Microbial Metabolism, Growth, and Control: Study Notes for Microbiology

Microbial Metabolism

Catabolism and Anabolism

Metabolism encompasses all chemical reactions in a cell, divided into two main types: catabolism and anabolism.

• Catabolism: Exergonic reactions that break down larger molecules, releasing energy. Often involves oxidation (removal of high-energy electrons).

• Anabolism: Endergonic reactions that build complex molecules from simpler ones, requiring energy input. Often involves reduction (insertion of high-energy electrons).

Enzymes control metabolic reactions by lowering activation energy and increasing reaction speed. They are highly specific proteins that bind substrates at their active sites.

• Types of enzymatic reactions:

  • Catabolic (hydrolases, lyases): Disassemble molecules

  • Anabolic (dehydration synthase, ligase): Assemble molecules

  • Isomerases: Change molecular structure

  • Transferases: Transfer functional groups between molecules

• Enzymes have optimal pH and temperature conditions and can be inhibited by various factors.

Reaction Coupling

Cells couple exergonic and endergonic reactions to drive essential processes.

• Exergonic reactions ($\Delta G < 0$): Release energy, spontaneous.

• Endergonic reactions ($\Delta G > 0$): Require energy input.

Catabolism of Macromolecules

Microbes digest macromolecules using secreted enzymes:

• Carbohydrates: Hydrolyzed into monomers (e.g., cellulose by fungi, chitin, agar, lignin, pectin by bacteria).

• Proteins: Proteolytic enzymes cleave polypeptides into amino acids; some enzymes (hyaluronidase, collagenase) cause tissue damage.

• Lipids: Fatty acids split from glycerol and transported into the cell.

• Nucleic acids: DNases degrade DNA, aiding bacterial invasion (e.g., Staphylococcus aureus).

Catabolism of Monomers

• Monosaccharides: Enter glycolysis, producing ATP and NADH. Pyruvate proceeds to the Krebs cycle or fermentation.

• Amino acids: Enter glycolysis, pyruvate oxidation, or Krebs cycle after deamination.

• Fats: Broken into acetyl groups for the Krebs cycle; glycerol enters glycolysis.

Glycolysis and Alternative Pathways

Glycolysis splits glucose into two three-carbon molecules, generating ATP and NADH. Occurs with or without oxygen.

• Electron carriers: NAD+ and NADP+ shuttle electrons during metabolism.

• Pathways:

  • Embden-Meyerhof (classic glycolysis): Used by most organisms.

  • Entner-Doudoroff: Used by some Gram-negative bacteria.

  • Phosphoketolase: Found in some prokaryotes and eukaryotes.

Fermentation

Fermentation regenerates NAD+ by transferring electrons to organic molecules, producing characteristic end products.

• Types:

  • Homolactic acid: Only lactic acid produced.

  • Heterolactic acid: Lactic acid, ethanol, and CO2.

  • Alcoholic: Ethanol and CO2.

  • Propionic, butyric, butanediol, mixed acid fermentations: Various acids and gases.

• Applications: Fermentation patterns help identify bacteria (e.g., phenol red, methyl red tests).

Krebs Cycle (Citric Acid Cycle)

A series of enzyme-catalyzed reactions that extract high-energy electrons from acetyl-CoA, producing ATP, NADH, and FADH2.

Electron Transport Chain and Chemiosmosis

• High-energy electrons from NADH and FADH2 are transferred through membrane-bound carriers, pumping H+ to create a gradient.

• ATP synthase uses the H+ gradient to generate ATP from ADP (oxidative phosphorylation).

• Oxygen is the final electron acceptor in aerobic respiration.

• Facultative anaerobes can switch between fermentation and respiration depending on oxygen availability.

Anaerobic Respiration

• Uses electron acceptors other than oxygen (e.g., nitrate, sulfate).

• Denitrifying bacteria reduce nitrate to nitrogen gas, important in the nitrogen cycle.

• Sulfate-reducing bacteria produce H2S, contributing to corrosion and odor in anaerobic environments.

Metabolic Diversity

• Energy sources:

  • Phototrophs: Use light energy.

