Handout on

Microbiology of sewage and wastewater

WHAT IS SEWAGE?

Sewage is the wastewater of a community. It may be purely domestic in origin or it may contain
some industrial or agricultural wastewater as well. Initially we will consider only domestic
sewage. This is composed of human body wastes (feces and urine) and sullage which is the
wastewater resulting from personal washing, laundry, food preparation and the cleaning of
kitchen utensils.

Sewage contains various types of micro-organisms and bacteria such as virus, protozoa, algae,
fungi etc. Some of these organisms are pathogens and are harmful for animal and human life.

DEFINITION OF MICROBIOLOGY

Microbiology (from Greak mkros, "small"; bios, life"; and logia is the study of microscopic
organism, those being unicellular(single cell multiceluller (cell colony), or acelluer (lacking
cells).

Microbiology is the study of microscopic organisms, such as bacteria, viruses, archaea, fungi and
protozoa. This discipline includes fundamental research on the biochemistry, physiology, cell
biology, ecology, evolution and clinical aspects of microorganisms, including the host response
to these agents.

BRANCHES OF MICROBIOLOGY

The branches of microbiology can be classified into

pure and
applied sciences.

Pure microbiology

Bacteriology (Study of bacteria)

Mycology (Study of fungi)

Protozoology (Study of protozoa)

Phycology

Parasitology

Immunology

Virology (Study of viruses)

Nematology
 Microbial cytology

Microbial physiology

Microbial ecology

Microbial genetics

Cellular microbiology

Evolutionary microbiology Generation microbiology

Systems microbiology

Molecular microbiology

Nano microbiology

Exo microbiology

Biological agent

Agrology (Study of algae)

Applied microbiology

Medical microbiology
Pharmaceutical microbiology
Industrial microbiology
Microbial biotechnology
Food microbiology
Agricultural microbiology
Plant microbiology and Plant pathology
Soil microbiology
Veterinary microbiology
Environmental microbiology
Water microbiology
Aeromicrobiology
MICROBES AND ITS IMPORTANCE IN SEWAGE TREATMENT

The important microbes are bacteria, viruses, algae and protozoa. Microbes have a more positive
role to play in sewage treatment. Microorganisms are beneficial for microbial biodegradation or
bioremediation of domestic, agricultural and industrial wastes and subsurface pollution in soils,
sediments and marine environments.

BACTERIA

The word bacteria is the plural of bacterium. Bacteria are tiny living beings microorganisms.
Bacteria are tiny single-cell microorganisms, usually a few micrometers in length that normally
exist together in millions.

Characteristics of Bacteria
1. Single celled organisms
2. Very small
3. Need a microscope to see
4. Can be found on most materials and surfaces
5. They are prokaryotic organism i.e. their nucleus are not bounded by membrane
6. Some are free-living organisms and some are parasitic. Free-living bacteria use flagella for
movement
7. They are unicellular organisms
8. Some are toxins
9. They can exist as single cells, in pairs, chains or clusters
Types of Bacteria

Bacteria are usually classified three into groups (according to the temperature they flourish)

(i) Psychrophilic This bacteria can persist at low temperature only. They normally flourish
within temperature range of 100C 200C.

(ii) Mesophilic bacteria this bacteria can thrive within temperature range of 200C-450C .

(iii) Thermophilic bacteria This bacteria is most effective in temperature range of 450C-650C.

According to the oxygen demand

Aerobic bacteria - Bacteria that need oxygen for their survival.

Anaerobic bacteria - Bacteria that do not require oxygen for survival.

Facultative bacteria This bacteria can live and multiply with or without oxygen.

According to process of their life

(i) Parasitic bacteria It is such a bacteria which lives and multiplies on or within
the body of a living organism.

(ii) Saprophytic bacteria This bacteria lives and multiplies on decaying organic
matters.

According to their disease producing characteristics

(i) Pathogenic bacteria This bacteria is the cause of diseases like typhoid, dysentery, cholera
etc. This is mostly intestinal bacteria. Their presence in excreta which ultimately joins the rivers
will definitely contaminate water of the river.

(ii) Non-pathogenic bacteria This is the bacteria which do not caused any disease. For
example coli form bacteria.

Coliform Bacteria

Coliform bacteria are present in the environment and feces of all warm-blooded animals and
humans. Coliform bacteria are unlikely to cause illness. However, their presence in drinking
water indicates that disease-causing organisms (pathogens) could be in the water system. Most
pathogens that can contaminate water supplies come from the feces of humans or animals.
Testing drinking water for all possible pathogens is complex, time-consuming, and expensive. It
is easy and inexpensive to test for coliform bacteria. If testing detects coliform bacteria in a
water sample, water systems search for the source of contamination and restore safe drinking
water.

