Metabolism and Energy Pathways

Nutritional Requirements of Microbes

Microbes that produce their own food are autotrophs, those that obtain energy from light are called photoautotrophs, and those that obtain energy from chemicals are chemoautotrophs. Heterotrophs must obtain carbon from biological sources.
Microbes can be classified according to how they obtain carbon and energy. Prefixes attached to the Greek root -troph are used to categorize organisms based on the carbon source used to acquire energy. An autotroph uses simple inorganic carbon sources such as carbon dioxide to make complex carbon based chemicals that store energy. A heterotroph obtains carbon from consuming complex organic (compounds containing carbon-hydrogen bonds) compounds, such as carbohydrates, that other organisms have generated. Energy for powering the cell's metabolism can come from light (photo-) or redox reaction involving chemicals (chemo-).

Microbial Nutrient Requirements

Classification Carbon Source Energy Source Example
Photoautotroph Carbon dioxide Light Algae, cyanobacteria
Credit: Matthew ParkerLicense: CC BY-SA
Tolypothrix
Photoheterotroph Organic carbon Light Purple nonsulfurous bacteria
Credit: Li et al.License: CC BY
Rhodopseudomonas palustris
Chemoautotroph Carbon dioxide Bonds in inorganic compounds Chemosynthetic bacteria
Credit: Boden et al.License: CC BY, CC0
Thermithiobacillus tepidarius
Chemoheterotroph Organic carbon Bonds in organic compounds Staphylococcus aureus, other human pathogens
Staphylococcus aureus

Microorganism may create their own energy autotrophically or obtain their energy heterotrophically. Organic carbon refers to compounds containing carbon-hydrogen bonds.

Glycolysis

Glycolysis is the metabolic pathway that breaks down glucose in the presence (aerobic respiration) or absence (anaerobic respiration) of oxygen to generate energy in the form of energy carriers (ATP and NADH) and pyruvate.

Glycolysis is a metabolic pathway in which glucose is broken down into pyruvate in the cytoplasm of the cell. The breakdown of glucose in the absence or presence of oxygen generates small amounts of ATP and pyruvic acid. In most organisms, including microbes, the type of glycolysis used is the Embden-Meyerhof-Parnas (EMP) pathway. The second most common glycolysis pathway is the Entner-Doudoroff (ED) pathway. In both the EMP and ED pathways, the initial reactant is glucose and the final products are pyruvic acid and energetic molecules, though the EMP yields more ATP. The EMP and ED pathways use different enzymes at three points early in the pathways, and because of this have two different intermediary reactants. Some gram-negative bacteria, such as Pseudomonas aeruginosa, utilize the ED pathway exclusively; some, such as E. coli, can use both the EMP and ED glycolysis pathways.

Glycolysis is the initial stage of turning glucose into ATP by both aerobic and anaerobic pathways. In glycolysis, glucose (a six-carbon sugar) is broken into pyruvate (a three-carbon sugar). Glycolysis occurs in the cytosol, the aqueous component of a cell.

There are two main phases to glycolysis: energy investing and energy harvesting. In the energy investing phase, two ATP are required to phosphorylate glucose by adding a phosphate group. This forms fructose 1,6-bisphosphate. Fructose 1,6-bisphosphate is broken down into glyceraldehyde 3-phosphate (G3P) and dihydroxyacetone phosphate (DHAP). DHAP has a similar structure to G3P and is readily converted to G3P.

In the energy harvesting phase, oxidation of G3P to form pyruvic acid results in ATP formation. NAD+ is used as a reducing agent, or donates an electron, in this process and yields NADH. Each G3P transfers a high-energy phosphate to an ADP phosphorylating it to form ATP. At the end of G3P oxidation, four ATP, two molecules of pyruvic acid, two NADH, and two molecules of water are produced. As two ATP were initially invested in this process, the net gain of ATP from glycolysis of one glucose molecule is two ATP. However, glycolysis also produces 10 molecules of NADH, which are used in the electron transport chain. In this sense, glycolysis is the initial phase of cellular respiration.

Glycolysis does not require oxygen and is a universal pathway utilized by both aerobic and anaerobic organisms. When oxygen is present (aerobic conditions), the two molecules of pyruvic acid produced are utilized in the citric acid cycle. When oxygen is not present (anaerobic conditions), the two molecules of pyruvic acid are used in fermentation. The resulting pyruvic acid can be utilized in several other pathways.

Steps of Glycolysis

Glycolysis is the first pathway in generating energy from sugars. It can be performed aerobically and anaerobically.

