HCDC of E. coli under FedBatch conditions
Whiffin Cooney Cord-Ruwisch
High cell density cultures
Chemostat and Batch culture have different advantages
and problems
Productivity R is higher in chemostat
Maintaining sterility is easier in batch. Contamination from
inside = highly productive mutants reverting to wild strain
For typical industry processes batch is preferred although it
is more difficult to control and has lower .
Batch is the only option for :
seondary metabolites
Process Control examples
Example
Measure
What
Act
By
Why
Chemostat
pH
Adding
NaOH
Avoid
Contamination
Anaerobic
digestion
H2
Adjusting loading Avoid
rate / recycle of
acidification
methanogens /
adding NaOH
SND
DO, OUR
Air supply
Avoid over/
underoxidation
HCDC
Starvation level
Feeding
Avoid acetate
build-up in cells
What is HCDC
High Cell density Cultures are essential for maximum
productivity of recombinant protein production (e.g.
pharmaceuticals)
After producing the biomass to the highest possible density
(e.g. OD 50) the expression of the recombinant product
(e.g. on mulitple plasmid copies) is induced.
To produce OD 50 (about 50 g/L of dry mass) more than
100 g of substrate (e.g. sugar) is needed.  Substrate
inhibition (almost “jam”).
What is HCDC
High Cell density Cultures are essential for maximum
productivity of recombinant protein production (e.g.
pharmaceuticals)
After producing the biomass to the highest possible density
(e.g. OD 50) the expression of the recombinant product
(e.g. on mulitple plasmid copies) is induced.
To produce OD 50 (about 50 g/L of dry mass) more than
100 g of substrate (e.g. sugar) is needed.  Substrate
inhibition (jam).
Why Fed-batch for HCDC?
To add the full amount of substrate needed at the
beginning  substrate inhibition
Solution: After an initial batch with less substrate, add
substrate sequentially (Fed batch)
Initial
Batch
Fed-batch
S
The fedbatch has no
outflow
Feed is added from a
concentrate (syrup)
Arrows show Saddition
Time
Need for precise feed addition
Too little glucose addition slows down the process too
much
Too high glucose concentrations  build up acetate even
at high DO concentrations when sugars are saturating
Initial
Batch
Controlled
sugar addit.
S
Time
Acetate buildup
interferes with growth
and expression of
recombinant proteins
Glucose should be
administered on
demand (avoid over
saturation and strong
substrate limitation)
Why does acetate build up?
246
103 20 20 103
01
20 82
82
TCA
01
20
8*20
Glycolysis faster than
TCA / ETC / OUR
Hence acetate (82)
builds up.
Metabolic overflow
Crabtree effect.
TCA bottleneck
ETC, OUR
Abbreviations: TCA- Tricarboxylic Acid Cycle. OUR = oxygen uptake rate, ETC = Electron Transport Chain, 24-6 = glucose, 10-3=
pyruvate
Strategies to administer glucose on demand
Administering feed ON DEMAND requires feedback loop
(measuring glucose demand – then increasing or
decreasing glucose flow rate to reactor).
No demand if glucose is saturating.
A number of strategies are possible to evaluate DEMAND
for glucose:
Strategy 1: Glucose stat:
measure glucose and only increase feedrate when glucose
is less than saturating levels. Problems: No online glucose
sensor, need for very precise measurements at very low
[glucose ](close to ks value of E.coli)
Strategies to administer glucose on demand
Strategy2: Acetate stat: measure acetate online and lower
glucose feedrate as soon as acetate builds up. Problem:
No online acetate sensor
Strategy 3: Monitor glucose saturation from changes in
OUR in response to feedrate changes. (“Starvostat”)
BTW. E.coli can take up acetate excreted acetate under
glucose starvation.
How to determine glucose demand of the
culture ?
Experience from our lab class (OUR) shows that a starving
culture responds to the addition of substrate by increasing
OUR.
If there is not effect by adding extra substrate  culture
was S-saturated.
Culture can provide information to operator whether
it is substrate saturated or limited.
Monitor glucose demand of the culture
Keep DO steady state by constant glucose feed rate
Do a step increase in feedrate
No decrease in DO (hence no increase in OUR)  go
back to old feedrate, as culture is feed saturated (no
demand)
Sign of demand
D.O.
Feed
Glucose feedrate
rate
Time
Decrease in DO 
culture was limited 
shows a higher
capacity for OUR, ETC
and TCA and demands
more glucose without
risk of acetate buildup
Feedback control to keep culture on the
border of substrate saturation
Keep testing for glucose demand.
If the culture shows glucose demand, increase the
feedrate by say 2% and keep testing for further glucose
demand.
As long as the culture will grow, its demand will increase
D.O.
S Glucose feedrate
Time
Problem with this
approach: If culture is
saturated the glucose
step increase 
acetate buildup. Better
approach?
Method published by V. Whiffin et
al.
Measure OUR-1
Interrupt feed for 30 sec.
Measure OUR-2
If OUR-2 < OUR-1,(=starving) then increase FR by
1 %, else decrease FR by 1%
Keep new FR for 2 min
Resume from the top
Feedback control to keep culture on the
border of substrate saturation
Keep testing for glucose demand.
If the culture shows glucose demand, increase the
feedrate by say 2% and keep testing for further glucose
demand.
As long as the culture will grow, its demand will increase
OUR
S Glucose feedrate
Time
Problem with this
approach: If culture is
saturated the glucose
step increase 
acetate buildup. Better
approach?