Day 4 | GIAN Course on “PRECISION FERMENTATION FOR SUSTAINABLE MANUFACTURING OF BIO-ACTIVES AND …

good morning everyone so yesterday we did discuss about the batch design and the FED batch design do you have any questions with respect to that no okay so in today’s session we are going to discuss about the Heat and mass transfer in a bioreactor and we are also going to discuss about the scale up of Biore reactors because you always start small at lab scale then you increase the volume and then slowly you move it on to your large scale for Mentor or the industrial skill for Mentor so we’ll start our conversation today with uh Mass transfer in bi reactors but before that I wanted to set up the base with respect to some of the basics of heat and mass transfer okay now let’s start with heat transfer I’m hoping all of you have had courses in heat transfer how many modes of heat transfer exist there are three modes so you have your conduction convection and radiation radiation is do you think sorry so do you think radiation would be applicable in bi reactors not to a greater extent the most important would be the conductions and the convection so conduction we do have the equation which is your fixed law where your K is your conductivity the a is your cross sectional area right so this is your cross sectional area and then you have your temperature gradient DT by DX in in case of your convection it is h a delta T where your H is your convective heat transfer coefficient the notation might change from textbook to textbook but H is the most common notification that I have always seen so H is your convective heat transfer coefficient the area in case of convection is cross-sectional or the surface it’s a surface area and again the temperature will be the temperature difference now keeping these These are the the most basic right and after this we get into the governing equation where you have your Inus out plus generation is equal to accumulation right so the accumulation term for the uh is always given as DT by DX is it accumulation is with respect to time right so that’s why when a steady state scenario you don’t consider the accumulation because there is no change in temperature with respect to time in practical sense do we use steady state scenarios or non-steady state scenarios or unsteady State scenarios it’s mostly unsteady State because the temperature is going to change with respect to time even in bioreactors when you’re going to work with the bioreactors what are the source of heat in the bioreactor the organisms are one source of heat right because as they are multiplying as they are going to grow their metabolic engineering sorry metabolic mechanism is working that is going to generate some amount of heat right so that is your generation that gets added in the Govern equation if you remember for your heat transfer what else is there with respect to your heat what adds the heat mixing right vegitation that will add a little bit of heat do you think the amount the amount of heat that is added through the biochemical process that mean the growth of the microorganism or through agitation is it very significant the delta T is that very significant in uh in case of a bi reactor it’s not very significant you’re right it’s not very very significant but it is still it still exist even a small variation in temperature can ruin your whole by reaction so it is very very important to understand how the reaction uh is moving forward how much of heat is generated from the biochemical process that means from the cell growth how much heat is being added through the agitation and how much heat you are adding to the system heat from external sources is most mostly used to maintain the temperature the optimum temperature that is required for the cell growth or for the fermentation process and it’s also used for sterilization so you can use the steam sterilization to clean your bi reactor at one point of time right so even that’s one way of transferring the heat but that is not when you have your material inside you don’t put the steam because otherwise you’re going to kill all your microbes so that’s the basics of heat transfer sorry before I move on to the mass transfer which of the mode of heat transfer is prominent in a bioreactor convection or conduction convection right so You’ be dealing with surface area majority of the times you’ll be dealing with surface area you’re going to see equations such as u a DT where your U is universal heat transfer coefficient a is your surface area of the inside of your Biore reactor and the DT would be the temperature gradient similarly you have mass transfer so what are the different modes of mass transfer diffusion and have you heard of the term dispersion dispersion is when you have I call it forced diffusion and then you have your convection so you have your diffusion which is your d d is the alphabet which is used to denote diffusivity so da a DC by DX but it’s always like D A to B DC by DX where your X is the distance that it has diffused the DC is a change in the concentration or the concentration difference a is your area but generally when we are working with mass we always work with flux so when we say flux is per unit area we go for that so that’s why you will see D DC by DX because a would have gone onto side of the flow to give you the flux so in case of heat let’s say it’s minus k a DT by DX Q by a Q dot is your flux heat flow per unit area is going to be your flux so mass flow per unit area is going to give you your flux so when we are working with Biore reactors we are working with flux the dispersion is given as e even that equation looks the same so e a DC by DX or E DC by DX this would be for your dispersion this would be for your diffusion the negative sign is going to say from higher concentration to the lower concentration for the heat the negative sign is from higher temperature to the lower temperature the other is h m a DC or Delta C where your hm is your convective mass transfer coefficient now for this h on your conduct on your heat transfer side that is your convective heat transfer coefficient the the HM which is convective mass transfer coefficient if I do provide you that information if I provide you with the HM value or the H value you’re okay to solve any type of scenario with respect to convection if you are not provided with the H value how do you estimate it you use equations for nles number right which is a function of renold’s number and panal number same thing in Mass transfer nles number is called as sharewood number which is a function of renold’s number and Schmid number this is the most important concept that you would be applying in heat and mass transfer in by reactor your ral’s number the ral’s number is given as U which is your velocity D that would be the distance right Row the density divided by the viscosity right now U the velocity of what use the velocity of what of the fluid right so U is the velocity of the fluid D is a distance at which you are trying to estimate your mass transfer or heat transfer so or the sorry the type of flow that you have whether it is going to be what sort of flows can R number tell you whether it is laminar or turbulent what what sort of float do you assume will be existing inside a Biore reactor you have your agitator moving so you’re going to have mostly turbulent the regular standing fan that we keep in summer in front of our face it’s like sturing air right is basically generating turbulence and it’s trying to push that turbulence towards you you have so that’s why it’s a because a turbulent flow you will feel Air at one point of time and then suddenly it goes away and then again you feel a gust of air on your face that’s a turbulency because it’s coming in bursts to you D sorry the road the density is also very important that’s also of the fluid the V is also of the fluid so fluid properties are important the fluid properties are very very important if you are trying to estimate your renals number now the renal number are required to estimate your sharewood number or your nles number which are part of your heat and mass transfer respectively sorry sharewood number is mass transfer nles number is heat transfer you have to have an understanding of the fluid properties so when we talk about the fluid properties what are those properties the viscosity the density you need to know all of those parameters before you go and start doing the calculations right so estimating those is also an important parameter in terms of assessing the values for your heat and mass transfer the other part is your planet number and Schmid number your planet number depends on what if you have the mic if you can answer in that that would be great the panel number would be dependent on is it is it for the fluid is it based for on the geometry okay do you remember a term called as Alpha which is your diffusivity what is the equation for Alpha I’m so sorry for putting you at the spot but do you remember you have your k k over row CP so your density of the material the CP is your heat capacity of the material K is your conductivity of the material does this Alpha has to do anything with PR the planet number you have your phone you can check what is the equation for Planet number if I remember correctly is p r a n D LT parel number V row VY row that is ktic is kemetic viscosity by density where is Alpha now where in the numerator or denominator what about number so your parate number is Chic viscosity divided by diffusivity heat what it spint number okay so what’s the value for spint number see I learned while teaching my students undergrads there that the best way because you all are so inclined towards your own technology to make you do the work that I’m supposed to do that is finding it so give me the value of of speci number V by D so V by dtic viscosity by diffusivity diffusivity diffusivity right is the density term there at all if Will convert it is the density if you will convert mu upon r r d okay kynetic viscosity will be converted to Dynamic this m upon Row D right so that’s your Smith number now the row the D the D is the diffusivity is a property of the fluid of the material through which your mass so whatever material sorry if a is diffusing right it is diffusing through with it is a fluid or any material is a property of the material right we talk about the diffusivity of a membrane it’s a property of the of the material so the material property comes into picture when we are working with SMI number and Planet number renal number provides you the fluid properties that’s why these two are very very important and we are going to use them when we get into the Heat and mass transfer proper discussion about of the Heat and mass transfer in the bi reactor so I hope this basic refresh of your heat and mass transfer concept are helpful later on okay so Mass transfer in bioreactor so mass is transferred under the influence of concentration gradient Mass can only move from a higher concentration region to a lower concentration region in case of a bioreactor gas liquid mass transfer is extremely important because in a bioreactor you’re looking at Mass transfer specifically the one that you are mostly interested about is your oxygen which you are sparging so when you’re sparging Air at the bottom of the bio reactor it is going to throw bubbles in the bioreactor in the medium and the mass transfer that means oxygen coming out from the gas molecule where it is in higher concentration into the medium where it is in lower concentration and then to the cells that transfers that’s it’s a two- stage transfer so you have your gas bubble so let’s say these are your cells here so your oxygen is going to come out of the gas bubble into the medium and then from The Medium it is going to go to your cells now if it is a small single cell the mass transfer would be different if it is a cluster of cells the mass transfer is going to be different so the type of host that you use the way they grow or the way you are mixing it how homogeneous your medium is inside is going to determine how much of mass transfer of oxygen is going to take place so this oxygen coming out of the gas bubble if you remember there were two terms that we came across yesterday one was OTR and O Ur OTR was oxygen transfer rate right and this was oxygen uptake so OTR is basically your this stage o are is this stage where the oxygen is being uptick by the cells to grow clear okay so in an anerobic process oxygen must first be transferred from gas bulk through a series sorry in an aerobic process not an aerobic an aerobic process you don’t transfer in oxygen you should have stopped me if I made a mistake in an aerobic process oxygen must first be transferred from gas bulk through a series of steps onto the surface of the cell this is your step so you have spared the oxygen so you have your reactor you have your Sparger it’s giving out the bubbles here as the bubbles are moving up in the medium medium they’re going to release their oxygen into the medium the solubility of oxygen within broth is actually very very poor we did talk about that because you’re trying to dissolve oxygen in the liquid that’s where the concept of Henry’s constant comes into picture now temperature plays a very important role in determining how much of gas is dissolved within a liquid the pressure also plays a very important control in determining how much of gas is dissolved in a liquid correct now let’s have a very quick conversation about this Henry’s constant what would be the best example in your day-to-day life of hen’s constant soda can you have carbon dioxide dissolve D in the soda or like in the liquid right so when you open it you hear that noise right so that’s like it’s under pressure within that pressure the the carbon dioxide is dissolved but the moment you open you will see that FY bubbles coming up so that means that your carbon dioxide is coming up the other would be uh we call it diverse Bend so if you do a deep sea diving you go pretty quick inside the pressure of the water is pretty high on you the nitrogen gets dissolved in your blood and if you come up very very quickly that nitrogen the pressure has been released on the body that nitrogen will become bubble and will stop the blood flow it can kill you okay so this is what happens the other part would be when you are moving from uh let’s say from an it you’re going all the way to Himalayas when you go up the mountain you feel that oxygen is less in the air is it that the concentration of oxygen in the air is Al itself less or less oxygen is dissolving in your blood it’s less oxygen which is dissolving the blood will the concentration of oxygen change from ground surface onto the mountain top or the air will have constant concentration of oxygen throughout that’s the question that I ask my undergraduate students every time it’s a homework think about it it it’s a pretty good uh it’s an interesting topic is basically the amount of oxygen that is dissolving in your blood is less when you go on a