Downloaded 735 times
More Related Content
Similar to Downstream processing
Downstream processing
- 1. DOWNSTREAM PROCESSING “BEGINNING AFTER THEEND” PRESENTEDBY: AYUSHJAIN (ALM3008)
- 2. AT WHICH STAGEARE WE IN THE PRODUCTION CYCLE ?? 18-03-2017Basic Science H.O.T. 2
- 3.
- 4. WHAT IS DOWNSTREAM PROCESSING??? The various process used for actual Recovery and purification of biosynthetic products from a fermentation or any other industrial process together constitute a downstream processing. 18-03-2017Basic Science H.O.T. 4
- 5. WHAT ARE THOSEPROCESSES ??? Removal of insoluble Isolation or extraction of product concentration of product Packaging of product 18-03-2017Basic Science H.O.T. 5
- 6. WHAT ARE THECLASSES OF PRODUCT OBTAINED??? Cell itself • Bakers yeast • Bio-fertilizers Extracellular type • Antibiotics • Alcohol Intracellular type • Enzymes • Vitamins 18-03-2017Basic Science H.O.T. 6
- 7. IF THE PRODUCTIS THE CELL ITSELF ??? Separation of particles • Filtration • Centrifugation • Flocculation and floatation Cooling and packaging 18-03-2017Basic Science H.O.T. 7
- 8.
- 9. IF THE PRODUCTIS EXCRETED OUTSIDE THE CELL ?? • Centrifugation or filtration • Flocculation and floatation Separation of Particles • Liquid-liquid extraction • Whole broth extraction • Aqueous Multiphase Extraction Extraction • Chromatographic techniques • Evaporation process • Crystallization • Adsorption Concentration 18-03-2017 Basic Science H.O.T. 9
- 10.
- 11. IF THE PRODUCTIS INTRACELLULAR ??? • Filtration • Centrifugation • Flocculation and floatation Separation of particles • Mechanical cell disruption • Drying • Lysis Disintegration of cells • Liquid-liquid extraction • Whole broth extraction • Aqueous multiphase extraction Extraction • Evaporation • Membrane filtration • Chromatography techniques • Adsorption Concentration 18-03-2017Basic Science H.O.T. 11
- 12. LETS UNDERSTAND VARIOUSTECNIQUNIES INVOLVED IN DOWNSTREAM PROCESSING 1. Filtration filamentous fungi filamentous bacteria Yeast 18-03-2017Basic Science H.O.T. 12
- 13. 18-03-2017Basic Science H.O.T.13 Surface filters Filtration on the basis of pore size of the membrane. Cross flow filters Filtration on the basis of pore size but pores are not clogged due to tangential flow of particles Rotatory flow filters Filtration due to sucking of liquid by vacuum when filter drum is rotated in the slurry. Depth filters Uses a porous filtration medium to retain particles through out the medium rather than just on Centrifugal filters Filtration by rapidly rotating slurry, so solids are retained on porous screen and liquid filter out
- 14. 2. CENTRIFUGATION •Works on sedimentation principle Thecentripetal acceleration causes denser substances and particles to move outward and less dense are displaced and move to the centre 18-03-2017Basic Science H.O.T. 14
- 15. 3. FLOCCULATION ANDFLOATATION •Flocculation is the process by which individual particles aggregate to form a clump •Due to addition of clarifying agent Floatation is the process of separation of particles based on relative buoyancy of particles Froth flotation Dissolve air flotation Induced gas flotation 18-03-2017Basic Science H.O.T. 15
- 16. 4. CELL DISRUPTION Ultra sound Beadmill Homogenisers •Drying Lysis by osmotic shock Frezzing Lytic enzymes 18-03-2017Basic Science H.O.T. 16 Mechanical cell lysis Non-mechanical cell lysis
- 17.
