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BIOPROCESS

ENGINEERING
CELL GROWTH

Bioprocess Engineering | Khairul Akli

Introduction
For microbes, growth is their most essential response to
their physiochemical environment.
Growth is a result of both replication and changes in cell
size.
In a suitable nutrient medium, organisms extract
nutrients from the medium and convert them into
biological compounds.
As a result of nutrient utilization microbial mass
increases with time and can be described simply by:
Substrates + cells
extracellular products + more cells
S + X P + n X

Bioprocess Engineering | Khairul Akli

Specific growth rate () is used to characterize the rate


of microbial growth,
X is cell mass concentration (g/l)
t is time (h)
is specific growth rate (h -1)

Bioprocess Engineering | Khairul Akli

BATCH GROWTH
Batch growth refers to culturing cells in a vessel with an
initial charge of medium that is not altered by further
nutrient addition or removal.

Bioprocess Engineering | Khairul Akli

Quantifying Cell Concentration


Quantifying cell concentration is useful to determine
kinetics and stoichiometry of microbial growth
The methods used: direct and indirect.
The direct methods are not feasible due to the
presence of suspended solids or other interfering
compounds in the medium.
Cell mass concentration is often preferred to the
measurement of cell number density.

Bioprocess Engineering | Khairul Akli

Determining cell number density


Using Petrof-Hausser slide or hemoctyometer, suitable
for nonaggregated cultures.
Using Petri dish with growth media/agar, more suitable
for bacteria and yeasts.
Using agar-gel medium in a small ring mounted in a
microscope slide.

Bioprocess Engineering | Khairul Akli

Another method is based on the relatively high electrical


resistance of cells.
It is more suitable for discrete cells in a particulate-free
medium and cannot be used for mycelial organisms.

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Determining cell mass concentration


Direct methods:
Determining cellular dry weight
Packed cell volume, in centrifuged fermentation
broth
Absorption of light by suspended cells in a culture
media, where the intensity of the transmitted light is
measured using a spectrometer
Indirect methods:
Mainly based on the measurement of substrate
consumption and product formation during the
course of growth

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Growth Patterns in Batch Culture

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1. The lag phase occurs immediately after inoculation and is a


period of adaptation of cells to a new environment.
2. The exponential growth phase: the cells have adjusted to
their new environment, and then multiply rapidly, and cell
mass and cell number density increase exponentially with
time.
3. The deceleration growth phase: the growth decelerates due
to either of one or more essential nutrients or the
accumulation of toxic by-products of growth.
4. The stationary phase starts at the end of the deceleration
phase, when the net growth rate is zero (no cell division) or
when the growth rate is equal to the death rate.
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Lag Phase
The age of inoculum culture has a strong effect on the
length of lag phase.
The age refers to how long a culture has been
maintained in a batch culture.
Usually, the lag period increases with the age of the
inoculum.
To minimize the duration of the lag phase, cells should
be adapted to growth medium and condition before
inoculation, and cells should be young (exponential
phase cells) and active, and the inoculum size should be
large (5-10% by volume).
The nutrient medium may need to be optimized, and
certain growth factor may need to be included to
minimize the lag phase
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Multiple lag phases may be observed when the medium


contains more than one carbon source, which is known
as diauxic growth.
This phenomenon is caused by a shift in metabolic
pathways in the middle of a growth cycle.
After one carbon source is exhausted, the cells adapt
their metabolic activities to utilize the second carbon
source.

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Exponential Growth Phase


During balanced growth (cell grow with all the same
rate), the specific growth rate determined from either
cell number or cell mass would be the same.
The exponential growth rate is first order:
Integration yields:
X and X0 are cell concentration at time t and t = 0.

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The time required to double the microbial mass,


where d is the doubling time of cells.
The exponential growth rate is characterized by a
straight line on a semilogarithm plot of ln X versus time.

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The deceleration growth phase


In deceleration phase, the stresses induced by nutrient
depletion or waste accumulation cause a restructuring of
the cell to increase the prospects of a cellular survival in
a hostile environment.
These observable changes are the result of the
molecular mechanisms of repression and induction.

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The stationary phase


During the stationary phase, the net growth rate is zero,
but cells are still metabolically active and produce
secondary metabolites.
Primary metabolites are growth-related products and
secondary metabolites are nongrowth-related.

