Alcohol Fermentation

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Introduction

Alcohol fermentation is a complex biotechnological process in which sugars such as glucose, sucrose, and fructose are converted into energy molecules and produce ethanol, carbon dioxide, and metabolic by-products during this process. These products contribute to the sensorial properties and chemical composition of the fermented foodstuff.

Alcohol fermentation also called ethanol fermentation is processed by yeast or some other microorganisms like bacteria. They use this process to make ATP. Yeast typically functions in the presence of oxygen or aerobic condition but can also perform their function in absence of oxygen or under anaerobic conditions.

During alcohol fermentation, the anaerobic pathway is adopted by yeast in the absence of oxygen. This process has a profound importance for the manufacturing of alcoholic beverages such as beer and wine. The process takes place in yeast cytosol in the absence of oxygen.

Fermentation

Fermentation is a metabolic process taking place in the absence of oxygen. Many beneficial microorganisms create desirable changes in beverages and food through the process of fermentation. The resulting products have better flavor and more life as they are preserved. They also provide many health benefits.

Let us first understand the various types of fermentation before going into the discussion of alcohol fermentation.

Classification of fermentation

There are three types of fermentation based on the end products obtained from pyruvate.

#1. Lactic acid fermentation

In this fermentation different sugars are converted into lactic acid by yeast strains and bacteria without heat preparation. Anaerobic chemical reactions occur in which pyruvic acid uses NADH to form lactic acid and NAD+.

During strenuous activity, lactic acid fermentation also occurs in the muscle cells of the human body. Muscles expend energy in the form of ATP at a faster rate than the rate of oxygen being supplied to muscle cells. During strenuous exercise, oxygen supply to the muscles is depleted. They shift to lactic acid fermentation. Lactic acid accumulates in the muscles and causes muscle cramps.

Lactic acid bacteria play a vital role in producing and preserving wholesome foods such as yogurt and pickles etc. These food products are important in the digestion process.

#2. Alcoholic fermentation

In this fermentation, yeast breaks pyruvate molecules (glycolysis output of glucose metabolism) into alcohol and carbon dioxide. Alcoholic fermentation produces beer and wine.

#3. Acetic acid fermentation:

In this fermentation, grains and fruit starch and sugars fermented acetic acid and vinegar. Acetic acid fermentation produces apple cider vinegar, and wine vinegar, etc.

Effect of oxygen on fermentation

Fermentation does not require oxygen as it is an anaerobic process. If oxygen is present, pyruvate will be completely oxidized to water and carbon dioxide molecules by some yeast species spiration.

On the other hand, yeast species will produce ethanol only in an anaerobic environment by a process called Pasteur Effect.

Rate of Fermentation

During fermentation, the rate of ethanol production is maximal in the beginning.  However, the rate of fermentation progressively falls due to ethanol being accumulated in the surrounding environment. It has been found that the removal of this accumulated ethanol does not restore fermentation activity and a continuous metabolic decline begins.

Many causes have been investigated to explain this decline in fermentation activity. It is believed that it is due to the damage caused by the fermentation process to the yeast cells.

By-products of fermentation

Unharvested byproducts are produced in ethanol fermentation such as heat, food for livestock, carbon dioxide, methanol, fuels, water, alcohol, and fertilizer. The cereal unfermented residues can be used in the biogas production or as livestock feed.

Process of alcoholic fermentation

The basic formula for alcohol fermentation indicates that the process begins with glucose (sugar) and ends with carbon dioxide and ethyl alcohol. For a better understanding of the process, it is divided into a series of steps.

Major Steps:

The process of alcohol fermentation can be divided into two major parts:

  1. The first part involves the breaking down of glucose into 2 pyruvate molecules in a process called glycolysis.
  2. The second part is called fermentation in which 2 pyruvate molecules are converted into 2 molecules of carbon dioxide and 2 ethanol molecules, otherwise known as alcohol.

Chemical formula:

Alcohol fermentation can be represented by the chemical formula as follows:

C6H12O6 → 2 C2H5OH + 2 CO2

Fermentation and glycolysis reactions occur in the cytoplasm of the eukaryotic cell. Purpose:

The major purpose of alcohol fermentation is to produce energy in the form of ATP that is used during cellular activities, under anaerobic conditions. However, from the yeast point of you, ethanol and carbon dioxide are waste products.

