Fermentation

Fermentation is an enzyme catalysed, metabolic process that produces chemical changes in

organic substrates. It is the primary means of making adenosine triphosphate (ATP) through the
degradation of carbohydrates anaerobically (in the absence of oxygen) in microorganisms. The
study of fermentation and its practical uses is named zymology and originated in 1856 when
French chemist Louis Pasteur demonstrated that fermentation was caused by yeast.
Fermentation occurs in the cytosol of cells.

E.g.: Mammalian muscle cells carry out fermentation during periods of intensive exercise when
oxygen is in short supply, resulting in the generation of lactic acid.

• A cell can recycle the coenzyme nicotinamide adenine dinucleotide (NAD+) from NADH
through the process of aerobic respiration. However, when oxygen amount is limited,
fermentation provides an alternate path to regenerate the electron carrier NAD+, which is
essential in glycolysis to release energy in the form of ATP. In the presence of O2, aerobic
respiration yields as many as 38 molecules of ATP per glucose molecule utilizing NADH
and pyruvate.
• In anaerobic environment, fermentation results in the production of a net of 2 ATP per
glucose molecule, why the process is rarely used when oxygen is available. However, in
certain types of bacteria and fungi that require an oxygen-free environment to live, known
as obligate anaerobes, in facultative anaerobes (organisms that make ATP by aerobic
respiration if oxygen is present, but is capable of switching to fermentation if oxygen is
absent) such as yeast, and also in muscle cells, fermentation occurs.

❖ Nicotinamide adenine dinucleotide (NAD) is a coenzyme that exists in two forms: an oxidized
and a reduced form, abbreviated as NAD+ and NADH, respectively.

Fig. 1: Nicotinamide adenine dinucleotide (NAD)

During fermentation, an organic electron acceptor, such as, pyruvate or acetaldehyde reacts with
NADH to form NAD+, and generates products like carbon dioxide, hydrogen, ethanol, or lactic acid.
Therefore, processes of fermentation are valuable to the food and beverage industries, with the
conversion of sugars into ethanol applied in making of alcoholic beverages, the release of CO2 by

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yeast used in the leavening of bread, and with the production of organic acids to preserve and
flavour vegetables and dairy products. Furthermore, in treatment of waste water fermentation
has become an effective tool. In activated sludge process, aerobic bacteria are employed to
ferment solid organic matters in waste water into carbon dioxide, water and mineral salts.

Fig. 2: Adenosine triphosphate (ATP)

Alcoholic Fermentation:

Ethanol fermentation or alcoholic fermentation, is a biological process that transforms sugars,

such as, glucose, fructose, and sucrose into cellular energy, and produces ethanol and carbon
dioxide as by-products. Because yeasts perform this conversion in the absence of oxygen,
alcoholic fermentation is considered as an anaerobic process.

Uses: In preparation of beers, wines, and other alcoholic beverages, ethanol fuel production, and
bakery.

The purpose of fermentation in yeast is the same as that in muscle and bacteria, to replenish the
supply of NAD+ for glycolysis, and the process occurs in two steps ―

i. The pyruvate produced via glycolysis is converted into acetaldehyde by the enzyme
pyruvate decarboxylase and releases CO2.
ii. Then, acetaldehyde is reduced by NADH to ethanol using alcohol dehydrogenase and
thereby regenerates NAD+ to be used in glycolysis.

Fig. 3: Ethanol Fermentation

Overall, the catabolic process of glycolysis furnishes two pyruvates from one molecule of glucose,
and the pyruvates are then converted into two molecules of carbon dioxide and two molecules of
ethanol in alcoholic fermentation.

