AN INTRODUCTION TO

FERMENTATION PROCESS
• Conversion of sugar to alcohol using yeast.

• Chemical conversion of carbohydrates into alcohols or acids.

• The process is often used to produce wine and beer

• Also employed in preservation to create lactic acid in sour foods such

as pickled cucumbers, kimchi and yogurt.

• The science of fermentation is known as zymology

 Biochemistry perspective

• Process that is important in anaerobic conditions when there is no

oxidative phosphorylation to maintain the production of ATP
(Adenosine triphosphate) by glycolysis.

• During fermentation pyruvate is metabolised to various different

compounds.

• Homolactic fermentation is the production of lactic acid from

pyruvate; alcoholic fermentation is the conversion of pyruvate into
ethanol and carbon dioxide

• Heterolactic fermentation is the production of lactic acid as well as

other acids and alcohols.
• Typical examples of fermentation products
are ethanol, lactic acid, and hydrogen.

• However, more exotic compounds can be

produced by fermentation, such as butyric
acid and acetone
 Cont.
• Fermentation is derived from the Latin Verb - Fevere - to boil

• Describing the appearance of the action of yeast on extracts of fruit or malted

grain.

• The boiling appearance is due to the production of carbon dioxide bubbles

caused by the anaerobic catabolism of the sugars present in the extracts.

• Fermentation have different meaning to biochemistry and industrial

microbiologists.

• Biochemical meaning relates to the generation of energy by the catabolism of

organic compounds

• Whereas meaning in industrial microbiology tends to be much broader.

• The catabolism of sugars is an oxidative process which results in the production of
reduced pyridine nucleotides which must be reoxidized for the process to continue.

• Under-aerobic conditions, reoxidation of reduced pyridine nucleotide occurs by electron

transfer, via the cytochrome system, with oxygen acting as the terminal electron
acceptor.

• Under anaerobic conditions, reduced pyridine nucleotide oxidation is coupled with the
reduction of an organic compound, which is often a subsequent product of the catabolic
pathway.

• In the case of the action of yeast on fruit or grain extracts, NADH is regenerated by the
reduction of pyruvic acid to ethanol.

• Different microbial taxa are capable of reducing pyruvate to a wide range of end
products.

• Thus, the term fermentation has been used in a strict biochemical sense to mean an
energy-generation process in which organic compounds act as both electron donors and
terminal electron acceptors.
• Industrial microbiologists have extended the term -
fermentation - to describe:

Any process for the production of product by the mass culture

of microorganism.

Brewing and the production of organic solvents may be

describes as fermentation in both sense of the word.
• NADH reduces pyruvic acid to form lactic acid.

• Another simple fermentation pathway involved a decarboxylation

reaction to form ethanol.

• This is representative fermentation products and the organism that

produce them.

• All of the organisms are bacteria except Saccharomyces, which is

yeast.

• Different organism will produced different end products.

 The range of fermentation
process.
There are five major groups of commercially important fermentations:

• Those that produce microbial cells (or biomass) as the product.

• Those that produce microbial enzymes

• Those that produce microbial metabolites.

• Those that produce recombinant products.

• Those that modify a compound which is added to the fermentation - the

transformation
 THE COMPONENT PARTS OF
A FERMENTATION PROCESS
(1) The formulation of media to be used in culturing the
process organism during the development of the inoculums
and in the production fermenter.

(2) The sterilization of the medium, fermenters and ancillary

equipment.

(3) The production of an active, pure culture in sufficient

quantity to inoculate the production vessel.
(4) The growth of the organism in the production fermenter
under optimum conditions for product formation.

(5) The extraction of the product and its purification.

(6) The disposal of effluents produced by the process.

Interrelationships between the six
component parts
Some important fermentation
products
 Enzymes
• Are biological catalysts!!
• Enzymes are applied to many fields of people’s
daily life
• Added or used to cause particular reaction
Advantages
• Natural, Nontoxic
• Catalyze specific reactions
• Active under mild conditions
• Active at low concentrations
• Can control rate of reaction
• Can be inactivated
Industrial enzymes and their use
 Classification
• Depending on the type of reaction catalyzed, enzymes are divided into six main classes:

• 1. Oxidoreductases. Transfer of electrons from one substrate molecule to another

(e.g., dehydrogenases, reductases, oxidases).

