Culture kinetics

PHE2032
AY20/21
Wan Ping LOH
Batch growth curve

Deceleration
phase

http://academic.pgcc.edu/~kroberts/Lecture/Chapter%206/growth.html

May 2021 Loh Wan Ping | PHE 2032 2

Batch growth curve

Deceleration
phase

Period of adaptation to new environment.

Cell mass may increase a little, without much
increase in cell number/density.
http://academic.pgcc.edu/~kroberts/Lecture/Chapter%206/growth.html

May 2021 Loh Wan Ping | PHE 2032 3

Batch growth curve

Deceleration
phase

Period of rapid balanced cell growth

Cells multiply rapid, with cell mass and
number increasing exponentially with time
http://academic.pgcc.edu/~kroberts/Lecture/Chapter%206/growth.html

May 2021 Loh Wan Ping | PHE 2032 4

Batch growth curve

Deceleration
phase

Period of growth deceleration.

Nutrient depletion or toxic by-products
accumulation causes growth to slow down.
http://academic.pgcc.edu/~kroberts/Lecture/Chapter%206/growth.html

May 2021 Loh Wan Ping | PHE 2032 5

Batch growth curve

Deceleration
phase

Net growth rate is zero (cell growth = cell

death). Production of secondary metabolites
initiates.
http://academic.pgcc.edu/~kroberts/Lecture/Chapter%206/growth.html

May 2021 Loh Wan Ping | PHE 2032 6

Batch growth curve

Deceleration
phase

Cell death increases.

Mostly due to drastic nutrient depletion and
accumulation of toxic product
http://academic.pgcc.edu/~kroberts/Lecture/Chapter%206/growth.html

May 2021 Loh Wan Ping | PHE 2032 7

 Biological processes in bioreactor

S S P
S P S
S P
S S S S S
S S P
S P S
S
S S
S
S S P Larger cell
S
S S
S
S S / new cell
S P
S S P

Cell
Substrate Product Dead cell

May 2021 Loh Wan Ping | PHE 2032 8

Kinetics in bioreactor

• Growth (𝜇, 𝑟𝑥 )
• Death (𝑘𝑑 , 𝑟𝑑 )
• Product formation (𝑞𝑝 , 𝑟𝑝 )
• Maintenance (𝑟𝑠𝑚 ) → difficult to
get a figure .

• Substrate utilization (𝑟𝑠 )

• Yield factor (𝑌𝑥/𝑠 ,𝑌𝑝/𝑠 )

May 2021 Loh Wan Ping | PHE 2032 9

Specific growth rate (𝜇)

Increase in biomass per unit viable biomass per unit

time

1 𝑑𝑋𝑉 In the exponential phase, cell

𝜇= . number may be used in place
𝑋𝑉 𝑑𝑡
of biomass for calculation of 𝜇
𝜇: Specific growth rate (h-1)
𝑋𝑉 : Viable cell concentration (kg viable cells/m3)

May 2021 Loh Wan Ping | PHE 2032 10

Growth modeling

Growth models based on:

• Limiting substrate – Monod equation
• Carrying capacity – Logistic equation
• Substrate inhibition
• Product inhibition
Assumptions made for
each growth model

May 2021 Loh Wan Ping | PHE 2032 11

Monod equation

Specific growth rate (𝜇) is associated with substrate

concentration Umwa
-

y
𝜇𝑚 𝑆 Specific growth rate
𝜇=
𝐾𝑆 + 𝑆 changes with concentration
of substrate S

𝜇𝑚 : Maximum specific growth rate (h-1)

𝑆: Substrate concentration (kg substrate/m3)
𝐾𝑆 : Substrate saturation constant (kg substrate/m3)

May 2021 Loh Wan Ping | PHE 2032 12

Monod equation
When S>>KS : 𝝁 = 𝝁𝒎
𝜇𝑚 𝑆 Specific growth rate is at maximum
𝜇= and does not change with S
𝐾𝑆 + 𝑆
𝟏
When S=KS : 𝝁= 𝝁
𝟐 𝒎
Specific growth rate is at half the
maximum specific growth rate when
S is equal to values of Ks

𝝁𝒎
When S<<KS : 𝝁= 𝐒
𝑲𝑺
Specific growth rate changes
linearly with changes in S

May 2021 Loh Wan Ping | PHE 2032 13

Logistic equation

Specific growth rate (𝜇) is negatively associated with

viable cell concentration this
be using
,

won't
µ Describes the scenario of
𝑋𝑉 growth inhibition
𝜇 =𝑘 1− When there are too many
𝑋𝑉𝑚
cells, growth is inhibited,
specific growth rate → 0

k: growth constant (h-1)

𝑋𝑉 : Viable cell concentration (kg viable cells/m3)
𝑋𝑉𝑚 : Maximum viable cell concentration (kg viable cells/m3)

May 2021 Loh Wan Ping | PHE 2032 14

Logistic equation *simple modelling .