C6H12O6 → 2C2H5OH + 2CO2

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Lactic acid Fermentation:

Lactic acid fermentation, an anaerobic reaction that converts the pyruvate to the lactate and
regenerates NAD+ in the process, allowing glycolysis to continue to make ATP in low-oxygen
conditions. Since, there is a limited supply of NAD+ available in any given cell, this electron
acceptor must be restored to maintain a steady production of ATP. To achieve this, NADH donates
its extra electrons to the pyruvate molecules, regenerating NAD+ and lactic acid is formed by the
reduction of pyruvate. Lactate dehydrogenase catalyzes the interconversion of pyruvate to lactate
with concomitant generation of NAD+ from NADH.

C3H3O3 (pyruvate) + NADH → C3H5O3 (lactate) + NAD+

Fig. 4: Lactic acid Fermentation

Sometimes even when oxygen is present and aerobic metabolism is happening in the
mitochondria, if pyruvate is building up faster than it can be metabolized, the fermentation will
happen anyway.

There are two types of lactic acid fermentation―

I. Homolactic fermentation, where, NADH reduces pyruvate directly to form lactate. This
process does not release gas, converts one molecule of glucose into two molecules of
lactate.
II. Heterolactic fermentation, some lactate is further metabolized and yields ethanol and
carbon dioxide in addition to lactic acid, in a process called the phosphoketolase pathway.

Lactic acid fermentation is primarily performed by certain types of bacteria and fungi. However,
this type of fermentation also occurs in muscle cells to produce ATP when the oxygen supply has
been depleted during strenuous exercise and aerobic respiration is not feasible.

Acetic acid Fermentation:

Hydrogen gas is produced in many types of fermentation (a process called fermentative hydrogen
production), as a way to recycle NAD+ from NADH. This is a two-step process where ultimately
glucose is transformed into acetate along with evolution of hydrogen. Vinegar is prepared
following this procedure.

Step-I: Formation of ethyl alcohol from sugar anaerobically using yeast.

Step-II: Oxidation of ethanol to acetic acid using acetobacter bacteria under aerobic condition.

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For example, Clostridium pasteurianum ferments glucose to butyrate, acetate, carbon dioxide, and
hydrogen gas. The reaction leading to acetate is:

C6H12O6 + 4H2O → 2CH3CO2− + 2HCO3− + 4H+ + 4H2

Aerobic Fermentation:

Fermentation does not necessarily have to be carried out in an anaerobic environment. For
instance, even in the presence of abundant oxygen, few strains of yeast greatly prefer
fermentation over oxidative phosphorylation, provided sugars are readily available for
consumption. This phenomenon where yeast forms ethanol (alcohol) in aerobic conditions at
high external glucose concentrations rather than producing biomass, is known as the Crabtree
effect. This is observed in most species of the Saccharomyces, Schizosaccharomyces,
Debaryomyces, Brettanomyces, Torulopsis, Nematospora, and Nadsonia genera of yeast. On
increasing the glucose concentrations glycolysis is accelerated, and that leads to a substantial
amount of ATP. This reduces the need of oxidative phosphorylation done by the TCA cycle and
hence, diminishes oxygen consumption. The phenomenon is believed to have evolved as a
competitive mechanism because of the antiseptic nature of ethanol.

In the presence of oxygen, the metabolic process through which cells metabolize sugars via
fermentation by repression of normal respiratory metabolism, is called aerobic fermentation. In
general, the process is shorter and more intense than anaerobic fermentation. Aerobic
fermentation does not produce ATP in high yield, instead, it allows proliferating cells to convert
nutrients like glucose and glutamine more efficiently into biomass by avoiding unnecessary
catabolic oxidation of such nutrients into CO2, preserving carbon-carbon bonds and promoting
anabolism.

Aerobic Respiration:

Aerobic respiration is the process by which organisms use oxygen to turn fuel, such as, fats and
carbohydrates, into chemical energy. The products of this process are carbon dioxide and water,

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and the energy transferred is utilized to cleave bonds in ADP to add a third phosphate group to
form ATP.
C6H12O6 + 6O2 → 6CO2 + 6H2O + 38 ATP

The complete process of aerobic respiration occurs in four different stages:

(i) Glycolysis:

It is the primary step of aerobic respiration, occurring within the cytosol of the cell under oxygen-
free conditions. During glycolysis, glucose undergoes a series of enzymatic transformations to get
converted into two molecules of pyruvate, alongside the production of two ATP.

