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TIL010 Basic Principles By Ray Layton & Greg Devantier
Microbiology TIL010 Basic Principles By Ray Layton & Greg Devantier
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Microbiology in Waste Water Treatment
Types of microbes Energy metabolism Environmental factors Testing Disinfection Case study
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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
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Microbial Diversity Bacteria Fungi Parasites Viruses
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Bacteria All single cell organisms Replication by binary fission
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
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Bacteria Electron micrograph of E. coli a gram negative rod shaped bacteria
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Bacteria E.coli strain 0157:H7 E.coli on selective agar, pink colouration from lactose fermentation
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Parasites Protozoa Multi cellular parasites
Single cell nucleated organisms Giardia sp. Entamoeba Multi cellular parasites Worms and Flukes
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Protozoa Two distinct nuclei Flagella for motility Giardia sp.
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Viruses 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
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Viral Structure Head Tail End plate Tail fibres
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Pathogens in Sewage (Bacteria) From Wastewater Treatment Table 7. 1
Pathogens in Sewage (Bacteria) From Wastewater Treatment Table Infection Agents Potentially Present In Raw Domestic Wastewater Escherichia coli Legionella pneumophila Leptospira sp. Salmonella typhi Shigella sp. Vibrio cholerae Yersinia entercolitica Diarrhoea Respiratory illness (acute) Fever jaundice Diarrhoea ulceration Small Intestine Dysentery Diarrhoea dehydration
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Pathogens in Sewage (viruses)
Adenoviruses Enteroviruses (polio) Hep A Norwalk like viruses Reoviruses Rotaviruses Respiratory disease GI cardiovascular meningitis Fever jaundice Vomiting (shell fish) Gastroenteritis
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Pathogens in Sewage (Protozoa)
Balantidium coli Cryptosporidium Entamoeba histolytica Giardia lamblia Dysentery Diarrhoea Diarrhoea abscesses SI Diarrhoea nausea indigestion
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Pathogens in Sewage (Helminths)
Ascaris lumbricoides Enterobius vericularis Fasciola hepatica Hymenolepis nana Taenia saginata T. solmon Trichuris trichiura Roundworm infestation Pinworm Sheep liver fluke Dwarf tapeworm Beef tapeworm Pork tapeworm Whipworm
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Biological Mechanisms
Energy and Metabolism in Microbes
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Why do Organisms Need Food
Growth and reproduction ENERGY
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Energy Production Aerobic respiration
Uses oxygen to produce energy and CO2 from nutrient breakdown Anaerobic respiration (anoxic environments) Nitrate reduction, Sulphate reduction, CO2 Fermentation Methane Hydrogen Sulphide Facultative organisms Breakdown compounds with/without O2
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Substrates for Energy Production
Carbohydrates (sugars and starches) Lipids (fats and oils) Proteins Carbon, sulphur and nitrogenous compounds
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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
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Conditions That Effect Microbial Growth
Temperature pH Water availability Oxygen/nutrient availability
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Temperature Psycrophiles Mesophiles Thermophiles Hyperthermophiles
Grow in extremely low temperatures 13°C Mesophiles Grow in moderate environments 38C Found in warm blooded animals and aquatic and terrestrial environments Thermophiles Growth in unusually hot environments 80C Hyperthermophiles Grow in Deep sea thermal/volcanic vents C
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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
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Water Availability Biological reactions require aqueous environments
Osmosis
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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
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Microbes in Waste Water Treatment
Microflora Selection Biodegradation
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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 micro-organisms
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Microflora Fungi Compete for food with bacteria
Higher numbers in biofilters Can become more prevalent if the pH becomes low (acidic)
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Microflora Algae Photosynthetic organisms (autotrophic)
Found mainly on the surface of biofilters Require good conditions eg. food and light
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Microflora Protozoa Common in biofilters
Eat bacteria, fungi, algae and suspended organic matter Low loaded activated sludge plants have higher numbers
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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
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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
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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
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Selection in Activated Sludge
Potential Organism Yes Activated sludge organism Aerobic Dies No Use 1 substrate No Use 2° substrate No Dies of starvation Settling/flocculation properties Yes Yes Survives temp Yes High enough growth rate Yes Freely suspended Flushed out No Dies of heat or cold Flushed out Dies
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Biodegradation Slowly degradable material Easily degradable material
Decay Hydrolysis Easily degradable material Biological growth Biomass Inert material
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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
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Sampling Location BOD/SS Thermotolerent Coliform Storage and Transport
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
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Tests and Analysis Laboratory tests Field Tests (see reading 11.2)
BOD-Biochemical oxygen demand Thermotolerent coliform Suspended solids Field Tests (see reading 11.2) Sludge volume 30 minutes (SV30) Sludge depths Dissolved oxygen pH Temperature Total chlorine Total nitrogen Total phosphorous
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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
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Coliforms A large group of facultative bacteria capable of fermenting lactose Natural flora of the human GIT
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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
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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
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Dissolved Oxygen Measured using an oxygen electrode
Indicator of the level of aeration within a system
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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
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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.
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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
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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
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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
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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
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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
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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
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Disinfectants used to Treat Wastewater Effluent
Chlorination Ozone UV light Sand polishing
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Chlorination Strong oxidising agent Good points Bad 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)
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Chlorine Chemistry Chlorine reacts with ammonia to form chloramines – much slower acting disinfectants Addition of chlorine needs to continue beyond “breakpoint”
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Breakpoint Chlorine added mg l-1 Destruction of chloramines
Formation of chloramines Breakpoint Combined residual Free residual Formation of free chlorine for disinfection Chlorine added mg l-1 Chlorine residual mg l-1 Destruction of chloramines
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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
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Dechlorination Sulphurdioxide Activated carbon
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
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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
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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
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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
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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
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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
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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
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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
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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)
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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
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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
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Disinfection method Effective against Comments Viruses Bacteria
Protozoa Ozone yes Yes Works well, expensive, generated on site. Ultra violet Ditto but affected by high S.S Sand Filter ? Yes? Size of organism? Wetlands Can be effected by environmental conditions Chlorine Not all Not effective against all organisms, cheap easy & familiar, harmful to the environment
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Effluent Disposal Recommendations from local authorities/designers depending on risk assessment Subterranean trenches/irrigation Sprinkler systems Mulch beds
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Trenches Low maintenance Convenient
Relatively safe in ideal conditions Expensive Not suitable for heavy clay soils Can contaminate ground water
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Sprinkler Systems Relatively inexpensive Easily maintained
Can Create aerosols Require disinfection of effluent Require compliant home owner
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Mulch Beds Discharge lines placed under mulch beds in gardens
No creation of aerosols Low maintenance Relatively low cost Require moderately compliant home owner
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Case Study Walkerton Canada May 2000
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The Events Walkerton Canada population 4700 Farming district
Town drinking water derived from wells Farmer located near well 5 spread manure on his fields as fertilizer
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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
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The Organisms The well was found to be contaminated with Campylobacter jejuni and E. coli 0157:H7
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Campylobacter jejuni Bacteria
Produces a cholera like enterotoxin that causes diarrhoea
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E.coli 0157:H7 E. coli are normal GIT flora and non-pathogenic
Phage mediated exchange of genetic material from Vibrio chloerae gives E. coli genes encoding enterotoxins Causes acute renal failure
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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
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Summary Important to understand Microflora of waste water treatment
Processes of biodegradation Disinfection methods Monitoring processes Possible outcomes
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Definitions Biofilm Biomass Hydrolysis Biological growth Decay Colloid
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
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