• 2. Transferases. Transfer of functional group from one substrate molecule to another

(e.g., glycosyl transferases, acetyl transferases, and aminotransferases).

• 3. Hydrolases. Transfer of functional group from substrate to water (e.g., glycoside

hydrolases, peptidases, esterases).

• 4. Lyases. Elimination of functional group from substrate with the formation of double
bonds. Thus, bonds are cleaved using a different principle than hydrolysis (e.g., pectate
lyases break glycosidic linkages by b-elimination).

• 5. Isomerases. Transfer of groups from one position to another in the same molecule
(e.g., glucose isomerase).

• 6. Ligases. Addition of function group to substrate usually coupled with ATP hydrolysis
(e.g., glycine–tRNA ligase).
 introduction
• key roles in numerous biotechnology products and processes that are commonly
encountered in the form of food and beverages, cleaning supplies, clothing, paper
products, transportation fuels, pharmaceuticals, and monitoring devices

• the most frequently used enzymes in biotechnology are hydrolases, which catalyze the
breakdown of molecules.

• Enzymes can display regio- and stereospecificity, properties that have been exploited
for asymmetric synthesis and racemic resolution.

• Chiral selectivity is employed to prepare enantiomerically pure pharmaceuticals,

agrochemicals, chemical feedstocks, and food additives.

• industrial enzyme market has expanded at a rate of about 10% annually, microbial
enzymes have largely replaced the traditional plant and animal enzymes, and most of
them are produced recombinantly.

• DNA technology has been used to modify substrate specificity and improve stability properties of
enzymes for increasing yields of enzyme-catalyzed reactions.
• used in metabolic engineering of cellular metabolism to increase
yields of fermentation products

• Enzymes are environmentally friendly; they work under moderate

conditions of temperature, pH, and pressure, their catalyzed
reactions rarely form wasteful side products, and the proteins
themselves are biodegradable and generally pose no threat to the
environment.
 Industrial Uses

1. Starch conversions
• Production of glucose syrup
• Production of high fructose corn syrup
• Production of high maltose conversion syrups
• Production of cyclodextrins
• Production of ethanol
2. Lignocellulosic Biomass conversions
• Cellulose conversion
• Hemicellulose conversion
• Lignin conversion
3. Enzymes in the Production of Functional Oligosaccharides and Other
Neutraceuticals
4. Enzymes in the Modification of Fats and Oils
5. Enzymes in the Animal Feed Industry
6. Enzymes in the Pulp and Paper Industry
7. Enzymes in the Fruit Juice Processing Industry
8. Enzymes in the Meat and Fish Processing Industry
9. Enzymes in the Dairy Industry
10. Enzymes in Detergents
11. Enzymes in the Leather Industry
12. Enzymes in the Production of Bulk and Fine Chemicals
13. Analytical Applications of Enzymes
14. Enzyme-Replacement Therapy
 Enzyme Technology
• An emerging field of biotechnology that utilizes enzymes for several
commercial applications.
 Pharmaceuticals
 Fine chemicals
 Foods and beverages
 Cosmetics
 Textile
 Paper
 Leather
 Animal Feed
 Goals of Enzyme-Tehnology
Integration of biology, chemistry and
engineering
To develop of novel alternative routes,
processes and services
To design innovative products and
processes that are not only competitive but
also meet criteria of sustainability
 Advantages of enzymes

 Efficient catalysis
 Selective (Enantio-and regio-selectivity)
 Specific in action (Less byproducts)
 Environmentally benign catalysis
 Non Toxic, Non-flammable,
 Completely biodegradable,
 Sustainable,
 Requires mild reaction conditions
 Recycled and reused when immobilized
 Enzyme Technology
Enzyme technology is concerned with the application of enzymes
as tools of industry, agriculture and medicine

Enzymes are biological catalysts that fulfil their role

by binding specific substrates at their active sites

This specificity is one property of enzymes that

makes them useful for industrial applications

The value of using enzymes over inorganic catalysts in the

technological field is their efficiency, selectivity and specificity

Enzymes are able to operate at room temperature, atmospheric

pressure and within normal pH ranges (around 7)
– all of which create energy savings for industry