Citric Acid Cycle

The citric acid cycle is a series of steps that transfers stored energy by reducing electron carriers to drive the synthesis of ATP.

The second stage of aerobic respiration, a chemical process in which energy is produced using oxygen, is the citric acid cycle, also known as the Krebs cycle or the tricarboxylic acid cycle. The citric acid cycle is a series of chemical reactions used by all aerobic organisms to release ATP. It takes the two molecules of pyruvate created during glycolysis to produce high-energy molecules of NADH, FADH2, and some ATP.

Substantial chemical energy from pyruvate is extracted in the citric acid cycle. Pyruvic acid must first be decarboxylated and attached to a carrier enzyme. The resulting complex, acetyl-coenzyme A (also called acetyl-CoA), holds acetate from pyruvate so it can be oxidized in the citric acid cycle. With acetyl-CoA produced, the citric acid cycle can now begin.

While the citric acid cycle produces two ATP, its primary purpose is to transfer energy from acetyl-CoA into electron carriers NAD+ and FAD, which drive the electron transport chain of oxidative phosphorylation. This takes place in a series of eight enzymatic reactions on a regenerating substrate.

Step 1: Citrate synthase attaches carbons from acetyl-CoA to oxaloacetic acid, forming citric acid.

Step 2: Aconitase isomerizes citric acid by moving a hydroxyl from carbon 3 to carbon 2. This results in isocitric acid.

Step 3: Isocitric acid is oxidized and decarboxylated to form the five-carbon α-ketoglutaric acid and produce one NADH.

Step 4: α-ketoglutaric acid is oxidized and decarboxylated. The resulting molecule is taken up by coenzyme A to form succinyl-CoA.

Step 5: Succinyl-CoA phosphorylates GDP to GTP, releasing the CoA unit and producing succinic acid. GTP then phosphorylates ADP, producing the ATP molecule of that cycle.

Step 6: Succinic acid is oxidized to fumaric acid, forming a molecule of FADH2.

Step 7: Fumarase hydrates fumaric acid to malic acid.

Step 8: Malic acid is oxidized to oxaloacetic acid, creating another NADH. The cycle is complete, and oxaloacetic acid is ready to receive another acetyl-CoA.

Citric Acid Cycle

The citric acid cycle is an aerobic pathway to further process sugar after its conversion to pyruvate by glycolysis, producing carbon dioxide, some ATP, and electron carriers used in oxidative phosphorylation (or the electron transport chain).
Because glycolysis produces two pyruvic acids from each glucose, the citric acid cycle runs twice for each glucose molecule. Therefore, in respiration, one molecule of glucose yields two ATP during glycolysis and two ATP in two turns of the citric acid cycle. One glucose also nets six NADH and two FADH2 from the cycle. These are energy dense compounds utilized in the electron transport chain.

Electron Transport and Oxidative Phosphorylation

Electron transport and oxidative phosphorylation serve as the final processing for electrons and hydrogen ions, yielding about 30 ATP per glucose molecule.

The electron transport chain is responsible for the vast majority of ATP produced in respiration. The electron transport chain is a series of electron transporters that move electrons from NADH and FADH2 to oxygen molecules; protons are pumped from the mitochondrial matrix to the intermembrane space, and oxygen is reduced to form water.

In eukaryotic cells, which have a nucleus and membrane-bound organelles, electron transport and oxidative phosphorylation occurs from the mitochondrial membrane to the intermembrane space. In prokaryotic cells this process occurs on folded invaginations of the plasma membrane in the cytoplasm. The electron transport chain relies on a membrane to establish chemiosmosis, which moves protons across the membrane. As electrons are passed from one electron carrier to the next, they eventually arrive at the final acceptor. In the case of aerobic respiration, the final electron acceptor is oxygen. In anaerobic respiration, the final electron acceptor is something other than oxygen, such as sulfate or nitrate. As protons are pumped across a membrane, the concentration of protons increases outside the membrane, and decreases inside the membrane. Ions diffuse to create equilibrium but are actively transported against it, which creates a proton motive force. A proton motive force is the accumulation of potential energy among protons on the concentrated side of a membrane, which, when allowed to equilibrate through controlled channels, establishes chemiosmosis. In the mitochondria of eukaryotic microbes, protons are moved from the intermembrane space to the mitochondrial matrix. In prokaryotic cells, protons are moved from outside the cell into the cell. Once oxygen absorbs the charge, it is reduced to form water.

