Fermentation

By– Dr. Ekta Verma

PG DEPARTMENT OF BOTANY ,
M.U. BODHGAYA
 Learning Objectives
Students should able to understand

Fermentation Technology

Fermentation Media

Bioreactor Design
 Fermentation

 Fermentation is the chemical transformation of organic

substances into simpler compounds by the action of enzymes,
complex organic catalysts, which are produced by
microorganisms such as molds, yeasts, or bacteria.

 Fermentation technology is the use of microorganisms to

produce food, pharmaceuticals and alcoholic beverages
on a large scale industrial basis.

 The science of fermentation is called “zymology”.

 Principle of Fermentation Process
 The basic principle involved in the industrial fermentation
technology is that organisms are grown under suitable
conditions, by providing raw materials, meeting all the
necessary requirements such as carbon, nitrogen, salts, trace
elements and vitamins.

 The end products formed as a result of their metabolism during

their life span are released into the media, which are extracted
for use by human being and that have a high commercial
value.
 Major fermentation Products
Group Product Organism
Industrial Ethanol Saccharomyces cerevisiae
chemicals Lactic acid Lactobacillus bulgaricus

Enzymes -amylase Bacillus subtilis

Proteases Bacillus species
Lipases Saccharomyces lipolytica
Antibiotics Penicillin Penicillium chrysogenum
Streptomycin Streptomyces griseus
Chlorampenicol Streptomyces venezuelae
Vitamins Riboflavin Ashbya gossypi
Vitamin B12 Pseudomonas dentrificians
Types Based On Respiration
1.Aerobic Fermentation:

Aerobic fermentation or aerobic glycolysis is a metabolic

process by which cells metabolize sugars via fermentation in the
presence of oxygen and occurs through the repression of normal
respiratory metabolism.

Aerobic fermentation means that oxygen is present. Wine, beer and

acetic acid vinegar (such as apple cider vinegar), need oxygen in the
“primary” or first stage of fermentation.

When creating acetic vinegar, for example, exposing the surface of

the vinegar to as much oxygen as possible, creates a healthy, flavorful
vinegar with the correct pH.
Aerobic
Fermentation
2.Anaerobic Fermentation:
Anaerobic fermentation is a method cells use to extract energy from carbohydrates
when oxygen or other electron acceptors are not available in the surrounding
environment.
This differentiates it from anaerobic respiration, which doesn‟t use oxygen but does
use electron-accepting molecules that come from outside of the cell.
The process can follow glycolysis as the next step in the breakdown of glucose and
other sugars to produce molecules of adenosine triphosphate (ATP) that create an
energy source for the cell.
Depending upon the end product formed,
fermentation can be categorized into various types
Homo fermentation: When only one type of product is
formed

Hetero fermentation: When more than one products are

formed
 1. Lactic Acid Fermentation
• Lactic acid is formed from pyruvate produced in glycolysis. NAD+ is
generated from NADH.
• Enzyme lactate dehydrogenase catalyses this reaction.
• Lactobacillus bacteria prepare curd from milk by 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 CO2 is released. In the next step,
ethanol is formed from acetaldehyde.

NAD+ is also formed from NADH which is reused 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 oxidised 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 of 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.

Sugar is first oxidised to pyruvate by the process of glycolysis and then pyruvate is
further oxidised to form acetyl-CoA by the oxidoreductase enzyme system with the
production of CO2 and H2. 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.
 Fermenter ?
“A specially designed vessel in which large quantity fermentation media
is added with fermentation microorganisms which provides best possible
environment control and process control for the biosynthesis of
fermentation products.”
Design of Fermenter
 FERMENTOR

 Closed vessels

 Used for fermentation

 Production of large scale process

 Fermenter is a closed vessels now it is called as bioreactor

 Fermenter is a old process used for cultivate the

microorganisms

 Fermenter used for cultivate Prokaryotic cells (fungi bacteria)

 Bioreactors used for cultivate eukaryotic cells

(mammalian and insects)
 1. Vessel

Function of a fermenter is to carryout process under appropriate

aseptic and pre-defined environmental conditions.

A fermentation vessel is designed in such a way that it requires

minimal labour operation and maintenance.

There are mainly two types of vessels base on the type

fermentation process :

Small scale fermenter (Laboratory scale fermenter) These

are made up of glass

 Large scale fermenter (Industrial scale fermenter)

As, stainless steel is the most satisfactory material, it is used to
manufacture vessels of high volume
 2.Impeller (Agitator)

 Mounted to a shaft through abearing in

the lid

Driven by an external power source

The agitator is required to achieve a number of mixing

objective.

