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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)
X = 2n. X0
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 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
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:
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:
*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 ):
• 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.