Introduction to Bioporcess Engineering c
- 1. Overview of fermentation process and media design Module-1
- 2. What is fermentation? • Fermentation – Definition • Fermentation is an enzyme catalysed, metabolic process whereby organisms convert starch or sugar to alcohol or an acid anaerobically releasing energy. The science of fermentation is called “zymology”. In fermentation, the first process is the same as cellular respiration, which is the formation of pyruvic acid by glycolysis where net 2 ATP molecules are synthesised. In the next step, pyruvate is reduced to lactic acid, ethanol or other products.
- 3. • Types of Fermentation • Homo fermentation: only one type of product formation Eg: production of lactic acid or lactate Glucose + 2 ADP + 2Pi → 2 lactate + 2 ATP The homofermentative bacteria can mainly produce lactic acid through the glycolytic pathway. The most common forms of bacteria that can perform this process include Lactococcus lactis, Streptococcus species, and thermobacteria species. • Hetero fermentation: more than one product formed Eg: production of end products like ethanol, CO2 and lactate Glucose + ADP + 2Pi → lactate + ethanol + CO2 + ATP Some examples of heterofermentative bacteria include Leuconostoc mesenteroides, Lactobacillus bifermentous, and Leconostoc lactis.
- 4. • Types of fermentation (based on end products) • Lactic Acid Fermentation • Lactic acid is formed from pyruvate produced in glycolysis. NAD+ is generated from NADH. Enzyme lactate dehydrogenase catalyses this reaction. Lactobacillus bacteria prepare curd from milk via this type of fermentation. During intense exercise when oxygen supply is inadequate, muscles derive energy by producing lactic acid, which gets accumulated in the cells causing fatigue.
- 5. • Alcohol Fermentation • This is used in the industrial production of wine, beer, biofuel, etc. The end product is alcohol and CO2. Pyruvic acid breaks down into acetaldehyde and CO2 is released. In the next step, ethanol is formed from acetaldehyde. NAD+ is also formed from NADH, utilized in glycolysis. Yeast and some bacteria carry out this type of fermentation. Enzyme pyruvic acid decarboxylase and alcohol dehydrogenase catalyse these reactions.
- 6. • Acetic acid Fermentation • Vinegar is produced by this process. This is a two-step process. • The first step is the formation of ethyl alcohol from sugar anaerobically using yeast. • In the second step, ethyl alcohol is further oxidized to form acetic acid using acetobacter bacteria. Microbial oxidation of alcohol to acid is an aerobic process. Butyric acid Fermentation This type of fermentation is characteristic of obligate anaerobic bacteria of genus clostridium. This occurs in retting of jute fibre, rancid butter, tobacco processing and tanning of leather. Butyric acid is produced in the human colon as a product of dietary fibre fermentation. It is an important source of energy for colorectal epithelium. Sugar is first oxidized to pyruvate by the process of glycolysis and then pyruvate is further oxidized to form acetyl-CoA by the oxidoreductase enzyme system with the production of H2 and CO2. Acetyl- CoA is further reduced to form butyric acid. This type of fermentation leads to a relatively higher yield of energy. 3 molecules of ATP are formed
- 8. • Type of fermentation (based on type of substrate) • Solid State fermentation • Submerged fermentation • Solid state fermentation (SSF) • SSF is also referred as surface fermentation. It the process in which the microbial growth and product formation occurs on the surface of solid substrate. The organisms grow on the moist substrate with little or no ‘free water’. This method is suitable for microorganisms that need lesser water for its growth. This method is economically viable, as the fermentation is carried out using agricultural byproducts. Several agro crops such as rice, wheat, maize, barley and industrial residues such as bran, straw, sugarcane bagasse, oil cakes, corn cobs, saw dust, fruit pulps etc. can be used as substrate. These natural raw materials serve as a source of carbon and energy. Few examples of solid state fermentations are mushroom cultivation, bread making, production of miso, tempeh and soy sauce, cocoa processing
- 9. • Submerged Fermentation (SmF) • Submerged fermentation occurs in the presence of liquid nutrient media seeded with microorganisms which trigger the fermentation process. The commonly used liquid medias are molasses, broth, corn steep liquor etc. This method is suitable for microorganisms that require high moisture. The substrates are utilized rapidly by the microorganisms and need to be constantly supplemented with nutrients. Also a steady flow of oxygen must be circulated throughout the process. As the microbe’s breakdowns the nutrients in the media, they produce enzymes. The significance of submerged fermentation is that purification of products is easier and primarily used in the extraction of secondary metabolites that need to be used in liquid form. This process is industrially used for the production of enzymes, citric acid etc • Type of fermentation (Based on feeding of substrate to fermenter) • Batch fermentation • Continuous fermentation • Fed-batch fermentation • Batch fermentation is a closed process system. In this method, the fermenter tank is filled with the materials such as substrate and inoculum. The process parameters such as temperature and pH are set and occasionally nutritive supplements are added to the substrate. Until the process comes to an end, neither substrate is added nor the product is removed from the fermenter. Fermentation proceeds and after desired period, the products are taken out from the fermenter. Once the process is over, the fermenter is cleaned and the process is repeated.
