Microbial Nutrition

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• Microbial Nutrition

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• I. The Common Nutrient Requirements of Microbial
  Cells

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gt95 of dry weight of bacterial cells is made up
of 10 major components

• g/l used for carbohydrates, proteins, lipids,
  nucleic acids
• Carbon (C)
• Oxygen (O)
• Hydrogen (H)
• Nitrogen (N)
• Sulfur (S)
• Phosphorous (P)

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• mg/l enzyme activity, heat-resistance of
  spores, co-factors, cytochrome components
• Potassim (K)
• Calcium (Ca)
• Magnesium (Mg)
• Iron (fe)

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Minor components

• mcg/l (mg/l) enzyme activity, co-factors,
  nitrogen fixation, vitamin components
• Manganese (Mn)
• Zinc (Zn)
• Cobalt (Co)
• Molybdenum (Mo)
• Nickel (Ni)
• Copper (Cu)
• Others (B, Se, )
• Usually enough in water sources to satisfy
  requirements

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Specialized Requirements

• Silica
• Diatoms need silicic acid for silica walls
  (H4SiO4)
• High sodium concentrations
• Halophiles

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• II. Requirements for Carbon, Hydrogen, and
  Oxygen-often satisfied together

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Terms and Definitions Categorization based on
nutritional requirements
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Carbon Source
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Autotroph

• CO2 principle carbon source
• Includes photosynthetic bacteria and those
  capable of oxidizing inorganic material for
  energy generation

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Heterotroph

• Utilize more reduced and complex carbon sources
  derived from other organisms (nourished by
  others)
• Organic compounds used to provide carbon

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Prototroph

• Utilizes same components as most members of the
  same species

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Auxotroph

• Mutated microbe that has lost the ability to
  synthesize critical precursors
• Must have nutritional precursors supplied

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Energy Source
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Phototroph

• Light energy harvested by photosynthetic
  processes
• Carbon from CO2

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Chemotroph

• Organic or inorganic compounds provide energy by
  oxidative processes

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Hydrogen or Electron Source
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Lithotrophs

• Use reduced inorganic compounds as electron
  source
• Rock eaters

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Organotrophs

• Use organic compounds as H and electron donors

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• III. Major Nutritional Microorganism Types

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Photolithhotrophic autotrophs (aka photautotrophs)

• Carbon and energy source
• CO2
• Light energy
• H/e- source inorganic donor
• e.g. H2O, hydrogen, H2S and elemental sulfur
• Examples
• Algae (eukaryotic)
• Cyanobacteria
• Purple and green sulfur bacteria

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Photoorganotroic heterotrophs

• Carbon and energy source
• CO2 and organic compounds
• Light energy
• H/e- source
• Organic donor
• Examples
• Purple non-sulfur bacteria
• Green non-sulfur bacteria

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Chemolithotrophic autotrophs (aka chemoautotrophs)

• Carbon and energy source
• CO2
• Inorganic compounds
• (a few chemolithotrophs get carbon from organic
  sources chemolithotrophic heterotrophs
  mixotrophic inorganic energy, organic carbon)
• H/e- source
• Oxidation of inorganic compounds
• H2S, S, NO2, H2, Fe2

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• Examples
• Sulfur oxidizers (Thiobacillus)
• Hydrogen bacteria
• Nitrifying bacteria (nitrites, ammonia)
  (Nitrobacter, Nitrosomonas)
• Iron bacteria (Siderocapsa)
• Play major role in ecological transformation of
  compounds (ammonia to nitrate sulfur to sulfate
• NH3 ? NO3-
• S ? SO42-

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Chemoorganotrophic heterotroph (aka
chemoheterotrophs)

• Carbon and energy source
• Organic
• H/e- source
• Organic donor
• Examples
• Protozoa
• Fungi
• Most non-photosynthetic bacteria
• Most pathogens (medically important bacteria
  chemoheterotrophs)

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• IV. Nitrogen, Phosphorous and Sulfur are needed
  for the basic building blocks of cells

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Nitrogen

• Amino acids
• Nucleic acids (purines and pyrimidines)
• Some carbohydrates and lipids
• Enzyme co-factors

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Phosphorous

• ATP
• Co-factors
• Nucleic acids (phosphodiester bonds)
• Phospholipids (lipid bilayer)
• Some proteins

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Sulfur

• S-containing amino acids
• Some carbohydrates
• Thiamine
• Biotin

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• Growth Factors

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• Organic compounds required by microorganisms for
  growth and NOT synthesized by that mircoorganism
• Obtain compounds or their precursors from the
  external environment
• Three major classes
• Amino acids, purines/pyrimidines, vitamins
• Minor classes
• Heme (H. influenzae), cholesterol (some
  Mycoplasma)

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• VI. Nutrient Uptake Specific Mechanimsms
  Utilizing Selective Permeability

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Facilitated Diffusion

• Requires large concentration gradient for
  efficient transport
• Differs from passive diffusion which utilizes
  osmosis to achieve transfer of small substances
  (glycerol, H2O, O2, CO2)

