Lecture 3: Continuous Cultures and Fed Batch Cultures

Question 1

How do you calculate yield?

Answer 1

biomassT1-BiomassT0 / SubstrateT0-SubstrateT1

Question 2

What does Qp mean?

Answer 2

Productivity

Question 3

How can fermentation be manipulated to increase yield?

Answer 3

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

Question 4

What is a continuous culture

Answer 4

1. constant nutrient medium supply
2. well-mixed culture
3. Products and cells are simultaneously withdrawn
4. Can maintain growth and product formation
5. At steady state, cell, product, and substrate concentrations remain constant

Question 5

What factors affect populatoin growth in a continuous culture?

Answer 5

1. relationship between organisms growth rate and limiting nutrient concentration
2. amount of bacteria prod per unit mass of nutrient (yield)
3. Concetnration of limiting nutrient feed
4. Flow rate and vessel volume

Question 6

What is the equation for the rate of dilution

Answer 6

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”`

Question 7

What is the chemostat theory?

Answer 7

The flow of medium into chemostat is described by the dilution rate

Question 8

What is the equation for net change in biomass?

Answer 8

Sigma x / Sigma t = ux - Dx

Question 9

How is a steady state achieved

Answer 9

When ux = Dx
(rate of biomass formation = rate of biomass loss)

Question 9

How is a steady state achieved

Answer 9

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)

Question 10

What happens at steady state?

Answer 10

1. increased growth rate = increased dilution rate
2. Slow the growth rate = slow the dilution rate
3. setting the correct dilution rate is therefore important in optimising product and biomass formation

Question 11

What is Dcrit?

Answer 11

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

Question 12

How do you calculate yield for continuous cultures

Answer 12

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

Question 13

What is the Monod Paramaters

Answer 13

U = D = UmS/Ks+S

-> S = KsD/Um-D

therefore concentration of the limiting substrate increases with dilution rate

Question 14

How can you predict Dcrit using Monod

Answer 14

Dcrit = Umax Cfs/Ks+Cfs

Increases nutrient feed concentration will increase Dcrit and U

Umax = max growth rate
Cfs = conc of feed stock
Ks

Question 15

What are the problems with continuous batch culture?

Answer 15

1. req constant monitoring
2. increase chance of contamination
3. increase chance of mutation
4. selective pressure for biofilms
5. hard to reconcile productivity with complete substrate conversion

Question 16

What is the fed-batch technique

Answer 16

Last 20 years its been increasingly explored for prod of recombinant proteins

Now many industrial fermentation processes are batch fed

Question 17

What are fed batches

Answer 17

1. Fed with substrate solution so that one substrate component is growth rate limiting
2. substrate feed is often sugar rather than complete medium
3. feed flow is low - therefore low dilution rate
4. substrate concentration is a high as possible (30-50%) to reduce voume increases
5. no withdrawl of culture (so volume DOES increase)

Question 18

Why use a fed-batch?

Answer 18

1. substrate limitation offers a tool for reaction rate control: control prod of specific product over another
2. Substrate limitation offers metabolic control: avoid catabolite repression, repression of gene expression due to intermediary catabolites
3. 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

Question 19

Why cant fed batch cultures reach steady state?

Answer 19

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

Question 20

How is the yield produced linked to the feed concentration in fed-batches

Answer 20

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

Question 21

What is Cfs with regards to chemostat

Answer 21

concentration of limiting nutrient

Question 22

hat is Cs with regards to chemostat

Answer 22

residual substrate

Question 23

What is X with regards to chemostat

Answer 23

change in biomass

Question 24

What is V with regards to chemostat

Answer 24

volume of vessel

Question 25

What is F with regards to chemostat

Answer 25

Medium flow rate

Question 26

What happens as the dilution rate goes up

Answer 26

the growth rate goes up as theres more available nutrients