Cells stop growing upon reaching the

M¥ssrbV maximum cell concentration.
-

When Xv=Xvm, specific growth rate = 0

𝑋𝑉
𝜇 =𝑘 1−
𝑋𝑉𝑚

May 2021 Loh Wan Ping | PHE 2032 15

Volumetric growth rate (𝑟𝑥 )

Increase in biomass per unit volume per unit time

𝑟𝑥 = 𝜇𝑋𝑉
𝑟𝑥 : Volumetric growth rate (kg viable cells/m3∙h)
𝜇: Specific growth rate (h-1)
𝑋𝑉 : Concentration of viable cells (kg viable cells/m3)

Monod Logistic
𝜇𝑚 𝑆 𝑋𝑉
𝑟𝑥 = 𝑋𝑉 𝑟𝑥 = 𝑘 1 − 𝑋𝑉
𝐾𝑆 + 𝑆 𝑋𝑉𝑚

May 2021 Loh Wan Ping | PHE 2032 16

Example 01 State assumptions

For bioreactor with the following specifications:

Cell property
Cell growth is substrate dependent
i. * ⇐
tests
=

:÷::÷÷
=3 333. 1h
𝜇𝑚 = 10 h-1 𝐾𝑆 = 10 kg/m3
2. rx =
te ✗ ✓ = 13.333 / h ) ( 0.5kg / m3)
Bioreactor conditions
=\ 667
Substrate concentration = 5 kg/m3
.

Ky Irish .

Inoculating cell concentration = 0.5 kg/m3

Determine the following:
1. Specific growth rate (𝜇)
2. Volumetric growth rate (rx)

May 2021 Loh Wan Ping | PHE 2032 17

 l

Volumetric death rate (𝑟𝑑 )

Increase in non-viable biomass per unit volume per

unit time

The higher the viable cell

𝑟𝑑 = 𝑘𝑑 𝑋𝑉 concentration, the higher
the cell death

𝑟𝑑 : Volumetric death rate (kg non-viable cells/m3∙h)

𝑘𝑑 : death rate constant (kg non-viable cells/kg viable cells∙h)
𝑋𝑉 : Concentration of viable cells (kg viable cells/m3)

May 2021 Loh Wan Ping | PHE 2032 18

Product formation

Products can be produced at different phases of cell

cycle:
• Growth associated F- same patter as cell grvwh .

Eg alcohol fermentation in yeast

at
→ ✗nducmy stationary
pupil
-

• Non-growth associated l

"

product generated
Eg penicillin production by Penicillium stationary phase
←
"
.

only @
chrysogenum i

varies .

• Mixed kinetics (Both growth and non-

:
growth associated)
"
Eg mAb production by hybridoma cells
!
May 2021 Loh Wan Ping | PHE 2032 19
Specific product formation rate (𝑞𝑝 )

Amount of product generated per unit viable

biomass per unit time
𝑞𝑝 = 𝛼𝜇 + 𝛽 Luedeking-Piret

/3∙h)
𝑞𝑝 : Specific product formation rate (kg product generated/m
𝛼: Growth associated product constant(-) Cells

𝜇: Specific growth rate (h-1)

𝛽: Non-growth associated product constant (h-1)

Growth associated product: 𝑞𝑝 = 𝛼𝜇

Non-growth associated product: 𝑞𝑝 = 𝛽
Mixed kinetics product: 𝑞𝑝 = 𝛼𝜇 + 𝛽
May 2021 Loh Wan Ping | PHE 2032 20
Volumetric product formation rate (𝑟𝑝 )

Amount of product generated per unit volume per

unit time
𝑟𝑝 = 𝛼𝑟𝑥 + 𝛽𝑋𝑉
𝑟𝑝 : Volumetric product formation rate (kg product generated/m3∙h)
𝛼: Growth associated product constant(-)
𝑟𝑥 : Volumetric growth rate (kg viable cells/m3∙h)
𝛽: Non-growth associated product constant (h-1)

