Microbial Metabolism

Metabolism, Anabolism, and Catabolism

Metabolism refers to all chemical reactions occurring within a cell. These reactions are divided into two main types: anabolism (building up) and catabolism (breaking down).

• Anabolism: Synthesis of complex molecules from simpler ones; requires energy. Example: Protein synthesis from amino acids.

• Catabolism: Breakdown of complex molecules into simpler ones; releases energy. Example: Glucose breakdown during glycolysis.

Oxidation and Reduction Reactions

Oxidation-reduction (redox) reactions involve the transfer of electrons between molecules, crucial for energy production.

• Oxidation: Loss of electrons.

• Reduction: Gain of electrons.

• Significance: Redox reactions drive ATP synthesis in cells.

Phosphorylation Mechanisms

Phosphorylation is the addition of a phosphate group to a molecule, important for ATP production.

• Substrate-level phosphorylation: Direct transfer of phosphate to ADP from a substrate.

• Oxidative phosphorylation: Uses electron transport chain and chemiosmosis to generate ATP.

• Photophosphorylation: Occurs in photosynthetic organisms, using light energy.

Enzymes: Structure and Function

Enzymes are biological catalysts that speed up chemical reactions without being consumed.

• Structure: Most are proteins; some require cofactors.

• Function: Lower activation energy, increase reaction rate.

• Examples: Amylase (breaks down starch), DNA polymerase (synthesizes DNA).

Cofactors and Enzyme Action

Cofactors are non-protein components required for enzyme activity.

• Inorganic cofactors: Metal ions (e.g., Mg2+, Fe2+).

• Organic cofactors (coenzymes): Vitamins or vitamin derivatives (e.g., NAD+, FAD).

Enzyme Denaturation and Factors Affecting Activity

Denaturation is the loss of enzyme structure and function due to environmental changes.

• Causes: High temperature, extreme pH, chemicals.

• Enzyme and substrate concentration: Higher concentrations increase reaction rate up to a saturation point.

Enzyme Inhibition

Enzyme activity can be regulated by inhibitors.

• Competitive inhibitors: Bind to the active site, blocking substrate.

• Non-competitive inhibitors: Bind to an allosteric site, changing enzyme shape.

• Allosteric control: Regulation by binding at sites other than the active site.

• Feedback inhibition: End-product inhibits pathway, preventing overproduction.

ATP: Structure and Function

ATP as Cellular Energy Currency

ATP (adenosine triphosphate) stores and transfers energy within cells.

• Structure: Adenine, ribose, three phosphate groups.

• Function: Energy released by hydrolysis of terminal phosphate.

Aerobic Glucose Catabolism

Stages of Aerobic Respiration

Aerobic respiration consists of three main stages, each with distinct substrates, products, and energy yields.

• Glycolysis: Occurs in cytoplasm; glucose → pyruvate; net yield: 2 ATP, 2 NADH.

• Krebs Cycle (Citric Acid Cycle): Occurs in mitochondria (eukaryotes) or cytoplasm (prokaryotes); pyruvate → CO2; net yield: 2 ATP, 6 NADH, 2 FADH2.

• Electron Transport Chain: Occurs in mitochondrial membrane (eukaryotes) or plasma membrane (prokaryotes); NADH/FADH2 → ATP; net yield: ~34 ATP.

Alternative Pathways to Glycolysis

Some microbes use alternative pathways for glucose catabolism.

• Pentose Phosphate Pathway: Generates NADPH and ribose-5-phosphate for biosynthesis.

• Entner-Doudoroff Pathway: Found in some bacteria; yields ATP and NADPH.

Fermentation vs. Aerobic Respiration

Fermentation is an anaerobic process that allows ATP production without oxygen.

• Fermentation: Pyruvate converted to organic products (e.g., lactic acid, ethanol); low ATP yield.

• Aerobic respiration: Requires oxygen; high ATP yield.

ATP Yield in Aerobic Respiration

Net ATP yield from aerobic respiration:

• Glycolysis: 2 ATP

• Krebs Cycle: 2 ATP

• Electron Transport Chain: ~34 ATP

• Total: ~38 ATP per glucose (prokaryotes); ~36 ATP (eukaryotes)

Anaerobic Respiration

Microbes may use alternative electron acceptors in the absence of oxygen.

• Electron acceptors: Nitrate (NO3-), sulfate (SO42-), carbonate (CO32-).

• Significance: Enables survival in oxygen-poor environments.

Comparison of Metabolic Pathways

Fermentation Products

Fermentation produces various products depending on the microbe.

