FERMENTATION

BY:
Sidra Sohail Asim
LEARNING OBJECTIVES

At the end of this topic, students will be able to:

 Process of cellular respiration
 Explain the process of fermentation
 Describe aerobic & anaerobic fermentation
 Identify the microorganisms used and the main stages
in the production of Penicillin, Cephalosporin,
Erythromycin, Tetracycline, Rifamycin, Griseofulvin,
Chloramphenicol by fermentation.
 Describe how Downstream processing is carried out to
extract and purify the end-product of fermentation.
ANTIBIOTICS

 Of all the microbial products manufactured

commercially, antibiotics are the most
important.
 Antibiotics are chemical substances
produced by microorganisms to kill other
microorganisms.
 They are used in the treatment of infectious
diseases.
CELLULAR RESPIRATION
 Cellular respiration is a series of chemical
reactions that break down glucose to
produce ATP, which may be used as energy
to power many reactions throughout the
body.
 It take place within the cell of organism.

 There are two main types

a. Aerobic cellular respiration
b. Anaerobic cellular respiration
AEROBIC CELLULAR
RESPIRATION
 Aerobic means ‘with air”. This type of respiration
needs oxygen for it to occur so it is called aerobic
respiration
 Glucose + oxygen Carbon dioxide + Water
+ Energy
The chemical equation is:
𝑪 𝟔 𝑯 𝟏𝟐 𝑶 𝟔 + 𝟔𝑶 𝟐 → 𝟔𝑪𝑶 𝟐 +𝟔 𝑯 𝟐 𝑶+𝟐𝟗𝟎𝟎 𝐤𝐣
 3 stages
 – glycolysis
 – citric acid cycle
 – electron transport chain
 STAGES OF AEROBIC CELLLULAR
RESPIRATION
 In glycolysis, a net of 2 molecules of ATP, or chemical
energy, are produced.

 The citric acid cycle (kreb’s cycle)produces another

2 molecules of ATP
 The electron transport chain produces 36 molecules
of ATP

 Oxygen is used in aerobic cellular respiration as the

final electron acceptor in the electron transport
chain, which is part of why it’s able to create so
much ATP https://
www.youtube.com/watch?v=eBl3U-T5Nvk
ANAEROBIC CELLULAR
RESPIRATION

It takes place when there is not enough oxygen

available for aerobic respiration
In anaerobic cellular respiration, the only step of
this process that occurs is glycolysis

In this type of respiration, glucose is converted into lactic

acid and energy is released.
https://www.youtube.com/watch?v=YbdkbCU20_M&t=51s
FERMENTATION
Fermentation is a metabolic process in which an
organism converts a carbohydrate, such as starch
or a sugar, into an alcohol or an acid. Fermentation
is a type of anaerobic respiration.
The science of fermentation is called “zymology”.
RANGES OF FERMENTATION
PROCESS
 Microbial cell (Biomass)
 Yeast
 Microbial enzymes
 Glucose isomerase
 Microbial metabolites
 Penicillin
 Food products
 Cheese, yoghurt, vinegar
 Vitamins
 B12, riboflavin
 Transformation reactions
 Steroid biotransformation
TYPES OF FERMENTATION

 1.Lactic acid fermentation

 2.Alcoholic fermentation
 LACTIC ACID FERMENTATION
 lactic acid is formed from pyruvate produced in
glycolysis.
 NAD+ is generated from NADH. Enzyme lactate
dehydrogenase catalyses this reaction.
 Lactobacillus bacteria prepare curd from milk
via this type of fermentation.
 During intense exercise when oxygen supply is
inadequate, muscles derive energy by producing
lactic acid, which gets accumulated in the cells
causing fatigue.
LACTIC ACID FERMENTATION
ALCOHOL FERMENTATION
 This is used in the industrial production of
wine, beer, biofuel, etc.
 The end product is alcohol and CO2. Pyruvic
acid breaks down into acetaldehyde and CO2
is released.
 In the next step, ethanol is formed from
acetaldehyde. NAD+ is also formed from
NADH, utilized in glycolysis.
 Yeast and some bacteria carry out this type
of fermentation. Enzyme pyruvic acid
decarboxylase and alcohol dehydrogenase
catalyse these reactions.
ALCOHOL FERMENTATION
PRODUCTS OF FERMENTATION
TECHNIQUES OF FEREMENTATION

