07 Growth Kinetics Flashcards
What is the overall goal of bioprocessing?
To produce our target biomolecule
Upstream processsing & examples
- growth & production >stock culture/ shake flasks >seed fermentor >sterilize fermentor & media >production fermentor
downstream processing & examples
- seperation & purification
>cell seperation
>product purification
bioprocess analysis
- upstream process
- determines the best means for achieving optimal yield during fermentation phase
Growth Associated Product Formation
- Product expression occurs as a consequence of growth
- Product formation is linked to energy metabolism
Non-Growth Associated Product Formation
Product expression occurs as a product of secondary metabolism, and is not linked to energy metabolism.
biomass
- can be an indicator of product formation
>if growth associated
substrate
- cells can only grow/ from bioproduct when sufficient substrate is available
- limiting it can inhibit growth
- over addition = less than ideal growth
Substrate uses:
- maintenance
> limiting substrates: carbon & oxygen - cell mass
- products
>synthesis of chemical products
cell yield formula
lChange in Cell Mass l
Yx/s = l———————————l
lSubstrate Consumed l
lchngeX l Yx/s = l-------------l
lchngeS l
Product yield formula
lchngeP l
Yp/s = l————- l
lchngeS l
P= delta product S= delta substrate
Growth kinetics formula
(lnN2-lnN1)
u = ——————
(t2-t1)
N = # of cells at instance 1 & 2
t = time at instance 1 & 2
** growth rate constant = k, u, [h^-1]
Doubling time formula
ln(2)
td = ———–
u
Growth rate & Doubling time formulas (OD)
ln(OD at time 2)-ln(OD at time 1)
Uod = ———————————————–
elapsed time 2-elapsed time 1
ln2 tD =------
u
Trypan Blue
- dye assay to determine cell viability
- more accurate than OD600nm
- Dead/Non viable cells are stained blue
- Living/Viable remain unaffected
- helps estimate volume of living and dead cells in culture
Hemocytometer
- thick glass slide with etched grid lines
- count # of cells in specific volume (area) of solution
Media nutrient review
- Carbon
- Carbs are primary energy source - Nitrogen
- makes amino acids, nucleic acids, & other molecules - Oxygen
- pH, Temp, Secondary factors
pH can change due to:
- utilization of substrate
- NH4 production
- Cellular by products
- Cellular lysis/ cell toxins
pH range: Bacteria Yeast Mammalian Plant
> 3-8
3-6
6.5-7.5
5-6
dissolved CO2
- 2 degree factor
- toxic at extremely high or low levels
Ionic strength
- 2 degree factor
- too high, inhibitory to normal cell membrane fxn
> membr transport
> osmotic balance
Additives that can also inhibit growth:
- Glucose > 100g/L
- EtOH > 10g/L
- NH4+ > 5g/L
dissolved oxygen (DO)
- O2 can be limiting substrate for aerobic ferment.
- not readily soluble in water
- too much can be detrimental to cellular production
Oxygen Transfer Rate (OTR)
Ro2 = Kla(C*-Cl)
- Ro2 = oxygen transfer rate
- Kla = volumetric transfer coefficient (constant)
- C* = saturated conc of oxygen (constant)
- Cl = actual DO
Oxygen Uptake Rate (OUR)
chngeO2
qo2X = ————–
chnge t
- mols O2 consumed/ time
The ___ the size of the bubble/globule, the ____ the SA available for mass transfer
- smaller
- larger
Impeller (Agitator)
- helps diminish the size of air bubbles
- Provide a large interaction area for oxygen transfer
- decrease the diffusion path
- maintain a uniform envmt
- shear damage
Operational Mode- Batch
- simplest ferment operation
- sterilization can be performed in reactor
- all nutrients added before inoculation
- max levels or C & N limited due to inhibition of cell growth
- cells harvested when levels start to decline
Continuous Flow rates:
Dilution > Growth
- culture washes out
Dilution < Growth
- culture overgrows
Dilution = Growth
- steady state culture
