Microbial Metabolism

Metabolism Basics

• Metabolism is the collection of controlled biochemical reactions within a microbe
• The ultimate purpose of metabolism is to reproduce the organism
• Metabolic processes follow eight elementary statements including nutrient acquisition, energy utilization, and cellular building

Metabolic Processes

• Cells require nutrients for building blocks and energy
• Metabolism needs energy obtainable from light or nutrient catabolism
• Energy is stored as adenosine triphosphate (ATP)
• Anabolic reactions use precursor metabolites, ATP energy, and enzymes
• Catabolism uses enzymes to break down nutrients, creating precursor metabolites
• Enzymes and ATP create macromolecules by polymerization
• Cells grow through the assembly of macromolecules like ribosomes and membranes
• Cells reproduce once they have doubled in size

Metabolism Overview

• Anabolism synthesizes complex molecules, like proteins, lipids, DNA and RNA, from simple molecules.
• Catabolism breaks down complex molecules into simple molecules.
• ATP provides energy for anabolism and is produced in catabolism.

Energy and Metabolism

• Metabolism includes catabolic and anabolic reactions
• Catabolic reactions release energy
• Anabolic reactions utilize biosynthesis

Microbial Growth

• Microbial growth refers to the growth of a microbe population
• Microbial growth comes from the reproduction of individual microbes
• The result is a discrete colony of cells from a single parent cell

Microbial Nutrient Requirements

• Organisms need various nutrients for energy and to construct organic molecules and cellular structures
• Carbon, oxygen, nitrogen, and hydrogen are common elements in nutrients
• Microbes get nutrients from various sources

Sources of Carbon, Energy, and Electrons

• Organisms are grouped by carbon source as autotrophs or heterotrophs.
• Organisms are grouped by energy source as chemotrophs, using chemical reactions, or phototrophs.

Nutritional Categories by Carbon and Energy Source

• Photoautotrophs are plants, algae, and cyanobacteria that use H2O to reduce CO2, producing O2.
• Green and purple sulfur bacteria do not use H2O or produce O2.
• Chemoautotrophs include hydrogen, sulfur, and nitrifying bacteria, and some archaea.
• Photoheterotrophs are green nonsulfur bacteria and purple nonsulfur bacteria, and some archaea.
• Chemoheterotrophs include aerobic respirators (most animals, fungi, protozoa, many bacteria), anaerobic respirators (some animals, protozoa, bacteria, and archaea) and fermenters (some bacteria, yeasts, and archaea).

Electron Sources

• Organotrophs use organic compounds as a source of electrons
• Lithotrophs use inorganic compounds as a source of electrons.

Oxygen Needs

• Oxygen is required for obligate aerobes
• Oxygen is deadly to obligate anaerobes
• Oxygen can be toxic in reactive forms, strong oxidizing agents, and resulting oxidation can damage cells

Toxic Oxygen Forms

• Toxic forms of oxygen include singlet oxygen (¹O₂), superoxide radicals (O₂⁻), peroxide anion (O₂²⁻), and hydroxyl radical (OH•).

Neutralizing Toxic Forms of Oxygen

• Enzymes can neutralize toxic oxygen species.
• Catalase reduces hydrogen peroxide into water and oxygen.
• Peroxidase uses NADH to reduce hydrogen peroxide.
• Superoxide dismutase converts superoxide radicals to hydrogen peroxide and oxygen.
• Superoxide reductase reduces superoxide.

Oxygen Requirements for Growth

• Aerobes need oxygen
• Anaerobes grow without oxygen
• Facultative anaerobes can live with or without oxygen
• Aerotolerant anaerobes tolerate oxygen
• Microaerophiles need specifically low amounts of oxygen

Thioglycolate Broth

• Thioglycolate broth separates microbes based on oxygen needs.
• The broth reacts with oxygen, so oxygen penetrates only the top

Nitrogen Requirements

• Anabolism stops when there is insufficient nitrogen
• Nitrogen comes from organic and inorganic nutrients
• Cells recycle nitrogen from amino acids and nucleotides
• Nitrogen fixation by bacteria is essential to life on Earth.

Chemical Requirements for Growth

• Phosphorus is necessary for growth
• Sulfur is necessary for growth
• Trace elements are required, but only in small amounts
• Growth factors are essential organic chemicals not synthesizable for some organisms

Microbial Growth Factors

• Growth factors are necessary organic chemicals that organisms can’t synthesize
• Amino acids are for protein construction
• Cholesterol is for use by mycoplasmas, a bacteria, to build cell membranes
• Heme functions as a component of cytochromes in electron transport
• NADH is an electron carrier
• Niacin (nicotinic acid, vitamin B3) is a precursor of NAD+ and NADP+
• Pantothenic acid (vitamin B5) is a component of coenzyme A
• Para-aminobenzoic acid (PABA) is a precursor of folic acid, involved in the metabolism of one-carbon compounds and nucleic acid synthesis
• Purines and pyrimidines are components of nucleic acids
• Pyridoxine (vitamin B6) is utilized in transamination syntheses of amino acids
• Riboflavin (vitamin B2) is a precursor of FAD
• Thiamine (vitamin B1) is utilized in some decarboxylation reactions

