Anaerobic & Anoxic Biotreatment of Waste

Group Presentation · Biochemistry

Anaerobic and Anoxic
Biotreatment of Waste

How microbes turn waste into resources — a journey through treatment technologies

Character A · 甲

Xiao Nong 小浓

High-strength organic wastewater.
Our protagonist on this journey.

Character B · 乙

Dr. Bio

Environmental engineer & guide.
Explains the science along the way.

02 — Foundation

What is Waste?

Not all wastewater is "high-potential" — the distinction matters for how we treat it

💧 Weak sewage

Low concentration

COD typically < 200 mg/L
Urban runoff, domestic sewage
Pollutant as nuisance
Goal: remove & discharge

🏭 Wastewater (High-Strength)

High concentration

COD typically > 1,000 mg/L
Breweries, food processing, landfill leachate
Pollutant as potential resource
Goal: treat AND recover

Key insight: High-strength "waste" contains chemical energy — COD = stored energy waiting to be harvested

Why biotreatment?

Microorganisms naturally break down organic compounds
Can be engineered to recover energy (biogas) and nutrients (N, P)
Lower energy input than chemical/physical treatment for high-strength waste

03 — Meet the Protagonist

Xiao Nong's Profile

High-strength wastewater from a brewery — high concentration sometimes can = high potential

⚠ High COD

5,000 mg/L

Organic matter — depletes O₂ in rivers, kills fish

→ Energy potential

⚠ High NH₄⁺

200+ mg/L

Ammonia nitrogen — toxic to aquatic life, causes eutrophication

→ Nitrogen resource

⚠ High P

30+ mg/L

Phosphorus — triggers algal blooms, eutrophication

→ Fertilizer potential

High concentration ——> High potential
The bigger the problem, the bigger the opportunity for resource recovery

04 — Core Concepts

Anaerobic vs Anoxic

Two oxygen-free environments — completely different microbial strategies

    Quick check — drag each label to the correct environment
  

DROP ZONE A

?

DROP ZONE B

?

ZONE A clues

Zero O₂ · produces CH₄ · electron acceptor: CO₂

ZONE B clues

No O₂ · has NO₃⁻ · produces N₂

      ANAEROBIC
    

      ANOXIC
    

⚫ ANAEROBIC

Zero O

No (strictly zero)
Electron acceptor: CO₂ / organic compounds
End product: CH₄ + CO₂ (biogas)
Carbon source: Organic matter (oxidised)
Use: COD removal & energy

🔵 ANOXIC

No O, has NO₃⁻

No (but NO₃⁻ present)
Electron acceptor: NO₃⁻ or NO₂⁻
End product: N₂ gas
Carbon source: Organic matter (as electron donor)
Use: Nitrogen removal

A²/O note: Real systems like A²/O combine Anaerobic + Anoxic + Oxic zones — no single environment handles everything alone

Route A · Stop 1

The Biogas Pit Era

The original anaerobic reactor — slow but foundational. CSTR (Continuously Stirred Tank Reactor) is its engineered upgrade.

📷 Biogas Pit — 沼气池

4-Stage Relay Reaction

📷 CSTR Reactor — 连续搅拌反应器

    Quick check — what does the stirrer actually do in a CSTR?
  

A · It pumps wastewater in and out of the reactor

B · It separates biogas from the liquid

C · It keeps the contents uniformly mixed — preventing dead zones and improving microbe-substrate contact

D · It heats the reactor to optimal temperature

CSTR upgrade: Adds mechanical mixing → more uniform environment, prevents dead zones, slightly faster than open pit

HRT: 20–30 days

COD removal: ~50–70%

Biogas: 60% CH₄

First proof that high-COD waste → biogas energy is possible
Established the 4-stage anaerobic pathway — still the basis of all anaerobic processes
Significant COD reduction without chemical inputs
Biogas can power the treatment plant itself

Conceptual breakthrough: Waste = energy source

HRT 20–30 days — enormous reactor volume needed
Microbes are not retained — wash out with effluent
No N or P removal — only handles COD
Low biomass concentration → low reaction rate

Core problem: Microbes leave the reactor — you can't keep them working

Comparison will grow as you learn more technologies

Parameter	Biogas Pit / CSTR
HRT	20–30 days
Biomass retention	❌ Poor (washout)
COD removal	50–70%
Reactor volume	Very large
N & P removal	❌ None

Route A · Stop 2

The UASB Revolution

Up-flow Anaerobic Sludge Blanket — Prof. Lettinga's 1970s breakthrough that changed everything

Key Innovation: Keep the Microbes

Innovation 1

Granular Sludge

Microbes self-aggregate into dense granules (1–3 mm). Dense enough to settle against upward flow — retained in reactor.