- 18. 5. EXTRACTION OFPRODUCT •Liquid-liquid extraction •Separation of compound based on there relative solubility's in two different immiscible liquids Whole broth Both cells and broth are extracted together Aqueous multiphase extraction Enzymes are extracted in aqueous polyethylene glycol- dextran mixture, which form separate phases 18-03-2017Basic Science H.O.T. 18
- 19.
- 20. 7. PURIFICATION •Crystallization •Impure substancesare dissolved in solvent to reach saturated stage at higher temperature which is then cooled to form pure crystals •Antibiotics like penicillin G, Chromatographic techniques 18-03-2017Basic Science H.O.T. 20
- 21. 18-03-2017Basic Science H.O.T.21 Paper chromatography Thin layer chromatography Column chromatography Ion exchange chromatography Affinity chromatography High performance liquid chromatography Reversed phase high performance liquid chromatography Gas chromatography-mass spectrometry Gel filtration – chromatography/ size exclusion chromatography Types of chromatography
- 22. PAPER CHROMATOGRAPHY 18-03-2017Basic ScienceH.O.T. 22 Stationary phase Solid (paper) Filter Paper Filter Towel Mobile phase Organic solvent Water, rubbing alcohol Characteristics Based on polarity And Size Amino Acids Mechanism If molecule to be separated are more attracted to stationary phase then it will move slowly and vise versa. Advantage 1.Cheaper 2. Both organic and inorganic compounds can be identified Disadvantage 1. Large quantity of sample cannot be applied 2.complex mixture cannot be separated 3. Less accurate
- 23.
- 24. THIN LAYER CHROMATOGRAPHY 18-03-2017BasicScience H.O.T. 24 Stationary phase Solid Glass slide with silica gel, Aluminum oxide Mobile phase Organic solvent Ethanol, water, hexane, pentane Characteristics Attraction of compound to solid or liquid phase Mechanism - Based on retention factor Rf - Higher the Rf higher the rate with which product has travelled Advantages - Simple - Cheap - Easy recovery of product Disadvantages - Plate length limited so separation up to limited length - Sample affected by Humidity and Temperature
- 25.
- 26. COLUMN CHROMATOGRAPHY 18-03-2017Basic ScienceH.O.T. 26 Stationary phase Silica gel Alumina Mobile phase Solvent according to stationary phase Organic solvents Characteristics Differential adsorption of substance by adsorbent or relative affinity Mechanism Based on retention factor Rf - Higher the Rf higher the rate with which product has travelled Advantages - Low cost -Disposability of stationary phase so less cross contaminatio n Disadvantages -Time consuming - Low separation power -Irreproducible result
- 27.
- 28. GAS CHROMATOGRAPHY-MS Stationary phase Volatile liquid Mobile phase Gas(carrier) Inert gas (He,Ar, Ne) Characteristics volatility Polarity Mechanism Separation due to equilibrium established between the solutes and stationary phase Advantages -Fast -Sensitive -High quality product obtained -More resolution Disadvantages -Apply to only volatile compound -Performed at higher temperature so thermally liable compounds cannot be analyzed -Detectors used are destructive 18-03-2017Basic Science H.O.T. 28
- 29. 18-03-2017Basic Science H.O.T.29 Retention time Time taken by compound to be detected Nature of stationary phase Solubility of gas in solid phase Length of column Temperature of column Flow rate of mobile phase
- 30.
- 31.
- 32.
- 33.
- 34.
- 35.
- 36.
- 37.
- 38. ADVANTAGES AND DISADVANTAGESOF HPLC 18-03-2017Basic Science H.O.T. 38 ADVANTAGES • Can be applied to any compound soluble in liquid phase ex. AA, Lipids, Carbohydrates • Performed at room temperature so thermally compounds can analyzed • More flexible • detectors can be reused i.e. they are non destructive Disadvantages • Slower technique than GC • Lower resolution when compared to GC.
