By Ranjan Kumar Sahoo

+3 Bsc
Botany DSE-4 Notes
(Industrial & Environmental
Microbiology)
ALL Units Full Syllabus Notes
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Unit-1

(Scope Of Microbes in Industry and environment)

A) Introduction

 What is microbiology?

Simply the branch of science that deals with the study microorganisms Called as Microbiology.

Or

Microbiology is the study of all living organisms that are too small to be visible with the naked eye and reffered as
microorganisms. This includes bacteria, archaea, viruses, fungi, prions, protozoa and algae, collectively known as
'microbes'.

- The Organisms that are too small tobe observed clearly by the unaided eye are called micro-organisms. Broadly
Speaking organism with a diameter of 1mm or less are called as microorganisms or microbes.

- Microbiology encompasses the study of some Organisms that fall under botany and zoology.

- The term microbiology was given by French chemist Louis Pasteur (1822-95).

- The term microbe was first used by Sedillot (1878).

 What is industrial Microbiology ?

Industrial microbiology is a branch of biotechnology that applies microbial sciences to create industrial products in
mass quantities.

Or

Industrial Microbiology is a branch of applied microbiology in which microorganisms are used for the production of
important substances, such as antibiotics, food products, enzymes, amino acids, vaccines, and fine chemicals.

 What is Environmental Microbiology?

Environmental microbiology is the study of microorganisms (such as bacteria, viruses, fungi, and protozoa) and
their interactions with their environment. Microorganisms are found almost everywhere on earth and play important
roles in a wide range of ecological processes, including decomposition, nitrogen fixation, and nutrient cycling.

B) Scope Of Microbiology :

- There is vast scope in the field of microbiology due to the advancement in the field of science and technology.

- The scope in this field is immense due to the involvement of microbiology in many fields like medicine, pharmacy,
diary, industry, clinical research, water industry, agriculture, chemical technology and nanotechnology.

- The study of microbiology contributes greatly to the understanding of life through enhancements and intervention
of microorganisms. There is an increase in demand for microbiologists globally.

- Following are the various roles of microbiology in various fields of science:

Genetics: Mainly involves engineered microbes to make hormones, vaccine, antibiotics and many other useful
products for human being.

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Agriculture: The influence of microbes on agriculture; the prevention of the diseases that mainly damage the useful
crops.

Food science: It involves the prevention of spoilage of food and food borne diseases and the uses of microbes to
produce cheese, yoghurt, pickles and beer.

Immunology: The study of immune system which protect the body from pathogens.

Medicine: deals with the identification of plans and measures to cure diseases of human and animals which are
infectious to them.

Industry: it involves use of microbes to produce antibiotics, steroids, alcohol, vitamins and amino acids etc.

i) Agricultural microbiology – try to combat plant diseases that attack important food crops, work on methods to
increase soil fertility and crop yields etc. Currently there is a great interest in using bacterial or viral insect
pathogens as substitute for chemical pesticides.

ii) Microbial ecology – biogeochemical cycles – bioremediation to reduce pollution effects

iii) Food and dairy microbiology – try to prevent microbial spoilage of food and transmission of food borne
diseases such as botulism and salmonellolis. Use microorganisms to make foods such as cheese, yogurt, pickles
and beers.

iv) Industrial microbiology – used to make products such as antibiotics, vaccines, steroids, alcohols and other
solvents, vitamins, amino acids and enzymes.

v) Microbial physiology and Biochemistry – study the synthesis of antibiotics and toxins, microbial energy
production, microbial nitrogen fixation, effects of chemical and physical agents on microbial growth and survival
etc.

vi) Microbial genetics and Molecular biology – nature of genetic information and how it regulated the development
and function of cells and organisms. Development of new microbial strains that are more efficient in synthesizing
useful products.

vii) Genetic engineering – It arisen from work of microbial genetics and molecular biology. Engineered
microorganisms are used to make hormones, antibiotics, vaccines and other products. New genes can be inserted
into plants and animals.

(C)Applications of Microbiology:-

- Microbiology is one of the largest and most complex of the biological sciences as it deals with many diverse
biological disciplines.

- In addition to studying the natural history of microbes, it deals with every aspects of microbe-human and
environmental interaction. These interactions include: ecology, genetics, metabolism, infection, disease,
chemotherapy, immunology, genetic engineering, industry and agriculture.

1) The environment:

- Microbes are responsible for the cycling of carbon, nitrogen phosphorus (geochemical cycles)

- Maintain ecological balance on earth

- They are found in association with plants in symbiotic relationships, maintain soil fertility and may also be used to
clean up the environment of toxic compounds (bio-remediation).

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- Some are devasting plant pathogens, but others act as biological control agents against these diseases.

2) Medicine:

-Disease causing ability of some microbes such as, Small Pox (Variola virus), Cholera (Vibrio cholera), Malaria
(Plasmodium, protozoa) etc.

They have also provided us with the means of their control in the form of antibiotics and other medically important
drugs.

3) Food:

- Microorganisms have been used to produce food, from brewing and wine making, through cheese production and
bread making, to manufacture of soy sauce.

- Microbes are also responsible for food spoilage.

4) Biotechnology:

Commercial applications include the synthesis of acetone, organic acids, enzymes, alcohols and many drugs.

Genetic engineering – bacteria can produce important therapeutic substances such as insulin, human growth
hormone, and interferon.

5) Research: Because of their simple structure they are easier to study most life processes in simple unicellular
organisms than in complex multicellular ones.

- Millions of copies of the same single cell can be produced in large numbers very quickly and at low cost to give
plenty of homogenous experimental material.

- Because they reproduce very quickly, they are useful for studies involving the transfer of genetic information.

D) Fermenter/Bioreacter : Definition, Types, Design And Application :-

 Defination :

A Fermenter is a device that is used to carry out the fermentation process utilising microorganisms, which
is why it is also known as a “Fermenter or Bioreactor.” It contains all of the components required for the
commercial synthesis of compounds such as antibiotics, enzymes, and drinks in a variety of sectors.

OR

A Fermentor can define as a closed cylindrical vessel which supports the biochemical and chemical activity
of the microorganisms to carry the conversion of raw material into some useful product.

Fermentation is a metabolic process in which an organism converts a carbohydrate, such as starch or a

sugar, into an alcohol or an acid. for example, yeast performs fermentation to obtain energy by converting sugar
into alcohol. bacteria perform fermentation, converting carbohydrates into lactic acid.

 Objectives of Fermentor:

- To produce microbial biomass

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- To produce microbial metabolites

- To produce microbial enzymes

- To produce recombinant products

 Ideal Properties of a fermentor:

- A fermentor should be made of a good quality material that can withstand all the conditions inside the vessel.

- It should give high productivity.

- It should be able to handle the stream sterilization pressure.

- There should be all the control parameters to monitor the fermentation process like pH electrode,
temperature probe etc.

- A material used in the fermentor should be cheap that could give satisfactory results.

 Design of Fermentor Or Components Of a typical Fermenter:-

The construction of fermentor includes the following components:

i) Basic elements :

Basic components are necessary for the construction of fermentor, which involves:

Top-plate: It is the cover that is generally made of stainless steel.

Inoculation pipe: It helps to port the inoculum inside the fermentor.

Drive motor: It drives the impeller shaft.

Impeller shaft: Holds the agitator centrally.

Impeller: Acts as an agitating device for mixing up the nutrients and microorganisms uniformly.

Stirrer: Mixes the gas bubbles throughout the liquid culture medium.

Baffle: Prevents the counterflow or vortex formation by breaking down the gas bubbles to improve aeration
efficiency.

Sparger: It supplies oxygen into the culture medium through the perforated tubes.

Drain point: Withdraws cells or medium for the continuous fermentation.

Cooling jacket: It is fitted externally to the fermentation vessel which allows the passage of steam or cold water to
balance the heat generated during the process.

ii) Controlling Elements :

Controlling elements monitor the parameters like (temperature, pH, acid, bases, oxygen supply, pressure etc.) that
are necessary for the product formation and it includes:

Pt-100: Monitors the temperature in the culture vessel.

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Foam probe: It senses foam formation.

pH electrode: Monitors the pH in the culture vessel.

Oxygen sensor: Maintains the dissolved oxygen content level.

Heating pad: Provides heat to the medium.

Cold finger: It is a pipe that passes cold water inside a vessel to cool the contents.

Rotameter: Provides variable airflow into the culture vessel.

Pressure valve: Maintains the pressure.

Air pump: Supplies air throughout the medium.

Peristaltic pump: It pumps acid, base and antifoam into the medium.

 Properties of a Fermentor

It should be reliable for long-term operation.

A fermentor should be capable of being operated aseptically or should provide sterile conditions.

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The bioreactor provides adequate aeration and agitation for uniform mixing of the contents in the vessel.

It should consume less power.

A fermentor must be equipped with controlling probes that can maintain the temperature, pH, oxygen level etc.

It facilitates the passage of inoculum and media into the vessel.

A bioreactor does not allow excessive evaporation loss.

It minimizes the labour input for the operation, harvesting, cleaning and maintenance.

 Types Of Fermenter :

A fermentor is mainly of five types:

i) Stirred tank fermentor

ii) Airlift fermentor

iii) Fluidised bed fermentor

iv) Packed bed fermentor

v) Photo fermentor

i) Stirred Tank Fermentor:

It consists of the motor driveshaft and a variable number of impellers (more than one). The impellers have a 1/3rd
diameter of the vessel. The height and diameter ratio of this bioreactor is between 3:5. Air passes into the culture
medium through a single orifice from the tube attached externally. It enables better distribution of the contents
throughout the vessel.

 Its basic functions include:

Homogenization

Suspension of solid material

Aeration to the medium

 Heat exchange

In stirred tank fermentor, rotating stirrer and baffle are found either at the top or the bottom. It mainly uses the
batch process of fermentation.

 Advantages

It is easy to operate and easy to clean.

Provides a good temperature control.

The construction of stirred tank fermentor is quite simple.

It has a low operating cost.

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It does not cause shear damage to the cells.

 Disadvantages

It cannot be used for immobilized cells or enzymes.

It has low volumetric productivity.

ii) Airlift Fermentor:

It consists of a single container inside which a hollow tube is present. This hollow tube called “Draft tube”. There is
a gas flow inlet present at the bottom of the fermentor, which allows the passage of oxygen. Gas flow inlet is
attached with the perforated disc or tube that allows continuous distribution of air.

This type of bioreactor lacks the mechanical stirring arrangements for agitation. As from the name airlift, it is clear
that the air lifts the medium upwards. There are internal liquid circulation channels, which enable continuous
circulatory motion of the medium. Here, the fermentation occurs at a fixed rate of volume and circulation.

 Advantages

It ensures adequate mixing.

Consumes less energy.

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It favours the growth of aerobic cultures.

Construction is simple.

