Transcript
UxzeCftgiuE • Webinar Series - Solid State Fermentation: Opportunities and Challenges by Prof. Ashok Pandey
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Kind: captions Language: en uh CP of uh Professor P so Professor asop Pand is currently distinguished scientist at the center for Innovation and translation research and also in the csrr Indian Institute of tech tech toxicology research in LNO India and also he is a executive director honorary at the Center for Energy and environmental sustainability India so he has published a lot of publication and said that now it's more more than uh 1, 400 it might be now more uh and also he uh wrote books uh book chapter and Edge index currently 96 and with the station more than 40,000 station I think so it could be more because this is long so he is also on the Stanford list a top 2% with rank number eight in the batt field of course number one uh in India I think uh without further Ado I would like to invite uh Professor Pand to uh give the lecture I think gur uh Professor Pand is already have a able to share the yes please for thank you for can you see my screen please uh yes C very good thank you uh thank you for uh uh invitation and I really feel sorry that I could not join your uh uh last meeting yes okay sorry thank because you know that uh it becomes very important and since it was annual review of The Institute my presence was a kind of mandatory yes so uh my index just to let you know is now 104 oh yes yeah cations are a little more than 48,000 yes yes and in on 18th November probably you might have uh seen or known uh every year this uh uh clate analytics they bring out the top 1% scientist in the world and that list is out now on 18th of November just a week ago and uh in that list uh uh obviously my name is there and uh uh apparently I am ranked number four in the world although they do not give a ranking but uh since uh they and they have taken my interview in fact this interview is there uh on the website of Cl I I I'll send you a link and I would want if you could uh share this interview with your uh younger it can be of course listened by anyone but could be interesting for your younger younger colleagues and students all right so uh I'm going to talk to you now uh about uh this lecture which is on solid state fermentation opportunities and challenges there's a title given to me by uh you uh so uh to begin with just uh briefly I'll introduce me my introduce all of you my Institutes so this is what you call csir map of India as you can see this map of physical map of India csir has uh uh 38 uh Research Institute which are spread in different part of the uh country and each of the laboratory has a very different mandate altoe 38 labor approxim to this is on the the photograph you see on the right side upper side uh Indian of Toxicology research this is in the city called city is called LNO which is the capital city of utar Pradesh the biggest uh state in India and in fact uh this is very close lakau city is very close to my hometown about 200 kilometers from here so it's very different after working 30 years coming back to North but then I'm happy about it so uh this is the uh uh few more detail about my current Institute that is um Indian insute of Toxicology research so this institute was established 55 years ago uh where the its main scope was uh uh uh industrial toxicology and uh slowly over the period of time uh occupational toxicology also became uh its activity and two decades we [Music] and then uh sorry have more than 200 Industries we have served in fact this is one of our mandate so more than 200 Industries we have served in the last uh three decades or something like that apart from that we participate in all the important National Mission program which have anything to do with toxicology and uh uh we also are heavily involved onto predictive risk assessment as well as um other kind of uh risk which are involved uh uh into uh industrial uh research so if you look at activities we have primarily four verticals the first one is basic plan this is one common in any organization then we are second vertical safety assessment and led by the in regology and if anyone to obtain it it is not only food material for example if someone has to put uh go for commercial ization of any uh cosmetic products or uh any product which is for human need or human use or any product which is to be uh left in environment then it is necessary to obtain regulatory clearance and we do the uh different levels of safety evaluation uh uh for that product then of course uh uh we are also as any research organization human resource development various kind [Music] this per se uh so we take up collaboration and partnership we have several uh it's more it's now four years and little more than three years I am uh taking care of the center and we have large number of starts up large number in the sense in last two years nine starts up have established have been established here and uh several industrial consultancy product including launching of two Industrial Products one of them is very uh useful that is oir it is a water purification system which uh does not require even uses of electricity and uh it has different modules IND collaboration with technological solutions so from our own Institute s or from other organization collabora Partnership of could be hiring of analytical facilities or what so different kind of options are there so this model is one of the new ncsr system and we are experimenting and it seems that it is working quite good so now I'll move on to the our lecture topic today solidate fermentation so essentially uh all of you know it's a it's a very classical uh kind of fermentation is a cultivation of microorganism under controlled or uncontrolled conditions in the absence or near absence of free water so the bottom line is that there should not be any free water and depending upon the kind of f stock if you f stock the water content could be anything betweener to 20 or even 30 so uh it's a classical fermentation has been used since have to D upon that but the again products Etc which are currently being produced commercially us think [Music] fermentation steroid in factal and sign so uh we generally know that it is ideal for growing filus fungi but then in science there are no limitations and we have proved that uh even uh different kind of bacterial culture could be well cultiv cated and uh exploited uh to get uh quite competitive yield in fact Superior than whatever has been reported in U uh liquid fermentation so traditionally it has been used for food fermentation we all know that uh the countries in uh not only in Asia not only in Africa uh Latin America but even Europe also have been using it for traditional food fermentation for example k fermentation and for Europe when we talk of it is essentially the cheese fermentation what the interest for solic fermentation it has a much lower water consumption water is one of the critical element for the sustainability in the world and we know that uh almost 60% part of the world are water stess today so if we could reduce the water utilization uh effectively that will also not only help in sustainable development but also lead to less production of industrial influent so these are the very important U consideration this is uh where the word again and again keeps looking at solidate fermentation so some other information that what are the specific applications uh where there is a lot of focus and uh interest for commercialization is in specifically in food now functional foods and food ingredients in agriculture areas by fertilizers in Pharmaceuticals so functional bioactive ingredients secondary metabolites and then specifically enzymes which have been you know one of the core uh commercial strength of solate fermentation so we are today looking at cellus production Alpha proteas Etc and on livestock probiotics probiotics for human consumption probiotics for animal consumption and if you look at uh uh uh the right side figure that uh which organism or which products are being utilized which cultures are you being utilized by which industry so it's very interesting and uh in spite of uh you know not being used currently as a high technology it continues to grow in different parts of the world uh for multiple applications so if look at the pros and cons when I say it continues to uh grow for commercialization or for research perspective in different parts of the world and advantages you all know because all of you have been working into this area that uh it has a low Capital cost it has reduced energy requirement it has a low affluent generation it does not have one of the sever problem what we face in some fermentation is foing and in fact foing is the one which often causes contamination in large scale fermentation Etc and uh also an important fact as a researcher you uh need to know that a question is often asked why we get high yelds and high productivities in solidate fermentation so uh it's it's only a speculation it's only hypothesis it has uh uh been proven to certain exent extent but not uh absolutely in not not in absolute terms that why there are higher uh yields and why there are higher productivities obvious apparently it is because in solate fermentation the microorganism which we use naturally are