higher altitude because of the pressure difference so enhancement of gas liquid mass transfer during aerobic fermentation is priority if you’re working on aerobic fermentation because oxygen is required for your cells to grow and to flourish you have to make sure that you are able to transfer the oxygen in appropriate amount that is required by the micro orms for their growth if you’re not able to provide that you’re going to see sell that okay so you’re going to see sell that if there is the oxygen is not about sorry so whenever the light goes off it breaks the continuity of the thought oxygen transfer is very very important in BIO process you have to make sure that you are providing appropriate amount of oxygen so that means the am the OU cannot be less than OTR can it be oh sorry o cannot be more than OT so oxygen uptake rate is higher OTR is less you won’t have any Oxygen for the uptake anymore right it’s the other way around I will say things which will be other way around just to check whether you’re listening to me or not and if you not saying yes that means you are just agreeing to everything I am saying think use your brain okay now mode of mass transfer which is common in a bio reactor is molecular diffusion so movement of molecules under the influence of concentration gradient is what is used for with respect to the diffusion now continuous diffusion that means supplying material to the high concentration region and removing it from the low concentration region is possible right if you continuously provide the if you maintain that concentration gradient you can maintain continuous diffusion process so if you maintain that OTR at a very high level your Ur will remain if you don’t have that OTR you cannot have your o you won’t have any Oxygen for to be taken by the by the microbes now continuous diffusion is exploited for Mass transfer operations in bioreactor the fixed law of diffusion which we already discussed is basically used in terms of estimating the amount of mass that has been transferred so you do need to know the diffusivity the concentration gradient and the distance through which it is diffusing through now mixing as we have already discussed in the very first slide when we were having conversation about the mass transfer is a very very important uh unit operation you can say in a bi reactor so mixing that means turbulence INF fluids produce bulk mixing to a Max up to the smallest edies formation so when you are having your agitation taking place you’re going to have small edies which will get generated you know what the edies are Ed is like a Vortex right they are very small vortexes which will form and those vortexes allow a very nice mixing ground it’s like have you ever seen a movie called twister no if you get a chance there is an new movie called twister watch it it’s really interesting or have you seen a tornado on TV it’s moving very very fast so it’s actually mixing everything just think about that at a very microscopic scale taking place inside the Biore reactor in an Eddie if you are in that particular Eddie what is going to happen your mixing is pretty quick so oxygen and the cell if the if you have the gas bubble up there and you have the cell also up there the oxygen transfer would be faster so within the small cies flow is largely streamline so that the further mixing must occur for diffusion of fluid components mixing on a molecular scale therefore completely relies on diffusion as the final step in the mixing process yes edes are there but diffusion is the main parameter through which the mass is transferred now Mass transfer between phases occurs often in biopress so in the case of your your gas bubble moving through let’s say a liquid media if I give you a column and I’m sparging oxygen from the bottom let’s say so the bubbles will move up right and this particular column is filled with water the bubble is going to move up from bottom it is going to move up what will happen to the size of the bubble will it increase decrease if decrease y if increase y if you have a fish tank at home or or an aquarium close your eyes remember how how the the bubble is moving up in the aquarium it remains in the same size or it increases in size or decreases in size it increases in size as it goes up right why because of pressure keeps on decreasing right from the depth and by the moment it has touched up it has become bigger now oxygen transform from gas bubbles to the fermentation broth will also follow the same principle that’s why when you’re sparging oxygen the design of the Sparger plays a very very important role you don’t want smaller or bigger bubble like you don’t want bigger bubbles to go through the medium you want very small bubbles because very small bubbles can be uniformly distributed and homogeneously mixed so you have to make sure that when it is moving up it is in a very small shape like the size of the B is very very small so design of the sparer itself is a very important parameter in the design of a bioreactor the other part would be what are the Restriction what would what are the phases that the oxygen has to go through so oxygen inside the bubble is in the gas phase now when you have a bubble it is inside a liquid media you have a film that generates at the surface right so the oxygen has to come out from the gas bubble go through this particular film into the and this film is because of the liquid media surface tension right the bubbles is going through the liquid medium so it’s going to be have a sort of like a restrictive film across it which will get generated and that particular film has to be crossed before the oxygen goes into the medium it’s a very important parameter to look at similar type of film does exist on the cell structure itself so you have now two films through which the oxygen has to go through the movement of oxygen in a bio reactor is also called as a two film Theory because of these two films that exist we’ll be discussing that in detail but I’m just giving you a bit of a basic idea to it so oxygen transfer from gas bubbles to the fermentation broth takes place uh goes through the the two phases so it is going from the gas bubbles to the fermentation broth Now product recovery from the Aquas to organic liquid the nutrient transfer from liquid medium into the cell is also going through phases right there are two phases from liquid to to the solid the cell is considered as a solid medium not as a liquid medium the fluid velocity near the phase interface is significantly decreased diff Fusion becomes crucial for Mass transfer across the phase interface why here comes the concept of convection so let’s say if I’m going back to convection ction for a moment if you have a flat plate now I’m going to talk about convection from the heat transfer perspective first then we’ll talk about the mass transfer perspec perspective so you have a flat plate the air is going to flow over the surface of this flat plate it is going to flow in layers right that will lead to formation of a velocity boundary layer right and a thermal boundary layer depending upon which particular whether the plate or the fluid has a higher temperature so the thermal boundary layer will exist and a velocity boundary layer will exist now what is this boundary layer if you remember correctly it used to be the 99.9% of the distance at which the fluid will have the same temperature as the surrounding fluid or the so if this is at higher temperature let’s say 100° C this one is at 20 C what is the temperature at the surface the plate is at 100° C the fluid which is moving over the surface of the plate is at 20° C what is the temperature at the surface 100 20 60 50 40 80 what is the temperature at the surface I given you so many options now we call it a film temperature at the surface the velocity of the layer of the air at the surface is zero what is the mode of heat transfer at the surface of that layer with the surface of this or of the heated surface what is the mode of heat transfer then conduction same concept in convection the fluid is flowing over the surface at the surface the velocity of the fluid is zero what would be the mode of convection then sorry what would be the mode of mass transfer diffusion as the bubble is moving up it’s moving up at the surface there is that layer through which the diffusion is going to take place the mode of is convective mass transfer but it has diffus diffusion at the interface the transfer is called convective Mass transfer it has diffusion at the interface just the way you have conduction at the interface now to answer that question what would be the temperature is the film temperature the film temperature is an average of your surface temperature and the fluid temperature so that means 60 so from 60 50° C you’re going to reach all the way to 20° C so with the distance at which the fluid temperature at the surface which is 60 it will keep on lowering down and it will reach the fluid temperature of 20° C that distance is called your thermal boundary layer the velocity of the fluid is at 1 m/s at the surface it is 0 m/s the distance at which the fluid layer goes back to 1 m/s is your velocity boundary layer same concept is followed in Mass transfer at the surface it’s always a surface phenomena it’s the most interesting thing generally you you have studied this uh concept of boundary conditions have you studied boundary conditions so let’s say I give you a membrane or like a a thick slab of membranes you have diffusion taking place right so it’s minus D DC by DX inside at the surface you have air blowing so here you’ll have hm a DC right or Delta C the flux Remains the Same Mass conservation so your minus D DC by DX is equal to h a Delta C or H ma a Delta C at the surface what is the boundary condition at xal to 0 minus D DC by DX is equal to hm a Delta C that’s what your boundary condition is so now coming back into your bio Reactor with the same concept what would be the boundary condition at the surface of the gas bubble is diffusion and if it is moving with a velocity it would be convection you’re getting my point if it is stationary diffusion only the flux has to be conserved Mass has to be conserved right the mass flux has to be conserved now the theories of interfase mass transfer there is a two film Theory and a penetration Theory the two film Theory the entire resistance to transfer is contained in two fous fous films on either side of the interface in which transfer occurs by molecular diffusion we will look at what that film Theory looks like later on so the KL which is your diffusivity constant or parameter would be given by D by Z where D is the diffusivity and the Z is the film thickness the mass transfer coefficient is the KL you would see there is a term called as KL a which is used in estimating your oxygen uptake in a bi reactor the a is your area D is your diffusivity the Z is a distance that it has moved it requires for the diffusion that combined gives you the KL and the a gives you the area the penetration theory on the other hand assumes turbulent edes travel from the bulk of the phase to the interface where they remain for a constant exposure time T the solute is assumed to penetrate into the Eddie during its stay at the interface by diffusion so the k for that particular case is given by this equation don’t get bothered by the equations that much but understand the conceptual part at the interface what is your boundary condition that matters the most if there is a thick layer at the interface the diffusion of the gas has has to come from inside which is at highest concentration it is going to diffuse through that thick layer and into the medium and then from The Medium it is going to go into the cells that thick layer will have its own resistance similar to resistances that you see in your mass transfer you remember right minus da a DC by DX the resistance is given by D is DX by minus da l/ da this is your resistance R diffusion resistance to diffusion remember this we also use a term called as K which is your distribution coefficient when we are working with mass transfer which has to be multiplied to your diffusivity for a specific product or a specific uh component which is diffusing through the membrane or anything like that this is mostly used when we are working with the membrane now the film Theory so this is your phase boundary you have your gas phase and then you have your liquid phase so the gas phase film resistance and then you have your liquid phase film resistance so the rate of mass transfer is direct ly proportional to the driving force for transfer which would be the concentration gradient because that’s the only driving force if the concentration gradient does not exist mass does not move and the available area for the transfer process to take place transfer rate is directly proportional to transfer area the transfer rate is also directly proportional to the driving force so d force is the concentration gradient and the a the area this K is your proportionality coefficient in case of heat transfer we generally use the K as conductivity but in this case we are not going to use it as conductivity we are going to call it as a mass transfer coefficient I have used the same as hm before but I said it is convective Mass Mass transfer coefficient but here we are using it as a k because diffusive values it the K is already estimating d by Z right so it already has the parameter of diffusivity in it so I’m not representing it as D I’m representing it as K this is because of the film Theory the K depends on the boundary layer the geometry the flow and the fluid properties why ral’s number Schmid number are involved right so number is going to give you the require all the fluid properties if the material is very viscous what would be the mass transfer will it be less will it be more it would be less diffusion would be less right if it is yeah flu viscosity is the most important fluid property other would be the speed at which the fluid is Flowing that will determine the rate of mass transfer the value of K what else can be taken into account the temperature the density density will Define not the temperature the density will Define change in temperature will change the density but density is the the parameter that will defin so density viscosity right all of these will come into the fluid properties that we are looking at so the gas liquid mass transfer the rate of mass transfer of a component a through the gas boundary layer for that means your gas B B layer is kga a c a minus C AI that this layer the distance the the concentration gradient similarly for the liquid side it would be KL c a i minus Cal so the thing is there’s always a resistance at the boundary at the phas right so when you have your concentration coming in it is at a lower concentration at the surface then the concentration that was there in the bulk then from there when it goes into another phase there’s a drop let’s say if I use a membrane there will be a drop in the concentration within the membrane if the