FE 462 BIOCHEMICAL ENGINEERING Sterilization Dr Ali Cokun

FE 462 BIOCHEMICAL ENGINEERING Sterilization Dr. Ali Coşkun DALGIÇ

INTRODUCTION Most industrial fermentations are carried out as pure cultures in which only selected strains are allowed to grow. If foreign microorganisms exist in the medium or any parts of the equipment, the production organisms have to compete with the contaminants for the limited nutrients. The foreign microorganisms can produce harmful products which can limit the growth of the production organisms. Therefore, before starting fermentation, the medium and all fermentation equipment have to be free from any living organisms, in other words, they have to be completely sterilized. Furthermore, the aseptic condition has to be maintained. Page 2 Dr. Ali Coşkun DALGIÇ

STERILIZATION METHODS Sterilization of fermentation media or equipment can be accomplished by destroying all living organisms by means of heat (moist or dry), chemical agents, radiation (ultraviolet or X-rays), and mechanical means (some or ultrasonic vibrations). Another approach is to remove the living organisms by means of filtration or high-speed centrifugation. ultrasonic‑light‑sterilization Page 3 Ozone Enriched Water Sterilization UV Sterilization Dr. Ali Coşkun DALGIÇ

Heat is the most widely used means of sterilization, which can be employed for both liquid medium and heatable solid objects. It can be applied as dry or moist heat (steam). Laboratory autoclaves are commonly operated at a steam pressure of about 30 psia, which corresponds to 121°C. Even bacterial spores are rapidly killed at 121 °C. Many cellular materials absorb ultraviolet light, leading to DNA damage and consequently to cell death. Wavelengths around 265 nm have the highest bactericidal efficiency. Sonic or ultrasonic waves of sufficient intensity can disrupt and kill cells. Filtration is most effectively employed for the removal of microorganisms from air or other gases. Chemical agents can be used to kill microorganisms as the result of their oxidizing or alkylating abilities. However, they cannot be used for the sterilization of medium because the residual chemical can inhibit thefermentation organisms. Page 4 Dr. Ali Coşkun DALGIÇ

THERMAL DEATH KINETICS Thermal death of microorganisms at a particular temperature can be described by first-order kinetics: where kd is specific death rate, the value of which depends not only on the type of species but also on the physiological form of cells. Integration of Eq. yields Page 5 Dr. Ali Coşkun DALGIÇ

which shows the exponential decay of the cell population. The temperature dependence of the specific death rate kd can be assumed to follow the Arrhenius equation: where Ed is activation energy, which can be obtained from the slope of the In(kd) versus 1/T plot. Page 6 Dr. Ali Coşkun DALGIÇ

DESIGN CRITERION From above equations, the design criterion for sterilization can be defined as which is also known as the Del factor, a measure of the size of the job to be accomplished. The Del factor increases as the final number of cells decreases. For example, the Del factor to reduce the number of cells in a fermenter from 1010 viable organisms to one is Page 7 Dr. Ali Coşkun DALGIÇ

Batch Sterilization of the medium in a fermenter can be carried out in batch mode by direct steam sparging, by electrical heaters, or by circulating constant pressure condensing steam through heating coil. The sterilization cycles are composed of heating, holding, and cooling. Therefore, the total Del factor required should be equal to the sum of the Del factor for heating, holding and cooling as The values of heat and cool are determined by the methods used for the heating and cooling. The value of hold is determined by the length of the controlled holding period. Page 8 Dr. Ali Coşkun DALGIÇ

The Design Procedure The design procedure for the estimation of the holding time is as follows: 1. Calculate the total sterilization criterion, total. 2. Measure the temperature versus time profile during the heating, holding, and cooling cycles of sterilization. a. For batch heating by direct steam sparging into the medium, the hyperbolic form is used: b. For batch heating with a constant rate of heat flow such as electrical heating, the linear form is used: Page 9 Dr. Ali Coşkun DALGIÇ

c. For batch heating with a isothermal heat source such as steam circulation through heating coil, the exponential form is used: d. For batch cooling using a continuous nonisothermal heat sink such as passing cooling water through cooling coil, the exponential form is used: 3. Plot the values of kd as a function of time. Page 10 Dr. Ali Coşkun DALGIÇ

4. Integrate the areas under the kd-versus-time curve for the heating and the cooling periods to estimate heat and cool‘ respectively. If using theoretical equations, integrate equation numerically after substituting in the proper temperature profiles. Then, the holding time can be calculated from Page 11 Dr. Ali Coşkun DALGIÇ

Example Page 12 Dr. Ali Coşkun DALGIÇ

Solution The direct injection of steam into the medium can be assumed to follow the hyperbolic temperature-time profile, which can be used to calculate the time required to heat the medium from 25°C to 122°C. From the steam table (Felder and Rousseau, 1986), the enthalpy of saturated steam at 345 k. Pa and water at 25°C is 2, 731 and 105 k. J /kg, respectively. Therefore, the enthalpy of the saturated steam at 345 k. Pa relative to raw medium temperature (25°C) is Page 13 Dr. Ali Coşkun DALGIÇ

Numerical integration of the preceding equation yields. Page 14 Dr. Ali Coşkun DALGIÇ

During the cooling process, the change of temperature can be approximated as Solving for t when the final temperature is 303°K yields 4. 38 hrs. Substitution of the previous equation gives Therefore, the Del factor for the holding time is Page 15 Dr. Ali Coşkun DALGIÇ

At 122°C, thermal death constant is 197. 6 hr-1. Therefore, the holding time is Page 16 Dr. Ali Coşkun DALGIÇ