Unit 4: Microbial Growth and Reproduction

I. Orientation

Microbial growth is the increase in cellular material and usually the number of cells in a population. In bacteria, growth commonly occurs through binary fission, while genetic variation can arise through horizontal gene transfer. Population growth depends on nutrient availability, environmental conditions, and the way cells are cultured.

• Governing principle: Under favorable conditions, one bacterial cell divides into two genetically similar cells; repeated divisions produce exponential population increase.
• Population versus individual growth: Individual growth involves increased cell mass; population growth is measured as increasing cell number, biomass, turbidity, or metabolic activity.
• Asexual division: Binary fission generally produces two daughter cells after chromosome replication and septum formation.
• Growth limitation: Nutrient depletion, toxic waste accumulation, unfavorable temperature, pH, oxygen conditions, or osmotic stress eventually slow growth.
• Genetic exchange: Transformation, transduction, and conjugation transfer DNA between cells without requiring reproduction of the recipient cell.
• Culture conventions: A batch culture is closed with respect to nutrient input, whereas a continuous culture receives fresh medium and removes culture at a controlled rate.

II. Microbial Growth — Population increase in culture

Microbial growth is analyzed by following changes in cell number or biomass over time. A typical bacterial population passes through recognizable phases because environmental resources and physiological conditions change during cultivation.

A. bacterial growth curve

The bacterial growth curve describes the sequential phases of population change in a closed batch culture, usually plotted as the logarithm of viable cell number against time.

• Lag phase: Cells adapt to the new medium, synthesize enzymes, repair damage, and increase in size; cell division is limited. For example, bacteria transferred from nutrient-poor conditions may require time to produce transport proteins.
• Log or exponential phase: Cells divide at a constant maximum rate under the available conditions. Cell number follows exponential growth:

N_t = N_0 × 2^n

• N_t: cell number at time t.
• N_0: initial cell number.
• n: number of generations.
  • Stationary phase: Net population size remains approximately constant because the rate of cell formation equals the rate of cell death. Nutrient exhaustion and accumulation of acids or other wastes are common causes.
  • Death or decline phase: Viable cells decrease, often exponentially, although some cells may remain dormant or persist in a non-culturable state.
  • Physiological significance: Secondary metabolites such as some antibiotics are often produced during late stationary phase, while primary metabolites such as amino acids are commonly associated with active growth.

B. generation time

Generation time is the time required for a population to double during exponential growth; it is a key measure of bacterial growth rate.

• Formula: If a culture increases from (N_0) to (N_t) through (n) generations, then:

n = (log10 N_t − log10 N_0) / log10 2
g = t / n

• n: number of generations.
• g: generation time.
• t: elapsed growth time.
• N_0 and N_t: initial and final cell numbers.
  • Worked example: If a population rises from (10^3) to (10^6) cells in 90 minutes, it undergoes about 10 generations because (10^6/10^3 = 10^3 = 2^n), approximately (n = 9.97). Thus, (g \approx 90/9.97 = 9.0) minutes.
  • Growth-rate constant: The specific growth rate, (\mu), is related to generation time by:

μ = ln(2) / g

• μ: growth-rate constant, usually expressed in time(^{-1}).
• g: generation time in the same time unit.
  • Interpretation: A shorter generation time means faster population growth, but it is not a fixed species-wide value; it changes with nutrients, temperature, pH, and oxygen availability.

C. synchronous batch and continuous culture

Culture design determines whether cells divide together, experience changing conditions, or remain near a steady physiological state.

• Synchronous culture: Cells are induced or selected to begin the cell cycle at approximately the same time, allowing division events to occur together.
  • Uses: Studying chromosome replication, septum formation, cell-cycle timing, and division proteins.
  • Limitation: Synchrony usually decreases after several generations because cells become physiologically heterogeneous.
• Batch culture: A fixed volume of sterile medium is inoculated and incubated without continuous nutrient replacement.
  • Characteristic: Conditions move from lag through exponential, stationary, and death phases.
  • Example: A flask containing glucose broth shows falling glucose concentration and increasing waste products during growth.
• Continuous culture: Fresh medium enters while an equal volume of culture leaves, maintaining an approximately constant volume.
  • Chemostat: Nutrient concentration and dilution rate are controlled; the limiting nutrient determines growth.
  • Turbidostat: Turbidity is monitored and medium flow is adjusted to maintain a chosen cell density.
• Dilution rate: In a continuous system:

D = F / V

• D: dilution rate, usually h(^{-1}).
• F: flow rate of fresh medium, such as liters per hour.
• V: culture volume, such as liters.
• At steady state, growth rate approximately equals (D); if (D) exceeds the organism’s maximum growth rate, washout occurs.

D. methods for measuring microbial growth

Growth can be measured directly, indirectly, or by estimating viable cells, and each method measures a different biological property.