  • Chemotrophs: Use chemical energy (chemoorganotrophs use organic, chemolithotrophs use inorganic compounds).

• Carbon sources:

  • Autotrophs: Use CO2 or other inorganic carbon.

  • Heterotrophs: Use organic carbon sources.

• Combined terms: e.g., photoautotrophs, chemoheterotrophs.

Biochemical Tests and Bacterial Identification

Common Biochemical Tests

• Phenol Red Fermentation Test: Detects acid production from sugar fermentation (positive: red color at pH ~6.8).

• Methyl Red Test: Detects strong acid production from glucose fermentation (positive: red at pH < 4.4).

• Citrate Agar Test: Identifies bacteria that use citrate as sole carbon source (positive: blue color change).

• Catalase Test: Detects catalase enzyme (breaks down H2O2 to O2).

• Oxidase Test: Detects cytochrome c oxidase (positive in Neisseria, negative in enterics).

• H2S Production: Black precipitate in agar indicates sulfide production.

• IMViC Tests: Indole, Methyl Red, Voges-Proskauer, Citrate tests distinguish enteric bacteria.

Bacterium	Indole	Methyl Red	Voges-Proskauer	Citrate
Escherichia coli	+	+	-	-
Enterobacter	-	-	+	+
Citrobacter freundii	-	+	-	+
Klebsiella	-	-	+	+

Growth and Culturing of Bacteria

Temperature, pH, and Moisture Requirements

• Cardinal temperatures: Minimum, optimum, and maximum for growth.

• Temperature classes:

  • Psychrophiles: -10°C to 25°C (optimum 10–20°C)

  • Mesophiles: 10°C to 45°C (optimum 20–40°C)

  • Thermophiles: 30°C to 80°C (optimum 45–80°C)

  • Hyperthermophiles: 80°C and above

• pH: Most bacteria grow best at pH 6.5–7.5. Acidophiles and alkalophiles tolerate extreme pH.

• Moisture: Water activity (aw) > 0.90 is generally required. Halotolerant and halophilic microbes tolerate or require low aw.

Oxygen Requirements

• Aerobes: Require oxygen.

• Microaerophiles: Require lower oxygen levels.

• Facultative anaerobes: Can grow with or without oxygen.

• Aerotolerant anaerobes: Do not use oxygen but tolerate it.

• Obligate anaerobes: Cannot grow in oxygen.

Culture Media

• Nondefined (complex) media: Contain extracts from animals, plants, or yeasts (e.g., nutrient broth, brain-heart infusion).

• Defined (synthetic) media: Exact chemical composition known (e.g., citrate agar).

• Enrichment media: Enhance growth of specific microbes (e.g., selenium for Salmonella).

• Selective media: Inhibit unwanted microbes (e.g., Sabouraud dextrose for fungi).

• Differential media: Distinguish microbes by colony appearance or metabolic products (e.g., blood agar for hemolysis).

• Selective & Differential media: Mannitol salt agar, MacConkey agar, EMB agar.

Measuring Bacterial Growth

• Binary fission: Main method of bacterial reproduction (not mitosis).

• Generation (doubling) time (g): Time required for population to double.

• Population growth curve: Four phases: lag, exponential, stationary, decline (death).

Equation for exponential growth:

$N = N_0 \times 2^{(t/g)}$

Where $N$ = final cell number, $N_0$ = initial cell number, $t$ = time, $g$ = generation time.

Counting Techniques

• Direct microscope count: Counts all cells (viable and non-viable).

• Viable cell count: Plate or membrane count; counts colony-forming units (CFUs).

• Turbidimetric measurement: Estimates cell density by light absorbance.

• Dry weight: Measures biomass.

Control of Microbial Growth

Key Terms

• Sterilization: Complete destruction of all life forms, including spores and viruses.

• Disinfection: Destruction of most life forms (not necessarily spores); disinfectants for surfaces, antiseptics for living tissue.

• Sanitizing: Reduces microbial load to safe levels.

• "-cides": Kill microbes (e.g., bactericide).

