VARIOUS STERILIZATION

TECHNIQUES FOR
FERMENTATION MEDIA

Presented – Patil Pratibha Pushkaraj

M.Sc II (Microbiology)
Shivaji University, Kolhapur
 Contents • What is Sterilization?
• Why is it necessary to sterilize
at a Glance fermentation media?

• Lab scale or Pilot scale sterilization

1. Use of moist heat- Autoclaving
2. filtration

• Industry scale sterilization process

1. Batch sterilization
2. Continuous sterilization
 What is meant by
What is meant by Steralization?
ELEMENTS
Sterilization ?
Sterilization is a broad term that refers to any process that removes
or kills all forms of microorganisms. This includes bacteria,
Atomsfungi,
viruses and protozoans including their spore forms which are usually
very resistant. This refers to life forms that are on the surface, within
Chemical
a fluid, medications, or compounds such as buffers and Testing
culture
media.

Formulas
Beakers

Why is it necessary to
INVENTOR
Pipettes

sterilize Y BY DAY
Test tubes

0%
fermentation media?
10% 20% 30% 40% 50% 60% 70% 80% 90% 100%

Monday Tuesday Wednesday

 They may reduce the yield of product by-
• The contaminants may utilize the nutrients thereby causing
depletion of nutrients in the medium.
• They can cause foaming problems and may change the pH.
• They can produce toxic compounds that inhibit growth of desired
organism.
• They can use the fermented product as substrate and destroy it.
 Lab scale or pilot
scale media
sterilization practices
 Use of Moist Heat
• Autoclaves provide a physical method for disinfection
and sterilization.

• They work with a combination of steam, pressure and

time. Autoclaves operate at high temperature and pressure
in order to kill microorganisms and spores.
 
• They are used to decontaminate certain biological waste
and sterilize media, instruments and lab ware.

• To be effective, the autoclave must reach and maintain a

temperature of 121° C for at least 30 minutes by using
saturated steam under at least 15 psi of pressure.
 Use of Membrane filters

• Membrane filters are the most common type of filters used for liquid
sterilization in the microbiology laboratory.
• Membrane filters are composed of high tensile strength polymers such
as cellulose acetate, cellulose nitrate, or polysulfone.
• Membrane filters are prepared as circular membranes of about 150μm
thickness and contain millions of microscopic pores of uniform
diameters;the size of which is adjusted based on requirements, during the
polymerization process.
• Porosities of membrane filters range from 0.1μm to 10μm and the most
commonly used membrane filter has the pore size of 0.22μm and 0.45μm
• The membranes are held in special holders and often preceded by depth
filters made of glass fibers to remove larger particles that might clog the
membrane filter. The solution is pulled or forced through the filter and is
collected in previously sterilized containers.
 Industry scale
media sterilization
practices
 Batch Sterilization
• Batch sterilization is one of the most commonly used methods for liquid media
sterilization.
• In this process, the fermenter vessel containing medium is heated using steam and held
at the sterilization temperature for a period of time.
• Usually employed for pilot projects.

• Batch sterilization is done by one of two methods:

1. Direct batch sterilization
By injecting steam into the medium.
2. Indirect batch sterilization
By first sterilizing the medium in the batch cooker and then transferring
the medium to the bioreactor or fermenter.
Direct batch sterilization:
The air inside the bioreactor is removed and
replaced by steam

Temperature is raised upto 1210C

Pressure is allowed to raise till 15lbs and these

conditions are held for 20 mins

After sufficient time it is allowed to cool by

passing cold water through the jacket

Inocula can now be added for fermentation

process to commence
 Bioreactor
Indirect batch sterilization:

Batch cooker

Interconnecting Pipe

Heating jacket
 Continuous Sterilization
• Continuous sterilization, particularly a high-temperature, short-exposure-time
process, can reduce thermal damage to the medium significantly compared with
batch sterilization, while achieving high levels of cell destruction.
• Employed in big industries.

• It can be of two types:

1. Direct continuous sterilization-
By injecting steam directly in the medium
2. Indirect Continuous sterilization-
By using heat exchangers (Spiral or plate heat exchangers)
 Direct Continuous sterilization
Raw material

Steam in Flash Cooler

Holding coil

Steam Bleed

Sterile Medium
Indirect Continuous sterilization
There are two types of heat exchangers:
i. Plate heat exchanger and ii. Spiral heat exchanger

Raw material is passed through a pipe

Steam is added to increase the temperature

The medium is held in holding section at 1400C for

100-120 sec

In the third chamber cold water is added to lower the

temperature to the temperature of fermenter
 Steam

Sterile medium

Cool exchanger Pre-heat

Heated with
Heat Exchangers 20-30 sec 90-1200C
steam 1400C

Holding section
100-120 sec

20-30 sec

Cold water Raw medium

THANK
YOU!