EVS033 Environmental Microbiology - Toxilogy PDF

School of Architecture, Science and Technology,
Yashwantrao Chavan Maharashtra Open University

Programmes:
V135: Diploma in Environmental Science
Environmental
& Programme
V136
M.Sc. Environmental Science
Microbiology V136: M.Sc. in

Environmental
and
Science
Toxicology
Sem 03
Semester 03 EVS033
Email: director.ast@ycmou.ac.in
Website: www.ycmou.ac.in
Phone: +91-253-2231473
EVS033: Environmental Microbiology and Toxicology Page 1
A S T, Y C M O U , N a s h i k – 4 2 2 2 2 2 , M H , I n d i a
Yashwantrao Chavan EVS033
Maharashtra Open University Environmental Microbiology

and Toxicology
BRIEF CONTENTS
Vice Chancellor’s Message ................................................ 4
Foreword By The Director ................................................ 5
Credit 01 ............................................................................. 6
Unit 01-01: Environmental Microbiology .................. 6
Unit 01-02: Microbial Diversity, Microbial Activities & Metabolism
............................................................................................ 17
Unit 01-03: Microbial Population & Community Dynamics 72
Unit 01-04: Effect & Measurement of Environmental Detriminants
.......................................................................................... 103
Credit 02 ......................................................................... 138
Unit 02-01: Bio Indicators ......................................... 138
Unit 02-02: Determination of Microbiological Quality 147
Unit 02- 03: Biosensor ............................................... 160
Unit 02-04: Bio-transformation, Bio-accumulation, Bio-magnification
.......................................................................................... 179
Credit 03 ......................................................................... 195
Unit 03-02: Environmental Toxicology-1 .............. 213
Unit 03-03 : Non-toxicity ........................................... 224
Unit03- 04: Carcinogenesis ....................................... 232
Credit 04 ........................................................................ 241
Unit 04-01: Environmental Toxicology – 2 ........... 241
Unit 04- 02: Safety regulations- legal control ...... 257
Unit 04-03: Pollution of ecosphere ......................... 265
Unit 04-04: Degradable & non-degradable toxic substances & food
chain ................................................................................ 273

EVS033: Environmental Microbiology and Toxicology
Yashwantrao Chavan Maharashtra Open University

Vice-Chancellor: Prof.Dr. E. Vayunandan
School of Architecture, Science and Technology
Programme Advisory Committee
Dr Sunanda More Dr Manoj Killedar Dr.ChetanaKamlaskar
Director(I/c) & (Retired) Associate Professor, Assistant Professor, School of
Associate Professor, School of School of Architecture, Science & Architecture, Science &
Architecture, Science & Technology, YCMOU, Nashik Technology, YCMOU, Nashik
Technology, YCMOU, Nashik
Prof. Jaydeep Nikam Dr.Pondhe G.M. Prof. Dr. Sharad Ratan Khandelwal
Director, School of Continuing Assot.Professor and Head, Professor & Principal
Education , YCMOU, Nashik Department of Environmental H.A.L. College of Science &
Science Commerce,
PadmshreeVikhe Patil College of Ozar Township Tal: Niphad Dist:
Arts, Science and Commerce, Nashik 422207
Pravaranagar, A/P-Loni-413713
Prof. Pravin Nalawade Dr.Anita V. Handore,
Assistant Professor and Head Department of
Environmental Science, KTHM College, Nashik – 2 Incharge, Research and Development Dept. Sigma
Wineries Pvt.Ltd.,Nashik-422112
Development Team
Course Coordinator and Book Writer Book Editor
Instructional Technology
Editor
Dr. Sunanda More Dr.Anita V. Handore Prof.Dr. Sharad R Khandelwal
Director(I/c) & M.Sc , M. Phil , PhD. M.Sc. , PhD
Associate Professor, School of Dr. Rajib Karmakar
Professor & Principal
Architecture, Science & Technology, M.Sc. , PhD. H.A.L. College of Science & Commerce,
YCMOU, Nashik Assistant Professor & Officer-In- Ozar Township Tal: Niphad Dist: Nashik
Shweta Mahendra More Charge, 422207
Academic coordinator, School of All India Network Project on Pesticide
Architecture, Science & Technology, Residues,
YCMOU, Nashik Directorate of Research, BCKV Dr.Anita V. Handore
M.Sc , M. Phil , PhD
This work by YCMOU is licensed under a CreativeCommons Attribution-

NonCommercial-ShareAlike 4.0 International License .
 Book Publication : 10-Jan-2022 Publication No: 2453
 Publisher : Dr. Dinesh Bhonde, Registrar, YCMOU, Nashik - 422 222, India
 ISBN No. 978-93-92982-58-3

V ICE C HANCELLOR ’ S M ESSAGE
Dear Students,
Greetings!!!
I offer cordial welcome to all of you for the Master’s degree programme of Yashwantrao Chavan
Maharashtra Open University.
As a post graduate student, you must have autonomy to learn, have information and knowledge regarding
different dimensions in the field of Environmental Science and at the same time intellectual development is necessary
for application of knowledge wisely. The process of learning includes appropriate thinking, understanding important
points, describing these points on the basis of experience and observation, explaining them to others by speaking
or writing about them. The science of Education today accepts the principle that it is possible to achieve
excellence and knowledge in this regard.
The syllabus of this course has been structured in this book in such a way, to give you autonomy to study easily
without stirring from home. During the counseling sessions, scheduled at your respective study centre, all your doubts
will be clarified about the course and you will get guidance from some qualified and experienced counsellors/
professors. This guidance will not only be based on lectures, but it will also include various techniques such as
question-answers, doubt clarification. We expect your active participation in the contact sessions at the study
centre. Our emphasis is on ‘self study’. If a student learns how to study, he will become independent in learning
throughout life. This course book has been written with the objective of helping in self-study and giving you autonomy
to learn at your convenience.
During this academic year, you have to give assignments, complete laboratory activities, field visits and the Project
work wherever required. You have to opt for specialization as per programme structure. You will get experience and
joy in personally doing above activities. This will enable you to assess your own progress and thereby achieve a
larger educational objective.
We wish that you will enjoy the courses of Yashwantrao Chavan Maharashtra Open University, emerge
successful and very soon become a knowledgeable and honorable Master’s degree holder of this university.
I congratulate “Development Team” for the development of this excellent high quality “Self- Learning
Material (SLM)” for the students. I hope and believe that this SLM will be immensely useful for all students of this
program.
Best Wishes!
- Prof. Dr. E. Vayunandan
Vice-Chancellor, YCMOU

F OREWORD B Y T HE D IRECTOR
Dear Students,
Greetings!!!
This book aims at acquainting the students with conceptualand applied fundamentals about
Environmental Sciencerequired at degree level.
The book has been specially designed for Science students. It has a comprehensive coverage of
environmentalconcepts and its application in practical life. The book contains numerous examples
to build understanding and skills.
The book is written with self- instructional format. Each chapter is prepared with articulated
structure to make the contents not only easy to understand but also interesting to learn.
Each chapter begins with learning objectives which are stated using Action Verbs as per the
Bloom’s Taxonomy.Each Unit is started with introduction to arouse or stimulate curiosity of learner
about the content/ topic. Thereafter the unit contains explanation of concepts supported by tables,
figures, exhibits and solved illustrationswherever necessary for better effectiveness and
understanding.
This book is written in simple language, using spoken style and short sentences. Topics of e ach unit
of the book presents from simple to complex in logical sequence. This book is appropriate for low
achiever students with lower intellectual capacity and coversthe syllabus of the course.
Exercises given in the chapter include MCQs, conceptual questions and practical questions so as to
create a ladder in the minds of students to grasp each and every aspect of a particular concept.
I thank the students who have been a constant motivation for us. I am grateful to the writers, editors
and the School faculty associated in this SLM development of the Programme.
Best Wishes to all of you!!!
- Dr. Sunanda More

Director,
School of Architecture, Science and Technology,
YCMOU

CREDIT 01
UNIT 01-01: ENVIRONMENTAL MICROBIOLOGY

LEARNING OBJECTIVES
After successful completion of this unit, you will be able -
 To understand the history and role of various scientists to develop knowledge in

microbiology
 To understand the mechanism and role of microorganisms
INTRODUCTION
What is microbiology?
Microbiology is the study of microscopic organisms, such as protists, bacteria, fungi. It also
includes the study of viruses, which are not technically classified as living organisms containing
genetic material.
Brief History of Microbiology
In the history of science, the discovery of living forms invisible to the naked eye was a significant
milestone.Microbiology has a long history which is enriched and developed with contribution of
several individuals. In the beginning, it was addressed in the causes of infectious diseases and
disorders but presently encompassing practical applications of the science.
The Discovery Era (https://microbenotes.com/history-of-microbiology/)
The historians are uncertain about the first person, who had first time observed the microorganisms.
However, the presence of microscope was reported during the mid ‐1600s.
 Robert Hooke
In the 17 th century this scientist has first time used a lens to observe the smallest unit of tissues
which he called ―cells.‖ He was the scientist who has first time observed fungal mycelia among the
specimens of cells he viewed.

(Adapted without modification from:https://fac.ksu.edu.sa/sites/default/files/140_mbio -
final_notes.pdf)
Figure.1.1. Robert Hooke and Early Microscopy. (A) A Drawing of the Microscope Used By
Robert Hooke In 1664
 Antonie van Leeuwenhoek
In 1670s Antonie van Leeuwenhoek has constructed the microscope using combination of lenses
and made careful observations of microscopic organisms, which he called animalcules.Until his
death in 1723, he revealed the microscopic world to scientists and contributed to microbiology by
observing bacteria and protozoa with specific drawings.Because of this extraordinary contribution
to microbiology, he is called as the “Father of Microbiology”.

final_notes.pdf)
. The lens is mounted in the brass plate adjacent to the tip of the adjustable focusing screw. ]
Figure.1.2. TheVan Leeuwenhoek Microscope- Replica
Controversy over concept of spontaneous generation (https://microbenotes.com/history-of-
microbiology/)

A group of scientist has stated that ―Microorganisms spontaneously arise from lifeless matter‖
There was a controversy regarding the concept of spontaneous generation.
The main aspect was to solve the controversy over spontaneous generation which includes
experimentations mainly of Francesco Redi, John Needham, Lazzaro Spallanzani and Nicolas
Appert, etc and to know the disease transmission which mainly includes the work of Ignaz
Semmelweis and John Snow.
 Francesco Redi (1626-1697)
The ancient belief in spontaneous generation was first of all challenged by Redi, an Italian
physician, who carried out a series of experiments on decaying meat and its ability to produce
maggots spontaneously.He has objected this concept.
 John Needham (1713-1781)
He was probably the greatest supporter of the theory of spontaneous generation. He proposed that
tiny organisms the animalcules arose spontaneously on his mutton gravy. He covered the flasks with
cork as done by Redi and even heated some flasks. Still the microbes appeared on mutton broth. H e
was strong supporter of this concept.
 Lazzaro Spallanzani (1729-1799):
He was an Italian Naturalist who attempted to refute Needham‘s experiment. He boiled beef broth
for longer period, removed the air from the flask and then sealed the container. Followed incubation
no growth was observed by him in these flasks. He showed that the heated nutrients could still grow
animalcules when exposed to air by simply making a small crack in the neck. Thus he objected this
concept.
 Nicolas Appert
He followed the idea of Spallanzani‘s work. He was a French wine maker who showed that soups
and liquids can be preserved by heating them extensively in thick champagine bottles. He has
supported this concept. Whereas, .Ignaz Semmelweis and John Snow were the two perso ns who
showed a growing awareness of the mode of disease transmission.
 Schulze (1815-1873) and Theodor Schwan (1810-1882)
These both have observed that air was the source of microbes and sought to prove this by passing
air through hot glass tubes or strong chemicals into boiled infusions in flasks. The infusion in both

the cases remained free from the microbes thus the scientist concluded that air is the source of
micobes, which are killed by boiling or heating.Thus they both have objected the concept.
 George Schroeder and Theodor Von Dusch (1854)
These were the first to introduce the idea of using cotton plugs for plugging microbial culture
tubes. These scientist first reported that t he cotton fibers retain the microorganisms thus acts as
microbial filters.
From the above observation,it is concluded that microorganisms do not arrive from lifeless matter.
Golden Era of Microbiology (https://microbenotes.com/history-of-microbiology/)
 Louis Pasteur-Father of Modern Microbiology / Father of Bacteriology
The Golden age of microbiology began with the work of Louis Pasteur and Robert Koch .More
important there was an acceptance of their work by the scientific community throughout the world
and willingness to continue and expand the work. During this period, we see the real beginning of
microbiology as a discipline of biology.
The concept of spontaneous generation was finally put to rest by the French chemist Louis
Pasteur in an inspired set of experiments involving a goose necked flask. When he boiled broth in a
flask with a straight neck and left it exposed to air, organisms grew. When he did this with his
goose-necked flask, nothing grew. The S-shape of this second flask trapped dust particles from the
air, preventing them from reaching the broth. By showing that he could allow air to get into the
flask but not the particles in the air, Pasteur proved that it was the organisms in the dust that were
growing in the broth.
Pasteur, thus in 1858 finally resolved the controversy of spontaneous generation versus
biogenesis and proved that microorganisms are not spontaneously generated from inanimate matter
but arise from other microorganisms.
 Louis Pasteur
He has introduced the concept of fermentation and reported that fermentation of fruits and grains,
resulting in alcohol, was brought about by microbes and also determined that bacteria were
responsible for the spoilage of wine during fermentation.
In 1862,he has suggested that mild heating at 62.8°C (145°F) for 30 minutes rather than boiling
was enough to destroy the undesirable spore bearing microorganisms without ruining the taste of
the product, the process was called Pasteurization. Pasteurization is defined as heating of

milk/product at 72.1 °C for 30 sec. or 62.8°C for 30 minutes in an properly operated equipment.
His work led to the development of the germ theory of disease.
 Robert Koch
Koch’s four postulates are:
 The organism causing the disease can be found in sick individuals not in healthy ones.
 The organism can be isolated and grown in pure culture.
 The organism must cause the disease when it is introduced into a healthy animal.
 The organism must be recovered from the infected animal and sh own to be the same as the
organism that was introduced.
The combined efforts of many scientists and most importantly Louis Pasteur and Robert Koch
established the Germ theory of disease. The idea that invisible microorganisms are the cause of
disease is called germ theory. This was another of the important contributions of Pasteur to
microbiology. It emerged not only from his experiments disproving spontaneous generation but also
from his search for the infectious organism (typhoid) that caused the deaths o f three of his
daughters.
 John Tyndall (1820 – 1893):
He has introduced the process of killing spore bearing microorganisms by a process known as
Tyndallisation. It is defined as intermittent heating of infusion broth for three consecutive days at
121 °C for 30 min.to kill 100 % spores.An English physicist, deal a final blow to spontaneous
generation in 1877. He conducted experiments in an aseptically designed box to prove that dust
indeed carried the germs. He demonstrated that if no dust was present, ster ile broth remained free of
microbial growth for indefinite period even if it was directly exposed to air. He discovered highly
resistant bacterial structure, later known as endospore, in the infusion of hay. Prolonged boiling or
intermittent heating was necessary to kill these spores, to make the infusion completely sterilized, a
process known as Tyndallisation.
Thus, looking at above observations, Louis Pasteur reported that the diseases causing bacteria of
Anthrax (Bacillus anthracis) and TB bacillus (Mycobacterium tuberculosis.)could be
successfullykilled byTyndallisation.
 Fanne Eilshemius Hesse (1850 – 1934)

He is one of Koch‘s assistant first proposed the use of agar in culture media. Agar was superior to
gelatin because of its higher melting (i.e. 96°C ) and solidifying (i.e. 40-45°C) points than gelatin
and was not attacked by most bacteria. Koch‘s another assistant Richard Petri in 1887 developed the
Petri dish (plate), a container used for solid culture media.
Thus contribution of Robert Koch, Fannie Hesse and Richard Petri made possible the isolation of
pure cultures of microorganisms and directly stimulated progress in all areas of microbiology.At the
beginning of 19 th century,the scientist learned that the microorganisms are not only responsible fo r
causing diseases but are important in the field of agriculture,fermentation and synthesis of valuable
products.This is an exciting time for microbiology.As the foundation of the biosphere and major
determinants of human health, microbes claim a primary, fundamental role in life on earth. Hence,
the study of microbes is pivotal to the study of all living things, and microbiology is essential for
the study and understanding of all life on this planet.
The Spectrum of Microbiology
(https://www.cliffsnotes.com/study-guides/biology/microbiology/introduction-to-microbiology/the-
spectrum-of-microbiology)
Classification highlights characteristics that are common among certain groups while providing
order to the variety of living things. T he science of classification is known
as taxonomyand taxon is an alternative expression for a classification category. Taxonomy displays
the unity and diversity among living things, including microorganisms. Among the first taxonomists
was Carolus Linnaeus. In the 1750s and 1760s, Linnaeus classified all known plants and animals
of that period and set down the rules for nomenclature.
Classification scheme of Microorganisms
The fundamental rank of the classification as set down by Linnaeus is the species. For organisms
such as animals and plants, a species is defined as a population of individuals that breed among
themselves. For microorganisms, a species is defined as a group of organisms that are 70 percent
similar from a biochemical standpoint.
In the classification scheme, various species are grouped together to form a genus. Among the
bacteria, for example, the species Shigella boydii and Shigella flexneri are in the
genus Shigella because the organisms are at least 70 percent similar. Various genera are then
grouped as a family because of similarities, and various families are placed together in
an order. Continuing the classification scheme, a number of orders are grouped as a class, and

several classes are categorized in a single phylum or division. The various phyla or divisions are
placed in the broadest classification entry, the kingdom.
Microorganisms i.e. bacteria are unicellular therefore placed in the group Prokaryotes (No
nucleus and organelles). The other organisms having membrane bound nucleus, multi-cellular and
having variety of organelles are placed in Eukaryotes .While other microorganisms such as fungi,
protozoa, and unicellular algae are eukaryotes (more complex organisms whose cells have a
nucleus and organelles). Acellular agents i.e. Viruses are neither prokaryotes nor eukaryotes
because of their simplicity and unique characteristics (Genomes contain either DNA or RNA; newer
agent is proteinaceous)
[Adapted without modification from: https://www.thoughtco.com/what-are-prokaryotes-and-eukaryotes-129478]

Figure.1.3. Prokaryotic and Eukaryotic Cell
Brief descriptions of Microorganisms
 Bacteria: These are relatively simple, prokaryotic organisms whose cells lack a nucleus or
nuclear membrane. The bacteria may appear as rods (bacilli), spheres (cocci), or spirals (spirilla
or spirochetes). Bacteria reproduce by binary fission, have unique constituents in their cell
walls, and exist in most environments on earth. For instance, they live at temperatures ranging
from 0° to 100°C and in conditions that are oxygen rich or oxygen free. A microscope is
necessary to see and study them.The extremophilic bacteria i.e. Archaebacteria are also included
in the true bacteria.
 Fungi: These are eukaryotic microorganisms that include multicellular molds and unicellular
(single‐celled) yeasts. The yeasts are slightly larger than bacteria and are used in alcoholic
fermentations and bread making. Certain yeasts such as Candida albicans are pathogenic
(disease causing). Molds are filamentous, branched fungi that use spores for reproduction. The
fungi prefer acidic environments, and most live at room temperature under oxygen ‐rich
conditions. The common mushroom is a fungus.
 Protozoa: These are eukaryotic, unicellular organisms. Motion is a characteristic associated

with many species, and the protozoa can be classified according to how they move: Some
protozoa use flagella, others use cilia, and others use pseudopodia. Certain spe cies are
nonmotile. Protozoa exist in an infinite variety of shapes because they have no cell walls. Many
species cause such human diseases as malaria, sleeping sickness, dysentery, and toxoplasmosis.
 Algae: This implies a variety of plantlike organisms. In microbiology, several types of

single‐celled algae are important. Examples are the diatoms and dinoflagellates that inhabit the
oceans and are found at the bases of marine food chains. Most algae capture sunlight and
transform it to the chemical energy of carbohydrates in the process of photosynthesis.
 Viruses: These are ultramicroscopic bits of genetic material (DNA or RNA) enclosed in a
protein shell and, sometimes, a membranous envelope. Viruses have no metabolism; therefore, it
is difficult to use drugs to interfere with their structures or activities. Viruses multiply in living
cells and use the chemical machinery of the cells for their own purpose. Often, they destroy the
cells in the process of replicating.
Microbial Identification
The process of microbial identification and placing them in a taxonomic scheme includes:
1. Microscopic morphology and colony appearance
2. Physiological/biochemical characteristics
3. Chemical analysis
4. Serological analysis
5. Genetic and molecular analysis
 G + C base composition
 DNA analysis using genetic probes
 Nucleic acid sequencing and rRNA analysis
 Phylogenetic analysis

Branches of Microbiology
Pure Microbiology Applied Microbiology Other Microbiology
 Bacteriology  Medical microbiology  Astro microbiology

 Mycology  Pharmaceutical  Biological agent
 Protozoology microbiology  Nano microbiology
 Phycology/algology  Industrial  Predictive
 Parasitology microbiology microbiology, etc
 Immunology  Microbial
biotechnology
 Virology
 Food microbiology
 Nematology
 Agricultural
 Microbial cytology
microbiology
 Microbial physiology
o Plant
 Microbial pathogenesis microbiology
 Microbial ecology o Plant pathology
 Microbial genetics o Soil microbiology
 Cellular microbiology  Veterinary
 Evolutionary microbiology
microbiology  Environmental
 Generation microbiology:
microbiology  Water microbiology (or
 Systems microbiology aquatic microbiology)
 Molecular  Aero-microbiology (or
microbiology air microbiology) etc.
 Phylogeny etc.
Scope of Microbiology (Moselio Schaechter et al.,2003)
Our knowledge and understanding of micro-organisms have led to the development of safe
food, clean water, novel foods, antibiotics, vaccines, healthier plants, animals and soils, and more,
which will defiantly support worldwide Sustainable Development.
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. The study of microbiology contributes greatly to the unde rstanding of life through
enhancements and intervention of microorganisms. Glob ally,there is an increase in demand for
microbiologists in the following branches of Microbiolgy.
 Identifying new and better drug targets for treating disease.

 Novel vaccine development and delivery systems.
 Exploring the mechanisms of spread of antimicrobial resistance.
 Using microbes, viruses, and phages as therapeutic agents for diseases, such as cancer and
congenital defects.
 Gaining a thorough understanding of the physiology of those microbes that have not been
cultivated in the laboratory.
 Determining the pools and fluxes of small molecules within cells.
 Exploring the variety of basic biological functions under extreme (low -high) temperatures.
 Determining how microbial cells in a population differ in structure and function.
 Exploring the diversity of microbial associations with hosts and the diversity of possible
microbe-host interactions. Determining how microbes drive the development and diversification
of macro-organisms.
 Unraveling the environmental causes for the rise of virulence factors, the crossing of the species
barriers of pathogens, and the ensuing emergence of novel diseases.
 Determining the fate of the 45,000 chemical compounds that humans use in daily life, most of
which are subject to microbially mediated processes.
 Uncovering the complete landscape of chemical reactions executed by bacterial enzymes and
their artificially evolved variants.
 Elaborating new concepts and methods for function assignment to orphan g enes and unknown
ORFs.
 Defining the stability or ―health‖ of naturally occurring microbial communities.
 Exploring the extent of microbial impacts on climate change and the effects of climate change
on microbes.
 Determining the extent to which bacterial viruses (phages) facilitate gene flux.
In this way, the future is bright for microbiology. Advancements in the study of infectious
disease, microbial ecology, plant and animal pathology, and biotechnology promise to improve
human life and the well being of the environment, and new opportunities have come about through
social and scientific changes. Progress on these synthetic activities will be hastened through
improvements in technology and through changes in education and training.

Limitations (Moselio Schaechter et al.,2003)
Several technological hurdles stand before today‘s microbiology researchers. To make progress,
science should not accept the limitations placed on discovery by traditional methods, conventional
approaches, or existing infrastructure. Particular attention should be focused on the technologies
that enable genomics, single-cell analyses, microbial cultivation, and establishment and
maintenance of microbiological databases.
A number of obstacles to progress in microbiology research lay out side the realm of technology. If
microbiological science is to progress at a desirable rate, the following must take place:
 Researchers must integrate their work with that of scientists in related fields.
 Computational scientists should become more famili ar with and integral to microbiology.
 Microbiology materials and data must be more carefully curated.
 Powerful, but expensive, modern equipment should be housed in community facilities, open to
researchers who might not otherwise have access to these techn ologies.
 Isolation and cultivation of uncultivable bacteria are still challenge to microbiologist
SOLVED PROBLEMS
1) Define microbiology. Discuss golden era of Microbiology.
a) Solution: Please refer the topic history of Microbiology
2) Enlist Koch postulates.
a) Solution -Please refer the topic history of Microbiology
3) Enlist ten different scopes of microbiology.
a) Solution- Please refer the topic scope of Microbiology
4) Discuss principle and method of Gram’s Staning.
a ) Solution: Please refer the topic Gram‘s Staning.
SELF-TEST
1) Who is called “Father of Microbiology?”
a) Robert Hook
b) Antonie Van Leeuwenhoek
c) John Needham
d) Lazzaro Spallanzani
[Answer Key: b) Antonie Van Leeuwenhoek]
2) Bacteria are placed in following group
a) Eukaryotes
b) Prokaryotes
c) Both a and b

d) None of above
[Answer Key: b) Prokaryotes]
3) Viruses are the...
a) Obligate intracellular parasite
b) Obligate extracellular parasite
c) Phototrophic particle
d) Saprophyte
[Answer Key :a)Obligate intracellular parasite]
SHORT ANSWER QUESTIONS -SAQ

SAQ 1 .Write note on: Antonie Van Leeuwenhoek
a) Solution-Please refer the topic history of Microbiology
SAQ 2 Write note on: Viruses
b) Solution-Please refer the topic Viruses
UNIT 01-02: MICROBIAL DIVERSITY, MICROBIAL ACTIVITIES &

METABOLISM
LEARNING OBJECTIVES
 To understand classification of microorganisms on nutrition, photosynthesis and Nitrogen

fixation
 To learn various methods of determination of microbial numbers
 To learn various bio-chemical tests for microorganisms
Microbial structure (https://fac.ksu.edu.sa/sites/default/files/140_mbio -final_notes.pdf)
Major groups of Microorganisms are as:
I. Bacteria
Bacteria are microscopic, single-celled organisms that exist in their millions, in every environment,
both inside and outside other organisms. These are prokaryotes,which means they have no nucleus.
A bacterial cell includes:
 Cell wall: A layer that is made of a polymer called peptidoglycan. The cell wall gives the
bacteria its shape. It is located outside the plasma membrane. The cell wall is thicker in some
bacteria, called Gram positive bacteria.

 Plasma membrane: Found within the cell wall, this generates energy and transp orts chemicals.
The membrane is permeable, which means that substances can pass through it
 Cytoplasm: A gelatinous substance inside the plasma membrane that contains genetic material
and ribosomes.
 Capsule: A layer found on the outside of the cell wall in some bacteria.
 Flagella: This is used for movement, to propel some types of bacteria. There are some bacteria
that can have more than one. The arrangement of flagella can be as:
(i) Monotrichous – single flagella on one side
(ii) Amphitrichous – single or tuft on both sides
(iii) Lophotrichous – tuft of flagella on one side
(iv) Peritrichous – surrounded by lateral flagella

final_notes.pdf)
Figure.1.4. Arrangement of Bacterial Flagella
 Pili: These hair-like appendages on the outside of the cell allow it to stick to surfaces and
transfer genetic material to other cells. This can contribute to the spread of illness in humans.

[Adapted without modification from :https://www.pinterest.com/pin/220043131763326948/ ]
Figure.1.5. Structure of Bacteria
 Spore: Some bacteria have the ability to form highly resistant resting stage called spores, which
helps them to overcome adverse environmental conditions that are unfavorable for vegetative
growth of cell. They are not a reproductive form and not a storage granule. These spores are
resistant to bactericidal agents and adverse physical conditions. Each spore can give rise to only
one endospore which plays a role in heat resistance. Spores consists of three layers namely core,
cortex and spore coat
 DNA: This contains all the genetic instructions used in the development and function of the
bacterium. It is located inside the cytoplasm.
 Ribosomes: This is where proteins are made, or synthesized. Ribosomes are complex particles
made up of RNA-rich granules.
Morphological distinction and Classification of bacteria
Principle of Gram Staining and Technique
Gram Staining technique was proposed by Christian Gram to distinguish the two types of bacteria
based on the difference in their cell wall structures. The gram -positive bacteria retain the crystal
violet dye, which is because of their thick layer of peptidoglycan in the cell wall.
This process distinguishes bacteria by identifying peptidoglycan that is found in the cell wall of the
gram-positive bacteria. A very small layer of peptidoglycan is dissolved in gram-negative
bacteria when alcohol is added.

Gram positive and gram negative are two differentiations found in bacteria, which can be used to
classify bacteria. The differentiation is based on the thickness of the peptidoglycan layer, which is
found in the cell wall. Peptidoglycan is found in both gram positive and gram negative bacteria. It
provides mechanical support and the characteristic shape to the bacteria. Peptidoglycan layer of
gram positive bacteria is multilayered. But, it is a monolayer in gram negative bac teria. Due to the
thickness of the peptidoglycan layer, gram positive bacteria is capable of retaining the gram stain,
crystal violet-Iodine complex, inside the cell wall. Hence, they can be visualized under the
microscope in purple color. However, gram ne gative bacteria are unable to retain the gram stain and
they can be stained by the counter stain safranin. On the other hand, gram negative bacteria
contains an outer membrane, which gives the antibiotic resistance to the bacteria. Some bacteria
like Mycoplasma species lack peptidoglycans in the cell wall and are unable to be distinguished as
gram positive or gram negative. These species bear some membrane structures of both gram
positive and gram negative bacteria. The main difference between gram positive and gram negative
bacteria is the thickness of cell wall peptidoglycan layer present in each bacteria.
Examples
-Gram Positive Bacteria: Lactobacillus spp., Actinomyces spp, Bacillus spp, Clostridium spp,
Corynebacterium spp, Staphylococci spp, and Strept ococci spp.
-Gram Negative Bacteria: Acetobacterspp.,Chlamydiaspp.,Borreliaspp.,, Bortadellaspp.,,

Burkholderia spp., Enterobacter spp.,, Escherichia spp.,, Helicobacter spp.,, Klebsiella spp., and
Neisseria spp.,
[Adapted without modificationfrom :https://byjus.com/biology/difference-between-gram-positive-

and-gram-negative-bacteria/]
Figure.1.6. Gram Staining of Bacteria

Classification of bacteria on the basis of shape
(https://www.ramauniversity.ac.in/online
studymaterial/pharmacy/bpharma/iiisemester/pharmaceuticalmicrobiology/lecture -2.pdf)
In the year 1872 scientist Cohn classified bacteria to 4 major types depending on their shapes are as
follows:
A) Cocci:These types of bacteria are unicellular, spherical or elliptical shape. Either they may
remain as a single cell or may aggregate together for various configurations. They are as follows:
 Monococcus: they are also called micrococcus and represented by single, discrete round
Example: Micrococcus flavus.
 Diplococcus: the cell of the Diplococcus divides ones in a particular plane and after division,
the cells remain attached to each other. Example: Diplococcus pneumonia.
 Streptococcus: here the cells divide repeatedly in one plane t o form chain of cells.
Example:Streptococcus pyogenes.
 Tetracoccus: this consists of four round cells, which defied in two planes at a right angles to
one another. Example: – Gaffkya tetragena. Staphylococcus: – here the cells divided into three
planes forming a structured like bunches of grapes giving and irregular configuration. Example:
– Staphylococcus aureus.
 Sarcina:in this case the cells divide in three planes but they form a cube like configuration
consisting of eight or sixteen cells but they have a regular shape. Example: –Sarcina lutea.
[Adapted without modification from:https://www.shutterstock.com/search/cocci+bacteria ]
Figure.1.7. Shape of CocciBacteria
B) Bacilli: These are rod shaped or cylindrical bacteria which either remain singly or in pairs.
Example: – Bacillus cereus.

[Adapted without modification from:https://www.offset.com/search/bacilli]
Figure.1.8. Shape of Bacilli Bacteria
C) Vibro:The vibro are the curved, comma shaped bacteria and represented by a single genus.
Example:–Vibro cholerae.
[Adapted without modification from:https://www.microscopemaster.com/vibrio-bacteria.html]
Figure.1.9. Shape of Vibro Bacteria
D) Spirilla: These type of bacteria are spiral or spring like with multiple curvature and termina l
flagella.Example: –Spirillum volutans.
[Adapted without modification from:https://www.alamy.com/stock-photo/spirillum-bacteria.html]

Figure.1.10. Shape of Spirilla Bacteria
E) A spirochaete or spirochete: These type of bacteria have long, helically coiled cells.
Spirochaetes are chemoheterotrophic in nature, with lengths between 3 and 500 μm and diameters
around 0.09 to at least 3 μm. Spirochaetes are distinguished from other bacterial phyla by the
location of their flagella, called endoflagella
Example: –Treponema pallidum
[Adapted without modification without modification from:

https://www.shutterstock.com/search/spirochetes]
Figure.1.11. Shape of Spirochaete or Spirochete Bacteria
Others:-
 Actinomycetes are branching filamentous bacteria, so called because of a fancied

resemblance to the radiating rays of the sun when seen in tissue lesions (from actis meaning
ray and mykes meaning fungus).
 Mycoplasmas are bacteria that are cell wall deficient and hence do not possess a stable
morphology. They occur as round or oval bodies and as interlacing filaments.
Classification on the basis of Mode of Nutrition
1.Phototrophs:
 Those bacteria which gain energy from light.
 Phototrops are further divided into two groups on the basis of source of electron.
 Photo-lithotrophs: these bacteria gain energy from light and uses reduced inorganic compounds
such as H 2 S as electron source. Eg. Chromatium okenii.

 Photo-organotrophs: these bacteria gain energy from light and uses organic compounds such as
succinate as electron source.
2. Chemotrophs:
 Those bacteria gain energy from chemical compounds.
 They cannot carry out photosynthesis.
 Chemotrops are further divided into two groups on the basis of source of electron.
 Chemo-lithotrophs: they gain energy from oxidation of chemical compound and reduce
inorganic compounds such as NH 3 as electron source. Eg. Nitrosomonas.
 Chemo-organotrophs: they gain energy from chemical compounds and uses organic compo und
such as glucose and amino acids as source of electron. eg. Pseudomonas pseudoflava
3. Autotrophs:
 Those bacteria which uses carbon dioxide as sole source of carbon to prepare its own food.
 Autotrophs are divided into two types on the basis of energy ut ilized to assimilate carbon
dioxide. i.e. Photoautotrophs and chemoautotrophs
 Photoautotrophs: they utilized light to assimilate CO 2 . They are further divided into two group
on the basis of electron sources. i.e. Photolithotropic autotrophs and Photo organotropic
autotrophs
 Chemoautotrophs: They utilize chemical energy for assimilation of CO 2.
4. Heterotrophs:
 Those bacteria which uses organic compound as carbon source.
 They lack the ability to fix CO 2.
 Most of the human pathogenic bacteria are heterotropic in nature.
 Some heterotrops are simple, because they have simple nutritional requirement. However there
are some bacteria that require special nutrients for their growth; known as fastidious
heterotrophs.
Classification of bacteria on the basis of Oxygen Requirement
Obligate Aerobes:

 Require oxygen to live.
Example: Pseudomonas spp, common nosocomial pathogen.
Facultative Anaerobes:
 Can use oxygen, but can grow in its absence.
 They have complex set of enzymes.
Examples: E. coli, Staphylococcus spp, yeasts, and many intestinal bacteria.
Obligate Anaerobes:
 Cannot use oxygen and are harmed by the presence of toxic forms of oxygen.
Examples: Clostridium spp. that cause tetanus and botulism.
Aerotolerant Anaerobes:
 Cannot use oxygen, but tolerate its presence.
 Can break down toxic forms of oxygen.
Example: Lactobacillus spp carries out fermentation regardless of oxygen presence.
Microaerophiles:
 Require oxygen, but at low concentrations.
 Sensitive to toxic forms of oxygen.
Example: Campylobacter spp.
Classification of bacteria on the basis of Temperature Requirement

Bacteria can be classified into the following major types on the basis of their temperatures response
as indicated below:
1.Psychrophiles:
 Bacteria that can grow at 0°C or below but the optimum temper ature of growth is 15 °C or
below and maximum temperature is 20°C are called psychrophiles
 Psychrophiles have polyunsaturated fatty acids in their cell membrane which gives fluid
nature to the cell membrane even at lower temperature.
 Examples: Vibrio psychroerythrus, vibrio marinus, Polaromonas vaculata, Psychroflexus.
2. Psychrotrops (facultative psychrophiles):
 Those bacteria that can grow even at 0°C but optimum temperature for growth is (20 -30)°C

3.Mesophiles:
 Those bacteria that can grow best between (25-40) °C but optimum temperature for growth
is 37°C
 Most of the human pathogens are mesophilic in nature.
Examples: E. coli, Salmonella, Klebsiella, Staphylococci.
4. Thermophiles:
 Those bacteria that can best grow above 45°C
 Thermophiles capable of growing in mesophilic range are called facultative thermophiles.
 True thermophiles are called as Stenothermophiles, they are obligate thermophiles
Thermophils contains saturated fattyacids in their cell membrane so their cell membrane does not
become too fluid even at higher temperature.Some thermophils are known as extreme thermophile
Examples:Streptococcus thermophiles, Bacillus stearothermophilus, Thermus aquaticus.
5. Hypethermophiles:
 Those bacteria that have optimum temperature of growth above 80°C
 Mostly Archeobacteria are hyperthermophiles.
 Monolayer cell membrane of Archeobacteria is more resistant to heat and they adopt to grow
in higher remperature.
 Examples: Thermodesulfo bacterium, Aquifex, Pyrolobus fumari, Thermotoga
Classification of bacteria on the basis of pH of Growth
Acidophiles:
 These bacteria grow best at an acidic pH.
 The cytoplasm of these bacteria is acidic in nature.
 Some acidopiles are thermophilic in nature, such bacteria are called Thermoacidophiles.
Examples: Thiobacillus thioxidans, Thiobacillus, ferroxidans, Thermoplasma, Sulfolobus
2. Alkaliphiles:
 These bacteria grow best at an alkaline pH.
Example: Vibrio cholerae optimum pH. of growth is 8.2.

3. Neutrophiles:
 These bacteria grow best at neutral pH (6.5-7.5).
 Most of the bacteria grow at neutral pH.
Example: E. coli
Classification of bacteria on the basis of Osmotic Pressure Requirement
Halophiles:
Require moderate to large salt concentrations.
 Cell membrane of halophilic bacteria is made up of glycoprotein with high content of

negatively charged glutamic acid and aspartic acids. So high concentration of Na+ ion
concentration is required to shield the –ve charge.
 Ocean water contains 3.5% salt. Most such bacteria are present in the oceans.
e.gArcheobacteria, Halobacterium, Halococcus.
Extreme or Obligate Halophiles:
 Require a very high salt concentrations (20 to 30%).
e.gBacteria in Dead Sea, brine vats.
Facultative Halophiles:
Do not require high salt concentrations for growth, but tolerate upto 2% salt or more. (Most of the
halophiles are found in Red sea)
Classification of bacteria on the basis of Number of Flagella
On the basis of flagella the bacteria can be classified as:
1. Atrichos: – These bacteria has no flagella.
Example: Corynebacterium diptherae.
2. Monotrichous: – One flagellum is attached to one end of the bacteria cell.
Example: Vibrocholerae.
3. Lophotrichous: – Bunch of flagella is attached to one end of the bacteria cell.
Example: Pseudomonas spp.
4. Amphitrichous: – Bunch of flagella arising from both end of the bacteria cell.

Example: Rhodospirillum rubrum.
5. Peritrichous: – The flagella are evenly distributed surrounding the entire bacterial cell.
Example: Proteus vulgaris
Classification of bacteria on the basis of Spore formation
1. Spore forming bacteria: Those bacteria that produce spore during unfavorable condition.
These are further divided into two groups:
i) Endospore forming bacteria: Spore is produced within the bacterial cell.
Examples. Bacillus spp., Clostridium spp, Sporosarcina spp. etc
ii) Exospore forming bacteria: Spore is produced outside the cell.
Example. Methylosinus spp.
2. Non sporing bacteria:
Those bacteria which do not produce spores.
Eg. Salmonella spp.
II.Archaebacteria
Archaea have special characteristics of surviving under extreme temperatures so they are known as
extremophiles. These are single-celled microorganisms as other bacteria with an undeveloped
nucleus and no organelle. Their genetic material is in the form of a loop which is called plasmid.
Anatomy and physiology of archaea are similar to eukaryotic organisms which are the distinctive
feature of this group. Archaea shows unique structure of cell membrane which is composed of
pseudo peptidoglycans Archaebacteria possess a single circular chromosome i n plasmid form which
divides asexually by budding, binary fission and fragmentation. Archaea have one flagellum which
helps in the locomotion and to identify unfavorable conditions for survival. They are mostly
metabolically active in the situation of acid s, pH, pressure, high temperature, and deep water.
Archaebacteria build the same structures as other organisms, but they build them from different
chemical components. For instance, the cell walls of all bacteria contain the chemical
peptidoglycan. Archaeal cell walls do not contain this compound, though some species contain a
similar one. Likewise, Archaebacteria do not produce walls of cellulose (as do plants) or chitin (as
do fungi). The cell wall of archaeans is chemically distinct.from true bacteria .The most striking
chemical differences between Archaebacteria and other living things lie in their cell membrane.

There are four fundamental differences between the archaeal membrane and those of all other cells:
(1) ether linkage. (2) isoprenoid chains. ( 3) branching of side chains (4) chirality of glycerol (5)
Arranging phospholipids as Tetra ethers to form Monolayer of cell membrane. (6) Formation of
Cyclopropane and Cyclohexane Carbon rings by Isoprenoid side chains
[Adapted without modification from: https://en.wikipedia.org/wiki/Archaea]
Figure.1.12. Archaea :Halobacterium spp.
Types of Archaebacteria
A.Crenarchaeota
Archaea having ability to tolerate extreme temperature and acidity is called crenarchaeota. This
type is further divided into two sub-types.
1. Thermophiles: Archaeans that live in extremely hot temperatures are called thermophiles.
They can grow and reproduce at temperature 100 o C, and sometimes even above. They are
found in acidic soils, hot springs and near volcano openings.
Example – Methanopyrus kandleri
2. Mesophiles: Archaeans that live in neither too hot nor too cold, but moderate temperature
are known as mesophiles. They grow and develop the best in temperature ranging from 20 -
40 o C. They are typically found in cheese, yoghurt, etc.
Example – Listeria monocytogenes
3. Psychrophiles: Archaeans that live in extremely cold temperatures are called psychrophiles
or cycrophiles. They can grow and reproduce in temperature ranging from -20 o C to +10 o C.
They are found in arctic and alpine soil, deep ocean water, high lati tude, glacier, snowfields,
etc.
Example – Chryseobacterium greenlandensis

B.Euryarchaeota
Methane producing and salt loving archaea is known as euryarchaeota. This type is further divided
into two sub-types.
1. Methanogens: Archaeans that release methane as waste during the process of digestion or
making energy. They play important role in carbon cycle and are often used in sewage
treatment plant.
Example – Methanocaldococcus jannaschii, Methanobrevibacter smithii
2. Halophiles: Archaeans that flourish in extremely saline locales are known as halophiles.
They can be found anywhere with concentration o f salt five times greater than that of the
ocean such as Great Salt Lake, Owens Lake, Dead Sea, etc.
Example – Dunaliella salina, Halobacterium salinarum.
C.Korarchaeota
According to the researches, this group of archaeans does not belong to the lineage of crenarchaeota
and euryarchaeota but is enriched with mixed culture of both the types. They are found only in high
temperature hydrothermal environment. However, they are not abundant in nature.
III. Fungi
The fungi constitute a unique kingdom of heterotrophic organisms. They show a great diversity in
morphology and habitat. These are the eukaryotic microorganisms that include yeasts, rusts, smuts,
mildews, molds, and mushrooms. There are also many fungus like organisms, including slime
molds and oomycetes (water molds) that do not belong to kingdom Fungi but are of ten called fungi.
Many of these fungus like organisms are included in the kingdom Chromista. Fungi are among the
most widely distributed organisms on Earth and are of great environmental and medical importance.
Many fungi are free-living in soil or water; others form parasitic or symbiotic relationships with
plants or animals. (https://ncert.nic.in/textbook/pdf/kebo102.pdf )
Structures of Fungi:
Fungi (Molds) are saprophytic microorganisms growing on decaying matter. They are non -
photosynthetic. There is substantial variation in the structure, siz e, and complexity of different
fungal species.The main body of most fungi is made up of fine, branching, and usually white,
cottony threads i.e. hyphae. Each fungus will have vast numbers of these hyphae, all intertwining
to make up a tangled web called the mycelium.

In coenocytic fungi, the cytoplasm passes through and among cells of the hypha uninterrupted by
cross walls. . Those fungi that have cross walls are called septate fungi, since the cross walls are
called septa.Yeasts are microscopic, unicellular fungi with a single nucleus and eukaryotic
organelles. They reproduce asexually by a process of budding. In this process, a new cell forms at
the surface of the original cell, enlarges, and then breaks free to assume an independent existence.
Some species of fungi have the ability to shi ft from the yeast form to the mold form and vice versa.
These fungi are dimorphic. (https://www.cliffsnotes.com/study-guides/biology/microbiology/the-
fungi/structure-and-physiology-of-fungi)
Classification of Fungi
Kingdom Fungi are classified based on different modes. The different classification of fungi is as
follows:
Based on Spore Formation
The morphology of the mycelium, mode of sp ore formation and fruiting bodies form the basis for
the division of the kingdom into various classes as:
A. The zygomycota(https://mycology.adelaide.edu.au/descriptions/zygomycetes/)
This group includes the fungi whose mycelium is aseptate.The aseptate hyphae is referred to as
coenocytic.This is the most important character used for classification of zygomycota group.The
Zygomycota, or conjugation fungi, include molds, such as those that invade breads and other food
products. The identifying characteristics of the Zygomycota are the formation of a zygospore during
sexual reproduction and the lack of hyphal cell walls except in reproductive structures. Many (~100
species) are known plant root symbionts.
The mycelia of Zygomycota are divided into three types of hyphae.
a.The rhizoids reach below the surface and function in food absorbtion. Above the surface,
sporangiophores bear the spore-producing sporangia. Groups of rhizoids and sporangiophores are
connected above the surface by stolons. Cell walls separat ing individual cells are absent in all but
reproductive structures, allowing cytoplasm and even nuclei to move between cells.
b.Sexual reproduction is by conjugation (fusing) hyphae from two different strains, followed by
plasmogamy, karyogamy and meiosis and the production of Zygospores.

c. Asexual reproduction in Zygomycota varies greatly among orders and species. Spores may be
formed by the separation and thickening of hyphal cells. They may also be produced in specialized
organs, whose structure is also widely varied.
[Adapted without modification from: https://www.pinterest.com/pin/506232814335566638/]
Figure.1.13. Microscopic Image of Rhizopus stolonifer
B. Ascomycetes
These are multicellular with the exception of yeast such as Saccharomyces spp. They are
saprophytic, decomposers, parasitic or coprophilous (growing on dung). Myceliumis branched and
septate. The asexual spores are conidia produced exogenously on the special mycelium called
conidiophores. Conidia on germination produce mycelium. Se xual spores are called ascospores
which are produced endogenously in sac like asci (singular ascus). These asci are arranged in
different types of fruiting bodies called ascocarps. Some examples are Aspergillus spp. Claviceps
spp and Neurospora spp. and penicillium spp .It is used extensively in biochemical and genetic
work. Many members like morels and truffles are edible and are considered delicacies.
(https://ncert.nic.in/textbook/pdf/kebo102.pdf)
[Adapted without modification from:

https://en.wikipedia.org/wiki/Ascomycota#/media/File:Trichoderma_harzianum.jpg]

[Figure.1.14. Microscopic Image of Trichoderma harzianum]
C. Basidiomycetes
They grow in soil, on logs and tree stumps and in living plant bodies as parasites, e.g., rusts and
smuts. Basidiomycetes commonly known forms of basidiomycetes are mushrooms, bracket fungi or
puffballs. The mycelium is branched and septate. The asexual sp ores are generally not found, but
vegetative reproduction by fragmentation is common. The sex organs are absent, but plasmogamy is
brought about by fusion of two vegetative or somatic cells of different strains or genotypes. The
resultant structure is dikaryotic which ultimately gives rise to basidium. Karyogamy and meiosis
take place in the basidium producing four basidiospores. The basidiospores are exogenously
produced on the basidium .The basidia are arranged in fruiting bodies called basidiocarps. Some
common members are Agaricus spp. (mushroom) .Ustilago spp. (smut) and Puccinia spp. (rust
fungus). (https://ncert.nic.in/textbook/pdf/kebo102.pdf )
[Adapted without modification

from:http://www.botany.hawaii.edu/faculty/wong/Bot201/Basidiomycota/Basidiomycota.htm ]
[Figure.1.15. Basidiospores Germinating from a Rust Teliospore]
D. Deuteromycetes/Imperfect fungi
Commonly known as imperfect fungi because only the asexual or vegetative phases of these fungi
are reported. When the sexual forms of these fungi were discovered they were moved into classes
they rightly belong to. It is also possible that the asexual and vegetative stage have been given one
name (and placed under deuteromycetes) and the sexual stage another (and placed under another
class). Later when the linkages were established, the fungi were correctly identified and moved out
of deuteromycetes. Once perfect (sexual) stages of members of dueteromycetes were discovered
they were often moved to ascomycetes and basidiomycetes. The deuteromycetes reproduce only by
asexual spores known as conidia. The mycelium is septate and branched. Some members are
saprophytes or parasites while a large number of them are decomposers of litter and help in mineral

cycling. Some examples are Alternaria, Colletotrichum etc.
(https://ncert.nic.in/textbook/pdf/kebo102.pdf)
[Adapted without modificationfrom :https://www.sciencedirect.com/topics/biochemistry -genetics-

and-molecular-biology/deuteromycetes]
Figure.1.16. Conidia of Aspergillus spp.
Based on Mode of Nutrition
Unlike green plants, which use carbon dioxide and light as sources of carbon and energy,
respectively, fungi meet these two requirements by assimilating preformed organic matter;
carbohydrates are the preferred nutrient source.
This kind of classification usually depends on assimilation and utilization of organic matter; and
carbohydrates.These fungi mostly metabolite carbohydrates such as glucose, xylose, sucrose and
fructose. These fungi specially metabolize insoluble carbohydrates like starches, cellulose,
hemicelluloses, and lignin. To do so, they must first digest these polymers extracell ularly. Saprobic
fungi obtain their food from dead organic material; parasitic fungi do so by feeding on living
organisms (usually plants), thus causing disease.
(a) Saprophytes(Decaying fungi):
These fungi live on dead organic materials produced by the d ecay of animal and plant tissues. These
fungi lives on dead organic matter or excreta of both plant and animal origin.Vegetative hyphae of
these fungi directly absorb food materials from organic matter. The mycelium may be ectophytic or
endophytic. In the case of Rhizopus the mycelium is ectophytic whereas the rhizoids remain
embedded in the substratum and said to be endophytic.
e.g. Saprolegnia spp., Mucor spp., Rhizopus spp.,, Penicillium spp.,, Morchella spp., , Aspergillus
spp.,, Agaricusspp., etc.

