EV4102 – ENVIRONMENTAL MICROBIOLOGY

UNIT – 1 FUNDAMENTALS OF MICROBIOLOGY

1. Microbiology:
• Microbiology is the study of all living organisms that are too small to be visible with the
naked eye.
• This includes bacteria, archaea, viruses, fungi, prions, protozoa, and algae, collectively
known as 'microbes'.
• These microbes play key roles in nutrient cycling, biodegradation/biodeterioration, climate
change, food spoilage, the cause and control of disease, and biotechnology.

2. Classification of Microorganisms:
The chart below shows how microorganisms are related,
• The three most general groups into which the organisms are placed are prokaryotes,
eukaryotes, and non-living organisms.
• Prokaryotes are more primitive organisms than eukaryotes.
• Only bacteria are prokaryotes; the rest of the organisms considered in this course are either
eukaryotes or viruses.

2.1 Prokaryotes:

Introduction:

• Prokaryotes are organisms which do not contain nuclei or membrane-bound organelles.

• All prokaryotes are unicellular, which means that each organism is made up of only one
cell.
• Another trait common to all prokaryotes is their small size - a typical cell is only about 2 um
long (A micrometer, abbreviated as um and sometimes known as a micron).
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 • It would take about 13,000 prokaryotes lying end to end to stretch the length of one
inch. Under a light microscope, bacteria are so small that they are usually visible only as
tiny dots.
• Although there are two kingdoms which contain prokaryotes (Eubacteria and
Archaebacteria), all prokaryotes are commonly known as bacteria. In the past, some
prokaryotes have been called blue-green algae, but these organisms are now known as
cyanobacteria.

Importance:

• Bacteria are present in large numbers in raw wastewater, in biological treatment plants, in
plant effluent, in natural waters, and throughout our environment.
• In the wastewater treatment plant, they form part of the slime on trickling filters and on the
discs of rotating biological contactors. They are also present in activated sludge.
• Bacteria are heterotrophs, meaning that they get their food from eating other organisms or
from eating organic matter. (In contrast, organisms like plants which make their own food
are known as autotrophs.)
• As a result, bacteria are important to the wastewater operator since the bacteria are able to
digest a large amount of the waste in wastewater.
• On the other hand, some bacteria get their food from living inside organisms such as humans,
in which case they can cause disease.

Cell Structure:

• A cell is the fundamental unit of all life. In the case of unicellular organisms, a cell is the body
of the organism. In the case of multicellular organisms (organisms which consist of more
than one cell), the cell is the building block from which the organism's body is made. The
diagram below illustrates a typical bacterial cell. As with every other kind of cell,
a membrane serves as a sac holding the parts of the cell together. The membrane also
regulates what passes into and out of the cell.

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 • Inside the membrane, the cell is filled with a fluid known as cytoplasm. Floating in the
cytoplasm are various organelles (subcellular structures with specific functions.) Notice
that the DNA, which contains the genetic material of the cell, is floating freely in a mass
within the cell. In addition to the main mass of DNA, the bacterial cell contains plasmids,
which are small loops of DNA which can be transferred to other bacteria, or in some cases
into other organisms. Ribosomes are the sites of protein synthesis.
• Outside the membrane, most bacteria are surrounded by two other layers. The first of these,
the cell wall, is a rigid layer made up of proteins, polysaccharides, and lipids. The cell wall
gives the bacterium a set shape. Outside the cell wall is the capsule, a gelatinous slime layer
which allows the bacterium to attach to surfaces and protects the bacterium. In the
treatment plant, bacterial capsules are responsible for clumping the organisms into flocs, or
aggregations, which can settle out of water. For disinfecting agents such as chlorine to be
effective, they must penetrate this protective slime layer.
• The bacterium can also have various appendages. Pili are hollow, hair-like structures which
allow the bacterium to attach to other cells. Flagella are longer projections which can move
and push the bacterium from place to place.

Endospores:

• Some bacteria can survive in harsh environments by forming endospores. Endospores are
small spores which develop asexually inside the bacterial cell. An endospore consists of the
bacterium's DNA surrounded by a protective cell wall.
• Once the endospore has formed, the parent cell bursts open and releases the endospore.

