Nutritional Requirements of Microbes

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.

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. 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.

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.