Molecules Involved

The following are the important molecules involved in the process of alcohol fermentation.

Pyruvate:

Pyruvate or pyruvic acid is a carboxylic acid that is used to make ethanol. 2 pyruvate molecules are formed by breaking down one glucose molecule in the first step. Electron carriers like NADH are also involved in this process.

Electron Carriers:

These are the molecules responsible for capturing the electrons that are released during a chemical reaction.

NAD is the main electron carrier involved in these reactions. It captures the electrons during the first step of fermentation (glycolysis) and gets reduced to NADH. This reduced form provides electrons during the conversion of pyruvate to ethanol.

Reactions

The various reactions taking place during alcohol fermentation are as follows.

Glycolysis

The Glycolytic process can be summarized by the following equation:

C6H12O6 + 2 ADP + 2 Pi + 2 NAD+ 2 CH3COCOO + 2 ATP + 2 NADH + 2 H2O + 2 H+

  • 2 CH3COCOO (pyruvate molecules)
  • 2 NADH  (acceptor molecule)
  • 2 Pi  (inorganic phosphate)

The overall products of these reactions are two pyruvate molecules, two NADH and two molecules of ATP. The pyruvate molecules are further p[rocessed in the absence of oxygen to form ethanol (alcohol).

Pyruvate to Ethanol Conversion

This conversion takes place in two steps:

  • In the first step, the carboxyl group of pyruvate is removed and released in the form of CO2. The product of this reaction is acetaldehyde (a 2 carbon molecule)
  • In the second step, the acetaldehyde molecule is reduced. One molecule of  NADH passes its electrons to acetaldehyde, forming ethanol. The NAD molecule is regenerated during this process.

Reaction 1:

CH3COCOO+ H+ CH3CHO + CO2

This reaction is catalyzed by (pyruvate decarboxylase)

Reaction 2:

CH3CHO + NADH + H+ C2H5OH + NAD+

This reaction is catalyzed by (alcohol dehydrogenase)

Key:

  • CH3CHO (acetaldehyde)
  • CO2   (carbon dioxide)
  • C2H5OH ­=ethanol (Alcohol)

Enzymes

The two enzymes that are involved in alcohol fermentation are as follows.

Pyruvate decarboxylase:

It is an enzyme that catalyzes the decarboxylation of pyruvic acid to carbon dioxide and acetaldehyde. This enzyme plays an important role during the fermentation process in anaerobic conditions that occur in yeast (especially in genus Saccharomyces) to produce ethanol by fermentation.

Alcohol dehydrogenase:

This enzyme is responsible for converting acetaldehyde to ethanol during alcohol fermentation. During the fermentation process, this enzyme works in the reverse direction.

The same enzyme is responsible for converting alcohol to acetaldehyde in our liver, reducing NAD to NADH during alcohol metabolism.

Role of Microorganisms

The following microbes are involved in ethanol fermentation:

  1. Yeast
  2. Schizosaccharomyces
  3. Saccharomyces cerevisiae
  4. Zymomonas mobilis (a bacterium)

Yeast

Yeast cells are categorized as unicellular fungi having a diameter in the range of micrometers. The size of these organisms is very small as compared to most of the fungi. They can also vary in size and shape.

Yeast is used in several processes like making bread, wine, and alcohol fermentation.

In the process of making bread, yeast performs alcohol fermentation on flour and makes the bread fluffy. Carbon dioxide produced during this process creates gas bubbles in the bread and expands it like a foam. After the baking process is complete, only 2% ethanol remains in bread. So, the bread will have a trace amount of alcohol in it.

Alcoholic drinks contain a large amount of ethanol prepared by alcoholic fermentation performed by yeast. Ethanol is also toxic to yeast just like it is to humans. Ethanol tolerance of yeast depends upon the environmental conditions as well as different yeast strains.

Bacteria

By utilizing the process of alcohol fermentation, bacteria can break down organic compounds in an anaerobic environment to obtain energy. The fermentation process produces different products in bacteria. The end product depends on the strain of bacteria being used.

For example:

  • Salmonella and Escherichia produce ethanol, lactic acid, acetic acid.
  • Lactobacillus, Bacillus, and Streptococcus produce lactic acid.

Louis Pasteur studied fermenting bacteria in 1860. He demonstrated that fermenting bacteria could contaminate beer and wine during the manufacturing process. He also showed that the flavor of wine and beer can be preserved by heating them that will kill bacteria. This process of heating is called pasteurization.

