🌱 1. From Sulfur to Nitrogen: The Early Journey

• Kuenen started with sulfur-oxidizing bacteria (like Thiomicrospira pelophila) to understand how microorganisms use inorganic compounds as energy.
• These early studies helped define chemostat culture as a tool for studying ecological niches — comparing obligate autotrophs (use CO₂ only) vs. facultative (can use organic C too).
• This laid the groundwork for later wastewater applications: using sulfur oxidizers to remove H₂S and recover elemental sulfur 🧪💨.

🧫 2. Sulfur + Nitrogen = New Possibilities

• Collaborations led to denitrifying sulfur-oxidizers that could remove both sulfide and nitrate from wastewater.
• One special bacterium, Thiosphaera pantotropha, could use oxygen or nitrate flexibly — a perfect example of metabolic versatility (mixotrophy).

💡 This success encouraged the team to explore nitrate, nitrite, and ammonium removal more deeply — paving the way for the next big surprise.

🚀 3. The Accidental Discovery of Anammox

💥 In 1986–1989, while running a denitrifying fluidized bed reactor, engineers noticed something strange:

Ammonium disappeared — even though there was no oxygen.

After testing with ¹⁵N-labelled ammonium, they saw formation of mixed ¹⁴,¹⁵N₂ gas → proof that ammonium was being oxidized anaerobically! This was the long-predicted but never-seen reaction: ext{NH}_4^+ + ext{NO}_2^- → ext{N}_2 + 2H_2O → The anammox (anaerobic ammonium oxidation) process was real 🎉

🧪 4. Growing the “Un-growable”

• It took months to years to cultivate anammox bacteria — they grew extremely slowly (doubling time ≈ 10 days).
• Success came when they used nitrite instead of nitrate, and the culture turned pink-red — a key visual clue.
• The anammox cells used only ammonium, nitrite, bicarbonate + vitamins — showing they were autotrophic.

🧬 Later, researchers discovered an intracellular organelle, the anammoxosome, responsible for the main reactions.

🧠 5. Unraveling the Metabolism

Inside the anammoxosome, reactions involve some truly wild chemistry:

1. Nitrite → nitric oxide (NO)
2. NO + ammonium → hydrazine (N₂H₄) 😱 — yes, the same compound used as rocket fuel!
3. Hydrazine → N₂ gas + electrons for CO₂ fixation

These electrons drive energy conservation and ATP production via a membrane system similar to mitochondria. This was one of the first known bacteria with a real organelle-like compartment.

🧫 6. Naming and Classifying the Bacteria

• The first one enriched was named Candidatus Brocadia anammoxidans.
• Later came Kuenenia stuttgartiensis, Scalindua, and others (five genera total today).
• They belong to the Planctomycetes phylum, famous for their compartmentalized cells.
• Their membranes contain unique ladderane lipids 🧩— never seen before in nature — that keep hydrazine safely contained.

🌊 7. Anammox in Nature

Marine scientists soon found anammox activity in:

• Aarhus Bay sediments (Denmark)
• The Black Sea’s oxygen minimum zone
• Ocean upwelling regions and sediments worldwide 🌍

Now we know anammox accounts for 40–50% of nitrogen loss from the oceans, making it a major player in the global N-cycle.

🧬 8. Genomics and Biochemistry

• The Kuenenia stuttgartiensis genome (4.27 Mb) revealed >200 cytochrome genes — explaining the red color.
• Metagenomics and structural studies (2006–2016) mapped the complete energy and CO₂-fixing pathways, confirming that NO (not hydroxylamine) is the key intermediate in most species.

🧩 The membrane proteins Nir, HZS, HDH, and NXR run the electron flow that powers ATP synthesis and CO₂ fixation (via the acetyl-CoA pathway).

🧱 9. From Discovery to Application

Researchers at Delft and Paques Company built the first pilot anammox reactors in Rotterdam 💧:

• Step 1: partial nitrification (ammonium → nitrite) under oxygen limitation.
• Step 2: anammox bacteria use remaining ammonium + nitrite → N₂.

This saves oxygen and avoids methanol addition used in classical denitrification → much cheaper and greener 🌿.

Now, granular sludge systems combine both steps in one reactor (CANON process), where nitrifiers grow on the surface and anammox bacteria in the core.

🌎 10. Ecology & Interactions

Anammox bacteria thrive where nitrite + ammonium overlap, such as:

• Sediment layers
• Biofilms and sludge granules
• Oxygen-limited interfaces

They interact and compete with nitrifiers, denitrifiers, and DNRA bacteria, forming tight nutrient cycles. Sometimes, anammox bacteria even generate their own substrates by reducing nitrate to ammonium internally — a neat self-feeding trick 🤯.

🔬 11. Beyond Anammox: Other Adventures

Kuenen’s curiosity didn’t stop there:

• He studied Thioploca in Chile — giant sulfur filaments storing nitrate intracellularly.
• Later, in California’s “The Cedars,” he explored hyper-alkaliphilic hydrogen-oxidizing bacteria (pH > 12!).
• Even in retirement, he remained active in denitrifier and N₂O-reducing research.

💭 12. Future Questions

Still open today:

• How exactly is the proton motive force generated inside the anammoxosome?
• What’s the full function of all those HAO-like enzymes?
• How are ladderane lipids made, and do they protect against hydrazine leakage?
• Can we ever grow a pure culture (no “Candidatus”)?

🧩 Final Takeaway

Anammox changed how we understand and treat nitrogen: 💧 Ecologically: It’s a key global N-loss pathway. 🏭 Technologically: It revolutionized wastewater treatment. 🔬 Biologically: It revealed the first bacterial organelle for energy metabolism and the strangest biochemistry imaginable — involving rocket fuel!