This study explored the implementation of mainstream partial denitrification with anammox (PdNA) in the second anoxic zone of a wastewater treatment process in an integrated fixed film activated sludge (IFAS) configuration. A pilot study was conducted to compare the use of methanol and glycerol as external carbon sources for an IFAS PdNA startup, with a goal to optimize nitrogen removal while minimizing carbon usage. The study also investigated the establishment of anammox bacteria on virgin carriers in IFAS reactors without the use of seeding, and it is the first IFAS PdNA startup to use methanol as an external carbon source. The establishment of anammox bacteria was confirmed in both reactors 102 days after startup. Although the glycerol-fed reactor achieved a higher steady-state maximum ammonia removal rate because of anammox bacteria (1.6 ± 0.3 g/m2/day) in comparison with the methanol-fed reactor (1.2 ± 0.2 g/m2/day), both the glycerol- and methanol-fed reactors achieved similar average in situ ammonia removal rates of 0.39 ± 0.2 g/m2/day and 0.40 ± 0.2 g/m2/day, respectively. Additionally, when the upstream ammonia versus NOx (AvN) control system maintained an ideal ratio of 0.40-0.50 g/g, the methanol-fed reactor attained a lower average effluent TIN concentration (3.50 ± 1.2 mg/L) than the glycerol-fed reactor (4.43 ± 1.6 mg/L), which was prone to elevated nitrite concentrations in the effluent. Overall, this research highlights the potential for PdNA in IFAS configurations as an efficient and cost-saving method for wastewater treatment, with methanol as a viable carbon source for the establishment of anammox bacteria. PRACTITIONER POINTS: Methanol is an effective external carbon source for an anammox startup that avoids the need for costly alternative carbon sources. The methanol-fed reactor demonstrated higher TIN removal compared with the glycerol-fed reactor because of less overproduction of nitrite. Anammox bacteria was established in an IFAS reactor without seeding and used internally stored carbon to reduce external carbon addition. Controlling the influent ammonia versus NOx (AvN) ratio between 0.40 and 0.50 g/g allowed for low and stable TIN effluent conditions.

The underlying adaptative mechanisms of anammox bacteria to salt stress are still unclear. The potential role of the anammoxosome in modulating material and energy metabolism in response to salinity stress was investigated in this study. The results showed that anammox bacteria increased membrane fluidity and decreased mechanical properties by shortening the ladderane fatty acid chain length of anammoxosome in response to salinity shock, which led to the breakdown of the proton motive force driving ATP synthesis and retarded energy metabolism activity. Afterward, the fatty acid chain length and membrane properties were recovered to enhance the energy metabolic activity. The relative transmission electron microscopy (TEM) area proportion of anammoxosome decreased from 55.9 to 38.9% under salinity stress. The 3D imaging of the anammox bacteria based on Synchrotron soft X-ray tomography showed that the reduction in the relative volume proportion of the anammoxosome and the concave surfaces was induced by salinity stress, which led to the lower energy expenditure of the material transportation and provided more binding sites for enzymes. Therefore, anammox bacteria can modulate nitrogen and energy metabolism by changing the membrane properties and morphology of the anammoxosome in response to salinity stress. This study broadens the response mechanism of anammox bacteria to salinity stress.

Low-energy nitrogen removal from ammonium-rich wastewater is crucial in preserving the water environment. A one-stage nitritation/anammox process with two inflows treating ammonium-containing wastewater, supplied from inside and outside the wound filter, is expected to stably remove nitrogen. Laboratory-scale reactors were operated using different start-up strategies; the first involved adding nitritation inoculum after anammox biomass formation in the filter, which presented a relatively low nitrogen removal rate (0.171 kg N/m3 · d), at a nitrogen loading rate of 1.0 kg N/m3 · d. Conversely, the second involved the gradual cultivation of anammox and nitritation microorganisms, which increased the nitrogen removal rate (0.276 kg N/m3 · d). Furthermore, anammox (Candidatus Brocadia) and nitritation bacteria (Nitrosomonadaceae) coexisted in the biofilm formed on the filter surface. The abundance of nitritation bacteria (10.5%) in the reactor biofilm using the second start-up strategy was higher than that using the first (3.7%). Thus, the two-inflow nitritation/anammox process effectively induced habitat segregation using a suitable start-up strategy.

