Lesson 8 note about EBPR

Environmental Biotechnology

🧫 1. Why do we test activated sludge?

Wastewater treatment depends on microbial populations that clean up pollutants. To understand or optimize these systems, scientists run batch activity tests — small experiments to see:

How fast and how well microbes remove contaminants 💧
Which types of bacteria dominate (e.g. PAO, GAO, AOO, NOO, Anammox, SRB)
How toxins or different conditions affect performance

➡️ These tests help determine stoichiometry (mass balances) and kinetic rates for modeling microbial activity.

⚙️ 2. Types of activity tests

Batch tests vary depending on the microbe and process studied:

OHO (Ordinary Heterotrophic Organisms): aerobic degradation of organics
dOHO: denitrifying organics users (use nitrate/nitrite instead of oxygen)
AOO: ammonia oxidizers 🧪
NOO: nitrite oxidizers
PAO: phosphorus removers 🧫
Anammox: anaerobic ammonia oxidizers
SRB: sulfate reducers

The book starts with EBPR since it combines anaerobic, anoxic, and aerobic stages — the most complex case!

🧪 3. Enhanced Biological Phosphorus Removal (EBPR)

EBPR removes phosphorus biologically rather than chemically, saving costs and energy ⚡. It relies on Polyphosphate-Accumulating Organisms (PAO).

💡 Why it’s useful:

High phosphorus removal efficiency
Low sludge production
Possibility to recover phosphorus (a valuable nutrient)
Lower operational costs

🧍‍♀️ The main players:

PAO (Polyphosphate-Accumulating Organisms) – the “good guys” who store P
GAO (Glycogen-Accumulating Organisms) – the “troublemakers” who compete but don’t remove P

🔄 4. The PAO metabolism — three stages

1️⃣ Anaerobic phase (no oxygen)

PAO take up volatile fatty acids (VFAs) (especially acetate and propionate).
They store these as poly-β-hydroxy-alkanoates (PHA) for later energy.
To do this, they hydrolyze polyphosphate (poly-P) and consume glycogen.
That releases orthophosphate (PO₄³⁻) into the water 💥
→ Result: Phosphorus goes up in liquid.

Main polymers used:

Poly-P → supplies energy (ATP)
Glycogen → provides reducing power (NADH)
PHA → carbon/energy storage for later

Example polymers: PHB, PHV, PH2MV — types of PHA depending on acetate or propionate used.

2️⃣ Aerobic phase (oxygen present)

PAO now consume PHA for energy, using oxygen.
They take up more phosphate from the water than they released before 🧲
P is stored again as poly-P inside the cell
They also rebuild glycogen, grow, and perform maintenance.
When the phosphorus-rich sludge is removed (WAS), phosphorus leaves the system 🌱

➡️ This is how EBPR actually removes phosphorus from wastewater.

3️⃣ Anoxic phase (no oxygen but nitrate/nitrite available)

Some PAO can also use nitrate or nitrite instead of oxygen — these are denitrifying PAO (DPAO).
This allows simultaneous removal of nitrogen and phosphorus 💪
Important for full nutrient-removal plants.

⚠️ 5. When things go wrong — GAO invasion

GAO (Glycogen-Accumulating Organisms) behave like PAO but don’t use poly-P and don’t remove phosphorus. They rely only on glycogen. If GAO dominate, EBPR fails ❌

GAO thrive under:

High temperature (> 20 °C)
Low pH (< 7)
High oxygen (> 2 mg/L)
Only one carbon source (like acetate only)

🧰 6. Experimental set-up (how to test EBPR)

To test EBPR performance, scientists use reactors (fermenters) under controlled conditions.

A good setup must:

Prevent oxygen intrusion (anaerobic/anoxic tests)
Maintain proper DO (> 2 mg/L) in aerobic stage
Ensure good mixing
Control temperature and pH precisely
Have sampling ports for gas/liquid addition

📸 The chapter shows vintage EBPR reactors from Delft University — early 1990s pioneering setups for Bio-P modeling!

🧬 7. Key concepts and models

TUDelft Bio-P model — describes metabolic pathways and balances for PAO and GAO.
Research evolved to understand:
- Temperature effects 🌡️
- Substrate competition (acetate vs propionate)
- Poly-P and glycogen kinetics
- Nitrite/nitrate inhibition and sulfide effects

These models help design modern EBPR plants that work efficiently even under complex conditions.

The reference list spans decades of foundational EBPR work — from Mino, Comeau, van Loosdrecht, Nielsen, and Oehmen, all the way to modern metabolic and molecular studies identifying Candidatus Accumulibacter phosphatis 🧫.

This body of work built today’s understanding of how activated sludge communities achieve biological phosphorus removal.

