Microbiology Revision

Main aims of wastewater treatment 

• Ensure water quality to protect public health

• Potential common source of infectious diseases

• Prevent pollutants including chemicals from industry, endocrine disrupting chemicals, disinfection by-products

Eutrophication

biological process where a body of water becomes excessively enriched with nutrients, like nitrogen and phosphorous, causing a rapid increase in algae and aquatic plant growth, known as an algal bloom.

Activated Sludge

• Pre-treatment: Screens & grit chambers remove debris; solids settle as primary sludge.

• Aeration Tank: Microbes feed on suspended organic matter, form flocs; biological oxidation → CO₂, H₂O, microbial biomass.

• Secondary Clarifier: Flocs settle → activated sludge; clarified effluent discharged.

• Sludge Return: Part of settled sludge returned to aeration tank → maintains high microbial concentration.

• Waste Sludge: Excess sludge removed → thickened, digested (anaerobic/aerobic), dewatered, then disposed or reused.

FLOCS

• Filamentous bacteria matrix

• Micro-colonies of bacteria and inorganic particles

• Embedded in gel-like exocellular polymeric substances = glue holding it all together

• Complex heterogenous structures

• Floc structure and shape determines how successfully the solids phase separate from the liquid phase in secondary clarifier, ultimately determines success of the process

FISH = (Fluorescent in Situ Hybridization)

• FISH uses fluorescent probes to identify specific genetic material in flocs.

• Probes bind to DNA or RNA, visualising different bacteria and their arrangement.

• Combined with PHA or polyP staining, it identifies phosphorus-removing bacteria in activated sludge.

Role of Protozoa

• Heterotrophs → require organic carbon

• Feed on bacteria → control bacterial numbers, reduce turbidity

Functions:

• Predators: regulate bacterial populations in suspension & flocs

• Indicators: type & abundance reflect system performance

• Floc support: attach to flocs, secrete EPS → bind particles, improve sludge–water separation

How is N removed from wastewater in activated sludge process?

• Inorganic forms of N = NH4 / NH3 = toxic to aquatic organisms, NH4 + NO3- contribute to ozone depletion + greenhouse gas effect

• Need to be removed and converted to N2 : 

• Sequential process of NITRIFICATION, followed by DENITRIFICATIOn

NITRIFICATION

• Process where reduced N compounds like NH3 are oxidised via nitrite -> nitrate to supply energy (ATP) for bacterial growth

• Provides very little energy so these are slow growing organisms - need long residence time in the system

• Bacteria that carry out ammonia oxidation are called = nitroso bacteria 

• Bacteria that oxidise nitrite to nitrate are called nitro bacteria

Ammonia-Oxidising Bacteria (AOB / Nitroso-bacteria) OVERVIEW

• Traits: Gram–, aerobic chemolithoautotrophs (β- or γ-Proteobacteria).

• Energy source: Oxidation of ammonia → Nitrite

• Carbon source: Inorganic carbon (CO₂).

• Occurs: In aerobic zones (aeration tanks).

Ammonia Oxidising Bacteria Process : 1. Ammonia oxidation

1. AMMONIA + OXYGEN + H -> HYDROXYLAMINE + WATER

   • Enzyme: Ammonia monooxygenase (cell membrane)

   • Oxygen source: Molecular O₂

   • No ATP produced

Ammonia Oxidising Bacteria Process : 2. Hydroxylamine oxidation

HYDROXYLAMINE + OXYGEN -> NITRITE + WATER + ATP

• Enzyme: Hydroxylamine oxido-reductase (periplasm)

• Oxygen source: Water

• ATP produced

• Energy released AOB usedto fix CO₂ → biomass.

2. Nitrite-Oxidising Bacteria (NOB / Nitro-bacteria)

• Traits: Gram–, chemoautotrophic (α-, γ-, or δ-Proteobacteria).

• Energy source: Oxidation of nitrite.

• Carbon source: Inorganic CO₂ (can use organics when available).

• Process:
  NITROGEN DIOXIDE + OXYGEN → NITRATE

  • Enzyme: Nitrite oxido-reductase (cell membrane)

  • Low energy yield → slow growth

  • Complex ATP production system adapts to conditions.

3. Nitrifiers in Activated Sludge

Nitroso (AOB):

• Form clusters of 100s of cells.

• Community composition depends on treatment plant type and operating conditions.

• Dominant species examples:

  • Nitrosomonas: Common; species diversity varies (1–5 spp./plant).

  • Nitrosococcus: Found in high-ammonia systems.

  • Nitrosospira: Rare in sludge (adapted to low-NH₃ environments).

Nitro (NOB):

• Also form clusters, located adjacent to AOB clusters.

• Syntrophy:

  • AOB produce nitrite (energy source for NOB).

