Microbial Growth

Microbiology

Basic Chemical Reactions Underlying Metabolism

• Metabolism: Sum of complex biochemical reactions within an organism.
• Divided into two major classes:
  • Catabolism:
    • Breakdown of larger (nutrient) molecules.
    • Releases energy (exergonic).
    • Energy stored in ATP.
  • Anabolism:
    • Synthesizes macromolecules.
    • Uses ATP energy (endergonic).
• Pathways: Series of reactions in assembly or disassembly of molecules.
  • Catabolic pathways: Enzymes catalyze breakdown of nutrients into precursor metabolites and smaller molecules.
  • Anabolic pathways: Precursor metabolites rearranged by polymerization to form macromolecules.
• Cells grow as they assemble large molecules into cell parts.

Ultimate Function of Metabolism & Metabolic Processes

• Ultimate function is to reproduce the organism.
• Metabolic processes are guided by the following:
  • Every cell acquires nutrients (building blocks and energy).
  • Metabolism requires energy (light or catabolism of nutrients).
  • Energy stored in ATP chemical bonds.
  • Cells catabolize nutrient molecules into precursor metabolites via enzymes.
  • Cells construct larger building blocks in anabolic reactions, using precursor metabolites, enzymes, and ATP.
  • Cells use enzymes and ATP to link building blocks into macromolecules via polymerization.
  • Cells grow by assembling macromolecules into cellular structures.
  • Cells typically divide into two after doubling in size.

Metabolism: Overview

• Organisms are constantly making and breaking bonds in molecules as they live and grow.
• Figure 5.1 illustrates the relationship between catabolism and anabolism:
  • Catabolism breaks down nutrients into precursor molecules, releasing energy (some lost as heat) and storing some energy in ATP.
  • Anabolism uses ATP energy and precursor molecules to create larger building blocks, macromolecules, and cellular structures, also losing energy as heat.
  • These components enable cellular processes like cell growth and division.

Oxidation and Reduction Reactions

• Many metabolic reactions involve electron transfer.
• Electrons carry energy.
• Electrons commonly transferred as part of a hydrogen atom.
• Oxidation-reduction (redox) reactions:
  • Involve electron transfer from an electron donor to an electron acceptor.
  • Substance losing electrons is oxidized; substance gaining electrons is reduced.
    • Mnemonic: OIL RIG (Oxidation Is Loss; Reduction Is Gain).
  • Electron acceptor is reduced; electron donor is oxidized.
  • Reduction and oxidation reactions always happen simultaneously.

Flow of Electrons and ATP Synthesis

• Electron flow from one molecule to another is used to perform cellular work, including ATP synthesis.
• This process is similar to a battery's function.
  • Batteries contain separate compartments with two metals (e.g., zinc and copper) with different electron affinities.
  • Electrons leave the metal with lower electron affinity (zinc) and flow toward the metal with higher electron affinity (copper).
  • Zinc is oxidized, and copper is reduced.
  • The flow of electrons through a device (light bulb, motor) provides electromotive force, performing work.
  • A battery “dies” when the metal with low electron affinity is completely oxidized.
• Cells contain “biological batteries” that work by the same principle.

Redox Reaction Definitions

• OXIDATION: Substance loses electrons or is oxidized.
• REDUCTION: Substance gains electrons or is reduced (positive charge is reduced).

Electron Carriers

• Cells use electron carriers to transfer electrons (often in H atoms).
• Electron Carriers: Molecules that transfer electrons from one molecule to another.
• Three important electron carriers:
  • Nicotinamide adenine dinucleotide (NAD+)
  • Nicotinamide adenine dinucleotide phosphate (NADP+)
  • Flavin adenine dinucleotide (FAD)

NAD+ and NADH

• NAD+ within a cell, along with two hydrogen atoms that are part of the food that is supplying energy for the body.
• NAD+ is reduced to NAD by accepting an electron from a hydrogen atom. It also picks up another hydrogen atom to become NADH.
• NADH carries the electrons to a later stage of respiration then drops them off, becoming oxidized to its original form, NAD+.