🧠 Summary cheat-view

Stage	Key Event	Energy Source	Result
Anaerobic	VFA uptake, PHA storage, poly-P hydrolysis	Poly-P + Glycogen	P released
Aerobic	PHA oxidation, P uptake, glycogen rebuild	Oxygen	P removed
Anoxic	Same as aerobic but with nitrate/nitrite	Nitrate/nitrite	Simultaneous P+N removal

Quiz

Score: 0/30 (0%)

Q0. What is the main purpose of batch activity tests in biological wastewater treatment?

To determine sludge color

To study microbial kinetics and stoichiometry

To increase pH stability

To remove heavy metals

Q1. Which organisms are primarily responsible for Enhanced Biological Phosphorus Removal (EBPR)?

Ordinary heterotrophic organisms

Polyphosphate-accumulating organisms

Glycogen-accumulating organisms

Ammonia-oxidizing organisms

Q2. During the anaerobic phase, PAOs use which intracellular compound to generate ATP for VFA uptake?

Polyphosphate

Nitrate

Nitrite

Ammonium

Q3. Which compounds are stored by PAOs as intracellular carbon and energy reserves during the anaerobic phase?

Polyhydroxyalkanoates (PHA)

Glycogen

Polyphosphate

All of the above

Q4. What happens to orthophosphate concentration in the bulk liquid during the anaerobic stage?

It decreases

It remains constant

It increases

It is oxidized

Q5. Which of the following VFAs mainly leads to PHB production in PAOs?

Propionate

Acetate

Butyrate

Lactate

Q6. What occurs during the aerobic stage of the EBPR cycle?

Poly-P is hydrolyzed

PHA is used for phosphate uptake

VFA uptake continues

Nitrate reduction occurs

Q7. What is the typical phosphorus content of PAO biomass compared to OHO?

0.03 gP/gVSS for PAO vs 0.35 for OHO

0.35 gP/gVSS for PAO vs 0.03 for OHO

0.7 gP/gVSS for both

Equal for both

Q8. Which factor does NOT favor GAO proliferation in EBPR systems?

High temperature (>20°C)

Low pH (<7)

High dissolved oxygen (>2 mg/L)

Low temperature (<15°C)

Q9. What makes GAOs problematic in EBPR systems?

They consume nitrate

They remove phosphorus too fast

They compete with PAOs for substrate but do not remove phosphorus

They increase sludge production

Q10. Which conditions must be maintained in reactors to ensure reliable EBPR activity tests?

Uncontrolled temperature

High turbulence only

Controlled DO, pH, and temperature

No mixing to allow settling

Q11. Which molecule acts as an electron acceptor during anoxic phosphorus uptake by denitrifying PAO?

Oxygen

Carbon dioxide

Nitrate or nitrite

Sulfate

Q12. Which Delft model was developed to simulate EBPR metabolism?

ASM3

TUDelft Bio-P model

Anammox-N model

BioWin

Q13. Which stage results in net phosphorus removal from wastewater?

Anaerobic

Aerobic

Anoxic

Settling

Q14. What is the main energy source for GAOs during the anaerobic stage?

Polyphosphate

Glycogen

Nitrate

Oxygen

Q15. Enhanced Biological Phosphorus Removal (EBPR) is performed by PAOs.

True

False

Q16. PAOs can store volatile fatty acids as PHA even under aerobic conditions.

True

False

Q17. During the anaerobic phase, PAOs release phosphate into the liquid.

True

False

Q18. In the aerobic phase, PAOs consume PHA to generate energy for phosphorus uptake.

True

False

Q19. GAOs enhance phosphorus removal efficiency in EBPR systems.

True

False

Q20. Denitrifying PAOs can use nitrate as an electron acceptor to remove phosphorus under anoxic conditions.

True

False

Q21. Polyphosphate hydrolysis provides reducing power for PHA formation.

True

False

Q22. The anaerobic uptake of acetate by PAOs depends on intracellular glycogen reserves.

True

False

Q23. High pH values favor GAO growth over PAO.

True

False

Q24. The EBPR process includes anaerobic, anoxic, and aerobic environments.

True

False

Q25. Polyhydroxybutyrate (PHB) and Polyhydroxyvalerate (PHV) are examples of PHA polymers.

True

False

Q26. Net phosphorus removal occurs when PAO release phosphate during anaerobic conditions.

True

False

Q27. Temperature has no effect on PAO and GAO competition.

True

False

Q28. The TUDelft Bio-P model was validated using full-scale plant batch tests in the Netherlands.

True

False

Q29. Anaerobic P release and aerobic P uptake form the metabolic basis of EBPR.

True

False