Flash Cards Revision Page

Main aims of wastewater treatment

1. ensure water quality

2. potential common source of infectious disease

3. prevent pollutants including chemicals from industry, endocrine disrupting chemicals, disinfection by-products

Eutrophication

biological process where water become excessively enriched with nutrients, like nitrogen and phosphorus, causing a rapid increase in algae and aquatic plant growth, (an algal bloom)

Activated Sludge

1. Screening and Grit Removal = Wastewater passes through screens & grit chambers to remove debris

2. Primary Sedimentation (Primary Clarifiers) =solids settle as primary sludge, liquid (primary effluent) moves on

3. Aeration tank: microbes feed on suspended organic matter, grow, and form FLOCs, organic matter → CO₂, water, new microbial biomass

4. Secondary Clarification (Secondary Settling Tank) = flocs settle as activated sludge, treated effluent overflows for discharge

5. Return Activated Sludge = portion of settled sludge returned to aeration tank → maintains high microbial concentration

6. Waste Activated Sludge = Excess sludge: removed as waste activated sludge, thickened, digested (anaerobic/aerobic), dewatered → disposal or reuse

FLOCs 

• Clusters of microorganisms, organic matter and particles

• Made of filamentous bacteria, small colonies & inorganic particles

• Held together by sticky exocellular material (“glue”)

• FLOC shape & structure determine how well solids settle in secondary clarifier

• Good FLOC structure → better settling → better wastewater treatment

FISH (fluorescent in Situ hybridisation)

• Uses fluorescently labelled probes to identify specific genetic material in flocs → reveals microbial communities & abundance.

• Probes bind to specific DNA/RNA sequences (e.g. 16S rRNA) → visualises bacteria types & arrangement under fluorescent microscopy.

• With staining for storage compounds (PHA, polyP), FISH helps identify phosphorus-removing populations in activated sludge.

Role of Protozoa

• Require organic carbon as their carbon source.

• Feed on bacteria, controlling bacterial numbers and reducing turbidity.

• Act as predators of suspended and floc-associated bacteria.

• Maintain bacterial balance → clearer effluent.

• Type and abundance indicate system performance.

• Attach to flocs and secrete extracellular polymeric substances (EPS) → help bind particles, improving sludge and water separation in the clarifier.

Nitrification

• Ammonia (NH₃) is oxidised → nitrite (NO₂⁻) → nitrate (NO₃⁻) to generate energy (ATP) for bacterial growth.

• Produces little energy, so nitrifying bacteria are slow-growing and need a long residence time in the system.

Nitroso bacteria

bacteria that carry out ammonia oxidation

nitro bacteria

bacteria that oxidise nitrite to nitrate

Aerobic Chemolithoautotrophs

• Obtain cell carbon from CO₂ and energy from inorganic nitrogen compounds.

• CO₂ fixation is energy demanding, so many use organic carbon sources when available.

Autotrophic ammonia or Nitroso-bacteria (AOB) 

• Gram-negative, aerobic chemolithoautotrophs (Betaproteobacteria or Gammaproteobacteria).

• Use inorganic carbon (CO₂) as carbon source and gain energy by oxidising ammonia (NH₃ → NO₂⁻).

• Oxygen-consuming process that occurs in aerobic zones of the aeration tank.

• Released energy is used to fix CO₂ into cell material during nitrification.

Ammonia Oxidation

1. Ammonia + oxygen +  —> Hydroxylamine + water 

• enzyme = ammonia monooxygenase, situated in cell membrane 

• source of oxygen = molecular oxygen 

• no ATP

2. Hydroxylamine + oxygen —> nitrite + water + ATP 

• enzyme = hydroxylamine oxido-reductase, situated in periplasm

• source of oxygen = water 

Nitrite Oxidation in nitrification process

1. 2NO2 + O2 —> 2NO3 

• enzyme = nitrite oxido reductase, situation in cell membrane 

• energy yield is low 

• complex system for ATP production which can change depending on conditions

Nitroso Bacteria in Activated Sludge

• Organised into clusters of hundreds of cells.

• Distribution of Nitrosomonas AOB varies with treatment plant type and operating conditions, reflecting adaptation to local conditions.