ATP Production and Energy Storage

• During catabolism, organisms release energy from nutrients that can be concentrated and stored in high-energy phosphate bonds of molecules such as ATP.
• Phosphorylation: Inorganic phosphate is added to a substrate.
• Cells phosphorylate adenosine diphosphate (ADP) to ATP in three ways:
  • Substrate-level phosphorylation: Transfer of phosphate to ADP from another phosphorylated organic compound.
  • Oxidative phosphorylation: Energy from redox reaction of respiration attaches phosphate to ADP.
  • Photophosphorylation: Light energy is used to attach phosphate to ADP.
• Anabolic pathways use some energy of ATP by breaking a phosphate bond.

Enzymes

• Enzymes are organic catalysts.
• Increase likelihood of a reaction but do not change in the process.
• Naming and classifying enzymes:
  • Often incorporates name of substrate, the molecule the enzyme acts on.
• Six categories of enzymes based on mode of action:
  • Hydrolases
  • Isomerases
  • Ligases or polymerases
  • Lyases
  • Oxidoreductases
  • Transferases

Enzyme Classification Based on Reaction Types

• Hydrolase: Catalyzes hydrolysis (catabolic). Example: Lipase (breaks down lipid molecules).
• Isomerase: Catalyzes rearrangement of atoms within a molecule (neither catabolic nor anabolic). Example: Phosphoglucoisomerase (converts glucose 6-phosphate into fructose 6-phosphate during glycolysis).
• Ligase or Polymerase: Catalyzes joining two or more chemicals together (anabolic). Example: Acetyl-CoA synthetase (combines acetate and coenzyme A to form acetyl-CoA for the Krebs cycle).
• Lyase: Catalyzes splitting a chemical into smaller parts without using water (catabolic). Example: Fructose-1,6-bisphosphate aldolase (splits fructose 1,6-bisphosphate into G3P and DHAP).
• Oxidoreductase: Catalyzes transfer of electrons or hydrogen atoms from one molecule to another. Example: Lactic acid dehydrogenase (oxidizes lactic acid to form pyruvic acid during fermentation).
• Transferase: Catalyzes moving a functional group from one molecule to another (may be anabolic). Example: Hexokinase (transfers phosphate from ATP to glucose in the first step of glycolysis).

Enzyme Makeup

• Many protein enzymes are complete in themselves.
• Others are composed of both protein and nonprotein portions.
• Apoenzymes are inactive if not bound to nonprotein cofactors (inorganic ions) or organic coenzymes.
• All coenzymes are either vitamins or contain vitamins
• Binding of apoenzyme and its cofactor(s) yields holoenzyme – active form.
• Some are RNA molecules called ribozymes.

Representative Cofactors of Enzymes

Enzyme Activity

• Enzymes catalyze reactions by lowering the activation energy, the amount of initial energy to trigger a chemical reaction.
• Each reaction is catalyzed by a specific enzyme.
• Enzyme activity is very specific to its target substrate.
• Enzyme has specific recognition for its personal substrate.
• Active site is the enzyme’s functional site and is complementary to the shape of the enzyme’s substrate.
• Induced fit model.

Enzymes and Activation Energy

• Figure 5.4: The effect of enzymes on chemical reactions.
• Enzymes lower the activation energy required for a reaction to proceed.

Enzymes Fitted to Substrates

• Figure 5.5: Enzymes fitted to substrates.
• Enzyme active sites are similar to the substrate's shape.
• Substrate binding induces an enzyme's active site to more closely match the shape of the substrate (induced fit).

Steps in an Enzyme Reaction

• Substrate binds to enzyme.
• Enzyme-substrate complex.
• Products are released.
• Enzyme is unchanged.

Factors influencing Enzyme Activity

• Temperature: Higher temperatures generally increase reaction rates due to increased molecular motion and collisions. However, exceeding an enzyme's stability point causes denaturation and loss of the active site.
• pH: Extremes of pH also denature enzymes. Each enzyme has an optimal pH.
• Enzyme and substrate concentrations: Enzymatic activity increases with substrate concentration until saturation is reached, where all active sites are bound.
• Presence of inhibitors: Substances that block enzyme activity are called inhibitors, which may be competitive or noncompetitive.

