1. STERILIZATION ☻Dry heat sterilization ☻Sterilization with filtration ☻Electron beam sterilization (radiation) ☻Chemical sterilization ☻Batch Sterilization ☻Continuous Sterilization ☻Introduction ☻High temperature sterilization ☻Sterilization of inoculation table ☻Sterilization of media for stock cultures ☻Sterilization of microbiological media ☻Sterilization of glass wares/Petri dishes

2. What is STERILIZATION? Is the action of eliminating microorganisms from a medium (Ghasem 2007) Reduction of any naturally occurring organisms in the substrate and air supply to a level where they are unable to compete effectively for the nutrients in the substrate with the organism being deliberately cultivated (Jackson 1990) Killing any microbes that is present in the media or passed through any filtering system (Layman)

5. Where is it being used? Food & dairy industries Pharmaceutical industries Research laboratories and etc. Autoclave → huge stainless steel vessel → steam → 105 kPa (15 psig) for 20-30 mins High temp & long duration → will kill microorganism / spores of fungus / bacteria Overheating → negative impact / media components destroyed / media pH ↓ / affected culture growth → acid pH sensitive to heating → carbohydrates will caramelize → difficult to solidify

6. High temperature sterilization High temp → fast sterilization Requires short holding/retention time Used in milk pasteurization Short retention time →used in media with heat sensitive proteins →may cause less damage to the biochemical composition of the media Recommended for 3 mins at 134 o C, 20 mins at 115 o C

7. Sterilization of inoculation table Laminar flow cabinet → various types of laminar flow available in the market → depend on the circulation of air Wipe with Clorox / ethanol (preferable) On the uv light overnight (show in class)

8. Sterilization of media for stock cultures Prepare slant → Mac Cartney bottle/ boiling tube/ test tube/ sterile container (market) Prepare the media of N, C and P sources & other nutrients → boil until dissolved → put it in the container → sterilized at 121 o C for 15- 20 mins. → cool → pour in sterile plastic and kept in the fridge if not used (show in class)

9. Sterilization of microbiological media Culture media (containing N,C and P sources) → need to be sterilized either at 121 o C/134 o C Must know the damage that could happen to the media if sterilized Media containing peptides, sugars, vitamins, minerals and metals should not be heated at high temp. → cause deterioration or there is reaction between components (caramelization) → or toxic compounds is produced → retard/inhibit the microbial growth How to solve this? ↓ damage to the ingredients of the media, ↓ heating/holding time of the sterilization process

10. Sterilization of glass wares/Petri dishes Oven/autoclave Sterilize separately → culture media & Petri dish → then pour the media into the Petri dish Wrap the Petri dish/glass wares with autoclavable paper / aluminium foil and heat it up to 100 o C for 20-30 mins – cool it down

11. Dry heat sterilization Usually pipettes that we want to use it over n over again Put in a pipette can/ wrap with aluminum foil Can be sterilized using an oven (at 100 o C) or autoclave (at 121 o C for 10-15 mins) Normally at the tip of both ends of the pipettes are inserted with just a small piece of cotton to prevent from any dust if it is to be used later

12. Sterilization with filtration Some media components are sensitive to heat → denatured → so must be added after the main media is being sterilized Example vitamins, proteins and etc Filter →1st 0.45 µm → then 0.22 µm (prevent bacteria from entering) Show how media for hyaluronic acid production is being prepared (Details next section)

13. Electron beam sterilization (radiation) Electron beam sterilization produced at high intensity in a vacuum Electrons intensities that is accelerated have potential as a bacteriocide Widely used in medical field and surgical appliances

14. Chemical sterilization Disinfecting chemicals → sodium hypochlorite / Clorox / ethanol Ethylene oxide (gas phase) → used for column bioreactors → volatile compound n strong oxidizing agent → boils at ambient temperature, so the solution must be stored at 4 o C → excellent oxidizing agent used for chemical sterilization of equipments (show in class)

15. Batch sterilization The medium is prepared and pumped to an agitated vessel fitted with either a heating jacket or heating coils. The contents of the vessel are then heated to a particular sterilization temperature (usually 121 o C, held for a certain length of time at that temperature, and then cooled down to the fermentation temperature. This time of process is carried out in the fermentor itself – in-situ sterilization (show the diagram & explain)

16. Temperature ( o C) Time (min) heating sterilization cooling

17. Continuous sterilization The medium is pumped at a constant flow rate through 2 heat exchangers. One heat the liquid and the other cools it down In between the two heat exchangers, the liquid is held either in a vessel or a large volumetric capacity pipeline to give the required time at the sterilizing temperature (show the diagram)

18. System for continuous sterilization To sterilize a large volume of medium in a reasonable time, the flow rate through the sterilizer must be high. For example, suppose that you wish to sterilize 100,000 liters in 100 minutes. The pumping rate would be 1000 l/m. If the holding time in the coil must be 2 minutes, the coil volume must be 2000 l. Use the applet to calculate the length of coil for various diameters.

