Kinetics of biomass formation, substrate utilization and

product formation in cell cultures

VIVEK R BITS Pilani, K K Birla Goa Campus

Biomass formation kinetics

VIVEK R BITS Pilani, K K Birla Goa Campus

 Natural growth Vs Controlled growth

Animals, Plants  Eukaryotes  Difficult to tune up as a whole (-) (Climate dependent)

Animal and plant cells  tunable  controlled growth (+)

Growth in
Yeasts and moulds  lower eukaryotes  controlled growth ( + + )
Bioreactors
Bacteria prokaryotes  controlled growth ( + + + )

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 Simple-complex-simple

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 Bacteria reproduce by binary fission, by asexual mode or by budding
 Binary fission one cell becomes two organisms

 The required time for each cell division is generation time ( can vary from days to less than 20 min)
 For example  if generation time is 30 min, one bacterium will form 224 = 16,777,216
after a period of 12 h

BITS Pilani, K K Birla Goa Campus

 Batch growth

Also called log/exponential phase

𝟏 𝒅𝒙
µ=
𝒙 𝒅𝒕
x  biomass concentration
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1) Exponential phase

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Rate of cell growth in growth and decline phases

rx = µ. x = dx/dt rx in kg m-3 h-1

µ in h-1; x in g L -1

Integrating with x=x0 at t=0

𝑑𝑥
= 𝜇 𝑑𝑡
𝑥

ln 𝑥 = ln 𝑥0 + 𝜇 𝑡

𝒙 = 𝒙𝒐 𝒆𝝁𝒕

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ln x and x vs t: use ln x vs t to calculate µ

VIVEK R BITS Pilani, K K Birla Goa Campus

Doubling time
Doubling time is the period of time required for the cells to double in weight/ number
When x0 is the starting concentration; at t=td x= 2x0
2𝑥0 = 𝑥0 𝑒 𝜇𝑡

𝐥𝐧 𝟐
𝒕𝒅 =
𝝁
Balanced growth
During balanced growth, the composition of the biomass remains constant.

rZ = µ z
where Z is a cellular constituent such as protein, RNA, polysaccharide, and so on, rZ is
the
volumetric rate of production of Z, and zVIVEK
is theR concentration of Z in the reactor volume
BITS Pilani, K K Birla Goa Campus
2) Stationary phase

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Endogenous metabolism in stationary phase

Some times µ is expressed as µ net when endogenous metabolism is considered

𝟏 𝒅𝒙
𝝁𝒏𝒆𝒕 =
𝒙 𝒅𝒕 𝜇𝑛𝑒𝑡 = 𝑛𝑒𝑡 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑔𝑟𝑜𝑤𝑡ℎ 𝑟𝑎𝑡𝑒, ℎ−1
𝜇𝑔 = 𝑔𝑟𝑜𝑠𝑠 𝑠𝑝𝑒𝑐𝑖𝑓𝑖𝑐 𝑔𝑟𝑜𝑤𝑡ℎ 𝑟𝑎𝑡𝑒, ℎ−1
𝝁𝒏𝒆𝒕 = 𝝁𝒈 − 𝒌𝒅 𝑘𝑑 = 𝑟𝑎𝑡𝑒 𝑐𝑜𝑛𝑠𝑡𝑎𝑛𝑡 𝑓𝑜𝑟 𝑒𝑛𝑑𝑜𝑔𝑒𝑛𝑜𝑢𝑠, ℎ−

In stationary phase, cells catabolize their cellular reserves for new building blocks,
called endogenous metabolism
𝒅𝒙
= −𝒌𝒅 𝒙 or 𝒙 = 𝒙𝒔𝟎 𝒆−𝒌𝒅𝒕
𝒅𝒕
xso  the cell mass concentration at the beginning of the stationary
phase.
VIVEK R BITS Pilani, K K Birla Goa Campus
3) Death phase

VIVEK R BITS Pilani, K K Birla Goa Campus

Death phase

The rate of death usually follows first-order kinetics

𝑑𝑁
= −𝑘′𝑑 𝑁
𝑑𝑡

−𝒌′ 𝒕
𝑵 = 𝑵𝑺 𝒆 𝒅

where Ns number concentration of cells at the end of the stationary phase

k’d first order death-rate constant.