- 39. ION-EXCHANGE CHROMATOGRAPHY 18-03-2017Basic ScienceH.O.T. 39 Stationary phase Solid Cellulose, Agarose Mobile phase liquid characteristics polarity Solubility Mechanism Charged molecules attaches to the oppositely charged stationary phase as a result the molecules are separated and the molecule of interest can be eluted by adding another compound which has more affinity for those charged sites Advantages More matrix tolerance Sensitive High quality product obtained Disadvantages Expensive less Reproducible
- 40.
- 41.
- 42. GEL- FILTERATION/ MOLECULAR EXCLUSIONCHROMATOGRAPHY 18-03-2017Basic Science H.O.T. 42 Stationary phase Beads with holes hydrocarbon like dextran Mobile phase Liquid Organic solvent characteristics Size Mechanism Based on size proteins can pass through the holes in beads as a result pass the column first and vice versa Advantages Good separation Biological activity of particles protected Less and defined time No sample loss Disadvantages Molecules with exact same size and weight cannot be separated
- 43.
- 44. SDS- PAGE •Proteins are purified •setup similar to gel electrophore sis Based on size Forces applied Frictional force and Electric force (Gravitation al force can be neglected) Velocity depends on Electric field, Charge on molecule and coefficient of fricition •Polyacramide gel is unreactive It has many pores to allow proteins to pass SDS anions and Beta mercaptoethanol denatures the protein by attaching to side chains. So net charge becomes negative on protein 18-03-2017Basic Science H.O.T. 44
- 45.
- 46. 8. PACKING •Drying SprayVaccum Freeze drying 18-03-2017Basic Science H.O.T. 46
- 47.
- 48. DOWNSTREAM PROCESS DEVELOPMENTIN BIOTECHNOLOGICALITACONIC ACID MANUFACTURING APPLIED MICROBIOLOGYAND BIOTECHNOLOGY ISSN: 0175-7598 (PRINT) 1432-0614 (ONLINE JANUARY 2017, VOLUME 101, ISSUE 1, PP 1–12 ANTONIO IRINEUDO MAGALHÃES JR. JÚLIO CESAR DE CARVALHO JESUS DAVID CORAL MEDINA CARLOS RICARDO SOCCOL ABSTRACT • The Most Important Uses Of This Biomonomer Is The Environmentally Sustainable Production Of Biopolymers • Separation Of Itaconic Acid From The Fermented Broth Has A Considerable Impact In The Total Production Cost. • Previous Studies On The Separation Of Itaconic Acid Relying On Operations Such As Crystallization, Precipitation, Extraction, Electrodialysis, Diafiltration, Pertraction, And Adsorption. • Despite Recent Advances In Separation And Recovery Methods, There Is Still Space For Improvement In Ia Recovery And Purification. 18-03-2017Basic Science H.O.T. 48
- 49. INTRODUCTION • Downstream Processes,Such As Recovery And Purification, Result In 30 To 40% Of The Final Product Cost (Straathof 2011). A Competitive Bioprocess Is Highly Dependent On The Development Of Efficient Recovery And Low-cost Processes • There Are Two Main Routes For The Production Of Biobased IA: The Direct Fermentation Into The Acid, Or The Production Of Citric Acid Followed By Its Conversion Into IA By Pyrolysis. • Direct Fermentation Of Glucose Into Ia Is More Efficient Than The Chemical Process. • The Price Of Ia Ranges Between Us$1.5 And 2.0/Kg And The Annual World Production Of IA Exceeded 80,000 T (Okabe Et Al. 2009). It Is Estimated That The World Market Of IA Was Valued At US$126.4 Million In 2014 And Will Reach US$204.6 Million By 2023 • Crystallization Is The Most Usual Unit Process For Recovery Of IA. 18-03-2017Basic Science H.O.T. 49