NADH and FADH2 are unique in that they donate the initiating charge. NADH and FADH2 donate protons to initiate the proton gradient, a pathway in which protons move from an area of higher concentration to an area of lower concentration. However, other molecules in the chain only pass electrons. These molecules include flavoproteins, ubiquinones, cytochromes, and metal-containing proteins. The exact composition of the chain varies somewhat by organism. These molecules alternate between reduced and oxidized states, moving electrons along the chain until they reach the final acceptor. As they do so, they pump protons increasingly against the gradient. These protons flow down their gradient through complex enzymes in the membrane called ATP synthases.

ATP synthases are proton-driven enzymes that phosphorylate molecules of ADP to ATP. Because it is ultimately derived from the oxidation of NADH and FADH2, this production of ATP through chemiosmosis in the mitochondria through an electron transport chain is known as oxidative phosphorylation. Oxidative phosphorylation generates up to 34 ATP for each molecule of glucose, compared to the direct generation of four from glycolysis or two from the citric acid cycle. However, it must be remembered that glycolysis produces most of the NADH oxidized in the electron transport chain.

Electron Transport Chain

The electrons delivered to the mitochondrial membrane are used to create a hydrogen ion concentration gradient. Facilitated diffusion of hydrogen ions through ATP synthase is used to produce ATP.

Energy Yield from Respiration

Pathway Carbon Reactants Products Electron Carriers Produced Net ATP in Pathway Net ATP from Oxidative Phosphorylation Maximum Theoretical ATP Yield
Glycolysis Glucose 2 acetyl-CoA 4 NADH 2 12 14
Citric acid cycle 2 acetyl CoA 4 CO2 6 NADH, 2 FADH2 2 22 24
Total Glucose 6 CO2 10 NADH, 2 FADH2 4 34 38

A single molecule of glucose that is fully oxidized through glycolysis and the citric acid cycle yields multiple types of useable, stored chemical energy.

Fermentation

Fermentation is the process in which pyruvate, the end product of glycolysis, is further processed to release energy in the absence of oxygen.

Organisms that are unable to utilize oxygen for respiration are unable to complete the electron transport chain because there is no oxygen to act as the final electron acceptor. For example, bacteria in the mammalian intestines or water saturated soils have no available molecular oxygen. Electron carrier molecules remain in their reduced states and are consequently unavailable for further reaction. Because the electron transport chain produces the majority of ATP available to an organism, its inactivation has a serious cost in lost potential energy. More importantly, the electron transport chain is the primary supply of oxidized NAD+ molecules that enable glycolysis and the citric acid cycle. Without an alternative pathway to supply NAD+, all cell activity would cease.

Fermentation is the anaerobic breakdown of a substance that produces a limited amount of ATP for a cell. There are several pathways in microbiology that are referred to as fermentation. Lactic acid fermentation reduces pyruvate (the last product of glycolysis) into lactic acid while oxidizing NADH to NAD+. The only product of fermentation by Lactobacillus and Streptococcus, used in the production of yogurt, is lactic acid, and this form of fermentation is referred to as homolactic fermentation. Heterolactic fermentation results in a mixture of lactic acid and ethanol or acetic acid. Leuconostoc mesenteroides uses heterolactic fermentation to sour vegetables, such as turning cucumbers into pickles. The most recognizable fermentation is alcohol fermentation. Alcohol fermentation is the reduction of sugar to ethanol, a reaction that entails CO2 as a waste product and facilitates the rising of bread, the brewing of beer, and the making of wine.

Commonly Used Fermentation Pathways

Fermentation Pathway End Product Example Microbe Products
Alcohol Ethanol, carbon dioxide Saccharomyces Beer, wine, bread, etc.
Lactic acid Lactic acid Streptococcus Yogurt, cheese, pickles, etc.
Acetone-butanol-ethanol Acetone, butanol, ethanol, carbon dioxide Clostridium acetobutylicum Acetone, gasoline alternative
Butyric acid Butyric acid, carbon dioxide, hydrogen gas Clostridium butyricum Butter
Mixed acid Acetic, formic, lactic, succinic acids; ethanol, carbon dioxide, hydrogen gas Escherichia Vinegar, vitamins

Several fermentation pathways have evolved to extract energy from organic compounds under anaerobic conditions.

Fundamentally, fermentation reduces organic molecules with carbon-hydrogen bonds from inside the cell to produce NAD+ and NADH instead of using external oxygen or another oxidizer (such as sulfur) in respiration. A lot of energy remains bound in the reduced metabolites such as ethanol and lactic acid that could have otherwise been phosphorylated. In contrast to the 36 to 38 ATP produced by aerobic respiration, fermentation only produces two ATP.