CHAPTER-THREE

Microbial Fermentation Technology

04/18/2024 Alena B. and Woinshet M. 1

 3.1. Fermentation
• Is a process in which microorganisms, such as fungi and bacteria, break down organic
substances, such as sugars, anaerobically and it produces substances such as alcohol and
organic acids.

• Fermentation also produces chemical energy, such as ATP, that is important for biological
processes.

• It is used to make products such as wine, beer and bread.

• There are many types of fermentation that produce different end products from pyruvate
or its derivatives.

• The two most commonly used fermentations by humans are ethanol and lactic
acid fermentation
04/18/2024 Alena B. and Woinshet M. 2
3.1.1. Types of Fermentation
 Fermentation has been classified into SSF and SmF mainly based on the type of substrate
used during fermentation.

1. Solid-State Fermentation (SSF)/New Method

 SSF utilizes solid substrates, like bran, bagasse, and paper pulp.
The main advantage of using these substrates is that nutrient-rich waste materials can be easily recycled as
substrates.

In SSF the substrates are utilized very slowly and steadily, so the same substrate can be used for long
fermentation periods. Hence, this technique supports controlled release of nutrients.

SSF is best suited for fermentation techniques involving fungi and microorganisms that require less
moisture content.
04/18/2024 Alena B. and Woinshet M. 3
2. Submerged Fermentation (SmF)/Traditional Method

 SmF utilizes free flowing liquid substrates, such as molasses and broths.

 Some common substrates used in submerged fermentation are soluble sugars, molasses, liquid media, fruit and
vegetable juices, and sewage/waste water.

 The substrates are utilized quite rapidly and need to be constantly replaced/supplemented with nutrients.

 This fermentation technique is best suited for microorganisms such as bacteria that require high
moisture content.

 In SmF technique purification of products is easier.

 SmF is primarily used in the extraction of secondary metabolites that need to be used in liquid form.

 The bioactive compounds are secreted into the fermentation broth. Various bioactive compounds such as

antibiotics, pigments, enzymes, hypercholestrolemic agents, antioxidants, antihypertensive agents,

4
Fermentation is the primary technique for the production of various enzymes.

 Both fungi and bacteria yield an invaluable array of enzymes when fermented on appropriate
substrates.

Both solid-state and submerged fermentation are used for enzyme production.

SmF is usually implemented in case of bacterial enzyme production, due to the requirement of
higher water potential.

SSF is preferred when enzymes have to be extracted from fungi, which require lesser water
potential.

• SSF is preferred over SmF as it is cost effective, eco-friendly and it delivers high yield of enzyme.
04/18/2024 Alena B. and Woinshet M. 5
SSF process utilizes agricultural and industrial wastes as solid substrate.
3.2. Bioreactors

 Bioreactors are cylindrical vessels with hemispherical top and bottom, made of
stainless steel and glass ranging in size from some liter to cube meters.

 It is an apparatus for growing organisms such as bacteria, viruses, or yeast that

are used in the production of d/t products.

 Under optimum conditions of gas flow rates, temperature, pH, dissolved

oxygen level, and agitation speed, the microorganisms or cells will reproduce
at04/18/2024
a rapid rate. Alena B. and Woinshet M. 6
 The sizes of the bioreactor can vary over several orders of magnitudes.

 The microbial cell (few mm³), shake flask (100-1000 ml), laboratory fermenter (1 – 50
L), pilot scale (0.3 – 10m³) to plant scale (2 – 500 m³) are all examples of bioreactors.

 The design and mode of operation of a fermenter mainly depends on the production
organism, the optimal operating condition, product value and scale of production.

 The design also takes into consideration the capital investment and running cost.

04/18/2024 Alena B. and Woinshet M. 7

• Large volume/large quantity/ and low value/lower priced products/ like alcoholic
beverages need simple fermenter and do not need aseptic condition.

• High value/higher-priced products and low volume /small amount/products require more
elaborate system of operation and aseptic condition. High value products Such as bioethanol,
biodiesel, biohydrogen, these cannot be produced at a very large scale from raw biomass,
they are produced in low amounts.
 Bioreactors differ from conventional chemical reactors in that they support and control
biological entities.

• Bioreactor must be designed to provide a higher degree of control over process upsets and
contaminations, b/cs organisms are more sensitive and less stable than Chemicals.

04/18/2024 Alena B. and Woinshet M. 8

The general requirements of the bioreactor are as follows:

1. The vessel should be robust and strong enough to withstand the various treatments.

2. The vessel should be able to be sterilized and to maintain stringent aseptic conditions over

long periods of the actual fermentation process.

3. The vessel should be equipped with stirrers or mixers to ensure mass transfer processes occur

efficiently.

4. It should have sensors to monitor and control the fermentation process.

04/18/2024 Alena B. and Woinshet M. 9
5. It should be provided with inoculation point for aseptic transfer in inoculum.

6. Sampling valve for withdrawing a sample for different tests.

7. Baffles should be provided in case of stirred fermenter to prevent vertex formation.

8. It should be provided with facility for intermittent addition of an antifoam agent.

9. In case of aerobic submerged fermentation, the tank should be equipped with the aerating

device.

10. Provision for controlling temperature and PH.

04/18/2024 Alena B. and Woinshet M. 10

3.3. Fermentor
 Fermentors may be grouped in several ways: shape or configuration, whether aerated or anaerobic and
whether they are batch or continuous.

1. Submerged fermentation system stirred tank reactors

Stirred tank reactor is the choice for many fermentation processes.

Stirred tank reactors have the following functions: homogenization, suspension of solids, dispersion of
gas-liquid mixtures, aeration of liquid and heat exchange.

The Stirred tank reactor is provided with a baffle and a rotating stirrer is attached either at the top or at the
bottom of the bioreactor.

The industry prefers stirred tanks because in case of contamination or any other substandard product
formation the loss is minimal.

 The Stirred tank reactors offer excellent mixing and reasonably good mass transfer rates.
11
04/18/2024 Alena B. and Woinshet M. 12
SmF methods:
Batch fermentation
Fed-batch fermentation
Continuous fermentation
Semi-continuous fermentation

A. Batch fermentation
 Considered to be a closed system.
 Is a process in w/c all substrate and nutrients are added at zero time or soon after
inoculation takes place.
 The sterilized media in the fermenter is inoculated with the microorganism.
 Incubation is allowed under the optimum conditions (aeration, agitation, temperature).
 no extra feeding is used from beginning to end of the process/extra nutrients are not
supplied till the end of the process.
 During entire fermentation nothing is added except air, antifoam and acid/base.
Alena B. and Woinshet M. 13
B. Fed-batch fermentation/modified version of batch fermentation

It is semi-open system. It yields a better result than batch fermentation.

After consumption of early substrate continuous and constant nutrition is added.

Continue adding the nutrients (feeding) in a small doses during the fermentation.

The method in controlling nutrients feeding process is by measuring methods.

Nutrients are added systematically in stages to maximize cell growth for an extended duration.

The main advantage of fed-batch fermentation is the elimination of catabolite repression (feed-

back 04/18/2024
inhibition). Alena B. and Woinshet M. 14
C. Continuous fermentation
 It is an open system.
 Continuously sterile nutrient is added and the converted nutrient is taken out from the
fermentor. Nutrients are added, and products are removed continuously.
 Continuous processes are designed to produce products constantly without interruption.
 Fresh medium is continuously added to the fermentor, while used medium and cells are
harvested at the same time.
 In continuous process cell loss as a result of outflow must be balanced by growth of the
microorganism.

04/18/2024 Alena B. and Woinshet M. 15

Important factors for continuous fermentation:

The system must be stable for at least 500 hours.

Maintaining sterile conditions for all period of fermentation time.

The composition of nutrients must be constant all the time.

Maintaining the strain stability for constant high production yield.

D. Semi-continuous fermentation

 Complex organic compounds are slowly decomposed into simple organic

compounds by enzymes present in micro-organisms
 Semi-continuous fermentations, in which a fraction of a fermentation is replaced with fresh
media at regular intervals.
04/18/2024 Alena B. and Woinshet M. 16
2. Airlift bioreactor

 Airlift fermenter (ALF) is generally classified as pneumatic reactors without any mechanical stirring
arrangements for mixing.

 It is a specialized type of bioreactor used for cultivating cells or microorganisms in a controlled

environment.

 Airlift bioreactors are tower reactors for large-scale aerobic cultures where the mixing of the
culture broth is done by the inserted gas via an airlift pump.

 The turbulence caused by the fluid flow ensures adequate mixing of the liquid. It is ideally suited for
aerobic cultures.

 The draft tube is provided in the central section of the reactor.

 The introduction of the fluid (air/liquid) causes upward motion and results in circulatory flow in the
Alena B. and Woinshet M.
entire04/18/2024
reactor. 17
 The air/liquid velocities will be low and hence the energy consumption is also
low.

 ALFs can be used for both free and immobilized cells.

 It utilizes gas sparging to create circulation within the reactor, eliminating the
need for mechanical stirring.

 It is ideally suited for aerobic cultures.

 ALFs can be used for both free and immobilized cells.

04/18/2024 Alena B. and Woinshet M. 19
3. Fluidized bed reactor

Fluidized bed bioreactors (FBB) have received increased attention in the recent years.

 In this type of reactor, a fluid is passed through a solid granular material at high speed to
suspend the solid and cause it to behave as though it were a fluid.

Most of the FBBs developed for biological systems involving cells as biocatalysts are three
phase systems (solid, liquid & gas).

Usually fluidization is obtained either by external liquid re-circulation or by gas fed to the
reactor.

In the case of immobilized enzymes the usual situation is of two-phase systems involving solid
and liquid but the use of aerobic biocatalyst necessitate introduction of gas (air) as the third
phase.
04/18/2024 Alena B. and Woinshet M. 20
04/18/2024 Alena B. and Woinshet M. 21
Fig.5 Fluidized bed bioreactors
04/18/2024 Alena B. and Woinshet M. 22
4. Bubble column reactor

A bubble column reactor is basically a cylindrical vessel with a gas distributor at the
bottom.

It is a simplest type of tower fermenter consisting of a tube which is air sparged at the
base. It consists of vertically-arranged cylindrical column filled with liquid.

It is an elongated non-mechanically stirred fermenter with an aspect ratio of 6:1.

The gas flow rate is introduced at the bottom of the column through a gas distributor. The gas

is supplied in the form of bubbles to liquid phase or a liquid-solid suspension . This type of
fermenter was used for citric acid production.

This type of fermenter was used for citric acid production.

04/18/2024 Alena B. and Woinshet M. 23
04/18/2024 Alena B. and Woinshet M. 24
Solid State Fermentation
There are many biotechnological processes that involve the growth of microorganisms
on solid substrates in the absence or near absence of free water.

The most regularly used solid substrates are cereal grains, legume seeds, wheat bran,
lignocellulose materials such as straws, sawdust or wood shavings, and a wide range of
plant and animal materials.

Most of these compounds are polymeric molecules, insoluble or sparingly soluble in

water, but are mostly cheap, easily obtainable and represent a concentrated source of
nutrients for microbial growth.

04/18/2024 Alena B. and Woinshet M. 25

 In SSF technique, microorganisms are grown on and inside the humidified solid
substrate.

 Many of the filamentous fungi basically live and grow on solid substrate.

 The efficiency of the SSF basically depends on: Energy, Economy and Environment.

 In SSF, substrate itself is the source of energy and requires no medium for growth of
micro-organism.

 It is more cost effective (smaller vessels lower water consumption, reduced

waste water treatment costs, lower energy consumption, and less contamination
problems).
04/18/2024 Alena B. and Woinshet M. 26
04/18/2024 Alena B. and Woinshet M. 27
Applications:

 Many high value products such as extra-cellular enzymes, primary metabolites, and
antibiotics could be produced in SSF.

 It is estimated that nearly a third of industrial enzyme produced in Japan is made by SSF
process.

 Production of organic and ethanol from starchy substrates.

 Digestibility of fibers and lignocelluloses materials for both human and animal consumption.

04/18/2024 Alena B. and Woinshet M. 28

Laboratory scale SSF bioreactor

•Laboratory-scale bioreactors typically have a working volume that varies from about
0.2 L to 20 L.

04/18/2024 Alena B. and Woinshet M. 29

Industrial scale SSF bioreactor

• For large scale production at industrial level, SSFr employs either tray type or drum type
fermenter.

• In large-scale industrial bioreactor cultivation volumes ranging from 10,000 to 500,000 L.

• A leading enzyme manufacturer in India, ‘BIOCON’ uses tray type fermenter for large
capacity production of immunosuppressants.

04/18/2024 Alena B. and Woinshet M. 30

 Fig. 7 drum bioreactor

31
Alena B. and Woinshet M.
04/18/2024
Fig. 8 Tray type bioreactors
04/18/2024 Alena B. and Woinshet M. 32
Similarity and difference between bioreactor and fermentor

• Bioreactor and fermentor are basically the same thing.

• Both Bioreactor and fermentor are closed systems that carry out biochemical reactions.

• A Fermentor used for cultivation of prokaryotic cells, while a Bioreactor grows the
eukaryotic cells (mammalian, insect cells).

• The key difference between bioreactor and fermentor is that: bioreactors are carry out any
type of biochemical reactions while fermentors only carry out fermentation

• Bioreactor can produce both cell mass and secondary metabolites, while the fermenter can
only produce primary metabolites.

• Bioreactor can be used in the production process of pharmaceutical products, antibodies,

and vaccines, while fermenter can be used in the production of lactic acid or ethanol.
 Upstream Processing and Downstream Processing
•What is Bioprocessing?
 Is a process w/c uses living cells or their components (e.g., bacteria, enzymes, chloroplasts) to obtain desired
products such as ethanol and biodiesel, therapeutic stem cells, gene therapy vectors, and new vaccines and etc
 Bioprocessing includes two important processes - Upstream and downstream processes.

 Upstream and downstream bioprocessing are the two main stages of a bioprocess or fermentation that involves
the production of biologics using host cell proteins.

Upstream Bioprocessing

 The process of converting raw materials into a form that can be used in a biologic manufacturing process.

 Deals with the identification, screening, culture, and growth of the organism in a bioreactor.
04/18/2024 Alena B. and Woinshet M. 34
 Upstream bioprocessing's goal is to create a high-quality starting material for downstream bioprocessing.
Downstream bioprocessing

The term "downstream bioprocessing" refers to the steps that take place after the
initial bioprocessing steps, which involve the production of a biological agent

Downstream bioprocessing deals with the harvesting, testing, purification, and

packaging of the product.

 The various steps of Downstream Processing involve: Separation; Cell

disruption; Extraction; Isolation; Purification; Drying; Separation of
particles.

Both stages require controlled conditions and quality assurance .

04/18/2024 Alena B. and Woinshet M. 35

 Similarities Between Upstream and Downstream Bioprocessing

 Both are the two main parts of a bioprocess w/ch are upstream and downstream bioprocessing.
 Both processes involve living organisms, particularly microorganisms.
 Both stages require controlled conditions and quality assurance
 When it comes to making bioproducts, both processes are crucial and During both processes,
contamination should be avoided.
 Both processes are carried out on bioproducts that are both industrially and medicinally
important.
Difference Between Upstream and Downstream Bioprocessing
 Product development happens in the upstream bioprocessing stage, while product
harvesting happens in the downstream bioprocessing stage. As a result, the key distinction
between upstream and downstream bioprocessing
04/18/2024
is this.
Alena B. and Woinshet M. 36
 ND
E
04/18/2024 BY: Alena B. and weinshet M. 37