Anammox and denitrifying bacteria and their nitrogen removal potential in lake sediments mediated by environmental changes

Abstract

Anammox and denitrification are key processes for nitrogen removal in lake sediments. However, how environmental changes mediate the community structure and functional genes of nitrogen removal bacteria in lakes remain unclear. Using metagenome and amplicon sequencing, we investigated the anammox and denitrifying bacteria and their nitrogen removing potentials in lakes experiencing significant spatiotemporal and environmental variations. The community structure of anammox and denitrifying bacteria exhibited stronger lake-wide spatial variations than that of seasonality, while only the denitrification-related functional genes showed substantial variations in both lakes. Anammox genes (e.g., hzsA/B/C and hdh) showed no significant spatial variations. However, the abundances of anammox and denitrifying genes were significantly higher in winter than in summer. The mesotrophic Lake Weishan demonstrated a greater capacity for complete denitrification in winter, while the eutrophic Lake Donghu exhibited a higher potential of anammox in summer. Differences in functional gene abundances between lakes were more pronounced than variations in phylogenetic diversity, indicating clear functional adaptations to local environments. The coupled nitrogen removal potentials also reflected ecological interactions among anammox and denitrifying genes. Importantly, anammox and denitrifying bacterial communities and their functional genes were primarily driven by dissolved organic carbon, total phosphorous and zinc (Zn). The dissimilarities of anammox and denitrifying bacterial communities increased with geographic distance, indicating a clear distance-decay effect. This study highlights the anammox and denitrifying bacteria and their nitrogen removal potentials in lake sediments that are mediated by both spatial and seasonal environmental changes.

Keywords

Anammox and denitrification / Eutrophic lake / Mesotrophic lake / Metagenomics / Nitrogen removal

References

[1] Abdullah AlM, ZhangD, LiuS, MingY, LiM, XingP, YuX, NiuM, KunW, XieW, HeZ, YanQ. Community assembly mechanisms of nirK- and nirS-type denitrifying bacteria in sediments of eutrophic Lake Taihu. China Cur Microbiol, 2025, 8253

[2] Abdullah AlM, XueY, XuJ, XiaoP, ChenH, MoY, ShimetaJ, YangJ. Community assembly of microbial habitat generalists and specialists in urban aquatic ecosystems explained more by habitat type than pollution gradient. Water Res, 2022, 220118693

[3] Baumann KBL, Thoma R, Callbeck CM, Niederdorfer R, Schubrt CS, Muller B, Lever MA, Burgmann H (2022) Microbial nitrogen transformation potential in sediments of two contrasting lakes is spatially structured but seasonally stable. mSphere 7:e01013–21.

[4] BolgerAM, LohseM, UsadelB. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics, 2014, 30: 2114-2120

[5] BromanE, Izabel-ShenD, Rodriguez-GijonA, BonagliaS, GarciaSL, NascimentoFJA. Microbial functional genes are driven by gradients in sediment stoichiometry, oxygen, and salinity across the Baltic benthic ecosystem. Microbiome, 2022, 10126

[6] BuchfinkB, XieC, HusonDH. Fast and sensitive protein alignment using DIAMOND. Nat Methods, 2015, 12: 59-60

[7] CaiM, YeF, WuJ, WuQ, WangY, HongY. Bias of marker genes in PCR of anammox bacteria in natural habitats. PLoS ONE, 2020, 15e0239736

[8] CaporasoJG, KuczynskiJ, StombaughJ, BittingerK, BushmanFD, CostelloEK, FiererN, PeñaAG, GoodrichJK, GordonJ. QIIME allows analysis of high-throughput community sequencing data. Nat Methods, 2010, 7: 335-336

[9] ChaumeilPA, MussigAJ, HugenholtzP, ParksDH. GTDB-Tk: a toolkit to classify genomes with the Genome Taxonomy Database. Bioinformatics, 2019, 36: 1925-1927

[10] ChenZ, ZhangT, ZhangZ, YueL, ZhangJ, ZhouS, ChaiB. Assembly mechanisms and driving factors of aerobic denitrifying bacteria community with different seasons and rarity in the sediments of Baiyangdian Lake. J Soils Sediments, 2023, 24: 1838-1853

[11] DingC, GongZ, ZhangK, JiangW, KangM, TianZ, ZhangY, LiY, YangY, QiuZ. Distribution and model prediction of antibiotic resistance genes in Weishan Lake based on the indication of Chironomidae larvae. Water Res, 2022, 222118862

[12] EdgarRC. UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods, 2013, 10: 996-998

[13] EdgarRC, HaasBJ, ClementeJC, QuinceC, KnightR. UCHIME improves sensitivity and speed of chimera detection. Bioinformatics, 2011, 27: 2194-2200

[14] GuiM, ChenQ, MaT, ZhengM, NiJ. Effects of heavy metals on aerobic denitrification by strain Pseudomonas stutzeri PCN-1. Appl Microbiol Biotechnol, 2017, 101: 1717-1727

[15] GutwińskiP, CemaG, Ziembińska-BuczyńskaA, WyszyńskaK, Surmacz-GórskaJ. Long-term effect of heavy metals Cr(III), Zn(II), Cd(II), Cu(II), Ni(II), Pb(II) on the anammox process performance. J Water Proc Enginer, 2021, 39101668

[16] HanS, JuT, MengY, DuY, XiangH, AihemaitiA, JiangJ. Evaluation of various microwave-assisted acid digestion procedures for the determination of major and heavy metal elements in municipal solid waste incineration fly ash. J Cleaner Prod, 2021, 321128922

[17] HyattD, ChenGL, LocascioPF, LandML, LarimerFW, HauserLJ. Prodigal: prokaryotic gene recognition and translation initiation site identification. BMC Bioinformatics, 2010, 11119

[18] KanehisaM, SatoY, KawashimaM, FurumichiM, TanabeM. KEGG as a reference resource for gene and protein annotation. Nucleic Acids Res, 2016, 44: D457-D462

[19] Kou Y, Liu Y, Li J, Li C, Yao M, Li X (2021) Patterns and drivers of nirK and nirS-type denitrifier community assembly along an elevation gradient. mSystems 6:e00667–21.

[20] KuypersMMM, MarchantHK, KartalB. The microbial nitrogen-cycling network. Nat Rev Microbiol, 2018, 16: 263-275

[21] LiD, LiuC, LuoR, SadakaneK, LamTW. MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph. Bioinformatics, 2015, 31: 1674-1676

[22] LiW, GodzikA. Cd-hit: a fast program for clustering and comparing large sets of protein or nucleotide sequences. Bioinformatics, 2006, 22: 1658-1659

[23] LiY, TianH, YaoY, ShiH, BianZ, ShiY, WangS, MaavaraT, LauerwaldR, PanS. Increased nitrous oxide emissions from global lakes and reservoirs since the pre-industrial era. Nat Commun, 2024, 15942

[24] LinJ, ChenN, YuanX. Impacts of human disturbance on the biogeochemical nitrogen cycle in a subtropical river system revealed by nitrifier and denitrifier genes. Sci Total Environ, 2020, 746141139

[25] LiuE, FanC, ZhaoM, JiangS, WangZ, JinZ, BeiK, ZhengX, WuS, ZengQ. Effects of heavy metals on denitrification processes in water treatment: a review. Sep Purif Technol, 2022, 299121793

[26] LiuJ, LiD, HeX, LiuR, ChengH, SuC, ChenM, WangY, ZhaoZ, XuH, ChengZ, WangZ, PedentchoukN, Lea-SmithDJ, ToddJD, LiuX, ZhaoM, ZhangX. A unique subseafloor microbiosphere in the Mariana Trench driven by episodic sedimentation. Mar Life Sci Technol, 2024, 6: 168-181

[27] LiuW, YaoL, JiangX, GuoL, ChengX, LiuG. Sediment denitrification in Yangtze lakes is mainly influenced by environmental conditions but not biological communities. Sci Total Environ, 2018, 616: 978-987

[28] MagočT, SalzbergSL. FLASH: fast length adjustment of short reads to improve genome assemblies. Bioinformatics, 2011, 27: 2957-2963

[29] MingY, Abdullah AlM, ZhangD, ZhuW, LiuH, YuX, WuK, NiuM, ZengQ, HeZ, YanQ. Insights into the evolutionary and ecological adaption strategies of nirS- and nirK-type denitrifying communities. Mol Ecol, 2024, 33e17507

[30] MyrstenerM, JonssonA, BergstromA-K. The effects of temperature and resource availability on denitrification and relative N₂O production in boreal lake sediments. J Environ Sci, 2016, 47: 82-90

[31] NingD, YuanM, WuL, ZhangY, GuoX, ZhouX, YangY, ArkinA, FirestoneM, ZhouJ. A quantitative framework reveals ecological drivers of grassland microbial community assembly in response to warming. Nat Commun, 2020, 114717

[32] OlmMR, BrownCT, BrooksB, BanfieldJF. dRep: a tool for fast and accurate genomic comparisons that enables improved genome recovery from metagenomes through de-replication. ISME J, 2017, 11: 2864-2868

[33] ParksDH, ImelfortM, SkennertonCT, HugenholtzP, TysonGW. CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes. Genome Res, 2015, 25: 1043-1055

[34] PatroR, DuggalG, LoveMI, IrizarryRA, KingsfordC. Salmon provides fast and bias-aware quantification of transcript expression. Nat Methods, 2017, 14: 417-419

[35] StegenJC, LinX, KonopkaA, FredricksonJK. Stochastic and deterministic assembly processes in subsurface microbial communities. ISME J, 2012, 6: 1653-1664

[36] UritskiyGV, DiRuggieroJ, TaylorJ. MetaWRAP-a flexible pipeline for genome-resolved metagenomic data analysis. Microbiome, 2018, 6158

[37] WoolwayRI, KraemerBM, LentersJD, MerchantCJ, O’ReillyCM, SharmaS. Global lake responses to climate change. Nat Rev Earth Environ, 2020, 1: 388-403

[38] WuS, WuZ, LiangZ, LiuY, WangY. Denitrification and the controlling factors in Yunnan Plateau Lakes (China): exploring the role of enhanced internal nitrogen cycling by algal blooms. J Environ Sci, 2019, 76: 349-358

[39] WuY, SimmonsBA, SingerSW. MaxBin 2.0: an automated binning algorithm to recover genomes from multiple metagenomic datasets. Bioinformatics, 2016, 32: 605-607

[40] Wu Z, Li J, Sun Y, Peñuelas J, Huang J, Sardans J, Jiang Q, Finlay JC, Britten GL, Follows MJ, Gao W, Qin B, Ni J, Huo S, LiuY (2022) Imbalance of global nutrient cycles exacerbated by the greater retention of phosphorus over nitrogen in lakes. Nat Geosci 15:464–468

[41] YanQ, StegenJC, YuY, DengY, LiX, WuS, DaiL, ZhangX, LiJ, WangC, NiJ, LiX, HuH, XiaoF, FengW, NingD, HeZ, Van NostrandJD, WuL, ZhouJ. Nearly a decade-long repeatable seasonal diversity patterns of bacterioplankton communities in the eutrophic Lake Donghu (Wuhan, China). Mol Ecol, 2017, 26: 3839-3850

[42] YangR, LiH, SuQ, ZhouW. Anammox bacteria are potentially involved in anaerobic ammonium oxidation coupled to iron(III) reduction in the wastewater treatment system. Front Microbiol, 2021, 12717249

[43] YaoL, ChenC, LiuG, LiuW. Sediment nitrogen cycling rates and microbial abundance along a submerged vegetation gradient in a eutrophic lake. Sci Total Environ, 2018, 616: 899-907

[44] YaoL, JiangX, ChenC, LiuG, LiuW. Within-lake variability and environmental controls of sediment denitrification and associated N₂O production in a shallow eutrophic lake. Ecol Eng, 2016, 97: 251-257

[45] YuanT, McCarthyAJ, ZhangY, SekarR. Impact of temperature, nutrients and heavy metals on bacterial diversity and ecosystem functioning studied by freshwater microcosms and high-throughput DNA sequencing. Current Microbiol, 2020, 77: 3512-3525

[46] YueY, YangZ, WangF, ChenX, HuangY, MaJ, CaiL, YangM. Effects of cascade reservoirs on spatiotemporal dynamics of the sedimentary bacterial community: co-occurrence patterns, assembly mechanisms, and potential functions. Microb Ecol, 2024, 8718

[47] ZhangD, LiMY, YangYC, YuH, XiaoFS, MaoCZ, HuangJ, YuYH, WangYF, WuB, WangC, ShuLF, HeZL, YanQ. Nitrite and nitrate reduction drive sediment microbial nitrogen cycling in a eutrophic lake. Water Res, 2022, 220118637

[48] ZhangD, LiuF, Abdullah AlM, YangY, YuH, LiM, WuK, NiuM, WangC, HeZ, YanQ. Nitrogen and sulfur cycling and their coupling mechanisms in eutrophic lake sediment microbiomes. Sci Total Environ, 2024, 928172518

[49] ZhangD, YuH, YangYC, LiuF, LiMY, HuangJ, YuYH, WangC, JiangF, HeZ, YanQ. Ecological interactions and the underlying mechanism of anammox and denitrification across the anammox enrichment with eutrophic lake sediments. Microbiome, 2023, 1182

[50] ZhangD, YuH, YuX, YangY, WangC, WuK, NiuM, HeJ, HeZ, YanQ. Mechanisms underlying the interactions and adaptability of nitrogen removal microorganisms in freshwater sediments. Adv Biotechnol, 2024, 221

[51] ZhangJ, KobertK, FlouriT, StamatakisA. PEAR: a fast and accurate Illumina Paired-End reAd mergeR. Bioinformatics, 2014, 30: 614-620

[52] ZhangL, BaiJ, ZhaiY, ZhangK, WangY, TangR, XiaoR, JorqueraMA. Seasonal changes in N-cycling functional genes in sediments and their influencing factors in a typical eutrophic shallow lake. China Front Microbiol, 2024, 151363775

[53] ZhaoY, ChenZ, WangQ, ZhangC, JiM. A new insight to explore toxic Cd(II) affecting denitrification: reaction kinetic, electron behavior and microbial community. Chemosphere, 2022, 305135419

[54] ZhouJ, NingD. Stochastic community assembly: does it matter in microbial ecology?. Microbiol Mol Biol Rev, 2017, 81: e00002-17

[55] ZhouZ, TranPQ, BreisterAM, LiuY, KieftK, CowleyES, KaraozU, AnantharamanK. METABOLIC: high-throughput profiling of microbial genomes for functional traits, metabolism, biogeochemistry, and community-scale functional networks. Microbiome, 2022, 1033

[56] ZhouZ, WeiQ, YangY, LiM, GuJD. Practical applications of PCR primers in detection of anammox bacteria effectively from different types of samples. Appl Microbiol Biotechnol, 2018, 102: 5859-5871

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