It can use both free and immobilized cells and enzymes.

Widely used in SCP production.

Avoids excessive heat generation.

 Disadvantages

Lacks mechanical stirring arrangements.

iii) Fluidized Bed Fermentor :

The top portion of this bioreactor is more expanded. This expansion reduces the velocity of the fluid. Its bottom
part is slightly narrow. It is designed in such a way where:

- The solid retain inside the vessel.

- And, liquid flows out.

An adequate amount of gas introduces into the medium to form a suitable gas-liquid-solid fluidised bed. By using
this kind of biofermentor one should note that the suspended particles should not be too light or too heavy and
secondly, there should be continuous recycling of the medium for good bioprocessing.

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 Advantages :

Mixes the contents uniformly.

Maintains the uniform temperature gradient.

It can be used for continuous operation.

Produces higher volumetric productivity.

There is little or no clogging of particles.

iv) Packed Bed Fermentor :

- It consists of a cylindrical vessel and a packed bed with biocatalysts. The solid matrix that is used for the packed
bed fermentor possess the following properties:

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 Porous or non-porous

 Highly compressible

 Rigid

- In this kind of biofermentor, a nutrient broth continuously flows over the immobilized biocatalyst. After that, a
product releases into the fluid at the bottom of the culture vessel and finally, it can be removed. Here, the flow of
fluid can be upward or downward.

 Advantages

It has a low operation cost.

Provides continuous operation.

Separation of the biocatalyst is easy.

It is widely used in wastewater engineering.

 Disadvantages

There are undesired heat gradients.

Poor temperature control.

There is an alternation in the bed porosity.

Involves higher risk of particles clogging.

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v) Photo Fermentor

This fermentor works under the principle of light energy that involves direct exposure to the sunlight or through
some artificial illumination. It is extensively used for the production of p-Carotene, astaxanthin etc. This type of
bioreactor is basically made of glass or plastic. Photo fermentor consists of:

- A single container

- Number of tubes or panels

Tubes act as “Solar light receivers or trappers”. In this, culture transfers through solar trappers with the help of a
centrifugal pump. Inside photo fermentor, adequate penetration of sunlight is maintained and after that, it is cooled
when there is a rise in temperature. Here, the microalgae and cyanobacteria are the common microorganisms that
are used. These microorganisms grow in the presence of solar light and then product forms during the night.

 Advantages :

It gives higher productivity.

Provides a large surface and volume ratio.

Provides better control over the gas transfer.

Maintains uniform temperature gradient.

 Disadvantages :

Expensive method to carry out.

There is a technical difficulty in the process of sterilization.

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8. Application Of A Fermenter Or Fermentation :

- A Fermenter is Use to carry out fermentation Process and major application are as follows :

i) Application in medicine :

Production of antibiotics

Production of insulin

Production of growth hormones

Production of vaccines

Production of interferon

ii) Application in the food industry :

Production of fermented foods as cheese, wine, beer, and bread to high-value products

Food grade bio preservatives

Functional foods/Neutraceuticals

Production of single-cell protein

iii) Other Applications :

It is also used for waste management such as biofuels production (biodiesels, bioethanol, butanol, biohydrogen,
etc).

It is also used to produce bio-surfactant, polymers production such as bacterial cellulose production.

Development of bioremediation processes (involving microbes or their isolated enzymes) for soils and wastewater
treatments.

(C) Fermentation & Factors Affecting Fermentation Process :-

(1) Fermentation:

Fermentation is a metabolic process in which an organism converts a carbohydrate, such as starch or a sugar,
into an alcohol or an acid. for example, yeast performs fermentation to obtain energy by converting sugar into
alcohol. bacteria perform fermentation, converting carbohydrates into lactic acid.

OR

Fermentation is a metabolic process that produces chemical changes in organic substrates through the action of
enzymes using microrganisms.

(2) Principle of fermentation :

- The main principle of fermentation is to derive energy from carbohydrates in the absence of oxygen.

- Glucose is first partially oxidized to pyruvate by glycolysis.

- Then pyruvate is converted to alcohol or acid along with regeneration of NAD+ which can take part in glycolysis to

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produce more ATP.

- Fermentation yields only about 5% of the energy obtained by aerobic respiration.

- Fermentation is an anaerobic biochemical process that is used for the production of energy from the partial
oxidation of glucose or other carbon sources.

- The oxidation of the substrate, which occurs through the Embden–Meyerhoff (EMP) or Entner–Doudoroff(ED)
pathways, results in the production of pyruvate, ATP, and NAD (P) H.

- In the absence of external electron acceptors, the pyruvate undergoes reduction with the regeneration of NAD+(P).

- This step is essential for the fermentation process to progress and it leads to the production of products (ethanol
and organic acids).

- ATP is the main product of fermentation, and it is generated by phosphorylation at the substrate level.

- NADH is then re-oxidized, reverting to NAD+ in the second phase of fermentation that reduces pyruvate to the
fermentation product, such as ethanol and lactate.

- For example, in the fermentation of glucose by Streptococcus lactis, the pyruvate is converted to lactic acid to
reform NAD+ coenzymes so two ATP molecules are produced

- In yeasts like Saccharomyces, when pyruvate is converted to ethyl alcohol (ethanol), NAD+ is reformed.

(3) Factors Regulating Or Affecting Fermentation Process :-

During the fermentation process, to be able to control the production process is necessary. It is also necessary to
regularly sample and determine the relevant process parameters or to conduct continuous measurement.

The parameters that reflect changes in the fermentation process can be divided into two categories: One is the
parameters that can be directly detected by a specific sensor. They include parameters that reflect changes in the
physical environment and chemical environment, such as temperature, pressure, stirring power, speed, foam,
fermentation broth viscosity, turbidity, pH, ion concentration, dissolved oxygen, matrix concentration, etc., which
are called direct parameters. The other is the parameters that are still difficult to detect with sensors, including cell
growth rate, product synthesis rate, and respiration. These parameters need to be calculated with the help of
computer calculations and specific mathematical models based on some directly detected parameters. Therefore
such parameters are called indirect parameters. Among the above parameters, temperature, pH, dissolved oxygen
concentration, etc. have a greater influence on the fermentation process.

• The main factors affecting the fermentation process are as follows :

1. Temperature :

- The effect of temperature on microorganisms is numerous.

- Temperature can affect the enzyme activity. In the optimum temperature range, as the temperature increases the
growth and metabolism of the bacteria accelerate, and the rate of the fermentation reaction increases.

- When the temperature exceeds the optimal temperature range as the temperature rises the enzymes are quickly
inactivated, the bacteria die the fermentation cycle is shortened and the output is fermentation reduced.

- Temperature can also affect the pathway of biosynthesis. For example, when the temperature of Streptomyces
aureus is below 30°C, the ability to synthesize chlortetracycline is stronger, but when the temperature exceeds
35°C, it only synthesizes tetracycline but not chlortetracycline.

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- In addition, the temperature will also affect the physical properties of the fermentation broth and the
decomposition and absorption of nutrients by the bacteria.

- Therefore, to ensure the normal fermentation process, it is necessary to maintain the optimum temperature.
However, the optimum temperature required for bacterial growth and product synthesis is not necessarily the same.

For example, the optimum growth temperature of Streptomyces griseus is 37℃, but the optimum temperature for
antibiotic production is 28℃. Generally, experiments must be conducted to determine the optimum temperature for
each fermentation stage of different strains, and segmented control is adopted.

2. pH :

- pH can affect the activity of enzymes and the charge status of cell membranes.

- If the charge status of the cell membrane changes, the permeability of the membrane will also change, which may
affect the absorption of nutrients and the secretion of metabolites by microorganisms.

- In addition, pH will also affect the decomposition of nutrients in the medium. Therefore, the pH of the
fermentation broth should be controlled.

- However, the optimum pH of different strains in the growth stage and the synthetic product stage is often
different and needs to be controlled separately.

- During the fermentation process, with the utilization of nutrients and the accumulation of metabolites by the
bacteria, the pH of the fermentation broth will inevitably change. For example, when urea is decomposed, the
concentration of NH+4 in the fermentation broth will rise, and the pH will also rise accordingly.

- In industrial production, it is often used to add a buffer system to maintain pH in the fermentation broth, or to
control pH by adding ammonia, urea, ammonium carbonate, or calcium carbonate in the middle.

- At present, domestic pH electrodes have been developed to detect the fermentation process, which is used to
continuously measure and record pH changes, and the pH controller adjusts the amount of acid and alkali added.

3. Dissolved oxygen Concentration :

- The supply of oxygen is a key factor for aerobic fermentation.

- From the perspective of oxygen demand for glucose oxidation, 1 mol of glucose is completely oxidized and
decomposed and 6 mol of oxygen is required; when sugar is used to synthesize metabolites, 1 mol of glucose
requires about 1.9 mol of oxygen.

- Therefore, aerobic microorganisms require a large amount of oxygen, but the bacteria can only use the dissolved
oxygen in the fermentation broth during the fermentation process, but the oxygen is difficult to dissolve in water.

- At 101.32 kPa and 25°C, the solubility of oxygen in water is 0.26 mmol/L.

Under the same conditions, the solubility of oxygen in the fermentation broth is only 0.20 mmol/L. And as the
temperature increases, the solubility will decrease.

- Therefore, a large amount of oxygen must be continuously added to the fermentation broth, and stirring can
increase the solubility of oxygen in the fermentation broth.

4. Foam Concentration :

- During the fermentation process, aeration and stirring, the metabolic process of microorganisms, and the

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decomposition of certain components in the culture medium, etc., may produce foam.

- It is normal to produce a certain amount of foam during the fermentation process, but too much persistent foam
is detrimental to the fermentation.

- Because the foam will occupy the volume of the fermentation tank affect the normal operation of aeration and
stirring and even cause abnormal metabolism, it is necessary to eliminate the foam.

- There are two types of commonly used defoaming measures: one is to install a defoaming baffle to promote the
collapse of the foam through strong mechanical oscillation; the other is to use a defoaming agent.

5. Concentration of nutrients :

- The concentration of various nutrients in the fermentation broth, especially the ratio of carbon to nitrogen,
inorganic salts, and vitamins, will directly affect the growth of bacteria and the accumulation of metabolites.

- For example, in the fermentation of glutamate, the change of NH+4 concentration will affect the metabolic
pathway. Therefore, during the fermentation process, a sufficient amount of nutrients are added for fermentation.

* The control of various parameters in the fermentation process is very important. At present, the direction of
fermentation process control is to turn to automatic control. Therefore, it is hoped that more and more effective
sensors can be developed for the detection of process parameters. In addition, the judgment of the fermentation
endpoint is equally important. Production cannot simply pursue high productivity without taking into account the
cost of the product. The two must be combined. The reasonable set-up time is determined by experiment, that is,
the productivity and product cost of the fermenter is calculated according to the product output obtained from
different fermentation times, and the time with high productivity and low cost is used as the set-up time. The
indicators to determine the tank release are product output and filtration speed. The content of amino nitrogen,
mycelial morphology, and pH value. The appearance and viscosity of the fermentation broth, etc. The determination
of the fermentation endpoint requires comprehensive consideration of these factors.

