Microbial Metabolism, Growth, and Laboratory Techniques: Study Notes

Catabolic and Anabolic Reactions

Metabolism

Metabolism encompasses all chemical reactions within a living organism, divided into catabolic and anabolic processes.

• Catabolism: Breaks down complex molecules, providing energy and building blocks for anabolism. These are typically hydrolytic reactions (require water).

• Anabolism: Uses energy and building blocks to synthesize complex molecules. These are biosynthetic reactions (require energy input).

ATP & Role: ATP is the intermediate between catabolism (produces ATP) and anabolism (uses ATP).

• Metabolic Pathways: Sequences of enzymatically catalyzed chemical reactions in a cell, determined by enzymes encoded by genes.

Enzymes

Collision Theory

Chemical reactions occur when atoms, ions, and molecules collide.

• Activation energy: Minimum energy required for a chemical reaction to occur.

• Reaction rate: Frequency of collisions with enough energy to bring about a reaction. Increased by enzymes, temperature, pressure, or concentration.

Enzymes & Chemical Reactions

• Catalyst: Speeds up chemical reactions without being altered.

• Enzyme-substrate complex: Substrate binds to the enzyme's active site, forming a complex.

• Mechanism:

  1. Substrate binds to active site.

  2. Enzyme catalyzes reaction and releases products.

  3. Enzyme remains unchanged and can react with other substrates.

Naming Enzymes

Enzymes are usually named with an -ase ending and grouped by reaction type:

• Oxidoreductase: Oxidation-reduction reactions.

• Transferase: Transfer functional groups.

• Hydrolase: Hydrolysis reactions.

• Lyase: Removal of atoms without hydrolysis.

• Isomerase: Rearrangement of atoms.

• Ligase: Joining of molecules; uses ATP.

Enzyme Structure

• Apoenzyme: Protein portion (inactive alone).

• Cofactor: Nonprotein component.

• Holoenzyme: Apoenzyme + cofactor (whole, active enzyme).

Factors Influencing Enzyme Activity

• Temperature

• pH

• Substrate concentration (saturation, max reaction rate)

• Inhibitors

Enzyme Inhibition

• Competitive inhibitors: Compete with substrate for the active site.

• Noncompetitive inhibitors: Interact with another part of the enzyme (allosteric site), changing the enzyme's shape.

• Feedback inhibition: End-product of a reaction allosterically inhibits an enzyme from earlier in the pathway.

Ribozymes

• RNA molecules that function as catalysts, not used up in the reaction.

• Frequently involved in cutting and splicing RNA and protein synthesis in ribosomes.

Energy Production

Oxidation-Reduction Reactions (Redox Reactions)

• Energy in nutrient molecules is associated with electrons. Catabolic reactions capture this energy in ATP.

• Oxidation: Removal of electrons (OIL - Oxidation Is Loss).

• Reduction: Gain of electrons (RIG - Reduction Is Gain).

• In biological systems, electrons and protons are often removed together (equivalent to a hydrogen atom).

Generation of ATP (Phosphorylation)

• Phosphorylation: Addition of a phosphate to a chemical compound.

• ATP generation methods:

  1. Substrate-level phosphorylation: Direct transfer of phosphate group to ADP from a phosphorylated compound.

  2. Oxidative phosphorylation: Electrons transferred from organic compounds to electron carriers (NAD+, FAD), then through an electron transport chain (ETC) to oxygen. Energy released is used to generate ATP via chemiosmosis.

  3. Photophosphorylation: Occurs in photosynthetic cells with light-trapping pigments (e.g., chlorophyll). Light energy is converted to chemical energy (ATP) during electron transport.

Metabolic Pathways of Energy Production

• Series of enzymatically catalyzed reactions that extract energy from organic compounds and store it in ATP.

Carbohydrate Catabolism

Overview

Most microorganisms oxidize carbohydrates, with glucose being the most common energy source. Two general processes: Cellular Respiration and Fermentation.

• Cellular respiration includes glycolysis, Krebs cycle, and electron transport chain.

Glycolysis (Embden-Meyerhof pathway)

• Oxidation of glucose to pyruvic acid.

