Microbial Nutrition and Growth

1.
2.
3.
4.
5.
6.
7.

Nutrient requirements
Types of microbiological media
Factors that influence growth
Measurement of growth
Growth in batch culture
Growth in continuous culture
Growth in colonies and biofilms

A. Nutrient Requirements
1. Energy Source
a) Phototroph

Uses light as an energy source

b) Chemotroph

Uses energy from the oxidation of reduced

chemical compounds

A. Nutrient Requirements
2. Electron (Reduction potential) Source
a) Organotroph

Uses reduced organic compounds as a source for

reduction potential

b) Lithotroph

Uses reduced inorganic compounds as a source for

reduction potential

A. Nutrient Requirements
3. Carbon source
a) Autotroph

Can use CO2 as a sole carbon source

(Carbon fixation)

b) Heterotroph

Requires an organic carbon source; cannot use CO2

as a carbon source

A. Nutrient Requirements
4. Nitrogen source
a) Organic nitrogen

Primarily from the catabolism of amino acids

b) Oxidized forms of inorganic nitrogen

Nitrate (NO32-) and nitrite (NO22-)

c) Reduced inorganic nitrogen

Ammonium (NH4+)

d) Dissolved nitrogen gas (N2) (Nitrogen fixation)

A. Nutrient Requirements
5. Phosphate source
a) Organic phosphate
b) Inorganic phosphate (H2PO4- and HPO42-)

A. Nutrient Requirements
6. Sulfur source
a) Organic sulfur
b) Oxidized inorganic sulfur

Sulfate (SO42-)

c) Reduced inorganic sulfur

Sulfide (S2- or H2S)

d) Elemental sulfur (So)

A. Nutrient Requirements
7. Special requirements
a) Amino acids
b) Nucleotide bases
c) Enzymatic cofactors or vitamins

A. Nutrient Requirements
8. Prototrophs vs. Auxotrophs
a) Prototroph

A species or genetic strain of microbe capable of

growing on a minimal medium consisting a simple
carbohydrate or CO2 carbon source, with inorganic
sources of all other nutrient requirements

b) Auxotroph

A species or genetic strain requiring one or more

complex organic nutrients (such as amino acids,
nucleotide bases, or enzymatic cofactors) for
growth

B. Types of Microbiological Media

1. Liquid (broth) vs. semisolid media
a) Liquid medium

Components are dissolved in water and sterilized

b) Semisolid medium

A medium to which has been added a gelling agent

Agar (most commonly used)
Gelatin
Silica gel (used when a non-organic gelling agent is
required)

B. Types of Microbiological Media

2.

Chemically defined vs. complex media

a)

Chemically defined media

The exact chemical composition is known

e.g. minimal media used in bacterial genetics experiments

b) Complex media

Exact chemical composition is not known

Often consist of plant or animal extracts, such as soybean meal,
milk protein, etc.
Include most routine laboratory media,
e.g., tryptic soy broth

B. Types of Microbiological Media

3. Selective media
a) Contain agents that inhibit the growth of certain
bacteria while permitting the growth of others
b) Frequently used to isolate specific organisms from
a large population of contaminants

4. Differential media
a) Contain indicators that react differently with
different organisms (for example, producing
colonies with different colors)
b) Used in identifying specific organisms

C. Factors that Influence Growth

1.

Growth vs. Tolerance

Growth is generally used to refer to the acquisition of
biomass leading to cell division, or reproduction
b) Many microbes can survive under conditions in which they
cannot grow
c) The suffix -phile is often used to describe conditions
permitting growth, whereas the term tolerant describes
conditions in which the organisms survive, but dont
necessarily grow
d) For example, a thermophilic bacterium grows under
conditions of elevated temperature, while a thermotolerant
bacterium survives elevated temperature, but grows at a
lower temperature
a)

C. Factors that Influence Growth

2.