Two broad fermentation techniques have emerged as a

result of this rapid development:
Solid State Fermentation (SSF).
Submerged Fermentation (SmF).

Solid State Fermentation Solid state (substrate)

fermentation (SSF) has been defined as the
fermentation process occurring in the absence or near-
absence of free water. Solid state fermentation (SSF) is
another method used for the production of enzymes,
which involves the cultivation of microorganisms on a
solid substrate, such as grains, rice and wheat. SSF
employs natural raw materials as carbon source such as
cassava, barley, wheat bran, sugarcane bagasse.
Submerged fermentation:
In the submerged process, the substrate used for fermentation is
always in liquid state which contains the nutrients needed for
growth.
The fermentor which contains the substrate is operated
continuously and the product biomass is continuously harvested
from the fermenter by using different techniques then the
product is filtered or centrifuged and then dried.

Submerged fermentation is a method of manufacturing bio

molecules in which enzymes and other reactive compounds are
submerged in a liquid such as alcohol, oil or a nutrient broth
FERMENTOR
 A fermentor (bioreactor) is a closed vessel
with adequate arrangement for aeration,
agitation, temperature and pH control, and
drain or overflow vent to remove the waste
biomass of cultured microorganisms along-
with their products.

• Bioreactor is an instrument, which

performs the process of fermentation on
large scale.
FERMENTOR
INDUSTRIAL FERMENTORS
VIEW LOOKING DOWN INTO A 125M3
STAINLESS STEEL FERMENTOR
INDUSTRIAL FERMENTORS
 125-250m3
 Conditions in the fermenter are carefully
monitored to regulate cell growth.
 Fermenter and all pipe work must be sterile
before fermentation begins
 This is usually achieved by flushing the whole
system with superheated steam before the
production begins.
  pH controller. Control pH of fermentor
• COMPONENTS OR PARTS OF FERMENTOR

 Temperature controller.
 Impeller paddles.: impeller paddles is a
components which maintains the
homogeneous phase
 Cooling jackets.
 Baffles.: reduces particles size and prevent
particles from sticking the wall.
 Diffuser.: diffuser is the part , which diffuses
the air from the bottom of the inlet for
maximum aeration.
 Step 1: microbial inoculum (microbial cell) is
• mixed with the nutrition which help to grow
FERMENTATION PROCESS:

the medium.
Step 2: mixture is placed in the fermenter
( the instrument for the fermentation
process occurs.)
Step 3: filter the mixture after the process in
the fermenter.
Step 4: extraction
Step 5: purification
Step 6: final product
 Schemic representation of fermentation
process

https://www.youtube.com/watch?v=5eKdZ0dVCCo
https://www.youtube.com/watch?v=VKpthcW1llU
MODES OF FERMENTATION
 Fermentation could be:

 Batch mode
 Fed batch mode
 Continuous mode
BATCH FERMENTATION
 Most fermentations are batch processes

 Nutrients and the inoculum are added to the sterile

fermenter and left to get on with it!

 Anti-foaming agent may be added.

 Once the desired amount of product is present in the

fermenter the contents are drained off and the product
is extracted.

 After emptying, the tank is cleaned & prepared for a

new batch.
CONTINUOUS FERMENTATION
 Some products are made by a continuous
culture system.
 Sterile medium is added to the fermentation
with a balancing withdrawal of broth for
product extraction.

