Production Of Enzymes By
Fermentation Method
What is fermentation?
• Pasteur’s definition: “life without air”, anaerobe
redox reactions in organisms.
• New definition: a form of metabolism in which the
end products could be further oxidized.
For example: a yeast cell obtains 2 molecules of
ATP per molecule of glucose when it ferments it
to ethanol.
What is fermentation?
Microorganisms, typically grown on a large
scale, to produce valuable commercial products
or to carry out important chemical
transformations. This process is commonly
referred to as Fermentation.
Types of fermentation process
•
There are two methods of fermentation used to
produce enzymes.
1. submerged fermentation
2. solid-state fermentation.
Submerged fermentation/ Solid-state
fermentation
• Submerged fermentation involves the production of
enzymes by microorganisms (e.g. bacteria, yeast) in a liquid
nutrient media (water content of the media: > 95%)
• Solid-state fermentation is the cultivation of
microorganisms on a solid substrate.
• Carbon containing compounds in or on the substrate are
broken down by the micro organisms, which produce the
enzymes either intra-cellularly or extra-cellularly.
Submerged fermentation/ Solid-state
fermentation Cont….
• The enzymes are recovered by methods such as
centrifugation, for extra cellularly produced enzymes
and lysing of cells for intracellular enzymes.
• Many industries are dependent on enzymes for the
production of their goods.
• Industries that use enzymes generated by fermentation
are the brewing, wine making, baking and cheese making.
Submerged Fermentation
Cont…
•
•
•
•
Advantages:
Measure of process parameters is easier than
with solid-state fermentation.
Bacterial and yeast cells are evenly distributed
throughout the medium.
There is a high water content which is ideal for
bacteria.
Disadvantages:
High costs due to the expensive media.
2. Solid State Fermentation
- Solid-state fermentation (SSF) is another method used
for the production of enzymes.
- In solid state, water content is 40~ 80%.
- Solid-state fermentation involves the cultivation of
microorganisms on a solid substrate, such as rice husk,
wheat bran, sugar beet pulp, wheat and corn flour.
Solid State Fermentation
Cont…
Advantages:
SSF has many advantages over submerged
fermentation. These include:
1. High volumetric productivity
2. Relatively high concentration of product
3. Less effluent generated
4. Simple fermentation equipment.
Fermentation
• Aerobic
• Anaerobic
Aerobic fermentation
• Adequate aeration.
• Bioreactors- adequate supply of sterile air.
• In addition, these fermentors may have a
mechanism for stirring and mixing of the medium
and cells .
eg. Antibiotics, enzymes, vitamins.
Anaerobic fermentation
• In anaerobic fermentation, a provision for
aeration is usually not needed.
e.g. Lactic acid, ethanol, wine
• When referring to fermentation regarding food,
there are no distinctions between
anaerobic and aerobic metabolism.
Cont….
•
Fermentation changes the characteristics of
the food by the action of the enzymes
produced by bacteria, mould and yeasts, which
can occur in aerobic or anaerobic conditions
Cont….
• The process of fermentation requires a food
source (e.g. glucose); enzymes form
bacteria or yeast and (depending on the product)
anaerobic or aerobic conditions.
Cont..
• 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.
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
Fermenter
• The heart of the fermentation process is the
fermentor.
In general:
• Stirred vessel, H/D  3
• Volume 1-1000 m3 (80 % filled)
• Biomass up to 100 kg dry weight/m3
• Product 10 mg/l –200 g/l
Types of fermentor
•
•
•
•
•
•
Simple fermentors (batch and continuous)
Fed batch fermentor
Air-lift or bubble fermentor
Cyclone column fermentor
Tower fermentor
Other more advanced systems, etc
The size is few liters (laboratory use) - >500 m3
(industrial applications)
View looking down into a 125m3
stainless steel fermentor
Cross section of a fermenter for Penicillin
production
Cross section of a fermenter for Penicillin
production
Fermentation could be:
• Batch mode
• Fed batch mode (continuous)
Batch fermentation
• Most fermentations are batch processes
• Nutrients and the inoculum are added to the sterile
fermentor and left to get on with it!
• Anti-foaming agent may be added.
• Once the desired amount of product is present in
the fermentor 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.
Fermentation Medium
Definition of medium  nutritional, hormonal, and
substrate requirement of cells
• In most cases, the medium is independent of
bioreactor design and process parameters
•
the
Type of medium: complex and synthetic medium
(mineral medium)
• Even small modifications in the medium could change
cell line stability, product quality, yield, operational
parameters, and downstream processing.
Medium Composition
Fermentation medium consists of:
• Macronutrients : (C, H, N, S, P, Mg sources 
water, sugars, lipid, amino acids, salt minerals)
• Micronutrients: (trace elements/ metals,
vitamins)
• Additional factors: growth factors, attachment
proteins, transport proteins, etc)
• For aerobic culture, oxygen is sparged
Inoculums
• Incoculum is the substance/ cell culture that is
introduced to the medium. The cell then grow in
the medium, conducting metabolisms.
• Inoculum is prepared for the inoculation before
the fermentation starts.
• It needs to be optimized for better performance:
• Adaptation in the medium
• Mutation (DNA recombinant, radiation, chemical
addition)
Required value generation in fermentors as
a function of size and productivity
Microbial rates of consumption or
production
C, N, P, S source
H2O
H
+
biomass
CO2
O2
product
heat
Microbes
Overview of bioreactors for
submerged system
-
Classification:
operation modes:
1. batch:
stirred tank
2. continuous: chemostat, fluidized-bed
3. modified types of the above modes:
fed-batch, chemostat with
recycle, multi-stage continuous reactors
Oxygen supply:
- aerobic: airlift
- anaerobic
Form of biocatalyst:
free cell (enzyme)
immobilized cell (enzyme)
packed-bed, membrane reactor
Industrial Bioreactor
World's Largest Industrial Fermenter (Chem. Eng.
News,10-Apr-78)
The fermenter is 200' high and 25 ft diam
The End