E) Different Types of. Fermentation process (As mentioned in your syllabus):-

1. Batch Fermentation:

A batch fermentation is a closed culture system, because initial and limited amount of sterilized nutrient medium is
introduced into the fermenter. The medium is inoculated with a suitable microorganism and incubated for a definite
period for fermentation to proceed under optimal physiological conditions. Oxygen in the form of air, an antifoam
agent and acid or base, to control the pH, are being added during the course of fermentation process (Fig. 2.11).

During the course of incubation, the cells of the microorganism undergo multiplication and pass through different
phases of growth and metabolism due to which there will be change in the composition of culture medium, the
biomass and metabolites. The fermentation is run for a definite period or until the nutrients are exhausted. The
culture broth is harvested and the product is separated.

Batch fermentation may be used to produce biomass, primary metabolites and secondary metabolites under
cultural conditions supporting the fastest growth rate and maximum growth would be used for biomass production.
The exponential phase of growth should be prolonged to get optimum yield of primary metabolite, while it should
be reduced to get optimum yield of secondary metabolites.

The used medium along with cells of microorganism and the product is drawn out from the fermenter. When the

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desired product is formed in optimum quantities, the product is separated from the microorganism and purified
later on.

It has both advantages and disadvantages which are detailed below:

(i) Merits:

(a) The possibility of contamination and mutation is very less.

(b) Simplicity of operation and reduced risk of contamination.

(ii) Demerits:

(a) For every fermentation process, the fermenter and other equipment are to be cleaned and sterilized.

(b) Only fraction of each batch fermentation cycle is productive.

(c) It is useful in fermentation with high yield per unit substratum and cultures that can tolerate initial high
substrate concentration.

(d) It can be run in repeated mode with small portion of the previous batch left in the fermenter for inoculum.

(e) Use of fermenter is increased by eliminating turn round time or down time.

(f) Running costs are greater for preparing and maintaining stock cultures.

(g) Increased, frequency of sterilization may also cause greater stress on instrumentation and probes.

(h) Fresh sterilized medium and pure culture are to be made for every fermentation process.

(i) Yield of the desired product may also vary.

(j) There will be a non-productive period of shutdown between one batch productive fermentation to the other,

(k) More personal are required.

2. Continuous Fermentation:

It is a closed system of fermentation, run for indefinite period. In this method, fresh nutrient medium is added
continuously or intermittently to the fermenter and equivalent amount of used medium with microorganisms is
withdrawn continuously or intermittently for the recovery of cells or fermentation products

As a result, volume of the medium and concentration of nutrients at optimum level are being maintained. This has
been operated in an automatic manner. The continuous fermenter has its maximum use that take long time to
reach high productivity, reduces down time and lowers the operating costs.

In continuous mode, starting medium and inoculum are added to the fermenter. After the culture is grown the
fermenter is fed with nutrients and broth is withdrawn at the same rate maintaining a constant volume of broth in
the fermenter. In continuous mode with cell cycle, the cell mass is returned to the fermenter using micro filtrations
with bacteria or screens with fungal mycelium.

A continuous fermentation is generally carried out in the following ways:

(a) Single stage fermentation

(b) Recycle fermentation

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(c) Multiple stage fermentation

(a) Single Stage Fermentation:

In this process, a single fermenter is inoculated and the nutrient medium and culture are kept in continuous
operation by balancing the input and output of nutrient medium and harvested culture, respectively.

(b) Recycle Fermentation:

In this method, a portion of the medium is withdrawn and added to the culture vessel. Thus, the culture is recycled
to the fermentation vessel. This method is generally adopted in the hydrocarbon fermentation process. The
recycling of cells provides a higher population of cells in the fermenter which results in greater productivity of the
desired product.

(c) Multiple Stage Fermentation:

In this process, two or more fermenters are employed simultaneously and the fermentation is operated in a
sequence. Different phases of fermentation process like growth phase and synthetic phase are carried out in
different fermenters. Generally, growth phase is allowed in the first fermenter, synthetic phase in the second and
subsequent fermenters.

This process is adapted particularly to those fermentations in which growth and synthetic activities of the
microorganisms are not simultaneous. Synthesis is not growth related but occurs when cell multiplication rate has
slowed down.

The process of continuous fermentation is monitored either by microbial growth activity or by product formation
and these methods are called:

(i) Turbidostat method, and

(ii) Chemostat method.

(i) Turbidostat Method:

In this method the total cell content is kept constant by measuring the culture turbidity at a regular interval of
fermentation process. By turbidity measurement it is possible to the fermenter to regulate both the nutrient feed
rate and the culture withdrawal rate.

Fermentation, in which this method is employed, must be carried out at a low maximum cell population which leads
to the usage of less amount of substrate and wastage of greater amount of substrate as unused and residual
medium, which is removed from the fermenter along with the harvested culture (Fig. 2.13).

(ii) Chemostat Method:

In this method nutrient feed rate and harvest culture withdrawal rate are maintained at constant value. This is
achieved by controlling the growth rate of the microorganism by adjusting the concentration of any one of the
chemicals of the medium, like carbon source, nitrogen source, salts, O2 etc. which acts as a growth limiting factor.

Apart from the above chemicals, sometimes the concentration of the toxic product generated in the fermentation
process, the pH values and even temperature also act as growth limiting factors. This method is employed more
often than turbidostat method because of fewer mechanical problems and presence of less amount of unused
medium in the harvested culture (Fig. 2.14).

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However, continuous fermentations have certain advantages and limitations which are as follows:

(a) Merits:

1. The fermenter is continuously used with little or no shutdown time.

2. Only little quantity of initial inoculum is needed and there is no need of additional inoculum.

3. It facilitates maximum and continuous production of the desired product.

4. There is optimum utilization of even slow utilizable substances like hydrocarbons.

(b) Demerits:

1. Possibility of contamination and mutation because of prolonged incubation and continuous fermentation, are
more.

2. Possibility of wastage of nutrient medium because of continuous withdrawal for product isolation.

3. The process becomes more complex and difficult to accomplish when the desired products are antibiotics
rather than a microbial cells.

4. Lack of knowledge of dynamic aspects of growth and synthesis of product by microorganism used in
fermentation.

(c) Applications:

Continuous culture fermentation has been used for the production of single cell protein, antibiotics, organic
solvents, starter cultures etc.

Pilot plants or production plants have been installed for production of beer, fodder yeast, vinegar, baker’s yeast. A
wide variety of microorganisms are used for this type of fermentation

3. Fed Batch Fermentation:

It is a modification to the batch fermentation. In this process substrate is added periodically in instalments as the
fermentation progresses, due to which the substratum is always at an optimal concentration. This is essential as
some secondary metabolites are subjected to catabolite repression by high concentration of either glucose, or
other carbohydrate or nitrogen compounds present in the medium.

- For this reason, the critical elements of the nutrient medium are added in low amount in the beginning of the
fermentation and these substrates continue to be added in small doses during the production phase. This method
is generally employed for the production of substances such as penicillin. Yoshida (1973) introduced this term for
the first time for feeding the substrates to the medium as the nutrients are exhausted, so as to maintain the
nutrients at an optimum level.

- The fed-batch fermentation may be of three types:

(i) Variable Volume Fed Batch Culture:

The same medium is added resulting in an increase in volume.

(ii) Fixed Volume Fed Batch Culture:

A very concentrated solution of the limiting substrate is added at a very little amount resulting in an insignificant

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increase in the volume of medium.

(iii) Cyclic Fed Batch Culture:

As it is not possible to measure the substrate concentration by following direct methods during fermentation,
which is necessary for controlling the feeding process, generally indirect methods are employed. For example – in
the production of organic acids, the pH value may be used to determine the rate of glucose utilization.

Advantages:

1. Production of high cell densities due to extension of working time (particularly growth associated products).

2. Controlled conditions in the provision of substrates during fermentation, particularly regarding the concentration
of specific substrates for e.g. the carbon source.

3. Control over the production of, by products or catabolite repression, effects due to limited provision of
substrates solely required for product formation.

4. The mode of operation can overcome and control deviations in the organism’s growth pattern as found in batch
fermentation.

5. Allows the replacement of water loss, by evaporation.

6. Alternative mode of operation for fermentations dealing with toxic substances or low solubility compounds.

7. Increase of antibiotic marked plasmid stability by producing the correspondent antibiotic during the time span of
the fermentation.

8. No additional special piece of equipment is required as compared with the batch fermentation.

9. It is an effective method for the production of certain chemicals, which are produced at optimum level when the
medium is exhausted like penicillin.

Disadvantages:

1. It is not possible to measure the concentration of feeding substrate by following direct methods like
chromatography.

2. It requires precious analysis of the microorganism. Its requirements and the under standing of its physiology
with productivity is essential.

3. It requires a substantial amount of operator skill for the set-up of fermentation and development of the process.

4. In a cyclic fed batch culture, care should be taken in the design of the process to ensure that toxins do not
accumulate to inhibitory levels and that nutrients other than those incorporated into the fed medium become
limited also, if many cycles are run. The accumulation of non-producing or low producing variants may result.

5. The quantities of components to control must be above the detection limits of the available measuring
equipment.

Fed-batch with recycle of cells can also be used for specific purpose such as ethanol fermentation and waste
water treatment.

At present following products are being produced under fed batch culture:

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1. Production of baker’s yeast.

2. Penicillin production.

3. Production of Thiostrepton by Streptomyces laurentii

4. Production of industrial enzymes, histidine, glutathione (Brevibacterium flavum), Lysine (Corynebacterium

glutamicum)

(c) Applications:

1. It facilitates in avoidance of repressive effect.

2. It has control over organisms growth rate and O2 requirement.

3. In maintaining concentration of both the biomass and non-limiting nutrient substrates constant.

4. Production phase may be extended under controlled conditions and overcome problems associated with the use
of repressive rapidly metabolized substrates.

5. Shift in growth rate may provide an opportunity to optimum product synthesis.

6. It facilitates to overcome viscosity problems or its toxicity at higher concentration.

4) Solid Fermentation and Liquid fermentation:-

 SOLID STATE FERMENTATION:-

Solid-state (substratum) fermentation (SSF) is generally defined as the growth of the microorganism on moist solid
materials in the absence or near the absence of free water. In recent years SSF has shown much promise in the
development of several bioprocesses and products, SSF has been ambiguously used as solid-state fermentation or
solid-substrate fermentation.

