Cell separation by centrifugation

Centrifugation technique and application in industrial processes

1. Cell separation by Centrifugation BY- Gautam Parmar Industrial Biotechnology

2. Preface

3. The various stages of processing that occur after the completion of the fermentation or bioconversion stage, including separation, purification, and packaging of the product. Downstream process

4. Centrifugation is used to separate materials of different density when a force greater than gravity is desired. Centrifugation method is utilized to separate the cellular debris from the released protein. Example-a key role in many industrial processes, including the production of insulin, is to separate liquid phases and solids from each other. It depends on particles size, density difference between the cells and the broth and broth viscosity. It is based on the behavior of particles in an applied centrifugal field. More dense components of the mixture move away from the axis of the centrifuge while less dense components of the mixture move towards the axis Introduction

5. 5 Principle Centrifugation is used to separate materials of different density when a force greater than gravity is desired. The particles will tend to sediment under the influence of gravity. If the particles suspended in a liquid are so small or have a density so close to that of the liquid, then the force of gravity fails to sediment the particles into a separate layer. So the basis of centrifugation techniques is to exert a larger force than the gravitational force to enhance the effective sedimentation force for the separating such particles from the liquid. Particles which differ in density, shape or size can be separated since they sediment at different rates in the centrifugal field, each particle sedimenting at a rate which is proportional to the applied centrifugal field.

6. The rate at which the sedimentation occurs in centrifugation is expressed in terms of sedimentation coefficient and is given by the formula: Where, V = sedimentation of the molecules ω = Rotation of the rotor in radians/sec. (angular velocity) r = Distance in cm, from the centre of the rotor S = V/ ω 2 r

7. Bowl Basket Centrifuge - Useful for separating mould mycelia or crystalline compounds. Perforated & Lined with a filter bag of nylon, cotton. Normally operated at speed of up to 4000 rpm for feed rate 50-300 dm³ min¯¹. Continuous feed is used & when the basket is filled with the filter cake it is possible to wash the cake before removing it. It may be considered to be a centrifugal filter and holding capacity is 50-300 dm³ Blinding with soft biological materials so that high centrifugal forces cannot be used. Industrial Centrifuges

8. Disc-stack bowl centrifuge- This type is common in bioprocess. The developed forces is 5000-15000 G with minimal density difference between solid and liquid is 0.01-0.03 kg/m 3 . Large particles have higher settling velocities than small particles Cellular debris ends up at the outer edge of the bowl Soluble intracellular material passes through with the clarified liquid The minimum particle diameter is 5 µm. The discs split the stream into a large number of very thin layers thereby improving separation. Solids flow downwards on bottom face of disc. Liquid flows upwards on top face of disc. The close packing of the discs assists rapid sedimentation and the solids then slides to the edge of the bowl. Smaller in size compared with a bowl without discs for given throughput. Requires in-situ steam sterilization and the discs arrangement makes this type of centrifuge laborious to clean. Feed rate 45-1800 dm³ min¯¹.

9. Tubular bowl centrifuge (Narrow tubular bowl centrifuge or ultracentrifuge, decanter centrifuge)-Used for particle size ranging from 0.1-200 µm . Simple and widely applied in food and pharmaceutical industry. Operates at 13000-16000 G, 10 5 -10 6 G for ultracentrifuge. Feed containing solids, heavy liquid phases are introduced by nozzle and are kept separate in their exit from the bowl by an adjustable ring. High centrifugal force, good dewatering and ease of cleaning and plastic liners can be used in the bowls to help improve batch cycle time. It can be employed for Light phase/heavy phase liquid separation, Solid/light-liquid phase/heavy-liquid phase separation. Disadvantages re limited solids capacity, difficulties in the recovery of collected solids, gradual loss in efficiency as the bowl fills, and foaming.

10. The clarification efficiency of centrifugation process is affected by harvest parameters such as centrifuge feed rate, G-force, bowl geometry, operating pressure, discharge frequency and ancillary equipment used in transfer of cell culture fluid. Peak cell density, total cell density and culture viability during the culture process and harvest will also affect separation performance. Particles of submicron size cannot be removed in the centrifuge, thus increases the burden on subsequent depth filtration. Disk stack continuous centrifuge removes cell debris from viable cells and liquid phase but some cells gets disrupted during process especially feedstock with low viability culture fluid Merits and demerits

11. Optimization of centrifugation process can be done at lab scale and pilot to select the feed rate and bowl rotational speed using the scaling factors of feed rate (Q) and equivalent settling area As lower the value of sigma factor the better will be the clarification and it should be govern that Q/ better will be the separation. Feed rate containing fragile cells, viable cells are to be separated at lower speed with constant value of sigma factor

12. Centrifuges are basically employed for separation of whole large cells from heterogeneous cell mixture. Can be used to separate viable cells from cell debris using disc stack centrifuge. clarification (removal of solid impurities from milk prior to pasteurization) skimming (separation of cream from skim milk) standardizing whey separation (separation of whey cream (fat) from whey) bactofuge treatment (separation of bacteria from milk) quark separation (separation of quarg curd from whey) butter oil purification (separation of serum phase from anhydrous milk fat). Disc-stack centrifuges used by some companies in the oil sands industry to separate small amounts of water and solids from bitumen. Large industrial centrifuges are commonly used in water and wastewater treatment to dry  sludges . The resulting dry product is often termed cake, and the water leaving a centrifuge after most of the solids have been removed is called  centrate . Large industrial centrifuges are also used in the oil industry to remove solids from the drilling fluid. Centrifuge are also used in whole blood separation into different components. Applications

13. Industrial centrifuges Tubular bowl Chamber Disc

14. Tube High centrifugal force Good dewatering Easy to clean Chamber Large solids capacity Good dewatering Bowl cooling possible Disc type Solids discharge No foaming Bowl cooling possible Limited solids capacity Foams Difficult to recover protein No solids discharge Cleaning difficult Solids recovery difficult Poor dewatering Difficult to clean Properties of industrial centrifuges

15. Bacteria Small cell size Resilient Yeast cells Large cells Resilient Filamentous fungi Mycelial Resilient Cultured animal cells Large cells Very fragile High speed required Low cell damage Lower speed required Low cell damage Lower speed required High water retention in pellet Very susceptible to damage Centrifugation properties of different cell types

16. 16 The centrifugation theory The terminal velocity during gravity settling of a small spherical particle in dilute suspension is given by Stoke’s law: Where u g is sedimentation velocity under gravity, ρ p is particle density, ρ f is liquid density, µ is liquid viscosity, D p is diameter of the particle, and g is gravitational acceleration. In the centrifuge: u c is particle velocity in the centrifuge, ω is angular velocity in rad/s, and r is radius of the centrifuge drum.

17. 17 The centrifugation theory Disc-stack bowl centrifuge N is number of disc, θ is half-cone angle of the disc. The r 1 and r 2 are inner and outer radius of the disc, respectively. Tubular-bowl centrifuge b is length of the bowl, r 1 and r 2 are inner and outer radius of the wall of the bowl.

18. Principle of Fermentation Technology by A. Whitaker, P.F Stanbury , S.J. Hall. Wikipedia.org. Slideshare.com NCBI.com Hamel & Hunter- Modeling and Applications of Downstream Processing. Downstream processing article- springer References

19. THANX A LOT