Microbial Nutrition and Growth

A. Nutrients
1. Macronutrients
a. They’re required in larger amounts. C, H, O, N, P, S and cations like Mg2+ Ca2+
and Fe2+ are added to the media as salts.
b. Carbon: organic sources include sugars and a.a. The inorganic source is CO2.
c. Nitrogen: organic sources include a.a and nucleotides. Inorganic sources
include ammonia (NH3) and nitrate (NO3-). A few species can fix N2
(Cyanobacteria).
d. Phosphorus: found in phosphate. Organic sources include phospholipids and
nucleotides. Inorganic sources are added to the media as salts.
e. Sulfur: organic sources include some a.a. Inorganic sources are added to the
media as salts.
2. Micronutrients: they’re required in very small amounts. Example: copper, nickel and
zinc. They’re not usually added to the media b/c there is enough in the water and on
the glassware., etc.
3. Growth Factors
a. GFs are pre-formed organic molecules that must be added to the media if the
bacteria does not have the genes that encode the enzymes that are required to
synthesize these molecules. The requirement of GFs varies with the bacterial
species.
b. There are 3 main types of GFs: vitamins, amino acids and purines and
pyrimidines.
c. Examples:
i. E. coli does not require any GFs
ii. Leuconostoc requires 20 a.a and 20 vitamins. B/c of their complex
nutritional requirements this genus is considered to be fastidious.
B. Culture Media
1. Selective media favours the growth of some bacteria and inhibits the growth of others.
Note: it may not kill them. Example: MacConkey Agar: it contains nutrients, it also
contains bile salts and crystal violet, which inhibit the growth of gm (+) bacteria.
2. Differential media can distinguish b/w differential bacteria based on differences in
metabolism. Example: MacConkey Agar: it is also a differential medium. It contains
lactose and pH indicator that changes to a red colour in acidic pH.
Note: a colony is a macroscopic clone of one cell growing on the surface of a solid
medium.
Results: pink colonies mean the gm (-) species can ferment lactose with an acidic
waste. Colourless colonies are gm (-) but they cannot ferment lactose with an acidic
waste.
C. Batch Culture (this section refers to the species that divide by binary fission)
1. Definitions:
a. Growth: an increase in cell number and or mass.
b. Generation time: (g) the amount of time it takes for the population to double.
c. Balance growth: the cellular components increase proportionally.
d. Unbalanced growth: the cellular components do not increase proportionally
 e. Batch culture: growth in a test tube or flask, containing a broth, it is usually a
pure culture.
f. Growth rate: the # of generations per hour

2. Batch cycle growth curve:

a. Lag phase: growth is unbalanced as cell adapt to new environment; lots of RNA
and proteins are synthesized but the cells are not dividing. The length of the lag
phase depends on the amount for adaptation required.
b. Log phase or exponential phase: the cells replicate exponentially. There is
balanced growth and the cells are dividing. The generation time is constant. The
length of the generation time is affected by environmental conditions like
temperature, type of media and the bacterial genetics. ie. Mycobacterium
tuberculosis (g) = 800 minutes. E. coli (g) = 20 min (under similar conditions).
c. Stationary phase: growth is affected by limiting nutrients like C, N and
accumulation of waste. During this phase some cells are growing and some are
dying. There is no change in cfu/ml.
i. Starvation response: during stationary phase, a few genera will produce
endospores. Most bacteria will synthesize starvation proteins that will
help the cell survive. Special proteins will bind to the DNA to help
protect it. Other proteins help prevent protein denaturation. There will
be an increase in the number of crosslinks in the peptidoglycan. This
results in the cell becoming tougher and more resistant to starvation,
damage by heat, osmotic pressure and some chemicals.
Note: gm stains are unreliable here
d. Death phase: there is an exponential decline in the cdu/ml

3. Metabolites:
a. Metabolism: all of the chemical reactions in a cell
b. Metabolite: any product of a chemical reaction in a cell
c. Primary metabolite: any metabolite required for log phase
d. Secondary metabolite: they’re not essential for growth. They’re often produced
in late log and stationary phase. ie. some bacteria synthesize antibiotics

4. Diauxic growth curve

a. E. coli in a medium with both glucose and lactose as a source of C , energy and
electrons.