- 50. PHYSICALAND CHEMICAL PROPERTIESOF ITACONIC ACID • IA Is A White, Crystalline, Monounsaturated Organic Diacid With Formula C5H6O4 And A Molar Mass Of 130.1 G/Mol • Solubility In Water Of 83.1 G/LAt 20 °C • The Melting And Boiling Points Are 167–168 And 268 °C, Respectively • IA Has Three Different Protonation States (H2ita, Hita−, And Ita2−) With Dissociation Constants In Aqueous Solutions Of 3.83–3.89 (Pka1) And 5.41–5.46 (Pka2) At 15 To 45 °C • Besides Its Two Carboxyl Groups, IA Has A Methylene Group. This Functional Diversity Allows A Variety Of Reactions, Such As Complexation With Metal Ions, Esterification With Alcohols, Production Of Anhydrides And Polymerization By Addition And By Condensation • The Solubility Of IA Is Highly Dependent On Temperature 18-03-2017Basic Science H.O.T. 50
- 51. RECOVERY METHODS • AfterConversion Of Substrates Into Itaconic Acid By Fermentation, The Resulting Broth Contains Also Biomass, Residues From The Fermentation Media, Other Organic Acids, And Minor Components • The Initial Step In The Downstream Process Usually Consists Of Biomass Removal, After Which The Acid Is Concentrated And Purified To The Desired Extent. An Initial Concentration (Recovery) Of The Acid From The Broth Is Central To IA Production: Lower Flows And High Final Concentrations Will Reduce Overall Operating And Equipment Costs CRYSTALLIZATION • The Classical Method Of IA Recovery Produced By Fermentation Processes Is Crystallization. • Ia Can Be Easily Recovered Through This Method By Cooling Or Evaporation-crystallization At Low Ph Values, But Both Treatments Do Not Separate Some By-products Of Fermentation, Such As Succinic, Malic, And Α-ketoglutaric Acid, Causing A Decrease In The Final Product Quality. • The Filtrate Is Concentrated By Evaporation To Achieve A Concentration Of 350 G/L, And The Crude Crystals Are Formed Using A Cooling Crystallizer At 15 °C. 18-03-2017Basic Science H.O.T. 51
- 52. PRECIPITATION • IA CanBe Recovered By Precipitation With Calcium And Lead Salts (Kobayashi And Nakamura 1971) • This Precipitate Is Filtered And Then Separated From The Liquid Phase. • The Lead Itaconate Can Be Regenerated By Adding Carbonate Or Bicarbonate Of Alkali Metals Or Ammonium To Obtain The Respective Itaconate Salts And Lead Carbonate. • To Isolate The Ia, It Is Necessary To Use A Cation Exchange Step • Lead Solutions Require Appropriate Chemical Processing Due To Its High Toxicity, Which May Cause The Increase Of The Final Cost Of The Product. • Another Ia Precipitation Method Uses Calcium Hydroxide. This Method Is Similar To The Recovery Of Other Organic Acids, Such As Citric Acid • In This Method, Calcium Itaconate Is Formed, And Since It Is Much Less Soluble Than The Acid, It Precipitates. The Solid Is Recovered By Filtration. The IA Can Be Regenerated By Reacting Calcium Itaconate With Sulfuric Acid 18-03-2017 Basic Science H.O.T. 52
- 53. LIQUID-LIQUID EXTRACTION • TheUse Of Conventional Solvents, Such As Long-chain Alcohols, Esters, And Alkanes Is Not Effective For The Recovery Of Organic Acids Due To The Low Distribution Coefficient Of The Acid, I.E., Its Higher Solubility In Water Than In Organic Solvents • The Distribution Coefficient May Be Altered Using Organophosphorus Compounds, Or Tertiary Or Quaternary Amines. This Technique Is Called Reactive Extraction (RE). • Organophosphates And Aliphatic Amines Have Been Studied As Extractants For The Separation Of Ia From Aqueous Phase Due To Their Thermal Stability And Their Ease Of Regeneration, Which Can Be Done By Simple Distillation • Tri-n-octylamine (TOA), A Long-chain Aliphatic Amine, Is More Effective In The RE Processes Of IA Than Tri-n-butylphosphate (TPB), An Organophosphate, Using Hexane As Diluent. • Re System Has Numerous Advantages Such As High Productivity, Increased Conversion Of Substrate To Product, Assists In Maintaining Ph Without The Addition Of Basic Solutions, And Can Make The Continuous Process And More Energy Efficient 18-03-2017Basic Science H.O.T. 53
