Chapter 35

30.12.
2012
LECTURE PRESENTATIONS
For BROCK BIOLOGY OF MICROORGANISMS, THIRTEENTH EDITION
Michael T. Madigan, John M. Martinko, David A. Stahl, David P. Clark
Chapter 35
Wastewater Treatment, Water
Lectures by
Purification, and Waterborne
John Zamora
Middle Tennessee State University Microbial Diseases
2012 Pearson Education, Inc.
I. Wastewater Microbiology and Water

Purification
35.1 Public Health and Water Quality
35.2 Wastewater and Sewage Treatment
35.3 Drinking Water Purification
2012 Pearson Education, Inc. Marmara University Enve303 Env. Eng. Microbiology Prof. BARI ALLI
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I. Wastewater Microbiology and Water

Purification
Water
Potential common source of infectious diseases
Can also be a source for chemically induced
intoxications
Ensuring water purity is essential for public health
Treatment and purification schemes use
microorganisms to identify, remove, and degrade
pollutants

Coliforms and Water Quality
Water can be sampled for the presence of
specific indicator microorganisms
The coliform group of microorganisms
Includes Enterobacter, Escherichia coli, and
Klebsiella pneumoniae
They are a widely used indicator for fecal
contamination in water
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Testing for Coliforms and E. coli
All tests assay the growth of organisms
recovered from the water samples
Common methods for enumerating the samples:
Most-probable-number (MPN) procedure
Membrane filter (MF) procedures (Figure 35.1)
E. coli can be distinguished from total coliforms
with defined substrates (e.g., MUG, IBDG;
Figure 35.2)
Figure 35.1 Coliform colonies growing on a membrane filter
A drinking water sample was passed through the filter. The filter was then placed on
eosinmethylene blue (EMB) medium that is both selective and differential for lactose-
fermenting bacteria (coliforms). The dark, shiny appearance of the colonies is characteristic of
coliforms. Each colony developed from one viable coliform cell present in the original sample.
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Figure 35.2 Total coliforms and Escherichia coli.
A filter exposed to a drinking water sample was incubated at 35oC for 24 hours on MI
media and examined under UV light. The single E. coli colony appears dark blue (arrow).
The other colonies are coliforms that fluoresce and appear white to light blue.

A commonly used method for performing
coliform counts is the IDEXX Colilert test
system (Figure 35.3)
Utilizes proprietary substrates
Sample with no coliforms remains clear
Coliforms turn media yellow
E. coli turns media fluorescent blue
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Figure 35.3 The IDEXX Colilert water quality test system
Colilert reagents are added to 100-ml water samples. After incubation for 24 h at 35-
37oC, the samples develop yellow color if they contain coliform bacteria (right). Samples
containing Escherichia coli develop yellow color and also develop blue fluorescence
(left). Samples negative for coliform bacteria remain clear (center).

Public Health and Drinking Water Purification
Effective water treatment practices that reduced
the incidence of waterborne disease were not in
place until the twentieth century (Figure 35.4)
Coliform-counting methods were developed and
adapted around 1906
Water purification started with filtration
About 1913, chlorine came into use as a
disinfectant for large water supplies
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Figure 35.4 The effect of water purification on the incidence of waterborne disease
10,000 Filtration, 1906
Number of typhoid cases
Chlorination, 1913
1000
100
1885 1895 1905 1915 1925 1935 1945
Year
The graph shows the incidence of typhoid fever in Philadelphia, Pennsylvania. Note
the dramatic reduction in the incidence of typhoid fever after the introduction of both
filtration and chlorination.

