Microbial growth kinetics

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 Components of kinetics
1. Cell Growth
– Increase in biomass (number of cells): Population growth
– Increase in size and density
2. Uptake of nutrients
3. Release of metabolic end products

The rate of these processes vary widely as growth occurs

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Factors determining cell population kinetics
Environment phase Biological phase

Cellular pathways are regulated by environmental factors 3

 Assumptions
Environmental factors
• Sufficiently high concentrations of growth media except one rate
limiting nutrient
• Constant environmental parameters like pH, temp. and dissolved
oxygen with time
Biological factors
• Cell-to-cell heterogeneity does not substantially influence kinetic
processes [average cell approximation]
• Average cellular composition is not affected by proliferation of the
population [balanced growth]

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Different perspective for cell population kinetics

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 Different perspective for cell population kinetics

Structured Unstructured
• Cells are considered as • Cells are considered as single
multicomponent component

Segregated Unsegregated
• Consideration of discrete and • Consideration of average cellular
heterogeneous cells properties

Models may be structured and segregated, structured and unsegregated, unstructured and segregated, and
unstructured and unsegregated

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 Contd…
• An unstructured model assumes fixed cell composition, which is equivalent to
assuming balanced growth.
• The balanced-growth assumption is valid primarily in single-stage, steady-state
continuous culture and the exponential phase of batch culture; it fails during any
transient condition.
• How fast the cell responds to perturbations in its environment and how fast these
perturbations occur determine whether pseudobalanced growth can be assumed.
• If cell response is fast compared to external changes and if the magnitude of these
changes is not too large (e.g., a 10% or 20% variation from initial conditions),
then the use of unstructured models can be justified, since the deviation from
balanced growth may be small.
• Culture response to large or rapid perturbations cannot be described satisfactorily
by unstructured models.

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 Ideal Batch Reactor
• Assumption: well-mixed system
𝑑
(culture vol)(molar conc. of component 𝑖) =
𝑑𝑡
moles 𝑖 formed by reaction
(culture vol)( )
unit culture vol×unit time
𝑑
𝑉𝑅 𝑐𝑖 = 𝑉𝑅 × 𝑟𝑓𝑖 [𝑉𝑅 is constant]
𝑑𝑡

𝑑
𝑐𝑖 = 𝑟𝑓𝑖
𝑑𝑡
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Ideal Continuous-Flow Stirred-Tank Reactor
(CSTR)

Steady state

Chemostats
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 Ideal Continuous-Flow Stirred-Tank Reactor
(CSTR)
Rate of addition to reactor - Rate of removal from reactor +
Rate of formation within reactor = 0
𝐹 𝑐𝑖𝑓 − 𝑐𝑖 + 𝑉𝑅 × 𝑟𝑓𝑖 =0
𝐹
𝑟𝑓𝑖 = 𝑐𝑖 − 𝑐𝑖𝑓 =D 𝑐𝑖 − 𝑐𝑖𝑓
𝑉𝑅
𝐹
Dilution rate, 𝐷 =
𝑉𝑅

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 Ideal Batch and Continuous Reactor
𝑑
𝑐𝑖 = 𝑟𝑓𝑖 (Batch) 𝑟𝑓𝑖 = D 𝑐𝑖 − 𝑐𝑖𝑓 (CSTR)
𝑑𝑡
• Time dependent concentration data are • Kinetics determinations are
needed straightforward
• Transient • Steady state
• Unbalanced growth • A state of balanced growth
• Not well defined state of cells
• Reproducible state of cells
• Conducted in flasks in a shaker
incubator • A sophisticated bioreactor is needed
• Quick process
• Achieving steady state takes hrs to days
• Less susceptible to contamination • More susceptible to contamination

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 Kinetic models
• Non structured Nor Segregated models
– Growth Models
– Models describing both growth and substrate uptake
– Models describing growth, substrate uptake and product generation
• Structured but Non Segregated
– Cell Models
– Metabolic Models
– Chemically Structured Models
• Non structured but Segregated
– Filamentous microorganisms
– Mixed culture

