Day3-PRECISION FERMENTATION FOR SUSTAINABLE MANUFACTURING OF BIO-ACTIVES AND INDUSTRIAL BIOCHEMICALS

Day 3 | GIAN Course on “PRECISION FERMENTATION FOR SUSTAINABLE MANUFACTURING OF BIO-ACTIVES AND …

what enzyme engineering is system biology is synthetic biology is now you can Implement those to tweak your host or even tweak your product or type of product that you are expecting after the fermentation process but the question would be what sort of reactor will you choose and uh what are the different types of reactors that are available why we actually look into bi reactors in one way or other and what would be the design principles that are involved when you’re are actually designing a bi reactor for a specific purpose now before I start the lecture for today I did receive a question with respect to the case study 2 that we were discussing yesterday do you all have any questions with respect to what you are going to do in your case study one or case study 2 what I have provided you with respect to the case studies was the product what you have to do is go backward develop the whole process okay now the product that I have stated might not be the final product it’s just it might be just the the the the product that would have received from the industry but it cannot be consumed let’s say monoline cannot be consumed if it is not heat stable right enzymes are also not heat stable so the alpha mileage that you will produce how do you want to sell it if you want to sell it do you want to encapsulate it do you don’t want to encapsulate it what would be the optimum way of producing the final product has to be decided by you not me okay let’s start the lecture for today so we are going to start our conversation with bioreactor design and Analysis but before we get into the design and Analysis part we will talk a bit more about the bi reactors we have covered majority part of the bi reactors how they are implemented or how they are used with respect to your cellular Agriculture and precision fermentation but we’ll re uh I say like we’ll go through it again so bi reactors as you can see from the picture here uh those are the huge ones and all those pipes that you see here those are the controls or any of the inlets that are providing the substate inside the bi reactor so it’s a pretty complex unit when you look at it it’s a very complex unit when you operate it actually looks very complex but it’s a very simple system in principle it’s a very simple system what it does is very simple but the way it has to be operated is very complex and that’s the reason why you have to have the engineering design principles involved here and we need to understand how to design a bi reactor now we are using enzymes plants and animal cells or we are even using microbes as our hosts uh enzymes if you’re just providing a substrate you put in the enzymes you can get the substrate converted into the product directly plant and animal cells we have already discussed multiple times they can be used as a host to produce your product of Interest microbes are widely used as a source uh as a host for to produce your product of Interest so then there are different types of products that we do developed so there will be the bio conversion products where you have the substrate being converted into product biot transformation where one product sorry one substate has been transformed into a product and the recombinate products is what we have discussed as part of the Precision fermentation where we are developing the product using Rec component DNA technology so we have genetically modified our host to produce the product of Interest so a bioreactor by definition is a device in which a substrate of low value is utilized by living cells or enzymes to generate products with of higher value now substrate of low value if you are using pure glucose or pure fructose or gcto they are not cheap they are abundant but they are not cheap when we do talk about low value uh substrates we are thinking about the feed stock so let’s say if we are able to utilize the agricultural waste so that’s the reason why I give this particular definition there but majority of the times the final product that you’re developing that is of super high value so that’s why the substrate cost doesn’t matter much even if you are using galactose sucrose fructose to produce your product if the final product has a way much more higher market value your subs cost will not be of any concern correct that’s the same case that you will have in your case study one if the monoline is able to capture the whole Market or the whole food industry Market with respect to the sweetener part that particular product is going to have a pretty high value a market value so your Capital cost involved in terms of choosing the substrate might be might not be of concern to you okay so be wise when you’re doing the technomic analysis you would be able to figure it out what should be your input what should be your output and how you you going to do that now the the bi reactors have been used to produce different types of products some of those that I have mentioned here so vitamin B12 uh that those are produced by through Genetically Enhanced microbes uh that develop it through one step fermentation process using vegetable oil as a feed stock and sugar as a nutrient we have Genetically Enhanced microbes producing baking enzymes used to enhance Rising strengthen D and prolong freshness we have already discussed how the enzymes will basically uh provide the sensory aspect to our products right microbes and fungi are Genetically Enhanced to produce enzymes that are added to as brightening and cleaning agent we did discuss about that uh the proteas enzymes and the lipas enzymes the lipases are mostly used to remove grease uh you would have heard about uh natural no not natural but bio compostable detergents they’re not uh not the regular detergent uh liquid detergents but they are like uh compatible more environmentally friendly they actually have more of these enzymes genetically enhanced enzymes or genetically produced enzymes now bassilus micros ferment corn sugar to make lactic acid which is heated to create biodegradable polymers for Wen fabrics and these are the ones which are used to make uh diapers for kids for babies now this example this particular slide shows different examples of fermentation system like how fermentation has been used but majority of the examples that I provided are are more inclined towards a recombinant DNA technology right always using a genetically modified organism to produce a product because currently that’s the norm the regular fermentation we are using uh Sach servy to convert barley into alcohol right those will be the regular fermentation the traditional fermentation so traditional fermentation is still going on they still use the same type of bi reactors even in there you have to choose whether you’re going to go for a specific B reactor which might be a batch reactor or fed batch or a continuous stir tank reactor or even a plug flow reactor so there are different types of reactors that you can use now what will you use for majority of your Precision fermentation processes that is what my interest was and that’s the reason why we have these examples okay so biochemical synthesis versus chemical synthesis uh why biochemical synthesis uh is better is because it is mild reaction conditions you’re not using very harsh reaction conditions if you remember uh from the slides of yesterday where we are producing the PTA at that time we were looking at Cobalt manganese being used those are harsh chemicals which are basically being used it has a very high Regio and stereo Selec uh selectivity because if you’re using enzymes you have the capability of this is sustainable if you are using a sustainable feed stock uh it has a better product yield we did compare if you remember we did compare the pet with the conventional p uh non-toxic and undesirable waste as uh byproducts so they are non-toxic in nature and the byproducts which are produced are not toxic either and they can be used further for other purposes if need B and it is more environment friendly so the ghg emissions and all those things are reduced because you are changing the feed stock by using a cheaper feed stock or a waste as a feed stock you can reduce the overall ghg and you can make it a very carbon neutral system you know what carbon neutrality is so let’s say you have plants now you use the plants to make fiber you use that fiber for making clothes now once the clothes uh like the production of the clothes the CO2 which is generated so let’s say this is the industry which is producing the clothes they are generating the CO2 now this CO2 is again used by the plant to make to grow right so basically it becomes a carbon neutral cycle so that is what I’m talking about so it’s more environment friendly High Regio and stereo selectivity if you’re using enzymes enzymes have the lock and key functionality right so depending upon the active sites it might and the substrate that you are going to use it would be very selective to that particular substrate so if you are using multiple substrate but you only want one substrate to be used you can actually use that so that that’s what it is trying to say so you have the regional and the IO selectivity thank you sir now this is an example of a typical bioprocess that you can see so you have your media and nutrients which are coming in through ultra filtration into a bioreactor you have water which is being added to the bio reactor through reverse osmosis air is being passed in to provide the oxygen to the bioreactor and then you have once the cells have grown to a specific concentration they are moved into membrane filteration now where the after the membrane filteration some of that cell go back as cell recycle or you can harvest the rest of the cell some you put back some you reh harvest and whatever product you have had after the membran filtration you can do the downstream process Downstream processing so this is your Downstream processing now this is where the maximum cost is now just a quick question why do we do a cell recycle any ideas sir when we operate at high dilution rate we do not want our C Mass biomass to decrease in a Biore reactor okay we have gone way much more in advance we have have not reached the dilution rate yet in our discussion but to a certain extent yes but more of to maintain the same culture you don’t want to change the batch of your culture right you want to maintain the consistency so what is the best example of cell recycle in your day-to-day life yeah yogurt and CD right at home so you keep the culture after you have finished the yogurt you keep a little bit of it so you can reuse it so that maintains a consistency cell recycle allows you to maintain that consistency okay oh I could have asked another question the media and the nutrients that are being added into the bioreactor they go through ultra filtration why you need them to be clean but you also need them to be sterile anything that is added into the bio reactor has to be sterile except the cells okay all the nutrients and the media that is added into the bioreactor at all times has to be sterile it cannot have any contamination because the moment you have a contamination you’re going to change the whole profile of your bioreactor inside right now this is a typical bioprocess I’m not saying that this is a bioreactor design that we are following but what type of bioreactor will this be it has an input and it has an output if it was a continuous input and output it becomes a cstr a continuous third tank reactor right if it has only an input it becomes a Fed batch if it has no input no output it becomes batch right you most of you have the background in B technology would have taken a course in BIO process so I know that you’re aware of it for those who don’t there three types batch fed batch continuous so design of a bio process depends on a couple of things the most important would be how fast will the process take place so the reaction kinetics what changes can be expected to occur like what would be the metabolic State what would be the cell physiology that would be reached and how will be the system be operated and controlled to provide the maximum yield and productivity so what sort of design of reactor we are going to use and what sort of instrumentation and control we are going to have so what would be the temperature profile what would be the pH profile how much oxygen is going to be purged what would be the speed of the impeller within the bi reactor all that has to be controlled that comes under the the topic of process control and instrumentation design if you’re looking into chemical engineering that would be process control instrumentation design so so another aspect of the design of a bioprocess would be how will be the products be separated or recovered with maximum Purity so the downstream processing now we have discussed to a certain extent in detail about Downstream processing uh we did talk a little bit about the temperature and the pH yesterday when we were discussing about the enzymes and how they might impact the the structure of the enzymes so if you if let’s say I am assuming that you are aware what a cell culture looks like okay so if you have a flask have you ever seen a flask with a cell in it in the microbiology if you have taken a course in microbiology you would have seen that right now if I heat it what will happen to the cells they will die right so temperature so the moment let’s say if the cell cultures were at 25° C that’s the normal room temperature right now okay so 25° C which is a normal room temperature if I slowly increase this temperature all the way to 70° c will they start dying the moment I start increasing the temperature or will they die after we have reached a specific threshold temperature so once we have reached a threshold temperature right so temperature actually sometimes will promote cell growth eventually once you have reached a threshold it will start dying so you will see that the cell will start growing and then and this would be the temperature at which or T Max that you can actually have now the reaction kinetics like how fast will the the process take place it is very very important if you’re looking it from the chemical engineering uh perspective the chemical reactors using chemicals as your substrate and the product that you get is also a chemical you are providing a catalyst you have to control the temperature and the pressure everything now some of the reactions are super fast some of the reactions take time right and that’s the reason why we add catalyst so that we can increase the speed of the reaction then some of the reactions are exothermic some of the reactions are endothermic right when they’re exothermic what does that mean thank you for the unity I would prefer if one person can answer that energy will be needed endothermic so when when you’re releasing the energy into the system that’s exothermic so that means you’re increasing the temperature of the system am I correct endothermic you are absorbing the energy does do that mean that I’m reducing the temperature of the system yeah EXO yes or no EXO will relase Endo will take so if I’m absorb if I’m