INDUSTRIAL STERILIZATION-new.pptx

Industrial sterilization

1. POST GRADUATE & RESEARCH CENTER DEPARTMENT OF MICROBIOLOGY (Government Aided) 22-04-2022 INDUSTRIAL STERILIZATION Submitted By, Saffana N II M.Sc., MICROBIOLOGY REG NO: 20181331303207 MAIL ID:[email protected] INDUSTRIAL STERILIZATION OF EQUIPMENT, PRODUCTION MEDIA AND AIR... SUBMITTED TO , Dr. S. Vishwanathan Assistant Professor Head of the Department PG and Research Department of Microbiology SPKC - Alwarkurichi .

2. 1. Introduction 2. Principles of Sterilization 3. Sterilization of Equipment 4. Sterilization of Production Media 5. Sterilization of Air

3. AIM... Is to provide a product that is safe and eliminates the possibility of contamination. WHY....? To reduce amount of contaminant's present in environment, on surface of container's, closure's as well as equipment's and to achieve better sterile condition.

4. 1. INTRODUCTION Usually, sterilization of equipment, medium, air etc. and the subsequent maintenance of sterility of the whole system undergoing fermentation are critical points in the success of the fermentation process. There are many sterilizing agents (e.g. steam, U.V. light, chemical agents, etc.) for bringing about sterilization. Steam is preferred to other agents, because it is cheaper for mass sterilization. Moreover, it can penetrate into small awkward sites, as in valves, for efficient sterilization. Sterility of the fermentation process is needed, since contaminants may affect the process adversely and sub- sequently lower yields of the fermentation product. In certain fermentations (e.g. antibiotic fermentations), sterility of the fermenting system is strictly to be controlled. Therefore, sterility tests are carried out during the fermentation to detect suspected contaminants.

5. PHYSICAL METHOD MOIST HEAT Autoclave Pasteurization Tyndallization DRY HEAT Hot Air Oven Red Heat Flaming

6. RADIATION Non-Ionizing Radiations: Ultraviolet (UV) Light Ionizing Radiations (Cold Sterilization): X-rays, Gamma Rays, Cathode Rays (Electron Beam Radiation) FILTRATION Depth Filter : Sintered Glass Filter Screen Filter : Filter Candle, Membrane Filter

7. SPECIFICATION MOIST HEAT STERILIZATION : Microorganisms destroyed by cellular protein coagulation. The objects to be sterilized are exposed to saturated steam under 1 atmosphere pressure at a minimum temperature of 121°C for 15 min. An autoclave is commonly used for moist heat sterilization. Because it does not require as high temperature, moist heat sterilization cause less product and equipment damage compared to dry heat sterilization.

8. 2. PRINCIPLES OF STERILIZATION Sterilization removes infecting micro-organisms which would otherwise interfere with the efficient fermentation process. It can also remove pathogenic micro-organisms or spoiling agents which could devalue the products of fermentation. There are two main methods employed for sterilization: 1. Destruction of Micro-organisms and 2. Removal of Micro-organisms

9. There are many process operations to achieve each method. Destructive methods make use of a lethal condition. This may be accomplished either by chemical or physical means. Moist heat is the most common physical agent. It allows for satisfactory industrial sterilization. It is also the least expensive and most reliable to design and operate. The severity of sterilization conditions that are needed depends on: ( i ) the type of fermentation process that is to be protected, (ii) the duration of the production campaign and (iii) the type and extent of contamination of the feed material.

10. Infecting micro-organisms may be a combination of yeasts, protozoa, bacteria, spores and phages. They are more or less resistant to sterilize agents. With sterilization using heat, there is a temprature /time relationship, determining the degree of sterility. Heat-sterilization also imparts some degree of cooking of the nutrients. This may be beneficial in so far as break-down and solubilization of complex starchy materials arc concerned rendering them more available for assimilation by the fermenting micro-organisms. On the other hand, some destruction of heat-sensitive compounds may take place, and this results in a loss in efficiency, impairment of product flavour or scaling in the heat exchanger by charring or precipitation. .

11. The other method of sterilization is the removal of infecting micro-organisms. This is done by filtration. Numerous types of filters arc available for this purpose. The statistical probability of eliminating infecting micro-organisms depends critically on ( i ) the size of the micro-organisms and (ii) the retention efficiency of the filter. Usually, sterilization of gases and biostatic fluids is done by filtration

12. 3. STERILIZATION OF EQUIPMENT Steam, a physical agent, is universally employed for the sterilization of fermentation equipment and production medium. There exists a linear relationship between time and temperature. In other words, high temperature application needs less time and vice versa. The seed tank and the fermentor are the central pieces of the equipment. These may be sterilized either in charged (with medium) condition or they may be sterilized empty. (a) Sterilization of fermentor and medium as a unit: Steam sterilization of the fermentor and the production medium together is a simple operation.

