Download Citric Acid Cycle

Bacterial Physiology
王淑鶯
微生物免疫學所
國立成功大學醫學院
分機: 5634
Email: [email protected]

Microbial metabolism

Microbial growth
•Reference:
Chapter 3 in Medical Microbiology
(Murray, P. R. et al; 6th edition)
What is Metabolism?

The Greek metabole, meaning change

It includes all the biochemical reactions
that occur in the cell.
- Catabolism
- Anabolism
Why do we must know the
metabolism of bacteria ?
Because we want to control their metabolism
and know how to inhibit or stop bacteria
growth
Why Study Metabolism?

Classification of bacteria



Oxygen Tolerance
Biochemical reactions
Fermentation Products

Food Products


Commercial Products


Yogurt, Sour Cream, Bread, Alcohol
Citric Acid
Environmental Cleanup

e.g. common soil bacteria could clean up
nuclear contamination
Common Soil Bacteria Could Clean Up Nuclear Contamination
COLUMBUS, Ohio, March 17, 2009 (ENS) - An international team of scientists has found a
common soil bacterium that might one day be used to clean up radioactive toxics left from
nuclear weapons production decades ago.
The bacteria's cleaning power comes from their ability to "inhale" toxic metals and "exhale" them
in a non-toxic form, explains team member Brian Lower, assistant professor in the School
of Environment and Natural Resources at Ohio State University.
Using a unique combination of microscopes, researchers at Ohio State University and scientists
from Austria, Sweden, Switzerland and the United States were able to see how the
bacterium Shewanella oneidensis breaks down metal to chemically extract oxygen.
The study, published online this week in the journal "Applied and Environmental Microbiology,"
provides the first evidence that the Shewanella bacterium maneuvers proteins within the
bacterial cell into its outer membrane to contact metal directly.
The proteins then bond with metal oxides, which the bacteria utilize the same way we use oxygen
- to breathe.
"We use the oxygen we breathe to release energy from our food. But in nature, bacteria don't
always have access to oxygen," said Lower. "Whether the bacteria are buried in the soil or
underwater, they can rely on metals to get the energy they need. It's an ancient form of
respiration."
Catabolism
Anabolism
Metabolites
degrade, break bonds, convert large
molecules into smaller component
often produce energy
synthesis of cell molecules and structures
usually requires the input of energy
compounds given off by the complex
networks of metabolism
Overview of cell metabolism
Microbes need three things to grow



Energy source
Nutrients (C)
Suitable environmental conditions
Nutrient Requirements

Energy Source

Phototroph
 Uses

light as an energy source
Chemotroph
 Uses
energy from the oxidation of reduced chemical
compounds
Nutrient Requirements

Carbon source
All bacteria require carbon for growth
 Bacteria can been classified on the basis of their
carbon source

 Autotroph

Can use CO2 as a sole carbon source
 Heterotroph

use more complex organic compounds such as carbohydrates and
amino acids as source of carbon
Energy/Carbon classification

Where microbes get their energy?



How do they obtain carbon?



Sunlight vs. Chemical
Photo- vs. Chemo- trophs
Carbon Dioxide (or inorganic cmpds.) vs.
Organic Compounds (sugars, amino acids)
Auto- vs. Hetero- trophs
Examples


Photoautotrophs vs. Photoheterotrophs
Chemoautotrophs vs. Chemoheterotrophs
Nutrient Requirements

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)
Nutrient Requirements

Inorganic nutrients (ions)


contain no carbon and hydrogen atoms :
phosphates, potassium, magnesium, nitrogen,
sulfur, iron and numerous trace metals
Organic nutrients

contain carbon and hydrogen atoms. Include
carbohydrates, lipids, amino acids, Nucleic
acids etc.
Nutrient Requirements

Carbohydrates


are used as the initial carbon source for many
biosynthetic pathways and as electron donors
(energy source) by many bacteria
Amino acids

are important source of carbon and nitrogen.
The nitrogen is converted to ammonia.
Nutrient Requirements

