Chapter 4: How Cells Grow

Prof. Zakaria Al-Qodah

Department of Chemical Engineering

School of Engineering

2nd Semester (2019-2020)

Presentation Outline: Lectures 5-8
❖ Introduction

❖ Batch Growth Characteristics

▪ Growth Stages
▪ Effects of Environmental Conditions
▪ Product Formation
▪ Mathematical Models

❖ Continuous Growth Characteristics

❖ Dilution Rate, Optimum Operation
Introduction
Cells can be used as catalyst in three modes

❖ Dead Cells

❖ Viable non growing cells

❖ Whole viable cells

❖ Cellular growth is the primary response of viable cells to

physiochemical environment including substrates and
nutrients.

❖ Product formation is a secondary response

 Introduction

Growing organisms extract nutrients from the medium then

convert them into the following

❖ biological compounds inside the cell.

❖ Energy needed for cellular activity

❖ Biosynthesis and product formation.

❖ Maintenance of cells bio compounds

substrates + cells extracellular products + more cells

S + X →  P + nX (6.1)
 Growth Rate

Two types of growth rates:

❖Net specific growth rate described as:

1 dX
 net = (6.2a)
X dt
net =  g − kd (6.2b)

❖ Microbial growth can also be described in terms of cell number

concentration, N, as well as X. In that case:

1 dN
R =
N dt (6.3)
 Batch Growth

Batch growth: culturing cells in a vessel with an initial charge of

medium with no further nutrient addition or removal.

Quantifying Cell Concentration in a culture medium is essential for the

determination of the kinetics and stoichiometry of microbial growth.
This can be done by:

❖ Direct methods which could be not accurate due to the presence of

suspended solids or other interfering compounds in the medium.

❖ Indirect methods measurement of substrate consumption and/or

product formation during the course of growth
Determining Cell Concentration

1.Cell number concentration

▪ Hemocytometer (Petroff-Hausser slide)

▪ Viable cell counts (petri dish)

▪ Electronic particle counter

A Petroff–Hausser slide or a hemocytometer

Used for direct cell counting

❖ A calibrated grid is placed over the culture chamber,

❖ The number of cells per grid square is counted using a

microscope.

❖ To be statistically reliable, at least 20 grid squares must

be counted and averaged.
Hemocytometer
Viable cell counts (petri dish)
The word viable used in this context means capable of
reproduction
❖ Culture samples are diluted and spread on the agar
surface and the plates are incubated.

❖ Colonies are counted on the agar surface following the

incubation period.

❖ The results are expressed in terms of colony-forming

units (CFU).
❖ If cells form aggregates, then a single colony may not
be formed from a single cell.
❖ This method is more suitable for bacteria and yeasts
and much less suitable for molds.
Viable Cell Count
Electronic particle counter

This method is based on the relatively high electrical

resistance of cells
▪ particle counters employ two electrodes and an
electrolyte solution.
▪ One electrode is placed in a tube containing an orifice.
▪ A vacuum is applied to the inner tube, which causes an
electrolyte solution containing the cells to be sucked
through the orifice.
▪ As cells pass through the orifice, the electrical resistance
increases and causes pulses in electrical voltage.
▪ The number of pulses is a measure of the number of
particles;
Electronic particle counter
 Determining Cell Concentration (cont.)

2- Cell mass concentration

❑ Direct methods

❖ Dry weight (filtration or centrifugation)

❖ Packed cell volume (centrifugation)

❖ Optical density (light scattering, 600-700 nm)

Determining Cell Mass Concentration –
Direct Methods

❖ Dry cell weight (DCW)

▪ A sample of fermentation broth is centrifuged, washed,
and dried at 80°C for 24hrs
▪ Off-line measurement; wet cell weights (WCW) can
performed in-process
❖ Packed cell volume
• Like wet cell weight, but measures cell pellet volume

❖ Optical density (OD)

▪ Turbidity –based on the absorption of light by
suspended cells in culture media
Determining Cell Mass Concentration –
Indirect Methods

