Teknik BioseparasiDina WahyuGenap/ Feb 2014

OutlineChemical Reaction Engineering

Pendahuluanmempelajari ruang lingkup teknik bioseparasi dan teknik “cel disruption”

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Teknik Pemisahan Secara Fisika 1

Koagulasi dan flokulasi Mengetahui teknik pemisahan dengan cara koagulasi dan flokulasi

Teknik Pemisahan Secara Fisika 2Mempelajari teknik pemisahan sedimentasi

Teknik Pemisahan Secara Fisika 3 Mempelajari teknik filtrasi pada bioseparasi

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Teknik Pemisahan Secara Fisika 1Mempelajari teknik sentrifugasi pada bioseparasi

Koagulasi dan flokulasi Mengetahui teknik pemisahan dengan cara koagulasi dan flokulasi

Adsorpsi Proses adsorpsi pada cairan dan gas, serta pengetahuan bahan adsorpsi

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Adsorpsi 2Kinetika Adsorpsi, Isotherm Adsorption7

the breaking open of cells and separation

Cell Fractionation Methods

the breaking open of cells and separation of the parts into pure fractions

involve the homogenization or destruction ofcell boundaries by different mechanical orchemical procedures, followed by theseparation of the subcellular fractionsseparation of the subcellular fractionsaccording to mass, surface, and specificgravity

Properties of Microbial Cell Envelopes

Lysing

Product of Interest: Intracellular or Extracellular ?

Method Technique PrincipleStress

on ProductCost Examples

Chemical Osmotic Shock Osmotic rupture of membrane

Gentle Cheap Rupture of red blood cells

Enzyme digestion Cell wall digested,providing disruption

Gentle Expensive Micrococcus lysodeikticus treated with egg lysosyme

Table 1. Cell Disruption Technique

with egg lysosymeSolubilization Detergents solubilize

cell membraneGentle Moderate-

expensiveBile salts acting on E. coli

Lipid dissolution Organic solvent dissolvesin cell wall, and sodestabilizes it

Moderate Cheap Toluene disruption of yeast

Alkali treatment Saponification of lipids solubilizes membrane

Harsh Cheap

Mechanical Homogenization(blade type)

Cells chopped in Waring blender

Moderate Moderate Animal tissue and cells

Grinding Cell ruptured by grinding with abrasives

Moderate Cheapwith abrasives

Ultrasonication Cells broken withultrasonic cavitation

Harsh Expensive Cell suspensions at least on small scale

Homogenization(orifice type)

Cells forced throughsmall hole are brokenby shear

Harsh Moderate Large scale treatment of cell suspensions, except of bacteria

Crushing inball mill

Cells crushed between glass or steel balls

Harsh Cheap Large scale treatment of cell suspensions and plant cells

• Chemical: alkali, organic solvents,

Cell Disruption

• Chemical: alkali, organic solvents,detergents

• Enzymatic: lysozyme, chitinase• Physical: osmotic shock, freeze/thaw• Mechanical: sonication, homogenization,

French pressFrench press

Chemical Methods (not popular in industry)

Enzyme digestion Enzyme cost

Alkali treatment Harsh condition, degradation(PHB separation)(PHB separation)

Osmotic ShockCells are put into pure water

Solutes in the cells cause an osmotic flow of water into the cell

(Note: Plant cell are difficult to be burst.)

Solubilization : by detergentConcentrated detergent solution is added to disrupts the cell membraneDetergent - Ex: SDS (Sod Dodecylsulfate)Solubilize cell wall lipid

Chemical Disruption

• Detergents such as Trition X-100 or NP40 can permeabilize cells permeabilize cells by solubilizing membranes.

• Detergents can be expensive, denature proteins, and must be removed after disruptionremoved after disruption

Figure 1. Chemical structures of selected surfactants.

Lipid Dissolution Lipid DissolutionA volume of solvent( toluene) about 10% of the biomass is added to a cell suspension

The cell wall lipid is solubilized.

Enzymatic Lysis

• dissolve the outer mannoprotein layerproduction of protoplasts from yeast and bacteria

• endo-β(1,3) glucanase• endo-β(1,3) glucanaseLytic protease

• Preparation of lytic enzymes• Lysis experiment for various enzymes -required

Cell disruption – Mechanical method

Laboratory techniques:

SonicationEnzyme treatmenttechniques: Enzyme treatment

Industrial technology:

Ball mill grinding

Homogenization (orifice type)

High mechanical shear

Heat generation

(orifice type)

*Chemical method + mechanical method combination

Chemical Permeabilization of Cell

Sonication

• A sonicator can beimmersed directlyinto a cell suspension.immersed directlyinto a cell suspension.

• The sonicator isvibrated and highfrequency soundwaves disrupt cells.waves disrupt cells.

Homogenization

• Cells are placed in a closed vessel (usually glass). A tight fitting closed vessel (usually glass). A tight fitting plunger is inserted and rotated with a downward force.

• Cells are disrupted as • Cells are disrupted as they pass between the plunger and vessel wall.

Mechanical Disruption

Homogenizer

Figure 2. Homogenizer Assembly. (a) A typical homogenizer and (b) a homogenization valve.