Wastewater
Domestic sewage or liquid industrial waste
Wastewater treatment
Relies on industrial-scale use of microbes for
bioconversion
Following treatment, the discharged treated
wastewater (effluent water) is suitable for
Release into surface waters
Release to drinking water purification facilities
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Wastewater treatment facility
Its goal is to reduce organic and inorganic
materials in wastewater to a level that no longer
supports microbial growth and to eliminate other
potentially toxic materials
The efficiency of treatment is expressed in terms
of a reduction in the biochemical oxygen demand
(BOD)
Amount of dissolved oxygen consumed by
microbes to completely oxidize all organic and
inorganic matter in a water sample

Wastewater treatment is a multistep operation
employing both physical and biological
processes (Figure 35.5)
Primary, secondary and sometimes tertiary
treatments are used
Primary treatment
Uses physical separation methods to separate
solid and particulate organic and inorganic
materials from wastewater
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Figure 35.5 Wastewater treatment processes
WASTEWATER
Screening
PRIMARY
treatment
Sedimentation
Anaerobic Aerobic Activated sludge/aeration SECONDARY

digestion oxidation Trickling filter treatment
Digested Disinfection Treated effluent to discharge

sludge: drying;
incineration;
use as fertilizer,
or burial
Effective water treatment plants use the primary and secondary treatment methods
shown here. Tertiary treatment may also be used to reduce BOD levels in effluent water
to undetectable levels.

Secondary treatment
Anaerobic secondary treatment involves a series
of digestive and fermentative reactions carried
out by various microbes under anoxic conditions
(Figure 35.7)
The process is carried out in large enclosed tanks
(sludge digesters or bioreactors)
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Figure 35.7 Anaerobic treatment
Anaerobic sludge digester. Only the top of the tank is shown; the remainder is underground.
Complex polymers
Gas outlet (polysaccharides, lipids, proteins)
Scum Hydrolysis by
CH4/CO2 removal microbial
enzymes
Sludge Scum layer
inlet Supernatant Monomers
Supernatant removal (sugars, fatty acids, amino acids)
Actively digesting Fermentation Fermentation

sludge
Stabilized sludge Acetate H2 CO2
Methanogenesis
CH4 CO2 CH4 H2O

Sludge outlet Major microbial processes in anaerobic sludge
Inner workings of a sludge digester digestion. Methane (CH4) and carbon dioxide (CO2)
are the major products of anaerobic biodegradation.

Secondary Treatment (contd)
Aerobic secondary treatment uses digestive
reactions carried out by microbes under aerobic
conditions to treat wastewater with low levels of
organic materials
Activated sludge (Figure 35.8a & b) and the
trickling filter (Figure 35.8c) are the most
common decomposition processes
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Figure 35.8a Aerobic wastewater treatment processes (aeration tank)
Aeration tank of an activated sludge system in a metropolitan

wastewater treatment plant. The tank is 30 m long, 10 m wide
and 5 m deep.
Figure 35.8b Aerobic wastewater treatment processes (activated sludge process)
Wastewater from primary treatment
Aeration tank Settling tank
Clear effluent
Activated
sludge
Air
Anaerobic
sludge
digester
Activated sludge return Excess sludge
Wastewater flow through an activated sludge system. Recirculation

of activated sludge to the aeration tank introduces microorganisms responsible
for oxidative degradation of the organic components of the wastewater
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Figure 35.8c Aerobic wastewater treatment processes (trickling filter)
Trickling filter process. The booms rotate, distributing wastewater

slowly and evenly on the rock bed. The rocks are 10-15 cm in
diameter and the bed is 2 m deep.

In the activated sludge process, wastewater is
mixed and aerated in large tanks and slime-
forming bacteria (e.g., Zoogloea ramigera; Figure
35.9) grow and form flocs
Most treatment plants chlorinate the effluent after
secondary treatment to further reduce the
possibility of biological contamination
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Figure 35.9 A wastewater floc formed by the bacterium Zoogloea ramigera
Floc formed in the activated sludge process consists of a large number of small, rod-
shaped cells of Z. ramigera surrounded by a polysaccharide slime layer, arranged in
characteristic fingerlike projections in this negative stain with India ink.

