F. Biotech L3 Flashcards
What does cell immobilization do
Changes cell biology
Methods of immobilizing cells
- Containment of cells in membrane
- Attach cells to solid substrate
- Entrapment of cells in gel
- Flocculation
Advantages of cell immobilization
- High cell density
- No wash out of cells ;easy to hold onto them)
- Reduce contamination; high cell conc, no need to reuse
- Reduce growth inhibition
- Separate cells and medium
- Increase product yield
- Faster speed of fermentation
Disadvantage of cell immobilization
- Difficult to maintain yeast viability; viability drops, cells die
- Stability over time (system breaks down)
Containment of cells in membrane bioreactors
Involves trapping of cells into a porous material eg dialysis tube. The medium goes in, ferments the product and is then released.
Problem with containment of cells
As cells replicate, membrane gets clogged
Attachment of cells to a solid substrate
Make use of gluteraldehyde to cross link. Yeast cells can then be added so they can attach to the surface
What is gluteraldehyde?
It is a preservative that makes surfaces sticky
Problem with attachment of cells to cell surface
Some cells are trapped and the rest bud off and float away
Entrapment or cells in alginate beads
Cells are trapped in sodium alginate, they are then added to CaCl2 solution and form beads.
Advantage of entrapping cells in beads
Beads are porous, stable, diffusable. They are used in a wide range of systems eg batch fermentation, cell recycle batch fermentation. The beads can be used free or packed in columns. As a result, there is increased ethanol production and faster fermentation.
Problem with entrapping cells in beads
Calcium chelating agents eg phosphate reacts with Ca and removes Ca from calcium alginate making it dissolve. Gas then builds up in the beads making it unstable
What other gels/ synthetic polymers can cells be trapped in?
Gel- Agar, gelatin
Synthetic polymer- Polyacrylamide
Flocculation
Cell walls become sticky, forming clumps of cells at a very high cell density. Some yeast cells flocculate naturally. Usually occurs in stationary phase but they can be tweaked to occur earlier
Downstream processing
Involves pulling out and concentrating products for the consumer. Difficult to do.
Product recovery of ethanol
A product with low level of ethanol is distilled and the ethanol is taken up and concentrated until you get a greater ethanol concentration (whiskey)
Product recovery of proteins
Isolate and purify
What is the main problem with downstream processing
Getting product out. If the product is in the cells, they have to be broken up to get the product out.
Ways in which a cell can be disrupted
- Sonification
- Agitation
- French press
- Cell wall degradation
- Controlled autolysis
Sonication
Use of sound waves to break cells
Agitation
Can be carried out with the use of glass beads to smash cells up
Cell wall degradation makes use of?
Enzymes to chop cells up
Isolation of intracellular enzymes can be carried out by:
- Removing nucleus acids from cell debris with the use of nuclease which chops up DNA
- Concentration of the product
- for soluble proteins, ammonium sulphate ppt can be used for salting out proteins. This is used for precipitation of enzymes
- for proteins present in membranes, PEG can be added to disrupt membranes to release things trapped inside.
This is used for microsomal enzymes
What does PEG stand for
Polyethyleneglycol
Cell breakage can be?
Mechanical or non-mechanical
Mechanical methods of cell breakage
- Grinding
- Pressure
- Ultrasound
Non-mechanical methods of cell breakage
- Dessication
- Lysis
Means of separating cells
- Size
- Diffusivity
- Ionic charge
- Solubility
- Density
Separation based on size can be carried out by?
- Ultracentrifugation
- Gel chromatography
Separation based on diffusivity can be carried out by?
Dialysis
Separation based on ionic charge can be carried out by?
Electrolysis
Separation based on solubility can be carried out by?
Solvent extraction
Separation based on density can be carried out by?
Centrifugation
Characteristics of downstream processing
- Maximum recovery of products
- Process must be reliable and continuous
- Large reductions in volume
- Final broth must be usable
- Processing commence as broth leaves fermenter
- Cost considerations
