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CHAPTER 6

Microbial Growth
Kinetics in Batch
Culture
SITI NOR SYAIRAH BINTI ANIS
FERMENTATION TECHNOLOGY
(BTC 661)
Definition of Batch Culture
& Cell Growth Phases in
Batch Culture
Definition of Batch Culture
• Batch culture technique is also called as closed system of cultivation.
• In this technique at first nutrient solution is prepared and it is
inoculated with inoculum (culture organism) and then nothing is
added in the fermentation tank except O2 (aeration), antifoam and
acid @ base (control pH).
• In batch culture, neither fresh medium is added nor used up media is
removed from the cultivation vessel. Therefore volume of culture
remains same.
• Since fresh media is not added during the course of incubation,
concentration of nutrition decreases continuously. Furthermore
various toxic metabolites also accumulates in the culture vessel.
Characteristics of a Batch
Fermentation System
• Simplest fermenter operation
• Sterilization can be performed in the
reactor
• All nutrients are added before inoculation
• Biomass production limited by C/N load
• Cell are harvested when biomass levels @
products level start to decline
 Substrate
 Microbial inoculum
 Close batch system
 Sufficient amount of time
 End time – harvest the product

Lab scale
Standardizing - pH, oxygen,
time & others factors
Growth pattern (minimize
lag phase)

Large scale
General Batch Curve
Cell Growth Phases in Batch Culture
The inoculated culture in batch
culture will pass through a
number of phases following a
growth curve. The growth curve
contains four distinct regions as :

 Lag Phase
 Exponential Phase
 Stationary Phase
 Death Phase
Lag Phase
• The first major phase of growth in a batch bioreactor
• A period of adaptation of the cells to their new environment
• Minimal increase in cell density
• The length should be reduces as much as possible (using a suitable inoculum)

Exponential / Log Phase


• Also known as the logarithmic growth phase
• Cells have adjusted to their new environment
• The cells are dividing at a constant rate resulting in an exponential increase in the
number of cells present.
Stationary Phase
The third major phase of microbial growth in a batch process occur
when the number of cells dividing and dying is in equilibrium and can
be the result of the following:
Depletion of one or more essential growth nutrients - the carbon and
energy source or an essential nutrient becomes completely used up
Waste products build up to a point where they begin to inhibit cell
growth or are toxic to cells.
 Growth in the stationary phase is unbalanced because it is easier for
the cells to synthesize some components than others.
Death Phase
• The final phase of the growth curve is the death phase, which is
characterized by a net loss of culturable cells
• The rate of cells dying is greater than the rate of cells dividing
Physiology and Kinetics
of batch culture
Growth Index (GI)
• Growth index (GI) is a relative estimation of such capacity as it correlates
the biomass data at the sampling time to that of the initial condition.
• It is calculated as the ratio of the accumulated and the initial biomass.
• The accumulated biomass corresponds to the difference between the final
and the initial masses.

• Where in this equation, GI is growth index


Wf is final cell mass
Wi is the initial cell mass.
(Both Wf and Wi are taken either as fresh or dry weight)
(Loyola-Vargas and V á zquez-Flota, 2006)
Exponential Cell Division.
• Each cell division results in a doubling of
the cell number.
• At low cell numbers the increase is not
very large; however, after a few
generations, cell numbers increase
explosively.
• If the initial cell number is X0, the
number of cells X after n (no. of
generations) doublings is

X = 2n. X0

* X0 can also be used to represent cell


mass, which is often more convenient to
measure than cell number.
Doubling time
The time it takes for a cell division to occur
@
the time required for the concentration of biomass of
a population of suspension cells to double is called the
generation time or the doubling time.
Example Calculation 1 - Generation Time

Problem:
If one starts with 10,000 (104) cells in a culture that has a
generation time of 2 h, how many cells will be in the culture
after 4, 24, and 48 h?
Use the equation X = 2n X0 , where X0 is the initial number of cells,
n is the number of generations, and X is the number of cells after n generations.

After 4 h, n = 4 h /2 h per generation = 2 generations:


X = 22 (104) = 4.0 x 104 cells

After 24 h, n = 12 generations:
X = 212 (104) = 4.1 x 107cells

After 48 h, n = 24 generations:
X = 224 (104) = 1.7 x 1011 cells

This represents an increase of less than one order of magnitude for the 4-h culture, four
orders of magnitude for the 24-h culture, and seven orders of magnitude for the 48-h
culture!
Growth Kinetics

If a viable inoculum is introduced into a medium that contains a carbon


source, suitable nitrogen source, other nutrients necessary for growth,
and physiologic temperature and pH are maintained, it will grow.
• The rate of biomass synthesis is proportional to biomass present.
• Describe cell growth during the exponential phase using the following
equation:
where, X is the number or mass of cells (mass/volume),
t is time,
µ is the specific growth rate constant (1/time)

• This equation also can be used to calculate the


generation time @ doubling time as well as the
specific growth rate using data generated from a
growth curve.
Specific Growth Rate (µ)

• The specific growth rate (µ = 1/time) refers to the steepness of a


curve, and it is defined as the rate of increase of biomass of a cell
population per unit of biomass concentration.
• It can be calculated in batch cultures, since during a defined period of
time, the rate of increase in biomass per unit of biomass
concentration is constant and measurable.
• This period of time occurs between the lag phase and stationary
phases.
• During this period, the increase in the cell population fits a straight-
line equation between ln x and t.
@ @

• Where, xo is the initial biomass (or cell density),


x is the biomass (or cell density) at time t,
µ is the specific growth rate.
• µ can be calculated from the above relationship, which is the slope of
the line between ln x and t.
@
Example Calculation 2 – Specific Growth Rate & Doubling Time

ln16 – ln2
µ?

