The document discusses the fermentation processes involved in the production of penicillin, streptomycin, and vitamin B12, highlighting the roles of specific microorganisms, conditions, and steps required for each process. It details the types of fermentation (aerobic and anaerobic), the use of bioreactors for cultivation, and the extraction and recovery methods for the antibiotics produced. Additionally, it outlines the biochemical processes of riboflavin production and the importance of nutrient sources in maximizing yields.

1. Production of
Penicillin
Streptomycin
Vitamin B12
Submitted By: Abutala
M. Pharm
Department of Pharmaceutical
Chemistry
SPER, Jamia Hamdard
F E R M E N T A
T I O N
F E R M E N T A
T I O N
F E R M E N T A
T I O N
F E R M E N T A T I O N
&

2. CONTENT
Fermentation;
Introduction
Aerobic Fermentation
Anaerobic Fermentation
Production of Penicillin
Production of
Streptomycin
Production Vitamin B2
Reference

3. The word fermentation originates from a latin verb
fervere which literally means to boil.
During the production of alcohol (the first truly
industrialized process), the gas bubbles (of CO₂) appear
at the surface of the boiling liquid.
Fermentation is the generation of energy by catabolism
of
organic molecules
INTRODUCTI
ON
"Any process mediated by or involving microorganism in
which a product of economic value is obtained" is called
fermentation
Definition

4.  "Aerobic" means 'in the presence of oxygen“
 Organism use oxygen for the conversion of the
complex organic compounds, but process is
known as aerobic respiration
 Oxygen is required for the production & growth of
microorganisms (yeast/ Bacteria etc)
 Yeast requires oxygen for a number of processes
essential for reproduction
 Most fermentation involves the initial introduction
of oxygen to ensure a strong yeast colony is
established & yeast will fermented without using
oxygen even if oxygen is available.
Aerobic Fermentation

5. Anaerobic Fermentation
Chemical breakdown of organic substrates into
ethanol or lactic acid by microorganism in the
presence of oxygen
Occurs in the cytoplasm
Occurs in yeast, parasites and bacteria
Does not used oxygen
Only 2 ATP is produced
Aerobic Fermentation
Set of chemical reaction involve in the
production of energy by completely
oxidizing food
Occurs in both mitochondria and
cytoplasm
Occurs in higher animals and plants
Uses molecular oxygen as the final
electron accepter in the electron
transport chain
36 ATP is produced

6. Time 
No.
of
Cells(log)

Lag
Phase
Acceleratio
n
Phase
Log
Phase
Stationary
Phase
Decline
Phase
Time  Time 
No.
of
Cells(log)

No.
of
Cells(log)

Batch Fermentation Continous
Fermentation
Product
Product
Substrates
Substrates

7. Bioreactors
A bioreactor is basically a device in which the organisms
(cells) are cultivated and motivated to form the desired
product(s).
Homogenously mixed bioreactors
Chemostat bioreactors
Turbidostat bioreactors

8. Fermenters
A fermenter usually refers to the containment system for the cultivation of prokaryotic
cells (bacteria, fungi), while a bioreactor grows the eukaryotic cells (mammalian,
insect).
Batch Fermenter Continous Fermenter

9. Penicillium chrysogenum

10. ► P. chrysogenum is high yielding strain and therefore most widely used as production strain.
Production of penicillin

11.  1) A starter culture is needed for inoculation.
 2) After getting growth on solid media, one or two growth
stages should allowed in shaken flask cultures to create a
suspension, which can be transferred to seed tanks for
further growth.
 3) After about 24-28 hours, the content of the seed tanks is
transferred to the primary fermentation tanks
 4) All the bio parameters like temperature, pH, aeration,
agitation etc. should be properly maintained.
near 6.5
26°C to 28°C
a continuous stream of
sterilized
air is pumped into it
(0.5 – 1.0 vvm)
baffles allow constant
agitation (200rpm)
Inoculum Preparatio

12. The lyophophilized culture of spores is cultivated for inoculum development
which is transferred to preferment and then to fermenter
Penicillin production is an aerobic process & continuous supply of O₂ to the
growing culture is very essential.
Preferment
ation
Carbon
Source
Nitrogen
Source
Precursors
4-5% dry
Corn
Steep
Liquor
Lactose
Yeast Soy
Meal Ammoni
um
Sulphate
Phenoxy acetic acid
 penicillin-G  penicillin-X
 penicillin-V
phenyl acetic acid
hydroxy phenyl
acetic acid
phenoxy acetic acid

13.  Continuous feeding of sugar is advantageous
for a good yield of penicillin.
 The growth of the organism from spores must
be in a loose form & not as pellets. The growth
phase is around 40hrs with a doubling time of 6
- 8 hrs.
 After the growth phase is stabilized, the
penicillin production exponentially increases
with appropriate culture condition.
 The penicillin production phase can be extended
to 150-180hrs.