doing an endothermic reaction does the temperature of the system decreases thermodynamics 101 tell me what will happen to the entropy of the system in an endothermic reaction it will reduce right the temperature might not there are different exceptional cases but temperature might not okay so this see your thermodynamics is now involved down here you have to have a background in thermodynamics if you want to do a b Rea design so engineering background is very much required now what changes can be expected to occur when we looking at the metabolic State the cell physiology so if we are aware of how the which metabolic pathway is going to be followed by our cells we we would be able to provide them with the right nutrients which would be required for that metabolic Pathway to get you the product of Interest okay so understanding that will also help uh understanding what is the growth uh rate or how quickly our cells will double what would be the physiology what would be the concentration of ourselves later on will also help in designing the body reactor understanding that the the thing that I’m trying to State here is that whether I’m going to choose an equal ey or whether I’m going to choose an yeast that depends on the product that I want to grow but it also depends on how well they grow right so understanding that is very very important when you’re designing a bioprocess now what is an efficient bioprocess when ever we talk about efficiency efficiency has to be defined in terms of time efficiency can be defined in terms of money if it is able to make you more money it’s an efficient process if it is able to save you money it is an efficient process if it takes less time makes you more product it’s an efficient process other than that it also depends on the production of organism it depends on optimal conditions for the desired products if you’re using the optimal condition for a desired product you’re going to have a very efficient bio process the product value will Define your efficiency of the bioprocess and the scale of production if you’re able to produce in Mass a grand scale or a large scale you would be able to make more money out of it and your process would be an efficient process like setting up a startup you should be able to make some money at the very end so this includes so when we are trying to do uh the efficiency calculation we have to look into the size of the reactor the type of the reactor and the method of operation that we are going to use which is which will give you the best for a given conversion so if you’re converting Pro subset a into product B you should be you should make the right choice in terms of choosing the right reactor the right size of the reactor right a a single person does not need an airplane a lot of people will require an airplane let’s say if I’m a billionaire I will definitely need an airplane but I’m just saying in terms of like as a single person I can I I might be happy with a bicycle to reach from point A to point B so depending upon your need you have to make the right choice in terms of choosing the right reactor the type of the reactor the size of the reactor because all of that will Define how much how energy efficient your process would be if you’re choosing a bigger reactor for a smaller reaction or for a smaller product size your your your efficiency is less okay now the bioreactor design uh there are a couple of other things that are involved in it so those would be your control and the positivity positively influence the the biological reactions and you have to prevent contamination the capital investment and the operating cost we have already discussed that so during fermentation maintenance of monos septic condition is very very important so anything and any everything that is added inside is sterile so the nutrients that are going in are sterile so you’re only growing one microb inside the or the microb that you have put in the culture that you have put in is the only thing that is growing inside the reactor you’re not making any you’re not adding any contaminant to it you’re not adding any other microbial uh colonies to it or any other microbial population to it so optimal mixing with low and uniform Shear rate now why do we need optimal mixing in terms of a bioreactor and why do we need a uniform Shear rate optimal mixing will help to homogeneously distribute the nutrient and and oxygen if you’re sparging oxygen uniform Shear rate if if you’re able to identify the optimum Shear rate or optimal movement of the Sparger or the rotational speed of the Sparger the mixing would be ideal you’re not going to break your cells if you make your SP sorry not the spares uh sorry your impeller if you make your impeller to run fast you might break your cells that will lead to cell lices then comes the areation uh surface or direct sparging or in direct so depending upon what will provide you the best and Optimum O2 distribution within the bi reactor if it is an aerobic uh if it requires aerobic conditions you need to identify the best way of providing that Iration into the bi reactor so maintenance of adequate heat and mass transfer or and the flow conditions so let’s say for a Fed batch we have to have uh an input let’s say for cstr you have to have an input and an output it has to be maintained in such a way you cannot have a lower output higher input because then your reactor will overflow correct so you have to maintain that optimal condition the balance so that your cell uh the the cultural growth or the silver growth is maintained within the reactor at all time the products and the byproducts removal is also there so to maintain the optimal conditions within now bi reactor uh for that we have to look at like it provides a control level so my mistake so what does B reactor do bioreactor provides you a controllable environment enabling the biochemical and the biomechanical requirements to create a desirable product so you are pro being provided with both the parameters to grow your product to the best of its cap abilities it enables close monitoring and control of the reaction parameters like internal and external Mass transfer heat transfer fluid velocity and sheer stress so one unit operation can provide you all of this control and then you can grow your cell culture within that unit operation this is a diagram of a typical bioreactor the previous one that I had shown that was a diagram for a typical bio process this is a Biore reactor so what are the parts and what are the most important parts for us would be your agitation system that means your impellers the design of your impeller will also be a parameter at one point of time the sensors the sensor probes would be for your pH and the temperature so you’re looking at your pH you’re looking at your temperature what else can I uh use the sensors for dissolve oxygen right do what else the level the no let’s say it’s a batch reactor so the level is maintained so you’re looking at a disolve oxygen you’re looking at the pH you’re looking at the temperature right these are the major sensors what else can be used what else can I sense inside a body conductivity conductivity conductivity why and in what sort of application will you use conductivity sensors majorly conductivity measure like uh for insulin production uh they measure the conduct to uh consider how much Sal Con conentration how much uh is available in or how much if you change your salt concentration that will change your pH also H yes but majorly in Industry level conduct also one of the parameter that can they they do use uh conductivity meters yes the connectivity sensors are there um I personally think if they have been used when you’re working with microwave fuel cells mostly uh cell counting sensors and n proofs have you ever seen I have seen them a bi reactor having that yeah I have I have seen that so when they’re doing the N proofs you said nir proofs I don’t know the full form but they are used to measure the cell like what is the cell count at particular time okay I haven’t had that experience yet so thank you so there are a lot of many sensors now that we can use inside a bi reactor so you have your reactor tank that’s a typical design of a bioreactor you have your feeding pump which is basically used to push in the nutrients inside uh you have your spargers which is going to provide you the areation you have your agitation system to maintain the homogeneous distribution of the nutrients and oxygen you have the thermal jacket now this thermal jacket is to maintain the temperature Within okay so this is a typical diagram so Parts would be agitator baffle now baffles are sometimes here in the corners of the bi reactors in this particular diagram it was not there now why do we add those baffles to remove the vortex right so because when you’re running your Sparger sorry when you’re running your uh stter or your impeller you generate the vortex to break that Vortex you’re going to use the baffles so you have your Sparger the cooling and heating jacket to control the temperature you have your Control Systems typical ones would be temperature pH and dissolve oxygen and thank you for the self count part I will add it now let’s get into the classifications of bioreactors now bioreactors can be classified based on the presence of or absence of oxygen uh before we start discussing that can you tell me the two types of bioreactors that we have actually discussed once uh on the day one I gave I asked you a question about if you have potato peels or let’s say you have uh Orange Peel what sort so either it would be a submerged bi reactor [Music] or or solid state right so that’s also another classification so whether you’re going to use have everything submerged inside or not then you have your in the in the presence of oxygen or in the absence of oxygen so anerobic fermentation so no Iration is required initial preparation of inum may require Iration because you want to grow the cells the gas released during the fermentation is sufficient to provide IDE the mixing so when you are in anerobic production you will have some gas generation within the reactor inside the reactor as the cells are growing and that gas bubble is going to move up as it is going to move up it is going to make the mixing mixing of the nutrients with the cell culture the product recovery may also require anerobic conditions majority of the times uh for if I if I remember correctly in the lab we provide an condition by purging with nitrogen for aerobic fermentation you have non-st erated reactors which would be airlift bubble column reactors in which you’re basically so there is no impeller so it’s mostly being mixed with the Iration uh stirred and irated reactors where you have the stirred tank reactors so cstr would be a part of that some of the batch reactors will be part of that fed batch are also part of that so on the right hand side you can see an example of airlift bi reactors where you have the draft tube internal Loop uh configuration a split cylinder device and then you also have an external loop system so you’ll see majority of these things are moving a very similar design for this would be if you have learned dehydration of food products there is a design of one of the dryer like that what is the name of the dryer where your product is fluidized we call it fluid bed dryer right so it’s it’s because you want to make sure that your air is it’s uniformly distributed your product will drive faster in that case right it’s very simple similar concept ccept applied here or this concept was applied there now based on the modes of operation now this is what we have been discussing I have actually stated these names multiple times so it can be a batch reactor it can be a Fed batch or a continuous dirt tank reactor now when it is a batch reactor no input no output it’s like pressure cooker we put everything inside close it let it run once it’s done give ref you the signal take your product out okay in a Fed batch we can add the nutrients there is no output but we keep on adding the nutrients so the intent of a Fed batch would be to reach the maximum cell Mass right we keep on adding the cell will keep on growing the cells will grow continuously till the time all the substrate that is present in inside the bioreactor is consumed if you keep on adding the substrate the cell mass will keep on growing so the intent of the FED batch reactor can be to have the maximum cell Mass so now from your own understanding and experience that we have had through conversation in the past couple of days where will you use a Fed batch if uh if the product is intracellular right developed intracellularly you want to have the maximum biomass that you can take out crush and take your product out that can be one application where else will you use it will I like to use it in uh let’s say if the product was extracellular and the cell can continuously produce it in that case I can go for a continuous thir tank reactor right so depending upon the type of product that you are going to develop you can choose the right type of reactor right so in the continuous thirdd tank reactor you have your s not and X not your s not is basically your your substrate concentration your substrate at time T that is initial substate concentration or the substrate that is coming inside then you have your X not that is your cell biomass concentration at time tal to Z your initial cell biomass concentration X S and P are your X would be your cell at the end of what is coming out s would be what the substrate that is coming out and P is the product now in a continuous third tank reactor if you see the output you have your cell you have your substrate and you have your product correct you still have your substrate so not 100% of the substrate has been used the cell that is coming out is going to reduce the cell count inside so you can do a recycle in that case that’s the reason in cstr we generally have a recycle so what are the design consideration for a bu reactor the most important for any startup in the world Capital similarly if you want to set up a bi reactor facility or any uh fermentational facility the very first thing that will come into picture would be your initial capital expenditure now out of all these three reactors which one do you think is the cheapest batch because of ease of operation or you can buy as big as you want you can put as much of your mic uh your substrate into that let it run and once it is done open it take your product out right so substrate if you see that’s why I’m saying like if let’s say I come to you and say that okay I need this particular product your choice of reactor will depend on many many things one of the thing is how much money I’m going to give you as your initial Capital right so your initial capital expenditure will Define the type of reactor that you’re going to choose the ease of operation would be the second most important parameter less downtime now what is your downtime what is downtime so you have a reactor all the reactions are done so let’s say it took uh you started it now it took three or four hour 4 hours for the whole process to end after 4 hours you removed you open it you remove your mask cell mask you clean it it takes you 2 hours to make it ready for the next batch the down time downtime