• Direct microscopic count: A counting chamber, such as a Petroff–Hausser chamber, estimates cells in a known volume.
  • Strength: Rapid and counts cells whether alive or dead.
  • Limitation: Cannot reliably distinguish viable cells without special stains.
• Viable plate count: A diluted sample is spread or poured onto agar, and colonies are counted as colony-forming units (CFU).
  • Calculation: CFU/mL = colonies × dilution factor ÷ volume plated.
  • Limitation: A clump may produce one colony, and cells unable to grow on the selected medium are missed.
• Membrane filtration: A measured liquid volume is passed through a membrane that retains cells; the membrane is placed on agar for colony development. This is useful for low microbial concentrations in water.
• Turbidity: Optical density, commonly measured near 600 nm, increases as cells scatter light.
  • Strength: Fast and nondestructive.
  • Limitation: Optical density must be calibrated against cell number and becomes unreliable at very high density.
• Biomass measurement: Cells can be collected, dried, and weighed as dry weight, or cellular nitrogen/protein can be measured.
• Metabolic indicators: Oxygen consumption, carbon dioxide production, ATP, acid formation, or substrate disappearance can estimate activity but may not equal cell number.

E. factors affecting the growth of bacteria

Bacterial growth requires compatible physical and chemical conditions; altering one factor can change enzyme activity, membrane function, and nutrient uptake.

• Temperature: Psychrophiles grow best at low temperatures, mesophiles commonly near 20–45°C, and thermophiles at higher temperatures. Excess heat denatures proteins, while cold slows membrane and enzyme reactions.
• pH: Most bacteria prefer near-neutral pH, approximately 6.5–7.5. Acidophiles and alkaliphiles require more extreme values; buffers reduce sudden pH changes.
• Water availability: Cells need water for transport and biochemical reactions. High salt or sugar lowers water activity, causing plasmolysis; halophiles are adapted to high salt.
• Oxygen: Obligate aerobes require oxygen, obligate anaerobes are harmed by it, facultative anaerobes use oxygen when present but grow without it, and microaerophiles require lower-than-atmospheric oxygen.
• Nutrients: Macronutrients include carbon, hydrogen, oxygen, nitrogen, phosphorus, and sulfur; trace elements such as iron, zinc, and manganese support enzymes.
• Osmotic pressure: Hypertonic surroundings draw water from cells, whereas hypotonic surroundings can cause swelling; bacterial cell walls help resist lysis.
• Inhibitory substances: Antibiotics, disinfectants, heavy metals, and metabolic wastes reduce growth by damaging DNA, membranes, ribosomes, or essential enzymes.

III. Bacterial Reproduction — Genetic exchange and cell multiplication

Bacterial reproduction normally occurs by binary fission, but bacteria also acquire new traits through horizontal gene transfer. The transferred DNA may encode antibiotic resistance, toxins, metabolic enzymes, or surface structures.

A. transformation

Transformation is the uptake of free DNA from the environment by a competent bacterial cell, followed by maintenance or incorporation of that DNA.

• Source of DNA: DNA commonly comes from lysed bacterial cells and may be present as chromosomal fragments or plasmid DNA.
• Competence: Naturally competent bacteria produce DNA-binding and uptake proteins; laboratory treatments such as calcium chloride and heat shock can make some cells competent.
• Process: A double-stranded DNA fragment binds the surface, enters partly as single-stranded DNA, and may recombine with a homologous chromosome.
• Stable inheritance: Integration by homologous recombination requires sufficient sequence similarity; an independent plasmid can replicate if its origin is functional in the recipient.
• Example: A nonencapsulated strain may acquire capsule genes from DNA released by an encapsulated strain, changing colony appearance and virulence.
• Importance: Transformation can spread antibiotic-resistance alleles, but free DNA is vulnerable to nucleases and transfer depends on recipient competence.

B. transduction

Transduction is bacterial DNA transfer mediated by a bacteriophage, a virus that infects bacteria.

• Generalized transduction: During a lytic infection, a phage may accidentally package a random fragment of bacterial DNA instead of phage DNA.
  • Consequence: Nearly any bacterial gene can potentially be transferred, depending on the packaging error.
• Specialized transduction: A temperate phage integrates at a specific bacterial chromosome site; inaccurate excision transfers particular neighboring bacterial genes.
  • Consequence: Only genes close to the prophage integration site are usually transferred.
• Process: The transducing phage attaches to a recipient, injects donor bacterial DNA, and the DNA may recombine with the recipient chromosome.
• Example: Phage-mediated movement of toxin genes can convert a normally less harmful bacterial strain into a toxin-producing strain.
• Importance and limitation: Transduction protects DNA from some environmental degradation, but it depends on suitable phage host range and packaging or excision events.

C. conjugation

Conjugation is direct DNA transfer between bacterial cells through cell-to-cell contact, usually involving a conjugative plasmid and a type IV secretion system or sex pilus.

• F plasmid: An F(^{+}) donor carries fertility genes, including transfer functions; an F(^{-}) recipient lacks the plasmid.
  • Transfer: One DNA strand of the plasmid moves through the mating bridge while complementary strands are synthesized in both cells.
  • Result: The recipient generally becomes F(^{+}).
• Hfr transfer: If the F plasmid integrates into the chromosome, the donor is called high-frequency recombination (Hfr).
  • Result: Chromosomal genes adjacent to the integrated plasmid can enter the recipient, although the complete F sequence rarely transfers before contact ends.
• F-prime transfer: An inaccurately excised plasmid may carry bacterial genes; this F′ plasmid transfers those genes at high frequency and can create partial diploids.
• Resistance plasmids: R plasmids may carry several resistance genes, such as genes for resistance to ampicillin and tetracycline, allowing rapid spread across bacterial populations.
• Importance and limitation: Conjugation transfers DNA efficiently between compatible cells, including across some species, but requires physical contact and compatible transfer machinery.