An endospore can survive in very harsh environments because it is in a dormant state and
does not attempt to eat, grow, and reproduce.
• Bacteria typically form endospores when they encounter an undesirable pH, electrolyte
content, amount of food, or amount of oxygen in the environment.
• Once the environmental conditions improve, the endospore is able to germinate and turn
back into a growing bacterial cell.

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Classification:

• There are thousands of species of bacteria on earth, many of which have not yet been
identified.
• When attempting to classify a bacterium, a variety of characteristics are used, including
visual characteristics and laboratory tests.
• Some bacteria can be identified through a simple visual perusal. First, the operator
considers the appearance of the bacterial colony (a group of the same kind of bacteria
growing together, often on a petri dish.) The operator also views individual bacteria under
a microscope, considering their shape, groupings, and features such as the number and
location of flagella.
• A variety of laboratory techniques can be used to narrow down the identity of a bacterial
species if a visual survey is not sufficient. The operator can stain the bacteria using a gram
stain or an acid-fast stain.
• The bacteria can be cultured on a specific medium which promotes the growth of certain
species, as in the membrane filter method of testing for coliform bacteria. Other tests can
detect bacterial by-products, while yet more advanced tests analyze the DNA of the bacteria.

Bacterial Shape:

The most basic method used for identifying bacteria is based on the bacterium's shape and cell
arrangement. The three morphological categories which all bacteria fall into - cocci, bacilli, and
spirilla. These categories are merely a way of describing the bacteria and do not necessarily refer
to a taxonomic relationship.

Cocci (or coccus for a single cell) are round cells, sometimes slightly flattened when they are
adjacent to one another. Cocci bacteria can exist singly, in pairs (as diplococci), in groups of four
(as tetrads), in chains (as streptococci), in clusters (as stapylococci), or in cubes consisting of
eight cells (as sarcinae.)

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Bacilli (or bacillus for a single cell) are rod-shaped bacteria. Since the length of a cell varies under
the influence of age or environmental conditions, you should not use cell length as a method of
classification for bacillus bacteria. Like coccus bacteria, bacilli can occur singly, in pairs, or in
chains. Examples of bacillus bacteria include coliform bacteria, which are used as an indicator of
wastewater pollution in water, as well as the bacteria responsible for typhoid fever.

Spirilla (or spirillum for a single cell) are curved bacteria which can range from a gently curved
shape to a corkscrew-like spiral. Many spirilla are rigid and capable of movement. A special group
of spirilla known as spirochetes are long, slender, and flexible.

2.2 Eukaryotes

Introduction

Except for bacteria and viruses, all other organisms are eukaryotes.

Eukaryotes are unicellular or multicellular organisms which contain a nucleus and membrane-
bound organelles. A nucleus is a membrane sac within the cell which holds all the cell's
DNA. Membrane-bound organelles within the cell can include chloroplasts, mitochondria, and
several other organelle types.

The diagram above shows some of the parts found in a typical eukaryotic cell. Since there are so
many kinds of eukaryotes, several of the parts shown in the cell above will not be present in certain
species. Also, some species may contain additional parts not shown above.

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As you can see, eukaryotic cells are always bounded by a membrane, just as prokaryotic cells
are. Some eukaryotic cells are also surrounded by a cell wall, but eukaryotic cells do not have
capsules. Although they are not shown in the diagram above, eukaryotic cells can have
protuberances such as flagella or cilia (tiny hairs which typically form a fringe all the way around a
cell.)

Like the prokaryotic cell, the eukaryotic cell is filled with cytoplasm. Ribosomes and various other
organelles can be found floating in the cytoplasm. The two additional organelles shown in the
diagram above are membrane-bound and are found only in eukaryotic
cells. Mitochondria (mitochondrion when referring to a single organelle) are present in nearly all
eukaryotic cells and produce the cell's energy by breaking down food.

Chloroplasts, in contrast, are present only in plants and algae and are used in photosynthesis, the
process through which the organism uses energy from the sun to build sugars.

The various types of eukaryotes typically found in water and wastewater,

Fungi:

Fungi are organisms which typically cannot move, which cannot make their own food
(heterotrophic), and which contain a chemical known as chitin in their cell walls. They can be
multicellular or unicellular, with the unicellular organisms having relatively large cells.