is 2 hours right run time 4 hours okay okay so we’ll be using these terms like downtime the T and the runtime or the the batch time would be your 4 hours downtime would be your 2 hours so TB would Define your batch time TD would be defining your downtime so using these parameters you will be able to understand the efficiency of your process that you’re working with or even the the productivity you can Define productivity not just on the terms of how much cell Mass you can produce or how much product you can produce you are also going to look at the total like if you’re if you’re running your reactor every day for 365 days of the year with a specific downtime in between your productivity will depend on how many times you have ran it how much of the downtime you have had in between because that will Define your total time so total time is batch time plus TD simply so how if I tell you like you are able to run two batches a day if you’re running your system for 365 days how many batches did you run 365 into 2 and that will give you the total mass you would have accumulated right over the year or what your yield have been over the year maintenance ease of Maintenance not just maintenance ease of maintenance is also a very very important parameter when you’re trying to choose a bi reactor or even design a bi reactor skill requirement there was a question I remember so skill level that is required to run a bio reactor depending upon the choice of bi reactor will differ if you add too many complexities you need somebody who’s highly skilled right if you have less complexities you can run it without any skill give me one very good example of a batch reactor not a con tank reactor or a or let’s say any of the reactors which have been operated by least skilled people a product that was developed by least skilled people they don’t have to have a degree in engineering come on you do that every day at home your mom does that yogurt good another in North America it would be moonshine have you heard about moonshine uh illegal liquor okay which you can do brewing in a jungle you don’t need a highly skilled engineer to be in a jungle hiding from the police brewing your fermentable alcohol that’s your moonshine in in north at one point of time it used to be illegal to actually brew moonshine and uh people were still doing it so that’s a very good example you don’t need to have skill you need to understand how the process works right skill is if you can operated technical skill is a different thing so when you start adding complexities where you have to estimate how much of dissolved oxygen is there are you able to maintain it you have to do some calculations yes technical skill is required at that time that might change the type of reactor or the choice of reactor for you if your choice is to make sure that a specific skilled level is utilized or employed for a process the other would be your Control Systems the type of controls that you would like to put if if if you only require a basic control that means temperature what is happening inside or you need your temperature you need your pH you need to dissolve oxygen you need to know your cell uh concentration you you you need to add all of those controls to design the B reactor then you have your quality of the culture the design consideration will require the quality of culture now quality of culture matters a lot what do I mean by the culture so if you remember when we were discussing about the process to reach the reactor stage we have to grow the cells in the lab so we use the flasks we grow in them in that but then we are what what are we trying to do we are trying to grow the culture or the cells in the nut nutrient Rich environment right so there so that their metabolic Machinery within the cell has aligned to that nutrient but that nutrient has to be the similar to the nutrients that they going to get in the reactor might not be in the same concentration but they will be getting in the reactor so that small scale pirate scale large scale that’s how it goes so you have to have that chain so you develop the culture you build the culture then you pass on the culture inside correct now that Machinery the metabolic Machinery which is within the cell that will will keep on that will change if you change the carbon sources so let’s say if I have given a carbon Source One to the to the cell culture in the lab and in the fermentor I’m giving it a carbon Source too what will happen will my cells grow yes or no if that carbon Source can be used by the cells it might grow if that carbon source is not to be used by my cells they will not grow they will die the quality of culture will also make a lot of uh impact because if you have an older culture again an example would be your CD and yogurts at home if you use an older culture you might not get a proper set card if you are using a new culture very new culture where you don’t have enough microbes you might not get a proper set card but you have to have the appropriate Optimum culture to have to grow in the fermented system right or in the fermentation process when do you reach it how do you reach it it has to be done outside the fermentation process okay now the classifications of bioreactors you have your solid state bioreactors the cultivations in the absence of free water of uh pre-treatment of the substrate and difficult to control process parameters now solid state sorry solid state by reactors the cultivation is in the absence of free water pre-treatment of of substrate and is required and difficult to control process parameters are involved in this it’s not as simpler as uh regular submerged by reactors like a batch process or a Fed batch or a control tank reactor application of these are mostly for antibiotics food additives biocontrol agents and bio Remediation in my research group we have used it for food additives uh specifically to get natural pigments natural pigments would be your natural colors from the food waste product so we were using onion peels we have used uh carrot in North America you get uh so you here you have your carrots with the top then you have your leaves right excuse my drawing when you go to the market you can buy the whole carrot here there they will cut this part and this part and what you get is in between and bunch of that so this goes as a waste with the leaf that we call it the crown and the tip the crown and tip is thrown away so my research group we did uh we mash it we make a pulp out of it put it in a solate reactor run it just to see if we are able to extract the carotenoid whatever is present in it now keratinoid is a natural pigment that will give you the natural orange color right now other classifications of bioreactors are immobilized cell bioreactors and the fluidized bed bioreactors we did discuss about the fluidized bi reactors a little bit uh immobilized cells bi reactors are where you reutilization of cells is required so your cells are immobilized into a different chamber or you can say a flow cells or flow yeah a flow cell and then you have your substrate being pumped into that flow cell and as it moves through the chamber your cells are interacting with the the substrate your product is formed it is pumped back and the and the and the substrate whatever conversion has taken place is pumped back so it reduces the contact time it increases the volumetric productivity uh the fluidize bed reactors you have your heat and mass transfer is efficient in this case because everything is fluidized so the transfer of temperature the transport of U or the mass movement would be appropriate effective mixing between liquid solids and gaseous phases and it has a very low Shear rate suitable for suitable for plant cells and mamal cells so depending upon the type of host so let’s say if you’re going to use Mamon cells as a host to grow something you would like to go for a fluidized bed by reactor okay now comes the main aspect of your bioreactor design and Analysis so what are uh couple of parameters that we have to discuss here sorry so for the bi reactor design analysis till now we have talked about batch reactor fed batch reactor continuous St tank reactor fluidized bed reactor imiz imized bed reactor there’s another type of bioreactor have you uh heard of this uh plug flow reactors you know what a plug flow reactor is those who know it uh please help me out okay so in the chamber do you have the cells immobilized or what do you have in the chamber because that’s a plug flow reactor right so you have pass in my understanding it is you’re passing the substrate as a plug so it is moving from one side to the other side it will get converted or the surate will get consumed and will go away right the product will be formed and that can come out so this particular chamber in a plug flow reactor does that have your immobilized cells yes or no in this particular design that I have made yes so in the plug flow reactor that is one way of uh passing it it’s a it’s a really nice design but I to be honest I don’t remember what exactly we use it for I forgot Dr Bala without yes there’s no axal missing mixing exal mixing so in the axis there is no mixing it’s moving from one direction to the other direction right okay so that’s one other uh so plug flow reactors that’s another type of reactor which is there fiz bed we have done imol s by reactors we have done okay so bi reactor design analysis so what size of reactor type of reactor or method of operations are best for a given product formation that’s the very first question that you’re going to ask yourself when you’re trying to choose the type of bi reactor for your own process now it depends it very much depends uh for your own project so not project for your own case studies depending upon the type of product that I have asked you to make you have to choose the right by reactor but that choice will depend on the host that you have chosen correct it will also depend on whether your product which is made is extracellular or intracellular what else would be there the host the product you need Iration or you don’t need Iration do you need mixing continuous mixing what else would be there somebody gave the answer environmental conditions okay process parameters what else would there will the amount of product that you want to develop be a condition that should be the primary condition the productivity right yes if if if you’re having difficulty go back to this particular slide ease of operation less downtime product variation variability should be less productivity or yield is also a very important parameter in choosing the right reactor for your process maintenance is another thing the skill requirements I’m not going don’t consider that for your own case studies that is not that won’t be necessary Control Systems yes would be very very important the type of feed stock that you’re going to use we’ll also Define now how do you define the performance at the very beginning of our lecture we we talked about from the monitary perspective and the time perspective right if you have to say if if a particular process is efficient enough I say how quickly I was able to get my product or how much product I was able to get to make the money out of it when you’re looking it from the reactor perspective you’ll be looking at the reaction time so how much time it takes for the conversion of the substrate into the product so it is a measure of processing rate in a batch reactor and so whenever we are working with a batch reactor the performance measurement is based on the reaction time when we are working with let’s say a flow reactor uh like a cstr continuous third tank reactor we are looking at space time and space velocity so space time and space velocity so space time is the time required to process one reactor volume of feed and the space velocity is the number of reactor volume treated in unit time to define the performance of a flow reactors so both space time and space velocity are used to define the performance of a flow reactor reaction time is only used to define the performance of a batch reactor before we go into the next set of slides which would be later I just wanted to go back uh on a few slides and have a bit of of a discussion with respect to your case studies that was the intent now when we are looking at your case studies now case study one was monoline please correct me if the spelling is correct or not and the case study two was Alpha mileage for monoline and Alpha mileage for monoline it’s a it’s a plant protein it’s a protein as a product now what will impact the quality of the protein or the product in your fermentation system your pH will impact it the temperature will impact so the process parameters right so you have to be very careful in terms of what reactor you’ll choose depending upon what will happen to my product at the very end so you want where you have maintained the temperature okay pH will change based on if the product is going to add to the medium if my product is acidic or basic that is going to change the medium pH the protein if that is extracellular is it going to change the pH of the medium guess yes or no so when you add protein to a media will the pH of the media change or any like say if you add protein in the water the the water PH changes you have your protein shakes right if you go to the gym does the pH of your protein shakes change when you mix water in the in the powder protein so let’s say it doesn’t let’s say it doesn’t I’m not agreeing with this or disagreeing with it just for the sake of this conversation I’m saying let’s say it doesn’t change the pH of your medium the only parameter that remains would be temperature right if the temperature is is also Optimum your interest is that you get maximum amount of protein generated right then you can decide what type of bioact would be be careful in that in case stud 2 Alpha milees that’s an enzyme again a protein whether it is produced extracellularly or intracellularly depending on the host that you choose depending on the process that you choose will Define the type of reactor you are going to use all so you have to be very very careful in uh in choosing the right reactor design for these type of processes the second part of our uh presentation is pretty long uh the FED batch and the batch reactor and this part was not too long so I would like to end the session here for this one and we’ll meet again at 2 to we start the second session both material huh e e good evening I hope you had a nice lunch okay uh there are a couple of things that before we start the presentation or like before we start the discussion on the design of the batch and the FED batch reactor I have to clarify a mistake that I made in the previous session when we were talking about uh oh oh the plug flow reactor I stated that the cells are immobilized and we are moving the the substrate that’s wrong we are actually sending the cell and the substrate as a plug so we sell we send like specific concentration of the cell and the substrate through the reactor so it gets completely consumed and by the time it comes out it is like you have the whole cell mass and then again you send the second plug the third plug it’s a continuous plugging of that it becomes like a continuous third tank reactor where you have the continuous growth