Lecture 5 | Sterilization (Microbiology) | Hvac

Sterilization

Chapter 6

Introduction

Most industrial fermentation are carried out as pure cultures-selected strains are allowed to grow. Foreign microorganisms exist in the medium or any parts of the equipment, the production organisms have to compete with the contaminants (limited nutrients). Foreign microorganism can produce harmful products-limit the growth of the production organism. B4 starting fermentation, medium + all fermentation equipment – free from any living organism (completely sterilized). The aseptic condition - maintained

Sterilization Methods
for fermentation media & equipment  Heat (moist & dry)  Chemical agents  Radiation (ultraviolet/X rays)  Mechanical (sonic/ultrasonic vibrations)  Filtration

Heat
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Widely used For liquid medium & heatable solid objects As dry or moist heat (steam) Moist heat-more effective Note: Because the intrinsic heat resistance of vegetative bacterial cells is greatly increased in a completely dry state. The death rate-much lower for the dry cells than for moist ones. Heat conduction in dry air-less rapid than in steam Dry heat-for sterilization of glassware/heatable solid materials

By pressurizing a vessel, the steam temperature increased significantly above the boiling point of water  Lab autoclave: steam pressure:30 psi,121°C (Bacterial spores rapidly killed at 121°C).

Chemical
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Kill microorganisms as the result of their oxidizing or alkylating abilities Cannot be used for the sterilization of medium-the residual chemical can inhibit the fermentation organisms Implies the treatment to remove or reducethe risk from pathogenic organisms Antimicrobial chemical: phenol & phenolic compounds (phenol, cresol) alcohol Halogens (iodine) Detergents Dyes Acids,alkalies

Radiation (UV/X Rays)
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Many cellular materials absorb UV light-leading to DNA damage & consequently to cell death. UV-very little ability to penetrate matter Limited to the reduction of microbial population in a room (sterility needs to be maintained) Ex: hospital operating rooms, clean chambers in a lab. X Rays-lethal to microorganisms & have penetration ability Impractical as sterilization tools-expense & safety concerns.

Sonic/Ultrasonic
Can disrupt & kill cells  Usually employed in the disruption of cells for the purpose of extracting intracellular constituents rather than as a sterilization technique.

Filtration
Most effectively-removal of microorganisms from air or other gases.  In the case of liquid solutions-used with medium or products which easily destroyed by heat: human & animal serums, enzymes.

Batch Sterilization
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Method:* steam sparging electrical heating steam heating (constant pressure condensing steam). Sterilization cycles are composed of heating, holding & cooling Total Del factor: Heating & cooling-the values by the methods used for heating & cooling Holding- the values by the length of the controlled holding period.

Continuous Sterilization
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Advantages: simplifies production planning, thus allowing max plant utilization & minimum delays provides reproducible conditions Can be operated at a high temperature (140°C instead of 121°C in batch sterilization)-the sterilization time can be shortened (holding time 1-2 min) requires less steam by recovering heat from the sterilized medium. require less cooling water easier to automate the process – less labor intensive Consist 3 main sections: heating, holding & cooling

Heating section: - 2 methods:  direct steam injection  indirect heating in the shell-and –tube or plateand-frame heat exchanger - Direct heating more effective-no barrier between the medium & heat source. - the steam injection heats the medium to the peak sterilization temperature quickly (sterilization during heating period is negligible)

- Indirect heating- the plate-and-frame HE more effective than shell-and-tube type for heat transfer due to its larger heat transfer area. - the plate-and-frame favorable for the sterilization of a high viscous system

Plate-and-frame

Shell-and-tube

Holding Section: - the heated medium passes through a holding section, usually composed of a long tube. - maintained in adiabatic conditions - if the heat loss in the section is negligible, the temperature can be assumed to be constant.

Cooling Section: - a quench cooler with adequate heat removal capacity is effective. - flash cooling- inject the hot medium through an expansion valve into the vacuum chamber - Both of these take a very short time - therefore, the sterilization during the cooling period can be assumed to be negligible. - shell-and-tube & plate-and-frame HE can also be employed for cooling.

Air Sterilization
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For aerobic fermentations, air needs to be supplied continuously Typical aeration rates for aerobic fermentation are 0.5-1.0 vvm (air volume per liquid vol per min). This requires an enormous amount of air Therefore, not only the medium but also the air must be free from microbial contaminants. All of the sterilization technique for medium can be employed for air. Sterilization of air by means of heat is economically impractical & ineffective due to the low heat transfer efficiency of air compared with liquids. Most effective technique for air sterilization- fibrous or membrane filters The cotton plug, routinely used as a closure for tubes or flask of sterile solution-good example of removal microorganisms from air by a fibrous filter.

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A simple air filter can be made by packing cotton into a column. With cotton filters the pressure drop is high and wetting can be a breeding ground for the contamination Glass fibers are favorable as filter medium- give a lower pressure drop & less liable to wetting or combustion Modern fibrous filter systems are cylinders made from bonded borosilicate microfibers With fibrous filters, airborne particles are collected by the mechanism of: Impaction Interception Diffusion

Sterilization of Fermenter

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The fermenter has to be sterilized separately b4 the sterile medium is added to it. By heating the jackets or coils of fermenter with steam and sparging steam into the vessel through all entries, apart from the air outlet from which steam is allowed to exit slowly. Steam pressure-15 psi for approximately 20 min Sterile air is sparged into the fermenter after cycle is complete & +ve pressure is maintained.

Sterilization of Feeds

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A variety of additive may be administered to a fermentation during the process & it is essential that these materials are sterile The sterilization method depends on the nature of the additive, the volume and feed rate at which it is administered. Ex: if fed in large quantities then continuous sterilization may be desirable. Batch sterilization of feed liquids normally involves steam injection into the material held in storage vessels.

Sterilization of Liquid Wastes

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Process organisms which have been engineered to produce ‘foreign’ products & therefore contain heterologous genes are subject to strict containment regulations. Thus, waste biomass of such organisms must be sterilized b4 disposal. By either batch or continuous. Batch sterilization- sparging the steam into holding tanks Cont sterilization- employ the type of heat exchangers

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