Genome-resolved metagenomic analysis reveals different functional potentials of multiple Candidatus Brocadia species in a full-scale swine wastewater treatment system

Highlight

● Four Ca. Brocadia species were observed during the spontaneously enrichment.

● Novel anammox species SW510 and SW773 dominated the full-scale ecosystem.

● Urease and cyanase genes were detected in the new anammox genomes.

● Functional differentiation potentially facilitated co-occurrence of anammox species.

Abstract

The increasing application of anammox processes suggests their enormous potential for nitrogen removal in wastewater treatment facilities. However, the functional potentials and ecological differentiation of cooccurring anammox species in complex ecosystems have not been well elucidated. Herein, by utilizing functional reconstruction and comparative genome analysis, we deciphered the cooccurring mechanisms of four Candidatus Brocadia species that were spontaneously enriched in a full-scale swine wastewater treatment system. Phylogenetic analysis indicated that species SW172 and SW745 were closely related to Ca. Brocadia caroliniensis and Ca. Brocadia sapporoensis, respectively, whereas the dominant species SW510 and SW773, with a total average abundance of 34.1%, were classified as novel species of the genus Ca. Brocadia. Functional reconstruction revealed that the novel species SW510 can encode both cytochrome cd₁-type nitrite reductase and hydroxylamine oxidase for nitrite reduction. In contrast, the detected respiratory pentaheme cytochrome c nitrite reductase and acetate kinase genes suggested that SW773 likely reduced nitrite to ammonium with acetate as a carbon source. Intriguingly, the presence of genes encoding urease and cyanase indicated that both novel species can use diverse organic nitrogen compounds in addition to ammonia and nitrite as substrates. Taken together, the recovery and comparative analysis of these anammox genomes expand our understanding of the functional differentiation and cooccurrence of the genus Ca. Brocadia in wastewater treatment systems.

Keywords

Anammox / Candidatus Brocadia / Functional potential / Cooccurring mechanisms / Swine wastewater treatment facilities

参考文献

[1] AliM, ShawD R, AlbertsenM, SaikalyP E. (2020). Comparative genome-centric analysis of freshwater and marine ANAMMOX cultures suggests functional redundancy in nitrogen removal processes. Frontiers in Microbiology, 11 : 1637

[2] AraiW TaniguchiT GotoS MoriyaY UeharaH TakemotoK OgataH TakamiH ( 2018). MAPLE 2.3.0: an improved system for evaluating the functionomes of genomes and metagenomes. Bioscience, Biotechnology, and Biochemistry, 82( 9): 1515– 1517

[3] AsnicarF, ThomasA M, BeghiniF, MengoniC, ManaraS, ManghiP, ZhuQ, BolzanM, CumboF, MayU. . (2020). Precise phylogenetic analysis of microbial isolates and genomes from metagenomes using PhyloPhlAn 3. 0. Nature Communications, 11( 1): 2500

[4] BrownC T, OlmM R, ThomasB C, BanfieldJ F. (2016). Measurement of bacterial replication rates in microbial communities. Nature Biotechnology, 34( 12): 1256– 1263

[5] CarranzoI V. (2012). Standard methods for examination of water and wastewater. Madrid: Complutense University of Madrid, 185

[6] CastelleC J, BanfieldJ F. (2018). Major new microbial groups expand diversity and alter our understanding of the tree of life. Cell, 172( 6): 1181– 1197

[7] ChaumeilP A MussigA J HugenholtzP ParksD H (2019). GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics (Oxford, England), 36(6): 1925–1927

[8] EmmsD M, KellyS. (2019). OrthoFinder: phylogenetic orthology inference for comparative genomics. Genome Biology, 20( 1): 238

[9] FengY, ZhaoY, JiangB, ZhaoH, WangQ, LiuS. (2019). Discrepant gene functional potential and cross-feedings of anammox bacteria Ca. Jettenia caeni and Ca. Brocadia sinica in response to acetate. Water Research, 165 : 114974

[10] GaneshS, BertagnolliA D, BristowL A, PadillaC C, BlackwoodN, AldunateM, BourbonnaisA, AltabetM A, MalmstromR R, WoykeT. . (2018). Single cell genomic and transcriptomic evidence for the use of alternative nitrogen substrates by anammox bacteria. ISME journal, 12( 11): 2706– 2722

[11] HouF, ZhangT, PengY, CaoX, PangH, ShaoY, LuX, YuanJ, ChenX, ZhangJ. (2022). Partial anammox achieved in full scale biofilm process for typical domestic wastewater treatment. Frontiers of Environmental Science & Engineering, 16( 3): 33

[12] Huerta-CepasJ SzklarczykD HellerD Hernández-PlazaA ForslundS K CookH MendeD R LetunicI RatteiT JensenLars J, .( 2018). eggNOG 5.0: a hierarchical, functionally and phylogenetically annotated orthology resource based on 5090 organisms and 2502 viruses. Nucleic Acids Research, 47( D1): D309− D314

[13] KartalB, KuenenJ G, van LoosdrechtM C. (2010). Sewage treatment with anammox. Science, 328( 5979): 702– 703

[14] KumarS, StecherG, LiM, KnyazC, TamuraK. (2018). MEGA X: molecular evolutionary genetics analysis across computing platforms. Molecular Biology and Evolution, 35( 6): 1547– 1549

[15] LawsonC E, NuijtenG H L, deGraaf R M, JacobsonT B, PabstM, StevensonD M, JettenM S M, NogueraD R, McMahonK D, Amador-NoguezD, LückerS. (2021). Autotrophic and mixotrophic metabolism of an anammox bacterium revealed by in vivo ¹³C and ²H metabolic network mapping. ISME journal, 15( 3): 673– 687

[16] LiD Liu C M LuoR SadakaneK LamT W ( 2015). MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics (Oxford, England), 31( 10): 1674– 1676

[17] LiuL, WangY, CheY, ChenY, XiaY, LuoR, ChengS H, ZhengC, ZhangT. (2020). High-quality bacterial genomes of a partial-nitritation/anammox system by an iterative hybrid assembly method. Microbiome, 8( 1): 155

[18] LiuS, GongZ, YangF, ZhangH, ShiL, FurukawaK. (2008). Combined process of urea nitrogen removal in anaerobic anammox co-culture reactor. Bioresource Technology, 99( 6): 1722– 1728

[19] MardanovA V, BeletskyA V, RavinN V, BotchkovaE A, LittiY V, NozhevnikovaA N. (2019). Genome of a novel bacterium “Candidatus Jettenia ecosi” reconstructed from the metagenome of an anammox bioreactor. Frontiers in Microbiology, 10 : 2442

[20] OkabeS, ShafdarA A, KobayashiK, ZhangL, OshikiM. (2021). Glycogen metabolism of the anammox bacterium “Candidatus Brocadia sinica”. ISME journal, 15( 5): 1287– 1301

[21] OkuboT, ToyodaA, FukuharaK, UchiyamaI, HarigayaY, KuroiwaM, SuzukiT, MurakamiY, SuwaY, TakamiH. (2021). The physiological potential of anammox bacteria as revealed by their core genome structure. DNA Research, 28( 1): dsaa028

[22] OlmM R, BrownC T, BrooksB, BanfieldJ F. (2017). dRep: a tool for fast and accurate genomic comparisons that enables improved genome recovery from metagenomes through de-replication. ISME journal, 11( 12): 2864– 2868

[23] OshikiM, SatohH, OkabeS. (2016). Ecology and physiology of anaerobic ammonium oxidizing bacteria. Environmental Microbiology, 18( 9): 2784– 2796

[24] ParkH, BrottoA C, van LoosdrechtM C M, ChandranK. (2017). Discovery and metagenomic analysis of an anammox bacterial enrichment related to Candidatus “Brocadia caroliniensis” in a full-scale glycerol-fed nitritation-denitritation separate centrate treatment process. Water Research, 111 : 265– 273

[25] ParksD H, ChuvochinaM, ChaumeilP A, RinkeC, MussigA J, HugenholtzP. (2020). A complete domain-to-species taxonomy for bacteria and archaea. Nature Biotechnology, 38( 9): 1079– 1086

[26] SchoutenS, StrousM, KuypersM M M, RijpstraW I C, BaasM, SchubertC J, JettenM S M, SinningheDamsté J S. (2004). Stable carbon isotopic fractionations associated with inorganic carbon fixation by anaerobic ammonium-oxidizing bacteria. Applied and Environmental Microbiology, 70( 6): 3785– 3788

[27] SongW, WemheuerB, ZhangS, SteensenK, ThomasT. (2019). MetaCHIP: community-level horizontal gene transfer identification through the combination of best-match and phylogenetic approaches. Microbiome, 7( 1): 36

[28] SuiQ, ZhengR, ZhangJ, DiF, ZuoF, ZhangY, WangX, ChenY, WeiY. (2021). Successful enrichment of anammox consortium in a single-stage reactor at full-scale: the difference in response of functional genes and transcriptional expressions. Chemical Engineering Journal, 426 : 131935

[29] TaoY, HuangX, GaoD, WangX, ChenC, LiangH, van LoosdrechtM C M. (2019). NanoSIMS reveals unusual enrichment of acetate and propionate by an anammox consortium dominated by Jettenia asiatica. Water Research, 159 : 223– 232

[30] UritskiyG V, DiRuggieroJ, TaylorJ. (2018). MetaWRAP: a flexible pipeline for genome-resolved metagenomic data analysis. Microbiome, 6( 1): 158

[31] WangS, PengY, MaB, WangS, ZhuG. (2015). Anaerobic ammonium oxidation in traditional municipal wastewater treatment plants with low-strength ammonium loading: widespread but overlooked. Water Research, 84 : 66– 75

[32] WangY, NiuQ, ZhangX, LiuL, WangY, ChenY, NegiM, FigeysD, LiY Y, ZhangT. (2019). Exploring the effects of operational mode and microbial interactions on bacterial community assembly in a one-stage partial-nitritation anammox reactor using integrated multi-omics. Microbiome, 7( 1): 122

[33] YangY, AzariM, HerboldC W, LiM, ChenH, DingX, DeneckeM, GuJ D. (2021). Activities and metabolic versatility of distinct anammox bacteria in a full-scale wastewater treatment system. Water Research, 206 : 117763

[34] YangY, PanJ, ZhouZ, WuJ, LiuY, LinJ G, HongY, LiX, LiM, GuJ D. (2020). Complex microbial nitrogen-cycling networks in three distinct anammox-inoculated wastewater treatment systems. Water Research, 168 : 115142

[35] ZhangL, NaritaY, GaoL, AliM, OshikiM, IshiiS, OkabeS. (2017). Microbial competition among anammox bacteria in nitrite-limited bioreactors. Water Research, 125 : 249– 258

[36] ZhangL, OkabeS. (2020). Ecological niche differentiation among anammox bacteria. Water Research, 171 : 115468

[37] ZhaoB, LiJ, LeuS Y. (2014). An innovative wood-chip-framework soil infiltrator for treating anaerobic digested swine wastewater and analysis of the microbial community. Bioresource Technology, 173 : 384– 391

[38] ZhengQ, ZhangM, ZhangT, LiX, ZhuM, WangX. (2020). Insights from metagenomic, metatranscriptomic, and molecular ecological network analyses into the effects of chromium nanoparticles on activated sludge system. Frontiers of Environmental Science & Engineering, 14( 4): 60

Supplemental materials

Microbial communities of the anammox consortia and the overall nitrogen cycle pathways were included in supplemental texts. Detailed descriptions of the comparative genomic analysis and the key genes involved in amino acid and vitamin biosynthesis were provided in supplemental tables and figures. Functional annotations refer to the four anammox species were listed in supplemental data.

Acknowledgements

This study was supported by the National Natural Science Foundation of China (Nos. 52000185, 51878675, and 51622813), the Natural Science Foundation of Guangdong Province (No. 2021A1515010512), and the Fundamental Research Funds for the Central Universities, Sun Yat-sen University (No. 2021qntd07).

Electronic Supplementary Material

Supplementary material is available in the online version of this article at https://doi.org/10.1007/s11783-023-1602-7 and is accessible for authorized users.

版权

2023 Higher Education Press