• Some plants are dominated by one species, others have up to five.

• Nitrosococcus common in plants with high ammonia loads.

• Nitrosospira rarely found in activated sludge — better adapted to low-substrate environments, so outcompeted in activated sludge systems.

Nitro Bacteria in Activated Sludge Systems

• also in clusters 

• physically located next to clusters of AOB 

• syntropy = combine metabolic capabilities to break down single substrate, neither can degrade alone 

• AOB supply their energy source nitrite 

• NOB remove toxic nitrite from AOB cells

How is N removed from wastewater in activated sludge process

1. Nitrification (aerobic step) 

• Ammonia oxidised to nitrate by bacteria 

• Nitrosomonas: converts ammonia —> nitrite (NO2-) 

• Nitrobacter: converts nitrite —> nitrate (NO3-)

2. Denitrification (anoxic step - no oxygen)

• nitrate converted to nitrogen gas by denitrifying bacteria

• release harmless N2 gas that escaped to the atmosphere

Denitrification

nitrate (NO3-) or nitrite (NO2-) changed into nitrogen gas (n2) that escapes to the air 

NO3- —> NO2- —> NO —> N2O —> N2 

• happens in anoxic zone (no oxygen, but nitrate present)

• done by heterotrophic bacteria (use organic C for energy) 

• needs and organic carbon source (like methanol or acetate) 

• Azoarcus and thauera common denitrifying bacteria 

• if N2O (nitrous oxide) forms instead of N2 = bad = its a greenhouse gas and air pollutant

ANAMMOX process (ANaerobic AMMonia OXidation)

• converts ammonia + nitrite —> nitrogen gas + water without oxygen

• done by plantomycetes in a special compartment called the anammoxosome

• anammoxosome has ladderane lipids that protect the cell from hydrazine

• energy-efficient, cheap N removal process

using ANAMMOX for treating wastewater

• suitable for N removal from wastes with high ammonium content and low C:N ratios

• required no C addition or aeration

• still needs nitrite as energy source, can be supplied by AOB

process using ANAMMOX for treating wastewater

• Nitrite (NO₂⁻) first reduced to NO by nitrite reductase.

• Hydrazine synthase and hydrolase form hydrazine (N₂H₄) from NO + NH₄⁺ by reducing NO and oxidising NH₄⁺.

• Hydrazine is then oxidised → N₂ by an enzyme similar to hydroxylamine oxidoreductase (like AOB).

• Very slow-growing chemolithotrophs that use CO₂ as carbon source.

use of FISH in wastewater treatment

• detects which bacteria are present (e.g., nitrifiers, denitrifies, filamentous bacteria)

• track population shifts during nitrification/ denitrification or sludge bulking events, monitors process health 

• observe where microbes live in FLOCs or biofilms to study spatial organisation (e.g., nitrifiers near surface, denitrifies deeper inside)

Microautoradiography (MAR)

• technique used to study metabolic activity of microorganisms

• uses radioactively labelled compounds that microbes incorporate when metabolically active (taking up and using substrates) 

Microautoradiography (MAR) use in wastewater treatment

• determines which bacteria are metabolically active in situ 

• shows which groups use carbon, nitrogen, or phosphorous compounds 

• confirms that a detected organism (via FISH) us actively performing a process (nitrification/ denitrification) 

• e.g., using MAR with C-bicarbonate confirms that autotrophic AOB and NOB in an activated sludge sample are actively fixing carbon during nitrification

1. method of P reduction from wastewater

1. chemical removal with lime or alum

• Expensive and increases sludge volume, may raise salinity.

• Chemicals react with dissolved phosphate → form insoluble compounds removed with sludge.

• Lime: raises pH, Ca²⁺ reacts with phosphate → insoluble calcium phosphate.

• Alum: Al³⁺ acts as coagulant, binds phosphate → insoluble aluminium-phosphate complex.

