Lecture 3: Continuous Cultures and Fed Batch Cultures Flashcards
How do you calculate yield?
biomassT1-BiomassT0 / SubstrateT0-SubstrateT1
What does Qp mean?
Productivity
How can fermentation be manipulated to increase yield?
Production of Biomass (primary product): Develop conditions for maximum cell yield in stationary phase, but consider factors such as the time required to realise the yield
Primary metabolites (growth-linked products): Develop conditions to extend exponential growth phase. E.g., use substrate for which the organisms has a low Ks
Secondary metabolites (non-growth-linked products): Develop conditions to reduce exponential phase and extend deceleration phase. E.g., use substrate for which organisms maintain a high Ks
What is a continuous culture
- constant nutrient medium supply
- well-mixed culture
- Products and cells are simultaneously withdrawn
- Can maintain growth and product formation
- At steady state, cell, product, and substrate concentrations remain constant
What factors affect populatoin growth in a continuous culture?
- relationship between organisms growth rate and limiting nutrient concentration
- amount of bacteria prod per unit mass of nutrient (yield)
- Concetnration of limiting nutrient feed
- Flow rate and vessel volume
What is the equation for the rate of dilution
D = F/V
D = rate of dilution
F = flow rate (L.h^-1)
V = volume of vessel (L)
Units: (L.h^-1/L) = h^-1
“THE RATE INWHICH WE ALTER THE CONTENSE OF THE VESSEL”`
What is the chemostat theory?
The flow of medium into chemostat is described by the dilution rate
What is the equation for net change in biomass?
Sigma x / Sigma t = ux - Dx
How is a steady state achieved
When ux = Dx
(rate of biomass formation = rate of biomass loss)
How is a steady state achieved
When ux = Dx
(rate of biomass formation = rate of biomass loss)
THEREFORE u = D (specific growth rate is the same/controlled by the dilution rate)
What happens at steady state?
- increased growth rate = increased dilution rate
- Slow the growth rate = slow the dilution rate
- setting the correct dilution rate is therefore important in optimising product and biomass formation
What is Dcrit?
If dilution rate is increased wash-out of cells can occur, as cells dont have enough time to ‘double’ before being washed out of overflow.
This point is referred to as the critical dilution rate (Dcrit)
STEADY STATE IS LOST
How do you calculate yield for continuous cultures
Ysp = P/(Cfs - Cs)
P = product
Cs = residual substrate from eluent
Cfs = concentration of substrate/feed
P and Cs can be determined from the eluent
Productivity can be measure by multiplying yield by the dilution rate
What is the Monod Paramaters
U = D = UmS/Ks+S
-> S = KsD/Um-D
therefore concentration of the limiting substrate increases with dilution rate
How can you predict Dcrit using Monod
Dcrit = Umax Cfs/Ks+Cfs
Increases nutrient feed concentration will increase Dcrit and U
Umax = max growth rate
Cfs = conc of feed stock
Ks
What are the problems with continuous batch culture?
- req constant monitoring
- increase chance of contamination
- increase chance of mutation
- selective pressure for biofilms
- hard to reconcile productivity with complete substrate conversion
What is the fed-batch technique
Last 20 years its been increasingly explored for prod of recombinant proteins
Now many industrial fermentation processes are batch fed
What are fed batches
- Fed with substrate solution so that one substrate component is growth rate limiting
- substrate feed is often sugar rather than complete medium
- feed flow is low - therefore low dilution rate
- substrate concentration is a high as possible (30-50%) to reduce voume increases
- no withdrawl of culture (so volume DOES increase)
Why use a fed-batch?
- substrate limitation offers a tool for reaction rate control: control prod of specific product over another
- Substrate limitation offers metabolic control: avoid catabolite repression, repression of gene expression due to intermediary catabolites
- Eg, bakers east grown on glucose - low glucose favours cell mass over alcohol
Glucose prevents cAMP formation, meaning cAMP cant bind to CAP, CAP affects transcription of a large number of operons, in most cases, it functions as an activator but there are also cases where it is a repressor
Why cant fed batch cultures reach steady state?
Maths is different to batch or contrinous as the volume constant changes.
D = (1/V) . (sigmaV/sigmaT)
Either: dilution rate exponentially decreases or the feed flow has to exponentially increase with time
How is the yield produced linked to the feed concentration in fed-batches
Ysx(Cfs-Cso)-X0/Ysx(Cfs-Cs)-X = V/V0
Ysx = yield
X0 & X = start & end biomasses
V0 & V = start & end culture volumes
A high Cfs will give high biomass for little change in volume
What is Cfs with regards to chemostat
concentration of limiting nutrient
hat is Cs with regards to chemostat
residual substrate
What is X with regards to chemostat
change in biomass
What is V with regards to chemostat
volume of vessel
What is F with regards to chemostat
Medium flow rate
What happens as the dilution rate goes up
the growth rate goes up as theres more available nutrients