Types of Coliform Bacteria

There are three groups of coli form bacteria

1.Total coli form bacteria

2.Fecal coli form bacteria

3.E.coli bacteria

Total coli form bacteria

Total coliform bacteria are common in the environment (soil or vegetation) and are generally
harmless. If a lab detects only total coliform bacteria in drinking water, the source is probably
environmental and fecal contamination is unlikely. However, if environmental contamination can
enter the system, pathogens could get in too. It is important to find and resolve the source of the
contamination.

Fecal coli form bacteria

Fecal coliform bacteria are a subgroup of total coliform bacteria. They exist in the intestines and
feces of people and animals. The presence of fecal coliform in a drinking water sample often
indicates recent fecal contamination. That means there is a greater risk that pathogens are present

E.coli bacteria

E. coli is a subgroup of the fecal coli form group. Most E. coli bacteria are harmless and exist in
the intestines of people and warm-blooded animals. However, some strains can cause illness. The
presence of E. coli in a drinking water sample usually indicates recent fecal contamination. That
means there is a greater risk that pathogens are present.

According to shape when viewed through powerful microscope

(i) Cocci bacteria these bacteria cells are round or spherical.

(ii) Bacilli bacteria Cells of these bacteria are rod-shaped.

(iii) Spirilla bacteria The cells of these bacteria are spiral- shaped
Gram staining bacteria are a method of differentiating bacterial species into two large
groups, which are based on their chemical and physical properties of their cell wall.

Gram positive bacteria: Those bacteria when they are stained in gram stain results in
purple colour.

Gram negative bacteria: Those bacteria when they are stained in gram stain results in
pink colour.

Shapes of Bacteria

Bacteria come in five main shapes:

1) Spherical (like a ball) (cocci)

2) Rod shaped (bacilli)

3) Spiral (spirilla)

4) comma (vibrios)

5) corkscrew (spirochetes)

Structure of Bacteria
BACTERIAL GROWTH

Growth of Bacteria is the orderly increase of all the chemical constituents of the bacteria.
Multiplication is the consequence of growth. Death of bacteria is the irreversible loss of ability to
reproduce. Bacteria are composed of proteins, carbohydrates, lipids, water and trace elements.

FACTORS REQUIRED FOR BACTERIAL GROWTH

1.Environmental Factors affecting Growth-

pH of the medium :

Neutrophiles grow best around neutral pH (7)

Acidophiles grow best at pH < 7

Alkophiles grow best at pH > 7

Acidotolerant grow best at pH 7 but can also grow at lower pH

Alkotolerant grow best at pH 7 but can also grow at higher pH

Temperature:

Enzymes, the machinery of the cell, are influenced by external factors and can be shown
to have a range where they function that includes an optimal value that produces the
highest activity.

The range of enzyme activity determines the range for growth of specific bacteria,
analogously leading to a value for optimal growth rate.

In the case of temperature, bacteria are divided into categories based on the temperature
range where they can grow and the temperature that provides optimal growth.

Water availability:

Liquid water is essential for life.

Aqueous solutions actually have different amounts of water available, depending on how
many solutes are dissolved in it. As a very simple model, consider two glasses, one full
of pure water, the other containing the same amount of water plus a sponge. Which one
would be easier to drink? On a much smaller scale, dissolved solutes act like a sponge,
making less water available.

Water activity (aw) can be decreased by the addition of any soluble molecule although
salt (NaCl) and sugars are probably the most common.
 Microbes that require a high water activity (near or at 1) are termed nonhalophiles.
(Halophile = salt-loving)

Some bacteria require salt to grow and are called halophiles. If a very high concentration
of salt is required (around saturation), the organisms are termed extreme halophiles.

A nonhalophile that can grows best with almost no salt but can still grow with low levels
of salt (~ 7%) is called halotolerant.

In general, fungi are more tolerant of low water activity. (Thats why your jelly is more
likely to get contaminated by fungi than bacteria.

Oxygen recuirements :

Oxygen is a very reactive molecule and can affect cells in several ways. The effect of
oxygen is often determined using thioglycollate broth, a special medium that contains a
reducing agent (thioglycollate) that removes oxygen so that a gradient occurs within the
tube.
Obligately aerobic bacteria can obtain energy only through aerobic respiration and have
to have oxygen available. Thus, they will grow only at the surface of thioglycollate
broth.
Obligately anaerobic bacteria die in the presence of oxygen and can only grow at the
bottom of thioglycollate broth. Some anaerobes are so sensitive to oxygen that even
thioglycollate broth is not anoxic enough to provide suitable anaerobic conditions.
Microaerophiles require oxygen for growth but the 20% in air is too toxic. As a result,
they grow near the top but beneath the surface of thioglycollate broth where the oxygen
concentration is typically 4 10%.
Facultative anaerobes can use oxygen for aerobic respiration but can switch to
fermentative metabolism in the absence of oxygen. As a result, they will grow
throughout thioglycollate broth. (Heavier growth at top.)
Aerotolerant anaerobes are anaerobic bacteria that can grow in the presence of air. (Not
shown in diagram.)

