Metagenomic Analysis of Five Phylogenetically Distant Anammox Bacterial Enrichment Cultures

Abstract

Anaerobic ammonium-oxidizing (anammox) bacteria are slow-growing and fastidious bacteria, and limited numbers of enrichment cultures have been established. A metagenomic ana­lysis of our 5 established anammox bacterial enrichment cultures was performed in the present study. Fourteen high-quality metagenome-assembled genomes (MAGs) were obtained, including those of 5 anammox Planctomycetota (Candidatus Brocadia, Ca. Kuenenia, Ca. Jettenia, and Ca. Scalindua), 4 Bacteroidota, and 3 Chloroflexota. Based on the gene sets of metabolic pathways involved in the degradation of polymeric substances found in Chloroflexota and Bacteroidota MAGs, they are expected to be scavengers of extracellular polymeric substances and cell debris.

The anaerobic ammonium oxidation (anammox) process in which NH₄⁺ is oxidized to N₂ gas with NO₂^– markedly contributes to the global nitrogen cycle (Kuypers et al., 2005), and has been installed in full-scale wastewater treatment plants as a cost-efficient and environmentally-friendly nitrogen removal process (van der Star et al., 2007; Ali and Okabe, 2015). Anammox bacteria were discovered in the mid-1990s, and belong to a deep-branching monophyletic group tentatively proposed in the order Brocadiales of the bacterial phylum Planctomycetota (Strous et al., 1999). The following five candidate genera have been identified in the order: Ca. Brocadia, Ca. Kuenenia, Ca. Jettenia, Ca. Anammoxoglobus, and Ca. Scalindua. Although many researchers have attempted to enrich and isolate anammox bacteria, a pure culture has not yet been obtained. This is somewhat surprising because anammox bacteria may be highly enriched in membrane bioreactors (<98% in total biomass) (Lotti et al., 2014), and a subsequent buoyant density separation technique enables the further enrichment of anammox bacteria (>99.9%) (Strous et al., 1999; Kartal et al., 2011). Therefore, several reasons for unsuccessful isolation attempts have been proposed, such as the occurrence of cell density-dependent anammox activity (Strous et al., 1999; Oshiki et al., 2020; Zhang and Okabe, 2020) and microbial interactions between anammox bacteria and coexisting microorganisms; e.g., symbiotic/cooccurring bacteria supply the micronutrients required for the growth of anammox bacteria (Kindaichi et al., 2004; Mee et al., 2014; Kim et al., 2021). Although the micronutrients required for the growth of anammox bacteria have not yet been identified, the supply of soluble organic matter from anammox bacteria was shown to support the growth of heterotrophs in an anammox bacterial enrichment culture (Ni et al., 2012). A previous metagenomic ana­lysis revealed microbial interactions in anammox bacterial enrichment cultures: 1) NO₂^– and/or NO₃^– reduction by heterotrophs to supply NH₄⁺ and/or NO₂^– to anammox bacteria, and 2) the vitamin and amino acid auxotrophy of coexisting heterotrophs (Lawson et al., 2017). However, (meta)genomic information on the microbes cooccurring with anammox bacteria remains limited, and metagenomic ana­lyses have investigated potential interactions between anammox bacteria and cooccurring bacteria (Speth et al., 2016; Lawson et al., 2017). The authors dedicated >10 years to the cultivation of phylogenetically different anammox bacteria, and obtained enrichment cultures of B. sinica (Oshiki et al., 2011), B. sapporoensis (Narita et al., 2017), J. caeni (Hira et al., 2012; Ali et al., 2015), K. stuttgartiensis (Oshiki et al., 2018), and Scalindua sp. husus a7 (Kindaichi et al., 2011). To the best of our knowledge, no other laboratory has maintained these phylogenetically diverse anammox bacterial enrichment cultures in parallel, and these cultures provide an excellent opportunity to examine the metabolic potential of anammox bacteria and cooccurring bacteria in a metagenomic ana­lysis.