14. Recovery of penicillin
 As the fermentation is complete, the broth containing about 1%
penicillin is processed for extraction. The mycelium is removed by
filtration.
 Penicillin is recovered by solvent (n-butyl acetate or methyl ketone)
extraction is low temperature (<10 °C) & acidic pH (<3.0).
 The chemical & enzymatic (bacterial penicillinase) degradation of
penicillin can be minimized.
 The penicillin containing solvent is treated with activated carbon to
remove impurities and pigments.
Penicillin can be recovered by adding potassium or sodium acetate. The
potassium or sodium salts of penicillin can be further processed (in dry solvent
such as n-butanol or isopropanol) to remove impurities.
As the water is totally removed, penicillin salt can be crystallized and dried
under required pressure. This can be processed to finally produce the
pharmaceutical dosage forms.
90% Yield

15. Application to
Biotechnology
It produces the hydrophobic ẞ-
lactam compound penicillin.
Penicillium chrysogenum remains the
primary producer of Penicillin G and
Penicillin V
P. chrysogenum has been used
industrially to produce Penicillin G
and Penicillin V and Xanthocillin X
Penicillium chrysogenum can be used to
assist crops to fight off other pathogenic
species.
to produce the enzymes polyamine oxidase,
phosphogluconate dehydrogenase, and glucose
oxidase.

16. Production of Streptomycin
The medium used for streptomycin usually consist of soy
meal or soy flour or corn syrup that can supply glucose at
a slow rate (amylase activity is poor in Streptomyces sp).
Medium
Carbon
Source
The initial supply of nitrogen (NH3) and phosphate is also
obtained from soy meal. This is required since glucose,
ammonia & phosphate in high quantities inhibit streptomycin
synthesis.
near 6.5 – 7.5
0.5 – 1.0 vvm
6 – 8 Days
27°C to 30°C

17. It take about 24 hours to 48 hours.
Rapid growth and formation of abundant mycelium occur
during this phase.
The pH rises to 8.0 due to release of ammonia into medium, due
to proteolytic activity of S.griseus.
Glucose is utilized slowly and little production of streptomycin is
witnessed
It lasts for 2days.
Streptomycin production takes place at a rapid rate without
increase in the mycelial growth.
The ammonia released in the first phase is utilized, which
results in the decrease of pH to 7.6 - 8.0, glucose and oxygen
are required in large quantity during this phase.
Cells undergo lysis, releasing ammonia & increase in the pH,
which falls again after a period of continuous streptomycin
production.
Requirement of oxygen decreases & the contents of the
medium including sugar get exhausted. The yield of 1200
microorganism per milliliter of streptomycin is obtained.
01
02
03
Phase
Phase
Phase

18.  Streptomycin or other amino glycoside are basic in
nature.
 They are recovered by weak cationic exchange resins
in an ion exchange column.
 Treatment with activated carbon is necessary to
remove impurities.
 Streptomycin can be precipitated in the form of sulfate
salt
Recovery of Streptomycin

19. Production of Vitamin B2
exerts its biochemical functions through the
coenzymes; flavin adenine dinucleotide (FAD) &
flavin mononucleotide (FMN).
worldwide requirement of riboflavin is
estimated to be around 2500 tone per year.
water soluble
vitamin
deficiency result in cheilosis (fissures at the
corner of mouth), glossitis (purplish
tounge) and dermatitis
Biotransformation
Chemical
Synthesis
Fermentation
There are three processes employed for the large scale production of
riboflavin.

20.  Industrial production of riboflavin is mostly carried out with the organism,
Ashbya gossypii by using simple sugar such as glucose & corn steep
liquor.
 Glucose can be replaced by sucrose or maltose for the supply of carbon
source.
 In recent year, lipids such as corn oil, when added to the medium for
energy purpose, have a profound influence on riboflavin production.
 Further, supplementation of the medium with yeast extract, peptones,
glycine, inositol, purines (not pyrimidines) also increase the yield of
riboflavin.
 The process is by submerged aerated fermentation.
near 6 – 7.5
0.3 vvm
5 – 7 days
26°C to 28°C

21. 01
02
03
Phase
Phase
Phase
This phase is characterized by rapid growth of the organism
utilizing glucose.
As pyruvic acid accumulates, pH becomes acidic.
The growth of the organism stops as glucose gets exhausted.
In phase I, there is no production of riboflavin.
Sporulation occurs in this phase, and pyruvate concentration
decreases.
Simultaneously, there is an accumulation of ammonia (due to
enhanced deaminase activity) which makes the medium alkaline.
Phase II is characterized by a maximal production of riboflavin.
But this is mostly in the form of FAD and a small portion of it as
FMN.
In this last phase, cells get disrupted by a process of
autolysis.
This allows release of FAD, FMN and free riboflavin into the
medium.

22. Candida sp can also produce riboflavin, but this fermentation process is
extremely sensitive to the presence of iron.
Consequently, iron or steel equipment cannot be used. Such equipment
have to be lined with plastic material.
Microorganism with corresponding yields of riboflavin
Microorganism Yield (mg/l)
Mycocandida riboflavin
Candida flareri
Eremothecium ashbyii
Ashbya gossypii
0.200
7.500
0.575
2.500

23.  JJ Nunes, R, Maharaj, Vijaya, Maharaj A Sedoo, LJ Fernandes, C.Holder, Waste
Paper to Antibiotics: A Design and Feasibility Study of a Penicillin Production
Facility in Trinidad and Tobago. Waste and Biomass Valorization (2020) 11:2581–
2589.
 CR Gadipelly, GD Yadav, A Perez-Gonzalez, I Ortiz, R Ibanez, VK Rathod, KV
Marathe, Pharmaceutical Industry Wastewater: Review of the Technologies for
Water Treatment and Reuse. Industrial & Engineering Chemistry Research
53(29):11571-11592.
 Riboflavin (Vit B2) Production, Dr. Ekta Khare, Department of Microbiology
Institute of Biosciences & Biotechnology, CSJM University, Kanpur

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