  • NOB remove toxic nitrite from AOB cells.

  • Mutual benefit allows efficient nitrification.

DENITRIFICATION

• Definition: Reduction of nitrate (NO₃⁻) and/or nitrite (NO₂⁻) to gaseous nitrogen compounds (NO, N₂O, N₂).

• Sequence:
  Nitrate -> Nitrite -> Nitric Oxide -> Nitrous Oxide -> Dinitrogen

• Carried out by: Chemoorganoheterotrophic microbes via anaerobic respiration.

• Products: Escape as gases to atmosphere.

  • Incomplete reduction (NO, N₂O) = air pollutants & greenhouse gases.

  • Complete reduction to N₂ = desired end product.

• Anoxic zone: No O₂ but high NO₃⁻.

• Organic carbon source: Needed as electron donor (e.g. methanol, acetate).

• Energy source: From oxidation of organic carbon (heterotrophic process)

ANAMMOX (Anaerobic Ammonia Oxidation)

• Reaction: Ammonia + Nitrite → Dinitrogen + H2O

• Nitrite converted to dinitrogen gas using ammonia as the electron donor

  • No need for oxygen for denitrification, cheap

• Carried out by: Planctomycetes (anaerobic, chemolithoautotrophs).

• Location: Inside anammoxosome (special intracellular compartment).

  • Membrane: Contains unique ladderane lipids to protect from toxic intermediates (e.g. hydrazine

  • Nitritre oxidised → NO (by nitrite reductase).

  • Reduce NO + Oxidise Ammonia → Hydrazine (by hydrazine hydrolase).

  • Hydrazine Oxidised → N2 (by hydrazine oxidoreductase, similar to AOB enzyme).

• No oxygen or organic carbon needed → cost-effective.

FISH = (Fluorescent in Situ Hybridization)

• Technique that uses fluorescently labelled DNA/RNA probes that bind to cUses fluorescently labelled DNA/RNA probes that bind to rRNA (e.g. 16S rRNA).

• Identifies and visualises specific microbes and their arrangement in flocs using fluorescence microscopy.

In Wastewater Treatment:

• Detects nitrifiers, denitrifiers, filamentous and P-removing bacteria.

• Tracks population changes and process health (e.g. sludge bulking).

• Shows spatial organisation in flocs/biofilms (nitrifiers at surface, denitrifiers deeper).

• Combined with PHA/polyP stains to identify phosphorus-removing microbes.

Microautoradiography (MAR)

• Uses radioactively labelled substrates to show which microbes are metabolically active (taking up and using compounds).

In Wastewater Treatment:

• Identifies active bacteria in situ.

• Reveals which groups use C, N, or P compounds.

• Confirms if microbes detected by FISH are functionally active (e.g. AOB/NOB fixing C during nitrification).

Models of alternating Anaerobic:Aerobic zones to achieve enhanced biological phosphorous reduction - CHEMICAL

(Lime or Alum)

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

• Lime : Raises pH → calcium ions from lime react with P → insoluble calcium phosphate compound 

• Alum : Al ions act as coagulant binding with phosphate → insoluble aluminium phosphate complexes.

• Advantages = 

• Disadvantages: Expensive, increases sludge volume and salinity, reduced bio-available P

Chemical P Reduction Advantages and Disadvantages

• Advantages = low initial cost, dose flexibility, improved clarifier performance

• Disadvantages: Expensive, increases sludge volume and salinity, reduced bio-available P

Models of alternating Anaerobic:Aerobic zones to achieve enhanced biological phosphorous reduction EBPR - MICROBIOLOGICAL ACTIVATED SLUDGE

1. Anaerobic Zone (no O₂ or NO₃⁻):

• PAOs break down poly-P → energy → use VFAs (e.g. acetate) → PHB

• Phosphate released into liquid.

• Biomass stains for PHB, not poly-P.

2. Aerobic Zone (O₂ present):

• PAOs oxidise stored PHB as carbon → energy + reducing power.

• Uptake phosphate from liquid → rebuild poly-P.

• Biomass stains for poly-P, not PHB.

• P removed when sludge (biosolids) is wasted.

3. Anoxic Zone (optional):

• Supports denitrification using stored carbon (PHB).

Key Microbe:

• Candidatus Accumulibacter (Rhodocyclus-like) 

EBFR Plants Often Fail

Glycogen Accumulating Organisms (GAOs)

• outcompeted by non-PAO bacteria = GAO, for substrates in anaerobic zones.

• Anaerobic: GAO Assimilate acetate → synthesize PHB.

• Aerobic: Use PHB → regenerate glycogen, no poly-P accumulation.

• Cannot be stained for glycogen in bacterial cells → not identifiable by microscopy.

