Biological nitrogen fixation at four Sphagnum-dominated peatlands in the Bohemian Massif: Spatial and temporal variability based on 15N2 moss incubation experiments and microbial community analysis

Accumulation of carbon and nitrogen in freshwater wetlands is affected by climatic change. Elevated temperatures and changes in precipitation patterns may lead to degradation and thinning of peat deposits resulting from higher rates of biogenic emissions of greenhouse gases. Higher atmospheric concentrations of greenhouse gases, mainly CO₂, CH₄, and N₂O, may then accelerate global warming. A number of recent studies have addressed the relationship between increasing deposition of reactive nitrogen (N_r, predominantly ammonium and nitrate)and stability of the N stock in peat. Under high atmospheric N_r inputs, peatlands may become a net source of N, rather than a net sink. Nitrogen inventories in ombrotrophic bogs may be influenced by biological N₂-fixation (BNF), the conversion of atmospheric molecular N₂ by diazotrophic microorganisms to bioavailable NH₄⁺. Because a high energy is required to break the triple bond in the N₂ molecule, microbial N₂-fixation may shut off when other N_r sources are available. To verify this assumption, we studied four Sphagnum-dominated peat bogs in the Czech Republic differing in N_r deposition by a factor of two. We hypothesized that the more N_r-polluted sites in the north, Velke jerabi jezero (VJ) and Cerny potok (CP), would exhibit lower BNF rates than the less polluted sites in the south, Cervene blato (CB) and Zdarecka slat (ZS). At the end of laboratory incubations of waterlogged peat in a ¹⁵N₂ atmosphere (t = 2-7 days), samples of living Sphagnum exhibited an increase in d¹⁵N values from about -3 ‰ typical of all sites to 27, 259, and 266 ‰ at VJ, CP and ZS, respectively. No significant change in d¹⁵N values was recorded at CB. At VJ, the estimated BNF rates reached 840 ng N per gram Sphagnum per day. At CP and ZS these rates were about 10 times higher. Microbial analysis revealed higher activity of diazotrophs at VJ than at CB. At VJ, autotrophic cyanobacterial diazotrophs of the Nosctocaceae family comprised 4.2 %, while at CB they were below 0.1 %. Repeated sampling at CP and ZS in spring and summer showed complex temporal trends in d¹⁵N shifts during ¹⁵N₂ moss incubations. At CP, the d¹⁵N shift and BNF rates were larger in summer, whereas at ZS, a larger d¹⁵N shift and higher BNF rates were observed in spring. Vertical trends in d¹⁵N values at the end of the ¹⁵N₂ incubations were also complicated. Out of four peat sampling depths (0, 10, 20 and 30 cm), the highest positive d¹⁵N shifts during incubation experiments were found 20 cm below surface at CP (561 ‰; spring) and 10 cm below surface at ZS (805 ‰; summer). Collectively, our data indicate that atmospheric N_r inputs were not the main control of BNF in the studied Central European peat bogs. Also large within-site and seasonal variability in BNF was observed. Other site characteristics, such as phosphorus availability, NH₄⁺/NO₃^- ratios, and moisture conditions served as important BNF drivers.