Sterilization of media (batch and continuous)

high pressure stream, principles of sterilization, batch and continuous sterilization process

1. BSC C 505C UNIT 2 B. Sterilization of media: Use of high-pressure steam: Principle, batch and continuous sterilization process BY Dr. PRASANNA V DHARANI S. P. T SCIENCE COLLEGE GODHRA SHRI GOVIND GURU UNIVERSITY 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 1

2. INTRODUCTION A fermentation product is produced by the culture of an organism in nutrient media If the fermentation media is contaminated then The media would have to support the growth of process organism and contaminate causing loss in productivity The contaminant may displace the process organism in continuous fermentation The contaminant may be present even in the final product e.g. single cell protein i.e. biomass Contaminant may produce compound which makes down stream process difficult. The contaminant may degrade the final product. For example degradation of beta lactam antibiotics by beta lactamase producing contaminant. Phage contamination may cause lysis of bacterial process organism. 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 2

3. Conatamination can be avoided by : Using pure inoculums to start fermentation Sterile media and sterile fermenters must be used Sterilizing all the additives to be added during the process Maintaining aseptic conditions during fermentation 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 3

4. Protected fermentation: Some of the fermentations are protected The media may be used by limited range of microbes. Otherwise the growth of the process organism may develop selectie growth conditions e.g. reduced pH as in brewing fermentation Hop resins that are used as media component inhibit growth of many microbes Brewing yeast reduces pH of the media So brewing worts are boiled but not sterilized. The fermenters are cleaned and disinfected and not sterilized. 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 4

5. Media may be sterilized by   1 ) Filtration, 2) Radiation, 3) Ultrasonic treatment, 4) Chemical treatment 5) Heat  Heat or steam is the widely used method for the sterilization of fermentation media. It involves a cycle of heating up, holding time and cooling period Factors influencing the efficiency of heat sterilization The number and types of microorganisms present The composition of the culture medium The pH value and the size of the suspended particle 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 5

6. Medium sterilization   Filtration is used for the sterilization of medium which is exception for the medium containing heat labile components Time for the sterilization is dependent on the type of the population. If the sensitive organisms are more in number than whole culture sterilization will be equal to that of the sensitive culture. But if the number of the resistant organisms is more than the sterilization of whole culture is equal to that of the sterilization of the resistant organisms. Contaminant may be by not a single type of organism but by different types of organisms. Sterilization of media require destruction of all types of organisms. The destruction of organisms in sterilization process is given by the factor called Del factor. Deindoerfer and Humphrey (1959) used the term In  N o  / N t    as a design criterion for sterilization, which has been variously called the Del factor, Nabla factor and sterilization criterion represented by the term ▼ Thus, the Del factor gives idea about fractional reduction in viable organism count produced by a definite heat in particular time 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 6

7. During sterilization reaction causes the decrease in nutrient value because of : Interactions between nutrient components of the medium Maillard -type browning reaction       discoloration of the medium as well as deterioration of nutrient value caused by the reaction of carbonyl groups and  amino groups from reducing sugars, and amino acids and proteins respectively Degradation of heat labile components Certain vitamins, amino acids and proteins may be degraded during a steam sterilization regime Thus heat labile compounds can be sterilized by filtration However, for the vast majority of fermentations these problems may be resolved by the judicious choice of steam sterilization regime The activation energy for thermal destruction of  Bacillus steareothermophilus   spores is more than for thermal destruction of nutrient. Thus it would be advantageous to employ high temperature for shorter period of time to achieve desired sterility causing minimum degradation of nutrients. Batch sterilization is not possible as high temperature cannot be kept for short period of time by this method So the solution to this problem is continuous stream sterilization 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 7

8. The Design of Batch Sterilization Processes Main aim of batch sterilization process is sterilization with the least change in nutrient value of the medium Continuous sterilization process is better than batch sterilization process in avoiding the damage of nutrients than a continuous sterilization process The maximum temperature in batch sterilization is 121.6°C High temperature and short time sterilization is attained by taking into consideration the heating and cooling time of the batch sterilization The following point should taken into consideration for a batch sterilization process How much temperature of the fermentation medium is increased during heating or decreased during cooling periods of the batch sterilization The initially number of micro-organisms in the medium The thermal death rate of the selected organism 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 8

