Glycolysis is the first phase of a series of reactions for the catabolism of carbohydrates. Catabolism is the breakdown of larger molecules into its respective smaller constituents. Glycolysis is the first part of cellular respiration that generates pyruvate to be used in either anaerobic respiration in the absence of oxygen or in the TCA cycle in aerobic respiration which yields useable energy for cells. This will be a general outline of the steps in glycolysis.

The whole process can be broken down into an energy investment phase where ATP is being used and an energy payoff phase where ATP is being generated. Fructose-1,6-biphosphate is where the energy investment phase ends. That is where the last ATP has to be used for energy to drive glycolysis.

A simple equation can be remembered as a summary of glycolysis.

Glucose + 2 ADP + 2 phosphate ions + 2 NAD+ —-> 2 Pyruvate + 2 ATP + 2 NADH + 2 H20 + 2 H+.


In the first step of glycolysis an addition of a high energy phosphate from ATP yields glucose-6-phosphate and ADP. This step is initialized by the enzyme hexokinase. G6P is more reactive than glucose.

In the next step, glucose-6-phosphate is converted to its isomer, fructose-6-phosphate by phosphoglucose isomerase.

In the third step of glycolysis, fructose-6-phosphate is converted to fructose-1,6-biphosphate by phosphofructokinase (PFK) and the addition of ATP. This is the committed step, meaning that fructose-1,6-biphosphate MUST be converted to pyruvate. This is also the end of the energy investment phase of glycolysis.

Fructose-1,6-biphosphate is converted to glyceraldehyde-3-phosphate (G3P) and dihydroxyacetone phosphate catalyzed by aldolase. Glyceraldehyde-3 phosphate maintains a reversible reaction with dihydroxyacetone phosphate through triode phosphate isomerase. The resulting reaction generates two molecules of glyceraldehyde-3-phosphate. A key point going forward is that two molecules of each substrate are produced.

Each G3P molecule gains an inorganic phosphate and with the addition of NAD+ to form the energized carrier molecules NADH. The resulting reaction catalyzed by glyeraldehyde-3-phosphate dehydrogenase generates two molecules of 1,3-bisphophoglycerate which yield two high energy phosphates.

Through the addition of two low energy ADP molecules and the enzyme phosphoglycerate kinase, the two molecules of 1,3-bisphophoglycerate are converted to 3-phosphoglycerate and yields two molecules of ATP. This reaction is called the break even reaction because at this point the energy input is equal to the energy output. Two molecules of ATP were expended and at this step there was a generation of two ATP molecules.

In the next step the two molecules of 3-phosphoglyercate are converted to 2-phosphoglycerate through the enzymatic properties of phosphoglycerate mutase.

The molecules of 2-phosphoglycerate are converted to phosphoenolpyruvate catalyzed by enolase. This step yields H20 molecules.

In the final step of glycolysis, the molecules of phosphoenolpyruvate are converted to pyruvate catalyzed by pyruvate kinase. ATP is generated from the addition of ADP and the two high energy phosphates from the molecules of phosphoenolpyruvate.

Upon the completion of glycolysis, the pyruvate molecules can be oxidized to carbon dioxide in cellular respiration to generate 28 molecules of ATP.

The NADH that is produced is turned back into NAD+ to drive further glycolysis. There are two ways to accomplish this. In the presence of oxygen NADH passes it electrons into the electron transport chain, which regenerates NAD+ for use in glycolysis. In the absence of oxygen, cells regenerate NAD+ by undergoing fermentation.



Triple Sugar Iron Agar (TSI)

The TSI is a multiple test medium. Its a slanted medium with a deep butt that is used to further investigate Gram-negative microorganisms. It differentiates the microbes by their ability to ferment glucose, lactose and/or sucrose with or without the production of gas and production of hydrogen sulfide.

The TSI medium contains three carbohydrates; 1.0% lactose, 1.0% sucrose and 0.1% glucose. Phenol red is added as a pH indicator. Ferric ammonium citrate and sodium thiosulfate are added as indicators for the production hydrogen sulfide.

There are multiple reactions that can be observed;

A reaction of alkaline/no change is denoted K/NC and it means that the organism can only catabolize peptones aerobically, hence only the slant exhibited a color change, usually a red/orange color. This means that no carbohydrates were utilized.

When the slant is alkaline after 18-24 hours of incubation it means that there was rapid depletion of glucose and there is a subsequent reliance on peptides for nutrients. This occurs because the concentration glucose is so low and therefore it is consumed quickly. Catabolism of peptones results in the release of ammonia (NH3) which yields an alkaline pH. The butt of the medium remains acid because the degradation of peptones occurs aerobically (i.e. in the slant).

Some organisms have the ability to ferment lactose and/or sucrose with glucose for their nutrients. This results in an acid slant and acid butt reaction denoted A/A and a color change of yellow/yellow will appear. Because the concentrations of lactose and sucrose are 10x the amount of glucose, therefore a large amount of acid is produced.

A TSI medium also is used to determine whether or not a microorganism can produce carbon dioxide and hydrogen gases from the fermentation of the carbohydrates present. Gas production is seen when a bubble forms, which splits the medium. A clear disc shaped area is seen within the medium.

Ferric ammonium citrate and sodium thiosulfate are both indicators that are added to view the presence of H2S hydrogen sulfide. A microorganism in an acidic environment acts on the sodium thiosulfate to produce H2S gas. H2S reacts with ferric ions to produce ions that produce ferrous sulfide which is an insoluble black precipitate.

It should be noted that the black precipitate of ferrous sulfide that indicates H2S production may mask an acidic condition in the butt of the tube. Since H2S is only produced under acidic conditions, when the butt of the tube is black, an acid butt exists as well even without the presence of the yellow color.

TSI Results K/K K/A A/A +g A/A +g,+H2S A/K +g, +H2S