Transcript lecture notes-separation and purification-2-cell

Recovery and Purification of Bio-Products
- Strategies to recovery and purify bio-products
Fermenter
Solid-liquid separation
Cell products
Cells
Cell disruption or
rupture
Supernatant
Recovery
Purification
Cell debris
Crystallization and drying
Cell Disruption
Disruption: the cell envelope is physically broken,
releasing all intracellular components into the
surrounding medium
Methods: Mechanical and non mechanical
- Mechanical
- Ultrasonication (sonicators)
bacteria, virus and spores
suspensions at lab-scale
Electronic generator→ultrasonic waves
→mechanical oscillation
by a titanium probe immersed
in a cell disruption.
http://www.biologics-inc.com/sd-models.htm
Cell Disruption
- Mechanical
Milling: continuous operation,
Dyno-Mill
(liquid)
Algae, bacteria and fungi
Large scale, up to 2000kg/h
liquid and solid
http://www.cbmills.com/Products/horizontalmills.htm
Principle of operation:
A grinding chamber filled with about 80% beads.
A shaft with designed discs or impellers is within the
chamber.
The shift rotates at high speeds, high shearing and impact
forces from the beads break the cell wall.
Cell Disruption
- Mechanical
Ball Mill: solid
Frozen cell paste, cells attached to or within a solid matrix.
Large scale
http://www.unitednuclear.com/mills.htm
Cell Disruption
- Mechanical
Homogenization: suspension, large scale
To pump a slurry (up to 1500 bar) through a restricted orifice
valve.
The cells disrupt as they are extruded through the valve to
atmosphere pressure by
- high liquid shear in the orifice
- sudden pressure drop upon discharge
i.e. French press & Gaulin-Manton: lab scale
Rannie high-pressure
Homogenizer (large scale)
High pressure
orifice
Cell Disruption
- Nonmechanical
- Chemicals: use chemicals to solubilise the components in
the cell walls to release the product.
Chemical requirements:
- products are insensitive to the used chemicals.
- the chemicals must be easily separable.
Types of chemicals:
- surfactants (solubilising lipids): sodium sulfonate,
sodium dodecylsulfate.
- Alkali: sodium hydroxide, harsh
- Organic solvents: penetrating the lipids and swelling
the cells. e.g. toluene.
e.g. Bacteria were treated with acetone followed by sodium dodecyl
sulfate extraction of cellular proteins.
Cell Disruption
- Nonmechanical
- Enzymes: to lyse cell walls to release the product.
gentle, but high cost
i.e. lysozyme (carbohydrase) to lyse the cell
walls of bacteria.
- Osmotic shock
Osmosis is the transport of water molecules from high- to a
low-concentration region when these two phases are
separated by a selective membrane.
Water is easier to pass the membrane than other
components.
When cells are dumped into pure water, cells can swell and
burst due to the osmotic flow of water into the cells.
Cell Disruption
Challenge: Damage to the product
- Heat denaturation
- Oxidation of the product
- Unhindered release of all intracellular
products
Recovery and Purification of Bio-Products
- Strategies to recovery and purify bio-products
Fermenter
Solid-liquid separation
Cell products
Cells
Cell disruption or
rupture
Supernatant
Recovery
Purification
Cell debris
Crystallization and drying
Separation of Soluble Products
Liquid-liquid extraction:
- Difference of solubility in two immiscible liquid
- Applicable: separate inhibitory fermentation products such
as ethanol and acetone-butanol from fermentation broth.
antibiotics (i.e. solvent amylacetate)
- Requirements of liquid extractants :
Light, YL
nontoxic, selective, inexpensive, immiscible with
Heavy, XH
fermentation broth and
high distribution coefficient: KD=YL/XH
YL and XH are concentrations of the solute in light and heavy
phases, respectively.
The light phase is the organic solvent and the heavy phase
is the fermentation broth. e.x. Penicillin is extracted from a
fermentation broth using isoamylacetate. KD could reach 50.
Separation of Soluble Products
• Liquid-liquid extraction:
When fermentation broth contains more than one component,
then the selectivity coefficient (β) is important.
βil = KD,,i/KD,j
KD,,I and KD,j are distribution coefficients of component i and j.
The higher the value of βil is, the easier the separation of i
from j.
pH effect
e.g. at low pH <4, Penicillin can be
separated from other impurities such as
acetic acid from the fermentation broth to
organic phase amylacetate.
Light, i, j
Heavy, I, j
Separation of Soluble Products
Precipitation
Reduce the product solubility in the fermentation
broth by adding chemicals.
Applicable: separate proteins or antibiotics from
fermentation broth.
Separation of Soluble Products
Precipitation
- Methods:
- salting-out by adding inorganic salts such as
ammonium sulfate, or sodium sulfate to increase
high ionic strength (factors: pH, temperature)
e.g. The solubility of hemoglobin is reduced with
increased amount of ammonium sulfate.
- added salts interact more stronger with water so
that the proteins precipitate.