D. Continuous culture
1.
a. A chemostat can be used for continuous culture. A reservoir containing sterile
medium is connected to a culture vessel. The operator can adjust the amount of
fresh medium being added to the culture vessel. The contents of the culture
 vessel, which include the medium, waste and bacteria are removed at the same
rate.
b. The medium contains a limiting amount of C or N, this means that the growth
rate is determined by the rate the fresh medium is being added.
2. Effect of dilution rate
D = F/V
D is the dilution rate = hr `1
F is flow rate = ml/hr
V is the culture vessel volume = ml

E. Environmental factors influencing growth

1. Descriptive terms:
a. phile: prefers and often means requires
b. tolerant: means it will grow in a wider range of conditions than non-tolerant
c. facultative: means optional mode
d. obligate: means an absolute requirement
e. hyper: means above
f. extreme: means outside of the normal range
2. Water availability is expressed as water activity or Aw. Solute in H2O interact with
water molecules and reduce their availability. Aw refers to H2O that is not bound to
solutes and is therefore available to bacteria. The higher the [solute] the lower the Aw.
Psoln
a. Aw = P = vapour pressure
P pure water
The values range from 0-1, pure H2O has an Aw of 1
b. Most bacteria have their maximum growth rate with Aw= ~ 0.995. As the Aw
decreases, the growth rate decreases, until it reaches a minimum Aw, thein it
cannot grow. A typical minimum Aw for bacteria is 0.95 - 0.98.
c. Bacterial cells in an environment with a high Aw will be protected by their
peptidoglycan.
d. Bacterial cells in an environment with an Aw lower than their minimum, will
become dehydrated. The cells will become dormant or undergo plasmolysis.
e. Osmotolerant bacteria are called halotolerant if the solute is NaCl. ie.
Staphylococcus species are halotolerant, their minimum Aw is 0.85.
f. Halophiles are adaptive to grow in the presence of NaCl, they require high
levels of salt to grow.
g. To prevent plasmolysis, osmotolerant, osmotolerant and halophilic bacteria
must keep their internal [solute] the same or higher of that of the environment.
They do this by the uptake or the synthesis of a “compatible solute” . Some
species accumulate ions or synthesize organic molecules like a.a or sugars.
h. Application of Aw to food preservation: Food can be dried or freeze dried,
sugar and or salt are also added to limit the amount of free water.

3. pH
a. Bacterial growth is affected by the environmental pH. The optimal pH can vary.
b. Acidophiles optimal pH is under 5.5
c. Most bacteria are neutrophiles and have an optimum pH by of about 7
ie. Helicobacter pylori is a neutrophile, it protects itself from the stomach acid
by secreting urease, the substrate of urease is urea. One product of this reaction
is ammonia which can bind H+ ions and therefore increase the pH.
d. Alkaliphiles optimum is over pH 8.
Application: some bacillus species are alkaliphiles, they secrete proteases and
lipases that are stable at a high pH. These enzymes are produced industrially,
added to laundry detergents.
e. The optimal pH refers to the external environment only, in most bacteria, the
cytoplasmic pH must remain neutral to prevent denaturation of proteins and
nucleic acids.
f. Acid tolerance: ie. E. coli O157:H7 has adapted to a lower pH. This helps it
survive a human’s acidic stomach.
4. Temperature
a. At the minimum temperature, enzymatic reactions are too slow to support
growth and membranes will solidify.
b. Above the maximum temperature, membranes will melt and proteins will
denature and the cell will die.
 c. The optimal temperature is closer to the maximum temperature than the
minimum temperature. Different bacteria have evolved so that their proteins
and membranes function at different temperatures.
d. The range b/w min and max temperature is typically ~ 30 C.
e. Graph
f. Examples of classifications:
i. Psychrotrophs: their optimal is about 21 C, they commonly cause food
spoilage and can grow in the refrigerator. Refrigerator is at 4 C.
ii. Mesophiles: the optimal temp for bacteria that live on the body is about
37 C. The optimal for soil organisms is ~ 30 C.
5. Oxygen Concentration
a. Classifications:
i. Obligate aerobes: require oxygen as the TEA of their ETC.
ii. Microaerophiles: require O2 as the TEA of their ETC, but are killed by
atmospheric levels of it.
iii. Facultative anaerobes: grow best with oxygen, O2 as TEA but can grow
without it.
iv. Aerotolerant anaerobes: don’t use O2 as TEA but aren’t killed by it.
v. Obligate anaerobes: don’t use O2 as TEA and are killed by toxic oxygen
derivatives
b. Toxic oxygen derivatives: oxygen is a highly reactive molecule:
O2 + e ----> O2- (superoxide radical)
O2 + e + 2H+ -----> H2O2 (hydrogen peroxide)
H2O2 + e + H -----> H2O + OH (hydroxyl radical)
These toxic products can damage cellular components by oxidizing them, and
thi will kill the cell.
c. Protective enzymes:
i. Superoxide dismutase (SOD):
O2- ----> H2O2
ii. Catalase:
H2O2 -----> H2O + O2
(Other enzymes can do this)

