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Bioprocess (Extraction Techniques (Supercritical fluid extraction…
Bioprocess
Extraction Techniques
Supercritical fluid extraction
Used for the recovery of essential oil from plants
Based on the use of carbon dioxide in supercritical phase
It is non toxic
It is nonflammable
Not expensive
Multi-step fractionation
First step: We obtain the fraction of the more soluble compounds
Second step: we have the recovery of less soluble components
On-line fractionation
Works in a cascade depressurization system to obtain the precipitation of several fractions
Properties
High diffusion
Low viscosity
Advantages
Chemically inert
Non flammable
Easy to recicle
You get pure extracts
Disasvantages
Limited availability
Only small amounts of a large specimen goes throng the column
Expensive to operate
Microwave-assisted extraction
Extractive method based on the utilization of microwave energy.
It can be classified according to the pressure it operates:
Higher than the atmospheric pressure
Lower than the atmospheric pressure
HPMAE (High pressure microwave assisted extraction)
Enhance the capacity of the solvent to incorporate the energy from radiation
Avoid large amount of solvent for the extraction
VMAE (Vacuum microwave assisted extraction)
Allows the reduction of the boiling point of the solvent
UMAE (Ultrasonic/microwave-assisted extraction)
Reduce extraction times
Reduce the amount of solvent
Advantages
Higher quality
Higher yields
Allows a gain of time
Cheaper than supercritical fluid extraction
Faster than ultrasonic-assisted extraction
Disadvantages
Less eco-friendly due to the use of organic solvents
It is more expensive than ultrasonic-assisted extraction
Not suitable for thermolabile compounds
Solid-Liquid extraction
Solids are in contact with a extraction solvent
Limitations
Size of the compound of interest
Hard to recover if our product is high molecular weight
Solvent specifications
It should not change the stability of the product
Solvent that facilitate the lixiviation process
It should permit the products
Sample preparation
Crushed with a blade mixer
Dried samples
Ionic liquids
Consists of a liquid organic salt that interacts with specific compounds.
Types of interactions
π-stacking
Ion-exchange
Hydrophobic interactions
Hydrogen bonds
Advantages
Quality and efficacy of extraction
Recovery of high yields
Properties
Non-flammability
Wide electro-chemical window
Negligible vapor pressure
Good chemical and thermal stability
Applications
Electrochemistry
Polymer science
Biological processes
Nano-chemistry
Enzyme-assisted extraction
Enzymes catalyze reactions in a specific way without operating under strong conditions
Some enzymes disrupt cell wall with the hydrolysis leading to a major permeability
Hemicellulases
Pectinases
Cellulases
Some other enzymes permits to reduce the use of solvent for extraction
Lipases
Proteases
Phospolipases
Limitations
Incomplete disruption of the cell wall
Complicated application in a commercial scale
Costo of enzymatic approach
liquid-liquid fractionation
Organic-aqueous systems
Solvent toxicity
aqueous two-phase systems
high recovery yield and is easily to scale up
industry and academia
economic and environment friendly
analytical tool
separation of metals and metal ions
polymer – salts system
high ionic strength
It is according to the solubility of a bioactive compound
Advantages
Limited cost
Simple equipment
Types of solvents to perform a good extraction
Non-polar
Organic
Polar
Inorganic
Pressurized liquid/liquid extraction
Operates at high temperatures
Reduction of viscosity
Operates at high pressure
Permits maintaining the solvent in the liquid phase
Requires water as solvent
Stream destillation extraction
Is a uniform, useful and representative method
Specifications of the product
Volatile
Thermostable
Low molecular weight
Applications
Cosmetic industry
Oil industry
Scents and actives
Process
The materials go through a condensation
Collection process
Steam vaporizes the materia'l volatile compounds
Advantages
Control the temperature and the amount of steam
Works well for types of substances that don't mix with water
inclusion body processing
characteristics
bacterial hosts
Ex.
Escherichia coli
0.2 to 1.5 μm
low yields
labor inttensive
at the poles of bacterial cell
cylindrical to ovoid shape
mostly composed of recombinant protein of interest
solubilization
aim
solubilize
inclusion body aggregates
preserve
native-like protein structure
denaturants and chaotropes
urea
advantages
no requirement of refolding step
disadvantages
works only for classical inclusion bodies
guanidine hydrochloride
β-mercaptoethanol or dithiothreitol
For proteins containing multiple cysteine residues
diaadvantages
disruption of protein structure
non-denaturing buffers
example
Tris–HCl buffer at pH 7
advantages
preserve the native-like protein structures
organic solvents
non-classical inclusion bodies
5% n-propanol
DMSO
High hydrostatic pressure
extreme pH
Continuous Centrifugation
insoluble protein inclusion bodies
depends on
centrifuge design
disk-stack
large scale
operational parameters
dilution buffer
subsequent steps
refolding
purification
factors that contribute to their formation
.