fungi or some kind of yeast and bacterial culture and since uh their hom Town what we often refer in our reference that Hometown that means the place where we are born where we are comfortable where we where we know the neighbors we know where we know the people so Hometown for this culture which we utilize are in solid state and then it has very hetrogeneous uh you know uh surroundings and this is what happens in case of solist fermentation that the substrate condition is solid and it is very heterogeneous in nature so apparently this could be you know related with their better performance for higher productivities for higher uh title so uh in spite of the fact that there are lot of advantages is specifically you know the the the in the area where we have very serious concern that is uh uh sustainability specifically for water and specifically for industrial affluent and also about the energy consumption in in uh several parts of the world uh including uh more specifically that developing countries so all these uh advantages add up for the use of solidate fermentation but then we have several uh uh negative point what you call disadvantages so it has less automation we all know and online monitoring is not possible what is happening inside the fermentation medium fermentation bed it is labor intensive that means because automation is less obviously it is a labor intensive mixing is difficult and this is one of the very strong negative point for solic fermentation specifically when you go for scale up because then Mass transfer uh is uh obviously not homogeneous and there is a heat built up and carbon dioxide accumulation so these two are one of the are the major negative points still not have been able to this is in fact a very strong chemical engineering uh uh problem and I can tell you that I have been collaborating still continue to collaborate with Scientists from uh uh one of the best institutes or universities in the world from us from France from Germany from UK and so on you know and uh I've been collaborating with chemical Engineers trying to tell them that this is the issue you have to take develop kinetic models and look at heat and mass transfer effects and try to understand how they could go be how this could be taken for a scale up engineering parameters and so and so forth but then uh none has been able to resolve this and until and unless we are able to understand this for scale up that how to tackle Mass transfer and heat built up removal without affecting the growth of microorganism without impacting their metabolic activity both are important is just not the growth but then their metabolic activity to form the desired product is also very critical and very important so that must uh be understood and then only we can go for scale up and once we are able to understand obviously there will be drastic change in commercialization of solid state fermentation so related aspect in this regard is a poor Iration because the bed is solid bed is heterogeneous so we we find that uh the uniform areation is very difficult it is related with the particle size it is related with the interparticle space and so on so this uh these are the related issues and uh which ultimately uh lead difficulties in scale up and uh then uh overall the process uh becomes very laborious or uh we need to develop large number of reactors requiring then higher space and so on so this is overall fermentation process we have preparatory step we have main fermentation we have manufacturing stages so raw material procurement uh normally these are this comprise agroindustrial residues this is one of the advantages in fact of solate fermentation that different kind of agroindustrial residues coming from uh different crops crop residues or for example paper and Pulp industry residues and uh uh other uh uh Industries which are essentially Agro based their residues could be utilized as CD stock for solate fermentation that is one of the advantage because by using them again we have two advantages that one is the cost we have lower cost and also second is that we help some way in development of sustainable green process by using them then we have uh the second one is the uh Water Act the microorganism example is given of your probiotic but depending upon the need uh to develop what kind of product we want to study or want to produce we have to look microorganism very carefully each of these are very important in fact most of the researcher have taken both of these aspect very casually and that is where we even are not able to publish high quality research paper in today's time when we talk of solicited fermentation because we need to put our mind into that and go into looking uh newer aspect and how to Define the research in a sense that it could have uh uh some novelty and it could have aspect of developing a process which has IPR POS generation possibility and which could be uh thought by someone uh for commercialization then water content is uh again very important at uh uh it is related aspect and macr growth is directly governed by water content in fact I will devop on these all details little in more detail little later then functional ingredients sorry there is a mistake in functional functional ingredients because depending upon kind of microorganism we are using we may need to provide a different kind of nutrients specifically for some uh products otherwise in most of the cases since we use industrial residues which are rich in organic uh know content and also which have which also provide or supplement uh uh you know uh growth factors that is nitrogen is there different kind of Trace metals are there so often it has been seen that we don't need to supplement many things in solate uh fermentation medium so but in certain cases we may require to add certain things then we have and looking at wind fermentation we have things to worry about the temperature of ambient temperature obviously the better temperature we need to motor and we need to we can try to control but it's not easy then we have issues of Iration and mixing as far as possible to you know uh do the Iration com comprising moist air as far as possible and if require we need to go for mixing but then this is a difficult issue because you know if you mix it for example if you take uh uh uh fungal culture it they are uh you know they have melium and if you do mixing along since they are uh interated with substrate particle mixing could lead and rather leads breaking of those Melia so uh and if the my are broken it it it impacts their growth and it impacts the product formation so usually mixing is uh desirable but need to be controlled at very very low rate that is could be 1 or 1.5 or two uh RPM and so on so it has to be done and it has to be uh thereafter uh uh uh you know evaluated we need to study the macral growth that how it is being impacted and it is not the growth only we need to also monitor the product fermentation and again water content as I said that uh there are Pockets sometime there are Pockets where there's a lot of condensation taking place and those places water content become very high and that leads uh you know uh poor growth or poor formation of product product so that is to be very carefully again monitored and there could be situation that we need to do in two stages or single stage in some product formation uh anerobic process becomes secondary and the first stage we grow the cells and thereafter in second stage we control the uh gases environment in order to uh produce the product in higher quantities then we have control as is mentioned that we need to look about water content uh temperature and mixing and then uh of course the product is there product isolation and packaging Etc so let us look at this uh points in little bit more detail that uh uh as I mentioned that previously that microorganism selection of microorganism is very very critical and very important uh for solate fermentation processes and then what we need to understand that uh what kind of St selection is very critical and very uh Paramount if we are not uh simply taking any strend from environment and simply making a study on some product is not a scientific study for a researcher who is intended it may be good for a uh you know to begin with uh someone starts master's degree look at the microorganism try to understand how a culture is isolated how in medium is produced how what is the way to you know look for a specific culture and so on so much but then for a specifically when you look at PS student all that they must first understand that even if you're going to isolate this St from where to isolate there are certain governing PL principle keeping into mind what is the main target one product is main target or to utilize a feed stock is main target uh or what so uh stain selection you need to look at at then discuss with a microbiologist specifically someone who is uh very ver with the taxonomy so we have to keep into mind product properties and taxonomy product properties and environment product part properties and the presence of substrate method of isolating uh organism it is a enrichment or it is a negative selection and then also we need to always keep into mind uh since there is a human involvement safety measures we need to ensure that we are isolating at first point uh generally recognize