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Applied Environmental Microbiology
CHAPTER OVERVIEW
This chapter discusses the uses of microorganisms in processes that are grouped under the heading of applied
environmental microbiology. Water is an excellent vehicle for the transmission of diseases, and the chapter
explores the measures taken to ensure the availability of safe drinking water. The chapter continues with a
discussion of the contamination of groundwaters by domestic and industrial wastes and the use of home
wastewater treatment systems.
CHAPTER OBJECTIVES
After reading this chapter you should be able to:
•
•
•
•
•
•
•
•
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list the major pathogens transmitted by water
describe the steps used to purify drinking water
describe the ideal characteristics of indicator organisms and how they are used to measure the
microbiological quality of water
describe the methods commonly used to measure the level of organic material in wastewater
discuss wastewater treatment systems
compare and contrast wastewater treatment systems to the natural purification processes observed in
waters
discuss the ways humans impact groundwater and surface water
describe home wastewater treatment systems
discuss the manipulation of microorganisms in the environment to control biodegradation
CHAPTER OUTLINE
I. Water Purification and Sanitary Analysis
A. Waterborne pathogens and water purification
1. Many human pathogens are transmitted by water (e.g., Vibrio spp., Giardia, Cryptosporidium)
2. Water purification is critical for public health and safety; common steps in water purification
are:
a. Sedimentation in a sedimentation basin removes sand and large particles
b. Coagulation with alum, lime, and/or organic polymers is followed by clarification in a
settling basin; removes many of the microorganisms (including many of the viruses),
organic matter, toxic contaminants, and suspended particles
c. Rapid sand filtration—physically traps particles and microbes
d. Disinfection with chlorine or ozone; chlorination can lead to formation of disinfection
by-products (DBPs) that may be carcinogens
3. Giardia cysts, Cryptosporidium oocysts, and viruses are not consistently and reliably removed
by the above procedures; slow sand filtration, which involves the slow passage of water over a
bed of sand, more consistently removes Giardia
4. The EPA sets maximum contaminant level goals for important pathogens in waters and this
influences the processes chosen for water purification
B. Sanitary analysis of water
1. Since intestinal pathogens gradually lose their ability to form colonies after release into aquatic
environments, microbiologists have generally monitored the presence and amount of indicator
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organisms as an index of fecal contamination of water; ideal indicator organisms have the
following characteristics:
a. Should be suitable for the analysis of all types of water
b. Should be present whenever enteric pathogens are present
c. Should survive longer than the hardiest enteric pathogen
d. Should not reproduce in the contaminated water
e. Should be detected by a highly specific assay
f.
Should be detected by a test that is easy to perform and sensitive
g. Should be harmless to humans (ensuring safety for laboratory personnel)
h. Concentration of indicator should directly reflect the degree of fecal pollution
2. Coliforms—the most commonly used indicator organisms
a. All are facultative anaerobic, gram-negative, nonsporing, rod-shaped bacteria that
ferment lactose with gas formation within 48 hours at 35°C (e.g., Escherichia coli,
Enterobacter aerogenes, and Klebsiella pneumoniae)
b. Detected by the following tests
1) Most probable number (MPN)—statistical estimation; does not distinguish
coliforms from fecal coliforms (those derived from intestines of homeothermic
animals; can grow at 44.5°C)
2) Membrane filtration technique—water is filtered, filter is placed on an absorptive
pad containing liquid medium; this is incubated and colonies are counted; detects
total coliforms, fecal coliforms, and fecal streptococci
3) Presence-absence (P-A) test—detects both coliforms and fecal coliforms
4) Defined substrate tests (e.g., Colilert) involve the production of a colored product
(for total coliforms) or a fluorescent product (for E. coli) from a specific growth
substrate
5) Molecular techniques are now being routinely used to detect E. coli and other
pathogens
II. Wastewater Treatment
A. Wastewater can contain high levels of organic matter and human pathogens; these can be removed
(or their amount decreased) by wastewater treatment; such treatment is one of the most important
factors in maintaining public health
B. Measuring water quality—the tests described below monitor organic carbon in wastewater but do
not address the levels of nitrate, phosphate, and sulfate; these also are of concern
1. Total organic carbon (TOC)—quantifies carbon concentration by oxidizing organic matter at
high temperatures and measuring the amount of carbon dioxide produced
