How Cellular Information is Altered Flashcards
(189 cards)
1
Q
HOW CELLULAR
INFORMATION IS ALTERED
A
Mutation and Selection
Natural Mechanisms for Gene Transfer and Rearrangement
Genetically Engineering Cells
Genomics
2
Q
We can alter cells by using mutation or genetic engineering.
A
HOW CELLULAR
INFORMATION IS ALTERED
3
Q
____________ is the purposeful transfer of DNA from one type of organism to another.
A
Genetic Engineering
4
Q
_________ is subjecting the cells to stress, causing changes in the genetic make-up.
A
Mutation
5
Q
___________ = mistakes in the genetic code (can arise from replication and/or damage)
A
Mutations
6
Q
____________ = organism with a genetic mutation
A
Mutant
7
Q
_________ = the organism without the genetic organism
A
Wild type
8
Q
______________ = genetic construction of an organism
A
Genotype
9
Q
_________ = characteristics expressed by an organism.
A
Phenotype
10
Q
__________ = usually refers to transcription + translation + post-translation processing.
A
Expression
11
Q
If the mutation is in the active site, there may be some _________ consequences.
A
enzyme activity
12
Q
If the mutation changes the amino acid to a ________, the resulting _______ will be ________ and probably ___________.
A
stop codon, protein, truncated, not active
13
Q
If the amino acid is the same as before the mutation there is _____________
A
no consequence
14
Q
If the amino acid is different, but not in the ________ of the active site, there _________
A
region, may be no consequences.
15
Q
confers upon the mutant an advantage for growth, survival or detection under a set of environmental conditions that the wild type does not have
A
Selectable mutation:
16
Q
Antibiotic resistance
Ability to grow on toluene
Inability to produce lysine
Ability to produce bioluminescence
Ability to produce more of an enzyme
Inability to grow at higher temperatures
A
SELECTION
17
Q
____________ mutations per cell conversion
A
10^-3 - 10^-9
18
Q
10^-6 = ________________
A
1 mutation/1,000,000 divisions
19
Q
_________: chemicals, radiation
A
Mutagens
20
Q
Lots of growth (i.e. lots of divisions)
A
INCREASE MUTATION RATES
21
Q
WHY DO WE WANT TO
INCREASE MUTATIONS?
A
We want a cell to develop specific characteristics that are advantageous for us.
22
Q
_____________: uptake of free DNA by a cell. The cell membrane has to be permeable to DNA.
A
Transformation
23
Q
_____________: DNA is carried into the call in a phage.
A
Transduction
24
Q
_________: Cell-to-cell transfer of DNA.
A
Conjugation
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Also called mating.
Conjugation
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Using ___________, engineers and microbiologists were able to increase penicillin from 0.001 g/L to 50 g/L.
mutation and selection
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Using natural mechanisms to purposefully manipulate DNA. The DNA is manipulated outside of the cell, and then sent into the cell.
GENETIC ENGINEERING
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__________: enzymes that cut DNA at specific sequences. Different enzymes will cut at different sequences.
Restriction enzymes
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______________: A method to detect what sizes of DNA a sample contains.
Gel electrophoresis (Southern Blot)
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______________: A process used to make many copies of a piece of DNA.
Polymerase chain reaction (PCR)
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_____________: self replicating, circular piece of DNA that can survive in a cell.
Plasmid
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________________recognition sequence cuts long DNA more frequently and produces smaller DNA fragments than a restriction enzyme with a six-nucleotide recognition sequence.
A restriction enzyme with a four-nucleotide
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Any given __________ occurs in DNA, on average, at a distance of 256 (4^4)nucleotides.
four nucleotide long recognition site
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Any given _________________ occurs, on average, at a distance of 4096 (4^6) nucleotides.
six nucleotide long sequence
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Any given __________________ occurs, on average, at a distance of 65536
(48) nucleotides.
eight nucleotide long sequence
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allows scientists to extract and analyze bits of microbial DNA from samples, meaning they don't need to find and grow whole cells.
PCR
37
is an essential element in DNA fingerprinting and in the sequencing of genes and entire genomes.
PCR
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Basically, it's like a technique to photocopy pieces of DNA. In a matter of a few hours, a single DNA sequence can be amplified to millions of copies
PCR
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lets scientists work with samples containing even very small starting amounts of DNA.