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 Kinetics of balanced growth
• Net rate of cell mass growth, 𝑟𝑥 = 𝜇 × 𝑥
• For CSTR (𝑟𝑓𝑖 = D 𝑐𝑖 − 𝑐𝑖𝑓 ):
• 𝑟𝑥 = 𝜇 𝑥 = D 𝑥 − 𝑥𝑓
D𝑥𝑓 = 𝑥 𝐷 − 𝜇
In the continuous culture: 𝑥𝑓 = 0
𝐷=𝜇

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 Monod Growth Kinetics
𝜇 = 𝑓 𝑥, 𝑠
• 𝑠 is the concentration of growth limiting substrate
𝜇𝑚𝑎𝑥 𝑠
𝜇=
𝐾𝑠 + 𝑠

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 Contd…
• Steady state mass balance on substrate:
𝑟𝑓𝑐 𝜇𝑥
• 𝑟𝑓𝑠 =D 𝑠 − 𝑠𝑓 [𝑟𝑓𝑠 = = ]
𝑌𝑥/𝑠 𝑌𝑥/𝑠
𝜇𝑥
• D 𝑠𝑓 − 𝑠 − =0
𝑌𝑥/𝑠
𝜇𝑚𝑎𝑥 𝑠𝑥
D 𝑠𝑓 − 𝑠 − =0
𝑌𝑥/𝑠 (𝐾𝑠 + 𝑠)
• Steady state mass balance on cells:
𝜇𝑚𝑎𝑥 𝑠
𝑥 − 𝐷 + D𝑥𝑓 = 0
𝐾𝑠 +𝑠

Monod chemostat model 15

 Contd…
• In the case of sterile feed: 𝑥𝑓 = 0
𝐷𝐾𝑠
𝑥𝑠𝑡𝑒𝑟𝑖𝑙𝑒 𝑓𝑒𝑒𝑑 = 𝑌𝑥/𝑠 𝑠𝑓 −
𝜇𝑚𝑎𝑥 − 𝐷
𝐷𝐾𝑠
𝑠𝑠𝑡𝑒𝑟𝑖𝑙𝑒 𝑓𝑒𝑒𝑑 =
𝜇𝑚𝑎𝑥 − 𝐷

For very slow slows at a given volume, D → 0

𝑥 = 𝑠𝑓 𝑌𝑥/𝑠
For D increases and D → 𝜇𝑚𝑎𝑥
𝑠𝑓 𝜇𝑚𝑎𝑥
𝐷𝑚𝑎𝑥 =
𝐾𝑠 + 𝑠𝑓

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Effect of dilution rate

𝐷𝐾𝑠
𝑥𝑆𝑡𝑒𝑟𝑖𝑙𝑒 𝑓𝑒𝑒𝑑 = 𝑌𝑥/𝑠 𝑠𝑓 −
𝜇𝑚𝑎𝑥 − 𝐷
𝐷𝐾𝑠
𝑠𝑆𝑡𝑒𝑟𝑖𝑙𝑒 𝑓𝑒𝑒𝑑 =
𝜇𝑚𝑎𝑥 − 𝐷

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 Contd…
• Steady state mass balance on product:
𝜇𝑥
D 𝑝𝑓 − 𝑝 + = 0; or
𝑌𝑥/𝑝
D 𝑝𝑓 − 𝑝 + 𝑌𝑝/𝑥 𝜇𝑥 = 0

𝑌𝑝/𝑥 𝜇𝑥
𝑝 = 𝑝𝑓 +
𝐷

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 Other similar models
𝜇𝑚𝑎𝑥 𝑠
• Monod 𝜇= 1942
𝐾𝑠 +𝑠

𝑠
−
• Tessier 𝜇 = 𝜇𝑚𝑎𝑥 (1 − 𝑒 𝐾𝑠 ) 1936

𝜇𝑚𝑎𝑥 𝑠 𝑛
• Moser 𝜇= 1958
𝐾𝑠 +𝑠 𝑛

𝜇𝑚𝑎𝑥 𝑠
• Contois 𝜇= 1959
𝐾𝑠 𝑋+𝑠

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 Unstructured Batch Growth Models
• Simplest approach to model batch culture

