Methanogens Are Major Contributors to Nitrogen Fixation in Soils of the Florida Everglades

Abstract

The objective of this study was to investigate the interaction of the nitrogen (N) cycle with methane production in the Florida Everglades, a large freshwater wetland. This study provides an initial analysis of the distribution and expression of N-cycling genes in Water Conservation Area 2A (WCA-2A), a section of the marsh that underwent phosphorus (P) loading for many years due to runoff from upstream agricultural activities. The elevated P resulted in increased primary productivity and an N limitation in P-enriched areas. Results from quantitative real-time PCR (qPCR) analyses indicated that the N cycle in WCA-2A was dominated by nifH and nirK/S, with an increasing trend in copy numbers in P-impacted sites. Many nifH sequences (6 to 44% of the total) and nifH transcript sequences (2 to 49%) clustered with the methanogenic Euryarchaeota, in stark contrast to the proportion of core gene sequences representing Archaea (≤0.27% of SSU rRNA genes) for the WCA-2A microbiota. Notably, archaeal nifH gene transcripts were detected at all sites and comprised a significant proportion of total nifH transcripts obtained from the unimpacted site, indicating that methanogens are actively fixing N₂ Laboratory incubations with soils taken from WCA-2A produced nifH transcripts with the production of methane from H₂ plus CO₂ and acetate as electron donors and carbon sources. Methanogenic N₂ fixation is likely to be an important, although largely unrecognized, route through which fixed nitrogen enters the anoxic soils of the Everglades and may have significant relevance regarding methane production in wetlands.IMPORTANCE Wetlands are the most important natural sources of the greenhouse gas methane, and much of that methane emanates from (sub)tropical peatlands. Primary productivity in these peatlands is frequently limited by the availability of nitrogen or phosphorus; however, the response to nutrient limitations of microbial communities that control biogeochemical cycling critical to ecosystem function may be complex and may be associated with a range of processes, including methane production. We show that many, if not most, of the methanogens in the peatlands of the Florida Everglades possess the nifH gene and actively express it for N₂ fixation coupled with methanogenesis. These findings indicate that archaeal N₂ fixation would play crucial role in methane emissions and overall N cycle in subtropical wetlands suffering N limitation.

Keywords: Everglades; methanogenesis; methanogens; nifH gene; nitrogen cycle; nitrogen fixation.

FIG 1

Box-and-whisker plots (top) and temporal profiles (bottom) of N-cycling gene copy numbers measured in WCA-2A soils collected in April 2010, August 2012, and December 2012 (from the left in temporal profiles). Error bars in the bar graphs (bottom) represent 1 ± standard error (SE; n = 3). Box-and-whisker plots were constructed from the data pool of temporal profiles. The different letters indicate a significant difference among the N-cycling genes (P < 0.05 by the Tukey-Kramer HSD test).

FIG 2

Maximum likelihood (ML) tree showing phylogenetic affiliation of NifH OTUs defined from translated nifH and nifH transcript sequences obtained from WCA-2A soils. a,g-Proteobacteria, Alpha- and Gammaproteobacteria; e-Proteobacteria, Epsilonproteobacteria; g,b-Proteobacteria, Gamma- and Betaproteobacteria; d-Proteobacteria, Deltaproteobacteria.

FIG 3

Composition and relatedness of N₂-fixing assemblages determined using nifH or nifH transcript sequences obtained from WCA-2A soils. (A) Relative abundance of individual taxa within each sample. (B) Percentage of methanogen-like N₂ fixers detected within individual samples. (C) PCoA biplot showing the relatedness between N₂-fixing communities occurring from individual samples. The soil samples used for determination of nifH gene and nifH transcript sequences are denoted within each panel. a-, b-, d-, e-, and g-Prot., Alpha-, Beta-, Delta-, Epsilon-, and Gammaproteobacteria, respectively; Clost., Clostridia; Euryarch., Euryarchaeota; Verruco., Verrucomicrobia.

FIG 4

Nitrogenase activities (top) and methane production (bottom) in incubation with WCA-2A soils. The soil was supplemented with 4 mM acetate (Act.) or H₂+CO₂ (80:20 [vol/vol]) (H₂) or was a control with no addition of substrate (CT). Soil used for this incubation was sampled in August 2012. The sampling days 3, 6, and 11 for determination of CH₄ production and nitrogenase activities are denoted in the x axis of the bottom graph. Error bars represent ±1 standard deviation (SD) from three replicates.

FIG 5

Composition and relatedness between nifH transcripts in the soil incubations. (A) Composition of nifH transcript in incubation with H₂+CO₂ or cellulose. (B) Percentage of methanogen-like nifH transcript sequences. (C) PCoA biplot representing relatedness between nifH transcript assemblages in incubations, which were also compared with those from field soils. For definitions of the abbreviations in panel A, see the legend to Fig. 3.

FIG 6

Nitrogenase activities (top) and methane production (bottom) in the incubation of WCA-2A soils sampled in January 2012. The soil incubation was supplemented with cellulose along with (+NH₄⁺) or without (−NH₄⁺) ammonium chloride (100 mM). The incubations without cellulose additions were used as the control treatment (CT). Methane concentrations and acetylene reduction (ARA) were determined for days 7 and 14 and are denoted on the x axis of the bottom graph. Error bars represent ±1 SD from three replicates. nd, not determined.