Name SOD Catalase

Aerobes + facultative + +
anaerobes

Microaerophiles Less Less (not as much)

Aerotolerant anaerobes + Other enzyme instead of

catalase

Obligate anaerobes - -

d. Application: Identification
 i. Catalase test: add H2O2 to slide and add some bacteria, if you see
bubbles, the test is catalase positive, if not it’s catalase negative.
ie. Streptococcus species are aerotolerant and catalase negative.

F. Growth in a natural environment

1.
a. It is complicated and not a pure culture.
b. In nature, bacteria naturally aggregate with other bacteria and form a biofilm.
c. Biofilms form on solid liquid interphases. Biofilms are composed of organized
regions of bacterial cells embedded in an exopolymeric substance or EPS. As
the biofilm matures it will develop channels to permit the exchange of nutrient
and wastes.
2. Benefits of living in a biofilm:
a. Prevents them from being washed away
b. It provides protection from dehydration, chemical and UV light.
c. It facilitates a food web, this means it allows different species to interact with
each other. ie. one will use others’ waste as a source of energy
d. They’re able to communicate within their population.

G. Quorum sensing:
1. Quorum (sufficient numbers) sensing is used by bacteria to assess population density.
At a high population density, it results in the coordination of the transcription of
specific genes.
2. Signaling molecule
a. Each species synthesizes a signalling molecule called autoinducer, in gram (-),
the autoinducer can diffuse freely in and out of the cell.
b. High concentration of the autoinducer inside the cytoplasm triggers the
transcription of specific genes.
3. Vibrio fischeri
a. Vibrio fischeri is a gram (-) bacterium that lives freely in seawater. It also lives
as synbiont in the light organs of some fish and squid.
b. Vibrio fischeri synthesizes an autoinducer that will diffuse down its
concentration gradient outside the cell. In free living bacteria, the [autoinducer]
inside the cell will always be low.
c. If the bacteria is living in a contained environment of a light organ, the
population density will increase. This means that the concentration of the
autoinducer outside the cell will increase. The rate of diffusion is reduced and
the intracellular concentration increases. This results in the expression of the
lux genes which convert chemical energy into light.

Control microbial growth by physical and chemical agents

A. Overview
1. Definition
a. Sterilization is a process that removes/kills all viable cells and endospores
 b. Disinfection is the killing of vegetative pathogens. Not all non-pathogenic
vegetative cells or endospores will be killed/removed.
2. The efficiency of the microbial agent is affected by:
a. Population size: the larger the population, the longer it takes to kill them
b. The growth stage of the cell: cells in stationary phase and endospores are
harder to kill
c. Temperature: A higher temperature increases the activity of most chemical
reactions
d. Environment: organic matters protect the bacteria from chemicals ie. biofilms,
vomit, blood and feces.

B. Physical Methods of Control

1. Moist heat
a. Moist heat works by denaturing proteins and nucleic acids
b. Boiling water for 10 minutes disinfects it
c. An autoclave uses moist heat and pressure to produce 121 C. It’s used to sterilize
liquid-like media.
d. Pasteurization: is a brief heating of a liquid to kill pathogens and reduce the
number of food spoilage organisms.
2. Dry Heat
It also works by the denaturation of proteins and nucleic acids, but it’s less effective. It
can be used to sterilize or disinfect. ie. bacticinirator, oven
3. Low Temperature
a. Freezing stops cells from growing, but doesn’t kill them
b. Refrigeration slows the growth of pathogen but not food spoilage
psychrotrophs.
4. Filtration
a. Fluids: a 0.2um pore filter removes vegetative cells from heat-sensitive fluids

b. Air
i. Mask filter air to protect the workers
ii. HEPA filters are used to filter air in a number of situations
5. Water activity (see above)

