AMJ30804

WASTEWATER ENGINEERING
Chapter 2 – Wastewater Microbiology
Semester II, 2022/2023

By: Ir. Ts. Siti Salwa Othman

[email protected]
Course Objective

Ability to analyze the

knowledge of wastewater
engineering
Course Outline
Wastewater Microbiology
 Why treat wastewater?
 Brief the wastewater treatment plant.
 Important microbial processes.
 Microbiol monitoring.
 Water Quality Standard and Water Quality Guidelines.
 Why We Need Wastewater
Treatment Plants
Goal of Wastewater Treatment
 Protech Health.
 Preserve Natural Resources.
 Prevent Ecological Damage.
How to Accomplish These Goals
 Use Wastewater Treatment Plants (WWTP).
 WWTP removes energy rich organic matter
before discharge into the environment.
 Uses technology to prevent/lower the
occurrence of water born diseases.
However, If Not Treated?
People can get sick from pathogen contaminated water.
Problem occur in developing nations.
Releasing wastewater directly into a stream leads to O2
depletion.
 This is caused by aerobic respiration linked to high
organic carbon loads in the water.
 Low O2 in water can cause fish kills.
The degree of O2 consumption in wastewater can be
quantified by BOD.
Video: Sewage Treatment Plant

 Video Sewage Treatment Plant:

 Sewage Treatment

Secondary Treatment
Primary Treatment
Decrease dissolved organic
Non biological treatment
carbon.
starting screening process.
Uses biological treatment
Removes solids and debris.
such as Aeration.
Waste has nutrient load
Aerobic and anaerobic
(e.g. Carbon and Nitrogen).
secondary treatment.
Sludge settle at the bottom
Oxidation pond is shallow,
tank via sedimentation
design at 2-40 feet.
process.
Sludge settle, collect and
dispose.
Microbiology of Anoxic Secondary
Treatment
Microbe is used for breaking down solid waste.
Done is an anoxic sludge digester.
Reaction: Solid waste are complex polymer such as cellulose
and fiber.
 Common polymer: Microbes Secret Lipase, Protease and Amylases.
Fermentation is the major metabolism in this treatment.
Lots of methane is produced by Methanogenic Achaea.
Wastewater Treatment Plant (WWTP) biogas digester: Acetate
utilizing methanogens can produce ~ 70% methane gas.
 CH4 can be collected and used to generate electricity.
Anoxic is a condition in
which aquatic (water)
environment does not
contain Dissolved Oxygen
(DO).
Which is called an oxygen
deficient condition.
 Aerobic Secondary
Waste Treatment

Trickling Filter Activated Sludge

 Convenient Technique Concept: Air bubble system through
 Trickling filter is a bed of crushed waste water in the aeration tank.
rocks where non treated sewage is After microbial reaction large flocs
trickled over it, give tots of surfaces are form and allow to settle out.
of microorganism to attach to. Zoogloea Ramigera is one of the key
 Complete mineralization of waste to species that form a slime that is the
CO2, ammonia, nitrate, sulfate and base of the flocs.
phosphate. Problem: Filamentous bacteria can
 Same process occurring in fish cause sludge bulking and sludge
aquarium. thickens, collect and dispose.
 Video: Aerobic Secondary Waste
Treatment

 Video Trickling Filter:

 Video Activated Sludge:
Type of Contaminant in Sewage
Type on contaminant or impurities present in sewage:
 Organic impurities.
 Inorganic impurities.
 Nutrients.
 Saprotrophic bacteria (soil bacteria).
 Disease causing bacteria.
Important Microbes in The Sewage
Treatment Plant
Nitrifying Bacteria
 Aerobes.
 Convert Nitrogenous waste into nitrate.
Denitrifying Bacteria
 Anaerobes.
 Convert Nitrate to N2.
Methanogens
 Generate methane from acetate.
 Or use H2 and CO2 to make methane.
 Mostly Archaea.
Nitrifying Bacteria
Ammonia is converted in nitrate.
Ammonia has a high BOD because NH3 oxidation requires O2.
Two group of microbes are involved:
 Ammonia oxidizing bacteria (AOB).
 Nitrate oxidizing bacteria (NOB).
AOB oxidize NH3 to NO2- in two (2) steps:
 Ammonia monooxygenase (AMO).
 Hydroxylamine oxidoreductase (HAO).
NOB oxidize NO2- to NO3-
 Uses NOR enzyme complex.
Both AOB and NOB respire oxygen.
Common Waterborne Pathogens
Organism Disease
Bacteria
 Salmonella Typhi Typhoid Fever, Enteric Fever
 Shigella Bacillary Dysentery
 Vibrio Cholerea Cholera
Virus
 Poliomyelitis Polio
 Other Viruses Meningitis, Hepatitis
Protozoa
 Entamoeba Histolytica Amoebic Dysentery
 Giardia Lamblia Giardia Enteritis
Helminths
 Echinococcus Echinococcosis
Sewage Treatment and Environmental
Monitoring
Monitoring effluents and surrounding environment is important.
 Assess the efficacy of the treatment process.
 Testing: C, N, P, metals, microbes, effluent toxicity.
It is often too difficult to directly monitor a specific pathogens
or virus and phage.
What is an Indicators Organism
An organism that can be readily cultured that indicates the presence
of a pathogenic microorganism or correlates to a health problem.
Widely used in determination and estimation of water contamination
correlated to the presence of pathogens.
Why used?
 Population large enough to isolate in small water samples
(100ml).
 Rapid.
 Inexpensive.
 Safe, not culturing pathogens.
U.S. Public Health Services adopted coliforms as indicators of
fecal contamination of drinking water in 1914:
 Coliforms are bacteria that live in the intestines of
warm-blooded animals and are excreted in high numbers
in feces.
 Indicate fecal contamination of drinking water.
 Presence demonstrates a breakdown of wastewater
treatment process.
Other uses:
 The food industry uses other indicator microorganisms to
evaluate the efficiency of food processing.
Criteria for an Indicators Organism
Should be useful for all types of water (drinking water, wastewater,
recreational water, sea water).
Should be present whenever enteric pathogens are present and absent.
Should survive longer in the environment than the toughest enteric
pathogen.
Should not grow in water.
Detection protocols should be easy and inexpensive.
Density of indicator microorganisms should correlate with the degree of
fecal pollution.
 Should be a member of the normal intestinal microflora of warm-
blooded animals.
Bacterial Indicator Organisms Common
Group
1) Total Coliforms
2) Fecal Coliforms
3) Fecal Streptococci
4) Anaerobic Bacteria
5) Bacteriophage
Estimated Levels of Indicator
Organisms in Raw Sewage
Organism CFU/100ml
Coliforms 107 - 109
Fecal Coliforms 106 - 107
Fecal Streptococci 105 - 106
Entercocci 104 - 105
Clostridium Perfringens 104
Staphylococcus 103
Psedudomonas Aeruginosa 105
Acid Fast Bacteria 102
Coliphages 102 - 103
Bacteroides 107 - 1010
Characteristic of Total Coliforms
Commonly used indicator for drinking water, wastewater treatment,
shellfish harvesting water and recreational water.
Aerobic for facultatively anaerobic.
Gram negative.
Non spore forming.
Rod shaped
Gas production during lactose fermentation withing 48 hours at
35°C.
Examples: Escherichia, Citrobacter, Klebsiella and Enterobacter.
High numbers (2 x 109 per capita per day ~ 218 per capita day) in
human and animal feces.
Disadvantage of Total Coliforms
Coliforms may grow in aquatic environments, particularly if organic
matter levels and temperature are elevated.
Coliforms may from biofilms in drinking water distribution systems – this is
a problem because for example E. coli is 2400 times more resistant to
chlorine in a biofilm than when planktonic.
Coliforms may recover from disinfectant injury.
Growth of heterotopic bacteria on media selective for coliforms can mask
coliform population in water (occurs when heterotrophic counts exceed
500/ml).
More vulnerable to disinfection and environmental trauma than enteric
viruses or parasite.
Do not necessarily indicate fecal contamination.
Characteristic of Fecal Coliforms
Subgroup of total coliforms.
