Microbial Community Structure of Mesophilic and Low-temperature Partial Nitrification-anammox Reactors: Distribution and Functional Roles of the Core Microbiome

Abstract

Microbial community structures in mesophilic and low-temperature anammox and partial nitrification–anammox reactors were exami­ned by a 16S rRNA–gene amplicon sequencing ana­lysis. The anammox bacterium, Jettenia sp., was dominant, and nitrifying bacteria, including Nitrosomonas sp. (aerobic ammonia–oxidizing bacterium) and Nitrospira sp., (nitrite–oxidizing bacterium) coexisted in the bioreactors. Core coexisting bacteria, such as Sulfurisoma sp. and Zeimonas sp., showed oxygen-scavenging and NO₃^– reduction potentials. Sulfurisoma-related bacteria are distributed across wastewater treatment plants worldwide, particularly in denitrification systems. These results underscore the ecological and functional importance of microbial consortia in enhancing nitrogen removal efficiency.

Anaerobic ammonium oxidation (anammox) is an eco-friendly biological nitrogen removal technology that utilizes nitrite (NO₂^–) supplied by partial nitrification (PN) or partial denitrification as an electron acceptor to oxidize ammonium (NH₄⁺) to dinitrogen (N₂) gas (Strous et al., 1999). The anammox process significantly reduces energy consumption for aeration (by approximately 60%), excess sludge production (by 80–90%), and the need for external organic carbon addition for denitrification, resulting in substantial cost savings and improved energy efficiency in wastewater treatment systems (Ali & Okabe, 2015; Jetten et al., 2001). Although full-scale single– and two–stage PN–anammox processes have been successfully applied to high-strength wastewater, such as livestock wastewater and reject water containing NH₄⁺ at several hundred mg NH₄⁺-N L^–1 (Lackner et al., 2014; Ali and Okabe, 2015), their application to low-strength wastewater, such as municipal sewage (NH₄⁺ available at several tens of mg NH₄⁺-N L^–1), and operation at low temperatures (e.g., 8–20ºC in cold regions and/or during the winter season) remain challenging (Cao et al., 2017; Wang et al., 2022). In low-strength wastewater, difficulties are associated with suppressing the growth of NO₂^–-oxidizing bacteria (NOB) and maintaining a sufficient supply of NO₂^– for the anammox process. Additionally, microbial activity generally decreases under low-temperature conditions, leading to a reduction in nitrogen removal rates (Hendrickx et al., 2012; Hu et al., 2013). To overcome these limitations and optimize the configuration of biological nitrogen removal using the anammox process, a more detailed understanding of the microbial ecology involved in anammox and PN processes is essential. However, the microbial community structure in anammox and PN-anammox reactors has not yet been fully exami­ned, particularly in bioreactors fed with low-strength wastewater and operated under low-temperature conditions. Therefore, the present study investigated the microbial community structures and core bacterial genera of anammox reactors and a PN–anammox reactor operated under different configurations and conditions by a 16S rRNA gene-amplicon sequencing ana­lysis. Furthermore, the distribution and abundance of the identified core bacterial genera coexisting with anammox bacteria were exami­ned in wastewater treatment plants worldwide using the MiDAS4 database (Dueholm et al., 2022).