Therefore, a metagenomic ana­lysis of these 5 anammox bacterial enrichment cultures was performed in the present study to retrieve the whole genome sequences of anammox bacteria and cooccurring bacteria and examine potential microbial interactions occurring in the enrichment cultures. Anammox bacterial cultures were maintained in membrane bioreactors (MBRs) as previously described (Oshiki et al., 2013; Zhang and Okabe, 2017). Inorganic basal media fed into the MBRs contained KH₂PO₄ (24.4‍ ‍mg L^–1), MgSO₄·7H₂O (60‍ ‍mg L^–1), CaCl₂ (51‍ ‍mg L^–1), and 0.5‍ ‍mL of trace element solutions I and II (van de Graaf et al., 1996). Equimolar amounts of NH₄(SO₄)₂ and NaNO₂ were supplemented into inorganic basal media at 5 to 10‍ ‍mM, and nitrogen loading rates were in the range of 0.1 to 0.6‍ ‍kg‍ ‍N‍ ‍m^–3‍ ‍d^–1. In cultivations of K. stuttgartiensis and Scalindua sp. husus a7, the artificial sea salt SEALIFE (Marine Tech) (Kindaichi et al., 2011) was supplemented into inorganic basal media at final concentrations of 10 and 28‍ ‍g L^–1, respectively. Anammox bacteria proliferated in the form of planktonic cells, which were harvested by centrifugation at 13,420×g for 10‍ ‍min for genomic DNA extraction. Total genomic DNA was extracted using the DNeasy Blood and Tissue kit (Qiagen) and then subjected to shotgun sequence library construction using the KAPA Hyper Prep Kit (for Illumina) (KAPA Biosystems) as described in Hirai et al. (2017). Between 3.4 and 5.7 M of 300-‍bp paired-end reads (corresponding to 1.0–1.7 Gb) were obtained for samples on the Illumina MiSeq sequencer. Reads were then subjected to adapter trimming using Trimmomatic version 0.36 (Bolger et al., 2014), de novo assembly by the CLC Genomics workbench (word size 64, bubble size 500), and binning of the assembled contigs using MyCC (Lin and Liao, 2016). Gene annotation and completeness checks of the metagenome-assembled genomes (MAGs) obtained were performed using the DDBJ Fast Annotation and Submission tool (DFAST) (Tanizawa et al., 2018). Average nucleotide identity (ANI) values were calculated in the DFAST pipeline and also using the OrthoANI tool (Lee et al., 2016). Fourteen high-quality MAGs with >83% and <15.7% of completeness and contamination, respectively, were obtained (Table 1). The taxonomy of these MAGs was examined using GTDB-Tk v1.7.0 with release 202 data (Chaumeil et al., 2019) and AnnoTree (Mendler et al., 2019). Five MAGs were affiliated to 4 anammox bacterial genera, Brocadia, Jettenia, Kuenenia, and Scalindua (Fig. S1a), while other non-anammox bacterial MAGs were affiliated to the phyla Planctomycetota (Fig. S1a), Bacteroidota (Fig. S1b), and Chloroflexota (Fig. S1c). Anammox bacterial MAGs were identified as genomes of B. sinica (the HBSIN01 MAG), B. sapporoensis (the HBSAPP01 MAG), J. caeni (the JETCAE04 MAG), K. stuttgartiensis (the HKUEN01 MAG), and Scalindua sp. (the SCALA701 MAG) because they had >99.4% ANI to reference anammox bacterial genomes (Table S1). The relative abundance of anammox bacterial MAGs in metagenomic sequencing data was calculated by dividing the read numbers assigned to anammox bacterial MAG by total read numbers, which were generally high (37–64%), except for the J. caeni and K. stuttgartiensis biomasses (17 and 9%, respectively) (Table 1). Apart from anammox bacterial MAGs, Chloroflexota (JETCAE01, JETCAE02, and HKUEN02) and Bacteroidota (HBSIN02, HBSAPP04, JETCAE03, and SCALA702) MAGs were obtained from anammox bacterial enrichment cultures. A previous metagenomic sequencing ana­lysis of anammox bacterial enrichment cultures also retrieved Chloroflexota (Table S2) and Bacteroidota (Table S3) MAGs (Speth et al., 2016; Bhattacharjee et al., 2017; Lawson et al., 2017; Mardanov et al., 2019; Okubo et al., 2021): e.g., Chloroflexota MAGs (JETCAE01 MAG) and Bacteroidota MAGs (JETCAE03 MAG and HBSAPP04 MAGs) obtained in the present study showed high ANI values with Anaerolineae and Ignavibacteria MAGs obtained from anammox bioreactors operated by other research groups (Zhao et al., 2019; Ali et al., 2020) (Table S1). This result implies that anammox bioreactors fed with inorganic media containing NH₄⁺ and NO₂^– enrich phylogenetically-defined bacterial members as a core microbiome, as previously suggested by Lawson et al. (2017).

Table 1. Metagenome-assembled genomes (MAGs) obtained from 5 anammox bacterial cultures. Taxonomic assignments were examined using GTDB-Tk and shown in Fig. S1.