- 54. MEMBRANE SEPARATION • DiafiltrationIs A Method Of Separation Or Removal Of Components Present In A Solution By Means Of Permeable Membranes And A Concentration Gradient • This Separation Process May Use Incorporated Membranes In Bioreactors For The Continuous In Situ Product Recovery (ISPR). • Carstensen Et Al. (2012) Developed A ISPR With Diafiltration Concept Called “Reverse-flow Diafiltration” (RFD). The RFD Process Yields A Product Stream Through A Hydrophilic Ultrafiltration Hollow-fiber Membrane Immersed In The Bioreactor • The Pertraction Technique Uses An Organic Solvent To Extract The Solute From The Aqueous Phase By A Procedure Similar To Liquid-liquid Extraction; However, The Solvents Are Separated By A Hydrophobic Membrane • Membrane Processes Have A Relatively High Capital Cost, Can Be Selective, And With The Increasing Options Of Membrane Modules On The Market, Is A Real Possibility For Itaconic Acid Downstream. 18-03-2017Basic Science H.O.T. 54
- 55. ADSORPTION • Ia CanBe Desorbed From The Resin, Which Can Be Reused Multiple Times. There Is A Wide Range Of Adsorbents For Adsorption Processes, Such As Alumina, Activated Carbon, Silica, And Several Kinds Of Synthetic Ion Exchange Resins. • Gulicovski Et Al. (2008) Found That The IAAdsorption On The Surface Of Alumina Is Extremely Ph Dependent, And The Maximum Adsorption Occurred At A Ph Value Of The First Dissociation Constant, Pka1 • Separation Of IA By Adsorption From Aqueous Solutions, Using Two Types Of Commercial, Strongly Basic Ion-exchange Resins: Purolite A-500PAnd PFA-300. The Resin PFA-300 Proved To Be More Efficient For IA Recovery. • In The Desorption Step, An Adequate Solution, The Eluent, Is Used To Induce The Desorption Of Ia And Carry It Out Of The Column. The Nature Of The Eluent Depends On The Type Of Adsorption: • Adsorption Has A Relatively High Capital Cost For The Production Of Bulk Chemicals, Because Of The Cost Of The Adsorbent. 18-03-2017Basic Science H.O.T. 55
- 56. FINAL REMARKS ANDFUTURE OUTLOOK • The Development Of An Efficient Process For Separating And Purifying Itaconic Is Challenging Due To The High Affinity Of This Hydrophilic Solute For Aqueous Solutions And The Complex Composition Of The Fermentation Broth. The Advantages And Weaknesses Of Each Separation Method For IA Can Be Compared Using Literature Yields And Regarding Technology Maturity, Scalability, Costs, Energy Consumption, By-product Formation, Yield, And Purity. Conceptual Separation Trains Relying On Each Recovery Technology Were Evaluated And Summarized In Fig. All Processes Start With The Fermentation Broth And End With IA Crystals, The Preferred Product Presentation, But With Different Yields And Purity According To The Recovery Options. • The Industrial Production Of Cheap, High-quality Itaconic Acid Depends On The Successful Recovery And Purification Of Itaconic Acid From Fermentation Broths With Low Demand Of Energy And Reagents. From The Analysis Of The Operations, It Is Clear That Further Research Laboratory To Pilot-scale Techno- economic Analysis And Environmental Impact Of Each Recovery Option Is Paramount 18-03-2017Basic Science H.O.T. 56
- 57.
- 58.
- 59.