Week 15

Wastewater Treatment, Water Purification & Waterborne Diseases

1 sub-topics · Pages 586–613

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1. Introduction

📖 Lecturer's Note

Wastewater treatment is applied microbial ecology at the largest scale: engineered microbial communities in activated sludge tanks, trickling filters, and anaerobic digesters remove biochemical oxygen demand (BOD), suspended solids, nutrients (N, P), and pathogens from sewage. Waterborne diseases — cholera, typhoid, giardiasis, cryptosporidiosis — remain major public health threats globally, claiming hundreds of thousands of lives annually. The interplay between microbial ecology, engineering, and epidemiology in water treatment represents one of the most important applications of environmental microbiology.

✏️ Fill in the Blank

1. The amount of dissolved oxygen consumed by microorganisms when decomposing organic matter in water is measured as _______.

Show Answer Biological Oxygen Demand (BOD)

2. The conversion of ammonium (NH₄⁺) to nitrate (NO₃⁻) by nitrifying bacteria in wastewater treatment is called _______.

Show Answer Nitrification

3. The combination of coagulation, flocculation, sedimentation, and disinfection steps used to produce safe drinking water from raw surface water is collectively called water _______.

Show Answer Treatment (purification)

4. The 1993 Milwaukee waterborne outbreak affecting 400,000 people was caused by the protozoan pathogen _______.

Show Answer Cryptosporidium parvum

5. E. coli and Enterococcus are used as faecal _______ organisms in water quality monitoring, indicating potential faecal contamination.

Show Answer Indicator

6. The aerobic biological treatment step in a conventional wastewater treatment plant that uses a mixed microbial community to oxidise dissolved organic matter is called the _______ process.

Show Answer Activated sludge

7. The protozoan parasite _______ forms oocysts that are resistant to chlorine disinfection and is a major cause of waterborne diarrhoeal disease.

Show Answer Cryptosporidium

8. The WHO guideline value for E. coli in drinking water is _______ CFU per 100 mL.

Show Answer 0 (zero)

🔘 Multiple Choice

1. The activated sludge process used in secondary wastewater treatment works by:

  • A) Adding iron salts to precipitate phosphorus
  • B) Aerating wastewater with a mixed culture of aerobic microorganisms that degrade dissolved organic matter and form settleable flocs
  • C) Using UV radiation to disinfect the final effluent
  • D) Passing wastewater through sand filters to remove pathogens by physical straining
Show Answer Correct: B) Aerating wastewater with a mixed culture of aerobic microorganisms that degrade dissolved organic matter and form settleable flocs

2. Cryptosporidium parvum is particularly problematic in drinking water treatment because:

  • A) It produces toxins that survive chlorination at any concentration
  • B) Its oocysts are highly resistant to standard chlorination but can be removed by filtration or inactivated by UV/ozone
  • C) It cannot be detected by standard water quality monitoring protocols
  • D) It grows rapidly in chlorinated water above 5°C
Show Answer Correct: B) Its oocysts are highly resistant to standard chlorination but can be removed by filtration or inactivated by UV/ozone

3. Indicator organisms (such as E. coli) are used in water quality monitoring because they:

  • A) Are themselves the primary cause of waterborne disease
  • B) Are easy to culture and their presence indicates potential faecal contamination and co-occurrence of pathogens
  • C) Produce visible colour changes in contaminated water
  • D) Are present only in industrially polluted water, not faecally contaminated water
Show Answer Correct: B) Are easy to culture and their presence indicates potential faecal contamination and co-occurrence of pathogens

4. Biological nitrogen removal in advanced wastewater treatment involves which sequential processes?

  • A) N₂ fixation followed by ammonification
  • B) Nitrification (aerobic) followed by denitrification (anoxic) to convert NH₄⁺ to N₂ gas
  • C) Anammox only, converting NO₃⁻ directly to N₂
  • D) Assimilation of NH₄⁺ by algae in treatment ponds
Show Answer Correct: B) Nitrification (aerobic) followed by denitrification (anoxic) to convert NH₄⁺ to N₂ gas

5. Primary wastewater treatment differs from secondary treatment in that primary treatment:

  • A) Uses microorganisms to degrade dissolved organic matter
  • B) Removes only suspended solids and large debris by physical processes (screening, sedimentation) without biological treatment
  • C) Removes nitrates and phosphates from the effluent
  • D) Disinfects the effluent using chlorine or UV
Show Answer Correct: B) Removes only suspended solids and large debris by physical processes (screening, sedimentation) without biological treatment

