The glycolysis pathway is nearly universal in biological systems. Glycolysis is the sequence of reactions that converts glucose to pyruvate with the concomitant formation of ATP. Three fates of this pyruvate produced exist. In this practical the production of pyruvate and acetaldehyde by fermentation of glucose is established. A series of test tubes was set up each containing glucose and yeast suspension in buffers at different pH values. These test tubes were incubated for an hour at 37?. Trichloro-acetic acid solution was then added to the first 2 of the 4 test tubes prior to centrifugation at 2500g.
Solid ammonium sulphate and freshly prepared sodium nitroprusside were added to these tubes and colour observations made. For the remaining 2 tubes sodium nitroprusside and aqueous pyrrolidine were added to the supernatant and colour changes observed. INTRODUCTION/LIERATURE REVIEW Three alternative catabolic routes are taken by the pyruvate formed by glycolysis. In aerobic organisms or tissues, under aerobic conditions, glycolysis constitutes only the first stage in the complete degradation of glucose.
Pyruvate is oxidized, with loss of its carboxyl group as CO2, to yield the acetyl group of acetylcoenzyme A, which is then oxidized completely to CO2 by the citric acid cycle. The electrons from these oxidations are passed to O2 through a chain of carriers in the mitochondrion, forming H2O. (Voet and Voet; 2011). The energy from the electron transfer reactions drives the synthesis of ATP in the mitochondrion. The second route for pyruvate metabolism is its reduction to lactate via lactic acid fermentation.
When a tissue such as vigorously contracting skeletal muscle must function anaerobically, the pyruvate cannot be oxidized further for lack of oxygen. Under these conditions pyruvate is reduced to lactate. Certain tissues and cell types (retina, brain, and erythrocytes) convert glucose to lactate even under aerobic conditions. Lactate (the dissociated form of lactic acid) is also the product of glycolysis under anaerobic conditions in microorganisms that carry out the lactic acid fermentation (www. antoine. frostburg. edu). The third major route for catabolism of pyruvate leads to ethanol.
In some plant tissues and in certain invertebrates, protists, and microorganisms such as brewer’s yeast, pyruvate is converted anaerobically into ethanol and CO2, a process called alcohol (or ethanol) fermentation. (Hames and Hopper; 2005) Pyruvate is an important metabolic intermediate in a variety of cellular processes that help create cellular energy. It is the simplest alpha-keto acid having the chemical formula CH3COCOO-. In humans, pyruvate is involved in converting sugar to energy, creating energy in the presence of oxygen, and even producing lactic acid, which occurs in muscle cells under low oxygen conditions.
In other organisms, pyruvate is an intermediate in the fermentation of alcohol from glucose (www. brilliantbiologystudent. com). Pyruvate is a three-carbon molecule. One carbon atom exists as a methyl CH3 group, the middle carbon atom is double-bonded to an oxygen molecule, creating the ketone group, and the third carbon is part of a carboxyl COO- group. Pyruvate is the carboxylate anion of pyruvic acid, having the formula CH3COCOOH. (Wrolstad; et al; 2005). Glycolysis is the process by which cells convert sugar into energy.
During glycolysis, the six-carbon sugar molecule called glucose is converted into two three-carbon pyruvate molecules through a series of steps. The process of glycolysis is not particularly efficient as a means of producing cellular energy. However, the resulting three-carbon pyruvate molecules are the beginning substrates needed to enter the citric acid cycle, which is a very efficient process of creating cellular energy. Pyruvate can also be converted into alcohol through the process of fermentation. In alcohol fermentation, two atoms of oxygen and one atom of carbon leave the pyruvate molecule as carbon dioxide.
This is known as pyruvate decarboxylation. This creates acetaldehyde, CH3CHO, a two-carbon aldehyde molecule. Acetaldehyde is then converted into ethanol, CH3COOH. (www. sciencedirect. com) This reaction occurs under low oxygen, or anaerobic conditions. Organisms that convert pyruvate to ethanol, or ferment under anaerobic conditions are called facultative anaerobes. Yeast are examples of facultative anaerobes that undergo alcohol fermentation. The carbon dioxide gas given off in this process is useful in the process of bread baking, causing the dough to rise. www. sciencedirect. com) Many bacteria are capable of converting pyruvate to lactic acid. This process is called lactic acid fermentation. It is a process similar to alcohol fermentation, but in this case, the three-carbon pyruvate molecule is converted to a three-carbon lactic acid molecule having the formula CH3CHOHCOOH. Unlike alcohol fermentation, no carbon dioxide is given off. A three-carbon molecule is simply converted to another three carbon molecule (www. livestrong. com) Some bacteria and yeasts organisms are unable to cope with the presence of oxygen.
These organisms use fermentation as a method of obtaining energy in the form of ATP. Because the production of lactic acid frees up NAD+, the process of glycolysis can continue. Lactic acid fermentation is the simplest type of fermentation. In essence, it is a redox reaction. In anaerobic conditions, the cell’s primary mechanism of ATP production is glycolysis. Glycolysis reduces – that is, transfers electrons to NAD+, forming NADH. However, there is only a limited supply of NAD+, available in a cell. For glycolysis to continue, NADH must be oxidized, that is, have electrons taken away to regenerate the NAD+.
This is usually done through an electron transport chain in a process called oxidative phosphorylation; however, this mechanism is not available without oxygen. Instead, the NADH donates its extra electrons to the pyruvate molecules formed during glycolysis. Since the NADH has lost electrons, NAD+, regenerates and is again available for glycolysis. Lactic acid, for which this process is named, is formed by the reduction of pyruvate. Lactic acid fermentation also occurs in animal muscle cells under conditions when oxygen is low. Extreme exercise would be an example of this.
