Cell DistruptionDr. N. Banu,

Associate Professor,Department of Biotechnology

Vels University

Cell disruption (1) energy-intensive and violent(2) without destroying interest protein(3) contain process ( bacteria, yeast, mammalian, plant, and insect

sources)(4) be controlled(5) be validated

Products requiring cell disruptionVaccines (tetanus, meningitis)GlucokinaseGlycerokinase Invertase Toxin (enterotoxin)Subcellular components (mitochondrai, chloroplast)Intracellular constituents (DNA, RNA preparation,

and virus-like particles)Recombinant insulinRecombinant growth hormoneProtein A and G.

Cell disruptionBiological products:1. Extracellular2. Intracellular3. PeriplasmicCells• Gram positive bacterial cells• Gram negative bacterial cells• Yeast cell • Mould cells• Cultured mammalian cells• Cultured plant cells• Ground tissue

Bacteria

Cell membraneCell wall Peptidoglyca

n

Cell membrane

Periplasm

Lipopolysaccharides + proteins

•Sub-micron to 2 microns in size•Have thick cell walls, 0.02-0.04 microns, peptidoglycan + polysaccharide+ teichoic acid•Phospholipid cell membrane present

•Sub-micron to 1 micron in size•Cell capsule present•Peptidoglycan layer is thin•Periplasmic space present•Mechanically less robust than gm+ bacteria•Chemically more resistant than gm+ bacteria

Yeast and mould•Yeast: 2-20 microns in size, spherical or ellipsoid•Moulds: Bigger and filamentous•Yeasts are unicellular while moulds are multicellular •Very thick cell walls are present in both•Cell wall is mainly composed of polysaccharides such as glucans, mannans and chitins•Plasma membranes are mainly made up of phospholipids

Animal and plant cells•Animal cells do not have cell walls•Animal cells are very fragile•Cultured animal cells are several microns in size•Spherical or ellipsoid

•Plant cells can be bigger•Plant cells have thick and robust cell walls mainly composed of cellulose•Plant cells are difficult to disrupt•Cultured plant cells are less robust than real plant cells

Delineating mechanisms of cell disruption(1) facilitate controlling(2) validating(3) reproducible(4) better design and optimize equipment(5) shear stress, turbulence by impingement

Choice of Disruption MethodThe method selected for large scale cell

disruption will be different in every case, but will depend on:Susceptibility of cells to disruptionProduct stabilityEase of extraction from cell debrisSpeed of methodCost of method

Ideal large scale cell disrupter(1) disrupt tough organisms(2) mechanism of cell disruption should be well understood(3) sterilizable(4) containable and validatable(5) amenable to being made automatic(6) continuous and compact(7) economical

Cell disruption goalObjective function (monetary value of product)Small scale cell disruption(1) ultrasonic(2) freeze thawing (3) force cells through very small orifices at high pressure(4) glass or porcelain ball mills

Cell disruption methodsPhysical methods• Disruption in bead mill• Disruption using a colloid mill• Disruption using French press• Disruption using ultrasonic vibrations

Chemical and physicochemical methods

• Disruption using detergents• Disruption using enzymes e.g. lysozyme• Disruption using solvents• Disruption using osmotic shock

Large scale cell disruptionHomogenizer Structure (positive displacement pump, pistons, nozzle)Mechanisms (1) Shear(2) Cavitation (3) impingement Condition(1) concentration (4-175 g dry wt/liter)(2) pressure (30-95 MPa)(3) passes number (~5time)(4) modeled by equation (5) wild-type E. coli cells more difficult to disrupt (heat induction at 42C)

Bead millCascading beads

Cells being disrupted

Rolling beads

•Disruption takes place due to the grinding action of the rolling beads and the impact resulting from the cascading ones •Bead milling can generate enormous amounts of heat •Cryogenic bead milling : Liquid nitrogen or glycol cooled unit•Application: Yeast, animal and plant tissue•Small scale: Few kilograms of yeast cells per hour•Large scale: Hundreds of kilograms per hour.