6. Giardia lamblia causes waterborne disease characterised by:

  • A) Severe bloody diarrhoea and systemic infection via bacteraemia
  • B) Prolonged watery diarrhoea, bloating, and malabsorption due to trophozoite attachment to the small intestinal epithelium
  • C) Cholera-like rice-water diarrhoea via enterotoxin (CT)
  • D) Liver abscess formation following haematogenous spread from the gut
Show Answer Correct: B) Prolonged watery diarrhoea, bloating, and malabsorption due to trophozoite attachment to the small intestinal epithelium

7. The BOD₅ test measures:

  • A) Total dissolved solids in a water sample over 5 hours
  • B) The oxygen consumed by microorganisms degrading organic matter in a water sample incubated at 20°C for 5 days
  • C) The chemical oxygen demand of industrial effluents using potassium dichromate
  • D) The concentration of faecal coliforms per 100 mL of water
Show Answer Correct: B) The oxygen consumed by microorganisms degrading organic matter in a water sample incubated at 20°C for 5 days

8. Disinfection byproducts (DBPs) such as trihalomethanes (THMs) form when chlorine is added to water containing:

  • A) Iron and manganese ions in solution
  • B) Natural organic matter (humic acids, fulvic acids) which react with chlorine to form chlorinated compounds
  • C) Nitrate above 50 mg/L
  • D) Coliform bacteria that produce organic acids during growth
Show Answer Correct: B) Natural organic matter (humic acids, fulvic acids) which react with chlorine to form chlorinated compounds

9. The anammox (anaerobic ammonium oxidation) process is used in modern wastewater treatment because:

  • A) It removes phosphorus from effluents without chemical addition
  • B) It converts NH₄⁺ and NO₂⁻ directly to N₂ gas under anaerobic conditions, requiring less aeration energy and no organic carbon source
  • C) It produces methane that can be used as an energy source for the treatment plant
  • D) It simultaneously degrades BOD and removes nitrogen in a single aerobic tank
Show Answer Correct: B) It converts NH₄⁺ and NO₂⁻ directly to N₂ gas under anaerobic conditions, requiring less aeration energy and no organic carbon source

10. The membrane bioreactor (MBR) process for wastewater treatment combines activated sludge with:

  • A) Anaerobic digestion in a separate reactor
  • B) Ultrafiltration or microfiltration membranes that replace secondary clarifiers, producing high-quality effluent with complete retention of biomass
  • C) Chemical precipitation to remove phosphorus before biological treatment
  • D) UV disinfection applied simultaneously with biological degradation
Show Answer Correct: B) Ultrafiltration or microfiltration membranes that replace secondary clarifiers, producing high-quality effluent with complete retention of biomass

11. Chloramination in drinking water treatment is preferred over free chlorine in some systems because:

  • A) It is more effective against Cryptosporidium oocysts
  • B) It produces fewer disinfection by-products (DBPs) and has a longer residual in distribution systems
  • C) It requires lower doses to achieve the same CT value
  • D) It kills all protozoan cysts in 30 seconds
Show Answer Correct: B) It produces fewer disinfection by-products (DBPs) and has a longer residual in distribution systems

12. Legionella pneumophila causes disease primarily when:

  • A) Ingested in contaminated drinking water
  • B) Inhaled as aerosols from contaminated warm water systems (cooling towers, showers)
  • C) It penetrates skin wounds during swimming
  • D) It is transmitted person-to-person by respiratory droplets
Show Answer Correct: B) Inhaled as aerosols from contaminated warm water systems (cooling towers, showers)

13. The BOD (biochemical oxygen demand) test measures:

  • A) The concentration of dissolved oxygen in treated effluent
  • B) The amount of oxygen consumed by microorganisms to biodegrade organic matter in a water sample over 5 days at 20°C
  • C) The toxic oxygen demand of chemical pollutants
  • D) The oxygen produced by algal photosynthesis in a sample
Show Answer Correct: B) The amount of oxygen consumed by microorganisms to biodegrade organic matter in a water sample over 5 days at 20°C