Phosphorus



Minerals



is present as phosphates salts
They function in energy metabolism and as
constituents of nucleic acids, phospholipids, teichoic
acids, ATP, etc
K, Mg, Ca, Fe are required in relatively high levels
Function as cofactors in enzyme reactions and as
cations they act as buffers within the cells
Vitamins

function as coenzymes
General Pathways of Metabolism
-- Catabolism -1. Breakdown of macromolecules to building blocks
protein
amino
acids
polysaccharide
glucose,
other sugars
lipid
glycerol,
fatty acids
nucleic acids
ribose,
bases,
phosphate
no useable energy yield here - only building blocks obtained
2. Breakdown of monomers to common intermediates
amino
acids
glucose,
other sugars
glycerol,
fatty acids
pyruvate
NH4+
acetyl CoA
citric acid cycle  ETS/Ox Phos  ATP
CO2
3. Breakdown of intermediates to CO2 and electrons
is accomplished through a central oxidative pathway:
The Citric Acid Cycle or TCA or the Krebs Cycle.
This cycle leads to the production of ATP by processes
called electron transport and oxidative phosphorylation.
--Anabolism-proteins
polysaccharides
lipids
amino
acids
glucose,
other sugars
glycerol,
fatty acids
NH4+
pyruvate
acetyl CoA
citric acid cycle
Anabolism, cont’d
1. utilization of critical Common Intermediates
including components of TCA cycle to make building
blocks
2. making building block requires energy = ATP
3. synthesis of macromolecules requires energy = ATP
-- Some General Principles -•Processes of metabolism are highly controlled
•Anabolism and catabolism are not necessarily balanced- one or the other may predominate in certain cells or at
different times depending on cell needs
•The pathway to synthesize a complex substance is not
simply the reverse of the degradative pathway.
Carbohydrate Catabolism



Microorganisms oxidize carbohydrates as
their primary source of energy
Glucose - most common energy source
Energy obtained from Glucose by:


Respiration
Fermentation
Aerobic Respiration




Glucose to Carbon dioxide + Water +Energy
C6H12O6 + O2  6CO2 + 6H2O + 38 ATP
Electrons released by oxidation are passed down an
Electron Transport System with oxygen being the Final
Electron Acceptor
Requires Oxygen
4 subpathways
1. Glycolysis
2. Transition Reaction
3. Kreb’s Cycle
4. Electron Transport System
1. Glycolysis
(splitting of sugar)

Oxidation of Glucose into 2 molecules of
Pyruvic acid
Embden-Meyerhof Pathway

End Products of Glycolysis:




2 Pyruvic acid
2 NADH2
2 ATP
2. Transition Reaction



Connects Glycolysis to Krebs Cycle
Pyruvic Acid  Acetyl - Co A + CO2 +
NADH
End Products:



2 Acetyl CoEnzyme A
2 CO2
2 NADH
3. Krebs Cycle (Citric Acid Cycle)

Metabolic Wheel



Series of chemical reactions that begin and end
with citric acid
Fats, amino acids, etc. enter or leave
Energy Produced:



2
6
2
ATP
NADH
FADH2
4. Electron Transport System


Occurs within the cell membrane of Bacteria
How 34 ATP from E.T.S. ?
3 ATP for each NADH
2 ATP for each FADH2
NADH

Glycolysis
FADH2

Glycolysis
R.
Krebs Cycle
2
2
6
Total
10
Total
T.
10
x 3 = 30 ATP
T.R.
Krebs
2
Cycle
x 2 = 4 ATP
0
0
2
2
Total ATP production for the
complete oxidation of 1 molecule
of glucose in Aerobic Respiration


Glycolysis
Transition Reaction
Krebs Cycle
E.T.S.