Sludge biomass was collected from three laboratory-scale anammox reactors and one PN–anammox reactor (Table 1). Two anammox reactors (AN–M1 and AN–M2, respectively) were operated under mesophilic conditions (33 or 37°C), while the remaining anammox reactor and the PN–anammox reactors (AN–L1 and PN–AN–L2, respectively) were operated at 10 or 7°C, respectively. Sludge biomass collected from a lab-scale PN–anammox reactor (Jo et al., 2020) was inoculated into the AN–M1 and AN–L1 reactors, whereas the biomass collected from a pilot scale PN–anammox reactor fed with reject water in Daegu was used as an inoculum for the AN–M2 and PN–AN–L2 reactors. Aeration was performed in the PN–AN–L2 reactor to supply dissolved oxygen (DO) required for PN, and the DO concentration was maintained at <0.5‍ ‍mg L^–1 using a DO controller. Synthetic wastewater containing NH₄⁺ (30–175‍ ‍mg N L^–1) (the detailed composition is available in Table S1) was fed into the bioreactors, and operated for >100 days under stable operational conditions. The anammox and PN–anammox reactors showed stable nitrogen removal performance, with NH₄⁺ removal efficiencies >76% and nitrogen removal efficiencies >60%. In the PN–AN–L2 (PN-anammox) reactor, 18% of influent NH₄⁺ was fully oxidized to NO₃^–, which resulted in lower nitrogen removal efficiency than the other anammox reactors.

Table 1.

Operational conditions and nitrogen removal performance of anammox and partial nitrification (PN)–anammox reactors. The compositions of the inorganic synthetic wastewater supplied to the bioreactors are provided in Table S1.

	AN–M1 (anammox) Sequencing batch reactor	AN–M2 (anammox) Upflow granular reactor	AN–L1 (anammox) Upflow granular reactor	PN–AN–L2 (PN–anammox) Baffled reactor
Temperature	33°C	37°C	10°C	7°C
Volume	10 L	22 L	1.1 L	5 L
Influent pH	7.7	7.4	7.7	7.5
Dissolved oxygen (mg O2 L–1)	n.a.	n.a.	n.a.	<0.5
Hydraulic retention time (h)	24	24	2	4
Nitrogen loading rates (kg N m–3 d–1)	0.23	0.38	0.84	0.26
Nitrogen removal rates (kg N m–3 d–1)	0.20	0.32	0.67	0.16
Nitrogen removal efficiency	87%	84%	79%	60%
Influent (mg N L–1)
NH4+	100	175	30	40
NO2–	130	231	40	—
Effluent (mg N L–1)
NH4+	n.d.	9.3	n.d.	10
NO2–	n.d.	6.6	4.9	n.d.
NO3–	28.9	44.2	7.7	6.99

n.a.; not applicable, n.d.; not detected

Genomic DNA was extracted from sludge samples by the bead beating method, and subjected to the PCR amplification of the 16S rRNA gene using the oligonucleotide primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) (Caporaso et al., 2012). PCR amplicon was subjected to 300-bp paired-end sequencing using Illumina MiSeq. Raw sequence reads‍ ‍were subjected to quality filtering using the fastx_barcode_splitter tool from FASTX-Toolkit (ver. 0.0.14), and 70,679–84,650 paired sequence reads per sample were analyzed using QIIME2 software (ver. 2024.2) (Caporaso et al., 2010). Reads were clustered into amplicon sequence variants (ASVs) using the DADA2 plugin (Callahan et al., 2016), and the phylogeny of ASVs was exami­ned using the blastn (ver. 2.9.0) program against the Greengene (ver. 13_8) and nr (accessed on 16^th December 2024) databases. The metabolic potential of ASVs was predicted using PICRUSt2 software (Douglas et al., 2020) and also by the manual genome annotation of closely-related species using the KAAS (Moriya et al., 2007) and DRAM annotation tools (Shaffer et al., 2020).