❖ In many fermentation processes, particularly with moulds,

direct methods cannot be used
❖ Indirect methods are therefore employed, based on the
measurement of substrate consumption and/or product
formation
▪ Intracellular components of cells such as RNA, DNA
and protein can be measured as indirect indicators of
cell growth
▪ Concentration of RNA/cell weight varies significantly
during a batch growth cycle, while DNA and protein
concentrations per cell weight remain fairly constant,
and can therefore be used as reasonable measures of
cell growth
Time-Dependent Changes in Cell
Composition and Cell Size

Azotobacter vinelandii Growth in Batch Culture

Determining Cell Mass Concentration – Indirect Methods

Measure biomolecule concentration and correlate to dry cell mass

concentration. (DNA, protein, ATP, NADH, product formation)

Example 1: NH4+ utilization during growth releases H+, amount of

OH- added is proportional to growth

Example 2: For a given organism, the intracellular ATP concentration

(mg ATP/mg cells) is approximately constant. Thus, as biomass
concentration increases in a fermentation broth the ATP concentration
increases.

The method is based on luciferase activity, which catalyzes oxidation of

luciferin at the expense of oxygen and ATP with the emission of light.

(6.4)
Batch Growth Curve

X = cell
Concentration
(cells/mL)
Growth Phases

1. Lag

2. Exponential

3. Deceleration

4. Stationary

5. Death/Decline
 Lag Phase

❖ No increase in cell numbers

❖ Induction of enzymes to utilized substrate(s)

❖ Very important to decrease lag period to increase productivity

▪ Inoculate with exponential phase cells

▪ Pre-acclimate inoculum in growth media

▪ Use high cell inoculum size (5-10% by volume)

Influence of [Mg2+] on Lag Phase Duration in E.
aerogenes Culture
❖ E. aerogenes requires Mg2+ to
activate the enzyme
phosphatase, which is required
for energy generation by the
organism

❖ The concentration of Mg2+ in the

medium is indirectly proportional
to the duration of the lag phase
 Exponential Growth Phase

 In this phase, the cells have adjusted to their new

environment
➢ At this point the cells multiply rapidly (exponentially)
➢ Balanced growth –all components of a cell grow at
the same rate
➢ Growth rate is independent of nutrient concentration,
as nutrients are in excess
➢ The first order exponential growth rate expression is:
dX
=  net X where X = X 0 at t = 0
dt
X
ln =  nett or X = X 0 e net t
X0
 Exponential Growth Phase (cont’d)

 An important parameter in the exponential phase is the

doubling time (time required to double the microbial
mass)
 A graph of lnX versus t produces a straight line on a
semi-logarithmic plot:

ln 2 0.693
d = =
max max
 Thedoubling time based on cell number isexpressed
as: ln 2
 d' =
R
Exponential Growth Phase (cont…)

t
 Deceleration Phase

❑ Very short phase, during which growth decelerates due to

either:
➢ Depletion of one or more essential nutrients, or,

➢ The accumulation of toxic by-products of growth (e.g.

Ethanol in yeast fermentations)

❑ Period of unbalanced growth: td=td’

❑ Cells undergo internal restructuring to increase their
chances of survival
❑ Followed quickly by the Stationary Phase
 Stationary Phase

❖ Starts at the end of the Deceleration Phase, when the net

growth rate is zero (no cell division, or growth rate is equal to
death rate)

❖ Cells are still metabolically active, and can produce

secondary metabolites
➢ Primary metabolites are growth-related products, while
secondary metabolites are non-growth-related
➢ Many antibiotics and some hormones are produced as
secondary metabolites
➢ Secondary metabolites are produced as a result of
metabolite deregulation
 Stationary Phase (cont’d)

 During the stationary phase, the cell catabolizes cellular

reserves for new building blocks and for energy-
producing monomers
This is called endogenous metabolism

 The cell must expend maintenance energy in order to stay

alive
The equation that describes the conversion of cellular
mass into energy, or the loss of cell mass due to lysis
during the stationary phase is:
dX −k d t
= −k d X or X = X SOe
dt
 Death Phase

 The death or decline phase is characterized by the

expression:
dN −k d' t
= −k d N or N = N S e
'

dt

 Where Ns is the concentration of cells at the end of the

stationary phase, and is the first-order death-rate
constant

 A plot of lnN versus t yields a line of slope –kd’

