Obtain pure samples of Ethanol (CH3CH2OH) and Ethanoic Acid (CH3COOH) from fermented Yeast (Saccharomyces Cerevisiae).

PREPARATION OF ETHANOL AND ETHANOIC ACID

AIM: To obtain pure samples of Ethanol (CH3CH2OH) and Ethanoic Acid (CH3COOH) from fermented Yeast (Saccharomyces Cerevisiae).

BACKGROUND INFORMATION:

The use of yeast in food production is the oldest and most extensive contribution made by any group of microorganisms. A most common substrate that yeast can work with is GLUCOSE.

Glucose is a monosaccharide, which are sweet crystalline sugars that dissolve easily in water to form sweet solutions. Monosaccharides have the general formula (CH2O)n and consist of a single sugar molecule. Glucose is the simplest and most common monosaccharide. It is a Hexose sugar and therefore has the formula C6H12O6.

Glucose can exist in two possible ring forms, known as the alpha (α) and beta (β) forms:

The hydroxyl group on Carbon atom 1 can project below the ring (α glucose) or above the ring (β glucose). Molecules like this, which have the same molecular formula but a different structural formula, are said to be ISOMERS of each other. One type of isomerism, called sterioisomerism, occurs when the same atoms or groups, are joined together but differ in their arrangement in space.

The existence of two isomers of glucose is important because it is linked to the way in which polysaccharides are formed and subsequently their roles. A glucose molecule can switch spontaneously from the open chain form to either of the two ring forms and back again.

As glucose is respiratory substrate, it is a very important source of energy in living organisms. Glucose is an important building block as it is used to build up more complex carbohydrates e.g. Starch, Glycogen and Cellulose. It is a substrate, which gets broken down by the enzymes in the yeast.

ENZYMES are defined as biological catalysts. A catalyst is a substance, which speeds up a chemical reaction, but remains unchanged itself throughout the whole reaction. Enzymes are biological because they are protein molecules made by living cells. An enzyme combines with its substrate to form a short-lived enzyme/substrate complex. This proximity of the enzyme with the substrate in the complex greatly increases the chance of a reaction occurring. Once the reaction has occurred, the complex breaks up into products and enzymes.

Enzymes are globular proteins and therefore they are soluble. Their presence does not alter the nature or properties of the end product(s) of the reaction however, they cannot survive temperatures above 40 degrees as they denature and lose their catalytic properties. At their optimum conditions the enzymes work more efficiently in the reaction(s). They have a special feature called the ACTIVE SITE, which is usually a depression in the enzyme to which a substrate molecule (glucose) can bind. In order for a reaction to start a little energy is needed to begin the reactor. This energy is called the ACTIVATION ENERGY (see diagram below). This is the energy required to make the substances react so when biochemical reactions take place, chemical bonds are either formed or destroyed.

The activation energy barrier is lowered when the enzymes become attached to the substrate so the reaction takes place more easily.

The enzyme is usually larger than the substrate. They have a particular shape into which the substrate(s) fit exactly. This if often referred to as the ‘lock and key’ hypothesis, where the substrate is imagined being like a key whose shape is complementary to the enzyme or the lock.

The site where the substrate binds in the enzyme is known as the active site and it is this, which has the specific shape. Once formed, the products no longer fit into the active site and escape into the surrounding medium, leaving the active site free to receive further substrate molecules.

One particular process where the yeast breaks down glucose is FERMENTATION.

Fermentation is a biological process, which occurs in the absence of oxygen, also known as anaerobic respiration. Much of modern biotechnology involves the large-scale production of substances by growing specific microorganisms in a large container known as a fermenter.

Biotechnology is the application of scientific and engineering principles to the production of materials by biological agents.

Fermenters are used for the anaerobic respiration to take place. Fermenters are vessels used for the growth of microoraganisms in liquid media (in this case the fermented yeast solution).

ALCOHOL FERMENTATION

In alcoholic fermentation the pyruvate from glycolysis is first converted to ethanal (acetaldehyde) through the removal of a carbon dioxide molecule.

CH3COCOOH CH3CHO + CO2

(Pyruvate) (Ethanal) (Carbon dioxide)

The ethanal then combines with the hydrogen ions, which are transported by the hydrogen carrier NAD, to form the alcohol, ethanol.

NADH + H+ NAD+

CH3CHO CH3CH2OH

(Ethanal) (Ethanol)

This form of fermentation occurs in yeast, where the alcohol produced may accumulate in the medium around the cells until its concentration rises to a level, which prevents further fermentation, and so kills the yeast. At the start of the reaction there are many glucose molecules. These collide often with the yeast enzymes and react, using up the glucose. As the glucose is consumed, there are fewer and fewer glucose molecules to collide with the yeast, so the reaction slows down. Eventually there are almost no glucose molecules left and the reaction stops. The ethanol cannot be further broken down to yield additional energy.

Brewing and wine making are chemical processes in which ethanol is formed by the fermentation of the glucose obtained from fruit or grain.

C6H12O6 (aq) 2CH3CH2OH (aq) + 2CO2 (g)

(Glucose) (Ethanol) (Carbon dioxide)

The reaction is catalysed by Zymase, an enzyme present in yeast. Yeast is a microorganism, which obtains its energy from what is available during the reaction. All alcoholic drinks are products of this reaction.

Fermentation processes are commercial or experimental processes in which microorganisms are cultured in containers called FERMENTERS or BIOREACTORS. They play an important role in modern industry.

Fermenters are chambers in which microorganisms are cultured in a liquid or solid medium. The processes, which take place in bioreactors, are referred to as fermentation. The term fermentation originally applied only to anaerobic processes but is now used broadly to include all the processes whether aerobic or anaerobic. The contents of most fermenters are stirred during operation, but this is not always the case as with the new discontinued production of the single cell protein Pruteen by ICI, where air introduced at high velocity at the bottom of the vessel was used to achieve mixing. In traditional laboratory fermenters a small piece of magnet is acting as the mixer as it moves around vigorously so it can prevent any accumulation of solids at the bottom. All operations must be carried out under sterile conditions to avoid contamination of the culture. In addition, it must be possible to keep all inlets and outlets of fermenter sterile. The fermenter and the medium used are sterilised before use, either together or separately. (See diagram of fermenter figure 1-pg8)