Advantages and Disadvantages of chemical removal with lime or alum

Advantages = low initial cost, dose flexibility, low energy use/ maintenance, improved clarifiers performance and reliable

Disadvantages = chemical sludge handling, high chemical costs, environmental damage and reduced bio-available P 

2. method of P reduction from wastewater

2. Microbiological using modified activated sludge system 

known as enhanced biological phosphorous removal (EBPR)

many designs but one in common = all have alternative anaerobic : aerobic zones

Advantages and Disadvantages of microbiological using modified activated sludge system in remove P from wastewater

Advantage = lower long term costs, only biosolids produced, environmentally more acceptable, higher reduction of P achievable and recyclable 

Disadvantages = high initial costs, relatively higher energy costs, larger foot-print, less reliable and potentially higher maintenance costs

EBPR (modified university of cape town process) - Zones

1. Anaerobic: no oxygen or nitrate 

2. Anoxic: No oxygen, nitrate present 

3. Aerobic 

4. Anoxic (sludge denitrification)

EBPR (modified university of cape town process) - PAO

phosphate accumulating organisms

1. anaerobic phase

• break down polyphosphate (Poly-P) for energy 

• take up volatile fatty acids (e.g., acetate) —> make PHB 

• release phosphate (P) into the liquid 

2. Aerobic phase 

• oxidise stored PHB for energy 

• take up phosphate —> resynthesise Poyl-P

outcome = phosphate removed when biomass (sludge) is wasted → P leaves as biosolids 

Key PAO

Rhodocyclus-like bacteria —> now called Candidatus, confirmed via FISH/MAR

EBPR failure

PAOs can be outcompeted by GAOs (glycogen accumulating organisms)

• GAOs also take up acetate anaerobically —> make PHB 

• aerobically, use pHB to make glycogen, no Poly-P, so P removal fails 

GAOs cannot be identified by normal staining

two main GAOs

1. Gammaproteobacteria: Candidatus Competibacter phosphatis - large oval cells

2. Alphaproteobacteria: Defluvicoccus related - tetrad forming

indicator organisms 

• Non-pathogenic, indicate faecal contamination → possible pathogen presence.

• Enter water with faeces but are easier to measure.

Key Qualities:

• Cannot replicate freely in environment → correlate with pathogens.

• Native to intestines of warm-blooded animals.

• Higher concentration than pathogens.

• Resistant to environmental stress.

• Rapidly detectable by simple methods.

types of indicator organisms

1. coliforms = facultatively anaerobic, gram -ve rods that ferment lactose with acid production at 35-37 degrees

2. thermotolerant coliform = form gas or produce a blue colony with 24hr at 44.5 degrees

3. E.coli = thermotolerant coliform, can produce indole from tryptophan at 44.5 degrees 

why are coliforms used to detect water contamination rather than directly quantifying pathogens

• Coliforms: common in intestines of warm-blooded animals → indicate faecal contamination and possible pathogen presence.

• Direct pathogen detection is difficult as there are Low numbers, Intermittent/irregular shedding, Expensive & complex methods, Multiple pathogen types

Advantages of Coliforms:

• Fast, easy, reliable to test

• Grow easily on simple media

• Short incubation period

• Quantitative tests available

• Non-pathogenic

Virus Monitoring:

• Bacteriophages used as surrogates for human viruses

• Detection via culture techniques or PCR

Cryptosporidium

techniques for detecting Cryptosporidium and Giardia cysts involving FITC-Antibody staining, PCR, or cell culture have been developed recently

• expensive and have limitations 

• staining alone is unable to distinguish between dead/ viable cysts, those that are pathogenic to humans and those that are not 

• low infective dose (10-30 cysts)

why is cryptosporidium difficult to remove using conventional water treatment

• cysts are tiny and highly resistant, can pass through standard sand or rapid filtration systems if not optimised 

• conventional water treatment (coagulation, flocculation, sedimentation, filtration) may reduce but not eliminate cysts 

• chlorine ineffective, making conventional disinfection ineffective

Cryptosporidium treatment and control

lack of effective therapies

• no fully reliable drug, usually self-limiting in healthy adults 

• supportive care: increase fluids to prevent dehydration 

• Nitazoxanide approved in US (FDA)