as sa grass cultures unless and until there is very critical need to look for some uh organism which even if have pathogenicity we could handle them provided the lab is provide is in having uh safety level two safety level three facilities so that is the first point to keep into mind when you start isolating the microorganism another important point is about strain development uh we know that by adaptation is one of the very important aspect for micro B cultures and by adaptation we can make several changes it is never necessary that we look only for genetic tools or molecular tools for modifying the microorganism uh because you have to keep in mind that uh again looking at uh human behavior uh you know if somebody is not behaving properly if child is not behaving properly by you know love and affection by some way or other by teaching uh uh in a proper manner by giving proper food by providing proper living condition we can change the nature of child or even of those are grown up including the researcher sometime we don't get every researcher on the same platform someone is not you know as serious as others so it is a teacher it is a success of teacher to adopt that uh bad boy or bad girl into right path and this is what exactly happens with the macrobial cultures we need to adopt them we need to train them we need to provide them proper feed on right time and so on then the culture start behaving the way you want obviously it it is practically impossible that the the microb is producing product a and we want it to produce product B that is probably not possible unless there is a broad relationship and then we need to adopt molecular tools to change this property then we also look have to look uh methods of strain development uh what is as I mentioned what is that we are trying to achieve and which method classical methods to begin are the best methods and we don't have to in fact keep looking uh always that for St development we need to look at uh molecular tools or genetic engineering tools I would not uh suggest that although if this collaboration is possible it is always good to keep running it as a parallel stream then St stream meant main this is again a very critical element and uh in my uh you know knowledge all over the world where I have been working or collaborating with the researcher majority of the people absolutely ignore this point at times often uh transfer from solid medium to solid medium or solid medium to liquid medium and maintaining on liquid medium is not good and at time too long preservation and thereafter transfer is taking place that also is not too good so depending upon the uh nature of microorganism the transfer should be uh done the stain maintenance what is the temperature of Maintenance uh and uh uh which kind of storage you require shortterm longterm medium-term accordingly the things should be selected and this information are normally available in classical uh microbiology book General microbiology book all this information one can find or in fact today is uh you know internet uh time if you Google it uh a lot information you can get but of course you also have to use your wisdom to decide the information which has come from Google which one is the best and which one to be used utilized not necessarily that uh whatever information you find on Google is all correct so uh it is also important that we need to uh choose right process I'll give a very specific example now in next this slide and next two three slides where a strain uh which has been uh which have been used using some method here example is shown by mutation and three kind of fermentation techniques have been utilized for them so his of ropas as you can see that four kind of muts were done and this St have been referred as R35 and so on right and when this Sumer fermentation was uh done for uh uh an enzyme production as you can see uh the four different strength gave four different kind of uh enzyme activities so the best was by the fourth one but then the same culture when it was used as surface culture but in sub much fermentation the enzyme units are substantially high if you look at the first culture they are almost 9 time more and so on but the fourth one was of course about 1.7 time more but then when the same culture was utilized in solid state fermentation if you look at the yields this was approximately know few hundred time and here if you look at the last one it is uh more than uh uh 124s so this is a judicious uh you know uh decision to be taken by researcher and uh nothing should be left unexplored and that is what what is called basic research so there should not be bias for so much fermentation there should not be bias for solic fermentation and a researcher anyone who would want to use one of these fermentation my strong suggestion would be that do not leave out any possibility any process because you never know where there is a brighter silver silver line right now look at this line This slide now we also again are working need to choose right uh process look at uh utilization of carbon Spectrum so uh there's a culture of again fungi that is this time is is tricoderma tricoderma V fermentation and uh so suppose uh you are exploring that you have a culture and you have been targeting to produce alpha males you know and that is how you started looking at isolation of the culture that is how you started looking at literature and then uh did the isolation and so on so if you have been using these the sources for enzy production you found that in uh uh different depending upon different carbon Source uh enzyme uh units varied hugely lactose which could be well expected because trma cannot utilize lactose but then trma definely can utilize sucrose depending on the concentration but it still produced only 17 units per milliliter Al amales and when corn starch was used in the medium the enzyme production was substantially high that because we were looking at Tic culture and obviously when there was starch was there the enzyme ructions are substantially high but then at the same time the same culture when used for Proteus production if you look at lactose so just now I said to you that we know that tricoda cannot produce cannot utilize substantially or in a preferred manner lactose but when uh you look at enzyme system and the metabolic activity of this culture we find that it is still produced 175 units you know uh proteas enzyme and when sukrus was the medium the activities were substantially high as as high as keeping into a maltic nature of the culture 351 units so equal quantity of uh uh proteas was produced whether F or the substrate was corn starch or substrat was sucrose and in in any case in all cases the proteas enzyme formation was far more High except when the maltose was substrate then the alpha males now the the point is that we were looking this culture for the production of alpha males but if you look at this enzyme formation we can understand the major focus of metabolic activity of trod vity was not on Alpha malus production rather it was on protus production an enzyme which was not not our targeted enzyme so carbon was wasted carbon is being wasted here metabolic activity is diverted for some on a for a product which we do not want to produce so that means use of this trodd for studies on alpha males is not worth to carry forward even if we ended up isolating this culture that means so uh what is the message here or what we can conclude from this slide that if you're working on enzyme production at least you must also look what are the related Associated activities the culture which we have isolated is go is producing this very important to understand in enzyme production Associated activities sometime have much more relevance uh than uh the main product I'll I I I'll tell you about this a little bit more when you move on to last part of uh the presentation so then comes the issue of micro organm this is one of the very very critical uh point I have mentioned earlier also to you uh that selection of right kind of macro organism is very important and how do we do it now normally we will refer to literature and uh suppose we are interested in production of alpha males and then if you look at uh literature you will find to know that oh my God I'm sorry it has lot of Animation more than uh uh you know three four dozen five dozen microorganisms are reported into literature and several of them in fact those which are shown in red fun have been claimed to produce in quantities which are commercially feasible there are several asperous species without any species then they also have been reported in literature as producing significant quantities which are commercially feasible acceptable quantities of Fales so this is another slide you know we reach up to uh several bacterial culture the previous one this is uh if you look at uh fungal culture normally they produce enzyme alpham which are food application while for other application laundry application detergent application uh leather industry application enzyme usually is from bacterial culture so if you look at here in this page also you can see at least three cultures have been uh defined or you can find their description for commercially commercial applications we continue the list and then we still continue the list so 64 micro organisms are reported or