14. In a conventional activated sludge system, what is the purpose of the return activated sludge (RAS)?

  • A) To supply oxygen to the aeration tank
  • B) To maintain adequate microbial biomass in the aeration tank by recycling settled sludge
  • C) To neutralise pH in the secondary clarifier
  • D) To remove phosphorus by chemical precipitation
Show Answer Correct: B) To maintain adequate microbial biomass in the aeration tank by recycling settled sludge

15. Membrane bioreactors (MBRs) offer advantages over conventional activated sludge in that they:

  • A) Require no energy for aeration
  • B) Combine biological treatment and membrane filtration, producing higher-quality effluent and eliminating secondary clarifiers
  • C) Do not produce any sludge
  • D) Only work for industrial wastewater, not municipal sewage
Show Answer Correct: B) Combine biological treatment and membrane filtration, producing higher-quality effluent and eliminating secondary clarifiers

16. Viable but non-culturable (VBNC) cells are a concern in water safety because:

  • A) They are always non-pathogenic
  • B) They may not be detected by standard culture-based monitoring methods yet can remain infectious
  • C) They produce more biofilm than culturable cells
  • D) They are resistant to UV disinfection
Show Answer Correct: B) They may not be detected by standard culture-based monitoring methods yet can remain infectious

💬 Open-Ended Questions

1. Describe the stages of conventional wastewater treatment — primary, secondary, and tertiary — explaining the role of microorganisms and the pollutants removed at each stage.

Hint / Guidance Primary: screening (removes rags, large solids); primary sedimentation (removes ~50–60% TSS, 30% BOD); no biology. Secondary: activated sludge — aerobic bacteria (Zoogloea, Nitrosomonas, Nitrobacter) in aeration tank degrade BOD; biofilm communities form flocs; secondary clarifier settles sludge; return activated sludge (RAS); removes ~85–95% BOD. Alternatives: trickling filters (biofilm), MBR, SBR. Tertiary: nutrient removal (nitrification-denitrification for N; enhanced biological P removal — Accumulibacter luxury P uptake; chemical precipitation with alum/iron for P); advanced filtration (sand, activated carbon); disinfection (Cl₂, UV, ozone). Sludge treatment: anaerobic digestion → biogas (60% CH₄); digested sludge → land application if metals/pathogens within limits.

2. Why is E. coli used as a faecal indicator organism in drinking water quality standards? Discuss its advantages and limitations, and suggest two additional indicators that address those limitations.

Hint / Guidance E. coli advantages: abundant in faeces of warm-blooded animals (10⁶–10⁸/g faeces); easily detected (ONPG/MUG fluorogenic reactions, Colilert® method); 24-h result; correlates with faecal contamination; risk model based on E. coli established (WHO guideline 0 E. coli per 100 mL drinking water). Limitations: (1) E. coli can grow in tropical soils/water without faecal input → false positives; (2) More resistant pathogens (Cryptosporidium oocysts) survive standard disinfection after E. coli eliminated → false safety; (3) Some enteric viruses removed differently than E. coli; (4) Cannot distinguish human vs animal faeces without source tracking. Additional indicators: (1) Enterococcus — more resistant to chlorine + UV, better predictor of marine water safety; (2) Coliphages (F-specific RNA coliphages, somatic coliphages) — viral surrogates; more conservative indicators of virus removal; used in EU Bathing Water Directive revision; (3) Clostridium perfringens spores — persist longer than E. coli, indicator of historical contamination and Cryptosporidium (similar resistance profile).

3. In 1993, Milwaukee, USA experienced a massive Cryptosporidium outbreak affecting over 400,000 people. Analyse how this could occur in a treated water system and propose engineering and microbiological solutions to prevent future outbreaks.

Hint / Guidance Cause analysis: Cryptosporidium oocysts (4–6 µm, resistant to routine chlorination) entered via spring runoff from cattle farms into Lake Michigan source water; coagulation-flocculation was inadequately optimised at South plant (turbidity not meeting standards); filtration breakthrough not detected in time; oocysts passed filtration into distribution system. Contributing factors: seasonal calving → peak oocyst shedding in watershed; inadequate turbidity monitoring (should be <0.3 NTU); no real-time Cryptosporidium monitoring. Prevention engineering: (1) Source water protection (agricultural buffer zones, cattle exclusion from waterways); (2) Optimised coagulation (jar testing, continuous turbidity monitoring <0.3 NTU as filtration proxy); (3) UV disinfection (highly effective: 3 log reduction at 40 mJ/cm²); (4) Ozone treatment; (5) Real-time qPCR/immunofluorescence monitoring of source water; (6) Watershed vulnerability assessment. Regulatory: USEPA Long-Term 2 Enhanced Surface Water Treatment Rule mandates Cryptosporidium monitoring and treatment (UV/ozone for high-risk sources).