Inside the fermenter, the exponential growth of

microorganisms is maintained by providing and
changing the conditions and nutrients.
FED BATCH FERMENTATION
 Fed-batch culture is, in the broadest sense,
defined as an operational technique in
biotechnological processes where one or
more nutrients (substrates) are fed
(supplied) to the bioreactor during
cultivation and in which the product(s)
remain in the bioreactor until the end of the
run.
 PRODUCTION OF
ANTIBIOTICS
 SOME ANTIBIOTICS PRODUCED
BY MICROORGANISMS
Antibiotic Producing microorganism
Cephalosporin Cephalosporium acrimonium
Chloramphenicol Streptomyces venezuelae
Erythromycin Streptomyces erythreus
Griseofulvin Penicillium griseofulvin
Penicillin Penicillium chrysogenum
Streptomycin Streptomyces griseus
Tetracycline Streptomyces aureofaciens
Gentamicin Micromonospora purpurea
 Thanks to work by Alexander Fleming (1881-1955), Howard
Florey ( 1898-1968) and Ernst Chain (1906-1979), penicillin was
first produced on a large scale for human use in 1943. At this
time, the development of a pill that could reliably kill bacteria
was a remarkable development and many lives were saved
during World War II because this medication was available.

A. Fleming E. Chain H. Florey

PRODUCTION OF PENICILLIN

 During world war II-

importance realized, as
penicillin had been
used to treat many
wounded soldiers.
A TALE BY A. FLEMING
 In 1928, Sir Alexander Fleming, a Scottish
biologist, observed that Penicillium notatum,
a common mold, had destroyed
staphylococcus bacteria in culture.
A TALE BY A. FLEMING
 He took a sample of the mold
from the contaminated plate.
He found that it was from the
Penicillium family, later
specified as Penicillium
notatum. Fleming presented his
findings in 1929, but they
raised little interest. He
published a report on penicillin
and its potential uses in the
British Journal of Experimental
Pathology.
MOA OF PENICILLIN
 All penicillin like antibiotics inhibit
synthesis of peptidoglycan, an essential
part of the cell wall.

 They do not interfere with the synthesis

of other intracellular components.

 These antibiotics do not affect human

cells because human cells do not have cell
walls.
SPECTRUM OF ACTIVITY
 Penicillins are active against Gram positive
bacteria
 Some members (e.g. amoxicillin) are also
effective against Gram negative bacteria but
not Pseudomonas aeruginosa
PENECILLIN
 Penicillin is not a single chemical compound,
but a group of compounds of related
structure and activity. There are six
penicillins –
 Penicillin G (benzyl penicillin)
 penicillin V (phenoxy-methyl penicillin)
 penicillin F (A2-pentcnyl penicillin)
 penicillin X (p-hydroxybenzyl penicillin)
 penicillin K (n-heptyl penicillin)
 penicillin O (allyl-mercaptomethyl
penicillin).
PRODUCTION OF PENICILLIN
 First antibiotic to have been manufacture in
bulk.

 Earlier media contained lactose, but it is no

longer used. The fermentation is carried out
under aerobic conditions, and nutrient
supply is maintained by regular feeding (fed-
batch culture). The fungus grows in a
submerged culture mostly as mycelial balls.
 When penicillin was first made at the end of
the second world war using the fungus
Penicillium notatum, the process made 1 mg
dm-3.
 Today, using a different species (P.
chrysogenum) and a better extraction
procedures the yield is 50 g dm-3.
 There is a constant search to improve the
yield.
  Improvement in composition of the medium
THE YIELD OF PENICILLIN CAN BE INCREASED BY:

 Isolation of better penicillin producing mold

sp. Penicillium chrysogenum which grow
better in huge deep fermentation tank
 Development of submerged culture
technique for cultivation of mold in large
volume of liquid medium through which
sterile air is forced.
PRIMARY AND SECONDARY
METABOLITES
 Primary metabolites are produced during
active cell growth, and secondary
metabolites are produced near the onset of
stationary phase.
COMMERCIAL PRODUCTION OF
PENICILLIN
 Like all antibiotics,
penicillin is a
secondary
metabolite, so is
only produced in the
stationary phase.
INDUSTRIAL PRODUCTION OF ANTIBIOTIC- PENICILLIN

 The industrial production of penicillin was

broadly classified in to two processes
namely,

 Upstream processing
 Downstream processing
UPSTREAM PROCESSING

 Upstream processing encompasses any

technology that leads to the synthesis of a
product.