the concentration on the left hand side of the material is pretty high than on the right hand side so there’s a drop because that would be determined by the diffusivity parameter permeability of that membrane how much of the material can be diffused through at a given time if it can only give let’s say if 10 molecule comes on the one side of the membrane so if this is a membrane 10 molecules have come from the bulk and they’re like hey I I need to go through I need to go through but this particular membrane can only allow two at a time or three at a time it will only allow three so there would be dip in the concentration from the surface within it at this surface you’ll have three now this three is still higher than what is present on the right hand side so that that dip is what you are seeing here got the concept in the most Len way I’m trying to explain there is a dip because of how much can go through at a given time that’s the resistance of the film that will determine now assume equilibrium exist at the interfaces cagi and CLI can be related equilibrium concentration in the gas phase is a linear function of the liquid concentration the steady state at interface there is no accumulation of component a so there is the K Star Distribution coefficient is not existing there the component a transported through Phase 1 must be transported through Phase 2 these are the assumptions that you have to make so your n a is equal to n l is equal to n a the total flux the mass is conserved or the flux is conser conserved at all times now oxygen Mass transfer so when you multiply equation 2 by m so your equation two is here on the liquid side and rearrange you get this particular equation here now dividing the equation by m we’ll provide you further so the overall the most important equation that you have to look into would be this or sorry this where the KL is overall Mass transfer coefficient so KL is c l star minus c a l this is the equation that determines the mass transfer you have your flux isal sorry you have your uh mass flow on one side you have your KL which is your overall Mass transfer coefficient a and the CL star minus CL where exactly will you have the Cal star at the interface now this this particular uh concept that is there so liquid phase Mass transfer resistant dominates and the kga is much larger than the klaa so liquid side Mass transfer resistance is more as compared to the gas side resistance which is an obvious thing liquid side will have a higher resistance as compared to the gas side so the kga would be higher in terms of the to maintain the flow so the rate of oxygen transfer from gas to liquid is of prime importance in an aerobic fermentation process because without oxygen the micros are not going to grow but understanding how much oxygen is required the oxygen demand of the microbes will be able you will provide you with the information which will be which will make you capable to estimate the flow rate that you will set up so that your oxygen transfer rate is always higher than the O oxygen uptake rate can you so let’s say if if I give you a bi reactor you are purging oxygen into it I ask you to stop it what will happen what is the worst case that will happen eventually the cells will die right but what if you reduce it only for a certain period of time and then again increase it will that impact the metabolic pathway of the the cells or how what type of product is being formed by the cells because for survival the cells will change their Machinery immediately and then again when they get it they are going to go into a different mode so that switching of the mode takes a bit of time so there is always some uh products which gets generated during that period so it’s always make you have to always make sure for an aerobic fermentation system you’re maintaining the oxygen demand that is required by the fermentation system other otherwise what the product that you are expecting the quality of the product that you’re expecting might not be there at high cell densities oxygen is quickly consumed in an aerobic culture and must be constantly replaced by sparging the low solubility of oxygen which is Max of 7 to 8 PPM at ambient condition now if you ask me what an ambient condition would be I will say 25° C that’s what an ambient condition is for us what would would the ambient condition in a bi reactor 25° C the ambient condition is ambient condition you don’t change it so the low solubility of oxygen limits the concentration difference the CL R by minus cl to very small value at all times what it means is like once the oxygen has come out of the bubble so now once the oxygen has come out of the gas bubble it has to be in the liquid medium but if it does not dissolve if it does not solubilize in the liquid medium it cannot be transported now the the the solubility is less the concentration in the liquid medium available of concentration of oxygen in the liquid medium is less as compared to that present in the gas that might be available for the cells to obtain so that means your oxygen concentration in the liquid medium is lower that’s an obvious thing as compared to what is there in the gas but because of lower solubility it goes even lower the then what is present like you have added let’s say 10 but only five or six would be available for the cells because the solubility is less now solubility is uh depends on the temperature solubility depends on the pressure solubility depends on the fluid properties so all of those parameters has to be taken into account when you’re trying to Define how much oxygen would get solubilized what can you do in that case if you want to improve the solubility of your fermentation system what will you do you can change the type of medium that you’re using that might be able to dissolve more oxygen in it or you can provide some sort of uh let’s say additives that will help dissolve oxygen but all of that will impact your fermentation process so you have to be very careful in choosing what you can do to maintain this oxygen demand so designing of the fermentors for aerobic operation takes these factors into account to provide Optimum Mass transfer conditions now factors affecting cellular oxygen demand now the rate of oxygen consumptions by cell determines the rate of oxygen from transfer from the gas to broth the OTR is dependent on o how much oxygen is being taken away that will determine what the OTR would be that means oxygen coming from the uh from the gas bubbles into the liquid medium now factors that influence this oxygen demand are the type of microbes the host the type of microb that you have chosen for the fermentation process the species some uh microbes require higher oxygen they have higher oxygen demand some don’t have that high as compared to the others the culture growth phase whether it is in the lack phase whether it is in the log phase early log phase or later log phase like in the de acceleration stage or in the stage phase the oxygen demand will change will it not when do you think in the case of the cell growth the oxygen demand is the highest the early growth phase to the the exponential phase as it being the constant phase where you have the constant growth going up your oxygen demand is high you need as it is doubling right at this particular stage when it is starting to e climatize you might not have a very high oxygen demand but the moment it will start dividing the oxygen demand will shoot up and you have to make sure during that period you’re providing appropriate amount of oxygen into your bi reactor and the nature of the carbon Source in the medium the type of source of carbon that you choose will also Define how much oxygen is there is is available because if the if the if the source itself interacts with the oxygen then less oxygen is available okay you have to be very cautious about the choice of your source now factors affecting cellular oxygen demand so in a batch culture the rate of oxygen uptake varies as the concentration of cells increases during the ctiv cultivation period this means that the rate of oxygen uptake is proportional to the number of cell present so as the number of cells will increase the rate of uptake of oxygen will INE increase so in a batch culture the rate of oxygen uptake varies as the concentration of cells increases during the cultivation period so as you are growing the bulk of the cell Mass keeps on increasing your oxygen demand will also keep on increasing now the specific oxygen uptake which is your Q2 or the specific oxygen uptake rate increases to a Max maximum value at early log phase and then it decreases gradually at early log phase it will start it increases and then it decreases gradually so you you can see that early loog phase it has increased for a certain period of time and then it has decreased gradually as the cell concentration has increase this is your X this is your Q2 okay and now this is where we were talking about the film theories type right so you have the gas liquid interface and the liquid cell interface so your OTR is basically the oxygen that is coming out from the gas into the liquid that is your oxygen transfer rate and then your o is from the liquid whatever oxygen is being taken away by the cell again the question would be single cells in a cluster things will change because this size of this interface will change question is this so let’s say you have a cluster of cells right now there is this let’s say a thin layer of interface over please excuse my drawing okay please excuse my drawing now this particular cell requires oxygen now the oxygen which is coming in has to pass through the other cells to reach that last cell right that’s why clusters are not good for your bi reactor because the cells will die they cannot get enough oxygen for their survival and that might lead to production of uh products that are of not your interest that is something that you don’t want so you always try to reduce the cluster what is the way that you can reduce the cluster agitation right not this type of agitation a single one you have an agitation rotating at a particular speed question here if I may so let’s say if I give you a bio reactor I have a sparer that is pumping I have set the flate of oxygen or the flow rate of the gas through the sparer let’s say okay volumetric fluorate would be like let’s say 50 ml per liter like 50 mL of oxygen per liter I’m just throwing it in will the speed of the agitate impact how much oxygen will get dissolved in the water if just the water is here if I take a dissolve oxygen probe at one end and put it inside of my in the in the fermentor or in the by reactor and I change the speed of my agitator do you think that will change the amount of oxygen that would be present in the water because it is going to break the bubbles into smaller bubbles diffusion changes smaller bubbles will have smaller interface layer thinner not smaller thinner interface layer so your mass transfer is higher larger the surface area more the catalysis we call it more smaller the bubbles is much better it is the easiest example to that would be um the the Taps that you see these days the old tabs were like just the liquid coming out this uh current time the tabs it gives you that sound sh and it comes out in like bubbles like in streamlines right it’s less water but it’s very stream line so that’s why it reduces the amount of water that you’re using same way you if you streamline if you basically break all the bubbles into very small bubbles it will diffuse very EAS easily in the medium so agitation does impact the OTR oxygen transfer rate it’s a very important parameter in that case and that’s why when you’re trying to think about scale up from a small scale to a larger scale you have to maintain that OTR depending upon because larger the fermentor more the OU are so you have to increase your OTR so what all you have to increase you have to increase your agitation system also and the amount of flow of oxygen the sparer design Everything Will Change that’s why scale up is not an easy process when we looking at the fermentation we think about it’s a small bottle I can go to a bigger bottle it doesn’t it does not replicate just by comparing okay in this small bottle I have 2 L for 2 L I used uh this much of oxygen so how much will I use for 4 L you don’t do that calculation anymore because if you do that your OTR is less you have to now think about what are the other parameters that were impacting my whole thing now the viscosity will change the density will change because I’m going into bigger system you have to take into account all the other parameters to estimate the scale up that’s why understanding Mass transfer is very very important before you understand scaleup any questions no excuse me now now if the qo which is your specific oxygen uptake rate is the oxygen sorry if the qo is oxygen uptake small qo is your specific oxygen uptake rate if the qo is oxygen uptake rate per volume of the broth and qo is your specific oxygen uptake rate the equation is your qo divided by X it has to be with respect to the X to give you your specific oxygen uptake rate so total of oxygen upt rate divided by per unit volume divided by the cell biomass so inherent demand of oxygen depends primary primarily on the biochemical nature of the cell and its nutritional environment now this is also another parameter that has to be taken into account when you’re trying to design a particular bioprocess what it means is that many of times when you’re designing a bioprocess the type of uh sources you use as your medium Source or the uh nutrient Source how is the bacteria going to utilize it it’s like how much water you’re going to drink if you’re eating something very very spicy it will change the amount of water intake for you depending on the type of food that you’re eating right same thing happens with the cells there’s not much of a difference in that sort of concept but the cells biochemical pathway will change and the amount of water demand or sorry oxygen demand will also change depending upon the type of carbon source that they are working with the other aspect would be the environment depending on the temperature outside the the temperature of the medium the cell metabolism might reduce the cell metabolism might increase right because if the temperature is at its Optimum the cell growth was Optimum the moment we cross that Optimum temperature threshold you remember from yesterday’s lecture the moment we cross this Optimum temperature threshold the cell starts to die but it will keep on increasing as I’m increasing the temperature so that means more and more metabolism is taking place more and more cell mass is growing my oxygen demand is higher so the surrounding environmental parameters also impact the amount of oxygen that would be required for the cell to grow you have to take into that take that into account when you’re trying to design your bi reactor again as the level of dissolved oxygen in the BR Falls below a certain point which is your critical oxygen