• Lactic acid: Lactobacillus

• Ethanol: Yeast

• Acetic acid, propionic acid, butyric acid: Various bacteria

Amphibolism

Amphibolism refers to metabolic pathways that serve both anabolic and catabolic functions.

• Example: Krebs cycle intermediates used for biosynthesis.

Processing of Lipids and Proteins

Microbes can catabolize lipids and proteins for energy.

• Lipids: Broken down by lipases; fatty acids enter beta-oxidation.

• Proteins: Broken down by proteases; amino acids deaminated and enter metabolic pathways.

Anabolic Reactions in Microbial Cells

Microbes synthesize essential biomolecules through anabolic pathways.

• Carbohydrate biosynthesis

• Amino acid biosynthesis

• Nucleotide biosynthesis

Interrelationships Between Catabolism and Anabolism

Catabolic reactions provide ATP and substrates for anabolic reactions.

• ATP: Energy for biosynthesis.

• Substrates: Building blocks for macromolecules.

Microbial Nutrition and Growth

Roles of Elements and Nutrients

Microbial growth requires various elements and nutrients.

• Carbon: Backbone of organic molecules.

• Oxygen: Electron acceptor, component of organic molecules.

• Hydrogen: Maintains pH, component of organic molecules.

• Nitrogen: Component of proteins, nucleic acids.

• Trace elements: Required in small amounts (e.g., Zn, Cu).

• Vitamins: Often serve as coenzymes.

Macronutrients vs. Micronutrients

Inorganic vs. Organic Nutrients

• Inorganic nutrients: Do not contain carbon-hydrogen bonds (e.g., water, salts).

• Organic nutrients: Contain carbon-hydrogen bonds (e.g., carbohydrates, proteins).

Composition of a Microbial Cell

A typical microbial cell is composed of water, proteins, nucleic acids, lipids, carbohydrates, and minerals.

• Water: ~70%

• Proteins: ~15%

• Nucleic acids, lipids, carbohydrates: Remaining cell mass

Categories of Organisms by Carbon and Energy Source

Oxygen Requirements and Microbial Types

Toxic Forms of Oxygen and Protection Mechanisms

Oxygen can form toxic free radicals harmful to cells.

• Free radicals: Singlet oxygen, superoxide radical, peroxide anion, hydroxyl radical.

• Protection: Enzymes such as superoxide dismutase and catalase detoxify radicals.

Environmental Factors Limiting Growth

• Temperature: Extremes can denature proteins.

• pH: Extreme pH affects enzyme activity.

• Osmotic pressure: High salt/sugar can inhibit growth.

• Hydrostatic pressure: Deep-sea microbes adapt to high pressure.

Quorum Sensing and Biofilms

Quorum sensing is cell-to-cell communication that regulates gene expression based on population density.

• Biofilms: Structured microbial communities; Pseudomonas aeruginosa forms biofilms.

Microbial Culturing and Growth Measurement

Definitions in Microbial Culturing

• Inoculum: Introduction of microbes into a medium.

• Medium: Nutrient material for growth.

• Culture: Microbes growing in a medium.

• Clinical specimen: Sample from patient.

• Mixed population: Multiple species.

• Pure culture: Single species.

Streak Plate vs. Pour Plate

• Streak plate: Isolates colonies by spreading on agar.

• Pour plate: Diluted sample mixed with agar, poured into plate.

Types of Media

Special Culturing Techniques

• Low-oxygen/anaerobic culture: Uses reducing agents.

• Animal culture: Used for viruses.

• Tissue culture: Used for obligate intracellular microbes.

Cell Division in Prokaryotes vs. Eukaryotes

• Prokaryotes: Binary fission; no mitosis or cell cycle.

• Eukaryotes: Mitosis and cell cycle.

Logarithmic Growth and Generation Time

Bacterial populations grow exponentially under optimal conditions.

• Logarithmic growth: Population doubles each generation.

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

• Formula:

Where: = final number of cells = initial number of cells = number of generations

• Short generation time: Escherichia coli (~20 min)

• Long generation time: Mycobacterium tuberculosis (~24 hours)

Bacterial Growth Curve

A typical growth curve has four phases:

• Lag phase: Adaptation, no growth.

• Log phase: Exponential growth.

• Stationary phase: Growth rate equals death rate.

• Death phase: Decline in population.

Measuring Bacterial Growth

• Direct methods: Plate counts, microscopic counts.

• Indirect methods: Turbidity, metabolic activity, dry weight.

Viable Plate Count Procedure

1. Serially dilute sample.

2. Plate aliquots on agar.

3. Incubate and count colonies.

4. Calculate original cell number using dilution factor.

Quantification Methods: Pros and Cons