However, it is proper to distinguish between two processes. Solid substrate fermentation should be used to define
only those processes in which the substrate itself acts as a carbon source occurring in the absence or near
absence of free water. On the other hand, solid-state fermentation is that fermentation that employs a natural
substrate as above or an inert substrate used as solid support. Solid substrate fermentation is normally many step
process involved.

 Liquid state Fermentation:-

- The process of Fermentation that is carried out in a totaly liquid state I'd called as liquid state Fermentation.

- Here the Fermentation process May be aerobic or anaerobic.

- Here the micro-organisms are grown on liquid Fermentation medium.

- This fermentation technique is best suited for microorganisms such as bacteria that require high moisture. An
additional advantage of this technique is that purification of products is easier.

- It is of two types :

 Stationary liquid Fermentation

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 Submerged Fermenter

1) Stationary liquid Fermentation :-It is a type of liquid Fermentation in stationary state may be without agitation
system.

2) Submerged Fermentation:-

A. Submerged fermentation is the technique where microorganisms are grown in the liquid medium which is
vigorously aerated and mostly agitated in contrast to the solid media. It uses free flowing substrates like molasses
and broths to achieve a quite rapid fermentation process. Thisfermentation process is suitable for microorganisms
that require high moisture content to grow during the fermentation process. Substrates selection is extremely
important as different organism reacts in a different way to each substrate and so does affects productivity. The
most common substrates used in submerged fermentation are molasses, soluble sugars, fruit and vegetable juices,
broth, and sewage/wastewater. During the fermentation process various medium ingredients and different
submerged culture conditions, such as temperature, pH, oxygen supply, incubation period, and inoculation rate,
have an effect on the production of fermented beverages. In this fermentation process, organisms growing in a
vigorously aerated and agitated liquid may be either batch or continuous type fermentation.

• Submerged fermentation is a method ofmanufacturing biomolecules in which enzymes and other reactive
compounds are submerged in

a liquid such as alcohol, oil or a nutrient broth. o Submerged Fermentation (SmF)/Liquid Fermentation (LF) SmF
utilizes free-flowing liquid substrates, such as molasses and broths.

• The process is used for a variety of purposes, mostly in industrial manufacturing.

Principles of Submerged Fermentations

Submerged fermentation involves the growth of the microorganism as a suspension in a liquid

medium in which various nutrients are either dissolved or suspended as particulate solids in many commercial
media.

• Submerged fermentation is a process involving the development of microorganisms in a liquid broth.This liquid
broth contains nutrients and it results in the production of industrial enzymes,antibiotics or other products.

• The process involves taking a specific microorganism such as fungi and placing it in asmall closed flask
containing the rich nutrient broth.

• A high volume of oxygen is also required for the process. The production of enzymes thenoccurs when the
microorganisms interact with the nutrients on the broth resulting in thembeing broken down.

• The bioactive compounds are secreted into the fermentation broth.

Methods for carrying out Submerged Fermentation

There are two common methods by which submerged fermentation takes place; they are batch-fed fermentation
and continuous fermentation.

• In batch-fed fermentation sterilized growth nutrients are added to the culture. It is most

common in bio-industries as it occurs during the growth of biomass in the fermenter. Ithelps raise the cell density
in the bioreactor and it is typically highly concentrated to stopdilution. The rate of growth in the culture is

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maintained by adding nutrients, this also reduces the risk of overflow metabolism.

• An open system is constructed for continuous fermentation. Then sterilized liquid

nutrients are slowly and continuously added to the bioreactor at the same rate at which the

converted nutrient solution is being recovered from the system. This results in a steadyrate production of the
fermentation broth.

In order to maintain a successful fermentation, certain variables must be monitored, for example,

temperature, pH, as well as oxygen and carbon dioxide levels.

Submerged Fermentation Substrate

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

 Advantages of Submerged Fermentations

• Submerged fermentation technology has the advantages of short period, low cost and high yield.

• Purification of products is easier In liquid culture the control of the fermentation is simpler and consequently
significant reductions in fermentation times can be achieved.

• In the same way, the use of submerged culture can benefit the production of many secondary metabolites and
decrease production costs by reducing the labour involved in solid-state methods.

 Limitations of Submerged Fermentations

• In recent years, many researchers have demonstrated that SSF has a large impact on productivity, leading to
higher yields and improved product characteristics compared to SmF

• Low volumetric productivity

• Relatively lower concentration of the products

• More effluent generation

• Complex fermentation equipment

Applications:

• Submerged Fermentation (SmF)/Liquid Fermentation (LF) SmF utilizes free flowing liquid substrates, such as
molasses and broths. The bioactive compounds are secreted into the fermentation broth.

• The substrates are utilized quite rapidly; hence need to be constantly replaced/supplemented with nutrients.

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

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• An additional advantage of this technique is that purification of products is easier.

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

Part-2

A) Microbes Involved In Fermentation:

- Micro organism are most widely used to carry out Fermentation process, in Industry to produce various Industrial
products. And they are also known as Industrial Micro-organisms.

- Micro-organisms Like Variety of algae, Fungi(like penicillium, Aspergillus,yeast,etc) Bacteria (lactobacillus, etc.
are used in Fermentation.

- Micro organism are widely used in Fermentation because they are simple structure, unicellularor multicellular, the
are rapidly multiply with very low amount of nutrients condition, and they are easily handle.

- Micro-organisms In Fermentation also known as Producer because the produce the substance that we want.

(B) Media Or Production Media :-

 A production or fermentation medium is a growth media intended for the culture of a production strain
and subsequent manufacture of either microbial cells or a biochemical product.

 Designing an appropriate production medium is one of the challenges associated with industrialising
fermentation. This is accomplished by trial and error.

 Typically, liquid production media are employed to aid the fermentation process.

 For a good output of product during a fermentation process, the selection of the optimal microorganisms
and fermentation media is crucial. As it provides nutrients and energy for the growth of microorganisms,
the quality of fermentation media is vital. This medium serves as a substrate for the production of a
product in a fermentor.

 The components of fermentation media comprise of major and minor components.

 Sources of Carbon and Nitrogen are among the primary components.

 Inorganic salts, vitamins, growth stimulants, anti-foaming chemicals, buffers, dissolved oxygen, other
dissolved gases, growth inhibitors, and enzymes are minor components.

Types of media:-

Growth media: Low concentrations of nutrients are present in the growth medium. It is useful for the production of
raw materials for further fermentation operations.

Fermentation media: High concentrations of nutrients are present in fermentation media. It is utilised in the
fermentation of final goods. For instance, yeast development requires 1% carbon. However, during alcohol
fermentation, yeast requires 12 to 13% carbon in the medium.

Types of Fermentation media

- There are two types of industrial fermentation media.

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1) Synthetic Media :

Media Whose composition are chemically defifined are termed as Synthetic Media. This media contain highly
purified organic and inorganic compounds that vary little from one source to another and have a molecular content
specified by means of an exact Formula.

2) Crude Media

Fermentation on an industrial scale typically utilises crude medium. Crude media contain a preliminary composition
of fermentation-required media. It produces a high yield of product and contains unidentified ingredient sources.

High levels of minerals, vitamins, proteins, growth factors, anti-foaming agents, and precursors are present in crude
media.

It is crucial to ensure that crude media does not contain any toxic substances that could hinder bacterial growth
and product yield.

 Characteristics Of an Ideal production or fermentation Medium

1. Chemical composition

The medium for production must have an appropriate chemical composition. The medium should typically contain
a carbon source, an nitrogen source, growth factors, and mineral salts.

2. Precursors

In particular fermentations (e.g., the fermentation of penicillin), the medium should provide the necessary
precursor for improved yields of a desired product.

3. Buffering capacity

Due to the accumulation of acidic and/or basic molecules, depending on the nature of the fermentation process, it
is crucial to keep the pH within the optimal range for the success of the process.

To regulate the pH of the medium, buffers (such as CaCO) should be introduced to the media.

In the pH range close to neutrality, media containing significant concentrations of proteins, peptides, and amino
acids have a high buffering capacity.

In this pH range, phosphates give additional buffering capacity (e.g. mono- and dihydrogen potassium or sodium
phosphates).

4. Avoidance of foaming

Foaming is a severe concern in the fermentation sector, since it can contribute to the contamination of the
fermentation medium and cause other issues.

For controlling foam, defoamers (such as lard oil combined with octadecanol for penicillin fermentations) should
be employed.

These defamers may be introduced at the stage of media preparation (before to sterilisation), after sterilisation, or
as needed during the fermentation process.

5. Toxicity

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Being an ideal production medium, it should be free from any toxic effect on culture or product formation.

6. Consistency

In aerobic fermentations, sterile air must be introduced into the medium. Under these conditions, agitated liquid
media permit the diffusion of air throughout the medium.

This explains why the majority of fermentation methods utilise liquid media. Additionally, fermentation media
should not be viscous, as the viscosity of the medium hinders the penetration of air into the interior of the medium.

7. Contamination

Some conditions of the manufacturing medium are useful for detecting contamination. For instance, low pH levels
in the synthesis of citric acid using Aspergillus niger keep contamination in check.

Furthermore, low pH media can be sterilised at low temperatures.

8. Recovery

Recovery of the targeted product is a crucial procedure that affects the product’s final cost.

Therefore, the medium’s components should be such that separation and extraction of the product are simple and
inexpensive.

9. Availability of raw materials

The necessary raw materials for the design of the production medium should be easily accessible and reasonably
priced.

This explains why non-synthetic media (or crude media) are widely used in large-scale manufacturing processes.

Synthetic media, on the other hand, are insufficient for research because they are expensive and offer low yields.

However, these media are repeatable, allowing researchers to examine the effect of various medium components
on culture and product development. Again, media can have simple or complicated compositions.

(C) DOWNSTREAM PROCESSING AND ITS STEPS

1) Introduction :-

The various procedure involved in the actual recovery of useful products after fermentation or any other process
together constitute Downstream Processing.

It is a very important step in the manufacture of different product in pharmaceutical industry (Such as antibiotics,
hormones, antibodies, vaccine, enzymes), Food industry etc.

In addition, the product is either present in the cell, in the medium or both.

The concentration of product is generally low, in either cases, and it is mixed with other molecules from which it
has to be separated.

2) Steps of downstream processing

The various steps of Downstream Processing involve:

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 Separation

 Cell disruption

 Extraction

 Isolation

 Purification

 Drying

i) SEPARATION OF PARTICLES :

- It is first step of DSP and usually involve the separation of solids substances, from the liquid media.

- It is generally achieved by following ways:

Filtration– it is used for filamentous fungi and bacteria.

- Different techniques of filtration are as follows:

- Surface filtration

- Depth filtration

- Centrifugal filtration

- Rotating drum vacuum filtration

Centrifugation– It may be used for bacteria, usually, protein precipitates.

Flocculation and flotation– It is used for small bacterial cells which are difficult to separate even by centrifugation.