9. Batch sterilization Methods Most nutrient media are sterilized in batch in bioreactors at 121.6 o C The sterilization time depends on nature of media, size of fermenter, fittings, valves, electrodes The sterilization time is long The sterilization procedure must be designed so that the exposure of medium to high temperature must be minimum To design the batch sterilization process the information required are: Profile of increase and decrease in temperature of fermentation media during heating and cooling periods of sterilization cycles Number of microbes present in the media Thermal death character of design microbe i.e B sterethermophilus By knowing the initial number of organisms in the medium and the danger of contamination . A ccepted threat of contamination is 1 in 1000, which means number of living organisms after time t is 0.001 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 9

10. For example if any unsterile broth contain 10 11  number of cells then Del factor for that situation is 32.2 The killing of cells take place during both the heating period and cooling period of the sterilization process in addition to during holding period at 121.6°C So, Del factor can be ▼ overall  =  ▼ heating  +  ▼ holding  +  ▼ cooling Knowing the temperature and time required to reach that temperature during heating period and cooling period of sterilization process it is possible to determine the overall Del factor by these periods Thus, from the Del factors contributed by heating and cooling periods, it is possible to estimate the holding time that may be required for overall Del factor One method of sterilization is to inject steam into fermentor mantle or interior coils. i.e indirect steam sterilization Another method is to inject steam into the nutrient broth i.e direct method , in which pure steam which is free of chemicals must be used Condensate accumulate in fermentor and the volume of liquid increases during sterilization The batch sterilization of the medium for a fermentation may be achieved by in the fermentation vessel in a separate mash cooker 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 10

11. Advantages of a separate medium sterilization vessel/mash cooker The medium may be sterilized in a cooker in a more concentrated form than would be used in the fermentation It is then diluted in the fermenter with sterile water prior to inoculation. This would allow the construction of smaller cookers One cooker may be used to serve several fermenters The medium may be sterilized as the fermenters are being cleaned and prepared for the next fermentation This saves time between fermentations Some of the fermentation medium is viscous during sterilization So the power requirement for agitation is not alleviated (lessen) by aeration. Fermenter equipped with a powerful motor providez sterile medium for several fermenters The fermenter are less likely to corrosion which may occur with medium at high temperature. 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 11

12. Advantages of batch sterilization over continuous sterilization Lower capital equipment costs Lesser  assets apparatus expenditure Less chance of contamination The processes require the aseptic inoculums transfer of the sterile broth to the sterile vessel Easier manual control Easier to use with media having a high amount of solid material Disadvantages of a separate medium sterilization vessel/cooker The cost of constructing a batch medium sterilizer is much the same as that for the fermenter If a cooker serves a large number of fermenters complex pipe work would be necessary to transport the sterile medium So there is a risk of contamination Mechanical failure in a cooker supplying medium to several fermenters may cause all the fermenters temporarily redundant (unneeded). The provision of contingency equipment may be costly 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 12

13. Continuous sterilization of media: Continuous sterilization includes heating period, holding time at desired temperatures and cooling period to reach fermentation temperatures The medium is heated to reach to the sterilization temperature (121 o C), holding this temperature to particular period of time and then cooling the medium to reach to the temperature of the fermentation process The temperature of the medium is increased in a continuous heat exchanger and is maintained for the holding time in an shielding serpentine winding holding coil The period of the holding time is decided by the coil length and the medium stream speed The medium after holding time is cooled to the temperature required for fermentation using two sequential heat exchangers The first used the coming medium as the cooling source and The second uses cooling water 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 13

14. Advantages of continuous sterilization over batch sterilization Media quality can be maintained Scale-up is easy Automatic control is easier The decrease of flow ability for steam Sterilization cycle time is shorter Under certain conditions, corrosion of fermentor is lesser In continuous process high temperature is used which reduce the holding time and nutrient loss The necessary Del factor required may be attained by the proper temperature and holding time which decrease the amount of nutrient loss Continuous process engage heating of small amount of medium and cooling of small amount of medium which is very less in contrast with batch system 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 14