Biomass1)	MAGs	Taxonomy2)	Total length	Contigs	GC	CDS	rRNA	tRNA	Completeness	Contamination	Abundance3)
BS	HBSIN01	Planctomycetota	3,980,744	87	42.4%	3,604	16S-1, 23S-1, 5S-1	48	96%	0.0%	64%
	HBSIN02	Bacteroidota	3,004,117	111	55.4%	2,565	16S-1, 23S-1, 5S-1	46	100%	0.0%	8%
BA	HBSAPP01	Planctomycetota	3,345,265	139	42.4%	2,758	16S-1, 23S-1, 5S-1	47	96%	0.0%	37%
	HBSAPP02	Planctomycetota	3,888,461	24	63.4%	3,148	16S-1, 23S-1, 5S-1	49	89%	0.8%	33%
	HBSAPP03	Planctomycetota	3,538,919	30	67.9%	3,045	16S-1, 23S-1, 5S-1	55	83%	1.2%	10%
	HBSAPP04	Bacteroidota	4,378,747	1118	47.1%	2,829	5S-1	44	100%	7.5%	4%
JC	JETCAE01	Chloroflexota	4,077,411	398	53.2%	3,598	23S-1, 5S-1	42	90%	8.3%	6%
	JETCAE02	Chloroflexota	3,195,621	90	60.9%	2,914	16S-1, 23S-1, 5S-1	45	91%	2.4%	9%
	JETCAE03	Bacteroidota	4,208,711	87	34.5%	3,744	16S-1, 23S-1, 5S-1	81	83%	15.7%	9%
	JETCAE04	Planctomycetota	3,935,265	95	40.0%	3,368	16S-1, 23S-1, 5S-1	46	96%	0.0%	17%
KS	HKUEN01	Planctomycetota	4,181,252	391	40.8%	3,539	16S-1, 23S-1, 5S-1	51	93%	12.5%	9%
	HKUEN02	Chloroflexota	2,777,596	539	52.8%	2,278	16S-1, 5S-1	43	100%	3.5%	3%
SC	SCALA701	Planctomycetota	4,498,465	120	41.1%	3,748	16S-1, 23S-1, 5S-1	43	96%	8.3%	52%
	SCALA702	Bacteroidota	4,901,315	164	38.8%	3,866	16S-1, 23S-1, 5S-1	42	100%	0.0%	12%

1) BS, BA, JC, KS, and SC correspond to cultures of Brocadia sinica, Brocadia sapporoensis, Jettenia caeni, Kuenenia stuttgartiensis, and Scalindua sp. husus a7, respectively.

2) Phylogenetic trees are available in Fig. S1. The closest reference genome and ANI scores are available in Table S1.

3) Relative abundance of the number of sequence reads assigned to each MAG to the total number of sequence reads.

The metabolic capabilities of MAGs for central nitrogen and carbon metabolism were examined by performing a blastKOALA search using the KEGG database (Kanehisa et al., 2016), and search hits were visualized using the KEGG Decoder (Graham et al., 2018). Known anammox bacterial genomes commonly harbor the gene sets required for the anammox process (nitrite reduction, hydrazine synthesis, and hydrazine oxidation) and CO₂ fixation via the Wood-Ljungdahl pathway (Strous et al., 2006; Oshiki et al., 2015, 2017) (Table S4). These gene sets are generally conserved on the anammox bacterial MAGs obtained. The nitrite reductase (Nir) of anammox bacteria is still controversial because the gene encoding a canonical Nir (cytochrome cd₁-containing NirS and copper-containing NirK) is often missing in Brocadia genomes (Oshiki et al., 2016; Okubo et al., 2021), and neither nirS nor nirK was found in HBSIN01 and HBSAPP01 MAGs (Table S4). The involvement of atypical hydroxylamine dehydrogenase (rHao) in anammox bacterial nitrite reduction has been proposed (Kartal et al., 2013; Oshiki et al., 2016), and rHao was recently purified and characterized from a K. stuttgartiensis culture (Ferousi et al., 2021). rHao lacks the tyrosine residue required for the crosslinking of catalytic haem 4 in Hao, and the gene encoding putative rHao was conserved among the anammox bacterial MAGs obtained in this study (Table S4). In addition, the SCALA701 MAG differed from the other known Scalindua genomes as follows: 1) SCALA701 MAG has nirK instead of Scalindua nirS (van de Vossenberg et al., 2013; Oshiki et al., 2017), and 2) SCALA701 HzsB and HzsG are encoded in each CDS as well as the known Brocadiaceae genomes, whereas the fusion protein of HzsBG is encoded in the genome of Scalindua profunda (van de Vossenberg et al., 2013). Functional difference(s) resulting from the presence of nirK and separated hzsBG remain unclear and, thus, warrant further study.

