🌍 Overview

The lecture explores how carbon (C), nitrogen (N), and phosphorus (P) are cycled, removed, and recovered in wastewater treatment plants (WWTPs). It connects microbial ecology with environmental engineering and sustainability.

🧪 Nitrogen Cycle & Removal

🔄 The N-Cycle

• Involves transformations between NH₄⁺, NO₂⁻, NO₃⁻, N₂, and organic N.
• Key microbial processes:
  • Nitrification (aerobic oxidation of NH₄⁺ → NO₂⁻ → NO₃⁻)
  • Denitrification (anaerobic reduction NO₃⁻ → N₂)
  • DNRA (nitrate → ammonium)
  • Anammox (anaerobic ammonium oxidation)

💧 Wastewater Composition & Regulations

Typical Danish domestic wastewater:

• COD: 300–600 mg/L → must be <70
• N: 30–50 mg/L → must be <8
• P: 10–20 mg/L → must be <0.5–1 One “person equivalent” = 21.9 kg organic matter, 4.4 kg N, 1 kg P per year.

⚙️ Nitrogen Removal & Recovery

♻️ Recovery options

• Direct recovery as ammonia gas (NH₃) or precipitate (struvite, MgNH₄PO₄).
• Recovery from sludge for fertilizers or single-cell protein.

🚰 N-Removal in WWTPs

Two routes:

1. Conventional nitrification–denitrification
2. Deammonification (partial nitrification + anammox)

🔬 Nitrification

• AOB (Ammonia-Oxidizing Bacteria): Nitrosomonas, Nitrosospira
• NOB (Nitrite-Oxidizing Bacteria): Nitrospira, Nitrotoga
• AOA (Archaea): minor role in WWTPs Reaction: NH₄⁺ + 2 O₂ → NO₃⁻ + H₂O + 2H⁺ → lowers pH, requires oxygen and long sludge age.

🧫 Nitrifier surprises

• Nitrospira can use hydrogen and organics (mixotrophic).
• Nitrotoga tolerates high oxygen and cold.
• Comammox Nitrospira can perform full ammonia→nitrate oxidation in one organism — a game-changer ⚡

🌍 Global Diversity

• Comammox dominates many Danish plants (high NH₄⁺ affinity, low N₂O production).
• AOB show high species diversity; NOB show low diversity.

🌬️ Denitrification

• Converts NO₃⁻ → NO₂⁻ → NO → N₂O → N₂.
• Occurs under anoxic conditions.
• Produces alkalinity (↑ pH). Main enzymes: Nar/Nap, Nir, Nor, Nos.

⚠️ Problem: N₂O (265× stronger than CO₂ as a greenhouse gas) often emitted from WWTPs.

🧫 ANAMMOX (Anaerobic Ammonium Oxidation)

• Reaction: NH₄⁺ + NO₂⁻ → N₂ + 2H₂O
• Carried out by Planctomycetes (e.g. Brocadia, Kuenenia).
• Uses anammoxosome with hydrazine intermediate.
• Very slow growth (T₂ ≈ 10–20 days).
• Found to dominate marine/sediment denitrification 🌊

⚗️ Advantages

• Needs 60% less oxygen and no organic carbon.
• Uses NH₄⁺ + NO₂⁻ instead of NO₃⁻.
• Ideal for low-C waste streams.

🧱 SHARON + ANAMMOX System

Two-step setup:

1. SHARON: partial nitrification to NO₂⁻.
2. ANAMMOX: converts NO₂⁻ + NH₄⁺ → N₂. Competition between AOB/NOB controlled by pH, O₂, temperature. Low O₂ suppresses NOB (“bad guys”), enabling stable partial nitritation.

🤝 Comammox interactions

• May disturb anammox by producing too much nitrate.
• Or support it by providing NO₂⁻ — outcome depends on conditions.

💡 Summary of N Removal Pathways

🧬 Phosphorus Cycle & Removal

🌎 P-Cycle Basics

• Exists as phosphate (PO₄³⁻).
• Essential for life and agriculture.
• Excess → eutrophication in lakes.
• Finite natural resources → “peak phosphorus” concern (major reserves: Morocco, China, USA).

⚗️ P Recovery in Denmark

Imports:

• 53 000 t via food
• 15–20 000 t via fertilizer Wastewater can supply ~20% of fertilizer need via:
• Sludge reuse
• Struvite crystallization
• Ash extraction after incineration

🧂 Struvite (MgNH₄PO₄·6H₂O) forms granules at high pH — can clog pipes but can be recovered as valuable fertilizer.

🧫 Enhanced Biological Phosphorus Removal (EBPR)

Uses polyphosphate-accumulating organisms (PAOs) that store P as intracellular poly-P.

🧬 PAO Process (Ca. Accumulibacter)

• Anaerobic phase: take up volatile fatty acids (acetate, propionate), release phosphate, store carbon as PHA.
• Aerobic phase: use stored PHA for growth and take up phosphate again → P-rich biomass.
• Sludge is then removed → P recovery ✅

🌈 Key PAOs

• Ca. Accumulibacter
• Ca. Phosphoribacter (Tetrasphaera)
• Axonexus (Dechloromonas)

Each has different substrate preferences (acetate, amino acids, glucose) and denitrification potential.

🧫 Dechloromonas species discovered as new PAOs (e.g. Ca. D. phosphoritropha, Ca. D. phosphorivorans) with varying denitrification genes.

🔍 Detection & Analysis

• FISH (Fluorescence In Situ Hybridization) identifies PAOs.
• Raman microspectroscopy quantifies poly-P, PHA, and glycogen in single cells.
• Combined FISH-Raman reveals both identity + function at single-cell level.

🧫 Storage Polymers

• Poly-P: energy and P storage.
• PHA (polyhydroxyalkanoates): carbon reserve.
• Glycogen: carbon and energy storage.

🧬 Enzymes in Accumulibacter:

• Ppk1/Ppk2 (poly-P kinases)
• Ppx (exopolyphosphatase)
• Adk (adenylate kinase)
• Pit/Pst (phosphate transporters)

🍬 GAOs (Glycogen-Accumulating Organisms)

• Do not store poly-P.
• Compete with PAOs for carbon.
• Main genera: Ca. Competibacter, Propionivibrio, Defluviicoccus, Micropruina. But: rarely problematic in full-scale plants.

⚙️ Danish EBPR Operations

~75 plants. Layouts: with/without Side-Stream Hydrolysis (SSH) SSH enhances substrate availability by fermenting sludge → more VFA for PAOs.

🧪 P-release test: Add acetate to anaerobic sludge, monitor soluble P. Typical ratio: 0.5 mg P released / mg acetate taken up.

⚠️ Common EBPR Issues

• Anoxic zones where anaerobic should be.
• Excess Fe addition (chemical P-removal).
• Insufficient carbon substrate.
• Overabundant GAOs.

✅ Final Conclusions

• PAOs (Accumulibacter, Tetrasphaera, Dechloromonas) dominate globally.
• GAOs often coexist, not necessarily harmful.
• Danish EBPR plants perform well but can improve in stability.
• Microbial diversity and ecology determine process success.
• Recovery of N and P is key for circular nutrient economy 🌍♻️