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Microbiology
TIL010 Basic Principles
By Ray Layton & Greg Devantier
Microbiology in Waste Water
Treatment
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Types of microbes
Energy metabolism
Environmental factors
Testing
Disinfection
Case study
Objective of Wastewater
Treatment
• Breakdown dissolved and solid organic
materials
• Produce discharge that is
• Low in nutrients
• Low in BOD
• Free of pathogens
• Dispose of the effluent in a safe and
sustainable manner
Microbial Diversity
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Bacteria
Fungi
Parasites
Viruses
Bacteria
• All single cell organisms
• Remain separate in fluid medium
• Form colonies on solid surfaces
• Replication by binary fission
• Simple cell division-mother cell produces two
daughter cells
• Many pose serious health risks
Bacteria
Electron micrograph of E. coli a gram negative rod
shaped bacteria
Bacteria
E.coli strain
0157:H7
E.coli on selective agar, pink colouration from lactose fermentation
Parasites
• Protozoa
• Single cell nucleated organisms
• Giardia sp. Entamoeba
• Multi cellular parasites
• Worms and Flukes
Protozoa
Two distinct
nuclei
Flagella for
motility
Giardia sp.
Viruses
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Non-cellular
No innate metabolic activity
Can only replicate within a host cell
The ultimate parasite
Simple structure consisting of capsid
proteins and nucleic acid
Viral Structure
Head
Tail
End plate
Tail fibres
Pathogens in Sewage
(Bacteria)
From Wastewater Treatment Table 7.1.13 Infection Agents Potentially
Present In Raw Domestic Wastewater
• Escherichia coli
• Legionella
pneumophila
• Leptospira sp.
• Salmonella typhi
• Shigella sp.
• Vibrio cholerae
• Yersinia entercolitica
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Diarrhoea
Respiratory illness (acute)
Fever jaundice
Diarrhoea ulceration
Small Intestine
• Dysentery
• Diarrhoea dehydration
• Diarrhoea
Pathogens in Sewage
(viruses)
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Adenoviruses
Enteroviruses (polio)
Hep A
Norwalk like viruses
Reoviruses
Rotaviruses
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Respiratory disease
GI cardiovascular meningitis
Fever jaundice
Vomiting (shell fish)
Gastroenteritis
Gastroenteritis
Pathogens in Sewage
(Protozoa)
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Balantidium coli
Cryptosporidium
Entamoeba histolytica
Giardia lamblia
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Dysentery
Diarrhoea
Diarrhoea abscesses SI
Diarrhoea nausea
indigestion
Pathogens in Sewage
(Helminths)
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Ascaris lumbricoides
Enterobius vericularis
Fasciola hepatica
Hymenolepis nana
Taenia saginata
T. solmon
Trichuris trichiura
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Roundworm infestation
Pinworm
Sheep liver fluke
Dwarf tapeworm
Beef tapeworm
Pork tapeworm
Whipworm
Biological Mechanisms
Energy and Metabolism in
Microbes
Why do Organisms Need
Food
• Growth and reproduction
• ENERGY
Energy Production
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Aerobic respiration
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Anaerobic respiration (anoxic environments)
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Uses oxygen to produce energy and CO2 from nutrient
breakdown
Nitrate reduction, Sulphate reduction, CO2
Fermentation
Methane
Hydrogen Sulphide
Facultative organisms
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Breakdown compounds with/without O2
Substrates for Energy
Production
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Carbohydrates (sugars and starches)
Lipids (fats and oils)
Proteins
Carbon, sulphur and nitrogenous
compounds
Substrates for Energy
Production
• Carbohydrates are an easier food source
• Fats and oils are more difficult for bacteria
to breakdown in domestic systems
(approximately 10%)
• Specialised Bacteria
• Grease Traps
• Larger primary tank see ASNZS 1547
Conditions That Effect
Microbial Growth
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Temperature
pH
Water availability
Oxygen/nutrient availability
Temperature
• Psycrophiles
• Grow in extremely low temperatures  13°C
• Mesophiles
• Grow in moderate environments  38C
» Found in warm blooded animals and aquatic and
terrestrial environments
• Thermophiles
• Growth in unusually hot environments 80C
• Hyperthermophiles
• Grow in Deep sea thermal/volcanic vents  88 - 105C
pH
• Most natural environments have pH
between 5 and 9
• Physiological pH is 7.4
• Biological Growth Range pH 6.8 to 7.2
• Some specialised organisms can grow
at extremes of pH <2 or >10
Water Availability
• Biological reactions require aqueous
environments
• Osmosis
Common Pollutants of Waste
Water
