Recovery and Purification of Products

1
Recovery and Purification of Products

• BKC4622 Bioprocess Engineering

2
Introduction

• Bioproducts are either cellular cell mass,
  intracellular proteins, inclusion bodies or
  extracellular amino acids, antibiotics, enzymes
• Separation is depends on the nature of the
  products

3
Principles of separation
4
General steps
Separation of insoluble products and other
solids centrifuge, cell disruption
Product preparation drying, lyophilization
Primary isolation or concentration of products
and removal of most water salt precipitation
Removal of contaminating chemicals ultrafiltratio
n, chromatography
5
Filtration

• Used for the separation of large solid particles
  and cells from fermentation broth (bakers yeast)

6
Centrifugation

• Used to separate particles of size between 100
  and 0.1?m by centrifugal forces

7
Coagulation Flocculation

• Usually used to form cell aggregates before
  centrifugation, settling or filtration
• Coagulation - formation of small flocs using
  coagulants (simple electrolytes)
• Flocculation agglomeration of small flocs into
  larger settlable particles using flocculants
  (polyelectrolytes or salts such CaCl2)

8
Cell disruptionMechanical methods

• Sonicators Used to disrupt cell wall and
  membrane of bacterial cells by ultrasonic waves.
  Can cause denaturation of sensitive enzymes.
• Gaulin-Manton French presses Hollow cylinder
  filled with cell paste and subjected to high
  pressure
• Homogenizers Dyno-Mill grinder better
  temperature control compared to pressure devices
• Hughes, X-press Used to disrupt frozen cells.

9
Cell disruptionNonmechanical methods

• Osmotic lysis Occurs in a hypotonic
  environment, where water diffuses into the cell,
  the cell grows larger, and will eventually burst
• Osmotic shock Concentration of either salts,
  substrates or any solute in the supernatant is so
  high that it draws water out of the cells
• Rupture with ice crystals Freeze-thaw,
  freeze-dry after treatment with acetone, butanol
  and buffers
• Lysozyme Carbohydrase, used to lyse cell wall
  of bacteria, cell treated with EDTA or
  freeze-thaw before treatment with lysozyme

10
Liquid-liquid extraction

• Commonly used to separate inhibitory fermentation
  products such as ethanol, acetone-butanol,
  antibiotics
• Liquid extractant should be nontoxic, selective,
  inexpensive and immiscible with fermentation broth

11
Aqueous two-phase extraction

• Extraction of soluble proteins between two
  aqueous phases containing incompatible polymers
  such as PEG and dextran
• Can also be used for the recovery of cell debris,
  polysaccharides and nucleic acids
• PEG and dextran can be recovered by
  ultrafiltration
• Fast, mild conditions of temperature, pressure
  and pH

12
(NH4)2SO4 Precipitation

• Separation is based on solubility of protein
• Solubility of most proteins is lowered at high
  salt concentrations
• As salt concentration is increased, a point is
  reached where the protein comes out of solution
  and precipitates (salting out)
• Precipitated protein can then be redissolved in a
  smaller volume of buffer
• Normally applied at early stage of protein
  recovery

13
Dialysis

• Removal of low-MW solutes such as organic acids
  (100ltMWlt500) and inorganic ions (10ltMWlt100)
• Separated through a semi permeable membrane such
  as cellophane (cellulose acetate)
• Often used to remove small molecules such as
  salts from a protein solution (normally after
  precipitation)
• At equilibrium, the concentration of small
  molecules inside a dialysis bag will be equal to
  that outside

14
Ultrafiltration

• Not fundamentally different from reverse osmosis,
  microfiltration or nanofiltration, except in
  terms of the size of the molecules it retains.
• Variety of membrane filtration in which
  hydrostatic pressure forces a liquid against a
  semipermeable membrane
• Used in industry and research for purifying and
  concentrating macromolecular (10-3 10-6 Da)
  solutions, especially protein solutions

15
Cross-flow filtration

• Pressure is applied parallel to the membrane to
  avoid gel formation
• Agitation or vibration on the membrane surface
  alleviate gel formation