[Adapted without modification from: https://www.researchgate.net/figure/Figure1 -Branched-Oyster-
Mushroom-Role-of-fungi-breaking-down-plant-and-woody-material_fig1_307475743]
Figure.1.17. Saprophytes :Branched Oyster Mushroom
(b) Parasites (Dependent Fungi) :
The parasitic fungi absorb their food material from the living tissues of the hosts on which they
parasitize. Such parasitic fungi are quite harmful to their hosts and cause many serious diseases and
great losses. They are again classified as:
 Obligate parasites -The parasites which survive on living hosts and only on living hosts are
called the obligate parasites. Such parasites cannot be grown upon dead organic culture
media, e.g., Puccini spp .,Peronospora spp., Melampsora spp.etc.
 Facultative saprophytes --The parasitic fungi which usually live on living hosts and
according to their need they adopt saprophytic mode of life for some time are called the
facultative saprophytes, e.g., Taphrina deformans and some smuts.
 Facultative parasites --some parasitic fungi usually pass saprophytic mode of life, but
under certain conditions they parasitize some suitable host and are called the facultative
parasites, e.g., Fusarium spp., Pythium spp., etc.
A.Corn smut B.Ant paras ite
[Adapted without modification from: mac122.icu.ac.jp/gen-ed/fungi-gifs/a07%20parasitic-

fungi.JPG]
Figure.1.18. Parasites: A.Corn smut B.Ant parasite

C. Symbionts (Friendly Fungi):
These fungi grow on or with other living organism but both of them are m utually benefited .Such
relationship is called the ‗symbiosis‘ and the participants the ‗symbionts‘. The most striking
examples are the Lichens spp. and Mycorrhiza. spp. The Lichens are the resultants of the symbiotic
association of algae and fungi. Here both algae and fungi are mutually benefited as fungi provide
shelter for algae and in reverse algae synthesis carbohydrates for fungi.Certain fungi develop in the
roots of higher plants and the mycorrhiza are developed which helps in the absorption of nutr ients
by the host plant. The mycorrhiza may be external or internal.
[Adapted without modification from: https://www.post-gazette.com/life/garden/2019/12/20/Why-

we-like-lichens-Penn-State-Extension-bark-statues/stories/201912200011]
[A.Lichen ]
[Adapted without modification from: https://sdhydroponics.com/2012/04/24/what -is-mycorrhizal-

fungi/]
[B. Mycorrhizal fungi]
Figure.1.19. Symbionts: A.Lichen B. Mycorrhizal fungi]

D) Predacious fungi (Trapping fungi):
These are animal trapping fungi which have developed ingenious mechanisms for capturing small
animals such as eelworms, rotifers or protozoa which they use for food. The most interesting of
these mechanisms is that which utilizes a rapidly constricting rin g around a nematode which holds
it captive while the hyphae sink haustoria into the body of the victim.E.g. Arthrobotrys spp.,
Dactylella spp., and Dactylaria spp., etc.
[Adapted without modification from:https://www.sciencephoto.com/media/156718/view/a rthrobotrys-oliospora]
Figure.1.20. Nematode-Trapping Loops of Fungi Imperfecti
IV.Protists
Protists are defined as member of a group of diverse eukaryotic, predominantly unicellular

microscopic organisms.This is a large complex grouping of mostly unicell ular eukaryotic
organisms. They are morphologically diverse and can be found in most terrestrial, aquatic,
and marine habitats as free-living forms and as parasites of other protists, of fungi, and of plants
and animals. With their nutritional modes restricted primarily t o osmo- and phago-heterotrophy
and phototrophy, protists are metabolically much less diverse than Bacteria and Archaea. Along
with various independent amoeboid groups, major groupings include the Alveolates, composed of
ciliates (e.g., Paramecium spp), dinoflagellates (e.g., Alexandrium spp),
and apicomplexans (e.g., Plasmodium spp), and the Stramenopiles, composed of the brown
and golden-brown algae, diatoms, chrysophytes, oomycetes, and distinct groups of slime molds,
among other groups. Cryptophytes, Rhodophytes, and Haptophytes are other major groupings of
protists. Along with these groups are the diplomonads, trichomonads, microsporidia, amoeba-
flagelates, and euglenoid (Paul V. Dunlap, in Encyclopedia of Biodiversity, 2001)

[Adapted without modification from: https://www.thoughtco.com/protista-kingdom-of-life-
4120782]
Figure.1.21. Microscopic image Paramecium
[Adapted without modification from: https://www.pinterest.com/pin/protist-amoeba--

531284087274695531/]
Figure.1.22. Microscopic Image Amoeba

[Adapted without modification from: https://www.britannica.com/science/Euglena]
Figure.1.23. Microscopic Image Euglena
Algae
Algae: It is an important group of Thallophyta , the primitive and simplest division of the plant
kingdom. These are a ubiquitous and diverse group ranging from unicellular to giant multicellular
forms. They are autotrophic, with simple thallus and lacks tissue differentiation. They are found in
different habitats from fresh to marine waters to extreme conditions. As they are diverse in structure
and habitat there is a lot of diversity in their reproduction and perennation strategies.
Groups of algae:
A. Classification of algae based on habitat: (https://www.biologydiscussion.com/algae/algae -

definition-characteristics-and-structure-with-diagram/46727)
The algae are ubiquitous (present every-where) in distribution, i.e., they are found in fresh water as
well as marine water, on soil, on rock, as epiphytes or parasites on plants and animals, in hot
springs, in desert, on permanent snow-fields etc. But they mainly dwell in aquatic environments.
I. Aquatic Algae:
Aquatic algae may be fresh water (when salinity is as low-as 10 ppm) or marine (when salinity is
33-40%). Again, certain algae grow in brackish water which is unpalatable for drinking, but less
salty than sea water. The fresh water algae usually grow in ponds, lakes, tanks, ditch-es etc.
 Fresh water algae: It may be termed as planktonic when they grow and remain suspended
on the upper part of water (e.g., Volvox, diatom), while the benthic algae are bottom -dwellers. The
algae that grow at air-water interface are called neustonic. The benthic algae may be epilithic, that
grow on stones; epipelic attached to sand or mud; epiphytic — growing on plants; and epizoic —
growing on animal body surface.
E.g.Chlamydomonas spp., Oscillatoria spp., etc.

[Adapted without modification from:https://www.nature.scot/plants-animals-and-
fungi/algae/freshwater-algae]
Figure.1.24. Fresh Water Algae
 The marine algae : It may be supralittoral or sub- aerial, as they grow above the water level
and in the spray zone. The intertidal algae grow in such a depth so that they are exposed
periodically due to tides. Other marine algae are sublittoral, meaning hat they are cons tantly
submerged at depths as great as 30-60 metres (100-200 ft).The supralittoral algae may be edaphic—
that grow in and on the soil, epilithic— growing on stones, epiphytic — growing on plants,
epizoic— growing on animal body surface, and corticolous — growing on tree barks and parasitic
on plants and animals. Some algae (e.g., Chlorella spp.) live endozoically in various protozoa,
coelenterates, molasses etc. Chlorella spp. is unique algae reported to have highest chlorine
E.g. Sargassum spp., Laminaria spp., Ectocarpus, spp., Polysiphonia spp.,, Caulerpa spp.,, Bangia
spp.,, Padina spp., etc.
[Adapted without modification from:http://ourmarinespecies.com/c-seaweed/marine-algae/]
Figure.1.25. Marine Algae
II. Terrestrial Algae:
Some algae are found to grow in terrestrial habitats like soils,‘ rocks, logs etc. The algae that grow
on the surface of the soil are known as saprophytes. Many blue -greens, on the other hand, grow
under the surface of the soil, and are called cryptophytes.
The algae growing in the desert soil may be typified as endedaphic (living in soil), epidaphic
(living on the soil surface), hypolithic (growing on the lower surface of the stones on soil),
chasmolithic (living in rock fissures) and endolithic algae (which are rock penetra ting).

E.g. Oscillatoria sancta, Vaucheria geminata, Chlorella lichina, Euglena sp., Fritschiella sp. and
Phormidium sp.
[Adapted without modification fromhttps://microscopesandmonsters.wordpress.com/tag/terrestrial -algae/]
Figure.1.26. Terrestrial Algae
III. Algae of Remarkable Habitats:
In addition to above mentioned habitats, some algae also occur in uncommon habitats and
termed as:
1. Halophytic Algae (or Eurhaline):
They grow in the highly concentrated salt lakes
e.g.Chlamydomonas ehrenbergli, Dunaliella spp and Stephanoptera spp.etc.
2. Symbiotic Algae:
They grow in association with fungi (Lichen), bryophytes, gymnosperms or angiosperms. The best
examples of symbiotic algae found in association with fungi are Nostoc spp., Gloeocapsa spp. etc.
3. Cryophytic Algae:
This group of algae growing on ice or snow provides attractive colours to snow-covered mountains.
e.g.Red colour - Haemotococcous nivalis; green colour -Chlamydomonas yellowstonensis, Black
colour -Scotiella nivalis and Raphidonema brevirostri , brownish purple - Ancyclonema
nordenskioldii
4. Thermophytes or Thermal Algae:
This group of algae occurs in hot water springs (50 - 70°C) where normal life is not possible. Many
blue-greens
e.g., Oscillatoria brevis, Synechococcus elongates, Heterohormogonium spp.

5. Lithophytes:
They grow on the moist surface of stones and rocks, e.g., Nostoc spp. Gloeocapsa spp etc.
6. Epiphytic Algae:
They grow on other plants including other algal members.
a. Algae on Algae:
Ptilota plumosa and Rhodymenia pseudopalmatta on Laminaria hyperborean .Also, Diatoms on
Oedogonium spp, Spirogyra spp. etc.
b. Algae on Bryophytes:
Blue-green algae like Nostoc spp, Oscillatoria, spp diatoms like Achnanthes spp. etc. grow on
different bryophytes.
c. Algae on Angiosperms:grows on the various angiosperms e.g Cocconis spp., Achnanthes spp.
etc.
7. Epizoic Algae:
The algae growing on animals e.g., Stigeoclonium spp. etc
8. Endozoic Algae:
They grow in the tissues of animals, e.g., Zoochlorella spp. etc
9. Parasitic Algae:
Some algae grow parasitically on different plants and animals.
e.g.Cephaleuros spp. , Rhodochytrium spp. ,Ceratocolax spp.
10. Psammon:
The algae which grow in sandy beaches
e.g., Vaucheria spp., Phormidium spp. etc.
Classification based on pigments

A. Diatoms (Bacillariophyta) : Bacillariophyceae
(https://www.daviddarling.info/encyclopedia/D/diatom.html)
A diatom is any member of a class of unicellular algae, known formally as Bacillariophyceae, that
live in cold waters of relatively low salinity. Diatoms come a wide variety of beautiful, symmetrical
shapes. All have shell-like, brittle cell walls made out of silica (glass) and pectin. The walls, which
are porous to allow materials in to and out of the cell, consist of two i nterlocking halves that fit
together like a pillbox.

Because they depend on sunlight for photosynthesis, diatoms generally live in the upper 200 m of
oceans and bodies of fresh water. Some species simply float in the water currents near the surface;
others attach themselves to larger floating objects or to the sea floor.
When diatoms die, they slowly sink to the seabed. The buildup of trillions of these shells forms
crumbly white sediment known as diatomaceous earth or diatomite, which is used in manufactur ing
pool filters and abrasives, including toothpaste.
[Adapted without modification from: https://www.daviddarling.info/encyclopedia/D/diatom.html]
Figure.1.27. Diatom
B. Red Algae: Rhodophyceae
They are commonly known as red algae due to the presence of a water soluble red pigment,
rphycoerythrin. The plant body may be unicellular (Porphyridium) or multicellular. The
multicellular forms: filamentous(Gonio-trichum),parenchymatous(Porphyra,Crinellia), feathery
(Polysiphonia) pseudoparenchymatous (Helmin - thocladia), or ribbon like (Chondrus) .
The flagellated motile stages are totally absent.The cell wall is two layered and consists of outer
pectic and inner cellulosic layer. The mucilaginous material of the outer layer mainly consists of
agar-agar and carrageenans and constitute major portion of dry weight of the cell wall. Sometimes
the cell wall contains iodine in it. The characteristic red colouration of the algae is due to the
sufficient presence of rphycoerythrin which completely masks the chlorophyl l- a

[Adapted without modification from: https://commons.wikimedia.org/wiki/File:Red_Algae_on_bleached_coral.JPG ]
Figure.1.28. Red Algae
C.Brown algae: Phaeophyceae.
These are a group of algae belonging to class Phaeophyceae.They are multicellular and the colour
depends on the ratio of chlorophyll and the pigment, fucoxanthin. E.g. Ectocarpus spp., Fucus
spp.,, giant kelps, Sargassum spp.,, etc.They are multicellular algae. They are filamentous and
branched. The plant body is thallus, i.e. they lack true roots, stem and leaves. The cell wall is made
up of two layers, outer gummy, made up of algin and inner cellulose, which provides strength.They
have a root-like structure called a holdfast, which anchors them to their substrate. The structure of
holdfast varies in different species. They do not take part in nutrient or water uptake like roots in
plants. They prevent algae from being flown away by the water current.There is a small stalk
present, which is more like a stem. It is called a stipe.They have a flattened structure called the
lamina, blade or frond, which resembles leaves. The surface of the lamina may be smooth or
wrinkled. Sometimes it is coated with slime to p revent attachment of epiphytes.Some of the brown
algae contain special gas-filled bladders called pneumatocysts.
[Adapted without modification from: https://www.dreamstime.com/photos-images/brown-

algae.html]
[Figure.1.29. Brown Algae]
D. Green algae: Chlorophyta: Chlorophyceae

Green algae, members of the division Chlorophyta, . The photosynthetic pigments (chlorophylls a
and b, carotene, and xanthophyll) are in the same proportions as those in higher plants.
Most Chlorophyta are unicelluar, but there are some multicelluar species. S ome are free-living,
some are colonial, others are coenocytic. Cell walls are made of cellulose. Filamentous sporophytes
have singluar lenticular nuclei, which are embedded in a thick cytoplasm. Chlorophyta usually have
biflagellated gametes and contain chlorophylls a and b, although the major pigment is chlorophyll
b. In addition, some tropical species are pigmented by siphonoxanthin and siphonein. They store
starches made from photosynthesis in double -membrane bounded
chloroplasts..(http://www.uobabylon.edu.iq/eprints/publication_12_3410_754.pdf)
[Adapted without modification from: https://raheemtabet.wordpress.com/2013/11/22/green -algae-

chlorophyta/]
Figure.1.30. Green Algae
V. Virus (Obligate Parasites)
Virus particle or virion: It is an infectious agent composed of protein shell (capsid) and, in some
cases, a lipid envelope and nucleic acid (RNA or DNA), Virions have full capacity for replication
when a susceptible host cell is encountered. Viruses depend on specialized host cells for
propagation , supplying the complex metabolic and biosynthetic machinery of eukaryotic or
prokaryotic cells. Viruses obtain energy by utilizing host metabolic system. As obligate intracellular
parasites, during replication, they fully depend on the complicated biochem ical machinery of
eukaryotic or prokaryotic cells. A complete vi rus particle is called a virion.The main purpose of a
virus is to deliver its genome into the host cell to allow its expression (transcription and translation)
by the host cell.
Some Useful Definitions in Virology

 Capsid: The symmetric protein shell that encloses the nucleic acid genome. Often, empty
capsids are by-products of the viral replicative cycle.
 Nucleocapsid: The capsid together with the enclosed nucleic acid. Structural units: The
basic protein building blocks of the capsid.
 Capsomeres: Morphologic units seen in the electron microscope on the surface of virus
particles.
 Capsomeres represent clusters of polypeptides, which when completely assembled form

the capsid.
 Virion: The complete infective virus particle, which in some instances (adenoviruses,
papovaviruses, picornaviruses) may be identical with the nlucleocapsid. In more complex
virions (herpesviruses, myxoviruses), this includes the nucleocapsid plus a surrounding
envelope.
 Detective virus: A virus particle that is functionally deficient in some aspect of

replication. Defective virus may interfere with the replication of normal virus.
 Pseudovirus: During viral replication the capsid sometimes encloses host nucleic acid
rather than viral nucleic acid. Such particles look like ordinary virus, particles when
observed by electron microscopy, but they do not replicate. Pseudovirions contain the
―wrong‖ nucleic acid.
Structure and Function
(Hans R. Gelderblom., Structure and Classification of Viruses,Medical Microbiology. 4th

edition,1996)
Viruses are inert outside the host cell. Small viruses, e.g., polio and tobacco mosaic virus, can even
be crystallized. As obligate intracellular parasites, during replication, they fully dep end on the
complicated biochemical machinery of eukaryotic or prokaryotic cells. The main purpose of a virus
is to deliver its genome into the host cell to allow its expression (transcription and translation) by
the host cell.
A fully assembled infectious virus is called a virion. The simplest virions consist of two basic
components: nucleic acid (single- or double-stranded RNA or DNA) and a protein coat, the capsid,
which functions as a shell to protect the viral genome from nucleases and which during infe ction
attaches the virion to specific receptors exposed on the prospective host cell. Capsid proteins are

coded for by the virus genome. Because of its limited size the genome codes for only a few
structural proteins (besides non-structural regulatory proteins involved in virus replication).
Capsids are formed as single or double protein shells and consist of only one or a few structural
protein species. Therefore, multiple protein copies must self assemble to form the continuous three -
dimensional capsid structure. Self-assembly of virus capsids follows two basic patterns: helical
symmetry, in which the protein subunits and the nucleic acid are arranged in a helix, and
icosahedral symmetry, in which the protein subunits assemble into a symmetric shell that c overs the
nucleic acid-containing core.
Some virus families have an additional covering, called the envelope, which is usually derived in
part from modified host cell membranes. Viral envelopes consist of a lipid bilayer that closely
surrounds a shell of virus-encoded membrane-associated proteins. The exterior of the bilayer is
studded with virus-coded, glycosylated (trans-) membrane proteins. Therefore, enveloped viruses
often exhibit a fringe of glycoprotein spikes or knobs, also called peplomers. In viru ses that acquire
their envelope by budding through the plasma or another intracellular cell membrane, the lipid
composition of the viral envelope closely reflects that of the particular host membrane. The outer
capsid and the envelope proteins of viruses are glycosylated and important in determining the host
range and antigenic composition of the virion. In addition to virus -specified envelope proteins,
budding viruses carry also certain host cell proteins as integral constituents of the viral envelope.
Virus envelopes can be considered an additional protective coat. Larger viruses often have a
complex architecture consisting of both helical and isometric symmetries confined to different
structural components. Small viruses, e.g., hepatitis B virus or the me mbers of the picornavirus or
parvovirus family, are orders of magnitude more resistant than are the larger complex viruses, e.g.
members of the herpes or retrovirus families.
A.Non-Enveloped virus B.Enveloped virus

[Adapted without modification from:https://microbenotes.com/viruses-structure-replication-and-
diagnosis/#structure-of-virus]
Figure.1.31. Structure of Virus
Viruses are small obligate intracellular parasites, which by definition contain either a RNA
or DNA genome surrounded by a protective, virus -coded protein coat. Viruses may be viewed as
mobile genetic elements, most probably of cellular origin and charac terized by a long co-evolution
of virus and host. For propagation viruses depend on specialized host cells supplying the complex
metabolic and biosynthetic machinery of eukaryotic or prokaryotic cells. A complete virus particle
is called a virion. The main function of the virion is to deliver its DNA or RNA genome into the
host cell so that the genome can be expressed (transcribed and translated) by the host cell. The viral
genome, often with associated basic proteins, is packaged inside a symmetric protein capsid. The
nucleic acid-associated protein, called nucleoprotein, together with the genome, forms the
nucleocapsid. In enveloped viruses, the nucleocapsid is surrounded by a lipid bilayer derived from
the modified host cell membrane and studded with an o uter layer of virus envelope
glycoproteins(https://gsbs.utmb.edu/microbook/ch041.htm)
Viruses do not reproduce by division, such as bacteria, yeasts or other cells, but they
replicate in the living cells that they infect. In them, they develop their genomi c activity and
produce the components from which they are made. They encode neither their own protein synthesis
machinery (ribosomes) nor energy-generating metabolic pathways. Therefore, viruses are
intracellular parasites. They are able to re -route and modify the course of cellular processes for the
optimal execution of their own reproduction. Besides the genetic information encoding their
structural components, they additionally possess genes that code for several regulatory active
proteins (such as transactivators) and enzymes (e.g. proteases and polymerases). (Modrow S et
al.,2013)
Classification of viruses
The most commonly and currently used system of virus classification was first developed by Nobel
Prize-winning biologist David Baltimore in the earl y 1970s. In addition to the differences in
morphology and genetics mentioned above, the Baltimore classification scheme groups viruses
according to how the mRNA is produced during the replicative cycle of the
virus.(Ref.https://opentextbc.ca/biology2eopenstax/chapter/viral-evolution-morphology-and-
classification/)

Table.1.1.Classification of Viruses
A.Baltimore Classification
Group Characteristics Mode of mRNA Production Example
I Double-stranded DNA mRNA is transcribed directly from the Herpes simplex

DNA template (herpesvirus)
II Single-stranded DNA DNA is converted to double-stranded Canine parvovirus

form before RNA is transcribed (parvovirus)
III Double-stranded RNA mRNA is transcribed from the RNA Childhood

genome gastroenteritis
(rotavirus)
IV Single stranded RNA Genome functions as mRNA Common cold

(+) (picornavirus)
V Single stranded RNA mRNA is transcribed from the RNA Rabies

(-) genome (rhabdovirus)
VI Single stranded RNA Reverse transcriptase makes DNA from Human

viruses with reverse the RNA genome; DNA is then immunodeficiency
transcriptase incorporated in the host genome; virus (HIV)
mRNA is transcribed from the
incorporated DNA
VII Double stranded DNA The viral genome is double-stranded Hepatitis B virus
viruses with reverse DNA, but viral DNA is replicated (hepadnavirus)
transcriptase through an RNA intermediate; the RNA
may serve directly as mRNA or as a
template to make mRNA
B. Classification by Genome Structure

Genome Structure Examples
 RNA  Rabies virus, retroviruses

 DNA  Herpes viruses, smallpox virus
 Single-stranded  Rabies virus, retroviruses

 Double-stranded  Herpes viruses, smallpox virus
 Linear  Rabies virus, retroviruses, herpes viruses, smallpox

virus
 Circular
 Papillomaviruses, many bacteriophages
 Non-segmented:  Parainfluenza viruses

genome consists of
 Influenza viruses
a single segment of
genetic material
 Segmented: genome
is divided into
multiple segments
C. Classification by Capsid Structure

Capsid Classification Examples
 Naked icosahedral Hepatitis A virus, polioviruses
 Enveloped icosahedral Epstein-Barr virus, herpes simplex virus, rubella virus,
yellow fever virus, HIV-1
 Enveloped helical Influenza viruses, mumps virus, measles virus, rabies virus
 Naked helical Tobacco mosaic virus
 Complex with many proteins; Herpes viruses, smallpox virus, hepatitis B virus, T4
some have combinations of bacteriophage
icosahedral and helical capsid
structures
Anaerobiosis

This phenomenon can be defined as the existence of or occurrence of microorganisms in the
absence or partial presence of oxygen.
In this method, environment free from O 2 is carried out either by displacement of O 2 by inert gas
or by absorption of O 2 by chemical or biological technique for isolation, identification and
preservation of anaerobic bacteria.Anaerobes do not require molecular oxygen for growth and
even die if free oxygen is present. Anaerobes may be unicellular or multicellular. Most fungi are
obligate aerobes, requiring oxygen to survive. However, some speci es, such as the Chytridiomycota
that reside in the rumen of cattle, are obligate anaerobes; for these species, anaerobic respiration is
used because oxygen will disrupt their metabolism or kill them.
Classification:
 Obligate anaerobes- These are harmed by the presence of oxygen.
e.g.Clostridium botulinum and the bacteria which live near hydrothermal vents on the deep -sea
ocean floor.
 Aerotolerant organisms-These cannot use oxygen for growth, but tolerate its
presencee.g.Lactobacilli spp. and Streptococci spp.
 Facultative anaerobes- These can grow without oxygen but use oxygen if it is present
However, recent research showed that human "obligate anaerobes" e.g. Finegoldia magna or
Methanogenic archaea , Methanobrevibacter smithii can be grown in aerobic atmosphere if the
culture medium is supplemented with antioxidants such as ascorbic acid, glutathione and uric
acid.(La Scolaet al.,2014 ; Khelaifia, S et al.,2016)
Microbial metabolism
Throughout earth's history, microbial metabolism has been a driving force behind the development
and maintenance of the planet's biosphere. Microbial metabolism is the means by which a microbe
obtains the energy and nutrients (e.g. carbon) it needs to live and reproduce. Microbes use many
different types of metabolic pathways and species can often be differentiated from each other based
on metabolic characteristics. The specific metabolic properties of a microbe are the major factors in
determining that microbe's ecological niche, and often allow for that mic robe to be useful in
industrial processes or responsible for biogeochemical cycles.
I.Metabolism (http://people.uleth.ca/~selibl/Biol3200/CourseNotes/MetabolismCh5.pdf)
• Metabolism is all of an organism's chemical processes i.e. an emergent property arisin g from

interactions of molecules in the orderly environment of the cell
• Metabolism is very important for the management of cellular material and energy resources
e.g.TCA cycle
 Metabolic reactions
• Metabolic reactions are organized into pathways of enzyme controlled chemical reactions.
• Cells need a supply of molecules and energy
• Cells need to get rid of waste products
e.g. Respiration
 Catabolic pathways
• Break down complex molecules into simple molecules

• Energy stored in complex molecules is made available to do work or transformed into readily
usable chemical forms (i.e., ATP)
• small molecules resulting from the catabolism of complex energy rich molecules may be used by
the cell to build new molecules
e.g., β- oxidation ,Mitochondrial ATP generation
 Energy stored in compounds can be used to perform cellular work
• Mechanical - movement of cilia, chromosomes, organelles
• Transport - movement of substances across membranes
• Chemical - endergonic reactions
e.g. Flagellar movement
 Anabolic pathways
• Use energy for the biosynthesis of complex molecules from simple molecules.
• Energy is obtained from usable chemical forms of energy (i.e., ATP) produced during catabolic
processes or from energy released during catabolic processes
• e.g. synthesis of macromolecules-Ribosomes
 Amphibolic pathways-These pathways may function both catabolically and anabolically

Table.1.2. Basic Principles of Microbial Metabolisms
Sr.No. On basis of On basis of obtaining the reducing On basis of obtaining

obtaining carbon equivalents (Hydrogen atoms or energy for living and
for synthesizing electrons) used either in energy growing:
cell mass conservation or in biosynthetic
reactions
1 Autotrophic – Lithotrophic – reducing equivalents are Phototrophic – energy

carbon is obtained obtained from inorganic compounds is obtained from light
from carbon
dioxide (CO2)
2 Heterotrophic – Organotrophic – reducing equivalents Chemotrophic –
carbon is obtained are obtained from organic compounds energy is obtained from
from organic external chemical
compounds compounds
II. Metabolic Diversity Among

Microorganisms(http://people.uleth.ca/~selibl/Biol3200/CourseNotes/MetabolismCh5.pdf)
• Life is based on organic molecules made of carbon skeletons
• Oxygen and hydrogen are important elements of organic compounds
• Electrons are needed i) for processes that provide en ergy (e.g., movement of electrons along
energy transport chains and during oxidation reduction reactions) for cellular work and ii) to
reduce molecules during biosynthesis
• Molecules that serve as a source of carbon may also provide a source of oxygen and hydrogen
• Microbes show an incredible ability to use organic molecules as carbon sources
Organisms can be classified based on their sources of carbon, energy and electrons
 Carbon Source
o Autotroph – CO 2 is the sole or principal carbon source
o Heterotroph – reduced, preformed, organic molecules from other organisms
 Energy Source

o Phototrophs – Light
o Chemotrophs – oxidation of organic or inorganic compounds
 Electron Source
o Lithotrophs – reduced inorganic chemicals
o Organotrophs – Organic molecules
Table.1.3.Major Nutritional Types of Microorganisms
III Heterotrophic (Chemoorganotrophic) Metabolism
• Conversion of organic substrate molecules to end products by a metabolic pathway that releases
sufficient energy for it to be coupled to the formation of ATP.
• Chemoorganotrophs have three options for generating ATP from organic molecules; the electron
acceptor used differentiates these processes:
i) Aerobic respiration, ii) Anaerobic respiration andiii) Fermentation

i. Respiration
An external terminal electron acceptor is present and is not derived from the organic substrate.
Respiration is a process yielding energy in the form of ATP.1 molecule of ATP on total breakdown
gives 16.4 Kcal energy.This process is brought about by various component s of electron transport
chain,where oxygen acts as terminal acceptor.This results in the generation of proton motive force
(PMF).This process is called as oxidative phosphorylation.
a) Aerobic - O 2 is the terminal electron acceptor.
C 6 H 12 O 6 + 6 O 2 → 6 CO 2 + 6 H 2 O + (ATP + Heat)
b) Anaerobic - compounds other than O 2 serve as electron acceptor (e.g., NO 3 , SO 4 -2, CO 3 -2
fumarate)
*Some microbes can carry out both aerobic and anaerobic respiration – dependent upon the
conditions
ii. Fermentation
Fermentation is another anaerobic (non-oxygen-requiring) pathway for breaking down glucose, one
that's performed by many types of organisms and cells. In fermentation, the only energy extraction
pathway is glycolysis, with one or two extra reactions tacked on at the en d.
Fermentation and cellular respiration begin the same way, with glycolysis. In fermentation,
however, the pyruvate made in glycolysis does not continue through oxidation and the citric acid
cycle, and the electron transport chain does not run. Because th e electron transport chain isn't
functional.(https://www.khanacademy.org/science/ap-biology/cellular-energetics/cellular-
respiration-ap/a/fermentation-and-anaerobic-respiration)
In this process, the ATP is predominantly generated by substrate level phospho rylation
Microbial Photosynthesis.
Microbial Photosynthesis is the process of conversion of light enegy i.e.Photons to chemical energy
i.e.sugar or ATP. Although photosynthesis is most commonly associated with plants, microbial
photosynthesis is also a significant supplier of chemical energy, fueling many diverse ecosystems. A
great variety of living things on earth, including all photosynthetic microorganisms, synthesize their
foods from simple molecules such as carbon dioxide and water. In microorganisms , photosynthesis
occurs in unicellular algae and in photosynthesizing bacteria such as cyanobacteria and green and
purple sulfur bacteria.

In all of the metabolic pathways, organisms obtain energy for cellular work by oxidizing organic
compounds. Basically, bacteria may catabolize compounds from dead plants and animals, or may
obtain nourishment from a living host. Other organisms synthesize complex organic compounds
from simple inorganic substances. The major mechanism for such synthesis is a process calle d
photosynthesis, which is used by plants and many microbes. Essentially, photosynthesis is the
conversion of light energy from the sun into chemical energy. The chemical energy is then used to
convert CO 2 from the atmosphere to more reduced carbon compoun ds, primarily sugars. The word
photosynthesis summarizes the process: photo means light, and synthesis refers to the assembly of
organic compounds. This synthesis of sugars by using carbon atoms from CO 2 gas is also called
carbon fixation. Continuation of life as we know it on Earth depends on the recycling of carbon in
this way Cyanobacteria, algae, and green plants all contribute to this vital recycling with
photosynthesis. Photosynthesis can be summarized with the following equations:
1. Plants, algae, and cyanobacteria use water as a hydrogen donor, releasing O 2.
6 CO2 + 12 H 2 O + Light energy C 6 H 12 O 6 + 6 H 2 O + 6O 2
2. Purple sulfur and green sulfur bacteria use H 2 S as a hydrogen donor, producing sulfur granules
6 CO 2 +12 H 2 S + Light energy C 6 H 12 O 6 +6 H 2 O +12 S
In the course of photosynthesis, electrons are taken from hydrogen atoms, an energy -poor molecule,
and incorporated into sugar, an energy-rich molecule. The energy boost is supplied by light energy,
although indirectly.
Photosynthesis takes place in two stages. (I) .Light -dependent (light) reactions, light energy is used
to convert ADP and to ATP. In addition, in the predominant form of the light -dependent reactions,
the electron carrier NADP + is reduced to NADPH. The coenzyme NADPH, like NADH, is an
energy-rich carrier of electrons. (II.)Light -independent (dark) reactions, these electrons are used
along with energy from ATP to reduce CO 2 to sugar.

[Adapted without modification from: https://s3-us-west-2.amazonaws.com/courses-images/wp-
content/uploads/sites/1094/2016/11/03164139/OSC_Microbio_08_06_LightDepIn.jpg ]
Figure.1.32. Figure.1.32.Reactions of Photosynthesis
I.The Light-Dependent Reactions:
Photophosphorylation is one of the important mechanism of ATP formation.and it occurs only in

photosynthetic cells. In this mechanism, light energy is absorbed by chlorophyll molecule s in the
photosynthetic cell, exciting some of the molecules‘ electrons. The chlorophyll principally used by
green plants, algae, and cyanobacteria is chlorophyll a. It is located in the membranous thylakoids
of chloroplasts in algae and green plants and i n the thylakoids found in the photosynthetic
structures of cyanobacteria. Other bacteria use bacterio -chlorophylls. The excited electrons jump
from the chlorophyll to the first of a series of carrier molecules, an electron transport chain similar
to that used in respiration. As electrons are passed along the series of carriers, protons are pumped
across the membrane, and ADP is converted to ATP by chemiosmosis.This happens by generation of
high proton gradient.
In cyclic photophosphorylation, the electrons eventually return tochlorophyll. In noncyclic

photophosphorylation, which is used in oxygenic organisms, the electrons released from chlorophyll
do not return to chlorophyll but become incorporated into NADPH . The electrons lost from
chlorophyll are replaced by electrons from H 2 O. To summarize: the products of noncyclic
photophosphorylation are ATP (formed by chemiosmosis using energy released in an electron
transport chain), O 2 (from water molecules), and NADPH (in which the hydrogen electrons and
protons were derived ultimately from water).

II.The Light-Independent Reactions:
The Calvin-Benson Cycle .The light-independent (dark) reactions are so named because they
require no light directly. They include a complex cyclic pathway called the Calvin -Benson cycle, in
which CO 2 is ―fixed‖—that is, used to synthesize sugars (C 6 H 12 O 6 )
The Calvin-Benson cycle (Autotrophic CO 2 Fixation)
(https://courses.lumenlearning.com/microbiology/chapter/photosynthesis/)
The light-independent reactions of the Calvin cycle can be organized into following three basic
stages:
 Fixation: The enzyme ribulose bisphosphate carboxylase (RuBisCO) catalyzes the addition
of a CO 2 to ribulose bisphosphate (RuBP). This results in the production of 3-
phosphoglycerate (3-PGA).
 Reduction: Six molecules of both ATP and NADPH (from the light -dependent reactions) are
used to convert 3-PGA into glyceraldehyde 3-phosphate (G3P). Some G3P is then used to
build glucose.
 Regeneration: The remaining G3P not used to synthesize glucose is used to regenerate
RuBP, enabling the system to continue CO 2 fixation. Three more molecules of ATP are used
in these regeneration reactions.
The Calvin cycle is used extensively by plants and photoautotrophic bacteria, and the enzyme
RuBisCO is said to be the most plentiful enzyme on earth, composing 30% –50% of the total soluble
protein in plant chloroplasts. However, besides its prevalent use in photoautotrophs, the Calvin
cycle is also used by many nonphotosynthetic chemoauto trophs to fix CO 2 . Additionally, other
bacteria and archaea use alternative systems for CO 2 fixation. Although most bacteria using Calvin
cycle alternatives are chemoautotrophic, certain green sulfur photoautotrophic bacteria have been
also shown to use an alternative CO 2 fixation pathway.

[Adapted without modification from: https://s3-us-west-2.amazonaws.com/courses-images/wp-
content/uploads/sites/1094/2016/11/03153350/OSC_Microbio_00_CC_CB.jpg]
Figure.1.33. Calvin-Benson Cycle
A. Respiration (http://people.uleth.ca/~selibl/Biol3200/CourseNotes/MetabolismCh5.pdf)
1. Glycolytic pathways
• Breakdown of sugars to pyruvate and similar intermediates (EMP pathway)
• Some production of ATP (substrate-level phosphorylation) and reducing power (reduced
coenzymes; NADH)
• Several pathways by which a cell can break down a sugar (sugars are the major substrates of
catabolic energy releasing reactions used in heterotrophic metabolism).
• Glycolytic pathways are typically anoxic processes that do not require oxygen
• The end-product of glycolysis is commonly pyruvate
COOH

C=O
CH3
i). Embden-Meyerhof pathway (EMP)
• Most common pathway - Central metabolic pathway for eukaryotic cells and many bacteria
(Net reaction --------------------------------------------------------------------------- 10 enzymatic steps)
Glucose + 2 ADP + 2 Pi + 2 NAD + → 2 pyruvate + 2 ATP + 2 NADH + 2 H2O
• ATP production - Substrate level phosphorylation
• Phosphofructokinase is key enzyme in regulating this process, catalyzing the conversion of
fructose – 6-P to fructose 1,6-bisphosphate
ii). Entner-Doudoroff pathway
• Mainly used by Gram negative soil bacteria and a few other Gram -negative bacteria as well
as some Archaea
• Lacks 6-phosphofructokinase (i.e., a key enzyme in Embden -Meyerhof pathway)
(Net reaction -------------------------------------------------------------------------- 9 enzymatic steps)
Glucose + ADP + Pi + NADP + + NAD + → 2 pyruvate + 1 ATP + NADPH + NADH + 1 H 2 O
*NADPH is usually used in biosynthetic pathways
iii). Pentose Phosphate pathway
• Can occur at the same time as the Embden -Meyerhof or the Entner-Doudoroff pathways
• connects the metabolism of 6-C and 5-C sugars
• Consumes 1 ATP
• Products = reducing power (NADPH) and small molecules required for biosynthesis
• 5-C sugars produced (e.g., ribose 5-phosphate; xylulose 5-phosphate)
• Erythrose 4-phosphate is used to synthesize aromatic amino acids and vitamin B6
• Intermediates of the pathway may be fed into the EMP to pr oduce ATP
iv) Methylglyoxal pathway

• Alternative to Embden-Meyerhof pathway during conditions of low phosphate availability
• Consumes 2 ATP but produces pyruvate that can be used to generate ATP
2. Oxidation of pyruvate to 3 CO 2 .
Tricarboxylic acid cycle (TCA cycle; also known as Citric acid cycle, Krebs cycle)
This cycle is discovered by Hans Krebs - 1930s.
Steps involved in the TCA cycle-
Pyruvate + NAD + CoA → Acetyl-CoA + NADH + CO 2
• Three step process mediated by multienzyme pyruvate dehydrogenase c omplex
• Acetyl CoA is a very unstable and reactive product.
• Acetyl CoA feeds it's acetate → TCA cycle
• Carbohydrates, fatty acids and amino acids may be converted into acetyl CoA during
aerobicRespiration
• 2 C enter in a relatively reduced form – acetate and two different C leave in a completely oxidized
form (CO 2 )
• Acetate joins the cycle by enzymatic addition to oxaloacetate (4 C) → formation of citrate
• Cyclical process resulting in the regeneration of oxaloacetate by the decomposition of citrate and
evolution of 2 CO2 per acetate
• Oxidation steps (transfer of electrons) of one acetate results in reduction of 3 NAD +
to 3NADH and 1 FAD to FADH 2 (like NADH it donates its e- to the electron transport chain butat a
lower energy level)
• One step for the production of GTP (substrate level phosphorylation; GTP can be convertedto
ATP)
pyruvate + 4 NAD + + FAD → 3 CO 2 + 4 NADH + 1 FADH 2 + 1 GTP
• TCA cycle source of key biosynthetic intermediates
e.g., oxaloacetate and α-ketoglutarate are precursors to a number of amino acids
The TCA cycle generates total 32 ATP. Acetyl -CoA is starting material for fatty acid biosynthesis.

3. Oxidative phosphorylation
• Reducing power (NADH and FADH 2 ) is used to generate a proton gradient (proton motiveforce)
• NADH - FADH 2 are oxidized – electron transport carrier proteins are reduced and in theprocess H +
are moved across the plasma membrane (prokaryotes) or inner mitochondrial membrane
(eukaryotes). This results in a proton gradient or proton motive force across themembrane. The
movement of H + across the membrane is not completely understood
Chemiosmosis
It is studied by Peter Mitchell (Nobel prize in Chemistry in 1978).This process generates PMF
(Proton Motive Force) which act as driving force for ATP synthesis .
ATP synthase (ATPase)
• F0 subunit – multimeric membrane spanning proton conducting channel
• F1 – multimeric headpiece → inside of the membrane
• Catalyze ADP + Pi → ATP (oxidative phosphorylation)
• Highly conserved throughout all domains of life
• Can also drive reverse reaction ATP → ADP + Pi
• Explains why some obligate fermenters have ATPase - creates proton gradients that can beused to
drive transport and flagella .Much evidence supporting the chemiosmotic theory comes from studies
using chemicals thatinhibit the aerobic synthesis of ATP
• Inhibitors, (e.g., CO, cyanide) inhibit ATP synthesis by blocking the electro n flow preventing
oxidative phosphorylation
• Uncouplers (e.g., dinitrophenol, dicumarol) allow protons to cross the membrane

withoutactivating ATP synthase and prevent ATP synthesis without affecting electron transport
Net ATP Production in Aerobic Respiration
Substrate-level phosphorylation
Glycolysis (EMP) 4 ATP
-2 ATP
Citric acid cycle (2 GTP →2 ATP) 2 ATP

Oxidative phosphorylation
2 NADH (from Glycolysis) 6 ATP
if membrane impermeable to NADH
then e- relayed across membrane
at the expense of 2 ATP 0 -2 ATP
8 NADH (from TCA cycle) 24 ATP
2 FADH 2 (from TCA cycle) 4 ATP
36-38 ATP
Types of organisms on basis of energy source
Autotrophs
Autotrophs are organisms that use energy directly from the sun or from chemical bonds. Commonly
called producers, they use energy and simple inorganic compounds to produce organic molecules.
Autotrophs are vital to all ecosystems because all organisms need organic molec ules and only
autotrophs can produce them from inorganic compounds. There are two basic types of autotrophs:
photoautotrophs and chemoautotrophs.
Photoautotrophs are organisms that use light energy and inorganic carbon to produce organic
materials. Eukaryotic photoautotrophs utilize absorb energy through the chlorophyll molecules in
their chloroplasts while prokaryotic photoautotrophs use chlorophylls
and bacteriochlorophylls present in their cytoplasm. All known photoautotrophs
perform photosynthesis.
E.g.algae, and cyanobacteria
Chemoheterotroph
If the heterotroph uses chemical energy, it is a chemoheterotroph .They obtain energy by

the oxidation of electron donors in their environments.These molecules can
be organic (chemoorganotrophs) or inorganic (chemolithotrophs). Chemotrophs can be
either autotrophic or heterotrophic. Chemotrophs can be found on ocean floors where sunlight
cannot reach or above ground, such as the case with iron bacteria.
e.g., fungi, certain bacteria and archaeans.

Heterotrophs
These are organisms that obtain energy from other living things. Like sea angels, they take in
organic molecules by consuming other organisms, so they are commonly called consumers.
Heterotrophs include all animals and fungi as well as many protists and bacteria Classification on
basis of source of food.
Photoheterotrophs
These are heterotrophic organisms that make use of light energy as their energy source. They also
cannot use carbon dioxide as their sole carbon source. They use organic compounds from the
environment. Heterotrophs are the consumers in the food chain, particularly the herbivores,
carnivores and omnivores.
E.g. purple non-sulfur bacteria, green non-sulfur bacteria, and helio-bacteria.
Microbial Enzymes
Introduction
Enzymes are the biocatalysts which enhances the formation of product from substrate by lowering
the activation energy of chemical reactions. All biochemical reactions are catalysed by enzymes
hence they play crucial role in the metabol ism and bioconversion processes. Chemically, enzymes
are protein or glycoprotein in nature and have definite structural conformations .The active site of
enzyme are involved in the catalytic activity. Enzymes shows specificity to the substrates which
means they are evolved to catalyse one reaction, or a particular class of reaction, and the level of
specificity will depend on the function of the particular enzyme .( Colin J. Jackson, et al.,2010).The
binding of enzyme to substrate involves the active site. This site consists of a unique sequence of
amino acid residues. The factors such as positions, sequences, structures, and properties of the
amino acid residues create a very specific chemical environment within the active site. The enzyme
substrate binding can be represented by three models viz. a) Lock and Key model b) The induced fit
model c) The transition state model.
Enzymes are miracle molecules having transforming capability. They can covert a substrate in to
different product based on its molecular activity. Due to this potential, enzymes have crucial role in
environment. Enzymes are used in bioremediation, biofuel production and various bioconversion
process. Microbes in the environment are subjected to consistent environmental changes and hence
gain capabilities to synthesis variety of biomolecules. This environmental microflora is naturally
trained to degrade or convert toxic compounds to less toxic form or other products by synthesising

various set of enzymes. Hence it is very necessary to stud y the microbial enzymes and their role in
environment.
Enzyme activity
Enzyme activity is the amount of enzyme that will catalyse the transformation of 1 μmol of
substrate/min under specified conditions of pH and temperature (https://doi.org/10.1016/B978-0-12-
397176-0.00001-7).The SI unit is the katal, where as commonly used value is enzyme unit (U) = 1
μmol min−1.The activity of enzyme is strongly influenced by Temperature, pH, Enzyme
concentration, Substrate concentration and presence of Enzyme Inhibitors .
Enzyme Classification
The enzyme classification system is a numerical system which classifies enzymes in group as per
the type of reactions they catalyse moreover they are systematically named describing the chemical
reaction catalysed by them..(ref. Boyce, S. and Tipton, K.F. (2001). This system helps in proper
identification of enzyme with respect to reactions they catalyse.Earlier enzymes were classified into
6 categories until 2018 where seventh enzyme was added by Biochemical Nomenclature Committ ee
of IUPAC and NC-IUBMB. Hence as per the latest system enzymes are classified according to
Table.1.4.A Enzyme Classes
Name Reaction catalysed
Oxidoreductases *AH2 + B = A +BH2
Transferases AX + B = BX + A
Hydrolases A-B + H2O = AH + BOH
Lyases A=B + X-Y = A-B

ǀ ǀ
XY
Isomerases A=B
†
Ligases A + B + NTP = A-B + NDP + P (or NMP + PP)
Translocases AX + B ǀǀ = A + X + ǀǀB
(side 1) (side 2)

*Where nicotinamide-adenine dinucleotides are the acceptors, NAD+ and NADH + H+ are used, by
convention. † NTP = nucleoside triphosphate. ( Ref. A Brief Guide to Enzyme Nomenclature and
Classification,Keith Tipton and Andrew McDonald )
Microbial Enzymes
Microorganisms are live factories which are omnipresent having capability to produce variety of
metabolites. Enzymes are one of the most exploited product from microbes. In environment,
microbes are naturally involved in various bioconversion processes by synthesising various
enzymes, hence they are principle source for industrial purpose. Around 50% of the industrial
enzymes are derived from Yeasts and molds and 30% from bacteria (http://www1.lsbu.
ac.uk/water/enztech/sources.html)). Table 2 and table 3 provides brief overview of microbial
enzymes and their environmental applications.
Table.1.4. B.List of Some Enzymes from Microbial Sources
Source Enzyme Microorganism

Bacteria Cellulase Bacillus sphaericus
Amylase Bacillus subtilis
Protease Bacillus intermedius
Xylanase Bacillus sp.
Lipase Bacillus megaterium
Esterase Bacillus lichenoformis
Pullulanase Bacillus sp.
Penicillinase Bacillus subtilis
Pyrophosphatase Bacillus subtilis
Fungi Amylase Aspergillus oryzae

Glucosidase Aspergillus flavus
Catalase Aspergillus terreus
Pectinase Aspergillus niger
Cellulase Trichoderma reesei
Lipase Rhizopus oryzae
Beta-xylosidase Aspergillus niger

Laccases Coriolopsis sp.
Yeast Lipase Candida rugosa

Lactase Kluyveromyces lactis
Invertase Saccharomyces cerevisiae
Uricase Aspergillus flavus
Ribonuclease Saccharomyces cerevisiae
(Adapted without modificationfrom ,https://doi.org/10.1007/s12033-019-00187-1)
Table.1.5.Applications of Microbial Enzymes in Environment and Industry

Sr.No Enzyme Substrate Reaction Applications
1 Oxidoreductase
1.1 Oxygenase
1.1.1 Monooxygenase Alkane, steroids, Incorporation Protein

fatty acid,and ofoxygen atom to engineering,
aromatic substrate and utilize bioremediation,
compounds substrate as reducing synthetic
agent. chemistry, and
Desulfurization, so forth.
dehalogenation,
denitrification,
ammonification, and
hydroxylation of
substrate
1.1.2 Dioxygenase Aromatic Introduction oftwo Synthetic

compounds oxygen atom to the chemistry,
substrate results in pharmaceutical
intradiol cleaving industry,
and extradiol bioremediation,
cleaving with the and so forth.

formation of
aliphatic product
1.2 Laccase Ortho and Oxidation, Food industry,

paradiphenols, decarboxylation and paper and pulp
aminophenols, demethylation of industry, textile
polyphenols, substrate. industry,
polyamines, nanotechnology,
lignins, and synthetic
aryldiamines chemistry,
bioremediation,
cosmetics, and
so forth.
1.3 Peroxidase
1.3.1 Lignin Halogenated Oxidation of Food industry,

Peroxidase phenolic substrate in the paper and pulp
compounds, presence of industry, textile
polycyclic cosubstrate H2O2 industry,
aromatic and mediator like pharmaceutical
compounds and veratryl alcohol. industry,
other aromatic bioremediation,
compounds and so forth.
1.3.2 Manganese Lignin and other In Food industry,

peroxidase phenolic thepresenceofMn2+ Paper and pulp
compounds and H2O2 the co- industry, textile
substrate catalyses industry,
oxidation ofMn2+ to pharmaceutical
Mn3+ which results industry,
in an Mn3+ bioremediation,
chelateoxalate, and so forth
which in turn
oxidizes the phenolic

substrates.
1.3.3 Versatile Methoxybenzenes The enzyme Industrial

peroxidase and phenolic catalyzes the biocatalyst,
aromatic electron transfer bioremediation,
from an oxidizable and so forth.
substrate, with the Control
formation and
reduction of
compound I and
compound II
intermediates.
2.Hydrolase
2.1 Lipase Organic The hydrolysis of Control ofoil

pollutants such as triacylglycerols to spills, detergent
oil spill glycerols and free- production,
fatty acids baking industry,
paper and pulp
industry,
personal care
products, and so
forth.
2.2 Cellulase Cellulosic Hydrolyses the Textile

substance substrate to simple manufacturing.
carbohydrates. detergent
production,
paper and pulp
industry,
bioremediation,
and so forth.
2.3 Protease Proteins Enzymes that Leather, laundry,

hydrolyze peptide biocatalyst,
bonds in aqueous bioremediation,
environment. and so forth.
(Adapted without modification from Karigar, Chandrakant & Rao, Shwetha.,2011)
Specific Enzyme assay

Isolation and production of enzyme from microbes: Eg.Cellulase
Cellulose is the major structural component of cell wall in plants, algae and Fungi. It represent the
most abundant form of biomass present on the planet as well as promising source of renewable
energy (Saha S et al.,2006;Klemm D, et al.,2005;Bhat M,2000).It is an ubiquitous organic
homopolymer of D-glucose units having β-1,4 glycosidic linkage. Due to its abundance, cellulose is
one of the cheap carbon source for industry utilization and also used in biofuel p roduction.
The degradation of cellulose is done by cellulase enzyme. Bacteria and fungi which utilizes
cellulose as a carbon source can synthesis cellulase enzymes, hence isolation and enzyme
production from these microbes is important from environmental and industrial perspective.
The common methodology for isolating microbes producing enzymes of interest consist of culturing
sample on enzyme specific substrate plate. Colonies forming zone of clearance are screened for
their enzyme producing activity. In this module we will shed light on isolating and testing of
cellulase producing microorganisms from Molasses.(Islam, F.,2018)
1. Isolation of Cellulase producing bacteria.
a. Serially dilute the molasses
b. Take 10 −6 diluted sample and spread on carboxymethyl c ellulose (CMC)
agar media plates with 0.5g KH2PO4, 0.25 g MgSO4, 0.25 g cellulose and 2 g gelatine
c. Incubate the plates overnight at 37 °C.
d. Screening of microbes can be performed by congo red, iodine solution and filter
paper degradation method.
2. Determination of enzyme activity of crude enzyme
a. Culture the isolate showing maximum zone of hydrolysis in LB broth medium with
incubation at 37 °C overnight
b. After incubation, centrifuge the culture and harvest the supernatant containing crude
enzyme.

c. Subject the crude enzyme for estimation of its activity by using dinitrosalisic acid
(DNS) reagent .The end point can be determined by colorimetric/ spectrophotometric
method.
DNS reagent measures the amount of reducing sugar formed by the activity of cellulase on
carboxymethyl cellulose (CMC) which is substrate for the enzyme.
d. The isolate exhibiting highest enzyme activity can be subjected to further
optimization studies and molecular characterization.
SOLVED PROBLEMS
1) Comment on Classification based on nutrition for bacteria.
a) Solution-Please refer the topic bacterial.classification
2) What are Archaebacteria? Give its important characteristics.
a) Solution-Please refer the topic Archaebacteria
3) Discuss classification based on various classes of fungi.
a) Solution-Please refer the topic fungi.
4) What are parasites? Discuss its types with suitable example.
a) Solution-Please refer the topic parsite
5) Discuss important characters of Chlorophyta and Brown Algae.
a) Solution-Please refer the topic Algae
6) Discuss structure and functions of enveloped and non enveloped viruses.
a) Solution-Please refer the topic viruses.
7) Define photosynthesis. Discuss photosynthetic cycle.
a) Solution-Please refer the topic photosynthesis
8) Discuss TCA cycle with its bioenergetics
a) Solution-Please refer the topic TCA cycle
9) Give protocol for microbial enzyme assay with suitable example.
a) Solution-Please refer the topic enzyme
SELF-TEST 02
MCQs
1) Fermentation is
a) Anaerobic process
b) Aerobic process
c) Both a and b
d) None of above
[Answer Key: a) anaerobic process]
2) Calvin-Benson cycle is related to
a) Nitrogen fixation
b) CO2 fixation
c) Ammonia fixation
d) None of above
[Answer Key: b) CO2 fixation]
3) Oxidase test is used to identify

a) Cytochrome oxidase
b) Catalase
c) Nitrates
d) Coagulase
[Answer Key: a) Cytochrome oxidase]
SAQ 01.Write note on Fermentation
a) Solution: Please rfere topic Fermentation
SAQ 02.Describe algae on basis of habitat
a) Solution Please rfere topic algae
UNIT 01-03: MICROBIAL POPULATION & COMMUNITY DYNAMICS

LEARNING OBJECTIVES
 To understand growth curve of bacteria
 To determine generation time of bacteria
 To understand various methods of enrichment and isolation of microorganisms
Determination of Microbial number

Methods for Measurement of Cell Numbers (http://textbookofbacteriology.net/growth_2.html)
Measuring techniques involve direct counts, and indirect viable cell counts.
Direct Microscopic Counts (DMC):
[Direct Counting. (2021, January 4). Retrieved June 6, 2021, from
https://bio.libretexts.org/@go/page/9186]
It includes microscopic counts using a hemocytometer or a counting chamber. The hemocytometer
works by creating a volumetric grid divided into differently sized cubes for accurately counting the
number of particles in a cube and calculating the concentration of the entire sample. One can also
quantify the number of cells in a culture by plating a known volume of the cell culture on a petri
dish with a growth medium, which is also known as a streak plate. If the cells are distributed on the
plate properly, it can generally be assumed that each cell will give rise to a single colony. The
colonies can then be counted and, based on the known volume of the culture that was spread on the
plate, the cell concentration can be calculated. Bacterial c olony counts made from plating dilutions
of bacteria are useful to estimate the strength of bacterial infections; for example, a urinary tract
bacterial infection.

