiotic fermentations involve centrally stirred tank reactors run
under aerobic batch conditions. Modifications in production processes may well follow on
from the novel fermenter designs that are gaining wider industrial acceptance (Smith, 2006).
Because of the increasing knowledge of the biosynthetic pathways in microorganisms it is
increasingly possible to involve genetic manipulation directly, rather than to rely mostly on
mutation and natural selection. The three major applications of genetic manipulation
technology to antibiotic production are; strain improvement programmes, producing novel
antibiotics by gene insertion, engineering microbial strains and enzymes relevant to the
production process (Hook, 2006).

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Most fungi sporulate on suitable agar media but a large surface area is required to
produce sufficient spores. A roll-bottle technique was used to produce spores of Penicillium
chrysogenum, with 300 ml of media containing 3% agar sterilised in a 1 litre cylindrical
bottle. After autoclave, the media was cooled at 45 C and rotated on a roller mill so that a
layer of agar formed on the cylinder wall. The inoculum was of spore suspension incubated at
24 C for 67 days. Submerged culture is common for sporulation of fungi such as P.
chrysogenum. The sporulation is induced by inoculating 300 ml in a 1 litre shaking flask with
spores from a well-sporulated agar culture and incubated for sufficient time. At this stage, a 2
litre fermenter was inoculated with a pure inoculum (300 ml) and harvested in the fast-
growing (logarithmic) phase, so that in the culture media a high cell density could be
obtained. The organism, P. chrysogenum, grows in a filamentous (hyphal) form, with
branching occurring to a greater or lesser extent (Gutierrez, Casqueiro, & Martn, 2014). The
B. Braun airlift fermenter was used. Pressurised filter air was used to circulate the mycelia in
the internal loop pattern. Air was continuously supplied. The bubbles lose oxygen as they rise
up the column. At the same time, carbon dioxide and other gaseous metabolites diffuse into
the media and are released in the overhead gas compartment. The production of penicillin G
is very sensitive to temperature, the tolerance being less than 1 C. Heat is generated by the
metabolism of nutrients. It has to be removed by a well-controlled cooling system. Cooling
coils are used for isothermal operation. The fermentation vessel is fitted with several probes
to detect foaming, to monitor temperature, to control media level and to record parameters
such as pH. The rate of air flow through the fermenter is measured, and the exhaust gases that
emerge from the top of the vessel may also be analysed. Originally all penicillin G was
manufactured using lactose in this way, and some manufacturers still prefer this technique
(Ghasem, 2007).
At harvesting time, the cells are removed. The penicillin G as the cell product is in the
solution that is extracellular with a range of other metabolites and medium constituents. The
first step is to remove the cells by filtration. This stage is done under conditions that avoid
contamination of filtrate with enzyme, which may destroy antibiotic. The -lactamase-
producing microorganisms could react with the antibiotics, which would cause serious or
total loss of product (Anon, 1999). The next stage is to isolate the penicillin G. Solvent
extraction is the generally accepted process. In aqueous solution at pH 22.5, there is a high
partition coefficient in favour of certain organic solvents such as amyl acetate, butyl acetate
and methyl isobutyl ketone. The extraction has to be done quickly since penicillin G is very
unstable at those low pH values. The penicillin is then extracted back into an aqueous buffer
at pH 7.5 (Gutierrez, Casqueiro, & Martn, 2014). The partition coefficient now strongly
favours the aqueous phase. The solvent is recovered by distillation for re-use.
The extracted antibiotic was used in an antibiogram test. Petri dishes of Bacillus subtilis
ATCC 6633 was cultured for bioassay of penicillin. Small circular paper filters (35mm) are
placed at different positions on the surface of the agar (Ghasem, 2007). The small circular
filters were sterilised earlier. A few drops of concentrated and extracted antibiotic were
poured on the filter. The small circular filter will hold antibiotic after incubation for 24 hours.
There will be a clear circle around the paper filter without any microbial growth. This
bioassay is called antibiogram. Normally, an antibiogram is used for clinical purposes to
identify a suitable antibiotic for infected patients. High-performance liquid chromatography
(HPLC) is commonly used for quantitative analysis. The carbohydrate concentration is
determined by the dinitrosalicylic acid method.8 Biomass was evaluated by measuring the
total solid concentration. Cell dry weight may represent the growth curve in the fermentation
broth. The samples were centrifuged at 4500 rpm for 20 min and the sediment washed with
distilled water and dried in an oven at 105 C to determine cell dry weight (Anon, 1999).