have been reported into literature which produce uh Alpha amales and these are the research or patent information so which one you are going to use if your target is Alpha Amal so you need to understand firstly the application if you are primarily looking food application then it is good to look at uh uh fungal culture and if it is for other application it is good to look at at uh bacterial culture and then thereafter we need to produce if you look at the couple of slide which I showed that strain selection slides that what are the parameters we need to consider then perhaps we would be able to make a right choice now we continue to this one that need to choose right microorganism this is one aspect I mentioned about the formation of one product but if you look at uh this one uh suppose we have a culture and we want to produce uh we want to use it example is sh enzymes produced by it's very strange kind of Animation sorry so asperous Niger has been uh if you look at literature again has been reported to produce at least 21 kinds of enzyme and those which are shown in red fun in the slide are the one where there are patients claiming that it produces commercially feasible or commercially acceptable uh quantities of uh uh enzymes these enzymes so how do we make a selection suppose we have a strain and if you start working only on um uh tnis production possibly it may produce better zenes or suppose our focus is z xenes production and we have a culture of as pilous Niger and we think that yes it is producing good quantities those that good is very in fact dangerous word in scientific research writing good significant High quantity these are very relative terms and unless we Define that how do we claim this good comparing with literature unless we Define this significant quanti is comparing with literature sorry it's not very good idea so again if you have taken into consideration that what is our main targeted product and if you try to keeping this slide into mind also evaluate at least Associated activities being produced by Asus Niger then perhaps you would be able to decide that whether my strain of asparagus Niger is good to produce xylon or not okay so we move next now we have uh uh understood all those Point very well so we look at process uh methodology now preparation of solid substrate sterilization process rising of suitable inoculum then inoculation of the moist substrate incubation in appropriate culture vessels or reactors maintenance maintenance of optimal conditions primarily the pH temperature Iration flow method Etc then harvesting of fermented solid drying extraction of product and further down string processing if necessary so selection of microorganism I have already uh gone into great detail and then we have uh substrate as I mentioned that uh it uh depends upon but normally agroindustrial residues should be taken are utilized as a uh P stock then we go on to process optimization looking at the basic aspect of process uh and then product isolation and purification in case of enzyme largely we feel that it should not be uh necessary at many time uh to to isolate the enam for example in talk of biofuel production the enzyme which are uh which are produced in ssf the W substrate medium directly should be mixed with uh the the pre-treated substrate for hydrolysis and and we are able to show it that it is far more effective and obviously it is far far more economical large number of process steps have been removed large number of losses have been uh the enzyme losses have been removed and uh uh obviously the cost have been removed and and then uses of water and the time Etc everything uh such uh everything has been removed so preparation of solid substrate we know that uh uh we need to add uh uh water and when you go for scale up we should try to avoid using even uh distill water depends upon if uh you have uh uh the Water property what is available from Municipal Supply your uh Institute what your pallet plant whatever if it has a pH desirable pH and much more than if it has uh not any heavy metallic impurities heavy metals or calcium and so much so on then uh we simply can utilize boiled hot water ization of substrate as say that we can go for normal aut caving method we require longer time uh because uh compared to liquid fermentation because it's a solid bed and depending upon the bed height the time of sterilization is fixed in fact this is one of the uh negative Point suppose we would like to or to CL a bed of 1 meter there are reactors one of the first such reactor was developed uh in France where they claim that they can do calization without having uh unsized Pockets uh effectively uh but then it could not still get commercialized because of some of the uh problems and of course one of them has been the high cost involved into its uh Fabrication in ulum uh preparation and this is again one of the uh uh positive point because uh uh most of the time when we use a fungal culture we you can utilize spores and in fact spores can be produced in large quantities and stored at uh uh you know low temperature so in lesser volume we can produce uh High High number of spores High number of inoculum store it and utilize depending upon as and when required even if it is a direct M inoculum this is also achievable in much easier way when compared to other processes then inoculation of the moist substrate so when it is a product formation for example enzyme or organic acid or estro Etc it is always good to use spores and their uh mixing is not a uh difficult issue it has been well achieved at larger scale but when it is a for example single cell protein usually then use or cage the normally we use inoculum of fungal Mycelia and then at times this also poses some issues in the Sens some places the M spread is not uniform because of the heterogenous nature of uh substrate then optimal condition uh pH is one point that must be taken very proper care because you know that if you use agroindustrial residu specifically if you use uh uh uh uh Solid State uh bed there is a there is a this this uh medium have very strong buffering capacity for example if you take wheat brand if you take rice straw rice brand all these have very strong buffering capacity so it will not be possible suppose unlike liquid fermentation where we take for one normal sodium hydroxide or one normal uh hydrochloric acid and set the pH of the medium in case of solidate fermentation that is not possible if you add one normal uh hydrochloric acid and thinking that by this formula uh adding this much uh uh quantity will bring the pH of the fermenting bed to medium to 4.5 that is not possible because of the buffering capacity of this uh agroindustrial residues primarily the crop residues so in this case if you have it is if it is essential to to change the pH we need to look at uh the pH solution for example if the idea is to make the pH of the uh with brand medium 5 maybe we have to prepare a pH solution of one and then add in some quantities and then see if the pH has changed or not same thing is if you want to check take pH to 9 fermenting medium that is solid bed medium then we have to use a very high alkaline solution to set the pH so in fact this should be taken as one point in favor of ssf and that is effectively what happens when you are concerned with fungal growth in uh medium for enzyme production Etc we don't have to precisely we don't have to worry about pH temperature is important aspect and temperature of fermenting bed is much more important and we we need to understand that there could be Pockets because of accumulation of carbon dioxide there is a uh because of the uh aerobic nature there is a respirometric action and then we uh today we know very clearly that how it happens and then uh depending upon the efficienty of the uh you know Iration the temperature could be well controlled limiting factor obviously is the bed height bed thickness and we cannot go uh in fact something like 7.5 cm today is considered as the maximum which could be utilized in a normal tree kind of fermentor and so on then harvesting of the fermented solids depends upon suppose if it is a single cell uh protein nutrient Rich protein for Cle feed Etc nothing is to be isolated for enzymes as I said to you today for biofuel application it is highly you know uh desirable that we use the solid wet fermented material that means the enzyme production should be carried out at in C2 wherever the P the plant itself should have a in module to produce enzyme and the fermented Mass should be directly added used for added into hydrolyzing medium then uh uh there are again situation where drying and extraction of product is required uh for example you want to produce enzyme and enzyme should be you know extracted using a different uh way normally water or with saline and they should be dried uh you there could be some filling material Etc these are commercial secret and nobody would like to uh know share them so normally uh these are the four cultures which are predominantly so far have been reported in uh product different prodct formation except some patented specific processes where they would not like to reveal the specific name or uh uh of the culture because by Patent regulation it is never necessary you can say biological agent specific biological agent property describes property