C6H12O6 + 2ADP + 2Pi + 2NAD+ → 2Pyruvate + 2ATP + 2NADH + 2H+ + 2H2O

Fig. 5: Glycolysis pathway that converts glucose (C6H12O6) into pyruvate (CH3COCO2‒).

(ii) Pyruvate Oxidation / Oxidative Decarboxylation of Pyruvate:

Pyruvate oxidation is sometimes referred to as the transition reaction because the process links
glycolysis to the citric acid cycle. Each pyruvate from glycolysis is transferred to the
mitochondrial matrix via a protein known as pyruvate translocase. There, the pyruvate is
combined with Coenzyme A to release a carbon dioxide molecule and forms Acetyl-CoA, that,
further fuels the citric acid cycle.

Pyruvate + Coenzyme A + NAD+ → Acetyl CoA + CO2 + NADH

(iii) Citric acid cycle (CAC) / Tricarboxylic acid cycle (TCA cycle) / Krebs cycle:

The Krebs cycle, is a series of redox reactions utilized by all aerobic organisms to release stored
energy through the oxidation of Acetyl-CoA. These reactions take place in the matrix of the
mitochondria of eukaryotic cells and in the cytoplasm for prokaryotic cells.
The overall reaction is as follows:

Acetyl CoA + 3NAD+ + FAD + ADP + Pi → CO2 + 3NADH + FADH2 + ATP + H+ + Coenzyme A
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The reaction occurs twice for each molecule of glucose, as there are two pyruvates and hence two
molecules of Acetyl CoA generated to enter the citric acid cycle. Both NADH and FADH2, another
carrier of electrons for the electron transport chain, are formed in this process.

Fig. 6: Outline of the Krebs cycle

(iv) Oxidative Phosphorylation/Electron Transport-Linked Phosphorylation/Terminal

Oxidation:

Oxidative phosphorylation is the primary energy providing stage of aerobic respiration. It uses
the folded membranes within the cell’s mitochondria to produce huge amounts of ATP. Almost all
aerobic organisms carry out oxidative phosphorylation.

ADP + Pi + NADH + 1/2 O2 + H+ → ATP + NAD+ + H2O

• In this process, NADH and FADH2 donate the electrons they obtained from glucose during
the previous steps of cellular respiration to the electron transport chain in the inner
mitochondrial membrane.
• The electron transport chain consists of a number of protein complexes (Complex I‒IV, Q,
and cytochrome C) that are embedded in the mitochondrial membrane and they serve to
pass electrons from higher to lower energy levels.
• As the electrons move down the chain, energy is released and used to pump protons out
of the matrix. This store of energy is tapped when protons flow back across the membrane
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 and down the potential energy gradient, through an enzyme called ATP synthase in a
process called chemiosmosis.

• The ATP synthase uses the energy to transform ADP into ATP, in a phosphorylation
reaction. This process is why mitochondria are referred to as “the powerhouses of the
cell.” The mitochondria’s electron transport chain makes nearly 90% of all the ATP
produced by the cell from breaking down food.
• It is often stated that 38 ATP molecules can be made per oxidized glucose molecule during
cellular respiration (2 from glycolysis, 2 from the Krebs cycle, and ~ 34 from the electron
transport system). However, due to leaky membranes, energy expenses to transfer
pyruvate and ADP into the mitochondrial matrix, this maximum yield is never quite
reached.
• Although oxidative phosphorylation is a vital part of metabolism, it produces reactive
oxygen species such as superoxide and hydrogen peroxide, which lead to propagation of
free radicals, damaging cells and contributing to disease and, possibly, aging and
senescence.