Death Phase
1. Cell lysis (spillage) may occur

2. Rate of cell decline is first-order

dX − kd t
dt
= −k d X or X = X oe (6.9)
X
ln = −k d t
Xo

3. Growth can be re-established by transferring to fresh

media
4. There is a distribution of the properties among the
individuals in the population
 Yield Coefficients
❑ Growth kinetics are generally further described by defining
stoichiometrically related parameters
❑ Yield coefficients are defined based on the amount of
consumption of a given material
❑ For example, the growth yield coefficient is:
X
YX / S =−
S
❑For organisms growing aerobically on glucose, Yx/s is
typically 0.4 to 0.6 g/g, for most yeast and bacteria; anaerobic
growth is much less efficient
Aerobic and Anerobic Growth Yields of S..faecalis
on Glucose
 Yield Coefficients

 At the end of a batch growth period, there is an

apparent or observed growth yield:
S = Sassimilation + Sassimilation + Sgrowth + Smaintence
into biomass into an energy energy
extracellular
product

 The apparent yield is not a true constant for compounds that

can be used as both a carbon and energy source, but the true
growth yield (YX/S) is constant ΔS
 Yield Coefficients
 Yield coefficients can also be defined for other
substrates or for product formation:
X
YX / O2 =−
O2
P
YP / S =−
S
 YX/O2 is typically 0.9 to 1.4 g/g for most yeast and
bacteria, but is much lower for highly reduced
substrates (e.g. methane, CH4)
Summary of Yield Factors for Aerobic Growth
 TheMaintenance Coefficient

The maintenance coefficient is used to describe the specific rate of

substrate
: uptake for cellular maintenance

m−
 dS / dtm
X
❖ However, during the Stationary Phase, where little external substrate
is available, endogenous metabolism of biomass components is
used for maintenance energy

❖ Maintenance energy is the energy required to repair damaged

cellular components, to transfer nutrients and products in and out of
cells, for motility, and to adjust the osmolarity of the cells’ interior
volume
 Microbial Products

 Microbial products can be classified into three

major categories
Growth-associated products
Non-growth-associated products
Mixed-growth-associated products
 Growth-associated products
❖ These products are produced simultaneously with
microbial growth
❖ Specific rate of product formation is proportional to
the specific growth rate, μg
❖ Note that μg is not equal to μnet, the net specific
growth rate, when endogenous metabolism is
occurring
Growth-Associated Products
 The rate expression for product formation in growth-associated
production is:

1 dP
qp = = YP / X  g (6.16)
X dt

 Where qp is the rate of product formation (h-1)

 The production of a constitutive (continuously produced, as
opposed to inducible) enzyme is an example of a growth-
associated product
Non-Growth-Associated Products

 Non-growth-associated product formation takes place

during the Stationary Phase, when the growth rate is zero

 Specific rate of product formation is constant:

qp =  = constant

 Many secondary metabolites, such as most antibiotics

(e.g. penicillin), are non-growth- associated products
 Mixed-Growth-Associated Products

 Mixed-growth-associated product formation takes place

during the Deceleration (slow growth) and Stationary
Phases
 The specific rate of product formation is given by the
Luedeking-Piret equation:
qp = g + 

 If α= 0, the product is completely non-growth associated; If

β= 0, the product is completely growth-associated

 Examples: lactic acid fermentation, production of xanthan

gum, some secondary metabolites
Product Yield Coefficients (cont…)

a) Growth-associated product formation

b) Mixed-growth-associated product formation
c) Non-growth-associated product formation
 Environmental Factors
 Patterns of microbial growth and product formation are
influenced by environmental factors such as temperature, pH
and dissolved oxygen concentration (D.O.)
 Microorganisms can be classified by their optimum growth
temperatures, Topt
i. Psychrophiles: (Topt< 20°C)
ii. Mesophiles: (20°C < Topt< 50°C)
iii. Thermophiles: (Topt> 50o°C)

 As the temperature increases towards Topt, the growth rate

doubles every ~10°C
Optimum Growth Temperature
Optimum Growth Temperature
 Effect of Temperature on CellGrowth
 Above Topt the growth rate decreases and thermal
death may occur
The net specific replication rate for temperatures above Toptsi
= (R − k d ) N
expressed by: dN ' '

Both  and k
' '
dt
 R d vary with temperature according to
the Arrhenius equation:
 ='
R
Ae −Ea / RT
k d' = −Ae−Ed / RT
Where:
 Ea =activation energy for growth ≈ 10-20

kcal/mol
 Ed =activation energy for death ≈ 60-80
Arrhenius Plot of Growth Rate of E.Coli

Legend:
(●) Growth on rich,
complex medium

(○) Growth on glucose-

mineral salts medium
 Effect of pH on Cell Growth
➢ pH affects the activity of enzymes, and therefore the
microbial growth rate
➢ Acceptable pH’s for growth are typically within 1 or 2
pH units of the optimum pH
➢ pH range varies by organism:
❖ bacteria (most) pH = 3 to 8

❖ yeast pH = 3 to 6

❖ plants pH = 5 to 6

❖ animals pH = 6.5 to 7.5

 Effect of pH on Cell Growth
The optimal pH for growth may be different from the
optimal pH for product formation (e.g. Pichia
pastoris)

Microorganism have the ability to control pH inside

the cell, but this requires maintenance energy

pH can change due to:

 Utilization of substrates; NH4+ releases H+, NO3-
consumes H+
Production of organic acids, amino acids, CO2, bases
Effect of pH on Cell Growth (cont…)
 Effect of Dissolved O2 on Cell Growth

 At high cell concentrations, the rate of oxygen consumption

may exceed the rate of O2 supply

 When oxygen is the rate-limiting factor, specific growth

rate varies with [DO] according to saturation (Michaelis-
Menten) kinetics

 Below a critical concentration, growth approaches a first-

order rate dependence on DO (oxygen is a limiting
substrate)
 Above a critical concentration, the growth rate becomes
independent of DO (oxygen is non-limiting))
Effect of Dissolved O2 on Cell Growth (cont…)

Obligate aerobic cells Facultative aerobic cells

Saturation kinetics Saturation kinetics

 Effect of Dissolved O2 on Cell Growth

 The saturated DO concentration for water at 25°C and 1

atm is ~7 ppm

The presence of dissolved salts and organics can alter

the saturation value
Increasing temperatures decrease the saturation value

 The critical oxygen concentration is about 5%-10% of the

saturated DO concentration for bacteria and yeast, and
about 10%-50% of [DO]sat for molds, since they grow as
large spheres in suspended culture (diffusion issues)
 Other Effects on Cell Growth
 Dissolved CO2 can have a profound effect on the performance
of microorganisms

❖ Very high DCO2 concentrations can be toxic to some cells

❖ On the other hand, cells require a certain minimum DCO2 level for
proper metabolic function

Ionic strength (I); too high dissolved salts is inhibitory to

membrane function (membrane transport of nutrients, osmotic
pressure):

Where: Ci = molar concentration of ion i Zi = ion charge

 Other Effects on Cell Growth
 The redox potential is an important parameter that affects the rate and
extent of many oxidative-reductive reactions
In fermentation media, the redox potential is a complex
function of DO, pH, and other ion concentrations, such as
reducing and oxidizing agents

 Substrate concentrations significantly above stoichiometric

requirements are inhibitory to cellular functions
➢ Inhibitory levels of substrates vary depending on cell type and
substrate
➢ Typical maximum non-inhibitory concentrations of some
nutrients are –glucose, 100 g/l; ethanol, 50 g/l for yeast, much
less for other organisms; ammonium, 5 g/l; phosphate, 10 g/l;
nitrate, 5 g/l
Cooling coils and Water Jacketed Fermenter
 Modeling Cell Growth
➢ Structured Models:

Model divides cell mass into components (by molecule or by element)

and predicts how these components change as a result of growth. These
models are very complex and not used very often.

➢ Unstructured Models:

Model assumes balanced growth where cell components do not change

with time. Much less complex and much more commonly used. Valid
for batch growth during exponential growth stage and also for
continuous culture during steady-state operation.
 Monod Equation
Similar to Michaelis-Menten Kinetics
➢ Assumes that a single enzyme system is responsible for the uptake
of substrate (S), and that S is limited (growth-dependent variable).

➢ This is the most common kinetic model for cell growth.

m S
➢ g = (6.30)
Ks + S

• μg = specific cell growth rate (hr-11)

• μm = maximum specific cell growth
rate (hr-1),
• S = substrate concentration (g/L)
•Ks = Saturation constant (g/L) = S when μ= 1/2 μ
 The Logistic Equation
❖ When plotted on arithmetic paper, the batch growth curve assumes a
sigmoidal shape (Fig. 6.3)

❖ This shape can be predicted by combining the Monod equation

(6.30) with the growth equation (6.2) and an equation for the yield
of cell mass based on substrate consumption.
1 dX m S dX m S
g = = →→ = X (6.50)
X dt Ks + s dt Ks + s
❖ The relationship between microbial growth yield and substrate
oX − X = Y (S − S ) →→→ X = X + Y (S − S )
X /S o o X /S o
consumption is
Y S + Xo − X (6.51)
X − X o = YX / S ( So − S ) →→→ S = X / S o
YX / S
Substituting: dX m (YX / S ( So + X 0 − X )
❖ = X (6.52)
dt K sYX / S + YX / S ( So + X 0 − X
 Batch Culture Growth Model (cont.)
❖ When plotted on arithmetic paper, the batch growth curve assumes a
sigmoidal shape (Fig. 6.3)

❖ This shape can be predicted by combining the Monod equation

(6.30) with the growth equation (6.2) and an equation for the yield
of cell mass based on substrate consumption.
1 dX m S dX m S
g = = →→ = X (6.50)
X dt Ks + s dt Ks + s
❖ The relationship between microbial growth yield and substrate
oX − X = Y (S − S ) →→→ X = X + Y (S − S )
X /S o o X /S o
consumption is
Y S + Xo − X (6.51)
X − X o = YX / S ( So − S ) →→→ S = X / S o
YX / S
Substituting: dX m (YX / S ( So + X 0 − X )
❖ = X (6.52)
dt K sYX / S + YX / S ( So + X 0 − X
 Batch Culture Growth Model (cont.)
❖ The integrated form of the rate expression in this phase is:
 Monod Equation Parameters
❖ Though the Logistic Equation qualitatively predicts the shape of
batch growth, it is not very useful when attempting to determine KS
and μmax from X versus time data.

❖ Generally, Ks = S when μg = ½ μmax

❖ μg = μmax for S >> Ks

❖ and μg = μmax /Ks)S for S < < Ks.

Kinetic of Growth
in Continuous
Culture
Advantages of Continuous Culture
Advantages of Continuous Culture
Cell growth in Continuous
Culture (Schematic of
Chemostat)
Chemostat as Tool
Chemostat Mass Balance
Ideal Constant- Stirred tank Reactor (Chemostat)
Ideal Constant- Stirred tank Reactor (Turbidostat)
MASS BALANCE STATEMENT FOR CELL
MASS
Steady State and Sterile Feed
Substrate Concentration
Bioreactor Washout Condition
With Endogenous Metabolism
Cell Concentration
Cell concentration (cont.)
Effect of Endogenous Metabolism
Measurement of Maximum Cell Yield and
Maintenance a Chemostat
Using a Chemostat to Determine µmax and KS
Productivity of a Chemostat
Product Mass Balance
 Substrate Mass Balance

At SS, and re-arranging:

 Product Mass Balance

μ = D + k d then

Then mass balance equation becomes:

FSo – FS – V (D +kd) X

At SS and rearranging:
The Chemostat as a tool

➢ The chemostat can be used as a tool to study the mutation and

selection of cultures

➢ To study the effect of changes in the environment on cell

physiology

➢ Natural or induced mutations can take place in a chemostat

culture.

➢ A chemostat culture can be used for the selection of special

organisms (Thermophiles, Acidophiles).

➢ Selection or enrichment nutrient media need to be used for this

purpose
Lnk2=lnA-Ea/RT
Deviation from Ideality

➢ fermentation broth may be highly viscous and it may be difficult

to maintain complete mixing.

➢ Also, certain cells tend to grow in the form of a film on solid

surfaces in fermenters, such as on fermenter walls or probe
surfaces

➢ The term “chemostat” is actually applicable only to perfectly

mixed vessels.

➢ Here we denote non-ideality by putting chemostat in quotation

➢ marks.
➢ A segregated reactor model can be used to analyze an
incompletely mixed continuous-flow fermenter.
lnk2=lnA-Ea/RT