and give its ACC number in patent depositories so uh now let us start with specific example that is we know that Co fermentation uh in fact it became so much phenomenal that for large number of years people have been talking ssf as a CO fermentation so all of you must be knowing that what is uh Co fermentation how it takes place and uh it is one of the very common in East Asian uh countries and uh commercialized uh centuries before and so on so the what are the processes the kind of setup some other details are given here and enzymes involved are essentially alpha males glucal and acid prote but then there are several Associated activities and that is how Koji from one industry to other industry is very different and the product is also very different and some product are are become very famous or liked much by uh the consumers and it is only because the associated activities which I mentioned in the beginning to you are very different and some are able to utilize are utilizing some cultures which produce very desirable Associated activities associated activi means production of different other enzymes in a small uh quantities so kif fermentation again uh uh that uh what are the other steps and uh uh more details refinement is also considered as one of the very very important aspect including what kind of fermentation or filtration process we are employing so in fact people still believe much more in Co process the traditional way rather than uh uh going for looking at for some uh uh newer aspect there are moderation there are developments but then uh it it is primarily governed by the traditional uh factors so if you look at this one the fermentation process creates over 200 different flavor compounds so this is what is very important for uh you know the successful production and commercialization commercialization of uh Ki so process parameters uh if you look at which are the process parameters we need to control at chemical factors as mentioned that nutrients very important growth factors temperature and oxygen and then biological factor microorganism we have already uh uh discussed this points and in uh in the process if you look at a specific characterization at solid Matrix different kind of f stock and uh of course rice BR wheat brand is the most common then we have liquid medium basically it is a mineral medium sometime nitrogen is uh also supplemented and depending upon the nature of rice BR is different saw dust is different sugar be p is different so depending upon their chemical composition about their minerals content we need to add additional uh uh uh nutrients inoculation that depends upon what kind of process you're using product formation using then uh uh uh we need to uh decide doses of inoculation temperature again depending on the product formation it could be as slow as 5 to 95° and also it is directly related with the microorganism which are using the moisture content 50 to 85% these are you know range given not necessary that everything will be primarily defined by these parameters only agitation it could be inter intermittent it could be no agitation and it could be a constant rotation depending on the kind of reactor system we are using then if you go for uh Iration 0.5 to to five vvm and uh duration could be up to 24 hour or fungal foration for example if you're looking some enzyme it could be as high as five days also it depends directly the kind of microorganism and product cellules production takes longer time and xinis by bacterial culture takes much shorter time and so on D stream processing or centrifugation uh for many applications process parameters so the size of substrate particle in fact this is one of the very important aspect because we have to keep into mind that when you use a smaller particle uh they provide larger surface area in solidate fermentation whatever microorganism we use they normally grow on the surface of the substrate part particle and so larger the surface area larger will be space available for uh the growth of those microorganism but then smaller particles even if they provide larger surface area they also create a compact bed and if the bed is compact there will be difficulty in Iration so it is uh not very good uh situation then then if you are not able to do Iration uh so air penetration will be one uh very big issue but then if you use uh bigger particle size the bed would be much more you know porous vo volume will be uh larger and Iration would be easier but then there will be lesser surface area available that means uh the space available for the uh growth of [Music] microorganism will be less so both are contradictive we want as large as possible surface area but then we also want proper Iration to take place so so from this we understand that uh we need to make a compromise and this is again one of the very critical scientific and wise decision to be taken by the investigator that what kind of substrate particle we have to use it has to be a mix of uh small to smaller and then big to bigger and this is where you have to do the experimentation and understand that uh uh the level of particle size and they're mixing we also have uh uh to understand substrate characteristic that excessive water tends to aggregate on solid particle making Iration difficult and when the Iration is difficult the growth will be impacted and low moisture environment fce growth and Eastern fungi so this could be well understood if you keep into mind the aspect of water activity if you look at Water activity of microorganism that is scale water activity is theoretically one of pure water and uh uh the the water activity Factor required for the fungal uh growth is uh much lower than uh bacteria so normally bacterial growth is well around 0.9 water activity that means the water should be free flowing and this is how the basic information or basic definitions and thoughts of solate mation were there in the olden time that bacterial cultures are usually not uh good for solic fermentation but then uh in science nothing is impossible and we have proven that uh by adaptation and by manipulating the growth condition and Engineering the medium we can very well cultivate bacterial culture into medium and produce different kind of products we have shown it for lactic acid product and we have sh it for different kind of other products uh uh using uh bacterial cultures so the yeast and uh uh fungal culture depending upon the the kind of strain we using the water activity could be Z between 0.5 to 0 7 or even uh in certain cases uh 0.8 or 0.85 is specifically we are looking at uh the uh cultures fungal cultures which grow on uh cellic feed stock rice straw uh wheat straw sugar can bagas and so on so in this kind of f fungal cultures have higher water reactivity requirement compared to the cultures fungal cultures which grow on a starch if it is St and same is true for East normally East 0.5 to 0.6 uh water activity so water control in the medium is important and this uh in fact is done by a uh spring uh intermittently water and if there is a mixing also then uh it can be well controlled and together both of them uh remove the heat as well as with foration we also remove moist Iration we also remove carbon di oxide so from this once we have understood that the microorganism the fedus RO that is substrate and the process parameters now I'll take uh another few minutes to talk about design of bioreactor and then we'll come to the last part of my talk about what we have been doing as briefly telling you what we did and where we have reached so uh ssf is mostly you know the process we know at least commercial skill are batch processes and different kind of Bio reactors have been utilized but then uh uh mostly uh it is a tree kind of ferment the concept still evolves around that it's a very traditional uh design of stay fermentor you can see here and this another one that uh usually small often simple reactors and small or medium scale because as I mentioned that we still do not know how to tackle uh heat built up carbon dioxide removal in larger bed size and also the still the issue of sterilization continues with larger bed size so Tre Bor is one of the most common common reactor we utilize an ssf uh also there are large number of react reports and uh uh this also has been uh commercialized in fact uh uh for food processes and other uh spefically food processes that is a kind of pad bed reactor we have been working in different kind of paded reactor horizontal vertical and so on and uh although there has been always issue even in this um uh reactor uh about the scalability in this mixing is uh very well possible and uh the traditional drum kind of reactor which resembles like a pack bed reactor I'll I'll talk about that also little later but then the biggest weakness is that bed depth we cannot go much High even in uh large reactors cylindrical uh drum kind of fermenter is the one which I mentioned and it has been uh also commercialized for multiple application in fact specifically for uh development of uh uh single cell protein and uh as you can see as the visual figure is there rotating drum and state drum this state drum kind of reactor we have you know air flow we have rotation uh and we have material so this ensures that with this pedals which are present into this system that uh the medium keeps uh