The pilot-scale simultaneous denitrification and methanation (SDM)-partial nitrification (PN)-anaerobic ammonia oxidation (Anammox) system was designed to treat anaerobic digestion effluent of kitchen waste (ADE-KW). The SDM-PN was first started to avoid the inhibition of high-concentration pollutants. Subsequently, Anammox was coupled to realize autotrophic nitrogen removal. Shortcut nitrification-denitrification achieved by the SDM-PN. The NO2--N accumulation (92 %) and NH4+-N conversion (60 %) were achieved by PN, and the removal of TN and COD from the SDM-PN was 70 % and 73 %, respectively. After coupling Anammox, the TN (95 %) was removed with a TN removal rate of 0.51 kg·m-3·d-1. Microbiological analyses showed a shift from dominance by Methanothermobacter to co-dominance by Methanothermobacter, Thermomonas, and Flavobacterium in SDM during the SDM-PN. While after coupling Anammox, Candidatus kuenenia was enriched in the Anammox zone, the SDM zone shifted back to being dominated by Methanothermobacter. Overall, this study provides new ideas for the treatment of ADE-KW.

N2O-reducing bacteria have been examined and harnessed to develop technologies that reduce the emission of N2O, a greenhouse gas produced by biological nitrogen removal. Recent investigations using omics and physiological activity approaches have revealed the ecophysiologies of these bacteria during nitrogen removal. Nevertheless, their involvement in‍ ‍anammox processes remain unclear. Therefore, the present study investigated the identity, genetic potential, and activity‍ ‍of N2O reducers in an anammox reactor. We hypothesized that N2O is limiting for N2O-reducing bacteria‍ ‍and an‍ ‍exogeneous N2O supply enriches as-yet-uncultured N2O-reducing bacteria. We conducted a 1200-day incubation of N2O-reducing bacteria in an anammox consortium using gas-permeable membrane biofilm reactors (MBfRs), which efficiently supply N2O in a bubbleless form directly to a biofilm grown on a gas-permeable membrane. A 15N tracer test indicated that the supply of N2O resulted in an enriched biomass with a higher N2O sink potential. Quantitative PCR and 16S rRNA amplicon sequencing revealed Clade II nosZ type-carrying N2O-reducing bacteria as protagonists of N2O sinks. Shotgun metagenomics showed the genetic potentials of the predominant Clade II nosZ-carrying bacteria, Anaerolineae and Ignavibacteria in MBfRs. Gemmatimonadota and non-anammox Planctomycetota increased their abundance in MBfRs despite their overall lower abundance. The implication of N2O as an inhibitory compound scavenging vitamin B12, which is essential for the synthesis of methionine, suggested its limited suppressive effect on the growth of B12-dependent bacteria, including N2O reducers. We identified Dehalococcoidia and Clostridia as predominant N2O sinks in an anammox consortium fed exogenous N2O because of the higher metabolic potential of vitamin B12-dependent biosynthesis.

Granular activated carbon (GAC), a porous carbon-based material, provides increased attachment space for functional microorganisms and enhances nitrogen removal by facilitating extracellular electron transfer in the anammox process. This study investigates the effects of GAC on the biosynthesis of microbial extracellular secretions (MESs) and explores the roles of these secretions in anammox activities. Four lab-scale reactors were operated: two downstream UASB reactors (D1 and D2) receiving effluents from the upstream UASB reactors (U1: no-GAC, U2: yes-GAC). Our results indicate that MESs were enhanced with the addition of GAC. The effluent from U2 exhibited a 59.62 % higher amino acid content than that from U1. These secretions contributed to an increase in the nitrogen loading rate (NLR) in the downstream reactors. Specifically, NLR in D1 increased from 130.5 to 142.7 g N/m3/day, and in D2, it escalated from 137.5 to 202.8 g N/m3/day, likely through acting as cross-feeding substrates or vital nutrients. D2 also showed increased anammox bacterial activity, enriched Ca. Brocadia population and hao gene abundance. Furthermore, this study revealed that D2 sludge has significantly higher extracellular polymeric substances (EPS) (48.71 mg/g VSS) and a larger average granule size (1.201 ± 0.119 mm) compared to D1 sludge. Overall, GAC-stimulated MESs may have contributed to the enhanced performance of the anammox process.

The enrichment of anammox bacteria is a key issue in the application of anammox processes. A new type of reactor － anaerobic baffle biofilm reactor (ABBR) developed from anaerobic baffle reactor (ABR) was filled with columnar packings and established for effective enrichment of anammox bacteria. The flow field analysis showed that, compared with ABR, ABBR narrowed the dead zone so as to improve the substrate transferring performances. Two ABBRs with different types of columnar packings (Packings 1 and Packings 2) were constructed to culture anammox biofilms. Packings 1 consisted of the single-form honeycomb carriers while Packings 2 was modular composite packings consisting of non-woven fabric and honeycomb carriers. The effects of different types of columnar packings on microbial community and nitrogen removal were studied. The ABBR filled with Packings 2 had a higher retention rate of biomass than the ABBR filled with Packings 1, making the anammox start-up period be shortened by 21.28%. The enrichment of anammox bacteria were achieved and the dominant anammox bacteria were Candidatus Brocadia in both R1 and R2. However, there were four genera of anammox bacteria in R2 and one genus of anammox bacteria in R1, and the cell density of anammox bacteria in R2 was 95% higher than that in R1. R2 has the advantage of maintaining excellent and stable nitrogen removal performance at high nitrogen loading rate. The results revealed that the packings composed of two types of carriers may have a better enrichment effect on anammox bacteria. This study is of great significance for the rapid enrichment of anammox bacteria and the technical promotion of anammox process.