- 10. • In continuous fermentation, the sterilized liquid nutrients are added continuously to the fermenter at a fixed rate as the end products are continuously removed. In this type of process, the growth of bacterial population can be maintained in a steady state over a long period of time. • Fed-batch fermentation employs both modes of operations such as batch and continuous process, where substrate is added at fixed time intervals during the fermentation process. • Type of fermentation (based on need of aeration) • Aerobic fermentation 2. Anaerobic fermentation • Most of large scale fermentation processes are carried out in presence of aerobic conditions. In aerobic fermentation, the materials in fermenter are agitated with the help of impeller or sterile air is forced into the fermenter. In this process, it is necessary to maintain the dissolved oxygen concentration above the specified minimal level. • The provision for aeration and mixing device is not needed in anaerobic fermentation. But in few cases, aeration and mixing may be needed in an initial period. Once the fermentation begins, the gas produced in the process generates sufficient mixing. The air present in head space should be replaced by CO2 or N2 or suitable combination
- 11. Range of fermentation process • Commercially important fermentation process can be divided into four groups. They are 1. Production of biomass or microbial cells (yeast, lactobacillus etc) 2. Production of extracellular metabolites Primary metabolites – produced during growth phase (ethanol, lactic acid, citric acid, nucleotides, vitamins and amino acids (lysine, tryptophan)) Secondary metabolites- Produced in stationary phase of microbial growth (antibiotics, antiseptics, fungicides) 3. Production of intracellular components cells are ruptured to extract the end product (eg. Lipase, cellulose, lactase, recombinant proteins and microbial oils) 4. Transformation of substrate process of conversion of raw material into valuable finished product via fermentation (eg. Production of vinegar, steroids, antibiotics, prostaglandins etc)
- 12. Fermentation industry • Industrial fermentation • Industrial fermentation processes begin with suitable microorganisms and specified conditions, such as careful adjustment of nutrient concentration. • The products are of many types: alcohol, glycerol, and carbon dioxide from yeast fermentation of various sugars; butyl alcohol, acetone, lactic acid, monosodium glutamate, and acetic acid from various bacteria; and citric acid, gluconic acid, and small amounts of antibiotics, vitamin B12, and riboflavin (vitamin B2) from mold fermentation. Ethyl alcohol produced via the fermentation of starch or sugar is an important source of liquid biofuel. • It is a multidisciplinary technology and includes the integrated application of disciplines such as biochemistry, microbiology, molecular genetics and process technology to develop useful processes and products.
- 13. The Fermentation process is now used for manufacturing different types of products in Industries. Not only the Biotechnology companies use the fermentation process lots of other companies like pharmaceuticals, Food & Beverages, Waste Management companies, Biofuel manufacturing companies, Distilleries, Nutraceutical companies, Biofertilizer manufacturing companies, etc. On the basis of the final product production at a commercial scale fermentation industries can be divided into the following major types. •Food & Beverages Industry •Ethanol Producing (Distilleries) •Pharmaceutical or Bio-Pharmaceutical •Waste and Sewage Treatment •Agricultural Feed Products •Enzymes Producing •Bio-fertilizer •Textile Manufacturing
- 14. • Product Depending on the type of microorganisms and its genetic modifications, a range of products can be synthesized. The most common products are listed and divided over two categories: (1) biomass, (2) bioproducts. In case of the latter, some products require complex genetic modifications. • Biomass- Single Cell Protein, Single Cell Oil, Baker's yeast, Lactic acid bacteria • Bioproducts • Enzymes- Proteases, Lipases, Amylases, Cellulases, Peroxidases • Biopolymers- Poly-hydroxyalkanoates (PHA), Polysaccharides: xanthan gum, dextran • Organic acids- Acetic acid, Lactic acid, Citric acid, Tartaric acid, Fumaric acid • Alcohols- Ethanol Butanol, Glycerol, Butanediol • Solvents- Acetone • Pharmaceuticals- Vitamins: vitamin C, B2, B12 ..., Antibiotics: aminoglycosides, penicillins, cephalosporins, tetracyclines ..., Hormones • Biocolorants- Cartenoids, astaxanthins • Biosurfactants and bioemulsifiers- Glycolipids, rhamnolipids • Amino-acids- monosodium glutamate (MSG), Lysine, Tryptophan, Phenylalanine
- 15. • The most common applications of industrial biotech fermentation fall into two categories: biomass fermentation, where the microbial cell biomass is the product itself (i.e., baker’s yeast or Quorn mycoprotein); and precision fermentation, where synthetic biology is used to engineer microbial cells to make non-native products, like recombinant proteins or other high-value molecules (i.e., whey or egg white proteins), which are most often secreted from the cell into the broth. • Most modern precision fermentation processes today are fed batch: the fermentation is started with an initial batch of nutrients and feedstock, and additional feedstock is fed to the fermentation to maximize production of the target protein or molecule. Once productivity plateaus, the fermentation is harvested, the fermenter is cleaned and sterilized, and the cycle is started again. This fed-batch process enables high product concentrations (titers) and tight control of fermentation performance by limiting the number of microbial generations. It also minimizes the risk of foreign microbe contamination due to relatively short fermentation durations (50-120 hours) with frequent sterilization cycles.