Major GAOs:

• Gammaproteobacteria: Candidatus Competibacter phosphatis, large oval cells

• Alphaproteobacteria: Defluvicoccus-related, tetrad-forming

EBFR Advantages and Disadvantages

Advantages = lower long term costs, only biosolids produced, higher reduction of P achievable

Disadvantages = high initial costs, high energy costs, larger foot-print

Indicator Organisms

Not pathogenic but signal faecal contamination → possible pathogen presence

Qualities =

• Not able to replicate freely in environment/correlate with pathogen presence

• Be native to intestine of warm blooded animals

• Present at higher conc than pathogens

• Resistant to environmental stress

• Rapidly detected by simple methods

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

• Present in intestines of warm-blooded animals like many pathogens → indicate faecal contamination.

• Pathogens are hard to detect: low numbers, intermittent, diverse, expensive.

Advantages of coliforms:

• Fast, easy, reliable detection

• Grow on simple media

• Short incubation

• Quantitative tests available

• Non-pathogenic

Cryptosporidum

Detection:

• Methods: FITC-antibody staining, PCR, cell culture

• Limitations: expensive, cannot distinguish viable vs dead, or human-pathogenic vs non-pathogenic

• Low infective dose: 10–30 cysts

Resistance & Treatment Challenges:

• Difficult to remove via conventional treatment (coagulation, flocculation, sedimentation, filtration)

• Chlorine-resistant: cysts have thick protective walls

• Effective methods: boiling, UV, ozone, enhanced filtration

• Therapy: no reliable drug; usually self-limiting in healthy adults

  • Nitazoxanide FDA-approved in US

  • Management: boil water alerts, shutdown of water treatment plant

Modern Control Measures:

• Enhanced filtration: physical removal

• UV disinfection: DNA damage

• Ozone: chemical inactivation

• Monitoring: FISH / immunofluorescence assays

Cryptosporidum Case Study 1 1994 North QLD

1994 Child Day Care Outbreak – North Qld

• Event: November 1994, diarrhoea outbreak in nursery

• Cases: 7/8 infants affected; Cryptosporidium cysts found in faeces

• Exposure: Only boiled water; no pool or pets → transmission via faecal–oral route (hands, nappies, surfaces)

• Features aiding transmission:

  • Low infectious dose (10 cysts)

  • Environmentally resistant cysts

  • Prolonged shedding

• Control measures:

  • Exclude infected children

  • Strict hygiene (gloves, dedicated nappy areas)

  • Disinfect surfaces and toys

• Duration: Diarrhoea ≥7 days

Cryptosporidium Case Study 2 Milwaukee 1993

• 400,000 illnesses caused, 70 deaths

• Stool samples positive for cryptosporidum 

• 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

Different Approaches to Disinfection - CHLORINE

• Used extensively as primary + secondary disinfectants

• Dissociates in water to form hypochlorous acid (very effective) and hypochlorite ion (less effective) 

• Effective against bacteria + giardia, moderately effective against viruses, not effective against cryptosporidium 

• Affected by turbidity (should be <1NTU), and pH

Different Approaches to Disinfection - ULTRAVIOLET

• Increasingly used in tertiary/recycled water systems across Aus 

• UV light damages microbial DNA/RNA preventing replication

• Effective against bacteria, viruses, giardia, cryptosporidium 

• Rapid treatment

Different Approaches to Disinfection - OZONE (O3)

• Powerful oxidising gas generated on site using electrical discharge 

• Destroys cell walls, oxidises enzymes/nucleic acids 

• Effective against bacteria, giardia, viruses, cryptosporidium 

• More powerful oxidant than chlorine

Different Approaches to Disinfection - Ideal Disinfectant

• Stable + easily measured residual

• Produce no unacceptable by-products 

• Be easily generated, safe to handle, suitable for widespread use

• Be cost effective

Waterborne Pathogens - E. Coli

• Gram neg rod, normal flora of GIT

• Enterotoxigenic, enteropathogenic, enteroinvasive, enterohaemorrhagic 

• faecal/oral transmission

• Travellers diarrhea 

Waterborne Pathogens - Legionella Pneumophila

• Pneumonia + respiratory failure

• Aerosol, aquatic reservoir

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

• Legionnaires disease 

  • Fever, pneumonia, diarrhea, death

Waterborne Pathogens - 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 for Estimating Disease Impact

DALY = Disability Adjusted Life Year, estimates disease impact by combining years of healthy life lost from premature death and years of healthy life lost from disability

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 - Refrigeration

• Low temp, slow microbial growth/spoilage

• Many microorganisms particularly bacteria grow at fridge temp

• In freezer slow growth still occurs in pockets of liquid water trapped within frozen food