• management during outbreaks: boil water alerts, temporary shutdown of water treatment plants 

modern control measured 

• enhanced filtration = physically removed cysts

• UV disinfection = damaged cyst DNA 

• ozone disinfection = highly effective chemical inactivation 

• regular monitoring = FISH or immunofluorescence assays

Cryptosporidium Case Study - 1994 QLD Daycare

• outbreak: 7/8 infants has diarrhoea; cysts detected in faeces

• children drank only boiled water; no other obvious sources 

• diarrhoea lasted more 7 days 

• transmission: fecal oral route via hands, nappies, or contaminated surfaces 

• factors aiding spread: low infectious dose (10 cysts, resistant cysts, prolonged shedding 

• Control: exclude infected children, strict hygiene, gloves/ dedicated nappy areas, disinfect surfaces/ toys

Cryptosporidium Case Study - Milwaukee 1993

• 400, 000 illness caused, 4, 400 hospitilised, 70 deaths

• stool samples positive for cryptosporidium 

• source not identified, possibly increased flows in rivers supplying lake Michigan carrying cysts from livestock/ human waste 

• turbidity of source water had deteriorated 

• treatment plant operation 

Pathogen Removal - Disinfection methods

Chlorine

• widely used (primary and secondary) 

• forms hypochlorous acid (very effective) and hypochlorite (less effective) 

• works against bacteria and giardia, moderately on viruses, not Cryptosporidium 

• effectiveness affected by turbidity (<1 NTU, and pH) 

UV: 

• damages microbial DNA/ RNA —> prevent replication 

• effective against bacteria, viruses, giardia, cryptosporidium 

• rapid treatment, used in tertiary/ recycled water 

Ozone:

• strong oxidant; destroys cell walls, enzymes, nucleic acids 

• effective against bacteria, viruses, giardia, cryptosporidium 

• more powerful than chlorine; generated on site 

ideal disinfectant 

• stable with measurable residual 

• minimal harmful by-products 

• safe, easy to generate, cost effective, suitable for wide use

waterborne pathogen - E.coli

• gram negative, normal flora of GIT 

• enterotoxigenic, enteropathogenic, enteroinvasive, enterohaemorrhagic

• faecal/ oral transmission

• travellers diarrhoea

waterborne pathogen - Leigonella pneumonia

• pneumonia + resp failure

• aerosol, aquatic reservoir

• more resistant than E.coli to chlorine and other environment antagonists

• legionnaires disease (fever, pneumonia, diarrhoea, death)

waterborne pathogen - salmonella

• all serotypes pathogenic to humans

• mild gastroenteritis to severe illness/ death

• S. typhi, S. paratyphi, S. enteritidis 

• Food-borne (beef/ poultry) or faecal/ oral 

• typhoid fever = bacterial infection caused by ingested food/ water contaminated with S. typhi (headaches, nausea, loss of appetite, liver damage) 

DALY

Disability Adjusted Life Year

• estimated disease impact by combining years of healthy life lost from premature death and years healthy life lost from disability 

• used as a standardised way to measure the health burden of disease

Food poisoning vs infection

poisoning = disease from ingestion of foods, water or products containing PREFORMED microbial toxins

infection = ingestion of pathogen contaminated food followed by growth of pathogen in host

Food preservation methods

slow/ stop microbial growth to prevent spoilage or disease

1. temp

• refrigeration = slows microbial growth; some bacteria still grow; freezing slows growth further but costly and may affect food quality 

• heat treatment = reduced or kills pathogens

  • Pasteurisation = reduces microbes, kills pathogens, not full sterilisation

  • UHT = near-sterilisation

  • Caning = sterilises food in sealed containers at correct temp/ time

• Moisture control 

  • drying/ adding salt or sugar = removes water to inhibit bacteria 

  • fungi can still cause spoilage

Food Preservation - Chemicals and Radiation - Chemicals

Chemicals

• antimicrobials: organic acids, nitrites, sulphites, propionate, benzoate, antibiotics 

• target cell wall, membrane, enzymes, DNA; also preserve texture, colour, flavour

Food Preservation - Chemicals and Radiation - Nitrites

• used in cured meats, pink colour (nitrosomyglobin) 

• inhibit C. botulinum, E. coli, antioxidants, enhance flavour/ texture 

• can form nitrosamines (carcinogens); sodium erythrobate/ isocorbate prevent this

