Changes in organic matter accumulation in wetlands are critical for climate dynamics. Different nitrogen (N) inputs in Sphagnum-dominated peat bogs can lead to varying rates of carbon (C) and N accumulation, influencing greenhouse gas emissions. We investigated how contrasting N deposition shapes microbial communities in two Czech peat bogs, focusing on biological N2 fixation (BNF) as a key N input in pristine wetlands. Higher N deposition resulted in a more active microbial community with increased enzyme activity and C acquisition, potentially accelerating decomposition and reducing C storage. Enhanced denitrification, indicated by active nosZ Clade I genes, suggests that higher N inputs may increase N losses through denitrification. In contrast, the lower N site showed a less active microbial community with slower decomposition, beneficial for C sequestration, though potentially less adaptable to future N increases. Experimental BNF rates were 70 times higher at the high N site, consistent with elevated diazotroph activity indicated by active nifH gene. Phosphorus (P) availability and NH4+/NO3- ratios appeared to drive BNF differences, emphasizing the need for managed N inputs to maintain peatland ecological functions.

Department of Ecosystem Biology University of South Bohemia in České Budejovice Branisovska 1645a 370 05 České Budejovice Czech Republic

Department of Environmental Geochemistry and Biogeochemistry Czech Geological Survey Geologicka 6 152 00 Prague 5 Czech Republic

Department of Rock Geochemistry Czech Geological Survey Geologicka 6 152 00 Prague 5 Czech Republic

See more in PubMed

Allison  SD, Martiny  JBH.  Resistance, resilience, and redundancy in microbial communities. Proc Natl Acad Sci. 2008;105:11512–9. 10.1073/pnas.0801925105. PubMed DOI PMC

Andersen  R.  Importance of microbes in peatland dynamics, restoration, and reclamation. In: Restoration and Reclamation of Boreal Ecosystems. Cambridge: Cambridge University Press, 2012.

Bååth  E, Anderson  T-H.  Comparison of soil fungal/bacterial ratios in a pH gradient using physiological and PLFA-based techniques. Soil Biol Biochem. 2003;35:955–63. 10.1016/S0038-0717(03)00154-8. DOI

Baldrian  P, Bell-Dereske  L, Lepinay  C  et al.  Fungal communities in soils under global change. Stud Mycol. 2022;103:1–24. 10.3114/sim.2022.103.01. PubMed DOI PMC

Bárta  J, Applová  M, Vaněk  D  et al.  Effect of available P and phenolics on mineral N release in acidified spruce forest: connection with lignin-degrading enzymes and bacterial and fungal communities. Biogeochemistry. 2010;97:71–87. 10.1007/s10533-009-9363-3. DOI

Bárta  J, Šlajsová  P, Tahovská  K  et al.  Different temperature sensitivity and kinetics of soil enzymes indicate seasonal shifts in C, N and P nutrient stoichiometry in acid forest soil. Biogeochemistry. 2014;117:525–37. 10.1007/s10533-013-9898-1. DOI

Basilier  K.  Moss-associated nitrogen fixation in some mire and coniferous forest environments around Uppsala, Sweden. Lindbergia. 1979;5:84–8.

Benner  JW, Vitousek  PM.  Development of a diverse epiphyte community in response to phosphorus fertilization. Ecol Lett. 2007;10:628–36. 10.1111/j.1461-0248.2007.01054.x. PubMed DOI

Borneman  J, Hartin  RJ.  PCR primers that amplify fungal rRNA genes from environmental samples. Appl Environ Microbiol. 2000;66:4356–60. 10.1128/AEM.66.10.4356-4360.2000. PubMed DOI PMC

Bragazza  L, Freeman  C, Jones  T  et al.  Atmospheric nitrogen deposition promotes carbon loss from peat bogs. Proc Natl Acad Sci. 2006;103:19386–9. 10.1073/pnas.0606629104. PubMed DOI PMC

Bragina  A, Oberauner-Wappis  L, Zachow  C  et al.  The PubMed

Buzek  F, Cejkova  B, Jackova  I  et al.  15. N study of the reactivity of atmospheric nitrogen in four mountain forest catchments (Czech Republic, central Europe). Appl Geochem. 2020;116:104567. 10.1016/j.apgeochem.2020.104567. DOI

Caporaso  JG, Lauber  CL, Walters  WA  et al.  Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc Natl Acad Sci. 2011;108:4516–22. 10.1073/pnas.1000080107. PubMed DOI PMC

