FE 462 BIOCHEMICAL ENGINEERING

1. FE 462 BIOCHEMICAL ENGINEERING Sterilization

2. INTRODUCTION Most industrial fermentations are carried out as pure cultures inwhich only selected strains are allowed to grow. If foreignmicroorganisms exist in the medium or any parts of the equipment, theproduction organisms have to compete with the contaminants for thelimited nutrients. The foreign microorganisms can produce harmfulproducts which can limit the growth of the production organisms.Therefore, before starting fermentation, the medium and allfermentation equipment have to be free from any living organisms, inother words, they have to be completely sterilized. Furthermore, theaseptic condition has to be maintained.

3. STERILIZATION METHODS • Sterilization of fermentation media or equipment can beaccomplished by destroying all living organisms by means of heat(moist or dry), chemical agents, radiation (ultraviolet or X-rays), andmechanical means (some or ultrasonic vibrations). Another approachis to remove the living organisms by means of filtration or high-speedcentrifugation. UV Sterilization ultrasonic‑light‑sterilization Ozone Enriched Water Sterilization

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

5. THERMAL DEATH KINETICS • Thermal death of microorganisms at a particular temperature can bedescribed by first-order kinetics: • where kd is specific death rate, the value of which depends not onlyon the type of species but also on the physiological form of cells. Integration of Eq. yields

6. which shows the exponential decay of the cell population. Thetemperature dependence of the specific death rate kd can be assumedto follow the Arrhenius equation: where Ed is activation energy, which can be obtained from the slope ofthe In(kd) versus 1/T plot.

7. DESIGN CRITERION • From above equations, the design criterion for sterilization canbe 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

8. Batch Sterilization • Sterilization of the medium in a fermenter can be carried out in batchmode by direct steam sparging, by electrical heaters, or by circulatingconstant pressure condensing steam through heating coil. Thesterilization cycles are composed of heating, holding, and cooling.Therefore, the total Del factor required should be equal to the sum ofthe 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.

9. The Design Procedure • The design procedure for theestimation of the holding time is as follows: 1. Calculate the total sterilization criterion,  total. 2. Measure the temperature versus time profile during theheating, holding, and cooling cycles of sterilization. a. For batch heating by direct steam sparging into themedium, the hyperbolic form is used: b. For batch heating with a constant rate of heat flow such aselectrical heating, the linear form is used:

10. c. For batch heating with a isothermal heat source such assteam circulation through heating coil, the exponentialform is used: 3. Plot the values of kd as a function of time. d. For batch cooling using a continuous nonisothermal heatsink such as passing cooling water through cooling coil, theexponential form is used:

11. 4. Integrate the areas under the kd-versus-time curve for theheating and the cooling periods to estimate heat and  cool‘respectively. If using theoretical equations, integrate equation numerically after substituting in the proper temperatureprofiles. Then, the holding time can be calculated from

12. Example

13. Solution The direct injection of steam into the medium can be assumed to follow the hyperbolic temperature-time profile, which canbe used to calculate the time required to heat the medium from 25°Cto 122°C. From the steam table (Felder and Rousseau, 1986), theenthalpy of saturated steam at 345 kPa and water at 25°C is 2,731 and105 kJ /kg, respectively. Therefore, the enthalpy of the saturatedsteam at 345 kPa relative to raw medium temperature (25°C) is

14. Numerical integration of the preceding equation yields.

15. During the cooling process, the change of temperature canbe 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

16. At 122°C, the thermal death constant is 197.6 hr-1.Therefore, the holding time is