Non-anammox bacterial Planctomycetota, Chloroflexota, and Bacteroidota MAGs have the gene sets required for fermentation (substrate-level phosphorylation; such as glycolysis) (Fig. 1) and respiration (cytochrome c oxidase and dissimilatory NO₃^– reduction), whereas the MAGS of known inorganic carbon fixation pathways are absent. JETCAE02 (Chloroflexota) MAG harbors some of the genes involved in the Wood-Ljungdahl pathway, whereas the genes encoding key enzymes, namely, formate dehydrogenase and formate-tetrahydrofolate ligase, are missing. These features suggest that non-anammox bacterial Planctomycetota, Chloroflexota, and Bacteroidota are heterotrophic bacteria, whereas inorganic basal media fed into the operated MBRs and the nutrients required for heterotrophic growth were not available in influents. Extracellular polymeric substances (EPS) (Ali et al., 2018), soluble microbial products (SMP) (Tsushima et al., 2007; Oshiki et al., 2011), and/or cell debris derived from anammox bacteria may be nutrient sources for heterotrophs. Anammox bacteria produce large amounts of EPS mainly composed of proteins and polysaccharides (Hou et al., 2015; Jia et al., 2017; Ali et al., 2018), and the anammox bacterial MAGs obtained have the genes encoding the bacterial type II secretion system involved in protein secretion (GspDEFGIK) and the ABC transporters of lipopolysaccharide (LptBFG) and lipoprotein (LolCDE) involved in the formation of the lipopolysaccharide layer. Chloroflexota and Bacteroidota MAGs have the genes required for the degradation of polysaccharide chains (alpha-amylase and beta-glucosidase) (Fig. 1), indicating their metabolic potential for the degradation of EPS. Chloroflexota bacteria belonging to the class Anaerolineae are obligately anaerobic bacteria (Yamada and Sekiguchi, 2009; Nunoura et al., 2013), and utilize a number of organic compounds, including sugars, with the production of short fatty acids and hydrogen gas (Sun et al., 2016). Chloroflexota bacteria in an anammox bioreactor assimilate sucrose, glucose, and N-acetyl-glucosamine, as confirmed by microautoradiography and fluorescence in situ hybridization (Kindaichi et al., 2012). The Chloroflexota MAGs obtained had genes encoding the thiamin transporter, but generally lacked the gene set required for thiamine biosynthesis (Fig. 1); i.e., thiFGHI required for the synthesis of 4-methyl-5-(β-hydroxyethyl) thiazole phosphate, thiCD for the synthesis of 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate, thiE for the synthesis of thiamine monophosphate, and thiL for the synthesis of thiamine pyrophosphate (Leonardi and Roach, 2004). On the other hand, Planctomycetota MAGs exhibited metabolic potential for thiamine synthesis, and these bacteria may supply thiamine to Chloroflexota bacteria in anammox bacterial enrichment cultures. The exchange of amino acids and vitamins between anammox bacteria and cooccurring bacteria was predicted based on the findings of previous metagenomic and metatranscriptomic ana­lyses (Lawson et al., 2017), and the present results are consistent with this hypothesis. Among Bacteroidota bacteria, those belonging to the genera Melioribacter and Ignavibacteria are facultative anaerobic heterotrophs (Iino et al., 2010; Podosokorskaya et al., 2013), and may scavenge contaminated O₂ in anammox bioreactors. Melioribacter roseus utilized a number of carbon compounds for fermentation, and proliferates with the production of acetate and H₂ gas or by respiration using oxygen or NO₂^– as an electron acceptor (Podosokorskaya et al., 2013). In the present study, Chloroflexota and Bacteroidota MAGs harbored the genes required for dissimilatory NO₃^– reduction to NO₂^– or dissimilatory NO₂^– reduction to NH₄⁺ (DNRA) (Fig. 1). Anammox bacteria oxidize NO₂^– to NO₃^– to gain the reducing power for CO₂ fixation (Kartal et al., 2013 and references therein), and NO₃^– concentrations are generally at >1‍ ‍mM in the operated MBRs. On the other hand, NH₄⁺ and NO₂^– are consumed by anammox bacteria in the MBRs, and may be a limiting substrate(s) of anammox bacteria after their depletion. Therefore, the production of NO₂^– and/or NH₄⁺ by Chloroflexota and Bacteroidota bacteria is beneficial for anammox bacteria. These interactions via NO_x^– in the anammox bacterial community were proposed in previous metagenomic studies (Speth et al., 2016; Lawson et al., 2017), and the metabolic potential of the Chloroflexota and Bacteroidota MAGs obtained further rationalize this hypothesis.