Although some fungi live in salt or fresh water, most fungi are terrestrial. Many species
are saprophytic, feeding on dead organic matter. Others are parasites which live inside or on host
animals, primarily feeding on plants though a few also live on animals. The aquatic fungi are
important in treating wastewater.
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Classification of fungi is based primarily on reproductive structures, with all the aquatic fungi being
found in the Mastigomycota group. We use several common names to refer to groups of fungi, but
these groupings refer only to morphology and not to any relationship or scientific
classification. Yeast are single-celled fungi, molds are filamentous fungi consisting of multiple cells
in threads known as hyphae, and mushrooms are the fruiting bodies of filamentous fungi.

Algae:

Introduction:

Algae are distinguished from animals, fungi, and protozoans by their ability to make their own food
through photosynthesis and are distinguished from plants by their relative simplicity of
structure. All algae contain the green pigment chlorophyll and the organelles chloroplasts, both of
which are essential for photosynthesis.

Algae may be either unicellular (in which case they are known as phytoplankton) or
multicellular. The algae which are important to water treatment are generally unicellular. All algae
contain a rigid cell wall and some also have sheaths (or thin gelatinous coatings) outside the cell
wall. Algae may be non-motile, but many are able to move using a flagella, in which case they are
known as flagellates (a term based on morphology rather than taxonomy.)

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Importance:

Most algae are aquatic, living in salt or fresh water, though a few live in soil or on the bark of trees. In
natural waters, algae are an important source of food for other organisms. They also produce oxygen
during photosynthesis, adding to the dissolved oxygen content of the water during the day.

Since algae require light for growth, they are restricted mostly to the top surfaces of trickling filters
and ponds. They are seldom found in large numbers except in tertiary treatment units, such as clear
wells, and usually are not important for water treatment. However, in oxidation ponds, the algae
may represent a substantial portion of the microorganism population and may play a significant role
in treatment.

Algae Bloom:

Algae can be problematic in nutrient-rich waters, especially those containing phosphorus, in which
case they often reproduce rapidly and produce colored water and mats of algae known as algae
blooms. In natural waters or treatment plants, algae blooms are problematic because they can
change many water characteristics.

One of the primary factors which algae blooms influence is the dissolved oxygen content of the
water. During the day, the masses of algae produce so much oxygen that the water becomes
supersaturated. Then, at night, the algae use up oxygen in the water and can cause such extremely
low dissolved oxygen levels that fish kills may result.

Algae blooms can also cause elevated pH levels in the water. They may raise the pH levels as high as
9.5, which will influence many of the natural processes occurring in the water.

In some cases, an algae bloom will consume itself. As the algae grow and reproduce, they use up
nutrients in the water. Eventually, the nutrient levels will drop so low that the algae will have no
nutrients and will die back. In this case, the dead algae bodies will often promote a bacterial bloom
as the bacteria respond to the abundance of food. This overabundance of bacteria can cause yet more
problems, depleting the dissolved oxygen levels in the water and causing the system to become
anaerobic.

The best method of dealing with algae blooms is prevention. By limiting the nutrient levels in
wastewater effluent, algae blooms in the receiving waters can be avoided. The limitation on
phosphorus in some wastewater effluents in New York State stems directly from this concern.

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Classification

Cellular properties, the nature of the cell wall, and the arrangement of flagella all influence the
classification of algae species. However, the most important factor in algae classification is the types
of photosynthetic pigments present in the cell. Although all algae contain chlorophyll, some species
also contain xanthophylls (which are yellow) or carotenoids (which are orange.) The three phyla
of algae which are commonly found in fresh water are described below.

Phylum Chlorophyta consists of green algae which typically do not contain pigments other than
chlorophyll. Chlamydomonas, shown in the picture above, is a typical green alga. Notice that this
species has two flagella and is thus able to swim freely through the water. (When looking through a
microscope, you should be aware that the algae's flagella are only visible under extremely high
magnification. Also note that some species in phylum Chlorophyta do not contain flagella and are not
mobile.) Each Chlamydomonas cell contains a single, large chloroplast which fills up most of the
cell. It also contains an eyespot which is a light-sensitive organelle which helps
the Chlamydomonas know how to swim toward the light.

Phylum Euglenophyta contains green or colorless flagellates commonly known as euglenoid

algae. Some scientists consider euglenoid algae to be protozoans because of the species which do not
contain chloroplasts and which are able to feed on organic matter and other microorganisms in the
water. A typical euglenoid is Euglena, shown above. Euglenoids usually have a single flagellum and
several chloroplasts.