Growth associated product: 𝑟𝑝 = 𝛼𝑟𝑥

Non-growth associated product: 𝑟𝑝 = 𝛽𝑋𝑉
Mixed kinetics product: 𝑟𝑝 = 𝛼𝑟𝑥 + 𝛽𝑋𝑉
May 2021 Loh Wan Ping | PHE 2032 21
 r

Example 02 State assumptions

N = 3.333 h /
r ,c=
1.667kg / wish
For bioreactor with the following specifications:
Cell property
Cell growth is substrate dependent
Product formation is growth associated with 𝛼 being 0.05
𝜇𝑚 = 10 h-1 𝐾𝑆 = 10 kg/m3 kd = 0.05 h-1
3.
rd =kd✗ , = lo.is/h)co.5kg1m3 )
Bioreactor conditions = 0.025 kg / m3h .

Substrate concentration = 5 kg/m3

"

-1¥
4.
rp= arx -1,1374
"

Inoculating cell concentration = 0.5 kg/m3

= (0-005) II. 667kg /msh )
Determine the following:
0.0834 kg / wish
=
.

3. Volumetric death rate (rd)

4. Volumetric product formation rate (rp)

May 2021 Loh Wan Ping | PHE 2032 22

Maintenance N) theoretical .

Energy requirement to maintain cell in viable state

but not for growth or product formation

Volumetric rate of consumption of substrate for

maintenance energy:
𝑟𝑠𝑚 = 𝑚𝑠 𝑋𝑉
𝑟𝑠𝑚 : Volumetric rate of substrate consumption for maintenance energy
(kg substrate/m3 ∙h)
𝑚𝑠 : Maintenance energy rate constant (kg substrate/kg viable cells∙h)

May 2021 Loh Wan Ping | PHE 2032 23

 Yield factor (𝑌𝑥/𝑠 )

Ratio of cells formed per unit of substrate S

consumed for cell growth

𝑟𝑥
𝑌𝑥/𝑠 =
𝑟𝑠𝑥

𝑌𝑥/𝑠 : Growth yield on substrate (kg viable cells/kg S consumed for growth)
𝑟𝑥 : Volumetric growth rate (kg viable cells/m3 ∙h)
𝑟𝑠𝑥 : Volumetric rate of substrate consumption for growth (kg S/m3 ∙h)

May 2021 Loh Wan Ping | PHE 2032 24

 Yield factor (𝑌𝑝/𝑠 )

Ratio of product P formed per unit of substrate S

consumed for product formation
glucose = carbonSorce

t
𝑟𝑝 growth
.

cell

𝑌𝑝/𝑠 =
𝑟𝑠𝑝

𝑌𝑝/𝑠 : Product yield on substrate (kg P/kg S consumed for product formation)
𝑟𝑝 : Volumetric production formation rate (kg P generated/m3 ∙h)
𝑟𝑠𝑝 : Volumetric rate of substrate consumption for product formation (kg S/m3 ∙h)

May 2021 Loh Wan Ping | PHE 2032 25

Substrate utilization

Substrate is essential for the respective processes:

• Cell growth
• Cell maintenance
• Product formation

Use of substrate Growth + Maintenance + Product

𝑟𝑠 = 𝑟𝑠𝑥 + 𝑟𝑠𝑚 + 𝑟𝑠𝑝

𝑟𝑠 : Volumetric rate of substrate consumption (kg substrate/m3 ∙h)
𝑟𝑠𝑥 : Volumetric rate of substrate consumption for growth
𝑟𝑠𝑚 : Volumetric rate of substrate consumption for maintenance energy
𝑟𝑠𝑝 : Volumetric rate of substrate consumption for product formation
May 2021 Loh Wan Ping | PHE 2032 26
Substrate utilization

𝑟𝑠 = 𝑟𝑠𝑥 + 𝑟𝑠𝑚 + 𝑟𝑠𝑝

𝑟𝑥 𝑟𝑝
𝑌𝑥/𝑠 = 𝑟𝑠𝑚 = 𝑚𝑠 𝑋𝑉 𝑌𝑝/𝑠 =
𝑟𝑠𝑥 , , 𝑟𝑠𝑝

𝑟𝑥 𝑟𝑝
𝑟𝑆 = + 𝑚𝑠 𝑋𝑉 +
𝑌𝑥/𝑠 𝑌𝑝/𝑠

May 2021 Loh Wan Ping | PHE 2032 27

Example 03 State assumptions
"

tl = 3.333h
rx
-

-1.667kg/ wish
For bioreactor with the following specifications: rd -0.025kg/ pih
-

rp= 0.0834kg
/ wish
Cell property
Cell growth is substrate dependent rg=rsx tismtrsp
Product formation is growth associated with 𝛼 being 0.05
-1¥
=
+ rsm

