Fermentation

Definition
Fermentation is an enzyme catalyzed, metabolic process whereby organisms convert
starch or sugar to alcohol or an acid anaerobically releasing energy. The science of
fermentation is called “zymology”.

Buchner was a scientist who identified that the yeast cells can carry fermentation even
after they are dead. It was due to the presence of an enzyme that carries fermentation.
Later, he named this enzyme zymase. Hence, fermentation is also known as Zymosis.

Process of Fermentation
Fermentation is an anaerobic biochemical process. In fermentation, the first process is
the same as cellular respiration, which is the formation of pyruvic acid by glycolysis
where net 2 ATP molecules are synthesised. In the next step, pyruvate is reduced to
lactic acid, ethanol or other products. Here NAD+ is formed which is re-utilized back in
the glycolysis process.
Types of Fermentation
 Homo fermentation: only one type of product formation

 Hetero fermentation: more than one product formed

On the basis of the end product formed, fermentation can be categorized as follows:

1. Lactic Acid Fermentation

Lactic acid is formed from pyruvate produced in glycolysis. NAD+ is generated by
NADH. Enzyme lactate dehydrogenase catalyses this reaction. Lactobacillus bacteria
prepare curd from milk via this type of fermentation. During intense exercise when
oxygen supply is inadequate, muscles derive energy by producing lactic acid, which
gets accumulated in the cells causing fatigue.
2. Alcohol Fermentation
This is used in the industrial production of wine, beer, biofuel, etc. The end product is
alcohol and CO2. Pyruvic acid breaks down into acetaldehyde and CO 2 is released. In
the next step, ethanol is formed from acetaldehyde. NAD+ is also formed from NADH,
utilized in glycolysis. Yeast and some bacteria carry out this type of fermentation.
Enzyme pyruvic acid decarboxylase and alcohol dehydrogenase catalyse these
reactions.

3. Acetic Acid Fermentation

Vinegar is produced by this process. This is a two-step process.

The first step is the formation of ethyl alcohol from sugar anaerobically using yeast.
In the second step, ethyl alcohol is further oxidized to form acetic acid using acetobacter
bacteria. Microbial oxidation of alcohol to acid is an aerobic process.

4. Butyric Acid Fermentation

This type of fermentation is characteristic of obligate anaerobic bacteria of genus
clostridium. This occurs in retting jute fibre, rancid butter, tobacco processing and
tanning of leather. Butyric acid is produced in the human colon as a product of dietary
fibre fermentation. It is an important source of energy for colorectal epithelium. Sugar is
first oxidized to pyruvate by the process of glycolysis and then pyruvate is further
oxidized to form acetyl-CoA by the oxidoreductase enzyme system with the production
of H2 and CO2. Acetyl-CoA is further reduced to form butyric acid. This type of
fermentation leads to a relatively higher yield of energy. 3 molecules of ATP are formed.

Advantages of Fermentation

Fermentation is suitable for all kinds of environments. It is one of the oldest metabolic
processes which is common to prokaryotes and eukaryotes. Fermentation is widely
used in various industries.
Using suitable microorganisms and specified conditions different kinds of fermentation
products are formed namely:-

 Wine

 Beer

 Biofuels

 Yoghurt

 Pickles

 Bread

 Sour foods containing lactic acid

 Certain antibiotics and vitamins

Fermentation can make food nutritious, digestible and flavoured. There are many
benefits of consuming fermented food.

 It improves digestion and helps to maintain intestinal bacteria

 It has an anti-cancer effect.

 Improves immune system

 Reduces lactose intolerance

Other than the food industry, there are many other areas where the fermentation
process is used. Methane is produced by fermentation in sewage treatment plants and
freshwater sediments.
 Fermenters
Fermenter, also known as bioreactors, are sterilised and enclosed vessels that are used
for the growth of microorganisms under optimal conditions. The microorganisms can be
grown in large quantities to produce metabolites for commercial uses. Fermenter are
equipped with special components for heating, mixing, and aeration. Its volume can be
as big as 500,000 litres for an industrial scale, or as small as 1 litre for laboratory uses.

Different Types of Fermenter

 Continuous Stirred-Tank Fermenter
The continuous stirred-tank reactor (CSTR) is composed of a vessel with pipes, pumps,
valves, agitator, motor, shaft, and impeller(s). The shaft is situated at the bottom of the
tank, and the number of impellers depends on the size of the bioreactor.