Fig. 1.

Metabolic potential of metagenome-assembled genomes (MAGs) obtained from anammox bacterial enrichment cultures. The figure includes MAGs in the present study (i.e., HBSIN, HBSAPP, JETCAE, HKUEN, and SCALA7 MAGs) and those obtained from a partial-nitritation anammox reactor (UTPRO, UTCFX, UTCFB, UTPLA, and UTAMX MAGs), a sequencing batch anammox reactor (OLB MAGs), and an up-flow column anammox reactor (the 317325 MAGs). MAGs with a parenthesis share >97% of the average nucleotide identity values. The heatmap indicates metabolic pathway completeness calculated using the KEGG Decoder. The taxonomic affiliations of MAGs are available at the top of the heatmap.

In summary, the present study provides metagenome sequencing data obtained from 5 phylogenetically different anammox bacterial enrichment cultures in addition to genomic information on 14 high-quality MAGs. Anammox bacteria appear to supply organic matter (in the form of EPS, soluble microbial products, and cell debris), vitamins, and NO₃^– to cooccurring heterotrophic bacteria. Cooccurring heterotrophic bacteria may scavenge contaminated O₂ and prevent the accumulation of organic matter, which suppresses anammox activity (Tsushima et al., 2007). Although the verification of microbial interactions by a culture-dependent ana­lysis is warranted (Murakami et al., 2022), the genome data obtained supports previously proposed microbial interactions between anammox bacteria and cooccurring bacteria (Lawson et al., 2017) and will advance our understanding of microbial interactions in anammox enrichment cultures. The clarification of these microbial interactions will provide insights into the specific reason(s) for unsuccessful isolation attempts of anammox bacteria, and metatranscriptomic and metaproteomic ana­lyses (Masuda et al., 2017) in addition to the isolation of cooccurring bacteria are required to reveal microbial interactions in anammox bacterial communities.

Data availability

Raw metagenomic sequence data obtained in the present study are available in the DDBJ nucleotide sequence database under the accession number DRA013237. The 14 assembled and annotated MAGs are deposited in the DDBJ nucleotide sequence database with the accession numbers shown in Table S5.

Citation

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.

https://doi.org/10.1264/jsme2.ME22017

Acknowledgements

We thank Dr. Yasuhiro Shimane for his technical support in bioinformatic ana­lyses. This work was supported by JSPS KAKENHI Grant numbers 19K05805 to M.O., 19H00776 to S.O, and JP19H05684 within JP19H05679 (Post-Koch Ecology) to T.N., the JST FOREST Program [JPMJFR216Z for M.O.], and the Nagase Science and Technology Foundation granted to M.O. Computations were partially performed on the NIG supercomputer at the ROIS National Institute of Genetics. The authors declare no conflicts of interest associated with this manuscript.