19. Thermal Death Kinetics The destruction of micro-organisms by heat at a fixed temperature (isothermal) follows a first order reaction, whereby the rate of destruction is given by Where k = number of organisms at time N=specific reaction (death) rate constant Rearranging, we have

20. And integrating between N o (the number of organisms initially) at time 0, and N at time gives In microbiology, the term decimal reduction time (D) is often used, and is the time taken at a particular temperature to reduce the initial population by a factor of 10. Thus

21. So the decimal reduction time (D) is related to the specific death rate constant (k) by The specific death rate constant (k) varies with temperature, and follows a relationship of the Arrhenius type: Where A = constant E = activation energy R = universal gas constant T = absolute temperature

22. Continuous sterilization From the basic equation for the thermal death of the organism So Where k is now a function of time because the temperature is varying with time Thus,

23. In order to design a sterilization process to give a desired results, Deindoerfer & Humphrey defined the term ( design criteria) which is a measure of the destruction ratio Deindoerfer & Humphrey assumed that the contribution of the sterilizing effect in each stage of the process is additive, so that

24. Methods to determine the values of (1)Experimental If laboratory experiments are carried out using different sterilizing holding times until an acceptable aseptic medium is obtained (i.e. a substrate subsequently proved suitable for the desired fermentation), the acceptable process can be analyzed to evaluate the design criterion. The value of can then be used for all sizes of batch (or flow rates in continuous sterilization)

25. (2) Theoretical Can be made using values from the literature to design the sterilization process. If data for the contaminating organism is not known, that for B. stearothermophilus may be used. In practice, values between 30 and 80 are used. Deindoerfer and Humphrey suggested that a value of 40 is suitable for most industrial fermentations.

26. Since = ln = 30 correspond to the ratio of ( ) = 1.0 X 10 13 = 40 correspond to the ratio of () = 1.0 X 10 17 = 80 correspond to the ratio of () = 1.0 X 10 34 In the food industry, the criterion for sterility in canning operations is the probability of the survival of 1 spore in 10 12 of Cl. Botulinum (correspond to a value for of 27.6)

27. Example 1 For the inactivation of B. subtilis spores, A = 9.5 X 10 37 min -1, and E =68.7 kcal/mol. Assuming that a liquid containing these spores is instantaneously sterilized at 115 o C, calculate the time required to give a destruction ratio of 10 6. At 115 o C, k = 9.5 X 1037 exp (- 68700/(1.9872 X 388)) k = 0.1922 But and ln(10 6 ) = 0.1922 = = 71.9 min

28. At 115 o C, k = 9.5 X 1037 exp (- 68700/(1.9872 X 388)) k = 0.1922 But and ln(10 6 ) = 0.1922 = = 71.9 min

30. Example 2 10,000 L of medium is sterilized in a fermenter at a temperature of 120 o C. The time/temperature profile of the sterilizing process from 100 o C is given in Table 2 below. Assuming that the contaminating species is B. stearothermophius. Given E = 67.48 kcal/mol and A = 4.93 X 1037. [a] calculate the design criterion for the process [b] What will be the destruction ratio N o /N?

31. TemperatureTimek 100540.0143 105610.0477 110690.154 115790.483 120911.47 1201011.47 1151040.483 1101070.154 1001140.0143

32. STERILIZATION OF GASES Aim: to reduce the viable organism population to an acceptable, non-competing level Ways sterilization of gases to be carried out mechanical collection (e.g. filtration) use of chemicals or irradiation application of heat For large volume of air – aerobic fermentation – fibre filters were used cartridge configuration

33. Filtration of gases size of m/o varies (0.5 – 2.0  m wide X 0.5 – 2.0  m long) – separation mechanism used for removal of m/o from flowing gases will be different filtration of solid from liquid suspension – - sterile filter cloth (muslin cloth) was used. - pores of filter cloth must be smaller than the m/o - separation took place on the surface of the cloth and on the bed of particles formed.

