Recovery and Purification of Bio-Products

1
Recovery and Purification of Bio-Products

• Strategies to recovery and purify bio-products

Fermenter
Solid-liquid separation
Cells
Cell products
Supernatant
Recovery
Cell disruption or rupture
Purification
Cell debris
Crystallization and drying
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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.

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Cell Disruption

• Mechanical
• Milling continuous operation,
• Algae, bacteria and fungi
• Large scale, up to 2000kg/h
• liquid and solid
• 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.

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Cell Disruption

• Mechanical
• Ball Mill solid
• Frozen cell paste, cells attached to or within a
  solid matrix.
• Large scale

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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
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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.

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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.

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Cell Disruption

• Challenge Damage to the product
• Heat denaturation
• Oxidation of the product
• Unhindered release of all intracellular products

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Recovery and Purification of Bio-Products

• Strategies to recovery and purify bio-products

Fermenter
Solid-liquid separation
Cells
Cell products
Supernatant
Recovery
Cell disruption or rupture
Purification
Cell debris
Crystallization and drying
10
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
• nontoxic, selective, inexpensive, immiscible
  with fermentation broth and
• high distribution coefficient KDYL/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.

Light, YL
Heavy, XH
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Separation of Soluble Products

• Liquid-liquid extraction
• When fermentation broth contains more than one
  component, then the selectivity coefficient (ß)
  is important.
• ßil KD,,i/KD,j
• KD,,I and KD,j are distribution coefficients of
  component i and j.
• The higher the value of ßil is, the easier the
  separation of i from j.
• pH effect
• e.g. at low pH lt4, Penicillin can be
• separated from other impurities such as
• acetic acid from the fermentation broth to
• organic phase amylacetate.

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Separation of Soluble Products
Precipitation Reduce the product solubility in
the fermentation broth by adding
chemicals. Applicable separate proteins or
antibiotics from fermentation broth.
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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, 10681074).

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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

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Separation of Soluble Products
CHALLENGE!
SCREENING ADSORBENTS THE MOST PROMISING
TYPES - high capacity - reusable
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Saturated uptake
Adsorbent 1
Adsorbent 2
Cs1
affinity
Cs2
C1
Adsorption Isotherms
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Separation of Soluble Products
Langmuir isotherm CsCsM CL/(KCL) 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.

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Separation of Soluble Products

• Membrane separation
• Microfiltration 0.1 - 10 µm, bacterial and yeast
  cells.
• Ultrafiltration macromolecules (2000 ltMWlt
  500,000)
• Dialysis removal of low-MW solutes organic
  acids (100ltMWlt500) and inorganic ions
  (10ltMWlt100).
• Reverse osmosis a pressure is applied onto a
  salt-containing phase, which drives water from a
  low to a high concentration region. MW lt 300.
• The common features of the above methods
• Use membrane
• Driving forces pressure

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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.
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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)
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Separation of Soluble Products
Chromatography

• Mechanism
• Similar to adsorption interaction of
  solute-adsorbent
• 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.

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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.
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Proteins Electrophoresis
http//fig.cox.miami.edu/cmallery/150/protein/SDS
.electrophoresis.jpg
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Recovery and Purification of Bio-Products

• Strategies to recovery and purify bio-products

Fermenter
Solid-liquid separation
Cells
Cell products
Supernatant
Recovery
Cell disruption or rupture
Purification
Cell debris
Crystallization and drying
25
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

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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

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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