 Bulk fluid and gas-phase mixing

 Air dispersion,
 Oxygen transfer,
 Heat transfer,
 Suspension of solid particles and maintain a uniform environment
throughout the vessel contents.
 3. Sparger
 A device that introduce air into medium
 Has a pipe with minute holes (1/64 - 1/32 inch or large)

 Hole – allows air under pressure to escape into medium

Depending on volume of medium in the fermentation vessel,

different types of spargers are installed in the fermenter.
1.Porous
2.Orifice
3.Nozzle
 4.Baffles
Baffles are metal strips roughly one-tenth of vessel diameter and attached
radially to the wall of bioreactor

 Generally four to eight baffles are incorporated.

They are normally incorporated into agitated vessels of all sizes to prevent
vortex and to improve aeration efficiency
 5.Temperature controlling (heating and cooling)
devices

Mechanical agitation and exothermic microbial metabolic activity

generates heat during the fermentation process.

Endothermic microbial metabolic activity lower down the temperature

of the fermentation medium

To maintain this temperature, heat is to be either added to or removed

from the system

The cooling system is used to remove excess heat from the system

Internal heating coils are used for providing heat (Note: In case of lab
scale process, the fermenter is placed in thermostatically controlled
bath)
 6.pH control: -

Certain microorganisms grow in particular pH only. In fermentation it

is very essential to control

 pH in order to grow the desired microorganisms for product

formation.

 pH control sensors are used in fermenter for periodically checking of

pH.
 7.Feed ports

Feed ports are the tubes ( for Lab scale fermenter) and
pipelines (for large scale fermenter) connected to the
nutrient reservoir

These tubes or pipelines are used to add nutrients and

acid/alkali in the fermenter before and during the
fermentation process

 They are heat sterilized in situ and /or ex situ with stem

It is advisable to sterilize after connection has been done

and before any additions are made
Monitoring and controlling parts of fermenter
are:
 Upstream Process
The pre-fermentation stage
 Isolation
 Improvement
 Producing of microorganisms
Screening method; isolate microbes to produce decreed products Two
methods;
primary screening checking the quality of microbes done in agar
plate.
Secondary screening checking the quntative of microbes done in
liquid media
 Upstream Process
• Microbes isolated from natural sources thus is improved to get product
• strains by using
 Recombination
 Mutations
 Cell fusion
 Gene cloning
• Media formulation : growth medium must have essential nutrients for
microbial growth for successful fermentation process
• Two kind media :
 Inoculum media : enrich the culture
 Production media: contain carbon and nitrogen
• Raw materials : corn molasses, cellulose, corn, streep liquor soybean,
sugar, beet molasses, malt extract etc.,
 Upstream Process
Upstream processing includes formulation of the fermentation
medium, sterilization of air, fermentation medium and the Fermenter,
inoculum preparation and inoculation of the medium.

The fermentation medium should contain an energy source, a carbon

source, a nitrogen source and micronutrients required for the growth
of the microorganism along with water and oxygen, if necessary.

A medium which is used for a large scale fermentation, in order to

ensure the sustainability of the operation, should have the following
characteristics;

1. It should be cheap and easily available

2. It should maximize the growth of the microorganism, productivity
and the rate of formation of the desired product
3. It should minimize the formation of undesired products
 Upstream Process
Usually, waste products from other industrial processes, such as molasses,
lignocelluloses wastes, cheese whey and corn steep liquor, after modifying
with the incorporation of additional nutrients, are used as the substrate for
many industrial fermentations.

Sterilisation is essential for preventing the contamination with any

undesired microorganisms.

Air is sterilised by membrane filtration while the medium is usually

heat sterilised.
Any nutrient component which is heat labile is filter-sterilised
and later added to the sterilised medium.
The fermenter may be sterilised together with the medium or
separately.
Inoculum build up is the preparation of the seed culture in amounts sufficient to be used in
the large Fermenter vessel.

This involves growing the microorganisms obtained from the pure stock culture in several consecutive
Fermenter.

This process cuts down the time required for the growth of microorganisms in the Fermenter,
thereby increasing the rate of productivity.

Then the seed culture obtained through this process is used to inoculate the fermentation
medium.
 Micro-organisms
 Micro-organisms used for fermentation process grow on or in growth medium
which satisfies the nutritional needs of microbes.
 Complete analysis is needed to be done to establish the most favourable
medium for the growth of the microbe used for fermentation.
 Formulating medium at lab scale can be done by adding main ingredients
like water, carbon source, nitrogen source, minerals and other supplements in
pure form and in required quantities is very easy which supports the growth
of the microbe whereas, the same may not support the satisfactory growth
of the same organism at industrial level
 Following criteria need to be satisfied for the material to be treated as
medium at industrial level.
 It should give maximum yield of product.
 It should give minimum yield of undesired product.
 It should be consistently available throughout the year.
 It should be cheap.
Fermentation Process
Types Of Fermentation
1.Batch fermentation:
 Nutrients are added in the fermentation for the single time only and
growth continues until the particular nutrients are exhausted
 In the batch process when the microorganism is added into a
medium which supports its growth, the culture passes through
number of stages known as „growth curve‟