15. There are two types of continuous sterilizer: The indirect heat exchanger The direct heat exchanger (steam injector) Indirect Heat Exchanger There are different types of heat exchangers: Double spiral type of heat exchangers It consists of two sheets of high-grade stainless steel (SS) They are mould around central axis in such a way that they form a double spiral For sterilization steam is passed through one spiral and media through the other in counter current i.e opposite direction Spiral heat exchangers are also used to cool the media after passing the holding coil Incoming unsterile media is used as cooling agent in the first cooler So the incoming media is preheated before it reaches the sterilizer so heat is conserved   Advantages of spiral heat exchangers: The two streams i.e media and steam or media and cooling liquid are separated by continuous stainless barrier so cross contamination of two streams is unlikely The spiral route traversed by the media allows sufficient clearance so it is self cleaning This reduces the risk of sedimentation, fouling and burning of media particles 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 15

16. Plate heat exchangers : It consists of alternating plates through which countercurrent streams are circulated The plates are separated by gasket Failures of gaskets can cause cross contamination between two stream Suspended solids in media may block exchamger so it is useful in sterilizing soluble media There are more adjustable as extra plates may be added to increase the capacity.   11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 16

17. Indirect heat exchanger for continuous sterilization of media: Advantages Indirect heat exchanger ( i )   Immediate heating up times (ii)  Media containing solids can be sterilized by this exchanger (iii) Less investment (iv) Easy to maintain and clean (v)  Efficient in using steam Disadvantages Indirect heat exchanger ( i )  Heating may cause foams (ii) Steam is in direct contact with medium, so medium should be enough concentrated and steam should be free from any agent responsible for anticorrosion 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 17

18. The direct heat exchanger (steam injector) The steam is injected directly in the unsterile broth The heating up of media is almost instantaneously It could be used for media containing suspended solids This is cheaper Steam is used efficiently Cleaning and maintenance is easy Steam injection may cause foaming of media during heating Condensate may dilute the media The steam used must be free from particles and anticorrosion additives The injection system is combined with flash cooling The sterilized media is cooled by passing it through the expansion valve in a vacuum chamber so cooking is almost instant 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 18

19. 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 19 Advantages of continuous steam injector It requires very short heating up time It may be used for media containing suspended solids It needs low capital cost It is easy to clean and maintain It has high seam utilization efficiency Disadvantages of continuous seam injector Foaming may occur during heating Direct contact of medium with steam require that allowance be made for condensate dilution and require ‘clean’ steam, free from anticorrosion additives For starch containing broths preheating is done by steam injection The plant is sterilized, before media sterilization by circulating hot wate through closed circuit Fermenters and pipelines are also steam sterilized Heat is conserved by using incoming media which will cool sterile medium, which in turn get preheated before reaching the sterilizer

20. FILTER STERILIZATION OF MIDIA AND AIR Suspended solids can be separated from fluid and gas by filteration This is due to Inertial impaction Diffusion Electrostatic attraction Interception Intertial impaction: Suspended particle that have momentum travel in straight line and become impacted upon the fibres where they remain Diffusion : Very small particles in fluid have Brownien motion So the particles change their direction from the fluid flow and get impacted on filter fibres. Electrostatic attraction: Charged particles are attracted by opposite charge on surface of filters Interception: The fiber of filter are woven to give pores of various sizes Particles that are larger than the filter pores are removed by direct interception Smaller particles can also be removed by interception because: More than one particle arrives at the pore simultaneously Irregular shaped particles may bridge the pore Pores that trap a particle may trap smaller particles   11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 20

21. Types of filters: There are two types of filters: Absolute filters or fixed pore size filters: These filters have pore size smaller than the particle which is removed These filters may be 100 percent efficient in removing microbes In fixed pore filters the pore size is controlled during manufacturing so that the absolute rating can be maintained as mentioned for the filter The removal of particular size particles is guaranteed by interception, diffusion , inertial impaction and attraction. Fixed pore filters are superior. Modern fixed pore filters are of cartridge and may be pleated to give large surface area and minimize pressure drop across filter. 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 21

22. Depth filters or nonfixed pore size filters: It consists of felts, yarn, asbestos pads, cotton, loosely packed fiber glass or glass wool Particles are removed by inertial impact, diffusion and electrostatic attraction There is a possibility that an organism may pass through the filtes as the fibers are not cemented in position So when the pressure increases the material may move producing channels through the filter. Increased pressure may also displace trap particles Filters should be Steam sterilized before and after use. The material must be stable at sterilization temperature. Should not adsorb protein as it gets fouled Should be hydrophilic   Steam used for filters sterilization must be filtered through stainless steel mesh filters of 1 micrometer . Nylon/polyester filters will minimize proteins adsorption and 0.2 micrometer absolute rating. 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 22