[Adapted without modification from:: https://courses.lumenlearning.com/boundless -
microbiology/chapter/counting-bacteria/]
Figure.1.34. Counting Colonies on Streak Plate
As with hemocytometers or counting chambers, cultures need to be heavily diluted prior to plating.
Otherwise, instead of obtaining single colonies that can be counted, a so-called ―lawn‖ of thousands
of colonies will form, all lying atop each other. Additionally, plating is the slowest method because
most microorganisms need at least 12 hours to form visible colonies. These methods of direct
counting do not require sophisticated instrumentation, so they can easily be performed in most
laboratories.
In this way, directly counting blood cells or tissue cells by using a hemocytometer can determine
the concentration of a known volume. Counting the number of colonies that arise on a pour plate
can calculate the concentration by multiplying the count by the volume spread on the pour plate.
Direct counting methods are easy to perform and do not require highly specialized equipment, but
are often slower than other methods.This metho d usually yields higher count because ut counts
living and nonliving cells.
Total Viable Cell (TVC) Counting

[Viable Cell Counting. (2021, January 4). Retrieved June 6, 2021, from
https://bio.libretexts.org/@go/page/9187]
There are a variety of ways to enumerate the number of bacteria in a sample. A viable cell count
allows one to identify the number of actively growing/dividing cells in a sample.
The plate count method or spread plate relies on bacteria growing a colony on a nutrient medium.
The colony becomes visible to the naked eye and the number of colonies on a plate can be counted.
To be effective, the dilution of the original sample must be arranged so that on average between 30
and 300 colonies of the target bacterium is grown. Fewer than 30 colonies makes the interpretation
statistically unsound and greater than 300 colonies often results in overlapping colonies and

imprecision in the count. To ensure that an appropriate number of colonies will be generated several
dilutions are normally cultured. The laboratory procedure involves making serial dilutions of the
sample (1:10, 1:100, 1:1000 etc.) in sterile water and cultivating these on nutrient agar in a dish
that is sealed and incubated.
[Adapted without modification from: from:https://courses.lumenlearning.com/boundless -

microbiology/chapter/counting-bacteria/]
Figure.1.35.Growth of Non-Target Bacteria on Selective Media
Typical media include Plate count agar for a general count or MacConkey agar to count gram -
negative bacteria such as E. coli.Typically one set of plates is incubated at 22°C and for 24 hours
and a second set at 37°C for 24 hours. The composition of the nutrient usually includes reagents
that resist the growth of non-target organisms and make the target organism easily identified, often
by a color change in the medium. Some recent methods include a fluorescent agent so that counting
of the colonies can be automated. At the end of the incubation period the colonies are counted by
eye, a procedure that takes a few moments and does not require a microscope as the colonies are
typically a few millimeters across.
The pour plate method is used when the analysis is looking for bacterial species that grow poorly in
air. The initial analysis is done by mixing serial dilutions of the sample in liquid nutrient agar
which is then poured into bottles. The bottles are then sealed and laid on their sides to produce a
sloping agar surface. Colonies that develop in the body of the medium can be counted by eye after
incubation. The total number of colonies is referred to as the Total Viable Count (TVC). The unit of
measurement is cfu/ml (or colony forming units per milliliter) and relates to the original sample.
Calculation of this is a multiple of the counted number of colonie s multiplied by the dilution used.
Examples of a viable cell count are spread plates from a serial dilution of a liquid culture and pour
plates. With a spread plate one makes serial dilutions in liquid media and then spreads a known

volume from the last tube in the dilution series. The colonies on the plate can then be counted and
the concentration of bacteria in the original culture can be calculated. In the pour plate method a
diluted bacterial sample is mixed with melted agar and then that mixture is pou red into a petri dish.
Again the colonies would be counted and the viable cell count calculated .
Table.1.6.Methods Used To Measure Bacterial Count
Method Application Comments
Direct microscopic count Enumeration of bacteria in Cannot distinguish living from

milk or cellular vaccines nonliving cells
Viable cell count (colony Enumeration of bacteria in Very sensitive if plating

counts) milk, foods, soil, water, conditions are optimal
laboratory cultures, etc.
Turbidity measurement Estimations of large numbers Fast and nondestructive, but

of bacteria in clear liquid cannot detect cell densities less
media and broths than 10 7 cells per ml
Measurement of total N or Measurement of total cell only practical application is in

protein yield from very dense the research laboratory
cultures
Measurement of Biochemical Microbiological assays Requires a fixed standard to

activity e.g. O 2 uptake CO 2 relate chemical activity to cell
production, ATP production, mass and/or cell numbers
etc.
Measurement of dry weight or Measurement of total cell probably more sensitive than
wet weight of cells or volume of yield in cultures total N or total protein
cells after centrifugation measurements
(Adapted from: http://textbookofbacteriology.net/growth_2.html )
Biochemical tests (https://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm)

Theseare the tests used for the identification of bacteria species based on the differences in
the biochemical activities of different bacteria. Bacterial physiology differs from one type of
organism to another.
Table.1.7. Biochemical Tests Used To Identify Bacteria
Tests for Gram Positive Bacteria Tests for Gram Negative Bacteria
 Catalase Test  Oxidase Test
 Mannitol Salt Agar (MSA)  Sugar (eg glucose) broth with
 Blood Agar Plates (BAP) Durham tubes
 Streak-stab technique  Methyl Red / Voges-Proskauer
 Taxos P (optochin sensitivity testing) (MR/VP)
 Taxos A (bacitracin sensitivity  Kliger‘s Iron Agar (KIA)
testing)  Nitrate Broth
 CAMP Test  Motility Agar
 Bile Esculin Agar  MacConkey agar
 Nitrate Broth  Simmon‘s Citrate Agar
 Spirit Blue agar  Urease test
 Starch hydrolysis test  Sulfur Indole Motility Media (SIM)
 Motility Agar
 Coagulase Test
Mannitol Salt Agar (MSA)
This type of medium is both selective and differential. The MSA will select for organisms such
as Staphylococcus species which can live in areas of high salt concentration (plate on the left in the
picture below). This is in contrast to Streptococcus species, whose growth is selected against by this
high salt agar (plate on the right in the picture bel ow).The differential ingredient in MSA is the
sugar mannitol. Organisms capable of using mannitol as a food source will produce acidic
byproducts of fermentation that will lower the pH of the media. The acidity of the media will cause
the pH indicator, phenol red, to turn yellow. Staphylococcus aureus is capable of fermenting
mannitol (left side of left plate) while Staphylococcus epidermidis is not (right side of left plate).

(Adapted without modification from:
https://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm )
Figure.1.36.Growth on Mannitol Salt Agar
Glucose broth with Durham tubes
This is a differential medium. It tests an organism's ability to ferment the sugar glucose as well as
its ability to convert the end product of glycolysis, pyruvic acid into gaseous byproducts. This is a
test commonly used when trying to identify Gram-negative enteric bacteria, all of which are gluco se
fermenters but only some of which produce gas.
Like MSA, this medium also contains the pH indicator, phenol red. If an organism is capable of
fermenting the sugar glucose, then acidic byproducts are formed and the pH indicator turns
yellow. Escherichia coli is capable of fermenting glucose as are Proteus mirabilis (far right)
and Shigella dysenteriae (far left). Pseudomonas aeruginosa (center) is a nonfermenter.
The end product of glycolysis is pyruvate. Organisms that are capable of converting pyruvate to
formic acid and formic acid to H2 (g) and CO2 (g), via the action of the enzyme formic hydrogen
lyase, emit gas. This gas is trapped in the Durham tube and appears as a bubble at the top of the
tube. Escherichia coli and Proteus mirabilis (far right) are both gas producers. Notice that Shigella
dysenteriae (far left) ferments glucose but does not produce gas.
*Note - broth tubes can be made containing sugars other than glucose (e.g. lactose and
mannitol). Because the same pH indicator (phenol red) is a lso used in these fermentation tubes, the
same results are considered positive (e.g. a lactose broth tube that turns yellow after incubation has
been inoculated with an organism that can ferment lactose).

Figure.1.37.Sugar Fermentation Test
Blood Agar Plates (BAP)
This is a differential medium. It is a rich, complex medium that contains 5% sheep red blood cells.
BAP tests the ability of an organism to produce hemolysins, enzymes that damage/lyse red blood
cells (erythrocytes). The degree of hemolysis by these hemolysins is helpful in differentiating
members of the genera Staphylococcus, Streptococcus and Enterococcus.
 Beta-hemolysis is complete hemolysis. It is characterized by a clear (transparent) zone

surrounding the colonies. Staphylococcus aureus, Streptococcus pyogenes and Streptococcus
agalactiae are -hemolytic (the picture on the left below shows the beta-hemolysis of S.
pyogenes).
 Partial hemolysis is termed alpha-hemolysis. Colonies typically are surrounded by a green,

opaque zone. Streptococcus pneumoniae and Streptococcus mitis are -hemolytic (the
picture on the right below shows the a-hemolysis of S. mitis).
 If no hemolysis occurs, this is termed gamma -hemolysis. There are no notable zones around
the colonies. Staphylococcus epidermidis is gamma-hemolytic.

Figure.1.38.Growth on Blood Agar Plates (BAP)
Streak-stab technique
Often when inoculating a BAP to observe hemoloysis patterns, investigators will also stab several
times through the agar using an inoculating loop. This stab allows for the detection of streptolysin
O, a specific hemolysin produced by Streptococcus pyogenes. This hemolysin is inactivated by
O 2 and is only seen subsurface (in an anaerobic environment) around the stab mark. Note the oval -
shaped areas of clearing around the stab marks in the picture below; these are caused by
streptolysin O.

Figure.1.39.Growth of Microaerophilic Bacteria
Bile Esculin Agar

This is a medium that is both selective and differential. It tests the ability of o rganisms to hydrolyze
esculin in the presence of bile. It is commonly used to identify members of the
genus Enterococcus (E faecalis and E. faecium).
The first selective ingredient in this agar is bile, which inhibits the growth of Gram -positives other
than enterococci and some streptococci species. The second selective ingredient is sodium azide.
This chemical inhibits the growth of Gram-negatives.
The differential ingredient is esculin. If an organism can hydrolyze esculin in the presence of bile,
the product esculetin is formed. Esculetin reacts with ferric citrate (in the medium), forming a
phenolic iron complex which turns the entire slant dark brown to black. The tube on the far right
was inoculated with E. faecalis (positive). The tube in the center was inoculated with a bilie esculin
negative organism and the tube on the left was uninoculated.

https://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm)
Figure.1.40.Esculin Hydrolysis test
Sulfur Indole Motility Media (SIM)
This is a differential medium. It tests the ability of an organism to do several things: reduce sulfur,
produce indole and swim through the agar (be motile). SIM is commonly used to differentiate
members of Enterobacteriaceae.
Sulfur can be reduced to H 2 S (hydrogen sulfide) either by catabolism of the amino acid cysteine by
the enzyme cysteine desulfurase or by reduction of thiosulfate in anaerobic respiration. If hyd rogen
sulfide is produced, a black color forms in the medium. Proteus mirabilis is positive for H 2 S
production. The organism pictured on the far left is positive for hydrogen sulfide production.

Bacteria that have the enzyme tryptophanase, can convert the amino acid, tryptophane to indole.
Indole reacts with added Kovac‘s reagent to form rosindole dye which is red in color (indole
+). Escherichia coli is indole positive. The organism pictured second from left is E. coli and is
indole positive. SIM tubes are inoculated with a single stab to the bottom of the tube. If an
organism is motile than the growth will radiate from the stab mark and make the entire tube appear
turbid. Pseudomonas aeruginosa and the strain of Proteus mirabilis that we work with are motile.

Figure.1.41.Sulphur Reduction Test
Kliger’s Iron Agar (KIA)
This is a differential medium. It tests for organisms‘ abilities to ferment glucose and lactose to acid
and acid plus gas end products. It also allows for identification of sulfur reducers. This media is
commonly used to separate lactose fermenting members of the
family Enterobacteriaceae (e.g. Escherichia coli) from members that do not ferment lactose,
like Shigella dysenteriae. These lactose nonfermenting enterics generally tend to be the more
serious pathogens of the the gastrointestinal tract.
The first differential ingredient, glucose, is in very short supply. Organisms capable of fermenting
this sugar will use it up within the first few hours of incubation. Glucose fermentation will create
acidic byproducts that will turn the phenol red indicator in the media ye lllow. Thus, after the first
few hours of incubation, the tube will be entirely yellow. At this point, when the glucose has been
all used up, the organism must choose another food source. If the organism can ferment lactose, this
is the sugar it will choose. Lactose fermentation will continue to produce acidic byproducts and the
media will remain yellow (picture on the far left below). If gas is produced as a result of glucose or
lactose fermentation, then fissures will appear in the agar or the agar will b e lifted off the bottom of
the tube.

If an organism cannot use lactose as a food source it will be forced to use the amino acids / proteins
in the media. The deamination of the amino acids creates NH 3 , a weak base, which causes the
medium to become alkaline. The alkaline pH causes the phenol red indicator to begin to turn red.
Since the incubation time is short (18 -24 h), only the slant has a chance to turn red and not the
entire tube. Thus an organism that can ferment glucose but not lactose, will produce a red slant and
a yellow butt in a KIA tube (second from the left below). These organisms are the more serious
pathogens of the GIT such as Shigella dysenteriae.
If an organism is capable of using neither glucose nor lactose, the organism will use solely a mino
acids / proteins. The slant of the tube will be red and the color of the butt will remain unchanged
(picture on the far right below). Pseudomonas aeruginosa is an example of a nonfermenter.
KIA tubes are also capable of detecting the production of H 2 S. It is seen as a black precipitate
(second picture from the right). Sometimes the black precipitate obscures the butt of the tube. In
such cases, the organisms should be considered positive for glucose fermentation (yellow
butt). Proteus mirabilis (pictured here, second from right) is a glucose positive, lactose negative,
sulfur reducing enteric.

Figure.1.42.Glucose and Lactose Fermentation Test
Nitrate reduction
This is a differential medium. It is used to determine if an organism is capable of reducing nitrate

(NO 3 - ) to nitrite (NO 2 - ) or other nitrogenous compounds via the action of the enzyme nitratase (also
called nitrate reductase). This test is important in the identification of both Gram-positive and
Gram-negative species.
After incubation, these tubes are first inspected for the presence of gas in the Durham tube. In the

case of nonfermenters, this is indicative of reduction of nitrate to nitrogen gas. However, in many
cases gas is produced by fermentation and further testing is necessary to determine if reduction of
nitrate has occurred. This further testing includes the addition of sulfanilic acid (often called nitrate
I) and dimethyl-alpha-napthalamine (nitrate II). If nitrite is present in the media, then it will react
with nitrate I and nitrate II to form a red compound. This is considered a positive result. If no red
color forms upon addition of nitrate I and II, this indicates that either the NO 3 - has not been
converted to NO 2 - (a negative result), or that NO 3 - was converted to NO 2 - and then immediately
reduced to some other, undetectable form of nitrogen (also a positive result). In order to determine
which of the preceding is the case, elemental zinc is added to the broth. Zinc will convert any
remaining NO 3 - to NO 2 - thus allowing nitrate I and nitrate II to react with the NO 2 - and form the red
pigment (a verified negative result). If no color change occurs upon addition of zinc then this means
that the NO 3 - was converted to NO 2 - and then was converted to some other undetectable form of
nitrogen (a positive result).
If the nitrate broth turns red (tubes pictured in the center) after nitrate I and nitrate II are added,
this color indicates a positive result. If instead, the tube turns red (tube pictured on the left) after
the addition of Zn, this indicates a negative result. If there is no color change in the tube after the
addition of nitrate I and nitrate II, the result is uncertain. If the tube is colorless (picture on the
right) after the addition of Zn this indicates a positive test.

Figure.1.43. Nitrate Reduction Test
Catalase Test
This test is used to identify organisms that produce the enzyme, catalase. This enzyme detoxifies
hydrogen peroxide by breaking it down into water and oxygen gas.

2H 2 O 2 2H 2 O+O 2
The bubbles resulting from production of oxygen gas clearly indicate a catalase positive result. The
sample on the right below is catalase positive. The Staphylococcus spp. and the Micrococcus spp.
are catalase positive. The Streptococcus and Enterococcus spp. are catalase negative.

Figure.1.44.Slide Catalase Test
Oxidase Test
This test is used to identify microorganisms containing the enzyme cytochrome oxidase (important
in the electron transport chain). It is commonly used to distinguish between oxidase
negative Enterobacteriaceae and oxidase positive Pseudomadaceae.
Cytochrome oxidase transfers electrons from the electron transport chain to oxygen (the final
electron acceptor) and reduces it to water. In the oxidase test, artificial electron donors and
acceptors are provided. When the electron donor is oxidized by cytochrome oxidase it turns a dark
purple. This is considered a positive result. In the picture below the organism on the rig ht
(Pseudomonas aeruginosa) is oxidase positive.

Figure.1.45.Positive Oxidase Test

Coagulase test
Coagulase is an enzyme that clots blood plasma. This test is performed on Gram -positive, catalase
positive species to identify the coagulase positive Staphylococcus aureus. Coagulase is a virulence
factor of S. aureus. The formation of clot around an infection caused by this bacteria likely protects
it from phagocytosis. This test differentiates Staphylococcus aureus from other coagulase
negative Staphylococcus species.
(Adapted without modification

from:https://www.uwyo.edu/molb2210_lab/info/biochemical_tests.htm )
Figure.1.46.Coagulase Test
Taxos A (bacitracin sensitivity testing)
This is a differential test used to distinguish bet ween organisms sensitive to the antibiotic bacitracin
and those not. Bacitracin is a peptide antibiotic produced by Bacillus subtilis. It inhibits cell wall
synthesis and disrupts the cell membrane. This test is commonly used to distinguish between
the -hemolytic streptococci: Streptococcus agalactiae (bacitracin resistant) and Streptococcus
pyogenes (bacitracin sensitive). The plate below was streaked with Streptococcus pyogenes; notice
the large zone of inhibition surrounding the disk.

Figure.1.47. Bacitracin Test

Taxos P (Optochin sensitivity testing)
This is a differential test used to distinguish between organisms sensitive to the antibiotic optochin
and those not. This test is used to distinguish Streptococcus pneumoniae (optochin sensitive
(pictured on the right below)) from other -hemolytic streptococci (optochin resistant
(Streptococcus mitis is pictured on the left below)).

Figure.1.48. Optochin Test
MacConkey agar
This medium is both selective and differential. The selective ingredients are the bile salts and the
dye, crystal violet which inhibit the growth of Gram -positive bacteria. The differential ingredient is
lactose. Fermentation of this sugar results in an acidic pH and causes the pH indicator, neutral red,
to turn a bright pinky-red color. Thus organisms capable of lactose fermentation such
as Escherichia coli, form bright pinky-red colonies (plate pictured on the left here). MacConkey
agar is commonly used to differentiate between the Enterobacteriaceae.
Organism on left is positive for lactose fermentation and that on the right is negative.
(Adapted without modificationfrom:
Figure.1.49. Lactose Fermentation Test

Simmon’s Citrate Agar
This is a defined medium used to determine if an organism can use citrate as its sole carbon source.
It is often used to differentiate between members of Enterobacteriaceae. In organisms capable of
utilizing citrate as a carbon source, the enzyme citrase hydrolyzes citrate into oxaoloacetic acid and
acetic acid. The oxaloacetic acid is then hydrolyzed into pyruvic acid and C O 2 . If CO 2 is produced,
it reacts with components of the medium to produce an alkaline compound (e.g. Na 2 CO 3 ). The
alkaline pH turns the pH indicator (bromthymol blue) from green to blue. This is a positive result
(the tube on the right is citrate positive). Klebsiella pneumoniae and Proteus mirabilis are examples
of citrate positive organisms. Escherichia coli and Shigella dysenteriae are citrate negative.

Figure.1.50. Citrate Utilization Test
Spirit Blue agar
This agar is used to identify organisms that are capable of producing the enzyme lipase. This
enzyme is secreted and hydrolyzes triglycerides to glycerol and three long chain fatty acids. These
compounds are small enough to pass through the bacterial cell wall. Glycerol can be converted into
a glycolysis intermediate. The fatty acids can be catabolized and their fragments can eventually
enter the Kreb‘s cycle. Spirit blue agar contains an emulsio n of olive oil and spirit blue dye.
Bacteria that produce lipase will hydrolyze the olive oil and produce a halo around the bacterial
growth. The Gram-positive rod, Bacillus subtilis is lipase positive (pictured on the right).The plate
pictured on the left is lipase negative.

[A.Lipase negative] [B.Lipase positive]

Figure.1.51. Lipase Test
Starch hydrolysis test
This test is used to identify bacteria that can hydrolyze starch (amylose and amylopectin) using the
enzymes α-amylase and oligo-1,6-glucosidase. Often used to differentiate species from the
genera Clostridium and Bacillus. Because of the large size of amylose and amylopectin molecules,
these organisms can not pass through the bacterial cell wall. In order to use these starches as a
carbon source, bacteria must secrete α-amylase and oligo-1,6-glucosidase into the extracellular
space. These enzymes break the starch molecules into smaller glucose subunits which can then enter
directly into the glycolytic pathway. In order to interpret the results of t he starch hydrolysis test,
iodine must be added to the agar. The iodine reacts with the starch to form a dark brown color.
Thus, hydrolysis of the starch will create a clear zone around the bacterial growth. Bacillus
subtilis is positive for starch hydrolysis (pictured below on the left). The organism shown on the
right is negative for starch hydrolysis.
[Adapted without modification from :https://www.microbiologyclass.com]

Figure.1.52. Starch Hydrolysis Test.

Methyl Red / Voges-Proskauer (MR/VP)
This test is used to determine which fermentation pathway is used to utilize glucose. In the mixed
acid fermentation pathway, glucose is fermented and produces several organic acids (lactic, acetic,
succinic, and formic acids). The stable production of enough acid to overcome the phosphate buffer
will result in a pH of below 4.4. If the pH indicator (methyl red) is added to an aliquot of the
culture broth and the pH is below 4.4, a red color will appear (first pict ure, tube on the left). If the
MR turns yellow, the pH is above 6.0 and the mixed acid fermentation pathway has not been
utilized (first picture, tube on the right). The 2,3 butanediol fermentation pathway will ferment
glucose and produce a 2,3 butanediol end product instead of organic acids. In order to test this
pathway, an aliquot of the MR/VP culture is removed and -naphthol and KOH are added. They are
shaken together vigorously and set aside for about one hour until the results can be read. The
Voges-Proskauer test detects the presence of acetoin, a precursor of 2,3 butanediol. If the culture is
positive for acetoin, it will turn ―brownish-red to pink‖ (tube on the left in the second picture). If
the culture is negative for acetoin, it will turn ―brown ish-green to yellow‖ (tube on the left in the
second picture). Note: A culture will usually only be positive for one pathway: either MR+ or
VP+. Escherichia coli is MR+ and VP-. In contrast, Enterobacter aerogenes and Klebsiella
pneumoniae are MR- and VP+. Pseudomonas aeruginosa is a glucose nonfermenter and is thus MR -
and VP-.
IMViC Test: MR/VP Test:
[A][B]
(Adapted from: https://laboratoryinfo.com/voges-proskauer-test/)
Figure.1.53. Comparison between A. Methyl Red Test and B.Voges Proskauer Test.

CAMP Test
CAMP factor is a diffusible, heat-stable protein produced by group B streptococci. This is a

synergistic test between Staphylococcus aureus and Streptococcus agalactiae. S.
agalactiae produces CAMP factor. S. aureus produces sphingomyelin C, which binds to red blood
cell membranes. The two bacteria are streaked at 90 o angles of one another. They do NOT touch.
The CAMP factor produced by S. agalactiae enhances the beta-hemolysis of S. aureus by binding to
already damaged red blood cells. As a result, an arrow of beta -hemolysis is produced between the
two streaks. The test is presumptive for S. agalactiae that produces CAMP factor.
In the picture here, Streptococcus agalactiae was streaked throughout the top region of the plate
and brought down toward the center of the plate. Staphylococcus aureus was streaked in a straight
line across the center of the plate. Rings of hemolysis are evident all around S. aureus, however the
hemolysis if greatly enhanced (in an arrow shape) where the S. agalactiae crosses the hemolysis
rings.
(Adapted without modification

from:https://www.uwyo.edu/molb2210_lab/info/bioch emical_tests.htm)
Figure.1.54. CAMP Diffusion Test
Urease test
This test is used to identify bacteria capable of hydrolyzing urea using the enzyme urease. It is
commonly used to distinguish the genus Proteus from other enteric bacteria. The hydrolysis of u rea
forms the weak base, ammonia, as one of its products. This weak base raises the pH of the media
above 8.4 and the pH indicator, phenol red, turns from yellow to pink. Proteus mirabilis is a rapid
hydrolyzer of urea (center tube pictured here). The tube on the far right was inoculated with a
urease negative organism and the tube on the far left was uninoculated.

Figure.1.55. Urea Hydrolysis Test
Motility agar
It is a differential medium used to determine whether an organism is equipped with flagella and
thus capable of swimming away from a stab mark. T he results of motility agar are often difficult to
interpret. Generally, if the entire tube is turbid, this indicates that the bacteria have moved away
from the stab mark (are motile). The organisms in the two tubes pictured on the right are motile. If,
however, the stab mark is clearly visible and the rest of the tube is not turbid, the organism is likely
nonmotile (tube pictured on the left).

Figure.1.56. Motility Test on semisolid Agar

Measurements of Microbial Mass (Measurements of Microbial Mass.,2021,
https://bio.libretexts.org/@go/page/9188)
There are several methods for measuring cell mass, including the gravimeter method which uses
ordinary balances to weigh a sample (dry weight/ml) after the water has been removed.
Microbiologists use turbidity as a measure of cell density within a culture sample. Microbiologists
use machines called photometers and spectrophotometers that shine different types of light through
culture samples to determine turbidity. The general assumption is that the h igher the turbidity, the
higher number of cells within the culture.
Turbidimetry is used as indirect method for calculating cell mass. Cell cultures are turbid: they
absorb some of the light and let the rest of it pass through. The higher the cell concent ration is, the
higher the turbidity. Spectrophotometers are electrical appliances that can measure turbidity very
accurately. The culture is placed in a translucent cuvette; the cuvette is placed in the machine and
the turbidity measured immediately. Simple mathematical formulae help convert the detected
turbidity to cell concentration. Using spectrophotometry for measuring the turbidity of cultures is
known as turbidometry.
In spectrophotometry, cultures usually do not need to be diluted, although above a certain cell
density the results lose reliability. Of all the electrical appliances used for counting cells, a
spectrophotometer is the cheapest and its operation the fastest and most straightforward. This has
made spectrophotometry the methods of choice for quick measurements of bacterial growth and
related applications. There are spectrophotometers in which several cuvettes can be inserted at one
time, reducing work time even more. Additionally, there are spectrophotometers that require
extremely small volumes of culture, as little as 1 microliter. This, combined with the stochastic
nature of liquid cultures, enables only an estimation of cell numbers.
An additional method for the measurement of microbial mass is the quantification of cells in a
culture by plating the cells on a petri dish. If the cells are efficiently distributed on the plate, it can
be generally assumed that each cell will give rise to a single colony. The colonies can then be
counted, and based on the known volume of culture that was sp read on the plate the cell
concentration can be calculated.
As is with counting chambers, cultures usually need to be heavily diluted prior to plating;
otherwise, instead of obtaining single colonies that can be counted, a so -called ―lawn‖ will form,
resulting in thousands of colonies lying over each other. Additionally, plating is the slowest method
of all: most microorganisms need at least 12 hours to form visible colonies.

In this way, calculating the dry weight of a sample enables us to calculate the ce ll count, but the
sensitivity is limited to samples containing more than 10E8 bacteria per milliliter.
Spectrophotometry is an indirect method for calculating cell concentrations by measuring the

changes in turbidity.
Bacteria can also be counted by using the plating method, which is based on the number of colonies
formed in Petri dishes containing specific growth media.
Microbial growth in closed and open Environment
(http://people.uleth.ca/~selibl/Biol3200/CourseNotes/GrowthCh6.pdf)
To understand the biology of an organism it is very essential to study the growth of any organism
which also helps for control of the microorganism
Basically, growth of microbe is steady increase in all the chemical components of that organism that
may result in an increase cell size, cell number or both
 In unicellular organisms cell growth results in increase in numbers
 In multicellular organisms cell growth results in an increase in organism size
I. Factors Affecting Growth
A. Chemical factors–conditions
• Nutrients are substances used in biosynthesis and energy release and are therefore requiredfor
growth
• Nutritional requirements are needed in order to cultivate the microbe in the laboratory
• Chemical factors are supplied by i) the culture medium (plain. media) that cont ains
substratesrequired for growth and ii) culture conditions (i.e., aerobic vs anaerobic conditions).
1. Macroelements (major elements - C, O, H, N, S, P, K, Ca, Mg, and Fe etc.)
• required in large amounts by the cell – >95% of cells are composed of macroelements
(Sometimes call macronutrients)
• C, O, H, N, S, P are components of macromolecules
2. Trace elements or Micronutrients
• Required in lesser or trace amounts.

• Critical to cell function
• Many are metals – structural role with many enzymes - cofactors
• Often trace elements present in medium components or water provide all that is required
forgrowth
• Co, Cu, Mn, Mo, Ni, and Zn are needed by most cells.
• Some cells require Cr, Se, W, and V
3. Oxygen
a) Aerobic organisms
• Growth at full atmospheric O 2 tensions (21% O 2 in the atmosphere)
• Facultative organisms (under appropriate nutrient and culture conditions) can grow undereither
aerobic or anaerobic condition
• Obligate aerobes - require O 2 for growth
• O 2 is poorly soluble - forced aeration is often used in culture systems to provide O 2
b) Anaerobic organisms
• Obligate (strict) anaerobes - grow only in the absence of O 2 ; sensitive to O 2 and brief
exposurewill kill these organisms; perhaps because these organisms are unable to detoxify s ome of
theproducts of O 2 metabolism
• Lack a respiratory system and can‘t use oxygen as a terminal electron acceptor
• These organisms do use oxygen found in cellular materials
Obligate anaerobiosis - prokaryotes and a few groups of fungi and protozoa
4. Other required elements
• Some microbes may have particular requirements that reflect their specific environment
(Halophiles require Na+) and morphology (Diatoms and Silicon dioxide based cell walls)
5. Growth Factors
• Some microbes have the enzymes and biochemical pathways needed to synthesize all cellular
components using minerals and sources of energy, carbon, nitrogen, phosphorus and sulfur.

• Other microbes lack one or more enzymes necessary to synthesize essential constituents – they get
these constituents or precursors from the environment
• Growth factors are organic compounds that are essential cellular components or precursors of
these components but cannot be synthesized by the organism
• Major Classes of Growth factors
Basically, Amino acids, Purine and Pyrimidines, Vitamins (e.g., thiamine, biotin, cobalamin,
pyridoxine), heme (nonprotein component of many cytochromes) or cholesterol are the major
classes of growth factors.
B. Physical (or environmental) Factors
Cardinal temperatures
• Depend on environmental factors such as pH and available nutrients
a) Minimum temperature - below which cells are inactive
• Reduced membrane fluidity – perhaps affects nutrient transport or proton gradient formation
b) Optimum temperature
• Highest rate of growth and reproduction, always nearer maximum temperature
c) Maximum temperature - above which growth is not possible
• Growth stops because of inactivation of one or more key prote ins, damages transport carriers or
other proteins, or thermal disruption of membrane
• Cardinal temperatures vary for different organisms
• Medium composition can have a slight affect
• Temperature optima usually vary from 0°C to 75°C
Growth Measurement
Different system or mode of microbial cultures: (https://www.biotechnologynotes.com/food-

biotechnology/microorganisms-in-food/different-systems-or-modes-of-microbial-cultures-
microorganism-biotechnology/14126)
1. Closed System – Batch Culture

2. Fed Batch Culture
3. Continuous Culture (Open System)

4. Synchronous Culture.
1. Closed System – Batch Culture:
Microbial cells growing in a tube or flask of liquid medium are said to be in a closed system. Here
no nutrients are added to the system and no metabolic waste products are removed. When nutrients
are added to the system, the cells initially divide by binary fission and the cell number increase for
a period of time. Once the nutrients are over in the medium the growth eventually stops and certain
metabolic waste products are accumulated in the medium.
Four characteristic phases of the growth cycle are recognized.
1. Lag Phase. Immediately after inoculation of the cells into fresh medium, the population remains
temporarily unchanged. Although there is no apparent cell division occurring, the cells may be
growing in volume or mass, synthesizing enzymes, proteins, RNA, etc., and increasing in metabolic
activity.
The length of the lag phase is apparently dependent on a wide variety of factors including the size
of the inoculum; time necessary to recover from physical damage or shock in t he transfer; time
required for synthesis of essential coenzymes or division factors; and time required for synthesis of
new (inducible) enzymes that are necessary to metabolize the substrates present in the medium.
2. Exponential (log) Phase. The exponential phase of growth is a pattern of balanced growth
wherein all the cells are dividing regularly by binary fission, and are growing by geometric
progression. The cells divide at a constant rate depending upon the composition of the growth
medium and the conditions of incubation. The rate of exponential growth of a bacterial culture is
expressed as generation time, also the doubling time of the bacterial population. Generation time
(G) is defined as the time (t) per generation (n = number of generations). Hen ce, G=t/n is the
equation from which calculations of generation time (below) derive.
3. Stationary Phase. Exponential growth cannot be continued forever in a batch culture (e.g. a
closed system such as a test tube or flask). Population growth is limited by one of three factors: 1.
exhaustion of available nutrients; 2. accumulation of inhibitory metabolites or end products; 3.
exhaustion of space, in this case called a lack of "biological space".
During the stationary phase, if viable cells are being counted , it cannot be determined whether some
cells are dying and an equal number of cells are dividing, or the population of cells has simply
stopped growing and dividing. The stationary phase, like the lag phase, is not necessarily a period

of quiescence. Bacteria that produce secondary metabolites, such as antibiotics, do so during the
stationary phase of the growth cycle (Secondary metabolites are defined as metabolites produced
after the active stage of growth). It is during the stationary phase that spore -forming bacteria have
to induce or unmask the activity of dozens of genes that may be involved in sporulation process.
4. Decline Phase. If incubation continues after the population reaches stationary phase, a death
phase follows, in which the viable cell population declines. (Note, if counting by turbidimetric
measurements or microscopic counts, the death phase cannot be observed.). During the death phase,
the number of viable cells decreases geometrically (exponentially), essentially the reverse of
growth during the log phase.
Figure.1.57. Bacterial Growth curve
A culture of microorganisms produced by inoculating a closed vessel containing a single batch of

medium is called a batch culture. Because no fresh medium is provided during incubation, nutrient
concentration decreases and concentration of waste products increases. The growth of
microorganisms reproducing by binary fission can be plotted on the logarithm of the number of
viable cells versus incubation time.
In the laboratory, under favorable conditions, a growing bacterial population doubles at regular
intervals. Growth is by geometric progression: 1, 2, 4, 8, etc. or 2 0 , 2 1 , 2 2 , 2 3 .........2 n (where n = the
number of generations). This is called exponential growth. In reality, exponential growth is only
part of the bacterial life cycle, and not representative of the normal pattern of growth of bacteria in
Nature.
Growth Rate and Generation Time

Bacterial growth rates during the phase of exponential growth, under standard nutritional conditions
(culture medium, temperature, pH, etc.), define the bacterium's generation time. Generation times
for bacteria vary from about 12 minutes to 24 hours or more. The generation time for E. coli in the
laboratory is 15-20 minutes, but in the intestinal tract, the coliform's generation time is estimated to
be 12-24 hours. For most known bacteria that can be cultured, generation times range from about 15
minutes to 1 hour. Symbionts such as Rhizobium tend to have longer generation times. Many
lithotrophs, such as the nitrifying bacteria, also have long generation times. Some bacteria that are
pathogens, such as Mycobacterium tuberculosis and Treponema pallidum, have especially long
generation times, and this is thought to be an advantage in their virulence.
Table.1.8.Generation times for some common bacteria under optimal conditions of growth.
Bacterium Medium Generation

Time (minutes)
Escherichia coli Glucose-salts 17
Bacillus megaterium Sucrose-salts 25
Streptococcus lactis Milk 26
Streptococcus lactis Lactose broth 48
Staphylococcus aureus Heart infusion broth 27-30
Lactobacillus acidophilus Milk 66-87
Rhizobium japonicum Mannitol-salts-yeast extract 344-461
Mycobacterium tuberculosis Synthetic 792-932
Treponema pallidum Rabbit testes 1980
Calculation of Generation Time
When growing exponentially by binary fission, the increase in a bacterial population is by

geometric progression. If we start with one cell, when it divides, there are 2 cells in the first
generation, 4 cells in the second generation, 8 cel ls in the third generation, and so on.
The generation time is the time interval required for the cells (or population) to divide.
G (generation time) = (time, in minutes or hours)/n(number of generations)

G = t/n

t = time interval in hours or minutes
B = number of bacteria at the beginning of a time interval
b = number of bacteria at the end of the time interval
n = number of generations (number of times the cell population doubles during the time
interval)
b = B x 2 n (This equation is an expression of growth by binary fission)
Solve for n:
logb = logB + nlog2
Figure.1.58. Generation time Determination
2. Fed Batch Culture(Chemostat):
In fed batch culture we add the growth limiting nutrient substrate to the culture. The fed batch
process is used in bio-industrial process to reach a high cell density in the bioreactor. Controlled

addition of the nutrient directly affects the growth rate of the culture and allows avoiding overflow
metabolism (formation of side metabolite such as acetate, lactic acid, etc.).
The substrate limitation offers the possibility to control the reaction rates to avoid technological
limitations connected to the cooling of the reactor and oxygen transfer. Substrate limitation also
allows the metabolic control, to avoid osmotic effects, catabolite repression and metabolism of side
products.
3. Continuous Culture of Bacteria
Bacterial cultures can be maintained in a state of exponential growth over long periods of time
using a system of continuous culture (Figure 4), designed to relieve the conditions that stop
exponential growth in batch cultures. Continuous culture, in a device called a chemostat, can be
used to maintain a bacterial population at a constant density, a situation that is, in many ways, more
similar to bacterial growth in natural environments.
In a chemostat, the growth chamber is connected to a reservoir of sterile medium. Once growth is
initiated, fresh medium is continuously supplied from the reservoir. The volume of fluid in the
growth chamber is maintained at a constant level by some sort of overflow drain. Fresh medium is
allowed to enter into the growth chamber at a rate that limits the growth of the bacteria. The
bacteria grow (cells are formed) at the same rate that bacterial cells (and spent medium) are
removed by the overflow. The rate of addition of the fresh medium determines the rate of growth
because the fresh medium always contains a limiting amount of an essential nutrient. Thus, the
chemostat relieves the insufficiency of nutrients, the accumulation of toxic substances, and the
accumulation of excess cells in the culture, which are the parameters that initiate the stationary
phase of the growth cycle. The bacterial culture can be grown and maintained at relatively constant
conditions, depending on the flow rate of the nutrients.

Figure.1.59. Schematic diagram of a chemostat
4. Synchronous Growth of Bacteria
Studying the growth of bacterial populations in batch or continuous cultures does not permit any
conclusions about the growth behavior of individual cells, because the distribution of cell size (and
hence cell age) among the members of the population is completely random. Information abo ut the
growth behavior of individual bacteria can, however, be obtained by the study of synchronous
cultures. Synchronized cultures must be composed of cells which are all at the same stage of
the bacterial cell cycle. Measurements made on synchronized cultures are equivalent to
measurements made on individual cells.
A number of clever techniques have been devised to obtain bacterial populations at the same stage
in the cell cycle. Some techniques involve manipulation of environmental parameters which induc es
the population to start or stop growth at the same point in the cell cycle, while others are physical
methods for selection of cells that have just completed the process of binary fission. Theoretically,
the smallest cells in a bacterial population are those that have just completed the process of cell
division. Synchronous growth of a population of bacterial cells is illustrated in Figure 5.
Synchronous cultures rapidly lose synchrony because not all cells in the population divide at
exactly the same size, age or time.

Figure.1.60. The synchronous growth of a bacterial population.
SOLVED PROBLEMS
1) Discuss direct microscopic count and give its importance.
a) Solution-Please refer topic microscopic count
2) Discuss direct viable count and give its importance.
a) Solution-Please refer topic direct viable count
3) Write notes on
i Nitrate reduction test
ii Esculin hydrolysis test
iii. ImViC test
a) Solution-Please refer topic Biochemical test
SELF-TEST -MCQS
1) Which factor is not abiotic factor
a) Pressure
b) Fungi
c) Temperature
d) Salinity
[Answer Key: b) Fungi]

2) Generation time of streptococcus lact is in milk is
a) 21 min
b) 26 min
c) 19 min
d) 10 min
[Answer Key: b) 26 min]
3) Which of the following is system/mode of microbial culture
a) Batch culture
b) Fed batch culture
c) c, Synchronous culture
d) All of the above
[Answer Key: d)All of the above]
SHORT ANSWER QUESTIONS
SAQ 01- Write note on -Synchronous Growth of Bacteria
a) Solution-Please refer topic Synchronous Growth of Bacteria
SAQ 02 -Write note on - Generation Time
c) Solution -Please refer topic Generation Time
UNIT 01-04: EFFECT & MEASUREMENT OF ENVIRONMENTAL

DETRIMINANTS
LEARNING OBJECTIVES
 To understand the importance of siderophores
 To understand the effect of measurement of environmental determinants
Environmental factors Terminology and definitions
Organisms respond to factors in the environment in which they live. These environmental factors
are part abiotic (nonliving) and part biotic (living). Th e abiotic factors are usually the governing
forces of environment, one organism ordinarily affecting others by its ability to modify the abiotic
environment. The abiotic factors of the environment influence the well -being and distribution of
organisms and the functions of the ecosystem

Temperature
Temperature is one of the most critical factors of the environment exerting strong influence on all
the physiological activities of organism. The temperature at which organisms can exist is referred to
as the physiological range. It varies in different organisms depending on local adaptations .On a
worldwide basis, temperature is perhaps the dominating factor affecting al distribution of
organisms.The environmental temperatures experienced by most organisms res ult directly or
indirectly from solar radiation reaching any point on Earth at any time and varying with the time of
year, slope, aspect, cloud cover, time of day, and other factors.
Radiation
DNA is the primary target for the induction of biological effec ts from radiation in all living
organisms. There are broad similarities in radiation responses from different organisms, and yet
wide differences in radiation sensitivity. The range in lethality from acute exposure to radiation
changes from organism to organism (Whicker and Schultz, 1982). Damage from radiation is
initiated by ionization. Ionization occurs if the radiation has sufficient energy to eject one or more
orbital electrons from the atom in which it interacts. Ionizing radiation is characterized by a large
release of energy (approximately 33 eV per event), an amount that is more than enough to break
strong chemical bonds (e.g. only 4.9 eV are required to break a C=C bond; IAEA 2010) The
ionization process and resulting charged particles can subseque ntly produce significant damage to
biological cells. Such damage is often referred to as direct effects. Much of the biological damage
from radiation, however, is due to indirect effects from free radicals. Radiation and the free radicals
produced can damage DNA by causing several different types of lesions (e.g. single strand breaks,
double strand breaks, base changes, and interstrand crosslinks).
Salinity
Salinity is the presence of salt in the land surface, in soil or rocks, or dissolved in water bodies.
Salinity can develop naturally, but where human intervention has disturbed natural ecosystems and
changed the hydrology of the landscape, the movement of salts into rivers and onto land has been
accelerated. The acceleration will continue until a new hydr ological equilibrium is reached. Salinity
can be categorized in a number of different ways: irrigation, dry land, urban, river and industrial
salinity etc. Plants and the associated microorganisms are highly affected due to Salinity .It not
only affects the physicochemical properties of soil but also the ecological balance of that area.
Several beneficial microorganisms colonizing the rhizosphere/endorhizosphere of such plants and

promote plant growth through various direct and indirect mechanisms can be used to decrease the
toxic effects caused by high salinity .
Moisture /water activity
Water is the major constituent of the planet, which determine the climate of that region and the
distribution of organisms. On geographical scale, moisture is more impor tant and its relationships
within an ecosystem is related to rainfall. Almost all microorganisms need moisture, or water, in a
"useable" or "available" form to grow and reproduce.
Redox potential
It is one of the most complex indicators of the physiologic al state of microbial cultures and its
measurement could be a useful tool for the qualitative and quantitative determination of
the microbial contamination.In environmental situations, it is common to have complex non -
equilibrium conditions between a large number of species, meaning that it is often not possible to
make accurate and precise measurements of the reduction potential. However, it is usually possible
to obtain an approximate value and define the conditions as being in the oxidizing or reducing
regime
Magnetism
It is a technique used to analyse the natural characteristics of materials. Environmental magnetism

utilises the mineral magnetic behavior of a material to interpret the environmental processes acting
upon it. The magnetic character is dependent upon the assemblage of minerals within a material.
Although all materials will respond to magnetic fields, iron oxides are particularly sensitive. The
near-universal occurrence of iron oxides in the environment e.g. hematite, magnetite and goethite,
provides many opportunities to apply the technique. The assemblage of magnetic minerals is
primarily dependent upon the parent material, specifically rock formation, and any subsequent
modification due to geomorphic processes e.g. transportation, depositio n and post depositional
processes. Due to the range of electron structures within different oxides, responses from each type
will differ when exposed to externally applied magnetic fields, and this enables discrimination
between contrasting sources and processes.Generally, environmental magnetism has been utilised in
sediment source tracing investigations. Sediment accumulations in impacted locations
estuarine,fluvial sediment source tracing, reconstruction of ice extent in glacial environments
,climatic reconstruction of loess sequences; reconstruction of paleo -environmental geomorphology
classification of soils, pollution studies to identify spatial patterns of vehicle pollution . Mineral
magnetism provides great utility for techniques such as source traci ng studies (Sophie C, 2014)

Biological interactions
Biological interactions are the interactions between organisms in a community. In the natural
world no organism exists in absolute isolation, and thus every organism must interact with the
environment and other organisms. An organism's interactions with its environment are fundamental
to the survival of that organism and the functioning of the ecosystem as a whole.
[https://www.onlinebiologynotes.com/microbial -interaction-and-types-mutualism-syntropism-proto-
cooperation-commensalism-antagonism-parasitism-predation-competition/]
Microbial interaction
 Microorganisms interacts with each other and can be physically associated with another
organisms in a variety of ways.
 One organism can be located on the surface of another organism as an ectobiont or located
within another organism as endobiont.
 Microbial interaction may be positive such as mutualism, proto -cooperation, commensalism

or may be negative such as parasitism, predation or competition
The nature and magnitude ofinteraction will depend on the types of microorganism present as well
as the abundance of themicroorganismsand types ofsensorysystems oftheindividual organisms
Types of microbial interaction
Several types of interaction of a microorganism with another microorganism. Following are the
specific examples of the processes associated with microbe–microbe interactions:
1. Positive interaction: mutualism, proto-cooperation, commensalism
2. Negative interaction: Ammensalism (antagonism), parasitism, predation, competition

[A–Plant-Microbe Interaction;B-Animal-Microbe Interaction]
Figure.1.61. Types of Positive (+) and Negative (-) Interactions of microorganisms

Table.1.9.Types of Interaction between Microorganisms and
Hosts Table.1.10.MicrobialInteractions
1. Mutualism

Mutualism defines as an obligatory association that provides some reciprocal benefit to
bothpartners. This is an obligatory relationship in which the mutualist and the host are
metabolicallydependenton each other.
 Mutualistic relationship is very specific where one member of association cannot be

replaced by another species.
 Mutualism require close physical contact between interacting organisms.
 Relationship of mutualism allows organisms to exist in habitat th at could not occupied by

either species alone.
 Mutualistic relationship between organisms allows them to act as a single org anism.
Examples of mutualism:
i. Lichens:
 Lichens are excellent example of mutualism.
 They are the association of specific fungi and certain genus of algae. In lichen, fungal
partner is called mycobiont and algal partner is called
 Phycobiont is member of cycanobacteria ad green algae (Trabauxua).
 Because phycobionts are photoautotrophs, the fungus get its organic carbon directly from
algal partner, in turn fungi protects the phycobiont from extreme conditions and also provide
water and minerals to algae.
 Lichen grow very slowly but are able to colonies habitat that do not permit the growth of
other organisms.
 Most lichens are resistant to high temperature and drying.
ii. Protozoan-termite:
 Protozoan-termite relationship is the classical example of mutualism in which flagellated

protozoan lives in the gut of termites.
 Theses flagellated protozoan feeds on diet of carbohydrates acquired as cellulose or lignin

by their host termites, metabolize into acetic acid which is utilized by termites.
iii. Paramecium-Chlorella:

 Paramecium (protozoa) can host Chlorella (algae) within its cytoplasm.
 The algae Chlorella provide the protozoan partner with organism carbon and O2, in turn
protozoa provide protection, mortility, CO2 and other growth factors.
 The presence of Chlorella within Paramecium helps to survive protozoa in anaerobic

condition as long as there is sufficient light.
2. Protocooperation (Synergism)
Protocooperation is a mutually beneficial relationship, similar to that which occurs in mutualism ,

butin protocol operation, this relationship is not obligatory.The beneficial complementary resources
are provided by each of the paired microorganisms.The organisms involved in this type of
relationship can be separated,and if the resources provided by the complementary microorganism
are supplied in the growth environment, each microorganism will function independently.
An example of this type of relationship is the association of Cellulomonas spp. and Azotobacter
spp. In thisrelationship, Azotobacter spp. Uses glucose provided by a cellulose-degrading
microorganism such as Cellulomonas spp., which uses the nitrogen fixed by Azotobacter spp.
Another example of this type of relationship is the association of Desulfovibrio and Chromatium,in
which the carbon and sulfur cycles are linked.The organic matter (OM) and sulfate required by
Desulfovibrio are produced by the Chromatium while reduction of CO 2 to organic matter and
oxidation of sulfide sulfate required to Chromatiumcarried out by Desulfovibrio. Syntrophism is a
protocooperative symbiotic process in which phototrophic and chemotrophic bacteria not only
exchange metabolites but also interact at thelevel of growth coordination.
Examples of syntrophism:
 i. Methanogenic ecosystem in sludge digester
 Methane produced by methanogenic bacteria depends upon interspecies hydrogen transfer by

other fermentative bacteria.
 Anaerobic fermentative bacteria generate CO 2 and H 2 utilizing carbohydrates which is then

utilized by methanogenic bacteria (Methanobacter) to produce methane.
 ii. Lactobacillus arobinosus and Enterococcus faecalis:

 In the minimal media, Lactobacillus arobinosus and Enterococcus faecalis are able to grow
together but not alone.
 The synergistic relationship between E. faecalis and L. arobinosus occurs in wh ich E.

faecalis require folic acid which is produced by L. arobinosus and in turn lactobacillus
require phenylalanine which is produced by Enterococcus faecalis.
3 Commensalism
The commensalistic relationship involves two microorganism here one partner (the commensal)
benefits while the other species (the host) is not harmed or helped.
There are several situations under which commensalism may occur between
microorganisms.Commensalistic relationships between microorganisms include situations in which
the waste product of microorganism is the substrate for another species.
An example is Nitrification, the oxidation of ammonium ion to nitrite by microorganisms such as

Nitrosomonas, and the subsequent oxidation of the nitrite to nitrate by Nitrobacter and
similarbacteria. Nitrobacter benefits from its association with Nitrosomonas because it uses nitrite
to obtain energy for growth.
(1) Commensalistic associations also occur when one microbial group modifies the environment to
make it more suited for another organism.
For example, in the intestine the common, nonpathogenic strain of Escherichia coli lives in
thehuman colon, but also grows quite well outside the host, and thus is a typical commensal.
Whenoxygen is used up by the facultatively anaerobic E. coli, obligate anaerobes such as
Bacteroides are able to grow in thecolon.
(2) One species releasing vitamins, amino acids and other growth factors that are needed by
asecondspecies.
B. Negative interaction: Conflictual Interactions .
1. Predation
Predation is a widespread phenomenon where the predator engulfs or attacks the prey. In theworld
of eukaryotes, it is common that the larger animal eats the smaller one; however,
withmicroorganisms the predator may be larger or smaller than the predator, and this normally
resultsinthedeath ofprey.
Examples.Bdellovibrio,Vampirococcus,and Daptobacter.