- inexpensive
- Isoelectric (IE) precipitation:
Precipitate a protein at its isoelectric point. E.g. The IE of
cytochrome cM (without histidine tag) is 5.6 (Cho, et.al., 2000,
Eur. J. Biochem. 267, 1068±1074).
Separation of Soluble Products
Adsorption
Adsorb soluble product from fermentation broth onto
solids.
Approaches: physical adsorption (activated carbon), ion
exchange (carboxylic acid cation exchange resin for
recovering streptomycin)
Adsorption capacity: mass of solute adsorbed per unit mass
of adsorbent
Affected by properties of adsorbents:
functional groups and their numbers, surface
properties
by properties of solution: solutes, pH, ionic
strength and temperature
- Difference of Affinity of product in the solid and liquid phase.
- Applicable: soluble products from dilute fermentation
Separation of Soluble Products
CHALLENGE!
SCREENING ADSORBENTS:
THE MOST PROMISING TYPES
- high capacity
- reusable
Equilibrium solute
concentation on solid
(mol/g adsorbent)
14
Saturated uptake
Adsorbent 1
12
10
Adsorbent 2
8
Cs1*
6
4
Cs2*
2
0
affinity
C1
0
10
20
30
40
50
Equilibrium solute concentration in liquid (mol/l)
Adsorption Isotherms
Separation of Soluble Products
Langmuir isotherm:
Cs*=CsM CL*/(K+CL*)
CsM is the maximum concentration of solute adsorbed on
the solid phase. K is a constant. Cs* and CL* are
equilibrium concentration of solute in solid and liquid
phases, respectively.
Freundlich isotherm:
Cs* =KF CL* 1/n
KF and n are empirical constants.
Separation of Soluble Products
Membrane separation:
- Microfiltration: 0.1 - 10 µm, bacterial and yeast cells.
- Ultrafiltration: macromolecules (2000 <MW< 500,000)
- Dialysis: removal of low-MW solutes: organic acids
(100<MW<500) and inorganic ions (10<MW<100).
- Reverse osmosis: a pressure is applied onto a saltcontaining phase, which drives water from a low to a high
concentration region. MW < 300.
The common features of the above methods:
- Use membrane
- Driving forces: pressure
Separation of Soluble Products
Chromatography
To separate the solutes based on the different rate of
movement of the solutes in the column with adsorbent
materials.
Principles:
Chromatographic processes involve a stationary phase and a
mobile phase.
Stationary phase can be adsorbent, ion-exchange resin,
porous solid, or gel usually packed in a cylindrical column.
Mobil phase is the solution containing solutes to be separated
and the eluant that carriers the solution through the
stationary phase.
Applicable for protein, organics separation.
Separation of Soluble Products
Chromatography
Method: A solution containing several solutes is injected at
one end of the column followed by the eluant carrying the
solution through the column.
Each solutes in the original solution moves at a rate
proportional to its relative affinity for the stationary phase
and comes out at the end of the column as a separated
band.
(M. Shuler, Bioprocess.
Eng. 2005)
Separation of Soluble Products
Chromatography
Mechanism:
Similar to adsorption: interaction of soluteadsorbent
Different to adsorption:
- Chromatography is based on different rate of
movement of the solute in the column
- Adsorption is based on the separation of one
solute from other constituents by being captured
on the adsorbent.
Separation of Soluble Products
Electrophoresis
To separate charged solutes based on their specific
migration rates in an electrical field.
Positive charged solutes are attracted to anode
and negative charged solutes to cathode.
Factors: electric field strength, electric charge of
the solutes, viscosity of liquid and the particles
size.
Applicable for protein separation.
Proteins Electrophoresis
http://fig.cox.miami.edu/~cmallery/150/protein/SDS.electrophoresis.jpg
Recovery and Purification of Bio-Products
- Strategies to recovery and purify bio-products
Fermenter
Solid-liquid separation
Cell products
Cells
Cell disruption or
rupture
Supernatant
Recovery
Purification
Cell debris
Crystallization and drying
Recovery and Purification of Bio-Products
- Crystallization: last step in producing highly purified
products such as antibiotics.
Supersaturated solution, low temperature,
Crystals are separated by filters.
- Drying
To remove solvent from purified wet product such as crystal
or dissolved solute.
Vaccum-tray dryers: pharmaceutical products
Freezing drying: by sublimation (from solid ice to vapor),
antibiotics, enzyme, bacteria
Spray dryer: heat-sensitive materials
Summary of separation and
purification
• Liquid-Solid Separation
- Filtration: rotary vaccum drum filter, micro- and
ultra- filtration
- Centrifugation
• Cell disruption
- Mechanical: ultrasonication, milling, homogenization
- Nonmechanical: chemicals, enzyme and osmotic
shock
Summary of separation and
purification
• Separation of soluble products
- Liquid-liquid extraction
- Precipitation
- Adsorption
- Membrane separation: ultrafiltration, dialysis,
reverse osmosis
- Chromatography
- Electrophoresis
• Crystallization and drying