𝜇𝑚 = 10 h-1 𝐾𝑆 = 10 kg/m3 kd = 0.05 h-1 Ybds ,

Bioreactor conditions / mish 0.0834kg /m}h

=\ 667kg
.
y, +

0.6kg/ kg oibkglkg
Substrate concentration = 5 kg/m3
Inoculating cell concentration = 0.5 kg/m3 =
3.06kg / wish .

𝑌𝑥/𝑠 = 0.6 𝑌𝑝/𝑠 = 0.3 ) (500ms ) ( 24h)

6. Amt = ( 3.06kg / mish

Determine the following: =

36720kg
5. Rate of substrate usage
6. Amount of substrate to add to the bioreactor (500 m3) to run it for 24 h
May 2021 Loh Wan Ping | PHE 2032 28
Culture kinetics – summary
Monod model
Growth (𝜇, 𝑟𝑥 ) 𝜇 = 1 . 𝑑𝑋𝑉 𝑟𝑥 = 𝜇𝑋𝑉
𝑋𝑉 𝑑𝑡 𝜇𝑚 𝑆 𝜇𝑚 𝑆
𝜇= 𝑟𝑥 = 𝑋
𝐾𝑆 + 𝑆 𝐾𝑆 + 𝑆 𝑉
Death (𝑘𝑑 , 𝑟𝑑 ) 𝑟𝑑 = 𝑘𝑑 𝑋𝑉

𝑞𝑝 = 𝛼𝜇 + 𝛽 𝑟𝑝 = 𝛼𝑟𝑥 + 𝛽𝑋𝑉
Product formation (𝑞𝑝 , 𝑟𝑝 ) Luedeking-Piret
Growth associated, non-growth associated, mixed kinetics
Maintenance (𝑟𝑠𝑚 ) 𝑟𝑠𝑚 = 𝑚𝑠 𝑋𝑉
Substrate utilization (𝑟𝑠 )
𝑟𝑥 𝑟𝑝
𝑟𝑠 = 𝑟𝑠𝑥 + 𝑟𝑠𝑚 + 𝑟𝑠𝑝 𝑟𝑠 = + 𝑚𝑠 𝑋V +
𝑌𝑥/𝑆 𝑌𝑝/𝑆

𝑟𝑥 𝑟𝑝
Yield factor (𝑌𝑥/𝑆 , 𝑌𝑃/𝑆 ) 𝑌𝑥/𝑆 = 𝑌𝑝/𝑆 =
𝑟𝑠𝑥 𝑟𝑠𝑝
May 2021 Loh Wan Ping | PHE 2032 29
Some notes on material balance✗
defends
on u

( r
cannot be
Constant
𝑑𝑋𝑉 dXV
.

= 𝑟𝑥 − 𝑟𝑑 ⇒ = (μ − k d )①
XV
𝑑𝑡 dt
1
න 𝑑𝑋𝑉 = න 𝜇 − 𝑘𝑑 𝑑𝑡
𝑋𝑉
𝑋𝑉
𝑙𝑛 = 𝜇 − 𝑘𝑑 𝑡 − 𝑡𝑂
𝑋𝑉𝑂

XV = XV0 . e(𝜇− kd )(𝑡− 𝑡0 )

Under certain assumptions, equation may be simplified. For eg,
-Monod model under substrate-rich condition (S >>> 𝐾𝑠 𝜇 → 𝜇𝑚 ),
-Negligible cell death (𝑘𝑑 = 0)
I
𝑋𝑉
𝑙𝑛 = 𝜇𝑚 𝑡 − 𝑡𝑂 ⇒ XV = XV0 . 𝑒 (𝜇𝑚 )(𝑡− 𝑡0)
𝑋𝑉𝑂
May 2021 Loh Wan Ping | PHE 2032 30
Some notes on material balance