Total



ATP
2
0
2
34
38 ATP
Anaerobic Respiration

Electrons released by oxidation are passed
down an E.T.S., but oxygen is not the final
electron acceptor

Nitrate (NO3-)
----> Nitrite (NO2-)
Sulfate (SO24-)
----> Hydrogen Sulfide (H2S)
Carbonate (CO24-) ----> Methane (CH4)


Anaerobic Respiration

Examples of anaerobic respiration




glucose + 3NO3- + 3H2O
glucose + 3SO42- + 3H+
glucose + 12S + 12H2O
6HCO3- + 3NH4+
6HCO3- + 3SH6HCO3- + 12HS- + 18H+
All of these terminal electron acceptors have smaller
reduction potentials than O2, so it is less energetically
efficient than aerobic respiration
Commercial applications of
anaerobic respiration

Anaerobic digestion
is a series of processes in which microorganisms
break down biodegradable material in the absence
of oxygen. Anaerobic digestion is a renewable
energy source because the process produces a
methane suitable for energy production helping
replace fossil fuels. Also, the nutrient-rich solids
left after digestion can be used as fertilizer.
http://www.wikipedia.com
Fermentation

Anaerobic process that does not use the
E.T.S. Usually involves the incomplete
oxidation of a carbohydrate which then
becomes the final electron acceptor.

Glycolysis - plus an additional step
Fermentation may result in numerous
end products
1. Type of organism
2. Original substrate
3. Enzymes that are present and active
1. Lactic Acid Fermenation



Only 2 ATP
End Product - Lactic Acid
Food Production




Yogurt
- Milk
Pickles
- Cucumbers
Sauerkraut - Cabbage
2 Genera:


Streptococcus
Lactobacillus
2. Alcohol Fermentation


Only 2 ATP
End products:





alcohol
CO2
Alcoholic Beverages
Bread dough to rise
Saccharomyces cerevisiae
(Yeast)
3. Mixed - Acid Fermentation


Only 2 ATP
End products- “FALSE”






formic acid
acetic acid
lactic acid
succinic acid
ethanol
Escherichia coli and other enterics
Propionic Acid Fermentation


Only 2 ATP
End Products:



Propionic acid
CO2
Propionibacterium
spp.
Saccharomycetes
Clostridium
Propionebacterium
E. coli
Enterobacter
Streptococcus
Lactobacillus
Summary of Respiration

Aerobic Respiration





Glycolysis
Transition Rx.
Kreb’s Cycle
Electron Transport Chain
Anaerobic Respiration

Pyruvate 





Lactic Acid
Mixed Acids
Alcohol + CO2
Recycle NADH
2 ATP / Glucose
Pentose phosphate pathway

Function

NADPH production

Reducing power carrier



Synthetic pathways
Role as cellular
antioxidants
Ribose synthesis

Nucleic acids and
nucleotides
Microbes need three things to grow



Energy source
Nutrients (C)
Suitable environmental conditions
Environmental factors

Oxygen Requirement

Obligate aerobes
The growth of bacteria is inhibited by absence of
oxygen
 Pseudomonas aeruginosa, Mycobacterium
tuberculosis


Obligate anaerobes
Growth is inhibited by the presence of oxygen
 Clostridium spp and Bacteriodes spp.

Environmental factors

Oxygen Requirement

Facultative anaerobes
are able to grow in the presence or absence of
molecular oxygen
 Staphylococci, Streptococci, E. coli, etc


Microaerophilic bacteria
grow best under increased carbon dioxide tension
 Neisseria gonorrhoeae, Haemophilus influenzae

Environmental factors

Oxygen Requirement

Aerotolerant bacteria
can survive (but not grow) for a short period of
time in the presence of atmospheric oxygen
 Tolerance to oxygen is related to the ability of the
bacterium to detoxify superoxide and hydrogen
peroxide produced as bye products of aerobic
respiration.

Enzymes that detoxify the toxic byproducts of
aerobic metabolism

Superoxide dismutase


which converts superoxide ( a toxic metabolite) into
hydrogen peroxide is present in aerobic and
aerotolerant bacteria but not in obligate anaerobes.
2O2− + 2H+
H2O2 + O2
SOD

Catalase


which converts hydrogen peroxide into water and
oxygen is also present in all aerobic bacteria but is
lacking in aerotolerant organisms. Strict anaerobes
lack both enzymes
2 H2O2 catalase 2H2O + O2
Environmental factors

Temperature

There are three critical temperature
ranges for growth:
(a) Minimum temperature
 (b) Maximum temperature
 (c) Optimum temperature

Environmental factors

Temperature

Psychrophiles:


Mesophiles:


Has optimum temperature below 15°C but capable
of growth at 0°C
grow at a range of 20° – 40°C. Include most
bacterial pathogens with optimum temp. at 37°C
Thermophiles:
microbes that has optimum temperature above
45°C with a general range of 45-80°C
 Most thermophiles form spores e.g. Bacillus
steareothermophilus

Environmental factors

pH


Optimum pH for most bacteria is near pH
7.0 (pH 6.5- pH 7.5)
Bacteria can be classified as alkalinophiles,
neutrophiles or acidophiles according to
their degree of tolerance to pH changes
Environmental factors

Ionic strength and osmotic pressure




When a microbial cell is in a
hypertonic solution cellular water
moves out of the cell through the cell
membrane to the hypertonic solution
This osmotic loss of water causes
shrinkage of the cell (PLASMOLYSIS)
In a hypotonic solution such as in
distilled water, water will enter the cell
and the cell may be lysed by such
treatment (PLASMOPTYSIS)
Halophiles

require high salt concentrations for
growth. Some bacteria can tolerate
15% salt. E.g. S. aureus
Bacterial growth




.
Replication of
chromosome
Cell wall extension
Septum formation
Membrane attachment
of DNA pulls into a
new cell.
Cell division

Generation or doubling time:

The average generation time for bacteria is 30-60
minutes under optimum conditions.

Most pathogens such as Staphylococcus aureus and
Escherichia coli double in 20 – 30 minutes.

The longest generation time requires days. E.g.
Mycobacterium leprae that causes leprosy doubles in
20 to 30 days.
The growth curve
The growth curve

The lag phase



Cells adjust to new environment.
There is no change in the number of cells but
metabolic activity is high leading to increase in
cellular components
The log or exponential phase



Bacteria multiply at the fastest rate possible under
the conditions provided.
Are susceptible to cell wall active antibiotics
Form metabolic end products
The growth curve

The stationary phase

there is an equilibrium between cell division and cell
death caused by :



decrease in nutrient
increase in cell population and accumulation of metabolic
waste /end products
Death or Decline phase

The number of death cells exceeds the number of
new cells formed due to lack of nutrients and
accumulation of toxic waste
Types of Culture media


Basic media
Rich media


Enrichment media


is used to encourage the growth of a particular organism in a mixed
culture
Selective media


contain additional nutrients to support the growth of fastidious
organisms. E.g. blood agar and chocolate agar
contains salts, dyes or other chemicals that inhibit the growth
unwanted microorganisms.
Differential media

contain chemicals that allow the distinction between different types
of organisms e.g. Lactose in MacConkey agar
MacConkey agar with lactose(left) and
non-lactose(right) fermenters
Cultivation methods

For microbiologic examination


For isolation of a particular organism




Enrichment culture
Differential medium
Selective medium
Isolation of microorganisms in pure culture



Use as many different media and conditions of
incubation as is practicable. Solid media are preferred;
avoid crowding of colonies.
Pour plate method
Streak method
For growing bacterial cells

Provide nutrients and conditions reproducing the
organism's natural environment.
Anaerobic cultivation methods
Excluding oxygen
Reducing agents
Anaerobic jar
Anaerobic glove chamber
Measurement of Microbial Growth

Viable cell counts


plating diluted samples (using a pour plate or
spread plate) onto suitable growth media and
monitoring colony formation
Serial Dilution




1. Carry out dilution series
2. plate known volumes on plates
3. count only plates with 30-300 colonies (best statistical
accuracy);
4. extrapolate to undiluted cell conc.
10-1
10-2
10-3
10-4
10-5
10-6
0.1 ml
> 1000
220
18
Bacterial concentration:
220 x 106 x 10 = 2.2 x 109/ml
10-7
Measurement of Microbial Growth

Turbidimetric measurements



can estimate cell numbers accurately by
measuring visible turbidity
Use a spectrophotometer to accurately
measure absorbance, usually at wavelengths
around 400-600 nm
Light scattered is proportional to number of
cells.
Summary



Metabolic Requirements

Energy source

Nutrients
Metabolism & the Conversion of Energy
- Glucose: Glycolysis (Embden-Meyerhof-Parnas pathway)
TCA cycles
Pentose phosphate pathway
Bacterial Growth

Environment factors

Growth curve

Measurement of growth