• Produces ATP and NADH.

• First step in carbohydrate catabolism.

• Preparatory stage: Glucose split into two 3-carbon molecules (glyceraldehyde 3-phosphate and dihydroxyacetone phosphate).

• Energy-conserving stage: 3-carbon molecules oxidized to 2 pyruvic acid molecules, producing 4 ATP (net 2 ATP) and 2 NADH.

Additional Pathways to Glycolysis

• Pentose phosphate pathway: Breaks down five-carbon sugars and/or glucose, operates simultaneously with glycolysis, produces NADPH, and is important for biosynthetic reactions.

• Entner-Doudoroff pathway: Produces NADPH, NADH, and ATP, found in some bacteria.

Cellular Respiration

• Oxidation of molecules liberates electrons to operate an ETC.

• Final electron acceptor comes from outside the cell and is inorganic.

• Aerobic respiration: Uses O2 as the final electron acceptor.

• Krebs cycle: Produces NADH, FADH2, and ATP; liberates CO2 as waste.

• Electron transport chain (ETC): In prokaryotes: plasma membrane; in eukaryotes: inner mitochondrial membrane.

Anaerobic Respiration

• Uses a molecule other than O2 as the final electron acceptor.

• Yields less energy than aerobic respiration.

• Examples of electron acceptors and products:

  • NO3- → NO2- + N2 + H2O

  • SO42- → H2S + H2O

  • CO32- → CH4 + H2O

Fermentation

• Releases energy from oxidation of organic molecules.

• Does not require oxygen.

• Does not use the Krebs cycle or ETC.

• Uses an organic molecule as the final electron acceptor.

• Produces small amounts of ATP.

• Homolactic fermentation: Produces lactic acid only.

• Alcohol fermentation: Produces ethanol and CO2.

Lipid and Protein Catabolism

Lipid Catabolism

• Lipids are broken down by extracellular proteases and peptidases into amino acids.

• Amino acids are deaminated, decarboxylated, and desulfurized to obtain molecules that can enter the Krebs cycle.

Biochemical Tests and Bacterial Identification

• Fermentation test: Detects acid production from carbohydrate/protein catabolism (pH indicator changes) and gas production (Durham tube).

• Oxidase test: Detects bacteria with cytochrome c oxidase (e.g., Pseudomonas).

• Detection of H2S production: (e.g., Salmonella).

Photosynthesis

Light-dependent (light) reactions

• Conversion of light energy into chemical energy (ATP and NADPH).

• Cyclic photophosphorylation: Electrons cycle among photosystem components.

• Noncyclic photophosphorylation: Electrons transferred to NADP+ (implied).

Light-independent (dark) reactions

• ATP and NADPH are used to reduce CO2 to sugar (carbon fixation) via the Calvin-Benson cycle.

Types

• Oxygenic: Plants, algae, cyanobacteria (produce O2).

• Anoxygenic: Purple sulfur/green sulfur bacteria (do not produce O2).

Metabolic Diversity Among Organisms

Nutritional Types

• Photoautotrophs: Energy source: Light; Carbon source: CO2. Examples: Oxygenic (cyanobacteria, plants); Anoxygenic (green/purple bacteria).

• Photoheterotrophs: Energy source: Light; Carbon source: Organic compounds. Examples: Green nonsulfur bacteria, purple nonsulfur bacteria.

• Chemoautotrophs: Energy source: Inorganic chemicals; Carbon source: CO2. Examples: Iron-oxidizing bacteria.

• Chemoheterotrophs: Energy source: Chemicals (organic compounds); Carbon source: Organic compounds. Examples: Fermentative bacteria, animals, protozoa, fungi, most bacteria.

Metabolic Pathways of Energy Use (Anabolism)

• Biosynthesis of polysaccharides, simple lipids, amino acids, and nucleotides.

The Integration of Metabolism

• Amphibolic pathways: Metabolic pathways that function in both anabolism and catabolism.

• Many pathways function simultaneously with common intermediates.

Microbial Growth

Physical Requirements

• Temperature:

  • Minimum growth temperature: Lowest temperature at which a species will grow.

