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Microbial Growth Kinetics

 Fermentation (more details in Chapter 6) may be

carried out as batch, fed-batch and continuous
processes.
 The mode of operation is to a large extent, dictated by
the type of product being produced.

CHAPTER 3
Microbial Growth Kinetics
By
Dr. Law Jeng Yih

Batch Culture Batch Culture

 Microbial cells use nutrients for growth, energy production and

product formation as indicated in the following expression;
Nutrients + microbial cells --> cell growth + energy + reaction products
 According the batch system shown in Figure 1. This container
initially contains a known growth substrate concentration S. The
container is well mixed and therefore the dissolved oxygen
concentration O2 does not become a limiting factor for microbial
growth.
 Initially a known concentration X of viable microbial cells (i.e.
inoculum) is added to the container and, with time, growth
substrate S is utilized for cell growth.
 We therefore over time will observe a decrease in S (negative
dS/dt) and a corresponding increase in X (positive dX/dt).

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Batch Culture Batch Culture

 Batch culture is a closed system in broth medium in

which no additional nutrient is added after inoculation
of the broth.
 Typically, a batch culture passes through four distinct
stages:
 Lag stage
 Logarithmic (exponential) growth
 Stationary stage
 Death stage

Batch Culture Batch Culture

1. Lag phase – “flat” period of adjustment, 3. Stationary phase – rate of cell growth equals
enlargement; little growth, synthesizes rate of cell death cause by depleted nutrients
enzymes & essential constituents, repairs any & O2, excretion of organic acids & pollutants
lesions from earlier injury e.g. freezing, drying,
heating; no cell-division occur. (waste & inhibitory products accumulate)

2. Exponential growth phase – a period of 4. Death phase – cells begin to die at a more
maximum growth will continue as long as cells rapid rate than that of reproduction;
have adequate nutrients & a favorable sometimes accompanied by cell-lysis;
environment; generation time (or time to exponential decline phase
doubling cell-number) is constant

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Exponential Growth Phase Exponential Growth Phase

 substrate and nutrients are abundant
Growth:
• Increase in number of cells  growth rate : proportional to the number of microorganisms
• Increase in microbial mass
 The exponential growth phase can be described as follows:
Specific growth rate (μ):
Change in the number of cells/ Data for a population that doubles every 30 min. dX X = concentration of microorganisms
unit of time  X
dt (microbial biomass) at time t
Generation (n): t = time
dX
Interval between two divisions   dt  = proportionality constant or
X specific growth rate, [time-1]
Generation time / doubling time dX/dt = microbial growth rate,
X
(td): ln( )  t [mass/volume time]
• Time for population to double X X0 = the original biomass concentration
during the exponential phase 0
• Time between two cell- t
divisions X  X e
Data plotted on an arithmetic and a logarithmic scale 0
The rate of growth of a microbial culture

Exponential Growth Phase

Exponential Growth Phase
• Exponential Growth:
 On taking natural logarithms, the equation (previous pg.) • Population doubles per unit of time
5 x 107
becomes: • # mo increases logarithmically:
1 → 2 → 4 → 8 → 16 → 32…2n)
mathematically exponential growth
 Thus, a plot of the natural logarithms of biomass g
concentration against time should yield a straight line, Nt = N0 2n => log Nt = log N0 + n log 2
the slope would equal to .
=> n = 3.3 (log Nt - log N0 )
 During the exponential phase, nutrients are in excess. • Nt = # cells at time t
Hence, the organism is growing at its maximum specific → td = 0.301/0.15 (slope) → td = 2 h
• N0 = # initial cells
growth rate, max.
• n = # of generations during time (t)
Method of estimating the generation
• Generation time (td): times (td) of exponentially growing
• directly from graph populations with generation times of
• derived from n  td = t / n (a) 6 h and (b) 2 h from data plotted
on semi logarithmic graphs.
• from the slope = 0.301/td

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Generation Time What is a Logarithm?

n = (log10Nt – log10No ) / .301  Logarithm is a function that gives the exponent in the
equation bn = x. It is usually written as logb x = n.
 For example:
n = Number of generations
Nt = Final Concentration of Cells
34 = 81 Therefore log3 81 = 4
No = Original concentration of cells
Log10 2 = 0.301

Example 1
Measure Culture at 9:00 a.m.
Measure Culture at 3:00 p.m.
Calculate N
n = (log10Nt – log10No ) / .301
n = (5-4)/0.301
n = 1/0.301
n = 3.33 Generations in 6 Hours
Exponential Growth Phase
10,000 cells / ml
100,000 cells / ml
 The biomass yield factor/coefficient (Y or Yx/s) is a
measure of the efficiency of conversion of substrate into
biomass. It can be used to predict the substrate
concentration required to produce a certain biomass
concentration.
 Y is not a constant. It will vary according to growth rate,
pH, temperature, the limiting substrate, and the substrate
in excess.

We Know: 6 Hours = 360 Minutes Y = mass of cell mass produced

mass of substrate consumed
Therefore: Generation Time = 360 Minutes / 3.33 Generations

= 108 Minutes to Generate

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Monod Kinetics Monod Kinetics

 The batch experiment can be repeated by varying initial  The zone A to B is equivalent to the exponential phase in
limiting substrate concentration S over a wide range of batch culture where limiting substrate concentration is in
values — resulting in observation of individual µ values excess and growth is at max.
which correspond to each substrate concentration. An
arithmetic plot of µ vs S will exhibit the general behavior
shown in following Figure.

