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Recovery and Purification of Bio-Products. Strategies to recovery and purify bio-products. Fementer. Solid-liquid separation. Cell products. Cells. Supernatant. Recovery. Cell disruption or rupture. Purification. Cell debris. Crystallization and drying. Cell Disruption. - PowerPoint PPT Presentation
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Recovery and Purification of Bio-Products
- Strategies to recovery and purify bio-products
Fementer
Solid-liquid separation
Recovery
Purification
SupernatantCellsCell products
Cell disruption or rupture
Cell debris
Crystallization and drying
Cell DisruptionDisruption: the cell envelope is physically broken, releasing all intracellular
components into the surrounding medium
Methods: Mechanical and non mechanical
- Mechanical
- Ultrosanication (sonicators)bacteria, virus and spores suspensions at lab-scale
Electronic generator→ultrasonic waves→mechanical oscillation by a titanium probe immersedin a cell disruption.
http://www.biologics-inc.com/sd-models.htm
Cell Disruption- Mechanical
Milling: continuous operation,
Algae, bacteria and fungiLarge 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.
http://www.cbmills.com/Products/horizontalmills.htm
Dyno-Mill(liquid)
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- MechanicalHomogenization: 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, Rannie high-pressure homogenizer
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
dodecylsulfate.
- Alkali: sodium hydroxide, harsh
- Organic solvents: penetrating the lipids and swelling the cells. e.g. toluene.
Cell Disruption
- Nonmechanical
- Enzymes: to lyse cell walls to release the product.gentle, but high cost
i
i.e. lysozyme (carbohydrase) to lyse the cell walls of bacteria.
- Osmotic shockOsmosis 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
Fementer
Solid-liquid separation
Recovery
Purification
SupernatantCellsCell products
Cell disruption or rupture
Cell debris
Crystallization and drying
Separation of Soluble ProductsLiquid-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: 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.
Heavy, XH
Light, YL
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, multi-stage extraction
Heavy, I, j
Light, i, j
Separation of Soluble Products
Precipitation
Reduce the product solubility in the fermentation broth by adding salts.
- Applicable: separate protein or antibiotics from fermentation broth
- Methods:- salting-out by adding inorganic salts such as ammonium sulfate, or sodium sulfate at high ionic strength (factors: pH, temperature)
- salts interact more stronger with water- cause little denaturation- inexpensive
Separation of Soluble Products
Precipitation
- Isoelectric precipitation:
Precipitate a protein at its isoelectric point.
- Solubility reduction at low temperature by adding organic solvents (T< -5oC)
Reduce the dielectric constant of the liquidMay cause protein denaturationSolvents: acetone, ethanol, methanol.
- HeatRemove undesired proteins
Separation of Soluble Products
Adsorption
Adsorb soluble product from fermentation broth onto solids.
Approaches: physical adsorption, ion exchange
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
0
2
4
6
8
10
12
14
0 10 20 30 40 50
Equilibrium solute concentration in liquid (mol/l)
Equ
ilibr
ium
sol
ute
conc
enta
tion
on
solid
(m
ol/g
ads
orbe
nt)
Adsorption Isotherms
Saturated uptake
affinity
C1
Cs1
Cs2
Adsorbent 1
Adsorbent 2
Separation of Soluble Products• Models of adsorption equilibrium
Freundlich isotherm:
Cs=KFCL1/n
Cs and CL are equilibrium concentration of solute in solid and liquid phases, respectively.
KF and n are empirical constants.Langmuir isotherm:
Cs=CsM
CL/(K+CL)
CsM
is the maximum concentration of solute adsorbed on the solid phase. K is a constant.
Limitation: the above model constants are dependent on the solutions seriously limiting the prediction capability of these models over more extended sorption conditions.
Modeling the effect of pH on Cr uptakes
00.10.20.30.40.50.60.70.80.9
1
0 2 4 6 8 10
Equilibrium Cr concentration (mM)
Cr U
ptak
e (m
mol
/g)
pH2.5
pH2.0
Langmuir model fitting curve
pH3.6
corr. coef.: 0.999relative dev.: 2.4%
error bar: 95% confidence interval
ANION BIOSORPTION EQUILIBRIUM MODEL DESCRIPTION
BNH + zH+ + HxMpLqZ- = BNH2
+ HxMpLqZ- H+
z-1
BNH + H+ + Cl- = BNH2+ Cl-
-log i = -0.507*Z2 ( )
[M]T = (speciation), mass & charge balance
ZS
ZZ
H
ZZ
sad
SHBNH
HSBNHK
][)]]([[
][ 12
II
I3.0
1
z
qi
pX LMH
Attributed all non-ideality to the liquid phaseConsidered the interference of Cl-
Ks: adsorption equilibrium constant s: metal or Cl speciesBNH: weak base binding sites HxMpLq
Z- : anionic metal species
i : activity coefficient of solution sp.i Z: charge of species
I: ionic strength in the solution [M]T: total metal concentration in the solution.
Modeling the effect of pH on Cr uptakes
00.10.20.30.40.50.60.70.80.9
1
0 2 4 6 8 10Equilibrium Cr concentration (mM)
Cr
Up
take
(mm
ol/g
)
pH2.5
pH2.0
model fitting curve
BNH + H+ + HCrO4 - = BNH2
+HCrO4 -
BNH + 2H+ + Cr2O7 2- = BNH2
+Cr2O72-H+
pH3.6
corr. coef.: 0.999relative dev.: 2.4%
error bar: 95% confidence interval
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 salt-containing 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