Chapter 4. Growth of cells
• Microorganisms makes biological compound from nutrients by absorbing
them in suitable growth medium.
• With time, increase the mass of microorganisms.
• Growth rate of the microorganism is identified by the specific growth rate,
this is defined as follows.
X : cell concentration(g/L), t : time(h) and μ : specific growth rate(
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).
• Batch growth, after filling the growth medium once initially
refers to culturing the cells in the reactor without removal and
supply of nutrients more. It is most simple culturing
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• Quantity of cell concentration – determination of cell number density
Petroff-Hausser slide
hemocytometer
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• Quantity of cell concentration – determination of cell number density
Particle counter
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• Determination of cell concentration (direct observation)
- By measuring the dry weight of the cells, the method most commonly
used to determine the concentration
- It is applicable only in the case of cells growth in growth medium there is no
solid.
- Rather than cells, if there is such a solid, inaccurate mass
measurements were.
- Usually, after filtration or centrifugation of the sample broth, it is
Washed by water or buffer. And weigh after dried the cells which were
washed in wet conditions for 24 hours at 80oC
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• Determination of cell concentration (spectrometer)
- It is based on a property of cells floating in the culture sample to absorb light.
- Measurement of the optical density or turbidity of growth medium makes it
possible to estimate the density of the cell easily when there is no substance
that absorbs light or other solid substance.
- Transmittance of light through the sample chamber is a function of the
thickness of the sample chamber and the cell density.
- If the cells ingested the dissolved components that absorb light, you must also
consider the background absorbance of the growth medium component.
- It use wavelength 600~700nm.
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• Determination of cell concentration (indirect observation)
- Indirect observation is mainly based on the measurement of the
amount of production resulting in the grouwth process and
consumption of substrate.
- By measuring intracellular components like RNA, DNA, protein,
it can be used to measuring growth of cells indirectly.
- In batch growth, the concentration of them is changed with time.
- It shows the time variation of the intracellular specific
components which in batch growth.
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• Determination of cell concentration (indirect observation)
Time changes in intracellular specific components in batch.
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• Growth from and reaction process in batch growth.
Log phase  Exponential  Stationary  Deceleration  Death or Decline
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• Growth from and reaction process in batch growth(log phase)
- Time of the cells to adapt to the new environment.
- sometimes cell mass is slightly increased without an increase in
cell number density.
- To minimize the log phase, the cells must be adapt to the growth
medium prior to inoculation, be young and need active and
inoculation amount must be plenty
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• Growth from and reaction process in batch growth(log phase)
- When the medium has a carbon source of one or more, many
group of log phase can be observed
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• Growth from and reaction process in batch growth
(Exponential phase)
- State the cells are adapted to the new environment already.
- It is possible to grow rapidly, the mass and number of cells
increases as a function of time exponentially.
- It is the time of growth balance. all components of the cell is
increased at the same rate.
- In this step, the growth rate is independent of concentration of
nutrients as high concentration of nutrients.
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• Growth from and reaction process in batch growth
(Exponential phase)
exponential growth rate is linear.
- as this equation is integrated
Here, X and X0 are cell concentration when time is t and t=0.
- The following equation is for doubling the mass of microorganisms.
(doubling time)
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• Growth from and reaction process in batch growth.
(Deceleration phase)
- In this step, glowth is slow. By depletion of essential nutrients of
one or more or by the accumulation of toxic by-products resulting
from growth.
- In exponential phase, in order to maximizing the reproductive
rate, it adapt to metabolic control system. The accumulation of
stress caused by nutrient depletion or waste reducer causes a
structural change of the cell to increase the possibility that cells
survive in harsh environments.
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• Growth from and reaction process in batch growth.
(stationary phase)
- Though net growth rate is 0, cells produce secondary metabolites
undergoing metabolically active.
- The primary metabolites are growth-associated products and the
secondary metabolites are non growth-associated products.
- The production of a particular metabolites may be promoted
when stationary phase when modulation of metabolites are
removed.
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• Growth from and reaction process in batch growth.
(stationary phase)
- During the stationary phase, the phenomenon of one or more of
the following occurs.
(1) The total cell concentration remains a constant value, but the number of
living cells decreases.
(2) When the phenomenon of hemolysis of cells occurs, mass of living cells is
reduced. There can be secondary growing phase, cells glows by eating
productions of other cells by their hemolysis
(3) The cells are not growth, but they have active metabolic activity, and produce secondary metabolic
production. When concentration specific metabolic materials(carbon, nitrogen, phospate) are low, the
adjustment mechanism of intracellular changes. As a result of metabolites deregulated, secondary products
are generated.
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• Growth from and reaction process in batch growth.
(stationary phase)
- The reason for the growth is stopped is the accumulation of toxic products and
depletion of essential nutrients.
if the production of inhibitory products are accumulated in the growth medium,
growth rate will be reduced to the extent that inhibitors are produced. The growth
stops at a concentration of inhibitor greater than or equal to the specific value.
