micro nutrition

Microbial Nutrition and Growth

1. Nutrient requirements

2. Types of microbiological media

3. Factors that influence growth

4. Measurement of growth

5. Growth in batch culture

6. Growth in continuous culture

7. Growth in colonies and biofilms

A. Nutrient Requirements

1. Energy Sourcea) 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) Sourcea) 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 sourcea) 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 sourcea) Organic nitrogen

• Primarily from the catabolism of amino acids

b) Oxidized forms of inorganic nitrogen• Nitrate (NO3

2-) and nitrite (NO22-)

c) Reduced inorganic nitrogen• Ammonium (NH4

+)

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

A. Nutrient Requirements

5. Phosphate sourcea) Organic phosphate

b) Inorganic phosphate (H2PO4- and HPO4

2-)

A. Nutrient Requirements

6. Sulfur sourcea) Organic sulfur

b) Oxidized inorganic sulfur• Sulfate (SO4

2-)

c) Reduced inorganic sulfur• Sulfide (S2- or H2S)

d) Elemental sulfur (So)

A. Nutrient Requirements

7. Special requirementsa) Amino acids

b) Nucleotide bases

c) Enzymatic cofactors or “vitamins”

A. Nutrient Requirements

8. Prototrophs vs. Auxotrophsa) 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 mediaa) 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 mediaa) 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 mediaa) Contain agents that inhibit the growth of certain

bacteria while permitting the growth of othersb) Frequently used to isolate specific organisms

from a large population of contaminants

4. Differential mediaa) 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. Tolerancea) “Growth” is generally used to refer to the acquisition of

biomass leading to cell division, or reproductionb) Many microbes can survive under conditions in which they

cannot growc) The suffix “-phile” is often used to describe conditions

permitting growth, whereas the term “tolerant” describes conditions in which the organisms survive, but don’t 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

C. Factors that Influence Growth

2. Obligate (strict) vs. facultativea) “Obligate” (or “strict”) means that a given condition is

required for growth

b) “Facultative” means that the organism can grow under the condition, but doesn’t 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. Temperaturea) Most bacteria grow throughout a range of

approximately 20 Celsius degrees, with the maximum growth rate at a certain “optimum temperature”

b) Psychrophiles: ~0 – 20ºC

c) Mesophiles: ~20 – 45ºC

d) Moderate thermophiles: ~45 – 70 ºC

e) Extreme thermophiles: ~70 – 100 ºC

C. Factors that Influence Growth

4. pHa) 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 concentrationa) 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 concentrationa) 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 countinga) 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).

ml) in plated, lumefactor)(vo (dilution

counted colonies # sample original in

ml

CFU

D. Measurement of Growth

3. Membrane filtrationa) Used for samples with low microbial

concentrationb) 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. Turbiditya) 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 determinationa) 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. A “batch culture” is a closed system in broth medium in which no additional nutrient is added after inoculation of the broth.

2. Typically, a batch culture passes through four distinct stages:

a) Lag stage

b) Logarithmic (exponential) growth

c) Stationary stage

d) Death stage

Lag Log Stationary Death

Time After Inoculation (min)

Bac

teri

al C

ells

/ml

1000

10000

100000

1000000

10000000

0 50 100 150 200 250 300

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

R2 = 0.9928

0.01

0.1

1

10

0 200 400 600 800 1000 1200 1400 1600

time, min

A42

5

F. Growth in Continuous Culture

1. 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.

2. This method is accomplished in a device called a chemostat.

3. 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. In nature, microbes often grow as colonies or biofilms attached to surfaces (such as soil particles, teeth, etc.)

2. The metabolic state and morphology of cells may vary significantly in different parts of the biofilm.

3. Frequently, there are several species interacting symbiotically in the biofilm, forming an ecological community.