Microbial Communities and Interactions of Nitrogen Oxides With Methanogenesis in Diverse Peatlands of the Amazon Basin

Abstract

Tropical peatlands are hotspots of methane (CH₄) production but present high variation and emission uncertainties in the Amazon region. This is because the controlling factors of methane production in tropical peats are not yet well documented. Although inhibitory effects of nitrogen oxides (NO _x ) on methanogenic activity are known from pure culture studies, the role of NO _x in the methane cycling of peatlands remains unexplored. Here, we investigated the CH₄ content, soil geochemistry and microbial communities along 1-m-soil profiles and assessed the effects of soil NO _x and nitrous oxide (N₂O) on methanogenic abundance and activity in three peatlands of the Pastaza-Marañón foreland basin. The peatlands were distinct in pH, DOC, nitrate pore water concentrations, C/N ratios of shallow soils, redox potential, and ¹³C enrichment in dissolved inorganic carbon and CH₄ pools, which are primarily contingent on H₂-dependent methanogenesis. Molecular 16S rRNA and mcrA gene data revealed diverse and novel methanogens varying across sites. Importantly, we also observed a strong stratification in relative abundances of microbial groups involved in NO _x cycling, along with a concordant stratification of methanogens. The higher relative abundance of ammonia-oxidizing archaea (Thaumarchaeota) in acidic oligotrophic peat than ammonia-oxidizing bacteria (Nitrospira) is noteworthy as putative sources of NO _x . Experiments testing the interaction of NO _x species and methanogenesis found that the latter showed differential sensitivity to nitrite (up to 85% reduction) and N₂O (complete inhibition), which would act as an unaccounted CH₄ control in these ecosystems. Overall, we present evidence of diverse peatlands likely differently affected by inhibitory effects of nitrogen species on methanogens as another contributor to variable CH₄ fluxes.

Keywords: Amazon peatlands; methanogens; microbial communities and interactions; nitrogen oxides; peat geochemistry.

FIGURE 1

Levels of carbon and nitrogen along soil profiles of three tropical peatlands (QUI, SJO, BVA). Two separately sampled (duplicate) profiles from Quistococha (QUI), San Jorge (SJO), and Buena Vista (BVA) were each set up to sample pore water and soil gas. Ions, total carbon and nitrogen abundances, and DOC were determined from pore water. Gas concentrations were measured in equilibrated gas samples. Liquid samples in BVA were not collected below 60 cm due to blocked sampling paths by a dense network of roots.

FIGURE 2

16S rRNA gene relative abundance heatmap of archaeal (left) and bacterial (right) taxa with known nitrogen metabolic potential. Archaeal 16S rRNA gene relative abundances were summarized on the phylum level with Bathyarchaeota, Euryarchaeota, and Thaumarchaeota consistently making up >90% of all archaeal taxa. Only bacterial taxa at the family level that were either most dominant (at least 2% of total OTUs), had the potential of a syntrophic relationship to methanogens, or were affiliated to known metabolic reactions involving NO_x are depicted. Confirmed metabolic capacities to produce NO_x, consume NO_x, or for a syntrophic lifestyle are marked with closed circles. Hypothesized metabolic capacities (unsupported by pure cultures) are marked with open circles. The number of unassigned sequences (no match in the SILVA database) increased notably in deeper layers. Relative abundances for individual cores and bacterial metabolic capacities at the family level are provided in Supplementary Table 1.

FIGURE 3

Redox gradient and carbon isotope composition of CH₄ and dissolved inorganic carbon along soil profiles of three tropical peatlands (BVA, QUI, SJO). Redox potential (E_h) is based on the ratio of the CH₄-CO₂ redox couple and soil pH-corrected. VPDB, Vienna Pee Dee Belemnite. Each data point corresponds to one replicate soil core.

FIGURE 4

McrA/archaeal 16S rRNA gene copy ratios along soil depth (left) and mcrA phylogeneticphylogenetic distribution of cumulative reads at all levels (right) in each peatland. Gene copy ratios are based on qPCR analysis and data from both soil cores are shown (dark and light gray lines). The gene ratios were used to better illustrate methanogen proportions relative to the total number of archaea, which explicitly revealed differences between peatlands. Absolute gene quantities ranged from 1.3 × 10⁴ (SJO) to 1.8 × 10⁹ (BVA) mcrA gene copies per g of dry soil and 8.7 × 10⁵ (SJO) to 7.3 × 10¹¹ (QUI) archaeal 16S rRNA gene copies per g of dry soil. Relative abundance percentages refer to the total number of OTUs recovered at each site. Higher level taxa were collapsed if these were comprised of 100% the lower-level taxa. Mr., Methanoregulaceae; Mst., Methanosaetaceae; Msr., Methanosarcinaceae; Mc., Methanocellaceae; Mb., Methanobacteriaceae; Mmc., Methanomassiliicoccaceae.

FIGURE 5

PCA ordination plots of microbial and environmental data from three soil profiles of contrasting Amazon peatlands. Samples were derived from QUI (red triangles), SJO (green circles) and BVA (blue squares) with abbreviations as in Figure 1. Methane concentration ([CH₄]), 16S rRNA gene OTU relative abundances, and mcrA gene copy number (A), and Bathyarchaeota alpha (Shannon) and beta (Unifrac) diversity indices, NO_x concentrations ([NO₂^–], [NO₃^–]) and pH (B) were evaluated against microbial distribution. We used Bathyarchaeota abundance and diversity indices as a not yet physiologically resolved close archaeal group important to evaluate but not as assumed methanogens.

FIGURE 6

Effect of NO₂^– amendment on methanogenic activity in peat soil incubated under anoxic conditions (A) and effect of N₂O on enrichment cultures (B). (A) For each site, two shallow (0–15 cm) and one deep (15–30 cm) soil slurry samples were incubated in triplicates. Residual activity denotes fractional CH₄ production relative to controls without additions. Dotted line for SJO’s deep soil denotes CH₄ levels below detection limit. Levels not reporting the same letter are significantly different (Student’s t-test, p = 0.05). (B) Nitrous oxide (full symbols) and CH₄ (open symbols) dynamics in enrichment cultures from SJO and BVA. Units are nmol per incubation vessel. Methane concentrations in SJO remained below detection throughout the illustrated incubation period. Gray arrowheads denote N₂O addition time. Error bars represent one standard deviation.