Dhandapani  S, Ritz  K, Evers  S  et al.  Are secondary forests second-rate? Comparing peatland greenhouse gas emissions, chemical and microbial community properties between primary and secondary forests in Peninsular Malaysia. Sci Total Environ. 2019;655:220–31. 10.1016/j.scitotenv.2018.11.046. PubMed DOI

Diáková  K, Biasi  C, Čapek  P  et al.  Variation in N DOI

Dise  NB, Verry  ES.  Suppression of peatland methane emission by cumulative sulfate deposition in simulated acid rain. Biogeochemistry. 2001;53:143–60. 10.1023/A:1010774610050. DOI

Dohnal  Z.  Czechoslovak Peatlands. Prague: Czechoslovak Academy of Sciences in Czech, 1965.

Dossa  GGO, Yang  Y-Q, Hu  W  et al.  Fungal succession in decomposing woody debris across a tropical forest disturbance gradient. Soil Biol Biochem. 2021;155:108142. 10.1016/j.soilbio.2021.108142. DOI

Edgar  RC.  UPARSE: highly accurate OTU sequences from microbial amplicon reads. Nat Methods. 2013;10:996–8. 10.1038/nmeth.2604. PubMed DOI

Fierer  N, Leff  JW, Adams  BJ  et al.  Cross-biome metagenomic analyses of soil microbial communities and their functional attributes. Proc Natl Acad Sci. 2012;109:21390–5. 10.1073/pnas.1215210110. PubMed DOI PMC

Freedman  Z, Zak  DR.  Atmospheric N deposition increases bacterial laccase-like multicopper oxidases: implications for organic matter decay. Appl Environ Microbiol. 2014;80:4460–8. 10.1128/AEM.01224-14. PubMed DOI PMC

Gaby  JC, Buckley  DH.  A comprehensive evaluation of PCR primers to amplify the nifH gene of nitrogenase. PLoS One. 2012;7:e42149. 10.1371/journal.pone.0042149. PubMed DOI PMC

Gallo  M, Amonette  R, Lauber  C  et al.  Microbial community structure and oxidative enzyme activity in nitrogen-amended north temperate forest soils. Microb Ecol. 2004;48:218–29. 10.1007/s00248-003-9001-x. PubMed DOI

Galloway  JN, Townsend  AR, Erisman  JW  et al.  Transformation of the nitrogen cycle: recent trends, questions, and potential solutions. Science. 2008;320:889–92. PubMed

GARDES  M, BRUNS  TD.  ITS primers with enhanced specificity for basidiomycetes—application to the identification of mycorrhizae and rusts. Mol Ecol. 1993;2:113–8. 10.1111/j.1365-294X.1993.tb00005.x. PubMed DOI

Godin  A, McLaughlin  JW, Webster  KL  et al.  Methane and methanogen community dynamics across a boreal peatland nutrient gradient. Soil Biol Biochem. 2012;48:96–105. 10.1016/j.soilbio.2012.01.018. DOI

Hallin  S, Philippot  L, Löffler  FE  et al.  Genomics and ecology of novel N PubMed DOI

Ho  A, Bodelier  PLE.  Diazotrophic methanotrophs in peatlands: the missing link?. Plant Soil. 2015;389:419–23. 10.1007/s11104-015-2393-9. DOI

Hobbie  SE.  Nitrogen effects on decomposition: a five-year experiment in eight temperate sites. Ecology. 2008;89:2633–44. 10.1890/07-1119.1. PubMed DOI

Houlton  BZ, Wang  Y-P, Vitousek  PM  et al.  A unifying framework for dinitrogen fixation in the terrestrial biosphere. Nature. 2008;454:327–30. 10.1038/nature07028. PubMed DOI

Hůnová  I, Novák  M, Kurfürst  P  et al.  Comparison of vertical and horizontal atmospheric deposition of nitrate at Central European mountain-top sites during three consecutive winters. Sci Total Environ. 2023;869:161697. 10.1016/j.scitotenv.2023.161697. PubMed DOI

Jaszczuk  I, Kotowski  W, Kozub  Ł  et al.  Physiological responses of fen mosses along a nitrogen gradient point to competition restricting their fundamental niches. Oikos. 2023;2023. 10.1111/oik.09336. DOI

