Lecture 3: Microbial metabolism, microbial growth, control of microbial
growth
Edith Porter, M.D.
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Microbial metabolism
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Microbial growth
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Overview
Enzymes and cofactors
Oxidation Reduction reactions
ATP generation
Respiration and fermentation
Biosynthesis
Physical requirements
Chemical requirements
Biofilm
Bacterial growth curve
Control of microbial growth
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Terminology
Microbial death rate and actions of microbial control agents
Physical methods
Chemical methods
Microbial resistance to control agents
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Metabolism is the sum of all
chemical reactions within a
living cell
Includes catabolism and
anabolism
Catabolism
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Complex organic molecules
converted to small simple
compounds
 Releases energy
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Anabolism
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Simple compounds
converted to complex
organic molecules
(biosynthesis)
 Consumes energy
Metabolism=
Catabolism
+
Anabolism
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Chemical reactions accelerated
by
 Temperature increase
 Enzymes
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Enzymes (xxx-ase)
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Mostly proteins
Specific for certain reactions
Not changed upon the reaction
Typically re-usable
Some require co-factor or coenzyme for activity
▪ Co-factor: Ions
(magnesium,calcium)
▪ Co-enzyme: organic molecule;
many are derivates from vitamines,
e.g. NAD+ and NADP+
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Temperature (if too high:
enzyme becomes
denatured)
pH (if too extreme:
enzyme becomes
denatured)
Substrate concentration
Inhibitors
 E.g. cyanide, arsenic,
mercury
 Block enzymes that require
metal ions
 Tie up metal ion activators of
enzymes
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Based on the chemical reaction
 Oxido-reductases: oxidation-reduction reaction in which oxygen and
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hydrogen are gained or lost
Transferases: transfer of functional groups
Hydrolases: cleavage of molecules with hydrolysis (addition of water)
Lyases: removal of groups of atoms without hydrolysis
Isomerases: rearrangement of atoms within a molecule
Ligases: joining of 2 molecules
Based on the target
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Protease
Lipase
DNAse
RNAse
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Basic reaction
: electron uptake
: electron removal
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Biological reaction
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Adenosine Tri Phosphate
 ADP + energy + phosphate
ATP contains energy that can be easily released (highenergy or unstable energy bond)
 Required for anabolic reactions
 Produced by
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 Substrate-level phosphorylation (fermentation): direct
transfer of phosphate group from one molecule to the next
C-C-C~ P + ADP  C-C-C + ATP
 Oxidative phosphorylation (respiration): involves electron
transport chain, oxidation-reduction reactions and inorganic
phosphate
ADP +
 ATP
P
C
D
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Important electron carriers
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NAD+
FMN
FAD
Important oxidase
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Cytochrome oxidases
E.g. cytochrome C oxidase
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The electron flow is coupled to H+
efflux via proton pumps
 H+ accumulates outside and a
chemical and charge based gradient
is generated (potential energy)
 Special protein channels allow H+
flux back into the cell
 Re-entering of H+ into the cell
generates energy for
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 ATP
 Motility
 Active transport
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2 ATP (energy
entrapped in organic
compounds)
36 ATP
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CO2 and H2 gas production!
Acid production will lower pH!!!
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Grapes and yeast: wine
Grain and yeast: beer
Milk and lactobacilli: yogurt
Milk and lactobacilli and propionibacteria:
swiss cheese
Ethanol and Acetobacter: vinegar
And many more…
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Part of electron transport chain
Membrane-bound, water soluble enzyme
Found in some bacteria like Pseudomonas
aeruginosa or Neisseria
Can be easily detected by adding to the
grown cultures a substrate that changes color
when oxidized
 “oxidase positive”
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Single sugar + protein + pH indicator+ Durham tube
Inoculate organism and incubate for 24- 48 h
Gas
Turbid = growth
Yellow/orange = acid
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Glucose: to test for ability to conduct
fermentation
Lactose: many intestinal pathogens are
lactose negative!
