Microbial Growth and Nutrition

Macronutrients and Micronutrients

Microorganisms require a variety of chemical elements for growth, which are classified as macronutrients and micronutrients.

• Macronutrients: Needed in large amounts. Include carbon, nitrogen, phosphorus, sulfur, oxygen, and hydrogen. Essential ions such as potassium, sodium, magnesium, calcium, and iron serve as enzyme cofactors and structural components.

• Micronutrients (Trace Elements): Required in small amounts. Include manganese (Mn), zinc (Zn), cobalt (Co), molybdenum (Mo), nickel (Ni), and copper (Cu). Often part of enzymes and cofactors.

Categorizing Microbes by Energy, Carbon, and Electron Source

• Energy Source:

  • Phototrophs: Use light as an energy source.

  • Chemotrophs: Obtain energy from oxidation of chemical compounds.

• Carbon Source:

  • Autotrophs: Use inorganic carbon (CO2) as their primary carbon source.

  • Heterotrophs: Use preformed organic molecules from other organisms.

• Electron Source:

  • Lithotrophs: Use reduced inorganic substances (e.g., H2, H2S, NH3).

  • Organotrophs: Use organic compounds as electron donors.

Importance of Nitrogen, Sulfur, and Phosphorus

• Nitrogen: Found in amino acids, nucleic acids, and other cell components. Acquired from ammonia, nitrates, or atmospheric N2.

• Sulfur: Present in amino acids (cysteine, methionine), coenzymes, and vitamins.

• Phosphorus: Essential for nucleic acids, phospholipids, and some proteins.

Growth Factors

Growth factors are organic compounds that an organism cannot synthesize but are essential for growth (e.g., amino acids, purines/pyrimidines, vitamins).

Nutrient Concentration and Growth

• High nutrient concentration leads to rapid growth.

• Low nutrient concentration slows the growth rate.

Oxygen and Microbial Growth

• Aerobes: Grow in the presence of oxygen.

• Obligate aerobes: Require oxygen for growth.

• Microaerophiles: Require oxygen but at lower concentrations than atmospheric levels.

• Anaerobes: Do not use oxygen; may be harmed by it.

• Aerotolerant anaerobes: Tolerate oxygen but do not use it.

• Obligate anaerobes: Oxygen is toxic to them.

• Facultative anaerobes: Prefer oxygen but can grow without it.

Reactive Oxygen Species (ROS) and Detoxification

• Oxygen can be reduced to toxic ROS (e.g., superoxide radicals, hydrogen peroxide).

• Microbes detoxify ROS using enzymes:

  • Superoxide dismutase (SOD): Converts superoxide radicals to O2 and H2O2.

  • Catalase: Breaks down H2O2 into water and oxygen.

  • Peroxidase: Reduces H2O2 without producing O2.

Anaerobic Work Environments

• Oxygen must be excluded (e.g., anaerobic jars, glove boxes, or chambers flooded with inert gases).

• GasPak systems or airlocks are used to maintain anaerobic conditions.

pH and Temperature in Growth

• pH Preferences:

  • Acidophiles: pH < 5.5

  • Neutrophiles: pH 5.5–8.0 (most human pathogens)

  • Alkaliphiles: pH 8.5–11.5

• Temperature Preferences:

  • Psychrophiles: Optimum ≤ 15°C

  • Psychrotrophs: Grow at 0–7°C, optimum 20–30°C

  • Mesophiles: Optimum 20–45°C (human body temp: 37°C)

  • Thermophiles: Optimum 55–65°C

  • Hyperthermophiles: Optimum 85–113°C

Solutes, Water Availability, and Adaptations

• High solute concentrations and low water availability can inhibit growth.

• Microbes adapt via compatible solute accumulation and specialized proteins/membranes.

Culture Media Types

• Synthetic (Defined) Media: Exact chemical composition is known.

• Complex Media: Contains ingredients of unknown composition (e.g., peptones, yeast extract).

• General Purpose: Supports many microbes (e.g., nutrient agar).

• Enriched: Supplemented with nutrients for fastidious organisms (e.g., blood agar).

• Selective: Favors growth of specific microbes, inhibits others (e.g., MacConkey agar).

• Differential: Distinguishes microbes based on biological characteristics (e.g., lactose fermentation on MacConkey).