• Organic Matter
– Any of a number of organic compounds
• Proteins
• Carbohydrates
• Simple
• Complex
• Fats, oils and grease
• Surfactants
• Large complex molecules that cause foaming in
treatment plants
Microbes in Waste Water
Treatment
• Microflora
• Selection
• Biodegradation
Microflora
• Bacteria
• Occur in large numbers in both biofilters AWTS
sand filters and activated sludge systems
• Primary transformation and degradation of
dissolved organic matter
• Degrade suspended organic matter by
producing extracellular enzymes
• Larger numbers relative to other microorganisms
Microflora
• Fungi
• Compete for food with bacteria
• Higher numbers in biofilters
• Can become more prevalent if the pH becomes
low (acidic)
Microflora
• Algae
• Photosynthetic organisms (autotrophic)
• Found mainly on the surface of biofilters
• Require good conditions eg. food and light
Microflora
• Protozoa
• Common in biofilters
• Eat bacteria, fungi, algae and suspended
organic matter
• Low loaded activated sludge plants have higher
numbers
Microflora
• Metazoa
• More complex multi cellular organisms
• Rotifers, crustaceans, animals and insects
• In biofilters and low loaded activated sludge
plants
• Rotifera are an indicator of good biological
treatment
Selection
• Selection is a process in which the
growth of one particular group of
species of microorganisms is favoured
over all others
• Low pH favours fungi
• Anoxic environments favour anaerobic
organisms
Activated Sludge
• Biologically active growths are mixed with incoming
biodegradable waste in an aerobic environment.
• Aerobic process except for the denitrifying stage
• Eventually sediment forms
• Activated sludge – biomass of microorganisms
• Efficiency dependent on the type of micro-organisms
present
• More compact organisms have better settling abilities
Selection in Activated Sludge
Potential
Organism
Aerobic
Use 1
substrate
Activated
sludge
organism
No
Dies
No
Use 2°
substrate
No
Dies of starvation
Yes
Yes
Yes
Settling/flocculation Yes
properties
Survives
temp
Yes
High enough
growth rate
Yes
Freely
suspended
No
Flushed out
Dies of heat or cold
Flushed out
Dies
Biodegradation
Slowly degradable material
Hydrolysis
Easily degradable material
Biological growth
Biomass
Decay
Inert material
Sampling
• Wastewater sampling and analysis
conducted to determine
• The physical, chemical and biological
characteristics
• Concentrations of constituents
• The best methods for reducing or removing the
pollutants
• Location
Sampling
• First outlet downstream of the treatment plant (often
taken from the pump chamber)
• BOD/SS
• Clean bottle fill to top (no air space)
• Thermotolerent Coliform
• Sterile bottle
• Chlorine neutralizing agent
• Air space in bottle
• Storage and Transport
• Transport at 4 degrees C and deliver to lab within
6hrs if possible 24hrs maximum. Microbes make for
a dynamic system – process samples promptly
Tests and Analysis
• Laboratory tests
– BOD-Biochemical oxygen demand
– Thermotolerent coliform
– Suspended solids
• Field Tests (see reading 11.2)
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Sludge volume 30 minutes (SV30)
Sludge depths
Dissolved oxygen
pH
Temperature
Total chlorine
Total nitrogen
Total phosphorous
BOD
• Biochemical Oxygen Demand
• Widely used parameter in monitoring pollution and
wastewater
• Sample seeded with bacteria
• BOD5 20 measurement of the dissolved oxygen used
over a five day period in the biochemical oxidation of
organic matter
• Must be performed at a constant temperature
• Generally at 20°C
• Over a five day period oxidation is between 60 and
70% complete
Coliforms
• A large group of facultative bacteria
capable of fermenting lactose
• Natural flora of the human GIT
Faecal coliforms
• Also known as thermotolerant coliforms
• Found in the GIT of humans and warm
blooded animals
• E. coli
• Indicator of faecal contamination
• Indicator of Overloaded systems
Testing for Faecal Coliforms
Membrane-Filter Method
• Sample drawn
through filter
membrane by
vacuum
• Put into contact with
selective agar
• Colonies counted
relative to the
volume of fluid used
Dissolved Oxygen
• Measured using an oxygen electrode
• Indicator of the level of aeration within a
system
Suspended Solids
• Can lead to sludge deposits and
anaerobic conditions when inadequately
treated water is discharged into aquatic
environments
• Hinders adequate disinfection
• Set volumes of fluid are filtered
(1.2 m)and the filter is dried then
weighed
Effulent compliance criteria
• BOD5
• 20 mg l-1
• SS
• 30 mg l-1
• Faecal coliforms
• 200cfu 100 ml-1
• Total chlorine
• Greater than or equal to 0.5ppm and less than 2.0 ppm in
four out of five samples taken
• Total N & P
• < 10 mg l-1 & 5 mg l-1 respectively
Data obtained from Queensland Plumbing and Wastewater Code.