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 Batch growth

Also called log/exponential phase

𝟏 𝒅𝒙
µ=
𝒙 𝒅𝒕
x  biomass concentration
VIVEK R BITS Pilani, K K Birla Goa Campus
Effect of substrate concentration on specific growth rate

VIVEK R BITS Pilani, K K Birla Goa Campus

 Effect of substrate concentration on specific growth rate

During balanced growth

Specific growth rate is related to the substrate concentration, often a rate limiting substrate

Monod Model 𝝁𝒎𝒂𝒙 𝑺

𝝁=
𝑲𝒔 + 𝑺
S  concentration of rate limiting substrate Zero order
Ks  substrate/ monod/saturation constant g/L or mg/L
𝝁𝒎𝒂𝒙  maximum specific growth rate T -1
First order
𝝁 independent of substrate concentration until S > 10 Ks

In batch growth, substrate concentration S >> Ks

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KS Values for Several Organisms

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Monod model limitations

The most frequently used expression relating growth rate to substrate concentration.

 Valid only for balanced growth and should not be applied when growth conditions
are changing rapidly

 Limited applicability at extremely low substrate levels.

Extra terms can be added to Monod expression when there is growth inhibition due to high
substrate and product concentration

VIVEK R BITS Pilani, K K Birla Goa Campus

Exponential vs Logistic biomass growth

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 Exponential Vs logistic biomass growth

 Starts rapidly  Starts slowly

 Rate proportional to the cells  Rate begins to slow down when organisms
which already exist are in competition for limited space
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The logistic equation

The sigmoidal shape of the growth curve can be predicted by combining Monod equation and
with the growth equation

𝜇𝑚𝑎𝑥 𝑆
𝜇= Eq 1
𝐾𝑠 + 𝑆

𝑑𝑥
= 𝜇𝑥 Eq 2
𝑑𝑡

𝑑𝑥 𝜇𝑚𝑎𝑥 𝑆
= 𝑥 Eq 3
𝑑𝑡 𝐾𝑆 + 𝑆

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 The relationship between microbial growth and substrate consumption is given by
𝑥 − 𝑥0
𝑌𝑋/𝑆 = Eq 4
𝑆0 − 𝑆
Substituting for S in Eq 3 from Eq 4

𝑑𝑥 𝜇𝑚 (𝑌𝑋/𝑆 𝑆0 + 𝑥0 − 𝑥)
= 𝑥 Eq 5
𝑑𝑡 (𝐾𝑆 𝑌𝑋/𝑆 + 𝑌𝑋/𝑆 𝑆0 + 𝑥0 − 𝑥)

The integrated form of rate expression is Eq 6

(𝑲𝑺 𝒀𝑿/𝑺 + 𝑺𝟎 𝒀𝑿/𝑺 + 𝒙𝟎 ) 𝒙 𝑲𝑺 𝒀𝑿/𝑺 𝒀𝑿/𝑺 𝑺𝟎 + 𝒙𝟎 − 𝒙

𝐥𝐧 − 𝐥𝐧 = 𝝁𝒎 𝒕
(𝒀𝑿/𝑺 𝑺𝟎 + 𝒙𝟎 ) 𝒙𝟎 (𝒀𝑿/𝑺 𝑺𝟎 + 𝒙𝟎 ) 𝒀𝑿/𝑺 𝑺𝟎

VIVEK R BITS Pilani, K K Birla Goa Campus

Eq 5 requires a predetermined knowledge about the maximum cell mass (x∞)
in a particular environment, also called as carrying capacity

Logistic equations are set of equation that characterize growth in terms of their carrying capacity

Usually the specific growth rate is related to the amount of unused carrying capacity
𝑥
𝜇 = 𝐾 (1 − ) Eq 7
𝑥∞

𝑑𝑥 𝑥 Eq 8
= 𝐾𝑥(1 − )
𝑑𝑡 𝑥∞

K  carrying capacity coefficient

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 Integrating with B.C: x(0) = x0

𝒙𝟎 𝒆𝒌𝒕
𝒙= 𝒙𝟎 Eq 9
𝟏− (𝟏 − 𝒆𝒌𝒕 )
𝒙∞

Eq 9 is represented by the growth curve

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 Cell mass concentration

Dry weight method Optical density

Centrifuge the broth Dilute the sample appropriately

using pre-weighed filter paper

Take optical density

Dry the paper in hot air over
OD 600 nm
At 80 °C till constant weight
(Vis spectrophotometer)

Calculate mass difference

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Quantifying cell concentration

 Cell number density

Haemocytometer
Cell count by serial dilution

 Cell mass concentration

Dry cell weight/wet cell weight
Optical density

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 Cell counting using haemocytometer

Volume enclosed by the cover slip for each large square = 0.1 mm3

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Viable cell count by serial dilution method

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