13. A jacket or coil is fitted to the fermentor and supplied with pressure steam. Water is required to provide a temperature of 120'C. for a period of at least 20 minutes and the subsequent cooling back to the fermentation temperature. There is provision of a vent for the air to come out from the fermentor . Also, the head-space above the medium is filled with steam. It is necessary to prevent the formation of a vacuum during the cooling of the system. It is, therefore, necessary to have a supply of sterile air to maintain positive pressure. The presence of a vacuum in a sterile system is always a hazard to sterility. This is due to the danger of drawing non-sterile air or liquid into the sterile system through the seals on the agitator shaft and the spindles of cocks and valves. Apart from sterilization and maintenance of sterile condition of the tanks ( fermentor and seed tank), it is equally important to sterilize and to maintain sterility of ancillary equipment attached to the fermentors . Ancillary equipment includes many extra-connections, namely:

14. ( i ) Agitator shaft passing through the lid of the vessel—There should be an adequate seal or gland for maintaining pressure in the vessel. Moreover, this prevents the entry of microbes to the vessel from outside (ii) Sparger, usually beneath the agitator and a pipe for expelling exhausted air from the fer- mentor. (iii) A connection between the defoamer tank and the fermentor . (iv) A connection for introducing the inoculum (v) A connection for drawing samples during the fermentation (vi) A connection for unloading the fermentor after the completion of the fermentation.

15. (b) Sterilization of tormentor and medium separately: The method for sterilizing an empty fermentor is exactly the same as described Above for the fermentor and production medium together. There are several methods of sterilizing the production medium separately from the fermentor . And this topic has been dealt with in the next section separately. Certain points are to be kept in mind while sterilizing and maintaining sterile conditions in the fermentation equipment. They are:

16. ( i ) No direct connection should be made permanently between the non-sterile and the sterile parts of the system. (ii) Welded construction should be used, if possible and convenient. (iii) Where joints have to he used, they should he of a high quality valve, using rubber or other impervious material as the seal. (iv) The type of valves used and service lines should be carefully selected, so that they can be easily sterilized and serviced when necessary. Moreover, they should be capable of maintaining pure culture conditions during the fermentation cycle. (v) After sterilization, all parts of the system which arc to be kept sterile should be kept under a positive pressure. This may be accomplished either with the sterile air or the sterile liquid used during the period of cooling and fermentation. (vi) Each part of the system should be capable of independent sterilization without interfering with the operation of the rest of the plant. (vii) Isolated danger spots which are difficult to sterilize at the start of a batch, should be pro- vided with pressure steam connection for continuous or intermittent use.

17. 4. STERILIZATION OF PRODUCTION MEDIA Usually, production media are sterilized before they are inoculated with the desired fermentative pure culture. Sterilization of these media is decided by the chemical composition of a particular Medium. For instance, media containing sugars cannot be sterilized by prolonged heating, since sugars undergo caramelization. Moreover, media containing sugars and phosphates together cannot be sterilized by prolonged heating, since sugars react with phosphates. Therefore, either the sugar or the phosphate is to be sterilized separately before adding it to the sterilized production medium.

18. Sterilization of production media may be done by one of the following three methods: (a) by boiling, (b) by passing live steam, or (c) by subjecting the medium to steam under pressure (i.e. autoclaving). It is to be kept in mind that heat treatment should be sufficient only to sterilize, avoiding overcooking of the medium. Overcooked media demonstrate poor growth or low yields as compared to properly sterilized media. The classical technique of making the medium sterile by the use of steam may be carried out in two ways: ( i ) batchwise in the fermentor and (ii) continuous sterilization.

19. fermentor (whether empty or with a charge of medium). It is a usual practice to supply each connection with its own presure air-line. This is advantageous, since the ancillary equipment can be steamed out, while the fermentor is held under steam pressure at a temperature of 120C. Such ancillary equipment is used in aerobic fermentations, requiring much more care for sterility of the system. These processes involve a considerable toss of production time, for it may require several hours to reach a temperature of 120`C. and subsequently cool the medium before inoculation. Steam

20. with it coil or jacket for heating and cooling. Also, the agitator may be fitted to aid heat-exchange. It is needed to raise the temperature of the whole system (i.e., vessel and medium) to 120°C with steam and to Maintain this for a period of 20 minutes before cooling the system. There is no interconnecting pipeline between a batch cooker and a fomenter for transferring the sterile medium from the cooker to the previously steam sterilized fomenter. Therefore. the interconnecting pipeline must also be sterilized before making use of it for transfer purpose. Advantage: The batch cooker method saves the production time. Since the fermentor is unoccupied between two fermentation runs.     Limitations: ( i ) It occupies increased plant (ii) It involves higher cost of additional equipment required. (iii) It involves increased steam usage .

21. The latter method, continuous sterilization, offers more. flexibility in the choice of time-temperature conditions to which the medium is exposed. Usually, this method involves passing of production medium through a heat-exchanger. a holding coil and a cooler. The temperature of the medium undergoing sterilization is raised to the desired level in the heat-exchanger. The medium then passes on to a holding coil, where it is maintained at the sterilizing temperature for a predetermined time period.   Finally, the medium is rapidly cooled by counter circulating it in the exchanger against the cool input medium, and then against cold water. The possibility of short holding time allows the use of sterilization temperatures higher than 120°C., without any adverse effects on the nutritional qualities of culture medium.  