2. Chemical oxygen demand (COD)—quantifies the amount of organic matter present (except
lignin) by reacting organic material with a strong acid
3. Biochemical oxygen demand (BOD)—amount of oxygen needed to utilize organic material as
growth substrates; indirectly measures the amount of organic material in a sample; can be
affected by presence of ammonia, so nitrification is inhibited by addition of nitrapyrin to the
sample
C. Water treatment processes
1. A controlled self-purification; usually involves the use of large basins where mixing and gas
exchange are carefully controlled
2. Conventional wastewater treatment
a. Primary (physical) treatment—removal of particulates; resulting solid material is called
sludge
b. Secondary (biological) treatment—removal of dissolved carbonaceous materials (90 to
95% of the BOD) and many bacterial pathogens; produces a sludge, which must be
further processed or disposed of; if not carefully monitored, these processes can produce
bulking sludge, which is not easily removed
1) Aerated activated sludge systems—involve horizontal flow of materials and the
addition of sludge, which acts as a source of microorganisms; the sludge
microorganisms oxidize the organic matter; the resulting biomass is later removed
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2)
Trickling filters—vertical flow over gravel on which microorganisms have
developed in surface films; the microorganisms degrade the organic matter
3) Extended aeration systems—reduce the amount of sludge produced by the process
of biological self-consumption (endogenous respiration)
4) All of the above secondary treatment processes as well as the primary treatment
produce sludge; anaerobic sludge digestion reduces the amount of sludge that must
be disposed of in landfills or by other means; also produces methane, which can be
used as a fuel for generation of electrical power; involves three steps: fermentation
of sludge components to form organic acids, production of methanogenic substrates
(acetate, carbon dioxide, and hydrogen), and methanogenesis
c. Tertiary treatment (physical, chemical and/or biological)—removes inorganic nitrogen,
phosphorus, recalcitrant organics, viruses, etc.
3. Constructed wetlands use floating emergent and/or submerged plants to provide nutrients for
microbial growth in their root zone; they help remove organic matter, inorganic matter, and
metals from waters
D. Home treatment systems
1. Groundwater—water in gravel beds and fractured rocks below the surface of soil; it is an
important source of water but the microbiological processes occurring in groundwater are not
well understood; it is known that disease-causing organisms and organic matter are removed
by adsorption and trapping as they move through the subsurface; microbial predators use
trapped pathogens as food
2. Home treatment in a conventional septic tank system mimics the natural adsorption-biological
predation process; septic systems are now being designed with nitrogen and phosphorus
removal steps; a conventional system is described below
a. Anaerobic liquefaction and digestion occurs in a septic tank
b. Aerobic digestion, adsorption, and filtration of organic material are accomplished by
drainage through suitable soil in a leach (drain) field; if drainage is too rapid, there is
little adsorption and filtration, with subsequent contamination of well waters and
groundwaters
3. Groundwater also can be contaminated by land disposal of sewage sludges, illegal dumping of
septic tank pumpage, improper toxic waste disposal, agricultural runoff, and deep-well
injection of industrial wastes
4. In situ treatment procedures for groundwater are under investigation; microorganisms are
critical in many of these remediation efforts
III. Biodegradation and Bioremediation by Natural Communities
A. Microorganisms can be used to carry out desirable processes in natural environments; in these
environments, complete control of the process is not possible; processes carried out in natural
environments include:
1. Biodegradation, bioremediation, and environmental maintenance processes
2. Addition of microorganisms to soils or plants for improvement of crop production
B. Biodegradation and bioremediation processes
1. Biodegradation has at least three definitions
a. A minor change in an organic molecule, leaving the main structure still intact
b. Fragmentation of a complex organic molecule in such a way that the fragments could be
reassembled
c. Complete mineralization
2. Bioremediation is the use of microbes to transform contaminants into nontoxic degradation
products
3. Degradation of a complex compound such as a halogenated compound occurs in stages
a. Dehalogenation often occurs faster under anaerobic conditions; humic substances may
facilitate this stage
b. Subsequent steps usually proceed more rapidly in the presence of oxygen
4. Structure and stereochemistry impact rate of biodegradation (e.g., meta effect and preferential
degradation of one isomer)
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5.
