Comprehensive Study Notes on Microbial Metabolism, Respiration, and Diversity

Fundamentals of Cellular Metabolism

• Metabolism Overview

  • Metabolism is defined as the buildup and breakdown of nutrients within a cell.

  • These chemical reactions provide energy and create essential substances that sustain life.

• Catabolism and Anabolism

  • Catabolism

  • Breaks down complex organic molecules into simpler ones.

  • Releases energy via exergonic reactions.

  • Provides energy and basic building blocks required for anabolic processes.

  • Involves the oxidation of molecules (e.g., glucose broken down into $CO_2 + H_2O$).

  • Anabolism

  • Uses energy and structural building blocks to synthesize complex macromolecules (e.g., combining amino acids to form proteins).

  • Consumes energy via endergonic reactions.

  • Synthesizes macromolecular structures that make up the cell.

• Role of ATP in Energy Coupling

  • Catabolism releases energy by oxidizing nutrient molecules, coupling this release to the phosphorylation of $ADP$ and inorganic phosphate ($P_i$) to form $ATP$:     $ADP + P_i + \text{Energy} \rightarrow ATP$

  • Anabolism consumes energy by hydrolyzing $ATP$ into $ADP$ and $P_i$, releasing energy to drive endergonic synthesis reactions:     $ATP \rightarrow ADP + P_i + \text{Energy}$

Enzyme Structure, Function, and Kinetics

• Metabolic Pathways and Catalysts

  • Metabolic pathways are organized sequences of enzymatically catalyzed chemical reactions occurring within a cell.

  • Enzymes determine the specific pathways that operate in a given cell.

  • Enzymes are proteins encoded by cellular genes.

  • Catalysts are chemical agents that speed up reaction rates without being consumed or permanently altered.

  • Enzymes serve as biological catalysts.

• Mechanism of Enzymatic Action

  • Enzymes act on specific molecules termed substrates and lower the activation energy ($E_a$) required for a reaction to proceed.

  • Activation Energy Comparison

  • Uncatalyzed reactions require a significantly higher initial activation energy to transform reactants into products.

  • Catalyzed reactions lower the required activation energy barrier, accelerating product formation without altering the initial energy level of reactants or final energy level of products.

  • Reaction Steps

  1. The substrate comes into contact with the enzyme's specific active site.

  2. An enzyme-substrate complex is formed temporary.

  3. The substrate molecule is transformed and rearranged into specific products.

  4. Products are released from the active site because they no longer fit the enzyme's binding pocket.

  5. The enzyme remains entirely unchanged by the reaction and is free to react with additional substrate molecules.

• Enzyme Specificity and Efficiency

  • Enzymes demonstrate high specificity for particular substrates based on chemical fit and active site conformation.

  • Turnover Number: Defined as the maximum number of substrate molecules an enzyme converts to product per second.

  • Turnover rates generally range between $1$ and $10,000$ molecules per second.

• Enzyme Naming and Classification

  • Enzyme names systematically end with the suffix -ase and are categorized into six functional classes based on the reaction type catalyzed:

  • Oxidoreductase: Catalyzes oxidation-reduction reactions.

  • Transferase: Catalyzes the transfer of functional chemical groups (e.g., amino or phosphate groups).

  • Hydrolase: Catalyzes hydrolysis reactions (cleavage of bonds by adding water).

  • Lyase: Catalyzes the removal of groups of atoms without hydrolysis.

  • Isomerase: Catalyzes the rearrangement of atoms within a molecule.

  • Ligase: Catalyzes the joining of two molecules using energy derived from $ATP$ breakdown.

• Enzyme Components

  • Apoenzyme: The protein portion of the enzyme, which is inactive by itself.

  • Cofactor: The nonprotein component required for enzyme activity (acts as an activator; e.g., metal ions).

  • Coenzyme: An organic cofactor.

  • Holoenzyme: The complete, active enzyme consisting of the apoenzyme plus its cofactor/coenzyme.

