advanced bioprocess engineering recovery and purification of products
DESCRIPTION
Advanced Bioprocess Engineering 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. - PowerPoint PPT PresentationTRANSCRIPT
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Advanced Bioprocess Engineering
Recovery and Purification of Products
Lecturer Dr. Kamal E. M. ElkahloutAssistant Prof. of Biotechnology
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General Approach1. Separation of insoluble products
or components.2. Primary isolation or concentration
and removal of water.3. Purification and removal of
contaminated chemicals.4. Product preparation.
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Factors that impact difficulty and cost of recovery
• Product 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 costs
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Insoluble Products or Components
•Filtration•Centrifugation•Coagulation and
Flocculation
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Filtration• Most cost-effective, most common in
industrial biotechnology.• Rotary vacuum precoat filters: traditional.
Penicillin mold.• Cross flow ultrafiltration: 0.02-0.2 µm
bacterial separations• Cross flow microporous filtration0.2-2 µm
for yeast
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Rotary vacuum precoat filters
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V = volume of filtrateA = surface area of filterp = pressure drop through the cake and filter
mediumu = viscosity of filtraterm= resistance of filter mediumrc = resistance of cake
)μr(rΔpAg
dtdV
cm
c
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• Substitute, integrate, linearize
• = specific resistance of cake, C = cake weight/volume filtrate
• Plot t/V vs. V, slope = 1/K, intercept = 2Vo
• Can find rm and
)2V(VK1
Vt Equation Ruth o
c
2m
o ΔpgαCμ2AK andA
αCr V
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• 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.
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Centrifugation• Used to separate solids of size 0.1 um to
100 um using centrifugal forces.• Being replaced by microfiltration.• Fc=2Uo• Fc= flow, Uo= free settling velocity• =centrifugation coefficient = re2Vc/gLe
• Re=radius of rotation, = angular velocity, Le=settling distance,
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Coagulation and Flocculation
• Pretreatment 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.
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Cell Disruption – Intracellular Products
• Mechanical Methods–Sonication–Bead beating–Pressing
• Non-Mechanical methods–Osmotic shock–Freeze-thaw–Enzymatic
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• 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.
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• 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.
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Separation of Soluble Products
• Liquid-liquid extraction• Aqueous two phase extraction• Precipitation• Adsorption• Dialysis• Reverse osmosis• Ultrafiltration and microfiltration• Cross-flow filtration and microfiltration• Chromatography• Electrophoresis• Electrodialysis
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Liquid-Liquid Extraction• Separate inhibitory fermentation
products from broth.• Based on solubility difference for the
compound between the phases.• Distribution coefficient = KD = YL/XH
• YL=concentration in the light phase• XN=concentration in the heavy phase
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• Mass balance assuming immiscibility yields…X1/X0 = 1/(1+E) where E = extraction factor = LKD/H
• Percent extraction = f(E and the number of stages)
• Antibiotics are extracted using liquid-liquid extraction.
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http://www.facstaff.bucknell.edu/mvigeant/field_guide/kandle01/
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http://www.liquid-extraction.com/
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Precipitation1. Salting out – inorganic salts
(NH4)2SO4 at high ionic strength2. Solubility reduction at low
temperatures (less than –5oC) by adding organic solvents
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Adsorption• Removal of solutes from
aqueous phase onto a solid phase.
• Chromatography is based on adsorption.
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Dialysis• Membrane 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.
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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.
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Ultrafiltration and Microfiltration
• Pressure 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 filter
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Cross-flow filtration
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Types of filtration equipment
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http://www.gewater.com/equipment/membranehousing/1193_Membrane_elements.jsp
http://www.lcsupport.com/home.htm
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http://www.gewater.com/equipment/membranehousing/1193_Membrane_elements.jsp
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Configurations of filtration equipment
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Effect of pressure and protein concentration
on flux
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Costs of filtration equipment
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Chromatography• Separates 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.
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http://sepragen.com/products/columns/process_columns.html
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Affinity Chromatography
Highly specific interaction between a ligand on the particle and a component in the mixture. Often based on antibodies.
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Electrophoresis
Separation of molecules based on size and charge in an electric field.
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Electrodialysis
Membrane separation to separate charged molecules from a solution.
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Finishing Steps
•Crystallization•Drying