 In this module, we will talk about sterilization of the fermenter, feeds, and the liquid waste. As we discussed in our previous module about the sterilization process in continuous process while dealing with the medium sterilization. As we discussed that there are two strategies for the medium sterilization. One is that we sterilize the medium after adding into the fermentation vessel. But in the second approach, when fermentation media or a medium sterilized in a separate tank and the fermenter vessel sterilize separately. So, in previous modules we focused just on the sterilization of the medium either inside the fermenter or outside in a extra container just like a fermenter vessel. So, in this module we will talk about first about if the fermenter is empty without the medium what are our choice for the sterilization. So, as concerned the fermenter if the medium is sterilized in a separate batch cooker or is sterilized continuously. Because if there is a continuous process of sterilization then we have to add the sterilized medium into the fermentation vessel which we called as body vessel. So, we should ensure first that the body vessel should be properly sterilized. So, that is why there is the choice we should assure ourselves that that body vessel should be properly sterilized. So, this is normally achieved by heating jacket or the coil of the fermenter directly or indirectly by using the steam. So, if the body vessel is covered with a heating coil and there is the movement of the steam or any other mobile phase circulating into those coils. The second approach is then we sparger the steam into the vessel directly. So, if we just sparge the steam into the vessel through all the entities and the pipe sump and dump lines apart from the air outlet from which the steam is out allowed to exit slowly. So, then when we sparge the body vessel then the steam pressure is held at 15 pound per square inch which we called as 15 psi in the vessel for approximate 20 minute. So, the time is very critical as we know when we sterilize the body vessel along with the medium. But in this case there is no more medium in that. So, there is no any probability of the nutrient loss because we are just going to sterilize the body vessel of the fermenter separately. So, it is recommended that by steam we maintain the pressure of 15 pound per square inch when we maintain such pressure automatically we can assess the temperature will be at 121 degree Celsius. So, we have to maintain that pressure for approximately 20 minute. So, it is essential that the sterilize or a sterile air is sparged into the fermenter. So, if first of all if we sparge the steam just to sterilize the air then if we allow the steam to exit and then the next step is we have to sparge the whole fermenter with a sterile air into the fermenter vessel when the sterilization cycle will be completed. And then the next important thing to do is to maintain the positive pressure inside the body vessel before entering or before adding the fermentation medium into that vessel. Because if the pressure inside the vessel will be the negative then there is a chance of contamination from the outside. So, if you remind that module in which we are talking about the aeration control then at that time we discussed that what is the factor and what is the advantages and disadvantages while dealing with the contamination of having the positive pressure inside the body vessel. So, as concerned after sterilizing the fermenter vessel and then we added the fermentation medium then the actual fermentation process will continue. So, during the fermentation process sometimes the fermentation process need to add some additives. Sometimes we have to run the fermentation as a fat batch in that case then we have to add all the materials all the nutrients all the medium separately sterilize and then add to that. But in some cases when the medium is not required when the fermentation is not fat batch, but we have to add some additives either precursors either some inducers into the fermentation vessel then there will be a proper sterilization of those additives is required. So, in this slide you can see that a variety of additives may be administrated to the fermentation during the process. So, it is essential that these materials should be sterilized. So, the sterilization method depend upon the nature of that additives and the volume and the feed rate at which we are adding those additives as a feed to the fermentation vessel. So, if the additive is fed in a large quantity then the continuous sterilization may be desirable same in case when we are adding the fermentation medium. But in batch sterilization of the feed liquid normally involve the direct injection of the steam into the material held in a storage vessel. So, whatever the sterilization system implied it is essential that all the inquiry equipment and the feed pipe works associated with the addition of these additives should be properly sterilized. As we discussed in our previous slide that if we are dealing with the sterilization of the fermentation vessel we directly sparse the steam into the fermentation vessel. By the same way we have to sparse those pipes and all the lines with the steam just to assure the sterilization of the whole transportation lines etcetera. As concern the third case when we are dealing with the sterilization of the liquid waste it mean when the fermentation process is just completed as you know that we divided the fermentation process into two major steps. The first step is upstreaming and the second one is the downstreaming. So, when the upstreaming is completed and then the downstreaming will start. So, when we are dealing with the downstreaming process sometime the process organism which had been engineered to produce the foreign products or we are dealing with such microorganisms which are pathogens we have to maintain the containment level. As we discussed the risk criteria in previous module. So, if such kind of the containment which we called as strict containment regulations are required then we have to assure the sterilization of the process organism as well as the liquid waste or a liquid residue which we have in result of the fermentation process. So, the sterilization may be achieved either by the batch or a continuous, but the whole process must be carried out under contained conditions very regulated conditions just to maintain the containment level. So, the batch sterilization involve this purging of the steams directly into the holding tanks whereas, the continuous process would imply the type of different heat exchangers. So, here you can see that mostly in a batch sterilization the autoclave is mostly used for sterilization purposes. So, what is the function of the autoclave that only maintain the pressure of mostly 15 pound pressure just to maintain the temperature of 121 degree Celsius. You can see in this slide by the red lines that how the steam entered into the autoclave chamber and then when the steam is just entering into the autoclave chamber the first the air that will be oozes out. And when this whole air is out oozes out then this whole container filled with the steam because the wet heat is required in autoclave process. So, if the air will remain in as a pocket then that space cannot be fully sterilized. So, as concern the sterilization in autoclave the holding vessel of the batch sterilized of the waste you can see in the next slide by the figure in which we can see that how the different batch sterilization waste is controlled. So, whichever method is implied in the effluent treatment but it should be the necessary because if we sterilize the liquid waste but we have to cool down that waste before discharge as a waste. So, the temperature required just to maintain at 60 degree Celsius. So, we have to need the cooling of that material. So, the sterilization process have to be validated and are designed using the Dell factor approach as we discussed in our previous modules. However, the kinetic characteristics used in the calculation would be those of the process organism rather than basillus stereothermophilus. As we know that our sterilization process is mostly designed keeping in mind the basillus stereothermophilus which is the indicator organism for sterilization. But as concern the death are the killing of the process organism we have to select the method according to that proper organism. So, you can see in this slide that what are the different steps involved in such liquid waste. You can see here is the head and tongue tank and this is the kill tank which we called as the treatment tank. These are the different lines and you can see here is the level control and temperature controls and different so. A vessel for the batch sterilization of the liquid waste for a contained fermentation. So, thus the sterilization regime used for the destruction of the process organism will be different from that of the sterilization process. So, because the sterilization process focused on and controlled with respect to the basillus stereothermophilus as I have already told you, but in this case we have to run the process according to the process organism.