C.
D.
Microbial communities change in response to addition of inorganic and organic substrates;
these can impact rate and extent of biodegradation (e.g., repeated contact with a herbicide
leads to the adaptation of the microbial community and a faster rate of degradation—
acclimation)
6. Waste materials can be degraded after incorporation into soil or as they flow across soil
surface
7. Biodegradation does not always reduce environmental problems (e.g., partial degradation can
produce equally hazardous or more hazardous substances)
8. Microbial fuel cells are now being applied to bioremediation; fuel cells provide an oxidant that
facilitates degradation; the transfer of electrons can produce an electrical current
Stimulating biodegradation
1. Engineered bioremediation—addition of oxygen or nutrients to stimulate degradation activities
of microorganisms
2. Stimulating hydrocarbon degradation in waters and soils—usually involves addition of
nutrients and substances that increase contact between microorganisms and substrate to be
degraded; also can involve aeration or creating anoxic conditions
3. Stimulating degradation with plants—phytoremediation is the use of plants to stimulate the
extraction, degradation, adsorption, stabilization, or volatilization of contaminants; transgenic
plants can be used
4. Stimulation of metal bioleaching from minerals—involves the use of acid-producing bacteria
to solubilize metals in ores; may require addition of nitrogen and phosphorus if they are
limiting
Bioaugmentation—addition of microorganisms to complex microbial communities
1. Impact of protective microhabitats
a. Often fails to produce long-lasting increases in rates of biodegradation; this may be due
to three factors:
1) Attractiveness of laboratory grown microbes as a food source for predators
2) Inability of microorganisms to contact the compounds to be degraded
3) Failure of the microorganisms to survive
b. “Toughening” microorganisms by starvation before they are added has increased
microbial survival somewhat, but has not solved the problem
2. More success is possible when considering protective microhabitats when adding
microorganisms by including materials that provide protection and/or supply nutrients
a. Living microhabitats—include surfaces of a seed, a root, or a leaf
b. Inert microhabitats—include microporous glass or “clay hutches”
TERMS AND DEFINITIONS
Place the letter of each term in the space next to the definition or description that best matches it.
____ 1.
____ 2.
____ 3.
____ 4.
____ 5.
____ 6.
____ 7.
____ 8.
____ 9.
Microbial biomass produced during wastewater treatment that does not settle properly; it results
from the development of massive amounts of certain filamentous organisms
Organisms that are used to indicate fecal contamination of water
Methods for treating groundwaters where they are located
Chemicals such as trihalomethanes (THMs) produced during water treatment as a result of the
reaction of chlorine with organic matter in the water
The active biomass that is formed when organic matter is oxidized and degraded by microorganisms
in a wastewater treatment system
Minor modifications in molecules that are carried out by nongrowing microbes
The decrease in the rate of biodegradation observed when a constituent of a molecule is in the meta
position rather than the ortho position
Describes molecules with the characteristic of being asymmetric
A process in which waste material is incorporated in soil or allowed to flow across the soil surface,
where degradation occurs
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____ 10. Stimulation of degradative activities of microorganisms by modifying the water or soil in which
biodegradation is occurring
____ 11. The stimulation of the degradation of recalcitrant molecules by the addition of easily degraded
organic molecules
____ 12. The use of plants to stimulate the degradation, transformation, or removal of compounds
____ 13. The addition of microbes with known activities to soils, waters, or other complex systems in an
attempt to speed up existing microbiological processes
____ 14. Precipitated solid matter produced during water and sewage treatment
____ 15. The amount of oxygen used by organisms under standard conditions to degrade oxidizable organic
matter present in wastewaters
____ 16. An organism whose presence suggests the quality of a substance or environment
____ 17. Used for anaerobic treatment in home sewage processes
____ 18. The breakdown of a complex chemical through biological processes
____ 19. The amount of oxidation required to convert organic matter in wastewater to carbon dioxide
____ 20. A process that removes chlorine from compounds such as PCBs under anaerobic conditions
____ 21. The number of pathogens that can be present in a water supply that will prevent adverse health
effects and provide a margin of safety
a.
