Recovery and Purification of Products Lecturer Dr Kamal E M Elkahlout Assistant P rof of Biotechnology General Approach Separation of insoluble products or components Primary isolation or concentration and removal of water ID: 626397
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Advanced Bioprocess Engineering Recovery and Purification of Products
Lecturer Dr. Kamal E. M. ElkahloutAssistant Prof. of BiotechnologySlide2Slide3Slide4Slide5
General ApproachSeparation of insoluble products or components.Primary isolation or concentration and removal of water.
Purification and removal of contaminated chemicals.Product preparation.Slide6Slide7Slide8
Factors that impact difficulty and cost of recoveryProduct can be biomass, intracellular or extracellular component.Fragile or heat sensitive.Concentration or titer in the broth.
Typically recovery and purification is more than 50% of total manufacturing costsSlide9Slide10Slide11Slide12
Insoluble Products or ComponentsFiltrationCentrifugationCoagulation and FlocculationSlide13
FiltrationMost cost-effective, most common in industrial biotechnology.Rotary vacuum precoat filters: traditional. Penicillin mold.
Cross flow ultrafiltration: 0.02-0.2 µm bacterial separationsCross flow microporous filtration0.2-2 µm for yeastSlide14
Rotary vacuum precoat filtersSlide15
V = volume of filtrateA = surface area of filterp = pressure drop through the cake and filter mediumu = viscosity of filtrater
m= resistance of filter mediumrc = resistance of cakeSlide16
Substitute, integrate, linearize = specific resistance of cake, C = cake weight/volume filtratePlot t/V vs. V, slope = 1/K, intercept = 2VoCan find r
m and
Slide17
Assumes incompressible cake.Fermentation cakes are compressible.Filter aid is added to decrease the cake resistance.pH and fermentation time can affect resistance.Heat treatment can reduce cake resistance.Slide18
CentrifugationUsed to separate solids of size 0.1 um to 100 um using centrifugal forces.Being replaced by microfiltration.Fc
=2UoFc= flow, Uo= free settling velocity=centrifugation coefficient = re
2
V
c
/gL
e
R
e
=radius of rotation,
= angular velocity, L
e
=settling distance, Slide19Slide20
Coagulation and FlocculationPretreatment to centrifugation, gravity settling or filtration to improve separation.Coagulation: formation of small flocs of cells using coagulating agents, electrolytes.
Flocculation: formation of agglomeration of flocs into settleable particles using flocculating agents, polyelectrolytes or CaCl2.Used wastewater treatment processes to improve clarification.Slide21
Cell Disruption – Intracellular ProductsMechanical MethodsSonicationBead beatingPressing
Non-Mechanical methodsOsmotic shockFreeze-thawEnzymaticSlide22
Ultrasound: disrupts cell membrane. Mostly used at the laboratory scale.Pressing: extrude cell paste at high pressure.Bead beating: grind cells with glass, metal beads.Heat dissipation is a problem with all of these methods.Slide23
Osmotic shock: Salt differences to cause the membrane to rupture. Common. Freeze-thaw: Causes cell membrane to rupture. Common.Enzymatic: Lysozyme attacks the cell wall.
Can use a combination of these methods.Slide24
Separation of Soluble ProductsLiquid-liquid extractionAqueous two phase extraction
PrecipitationAdsorptionDialysisReverse osmosisUltrafiltration and microfiltrationCross-flow filtration and microfiltrationChromatographyElectrophoresisElectrodialysisSlide25
Liquid-Liquid ExtractionSeparate inhibitory fermentation products from broth.Based on solubility difference for the compound between the phases.Distribution coefficient = K
D = YL/XHYL=concentration in the light phaseXN=concentration in the heavy phaseSlide26
Mass balance assuming immiscibility yields… X1/X0 = 1/(1+E) where E = extraction factor = LKD/HPercent extraction = f(E and the number of stages)
Antibiotics are extracted using liquid-liquid extraction.Slide27
http://www.facstaff.bucknell.edu/mvigeant/field_guide/kandle01/Slide28
http://www.liquid-extraction.com/Slide29Slide30
PrecipitationSalting out – inorganic salts (NH4)2SO4
at high ionic strengthSolubility reduction at low temperatures (less than –5oC) by adding organic solventsSlide31Slide32
AdsorptionRemoval of solutes from aqueous phase onto a solid phase.Chromatography is based on adsorption.Slide33Slide34
DialysisMembrane separation used to remove low molecular weight solutes.For example, removal of urea from urine medical treatment ‘dialysis’ for diabetic patients.
Used to remove salts from protein solutions.Transport occurs due to a concentration gradient driving force.Slide35Slide36
Reverse Osmosis (RO)Osmosis: Transport of water molecules from a high to a low concentration pure water to salt water.In RO, pressure is applied to salt phase causing water to move against a concentration gradient.
Salt phase becomes more concentrated.Slide37Slide38
Ultrafiltration and MicrofiltrationPressure driven molecular sieve to separate molecules of different size.Dead end filtration: retained components accumulate on the filter. Gel layer formed on the filter.Cross flow filtration: retained components flow tangentially across the filterSlide39Slide40
Cross-flow filtrationSlide41Slide42Slide43
Types of filtration equipmentSlide44Slide45Slide46
http://www.gewater.com/equipment/membranehousing/1193_Membrane_elements.jsp
http://www.lcsupport.com/home.htmSlide47
http://www.gewater.com/equipment/membranehousing/1193_Membrane_elements.jspSlide48
Configurations of filtration equipmentSlide49Slide50Slide51Slide52Slide53
Effect of pressure and protein concentration on flux Slide54Slide55Slide56Slide57
Costs of filtration equipmentSlide58Slide59Slide60
ChromatographySeparates mixtures into components by passing the mixture through a bed of adsorbent particles.Solutes travel at different speeds through the column resulting in the separation of the solutes.Slide61Slide62Slide63Slide64Slide65
http://sepragen.com/products/columns/process_columns.htmlSlide66
Affinity ChromatographyHighly specific interaction between a ligand on the particle and a component in the mixture. Often based on antibodies.Slide67Slide68Slide69
ElectrophoresisSeparation of molecules based on size and charge in an electric field.Slide70
ElectrodialysisMembrane separation to separate charged molecules from a solution.Slide71
Finishing StepsCrystallizationDrying