Biology 261

Chapter 4
- Microbial nutrition
o Metabolism: sum total of all chemical reactions that occur in a cell
o Catabolic reactions: energy-releasing metabolic reactions (digestive reaction)
 Break down macromolecules into monomers that are used to make
macromolecules needed for cell growth
o Anabolic reactions: energy-requiring metabolic reactions (synthetic reaction)
- Substrates (starches, lipids, proteins) are what enzymes act on which are broken down to
macromolecules like glucose, fatty acids, and amino acids (catabolism) and energy (ATP) is
generated. Cell then uses monomers to make the macromolecules that the cell needs to grow
in anabolic reaction which require energy
- Culture media: nutrient solution used to grow microbes in the laboratory
o Two broad classes
 Defined media: precise chemical composition in known (we know all the chemical
structures)
 Complex media: composed of digests of chemically undefined substances (yeast,
meat extracts, etc) (we do not know the exact chemical structure of ingredients)
o Selective media: contains compounds that selectively inhibit growth of some microbes
but not others
o Differential media: contains an indicator, usually a dye, that detects particular chemical
reactions occurring during growth (yes or no result)
o For successful cultivation of a microbe it is important to know the nutritional
requirements and supply them in proper form and proportions in a culture medium
- Lab culture of microorganisms
o Pure culture: culture containing only a single kind of microbe
o Contaminants: unwanted organisms in a culture
o Cells can be grown in liquid or solid culture media
 Solid media are prepared by addition of agar as a gelling agent
 When grown on solid media, cells form isolated masses (colonies)
o Microbes are everywhere, so sterilization of media prior to inoculation is critical, as in
the use of aseptic technique
o Pure culture technique
 Streak plate: isolated colonies
 Transferring the bacteria from the culture in the broth to the agar media in
the petri dish to do a quadrant for isolation
 Once you incubate the plate and the bacteria have a chance to grow this is
what the plate will look like. Each one of the isolated colonies came from one
bacteria that underwent binary fission to make millions of cells that we can
see as a bacterial colony
 Pour and Spread plate: done when you are doing plate counts to determine the
number of bacteria present in a sample
 Pour plate: pour media on top of a dilution of bacteria as a result the
bacteria grow all throughout the media depending on their oxygen
requirements
 Spread plate: dilution is spread on the surface of an agar plate and the
bacterial colonies grow on the surface of the media
- Catalysis and Enzymes
o Activation energy: energy required to bring all molecules in a chemical reaction into the
reactive state
 A catalysis is usually required to breach activation energy barrier
o Catalyst: substance that -
 Lowers the activation energy of a reaction
 Increases reaction rate
 It is also not changed or consumed in the reaction
 o Enzymes
 At on substrate and where the bond is made or broken is the active site
 Biological catalyst
 Typically proteins (some RNAs)
 Highly specific
 Generally larger than substrates
 Typically rely on weak bonds
 Active site: region of enzyme that binds to substrate
 Increase rate of chemical reactions
- Energy rich compounds and energy storage
o Chemical energy released in redox reactions is primarily conserved in certain
phosphorylated compounds
 ATP: the prime energy currency
 Coenzyme A
o Long term energy storage involves insoluble polymers that can be oxidized to generate
ATP
 Pro: Glycogen, poly-B-hydroxybutyrate and other polyhydroxyalkanoates, and
elemental sulfur
 Eu: starch, lipids
- Energy conservation
o Two reaction series are linked to energy conservation in Chemoorganotrophs:
fermentation and respiration
o Differ in mechanism of ATP synthesis
 Fermentation: substrate-level phosphorylation; ATP directly synthesized from an
energy-rich intermediate
 Starts with glycolysis and then the pyruvic acid is converted to various acids,
gases and alcohols
 Respiration: oxidative phosphorylation; ATP produced from proton motive force
formed by transport of electrons from organic or inorganic electron donors
(completely breakdown of glucose) – produces more ATP than fermentation
 Starts with glycolysis, which feeds into the Krebs cycle (Citric acid cycle) and
finally the electron transport chain, which is in the membrane and is where
the most ATP is produced. Oxygen is the final electron acceptor to make
water so this is aerobic respiration