Blood Agar vs. MacConkey Agar

Colony Isolation Techniques

• Streak Plate: Dilutes cells across agar to isolate colonies.

• Spread Plate: Spreads diluted sample evenly on agar surface.

• Pour Plate: Mixes diluted sample with molten agar, poured into Petri dish.

Unculturable Bacteria and Microbial Consortia

• Most microbes cannot be cultured in standard lab media.

• Studied using metagenomics, transcriptomics, and single-cell genomics.

• Microbial consortia: Communities where microbes depend on each other's metabolic products.

Counting Microbes

• Direct Counts: Counting chambers or flow cytometry; counts live and dead cells.

• Viable Counts: Dilution and plating; counts only living cells (CFUs).

• Turbidimetry: Measures optical density; estimates total biomass.

Colony Forming Units (CFU) and Calculation

• CFU = Number of colonies / (dilution factor × volume plated)

Microbial Growth Curve

• Lag Phase: Adaptation, enzyme synthesis, no immediate cell division.

• Exponential (Log) Phase: Maximum, constant cell division rate.

• Stationary Phase: Growth rate equals death rate; nutrients deplete, waste accumulates.

• Death Phase: Exponential cell death due to starvation and damage.

VBNC and Programmed Cell Death

• VBNC: Viable but non-culturable; cells alive but not growing on standard media.

• Programmed Cell Death: Some cells self-destruct, releasing nutrients for survivors.

Balanced vs. Unbalanced Growth

• Balanced: All cell components synthesized at constant rates (log phase).

• Unbalanced: Synthesis rates vary (e.g., after nutrient shift).

Growth Curve Calculations

• Generation Time (G): Time for population to double.

• Formula:

• Semi-log Plots: Logarithmic plotting linearizes exponential growth for easier calculation.

Open vs. Closed Systems

• Open System (Continuous Culture): Fresh medium added, waste removed; cells remain in log phase (e.g., chemostat, turbidostat).

• Closed System (Batch Culture): Finite nutrients, waste accumulates; classic 4-phase growth curve.

Chemostat vs. Turbidostat

Microbial Control Methods

• Sterilization: Destroys all microbes, including spores and viruses.

• Disinfection: Reduces pathogens on inanimate objects.

• Sanitization: Lowers microbial counts to safe levels.

• Antisepsis: Destroys/inhibits microbes on living tissue.

• Chemotherapy: Internal use of chemicals to kill/inhibit pathogens.

Factors Affecting Antimicrobial Efficacy

• Population size and composition

• Agent concentration/intensity

• Exposure duration

• Temperature

• Local environment (e.g., organic matter, biofilms)

Physical Control Methods

• Heat: Moist (autoclaving) and dry (incineration, hot air ovens)

• Low Temperature: Refrigeration/freezing (bacteriostatic)

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

• Radiation: Ionizing (deep penetration, DNA breaks) vs. non-ionizing (UV, surface sterilization)

Chemical Control Methods

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

• Alcohols: Dissolve lipids, denature proteins (e.g., ethanol, isopropanol)

• Halogens: Oxidize cell components (e.g., chlorine, iodine)

• Heavy Metals: Inactivate proteins (e.g., silver nitrate)

• Surfactants: Disrupt membranes, remove microbes (e.g., soaps, quats)

• Aldehydes: Cross-link proteins/nucleic acids (e.g., glutaraldehyde)

Sterilizing Gases

• Ethylene Oxide (EtO): Alkylates proteins/DNA; used for heat- and moisture-sensitive items.

Bacterial Cell Structure and Function

Bacterial Morphology

• Cocci: Spherical

• Bacilli: Rod-shaped

• Vibrio: Comma-shaped

• Spirilla/Spirochetes: Spiral-shaped

• Pleomorphic: Variable shapes

Surface Area-to-Volume Ratio (S/V)

• Smaller cells have higher S/V ratios, allowing efficient nutrient uptake and waste removal, supporting faster growth and division.

Cytoplasmic Components

• Nucleoid: Region containing the bacterial chromosome (not membrane-bound).

• Plasmids: Small, circular DNA molecules carrying non-essential genes (e.g., antibiotic resistance).

