CELL GROWTH AND CELL

CULTIVATION (BATCH)
Engr. Patrick Luis Z. Soriano
Chemical Engineering Department
Adamson University
 Outline
• Batch Growth
• Biomass Yield
• Effects of Environmental Conditions to Growth Rate
• Oxygen-Demand For Aerobic Microorganisms
• Monod Equation
• Growth Inhibitors
• Batch Culture
 Batch Growth
• When a liquid nutrient medium is inoculated with
a seed culture (inoculums), the organisms
selectively take up dissolved nutrients from the
medium and convert them into biomass.
𝑆+𝑋 → 𝑃 + 𝑛𝑋
 Batch Growth
• A typical batch growth curve includes the
following phases:
– lag phase
– logarithmic or exponential growth phase
– deceleration phase
– stationary phase
– death phase.
Batch Growth
• A typical batch
growth pattern
Batch Growth
• lag phase
– occurs immediately
after inoculation and
is a period of
adaptation of cells to
a new environment.
Batch Growth
• lag phase
– diauxic growth
• Multiple lag phases
may be observed
when the medium
contains more than
one carbon source.
Batch Growth
• exponential growth
phase or logarithmic
growth phase
– Also known as
maximum growth
phase
Batch Growth
• exponential growth
phase or logarithmic
growth phase
– In this phase, the cells
have adjusted to their
new environment.
Batch Growth
• exponential growth
phase or logarithmic
growth phase
– Malthus growth
model
𝒅𝑿
= 𝝁𝒏𝒆𝒕 𝑿
𝒅𝒕
Batch Growth
• deceleration growth
phase
– follows the maximum
growth phase
Batch Growth
• deceleration growth
phase
– A modification of the
Malthus model by
Verhulst in 1844
𝑑𝑋 𝑋
= 𝑘𝑋 1 −
𝑑𝑡 𝑋∞
Batch Growth
• deceleration growth
phase
– Logistic Equation
𝑿𝒐 𝒆𝒌𝒕
𝑿=
𝑿𝒐
𝟏− (𝟏 − 𝒆𝒌𝒕 )
𝑿∞
Batch Growth
• Ethanol formation from
glucose is accomplished in
a batch culture of
Saccharomyces cerevisiae
with a certain amount of a
complex medium
(nitrogen, energy and
minerals) to produce
biomass from the small
inoculums. The following
data (Table P11.11) were
obtained:
Batch Growth
• Correlating the data
with the Logistic
Equation,
determine the
carrying capacity
• and carrying
capacity coefficient.
Batch Growth
• Consider that glucose is the
only substrate for the product
(ethanol) generation while
some glucose is also
consumed for the cell growth.
Correlate the ethanol product
rate with the Monod model.
Determine the specific
maximum product formation
rate, saturation coefficient,
and the yield factors YFP/S
and YFX/S.
Batch Growth
• 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.
 Batch Growth
• 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 mass may drop. A
second growth phase may occur and cells may grow on
lysis products of lysed cells (cryptic growth).
– Cells may not be growing but may have active metabolism
to produce secondary metabolites.
Batch Growth
• stationary phase
– The reason for
termination of growth
may be either
exhaustion of an
essential nutrient or
accumulation of toxic
products
Batch Growth
• death phase (or
decline phase)
– follows the stationary
phase
– The death rate can be
thought of as a first-
order reaction.
Batch Growth
• death phase (or
decline phase)
– The death rate can be
thought of as a first-
order reaction
𝒅𝑿
= −𝒌𝑫 𝑿
𝒅𝒕
 Batch Growth
• Mass balance of the cell biomass in the batch
reactor leads to:
𝒅(𝑿𝑽)
−𝒌𝑫 𝑿𝑽 =
𝒅𝒕

𝑋 = 𝑋𝑆𝑂 𝑒 −𝑘𝐷𝑡
 Biomass Yield
• Yield factors are defined based on the amount
of consumption of another material.
• Apparent Growth Yield
∆𝑿
𝒀𝑭𝑿/𝑺 ≡ −
∆𝑺
 Biomass Yield
• Yield factors based on other substrates or
product formation may be defined
𝒅𝑿
𝒀𝑭𝑿/𝑶𝟐 =
−𝒅[𝑶𝟐 ]

𝒅𝑷
𝒀𝑭𝑿/𝑶𝟐 =
−𝒅𝑺
Biomass Yield
Biomass Yield
 Biomass Yield
• The differential selectivity (or instantaneous
selectivity) is defined as:
𝒓𝑿
𝒔𝑿/𝑺 =
−𝒓𝑺