𝑑𝑃 d𝑃
= 𝑟𝑝 = 𝛼𝜇𝑋𝑉 + 𝛽𝑋𝑉 ⇒ = (𝛼𝜇 + 𝛽) XV
𝑑𝑡 dt

න 𝑑𝑃 = 𝛼𝜇 + 𝛽 න 𝑋𝑉 𝑑𝑡

𝑑𝑆
= − 𝑟𝑠 = −(𝑟𝑠𝑥 + 𝑟𝑠𝑚 + 𝑟𝑠𝑝 )
𝑑𝑡

𝑑𝑆 𝜇𝑋𝑉 𝛼𝜇𝑋𝑉 + 𝛽𝑋𝑉

⇒ =− ( + 𝑚𝑠 𝑋𝑉 + )
𝑑𝑡 𝑌𝑥/𝑠 𝑌𝑝/𝑠

𝜇 𝛼𝜇 + 𝛽
න 𝑑𝑆 = − ( + 𝑚𝑠 + ) න 𝑋𝑉 𝑑𝑡
𝑌𝑥/𝑠 𝑌𝑝/𝑠

May 2021 Loh Wan Ping | PHE 2032 31

References

Bioprocess Engineering: Basic Concepts

Author: Michael L. Shuler, Fikret Kargi, Matthew
Delisa
Edition: 3rd Ed, 2017
Publisher: Prentice hall

May 2021 Loh Wan Ping | PHE 2032 32

Example 04 State assumptions

Your company BPE-DrinkHealth is setting up a bioreactor to grow

lactobacillus for incorporation into their latest health drink. Substrate is
added in large excess to ensure sufficient nutrient for cell growth. The 𝜇𝑚
and 𝑘𝑑 for the strain of lactobacillus is determined to be 0.9 h -1 and 0.1 kg
non-viable cells/kg viable cells.h respectively. The bioreactor was
inoculated with 0.5 kg/m3 of lactobacillus. Determine the viable cell
concentration after 8 h.
You may assume lactobacillus follow substrate dependent growth (i.e.
Monod’s equation).

May 2021 Loh Wan Ping | PHE 2032 33

Example 05 State assumptions

Your company BPE-Therapeutics has recently developed a NS0 cell line

producing a novel recombinant protein CreOxin-3 and is testing out the
production in a lab scale batch bioreactor (10L). Cells were inoculated at
the exponential phase, and the following viable cell concentration data
were obtained.
Time (h) VCD (10^9 cells/L)
0 0.320
4 0.341
22 0.588
45 1.054
71 2.256
94 3.613
Calculate
a) the specific growth rate of the cells
b) the doubling time of the cells (tD)

May 2021 Loh Wan Ping | PHE 2032 34

Example 06 State assumptions

Your company BPE-Therapeutics has recently developed a NS0 cell line

producing a novel recombinant protein CreOxin-3 and is testing out the
production in a lab scale batch bioreactor (10L). Cells were inoculated at
the exponential phase, and the following viable cell concentration data
were obtained.
Time (h) VCD (10^9 cells/L)
0 0.320
4 0.341
22 0.588
45 1.054
71 2.256
94 3.613
Calculate
c) the approximate amount of product obtained at 94h assuming growth
associated product formation at = 1.84g product/10^9 cells

May 2021 Loh Wan Ping | PHE 2032 35

Example 07 State assumptions

After testing the batch culture sample at 94h, no CreOxin-3 was detected.
You realize that formation of CreOxin-3 is a secondary metabolite
(formation is non-growth associated). You retrieved the data for the rest of
the bioreactor run:

Time (h) VCD (10^9 cells/L) Time (h) VCD (10^9 cells/L)
0 0.320 116 5.285
4 0.341 141 5.999
22 0.588 164 6.183
45 1.054 188 6.183
71 2.256 209 6.183
94 3.613 232 6.183

Calculate
d) if product concentration at 232h is 7.39g/L

May 2021 Loh Wan Ping | PHE 2032 36

Example 08 State assumptions

A Chinese hamster ovary cell line is used to produce human erythropoietin

(EPO). Growth in batch culture is monitored with results shown in Table 1.
Table 1. Cell and substrate
concentrations over time
Time (h) X (cells/mL) S (g/L)
0 2.03E+05 8.00
24 2.13E+05 7.97
48 2.48E+05 7.88
72 3.21E+05 7.70
96 6.17E+05 7.35
120 1.224E+06 6.72
144 2.309E+06 5.56
168 3.533E+06 3.48
192 4.020E+06 0.00
216 2.563E+06 0.00
240 4.68E+05 0.00
Determine
a) Determine the maximum specific growth rate (𝜇𝑚 )
b) Determine the maximum growth yield (𝑌𝑥/𝑆 )
May 2021 Loh Wan Ping | PHE 2032 37