level the specific rate of oxygen uptake is also dependent on the oxygen concentration in the broth so you have to make sure that you have provided enough oxygen beyond the threshold not less than the threshold beyond the Threshold at all times to maintain enough oxygen in the broth the broth that we keep on talking about is the medium you need to maintain enough oxygen in the broth for the cells to grow it’s the same concept again and again you have to maintain the oxygen you have to maintain the oxygen but that maintenance of oxyen depends on the broth parameters like the type of sugar that you are taking the species the cell species that you have inside the what else the properties of the fluid that you are using so to eliminate dissolved oxygen just give me one second oh yeah so to eliminate dissolved oxygen limitations and allow cell metabolism to function at its Optimum the dissolved oxygen concentration at every point in the fermentor must be above the C critical so you don’t want to be below the C critical at any given point as the fermentor is running estimation of the C critical is very very important how will you estimate this C critical just give me an idea on that how will you estimate the C critical value through experimentation you’ll try to see the optimum growth right you’re going to you never go to the fermentor directly you always go to the seed culture you have a small scale fermentation unit first that is what you’re trying to optimize first and then only then you go into the industrial scale fermenter so you try everything there understanding the relationship between all the process parameters with your cell growth and only then you replicate that in a large scale formentor you can estimate through that through experiment ation that’s the only way you can do it the exact value of C critical depends on the organism but under average condition operating conditions usually Falls between 5 to 10% of air saturation so other way to circumvent the circumvent the C critical is pump as much as you can just pump as much as you can but that is going to add to the cost that’s why understanding the SEC iCal is important because when you are working with Precision fermentation you are always giving every parameter at the precise value at the specific value nothing more nothing less you’re trying to optimize your productivity right improve your productivity with the least amount of resources oxygen is not a cheap substrate that is added to your fermentation system so you have have to be very careful in estimating the C critical for cells with relatively high C critical level the task of transferring sufficient oxygen to maintain the concentration on the liquid side the Cal greater than the C critical now what is this C critical C critical is the concentration of oxygen that should be there available for the cells to take right so that means I’m not bothered about how much oxygen is there in the bubble I’m more bothered about how much oxygen is dissolved in the liquid medium at all times so when you are putting your dissolved oxygen probe you’re estimating how much oxygen dissolved in the medium that gives you the idea so you’re trying to maintain that Cal that me the concentration in the liquid medium over the C critical value it’s always more challenging than for the cultures with a low C critical so for cells with relatively high C critical level the task of transferring sufficient oxygen to maintain Cal is always more challenging than for cultures with low cal or C critical why if I have cells which have a very high Ur oxygen uptake rate or the demand is pretty high you have to maintain a pretty high concentration of Cal right why it is a challenging thing fluid properties like how much oxygen can you dissolve there’s a limitation to it right you cannot increase the temperature you cannot change the pressure what do you change you change your agitation you Chang how much oxygen you are pumping in those are the very few parameters that you can actually handle and control you cannot play with the temperature and pressure that much because the moment you change the temperature you’re going to change the growth rate you don’t want to go into the thermal rate uh thermal death rate for uh for your micros right you want to maintain it below that but there are only few parameters that are manageable or can be maintained by you so maintaining any so choosing the right host which will require less and less oxygen and still give you your product might be an ideal choice to reduce the overall cost of production if you’re choosing something which is of higher oxygen demand you have to provide more oxygen at all times and that is going to increase your production cost so a lot of parameters gets affected by this the choice of substrate for the fermentation can also significantly affect your oxygen demand as glucose is generally consumed more rapidly than other sugars or carbon containing substrates rate of oxygen demands are higher when glucose is used now you see the relationship between the substrate and oxygen demand I’m hungry or I get to see gulab jamun I love gulab Jam I’ll eat a lot of it but I need to drink water too you all depends on your choice right when you’re when they are basically consuming glucose that means your metabolic pathway is consuming glucose continuously it is going to need because it it cells don’t say to themselves you know what my tummy is full I’m going to sit now I W I don’t want that extra glucose right now I’m good I’m good no they they will continue dividing they’ll continue consuming it they don’t stop like us they continue consuming it that creates the situation in which your oxygen is being continuously utilized for the growth of your microbes so for that you have to maintain it depending on the choice of the substrate that you have used you might want to change glucose to galactose or fructose something which the micros might not like that much or the the demand of oxygen for the processing of that uh sugar would be less so you have to choose the right carbon source for the growth of your microbes now the step of Transport for the gas uh for of oxygen from the gas bubble to the cell I have already discussed this a little bit let’s look at it so let’s say this is your gas bubble inside this is your gas liquid interface so at the gas liquid interface I said that this is an imaginary or line or imaginary thickness or layer which is present and that oxygen has to cross through this layer and only then it will reach the cells so oxygen molecules must overcome a series of Transport resistances before being utilized by the cell what about this cell or the cell here it might get the oxygen from the other sides but I’m just saying that there are other cells around it’s a cluster it will has to go through now other barriers so you will keep on increasing the resistance for the oxygen transport you’re going to reduce the amount of oxygen that is going to be transported the stagnant medium or the stagnant region is the uh the the the film that we have talked about the two films on the liquid side and on the gas side you you should have heard about this in certain biomaterials porous biomaterials which are used uh to immobilize cell for cell growth or they can be used for as uh as a scaffold for the cells to grow properly and they poor in nature for the nutrients to get transported for the oxygen to get transported quite easily to the microbes those are also utilized these days in the fermentation process to improve the amount of oxygen that is transported into the system it’s like uh it’s a very porous media and the cells are growing on the surface of these pores or on the surface and then because it’s Hollow and porous the liquid medium with the nutrient and the oxygen can go through the distance that they have to now move for the oxygen transport is less so that reduces the amount of resistance it’s it’s like imagine this as a pen this has a hollow cylinder but it’s a very small section of it so there is a small hole in the center okay and this whole structure the cylinder part is porous in nature the cells will be growing or immobilized on on the surface am I correct now the nutrient can flow through the hole in the center on the like axially and the transport of nutrients can happen through the pores inside so it’s a porous media the cells are growing on the surface that is sometimes used for cell culture basically we use that for cell culture but those type of materials are or the beads we call them are generally used in the fermentation system even nanomaterials are also being used in the fermentation system okay this is just to improve the overall fermentation efficiency I want more cell Mass but I want to reduce the overall cost so if I can do that why not okay so steps for the oxygen transport from gas bubble to the cell so transfer through the bulk gas phase in the bubble is relatively fast the gas liquid interface itself contr attributes negligible resistance the gas liquid interface is negligible resistance the liquid film around is a major resistance for the transport so you have your gas bubble it has released the oxygen into the liquid and then it goes towards the cell that’s the major hindrance or the resistance in a well mixed fermentor concentration gradients in the bulk liquid are minimized and the mass transfer resistance in this region is small so if you are well mixed the distance imagine it this way the cell cluster is here the distance of the bubble is not too far it might be available that the distance the resistance is less that it has to go through single cells are much smaller than the gas bubbles hence the liquid film surrounding each cell is much thinner than the around the bubbles and it effects on mass transfer can be neglected completely if the cells from large clums if they form large clums liquid film resistance can be significant now the other steps for oxygen transport from gas bubble to the cell is resistance at the cell liquid interface is generally neglected resistance at the cell liquid interface is generally neglected not the liquid so the resistance at at the cell and the liquid interface if this is your cell the cell and the liquid interface is generally neglected so when the cells are in clumps intra particle resistance is likely to be significant as oxygen has to diffuse through solid pellets like one cell to another cell to reach the interior cells the magnitude of this resistance depends on the size of the clumps larger the size more the resistance smaller the size less the resistance intercellular oxygen transfer resistance is negligible because of small distance involved we don’t consider the resistance at that time when we are trying to estimate the the movement of oxygen because it’s not too like what is the size of a micro microns so it’s not too much of a distance that the oxygen has to get transferred that’s the reason so the mass balance for oxygen at a steady state will involve the k that means CL star minus CL the CL is a concentration at the liquid side CL star is on the interface where your A a is a by V A is your interfacial area interfacial transfer area and the V is the broth volume the K is your volumetric mass uh transfer coefficient this equation above can be used to predict the effect of cl to changes in the mass trans transfer operating conditions for example if K is increased by raising the stir speed to reduce the thickness of the boundary layer around the bubbles the dissolved oxygen concentration that is clal will rise for equilibrium if you can reduce the film resistance thickness you will have more CL more oxygen in the liquid similarly if the rate of oxygen consumption by the cell accelerates while K is unaffected the Cal will decrease that means the concentration of oxygen on the liquid side will decrease because now uptake has increased so you have to maintain CL whatever you do maintain CL at all cost above the C critical now the maximum cell concentration that can be supported by by the fermentate uh fermented oxygen transfer system is an important parameter to look at so the way we estimate that is for a given set of operating condition the maximum rate of oxygen transfer occurs when the concentration difference driving force that is C star CL star minus CL is highest so higher the concentration gradient more the transfer that is when the concentration of dissolved CL is zero that means the the concentration gradient would be highest when the CL is zero under what condition will the CL be zero when your OT R is equal to O so whatever oxygen comes out from the gas is consumed comes out consumed comes out consumed there is no oxygen in the medium so your C concentration gradient will always be maximum so because of that your mass transfer would be maximum so maximum cell concentration can be supported by the mass transfer function of the reactor and this is the equation that is used so your x max that means the amount of cell the maximum amount of cell that can be generated you if you know the K you know the Cal divide it with Q which is your specific oxygen uptake rate another important parameter is the max is the minimum k a required to maintain Cal over C critical in the fermentor this can be determined as CLA critical is equal to QX / CL star minus C critical this is like to maintain the cells that I have right now what is the minimum K value that I have to have this is the equation that I’m trying to use at that time to maintain what I have like what is required above or how much I can get with the respective Kaa would be the xmax value if I know what my xmax value is and I want to know how much Kaa I need to have to maintain that x max is what I will use the second equations this is to get what xmax I want I can get second is to what K I should have to maintain it now determining the mass transfer coefficient the KL the k and a can be calculated using empirical correlations and experimental me measurements experimental measurements are the most common way of estimating the K value separate determination of KL and a is tedious it is convenient to directly evaluate the product Kaa The combined term Kaa is referred as a volumetric mass transfer coefficient the objective of the ferment design is to maximize oxygen transfer rate with minimum power consumptions necessary to agitate the fluid and also minimum air flow rate you got the drift least amount of air flow least amount of agitation but I still want growth right it sounds like Industries I’ll give you least amount of money but I want the product right the boss will come and give you the work and they’ll say like okay I’m not giving going to give you enough salary but you have to do it it’s exactly like that so you the reasoning for that is because all of these agitation requires power power means energy is consumed energy is consumed means what amount of money that is we are going to spend on energy it becomes an energy intensive process you don’t want it to be if you’re purging in a lot of uh air inside you’re