PCR
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The technique makes use of the DNA repair enzyme polymerase. This enzyme, present in all living things, fixes breaks or mismatched nucleotides in the double- stranded DNA helix. These breaks or mismatches could cause genes to malfunction if left unfixed.
PCR
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Polymerase uses the intact half of the DNA molecule as a template and attaches the right nucleotides, which circulate constantly in the cell, to the complementary nucleotide at the site of the break. (DNA consists of two strands of nucleotide bases, which are represented as A, G, C, and T. In the laws of DNA base-pairing, A joins with T and G with C.)
PCR
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Not all polymerases are created equal, however. Many fall apart in high heat.
PCR
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PCR was developed in ______ following the discovery of an unusual heat-loving bacterium called ________ in a hot spring in __________
1985, Thermus aquaticus, Yellowstone National Park.
44
This bacterium's polymerase, dubbed Taq, does its job of matching and attaching nucleotides even in the high heat generated by the successive_________ cycles required during PCR.
"photocopying"
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______ made PCR possible.
Taq
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_________: changing conditions - transient (S, X, growth rate), high initial substrate, different phases of growth.
Batch
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________: steady-state, constant low concentration of substrate, constant growth ratethat can be set by setting the dilution rate (ie. the feed flow rate).
Chemostat
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__________ is more efficient.·
Chemostat
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____________ is more common.
Batch
50
CHOICE OF CONTINUOUS VS
BATCH PRODUCTION
Productivity
Flexibility
Control
Genetic stability
Operability
Economics
Regulatory
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_________: rate of product per time per volume. Chemostat is better for growth-associated products. Wasted time in batch process.
Productivity
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____________: the ability to make more than one product with the same reactor. Batch better.
Flexibility
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___________: maintaining the same conditions for all of the products produced. In theory, the chemostat is better, steady state. In reality???
Control
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____________: maintaining the organism with the desired characteristics. Chemostat selects for fast-growing mutants that may not have the desired characteristics.
Genetic stability
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___________: maintaining a sterile system. Batch better.
Operability
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____________: validating the process. Initially, many process batch, too expensive to re-validate after clinical trials.
Regulatory
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Consider the production of a growth-associated product (like cell mass) in _________.
suspension culture
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__________ of a chemostat is detrimental to engineered organisms.
Selective pressure
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______ is more mechanically reliable.
Batch
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________ system is more more flexible.
Batch
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SPECIALIZED REACTORS
Chemostat with recycle
Multistage chemostat
Fed-batch
Perfusion
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_________ under the control of an inducible promoter
Recombinant product
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________ at the same rate if the recombinant product is not expressed.
Recombinant strain and wild type grow
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If the __________is expressed, the _____________ grows much slower.
recombinant product, recombinant strain
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First chemostat is fed with a non-inducing growth substrate, allowing the recombinant strain to be produced.
MULTISTAGE CHEMOSTAT
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The effluent from the first chemostat feeds a second chemostat that is fed inducer, and the product is produced.
MULTISTAGE CHEMOSTAT
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Note: new recombinant cells are continually added to the second chemostat not allowing take-over by a fast growing mutant.
MULTISTAGE CHEMOSTAT
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_________ reactors gain some advantages of a CSTR, retain some disadvantages of batch
Fed-batch
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Reduces substrate inhibition or catabolic repression, allows for high conversion, and the extension of stationary phase.
Fed-batch
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______ nature usually leads to higher operation cost and batch variability.
Semi-batch
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________ cultures are started as batch cultures and grown to an initial cell concentration
X, after which fed-batch operation begins.
Fed-batch
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Substrate is consumed at the same rate it is added.
QUASI-STEADY STATE
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Usually, fed-batch cultures are taken through many feedings cycles, with each feeding cycle followed by a harvest cycle during which the volume is drawn back down to V0 and the cycle begun again.
REPEATED FED-BATCH
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PERFUSION CULTURE
Animal Cell culture
Constat medium flow
Cell retention
Selective removal of dead cells
Removal of cell debris, inhibitory by products
High medium use, costs raw materials and sterilization.