Disinfection
• A process that facilitates the removal of
pathogenic organisms
• A disinfectant is an agent that kills
microorganisms but may also be
harmful to animal tissue
• Some microorganisms are very
resistant to disinfection
Desirable Characteristics of
Disinfectants
• Toxic to microorganisms
– High toxicity at high dilutions
• Soluble
– Must be soluble in water and cell tissue
• Nontoxic to higher life forms
– Limited effects on humans and other mammals
• Homogeneity
– Solutions must be uniform in composition
• Interactions with extraneous material
– Should not be absorbed by organic mater other than
bacterial/Viruses/cells
Desirable Characteristics of
Disinfectants
• Toxicity at ambient temperatures
– Must retain toxicity at working temperatures
• Penetration
– Should penetrate surface
• Noncorrosive
– Should not disfigure metals or clothing
• Deodorising
– Should remove odours while disinfecting
• Availability
– Should be freely available and reasonably priced
Factors Effecting Disinfection
• Contact time
• Concentration and type of chemical
agent
• Temperature
• Intensity and nature of physical agents
• Type and number of organisms
• Nature of suspending fluid
Factors Effecting Disinfection
• Contact time
• Generally, the observation is that for a given
concentration, the longer the contact time the greater the
kill rate
• Concentration of chemical agent
• Higher concentration require less time for microbial killing
• Numbers of organisms
• Greater numbers of organisms take longer to kill
Factors Effecting Disinfection
• Types of organisms
• Spore forming organisms are extremely difficult
to kill with chemical means. Also heat stable
and not prone to dehydration (Bacillus sp.)
• Suspending liquid
• Liquid high in organic material will reduce the
effectiveness of oxidising disinfectants
Disinfectants used to Treat
Wastewater Effluent
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Chlorination
Ozone
UV light
Sand polishing
Chlorination
• Strong oxidising agent
• Good points
• High toxicity, soluble, stable, homogeneous,
temp, high penetration, good deodoriser and
low cost
• Bad points
• Highly toxic to higher life forms, oxidises
organic matter and highly corrosive (see Report
department of environment)
Chlorine Chemistry
• Chlorine reacts with ammonia to form
chloramines – much slower acting
disinfectants
• Addition of chlorine needs to continue
beyond “breakpoint”
Breakpoint
Destruction of
chloramines
Chlorine
residual
mg l-1
Formation
of
chloramines
Formation of free
chlorine for disinfection
Free residual
Breakpoint
Chlorine added mg l-1
Combined residual
Other Factors Reducing
Chlorination Efficiency
• Acid Generation
–H+ produced reduces the pH
–Chlorine only active between
pH 6.5 and pH 8.2
–Only 5% efficiency outside this
range
Dechlorination
• Sulphurdioxide
• Reacts with both chlorine and chloramines
• Instantaneous reaction means residual times
are not a critical factor
• Activated carbon
• Complete removal of both combined and free
residual chlorine
• Gravity or pressure filter bed
• Expensive
Ozone Disinfection
• Primarily used to control taste, odour
and colour producing agents
• Improved ozone generation techniques
now allow it to be used for wastewater
disinfection
• Oxygen radicals are strong oxidising
agents
Ozone Generation
• Ozone unstable so must be generated
on site from air or pure oxygen
• High voltage applied across a narrow
gap between two electrodes
• Free radicals HO2 and HO
Mode of Action
• Strong oxidising radicals destroy the cell
wall of bacteria causing cell lysis
• Oxidation of protein coat of viral capsids
and is more effective than chlorine
Other Considerations
• Mainly beneficial effects in the
environment
• Ozone residuals are highly unstable and
degrade quickly
• No residuals in effluent
• Decomposition results in higher
dissolved oxygen levels
UV Light Disinfection
• Electromagnetic radiation (light)
• Can be explained in terms of a wave or a
particle
• All chemical compounds have a maximum
absorption at a specific wavelength
• The light energy absorbed by a compound
can be given off as light or heat
• Light energy can also be used to modify
chemical compounds
UV Light Disinfection
• UV light has wave lengths below 300
nm
• Generated by Hg vapour lamps for
monochromatic output
• UV light is very high energy
