SEM 1 2012/13

CHAPTER 3:
KINETICS OF GROWTH
IN BATCH AND
CONTINOUS CULTURE

ERT 317 BIOCHEMICAL ENGINEERING

Lecture Outline

Introduction
Batch Growth Characteristics
Growth

Stages, Effects of Environmental Conditions,

Product Formation, Mathematical Models

Continuous Growth Characteristics

Dilution

Rate, Optimum Operation

Cell growth
Substrate Cells Extracellu lar Products More Cells

S X P nX

Microbial growth is an autocatalytic reaction

The

rate of growth is directly related to cell concentration

Characterized by the net specific growth rate:

net

1 dX

X dt

Cell growth

The net specific growth rate is the difference between

a gross specific growth rate (g, h-1) and the rate of
loss of cell mass due to cell death (kd, h-1):

net g kd

Microbial growth can also be described in terms of

cell number, N:

1 dN
R
N dt

where R is the net specific replication rate (h-1)

Batch Growth Determining Cell

Number Density

Hemocytometer

Agar plates

Direct microscopic count

Counts all cells present (viable and non-viable)
Immediate result
Counts only living cells
Delayed result
Assumption: each viable cell will yield 1 colony
Results expressed in CFUs(colony-forming units)

Particle counters

Counts all cells present (viable and non-viable)

Suitable for discrete cells in a particulate-free medium
Can distinguish between cells of different sizes

Hemocytometer

Viable Cell Count

Coulter Particle Counter

Determining Cell Mass Concentration Direct

Methods

Dry cell weight (DCW)

A

sample of fermentation broth is centrifuged, washed,

and dried at 80C 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

Batch Growth

Batch Growth Curve

Growth Phases
1. Lag
2. Exponential
3. Deceleration
4. Stationary
5. Death/Decline

Lag Phase

Occurs immediately after inoculation and is a

period of adaptation for the cells to their new
environment
New

enzymes are synthesized, synthesis of other

enzymes is repressed
Intracellular machinery adapts to the new conditions
May be a slight increase in cell mass and volume, but
no increase in cell number
The lag phase can be shortened by high inoculum
volume, good inoculum condition (high % of living cells),
age of inoculum, nutrient-rich medium

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 nett
X0

Exponential Growth Phase (contd)

An important parameter in the exponential phase is

the doubling time (time required to double the
microbial mass)
A graph of ln X versus t produces a straight line on a
semi-logarithmic plot:
ln 2 0.693
d

max

max

The doubling time based on cell number is expressed

as:
ln 2
'
d

Exponential Growth Phase (cont)

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 (contd)

During this phase, one or more of the following

phenomena may occur:
Total

cell mass concentration may stay constant, but the

number of viable cells may decrease
Cell lysis may occur, and viable cell mass may drop. A
second growth phase may occur as cells grow on lysis
products from the dead cells (cryptic growth)
Cells may not be growing, but may have active
metabolism to produce secondary metabolites

Stationary Phase (contd)

During the stationary phase, the cell catabolizes cellular

reserves for new building blocks and for energyproducing 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
kd t
kd t or X X SOe
dt

Death Phase

The death or decline phase is characterized by the

expression:
dN
k d' t
'
kd t 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 ln N versus t yields a line of slope kd

Death Phase
1.
2.

Cell lysis (spillage) may occur

Rate of cell decline is first-order

where:

3.

kd = 1st order death rate constant,

Xs = conc. of cell at end of stationary phase

Growth can be re-established by transferring to fresh media

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:

YX / 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 S maintence

into biomass

into an
extracellular
product

energy

energy

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 S

Yield Coefficients

Yield coefficients can also be defined for other

substrates or for product formation:

YX / O2
YP / S

O2

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

The Maintenance Coefficient

The maintenance coefficient is used to describe the specific

rate of substrate uptake for cellular maintenance:

dS / dt m
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, g
Note that g is not equal to net, 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
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:

q p constant

Many secondary metabolites, such as most

antibiotics (e.g. penicillin), are non-growthassociated 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:

q p g

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

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) Non-growth-associated product formation
c)

Mixed-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
Psychrophiles: (Topt< 20C)
Mesophiles: (20C < Topt< 50C)
Thermophiles: (Topt> 50oC)

As the temperature increases towards Topt, the growth

rate doubles every ~10C

Optimum Growth Temperature

Optimum Growth Temperature

Effect of Temperature on Cell Growth

Above Topt the growth rate decreases and thermal death

may occur

The net specific replication rate for temperatures above Topt is

dN
expressed by:
'
'

dt

R kd N

Both and k vary with temperature according to

the Arrhenius equation:
'
R

'
d

Ae
'
R

Ea / RT

k Ae
'
d

Ea / RT

Where:
Ea =activation energy for growth 10-20 kcal/mol
Ed =activation energy for death 60-80 kcal/mol

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 pHs 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+, NO3consumes 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

Saturation kinetics

Facultative aerobic cells

Saturation kinetics

Effect of Dissolved O2 on Cell Growth

The saturated DO concentration for water at 25C

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 moulds,
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

Heat Generation by Growth

About 40% to 50% of the energy stored in a carbon and energy

source is converted to biological energy (ATP) during aerobic
metabolism, and the rest of the energy is released as heat

For actively growing cells, the maintenance requirement is low, and heat
evolution is directly related to growth
The heat of combustion of the substrate is equal to the sum of the metabolic
heat and the heat of combustion of the cellular material:

Where HS is the heat of combustion of the substrate (kJ/g substrate), HC

is the heat of combustion of cells, and 1/YH is the metabolic heat evolved
per gram of cell mass produced (kJ/g cells)

Energy Balance on Microbial Utilization

of Substrate

Heat Generation by Growth

The above equation in heat generation can be

rearranged to become:

HS and HC can be determined from the combustion of

substrate and cells
Typical HC values for bacterial cells are 20-25 kJ/g cells
Typical values of YH are: glucose, 0.4 g/kcal; malate, 0.3
g/kcal; acetate, 0.21 g/kcal; ethanol, 0.18 g/kcal; methanol,
0.12 g/kcal; and methane, 0.061 g/kcal
Clearly, the degree of oxidation of the substrate has a strong
effect on the amount of heat released

Heat Generation by Growth (cont)

For substrates:
Substrate, S

Hs (kJ/g S)

YH (g dcw/kJ)

Glucose

15.64

0.072

Methanol

22.68

0.029

Ethanol

29.67

0.043

n-Decane

47.64

0.038

Methane

55.51

0.015

The oxidation state of S has a large effect on 1/ YH

Rate of Heat Generation by Growth,

Q
Gr

The total rate of heat evolution in a batch

fermentation is:

where: VL = liquid volume

In aerobic fermentations, the rate of metabolic heat

evolution can roughly be correlated to the rate of
oxygen uptake:
where:

QGR is in kcal/h, and QO2 is in mM of O2/h

Heat Generation by Microbial Growth

Metabolic heat released during a fermentation can

be removed by circulating cooling water through a
cooling coil within the fermenter, or a cooling jacket
surrounding the fermenter
Temperature control is a critical limitation on reactor
design
The ability to estimate heat removal is essential to
proper reactor design

Cooling coils and Water Jacketed Fermenter