- 16. General requirements of fermentation process • Selection of bioreactor The selection of reactor is based on the type of product produced and the type of organism. For eg. To produce mushroom solid state fermentation is more suitable and hence solid type fermenter can be selected • Microorganism Selection and usage of a suitable organism plays a major role in fermentation process. For eg. To produce beer Sacchaormyces sp. is most commonly employed • Temperature control The fermenter should be equipped with suitable temperature control systems in order to sterilize media and maintain the temperature • Water All fermentation systems except solid states requires vast quantities of clean water
- 17. • Media: The microorganisms require nutrient sources like carbon, nitrogen and other micronutrients for their growth • Oxygen: Depending upon the amount of oxygen required by the organism, it may be supplied in the form of air containing 21%v/v oxygen • buffers: Buffers are used to control drastic changes of pH .for example:-protein, peptides, amino acids act as good buffers at neutral pH • Growth factors: Crude media constituents provide enough amounts of growth factors so no extra addition of growth factor is required. If there is a lack of any kind of vitamins or nutrients, growth factor can be added to media for example:-Yeast extract and beef extract • Chealators: Many media can be prepared without precipitation during autoclaving for example:-EDTA and citric acid General requirements of fermentation process
- 19. Basic configuration of fermenter
- 20. a) Disk turbine b) Vaned disc c) Open turbine of variable pitch d) Marine propeller
- 22. Continuous stirred tank fermentor •A continuous stirred tank bioreactor is made up of a cylindrical vessel with a central shaft controlled by a motor that supports one or more agitators (impellers). •The sparger, in combination with impellers (agitators), allows for improved gas distribution throughout the vessel. •A stirred tank bioreactor can be operated continuously in the fermentor, temperature control is effortless, construction is cheap, easy to operate, resulting in low labor cost, and it is easy to clean. •It is the most common type of bioreactor used in industry.
- 23. Main parameters monitored and controlled in fermentation process • In fermenter defined conditions should be maintained to achieve maximum biomass and product formation. Hence some parameters like temperature, pH, degree of agitation, oxygen concentration, foaming etc. requires careful monitoring • Physical parameters- Temperature, Pressure, Agitator shaft power, foam • Chemical parameters- pH, redox, oxygen, exit gas, medium analysis etc. Three main classes of sensor used in fermenters 1. Penetrate into the interior of the fermenter eg. pH electrodes, dissolved oxygen electrodes 2. Operate on samples withdrawn from the fermenter eg. Exhaust-gas analyzers 3. Do not come into contact with the fermentation broth or gases eg: tachometers, load cells
- 24. • Temperature: One of the important parameter to be monitored & controlled. Can be monitored via mercury-in-glass thermometers, bimetallic thermometers, pressure bulb thermometers, thermocouples, metal-resistance thermometers or thermistors. Can be controlled via boilers and chillers by hot or cold water circulation (PID control systems) • pH : It can be monitored via pH probes inserted inside a reactor and controlled by pumping acid or base as required via peristaltic pump (operated through PLC controller) • Pressure: crucial for safety concerns. Can be monitored via Bourdon tube pressure gauge, diaphragm gauge, piezoelectric transducer Can be controlled by employing safety & regulatory valves (PID control systems) • Agitation: can be monitored by the rpm speed recorded & displayed on screen. Can be controlled via controls from PLC unit (Programmable logic controllers) • Aeration and oxygen: can be monitored via D.O probes present inside the reactor & controlled by adjusting the aeration speed via D.O controller For measuring oxygen- galvanic electrodes, polarographic electrodes, fluorometric sterilizable oxygen sensor • Foam sensing & control: A probe is inserted into the reactor to monitor foam & it can be controlled by dispersal of antifoam via plc unit
- 26. What is culture media? • Culture media is a gel or liquid that contains nutrients and is used to grow bacteria or microorganisms. They are also termed growth media. Different cell types are grown in various types of medium. Nutrient broths and agar plates are the most typical growth media for microorganisms. Some microorganisms or bacteria need special media for their growth.