Each of these has unique mode of attack against a susceptible bacterium.
(1) Epibiotic predator with growth on the surface of the prey.Ex.–Vampirococcus
(2) periplasmic
Predator with growth in between inner and outer membrane of bacteria.
Examples Bdello vibrio cytoplasmic predator with growth in the cytoplasm of prey.Ex,
Daptobacter.
2. Parasitism
Parasitism occurs when one species obtain nutriients from another for the purpose of cell
growth.Parasites display two types: (1) direct lifecycle that does not require an intermediate host
and(2) indirect lifecyclethat requires anintermediatehost.In this parasite get benefits from the
host and there is a degree of coexistence between the host and parasite that can shift to a
pathogenic relationship (a type ofpredation).Thehost maybemicrobes, plantsoranimals.
Microbe-microbeparasitism
(1) Mycoparasitism (Fungus-Fungus Interaction)
When one fungus is parasitized by the other fungus, this phenomenon is called mycoparasitism.The
parasitizing fungus is called hyper parasite and the parasitized fungus as hypoparasite.Barnett and Binder
(1973) divided mycoparasitism into (i) necrotrophic parasitism, in which therelationships result in death of
the host thallus, and (ii) biotrophic parasitism, in which the development of the parasite is favored by a
living rather than a dead host structure.The antagonistic activity of necrotrophic mycoparasites is attributed
to the production of antibiotics,toxins,and hydrolytic enzymes.It is used as biocontrolagent.Ex.-The fungal
genus,Trichoderma produces enzymes such as chitinases which degrade the cell walls of other fungi
(2) Mycophagy
Mycophagy or Fungivory is the process of organisms consuming fungi. Bacterial mycophagy-mechanisms by

which bacteria feed on fungi. Ex.-Bacteria Aeromonascaviae feed on fungus Rhizoctonia solani and
Fusariumoxysporum. Many amoebae are also known to feed on pathogenic fungi.The antagonistic oil amoebae
are Arachnula, Gephyramoeba, Geococcus, Saccamoeba, and Vampyrellaetc.

(1) Bacterivores
Bacterivores are free-living, generally heterotrophic organisms, exclusively microscopic,

whichobtain energy and nutrients primarily or entirely from the consumption of bacteria. Many
species of amoeba are bacterivores,as well asother types of protozoans. i.e. Vorticella
(2) Bacteriophage
A bacteriophage, also known as a phage, is a virus that infects and replicates within Bacteria and
Archaea. i.e. ds DNA phases (T4 – phase, lambda phase), ssDNA phase (M13 phase, ΦX174).
Amensalism (antagonism):
Amensalism describes the negative effect that one organism has on another organism. This is
aunidirectional process based on the release of a specific compound by one organism which has a
negative effect on another organism. In this interaction, basically
 One microbial population produces substances that is inhibitory to other microbial

population then this inter population relationship is known as Ammensalism or Antagonism.
 The first population which produces inhibitory substances are unaffected or may gain a
competition and survive in the habitat while other population get inhibited. This chemical
inhibition is known as antibiosis. Production of antibiotics that can inhibitor kill a susceptible
microorganism.
Ex. - the destructive effect of the bread mold Penicillium spp.on certain bacteria by the
secretion of penicillin.,Fatty acid produced by skin flora inhibits many pathogenic bacteria in
skin, Thiobacillus thioxidant produces sulfuric acid by oxidation of sulfur which is responsible
to lowering of pH in the culture media which inhibits the growth of most other bacteria.
Competition
When two or more species use the same nutrients for growth, some of the populations will be
compromised.Competition between microbial species may be attributed to availability of nitrogen
source, carbon source, electron donors and acceptors, vitamins, light, and water.Microbes also
compete with their neighbors for space and resources. Competition for a limiting nutrient among
microorganisms leads to exclusion of slower growing population.
Examples of competition:

 Competition between Paramecium cadatum and Paramecium aurelia:
 Both species of Paramecium feeds on same bacteria population when these protozoa are
placed together.
 P. aurelia grow at better rate than P. caudatum due to competition.
Plant–microbeInteractions
Interactions between plants and microorganisms occur in many different ways and on many
different levels. Virtually all organs of the plant inte ract with microorganisms at a certain stage of
their life, and this interaction is not necessarily negative for the plant. Indeed, there are plenty of
interactions where the plant benefits either through direct or through indirect effects of the
associated microbes.
Root-microbe interaction
Plant roots have a lot of extensions from the central root, and bacteria may become localized
onthe root surface. In general, plants benefit from two types of microbe–plant associations: (1)
highly specialized interaction,where there is considerable specificity found in mutalistic activities;
and (2) commensalism resulting from nutrient secretion from plants when bacteria and fungi grow
in close proximity to the roots.
The three best characterized symbiotic systems are the fungus–root system (Mycorrhizae),
bacterium–root nodule system and cyanobacteria-root system.
Mycorrhizae
Mycorrhizae are fungus-root associations, first discovered by Albert Bernhard Frankin 1885. The
term ―mycorrhizae‖ comes from the Greek words meaning fungus and roots.These
microorganisms contribute to plant functioning in natural environments, agriculture, and
reclamation. The roots of about 95% of all kinds of vascular plants are normally involved in
symbiotic associations with mycorrhizae.
Symbiosis begins when fungal spores germinate and emerging thread like structures,called
hyphae, enter the epidermis of plant roots. After colonization of the root, the fungus sends out
avast network of hyphae throughout the soil to form a greatly enhanced absorptive surface
area.This results in improved nutrient acquisition and uptake by plant roots, particularly elemental
phosphorus (P), zinc (Zn), manganese (Mn) and copper (Cu) and water. In return, the plant
provides carbohydrates for the fungi.
Rhizospheric interactions of terrestrial fungi are influenced by the number of microorganisms,

type of microorganisms, and specific plant root exudates. Mycorrhiza is mainly of two types, endo
mycorrhizae and ectotrophic mycorrhize.Growth on the exterior of the root is characteristic of

endomycorrhizae or ectotrophic mycorrhizae, while growth inside the root is attributed to the
endomycorrhizae or endotrophic mycorrhizae.
(1) Ectomycorrhizae
Ectomycorrhizae are commonly found on tree roots of gymnosperms or woody angiosperms, and
fungal partners include members of then on septate fungi.They form a mantle of varying
thickness.Such ectomycorrhizae, including Cennococcum, Pisolithus and Amanita, form irregular
structures that are easy to recognize.The fungal hyphae extend into the soil, where they sequester
phosphorus, nitrogen, sulfur, calcium, zinc, iron, and numerous minerals for fungal growth. When
the plant becomes mineral-deficient or when the fungal cellsdie, the appropriate ion will be passed
on to the roots.

https://bmcbioinformatics.biomedcentral.com/articles/10.1186/1471 -2105-6-178)
Figure.1.62. Ectomycorrhizal Symbiosis :Amanita spp.
(1) Endomycorrhizae
In this association the fungalhyphae penetrate the outer corticalcellsofthe plantroot, wherethey
grow intracellularly and form coils, swellings, or minute branches. The fungi penetrate the root
with growth between epidermal cellsorthroughroothairsandinvadecortexcells.Endomycorrhizae
are found on most herbaceous plants, including lower vascular plants such asfernsand bryophytes.
Vesicular-arbuscular mycorrhizae (VAM) are a type of endomycorrhizal association, where
bothvesicles and arbuscles are developed together. VAM is by far the commonest of all
mycorrhizaeandhas been reported in morethan 90% of land plants. The hyphae enter the root and
create swellings (vesicles) for nutrient storage structures where nutrients are transferred between
fungus and plant (arbuscules).

(Adapted without modification from: https://www2.nau.edu/~gaud/bio300/mycorrhizae.htm)
Figure.1.63. Cross Section of Roots Showing Endomycorrizae
Mutualism relationship of aerial plant surfaces with

microorganismPhyllosphereMicroorganisms
A wide variety of microorganisms are found on and in the aerial surfaces of plants, called the
phyllosphere. These include microorganisms that have interactions with leaf and sometimes stem
at various stages of development.The plant leaves and stems release organic compounds,and this
can lead to massive development of microbes.
E.g.Sphingomonas, Pseudomonas, and Methylobacterium.
Biological nitrogen fixation (BNF)
The conversion of atmospheric nitrogen into the nitrogenous compounds through the agency
ofliving organisms is called biological nitrogen fixation. The process is carried out by symbiotic
and ―freeliving‖ornon-symbiotic microorganisms. In this process, atmospheric nitrogen is reduced
to ammonia in the presence of ‗Nitrogenase‘ which is a biological catalyst found naturallyonly in
certain microorganisms such as the symbiotic Rhizobium and Frankia, or the free-
livingAzospirillumand Azotobacter and blue greenalgae(BGA).
Nitrogen Fixers /Diazotrophs.

Few prokaryotes like bacteria and cyanobacteria shows ability to fix atmosphere nitrogen.They
are called nitrogen fixers or Diazotrophs. They can be symbiotic or asymbiotic (freeliving) such as
given below:
(1) SymbioticNitrogen FixingBacteria
Rhizobium is aerobic, gram negative nitrogen fixing bacterial symbionts of leguminous

roots.Several species of Rhizobium live in the soil but are unable to fix nitrogen by
themselves.They do soonly as symbionts in the association of roots of legumes.

Several other genera are also able to form nitrogen-fixing nodules with legumes.These include
Allorhizobium, Azorhizobium, Bradyrhizobium, Mesorhizobium and Sinorhizobium
Noduleformation
It involves multiple interactions between free-living soil Rizobium and roots of the host plant.The
roots of young leguminous plants secrete a group of chemical attractants like flavonoids and
betaines which resulted into formation of nodules.
Stages in root nodule formation (http://eagri.org/eagri50/AMBE101/lec18.htm)
The stages in the infection and development of root nodules are not fairly well understood. They
include
1. Recognition of the correct parameter on the part of both plant and bacterium and attachment of
the bacterium to the root hairs.
2. Excretion of nod factors by the bacterium.
3. Invasion of the root hairs by the bacterial formation of an infection thread.
4. Travel to main root via the infection thread.
5. Formation of deformed bacterial cells, bacteroids, within the plant cells and development of the
nitrogen fixing state.
6. Continued plant and bacterial division and formation of the mature rootnodule.
Factors affecting nodulation
 Temperature and light
 Combined Nitrogen
 Hydrogen iron concentration
 Mineral nutrition-Co,Mo,P,Ca
 Genetic factors
 Ecological factors
 Salinity and alkalinity

[Adapted without modification from: http://eagri.org/eagri50/AMBE101/lec18.html]
Figure.1.64. RootNodule FormationbyRhizobium
MechanismofNitrogenfixation
The nodule serves as site for N2 fixation. It contains all the necessary bio-chemicals such as the
enzyme complex called nitrogenase and leghaemoglobin (leguminous haemoglobin).The
nitrogenase has 2 components i.e.Mo-Feprotein (molybdoferredoxin) and Fe-protein
(azoferredoxin). The nitrogenase catalyzes the conversion of atmosphere di-nitrogen (N2)
to2NH3.The ammonia is the first stable product of nitrogen fixation.
During nitrogen fixation, the free di-nitrogenfirst bound to MoFe protein and is not releaseduntil
completely reduced to ammonia. The reduction of di-nitrogen is a stepwise reaction in which
many intermediates are formed to form ammonia (NH3) which is protonated at physiological pH
to form NH4+. In this process ferredoxin serves as an electron donor to Fe-protein (nitrogenase
reductase) which in turn hydrolyzes ATP and reduce MoFe protein, the Mo Fe protein in Turn
reduce the substrateN2.The electrons and ATP are provided by photosynthesis and respiration of
the host cells.
The ammonia produced by nitrogenase is further used in synthesis of amino acid by assimilation
of ammonia and again in nitrate assimilation i.e. Nitrogen cycle

(1) FreeLivingNitrogen FixingBacteria
Azotobacter, Beijerinckia (both aerobic) andClostridium (anaerobic) are saprophytic bacteria that
perform nitrogen fixation. Desulphovibrio is chemotrophic nitrogen fixing bacterium.
Rhodopseudomonas, Rhodospirillum and Chromatium are nitrogen fixing photoautotrophic free
living bacteria.
(2) Free living Nitrogen Fixing Cyanobacteria
Many free living blue-green algae (also called cyanobacteria) perform nitrogen fixation,
e.g.,Anabaena, Nastoc, Aulosira, Cylmdrospermum etc. These are also important ecologically as
they live in water-logged sods where denitrifing bacteria can be active. Aulosira fertilissima is the
most active nitrogen fixer in Rice fields, while Cylindrospermum is active in sugarcane and maize
fields.
(3) SymbioticNitrogen FixingCyanobacteria
Anabaena and Nostoc species are common symbionts in lichens, Anthoceros, Azolla and
cycadroots. Azolla pinnata (a water fern) has Anabaena azollae in its fronds. It is often inoculated
to Rice fields for nitrogen fixation.
Some of the bacteria are capable of digesting proteins, lipids and starch as well.Lignin fraction of
plant remains undigested.
The rumen bacteria ferment proteins and lipids and produce hydrogen and carbondioxidesgase,
which in turn is converted into methane byMethanobacteriumruminantium.The bacteria of rumen
multiply into a large population. However, most of them are passed into stomach along with
undigested material where they are killed by proteases and other enzymes. The fatty acids in
rumen are absorbed and gases are passed out.
Nematophagus fungi
Nematophagous fungi are carnivorous fungi which depend on nematodes. The nematophagous
fungi are of three main types on the basis of ecological habit as-

Table.1.11.Types of Nematophagusfungi
Nematode-TrappingFungi EndoparasiticFungi EggParasites
Fungi capturing nematodes These fungi attack nematodes Therefungiattackonnematodeeggs

are called nematode- through many modifications by developing the swollen
trapping fungi.Such fungi brought about in conidia. structure at at the point of contact
have evolved structural and attached to the egg where
adaptations to trap or from a narrow infectious tube
penetrate their prey.They develops that penetrates the shell
may be predatory or ofthe egg.
endoparasites.
e.g. Pleurotus ostreatus,. e.g. Cephalosporium spp., e.g. Dactyllela oviparasitica and
Arthrobotrys dactyloides Meria spp.,Verticillium Paecilomyces lilacinus etc.
,Arthrobotrys oligospora etc. spp.etc
Luminescentbacteria
Luminous bacteria are the most widely distributed light-emitting organisms with the
majorityexisting in seawater and the remainder living in the terrestrial or freshwater environment.
Whilemost species of luminescent bacteria are capable of living free, the majority are found in
natureassociatedinsymbiosiswithhostorganisms i.e.,fishes,squids, crabs,nematodes,etc
In symbiosis, the bacteria are nourished with readily available food sources forgrowth, and atthe
same time the host utilizes the adopted illumination to communicate, to attract prey, and
tomasqueradeitselffrom predators.
Siderophore (Iron Chelating Agent)
Iron is an essential cofactor for conducting numerous metabolic processes of organisms. It plays
vital role by regulating biosynthesis of porphyrins, vitamins, antibiotics, toxi ns, cytochromes,
pigments, and aromatic compounds, nucleic acid synthesis and microbial biofilm formation by
regulating the surface motility of microorganism. At physiological pH (7.35 –7.40), the ferrous form
(Fe 2+) of iron is soluble, while the ferric form (Fe 3+) is insoluble. It is reported that at this condition,
the concentrations of dissolved ferrous iron has found to be approximately 10 −10 M to 10 −9 M
whereas the required level of ferrous iron by living organisms is around 10 −7 M to 10 −5 M .
Therefore, in order to survive under such iron-depleted condition, most of the
microorganisms produces certain organic compounds with low molecular masses called as

siderophores.Although, large number of siderophores produced by different microorganisms have
been documented but great structural variation has been observed in siderophores produced by
bacteria.
Classification of Siderophore (Handore A.V, Khandelwal S.R et al.,2019)
These siderophores are classified on the basis of co-ordinating groups that chelate the Fe (III) ion.
The most common co-ordinating groups are catecholates, hydroxamates and carboxylates
1. Catecholate Siderophore
The siderophore having binding groups of phenolate or 2,3 -dihydroxy benzoate belongs to the
catecholate type of siderophore. Each catecholate group supplies two oxygen atoms for chelation
with iron in order to form a hexadentate octahedral complex. It is reported that Enterobactin with
molecular formula C 30 H 27 N 3 O 15 is one of the strongest catecholate type siderophore exhibiting
significant potential to chelate iron even from the environment where concentration of iron is low
2.Hydroximate Siderophore
Most of hydroxamate groups consist of C (=O) N -(OH) R, where, ‗R‘ is either an amino acid or its
derivative. In this,two oxygen molecules from each hydroxamate group forms a bidentate ligand
with Fe +2 as a result, each siderophore is able to form hexadentate octahedral complex with Fe 3+ .
This siderophore family includes Rhodotorulic acid, Dimerium acid, Alcaligens and Putr ibactin.
3.Carboxylate Siderophore
Members of this class do not possess hydroxamate /phenolate li gands .However, iron binding is
achieved by hydroxyl carboxylate and carboxylates . It is found that Rhizoferrin is the best
characterized carboxylate type siderophore .
Bacterial Siderophore
Bacteria can directly antagonize pathogens by competition for root niches or by producing
allelochemicals like siderophores, antibiotics, biocidal, lytic enzymes, and detoxification enzymes
by pathogen virulence factors degradation or by the interference with pathogens quorum sensing .
Bacterial siderophores trap traces of iron in the form of very stable complexes ,which are
internalised into the cell by specific cell receptors Microorganisms like bacteria uses siderophor es
to fulfill their iron requirements. To transport iron in the cytoplasm, bacteria capture iron -loaded
siderophores at the cell surface and transport them into the cytosol. The binding constants of

siderophores for Fe (III) are extremely high, implying th at these compounds can effectively
scavenge Fe (III) from a variety of complexes found in natural environment (Handore A.V,
Khandelwal S.R et al.,2019)
Fungal Siderophore
It is reported that the fungal cells uses different mechanisms to extract iron: i) re ductive iron
assimilation ii) siderophore „shuttle‟ mechanism and iii) taxicab‟ mechanism ,iv)hydrolytic or
degradation .Depending of the iron availability, fungi use one or several of the four mainly iron
gathering mechanisms. These different modes of fu ngal iron transport provide a real advantage to
grow in different environments. (Handore A.V, Khandelwal S.R et al.,2019)
Method for Siderophore Production(Handore A.V, Khandelwal S.R et al.,2019)
A. Preparation of CAS Reagent:
1.Prepare 2mM CAS stock in distlled water
2. Prepare 1mM Ferrous Stock
3. Prepare Piperazine Buffer by dissoving Piperazine (4.307 g) in 30ml D/W and pH was adjusted
to 5.6 by addition of concentrated HCl
4. Preparation of HDTMA Solution: HDTMA(0.0219 g) was dissolved in 50 ml D/W
5.CAS Reagent Preparation: In the CAS solution (7.5ml), freshly prepared ferrous solution
(1.5ml) was slowly added and properly mixed .Then this mixture was slowly added to HDTMA
(30ml) in the mixing cylinder (gentle stirring was carried out so that t here should be no
foaming).Then, piperazine solution was slowly added and volume was made up to 100 ml with
double D/W.The reagent was stored in fridge by covering with black paper .
B.Preparation of Deferrated Media
Mayer and Abdullah (MA) media was prepared by using KH 2 PO 4 ,(6gm), K 2 HPO 4 (3gm),(NH 4 ) 2 SO 4
(1gm ), MgSO 4 (0.2 gm) and Succinic acid (4 gm) in 1000 ml .D/W. Deferration of media was
carried out by addition of 8 – hydroxyquinoline dissolved in chloroform (5ml/L). Phase separation
was carried out by shaking the media vigorously in the separating funnel .After the phase
separation, chloroform layer was removed and the medium was repeatedly washed with chloroform
to ensure the complete removal of iron complexes and any residues of 8 – hydroxyquinoline, which
could inhibit the growth

C.Preparation of Fermentation Medium for Siderophore Production
The bacteria under study which were grown in the Nutrient broth and fungal cultures in Potato
dextrose broth were further inoculated in autoclaved iron deficient succinate medium and
incubated at room temperature on a rotary shaker (110 rpm)
D.Detection of siderophore by Chrome Azurol Sulphonate (CAS) Assay
Fermented broth was centrifuged (3500 rpm, 15 min). Then, every after 4 th day, the cell free
supernatant was subjected to siderophore detection test by CAS assay. Development of pink/orange
colouration indicates synthesis of siderophore.
[P-Positive test , N-Negative test]
[Figure.1.65. Siderophore detection by CAS Assay]
(Adapted without modification from: Handore A.V, Khandelwal S.R et al.,2019)
E.Quantification of Siderophores
Percent siderophore unit were find out by adding the supernatant to CAS solution (1:1) and mixture
was allowed to incubate for 1hr at room temperature .Ab sorbance‘s were recorded at 630nm. and
percentage of siderophore units were calculated by formula, [A r − A s /A r ] × 100.where, Ar is
Absorbance of reference (Uninoculated media with CAS reagent)
F. Extraction and Purification of Siderophore

Cell free supernatant was acidified to pH 3.00 by addition of HCl (12M).Then the supernatant was
mixed with ethyl acetate (10:10 v/v) the mixture was vigorously shaken for 15 minutes. Further

organic phase was separated and allowed to evaporate till drynes s. The extract was resuspended in
deionized water. (1mg/ml)
G.Characterization of Siderophore
i) FeCl 3 Test
Presence of siderophore was detected by addition of freshly prepared 0.5 ml aqueous FeCl 3 solution
(2%) to 0.5 ml of culture filtrate. Appearance of reddish brown colour indicates presence of
siderophore

Figure.1.66. FeCl 3 Test
ii) Tetrazolium Test for Hydroxamate Type of Siderophores
To a pinch of tetrazolium salt, 150 μL of NaOH solution (2N ) was added with 2 mL culture filtrate.
Appearance of a cherry red colour indicates presence of hydroxymate type of siderophore

Figure.1.67. Tetrazolium Test

iii)Arnow’s Testfor Catecholate Type of Siderophores
1 ml of 0.5 N HCl and 1 ml of Nitrite- molybdate reagent was added to 1 ml of cell -
freesupernatant.Yellow colour formation showed presence of catechols

Figure.1.68. Arnow’s Test
iv) Test for Carboxylate Type of Siderophores
Three drops of NaOH (2N) was added to 1 drop of phenolphthalein and some water was added till
the light pink colour was developed. On addition of culture filtrate, disappearance of the colour
indicated presence of a carboxylate siderophore.
[P-Positive test, N-Negative test]

Figure.1.69. Phenolphthalein Test

Applications of Siderophores
Bacterial Siderophores :
Bacterial siderophores have promising applications in various fields,they have ability to accelerate
the plant growth by direct or indirect mechanism of action. Several bacteria were identified which
produces a variety of siderophores having a peptide backbone with several non - protein amino acid
. therefore plays important role in sustainable agriculture .Siderophores tightly bind with iron and
reduces the bioavailable iron for the plant pathogens, thus facilitating the killing of phyto -
pathogens .They plays significant role in bioremediation as they are extremely effective in
solubilizing and increasing the mobility of a wide range of metals such as Cd, Cu, Ni, Pb, Zn, and
the actinides Th (IV), U(IV) and Pu(IV) .Some bacterial siderophores have been considered as a
promising agent for the construction of biosensors. Vectorization of bactericide compounds by
siderophores is a promising strategy able to considerably increase the efficacy of drugs. (Handore
A.V, Khandelwal S.R et al.,2019)
Fungal siderophores
Fungal siderophores also leds to significant application in ant biotherapy. Siderophore can mediate
selective delivery of antibiotics to antibiotic -resistant bacteria by the Trojan horse strategy. This
strategy exploits the iron transport abilities of siderophores to carry drugs into cells by preparation
of conjugates between siderophores and antimicrob ial agents . Other major clinical applications of
siderophore include treatment of diseases like hemochromatosis, thalassemia, and dialysis
encephalopathy, removal of transuranic elements such as aluminum and vanadium and anti -malarial
activity.etc. .Therefore, siderophore production ability of different endophytic bacterial and fungal
isolates was studied.(Handore A.V, Khandelwal S.R et al.,2019)
Effect and measurement of environmentally determinants temperature salinity moisture

activity redox potential magnetism.pdf
Temperature
It is among the most pervasive and important physical factors in the environment of an organism.
The organism must either compensate for the rate changes induced by changes in temperature
(acclimation or acclimatization), or it must try to prevent or minimize changes in its body
temperature (thermoregulation). A combination of these strategies can also be employed.

Recent Advances of Bio surfactants in Environmental Applications
Bio surfactants are a structurally diverse group of surface -active amphiphilic compounds substances
produced by numerous microorganisms such as , bacteria, fungi and yeast. All bio surfactants
consist of two parts—a polar (hydrophilic) moiety and non -polar (hydrophobic) group. A
hydrophilic group consists of mono-, oligo- or polysaccharides, peptides or proteins and a
hydrophobic moiety usually contains saturated, unsaturated and hydroxylated fatty acids or fatty
alcohols (Lang S.2002). A characteristic feature of bio surfactants is a hydrophilic-lipophilic
balance (HLB) which specifies the portion of hydrophilic and hydrophobic constituents in surface -
active substances.
Biosurfactants are categorized by their chemical composition, molecular weight, physico -chemical
properties and mode of action and microbial origin. Based on molecular weight they are divided
into low-molecular-mass biosurfactants including glycolipids, phospholipids and lipopeptides and
into high-molecular-mass biosurfactants/bioemulsifiers containing amphipathic polysaccharides,
proteins, lipopolysaccharides, lipoproteins or complex mixtures of these biopolymers.
Classification of Bio Surfactants
Bio surfactants are mainly categorized on basis of their chemical composition and microbial or igin.
In general, their structure includes a hydrophilic moiety consisting of amino acids or peptides
anions or cations; mono-, di-, or polysaccharides; and a hydrophobic moiety consisting of
unsaturated, saturated, or fatty acids. Accordingly, the major c lass of biosurfactants are as follow
Glycolipids
These are carbohydrates in combination with long -chain aliphatic acids or hydroxyaliphatic acids.
Among the glycolipids, the well known examples are rhamnolipids, trehalolipids, and
sophorolipids.and mannosylerythritol lipids etc. Except these, some other types like
glycoglycerolipid,( Nakata K. et al.,2000), sugar-based bioemulsifiers, (Van Hoogmoed et
al.,2000), mannosylerythritol lipid A and many different hexose lipids. (Golyshin Pft, et al.,1999)
Lipopeptides and Lipoproteins
Lipopeptides are amphiphilic molecules which incorporate one or more lipid chains attached to a
peptide head group. In this self-assembly is observed on basis of hydrophile/ lipophile balance of

the molecules as well as interactions between the peptide units (Ian et al.,2015) .It includes,
Surfactin Iturin,Fengycin,Lichenysin etc.
Fatty Acids, Phospholipids, and Neutral Lipids
The fatty acid bio surfactants are saturated fatty acids in the range of C12 to C14 and complex fatty
acids containing hydroxyl groups and alkyl branches (Kretschmer et al.,1982). Number of bacteria
and yeasts produces large amounts of fatty acid and phospholipid surfactants during growth on n -
alkanes .It includes Corynomycolic Acid groups
PolymericBio Surfactants
Most polymeric bio surfactants has a backbone of three or four repeating sugars with fatty acids
attached to the sugars (Rosenberg and Ron 1997).The best -studied polymeric biosurfactants are
emulsan, liposan, mannoprotein, and other polysaccharide - protein complexes. .It includes Emulsan
,Biodispersan, AlasanLiposan, Emulsifying Biopolymer from Fungus, Emulsifying Protein etc.
Particulate bio Surfactants
These are of two types, extracellular vesicles and whole microbial cell. Extracellular membrane
vesicles partition hydrocarbons to form micro -emulsions, which play key role in hydrocarbon
uptake by microbial cells. Sometimes the whole bacterial cel l itself can work as surfactant.It
includes Vesicles,Whole Microbial Cells etc.
The bio surfactants have several benefits over chemical surfactants, such as a higher degree of
biodegradability, foaming, selectivity, and specific activity. Microbial surfactants can be labeled as
―green‖ because of their less toxicity, coupled with better environmental compat ibility, working
amicably on a wide range of physicochemical environments, and also contribution to limiting the
greenhouse effect. Hence, bio surfactants are expected to be promising alternatives for synthetic
surfactants for industrial, environmental, an d domestic applications .Notably, the bio surfactants
exhibit competent and feasible applications in different environmental avenues (Mulugeta et
al.,2021)
Numerous technologies have been established on basis of the capability of bio surfactants to
interact with hydrophobic substrates causing reduction of surface and interfacial tensions,
emulsification, solubilisation, dispersion, desorption and wetting. Generally chemically synthesized
surfactants are non-biodegradable and causes various hazardous impact towards Environment and

health of society. In this context demand of bio surfactants has been increased in various sectors
due to their ecofriendly and health supporting potential. Bio surfactants have recently became one
of the promising products of bio economy with wide variety of applications .
Bio Surfactants Applications
Hydrocarbons Degradation/Remediation
The extensive production and use of hydrocarbons has resulted in widespread environmental
contamination .Due to their toxicity, persistent and neg ative influence on living organisms it is
important to clean-up the polluted sites. Hydrocarbons, as the hydrophobic organic chemicals,
exhibit limited solubility in groundwater and tend to partition to the soil matrix. This partitioning
can account for as much as 90–95% or more of the total contaminant mass. As a consequence, the
hydrocarbon contaminants exhibit moderate to poor recovery by physico -chemical treatments;
limited bioavailability to microorganisms; and limited availability to oxidative and red uctive
chemicals when applied to in-situ and/or ex-situ applications
A promising method that can improve bioremediation effectiveness of hydrocarbon contaminated

environment is the use of bio surfactants. They can enhance hydrocarbon bioremediation by two
mechanisms. The first comprises the increase of substrate bioavailability for microorganisms, while
the other includes interaction with the cell surface which increases the hydrophobicity of the
surface allowing hydrophobic substrates to associate more eas ily with bacterial cells . By reducing
surface and interfacial tensions, bio surfactants increase the surface areas of insoluble compounds
leading to increased mobility and bioavailability of hydrocarbons. In consequence, bio surfactants
enhance biodegradation and removal of hydrocarbons. Addition of bio surfactants can be expected
to enhance hydrocarbon biodegradation by mobilization, solubilization or emulsification.
(Magdalena Pacwa et al.,2011)
Bio surfactants and Bio remediation
An alternative and eco-friendly method of remediation technology is the use of bio surfactants and
bio surfactant-producing microorganisms to control environmental pollution. At higher
concentration heavy metals are found to be the main culprit for plant growth due to formation of
free radicals and cause oxidative stress. This problem can be overcome by using both metal
resistant and bio surfactant producing microorganisms. Biosurfactants can be applied to a small part
of contaminated soil in which soil is put in a huge cement mi xer, biosurfactant-metal complex is

flushed out, soil deposited back, and biosurfactant -metal complex treated to precipitate out
biosurfactant, leaving behind the metal. The bond formed between the positively charged metal and
the negatively charged surfactant is so strong that flushing water through soil removes the
surfactant metal complex from the soil matrix. (Magdalena Pacwa et al.,2011)
Application of bio surfactants not only allow the recovery of oil from oily sludge but also allows to
reuse or recycle the valuable hydrocarbons recovered from the oily sludge. Oily sludge generated
from petroleum processing plants is another source of environmental pollution . It is reported that
bio surfactants plays key role in oil mobilization by emulsification in microbial enhanced oil
recovery (Sen et al.,2008).For the sustainable environment, there is need to treat and stabilize oily
sludge prior to disposal as it reduces the environmental impact. Moreover, the recovered oil can
also serves as an energy source which has led to the renewed and intensified efforts to treat the oily
sludge.
Sustainable Agriculture
These days bio surfactants can be widely exploited in agriculture to eliminate plant pathogens and
indirectly used for plant growth promotion due to thei r antimicrobial potential increasing
theplantmicrobeinteractionbeneficialforplant.
Improvement of Soil Quality
It is reported that the desorption of hydrophobic pollutants which are tightly bound to soil particles
can be acceleratedby bio surfactants. Besides, they can degrade certain chemical insecticides
accumulated in the agricultural soil. Bio surfactant can efficiently remove the organic insoluble
pollutants from soil as compared to synthetic surfactants .The requirement of iron for increased
production of biosurfactant by Pseudomonas sp. and enhancement of bioavailability of poly
aromatic hydrocarbons has also been reviewed (Dhara et al.,2013).
There are several reports on potential properties of bio surfactants produced by Pseudomonas
sp,Bacillus sp., and Acinetobacter sp. in respect of removal of heavy metals from contaminated soil
and even acceleration of biodegradation of pesticides (Pacwa Plociniczak et al. 2011; Kassab and
Roane 2006).Biosurfactant such as rhamnolipid and surfactin are known to remove heavy metals
such as Ni, Cd, Mg, Mn, Ca, Ba, Li, Cu, and Zn (ions) from soil with a new method of foaming -
surfactant technology (Mulligan and Wang 2004; Mulligan et al. 2001;Dhara et al.,2013).
Plant Pathogen Elimination

Antimicrobial activity of bio surfactants against various plant pathogens makes them a promising
biocontrol molecule to achieve the sustainable agriculture. They can facilitates the biocontrol
mechanism of plant growth promoting microbes like parasitism, antibiosis, competition, i nduced
systemic resistance, and hypovirulence (Singh et al. 2007). It is reported that some bio surfactants
are known to have antagonist properties and used to enhance the antagonistic activities of microbes
and microbial products in agriculture (Jazzar an d Hammad 2003; Kim et al. 2004;Nihorimbereet al.
2011). It is also reported that the plant growth -promoting P. putida can produces biosurfactant
causing lysis of zoospores of the oomycete pathogen .Besides, lipopeptide biosurfactant produced
by Bacillus spp. inhibit growth of phytopathogenic fungi like Fusarium spp., Aspergillus spp., and
B.sorokiniana etc. Moreover, biosurfactant produced P.fluorescens shows significant antifungal
properties which are found to inhibit the growth of fungal pathogens like P.ultimum ,F. oxysporum ,
P.cryptogea , V.microsclerotia etc (Dhara P et al.,2013).
Plant Microbe Interaction
Generally, Microbial factors such as ability to form biofilm on root surface and release of quorum
sensing molecules ,motility, is important for establishment of association with any plant. It is
reported that these molecules affects the motility of microorganisms, participate in signaling and
differentiation as well as in biofilm formation (Ron and Rosenberg 2001; Berti et al. 2007; Van
Hamme et al 2006; Kearns and Losick 2003). Moreover, the biosurfactant (rhamnolipid) produced
by Pseudomonas spp. can efficiently regulates the process of quorum sensing (cell to cell
communication (Dusane et al.,2010).The biosurfactants produced by rhizobacteria can improve the
bioavailability of hydrophobic molecules which can serve as nutrients. Also, the biosurfactants
produced by soil microbes provide wettability to soil and support proper distribution of chemical
fertilizers in soil thus supporting the plant growth promotion (Dhara et al.,2013).
Bio pesticides
At present bio surfactants has been promisingly used as bio pesticides due to their low toxicity,
high degree of biodegradability, optimal activity at extreme environmental conditions, and
environmental friendly nature. Among various bio surfactant producing microbes, Bacillus spp. are
one of the prominent biosurfactant-producing bacteria producing the broad -spectrum lipopeptides
such as surfactin, iturin, bacillomycin, fengycin, and lichenysin etc. (Mu kherjee and Das,2005).
Petroleum Industry

In the petroleum industry, biosurfactants have been applied effectively for the exploration of heavy
oil, offering advantages over their synthetic counterparts throughout the entire petroleum
processing chain (extraction, transportation and storage). These biomolecules are efficiently used
in microbial-enhanced oil recovery, the cleaning of contaminated vessels and to facilitate the
transportation of heavy crude oil by pipeline .Biosurfactants have several potential applications for
use across the oil processing chain and in the formulations of petrochemical products such as
emulsifying/demulsifying agents, anticorrosive, biocides for sulfate -reducing bacteria, fuel
formulation, extraction of bitumen from tar sands, and many other innovative applications. Due to
their versatility and proven efficiency, biosurfactants are often presented as valuable versatile tools
that can transform and modernize petroleum biotechnology in an attempt to provide a true picture of
state of the art and directions or use in the oil industry.
Although bio surfactants exhibits potential applications with diverse characteristics and properties
over synthetic surfactants, they are not able to compete with synthetic molecules due to their
limitations, such as high production cost, l ess production yield, bioreactor design and its related
complications, inefficient downstream processes, limited availability of substrate, etc. These
precincts can overcome with the development of better reactor design, product recovery technology,
ecologically and environmentally favored media, and decreased production cost. (Mulugeta et al.,
2021)
SOLVED PROBLEMS
1) Discuss growth curve of bacteria.
a) Solution: Please refer the topic: characteristic phases of the growth cycle
2) What is generation time? How it is calculated? Give protocol.
a) Solution: Please refer the topic: generation time
3) What is symbiotic relationship. Discuss with suitable example.
a) Solution: Please refer the topic: symbiotic relationship
4) What are siderophores? Discuss its types with importance.
a) Solution: Please refer the topic: Siderophore as Iron chelating agent
5) Comment on : CAS test
a) Solution: Please refer the topic: Siderophore as Iron chelating agent
SELF-TEST-MCQS
1) Nematophagus fungi depends ons

a) Namatodes
b) Plants
c) Microorganism
d) None of the above
[Answer Key: a) Namatodes]
2) Bio surfactant are categorised on basis of
a) Biological composition and microbial origin
b) Chemical composition and microbial origin
c) Both a and b
[Answer Key: b)Chemical composition and microbial origin]
3) In CAS assay following colour indicates synthesis of siderophore
a) Yellow/ white
b) Pink/ orange
c) Green/ yellow
d) Red/ purple
[Answer Key: b) Pink/ orange]

1) SAQ 01 Write note on application of siderophores
a) Solutionplease refer the topic: siderophores
2) SAQ 2 Write note on application of Biosurfactant
a) Solutionplease refer the topic: Biosurfactant
SUMMARY
First section of this unit presents the overview of Environmental Microbiology along with.brief
History of Microbiology and controversy over concept of spontaneous generation.their significance
and comprehensive classification ofmicroorganisms. Various methods of identification of
microorganisms. This unit also discuss some branches of Microbiology It also describes the scope
of Microbiology and its limitations.
Second section of this unit presents the overviewof microbial diversity and metabolism in detail. It
gives description of major groups of microorganisms like bacteria, principle of Gram Staining
technique,their structure and classification on basis of different parameters . Besides, it illustrates
various microbial processes such as anaerobiosis, chemolithotrophy, photosynthesisand Nitrogen

Fixation. Determination of microbial numbers, biomass and activities, Microbial numbers,
Directand viable count procedures, biochemical methods, microbial biomass. Furthermore, it
highlights various microbial activities such as, Photosynthesis, respiration, heterotrophic
potential, and specific enzyme.
Third section of this unit presents the overviewof microbial population and community dynamics in
detail. It gives description of various techniques for determination of microbial number,
measurements of microbial mass. Besides, it illustrates the microbial growth in closed and open
environments, characteristic phases of the growth cycle. It highlights the generation times for some
common bacteria under optimal conditions of growth .Methods for enriching, isolating and
analyzing microbial communities in laboratory system.
Fourth section of this unit presents the overview of effects and measurement of environmental
determinants. Explaining various environmental fact ors along with their definitions including
temperature, radiation, salinity, moisture activity, redox potential, magnetism, etc. It also describes
different biological interactions, microbial interactions and their interactions with plants and
animals. It highlightson iron chelating compounds i.e. siderophores,their types, the method for their
identification along with their applications. It also illustrated the recent advances of Bio surfactants
in environmental applications.
KEY WORDS
Spontaneous Generation, Microbial, Anaerobiosis, Chemolithotrophy, generation
Redox Potential, Magnetism, Siderophores Bio Surfactants
REFERENCES
 Sophie C. Sherriff, Environmental Magnetism: Sediment Source Tracing, Geomorphological
Techniques, Chap. 1.4, Sec. 1 (2014)
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CREDIT 02
UNIT 02-01: BIO INDICATORS

LEARNING OBJECTIVES
After successful completion of this unit, you will be able to
 To understand the types and importance of bio indicators

 To understand the importance of diversity index
INTRODUCTION
Bioindicators
These are the living organisms such as plants, planktons, animals, and microbes, which are
utilized as an important tool to screen the changes in the environment, either positive or negative,
and their consequences on natural ecosystem in the environment and the human society
They are used to evaluate environmental health and biogeographic changes taking place in the
environment. Each organic entity inside a biological system provides an indication regarding t he
health of its surroundings .They are used to predict the natural state of a certain region or the
level/degree of contamination (Khatri & Tyagi 2015).
The advantages associated with using Bio indicators are as follows:
Bio indicators can be used to determine the biological impacts and help to monitor the synergetic
and antagonistic effects of pollutants on ecosystem and its components. It can be used to monitor
early stage diagnosis and the adverse impacts of toxins on organisms.It is very easily c ounted due
to their prevalence.Moreover, it can be used as economically viable alternative than the specialized
measuring systems.
Plankton Community as Indicator of Water Pollution(Trishala Ket al.,2016)
Plankton is the productive base of both marine and freshwater ecosystems, providing food for
larger animals and indirectly for humans, whose fisheries depend upon plankton.Plankton are
primarily divided into following broad functional groups :
i. Phytoplankton :These are the autotrophic, prokaryotic, or eukaryotic algae live near the
water surface where there is sufficient light to suppo rt photosynthesis. Among the more
important groups are the diatoms, cyanobacteria, dinoflagellates, and
coccolithophores.Algae are quite sensitive to contamination, and this may be reflected in

their population levels and/or rates or photosynthesis Affects development of population or
photosynthesis, for the most part, algae are as sensitive to contaminations as other species.
When there is change in the diversity of phytoplankton species, it may indicate pollution of
the marine ecosystem(Trishala Ket al.,2016)
ii. Zooplankton :These are the microscopic animals living near to the surface of the water
body. They are poorswimmers, instead relying on tides and currents as a transport
mechanism .e.g
iii. . small protozoans or metazoans (e.g. crustaceans and other animals ) that feed on other
plankton and telonemia. Some of the eggs and larvae of larger animals, such as fish,
crustaceans, and annelids etc. They play key role for indicating water quality,
eutrophication, and production of a freshwater body. In order to dete rmine the status of a
freshwater body it is necessary to measure seasonal variations and presence of zooplanktons.
Differing varieties of species, biomass diversity and wealth of zooplankton groups can be
utilized to determine the strength of a biological system. The potential of zooplankton as a
bio-indicators species is high on the grounds that their development and conveyance are
subject to some abiotic (e.g. temperature,saltiness, stratification, and pollutants) and biotic
parameters (e.g. limitation of food, predation, and competition)(Trishala K et al.,2016)
In several water bodies like seas, lakes, streams, and swamps, significant biological
production is carried out by plankton. These planktons consist of communities that float
along currents and tides, yet they fuse and cycle important quantities of energy that is then
passed on to higher trophic levels They can rapidly react to ecological changes and viewed
as excellent indicators of water quality and trophic conditions due to their short time and
rapid rate of reproduction. Under the natural conditions, the occurrence of planktonic
organisms is identified with the resistance range in relation to abiotic ecological components
i.e.Oxygen fixation, Temperatureand pH etc.and the biotic interactions am ong the
organisms. The changes that occur within the communities of planktons provide the platform
to determine the trophic state of water bodies (Figure 2.1) (Pradhan et al. 2008;Trishala Ket
al.,2016)

Lake
Presence of planktons
Higher concentration of Nitrogen and Phosphorus
Plankton Reproduction
Poor water quality
Effect on living organisms
Figure.2.1.Flow Chart of Planktons Indicating Pollution of Lake Condition
Since planktons are profoundly sensitive to natural change they are best markers of water quality
and particularly lake conditions. One of the reasons planktons are being considered in lakes is to
monitor the water quality of the lake when there are high c entralizations of phosphorus and
nitrogen; these centralizations may be indicated by certain planktons reproducing at an increased
rate. This is evidence of poor water quality that may influence other organisms living in the water
body. In addition to being a health indicator, planktons are also the fundamental sustenance for
many larger organisms in the lake. Thus the plankton is key to the marine organisms, as both an
indicator of water quality and as the main food source for many fish (Thakur et al. 2013 ). Plankton
also plays an important role in biological deterioration organic matter; but if plankton populations
are too large this creates other problems in managing the water body. Fish at this critical stage of

ecological process play an important role by grazing the planktons. The two roles played by fish are
very crucial as they help in maintaining the proper balance of planktons in the pond and convert the
nutrient available in wastewater into a form which is consumable by humans. Additionally, certai n
planktons such as cyanobacteria produce toxins which are harmful for fish growth. Thus planktons
can be termed as useful or harmful, with respect to wastewater fed production of fish es ) (Table 2.1
and Table 2.2).( (Pradhan et al. 2008Trishala Ket al.,2016)
Table.2.1.Zooplanktons as Bio indicator Table.2.2. Phytoplankton as Bio indicator
(Adapted without modification from: Trishala Ket al.,2016)
Use of Diversity Index in evaluation of water Quality
Exploitation of natural water resources requires understanding of the quantity and its quality
because, the water resources are the ultimate recipients of pollution caused due to several natural
and human activities . Water in its path normally carries various materials .Thereby, the water
quality depending on the length and frequency of dissolution material in the path can be found very
different in different p Therefore, for preservation of the water resources, accurate identification of

the physical ,chemical and biological properties of these resources and determining the water
quality reservoirs is needed.
Nowadays for monitoring and control the quality of surface water, water quality indices are used.
Various water quality indexes have been developed in the past 40 years while one of the earliest
efforts to develop a WQI was done in association with the National Sanitation Foundation (NSF)
that the express results in simple language and understandable important role play in the study of
water quality. the application of this method, based on the parameters such as BOD, PH, DO, TS,
turbidity, nitrate, temperature, phosphate and is fecal coliform, using this index very popular, for
classification of surface water quality for drinking is complete and co mprehensive index. For
assessing the quality classification of various rivers some indices were used and its results showed
the effectiveness those methods .
The Water Quality Index (WQI)
Water quality index (WQI) is a number to express the overallwater qu ality of a certain location and
transforms the complexphysicochemical parameters into information that is usable
andunderstandable by general public. It is one of the most effective tools tocommunicate the water
quality information between policy makers andgeneral public.WQI represents a numerical
expression which has the purpose of establishing the ecological state of a body of water . For the
general calculation of this index, it is usually necessary to determine the following physical,
chemical and biological parameters: temperature, the pH, the electrical conductivity, dissolved
oxygen, the total dissolved solids (TDS), alkalinity, the biochimic consumption of oxygen the
chemic consumption of oxygen , nitrogenous (NO 3 -), nitrites, ammonium (NH 4+ ), clorides, metals
(Cr-total, Pb 2+ , Cd 2+ , Ni 2+ , Fe-total, Mn-total, Zn 2+, As 2+) and hardness . This method does not
usually limit the concomitant calculation of all physicochemical and biological parameters together,
they also can be calculated separately. It is very important to consider, when using this method, the
variation of physico-chemical parameters which exists mostly due to the anthropic factors, but also
due to natural processes such as the hydrological, topographical, litholology, climate, the
hydrographic basin, geological rocks and, last but not least, to erosion . Accor ding to the resulting
value obtained by the calculation of the WQI index, the water samples to be studied could be
framed in one of the following water quality categories: excellent, good, poor, very poor,
undrinkable .The WQI index was first developed in the United States in 1965 by Horton . Since
then, more methods of calculus have been invented to obtain the same water quality index (WQI) .

This is mainly based on the different approach regarding the selection of parameters and on their
influence on the calculation and final value of the indexes .(Valentina et al., 2018)
Case study: Application of Water Quality Index and Diversity Index for Pollution Assessment
of Kankaria Lake , Ahmedabad, India (Kumar A, Sharma MP ,2014)
Kankaria Lake is the biggest artificial lake of Ahmedabad located in south-eastern part of the city
.It was studied on basis of the 1 year literature survey and data of water quality parameters and
phytoplankton collected .The data was categorized into summer, monsoon and winter season s of the
year
The physicochemical data and phytoplankton data collected by Verma et al. [5] on Kankaria Lake
from March 2009 to February 2010 issued for the assessment of water quality. The assessment is
done in terms of seasonal variation in water quality index and phytoplankton diversity index. Apart
from these assessments, the importance of lake water to the people is also studied.
National Sanitation Foundation Water Quality Index (NSFWQI)
This index is useful tools for expression easier, water quality in order to public awareness and
managerial purposes and summarizes large amounts of data on water quality and in a word
is good or bad water quality. The parameter s used in this index include: The biochemical
oxygen demand (BOD), dissolved oxygen (DO) fecalcoliform, nitrate, PH, changes in water
temperature, total dissolved solids, total phosphorus and turbidity. In order to use the index,
the above-mentioned parameters were measured by standard methods at different stations, and
then results were analyzed by using the index National Sanitation Foundation Water Quality Index
(NSFWQI) of USA is one of the most acceptable and convenient WQI, pro posed by Horton in 1965
. This index is useful tools for expression easier, water quality in order to public awareness and
managerial purposes and summarizes large amounts of data on water quality and in a word is good
or bad water quality. The parameters used in this index include: The biochemical oxygen demand
(BOD), dissolved oxygen (DO) fecal coliform, nitrate, PH, changes in water temperature, total
dissolved solids, total phosphorus and turbidity. In order to use the index, the above -mentioned
parameters can be measured by standard methods at different stations, and then results were
analyzed by using the index.

The water quality data of several parameters like pH, total solids (mg/l), turbidity (NTU), D.O
(mg/l), B.O.D (mg/l), nitrate (mg/l), and t otal phosphate (mg/l)for the period March 2009 to
February 2010 is categorized into three season i.e. summer, monsoon and winter. NSFWQI for these
seasons can be calculated using standard software.
Shannon-Weiner Diversity Index (SDI)
Diversity index is the mathematical tool to express number of species in a biological community i.e.
to understand community structure. It provides more information about community composition
than simply species richness by taking the relative abundances of different species into account .A
widely used diversity index is the Shannon -Weiner Diversity Index (SDI). The mathematical
expression of SDI is expressed as follows:
Where, Pi –Proportional abundance of each species which is expressed as abundance of

species/total abundance.
Phytoplankton data of class Cyanophyceae (13 genera), Chlorophyceae (17 genera),

Bacillariophyceae (6 genera) and Euglenophyceae (3 genera) for the period March 2009 to February
2010 is categorized into three season namely summer, monsoon and winter. On the basis of these
data, their respective abundance ratio is computed . These abundance ratios are further used for the
computation of final SDI .
Results
NSFWQI between 70-90 are indications of good water quality in the lake throughout the year which
is attributed to the diversion of sewage away from the lake as well as the suitable lake management
adopted or strategies being adopted.