Control of Enzymatic Activity

• Activators: An enzyme can be activated by the binding of a cofactor to the enzyme at a site located away from the active site, a site called an allosteric site.
• Inhibitors: Include competitive and noncompetitive inhibitors.
  • Competitive inhibitors: Shaped such that they fit into an enzyme’s active site and thus prevent the normal substrate from binding. Can be permanent or reversible, if reversible can be overcome by increased substrate concentration.
  • Noncompetitive inhibitors: Do not attach to the active site but instead bind to an allosteric site on the enzyme. This binding alters the shape of the active site so that enzymatic activity is reduced or blocked completely.
• Feedback inhibition (negative feedback or end-product inhibition): The end-product of a metabolic pathway inhibits the initial step, shutting down the pathway.

Competitive Inhibition

• Figure 5.10: Competitive inhibition of enzyme activity.
• A competitive inhibitor binds to the active site, preventing substrate binding.
• Increased substrate concentration can overcome reversible competitive inhibition.

Noncompetitive Inhibition

• Figure 5.11: Noncompetitive inhibition at an allosteric site.
• A noncompetitive inhibitor binds to an allosteric site, distorting the active site and reducing or stopping enzymatic activity.

Carbohydrate Catabolism

• Many organisms oxidize carbohydrates as the primary energy source for anabolic reactions.
• Glucose is used most commonly.
• Glucose is catabolized via one of two processes:
  • Cellular respiration: Complete breakdown of glucose to carbon dioxide and water.
  • Fermentation: Results in organic waste products.
• Both cellular respiration and fermentation begin with glycolysis, catabolizing a single glucose molecule into two pyruvic acid molecules and producing small amounts of ATP.
• Cellular respiration continues with the Krebs cycle and then an electron transport chain.
• Fermentation converts pyruvic acid into other organic compounds.
• Fermentation does not yield as many ATP molecules as cellular respiration.

Summary of Glucose Catabolism

• Figure 5.13: Summary of glucose catabolism.
• Glucose -> Glycolysis
• Products of glycolysis can then enter fermentation or respiration.
• Respiration continues to the Krebs Cycle and finally the Electron Transport Chain (ETC).
• Fermentation proceeds through the Formation of fermentation end-products.

Glycolysis

• Occurs in cytoplasm of most cells.
• Involves splitting a 6-carbon glucose into two 3-carbon sugar molecules.
• Substrate-level phosphorylation: Direct transfer of phosphate between two substrates.
• 10-step process results in a net gain of two ATP molecules, two molecules of NADH, and the precursor metabolite pyruvic acid.
• Divided into three stages involving 10 total steps:
  • Energy-investment stage: 2 ATP molecules are invested to phosphorylate glucose and rearrange its atoms to form fructose 1,6-biphosphate.
  • Lysis stage: Fructose 1,6-biphosphate is cleaved and eventually 2 glyceraldehyde-3-phosphate (G3P) molecules are produced.
  • Energy-conserving stage: Each G3P is oxidized to pyruvic acid, yielding 2 ATP molecules each.
• Cells can use many of the glycolysis intermediate as precursor metabolites.

Glycolysis Stages

• Preparatory Stage (Energy-Investment Stage)
• Energy-Conservation Stage

Pyruvic Acid

• After glucose has been oxidized via glycolysis or one of the alternative pathways, a cell uses the resultant pyruvate molecules to complete either cellular respiration OR fermentation.
• Pyruvic acid molecules complete either cellular respiration or fermentation.

Cellular Respiration

• Metabolic process that involves the complete oxidation of substrate molecules and then production of ATP by a series of redox reactions.
• Three stages of cellular respiration:
  • Synthesis of acetyl-CoA
  • Krebs cycle
  • Final series of redox reactions (electron transport chain) that pass electrons to a chemical not derived from cellular metabolism.