Bead Mill

ktRR

R

m

m

ln

First Order

k is a function ofRate of agitation

(1500-2250 rpm)Cell concentration

(30-60% wet solids)Bead diameter (0.2 -

1.0 mm) Temperature

Cascading beads

Cells being disrupted

Rolling beads

Colloid mill

Cell suspension

Rotor

Stator

Disrupted cells

•Typical rotation speeds: 10,000 to 50,000 rpm•Mechanism of cell disruption: High shear and turbulence•Application: Tissue based material•Single or multi-pass operation

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Cell disruption

Mechanical cell disruption methods•French press (pressure cell) and high-pressure homogenizers. In these devices, the cell suspension is drawn through a check valve into a pump cylinder. At this point, it is forced under pressure (up to 1500 bar) through a very narrow annulus or discharge valve, over which the pressure drops to atmospheric. Cell disruption is primary achieved by high liquid shear in the orifice and the sudden pressure drop upon discharge causes explosion of the cells.•Ultrasonic disruption. It is performed by ultrasonic vibrators that produce a high-frequency sound with a wave density of approximately 20 kHz/s. A transducer convert the waves into mechanical oscillations via a titanium probe immersed in the concentrated cell suspension. For small scale

French press

Plunger

CylinderCell suspensio

n

Impact plate

JetOrifice

•Application: Small-scale recovery of intracellular proteins and DNA from bacterial and plant cells•Primary mechanism: High shear rates within the orifice •Secondary mechanism: Impingement•Operating pressure: 10,000 to 50,000 psig

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Cell disruption

                        

HomogeniserLarge scale, widely usedVariables in design

type of valve pressure single or two-stage surfactant type and content viscosity temperature

Disruption occurs through turbulence and cavitation

High pressure (400-600 bar)High solids (50% solids feed)High heat generation (~1.5°C/1000 psi)Throughput 4-21 kg dry yeast/hr

Homogeniser

a

m

m knPRR

R

ln

Design equation

k (release rate constant) is a function of temperature

a is of order 1.5-3

Ultrasonic vibrations

Cell suspension

Ultrasound tip

Ultrasound generator

•Application: Bacterial and fungal cells•Mechanism: Cavitation followed by shock waves•Frequency: 25 kHz•Time: Bacterial cells 30 to 60 seconds, yeast cells 2 to 10 minutes•Used in conjunction with chemical methods

SonicationHighly effective at lab scale (15-300W)Poor at large scales

High energy requirementsSafety issues - noiseHeat transfer problemsNot continuousProtein lability

Cell suspension

Ultrasound tip

Ultrasound generator

Chemical and physicochemical methods•Detergents•Enzymes•Organic solvents•Osmotic shock

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Cell disruptionNon mechanical cell disruption methodsAutolysis, use microbe own enzyme for cell disruption.lysozyme produced from egg-white catalyses the hydrolysis of β-1,4, glycosidic bonds in the polysaccharide portion of bacterial cell wall. Enzyme extracts from leucocytes, Streptomyces sp., Micromonospora sp. , Penicillium sp., Trichoderma sp. And snails shows lysozyme activity. Eg. Glucose isomerase from Streptomyces. Sp.Osmotic shock. Equilibrating the cells in 20% w/v buffered sucrose, then rapidly harvesting and resuspending in water at 4oC.Detergents: Anionic (sodium lauryl sulphate) cationic (cetyltrimethylammonium bromide), non-ionic (Tweens, spans, tritons). Detergents combined with lipoproteins and solubilizes the lipoprotein consituents of the cell wall and released the enzyme.eg. Cytochrome oxidase from beef heart, chloresterol oxidase from Nocardia.

Osmotic Shock

ions all i.e.molar in ion concentrat solute totalis cRTc

Dramatic change in the solute concentration of the liquid surrounding the microorganism – can cause the cell to burst

Pressure is calculated for dilute solutions from:

Sensitivity of Cells to DisruptionSonic Agitation Liquid

PressingFreeze

pressingAnimal cells 7 7 7 7

Gram –ve bacilli & cocci

6 5 6 6

Gram +ve bacilli 5 4 5 4

Yeast 3.5 3 4 2.5

Gram +ve cocci 3.5 2 3 2.5

Spores 2 1 2 1

Mycelia 1 6 1 5

Adverse Factors during DisruptionHeatShear Proteases Particle sizeDNA, RNAChemicalFoaming Heavy-metal toxicity