34. Suspension Filter cake Filter medium Filtrate Filtration applies to the separation of a solid suspension using a porous filter medium to retain the solid whilst allowing the liquid to pass through the bed of particles formed and the filter medium

35. if the above process is to be applied to gases, the pore size of the filter would be smaller than the m/o (0.5-2.0  m) for large volumetric flow rates of gases, the high pressure drop across the filter would imposed uneconomic use of energy due to the pumping cost – compressed air (? Horse power) one mechanism used for the removal of m/o from gases is to trap the m/o inside the filter medium (not on the surface).

36. The type of filter used is normally a fibrous mat, relatively thick, consisting of fibers of materials fabricated in random fashion so that the gas must follow a tortuous path in order to pass from one side of the filter to the other (Refer figure below). Collection of the m/o takes place by collision of the m/o with one of the fibers of the mat.

37. Figure 2.3 Path of gas (and m/o) through a fiber filter. Fibrous filters are dependent on the velocity of the gas for their collection efficiency, the variation of retention efficiency with gas velocity being similar to that shown in the figure below:

38. Bacteria Phages Filtration efficiency ln (N o /N) Gas velocity

39. The minimum velocity of No/N for bacterial spores occurs at a gas velocity of ~ 0.4 m/s. Above this critical value there is a dramatic increase in the collection efficiency (the higher the velocity the more chance of collision of the organisms with the fibers) For phages, where the size (and hence mass) is much smaller even than for spores, the gas flow must be kept low to improve the chances of collection, otherwise the lighter phages are carried past the fibers with the air stream.

40. The minimum velocity of No/N for bacterial spores occurs at a gas velocity of ~ 0.4 m/s. Above this critical value there is a dramatic increase in the collection efficiency (the higher the velocity the more chance of collision of the organisms with the fibers) For phages, where the size (and hence mass) is much smaller even than for spores, the gas flow must be kept low to improve the chances of collection, otherwise the lighter phages are carried past the fibers with the air stream. ln = KL where L = filter thickness K = filtration constant The filtration constant K is a function of gas velocity, fiber size and the density of the m/o to be removed, and it is a normal practice to express K in terms of the filter thickness required to remove 90 % of the organisms in the gas (L 90 ) ln = ln= KL 90

41. and K = Knowing L 90 for a particular material (fibre) at a particular gas velocity will enable a filter thickness to be specified for any value of No/N.

42. For the removal of B. subtilis spores from an air stream flowing at 1.54 m/s through a filter composed of 16  m glass fibre, what thickness of filter will be required for removal ratios of: 1000 : 1 10,000 : 1 10 12 : 1 2.3 X 10 17 : 1 Solution For this type of filter at a gas velocity of 1.54 m/s, L 90 = 1.52 cm (Table 2.4 will be given in class). Since the filter constant K = 2.303/L90 = 1.515 And ln (No/N) = 1.515 L (a) ln (1000) = 1.515 L; L = 6.908/1.515 = 4.56 cm (b) L = 6.08 cm (c) L = 18.24 cm (d) L = 26.4 cm Example

43. Availability of commercial filters fiber mats - from glass fiber and rock wool mats of glass wool (much finer than spun glass fiber natural fibers like cotton and wool should be avoided (when wet, the size of the pores through the material tend to be enlarged on drying, and the reproducibility of collection efficiency suffers) plastic filters (polyethylene, PTFE, PVC) – though problems may arise in sterilization of the filter due to the relatively low softening temperature. sintered metals (stainless steel)

44. Problems arises placing of the filter in the air flow channel causes the pressure to drop the denser the fiber mat, the greater the resistance to the flow of air – thus the cost of pumping the air through the filter rises, and an economic balance must be achieved between the cost of the filter and the cost of pumping

45. Example : a 30 cm mat of glass fiber or rock wool may be more economic than 7 cm of sintered stainless steel (5-6  m aperture) Filtration of organisms does not of course destroy the organism, and after a long period of operation some m/o will penetrate the filter because successive layers of fiber cannot filter anymore. So thermal methods should be used for sterilization

46. Filters can be sterilized in 2 ways: Treatment with “life steam” (121 o C up to 30 min.) will ensure that the majority of the trapped spores will be inactivated, but the filter material must be capable of withstanding the temperature, and a number of plastics cannot tolerate 121 o C. Cotton and wool (natural) are also wetted and are not suitable for sterilization by this method. “dry” heat can also be used (using electrical heating elements).