A total of 140 ASVs (>0.2% of relative abundance in at least one sludge biomass sample) were found in the bioreactors exami­ned (Fig. 1a). Dominant ASVs were affiliated into the bacterial phyla Planctomycetota (including anammox bacteria), Pseudomonadota, Chloroflexota, Chlorobiota, and Nitrospirota. Detailed phylogenetic affiliations of the dominant ASVs, including functional microbial groups (i.e., anammox, aerobic ammonia–oxidizing bacteria [AOB] and NOB), are shown in Fig. S1. Jettenia sp. ASV326 was the dominant anammox bacterium (26.4–50.9% in total biomass), and other anammox bacteria related to the genera Brocadia and Anammoxoglobus coexisted in the AN–M2 and PN–AN–L2 reactors. The bacterial genus Candidatus Jettenia represents a lineage of freshwater anammox bacteria with a physiological temperature range of 20–42.5°C (Ali et al., 2015). The presence and distribution of Jettenia sp. ASV326 in the low–temperature bioreactors (AN–L1 and PN–AN–L2) suggested that this bacterium is capable of acclimating to low-temperature conditions, as previously reported by a proteomic study (Lin et al., 2018). However, the adaptation mechanisms of Jettenia bacteria to low temperatures remain unclear (Kouba et al., 2022), and warrant further study. Nitrosomonas sp. ASV917 (99.6% sequence identity with the Nitrosomonas europaea ATCC25978 16S rRNA gene) is an AOB that was abundant in the low-temperature bioreactors (0.9 and 2.5% of the total biomass in the AN–L1 and PN–AN–L2 reactors, respectively). The high abundance of Nitrosomonas sp. ASV917 in the PN–AN–L2 reactor indicated that this bacterium was responsible for PN and supplying NO₂^– to Jettenia sp. ASV326. Nitrospira bacteria (ASV496, ASV498, and ASV499) are canonical NOB, whereas the sequence read affiliated into the phylogenetic clade of complete ammonia oxidation (comammox) Nitrospira (Daims et al., 2015; van Kessel et al., 2015) was not detected in the bioreactors exami­ned. Phylogenetically diverse NOB Nitrospira clades have been described, exhibiting a wide range of physiological characteristics, such as affinity for and tolerance to NO₂^– (Fujitani et al., 2013; Ushiki et al., 2013). Nitrospira sp. ASV496 found in the AN–M2 and PN–AN–L2 reactors (1.0 and 1.8%, respectively) related to Nitrospira tepida DNF (100% of sequence identity). N. tepida has been characterized as a moderately thermophilic bacterium with an optimal growth temperature range of 37–45°C (Keuter et al., 2023), and the present study expanded its known temperature range by 7°C, as observed in the PN–AN–L2 reactor. Additionally, Nitrospira bacteria prefer microaerobic conditions over fully aerobic conditions (Lücker et al., 2010), and the DO concentration in the PN–AN–L2 reactor (<0.5‍ ‍mg L^–1) is favorable for their proliferation. The overgrowth of Nitrospira sp. is detrimental to the nitrogen removal efficiency of the PN–anammox process because these bacteria oxidize NO₂^–, a substrate of anammox bacteria, to NO₃^–, thereby reducing the availability of NO₂^–. Therefore, the growth of the detected Nitrospira sp. ASVs needs to be suppressed in order to improve the nitrogen removal performance of the PN–AN–L2 reactor.

Fig. 1.

Microbial community structure and core microbiome in partial nitrification (PN)-anammox bioreactors. AN–M1 and AN–M2: mesophilic anammox bioreactors; AN–L1: anammox bioreactor operated at 10°C; PN–AN–L2: PN-anammox bioreactor operated at 7°C (Table 1). (a) Relative abundance (%) of 16S rRNA gene reads at the phylum level in each bioreactor. (b) Similarity of microbial community structures based on a principal component ana­lysis (PCA) performed using R software (version 4.2.0). The cumulative contributions of the PC1 and PC2 axes were 92.7 and 96.9%, respectively. (c) The core microbiome identified based on the mean relative abundance and coefficient of variation (CV) values. Phylogenetic affiliations of ASVs are shown in Fig. S1.