b.
c.
d.
e.
f.
g.
h.
i.
j.
k.
l.
m.
n.
o.
p.
q.
r.
s.
t.
u.
activated sludge
bioaugmentation
biochemical oxygen demand
biodegradation
biotransformations
bulking sludge
chiral
chemical oxygen demand
coliforms
cometabolism
disinfection by-products (DBPs)
engineered bioremediation
indicator organism
in situ treatment
land farming
maximum containment level goal
meta effect
phytoremediation
reductive dehalogenation
septic tank
sludge
FILL IN THE BLANK
1.
2.
3.
Three methods can be used to monitor the amount of organic matter present in wastewater as it is
processed in a wastewater treatment facility. One method determines the amount of a strong acid
neutralized by the organic matter in a sample; it is referred to as the
.
Another method reacts the organic matter with oxygen at a high temperature and measures the amount of
carbon dioxide produced; this is referred to as
. The third method is the most
commonly used method. It measures the amount of oxygen consumed when the organic material is used
as a growth substrate; it is referred to as the
). BOD is impacted by
nitrification (
), which can be inhibited by adding nitrapyrin to the sample.
The addition of chemicals such as alum and lime during water purification precipitates material in the
water. These settle to the bottom of
basins. This process is called
.
The solid material collected by primary treatment of wastewater is called
. This term is also
applied to the microbial biomass produced by aerobic secondary treatment of wastewater. Although the
amount of excess microbial biomass generated can be decreased by a process called
, it
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4.
5.
6.
7.
ultimately is further treated by
, which yields methane.
Home systems for treatment of wastewater include an anaerobic liquefaction and digestion step that
occurs in a
, followed by aerobic degradation in the leach field.
Wastewater treatment is a complex process that resembles the natural purification processes that occur in
streams and other bodies of water. The first step, called
treatment, physically removes
particulate material by screening, precipitation, and settling. This is followed by
treatment in which
dissolved organic matter is removed by biological activity. There are several forms of this treatment. One
commonly used system is the
system, which involves a horizontal flow of materials
and the introduction of sludge to the wastewater; this sludge is the source of microorganisms that will
degrade the organic matter, creating more sludge that later is removed in settling basins. A second
commonly used method is the
method. In this method, wastewater is passed over rocks
or other solid materials upon which microbial films have developed; the microbial community in these
films degrades the organic waste. The last type of treatment is more expensive and is not used by all
wastewater treatment facilities. It is
treatment, and it can use physical, chemical, or biological
methods to remove nitrogen or phosphorus.
Water that is safe to drink (
water) is the product of water purification systems. These
systems typically use
to partially clarify water,
to further clarify the water by adding
alum or other chemicals that cause impurities to precipitate out,
to trap fine particles and remove
most bacteria, and
with chlorine or ozone to destroy pathogens.
