PTT203

BIOCHEMICAL ENGINEERING

RECOVERY AND
PURIFICATION OF
PRODUCTS
PUAN NURUL AIN HARMIZA ABDULLAH
Lecture Outline
1.0 Introduction
2.0 Separation of Insoluble Products
Filtration, centrifugation, coagulation &flocculation
3.0 Cell Disruption
Mechanical & Nonmechanical metods
4.0 Separation of Soluble Products
Liquid-liquid extraction, aqueous two-phase extraction,
precipitation, adsorption, dialysis, reverse osmosis,
ultrafiltration &microfiltration, chromatography,
electrophoresis, electrodialysis
5.0 Finishing Steps for purification
Crystallization, Drying
6.0 Integration of Reaction and Separation
1.0 Introduction

• Recovery and purification of a fermentation product is

essential to any commercial process.
• Difficulty entailed heavily on the nature of the products
• Since the chemical nature of a fermentation broth is quit
complex and extremely high purity is required for some
products, recovery and purification often required many
processing steps and in many cases represent a manufacturing
cost higher than that involved in producing the products.
Figure 11.1
Major Steps Involved in the Separation and
Purification of Intracellular Enzymes.
Strategy in Separation and Purification
of Intracellular Products
1. Separation of insoluble products and other solids.
2. Primary isolation or concentration of product and removal
of most of the water.
3. Purification or removal of contaminating chemicals.
4. Product preparation (eg.: Drying).
1. Separation of Insoluble Products
• Separation of solids such as biomass, insoluble particles, and
macromolecules from the fermentation broth.
• Usually the first step in product recovery.
1.1 Filtration
• Most cost effective method for the separation of large solid
particles and cells from fermentation broth.
• Fermentation broth is passed through a filter medium and
filter cake is formed as a result of deposition of solids on the
filter surface.
• The most widely used is continuous
rotary filters
• After pre-coat has been applied, the liquid
to be filtered is sent to the tub below the
drum. The drum rotates through the liquid
and the vacuum sucks liquid and solids
onto the drum pre-coat surface, the liquid
portion is "sucked" by the vacuum
through the filter media to the internal
portion of the drum, and the filtrate
pumped away.
• The solids adhere to the outside of the
drum, which then passes a knife, cutting
off the solids and a small portion of the
filter media to reveal a fresh media
surface that will enter the liquid as the
drum rotates. The knife advances
automatically as the surface is removed.
1.2 Centrifugation
• Separate particles
size between 100 and
0.1 µm from liquid by
centrifugal process
• The theory of solid-
liquid separations in
a gravitational field
should be clearly
understood
1.3 Coagulation and Flocculation
• Used to formed cell • Coagulation –
aggregates before formation of small
centrifugation, flocs from disperesed
gravity settling, or colloids using
filtration to improve coagulating agents.
the performance of • Flocculation-
these separation agglomeration of
processes these small flocs into
larger settable
particles using
flocculating agents.
2. Cell Disruption
2.1 Mechanical methods
• Ultrasonic
vibrators/Sonicators
Used to disrupt the cell
wall and membrane of
the bacterial cells.
An electronic
generator is used to
generate ultrasonic
waves, an a transducer
converts these waves
into mechanical
oscillations
2.2 The Gaulin-Manton and French Press
2.3 The Ribi fractionator
2.4 The Rannie high-pressure homogenizer
2.5 The Dyno Mill
2. Cell Disruption
2.2 Non-Mechanical
methods
• Osmotic shock &
rupture with ice crystal
are commonly used.
• slowly freezing and the
thawing a cell paste, the
cell wall and membrane
may be broken,
releasing enzymes into
the media.
2. Cell Disruption
2.2 Non-Mechanical
methods
• Lysozyme
• Enzymatic lysis of cell
walls of bacteria.
• Expensive method.
3.0 Separation of Soluble Products
Example of soluble and extracellular products: antibiotics,
organic acids, solvents, amino acids, extracellular enzymes
3.1 Liquid-liquid Extraction
• Separate inhibitory products
such as ethanol and acetone-
butanol from fermentation
broth
• Ideally, the liquid extractant
should be nontoxic, selective,
inexpensive, and immiscible
and should have a high
distribution coefficient for the
product.
3.0 Separation of Soluble Products
3.2 Aqueous Two-Phase Extraction
• Separation of soluble proteins between TWO
aqueous phases containing incompatible polymers
(PEG/dextran).
3.0 Separation of Soluble Products
3.3 Precipitation TWO major methos for
• Proteins in protein precipitation:
fermentation broth can 1. Salting out by adding
be separated from inorganic salts
other components by (ammonium
using certain salts. sulphate).
2. Solubility reduction at
low temp (T<-5deg)
by adding organic
solvents.
3.0 Separation of Soluble Products
What is Salting out ?
• Achieved by increasing
the ionic strength of
protein-containing
solution by adding salts.
• The added ions interact
with water more
strongly causing protein
molecules to
precipitate.
3.0 Separation of Soluble Products
3.3 Adsorption • In physical
• Adsorption of solutes adsorption-weak
from liquid media forces, such as van
onto solids is a der Waals are
common practice in dominant.
separating soluble • In ion-exchange
materials from adsorption- strong
fermentation broth. ionic bonds are
utilized.
3.0 Separation of Soluble Products

A process in which molecules of gas, of dissolved substances in liquids,

or of liquids adher) in an extremely thin layer to surfaces of solid
bodies with which they are in contact. Adsorption is used in gas masks
and to purify and decolorize liquids.
3.0 Separation of Soluble Products
3.4 Dialysis • The dialysis membrane
• Membrane separation separates two phase
operation used for the containing low-MW
removal of low-MW and high-MW solution
solutes such as organic
acids and inorganic
ions from a solution.
• A well-known example-
used dialysis
membrane to remove
urea from urine in
artificial kidney
3.0 Separation of Soluble Products
• Surrounding the membrane is dialysis fluid. This
contains chemicals, which should be in the blood
plasma (glucose, amino acids & salts), in the correct
concentrations.
• The urea moves from the blood to the dialysis fluid
by diffusion. Other small particles diffuse from the
blood to the dialysis fluid too. As glucose diffuses
out of the blood, glucose also diffuses into the blood
from the dialysis fluid. This keeps the concentration
of important chemicals in the blood constant.
• Excess salt diffuses out of the blood, keeping it at
the right levels. Osmoregulation occurs in much the
same way. If there is too much water in the blood it
will enter the dialysis fluid by osmosis. The reverse
occurs if the blood is too concentrated.
• The blood is kept at the correct temperature while it
passes through the machine. The blood then returns
to the body.
3.0 Separation of Soluble Products
3.5 Reverse Osmosis
• Osmosis-transport of
water molecules from
high to low conc.
Region.
• Reverse osmosis- a
pressure is applied
onto salt containing
phase, which drives
water molecules from
low to high conc.
Region.
3.0 Separation of Soluble Products
3.6
Chromatography
• Separates mixture
into components by
passing a fluid
mixture through a
bed of adsorbent
material
3.0 Separation of Soluble Products
Some important types of
chromatographic methods
1. Adsorption chromatography (ADC)
2. Liquid-liquid partition chromatography (LLC)
3. Ion-exchange chromatograpHy (IEC)
4. Gel filtration chromatography(GFC)
5. Affinity chromatography(AFC)
6. Hydrophobic chromatography (HC)
7. High Pressure Liquid Chromatography (HPLC)
3.7 Electrophoresis
• Used for the separation
of charged biomolecules
according to their size
and charge in an electric
field.
• In an electric field, the
drag force on a charged
particles is balanced by
electrostatic forces when
the particles is moving
with a constant terminal
velocity
4.0 Finishing steps for Purification
4.1 Crystallization
• Operates at low
temperature, which
minimize thermal
degradation of heat-
sensitive materials
• High purity crystal are
recovered by using batch
Nutsche-type filter or
centrifugal filters
• After washing, the
crystals are discharged
for drying
4.2 Drying
• Removal of solvent from purified wet product (crystal) usually
achieved by drying.
Major type of driers

1. A vacuum tray drier

2. Freeze drying
3. Rotary drum driers
4. Spray dryers
5. Pneumatic conveyor
driers
Thank you