still spending a lot of money you don’t want that you want to reduce the overall cost so the idea is always you want to design a fermentor you want to make sure that you are going to use least amount of vegetation and the least amount of air flow so you have to be careful so what will you do if I want this like minimum air flow is required least amount of agitation is required what what what will you do how will you take care of it use the mic so when whenever we are selecting the media so the media which having the higher water sorry oxygen uh dis dissolving property so you will select a media that already has oxygen no no which which have the higher oxygen dissolving property because the viscosity matter the higher oxygen dissolving property that means it is able to dissolve enough oxygen in it okay got the idea but uh what media will that be it should be less viscous first of it has to be less vus okay what is the best media that we use in the ferment system what do you think is the liquid media that generally we use in the fermenter water water water right water is the only one thing that we use what other parameter can you change lower lower temperature the dissolution of carbon dioxide is higher definitely hopefully carbon oxin also higher so we can control the metabolism of the organism which can produce maximum yield at a lower temperature or we can modify the metabolism according to genetically modify the organism according to okay now let me put forward a question to you so let’s say if your intent for your fermentor is to get the maximum cell density and the cell mass that means you want to grow and grow and grow and grow and grow as much as you can okay what will happen to your Ur and O OT are even uh the oxygen uptake will be more than the oxygen transfer rate right that means you have to continuously increase your agitation yes yes so then again it is going to increase your cost what will you do at that time your intent is still to get the maximum yield but your intent is to use the least amount of agitation so you might want to go for a different type of fermentation system right yes rather than going for let’s say AIC uh a Fed batch or a batch where you have a stir you don’t want to use a stir you can go for a fluidized system in the fluidized system the cell is mixed because of the movement of the medium itself okay and mixing is taking place you might be able to get more oxygen transfer there has to be a solution I’m just trying to throw ideas I might be wrong in my idea but I’m just trying to throw to give you some type of solution you have to find me instead of agitation we can supply the oxygen as even for example uh sparge this noil can be the size of the noil can be reduced in order to reduce the size of a gas bubbling okay so that means you want spares with smaller holes like atomizer okay atomizers very good that can be used that is actually used what else you have some stabilizers can use stabilizers for what like uh even for example in certain industry uh for example in ice cream industry and all they are using certain stabilizer which will stabilize the dissolution of oxygen but I don’t know you’re comparing an Emulsion with uh with fermentation a stabilizer for an imulsion I can understand because you want to maintain them into their ulive state like dispersed State I I don’t know whether it’s work this no I I I really appreciate your effort that at least you’re you’re thinking out of the box I really appreciate that but think again it would be so funny using a stabilizer what are what are the most common stabilizer that you use like alides and if you add you’ll get clumps so the even the media uh which is used like whatever the substrate we are used for the fermentation uh which also should improve the oxygen dissolution see we we are we are we are having the same conversation again and again we are we are coming back to the media property we more or less something has to be done neither you are aware of it neither I aware of it it has to be done experimentally you have to choose right you have to go through that whole rigorous experimental process that’s the reason if you go back to my very first presentation on the fermentation when I started I showed you that diagram and I said like it has so many pipes it looks very dangerous it’s a very simple process fermentation is a very simple process but it’s very complex when you go deep into it it’s very very complex because but once you have optimized it you come you switch it on you forget about it you go back home you have your product ready that’s the way it works for this yes there there are solutions the beads I talked about that’s one of the solution where you might be able to keep your cells suspended without them clumping together they might you might give them extra surfaces so you’re increasing the surface area area but you’re also helping to improve the nutrient delivery so you’re not going to have like a thick cell mass in which you have let’s say an agitator trying the fan blade trying to move if if if your cell M was filamentous you you cannot have an agitation if you’re growing a filamental like a like a fungi you cannot have agitation it will break right the filaments so there are things that can be done there are a lot of research that keeps on going in the fermentation area even if it is the century old technique you will still find people doing research in this area just because of this it is such an interesting area okay let’s get into the final few slides of the oxygen supply sorry oxygen Mass transfer uh the factors that affect oxygen supply rate we have already discussed a few of them temperature is one of those pressure is another diffusivity now the diffusivity of what diffusivity of oxygen in the medium or diffusivity in the cell side it’s all about the CLA right in the medium the diffusivity of oxygen in the medium the viscosity of the medium density of the medium surface tension for the air bubbles surface tension will Define if I am correct the thickness of the of the film presence of surfectants surfectants can impact oxygen transfer the ionic strength can impact the oxygen transfer the concentration of solids can impact oxygen transfer power input less power input that means less agitation more power input higher agitation Iration rate higher the Iration rate you’ll be maintaining more OTR geomet of the Biore reactor you will never like to use a Biore reactor of this geometry where only you have a small like impeller in the center the mass is everywhere else you’re not going to have enough mixing so your design of the bi reactor has to be optimized based on mixing homogeneity has to be maintained homogeneous mixing of your nutrients homogeneous mixing of oxygen and it should also be capable of maintaining your cell growth and it ease of operation ease of cleaning all of those things come into picture anyway but your design has to be maintained that way now the variables that can be manipulated majority of the times are your pressure so if you increase the pressure you’re basically going to increase your OT R your Shear rate and power input that means more agitation you are increasing the OT we have already discussed this idea eration velocity will increase your OTR so there is a relationship between Iration rate and your uh agitation rate that can be studied for different mediums before I put the cell if I can study how that will change then I can provide my my cell growth into it even that will lead to changes into it but it will allow me to start with a certain SEC critical and then I can increase it depending upon how the cell is growing I know how much oxygen is required for the cells to grow I’ll keep on increasing it depending upon the demand so other parameters are for given geometry of the bioreactor depends on the nature of the fermentation process I will take a break of 15 minutes and then we’ll start the session on heat transfer operations in the Biore reactor okay e welcome back so now that we have gone through the mass transfer operations for a bioreactor we are going to go through the heat transfer operations in a bioreactor similar to mass transfer we did go through the concept of heat transfer at the very beginning of the lecture so similar to mass transfer heat transfers is also a very important parameter when you’re looking at the cell growth we have already discussed about this a little bit in detail yesterday when we were talking about the thermal depth or the growth of microbes or how the cell growth is related to the temperature so if you increase the temperature the cell growth will increase to a level which is your Optimum temperature value and below this particular temperature value the cell growth will reduce or they will die but heat transfer application is not just for the cell growth in terms of application of heat transfer in uh let’s say in molecular biology or cellular biology what do you use uh heat transer or heat for auto caving like sterilization so any time you are working with microbes let’s say any in any utensil if whether it is be a fermentor a batch fermentor or fed batch fermentor or cstr or even if you are uh let’s say curling milk any container if you have to clean it you will be applying heat to kill the microbes so that is part of the heat transport or heat application in case of uh fermentation system we need to have a sterile medium it should be free of any other micro but not free of sucrose or the nutrients that it has it has to have but it has to be free of any micro so what do you do when you’re growing uh let’s say micros on an agar plate what is the very first thing that you do with your agar gel thear you Auto cleave it heat it and then you pour you let it set then you streak your organism into it you see all the growth and everything and then when you have to get rid of it auto cleave again right so in C2 bath sterilization of liquid medium is one of the applications of uh heat transfer in by reaction or in by reactor in fermentation so in this process the fermenter vessel containing medium is heated using steam and held at sterilization temperature for a period of time it’s just like pasturization cooling water is then used to bring the temperature back to normal operating condition so you put in everything you use the steam to heat the ster up to the sterilization temperature you bring it back to an Optimum temperature that would be appropriate for your cells to grow that’s when you add your seed culture into it and then you let it ferment okay so temperature control during reactor operation is very very important to manage a few things that would be the metabolic activity of the cells generate heat because we have to have a temperature control because as the cells are growing they are also going to generate heat the metabolic activity within the cell is generating heat we have already had this discussion at the very beginning of the lecture today or the session today so some microorganisms need extreme temperature conditions example would be cyrilic thermophilic microorganisms for those we have to have we have to maintain a very high temperature now when you’re maintaining these type of organisms how will you work with your oxygen transport that will also change depending on the temperature of the medium the oxygen transport rates will also change so depending on the organism we are looking at the OTR so you study the organism you find out what OTR they require and only then you build the rest of the system on that but the generation of heat because of the metabolic or the microbial biochemical Pathways is an important parameter that you you have to take into account understanding what microbes you’re going to use for your process how they grow and how much heat do they produce is an important parameter now the heat transfer configuration for bu reactors include jacketed vessels external coils internal coils and external heat exchangers so you you will have like a jacket around the vessel that will add the heat to the vessel or you can have external coil into it and then you have your internal coils which are inside not on the outside of the fermentation system but on the inside of the fermentation system have you seen a ferment I think in today’s uh in the afternoon you’re going to see uh a bi reactor in the lab not today we don’t have the lab today okay sorry you if if you do get an opportunity go to the bio bio technology department here you should be able to see a bioreactor if you have had a course in bioprocessing if you have had a lab in that you should have seen a bio reactor so if you’re using a glass bioreactor the only way you can provide the temperature is by using a jacket around it and then that particular jacket has heating elements into it and then you pass on the heat to the fermentation system so you only jacket it to the level that you put in your fluid so that you’re not not providing excessive heat just to the fluid that is of your interest and then you have your external heat exchanger I always like these external heat exchanges they remind me of the old way of heating your water in the morning of winter you switch on and there is a probe that you put in the bucket and then you let the water heat take it out but that can also be used to murder people so don’t use that anyways so external heat exchangers are also used to maintain the temperature within a fermentation system so heat exchanger configurations so I will go with each and every one of them so external jackets and coil give low heat transfer area they are rarely used at industrial scale they are widely used at lab scale depending upon how how much money you have spent on buying your bioreactor or how much money you have for your B reactor in my research group we have this jacket ones to heat our fermentation system then you have your internal coils which are frequently used in the production vessels the coils give relatively large heat transfer area because you need more surface area for the transfer of the heat the coil interfere with the mixing in the vessel and make cleaning of the reactor difficult so if you have internal coils because they’re inside it everything is going to go over the coil have you heard about uh the term fowling right or plaque generation over the surface so if your if the heating elements are not cleaned properly or the fermentor is not cleaned properly heat exchange would be impacted so design of the heat exchanger where you have a cross flow parallel flow heat exchangers when we go into those type of design even there you have to take into account the fing process right the other would be the coil interfer sorry another problem is the growth of cells as film on the heat transfer surfaces again fing so those biofilms that get generated on the heat transfer surfaces so you have to make sure that you clean these heat transfer surfaces these heat transfer surfaces to maintain an appropriate heat transfer level external heat exchanger unit is independent of the reactor easy to scale up and provides the