The emerging nitrogen removal process known as CANDAN (Complete Ammonium and Nitrate removal via Denitratation-Anammox over Nitrite) has been developed in Sequencing Batch Reactors (SBRs). Yet, starting up and maintaining stability in continuous-flow reactors remain challenging. This study explores the feasibility of transitioning the CANDAN process from an anammox-dominated process by introducing appropriate external organics to facilitate indigenous nitrite-producing denitrification community in an Upflow Anaerobic Sludge Blanket (UASB) reactor. 150-day operation results indicate that under feeding rates of domestic wastewater at 0.54 L/h and nitrate-containing wastewater at 1.08 L/h, excellent N removal was achieved, with effluent TN below 10.0 mg N/L. Adding external sodium acetate at a COD/NO3--N = 2.0 triggered denitratation, ex-situ denitrification activity tests showed increased nitrite production rates, maintaining the nitrate-to-nitrite transformation ratio (NTR) above 90 %. Consequently, anammox activity was consistently maintained, dominating Total Nitrogen (TN) removal with a contribution as high as 78.3 ± 8.0 %. Anammox functional bacteria, Brocadia and Kuenenia were identified and showed no decrease throughout the operation, indicating the robustness of the anammox process. Notably, the troublesome of sludge flotation, did not occur, also contributing to sustained outstanding performance. In conclusion, this study advances our understanding of the synergistic interplay between anammox and denitrifying bacteria in the Anammox-UASB system, offering technical insights for establishing a stable continuous-flow CANDAN process for simultaneous ammonium and nitrate removal.

The stability of the two-stage partial nitrification-anammox (PN/A) system was compromised by the inappropriate conversion of insoluble organic matter. In response, a sludge redistribution strategy was implemented. Through the redistribution of PN sludge and anammox sludge in the two-stage PN/A system, a transition was made to the Anammox-single stage PN/A (A-PN/A) system. This specific functional reorganization, facilitated by the rapid reorganization of microbial communities, has the potential to significantly decrease the current risk of suppression. The results of the study showed that implementing the sludge redistribution strategy led to a substantial enhancement in the total nitrogen removal rate (TNRR) by 87.51%, accompanied by a significant improvement of 34.78% in the chemical oxygen demand removal rate (CRR). Additionally, this approach resulted in a remarkable two-thirds reduction in the aeration requirements. High-throughput sequencing revealed that the strategy enriched anammox and ammonia-oxidizing bacteria while limiting denitrifying bacteria, as confirmed by quantitative polymerase chain reaction analysis. Furthermore, the principal component analysis revealed that the location and duration of aeration had direct and indirect effects on functional gene expression and the evolution of microbial communities. This study emphasizes the potential benefits of restructuring microbial communities through a sludge redistribution strategy, especially in integrated systems that encounter challenges with suppression.

The nitrate denitrifying anaerobic methane oxidation-anaerobic ammonia oxidation (DAMO-anammox) can accomplish nitrogen removal and methane (CH4) reduction. This process greatly contributes to carbon emission mitigation and carbon neutrality. In this study, we investigated the electron transfer process of functional microorganisms in the iron-mediated DAMO-anammox system. Fe3+ could be bound to several functional groups (-CH3, COO-, -CH) in extracellular polymeric substance (EPS), and the functional groups bound were different at different iron concentration. Fe3+ underwent reduction reactions to produce Fe2+. Most Fe3+ and Fe2+ react with microorganisms and formed chelates with EPS. Three-dimensional fluorescence spectra showed that Fe3+ affected the secretion of tyrosine and tryptophan, which were essential for cytochrome synthesis. The presence of Fe3+ accelerated c-type cytochrome-mediated extracellular electron transfer (EET), and when more Fe3+ existed, the more cytochrome C expressed. DAMO archaea (M. nitroreducens) in the system exhibited a high positive correlation with the functional genes (resa and ccda) for cytochrome c synthesis. Some denitrifying microorganisms showed positive correlations with the abundance of riboflavin. This finding showed that riboflavin secreted by functional microorganisms acted as an electron shuttle. In addition, DAMO archaea were positively correlated with the hair synthesis gene pily1, which indicated that direct interspecies electron transfer (DIET) may exist in the iron-mediated DAMO-anammox system.