Synthesis of Acetyl-CoA

• Before pyruvate can enter the Krebs cycle for respiration, it must first be converted to acetyl-coenzyme A or acetyl-CoA which links glycolysis to the Krebs cycle.
• This step is carried out by a multi-enzyme complex that catalyzes three reactions:
  • Oxidation of pyruvate and release of CO2
  • Reduction of NAD+ to NADH
  • Combination of the remaining two-carbon fragment and coenzyme A to form acetyl CoA
• Acetyl CoA has high potential energy which is used to transfer the acetyl group to a molecule in the Krebs cycle, à highly exergonic reaction
• Remember, glycolysis produced two molecules of pyruvate from one molecule of glucose, therefore this stage produces:
  • two molecules of acetyl-CoA
  • two molecules of CO2
  • two molecules of NADH

The Krebs Cycle

• At this point in the catabolism of a molecule of glucose, a great amount of energy remains in the bonds of acetyl-CoA.
• The Krebs cycle is a “circular” series of 8 enzymatically catalyzed reactions that transfer much of this stored energy via electrons to NAD+ and FAD.
• Occurs in the cytosol of prokaryotes and in the matrix of mitochondria in eukaryotes.
• Further 2 CO2 molecules are released.
• The cycle oxidizes organic fuel derived from pyruvate, generating 1 ATP, 3 NADH, and 1 FADH2 per turn.
• 2 acetyl CoA’s therefore 2 turns per one glucose molecule. Total: 2 ATP, 6 NADH, 2 FADH2.
• Cells also use the Krebs cycle for the catabolism of lipids and proteins.
• Many of the Krebs cycle metabolites are also precursor metabolites.

The Krebs Cycle Image

• Figure 5.17

Overview of The Krebs Cycle

• Pyruvic acid -> Acetate -> Acetyl CoA
• Citric acid cycle is one of the three stages of cellular respiration.

Electron Transport

• The most significant production of ATP occurs through the stepwise release of energy from the series of redox reaction in the electron transport chain.
• A series of redox reactions that pass electrons from one membrane-bound carrier to another and then to a final electron acceptor.
• The energy from these electrons is used to pump protons across the membrane.
• This established a proton gradient that then generates ATP via a process called chemiosmosis.
• Aerobes use oxygen atoms as final electron acceptors in their electron transport chains in a process known as aerobic respiration.
• Whereas, anaerobes use other inorganic molecules (such as sulfate, nitrate, and carbonate) as the final electron acceptor in anaerobic respiration.
• Located in the inner mitochondrial membrane of eukaryotes and in the cytoplasmic membranes of prokaryotes.

Prokaryote Electron Transport Chain

• Figure 5.18b

Chemiosmosis

• General term for the use of ion gradients to generate ATP.
• Cells use energy released during redox reactions of ETC to actively transport protons (H+) across a membrane.
• This creates an electrochemical gradient called the proton gradient which has potential energy known as the proton motive force.
• Protons flow down the electrochemical gradient through ATP synthases (since protons are impermeable to the cytoplasmic membrane) that phosphorylate ADP to ATP.
• Called oxidative phosphorylation because the proton gradient is created by oxidation of components of ETC.
• Total of ~34 ATP molecules formed from one molecule of glucose with ETC and chemiosmosis.

Factors Affecting ATP

• Lack of oxygen.
• Add cyanide.
• Add uncoupling protein.

Ideal Prokaryotic Aerobic Respiration Summary

• Total of ~34 ATP molecules formed from one molecule of glucose with ETC and chemiosmosis.
• So net total of cellular respiration is ~38 ATP molecules per glucose molecule.

Glucose Breakdown in Metaphorical/Schematic Terms

• Insert 1 glucose.
• Glycolysis = 2 ATP, 2 NADH.
• Krebs cycle = 6 NADH, 2 ATP, 2 FADH2.
• Electron transport chain = 34 ATP + H20.
• 38 ATP maximum per glucose molecule.

Metabolic Diversity

• Entner-Doudoroff (ED) pathway
  • Some bacteria substitute this pathway for the EMP pathway.
  • Discovered only in prokaryotes.
  • ED pathway produces only one ATP, NADH, and NADPH.
• Pentose phosphate pathway
  • Alternative to glycolysis
  • Less energy efficient than glycolysis
  • Produces precursor metabolites and NADPH
  • Used to make DNA nucleotides, steroids, fatty acids

Fermentation

• Sometimes cells cannot completely oxidize glucose by cellular respiration.
  • For instance, they may lack sufficient final electron acceptors, like a bacterium that lacks oxygen in the anaerobic environment of the colon.
• Electrons cannot flow down an electron transport chain unless oxidized carrier molecules are available to receive them at the end.
• Fermentation is the partial oxidation of sugar (or other metabolites) to release energy using an organic molecule from within the cell as the final electron acceptor.
• Obtains its “fresh” supply of NAD+ (that can be reduced to NADH) by reducing organic molecules (so that NADH à NAD+)
• Energetically less efficient than respiration, but fermentation has no need for electron acceptors from the environment.
• 2 common fermentation pathways.

