FACULTY OF ENGINEERING & TECHNOLOGY

DEPARTMENT OF BIOTECHNOLOGY
 MEDIA STERILIZATION

 Sterilization is defined as the complete destruction or elimination of all viable

organisms (in or on an object being sterilized). There are no degrees of sterilization: an
object is either sterile or not.

 Sterilization procedures involve the use of heat, radiation, chemicals or physical

removal of cells. Media for industrial fermentations are usually sterilized. In some cases
the economics of the fermentation makes it unrealistic to sterilize.

The fermentations can proceed, however, these fermentations employ low pH and other
contamination inhibitors (lactic acid) to hold in check the numbers of contaminating
microorganisms. In other cases, sterilization is not required as the media components are
poorly utilized by contaminating microorganisms.

Fermentation media are sterilized by the use of: filtration, radiation, ultrasonic
treatment, chemical treatment or heat (boiling or passing live steam through the medium,
or by subjecting the medium to steam under pressure -autoclaving). Steam is used almost
universally for the sterilization of fermentation media.

The major exception is the use of filtration for the sterilization of animal cell.
Heat:
Heat is the most important and widely used method. For sterilization, the type of heat,
time of application and temperature required to ensure destruction of all
microorganisms must always be considered. Endospores of bacteria are the most
thermo-resistant of all cells so their destruction usually guarantees sterility.

Incineration:
In this process, organisms are burned and physically destroyed. It is widely used for
needles, inoculating wires, glassware, tubes etc.and objects that can not be destroyed
in the incineration process.

Boiling:
Boiling is done at >100 ̊C for 20-30 min. It kills everything except for some
endospores. To kill endospores and therefore perfectly sterilize the solution, very long
or intermittent boiling is required.

Autoclaving:
Autoclaving is the process of using steam under pressure in an autoclave or pressure
cooker. It involves heating at 121 ̊C for 15-20 min under 15 psi pressure and can be
used to sterilize almost anything. However heat labile substances will be denatured or
destroyed. Sterilization of nutrient media is usually done using this process.
Dry Heat (Hot Air Oven):

The process involves heating at 160 ̊Cfor 2 hours or at 170 ̊Cfor 1 hour. It is used for
glassware, metal and objects that will not melt.

Sterilization in industry-scale fermenters (or bioreactors)is more complex. Steam is used

to sterilize fermentation media. The medium can be sterilized in situ within the
bioreactor. However, if the medium is sterilized in a separate vessel, the bioreactor
needs to be sterilized before the sterile medium is added to it.

Bioreactors are sterilized by passing steam through spargers. Spargers are devices that
distribute gas bubbles (usually sterile air or steam) in a liquid phase. They have
particular design criteria, e.g., providing small sized bubbles (the sparger breaks the
incoming air into small bubbles).Various designs can be used such as porous materials
made of glass or metal. However, the most commonly used type of sparger used in
modern bioreactors is the sparge ring. A sparge ring consists of a hollow tube in which
small holes have been drilled and is easier to clean than porous materials and is also less
likely to block during fermentation. During sparging, steam pressure is held at 15 psi in
the vessel for 20 min.
 Batch sterilization

•Batch sterilization is the reduction of contaminant organisms through the heating of

a vessel.

•The entire volume of media is sterilized at once through the use of thermal or
radiation techniques. When running a thermal batch sterilization, a system goes
through 3 steps: heating, holding, and cooling.

•Heating requires the addition of energy throughout the entire medium volume. This
can be done by adding heat through a jacket on the vessel.

•The temperature is increased until it reaches the sterilization temperature where it is

held for a set period of time.

•During this phase, most of the unwanted microorganisms are destroyed. Finally, the
system is cooled to bring the sterile media back to the desired temperature.

•For radiation sterilization, the process is similar to above, although it uses radiation
intensity instead of heat.
t = time
No = initial spore concentration
Nf = final spore concentration
Vo = initial batch volume
Vf = final batch volume
k = death rate constant
By the Arrhenius equation, the death kinetics are k = Ae -E/RT.

In order to sterilize a batch, calculate the total area underneath the curve.
Therefore, model death using first order kinetics and integrate as seen above. This
will yield a temperature and the corresponding duration of time needed to
sterilize the media.

Advantages:
Most widely used technique
Simple operation
No additional materials are added to the media itself

Disadvantages:
More expensive heat requirements than continuous sterilization
Best results occur in well-mixed closed vessels
 Continuous sterilization

Continuous sterilization is the rapid transfer of heat to medium through steam

condensate without the use of a heat exchanger. Once the media is in a holding
loop, steam is injected to the system via a nozzle.

The medium stays in this loop for a predetermined holding time until the entire
medium is sterile. This is more efficient than batch sterilization because instead
of expending energy to heat, hold, and cool the entire system, small portions of
the inlet streams are heated at a time.

By looping sterile media tubes (which are at higher temperatures) past inlet
tubes, the difference in temperature is used to help heat the unsterile medium. So
instead of having a cold-water stream cool the sterile media, the lower
temperature unsterile media stream absorbs heat from the warm stream, cooling
the sterile media.

Finally, the sterile media is flash cooled through an expansion valve to adjust
the temperature to meet process parameters.
Advantages:
Uniform steam requirements throughout the duration of the sterilization
Simplified process control
Shorter sterilization time means less thermal degradation of medium
Disadvantages:
High demand for steam in a shorter period of time than batch
Concentration of media becomes dilute due to steam condensation
Since steam is actually dispersed in media, steam must be clean to avoid contamination