the effect of ethanol on the rate of anaerobic respiration

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Anabel Kersten                                                                                                                      November 08

Skill P: The effect of ethanol on the rate of anaerobic respiration of sucrose by yeast

Aim:

This is an experiment to investigate at which rates the different sugar-yeast-ethanol-mixtures move to the end of the 1mm capillary tube using five different ethanol concentrations.

Background theory:

Saccharomyces cerevisiae is a species of budding yeast. Yeast is used in bread making and brewing. In brewing yeasts ferment fruit or grain to produce alcoholic drinks. In bread making yeasts produce carbon dioxide which helps the bread to rise. The alcohol evaporates during baking. It is believed that yeast was originally isolated from the skin of grapes. Yeast contains many enzymes including sucrase which breaks down sucrose into glucose and fructose. The official name for sucrase is beta-fructofuranosidase (1, 11).

Carbohydrates are a group of substances that are important in many biological processes. They provide energy-rich nutrients to organisms and are used to build up their body structures; all carbohydrates contain hydrogen, carbon and oxygen. The carbohydrate sucrose is a disaccharide of one glucose sugar and one fructose sugar joined by a glycosidic bond. Glucose and fructose are both monosaccharides, and structural isomers and both respire to give off the same products (2).

When oxygen is not available, oxidative phosphorylation and the Krebs cycle cannot take place. This is also true for organisms or cells which don’t contain the machinery to carry these out. In these circumstances, respiration takes place without oxygen- anaerobic respiration. In anaerobic respiration, glycolysis takes place as usual, producing pyruvate and a small yield of 2 ATP (3).

Glycolysis is the lysis of glucose. It is a multi-step process in which a glucose molecule with six carbons is broken down into two molecules of pyruvate, each with three carbon atoms. Energy from ATP is needed in the first steps, but energy is released in later steps, when it can be used to make ATP. There is a net gain of two ATP molecules per molecule of glucose broken down. Glycolysis takes place in the cytoplasm of the cell. In the first stage, phosphorylation, glucose is phosphorylated using ATP and the use of different enzymes, including phosphofructose kinase and hexose kinase. As glucose is energy-rich, but doesn’t react easily energy must first be used to make the reaction easier. Two ATP molecules are used for each molecule of glucose to make hexose biphosphate, which breaks down to produce two molecules of triose phosphate. Hydrogen is then removed from triose phosphate and transferred to the carrier molecule NAD (nicotinamide adenine dinucleotide). Two molecules of NAD are produced for each glucose molecule entering glycolysis. The hydrogen carried by the reduced NAD can easily be transferred to other molecules and are used in oxidative phosphorylation to generate ATP (3, 4, and 5).

The reduced NAD which is produced in glycolysis must be oxidised back to NAD again or the cell would soon run out of it, bringing ATP production to halt. There are two different solutions to the problem, both of which get rid of pyruvate, and regenerate NAD. These are alcoholic fermentation, which is used by fungi and plants, and lactic fermentation, which is used by animals.

Ethanol for use in alcoholic beverages, and the vast majority of ethanol for use as fuel, is produced by fermentation. When Saccharomyces cerevisiae metabolizes sugar in the absence of oxygen, they produce carbon dioxide and ethanol (11).

C6H12O6 → 2 C2H5OH + 2CO2

This process is called alcoholic fermentation in which yeast converts pyruvate to ethanol. First, carbon dioxide is removed from pyruvate to produce ethanal. Next, alcohol dehydrogenase converts ethanal to ethanol. Ethanol’s toxicity to yeast limits the ethanol concentration obtainable by brewing (12).

Magnesium is an essential co-factor for many of the glycolytic enzymes and has also been identified as a limiting nutrient in fermentation broth containing peptone and yeast extract. Magnesium protects yeast cells by preventing increase in cell membrane permeability elicited by ethanol and temperature-induced stress (8).

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Modification/ Previous experiment (6):

Having carried out the previous experiments” An investigation into rates of fermentation by yeast “and “The effect of ethanol on the rate of anaerobic respiration of glucose by yeast”, we have seen that sucrose is the best respiratory substrate and the ethanol concentration of 25% is too high resulting in a reaction with infinity.  In the experiment “An investigation into rates of fermentation by yeast” we investigated at which rates the different sugar-yeast mixtures, using the four sugars glucose, sucrose, maltose and lactose, move to the end of the 1mm capillary tube. The mixture containing lactose ...

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