1. MIC 303 INDUSTRIAL AND ENVIRONMENTAL MICROBIOLOGY
MICROBIAL GROWTH KINETICS
Mdm Aslizah Mohd Aris

2. TYPE OF MICROBIAL GROWTH SYSTEM
3 major type:
Batch culture
Continuous or Chemostat Culture
Fed-batch Culture

3. BATCH
CULTURE

4. BATCH CULTURE
Definition: A closed system with limited amount of nutrient (no additional of medium added)
Cells grows through several phases:
i) Lag phase
ii) Log phase
iii) Deceleration
phase
iv) Stationary
v) Death phase

5. i) Lag phase
Time of adaptation of cell to the environment or medium (reorganization of micro molecular contituents).
Length of lag phase may vary (depend on specific circumstances).
Shorter lag time is recommended for industry, can be achieved using suitable inoculum (active or not) and environmental condition.
Synthesis or inhibition of the enzyme or cell structure components may occur.
Typical effects:
Low cell number / cell concentration.
No changes in substrate pH.
No changes in substrate concentration.
No product formation.

6. ii) Log phase
Illustrate by a linear line of the plot of log cell mass vs time.
During this phase, a growth at steady state where specific growth rate, µ are fixed.
Cell growth at maximum attainable rate.
Typical effects in log phase:
Rapid increase in cell concentration.
Rapid changes in substrate pH.
Substrate concentration decreased.
Product formation starts.

7. iii) Deceleration phase
Growth rate slowly decreases due to:
Consumption of nutrient or essential nutrient
become depleted (substrate limitation).
Accumulation of toxic product.

8. iv) Stationary phase Start when the growth rate begins to decrease
Nutrients have been used (finished)
Accumulation of product that inhibit the growth
Growth rate become zero – cell stop dividing
At equilibrium growth rate = death rate
Typical effects:
Microbial deaths balance production of new cells, cell mass may be constant.
The number of viable cell decreased, lysis cell may occur so the biomass will decreased .

9. iv) Death phase Typical effects: Substrate depletion
Rapid decline in cell numbers / biomass concentration.
Substrate concentration became until zero

10. GROWTH VS MULTIPLICATION CURVE

11. COMPARISON BETWEEN GROWTH AND MULTIPLICATION CURVE
GROWTH CURVE
MULTIPLICATION CURVE
A plot of biomass concentration against Incubation time.
A plot of cell number against time.
Lag phase is shorter than the multiplication curve since the growth rate begins to increase earlier than the multiplication rate.
Lag phase longer.
The log phase is substantially longest.
Shorter log phase.
Section 3 of both curve are identical.
Death phase sets in earlier.
Death phase sets in later.
Stationary phase is much longer.
Shorter stationary phase.

12. GROWTH CURVE X = Biomass concentration Individual phases: 1: Lag phase
2: Accelaration
phase
3: Balanced growth
4: Deceleration
5: Stationary phase
6: Death phase

13. MULTIPLICATION CURVE N = No of cell Individual phases: 1: Lag phase
2: Accelaration
phase
3: Balanced growth
4: Deceleration
5: Stationary phase
6: Death phase

14. PRODUCTION KINETICS

15. To determine the metabolic parameters
Need data on:
substrate uptake with time
with and without product formation
product generation with time
with and without cell growth
cell growth with time

16. Specific growth rate:
Where:
dx = Change in biomass concentration.
dt = Change in incubation time.
x = biomass concentration.
Specific growth rate, µ expressed in reciprocal time unit (h-1).

17. Cultivation Temperature
During batch cultivation, specific growth rate changes
continuosly from zero to the max value µmax.
µmax depends on microorganisms, physical, chemical
conditions.
Typical values of µmax:
Microorganisms
Cultivation Temperature
µmax (h-1)
Bacteria
37ºC
Yeast
30ºC
Actinomycetes
28ºC
Fungal

18. By plotting the growth curve of the microorganisms, then determine the instavenous µ value at each sampling time by ascertaining the tangent at the point of contact on the growth curve.
The highest value obtained (from 24-72h) is the µmax.

19. The Yield Coefficient (Y)
A measure of the overall efficiency of the conversion of substrate to cell mass or specific product:
Y is not constant, will vary depending on organism, pH, temperature and substrate
Parameter
Equation
Cell (Y x/s)
ΔX / ΔS
Product (Y p/s)
ΔP / ΔS
Product (Y p/x)
ΔP / ΔX

20. Substrate Utilization and Product Formation (Yp/s)
g/l product produced
g/g carbon sources utilized
= g/g
Economic yield (Yp/x)
YP/X =
g/l product produced
g/L biomass formed
= g/g

21. Batch Productivity
Productivity – a measure of product ( or biomass) produced per unit time (g/L/h).
Product formation of growth – link product is closely related with growth rate.
Productivity in batch culture will be a greatest when growth rate max (µmax).