Enzymes possess specifically shaped active sites for reacting with

one specific substrate thereby generating pure products
free from unwanted by-products

Enzymes are biodegradable and, unlike many inorganic

catalysts, cause less damage to the environment
 Products of Enzyme Technology
Micro-organisms have been
used for thousands of years
for making products such as
wine, beer, vinegar, soy sauce,
bread and cheese

The micro-organisms
(such as yeast) are really used as
a source of enzymes during the
manufacture of these products
of biotechnology

Many industrial processes now make use of pure sources of enzymes, i.e.
the enzymes have been ISOLATED from the micro-organisms before use
 Microbial enzymes are ISOLATED from a variety of sources
and these include bacteria, fungi and yeast cells

Electron micrograph of bacteria (Bacillus)

Micro-organisms produce enzymes that function inside their cells

(intracellular enzymes) and they may also produce enzymes that are
secreted and function outside the cells (extracellular enzymes)
 Large Scale Production of Enzymes
The large scale production of enzymes involves culturing micro-organisms
in chambers called FERMENTERS or BIOREACTORS

Micro-organisms are suitable for use in the large scale production of

enzymes in fermenters because:

• They have rapid growth rates and are able to produce larger numbers of
enzyme molecules per body mass than many other organisms

• Micro-organisms can be genetically engineered to improve the strain and

enhance yields

• Micro-organisms are found in a wide variety of different habitats such that

their enzymes are able to function across a range of temperatures and pH

• Micro-organisms have simple growth requirements and these can be

precisely controlled within the fermenter

• Micro-organisms can utilise waste products such as agricultural waste

as substrates
The Biotechnological Process of Enzyme Production
SCREENING – choosing an INDUSTRIAL SCALE
appropriate micro-organism FERMENTATION
for the desired enzyme

MODIFICATION – possible
application of genetic
engineering to improve
the microbial strain

LABORATORY SCALE PILOT

– to determine the optimum
conditions for growth of the
Micro-organism

PILOT PLANT – small scale

fermenter to clarify optimum
operating conditions
 Commercial Enzyme Production - An Example
PRODUCTION OF PECTINASE

Pectin is an insoluble substance found in the cell walls of plants

In the drinks industry, juice extracted from fruits

appears cloudy due to the presence of pectin

Pectinase is an enzyme that is used in the industry to break down the pectin

The effect of pectinase is to clarify the fruit juice and to make it flow more freely

Pectinase is obtained from the fungus Aspergillus niger

Aspergillus niger produces pectinase as an extracellular enzyme

 PRODUCTION OF PECTINASE

Filtration or centrifugation to obtain

a cell-free system containing
pectinase in solution

Evaporate to concentrate
the enzyme

Aspergillus niger is grown in

a fermenter with a source of Precipitate the pectinase
nitrogen, with sucrose as the out of the solution and
carbon source and the substrate filter the solid
pectin to stimulate pectinase
production by the fungus

Pure, powdered Dry and purify the crude

pectinase pectinase
 Enzymes in Biotechnology
Enzymes are used in industrial processes and as analytical
reagents in medicine

Thermostability and an ability to

withstand extremes of pH are
essential properties for enzymes used
in many industrial processes

Immobilisation of enzymes is an important technique used

in industry as it enables economical operation of a process
and protection of enzymes during their use

Because of their sensitivity and specificity, enzymes are

used as analytical reagents in systems such as the detection
of glucose in human blood and urine
Thermophilic bacteria inhabit hot springs
Thermophilic bacteria inhabit volcanic vents
 Immobilised Enzymes
The costs associated with the use of enzymes for industrial
purposes can also be reduced by immobilising the enzymes

Enzymes for industrial processes are more valuable when

they are able to act in an insolubilised state rather than in solution

Enzymes are immobilised by binding them to, or trapping

them in a solid support

Various methods for immobilising enzymes are available

Methods for Immobilising Enzymes

Enzymes are held on to a solid

support (matrix) by weak forces
such as hydrogen bonding

Enzymes are trapped within

the structure of a solid polymer
(usually in the form of beads)
– the enzyme is trapped rather
than bound
 Enzymes are covalently bonded
to a matrix such as cellulose
or collagen