In this type of Fermenter, a structure called sparger is found that keeps adding air to the
culture medium. It is a ring-like structure with many holes. The sparger, along with the
impellers, distribute gas in the entire vessel. The impellers break down the bubbles into
smaller ones that are homogeneously distributed in the bioreactor.

 Airlift Fermenter
The airlift bioreactors contain a baffle or a draft tube in the middle through which air is
pumped into the vessel. There are two types of airlift Fermenter:
  Internal loop airlift bioreactor: It has a single central draft tube that provides
inner circulation channels.

 External loop airlift bioreactor: It contains external loops that separate the
liquids from flowing into independent channels.

 Packed Bed Fermenter

In a packed bed Fermenter, a hollow tube or pipe is packed with a biocatalyst. The bed
is immobile in nature. The culture medium flows through the biocatalyst, which produces
the metabolites continuously in the broth. These bioreactors are easy to operate but are
often blocked due to poor oxygen circulation.

 Fluidized Bed Fermenter

In this type of reactor, a solid granular bed that is usually made up of a biocatalyst is
present. The fluid, that is, liquid or gas, is passed through the solid bed at high speeds,
such that the suspended solid behaves like a fluid. This type of Fermenter is used for
microbial flocs, immobilized cells, and enzymes.

 Membrane Fermenter
Membrane bioreactors work in conjugation with ultrafiltration and microfiltration. This
type of Fermenter is used for the biological treatment of wastewater. There are two
types of membrane bioreactors:

 Submerged membrane bioreactor: In this type of Fermenter, the membrane is

found inside the vessel submerged in the wastewater.

 Side-stream membrane bioreactor: In this type of Fermenter, the membrane is

found outside the reactor and filtration by the membrane is an additional step in
the whole process.

 Bubble Column Fermenter

A bubble column Fermenter is equipped with a cylindrical column that is filled with
liquid, and gas is inserted into it from the bottom. It is vertically arranged, such that the
introduction of gas from the bottom creates a turbulent stream and allows optimum gas
exchange. The sparger mixes the contents of the vessel. The liquid flows either in a
parallel direction or in a counter-current direction.
 Modes of Fermentation
There are four modes of fermentation: batch, fed-batch, continuous, and open
fermentation.

Batch Fermentation
In a batch fermentation process, all the ingredients are combined at once, and then
undergo reaction without any further input from outside. This type of fermentation goes
through three phases – lag phase, where the microbial cells adapt to the environment,
exponential phase, where the cells grow in size and then comes a stationary phase,
where all the nutrients have been consumed, and the cells die. It is used in industries
for the production of wines and bread.

 A batch fermentation system is a closed system.

 At time t = 0, the sterilized nutrient solution in the fermenter is inoculated with

microorganisms, and incubation is allowed to proceed at a suitable temperature
and gaseous environment for a suitable period.

 In the course of the entire fermentation, nothing is added, except oxygen (in the
case of aerobic microorganisms), an antifoam agent, acid, or base to control pH.

 The composition of the medium, the biomass concentration, and the metabolite
concentration generally change constantly as a result of the metabolism of the
cells.

Continuous Fermentation
Continuous fermentation is a process where ingredients are added throughout the
process as per the need, and the products are removed as they are formed. The
exponential phase is usually prolonged in this type because of the continuous addition
of nutrients. The two types of continuous culture are: open continuous culture and
closed continuous culture.

The advantages of continuous fermentation are the following:- it gives more product,
doesn’t require cleaning of the fermenter after every product extraction, and is very
efficient. Continuous fermentation has the disadvantage of getting contaminated very
easily.

Principle of Continuous Culture

 A continuous flow system consists of a reactor into which reactants are pumped
at a steady rate and from which products are emitted.
  The factors governing their operation are:

 how material passes through the reactor (which depends upon its design);

 the kinetics of the reaction taking place.

 In continuous culture, growth-limiting nutrients can be maintained at steady-state

concentrations, which permits microorganisms to grow at submaximal rates.

 In a steady state, the cellular growth rate and environmental conditions, like the
concentrations of metabolites, stay constant

 Moreover, in continuous culture, parameters such as pH, oxygen tension, the

concentration of excretion products, and population densities can easily be
monitored and controlled.

