1. Biochemical EngineeringCEN 551 Instructor: Dr. Christine Kelly Chapter 11: Recovery and Purification of Products

2. Schedule • Thursday 3/4 • chapter 11HW due • take home exam • homework solutions • Tuesday 3/16 – Haowen, Ashutosh, Nilay • Thursday 3/18 – take home exam due

6. General Approach • Separation of insoluble products or components. • Primary isolation or concentration and removal of water. • Purification and removal of contaminated chemicals. • Product preparation.

9. 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

13. Insoluble Products or Components • Filtration • Centrifugation • Coagulation and Flocculation

14. Filtration • Most cost-effective, most common in industrial biotechnology. • Rotary vacuum precoat filters: traditional. Penicillin mold. • Cross flow ultrafiltration: 0.02-0.2 um bacterial separations • Cross flow microporous filtration0.2-2 um for yeast

15. Rotary vacuum precoat filters

16. V = volume of filtrate A = surface area of filter p = pressure drop through the cake and filter medium u = viscosity of filtrate rm= resistance of filter medium rc = resistance of cake

17. 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 

18. 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.

19. 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 = re2Vc/gLe • Re=radius of rotation,  = angular velocity, Le=settling distance,

21. 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.

22. Cell Disruption – Intracellular Products • Mechanical Methods • Sonication • Bead beating • Pressing • Non-Mechanical methods • Osmotic shock • Freeze-thaw • Enzymatic

23. 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.

24. 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.

25. 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

26. 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

27. 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.

28. http://www.facstaff.bucknell.edu/mvigeant/field_guide/kandle01/http://www.facstaff.bucknell.edu/mvigeant/field_guide/kandle01/

29. http://www.liquid-extraction.com/

31. Precipitation • Salting out – inorganic salts (NH4)2SO4 at high ionic strength • Solubility reduction at low temperatures (less than –5oC) by adding organic solvents

33. Adsorption • Removal of solutes from aqueous phase onto a solid phase. • Chromatography is based on adsorption.

35. 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.

37. 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.

39. 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

41. Cross-flow filtration

44. Types of filtration equipment

47. http://www.lcsupport.com/home.htm http://www.gewater.com/equipment/membranehousing/1193_Membrane_elements.jsp

48. http://www.gewater.com/equipment/membranehousing/1193_Membrane_elements.jsphttp://www.gewater.com/equipment/membranehousing/1193_Membrane_elements.jsp

49. Configurations of filtration equipment