Jones  CM, Stres  B, Rosenquist  M  et al.  Phylogenetic analysis of nitrite, nitric oxide, and nitrous oxide respiratory enzymes reveal a complex evolutionary history for denitrification. Mol Biol Evol. 2008;25:1955–66. 10.1093/molbev/msn146. PubMed DOI

Keller  JK, Bauers  AK, Bridgham  SD  et al.  Nutrient control of microbial carbon cycling along an ombrotrophic-minerotrophic peatland gradient. J Geophys Res Biogeosci. 2006;111. 10.1029/2005JG000152. DOI

Knorr  K, Horn  MA, Borken  W.  Significant nonsymbiotic nitrogen fixation in Patagonian ombrotrophic bogs. Glob Chang Biol. 2015;21:2357–65. 10.1111/gcb.12849. PubMed DOI

Koerselman  W, Meuleman  AFM.  The vegetation N:P ratio: a new tool to detect the nature of nutrient limitation. J Appl Ecol. 1996;33:1441. 10.2307/2404783. DOI

Kõljalg  U, Nilsson  RH, Abarenkov  K  et al.  Towards a unified paradigm for sequence-based identification of fungi. Mol Ecol. 2013;22:5271–7. 10.1111/mec.12481. PubMed DOI

Kolton  M, Weston  DJ, Mayali  X  et al.  Defining the DOI

Kox  MAR, Aalto  SL, Penttilä  T  et al.  The influence of oxygen and methane on nitrogen fixation in subarctic PubMed DOI PMC

Kox  MAR., Lüke  C, Fritz  C  et al.  Effects of nitrogen fertilization on diazotrophic activity of microorganisms associated with Sphagnum magellanicum. Plant and Soil. 2016;406:83–100. 10.1007/s11104-016-2851-z DOI

Lamers  LPM, Bobbink  R, Roelofs  JGM.  Natural nitrogen filter fails in polluted raised bogs. Glob Chang Biol. 2000;6:583–6. 10.1046/j.1365-2486.2000.00342.x. DOI

Larmola  T, Leppänen  SM, Tuittila  E-S  et al.  Methanotrophy induces nitrogen fixation during peatland development. Proc Natl Acad Sci. 2014;111:734–9. 10.1073/pnas.1314284111. PubMed DOI PMC

Lett  S, Christiansen  CT, Dorrepaal  E  et al.  Moss species and precipitation mediate experimental warming stimulation of growing season N PubMed DOI

Limpens  J, Berendse  F, Blodau  C  et al.  Peatlands and the carbon cycle: from local processes to global implications—a synthesis. Biogeosciences. 2008;5:1475–91. 10.5194/bg-5-1475-2008. DOI

Limpens  J, Berendse  F, Klees  H.  How phosphorus availability affects the impact of nitrogen deposition on DOI

Lin  Y, Hu  H-W, Deng  M  et al.  Microorganisms carrying nosZ I and nosZ II share similar ecological niches in a subtropical coastal wetland. Sci Total Environ. 2023;870:162008. 10.1016/j.scitotenv.2023.162008. PubMed DOI

Liu  L, Greaver  TL.  A review of nitrogen enrichment effects on three biogenic GHGs: the CO PubMed DOI

Liu  Q, Qiao  N, Xu  X  et al.  Nitrogen acquisition by plants and microorganisms in a temperate grassland. Sci Rep. 2016;6:22642. 10.1038/srep22642. PubMed DOI PMC

Ma  X, Song  Y, Song  C  et al.  Effect of nitrogen addition on soil microbial functional gene abundance and community diversity in Permafrost peatland. Microorganisms. 2021;9:2498. 10.3390/microorganisms9122498. PubMed DOI PMC

McDowell  WH, Zsolnay  A, Aitkenhead-Peterson  JA  et al.  A comparison of methods to determine the biodegradable dissolved organic carbon from different terrestrial sources. Soil Biol Biochem. 2006;38:1933–42. 10.1016/j.soilbio.2005.12.018. DOI

Muyzer  G, de Waal  EC, Uitterlinden  AG.  Profiling of complex microbial populations by denaturing gradient gel electrophoresis analysis of polymerase chain reaction-amplified genes coding for 16S rRNA. Appl Environ Microbiol. 1993;59:695–700. 10.1128/aem.59.3.695-700.1993. PubMed DOI PMC

Nguyen  NH, Song  Z, Bates  ST  et al.  FUNGuild: an open annotation tool for parsing fungal community datasets by ecological guild. Fungal Ecol. 2016;20:241–8. 10.1016/j.funeco.2015.06.006. DOI