Another more expensive method involves

enzymes which are both covalently bonded
to, and cross-linked within, a matrix

Cross-linking and covalent bonding may

cause some enzymes to lose their catalytic
activity especially if the active site is involved
in forming the linkages
Advantages of Immobilising Enzymes
Compared with free enzymes in solution, immobilised enzymes
have a number of advantages for use in industrial processes

The stability of many enzymes is increased when they are in an

immobilised state; they are less susceptible to changes in
environmental conditions such as temperature and pH fluctuations

Immobilised enzymes can be recovered and re-used,

reducing overall costs

The products of the reaction are not contaminated with enzyme

eliminating the need to undertake costly separation of
the enzyme from the product

Immobilising enzymes allows for continuous production

of a substance with greater automation
 Enzyme Immobilisation and Thermostable Enzymes in
The Production of High Fructose Syrup
This industrial process involves the conversion of cheap corn starch into a
high fructose syrup for use as a sweetener in confectionary and drinks
Starch paste is incubated with the
Starch Paste thermostable enzyme alpha amylase
at 90oC for a couple of hours

Alpha amylase catalyses the hydrolysis of the starch

into short glucose chains called dextrins

Dextrins
The temperature is raised to 140oC to denature the
(short chains amylase and then lowered to around 55oC before
of glucose adding the fungal enzyme amyloglucosidase

molecules)
Amyloglucosidase catalyses the hydrolysis of
dextrins into glucose molecules

Glucose
 The final stage involves
the conversion of glucose
syrup into the much sweeter
fructose syrup using the
enzyme glucose isomerase

Glucose isomerase is immobilised

in rigid granules and packed into
a column

Glucose syrup is poured into

the top of the column and is
hydrolysed as it contacts the
immobilised enzyme

Fructose syrup emerges

from the end of the column
free from contamination
with enzyme
 Enzymes as Analytical Agents
The sensitivity and specificity of enzymes makes them useful
tools in medicine for the detection and measurement of chemicals
in fluids such as blood and urine

Because of their specificity, enzymes will bind to only one substrate

– they can therefore be used for the identification
of a specific substance in a biological sample

Because of their sensitivity, enzymes are able to detect the

presence of specific molecules even when they are
present at very low concentrations

The enzyme glucose oxidase is used in an immobilised form

for the detection of glucose in biological fluids
Glucose Measurement using 'Clinistix'
This method relies upon the specificity of the enzyme glucose oxidase,
allowing glucose to be detected in the presence of other sugars
N.B. Benedict's test is not specific for glucose
as it gives a positive reaction with ALL reducing sugars
This test uses a plastic strip (clinistix) for the
detection of glucose in the urine of diabetics
When the clinistix is dipped into a urine sample
(containing glucose), the glucose oxidase catalyses
the conversion of glucose to hydrogen peroxide:
Glucose + O2 gluconic acid + hydrogen peroxide (H2O2)

In the presence of the enzyme peroxidase, the

chromagen dye is oxidised by the hydrogen peroxide
to produce a colour change on the fibre pad
DH2 (chromagen dye) + H2O2 2H2O + D
The amount of coloured compound (D) produced is a direct
measure of the amount of glucose in the sample
At the tip of the clinistix is a cellulose fibre pad on to which glucose
oxidase, peroxidase and a chromagen dye are immobilised
Glucose Measurement using 'Clinistix'

The colour of the pad on the clinistix is compared with

a colour chart to determine the amount of glucose
present in the sample

No
glucose Increasing amounts of glucose
 Biosensors

Biosensors are
electronic monitoring
devices that make
use of an enzyme’s
specificity and the
technique of enzyme
immobilisation
 Biosensors
Biosensors are electronic monitoring devices that make use of an
enzyme’s specificity and the technique of enzyme immobilisation

Amplifier Read-out

Transducer

Immobilised The enzyme The electrical

enzymes bind reaction brings signal is amplified
with specific about a change and gives a
molecules that is converted read-out on a
even when they into an electrical small display
are present signal by a screen
in very low transducer
concentrations
 Biosensors
A biosensor has been developed for detecting
glucose in the blood of diabetics