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IMMOBILIZED CELL SYSTEMS
High cell concentrations
Cell reuse
Eliminates cell washout at high dilution rates
High volumetric productivities
May provide favorable microenvironment
Genetic stability
Protection from shear damage
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Mass transfer (diffusional) resistance
MAJOR LIMITATION
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Whole cells provide cofactors, reducing power, energy that many enzymatic reactions require.
ADVANTAGE OVER
IMMOBILIZED ENZYMES
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_____________: similar to enzyme immbolization. Entrapment and binding.
Active Immobilization
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_____________: Biofil-multilater growth on solid surfaces.
Passive Immobilization
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PHYSICAL ENTRAPMENT
Widely used method of cell immobilization.
Various matrices: porous polymers (agar, alginate, carrageenan, polyacrylamide. Chitosan, gelatin, collagen)
Porous metal screens
Polyurethane
Silica gel
Polystyrene
Cellulose triacetate
Polymer beads are also typically used
Encapsulation
Macroscopic membrane-based reactors (hallow fiber)
81
Method of preparing polymer beads:
Gelation of polymers
Precipitation of polymers
Ion exchange gelation
Polycondensation
Polymerization
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There is direct contact between nutrient and suppor materials.
PHYSICAL ADSORPTION
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High cell loadings
PHYSICAL ADSORPTION
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Disadvantages: porous support materials causes intraparticle pore diffusion (at high cell densities) and hard to control microenvironmental conditions
PHYSICAL ADSORPTION
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Selective of suitable support materials is highly based on adsorption capacity and strength of binding
PHYSICAL ADSORPTION
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In general, good support materials should be rigid and chemically inert, should bind cells firmly, high loading capacity.
COVALENT BINDING
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Widely used for enzymes but not for cells.
COVALENT BINDING
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Multilayer growth of cells on solid support surfaces
PASSIVE IMMOBILIZATION:
BIOLOGICAL FILMS
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Support materials can be biologically active or inert
PASSIVE IMMOBILIZATION:
BIOLOGICAL FILMS
90
Common in waste water treatment and mold fermentations
PASSIVE IMMOBILIZATION:
BIOLOGICAL FILMS
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DIFFUSIONAL LIMITATION
Analysis similar to immobilized enzymes.
Damkoler number
Effectiveness factor
Thiele modulus.
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___________: feed flows through a column packed with immobilized cells. Similar to a plug flow reactor. Can be recycles chamber.
Packed-column
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_________: feed flows up through a bed of immobilized cells, fluidizing the immobilized cell particles.
Fluidized-bed
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________: air bubbles suspend the immobilized cell particles in a reactor.
Airlift
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SOLID-STATE FERMENTATIONS
Fermentation of solid materials
Low moisture levels or water activities
Agricultural products or foods
Smaller reactor volume
Low contamination due to low moisture
Easy product separation
Energy efficiency
Differentiate microbiological structures
96
Cellular metabolism produces heat, removed by internal coils or reactor jackets.
Heat Removal
97
Cellular metabolism produces compounds that promote foaming. Controlled by mechanical foam breakers and chemical additives.
Foam Control
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Cellular respiration requires oxygen. Sparged air, impeller makes smaller bubbles and increases residence time.
Providing Oxygen
99
Single organism desired. Steam and filtering.
Sterilization
100
Good oxygen mass transfer
High energy requirement for mixing
Seal to maintain, keep sterile.
Agitated Tank
101
Low shea environment
No seal needed
Restricted to low viscosity
Less mixing than agitated tank
Bubble coalescence limits air flow rate
Bubble Column
102
Better mixing than bubble column with same low shear and energy requirements and lack of seal.
Work with higher viscosity liquids than bubble columns.
Still less mixing than agitated tank
Loop reactors
103
WHAT FACTORS LIMIT
SIZE OF REACTORS
Ability to provide oxygen and remove heat.
104
REACTOR TYPES
Stirred-tank
Bubble Column
Airlift
Propeller Loop
Jet Loop
105
Impeller breaks bubbles into smaller ones to provide for better oxygen mass transfer
AGITATED TANKS
106
are typically glass, commercial fermentors are typically stainless steel
Bench-top tanks
107
Heat removal/addition is typically by coils along the wall, or a water jacket around the tank
AGITATED TANKS
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prevents foaming problems, but can cause additional mass transfer resistance
Antifoam
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volume of liquid in tank; does not include head space
"working volume"
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must not allow contamination
Seal for agitator shaft
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are used to augment mixing and gas dispersion
Baffles
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__________: disc with 6 to 8 blades. Pumps fluid in a radial direction. Compartmentalization with multiple impellers on a shaft.