In winter season, the WQI is between good and medium water quality which is an alarming signal
for lake management authority (LMA) to take preventive measures to improve the quality. However,
the overall water quality trend shows relatively better trend in monsoon compared to other seasons
which might be due to the inflow of storm water to the lake, thereby diluting the p ollutant
concentration. Evident from the Indian standard of lake water quality, the lake water if found within
the limit.
The SDI of phytoplanktons of lake water is found within the range of 3.4 -3.5 Table 2.3 which shows
slight pollution level in lake water (Table 2.5).SDI for 39 genera of phytoplanktons calculated as
3.5, 3.4 and 3.4 for summer, monsoon and winter seasons respectively. The result shows that genera
diversity in summer is more than the monsoon and winter seasons.
The NSFWQI for monsoon holds maximum score followed by summer and winter. At the same
time, SDI for summer holds the maximum score. Therefore, it can be concluded that good water
quality among three seasons vary somewhere between summer and monsoon.
Table 2.3: Seasonal Variations in NSFWQI, Pollution Level and SDI.
Sr. Parameters Season

No. Summer Monsoon Winter
1 NSFWQI 73 80 70
2 WaterQuality Good Good Good
3 SDI 3.5 3.4 3.4
4 PollutionLevel Slight Slight Slight
Table 2.4:PhysicochemicalDataofKankariaLake
Sampling Season Indian Standard of

LakeWaterQuality
Sr. Parameters Summer Monsoon Winter Class-B of DBU
No Mean Mean Mean SystemofCPCB
.
1 pH 8.3 7.9 8.2 6.5-8.5
2 D.O(mg/l) 5.6 6.8 6.2 >5.0
3 B.O.D 2.1 1.3 1.8 <3.0

(mg/l)
Table 2.5: RelationsbetweenShannonDiversityIndexandPollutionLevel.
DiversityLevel ShannonDiversityIndex Pollution Level
High 3.0–4.5 Slight
Moderate 2.0–3.0 Light
Less 1.0–2.0 Moderate
Veryless 0.0–1.0 Heavypollution
The results indicated that NSFWQI from 70 -80 while SDI from 3.4-3.5 for summer, monsoon and
winter season is indication of slight pollution (Table 2.5) of the lake. A comparison with Indian lake
water quality standard, it is found that Kankaria Lake is fit for outdoor bathing. Through NSFWQI
and SDI, it can also be concluded that physicochemical and biological characteristic of Kankaria
lake water shows sign of further improvement somewhere between summer and monsoon .
SOLVED PROBLEMS
1) What are Bio-indicators? Discuss water pollution indicators with suitable examples.
a) Solution-Please refer topic: Bio-indicator
2) How diversity index is applied in water quality evaluation? Discuss.
a) Solution--Please refer topic:Diversity index
SELF-TEST -MCQS
1) The effort to develop a WQI was done in association with following
a) PTS
b) NSF
c) BGT

d) RSM
[Answer Key: b) NSF]
2) NSF WQI is related to
a) Air quality
b) Water quality
c) Soil quality
[Answer Key: b)Water quality]
3) Following diversity index is the mathematical tool to understand the community structure
a) SDI
b) NSFWQI
c) WQI
d) All of the above
[Answer Key: a) SDI]

1) SAQ :Write note on: Shannon-Weiner Diversity Index (SDI)
a.Solution--Please refer topic:Shannon-Weiner Diversity Index
2) SAQ :Write note on:National Sanitation Foundation Water Quality Index
b.Solution--Please refer topic:National Sanitation Foundation Water Quality Index
UNIT 02-02: DETERMINATION OF MICROBIOLOGICAL QUALITY

LEARNING OBJECTIVES
After successful completion of this unit , you will be able to
 To understand the importance of water quality in polluted area

 To understand importance of potable water and role of indicator organisms
 To understand importance of E.coli, Stereptococci spp and Clostridia spp as pollution
indicators
 To understand different types of bacterial plate counts
Waters is an important disease transmission vehicle due to the exposure of users to pathogenic
organisms present in the water bodies. The health risk linked to the presence of these
microorganisms depends on the pathogen form, type and concentration. A large variety of
pathogenic forms may occur, including indigenous and external microorganisms, usually with
considerable fecal content. The major cause of the presence of the pathogenic organisms in
recreational and potable waters is anthropogenic activities, land use, and fecal pollution sources.
Microorganisms like, Bacteria, protozoa and viruses, have been identified as the primary source of
microbiological water contamination usually responsible for w aterborne diseases However,
additional groups, such as helminthes worms and fungi, appear less frequently, especially at
significant levels of contamination. Routes of exposure to pathogens in such water are direct dermal
contact, ingestion and inhalation resulting in adverse health effects In this context, safety of the
recreational and potable water is a priority for managing the resources of water. (Carla Rodrigues
et al.,2017)
Indicator Organisms (https://www.ncbi.nlm.nih.gov/books/NBK234164/)
The term "indicator organism are the microbes whose presence is evidence that pollution
(associated with fecal contamination from man or other warm -blooded animals) has occurred.
Indicator organisms may be accompanied by pathogens, but do not necessarily cause di sease
themselves.
Generally, pathogens are usually more difficult to grow, isolate, and identify than indicator
organisms, and often require special media and procedures. Indicator organisms, rather than the
actual pathogens, are used to assess water quali ty because their detection is more reliable and less
time-consuming. Pathogens appear in smaller numbers than indicator organisms and are therefore
less likely to be isolated. An indicator organism should have the following characteristics:
 Applicable to all types of water.
 Present in sewage and polluted waters when pathogens axe present.
 Number is correlated with the amount of pollution.
 Present in greater numbers than pathogens.
 No after growth in water.
 Greater survival time than pathogens.
 Absent from unpolluted waters.
 Easily detected by simple laboratory tests in the shortest time consistent with accurate
results.
 Has constant characteristics.
 Harmless to man and animal.

No organism or group of organisms meets all these criteria, but the "coliform group" of organisms
fulfills most of them.
Escherichia Coli and the ColiformGroup
(https://www.ncbi.nlm.nih.gov/books/NBK234164/)
Escherichia coli is commonly found in the human intestine. It is not normally a pathogen, although
pathogenic strains are known. Physiologically, E. coli and members of the genera Salmonella and
Shigella are quite similar. All are classified as enteric bacteria of the family Enterobacteriaceae
(Cowan, 1974). They are facultatively anaerobic, and are able to ferment sugars with the production
of organic acid and gas. These three genera carry out a type of fermentation called "mixed -acid
fermentation," but differ in a number of physiological characteristics. Many physiological
differences between various enteric bacteria axe known (Ewing and Martin, 1974), bu t at the
beginning of the twentieth century this was not so. In the early days of water bacteriology, some
simple operational distinctions were necessary. The lactose -fermentation test became the prime
diagnostic tool: E. coli ferments lactose with the formation of acid and gas; Salmonella spp.and
Shigella spp.do not ferment lactose.
One source of confusion is the necessity to distinguish between E. coli and the "coliform group" of
bacteria. Although the taxonomy of bacteria is constantly undergoing revision, Escherichiaspp. is
well defined. It is distinguished from other mixed -acid fermenters of the Enterobacteriaceae
primarily on the basis of sugar-fermentation reactions, motility, production of indole from
tryptophan, lack of urease, inability to utilize citrate as sole carbon source, and inhibition of growth
by potassium cyanide. However, the "coliform group" is not so precisely defined. The "coliform
group," as defined in Standard Methods , American Public Health Association, 1975, comprises all
"aerobic and facultative anaerobic, gram-negative, non-spore-forming, rod-shaped bacteria which
ferment lactose with gas formation within 48 hr at 35 C." This is not a taxonomic grouping, but an
operational one that is useful in water-supply and sewage-treatment practice. It includes organisms
in addition to E. coli, most importantly Klebsiella pneumoniae and Enterobacter aerogenes, which
are not mixed-acid fermenters. The entry of the term "coliform'' into sanitary bacteriology was
associated with a policy established by H. E. Jordan when he became editor of the Journal of the
American Water Works Association ; he stated that he would substitute "coliform'' for " E. coli" in
papers submitted to him (Jordan, 1937).

Although most isolates classifiable as Escherichia by modern methods ferment lactose, about 5-9%
of them do not (Ewing and Martin, 1974). No isolates of the genus Salmonella, either in the species
S. typhi or in other species, produce gas from lactose ,therefore, a water sample containing
Salmonella and a lactose-negative E. coli would be negative on the coliform test and would
probably be discarded without further examination, because of the definition of "coliform." Even if
glucose were substituted for lactose in a coliform analysis, a significant fractio n of organisms
would be missed, inasmuch as about 9% of isolates of Escherichia do not form gas from glucose
(Ewing and Martin, 1974).
Because there are two procedures—the multiple-tube-dilution or most-probable-number (MPN)

technique, and the membrane-filter (MF) technique—the coliform group of organisms requires two
definitions (American Public Health Association, 1975). On the basis of the MPN technique, the
group consists of all aerobic and facultatively anaerobic, gram -negative, non-spore-forming, rod-
shaped bacteria that ferment lactose with formation of gas within 48 h at 35ºC. On the basis of the
MF technique, the group consists of all organisms that produce a dark colony (generally purplish -
green) with a metallic sheen within 24 h of incubation on t he appropriate culture medium; the sheen
may cover the entire colony or appear only in a central area or on the periphery. These two groups
are not necessarily the same, but they have the same sanitary significance.
If the coliform group is to be used as an indicator of fecal pollution of water, it is important to

know that the coliforms do not lose viability in the water environment faster than pathogenic
bacteria, of salmonella spp. and shigella spp.. Little information exists on the survival of bacteria
in finished water, and the data on other types of water are scattered and fragmentary. McFeters et al.
(1974) recently reviewed previous work and presented their own data on die -off of intestinal
pathogens in well water. It is found that die-off rates for pathogens and coliforms are
approximately the same. Earlier work on the survival of salmonellae in water was reviewed by
McKee and Wolf (1963).
Thermo tolerant coliform:
It isproposed that a faecal coliform: faecal streptococci ratio of four or greater may indicate human
pollution, whereas ratios of two or less may indicate animal pollution. There are many factors,
however, that can jeopardise the usefulness of this ratio. Foremost are the quicker die -off of
coliforms in the environment and different count s from various media used for bacterial isolation

.Hence, the use of this ratio is no longer recommended unless very recent faecal pollution is being
monitored. (Nicholas J.et al.,2001)
Sulphite-reducing clostridia and other anaerobes
Until bifidobacteria were suggested as faecal indicators ,C. perfringens was the only obligately
anaerobic, enteric micro-organism seriously considered as a possible indicator of the sanitary
quality of water . Despite the first isolation of bifidobacteria in the late 1800s T issier 1889) and
very high numbers in human faeces (11% of culturable bacteria), their oxygen sensitivity has
limited their role as useful faecal indicators in waters . The anaerobic sulphite -reducing clostridia
sp..are much less prevalent than bifidobacteria in human faeces, but their spore-forming habit gives
them high environmental resistance. C. perfringens is the species of clostridia most often associated
with the faeces of warm-blooded animals .however, is only present in 13–35% of human faeces
.C.perfringens as a faecal indicator persist for long time in the environment than the enteric
pathogens. In this context, suggested that all sulphite-reducing clostridia in receiving waters are not
considered as indicators of faecal pollution, however consider ed as appropriate indicator. (Nicholas
J.et al.,2001)
Other Indicator Organisms(https://www.ncbi.nlm.nih.gov/books/NBK234164/)
Because of certain limitations of the coliform group as general indicators of water quality, workers
have continually searched for better indicator organisms. No other organism has been found that is
better than the coliform group, but it is pertinent to mention briefly some of these other indicators
(Geldreich, 1975). Fecal coliforms, fecal streptococci (defined as those organisms which develop
on media incubated at 44.5ºC) have frequently been used in stream and lake pollution work, but are
not suitable as indicators of drinking water quality because the number of fecal coliforms is
considerably lower in source waters than total coliforms, making the test less sensitive. Fecal
streptococci (defined as those organisms able to grow on medium containing sodium azide) have
been used in water pollution work, but have not proven suitable for drinking water analysis because
of low recovery rates, poor agreements between various methods, and uncertainty as to their
significance in water. Several other organisms have been suggested as indicators but have found
even less acceptance: Clostridium perfringens, Bifidobacterium spp, Pseudomonas spp ,
Staphylococcus spp. It would be undesirable and extremely risky to substitute any organism for the
coliform group now, although research studies that compare other indicator organisms with
coliforms are warranted.

Method for determination of Microbiological Quality (Daiva Staradumskytė and Algimantas
Paulauskas,2012)
Confirmation of E.coli :
1. Collect the Samples from different sources such as tap water, well water, borehole water,
recreational water etc.
2. Samples should be transported to the laboratory i mmediately after collection. Various

dilutions and volumes should be filtered through membrane.
3. A 100 ml volume of water sample is drawn through a membrane filter (0.45 µm pore size)
using a vacuum pump.
4. The filter should be placed on a petri dish contai ning selective agar.
5. Membranes should be transferred on Tergitol 7 agar (T7A) which is used selectively for
coliform bacteria, when incubated at elevated temperatures (36 ± 2 °C, 44 ± 0.5 °C, 24 h).
6. All red, yellow, orange and other colonies should be cu ltivated for confirmation in Tryptone
water for indole test and Tryptone soy agar for oxidase test.
e.g.When oxidase test is negative, indole is negative ,it confirm as coliform bacteria, and when ,
oxidase test is negative, indole test is positive ,it co nfirmed as Escherichia coli (LST 9308-1,
2001).
To identify coliform bacteria at species level biochemical tests such as Triple Sugar Iron Agar,
Lysine Iron Agar, Urease Test Broth, RapID oneE system etc are used. SlanetzBartley agar which is
used selectively for fecal Streptococcus, when incubated at elevated temperatures (36 ± 2 °C, 48 h),
and Bile Esculin agar is used for fecal Streptococcus confirmation (44 ± 0.5 °C, 2 h) (Fig. 2 a, b).
All red or brown colonies on the membrane from Slanetz -Bartley agar are calculated and membrane
is transferred on Bile Esculin agar, if colonies are black -brown surrounding the medium after two
hours, it is a confirmation of fecal Streptococcus (LST 7899 -2, 2001) (Daiva Staradumskytė and
Algimantas Paulauskas,2012)
The Heterotrophic Plate Count (HPC)
It was formerly known as the standard plate count, is a procedure for estimating the number of live
culturable gheterotrophic bacteria in water

Heterotrophic plate count (HPC) bacteria represent those microbes isolated by a particular method,
whose variables include media composition, time of incubation, temperature of incubation, and
means of medium inoculation
According to Reasoner (1990), HPC is a useful tool for monitoring the efficiency of the water
treatment process, including disinfection; assessing changes in finished-water quality during
distribution and storage and distribution system cleanliness; Assessing microbial growth on
materials used in the construction of potable water treatment and dis-tributionsystems;monitoring
bacterial population changes followingtreatment modifications such as a change in the type of
disinfectant used etc.
HPC media and methods (Martin J et al.,2004)
Through the years, many ‗‗standard methods‘‘ have been used to enumerate the very broad range of
genera that comprise HPC populations in drinking water.
Although often referred to as non-selective media, all media used for HPC determinations, along
with respective time and temperature conditions, are ‗‗ selective‘‘ for those bacteria that can grow
under those specific conditions. Generally, there is no single medium or method to recover or
enumerate all bacteria in the water being analyzed.It is found that several heterotrophic bacteria
present in water are not culturable .The choice of culture medium, temperature, and incubation time
are important w.r.t. HPC results from a given water sample. HPC determination can be carried out
using high-nutrient and low-nutrient media also. However, high-nutrient media are comparatively
good for enumeration of bacteria from animals and humans.. Whereas, low -nutrient media are
comparatively good for enumeration of water-based bacteria, including drinking water. The most
commonly employed heterotrophic medium is R2A. New methods that employ fluorescent
substrates have been developed (Jackson et al., 2000). Fluorescence permits more rapid results and
has the potential for automation..Low-temperature incubation (20 – 28 jC) and longer incubation
time (5 – 7 days) favor the growth of water-based bacteria.
Generally, all bacterial pathogens and opportunistic pathogens are heterotrophic bacteria, some of
which can grow on media used for determining standard plate counts or heterotrophic plate counts
in drinking water. However, it is necessary to use selective ordifferential media to distinguish
pathogens and opportunistic pathogens from non -pathogens.
There are four prescriptive test method as:

(https://www2.gov.bc.ca/assets/gov/environment/research -monitoring-and-
reporting/monitoring/emre/methods/heterotrophic_plate_count_by_membrane_filtration_2017dec08
_edits.pdf)
A. Pour Plate:
The procedure is simple to perform and can accommodate volumes of sample or diluted sample
ranging from 0.1 to 2.0 mLs. The colonies produced are relative ly small and compact, and less
likely to encroach on each other than those produced by surface growth. However, submerged
colonies can be slower growing and difficult to transfer. A thermostatically controlled water bath is
essential for tempering the agar, and care is needed to prevent heat shocking the bacteria when
dispensing the hot agar. Replicating every volume and dilution plated analyses is not required. The
pour plate method generally yields lower bacterial counts regardless of medium or time of
incubation and is generally limited to 0.1 to 1.0 ml sample volume
B. Spread Plate:
This procedure causes no heat shock and all colonies are on the agar surface where they can be
easily distinguished from particles and bubbles. Colonies can be quickly transferr ed and
morphology easily discerned. However, this method is limited by the small volume of sample or
diluted sample that can be absorbed by the agar: 0.1 to 0.5 mLs depending on the degree to which
the pre-poured plates have been dried. A supply of pre -dried, absorbent agar plates must be
maintained to use this procedure. The spread plate method generally yields higher counts than other
methods but is limited to a 0.1 to 1.0 ml sample volume.
C. Membrane Filtration:
This procedure permits testing of large volu mes of low-turbidity water. It produces no heat shock.
Disadvantages include the expense for the membrane filtration equipment, the smaller display area
of the filter, the need to detect colonies by reflected light against a white background if coloured
filters or contrast stains are not used, possible damage to cells by excessive filtration pressures, and
possible variations in membrane filter quality. The membrane filtration method is more flexible
because it allows for the analysis of sample volumes grea ter than 1.0 ml.
D. Enzyme Substrate:

This procedure can be used with samples having a wide range of bacterial concentrations. The
method uses a substratebased medium in which the substrates are hydrolyzed by microbial enzymes
causing the release of 4-methylumbelliferone maximally after 48 hours of incubation at 35°C. 4 -
Methylumbelliferone fluoresces when exposed to long -wavelength (365 nm) ultraviolet light. The
number of fluorescing wells corresponds to a most probable number (MPN) of bacteria in the
original sample. This test produces no heat shock and is comparable in performance to the pour
plate method.
Table.2.6. Common HPC genera in drinking water
(Adapted without modification from Martin J et al.,2004)

Lichen asbio indicators of Air Pollution
Lichens are mutual associates of a fungus and an alga or Cyanobacterium and occur as crusty
patches or bushy growths on trees, rocks and bare ground. The names given to lichens strictly refer
to the fungal partner; the algae have separate names. They are v ery sensitive to sulfur dioxide
pollution in the air. Since lichens have no roots, they absorb much of their raw materials directly
from the air and moisture around them. This makes them very sensitive to air pollution and acid
rain and since lichens have no way to excrete the pollutants they absorb, these materials stay inside
of their cells. Since pollutants build up inside them, lichens can be used to monitor the long -term
accumulation of pollutants. Scientists collect and analyze lichens near sources of pollution to
determine how far the pollution has spread. .
Lichens were recognized as potential indicators of air pollution as early as the 1860's in Britain
and Europe (Hawksworth and Rose, 1976). Since then, lichens have played prominent roles in air
pollution studies throughout the world because of their sensitivity to different gaseous pollutants,
particularly sulfur dioxide. They have also been found to act as accumulators of elements, such as
trace metals, sulfur, and radioactive elements (Stolte et al., 1993). The lichen species best suited as
bio-monitors are foliose (having a lobed, leaf-like shape) and fruticose (having upright or
pendulous branches) epiphytic lichens. The properties that make them suitable for monitoring
purposes are the weakly developed cuticle and vascular bundles, absence of real roots, their slow
growing nature and long life cycle and their broad distribution (Wolterbeek et al., 2003)
There are two primary characteristics of lichens that make them particularlyusefulto charact erize
atmospheric deposition:
1. Unlike vascular plants, lichens lack a cuticle or specialized guard cells to control the
exchange of water, nutrients, gases, and particles with the external environment.This is an
adaptive mechanism that allows lichens to obtain sufficient nutrients from precipitation
and other atmospheric sources.However, this physiology also prevents lichens from
mediating their up take of atmospheric pollutants.Consequently,elemental content in
lichen reflectsatmospheric sources of nutrients and contaminants.
2. Lichens lack a vascular system and roots and are therefore not influenced by elements in
soils.This lack of interaction with the soil environment removes one of the confounding
factors presentwhen using vascular plants as bio monitor s for air pollution.This

characteristic of lichens isparticularly important when studying ecosystems in which there
is the potential for both naturalsoil-borne elemental enrichment and anthropogenic
airborne enrichment; for example, this is thetypicalcase around metalmining operations.
Responses of Lichens to Air Pollution
Lichens have been used as receptor-based bio-monitors in air quality studies. Several experimental
studies w.r.t effects of sulfur dioxide, nitrogen compounds, ozone, heavy metals and oth er
atmospheric pollutants on the morphology and physiology of lichens .Historically, lichens have
been used in a qualitative way, with observations of population changes and morphological effects
serving as indicators of pollutants. In the last few decades , quantitative measurements of the
chemical content of lichens and sensitive physiological processes have increasingly been used to
indicate pollutants. Possible responses to air pollution stress include chlorophyll degradation,
changes in photosynthesis and respiration, alterations in nitrogen fixation, membrane leakage,
accumulation of toxic elements, and possible changes in spectral reflectance, lichen cover,
morphology, community structure and reproduction. Microscopic and molecular effects include
reduction in the number of algal cells in the thallus, ultrastructural changes of the thallus , changes
in chlorophyll fluorescence parameters , degradation of photosynthetic pigments , and altered
photosynthesis and respiration rates (Rupnarayan Sett et al.,2 016)
Heavy metals
The action of rain, surface water and passive upward diffusion from the substrate likely bring
dissolved minerals in contact with lichen thalli . The amount of each type of metal ion that can be
accumulated by lichen is dependent upon the uptake characteristics of that particular species and
the amount and availability of metal ions in the surrounding environment. Extracellular uptake of
metal ions is essentially a passive process of ion exchange determined by the character of the
ligands in the fungal cell walls. Intracellular uptake is limited by the nature of the metal ion, cell
membrane permeability, and the concentration of extracellular ligands with affinity for cations
Lichens have been proven to be good accumulators of heavy metals, and that the concentrations
correlate well with the concentrations measured in deposition. The concentrations have been very
high near the emission sources, and they have decreased exponentially with increasing distance
from the emission sources . (Rupnarayan Sett et al.,2016)
Metal toxicity in lichens is evidenced by adverse effects on cell membrane integrity, chlorophyll
content and integrity, photosynthesis and respiration, potential quantum yield of photo -system II,
stress ethylene production, microstructure, spectral reflectance responses, drought resistance, and

synthesis of various enzymes, secondary metabolites, and energy transfer molecules (Garty, 2001
;Rupnarayan Sett et al.,2016)
Sulfur Compounds
It is found that fumigation of SO 2 may cause interference in the flow of carbohydrates causing
symbiotic damage. Protein biosynthesis may be interrupted due to damage of membrane protein due
to SO 2 . As lichens have a tendency to lose moisture under highly polluted conditions the evaluation
of dry weight to fresh weight ratio can be used as bio -monitoring purpose. Production of ethylene is
another indicator of stress. Besides, this acidic environment alters the substrate chemistry, thereby
affecting species diversity and composition at the c ommunity level. (Rupnarayan Sett et al.,2016)
Table.2.7. General Mechanisms of SO 2 Toxicity
Type of reaction Observed / expected response or injury

Enzyme deactivation: (e.g. sulfitolysis) - Reduced metabolic activity; loss of membrane
Chemical modification -Binding to metal integrity, membrane function and cell
centers osmolality,
Stimulation of enzyme systems: (Often in Use of increases in enzyme activity as a
response to low pollution levels) -Glucose- bioindicator for non-visible injury and
6-phosphate-dehydrogenase, -Increases in detoxification of metabolism absorbed SO 2
glutathione and total protein SH
Reaction with reactive bio-molecules: - Modification of metabolic precursors and
Chemical (bisulfite adducts) -Redox (acts products; interference with electron flow in
as electron acceptor / donor at pH 7) photosynthetic and respiratory electron
transport chains
(Adapted without modification from: Hutchinson et al., 2006, Online document

http://www.fs.fed.us/r6/aq/lichen/almanac.htm# Physiological%20Responses.)
Effects of Fluorine
The effect of fluorine includes decrease in respiration and photosynthesis, an increase in membrane
and thallus permeability with a concomitant loss of ions, and changes in cellular ultra -structure.
Damage to lichens begins at levels of 50-70 ppm .Above 80 ppm, fluorine concentration chlorosis

was observed. The ability of lichens to accumulate F is a function of relative humidity, which
determines the moisture conditions of the thallus ((Gilbert, 1986 ; Rupnarayan Sett et al.,2016)
Effects of Photochemical Toxins

The effect of photochemical oxidants like Ozone and PAN includes decreases in photosynthesis,
decrease in species distribution, morphological and ultra -structural changes. Photochemical toxins
may have synergistic effects when combined with other pollutants, under low pH conditions
Fumigated lichen shows bleaching and discoloration at 300 ppm ozone exposure for 34 days (Ruoss
and Vonarburg, 1995 ;Farmer et al., 1992;Rupnarayan Sett et al.,2016)
SOLVED PROBLEMS
1) Discuss with suitable example, how indicator organisms are used to check potability of
water?
a) Solution:Please refer topic water potability
2) Discuss the importance and significance of E.coli and Streptococci spp for water quality.
a) Solution:Please refer topic Water Quality Index
SELF-TEST -MCQS
1) Following Ratio of fecal coliform indicate the human pollution
a) 2
b) 4
c) 3. 5
d) 2.8
[Answer Key: b) 4]
2) Standard plate count is known as
a) HPC
b) LHP
c) TPC
d) PTC
[Answer Key: a)HPC]
3) Lichen are the bio indicator of
a) Water pollution
b) Air pollution
c) Soil pollution
d) Noise pollution
[Answer Key : b)Air pollution]

1.SAQ: Write note on role of microorganism as a bioindicator
Solution –Please refer Topic- Bioindicator
2.SAQ: Write note on Lichen as bioindicator
Solution –Please refer Topic- Bioindicator
UNIT 02- 03: BIOSENSOR

LEARNING OBJECTIVES
 To understand the construction and role of bio -sensor

 To understand the role of bio catalyst
 To understand the importance of bio-sensor in industries
Since last few decades, the progress of sensor technology has become increasingly important, owing
to various applications, such as environmental and food quality monitoring, medical diagnosis and
health care, automotive and industrial manufacturing, as well as space, defense, and security.A
biosensor is an analytical device that combines a biological sensing element with a transducer to
produce a signal proportional to the analyte concentration. This signal can result from a change in
protons concentration, release or uptake of gases, light emission, absorption and so forth, brought
about by the metabolism of the target compound by the biological recognition element. The
transducer converts this biological signal into a measurable response such as current, potential or
absorption of light through electrochemical or optical means, which can be further amplified,
processed and stored for later analysis. (Y. Lei et al.2006)
Design and Principle of Biosensor
A biosensor is a device or probe that integrates a biological element, such as an enzyme or

antibody, with an electronic component to generate a measurable signal. The electronic component
detects, records, and transmits information regarding a physiological change or th e presence of
various chemical or biological materials in the environment. Biosensors come in different sizes and

shapes and can detect and measure even low concentrations of specific pathogens, or toxic
chemicals, and pH levels.
Working Principle of Biosensor
(https://www.elprocus.com/what-is-a-biosensor-types-of-biosensors-and-applications/)
Usually, a specific enzyme or preferred biological material is deactivated by some of the usual
methods, and the deactivated biological material is in near conta ct with the transducer. The analyte
connects to the biological object to shape a clear analyte which in turn gives the electronic reaction
that can be calculated. In some examples, the analyte is changed to a device that may be connected
to the discharge of gas, heat, electron ions, or hydrogen ions. In this, the transducer can alter the
device linked convert it into electrical signals which can be changed and calculated.
Components of Biosensor
(a) Analyte: A substance of interest whose constituents are being identified or detected (e.g.,
glucose, ammonia, alcohol, and lactose).
(b) Bioreceptor: A biomolecule (molecule) or a biological element that can recognize the target
substrate (i.e., an analyte) is known as bioreceptor (e.g., enzymes, cells, aptamers , deoxyribonucleic
acid (DNA or RNA), and antibodies). The process of signal production (in the form of light, heat,
pH, charge or mass change, plant or animal tissue, and microbial products) during the interaction
between bioreceptor and analyte is called bio recognition.
(c) Transducer: A device that transforms energy from one form to another. The transducer is a key
element in a biosensor. It converts the bio recognition event into a measurable signal (electrical)
that connects with the quantity or in the presence of a chemical or biological target. This process of
energy conversion is known as signalization. Transducers generate either optical or electrical
signals proportional to the number of analyte –bioreceptor interactions. According to the operating
principle, transducers are broadly categorized as electrochemical, optical, thermal, electronic, and
gravimetric transducers
(d) Electronics: The transduced signal is processed and prepared for the display. The electrical
signals obtained from the transducer are amplified and converted into digital form. The processed
signals are quantified by the display unit.

(e) Display: The display unit is composed of a user interpretation system, such as a computer or a
printer that generates the output so that the c orresponding response can be readable and
understandable by the user. Depending on the end -user prerequisite, the output can be in the form of
a numerical, graphical, or tabular value, or a figure.
(Adapted without modification from: Naresh, V et al.,2021)
Figure.2.2 .Schematic Diagram of Typical Biosensor

Characteristics of Biosensors (Naresh, V et al.,2021)
To develop a highly effective and capable biosensor system, certain static and dynamic
requirements are necessary. Based on these specifications, the performance of the biosensors can be
optimized for commercial uses.
(a) Selectivity: Selectivity is a crucial feature to consider when selecting a bioreceptor for a
biosensor. A bioreceptor can detect a particular target analyte molecule in a sample co mprised of
admixture spices and unwanted contaminants.
(b) Sensitivity: The minimum amount of analyte that can be correctly detected/identified in a
minimum number of steps and in low concentrations (ng/mL or fg/mL) to verify the existence of
analyte traces in the sample.
(c) Linearity: Linearity contributes to the accuracy of the measured results. The higher the linearity
(straight line), the higher the substrate concentration detection.

(d) Response time: The time is taken for obtaining 95% of the results.
(e) Reproducibility: Reproducibility is characterized by precision (similar output when the sample
is measured more than once) and accuracy (capability of a sensor to generate a mean value closer to
the actual value when the sample is measured every time). It is the ability o f the biosensor to
produce identical results whenever the same sample is measured more than once.
(f) Stability: Stability is one of the key characteristics in biosensor applications where continuous
monitoring is required. Stability is the extent of vulne rability to environmental disturbances inside
and outside the bio sensing device. The factors that affect stability are the affinity of the
bioreceptor (the extent of binding of the analyte to the bioreceptor) and the degradation of the
bioreceptor over time.
Advantages of Biosensor(Shruthi G et al.,2014; Strehlitz, B et al.,2008)
 High specificity of the biological elements used in the system. Such is the case for a number
of enzymes which are able to catalyze only the reaction of a specific isomer or the e xtremely
specific binding of antibody with antigen.
 Fast movement is obtained in a direct way and based on an electrical or optical signal. This
also has the additional advantage that a large number of samples can be analyzed in a
relatively short period of time and therefore cost can be reduced.
 The method is simple as sample preparation and manipulation are reduced to a minimum.
Very low reagent usage as these are only necessary for calibration, maintenance of optimum
conditions and, when necessary, sample dilution.
 The biological element can be re-used as this is usually immobilized. On line measurements
are possible, an indispensable requirement when continuous recording or automation of a
process is the goal.
 Many processes could be rationally optimized if sensors measuring certain crucial
parameters could be configured such that they permitted automatic process control.
 Enzyme based sensors are highly selective and fairly fast acting.
 Have catalytic activity, thus improving sensitivity. Antigen/Ant ibody and Nucleic acids they
are highly selective.
 They are ultra-sensitive .They bind very powerfully. Tissue materials sensors have longer
lifetime.

 enzymes more stable as they exist in their natural environment so less subject to degradation
cheaper than purified enzymes. Cheaper than purified enzymes
Limitations(Shruthi G et al.,2014; Strehlitz, B et al.,2008)
 Enzyme based transducers are expensive, it‘s cost of source, extraction, isolation and
purification is very high.
 Activity may be lost when immobilised on a transducer.
 Tend to lose activity, due to deactivation after a relatively short period of time (unless stored
under appropriate conditions).
 Tissue materials used contains a multiplicity of enzymes so may not be as selective as

purified enzymes decreased substrate specificity.
 Response time is slower.
 More tissue material for substrate to diffuse through; this may also dilute effect of enzymes.
 Whole cells sometimes have longer response times but the substrate needs to be transported
into cytoplasm.
 Longer recovery times (cells need to be re-energized).
 Less selective (contain many enzymes like tissues) antibody/antigen and nucleic acids have
no catalytic effect (only a binding reaction occurs on contact with its antigen.
 Binding reaction is very strong and very harsh conditions needed to reverse the reaction and
so the biosensor can be used only once (disposable strips)
Types of Biosensors
Biosensors can be grouped according to their biological element or their transduction element.
Biological elements include enzymes, antibodies, micro-organisms, biological tissue, and
organelles. The method of transduction depends on the type of physicoche mical change resulting
from the sensing event. Primarily biosensors based on transducer element are mass based

(piezoelectric, etc), electrochemical biosensors (potentiometric, amperometric, etc), and optical
types of biosensors (fiber optics, etc)
Depending on the mechanism of transduction, biosensors are classified as follows:
A.Resonant Biosensor- In this type of biosensor, an acoustic wave transducer is coupled with an
antibody (bio- factor). The analyte molecule (or antigen) gets added to the membrane, the mass of
the membrane diversities. Resulting diversification in the mass subsequently diversities the
resonant frequency of the transducer. This frequency change is then measured . (Reza K.et al.,2013)
B.Optical biosensors -The output transduced signal that is measured is light for this type of
biosensor. The biosensor can be made based on optical diffraction or electrochemiluminescence.
Optical transducers are particularly attractive for application to direct (label -free) detection of
bacteria. These sensors are accomplished to discover minute conversions in the refractive index or
thickness which happens when cells fasten to receptors immobilized on the transducer surface. They
correlate changes in concentration, mass or number of molecules to direct c hanges in characteristics
of light. Several optical techniques have been reported for detection of bacterial pathogens
including: monomode dielectric waveguides, surface plasmon resonance (SPR), ellipsometry, the
resonant mirror and the interferometer etc. (Reza K.et al.,2013)
 Surface plasmon resonance (SPR) biosensor- This is an evanescent area based optical
sensors applying thin gold film for sensing approaches. The interaction between analyte
flowing over immobilized interactant on gold surface is probed through the detection of
reflection minima on photo-detector array sensors. SPR has successfully been applied to the
detection of pathogen bacteria by means of immunoreactions
 Piezoelectric biosensors Piezoelectric (PZ) biosensor –It offers a real-time output,

simplicity of use and cost effectiveness. The chief idea is based on coating the surface of the
PZ sensor with a selectively binding material, for instance, antibodies to bacteria, and then
locating it in a solution containing bacteria. The bacteria will bind to the antibodies and the
mass of the crystal will increase while the resonance frequency of oscillation will decrease
proportionally
C. Thermal Biosensors -This type of biosensor is exploiting one of the fundamental properties of
biological reactions, namely absorption or production of heat, which in turn changes the
temperature of the medium in which the reaction takes place. They are combined by combining

immobilized enzyme molecules with temperature sensors. When the analyte comes in contact with
the enzyme, the heat reaction of the enzyme is measured and is calibrated against the analyte
concentration. Common applications of this type of biosensor include the detection of pesticides
and pathogenic bacteria (Reza K.et al.,2013)
D.Electrochemical Biosensors -Electrochemical biosensors are mainly used for the detection of
hybridized DNA, DNA-binding drugs, glucose concentration, etc. Electrochemical biosensors can
be classified based on the calculating electrical guidelines as: (i) conductimetri c, (ii) amperometric
and (iii) potentiometric. Contrasted to optical approaches, electrochemistry gives the analyst to act
with turbid samples, and the capital cost of equipment is much lower. On the other hand,
electrochemical methods present slightly mor e limited selectivity and sensitivity than their optical
counterparts. (Reza K.et al.,2013)
 Conductimetric Biosensors -The measured parameter is the electrical

conductance/resistance of the solution. When electrochemical reactions create ions or
electrons, the overall conductivity or resistivity of the solution has been altering. This
convert is ended and calibrated to an appropriate degree. Conductance measurements have
relatively low sensitivity
 Amperometric Biosensors -This is maybe the most common electrochemical discovery

approach applied in biosensors. This high sensitivity biosensor can discover electroactive
species present in biological test samples. Amperometric biosensors produce a current
proportional to the concentration of the substance to be detected. The most common
amperometric biosensors use the Clark Oxygen electrode
 Potentiometric Biosensors -These are the least common of all biosensors, but different
strategies may be found nonetheless in this category of sensor the calculated guideline is
oxidation or reduction potential of an electrochemical reaction. The working basis relies on
the truth that when a voltage is applied to an electrode in solution, a current flow occurs
because of electrochemical reactions. The voltage at which these re actions occur indicates a
particular reaction and particular species
E.Bioluminescence sensors- Recent advances in bioanalytical sensors have led to the utilization of
the ability of certain enzymes to emit photons as a byproduct of their reactions. This phenomenon is
known as bioluminescence. The potential applications of bioluminescence for bacterial detection

were begun by the development of luciferase reporter phages. The bacterial luminescence lux gene
has been broadly exercised as a reporter either i n an inducible or constitutive mechanism. In the
inducible manner, the reporter lux gene is fused to a promoter regulated by the concentration of a
combine of interest. As an effect, the concentration of the compound can be quantitatively assayed
by determining the bioluminescence intensity. Bioluminescence systems have been used for
detection of a wide range of microorganisms (Reza K.et al., 2013)
F.Nucleic Acid-based Biosensors -A nucleic acid biosensor is an analytical instrument that
integrates an oligonucleotide with a signal transducer. The nucleic acid probe is immobilized on the
transducer and behaves as the bio-recognition molecule to detect DNA/RNA fragments. (Reza K.et
al.,2013)
G.Nanobiosensors- Nanosensors can be described as sensors based on n anotechnology.
Development of Nano biosensor is one of the most current advancement in the area of
Nanotechnology. The silver and certain other noble metal nanoparticles have many important
applications in the field of bio labeling, drug delivery system, f ilters and also antimicrobial drugs,
sensors. (Reza K.et al.,2013)
Classes of biosensors
A) Catalytic biosensors:
kinetic devices that measure steady-state concentration of a transducer-detectable species
formed/lost due to a biocatalytic reaction
Monitored quantities:
i) rate of product formation
ii) disappearance of a reactant
iii) inhibition of a reaction
Biocatalysts used:
i) enzymes
ii) microorganisms
ii) organelles
iv) tissue samples
B) Affinity biosensors:
The devices in which receptor molecules bind analyte molecules ―irreversibly‖, causing a
physicochemical change that is detected by a transducer
Receptor molecules: i) antibodies

ii) nucleic acids
iii) hormone receptors
Biosensors are most often used to detect molecules of biological origin, bas ed on specific
interactions.
Microorganism based Biosensors(Somayeh et al.,2012)
A microbial biosensor consists of a transducer in conjunction with immobilized viable or non -viable
microbial cells. Non-viable cells obtained after permeabilization or whole cells containing
periplasmic enzymes have mostly been used as an economical substitute for enzymes. Viable cells
make use of the respiratory and metabolic functions of the cell, while the analyte to be monitored is
either a substrate or an inhibitor of these processes. Bioluminescence-based microbial biosensors
have also been developed using genetically engineered microorganisms constructed by fusing the
lux gene with an inducible gene promoter for toxicity and bioavailability testing.
Microbes have a number of advantages as biological sensing materials in the fabrication of

biosensors. They are present ubiquitously and are able to metabolize a wide range of chemical
compounds. Microorganisms have a great capacity to adapt to adverse conditions and to develo p the
ability to degrade new molecules with time. In this respect, the utilization of whole cells as a source
of intracellular enzymes has been shown to be a better alternative to purified enzymes in various
industrial processes.
It avoids the lengthy and expensive operations of enzyme purification, preserves the enzyme in its
natural environment and protects it from inactivation by external toxicants such as heavy metals.
Whole cells also provide a multipurpose catalyst especially when the process require s the
participation of a number of enzymes in sequence. Another mechanism used for the viable microbial
biosensor involves the inhibition of microbial respiration by the analyte of interest, like
environmental pollutants.
The major limitation to the use of whole cells is the diffusion of substrate and products through the
cell wall resulting in a slow response as compared to enzyme - based sensors. The cells can be
permeabilised using physical (freezing and thawing), chemical (organic solvents/detergents) a nd
enzymatic (lysozyme, papain) approaches.
The most common technique uses organic solvents. Such chemical treatment creates minute pores
by removing some of the lipids from the cell membranes, thereby allowing for the free diffusion of

small molecular weight substrates/ products across the cell membrane. These types of approaches
may have major significance in the future, especially for sensors like BOD where in polymers such
as protein, starch, lipid etc have to be broken down to monomers before they can be metabolized.
Recently, genetically engineered cells have been obtained for expression of cellulase activity on the
cell surface. Such modified whole cells have been shown to hydrolyze the cellulose from the media
and can replace the use of acidinduced breakdown of biological polymers prior to biosensor
analysis.
Another limitation in using whole cells is the low specificity as compared to biosensors containing
pure enzymes. Several approaches are being investigated to minimize such non -specific reactions.
Permeabilisation of the cell empties most of the small molecular weight cofactors etc, thus
minimizing the unwanted side reactions. Another approach that is of significance in viable
cellbased biosensors is the blockage of unwanted metabolic pathways or transport systems.
Developments in recombinant DNA technology may help in producing whole cells rich in the
enzyme of interest and also engineered to have minimal amounts of enzymes that might catalyze
side reactions. Adaptation of a microbe for induct ion of desirable metabolic pathways and uptake
systems by cultivation in medium containing appropriate substrates may often be desirable.
Microbial biosensors based on light emission from luminescent bacteria are being applied as a
sensitive, rapid and non-invasive assay in several biological systems. Bioluminescent bacteria are
found in nature, their habitat ranging from marine (Vibrio fischeri) to terrestrial (Photorhabdus
luminescens) environments. Bioluminescent whole cell biosensors have also been dev eloped using
genetically engineered microorganisms (GEM) for the monitoring of organic, pesticide and heavy
metal contamination.
The microorganisms used in these biosensors are typically produced with a constructed plasmid in
which genes that code for luciferase are placed under the control of a promoter that recognizes the
analyte of interest. When such microbes metabolize the organic pollutants, the genetic control
mechanism also turns on the synthesis of luciferase, which produces light that can be dete cted by
luminometers. A useful reporter system responsible for light emission is made up of five structural
genes, luxCDABE, of the bioluminescence operon derived from marine bacterium V. fischeri. The
luxCDE genes encode an enzyme complex (fatty acid redu ctase, synthetase and transferase) that

synthesizes the substrate (a fatty aldehyde) for luciferase, using precursors from the fatty acid
cycle. The luxAB genes encode the luciferase enzyme. (Tag K et al.,1999)
Bacterial strains that increase light production in the presence of specific chemicals have been
constructed using bioluminescence genes (lux) as reporters of transcriptional responses. A
complementary approach, not requiring prior knowledge of expected contaminants, uses less
specific stress responses as general indicators of deleterious conditions. Cellular organelles can be
considered to be multifunctional biocatalysts, intermediate in complexity between whole cells and
enzymes.
Table.2.8. Microbial Sensors and Their Applications in Biomedicine
Biomarkers Designprinciples Applications
The transcriptional activator N or R Detection of

Nitric oxide binds nitricoxide to trigger the inflammation in the
permanent activation of a DNA gut
switch that expresses a
Fluorescentre porter.
Thetwo-component system
ThsSRactivates
The expression of a fluorescentre
Thiosulfate porter upon thiosulfate detection.
Thetwo-
componentsystemTtrSRactivates
the expression of a fluorescent
Tetrathionate reporter upon tetrathionate
detection,either directly
or through a toggleswitch
Extracellular hemeis internalized
through the
Heme outer membrane transporter ChuA
andinteractswiththe transcriptional
repressor HtrR to allow expression of
the bacterial luciferase
operonluxCDABE.
ThetranscriptionalactivatorsSoxRand
OxyR
Are activated by the
ROS inducers(paraquatandH2O2)and
trigger the expression of
fluorescentre porters either directly or
through an analog-to-digital
recombinasesystem;ananalogcompen

satingsystemisincludedinReference
Syntheticphenylketonuria- Atherapeutic
therapeuticbacteria,SYNB161 microbialgenecircuit
Oxygen 8,expressPheP,a for phenylketonuria
Arabioe high- inthegut
affinityPhetransporterthatcanbringP
heintothecell;Pheammonialyase,wh
ich converts Phe into trans-
cinnamate;andL–
aminoaciddeaminase,a
membrane-
associatedenzymethatconvertsPhe to
phenylpyruvate. Regulation of
thesecomponentsiscarriedoutbyane
robic-andL-arabinose-
induciblepromoterstoenableactivatio
ninthehumanguttometabolize it.
Multipletranscriptionalactivatorswere Marionette is a single

usedtooptimizecellularresourcesby array of high-
2,4-Diacetylphophloroglucinol simultaneous selection for lower performancesensors
Cuminic acid basalexpression levels, high
3OC6-AHL dynamic
Vanillicacid range,increasedsensitivity,andlowcr
Isopropyl-β-d-thiogalactoside osstalk,allowing complex profiling
AnhydrotetracyclineHCl of multiplesmall-
moleculesignalsinonecell.
L-Arabinose
Cholinechloride
Naringenin
3,4-Dihydroxybenzoicacid
Sodiumsalicylate
3OHC14:1-AHL
Acrylicacid
Erythromycin
Twofamiliesoftranscriptionalrepressors Hosttemperaturedetectio
(TlpA and TcI) provide switch-like nforfevermonitoring,ki
controlof reporter gene expression llswitches,orfocusultras
Temperature at oundtherapeuticactivati
thresholdsspanningthebiomedically on
relevantrangeof
32–46°C.

CqsS- Probiotic-
NisKfusionvariantsexpressedinL basedstrategyforcholerad
actococcuslactistriggerexpressionof etectionandtreatment
Choleraautoinducer anenzymaticreporteruponbinding
quorum-sensingsignals ofVibrio
cholerae
thatisreadilydetectedinfecalsamples.
Arabinogalactan Diet- Diet sensor in the
Isopropyl-β-d-thiogalactoside induciblepromoters(specificforarabi context of
Rhamnose nogalactan, IPTG, rhamnose, acomplexmicrobiota
Lactose andlactose) drive the expression of
NanoLucreporter or a recombinase
system
incommensalBacteroidesthetaiotaomicr
on
orStreptococcusthermophilus.
Escherichia coli cells were Scalablebiologicalstatema
programmed torespond chine
totheantibiotic aTcor
aTcorarabinose arabinosewithafluorescentreporter.
Differentoutputfluorescencepatterns
wereproduceddepending on whether
the cells
wereexposedtoaTcfollowedbyarabino
seoran
initialinputofarabinosefollowedbyaT
c.
Chimeraswerebuilt,consistingofthe Multi-
DNA-bindingdomain of inputANDgateelimin
Isopropyl-β-d-thiogalactoside LacIandtheligand- atestheneedforligand-
Fructose bindingdomainofrepressorsthatresp induciblepromoterstoc
Fucose ond to different sugars. Since ontroleachinput,simpl
Ribose eachchimeracanregulatethesamep ifyingthecircuit
Trehalose romoter,simpletranscriptional
and gatingis
achievedbycoexpressingmultiplech
imericrepressors; the ligand for
each
chimeramustbepresenttoallowdo
wnstream
transcriptiontooccur.
(Adapted from:https://www.annualreviews.org/doi/10.1146/annurev -micro-022620-081059)
Applications of Biosensor

(https://www.elprocus.com/what-is-a-biosensor-types-of-biosensors-and-applications/)
Biosensors are devices comprising a biological element and a physiochemical detector that are used
to detect analytes. These instruments have a wide range of applications . Moreover, these are highly
important devices to measure an extensive spectrum of analytes like gases, organic compounds,
bacteria & ions.In recent years, these sensors have become very popular, and they are applicable in
different fields as below:
 Industrial & Environmental  Common healthcare

Applications checking
 Study & Interaction of  Metabolites Measurement
Biomolecules  Screening for sickness
 Development of Drug  Insulin treatment
 Detection of Crime  Clinical psychotherapy &
diagnosis of disease
 Medical Diagnosis
 In Military
 Monitoring of Environmental
 Agricultural, and
Field Veterinary applications
 Quality Control  Drug improvement, offense
 Process Control in Industries detection
 Pharmaceuticals Manufacturer  Processing & monitoring in
& Organs Replacement Industrial
 Ecological pollution control
 Diagnostic & Clinical
Applications of Biosensor in Environment monitoring
Environmental monitoring is the premise of pollution, and it provides scientific basis for
environmental management and protection.. Biosensors for environmental monitoring, represent
analytical devices which use for a sensing element biomaterial, chemical element or a combination
of both . Biosensors used for environmental monitoring have several advantages over conventiona l
systems and methods, some of which are: (a) their ability for portability; (b) miniaturization; (c)
measurement of a pollutant with minimal samples. (E. Gieva et al.,2014)
Following are the applications of Biosensor for environmental monitoring:
 BOD Measurement :

Biochemical oxygen demand (BOD or BOD5) is a parameter widely used to indicate the amount of
biodegradable organic material in water. Its determination is time consuming, and consequently it is
not suitable for online process monitoring. Fas t determination of BOD could be achieved with
biosensor-based methods. Most BOD sensors rely on the measurement of the bacterial respiration
rate in close proximity to a transducer, commonly of the Clark type. With this system the real time
analysis of multiple samples was possible. These handy devices have been marketed primarily for
food and pharmaceutical industries. Moreover, an optical biosensor for parallel multi -sample
determination of biochemical oxygen demand in wastewater samples has been develope d by Kwok
et al. The biosensor monitors the dissolved oxygen concentration in artificial wastewater through an
oxygen sensing film immobilized on the bottom of glass sample vials. Then, the microbial samples
were immobilized on this film and the BOD value was determined from the rate of oxygen
consumption by the microorganisms in the first 20 minutes n Escherichia coli Electrochemical
Wastewater Zinc, cobalt and copper.
 Heavy Metal Measurement:
Heavy metals are currently the cause of some of the most seriou s pollution problems. Even in small
concentrations, they are a threat to the environment and human health because they are non -
biodegradable. People are constantly been exposed to heavy metals in the environment. The dangers
associated with heavy metals are due to the ubiquitous presence of these elements in the biosphere,
their bioavailability from both natural and anthropogenic sources, and their high toxicity. Thus,
there are several cases described in the literature where exposure of populations to thes e pollutants
has resulted in severe damage to their health, including a significant amount of deaths. Many of the
bacterial biosensors developed for analysis of heavy metals in environmental samples, make use of
specific genes responsible for bacterial resistance to these elements, such as biological receptors.
Bacterial strains resistant to a number of metals such as zinc, copper, tin, silver, mercury and cobalt
have been isolated as possible biological receptors
 Nitrogen compound measurement:
Nitrites are widely used for food preservation and for fertilization of soils. However, continuous
consumption of these ions can cause serious implications on human health, particularly because it
can react irreversibly with hemoglobin . The increasing levels of nitra te found in groundwater and
surface water are of concern because they can harm the aquatic environment. Developed a biosensor
for amperometric determination of nitrite using cytochrome c nitrite reductase (ccNiR) from