4. Compare the mechanisms of action, effectiveness, and limitations of chlorination, UV irradiation, and ozonation as water disinfection methods.

Hint / Guidance Chlorination: HOCl/OCl⁻ penetrates cells → inactivates enzymes, oxidises SH groups, attacks DNA; effective against bacteria/most viruses; low cost; residual protection in distribution; ineffective against Cryptosporidium/Giardia at CT values; forms THM/HAA byproducts (carcinogenic) with NOM; taste/odour. UV irradiation: 254 nm → thymine dimers → prevents DNA replication; effective against all pathogens including Cryptosporidium (highly sensitive); no chemical residual; no byproducts; does not kill spores outright (prevents reproduction); higher cost; requires clear water (turbidity limits penetration); no residual → must combine with Cl₂ for distribution. Ozonation: O₃ → OH radicals → oxidise cell membranes, DNA; highly effective (broadest spectrum including viruses, Cryptosporidium); removes taste/odour; no persistent residual (decomposes rapidly); may form bromate byproduct (carcinogenic) if bromide present; higher cost; requires on-site generation. Best practice: multi-barrier (coagulation + filtration + UV/ozone + Cl₂ residual).

5. Describe the waterborne diseases caused by Vibrio cholerae, Giardia lamblia, and Cryptosporidium parvum. For each, include the causative agent, transmission route, infectious dose, pathogenesis, and prevention.

Hint / Guidance Vibrio cholerae: Gram-negative curved rod; O1/O139 serogroups; faecal-oral via contaminated water (sewage contamination); low ID (10⁶–10⁸ in healthy adults); CT (cholera toxin) — ADP-ribosylates Gsα → constitutive adenylyl cyclase activation → cAMP → CFTR activation → massive Cl⁻/water secretion → rice-water diarrhoea (20 L/day); dehydration; IFR ~50% untreated, <1% with rehydration; prevention: safe water supply, ORT. Giardia lamblia: flagellated protozoan; cysts in faeces; ID 10 cysts; trophozoites attach to duodenum villus → mechanical disruption + immune response → malabsorption/diarrhoea; bloating, greasy stool; self-limiting or metronidazole treatment; waterborne (resists Cl₂ at standard doses); filtered water or boiling effective. Cryptosporidium parvum: apicomplexan; oocysts (4–6 µm); ID <10 oocysts; sporozoites penetrate enterocyte brush border (intracellular but extracytoplasmic); villous atrophy; watery diarrhoea; self-limiting in immunocompetent; severe in HIV+ (life-threatening); no effective drug (nitazoxanide limited); resistant to Cl₂; UV/ozone effective; filtration removes.

6. Explain the concept of Chemical Oxygen Demand (COD) vs. Biological Oxygen Demand (BOD). Why are both measured in wastewater, and what does a high BOD:COD ratio indicate?

Hint / Guidance BOD₅: O₂ consumed by microorganisms degrading biodegradable organic matter over 5 days at 20°C; reflects biologically available organic load; expressed in mg O₂/L. COD: O₂ equivalent of total organic matter (biologically available + refractory) oxidised by potassium dichromate under reflux at 150°C; faster (2–4 h); includes industrial recalcitrant organics (lignin, detergents). Both measured: COD for total organic load (regulatory compliance, rapid); BOD for biological treatability assessment; COD-BOD difference indicates refractory organic content. High BOD:COD ratio (>0.5): mostly biodegradable organic matter → suitable for biological treatment (activated sludge). Low BOD:COD ratio (<0.3): significant refractory fraction (industrial effluents with phenols, pesticides, heavy metals) → biological treatment alone insufficient; need chemical oxidation (ozone, Fenton), adsorption, or co-treatment with domestic sewage to dilute toxics.

7. What is the role of anaerobic digestion in modern wastewater treatment? Describe the four microbial stages and explain how biogas produced can contribute to energy self-sufficiency of a treatment plant.