Obligate (strict) vs. facultative

a)

Obligate (or strict) means that a given condition is

required for growth
b) Facultative means that the organism can grow under the
condition, but doesnt require it
c) The term facultative is often applied to sub-optimal
conditions
d) For example, an obligate thermophile requires elevated
temperatures for growth, while a facultative thermophile
may grow in either elevated temperatures or lower
temperatures

C. Factors that Influence Growth

3. Temperature
a) Most bacteria grow throughout a range of
approximately 20 Celsius degrees, with the
maximum growth rate at a certain optimum
temperature
b) Psychrophiles: ~0 20C
c) Mesophiles: ~20 45C
d) Moderate thermophiles: ~45 70 C
e) Extreme thermophiles: ~70 100 C

C. Factors that Influence Growth

4. pH
a) Acidophiles:

Grow below ~pH 6.0;

generally between pH 2 6

b) Neutrophiles

Grow between pH 6 8

c) Alkalophiles

Grow above pH 8;
generally between pH 8 9.5

C. Factors that Influence Growth

5. Salt concentration/sugar concentration
a) Halophiles grow at elevated salt
concentrations; often 1 M ionic strength or
greater
b) Osmophiles, grow at elevated sugar
concentrations.

C. Factors that Influence Growth

6.

Oxygen concentration
a) Strict aerobes: Require oxygen for growth (~20%)
b) Strict anaerobes: Grow in the absence of oxygen; cannot
grow in the presence of oxygen
c) Facultative anaerobes: Grow best in the presence of oxygen,
but are able to grow (at reduced rates) in the absence of
oxygen
d) Aerotolerant anaerobes: Can grow equally well in the
presence or absence of oxygen
e) Microaerophiles: Require reduced concentrations of oxygen
(~2 10%) for growth

D. Measurement of Growth
1. Microscopic cell counts
2. Serial dilution and colony counting
a) Also know as viable cell counts
b) Concentrated samples are diluted by serial dilution

D. Measurement of Growth
c) Diluted samples are spread onto media in petri
dishes and incubated
d) Colonies are counted. The concentration of
bacteria in the original sample is calculated
(from plates with 30 300 colonies).
CFU
# colonies counted
in original sample
ml
(dilution factor)(volume plated, in ml)

D. Measurement of Growth
3. Membrane filtration
a) Used for samples with low microbial
concentration
b) A measured volume (usually 1 to 100 ml) of
sample is filtered through a membrane filter
(typically with a 0.45 m pore size)
c) The filter is placed on a nutrient agar medium
and incubated
d) Colonies grow on the filter and can be counted

D. Measurement of Growth
4. Turbidity
a) Based on the diffraction or scattering of light
by bacteria in a broth culture
b) Light scattering is measured as optical
absorbance in a spectrophotometer
c) Optical absorbance is directly proportional to
the concentration of bacteria in the suspension

D. Measurement of Growth
5. Mass determination
a) Cells are removed from a broth culture by
centrifugation and weighed to determine the
wet mass.
b) The cells can be dried out and weighed to
determine the dry mass.

6. Measurement of enzymatic activity

E. Growth in Batch Culture

1.

2.

A batch culture is a closed system in broth

medium in which no additional nutrient is added
after inoculation of the broth.
Typically, a batch culture passes through four
distinct stages:
a)
b)
c)
d)

Lag stage
Logarithmic (exponential) growth
Stationary stage
Death stage

Lag

Log

Stationary

Death

Bacterial Cells/ml

10000000
1000000
100000
10000
1000
0

50

100

150

200

Time After Inoculation (min)

250

300

E. Growth in Batch Culture

1. The rate of growth, or mean generation
time, is the amount of time required for the
concentration of cells to double during the
log stage.

Growth of E. coli 23716,

9-20-01 batch culture

y = 0.0187e

0.0069x

R = 0.9928
10

A425

0.1

0.01
0

200

400

600

800
time, min

1000

1200

1400

1600

F. Growth in Continuous Culture

1.

2.
3.

A continuous culture is an open system in

which fresh media is continuously added to the
culture at a constant rate, and old broth is
removed at the same rate.
This method is accomplished in a device called a
chemostat.
Typically, the concentration of cells will reach an
equilibrium level that remains constant as long as
the nutrient feed is maintained.

Basic Chemostat System

Our Chemostat System

G. Growth in Colonies and Biofilms

1.

2.
3.

In nature, microbes often grow as colonies or

biofilms attached to surfaces (such as soil
particles, teeth, etc.)
The metabolic state and morphology of cells may
vary significantly in different parts of the biofilm.
Frequently, there are several species interacting
symbiotically in the biofilm, forming an ecological
community.