Desulfovibrio desulfuricans immobilized and electrically connected on a glassy carbon electrode by
entrapment into redox active [ZnCr- AQS] double layered hydroxide containing anthraquinone -2-
sulfonate (AQS). The instrument showed a fast response to nitrite (5 seconds) with a linear range
between concentrations of nitrite 0.015 and 2.35 μM and a detection limit of 4nM. A highly
sensitive, fast and stable conduct metric enzymatic biosensor for the determination of nitrate in
water.
 PCBs Measurement:
The level of PCBs in the environment depends on the matrix where it originated. There are 209
polychlorinated biphenyl congeners that persist worldwide in the environment and food chain.
These congeners are divided into three classes based upon the orientation of the chlorine moieties,
i.e., coplanar, mono-ortho coplanar, and non-coplanar. Conventional techniques used for the
analysis of PCBs are generally based on gas chromatography coupled with mass spectrometry
(GC.MS) Moreover, immunoassays are simple, sensitive, reliable, and relatively selective for PCBs
testing. Among several immunoassay techniques, the enzyme -linked immunosorbent assay (ELISA)
combined with colorimetric endpoint detection are the most popular. Another interesting approach
is the use of immunosensor technology . Imunosensors are a class of biosensors that use as
biological recognition elements, antibodies or antigens . Pribyl et al. developed a novel
piezoelectric immunosensor for determination of PCB congeners in the range of concentrations
usually found in real matrices (soil).
Multi-analyte determination:
Sensors capable of determining several analytes simultaneously allow a reduction in time and
sample volume and other reagents required and thus constitute a valuable tool for environmental
monitoring. Large-scale biosensor arrays, composed of highly miniaturised signal transducer
elements, enable the real-time parallel monitoring of multiple species and are an important driving
force in biosensor research . In recent years, several examples of multi -analyte determinations have
appeared in the literature, such as a portable SPR immunosensor designed for on -site analysis,
which was applied to the simultaneous determination of benzopyrene and 2 -hydroxybiphenyl , and
another SPR biosensor that enabled the division of wavelengths on serial sensing channels by
means of a specially designed SPR prism element . A planar array immunosensor, equipped with a
charge-coupled device (CCD) as a detector and a diode laser as light source, has been also
developed and applied to either the determination of multiple compounds, such as viruses, toxins

and bacterial spores, in a single sample analysis or a single analyte in multiple samples
simultaneously.
Table.2.9. Application of Biosensor for Environmental monitoring
Biosensor for environmental application
Compoundclass Biologicalsensing Physicaltransdu Application

element cer
Heavy Enzyme, Electrode, Water,
metals microbe optical Soil
Nitrogen Enzyme Electrode Water,Soil,waste
Compounds water
Pesticides Antibody,Enzyme, Optical,electrode Water,Soiland
microbe Air
Herbicides Antibody, Optical,electrode Water,
Enzyme,microbe Soil andAir
Dioxins Microbe, Optical, Water,
slimmode electrode Soil,Air
Phenolic Enzyme, Optical, Water,
compounds microbe electrode Soil
HeavyMetalDetermination
Analyte Recognition Method

biocatalyzer
Mercury,cadmium Ureaseenzyme Electrochemical
andarsenic
Cadmium DNA, Electrochemical,Op
Phytochelatins tical
Cadmium,copper,lead Sol-gel-immobilizedurease Electrochemical
Nickelions Bacillussphaericus Electrochemical
strain
Zinc,copper, Enzyme Optical
Cadmiumandnickel
Mercury(II) andlead(II)ions DNA Optical
Copper (I)and (II)ions Fluorescentprotein Optical
Biosensors for Nitrogen Compounds Determination

biocatalyzer
Nitrite CytochromeC Amperometric

Nitrate Viologenmediator Electrochemical
BiosensorUsedInTheDetectionOfPesticides
biocatalyzer
Paraoxon Alkaline Optical

phosphatase
Isoproturon Antibodyencapsulate Fluorescence
Parathion Parathionhydrolase Electrochemical/A

mperometric
Carbaril Acetilcolinesterase Electrochemical/A

mperometric
Simazina Peroxidase Electrochemical/Po

tentiometric
Biosensors Used In The Detection Of Herbicides
Analyte Type of Recognition
interaction biocatalyzer
Dichloro-fenoxiacetic Immuno-analysis Acetil-
colinisterase
Diuron, Paraquat Biocatalitic Cyano bacterial
BiosensorUsedInTheDetectionOfDioxins
Analyte Typeof interaction and Method
Recognition
biocatalyzer
Dioxin Immuno-Analysis Biomimetic
And cell
Dioxin-likepolychlo- Immuno-analysisAnd cell Biomimetic
rinatedbiphenyls
BiosensorsFor DetectionOfPhenolicCompounds

biocatalyzer

Binarymixtures:phenol/cloroph
enol,catechol/phenol,cresol/clo Laccaseandtyrosinase Amperometricmuli
rocresol canal
andphenol/cresol
m-cresolorcatechol DNA Amperometric
Phenol Mushroomtissue
(tyrosinase) Amperometric
Phenol,p-cresol,m-cresoland Polyphenoloxidase Amperometric

catechol
(Adapted without modification from: E.Gieva,et al.,2014)
SOLVED PROBLEMS
1) What is Bio-sensor? How it is constructed. Give its importance in detection of bio
products.
a) Solution:please refer topic: Bio-sensor
2) Give various advantages of Bio sensor.
SELF-TEST -MCQ
1) Which of the following is device/probe which integrates a biological element with electronic
component to generate a measurable signal
a) Biosensor
b) Bio indicator
c) Both an and b
[Answer Key : a) Biosensor]
2) Following Biomarkers have application in biomedicine
a) Thiosulphate
b) Heme
c) Ros
d) All of the above
[Answer Key: d) All of the above]
3) Which is the least common biosensor

a) Bioluminescence biosensor
b) Potentiometric biosensor
c) Conductimetric biosensor
d) Amperometric biosensor
[Answer Key : b) Potentiometric biosensor]

1) SAQ.Write note on:Limitation of Biosensor
2) SAQ Write the components of biosensors
UNIT 02-04: BIO-TRANSFORMATION, BIO-ACCUMULATION, BIO-

MAGNIFICATION
LEARNING OBJECTIVES
 To understand the mechanism of bio-transformation, bio-accumulation

 To understand the principle of bio-mangenification
 To understand importance of xenobiotics and toxico -genomicspharmaco-genomics
Xenobiotics
These are the chemicals found but not produced in organisms or the environment. Some naturally
occurring chemicals (endobiotics) become xenobiotics when present in the environment at
excessive concentrations

Figure.2.3 .Fate of Xenobiotics
(Adapted without modification from:https://gurunanakcollege.edu.in/files/science/metabolism -of-

xenobiotics.pdf)
Bioconcentration
Bioconcentration may be defined as a process whereby a xenobioticenters the body of organisms
from the surrounding medium and accumulates in certain tissues. orIt is the intake and retention of
a substance in an organism entirely by respiration from water in aquatic ecosystems or from air in
terrestrial
ones. (https://www.lkouniv.ac.in/site/writereaddata/siteContent/202004061923051615omkar_zool_
Bioaccumulation.pdf)
Biomagnification
It is a broader term, which refers to the entireprocess of bio concentration and bioaccumulation. In
addition, it takes into account the gradualincrease in the concentration of chemical in the tissues of
organisms as it passes through the foodchain. It indicates that the level of chemical increases as the
position of the organism increases inthe food-chain. That is, organisms occupying higher trophic
levels gradually accumulate moreamounts of xenobiotics in their tissues .Generlly, it occurs when
the chemical is passed up the food chain to higher trophic levels, such that in predators it exceeds

the concentration to be expected where equilibrium prevails between an organism and its
environment .Thus the fatty tissues of animals may accumulate residues of heavy metals or other
compounds. These are passed up the food chain and reach greater, possibly harmful, concentr ations
at high trophic levels among top predators .
There are several ways in which to measure and assess bioaccumulation and bioconcentration.
These include: octanol-water partition coefficients (KOW), bioconcentration factors (BCF),
bioaccumulation factors (BAF) and biota-sediment accumulation factor (BSAF). Each of these can
be calculated using either empirical data or measurements as well as from mathematical
models(Arnot et al.,2004).Following are the formulas :
Where, Co is the chemical concentration in the organism (µg/kg wet weight), fR is the lipid fraction
of the organism (g lipid/g wet weight), Cs is the chemical conc entration in surficial sediment (µg/kg
dry weight) and fsoc is the fraction of the sediments as organic carbon (g organic ca rbon/gdry
weight).
Biotransformation

Biotransformation means chemical alteration of chemicals such as nutrients, amino acids, toxins,
and drugs , xenobiotics in the body.e.g. The metabolism of a drug /toxin in a body.The body
typically deals with a foreign compound/ xenobiotics by making it more water-soluble, to increase
the rate of its excretion through the urine. There are many different processes that can occur; the
pathways of drug metabolism can be divided into:
 phase І
 phase II
Drugs can undergo one of four potential biotransformations: Active Drug to Inactive Metabolite,
Active Drug to Active Metabolite, Inactive Drug to Active Metabolite, Active Drug to Toxic
Metabolite (biotoxification).
Phase І reaction
Includes oxidative, reductive, and hydrolytic reactions.
 In these types of reactions, a polar group is either introduced or unmasked, so the drug
molecule becomes more water-soluble and can be excreted.
 Reactions are non-synthetic in nature and in general produce a more water-soluble and less
active metabolites.
 The majority of metabolites are generated by a common hydroxylating enzyme system

known as Cytochrome P450.
Phase II reaction
 These reactions involve covalent attachment of smal l hydrophilic endogenous molecule such
as glucuronic acid, sulfate, or glycine to form water-soluble compounds that are more
hydrophilic.
 This is also known as a conjugation reaction.
 The final compounds have a larger molecular weight.
Biotransformation of xenobiotics
(https://www.lkouniv.ac.in/site/writereaddata/ siteContent/202004061923052084omkar_zool_biotran
sformation.pdf)

Factors Affecting Biotransformation of Xenobiotics
It is obvious that biotransformation of xenobiotics may take place by different pathways involving
phase I and II reactions and they may give rise to different biotransformation products. Various
factors related to the chemical, the environment and the physiological state of organisms affect the
rate and relative importance of biotransformation reactions of xenobiotics.
These factors can thus be divided into three categories:
(1) Chemical factors,
(2) Environmental factors, and
(3) Factors related to organisms.
Sites of Biotransformation
The liver is the main site of biotransformation. All xenobiotics taken up from the intestine are
transported to the liver by a single blood vessel (vena porta). If taken up in small quantities a
foreign substance may be completely metabolized in the liver before reaching the general
circulation and other organs (first pass effect). Inhaled xenobiotics are d istributed via the general
circulation to the liver. In that case only a fraction of the dose is metabolized in the liver before
reaching other organs.
Liver cells contain several enzymes that oxidize xenobiotics. This oxidation generally activates the
compound—it becomes more reactive than the parent molecule. In most cases the oxidized
metabolite is further metabolized by other enzymes in a second phase. These enzymes conjugate the
metabolite with an endogenous substrate, so that the molecule becomes larg er and more polar. This
facilitates excretion.
Enzymes that metabolize xenobiotics are also present in other organs such as the lungs and kidneys.
In these organs they may play specific and qualitatively important roles in the metabolism of
certain xenobiotics. Metabolites formed in one organ may be further metabolized in a second organ.
Bacteria in the intestine may also participate in biotransformation.
Metabolites of xenobiotics can be excreted by the kidneys or via the bile. They can also be exhaled
via the lungs, or bound to endogenous molecules in the body.

The relationship between biotransformation and toxicity is complex. Biotransformation can be seen
as a necessary process for survival. It protects the organism against toxicity by preventing
accumulation of harmful substances in the body. However, reactive intermediary metabolites may
be formed in biotransformation, and these are potentially harmful. This is called metabolic
activation. Thus, biotransformation may also induce toxicity. Oxidized, inte rmediary metabolites
that are not conjugated can bind to and damage cellular structures. If, for example, a xenobiotic
metabolite binds to DNA, a mutation can be induced . If the biotransformation system is
overloaded, a massive destruction of essential pr oteins or lipid membranes may occur. This can
result in cell death .
Bioaccumulation
(https://www.lkouniv.ac.in/site/writereaddata/siteContent/202004061923051615omkar_zool_Bioacc
umulation.pdf)
Bioaccumulation is a wide term. Bioaccumulation is the intake of a chemical and its concentration
in the organism by all possible means, including contact, respiration and ingestion. Or It considers
the accumulation ofxenobiotics both from the medium and through the consumption of food. Thus,
it takes intoaccount the uptake of chemicals dissolved or suspended in the water and from ingested
food andsediment residues. Persistent hydrophobic xenobiotics may get bioaccumulated in aquatic
organisms through different mechanisms, like bioconcentration, ingestion and biomagn ification.
Bioaccumulation should be regarded as a hazard criterion in itself even if the subchronic, chronic or
acute effects are not visible, as some hazardous effects may only be recognized in a later phase of
life. Bioaccumulation of xenobiotics in biota may be a prerequisite for adverse effects on
ecosystems
Process of Accumulation
Bioaccumulation takes place when the rate of uptake of chemical exceeds the rate of its elimination.
The process of uptake of chemical from water, sediment and food has been worked out more in the
aquatic systems, hence the general process of bioaccumulation applicable for the aquatic systems is
being described as:
1. Uptake of xenobiotics from water- Various workers have reported direct uptake of
chemicals from the water by the aquatic organisms (such as, algae, annelids, arthropods,
mollusks, fish, etc.). The mechanism of uptake process of xenobiotics from water will be

concentrated on the following three transport processes such as : (a) Diffusion, (b) Special
transport, and (c) Adsorption.
a. Diffusion :
Various xenobiotics have been reported to enter the body of organisms by the process of passive
diffusion. It is a physical process. It occurs acr oss any permeable or semipermeable barriers against
a concentration gradient (C). It is an energy independent process. That is it does not require
expenditure of energy. For instance, the gills of fishes are particularly vulnerable against chemical
insults. 2-4m thin gill membranes possess 2-10 times the surface area of the body, which
facilitates the diffusion of xenobiotics. The lipid bilayers of semipermeable membranes of gills and
lining of mouth and gastrointestinal tract of fishes permit the rapid p assage of lipophilic chemicals.
Weakly acidic and basic substances pass across biological membranes in unionized form. The water
and other small ions upto molecular weight of 100 pass through proteinaceous pores present in the
biological membranes. A few workers have demonstrated the diffusion of dieldrin and cadmium
across isolated perfused gills of trout and Mytilus, etc.. Similarly, several investigators have
reported in vivo uptake of various xenobiotics (such as, DDT, methyl mercury, mercuric chloride,
copper, etc.) through the gills of fishes. Considerable proportion of xenobiotics passively diffuse
across general body surface of various arthropods.
b. Special transport:
Uptake of xenobiotics across biological membranes may also occur by special transpor t
mechanisms including active and facilitated transports. The active transport takes place against
concentration gradient and it requires energy to move xenobiotics. Therefore,it is considered as a
true concentrating mechanism. The facilitated transport do es not requireenergy input and does not
concentrate xenobiotics against any concentration gradient. Metalsaccumulate by both these
transport processes. But, it is not known as to which mechanism ismore common. Usually metal
uptake in aquatic organisms is directly proportional to the metalconcentration in the water. Low
water salinity enhances the rate of uptake of xenobiotics. Theplasma proteins are known to
facilitate the transport of xenobiotics into the blood.
C.Adsorption:
Adsorption is also a physical process and a surface phenomenon. Xenobiotics are often adsorbed
either to the body surface of arthropods or to the gill surface ofother aquatic organisms by covalent,

electrostatic and molecular forces. Adsorption isparticularly important as initial ste p in the
accumulation process. The xenobiotics binding to thebody surface generally do not contribute to the
uptake upto toxic effect level within the body oforganisms, but certainly contribute to the total body
burden and affect the vulnerable functions o fthe epithelium. The process of adsorption is especially
important for microorganisms owing totheir high surface to volume ratio.
(ii) Bioavailability of xenobiotics in water:
The uptake of xenobiotics by the organisms largely depends on their availability in dissolved form
in the surrounding water. Therefore, factors affecting the concentration of chemicals in true
solutions also affect the uptake of xenobiotics by the organisms owing to their effect on
concentration of xenobiotics in the water. The import ant processes reducing the bioavailability of
xenobiotics in water are adsorption to suspended solids, sediments, humic acids and other
macromolecules, formation of colloidal suspension, chelation, complexation and ionization.
Lipophilic xenobiotics having strong tendency of bioconcentration also partition into the organic
fraction of sediment or suspended solids. Thus, the nature of sediments profoundly affects the
availability of xenobiotics in the surrounding water. For instance, amphipods exposed to hex achloro
biphenyl in the sediments of stream microcosm accumulate substrate -desorbed residues directly
from water. The greatest bioaccumulation of xenobiotics has been recorded in amphipods exposed
to sediments with least organic content and largest particl e sizes. Suspended particulates and
adsorbents, such as humic acids, reduce the uptake of lipophilic xenobiotics by reducing their
concentration in water. For instance, uptake of highly lipophilic chemical, benzo (a) pyrene, from
the water by sunfish has been reported to be reduced due to the presence of humic acids whereasthe
uptake of anthracene, a less lipophilic chemical remained unaffected.
Uptake of xenobiotics from food:
The xenobiotics absorbed through the gill surface and the integument are also re adily absorbed by
the gastrointestinal tract by similar mechanisms of diffusion and transport. Lipophilic xenobiotics
present in food are efficiently absorbed owing to long -term contact between the food and the
membranes. Weak acids and bases are absorbed in unionized form. The stomach pH favours the
diffusion of weak acids whereas the intestinal pH favours the absorption of neutral or weakly basic
xenobiotics. Uptake of metals from food depends on their forms (e.g. free or bound). Xenobiotics of
high molecular weight, viz. corrageenans (40,000A ) and polystyrene (2200A ), are absorbed in
the intestine by the process of pinocytosis.

Absorption ofXenobiotics bythe Lungs
(https://olemiss.edu/courses/phcl675/gentox2.pdf)
Disposition and retention of inhaled gases and aerosols are influenced by many features of the
respiratory tract, including lung volume, alveolar surface area, and structure and special
relationships of conducting airways. Lungs have between 30 and 100 m2 of surface area, large
blood flow of 2000 km of capillary beds; thin physical chemical barrier between air and blood of
0.8 ~m - nice barrier for toxicants. Regions - nasopharyngeal, tracheobronchial, pulmonary
Toxicogenomics
(https://www.news-medical.net/health/What-is-Toxicogenomics.aspx)
Toxicogenomics is the study of the response of a whole genome to toxicants or environmental

stressors and thereby providing the potential to accelerate the discovery of toxicant pathways,
modes of action, specific chemical and drug targets (Waters and Fost el, 2004)
Toxicogenomics has provided an avenue for advancing a joining of multidisciplinary sciences

including engineering and informatics in traditional toxicological research. It includes technologies
such as genome sequence analysis, proteomics, metabo lomics, and bioinformatics to detect toxic
agent-induced alterations in genetic expression, protein expression, and metabolite production;
moreover, effects of these alterations on phenotypic expressions at the cellular, tissue, and organism
levels is also studied. Such information provides a deeper understanding of how biologic pathways
respond to toxic substances.
Branches of Toxicogenomics
Toxicogenomics can be divided into following branches:
A.Mechanistic:
It helps to understand how a toxin acts by us ing a known end-point and a test system to provide
data on indirect markers responsible for particular phenotypes. These may be used to identify
hundreds of genes that are modulated by a single toxin. A combination of transgenic and knockout
models could expand the data by identifying specific genes that contribute in this process.
B. Predictive: A class of compounds which share the same type of toxicity may be identified by the
characteristic manner in which gene expression changes occur following exposure to any of them.
This is the rationale behind predictive toxicogenomics, which uses in vitro systems treated with

known toxicants to detect diagnostic patterns of toxicity. Custom -built miniarrays are then used to
detect these end-points. The availability of advanced mathematical and statistical analysis
techniques makes these results more valuable. Even with many possible limitations in extrapolating
the results of such in vitro tests, their contribution to preclinical safety testing is substantial.
Proposed benifits
Toxicogenomics is of great importance in drug development as it plays significant roles as:
 It contribute to the current state of knowledge on the actions of specific drugs at molecular
level
 It identify indirect markers of sufficient sensitivity to allow multiple (and perhaps hitherto
unknown) effects of clinical-trial stage drugs to be discovered
 It select new candidate drugs with a good efficacy and safety profile
 It increases the value of extrapolation of animal data to human biolog y
 It provides insight into how genetics affects the outcome of toxicological exposure
Challenges
 Unavailability of sufficiently powerful analytical tools to handle low doses with great
accuracy, as well as to distinguish possibly irrelevant outcomes from r elevant ones.
 Finding the level at which a change in transcription or protein expression may be significant
in terms of actual adverse outcome is another challenge to be faced. Adequately powered
studies are also needed to draw proper conclusions about the effects observed without any
undue influence of individual variability.
 Proper application and development of toxicogenomics with an understanding attitude from

regulatory agencies has the potential to transform the way drug toxicity is assessed and to
speed up the drug development process immensely.
Pharmacogenomics
(https://www.news-medical.net/health/What-is-pharmacogenomics.aspx)
Pharmacogenomics is the identification and study of is the identification and study of genes and
their corresponding products which influence genes and their corresponding products which

influence individual variation in the efficacy and/or toxicity of individual variation in the efficacy
and/or toxicity of therapeutic products, and the application of genomic therapeutic produc ts, and the
application of genomic information to help inform therapeutic product information to help inform
therapeutic product development and/or clinical application. Pharmacogenomics combines
biochemistry and other pharmaceutical sciences with an enhan ced understanding of DNA variations
in the human genome to target the differences with pharmacological treatments.
It includes:
 choosing the most appropriate therapeutic product for a patient; choosing the most
appropriate therapeutic product for a patient;
 selecting optimal dose; and/or selecting optimal dose; and/or
 identifying those at risk for unexpected or more frequent identifying those at risk for
unexpected or more frequent adverse drug reactions adverse drug reactions
Proposed benefits
The ability to use targeted genetic therapies has the potential to improve medical treatment as it,
increases its effectiveness and decreases the risk of adverse effects. The benefits may include:
 Targeted therapies for specific diseases with optimal therapeutic ef fects
 Reduced damage to surrounding healthy cells
 Faster recovery time
The doses could be tailored to the individual‘s genetic makeup and how their body processes the
drug.
Challenges
 Response of most drugs is determined by several different genetic variants, which can make
it difficult to predict the exact response of an individual. Additionally, other factors such as
the environment and situation may influence the patient response to the medication.
 Integration of pharmacogenomics into healthcare se rvices will require adjustments to the
current system, including new ways to make decisions about choice of therapy.
SOLVED PROBLEMS
1) Discuss various types of Bio transformations with suitable example.
a) Solution:Please refer topic: Bio transformations
2) Comment on
i. toxico-genomics
ii. Pharmaco-genomics

a) Solution:Please refer topic: toxico-genomics
b) Solution:Please refer topic: Pharmaco-genomics
SELF-TEST-MCQS
1. Which of the following is the chemical found but not produced in organism or environment
a) Xenobiotics
b) Xenoabiotics
c) Both a and b
[Answer Key: a) Xenobitics]
2) Which is the main site of biotransformation
a) Liver
b) Mouth
c) C. Intestine
d) Skin
[Answer Key : a) Liver]
3) Following are branches of toxicogenomics
a) Mechanistic
b) Predictive
c) Both a and b
[Answer Key : c) Both a and b]
1) SAQ.Write note on Branches of Toxicogenomics
a) Solution:Please refer topic: toxico-genomics
2) SAQ Write note on Bioaccumulation
a) Solution:Please refer topic: Bioaccumulation
SUMMARY
First section ofthis unit presents the overview of Bio -indicators and the advantages associated with
using the bio indicators.It gives detail description of broad functional groups of plankton and their
significance as a asindicatorsofwater pollution . It highlights the use of Diversity Index in
evaluation of water Quality. Besides, it illustrates the Case study regarding application of Water
Quality Index and Diversity Index for Pollution Assessment of KankariaLake ,Ahmedabad, India
Second section of this unit presents the overview of determination of microbiological quality of
recreational and potable waters in detail. It gives description of indicator microorganisms like
coliforms and E.coli, fecal streptococci, clostridia spp. along with other Indicator Organisms.
Furthermore, it highlights on various methods for determination of Microbiological
Quality.Finally it illustrates Lichens as bio indicators of Air Pollution .

Third section of this unitpresents the overview of Biosensor,its Compon ents ,advantages and
limitations in detail. It gives detail description of various types of Biosensors. It highlights on the
applications of biosensorsinenvironmentalmonitoring along with the microbial sensors and their
applications in biomedicine
Fourth section of this unit presents the overview of Bio -transformation, bio-accumulationand bio-
magnification.Defining the xenobiotics ,principles, receptor sites of biotransformation, absorption
and storage of xenobiotics, and fate of xenobiotics along with its biotransformation. It highlights
on different factors affecting biotransformation of xenobiotics and bioavailability of xenobiotics in
water. Further, it gives detail description of Toxicogenomics and Pharmacogenomics alongwith its
proposed benefits and challenges.
KEY WORDS
Bio-indicators ,plankton ,Diversity Index , ,Water Quality Index,,Lichens, Biosensor,
Microbial Sensors ,Biomedicine, Toxicogenomics,Pharmacogenomics
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CREDIT 03
UNIT 03-01: TOXICOLOGY
LEARNING OBJECTIVES
 To understand detailed knowledge of acute and chronic toxicity
 To understand pros and cons of synergism and antagonism
 To understand the effects of toxic chemicals .
INTRODUCTION :
Toxicology is "the science of poisons." or "the study of the adverse effects of chemicals or physical
agents on living organisms". It can also defined as the study of poisons, or, more comprehensively,
the identification and quantification of adverse outcomes associated with exposures to physical
agents, chemical substances and other conditions. As such, toxicology draw s upon most of the basic
biological sciences, medical disciplines, epidemiology and some areas of chemistry and physics for
information, research designs and methods. It ranges from basic research investigations on the
mechanism of action of toxic agents through the development and interpretation of standard tests
characterizing the toxic properties of agents. It provides important information for both medicine
and epidemiology in understanding aetiology and in providing information as to the plausibility o f
observed associations between exposures, including occupations, and disease. By convention,
toxicology also includes the study of harmful effects caused by physical phenomena, such as
radiation of various kinds, noise, and so on.
Environmental Toxicology
It is concerned with the study of chemicals that contaminate food, water, soil, or the atmosphere. It
also deals with toxic substances that enter bodies of waters such as lakes, streams, rivers, and
oceans. This sub-discipline addresses the question of how various plants, animals, and humans are
affected by exposure to toxic substances.
Scope of toxicology( I. Hodgson,2010)
In modern society, toxicology has become an important element in environmental and occupational
health. This is because many organizations, governmental and non-governmental, utilize
information from toxicology to evaluate and regulate hazards in the workplace and non -

occupational environment. As part of prevention strategies, toxicology is invaluable, since it is the
source of information on potential hazards in the absence of widespread human exposures.
Toxicological methods are also widely used by industry in product development, to provide
information useful in the design of specific molecules or product formulations
Toxicology can be divided into standard disciplines, such as clinical, forensic, investigative and
regulatory toxicology; it can be considered by target organ system or process, such as immune
toxicology or genetic toxicology; toxicology can be presented in functional ter ms, such as research,
testing and risk assessment.
Modern toxicology goes beyond the study of the adverse effects of exogenous agents by
assimilating knowledge and techniques from most branches of biochemistry, biology, chemistry,
genetics, mathematics, medicine, pharmacology, physicology, and physics and applies safety
evaluation and risk assessment to the discipline. In all branches of toxicology, scientists explore the
mechanisms by which chemicals produce adverse effects in biological systems. Activitie s in these
broad subjects complement toxicologic research, thereby contributing to the application of this
knowledge to the science and art of toxicology. The study of toxicology serves society in many
ways, not only to protect humans and the environment f rom the deleterious effects of toxicants, but
also to facilitate the development of more selective toxicants such as anticancer and other clinical
drugs, pesticides, and so forth.Due to overlapping of mechanisms as well as use and chemical
classes of toxicants, clear division into subjects of equal extent or importance is not possible.
Table.3.1.(I. Hodgson,2010)
Table.3.1. Aspects Encompassing the Essentially Of Toxicology and Its Scope

Sr.No. Significant aspects Scope in different fields
A. Integrative Approaches 1. Bioinformatics
2. Systems Biology
B. Modes of Toxic Action. 1. Biochemical and molecular toxicology
2. Behavioral toxicology
3. Nutritional toxicology

4. Carcinogenesis.
5. Teratogenesis
6. Mutagenesis
7. Organ toxicity
C. Measurement of Toxicants and 1.Analytical toxicology

Toxicity.
2. Genomics.
3. Proteomics
4. Metabolomics
5. Toxicity testing
6. Toxicologic pathology
7. Structure - activity studies
8. Biomathematics and statistics
9. Epidemiology
D. Applied Toxicology. 1. Clinical toxicology
2. Veterinary toxicology
3. Forensic toxicology
4. Environmental toxicology
5. Industrial toxicology
E. Chemical Use Classes. 1. Agricultural chemicals.
2. Clinical drugs
3. Drugs of abuse
4. Food additives
5. Industrial chemicals

6. Naturally occurring substances
7. Combustion products
F. Regulatory Toxicology. 1. Legal aspects
2. Risk assessment
Important Terms
LC50
LC stands for "Lethal Concentration". LC values usually refer to the concentration of a chemical in
air but in environmental studies it can also mean the concentration of a chemical in water.
According to the (Organization for Economic Cooperation and Development) (OECD) Guidelines
for the Testing of Chemicals, a traditional experiment involves groups of animals exposed to a
concentration (or series of concentrations) for a set period of time (usually 4 hours). The animals
are clinically observed for up to 14 days.
The concentrations of the chemical in air that kills 50% of the test animals during the observation
period is the LC50 value. Other durations of exposure (versus the traditional 4 hours) may apply
depending on specific laws.
LD50
LD stands for "Lethal Dose". LD50 is the amount of a material, given all at on ce, which causes the
death of 50% (one half) of a group of test animals. The LD50 is one way to measure the short -term
poisoning potential (acute toxicity) of a material.
Toxicologists can use many kinds of animals but most often testing is done with rats and mice. It is
usually expressed as the amount of chemical administered (e.g., milligrams) per 100 grams (for
smaller animals) or per kilogram (for bigger test subjects) of the body weight of the test animal.
The LD50 can be found for any route of entry o r administration but dermal (applied to the skin) and
oral (given by mouth) administration methods are the most common.
The actual LD50 value may be different for a given chemical depending on the route of exposure
(e.g., oral, dermal, inhalation)

Acute Toxicity (I. Hodgson,2010)
Acute toxicity refers to those adverse effects occurring following oral or dermal administration of a
single dose of a substance, or multiple doses given within 24 hours, or an inhalation exposure of 4
hoursor the toxicity elicited as a result of short - term exposure to a toxicant. In the environment,
the incidences of acute toxicity are commonly associated with the accidents e.g., leakage of a
chemical into a river or aerial drift of a pesticide to non -target areas. The Acute toxicity of
environmental chemicals is experimentally determined by selection of species which serve as
representatives of particular levels of trophic organization within an ecosystem e.g., mammal, bird,
fish, invertebrate, vascular plant, algae.
Basically, the acute toxicity of a chemical is commonly quantified as the median lethal
concentration (LC 50) or median lethal dose (LD 50). These measures do not provide any insight
into the environmentally acceptable levels of contaminants (a concentration that kills 50% of the
exposed organisms is hardly acceptable). However, LC 50 and LD 50 values do provide statistically
sound, reproducible measures of the relative acute toxicity of chemicals. LC 50 and LD 50 ranges
for aquatic and terrestrial wildlife, respectively as per Table.3.2.
Classification and Labelling Summary
(https://www.ilo.org/legacy/english/protection/safework/ghs/ghsfinal/ghsc05.pdf)
Does the substance/mixture meet the criteria below?
No classified as a acutely toxic.
Yes
Table.3.2.Category and Criteria of Hazardous Chemicals

Category Criteria HazardCommunicationElements
Category1 LD 50 # 5 mg/kg (oral)LD 50 #50 mg/kg SignalWord Danger

(dermal)LC 50 # 100 ppm
Symbol SkullandCrossbones
(gas)LC 50 #0.5(mg/l)(vapour)
LC 50 #0.05(mg/l) (dust,mist) HazardStat Fatal if swallowed.

ement (oral)Fatal in contact
with skin(dermal)

Fatal if inhaled (gas,
vapour,dust, mist)
Category2 LD 50 between 5 and less than 50 mg/kg SignalWord Danger

(oral)LD 50 between 50 and less than
200 50 mg/kg(dermal)
LC 50 between 100 and less than 500 HazardStat Fatal if swallowed.

ppm(gas) ement (oral)Fatal in contact
with skin(dermal)
LC 50 between 0.5 and less than2.0
(mg/l)(vapour) Fatal if inhaled (gas,
vapour,dust, mist)
LC 50 between 0.05 and less than 0.5
(mg/l)(dust,mist)
Category3 LD 50 between 50 and less than 300 SignalWord Danger

mg/kg(oral)
LD 50 between 200 and less than 1000
mg/kg(dermal) HazardStat Toxic if swallowed.
ement (oral)Toxic in contact
LC 50 between 500 and less than 2500
with skin(dermal)
ppm(gas)
Toxic if inhaled
(gas,vapour, dust,mist)
(mg/l)(vapour)

(mg/l)(dust,mist)
Category4 LD 50 between 300 and less than 2000 SignalWord Warning

mg/kg(oral)
Symbol ExclamationMark

LD 50 between 1000 and less than 2000
HazardStat Harmful if swallowed.
mg/kg(dermal)
ement (oral)Harmful in
LC 50 between 2500 and less than contact with
5000ppm(gas) skin(dermal)
LC 50 between 10.0 and less than 20.0 Harmful if

(mg/l)(vapour) inhaled(gas,vapour,
dust, mist)
(mg/l)(dust,mist)
Category5 LD 50 between2000and5000(oral) SignalWord Warning
Symbol Nosymbol
Seealsotheadditionalcriteria(paraxx)
HazardStat May be harmful
ement ifswallowed(oral)
Maybeharmfulincontact
withskin (dermal)
Maybeharmfulifinhaled(
gas,vapour,dust,mist)
(Adapted without modification from,

https://www.ilo.org/legacy/english/protection/safework/ghs/ghsfinal/ghsc05.pdf )
Basically, a substance is classified in Category 5, if assignment to a more hazardous class is not

warranted, through extrapolation, estimation or measurement of data if:
-reliable information is available indicating significant toxicity effects in humans
-any mortality is observed when tested up to Category 4 values by the oral, inhalation or dermal
routes.
-where expert judgment confirms significant clinical signs of toxicity, whentested up to Category 4
values, except for diarrhea, piloerection

-where expert judgement confirms reliable i nformation indicating the potential for significant acute
effects from other animals.
Chronic Toxicity
Chronic toxicity is the toxicity elicited as a result of long - term exposure to a toxicant. Sub-lethal
end points are generally associated with chronic toxicity. It includes reproductive, immune,
endocrine, and developmental dysfunction. However, chronic exposure also can result in direct
mortality not observed during acute exposure. E.g. chronic exposure of highly lipophilic chemicals
can result in the eventual bioaccumulation of the chemical to concentrations that are lethal to the
organisms, or as discussed previously, mobilization of lipophilic toxicants from lipid compartments
during reproduction may result in lethality. It is important to recogni ze that, while theoretically, all
chemicals elicit acute toxicity at sufficiently high doses, all chemicals are not chronically toxic.
The following must always be considered when assessing the chronic toxicity of a chemical :
A. Simple numerical interpretations of chronic toxicity based upon acute: chronic ratios serve only
as gross indicators of the potential chronic toxicity of the chemical. Laboratory exposures designed
to establish chronic values.most often focus upon a few g eneral end points such as survival, growth,
and reproductive capacity. Examination of more subtle end points of chronic toxicity may reveal
significantly different chronic values.
B. Laboratory exposures are conducted with a few test species that are amena ble to laboratory
manipulation. The establishment of chronic and acute : chronic ratio values with these species
should not be considered absolute. Toxicants may elicit chronic toxicity in some species and not in
others.
C. Interactions among abiotic and biotic components of the environment may contribute to the
chronic toxicity of chemicals, while such interactions may not occur in laboratory assessments of
direct chemical toxicity. These considerations are exemplified in the following incidence of chronic
toxicity of chemicals in the environment.
Sub chronic toxicity:( I. Hodgson,2010)

The toxicity due to chronic exposure to quantities of a toxicant that do not cause any evident acute
toxicity for a time period that is extended but is not so long as to con stitute a substantial part of the
life span or the species in question. In sub chronic toxicity tests using mammals, a 30 – 90 day
period is considered appropriate.
Selective toxicity/ selectivity:( I. Hodgson,2010)
It is a characteristic of the relationship between toxic chemicals and living organisms whereby a
particular chemical may be highly toxic to one species but relatively innocuous to another. The
search for and study of selective toxicants is an important aspect of comparative toxicology because
chemicals toxic to target species but innocuous to non -target species are extremely valuable in
agriculture and medicine. The mechanisms involved vary from differential penetration rates through
different metabolic pathways to differences in recep tor molecules at the site of toxic action.
Example
 Insecticide is lethal to insects but relatively nontoxic to animals
 Antibiotics are selectively toxic to microorganisms while virtually nontoxic to humans
 Age may be important in determining the response t o toxicants. Some chemicals are more
toxic to infants or the elderly than to young adults. For example:
e.g. Parathion is more toxic to young animals ,nitrosamines are more carcinogenic to newborn or
young animals
 Toxic responses can vary depending on sex.
e.g. male rats are 10 times more sensitive than females to liver damage from DDT ,female rats are
twice as sensitive to parathion as male rats .
Synergism and Antagonism
Synergism :
In toxicology, synergism refers to the effect caused when exposure to two or more chemicals at one
time results in health effects that are greater than the sum of the effects of the individual chemicals.
It can be in two forms: summation (additive) and potentiation. First one is a simple sum (addition)
of effects, e.g., combination of agents for narcosis. Potentiation is an interaction between two or
more drugs or agents resulting in a pharmacologic response greater than the sum of individual

responses to each drug or agent, e.g. combination of sedative drugs with alcohol. Syner gism may be
direct, when drugs act on the same substrate, and indirect, when drugs act on different
substrates.When chemicals are synergistic, the potential hazards of the chemicals should be re -
evaluated, taking their synergistic properties into considera tion.( I. Hodgson,2010)
Antagonism
Antagonism is the opposite of synergism. It is the situation where the combined effect of two or
more compounds is less toxic than the individual effects It can be physiological, chemical, physico -
chemical and physical. Physiological antagonism appears on level of biological substrate. It may be
direct and indirect. Interaction of cholinomimetic and cholinolytic is an example of direct
antagonism. Indirect antagonism is stipulated by interaction of drugs with different m echanism of
action. For example, pilocarpine narrow pupil through the stimulation of M -cholinoreceptors of
sphincter pupil; adrenaline widens pupil due to stimulation adrenoreceptors of pupil's radial muscle.
Chemical antagonism is a type of chemical react ion that results in loosing of initial
pharmacological activity of agents and in formation of non -active substance (e.g., binding of
unithiol with metals). Example of physico -chemical antagonism is neutralization of heparin
(anticoagulant) by protamine sulfate, in result of electrostatic interaction. Physical antagonism is
stipulated by physical features of drug, e.g., activated carbon adsorb on own surface molecules of
many substances. (https://en.wikiversity.org/wiki/Pharmacodynamics)
Dose
The dose is the actual amount of a chemical that enters the body. The dose received may be due to
either acute (short) or chronic (long-term) exposure. An acute exposure occurs over a very short
period of time, usually 24 hours.
Chronic exposures occur over long periods of time such as weeks, months, or years. The amount of
exposure and the type of toxin will determine the toxic effect.
Dose-response
It is a relationship between exposure and health effect, that can be established by measuring the
response relative to an increasing dose. This relationship is important in determining the toxicity of
a particular substance (2). It relies on the concept that a dose, or a time of exposure (to a chemical,
drug, or toxic substance), will cause an effect (response) on the expos ed organism. Usually, the

larger or more intense the dose, the greater the response, or the effect. This is the meaning behind
the statement ―the dose makes the poison.‖
Threshold dose /No observed adverse effect level (NOAEL)/ No effect level (NEL).
The dose or exposure level below which the harmful or adverse effects of a substance are not seen
in a population , such dose is referred to as the ‗threshold dose‘. This dose is also referred to as the
no observed adverse effect level (NOAEL), or the no effe ct level (NEL). These terms are often used
by toxicologists when discussing the relationship between exposure and dose. However, for
substances causing cancer.
Toxic Chemicals in the Environment
Environmental toxicology:
It is a multidisciplinary field of science concerned with the study of the harmful effects of various
chemical, biological and physical agents on living organisms.
Heavy metals
There is no specific definition of a heavy metal, however, it is defined as a naturally occurring

element having a high atomic weight and high density which is five times greater than that of water
(Banfalvi G et al.,2011).Among all the environmental toxicants, heavy metals have received a
utmost attention as many of them are toxic even at very low concentrations. ( Herawati N et al.,
2000). Many of such metallic elements play an vital role in the function of living organisms; they
constitute a nutritional requirement and fulfill physiological role. However, their over abundance
and particularly their substitution by nonessential metallic elements can cause toxicity symptoms
or death. (Vhahangwele Masindi and Khathutshelo L. Muedi,,2018)
Sources of heavy metals
Heavy metals can emanate from both natural and anthropogenic processes and end up in different
environmental compartments (soil, water, air and their interface) (Vhahangwele Masindi and
Khathutshelo L. Muedi,2018)

Figure 3.1. Sources of Heavy Metals and Their Cycle in the Environment
(Adapted without modification from: Brady D et al., 1994)
Arsenic(As)
Arsenic is a ubiquitous element that is detected at low concentrations in virtually all environmental
matrices. Environmental pollution by arsenic occurs as a result of natural phenomena such as
volcanic eruptions and soil erosion, and anthropogenic activi ties.(Tchounwou et al.2014)
The toxicity symptoms depend on the chemical form ingested. Arsenic acts to coagulate protein,
forms complexes with coenzymes and inhibits the production of adenosine triphosphate (ATP)
during respiration. It is possibly carcinogenic in com-pounds of all its oxidation states and high -
level exposure can cause death. Arsenic toxicity also presents a disorder, which is similar to, and
often confused with Guillain-Barre syndr-ome, an anti-immune disorder that occurs when the
body‘s immune system mistakenly attacks part of the PNS, resulting in nerve inflammation that
causes muscle weakness (Singh J and Kalamdhad A,2011)
Cadmium (Cd):
It is a well known heavy metal toxicant with a specific gravity 8.65 times greater than water. The
target organs for Cd toxicity have been identified as liver, placenta, kidneys, lungs, brain and bones
On basis of severity of exposure its symptoms includes nausea, vomiting, abdominal cramps,
dyspnea and muscular weakness. Severe exposure may result in p ulmonary odema and death.
Pulmonary effects (emphysema, bronchiolitis and alveolitis) and renal effects may occur following
subchronic inhalation exposure to cadmium and its compounds . The Itai -itai disease in Japan

brought the dangers of environmental Cd to world attention. Cd has been associated to a lesser or
greater extent with many clinical conditions including anosmia, cardiac failure cancers,
cerebrovascular infarction, emphysema, osteoporosis, proteinuria cataract formation in the eyes.
Yet, it has been difficult to tie down obvious links of environmental exposures with morbidity and
mortality (Singh J and Kalamdhad A,2011)
Lead (Pb)
It is physiological and neurological toxic to humans. Acute Pb poisoning may results in a

dysfunction in the kidney, reproduction system, liver and brain resulting in sickness and death .Pb
heads the threats even at extremely low concentrations . A notablyserious effect of lead toxicity is
its teratogenic effect. Lead poisoning also causes inhibition of the synthesis of haemoglobin;
cardiovascular system and acute and chronic damage to the central nervous system (CNS) and
peripheral nervous system (PNS). Its other chronic effects include anemia, fatigue, gastrointestinal
problems and anoxia. Lead can causes difficulties in pregnancy, high blood pressure, muscle and
joint pain. Other effects include damage to the gastrointestinal tract (GIT) and urinary tract
resulting in bloody urine, neurological disorder and can cause severe and permanent brain damage.
While inorganic forms of lead, typically affect the CNS, PNS, GIT and other biosystems, organic
forms predominantly affect the CNS. Lead affects children; particularly in the 2 -3 years old range
by leading to the poor development of the grey matter of the brain, thereby re sulting in poor
intelligence quotient (IQ). Its absorption in the body is enhanced by Ca and Zn deficiencies (Singh
J and Kalamdhad A, 2011)
Chromium (Cr)
It is the 10th abundant element in the earth‘s mantle and persists in the environment as either Cr
(III) or Cr (VI). Cr (VI) is toxic to plants and animals, being a strong oxidizing agent, corrosive,
soluble in alkaline and mildly acidic water, toxic and potential carcinogens. The toxicity of Cr (VI)
derives from its ability to diffuse through cell membra nes and oxidize biological molecules.
Mercury is toxic and has no known function in human biochemistry and physiology. Inorganic
forms of mercury cause spontaneous abortion, congenital malformation and gastrointestinal
disorders (like corrosive esophagitis and hematochezia). Poisoning by its organic forms, which
include monomethyl and dimenthylmecury presents with erethism (an abnormal irritation or
sensitivity of an organ or body part to stimulation), acrodynia (Pink disease, which is characterized

by rash and desquamation of the hands and feet), gingivitis, stomatitis, neurological disorders, total
damage to the brain and CNS and are also associated with congenital malformation (Singh J and
Kalamdhad A,2011)
Copper (Cu)
It is an essential element in mammalian nutrition as a component of metalloenzymes in which it

acts as an electron donor or acceptor. Conversely, exposure to high levels of Cu can result in a
number of adverse health effects. Exposure of humans to Cu occurs primarily from the consumption
of food and drinking water. Acute Cu toxicity is generally associated with accidental ingestion;
however, some members of the population may be more susceptible to the adverse effects of high
Cu intake due to genetic predisposition or disease . Excessive huma n intake of Cu may leads to
severe mucosal irritation and corrosion, widespread capillary damage, hepatic and renal damage and
central nervous system irritation followed by depression. Severe gastrointestinal irritation and
possible necrotic changes in the liver and kidney can also occur. The effects of Ni exposure vary
from skin irritation to damage to the lungs, nervous system, and mucous membranes (Singh J and
Kalamdhad A,2011)
Mercury(Hg)
It is toxic and has no known function in human biochemistry and p hysiology. Inorganic forms of
mercury cause spontaneous abortion, congenital malformation and gastrointestinal disorders (like
corrosive esophagitis and hematochezia). Poisoning by its organic forms, which include
monomethyl and dimenthylmecury presents wi th erethism (an abnormal irritation or sensitivity of
an organ or body part to stimulation), acrodynia (Pink disease, which is characterized by rash and
desquamation of the hands and feet), gingivitis, stomatitis, neurological disorders, total damage to
the brain and CNS and are also associated with congenital malformation (Singh J and Kalamdhad
A,2011)

(Adapted without modification from:Vhahangwele M. and Khathutshelo L. Muedi, 2018)
Figure 3.2. Mechanisms for the removal of heavy metals

Disruption of metabolic functions by Heavy metals:
 They accumulate and thereby disrupt function in vital organs and glands such as the heart,
brain, kidneys, bone, liver, etc.
 They displace the vital nutritional minerals from their original place, thereby, hindering
their biological function. It is, however, impossible to live in an environment free of heavy
metals. There are many ways by which these toxins can be introduced into the body such as
consumption of foods, beverages, skin exposure, and the inhaled air.( Singh MR,2009)
Other Toxic Chemicals in the Environment
Carbon monoxide(CO):
Carbon monoxide (CO) is one of the most serious toxic chemical polluting Environment and highly
poisonous to living beings because of its ability to block the delivery of oxygen to the organs and
tissues. It is mainly released into the air by automobile exhaust, incomplete combustion of coal,
firewood, petrol, etc. Carbon monoxide is poisonous as it binds to haemoglobin to form carboxy -

haemoglobin, which is about 300 times more stable t han the oxygen-haemoglobin complex. In
blood, when the concentration of carboxy-haemoglobin reaches about 3–4 per cent, the oxygen
carrying capacity of blood is greatly reduced. This oxygen deficiency, results into headache, weak
eyesight, nervousness and cardiovascular disorder. This is the reason why people are advised not to
smoke. In pregnant women who have the habit of smoking the increased CO level in blood may
induce premature birth, spontaneous abortions and deformed babies.
(https://ncert.nic.in/textbook/pdf/kech207.pdf )
Ozone(O3)
Ozone is a unstable blue gas Ozone (O3) is the major oxidant of photochemical smog..It shows
adverse impacts on the sensitive vegetation and ecosystems, including forests, parks, wildlife
refuges and wilderness areas. Ozone causes various plant diseases and disorders .It can reduces the
rate of photosynthesis affecting plant‘s growth. It also shows negative impacts on ecosystems,
changes to water and nutrient cycles,loss of species diversity etc.
Its biological effect is attributed to its ability to cause oxidation or peroxidation of biomolecules
directly and/or via free radical reactions. A sequence of events may include lipid peroxidation and
loss of functional groups of enzymes, alteration of membrane permeability, and cell injury or death.
An acute exposure to O3 causes lung injury involving the ciliated cell in the airways and the type 1
epithelial cell in the alveolar region. A chronic exposure to O3 can cause or exacerbate lung
diseases, including perhaps an increased lung tumor incidence in susceptible animal models. Ozone
exposure also causes extra pulmonary effects involving the blood, spleen, central nervous system,
and other organs. A combination of O3 and NO2, both of which occur in photochemical smog, can
produce effects which may be additive or synergistic. A synergistic lung injury occurs possibly due
to a formation of more powerful radicals and chemical intermediates. (Mustafa MG,1990)
Ozone is a powerful oxidant, and, as such, it can react with a wide range of cellular components and
biological materials. Ozone exerts its action through several mechanisms:
 Reactions with sulfhydryl groups, aldehydes, and amino groups of low molecular weight;
 Reactions with antioxidants such as vitamins E and C; other direct scavengers include
broncho alveolar lavage (BAL) uric acid and intracellular taurine; and

 Reactions with polyunsaturated fatty acids, the stable reaction products of which include
hydrogen peroxide and aldehydes with some ozonides and lipid hydroperoxides; these
reactions may lead to the formation of free radicals.
Peroxyacetyl Nitrate (PAN)
PANs are secondary pollutants i.e. they are not directly emitted as exhaust from power plants or
internal combustion engines, but they are formed from other pollutants by chemical reactions in the
atmosphere. Free radical reactions catalyzed by ultraviolet light from the sun oxidize unburned
hydrocarbons to aldehydes, ketones, and dicarbonyl compounds, whose secondary reactions create
peroxyacyl radicals, which combine with nitrogen dioxide to form peroxyacyl nitrates. PANs are
highly unstable compounds in pure condensed form they are relatively stable in vapor phase in the
atmospher .The acute toxicity of PAN is less than that of ozone, similar to NO 2 and higher than
SO 2 .. PANs are both toxic as they dissolve more readily in water than ozone. They are
lachrymators, causing eye irritation at concentrations of only a few parts per billion. At higher
concentrations they acts as phytotoxins, and bacterial mutagens. The most serious biological effects
of PANs are of a phytotoxic nature resulting in injury to plants and vegetation. Both PANs and their
chlorinated derivates are said to be mutagenic, as they can be a f actor causing skin cancer.
Pesticides
Increasing world population has put a tremendous pressure on the agriculture system to obtain the
sufficient food from existing resources such as, water, soil etc. To increase the crop production,
various synthetic fertilizers, fungicides, insecticides, herbicides and pesticides have been
excessively applied. Since last few decades, pesticides have become an integral part of our modern
life. Although, pesticides were used initially to benefit human life through increase in agricultural
productivity and by controlling infectious disease, their adverse effects have overweighed the
benefits associated with their use. The above discussion clearly highlights the severe consequences
of indiscriminate pesticide use on different environmental components. Some of the adverse effects
associated with pesticide application have emerged in the form of increase in resistant pest
population, decline in on beneficial organisms such as predators, pollinators and earthworms,
change in soil microbial diversity, and contamination of water and air ecosystem. (H Gill and H
Garg, 2014). The persistent nature of pesticides has impacted our ecosystem to such an extent that
pesticides have entered into various food chains and into the higher troph ic levels such as that of

humans and other large mammals. Some of the acute and chronic human illnesses have now
emerged as a consequence of intake of polluted water, air or food. Pesticide drift from agricultural
fields, exposure to pesticides during appl ication and intentional or unintentional poisoning
generally leads to the acute illness in humans (Dawson et al., 2010; Lee et al., 2011b). Several
symptoms such as headaches, body aches, skin rashes, poor concentration, nausea, dizziness,
impaired vision, cramps, panic attacks and in severe cases coma and death could occur due to
pesticide poisoning (Pan-Germany, 2012). Human population is also affected especially due to
contaminated food and water or pesticides drift from the fields (Pan -Germany, 2012)The severity of
these risks is normally associated with toxicity and quantity of the agents used, mode of action,
mode of application, length and frequency of contact with pesticides and person that is exposed
during application (Richter, 2002).Continued expo sure to sub-lethal quantities of pesticides for a
prolonged period of time (years to decades), results in chronic illness in humans (Pan -Germany,
2012). Pesticides shows adverse impacts on nervous, reproductive, renal, cardiovascular, and
respiratory systems (Mostafalou and Abdollahi, 2012)
SOLVED PROBLEMS
1) What is chronic and acute toxicity? Discuss with suitable example.
a) Solution:Please refer topic:acute toxicity
2) Compare and contrast synergism and antagonism.
a) Solution:Please refer topic: synergism and antagonism
3) Justify judicious use of pesticides
a) Solution:Please refer topic: pesticides
SELF-TEST -MCQS
1) Toxicology is the science of
a) Microbes
b) Poisons
c) Chemicals
d) Nutrients
[Answer Key : b) Poisons]
2) Followings are used for measurement of toxicants and toxicity
a) Genomics
b) Toxicity testing

c) Toxicology pathology
d) All above
[Answer Key : d)All above]
3) Following is considered as the way to measure acute toxicity
a) LD 50
b) LD 10
c) LD 90
d) LD 80
[Answer Key : a) LD 50]
1) SAQ –Write note on Synergism
a.Solution: Please refer topic: Synergism
2. SAQ-Write note on-Antagonism
a. Solution: Please refer topic: Antagonism
UNIT 03-02: ENVIRONMENTAL TOXICOLOGY-1

LEARNING OBJECTIVES
 To understand LC50 and ILD50
 To understand pharmacological basis of toxicity
 To understand neurotoxicity, nephrotoxicity and radio -active toxicity
 To understand types and effects of various mutagenic agents
INTRODUCTION-
In toxicology, dose descriptor is the term used to identify the relationship between a specific effect
of a chemical substance and the dose at which it takes place. The dose descriptors will be used later
for deriving the no-effect threshold levels for human health (i.e, DNEL or reference dose RfD) and
the environment (PNEC). Dose descriptors are determined in the toxicological studies on the
hazards of the substance and are usually expressed as LC50, LD50, NOAEL etc. They are used for
GHS (Globally Harmonized System) hazard classification and risk assessment.
Important Terms

LC50
LC stands for "Lethal Concentration". LC values usually refer to the concentration of a chemical in
air but in environmental studies it can also mean the concentration of a chemical in water.
According to the (Organization for Economic Cooperation and Development) (OECD) Guidelines
for the Testing of Chemicals, a traditional experiment involves groups of animals exposed to a
concentration (or series of concentrations) for a set period of time (usually 4 hours). The animals
are clinically observed for up to 14 days.
The concentrations of the chemical in air that kills 50% of the test animals during the observation
period is the LC50 value. Other durations of exposure (versus the traditional 4 hours) may apply
depending on specific laws.
LD50
LD stands for "Lethal Dose". LD50 is the amount of a material, given all at once, which causes the
death of 50% (one half) of a group of test animals. The LD50 is one way to measure the short -term
poisoning potential (acute toxicity) of a material.
Toxicologists can use many kinds of animals but most often testing is done with rats and mice. It is
usually expressed as the amount of chemical administered (e.g., milligrams) per 100 grams (for
smaller animals) or per kilogram (for bigger test subjects) of the body weight of the test animal.
The LD50 can be found for any route of entry or administration but dermal (applied to the skin) and
oral (given by mouth) administration methods are the most common.
The actual LD50 value may be different for a given chemical depending on the route of exposure
(e.g., oral, dermal, inhalation)
No-Observed-Effect Level (NOEL)
It is the greatest concentration or amount of a substance, found by experiment or observation, that

causes no alteration of morphology, functional capacity, growth, development, or lifespan of the
target organism distinguishable from those observed in normal (control) organisms of the same
species and strain under the same defined conditions of exposure.
The value of the NOAEL depends strongly on the following characteristics of the study design:

● Group size. The power to detect a NOAEL at some dose levelis directly dependent on the sample
sizes chosen at those doselevels. The larger the group size, the smaller th e possible undetected
effect size at the NOAEL.
● Dose selection. The NOAEL must be one of the doses actually applied in the study. If the true
threshold is higher than the
NOAEL, the distance between the two can be expected to be limited (related to the d ose spacing
used), whereas if the true threshold is lower than the NOAEL, the distance between the two
ispotentially unlimited.
● Experimental variation. Experimental variation comprises biological (e.g. genetic) variation

between subjects, variation in experimental conditions (e.g. time of feeding, location in
experimentalroom, time of section or interim measurements) and measurementerrors. Larger
experimental variation between subjects will resultin lower statistical power, and hence higher
NOAELs.