Rushton impellers
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_______________: pumps liquid in a vertical direction. Lower energy for the same oxygen mass transfer. Lower shear rates.
Axial flow impeller
114
OXYGEN MASS TRANSFER
Bulk gas phase oxygen concentration
Tranfer across stagnan gas layer
Partitioning into the liquid phase (C* at saturation)
Transfer actress stagnant liquid layer
Bulk liquid concentration (
CL)
Transfer across stagnan liquid layer to cell.
115
Transfer rate at _________ is determined by the slowest rate (just like on a highway)
steady state
116
______ is not the rate at which you provide air to the reactor. You will actually provide much more oxygen to the reactor than is transferred to the cells.
OTR
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Utilizes a ________ with actively growing cells.
fermentor
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The air to the fermentor is shut off, and the DO decreases due to consumption by the microorganisms. The air is then turned on, and the the DO increases.
DYNAMIC METHOD
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Make the controlling regime the same on the small scale as on the large scale.
SCALE-UP
120
Empirical
SCALE-UP
121
SCALE-UP CRITERION
Power Input - OTR
Liquid circulation rate - mixing time
Tip speed - shear
Reynolds number - geometry
122
________ by requiring characteristic times to be constant between the small and large scale.
Scale-up
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Many types of characteristic times are related to mixing, diffusion, oxygen mass transfer, reaction, and growth.
ANOTHER METHOD
124
COMMON ON-LINE INSTRUMENTATION
pH
Temperature
Dissolved oxygen
Foam
Flow Rates
Level
Off-gas composition (CO2,O2,VOCs)
125
is generally not as sophisticated as chemical production process control due to a lack of on-line sensors.
Fermentation process control
126
Each probe into the ____________ the probability of contamination, difficult to sterilize some probes, probe fouling, probe placement (gradients within the fermentor).
fermentorincreases
127
Form a group of three and describe 5 control loops based on the most common instrumentation.
TYPICAL FERMENTOR
CONTROL SCHEMES
128
Identify the measured variable, and the controlled variable-specifying what is the final control element (ie. valve, pump, etc.)
TYPICAL FERMENTOR
CONTROL SCHEMES
129
the absence of detectable, viable organisms.
Sterilization
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reduction in the amount of detectable, viable organisms.
Disinfection
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__________: some portion of the population is more resistant to sterilizing agents than other portions.
Sterilization is probabilistic
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______: Heat-sensitive liquids and gases. Most common for gases - P important.
Filter
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_______: Most common for liquids and equipment. Steam. Typically 121°C.Time and T are important. Risk degrading medium components.
Heat
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_____: Surfaces.
Radiation
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_____: Risk of toxic residues.
Chemical
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______ - a faster growing contaminating organism can outgrow the desired organism and cause a washout of the desired organism.
Chemostat
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_________ - the product can be biologically contaminated (could be lethal) or the purity profile could be significantly effected (indust. fermentations 100 kl).
Batch
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to clean with the purpose of removing possible biological and nonbiological threats to human health.
Sanitize
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to greatly reduce the number of living organisms.
Disinfect
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to eliminate all viable organisms present (often our goal).
Sterilize
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(filtration equipment, reactors, etc.) can be sterilized by heat, microfiltration, radiation, chemical agents, and UV light.
Fluids and process equipment
142
a cell, spore, or virus that is dead will not reproduce (cells and viruses) or germinate (spores) under conditions favorable for growth (opposite is “viable”)
Death
143
is a common method.
Thermal sterilization
144
is common for the insides of reactors that can't be heat or steam sterilized.
Ethylene oxide
145
(heat labile vitamins, proteins, sugars) must be filter sterilized using filters with narrow pore-size distributions.
Media that can't be heat sterilized
146
70% v/v ETOH in water with HCl to pH 2 is a ____________________.
good sterilizing fluid
147
is commonly used to sterilize filtration equipment.
Weak (3%) sodium hypochlorite solution
148
GENERAL APPROACH
1.Separation of insoluble products or Components.