Rather than test directly for pathogens in drinking water, microbiologists monitor the presence and
numbers of
organisms that serve as an index for fecal contamination of water. The
are the most commonly used
organisms. They are defined as facultatively anaerobic, gramnegative, nonsporing, rod-shaped bacteria that ferment lactose with gas formation within 48 hours at
35 °C. Unfortunately, this description includes a wide variety of bacteria, including those that are not
inhabitants of the intestinal tract. Therefore in testing drinking water quality, the presence of
is also determined. These differ from the above definition in that they can grow at the more
restrictive temperature of 44.5 °C.
MULTIPLE CHOICE
For each of the questions below select the one best answer.
1.
2.
3.
Secondary wastewater treatment removes
organic material by which of the following?
a. biological processes
b. physical processes
c. chemical processes
d. All of the above are correct.
During one type of secondary wastewater
treatment, the wastewater flows horizontally
through an agitated aeration tank. What is
this called?
a. lagooning
b. activated sludge treatment
c. trickling filter processing
d. endogenous respiration
Which of the following is NOT an advantage
of anaerobic digestion?
a. Most of the biomass produced
aerobically is utilized for methane
production.
b. The remaining sludge can be dried
easily before disposal.
c. Heavy metals are concentrated in the
4.
5.
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sludge.
All of the above are advantages to
anaerobic sludge digestion.
Which of the following does NOT contribute
to the removal of organic material in leach
fields of home sewage treatment systems?
a. aerobic digestion as the waste
percolates through the soil
b. adsorption of organic material to soil
particle surfaces
c. entrapment of microbes in the pores of
the leach field
d. All of the above contribute to the
removal of organic material.
Which of the following is true about the
Colilert defined substrate test?
a. It can detect total coliforms but not E.
coli.
b. It can detect E. coli but not total coliforms.
c. It can detect total coliforms and E. coli
simultaneously and independently.
d. None of the above is correct.
d.

6.
Constructed wetlands use plants and their
associated microorganisms to remove which
of the following?
a. organic material
b. inorganic material
c. metals
d. All of the above are correct.
7. Which microorganism can usually be
effectively removed by slow sand filtration?
a. Giardia lamblia
b. Cryptosporidium parvum
c. Both (a) and (b) are correct.
d. Neither (a) nor (b) is correct.
8. During which step of wastewater treatment
are bacterial pathogens primarily removed?
a. primary sewage treatment
b. secondary sewage treatment
c. Both (a) and (b) contribute
equally to pathogen removal.
d. Neither (a) nor (b)
significantly remove
bacterial pathogens.
9. Which of the following is used during water
purification to remove large particles such as
sand from raw water?
a. sedimentation basins
b. settling basins
c. rapid sand filters
d. slow sand filters
10. Which of the following organisms is not
readily removed or inactivated to acceptable
levels by conventional water purification and
chlorination?
a. Cyclospora
b. Cryptosporidium
c. viruses
d. None of the above is effectively
removed or inactivated to acceptable
levels.
11. Why is chlorination of water during water
purification increasingly being replaced by
ozonation?
a. because ozonation is particularly
effective at destroying Cryptosporidium
oocysts
b. because chlorination can lead to the
formation of disinfection by-products
(e.g., trihalomethanes), which may be
carcinogenic
c. Both (a) and (b) are correct.
d. Neither (a) nor (b) is correct.
12. Which step of wastewater treatment
physically removes particulate material from
wastewater?
a. primary treatment
b. secondary treatment
c. tertiary treatment
d. All of the above physically remove
particulate matter.
13. Which step of wastewater treatment uses
biological processes to remove dissolved
organic matter from wastewater?
a. primary treatment
b. secondary treatment
c. tertiary treatment
d. All of the above use biological
processes to remove dissolved organic
matter.
14. Why have genetically engineered
microorganisms used in natural environments
not been as effective as was originally
anticipated?
a. They are used as a food source by
protozoa.
b. They frequently do not come in contact
with the materials to be degraded.
c. They do not survive well once released
and cannot compete with indigenous
microbes.
d. All of the above are correct.