  • Key Coenzymes (Electron Carriers)

  • Nicotinamide adenine dinucleotide ($NAD^+$)

  • Nicotinamide adenine dinucleotide phosphate ($NADP^+$)

  • Flavin adenine dinucleotide ($FAD$)

  • Coenzyme A ($CoA$)

Factors Influencing Enzymatic Activity and Inhibition

• Environmental and Physical Factors

  • Temperature

  • Reaction rate increases as temperature rises up to an optimal point.

  • Beyond the optimum temperature, thermal denaturation occurs: the tertiary structure of the protein unfolds, inactivating the enzyme, causing reaction rates to fall steeply.

  • Plotted across a range (e.g., $10^\text{o}\text{C}$ to $50^\text{o}\text{C}$), enzymatic activity peaks at an optimal temperature (e.g., around $35^\text{o}\text{C}$ to $40^\text{o}\text{C}$) before dropping sharply to zero.

  • pH

  • Enzymes exhibit optimal activity within a narrow pH range (e.g., optimal at approximately pH $5.0$ for specific hypothetical enzymes).

  • Extreme high or low pH values alter ionic bonds and denature proteins, sharply decreasing activity.

  • Substrate Concentration

  • At low substrate concentrations, the rate of reaction increases proportionally with increasing substrate concentration.

  • At high substrate concentration, all active sites become occupied simultaneously (saturation point).

  • Once saturated, the enzyme catalyzes at its theoretical maximum rate, and further increases in substrate concentration produce no additional increase in rate.

• Enzyme Inhibition

  • Competitive Inhibitors

  • Structurally resemble the substrate and directly fill or block the active site of the enzyme.

  • Compete head-to-head with the substrate for active site binding.

  • Noncompetitive Inhibitors

  • Bind to an alternative site on the enzyme known as the allosteric site.

  • This process, called allosteric inhibition, induces a conformational change in the enzyme that alters the active site, preventing the substrate from binding effectively.

  • Feedback Inhibition (End-Product Inhibition)

  • A biochemical control mechanism where the final end-product of a metabolic pathway allosterically inhibits an enzyme operating early in the pathway (typically Enzyme 1).

  • Mechanism:

    1. Substrate enters pathway at Enzyme 1.

    2. Pathway generates Intermediate A (via Enzyme 1), Intermediate B (via Enzyme 2), and ultimate End-product (via Enzyme 3).

    3. As the End-product accumulates, it binds to the allosteric site of Enzyme 1.

    4. Conformation of Enzyme 1's active site changes, shutting down the entire pathway to prevent overproduction.

Bioenergetics: Redox Reactions and ATP Generation

• Oxidation-Reduction (Redox) Reactions

  • Oxidation: The removal or loss of electrons ($e^-$) from an atom or molecule.

  • Reduction: The gain of electrons ($e^-$) by an atom or molecule.

  • Oxidation and reduction are always coupled together as a paired redox reaction.

  • Biological Oxidations

  • In biological systems, electrons are usually transferred simultaneously with protons ($H^+$), which is equivalent to transferring whole hydrogen atoms ($H$).

  • Consequently, biological oxidations are commonly referred to as dehydrogenation reactions.

  • General Reaction Scheme:       $\text{Organic molecule } (2H) + NAD^+ \rightarrow \text{Oxidized organic molecule} + NADH + H^+$

    • Here, an organic substrate containing two hydrogen atoms is oxidized, transferring two electrons and one proton to $NAD^+$ to yield $NADH$, leaving one free proton ($H^+$).

• Mechanisms of ATP Generation

  • ATP is generated by adding an inorganic phosphate ($P_i$) to $ADP$, which requires an input of energy.

  • Oxidative Phosphorylation

  • Electrons are transferred from organic compounds through a sequence of electron carriers along an electron transport chain (system) embedded in a membrane.

  • Energy released during electron transfers is harnessed to generate ATP via chemiosmosis.

  • Photophosphorylation

  • Occurs exclusively in light-trapping photosynthetic cells.

  • Light energy excites electrons in chlorophyll; as these electrons pass down a transport chain of carrier molecules, energy is harnessed to synthesize ATP.

