Anammox reactors are not pure cultures, and most failures are microbiological before they are hydraulic. Metagenomic screening can distinguish washout from inhibition from competition — three failures that look identical on an effluent chart and demand opposite responses.
Which organism you actually have
Four genera of Planctomycetota appear in engineered systems, and they occupy different niches. Knowing which one dominates a given reactor tells you how it will behave when the temperature or the load moves.
| Genus | Niche | Where it turns up |
|---|---|---|
| Candidatus Brocadia | 25–35 °C, ammonium above ~100 mg N/L | The usual dominant in sidestream reject water treatment |
| Candidatus Kuenenia | Below 20 °C, lower substrate concentrations | Mainstream systems; the model organism for genomic work |
| Candidatus Scalindua | Marine, low temperature | Rare in engineered systems |
| Candidatus Jettenia | Thermophilic, 40–50 °C | Niche |
Why 16S sequencing misleads on abundance
The 16S rRNA gene copy number varies between one and two per genome across these organisms, so a 16S-based quantification can be out by a factor of two purely on gene dosage before any other error. The hydrazine synthase gene hzsA is present as a single copy in all Anammox bacteria, which makes it the better normalisation target and the more reliable abundance marker.
The functional genes worth measuring
Metagenomics resolves the full metabolic pathway, but four gene targets carry nearly all the diagnostic value.
| Gene | Product | Role |
|---|---|---|
| hzsABC | Hydrazine synthase | The signature enzyme: NH4+ + NO → N2H4 |
| hdh | Hydrazine dehydrogenase | Oxidises hydrazine to nitrogen gas |
| nirS | Cytochrome cd1 nitrite reductase | Reduces nitrite to nitric oxide — the step feeding hydrazine synthase |
| dsrAB | Dissimilatory sulphite reductase | Not Anammox — a marker for sulphate reducers, whose sulphide inhibits |
What transcription shows under stress
RNA sequencing distinguishes a population that is absent from one that is present but suppressed — a distinction that matters enormously for the response.
- Cold stress at 10 °C: chaperones and cold-shock proteins upregulate while hzsA expression falls by around 60 per cent. The organisms are there and alive; they are simply not working.
- Oxygen above 0.5 mg/L: catalase and superoxide dismutase upregulate, and hydrazine synthase expression is inhibited — but reversibly. Anammox inhibition by oxygen is not the death sentence it is sometimes described as, provided exposure is brief.
- Organic shock: heterotrophs bloom on the added carbon and outcompete Anammox for nitrite. The Anammox population does not die, it starves.
The community around them
Four groups routinely coexist with Anammox, and only one is unambiguously helpful.
| Group | Effect |
|---|---|
| Denitrifiers (Comamonas, Thauera) | Compete for nitrite, but scavenge trace oxygen — can be net beneficial |
| DNRA organisms (Geobacter, Shewanella) | Reduce nitrate back to ammonium, raising the ammonium load the process must handle |
| Sulphate reducers (Desulfovibrio) | Produce sulphide, which inhibits Anammox from around 0.2 mM H2S |
| Nitrite oxidisers (Nitrospira) | The principal competitor in mainstream systems; consume the nitrite Anammox needs |
Using genomics as a diagnostic
The practical value is discriminating between failure modes that present identically — rising effluent ammonium, falling removal — but need opposite responses.
| Observation | Diagnosis | Response |
|---|---|---|
| hzsA gene abundance falling over successive samples | Population is washing out | Check sludge age and biomass retention — a hydraulic problem, not a chemical one |
| hzsA abundance stable but expression down | Population present and suppressed | Look at temperature, oxygen exposure and inhibitors; do not reseed |
| nirS expression down, nitrite accumulating | Nitrite reduction is limiting | Check pH is above 7.0, and temperature |
| dsrAB abundant | Sulphate reduction generating sulphide | Precipitate sulphide with ferrous dosing; check for saline infiltration or co-digestion feedstock |
Quarterly screening is proportionate for works above about 100,000 PE or industrial installations with high process risk — particularly co-digestion sites, where sulphide is a live hazard. Related: Anammox deammonification, hydrogen sulphide control, biological nutrient removal.
Frequently asked questions
Why use hzsA rather than 16S rRNA to quantify Anammox?
Because 16S copy number varies between one and two per genome across the Anammox genera, so abundance can be out by a factor of two on gene dosage alone. Hydrazine synthase (hzsA) is single-copy in all Anammox bacteria and unique to them, which makes it both a cleaner quantitative marker and an unambiguous presence test.
Is oxygen exposure fatal to Anammox?
Not usually. Above about 0.5 mg/L, hydrazine synthase expression is inhibited and the cells upregulate oxidative stress defences — but the inhibition is reversible if exposure is brief. Persistent exposure is a different matter. This is why transcription data is more useful than abundance data after an aeration upset.
What sulphide concentration inhibits Anammox?
Inhibition sets in from around 0.2 mM hydrogen sulphide. That makes sulphate-reducing activity a real risk at co-digestion works and at sites with saline infiltration. Ferrous dosing to precipitate sulphide is the usual control, and dsrAB abundance is the marker that tells you whether it is needed.
Is quarterly metagenomic screening worth the cost?
At works above roughly 100,000 PE, or on industrial installations where an upset is expensive, yes — principally because it separates washout from suppression, which conventional monitoring cannot. On a small, stable sidestream reactor it is hard to justify against routine respirometry.