Presentation on Sterilization of air & media

By: neitho-o Roll: 080217Sem: 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 treatment1.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 oc3.The temperature maintained for between 15-

30 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 = constantIntegrating (1) over the length of filterN/No = e-kx ……(2)Where, No = no. of particles entering the filter N = no.of particles leaving the filterTaking log, (2) becomesln(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 treatmentK=reaction rate constant of the reaction/specific death rate.Integration of (1)Nt /N0= e-kt …………(2)Where N0= no. of viable organisms present at the start of sterilization treatment 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 equationd ln k/dT = E/RT2 -------(4)Where, E = activation energyR= gas constantT= absolute temperatureIntegration of (4)K= Ae -E/RT ----------(5) where A=Arrhenius constantTaking 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 )

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.

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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 sterilizationk = 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 medium2. 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