Upstream includes the

 Exploration
 Development
 Production.
DOWNSTREAM PROCESSING
 The extraction and purification of a
biotechnological product from fermentation
is referred to as downstream processing.
UPSTREAM PROCESSING
INOCULUM PREPARATION
 The medium is designed to provide the
organism with all the nutrients that it
requires.

 Inoculation method- submerged technique

 Spores -major source of inoculum

RAW MATERIALS
 CARBON SOURCES:
Lactose acts as a very satisfactory carbon compound, provided that is
used in a concentration of 6%. Others such as glucose & sucrose may
be used.
NITROGEN SOURCES:
 Corn steep liquor (CSL)
 Ammonium sulphate and ammonium acetate can be used as
nitrogenous sources.
MINERAL SOURCES:
Elements namely potassium, phosphorus, magnesium, sulphur, zinc
and copper are essential for penicillin production. Some of these are
applied by corn steep liquor.

 Calcium can be added in the form of chalk to counter the natural

acidity of CSL

 PAA- precursor
FERMENTATION PROCESS
 The medium is inoculated with a suspension
of conidia of Penicillium chrysogenum.
 The medium is constantly aerated and
agitated, and the mould grows throughout as
pellets.
 After about seven days, growth is complete,
the pH rises to 8.0 or above, and penicillin
production ceases
STAGES IN DOWNSTREAM
PROCESSING
Removal of cells

 The first step in product recovery is the

separation of whole cells and other insoluble
ingredients from the culture broth by
technique such as filtration and
centrifugation.
ISOLATION OF BENZYL PENICILLIN

 The PH is adjusted to 2-2.5 with the help of phosphoric

or sulphuric acids.
 In aqueous solution at low PH values there is a partition
coefficient in favor of certain organic solvents such as
butyl acetate.
 This step has to be carried out quickly for penicillin is
very unstable at low PH values.
 Antibiotic is then extracted back into an aqueous buffer
at a PH of 7.5, the partition coefficient now being
strongly in favor of the aqueous phase. The resulting
aqueous solution is again acidified & re-extracted with
an organic solvent.
 These shifts between the water and solvent help in the
purification of penicillin.
 The treatment of the crude penicillin extract varies
according to the objective, but involves the
formation of an appropriate penicillin salt.
 The solvent extract recovered in the previous stage is
carefully extracted back with aqueous sodium
hydroxide.
 This is followed by charcoal treatment to eliminate
pyrogens and by sterilization.
 Pure metal salts of penicillin can be safely sterilized
by dry heat, if desired. Thereafter, the aqueous
solution of penicillin is subjected to crystallization.
FURTHER PROCESSING
 For parental use, the antibiotic is packed in
sterile vials as a powder or suspension.
 For oral use, it is tabletted usually now with
a film coating.
 Searching tests (ex: for purity, potency) are
performed on the appreciable number of
random samples of the finished product.
 It must satisfy fully all the strict government
standards before being marketed
THE MAIN STAGES OF
PENICILLIN PRODUCTION ARE:
PRODUCTION OF PENICILLIN V
 Phenoxy methyl penicillin

 Addition of different Acyl groups to the medium.

