Centrifugation

1. Centrifugation A centrifuge is a device for separating particles from a solution according to their size, shape, density, viscosity of the medium and rotor speed. In biology, the particles are usually cells, subcellular organelles, viruses, large molecules such as proteins and nucleic acids. Analytical centrifugation Analytical centrifugation involves measuring the physical properties of the sedimenting particles such as sedimentation coefficient or molecular weight. Preparative centrifugation In preparative centrifugation objective is to isolate specific particles which can be reused. There are many type of preparative centrifugation such as rate zonal, differential, isopycnic centrifugation.

2. Centrifugation classification based on speed Another system of classification is the rate or speed at which the centrifuge is turning. Ultracentrifugation is carried out at speed faster than 20,000 rpm. Super speed centrifugation is at speeds between 10,000 and 20,000 rpm. Low speed centrifugation is at speed below 10,000 rpm.

3. Differential centrifugation A third method of defining centrifugation is by the way the samples are applied to the centrifuge tube. In moving boundary (or differential centrifugation), the entire tube is filled with sample and centrifuged. Through centrifugation, one obtains a separation of two particles but any particle in the mixture may end up in the supernatant or in the pellet or it may be distributed in both fractions, depending upon its size, shape, density, and conditions of centrifugation.

4. Denisty gradient centrifugation It allows separation of many components in a mixture by creating density gradient during centrifugation There are two forms of density gradient centrifugation: rate zonal and isopycnic

5. Rate Zonal Centrifugation (also termed sedimentation velocity, zone centrifugation) In rate zonal centrifugation, the sample is applied in a thin zone at the top of the centrifuge tube on a density gradient. Under centrifugal force, the particles will begin sedimenting through the gradient in separate zones according to their size shape and density. The run must be terminated before any of the separated particles reach the bottom of the tube. Swing bucket rotor

6. Isopycnic centrifugation (also termed sedimentation equilibrium centrifugation) During the centrifugation, the CsCl generates a gradient (“self-generating gradient”), and the molecules move to the position in the gradient where their density is the same as the gradient material. Isopycnic means “same density,” so the molecules move to their “isopycnic position.” Fixed angle or swing bucket rotor

7. Gradients Sucrose Cscl2 Glycerol Dextran

8. Centrifugation The performance of a centrifuge is characterised by: Vc = dp2 . (ds - dl) . W2 r / 18 n where Vc = centrifugal sedimentation rate (gs-1), dp = particle diameter, ds = density of solid, dl= density of liquid, w= angular speed, r = distance of the particle to the axis of rotation and n = viscosity of medium.

9. Major forces acting on solid particle during settling- Gravitational force (FG) Drag force (FD) Buoyant force (FB) When the particles reach a terminal settling velocity, forces acting on a particle balance each other, resulting in zero net force. That is FG = FD + FB

10. Nomogram for convertingmaximum relative centrifugal force (RCF, i.e., g-force) to RPM

11. THE END

12. R I Isolation/Extraction P P

13. Release of protein from biological host • To gain access to the product • Access to the product is simple and inexpensive when the • protein is produced extracellularly • Microbial sources are preferred • Mammlian cell hosts are preferred when posttranslational • modification is essential for the function of eukaryotic • proteins • Bulk enzymes are invariably produced extracellularly by • Bacillus species & fungi, as are the proteins produced • by mammalian cell culture

14. Mannan partially cross- linked by phosphodiester bridges Lipopolysaccharide membrane Peptidoglycon layer Glucan layer with proteins Cytoplasmic membrane Gram-positive Gram-negative Yeasts bacteria bacteria Cell envelops of bacteria and yeast Animal cells: no cell wall, thus fragile in breaking Plant cells: composed of cellulose and other polysaccharides

15. Cell Disintegration Techniques Technique Example Principle Gentle Cell lysis Erythrocytes Osmotic disruption of cell membrane Enzyme digestion Lysozyme treatment Cell wall digested, leading of bacteria to osmotic disruption Chemical solubilization/ Toluene extraction Cell wall (membrane) par- autolysis of yeast tially solubilized chemically; lytic enzymes released complete the process Hand homogenizer Liver tissue Cells forced through narrow gap, disrupts cell membrane Minicing (grinding) Muscle etc. Cells disrupted during minicing process by shear force

16. Cell Disintegration Techniques Technique Example Principle Moderate Blade homogenizer Muscle tissue, most Chopping action breaks up (waring type) animal tissues, plant large cells, shears apart tissues smaller ones Grinding with abrasive Plant tissues, bacteria Microroughness rips off (sand, alumina) cell walls Vigorous French press Bacteria, plant cells Cells forced through small orfice at very high press- ure; shear forces disrupt cells Ultrasonication Cell suspensions Micro-scale high-pressure sound waves cause dis- ruption by shear forces and cavitation