Flocculation: It involves the aggregation of cells which may be induced by inorganic salt, minerals, hydrocolloids
and organic polyelectrolytes.

If flocculation isn’t effective than very minute gas bubbles is made by sparging, release of over pressure or
electrolysis.

Flotation: in any case, the gas bubble need to adsorb and surround the cell, raising the gas bubbles to the surface
of media in the form of foam.

ii) CELL DISRUPTION

- Disruption of microbial cell is usually difficult because of their small size, rigid cell walls and high osmotic
pressure inside the cells.

- Disruption of cell is generally achieved by mechanically, lysis or drying:

Mechanical cell disruption: this involves the uses of shear, E.g.-, colloid mill, ball mill grinder etc., homogenizer and
ultrasound.

Drying: it involves the drying of cells by adding the cells into a huge amount of cold acetone and extracted using
buffer or salt solution.

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Lysis: lysis of microbial cells may be achieved by chemical means, e.g., salt or surfactants, osmotic shock, freezing,
or Lytic enzymes, e.g., lysozyme, etc.

iii) EXTRACTION

- It involves the recovery of a compound or a group of compounds from a mixture or from cells into a solvent phase.

- It usually achieved both separation of particles as well as concentration of the product.

- The process of extraction is frequently useful for the recovery of Antibiotics and most of the lipophilic substances.

- The process of extraction is achieved by:

Liquid-liquid extraction

Whole broth (medium + cells) extraction

Aqueous multiphase extraction

iv) ISOLATION

- Some of the product concentration may occur during the extraction step.

- It is generally achieved by the following:

Evaporation: E.g.- continuous flow evaporator, falling film evaporator, thin film evaporator, spray dryers.

Membrane filtration:g.- Microfiltration, ultrafiltration, reverse osmosis and electrodialysis.

Ion exchange resins:g.- Dextran cellulose, polyamine, acrylate etc.

Adsorption resins: These may be:

Polar – Sulfoxide, amide.

Apolar – Styrene-divinyl benzene.

Semipolar – Acrylic ester.

v) Purification

- It aims at recovery of the product in a highly purified state.

- Purification is achieved by the following procedures:

Crystallization: This is used for the low molecular mass compound like antibiotics.

Chromatographic methods: it is generally, used for the purification of low molecular mass compound from mixture
of similar molecules, e.g., antibiotics(homologous) and macromolecules (enzymes).

- The different chromatographic procedures are:

Adsorption

Ion exchange

Gel filtration

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Hydrophobic

Affinity

Partition chromatography.

Vi) DRYING :

- Drying is the most important step in downstream processing which makes the product suitable for handling and
storage.

- The most frequent approaches of drying are:

 Vacuum drying

- Vacuum drying is the mass transfer operation in which the moisture present in a substance, usually a wet
solid, is removed by means of creating a vacuum. In

 Spray drying :

Spray drying is utilised to dry vast quantities of liquids.

Spray drying involves passing small droplets of liquid containing the product through a nozzle that directs them
over a stream of hot gas.

The water evaporates, leaving behind the solid particles.

 Freeze-drying

Freeze-drying or lyophilization is the preferred procedure for drying and formulating a vast array of items, including
medications, foodstuffs, diagnostics, bacteria, and viruses.

This is mostly due to the fact that freeze-drying does not typically result in the loss of biological activity in the
intended product.

The principle underlying lyophilization is the sublimation of a liquid from a frozen condition. In the actual procedure,
the product-containing liquid is frozen and then vacuum-dried in a freeze-dryer.

Now that the vacuum can be released, the vials containing the product can be sealed; for instance, penicillin can be
freeze-dried directly in ampules.

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UNIT-2

A) Enzyme Immobilization

 What is Enzyme Immobilization?

 What is a Support Matrix?

It is a substance that facilitates the entrapment of an enzyme. For effective immobilization, a support matrix must
possess the properties outlined in the following section.

 Properties of Support Matrix

- deal Properties of Support Matrix

 Mechanical durability.

 Biocompatible.

 Stable.

 Inertness.

 Regenerability.

 Ease of differentiation

 Expanding the specificity of enzymes.

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 Product inhibition reduction.

 Decrease in microbial contamination.

 Cost-effective.

# Classification of a Matrix:-

-Support material are classified into two categories based on their morphology

 Non porus support: eg glass

 Porus support: eg silica

Porous support has a greater surface area than nonporous support.

- Support material are classified into two categories based on their chemical nature

 Organic

 Inorganic

Two types of support matrices exist based on their chemical composition.

1. Organic matrix: There are natural polymers and synthetic polymers.

Natural polymers: It has a favourable affinity for proteins. Natural polymers include polysaccharides (Cellulose,
dextran, agar, agarose, chitin, alginate, etc.), proteins (Collagen, albumin), and carbon.

Synthetic polymers: It is chemically and mechanically stable. Synthetic polymers include polystyrene, polyacrylate,
polyacrylamide, and polyamides, among others.

2. Inorganic matrix: It separates minerals into Natural and Processed categories.

Natural minerals: E.g.Bentonite, celite, centolite, silica, charcoal etc.

Processed materials: E.g.Porous glass, metals and metal oxides.

Examples of Support Matrix

Some of the most often employed polysaccharides include

 Alginate: are polymers of n-acetyl glucuronic acid with a readily derivatizable hydroxyl group.

 Chitosan and chitin: Chitosan is chitin that has been deacetylated.

 Carrageenan: A linear sulfated polysaccharide, carrageenan is a carrageenan.

 Cellulose: Cellulose is a linear polymer composed of D-glucose units (two are depicted) connected by
β(1→4)-glycosidic linkages.

 Starch: Starch is a linear glucose polymer.

 Pectin: Pectin is a galacturonic acid-based heteropolysaccharide found in plant cell walls.

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 Some commonly used Proteins include:

 Collagen.

 Gelatin

As a result of their physical and chemical characteristics, synthetic supports are the most abundant
support materials for protein immobilization.

 DEAE cellulose

 polyvinyl chloride (PVC)

 UV-activated polyethylene glycol (PEG)

 glutaraldehyde-activated nylon

 cyclodextrin glucosyltransferase

 Inorganic materials as supports;

 Zeolites: Zeolites are crystalline microporous materials with well-defined features.

 Ceramics: Ceramics are inorganic and metal-free

 Celite: Celite is a very porous diatomaceous substance.

 Silica: oxide of silicon

 Glass: Glass is an extremely viscous fluid.

 Activated carbon: Activated carbon is derived from carbonaceous sources, including coal, coconuts,
nutshells, peat, wood, and lignite.

 Charcoal: Charcoal, a carbon allotrope, has exceptional adsorption capability.

Physical Characteristics of the Matrices

Mean particle size: Mean particle size is the parameter that determines a carrier’s porosity. The porous matrix is
preferred over the non-porous matrix because it improves the surface area, hence increasing the enzyme’s loading
capacity. This matrix feature defines the total surface area and influences a catalyst’s binding capacity. A porous
material should have optimal pore distribution in order to maximise particle flow.

Hydrophilic character: It determines the activity level of an immobilised enzyme.

Mechanical strength: Mechanical strength can be defined as the binding of an enzyme that is inversely proportional
to its ease of entrapment.

# Enzyme immobilization Techniques / Methods of Immobilization:-

Based on the binding property, it is classified as either physical or chemical.

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 Physical Methods

 Adsorption

 Entrapment

 ENcapsulation

 Chemical Methods

 Covalent Binding

 Cross Linking

A. Physical Methods of Enzyme immobilization

1. Adsorption

This is the earliest and simplest technique. Enzyme clings to the surface of the water-insoluble carrier matrix in this
form.

Similar to electrostatic or hydrophobic affinity binding to a particular ligand, the binding is nonspecific.

Typically, the bond between enzymes and the carrier matrix is strong, but it can be reduced by numerous causes,
including:

Addition of substrate

pH or ionic strength

In enzyme adsorption, the bonding is accomplished via weak forces, such as the hydrogen bond and the Vander
Waal force.

The matrix employed The particle size of the matrix must be modest (500Å-1mm D).

Examples: In this type, various carrier materials are used, including:

Mineral support (E.g. Aluminium oxide, clay)

Organic support (E.g. Starch)

Modified sepharose and ion exchange resins

 . Methods of Immobilization by Adsorption

Static method: The static approach is an effective yet time-consuming procedure. It involves agitation-free
immobilisation of enzyme and carrier molecule.

Dynamic process: This dynamic procedure requires the continual mixing of an enzyme with the carrier.

Reactor loading: Reactor loading entails introducing both the enzyme and the carrier into the reactor while stirring
the entire material. It is commonly employed in the industrial manufacturing of immobilised enzyme.

Electro-deposition: Electro-deposition involves keeping a carrier close to the electrode in an enzyme bath and then
passing an electric current through it. This causes an enzyme to travel toward the carrier. The enzyme is finally
deposited on the surface.

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Advantages of Adsorption

 It has no pore diffusion limitation.

 It is a straightforward and economical method.

 In this method, no reagents are necessary.

 There is only a slight decline in enzyme activity.

 It has less of an effect on an enzyme.

 It has minimal activation requirements.

 The enzyme that has been adsorbed can be recycled, regenerated, and reused.

 It has a high enzyme loading efficiency.

 Disadvantages of Adsorption

 It offers a small surface area for enzyme binding.

 Typically, desorption of an enzyme from its carrier occurs.

 The yield is also modest.

2. Entrapment

This method entraps an enzyme within a porous polymer or gel matrix. Also known as lattice entrapment. Covalent
or noncovalent bonds can exist between an enzyme and its matrix.

The matrix employed It is water-soluble and its nature varies depending on the enzymes present.

Examples: polyacrylamide gels, cellulose triacetate, agar, gelatine, alginate, etc. are utilised as carrier materials.

 Methods of enzyme entrapment

It involves the incorporation of an enzyme into the matrices listed below:

 Gels: This process involves entrapping an enzyme within the gel matrix.

 Fibres: Entrapment of an enzyme within the matrix of a fibre.

 Microcapsules: Involves trapping inside a microcapsule.

Advantages of Entrapment Method

 It has a high enzyme loading capacity.

 It is a quick method.

 Here, enzyme distortion is minimal.

 It is simple to implement.

 Disadvantages of Entrapment Method

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 Substrate and product diffusion creates difficulties.

 It results in the loss of low-molecular-weight enzymes.

 There may be the possibility of microbial contamination.

 In addition, it causes enzyme inactivation and sometimes enzyme activity loss.

 It has few industrial applications.

3. Encapsulation

It involves membrane confinement. In an aqueous solution, an enzyme is contained within the semipermeable
membrane of a capsule.

This procedure permits the exchange of substrate and product, but not an enzyme. The efficiency of encapsulation
depends on the stability of the enzyme.

