The Diverse Microbiome of Anammox Reactors
Beneath the surface of modern wastewater treatment plants, a silent revolution is taking place. Instead of relying on energy-intensive machinery and chemicals, a consortium of remarkable bacteria is working in harmony to purify water.
The anammox process represents one of the most significant discoveries in wastewater treatment in recent decades. Discovered in the 1990s, this biological process allows certain bacteria to convert ammonium and nitrite directly into nitrogen gas under anaerobic conditions, removing nitrogen from wastewater without the need for oxygen or organic carbon sources 2 .
Reduction in aeration energy consumption
Reduction in sludge production
Organic carbon additives required
This makes it exceptionally environmentally friendly and cost-effective, particularly for treating nitrogen-rich wastewater from sources like landfill leachate, pharmaceutical manufacturing, and rare earth mining operations 4 9 .
NH₄⁺ + 1.32NO₂⁻
1.02N₂ + 0.26NO₃⁻ + 2.03H₂O
Stoichiometry of the anammox process 2
What makes this process truly remarkable isn't just the capabilities of the anammox bacteria themselves, but the complex microbial communities they form with other bacteria—consortia whose composition shifts dramatically depending on whether they're fed precisely controlled synthetic wastewater or the chemically complex reality of industrial effluent.
Anammox bacteria belong to the phylum Planctomycetota and are currently represented by six known Candidatus genera: Brocadia, Kuenenia, Jettenia, Anammoxoglobus, Scalindua, and Anammoximicrobium 6 . These bacteria are the star performers in anammox reactors, but they don't work alone. They're supported by a diverse cast of microbial partners that create stable operating conditions and help break down interfering substances.
Recent research has dramatically expanded our understanding of this microbial diversity. A groundbreaking 2024 study compiled a comprehensive catalog of 1,376 species-level genomes from various anammox systems, revealing an astonishing complexity previously underappreciated by scientists 3 . This catalog provides unprecedented resolution for understanding the functional capabilities of different community members.
The anammox bacteria themselves, specializing in the core nitrogen conversion process.
Often the most abundant phylum alongside Planctomycetota, these filamentous bacteria provide structural support for granular sludge formation.
Including ammonia-oxidizing bacteria (AOB) that convert ammonia to nitrite in one-stage systems, and various heterotrophic bacteria that perform auxiliary functions.
Acidobacteriota, Bacteroidota, and Actinobacteria contributing to various metabolic functions and community stability.
The relative abundance of these phyla shifts significantly between reactors treating synthetic versus complex wastewater, with Chloroflexi often dominating in systems treating real wastewater where structural integrity becomes more critical for dealing with variable conditions .
The type of wastewater feeding an anammox reactor profoundly shapes its microbial composition and functional capabilities. Understanding these differences is crucial for optimizing treatment systems for different applications.
Typically contains precisely controlled concentrations of ammonium, nitrite, and essential minerals in balanced ratios. This controlled environment favors communities dominated by anammox bacteria with specific supporting cast members adapted to stable conditions.
From sources like pharmaceutical production, landfill leachate, or rare earth mining, contains unpredictable mixes of organic carbon, heavy metals, antibiotics, and other compounds that can inhibit anammox bacteria 4 9 .
| System Characteristic | Synthetic Wastewater Reactors | Complex Wastewater Reactors |
|---|---|---|
| Microbial Diversity | Lower diversity, clearer dominance patterns | Higher diversity with more species evenness |
| Key Phyla | Planctomycetota > Chloroflexi | Chloroflexi ≈ Planctomycetota |
| Network Complexity | Less modular, simpler interactions | Highly modular (modularity index ~0.56) 4 |
| Functional Redundancy | Lower - specialized functions | Higher - multiple species with similar capabilities |
| Stability Mechanism | Controlled conditions | Metabolic versatility and cooperative interactions |
A 2024 study conducted by researchers at Lanzhou Jiaotong University provides fascinating insights into how anammox bacterial communities can be rapidly enriched and studied . This experiment is particularly valuable for understanding the practical aspects of microbial community development in anammox systems.
The research team employed an up-flow anammox biofilm reactor filled with polyurethane porous material as a growth carrier. This material was selected for its high porosity, large specific surface area, and strong hydrophilicity—ideal properties for bacterial attachment and biofilm development.
The reactor was inoculated with sludge from a laboratory anammox reactor that had been operating stably for one year.
The reactor operated for 73 days with initial NH₄⁺-N and NO₂⁻-N concentrations of 30 mg/L and 40 mg/L respectively, and a hydraulic retention time (HRT) of 8 hours.
