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Assignments:
1. HW #3:
2. Read:
Chapter 3
Class notes
Cell Removal Liquid for Cell Removal Cell Removal Cells for
Liquid for Filtration Filtration
disposal Filtration disposal disposal
or recycle Microfiltration or recycle Microfiltration Microfiltration or recycle
Centrifugation Centrifugation Centrifugation
Physical
Chemical
Mechanical
Cell Structure
Prokaryotic
Gram positive peptidoglycan and plasma membrane
Gram negative outer (lipopolysaccharide) membrane,
peptidoglycan layer, and plasma membrane
Gram negative
Eukaryotic Cells
1. Animal cells thin cell coat, plasma membrane, and membrane
enclosed organelles
2. Plant cells complex with strong cell wall
Membrane
Chemical Methods
2. Solubilization
Stability in the delivered pressure is achieved through an attenuation volume between the pump and
the valve or the use of two or more reciprocating plungers and an overlapping algorithm control.
In a typical valve setup a zirconium or tungsten carbide needle-seat valve or ball-seat valve is used,
with homogenization pressure being controlled by the force (F) exerted over the needle blocking
the fluid flow (Figure B-C).
Common high pressure homogenization valves: (B) ceramic needle and seat; (C) ceramic ball
and seat; (D) diamond, sapphire, or ruby nozzle;
The nozzle orifice is usually <0.35 mm and the specific gemstone depends on the maximum
pressure and required nozzle life-span, with diamond being the most resistant and expensive option.
In the nozzle setup, homogenization pressure is determined by the pump pressure and/or a
diversion to flow.
Kinetics of cell disruption 1st order reaction
= k (Rm R )
dR
dt
k -- 1st order rate const.
Rm -- max releasable protein
R -- amt. of soluble protein released at time t
ln (1 R ) = k N P a (3.3.2)
where,
Feed
Pressure
(PSI
2. Jet-type microfluidics high-pressure
homogenizer (jet-type)
In the microfluidics system (Figure A), the flow stream is split in two or more channels
that are redirected over the same plane but in right angles and propelled into a single
flow stream. The pressure driving pump (up to 300 MPa) promotes a high speed at
crossover of the two flows which results in high shear, turbulence, and cavitation over
the single outbound flow stream.
Both (valve and jet) designs rely on the conversion of pressure to kinetic energy in a convergent flow
region, yielding a high - velocity liquid stream that ultimately undergoes an approximate 90
change in flow direction
Microfluidics High Pressure Homogenizer
Eq. (3.3.3) is a modification of eq. (3.3.2) describing shear and collision effect on cell breakage
in Microfluidics high-pressure homogenizer :
where,
ln(1 R) = k N b P a (3.3.3)
k & a -- parameters that depend on type of cells
Eqns. (3.3.4) and (3.3.5) are the governing correlations for batch and
continuous operation
Disadvantages
Not effective against all organisms
Expensive reagents
Enzyme must be approved for usage (FDA)
Subsequent process steps are needed
Comments on Cell Disruption Methods
Solvents
Advantages
Effective to permeabilize cell membrane
Used often in cell free systems
Disadvantages
Solvent toxicity
Not effective for all organisms
Potential damage to proteins
EPA and OSHA considerations
Comments on Cell Disruption Methods
Osmotic Shock
Advantages
Relatively simple
No equipment involved
Operates at low T
Does not effect protein activity / structure
Disadvantages
Not effective for all organisms (good for animal and red blood cells)
Requires subsequent removal of salts
Comments on Cell Disruption Methods
Sonication
Advantages
Efficient 95% disruption)
Rapid (sec to min)
Effective for many organisms
Simple
Small volumes
Disadvantages
Large amts. of heat
No scalable
Use with low cell concentration
Comments on Cell Disruption Methods
Homogenization
Advantages
Good efficiency with multiple passes
Effective for variety of microorganisms
Tolerates moderate cell concentrations
Scalable (scale up parameters available)
Continuous
Large throughputs
Disadvantages
High pressures
Multiple passages
Possible containment problems: seal leaks
High viscosity fluid due to DNA release
Needs often valve replacement (due to valve erosion)
Comments on Cell Disruption Methods
Ball / Bead Mills
Advantages
High disruption efficiency
Effective for variety of microorganisms
Low pressures: easy containment
High cell concentrations (as high as 500 g/L)
Scalable (scale up parameters available)
Continuous and batch
Low maintenance: low P, few moving parts
Disadvantages
Attrition of beads
Possible product contamination
Example: Homogenization of
Chlorella sp.
Assessment of Chlorella Cell Disruption
100.00
90.00
80.00
Turbidity (%) 70.00
60.00
50.00 pH 6.8
40.00 pH 11.75
30.00
20.00
10.00
0.00
Initial samplePass 1 Pass 2 Pass 3 Pass 4 Pass 5
100.00
Cell count (%)
80.00
60.00
pH 6.8
40.00
pH 11.75
20.00
0.00
Initial samplePass 1 Pass 2 Pass 3 Pass 4 Pass 5
Original Pass 1 Pass 2
sample
60.00
50.00
40.00
30.00 pH 6.8
20.00 pH 11.75
10.00
0.00
Initial Pass 1 Pass 2 Pass 3 Pass 4 Pass 5
sample
Problem for HW # 3.
Find coefficients a and k for Chlorella sp. homogenized by a valve-type homogenizer.
Experimental data are given below. Assume that total soluble protein (TSP) released using
16,000 psi and N = 5 is the maximum extractable protein (Rm =2520 g/ml). Wet biomass
concentration in this experiment was 10% w/w.