𝝁𝒏𝒆𝒕 𝑿
𝒔𝑿/𝑺 =
𝝁𝑮 + 𝝁𝒆 𝑿/𝒀𝑭𝑿/𝑺
Biomass Yield
• A strain of mold was grown in a batch
culture on glucose and the following
data were obtained as shown in Table
E11.1. Do the following:
– Calculate the maximum net specific growth
rate.
– Calculate the apparent growth yield.
– What maximum cell concentration could one
expect if 150 g of glucose were used with the
same size inoculum?
– How many generations of cells are there in
the culture for part (c)?
Effects of Environmental Conditions to
Growth Rate
• Temperature is an important factor affecting the
performance of cells.
– As the temperature is increased toward optimal
growth temperature, the growth rate approximately
doubles for every 10 °C increase in temperature.
– Above the optimal temperature range, the growth
rate decreases and thermal death may occur.
Effects of Environmental Conditions to
Growth Rate
Effects of Environmental Conditions to
Growth Rate
• At high temperatures, the thermal death rate
exceeds the growth rate, which causes a net
decrease in the concentration of viable cells:
𝒅𝑿
= (𝝁𝑹 − 𝒌𝑫 )𝑿
𝒅𝒕
Effects of Environmental Conditions to
Growth Rate
Effects of Environmental Conditions to
Growth Rate
• A hydrogen-ion concentration (pH) affects the
activity of enzymes and therefore the
microbial growth rate.
• The optimal pH for growth may be different
from that for product formation.
Effects of Environmental Conditions to
Growth Rate
• The pH optimum for many bacteria ranges
from pH 3–8
• for yeast pH 3–6
• for molds pH 3–7
• for plant cells pH 5–6
• animal cells pH 6.5–7.5.
Effects of Environmental Conditions to
Growth Rate
• pH can change because of the production of
organic acids, the utilization of acids
(particularly amino acids), or the production
of bases.
• The evolution or supply of CO2 can alter pH
greatly in some systems (eg, seawater or
animal cell culture).
Effects of Environmental Conditions to
Growth Rate
 Oxygen-Demand For Aerobic
Microorganisms
• When oxygen is the rate-limiting factor, the
specific growth rate varies with the DO
concentration according to Monod equation,
just like any other substrate-limited case.
Oxygen-Demand For Aerobic
Microorganisms
 Oxygen-Demand For Aerobic
Microorganisms
• The dependence of DO for aerobic and
facultative organisms on cell growth follows
the Monod growth equation. For aerobic
organisms:
𝝁𝒎𝒂𝒙 𝑫𝑶
𝝁 =
𝑲𝑫𝑶 + 𝑫𝑶
 Oxygen-Demand For Aerobic
Microorganisms
• For facultative organisms:
(𝝁𝒎𝒂𝒙 − 𝝁𝒎𝒂𝒙𝟎 )𝑫𝑶
𝝁 = 𝝁𝒎𝒂𝒙𝑶 +
𝑲𝑫𝑶 + 𝑫𝑶
 Oxygen-Demand For Aerobic
Microorganisms
• The critical oxygen concentration is about 5–
10% of the saturated DO concentration for
bacteria and yeast
• About 10–50% of the saturated DO
concentration for mold cultures, depending on
the pellet size of molds.
Monod Equation
• A comparison of the full
solution and approximate
solution (single-step
Michaelis-Menten
equation) for the reaction
network as shown. The
solid lines are based on the
full solution, while the
dotted lines are based on
the approximated model.
 Monod Equation
• Monod Equation:
𝒓𝑿 𝝁𝒎𝒂𝒙 𝑺
𝝁𝑮 = =
𝑿 𝑲𝒔 + 𝑺
 Monod Equation
• For extracellular product formation:
𝒓𝑷 𝝁𝑷𝒎𝒂𝒙 𝑺
𝝁𝑷 = =
𝑿 𝑲𝑷 + 𝑺
 Monod Equation
• Monod Equation:
𝒓𝑿 𝝁𝒎𝒂𝒙 𝑺