- 27. Culture Media design for fermentation process
- 28. • Nutrients Most fermentations require liquid media, often referred to as broth although some solid substrate fermentations are operated Fermentation media must satisfy all the nutritional requirements of the microorganism and fulfil the technical objectives of the process All microorganisms require water, sources of energy, carbon, nitrogen, minerals, vitamins and oxygen (if aerobic) The nutrients should be formulated to promote the synthesis of the target product, either cell biomass or a specific metabolite In most industrial fermentation process there are several stages where media are required. They may include several inoculum propagation steps, pilot scale fermentations and the main production fermentation.
- 29. Criteria for Ideal Production medium Ideal Media should satisfy the following criteria: • Maximum yield of product • Maximum yield of Biomass per gram of substrate used • Maximum rate of product formation • Minimum yield of undesired products • Consistent quality and readily available throughout the year • Minimum problem causing during preparation / Sterilization (foam etc) • Minimum problem causing in aeration / agitation / extraction, purification, waste treatment • Fewer problems causing in downstream operations • Media should have buffering capacity of optimum pH (organic acids may alter) • Media with vegetable oil might cause no foam • Medium should be free from any toxicity on the culture • Low density media will have better OTR • Media raw materials must be cheap (agricultural wastes will have good calorific value and cheap) • Medium should be suitable for scale up from lab to production • High viscous medium needs high power for mixing • Medium should protect the morphology of organism also
- 30. Types of Media • Defined vs undefined media • Common broadly defined culture media Nutrient media; minimal media; selective media; differential media; transport media, enriched media Undefined Media: (complex media, Natural media) An undefined medium has some complex ingredients, such as yeast extract, which consists of a mixture of many, many chemical species in unknown proportions. Undefined media are sometimes chosen based on price and sometimes by necessity – some microorganisms have never been cultured on defined media. • Complex natural sources • Mostly agro source media • All along undefined, cheaper natural sources used • Showed variation in Biomass and product formation (batch to batch variation) • Small yield difference also difficult to find out • Not all the nutrients are utilized by organism, hence difficult in effluent treatment • Residual organic sources increases the BOD level and interfere the product recovery • Plasmid instability will be higher in natural medium
- 31. Defined Media: (Simple media, synthetic media) • A defined medium will have known quantities of all ingredients. For microorganisms, it provides trace elements and vitamins required by the microbe and especially a defined carbon and nitrogen source. Glucose or glycerol are often used as carbon sources, and ammonium salts or nitrates as inorganic nitrogen sources. • Manufactures are reluctant to use defined medium because its costly • It gives predictable results • No batch to batch variations in results • Purification and Effluent treatment is simple (no color and unwanted / unutilized nutrients) • Maintains the cell morphology • Recombinant organisms can grow well with protein production Types of Media
- 32. Media components Water: Major component of Fermentation media • Needed for ancillary services such as heating, cooling, cleaning and rinsing etc • Clean water with consistent quality and quantities required from permanent sources • pH, dissolved solids, contamination etc to be checked before use • For brewing – mineral content is important • Especially in mashing process Historically sites of breweries influenced in beer qualities • Water with High CaSO4 concentration gives Hard water (produces bitter beers) Water with high CO3 concentrations yields Darker bears • Nowadays, purified and deionized water is used and does not influence • Reuse of water – after effluent treatments decreases the cost of product and effluent load
- 33. • Carbohydrate sources used in media design • The rate at which Carbon source is metabolized can often influence the formation of Biomass / Production of Primary and Secondary metabolites • Rapid utilization of Carbohydrates results in fast biomass formation, but decreases the secondary metabolites production. Hence slow feeding of Glucose or Lactose will be ideal for slow utilization • The cost of the Carbon sources also helps deciding the cost of the final product • Purity of Carbon sources also dictates the product purity • Sterilization during fermentation, might affect the suitability of carbon source • The Amines of amino group react with Carbonyl group of the carbohydrates, to form black nitrogen containing compounds, which will inhibit the growth of organisms (technically called Caramalization) • Starch solution when heated becomes gelatinous and less viscous • Manufacturers keep their production site near the carbohydrates available areas, to minimize transportation costs • Examples of Carbohydrates sources: For large scale uses – Starch obtained from Maize grains, Potatoes or other Cereals used as partially ground state. • Starch is readily hydrolyzed by dilute acids and enzymes to give a variety of glucose syrups. • Barely is particularly used after germination and heat treated to malt, which contains other sugars along with starch for brewing • Sucrose is obtained from sugarcane, beet molasses (residues left after crystallization of the sugar extraction) • Molasses used for high volume products such as ethanol, SCP, Organic acids etc • Molasses has many impurities, which complicates the downstream process (especially color) Types of Media