2.Primary isolation or concentration and removal of water.
3.Purification and removal of contaminated chemicals.
4.Product preparation.
149
FACTORS THAT IMPACT DIFFICULTY
AND COST OF RECOVERY
1.Product can be biomass, intracellular, or extracellular components.
2.Fragile or heat sensitive.
3.Concentration or titer in the broth.
4.Typically, recovery and purification are more than 50% of total manufacturing costs.
150
INSOLUBLE PRODUCTS OR COMPONENTS
Filtration
Centrifugation
Coagulation and Flocculation
151
Most cost-effective, most common in industrial biotechnology.
Filtration
152
Rotary vacuum precoat filters: traditional. Penicillin mold.
Filtration
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Cross-flow ultrafiltration: 0.02-0.2
um bacterial separations.
Filtration
154
Cross-flow microporous filtration 0.2-2
um for yeast.
Filtration
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Used to separate solids of size. 01 um to 100 um using centrifugal forces.
Centrifugation
156
Being replaced by microfiltration
Centrifugation
157
Pretreatment to centrifugation, gravity settling, or filtration to improve separation.
Coagulation and Flocculation
158
________: formation of small flocs of cells using coagulating agents electrolytes.
Coagulation
159
________: formation of agglomeration of flocs into settleable particles using flocculating agents, polyelectrolytes, or
CaCl2
Flocculation
160
Used wastewater treatment processes to improve clarification.
Coagulation and Flocculation
161
Mechanical Methods
Sonication
Bead beating
Pressing
162
Non-Mechanical Methods
Osmotic shock
Freeze-thaw
Enzymatic
163
_______: disrupts cell membrane. Mostly used at the laboratory scale.
Ultrasound
164
_________: extrude cell paste at high pressure.
Pressing
165
__________: grind cells with glass, metal beads.
Bead beating
166
________ is a problem with all of these methods.
Heat dissipation
167
____________: salt differences to cause the membrane to rupture. Common.
Osmotic shock
168
________: Causes cell membrane to rupture. Common.
Freeze-thaw
169
_________: Lysozyme attacks the cell wall.
Enzymatic
170
SEPARATION OF SOLUBLE PRODUCTS
Liquid-liquid extraction
Aqueous two-phase extraction
Precipitation
Adsorption
Dialysis
Reverse osmosis
Ultrafiltration and microfiltration
Cross-flow filtration and microfiltration
Chromatography
Electrophoresis
Electrodialysis
171
Separate inhibitory fermentation products from broth.
LIQUID-LIQUID EXTRACTION
172
Based on the solubility difference for the compound between the phases.
LIQUID-LIQUID EXTRACTION
173
PRECIPITATION
Solubility reduction at low temperatures (less than - 5°C) by adding organic solvents.
174
- inorganic salts (NH4)2SO4 at high ionic strength.
Salting out
175
Membrane separation is used to remove low molecular weight solutes.
DIALYSIS
176
Used to remove salts from protein solutions.
DIALYSIS
177
Transport occurs due to a concentration gradient driving force.
DIALYSIS
178
Sal phase becomes more concentrated.
REVERSE OSMOSIS (RO)
179
pressure is applied to the salt phase, causing water to move against a concentration gradient.
REVERSE OSMOSIS (RO)
180
_________: Transport of water molecules from a high to a low concentration of pure water to salt water.
Osmosis
181
Pressure-driven molecular sieve to separate molecules of different sizes.
ULTRAFILTRATION AND MICROFILTRATION
182
____________: retained components accumulate on the filter. Gel layer formed on the filter.
Dead end filtration
183
_____________: retained components flow tangentially across the filter.
Cross filtration
184
Separates mixtures into components by passing the mixture through a bed of adsorbent particles.
CHROMATOGRAPHY
185
Solutes travel at different speeds through the column resulting in the separation of the solutes.
CHROMATOGRAPHY
186
Highly specific interaction between a ligand on the particle and a component in the mixture. Often based on antibodies.
AFFINITY CHROMATOGRAPHY
187
Separation of molecules based on size and charge in an electric field.
ELECTROPHORESIS
188
Membrane separation to separate charged molecules from a solution.
ELECTRODIALYSIS
189
FINISHING STEPS
Crystallization
Drying