A principal component ana­lysis (PCA) was performed to examine similarities in microbial community structures among the bioreactors (Fig. 1b). The PC1 axis accounted for a cumulative contribution of 92.7%, and the microbial community structures in the low-temperature bioreactors (AN–L1 and PN–AN–L2) were distinct from those in the mesophilic bioreactors (AN–M1 and AN–M2) along the PC1 axis. This differentiation showed the impact of temperature in shaping anammox bacterial community structures (Sonthiphand et al., 2014; Oshiki et al., 2016). To identify a core microbiome shared across all temperature and operational conditions, the mean relative abundance and coefficient of variation (CV) values were calculated in the sludge samples exami­ned (Fig. 1c). The following 5 ASVs showed high abundance and wide distributions: Jettenia sp. ASV326, Sulfurisoma sp. ASV867 (classified as the genus Dok59 in the Greengene database ver. 13_8), Zeimonas sp. ASV884, Phycisphaerales sp. ASV505, and Anaerolineae sp. ASV027. Phycisphaerales and Anaerolineae bacteria have often been detected as coexisting bacteria in anammox bacterial cultures and their potential function (e.g., the degradation of extracellular polymeric substances [EPS]) has been exami­ned using metagenomic ana­lyses (Speth et al., 2016; Lawson et al., 2017; Ali et al., 2020; Oshiki et al., 2022), whereas limited information is available for Sulfurisoma sp. ASV867 (97.6% identity to Sulfurisoma sediminicola BSN1) and Zeimonas sp. ASV884 (98.4% identity to Zeimonas arvi CC–CFT501). S. sediminicola and Z. arvi were isolated from freshwater lake sediment and a maize field as a sulfur-oxidizing bacterium and a bacterium harboring biphenyl- and phenolic acid-metabolizing genes, respectively (Kojima and Fukui, 2014; Lin et al., 2021). As shown in Fig. S2, the S. sediminicola BSN1 and Z. arvi CC–CFT501 genomes (accession numbers: GCF_003865015.1 and GCF_008039575.1, respectively) contain the genes involved in aerobic respiration (including terminal cytochrome c oxidases, such as high-affinity cbb₃-type terminal oxidase) and NO₃^– respiration. These bacteria are capable of aerobic growth (Kojima and Fukui, 2014; Lin et al., 2021). Their genomic and physiological traits suggest that these bacteria play a role in anammox and PN–anammox reactors as O₂ scavengers. Notably, these bacteria are able to grow by NO₃^– reduction, and S. sediminicola and Z. arvi cells reduced NO₃^– to dinitrogen (N₂) gas (Kojima and Fukui, 2014) and NO₂^– (Lin et al., 2021), respectively, with the potential to produce nitric oxide (NO), as suggested from their metabolic potential (Fig. S2). Therefore, Zeimonas sp. ASV884 provides additional NO₂^–/NO to anammox bacteria through partial denitrification (Sumino et al., 2006; Waki et al., 2013; Du et al., 2015). S. sediminicola and Z. arvi utilized a number of carbon sources (e.g., acetate, lactate, propionate, and pyruvate for S. sediminicola and L-arabinose, citric acid, L-malic acid, and sodium butyrate for Z. arvi) and also H₂ for NO₃^– reduction (Kojima and Fukui, 2014; Lin et al., 2021). Although the anammox and PN–anammox reactors were operated with inorganic media, organic matter and H₂ may have been available in the biomass, generated through the degradation of EPS and/or cell debris and fermentative reactions mediated by coexisting Chloroflexota bacteria (Kindaichi et al., 2012; Bovio-Winkler et al., 2023). However, the metabolic potentials and interactions in the anammox biomass need to be further exami­ned using cultivation-based ana­lyses in future studies due to the current limitations of metabolic profile predictions using 16S rRNA gene amplicon data (Sun et al., 2020; Toole et al., 2021).

