This document provides an overview of fermentation technology and downstream processing. It defines fermentation as the production of a product by microorganism mass culture. It describes the basic stages of batch fermentation including lag, log, stationary and death phases. It then outlines the main steps in downstream processing including removal of insolubles, product isolation, purification, polishing and packaging. Specific unit operations used at each stage like centrifugation, filtration, chromatography are also explained. The document emphasizes that the level of downstream processing depends on the target product and its end use.

1. Fermentation technology
Down Stream Processing
Processing
Basics of
Fermentation
By
Chanakya Pachi

2. Basics of Fermentation
and Its Technology

3. Fermentation
• Fermentation is the term used by
microbiologists to describe any process for
the production of a product by means of the
mass culture of a microorganism.
• The extraction and purification of a
biotechnological product from fermentation.

4. Fermentation Basics
The product can either be:
A
microorganisms
’ own
metabolite
• Referred to as a
product from a
natural strain.

5. Batch Fermentation
• A batch fermentation can be considered to be a closed
system.
• At time t=0 the sterilized nutrient solution in the
fermenter is inoculated with microorganisms and
incubation is allowed to proceed.
• In the course of the entire fermentation, nothing is
added, except oxygen (in case of aerobic
microorganisms), and acid or base to control the pH

6. Batch Fermentation
• The composition of the culture medium, the
biomass concentration, and the metabolite
concentration generally change constantly as a
result of the metabolism of the cells.
• After the inoculation of a sterile nutrient solution
with microorganisms and cultivation under
physiological conditions, four typical phases of
growth are observed

8. Growth Phases
Lag phase
• Physicochemical equilibration between
microorganism and the environment.
Log phase
• Growth of the cell mass can now be described
quantitatively as a doubling of cell number per unit
time for bacteria.

9. Stationary phase
• As soon as the substrate is metabolized or toxic
substances have been formed, growth slows down or
is completely stopped.
Death phase
• In this phase the energy reserves of the cells are
exhausted.

10. Bio Reactors…

12. Down
StreamProcessing

13. General Steps in Downstream
Purification

14. Downstream processing
• The various stages of processing that occur after
the completion of the fermentation or
bioconversion stage, including separation,
purification, and packaging of the product.

15. Stages of Downstream
Processing
Removal of Insolubles
Product Isolation
Product Purification
Product Polishing
To markets

16. Stages in Downstream
Processing
• A few product recovery methods may be
considered to combine two or more stages.
• For example, expanded bed adsorption
accomplishes removal of insolubles and product
isolation in a single step. Affinity chromatography
often isolates and purifies in a single step.

17. Crystallization
Filtration
Gel
chromatography
Drying
Finishing/pack
aging
Chromatograph
y
Electrophoresis
Dialysis
High resolution
techniques
Precipitation
Chromatograph
y
Ultrafiltration
Partitioning
Distillation
Concentration
Centrifugation
Filtration
Clarification
Homogenizers
Hydrolytic
enzymes
Cell disruption
Flocculation
Centrifugation
Filtration
Cell Separation
Unit operations in downstream processing

18. Separation of cells and medium
• Recovery of cells and/or medium
(clarification)
• For intracellular enzyme, the cell fraction is
required
• For extracellular enzymes, the culture medium
is required
• On an industrial scale, cell/medium
separation is almost always performed by
centrifugation
• Industrial scale centrifuges may be batch,
continuous, or continuous with desludging

19. Solid liquid separation
• Following steps are involved:
• Floatation- gas is introduced into the liquid it
forms bubble. Cells and other solid particles are
absorbed and removed.
• Flocculation -cells and debris from large
aggregates and settles down. Addition of
flocculating agents are often done.
• Filtration- most commonly used to separate
biomass in culture medium.

20. Types of filtration used
Depth filters: they composed of filamentous matrix.
Particles are trapped in the matrix and fluid passes
out.
Absolute filters: the are of specific pore size than the
particles to be removed. Mostly used to remove
bacteria.
Rotatory drum vacuum filters: consists of rotating
drum immersed in tank of broth. As the drum
rotates it picks up the mass which forms a cake.
Membrane filters:

21. Centrifugation
• Tubular bowl centrifuge: small, commonly
used in pilot plants, can be run at high speed
and can be run in both batch or continuous
fermentation.
• Disc centrifuge: consists of several discs that
separate bowl into settling zones. Slurry is fed
through the centre.
• Multichamber centrifuge: modification of
tubular bowl type. consists of several
chambers which allows zigzag movement of
feed.

22. Release of intracellular products
Physical methods of cell disruption:
• Ultrasonication
• Osmotic shock: used to separate hydrolytic
enzymes and binding proteins.
• Heat shock:
• High pressure homogenization:
• Grinding with glass:

23. Chemical methods
• Alkali treatment:
• Organic solvents: toulene is very oftenly used. It
dissolves the membrane phospholipids.
• Detergents: triton x-100 or tweed is used

24. Enzymatic method
• Lysozymes: most frequently used for gram +ve
bacteria, for –ve in combination with EDTA it is
used.