➢Malthus Law
➢Logistic Equation
➢Monod equation

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Contd…

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 Malthus Law
𝑑𝑥
= 𝑟 = 𝜇𝑓(𝑥)
𝑑𝑡
𝑑𝑥
= 𝑟 = 𝜇𝑥
𝑑𝑡
• Growth rate characteristic of exponential growth
• Unable to describe the stationary phase

𝜇𝑡
𝑋 = 𝑋0 𝑒
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 Logistic Equation
𝑑𝑥
= 𝑟 = 𝜇𝑓(𝑥)
𝑑𝑡
𝑑𝑥 𝑥
= 𝑟 = 𝜇𝑥 1 −
𝑑𝑡 𝑥𝑚𝑎𝑥
𝑑𝑥 𝑥
= 𝜇𝑥 1 − 𝛽𝑥 [ = β]
𝑑𝑡 𝑥𝑚𝑎𝑥

𝑋0 𝑒 𝜇𝑡
𝑋=
1 − 𝛽𝑥0 (1 − 𝑒𝜇𝑡 )

✓ Predicts exponential and stationary phases, but does not consider the
influence of the substrate.
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 Monod equation
𝑑𝑥
= 𝑟 = 𝜇𝑓(𝑥)
𝑑𝑡
𝑑𝑥 𝜇𝑚𝑎𝑥 𝑠
= 𝜇𝑥 = 𝑥
𝑑𝑡 𝐾𝑠 + 𝑠
• Hyperbolic kinetics
• Applies under limiting substrate condition

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 Kinetic models
• Non structured Nor Segregated models
– Growth Models
– Models describing both growth and substrate uptake
– Models describing growth, substrate uptake and product generation
• Structured but Non Segregated
– Cell Models
– Metabolic Models
– Chemically Structured Models
• Non structured but Segregated
– Filamentous microorganisms
– Mixed culture

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 Growth of Filamentous Organisms
• Filamentous organisms such as molds often form microbial pellets at high cell densities
in suspension culture and cell growth inside pellets may be subject to diffusion
limitations
• The growth models of molds should include the simultaneous diffusion, and
consumption of nutrients and toxic metabolic by-products within the pellet at large
pellet sizes
• Mass and morphology of molds and other filamentous organisms grow [rich medium]
𝒅𝑹
• = 𝒌𝒈 = 𝐂𝐨𝐧𝐬𝐭𝐚𝐧𝐭
𝒅𝒕
𝟒
• 𝑴 = 𝝆 𝝅𝑹𝟑
𝟑
𝒅𝑴 𝟐 𝒅𝑹
• = 𝝆𝟒𝝅𝑹 = 𝒌𝒈 𝟒𝝆𝝅𝑹𝟐
𝒅𝒕 𝒅𝒕
𝒅𝑴
• = 𝜸𝑴𝟐/𝟑 𝜸 = 𝒌𝒈 (𝟑𝟔𝝆𝝅) 𝟏/𝟑
𝒅𝒕
𝜸𝒕 𝟑
• 𝑴=
𝟏/𝟑
𝑴𝟎 +
𝟑
𝑴 ∝ 𝒕𝟑 26
 Structured Kinetic Models
• Unstructured models do not represent the quality of bio-phase
• When cell population changes significantly and these
composition changes influence kinetics: Structured model
should be used
• The bio-phase variables employed in the structured model are
typically: mass (𝑥𝑗 ) or molar (𝑐𝑗 ) concentrations