 Aerobic respiration: final electron acceptor is oxygen
 Anaerobic respiration: something other than oxygen
- Glycolysis
o Glucose is transported into the cell by facilitated diffusion and is immediately
phosphorylated by a enzyme called hexokinase. Glucose and fructose are isomers
 A enzyme is involved in each step. It acts on a substrate to produce to product
which in turn acts as a substrate for the next enzyme)
 Glycolysis ends with the production of pyruvate or pyruvic acid
o Fermented substance is both an electron donor and electron acceptor
o Glycolysis (Embden-Meyerhof pathway): a common pathway for fermentation of glucose
 Anaerobic process
 Three stages
o Glucose consumed
o Two ATP’s produced  4 made, 2 used  2 net
o Fermentation products generated (some harnessed by humans for consumption)
- Citric acid cycle
o Pathway through which pyruvate us completely oxidized to CO2
 Initial steps (glucose to pyruvate) same as glycolysis
 Per glucose molecule, molecules released and 38 ATP generated
 Plays a key role in catabolism and biosynthesis
o Generates many compounds available for biosynthesis purposes
- Electron transport system
o Membrane associated
o Mediate transfer of electrons from primary donor to terminal acceptor
o Conserve some of the energy released during transfer and use to synthesize
o Many oxidation-reduction enzymes are involved in electron transport
o Takes place in cytoplasmic membrane and electron transport chain so that as electrons
are transported, protons are separated
o Most ATP is produced in the ETC – series of oxidation reduction reactions in which
electrons are passed from one protein or cytochrome to the next
o Carriers in electron transport chain arrange in membrane in order of their increasingly
positive reduction potential
o The final carrier in the chain donates the electrons and protons to the terminal electron
acceptor
o During electron transfer, several protons are released causing a slight acidification of
the external surface of the membrane
 Protons originate from NADH and the dissociation of water
o Results in generation of pH gradient and an electrochemical potential across the
membrane (the proton motive force)
 The inside surface membrane becomes electrically negative and alkaline; the
outside electrically positive and acidic
- Respiration and membrane-associated electron carriers
o Aerobic respiration (complete breakdown of glucose to carbon dioxide and water and
oxygen is the final electron acceptor)
 Oxidization using O2 as the terminal electron acceptor
 Higher ATP yield than fermentations
 ATP produced at the expenses of the proton motive force which is generated
by electron transport
o During electron transfer, several protons are released causing a slight acidification of
the external surface of the membrane
 Protons originate from NADH and the dissociation of water
o Results in generation of pH gradient and an electrochemical potential across the
membrane (proton motion force)
 The inside surface membrane becomes electrically negative and alkaline; the
outside electrically positive and acidic
- Catabolic diversity
o Microorganisms demonstrate a wide range of mechanisms for generating energy
 Fermentation (test tube experiment)
 Red  neutral
 Yellow  acidic
 If bacteria ferments it makes acid (lower pH) yellow color
 Examples:
o CHO = glucose
o Phenol Red = pH indicator
o Red = neutral
o Yellow = acid
o Pink = alkaline
 Inverted durham tube = gas production
 Turbidity = growth (cloudy)
 Aerobic respiration
 Anaerobic respiration
 Chemolithotrophy
 Phototrophy
 Anaerobic Respiration
- Regulation of activity of biosynthetic enzymes
o Two major modes of enzyme regulation
  Amount: regulation at the gene level
 Activity: temporary inactivation of the protein through either covalent or
noncovalent changes in enzyme structure
o Feedback inhibition: mechanism for turning off the reactions in a biosynthetic pathway
(controlled mechanism in which the cell is able to control whether or not it makes an
end product – when cells have enough amino acids, feedback inhibition kicks in, amino
acid binds to one of the enzymes at beginning of the pathway and keeps it from
reacting which in turn stops the pathway)
 End product of the pathway binds to the first enzyme in the pathway, thus
inhibiting its activity
 The inhibited enzyme is an allosteric enzyme (shape)
 Two binding sites: active and allosteric
 Lysin binds to allosteric site site on enzyme causing active site’s shape to
change
 Reversible reaction
o Some pathways controlled by feedback inhibition use isoenzymes
 Isoenzymes: Different enzymes that catalyze the same reaction but are subject to
different regulatory controls