Mannitol: used to screen for Staphylococcus
aureus which is able to ferment mannitol
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Group of important organisms that are under
no circumstances able to perform
fermentation
Only respiration is possible
Example: Pseudomonas aeruginosa
Non-fermenters play a role as opportunistic
pathogens in the hospital setting
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Respiration
 Complete oxidation of glucose to carbon dioxide and water while ATP
is generated
 Involves glycolysis, krebs cycle and extensive electron transport chain
 Higher energy yield, faster growth
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Fermentation
 Anaerobic process
 Involves glycolysis and production of organic compound
 Low energy yield, slower growth
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Physical
 Temperature
 Osmotic pressure
▪ Salt: halophil
▪ High salt (Halobacterium spec. requires 30% !!)
 pH
▪ Low pH (1.0 -2.0): acidophil
▪ High pH (> 8.0): alkaliphil
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Chemical
 Elements:
▪ Macroelements: C, N, S, P
▪ Trace elements: iron, copper, zinc
 Atmosphere
▪ Oxygen
▪ CO2
-PHIL MEANS MUST HAVE!!!
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Psychrophiles: -10 to 20C
Psychrotrophs: 0 to 30 C
Mesophiles: 10 to 48C
Thermophiles: 40 to 72C
Hyperthermophile: 65 to 110C
Only Archaea can grow above 95C!
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Some pathogens can multiply in the
refrigerator: Listeria monocytogenes
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Similar effect with sugars
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Oxygen is readily converted into radicals
(singlet oxygen, superoxide, hydrogen
peroxide, hydroxyl radical)
Most important detoxifying enzymes are
superoxide dismutase and catalase
Cells differ in their content of detoxifying
enzymes and hence, ability to grow in the
presence of oxygen
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Type of Bacteria Catalase Superoxide
Dismutase
Oxygen and Growth
Obligate aerobes
+
+
Require oxygen
Facultative
anaerobes
+
+
Can proliferate with and
without oxygen
Obligate anaerobes
-
-
Cannot survive oxygen,
must have anaerobic
conditions
Aerotolerant
anaerobes
-
+
Survive oxygen but
cannot use it for growth
(+)
(+)
Microaerophiles
Require low levels of
oxygen
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Classification of gram-positive cocci
 Staphylococci are catalase +
 Streptococci are catalase -
Staphylococci
Streptococci
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Enhanced CO2 concentration (5%)
 Capnophile
 Many mucosal pathogens are capnophile
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Most pathogens
 Are mesophiles
 Require moderate pH
 Require physiological salt
concentrations
 Require an atmosphere
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To prevent spoilage
 Refrigeration
 Acidity
 Add salt or high
concentrations of sugar
 Vacuum package
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Average reproduction rate of E. coli: ~ 20 minutes
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Lag phase
 Bacteria adjust to new medium
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Log phase:
 Logarithmic growth, all cells in the same growth phase
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Stationary phase:
 Nutrients limited, population very inhomogeneous,
 Bacilli/Clostridia: sporulation;
 Some pathogens upregulation of virulence factors
 Biofilm production
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Decline phase:
 Accumulated toxic products, nutrients exhausted
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XX-phile
 Requires XX for growth
Ability to survive oxygen depends on the presence of
enzymes that detoxify oxygen radicals
 The typical growth curve of bacteria includes lag, log,
stationary and decline phase
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Disinfection: removal of potential vegetative pathogens on
in-animated objects (disinfectants)
Antisepsis: removal of potential vegetative pathogens on
tissues (antiseptics)
Sterilization: eliminates all forms of microbial life (and
prions)
Commercial sterilization: killing of C. botulinum endospores
Sanitization: generates safe conditions for the public
Degerming: modified antisepsis, mechanical removal of
microbes with alcohol patch
Pasteurization: eliminates pathogens and spoilage microbes
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-cidal: to kill, reduce numbers of viable
microbes
-static: to prevent growth and proliferation
Add
antimicrobial
CFU/ml
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Time [h]
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Alteration of Membrane permeability
Damage to Proteins
 Disulfide bridges
 Hydrogen bonds
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Damage to Nucleic acids
 Strand brakes
 Dimerization
Loss of activity
Errors in proteins
with loss of function
or no protein at all
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Microbial population
 number, composition
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Concentration of agent
Exposure time
Environment
70% EtOH is more
effective than 95%
EtOH
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Heat works better at
low pH!