Fed – Batch Fermentation

In simple words, fed-batch culture is a modification to batch fermentation. In fed-batch
cultivation, nutrients are added aseptically; it is a semi-open system, and the volume of
liquid culture in the bioreactor increases as the culture is systematically added. A fed-
batch culture is more productive, yields better with controlled sequential additions of
nutrients, enables higher cell densities, and prolongs product synthesis.

The main advantages of fed-batch over batch culture are:

1. Long-term synthesis of products,

2. By increasing the number of cells and thereby increasing the amount of product,
which is proportional to the concentration of the biomass, greater efficiencies can
be achieved in the process,

3. Increasing yield and productivity with controlled sequential additions of nutrients,

4. It allows the bioreactor to be used for production for non-profitable periods when
it would normally be prepared for the next batch.

In a fed-batch process, the broth does not generally need to be removed during the
entire fermentation process, but the amount of limiting nutrients added determines the
rate of reaction.
The graph shows the principle of a substrate limited fed-batch cultivation with an initial
batch phase. After consumption of the initial substrate, a continuous and constant feed
of the substrate is started.

Fed-batch culture has the following advantages:

1. Catabolite repression and Crabtree effects can be managed by limiting the

substrate concentration.

2. The high cell density of the cells could be achieved

3. It is possible to increase the production of non-growth-related metabolites.

4. When necessary, the broth viscosity can be reduced.

5. It allows the replacement of water loss by evaporation.

Principle of Fed – Batch Culture

 The initial process is similar to batch culture; all of the substrates are added at
the beginning of the fermentation.

 The culture broth is harvested usually only at the end of the operational period,
either fully or partially (the remainder serving as the inoculum for the next
repeated run)

 In the fed-batch culture, a substrate is added in increments as the fermentation

progresses.
  These substrates are added in small concentrations during the production phase.

 In the course of operation, there are one or more feed streams but no effluent.

 To control the feeding process, many indirect parameters that are related to the
metabolism of a substrate are measured since it is not possible to measure the
substrate concentration directly and continuously.

 Therefore, the culture volume increases during the course of operation until the
volume is full.

Batch Fermentation Continuous Fermentation

Type of Fermentation

It is a closed batch. It is an open batch.

Nutrient Addition

All the nutrients are added at once in Nutrients are added throughout the batch.
the beginning.

Product Extraction

The product is extracted at the end of The product is continuously removed while the
the batch. batch is operating.

Turnover Rate

The turnover rate (formation of The turnover rate is high.

product) is low.
 Type of Product

It is used to extract secondary It is used to extract primary metabolites such as

metabolites such as antibiotics. amino acids and proteins.

Growth Conditions inside the Fermenter

The growth conditions can be changed The growth conditions have to be maintained
by altering pH and dissolved oxygen. throughout the operation of the batch.

Size of the Fermenter

Large fermenters are used. Small-size fermenters are used.

Chances of Contamination

It has less chance of contamination. It has a high chance of contamination.

Microbial Phases

The microbes go through three phases The microbes go through a very short lag
– lag, exponential and stationary. phase and then maintain themselves at the
exponential phase.

Nutrient Consumption

Nutrients are consumed at a slower Nutrients are consumed rapidly.

rate.
 Media Requirements for Fermentation
To obtain a good product from fermentation, the medium in which the microorganisms
are grown must be supplied with enough energy sources and nutrients. Several factors
must be kept in mind before designing or choosing the growth medium for fermentation.
To obtain primary metabolites such as citric acid and ethanol, the media should be rich
in components that support good growth. Similarly, for secondary metabolites such as
alkaloids and antibiotics, the substrate requirement for product formation must be kept
in mind.

While doing fermentation on a small scale, such as in laboratories, pure graded

chemicals are used that are expensive. However, in large scale industrial fermentation,
cheaper and unrefined chemicals are used. Therefore, the choice of media for
fermentation is a crucial step that requires a lot of thought processes.

Media Components
The fermentation media can either be liquid, known as broth, or it can be a solid-state
fermentation. The media should satisfy all the nutritional requirements of the
microorganism and should also obtain the target molecule. A typical media requires a
carbon source, a nitrogen source, salts, water and micronutrients. Let us look at them
one by one.