- 34. • Lactose is also used (limited to only few organism which can utilize) • Molasses: Water – 20%, Ash – 8%, Total sugar – 40 -60%, Total Nitrogen – 3%, Gums – 2%, Free acids 2% • Fruit juices: Contains soluble sugar Glucose / Fructose (Grapes juice for wine production) • Cheese whey: Straw colored, off smelled liquid, used in Lactic acid production and SCP production • Sulphite waste liquor: Paper and pulp industry, during wood digestion process – Hydrolysis of wood chips by calcium bisulphate with increased heat and pressure • The spent liquor has 10-12% of solids of cellulose and 2% of sugar (Hexoses / Pentoses) used in ethanol fermentation • Rice straw: used in Mushroom cultivation; SCP production Malt Extract: malted barley (contains 90-92% carbohydrates) • Oils / Fats: Oils were first used as antifoam in antibiotics fermentation (olive oil, Maize oil, cotton seed oil etc) – The presence of fatty acids (Linoleic / Linolenic acid) makes them costly to use – Animal fat (tallow) is also used - The oil source contains ~ 2.4 times more energy of glucose on per weight basis – Less space is required compared to glucose – Oil itself is antifoam – no problem of foam formation – Glycerol trioleate is used still as carbon source in Cephalosporin production
- 35. Nitrogen sources used in medium design • Organisms can utilize Inorganic / organic sources of Nitrogen • They are energy supplements for the biomass and Production • Inorganic nitrogen sources: Ammonia gas, ammonium salts and nitrates • When Ammonium salts, Nitrates are used - the free acid will be liberated which may alter the pH. • Antibiotic production starts only after complete nitrogen source is consumed by the organisms • In Shake flasks – weak acids salts (ammonium succinate) is used as Nitrogen sources, since it does not influence in pH change • Ammonium nitrate - forms first ammonia assimilation then nitrate assimilation • When ammonium nitrate is used in the medium, the enzyme ‘Nitrate Reductase’ is repressed by ammonia till its presence in medium, once diminished then nitrate assimilated. • Hence mixture of two nitrogen sources regulates other’s intake. • Organic nitrogen sources: Supplied as amino acid, protein, urea. Growth will be faster with a supply of organic nitrogen. Protein, nitrogen compounds can be obtained from sources like corn steep liquor, soya bean meal, peanut meal, cotton seed meal, yeast extract etc. But its storage is affected by moisture, temperature
- 36. • Nitrogen sources examples: • Corn Steep Liquor: Steep – Extraction of soluble substrates of corn - Used in the production of Starch / gluten • It’s a byproduct subjected to 50% solids, Photosensitive and stored in dark colored bottles • Used in penicillin and beta lactam antibiotics as precursor • Soya bean meal: soya bean seeds are de-oiled (residue is Oil cake) • The final residue is Meal contains 8% w/w of Nitrogen • Used in Streptomycin production Pharma Media: • Cotton seeds meal after oil recovery process – the embryo we get as finely ground powder • Contains 56% protein and 24% carbohydrates – Used in Tetracycline production • Yeast Extract: • Baker’s yeast subjected to Autolysis at 50 – 55 deg C. • Contains Amino acids, Peptides and Vitamins
- 37. • Minerals: Magnesium, Phosphorus, Potassium, Sulphur, Calcium, Chloride are more essential than other minerals defined media – it is added externally, in Natural media it is expected to be present • Phosphates used as buffer - to stabilize pH in shake flasks, where there is no control to it • Chelators: Media after autoclaving - will result in white precipitate (insoluble) They are metal phosphates that form as white precipitate eg. Iron phosphates, Calcium phosphates, Zinc phosphates This could be avoided by adding low concentration of chelating agents (EDTA – Ethylene Diamine Tetra Acetic acid) • EDTA preferably form metal complex with metals in the medium. These EDTA complexes, later gradually utilized by the organisms However, care to be taken as, EDTA concentration should not cause growth retardation In large scale media preparations – Yeast extract and Protease peptone will form complex with metals instead of EDTA
- 38. • Growth factors: Chemicals that are added to influence to form normal growth of microbes Some organisms – not capable of producing full composition of cell components Externally preformed compounds (growth compounds) are added eg. Vitamins Mostly Vitamin sources could be added in medium eg. Calcium pantothenate • Precursors Chemicals added to incorporate in to the product formation They become part and parcel of the product. eg. CSL added to penG production increases 20 units to 100 units/cm3, CSL found to be containing phenyl ethyl amine, which forms as seed in PenG molecules to yield more