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.,  Zhang,  L.,  Haroon,  M.F.,  Narita,  Y.,  Emwas,  A.H., et al. (2018) Aggregation ability of three phylogenetically distant anammox bacterial species. Water Res 143: 10–18.
•  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.
•  Bhattacharjee,  A.S.,  Wu,  S.,  Lawson,  C.E.,  Jetten,  M.S.M.,  Kapoor,  V.,  Domingo,  J.W.S., et al. (2017) Whole-community metagenomics in two different anammox configurations: Process performance and community structure. Environ Sci Technol 51: 4317–4327.
•  Bolger,  A.M.,  Lohse,  M., and  Usadel,  B. (2014) Trimmomatic: A flexible trimmer for Illumina sequence data. Bioinformatics 30: 2114–2120.
•  Chaumeil,  P.A.,  Mussig,  A.J.,  Hugenholtz,  P., and  Parks,  D.H. (2019) GTDB-Tk: A toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics 36: 1925–1927.
•  Ferousi,  C.,  Schmitz,  R.A.,  Maalcke,  W.J.,  Lindhoud,  S.,  Versantvoort,  W.,  Jetten,  M.S.M., et al. (2021) Characterization of a nitrite-reducing octaheme hydroxylamine oxidoreductase that lacks the tyrosine cross-link. J Biol Chem 296: 100476.
•  Graham,  E.D.,  Heidelberg,  J.F., and  Tully,  B.J. (2018) Potential for primary productivity in a globally-distributed bacterial phototroph. ISME J 12: 1861–1866.
•  Hira,  D.,  Toh,  H.,  Migita,  C.T.,  Okubo,  H.,  Nishiyama,  T.,  Hattori,  M., et al. (2012) Anammox organism KSU-1 expresses a NirK-type copper-containing nitrite reductase instead of a NirS-type with cytochrome cd₁. FEBS Lett 586: 1658–1663.
•  Hirai,  M.,  Nishi,  S.,  Tsuda,  M.,  Sunamura,  M.,  Takaki,  Y., and  Nunoura,  T. (2017) Library construction from subnanogram DNA for pelagic sea water and deep-sea sediments. Microbes Environ 32: 336–343.
•  Hou,  X.,  Liu,  S.,  Zhang,  Z. (2015) Role of extracellular polymeric substance in determining the high aggregation ability of anammox sludge. Water Res 75: 51–62.
•  Iino,  T.,  Mori,  K.,  Uchino,  Y.,  Nakagawa,  T.,  Harayama,  S., and  Suzuki,  K. (2010) Ignavibacterium album gen. nov., sp. nov., a moderately thermophilic anaerobic bacterium isolated from microbial mats at a terrestrial hot spring and proposal of Ignavibacteria classis nov., for a novel lineage at the periphery of green sulfur bacteria. Int J Syst Evol Microbiol 60: 1376–1382.
•  Jia,  F.,  Yang,  Q.,  Liu,  X.,  Li,  X.,  Li,  B.,  Zhang,  L., and  Peng,  Y. (2017) Stratification of extracellular polymeric substances (EPS) for aggregated anammox microorganisms. Environ Sci Technol 51: 3260–3268.
•  Kanehisa,  M.,  Sato,  Y., and  Morishima,  K. (2016) BlastKOALA and GhostKOALA: KEGG tools for functional characterization of genome and metagenome sequences. J Mol Biol 428: 726–731.
•  Kartal,  B.,  Geerts,  W., and  Jetten,  M.S.M. (2011) Cultivation, detection, and ecophysiology of anaerobic ammonium-oxidizing bacteria. Methods Enzymol 486: 89–108.
•  Kartal,  B.,  Almeida,  N.M. de,  Maalcke,  W.J.,  Op den Camp,  H.J.M.,  Jetten,  M.S.M., and  Keltjens,  J.T. (2013) How to make a living from anaerobic ammonium oxidation. FEMS Microbiol Rev 37: 428–461.
•  Kim,  S.,  Kang,  I.,  Lee,  J.W.,  Jeon,  C.O.,  Giovannoni,  S.J., and  Cho,  J.C. (2021) Heme auxotrophy in abundant aquatic microbial lineages. Proc Natl Acad Sci U S A 118: e2102750118.
•  Kindaichi,  T.,  Ito,  T., and  Okabe,  S. (2004) Ecophysiological interaction between nitrifying bacteria and heterotrophic bacteria in autotrophic nitrifying biofilms as determined by microautoradiography-fluorescence in situ hybridization. Appl Environ Microbiol 70: 1641–1650.
•  Kindaichi,  T.,  Awata,  T.,  Suzuki,  Y.,  Tanabe,  K.,  Hatamoto,  M.,  Ozaki,  N., and  Ohashi,  A. (2011) Enrichment using an up-flow column reactor and community structure of marine anammox bacteria from coastal sediment. Microbes Environ 26: 67–73.