23. Filter sterilization of fermentation media: Animal cell media cannot be sterilized by steam as it has heat labile proteins So the media is sterilized by filtration and absolute filters are preferred The media to be filtered must be free of fungal, bacteria and mycoplasma contaminations. Adsorption of protein to filter surface must be minimum Filtered media must also be free of endotoxins Absolute filters are generally used They are made of membranes that are steam sterilizable and hydrophilic. The membranes are in form of cartridge which are fitted in stainless steel steam sterilizable modules It is difficult t construct on filters that can remove all forms of microbes and toxins. So series of filters are used. The first filter is positively charged polypropylene prefilters with an absolute rating of 5 micrometer to remove coarse materials, clots, etc The second filter is also positively charged polypropelene and its absolute rating is 0.5 microns . It can remove microbes, gels, endotoxins etc. The third filter is a single layered nylon or polyester filter which has positive charge and absolute rating of 0.1 microns. It can remove microbes and endotoxins. The fourth filter is of 0.1 microns with double layer nylon/polyester layer with a positive charge. It removes mycoplasma and endotoxins. To remove virus a fifth filters of 0.04 microns made of nylon or polyester could be used.   11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 23

24. Filters can also be used for down stream process to separate cell and cell debris for the fermented broth and for purification of desired products. Filters of 0.1 microns rating and of polypropelene and the second filter if used is of hydroxyl modified is used to remove cell debris from animal cell broth. 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 24

25. Sterilization of air by filtes (air sterilization filters)   Sterile air is required during fermentation for: Aerobic fermentation For cooling of sterile fermenter and pipelines For maintaining positive pressure in sterile fermenter and during the process For transferring of sterile additives into fermenters during the process For sterilization of exhaust air 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 25

26. Air can be sterilized by heat but it is costly. Fixed pore filters with absolute rating are used. The filters consist of pleated membrane cartridges and placed in stainless steel modules. Material used for making filters are of polytetrafloroethelene (PTFE) It is hydrophobic so is resistant to wetting. It can be steam sterilized. Sometimes ammonia is injected into air stream for pH control, so these filters are also resistant to ammonia. Pre filters can be used prior to filters to remove dust, oil, carbon, pipeline scales, rust, moisture etc. 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 26

27. Sterilization of fermenter exhaust air In traditional fermentation exhaust gas from fermenter is vented out without sterilization Since the use of genetically modified microbes i.e. GMM or recombinant microbes, emissions of allergens, the containment of exhaust air has become important. Fixed pore membrane that could sterilize water saturated air of comparatively high temperature and having high level of contaminants are used. Their pore size are 0.2 microns. Foam may also enter the filters, when the fermenter overflows, so pre filters or pre treatment of exhaust air can be done. For pre treatment hydrophobic pre filters or mechanical separator to remove moisture particles and foam can be used. 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 27

28. Questions Explain ‘protected fermentation’. Why is sterilization in fermentation process important? Types of heat exchanges/steam sterilization methods Types of filters used? How is air/exhaust air/ animal cell culture media sterilized? Describe sterilization cycle? Del factors?? Factors affecting sterilization process?? Effect of media due to sterilization??? Reference: Stanbury P F, Whitaker A, And Hall S J, (1995). Principles Of Fermentation technology , 2nd Edn , Pergamon Press, London, Uk Waites M J, And Morgan N L,(2002). Industrial Microbiology: An Introduction Blackwell Science Crueger W And Crueger A, (2000), Biotechnology: A Text Book Of Industrial Microbiology , 2nd Edn , Panama Publishing Corporation, New Delhi, India Trevan M D, Boffey S, Goulding K H, And Standury S, ( Eds ), (1987), Biotechnology: The Biological Principles , Tata Mcgraw-Hill , New Delhi, India Casida L E, Jr. (1968). Industrial Microbiology , Wiley Eastern Ltd, New Delhi, India 11 June 2020 Dr. P V DHARANI S P T SCIENCE COLLEGE, SHRI GOVIND GURU UNIVERSITY 28 THANK YOU