Week 5 Downstream Flashcards
Downstream Processing
-Encompasses all processes following the fermentation; the aim is to efficiently, reproducibly and safely recover the target product. This needs to be done while maximizing recovery yield and minimizing costs.
-Influenced by product concentration and stability. Other considerations are yield at each step, process costs and purity requirements
-Depending on what the target product is, DSP may require processing the cells (intracellular) or processing the spend medium (extracellular).
-In some cases, it will also be necessary to maintain a certain level of biological activity- for enzymes, the purity is measured by unit of enzyme activity per unit protein.
Type of Downstream Processing and degree to which it takes place is dependent on?
-The preceding fermentation, consider the case of using a cheap but impure substrate on the DSP
The factors that affect Downstream Processing that need to take place include:
Properties of microorganisms
-Morphology
-Flocculation characteristics
-Size of cells
-Cell rigidity
They have influence on filterability, sedimentation and homogenization
Why are Downstream processes divided into a series of Unit processes?
Downstream processes are divided into a series of Unit Processes - separate operations themselves that are linked together to achieve product purifications. Typically, the number of steps is kept to a minimum as each subsequent step is costly and there is always some loss of yield.
Purification of a soluble intracellular bacterial protein
Includes:
-Cell harvesting (ex. centrifugation and then supernatant)
-Disruption of recovered cells (ex. liquid shear homogenization)
-Cellular debris separation (ex. centrifugation and then cell debris)
-Protein precipitation of the cell-free extract (ex. ammonium sulphate)
-Recovery of protein by centrifugation
-Desalting of resuspended protein pellet (gel filtration)
-Ion-exchange chromatography (ex. DEAE cellulose)
-Dialysis
-Lyophilization
Purification of an organic acid from a fungal culture?
-Broth clarification (ex. rotary vacuum filtration and then mycelium)
-Clarified broth decolorization (ex. activated charcoal treatment)
-Evaporation
-Crystallization
-Recovery of crystals (ex. filtration)
-Crystal cleaning
-Crystal drying
What will integrating the Downstream processes in the fermenter do
This will allow for one or more fewer unit processes and will increase overall yields. This can be achieved by placing separation units within the fermenter or by directly linking the two systems.
-In situ removal of products
-Ex situ removal of products
Downstream processing can fall into 3 broad categories?
-Cell harvesting
-Cell disruption
-Product recovery
Cell harvesting
-To remove cells from spent medium
-The first step in downstream processing of suspended cultures is a solid-liquid separation to remove cells from spent media.
-Both the solid and the liquid will then need to be processed further, depending on where the desired product is.
Cell disruption
-If the product is intracellular; breaking cell walls and cytoplasmic membrane
Product recovery
-Of extracellular material (proteins) from clarified medium, whereas disrupted cell preparations are used for both intracellular proteins and proteins from periplasmic space.
The type of solid-liquid separation that takes place depends on a number of factors including:
The type of solid-liquid separation that takes place depends on a number of factors including:
-Economics
-Required purity
-Yield obtained at each step
-Safety
Before the separation process, some___________ may need to take place.
Broth conditioning
What does broth conditioning include?
-This would include adding agents that would encourage the solid to either precipitate to the bottom or float to the top, though the former is much more common.
Because the majority of products are for human consumption or usage, the additions that are made need to be simple, safe agents
Safe agents?
Are agents that need to act as coagulants and flocculants. Typical agents include:
-Air bubbles (flotation)
-Egg albumen to ppt yeast cells
-Coagulation: electrolytes, acids, basis, salts, ions, polyelectrolytes; inorganic salts (CaCl2), polyacrylamide, polystyrene salts
What are the 3 main separation processes associated with cell harvesting?
Sedimentation, centrifugation, and filtration
What is sedimentation?
Sedimentation relies on particles of a very large size (>100um diameter) and density settling out from a quiescent liquid. It is a very important process for removal of yeasts from the production of beer & spirits and in wastewater treatment facilities. The rate at which a particle settles is dependent on Stoke’s Law: governed by gravity g=9.81 m/s2
What is centrifugation?
-Instead of relying on gravitational forces, centrifugation relies on centrifugal forces. With this, much smaller particles- in the range of 0.1 um can be removed.
-The terminal (settling) velocity of a particle is calculated from:
What are the advantages of centrifugation?
-Continuous process
-Sterilizable
-No consumable costs
What are the disadvantages of centrifugation?
-High initial cost
-Noise
-Cost of electricity
-Physical rupture of cells (high shear)
-Temperature-denaturation
What is Filtration?
-Filtration is a solid-liquid separation and can be both the separation of undissolved solids or dissolved solids from the spent media.
-For undissolved solids, the 2 most common methods are plate & frame filter presses and rotary vacuum filters. The plate & frame filter press is a batch process with cloths held in place between the plates and frames.
-Typical applications are: removal of yeasts, protein precipitates, and sewage sludges.