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During the course of the stationary phase, one or more of


the following phenomena may take place:
Total cell mass concentration may stay constant, but
the number of viable cells may decrease
Cell lysis may occur and viable cell may drop.
Cells may not be growing but may have active
metabolism to produce secondary metabolites.
Cellular regulation changes when concentration of
certain metabolites (carbon, nitrogen, phosphate) are
low. Secondary metabolites are produced as a result of
metabolite deregulation.

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The Death Phase (Decline Phase)


The rate of death:
N is the concentration at the end of the stationary
phase
kd is the first-order death rate constant.

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Growth Yield
The growth yield in a fermentation,
Yield coefficients based on other substrates or product
formation:

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For organisms growing aerobically on glucose, Y X/s is


typically 0.4 to 0.6 g/g for most yeast and bacteria, while
YX/O2 is 0.9 to 1.4 g/g.
Anaerobic growth is less efficient and the yield
coefficient is substantially reduced.

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Classification of microbial products


Growth-associated products are produced simultaneously to
microbial growth. The specific rate of product formation is
proportional to the specific rate of growth.

Example: the production of constitutive enzyme


Nongrowth-associated product formation takes place during
the stationary phase when the growth rate is zero. The
specific rate of product formation is constant.
Examples: antibiotics (penicillin)
Mixed-growth-associated product formation takes places
during the slow growth and stationary phases.
Examples: Lactic acid fermentation, xanthan gum
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Kinetic pattern of growth and product


formation

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Examples
A strain of mold was grown in a batch culture on glucose and the
following data were obtained:
Time
(h)

Cell
Concentration
(g/l)

Glucose
Concentration (g/l)

1.25

100

2.45

97

16

5.1

90.4

23

10.5

76.9

30

22

48.1

34

33

20.6

36

37.5

9.38

40

41

0.63

a. Calculate the maximum growth rate


b. Calculate the substrate yield
c. What is the maximum cell concentration one could expect if 150
g of glucose was used with the same size inoculum?
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Solution:

A plot of ln X versus t yields a slope of 1.0 h.

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Temperature Effects to Growth Kinetics


According to their temperature optima, organisms can be
classified in three groups:
1.

Psychrophiles (Topt < 20oC)

2.

Mesophiles (T opt = between 20 o to 50oC)

3.

Thermophiles (T opt 50oC)

As the temperature increases to the optimal growth


temperature, the growth rate approximately doubles for every
10o increase in temperature.
Above the optimal temperature range,
decreases and thermal death may occur.

the

growth

rate

Temperature also affects the product formation, yield


coefficient and the rate-limiting step in a fermentation process.
At high temperatures, the rate of bioreaction might become
higher than the diffusion rate and diffusion would then become
the rate-limiting step.
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pH Effects to Growth Kinetics


Hydrogen ion concentration (pH) affects the activity of
enzymes and therefore the microbial growth rate.
Different organisms have different pH optima: the pH
optimum for many bacteria ranges from pH = 3 to 8; for
yeast, pH = 3 to 6; for molds, pH = 3 to 7; for plant cells,
pH = 5 to 6; for animal cells, pH = 6.5 to 7.5.
When pH differs from the optimal value, the maintenance
energy requirements increase.
The nature of nitrogen source can affect the pH change,
e.g. if ammonium is the source, pH decreases. If nitrate is
the source, pH increases.
pH change also can be affected by the production of
organic acids, the utilization of acids or the production of
bases.
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Dissolved Oxygen Concentration Effects to Growth Kinetics

Dissolved oxygen (DO) is an important substrate in aerobic


fermentations and may be a limiting substrate, since
oxygen is a sparingly soluble gas in water.
At high concentration, the rate of oxygen consumption may
exceed the rate of oxygen supply, leading to oxygen
limitations.
When oxygen is the rate-limiting factor, specific growth rate
varies with dissolves oxygen concentration according to
saturation kinetics; below a critical concentration, growth or
respiration approaches a first-order rate dependence on the
dissolved oxygen concentration.
Above a critical oxygen concentration, the growth rate
becomes
independent
of
the
dissolved
oxygen
concentration.
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Heat Generation by Microbial Growth


The heat generated during the microbial growth can be
calculated using the heat of combustion of the substrate
and of cellular material.
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.