•  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.
•  Kuypers,  M.M.M.,  Lavik,  G.,  Woebken,  D.,  Schmid,  M.,  Fuchs,  B.M.,  Amann,  R., et al. (2005) Massive nitrogen loss from the Benguela upwelling system through anaerobic ammonium oxidation. Proc Natl Acad Sci U S A 102: 6478–6483.
•  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.
•  Lee,  I.,  Kim,  Y.O.,  Park,  S.C., and  Chun,  J. (2016) OrthoANI: An improved algorithm and software for calculating average nucleotide identity. Int J Syst Evol Microbiol 66: 1100–1103.
•  Leonardi,  R., and  Roach,  P.L. (2004) Thiamine biosynthesis in Escherichia coli. J Biol Chem 279: 17054–17062.
•  Lin,  H.H., and  Liao,  Y.C. (2016) Accurate binning of metagenomic contigs via automated clustering sequences using information of genomic signatures and marker genes. Sci Rep 6: 24175.
•  Lotti,  T.,  Kleerebezem,  R.,  Lubello,  C., and  van Loosdrecht,  M.C.M. (2014) Physiological and kinetic characterization of a suspended cell anammox culture. Water Res 60: 1–14.
•  Mardanov,  A.V.,  Beletsky,  A.V.,  Ravin,  N.V.,  Botchkova,  E.A.,  Litti,  Y.V., and  Nozhevnikova,  A.N. (2019) Genome of a novel bacterium “Candidatus Jettenia ecosi” reconstructed from the metagenome of an anammox bioreactor. Front Microbiol 10: 2442.
•  Masuda,  Y.,  Itoh,  H.,  Shiratori,  Y.,  Isobe,  K.,  Otsuka,  S., and  Senoo,  K. (2017) Predominant but previously-overlooked prokaryotic drivers of reductive nitrogen transformation in paddy soils, revealed by metatranscriptomics. Microbes Environ 32: 180–183.
•  Mee,  M.T.,  Collins,  J.J.,  Church,  G.M., and  Wang,  H.H. (2014) Syntrophic exchange in synthetic microbial communities. Proc Natl Acad Sci U S A 111: E2149–E2156.
•  Mendler,  K.,  Chen,  H.,  Parks,  D.H.,  Lobb,  B.,  Hug,  L.A., and  Doxey,  A.C. (2019) AnnoTree: Visualization and exploration of a functionally annotated microbial tree of life. Nucleic Acids Res 47: 4442–4448.
•  Murakami,  C.,  Machida,  K.,  Nakao,  Y.,  Kindaichi,  T.,  Ohashi,  A., and  Aoi,  Y. (2022) Mutualistic relationship between Nitrospira and concomitant heterotrophs. Environ Microbiol Rep 14: 130–137.
•  Narita,  Y.,  Zhang,  L.,  Kimura,  Z.,  Ali,  M.,  Fujii,  T., and  Okabe,  S. (2017) Enrichment and physiological characterization of an anaerobic ammonium-oxidizing bacterium ‘Candidatus Brocadia sapporoensis’. Syst Appl Microbiol 40: 448–457.
•  Ni,  B.-J.,  Ruscalleda,  M.,  Smets,  B.F. (2012) Evaluation on the microbial interactions of anaerobic ammonium oxidizers and heterotrophs in Anammox biofilm. Water Res 46: 4645–4652.
•  Nunoura,  T.,  Hirai,  M.,  Miyazaki,  M.,  Kazama,  H.,  Makita,  H.,  Hirayama,  H., et al. (2013) Isolation and characterization of a thermophilic, obligately anaerobic and heterotrophic marine Chloroflexi bacterium from a Chloroflexi-dominated microbial community associated with a Japanese shallow hydrothermal system, and proposal for Thermomarinilinea lacunofontalis gen. nov., sp. nov. Microbes Environ 28: 228–235.
•  Okubo,  T.,  Toyoda,  A.,  Fukuhara,  K.,  Uchiyama,  I.,  Harigaya,  Y.,  Kuroiwa,  M., et al. (2021) The physiological potential of anammox bacteria as revealed by their core genome structure. DNA Res 28: dsaa028.
•  Oshiki,  M.,  Shimokawa,  M.,  Fujii,  N.,  Satoh,  H., and  Okabe,  S. (2011) Physiological characteristics of the anaerobic ammonium-oxidizing bacterium “Candidatus Brocadia sinica”. Microbiology 157: 1706–1713.
•  Oshiki,  M.,  Awata,  T.,  Kindaichi,  T.,  Satoh,  H., and  Okabe,  S. (2013) Cultivation of planktonic anaerobic ammonium oxidation (anammox) bacteria by using membrane bioreactor. Microbes Environ 28: 436–443.
•  Oshiki,  M.,  Shinyako-Hata,  K.,  Satoh,  H., and  Okabe,  S. (2015) Draft genome sequencing of anaerobic ammonium oxidizing bacterium, “Candidatus Brocadia sinica”. Genome Announc 3: e00267-15.