22. Productivity
(R batch) =
X max - Xo
T final – T initial
Where;
X max = maximum cell concentration at stationary phase
Xo = initial cell during inoculation
T final = time during which organism growing at µmax
T initial = time which organism not growing at µmax, including lag phase, deceleration phase period of batching, sterilizing and so on.

23. Incubation Time (h) dt 1/t [Cell] (g/L) dx 1/x dx/dt µ (h-1)24
0.042
0.1
0.004 48
0.021
0.2
10.0
0.04 72
0.014
0.3
5.0 96
3.33
120
144
168
0.05
192
0.02

24. CONTINUOUS CULTURE

25. CONTINUOUS CULTURE
Fresh fermentation media is continuosly added to the reactor while fermenter broth containing biomass, products and unused nutrient are continuosly removed.
Exponential growth in batch culture may be prolonged by the addition of fresh medium to the vessel.
Growth can be maintained for long duration
Continuous feeding to a culture at a suitable rate formation of new biomass by the culture is balanced by the loss of cell from the vessel STEADY STATE.

26. Application of Continuous Culture
Biomass production
Growth associated product or primary metabolite – e.g: ethanol, citric acid
Not suitable for non-growth associated or secondary metabolite – e.g: antibiotic

27. Important
When referring to continuous culture systems, the terms used in batch culture (lag, exponential, stationary, death phase)
have no meaning because the system is operating continuously and growth cannot segregated into phases

28. BATCH CULTURE
CONTINUOUS CULTURE
Nutrients added only at start
Nutrients added continuously
Product removed when fermentation stops.
Product continuously removed .
Growth rates and product formation are slower because limiting factors
ex: substrate levels/ build up of toxins.
Organism held in exponential growth phase giving higher productivity so can be on a smaller scale.
Slower growth rates = Larger vessels are used.
Easy to set up and maintain.
Can be very difficult to maintain conditions so that exponential phase is maintained. Foaming, clumping and blocked inlet pose problems.
If contamination occurs only one batch is wasted.
Contamination can afferct a huge volume of product/ organism.
Less efficient / more time wasted shutting down removing product and starting up again.
Continuous, therefore more efficient use of time.
Product quality can vary between batches.
Product quality more consistent.

29. FED-BATCH
CULTURE

30. FED BATCH CULTURE
Extending the batch culture by feeding continuously or periodically with medium with no removal of culture from the vessel.
Somewhere between batch and continuous culture.
A volume of medium is inoculated with the organism and allowed to grow for a batch period of time.
Subsequently, a feed is initiated into the fermenter when a “quasi steady state” is obtained.
Quasi steady state: when the growth limiting substrate has depleted.

31. PRODUCT FORMATION

32. Production kinetics
Classified based on the relationship between product synthesis and energy generation in the cell
Growth associated.
Non-growth associated.
Mixed-growth associated.

33. Products Growth-associated. produced at the same time as cell growth.
constitutive enzymes (ones that are normally present).
glucose isomerase.
metabolic intermediates.
pyruvate, citrate, acetate.
Non-growth-associated.
takes place during the stationary phase (m=0)
secondary metabolites.
Antibiotics.
Mixed - growth associated / Partially growth associated.
takes place during growth and stationary phases.
metabolic by-products.
lactate, ethanol.

34. Garden’s Law of Product Formation.
Growth-associated
Mixed-growth associated
Non-growth associated

35. Primary Metabolite
Released as a result of metabolic processes which are essential for the life of the micro-organism e.g. ethanol from Saccharomyces cerevisiae.
Thus, primary metabolites are produced throughout the growth of the micro-organism, especially through the exponential phase.

36. Secondary Metabolite
A substance which is not essential for the life of the micro-organisms and which is not produced as a result of the growth process e.g. penicillin.
Secondary metabolites are produced after the exponential growth phase has stopped.
This is important because it means that secondary metabolites such as penicillin cannot be produced in continuous fermenters – which deliberately maintain the micro-organism in the exponential growth stage.

37. Production of secondary metabolite starts when exponential growth stops and growth of cells starts to slow.
Adding a lot of extra nutrients at time T (see graph) will simply increase the growth of the micro-organism but not formation of the product.
However, by adding a small amount of extra nutrients at this time, the amount of product formed can be increased.