Air sterilization, Methods of sterilization, depth filter, Membrane cartridge filters, sterilization of culture media

1. R. NITHYA, M. Sc., M. Phil., Ph. D
ASSISTANT PROFESSOR
DEPARTMENT OF BIOTECHNOLOGY
SRI ADI CHUNCHNAGIRI WOMENS COLLEGE, CUMBUM
THENI DT, TAMIL NADU

2. STERILIZATION OF AIR
Industrial fermentation are carried out under vigorous
and continuous aeration.
For an effective fermentation, the air should be
completely sterile.
Free from all microorganisms and
suspended particles
Quantity of suspended particles in air = 20 - 10,000/m3
Microorganisms may range from 10 - 2000/m3.
Among Microbes in air, fungal spores(50%) and
Gram negative bacteria (40%) dominate.

3. METHODS OF STERILIZATION
Air or other gases can be sterilized by
Filtration
Heat
UV radiation
gas scrubbing
Heat and filtration are most commonly used.
Sterilization by Heat
Air was passed over electrically heated elements and
sterilized
Expensive method

4. STERILIZATION BY HEAT

5. STERILIZATION BY FILTRATION
Most commonly used method in fermentation
industries.
Depth filters
Membrane cartridge filters
DEPTH FILTERS
Air is passed through a glass wool containing
depth filters the particles are trapped and removed

6. DEPTH FILTERS

7. DEPTH FILTERS
Filtration primarily involves physical effects
inertia
blocking
gravity
electrostatic attraction
Diffusion
Glass wool filters are subjected to seam sterilization and reused.
Limitation
During steam sterilization the glass wool shrinks and solidifies.
Nowadays glass fiber cartridges are being used

8. GLASS FIBER SHEET

9. MEMBRANE CARTRIDGE FILTERS
These are removable pleated
membrane filters made up of
cellulose ester nylon or polysulfone
Smaller in size
Simpler for operation and replacement

10. MEMBRANE CARTRIDGE FILTERS

11. LIMITATION OF AIR
STERILIZATION
There is no filter that can remove
bacteriophages.
Bacteriophages are capable of
crippling the industrial fermentation.
E.g. Bacteriophages interfere in the
production of glutamic acid by
Corynebacterium glutamicum

12. STERILIZATION OF CULTURE MEDIA
The constituents of culture media, water and containers
contribute to contamination by vegetative cells and spores.
The media must be free from contamination.
Sterilization is done by
Applying heat
Physical methods
Chemical treatment
Radiation

13. Heat sterilization
- Most widely used technique.
- Vegetative cells are destroyed at lower temperature in a
short time - around 60 C in 5 -10 minutes.
- Spores are destructed at higher temperature. It required
longer time (around 80 C for15-20 minutes).
-Spores of Bacillus stearothermophilus are the most heat
resistant.

14. Heat sterilization

15. PHYSICAL METHODS
The physical methods such as
Filtration
Centrifugation and
Adsorption are in use.
Filtration
Certain components like Vitamins, blood components, antibiotics
of culture media are heat labile.
These components are completely dissolve and then subjected to
filter sterilization.

16. FILTRATION

17. LIMITATIONS
Application of high pressure infiltration is unsuitable for
industries.
Some media components may be lost from the media
during filtration
Combination of filtration and heat sterilization are applied
sometimes.
For example
Water used for media preparation is filtered.
Concentrated nutrient solution is subjected to heat
sterilization.
Then the filtered water is used for dilution of the media.

18. CHEMICAL METHODS AND RADIATION
These chemical methods (using disinfectants)
and radiation methods (using UV rays, Y rays, X rays) are
not commonly used for media sterilization

19. BATCH STERILIZATION
The culture media are subjected to sterilization at 121 C in batch volumes in
the bioreactor.
Two methods
Direct method – Sterilization is done by injecting steam into the medium.
The steam should be pure and free from all chemical additives
Indirect method - Injecting the steam into interior coils

20. DISADVANTAGES OF BATCH STERILZATION
Damage to culture media
Alteration in nutrients, change in pH and discolouration of culture
media are common.
High energy consumption
Sterilization takes few hours (2- 4 hrs) for the entire contents of the
bioreactor to attain the requisite temperature (i.e. 120 C).
Another 20- 60 minutes for the actual sterilization.
Then cooling taken 1 – 2 hrs.
All this process involve the wastage of energy.

21. CONTINUOUS STERILIZATION
- Carried out at 140 C for a very short period of time ranging
from 30 – 120 seconds.
- The time required is shorter at higher temperature.
- It is carried out by directly injecting the steam or by means of
heat exchangers.
- In both cases the temperature is very quickly raised to 140 C
and maintained for 30 – 120 seconds.

22. Different stages in continuous sterilization process in
relation to temperature are
Exchanger,
Heater,
Heat maintenance unit,
Recovery of residual heat,
Cooling and
Fermenter.

23. HEAT EXCHANGERS
Three types of heat exchangers are used.
First exchanger - raises temperature to 90 - 120 C within 20-30 seconds
Second exchanger - raises temperature to 140 C and Maintains for 30 -120 seconds.
Third exchanger - brings down the temperature by Cooling in the next 20 - 30 seconds.

24. Advantages of continuous sterilization
Main advantage is that about 80- 90 % of the energy is
conserved.
Disadvantage
Certain compounds in the medium precipitate due to high
temperature.
e.g. calcium phosphate, calcium oxalate