• -20 degrees = expensive, affects food appearance/consistency/taste

Heat Treatment

• Reduce bacteria load, sterilise food products

• Useful for liquids and high moisture foods

• Pasteurisation = not sterilise liquid but reduce microbial number and eliminate pathogens

• UHT = Ultra High Temp processing, almost sterilise foods

• Canning = sterilise foods, require processing in sealed container at correct temp and time

Moisture Treatment

• Drying = physically removing water or adding solutes such as salt/sugar

• Prevent bacteria growth

• Spoilage can still occur, especially from fungi

Food Preservation Methods - Chemicals

• Antimicrobial chemicals = organic acids, nitrites, sulfites, sulfur dioxide, propionate, benzoate, antibiotics

• Target cell wall, cell membrane, enzymes, genes

• Enhance or preserve food texture, colour, freshness, flavour

Nitrites

• Used in cured meat, bacon, hot dogs, ham

• Nitrosomyoglobin makes cured meat pink

• Inhibits c. botulinum, e.Coli

• Antioxidant

• Contribute to flavour, texture

• Nitrite reacts with amines from meat protein -> nitrosamines (carcinogens)

• Sodium erythrobate or isoascorbate inhibit nitrosamine formation

Sulfites

• Sulfur dioxide/salts used since ancient times

• Used primarily in fruit/veg

• Antimicrobial activity = pH

Bacteriocins

• Antimicrobial proteins made by bacteria

• Bactericidal to a closely related group of bacteria

• Do not kill bacteria that produce them

• Nisin = LAB bacteriocin, nisin A/nisin Z, added to milk, cheese, sauces, salad dressings

• Target cell membrane of sensitive bacteria, disrupt by making pores

• Applications in foods = add LAB to produce bacteriocin or add bacteriocin

Radiation

• Ionising radiation including X-rays, gamma rays

• DNA destruction by ions/reactive molecules

• Ionising radiation is effective means for reducing microbial contamination

Food preservation methods - Fermentation

• Food preservation via microbial metabolism producing preservative chemicals

• Destroys toxins & undesirable components

Types of Microbes:

• Organic acid bacteria → lactic acid

• Yeast → alcohol

Metabolism:

• Anaerobic catabolism: organic compounds act as both electron donors & acceptors → redox balance without external electron acceptors

• Homofermentative bacteria: produce only lactic acid

• Heterofermentative bacteria: produce lactic acid + ethanol + CO₂ + acetic acid → flavour

Energy & Redox:

• ATP/ADP: ATP = energy for cell; ADP → ATP stores energy

• NAD⁺/NADH: coenzymes cycle electrons in redox reactions

Glycolysis:

• Glucose → ATP + fermentation products

• ATP = primary benefit for microbes; products = “waste” for them, but food/beverage products for humans

Other Fermentations:

• Without glycolysis:

  • Clostridium ferments amino acids, purines, pyrimidines

  • Some anaerobes ferment aromatic compounds

Fermentation in Food Production - Swiss Cheese

• Propionobacterium freudenreichii

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

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

Fermentation in Food Production - Yoghurt

• Streptococcus thermopulius, Lactobacillus bulgaricus

• Concentrate milk by 25% using vacuum dehydrator

• Add milk solids

• Heat mixture to 90 degrees for 30-90 mins

• Cool mixture to 45 degrees

• Add starter culture, incubate for 3-5 hours

Fermentation in food production - Vegetables

• Lactic acid bacteria (e.g. Lactobacillus brevis, plantarum) and yeasts

• Mainly rely on indigenous microbiota 

• Salt = help creates anaerobic conditions, has selective effect on vegetables natural microbiota

  • Higher salt concentrations favour homofermentative species, accelerating fermentation 

Fermentation in food production - Meat

• Lactic acid bacteria (mainly Lactobacillus such as curvatus), coagulase negative cocci (staphylococcus such as xylosus, saprophyticus, equorum)

• Fermented meats = dry and semidry fermented sausages, salami, ham

• Nitrites added to inhibit clostridium botulinum, convert myblobin to nitrosomyoglobin to give pink colour of cured meat

• Fermentable sugars added as meat does not contain much fermentable carbohydrate

Fermentation - Bread

• Saccharomyces cerevisiae

• Produce carbon dioxide that makes bread rise

• Produce amylase to break down starch to more fermentable glucose

Fermentation - Beer

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

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

Fermentation - Wine

• S. cerevisae or Saccharomyces bayanus 

• White wine = 1-2 weeks at 18 degrees

• Red wine = 1 week at 20-30 degrees to extract red colour

• Fermentation products = ethanol, carbon dioxide, glycerol (smooths taste + imparts viscosity), esters and aldehydes (flavour compounds)

• Aging = white wine usually none, red wine 1-2 years in barrel for flavour development