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• Facilitative diffusion employs carrier proteins
  called permeases to transfer components
  selectively across the PM
• No metabolic energy needed
• Works effectively even in low concentration
  gradients
• Requires concentration gradient to facilitate
  uptake
• Equilibrium will be established
• But substance is NOT accumulated against a
  gradient

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• Probably involves a conformational change of
  carrier to deliver components across the lipid
  bilayer
• Therefore effective for lipid-insoluble material
• Not utilized much by bacteria but it does occur
  (e.g glycerol uptake by E. coli)

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• Active Transport
• Transport of molecules AGAINST a concentration
  gradient
• Material is more concentrated on the inside of
  the cell than on the outside
• Ability to concentrate solutes in dilute
  environments
• Metabolic energy required
• ATP hydrolysis or
• Proton motive forces (proton gradients generated
  by electron transport)

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• Carrier proteins utilized ? energy dependent in
  PM (ATP)
• Membrane-bouond
• Multi-subunit
• Form a pore
• AKA permeases
• May associate with substrate binding proteins in
  the periplasmic space of Gram-negative bacteria
  where substrate is handed over for entry across
  PM (e.g. arabinose, lactose, maltose, galactose,
  robose, glutamate, histidine, leucine)

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• Types of active transport
• Symport is the linked transport of two substances
  in the same direction
• Antiport is the linked transport of two
  substances in opposite directions

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• Group Translocation
• Transfer of solutes coupled with chemical
  modification
• Example
• Phosphoenolpyruvate (PEP) Sugar
  phosphotransferase system (PTS)
• Sugars are transported ad phosphorylated using
  PEP as the phosphate donor
• Glucose, fructose, mannitol, sucrose, N-acetyl
  glucosamine, cellobiose, and other solutesn
• PTS proteins cann also serve as chemoreceptors in
  chemotaxis

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• Iron uptake
• Ferric iron (Fe3) is insoluble uner aerobic
  conditions
• Bacteria must transport iron across PM to use in
  cytochromes and many enzymes
• the organism secretes siderophores that complex
  with the very insoluble ferric ion, which is then
  transported into the cell
• Siderophores iron chelators

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• Types of siderophores
• Hydroxamates (e.g. ferrichrome used by fungi)
• Catecholates (e.g. enterobactin used by E. coli)

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• Iron handed off to the cell after siderophore
  binds to siderophore receptor protein on the
  microorganism

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• VII. Types of Culture Media

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• When media component are known Defined Media
  (synthetic media)

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• When exact composition of some components is not
  nown Complex Media (enriched, artificial,
  crude)
• Required by fastidious organisms
• Fastidious organisms are difficult to culture on
  ordinary media because of its need for secial
  nutritional factors (stringent physiological
  requirements for growth and survival)

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• Complex media often contains blood or serum
• Sometimes blood must be lysed (chocolate agar) to
  release hemin and NAD (e.g Haemohilus and
  Neisseria which do not produce siderophores)
• Other undefined components
• Peptones (hydrolyzed protein)
• Meat extracts or infusions (lean meat) amino
  acids, peptides, nucleotdes, vitamins, mnerals
  and organic acids
• Yeast extract (Brewers yeast B vitamins,
  nitrogen and carbon compounds)

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• Agar added if solid medium is required
• Agar complex polysaccharide from red algae
• General purpose media favors the growth of a
  variety of microbe types
• Example Tryptic soy broth
• Can be enriched with blood components

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• Enriched media are supplemented by blood or other
  special nutrients to encourage the growth of
  fastidious heterotrophs

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• Selective Media supports the growth of particular
  microorganisms while inhibiting the growth of
  others
• Examples
• Bile salts and dyes suppress Gram-positive
  bacteria while favoring the growht of
  Gram-negative bacteria
• Can select based on enzymes e.g. cellulose
  utilization requires cellulase
• Antibiotic resistance (plasmid-encoded, R-plasmid)

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• Differential Media distinguished different
  bacterial groups
• Examples
• Blood agar hemolysis (alpha, beta or gamma
  hemolysis)
• Eosin methylene blue agar (EMB) used to
  identify lactose fermenters (colony turns dark
  purple)

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• Some media can exhibit characteristics of more
  than one type
• blood agar is enriched and differential, and
  distinguishes between hemolytic and nonhemolytic
  bacteria

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• VIII. Culturing Techniques Isolating Pure
  Cultures

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• a population of cells arising from a single cell
• can be accomplished from mixtures by a variety of
  procedures
• spread plates
• streak plates
• pour plates

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• Spread plate
• 100-200 bacterial cells are placed on the center
  of an agar surface and spread evenly with a glass
  rod
• Every cell grows into a separate colony
• Each colony pure culture
• Useful for quantitative purposes

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• Streak plate
• Inoculating loop is used to streak cultures
• Dilutions made with different streaks, flaming
  between streaks

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• Pour plate
• Diluted sample series is mixed with molten agar
  and poured immediately
• Cells become embedded in the agar and on top ?
  forming discrete colonies

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• Colonies are macroscopically visible growths or
  clusters of microorganisms on solid media
• Colony growth is most rapid at the colony's edge
  because oxygen and nutrients are more available
  growth is slowest at the colony's center
• Colony morphology helps microbiologists identify
  bacteria because individual species often form
  colonies of characteristic size and appearance