best heat transfer capability sterility must be maintained the cells must be able to withstand the sheer forces imposed during pumping during aerobic fermentation the residence time in the heat exchanger must be small enough to ensure that Medium does not become depleted of oxygen if you’re using an external Heat exchanger you want to make sure that the medium does not get depleted of oxygen if you’re passing the heat from another source sterility has to be maintained because sterility is the most important parameter in a fermentation process because if it was internal I know everything is inside whatever is there inside is what I’m working with if I’m using something from another location I have to make sure the sterility is maintained let’s say even if uh if I’m using some sort of like steam to heat my uh system if the steam is being produced at different location and the steam is going to be passed through the pipes right when it is going to be passed through the pipes those pipes have to be clean enough that they don’t contain anything the steam before it enters in my fermentation system I have to check that is free of any microbes right so all that uh controls have to be in place if you’re working with an external heat exchanger excuse me so common heat transfer operations uh removing heat generated during the fermentation process so the operations that invol are involved in the fermentation process would be removing the heat generated during fermentation removing heat generated due to mixing almost 90% of the time the Heat generated due to mixing is higher than the heat of reaction now this heat of reaction is not with respect to the chemical reactors I’m talking about this heat of reaction from the microbes perspective how much energy is being produced by the microbes during the growth okay so that heat is less compared to the heat generated by mixing sterilization operation is a major heat transfer operation in a fermentation system sterilization is a major heat transfer operation in a fermented system coming back to this uh what sort of mode of heat transfer is this is it convection or we are looking at convection but we looking at mostly Heat exchangers if it is internal coils we are looking at heat exchangers it’s like a heat exchanger the the heat transfer the lmtd has to be included you remember that so you have to take that into account if it is just from the outside surface and the heat is being provided by the surface of the fermentor it would be conduction but if the fluid is moving over the surface it’s conduction at the interface but there is a flow of the fluid so convection has to be taken into account then again it will depend on the fluid properties the viscosity you have to take uh you have to look into the table get the values at a specific temperature what’s the viscosity of the medium the density of the medium and then you’re going to use your nles sorry nles number equations to estimate the value so microwave growth involves complex Network for metabolic reactions now I’m talking about the heat generation because of the microbes during cell growth cell derive energy from catabolic reaction and use them to drive anabolic reactions some energy is lost to the surrounding as heat if the bioreactor temperature is to be maintained then this heat must be removed hence the first objective is to quantify the heat released due to the growth so when you are going into designing a bi reactor if you have your growth of microbes being if you’re if you’re estimating the growth rate you can also estimate the amount of heat that would be generated by that particular microbes once you estimate that value it becomes easier for you to identify what sources will you choose to remove that heat from the peration system this part is mostly numerical there are equations that are available to you to do that I’ll give you some of the examples like what would be the heat of combustion for a biomass like KJ per kg if you’re looking at an eoli you’re looking at 23.03.2013 on different biomasses you’re going to have different heat of combustions once you know your heat of communions if you do your in minus out plus generation is equal to accumulation where will this particular value go the heat generated by the micros will that be an in or will that be considered as a generation in would be anything that you’re providing out is whatever you’re taking away generation can be from the microbes that will lead to the change in the temperature with respect to time so when you do that particular balance you can take into account all the heat sources which are going to add to the system now because it generation is adding to the system is positive if it was removal of the heat there it would have been a negative value right that’s how simple it is now the model equation for the batch system for heat transfer if you remember Q is minus k a DT by DX now DX is the distance DT is the time or sorry DT is the temperature difference DX is the distance you have to have that gradient minus K is your conductivity sorry K is your conductivity is your cross-sectional area but here you’re going to look at the QR value with respect to the microbial growth so this is the equation which is used with respect to microbial growth this QR is for the microbial growth VL is the volume okay change in biomass concentration with respect to time DX by DT is your biomass con uh concentration with respect to time so the I take that into account the yield you’ll be able to estimate how much would be the heat of combustion coming out so if you know the heat of combustion you know the biomass concentration you can estimate the amount of heat that is coming out for that particular biomass in a given period of time so the total heat that has to be removed would be this QR the heat of combustion or the heat generated by the biomass and the heat added by mixing so Q mixing is a heat generated due to agitation where we are converting the mechanical energy to the heat energy assuming no heat loss to the surrounding this amount of heat must be removed from the system to maintain constant temperature in the bioreactor so if you have a bioreactor this agitation as it is going around you it is going to generate heat because of the sheer stress like there’s a friction as is moving through you that heat which is generated my major concerns this is my own personal opinion to this my major concern with respect to the agitation has been that it does not generate a very huge amount of heat even if you keep on rotating your hand inside of water you won’t be generating a lot of heat yes you’ll warm it up a little bit maybe by a degree or two but even that small degree or two can impact your fermentation process because it’s a very controlled precise system so heat transfer area should be designated sorry designed accordingly so you’re what you’re looking for from the Q mixing is the UA delta T U is your overall heat transfer coefficient a is your surface area delta T is the temperature difference so the heat accumulation now would be the heat generation minus the heat removed so your DT by DT is the term with respect to your accumulation the CP is your heat capacity density and the volume then you’re looking at the Q and you’re looking at the UA delta T the QR is your for the biomass you have it for your mixing you add both of them you get the value for the total amount of heat that is going to be generated that needs to be removed how do you remove heat in uh fermentation system if you’re using that internal coil those are not heated coils let’s say those coils are hollow tubes and you’re passing steam through them if you pass cold water you’re going to reduce the temperature right if you have a jacket you pass cold water you’re going to reduce the temperature so you always use a medium that is able to extract the heat from the medium of the fermentation medium fairly quickly because you don’t want to have uh spend too much time in that right longer the medium will remain at a higher temperature it might lose its viability so you want to reduce amount of time that is required for the removal of the Heat and reach the optimum temperature that is required for your cells to grow that balance is very very important okay is there any other idea that you can actually use for removal of heat you have have your uh you can use some condensation column like you circulate it the circulation can be done the media can be circulated the media can be so you’re taking the media out of the fermentation system no like when there is a for example in pools and all there is a water purification the same way you’ll be like one time it’s circulating like there’s a pipe should be there from the side and it’s circulating like see you forgetting two things so yes there is a heat which is being generated by the cell uhhuh right yes there is a heat which is generated by the mixing now from where are you actually taking away from this heat from the surface right you’re not taking the heat from inside the broth or the medium you’re taking the heat away from the surface from the surface so if you increase the surface area reduce you are able to remove more heat heat that’s one way of doing it by increasing the surface area so baffles sometimes are not only used to get rid of the edies but they are also used to increase the overall heat transfer surface area question for you in this case this is just from the conceptual perspective so you have your medium so heat is going to pass from your medium to your surface right that is conduction so you do you remember the unsteady State heat transfer in unsteady State heat transfer we have something called as uh High SL char charts High SL charts you don’t remember that for different types of geometry if you have like a slab you have a cylinder or a hollow fiber or a sphere you can use these charts to estimate what would be the heat transfer specifically for cylinder slab and uh Sphere not for the hollow fibers I haven’t seen that but cylinder slab sphere you can use this high but the condition for those ISO chart was the surface the condition at the surface the B at the surface is maintained throughout the process it is not changing but here the things are changing so that mean what I’m what it means is like let’s say if this is a slab we’ll always look at the Symmetry and then if this is your x equal to 0 this becomes your x equal to l the length of least resistance right the length of least resistance that the least amount of distance that the heat has to travel to get out so if you’re looking at the Burger let’s say a burger patty the Alo Patty or potato Patty that you get the heat transfer if you’re doing conduction what would be the the L value in this case it would be the thickness not the length because this is the least resistance the heat is going to transfer this way why why I’m bringing this is I’m trying to imagine in our conversation with each other the medium in a cylindrical in a cylindrical uh fermentor system can be considered as a cylinder of uniform conductivity it can be considered as a cylinder of a uniform conductivity if it is homogeneously mixed if it is homogeneously mixed can I consider it to be a uniform distributed conductive material a cylindrical now the heat is going from inside to outside if the heat is being removed from the surface right heat is going from inside to outside now as you increase the size of the fermentor you’re increasing the surface area right as in the cylinder like in a slab the heat transfer is linear in a cylinder the Heat transfer is not linear it’s parabolic because of an increase in the area in the radius the surface area keeps on increasing the heat transfer the Q will keep on increasing in this case how will that impact if I consider that removal of the heat from the surface do you think that will have any impact if the material is stationary and if the material is moving homogeneous nature is maintained I have my material heated at a specific value I have to remove the heat so then it becomes if I am passing cold water on the surface of the jacket I’m basically doing convection convective heat transfer removal right but that would be from a cylindrical surface it would will it be considered as a force convection over a cylindrical surface there is an equation for force convection over a cylindrical surface right which can be used that nles number equations so those nles number equations can be used if you def if you have a defined geometry of your bio reactor when you’re trying to cool it down heat it up you’re using those defined nusle number equations to estimate the amount of heat transferred yes you’re getting my idea of the drift because I’m considering the whole bi reactor only the medium part as a cylinder of homogeneous constant conductivity there is water flowing over the surface that means through convection the water jacket is Flowing so will that be considered as a convective heat trans transer that is what my question is will that be considered as a convective heat transfer will that be considered as a heat exchanger will you consider it to be like a uh like a conduction a basic conduction because water is Flowing so convection is there how will you take it how will you take it like can you take it as convection can you take it as basic so convection within the uh media and then from the film the film which is at the boundary layer from there to the uh like jacket as conduction if the medium is moving then convection yeah right I’m considering the medium as stationary it’s like a solid cylinder of uniform conductivity because of the homogenity being maintained can I consider the heat that is coming out of that Medium because I’m trying to remove it from the surface and if I’m passing the water in tubes will that be considered as a convection or will that be considered as a heat exchanger as a heat exchanger as a heat exchanger so we generally end up using heat exchanger equations to solve the amount of heat that is being removed from the peration system but we use convective equations to estimate the amount of heat that we are generating within the fermentation system okay so don’t get confused in those two now design equation for a continuous system so the heat accumulation is equal to the heat input due to flow heat out due to flow heat generated and the heat removed by cooling so your accumulation is this ter so you have your VL row CP DT by DT you know what is v why VL row volumetric flate or the mCP delta T V is what is mass volume into density right so you have your volume into density CP delta T So mCP delta T is the amount of heat which is there accumulated within the system clear so this term is your mCP delta T similarly break it down for yourself first thing is heat input due to flow f is your flow row CP now this becomes volumetric flow f is your volumetric flow so that becomes your mass flow rate this is your heat generated because of biomass this is the heat added because of mixing this is the heat being removed this TC is the cold