Innovation 2

Three-Phase Separator

V-shaped baffle at top: separates gas ↑, liquid →, sludge ↓ simultaneously — no moving parts.

HRT: 6–12 hours

Speed-up: ~40×

COD removal: 70–90%

Solves biomass washout — granules stay, treatment is continuous
HRT: 6–12 hours vs 20–30 days — same volume, 40× faster
High biomass concentration → high reaction rate
No moving parts in separator — low maintenance

The insight: Separate HRT from SRT (sludge retention time) — keep microbes long, process water fast

Granule formation can take months — long start-up
Sensitive to high suspended solids (can clog sludge bed)
Still no N or P removal
Performance drops with very high-strength or toxic waste
Maximum up-flow velocity limited → throughput ceiling

Next challenge: Push velocity higher, handle more concentrated waste → EGSB

Parameter	Biogas Pit / CSTR	UASB
HRT	20–30 days	6–12 hours ✅
Biomass retention	❌ Poor	✅ Granular sludge
COD removal	50–70%	70–90% ✅
Up-flow velocity	~0.5 m/h	1–3 m/h
N & P removal	❌ None	❌ None
Start-up time	Weeks	Months (granule formation)

Route A · Stop 3

The EGSB — Expanding the Blanket

Expanded Granular Sludge Bed — higher velocity, taller reactor, expanded sludge bed

Same principle, higher velocity

UASB — real installation

EGSB — real installation

Expanded bed = better mixing — granules are in constant gentle motion → better contact between wastewater and microbes → faster degradation

HRT: 2–6 hours

Up-flow: 4–10 m/h

COD removal: 80–95%

Handles: Very high-strength

Handles very high COD waste (up to 50,000 mg/L) — UASB struggles above ~15,000
Better mass transfer → higher volumetric loading rate
Less clogging — expanded bed self-cleans
Smaller footprint than UASB for same throughput

Requires effluent recycle pump — energy cost
More complex operation — velocity must be carefully controlled
Granule loss possible if velocity too high
Still no N or P removal
Membrane fouling not addressed — biomass can escape

Next challenge: Retain even the finest biomass → couple with membrane → AnMBR

Parameter	Biogas Pit	UASB	EGSB
HRT	20–30 days	6–12 h	2–6 h ✅
Up-flow velocity	~0.5 m/h	1–3 m/h	4–10 m/h ✅
Max COD load	Low	~15,000 mg/L	~50,000 mg/L ✅
Sludge bed state	Settled	Packed blanket	Expanded (fluidised)
COD removal	50–70%	70–90%	80–95% ✅

Route A · Stop 4

EGSB + AnMBR

Anaerobic Membrane Bioreactor — retain everything, lose nothing

EGSB + Membrane Filtration

Result: Run at very high biomass concentrations (20–40 g VSS/L vs 8–12 in EGSB) → fastest degradation rate yet

HRT: < 2 hours

Biomass: 20–40 g/L

COD removal: >95%

Effluent SS: ~0

Ultimate biomass retention — no cell loss
Extremely high loading rates — smallest reactor footprint of all anaerobic systems
Excellent effluent quality for reuse or downstream treatment
Can handle toxic or inhibitory compounds — slow-growing specialised microbes are retained

Membrane fouling — requires periodic cleaning (energy + chemicals)
Biogas can get trapped in membrane → needs degassing system
Still no N or P removal — must be followed by Route B treatment

Route A conclusion: COD → handled. Energy → recovered. Now we need Route B for nitrogen and phosphorus.