The above core genera coexisting with Jettenia sp. ASV326 have been detected in other lab-scale anammox bioreactors (e.g., Zeimonas bacteria from a semi–continuous stirred tank reactor) (Ude et al., 2023), while the distribution of these bacteria in wastewater treatment plants (WWTPs) has not yet been investigated. In the present study, the distribution of core ASVs was assessed using the MiDAS4 database, which contains full-length 16S rRNA gene sequences obtained from >740 WWTPs worldwide (Dueholm et al., 2022). The distribution and abundance of Sulfurisoma-, Zeimonas-, Phycisphaerales-, and Anaerolineae-related ASVs, which exhibit more than 97% sequence similarity with MiDAS V4 ASV (Table S2), were exami­ned from the MiDAS4 database. The Sulfurisoma-related ASV was abundant (mean; 0.52%, n=651) and widespread in WWTPs (Fig. 2). The relative abundance of Sulfurisoma-related ASV was significantly higher in WWTPs operated for denitrification (the C, N, and DN types in Fig. 2). The higher abundance in WWTPs operated for denitrification suggests the involvement of Sulfurisoma-related bacteria in nitrogen removal (i.e., denitrification) in WWTPs, which is consistent with the findings of a previous MiDAS4 survey showing that Sulfuritales bacteria related to the Sulfurisoma-related ASV were identified as a common denitrifier (Dueholm et al., 2022). Additionally, the physiological traits of S. sediminicola (i.e., NO₃^– reduction to N₂) support the role of Sulfurisoma-related bacteria in denitrification (Kojima and Fukui, 2014). On the other hand, no distinct correlation was found between the abundance of Sulfurisoma-related ASV and temperature (Fig. 2), suggesting that Sulfurisoma-related bacteria adapt to a broad range of temperatures.

Fig. 2.

Global distribution of core bacteria associated with anammox bacteria in wastewater treatment plants (WWTPs). The MiDAS4 database, based on a worldwide survey of more than 740 WWTPs using full-length 16S rRNA gene sequences (Dueholm et al., 2022), was analyzed to examine the distribution of core bacteria. The distribution of Sulfurisoma-, Zeimonas-, Phycisphaerales-, and Anaerolineae-related ASVs (refer to Table S2 for a list of ASVs) grouped by process types (upper panel) and as a function of temperature (bottom panel). Process types in the MiDAS4 database are classified as C (WWTPs for carbon removal, e.g., biological oxygen demand, BOD), N (WWTPs for nitrification), DN (WWTPs for denitrification), and P (WWTPs for phosphorus removal). Black plots represent the relative abundance of ASVs in the WWTP samples analyzed from the MiDAS4 database, while red plots denote outliers. Mean values were compared using Welch’s t-test corrected with the Bonferroni-Holm method. Asterisks indicate significant differences in the means (**: P<0.01; *****: P<0.00001).

In summary, the microbial community structures of mesophilic and low-temperature anammox and PN–anammox reactors were exami­ned to elucidate their ecological and functional roles in nitrogen removal. Jettenia sp. ASV326 was identified as the dominant anammox bacterium, and other core coexisting bacteria, such as Sulfurisoma sp. ASV867 and Zeimonas sp. ASV884, were suggested to contribute to oxygen scavenging and NO₃^– reduction. Notably, Sulfurisoma-related bacteria are widely distributed in full-scale WWTPs globally, particularly in those operated for denitrification, where they may contribute to nitrogen removal. These results provide insights into microbial consortia that contribute to biological nitrogen removal in PN–anammox processes.

Data availability

The raw sequence reads of 16S rRNA gene amplicons are available in the DDBJ nucleotide sequence database under the accession number PRJDB18291.

Citation

Oshiki, M., Takahashi, K., Kawasaki, S., Choi, H., Park, J., Kim, K., et al. (2025) Microbial Community Structure of Mesophilic and Low-temperature Partial Nitrification-anammox Reactors: Distribution and Functional Roles of the Core Microbiome. Microbes Environ 40: ME25001.

https://doi.org/10.1264/jsme2.ME25001

Acknowledgements

This work was supported by JSPS KAKENHI (grant numbers: 23H02114 and 24KK0196 for M.O., 24KJ0001 for K.T., and 23H00192 for S.O.), and the JST FOREST Program (JPMJFR216Z for M.O.). Computations were partially performed on the NIG supercomputer at the ROIS National Institute of Genetics.