1. Carbon Source

Typically sugars and carbohydrates are used as carbon sources, but alcohols may also
be used in making products such as vinegar. For laboratory uses, refined and pure
carbon sources such as glucose, sucrose and glycerol are used that give a uniform
product. However, in the case of industrial fermentation, inexpensive sources such as
whey, malt extract, molasses, corn steep liquor or sugar cane juice are used.

2. Nitrogen Source

The nitrogen source for microorganisms may be used in the form of organic or inorganic
compounds. Inorganic sources include ammonium salts or the free form of ammonia.
Inexpensive nitrogen sources are used for bulk production, such as tryptone, peptone,
soy meal, corn steep liquor and yeast extract.

3. Growth Factors

Trace salts and growth factors are important components in the fermentation media.
Yeast extract is a good source of vitamins and macronutrients. Trace elements such as
copper, zinc, iron, cobalt, molybdenum, manganese are all usually found in the
unrefined nitrogen sources but may need to be added when using pure sources.

4. Miscellaneous

The process of fermentation sometimes produces a large amount of gas that forms a
layer of foam and hinders the process. To get rid of this problem, antifoaming agents
are also added to the fermentation medium. To stabilise pH of the media, mineral
buffering salts such as phosphates and carbonates are also added. The addition of
chelating agents may also be required when high concentrations of metals are present
in the media.

Optimization of the Fermentation Medium

Optimization is the process of developing a fermentation medium that gives the best
quality and quantity of the target product. One of the most common methods to optimise
a media is to optimize one factor at a time (OFAT). In this method, only one component
of the media is changed while others are kept constant, and the results are observed.
Similar observations can be done for all the components in a group of experiments, and
the best optimized media can be prepared by analyzing the results.
 Alcoholic Fermentation
Alcoholic fermentation is the anaerobic transformation of fructose and glucose (sugars)
into ethanol and carbon dioxide. The process is conducted by yeasts and a few bacteria
(Zymomonas mobilis).

The process of alcoholic fermentation regenerates the NAD+ taken up at the time of
glycolysis and provides yeast with an energy gain of 2 ATP molecules through the
metabolized hexose.

When grape juice is fermented, Saccharomyces cerevisiae (Species of yeast), primarily

directs the pyruvate for the production of ethanol to regenerate NAD + consumed by the
process of glycolysis. This phenomenon is referred to as alcoholic fermentation.

Steps of Alcoholic Fermentation

The process of alcoholic fermentation can broadly be divided into two main parts –

 Glycolysis – glucose is broken down into 2 pyruvate molecules

 Fermentation – pyruvate molecules are converted into 2 molecules of carbon

dioxide and 2 ethanol molecules

Where does Alcoholic Fermentation Occur?

The process of alcoholic fermentation occurs within the cytoplasm.

Agent of Alcoholic Fermentation

It is a well-known fact that the most widely used agent for the process of alcoholic
fermentation is S. cerevisiae. This yeast is commonly used as a microbial starter in
different fermentation industries.

At the time of alcoholic fermentation of fruits and juices, the S. cerevisiae becomes a
dominant species as a result of their strong selective environment, given the low pH and
high ethanol and sugar concentrations with anaerobic conditions.

Equation for Alcoholic Fermentation

The reaction occurring in alcoholic fermentation can be summarized as follows –

The process of alcoholic fermentation converts one mole of glucose into 2 moles of
ethanol and 2 moles of carbon dioxide, thus producing 2 moles of ATP in the process.
The process of alcoholic fermentation is a complex process. As the reaction proceeds,
various biochemical, physicochemical, and chemical processes occur, hence turning
grape juice into vine.

Products of Alcoholic Fermentation

Initially, pyruvate is decarboxylated into ethanal by pyruvate decarboxylase. The
enzyme requires cofactors in the form of magnesium and thiamine pyrophosphate.
Hence, alcohol dehydrogenase reduces ethanal to ethanol, thus recycling NADH to
NAD+.

In Saccharomyces cerevisiae, there are three isoenzymes of alcohol dehydrogenase,

however, isoenzyme I is mainly involved in the conversion of ethanal to ethanol. Zinc is
used as a cofactor by Alcohol dehydrogenase.