Able to ferment lactose and produce both acid and gas at 44.5°C
in 24 hours.
Include Escherichia and Klebsiella which are exclusively fecal.
Disadvantage of Fecal Coliforms
Same disadvantage as for total coliforms.
Indicates fecal contamination for sure but can’t distinguish
between animal and human feces.
Can survive and grow for extended periods of time in tropical
waters.
Characteristic of Fecal Streptococci
Do not multiply in water.
Are more resistant to stress and disinfection.
Last longer in environment.
Used as indicators of enteric viruses and gastroenteritis for
swimmers.
Members of the lactic acid bacteria.
Gram positive, non motile, non spore forming, aerotolerant
anaerobic bacteria that ferment sugars to lactic acid.
Disadvantage of Fecal Streptococci
FC/FS ratio: Ratio of fecal coliform counts to fecal strep counts.
FC/FS > 4: Fecal contamination of human origin.
FC/FS < 0.7: Fecal contamination of animal origin.
This relationship is only valid for recent fecal contamination
(within the last 24 hours).
Characteristic of Anaerobic Bacteria
Clostridium Perfringens as one example.
Gram positive, anaerobic spore forming rod shaped bacterium.
Spores are heat resistant (can survive 75°C for 15 min), resist
disinfection can remain viable in the environment for a long time.
Used as indicator of resistant pathogens (viruses, parasites), past
fecal contamination or tracing fecal contamination in a marine
environment.
Disadvantage of Anaerobic Bacteria
A common soil bacterium may not necessarily indicate fecal
contamination.
Pathogenic (causes gas gangrene if it infects wounds, produces
enterotoxin in small intestine causing gastroenteritis)
Anaerobic culture is difficult.
Characteristic of Bacteriophage
Coliphage as one example.
Bacteriophage that infect coliforms, particularly E. Coli.
Similar enteric viruses in size, morphology and performance in
environment.
Found in higher numbers than enteric viruses in wastewater and
other waters.
Rapid and easy detection methods available.
Survive for 7 days in shellfish without increasing in numbers.
Routinely used as indicator microorganisms to determine the
effectiveness of wastewater treatment process.
Resistant to disinfection.
Detection Methods
Most Probable Number (MPN).
Membrane Filter Test.
Presence Absence Test such as Colilert.
Test Heterotrophic Plate Counts (HPC)
Plate Assay.
Most Probable Number (MPN)
Used to detect coliforms.
MON is used to monitor waste effluents, drinking water system and
recreational waste.
High MPN results can lead to beach closures.
In a drinking water system, a positive for coliform is huge deal. This
would set off a series of events to find the sources of coliform
contaminant.
MPN test consists of three (3) steps:
 Presumptive Test.
 Confirming Test.
 Completed Test.
 Presumptive Test: Dilute Water Sample
 Inoculate 3 to 5 tubes of lauryl sulfate tryptose lactose broth
containing upside down Durham tubes with water dilutions.
 Incubate at 35°C for 48 hours.
 Determine number of tubes at each dilution that are positive for
gas production (contain bubble in Durham tube).
 Confirming Test:
 Select a positive tube and inoculate a Levines EMB agar and
Endo Agar plate
 Completed Test:
 Inoculate a colony back into MPN media and confirm acid and
gas production.
Sample (MPN) Table
Membrane Filter Test
Used to detect Coliforms.
Filter 100 ml water through a 0.45 μm filter.
Incubate filter on pad soaked with differential medium (Endo
Medium; contains lactose and Basic Fuchsin dye) at 35°C for hours.
Count colonies that grow filter.
 Coliforms will be dark red with metallic gold sheen.
 To enumerate Fecal Streptococci, grown on Streptococcus agar at
37⁰C for 24 hours. Fecal streptococcic reduce 2,4,5-triphenyl
tetrazolium chloride to formazan, which makes colonies appear
red.
 Much quicker and easier than MPN method.
Conclusion
Because on their diverse capabilities (microbes) to degrade organic
materials, microorganisms are exploited for the treatment of wastewater
(sewage).
Secondary wastewater treatment relies on the power of microorganisms
to breakdown solid waster into smaller molecules that other microbes can
convert into nutrients.
Nutrients are further converted either by nitrifying bacteria into nitrate
then denitrifies converts nitrate to N2 gas.
The effluents released into the environment should have a low BOD and
low number of pathogens.
Monitoring is done to quantify indicators of pathogens and BOD to make
sure the environment is safe for human and wildlife.
 Thank you
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