The capsule’s matrix consists of a semi-permeable membrane, which may be polymeric, lipoid, non-ionic, etc.

Examples: It includes nitrocellulose, nylon semi-permeable matrix etc.

 Methods of encapsulation

It can be attained through the following methods:

Encapsulation in a reaction vessel: Encapsulation in a reaction vessel entails separating a chamber with a
semipermeable membrane. In one compartment are enzymes, while in the other are substrate and product.

Encapsulation by hollow fibre membrane: It involves trapping an enzyme within a semipermeable matrix (cellulose,
triacetate etc.). Here, an enzyme is captured within the matrix.

Microencapsulation: Utilizing 1-6-diaminohexane, enzyme molecules are enclosed within a microcapsule through
chemical polymerization.

Encapsulation by liposomes: Using phospholipid, an enzyme attaches to the concentric lipoidal membrane of the
liposome.

Advantages of Encapsulation

 There is no leakage of enzymes.

 It has no impact on enzyme activity.

 It’s a straightforward procedure.

 It possesses a high enzyme loading efficiency.

Disadvantages of Encapsulation

 Utilizes a carrier with restricted pore size.

 It is not very economical.

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B. Chemical Methods of Enzyme immobilization

1. Covalent Binding

It is a common practise. An enzyme molecule forms a covalent link with the carrier during this procedure.

Here, the binding strength is strong, or a complex structure is stable due to this bonding.

Additionally, no enzymes are lost during the procedure. The functional group of an enzyme’s active component
forms a covalent bond with the carrier molecule.

The functional groups involved in the binding process include -NH2, -NH3, -COO, -OH, -SH, -O, and -S, among others.
The charged condition of these functional groups determines the order of their responsiveness to the carrier: -S– >
-SH > -O– > -NH2 > -COO– > -OH >> -NH3+

The following are examples of polymeric carriers employed in covalent bonding:

Carboxylic acid and related polyglutamic acid groups

Amide group of a polypeptide Amino and similar polysaccharide groups

Polysaccharides (celluloses, agarose, sepharose, etc.), polyvinyl alcohol, silica, and porous glasses are among the
most frequently used polymers.

Covalent coupling

Covalent coupling

Methods used for Covalent Binding

The subsequent procedures are involved.

Diazoation: It is based on the diazo bond between protein and aryldiazonium electrophilic groups of the matrix.

Development of Peptide bond: formation of a bond between the amino/carboxyl groups of the support and the
amino or carboxyl groups of the enzyme.

Poly functional reagents: Use of a bi-functional or multi-functional reagent (glutaraldehyde) that establishes a link
between the amino group of the support and the amino group of the enzyme.

Amidination reaction: The matrix containing amido ester functional groups can be employed to immobilise proteins
through the amidination process.

Thiol–disulphide exchanged reaction: This technique is utilised for protein bonding through the thiol groups of both
the carrier and the protein.

Akylation and arylation: This approach is based on the alkylation of the amino, phenolic, and thiol groups of
proteins with a matrix including reactive halides, vinyle, sulphonile, etc.

Advantages of Covalent Binding

 It provides an enzyme and a carrier with a strong binding force.

 There is no leaking of enzymes.

 It’s a straightforward and popular method.

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 pH and ionic strength have no impact on the process.

Disadvantages of Covalent Binding

 Enzyme is altered chemically, resulting in functional conformational loss.

 When active site reactions occur, conformational changes inactivate the enzyme.

 This can be circumvented by immobilising the enzyme in the presence of substrate or a competitive
inhibitor.

 Strong connection between enzyme and support.

 No leakage or desorption problem.

 Method that is comparatively straightforward.

 Different functional group assistance options are offered.

 Wide applicability

2. Cross Linking

It is often referred to as “Copolymerization.” Using polyfunctional reagents, the immobilised enzymes form
covalent bonds with the different groups of an enzyme. It does not require a support matrix. Cross-linking results in
the development of three-dimensional cross-linked aggregates. The most frequently employed polyfunctional
agents include glutaraldehyde and diazonium salts, among others.

Advantages of Cross Linking Method

 There is minimal or no enzyme loss.

 It results in a highly stable enzyme.

 It is a simple and inexpensive method to implement.

 It has a broad range of applications in the commercial production of the enzyme.

Disadvantages of Cross Linking Method

 It inhibits enzyme activity.

 In general, the polyfunctional chemicals utilised in this technique denature enzymes.

 It is not very economical.

 Advantages of Enzyme immobilization

 Stable and more functionally efficient

 Can be used several times.

 The products lack enzymes.

 Ideal for enzyme-based reaction systems.

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 Easy regulation of enzyme action.

 Fit for industrial and medical applications.

 Minimize wastewater disposal problems.

 high ratio of substrate to enzyme

 Minimum response time

 Continuous enzyme utilisation

Disadvantages of Enzyme immobilization

 The potential of an enzyme losing its biological function through immobilisation or usage.

 Often, immobilisation is a costly endeavour needing sophisticated technology.

 Some enzyme become unstable following immobilisation.

 Occasionally, enzymes are rendered inactive by the system’s generated heat.

Applications of Enzyme Immobilization

 Industrial production: Used extensively in the commercial manufacture of industrial-grade antibiotics,

drinks, amino acids, and secondary metabolites, etc.

 Biomedical applications: Immobilized enzymes are most frequently utilised in rapid diagnostic tests, such
as ELISA, and in the treatment of numerous pathogenic disorders.

 Food industry: Enzymes such as Pectinases, Cellulases, and Amylases are immobilised on suitable
carriers or matrices and successfully employed in the commercial manufacturing of fruit and vegetable
jams, jellies, and syrups. Lactase is immobilised on cellulose fibres, allowing milk and whey to make
lactose-free milk.

 Large Scale Using bioreactors for the production of bio-diesel from vegetable oils. They provide a
continuous procedure that cuts costs in half.

 Waste water management: Treatment of sewage and industrial effluents via packed bed reactors

 Textile industry: The textile industry, such as scouring and bio-polishing.

 In the detergent industry: In the detergent industry, immobilisation of lipases to breakdown lipids found in
stains or dirt is commonplace.

 Immobilized enzymes for bioremediation: Enzymes immobilised for bioremediation Bioremediation is a

technology that utilises biological organisms and enzymes to remove pollutants from a contaminated
place.

 Biodiesel production: Biodiesel manufacturing has acquired significance due to its ability to replace fossil
fuels that are expected to run out within a century.

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B) Large Scale Application Of Glucose Isomearse:-

- Glucose isomearse, also known as xylose isomerase, D-xylose isomerase, D-xylose keto isomerase, and D-xylose
ketol-isomerase, is an isomerase that isomerizes aldose, such as D-xylose, D-glucose, D-ribose, etc., to the
corresponding ketose. The systematic name of this enzyme class is D-xylose aldose-ketose-isomerase. Glucose
isomerase has a wide range of sources, including microorganisms, such as bacteria, fungi and actinomycetes, as
well as plants and animals. Glucose isomerase is a key enzyme in the industrial production of high fructose corn
syrup and fuel ethanol.

The most widely used application of this enzyme is in the conversion of glucose to fructose to produce high
fructose corn syrup (HFCS). Xylose isomerase is one of the enzymes used by bacteria in nature to use cellulose as
food and another focus on industrial and academic research, has been developing versions of xylose isomerase
that could be useful in the production of biofuel.

C) Application Of Penicillium Acrylase (PGAs):-

Penicillin G acylase is an enzyme that catalyzes the conversion of penicillin G to penicilloic acid. It is a monoclonal
antibody that recognizes the bacterial enzyme, penicillin G acylase. The antibody is immobilized on a solid carrier,
and the substrate analogue, penicilloyl-L-alanine, is used to determine the kinetic parameters of the enzyme.
Penicillin G acylase has shown activity against most Gram-positive bacteria tested. The rate of reaction increases
with increasing substrate concentration and decreases with increasing pH. There are no apparent substrate
inhibition or product inhibition effects.

The most widespread use of PGAs is in the production of 6-APA (6- aminopenicillanic acid) from both Penicillin G
and Penicillin V. Immobilized PGA en-zymes mainly from E. coli, B. megaterium and A. faecalis are available from a
number of commercial suppliers. Reac-tionsare carried out at >5,000L scale under controlled condi-tions, the pH
being eithercontrolled at approximately 8.0, or slowly ramped from 7.0 to 8.5, depending upon thecatalyst, as high
as 8.5. Exposure to high temperature (>30 οC) and pH (>8.0) isminimized to reduce inactivation of the enzyme and

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retain high product yield of the otherwise relatively unstable 6-APA.

The use of PGA in large scale production of semi synthetic penicillins and cephalosporins is also widespread.
These processes are focused on the condensation of an ap-propriate D-amino acid derivative with a β-lactam
nucleus in a PGA catalyzed reaction. This involves the direct acylation of nucleophiles such as 6-APA or 7-ADCA
with free acids.

Unit-3

A) Isolation Of Micro-organisms From Soil :-

1) Introduction : Soil is reservoir of micro-organism where varieties of micro-organism were found.

- Soil contain different types of microorganisms which are listed below;

 Bacteria: Bacteria is the key soil workforce. They are the last stage in the process of breaking down
nutrients before releasing them into the root zone of the plant. In fact, they are the final stage of breaking
down nutrients. Food and Agriculture Organization once declared that “Bacteria may well be the most
valuable of life forms in the soil.”

 Actinomycetes: These were classified as fungi and perform similar roles in soil. However, certain
actinomycetes are predators that can hurt the plant, while other species that live in the soil may act as anti
-biotics for plants.

 Fungi: Similar to bacteria, fungi are also found in the root zone and provide nutrients to plants. For
instance, Mycorrhizae is a fungus that aids in the absorption of nutrients and water by plants and roots to
supply amino acids, and other nutrients.

 Protozoa: Protozoa are bigger microbes that are awe-inspiring to consume and be at the mercy of bacteria.
Actually, the nutrients consumed by the bacteria get released into the air when protozoa eat bacteria.

 Nematodes: Nematodes are microscopic worms that reside around or within the plant. Some nematodes
can be predators but others have a beneficial effect, consuming pathogenic nematodes and releasing

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nutrients to the plant.

2) Principle of Isolation of Microorganisms from Soil

During this process, the soil sample is collected from the study site. After that, the soil sample is dissolved in
distilled water and then serial dilution is performed. Then the spread-plate/streak plate method is performed on the
sample with the highest dilution factor. The spread plate is incubated at an optimum temperature (based on the
type of microorganism you want to isolate). After incubation microbial colony will form of your desired
microorganisms.

3) Procedure

 First of all, collect the soil sample from the study site (it will be best if you collect more than one soil
sample from the study site). Then transfer it to the laboratories.

 In laboratories, removes the other debris or rocks, or large particles from the soil sample.