A typical growth curve consists of following stages

a) Lag phase
b) Acceleration phase
c) Log or exponential phase
d) Deceleration phase
e) Stationary phase
f) Death phase
(a) Lag phase:
Immediately after inoculation, there is no increase in the numbers of the microbial cells
for some time and this period is called lag phase. In this is phase the organisms adjust
to the new environment in which it is inoculated into.
(b) Acceleration phase:
The period when the cells just start increasing in numbers is known as acceleration
phase.
(c) Log phase:
This is the time period when the cell numbers steadily increase.
(d) Deceleration phase:
The duration when the steady growth declines.
(e) Stationary phase:
The period where there is no change in the microbial cell number is the stationary
phase. This phase is attained due to depletion of carbon source or accumulation of the
end products.
(f) Death phase:
The period in which the cell numbers decrease steadily is the death phase.
This is due to death of the cells because of cessation of metabolic activity and depletion
of energy resources.
Depending upon the product required the different phases of the cell growth are
maintained. For microbial mass the log phase is preferred. For production of secondary
metabolites i.e. antibiotics, the stationary phase is preferred.
 Growth kinetics of batch culture

The number of living cells (population of growth rate

dN/dt)varies with time in a batch system as shown below:
2.Feb-batch fermentation:
 In this type of fermentation, freshly
prepared culture media is added at regular
intervals without removing the culture
fluid.
 This increases the volume of the
fermentation culture.
 This type of fermentation is used form
production of proteins from recombinant
microorganisms.
 The total amount of the biomass in the
vessel increases but biomass concentration
is maintained constant
3.Continuous fermentation:
 The growth rate and physiological
conditions of microorganisms can be
maintained by using a process of
continuous culture (chemostat )
 In this the products are removed
continuously along with the cells and
the same is replenished with the cell
girth and addition of fresh culture
media.
 This results in a steady or constant
volume of the contents of the
fermenter.
 type of fermentation is used for the
production of single cell protein (S.S.P),
antibiotics and organic solvents.
 Advantages and disadvantages of batch and
continuous operations

CONTINUOUS SYSTEMS
BATCH SYSTEMS
 degeneration of
 Easy To Operate And Control
biocatalyst
 Genetic Stability Of Organism
 higher contamination risk is a
Could Be Controlled If It Is
disadvantage
Genetically Engineered Biocatalyst.
 efficient, higher
 Lower Contamination Risk
productivity
 Non-productive Down Time Is A
 product is obtained with
Disadvantage
uniform characteristics; quality
 Batch To Batch Variability Is Problem
of the product is almost same
 Accumulation Of Inhibitory
from time to time
Products Is Problem
 no accumulation of
 Down stream Process
when fermentation is over, the desired
product is recovered from the growth
medium.

Then the product is Extraction Purification

and Packed of a biotechnological product
from fermentation is refered to as DSP or
product recovery or downstream processing.

The end products include Antibiotics, Amino

acid, Vitamins,Organic acid, Industrial
enzyme, vaccines etc.

It is complex and important as fermentation

process
 CELL -DISRUPTION OR DISINTEGRATION
Mechanical methods
 Shear forces in solid matter and solution.
Non mechanical methods:
Lysis
Physical – freezing and thawing : now high
osmotic pressure, shock
 Chemical – surface active agents, solvents,
antibiotics etc.,
 Enzymatic – lysozyme
Drying
Freeze-drying, Air drying, Pressure release,
Drying with solvents
 SOLID-LIQUID SEPARATION OR CLARIFICATION
 Primary operation
 Separation of whole cells
 Removal of cell debris
 Collection of protein precipitate
 Collection of inclusion bodies etc.,
Solid-liquid separation (clarification)
 Coagulation
 Colloids into small flocs using simple electrolytes
 Flocculation
 Agglomeration of these small flocs into larger settle-able particles using
polyelectrolytes.
 Flotation
 Enrichment of microorganisms
 Filtration
 The separation of suspended particles from liquid
 Centrifugation
 Gravitational force used for separate the particles
 CONCENTRATION METHODS

• The purity or concentration of metabolite

 Evaporation –steam as heat source

Extraction – the cell mass and more or less clear solution obtained

Adsorption – special polymer resins (chemical) used for the isolation

of hydrophilic metabolites that cannot be extract with organic solvents.

Filtration – separation of biomolecules and particles [pore size]

 Precipitation – removal of product from the solvent

 Dialysis – semi permeable membrane
 PURIFICATION

• Purify relatively low concentration of metabolic

products
• The chromatography technique are used
• Purification is a main process in fermentation
• Desired product purification is important
• Many techniques are used for the purification
Thank You