Cell passing through this valve areruptured by both shear and mechanical stress.

(a)(b)

FumaraseActivity

ApparentParticle Size

Time, min

Alcohol DehydrogenaseActivity

Figure 3. Homogenization versus time. Mechanical disruption of cells reducesparticle size but some may also denature some of the products in the cell.

*How long for operation?

min

Ball Mill (Sand Grinder)

screen

Feed in

Cooling Jacket

Sand (bead)

Figure 4. Schematic diagram for ball mill equipment.Figure 4. Schematic diagram for ball mill equipment.

*batch, continuous type*paint, dyestuff industry

Cells are placed in astainless steel container.A tight fitting piston is

French Press

A tight fitting piston isinserted and highpressures are applied toforce cells through asmall hole.

Principles of the French PressPiston

Flow valve

Cylinder body

Figure 3. Diagram of French Press Cylinder.

Flow valve

Outlet tube

Figure 2. French Press Cylinder.

Sample

Bead mill

Media volume 5.5L

Laboratory bead mill (Dyno mill and DMQ-07)

Media volume 0.4L

Production machine (DMQ-10)

Scale-up & Application• Laboratory Scale-up

Bead mill – Grinding media• Weaknesses of the sand mill

• Transition to closed mills• ottawa sand grinding beads• ottawa sand grinding beads

• Grinding media (beads)• Steel, zirconium oxide, aluminum oxide, Si/Al/Zr mixed oxide

(SAZ), steatite (modification of talc), glass and plastics• Diameter lies in the range from 0.1 to 3 mm.• The harder the beads, the greater the intensity of dispersion.• The number of beads is proportional to 1/d3 use smallest

beads possible.The number of beads is proportional to 1/d use smallest beads possible.

• Translational and rotational movement: compressive stress and shear

Application in research• Horizontal bead mill used for cell rupture

(a) General view, (b) details of (i) stainless steel and (ii) polyurethane impellers

Where C = concentration of released product (kg/m3)Cmax = maximum concentration of released material (kg/m3)(kg/m3)t = time (s) θ = time constant (s)

The time constant θ depends on the processing conditions, equipment and the properties of the cells being disrupted.For multiple passes, the For multiple passes, the following relation can be used:

• Example:• A batch of yeast cells was disrupted using • A batch of yeast cells was disrupted using

ultrasonic vibrations to release an intracellular product.

• The concentration of released product in the solution was measured during the process:process:

Centrifugation

• Centrifuge themost versatiletools of moleculartools of molecularbiology

• to characterizesubstances

• to separate them

Swinging –Arm Centrifugation

Differential Centrifugation

Analytical ultracentrifuge• information concerning the mass and • (in a limited way) the shape of a molecule• (in a limited way) the shape of a molecule

Preparative centrifuge• permit one to use those parameters to

separate molecular types.separate molecular types.

Centrifugal fields

• The force that any particle experiences in a spinning rotor

F = m*w2 rF = m*w2 r• m* = buoyant mass of the particle (i.e., its mass less than

the mass of solvent it displaces)• w = the velocity of the rotor in radians/sec • r = the distance to the particle from the center of the

rotor. • w2 r = radial acceleration or centrifugal acceleration

• at 70,000 rpm, a particle 7 cm from the center• a = (70000 rpm x 2phi rad/rev x 1/60 min/s)2(7cm)

= (7329)2 s-2x 7cm= (7329)2 s-2x 7cm= 3.76 x 108 cm/s2

• normal acceleration of the earth’s gravity (g) = 980 cm/s• a ={ (3.76 x 108 ) / 980 } x g• a ={ (3.76 x 108 ) / 980 } x g

= 384,000 x g

Sedimentation Velocity

• Any molecule or particle that is not isodensewith the fluid it displaces will tend to float orwith the fluid it displaces will tend to float orsink, depending on whether it is lighter orheavier than the surrounding fluid.

• The velocity, v, at which a particular substancemoves toward the top or bottom of a liquidmoves toward the top or bottom of a liquidcolumn alpha the acceleration.

The constant of proportionality = sedimentation coefficient, S:sedimentation coefficient, S:

v = sw2r

S = velocity/unit accelerationS = velocity/unit acceleration

• e.g. g-globulin - has a component that• sediments @velocity of 2.6 x 10-4cm/s sediments @velocity of 2.6 x 10 cm/s

( 0.95 cm/h) @ centrifugal field 384,000 x g

• Sedimentation coefficient (S)• = ( 2.6 x 10-4 cm/s) / (3.8 x 108cm/s )• = ( 2.6 x 10 cm/s) / (3.8 x 10 cm/s )

= 7 x 10-13 s

Gradients is formed

material is layered on top

particles reach equilibrium with the gradientisopyknic (equal density) centrifugation

• use: Zonal rotors form density gradient while the rotor is spinning

• i.e. the density at which it will reach an equilibrium withthe suspending medium.

• sample is layered and centrifuged until the isopyknic zonal layer of the particles is reached.

• Buoyant density of macromolecule

the suspending medium.

• e.g. DNA 2 sources diff buoyant density

band at diff spots in CsCl gradient.

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