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Phylum Chrysophyta contains golden-brown algae which contain carotenoids as a major
pigment. Although there are several different kinds of algae in phylum Chrysophyta, one group - the
diatoms - is especially important in water treatment. Diatoms are algae which form a variety of
intricate shapes and which contain silicon in their cell walls. Although other algae in the phylum
Chrysophyta have flagella, diatoms are either nonmobile or glide slowly along surfaces.

In addition to the types of algae mentioned above, there are several other phyla which are found
primarily in salt water. Phylum Pyrrhophyta contains algae known as dinoflagellates, phylum
Phaeophyta contains brown algae, phylum Rhodophyta contains red algae, and phylum Cryptophyta
contains blue and red flagellates.

Protozoa:

Introduction

Protozoa are unicellular organisms which are heterotrophic and are mobile at some stage in their
life. They do not have a cell wall, although their membrane is often surrounded by a pellicle (a thin,
flexible, protective coating). A few protozoa give their cells rigidity by producing shells made out of
calcium carbonate or silicon.

Protozoa are important in both water and wastewater treatment. They are responsible for several of
the water-borne diseases. In addition, protozoa help break down waste in aerobic wastewater
treatment plants.

Classification

Protozoa are divided into four groups based on their method of locomotion. Scientists initially
believed that these groups actually represented taxonomic relationships, but now many scientists
suspect that the taxonomy of protozoa is much more complicated. For the sake of simplicity, we will
use the old method of classification here, based on mode of locomotion.

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Amoebae, like those shown above, are protozoans which move by extending finger-like protrusions
of their cells called pseudopodia. An amoeba can also use its pseudopodia to engulf a food particle
in a process known as endocytosis, bringing the food inside the cell where it can be digested. You
can see several engulfed food particles as circles within each amoeba cell above. Although most
amoebae are free-living, one species is the cause of amoebic dysentery.

Flagellates are protozoa which move using flagella. This is a very diverse group which is considered
by some scientists to include the euglenoid algae. An example of a flagellate is Giardia which is found
in many natural waters and causes giardiasis when ingested. As you can see in the picture
above, Giardia contains two nuclei, a trait shared by several other protozoa.

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Ciliates are protozoa which use the motion of tiny hairs, called cilia, to propel them through the
water. Ciliates are usually found in large numbers in natural waters and in sewage where they act as
predators and scavengers, ingesting food through "mouths." A few species are parasitic, living inside
hosts. The image above is an example of a Paramecium, which is a typical ciliate protozoan. Notice
that the Paramecium has both a large and a small nucleus, a trait typical of ciliates.

2.3 Animals:

Introduction:

In addition to the microorganisms mentioned above, a few multicellular organisms are important in
water and wastewater treatment. In this section, we will consider two types of important
multicellular organisms - rotifers and worms.

Rotifers:

Rotifers are extremely simple multicellular animals like those shown above. Although a few rotifers
can be found in saltwater, most are found in fresh water. Each rotifer is made up of about a thousand
cells and is no larger than some unicellular organisms.

Rotifers can be identified based on the wheel of cilia found at one end of their bodies. These cilia move
in a circular motion in order to draw food into the rotifer's mouth. Some species live out their lives
attached to a surface while others use the movement of their cilia to propel them through the water or
use the their foot to creep along surfaces.

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Rotifers are quite common in aerobic, biological wastewater treatment processes. They can be found
either grazing on smaller microbes or attached to debris by their forked tail. Since rotifers eat free-
swimming bacteria and organic matter, they help clean water in activated sludge processes.

Worms:

Worm is a term used to refer to several unrelated organisms with a long, skinny body shape. The two
types of worms commonly found in water are nematodes and flatworms.

Nematodes can be distinguished from other aquatic worms by their cylindrical body and by the typical
S-shaped, wriggling movement which they use to propel themselves through the water. Most are
microscopic, but a few can grow much larger, up to several feet in length. Nematodes may live in water,
in soil, or as parasites. There are several species of parasitic nematodes which attack humans, but these
species are believed to be associated with contaminated food rather than with contaminated water.

