Microbial Growth and Measurement

• Inclusion bodies store fats.
• Magnetosomes are microcompartments.
• Bacteria motility is directed by local geomagnetic fields.

Microbial Growth

• Microbial growth considers the increase in microbial population.
• Bacterial growth can be determined my measuring turbidity.
• Turbidity is the cloudiness of a liquid.
• Spectrophotometry measures the turbidity of a culture.

Measuring Absorbance

• Absorbance, also called optical density, can be measured at a set wavelength.
• Bacterial cells scatter incident light, reducing detected light.
• Microbial cells can be counted over time using a microscope.
• A grid cytometer can deduce the number of cells per unit area.
• The total number of cells in the whole suspension can be estimated.

Flow Cytometry

• Microbial cells can be counted in a sample with a flow cytometer.
• Droplets of individual cells pass through a capillary with a laser beam.
• Light scatters when cells pass through based on cell size, shape, surface topography, and internal complexity.
• Fluorescence can further sort cells based on structural, genetic, or biochemical characteristics.

Viable Cells

• Determining the number of living cells in a population is measuring viable cells.
• Dilution plating is a method of measuring viable cells.
• Each colony is assumed to arise from a single cell.
  • A colony-forming unit (CFU) is a clonal population.
• Distinguishing between live and dead cells is useful when looking at different treatments where survival rate is important.

Phases of Microbial Growth

• Microbes alternate between periods of growth and non-growth.
• Physiology changes with each stage.
• The phases include:
  • Lag phase.
  • Exponential phase.
  • Stationary phase.

Balanced Growth

• Cell constituents increase by the same proportion over an interval of time.
• Doubling of cell constituents includes DNA, ribosomes, enzymes, and membranes.
• Balanced growth is achieved during the exponential phase.
• A chemostat allows continuous culture to facilitate balanced growth, maintaining constant growth over time.

Environmental Factors

• Microbes can grow wherever there is liquid water.
• Liquid H2O exists along a range of temperatures on Earth.
• Most microbes cannot grow across a range of temperatures.
• Microbes have an optimum temperature.
• Optimal conditions support growth.
• Hydrophobic interactions are less successful below the optimum temperature.
• Proteins begin denaturing above the optimum temperature.

Optimal Temperature

• Microbes can be categorized by optimum temperature.
  • Psychrophiles: less than 18°C.
  • Psychotrophs: 0-30°C.
  • Mesophiles: 20-45°C.
  • Thermophiles: 45-80°C.
  • Hyperthermophiles: greater than 80°C.
• Microbes have an optimum pH and can be categorized by the pH where they preferably grow.
  • Acidophiles: pH 0-5.
  • Neutrophiles: pH 6-8.
  • Alkaliphiles: pH 10-13.

Microbial Survival

• Some microbes have a protective outer layer.
  • Capsule
  • Slime layers
    • Composed of high molecular weight polysaccharide or amino acids.
    • Synthesized only under certain environmental conditions.
• Microbes have specialized proteins to survive/thrive in extreme environments.
  • Heat shock proteins HSPs protect other proteins from heat denaturation and stabilize membranes.
  • Chaperones facilitate folding and refolding.
  • DNA binding proteins prevent DNA melting at high temperatures and reverse gyrase introduces supercoils.
• Microbes grow via cell division.
  • Binary fission is how most bacteria and archaea grow.
  • Process involves invagination of cell membrane, cell wall.

Cell Division

• Grum- construction of envelope mid cell.
• Grint = no apparent construction; septum formation.
  • All result in 2 identical, symmetrical progeny cells
• FtsZ is an important temperature-sensitive protein.
  • Multiple research groups are determining the FtsZ role in septum formation.
  • FFSA is thought to potentially interact with FtsZ during septal development.

FtsZ and Cell Division

• FtsZ facilitates the formation of the Z ring.
• FtsZ proteins polymerize to form short filaments.
  • It forms aggregates
  • Associates with FtsA near the cell membrane.
• Min proteins direct FtsZ polymerization.
  • Prevents FtsZ polymerization at polar sites.
  • Min O polymerizes at one pole of the cell.
  • MinC binds MinO and depolymerizes cytoplasmic FtsZ filaments.
  • MinE forms a ring near polar regions to block MinCO from polymerizing in central region.