It is possible to allow cell proliferation followed by eliminating the adverse effects
of toxic substances through dilution of toxic substances or the addition of nonmetabolizable chemicals to form a toxic substances and a complex compound.
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• Growth from and reaction process in batch growth.
(Death or decline)
- As death of some cells can start at stationary phase, it is not able
to build a clear boundary between these two.
In some cases, during the stationary phase living cells use
nutrients released into the culture medium as dead cells are
hemolysis.
At the end of stationary phase, death phase starts by
accumulation of toxic products and depletion of nutrients
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• Form of the product and the growth of microorganisms are
affected by the environmental conditions of temperature, PH,
and the dissolved oxygen concentration.
- Temperature is an important factor that affects the function of cells.
- Microorganisms can be divided into psychrophilic organisms, mesophilic
organisms, thermophilic organisms according to the optimal production
temperature.
- Growth rate is doubled every 10oC. Growth rate decreases at range higher than
optimal temperature and extinction phenomenon can occur according to
temperature.
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• The following equation : the net growth rate.
• Growth:
activation energy: 10~20kcal/mole
• Death:
activation energy : 60~80kcal/mole
• Death is more sensitive than growth to temperature.
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• Typical changes of the growth rate due to the temperature
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• pH affects on the growth rate of microorganisms by effecting on
activation of enzymes. Optimal pH necessary for growth can differ
to optimal pH for products
• pH of generally accepted is in the range of pH ±1~2 around the
optimum point. Each microorganisms has a different optimum pH
• It has mechanism that intracellular pH comparatively remains steady
even though surrounding pH changes.
• Important matters of the one obtained in terms of having each
different optimum pH is that pH of medium can be used for
screening microorganisms.
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• In general fermentation, pH can vary considerably. pH can vary by
property of nitrogen source, production of organic acid, consumption
of acid, production of basic material.
• Sometimes supply or generation of CO2 significantly alters the pH.
• Thereby pH control is important that use buffers or pH control
system.
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• Changes in the non-growth rate in response to the pH is as shown in
the following figure.
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• Dissolved oxygen(DO) is a substrate important to aerobic fermentation,
there is a possibility that the dissolved oxygen is the limiting substrate
as oxygen is not soluble in water.
• At high cell density, it is possible that oxygen consumption rate
exceeds the supply rate, oxygen shortage was caused by this.
• When the rate-limiting factor, non-growth rate shows changed saturated
phenomenon according to the dissolved oxgen concentration.
• In the critical concentration below, dependent form of growth or
respiration rate about dissolved oxygen concentration is close to the
first-order equation.
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• In the critical concentration or higher, growth rate is independent to
dissolved oxygen concentration..
• The figure below shows the change in non-growth rate in response to
the dissolved oxygen concentration.
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• When DO concentration is below critical value, oxygen is the growth
limiting factor.
• In this case, another component in media limits growth extent.
• During the incubation time of most while the growth rate depends on
DO, the extent of growth(mass of cells generated) is dependent on
the amount of glucose.
• In saturated concentration of oxygen there are salts and dissolved
organism and these can be varied.
• Saturation value decreases as the temperature rises.
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• 40~50% of energy stored in the energy source and carbon aerobic
metabolic changes in ATP, the rest is released as heat.
• If the cells are growing actively, for request of status quo is low,
there is a direct relationship between growth and release of heat.
• Metabolic heat emitted during fermentation can be eliminated by
circulating cooling water through the cooling jacket in the fermenter
cooling coil.
• In many cases, temperature control is an important constraint in the
design of the reactor.
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• Continuous culture is that flesh culture medium is supplied into wellstirred reactor continually, simultaneously cells emerge from reactor.
• Continuous culture can be maintained for a long time.
• Generally, after a certain amount of time the system reached a steady
state. In this state, the concentration of cells, the product, a subtrates are
kept constant.
• Continuous culture provide a constant environmental conditions
necessary for the generation of the product and growth and products that
has uniform quality.
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• Specific equipment for the continuous culture
- chemostat: cell proliferation is limited by one essential nutrient and
other nutrients are present in an excess.
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• Specific equipment for the continuous culture
- turbidostat : As concentration of cells are maintained, it may be very useful in
selecting the lower group that can withstand the stress under favorable
environment conditions(ex. High concentration ethanol)
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• Specific equipment for the continuous culture
- turbidostat : It helps keep a constant density through monitoring of optical
density and control of material flow.
Pump is running when the turbidity of the medium exceeds the set value, and
added to the fresh medium. By removing the culture medium of the same
amount of volume culture medium is kept constant.
because turbidostat requires more efforts than chemostat, it isn’t used often.
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• PFR: Plug Flow Reactor
- It can be used for continuous culture..
- Because there is no back-mixing in ideal PFR, the part that is in a different
position on the axial direction will not be inoculated by the part where cells are
alive.
- Recycling of culture medium is necessary for continuous vaccination.
- In PFR cell concentration and substrate is changed in accordance with the axial
position. Ideal PFR is same to batch reactor, distance defined along the fermenter
corresponds to the incubation time in batch culture.
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Thank
You