References

•  Ali,  M., and  Okabe,  S. (2015) Anammox-based technologies for nitrogen removal: Advances in process start-up and remaining issues. Chemosphere 141: 144–153.
•  Ali,  M.,  Oshiki,  M.,  Awata,  T.,  Isobe,  K.,  Kimura,  Z.,  Yoshikawa,  H., et al. (2015) Physiological characterization of anaerobic ammonium oxidizing bacterium “Candidatus Jettenia caeni.” Environ Microbiol 17: 2172–2189.
•  Ali,  M.,  Shaw,  D.R.,  Albertsen,  M., and  Saikaly,  P.E. (2020) Comparative genome-centric ana­lysis of freshwater and marine ANAMMOX cultures suggests functional redundancy in nitrogen removal processes. Front Microbiol 11: 1637.
•  Bovio-Winkler,  P.,  Guerrero,  L.D.,  Erijman,  L.,  Oyarzúa,  P.,  Suárez-Ojeda,  M.E.,  Cabezas,  A., and  Etchebehere,  C. (2023) Genome-centric metagenomic insights into the role of Chloroflexi in anammox, activated sludge and methanogenic reactors. BMC Microbiol 23: 45.
•  Callahan,  B.J.,  McMurdie,  P.J.,  Rosen,  M.J.,  Han,  A.W.,  Johnson,  A.J.A., and  Holmes,  S.P. (2016) DADA2: High resolution sample inference from Illumina amplicon data. Nat Methods 13: 581–583.
•  Cao,  Y.,  van Loosdrecht,  M.C.M., and  Daigger,  G.T. (2017) Mainstream partial nitritation–anammox in municipal wastewater treatment: Status, bottlenecks, and further studies. Appl Microbiol Biotechnol 101: 1365–1383.
•  Caporaso,  J.G.,  Kuczynski,  J.,  Stombaugh,  J.,  Bittinger,  K.,  Bushman,  F.D.,  Costello,  E.K., et al. (2010) QIIME allows ana­lysis of high-throughput community sequencing data. Nat Methods 7: 335–336.
•  Caporaso,  J.G.,  Lauber,  C.L.,  Walters,  W.A.,  Berg-Lyons,  D.,  Huntley,  J.,  Fierer,  N., et al. (2012) Ultra-high-throughput microbial community ana­lysis on the Illumina HiSeq and MiSeq platforms. ISME J 6: 1621–1624.
•  Daims,  H.,  Lebedeva,  E.V.,  Pjevac,  P.,  Han,  P.,  Herbold,  C.,  Albertsen,  M., et al. (2015) Complete nitrification by Nitrospira bacteria. Nature 528: 504–509.
•  Douglas,  G.M.,  Maffei,  V.J.,  Zaneveld,  J.R.,  Yurgel,  S.N.,  Brown,  J.R.,  Taylor,  C.M., et al. (2020) PICRUSt2 for prediction of metagenome functions. Nat Biotechnol 38: 685–688.
•  Du,  R.,  Peng,  Y.,  Cao,  S.,  Wang,  S., and  Wu,  C. (2015) Advanced nitrogen removal from wastewater by combining anammox with partial denitrification. Bioresour Technol 179: 497–504.
•  Dueholm,  M.K.D.,  Nierychlo,  M.,  Andersen,  K.S.,  Rudkjøbing,  V.,  Knutsson,  S., MiDAS Global Consortium, et al. (2022) MiDAS 4: A global catalogue of full-length 16S rRNA gene sequences and taxonomy for studies of bacterial communities in wastewater treatment plants. Nat Commun 13: 1908.
•  Fujitani,  H.,  Aoi,  Y., and  Tsuneda,  S. (2013) Selective enrichment of two different types of Nitrospira-like nitrite-oxidizing bacteria from a wastewater treatment plant. Microbes Environ 28: 236–243.