Examples of Fermentation

• Lactic acid fermentation
• Alcohol fermentation

Fermentation Products

• Microorganism produce a variety of fermentation products depending on the enzymes and substrates available to them.
• Though fermentation products are wastes to the cells that make them, many are useful to humans, including ethanol (drinking alcohol) and lactic acid (used in the production of cheese, sauerkraut, and pickles).

Comparison of Aerobic Respiration, Anaerobic Respiration, and Fermentation

Other Catabolic Pathways

• Microorganisms can use other molecules as energy sources as well.
• Lipids and proteins have abundant energy in their chemical bonds and can be catabolized into smaller molecules, which can be used as substrates for glycolysis and the Krebs cycle.
• Lipid catabolism: bonds attaching glycerol and fatty acids are broken.
  • Glycerol is converted to a substrate of glycolysis.
  • Beta-oxidation is a catabolic process in which enzymes split pairs of hydrogenated carbon atoms from a fatty acid and join them to coenzyme A to form acetyl-CoA which then joins the Krebs cycle.
• Protein catabolism:
  • Proteases secreted by microorganisms digest proteins outside the microbes’ cell walls.
  • The resulting amino acids are moved into the cell and used in anabolism or are deaminated (amine group removed) where the resulting molecules enter the Krebs cycle and are catabolized for energy.

Catabolism of a Triglyceride Molecule

• Glycerol and fatty acid chains.
• Hydrolysis and Beta-oxidation

Protein Catabolism

• Polypeptide -> Amino Acids -> Deamination -> Krebs Cycle

The Catabolism of Various Molecules from Food

• Proteins, Carbohydrates, and Fats turn into Amino acids, Sugars, Glycerol, and Fatty acids.
• They converge into glycolysis and the citric acid cycle to oxidative phosphorylation.

Photosynthesis

• Many organisms synthesize their own organic molecules from inorganic carbon dioxide.
• Most of these organisms capture light energy and use it to synthesize carbohydrates from CO2 and H2O by a process called photosynthesis.
• Few examples of photosynthetic organisms are cyanobacteria, green plants, and purple sulfur bacteria.

Stages of Photosynthesis

• Photosynthesis consists of two stages:
  • LIGHT–Dependent Reactions (the “photo” part)
  • Light-independent reaction or the Calvin cycle (the “synthesis” part)

Chemicals and Structures

• Chlorophylls: Important to organisms that capture light energy with pigment molecules. Many other kinds of light-capturing pigments.
• Photosystems: Arrangement of molecules of chlorophyll and other pigments to form light-harvesting matrices.
  • Embedded in cellular membranes called thylakoids.
    • In prokaryotes—invagination of cytoplasmic membrane
    • In eukaryotes—formed from the inner membrane of chloroplasts
  • Arranged in stacks called grana.
  • Stroma is the space between the outer membrane of grana and the thylakoid membrane.
• Two types of photosystems:
  • Photosystem I (PS I)
  • Photosystem II (PS II)
• Photosystems absorb light energy and use redox reactions to store energy in the form of ATP and NADPH.
• Light-dependent reactions depend on light energy.
• Light-independent reactions synthesize glucose from carbon dioxide and water.

Light-Dependent Reactions

• Light energy is absorbed by pigments, this energy excites electrons. As electrons move down the chain, their energy is used to pump protons across the membrane.
• Photophosphorylation uses proton motive force to generate ATP. Photophosphorylation can be cyclic or noncyclic.
  • Cyclic: The final electron acceptor is the original reaction center chlorophyll that donated the electron
    • Light excited the electron in PS I à the electron pass down ETC and then return to PS I
    • The energy from the electron in ETC establishes a proton gradient and ATP is produced through chemiosmosis
  • Noncyclic: The Light excited the electron in PS II à the electron pass down an ETC and then go to PS I, PS I further energizes the electron with additional light energy à the electron is passed through ETC to the final electron acceptor NADP+ (becomes NADPH)
    • A cell replenished the electron supply in PS II by deriving electron from water or even an inorganic compound dissociation