Hint / Guidance Purpose: stabilise primary/secondary sludge; reduce pathogen load; reduce sludge volume; produce biogas. Four stages: (1) Hydrolysis: extracellular enzymes (cellulases, proteases, lipases) from Firmicutes/Bacteroidetes break polymers to monomers; rate-limiting for complex substrates. (2) Acidogenesis: fermentative bacteria produce acetate, propionate, butyrate, H₂, CO₂. (3) Acetogenesis: syntrophic acetogens convert propionate/butyrate → acetate + H₂ (requires H₂ removal by methanogens — syntrophic coupling). (4) Methanogenesis: hydrogenotrophic (Methanobacterium: H₂ + CO₂ → CH₄) and acetoclastic (Methanosarcina/Methanosaeta: CH₃COO⁻ → CH₄ + CO₂). Biogas: 55–70% CH₄; ~21 MJ/m³; used in combined heat and power (CHP) units → electricity + heat. Energy self-sufficiency: modern WWTPs can achieve 50–100% energy from biogas (Amsterdam produces surplus electricity from sludge AD). Co-digestion with food waste/FOG increases biogas yield substantially.

8. Discuss the emerging challenge of antibiotic-resistant bacteria (ARB) and antibiotic resistance genes (ARGs) in wastewater treatment plants. How do WWTPs contribute to ARG dissemination, and what advanced treatment options are available?

Hint / Guidance Problem: WWTPs receive antibiotics, ARB, and ARGs from hospitals, households, animal farms; conventional treatment does not completely remove ARGs; secondary effluent still contains 10⁵–10⁸ ARB/mL and ARGs at µg DNA/L levels; discharge to receiving water → ARG spread to environmental microbiome. Contributing factors: (1) Activated sludge biofilm concentrates ARB from dilute influent; (2) HGT enhanced in biofilm (conjugation, transformation from lysed cells); (3) Sub-inhibitory antibiotic concentrations in effluent maintain selection; (4) Sludge land application spreads ARGs directly to soil. Advanced removal: (1) Ozonation (oxidative damage to DNA including ARGs, kills ARB); (2) UV (effectively inactivates ARB without byproducts, but DNA fragments may persist); (3) MBR (ultrafiltration physically retains bacteria; lower ARG discharge); (4) Activated carbon adsorption; (5) Anaerobic digestion + thermal hydrolysis reduces ARGs by ~2–3 log in sludge. Policy: WHO calls for surveillance of ARGs in wastewater as early warning system; integrated One Health approach needed.

9. Explain how an outbreak of typhoid fever (Salmonella Typhi) could occur through a municipal water supply in a low-income country. What would you do as a public health microbiologist to investigate the outbreak and recommend control measures?

Hint / Guidance Mechanism: Salmonella Typhi (faecal-oral, ID ~10³); sewage infiltrates drinking water distribution due to intermittent supply (negative pressure draws sewage into pipes), illegal connections, or contamination at source. Outbreak investigation: (1) Confirm diagnosis: blood/bone marrow culture (bacteraemia), faecal culture (Widal test unreliable); (2) Case-control study: identify water source, time-space cluster, common exposure; (3) Water sampling: culture (no S. Typhi by culture normally unless very high contamination) + indicator organisms (E. coli, thermotolerant coliforms) from multiple system points; (4) Network analysis: identify contamination point by comparing indicator levels at different nodes. Control: (1) Immediate: boil-water advisory; chlorination of distribution system; identify/repair leak; (2) Treatment of cases: fluoroquinolones or azithromycin (note rising resistance); (3) Long-term: chlorinated water supply; sewage system upgrade; typhoid conjugate vaccine for community; hygiene promotion. Report to WHO under IHR if outbreak exceeds national capacity.

10. Describe the complete pathway of a waterborne cholera outbreak from environmental source to epidemic. Include the causative organism, transmission, pathogenesis, and public health response.

Hint / Guidance Agent: Vibrio cholerae O1/O139; environmental reservoir: estuarine/coastal water (attached to copepods). Transmission: ingestion of contaminated water/food (ID ~10⁸ for healthy individuals, lower in low-acid stomach). Pathogenesis: V. cholerae colonises small intestine; CT (cholera toxin) B subunit binds GM1 ganglioside; A subunit activates adenylyl cyclase → ↑cAMP → constitutive activation of CFTR Cl⁻ channel → massive Cl⁻/water secretion → 'rice-water' stools (up to 20 L/day) → severe dehydration and death (50% untreated → <1% with treatment). Public health: (1) ORS (oral rehydration salts) — case management; (2) Cholera vaccines (Shanchol); (3) Water chlorination/boiling; (4) Sanitation infrastructure; (5) Epidemiological investigation to identify source; (6) WASH interventions; WHO reporting under IHR.

