BACTERIAL GROWTH
Industrial Microbiology
Bacterial growth



Binary fission
Generation time
Phases of growth
4-2
Binary fission
Figure 4.2
1.
Prokaryote cells grow
by increasing in cell
number (as opposed to
increasing in size).
2.
Replication is by binary
fission, the splitting of
one cell into two
3.
Therefore, bacterial
populations increase by
a factor of two (double)
every generation time.
Generation time



The time required to for a population to double (doubling
time) in number.
Ex. Escherichia coli (E. coli) double every 20 minutes
Ex. Mycobacterium tuberculosis double every 12 to 24
hours
4-4
Principles of Bacterial Growth

Growth can be calculated

Nt = N0 x 2n




(Nt ) number of cells in population
(N0 ) original number of cells in the population
(n) number of divisions
Example





N0 = 10 cells in original population
n = 12
 4 hours assuming 20 minute generation time
Nt = 10 x 212
Nt = 10 x 4,096
Nt = 40,960
Growth in Batch Culture
1.
Bacteria growing in batch culture produce a growth curve with up to four
distinct phases.
2.
Batch cultures are grown in tubes or flasks and are closed systems where no
fresh nutrients are added or waste products removed.
3.
Lag phase occurs when bacteria are adjusting to them medium. For example,
with a nutritionally poor medium, several anabolic pathways need to be turned
on, resulting in a lag before active growth begins.
4.
In log or exponential phase, the cells are growing as fast as they can, limited
only by growth conditions and genetic potential. During this phase, almost all
cells are alive, they are most nearly identical, and they are most affected by
outside influences like disinfectants.
5.
Due to nutrient depletion and/or accumulation of toxic end products,
replication stops and cells enter a stationary phase where there is no net
change in cell number.
6.
Death phase occurs when cells can no longer maintain viability and numbers
decrease as a proportion.
Growth in Batch Culture
Mean Generation Time
and Growth Rate

The mean generation time (doubling time) is
the amount of time required for the
concentration of cells to double during the log
stage. It is expressed in units of minutes.
1
-1
 Growth rate (min ) =
mean generation time

Mean generation time can be determined
directly from a semilog plot of bacterial
concentration vs time after inoculation
Mean Generation Time
and Growth Rate
Basic Chemostat System
Lab Chemostat System
Environmental factors
Temperature
 Oxygen requirement
 pH
 Water availability

4-13
Temperature
 Enzymes, the machinery of the cell, are influenced
by external factors and can be shown to have a
range where they function that includes an optimal
value that produces the highest activity.
 The range of enzyme activity determines the range
for growth of specific bacteria, analogously leading
to a value for optimal growth rate.
 In the case of temperature, bacteria are divided
into categories based on the temperature range
where they can grow and the temperature that
provides optimal growth.
4-14
Temperature

Psychrophile


0o to 18o C
Psychrotroph

20°C to 30°C


Mesophile

25°C to 45°C



More common
Disease causing
Thermophiles

45°C to 70°C


Important in food spoilage
Common in hot springs and hot water heaters
Hyperthermophiles

70°C to 110°C



Live at very high temperatures, high enough where water threatens to become
a gas
Usually members of Archaea
Found in hydrothermal vents
Oxygen requirements
•
•
•
•
•
•
Oxygen is a very reactive molecule and can affect cells in several ways. The effect of oxygen is often determined using
thioglycollate broth, a special medium that contains a reducing agent (thioglycollate) that removes oxygen so that a
gradient occurs within the tube.
Obligately aerobic bacteria can obtain energy only through aerobic respiration and have to have oxygen available. Thus,
they will grow only at the surface of thioglycollate broth.
Obligately anaerobic bacteria die in the presence of oxygen and can only grow at the bottom of thioglycollate broth.
Some anaerobes are so sensitive to oxygen that even thioglycollate broth is not anoxic enough to provide suitable
anaerobic conditions.
Microaerophiles require oxygen for growth but the 20% in air is too toxic. As a result, they grow near the top but
beneath the surface of thioglycollate broth where the oxygen concentration is typically 4 – 10%.
Facultative anaerobes can use oxygen for aerobic respiration but can switch to fermentative metabolism in the absence
of oxygen. As a result, they will grow throughout thioglycollate broth. (Heavier growth at top.)
4-16
Aerotolerant anaerobes are anaerobic bacteria that can grow in the presence of
air. (Not shown in diagram.)
pH