16
Reverse osmosis

• process of forcing a solvent from a region of
  high solute concentration through a membrane to a
  region of low solute concentration by applying a
  pressure in excess of the osmotic pressure

17
Chromatography gel filtration

• Normally used for determining molecular weight
  (based on size)
• Sometimes applied at early stage of recovery to
  remove larger solutes

18
Ion exchange chromatography

• Separation based on charge
• Sometimes applied at early stage because does not
  separate all proteins

19
Affinity chromatography

• Separation based on attraction of product to
  other molecules
• Because of high cost, normally it is applied at
  secondary step

20
Electrophoresis

• Important analytical separation technique
• Scale-up is problematic due to thermal convection
  (electrical heating)
• Native polyacrylamide gel electrophoresis (PAGE)
  proteins are applied to a porous polyacrylamide
  gel and separated in an electric field on the
  basis of their net negative charge and size.
  Small/more negatively charged proteins migrate
  further through the gel

21
Electrophoresis

• SDS-PAGE or denaturing PAGE proteins are
  treated with reducing agent (2-mercaptoethanol)
  to break disulfide bonds and then with the
  anionic detergent sodium dodecyl sulfate (SDS)
  which denatures the proteins and covers them with
  an overall negative charge
• Identical charge to mass ratio - separated on the
  basis of their mass. The smallest proteins move
  farthest
• Used to determine the degree of purity of a
  protein, molecular mass of a protein and number
  of polypeptide subunits in a protein.

22
Electrophoresis
23
Electrophoresis (Staining and Analysis)
Silver staining
Comassie Brilliant Blue R250 staining
24
Isoelectric focusing (IEF)

• Precipitation of proteins in a pH gradient at
  their isoelectric point (pI)

25
Isoelectric focusing (IEF)

• 2D-IEF The protein sample is first subjected to
  isoelectric focusing in one dimension and then to
  SDS-PAGE in the second dimension

26
Electrodialysis

• Used to transport salt from one solution, the
  diluate, to another solution, the concentrate, by
  applying an electric current
• Concentrate and diluate are separated by the
  membranes
• Electric current is applied, moving the salt over
  the membranes
• Alternative method instead of RO - desalination
  of brackish water or seawater

• Electrodialysis moves the ions, RO moves the
  water to obtain the desalinated product

27
Finishing steps crystallization

• Operates at low temperatures (to minimize thermal
  degradation), high concentrations
• Producing highly purified products such as
  antibiotics

28
Finishing steps drying

• Vacuum tray drier Consists of heated shelves,
  usually used in pharmaceuticals products, good
  for small batches of expensive materials
• Freeze-drying/Lyophylization Water is removed
  by sublimation, used for antibiotics, enzyme
  solutions and bacterial suspensions
• Rotary-drum drier Water is removed by heat
  conduction over a thin film of solution, dried
  product is scraped from drum, not good for
  crystal solutions

29
Finishing steps drying

• Spray drier Atomization and spraying of product
  solution into a heated chamber through a nozzle,
  dried particles are separated by cyclones,
  preferred for heat-sensitive materials
• Pneumatic conveyor drier Use hot air stream to
  suspend and transport particles, short retention
  time in gas stream, well suited for
  heat-sensitive materials, easily oxidized
  materials, food processing industries

30
Conclusions

• Primary purification removal of large particles
  such cell mass, cell debris filtration,
  centrifugation, coagulation, flocculation
• Secondary purification separation of soluble
  products such as proteins, enzymes, antibiotics,
  organic acids, amino acids, etc. extraction,
  ultrafiltration, RO, chromatography,
  electrophoresis, IEF
• However, there is no clear boundary on
  classifying between primary and secondary
  purification, it is all depend on the purpose of
  purification

31

• Thank you and
• GOOD LUCK!!

Say (O Muhammad SAW to mankind) If the sea
were ink for (writing) the Words of my Lord,
surely, the sea would be exhausted before the
Words of my Lord would be finished, even if We
brought (another sea) like it for its
aid. al-kahfi 109