Advanced Bioprocess Engineering Recovery and Purification of Products Lecturer Dr. Kamal E. M. Elkahlout Assistant Prof. of Biotechnology

General Approach 1.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: µm bacterial separations Cross flow microporous filtration0.2-2 µm for yeast

Rotary vacuum precoat filters

V = volume of filtrate A = surface area of filter  p = pressure drop through the cake and filter medium u = viscosity of filtrate r m = resistance of filter medium r c = resistance of cake

Substitute, integrate, linearize  = specific resistance of cake, C = cake weight/volume filtrate Plot t/V vs. V, slope = 1/K, intercept = 2V o Can find r m and 

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. F c =2U o  F c = flow, U o = free settling velocity  =centrifugation coefficient = r e  2 V c /gL e R e =radius of rotation,  = angular velocity, L e =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 CaCl 2. 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 = K D = Y L /X H Y L =concentration in the light phase X N =concentration in the heavy phase

Mass balance assuming immiscibility yields… X 1 /X 0 = 1/(1+E) where E = extraction factor = LK D /H Percent extraction = f(E and the number of stages) Antibiotics are extracted using liquid- liquid extraction.

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Precipitation 1.Salting out – inorganic salts (NH 4 ) 2 SO 4 at high ionic strength 2.Solubility reduction at low temperatures (less than –5 o C) 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

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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.

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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.

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