  • Cyclic Photophosphorylation: Excited electrons from Photosystem I pass through an electron transport chain generating ATP and return directly back to Photosystem I.

  • Noncyclic Photophosphorylation:

    • Light excites electrons ($2e^-$) in Photosystem II.

    • Electrons pass along an electron transport chain, releasing energy for ATP synthesis, and enter Photosystem I.

    • Electrons in Photosystem I are re-excited by light and transferred to $NADP^+$ to form $NADPH$.

    • Electrons lost from Photosystem II are replaced by splitting water molecules ($H_2O \rightarrow 2H^+ + 2e^- + \frac{1}{2}O_2$).

Carbohydrate Catabolism: Glycolysis

• Overview of Carbohydrate Catabolism

  • The primary process for energy release from carbohydrates involves three major integrated pathways:

  1. Glycolysis

  2. Krebs Cycle

  3. Electron Transport Chain (System)

• Glycolysis (Embden-Meyerhof Pathway)

  • The oxidation of glucose ($C_6H_{12}O_6$) into two molecules of pyruvic acid ($C_3H_4O_3$), producing net $ATP$ and $NADH$

  • Does not require oxygen and can occur under aerobic or anaerobic conditions.

  • Preparatory Stage (Steps 1–5; Energy Investment)

  1. Glucose enters the cell and is phosphorylated using $1$ molecule of $ATP$ to yield glucose 6-phosphate.

  2. Glucose 6-phosphate is rearranged to form fructose 6-phosphate.

  3. A second $ATP$ molecule is invested to phosphorylate fructose 6-phosphate into fructose 1,6-diphosphate (total investment = $2$ ATP).

  4. An enzyme splits fructose 1,6-diphosphate into two distinct three-carbon sugars: dihydroxyacetone phosphate (DHAP) and glyceraldehyde 3-phosphate (GP).

  5. DHAP is readily converted into GP by an isomerase enzyme (reactions downstream utilize GP).

  • Energy-Conserving Stage (Steps 6–10; Energy Payoff)

  1. Two GP molecules are oxidized; two hydrogen atoms are transferred to $2$ $NAD^+$ to form $2$ $NADH + 2H^+$. The enzyme couples this oxidation with the attachment of an inorganic phosphate group, forming two molecules of 1,3-diphosphoglyceric acid.

  2. High-energy phosphate groups are transferred from two molecules of 1,3-diphosphoglyceric acid to $2$ $ADP$, generating $2$ $ATP$ molecules via substrate-level phosphorylation and producing two molecules of 3-phosphoglyceric acid. (Repays initial $2$ ATP investment).

  3. An enzyme relocates the remaining phosphate group from the 3rd carbon to the 2nd carbon, converting 3-phosphoglyceric acid to 2-phosphoglyceric acid.

  4. A water molecule ($H_2O$) is lost from each 2-phosphoglyceric acid, forming phosphoenolpyruvic acid (PEP) and converting the phosphate bond into a high-energy bond.

  5. High-energy phosphate is transferred from two PEP molecules to $2$ $ADP$, forming $2$ $ATP$ molecules and two molecules of pyruvic acid.

  • Chemical Summary and Net Balance of Glycolysis

  • Overall Equation:       $\text{Glucose} + 2 ATP + 2 ADP + 2 PO_4^- + 2 NAD^+ \rightarrow 2 \text{ Pyruvic Acid} + 4 ATP + 2 NADH + 2 H^+$

  • Net Gain: Oxidation of $1$ molecule of glucose yields a net gain of $2$ molecules of $ATP$, $2$ molecules of $NADH$, and $2$ molecules of pyruvic acid.

• Alternative Pathways to Glycolysis

  • Pentose Phosphate Pathway

  • Operates simultaneously with glycolysis.

  • Uses five-carbon sugars (pentoses) and glucose to produce $NADPH$

  • Yields intermediate pentoses used in nucleic acid synthesis.

  • Entner-Doudoroff Pathway

  • Produces $NADPH$ and $ATP$ without utilizing glycolysis or the pentose phosphate pathway.