17. Cell Disintegration Techniques Technique Example Principle Bead mill Cell suspension Rapid vibration with glass beads rips cell walls off Manton-Gaulin Cell suspension As for French press, but on homogenizer a larger scale

18. Mechanical Methods • Mechanical methods can be applied to a liquid or solid medium • Most common mode, despite higher capital and operating costs • Disruption is based primarily on liquid or solid shear forces • Liquid shear cell disruption is associated with cavitation phenomenon • that involves formation of vapor cavities in liquid due to local reduction • in pressure that could be affected by ultrasonic vibrations, local increase • in velocity, etc. Collapse and rebound of the cavities will occur until an • incresae in pressure causes their destruction • On the collapse of the cavitation bubble, a large amount of energy is released • as mechanical energy in the form of elastic waves that disintegarte into • eddies which impart motions of diferent intensities to the cell, creating • pressure difference across the cell. • When the kinetic energy content of the cell exceeds the cell wall strength, • the cell disintegrates.

19. Mechanical Methods • Ultrasonic vibrators(sonicators) are used to disrupt the cell wall and membrane • of bacterial cells. An electronic generator is used to generate ultrasonic waves, • and a transducer converts these waves into mechanical oscillations by a • titanium probe immersed in a cell suspension. Wave density is usually around • 20 kc/s. • Rods are broken more readily than cocci, and gram negative cells more easily • than gram positive cells • The technique is not used at industrial scale primarily because the ultrasonic • energy absorbed into suspension ultimately apears as heat, and good • temperature control is necessary • In some cases results in denaturation of sensitive enzymes and fragmentation • of cell debries

20. Mechanical Methods • High-Pressure homogenization: • French Press: The french press is a hollow cylinder in a stainless-steel • block that is filled with cell paste and subjected to high pressure. The • cylinder has a needle value at the base, and the cells disrupt as they are • extended through the value to atmospheric pressure. The flow restriction • in the value assembly drives up pressure (in the range of 50 and 120 MPa) • Disruption follows first-order process at a given pressure in a high- • pressure homogenizer. The extent of protein release is represented • by • Rm • ln Rm – R • Where Rm and R are the maximal amount of protein available for • release and the protein amount released at a certain time, respectively • (kg protein/kg cells), k is the first-order rate constant, N the number • of passages, P the operating pressure. = kNP

21. Mechanical Methods • High-Pressure homogenization: • Manton-Gaulin homogenizer: Traditional form of high-pressure • homogenizer, works as french press but on a larger scale. • Bead Mill Disruption:Stirring a cell suspension with glass beads • is an effective method of disruption of organisms. The process • is normally performed in a bead mill, such asDyno-Mill • The principle of operation is to pump the cell suspension through • a horizontal grinding chamber filled with about 80% beads. • Within the grinding chamber is a shaft with specially designed • discs. When rotated at high speeds, high shearing and impact • forces from millions of beads break cell walls. • Can be used effectively at large scale • Available in sizes upto 275 l and can process 2000 kg/h of a cell • suspension or about 340 kg dw/h of yeast • Can work with algae, bacteria and fungi • Better temperature control

22. Mechanical Methods • Limitations • High risk of damage to the product • Heat denaturation a major problem • The release of proteases from cellular compartments can • lead to enzymatic degradation of the product • Bead mill have comparatively long residance times, products • released early may be damaged • Products released encounter an oxidizing environment, that • can cause denaturation and aggregation

23. Non-Mechanical Methods Physical Rupture of Microbial Cells Desiccation: by slow drying in air, drum drying, etc followed by extraction of the microbial powder Osmotic shock: Changes in the osmotic pressure of the medium may result in the release of certain enzymes, particularly periplasmic proteins in gram negative cells. Suspending a cell suspension in a solution with high salt concentration High temperature: Exposure to high temperature can be an effective approach to cell disruption but is limited to heat-stable products. Heating to 50 – 55 ºC disrupts outer membrane, releases periplasmic proteins. Heating at 90 ˚C for 10 min can be used for releasing cytoplasmic proteins

24. Non-Mechanical Methods Physical Rupture of Microbial Cells Freeze-thawing:Rupture with ice crystals is commonly used method. By slowly freezing and then thawing a cell paste, the cell wall and membrane may be broken, releasing enzymes into the media Nebulization: In nebulization gas is blown over a surface of liquid through a neck. Because of the differential flow within the neck, the cells are sheared Decompression:When pressurized, the microbial cells are gradually penetrated and filled with gas. After saturation by the gas, the applied pressure is suddenly released when the absorbed gas rapidly expands within the cells leading to rupture Note: Methods produce low protein yields and require long process time