Cell separation by Centrifugation

BY-Gautam ParmarIndustrial Biotechnology

Introduction

Principle

Types of Industrial Centrifuge

Merits Demerits

Applications

Stoke’s law

References

Preface

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

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

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.

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/ω2r

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

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/m3.

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.

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, 105-

106 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.

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

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

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

Industrial centrifuges

Tubular bowl Chamber Disc

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

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

gDu pfp

g2

18

rDu pfp

c22

18

The centrifugation theoryThe terminal velocity during gravity settling of a small spherical particle in dilute suspension is given by Stoke’s law:

Where ug is sedimentation velocity under gravity, ρp is particle density, ρf is liquid density, µ is liquid viscosity, Dp is diameter of the particle, and g is gravitational acceleration.In the centrifuge:

uc is particle velocity in the centrifuge, ω is angular velocity in rad/s, and r is radius of the centrifuge drum.

17

31

32

2

tan312 rr

gN

21

22

2

32

rrgb

The centrifugation theoryDisc-stack bowl centrifuge

N is number of disc, θ is half-cone angle of the disc. The r1 and r2 are inner and outer radius of the disc, respectively.

Tubular-bowl centrifuge

b is length of the bowl, r1 and r2 are inner and outer radius of the wall of the bowl.

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

THANX A LOT