Teknik Bioseparasi

Teknik Bioseparasi
Dina Wahyu
Genap/ Feb 2014
Outline
Chemical Reaction Engineering
1
Pendahuluan
2
Teknik Pemisahan Secara Fisika 1
3
Teknik Pemisahan Secara Fisika 2
4
Teknik Pemisahan Secara Fisika 3
5
Koagulasi dan flokulasi
6
Adsorpsi
7
Adsorpsi 2
mempelajari ruang lingkup teknik bioseparasi dan teknik “cel disruption”
Mempelajari teknik sentrifugasi pada bioseparasi
Mempelajari teknik pemisahan sedimentasi
Mempelajari teknik filtrasi pada bioseparasi
Mengetahui teknik pemisahan dengan cara koagulasi dan flokulasi
Proses adsorpsi pada cairan dan gas, serta pengetahuan bahan adsorpsi
Kinetika Adsorpsi, Isotherm Adsorption
Cell Fractionation Methods
the breaking open of cells and separation
of the parts into pure fractions
involve the homogenization or destruction of
cell boundaries by different mechanical or
chemical procedures, followed by the
separation of the subcellular fractions
according to mass, surface, and specific
gravity
Properties of Microbial Cell Envelopes
Lysing
Product of Interest: Intracellular or Extracellular
Table 1. Cell Disruption Technique
?
Stress
Method
Technique
Chemical
Osmotic Shock
Enzyme digestion
Solubilization
Lipid dissolution
Alkali treatment
Mechanical Homogenization
(blade type)
Grinding
Ultrasonication
Homogenization
(orifice type)
Crushing in
ball mill
Principle
Osmotic rupture of
membrane
Cell wall digested,
providing disruption
Detergents solubilize
cell membrane
Organic solvent dissolves
in cell wall, and so
destabilizes it
Saponification of lipids
solubilizes membrane
Cells chopped in
Waring blender
Cell ruptured by grinding
with abrasives
Cells broken with
ultrasonic cavitation
Cells forced through
small hole are broken
by shear
Cells crushed between
glass or steel balls
on Product
Gentle
Cost
Examples
Cheap
Gentle
Expensive
Gentle
Moderate
Moderateexpensive
Cheap
Rupture of red blood
cells
Micrococcus
lysodeikticus treated
with egg lysosyme
Bile salts acting on E.
coli
Toluene disruption of
yeast
Harsh
Cheap
Moderate
Moderate
Moderate
Cheap
Harsh
Expensive
Harsh
Moderate
Harsh
Cheap
Animal tissue and
cells
Cell suspensions at
least on small scale
Large scale
treatment of cell
suspensions, except
of bacteria
Large scale
treatment of cell
suspensions and
plant cells
Cell Disruption
•
•
•
•
Chemical:
alkali,
organic
solvents,
detergents
Enzymatic: lysozyme, chitinase
Physical: osmotic shock, freeze/thaw
Mechanical: sonication, homogenization,
French press
Chemical Methods (not popular in industry)
Enzyme digestion
Enzyme cost
Alkali treatment
Harsh condition, degradation
(PHB separation)
 Osmotic Shock
Cells 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 detergent
Concentrated detergent solution is added to disrupts the cell membrane
Detergent - Ex: SDS (Sod Dodecylsulfate)
Solubilize cell wall lipid
Chemical Disruption
•
•
Detergents such as Trition
X-100 or NP40 can
permeabilize cells
by solubilizing
membranes.
Detergents can be
expensive, denature
proteins, and must be
removed after disruption
Figure 1. Chemical structures of selected surfactants.
 Lipid Dissolution
A 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 layer
production of protoplasts from yeast and bacteria
• endo-β(1,3) glucanase
Lytic protease
• Preparation of lytic enzymes
• Lysis experiment for various enzymes -required
Cell disruption – Mechanical method
Laboratory
techniques:
Industrial
technology:
Sonication
Enzyme treatment
Ball mill
grinding
High mechanical shear
Heat generation
Homogenization
(orifice type)
*Chemical method + mechanical method combination
Chemical Permeabilization of Cell
Sonication
•
•
A sonicator can be
immersed
directly
into a cell suspension.
The
sonicator
is
vibrated and high
frequency
sound
waves disrupt cells.
Homogenization
•
•
Cells are placed in a
closed vessel (usually
glass). A tight fitting
plunger is inserted and
rotated with a
downward force.
Cells are disrupted as
they pass between the
plunger and vessel wall.
Mechanical Disruption
 Homogenizer
(a)
(b)
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.
Apparent
Particle
Size
Fumarase
Activity
Alcohol
Dehydrogenase
Activity
Time,
min
Figure 3. Homogenization versus time. Mechanical disruption of cells reduces
particle size but some may also denature some of the products in the cell.
*How long for operation?
 Ball Mill (Sand Grinder)
screen
Cooling Jacket
Sand (bead) Feed in
Figure 4. Schematic diagram for ball mill equipment.
*batch, continuous type
*paint, dyestuff industry
French Press
Cells are placed in a
stainless steel container.
A tight fitting piston is
inserted
and
high
pressures are applied to
force cells through a
small hole.
Principles of the French Press
Piston
Cylinder body
Flow valve
Outlet
Sample
tube
Figure 2. French Press Cylinder.
Figure 3. Diagram of French Press Cylinder.
Bead mill
Media
volume 5.5L
Media
volume 0.4L
Laboratory bead mill (Dyno mill and DMQ-07)
Production machine (DMQ-10)
Scale-up & Application
• Laboratory
Scale-up
Bead mill – Grinding media
•
Weaknesses of the sand mill
•
•
•
Grinding media (beads)
•
•
•
•
•
Transition to closed mills
ottawa sand  grinding 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.
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)
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
following relation can be used:
•
•
•
Example:
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:
Centrifugation
•
•
•
Centrifuge
the
most
versatile
tools of molecular
biology
to
characterize
substances
to separate them
Swinging –Arm Centrifugation
Differential Centrifugation
Analytical ultracentrifuge
•
•
information concerning the mass and
(in a limited way) the shape of a molecule
Preparative centrifuge
•
permit one to use those parameters to
separate molecular types.
Centrifugal fields
•
•
•
•
•
The force that any particle experiences in a spinning
rotor
F = 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
= 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
= 384,000 x g
Sedimentation Velocity
•
•
Any molecule or particle that is not isodense
with the fluid it displaces will tend to float or
sink, depending on whether it is lighter or
heavier than the surrounding fluid.
The velocity, v, at which a particular substance
moves toward the top or bottom of a liquid
column alpha the acceleration.
The constant of proportionality =
sedimentation coefficient, S:
v = sw2r
S = velocity/unit acceleration
•
•
•
•
e.g. g-globulin - has a component that
sediments @velocity of 2.6 x 10-4cm/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 )
= 7 x 10-13 s
Gradients is formed
material is layered on top
particles reach equilibrium with the gradient
isopyknic (equal density) centrifugation
•
use: Zonal rotors
form density gradient while the
rotor is spinning
sample is layered and centrifuged until the
isopyknic zonal layer of the particles is
reached.
•
•
Buoyant density of macromolecule
i.e. the density at which it will reach an equilibrium with
the suspending medium.
•
e.g. DNA
•
2 sources
band at diff spots in
CsCl gradient.
diff buoyant density
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