25. Combination metods
• Concentration: the filtrate free from suspended
particles contains 80% water. The water is
removed to achieve concentration. Commonly used
techniques are:
• Evaporation
• Liquid-liquid extraction
• Membrane filtration
• Precipitation
• adsorption

26. Evaporation
• The evaporators in general , have a heating device
for supply of steam and unit for the seperation of
concentrated product and vapour, a condenser,
accessories and control equipment.

27. Dewatering
• Precipitation
• Salting out – addition of a high concentration of a soluble salt
(typically ammonium sulphate) causes proteins to aggregate
and precipitate.
• Addition of organic solvents
• Ultrafiltration
• The solution is forced under pressure through a membrane
with micropores, which allows water, salts and small
molecules to pass but retains large molecules (e.g., proteins)
• Spray drying
• Requires use of heat to evaporate water – unsuitable for most
proteins

28. Protein purification
• Adsorption chromatography
• Ion exchange chromatography – binding and
separation of proteins based on charge-charge
interactions
• Proteins bind at low ionic strength, and are eluted at
high ionic strength
+
+
+
+
+
+
+ +
+
+
-
- -
-
+
+
+
+
+
+
+
+
+
+
-
- -
+
Positively charged
(anionic) ion
exchange matrix
Net negatively
charged (cationic)
protein at selected pH
Protein binds to matrix

29. Typical ion exchange protein separation
Loading starts
Loading ends,
Low salt wash begins
Protein absorbance
Peak of
unbound
protein
Salt gradient
0
1M
Salt gradient
begins
Salt gradient
ends
Eluted peaks of weakly bound (I),
moderately bound (II)
and tightly bound (III) proteins
II
III
I

30. Affinity chromatography
• Binding of a protein to a matrix via a protein-
specific ligand
• Substrate or product analogue
• Antibody
• Inhibitor analogue
• Cofactor/coenzyme
• Specific protein is eluted by adding reagent which
competes with binding

31. Affinity chromatography
Matrix Spacer arm
Affinity
ligand
+
Active-site-bound enzyme
1. Substrate analogue affinity chromatography
Matrix Spacer arm
Antibody
ligand
+
Antibody-bound enzyme
2. Immunoaffinity chromatography
Protein epitope
Enzyme

32. Gel permeation chromatography
(GPC)
• Also known as ‘size exclusion chromatography’
and ‘gel filtration chromatography’
• Separates molecules on the basis of molecular size
• Separation is based on the use of a porous matrix.
Small molecules penetrate into the matrix more,
and their path length of elution is longer.
• Large molecules appear first, smaller molecules
later

33. Precipitation
• Formation of a solid in a solution during a chemical
reaction.
• Solid formed is called the precipitate and the liquid
remaining above the solid is called the supernate.

34. Product Purification
• To separate contaminants that resemble the
product very closely in physical and chemical
properties.
• Expensive and require sensitive and sophisticated
equipment.

35. Crystallization
• Process of formation of solid crystals precipitating
from a solution, melt or more rarely deposited
directly from a gas.
• Chemical solid-liquid separation technique, in
which mass transfer of a solute from the liquid
solution to a pure solid crystalline phase occurs.

36. Product Polishing
• Final processing steps which end with packaging
of the product in a form that is stable, easily
transportable and convenient.
• Crystallization, desiccation, lyophilization and
spray drying are typical unit operations

37. lyophilization
• Freezing the material
• Reducing the surrounding pressure and adding
enough heat to allow the frozen water in the
material to sublime directly from the solid phase to
gas.

38. Downstream processing should be
modified based on target product
1. Enzyme preparations for animal feed
supplementation (e.g., phytase) are not
purified
2. Enzymes for industrial use may be
partially purified (e.g., amylase for starch
industry)
3. Enzymes for analytical use (e.g., glucose
oxidase) and pharmaceutical proteins
(e.g., TPA) are very highly purified

39. Fermentation
Culture supernatant
Centrifugation
to remove cells
Liquid preparation
to animal feed
market
Animal feed enzyme

40. Fermentation
Culture supernatant
Centrifugation
to remove cells
Protein
precipitation
Protein fraction
1 or 2 purification
steps
Semi-purified
protein
Lyophilisation
Bottling
To chemicals market
Analytical enzyme

41. Fermentation
Cell pellet
Intracellular fractionTherapeutic
Protein
Centrifugation
to remove
medium
Cell lysis Centrifugation
Protein
precipitation
Protein fraction
3-4 purification
steps
Homogeneous
protein
Sterile
bottling
To pharmaceuticals market

43. Reference
s
Wikipedia
Microbial
Biotechnology
• Glazer
Lecture Notes
• Dr.B.S.Vijayakumar
• Head
• Dept. of Biosciences