  • Optimum growth temperature: Temperature at which a species grows best.

  • Maximum growth temperature: Highest temperature at which a species will grow.

  • Classifications:

    • Psychrophiles: Cold-loving microbes.

    • Mesophiles: Moderate-temperature-loving microbes.

    • Thermophiles: Heat-loving microbes.

• pH: Most bacteria grow best between pH 6.5 and 7.5; molds and yeasts grow between pH 5 and 6.

• Osmotic Pressure:

  • Hypertonic environments: Cause plasmolysis (cell shrinkage).

  • Extreme or obligate halophiles: Require high salt concentrations (up to 30% NaCl).

  • Facultative halophiles: Tolerate high salt concentrations (2–10% NaCl).

Chemical Requirements

• Carbon: Structural backbone of organic molecules. Chemoheterotrophs use organic molecules; autotrophs use CO2.

• Nitrogen: Component of proteins, DNA, and ATP. Most bacteria decompose protein material; some use NH4+ or NO3-; some perform nitrogen fixation using N2.

• Sulfur: Used in amino acids, thiamine, and biotin. Most bacteria obtain sulfur by decomposing protein.

• Phosphorus: Used in DNA, RNA, and ATP; found in membranes.

• Trace Elements: Usually required in small amounts (e.g., iron, copper, molybdenum, zinc).

• Oxygen: Classifications based on oxygen requirements:

  • Obligate aerobes: Require oxygen.

  • Facultative anaerobes: Can grow with or without oxygen; use fermentation or anaerobic respiration.

  • Microaerophiles: Require oxygen concentration lower than air.

• Toxic forms of oxygen: Superoxide radicals, hydrogen peroxide. Organisms that tolerate oxygen have strategies to detoxify these forms (e.g., catalase, peroxidase).

Culture Media

Culture Media Types

• Chemically Defined Media: Known chemical composition; used for autotrophic organisms.

• Complex Media: Extracts and digests of yeasts, meat, or plants; chemical composition varies. Examples: Nutrient broth, nutrient agar.

• Agar: Generally polysaccharide used as a solidifying agent for culture media; not metabolized by microbes; liquefies at 100°C, solidifies at ~40°C.

Specialized Culture Techniques

• Anaerobic Growth Media and Methods: Reducing media contain chemicals (e.g., sodium thioglycolate) that combine with O2 to deplete it; used for cultivating anaerobic bacteria.

• Special Culture Techniques (for difficult-to-grow microbes): Some bacteria cannot grow on artificial laboratory media (e.g., Mycobacterium leprae grows in armadillos; Rickettsia, Chlamydia grown in tissue culture). Capnophile: Microorganisms requiring higher CO2 for growth.

Functional Classification of Culture Media

• Fermentation test: Detects acid and gas production from carbohydrate/protein catabolism.

• Oxidase test: Detects bacteria with cytochrome c oxidase.

• Detection of H2S production: Detects sulfur reduction.

Summary Table: Enzyme Types and Functions

Enzyme Type	Function	Example
Oxidoreductase	Oxidation-reduction reactions	Dehydrogenase
Transferase	Transfer functional groups	Transaminase
Hydrolase	Hydrolysis reactions	Protease
Lyase	Removal of atoms without hydrolysis	Decarboxylase
Isomerase	Rearrangement of atoms	Phosphoglucoisomerase
Ligase	Joining of molecules; uses ATP	DNA ligase

Key Equations

• ATP Generation: $\text{ADP} + \text{P}_i \rightarrow \text{ATP}$

• Redox Reaction: $\text{NAD}^+ + 2\text{H} \rightarrow \text{NADH} + \text{H}^+$

• Glycolysis Net Reaction: $\text{Glucose} + 2\text{NAD}^+ + 2\text{ADP} + 2\text{P}_i \rightarrow 2\text{Pyruvate} + 2\text{NADH} + 2\text{ATP} + 2\text{H}_2\text{O}$

Additional info: Some details, such as specific diagram descriptions and certain test procedures, were inferred from standard microbiology curriculum.