Doubling time?
ln16 – ln2
• The stationary phase in a batch culture can be defined as a state of no net
growth, which can be expressed by the following equation:

Growth rate = Death rate

• The death phase can be described by the following equation:

Where - kd is the specific death rate. In closed system, rate of cell death is
equal to the rate of decrease in cell number.
Example Calculation 3 – Specific Growth Rate
Problem: The following data were collected using a culture of
Pseudomonas during growth in a minimal medium containing salicylate
as a sole source of carbon and energy. Using these data, calculate the
specific growth rate for the exponential phase.
• The times to be used to determine the
specific growth rate can be chosen by visual
examination of a semi log plot of the data
(see figure).
• Examination of the graph shows that the
exponential phase is from approximately 6 to
10 hours .
• From the data given, the slope of the graph
from time 6 to 10 hours is:

 It should be noted that the specific growth rate and generation time calculated for growth
of the Pseudomonas on salicylate are valid only under the experimental conditions used.
 For example,
- at a higher temperature, the specific growth rate would be expected to increase.
- at a lower temperature, the specific growth rate would be expected to decrease.
Effect of Substrate Concentration
in Batch Culture
• During the growth and decline phase of batch culture, the specific growth
rate (µ) of cells is dependant on the concentration of nutrients in medium.
• Often, a single substrate exerts a dominant influence on rate of growth, this
component is known as the growth-limiting-substrate.
• The growth-limiting-substrate is often C @ N source (some cases O2).
• In general, when substrate, S is limiting growth, Monod (1949) reported that
growth rate variations can be expressed as

s
• A homologue of the Michalis-Menten expression
The specific growth rate (1/time) (µ) is generally found to be a function
of 3 parameters:

1. The substrate concentration (mass/volume), S


2. The maximum specific growth rate (1/time) for the culture, µmax
3. The half-saturation constant (mass/volume) also known as the
affinity constant, Ks (typical value are very small – mg/ L @ µg/ L)

*During µ = µmax , S is greater than 10Ks, so that this equation have limited
applicability at extremely low substrate level.
• This equation was developed by Monod - showed that at low substrate
concentrations, growth rate becomes a function of the substrate concentration.
• Describe the relationship between the specific growth rate and the substrate
concentration in cell culture
• Substrate limitation slows down metabolism
• There are two constants in this equation:
1. µmax, the maximum specific growth rate,
2. Ks, the half-saturation constant (which is defined as the substrate
concentration at which growth occurs at one half the value of µmax).
• Since S and µ can be measured as the Monod equation can be
linearized by inverting and factoring out µmax.
• the Monod equation can be expressed in terms
of cell number or cell mass (X ):

• The Monod equation has two limiting cases:

1) The first case is at high substrate


concentration where S>>Ks.
2) The second limiting case occurs at low
substrate concentrations where S<<Ks.
S>>Ks :
• The specific growth rate µ is essentially equal to µ max .
• This simplifies the equation and the resulting relationship is zero order or
independent of substrate concentration:

• Under these conditions, growth will occur at the maximum growth rate.
• There are relatively few instances in which ideal growth as described by this
equation can occur:
-One such instance is under the initial conditions found in pure culture in a batch
flask when substrate and nutrient levels are high.
-Another is under continuous culture conditions, which are discussed further in
other chapter.
S<<Ks :
• In this case there is a first order dependence on substrate concentration.

• When the substrate concentration is low, growth (dX/dt ) is dependent on the


substrate concentration.
• Since the substrate concentration is in the numerator, as the substrate
concentration decreases, the rate of growth will also decrease.
• This type of growth is typically found in batch flask systems at the end of the
growth curve as the substrate is nearly all consumed.
• The Monod equation can also be expressed as a function of substrate
utilization given that growth is related to substrate utilization by a constant
called the cell yield

• Where Y is the cell yield (mass/mass).


• The cell yield coefficient is defined as the unit amount of cell mass
produced per unit amount of substrate consumed.
• Thus, the more efficiently a substrate is degraded, the higher the value of
the cell yield coefficient
Limitation and
Application of Batch
culture
The Advantages & Disadvantages - Batch Culture
Advantages Disadvantages
Very basic Batch variability (different quality & quantity
of product)
Inexpensive (simple & easy to operate) Downtime @ high proportion unproductive
time is associated (results in significant
periods of idle time between batches,
comprising the charge and discharge of the
fermenter vessel, the cleaning, sterilization
and re-start process)
The fermenter can be used for different Gives low product yield
reactions with each separate use
Chance of contamination of culture is Resulting in higher costs and not economic
minimum because it is closed system of procedure
cultivation.
Application 1: Lactic acid fermentation

Lactic acid bacteria -


Lactobacillus acidophilus
Application 2: Deep-tank batch fermentation in
the mass production of penicillin
• The antibiotic penicillin can be mass produced via the use of deep-
tank batch fermentation
• Large industrial fermenters are constructed that have the capacity to
hold thousands of litres
• Penicillium mold is grown in the deep-tank batch fermenters following
the addition of sugars and other key ingredients
• The production process typically lasts 6 – 8 days, with the fermenter
drained at the end of the fermentation cycle
• Penicillin is separated from the solution and purified via downstream
processing to improve its antibiotic potential

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