Reduced environment of bacterial cytosol
lack of eukaryotic chaperones
lack of post-translational machinery
High copy number of target gene
strong promoter system
high inducer concentration
expression process
temperature
pH
quality
cell disruption method
Processes
ultracentrifugation
centrifugation
denaturing agents
detergents
Tritón X-100
Sarcosyl
Tween 80
microfiltration
Methods
Sample preparation
The biological sample preparation depends on the method of detection
Staining
Dehydration
Freeze substitution
Fixation
Freezing techniques
Using chemicals
Embedding
Prevents collapse
Staining
Enhaces contrast
Sectioning
Facilitates the process
Purpose of Primary Recovery
Is designed to capture target biomolecules
Eliminates big contaminants
adsorption
ion exchange
affinity
chromatography
expanded bed
packed bed liquid
monolith
magnetic fishing
Expanded Bed Chromatography
#
Recover proteins from unclassified feedstocks
Aplications
Biocatalysis
Bioanalysis
Disadvantages
Insufficient adsorbent density
Unfavourable biomass-adsorbent particles
Advantages
Has widespread applications in the large scale purification
biomass recovery techniques
.
induced precipitation
filtration
under pressure or vacuum
fail to recover organisms approaching bacterial dimensions
membranes
ultrafiltration
0.02 μm approx
2 to 10 bar
separation of molecules
microfiltration
0.1 to 2 bar
separation of phases
of 0.1 to 10 μm
permeability
pores size
permeability coefficient (P)
mol/(m･s･Pa)
constant cleaning process
nanofiltration
pressure-driven
liquid-phase separations
solution-diffusion mechanism
size of pores(nominally ∼ 1 nm)
Advantages
Simple to scale up
No phase change involved
Energy requirements are low
It can be used in:
Concentration
Purification
Separation
Disadvantages
Equipment cost can be high
Shear in sensitive materials
Membrane fouling
centrifugation
large volumes
energy intensive
preferred method of
recovering algal cells
high-value products
Limitations
no protection against noise
High maintenance costs
Heavy polymer consuption
Flocculation
to increase the effective ‘‘particle’’ size
metal salts
ferric chloride
aluminum sulfate
ferric sulfate
cationic polymers
Electrocoagulation-electroflotation
#
algae/microalgae
wastewater
electro oxidation
coagulating ions
aluminum anodes
introduce the flocculation agent into the effluent
sedimentation
sedimentation tank
settling ponds
dilute
Forced sendimentation
Magnetic forced sedimentation
iron oxide
Solutions for even smaller diameters of 10-6 cm.
centrifugal force
induced speeds
Natural sedimentation
gravity
Suspensions up to diameters of 10-4 cm.
Does not require power for separation
sedimentation rate
perfusion techniques
Bioreactors
extracelullar products
cell separation methods
GE Healthcare’s Hollow Fiber Microfiltration Cartridges
ATMI’s iCELLis Single Use Fixed Bed
Centrifuge method
factors
cells size
volume
level of moisture in the product
purpose
recover extracellular products
recycle biomass
cell rupture techniques
recovery of intracellular products
chemical
solvents
alcohol
ether
chloroform
detergents
solubilizing lipids and proteins in the membrane
types
ionic
dodecyl sulphate
non-ionic
Triton X-100
zwitterionic
3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate
acid/bases
biochemical
short scale
enzymes
breaking down cell walls
examples
protease
cellulases
protoplasts
hydrolases
glycosidases
endopeptidases
amidases
Physical
temperature
osmotic pressure
freeze-thaw
Mechanical
sonication
probe sonication
improve bubble formation
keep the system as cold as possible
diasdvantages
Denaturing of proteins
high temperature
excessive shear
Immersion sonication
bead milling
bead size
beads quantity
shaking the entire container
tissues
free cells
extremely small samples of 0.2 ml to somewhat
larger quantities of 50 ml
batch operation
limiting the amount
of materials than can be processed
advantages
low cost
durable
minimal training
spinning agitator
within a container
High pressure (French press)
up to 275 MPa
suitable for a variety of free cells
bacteria
yeast
mammalian cells
not suitable for
seeds
tissue samples
plant materials
optical lysis
laser pulses
cavitation bubble
tuning of laser
selectively lyse single cells
large scale