• Ribosomes: Sites of protein synthesis (70S: 50S + 30S subunits).

• Inclusion Bodies: Storage granules for nutrients or energy reserves (e.g., glycogen, polyphosphate, sulfur, gas vacuoles).

Cytoskeleton Proteins

• FtsZ: Forms Z-ring for cell division (tubulin homolog).

• MamK: Positions magnetosomes in magnetotactic bacteria.

• MreB/Mbl: Determines rod shape, directs peptidoglycan synthesis (actin homolog).

• CreS: Confers curved shape (intermediate filament homolog).

• ParM/ParR: Involved in plasmid segregation during division.

Plasma Membrane

• Functions: Permeability barrier, energy generation (ETC, proton motive force), anchor for transport proteins.

• Fluid Mosaic Model: Phospholipid bilayer with embedded proteins; dynamic structure.

• Lipids: Bacterial membranes lack cholesterol but may contain hopanoids for stability; fatty acids linked via ester bonds to glycerol.

Transport Mechanisms

• Passive Diffusion: Movement down concentration gradient (e.g., O2, CO2).

• Facilitated Diffusion: Uses carrier proteins; no energy required.

• Active Transport: Moves substances against gradient using energy (ATP or proton motive force); includes ABC and MFS transporters.

• PTS System: Group translocation; transports and phosphorylates sugars using phosphoenolpyruvate (PEP).

• Siderophores: Secreted molecules that bind and import iron via specific transporters.

Protein Secretion

• Sec System: Universal pathway for translocating unfolded proteins across the plasma membrane (ATP-dependent).

• Gram-Negative Secretion Systems: Multi-protein complexes exporting proteins (e.g., toxins, enzymes) across both membranes.

Cell Wall Structure and Synthesis

• Function: Provides shape, strength, and prevents osmotic lysis.

• Peptidoglycan: Rigid mesh of alternating N-acetylglucosamine (NAG) and N-acetylmuramic acid (NAM) cross-linked by peptides.

• Lysozyme: Cleaves β-1,4-glycosidic bonds between NAG and NAM (especially effective against Gram-positives).

• Lysostaphin: Cleaves pentaglycine bridges in Staphylococcus peptidoglycan.

• β-lactam Antibiotics: Inhibit penicillin-binding proteins, blocking peptidoglycan cross-linking (effective only on actively growing cells).

Antibiotic Mechanisms

• Penicillin: Inhibits transpeptidation, preventing cell wall cross-linking.

• Augmentin: Combines amoxicillin with clavulanic acid (β-lactamase inhibitor) to overcome resistance.

• Vancomycin: Binds D-Ala-D-Ala termini, blocking cross-linking enzymes.

Microbe Comparison Table

Guest Lecture: Methanogens (Archaea)

General Characteristics

• Methanogens: Methane-producing archaea, primarily in Euryarchaeota.

• Oxygen Sensitivity: Strict anaerobes, highly sensitive to O2.

• Ecological Role: Major contributors to global methane (CH4) production; important in nutrient cycles and as biofuel sources.

Metabolism and Substrates

• Energy Conservation: Methanogenesis via oxygen-independent electron transfer; CoM-S-S-CoB is the final electron acceptor.

• Substrates: CO2, H2, formate, acetate, methylated compounds, long-chain alkanes.

• Pathways: CO2-reducing, methylotrophic, alkanotrophic, acetoclastic; can be hydrogenotrophic or formatotrophic.

Model Organisms and Research Highlights

• Methanosarcina acetivorans: Model for methanogenic ecophysiology; growth inhibited by visible light; forms biofilms under stress.

• Methanobrevibacter smithii: Dominant in human gut; adapts for host colonization, forms aggregates, utilizes formate for growth.

One Health and Host Associations

• Ruminants: Methanogens in rumen produce large amounts of methane (e.g., cows emit 500–700 L/day).

• Human Microbiome: Methanogens help balance gases and short-chain fatty acids via syntrophy with bacteria.

• Infections/Dysbiosis: Methanogens detected in periodontitis, endodontic infections, brain abscesses, UTIs, and bacterial vaginosis.

Additional info: Methanogens are a key group within the Archaea, distinct from bacteria and eukaryotes, and play a critical role in anaerobic environments and the global carbon cycle.