moving and then there is no aeration of the medium particle and also there is no uh uh blocked of carbon dioxide Etc uh here we have two ways to control the temperature one is that we keep uh running in a in a jacket cooling or hot water and the other one is that we keep passing in this Central pipe where there is there moist and sterile air so that also takes care of bed drying so this has some automation that you have possibility to keep reading at what is the relative humidity at real time and what is the uh know temperature temperature of bed can be monitored continuously temperature of the gases uh environment in the chamber can be monitored and those gases can be you know uh can be uh utilized for determining the growth of the culture using respir respirometric principle which are very common today to determine the growth in solate fermentation so some other that is primarily what we refer as Eed erated Biore reactors that uh we have uh the first one is looked at estad Ed bi reactor the gas solid fluidized bio reactor uh and then we have a rocking drop kind of bi reactor and on all of them there could be possibility of force areation with moisture which can take care of heat accumulation carbon di accumulation and also uh possibility of sprinkling the uh water in the sense that uh uh the can be moined and so on this are other example that bi reactors that rocking drum bi reactor this is also available commercially in a small size the first one is uh usual that how it is there and then second one is um uh you know uh what is happening inside that rollers Etc are there and so this is also this being manufactured but only on uh a specific order given by the client and this is very compact as you can see this is how it uh looks like so uh when we want some Automation and we want to move from laboratory process to some level of uh scale up this is one of the important uh development and this reactor could be utilized for enzyme production for other product formation Etc this basic design in all kind of uh uh this 34 uh reactor I showed to you that uh and with much more automation we have exhaust gas analysis uh no possibility online monitoring we have liquid Supply possibility we can spray it we can pass through the uh Mo then we have in the center pedal type for mixing because this is the one which is considered most efficient even not to damage The Melia and if it is a uh in fact in real time we can also continue to uh extract the product when it is formed into the medium by you know simple passing through the liquid that it could be s plan it could be just water and then uh uh monitoring of growth monitoring of uh temperature monitoring of uh uh uh medium uh humidity can be online so this in a setup how it looks like that what are the things we require in a laboratory that we require a obviously to stock the culture in a cold we require cold room for fedock ETC clean bench for a septic work incubator and then different kind of other fermentor Etc and uh uh what kind of fermentor you can you would want to buy and what are the Avenues for you to procure it several P kind of P plant kind of reactors are available in the market or they can be constructed according to a specific requirement of the Institute or the group so look at the scope that uh still the question comes the potentially many high value product including primary and secondary metabolite are still being exploited all over the world and there are different kind of successes uh depending upon uh the interest and report a large number of patents are still being filed continuously on high value products and secondary metabolites but then we could increase the scope to bi remediation and biodegradation of hazardous compounds biological detoxification of AC industri residues by transformation of prop residues Etc by pulping and in fact biopulping again has been adopted by some of the paper and Pulp Industries the proposed uh configuration that uh specifically when you talk of second generation uh uh banol and when you talk of inc2 uh utilization of the product as I mentioned to you that uh after you know fermentation we don't have to follow all this step that the cultivated fungal culture directly can be ad mixed with biomass for sacrification and this is one of the thing already being you know put in some places I know some uh places in Hungary some place in which is a facility of no n s they have put it and uh it is being put uh in India as well and in Mexico also one of the altic uh Manufacturing Services which has put uh which is enzyme manufacturing which has put a demo plant for ethanol banol and they are also using that fungal cultivated directly uh this entire biomass mixing with the uh feed stock for sacrification bioanal production so with this uh I'll now go to the last part that what we have been doing that uh solate ferment research and technological development and IND industrial linkages what you have been doing at my previous Institute and to some extent I continue at my current Institute so basic studies we need to do that this example I'm showing that this was one of the first study we could show that CR trod herzum it which produces kenas enzyme and which arrested the growth of heterogenic pathogen col Tri com by breaking the high fee so this is the this control one and this one you can see when it is growing the my are broken into different places so so this was very good example to show by sem and then uh uh we carried out large scale production and very positive result and then we did with industry actually field work uh for its commercialization that's one example I have shown but uh uh we have several patents on ssf we have commercialized several and then uh many enzyme and Lucin production by stasis one specific strain then methine amop pepti production by againto strain and then figh for Feed application this has been commercialized by asparagus Niger uh food grid alpha males using asparagus nigers again commercialized transport industry then paper and Pulp industry specific requirement for zines again uh commercialized is it was through basus culture then uh we have also done inulin by st stop stopus stopus cous species then uh fungal spose that is how we showed that Sports could be cultivated in large quantities and stored in the required conditions and uh glucal this was of course the first enzyme we started to work onto this one these are the four Technologies we have transferred to Industries alpha males for food application in fact the as a several time I mention to you that Associated activities so in this particular case it was the uh the associated activity which imparted very you know desirable uh uh taste and and uh you know order or whatever you can say the aesthetic value of Bakery product increased substantially by the industry and that is how it was taken not that the only the enzyme yields and productivities which are important so Associated activities are very important and if you work on enzyme you should never ignore them then we transfer that xanes which are cellulous free for uh for uh paper and Pulp industry and then we again transfer the feed enzyme to for which is produced by as Niger for feed industry and uh uh the next we transferred was a specific cellules for de inking application using a fungal culture it is it's a mutant developed by us but mutation doing traditional uh uh xray uh mutation we have done uh several other consultancy project for enzymes kitus as I mentioned at Large Scale field trials induan is for another uh uh paper and Pulp industry and nit and lipases for a specific application uh by a Biotech Industry this I have already mentioned to you so in fact if you do uh keyword base uh index Sear you'll find that on solidate fermentation we are the uh number one in terms of seci Publications this what is shown into scopus we have put a pilot plant in our Institute that is at Center for biofuel in uh nist and this is completely automated modular system depending upon the requirement we can use one module or two module and the best size is 50 kg material it is essentially based on tree that is we utilized perforated tree made of his um aluminium and as you can see this is a uh control system it is completely uh the entire uh foration system can be controlled uh and monitored for uh parameters which I mentioned using uh computers and uh one of the strongest thing that we have all over the world we had uh we have this kind of uh uh collaboration in started with Brazil and then France and then everywhere into World more than uh a dozen countries or more than 15 countries we have collaboration somewhere it is over somewhere it is still continues on solate ferment depending upon the product and uh interest are very different but it continues so the last slide before I thank professori that science happens when people seek to discover their place in the world solic foration has been researched by hundreds of thousands of people people keep looking still but only those who want to make a place in the world and then from this uh perspective they look at as in a very different uh manner keeping into mind mind the basic aspect which I have shown you about the microorganism about the medium and so on into consideration so then only you'll be able to uh develop a novel aspect or new aspect in ssf otherwise it continues