METHODS FOR EVALUATING MICROBIAL CHARACTERISTICS OF WATER
For each of the descriptions below, indicate which of the following tests fits the description: most probable
number test (MPN), membrane filtration technique (MFT), presence-absence test (P-AT), defined substrate
test (DST), and polymerase chain reaction tests (PCR).
1. Uses a filter to trap microbial contaminants in the water sample
2. Can be used to detect total coliforms
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3. Can be used to detect fecal coliforms or E. coli
4. Can be used to detect fecal streptococci
5. Can be used to detect fecal viruses
6. Tubes containing test medium are inoculated with different volumes of
water
7. Test medium is inoculated with 100 ml of water
8. Test medium contains ONPG and MUG as the only nutrients
9. Can be used to differentiate nonpathogenic and enterotoxigenic strains of
E. coli
10. Results are adversely impacted by high turbidity, large populations of
noncoliform bacteria, metals, and phenol
11. Relatively rapid (i.e., < 4 days to complete)
TRUE/FALSE
____ 1.
____ 2.
____ 3.
____ 4.
____ 5.
____ 6.
____ 7.
____ 8.
Wastewater treatment uses unique processes that are unlike any that occur in aquatic environments.
Anaerobic sludge digestion resulting in methane production is dependent on the presence of carbon
dioxide, hydrogen, and organic acids to initiate the reaction.
Because of our great dependence on groundwater as a drinking water supply, we have developed a
tremendous understanding of the microorganisms and microbiological processes occurring in this
environment.
If a leach field floods, it becomes anaerobic and effective treatment ceases.
Inadequately treated municipal waste is considered to be a nonpoint pollution source.
Potable water is unfit for consumption or recreation because of the high levels of microbial
contaminants present in it.
Cryptosporidium has recently become of greater concern than Giardia because it is harder to
remove from water.
The survival of microorganisms added to complex microbial communities in soil or water can be
improved somewhat if they are added with materials that provide microhabitats.
CRITICAL THINKING
1.
Describe the primary, secondary, and tertiary (if any) processes used by the sewage treatment facility in
your community. At each stage, describe the method used and the basis for its activity. If your residence
utilizes a septic tank, then describe the functioning of that system.
2.
Discuss the ideal characteristics of an indicator organism for fecal contamination of water. Using these
characteristics, discuss the use of total coliforms, fecal coliforms, and fecal streptococci as indicators.
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Under what circumstances is one a better choice than the others? Why are none of these organisms
particularly useful as indicators of enteric viruses or protozoans?
ANSWER KEY
Terms and Definitions
1. f, 2. i, 3. n, 4. k, 5. a, 6. e, 7. q, 8. g, 9. o, 10. l, 11. j, 12. r, 13. b, 14. u, 15. c, 16. m, 17. t, 1 8. d, 19. h, 20. s,
21. p
Fill in the Blank
1. chemical oxygen demand (COD); total organic carbon (TOC); biochemical oxygen demand (BOD); nitrogen
oxygen demand 2. settling; coagulation 3. sludge; extended aeration; anaerobic digestion 4. septic tank
5. primary; secondary; activated sludge; trickling filters; tertiary 6. potable; sedimentation; coagulation;
filtration; disinfection 7. indicator; coliforms; indicator; fecal coliforms
Multiple Choice
1. a, 2. b, 3. c, 4. d, 5. c, 6. d, 7. a, 8. b, 9. a, 10. d, 11. b, 12. a, 13. b, 14. d
Methods for Evaluating Microbial Characteristics of Water
1. MFT 2. MPN; MFT; P-AT; DST 3. MFT; P-AT; DST; PCR 4. MFT 5. None 6. MPN 7. P-AT; DST 8. DST
9. PCR 10. MFT 11. MFT; P-AT; DST; PCR
True/False
1. F, 2. T, 3. F, 4. T, 5. F, 6. F, 7. T, 8. T
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