• "-stats": Inhibit growth (e.g., bacteriostat).

Physical Methods of Control

• Heat: Denatures proteins and nucleic acids.

  • Thermal death point: Lowest temperature to kill all microbes in 10 min.

  • Thermal death time: Minimum time to kill at a given temperature.

  • Autoclaving: 121°C under pressure.

  • Pasteurization: ~65°C for 30 min.

  • Dry heat: Flaming, hot air.

• Filtration: Removes microbes from liquids/air (0.22 µm filters).

• Low temperature: Slows growth, may kill over time.

• High pressure: Used in food preservation.

• Desiccation and osmotic pressure: Remove water or increase solute concentration to inhibit growth.

• Radiation: Damages DNA (UV, ionizing).

• Ultrasonic energy: Disrupts cells.

Chemical Methods of Control

• Phenols/Phenolics: Disrupt membranes, denature proteins (e.g., Lysol™).

• Bisbiguanides: e.g., chlorhexidine in surgical soap.

• Essential oils: Contain phenols and terpenes.

• Halogens: Disrupt protein function (e.g., chlorine, iodine).

• Alcohols: Disrupt proteins and lipids (ethanol, isopropanol).

• Heavy metals: Bind sulfhydryl groups, denature proteins.

• Surface active agents: Soaps, detergents, quaternary ammonium compounds (quats).

• Aldehydes: Disrupt proteins (formaldehyde, glutaraldehyde).

• Chemical sterilants: Hydrogen peroxide (vaporized), ethylene oxide.

• Plasmas, supercritical fluids, peroxygens: Advanced sterilization methods.

Antimicrobial Drugs

History and Key Terms

• Early use: Moldy bread for wounds (ancient), Paul Ehrlich (Salvarsan), Alexander Fleming (penicillin), Gerhard Domagk (sulfa drugs).

• Antibiotic: Natural antimicrobial produced by organisms.

• Antimicrobial drug: Any agent (natural, synthetic, or semisynthetic) for microbial diseases.

Attributes of Effective Antimicrobials

• Soluble in body fluids, non-toxic, stable, selective toxicity, non-allergenic, minimal disruption of normal flora, low resistance development, long shelf-life, reasonable cost.

• Spectrum: Broad-spectrum (many types) vs. narrow-spectrum (specific).

Mechanisms of Action

• Inhibit cell wall synthesis: β-lactams (penicillins, cephalosporins), carbapenems, vancomycin, isoniazid (for mycobacteria).

• Inhibit protein synthesis: Chloramphenicol, aminoglycosides (streptomycin), tetracyclines, macrolides (erythromycin).

• Injure plasma membrane: Lipopeptides (daptomycin), polymyxin B, bacitracin.

• Inhibit nucleic acid synthesis: Rifamycins (rifampin), quinolones/fluoroquinolones (ciprofloxacin).

• Inhibit synthesis of metabolites: Sulfonamides (folic acid synthesis inhibitors).

• Antifungal drugs: Target ergosterol (nystatin, amphotericin), β-glucan (echinocandins), nucleic acid analogs (flucytosine).

• Antiviral drugs: Block entry, uncoating, nucleic acid synthesis, assembly, or exit; include interferons and antiretrovirals.

• Antiprotozoan/antihelminthic drugs: Various mechanisms (not detailed here).

Assessment of Antimicrobial Susceptibility

• Disc diffusion (Kirby-Bauer): Zone of inhibition indicates susceptibility.

• E test: Determines minimal inhibitory concentration (MIC).

• Broth dilution: Serial dilutions to find MIC.

• Serum killing power: Tests patient blood after antibiotic administration.

• β-lactamase testing: Detects resistance enzymes.

Resistance to Antimicrobial Drugs

• Mechanisms: Enzymatic inactivation (β-lactamases), reduced entry, altered targets, alternative pathways, drug efflux.

• Types: Intrinsic, mutation-based, or acquired resistance.

• Notable resistant strains: MRSA, MDR tuberculosis, artemisinin-resistant malaria, cephalosporin-resistant gonorrhea.