7.1 Microbial growth and cultivation

Microbial growth and cultivation are key to understanding how microbes multiply and thrive. This knowledge is crucial for harnessing their potential in biotechnology and fermentation processes. From growth phases to cultivation methods, these concepts form the foundation of microbial applications.

Bioreactors play a vital role in scaling up microbial processes for industrial use. By controlling growth conditions and optimizing nutrient delivery, these systems enable large-scale production of valuable products like antibiotics, enzymes, and biofuels.

Microbial Growth Phases

Growth Curve and Phases

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• Microbial growth curve represents the changes in cell number over time in a growing population
• Lag phase is the initial period of slow growth as microbes adapt to a new environment and synthesize necessary components for cell division
• Exponential phase (log phase) is characterized by rapid cell division and doubling of the population at a constant rate until nutrients become limited or waste products accumulate
• Stationary phase occurs when the growth rate slows and the number of cells remains relatively constant due to nutrient depletion or accumulation of toxic metabolites
• Death phase is the period of decline in cell numbers as the rate of cell death exceeds the rate of cell division, often due to nutrient exhaustion or buildup of toxic waste products

Growth Rate and Generation Time

• Growth rate is the increase in cell number per unit time during the exponential phase, often expressed as the number of doublings per hour
• Generation time (doubling time) is the time required for a population to double in number during the exponential phase, typically ranging from 20 minutes to several hours depending on the species and growth conditions
• Factors influencing growth rate and generation time include nutrient availability, temperature, pH, oxygen levels, and the presence of growth-promoting or inhibiting substances
• Shorter generation times lead to faster population growth and higher cell densities, which is advantageous for industrial applications such as fermentation and biotechnology

Microbial Cultivation Methods

Batch and Continuous Culture

• Batch culture involves growing microorganisms in a fixed volume of nutrient medium until nutrients are depleted or growth is inhibited by waste products, resulting in distinct growth phases
• Advantages of batch culture include simplicity, ease of monitoring growth phases, and suitability for small-scale experiments or industrial processes with defined endpoints
• Continuous culture maintains a constant population size by continuously adding fresh nutrient medium and removing an equal volume of culture, allowing for steady-state growth and product formation
• Advantages of continuous culture include higher productivity, reduced downtime, and the ability to maintain optimal growth conditions for extended periods, making it suitable for large-scale industrial processes (biopharmaceuticals, biofuels)

Nutrient Media and Sterile Technique

• Nutrient media provide the necessary nutrients, energy sources, and growth factors for microbial growth, and can be formulated as liquid broths or solid agar plates
• Types of media include defined media with known chemical composition (minimal media), complex media with undefined components (nutrient broth, lysogeny broth), and selective media that favor the growth of specific microbes (mannitol salt agar for Staphylococcus)
• Sterile technique involves methods to prevent contamination of cultures by unwanted microorganisms, such as autoclaving media and equipment, using aseptic transfer techniques (laminar flow hoods, flame sterilization of loops), and disinfecting work surfaces
• Proper sterile technique is critical for maintaining pure cultures, ensuring reproducibility of experiments, and preventing the introduction of contaminants in industrial processes

Bioreactor Systems

Bioreactor Design and Applications

• Bioreactors are vessels used for growing microorganisms or cells under controlled conditions for the production of biomass, metabolites, or recombinant proteins
• Key components of bioreactors include a vessel for containing the culture, a system for aeration and agitation (spargers, impellers), sensors for monitoring pH, temperature, and dissolved oxygen, and ports for sampling and addition of nutrients
• Stirred-tank bioreactors are commonly used for aerobic processes and consist of a cylindrical vessel with a motor-driven impeller for mixing and a sparger for introducing air or oxygen
• Airlift bioreactors use the injection of air at the bottom of the vessel to achieve mixing and aeration, and are suitable for shear-sensitive cells or tissues
• Applications of bioreactors include the production of antibiotics (penicillin), enzymes (amylases, proteases), organic acids (citric acid), biofuels (ethanol), and recombinant proteins (insulin, monoclonal antibodies)
• Scale-up of bioreactor processes from laboratory to industrial scale requires optimization of parameters such as agitation rate, aeration rate, and feeding strategies to maintain optimal growth conditions and product formation