C. Chemical Methods of Control

1. Activity level of a chemical is rated as high, intermediate or low. The choice of a
chemical weighs its activity and toxicity.
2. Activity Level
a. High activity kills all vegetative cells and all endospores. ie. ethylene oxide is a
gas that is used to sterilize heat sensitive plastics like petri dishes and medical
implants. Second example: aldehydes like formaldehyde, the problem is
toxicity.
b. Intermediate activity kills all vegetative cells: for example, 70% ethanol is used
to disinfect surfaces. Second example: halogens: chlorine is used to disinfect
water. It can be toxic or irritant.
 c. Low activity kills all vegetative cells except, M. tuberculosis. For example:
quaternary ammonium compounds (QUATS), these are cationic detergents
thats are used in hospitals. Detergents form micelles and can remove microbes
from the environment. They are also disinfectants.
D. Selection of antimicrobial procedure home vs. hospital
1. Consider:
a. The type of microbe expected
b. The environment
c. The expected number of bacteria
d. The toxicity of antimicrobial agent

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L. Chemotaxis
1. Signal transduction systems
a. Bacterial cells can respond to extra-cellular signals using two component signal
transduction system.
b. Components:
i. Sensor kinase
ii. Cytoplasmic response regulator
c. Function: the sensor kinase detects an environmental signal,
autophosphorylates and then activates the response regulator by
phosphorylating it.
d. The activated response regulator mediates the required change in the cell.
e. Some signal transduction systems like chemotaxis require additional proteins.
2. Note
a. We will only look at response to attractants, the attractants include sugars and
amino acids.
b. Motile means capable of moving (different than mobile)
3. Chemotaxis components location
a. Methyl-accepting chemotaxis proteins (MCP) are integral PM proteins.
b. CheA is associated with the cytoplasmic side of the MCP
c. CheB, CheR, CheY, and CheZ are cytoplasmic
4. Chemotaxis components basic function:
a. CheA is a sensor kinase
b. CheB is a response regulator
c. CheY is a response regulator
d. CheR methylates the MCP
e. CheZ dephosphorylates CheY-P
5. MCP function
a. The MCP regulates the rate of CheA autophosphorylation
b. The periplasmic side of MCP can bind attractants temporarily. When an
attractant is bound to MCP, a signal is sent inside to CheA, inhibiting its
autophosphorylation. The filament rotates CCW and the cell will run.
6. Autophosphorylation of CheA
a. If not attractant is bound to MCP, CheA will use ATP to autophosphorylate, then
CheA-P will phosphorylate CheY and CheB.
7. CheA-P
a. CheA-P interacts with Fli protein. This causes the filament to change direction
and turn CW. The cell will tumble.
b. After a few seconds, CheZ will dephosphorylate CheY-P. The filaments will
turn CCW and the cell will run.
8. CheB-P
a. CheR steadily methylates the MCP. Each MCP can have several methyl groups
attached.
b. CheB-P demethylates MCP if no attractant is bound to MCP. Lots of CheB is
being phosphorylated and the level of MCP methylation is low.
Note: two things are going on at the same time. The filament is going CW/CCW
and the MCP is being methylated/demethylated. Both are affected by CheA
autophosphosylation.
9. Random Walk
a. If the E. coli is not in the gradient of attractant, it moves in a random walk
composed of short runs and then tumbles repeatedly.
10. Chemotaxis
a. Chemotaxis occurs in the gradient of attractant
b. If the concentration of attractant increases, the lengths of the run will increase,
this is because attractant is bound more often to the MCP and CheA
autophosphorylation is inhibited.
c. The lengths of the run will decrease if the cell moves down an attractant
gradient. Attractant will be bound less often to the MCP, CheA will
autophosphorylate and in turn phosphorylate CheY and CheB. CheA-P signals
cell to tumble.
11. Adaptation
a. If the level of attractant is high it is to the cells advantage to remain in the area
and not continue running out of it.
b. CheR continues to methylate MCP but now CheB is not being phosphorylated,
because there are lots of attractants bound to MCP, this means that the level of
methylation of MCP is high. This results is a conformational change of MCP, this
causes a signal to be sent to CheA to autophosphosylate and in turn
phosphorylate CheY (and CheB). This results in a tumble and the cell will remain
in the area.
12. E. coli mutants
a. This E.coli mutants have a non-functional MCP. They don’t signal CheA to
autophosphosylate. However, these cells still have normal chemotaxis of sugars
taken up by phosphotransferase system.