Neutrophiles grow best around neutral pH (7)
Acidophiles grow best at pH < 7
Alkophiles grow best at pH > 7
Acidotolerant grow best at pH 7 but can also grow at
lower pH
Alkotolerant grow best at pH 7 but can also grow at
higher pH
4-17
Water Activity
 Liquid water is essential for life.
 Aqueous solutions actually have different amounts of water
available, depending on how many solutes are dissolved in it.
As a very simple model, consider two glasses, one full of pure
water, the other containing the same amount of water plus a
sponge. Which one would be easier to drink? On a much
smaller scale, dissolved solutes act like a sponge, making less
water available.
 Water activity (aw) can be decreased by the addition of any
soluble molecule although salt (NaCl) and sugars are probably
the most common.
Water Activity




Microbes that require a high water activity (near or at 1) are
termed nonhalophiles. (Halophile = salt-loving)
Some bacteria require salt to grow and are called halophiles. If a
very high concentration of salt is required (around saturation), the
organisms are termed extreme halophiles.
A nonhalophile that can grows best with almost no salt but can still
grow with low levels of salt (~ 7%) is called halotolerant.
In general, fungi are more tolerant of low water activity.
(That’s why your jelly is more likely to get contaminated by
fungi than bacteria.)
Nutritional Requirements


Growth of prokaryotes depends on nutritional
factors as well as physical environment
Main factors to be considered are:
 Required
elements
 Growth factors
 Energy sources
 Nutritional diversity
Nutritional Requirements

Major elements (CHONPS + K, Mg, Fe, Ca)

Carbon, oxygen, hydrogen, nitrogen, sulfur, phosphorus,
potassium, magnesium, iron, and calcium


Organisms classified based on carbon usage

Heterotrophs


Use inorganic carbon (CO2) as carbon source
Trace elements (Co, Cu, Ni, Zn, Se, Mg, Wo)

Cobalt, zinc, copper, molybdenum and manganese



Use organism carbon as nutrient source
Autotrophs


Essential components for macromolecules
Required in minute amounts
Assist in enzyme function
Nutritional diversity

Different organisms require the same nutrients but may require
different forms of the nutrients
Major elements
Element
% dry wgt
Source
Carbon
50
organic compounds or CO2
Oxygen
20
H2O, organic compounds, CO2, and O2
Nitrogen
14
NH3, NO3, organic compounds, N2
Hydrogen
8
H2O, organic compounds, H2
Phosphorus
3
inorganic phosphates (PO4)
Sulfur
1
SO4, H2S, So, organic sulfur compounds
Potassium
1
Potassium salts
Magnesium
0.5
Magnesium salts
Calcium
0.5
Calcium salts
Iron
0.2
Iron salts
Carbon Source

Organic molecules
 Heterotrophs

Inorganic carbon (CO2)
 Autotrophs
Nitrogen Source

Organic nitrogen
 Primarily

Oxidized forms of inorganic nitrogen
 Nitrate

from the catabolism of amino acids
(NO32-) and nitrite (NO2-)
Reduced inorganic nitrogen
 Ammonium

(NH4+)
Dissolved nitrogen gas (N2) (Nitrogen
fixation)
Phosphate Source


Organic phosphate
Inorganic phosphate (H2PO4- and HPO42-)
Sulfur Source


Organic sulfur
Oxidized inorganic sulfur
 Sulfate

Reduced inorganic sulfur
 Sulfide

(SO42-)
(S2- or H2S)
Elemental sulfur (So)
Growth Factors

Some bacteria cannot synthesize some cell
constituents

These must be added to growth environment


Organisms can display wide variety of factor
requirements

Some need very few while others require many


Referred to as growth factors
These termed fastidious
Typical molecules
Amino acids
 Nucleotide bases
 Enzymatic cofactors or “vitamins”

Culture Media





Complex (contains undefined components)
Chemically defined (all concentrations are known)
Selective (favors the growth of a particular organism or
group of organisms)
Differential (has reactions that give isolates different
appearance)
Anaerobic (oxygen-free)
4-30
4-31
Characteristics of Media