Molybdenum-Based Diazotrophy in a Sphagnum Peatland in Northern Minnesota

Abstract

Microbial N₂ fixation (diazotrophy) represents an important nitrogen source to oligotrophic peatland ecosystems, which are important sinks for atmospheric CO₂ and are susceptible to the changing climate. The objectives of this study were (i) to determine the active microbial group and type of nitrogenase mediating diazotrophy in an ombrotrophic Sphagnum-dominated peat bog (the S1 peat bog, Marcell Experimental Forest, Minnesota, USA); and (ii) to determine the effect of environmental parameters (light, O₂, CO₂, and CH₄) on potential rates of diazotrophy measured by acetylene (C₂H₂) reduction and ¹⁵N₂ incorporation. A molecular analysis of metabolically active microbial communities suggested that diazotrophy in surface peat was primarily mediated by Alphaproteobacteria (Bradyrhizobiaceae and Beijerinckiaceae). Despite higher concentrations of dissolved vanadium ([V] 11 nM) than molybdenum ([Mo] 3 nM) in surface peat, a combination of metagenomic, amplicon sequencing, and activity measurements indicated that Mo-containing nitrogenases dominate over the V-containing form. Acetylene reduction was only detected in surface peat exposed to light, with the highest rates observed in peat collected from hollows with the highest water contents. Incorporation of ¹⁵N₂ was suppressed 90% by O₂ and 55% by C₂H₂ and was unaffected by CH₄ and CO₂ amendments. These results suggest that peatland diazotrophy is mediated by a combination of C₂H₂-sensitive and C₂H₂-insensitive microbes that are more active at low concentrations of O₂ and show similar activity at high and low concentrations of CH₄ IMPORTANCE Previous studies indicate that diazotrophy provides an important nitrogen source and is linked to methanotrophy in Sphagnum-dominated peatlands. However, the environmental controls and enzymatic pathways of peatland diazotrophy, as well as the metabolically active microbial populations that catalyze this process, remain in question. Our findings indicate that oxygen levels and photosynthetic activity override low nutrient availability in limiting diazotrophy and that members of the Alphaproteobacteria (Rhizobiales) catalyze this process at the bog surface using the molybdenum-based form of the nitrogenase enzyme.

Keywords: Alphaproteobacteria; Sphagnum; acetylene; diazotrophy; methanotrophs; molybdenum; nitrogen cycle enzymes; nitrogen fixation; peatland; vanadium.

FIG 1

Depth profiles of NH₄⁺ concentrations (black circles) and nifH gene copies (white squares) and transcripts (white triangle) in units of copies per gram (dry weight) for the S1 bog T3 middle site. Ammonium concentrations are means from measurements from May, June, and September 2014. nifH copy numbers are from July 2013. Error bars are standard errors. nifH transcripts were not detected at 20-, 30-, and 75-cm depths. Surface (0 to 10 cm) and deep (10 to 30 cm) peat depth intervals used for rate measurements are designated by dashed lines. The maximum water table depth at the S1 site in July 2013 was 2 cm below the hollow surface (dotted line) (97).

FIG 2

Dissolved molybdenum (white) and vanadium (gray) concentrations in porewater from three depths in S1 peat hollows (middle and far sites along T3 transect) from June 2015. Pie charts show the relative abundances of genes encoding the five nitrogenase H subunit clusters from metagenomes for each depth; clusters I and III encode Mo-Fe nitrogenases (nifH); cluster II encodes alternative (vnfH, anfH) nitrogenases; cluster IV encodes nitrogenase paralogs. Deepest metagenomes were from 75 cm; insufficient numbers of nitrogenase H subunit sequences were recovered from the far site for cluster analysis.

FIG 3

Numbers of cDNA amplicon sequence reads for nifH and nifD alphaproteobacterial transcripts. Primer sets were polF/polR and nifD820F/nifD1331R for nifH and nifD, respectively.

FIG 4

Acetylene reduction rates for hummocks (90 to 91% water content) and hollows (93 to 96% water content) at the S1 bog, T3 transect (0- to 10-cm depth incubated in the light at 25°C for 7 days). ARA units are μmol ethylene produced per gram (dry weight) per hour. Photo insets show dominant Sphagnum species in hummocks (S. magellanicum) (a) and hollows (S. fallax and S. angustifolium) (b). Photo inset (c) shows Sphagnum greening after incubation of hollow samples in the light for 7 days at 25°C; bottle B2* (April 2014) received air headspace with 1% CH₄ and D2 (Sept 2013) received air headspace without CH₄. The vertical dotted line divides the hummock samples (dominated by S. magellanicum) from the hollow samples (dominated by S. fallax/angustifolium) in terms of water content (92%).

FIG 5

Effects of 1% C₂H₂ on¹⁵N₂ incorporation and CH₄ consumption for S1 bog surface peat. Rates were measured for samples collected from the northwest (NW) S1 bog transect in September 2014. ¹⁵N₂ incorporation treatment conditions were 80% N₂ plus 20% CO₂ with and without 1% CH₄ (shaded bars) or 80% N₂ plus 20% O₂ with and without 1% CH₄ (white bars) with (a) and without (b) 1% C₂H₂; units are nmol ¹⁵N₂ incorporated per gram (dry weight) per hour. CH₄ consumption treatments were 80% N₂ plus 20% CO₂ plus 1% CH₄ (black circles) with (a) and without (b) 1% C₂H₂; units are nmol CH₄ consumed per gram (dry weight) per hour. Error bars are standard errors. Lowercase letters indicate statistically different elemental contents (P < 0.05 based on Tukey-Kramer HSD test).