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GROWTH KINETICS
For substrate-limited growth, the kinetics is described by
Monod equation:

Where:
m is the maximum growth rate when S >> Ks.
Ks is the saturation constant or half velocity constant (Ks
= S when m = max )
The Monod equation describes substrate-limited growth
only when growth is slow and population density is low.
If the consumption of a carbon-energy substrate is rapid,
then the release of toxic waste products is more likely.
At high population levels, the buildup of toxic metabolic byproducts becomes more important.
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The
alternatives for Monod equation:
Blackman equation:

Tessier equation:
Moser equation:
Contois equation:

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CELL GROWTH IN CONTINUOUS CULTURE


Growth, product formation, substrate utilization terminate
after a certain time interval in a batch culture, whereas, in
continuous culture, fresh nutrient medium is continually
supplied to a well-stirred culture and products and cells are
simultaneously withdrawn.
Growth and product formation can be maintained for
prolonged periods in continuous culture.
Continuous culture provides constant environmental
condition for growth and product formation and supplies
uniform-quality product.

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Devices for Continuous Culture


Chemostat

Cellular growth is usually limited by one of one essential nutrient and other
nutrients are in excess.

At steady state, the nutrient, the product and cell concentration is constant.

Turbidostat

The cell concentration is maintained constant by monitoring the optical


density of the culture and controlling the feed flow rate.

When the turbidity of the media exceeds the set point, a pump is activated
and fresh medium is added.

The culture volume is kept constant by removing an equal amount of culture


fluid.

Plug Flow Reactor (PFR)

There is no backmixing in an ideal PFR, so that the fluid elements containing


active cells cannot inoculate other fluid elements at different axial positions.

Liquid recycle is required for continuous inoculation of nutrient media.

Substrate and cell concentrations vary with axial position in the vessel.

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Continuous culture device (chemostat)

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Turbidostat

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Calculation for Chemostat

Material balance on the cell concentration:


where:
F = the flow rate of nutrient solution
VR = the culture volume (l), assume constant
= growth rate constants (h -1)
kd = endogenous/death rate constants (h -1)
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The balance becomes:


where D is the dilution rate and D = F/VR, D is the reciprocal
of residence time.
In chemostat, cells are removed at a rate equal to their
growth rate and the growth rate is equal to the dilution rate.
Usually the feed media is sterile , X0 = 0, and if the
endogenous metabolism (death rate) is negligible compared
to the growth rate (kd << ) and if the system is at the
steady state (dX/dt = 0), and substituning into Monod
equation:
The effluent substrate concentration to dilution rate for D <
m

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Amaterial balance on the limiting substrate in the absence of


endogenous metabolism yields

Where:
S0 and S are feed and effluent substrate concentration (g/l)
qp is the specific rate of extracellular product formation (g
P/G cells h)
YMX/S and YP/S are yield coefficients (g cell/g S and g P/g S)

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When extracellular product formation is negligible and at the


system is at the steady state (dS/dt = 0),

Since = D at steady state,

The steady-state cell concentration:

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Considering the effect the inclusion of endogenous


metabolism:

So the substrate balance;

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Example 6.4
A new strain of yeast is being considered for biomass
production. The following data were obtained using a
chemostat. An influent substrate concentration of 800 mg/l and
an excess oxygen were used at a pH of 5.5 and T = 35oC. Using
the following data, calculate m, Ks, YMX/S, kd and ms, assuming
= mS/(Ks+S) kd.
Dilution rate
(h-1)

Carbon substrate
concentration
(mg/l)

Cell
concentration
(mg/l)

0.1

16.7

366

0.2

33.5

407

0.3

59.4

408

0.4

101

404

0.5

169

371

0.6

298

299

0.7

702

59

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Example 6.5
The
specific growth rate for inhibited growth in a chemostat is
given by the following equation:

Where:
S0 = 10 g/l ; Ks = 1 g/l ; I = 0.05 g/l ;
subs
X0 = 0 ; KI = 0.01 g/l ; m = o.5 h-1

YX/S = 0.1 g cells/g

a. Determine X and S as a function of D when I = 0


b. With inhibitor added to a chemostat, determine the
effluent substrate concentration and X as a function of D.
c. Determine the cell productivity DX as a function of dilution
rate.

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