The final products of alcoholic fermentation are ethanol and carbon dioxide. Both are
transported to the exterior of the cell by the process of simple diffusion. Apart from
ethanol, some other compounds are generated all through the process of alcoholic
fermentation, such as esters, higher alcohols, succinic acid, glycerol, 2,3-butanediol,
diacetyl, acetoin.

In this process, the NADH donates their electrons to pyruvate’s derivative, thus
producing ethanol.

Conversion from pyruvate into ethanol occurs in two steps –

 Production of acetaldehyde – carboxyl group eliminated from pyruvate and

released as carbon dioxide, hence producing acetaldehyde, a two-carbon
molecule

 NADH passes their electrons to acetaldehyde, thus regenerating NAD + thus

forms ethanol

The alcoholic fermentation by yeasts generates ethanol seen in alcoholic beverages.

More Sub-products of Alcoholic Fermentation

As aforementioned, in addition to mainly producing ethanol and glycerol, some other
substances too are produced as a result of fermentation, which is the result of their
complex process.

Some of the sub-products of alcoholic fermentation are –

 Acetic acid
  Diacetyl, acetoin and 2,3-butanediol

 Ethanal/acetaldehyde

 Esters

 Higher alcohols

 Succinic acid

Why Fermentation Reduces Towards the End?

At times the process of alcoholic fermentation slows down as it reaches the end. The
yeasts dramatically decrease their consumption of sugar and fermentation can cease
much before the fermentable sugars are completely metabolised. In this event, there
could occur two scenarios –

 Wine is incomplete, something must be done to complete it

 High risk of bacterial spoilage

Some causes of sluggish fermentation are –

 Extremities of temperature

 Complete anaerobiosis

 Presence of medium-chain fatty acids

 Extreme levels of sugar concentrations

 Presence of antifungal substances

 Antagonism between microorganisms

 Nutrient deficiencies

These causes, usually a synergistic combination of a few of these causes, can restrict
the correct development of alcoholic fermentation. If the process of fermentation stops
finally, the yeast should be reinoculated

Hence, alcoholic fermentation is a complex process involving the transformation of

sugars into ethanol and other subproducts.
Glycolysis & Alcoholic Fermentation
When there is a dearth of oxygen supply during prolonged and heavy exercises, the
muscles derive their energy from glycolysis. Under anaerobic conditions, yeasts gain
their energy from a similar process referred to as alcoholic fermentation.

In glycolysis, there is a chemical breakdown of glucose into lactic acid, hence making
energy available for cellular activity in the form of ATP. Except for the final phase,
alcoholic fermentation is just like the process of glycolysis. The pyruvic acid in alcoholic
fermentation is disintegrated into carbon dioxide and ethanol. The lactic acid gained
from the process of glycolysis leaves a sense of fatigueness, while the products of
alcoholic fermentation have been used in brewing and baking for long.

Glycolysis and alcoholic fermentation both are anaerobic processes that start with
glucose. This process of glycolysis necessitates 11 enzymes that transform glucose to
lactic acid. For the initial 10 steps, the process of alcoholic fermentation follows the
same enzymatic route. The lactate dehydrogenase, the last enzyme of glycolysis, is
substituted by two enzymes in the process of alcoholic fermentation. Alcoholic
dehydrogenase and Pyruvate decarboxylase, both these enzymes convert pyruvic acid
into carbon dioxide and ethanol in the process of alcoholic fermentation.

Hence, neither the process of alcoholic fermentation nor glycolysis recognises any gain
in energy (ATP) till the tenth enzymatic disintegration.
 Lactic Acid Fermentation
Some bacteria produce lactic acid from pyruvic acid. This metabolic process is also
seen in the muscle cells of animals. There can be inadequate oxygen supply for
respiration in muscle cells during physical exercise. Thus, in anaerobic conditions,
pyruvic acid is reduced to lactic acid. This happens in the presence of the enzyme
lactate dehydrogenase. Also, a reducing agent like NADH+H + is reoxidised to NAD+.

It is a fermentation process where much of the energy is not released. Here, less than
seven per cent of the glucose energy is released. Also, not all of it is trapped as high
energy bonds of ATP.

Steps Involved in Lactic Acid Fermentation

 The glucose or 6-carbon molecule is broken down into Glyceraldehyde 3-

phosphate, and then to 3-Phosphoglyceric acid.