 Dissolve the soil sample in sterile distilled water, and make sure the soil sample is uniformly dissolved in
water.

 After that perform the serial dilution.

 Prepared the agar plate. Select the media based on the types of microorganisms you want to isolate.

 Nutrient media, such as tryptone yeast extract, yeast extract mannitol, and rhizobial minimal media have
been found to be extremely suitable for the growth of rhizobia.

 EMB Agar is utilized in the detection and removal of pathogenic bacteria. It has digested meat proteins as
the source of organic nutrients.

 To isolate fungal media, antibiotics must be utilized to prevent bacterial contamination. Sabouraud’s
Dextrose Agar and potato-dextrose agar are popular media.

 Select the highest dilution sample and spread it over the agar plate.

 Incubate the inoculum plate in an incubator at an optimum temperature using an incubator (temperature
may vary based on the types of microorganisms you want to isolate).

 After incubation, the colony will form of your desired microorganism.

 Perform further tests for the identification of the strain, e.g. staining method.

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4) What is Serial Dilution:-

A serial dilution is the stepwise dilution of a substance in solution.

Serial dilution is a laboratory technique, in which a stepwise dilution process is performed on a solution with an
associated dilution factor. In the laboratory, this method is used to decrease the counts of viable cells within a
culture to simplify the operation.

In serial dilution, the cell count or density gradually decreases as the serial number increases in each step. This
makes it easier to calculate the cell numbers in the primary solution by calculating the total dilution over the whole
series.

Serial dilution only reduces the number of bacteria/viable cells but doesn’t separate them like in other techniques
like Flow Cytometry.

The main objective of serial dilution is to reduce the concentration of a substance, typically a chemical or biological
sample, in a series of steps.

 Serial Dilution Method Procedure

The following is the serial dilution procedure for a ten-fold dilution of a sample to a dilution factor of 10-6:

 Take 7 sterile and clean test tubes.

 The selected sample is taken into a test tube and the remaining 6 test tubes are filled with 9 ml of sterile
diluent such as distilled water or 0.9% saline.

 Take a sterile pipette.

 Drawn 1ml of sample into the sterile pipette. The sample must be properly mixed, if necessary use a

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vortex meter.

 Then transfer this 1ml sample within the first test tube to make the total volume of 10 ml. It provides an
initial dilution of 10-1. Make sure during the transfer, the tip of pipette doesn’t touch the wall of test tube
or no amount of sample remains at the tube wall.

 Mix the sample properly with the diluent by shaking the tube.

 Now discard the pipette tip and add a new pipette tip to the pipette.

 Transfer 1 ml of mixture sample from the 10-1 dilution to the second tube by using pipette. The 2nd tube
now has a total dilution factor of 10-2.

 Repeat step 8 for the remaining tubes, transfer 1 ml from the previous tube to the next 9 ml diluents.

 The diluted sample for the bacteria/viable cells in the last test tube will be 10-6 (1 in 1,000,000).

B) Isolation Of Micro-organisms From Air :-

All the major groups of microbes remain suspended within the atmosphere, which are ranges from various algae to
viruses.

The microbial flora of the atmosphere is transient and variable.

The microbial content of the outer atmosphere is changing with the season, it contains various pollen, algae,
mosses, grains, spores of fungi, ferns, bacteria, and viruses.

These air-spora or air-born particles can cause disease in living beings such as animals, plants, and humans. They
are responsible for influenza, colds and mycoses, etc. The term air spora was first introduced by Gregory.

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Among these air-borne particles, the fungal spore is considered as the most important air-spora.

The air is a good carrier for microorganisms but not a good medium for their growth.

The numbers and the types of suspended microorganisms on-air varies based on source and location.

 Principle

The Isolation of Microorganism From Air is performed by using the settle-plate technique. In this method a suitable
medium is poured over a sterile petri dish and then allow it to slidify. After that the plate is exposed to the open air
for a few minutes. Then the plates are incubated for the formation of microbial colonies on the plate. Observe the
colonies by using a microscope.

Procedure

 Isolation of Microorganism From Air

 Isolation of Microorganism From Air

 Prepared the Czapek-dox agar medium and NA medium.

 Pour the melted Czapek-dox agar medium with streptopenicillin and NA medium in a petri dish.

 Keep them at room temperature in a steady condition to allow them to solidify.

 After solidification remove the covers of petri plates and expose them to the air for 5-10 minutes.

 After 5-10 minutes cover these plates.

 Incubate the Czapek-dox agar plates at 25 degrees centigrade for 2-7 days whereas NA plates at 37
degree centigrade for 24-48 hours.

 After incubation, observe the colony formation on both plates (Czapek-Dox medium and NA medium) and
count them.

 Perform further biochemical tests for the identification of those microbial colonies.

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C) Role Of Micro-organisms In sewage water treatment :-

What is sewage Water Treatment?

Sewage treatment is a type of wastewater treatment which aims to remove contaminants from sewage to produce
an effluent that is suitable to discharge to the surrounding environment or an intended reuse application, thereby
preventing water pollution from raw sewage discharges.

Sewage Treatment refers to the process of removing contaminants, micro-organisms and other types of pollutants
from wastewater.

Sewage water is wastewater from people living in a community. It is the water released from households after use
for various purposes like washing dishes, laundry, and flushing the toilet, thus the name wastewater. The used
water moves from the houses through pipes installed during plumbing. The sewage water then moves into sewers,
either constructed by the house owner, or into a sewer facility set up by the municipality.

Wastewater can be detrimental to the environment if left untreated. That’s because waste from humans and pets
are a source of several types of waterborne diseases and bacterial contamination.

Thanks in part to microorganisms, treating wastewater and sewage is possible. The role of microorganisms in
wastewater treatment helps to treat and purify wastewater and make it less harmful to the environment.

While there are many different microbes used in sewage treatment, there are three well-known microbes that play
an instrumental role in keeping sewage clean. Each of these types of bacteria help the treatment process in a
unique way to ensure there is little to no impact on the surrounding environment.

COMMON MICROORGANISMS USED IN WASTEWATER TREATMENT

Here is a list of bacteria used in sewage treatment you can reference.

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 AEROBIC BACTERIA

Aerobic bacteria are mostly used in new treatment plants in what is known as an aerated environment. This
bacterium uses the free oxygen within the water to degrade the pollutants in the wastewater and then converts it
into energy that it can use to grow and reproduce.

For this type of bacteria to be used correctly, it must have oxygen added mechanically. This will ensure the bacteria
are able to do their job correctly and continue to grow and reproduce on its food source.

 ANAEROBIC BACTERIA

Anaerobic bacteria are used in wastewater treatment on a normal basis. The main role of these bacteria in sewage
treatment is to reduce the volume of sludge and produce methane gas from it.

The great thing about this type of bacteria and why it’s used more frequently than aerobic bacteria is that the
methane gas, if cleaned and handled properly, can be used as an alternative energy source. This is a huge benefit
considering the already high wastewater treatment energy consumption levels.

Unlike aerobic bacteria, this type of bacteria is able to get more than enough oxygen from its food source and will
not require adding oxygen to help do its job. Phosphorus removal from wastewater is another benefit of anaerobic
microbes used in sewage treatment.

 FACULTATIVE

Facultative microorganisms in sewage treatment are bacteria that can change between aerobic and anaerobic
depending on the environment they are in. Note that these bacteria normally prefer to be in an aerobic condition.

D) Microbes or Micro-organisms as indicator of water quality:-

Water is the most important commodity in the world. Over the large parts of world, humans have inad-equate
access to potable water. Since the inception of industrial revolution different toxic compounds have entered in the
water bodies due to leakage, improper disposal or accidents and caused great harms to rivers and various water
bodies and imposed major health risks on human beings. Water pollution is measured by variety of physical,
biological and chemical methods. Microbiological tests have proven to be indispensable part of environmental
contamination detection.

Micro-organisms are ubiquitous in both terrestrial and aquatic habitat and they are good indicator of water quality.

The ideal indicator for assessing contamination in the aquatic environment should have following characteristics.

 The organism should be useful for all types of water•

 The organism should have a longer survival time than the hardiest enteric pathogens•

 There should be a specific dose-responsiveness to specific stressors.•

 Should have wide temporal and spatial distribution.•

 The organism should be found in warm-blooded animals’ intestines.•

 It should occur in much greater numbers than the pathogens.•

 It should be absent, or at least very few numbers in clean waters.•

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 It should respond to natural environmental stress and wastewater treatment processes and disinfec-tants
in a manner similar to the pathogens of interests.•

 Indicator density should bear some relation to the degree or extent of pollution.•

 It should be easy to isolate, identify, and enumerate by routine laboratory procedures, cost effective.•

 It should be present with the pathogen, numbers correlating with the pathogens.•

 Its die off rate should be parallel to pathogen.•

 Its Growth requirements/rate should also be equal to pathogen.•

 It should have a history of association with the pathogen i.e.The organism should be present
whenever enteric pathogens are present•

 It should be more resistant to environmental stress or treatment and persist for greater length of time
than pathogen

* None of the types of indicator organisms that are currently in use fit all of these criteria perfectly, i.e., there is no
universal indicator however, when cost is considered, use of indicators becomes necessary.

 Types of Bio Indicators

Bioindicators are organisms/ chemical markers or biological processes whose change points can be observed
to identify and quantify the effects of pollutants on the environment1.

1. Accumulation Bioindicators :- That Store pollutants without any evident changes in their metabolism

2. Response Bioindicators:- There are visible/apparent cell changes or signs of damage even at a slight
increase in the quantity of harmful substances (Jain et al.,)

 Microbes as Bioindicators

Presence of some microbes can be correlated with particular type of pollution. Some bacteria produce stress
proteins in response to contaminants like cadmium and benzene. Monitoring for bacterial indica-tors in water has
following major purposes:

• To identify source of fecal contamination of waters• To demonstrate that treatment and /or disinfection process
are working effectively

• To monitor the general system cleanliness

• To alert for possible cross contamination and contamination from open storages

WHO (2002) has recognized the following three groups in order to elucidate the term microbial indicator:

1. General (process) microbial indicators,

2. Faecal indicators (such as E. coli)

3. Index organisms and model organisms.

 Major Microbial Indicators Used Are

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 1. Total Coliforms: Gram-negative, non-spore-forming, oxidase-negative, rod-shaped facultative

anaerobic bacteria that ferment lactose (with β-galactosidase) to acid and gas within 24– 48h at 36±2°C.
These include E.coli, citrobacter, Klebsiella and Enterobacter. However, evidences are there to suggest
that some coliforms may multiply in water in the presence of organic matter.

 a. Fecal Coliforms: It is a subgroup of total coliform group, a much specific fecal pollution
indicator. They are widely used to test recreational waters and are approved as an indicator by the U.S.
Food and Drug Administration’s National Shellfish Sanitation Program (NSSP) for classifying
shellfishing waters. They are frequently present along with other inorganic pollutants due to runoff
from livestock farms to nearby water body. However, even in this group, some species have a
nonfecal origin (e.g., Klebsiella pneumoniae).

 b. Thermotolerant Coliforms: Coliforms that produce acid and gas from lactose at 44.5± 0.2°C within
24±2h, also known as faecal coliforms due to their role as faecal indicators.