Tertiary treatment
Any physiochemical or biological process
employing bioreactors, precipitation, filtration, or
chlorination procedures similar to those used for
purification of drinking water
Reduces the levels of inorganic nutrients (e.g.,
phosphate, nitrate, nitrite)
Most complete method of treating sewage but
not widely adopted due to costs
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Wastewater treated by secondary methods is
not yet potable, or safe for human consumption
It requires further treatment to remove
pathogens, eliminate taste and odor, reduce
chemicals (e.g., iron, manganese), and
decrease turbidity
A typical drinking water treatment installation
purifies raw (untreated) water (Figure 35.10a)
Figure 35.10a Water treatment plant
River pumping station

Ohio River
Sedimentation
Coagulation basin basins
Chlorination
Underground Filter buildings
clear-water
reservoir
Aerial view of a water treatment plant. The arrows indicate direction of flow of
water through the plant.
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Purification involves many steps (Figure 35.10b)
Sedimentation to remove particles
Coagulation and flocculation form additional
aggregates, which settle out
Filtration
Disinfection (typically with chlorine gas or UV
radiation)
Figure 35.10b Water treatment plant
Remove
sand, gravel,
large Raw water
particulates Sedimentation
Form and remove floc,
Coagulation containing insoluble
material and
microorganisms
Remove remaining
particulates and most
Filtration organic and inorganic
compounds
Kill remaining micro-

Chlorination
organisms and
prevent growth of
Storage new inocula
Finished water
Distribution
Schematic overview of a typical community water
purification system
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II. Waterborne Microbial Diseases

35.4 Sources of Waterborne Infection
35.5 Cholera
35.6 Giardiasis and Cryptosporidiosis
35.7 Legionellosis (Legionnaires Disease)
35.8 Typhoid Fever and Other Waterborne
Diseases

Common sources of waterborne disease
transmission include
Potable water used for drinking and cooking
(Figure 35.11a)
May be improperly treated or of low quality
May be from nonregulated sources (e.g., private
wells)
Recreational water from public ponds, lakes,
swimming pools, etc. (Figure 35.11b)
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Figure 35.11a Waterborne disease outbreaks
60
Drinking water outbreaks
Number of outbreaks
50
40
30
20
10
0
1974 1977 1980 1983 1986 1989 1992 1995 1998 2001 2004
Year
Data were provided by the Centers for Disease Control and Prevention, Atlanta,
Georgia, USA. Reported drinking water disease outbreaks from 1974 to 2006. Of 729
outbreaks, about 90% were due to biological agents (bacteria, viruses and protists)
Figure 35.11b Waterborne disease outbreaks
45
Recreational water outbreaks
Number of outbreaks
30
15
0
1980 1984 1988 1992 1996 2000 2004
Year
Reported recreational water outbreaks from 1980 to 2006. Of 544 total outbreaks,
almost all were due to biological agents.
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Microorganisms transmitted in water generally
grow in the intestines and leave the body in feces
Feces then pollute water
Numerous bacterial and protozoan pathogens
can be transmitted in drinking water
In the U.S. the number of disease outbreaks due
to drinking or recreational water contamination is
low
Lack of adequate water treatment facilities and
access to clean water contribute to the spread of
infectious disease
35.5 Cholera
Cholera
Severe diarrhea
Caused by Vibrio cholerae (Figure 35.12)
Typically transmitted through ingestion of
contaminated water
Largely restricted to developing countries
Endemic in Africa, Southeast Asia, the Indian
subcontinent, and Central and South America
Can be controlled by application of water
treatment
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Figure 35.12 Cells of Vibrio cholerae
This colorized scanning electron micrograph shows a rod to curved rod morphology.
The organism is about 0.3 m in diameter and up to 2 m in length.
35.5 Cholera
Pathogenesis
V. cholerae attaches to epithelial cells in the
small intestine, where it grows and releases
cholera toxin
This enterotoxin causes severe diarrhea
Can result in dehydration and death unless
treated
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35.5 Cholera
Diagnosis
Presence of V. cholerae bacilli in the feces of
patients
Prevention
Immunization not recommended
Public health measures are very important
Treatment
Oral rehydration and electrolyte replacement
therapy