 During this, NAD+ is converted into NADH+H+.

 The 3-Phosphoglyceric acid forms Phosphoenol pyruvic acid, which later forms
the Pyruvic acid.

 Net 2 ATP molecules are formed in this process (glycolysis).

 This Pyruvic acid is reduced to Lactic acid with the help of reducing agent
NADH+H+, which reoxidizes to NAD+.
  This process produces two lactate/lactic acid molecules from two
pyruvate/pyruvic acid molecules. This reaction happens in the presence of the
enzyme lactate dehydrogenase.

Applications
Lactic acid fermentation is the best method used for food preservation. Lactobacillus is
the most commercially used bacteria for this process. They are used in the production
of pickles, sour beer, fermented fish, yoghurt, etc.

What is homolactic & heterolactic fermentation?

When one glucose molecule is converted into two molecules of lactate or lactic acid, it is
termed homolactic fermentation. Here, only lactic acid is the final byproduct. In
heterolactic fermentation, other byproducts like CO2 and ethanol are formed along with
lactic acid.
 Vinegar Production
Vinegar is an acetic acid solution that is mild, aqueous and flavorful. In a two-step
process, sugar is first transformed into ethanol by yeast, and then Acetobacter oxidizes
the ethanol to produce acetic acid. This acetic acid is later used to make vinegar.

Vinegar production is an oxidative fermentation process in which Acetobacter bacteria

uses airborne oxygen (O2) and diluted ethanol solutions to produce water and acetic
acid.

Composition of Vinegar
Vinegar consists of an aqueous solution of acetic acid, water and a trace amount of
other chemicals. The concentration of acetic acid might vary. By volume, vinegar
contains approximately 5-8% of acetic acid.

Step 1 – Alcohol Fermentation

There are several different ingredients that can be used to manufacture vinegar, such
as grapes, apples, oats, malted barley, sugar, beer, rice and other ingredients.
However, vinegar was probably originally created from wine as a commercial
commodity.

Sugar is present in fruit juice or other liquid, and yeast enzymes use this sugar to
produce alcohol (ethanol) and carbon dioxide (CO2) gas.

(Yeast)

Sugar (Fructose, Glucose) → Ethanol + 2 CO2

Step 2 – Acetic Acid Fermentation

The two well-known acetic acid bacteria are –

Gluconobacter and Acetobacter. Acetobacter bacteria are comparatively better acid
producers. By the action of the bacteria Acetobacter, the alcohol produced in the
previous process (ethanol) reacts with atmospheric oxygen to produce acetic acid and
water.

Usually, the different flavors and aromas of vinegar are caused by the presence of
organic acids and esters that are derived from the fruit or other source material.

(Acetobacter)

Ethanol → Acetic acid + H2O

Different Methods of Vinegar Production
Vinegar manufacturing techniques can range from conventional ones using surface
culture and wooden casks (Orleans Process) to submerged fermentation. Many foods
contain vinegar as a key ingredient. The necessity for huge quantities of vinegar
requires the use of industrial fermentation systems that can reliably produce controlled
volumes of vinegar. To enhance the industrial production of vinegar, numerous
technological innovations have been made. In general, these enhancements speed up
the process whereby ethanol is converted into acetic acid when acetic acid bacteria are
present.

Vinegar Production Flow Chart

Fruit extract + Yeast

↓
Alcohol Fermentation

↓
Acetic Acid Fermentation

↓
Ripening

↓
Filtration

↓
Pasteurization

↓
Bottling of Vinegar

What is the pasteurization of vinegar?

Vinegar should be heated before pouring into a sterilized bottle. The vinegar is placed in
a hot water bath and the temperature is maintained between 140 to 160 ℉ for 30
minutes. Then the final product must be put into a glass or plastic bottle as the
penultimate step.
What are the different types of vinegar?

White vinegar, balsamic vinegar, apple cider vinegar, rice vinegar, coconut vinegar,
cane vinegar, distilled vinegar and fruit vinegar are some different types of vinegar.

What are some uses of vinegar?

Vinegar can be used to bake, cook, clean, and also used in weed control. It may also
aid in the process of weight loss and lowers cholesterol and blood sugar. Moderate
consumption is safe, but high doses of vinegar can be harmful.