Chemical Pharmacological basis of toxicity, Toxic effects at cell, tissue organ level
Figure 3.3. Representation of Mechanisms of Toxicity
(Adapted without modification from:http://www.ilocis.org/documents/chpt33e.htm)
Cell toxicity
It is caused by exogenous toxicant which can damage cells, especially when the toxicant can cause
cell death and serious organ dysfunction. The effects of a toxicant are usually dose -dependent and

species–specific. Cell toxicant include chemical agent, environment pollutant, natur al plants extract
and pharmaceutical drugs.

https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6206310/figure/Fig1)
Figure 3.4.The Mechanism of Cell Toxicity
Tissue /Organ specific /Organ toxicity studies
Neurotoxicity(https://www.news-medical.net/health/What-is-Neurotoxicity.aspx)
It refers to damage to the brain or peripheral nervous system caused by exposure to natural or man -
made toxic substances.These toxins can alter the activity of the nervous system in ways that can
disrupt or kill nerves. Nerves are essential for transmitting and processing information in the brain,
as well as other areas of the nervous system.
Due to their high metabolic rate, neurons are at the greatest risk of damage caused by neurotoxins.
This is followed, in order of risk, by oligodendrocytes, astroocytes, microglia and capillary
endothelium cells.Depending on a neurotoxin‘s chemical profile, it will cause damage to certain
parts or particular cellular elements of the nervous system. Non -polar substances are more soluble

in lipids and can therefore access the nervous tissue more easily than polar compounds, which are
less soluble in lipids. The body‘s response to neurotoxins is influenced by factors such as the
neurotransmitter affected, cellular membrane integrity and the presence of detoxifying mechanisms.
Following are the substances that can be neurotoxic to humans :
 Chemotherapy drugs that are used to kill fast growing cells

 Radiation
 Drug therapies or drugs of abuse
 Heavy metals such as mercury and lead
 Certain foods and food additives
 Insecticides/pesticides
 Cosmetics
 Industrial and cleaning solvents
Some examples of neurotoxic substances our environment has become polluted with and that it is
difficult for people to avoid exposure to in clude:
 Mercury
 Cadmium
 Lead
 Insecticides
 Solvents
 Car exhaust
 Chlorine
 Formaldehyde
 Phenol
Effects of Neurotoxicity
Some of the effects of neurotoxicity may appear immediately, while others can take months or years
to manifest.The effects of neurotoxicity depends on various different factors such as the
characteristics of the neurotoxin, the dose a person has been expos ed to, ability to metabolize and
excrete the toxin, the ability of affected mechanism and structures to recover and vulnerability of a
cellular target
 Some of the symptoms of neurotoxicity include:
 Paralysis or weakness in the limbs

 Altered sensation, tingling and numbness in the limbs
 Headache
 Vision loss

 Loss of memory and cognitive function
 Uncontrollable obsessive and/or compulsive behavior
 Behavioral problems
 Sexual dysfunction
 Depression
 Loss of circulation
 Imbalance
 Flu-like symptoms
Organ Specific Toxicity
The kidney is the main organ required by the human body to achieve and perform different
important functions including detoxification, regulation of extracellular fluids, homeostasis, and
excretion of toxic metabolites.(Stevens LA,et al.,2006)
Nephrotoxicity
It is the rapid deterioration in the kidney function due to toxic effect of medications and chemicals.
There are various forms, and some drugs may affect renal function in more than one way
There are various forms, and some drugs may affect renal function in more than one way.
Nephrotoxins are substances displaying nephrotoxicity. Nephrotoxicity should not be confused with
the fact that some medications have a predominantly renal excretion and need their dose adjusted
for the decreased renal function (e.g., heparin). The nephrotoxic effect of most drugs is more
profound in patients already suffering from kidney failure. About 20% of nephrotoxicity is induced
and caused by drugs.Aminoglycoside causes nephrotoxicity, which particularly affects the proximal
tubule epithelial cells due to selective endocytosis and accumulation of aminoglycosides via the
multi-ligand receptor megalin.
Different mechanisms lead to nephrotoxicity are renal tubular toxicity, inflammation, glomerular
damage, crystal nephropathy, and thrombotic microangiopathy. Although, there are traditional
markers of nephrotoxicity and renal dysfunction such as blood urea and serum creatinine which are
regarded as low sensitive in the detection of early renal damage, Kidney injury mol ecule-1,
Cystatin C, and neutrophil gelatinase-associated lipocalin sera levels are more sensitive than blood
urea and serum creatinine in the detection of acute kidney injury during nephrotoxicity.(Al -Naimi et
al.,2019)

Hepatotoxicity, or liver damage
Hepatotoxicity is the injury or liver damage caused by chemicals, dietary supplements,

pharmaceutical drugs, and medicinal plants. It is an adverse drug reaction that may be uncommon
but serious. The hepatic injury can be classified into hepatocellular, ch olestatic and mixed, caused
by increase in alanine aminotransferase and alkaline phosphatase than upper limit of normal.
Table.3.3.Causative Agents of Hepatotoxicity
CausativeAgent Product TypeofHepatotoxicity
Antibiotic Amoxicillinclavulanat hepatocellular,cholestaticormixedhepatocellular

e -cholestatic hepatitis
cholestaticpatternofinjurywithevidenceofportal
Antibiotic Macrolidesketolideser
andbullousinflammation,eosinophiliaandmildh
ythromycin
epatocellularnecrosis
Antibiotic Pyrazinamide Centrolobularcirrhosis and cholestasis
Antibiotic Rifampicin Cholestatichepatitis
IndustrialToxin Carbontetrachloride Centrilobular necrosis
IndustrialToxin Mercury Interferenceofbileexcretionanddestructionofhe
moglobin
MedicinalPlant Larreatridentata Fulminanthepatitis,subacutehepaticnecrosis,ch
olestatichepatitis,acuteliverfailure
Corticosteroids or
Pharmaceutica
glucocorticoids and Glyogenstorageinliver,enlargedliver
lDrug
anabolicandrogenic
steroids
Pharmaceutica
lDrug Non-steroidalanti- Acute,cytolytic,cholestaticormixed hepatitis
inflammatorydrugs
(Adapted without modification from:Thompson M et al.,2017)
Symptomsof Hepatotoxicity
 Yellowing of the skin and whites of the eyes (jaundice)

 Itching.
 Abdominal pain in the upper right portion of the abdomen.

 Fatigue.
 Loss of appetite.
 Nausea and vomiting.
 Rash.
 Fever.
(Adapted without modification from:Thompson M et al.,2017)
Figure 3.5. Mechanisms and Treatments for Hepatoprotection and Hepatotoxicity

Radioactive toxicity
(https://www.radioactivity.eu.com/site/pages/Radioactive_Toxicity.htm)
The danger presented by a radioactive substance is often rated through an indicator called "potential
radioactive toxicity" or radiotoxicity. This radiotoxicity concerns internal expos itions, external
expositions being considered separately. Internal expositions are the more harmful since we are not
protected against alpha and beta emitters which are settled durably in our bodies. Gamma are less
dangerous because depositing their energy in a diluted form while a substantial fraction may escape
without interacting.

Potential radiotoxicity is an indicator mostly used in radioactive waste management to assess the
potential hazard associated with ingestion. For example, a spent fuel discharg ed from a reactor
contains kilograms of plutonium. What would be the dose resulting from the ingestion of these
kilograms of plutonium by a population ? Since such an ingestion would not happen with a good
waste management, the potential radio toxicity ove restimates much in this case the risk. But the
indicator is useful for comparing the solutions envisaged for the very long -term storage of
radioactive waste.
No humans have ever died from acute toxicity due to plutonium uptake.4 Nevertheless, lethal
doses5 have been estimated from research on dogs, rats, and mice. Animal studies indicate that a
few milligrams of plutonium perkilogram of tissue is a lethal dose. For example, the LD50(30) for
dogs after intravenous injection of plutonium is about 0.32 milligr am per kilogram of tissue.
Assuming this animal dose also applies to humans, an LD50(30) by intravenous injection for an
average human of 70 kilograms would be about 22 milligrams. By inhalation, the uptake would
have to be about 4 times higher.(https://fas.org/sgp/othergov/doe/lanl/pubs/00818013.pdf)
Like radioactive activity, potential radiotoxicity is a useful risk indicator. However radioactive
elements must enter in our body to exercise their damages, like the poisons of Locust, the famous
roman poisoner. Fortunately the chances of this happening are, barring accidents, very small. The
qualifier "potential" being generally forgotten, potential radiotoxicity is often mistaken for a true
toxicity, generating misunderstandings and fears.
Ingestion or inhalation of a radioactive element results in a so -called committed dose, because its
effects are oncoming and would be spread over long periods of time , may be up to our whole
lifetime. One must take into account how radioactive materials are eliminated, how they are fixed
by our body, the type of radiation and the radioactive decay half -life.
There is a wide hierarchy in radio toxicities. Tritium is the least radiotoxic radioisotope. On the
contrary, most of the heavier nuclei, uranium, plutonium and minor a ctinides are very radiotoxic
because they emit short range alpha particles Plutonium -239 is thus 14,000 times more radiotoxic
than tritium.
The very low nuisance tritium nuisance is due to the particularly small energy of its beta electrons
(0.018 MeV maximum) and to the fact that this isotope of hydrogen is usually eliminated from the
body before having disintegrated (its biological half -life is only 10 days much shorter than its 12
years radioactive half-life). Instead, the plutonium is radiotoxic because this alpha emitter can be
fixed in the bones and liver for a long time. Fortunately, plutonium and minor actinides atoms have

a very low mobility in nature, which reduces their likelihood of bein g ingested or inhaled by
humans.
SOLVED PROBLEMS
1) Discuss concept and significance of LC.50.
a) Solution: Please refer topic: LC.50.
2) Comment on
i. Neuro-toxicity
ii. Hepato-toxicity
a) Solution: Please refer topic: Neuro-toxicity and Hepato-toxicity
SELF-TEST -MCQS
1) Following dose descriptors are determined in the toxicological studies
a) LC 50
b) LD 50
c) NOEL
d) All above
[Answer Key: d) All above]
2) Neurotoxicity refer to damage of
a) Kidney
b) Brain
c) Lungs
d) Heart
[Answer Key: b) Brain]
3) Nephrotoxicity in the rapid deterioration in function of
a) Lungs
b) Brain
c) Intestines
d) Kidney
[Answer Key: d)Kidney]
SHORT ANSWER QUESTIONS -SAQ.

1) SAQ.Write note on Nephrotoxicity
Solution:Please refer topic Nephrotoxicity

2) SAQ Write note on No-observed-effect level (NOEL)
Solution:Please refer topic NOEL
UNIT 03-03 : NON-TOXICITY

LEARNING OBJECTIVES
 To understand types and effects of various mutagenic agents
 To understand the structure of DNA
 To understand the structure of chromosome
Environmental Toxicants
Our environment is contaminated with lots of toxic chemicals from various sources. These toxicants
includes pesticides, some biological agents and inorganic pollutants. The toxic waste can be from
point sources like factories or diffused in the form of heavy metal and rubber from car tyre also
known as non-point sources. The environmental toxicants include lead, mercury, radon,
formaldehyde, benzene, cadmium, BPA, phthalates, pesticides etc. These pollutants have hazardous
effect on health of human being causing cancer, endocrine disrupts, developmental problems and
organ failure. Hence proper detection and assessment of these effluents is paramount.
Important Terms to Understand Toxicity.
Mutation
DNA is basic genetic material of all living organism in this universe. The central dogma of life says
DNA to RNA to protein where DNA & RNA are the genetic component while protein forms the
phenotypic part of living being. DNA also known as Deoxyribonuclei c acid consist of chemically
defied sequence of nitrogenous bases of purines (Adenine (A) and Guanine(G) ) and pyrimidine
(Cytosine (C) and Thymine (T)) having phosphodiseter backbone . DNA is double stranded
molecule where nitrogen bases are connected th rough hydrogen bonding. Where as Ribonucleic acid
(RNA) is single strand consisting of similar pure and pyrimidine except thymine is replaced by
Uracil. The unique arrangement of this sequence of ATCG in different permutation and combination
in DNA defines the genetic code.

Mutation can be defied as change in the DNA sequence with respect to the original sequence. The
alteration can be caused due to error in replication during cell division, effect of ionizing
radiations, mutation causing chemicals called mutagens or viral infection. Mutations occurring in
eggs & sperms which can be passed on to offspring are termed as germ line mutation while those in
body cells are known as somatic mutations. Somatic mutation can not be passed to next generation.
Types of mutation:
(Mutations and Health” by U.S. National Library of Medicine is in the Public Domain )
1. Missense mutation: Change in one DNA base pair that results in the substitution of one
amino acid for another in the protein made by a gene.
2. Nonsense mutation: It is also a change in one base pair but Instead of substituting one
amino acid for another, the altered DNA sequence to form stop codon (UGA, UAA, and
UAG )that prematurely signals the cell to stop building a protein. This type of mutation
results in a shortened protein that may function improperly or not at all.
3. Silent mutation: The mutational changes in DNA base that do not effect on the sequence of
amino acids in the protein are silent mutation. Their name is so because they do not affect
the structure or function of the protein as there is change in the resultant amino acid
sequence.
4. Insertion or Deletion: An insertion changes the number of DNA bases in a gene by adding a
piece of DNA. A deletion removes a piece of DNA. Insertions or deletions may be small up
to one or a few base pairs within a gene or large which can be an entire gene, several genes,
or a large section of a chromosome. In any of these cases, the protein made by the gene may
not function properly.
5. Duplication: A duplication consists of a piece of DNA that is abnormally copied one or

more times. This type of mutation may alter the function of the resulting protein.
6. Frameshift mutation: This type of mutation occurs when the addition or loss of DNA bases
changes a gene‘s reading frame. A reading frame consists of groups of 3 bases that each code
for one amino acid. A frameshift mutation shifts the grouping of these bases and changes the
code for amino acids. The resulting protein is usually nonfunctional.
However insertions, deletions, and duplications can all be frameshift mutations.

Mutagenic agents and mechanism of mutations:
James and Elizabeth Miller showed in the beginning in of1940s, that environmental mutagens are
converted to reactive products in the body(Miller J,1970; Hemminki K et al.,1994; Kadlubar F et
al.,1987)
The DNA adducts lead to mutations during DNA replication leading to cell death, aging, birth
defects, and cancer (Liu, B et al.,2016). Mutagens are any kind of factors that deliberately alters
genetic material of organism leading to mutations. Usually mutations caused by mutagenic agents
are induced mutations.
Types of mutagens and their mechanism:
Mutagen can be physical, chemical or a biological agent .

 Physical: Radiation
 Chemical : Base analogs, Alkylating agents, Intercalating agents, etc
 Biological agents: Viruses, transposons
Physical Mutagens
1. Radiations: It was reported in 1920 as one of the first mutagenic agent. It consist of UV
rays, X-rays, alpha rays, neutrons, and other ionizing and non -ionizing (Electromagnetic
radiation). Due to high penetration power, these mutagens causes direct damage to DNA or
nucleotide structure.
a. Electromagnetic Radiation: Visible light , electric and magnetic waves are types of
electromagnetic radiation. The component of sunli ght which is biologically
significant is UV and higher energy radiation. UV radiation is not ionizing but can
react with DNA and other biological molecules. UV radiation isn't ionizing but can
react with DNA and other biological molecules. UV radiation are responsible of
Cyclobutane pyrimidine dimers, Thymidine dimers (T-T).
b. Ionizing Radiations: X- and gamma-rays, corpuscular radiation produce free

radicals which react with biological molecules which damages the base and sugar
residues. These radiations acts upon rapidly dividing cell types (blood cell -forming
areas of bone marrow, gastrointestinal tract lining) .The severity of the effects
depends upon the dose of non ionizing radiations .

Chemical Mutagens :The mutagenic effect of the nitrogen mustard was reported by charlotte
Auerbach in 1942,which happened to be the first reported chemical mutagen. There are mainly four
groups of the chemical mutagen based on their effect of DNA.
1. Base analogs:
These are the chemicals which are analogous to purine and pyrimidine bases of DNA. Due to the
similarity instead of normal bases analogs, get incorporated in DNA during the replication process.
Bromouracil and aminopurine are example of base analog. 5 -bromouracil is chemically synthesised
analog of thymine which contains Br resembling to the thymine. It pairs with the adenine and
produces the mutation. Amino purine is similar to the adenine and can pair with either T or C,
however pairing with C is rare. It causes AT to GC or GC to AT transition during the rep lication.
Acridines (e.g., proflavin) are positively charged molecules. They may be inserted between two
DNA strands, thereby altering DNA's structure and rigidity. As a result, DNA replication will not be
faithful.
2. Base modifying Agents:
Some mutagens aren't incorporated into the DNA but instead alter a base, causing specific
mispairing eg.Ethylmethanesulfonate (EMS) and nitrosoguanidine (NG) .Alkylating agents are
chemicals that add an alkyl group to a different molecule. Alkylation of a base may change t he
traditional base pairing. For example, the alkylating agent EMS converts guanine to 7 -ethylguanine
which pairs with thymine. The mispairing will lead to mutation. Some alkylating agents can also
cross-link DNA, leading to chromosome breaks.Nitrous acid is one of the deaminating agent that
converts cytosine to uracil, adenine to hypoxanthine, and guanine to xanthine. The hydrogen -
bonding potential of the modified base is altered, leading to mispairing.
Hydroxylamine and free radicals modify base structur es, which causes mispairing.
Biological agents
Viruses insert there genetic material in to the host genome which can cause mutagenic changes.
Transposons also known as jumping genes can influence the gene function.
Detection of non toxicity:

1. Cytotoxicity Tests
Evaluation of cytotoxicity or cell viability is essential for the in vivo investigation of toxicity.
Different cell lines , such as colon or lung cancer cells, and can be used to predict the acute
toxicity, by the evaluation of the LC50 (lethal concentration for 50% of the cells. Most of the
cytotoxicity studies are colorimetric assays which includes the optical activity of organic dyes.
A. Trypan blue exclusion Assay: It is an azo dye which exclusively stains only dead cells,
hence damaged cells take up the dye and the ratio of damaged cells to that of living cells can
be counted in treated and non treated samples whose toxicity needs to be tested. This assay
detects cell membrane damage and not cell death.
B. The lactate dehydrogenase (LDH) Assay: In this assay, LDH enzyme activity is measured.
It is a cytosolic enzyme present in cells that is released outside the cells due to damage in
plasma membrane. This assay also measures cell membrane damage and not total cell death.
C. Tetrazolium Assays : MTT, XTT, and WST tests are based on enzymatic formation of
formazan salts. Cellular dehydrogenases catalyze conversion of the tetrazolium salt into
formazan which results in color change, this can be evaluated by spectrophotometric
technique. Since the assays occur inside cells, it measures metabolic activity corresponding
to cell viability.
D. Alamar Blue Assay: In this assay a drak blue, nonflurosent dye called resazurin is
converted to pink and fluorescent dye known as resorufin as a result of redo x reaction.The
intensity of fluorescence correlates with the cellular metabolic activity and in proportion to
the number of living cells.The damged and dead cells exhibit minimal innate metabolic
activity and thus results into low signal as compare to the viable cells.
2. Genotoxicity Tests
This test evaluates the damage caused to the genetic material to the cell rather than just the
cytosolic component. Genotoxins are mutagenic agents that causes genotoxicity leading to the
DNA damage or chromosomal material thus causing mutation.
A. Ames test (Bacterial reverse mutation test): This test was developed by Ames thus
named as Ames test.The amino acid requiring strains of Salmonella typhimurium and
Escherichia coli are used to detect the mutation points which may involve

substitution, deletion or addition of one or few of base pairs of DNA . It is based on
induction of reverse mutation in the histidine gene, which enables the bacteria to
synthesize histidine and form visible colonies in minimal histidine medium.In this
test the reverent microbial cells are identified by the ability of the pa rent test strain
to grow in the absence of amino acids. The bacterial reverse mutation test are rapid,
less expensive and easy to perform.
B. Mammalian chromosome aberration test: This test is used to identify the agents
which can cause structural aberration in chromosomes or chromatids in cultured
mammalian somatic cells. Changes like polyploidy and duplication can also be
detected in this test. A positive test result indicates the potential of test agent to be
carcinogenic. This test can be performed on var ious cell lines, such as Chinese
Hamster Ovary (CHO), Chinese Hamster Lung V79, Chinese Hamster Lung
(CHL)/IU, TK6) or primary cell cultures, including human or other mammalian
peripheral blood lymphocytes. The Cell cultures are treated with the test chem ical
with various concentration , both with and without metabolic activation during about
1.5 normal cell cycle lengths. After fixed intervals of exposure to the test chemical,
the cells are treated with a metaphase -arresting substance, then stained and observed
under microscope for chromosomal structural changes. If the dose dependent
increase in aberrant cells is observed as compared to the negative control data ,then
the test chemical is considered as genotoxic leading to chromosomal aberrations.
C. Mammalian cell gene mutation test: In this test chemicals are tested for their
potential to cause gene mutation.The L5178Y mouse lymphoma cells, the CHO,
CHO-AS52 and V79 lines of Chinese hamster cells, and TK6 human lymphoblastic
cells are commonly used cell lines for this purpose. The end point is detection of
mutations in thymidine kinase (TK) and hypoxanthine -guanine
phosphoribosyltransferase (HPRT), and a transgene of xanthineguanine
phosphoribosyltransferase (XPRT).
D. In-vivo comet assay:It is one of the most widely used test for detection of genotoxic
potential of a substance. It is used to detect DNA strand breaks in eukaryotic cells.
The Cells are embedded in agarose gel on a microscope slide which are lysed with
detergent and high salt to form nucleoids containing supercoiled loops of DNA

linked to the nuclear matrix. Upon electrophoresis at high pH, it results in structures
resembling comets, which can be observed by fluorescence microscopy; the intensity
of the comet tail relative to the head reflects th e number of DNA breaks. This is due
to ,the loops containing a break lose their supercoiling and become free to extend
toward the anode. This assay has applications in testing chemicals for genotoxicity,
monitoring environmental contamination with genotoxi ns, human biomonitoring and
molecular epidemiology, and fundamental research in DNA damage and repair. The
sensitivity and specificity of the assay can be enhanced if the nucleoids are
incubated with bacterial repair endonucleases which recognize specifi c kinds of
damage in the DNA and convert lesions to DNA breaks thus increasing the amount of
DNA in the comet tail. The DNA repair can be assessed by incubating cells after
treatment with damaging agent and measuring the damage remaining at intervals. In
the other way, the repair activity in a cell extract can be measured by incubating it
with nucleoids containing specific damage.( Collins AR. The comet assay for DNA
damage and repair: principles, applications, and limitations. Mol Biotechnol. 2004
Mar;26(3):249-61. doi: 10.1385/MB:26:3:249. PMID: 15004294 ).The test being
sensitivity to even low level of DNA damage it requires only small amount of cells
per sample and it can be completed in a short duration .
E. In-vivo micronuclei test:This test is performed to identify the damaged chromosome

or spindles. The mutagens when acted upon the cell ,can cause the damage due to
which during the cell division an additional micronucleus will be formed along with
the main nucleus. The principle of the test as follows, wh en a bone marrow
erythroblast develops into a polychromatic erythrocyte, the main nucleus is extruded;
any micronucleus that has been formed may remain behind in the otherwise
anucleated cytoplasm. An increase in the frequency of micronucleated polychromat ic
erythrocytes in treated animals is an indication of genotoxicity.(https://www.nucro-
technics.com/services/genetic-toxicology/invivo-mammalianmicronucleus/)

SOLVED PROBLEMS
1) Define mutation. Discuss any two mutagenic agents with their action.
a) Solution:Please refer topic mutation.
2) Draw schematic structure of bacterial DNA.
a) SolutionPlease refer topic bacterial DNA
SELF-TEST- MCQS
1) Which are the types of mutation
a) Nonsense mutation
b) Silent mutation
c) Missense mutation
d) All above
[Answer Key : d) All above]
2) Which is the mutagen is biological agent
a) Virus
b) Radiation
c) Fungi
d) Temperature
[Answer Key : a) Virus]
3) Cytotoxicity study can be carried out by which assay
a) Trypan blue exclusion assay
b) LDH assay
c) Alamar blue assay
d) All above

1) SAQ :Write note on toxicity detection
a) Solution:Please refer toxicity
2) SAQ: Write note on In-vivo micronuclei test
b) Solution:Please refer topicIn-vivo micronuclei test

UNIT03- 04: CARCINOGENESIS
LEARNING OBJECTIVES
 To understand relationship between mutagenesis and carcinogenesis
 To understand evaluation of toxicity routes
 To understand scope of toxicology
Mutagenesis &Carcinogenesis
(https://www.edcan.org.au/edcan-learning-resources/supporting-resources/biology-of-
cancer/defining-cancer/carcinogenesis)
The process by which normal, healthy cells transform into cancer cells is termed carcinogenesis or
oncogenesis. The development of a malignant tumour in otherwise healthy tissue is the result of a
complex series of events beginning with a single cell that has acquired malignant properties through
cellular DNA damage.
Errors in the DNA sequence interrupt the genetic codes that govern the structure and function of the
affected cell. The survival and proliferation of a cell with DNA damage, d ividing to give rise to two
daughter cells, each then capable of dividing, eventually results in a population of clones with
similar genetic errors and malignant properties.
Before malignant cells can cause symptoms or be detected, successive generations of daughter cells
must divide and double the size of the clonal population approximately 30 times. At this point, the
tumour will likely measure one cubic centimetre, weigh about one gram and comprise one billion
cells. 5, 7
Most current theories of carcinogenesis characterise it as a multi-step process involving initiation,

growth, promotion, conversion, propagation, invasion and metastasis.
Carcinogens
Carcinogens are defined as agents capable of initiating the development of malignant tumours by
inducing cellular genetic changes. The transformation of a normal cell to a malignant cell is thought
to be due to successive and cumulative exposures to carcinogens and other factors over the course
of decades. Most human cancers result from exposure to environmen tal (or exogenous) carcinogens.
Other carcinogens that cause malignant transformation include a broad group of factors from within
the body, termed endogenous factors.

Some Organizations working on CancerResearch
(https://www.cancer.gov/about-cancer/causes-prevention/risk/substances/carcinogens)
Two organizations—the National Toxicology Program (NTP), an interagency program of the U.S.
Department of Health and Human Services (HHS), and the International Agency for Research on
Cancer (IARC), the cancer agency of the World Health Organization —have developed lists of
substances that, based on the available scientific evidence, are known or are reasonably anticipat ed
to be human carcinogens.
Specifically, the NTP publishes the Report on Carcinogens every few years. This congressionally
mandated publication identifies agents, substances, mixtures, or exposures (collectively called
―substances‖) in the environment that may cause cancer in humans. The 2016 edition lists 62 known
human carcinogens and includes descriptions of the process for preparing the science-based report
and the criteria used to list a substance as a carcinogen.
IARC also produces science-based reports on substances that can increase the risk of cancer in
humans. Since 1971, the agency has evaluated more than 1,000 agents, including chemicals,
complex mixtures, occupational exposures, physical agents, biological agents, and lifestyle factors.
Of these, more than 500 have been identified as carcinogenic, probably carcinogenic, or possibly
carcinogenic to humans.
IARC convenes expert scientists to evaluate the evidence that an agent can increase the risk of
cancer. The agency describes the principles, procedures, and scientific criteria that guide the
evaluations.ExitDisclaimer. For instance, agents are selected for review based on two main criteria:
(a) there is evidence of human exposure and (b) there is some evidence or suspicion of
carcinogenicity.
Decision of NTP w.r.t including a substance on its list of known human carcinogens
As new potential carcinogens are identified, they are evaluated scientifically by the NTP‘s Board of
Scientific Counselors and the NTP Director. Next, a draft Report on Carcinogens monograph is
prepared, which is reviewed by other scientific experts as needed, the public, and other federal
agencies. The draft monograph is then revised as necessar y and released for additional public

comment and peer review by a dedicated panel of experts. Lastly, a finalized monograph and
recommendation for listing is sent to the HHS Secretary for approval.
Toxicology
(https://www.niehs.nih.gov/health/topics/science/toxicology/index.cfm )
Toxicology is a field of science that helps us understand the harmful effects that chemicals,
substances, or situations, can have on people, anima ls, and the environment. Some refer to
toxicology as the ―Science of Safety‖ because as a field it has evolved from a science focused on
studying poisons and adverse effects of chemical exposures, to a science devoted to studying safety.
Toxicology uses the power of science to predict what, and how chemicals may cause harm and then
shares that information to protect public health. When talking about toxicology it is important to
keep a few things in mind.
 Not everyone will respond to substances in exactly t he same way. Many factors, including
the amount and duration of exposure, an individual‘s susceptibility to a substance, and a
person‘s age, all impact whether a person will develop a disease or not. There are times in a
person‘s life when he or she may be more susceptible to chemicals. These times may include
periods of active cell differentiation and growth in the womb and in early childhood, as well
as during adolescence, when the brain is continuing to develop. Just because someone is
exposed to a harmful substance, does not always mean they will get sick from it.
 The dose of the chemical or substance a person is exposed to is another important factor in
toxicology. All substances have the potential to be toxic if given to humans and other living
organisms in certain conditions and at certain doses or levels. For example, one or two
aspirins may be good for you, but taking a bottle of aspirin may be harmful. The field of
toxicology tries to understand and identify at what dose and through what exposure a
substance poses a hazard.
 Toxicologists also realize that even low-dose exposures that may seem insignificant may
have biological meaning or lead to an adverse health effect if the exposure is continuous or
happens during a critical window of development.
Evaluation of toxicity routes of exposure(https://www.atsdr.cdc.gov/hac/phamanual/ch6.html )

A critical early step in the public health assessment process is evaluating exposure pathways. The
goal of exposure pathway evaluations is to identify likely site -specific exposure situations and
answer the questions: Is anyone at a given site exposed to environmental contamination? Under
what conditions does this exposure occur?
Exposure Routes
In general, individuals may be exposed to contaminants in environmental media in one or more of

the following ways:
 Ingestion of contaminants in groundwater, surface water, soil, and food.
 Inhalation of contaminants in air (dust, vapor, gases), including those volatilized or

otherwise emitted from groundwater, surface water, and soil.
 Dermal contact with contaminants in water, soil, air, food, and other media, such as exposed
wastes or other contaminated material.
 External exposure to radiation. Gamma radiation is unique in comparison to chemical

contaminants because it travels beyond the source. Therefore, direct contact is not necessary
for exposure to occur. In fact, radiation can easily penetrate solid materials such as soils,
drums, and even lead. Gamma radiation, in particular, can travel great distances before
losing strength. External exposure to radiation also includes exposure to beta particles from
many radioactive materials. These, too, can easily penetrate certain materials and travel
several meters prior to loss in energy.
Toxicity Evaluation
Different people have different ways of evaluating exposure pathways at their sites, but a common
approach involves developing a site conceptual model, which helps you envision how people might
come into contact with environmental contamination. Regardless of the site -specific nuances,
developing a site conceptual model will ultimately help you visualize how contaminants move in
the environment at your site and how people might come into contact with these contami nants.

(Adapted without modification from https://www.atsdr.cdc.gov/hac/phamanual/ch6.html)
Figure 3.6. Site Conceptual Model – Exposure Pathway Evaluation
This type of diagram more explicitly outlines examples of some factors you should consider when
analysing the exposure pathways at your site: What media are affected? What media transport
contaminants from the source to exposure points? Where are the exposure points? What are the
potentially exposed populations?
This outline the thought process for evaluating the five elements of exposure pathways, but having
a detailed site conceptual model will help in these evaluations.
SOLVED PROBLEMS
1) What is carcinogen? Discuss its effect with suitable example.
a) Solution:Please refer topic:Carcinogen

2) Which environmental exposure causes cancer in humans? Discuss with suitable example.
a) Solution:Please refer topic:Carcinogens and environmental exposure
SELF-TEST -- MCQS
1) Which is the International agency for research on cancer
a) IARC
b) ICMR
c) IRCT
d) IIMC
[Answer Key : a) IARC ]
2) Which of the following are the exposure routes of contaminants
a) Ingestion
b) Inhalation
c) Dermal contact
d) All above
3) Which radiation can travel great distance before losing strength
a) Alpha
b) Beta
c) Gamma
d) All above
[Answer Key: c) Gamma ]
SHORT ANSWER QUESTIONS :SAQ

1) SAQ Write note on carcinogen
Solution:Please refer topic: carcinogenesis
2) SAQ: What is the relation between Mutagenesis and Carcingenesis
Solution:Please refer topic: Mutagenesis and Carcingenesis
SUMMARY
First section of this unit presentsthe overview of Toxicology,its definition and scope, acute
and chronic toxicity, selective toxicity, Besides, it illustrates the classification and Labelling
Summary does synergism and antagonism .It also gives detail description of Toxic chemicals in

the environment ,Sources of heavy metals and their cycle in the environment and biochemical
aspects of As Cd, Pb, Hg, CO, O3, PAN, pesticides.
Second section of this unit presentsthe overview of Environmental Toxicolog y .In detail.,it
gives description of various concepts and significance ,T acute, chronic toxicity,LC 50 ,LD 50 ,
NOEL and their estimation, Chemical Pharmacological basis of toxicity,Toxic effects at cell, tissue,
organ level,Some organ specific toxicity studies -Neurotoxicity, Nephrotoxicity, Hepatotoxicity,
Radioactive toxicity.
Third section of this unit presentsthe overview of Nontoxicity - Mutations, mutagenic

agents, mechanism of mutagenesis, Detection of nontoxicity - DNA, Gene, Chromosome Level. It
gives detail description of Tissue /Organ specific /Organ toxicity studies.
Fourth section of this unit presents the overview of Carcinogenesis along with the relation
between mutagenesis and carcinogenesis.It highlights on Environmental carcinogens, to xicology-
scope, Definition, Further, it gives detail description evaluation of toxicity routes of exposure
KEY WORDS
Toxicology, AcuteToxicity, Chronic Toxicity, Synergism, Antagonism, PAN, Pesticides, LC 50,
LD 50, NOEL, Mutations, Carcinogens, Mutagenesis, Toxicity routes
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impacts-and-management-strategies
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CREDIT 04
UNIT 04-01: ENVIRONMENTAL TOXICOLOGY – 2

LEARNING OBJECTIVES
After successful completion of this unit, you will be able toTo understand effects of pesticides
on environment and human health
 To understand types and importance of degradable toxic substances
 To understand types and importance of non -degradable toxic substances
 To understand rotation of toxic substances in the food chain
INTRODUCTION
Toxic Agents in Environment
Agrochemicals(https://cabb-chemicals.com/what-are-agrochemicals/)
Modern agriculture fundamentally relies on an extensive use of agrochemicals to enha nce the crop
productivity by controlling harmful pests, pathogens, and undesirable weeds. However, currently,
we have reached a stage where several threats are emerging on food security, human and
environmental health, maintenance of ecological balance, an d conservation of the soil biodiversity.
Long-term unbalanced use of agrochemicals may lead to community shift of beneficial
microorganisms with dangerous consequences such as the development of antimicrobial resistance.
Agrochemicals usage in farming systems may adversely influence soil microorganisms that are
mainly involved in nutrient cycling processes, such as nitrogen fixation, phosphorus solubilization,
and other essential nutrient biotransformation. The recent report established that some
agrochemicals reduce biochemical reaction and activities of soil enzymes that are key indicators of
soil microbiological health. This chapter focuses on the effects of applied agro -pesticides on soil
microbiological and biochemical health attributes under different cropping systems.(AsitMandalet
al.,2020)
Types of Agrochemicals
There is a concerted effort to actively and conscientiously implement a broad range of
agrochemicals to safely maintain and control the global food supply to ensure consistently high
quality for widespread consumption. Types of agrochemicals include:

 Pesticides, or chemicals engineered to destroy insects and other organisms, weeds, and
funguses that could spoil crop yields;
 Synthetic fertilizers, for example ammonium nitrate (NH 4 NO 3 ), which is designed to
encourage crop growth by saturating soils with nutrients;
 Acidifiers and liming agents, engineered to alter the pH levels of soils to suit the planting
properties of given crops;
 Soil conditioners, for example gypsum (CaSO 4 ·2H 2 O), which is designed to condition soils
with high sodium (Na) contents to improve planting conditions;
 Growth hormones, or synthetic chemicals designed to increase growth rates in animals and
crops.
Among the agrochemicals, pesticides shows adverse impact on environment and health of
organisms.
Pesticides
These are the group of chemicals that are purposely applied to the environment with aim to
suppress plant and animal pests and to protect agricultural and industrial products.
Increase in food production is the prime -most objective of all countries, as world population is
expected to grow to nearly 10 billion by 2050. This increasing world population has resulted into
tremendous amount of pressure on the existing agricultural system to meet the demand and supply
of food by use of current resources such as land, water etc. To increase the crop production
different types of agrochemicals like herbicides, insecticides, fungicides, nematicides, fertilizers
and soil amendments has been tremendously applied past. (Harsimran Kaur Gill and Harsh Garg,
2014)
Since last few decades, pesticides have become an integral part of the modern life and continuously
applied to protect agricultural land, stored grain, flower gardens as well as t o eradicate the pests
transmitting dangerous infectious diseases. It has been estimated that globally nearly $38 billion are
spent on pesticides each year (Pan-Germany, 2012). Manufacturers and researchers are designing
new formulations of pesticides to meet the global demand.
Since the middle of the 19th century, pesticides have been commonly used to control pests
(Timmons F, 1970; ChauvelB,1944) causing a widespread release of these xenobiotics into the
environment .(Toccalino P et al.,2014) The intensive use of pesticide leads to an increased risk of

contamination of the environment and harmful effects on biodiversity, food security, and water
resources (Malaj E et al.,2014 ; Queyrel W et al.,2016). It is evidenced that partof pesticides
sprayed on crops will remain in farmland, but some of them enters the surrounding environment i.e.
soil, water, and air etc. Synthettic pesticidescan remain in the environment for many years and may
be transported over a long distance .Pesticide residues in soil and water are significant environment
threats and have beenclassified as carcinogen pollutants in many countries . Hence, the excessive
applicationof these compounds over the past half -century has posed serious risks to human health
.(Arzuozkara, et al., 2016)
Classification of Pesticides
In general, there are three main ways to classify pesticide : classification based on the (i) mode of
action, (ii) targeted pest species, and (iii) chemical composition of pesticides .
I) Classification based on the mode of action
Under this type of classification, pesticides are classified as non -systemic and systemic pesticides.
Non-systemic pesticides are those that do not appreciably penetrate plant tissues and consequently
not transported within the plant vascular system. Where as, systemic pesticides are those which
could effectively penetrate the plant tissues and transported within the plant vascular system to
bring about the desired effect . (Arzuozkara, et al., 2016)
II) Classification based on the targeted pest species
Classification by target pest is perhaps the most accustomed. For example, insecticides are
pesticides that target insects, and herbicides target plants. The others are rodenticides, fungicides,
acaricides and miticides, molluscicides, bactericides, avicides, and virucides etc. (Arzuozkara, et
al., 2016)
III) Classification based on the chemical composition (Arzuozkara, et al., 2016)
In this type of classification, pesticides are characterized as per their chemical nature andactive
ingredients. On basis of chemical properties, pesticides can be generally divided into following
types as:
A.Organochlorines- These are organic compounds with five or more chlorine atoms. These
pesticides generally have a steady chemical structure and often accumulate and persist in the
environment. Most of them are widely used as insecticides for the control of a wide range of

insects. Organochlorine insecticides act as nervous system disruptors leading to convulsions and
paralysis of the insect and its eventual death. They can caus e serious endocrine disorders in
mammals, fish, and birds, so most of them have been banned in agriculture worldwide.
(Adapted without modification from: https://doi.org/10.2166/wqrj.2001.012)

Figure.4.1. Structure of Organochlorine Pesticides
B.Organophosphorus- These are highly toxic pesticides that contain a phosphate group and
occupied up to 48.6% of all pesticides These chemical compounds inhibit the acetyl cholinesterase
enzyme, which hydrolyses acetylcholine in the nervous system of a number of species, including
humans .Although they are easier to be degraded than organochlorines, organophosphate pesticide
residues is one of the biggest threats to the ecosystem and food industry because their acute
toxicities are irreversible .Many people are exposed to pesticides occupationally, and pesticide self -
poisoning is a major public health problem. Therefore, the usage of organophosphates has been
restricted or banned all over the world.

(Adapted without modification from: https://www.semanticscholar.org/paper/Organophos phate-
pesticides%3A-A-general-review-Kazemi-
Tahmasbi/587b1727c1874b13b93082bb747a7f18be079b32/figure/1 )
Figure.4.2. Structure of Organophosphorus Pesticides
C.Carbamates- These are organic pesticides, reversibly inactivating the enzyme acetyl
cholinesterase; these pesticides are derived from carbamic acid. The cholinesterase inhibition of
carbamates differs from that of organophosphates in that it is species specific and i s reversible .
(Adapted without modification from:http://www.doiserbia.nb.rs/Article.aspx?ID=1820 -

39491904193T#.YQucihQzbIU)
Figure.4.3. Structure ofCarbamates
D.Pyrethroids-These are the synthetic analogues of the naturally occurring pyrethrins, a produ ct of
flowers from pyrethrum plant (Chrysanthemum cinerariaefolium), and were detected in the 1980s to
mimic the insecticidal activity of the natural pyrethrum. Pyrethroids are acknowledged for their fast
knocking down effect against insect pests, facile b iodegradation, and low mammalian toxicity .
These pesticides are nonpersistent sodium channel modulators and are much less toxic than
carbamates and organophosphates to mammals.However, they are found to be are highly toxic to
aquatic organisms such as mollusks, fish, and arthropods .
(Adapted without modification from:https://en.wikipedia.org/wiki/Pyrethroid)
Figure.4.4. Chemical Structure of Permethrin

E.Amides- These herbicides are widely used in recentyears. Among the herbicides like, acetochlor,
butachlor, and metolachlor, butachlor can persist in the environment for up to 10 weeks, and what‘s
evenworse is that butachlor and metolachlor have been identified as mutagens.
(Adapted without modification from :https://en.wikipedia.org/wiki/Amide)
Figure.4.5. Chemical Structure of Amide
F.Anilinsand dinitroaniline---Among these types, Trifluralin and pendimethalin are widely used .
These pesticides show high toxicity to aquatic organisms and they c animpair the thyroid gland and
liver. Hence, these two aniline herbicides have been banned inmany European countries.
[A][B]
Figure.4.6.[A].Structure of Aniline[B].Structure of 2,4-Dinitroaniline
A.( Adapted without modification from: https://commons.wikimedia.org/wiki/File:Aniline.svg
B.( Adapted without modification from: https://en.wikipedia.org/wiki/2,4-Dinitroaniline)
G.Azotic heterocyclic compounds.-Nitrogen-containing heterocyclic compounds, especially for

imidazole and triazole heterocyclic chemicals, have become the hotspot for new pesticide

development. In the last 10 years, they occupied no less than 70% of all the newly developed
chemical pesticides
(Figure.4.7. Imidazole andtriazole Heterocyclic Chemical)
(Adapted without modification from: https://www.researchgate.net/figure/Chemical -structure-of-

imidazole_fig1_338480383)
Other than the above mentioned classification, pesticides are also classified on basis of different
parameters as:
 Classification on basis of mode of formulation-wettable powders, emulsifiable

concentrates, baits, granules, dusts, and fumigants. (Arzuozkara, et al., 2016)
 Classification on basis of active spectrum-broadspectrum pesticides (designed to kill a

wide range of pests and other non-target organisms), selective pesticides. (designed to kill
only specific pests) (Arzuozkara, et al., 2016)
 Classification on basis of toxicity level- the World Health Organization (WHO) has
developed a classification system that group pesticides according to th e potential risks to
human health and they are grouped into the following classes: class Ia=extremely
hazardous, class Ib=highly hazardous, class II=moderately hazardous, class III=slightly
hazardous, and class IV=products unlikely to present acute hazards in normal use (Tano
Z,2011;Arzu ozkara, et al., 2016)
Impacts of Pesticides On Health And Environment
Impacts on environment

The ecological impacts of pesticides are varied and are often inter-related. Pesticides can
contaminate soil, water, turf, and other vegetation. In addition to killing insects or weeds, pesticides
can be toxic to a host of other organisms including birds, fish, beneficial insects, and non -target
plants. Ecological effects of pesticides extend beyond individual organisms and can exten d to
ecosystems.
i) Surface and ground water contamination:
Different categories of pesticides have different types of effects on living organisms. The principal
pathway that causing ecological impacts is that of water contaminated by pesticide runoff.
Pesticides can reach surface water through runoff from treated plants and soil. Contamination of
water by pesticides is widespread. Similarly, groundwater pollution due to pesticides is a worldwide
problem .During one survey in India, 58% of drinking water s amples drawn from various hand
pumps and wells around Bhopal were contaminated with Organo Chlorine pesticides above the EPA
standards . Once ground water is polluted with toxic chemicals, it may take many years for the
contamination to dissipate or be cleaned up. Cleanup may also be very costly and complex, if not
impossible . (Gyawali K,2018)
ii) Soil contamination - Overuse of pesticides in soil treatment and also large number of
transformation products (TPs) from a wide range of pesticides affects decrease in populations of
beneficial soil microorganisms resulted into low fertility of the soil, soil degradation w hich could
be responsible for poor production and productivity of crops and ultimately enhances hunger and
poverty by reducing the farm income.
iii) Contamination of air and non-target vegetation
Pesticide sprays can directly hit non-target vegetation, or can drift or volatilize from the treated
area and contaminate air, soil, and non-target plants. Some pesticide drift occurs during every
application, even from ground equipment (Glotfelty and Schomburg, 1989;Gyawali K,2018). Drift
can account for a loss of 2 to 25% of the chemical being applied, which can spread over a distance
of a few yards to several hundred miles. As much as 80 –90% of an applied pesticide can be
volatilized within a few days of application (Majewski, 1995;Gyawali K,2018). This contam ination
will make negative effect to non-targeted fauna and flora and disturb the ecosystem. This
imbalanced ecosystem influences socio economic aspect of human beings along -with health status
The two principal mechanisms related to pesticides are are bio concentration and bio magnification

Bio concentration:
Bio concentration is the ability of an organism to accumulate a chemical from the ambient
environment. It is quantitatively expressed in terms of the bio concentration factor (BCF) which is
the ratio of the concentration of the chemical in the organism to its concentration in the ambient
environment. The primary "sink" for some pesticides is fatty tissue ("lipids"). Some pesticides, such
as DDT, are "lipophilic", meaning that they are soluble in, and ac cumulate in fatty tissue such as
edible fish tissue and human fatty tissue. Other pesticides such as glyphosate are metabolized and
excreted.(Gyawali K, 2018)
Bio magnification:
It isthe increase in concentration of pesticides due to its persistent and non -biodegradable nature in
the tissues of organisms at each successive level of food chain .
Generally, organisms at the higher levels of food chain experience greater harm as compared to
those at lower levels. Several studies have been undertaken that demon strate enhanced amount of
toxic compounds with increase in trophic levels. Some of the adverse effects of pesticides on non -
target organisms such as fish, amphibians and humans have also occurred as a result of bio
magnifications of the toxic compounds. For example, reproductive failure and population decline in
the fish-eating birds (e.g., gulls, terns, herons etc.) was observed as a result of DDE induced
eggshell thinning (Grasman et al., 1998). The extent of bio magnifications increases with increase
in persistence and lipophilic (fat-loving) characteristics of the particular pesticide. As a result of
this, organochlorines are known to have higher biomaginification rate and are more persistent in a
wider range of organisms as compared to organophsphates (Favari et al., 2002). It is important to do
the risk assessments associated with the pesticides on the basis of their bioaccumulation and bio
magnifications before considering them for agricultural purposes.
Pesticides are designed to kill pests, but some pesticides can also cause negative health impacts to
human and other organisms resulted into damage of ecosystem. Pesticide residues absorbed by
inhalation, ingestion, and dermal contact can lead to acute and chronic toxicity. Such kinds of the
toxicity depend on types of pesticides, port of entry, dose, metabolism, accumulation and so on.
Acute toxicity is due to short-term exposure and happens within a relatively short period of time,
whereas chronic toxicity is due to repeated or long -term exposure and happens over a longer period.
Mainly it interrupts the metabolic and systemic functions of the human body. The chemical

compound of pesticide disrupts the neurological function. It is injurious to the immune and
endocrine systems as well (Wesseling, et al., 1997). Wide use of these pesticides can cause both
acute and chronic adverse health effects in human. Studies in the past have revealed the association
of organochlorine and organophosphate with diabetes mellitus (Paudayl, 2008). Organophosphate
inhibits the neurotransmitter acetyl cholinesterase and can affect the central and autonomic
nervoussystem. Few leading symptoms related to the autonomic nervous system are abdominal
cramps; nausea, diarrhea, salivation, miosis and symptoms related to the central nervous system are
dizziness, tremor, anxiety, and confusion. Symptoms usually occur within hours of exposure and
typically disappear within days or weeks as new cholinesterase is synthesized (Aryalet.al., 2016). In
many developing countries like Nepal, most pesticides are associated with adverse effects on human
health and environment due to inappropriate use and handling of pesticides by inadequately trained
farm workers (Gyawali K,2018).Majority of pesticides users, being unaware of pesticide types,
their mode of action, potential hazards and safety measures, and the problem is becoming more
havoc. The pesticides are widely applied in agriculture sector .Although, farmer are aware of the
health impacts of pesticid, they did not adopt the safety precautio n resulting higher risk of exposure
with pesticide toxicity.. As a result, more than 50% farmers have been experienced an acute toxicity
syndrome of pesticides and one of ten farmers reported several kinds of chronic diseases of which
24% farmers had chronic neuropathic diseases (Aryalet.al., 2016).
The other major harmful effects of pesticide in human and environment are direct impact on human
,including the enhanced economic potential w.r.t. increased production of food and fiber, and
management of vector-borne diseases, and then their debits have resulted in serious health
implications and unwanted adverse impacts on environment .(Gyawali K,2018 ;Aktar et.al., 2009)
Therefore, this is the time that imposes the proper use of pesticides to protect our environment .In
this cotext,alternative pest control strategies like, IPMthat deploys a combination of different
control measures such as cultural control, use ofresistant genotype, physical and mechanical
control, and rational use of pesticide could red ucethe number and amount of pesticide applications.
Further, advanced approaches such asbiotechnology and nanotechnology could facilitate in
developing resistant genotype orpesticides with fewer adverse effects. Community development and
various extensionprograms that could educate and encourage farmers to adopt the innovative IPM
strategieshold the key to reduce the deleterious impact of pesticides on our environment.

