PTT203 BIOCHEMICAL ENGINEERING RECOVERY AND PURIFICATION OF PRODUCTS

1. PTT203BIOCHEMICAL ENGINEERINGRECOVERY AND PURIFICATION OF PRODUCTS WEEK 14 MADAM NOORULNAJWA DIYANA YAACOB SCHOOL OF BIOPROCESS ENGINEERING

2. Lecture Outline 1.0 Introduction 2.0 Separation of Insoluble Products Filtration, centrifugation, coagulation &flocculation 3.0 Cell Disruption Mechanical & Nonmechanicalmetods 4.0 Separation of Soluble Products Liquid-liquid extraction, aqueous two-phase extraction, precipitation, adsorption, dialysis, reverse osmosis, untrafiltration &microfiltration, chromatography, electrophoresis, electrodialysis 5.0 Finishing Steps for purification Crystallization, Drying 6.0 Integration of Reaction and Separation

3. 1.0 Introduction • Recovery and purification of a fermentation product is essential to any commercial process. • Difficulty entailed heavily on the nature of the products • Since the chemical nature of a fermentation broth is quit complex and extremely high purity is required for some products, recovery and purification often required many processing steps and in many cases represent a manufacturing cost higher than that involved in producing the products.

4. Figure 11.1

5. 2.0 Separation of Insoluble Products • Separation of solids such as biomass, insoluble particles, and macromolecules from the fermentation broth • Usually the first step in product recovery

6. 2.1 Filtration • Most cost effective method for the separation of large solid particles and cells from fermentation broth. • Fermentation broth is passed through a filter medium and filter cake is formed as a result of deposition of solids on the filter surface.

7. The most widely used is continuous rotary filters • The drum is pre-coated with a filter aid, typically of diatomaceous earth (DE) or Perlite. • After pre-coat has been applied, the liquid to be filtered is sent to the tub below the drum. The drum rotates through the liquid and the vacuum sucks liquid and solids onto the drum pre-coat surface, the liquid portion is "sucked" by the vacuum through the filter media to the internal portion of the drum, and the filtrate pumped away. • The solids adhere to the outside of the drum, which then passes a knife, cutting off the solids and a small portion of the filter media to reveal a fresh media surface that will enter the liquid as the drum rotates. The knife advances automatically as the surface is removed.

8. 2.2 Centrifugation • Separate particles size between 100 and 0.1 µm from liquid by centrifugal process • The theory of solid-liquid separations in a gravitational field should be clearly understood

9. 2.3 Coagulation and Flocculation • Coagulation – formation of small flocs from disperesed colloids using coagulating agents. • Flocculation- agglomeration of these small flocs into larger settable particles using flocculating agents. • Used to formed cell aggregates before centrifugation, gravity settling, or filtration to improve the performance of these separation processes

11. 3.0 Cell Disruption 3.1 Mechanical methods • Ultrasonic vibrators/Sonicators Used to disrupt the cell wall and membrane of the bacterial cells. An electronic generator is used to generate ultrasonic waves, an a transducer converts these waves into mechanical oscillations

12. The Gaulin-Manton and French Press • The Ribifractionator • The Rannie high-pressure homogenizer • The Dyno Mill

13. 3.2 Nonmechanical Methods • Osmotic shock & rupture with ice crystal are commonly used(slowly freezing and the thawing a cell paste, the cell wall and membrane may be broken,releasing enzymes into the media)

14. 4.0 Separation of Soluble Products 4.1 Liquid-liquid Extraction Separate inhibitory products such as ethanol and acetone-butanol from fermentation broth Ideally, the liquid extractant should be nontoxic, selective, inexpensive, and immiscible and should have a high distribution coefficient for the product.

15. 4.2 Precipitation The first step in the purification of intracellular protein after cell disruption is usually precipitation. Proteins in fermentation broth can be separated from other components by using certain salts 2 major methos for protein precipitation: • Salting out by adding inorganic salts • 2. Solubility reduction at low temp. by adding organic solvents

16. What is Salting out Achieved by increasing the ionic strength of protein-containing solution by adding salts. The added ions interact with water more strongly causing protein molecules to precipitate

17. 4.3 Adsorption Adsorption of solutes from liquid media onto solids is a common practice in separating soluble materials from fermentation broth. In physical adsorption-weak forces, such as van der Waals are dominant In ion-exchange adsorption- strong ionic bonds are utilized

18. A process in which molecules of gas, of dissolved substances in liquids, or of liquids adher) in an extremely thin layer to surfaces of solid bodies with which they are in contact. Adsorption is used in gas masks and to purify and decolorize liquids.

19. 4.4 Dialysis Membrane separation operation used for the removal of low-MW solutes such as organic acids and inorganic ions from a solution. A well-known example- used dialysis membrane to remove urea from urine in artificial kidney The dialysis membrane separates two phase containing low-MW and high-MW solution

20. Surrounding the membrane is dialysis fluid. This contains chemicals, which should be in the blood plasma (glucose, amino acids & salts), in the correct concentrations. • The urea moves from the blood to the dialysis fluid by diffusion. Other small particles diffuse from the blood to the dialysis fluid too. As glucose diffuses out of the blood, glucose also diffuses into the blood from the dialysis fluid. This keeps the concentration of important chemicals in the blood constant. • Excess salt diffuses out of the blood, keeping it at the right levels. Osmoregulation occurs in much the same way. If there is too much water in the blood it will enter the dialysis fluid by osmosis. The reverse occurs if the blood is too concentrated. • The blood is kept at the correct temperature while it passes through the machine. The blood then returns to the body.

21. 4.5 Reverse Osmosis Osmosis-transport of water molecules from high to low conc. Region. Reverse osmosis- a pressure is applied onto salt containing phase, which drives water molecules from low to high conc. Region.

22. 4.6 Chromatography Separates mixture into components by passing a fluid mixture through a bed of adsorbent material • Elution chromatography Typically column is packed with adsorbent particles. A mobile phase is injected. This pulse is followed by a solvent eluent The pulse enters as a narrow concentrated peak, but exits dispersed and diluted by additional solvent

24. Some important types of chromatographic methods • Adsorption chromatography (ADC) • Liquid-liquid partition chromatography (LLC) • Ion-exchange chromatograpHy (IEC) • Gel filtration chromatography(GFC) • Affinity chromatography(AFC) • Hydrophobic chromatography (HC) • High Pressure Liquid Chromatography (HPLC)

25. 4.7 Electrophoresis Used for the separation of charged biomolecules according to their size and charge in an electric field. In an electric field, the drag force on a charged particles is balanced by electrostatic forces when the particles is moving with a constant terminal velocity

26. 5.0 Finishing steps for Purification 5.1 Crystallization Operates at low temperature, which minimize thermal degradation of heat-sensitive materials High purity crystal are recovered by using batch Nutsche-type filter or centrifugal filters After washing, the crystals are discharged for drying

27. 5.2 Drying Removal of solvent from purified wet product (crystal) usually achieved by drying.

28. Major type of driers • A vacuum tray drier • Freeze drying • Rotary drum driers • Spray dryers • Pneumatic conveyor driers

29. 6.0 Integration of Reaction and Separation • Separate optimization of fermentation and recovery does not necessarily yield the optimal process. • One form of integration is try to couple some aspects of recovery and purification with the bioreactor

30. Thank you