The free-living aquatic nematodes eat bacteria, rotifers, and other worms and can be very important in
wastewater treatment. Nematodes are abundant in sludge and are especially common in percolating
filters. They help stabilize sludge as well as controlling film accumulation in percolating filters. In
general, nematodes are very good at dealing with moderate pollution.

Flatworms, on the other hand, are worms with flattened bodies which glide along surfaces using tiny
cilia. The flatworm illustrated at the beginning of this section is a planaria, which is a common type of
flatworm. Notice the two eyespots found on the planaria - this is another feature which can be used to
help identify planaria.

Flatworms are usually found at the lower depths of ponds. They range in size from a fraction of a
millimeter to several centimeters in length. Some flatworms live as parasites while other are free-
living, feeding principally on algae.

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2.3 Viruses

Introduction

Viruses are non-living organisms which can only reproduce in a living host cell. As a result, all viruses
are obligate parasites, and all cause some sort of disease. Infectious hepatitis, polio, influenza, smallpox,
AIDS, and a variety of intestinal disturbances are all caused by viruses.

Viruses can attack many kinds of organisms ranging from bacteria through plants and animals, though
each type of virus is specific in its type of host.

For example, a plant virus will not attack an animal and a dog virus is unlikely to attack a human.

Viruses are too small to be to be seen with a light microscope, so their presence is usually recognized
only by the harm they cause. They often enter water in animal feces and are thus expected to be present
in domestic wastes.

In addition, viruses can often survive for long periods of time in natural waters. Viruses are a public
health concern in water and wastewater treatment since many are not removed by conventional
treatment methods such as disinfection.

Structure of viruses

Viruses are very simple organisms consisting primarily of genetic material (which can be either DNA
or RNA) enclosed within a protein coat called a capsid. The genetic material can have a variety of
forms, being either double-stranded or single-stranded and either circular or linear. The capsid coat
can have several shapes, including spherical and icosahedral (20-sided), and may further be
surrounded by an envelope. The envelope is made up of lipids and is usually imbedded with envelope
proteins which help the virus recognize its host cell.
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As you can see in the picture above, there are several different kinds of viruses. The virus on the left is
a typical bacteriophage, which is a virus that infects bacteria. Bacteriophages have complex tails
which are used to attach to and inject DNA or RNA into the host cell. The cell on the right is a typical
animal virus and has a much simpler structure.

Reproduction:

The animation above shows how a virus reproduces. First, the virus attaches to receptors on the host
cell using its envelope proteins. Then the virus inserts its DNA or RNA into the host cell. This second
step can be achieved through fusion (as shown in the animation), through endocytosis (which is a
process in which the host cell's membrane engulfs the virus), or through direct penetration (a common
process in bacteriophages in which the virus's DNA or RNA is injected into the host cell through the
virus's tail.)

If the virus's genetic material is RNA, then the RNA must be turned into DNA in the host cell's
cytoplasm. The viral DNA then makes its way into the host cell's nucleus. There, the viral genetic
materials sliced in to the host cells DNA.

A cell's DNA tells the cell how to perform cell processes, such as making proteins and new DNA. So,
once the virus's DNA has been inserted into the host's DNA, the viral DNA can tell the cell to produce
new viral parts - DNA, proteins, and lipids.

After the cell produces the viral parts, the virus DNA tells the cells to assemble the viral parts in to new
viruses known as virions.

Once the cell is full of virions, the virions are released into the environment. The animation shows one
method of virion release common in simple host cells, in which the host cell lyses (or breaks apart). In
other types of host cells, the virions may simply bud out of the cell. In either case, the new virions float
through their environment until they find a new host and can repeat the reproductive cycle.

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In some cases, the virus's reproductive cycle also has a lysogenic phase in which the virus is dormant
within the host cell for a time. The viral DNA enters the host cell and splices into the host DNA, but does
not immediately begin producing new viruses. Instead, the host cell is able to grow and reproduce
normally. As the host cell reproduces, however, it copies the virus's DNA as well as its own DNA. After
the host cell has reproduced several times, all of the daughter cells begin to produce new viruses.

As you read through the description of viral reproduction above, you may have noticed that the virus
was not active in much of the reproduction process. After the virus inserted its genetic material into
the host cell, the host cell did all of the work of making new viruses. This dependence on a living
organism for reproduction is one of the reasons that many scientists consider viruses to be non-living.

Reference: Lesson 2: Classification of Microorganisms (mecc.edu)

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