Monod Kinetics Monod Kinetics

 The most widely used expression for describing specific  By combining the following two equations, we can write
growth rate as a function of substrate concentration is the following expression for time-rate-of-change of
attributed to Monod (1942, 1949). This expression is: biomass:

(this equation derived from previous slide)

(Monod equation)

(time-rate-of-change of biomass)

max = maximum specific growth rate [day-1]

S = concentration of limiting substrate [mg/L]
Ks = Monod, or half-velocity constant [mg/L]

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Monod Kinetics Continuous Culture

 Similarly, by combining following two equations, we can
write an expression for substrate utilization rate:
(time-rate-of-change of biomass)
 The importance of continuous culture is to keep a culture
growing indefinitely.
 Exponential growth in batch culture may be prolonged by
the addition of fresh medium, provided that the medium is
designed to be substrate-limiting.
 If the vessel is designed with an overflow mechanism, such
that the added medium displaced an equal volume of
spent medium, then continuous culture of cells can be
achieved.

(substrate utilization rate)

Continuous Culture Continuous Culture

 A steady state will be achieved if the medium is fed  The net change in cell concentration over time may be
continuously at a suitable rate, i.e., formation of new expressed as:
biomass (cells) by the culture is balanced by the loss of
 dx/dt = growth – output
biomass from the vessel. The flow of medium is related to
the volume of the vessel by the dilution rate (D) as follows:  or dx/dt = µx – Dx
 D = F/V  Under steady state conditions, the cell concentration
remains constant, therefore
 Where F is the flow rate (L/h) and V is the volume (L).
 dx/dt = 0 and µx = Dx and µ = D
 Dilution rate is the rate of nutrient exchange. It is defined
as the flow of medium per unit of time (h-1).  Thus, under steady state conditions, the specific growth
rate is controlled by the dilution rate, which is an
experimental variable.

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Continuous Culture Continuous Culture

Fyi: (µmax = µm)

(Monod equation)
 A continuous culture may be operated only at dilution 
rates below the maximum specific growth rate. Thus, the
 AT steady state, µ=D
dilution rate may be used to control the growth rate of the
culture.  Therefore,
 Cell growth in such a continuous culture is controlled by  where is the steady state concentration of substrate in
the availability of the growth limiting substrate and the the chemostat.
system is referred to as a chemostat.  Rearranging the equation:
 The mechanism underlying the controlling effect of the (Please try to derive the
equations by your own)
dilution rate is essentially the relationship expressed in
the Monod equation: continue……  This eqn. predicts that the substrate concentration is
determined by the dilution rate. The growth of the cells
depleting the substrate to a concentration that supports
the growth rate equal to the dilution rate. If the substrate
becomes depleted below the level that supports the
growth rate dictated by the dilution rate, the following
would occur: continue……

Continuous Culture How Cells Grow in Continuous

 Continue……
 The growth rate of the cells will be less than the dilution
rate and they will be washed out of the vessel at a rate
greater than they are being produced resulting in a
decrease in biomass concentration.
 The substrate concentration in the vessel will rise because
fewer cells are left in the vessel to consume it.
 The increased substrate concentration in the vessel will
result in the cells growing at a rate greater than the
dilution rate and biomass concentration will increase.
 The steady state will be re-established.
Culture
 Fresh medium continually supplied to well-stirred culture.
 Product continuously withdrawn.
 Accumulated cells and waste products are also removed.
 Conditions such as temperature and pH are kept at their
optimum values.
 During cultivation, growth & product formation can be
prolonged.
 At steady state: cell, product and substrate concentrations
remain constant.
 An essential nutrient is in limiting quantities.

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Fed-Batch Culture
 In fed-batch culture, medium are fed
continuously, or sequentially, without the
removal of culture fluid.
Fed-Batch Culture
 Fed-batch system employing strategies (i) & (ii) are
described as variable volume.
 Fed-batch system employing strategy (iv) is described as
fixed volume.
 Fed-batch system employing strategy (iii) gives a culture in
between the variable and fixed volume.
3/4/2020
Fed-Batch Culture
 A fed-batch culture is established initially in batch mode
and then fed according to one of the following feed
strategies:
 (i) the same medium used to establish the culture is
added, resulting in volume increase.
 (ii) a solution of the limiting substrate at the same
concentration as that in the initial medium is added,
resulting in volume increase.
 (iii) a concentrated solution of the limiting substrate is
added at a rate less than in (i) & (ii), resulting in volume
increase.
 (iv) a very concentrated solution of the limiting substrate
is added at a rate less than in (i), (ii) & (iii), resulting in an
insignificant volume increase.
Variable Volume Fed-Batch
Culture
 Consider a batch culture in which growth is limited by the
concentration of one substrate, the biomass at any point in
time will be described by the equation:

 Y is the yield factor (g biomass produced per g substrate
utilized), SR is the initial substrate concentration and S is
the residual substrate concentration.
 When s = 0, the final biomass concentration produced is
xmax, provided x0 is small compared with xmax.
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Variable Volume Fed-Batch Variable Volume Fed-Batch

Culture
 If, at the time when x = xmax, a medium feed is started
such that the dilution rate is less than maximum specific
growth rate ( ), virtually all the substrate will be
consumed as fast as it enters the culture, thus:

x is the cell concentration
Culture
 As time progresses the dilution rate will decrease as the
volume increases and D is given the expression:
 Where V0 is the original volume.
 The major difference between the steady state of a
chemostat and the quasi steady of a fed-batch culture is
that specific growth rate is constant in the chemostat but
decreases in the fed-batch.

 From the equation, it may be concluded that input of

substrate is equaled by consumption of substrate by the
cells. Thus, ds/dt = 0.

Thank you