WEEK 14
MADAM NOORULNAJWA DIYANA YAACOB
SCHOOL OF BIOPROCESS ENGINEERING
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,
untrafiltration &microfiltration, chromatography,
electrophoresis, electrodialysis
5.0 Finishing Steps for purification
Crystallization, Drying
6.0 Integration of Reaction and Separation
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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.
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Separation of solids such as biomass,
insoluble particles, and macromolecules from
the fermentation broth
Usually the first step in product recovery
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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.
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The most widely used is continuous
rotary filters
The drum is pre-coated with a filter
aid, typically of diatomaceous earth
(DE) or Perlite.
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.
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Separate particles
size between 100
and 0.1 µm from
liquid by centrifugal
process
The theory of solidliquid separations
in a gravitational
field should be
clearly understood
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Used to formed cell
aggregates before
centrifugation,
gravity settling, or
filtration to improve
the performance of
these separation
processes
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Coagulation –
formation of small
flocs from disperesed
colloids using
coagulating agents.
Flocculationagglomeration of
these small flocs into
larger settable
particles using
flocculating agents.
3.1 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
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The
The
The
The
Gaulin-Manton and French Press
Ribi fractionator
Rannie high-pressure homogenizer
Dyno Mill
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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)
4.1 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.
4.2 Precipitation
The first step in the
purification of
intracellular protein
after cell disruption
is usually
precipitation.
Proteins in
fermentation broth
can be separated
from other
components by using
certain salts
2 major methos for
protein precipitation:
1. Salting out by
adding inorganic
salts
2. 2. Solubility
reduction at low
temp. by adding
organic solvents
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
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4.3 Adsorption
Adsorption of solutes
from liquid media
onto solids is a
common practice in
separating soluble
materials from
fermentation broth.
In physical
adsorption-weak
forces, such as van
der Waals are
dominant
In ion-exchange
adsorption- strong
ionic bonds are
utilized
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.
4.4 Dialysis
Membrane separation
operation used for the
removal of low-MW
solutes such as organic
acids and inorganic
ions from a solution.
A well-known exampleused dialysis
membrane to remove
urea from urine in
artificial kidney
The dialysis membrane
separates two phase
containing low-MW
and high-MW solution
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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.
4.5 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.
4.6 Chromatography
Separates mixture into
components by
passing a fluid mixture
through a bed of
adsorbent material
Elution
chromatography
Typically column is
packed with adsorbent
particles.
A mobile phase is
injected. This pulse is
followed by a solvent
eluent
The pulse enters as a
narrow concentrated
peak, but exits
dispersed and diluted
by additional solvent
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Adsorption chromatography (ADC)
Liquid-liquid partition chromatography (LLC)
Ion-exchange chromatograpHy (IEC)
Gel filtration chromatography(GFC)
Affinity chromatography(AFC)
Hydrophobic chromatography (HC)
High Pressure Liquid Chromatography (HPLC)
4.7 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
5.1 Crystallization
Operates at low
temperature, which
minimize thermal
degradation of heatsensitive materials
High purity crystal are
recovered by using
batch Nutsche-type
filter or centrifugal
filters
After washing, the
crystals are discharged
for drying
5.2 Drying
Removal of solvent from purified wet product
(crystal) usually achieved by drying.
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3.
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5.
A vacuum tray drier
Freeze drying
Rotary drum driers
Spray dryers
Pneumatic conveyor
driers
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Separate optimization of fermentation and
recovery does not necessarily yield the
optimal process.
One form of integration is try to couple some
aspects of recovery and purification with the
bioreactor
Thank you