Association of Novel and Highly Diverse Acid-Tolerant Denitrifiers with N2O Fluxes of an Acidic Fen

Wetlands are sources of denitrification-derived nitrous oxide (N₂O). Thus, the denitrifier community of an N₂O-emitting fen (pH 4.7 to 5.2) was investigated. N₂O was produced and consumed to subatmospheric concentrations in unsupplemented anoxic soil microcosms. Total cell counts and most probable numbers of denitrifiers approximated 10¹¹ cells·g_DW⁻¹ (where DW is dry weight) and 10⁸ cells·g_DW⁻¹, respectively, in both 0- to 10-cm and 30- to 40-cm depths. Despite this uniformity, depth-related maximum reaction rate (v_max) values for denitrification in anoxic microcosms ranged from 1 to 24 and −19 to −105 nmol N₂O h⁻¹· g_DW⁻¹, with maximal values occurring in the upper soil layers. Denitrification was enhanced by substrates that might be formed via fermentation in anoxic microzones of soil. N₂O approximated 40% of total nitrogenous gases produced at in situ pH, which was likewise the optimal pH for denitrification. Gene libraries of narG and nosZ (encoding nitrate reductase and nitrous oxide reductase, respectively) from fen soil DNA yielded 15 and 18 species-level operational taxonomic units, respectively, many of which displayed phylogenetic novelty and were not closely related to cultured organisms. Although statistical analyses of narG and nosZ sequences indicated that the upper 20 cm of soil contained the highest denitrifier diversity and species richness, terminal restriction fragment length polymorphism analyses of narG and nosZ revealed only minor differences in denitrifier community composition from a soil depth of 0 to 40 cm. The collective data indicate that the regional fen harbors novel, highly diverse, acid-tolerant denitrifier communities capable of complete denitrification and consumption of atmospheric N₂O at in situ pH.Nitrous oxide (N₂O) is a potent greenhouse gas with a global warming potential that is 300-fold higher than that of CO₂, and its concentration increased from 270 ppb in 1750 to 319 ppb in 2005 (17). N₂O can be produced in soils during denitrification, nitrification, the dissimilatory reduction of nitrate to nitrite and/or ammonium (hereafter referred to as dissimilatory nitrate reduction), or the chemical transformation of nitrite or hydroxylamine (5, 7, 49). The percentage of N₂O produced in any of these processes is variable, depending mainly on the redox potential, pH, and C/N ratio (49). In anoxic ecosystems such as waterlogged soils, most of the N₂O is considered to be denitrification derived (7, 9). Complete denitrification is the sequential reduction of nitrate to dinitrogen (N₂) via nitrite, nitric oxide (NO), and N₂O (75). The main product of denitrification varies with the organism and in situ conditions and is usually either N₂O or N₂ (68). N₂O can occur as a by-product during dissimilatory nitrate reduction when accumulated nitrite interacts with nitrate reductase to form N₂O (59). The production of N₂O by dissimilatory nitrate reducers is favored in environments with large amounts of readily available organic carbon (65). Thus, their contribution to nitrate-dependent production of N₂O in soils is likely insignificant compared to that of denitrifiers.The oxidoreductases involved in denitrification are termed dissimilatory nitrate reductase (Nar, encoded by narGHJI, or Nap, encoded by napEDABC), nitrite reductase (Nir, encoded by nirK and nirS), NO reductase (cNor and qNor, encoded by norBC and norB, respectively), and N₂O reductase (Nos, encoded by nosZ) (75). Nitrate reductase is also found in dissimilatory nitrate reducers (60). narG can therefore be used as a molecular marker to assess both denitrifiers and dissimilatory nitrate reducers, whereas nosZ is specific for the assessment of denitrifiers (25, 43, 48).Denitrification in soils is regulated by temperature, pH, substrate (i.e., carbon) availability, and water content (10, 24, 66). Although denitrification increases with increasing temperature, it can still occur at temperatures below 0°C (10, 24). Low temperatures appear to limit the activity of N₂O reductase more severely than other enzymes involved in denitrification and thus yield higher relative amounts of denitrification-derived N₂O (24). Although denitrification activity usually decreases under acidic conditions, the relative percentage of N₂O to total denitrification-derived nitrogenous gases increases with increasing acidity, a result attributed to the sensitivity of N₂O reductase to low pH (27, 70). However, denitrifier communities can be adapted to the in situ pH of the system (40, 58, 73).Wetlands are ecosystems in which denitrification is likely a dominant source of emitted N₂O (7, 44, 45). The identification and analysis of main drivers for N₂O production (i.e., the microbiota catalyzing N₂O production and consumption) is thus of major concern in such environments. Fens are specialized wetlands characterized by soil acidity (67). However, information on acid-tolerant denitrifier communities of such wetlands is scarce. It is hypothesized that fens harbor a diverse, hitherto unknown, denitrifier community that is adapted to in situ conditions and associated with N₂O fluxes (i.e., fen denitrifiers are acid tolerant and have a high affinity for nitrate and N₂O). Thus, the main objectives of the present study were to evaluate the capacities of denitrifier communities of an N₂O-emitting fen (20) to produce or consume N₂O and to determine if a novel and diverse denitrifier community was associated with these capacities.