in it right okay so now that I have fixed that mistake I can start so design of the batch and the FED batch by reactors so we were discussing about the design analysis so the idea is whenever you’re trying to develop a bi reactor or whenever you’re trying to develop a particular process which is going to use a bi reactor there’s couple of questions that you have to answer one of the question would be the size of the reactor the type of the reactor and the method of operation that you’re going to choose and which of those reactors and the method are better for your product formation so keeping in mind with respect to our case studies 1 and two you can actually you’re actually going to go through this exercise yourself but to give you a bit more information of how we actually design these reactors how do we understand that this reactor is better for our product or that reactor is better for our product we’ll get into the details of it now we have also discussed about the the performance measures so we when we are looking at the reaction time that is what is used for the batch reactors if it is the space time the space velocity we looking at The Continuous Flow reactor so that would be your continuous third tank reactors so batch reactor design the there has to be why we are doing it right so when we are starting the starting question for a batch reactor design would be like for a given reaction kinetics and the desired substrate conversion what should be the volume of the reactor to meet the product ction Target now for a batch reactor now if I have to give you the most lame in answer let’s say if you have to cook rice for yourself how many glasses of rice will you put in the pressure cooker and if you let’s say you have a party of four or five coming to your house how many glasses of rice will you put into the pressure cooker will a 2 L pressure cooker be good enough for you whether a 5 L pressure cooker would be good enough for you that sort of question is what we are trying to ask okay so what would be the size of the reactor if we want a specific desired level of product formation or the cell Mass concentration what should be the size of the volume of the reactor that we should be using as part of the batch reactor the other part would be to calculate the productivity of the given reactor volume and the process kinetics and the conversions so these are the two questions that we would like to answer as part of our batch reactor design the other how will we do that so the way we will go forward with that would be that we have to develop an equation between the cell Mass generation which is going to be denoted as X the product that we are generating would be p and the substate that we are consuming as an S so we have to find the relationship between the cell Mass the product and the substrate so there is a relationship between the cell mass and the substrate there has to be relationship between the cell mass and the product generation sometimes if you have too much of substrate that is not good for your cell Mass too less of substrate is also not good for your cell Mass if there is a substrate inhibition what would be the situation if there is a product inhibition what is the situation so we’ll be looking at all those type of equations or relationships in terms of the batch reactor design now these equations are used to predict the batch time for a desired conversion so once we have understood the relationship between the parameters the cell the product and the substrate we can use that to understand how much time will it take to get the specific product or the specific cell concentration using the specific amount of substrate a given volume of the reactor and we will also be able to estimate the volumetric productivity of the system through our bat reactor design now the growth kinetics the growth kinetics or when we are talking about the cell growth if you remember in the very first uh conversation that we had on the cellular agriculture we did talk about the sigmoid curve right the cell has a growth phase sorry lag phase the log phase and the stationary phase and then you also have a death phase even ually right so that is basically your cell concentration x with respect to the time you’re looking at how the cell mass is growing now how do you actually quantify this cell concentration so there are two methods you have the direct methods in uh in which you’re looking at no suspended solids or interference compounds and uh then there is a preferred method which is actually your dry weight uh value so if you remember when you’re doing experimentations as part of your molecular sorry microbiology course if you’re growing a cell in a media in a Shaker flask you’ll be continuously taking out at least an mL of the media and checking its turbidity and that is what you’re going to plot to check your growth curve right so that is what we do the optical density so you can take the dry weight and optical density which is uh estimated between 600 nanom to 700 nanom or you can use the cell number density or you can estimate the cell number density using uh hemocytometer or a plate count or a cter counter so using these techniques you can actually estimate how much of the cell has grown in your medium now the indirect methods are preferred when the direct methods are inapplicable that would be specifically for the solid state mold fermentation so solid state fermentation technique is widely used when you’re trying to grow melium or fungus for your own product as a product so solid state fermentation uh system is used so we basically used the indirect methods for that so the cell Mass can be determined indirectly by measurement of the protein or the DNA or the ATP or or the nadh now if you’re looking at the ATP and nadh these are products of the metabolic pathways right either gra cycle or TCA cycle you do get these produced so if you’re able to if you’re aware of how much of ATP would be generated by the biomass or by a given biomass you can estimate by estimating the Total ATP amount so for example the the the example that is given to you is like assume that 1 mgram of ATP per gram dry weight of bacterial cell is produced so if you have 100 Mig of ATP per liter then you can estimate your cell Mass concentration to be 100 G of dry weight cells per liter so you’re estimating it indirectly how much of the cell mass is there by estimating a product that has been generated out of the cell or within the cell as part of of its metabolic pathway or even if the protein is being produced how much protein is being produced per cell if you know that then you can go back and estimate how much your cell concentration would be so cell so you have the direct methods so direct method is the terity method that’s the preferred method the indirect method would be when we are looking at the DNA the ATP NL and the nadh now with respect to the design and operation of the fermentation system the cell growth kinetics plays a very important role and understanding the cell growth kinetic is also very very important so cell growth kinetics is the rate of cell growth and how it is affected by the various chemical and the physical conditions now when we are talking about the chemical and the physical conditions the physical conditions would be if you can give me an example of physical condition use the mic if you can if you can use the mic that would be really great like this pH and these environmental conditions the chemical would be that okay okay physical what would be the physical see the change in temperature change in PH will affect your growth kinetics for sure right so that is where we are looking at most of the time so when we are looking at the cell concentration with the the change in the cell concentration with respect to the temperature and the pH my question to you was like if when we are talking about various chemicals and the physical conditions what would be one of the physical conditions that would be the temperature chemical conditions pH we can take that way right now to when we are having the growth kinetics as we said like the growth curve looks more or less like this you have your death phase at the very end you have your lag phase this is your log or the exponential phase and this is your stationary phase there has to be a mathematical equation that can actually Define this relationship right with respect to time the concent change in concentration of the cells or the X with respect to time there has to be something now how do you accurately model this mathematically model the cell growth it is actually very very difficult to do that because the growth is dependent on various biochemical reactions and transport phenomenas so let’s say if you mixing is not proper and it is not able to get enough oxygen or the nutrients the cell growth will vary at different parts of the reactor if it is a batch reactor it is changing at very different parts of the reactor so mathematically you have to assume that there is a homogeneity the completely homogeneous environment is maintained there is no difference the nutrient content on the top and the bottom so you have to assume a lot of it to be able to mathematically design an equation that can Define your growth kinetics a hetrogeneous mixture of young and old cells are present at all times if you have this type of scenario your growth kinetics cannot be modeled mathematically because younger ones will have a different model the older ones will are already at the stationary or the death phase it changes because you have a mix of it so the mathematical models that are there which can actually explain cell growth fall under the unstructured and distributed empirical models now there are certain assumptions that have been made for these empirical models and we did discuss about this assumptions in the previous slide so assumptions concerning cell components and the population that as the cell is increasing the cell components are also increasing at the same time the cell kinetic equations derived from these models are used for the analysis and design of Ideal fermentors and that because whenever we work on the design of uh bi reactors or design of fermentor systems whether we are going to do the B uh the batch or the FED batch we are always going to design for an ideal batch reactor or or an ideal fed batch reactor or even an ideal cstr if you go up to there so the assumptions are the cells represented by a single component such as the cell weight or the cell number assuming balanced growth that means the cell weight and the cell number is increasing at the same rate as its components the second assumption is the cell suspension is homogeneous uniformly distributed okay the nutrients have been uniformly distributed if the oxygen is being added to the to the bioreactor it is uniformly distributed so all the parameters that are required for the cell to grow are uniformly distributed but when we are only considering the cell so the cell are homogeneously distributed only one medium component is limiting the reaction rate other components are in excess now when we talk about the limiting component for a cell growth what would they be what does a cell require to grow just give me two on in the mic in the mic that would be great sir carbon and nitrogen Source those are your nutrients right so nutrient or the the substrate that is coming in is one yes sir what is the next one what can inhibit a cell growth what can change the cell growth oxygen oxygen right so you have your nutrient concentration and the oxygen concentration so if you change either of it you will be varying the way the cell is going to behave or the cell growth is going to behave in the fermentation system in this case we are assuming all of the other things are constant only one is changing either we are changing the substate or we are changing the oxygen majority time s we are changing the substrate the substrate is your limiting factor if it has to be the concentration of the substrate that is going to be added to the reactor would be your limiting factor other than that nothing else changes because we have defined because when I said that homogeneous nutrient the homogeneous distribution of the cell homogeneous distribution of the nutrient homogeneous distribution of the oxygen for the cell growth I’m only changing the nutrients rest all the Remains the Same so monod model is one of those empirical models which is commonly used to define your cell growth now in this particular case you have mu which is your specific growth rate so your me is equal to Mu Max s/ s + KS now this is your monod saturation constant this is your limiting factor because we have to make that Assumption of the limiting factor right so this is your limiting factor in this case so monod’s model the s/ ks + S is your limiting factor now how do you define your mu your specific growth rate DX by DT would be the change in the cell concentration with respect to time right if you do normalize it with respect to the cell concentration X that is what is going going to give you your mu the specific so mass of cell produced divided by the original mass of cell into time so if you rewrite this equation that is what has been done in the last line here so DX by DT becomes mux or mux X is your initial cell concentration and we know that mu is mu Max s/ KS + S you just substitute that so this is the equation that you’ll get so further increase in the nutrient concentration mu after it reaches Max does not affect the con uh does not affect the MU so what it means is as you increase the s or S tends to go to Infinity just for now I’m just in inre keep on increasing the S the cell concentration will increase and it will become flatten out this is where your mu Max is and if you remember your micless mum constant from your enzyme kinetics when we are doing that so the half of your enzymes the same thing is used here so the half of mu Max by 2 is your KS the constant the monard saturation constant okay clear till now any questions these are the basics if you have already done that I’m sorry I’m going to repeat it anyway now this particular graph here is representing your growth kinetics so you have your lack phase you have your acceleration phase the growth phase and the decline that goes into your stationary and then you have your death phase just a question for you when the cell is in its lag phase what does that mean use the mic please if you have the mic please answer in that try to it’s on on it’s try to adjust to that environment it is trying to adjust to the environment adjust the envir why is it trying to adjust to the environment it has to utilize the carbon Source it will take some time but if I remember correctly when we were discussing about the fermentation system so we have a seed fermentor where we have grown the biomass I have acclimatized it to the nutrients that are going to be used in the larger biomass will it still require that lag phase we can reduce the lag phase timing sir if you’re using a seed culture culture we can reduce lack phase so if you use a seed culture many of times this particular lack phase will not even exist so when you don’t use it it is obvious because the cell is growing still cell is still growing it’s it’s not doubling it’s just growing in volume okay the cell