Giardia intestinalis causes giardiasis, an acute
gastroenteritis
Cryptosporidium parvum causes cryptosporidiosis
These organisms are found in nearly all surface
waters and are highly resistant to chlorine due to
cyst formation
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Giardia intestinalis (Figure 35.16)
A flagellated protist that is usually transmitted to
humans in fecally contaminated water
The protist cells produce a resting stage (cyst)
that is resistant to desiccation and chemical
disinfection
Following ingestion, cysts germinate and attach
to the intestinal wall, causing symptoms of
infection
Many individuals exhibit no symptoms and can
act as carriers
Figure 35.16 The parasite Giardia
Scanning electron micrographs.
A motile trophozoite*. The trophozoite is about

15 m in length.
A giardial cyst. The cyst is

about 11 m in length.
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Cryptosporidium parvum (Figure 35.17)
Protist that lives as a parasite in warm-blooded
animals
Produces thick-walled cells (oocysts) that are
shed in the feces of infected animals
The oocysts are transmitted in fecally
contaminated water
Oocysts are highly resistant to chlorine and UV
radiation; thus sedimentation and filtration
methods are most effective at removal
Figure 35.17 Cryptosporidium
The arrows point to two of the The thick-walled oocysts are

many intracellular trophozoites 3-5 m in diameter in this fecal
embedded in human gastro- sample.
intestinal epithelium. The
trophozoites are 2-5 m.
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Cryptosporidiosis is usually a self-limiting mild
diarrhea
But can lead to more serious disease in
compromised individuals

Legionella pneumophila
Causes legionellosis
Normally transmitted in aerosols
Gram-negative bacterium with complex
nutritional requirements (Figure 35.18)
Common in terrestrial and aquatic habitats
Relatively resistant to heating and chlorination
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Figure 35.18 Legionella pneumophila
Colorized scanning electron micrograph of L. pneumophila cells. Cells are

0.3-0.6 m in diameter and up to 2 m in length.

Legionella pneumophila (contd)
Common in cooling towers and evaporative
condensers of large air conditioning systems
The pathogen grows in the water and is
disseminated in humidified aerosols
Infection is not spread from person to person
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The prevalence of legionellosis is increasing and
infections are often underreported (Figure 35.19)
Treatment with antibiotics is effective
Prevention can be accomplished by improving
maintenance and design of water-dependent
cooling and heating systems and water delivery
systems
Figure 35.19 Incidence of Legionnaires disease in the United States
12
Cases per one million people
10
0
1992 1997 2002 2007
Year
In 2007, there were 2716 reported cases. Data are from the Centers for Disease
Control and Prevention, Atlanta, Georgia
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Diseases
Worldwide, the most important pathogenic
bacteria transmitted by water are Salmonella
enterica serovar Typhi and Vibrio cholerae
S. enterica ser. Typhi causes typhoid fever
and has been virtually eliminated in
developed countries due to water treatment

Diseases
Viruses can also be transmitted in water and
cause human disease
For example, enteroviruses such as poliovirus,
norovirus, and hepatitis A are shed into water
from feces
Viruses can survive in water for relatively long
periods but are inactivated by disinfection with
chlorine
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Diseases
Entamoeba histolytica (Figure 35.20)
A pathogenic protist transmitted to humans
primarily through contaminated water and
sometimes food
Anaerobic and produces resistant cysts
Infection can be asymptomatic or lead to
diarrhea and/or dysentery
If untreated, invasive cells can invade the liver
and occasionally the lungs and brain
Amoebicidal drugs are not universally effective
Figure 35.20 The trophozoite of Entamoeba histolytica, the amoeba that causes amebiasis
The small red structures are red blood cells. The trophozoites range from
12 to 60 m in length.
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Diseases
Naegleria fowleri (Figure 35.21)
A free-living amoeba found in soil and water runoff
that can also cause amebiasis
Infections usually result from swimming in warm,
soil-contaminated water sources (e.g., hot springs
or lakes)
Enters the human body through the nose and
burrows directly into the brain, causing extensive
hemorrhage and brain damage
(meningoencephalitis)
Drug treatment is effective if infections are
identified early
Figure 35.21 Trophozoites of Naegleria fowleri in brain tissue
This amoeba causes meningoencephalitis. Oval to round and amoeboid (irregularly