Temperature
pH
Pressure
Presence of organic material
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Physical
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Chemical
 Heat
 Liquids
 Cooling
 Gas
 Filtration
 Pressure
 Desiccation
 Radiation
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Moist sterilization under pressure
 Exposure time: 15 min 121° C at 15 psi
 High pressure and high heat
 Special training is required to use an autoclave
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How can you prove that the autoclave is
properly functioning and fulfills the
requirements to
 Eliminate ALL microbial life forms?
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Dry-heat sterilization
 2 – 3 hrs 160 – 170° C
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Prevents corrosion
Suited also for powders
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Kills pathogens, reduces spoilage organisms
Introduced by L. Pasteur in 1860s in wine
production
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Today: 30 min 63° C
Flash: 15 sec 72° C
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30 min 55 – 60 ° C
Better taste
Ultra high temperature treatment for milk
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1 – 3 sec 140 – 150 ° C
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0.2 (0.45) mm pore size
Limitations
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Cell wall less microbes (e.g.
mycoplasma) are not removed
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Viruses, nanobacteria not removed
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Problem in cell culture laboratories
Specialty filters with 0.01 mm pore
size
Other material may adhere to the
filters
HEPA filter with 0.3 mm pore size
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filter air that go into special rooms
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Most pathogenic bacteria do not replicate at
4C (static effect)
 Exception: Listeria monocytogenes
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Freezing: most damage occurs during
thawing
 Some worms are killed during storage at subzero
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High atmospheric pressure
 Prevent spoilage and preserve taste
 Fruit juices
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Osmotic pressure
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Hypertonic
High salt or high sugar
Used in food preservation
However, often molds can still grow
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Prevents typically
proliferation but does not
kill
 Exception: Neisseria
gonorrhoeae
 Bacterial spores in
particular resistant to
desiccation
 Survive for thousands
of years
 Problem: dried pus,
urine, feces in hospital
setting (mattresses…)
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http://prokariotae.tripod.com/Nei
sseria_gonorrhoeae.jpg
http://www.acmp.com.au/portfolios/mischk
ulnig/images/hospital-bed.jpg
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Ionizing (< 1nm wavelength)
 Gamma-rays (used for spices), x-rays, high-energy electron beams
 Ionize water hydroxyl radicals damage of DNA and other
molecules
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Non-ionizing
 UV light (1 – 400 nm, 260 nm!)
 Thymine dimerization
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Ethylene oxide
 Denatures proteins, attacks SH-, COOH- OH-
groups
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Highly penetrating
Sterilize in closed chamber 4 – 18 hours
Medical supplies, space crafts, mattresses
Caution: cancerogenic
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Agent
Mechanism
Preferred Use
Examples
Phenol based
Disruption of membrane,
protein denaturation
Hospitals, work well in the
presence of organic material,
Mycobacteria
Amphyl
Triclosan
Biguanide
Disruption of membrane
Surgical scrubs
Chlorhexidine
Halogenes
Strongly oxidizing
Cellular function and
structures altered
Wound treatment (I2)
Household (CL2)
Povidoneiodine
Chlorox
Alcohol
Protein denaturation
Dissolution of membrane
Thermometer
Skin scrubbing (alcohol pads)
Ethanol
Isopropanol
Aldehydes
Protein cross linker
Fixative
Surgical Instruments
Formalin
Glutaraldehyde
Peroxygenes
Oxidation
Deep wounds with anaerobes
Peracetic acid
Detergents
Membrane disruption
Protein denaturation
Industrial
Instrument sanitizers
Soap
Zephiran
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Organic acids in food and cosmetics
 Sorbic acid
 Benzoic acid
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Heavy metals (copper, silver, zinc)
 Copper: as algicide, copper coated cell incubators
 Silver nitrate: prevention of ophthalmica neonatarum,
wound treatment
 Zinc chloride: wound treatment
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Antibiotics (NOT as DRUG!!)
 Nisin and natamycin in cheese
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Major concern
 Endospores
 Mycobacteria
 Prions
▪ 134C autoclaving
and sodium
hydroxide not 100%
effective
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You try .....
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Look in your household
and identify
antimicrobial additives.
Bring a list describing 2
items, the antimicrobial
additives incorporated
(must be 2 different
ones), and their mode of
action in table format (as
shown to the left) to
class.
Complete tables will be
worth 5 points
Item
Antimicrobial
Additive
Mode of
Action
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