is still growing it’s not doubling it’s not multiplying what it is doing is it is changing the internal Machinery to get used to the nutrients and the environment that you have put it into once it get used to that it knows now I’m ready let’s double that’s where your acceleration phase starts and then gets into your exponential phase of cell growth once it is in the exponential uh phase of cell growth it will keep remain it will remain in the exponential phase of cell growth if you continuously provide it with substrate am I right with this statement if I continue to provide substrate will the cell continue to be in its exponential growth phase not I’m not saying a cell I’m talking about the the whole reactor if I if I continuously keep on adding the subate can I maintain the exponential phase for as long as I want no sir no so you you won’t be able to maintain the growth phase at all or the exponential phase the loog phase okay when will the cell get into the stationary phase use the mic please when will the cell get into the stationary phase uh substrate limitation or the the cell will produce tox toxic components okay let’s assume the cell is not producing any toxic components okay and there is no substate limitation okay will the cell reach its stationary phase like do sell have a biological clock like humans they have a life cycle like okay I’m only going to be alive for 1 hour or two hours or they will continue to keep on growing and multiply you’re talking about bacterias will the cell biomass keep on increasing that’s what my con my question is I’m not asking you anything else will my cell concentration be in an exponential phase or continue to grow if I continue to provide it with substrate in a batch reactor that’s not the situation right in a batch reactor you have given it enough substrate you have given the seed the substrate grows done you let it grow once the substrate is completely consumed it has been in the stationary phase like like it has reached the max that it can be in that mu Max stage and once it has completely consumed your substrate what will happen started to then your decline uh your death phase will start but that before the death phase the cell will still catabolize itself to grow it will kill other cells to to get the component for it to grow we will be reading we will be we will learning more about that later but I’m just remember this question that I asked you if you continue to provide the substate will the cell will continue to grow or not just remember this question okay will you will be able to answer it later on now let’s get into the monod equation so there are different uh so monod equation is with respect to your cell growth right cell kinetics there are different versions of monod equation that have been exist I think this is your uh long model this is to represent the substrate inhibition if the substrate is the one which is inhibiting the cell growth itself how do you represent that you’ll be representing it with this equation it’s a it’s a upgraded version of your monard model and this is the only part which has been added because what is your mual to Mu Max ss+ s right that’s what your monot model is if you add this 1 minus s – s m to the power a you adding the substrate inhibition aspect so this is your inhibition from the substrate side now when the substate inhibition takes place so let’s say if I have an equation with mu and subrate so there is let’s let’s let’s make a graph so when we are talking about inhibition there are two ways the the MU will behave with respect to the substate concentration either it will follow an astoic curve or it will follow a straight line a declining straight line or with a negative slope so when you’re looking at this declining straight line with a negative slope the location where it has intersected the substraight line the xais that is your SM so what it means is luong model is valid if you are provided with the concentration of the substrate that is actually going to inhibit your growth your mu so if you’re aware of the concentration of the substrate at which my growth will get inhibited you follow the luong model if you are not aware of what my substrate concentration is going to be at which the inhibition will take place that means your mu will become zero you don’t use the Mong model you use the second one the relationship the asymptotic Rel relationship between mu and S is represented by the equation two if this is your equation one that one is only valid if you are aware or if you are given with the value of the substate concentration that will inhibit your growth or your mu will become zero now if multiple substrates are present an upgraded version of the same model would be used z b now if you are looking at product inhibition so I have substrate inhibition sometimes if you have growth of product product might also inhibit the cell growth right can you give me an example to that ethanol sir if you have more ethanol generated it it will inhibit the cell growth eventually right so if if the product is the one which is going to inhibit I’m going to ask you a simple question which of these if I’m looking at mu and the P which of these will follow the negative slope or the asymptotic behavior one or two so which one will follow the negative slope so you know the concentration of your product at which your inhibition will take place so your number two is a straight line and your number one is going to give you the asymptotic behavior same equations similar equations for the subset and the product these are the upgraded versions of monod kinetics monod’s model the monot model is used to define your growth kinetics if you have inovations associated with it you have to use the monot model but a different upgraded version of it and that is what they did any questions now let’s come back to our discussion on the different uh phases of the growth of in the batch culture so you had your lag phase so we did discuss that the cell is going through the machinary changes right but it will increase in the volume but it’s not going to increase in number in the acceleration phase and growth phase it’s going to increase in the in the number it’s going to multiply once it has reached the decline of the stationary phase that’s the time when the relationship has become the that it is about to finish off all the subrate that is available right the balance has been reached and once all the substrates are gone what happens death and that is where I did state that it might start eating itself right not cannibalism catabolism cell catabolism starts to take place or endogenous uh I think I forgot I think I have it that in the slid so we’ll go there so this is your uh cell growth kinetics we did discuss a bit about your lags the growth and the stationary phase now do cells produce products as they’re growing so in their growth phase what sort of product will a cell produce primary metaboli secondary metabolize comes in the stationary phase your antibiotics vitamins minerals they come out in stationary phase in the growth phase is mostly the proteins and anything that is required for its own growth right so anything which is within the cellular structure of the of the biomass like the cell so lag phase is a period of adaptation we are just going to repeat a little bit for the cells to their new environment thank you for answering that question at that time so new enzymes are synthesized a slight increase in cell mass and volume but no increase in the cell number there is a prolong it can be prolonged you can prolong your lack phase if you have put less inoculum you can do that you can also prolong your lack phase if you have a poor inoculum that means either you have like a mix of uh dead cells with your VI like viable cells it’s like it’s an old culture if you take that the age of inoculum is older then you’re going to prolong your lack phas or your medium in which you have put your seed culture is not nutrient enough so it is going to remain in the lack phase for a longer period of time so if the medium contains more than one or more carbon sources you will be uh observing multiple lag phases and that is called call us dioic growth now what happens in that we did discuss about it so let’s say let’s say if your in the recombinant DNA technology when we are talking about the if you remember for one of the examples there were two plasmids that were added into the cell into into the into the bacterial cell I don’t remember exactly which example was that if I I think it was P so it was being grown in the using the cell there were two different plasmids let’s say there were two different plasmids now one plasmid happily works or will allow will make the modifications to the cell in such a way that you can use one carbon Source the second one requires a different carbon Source or the there are two different carbon sources that are present in the cell forget about the plasma for a moment so if there are two different carbon sources which one will the cell choose first I asked that question right C1 and C2 it will choose the one that it likes the most because everybody wants to be in their own comfort zone cell also wants to be in its own comfort zone so it’s going to choose the one which it likes the most so you will have a growth with a like small lag you’ll have the growth once it has completely consumed that particular carbon Source now it has to rearrange its metabolic pathways to start consuming the second carbon source which it might not like but it has to eat it’s like when you go to the hostel you might not like the food but you have to eat it right for survival so that’s exactly what happens so you have that is called your dioic growth so when you’re using multiple uh carbon sources it will continue to use the carbon source that it likes the most then the second one then the third one if you keep on adding you can keep on having that growth multiple lack phases the exponential growth in this particular phase the cell has adjusted to their new environment and it will start multiplying rapidly exponentially okay so it does a balanced growth all components of the cell grow at the same time and the growth rate is independent of nutrient concentration as the nutrient are in excess the growth rate is independent of the nutrient concentration for the exponential phase because the nutrient present is in excess so I will ask my question again what if I maintain my nutrient concentration in excess yes will my cell remain in the exponential growth I’m not asking you to answer right now it might be a trick question so think wisely whenever you get the idea to answer this think wisely if I maintain that excessive nutrient concentration will my cell remain in the exponential growth phase now before we get into the design equations for the batch reactor and the FED batch reactor we have to go through certain terminologies or we have to understand certain terminologies which will be used more or less every time we try to solve so in our conversation on the cell biomass and when we are looking at the micro microbial growth we’ll be using a terminology called a CX which is your cell concentration is your dry cell weight per unit volume your dcx by DT is your change in your cell concentration with respect to time so when we try to do if you remember uh mass balance right in minus out plus generation minus consumption is equal to accumulation your dcx by DT is your accumulation term in a batch reactor do you have an in everything is already inside right there is no Inlet there’s no Outlet is there any consumption consumption of what but am I balancing substrate or am I balancing the cell am I consuming the cell so the only thing that you will have is generation right so let’s say that I’ll give as RX but it is a volumetric generation volumetric accumulation because the V remains constant got the idea this is actually Your Design equation for your batch reactor I have given you way in advance but I’m just trying to explain how we are going to use these terminologies later on okay so your Rx is your growth rate of the cell biomass so what is the difference between dcx by DT and RX dcx by DT is a change in the cell concentration RX is your growth rate the difference between these two is rx is 1xx dcx by DT you have to have that term of X because the rate of growth so it will appear that both the terms one like the two and the three are the same but the two may include the effect of input and output flow rates and the cell cycle because you might do a cell cycle it might keep on increasing it now these the these terminologies can be used for batch reactor it can be used for fed batch reactor it can be used for CSR these are not specific to batch only but these are the terminologies that are basically used when we are going to go forward in our conversation here three is your actual growth rate of the cell three which is your Rx is your actual growth rate of the cell two is only equal to three for the batch operations 2 is only equal to three for your batch operations now the other terminology would be that growth rates based on cell number and cell weight can be assumed assumed to be equal in exponential growth phases with a balanced growth assumption that all the components are also growing at the same time and the microbial growth the product formation and the substrate utilization rates are usually expressed in the form of specific rates so the growth would be represented as Mu which is specific growth rate uh the substrate will also be represented Sim similarly in a specific we’ll have the product you will see QP for the product or the specific uh growth of the product we are always going to represent it in terms of the specific rate now specific rate are always normalized with respect to the initial concentration so that X has to always be there for specific growth so what was the equation for Mu that’s your specific growth the change in concentration with respect to the initial concentration multiplied by time there’s another terminology which is your division rate now we all understand so if let’s say a particular cell is going to divide it into two at a time right so if let’s say your cell c not CN not is your cells dividing n times in time T the total number of cells CN After Time T would be so your average division rate Delta is your n / T now this is the equation that comes with from the average division rate now if if you keep on rearranging the previous equations that we have been looking at what you will end up with is this this is the most important equation in this whole list of equations here so your RN or your Rx was DX by DT right or your dcn in this case so dcn by DT and they have done a lawn of it and the law of the N values and they have just rearranged it and put it back into the equation for Mu so the division rate is constant in exponential phase while growth rate is not what it means is as you are in your how do I go back so in this growth phase the division rate is constant okay growth rate is not constant the slope of this is your constant n the other terminology that we are going to use would be your doubling time so your DX by DT is given as mux or mu is 1xx DX by DT right so this is what the equation is where your mu is your specific growth rate X is your concentration