𝝁𝑮 = =
𝑿 𝑲𝒔 + 𝑺
Monod Equation
 Monod Equation
• The death rate of cells is proportional to the
cell population; thus, the specific death rate is
not a function of cell population of
concentration, which is confirmed by the
experimental observation
𝝁𝑫 = 𝒌𝑫
 Monod Equation
• The net-specific cell growth rate is the specific
growth rate minus the cell death rate; that is:
𝜇𝑛𝑒𝑡 = 𝜇𝐺 − 𝑘𝐷
Announcement
 Growth Inhibitors
• Substrate Inhibition
– At high substrate concentrations, microbial growth
rate is inhibited by the substrate.
– As in enzyme kinetics, substrate inhibition of
growth may be competitive or noncompetitive.
 Growth Inhibitors
• Substrate Inhibition
– Since the Monod equation is an approximate
growth rate equation, one is not able to
differentiate the maximum growth rate and
saturation constant from the noninhibited case.
– The kinetic constants are merely correlation
coefficients.
 Growth Inhibitors
• Substrate Inhibition
– Therefore, only the noncompetitive substrate
inhibition is presented here:
𝜇𝑚𝑎𝑥
𝜇𝐺 =
𝐾𝑆 𝑆
1+ 1+
𝑆 𝐾𝐼
 Growth Inhibitors
• Product Inhibition
– A high concentration of extracellular product can be
inhibitory for microbial growth.
– Product inhibition may be competitive or
noncompetitive, and in some cases when the
underlying mechanism is not known, the inhibited
growth rate is approximated to exponential or linear
decay expressions.
 Growth Inhibitors
• Product Inhibition
– Competitive Product Inhibition
𝜇𝑚𝑎𝑥
𝜇𝐺 =
𝑃
𝐾𝑆 1 + +𝑆
𝐾𝑃
– Noncompetitive Product Inhibition
𝜇𝑚𝑎𝑥
𝜇𝐺 =
𝑃
𝐾𝑆 + 𝑆 1 +
𝐾𝑃
 Growth Inhibitors
• Cell Inhibition
– In most cases, there is no limitation on the space
for the cells to occupy; however, it can become an
issue for batch growth.
– Before cells can divide, there needs to be space
available for the new cells.
 Growth Inhibitors
• Cell Inhibition
– Logistics Equation
𝜇𝑚𝑎𝑥 𝑆 𝑋
𝜇𝐺 = 1−
𝐾𝑆 + 𝑆 𝑋∞
 Growth Inhibitors
• Inhibition by Toxic Compounds
– Toxic compounds can affect the metabolic
behavior of cells via poisoning and/or rendering
the necessary enzymes less effective.
 Growth Inhibitors
• Inhibition by Toxic Compounds
– Competitive Inhibition
𝜇𝑚𝑎𝑥
𝜇𝐺 =
𝐼
𝐾𝑆 1 + +𝑆
𝐾𝐼
– Noncompetitive Inhibition
𝜇𝑚𝑎𝑥
𝜇𝐺 =
𝐼
𝐾𝑆 + 𝑆 1 +
𝐾𝑃
 Cell Cultivation
• Batch cultivation
– is a traditionally acceptable “home” operation.
– It is commonly used today in pharmaceuticals, food
processing, and in laboratory studies.
– It is easy to operate and minimum “control” is
necessary: temperature, moisture, and pH.
– Other than the environmental conditions and initial
materials loading, batch culturing can be classified as
no control.
Cell Cultivation
• Batch
cultivation
– a schematic
of a
laboratory
bioreactor
setup
 Cell Cultivation
• Batch cultivation
– Mass balance for the cell biomass leads to:
𝑑(𝑋𝑉)
0 − 0 + 𝑟𝑋 𝑉 =
𝑑𝑡
– Rate law for biomass is usually expressed as:
𝑟𝑋 = 𝜇𝑛𝑒𝑡 𝑋 = 𝜇𝐺 − 𝑘𝐷 𝑋
 Cell Cultivation
• Batch cultivation

𝑑𝑋
𝑟𝑋 = = 𝜇𝐺 − 𝑘𝐷 𝑋
𝑑𝑡
 Cell Cultivation
• Batch cultivation
– Mass balance for the limiting substrate leads to:
𝑑(𝑆𝑉)
0 − 0 + 𝑟𝑆 𝑉 =
𝑑𝑡
– For simplicity, we revert to the biomass yield
factor:
𝜇𝐺 𝑋
𝑌𝐹𝑋/𝑆 =
−𝑟𝑆
 Cell Cultivation
• Batch cultivation

𝑑𝑆 𝜇𝐺 𝑋
=−
𝑑𝑡 𝑌𝐹𝑋/𝑆