In this way, there are severe consequences of indiscriminate use of pesticides .Few of the negative
impacts associated with pesticide application have emerged in the form of increase in resistant pest
population, decline in on beneficial organisms such as predators, pollinators and earthworms,
change in soil microbial diversity, and contami nation of water and air ecosystem. The persistent
nature of pesticides has impacted the ecosystem to such an extent that pesticides have entered into
various food chains and into the higher trophic levels such as that of humans and other large
mammals. Some of the acute and chronic human illnesses have now emerged as a consequence of
intake of polluted water, air or food.
Industrial chemicals and their impact on Health and Environment (https://ipen.org/toxic-

priorities/industrial-chemicals)
Industrial chemicals are chemicals that are developed for use in the industrial processing of
chemicals. Some industrial chemicals are only used in industrial production processes while many
others are used as ingredients in the commercial products that appear in consumer markets. The
class of industrial chemicals is broad, including: solvents, reactants, lubricants, coatings, dyes,
colorants, inks, mastics, stabilizers, plasticizers, fragrances, flame retardants, conductors and
insulators. Significant exposures to many of these chemicals can result in harmful effects to people
or the environment.
Some industrial chemicals are Persistent Organic Pollutants (POPs). Persistent organic pollutants
(POPs) are considered as the silent killers due to their bio -accumulative and long persistent natures.
These are present everywhere in our environment including plants, animals and human beings.
These are responsible for various lethal diseases and environmental problems. The different
diseases due to POPs are diabetes, obesity, endocr ine disturbance, cancer, cardiovascular,
reproductive diseases, skin irritation, dizziness and headaches to chronic effects on the immune,
reproduction, nervous and endocrine systems. Some industrial POPs are also recognized as cancer
causing agents.
All share the common POPs characteristics as :
 remain intact for exceptionally long periods of time (many years);

 become widely distributed throughout the environment as a result of natural processes
involving soil, water and, most notably, air;

 accumulate in the fatty tissue of living organisms including humans, and are found at higher
concentrations at higher levels in the food chain; and
 are toxic to both humans and wildlife.
e.g. Persistent Organic Pollutants (POPs) are:
 Hexachlorobenzene
 Hexachlorobutadiene
 Polychlorinated Biphenyls (Pcbs)
 Polychlorinated Naphthalenes
 Short-Chain Chlorinated Paraffins (Sccps)
 Hexabromobiphenyl
 Hexabromodiphenyl Ether and Heptabromodiphenyl Ether
 Hexabomodyclododecane (Hbcdd)
 Pentachlorobenzene
 Perfluorooctane Sulfonic Acid, Its Salts And PerfluorooctaneSulfonyl Fluoride
 Tetrabromodiphenyl Ether And PentabromodiphenylEtherchlordane
 Decabromodiphenyl Ether (Commercial Mixture, C -Decabde) etc.
There is a profound relationship among air, water, animals and humans in the environm ent. The
affects on these identities directly or indirectly disturb the environment. Therefore, the environment
is being affected by POPs through biotic, abiotic, social, cultural and technological interferences.
The pollution of POPs degrade ecological balance; threatening the environment and health of all
organisms. POPs accumulated in marine animal leading to their death. This results into imbalance
in sea ecology.
Food additives
These are the substances added to food to preserve flavor or enhance taste, appearance, or other
sensory qualities. Some additives have been used for centuries as part of an effort to preserve food,
. With the advent of processed foods in the second half of the twentieth century, many additives
have been introduced, of both natural and artificial origin. Food additives also include substances
that may be introduced to food indirectly in the manufacturing process, through packaging, or
during storage or transport.
Food additives are among the safest chemicals in food due to their l ow toxicity, rigorous safety
testing, and control of use by the law. The permission to use specific food additives is

recommended by the Codex Alimentarius Commission and approved by national legislation. The
use of food additives is subject to strict cont rols, underpinned by scientific studies to demonstrate
their safety to human health. Their use brings many benefits including increased safety, and greater
choice of food products.
Table. 4.1. Types of Food Additives and TheirSignificance
Types of Food Significance
Additives
Acidulants It confer sour or acid taste. Common acidulants
include vinegar, citric acid, tartaric acid, malic acid, fumaric
acid, and lactic acid.
Acidity regulators These are used for controlling the pH of foods for stability or to
affect activity of enzymes.
Anticaking agents It keep powders such as milk powder from caking or sticking.
Antifoaming and foaming agents Antifoaming reduce or prevent
foaming in foods. Foaming agents do the reverse.
Antioxidants These are preservatives by inhibiting the degradation of food
by oxygen
Bulking agents These are additives that increase the bulk of a food without
affecting its taste.
Food coloring These are added to food to replace colors lost during preparation or
to make food look more attractive.
Fortifying agents These are used to increase the nutritional value
Color retention These are used to preserve a food's existing color.
agents
Emulsifiers These allow water and oils to remain mixed together in
an emulsion, as in mayonnaise, ice cream, and
homogenized milk.
Flavors These are additives that give food a particular taste or smell, and
may be derived from natural ingredients or created artificially. *In
EU flavors do not have an E-code and they are not considered as
food additives.

Flavor enhancers It enhance a food's existing flavors. A popular example
is monosodium glutamate. Some flavor enhancers have their own
flavors that are independent of the food.
Flour treatment These are added to flour to improve its color or its use in baking.
agents
Glazing agents These provide a shiny appearance or protective coating to foods.
Humectants These prevent foods from drying out.
Tracer gas It is used for package integrity testing to prevent foods from being
exposed to atmosphere, thus guaranteeing shelf life.
Preservatives These prevent or inhibit spoilage of food due
to fungi, bacteria and other microorganisms
Stabilizers These are used to give foods a firmer texture. While they are not
true emulsifiers, they help to stabilize emulsions
Sweeteners These are added to foods for flavoring. Sweeteners other
than sugar are added to keep the food energy (calories) low, or
because they have beneficial effects regarding diabetes
mellitus, tooth decay, or diarrhea
Thickening agents These are substances which, when added to the mixture, increase
its viscosity without substantially modifying its other properties.
Packaging Bisphenols, phthalates, and perfluoroalkyl chemicals (PFCs) are
indirect additives used in manufacturing or packaging.
(Adapted with modification from: https://en.wikipedia.org/wiki/Food_additive)
Drugs
A drug is any substance that causes a change in an organism's physiology or psychology when
consumed. Drugs are typically distinguished from food and substances that provide nutritional
support. Consumption of drugs can be via inhalation, injection, smoking, ingestion, absorption via
a patch on the skin, suppository, or dissolution under the tongue.
In pharmacology, a drug is a chemical substance, typically of known structure, which, when

administered to a living organism, produces a biological effect.A pharmaceutical drug, also called a

medication or medicine, is a chemical substance used to treat, cure, prevent,
or diagnose a disease or to promote well-being. Traditionally drugs were obtained through
extraction from medicinal plants, but more recently also by organic synthesis. Pharmaceutical drugs
may be used for a limited duration, or on a regular basis for chronic disorders.
Pharmaceutical drugs are often classified into drug classes—groups of related drugs that have
similar chemical structures, the same mechanism of action (binding to the same biological target), a
related mode of action, and that are used to treat the same disease.. The Anatomical Therapeutic
Chemical Classification System (ATC), the most widely used drug classification system, assigns
drugs a unique ATC code, which is an alphanumeric code that assigns it to specific drug classes
within the ATC system. Another major classification system is th e Biopharmaceutics Classification
System. This classifies drugs according to their solubility and permeability or absorption properties
Environment and Health implications of Drugs and food Additives
The issue of pharmaceuticals in the environment is gaining more attentio n around the world
Impacts of drugs
 During the manufacturing process, drug residue may infiltrate surface waters.
 Drugs are metabolized by humans and then excreted in trace quantities into the sewage
system. This pollution inevitably finds its way into th e water supply after going through
treatment systems.
 Veterinary pharmaceuticals are excreted in soils and surface waters by pasture animals.
 The drugs used in livestock can be distributed using manure as a fertilizer.
 Unused drugs are often dumped into public water supplies through sinks, toilets, and
landfills.
 During the manufacturing process, direct pollutants are emitted into the atmosphere.
 The pollution of drugs into the environment also adversely affects the lie of organisms
Impacts of additives
Manufacturing, processing of food additives contributes to Air, water soil pollution and could affect
the environment as discussed above. Chemical contaminants of food including food additives in
excess amount are strongly linked with severe consequences, lack of personal control, and long-

term effects. Food contaminants are also a leading cause of cancer ,it can cause neural and kidney
damage, congenital disabilities, reproductive problems, and can prove to be carcinogenic There is
also the risk of neurodevelopmental disorders like attention deficit disorders, autism, cerebral palsy
and mental retardation caused by fortified industrial chemicals .
SOLVED PROBLEMS
1) Enlist various types of agro chemicals. Give its significance.
a) Solution:Please refer topic agro chemicals
2) Discuss impact of pesticides on Human Health and Environment .
a) Solution:Please refer topic impact of pesticides
3) What are industrial chemicals? Discuss its impact on Human Health and Environment.
a) Solution:Please refer topic industrial chemicals
4) Discuss Biomangiminafication and gives its importance.
a) Solution:Please refer topic Biomangiminafication
5) What are food additives? Enlist any five and give its significance.
a) Solution:Please refer topic food additives
SELF-TEST -MCQS
1) Modern agriculture fundamentally relies on use of
a) Bio pesticides
b) Agrochemicals
c) Bio stimulants
d) None of above
[Answer Key : b) Agrochemicals]
2) Pesticides are classified on the basis of
a) Mode of action
b) Targeted pest species
c) Chemical compositions
d) All above
3) Following are the different types of food additives
a) Antioxidants
b) Fortifying agents
c) Color retention agents
d) All above
[Answer Key :d) All above]
SHORT ANSWER QUESTIONS -SAQS

1) SAQ - Discuss Bioaccumulation
b.Solution: Please refer topic Bioaccumulation
2) SAQ - Discuss with suitable example how pesticides are classified.
b.Solution: Please refer topic pesticides
UNIT 04- 02: SAFETY REGULATIONS- LEGAL CONTROL

LEARNING OBJECTIVES
After successful completion of this unit, you will be able toTo understand effects of pesticides
on environment and human health
 To understand the detailed knowledge of safety regulations and legal control
 To understand population monitoring for toxic end points
 To understand the detailed knowledge of ecological factors
 To understand concept of green chemistry, advantages and disadvantages.
Toxicological Endpoints
These are the values derived from toxicity tests resulted from specific measurements made during
or at the conclusion of the test. Two broad categories of endpoints widely used are assessment and
measures of effect. Assessment endpoints refer to the population, community, or ecosystem
parameters that are to be protected (e.g., population growth rate, sustainable yield). Measures of
effect refer to the variables measured, often at the individual level, that are used to evaluate the
assessment endpoints. The measures of effect describe the variables of interest for a given test. The
most common measures of the effect include descriptions of the effects of toxic agents on survival,
growth, and reproduction of a single species. Other measures of effect include descriptions of
community effects (respiration, photosynthesis, diversity) or cellular effects such as
physiological/histopathological effects (backbone collagen levels, ATP/ADP levels, RNA/DNA
ratios, biomarkers, etc.). In each case the endpoint is a variable that can be quantitatively measured
and used to evaluate the effects of a toxic agent on a given individual, population, or communit y.
The underlying assumption in making toxicological endpoint measurements is that the endpoints
can be used to evaluate or predict the effects of toxic agents in natural environments. EPA risk -
assessment guidelines provide information on how endpoints can be used in the environmental risk-
assessment process

Endpoints most often measured in acute toxicity tests include a determination of the LC or EC50
(median effective concentration), an estimate of the acute no -observed effect concentration
(NOEC), and behavioral observations. The primary endpoint is the LC or EC50. The LC50 is a
lethal concentration that is estimated to kill 50% of a test population. An EC50 measures
immobilization or an endpoint other than death. The LC and EC50 values are measures of ce ntral
tendency and can be determined by a number of statistical approaches. The Litchfield -Wilcoxen
approach is most often used and consists of plotting the survival and test chemical concentration
data on log-probability paper, drawing a straight line thr ough the data, checking the goodness of fit
of the line with a chi-squared test, and reading the LC or EC50 directly off the graph. Various
computer packages are also available to perform this calculation. Other common methods include
the moving-average and binomial methods.
The NOEC (no-observed effect concentration — acute and chronic tests) is the highest
concentration in which there is no significant difference from the control treatment. The LOEC
(lowest observed effect concentration — acute and chronic tests) is the lowest concentration in
which there is a significant difference from the control treatment. The NOEC and LOEC are
determined by examining the data and comparing treatments against the control in order to detect
significant differences via hypothesis testing. The effects can be mortality, immobilization, reduced
cell count (algae), or behavioral observations. These endpoints are typically determined using t -
tests and analysis of variance (ANOVA) and are most often associated with chronic tes ts.
NOECs/LOECs are concentration-dependent and do not have associated confidence intervals.
Sebaugh et al. demonstrated that the LC10 could be used as a substitute for the observed no -effect
concentration for acute tests..This provides a statistically val id approach for calculating the
endpoint and makes it possible to estimate when the lowest concentration results in greater than
10% effects. It should be noted, however, that the confidence in the estimated LC decreases as one
moves away from 50%. Regression analysis, as opposed to hypothesis testing, is gaining favor as a
technique for evaluating both acute and chronic data. The advantage is that it allows for the
calculation of a percentage of the population of test organisms affected, as opposed to ANOVA,
which simply determines whether or not a given response varies significantly from the control
organisms. EC and LC values are readily incorporated into risk -assessment models and are
particularly useful in probabilistic risk assessments.

There are major types of toxicity tests conducted as part of the hazard assessment which may be
required by regulatory agencies/authorities for chemicals, drugs, pesticides, household products,
and/or other substances.
Influence of ecological factors on effect of toxici ty
Ecological factors
These are the environmental factors such as temperature, climate, soil, topography and other related
organisms .Such factorscan significantly affect the toxicity of pollutant - chemicals on living
organisms, which can differaccording to region. Therefore. Ecologi cal risk assessment should be
included with environmentalcharacteristics related to each region.
Assessing the risk of pollutants to animal organisms is typically based on laboratory tests. Usually,
the tests are conducted for single chemical compounds. In reality, conditions in nature vary widely,
leading to inconsistencies between laboratory tests and how organisms are affected by pollutants in
the natural environment. It is known that natural environmental conditions, such as temperature or
drought, can influence how chemical contaminants affect different organisms. Standardized toxicity
tests can therefore only account for a narrow range of possible environmental conditions found
throughout the world. In addition, mixtures of chemicals can interact with each other, and can be
either more or less toxic than if each chemical were to act alone
Basically, bioassay experiments has to be conducted to evaluate the impact of Ecological factors on
effect of toxicity.
e.g,Evaluation of impact oftemperature and har dness on heavy metal toxicity to fresh water
cladoceran i.e. water fleas, Moinamicrura Kurrz,1874. i.e. Filtering grazers of small phytoplankton
.During the study, they have found the toxicity of the tested metals viz.Cd, Cu,Hg ,Zn and Cr
showed a direct correlation with temperature,and an inverse correlation with hardness. However in
case of Hg, the impact was less than that of other metals. (R.Jindal and B.Kaur, 2005)
Concept of Green chemistry
According to the EPA definition, green chemistry is defined as a chemistry that designs chemical
products and processes that are harmless to the environment, thus preventing the formation of
pollution. Chemical products should be made so that they do not remain in the environment at the
end of their application and that they are broken down into components that are harmless to the

environment. Saving based on efficient synthesis without the use of "exotic" reagents, reducing the
required energy, and replacing organic solvents with water are significant even at the l aboratory
level, while in industrial scale possible millions of savings .(Vojvodić, V.2009)
Trends in Green Chemistry (Jukić, M et al.,2005)
Achievement of the main goals of the green program comes through some dominant trends as :
 Research in the field of catalytic and bio catalytic reactions in order to obtain highly
selective, pure compounds without the formation of toxic byproducts;
 Seeking new raw materials, harmless and renewable, such as biomass;
 Designing less toxic eco-compatible chemicals;
 Finding and testing new alternative, non-toxic and renewable reaction media such as water,
ionic liquids and supercritical fluids
 Finding and testing new alternative reaction conditions, such as microwave, ultrasound and
light reacting
 Exploration of alternative routes for the purification of poisoned air and water to improve
their quality, such as photo catalytic reactions
Twelve Principles of Green Chemistry
(https://www.compoundchem.com/2015/09/24/green -chemistry/)
1: Waste Prevention
This tenet simply states that chemical processes should be optimized to produce the minimum
amount of waste possible. A metric, known as the environmental factor (or E factor for short), was
developed to gauge the amount of waste a process created, and is calculated by simply dividing the
mass of waste the production process produces by the mass of product obtained, with a lower E
factor being better. Drug production processes historically had notoriously high E factors, but the
application of some of the other green chemistry principles can help to reduce this. Other methods
of assessing amounts of waste, such as comparing the mass of the raw materials to that of the
product, are also used.
2: Atom Economy

Atom economy is a measure of the amount of atoms from the starting material that are present in
the useful products at the end of a chemical process. Side products from reactions that aren‘t useful
can lead to a lower atom economy, and more waste. In many ways, atom economy is a better
measure of reaction efficiency than the yield of the reaction; the yield compares the amount of
useful product obtained compared to the amount you‘d theoretically expect from calculations.
Therefore, processes that maximize atom economy are preferred.
3: Less Hazardous Chemical Synthesis
Ideally, we want chemicals we create for whatever purpose to not pose a health hazard to humans.
We also want to make the synthesis of chemicals as safe as possible, so the aim is to avoid
using hazardous chemicals as starting points if safer alternatives are ava ilable. Additionally, having
hazardous waste from chemical processes is something we want to avoid, as this can cause
problems with disposal.
4: Designing Safer Chemicals
This principle links closely to the previous one. Chemists must aim to produce chemic al products
that fulfil their role, be that medical, industrial, or otherwise, but which also have minimal toxicity
to humans. The design of safer chemical targets requires a knowledge of how chemicals act in our
bodies and in the environment. In some cases, a degree of toxicity to animals or humans may be
unavoidable, but alternatives should be sought.
5: Safer Solvents & Auxiliaries
Many chemical reactions require the use of solvents or other agents in order to facilitate the
reaction. They can also have a number of hazards associated with them, such as flammability and
volatility. Solvents might be unavoidable in most processes, but they should be chosen to reduce the
energy needed for the reaction, should have minimal toxicity, and should be recycled if possible.
6: Design for Energy Efficiency
Energy-intensive processes are frowned upon in green chemistry. Where it is possible, it is better to
minimize the energy used to create a chemical product, by carrying out reactions at room
temperature and pressure. Considerations of reaction design also have to be made; removal of
solvents, or processes to remove impurities, can increase the energy required, and by association
increase the process‘s environmental impacts.

7: Use of Renewable Feedstock
The perspective of this principle is largely towards petrochemicals: chemical products derived from
crude oil. These are used as starting materials in a range of chemical processes, but are non -
renewable, and can be depleted. Processes can be made more sustaina ble by the use of renewable
feedstock, such as chemicals derived from biological sources.
8: Reduce Derivatives
Protecting groups are often used in chemical synthesis, as they can prevent alteration of certain
parts of a molecule‘s structure during a chemi cal reaction, whilst allowing transformations to be
carried out on other parts of the structure. However, these steps require extra reagents, and also
increase the amount of waste a process produces. An alternative that has been explored in some
processes is the use of enzymes. As enzymes are highly specific, they can be used to target
particular parts of a molecule‘s structure without the need for the use of protecting groups or other
derivatives.
9: Catalysis
The use of catalysts can enable reactions with higher atom economies. Catalysts themselves aren‘t
used up by chemical processes, and as such can be recycled many times over, and don‘t contribute
to waste. They can allow for the utilization of reactions which would not proceed under normal
conditions, but which also produce less waste.
10: Design for Degradation
Ideally, chemical products should be designed so that, once they have fulfilled their purpose, they
break down into harmless products and don‘t have negative impacts on the environment. Persiste nt
organic pollutants are products which don‘t break down and can accumulate and persist in the
environment; they are typically halogenated compounds, with DDT being the most famous
example. Where possible, these chemicals should be replaced in their uses with chemicals that are
more easily broken down by water, UV light, or biodegradation.
11: Real Time Pollution Prevention
Monitoring a chemical reaction as it is occurring can help prevent release of hazardous and
polluting substances due to accidents or unexpected reactions. With real time monitoring, warning
signs can be spotted, and the reaction can be stopped or managed before such an event occurs.

12: Safer Chemistry for Accident Prevention
Working with chemicals always carries a degree of risk. Howeve r, if hazards are managed well, the
risk can be minimized. This principle clearly links with a number of the other principles that
discuss hazardous products or reagents. Where possible, exposure to hazards should be eliminated
from processes, and should be designed to minimize the risks where elimination is not possible.
Disadvantages of Green Chemistry
The basic task of green chemistry is designing such chemical products and processes that reduce or
completely eliminate the use or creation of harmful and dangerous substances. This goal is also the
biggest handicap-lack of green chemistry that is reflected in time, costs and lack of information.
More specifically, switching from an old, conventional product or process to a new "green" product
or process requires a lot of time, design or redesign of a new product and process is often difficult
and quite expensive, and there is also a lack of unity on what is considered safe.
With the high cost of implementation and the lack of information, the lack of green chemistry is
also the fact that there is no known alternative to used chemical raw materials or alternative
technologies for green processes. In addition, there is also a lack of human resources and skills. The
risks of switching to green products and processes are not divided within the supply chain, and
there is a lack of resources for further research. Ionic liquids are considered to be the future of
green chemistry. Although there is no doubt that those are useful in chemical synthesis, the question
is increasingly raised whether they meet expectations. When applying 12 principles that describe
green chemicals, ionic liquids do not look particularly green. There is an opinion that at the present
stage of science progress it is unrealistic to expect that in the next ten years a wide application of
ionic liquids will be seen. Although, as is well known, ionic liquids are slightly volatile due to the
low vapor pressure, yet it is only one of the many things that make a substance really green. For
example, ion based, imidazole-based and fluoro-anion-based liquids are likely to be poisonous but
can not reach the environment by evaporation. The problem is that most ionic liquids are water -
soluble and can easily reach the biosphere through that pathway (Bharadwaj , M. and Neelam,2015;
Anita Ivanković et al.,2017)
The Future of Green Chemistry
Though the tenets of green chemistry might seem simple to implement, improvements can still be
made in a large number of chemical processes. A lot of the chemical products we all utilize come

from processes that still fail to meet a number of these principles; plenty of these products are still
derived from chemicals from crude oil, and many still produce large amounts of waste. There are,
of course, challenges involved in meeting some of the principles in a large number of processes, but
it can also drive new research and the discovery of new chemistry. It is to be hoped that, in the
coming years, many more processes will be adapted with these principles in
mind.(https://www.compoundchem.com/2015/09/24/green -chemistry/)
SOLVED P ROBLEMS
1) What are ecological factors? How it affects toxicity of pollutant.
a) Solution:Please refer topic influence of ecological factors on toxicity
2) What are safety regulations
a) Solution Please refer topic safety regulations
3) Discuss concept of Green Chemistry.
a) Solution Please refer topic Green Chemistry
4) What are disadvantages of Green Chemistry.
a) Solution Please refer topic disadvantages of Green Chemistry
SELF-TEST- MCQS
1) Values derived from toxicity tests resulted from specific measurements made during conclusion of
tests are called as
a) Toxicity constant
b) Toxicological end point
c) Both a and b
d) None of above
[Answer Key: b) Toxicological end point]
2) A chemical which designs chemical products and process for preventing environmental pollution is
called
a) Green chemistry

b) Yellow chemistry
c) Orange chemistry
d) None of above
[Answer Key: a) Green chemistry]
3) Green chemistry has how many principles
a) 10 b) 12 c) 21 d) 8
[Answer Key : b) 12]
SHORT ANSWER QUESTIONS -SAQS
1.SAQWrite note on toxicological end points.
Solution Please refer topic toxicological end points
2.SAQmention all the Principles of Green Chemistry
Solution Please refer topic Green Chemistry
UNIT 04-03: POLLUTION OF ECOSPHERE

LEARNING OBJECTIVES
 To understand industrial pollution of ecosphere by industrial emission

 To understand hazards of dispersion of toxic substances in the environment
 To understand effect of industrial pollutants on health and environment
Industry contributes various kinds of pollutants to the ecosphere Different countries in the world
are facing different types of industrial pollution problems. Industry produces both traditional
pollutants such as organic substances, sulfur dioxide, particulates and nutrients, etc., and newly -
recognized pollutants such as dioxin and other specific toxic substances. The pollutant s are mainly
in gas, water, and solid forms that can cause serious damage to the ecosphere. Industrial pollution
has attracted a lot of attention. In recent years, the sustainable development concept has been
widely recognized, which has promoted the imple mentation of integrated management of industrial
production. The development of ―industrial ecology‖ aims to provide theories and methods to
harmonize the industrial sectors with the biosphere that may bring solutions of sustainable
development to the industry and society. (http://www.eolss.net/sample-chapters/c09/e4-11-02-
00.pdf)

Table. 4.2.Pollutantsfrom Different Industries
Pollutants
Industrialsectors Gas Solid waste Water Others
andsoils
Iron andSteel SO x , NO x , Slag, BOD,COD,oil, Noise,particulat
HC,CO,H 2 S wastes,sludge metals, e
Toxicchemic fromeffluenttr acids,phenol,cy
als eatment anide
Textiles SO x ,HC Sludge BOD,solids, Odor,
andleather (chromium) sulfates noise,particulat
fromeffluenttr andchromium,d e
eatment yes
Pulp andpaper SO x ,NO x sludge BOD,COD, Noise,
fromeffluenttr solids,chlorinat odor,particulate
eatment edorganic
compounds
Petrochemicals,ref SO x , NO x , Spent BOD,COD, oil, Noise,
ineries HC,CO,H 2 S catalysts,tars, phenols odor,particulate
Toxicchemic sludge andchromium
als
Chemicals Organicche Sludge COD, Odor,
micals frompollutio organicchemic toxicchemicals
ntreatment als,
andprocessw heavymetals,
aste solids
andcyanide
(Adapted from: http://www.eolss.net/sample-chapters/c09/e4-11-02-00.pdf)
Table. 4.3.Industrial Emission Sources and ParticulateType

Source Particulate types Origin, occurrence S/U
Fertilizerindustry phosphates, pulverizing,processing,
urea,potassium drying,sintering, WSCy/Rc
chloride,anhydrite, granulating,gases
and othersulfates
Carbo-chemistry coal + Degasifying,gasifying
cokeparticulates, pulverizing BP/En
soot,condensedpro
ducts
Electro-chemistry Metal + Electrolysis in the
oxideparticulates drymethod EP
Calcium-carbide coke, lime, coke pulverization
calciumhydroxide +drying,limesintering Cy/Co
Paintindustry ocher earth, + Pulverizing,dispersing
otherparticles, CyFS
heavy
metalcompound
Biocide-industry insecticides,herbici drying,mixing
des, carriermatter Cy/Rc
Detergentindustry sodium Mixing,

phosphate,soda, dispersing,granulating Cy/Rc
Na-borates
Rubber rubber + Mechanicaltreatment,
andplastics plasticparticles, extracting, FS/FM
talcum,soot; dispersing
other filler
Smelting Ores, coke, metal- Pulverizing,sintering,
,metal oxide-, and throatgas Cy/Cm
slagparticles

Metalprocessing Metalandmetaloxid Converter and
e smelting furnace,waste Cy/Rc
gas
Foundries Metal and metal Smelting furnace
oxidedust,silicates waste gas, moulding FS/Rc
foundry sand
treatment
Bondingagent Raw meal and Raw material WS
andconstruction cementdust, rock extracting,pulverizing, EPCy/Rc
material and mineraldust transporting,firing
Ceramicsand Quartz and Processing +treatment
glassindustry silicates,metal and processesWS FSCy
non-metaloxides
Wood Wooddust,sanding Grinding,sawing,
S = separation: EP = electrostatic precipitator, WS = wet separator Cy = cyclone, FS

=filtrationseparator
U = utilization: Rc = recycling/recirculation, En = Energy generation, FM = fillermaterial, CM =
cement manufacture, Co = construction material, C = composting, A =auxiliaryfiltration material
(Adapted from: http://www.eolss.net/sample -chapters/c09/e4-11-02-00.pdf)
Global Dispersion of Toxic substances
The dispersion of toxic substances in the environment related to accidental releases of hazardous or
toxic chemicals escaping from industrial facilities such as oil refineries, petrochemical plants,
natural gas processing plants, oil and gas transportatio n pipelines, chemical plants, and many other
industrial activities in the event of a sever accident, and its security, social and economic
consequences.
Dispersion and Circulating mechanism of pollutants
Pollutants in the Atmosphere(http://www.eolss.net/s ample-chapters/c07/e2-09-03-05.pdf)
Dispersion is the spread and movement of pollutants. Pollution dispersion depends onfollowing
Factors like wind speed and direction, plume rise, andatmospheric turbulence.

Pollutants disperse with time by mixing with the surrounding cleaner air, resulting in an
increasingly dilute mixture within a spreading smoke plume. Wind and turbulence are
characteristics of the ambient atmosphere, as were described in earlier chapters. While emissions
out of the top of the stack often have strong internal turbulence, this quickly decays, leaving the
ambient atmosphere to do the majority of the dispersing. The direction that the effluent travels is
controlled by the local, synoptic, and global -scale winds. Pollutant destinations from known
emission sources can be found using a forward trajectory along the mean wind, while source
locations of polluted air that reach receptors can be found from a backward trajectory. The goal of
calculating dispersion is to predict or diagnose the polluta nt concentration at some point distant
from the source. Concentration ‗C‘ is often measured as a mass per unit volume viz. µg m –3. It can
also be measured as volume ratio of pollutant gas to clean air,i.e.parts per million (ppm). (Roland
Stull, 2017)
Pollution dispersal in the air
Meteorological conditions (especially wind speed, wind direction and atmospheric stability),the
emission height (e.g. ground level sources such as road traffic or high level sources such as tall
chimneys),local and regional geographical features,the source (e.g. fixed point, such as a chimney,
or a diffuse number of sources such as cars and solvents).
During dispersion pollutants undergo a wide array of changes and transfers. Dilution occurs owing
to mixing into the air. Separation or accumulation of pollutants occurs on the basis of physical
characteristics of the pollutant. Chemical reactions occur, breaking down the original pollutant or
converting it into new compounds. Some pollutants can also be removed from the transporting
medium through deposition, for example, by settling out under the effects of gravity, by rain -wash
or by interception (scavenging) by plants and other obstructions.
Many pollutants therefore show extremely complex dispersion patterns, especially in environ ments
such as cities and towns where there are a large number of emission sources and major variations in
environmental conditions. This complexity means that it is often very difficult to model or measure
pollutant patterns and trends, and thus to predict levels of human exposure.
Temporal variations in pollution levels are important. In many cases long -term trends exist,
reflecting underlying changes in the rates of emission (e.g. as a result of technical or economic
changes, or due to policy intervention). Superimposed upon these there may be annual variations,

reflecting year-to-year differences in climate or source activity. Many pollutants also show marked
seasonal, weekly and daily patterns, owing to cycles of activity and short -term climatic and other
effects. Major, short-term pollution episodes may also occur as a result of sudden, accidental
releases.
Therefore, measurements of exposure will change according to when, where and for how long air
monitoring is carried out. (https://www.eea.europa.eu/ publications/2599XXX/page005.html)
Pollution dispersal in water
The problem under consideration is the mixing of conservative, nonbuoyant pollutants released into
a water bodies. Such pollutants are mixed with the water by different mechanisms, like, mole cular
diffusion, turbulent diffusion, advective transport and
dispersion.(https://doi.org/10.1080/02626668709491162)
Molecular diffusion (http://www.eolss.net/sample -chapters/c07/e2-09-03-05.pdf)
Molecular diffusion is caused by random molecular motion due to the thermal kineticenergy of the
solute. The molecular motion in liquids is smaller than in gases but largerthan in solids. The
coefficient of molecular diffusion is smaller for liquids in a porousmedium than in a pure liquid
because a collision with the solids of the groundwatermedium hinders diffusion. The value of the
coefficient of molecular diffusion dependson the type of solute in the groundwater medium, but for
major anions and cations it usually ranges between 10 -10 and 10 -11 m2 s -1
Turbulent diffusion
It is the transport of mass, heat, or momentum within a system due to random and chaotic time
dependent motions(Hideto Yoshida,2016). It occurs when turbulent fluid systems reach critical
conditions in response to shear flow, which results from a combination of steep concentration
gradients, density gradients, and high velocities. It occurs much more rapidly than molecular
diffusion and is therefore extremely important for problems concerning mixing and transport in
systems dealing with combustion, contaminants, dissolved oxygen, and solutions in industry. In
these fields, turbulent diffusion acts as an excellent process for quickly reducing the concentrations
of a species in a fluid or environment, in cases where this is needed for rapid mixing du ring
processing, or rapid pollutant or contaminant reduction for safety.

However, it has been extremely difficult to develop a concrete and fully functional model that can
be applied to the diffusion of a species in all turbulent systems due to the inabili ty to characterize
both an instantaneous and predicted fluid velocity simultaneously. In turbulent flow, this is a result
of several characteristics such as unpredictability, rapid diffusivity, high levels of fluctuating
vorticity, and dissipation of kinetic energy.( Roberts, P. and Webster, D ,2002)
AdvectiveTransport (http://www.eolss.net/sample-chapters/c07/e2-09-03-05.pdf)
Thetermadvectionreferstothetransportofasolutebythebulkmovem entofgroundwater, the movement of

particles within flowing water. The velocity of the bulkmovement of groundwater is nothing else
than the average linear groundwater
velocity,alsocalledtheadvectivevelocitywhenreferringtothetransportofsolutesinagroundwater
medium. The one-dimensional flux of a solute through a porous
mediumcanbeexpressedbytheequationbelow:
JvxCne
where:
J=mass fluxper unitarea perunit time
vx=averagelineargroundwatervelocityinthedirectionofflow
C=concentrationinmassper unitvolume ofsolution
n e =effectiveporosityofthegeologicalmedium
Dispersion and Diffusion (http://www.eolss.net/sample-chapters/c07/e2-09-03-05.pdf)
Mechanical dispersion is caused by the different flow paths water particles take in ageological
medium (Figure 1). Some of the flow paths are faster because they follow amore direct path or
because they are going through larger pores or through the center of pores in which waterflows
faster because less friction is involved.Other flow paths may be slower because they are closer to
the grain boundaries, thus being exposed tomore friction in the pore throat, slowing down the water
particles. The different flow paths of the water particles cause the mechanical dispersion, a
mechanical mixing and dilution of the solute within the bulk movement of groundwater.
The numerical value of mechanical dispersion is the product of advective ground water velocity and
the dispersivity. The dispersivity is a characteristic property of the geological
medium,and differs in value for each of the spatial components.Forexamples, if all
the pores are nearly the same size, dispersivity of the rock or sediment would below.Dispersivity in

the direction flow is referred to as longitudinal dispersivity(inx-direction),dispersivity
perpendicular to flow is referred to as transverse dispersivity both in a horizontal plane to flow(iny-
direction)and in avertical plane to flow (in z-direction,flow up ordown in a groundwater medium).
It is generally assumed that longitudinal dispersivity is about 10times larger than transverse
dispersivity because the local variation in the velocity field is much moredominant in the direction
of flow rather than perpendicular to it. Transverse dispersivity is primarily caused by flowpaths that
branchout from the center line of solute movement due to the tortuosity of the geological medium.
Concentration distributions(plumes) of a point source form ellipsoids of revolution in three
dimensions. If vertical transverse dispersivity is smaller than horizontal transverse dispersivity, as
is often the case in layered sedimentary rocks,the plumes take on asurf board shape.
SOLVED PROBLEMS
1) Discuss the phenomenon of Diffusion.
a) Solution Please refer topic Diffusion.
2) Discuss the phenomenon of Dispersion
a) Solution: Please refer topic dispersion
3) Discuss phenomenon of Molecular diffusion.
a) Solution: Please refer topic dispersion
SELF-TEST - MCQS
1) The phenomenon of accidental releases of hazardous chemical is called
a) Dispersion
b) Diffusion
c) Adjective transport
d) None of above
[Answer Key : a) Dispersion]
2) The transport of mass, heat or momentum within a system due to random and chaotic time dependent
motions
a) Turbulent diffusion
b) Molecular diffusion
c) Adjective transport
d) None of above
[Answer Key: a) Turbulent diffusion]
3) Molecular diffusion is caused by which motion
a) Molecules
b) Atom
c) Nucleus
d) None of above

[Answer Key : a) Molecules]
SHORT ANSWER QUESTIONS:SAQS
1) SAQ -Write note onTurbulent diffusion
a) Solution: Please refer topic Turbulent diffusion
2) SAQ -Write note on Adjective transport
Please refer topic Adjective transport
UNIT 04-04: DEGRADABLE & NON-DEGRADABLE TOXIC SUBSTANCES &

FOOD CHAIN
LEARNING OBJECTIVES
 To understand effect of industrial pollutants on health and environment

 To understand types and importance of degradable toxic substances
 To understand types and importance of non -degradable toxic substances
 To understand rotation of toxic substances in the food chain
Toxic substances: These can be defined as broad group of chemicals capable of causing harm to
plants and animals including humans as follow:
Table. 4.4. Groups of Toxic Substances
Groups of toxic Details

substances
Atmospheric Contaminants that are emitted to the atmosphere and

deposited upon Vermont‘s watersheds
Organic / Inorganic These are directly or indirectly discharged from

Contaminants (PCBs, municipal and industrial wastewater treatment facilities,
PAHs, Heavy Metals) hazardous waste sites, landfills, storm water runoff, and
historic or ongoing discharges from manufacturing, fuel

and roads
Pesticides Insecticides, herbicides, fungicides, algicides, biocides

used to control nuisances or pests that are applied to land
or directly to waters.
Contaminants of Mostly unmonitored and unregulated chemicals which

Emerging Concern have been recently ―discovered‖ in wastewater
(CECs) discharges, ambient receiving waters, and drinking water
supplies (e.g. pharmaceuticals, personal care products,
industrial and household compounds, nano -technology
products)
Biological Toxic compounds that are produced in nature (e.g.

cyanotoxins)
Biodegradable substance: These substances can be chemically decomposed (broken down to

simpler components) by natural biological processes (e.g. soil bacteria, weather, plants, animalsand
other living organisms). The process by which substanc es biodegrade is called biodegradation. In
this process, biodegradable substances can be broken down into carbon dioxide, water, methane or
simple organic molecules by micro-organisms and other living things by composting, aerobic
digestion, anaerobic digestion or similar processes.

e.g. .Paper and food waste, Human waste, Manure, Sewage sludge, Hospital waste, Slaughterhouse
waste, Dead animals and plants, Food waste etc.
Figure.4.8.Classification Scheme of Biodegradable Materials

Non-biodegradable substances
Non-biodegradable substances cannot be broken down by natural organisms. It cannot be broken

down by the naturally occurring agents, and continue existing on the surface of the earth for a large
number of years. Most of the inorganic substances are n on-biodegradable. Therefore may act as a
source of pollution. The non-biodegradable substances poses a serious threat to the environment and
surroundings also due to their entry in food chain they shows adverse impact on ecosystem and all
the concern organisms.
e.g.Glass,Plastic, Metals, synthetic chemicals, Pesticides,Fibers, E -waste, synthetic rubber-

polymersetc.
Harmful effects of non-biodegradable substances
The non-biodegradable substances are not dismantled and therefore, remain in the environment.
Their accumulation releases harmful gases,
The accumulation of non-biodegradable toxic substances have the following harmful effects:
 They modify the properties of the medium. In the case of landfills the properties of the soil
change with respect to its pH value and fertility.

 It causes an adverse impact on the flora and fauna and ecosystem
 Toxic substances in the water dissolve in the soil profile and then are mixed with the
groundwater. This causes adverse effects on life (plants and animals as well as humans) as
well pollutes the groundwater.
 It affects the water quality in the terms of aquatic life and water micro flora as well as
affects the water index. Therefore, the water becomes unfit for both consumption and neither
is portable.
 Poisonous substances like D.D.T when enter any food chain, being non -biodegradable, they
keep on accumulating progressively at each trophic level. Since humans occupy the topmost
trophic level in any food chain, the maximum concentration of these chemicals can be tr aced
in human bodies due to bio magnification.
Monitoring and assessment Steps for toxic substances to avoid their entry in food chain
 Maintain a database of water bodies with reported cyanobacteria blooms and/or cyanotoxins.
 Expand the cyanobacteria monitoring network to additional waterbodies
 Consider implementation of Advisory Committee on Mercury or other heavy metal pollution

recommendations regarding a fish or other aquatic animal -tissue monitoring program
 Develop a strategy for monitoring / surveyin g CECs and addressing those that reach a
certainthreshold of concern
 Evaluate mechanisms to monitor residential pesticide sales as proxy for use
 Develop a routine monitoring and assessment process for municipal and industrial
wastewater effluents and receiving waters
 Work with USEPA, USGS, and other State ,National/International agencies and partners to
conduct further CEC monitoring.
 Involve a site manager representative from DEC Hazardous Materials Section, and DEC
Solid
 Waste Section in the meetings as a tactical basin plan is started to address the need for
potentially focusing on sediment monitoring, screening salvage yards and other monitoring

needs with respect to sites and toxics in that basin. Include new information gained from
this collaboration into the CEC strategy.
 Evaluate sediments behind dams slated for removal
 Ensure that macro invertebrate and fish community sampling occurs below known
contaminated sites
Food Chain
Every ecosystem works in a systematic manner under natural conditions. It re ceives energy from
thesun and passes it to various biotic components. All ecosystems have a feeding hierarchy which
startswith the energy source i.e. the sun and then followed by producers, consumers and
decomposers.
These components are dependent on one another .In an ecosystem, numerous interactions between
organisms have been resulted into a flow of energy and cycling of matte r. Food chains are examples
of such interactions.
The food chain is the sequence of steps through which the process of energy tr ansfer occurs
in an ecosystem. All organisms need a continuous supply of energy. Energy flows through an
ecosystem in one direction— through food chains
Food chains illustrate the way of energy flows through a sequence of organisms, and transfer of
nutrients from one organism to another.
Food chains usually consist of producers, consumers, and decomposers. If a food chain has more
than one consumer level, its consumers are defined as primary, secondary, or tertiary consumers.
Primary consumers eat plants, secondary consumers eat primary consumers, and tertiary consumers
eat secondary and primary consumers.
The transfer of food energy through a sequence of eating and being eaten by the organisms in an
ecosystem can be termed as the food chain. For example, i n a grassland ecosystem, grass fixes the
light energy from the sun into chemical energy via synthesis of food and eaten up by a grasshopper,
which in turn is eaten by a frog and the frog itself is eaten up by a snake. So, grass is food for
grasshopper, grasshopper is food for frog and the frog is food for snake. Thus, grass, grasshopper,
frog and snake make a food chain, through which energy contained in food is transferred from one
organism to another. In a pond ecosystem, the big fishes eat the small fish es which eat the
zooplanktons which in turn consume phytoplanktons. The later fixes the light energy from the sun

into chemical energy. All the organisms, whether live or dead, are potential food for other
organisms. So, essentially there is no waste in an ecosystem. The sun is the ultimate source of
energy for all food chains. Through the process of photosynthesis, plants use light energy from the
sun to make food energy. Energy flows, or is transferred through the system as one organism
consumes another.
Types of Food Chains:
A. Grazing Food Chain:
The grazing food chain is the major food chain dominantly occurring in ecosystems It starts from
the green plants, the major source of energy for this chain is taken from the sun as plants carry out
the process of photosynthesis in presence of sunlight. The green plants are the primary producer
and eaten up by herbivores, which in turn are eaten up by carnivores. This food chain doesn‘t
consist of microbes or other decomposers; it is carried out by the macroscopic organisms.
Examples Food chain of Forest Ecosystem and Grassland Ecosystem
 Grass → Grasshopper → Frog → Snake → Eagle-------- Grassland
Ecosystem
 Tree → Fruit eating Birds → Eagle---------------------------- Forest
Ecosystem
B. Detritus Food Chain: -
It starts from the dead organic matter such as dead bodies of animals or fallen leaves, which are
eaten by microorganisms and then followed by detritus feeding organisms (and their predators. This
food chain has the remains of detritus as the major source of energy, and this process gets
completed by the subsoil organisms, which can either be macroscopic or microscopic. Thus, these
food chains are less dependent on direct solar energy.
For example, the food chain operating in the decomposing accumulated litter in a temperate forest
ecosystem is a detritus food chain,
Leaf Litter → Bacteria → Protozoa → Small fish → Big fish
The grazing and detritus food chains are shown as separate flows in a Y-Shaped or two channel
energy flow model Fig.4.8. One arm represents the grazing food chain and the ot her arm represents
detritus food chain. The Y-shaped energy flow model further indicates that the two food chains are
in fact, under natural conditions, not completely isolated from one another. For example, dead

bodies of small animals that were once part of grazing food chain become incorporated in the
detritus food chain as do the faeces of grazing food animals. The importance of two food chains
may differ in different ecosystems, in some cases, grazing is more important and in others, detritus
is more important.
Figure.4.8. The Y-Shaped Energy Flow Model Showing Linkage betweenGrazing and
DetritusFoodChain.
Significance of Mechanism of bio magnification or bioaccumulation Bio concentration:
 Bio magnification:It is the process whereby certain substances such as pesticides orheavy
metals become concentrated in successive trophic levels in a food chain.
 Bio accumulation: It occurs within a trophic level and is the increase in the concentration
of a substance in certain tissues of organisms‘ bodies due to absorption from food and the
environment.
 Bio concentration-It occurs when uptake from the surrounding environment isgreater than
excretion.
Generally, fat soluble substances (e.g. DDT,Mercur y, Lead etc.)cannot be diluted or broken down
and accumulate in fatty tissues of anorganism.When it is consumed by another organism,fats along
with the toxic substances are absorbed in the gut,and get accumulated in the fats of the
predator.Since at each level of the food chain there is a lot of energy loss,a predator must consume
many preys,including all of their fat soluble substances.
Significance of FoodChains

The food chains help understand the feeding relationships and the interactions between organisms
in any ecosystem.
1. Nutrient cycling and energy flow in an ecosystem takes place through food chains
2. It keeps a check on the population size of different organisms. For example, in a

foodchain in grassland, if deer population increases, there will be more food for the
carnivores, their population will increase, which in turn will reduce the deer population.
If there are less deer, some of the carnivores will starve and die, letting the deer
population to grow.
3. Each species of any ecosystem is kept under a natural check so that the system remains
balanced.For instance ,if the primary consumers (herbivores)had not been in nature the
producers would have been perished due to overcrowding and competition.Similarly,the
survivalof primary consumers is linked with the secondary consumers(carnivores)and so
on.
4. The study of food chain helps us to understand the problems of bio magnifications.
Sometimescertain toxic substances (biodegradable/ non -biodegradable toxic substances),
instead of dispersing, get concentrated at each level in the food chainandarereferred toas
bio-magnifications orbio-accumulation.
SOLVED PROBLEMS
1) What are toxic substances? Comment on bio degradable substances .
a) Solution:Please refer topic food
2) Discuss food chain and give its importance.
a) Solution:Please refer topic food chain
3) Discuss assessment steps to avoid entry of toxic substances in food chain.
a) Solution: Please refer topic food chain
SELF-TEST -MCQS
1) The substance which can be chemically decomposed by natural biological process are referred as
a) Biodegradable substance
b) Non-biodegradable substance
c) Both a and b
d) None of above
[Answer Key: a) Biodegradable substance]

2) The sequence of steps through which the process of energy transfer occurs in the ecosystem is defined
as
a) Food web
b) Food chain
c) Both a and b
d) None of above
[Answer Key: b) Food chain]
3) When uptake of toxic substance from surrounding environment is greater than excretion it is called as
a) Bio accumulation
b) Bio magnification
c) Bio concentration
d) None of above
[Answer Key: c) Bio concentration]
Short Answer Questions SAQs
1) SAQ -Write note on Bio magnification
a.Solution: Please refer topic Bio magnification
2) SAQ -Give the Classification scheme of Biodegradable materials
a. Solution: Please refer topic Biodegradable materials
SUMMARY
First section of this unitpresents the overview of environmental toxicology describing various toxic
agents in environment pesticides, agrochemicals and their classification along with their chemical
structure and function relationship. It also highlights on the industrial chemicals, drugs and their
impacts on health and environment .It illustrates various types of food additives and their
significance.
Second section of this unit presents the overview of Safetyregulations, legalcontrol,

population monitoring for toxic end points .It gives brief description of influence of various
ecological factors on effect of toxicity. It highlights on the trends in Green Chemistry along with
the twelve principles of Green Chemistry, disadvantages of Green Chemist ry alongwithits future
scope .
Third section of this unitpresents the overview of Pollution of the ecosphere by industries
with detail information of pollutants from different industries .It also highlightes on few important
industrial emission sources and particulate type.It gives description of dispersion and circulating
mechanisms of pollutants. Besides it explains the pollution dispersal in water and air.

Fourth section of this unit presents the overview of Degradable and non - degradable toxic
substances alongwith the Classification scheme of Biodegradable materials .It explain various
groups of toxic substances and their harmful effects. It illustrates the monitoring and assessment
steps for toxic substances to avoid their entry in food chain. At t he end it gives illustration of
different types of food chains along with their significance.
KEY WORDS
Agrochemicals ,Drugs, Food Additives, Safety Regulations, ToxicEndPoints, Green Chemistry ,
Ecosphere,Dispersion ,DiffusionFood Chain, BioMagnification
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Acknowledgement
Authors are grateful to Mr.Dilip V.Handore, Head of Department, Research
andDevelopmentDepartment, Sigma Wineries Pvt. Ltd. Nashik andAssit.Prof.Mr.Pushkar Padekar,
H.A.L. College of Science & Commerce, Ozar, Nashik. for technical assistance
Our Special thanks to Ms. Vinita S. Jagtap,Scientific Officer, Medical Oncology Molecular Laboratory,
and Tata Memorial Hospital.,Mr. V.C. Handore, Mrs. Hira V. Handore, Mr.Abhijeet Jagtap and
Ms.Mrunal Ghayal forconsistent valuable support.

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School of Architecture, Science and Technology,

Yashwantrao Chavan Maharashtra Open University

V135: Diploma in Environmental Science

M.Sc. Environmental Science

Microbiology V136: M.Sc. in

EVS033: Environmental Microbiology and Toxicology Page 1

Maharashtra Open University Environmental Microbiology

EVS033: Environmental Microbiology and Toxicology Page 2

Yashwantrao Chavan Maharashtra Open University

This work by YCMOU is licensed under a CreativeCommons Attribution-

EVS033: Environmental Microbiology and Toxicology Page 3

EVS033: Environmental Microbiology and Toxicology Page 4

- Dr. Sunanda More

EVS033: Environmental Microbiology and Toxicology Page 5

UNIT 01-01: ENVIRONMENTAL MICROBIOLOGY

 To understand the history and role of various scientists to develop knowledge in

 To understand the mechanism and role of microorganisms

EVS033: Environmental Microbiology and Toxicology Page 6

(Adapted without modification from:https://fac.ksu.edu.sa/sites/default/files/140_mbio -

EVS033: Environmental Microbiology and Toxicology Page 7

 Francesco Redi (1626-1697)

 John Needham (1713-1781)

 Lazzaro Spallanzani (1729-1799):

 Schulze (1815-1873) and Theodor Schwan (1810-1882)

EVS033: Environmental Microbiology and Toxicology Page 8

 George Schroeder and Theodor Von Dusch (1854)

Golden Era of Microbiology (https://microbenotes.com/history-of-microbiology/)

 Louis Pasteur-Father of Modern Microbiology / Father of Bacteriology

EVS033: Environmental Microbiology and Toxicology Page 9

Koch’s four postulates are:

 The organism can be isolated and grown in pure culture.

 John Tyndall (1820 – 1893):

 Fanne Eilshemius Hesse (1850 – 1934)

EVS033: Environmental Microbiology and Toxicology Page 10

The Spectrum of Microbiology

Classification scheme of Microorganisms

EVS033: Environmental Microbiology and Toxicology Page 11

[Adapted without modification from: https://www.thoughtco.com/what-are-prokaryotes-and-eukaryotes-129478]

 Protozoa: These are eukaryotic, unicellular organisms. Motion is a characteristic associated

 Algae: This implies a variety of plantlike organisms. In microbiology, several types of

EVS033: Environmental Microbiology and Toxicology Page 13

Pure Microbiology Applied Microbiology Other Microbiology

 Bacteriology  Medical microbiology  Astro microbiology

Scope of Microbiology (Moselio Schaechter et al.,2003)

 Identifying new and better drug targets for treating disease.

EVS033: Environmental Microbiology and Toxicology Page 14

 Exploring the mechanisms of spread of antimicrobial resistance.

 Determining the pools and fluxes of small molecules within cells.

 Determining how microbial cells in a population differ in structure and function.

 Defining the stability or ―health‖ of naturally occurring microbial communities.

EVS033: Environmental Microbiology and Toxicology Page 15

 Microbiology materials and data must be more carefully curated.

 Isolation and cultivation of uncultivable bacteria are still challenge to microbiologist

EVS033: Environmental Microbiology and Toxicology Page 16

SHORT ANSWER QUESTIONS -SAQ

UNIT 01-02: MICROBIAL DIVERSITY, MICROBIAL ACTIVITIES &

 To understand classification of microorganisms on nutrition, photosynthesis and Nitrogen

 To learn various methods of determination of microbial numbers

 To learn various bio-chemical tests for microorganisms

Microbial structure (https://fac.ksu.edu.sa/sites/default/files/140_mbio -final_notes.pdf)

Major groups of Microorganisms are as:

A bacterial cell includes: