INDUSTRIAL FERMENTATION

Prepared By :
Mahendra G
S
M-
Pharm,Pharm
aceutical
Chemistry
JSSCP,
MYSURU
 Introduction
• Fermentation
– Process involving the biochemical activity of organisms,
during their growth, development, reproduction, even
senescence and death.
• Fermentation Technology
– Involves the use of microorganisms and enzymes for
production of compounds which have application in the
energy, material, pharmaceutical, chemical and the food
industry.
 Basic Principle of Industrial Fermentation

• Oganisms are grown under suitable conditions, by providing raw

materials meeting all the necessary requirements such as carbon,
nitrogen, salts, trace elements and vitamins.
• The end products formed as a result of their metabolism during
their life span are released into the media, which are extracted for
use by human being and that have a high commercial value.
• The major products of fermentation technology produced
economically on a large scale industrial basis are
– wine, beer, cider, vinegar, ethanol, cheese, hormones, antibiotics,
complete proteins, enzymes
 Fermentation Methodology

• Fermentation process is carried out in a container

called the fermentor or bioreactor.
• The design and nature of the fermentor varies
depending upon the type of fermentation carried
out.
• Invariably all the fermentors have facilities to
measure some of the fermentation
parameters
– like temperature, pressure, pH, elapsed fermentation
time, liquid level, mass etc.
Typical Fermenter
 Six Phases of The Microbial Growth

1. Lag phase: Immediately after inoculation, there is no

increase in the numbers of the microbial cells for some
time and this period is called lag phase. This is in order
that the organisms adjust to the new environment they
are inoculated into.
2. Acceleration phase: The period when the cells just start
increasing in numbers is known as acceleration phase.
3. Log phase: This is the time period when the cell numbers
steadily increase.
 Six Phases of The Microbial Growth
4. Deceleration phase: The duration when the steady growth declines.

5. Stationary phase: The period where there is no change in the microbial

cell number.
This phase is attained due to depletion of carbon source or
accumulation of the end products.

6 Death phase: The period in which the cell numbers decrease steadily.
This is due to death of the cells because of cessation of metabolic
activity and depletion of energy resources.
Depending upon the product required the different phases of the cell
growth are maintained.
– For microbial mass the log phase is preferred.
– For production of secondary metabolites i.e. antibiotics, the
stationary phase is preferred.
 Procedure of Fermentation

a) Depending upon the type of product required,

a particular bioreactor is selected.
b) A suitable substrate in liquid media is added at
a specific temperature, pH and then diluted.
c) The organism (microbe, animal/plant cell, sub-cellular
organelle or enzyme) is added to it.
d) Then it is incubated at a specific temperature for
the specified time.
e) The incubation may either be aerobic or anaerobic.
 Procedure of Fermentation
Aerobic conditions are created by bubbling oxygen through
the medium.
Anaerobic conditions are created by using closed vessels,
wherein oxygen cannot diffuse into the media and the oxygen
present just above is replaced by carbon dioxide released.
e) After the specified time interval, the products are removed,
as some of the products are toxic to the growing cell or at
least inhibitory to their growth.
The organisms are re-circulated. The process of removal of
the products is called downstream processing.
Penicillin Production
 Production
• Penicillium chrysogenum that produce antibiotics, enzymes
or other secondary metabolites frequently require
precursors like purine/pyrimidine bases or organic acids
to produce said metabolites.
 Primary metabolism is the metabolism of energy production
for the cell and for its own biosynthesis.
 Typically, in aerobic organisms (Penicillium chrysogenum) it
involves the conversion of sugars such as glucose to pyruvic
acid and the production of energy via the TCA cycle.
 Secondary metabolism regards the production of
metabolites that are not used in energy production for
example penicillin from Penicillium chrysogenum.
 Production
 Secondary metabolite being utilized as a
is against other
microorganisms
defence in the environment.
 mechanism
In essence Penicillium chrysogenum can kill off
the competition to allow itself to propagate
efficiently.
 It should be noted that these secondary
metabolites are produced in times of
stress whenonly resources are low and the
organism must produce these compounds to kill
off its competitors to allow it to survive.
 Media Formulation:
• Lactose: 1%
• Calcium Carbonate: 1%
• Cornsteep Liquor: 8.5% Corn steep liquor is a by-
product of corn wet-milling. A viscous concentrate of
corn solubles which contains amino acids, vitamins and
minerals, it is an important constituent of some growth
media.
• Glucose: 1%
• Phenyl acetic acid: 0.5g
• Sodium hydrogen phosphate: 0.4%
• Antifoaming Agent: Vegetable oil
 Inoculums Development

• Development of active logarithmic microbial culture

that is suitable for the final industrial production level
is known as inoculum development.
• Inoculum we use for industrial fermentations should
be •In its active, healthy and exponential growth
phase. •
• Free from contamination and required large
volumes. •
• Retain its capability of formation of desired product
formation.
Penicillium chrysogenum
growth
Production of streptomycin
Cyanocobalamin, B12
Distiller solubles: disyUnfermented grain residues (protein, fibre, fat and up to
70% moisture)
Vitamin B2/Riboflavin
 Production process of riboflavin
• Industrial production of riboflavin is mostly carried out with the organism, Ashbya
gossypii by using simple sugars such as glucose and corn steep liquor.

• Glucose can be replaced by sucrose or maltose for the supply of carbon source.

• In recent years, 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 pyrimidine’s) also increase the yield of riboflavin.

• It is essential to carefully sterilize the medium for good yield of riboflavin.

• The initial pH of the culture medium is adjusted to around 6-7.5.

• The fermentation is conducted at temperature 26-28°C with an aeration rate

0.3 vvm.

• The process is carried out for about 5-7 days by submerged aerated
fermentation.
 Production process of riboflavin

• Fermentation through phases:

• Phase I: 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.
• Phase II: 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.
 Production process of riboflavin

• Phase III: In this last phase, cells get disrupted

by a process of autolysis. This allows release of
FAD, FMN and free riboflavin into the medium.
• Recovery: Riboflavin is found in fermentation
broth and in a bound form to the cells. The latter
can be released by heat treatment i.e. 120°C for
about 1 hour.
• The cells can be discarded after filtration or
centrifugation. The filtrate can be further purified
and dried, as per the requirements.
 Introduction
 Higher LDL levels/hypercholesterolemia, atherosclerosis
 Angina, coronary artery diseases, stroke.

 Cholesterol-controlling medications- Statins

 Statins are the world's most prescribed drug in world to
combat hypercholesterolemia.
 Merck in1979 reported Lovastatin from Aspergillus terreus-,1987
FDA Approval.
 An estimated 30 million people worldwide take statins
Atorvastatin
 Lovastatin
Fluvastatin
Semi-synthetic forms
Compactin Natural statins   Pravastatin
Rosuvastatin
Simvastatin
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Mode of action of lovastatin

60
 Diverse applications of lovastatin

 Cardio-vascular: Cholesterol level-Artherosclerosis (Praveen et al., 2014).

 Bones: Osteoporosis, fractures (Li et al., 2003).
 Neuro-degenerative: MS/Parkinsons/Alzheimers (Schuster et al., 2009)
 Rheumatoid arthritis (Doornum et al., 2004).
 Antifungal activity: Candida sp., Cryptococcus neoformans
Saccharomyces cerevisiae (Chamilos et al., 2003)
 Anti-cancerous: Propoptotic (Masa et al., 2004, Elena et al., 2008, Julie et al., 1997,
Linda et al., 2010).
 Antioxidant: Epithelial cell damage-
 NADP(H), OH-, ROS, ONOO-NO- scavenger
 (Mohan-Kumari et al., 2011)

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 Lovastatin as anticancer agents

Lovastatin is reported to reduce proliferation of

• Lung cancer (Elena et al., 2008)

• Breast cancer (MCF-7), (Julie et al., 1997)

• Liver cancer (HepG2) (Linda et al.,

2010) and

• Cervical cancer (HeLa cells): is the second most common cancer in

women worldwide and thus is one of the leading causes of
mortality in women (Fritz et al., 2003).

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 Fungal organisms reported for lovastatin
production

 Aspergillus spp. (Lopez et al., 2003, Praveen et al., 2014)

 Penicillium spp. (Latha et al., 2011)
 Monascus spp. (Sayyad et al., 2007)
 Trichoderma spp. (Siamak et al., 2003)
 Pleurotus spp. (Julio et al., 2003) are widely reported soil
fungi capable of lovastatin production.

 However, Commercial production of lovastatin employs

A. terreus (ATCC-20542) (a soil fungus)

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1. Materials and Methods
1.1 Culturing of A.terreus by Solid State
Fermentation (SSF)

Wheat bran (40g) as substrate

Inoculated with spore suspension (107/8ml spores) of A. terreus

(KM017963)

Incubated at 280C for 7 days

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 1.2 Extraction

Inoculated substrate was dried at 40◦C for 24h

Crushed to powder

Ethyl acetate (150 ml) was added

Filtrate was dried using rotary

vacuum evaporator
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 1.3 Purification of lovastatin

One gram of dried crude lovastatin extract was loaded

on to pre-packed silica gel column

Elution with benzene (100%),

And combination of Benzene: Acetonitrile in the following ratio
95:5, 90:10, 85:15, 80:20
Acetonitrile (100%)

Thin Layer Chromatography (TLC)

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1.4 Detection of Lovastatin by Thin Layer
Chromatography (TLC)

Organic phase (20 µl) was spotted on TLC

plate

Dichloromethane: Ethyl acetate (70:30)

Rf comparison with standard Lovastatin

(Sigma)

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 Simvastati
•
n
Simvastatin, a semisynthetic derivertive of lovastatin, is
an important drug for the treatment of
hypercholesteromia, and is traditionally prepared by
direct alkylation of lovastatin.
• Chemical reaction conditons are very rigid, and
the final product is difficult to purify, also the
pressure of labor protection and environment
protection is very high.
• Recently, with the devolpement in the research of
lovastatin biosynthesis, more and more attention has
been paid to simvastatin biosynthesis.