Presentation on Sterilization of air & media

By: neitho-o Roll: 080217 Sem: 7th Dept: Biotechnology

Sterilization
Term referring to any process that eliminates (removes) or kills all life forms of microbial life including transmissible agents (fungi, bacteria, viruses, spore forms etc.) present on a surface, contained in a fluid, in medication, or in a compound such as biological culture media

Sterilization of air
Heat treatment Ultraviolet light treatment Filter sterilization Ozone gas treatment Chemical treatment

Heat treatment
1.Dry heat sterilization

2. Moist heat sterilization

Dry heat sterilization

Circulation of heated air within the chamber of the oven can be of two processes: a. Gravity convection process: Produces inconsistent temperatures within the chamber and has a very slow turn over. b. Mechanical convection process: the oven contains a blower that actively forces heated air throughout the chamber. It ensures uniform temperatures and the equal transfer of heat throughout the load.

Moist heat sterilization

Processes/steps: 1.Chamber closed and heated so that steam forces air out of the vents or exhausts. 2. Pressure applied so that the interior temperature reaches 121 oc 3.The temperature maintained for between 1530 minutes.

UV-light treatment
Short wave length(254nm) Destroys the nucleic acids (disruption of DNA) of microorganisms resulting in reproductive incapability and dead. Used in medical sanitation and sterile work facilities

Advantages of UV-light treatment

It is rapid Low cost Does not need special operator training

Disadvantages of UV-light treatment

Must be operated in a closed system Needs careful monitoring

Filter sterilization of air

Most commonly used sterilization process fixed pore filter membrane (0.2-0.3 micrometer)widely used PTFE(hydrophobic) most common construction membrane used

Sterilization of fermenter exhaust air

Fixed pore membrane modules are also used Pretreatment of exhaust gas is necessary before it enters the absolute filter. Pretreated air is then fed to a 0.2 micrometer hydrophobic filter.

Basic principles of filter design

Reduction of particle entering the filter is: dN/dx = -KN.(1) Where, N =concentration of particles entering the filter K = constant Integrating (1) over the length of filter N/No = e-kx (2) Where, No = no. of particles entering the filter N = no.of particles leaving the filter Taking log, (2) becomes ln(N/No ) = -kx..(3) { log penetration relationship} Efficiency of filter , E = (No - N)/ No Or, E= 1 - e-kx

Ozone gas treatment

Ozone gas is most efficient, very reactive and it can decompose back to oxygen without leaving a trail. broad- spectrum Concentrations of 0.05-0.08 ppm. required

Advantages of ozone gas treatment

Faster sterilization No requirement of chemicals Improved shelf life of products. Higher preservation duration

Media sterilization
Media may be sterilized by filtration , radiation, ultrasonic treatment, chemical treatment or heat.

Kinetics of sterilization
Destruction of micro-organisms by steam (moist ) may be described by 1st order chemical reaction: -dN/dT= kN.(1) Where, N=no. of viable organism present, t = time of sterilization treatment K=reaction rate constant of the reaction/specific death rate. Integration of (1) Nt /N0= e-kt (2) Where N = no. of viable organisms present at the start of sterilization treatment 0 Nt = no.of viable organisms present after a treatment period t. Taking log, ln(Nt /N0) = -kt (3)

Relationship between T & k(reaction rate constant)

Arrhenius equation d ln k/dT = E/RT2 -------(4) Where, E = activation energy R= gas constant T= absolute temperature Integration of (4) K= Ae -E/RT ----------(5) where A=Arrhenius constant Taking natural log in (5) ln k = ln A E/RT --------(6)

Heat sterilization of a pure culture at constant temperature

Combination of eqn (3) & (5) ln(Nt /N0) = A*t*e -E/RT .........(7)

Del factor ( )or Nabla factor (i.e.,Nt /N0)

= measure of fractional reduction in viable organism count produced by a certain heat and time regime . = ln(No /Nt) since Kt = A*T*e (-E/RT) = ln(No /Nt) so, = A*t*e (-E/RT) (8) rearranging (8)
ln t = E/RT + ln ( /A )

69

23 4.6

The same degree of sterilization may result from treatment at a higher temperature for a short time as from a low temperature for a long time.
2.3

Deleterious effect of increasing medium sterilization on the yield of product

Destruction of essential media due to heat: Xt /x0 = e-kt
Where,

xt = concn. of nutrient after a heat treatment period,t x0 = original concn. of nutrient at the onset of sterilization k = reactn. rate constant

Two types of reaction contributing to the loss of nutrient quality during sterilization
Interactions between nutrient components of the medium Degradation of heat liable components.

Design of Batch Sterilization Process

Information needed for the design:
1. Temperature of the fermentation medium 2. Number of micro-organism originally present in the medium. 3. Thermal death characteristics of the design organism(example Bacillus stearothermophilus)

Calculation of Del factor in Batch culture

overall= heating+ holding+ cooling

i.e.,
= A*T*e (-E/RT)

sum of the values of Del factor corresponding to each time increment is equal to the overall Del factor

Methods of batch sterilization

Fermentation vessel Separate Mash cooker

Design of continuous sterilization processes

Approach is same as batch sterilization processes Includes a time period during which the medium is heated to the sterilization temperature, a holding time at the desired temperature and a cooling period to restore the medium to the fermentation temperature

Eg. Spiral heat exchanger

The plant is sterilized prior to sterilization of the medium by circulating hot water through the plant in a closed circuit The fermenter and the pipe work between the fermenter and the sterilizer are steam sterilized. In coming unsterile medium partially heated before reaching the sterilizer

Filter sterilization of fermentation media

Essential/suitable for heat-labile proteins(animal-cell culture)

Advantages of continuous sterilization over batch sterilization

Superior maintenance of medium quality Ease of scale up Easier automatic control Reduction of sterilization cycle time.

Advantages of batch sterilization over continuous sterilization

Lower capital equipment costs Lower risk of contamination Easier manual control Easier to use with media containing a high proportion of solid matter.

Reference :
1. Principles of fermentation technology by P.F. STANBURY A. WHITAKER & S.J.HALL 2. www.creative ozone.com 3. sterilization of air by R.C telling & J .W.S. Ford

Thank you