to be uh you know simple uh you know ssf studies which have no novelty so think a bit more read bit more unless you read a lot you would not be able to understand which way to design the uh your ssf in order to uh look at IPR issues and in order to uh think of application of product for commercialization which is a great need today all over the world and much more so for developing countries and including Indonesia of course including India as well so I'd like to thank uh Prof sadii for uh uh giving me this opportunity to share uh My Views and some scientific uh discussion on solidate fermentation so thank you very much and if you have any questions or anything you want to discuss I'm available thank you thank you P so uh we will have some uh I think it's a question uh I think it's a g to could help us yes please so that one is the the question uh from the audience this one from hro chrisanto so I'm interested in boping research or biod deification youing S so so hro this is a you know it's a question which you have to tell them pros and cons Biol delignification obviously is is slow process it can be fastened by increasing the quantity of enzyme of course it is not possible that use x quantity and then make it 10x and thinking that the pro the speed of pulping will also increase 10x there is a limit but then this is the first aspect so what are the advantage biopulping process will offer this they have to keep and one of the major aspect is the Environmental sustainability if the industry has no interest into that one it will be practically impossible for you to to you know make them understand that or make them interested and uh so environmental sustainability is one parameter which cannot be you know uh taken into account in quantitative terms it can be taken up it is being taken up in European countries where carbon footprint is being looked into the greenhouse gases emission is being looked into and the impact on environment by discharging hazardous a FL fluent is being lived into so uh you have to make them understand that what are the basic advantages it is just not only one consideration of economic economics and it is just not consideration of only the the time which is being taken because you can modulate your process into batches according to size of production of the industry and then similar way you uh uh in similar proportionate way like uh one single stage and two stage system you can develop and the biopulping process can State into many modules no today you start with one batch tomorrow you go for the second batch and then they may take a longer time but even then taking into account the size of Industries industrial production and fractionating into some part and then making biopulping process batch process uh taken into account your residence time for the treatment so these are the two options available to you there is no other option and this is where if the industry is cooperative industry is able to follow it the scenario will change but then you have to try taking these two scientific component as well into mind right okay just move on to the second one second one is a kinetic model how to retry is a simple model which you have to utilize for the mass of fungus you don't have to separate biomass from it is impossible no one can separate any kind of biomass whether it is a fungal yeast or bacterial and indirect estimations are the one which are taken place so you can also continue to use the traditional method of looking at uh uh you know uh uh estimation chemical estimation but if you are able to uh create this uh uh uh simple system which are described into literature that is the most accurate way to determine the fungal biomass okay then uh the third one is about the iner function yes inducer function on fungal man inducer function it depends upon firstly what is the culture you're using what is the is it for example if it is a uh product uh and if it is a cult which requires some induction if it is a inducive enzyme and if the medium does not induce it medium component do not uh uh induce it then obviously you have to look at uh some inducing uh product but uh uh if you can have if you can tell me your specific question that what enzyme you are looking what product you are looking what culture you using what substrate you using probably I can uh tell you something more okay it may be you may so that uh I think said Dia oh you like to X more uh yeah yeah yeah you can ask me even later you can uh give your question to and you can send it to me you can Conta it's not that it is just today we can continue yes yes okay prob next uh probably we move on to the next uh question yeah uh G please uh that one is from Mustafa yeah with there is a there is a direct relationship between the master content but you have you cannot take Master content stand alone you have to also take into account water activity that's is much more important as aspect because both are Interlink it is the gaseous moisture content which is in equilibrium with the substrate moer content that is what is uh uh taken into account and that is what uh together governs the microbial growth in solid medium so water activity is not a very difficult aspect to determine but those of you who work on ssf regularly you must optain in equipment that is water activ meter it is a simple equipment and you just need to put into your you know uh system and this will give you direct reading it is a handheld system very it's not expensive I do not exactly remember the cost but there are several companies today into uh manufacturing so please consider water activity together with the moner content because that is what it is not simply that 5 medium and uh that will tell you the moisture content present in the medium but not it will correlate with the fungal growth okay okay and uh the next question from REI about the solic for the pharmaceutical products uh let me tell you that let me go to the history back the first solate ferment first antibiotic in the world it was produced by solate fermentation only that was when we learned about what you call wonder drug pencillin huh which was uh it was referred as under drug because in second world war it was used like you know a what you call as a magic drug because the there were people Soldier soldiers were getting infection and by application of uh penicillin practically everybody was getting cured so that is how it was called Magic drug or wonder drug and uh this was produced interestingly by soled fermentation but then it was the uh it were the scientist and the researcher of that time for some reason never inscribed into literature they started working more on submer fermentation and then the whole world moved towards submerge fermentation and S got neglected but then to we know that secondary metabolite are the the very interesting product to be produced using solate fermentation so there are large number of of patent still as I mentioned to you being taken and uh what is very interesting that uh many product which includes primarily secondary metabolites and much more so is enzyme it is never necessary for the industry to reveal which method of production has been utilized and so they were never so they never reveal that uh which is the production system they have utilized in fact if you look at enzy Industries all over the world the biggest industry which is Novo Nova has the most modern ssf reactors and of course they have some much fermentation but they never reveal which enzyme has been produced by which method because it is not required by any law or any you know uh uh regulation by any country to reveal the method of production and it is so most secrety because it is a very very uh you know competitive word so nobody would like to reveal I know many Industries I I have visited Altech industri the US based Industries all over the world Novo have visited many indust Industries every of them is having uh some much fermentation as well as solic fermentation and they don't reveal exactly what they are using so uh this is very interesting that it's very good that your topic of secondary metabolite but then you have to be very specific which product you want to look at and why you want to look that one and what are the microorganism you have available or you're going to Sol unless you have a a you know reasonably good microorganism you know it's not very good good idea to move on to such processes techn thank you and then the next from Brian is uh the industrial practice in disposing the solid phase from the SSI this is in fact there is no disposal this is one of the advantage because in practically every solic fermentation we utilize only grass culture generally recognize as safe so uh when it is a fungal culture you can utilize this for multiple application you can otherwise uh if nothing is there multiple application if it if there is no you know inhibitory nuclic acid content it can go as a feed stock or it can be further used as uh for composting and so on there there is no waste generated from Solid State uh fermented material okay thank you sir uh good could you the next page please uh that's from from Ry uh it's a difficulty to grow the in plastic media you not have difficulty there again here you need to look at the specifically the surface area and there this the the uh very if you look at uh large