The pasteurization process is now being used to kill bacteria present in milk and other products.

Inhibition

The fermentation process can be inhibited by an increased concentration of alcohol in the medium. It has already been mentioned that alcohol is toxic to yeast and bacteria. When the concentration of alcohol increases to a certain limit, yeast growth is immediately stopped. However, the fermentation activity is not completely inhibited.

The ethanol sensitivity of yeat depends upon the following factors:

  • Temperature
  • pH
  • Medium composition
  • Plasma membrane modifications
  • Action of some enzymes

All of these factors enhance intracellular ethanol concentration.

Ethanol sensitivity also depends on the balance between ethanol excretion and production. Increased levels of ethanol retention inside the cell cause inhibitory effects on growth rate and alcohol production.

Benefits of fermentation

The following are some of the major benefits od fermentation to humans.

  • Fermented food is composed of beneficial microorganisms and probiotics that help to maintain a healthy gut by extracting nutrients from food.
  • Fermentation also helps in the neutralization of anti-nutrients like phytic acid that present in nuts, seeds, legumes, and grains. It can cause mineral deficiency in the body if left unchecked.
  • Lactic acid fermentation by the gut bacteria helps to convert ammonia into ammonium ions. It saves the body from the damaging effects of ammonia on the brain. This fermentation process plays a key role in preventing hepatic encephalopathy.

Alcoholic beverages

The following alcoholic beverages are prepared by the process of alcohol fermentation in the industry.

  • Wine is produced by the fermentation of natural sugars present in the grapes.
  • Perry and cider are produced from natural sugar in pear and apple by a similar fermentation process.
  • Eaux de vie and brandy are produced by fruit fermented beverages distillation.
  • Mead is produced by natural sugar fermentation present in honey.
  • Whiskey, vodka, and beer are produced by grain starches fermentation that has been converted into sugar by amylase enzyme. 
  • Rums are produced by distillation and fermentation of sugarcane product molasses.

In all these processes, fermentation must take place in a vessel that allows carbon dioxide to escape but prevents air from coming inside. It will help reduce the contamination risk by unwanted bacteria or mold as carbon dioxide creates a risk of vessel rupture.

Summary

Fermentation is an anaerobic process in which pyruvic acid is converted into different products by the action of some microorganisms.

Three types of fermentation are;

  • Alcohol fermentation
  • Lactic acid fermentation
  • Acetic acid fermentation

Alcohol fermentation involves the conversion of pyruvate into ethanol and carbon dioxide. It is a two-step process;

  • In the first step, glucose is converted to pyruvate by glycolysis
  • In the second step, pyruvate is converted to ethanol and carbon dioxide by using a molecule of NADH

The process of fermentation is augmented by some types of yeast and bacteria.

The fermentation process is being used in the industry for making bread, alcohol, vinegar, and other products.

Fermented foods are rich in prebiotics and have several health benefits.

The fermentation by gut bacteria protects the body from the harmful effects of ammonia.

The fermentation process is used in the beverage industry to make a variety of alcoholic beverages.

References

  1.  Dashko, Sofia; Zhou, Nerve; Compagno, Concetta; Piškur, Jure (2014-09-01). “Why, when, and how did yeast evolve alcoholic fermentation?”. FEMS Yeast Research. 14 (6): 826–832. doi:10.1111/1567-1364.12161ISSN 1567-1364PMC 4262006PMID 24824836.
  2. Aren van Waarde; G. Van den Thillart; Maria Verhagen (1993). “Ethanol Formation and pH-Regulation in Fish”. Surviving Hypoxia. pp. 157−170. hdl:11370/3196a88e-a978-4293-8f6f-cd6876d8c428ISBN 978-0-8493-4226-4.
  3. Stryer, Lubert (1975). Biochemistry. W. H. Freeman and Company. ISBN 978-0-7167-0174-3.
  4. Raj SB, Ramaswamy S, Plapp BV. “Yeast alcohol dehydrogenase structure and catalysis”. Biochemistry. 53: 5791-803. doi:10.1021/bi5006442PMC 4165444PMID 25157460

Image sources

  1. https://commons.wikimedia.org/wiki/File:Lactic_acid_fermentation.png
  2. https://commons.wikimedia.org/wiki/File:Alcohol_fermentation_process.png