•  Hendrickx,  T.,  Wang,  Y.,  Kampman,  C.,  Zeeman,  G.,  Temmink,  H., and  Buisman,  C. (2012) Autotrophic nitrogen removal from low strength waste water at low temperature. Water Res 46: 2187–2193.
•  Hu,  Z.,  Lotti,  T., Kreuk, M. de,  Kleerebezem,  R., Loosdrecht, M. van,  Kruit,  J., et al. (2013) Nitrogen removal by a nitritation-anammox bioreactor at low temperature. Appl Environ Microbiol 79: 2807–2812.
•  Jetten,  M.,  Wagner,  M.,  Fuerst,  J.,  van Loosdrecht,  M.,  Kuenen,  G., and  Strous,  M. (2001) Microbiology and application of the anaerobic ammonium oxidation (‘anammox’) process. Curr Opin Biotechnol 12: 283–288.
•  Jo,  Y.,  Cho,  K.,  Choi,  H., and  Lee,  C. (2020) Treatment of low-strength ammonia wastewater by single-stage partial nitritation and anammox using upflow dual-bed gel-carrier reactor (UDGR). Bioresour Technol 304: 123023.
•  Keuter,  S.,  Koch,  H.,  Nowka,  B.,  Lipski,  A.,  Kruse,  M.,  Lücker,  S., and  Spieck,  E. (2023) A novel Nitrospira lineage isolated from activated sludge using elevated temperatures. FEMS Microbiol Lett 370: fnad035.
•  Kindaichi,  T.,  Yuri,  S.,  Ozaki,  N., and  Ohashi,  A. (2012) Ecophysiological role and function of uncultured Chloroflexi in an anammox reactor. Water Sci Technol 66: 2556–2561.
•  Kojima,  H., and  Fukui,  M. (2014) Sulfurisoma sediminicola gen. Nov., sp. Nov., a facultative autotroph isolated from a freshwater lake. Int J Syst Evol Microbiol 64: 1587–1592.
•  Kouba,  V.,  Bachmannová,  Ch.,  Podzimek,  T.,  Lipovová,  P., and  van Loosdrecht,  M.C.M. (2022) Physiology of anammox adaptation to low temperatures and promising biomarkers: A review. Bioresour Technol 349: 126847.
•  Lackner,  S.,  Gilbert,  E.M.,  Vlaeminck,  S.E.,  Joss,  A.,  Horn,  H., and  Loosdrecht,  M.C.M. van. (2014) Full-scale partial nitritation/anammox experiences—An application survey. Water Res 55: 292–303.
•  Lawson,  C.E.,  Wu,  S.,  Bhattacharjee,  A.S.,  Hamilton,  J.J.,  McMahon,  K.D.,  Goel,  R., and  Noguera,  D.R. (2017) Metabolic network ana­lysis reveals microbial community interactions in anammox granules. Nat Commun 8: 15416.
•  Lin,  S.-Y.,  Hameed,  A.,  Tsai,  C.-F., and  Young,  C.-C. (2021) Zeimonas arvi gen. Nov., sp. Nov., of the family Burkholderiaceae, harboring biphenyl- and phenolic acid-metabolizing genes, isolated from a long-term ecological research field. Antonie van Leeuwenhoek 114: 2101–2111.
•  Lin,  X.,  Wang,  Y.,  Ma,  X.,  Yan,  Y.,  Wu,  M.,  Bond,  P.L., and  Guo,  J. (2018) Evidence of differential adaptation to decreased temperature by anammox bacteria: The proteome response of anammox bacteria to low temperature. Environ Microbiol 20: 3514–3528.
•  Lücker,  S.,  Wagner,  M.,  Maixner,  F.,  Pelletier,  E.,  Koch,  H.,  Vacherie,  B., et al. (2010) A Nitrospira metagenome illuminates the physiology and evolution of globally important nitrite-oxidizing bacteria. Proc Natl Acad Sci U S A 107: 13479–13484.
•  Moriya,  Y.,  Itoh,  M.,  Okuda,  S.,  Yoshizawa,  A., and  Kanehisa,  M. (2007) KAAS: an automatic genome annotation and pathway reconstruction server. Nucleic Acids Res 35: W182–W185.