Over 103 days, researchers progressively increased influent substrate concentration to 60 mg/L NH₄⁺-N and 80 mg/L NO₂⁻-N while decreasing HRT to 4 hours.
Researchers documented physical changes in the biofilm and collected samples for microbial community analysis using Illumina MiSeq sequencing of the 16S rRNA gene.
After the enrichment period, the system achieved impressive removal rates of 97.87% for ammonia and 99.96% for nitrite. Physically, the development of brick-red anammox biofilms and granules provided visual confirmation of successful anammox bacteria enrichment.
Microbial analysis revealed a community dominated by Planctomycetota (25.25%), Chloroflexi (29.41%), and Proteobacteria (14.3%), with Candidatus Brocadia as the dominant anammox genus at 20.44% of the total community .
| Phylum | Relative Abundance (%) | Primary Function |
|---|---|---|
| Planctomycetota | 25.25 | Core anammox process |
| Chloroflexi | 29.41 | Structural support, granule formation |
| Proteobacteria | 14.30 | Various auxiliary functions |
| Other Phyla | 31.04 | Diverse metabolic contributions |
This experiment demonstrates that appropriate carrier materials can significantly accelerate anammox bacteria enrichment, reducing the typically lengthy start-up time from years to months. The porous polyurethane created ideal conditions for biofilm development, proving that physical environment design is as important as chemical parameters in shaping microbial communities.
While knowing which bacteria are present is valuable, understanding what they're capable of doing is far more important for predicting and optimizing reactor performance. Modern metagenomic approaches allow scientists to move beyond simple taxonomic identification to functional characterization of the entire microbial community.
The comprehensive catalog of 1,376 species-level genomes from anammox systems revealed 64 core genera that appear across different reactor types, suggesting these taxa perform essential functions that maintain ecosystem stability 3 . Beyond these core members, systems treating complex wastewater contain numerous conditionally rare taxa that may become important when dealing with specific contaminants or operational upsets.
The abundance and expression levels of these functional genes provide better predictors of reactor performance than simple taxonomic composition. For instance, reactors with higher copies of hzsA and hdh genes typically show higher nitrogen removal rates, regardless of which anammox genus is dominant 7 .
| Reagent/Solution | Function | Typical Composition |
|---|---|---|
| Trace Element Solution I | Provides iron and chelating agent | EDTA 5,000 mg/L, FeSO₄·7H₂O 5,000 mg/L |
| Trace Element Solution II | Supplies micronutrients | EDTA 15,000 mg/L, ZnSO₄·7H₂O 430 mg/L, CoCl₂·6H₂O 240 mg/L, CuSO₄·5H₂O 250 mg/L, NiCl₂·6H₂O 190 mg/L, Na₂MoO₄·2H₂O 220 mg/L, MnCl₂·4H₂O 990 mg/L |
| Synthetic Wastewater Base | Creates controlled experimental conditions | NH₄Cl, NaNO₂, NaHCO₃, KHCO₃, KH₂PO₄, MgSO₄·7H₂O, CaCl₂·2H₂O 4 |
| Lysis Buffers | DNA/RNA extraction for molecular studies | Various commercial kits using detergent-based lysis |
| PCR Master Mixes | Amplification of target genes | Primers specific for anammox 16S rRNA, hzsA, hdh genes 7 |
Understanding the diversity and functional capabilities of anammox microbiomes has profound practical implications. First, it allows engineers to design better inoculation strategies for wastewater treatment plants, selecting seed sludge with microbial compositions suited to specific wastewater types. Second, it enables the development of more robust monitoring protocols that track not just which bacteria are present, but what functions they're performing.
To predict reactor performance based on microbial community data 6 .
Combining metagenomics, metatranscriptomics, and metaproteomics for a complete picture of community function 5 .
Designed for specific industrial wastewater compositions.
Through conductive materials to boost metabolic rates 6 .
As research continues, the invisible workforce in anammox reactors will become increasingly efficient at handling the complex waste streams of modern industry, transforming environmental liability into sustainable water resource management.
The diverse microbiome of anammox reactors represents a remarkable example of nature's ingenuity harnessed for environmental protection. These complex microbial communities, whether nurtured on synthetic media or battling complex industrial waste, demonstrate astonishing adaptability and functional sophistication.
As we deepen our understanding of their composition and interactions, we move closer to truly engineered biological treatment systems that can handle the ever-changing challenges of wastewater purification—all powered by nature's invisible workforce.