 Phenoxyacetic acid as precursor instead of

phenyl acetic acid.

https://
www.youtube.com/watch?v=SsozxmGX6cM
 CEPHALOSPORINS
 Cephalosporins are beta-lactam antibiotics that are
derived from Acremonium fungus. It was first
isolated in 1952
 Like other beta-lactams, these antibiotics work by
inhibiting bacterial cell wall synthesis.
 There are traditionally five generations of
cephalosporins classified based on their
antimicrobial characteristics.
 Each new generation provides a more extended
spectrum and has greater gram negative
bactericidal properties than the previous
generation.
 CEPHALOSPORIN PRODUCTION
 Cephalosporin is produced through fermentation
of A. chrysogenum under the presence of oxygen
(aerobic conditions), carried out in a
conventional method using free or immobilized
fungi under solidstate or surface-liquid
fermentation that is operated in a bioreactor
 The fermentation process is carried out in a
chemically defined medium with the presence of
carbon sources, usually glucose and/or sucrose.
Acremonium chrysogenum through fermentation
process, either by batch or fed-batch mode.
FERMENTATION OF
CEPHALOSPORIN
 Acreminium medium(ammonium chloride,
peptone, meat extract)
 Temp (28C)
 ph. 7 (cultivated)
 4 days
 extraction (by activated carbon or resin
other than solvent)
 filtration (ion exchange resin, gel filtration
activated)
 purification
 product
 CEPHALOSPORIN
 Organism use is cephalosporin acremonium .
fermentation

Cephalosporin C (not potent)

transform
adipic acid (removal of α-amino)

7-α amino cephalosporin acid

further modification by adding side

chain
GENTAMYCIN
 Gentamicin is naturally produced by the
bacterium Micromonospora purpurea, was
patented in 1962, approved for medical use
in 1964.

 The antibiotic is collected from the culture

of the Micromonospora by perforating the
cell wall of the bacterium
 Inoculation method: submegered technique
FERMENTATION OF GENTAMYCIN
M.purpurae+ Medium(sucrose< maltose, starch,
dextrin, glucose, nitrogen, CaCO3, MgSO4,
K2HPO4, Iron) (culture)

fermentation+(Medium)

Extraction (ethanol) acidified with H2SO4

filtration

purification

product
ERYTHROMYCIN
 Erythromycin is produced from Streptomyces
and Micromonospora.

 Erythromycin mainly produced by submerged

fermentation.

 During fermentation different types of

erythromycin are produced like A,B,C and D.
ERYTHROMYCIN PRODUCTION
Production Process 2 steps

1. Biomass accumulation
The sporulating culture of Streptomyces
erythraeus is done on Trypton agar slant. •
Cells are harvested from agar plates and
the suspension is taken in Sterile water. •
The suspension is then stored at 4C.
2. Production Phase
Media which used in a stirred tank fermenter
consists of,
• Sucrose – 5%
• Corn steep liquor – 0.5%
• Soyabean oil meal – 1.5%
• Yeast – 1.0%
• NaCl – 0.5%
• CaCo3 precipitate – 0.3%
• pH – 7.0 -7.2
Production of Erythromycin production occurs when
froth reaches stationary phase.
• Addition of n-propanol as precursor increases the
production of erythromycin.
TETRACYCLINE
 First prepared by catalytic dehydrogenation
of chlortetracycline synthetically and later
using streptomyces aureofacience in 1954.
Medium : aerated submerged culture.
Composition:
 Vegetable oil – antifoaming agent
 Glycerol
 Starch - carbon source
 Sucrose
 Ammonium salt - Nitrogen source
 Peanut oil
 Cotton seed oil
 Corn seed oil
 Buffer to maintain pH

PRODUCTION:
Production take place in three phases

1st phase:
 Rapid consumption of nutrients and increase
in cell mass of Streptomyces aureofacience
2nd phase
 Here growth of microorganism decreases
and same time ceases.
 Maximum quantity of antibiotic produces.

3rd phase:
 decreased antibiotic production.
 lysis of mycelium occur, phosphate ions
decreases the fermentation.

