Actinobacterial nitrate reducers and proteobacterial denitrifiers are abundant in N2O-metabolizing palsa peat

Abstract

Palsa peats are characterized by elevated, circular frost heaves (peat soil on top of a permanently frozen ice lens) and are strong to moderate sources or even temporary sinks for the greenhouse gas nitrous oxide (N(2)O). Palsa peats are predicted to react sensitively to global warming. The acidic palsa peat Skalluvaara (approximate pH 4.4) is located in the discontinuous permafrost zone in northwestern Finnish Lapland. In situ N(2)O fluxes were spatially variable, ranging from 0.01 to -0.02 μmol of N(2)O m(-2) h(-1). Fertilization with nitrate stimulated in situ N(2)O emissions and N(2)O production in anoxic microcosms without apparent delay. N(2)O was subsequently consumed in microcosms. Maximal reaction velocities (v(max)) of nitrate-dependent denitrification approximated 3 and 1 nmol of N(2)O per h per gram (dry weight [g(DW)]) in soil from 0 to 20 cm and below 20 cm of depth, respectively. v(max) values of nitrite-dependent denitrification were 2- to 5-fold higher than the v(max) nitrate-dependent denitrification, and v(max) of N(2)O consumption was 1- to 6-fold higher than that of nitrite-dependent denitrification, highlighting a high N(2)O consumption potential. Up to 12 species-level operational taxonomic units (OTUs) of narG, nirK and nirS, and nosZ were retrieved. Detected OTUs suggested the presence of diverse uncultured soil denitrifiers and dissimilatory nitrate reducers, hitherto undetected species, as well as Actino-, Alpha-, and Betaproteobacteria. Copy numbers of nirS always outnumbered those of nirK by 2 orders of magnitude. Copy numbers of nirS tended to be higher, while copy numbers of narG and nosZ tended to be lower in 0- to 20-cm soil than in soil below 20 cm. The collective data suggest that (i) the source and sink functions of palsa peat soils for N(2)O are associated with denitrification, (ii) actinobacterial nitrate reducers and nirS-type and nosZ-harboring proteobacterial denitrifiers are important players, and (iii) acidic soils like palsa peats represent reservoirs of diverse acid-tolerant denitrifiers associated with N(2)O fluxes.

Fig 1

Effect of nitrate and ammonium fertilization on in situ fluxes of N₂O from palsa peat soil within 3 h following fertilization (see Materials and Methods for details). Mean values and standard errors of four replicates are shown for unfertilized controls (C) and for treatments fertilized with a solution of 20 mM NaNO₃ (N) or 20 mM NH₄Cl (A).

Fig 2

Production and consumption of N₂O by unsupplemented palsa peat soil. Squares and circles represent 0- to 20-cm and below-20-cm palsa peat soils, respectively. Closed and open symbols represent microcosms with and without acetylene, respectively. Mean values and standard errors of three replicates are shown. Time points at which N₂O concentrations in below-20-cm soil with acetylene differed significantly (P < 0.05) from N₂O concentrations in below-20-cm soil without acetylene (*) or in 0- to 20-cm palsa peat soil with acetylene (+) are indicated. The horizontal line indicates the atmospheric N₂O concentration (319 ppb).

Fig 3

Apparent Michaelis-Menten kinetics of nitrate-dependent (A) and nitrite-dependent (B) N₂O production in the presence of acetylene and N₂O consumption (C) in anoxic palsa peat soil microcosms. Squares, circles, and triangles represent microcosms supplemented with nitrate, nitrite, and N₂O, respectively. Closed and open symbols represent 0- to 20-cm and below-20-cm palsa peat soils, respectively. The x axis displays the amount of supplemented (i.e., additional) nitrate, nitrite, or N₂O. Mean values and standard errors of three replicate microcosms are shown. Solid and dashed lines are indicative of 0- to 20-cm and below-20-cm palsa peat soils, respectively. Curves represent Michaelis-Menten fits of the data. Horizontal and vertical lines represent v_max and K_m values, respectively. Letters indicate the following values: (i) nitrate-dependent v_max (a, 2.75; b, 1.4) and K_m (c, 8.1; d, 17.3), (ii) nitrite-dependent v_max (e, 14.6; f, 3.0) and K_m (g, 12.3; h, 39.1), and (iii) N₂O-dependent v_max (i, −17.0; j, −18.9) and K_m (k, 2.5; l, 4.0).

Fig 4

Relative abundances of OTUs derived from narG forward reads (A), nirK (B), nirS (C), and nosZ forward reads (D) retrieved from palsa peat soil at species level (left bars) and at 3% (right bars) threshold distances from rarified data sets. OTUs were rarified at sampling depths of 1,000, 3,000, 80, and 1,000 for narG, nirK, nirS, and nosZ, respectively. OTUs called at species-level threshold distances were enumerated 1 through 10; subclusters of those OTUs called at 3% threshold distance were enumerated 1.1 through 6.1. All OTUs that had relative abundances below 2% in both layers were grouped. Please note that the same color coding for different structural genes does not indicate whether such genes were derived from the same organisms.