Physical Requirements for Growth

• Temperature affects growth
• PH affects growth
• Organisms live in association with different species
• Physical effects of water influence growth

Bioenergetics

• Energy is measured in kilojoules (kJ), a unit of heat energy.
• Energy is lost as heat during chemical reactions
• Free energy (G) is energy released that is able to do work
• The change in free energy is referred to as ΔG°'

Free Energy

• Reactions with negative ΔG°' release free energy (exergonic)
• Reactions with positive ΔG°' require energy (endergonic)
• Free-energy yield is measured from the free energy of formation (Gºf)
• Gºf is the energy released or needed during formation of a given molecule from the elements
• ΔG°' is not always a reliable predictor of free-energy fluctuations
• Free energy (ΔG) occurs at actual conditions in the reaction

Calculating Free Energy

• Calculating free energy for a reaction is: ΔG = ΔG°' + RT ln(K)
• R and T represent physical constants; K represents equilibrium constants

Catalysis and Enzymes

• Activation energy is the energy needed to induce a reactive state in chemical reactions
• Catalysis is often needed to overcome the activation energy barrier

Catalysts

• Catalysts lower activation energy
• They increase reaction rate
• Catalysts are not consumed or transformed by the reaction
• They do not affect energetics or equilibrium

Electron Donors and Acceptors

• Energy from oxidation-reduction (redox) reactions synthesizes energy-rich compounds like ATP
• Redox reactions occur in pairs with half reactions
• An electron donor is oxidized in a redox reaction
• An electron acceptor is reduced in a redox reaction

Redox Tower

• The redox tower shows the range of reduction potentials.
• Reduced substances at the top donate electrons
• The oxidized substances at the bottom accept electrons
• The more electrons "drop" results in greater energy released

Redox Reactions

• Redox reactions are usually are mediated by intermediates or carriers
• Electron carriers are divided into prosthetic groups or coenzymes

Energy-Rich Compounds

• Chemical energy released during redox reactions is primarily stored in phosphorylates that include ATP, phosphoenolpyruvate, and glucose-6-phosphate
• Chemical energy is also kept in coenzyme A
• Long-term energy is in insoluble polymers oxidizable for ATP

Long-Term Energy Storage

• Examples in prokaryotes for long-term energy storage are glycogen, poly-β-hydroxybutyrate, and elemental sulfur
• Examples in eukaryotes are lipids and starch

Glycolysis

• Two reaction series are linked to energy conservation in fermentation and respiration
• ATP synthesis differs between fermentation and respiration
• Fermentation is substrate-level phosphorylation where ATP comes from an intermediate energy-rich molecule
• Respiration is oxidative phosphorylation where electron transport creates a proton motive force that produces ATP

Glucose Catabolism

• Summary of the flow of cellular respiration:
• Glucose enters glycolysis, producing ATP and NADH and 2 pyruvic acid
• Releases acetyl-CoA, and undergoes Krebs Cycle releasing NADH and FADH2
• Electrons are added from the electron transport chain
• Releases ATP and ADP, ultimately forming a final electron acceptor

Glycolysis Details

• Fermentation uses a fermented substance as both the electron donor and acceptor
• Glycolysis, the Embden–Meyerhof pathway, is a shared pathway for catabolizing glucose
• Glycolysis is an anaerobic process across three stages

Glycolysis Key Facts

• Glycolysis consumes glucose
• Two ATPs are produced
• Fermentation products are generated

Alternatives to Glycolysis

• Alternatives to glycolysis include the pentose phosphate and the Entner-Doudoroff pathway
• The pathways yield fewer ATP molecules
• The pathways reduce coenzymes and yield different metabolites needed in anabolic pathways

Fermentation Types

• Fermentations are classified by products formed
• These include ethanol, lactic acid, propionic acid, mixed acids, butyric acid, and butanol
• Fermentations are also classified by substrate.
• This substrate is not usually glucose
• Alternate substrates include amino acids, purines, pyrimidines, and aromatic compounds
• Saccharomyces cerevisiae can carry out the most beneficial of fermentation or respiration

Cellular Respiration

• Resultant pyruvic acid through a series of redox reactions to produce ATP
• Three stages of cellular respiration: synthesis of acetyl-CoA, Krebs cycle, and the final series of a redox reactions (electron transport chain)

Citric Acid and Glyoxylate Cycle

• A.k.a. CAC: is a pathway in which pyruvate is completely oxidized to CO2 with these steps:
  • (glucose to pyruvate) same as glycolysis
  • 6 CO2 molecules releases and NADH and FADH generated (per glucose molecule)
  • plays a major role in catabolism AND biosynthesis
• Catabolism of C2-C3 organic acids typically involves production of oxaloacetate via glyoxylate cycle.
• Glyoxylate is a variation of the citric acid cycle.
• Glyoxylate is a key intermediate

Aerobic Respiration

• O2 is used for oxidation as the terminal electron acceptor
• Has a higher ATP yield than fermentations
• ATP is produced using proton motive force, which comes from electron transport