Novak  M, Buzek  F, Jackova  I  et al.  Isotope composition of dissolved organic carbon in runoff and peat leachates from a Central European wetland: temporal and spatial variability in DOC sources. Catena. 2019;173:217–25. 10.1016/j.catena.2018.10.011. DOI

Novak  M, Stepanova  M, Jackova  I  et al.  Isotopic evidence for nitrogen mobility in peat bogs. Geochim Cosmochim Acta. 2014;133:351–61. 10.1016/j.gca.2014.02.021. DOI

Novak  M, Zemanova  L, Buzek  F  et al.  The effect of a reciprocal peat transplant between two contrasting Central European sites on C cycling and C isotope ratios. Biogeosciences. 2010;7:921–32. 10.5194/bg-7-921-2010. DOI

Osono  T, Takeda  H.  Fungal decomposition of Abies needle and Betula leaf litter. Mycologia. 2006;98:172–9. 10.1080/15572536.2006.11832689. PubMed DOI

Oulehle  F, Fischer  M, Hruška  J  et al.  The GEOMON network of Czech catchments provides long-term insights into altered forest biogeochemistry: from acid atmospheric deposition to climate change. Hydrol Process. 2021;35. 10.1002/hyp.14204. DOI

Oulehle  F, Kopáček  J, Chuman  T  et al.  Predicting sulphur and nitrogen deposition using a simple statistical method. Atmos Environ. 2016;140:456–68. 10.1016/j.atmosenv.2016.06.028. DOI

Parks  DH, Tyson  GW, Hugenholtz  P  et al.  STAMP: statistical analysis of taxonomic and functional profiles. Bioinformatics. 2014;30:3123–4. 10.1093/bioinformatics/btu494. PubMed DOI PMC

Philippot  L, Hallin  S, Schloter  M.  Ecology of denitrifying prokaryotes in agricultural soil. Adv Agron. 2007;96:249–305.

Quast  C, Pruesse  E, Yilmaz  P  et al.  The SILVA ribosomal RNA gene database project: improved data processing and web-based tools. Nucleic Acids Res. 2012;41:D590–6. 10.1093/nar/gks1219. PubMed DOI PMC

R Core Team . R: A Language and Environment for Statistical Computing. Vienna: R Foundation for Statistical Computing, 2024.

Rich  JJ, Heichen  RS, Bottomley  PJ  et al.  Community composition and functioning of denitrifying bacteria from adjacent meadow and forest soils. Appl Environ Microbiol. 2003;69:5974–82. 10.1128/AEM.69.10.5974-5982.2003. PubMed DOI PMC

Rydin  H, Jeglum  JK.  The Biology of Peatlands. Oxford: Oxford University Press, 2013.

Saiz  E, Sgouridis  F, Drijfhout  FP  et al.  Chronic atmospheric reactive nitrogen deposition suppresses biological nitrogen fixation in peatlands. Environ Sci Technol. 2021;55:1310–8. 10.1021/acs.est.0c04882. PubMed DOI

Sgouridis  F, Yates  CA, Lloyd  CEM  et al.  Chronic atmospheric reactive N deposition has breached the N sink capacity of a northern ombrotrophic peatbog increasing the gaseous and fluvial N losses. Sci Total Environ. 2021;787:147552. 10.1016/j.scitotenv.2021.147552. PubMed DOI

Shabbir  S, Qian  C, Faheem  M  et al.  New insights into the spatial variability of microbial diversity and density in peatlands exposed to various electron acceptors with an emphasis on methanogenesis and CO PubMed DOI PMC

Sinsabaugh  RL, Carreiro  MM, Repert  DA.  Allocation of extracellular enzymatic activity in relation to litter composition, N deposition, and mass loss. Biogeochemistry. 2002;60:1–24. 10.1023/A:1016541114786. DOI

Sirová  D, Šantrůček  J, Adamec  L  et al.  Dinitrogen fixation associated with shoots of aquatic carnivorous plants: is it ecologically important?. Annals of Botany. 2014;114:125–33. 10.1093/aob/mcu067 PubMed DOI PMC

Song  L, Lu  H-Z, Xu  X-L  et al.  Organic nitrogen uptake is a significant contributor to nitrogen economy of subtropical epiphytic bryophytes. Sci Rep. 2016;6:30408. 10.1038/srep30408. PubMed DOI PMC