Temperature : 28-29˚C.
pH : 5.5-6.5
GRISEOFULVIN
 It is an antifungal antibiotic.
 It is less toxic.
 Use orally for the treatment and control of
skin, nail and hair fungal problems.
 Organism used is Penecillium griseofulvin.

Medium : submerged aerobic culture.

 PRODUCTION
 For production of grisofulvin nutrient level in
fermentor is limitation factor.
 For fermentation airation and control of both
pH and nitrogen availibility is maintained.
FOR GOOD YIELD
 When carbohydrates level is sufficient.

 PO4 ions is 0.4-0.5%,so good yield is achieve.

 Chloride ions are required.

If chloride ions are insufficient in fermentor

then the product will be devoid of antifungal
activity means no antifungal property.
 Glucose syrup 50% is added step wise after
the 1st 16-20 hours that mean continuous
addition of glucose done
 pH maintain 6.0-7.2
 Temperature 20c
 If nutrient is proper, so at end the result and
yield should be 6.8gm/l and time period is 10
days for fermentation.
 If we add methyl donors in fermentor so it
can increase yield that is chlorine salts,
methyl zentate and folic acid so yield will be
12.15g/l.
EXTRACTION AND PURIFICATION.
 Antibiotic is dissolve in organic solvent for

Extraction.
 filtration to separate from cells.
 it should be treated with butyl acetate.
 Evaporation process done. After that we

Obtain crude griseofulvin.

 Crude griseofulvin will washed with
chloroform and re-crystallized in aqueous
method.
 This whole procedure gives 58% yield of
Griseofulvin .
 This process is to improve yield.
 If extraction done with methylene chloride
as organic solvent, so yield increase to 95%
and product purity will be 88%.
 When recrystallization is done with acetone
so 99% pure product will obtain yield
will be same.
RIFAMYCIN
 It is a new group of antibiotic.
 Obtain by different species like
streptomyces, micromonospora.
 Highly used strain are Nocardia mediterranai
 It is isolated in 5 different active form.
 Rifamycin A
 Rifamycin B (most active)
 Rifamycin C
 Rifamycin D
 Rifamycin E
 Rifamycin B can be change to more active
antibiotics called as rifamycin O,S and .
 Rifamycin are microbiological more active
antibiotics.
 Rifamycin S and V effective against gram
+ve.

INOCULUM: stock cultures of Nocardia

mediterranei safe in sealed ampules ad they
are lyophilized.
Frozen culture is highly stable over a long
period of time.
Medium composition:
 Peanut oil
 Soyabean meal
 Ammonium sulphate
 Propylene glycol
 Glucose
 Glycerol
 Fish meal

The best yield can be obtained when 5%

inoculum of pre-culture grown for 24hrs. Or
With a 1.2% inoculum of 40hrs old culture is
used in aerated fermenter at 28˚C.
RANGES OF FERMENTATION
Production of Biomass:
Microbial biomass is produced commercially as
single cell protein (SCP) for human food and
animal feed and as a viable yeast cells to be
used in the baking industry.
Production of microbial enzymes:
Enzymes may be produced by animals and
plants as well as microbial sources but the
production by microbial fermentation is the
most economical and convenient method.
Production of microbial metabolite:
 Primary and secondary metabolite produced
during fermentation.
 Primary metabolite and their commercial
significance :
Prim.metabolite Micro-organism significance
ethanol Saccharomyces cervisiae Alcoholic
beverages
Citric acid Aspergillus niger Food industry
Glutamic acid Corynebacterium Flavor enhancer
glutamicum
Lysine Corynebacterium Feed additive
glutamicum
Polysaccharide Xanthamonas spp. Enhaced oil
recovery
 Secondary metabolite and their commercial
significance: penicillin, cephalosporin,
gentamycin used as antibiotic.

Transformation of different chemical

substances by micro-organism:
In fermentation micro-organism covert
substances into entirely different substances
in single step.
e.g: conversion of ethanol into acetic acid
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