  • Found in specific gram-negative bacteria, including Pseudomonas, Rhizobium, and Agrobacterium.

Cellular Respiration: Aerobic vs. Anaerobic

• General Principles of Cellular Respiration

  • Cellular respiration is an energy-yielding process in which substrate molecules are oxidized to liberate electrons to run an electron transport chain.

  • The final electron acceptor comes from outside the cell and is an inorganic compound.

  • ATP is generated primarily via oxidative phosphorylation.

• Aerobic Respiration

  • Requires molecular oxygen ($O_2$) as the ultimate final electron acceptor.

  • The Intermediate / Transition Step

  • Pyruvic acid produced in glycolysis enters respiration and undergoes decarboxylation (loss of a $CO_2$ molecule).

  • The resulting two-carbon acetyl group is oxidized while reducing $NAD^+$ to $NADH$.

  • The two-carbon acetyl group attaches to Coenzyme A ($CoA$) to form Acetyl CoA.

  • The Krebs Cycle (TCA / Citric Acid Cycle)

  • A cyclic pathway of enzymatic reactions that completely oxidizes acetyl groups to $CO_2$

  • Step-by-Step Cycle Mechanics:

    1. Acetyl CoA drops off its two-carbon acetyl group, which combines with oxaloacetic acid (a 4-carbon compound) to form citric acid (a 6-carbon compound).

    2. Citric acid is rearranged into its isomer, isocitric acid.

    3. Isocitric acid is oxidized, reducing $NAD^+$ to $NADH$, and decarboxylated (losing $CO_2$) to produce $\text{α-ketoglutaric acid}$ (a 5-carbon compound).

    4. $\text{α-ketoglutaric acid}$ undergoes oxidation (reducing $NAD^+$ to $NADH$) and decarboxylation (losing $CO_2$), and combines with $CoA$ to form succinyl CoA (a 4-carbon compound).

    5. $CoA$ is released from succinyl CoA; the energy released generates $GTP$, which transfers its phosphate to $ADP$ to form $ATP$ via substrate-level phosphorylation, leaving succinic acid (4-carbon compound).

    6. Succinic acid is oxidized by transferring two hydrogen atoms to $FAD$, forming $FADH_2$ and fumaric acid.

    7. Water ($H_2O$) is added to fumaric acid to form malic acid.

    8. Malic acid is oxidized, reducing $NAD^+$ to $NADH$, regenerating oxaloacetic acid to begin another turn of the cycle.

  • Electron Transport Chain (ETC) and Chemiosmosis

  • Location: Plasma membrane of prokaryotes; inner mitochondrial membrane of eukaryotes.

  • Carriers: Made of sequential electron carriers including flavoproteins (e.g., FMN), cytochromes (cyt $b$, cyt $c_1$, cyt $c$, cyt $a$, cyt $a_3$), and ubiquinones (coenzyme Q).

  • Proton Pumping: Complexes (NADH dehydrogenase complex, Cytochrome $b-c_1$ complex, Cytochrome oxidase complex) pump protons ($H^+$) across the membrane as electrons are transferred.

  • Chemiosmotic Mechanism:

    • High concentration of $H^+$ accumulates in the periplasmic space (prokaryotes) or intermembrane space (eukaryotes).

    • Low concentration of $H^+$ remains in the cytoplasm (prokaryotes) or mitochondrial matrix (eukaryotes).

    • Protons flow down their electrochemical gradient back through the membrane bound ATP synthase complex.

    • Energy released by proton movement synthesizes $ATP$ from $ADP + P_i$ ($3 ADP + 3 P_i \rightarrow 3 ATP$ per gradient cycle).

  • Overall Energy Yield of Aerobic Respiration

  • Oxidation of $1$ molecule of $NADH$ in the ETC generates $3$ molecules of $ATP$

  • Oxidation of $1$ molecule of $FADH_2$ in the ETC generates $2$ molecules of $ATP$

  • Complete Balance Equation for Aerobic Respiration in Prokaryotes:       $\text{C}_6\text{H}_{12}\text{O}_6 + 6 \text{O}_2 + 38 \text{ADP} + 38 \text{P}_i \rightarrow 6 \text{CO}_2 + 6 \text{H}_2\text{O} + 38 \text{ATP}$

• Anaerobic Respiration

  • The final electron acceptor in the electron transport chain is an inorganic substance other than $O_2$

  • Yields less ATP than aerobic respiration because the energy yield is lower without oxygen as the terminal acceptor.