- 39. • Inhibitors: Certain inhibitors are added to block / form certain specific products eg. Production of Glycerol • Glycerol Production depends on modified Ethanol production by removing acetaldehyde • Glucose ---- Acetaldehyde --- oxidized to form Ethanol • Addition of ’Sodium bisulfate’ forms a complex to form Acetaldehyde bisulfate • When acetaldehyde is no longer available for the oxidation to form ethanol, metabolism diverted to Glycolysis to form Glycerol – 3- phosphate which is later converted to Glycerol • All Industrial Enzymes are inducible They are synthesized only in the presence of certain chemicals (Substrates / Substrate Analogues) Eg Starch, dextrin for Amylase; maltose for Pullulanase; Pectin for Pectinases; Fatty acids for Lipase etc
- 40. • Oxygen Requirements in Media Design • Oxygen required for the better growth during aerobic organisms The factors which are influencing the oxygen availability in the medium are Fast Metabolism: Rapidly metabolizing carbon source will increase the Oxygen demand in the medium • To satisfy the demand, there is increase in design of OTR Penicillin require high OTR, to overcome this Carbon source is limited and added later as Fed Batch process • Rheology of Medium: Compounds influence in Viscosity of medium. Viscosity influence on OTR Addition of Polymers (Starch, Polysaccharides) alters the Viscosity Polysaccharides when degraded the rheological property is getting changed, which in-turn affects O2 uptake
- 41. • Addition of Antifoams: Antifoam readily reduce the surface tension so that foaming can be suppressed Presence of Surface Active reagents reduces the OTR CSL, Proteins, Pharma media etc, always foams more Foams leads to autolysis of cells and decrease OTR • Antifoam should not be assimilated by the organism Should be easily removed during downstream processing Antifoams should be metabolized, non toxic, heat stable to withstand sterilization Eg. Fatty acids, Glycerides (Cotton seed oil), Linseed oil, Olive oil, Silicones, Polypropylenes, Glycerol etc
- 42. • Animal Cell media • In vitro culturing of mammalian cell line Vaccine production, monoclonal antibodies (mAb), Interferons and other recombinant / therapeutic proteins • Serum based media: Serum is used particularly in the production of recombinant proteins, antibody based products through in-vitro culturing • Earlier animal cell media had 10% calf blood serum (extracted from foetal calf) + other organic and inorganic components Blood serum is complex mixture more than 1000 compounds Contains Organic, inorganic salts, amino acids, vitamins, carbon sources, hormones, growth factors, hemoglobin, albumin and other compounds Most of them are not essential / completely utilized for Cell growth and Differentiation According to FDA - Foetal Calf Serum components must be completely eliminated during product purification steps; Product should be free from BSE – Bovine spongiform encephalitis Serum should be free from Bacterial, Viral or even Pyrogens contamination, hence costs of these products are getting increased
- 44. • Serum free media supplements: Reduced amount of Serum in media used / Optimized medium formulated - with serum free Media formulated with the addition of Albumin Insulin Selenium Beta- mercapto- ethanol Ethanolamine Transferin • Advantages: More consistent results achieved Reduced batch to batch variation Sterility is achieved easier (no potential contamination) Cheaper components Simple steps of downstream operations
- 45. • Protein Free media Elimination of Protein in the medium – reduces foam formation Instead of Protein – only Amino acids are added • Osmolality: Optimum range of Osmotic Pressure is maintained in the medium - Isotonicity Maintains Cellular morphology and functions Bacteriological Saline - NaCl addition (0.85%) Non Nutritional Media Supplements: To minimize the mechanical damage caused by the shear-force of the impeller tips Sodium Carboxymethyl Cellulose (0.1%) is added in the medium • Mammalian cells are more susceptible to shear-force (has delicate plasma membrane) Tip Velocity : (Ts = rpm X Impeller Dia X pi) Step down Geared motors are sued (1/3 of normal speed ~ 10 rpm) Sometime Vanned disc or Vibrometer impellers are used – Magnetic pellets are used instead of Stirrer No sparger is used to avoid shear – instead surface aeration given at overhead space Pure oxygen supplied instead of air- enhance more OTR Cyclone column reactor used / encapsulation immobilization is adapted
- 46. • Essential Stage of Process development Constituents of the medium must satisfy the requirements of Cell biomass Metabolic Production Supply of energy for Cell maintence and biosynthesis of cellular organelles • Stoichiometry for Growth and Product formation for aerobic fermentation Carbon and energy sources + Nitrogen sources + O2 + other requirement ---------- Biomass + Products + CO2 + H2O + heat This equation should be expressed in quantities, for economic design of media and to keep component wastage to be minimal Minimal medium In fermentation of microbial cell should have minimum quantities of media components, for economic design of media. These minimal quantities of nutrients should be calculated for specific production of biomass and product without wasting. It is not always easy to quantify all the factors very precisely Knowledge of elemental composition of micro organism is necessary for media design