shaped) trophozoites (arrows) are present as dark-stained structures with densely
stained nuclei. There is extensive destruction of the surrounding brain tissue. Individual
trophozoites are 1035 m long.
27

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30.12.

I. Wastewater Microbiology and Water

I. Wastewater Microbiology and Water

35.1 Public Health and Water Quality

35.1 Public Health and Water Quality

Figure 35.1 Coliform colonies growing on a membrane filter

Figure 35.2 Total coliforms and Escherichia coli.

35.1 Public Health and Water Quality

Figure 35.3 The IDEXX Colilert water quality test system

35.1 Public Health and Water Quality

10,000 Filtration, 1906

Number of typhoid cases

35.2 Wastewater and Sewage Treatment

35.2 Wastewater and Sewage Treatment

35.2 Wastewater and Sewage Treatment

Figure 35.5 Wastewater treatment processes

Anaerobic Aerobic Activated sludge/aeration SECONDARY

Digested Disinfection Treated effluent to discharge

35.2 Wastewater and Sewage Treatment

Figure 35.7 Anaerobic treatment

Actively digesting Fermentation Fermentation

Stabilized sludge Acetate H2 CO2

CH4 CO2 CH4 H2O

35.2 Wastewater and Sewage Treatment

Figure 35.8a Aerobic wastewater treatment processes (aeration tank)

Aeration tank of an activated sludge system in a metropolitan

Figure 35.8b Aerobic wastewater treatment processes (activated sludge process)

Wastewater from primary treatment

Aeration tank Settling tank

Wastewater flow through an activated sludge system. Recirculation

Figure 35.8c Aerobic wastewater treatment processes (trickling filter)

Trickling filter process. The booms rotate, distributing wastewater

35.2 Wastewater and Sewage Treatment

Figure 35.9 A wastewater floc formed by the bacterium Zoogloea ramigera

35.2 Wastewater and Sewage Treatment

35.3 Drinking Water Purification

Figure 35.10a Water treatment plant

River pumping station

35.3 Drinking Water Purification

Figure 35.10b Water treatment plant

Kill remaining micro-

II. Waterborne Microbial Diseases

35.4 Sources of Waterborne Infection

Figure 35.11a Waterborne disease outbreaks

Figure 35.11b Waterborne disease outbreaks

35.4 Sources of Waterborne Infection

Figure 35.12 Cells of Vibrio cholerae

35.6 Giardiasis and Cryptosporidiosis

35.6 Giardiasis and Cryptosporidiosis

Figure 35.16 The parasite Giardia

Scanning electron micrographs.

A motile trophozoite*. The trophozoite is about

A giardial cyst. The cyst is

35.6 Giardiasis and Cryptosporidiosis

Figure 35.17 Cryptosporidium

The arrows point to two of the The thick-walled oocysts are

35.6 Giardiasis and Cryptosporidiosis

35.7 Legionellosis (Legionnaires Disease)

Figure 35.18 Legionella pneumophila

Colorized scanning electron micrograph of L. pneumophila cells. Cells are

35.7 Legionellosis (Legionnaires Disease)

35.7 Legionellosis (Legionnaires Disease)

Figure 35.19 Incidence of Legionnaires disease in the United States

35.8 Typhoid Fever and Other Waterborne