  • Electron Acceptors and Products Table:

  • Final Electron Acceptor: $NO_3^-$ (Nitrate) $ ightarrow$ End Products: $NO_2^-$ (Nitrite), $N_2 + H_2O$

  • Final Electron Acceptor: $SO_4^{2-}$ (Sulfate) $ ightarrow$ End Products: $H_2S + H_2O$

  • Final Electron Acceptor: $CO_3^{2-}$ (Carbonate) $ ightarrow$ End Products: $CH_4 + H_2O$

Fermentation

• General Characteristics of Fermentation

  • Releases energy from the oxidation of organic molecules.

  • Does not require oxygen (can occur in its presence or absence).

  • Does not use the Krebs cycle or an electron transport chain.

  • Uses an organic molecule generated within the cell as the final electron acceptor.

  • Produces only small quantities of ATP (derived exclusively from glycolysis).

• Major Pathways and Types of Fermentation

  • Lactic Acid Fermentation

  • Glycolysis oxidizes glucose to $2$ pyruvic acid molecules, producing $2$ $ATP$ and $2$ $NADH$

  • $2$ pyruvic acid molecules are reduced by $2$ $NADH$ to form $2$ molecules of lactic acid ($CH_3CHOHCOOH$).

  • Organisms: Streptococcus, Lactobacillus, Bacillus.

  • Alcohol Fermentation

  • Glycolysis oxidizes glucose to $2$ pyruvic acid molecules.

  • $2$ pyruvic acid molecules are decarboxylated to form $2$ molecules of acetaldehyde and $2$ $CO_2$

  • $2$ acetaldehyde molecules are reduced by $2$ $NADH$ to form $2$ molecules of ethanol ($CH_3CH_2OH$).

  • Organisms: Saccharomyces (yeast).

  • Summary of Microbial Fermentation End-Products:

  • Streptococcus, Lactobacillus, Bacillus: Lactic acid.

  • Saccharomyces: Ethanol and $CO_2$

  • Propionibacterium: Propionic acid, acetic acid, $CO_2$, $H_2$

  • Clostridium: Butyric acid, butanol, acetone, isopropyl alcohol, $CO_2$

  • Escherichia, Salmonella: Ethanol, lactic acid, succinic acid, acetic acid, $CO_2$, $H_2$

  • Enterobacter: Ethanol, lactic acid, formic acid, 2,3-butanediol, acetoin, $CO_2$, $H_2$

Lipid and Protein Catabolism

• Lipid Catabolism

  • Lipases hydrolyze lipids into glycerol and fatty acids.

  • Glycerol is converted into glyceraldehyde 3-phosphate and fed into glycolysis.

  • Fatty acids undergo beta-oxidation to yield acetyl CoA, which enters the Krebs cycle.

• Protein Catabolism

  • Extracellular proteases and peptidases break down large proteins into constituent amino acids so they can cross cell membranes.

  • Amino acids are enzymatically modified before entering the Krebs cycle:

  • Deamination: Removal of an amino group ($-NH_2$).

  • Decarboxylation: Removal of a carboxyl group ($-COOH$).

  • Dehydrogenation: Removal of hydrogen atoms.

  • Desulfurization: Removal of a sulfhydryl group ($-SH$).

  • Resulting organic acids enter the Krebs cycle directly to generate cellular energy.

Biochemical Tests and Bacterial Identification

• Enzyme-Based Identification

  • Laboratory identification of bacteria relies on detecting specific enzymatic activities (e.g., enzymes responsible for amino acid decarboxylation or desulfurization).

• Specific Test Methods

  • Fermentation Test

  • Contains a protein/carbohydrate medium, a pH indicator, and an inverted Durham tube for trapping gas.