- 48. • Initial medium recipe: C, N, S, P, Mg, K Additional requirements: Fe, Zn, Cu, Mn Some organisms cannot synthesis specific nutrients eg. Amino acids, Vitamins or Nucleotides etc (to be added) The carbon substrate has dual role in biosynthesis and energy generation The carbon requirement may be estimated from Cellular yield coefficient (Yx/s)
- 49. • For bacteria with Yx/s for Glucose = 0.5 means (0.5g of cell / g of glucose) • The concentration of glucose needed to get 30g / dm3 of cell will be (30/0.5 = 60g/dm3 of glucose) For Pen G production This bases is theoretically calculated for Penicillin = 1.1g of penicillin G / g of glucose Maximum experimental yield is 0.052g of Pen G/ g of glucose (theoretical value is much higher than practical values) • Under batch process: added glucose contributes as 28% for Cell mass; 61% for Maintenance; 11% for Pen G production • Under Fed batch Process: added glucose contributes as 26% for Growth; 70% for Maintenance; 6% for Production It was found, that the nutrient requirement increases O2 also
- 50. • Medium Optimization Optimization should meet all the characteristics of ideal production medium The optimized composition should efficiently grow biomass and production with high productivity Different combinations and sequence of process conditions needed to be optimized
- 51. • Classical method: • By changing one independent variable (nutrient, antifoam, pH, Temperature etc) • It is extremely time consuming and expensive in large number of variables i.e : Xn ; (Where X is number of levels and n is the number of variables) • may be possible for 3 variables with 2 concentrations : 23 trials = 8 trials • if 36 leads to 729 trials (tedious and time consuming) Industrial aim is to perform minimum number of experiments to determine best results
- 52. • Plackett-Burman design of media optimization • When more than five independent variable are investigated; • To find the most important variable in a system and it is further optimized • Technique allows use of X no. of experiments with X-1 variable X must be 4 or multiples of 4 (8, 12, 16, 20, 24…) • If number of variables are not matching with experiment then concept of dummy variable to be introduced • Construct a chart with 7 variables with High and Low concentrations of each variable
- 53. • This design is for 7 variables (A to G) at high and low levels 2 factors E & G are designated as Dummy Variables (might be used for calculating experiment error) Each Horizontal row represents a Trial (experiment) Each Vertical column represents High & Low values of one variable in the all the experiments The design says that there should be equal number of H & L concentrations in the given column
- 54. • Consider variable A: For the trials in which A is high (4) – B is High in two of the trials and Low in two of the trials Similarly C will be High in 2 trials and Low in 2 trials with respect to B and A In the same way, for those trials (4) in which A is low; B will be higher in 2 trials and Lower in 2 trials • Thus, effect of changing other variable cancels out when determining the effect of A The same logic applied for each variable • The difference between High & Low values should be large to ensure the optimum H & L response Caution must be taken in fixing sensitive variables, Since this large difference will influence/mask the other variables effect • The effect of dummy variables are calculated same way as experiment variables; • if it is ‘0’ implies that there is no interaction / no error in measuring responses • If it is not equal to ‘0’ ; it is assumed as lack of experimental precision and analytical error in measuring the response • This procedure will identify the important variable and allow them to be ranked in order to investigate further to arrive optimum concentration
- 55. • In the same way for each variable the F test is to be found by dividing with experimental mean square (0.0325) It is found A = 465.4 B = 400.2 C = 3.255 D = 0 F = 325.6 • Result shows: A, B, F show large effect (Significant) C shows very low effect (in-significant) D shows no effect (E & G need not to be calculated, since they are Dummy variables) The next stage would be to optimize the concentrations of more significant variables by Response surface methods
- 56. • Response Surface methods of optimization • According to Plackett-burman design, a key nutrient is being identified (rate limiting nutrient) • It ranks the nutrients according to its relative importance • The next stage is to determine the Optimum levels of each Key independent variable • This optimum level is done by RS methods (developed by Box & Wilson 1951 & later developed by Hendrix 1980) • Central Composite Design - CCD / Box - Behan design - BBD Number of experiments = 2 k + 2k + n0 Where k = levels (high, mid, low levels) ; n = no of centre points If k is 3 and n is 6 then = 23 + 2x3 + 6 = 8+ 6+ 6 = 20 experiments