22. A slightly modified system involves the injection of high temperature steam into the medium. Here, the required temperature (< 140°C.) is reached in microseconds. The cooling of the medium a carried out in the flash cooler in a vacuum chamber.   A continuous retention tube sterilizer is also available. It is widely employed in the antibiotic field. Advantages: ( i ) It saves both production time and plant space.   (ii) It gives improved quality of the medium. For instance, an increase of 160', in the yield has been secured in the manufacture of vitamin B15 (iii) It involves some economy in steam costs, provided the heat-exchange principle is applied, and   (iv) It allows the use of lower sterilizing temperature or shorter holding periods provided the medium has a low pH, or some other characteristic aiding the sterilization  

23. Synthetic media require a relatively shorter sterilization time period. The crude media often require a considerably longer sterilization time period because of the greater viscosity These media, making heat-penetration difficult. In addition, relatively heat-resistant bacterial spores may be present in some of the components of such media. Therefore, it is necessary to determine the conditions (i.e. temperature and time) for satisfactory sterilization, without over-cooking the medium. Certain medium components (e.g. vitamins and enzymes) do not allow heat sterilization of the media. These components can be sterilized separately by filtration (e.g. Seitzfilter ). They are then added to the previously sterilized medium. It is to be brought to one's notice that, some fermentations are carried out without the sterilization of the media (e.g. certain yeast fermentations that are conducted at relatively low pH values).

24. 5. STERILIZATION OF AIR     With aerobic fermentations, continuous supply of sterile air is vital for successful fermentation. Air can be sterilized by many methods namely: Filtration, Heat, Electrostatic Precipitation, U.V. light or (v) Chemical agents.

25. The sterilization of air in fermentation industries is widely carried out by the filtration methods.   Other methods, for some reasons, arc not employed, for example, sterilization of air by heat is carried out owing to high cost involved. Therefore, the only method for sterilize the air for industrial use is filtration.   Filtration Method for sterilizing very large volumes of air is studied by Terjesan and cherry. These scholars performed slab of slag wool 3-inches thick, packed a density of at least 17lb./ft.., and of mean fibre diameter less than 6u. The air velocity through the slab was kept below lIt. /sec. to avoid channelling through the slag wool material. Techniques for evaluating the sterility developed by Bourdillon et al were used by Terjesen and Cherry for assuring the efficiency each method under experimental conditions. After many experiments, they made following suggestions.

26. (a)The slab to be Initialled in the filtration system, must follow certain specifications (e.g., fibre uniformity, density of the slab, etc.,) (b) Attention should to the air which may escape by passing, around the edges of the filtering medium. (c) No holes should be drilled less than 3 inches from the edges of the perforated supporting plates. (d) A non-continuous sponge-rubber gasket should be inserted between the slab and the 3 inches blank annulus of the supporting plates. (e) The rubber gasket around the edges of the supporting frame can he eliminated, provided the Metal faces around the edges of the supporting frame are very smooth. (f) Air velocities higher, tutu 0'5 ft./see. should not be used, since there is an increased pressure drop across the filtering medium and an increased danger of channelling. (g) Sterilization of the slabs and the body of the filter should be practised by means of dry heating at 160 to 180'C. for 2 hours. (h) Prefilter, should he used to remove dust particles and related suspended matter, thus increasing the working life of the sterilizing filter.   It is to be noted that other fibrous materials (e.g., glass wool) may be used for sterilizing the air.  

27. EQUIPMENT VALIDATION/ QUALIFICATION Equipment validation is divided into Design qualification Installation qualification Operational qualification Performance qualification

28. DQ It define the functional & operational specification of the instrument & details for the continues design in selection of supplier. IQ Demonstrates that the process or equipment meets all specifications, is installed correctly, and all required components and documentation needed for continued operation are installed and in place.

29. REFERENCES: Industrial Microbiology A.H patel Industrial microbiology l.c. casida https://www.google.com/search?q=Industrial+Microbiology+L.+E.+Casida&stick=H4sIAAAAAAAAAFWTzWsTQRjGd1eapttWk61FDRTSaCH00GQ3mc2uHiq0FYQGoVboQQzZ2c9kdif74X7kIghevCgeiuDB4knQf0BBW7zZgwfFHqV4E- xVxIO11ma2Hp955v29XzPZodJYxajwvAj4CPmiMGU56m0_8Kw2KtoW9LBiYYSNpKhg3PW36f9uH0lBccwaFMNtevzQNVpxPwC6u02zFbtShXVR8rtHQoTABxIJtGOzZhqDQAhqnVAOiNaBazv1RBpoV3LtDpLlNBy2lJaflpF4Ylgl0XGMRDsyBkXbkRJ3AIErDRfqCHhEJ0j1QlBN4XWnL5iEpqPIa8HaoCk-\

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