Stepanova  M, Novak  M, Cejkova  B  et al.  Contrasting potential for biological N DOI

Sutton  MA, Oenema  O, Erisman  JW  et al.  Too much of a good thing. Nature. 2011;472:159–61. 10.1038/472159a. PubMed DOI

Tedersoo  L, Bahram  M, Põlme  S  et al.  Global diversity and geography of soil fungi. Science. 2014;346. 10.1126/science.1256688. PubMed DOI

Too  CC, Keller  A, Sickel  W  et al.  Microbial community structure in a Malaysian tropical peat swamp forest: the influence of tree species and depth. Front Microbiol. 2018;9. 10.3389/fmicb.2018.02859. PubMed DOI PMC

Turetsky  MR, Benscoter  B, Page  S  et al.  Global vulnerability of peatlands to fire and carbon loss. Nat Geosci. 2015;8:11–4. 10.1038/ngeo2325. DOI

van den Elzen  E, Kox  MAR, Harpenslager  SF  et al.  Symbiosis revisited: phosphorus and acid buffering stimulate N

van den Elzen  E, van den Berg  LJL, van der Weijden  B  et al.  Effects of airborne ammonium and nitrate pollution strongly differ in peat bogs, but symbiotic nitrogen fixation remains unaffected. Sci Total Environ. 2018a;610–611:732–40. 10.1016/j.scitotenv.2017.08.102. PubMed DOI

Vile  MA, Kelman Wieder  R, Živković  T  et al.  N DOI

Vitousek  PM, Aber  JD, Howarth  RW  et al.  Human alteration of the global nitrogen cycle: sources and consequences. Ecol Appl. 1997;7:737–50.

Waldrop  MP, Zak  DR, Sinsabaugh  RL  et al.  Nitrogen deposition modifies soil carbon storage through changes in microbial enzymatic activity. Ecol Appl. 2004;14:1172–7. 10.1890/03-5120. DOI

Wang  C, Zheng  M, Song  W  et al.  Impact of 25 years of inorganic fertilization on diazotrophic abundance and community structure in an acidic soil in southern China. Soil Biol Biochem. 2017;113:240–9. 10.1016/j.soilbio.2017.06.019. DOI

Wang  Y, Lett  S, Rousk  K.  Too much of a good thing? Inorganic nitrogen (N) inhibits moss-associated N DOI

Widdig  M, Schleuss  P-M, Weig  AR  et al.  Nitrogen and phosphorus additions alter the abundance of phosphorus-solubilizing bacteria and phosphatase activity in grassland soils. Front Environ Sci. 2019;7. 10.3389/fenvs.2019.00185. DOI

Wille  EA, Lenhart  CF, Kolka  RK.  Carbon dioxide emissions in relation to water table in a restored fen. Agric Environ Lett. 2023;8. 10.1002/ael2.20112. DOI

Xiang  X, Wang  H, Tian  W  et al.  Composition and function of bacterial communities of bryophytes and their underlying sediments in the Dajiuhu Peatland, Central China. J Earth Sci. 2023;34:133–44. 10.1007/s12583-020-1391-x. DOI

Xiao  D, Tan  Y, Liu  X  et al.  Responses of soil diazotrophs to legume species and density in a Karst grassland, southwest China. Agric Ecosyst Environ. 2020;288:106707. 10.1016/j.agee.2019.106707. DOI

Yang  Q, Liu  Z, Bai  E.  Comparison of carbon and nitrogen accumulation rate between bog and fen phases in a pristine peatland with the fen-bog transition. Glob Chang Biol. 2023;29:6350–66. 10.1111/gcb.16915. PubMed DOI

Yayi  N, Yulong  D, Yuqiang  L  et al.  Soil microbial community responses to short-term nitrogen addition in China's Horqin Sandy Land. PLoS One. 2021;16:e0242643. 10.1371/journal.pone.0242643. PubMed DOI PMC

Zhang  W, Hu  Z, Audet  J  et al.  Effects of water table level and nitrogen deposition on methane and nitrous oxide emissions in an alpine peatland. Biogeosciences. 2022;19:5187–97. 10.5194/bg-19-5187-2022. DOI

Zhao  B, Zhuang  Q.  Nitrogen cycling feedback on carbon dynamics leads to greater CH DOI

Živković  T, Helbig  M, Moore  TR.  Seasonal and spatial variability of biological N DOI