Light-Independent Reactions

• Do not require light directly.
• In prokaryotes, it occurs in the cytoplasm; in eukaryotes in the stroma of chloroplasts.
• Use ATP and NADPH generated by light-dependent reactions
• Key reaction is carbon fixation by the Calvin-Benson cycle.
• Three steps:
  • Fixation of CO2 – enzymes attach 3CO2 carbons onto RuBP ( a 5C organic compound already present in the cell).
  • Reduction - NADPH is reduces the present molecules to 6G3P (ATP is needed here).
  • Regeneration of RuBP – of the 6 G3P one is released and is then used to synthesis glucose, the other 5 are used to regenerate RuBP.

Other Anabolic Pathways

• Anabolic reactions are synthesis reactions requiring energy and a source of precursor metabolites.
• Energy derived from ATP from catabolic reactions
• Many anabolic pathways are the reverse of catabolic pathways.
• Reactions that can proceed in either direction are amphibolic - can operate catabolically or anabolically.

Carbohydrate Biosynthesis

• Carbon fixation in the Calvin cycle
• G3P is used as a starting point for the synthesis of sugars and complex polysaccharide
• Some cells are able to synthesize glucose from amino acids, glycerol, and fatty acids via a process called gluconeogenesis.

Lipid Biosynthesis

• Huge variety of lipids and so they are synthesized by a variety of routes
• Usually synthesized in anabolic reactions which are the reverse of catabolism
• Ex. Triglycerides – polymerization of glycerol (derived from G3P) and 3 fatty acids (produced from linkage of 2 acetyl-CoA to one another)

Amino Acid Biosynthesis

• Amino acids synthesize from precursor metabolites from glycolysis, Krebs and the pentose phosphate pathway
• Can also synthesize many amino acids from other amino acids
• Amine group can be added onto precures metabolite in one of two ways
  • Amination –amine group from ammonia
  • Transamination – move one amine group from one amino acid to the metabolite making a different amino acid
• Ribozymes of ribosomes polymerize amino acids into proteins

Nucleotide Biosynthesis

• Nucleotides made from precursor metabolites of glycolysis and Krebs
• Pentose part is derived from the pentose phosphate pathway
• Phosphate is derived from ATP
• Purines and pyrimidines are synthesized from glutamine and aspartic acid, ribose- 5-phosphate and folic acid

Gluconeogenesis

• The role of gluconeogenesis in the biosynthesis of complex carbohydrates.
• Starch, cellulose, Glycogen, Peptidoglycan Glucose

Biosynthesis of a Triglyceride Fat, a Lipid

• Glycolysis and the Calvin-Benson cycle.
• Acetyl-CoA -> Fatty acids
• Glyceraldehyde 3-phosphate (G3P) -> Glycerol -> Triglycerides/Fats

Amino Acid Synthesis

• Examples of the synthesis of amino acids via amination and transamination.

Nucleotide Biosynthesis

• DNA and RNA and the molecules needed.

Integration and Regulation of Metabolic Function

• Cells regulate metabolism in a variety of ways to maximize efficiency in growth and reproductive rate.
• Two Types of Regulatory Mechanisms
  • Control of gene expression; Cells control the amount and timing of protein (enzyme) production.
  • Control of metabolic expression; Cells control activity of proteins (enzymes) once produced.
• Cells synthesize or degrade channel and transport proteins.
• Cells often synthesize enzymes only when the substrate is available.
• Cells catabolize the more energy-efficient choice if two energy sources are available.
• Cells synthesize metabolites they need, typically cease synthesis if the metabolite is available.
• Eukaryotic cells keep metabolic processes from interfering with each other
• Cells use inhibitory and excitatory allosteric sites on enzymes to control activity of enzymes
• Feedback inhibition slows/stops anabolic pathways when product is in abundance.
• Cells regulate catabolic and anabolic pathways that use the same substrate by requiring different coenzymes for each pathway.

Integration of Cellular Metabolism

• Metabolic pathways for the polymerization of macromolecules. For example Proteins, Nucleic acids, Polysaccharides, Lipids, etc…