47. Application of heat Rate of destruction of m/o spores using heat follows the 1st order reaction: ln = The specific reaction rate constant (k) is also a function of temperature k = A exp

48. The sterilization of culture medium is a “wet” heat process, and the mechanism of destruction being due to protein destruction or “denaturation”. For the sterilization of gases it is possible to use a “dry” heat process. For “dry” heat destruction it is essential that oxygen is present because the destruction mechanism is one of an oxidation process. It is also essential that the air is not stagnant, since a continuous replenishment of oxygen to the organism is necessary.

49. For this type of “dry” process Ed = 12,000 – 24,000 cal/mol For a “wet” process Ew = 40,000 – 80,000 cal/mol The value of A in the Arrhenius expression is the same for wet and dry processes (typically 1037 – 1039).

50. Availability of commercial filters fiber mats - from glass fiber and rock wool mats of glass wool (much finer than spun glass fiber) natural fibers like cotton and wool should be avoided (when wet, the size of the pores through the material tend to be enlarged on drying, and the reproducibility of collection efficiency suffers) plastic filters (polyethylene, PTFE, PVC) – though problems may arise in sterilization of the filter due to the relatively low softening temperature. sintered metals (stainless steel)

51. Problems arises placing of the filter in the air flow channel causes the pressure to drop the denser the fiber mat, the greater the resistance to the flow of air – thus the cost of pumping the air through the filter rises, and an economic balance must be achieved between the cost of the filter and the cost of pumping

52. Example : a 30 cm mat of glass fiber or rock wool may be more economic than 7 cm of sintered stainless steel (5-6  m aperture) Filtration of organisms does not of course destroy the organism, and after a long period of operation some m/o will penetrate the filter because successive layers of fiber cannot filter anymore. So thermal methods should be used for sterilization.

53. Filters can be sterilized in 2 ways: Treatment with “life steam” (121 o C up to 30 min.) will ensure that the majority of the trapped spores will be inactivated, but the filter material must be capable of withstanding the temperature, and a number of plastics cannot tolerate 121 o C. Cotton and wool (natural) are also wetted and are not suitable for sterilization by this method. “dry” heat can also be used (using electrical heating elements).

54. Application of heat Rate of destruction of m/o spores using heat follows the 1st order reaction: ln = The specific reaction rate constant (k) is also a function of temperature k = A exp

55. The sterilization of culture medium is a “wet” heat process, and the mechanism of destruction being due to protein destruction or “denaturation”. For the sterilization of gases it is possible to use a “dry” heat process. For “dry” heat destruction it is essential that oxygen is present because the destruction mechanism is one of an oxidation process. It is also essential that the air is not stagnant, since a continuous replenishment of oxygen to the organism is necessary. For this type of “dry” process E d = 12,000 – 24,000 cal/mol For a “wet” process E w = 40,000 – 80,000 cal/mol The value of A in the Arrhenius expression is the same for wet and dry processes (typically 1037 – 1039).

56. Pipeline sterilization Pipelines are usually sterilized by passing steam through them, or, in the case of dry filters, by passing heated air through the line.

57. Example 5 For the destruction of B. stearothermophilus spores using both “wet’ and “dry” processes, the following values apply in the Arrhenius equation: A = 4.93 X 1037 (both “wet” and “dry”) E d = 24 kcal/mol (“dry oxidation process”) E w = 67.5 kcal/mol (“wet” process) Calculate the value of the specific reaction rate for both processes at 105 o C.

58. Solution (a) Wet process k = A exp = 4.93 X 1037 exp (- 67500/1.987(273+105)) = 0.0477 min-1 (b) Dry process = 4.93 X 1037 exp (- 24000/1.987(273+105)) = 6.6 X 1023 min-1

59. Effects of sterilization on mixing in a batch bioreactor Good mixing will allow the broth to both heat-up and cool-down quickly, thus minimizing the degradation of vitamins and proteins and the caramelization of sugars Good mixing will ensure that all of the broth reaches 121 o C and that the broth is maintained at 121 o C. If there is incomplete mixing, parts of the broth may not reach 121 o C and a longer sterilization time will be needed to ensure sterility Scale up alters the mixing, thus the heat-up, sterilization and cool down times will change with scale-up Some industrials air lift bioreactors are equipped with small turbine impellers to facilitate good mixing during sterilization.