• In spite of high energy, only effective
through relatively short distances in
aqueous media
Mode of Action
• UV at 254 nm penetrates cell walls of
bacteria and viral capsids
• Nucleic acids DNA and RNA have a
maximum absorption at 260 nm
• UV irradiation damages genetic material
causing the inefficient replication of cells
or cell death
Other Considerations
• Physical rather than chemical means of
disinfection
• No toxic residues
• Process must be optimised – thin film
method of passing effluent over lamps
(see reading 10.11)
Sand Polishing
• Intermittent sand filtration
• Excess SS and other organic matter is
removed by physical straining and biological
degradation
• Filtration achieved due to particle size of sand
• Particulate nature of sand produces high
surface area to volume ratio for biofilms
Risk Assessment
• Hazard level to occupants
• Hazard level to community
• Hazard level to environment
Control measures
– Separation distances (see guide lines)
– Secondary treatment
– Advanced treatment Nutrient Removal (see
wastewater biology)
– Disifection
– Subsurface, Covered surface, Spray Irrigation
Disinfection
method
Effective against
Viruses
Comments
Bacteria
Protozoa
Ozone
yes
yes
Yes
Works well,
expensive, generated
on site.
Ultra violet
Yes
Yes
Yes
Ditto but affected by
high S.S
Sand Filter
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Yes?
Yes
Size of organism?
Wetlands
Yes?
Yes?
Yes?
Can be effected by
environmental
conditions
Chlorine
Not all
Yes
?
Not effective against
all organisms, cheap
easy & familiar,
harmful to the
environment
Effluent Disposal
• Recommendations from local
authorities/designers depending on risk
assessment
• Subterranean trenches/irrigation
• Sprinkler systems
• Mulch beds
Trenches
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Low maintenance
Convenient
Relatively safe in ideal conditions
Expensive
Not suitable for heavy clay soils
Can contaminate ground water
Sprinkler Systems
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Relatively inexpensive
Easily maintained
Can Create aerosols
Require disinfection of effluent
Require compliant home owner
Mulch Beds
• Discharge lines placed under mulch
beds in gardens
• No creation of aerosols
• Low maintenance
• Relatively low cost
• Require moderately compliant home
owner
Case Study
Walkerton Canada
May 2000
The Events
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Walkerton Canada population 4700
Farming district
Town drinking water derived from wells
Farmer located near well 5 spread
manure on his fields as fertilizer
The Results
• Well 5 became contaminated with
coliforms from the fertilized field
• 2300 people became ill
• Several hundred suffered renal
infections requiring dialysis
• Many suffered permanent renal damage
• Seven people died as a result of the
contamination
The Organisms
• The well was found to be contaminated
with Campylobacter jejuni and E. coli
0157:H7
Campylobacter jejuni
• Bacteria
• Produces a cholera like enterotoxin that
causes diarrhoea
E.coli 0157:H7
• E. coli are normal GIT flora and nonpathogenic
• Phage mediated exchange of genetic
material from Vibrio chloerae gives
E. coli genes encoding enterotoxins
• Causes acute renal failure
The Reason
• Inadequate sampling of water quality at
the wells
• No total colifrom testing carried out
• No chlorination at the well
• It was estimated that E. coli 0157:H7
could survive at least 25 days in the
soil, 50 days in cooler weather
Summary
• Important to understand
– Microflora of waste water treatment
– Processes of biodegradation
– Disinfection methods
– Monitoring processes
– Possible outcomes
Definitions
– Biofilm
» Microbial colonies encased in an adhesive, usually polysaccharide,
attached to a surface
– Biomass
» Total quantity of living matter within a habitat expressed as weight for
unit area or volume
– Hydrolysis
» Breakdown of larger molecules into smaller directly degradable
molecules by the addition of water – digestion
– Biological growth
» The rate of increase in living microbes for a given unit area or volume
– influenced by environmental factors
– Decay
» Reduction in microbial numbers through cell death and predation
(endogenous)
– Colloid
» Suspension of matter in one phase in another – smoke, mist,
turbid fluid
– Flocculation
» The aggregation of suspended particles to form a precipitate