G per liter time is a time in hours as you rearranging it so if you make it DX by X mu DT and if I integrate it I’ll be left with Mt like 0 to T this would be X to X this will become l x by X equ to Mt right that is what they are doing this equation you have your doubling time right so when your X becomes 2 time of X that is where your T becomes TD so the equation for the TD is long 2 by mu so your TD your doubling time is basically a reciprocal of division rate this a reciprocal of your division rate so growth rate and the division rate defined by the cell numbers are are different entities the division rate is constant during the exponential period while growth is not this is the most important concept that you have to take into account division rate is constant during the exponential period while the growth is not now exponential growth phase the balance cell the of the cell mass in the culture gives is given as excuse me so the balance of the cell mass in the batch culture gives is the DX by D DX by DT is mux X is X at time tal to zero or when it is starting and the integration of this is given as your DX by X Mt right sorry mu DT sorry my mistake mu DT 0 to t x to X so lwn X by X KN is become mu t now if you put the equation of x x is e to^ mu T if I give you this equation you know let’s say if I give you the initial concentration that came into the batch reactor and I give you the time of the total batch or the batch time you would be able to use this equation given that I have provided you a specific growth rate if I provide you all of those parameters you’ll be able to tell me what the cell Mass concentration would be at the end of the time t with the given specific growth rate you will see that we’ll be using these equation to solve a few questions later on okay so this particular equation is an important equation in terms of your batch growth kinetics and batch operations this is what we are looking at mostly in the exponential phase right the slope of this would be constant this is a constant slope so the MU remains constant in this phase in the exponential phase it remains constant now stationary and the death phase exhaustion of the nutrients that means your s has been consumed completely and the buildup of the waste and the secondary metabolic uh metabolic products inhibits the growth the cells may have Active Metabolism to produce secondary metabolites that’s where in the stationary phase it happens endogenous metabolism occurs by catabolizing cellular reserves for new building blocks and energy produc proding monomer to for the maintenance energy so that means the old cells or the dying cells will be providing the materials for maintenance of whatever cells are remaining or that are still there okay the dying cells or the dead cells or the ended cells are the ones which are providing the maintenance materials the cell liis may occur and the viable cell mass will drop in the stationary and the death phase so the growth kinetics with respect to your stationary phase is given with instead of mu we have this minus KD this minus KD is the rate constant for endogenous metabolism because now the growth is endogenous in nature to catabolism we looking at KD rather than mu which is your specific growth rate it is related to specific growth rate as Mu net because you have your mu and then you have your KD so total mu net would be mu minus KD or sorry mu into minus KD where your mu is your gross specific growth rate so endogenous metabolism is the cell catabolizes cellular reserves for new building blocks and for energy producing monomers which are used for many maintenance okay it is used for maintenance now let’s get into other uh some of the terminologies which are your yield coefficients so yield coefficients are always represented as with Y so it can be YX by S this will represent the yield of X with respect to S so that means the cell with respect to substrate you will have y p by S production of product with respect to substrate and Y x/ O2 with with respect to how much O2 is consumed now y x by S simple equation would be x – x ided by S – s s is your initial subset concentration and S is your final subet con concentration X notot is your initial cell concentration X is your final cell concentration so the ratio of those two will provide you your yield now majority of the slides that I’m using for your batch analysis these were taken from uh an nptl course I I remember uh Dr Smita sasta from I Madras because when I saw that same presentation I teach the same course back in Canada it was pretty much the same thing that I wanted to teach so we used I used more or less her slides or her Concepts but I have added my own flavors to it the way I explain that would be it it reduces workload but I would like to acknowledge her and thank you for providing these slides okay so yield is clear how we write it the notations of it we’ll get into how the equation works out you will be seeing how we’ll be rearranging the equations so if you have to have X if you know the yield let’s say if you’re provided with the yield you have your YX by S right multiplied by S not by S Plus X so let’s say if I give you the initial cell concentration I give you what the yield of the cell biomass is and what was the substrate concentration before and what remained after so let’s say I’m asking you what would be the cell concentration after 70% of your substrate has been used if the substrate that was being put was 10 mg or 10 G per liter or this much of the subrate was added you would be able to use the same rearranged equation to estimate the amount of X okay now these are for your batch process only for batch not fed batch batch batch growth kinetics for most bacteria and the yeast the yield YX by S for glucose is ranges between 0.4 and 0.6 this is your in your Schuler and Ki textbook okay which is like uh the Bible for bioprocessing similarly YX by O2 would be 0.9 to 1.4 G per G O2 so how much cell is produced by how much oxygen is is being consumed so during the batch growth the measured yields are apparent due to endogenous metabolism occurring that is the KD is greater than zero so let’s say what ha what what it means is your cell grew reached the stationary phase right in the stationary phase we did had this conversation that the cell will maintain by killing other cells or other cells will lies and whatever content of that cell came out can be used by the other cells to keep themselves alive you’re getting my point right that if that is higher that means your KD instead of that mu if the KD term is higher or greater than zero the yield that you’re getting at the very end of the batch reactor that means I have done everything so let’s say if this is my reactor I put up everything I was supposed to open it uh after one one hour I got delayed by 1 hour let’s say by 5 minutes and I open it and I took out everything my yield will not be the same you getting my point my apparent the actual yield that I will get is different than my theoretical yield that I would have calculated okay so my Yi yield varies depending upon if my endogenous metabolism took place because some of my cells are gone for the maintenance of whatever remained because I I was delayed by let’s say 5 minutes that’s the best lame and way of understanding it because it has finished all the substrates now the cells are like okay we are done take us out I didn’t they kept on knocking I didn’t after third minutes they’re like dude we have to survive let’s kill people people endogenous metabolism takes place my cell concentration is going to go down a little bit right my yield will reduce little bit very little but not significantly very little if my KD is way too high it will be a significant inre decrease you got the idea how it work looks like so at the end of the batch fermentation we have apparent growth yield which can change with culture conditions so it is not truly constant now comes the design equation so you remember when we were doing the accumulation input output generation consumption so no consumption because we are balancing the cells there’s a difference between heat energy balance and material balance in material balance you always balance each and every component of a mixture your cell in a suspension is one of the component in a mixture so you’re going to only either balance your cell concentration or your substrate concentration or your product concentration you cannot balance all of them at the same time you can only balance one component at a time so one mass balance equation for one component clear that is the reason why we are only using the cell concentration here because that is what our interest is we have already done this so DX by DT will end up to being uh so V DX by DT becomes V RX RX is mux so r X is your mux so now we know DX by DT is mux now mu using the monot model is Mu Max s s + KS into X right so this is the equation that’s how it came so your monot model came up here for the value of mu in the batch the nutrients are added at the beginning and the product is withdrawn at the very end the cell balance equation in a batch reactor is well we have already sorry there’s two of the same thing my mistake so your DX by DT is your mux okay we have already estimated that we substituted mu to your mux s / k + S sorry S Plus KS you remember this equation YX by S is x – x / s – s so X can be written as y XY s s – s + x so that’s your equation clear you know your mu so we are putting both of these equations down there and then we are integrating it so when you integrate it the equation that you will get is this this is called Your Design equation so it is T mu Max a lwn of x/ x + B lwn of s/ s this is your design equation where your a is KS YX by S divided by x + S YX by S + one similarly your B is given so these are the two equations that are used but this is what is called Your Design equation for the batch reactor problem now we have discussed about the growth kinetics now let’s discuss about the product kinetics now let’s say sorry so your growth Associated products are the products produced simultaneously with growth example are constitutive enzymes now what are these constitutive enzymes are the enzymes that are produced by the cell for their own own growth okay these are the ones which are produced during your exponential growth phase in the non- growth Associated product formation that takes place in the stationary field because there’s no growth taking place is stationary that time your QP now what is your QP specific product formation rate right specific growth rate mu QP is your specific product formation rate I did this I did give you a little bit of a hint on that so let’s say go into this so if your p is your product your DP by DT is a change in product concentration with respect to time so your QP would be 1X X DP by DT now what is your y p byx y p byx is a change in the product concentration with respect to sorry yield of product with respect to the cell concentration so that means change in product or d p over d x I can rewrite your YP by X as DP by DT DX by DT now let’s say this is your equation one we know mu is 1xx d d p by DT this is your equation number two using both your equation 1 and two you’ll get QP sorry is Mu y p by X or 1 by X DP by DT you don’t have to write all of these things it’s just for you to understand how we are looking so there is a specific product formation rate there’s a specific growth rate mu QP is your specific product formation rate so the equation that we have just developed is this for the growth similarly for the non- growth Associated we are going to have the QP which is equal to constant which is beta here and during the non-growth associated product so non- growth Associated phase non- growth Associated product formation would be your secondary metabolites the mixed growth product formation takes place during the growth and the stationary phase and that would be your example of lactic acid fermentation and some of the secondary metabolites that are being produced commercially if you’re are looking at from the commercial perspective we try to be in the mixed growth phase if you’re looking at both now this is how your growth Associated product formation the mixed growth Associated product formation and the non-growth associated product formation looks like the primary metabolites are your growth related example ethanol by sacr Services the secondary metabolites are non- growth related like antibiotics and the pigments so if you’re looking at the natural color being produced you’re looking at it in the non- growth Associated phase if you’re looking at antib IC production it is in the stationary phase again and this is where your mixed growth sorry this is non growth and this is where your mixed growth uh phase takes place performance equations for the batch fermentor again we are just going to go through the equations very quickly these are the equations that we going to use when we going to solve a few questions so your dcx by DT is your Rx or mu CX because RX is mux right we have already done this same thing what we are doing is rearranging the thing so dcx by RX I will have mu DT now if I sorry no I will just have the DT now you integrate it your T KN to t x KN to X or CX to CX you’ll end up with this equation here okay now if you take this equation and let’s say in this case if I plot 1 by RX over CX 1 by RX over CX I’m just looking at this particular equation here so if I take that and I’m plotting this 1 by RX / CX I will get this is where sorry so this is where I have harvested my cell biomass this is the concentration at which I have harvested Ed my cell biomass this is my concentration so this is my CX and this is my CX not the Shaded region under this particular graph is your batch time or the batch growth time is the area under the curve now T not is your starting time so if you have had a lag phase there would be a value for T not if you have not had any lag phase if you had your seed culture and it started growing immediately it will be zero okay so let’s say you have your substrate which is being consumed by the X you get your X and that X is also giving another X so X is basically being uh the Catalyst here so the rate of reaction is slow at the start of the concentration of X as the concentration of X increases the rate of reaction increases so it increases as cell multiply and reaches a maximum rate so as a substrate deplete the toxic products accumulate and decreases to a low value this is what we have already discussed so you have one by RX CX the this shaded area is basically your batch growth time it is useful for comparing the performance of various fermented types so if you’re looking at the batch reactor the fair batch reactor the cstrs the plug flow reactors you can actually develop this equation for that and when you compare how much would be the shaded areas for all of those formentor you would be be able to say this one is better or that one is better these are some final slides and then we get into couple of other parameters you want to take a break we we’ll continue for some time and then we’ll take the break and then we have to get into fed batch okay so performance of the equations for the batch fermented so monod parameter cannot be estimated by series of batch runs as done for