Food Preservation - Chemicals and Radiation - Sulfites

used in fruits/ vegetables; antimicrobial activity depends on pH

Food Preservation - Chemicals and Radiation - Bacteriocins

• antimicrobial proteins from bacteria, kill closely related bacteria 

• example: Nisin (LAB) = added to milk, cheese, sauces 

• mechanisms = disrupts cell membrane by forming pores

Food Preservation - Chemicals and Radiation - radiation

• ionising radiation (X-rays, gamma rays) = destroys DNA via ions/ reactive molecules

• effective for reducing microbial contamination

Fermentation

• uses microbes to produce preservative chemicals (organic acids, alcohols)

• destroys toxins and undesirable components in raw materials

• anaerobic metabolism: microbes maintain redox balance without external electron acceptors

• homofermentative bacteria: produce only lactic acid

• heterofermentative bacteria: produce lactic acid, ethanol, CO2, acetic acid —> contribute flavour

NAD+/ NADH cycling

redox reactions facilitated by coenzymes

ATP + ADP

ATP = energy used by organism in daily operatives 

ATP —> ADP = energy release

ADP —> ATP = energy storage

Glycolysis

• glucose consumed —> ATP made —> fermentation products generated 

• for microorganisms = ATP is crucial product, fermentation products are waste and must be discarded 

• for human = fermented products are foundation of baking/ fermented beverage industries 

• sugars = glucose, hexose, disaccharides

• polysaccharide = cellulose, starch

fermentation without glycolytic reactions

clostridium ferment amino acids + purines + pyrimidines, the product of nucleic acid degradation

some anaerobes ferment aromatic compounds

how fermentation is exploited/used in food production, including detail of specific organisms for specific products: Dairy - Swiss cheese

• Propionobacterium freudenreichii

• Convert L-lactic acid to carbon dioxide -> holes

• Convert citric acid to glutamic acid -> natural flavour enhancer

how fermentation is exploited/used in food production, including detail of specific organisms for specific products: Yoghurt

• Organisms: Streptococcus thermophilus, Lactobacillus bulgaricus

• Process:

  1. Concentrate milk 25% using vacuum dehydrator

  2. Add milk solids

  3. Heat to 90°C for 30–90 min

  4. Cool to 45°C

  5. Add starter culture, incubate 3–5 hours

how fermentation is exploited/used in food production, including detail of specific organisms for specific products: Vegetables

• Organisms: Lactic acid bacteria (Lactobacillus brevis, L. plantarum) and yeasts

• Mainly use indigenous microbiota

• Salt: creates anaerobic conditions and selectively affects natural microbiota

• Higher salt favors homofermentative species, accelerating fermentation

how fermentation is exploited/used in food production, including detail of specific organisms for specific products: meat

• Organisms: Lactic acid bacteria (Lactobacillus curvatus), coagulase-negative cocci (Staphylococcus xylosus, S. saprophyticus, S. equorum)

• Products: Dry & semidry fermented sausages, salami, ham

• Nitrites: inhibit Clostridium botulinum, convert myoglobin → nitrosomyoglobin (pink cured meat colour)

• Fermentable sugars added as meat is low in carbohydrates

how fermentation is exploited/used in food production, including detail of specific organisms for specific products: bread

• Saccharomyces cerevisiae

• Produce carbon dioxide that makes bread rise

• Produce amylase to break down starch to more fermentable glucose

how fermentation is exploited/used in food production, including detail of specific organisms for specific products: beer

• S. cerevisiae used for ales = grow on top of fermentation mix (top fermentation)

• S. carlsbergensis used for lagers = settle at bottom (bottom fermentation)

how fermentation is exploited/used in food production, including detail of specific organisms for specific products: wine

• Organisms: Saccharomyces cerevisiae, S. bayanus

• White wine: 1–2 weeks at 18°C

• Red wine: 1 week at 20–30°C to extract colour

• Fermentation products: ethanol, CO₂, glycerol (smooths taste & adds viscosity), esters & aldehydes (flavour)

• Aging: White wine = usually none; Red wine = 1–2 years in barrel for flavour development