Electron Transport

• Electron transport systems are the membrane-associated that mediate electron transfer
• Transfer conserves energy that is used to synthesize ATP
• Oxidation-reduction enzymes used during electron transportation include:
• NADH dehydrogenases
• Flavoproteins
• Iron-sulfur proteins
• Cytochromes

Electron Carriers in Respiration

• NADH dehydrogenases are proteins bound to the inside surface of the cytoplasmic membrane; the active site binds NADH and 2 electrons and 2 protons are passed to flavoproteins
• Flavoproteins contain a prosthetic flavin group accepting 2 electrons and 2 protons, transferring the electrons to the next protein in the chain

Cellular Respiration Carriers

• Four groups of carrier molecules are used
• Flavoproteins
• Ubiquinones
• Metal-containing proteins
• Cytochromes
• Aerobic respiration: oxygen is the final electron acceptor
• Anaerobic respiration: a molecule other than oxygen is the final electron acceptor

Catabolic Diversity

• Microorganisms can generate energy many ways
• Methods include fermentation, aerobic and anaerobic respiration, chemolithotrophy, and phototrophy

Anaerobic Respiration

• Anaerobic respiration performs electron acceptation by:
• Nitrate
• Ferric iron
• Sulfate
• Carbonate
• Certain organic compounds
• Releases less energy compared to aerobic respiration, but requires the support of electron transport and motive force

Chemolithotrophy

• Uses inorganic chemicals such as hydrogen sulfide or hydrogen gas as electron donors
• Typically aerobic
• Oxidation of starting inorganic electron donors begins
• An electron transport chain and proton motive force are utilized
• Autotrophic, using CO2 as a carbon source

Phototrophy

• Light used for energy
  • ATP from light-mediated photophosphorylation
  • Photoautotrophs use ATP for assimilation of CO2 for biosynthesis
  • Photoheterotrophs use ATP for assimilation of organic carbon for biosynthesis

Biosynthesis of Sugars and Polysaccharides

• Prokaryotic polysaccharides are synthesized from activated glucose, e.g., uridine diphosphoglucose (UDPG) or adenosine diphosphoglucose (ADPG).
• When the cell grows on other carbon compounds, glucose is synthesized (gluconeogenesis).

Amino Acids and Nucleotides

• Biosynthesis of amino acids and nucleotides requires long, multistep pathways
• Carbon skeletons for amino acids come from intermediates of glycolysis or the citric acid cycle
• Purine and pyrimidines are constructed from carbon and nitrogen. Once synthesized and attached to ribose, they are ready to be incorporated into DNA

Complex Biosynthesis

• Purine and pyrimidine biosyntheses are complex
  • Purines = Adenine and guanine via inosinic acid intermediate
  • Pyrimidines = Thymine via cytosine or orotic acid

Biosynthesis of Fatty Acids and Lipids

• Fatty acids are made at two-carbon increments by protein, i.e., acyl carrier protein (ACP)
• In Bacteria and Eukarya, the final assembly of lipids is the addition of fatty acids to glycerol
• In Archaea, lipids have phytanyl side chains, not fatty acids

Nitrogen Fixation

• Only certain prokaryotes can fix nitrogen.
  • Some nitrogen fixers are symbiotic, while others are free-living
  • Nitrogenase catalyzes the reaction and is sensitive to oxygen
  • Metal cofactors compose a wide variety of nitrogenases
  • Electron flow in nitrogen fixation follows this order: electron donor to dinitrogenase reductase to dinitrogenase to N2
• Ammonia is the final product
• Requires 16 ATP to reduce N2 to 2 NH3

Photosynthesis Types

• Many organisms can synthesize organic molecules from inorganic carbon dioxide
  • Most can capture light and build carbohydrates of CO2 and H2O thru photosynthesis

Structures in Photosynthesis

• Chlorophylls are a must have
  • They have pigmented molecules for photosynthetic usage
  • Made from hydrocarbons with light-absorbing active sites at the magnesium
  • Active sites look similar to cytochrome, with different absorption rates
  • Arrangement of chlorophyll/pigments is required to construct harvesting matrices
• In prokaryotes, cellular membranes called thylakoids are present
• In eukaryotes, inner membranes of chloroplasts are present
• Stacked inside the stacks called grana
• The space between the outer layer and thylakoid membranes known as the stroma can be used for different types of photosystems
• Two photosystem types
• I (PSI), II (PSII)
  • Photosystems use light energy to store ATP and NADPH
  • The dependent reaction on light energy is then used on the independent reaction of carbs and water

How Light-Dependent Reactions Work

• Electrons move down the chain, their energy is for pumping protons across the membrane
• Photophosphorylation uses PMT for ATP
• Photophosphorylation is both cyclic and non-cyclic

Light-Independent Reactions

• Do not require light directly
• Use ATP and NADPH from the earlier light-dependent reactions
• Critical because of carbon fixation from the Calvin-Benson cycle
• Requires three actions: fixation, reduction, and regeneration

Other Anabolic Pathways

• Anabolic reactions use precursor metabolites for energy
• Energy requires ATP
• Most are just reverse catabolic pathways
• Reactions proceed each way and are then labeled amphibolic