micel constant parameters so initial rate of reaction is always zero for cell cultivation unlike enzyme reaction so there’s an extra CX terms because uh when you’re looking at this this is what they’re talking about so when you’re looking at the the cell and when you’re looking at the product the difference this is the CX term which has to have which is always there because because mu is 1 by X DX by DT right so that’s where it will come so that X goes back to the CX is that 1X x 1 by CX on this side so that terms always that term always exists and because of that it can never be zero so it’s like you’ll always get uh if I remember correctly the other way around wait I’m not going to draw the graph because I might make a mistake in there but anyways okay so effect of temperature on the growth kinetics we’ll just skip the slides uh pretty quickly these are some things that we have already discussed so Optimum medium pH temperature oxygen supply differs for various microorganisms so every microorganism will have different requirements for temperature pH and oxygen demand so according to Optimum temperature for the growth can be classified for bacterias if you’re looking at the Cyril the optimum temperature is less than 20° C for misiles it’s anywhere between 20° C and 50° C for thermophiles it has to be greater than 50° C so if you choose a thermophile for your product formation you need to maintain a fermentation temperature of greater than 50° C so above the optimum temperature the growth rate decreases and the thermal depth starts to occur so just like that endogenous catabolism that we are talking about we’ll have thermal death that will take place your mun net will change so the growth rate increases to a maximum with an increase in temperature when it is T Optimum so what you’ll have is if you remember in the morning I did draw this so at particular stage you have your t Optimum temperature your growth will continue to increase once you cross the T Optimum in your temperature profile your growth is gone your cells will start to die so at a higher temperature thermal death rate exceeds growth rate which can cause net decrease in concentration of your viable cells and the equations that can be used is this is for your uh the thermal growth a e arous equations have to be used here so a e to^ e^ a minus RT so when it is e a it is thermal growth if it is Ed it is death that’s a big difference between these two so temperature plays a very important role when you’re looking at batch or any of the fermentation processes Now power formation and the yield coefficients are also affected by temperature as the temperature increases Beyond Optimum value the maintenance requirements that means the coefficients of the culture increases as a result the yield will decrease if you remember we already had the YX by S the apparent yield and the actual theoretical yield so diffusional rate should be considered which might become rate limit step as the bioreaction rate increases with temperature now temperature also changes something uh in the bioreactor What will What will what will the temperature increase due to the let’s say the viscosity of the medium it will change the viscosity of the medium if the viscosity of the medium changes it is also going to change the mass transfer coefficients of how oxygen is going to diffuse how the nutrients are going to diffuse so in most fermentation pH can vary substantially depending on the nature of the nitrogen Source production or utilization of the organic acids and the evolutions or the supply of CO2 by changing the CO2 the pH is also getting affected and increase in PH it will affect the activities of the enzymes and therefore it will affect the growth rate now do which is your dissolved oxygen is an important substrate in anerobic sorry in Aerobic fermentation and may be limiting substate as it is sparingly soluble in water do you remember I’m not sure if you uh in Mass transfer you would have studied something called as Henry’s constant right that deals with dissolving gas into liquid at equilibrium right in a ferment in the in the fermentation system can you change the pressure because changing the pressure will change how much oxy how much gas will get dissolved in the liquid right so we cannot do those type of things in the fermentation process so don’t bring that heat transfer concept here that remains in the heat transfer sorry in the mass transfer okay so at higher cell concentration the rate of oxygen consumption May exceed the rate of oxygen supply leading to oxygen limitation so if the cell concentration is higher then what oxygen demand is if the oxygen demand is higher than the supply then oxygen becomes a limiting factor the specific growth rate in that case varies with respect to the do concentration to the saturation kinetics and below the critical concentration the growth respirator follows first order kinetics with respect to the do concentration and above the critical value the growth rate is independent of the do concentration what it means is what is a zero order reaction and the first order reaction which one is substate dependent and which one is not substate dependent first order is substrate dependent zero order is not dependent independent of it can you see the relationship if your concentration impacts the uh the if the do concentration or dissolve oxygen is impacting your cell concentration is a first order reaction right so below the critical concentration below the critical concentration the growth respirator follows first order kinetics above the critical con ation that you you have enough oxygen the self will happily grow so oxygen transfer from gas bubbles to cells is limited by oxygen transfer through liquid fil surrounding the gas bubbles when we get into the Heat and mass transfer you will see how the oxygen is transported so let’s say if this is your cell Mass like they might be in a clump right so your oxygen is coming out from your oxy like the the Sparger gives the bubble the bubble will give out the oxygen so there is a thin layer that it has to cross then there is a medium through which it has to pass and then there is a layer over a cell Mass where it has to pass and only then it can be consumed by the cell right so it becomes like uh you have mass transfer through multi mediums with different diffusivities so the diffusivity here is different the diffusivity here is different the diffusivity here would be different it’s like a series resistances which will get accumulated so it changes now if the Sparger was working properly there would be gas bubbles around the cell biomass so the the transport might be direct so that will improve the overall oxygen transport so DCL by DT the concentration with respect to change in uh the time is your K C Star by CL this is your oxygen transfer rate and then you have your oxygen uptake rate so which is your q2x is equal to x / y x by O2 this is your yield with respect to O2 so when oxygen transfer is your rate limiting your OTR is equal to your OU which means oxygen transfer rate is equal to your oxygen uptake rate and this is the equation that you will get at the sorry this is the equation that you get at the very end so the growth rate varies linearly with the do concentration under oxygen limitation these are just the conceptual parts that you should be aware of another uh would be your pH so the reduxx potential affects the extent of oxidative reductive uh reactions if the function of the dissolve oxygen the ph and the other is other ion concentration so it’s a function of that so you’re changing if you change the pH if you change the disolve oxygen if you change the ion concentration you’re going to change the detox potential of your system and that will create changes to the uh oxidated reductive reactions so if you CO2 gets uh added to your fermentation system CO2 can be toxic so that will impact your cell growth the presence of ions affects the transport of nutrients across cell and it also impacts the metabolic function and the solubility of dissolved oxygen we are going to take a break now for 15 minutes and then we will get back what do you think or do you want me to continue I can finish it off you want me to continue sure okay so let’s solve this particular question if you want to so what you have here is your single cell protein is produced from methanol using a, M Cub bioreactor the biomass yield from substrate is 0.42 G per gr biomass yield from substrate is 0.42 G per G the ks is 0.7 MGR per liter your mu Max is 0.45 per hour that means you’re given with your mu Max the medium contains 4% weight by volume methanol and a substrate conversion of 98% is desirable in the process now methanol is being used to produce single cell protein the medium contains 4% methanol weight by volume okay understand this difference this this this ratio so for batch mode of operation the initial cell concentration is 0.1 1 G per liter that is your X knot and the downtime between the batches is 5 hours your t d is given you need to calculate the annual biomass production you also need to calculate if the desired annual biomass production is to be 10,000 tons per year what should be the volume of the reactor okay now let’s say so what is the total mass of cell produced during the batch reactor for the batch uh culture X is XF minus X into V this is your total mass of cell produced during the batch culture and the total time for the culture is your t t total if you remember we did discuss T batch time plus T downtime TB plus TD how many number of batches are they uh doing in a given year so the number of batches would be 365 into how many days are there 365 days how many hours are there 24 if you divided by the total time you’ll be able to get the number of batches that are there so the number of batches carried out in one year is 365 into 24 divided by TT and the annual biomass production which is your tons per year would be X into number of bches clear till now now out of all of these what are the parameters that you have and what are the parameters that you don’t have are the equations that we are going to look for and we are going to choose and we are going to solve if you go back a couple of slides let’s say sorry if we go back a couple of slides for no we don’t need to go back couple of SES for this so what you have here the very first thing that let’s start with what what your mu is so mu is your mu Max s/ s + KS right and your me will become mu Max under a specific scenario when your XS is very very very very very very very less than your substrate so in this particular scenario what is your KS which is your 0.7 MGR per liter your s is pretty huge value as compared to your KS so in this particular case we can assume that to happen happen so your YX by S would be x – x / s – s your X becomes y x by s s – s + x not I have to keep the question open here side okay now now if you remember the equation where X was x e to^ Mt so if I do l x by X is Mu Max t b this is your batch time the T that we had here was your batch time so I’m rewriting that equation going backward into that okay so your TB will become 1 by mu Max lwn X by X right now X is X not sorry uh X is y x by s s – s + x this is the equation that we have identified right so your TB will become 1 by mu Max lawn 1 + YX by s x s minus SF so this is how we will estimate the value of s sorry TB now what are the parameters what are the values that are provided to us in this particular question so if you remember if we go back to the question what would be your s not or S initial 4% weight by volume methanol and a subate conversion is 98% is desirable right so 4% weight by volume so your si or S not is 4% weight by volume so let’s say if it is that means 4 G per 100 ml right that then only you will get weight by volume right in percentage 4 G per 100 ml so that means 40 G per liter okay so now you have your s not what is the SF s final 98% conversion of 98% is desirable in the process so 0.02 2% is still left so your final concentration that would be left 98% has been convered inverted 2% is left so 0.02 into 40 G per liter will give you 0.8 G per liter right so now you have your s not you have your SF I have this I have this do I have YX by S already the biomass yield from substrate is 0.42 G per G YX by S is 0.42 G per gam is given you’re given with your mu Max so I have my mu Max I have my YX by S do I have my X not initial cell concentration is 0.1 G per liter I have my X knot so once you put in all of those values you’ll get your TB to be 11.35 hours so your X final would be x e power mu Max TB so your X final would be 16.5 G per liter this is your final cell concentration per liter excuse me clear till now the very first slide we had that the total mass of cell produced during the cell culture was XF – x into V your XF is 16.5 X KN is 0.1 that means 16.5 – 0.1 that becomes 16.4 into V what is your V 1,000 so your total cell Mass would be 16 ,400 kgs right 16.5 – 0.1 it’s already given we have estimated our XF what is the downtime is 5 hours right so your total time is 5 + uh 11.3 5 that becomes 16.35 hours right so now 365 into 24 / 16.35 will give me 536 batches and I know my X I know my number of batches that will give me around 8,790 20.4 tons per year this is how we use these equations sorry for the mess I just didn’t want it to lose the the slide of the question because we have to come back to it again and again that’s why I’m using all the space provided good idea now what was the last question so if the desired annual biomass production is to be one is to be 10,000 tons per year what should be the volume of the reactor now if 1,000 gives you 87904 tons per year right how much will give you 10,000 that’s rearrangement so your answer that you should get is 1,137 6 M Cub so this should be the volume that should give you 10,000 tons per year simple I had given this Extra Spaces to solve this one is something that might relate to what you are going to do would you like to try solving this by yourself it’s a pretty straightforward so strain ofi or equoli has been genetically engineered to produce human protein a batch culture is started by inoculating 12 G of cells into 100 lit fermenter so your volume is L 12 G of cells is your inoculation that is your X KN and the fermentor containing 10 G per liter glucose so the nutrient content is 10 G per liter the maximum specific growth rate of the culture is 0.9 per hour assuming that the culture is growing at the maximum specific growth rate in the exponential phase it remains constant mu is equal to Mu Max the biomass yield from glucose is 0.575 G per gam so you’re given with YX by S you’re given with your mu Max x notot s anything else that I missed the V you have pretty much all the parameters so estimate the time required to read reach ex stary reach stationary phase your X notot is 12 G per 100 L which is your 0.12 G per liter right we did that equation in the previous question was TB is 1 by mu Max long 1 + YX by s x s minus SF this was your equation that we have used for the the batch time are you provided with your s not you provided with your s not right the we are going to assume all the substate was consumed so you’ll find your TB in this case to be 4.3 hours the other aspect is the what will be the final cell density