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 Contd…
• For a well mixed reactor, material balance on component j
𝑑 1 1 1
• 𝑉𝑅 𝑥𝑐𝑗 = 𝑉𝑅 𝑥𝑟𝑓𝑖 + Φ𝑥 𝑐𝑗
𝑑𝑡 𝜌𝑐 𝜌𝑐 𝜌𝑐
𝑑𝑐𝑗 1 𝑑𝑥 Φ𝑥
• = 𝑟𝑓𝑖 − 𝑐𝑗 + 𝑐𝑗
𝑑𝑡 𝑥 𝑑𝑡 𝑥𝑉𝑅
𝑑𝑐𝑗
= 𝑟𝑓𝑖 − 𝜇𝑐𝑗 [Φ𝑥 =0 for a batch reactor]
𝑑𝑡
𝝆𝒄 = mass density of cells
𝑟𝑓𝑖 = molar rate of formation of component j
𝜱𝒙 = mass of cell added to the reactor with feed flow
𝒙 = cell mass concentration
𝒄𝒋 = Moles j per unit volume
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 Compartmental models
• The biomass is compartmentalized into small number of
components. For example:
• Synthetic (RNA) and structural (DNA, proteins) components
• Assimilatory and synthetic components
• Based on the system dynamics: molecular collisions, chemical
reactions, diffusion, turnover of RNA, protein synthesis,
increase in the cell number, completion of batch process,
spontaneous mutation

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 Contd…
• Williams two–compartments model
1. A synthetic pool (“K-compartment”): RNA and Small
metabolites
2. A genetic pool (“G-compartment”): DNA and Proteins
𝒌𝟏 𝒌𝟐
S K G

𝑑𝑠 1 𝝆𝟏 + 𝝆𝟐 = 𝝆𝒄 = 𝐂𝐨𝐧𝐬𝐭𝐚𝐧𝐭
• = − 𝑘1 𝑠𝑥
𝑑𝑡 𝑌
𝑑𝑥
• = 𝑘1 𝑠𝑥 [𝑘1 𝑠 = 𝜇] : Follow logistic equation
𝑑𝑡

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 Contd…
• Mass balance on G:
𝑑𝜌2 𝑋
• = 𝑘2 (𝜌2 𝑋)𝜌1
𝑑𝑡
𝑑𝜌2 1 𝑑𝑋
• + 𝜌2 = 𝑘2 (𝜌2 )𝜌1
𝑑𝑡 𝑋 𝑑𝑡
𝑑𝜌2
= 𝑘2 𝜌1 𝜌2 − 𝜇𝜌2
𝑑𝑡
𝑑𝜌2
• = 𝑘2 𝜌1 𝜌2 − 𝑘1 𝑠𝜌2 [𝑘1 𝑠 = 𝜇]
𝑑𝑡

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 Contd…
• Mass balance on K:
𝑑𝜌1 𝑋
• = 𝑘1 𝑠𝜌𝑐 𝑥 − 𝑘2 𝜌1 𝜌2 x
𝑑𝑡
𝑑𝜌1 1 𝑑𝑋 𝝆𝟏 + 𝝆𝟐 = 𝝆𝒄
• + 𝜌1 = 𝑘1 𝑠 𝜌𝑐 − 𝑘2 𝜌1 𝜌2
𝑑𝑡 𝑋 𝑑𝑡
𝑑𝜌1
= 𝑘1 𝑠 𝜌1 + 𝜌2 − 𝑘2 𝜌1 𝜌2 - 𝜇𝜌1
𝑑𝑡
𝑑𝜌1
• = 𝑘1 𝑠 𝜌1 + 𝜌2 − 𝑘2 𝜌1 𝜌2 -𝑘1 𝑠𝜌1
𝑑𝑡 [𝑘1 𝑠 = 𝜇]
𝑑𝜌1
= 𝑘1 𝑠 𝜌2 − 𝑘2 𝜌1 𝜌2
𝑑𝑡
𝒅𝝆𝟏 𝒅𝝆𝟐 𝑑𝜌2
=− = 𝑘2 𝜌1 𝜌2 − 𝜇𝜌2
𝒅𝒕 𝒅𝒕 𝑑𝑡
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 Contd…
• Cell number:
𝜌1 𝑥
• 𝑁∝
𝜌𝑐
𝜌1 𝑥
𝑁 = 𝑘3
𝜌𝑐

• Cell volume:
(𝜌1 +𝜌2 )
• 𝑉∝
𝜌1
𝜌𝑐
𝑉𝑐𝑒𝑙𝑙 = 𝑘4
𝜌1

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