- 57. • RS method is a graphical representation of experimental results as Contour Plots (2D) or Topographical Plots (3D) Contour plots: show lines of constant values Topographical plots: Show lines of constant elevations These contour / topography shows “identical responses” Response means the result of an experiment, carried out at a particular level of variable The ‘axes’ (x axes / y axes) of these plots are experimental 2 variables The area within the axes is termed as ‘Response Surface’
- 58. • Construction of RS plots: The results (responses) of the series of experiments with different x , y variable values are inserted in Surface of the Plot The points giving same results (equal responses) are joined together to make a contour lines 2 variables are changed in experiment rather than one being maintained constant Three factors are also taken for experiment and responses surface of Topographical maps with constant elevation values. • This profile is made by fixing X1 and Changing X2 Keep Best X2 value and Change X1 value The optimum is then fixed with X1 and X2 variables To construct a contour plot, the responses of series of experiment, employing different conditions combinations are inserted on the surface of plot.
- 59. • Hendrix Proposed the Strategy to arrive Optimum as follows 1. Define the space on the plot to be explored 2. Run 5 random experiments in the space 3. Define a new area of space in optimal centre 4. Run 5 more random experiments in the new space 5. Continue doing this until no further improvements is observed • Applications of RS techniques: To use Mathematical model to understand the relationship between “Responses” (results) and “Variables” These models helps in rapid optimization with fewer experiments and develops several contour maps Software packages are available – to analyze optimal response surfaces
- 62. • Simplex Optimization method: It is type of Model where mathematical simplexes (Equilateral Triangles) are constructed according to the Reponses • Example: Antibiotic Production X axes fixed as Carbon source variable & Y axes fixed as Nitrogen source • A is the first experimental point where best antibiotic production with optimal 2 variables B is the second experimental point, where moderate production found D is the point where worst response is found When 3 points (A, B, D) are known, then C can be constructed mathematical simplex with reflection, Expansion, Contraction etc Centroid point is thus determined mathematically from Simplexes
- 64. • A series of “Simplexes” are constructed moving in a Crabwise way An optimum point, the simplex begins to circle on itself shows optimum concentration This procedure is continued until the optimum is located
- 65. • RSM is a sturdy, robust and efficient mathematical approach which includes statistical experimental designs and multiple regression analysis, for seeking the best formulation under a set of constrained equations • RSM employs several phases of optimization and it can be performed in three basic steps, i.e., experiments designed for the screening of the factors followed by the path of steepest ascent/descent and finally quadratic regression model is fitted
- 66. • No interactio ns- 1st order • Interactio ns- 2nd order
- 67. • Since, the theoretical relationships between the independent and dependent variables are not clear, multiple regression analysis can be applied to predict the dependent variables on the basis of a second- order equation. • Where Y = predicted response, a0 = intercept coefficient, aiXi = linear terms, aijXiXj = interaction terms and aiiX 2 = square terms. • It has been shown that the RSM model is simple, efficient, less time consuming and capable of predicting the optimization of various processes of metabolite production
- 73. • One of the important inputs of RSM is representation of the yield, as a surface plot. It can provide multiple responses at the same time by considering the interactions between the variables, which is utmost necessary for designing and process optimization
- 74. • This method helps us to determine, how a specific response is affected by changes in the level of the factors over the specified levels of interest and to achieve a quantitative understanding of the system behavior over the region tested.
- 78. • Even though widely employed with much success, some limitations are associated with RSM, • for e.g., the prediction of responses based on second-order polynomial equation is often limited to low levels and results in poor estimation of optimal formulations (Baishan et al., 2003). • Another important limitation is the metabolic complexity of the microorganisms. When a large number of variables are involved, the development of rigorous models for a given biological reaction system on physical and chemical basis is still a critical challenge. This is probably due to the non-linear nature of the biochemical network interactions and in some cases the incomplete knowledge about the kinetics involved in such systems (Franco-Lara et al., 2006). • Also, it is quite complicated to study the interactions of more than five variables and large variations in the factors can give misleading results possibly due to error, bias, or no reproducibility