•  Oshiki,  M.,  Ali,  M.,  Shinyako-Hata,  K.,  Satoh,  H., and  Okabe,  S. (2016) Hydroxylamine-dependent anaerobic ammonium oxidation (anammox) by “Candidatus Brocadia sinica”. Environ Microbiol 18: 3133–3143.
•  Oshiki,  M.,  Mizuto,  K.,  Kimura,  Z.,  Kindaichi,  T.,  Satoh,  H., and  Okabe,  S. (2017) Genetic diversity of marine anaerobic ammonium-oxidizing bacteria as revealed by genomic and proteomic ana­lyses of ‘Candidatus Scalindua japonica’. Environ Microbiol Rep 9: 550–561.
•  Oshiki,  M.,  Masuda,  Y.,  Yamaguchi,  T., and  Araki,  N. (2018) Synergistic inhibition of anaerobic ammonium oxidation (anammox) activity by phenol and thiocyanate. Chemosphere 213: 498–506.
•  Oshiki,  M.,  Hiraizumi,  H.,  Satoh,  H., and  Okabe,  S. (2020) Cell density-dependent anammox activity of Candidatus Brocadia sinica regulated by N-acyl homoserine lactone-mediated quorum sensing. Microbes Environ 35: ME20086.
•  Podosokorskaya,  O.A.,  Kadnikov,  V.V.,  Gavrilov,  S.N.,  Mardanov,  A.V.,  Merkel,  A.Y.,  Karnachuk,  O.V., et al. (2013) Characterization of Melioribacter roseus gen. nov., sp. nov., a novel facultatively anaerobic thermophilic cellulolytic bacterium from the class Ignavibacteria, and a proposal of a novel bacterial phylum Ignavibacteriae. Environ Microbiol 15: 1759–1771.
•  Speth,  D.R.,  In’t Zandt,  M.H.,  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.
•  Strous,  M.,  Pelletier,  E.,  Mangenot,  S.,  Rattei,  T.,  Lehner,  A.,  Taylor,  M.W., et al. (2006) Deciphering the evolution and metabolism of an anammox bacterium from a community genome. Nature 440: 790–794.
•  Sun,  L.,  Toyonaga,  M.,  Ohashi,  A.,  Matsuura,  N.,  Tourlousse,  D.M.,  Meng,  X.Y., et al. (2016) Isolation and characterization of Flexilinea flocculi gen. nov., sp. nov., a filamentous, anaerobic bacterium belonging to the class Anaerolineae in the phylum Chloroflexi. Int J Syst Evol Microbiol 66: 988–996.
•  Tanizawa,  Y.,  Fujisawa,  T., and  Nakamura,  Y. (2018) DFAST: A flexible prokaryotic genome annotation pipeline for faster genome publication. Bioinformatics 34: 1037–1039.
•  Tsushima,  I.,  Ogasawara,  Y.,  Kindaichi,  T.,  Satoh,  H., and  Okabe,  S. (2007) Development of high-rate anaerobic ammonium-oxidizing (anammox) biofilm reactors. Water Res 41: 1623–1634.
•  van de Graaf,  A.A.,  de Bruijn,  P.,  Robertson,  L.A.,  Jetten,  M.S.M., and  Kuenen,  J.G. (1996) Autotrophic growth of anaerobic ammonium-oxidizing micro-organisms in a fluidized bed reactor. Microbiology 142: 2187–2196.
•  van de Vossenberg,  A.,  Woebken,  D.,  Maalcke,  S.W.J.,  Wessels,  H.J.C.T.,  Dutilh,  B.E.,  Kartal,  B., et al. (2013) The metagenome of the marine anammox bacterium ‘Candidatus Scalindua Profunda’ illustrates the versatility of this globally important nitrogen cycle bacterium. Environ Microbiol 15: 1275–1289.
•  van der Star,  W.R.L.,  Abma,  W.R.,  Blommers,  D.,  Mulder,  J.W.,  Tokutomi,  T.,  Strous,  M., et al. (2007) Startup of reactors for anoxic ammonium oxidation: Experiences from the first full-scale anammox reactor in Rotterdam. Water Res 41: 4149–4163.
•  Yamada,  T., and  Sekiguchi,  Y. (2009) Cultivation of uncultured Chloroflexi subphyla: Significance and ecophysiology of formerly uncultured Chloroflexi “subphylum I” with natural and biotechnological relevance. Microbes Environ 24: 205–216.
•  Zhang,  L., and  Okabe,  S. (2017) Rapid cultivation of free-living planktonic anammox cells. Water Res 127: 204–210.
•  Zhang,  L., and  Okabe,  S. (2020) Ecological niche differentiation among anammox bacteria. Water Res 171: 115468.
•  Zhao,  Y.,  Feng,  Y.,  Chen,  L.,  Niu,  Z., and  Liu,  S. (2019) Genome-centered omics insight into the competition and niche differentiation of Ca. Jettenia and Ca. Brocadia affiliated to anammox bacteria. Appl Microbiol Biotechnol 103: 8191–8202.