11. What is the concept of 'CT value' in water disinfection? How is it used to ensure adequate inactivation of specific pathogens?

Hint / Guidance CT = concentration of disinfectant (mg/L) × contact time (min). Effectiveness proportional to CT; different pathogens require different CT for 1-log, 2-log, 4-log reduction. Examples (free Cl₂, pH 7, 20°C): E. coli: CT 0.1 for 4-log; Giardia cysts: CT 65 for 3-log; Cryptosporidium oocysts: essentially chlorine-resistant (CT >7200 impractical) → requires UV (CT ~10 mJ/cm²) or ozone. Regulators (US EPA Surface Water Treatment Rule) set CT requirements based on source water quality and pathogen risk. Practical use: operators measure residual Cl₂ in treated water + calculate contact time (T₁₀ = time for 10% tracer breakthrough) → ensure CT target met for Giardia inactivation; separate UV log-credit for Cryptosporidium.

12. Explain how antibiotic resistance genes (ARGs) enter aquatic environments and what risks they pose to human health. What measures can reduce their spread?

Hint / Guidance Sources: hospital effluent (high antibiotic concentrations + resistant pathogens); agricultural runoff (veterinary antibiotics + ARGs from livestock gut); wastewater treatment plants (WWTP) — activated sludge concentrates ARGs; biosolid application to land; aquaculture. Entry into environment: conventional WWTPs reduce but don't eliminate ARGs; resistant bacteria and naked DNA (integrons, plasmids) discharged in effluent or biosolids. Risks: environmental ARG reservoirs (resistance integrons in soil/water bacteria) → HGT transfers resistance to pathogens (e.g., ESBL E. coli from river water); drinking water treatment rarely eliminates ARGs completely. Reduction measures: advanced treatment (UV + ozone + activated carbon in WWTP effluent); reduce antibiotic use in agriculture (ban growth promoters); hospital effluent pre-treatment; wastewater surveillance as early warning; phage therapy as alternative to antibiotics.

13. Describe the role of Nitrosomonas, Nitrobacter, and Anammox bacteria in biological nitrogen removal from wastewater. Why is nitrogen removal important?

Hint / Guidance Importance: excess N in effluent causes eutrophication (algal blooms, O₂ depletion, fish kills); NO₃⁻ in drinking water causes methaemoglobinaemia in infants (blue baby syndrome); regulatory limit typically 10–15 mg TN/L. Processes: (1) Nitrification (aerobic): Nitrosomonas: NH₄⁺ → NO₂⁻; Nitrobacter/Nitrospira: NO₂⁻ → NO₃⁻; slow-growing (μmax ~0.05/h), sensitive to low DO, temperature, pH; long SRT required (>10 days at 15°C). (2) Denitrification (anoxic): Paracoccus, Pseudomonas use NO₃⁻ as electron acceptor with organic C; NO₃⁻ → N₂. (3) Anammox (SHARON-ANAMMOX): partial nitritation (NH₄⁺ → NO₂⁻, Nitrosomonas); Candidatus Brocadia converts NH₄⁺ + NO₂⁻ → N₂; saves 60% aeration energy + no organic C needed; increasingly used for sidestream treatment.

14. How is microbial source tracking (MST) used to identify the source of faecal contamination in recreational water bodies? Describe at least two methods.

Hint / Guidance MST: determine origin of faecal contamination (human, bovine, poultry, avian, etc.) to target corrective action. Methods: (1) Host-specific genetic markers — qPCR targeting host-specific 16S rRNA gene markers; e.g., HF183 (Bacteroides thetaiotaomicron marker, human-specific); CF128 (dog); BacR (ruminant Bacteroides); highly specific and sensitive; quantitative. (2) Bacteriophage typing — somatic coliphages (F-specific RNA phages) cross-react with all hosts; but male-specific phages (F+) more human-associated. (3) Chemical markers — caffeine + pharmaceuticals (human); sterols (coprostanol human vs. β-sitosterol plant); (4) Metagenomic approaches — microbial community composition of faeces differs by host; machine learning classifiers. Regulatory use: identify non-compliant agricultural discharge vs. sewage overflow, triggering targeted remediation.
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