Parameter	CSTR	UASB	EGSB	EGSB+AnMBR
HRT	20–30 d	6–12 h	2–6 h	<2 h ✅
Biomass (g/L)	~3–5	~15–20	~15–25	20–40 ✅
COD removal	50–70%	70–90%	80–95%	>95% ✅
Effluent SS	High	Low	Low	~0 ✅
Complexity	Low	Medium	Medium-High	High

Route B · Stop 1

The A²/O Process

Anaerobic–Anoxic–Oxic: three zones, three jobs — simultaneous C, N, P removal

    Quick check — drag to arrange the A²/O zones in the correct order
  

1st

2nd

3rd

Anaerobic zone

PAOs release P
Store VFAs as PHA

EQUATIONS

                  CH₃COO⁻ + PAO → PHA

                  Poly-P → Pi (released)

                  C₆H₁₂O₆ → 2C₂H₅OH + 2CO₂

Anoxic zone

Denitrifiers use PHA
NO₃⁻ → N₂ gas

EQUATIONS

                  6NO₃⁻ + 5CH₃OH →

                  3N₂ + 5CO₂ + 7H₂O + 6OH⁻

                  (PHA as electron donor)

Oxic zone

Nitrification: NH₄⁺→NO₃⁻
PAOs over-absorb P

EQUATIONS

                  NH₄⁺ + 2O₂ → NO₃⁻ + 2H⁺ + H₂O

                  PO₄³⁻ + PAO → Poly-P

                  (over-absorption)

First system to achieve simultaneous C, N, P removal
Biological phosphorus removal — no chemicals needed
Spatial separation → each microbial community thrives in its optimal zone
Internal recycle links the oxic and anoxic zones — elegant engineering

Breakthrough: Wastewater treatment becomes integrated — one system, three problems solved

The Achilles' Heel: Carbon Competition

PAOs (anaerobic zone) need organic carbon to store as PHA
Denitrifiers (anoxic zone) need organic carbon as electron donor
Low C:N ratio wastewater → not enough carbon for both
Adding external carbon (methanol, acetate) = extra cost
Still requires aeration energy for oxic zone

Key question: Can we remove nitrogen without needing organic carbon at all?

Route B starts here — comparison will grow

Parameter	A²/O
C removal	✅ Yes
N removal	✅ Yes (via nitrification/denitrification)
P removal	✅ Yes (biological)
Carbon source needed	⚠ Yes — for denitrification & P release
Aeration energy	⚠ High (oxic zone)
Sludge production	Moderate

Route B · Stop 2

The Anammox Revolution

Anaerobic Ammonium Oxidation — no carbon, no oxygen, no problem

NH₄⁺ + 1.32 NO₂⁻ → 1.02 N₂ + 0.26 NO₃⁻ + 2H₂O

Who does this?

Anammox bacteria

Autotrophic — fix CO₂, no organic C
Strictly anoxic environment
Deep red colour (haem protein)
Doubling time: ~11 days (very slow)

What's needed?

Partial nitritation

Need NO₂⁻, not NO₃⁻
AOB convert: NH₄⁺ → NO₂⁻
Stop nitrification at half-way
Ratio: NH₄⁺ : NO₂⁻ ≈ 1 : 1.32

Multi-Zonal Anammox (Prof. Yongzhen Peng, Beijing Univ. of Technology): Anammox bacteria distributed across all three zones of A²/O — more stable, broader applicability

🔋 Carbon saved

~100%

No organic carbon needed for N removal — solved the carbon competition problem completely

⚡ Energy saved

~60%

No need to fully nitrify NH₄⁺ → NO₃⁻ → massive aeration energy reduction

🧪 Sludge reduced

>90%

Autotrophic, slow-growing bacteria produce far less excess sludge

🌍 N₂O reduced

Lower

Fewer nitrification steps → less N₂O (300× CO₂ warming potential)

Anammox bacteria grow extremely slowly (doubling time ~11 days) — months of start-up
Sensitive to temperature (optimal 30–40°C), dissolved oxygen, and pH
Requires precise partial nitritation control — stopping at NO₂⁻ is tricky
Mainly applied to high-ammonia sidestreams (digester reject water) — mainstream application still challenging
P removal still needed separately

Future: Multi-zonal strategy aims to make Anammox work in mainstream wastewater → carbon-neutral WWTPs

Parameter	A²/O	Anammox
N removal pathway	Nitrification + Denitrification	Direct NH₄⁺ + NO₂⁻ → N₂
Carbon needed?	⚠ Yes	✅ No
Aeration energy	High	~60% less ✅
Sludge production	Moderate	>90% less ✅
P removal	✅ Yes (biological)	⚠ Not included
Start-up time	Weeks	⚠ Months
Operational complexity	Medium	High

Final — Synthesis

Anaerobic vs Anoxic: Full Picture

After seeing all the technologies — how do these two strategies compare and complement each other?