temperature right it is the heat being removed u a delta T U is your overall Heat transfer coefficient but we are looking it as from conduction perspective or convective perspective convection UA delta T okay we’re looking from the convection perspective so understand like mCP delta T is the key here so don’t get confused by the overall uh these big equations mCP delta T is what the VL row CP is the volume into density will give you the mass the CP then the delta T So model equation should be able to predict what heat transfer area is needed to achieve the required temperature time profile so if you are able to identify these equations once you solve this equation you’ll be able to predict what heat transfer area is required or is needed to achieve the required temperature time profile okay so if you’re designing your Biore reactor if you know how much heat is being generated by the biomass what would be the removal of the heat you can design inside again the area so you can add baffles just in case if you want to improve the overall removal of heat you can add baffles if you don’t need you can change the number of coils that you’ll have so you can do a lot of those modifications to your design of the bioreactor depending upon what heat requirement is or what heat removal requirement is then comes the topic of scaleup okay so Mass transfer done heat transfer is mostly for sterilization purposes heat transfer application is mostly for sterilization purposes I’m not going to teach you that log 10 deductions okay that is not part of this or it’s not in the scope of our conversation today you can check when you’re working on the calization pasturization purposes for your own courses the idea of heat transfer in the Forma system is because of the sterilization of the media because everything has to be sterile to start with once you add your culture to it or the seed culture to it that’s when it will start growing the other aspect of the heat transfer was to maintain the heat for the optimal growth of your microbes that is by your mixing the heat that is being added the generation of the biomass the heat that is being added and how much you can remove UA delta T right so that’s was what was involved now look at the scale up now you have understand you have now you have understood your mass transfer and the heat transfer now keep those Concepts in mind and then think about your scaleup all of those parameters have to be transferred if you’re moving from a small fermentor to a larger fermentor your OTR Ur has to be maintained your removal of heat has to be maintained the agitation speed that you were using in a smaller fermentor might not be the same as a larger fermentor the amount of power input for that small that agitation will differ if you’re working on a larger fermentor so all of that power ratio has to be maintained when you’re looking at scaleup so what is the scaleup in practice scaleup it is the study of problems associated with the transfer of experimental data from Laboratories and pilot plants equipments to a large scale industrial equipment so you’re starting from a test tube to a 5 ml flask 35 ml flask to 200 mL flask then you go to a 5 lit bioreactor move to a 30 L bioreactor 220 L bioreactor I personally have seen up to 1,000 L bioreactors then you go to 10,000 L bi reactors we have already scaled up these bio reactors up to 600,000 L now there are bi reactors of capabilities of 600,000 L think about it now it’s a very scaling it’s a process that can be scaled up quickly but it has to be done in a proper way because there are so many parameters that depends on the scale of process itself your heat transfer Mass transfer Mass transfer specifically your oxygen whenever you think about Mass transfer in a fermentation system it’s all about oxygen when you think about heat transfering for itation system the heat generated by the biomass plus the stiring is not that significant it’s all about sterilization the most important part will be the sterilization that’s the most important heat transfer application in the fermentation process what else will you take into account if you’re thinking about the scaleup the geometry of your bioreactor that has to be taken into account right the diameter the height all of that has to be taken into account the third most important the nature of your organism has to be taken into account the fourth so nature of the organism heat and mass what else was there this agitation is going to impact Mass it’s going to impact heat what else Iration agitation is going to impact Iration aration is going to impact mass aration is also going to impact heat can I now Iration is mostly for the mass transfer applications okay what else will you take into account when you’re looking at scale up you have taken your bioprocess course come on thinky thinky I can see people’s mouth moving but I cannot hear anything honestly give it a try there is no wrong answer let her answer first just three parameters we mainly consider about one is uh K okay that is your a m sir and imp tip speed tip speed that is your agitation yes sir you written everything sir then what else is there and the geometry right these are the three we take care because power power conception power conception is also agitation yes sir py yeah but then it is used for what power is used for what again comes to the and agitation but these are the three things mainly but you have written already yes but I’m seeing it from a different perspective right as a faculty my intent is that you just don’t give me the bookish example of the tips paid and all those things you give me from the conceptual whatever you have understood till now we have discussed batch reactors we have discussed fed batch reactor we have discussed how to design a batch reactor we have discussed how to design a Fed batch reactor you know none of you have talked to me about when you’re changing the geometry you’re also going to change the flow of your feed your feed rates will change there are so many parameters change the moment you change the design of your bioreactor or even when you scale it up there are a lot of things that come into picture the most important that are used to take if I have to take this pen make a bigger pen I will will take the things that I can control the first thing is the geometry that I can actually control and pass it on that’s why you were taking about talking about that now tip speed and all those things affect your agitation affect your K values how many let’s say for a small fermentor oh yeah see for a small fermentor I will can use only one impeller but what was the design of the pill that I was using for a small fermentor might not work if I move it to a bigger fermentor what will you do then the heat the agitation will change right so it requires some consistency but there are also certain parameters where variability exist that variability is what matters the most but for fermentation is a very forgiving unit operation that’s why I said it’s a very simple system it’s like it’s like one of the nicest unit operation that you can work with it’s also very forgiving you add everything you will get something at least but if you are able to put a little bit of effort in understanding each and every intricacies and then design it it will give you the best of it that’s what the intent is in our conversation I can understand what you told me the tips paid I have all of that in my slides next but I just wanted to get a bit more from you what else will you change if you are moving from one smaller fermentor to a larger fermentor what else will you change are we just focusing on the batch if we are only focusing on the batch I will not care about the feed will I care about the feed I won’t what if I’m trying to scale up on a Fed batch level I have to think about my feed rates my flow rates so that has to be scaled up depending upon how much I want to grow but that would also come with a different set of calculation but that has nothing to do with the design of the fermentor you’re getting my point that has to do with my working but it has nothing to do with my design of the ferment that’s the reason why feeds and all those things are not taken into account that will only depend on the growth productivity that you want productivity has nothing to do with the respect to the design in terms like in our conversation I’m just saying it’s just physical conversion of a small thing into bigger thing that is what we are talking about so we’ll get more into this so what is the exact aim of scaling up the objective is to keep the cellular environment constant your cellular environment has to be constant across scales to achieve comparable growth I’m not using the term exact I wish I can use the term exact but I’m using a comparable you might not get an exact growth but you’ll get a comparable growth relatively comparable growth metabolism productivity and product quality sometimes like people make fun of these things where they’ll say it’s like a onetime Wonder right your thing worked at the lab skill the moment you scaled it up it didn’t there is something wrong in the input for your large skill that’s what the problem is there’s nothing wrong at a small skill but there is something which is missing at the large scale understanding that from the biochemical perspective everything will remain the same it’s mostly the physical that thing has changed from a small fermentor I became a bigger fermentor that’s it I used to be a thin guy now I’m a big guy it changes but inside I remain the same I used to be the same person 10 years back I’m the same person 10 years now or I will be the same person 10 10 years after except I would be much more older but my body will change that’s what the scale up is all about is you’re changing the physical aspect but internal has to be constant you’re trying to maintain it as much as possible so which are the most important criteria similarity criteria now we talked about that and thank you for bringing all of that answers so similarity criteria that you have to maintain would be your mass transport which is your O2 and the CO2 the CO2 which is being generated has to be removed larger the fermentor more the biomass more the CO2 more the removal larger the fermentor more the biomass more the O2 mode is required mechanical stress on the cells larger the biomas bigger the agitators and the impellers the design of the impellers more the stress sheer stress larger the population too many people right it’s just like going in an autoa or you’re going in a crowded bus scaled up stress right from the sales perspective mixing time that would have been required at a small scale would be very different than the mixing time that would be required at a larger scale so how quickly can you reach the homogeneous or homogeneity is very very important when you’re trying to do scaleup now these are the most important parameters from the industrial perspective think about I’m so proud that you all are aware of the basics of scaleup I’m really really proud of that think it from a completely I said you keep an open mind think it from your own project your own uh experiences how are you going to do the scale of what are the parameters that will get impacted the most that has to be taken into account oh sorry okay yeah so the scale of principles uh involv ensuring circulation of nutrients dissolved gases and removing metabolites is an important in B reactor agitation and Iration allows for the bulk liquid mixing and sheer stress agitation adds to that oxygen transport and pco2 removal is through the areation aspect mixing is more difficult as scales increases mixing does become an issue as the scale increases so on the right hand side you can see the process definitions uh I would like you all to look at this particular link to get the same uh graph that I’m using here so temperature control so temperature is directly related to cell growth and viability okay then temperature control also regulates the O2 and the CO2 solubilities then you have your pressure control pressure control basically impacts your CO2 and O2 solubilities and that impact imps your cellular environment nutrient addition your glucose glutamine and the feed addition nutrient concentration that will impact your cellular uh environment if you’re looking at the pH you have your base addition now why do you add a base to maintain the pH right so now the base if the base and the substrate will interact or some other combinations get generated that is going to impact your cellular environment the one comes here is the focus the dissolve oxygen control so dissolve oxygen control is through agitation gas flow rates and the Sparger type the type of sparer that you’re going to use at a small scale I could have had a ring Sparger at a large scale what will I do if I have a larger like as big as this if I have a larger Sparger here the bubbles are going only in the quter nothing is going to come in the center so I have to Define I have to design a sparer which is multi-layer right so you have to think about the design from that perspective nobody talks about increase scaling up of a spon people are talking about the scaling up of Iration the O2 but that will lead to the change in the design of the Sparger so as an engineer I understand the O2 that what biochemical guys or biochemical engineer is interested in the O2 he or she will come and say hey bro I need this I say okay fine I have to redesign the sparer as an engineer I would love to do that that’s my area of Interest how to redesign the sparer to make sure that you able to maintain the O2 that you require now if the cellular environment is maintained that will impact your cell growth viabilities nutrient uptake and waste accumulation that will also look into your product concentration and the product quality maintaining all of this is your primary motor with respect to scale up now determining the derived parameters in reference to those would be your calculate your agitation regime so you have to look at your Ed size the power density the tip speed the mixing time the K and the turbulent Shear Force then you calculate so you once you have estimated you again calculate at your Iration regime you have your porosity spargers air flow spargers oxygen flow spargers then you have another second round of perity spargers or air flow spargers you have if you need more and a eration sorry agitation Iration regime for a targeted scale determination has to be determined depending upon O2 CO2 removal mixing time tip speed these are the main ones this is an example so if you are scaling up a 1 lit bioreactor to 100 lit volume based bioreactor so the volume is pi T1 2 by 4 so T1 is your diameter for the smaller one so Pi d by 4 right d ² by 4 H1 H1 is your height for a small one so you’re looking at s this s is the parameter that we use for scale up this is also called a scale up parameter from the geometry perspective so you’re looking at scale up from uh from the diameters of the spargers sorry of the SP