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
Advanced Bioprocess Engineering
Recovery and Purification of Products
Lecturer Dr. Kamal E. M. ElkahloutAssistant Prof. of Biotechnology
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.
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
Insoluble Products or Components
•Filtration•Centrifugation•Coagulation and
Flocculation
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
Rotary vacuum precoat filters
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
• 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
• 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.
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,
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.
Cell Disruption – Intracellular Products
• Mechanical Methods–Sonication–Bead beating–Pressing
• Non-Mechanical methods–Osmotic shock–Freeze-thaw–Enzymatic
• 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.
• 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.
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
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
• 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.
http://www.facstaff.bucknell.edu/mvigeant/field_guide/kandle01/
http://www.liquid-extraction.com/
Precipitation1. Salting out – inorganic salts
(NH4)2SO4 at high ionic strength2. Solubility reduction at low
temperatures (less than –5oC) by adding organic solvents
Adsorption• Removal of solutes from
aqueous phase onto a solid phase.
• Chromatography is based on adsorption.
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.
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.
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
Cross-flow filtration
Types of filtration equipment
http://www.gewater.com/equipment/membranehousing/1193_Membrane_elements.jsp
http://www.lcsupport.com/home.htm
http://www.gewater.com/equipment/membranehousing/1193_Membrane_elements.jsp
Configurations of filtration equipment
Effect of pressure and protein concentration
on flux
Costs of filtration equipment
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.
http://sepragen.com/products/columns/process_columns.html
Affinity Chromatography
Highly specific interaction between a ligand on the particle and a component in the mixture. Often based on antibodies.
Electrophoresis
Separation of molecules based on size and charge in an electric field.
Electrodialysis
Membrane separation to separate charged molecules from a solution.
Finishing Steps
•Crystallization•Drying