Mutation

• Improper cell division: irregular cells( nubbim mini cells); truncated DNA,
• FtsZ possesses tubulin homology
• FtsA possesses actin homology

Microbial Energy

• To grow, microbes must acquire nutrients and energy from their environment.
• Highly ordered, life-generating processes and pathways.
  • Known as growth metabolism.
  • Basis of cellular metabolism.
• 4 over-arching principles.
• Enzyme Catalysis.
  • Accelerate chemical reactions by bringing reactant molecules together
  • Lowers activation energy required for reaction to occur
• Energetically Coupled reactions.
  • A highly energetically favorable reaction can drive an energetically unfavorable reaction.

Phosphorylation

• Energy harvesting from redox reactions.
  • Harvest energy from different types of redox reactions:
    • Organic
    • Inorganic
    • Photochemical
    • Generation of high energy molecules (ATP) & reducing power

Energy and Membranes

• Transduction of energy: Gradients ATP
• Membranes act as a barrier to form ion gradients.
• Fueling building blocks macromolecules structures products
• Microbial strategies for fueling are so diverse
  • Group microbes based on carbon & energy source
  • First distinction: nature of the carbon source
    • Heterotroph
      • consume
      • natural
      • Carbon
    • Autotroph
      • inorganic
      • carbon
      • source
      • CO2
    • Second distinction: nature of energy source or reducing power
      • Chemotroph
        • chemical
        • sources
      • Phototroph
        • light

Metabolism

• Combine names to describe microbial fueling lifestyle.
• Energy harvesting differs in heterotrophs vs autotrophs.
• Heterotrophs intertwine carbon & energy metabolism.
• Autotrophs have separate pathways for carbon metabolism & energy harnessing.
• Both use ATP & reducing power to process precursor metabolites.
• ATP generation is achieved via one of two mechanisms:
  • Substrate-level phosphorylation.
  • Transmembrane ion gradients.
  • Highly conserved among all extant organisms.

Substrates

• Low energy substrate bond.
• Result of a hydrolytic event.
• Converts high energy phosphoryl bond. - Hydrolysis - Production of ATP - High energy
• For example glycolysis.
  • Net gain of 2 ATP.

Fermentation

• Fermentation in microbes generates all the ATP needed via substrate-level phosphorylation.
• They cannot regenerate NAD+.
  • NADH accumulates.
  • Relies on cyclical pattern, stops in trucks if NAD+ not regenerated!
  • Fermentation microbes "dump" their excess reducing power to facilitate recycling of NADH to NAD+.
• Pyruvate acts as a hub for auxiliary fueling pathways:
  • Homolactic to lactate
  • Heterolactic to:
    • Lactate
    • EtOH
    • Acetic acid
    • CO2.
• Industrial scale fermentations enable production of biofuels
• Many foods & beverages are made using fermentation

Gradients

• Transmembrane ion gradients - For gradients can facilitate greater energy capture.
• Membrane proteins form a "chain".
  • Electrons are transferred sequentially.
  • Protons are exported across membrane.
    • Proton motive force (PMF)
• ATP CF1FO) synthase uses the ion gradients to energize phosphorylation of ADP.
• Four ways to generate ion gradients: - Respiration. - Photosynthesis. - Enzyme Pumps. - Scalar reactions.

Respiration

• Electrons are passed from e- donor to e- acceptor via redox reactions.
  • e- pulls down redox potential of electron carriers in the chain.
  • Generates proton gradient powers ATP synthase.
• e- eventually lands on terminal electron acceptor
• 30 molecules of ATP generated
• Aerobic vs Anaerobic Respiration
• Terminal electron acceptor indicates whether respiration is aerobic or anaerobic - Aerobic: O2 - Anaerobic: NO3-, NO2-, SO42-, etc
• Not all are equal in how much ATP is generated by terminal electron acceptors.
• Some microbes are capable of using e- acceptors.
• Facultative microbes can respire both aerobically or anaerobically.
• Why would a microbe ever perform fermentation rather than respiring glucose? - Microbial community: - syntropy: microbe wants to use others outputs

Inheritance

• DNA stores all of a cell's genetic info & is often replicated w/high fidelity but not always...
• Why does genetic variation matter? - inbreeding could aur causing deleterious alleks - Important for community surviναι

Test your understanding of microbial growth, including balanced growth, chemostat use, and growth phases. Explore temperature effects on microbes and methods of bacterial quantification such as spectrophotometry and dilution plating.