See picture

b.
i. EIIB-P can phosphorylate the sugars being transported into the cell or
CheA.
ii. The more sugar that is taken up and phosphorylated, the fewer CheA
will be phosphorylated. This produces runs.
 iii. When there is no sugar bring phosphorylated. CheA would be
phosphorylated and the cell will tumble and change direction.
M. Endoflagella
1. The corkscrew shaped Spirochetes (phylum, no italics) are gm (-) bacteria that have an
unusual method of motility. They use endoflagella.
2. Structure:
a. Like flagella, endoflagella are composed of a basal body, a hook and a filament.
However, the basal body lacks the P+L rings and does not cross the OM. the
filaments are located in the periplasm b/w peptidoglycan and the OM.
b. Equal numbers are endoflagella are attached near each pole of the cell. The
filaments form a bundle which wraps around the helical cell. They usually
overlap in the middle.
3. Function:
a. Like flagella, they use the PMF as a source of energy. In some species, rotation of
the filaments produces a helical wave down the length of the cell, resulting in a
corkscrew movement.
b. This method of motility is most effective in highly viscous fluids.

N. Endospores
1.
a. They are found in domain bacteria only. They are only synthesized by a few
genera of gm(+) rod ie. Bacillus and Clostridium both live in the soil.
b. Function: endospores are very tough, they’re resistant to heat, UV and gamma
radiation, desiccation and disinfectants. They enable the organism to survive
during times of nutrient depletion. They can remain viable for thousands of
years. Some can survive boiling for more than an hour. Some are dangerous
pathogens that can cause anthrax and botulism.
c. Structure: the core contains the chromosome, ribosomes, enzymes, etc. It has a
low water content, which means endospores are inert (there is no metabolism).
It is covered by layers of peptidoglycan and proteins.
2. Sporulation
a. Sporulation is the process of a vegetative or metabolizing cell, producing one
endospore. It is a very complex process that takes about 200 genes and 10
hours. It is triggered when the vegetative cell becomes depleted of a source of C
or N.
b. Sporulation simplified
i. The chromosome is replicated
ii. The PM form a septum that divides the cytoplasm asymmetrically into
two. The larger part is called the mother cell. The smaller is called the
forespore. Each one has a copy of the chromosome.
iii. The mother cell engulfs the forespore. The forespore is now a protoplast
with two layers of membranes.
iv. Peptidoglycan is added b/w the two membranes of the forespore.
v. A thick layer of proteins is also added on the outermost membrane
 vi. The endospore matures and becomes refractile, heat resistant, etc.
Refractile means it looks bright under the microscope.
vii. The mother cell lyses, releasing the endospore.
3. Conversion to a vegetative cell
a. This process is also complex, it uses different genes and only takes about 90
minutes.
b. Steps
i. Activation: a trigger like heat or a change in pH is required to break the
dormant state.
ii. Germination: it is triggered by the presence of nutrients like sugars and
amino acids. A series of changes occur. Water enters the core and the
enzymes become active.
iii. Outgrowth: the return to the vegetative state.
4. Identification: the endospore stain is used to identify endospores using a microscope.
It is useful b/c there is only a few genera of gm(+) rods that produce endospores.

5. Bacillus thuringiensis
a. During sporulation, this species synthesizes an endospore and a crystal made
from multiple subunits of a protein, protoxin.
b. In nature, insects with an alkaline gut are susceptible to the toxic protein. If
they consume the endospore and the crystal, the high pH of their gut
solubilizes the protoxin; an insect digestive protease, cleaves the protoxin,
producing an active form.
c. Multiple subunits of the toxin insert into the PM of the cell lining in the insect’s
gut and form a pore. This causes the cell’s contents to leak out, providing the
nutrients for the endospore.
d. The endospore will get activated by the high pH, the leaked nutrients promote
germination and outgrowth. The insect will die.
e. Industrial application: the insecticide called Bt
i. Large quantities of this species might be grown (a chemostat) and trigger
to undergo sporulation. The endospores and crystals are isolated and
marketed as Bt. Bt can be sprayed on plants to kill certain insects.
ii. It is much safer to use than a chemical insecticide b/c it only targets
some insects and not animals. Also, b/c the crystals are made of
proteins, it is easily degradable in the environment.

O. Archaea Overview only

1. Size and shape
a. Like the domain bacteria, most Archaea are cocci or rods. Unique shapes
include branched cell. There is a species that is flat square shape.
b. Like bacteria, most Archaea are 1-3um in diameter
2. PM Lipids
a. They differ from those of bacteria and eukarya.