Nutrients: Nutrients in growth media must contain all the elements necessary for the
synthesis of new organisms. In general the following must be provided :

(a) Hydrogen donors and acceptors, (b) Carbon source, (c) Nitrogen source, (d) Minerals
: sulphur and phosphorus, (e) Growth factors: amino acids, purines, pyrimidines;
vitamins, (f) Trace elements: Mg, Fe, Mn.
(b) Growth Factors: A growth factor is an organic compound which a cell must contain
in order to grow but which it is unable to synthesize. These substances are essential
for the organism and are to be supplied as nutrients. Thiamine, nicotinic acid, folic
acid and para-aminobenzoic acid are examples of growth factors.
 (c) Essential Metabolites: These metabolites are essential for growth of bacterium.
These must be synthesized by the bacterium, or be provided in the medium. Mg, Fe
and Mn are essential trace elements.
1. Autotrophs: live only on inorganic substances, i.e. do not require organic nutrients for
growth. They are not of medical importance.
2. Heterotrophs: require organic materials for growth, e.g. proteins, carbohydrates,
lipids as source of energy. All bacteria of medical importance belong to heterotrophs.
(d) Carbon dioxide: All bacteria require CO2 for their growth. Most bacteria produce
CO2. N. gonorrhoeae and N. meningitides and Br abortus grow better in presence of
5 per cent CO2.

Culture Media:
Complex (contains undefined components)
Chemically defined (all concentrations are known)
Selective (favors the growth of a particular organism or group of organisms)
Differential (has reactions that give isolates different appearance)
Anaerobic (oxygen-free)

Light: Optimum condition for growth is darkness.

2. Ionic strength and osmotic pressure : Bacterial growth depend upon Ionic strength and
osmotic pressure.
BATCH CULTURE CURVE OF BACTERIA

The Five Stages of Bacterial Growth :

Lag Phase.

Exponential Phase or Logarithmic (Log) Phase

Maximal Stationary Phase.

Decline phase or death phase

Survival phase

The growth of bacteria (or other microorganisms as protozoa, or yeasts) in batch culture
can be modeled with five different phases

Log/Exponential Phase

Rapid growth of bacteria, they start multiplying exponentially at an accelerated pace doubling in
number every few minutes. Conditions are optimal for growth.

Lag Phase

An adjustment period where the bacteria adapt to the new environmental conditions (pH,
nutrients, temperature.). There is no significant increase in numbers with time.
Stationary Phase

Equal rate of growth and death so that overall bacterial numbers stay the same. At this point
bacteria are competing for food, space, moisture and oxygen. There is also a buildup of wastes.

Death Phase

Rapid cell death usually due to the cells bursting open. This is due to a lack of nutrients and a
toxic environment

Survival Phase

A small number survive as end spores

VIRUS

Viruses are peculiar microbes in that they do not directly use organic or inorganic compounds
during growth; they reproduce by invading a host cell whose reproductive processes they redirect
to manufacture more virus particles. Viruses are tiny organisms that may lead to mild to severe
illnesses in humans, animals and plants. This may include flu or a cold to something more life
threatening like HIV/AIDS.

Viruses enter the body from the environment or other individuals from soil to water to air via
nose, mouth, or any breaks in the skin and seek a cell to infect.

Structure of a virus

Nucleic acid

Protein Coat (capsid)

ALGAE

Algae are mostly multicellular photosynthetic organisms, which are extremely varied in their
shapes and sizes. Carbon dioxide is used as the source of carbon for the synthesis of new cells
and oxygen is evolved from water by the classic mechanism of plant photosynthesis:

6CO2+ 12H2O light C6H 12O6 +6H2O+6O2

In darkness algae need oxygen for respiration and organic compounds for growth. Their growth
(whether in darkness or light) is greatly stimulated by phosphates and nitrates which are usually
present in sewage effluents. In the operation of stabilization ponds algal blooms are an essential
part of treatment process.
PROTOZOA

Protozoa are multicellular protists to the animal kingdom. There are three main groups of
protozoa: amoebae, ciliates and flagellates. Amoebae and flagellates are not very important in
sewage treatment, but amoeba Entamoeba histolytica is an important human pathogen causing
amoebic dysentery (amoebiasis). The ciliates are more important, being extremely common in
sewage treatment works where they consume considerable numbers of bacteria; their numbers
are about 103_104 per ml.