 .c. Thermophilic Coliforms that produce indole from tryptophan, but also defined now as coli-forms
able to produce β-glucuronidase. These are the most appropriate group of coliforms to indicate faecal
pollution from warm-blooded animals.

 2. Faecal Streptococci (FS): A group of Gram-positive coccoid bacteria represented by various

Enterococcus spp., Streptococcus bovis and S. equinus. These were initially investigated as impor-tant
pollution indicator bacteria but gained popularity only after development of selective medium. These never
multiply in water but disappear rather rapidly, so indicate recent and dangerous pol-lution. Four key points
in favour of the faecal streptococci were:

 a. Relatively in high number in the excreta of humans and other warmblooded animals.

 b. Presence in wastewaters and known polluted waters.

 c. Absence from pure waters, virgin soils and environments having no contact with human and
animal life.

 d. Persistence without multiplication in the environment

 3. Enterococci: The enterococci are a subset of faecal streptococci that grow at pH 9.6, 10° and 45°C and
in 6.5% NaCl, capable of aerobic growth at 44±0.5°C and of hydrolysing 4-methlumbelliferyl-β-D-glucoside
(MUD) in the specified medium. The predominant enterococci are E. faecalis, E. faecium, E. durans and E.
hirae

 .4. Sulphite-Reducing Clostridia (SRC): Gram-positive, spore-forming, non-motile, strictly anaerobic rods
that reduce sulphite to H2S.

 5. Clostridium Perfringens: As for SRC, but also ferment lactose, sucrose and inositol with the
production of gas, produce a stormy clot fermentation with milk, reduce nitrate, hydrolyse gelatin and
produce lecithinase and acid phosphatase.

 6. E.coli: E. coli is considered a more suitable indicator as it is almost exclusively fecal in origin and
indicates recent faecal contamination. The mere presence of E. coli in food or water does not indicate the
presence of pathogenic microorganisms directly but it increases the probability and risk of the presence
of other pathogenic microorganisms.

 7. Phages: The use of phages as models for indicating the likely presence of pathogenic enteric bacteria
first appeared in the 1930s, and direct correlations between the presence of certain bacteriophages and

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the intensity of faecal contamination were reported.

# Measurement Of BOD (Biological Oxygen Demand) Of Water :-

Biological Oxygen Demand (BOD) is an important parameter used to measure the amount of oxygen consumed by
microorganisms in wastewater treatment plants. It is measured as mg/L or mg/l.

BOD is a procedure for determining the amount of dissolved oxygen required by aerobic biological organisms in a
body of water to break down organic material present in a given water sample at a specific temperature over a
specific time period.

 Sources of biochemical oxygen demand

Top soil, leaves, and woody debris; animal manure; effluents from pulp and paper mills, wastewater treatment
plants, feedlots, and food-processing plants; failing septic systems; and urban stormwater runoff are all sources of
biochemical oxygen demand.

 Formula For BOD :-

Biological oxygen demand (BOD) is a measure of how much oxygen is consumed by microorganisms in
water bodies. BOD is measured in milligrams per liter (mg/L). BOD is commonly used to determine if
wastewater treatment systems are working properly. If the amount of BOD in the water is high, then the
system may not be removing enough pollutants.

The calculation for BOD is based on the number of organisms present in the sample multiplied by their
metabolic rate. Metabolic rate is the speed at which they consume oxygen.

* To calculate BOD, multiply the number of organisms by their metabolic rate.

Metabolic Rate = Number of Organisms x 0.039 mg O2 / L

For example, if you have 1 million bacteria in a sample, and each consumes 0.039 mg of oxygen per hour,
then the BOD would be:

BOD = 1,000,000 * 0.039 = 39,000 mg/L

This means that the BOD concentration is 39,000 mg/l.

Determination Of COD Of Water:-

Chemical Oxygen Demand (COD) Chemical oxygen demand (COD) is used to determine the quantity of
pollution in water after wastewater treatment. The higher value of chemical oxygen demand indicates the
higher organic pollution in the water sample. Only chemically digest able matter can be determined by the
COD test. COD determination takes less time than the Biological Oxygen Demand test. COD is
recommended where the polluted water has toxicity and organic matter can’t be determined by biological
oxygen demand and useful in water effluent treatment plants. Principle: The organic matter, present in the
water sample is oxidized by potassium dichromate in the presence of sulfuric acid, silver sulfate and
mercury sulfate to produce carbon dioxide (CO2) and water (H2O). The quantity of potassium dichromate
used is calculated by the difference in volumes of ferrous ammonium sulfate consumed in blank and
sample titrations. The quantity of potassium dichromate used in the reaction is equivalent to the oxygen
(O2) used to oxidize the organic matter of wastewater. Preparation of Potassium dichromate (K2Cr2O7)
Solution: Add 6.13 gm Potassium dichromate (previously dried at 105 °C for at least two hours) into 800
ml distilled water. Shake the flask well to dissolve the content and make up the solution to 1000 ml and
mix well. Preparation of Silver sulfate-Sulfuric acid Solution: Dissolve 10 gm Silver sulfate (Ag2SO4) in 500

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ml concentrated sulfuric acid and make up the solution to 1000 ml swirl the flask to mix well. Allow
standing the solution for 24 hours before use. Preparation of Mercury sulfate Solution: Dissolve carefully
0.1 gm of HgSO4 in 5 ml of concentrated Sulfuric acid.

Preparation of Ferrous ammonium sulfate Solution (0.025 M): Dissolve 9.8 g ferrous ammonium sulfate in
a solution of 100 ml of distilled water and 20 ml concentrated Sulfuric acid. Cool the solution and make up
the solution to 1000 ml of distilled water. Standardize the solution to determine the actual concentration
to calculate the chemical oxygen demand. Preparation of Ferroin Indicator: Add 3.5 gm of Iron Sulfate
heptahydrate and 7.5 gm of Phenanthroline monohydrate to 400 ml of distilled water. Mix well to dissolve
and make up to 500 ml of distilled water.

Test for Chemical Oxygen Demand:

1. Take 10 ml of sample into a round bottom reflex flask.

2. Add some glass beads to prevent the solution from bumping into the flask while heating.

3. Add 1 ml of Mercury sulfate (HgSO4) solution to the flask and mix by swirling the flask.

4. Add 5 ml of Potassium dichromate (K2Cr2O7) solution.

5. Now add slowly and carefully 15 ml Silver sulfate- Sulfuric acid solution.

6. Connect the reflex condenser and digest the content using a hot plate for 2 hours.

7. After digestion cools the flask and rinses the condenser with 25 ml of distilled water collecting in the
same flask.

8. Add 2-4 drops of ferroin indicator to the flask and titrate with 0.025 M ferrous ammonium sulfate
solution to the endpoint.

9. Make the blank preparation in the same manner as sample using distilled water instead of the sample.

Calculation Of COD:-

Calculate the chemical oxygen demand by following formula:

COD = 8x1000xDFxMx(VB - VS)/ Volume of sample (in ml)

Where, DF – Dilution Factor (if applicable)

M – Molarity of standardized Ferrous Ammonium Sulfate solution

VB – Volume consumed in titration with blank preparation

VS – Volume consumed in titration with sample preparation

 Example Calculation:

Volume of ferrous ammonium sulfate for Sample (VS) = 23.8 ml

Volume of ferrous ammonium sulfate for Blank VB) = 25.6 ml

Dilution Factor (DF) = 1 (sample used as it is)

COD = 8x1000x1x0.025x(25.6-23.8) ÷ 10 = 8000x0.025x1.8 ÷ 10 = 360÷10 = 36 mg/lit or ppm

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#Determination of TOC Of Water :-

- Total Organic Carbon (TOC) is a measure of the total amount of carbon in organic compounds in pure water and
aqueous systems.

- What is measured when calculating Total Organic Carbon?

When completing TOC analysis, the following is measured:

• TC – Total Carbon

• TIC – Total Inorganic Carbon

• POC – Purgeable Organic Carbon

• NPOC – Non-Purgeable Organic Carbon

• DOC – Dissolved Organic Carbon

• NDOC – Non-Dissolved Organic Carbon

To calculate TOC, you can subtract the total amount of inorganic carbon from total carbon found. Alternatively, you
can add Purgeable and Non-Purgeable Organic Carbon, or Dissolved and Non-Dissolved Organic Carbon. As sums,
they look like:

 TOC = TC - TIC

 TOC = POC + NPOC

 TOC = DOC + NDOC

# Determination Of TDS (Total Dissolved Solids) Of Water:-

- Total dissolved solids (TDS) are the amount of organic and inorganic materials, such as metals, minerals, salts,
and ions, dissolved in a particular volume of water

- TDS in water is due to the dissolved salts and minerals in water which are usually present in the form of ions; ex-
sodium, potassium, carbonates, sulphates etc. Sometimes these dissolved solids can be toxic and also causes
formation of scales in pipes and hence determination of the same is essential.

 TDS can be determined by two methods:

1. Gravimetric analysis: This method is a laboratory method and is time taking but results are accurate. Here, water
sample is prepared by filtering water by 1.5 micron filter so as to separate suspended soilds from the water.

 Procedure-

Step1- Take an empty beaker and note down it’s weight, say- 20 g

Step2- Put water sample in the beaker and take weight again, say-220 g which, means weight of water=220 g – 20
g=200 g.

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Step3- Heat the beaker to evaporate water and once all the water is evaporated take the weight of beaker along
with the residue, say-21 g which means weight of residue is= 21g – 20 g=1 g.

weight of residue= 1 g= 1000 mg

weight of water=200 g

Volume of water=200/1(density of water= 1 g/cc) =200 cc=200 mL=0.2 litres

TDS=1000 mg/0.2 Litres= 5000 mg/L=5000 ppm.

*Note- when water is evaporated, dissolved solids in the form of ions combines to form solid residue.

2. TDS meter: Dissolved solids are usually present in water in the form of ions and ions conduct electricity. This
principle is utilized in finding the TDS of water. TDS meter tip is dipped inside water which measures the amount of
electricity getting conducted and this electricity value is calibrated to TDS value in ppm or mg/L. This method is
very quick to use and is widely popular. Results of this test are approximate because all the dissolved solids
present in water are not present as ions.

Unit-4

1) For Micorrhizae & Arbiscular Mycorrhiza colonization See Core-3 Unit-3 Botany Notes

2) Biological Nitrogen Fixation Is In Core-13 Unit-4 Botany Notes.

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