if the fermentation is stopped after only 70% of the substrate is consumed so your XF so if if only the 70% is consumed your batch time will change if only 70% was consumed your batch time will change so you have to estimate your batch time again and when you will do that you will get because your SF is now 0.3 of s not your s not was 12 G 12 G no uh 10 G so it becomes 3 G per liter right so SF becomes 3 G per liter at that time your TB should come around 3.94 hours so because we changed the substrate how much substrate was consumed my batch time changed so your XF is X KN E power mu Max DB your final concentration of XF would be 4.1 16 G per liter excuse me the B sorry oh you’re still writing you don’t have to write these these these these particular questions are available in your sh and Kari if you have solved that in the past see the reason I gave you this particular question in the previous one the intent is for you to understand what TB is how we estimate those TB how this equation that xal to sorry xal to x e power UT comes into picture what your me is what your me Max is now what we are left with is the Fed batch cultivation we can start the FED batch cultivation after taking a 10 minutes break if that be okay e e okay welcome back I hope the break was long enough or not long enough for many other people who are still missing here okay so we have discussed batch uh design of the batch reactor in detail so now we are going to get into the FED batch so we we when we started our conversation today in the morning we were looking at three different modes of uh cultivation one was the batch cultivation fed batch cultivation and then third was the cstr or the contr thir tank reactor now fed batch just like is an opposite I will not say it’s an opposite so in a batch reactor you have no input output in fed batch at least at least you have an input so that’s the only difference between the patch and the FED batch it has an input now what it does for us is that it gives us certain control you will see in the further slides but I will discuss a little bit about those type of controls so let’s say if I want to if if too much of substrate is injurious to the health of my cells I can limit the flow of my substrate right so it provides me some form of control if I have to have multiple types of substrate to produce uh for my cell growth I can do that or if I can regulate the the the growth factors that are required I can do that so that’s why the FED batch has a very versatile application in terms of uh where you can apply and that is where we were discussing like in some of the recent DNA applications that we have discussed in as part of the synthetic biology you might be able to go back into those references and see that they have used fed batch as the growth process or the cultivation process of choice so fed batch culture is a semi batch operation so nutrients are fed either intermittently or continuously during the course of the operation so if you have let’s say this is a reactor you have an input coming in there is a limit to which we go in terms of adding the the substrate concentr or the substrate of the total biomass would be there so the volume is at least up to 80% not more than that we still want to keep a little bit of head space any idea we keep that head space for gas mixing like or the for the transfer so nutrients uh so we the culture both is harvested only at the end of the operational period once we have reached the full volume either fully or partially so we once it has reached the volume I can remove the cell cultures and then I can restart so repeated fed batch till the cells remain fully viable and productive so we can keep on repeating it so one of the more feed streams but no affluent we can have more than one feed Stream So as I said if more than one carbon sources are required or if more than one nutrient is required I can have that and if the nutrients are my rate limiting I can change the flate I can control them so fed batch at least provides me some form of control so if in your work if you require control for your product formation fed batch would be a very good option the culture volume increases continuously because you keep on adding the nutrients right so the culture volume is increasing you’re not exiting out anything you keep on adding so a Fed batch culture is a dynamic operation the concentration of the limiting nutrients in the culture can be manipulated and the neutrient conent sorry Neutron concentration profile can remain at a constant level or to follow a predetermined profile so either you keep it constant so let’s say if this is your reactor you keep the nutrient concentration or the flow rate constant or you have put it as such that okay first half an hour this would be the flow rate the second half half an hour there will be another type of flow like the flow rate will increase or decrease depending upon what you’re trying to do sometimes if you give less substrate to your host or to the micros they might be developing a different type of metabolites right because metabolic pathways will change they will try to adjust themselves a batch mode is used to end the fermentations once it has reached that 0 % volume close everything you run it as a batch that is what it is used it ends as a batch so manipulation of one or more feed rates uh a mean of regulating the nutrient concentration controlling the key reaction rates uh what compounds should be fed and how they should be fed or how they should be added those are the controls that we have this benefits When You’re let’s say if if there is a cell as I was explaining at that time if there is a cell that can actually use two three different types of carbon sources and depending upon the type of carbon Source it can develop different types of products and your intention is to get all those products so let’s say if I okay help me out here if my intention is to generate natural uh natural color natural color with a different product also like both of them have to be done at the same time with the host whichever host I’m choosing for the natural color the host prefers a specific carbon Source or if I have the carbon Source if it is less in concentration it it tends to go and produce only the product a if I have more of my uh substrate it produces product a but it also produces my natural color so if my intent is to get more of my natural color what will I do I’ll keep on in like I’ll keep my flu rate high right I’ll provide it more nutrient if it was other way around I’ll keep my nutrient fluorate low so that only my natural is produced more so those type of controls are possible in a Fed batch reactor it’s just an example okay so you can maximize the cell formation rate for a constant cell Mass yield the subsid concentration maintained at a value that maximizes the specific growth rate now the MU Max is the max at the the concentration at which we can reach if I can keep on changing I can keep on maintaining my muax or I’ll keep on increasing it it will always be in increasing order once it has not reached the plateau the feed rate regulated to hold the substate concentration constant at the desired value until the reactor is full once the ferment is full it is run as a batch mode the advantages of the FED batch intermittent feeding of substrate and maintains low substrate concentration that can help in substrate inhibition if my substrate is the inhibiting factor I’ll maintain a lower substrate concentration so no inhibition I can get higher cell density in fed batch as compared to batch uh there is this catabolite repression uh example would be a penicillin fermentation process uh if you are changing the concentration of your subate it might lead in the in in the penicil fermentation the presence of the glucose basically lead to uh catabolite repression and the same thing happens with respect to the bakery eats fermentation if too much glucose is available ethanol generation can take place because of the glucose phosphorilation so that is considered as a crafty effect so if you are able to control your flow of your substrate in the fermenter you’re going to avoid these two types of issues so if you’re are going for penicillin fermentation what should be the reactor that you’re going to choose or the cultivation mode that you’re going to choose fed batch because that gives you the control right to avoid catabolite repression if you’re going for baker’s yeast fermentation fed batch auxotrophic mutants or expressions of control genes uh inducers and repressors so let’s say if you if you want to have that mutated uh versions of uh no not mut if there are Gene selective gen that needs to be activated or metabolic pathways that needs to be activated in the ecoli or any of the microbes that you’re putting into the FED batch by changing the repressors or or the the promoters that you’re going to send through as part of your nutrient you can change the expression so extension of the operating time supplement of water lost by evaporation and decreasing viscosity of the cultured broth comes now it come now comes the whole design equation and this would be the main one so for the FED batch now the whole equation will change if you remember we had in minus out plus generation minus consumption is equal to accumulation right so in this case again your DX by DTV is is your accumulation term you have an N this time let’s say so there is an in for the feed and there is a in for the substrate so if I’m only looking looking at the cell Mass concentration I’m looking at FX KN so that’s my feed in anything out nothing out def definitely there is a generat which is your V mu net X this is a simple mass balance okay this is a very simple mass balance so since DX by DT can be written as because the volume is not constant in this case in the case of the batch reactor the volume was constant in the case of fed batch the volume is not constant it’s changing by the time you’re adding your substrate so that’s why you will be differentiating that also and that is the reason why you have this V DX by DT and X DV by DT because you’re changing the volume with respect to time with respect to time then your FX not uh plus v muxx is equal to V DX by DT plus X DV by DT in the end the equation sorry I’ll keep on repeating this is the equation that is the most important your D is f over V now your D here is dilution rate f is your feed V is your total volume mu is f/ V so F and V is V plus ft the same equation here and the equation for the munet is given as mon not growth Model A different model of that application what is St State when things don’t change with respect to time right what is quasi steady state when T tends to zero when T tends to zero delt tends to zero explain it in lame and way to me instantaneous uh instantaneous yeah when the change is very very small so let’s say in a Fed batch your cell growth will continue till the time you have your substrate right the the time it is about to finish you start the tap you add the substrate so your cell Mass again starts growing up is it in a Cy steady state it is in a costy steady state scenario if but that is not the the reasoning of it to be in a costy stady state scenario your mu Max is pretty much equal to your dilution rate that is what happens in a CI stady state in this case it will all it will be almost equal to your dilution rate so muum net is equal to d right this is what happens in a CI steady state that’s the reason I asked now the total biomass the final equation that we get in terms of if you remember we had that xal to x e to^ minus Mt in case of the batch same equation here would be XT is XT KN or YX by s s f into T So balance on rate limiting substrate the DS by DT is FS not mu XT by YX by S at c c steady state essentially all the substrate is consumed so no significant level of substrate can accumulate hence as CI stady state is reaches all the subset is consumed more subsets comes in no accumulation then the new thing starts so no significant level of substate can accumulate hence FS not is equal to MXT by YX by S this is an important parameter here the most important equation would be this and this the product formation in terms of the FED batch reactor the final equation that if you do the mass balance for the product the final equation that we’ll end up with would be the P equal to p z v ided by V ft QX qpx QP you remember right 1X X DP by DT the specific product growth rate let’s solve this question very quickly it’s a very simple one so what you have is in a batch culture operating with intermittent addition of glucose solution values of the following parameters are given at time t equal to 2 hours so your time is given as 2 hours so when the system is at quy steady state so at Cy steady state so at quity steady state so V is given as 1,000 ml s not is given to you the ks value is given to you X not at time T is also given to you as uh sorry X not s not is given f is given DV by DT which is 200 mL per hour mu M which is your mu Max is given as 0.3 per hour y of M x/ S is given 0.5 G dry weight cells per gram glucose you need to determine V now which equation will you use for the V do you see any equations here your V is V + ft right your V is V plus ft your V is already given as 600 ml you can estimate right V is given f is given T is given you can estimate your V not to be 600 mL and your D the dilution is f/ V which will come around to be 0.2 per hour now for your second part your s is KSD by mu M minus D why where am I getting this equation from s is given as how much what is your KS KS is given to you 0.1 D is already known muum is known your s will come out to be 2 G per liter so you have your s you have your D how do you estimate your XT see it is asking you to find s x s XT and P at Cy steady state if QP is given us 0.2 G and a product per gram and the cells per hour is p not is zero there is no product at time t equal to zero so I have found my S I have found my I need to find my XT now XT from the equation would be XT + YX by s s ft I’m given by YX by S so XT + f YX by s s not t this will give me a value of 50 g similarly I have found my XT XS the P I’ll be using the equation here this one the moment I put all the values because I do have the F value I do have the FN value I have the Q P value I have the XM value do I have the XM value this is how you estimate the M value so your p with equation would come around to be 16 G per liter you don’t have to understand how we solved this sorry what this question question was sorry you do need to understand how we solved it by just understanding what were the equations we used but the most important part that you you should understand from this whole lecture today is what is the main difference between the batch and the FED batch what is the importance of the FED batch what are the benefits that you get out of fed batch and what are the benefits and advantages you get out of batch because depending on that plus depending upon what you’re trying to grow you would be able to design your own process once you get into your case studies okay we’ll end the lecture today here and uh we’ll meet tomorrow again for heat and mass transfer and the scaleup those are another set of equations and equations and equations see you all tomorrow thank you how do you find the XM