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Growth and Cultivation of bacteria
1
Growth requirements
•
•
•
•
•
oxygen (or absence)
energy
nutrients
optimal temperature
optimal pH
2
Oxygen requirements
Obligate aerobes
• grow in presence of oxygen
• no fermentation
• oxidative phosphorylation
4
Obligate anaerobes
• killed by oxygen
• fermentation
• no oxidative phosphorylation
• lack certain enzymes:
superoxide dismutase
O2-+2H+ => H2O2
catalase
H2O2 => H20 + O2
peroxidase
H2O2 + NADH + H+ => 2H20 + NAD
5
Aerotolerant anaerobes
not killed by oxygen
• respire anaerobically
6
Facultative anaerobes
• fermentation
• aerobic respiration
• survive in oxygen
7
Microaerophilic bacteria
• grow
– low oxygen
• killed
– high oxygen
8
Temperature
Optimal growth temperature
• Mesophiles:
– human body temperature
* pathogens
* opportunists
• pyschrophile
– close to freezing
• thermophile
– close to boiling
10
pH
• Many grow best at neutral pH
• Some can survive/grow
- acid
- alkali
11
Nutrient Requirements
•
•
•
•
•
Carbon
Nitrogen
Phosphorus
Sulfur
Metal ions (e.g. iron)
12
Siderophores (S)
Receptor
Transport of iron
Fe 2+/S
Fe 2+/S
13
Measuring bacterial mass (live + dead)
in liquid culture
Turbidity
(Cloudiness)
14
Measuring viable bacteria
Colony forming units
colony
15
Growth Curve
Stationary
COLONY
FORMING
UNITS
Death
Log
Lag
TIME
16
Growth Curve
Stationary
TURBIDITY
(cloudiness)
Autolysis
Log
Lag
TIME
17
Generation time
• time for bacterial mass to double
• Example
100 bacteria present at time 0
If generation time is 2 hr
After 8 hr mass = 100 x 24
18
SUGAR CATABOLISM
• Glycolysis
– Embden Meyerhof Parnas Pathway
– most bacteria
– also animals and plants
19
Other pathways for catabolizing
sugars
• Pentose phosphate pathway (hexose
monophosphate shunt)
– generates NADPH
– common in plants and animals
• Entner Doudoroff Pathway
– a few bacterial species
20
Glycolysis
NAD
NADH
Glucose
Pyruvate
C6
C3
ADP
ATP
21
Fermentation
NADH
Pyruvate
NAD
Short chain alcohols,
fatty acids
(C3)
(C2-C4)
22
Anaerobic Respiration =
Glycolysis + Fermentation
NAD
NADH
ATP
NADH
NAD
23
Krebs Cycle (C4-C6 intermediate compounds)
NAD
Pyruvate
NADH
3CO2
(C3)
(C1)
Oxidative phosphorylation
NADH
NAD
O2
H2O
ADP
ATP
24
Aerobic Respiration =
Glycolysis +
Krebs Cycle/oxidative phosphorylation
•
Pyruvate to CO2
–
NAD to NADH
–
glycolysis
– Krebs cycle
•
Oxidative phosphorylation
–
NADH to NAD
– ADP to ATP
25
Oxidative phosphorylation
• converts O2 to H20 (oxidative)
• converts ADP to ATP (phosphorylation)
• electron transport chain
• ubiquinones/cytochrome intermediates
26
The Krebs cycle
-CO2
C2
Acetate Citrate
Isocitrate
C6
+
C
X
-CO2 NADH
Alpha-keto
glutarate
Oxaloacetate C4
-CO2 NADH
Pyruvate
x
Succinate C
Malate
Fumarate
27
Krebs Cycle - sugar as sole
carbon source
-CO2
Pyruvate
C3
C
Acetate +
Oxalo
C2
C4 acetate
X
+ CO2
Pyruvate
C3
C
Citrate
C6
-2CO2
BIOSYNTHESIS
Oxalo
acetate
Krebs
cycle
ENERGY
STORAGE
Aspartic acid
Oxaloacetate
C4
28
Krebs Cycle – fatty acids as
sole carbon source
ENERGY
Acetate
+ Oxalo
acetate
BIOSYNTHESIS
Fatty acids
x
Oxalo
acetate
Krebs
cycle
Citrate
-2CO2
Aspartic acid
C2
Isocitrate
Succinate + Glyoxylate
-2CO2
C6 Krebs cycle
C2
+ Acetate
C4
Malate
29
C4
The Glyoxylate and Krebs cycles
Isocitrate
Citrate
Oxaloacetate
Malate
1
Glyoxylate
2
+ Acetate
Alpha-keto
glutarate
Succinate
Fumarate
Krebs and Glyoxylate cycles
Krebs cycle only
Glyoxylate cycle only
30
Krebs Cycle
– biosynthetic
– energy storage
• Removal of intermediates
– must be replenished
• Unique enzymatic replenishment pathways
– sugars
– fatty acids
31
Major nutritional types of
procaryotes
Nutritional Type
Energy Source
Carbon Source
Examples
Photoautotrophs
Light
CO2
Cyanobacteria,
some Purple
and Green
Bacteria
Photoheterotrophs
Light
Organic compounds
Some Purple and
Green Bacteria
Chemoautotrophs
or Lithotrophs
(Lithoautotrophs)
Inorganic
compounds, e.g.
H2, NH3, NO2,
H2S
CO2
A few Bacteria and
many Archaea
Chemoheterotrophs
or Heterotrophs
Organic compounds Organic compounds
Most Bacteria,
some Archaea