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CLASSICAL METHOD IN POTENTIAL ANTIBIOTIC FINDING

The classical method for the search for new antibiotics is by random search in the soil.
Although the first important commercially produced antibiotic was discovered by chance,
most present day antibiotics were discovered by systematic search. The soil is a vast
repository of microorganisms and it is to the soil that search is turned when antibiotics are
being sought. The classic methods will do step by step which are primary screening,
secondary screening, phytochemical test, and shake flask fermentation.
The most important are:
(i) Primary screening:
Several methods have been employed in primary screening such as crowded plate
method, direct-soil-inoculation method, cross-streak method, agar plug method, and replica
plating method. Besides all the methods, the most effective and preferable methods is
crowded plate method incorporate with cross-streak method. The crowded plate is used to
isolate soil organisms able to produce antibiotics against other soil organisms. A heavy
aqueous suspension (1:10; 1:100) of soil is plated on agar in such a way as to ensure as much
as possible the development of confluent growth. Organisms showing clear zones around
themselves are isolated for further study. Different groups of organisms could be encouraged
to develop by altering the media used (Handelsman, Rondon, Brady, Clardy, Goodman,
1998). This method has the disadvantage that slow-growing antibiotic-producing organisms
such as actinomycetes are usually over grown and are therefore hardly isolated. Furthermore,
the test organisms used in this method are soil organisms. The susceptibility of soil organisms
to the antibiotics produced in the test may therefore be unrelated to the susceptibility of
clinically important organisms. The cross-streak method is used for testing individual
isolates, especially actinomycetes which may be obtained from soil without any previous
knowledge of their antibiotic-producing potential. The organism may come from one of the
two methods already indicated above. The purified isolate is streaked across the upper third
of plate containing a medium which supports its growth as well as that of the test organisms.
A variety of media may be used for streaking the antibiotic producer. It is allowed to grow for
up to seven days, in which time any antibiotic produced would have diffused a considerable
distance from the streak. Test organisms are streaked at right angles to the original isolates
and the extent of the inhibition of the various test organisms observed (Golub, 1994).

Figure 3.1: The cross streak method for the primary search of antibiotic producing organisms
Source:
http://slideplayer.com/slide/4732093/15/images/42/The+Cross+Streak+Method+for+the+Pri
mary+Search+of+Antibiotic+Producing+Organisms.jpg

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(ii) Secondary screening

Organisms showing suitably wide zones of clearing against selected target organisms
are cultivated in broth culture in shake flasks using components of the solid medium in which
the isolate grew best. Crude methods of isolating the active antibiotic are developed by
extracting the broth using a wide range of extractive methods. With each extraction the
resultant material is assessed for activity against the target organisms at various dilutions. The
extract is either spotted on filter paper discs placed on agar seeded with the test organism or
introduced into wells dug out from the seeded agar with sterile cork borers. In this manner the
most efficient extractive methods and the spectrum of activity of the organisms are
determined (Handelsman et al. 1998). Secondary screening is aimed at eliminating at an early
stage any antibiotic which does not appear promising either by virtue of low activity, other
undesirable properties or because it has been discovered previously.

Figure 3.2: Replica plating method of testing antibiotic producing colonies

Source: Okafor, 2007
(iii) Phytochemical Analysis
The other qualities of the antibiotic outside anti-microbial activity depend on its
intended use. For antibiotics meant for clinical use, information on a number of the following
may be sought at this stage (Hill, Wrigley, & Nisbet, 1998).
(a) Toxicity to mammals, determined by intra peritoneal injection into animals;
(b) Haemolysis is tested by observing the effect on blood agar;
(c) Serum binding is tested by adding serum to the broth before testing against
susceptible organisms;
(d) The inactivation of the antibiotic by several enzymes from various organs is tested
by exposing the antibiotic to them;
(e) Acid stability is tested if the antibiotic is meant for oral fermentation
(f) Tetragonicity tests, which determine the effect on the unborn are carried out on
laboratory animals;
(g) For plant antibiotics, phytotoxicity as shown by damage to leaves in the laboratory
and in the green house, is determined;
(h) For feed antibiotics, low absorbability and low toxicity are desirable and are tested.
(iv) Shake Flask Fermentation
Further laboratory evaluation after the antibiotics is promising, then further
experimentation is done in shake flasks as a preparation for pilot production. The optimal
conditions of growth are determined; the most suitable medium, optimal pH, temperature,
length of fermentation etc. are all determined (Hook, 2006).

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IMPROVEMENT OF ANTIBIOTIC PRODUCING STRAIN

This method is done to get high yield and fast growing strains to decrease the cost and
duration of antibiotic production. Before do this method, we should have vast knowledge on
the biosynthetic pathways of the particular microorganisms. For this step, we can use genetic
engineering tools such as direct genetic manipulation, rather than to rely mostly on mutation
and natural selection. The genetic manipulation or insertion of gene to produce novel
antibiotic secreting strain will create a transgenic microorganism. As in all cases, it is
necessary to isolate the desired DNA sequence that codes for the desired protein together
with an understanding of promoter, terminator and regulatory regions of the gene. The
linearized DNA is then ready for transfer into the strain (Veenstra, van Solingen, Bovenberg,
& Van der Voort, 1991). Make sure that the genetic tool does not affect the antibiotic
production and alter the antibiotic originality.