for the impeller you’re looking at the the diameter of the vessel you’re also looking at the distance of the impeller the tip of the impeller and the base does that impact the speed of the impeller the distance at which it is placed that will impact your mixing that will impact your mixing so a single scale ratio s this is called your single scale ratio s defines the relative magnitude of all linear Dimensions between the large and the small scale okay so volume one is given as this volume two is for the larger one if you take a ratio of volume two and volume 1 the S value for your case should be 100 to^ 1X 3 if you make it a 10 lit let’s say every place where you have 100 will become a 10 so this will become 10 1×3 10 are easier to work for one you go to 10 10 to to 100 100,000 an increment of 10 makes your life much more easier let good any questions no question now the scale of strategies deep speed so tip speed is defined as the tangential velocity of an impeller at a point on its tip the tip speed is a function of the RPM revolutions per minute and the DI diameter of the impeller so diameter of the impeller or the size of the impeller will Define the tip speed so the tip speed is pi DN the D is your impellar diameter and N is the impellar agitation rate so the RPM that you’re talking about the criteria is you ensure the L by D the length by diameter to be the same in both cases so L by D in a small scale L by D in the large scale the agitators should be identical should be identical they might not be identical depending upon the how big an agitation system you go to or what type of design you all had before it doesn’t take into account the impeller design at all this one doesn’t take into account the impeller design so if you have a specific impeller with a twisted hook at one end you might want to have the same in the other one sometimes it’s possible sometimes it’s not possible so when that happens you have to redesign your smaller one with a different impeller and then see if that can be used or you have to compare again so you take that Twisted one you still think that it’s a flat and then you use the flat one on the other side and then compare the performance the productivity if it is comparable you’re good to go if it is is not comparable don’t go with it now the other scaleup strategy involves the power per unit volume the W per meter cube the power per unit volume sometimes referred to as power density is a power output for a given volume of culture so impeller design fluid density and the agitation rates are considered in the power per unit volume so your NP is the impellar power in number row is your fluid density N3 or n to^ 3 n is your impeller agitation rate the D is your impeller diameter the V is a liquid working volume and the P not is a ungassed power input this power input is just with respect to ration or agitation it’s just agitation okay there’s no irration yet it’s just agitation so larger the fermentor larger the agitation system that you use larger the amount of power that you’re going to consume redesign it you might be able to make it cheaper questions now shall I continue the other scale of strategy comes for the K so she was right you have K tip speed power density I’m going to put you on place don’t worry about that so tip speed power density k k is for the areation now tip speed power density is mostly for the agitation or the design of the geometry tip speed for with respect to Geometry also power density is with the impeller the agitation purpose K is with respect to oxygen Mass transfer coefficient so this is a parameter that controls the rate of how oxygen transitions from the gas phase into the liquid phase the intent is sufficient o or air delivery is required to support cell growth the metabolic pathways or cell metabolism itself the protein Productions protein Productions requires oxygen if you’re using the host that requires oxygen it is also to prevent excessive CO2 in the media which can impact critical performance end points So Physical K are determined by various Power by volume uh ratios you can use and the gas flow rates the vbms using the static gasing out method you know what the static gasing out method is what is it you have the mic wait somebody give the mic uh first of all we Supply the gas then we allow the bacteria to or microb to utilize the complete amount of gas and then we sparge again then we know how much gas was utilized okay now there are different way methods to measure the K one method I remember is you estimate the amount of oxygen that is there at the beginning of the fermenter as you passing in and then you measure how much is coming out so that will give you the difference of how much oxygen is required another one is where you pass in the oxygen you let the uh the growth and then you pass mer the nitrogen to remove all that oxygen and then you estimate then you pass the oxygen again that’s the static Dynamic guessing that’s the one right the static and dynamic one okay and also there is sodium sulfite oxidation method that I’m not aware of I know about these two that’s what I have read in my undergrad I have not used them yet but those are the ones we oh no for the K we have used the static one anyways so so the K is defined as oxygen transfer rate divided by the do which is your dissolve oxygen dissolve oxygen saturation concentration so OTR divided by the C star biological Kaa depends on the Clone and maximum viable cell concentration so you remember that x value that we have looked at how we estimate how much biomass we can generate with respect to the oxygen concentration that we have and if we have a specific biomass concentration how to estimate how much oxygen we require we did that back equation it was also there so K bio specifically for biological purposes would be your oxygen uptake rate divided by the dissolve oxygen saturation concentration o Max divided by the C star the oxygen uptake rate is not not known at all times sometimes it’s not known what will you do in that case you run the system is is learned by experience so you have to estimate it experimentally that’s why fermentation is not successful in the very first go that you go through it takes time for the fermentation to build up once you are set with all the parameters for your fermentation system you’re in the best condition possible so to prevent oxygen limitation available k for a bioreactor design should be superior to the biological K demand of the cell lines in culture that means your OTR should always be higher than your Ur as simple as that your OTR has to be greater than Ur clear up to here now what are the difficulties with respect to your scaleup if I’m looking from the geometric similarity large bio reactors are physically different from small ones they are physically different from the small world so it’s there’s nothing else I can talk about in that they are just physically different so sometimes it’s not that viable when you’re trying to do the the the scale up it’s not easy mixing time the fluid dynamics V vary in large scale bi reactors causing differences in mixing times due to size and geometry variations the power input per volume difficult to maintain consistent power input per unit volume due to the equipment constraints and the fluid property differences it changes the fluid properties will change depending on the volume oxygen Mass transfer coefficient larger bi reactors can have reduced oxygen Mass transfer coefficients why limitation in gas liquid interaction impacting cell growth and product yield so finding the appropriate size it’s not like that hey I have been successful in this 10 lit bi reactor in my lab let’s go and make 100,000 lit by reactor it doesn’t work like that you go from 10 L Max you go up to 200 L you try in that if it works perfect if you can scale that up then you go for 600 L you don’t go 100,000 immediately you have to be very cautious because as you scale up the difficulties will increase so you only go up to the extent where you know that you’re going to get the maximum productivity okay the tip speed the larger bio reactors require larger impellers resulting in variations in tip speed compared to small vessels affecting the sheer stress and the cell Behavior it will not mix properly cell clusters might appear homogeneity cannot be maintained all of this comes into picture when you’re looking at from the difficulty for the scale of purpose now this is an example where you’re looking at scaleup strategies for let’s say if you’re doing antibi antibodies uh production or the viral proteins or the cell production so if you’re looking at antibodies so production of monoclonal or polyclonal antibodies for therapy diagnosis or research you use Mamon cells example would be the CH cells which are your Mamon cells the process involves cell growth antibody expression and purification the emphasis for this type of fermentation is on the mass transfer for nutrient and oxygen supply so that’s our core focus when we are looking at mumon cells sheer force management to protect fragile antibody producing cells so your agitation you have to be very careful about it because it might break the cells which are going to produce your antibodies so depending on the type of product you’re going to make your scaleup practices will also change clear or the choice of design of B reactor will also change let’s go on the virus side so virus production involves the manufacturing of viral vectors which are used in gene therapy to deliver genetic materials into the target cells production of viral vectors often using aderant or suspension cell cultures example is given to you here the process involves involves transection viral replication and purification steps crucial focus is on controlling Mass transfer for efficient nutrient Supply sheer force management is essential to preserve the integrity and productivity of the viral vectors again agitation has to be very controlled in this case but you do need the mass transfer right so agitation has to be there to make sure the mixing is there is mix exist for the oxygen to get your OTR to be higher than your Ur similarly on the cell side so cell production for therapies like K cells or the stem cells involves cell expansion and differentiation to generate sufficient numbers of theraputic cells if you looking from the cellular agriculture if I’m looking at stem cells stem cells get differentiated into different types of cells that’s where this type of situation will come if you’re looking from cellular agriculture so during cell production is Cru is critical to optimize Mass transfer for adequate nutrients and oxygen delivery to maintain cell health and function throughout the expansion process additionally managing sheer forces is vital to prevent damage to the cultured cells ensuring they remain viable and functional so for almost all of them O2 agitation is important if you go from small scale to large scale you have to make sure the O2 and the agitation can be maintained you maintain the agitation you maintain your O2 but O2 also involves the sparer right it also involves uh putting in the oxygen in the system mixing will allow to better the OTR right it will also help for the viable growth of the cells so you have to make sure the agitation is proper so that mixing takes place now there are also regulatory aspects to bioprocess design this is the I think the last slide for us today so the regulatory aspects for the bioprocess design involved our product quality and Purity the sterility Assurance the validation and qualification and the scaleup validation these are the three that are the most important so what is about the product quality and Purity consistent bioprocess design is required to achieve rigorous control over the quality and purity of the therapeutic products the core focus of the fermentation industry is not only on food but fermentation is also widely utilized for therapeutic drugs from that perspective the Purity and the quality is primary requirement okay because that’s where the regulatory people will come into picture because the Purity and the quality has to be maintained if it is being used for therapeutic purposes for medicinal purposes the sterility Assurance the maintaining sterility to prevent contamination you have to maintain stability to prevent contamination you cannot expect any contamination anywhere traceability documentation and traceability of each steps in the bio process for product safety and Regulatory Compliance if you’re making any changes to the process parameters those have to be documented because that will lead to changes to the product quality If the product quality changes you have a written documentation to know why you would be able to trace the reasoning for it okay validation and qualification validating and qualifying the bioprocess in producing safe and effective therapies consistently so whenever you’re doing any sort of these bioprocessing they have to be consistent and you can validate them scale of validation requires the scale of procedures are validated and meet quality and safety standards so it’s not like I have one bottle in my room and I go and buy a drum and put everything inside and start doing fermentation in it you have to take into account the safety regulations because if you’re working with microbes some of the microbes that you would be working on let’s say if you’re doing fermentation they’re recombinantly uh generated right so they are genetically modified so if those microbes if God forbid if they are pathogenic in nature what will happen things can go wrong so you have to very careful when you’re trying to do the design of these Z cool I hope you have learned something with respect to your bioreactor design and Analysis till now with respect to scaleup the batch design the FED batch design the mass transfer the heat transfer we also had an amazing time discussing About Cellular agriculture the Precision fermentation and not so amazing time thinking about synthetic biology systems biology and enzyme engineering why I said not so wonderful because I felt that I would have given you more information I should have been more prepared for it but I felt that that way that I should have been more prepared but I hope you enjoyed okay I wish that you can work on your case studies case study one and case study two and please try to submit it today in the evening so that we can assemble all the presentations and that would be so we we have to assemble it with respect to the case study one and the case study 2 when you come tomorrow you’ll be presenting all the case study one in one of the sessions and all the case study two in the other session and then we’ll have this type of conversation where we’ll have back and forth why you chose this particular host what was the requirement of this particular host with this is necessary that is necessary we’ll be looking at all of that any questions for me before we end the session today you all look hungry so thank you that will be the end for today’s session