Amplifier

Transducer

Glucose Glucose Glucose oxidase The current generated is

molecules oxidase oxidises any glucose
in the blood present in the blood to
release electrons – these
are detected by the
transducer and converted
into an electrical current
proportional to the amount
of glucose present in the
sample and this is displayed
as a digital read-out
 Enzymes Application
Detergents
• Bacterial proteinases are still the most important detergent enzymes.
Some products have been genetically engineered to be more stable in
the hostile environment of washing machines with
• several different chemicals present. Lipid degrading enzymes, lipase,
were used in powder and liquid detergents to decompose fats.
• Amylases are used in detergents to remove starch based stains.
• Cellulases have been part of detergents since early 90s.An enzyme
complex capable of degrading crystalline cellulose to glucose.
 Cont..
Drink industry.
The use of chymosin in cheese making to
coagulate milk protein.
• Beta-galactosidase (lactase) Splits milk-
sugar lactose into glucose and galactose is
also used in milk industry.
This process is used for milk products that
are consumed by lactose intolerant
consumers.
 Enzyme applications:

Drink industry
 Fruit juice manufacturing:
 Pectins are substances in fruit lamella and cell walls which contain
also hemicelluloses and cellulose.
 Pectinase, xylanase and cellulase improve the liberation of the juice
from the pulp.
 Pectinases and amylases are used in juice clarification.
 Wine production: Enzymes are widely used to obtain a better
extraction of the necessary components and thus improving the yield.
 conti
Textile industry
• The use of enzymes in textile industry is one of the
most rapidly growing fields in industrial enzymology.
• Starch has for a long time been used as a protective glue of fibers in
weaving of fabrics. This is called sizing.
• Enzymes are used to remove the starch in a process called desizing.
• Amylases are used in this process since they do not harm the textile
fibers
• Laccase – a polyphenol oxidase from fungi is used to degrade lignin the aromatic
polymer found in all plant materials .
• Cellulases remove cellulose microfibrils, which are formed during washing.
 Cont…
Animal Feed
The net effect of enzyme usage in feed has been increased animal weight.
The first commercial success was addition of betaglucanase into barley
based feed diets.
• Barley contains beta-glucan, which causes high viscosity in the
chicken gut.

• In addition to poultry, enzymes are used in pig feeds and turkey feeds.
Baking
• Alpha-amylases have been most widely studied in connection with
improved bread quality and increased shelf life.
• Both fungal and bacterial amylases are used in bread making and excess
may lead to sticky dough
Pulp and paper
• xylanases in pulp bleaching for paper
Medicines
• As drugs for treatment of diseases
• In diagnosis
• In preparation of medicines
• To remove toxic substances
• Making lactose free products for patients suffering
from lactose intolerance (lactase breaks lactose to glucose and
galactose
Theraputic Enzymes
• Asparaginase For leukaemia
• Collagenase For Skin ulcers
• Glutaminase For Leukaemia
• Hyaluronidase For Heart attack
• Lysozyme For Infection
• Ribonuclease As Antiviral
• 6-Lactamase For Penicillin allergy
• Streptokinase For dissolving Blood clots
• Trypsin For Inflammation
• Uricase For Gout
• Urokinase For dissolving Blood clots
Leather
• Leather industry uses proteolytic and lipolytic enzymes in
leather processing.
• Enzymes are used to remove animal skin, hair,and any
unwanted parts. Lipases are used in this phase or in bating
phase to specifically remove grease.
Biofuel: Bioethanol is a biofuel used in cars.
It can be produced from starchy plant materials.enzymes are used to convert starch
to bioethanol. At present, corn is a widely used source of starch.
• Other plants including wheat, bamboo, or other grasses can be used as sources
of starch for bioethanol production
Enzymes for Genetic Engineering
• DNA polymerases
• •DNA ligases
• Dnases
• Rnases
• RNA polymerases
• Reverse transcriptase
 Reading assignment
• Industrial drug products
• Biocatalysts
• Properties of enzymes
• Enzyme immobilization Techniques
 Q?
• Thank you