  • Bacteria that ferment carbohydrates produce acid, triggering a color change in the pH indicator.

  • Gas production is indicated by a bubble inside the inverted Durham tube.

  • Oxidase Test

  • Used to identify bacteria that possess cytochrome c oxidase (e.g., distinguishing Pseudomonas from other gram-negative rods).

Photosynthesis

• Overview of Photosynthesis

  • Light-Dependent (Light) Reactions: Conversion of light energy into chemical energy in the form of $ATP$ and $NADPH$

  • Light-Independent (Dark) Reactions: Calvin-Benson cycle uses stored $ATP$ and $NADPH$ to reduce $CO_2$ to synthesize sugars (carbon fixation).

• Chemical Reactions of Photosynthesis

  • Oxygenic Photosynthesis:     $6 \text{CO}_2 + 12 \text{H}_2\text{O} + \text{Light energy} \rightarrow \text{C}_6\text{H}_{12}\text{O}_6 + 6 \text{H}_2\text{O} + 6 \text{O}_2$

  • Anoxygenic Photosynthesis:     $6 \text{CO}_2 + 12 \text{H}_2\text{S} + \text{Light energy} \rightarrow \text{C}_6\text{H}_{12}\text{O}_6 + 6 \text{H}_2\text{O} + 12 \text{S}$

• The Calvin-Benson Cycle

  • Input: $3$ molecules of $CO_2$

  • Step-by-Step Mechanism:

  1. Carbon Fixation: $3$ $CO_2$ molecules combine with $3$ molecules of ribulose diphosphate (RuBP; a 5-carbon sugar) to produce $6$ molecules of 3-phosphoglyceric acid (3-carbon).

  2. Phosphorylation: $6$ $ATP$ molecules are consumed to convert 3-phosphoglyceric acid into $6$ molecules of 1,3-diphosphoglyceric acid.

  3. Reduction: $6$ $NADPH$ molecules reduce 1,3-diphosphoglyceric acid to generate $6$ molecules of glyceraldehyde 3-phosphate (G3P).

  4. Output: $1$ molecule of G3P leaves the cycle to synthesize glucose and other carbohydrates.

  5. Regeneration: The remaining $5$ molecules of G3P consume $3$ additional $ATP$ molecules to regenerate $3$ molecules of ribulose diphosphate (RuBP), completing the cycle.

Metabolic Diversity and Integration

• Nutritional Classification of Organisms

  • Organisms are categorized by their primary energy source and carbon source:

  • Photoautotrophs

    • Energy Source: Light

    • Carbon Source: $CO_2$

    • Oxygenic Example: Cyanobacteria, plants, algae (use $H_2O$ to reduce $CO_2$).

    • Anoxygenic Example: Green bacteria, purple bacteria (use $H_2S$ or non-water donors to reduce $CO_2$).

  • Photoheterotrophs

    • Energy Source: Light

    • Carbon Source: Organic compounds

    • Examples: Green nonsulfur bacteria, purple nonsulfur bacteria.

  • Chemoautotrophs

    • Energy Source: Inorganic chemicals (e.g., $H_2$, sulfur, iron, nitrogen, carbon monoxide).

    • Carbon Source: $CO_2$

    • Examples: Iron-oxidizing, sulfur-oxidizing, and nitrifying bacteria.

  • Chemoheterotrophs

    • Energy Source: Organic chemicals

    • Carbon Source: Organic compounds

    • Subgroups by Final Electron Acceptor:

    • $O_2$: All animals, most fungi, protozoa, aerobic bacteria.

    • Not $O_2$ (Inorganic compound): Anaerobic respiration (e.g., Clostridium).

    • Organic compound: Fermentative bacteria (e.g., Streptococcus).

• Integration of Metabolism (Amphibolic Pathways)

  • Amphibolic Pathways: Dual-purpose metabolic pathways that function in both catabolism and anabolism.

  • Diverse pathways operate simultaneously within cells, sharing common metabolic intermediates to efficiently direct cellular resources based on current energy and structural needs.