Feature	Anaerobic	Anoxic
O₂	Strictly zero	Zero (but NO₃⁻ present)
Electron acceptor	CO₂ / organic C	NO₃⁻ or NO₂⁻
Main product	CH₄ (energy!)	N₂ (harmless gas)
Primary role	COD removal + energy recovery	Nitrogen removal
Technologies	CSTR → UASB → EGSB → AnMBR	A²/O → Anammox
Carbon requirement	Consumes organic C	Needs C (A²/O) or none (Anammox)
Energy	Produces energy	Consumes energy (aeration in A²/O)
Sludge	Moderate	High (A²/O) / Very low (Anammox)

They are complementary, not competing
Real wastewater treatment plants use both — Anaerobic first (remove COD, recover energy), then Anoxic/Oxic (remove N & P)

The evolution in one sentence:
From "treat waste and discard" → to "recover energy, nitrogen, phosphorus, and water — leave nothing behind"

References

References

[1]

Zieliński, M., Kazimierowicz, J., & Dębowski, M. (2023). Advantages and limitations of anaerobic wastewater treatment — technological basics, development directions, and technological innovations. Energies, 16(1), 83. https://doi.org/10.3390/en16010083

[2]

Zhang, X., Fan, Y., Hao, T., Chen, R., Zhang, T., Hu, Y., Li, D., Pan, Y., Li, Y. Y., & Kong, Z. (2024). Insights into current bio-processes and future perspectives of carbon-neutral treatment of industrial organic wastewater: A critical review. Environmental Research, 241, 117630. https://doi.org/10.1016/j.envres.2023.117630

[3]

Zhang, X., Hao, T., Zhang, T., Hu, Y., Lu, R., Li, D., Pan, Y., Li, Y. Y., & Kong, Z. (2024). Towards energy conservation and carbon reduction for wastewater treatment processes: A review of carbon-neutral anaerobic biotechnologies. Journal of Environmental Chemical Engineering, 12(3), 112448. https://doi.org/10.1016/j.jece.2024.112448

[4]

Mariraj Mohan, S., & Swathi, T. (2022). A review on upflow anaerobic sludge blanket reactor: Factors affecting performance, modification of configuration and its derivatives. Water Environment Research, 94(1), e1665. https://doi.org/10.1002/wer.1665

[5]

D'Bastiani, C., Kennedy, D., & Reynolds, A. (2023). CFD simulation of anaerobic granular sludge reactors: A review. Water Research, 242, 120220. https://doi.org/10.1016/j.watres.2023.120220

[6]

Parihar, R. K., Burnwal, P. K., Chaurasia, S. P., et al. (2024). Unveiling the evolution of anaerobic membrane bioreactors: applications, fouling issues, and future perspective in wastewater treatment. Reviews in Environmental Science and Bio/Technology, 23, 949–988. https://doi.org/10.1007/s11157-024-09710-6

[7]

Elmoutez, S., Abushaban, A., Necibi, M. C., Sillanpää, M., Liu, J., Dhiba, D., Chehbouni, A., & Taky, M. (2023). Design and operational aspects of anaerobic membrane bioreactor for efficient wastewater treatment and biogas production. Environmental Challenges, 10, 100671. https://doi.org/10.1016/j.envc.2022.100671

[8]

Deng, Z., Sun, C., Ma, G., Zhang, X., Guo, H., Zhang, T., Zhang, Y., Hu, Y., Li, D., Li, Y. Y., & Kong, Z. (2025). Anaerobic treatment of nitrogenous industrial organic wastewater by carbon-neutral processes integrated with anaerobic digestion and partial nitritation/anammox: Critical review of current advances and future directions. Bioresource Technology, 415, 131648. https://doi.org/10.1016/j.biortech.2024.131648

The End

Thank You — Q&A

The magical journey of waste continues...

Route A Summary

Biogas Pit → concept proven
UASB → biomass retained
EGSB → velocity + load up
AnMBR → ultimate retention

Route B Summary

A²/O → C+N+P together
Anammox → no carbon needed
Multi-Zonal → real-world scale

Waste is not waste — it's a misplaced resource

📖 Glossary