•  Oshiki,  M.,  Satoh,  H., and  Okabe,  S. (2016) Ecology and physiology of anaerobic ammonium oxidizing (anammox) bacteria. Environ Microbiol 18: 2784–2796.
•  Oshiki,  M.,  Takaki,  Y.,  Hirai,  M.,  Nunoura,  T.,  Kamigaito,  A., and  Okabe,  S. (2022) Metagenomic ana­lysis of five phylogenetically distant anammox bacterial enrichment cultures. Microbes Environ 37: ME22017.
•  Shaffer,  M.,  Borton,  M.A.,  McGivern,  B.B.,  Zayed,  A.A.,  La Rosa,  S.L.,  Solden,  L.M., et al. (2020) DRAM for distilling microbial metabolism to automate the curation of microbiome function. Nucleic Acids Res 48: 8883–8900.
•  Sonthiphand,  P.,  Hall,  M.W., and  Neufeld,  J.D. (2014) Biogeography of anaerobic ammonia-oxidizing (anammox) bacteria. Front Microbiol 5: 399.
•  Speth,  D.R.,  Zandt,  M.H. in ’t,  Guerrero-Cruz,  S.,  Dutilh,  B.E., and  Jetten,  M.S.M. (2016) Genome-based microbial ecology of anammox granules in a full-scale wastewater treatment system. Nat Commun 7: 11172.
•  Strous,  M.,  Fuerst,  J.,  Kramer,  E.,  Logemann,  S.,  Muyzer,  G.,  van de Pas-Schoonen,  K., et al. (1999) Missing lithotroph identified as new planctomycete. Nature 400: 446–449.
•  Sumino,  T.,  Isaka,  K.,  Ikuta,  H.,  Saiki,  Y., and  Yokota,  T. (2006) Nitrogen removal from wastewater using simultaneous nitrate reduction and anaerobic ammonium oxidation in single reactor. J Biosci Bioeng 102: 346–351.
•  Sun,  S.,  Jones,  R.B., and  Fodor,  A.A. (2020) Inference-based accuracy of metagenome prediction tools varies across sample types and functional categories. Microbiome 8: 46.
•  Toole,  D.R.,  Zhao,  J.,  Martens-Habbena,  W., and  Strauss,  S.L. (2021) Bacterial functional prediction tools detect but underestimate metabolic diversity compared to shotgun metagenomics in southwest Florida soils. Appl Soil Ecol 168: 104129.
•  Ude,  E.O.,  Haas,  J.,  Kaiyoum,  M.K.,  Ding,  C., and  Adrian,  L. (2023) Effects of reducing, stabilizing, and antibiotic agents on “Candidatus Kuenenia stuttgartiensis.” Appl Microbiol Biotechnol 107: 1829–1843.
•  Ushiki,  N.,  Fujitani,  H.,  Aoi,  Y., and  Tsuneda,  S. (2013) Isolation of Nitrospira belonging to sublineage II from a wastewater treatment plant. Microbes Environ 28: 346–353.
•  van Kessel,  M.A.H.J.,  Speth,  D.R.,  Albertsen,  M.,  Nielsen,  P.H.,  Op den Camp,  H.J.M.,  Kartal,  B., et al. (2015) Complete nitrification by a single microorganism. Nature 528: 555–559.
•  Waki,  M.,  Yasuda,  T.,  Fukumoto,  Y.,  Kuroda,  K., and  Suzuki,  K. (2013) Effect of electron donors on anammox coupling with nitrate reduction for removing nitrogen from nitrate and ammonium. Bioresour Technol 130: 592–598.
•  Wang,  Z.,  Zheng,  M.,  Duan,  H.,  Yuan,  Z., and  Hu,  S. (2022) A 20-year journey of partial nitritation and anammox (PN/A): From sidestream toward mainstream. Environ Sci Technol 56: 7522–7531.