In this situation, the lactate is carried away by the circulatory system to the liver, where it is converted back to pyruvate through the Cori cycle. (Voet and Voet; 2011). 2,4-Dinitrophenylhydrazine (DNPH, Brady’s reagent) is the chemical compound C6H3(NO2)2NHNH2. It is a red to orange solid, usually supplied wet to reduce its explosive hazard. It is a substituted hydrazine, and is often used to qualitatively test for carbonyl groups associated with aldehydes and ketones. A positive test is signalled by a yellow, orange or red precipitate (known as a dinitrophenylhydrazone. If the carbonyl compound is aromatic, then the precipitate will be red; if aliphatic, then the precipitate will have a more yellow colour. This reaction can be described as a condensation reaction, with two molecules joining together with loss of water. It is also considered an addition-elimination reaction: nucleophilic addition of the -NH2 group to the C=O carbonyl group, followed by the removal of a H2 O molecule (www. lsbu. ac. uk). A centrifuge is a piece of equipment, generally driven by an electric motor (some older models were spun by hand), that puts n object in rotation around a fixed axis, applying a force perpendicular to the axis. The centrifuge works using the sedimentation principle, where the centripetal acceleration causes denser substances to separate out along the radial direction (the bottom of the tube). By the same token lighter objects will tend to move to the top (Koolman and Roehm; 2005). The core principle of centrifuge operation is centrifugal force. If a bucket half-filled with water is spun quickly in a circle, over the head and back down to the ground, centrifugal force created by the rotation of the bucket forces the water towards the bottom.
This is what keeps the water in the bucket even when it’s upside down. Most centrifuges harness this force in a similar way, and consist of a casing with a lid and a driven central rotor. The rotor has a row of holes around its circumference into which the containers, typically test tubes, of solution are placed. Once the machine lid is closed and the centrifuge switched on, the rotor spins at high speed. Centrifugal force causes any matter in the solution denser than the liquid to be forced against the outer walls of the tubes, separating it from the fluid in the process (www. io. net). METHOD Test tubes were set up all including 5ml of 10% glucose solution. The first of these test tubes also contained 5ml of yeast suspension in Na2HPO4 at pH 8, the second with the same yeast suspension in KH2PO4 at pH 6. The third and fourth test tubes both contained 5ml yeast suspension in distilled water. All the 4 test tubes were incubated at 37? for an hour. Trichloro-acetic acid solution was added to the first and second test tube with a volume of 2ml and the solutions thoroughly mixed. All the tubes were centrifuged at 2500g for 10 minutes.
From tubes 1 and 2, 2ml of aliquots were taken and then boiled. Solid ammonium sulphate was added and so were 2 drops of 5% sodium nitroprusside. Concentrated ammonia was then run down the sides of the test tubes to form 2 layers. For tubes 3 and 4, 0. 5ml of freshly prepared sodium nitroprusside was added to 2ml of the supernatant, followed by 2ml of aqueous pyrrolidine. The solutions were mixed and colour change observations made. RESULTS Pyruvate was present in test tube 1 which contained 5ml of 10% glucose and yeast suspension in Na2HPO4 at pH 8. No pyruvate was present in the second est tube containing 5ml of 10% glucose and yeast suspension in KH2PO4 at pH 6. Acetaldehyde was present in both the third and fourth test tube which contained 5ml of 10% glucose solution and yeast suspension in water. DISCUSSION Fermentation is a process used by cells to generate energy where a suitable substrate is metabolized to make ATP by Substrate Level Phosphorylation (SLP). Fermentation pathways operate under anaerobic cell growth conditions when electron acceptors are unavailable to support cellular respiration. During anaerobic conditions, glucose is initially metabolized to pyruvate via the glycolysis pathway. lucose > 2 pyruvate These reactions also generate 2 molecules of NADH and 4 molecules of ATP. Since two ATP are consumed in early steps of the pathway, a net total of 2 ATP are produced per molecule of glucose consumed. The presence of this pyruvate produced is tested in the sodium nitroprusside test. If pyruvate is present a green or blue ring forms at the junction of the ammonia and nitroprusside solution. A transient pink ring may also form at the junction due to the presence of thiol groups. In this practical, the tube under slightly alkaline conditions had a pink junction and a green top.
There are thus thiol group present in the reaction vessel. The green colour is due to pyruvate produced. In alkaline conditions the enzyme that converts pyruvate to acetaldehyde, pyruvate decarboxylase, is inactive. Pyruvate thus accumulates in yeast suspension at pH 8. For the test tube in acidic conditions pyruvate decarboxylase is activated. There is thus conversion of pyruvate to acetaldehyde in the reaction mixture. The slightly positive sodium nitroprusside test alludes to the fact that pyruvate is a transition intermediate in the conversion to acetaldehyde.
In alcohol fermentation, two atoms of oxygen and one atom of carbon leave the pyruvate molecule as carbon dioxide. This is known as pyruvate decarboxylation and is catalysed by the enzyme pyruvate decarboxylase. This creates acetaldehyde, CH3CHO, a two-carbon aldehyde molecule. The presence of acetaldehyde is tested by adding sodium nitroprusside to the supernatant and pyrrolidine. If acetaldehyde is present a blue of green colour is observed. Both the third and fourth test tubes contained acetaldehyde as proved by the by the green colours observed. There is however more acetaldehyde present in the fourth ube which had sodium sulphite. This reagent traps the acetaldehyde produced.