number of Studies have utilized the plastic media which have you know what you call Pings that means they should have hole in between and the medium should still require some kind of support you cannot simply power your liquid medium on this one otherwise uh uh so it should it could have for example you can supplement it with uh some G nitrogen agent which do not have which the microorganism itself cannot utilize and then uh put into liquid medium you can use for you can use uh agar for example H melt it at you have to use a specific V which melts at 32 or not more than 30 37° put your um pre-inoculated uh nutrient or the medium liquid medium into uh agar medium 1% augar or little less 0.7% agar and then power onto plastic medium then trickling medium then it should grow well okay then it is from pya uh regarding the amulet so the question is it has an Associated activity and attent uh come to the additional uh selling valure for the industri so that it may there's another values not only selling the ulist haha this I cannot tell you this is trade [Laughter] secret yeah yeah yeah yeah it is I'll tell you that how we have to do it firstly we need to look at enzyme Spectrum this not very difficult thing you need to look at uh what look at literature what are the enzymes reported and thereafter look at uh your own products Spectrum with different kind of uh carbon sources and this will give you good clue that uh what is the metabolic activity uh what how much metabolic activity is being targeted onto men product formation and then which are the other enzymes being produced even if they're in Tres and then that is how you have to look a specific application whether it could go for paper and Pulp industry whether it could go for food application and so on okay okay then from gundur it should add the the question is about so the effect of the Iration irration in the solid state fermentation oh how yes this is a this is a good question but then uh in order to determine that how efficient is the method you have a control system a flask always running parall or in a system where you have not done irration control system without irration and if you look at a total carbon present in the medium then you have some idea that what are the Els and what are the productivities and then uh you have to start looking at 0.1 vbm and so on obviously you cannot do a large number of experiment but if you go onto that factoral design kind of experiment and develop the models and to see that how many runs can be done and then minimize the effective runs to ensure taking into consideration which Iration rates are significant and that is how it is determined okay then thank you uh it's still I think a question G that's one is a uh what is the isest and the cheapest uh biion technique for the management of poed uh so so that that's one for the soil bio remediation this is again very important and this is where we have uh quite good success in fact we have been able to remediate uh even uh uh highly toxic soil in my own City we had Industries which have been making pesticides you know and pesticide uh had been simply disposed into open environment and there have been no regulations and inside industry nobody bothered and also you know the problem of uh not abiding by the law and bribing the uh inspectors and so on so there's large area more than uh uh close to 100 acres and all that which is simply now uh closed with no entry for the people and uh with the intervention of Supreme Court then we could get the sample we did the studyed and uh since last three years we are doing field work on remediating this uh so we prepare precisely we prepare a bacterial Consortium of uh different pomas species and uh we make a small field plot of for example 50 by 5050 and then increase it to 200 by 200 feet and uh we spray this culture on the top layer and then we mix it and we do not do any ariation and we leave it but then we keep uh uh controlling the moisture we do moisture supplementation in between we don't supplement any other kind of nutrient and uh after uh every 10 days we keep looking at the uh residual uh this pesticide and now we find that uh in approximately 30 days time we are able to remove close to 75 to 80% but that is the limit even after 40 days it remains still the same so even if you able to receive 70 or 75 this is great relief and then uh this top soil could there after be removed and then we can go onto looking some other way to remediate remaining you know so this is very important uh you know topic and if you could uh work onto this rning it will be great uh uh help for you for the nation you must try it in a larger way but then look at a specific uh chemical which you want to remediate and then accordingly from the literature select culture C if you have your own culture that's the best thing otherwise look at cultures available in culture collection where there is no uh you know IPR restriction then develop your own Consortium it's pure cult is very difficult Consortium only and uh look at if the soil which are going to remediate if it is it has still any culture if it has still any culture that would the best to isolate enrich and adopt because that means the culture is growing under the adverse condition and probably using that that uh pollutant as carbon source for its growth and survival so that is how you have to look into this one okay thank you then uh that's one is from rainy again so that one is to decolorize the textile V water using the ssf uh with the white Ro fungi to remove the aodi the color was cly removed but the scod was increased any suggestion in fact when say color was removed it is in fact the first you need to look that what product were formed we know several time not all the time but several time we know that uh when we degraded degrade this kind of aod there formation of a smaller molecules and in fact sometimes they may be more toxic even than the isodi itself so the first we need to determine what it it is not that the color was uh uh I know uh removed color removal is one parameter but what were the product which were form you need to determine first that and I I cannot understand this one how Cod was uh increased because this must be in my perception an experimental because experimental error the carbon content cannot change the total carbon content would remain the same whether it is a aodi or if it is something else in fact it should reduce if the culture is aerobic and if it is utilizing asiz as carbon source for its growth so some carbon will be used for biomass C biomass formation but it very small quantity but then definitely if it is aerobic culturing uh carbon will be utilized for carbon dioxide formation so C cannot increase this is this is a big question so you need to look into this again and try to see that uh uh where something is going wrong I don't know which Cod method you looking one solution could be that you look at total carbon at control that time zero and thereafter carbon at different time of intervals which will give you a good clue whether uh your Cod removal analysis C analysis is correct or not okay I think sir one is the the last question uh G it may be we already almost up to the time but we uh the last question okay wait a bit oh so that uh from francisa yeah so you when you work on uh uh shell wte you need to First Look at uh what's what are its chemical composition what are the uh you know and accordingly you have to first find a culture which could utilize them primarily as its growth for its growth so the idea is what is your idea you want to utilize a shell waste for any product formation or to you know or to Simply it's a disposal that's the first point you to determine or you want to use the shell uh uh waste for formation of some other product specific product so that is how you have to look at the uh AAL culture for uh growth and activity do you have a do you understand my point yeah uh do the chemical analysis of the waste look at what are the chemical components of this waste and thereafter determine what is the kind of carbon Source from in this waste which can be utilized directly by the culture if the the carbon Source cannot be utilized directly you might need to supplement some additional carbon Source in order to initiate the growth of culture and thereafter see if the culture can utilize the carbon Source present in the uh in the waste or grow a culture which uh supplementing small quantities of carbon source which produces some enzyme or some organic acid which can decompose or grade shell waste to a smaller molecule where some carbon Source may be available for growth and activity of the culture okay I think uh we are already close to the 12 in our time so I think I would I would like uh to thank you to Professor uh Pand it seem that uh the the the the interest of from the student and also from the our faculty members is very high it maybe next year I would like uh to ask you again to uh give more Technical and or more uh research regarding the uh solid state fermentation or will be uh your idea in the in in in the more technical one so we I would like to to thank you to Prof uh P probably we will take a picture before we leave and uh could you uh G could you help us how to take the picture yes but okay um I will I will stop the YouTube stream first [Music] yes so