Figure 2.1: Example of Gene Insertion

Source: https://image.slidesharecdn.com/13geneticengineeringbw-120504133440
phpapp02/95/13-genetic-engineering-bw-15-728.jpg?cb=1336138594

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COMMERCIAL PRODUCTION OF ANTIBIOTIC BY FERMENTATION

Fermentation of antibiotic is operated as batch fermentations, in which a volume of
sterile medium in a vessel is inoculated. The broth is fermented for a defined period. The tank
is then emptied and the products are separated to obtain the antibiotic. The vessel is then
recharged for batch operation with medium and the sequence repeated, as often as required.
Continuous fermentation is not common practice in the antibiotics industry. The antibiotic
concentration will rarely exceed 20 g l-1 and may be as low as 0.5 g l-1. Before the antibiotic
produce in industrial scale certification of the antibiotic should be given. A government
agency must approve the antibiotic before it becomes available for general use. In the US, it
is the Food and Drug Administration (Chater & Bibb, 1997).

Figure 3.1: Fermenter shows production of antibiotic

Source: http://www.srmuniv.ac.in/sites/default/files/files/PENCILLIN.pdf

(i) Starting The Culture

Before fermentation can begin, the desired antibiotic-producing organism must be
isolated and its numbers must be increased by many times. To do this, a starter culture from a
sample of previously isolated, cold-stored organisms is created in the lab. In order to grow
the initial culture, a sample of the organism is transferred to an agar-containing plate. The
initial culture is then put into shake flasks along with food and other nutrients necessary for
growth (Okafor, 2007). This creates a suspension, which can be transferred to seed tanks for
further growth. The seed tanks are steel tanks designed to provide an ideal environment for
growing microorganisms. They are filled with the all the things the specific microorganism
would need to survive and thrive, including warm water and carbohydrate foods like lactose
or glucose sugars. Additionally, they contain other necessary carbon sources, such as acetic
acid, alcohols, or hydrocarbons, and nitrogen sources like ammonia salts. Growth factors like
vitamins, amino acids, and minor nutrients round out the composition of the seed tank
contents. The seed tanks are equipped with mixers, which keep the growth medium moving,
and a pump to deliver sterilized, filtered air. After about 24-28 hours, the material in the seed
tanks is transferred to the primary fermentation tanks (Shah Amran, 2012).

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(ii) Fermentation
The fermentation tank is essentially a larger version of the steel, seed tank, which is
able to hold about 30,000 gallons. It is filled with the same growth media found in the seed
tank and also provides an environment inductive to growth. Here the microorganisms are
allowed to grow and multiply. During this process, they excrete large quantities of the desired
antibiotic. The tanks are cooled to keep the temperature between 73-81 F (23-27.2 C). It is
constantly agitated, and a continuous stream of sterilized air is pumped into it (Shah Amran,
2012). For this reason, anti-foaming agents are periodically added. Since pH control is vital
for optimal growth, acids or bases are added to the tank as necessary.
(iii) Isolation and Purification
After three to five days, the maximum amount of antibiotic will have been produced
and the isolation process can begin. Depending on the specific antibiotic produced, the
fermentation broth is processed by various purification methods. For example, for antibiotic
compounds that are water soluble, an ion-exchange method may be used for purification. In
this method, the compound is first separated from the waste organic materials in the broth and
then sent through equipment, which separates the other water-soluble compounds from the
desired one. To isolate an oil-soluble antibiotic such as penicillin, a solvent extraction method
is used. In this method, the broth is treated with organic solvents such as butyl acetate or
methyl isobutyl ketone, which can specifically dissolve the antibiotic (Pelczar, Chan, &
Krieg, 1993). The dissolved antibiotic is then recovered using various organic chemical
means. At the end of this step, the manufacturer is typically left with a purified powdered
form of the antibiotic, which can be further refined into different product types.
(iv) Modify Produced Antibody Chemically
The production of a semi-synthetic antibiotic involves the use of a fermentation-derived
antibiotic, which is then modified by the addition of side chain to give rise to an antibiotic
with new properties. Another example achieved in a different manner is the chemical
alteration of specific sites in an antibiotic in order to render the antibiotic immune to an
enzyme which destroys it, as is done with streptomycin (Allsop, & Illingworth, 2002).
(v) Marketing and Financing
The marketing and financing of the business are of paramount importance since the aim
of the producing firm is profit maximization (Okafor, 2007).

CONCLUSION
Since the development of a new drug is a costly proposition, pharmaceutical companies
have done very little research in the last decade. However, an alarming development has
spurred a revived interest in the development of new antibiotics. It turns out that some of the
disease-causing bacteria have mutated and developed a resistance to many of the standard
antibiotics. This could have grave consequences on the world's public health unless new
antibiotics are discovered or improvements are made on the ones that are available. This
challenging problem will be the focus of research for many years to come.

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