Burkholderiaceae Are Key Acetate Assimilators During Complete Denitrification in Acidic Cryoturbated Peat Circles of the Arctic Tundra

Abstract

Cryoturbated peat circles (pH 4) in the Eastern European Tundra harbor up to 2 mM pore water nitrate and emit the greenhouse gas N₂O like heavily fertilized agricultural soils in temperate regions. The main process yielding N₂O under oxygen limited conditions is denitrification, which is the sequential reduction of nitrate/nitrite to N₂O and/or N₂. N₂O reduction to N₂ is impaired by pH < 6 in classical model denitrifiers and many environments. Key microbes of peat circles are important but largely unknown catalysts for C- and N-cycling associated N₂O fluxes. Thus, we hypothesized that the peat circle community includes hitherto unknown taxa and is essentially unable to efficiently perform complete denitrification, i.e., reduce N₂O, due to a low in situ pH. 16S rRNA analysis indicated a diverse active community primarily composed of the bacterial class-level taxa Alphaproteobacteria, Acidimicrobiia, Acidobacteria, Verrucomicrobiae, and Bacteroidia, as well as archaeal Nitrososphaeria. Euryarchaeota were not detected. ¹³C₂- and ¹²C₂-acetate supplemented anoxic microcosms with endogenous nitrate and acetylene at an in situ near pH of 4 were used to assess acetate dependent carbon flow, denitrification and N₂O production. Initial nitrate and acetate were consumed within 6 and 11 days, respectively, and primarily converted to CO₂ and N₂, suggesting complete acetate fueled denitrification at acidic pH. Stable isotope probing coupled to 16S rRNA analysis via Illumina MiSeq amplicon sequencing identified acetate consuming key players of the family Burkholderiaceae during complete denitrification correlating with Rhodanobacter spp. The archaeal community consisted primarily of ammonia-oxidizing Archaea of Nitrososphaeraceae, and was stable during the incubation. The collective data indicate that peat circles (i) host acid-tolerant denitrifiers capable of complete denitrification at pH 4-5.5, (ii) other parameters like carbon availability rather than pH are possible reasons for high N₂O emissions in situ, and (iii) Burkholderiaceae are responsive key acetate assimilators co-occurring with Rhodanobacter sp. during denitrification, suggesting both organisms being associated with acid-tolerant denitrification in peat circles.

Keywords: 16S rRNA stable isotope probing; climatechange; isotope tracing; nitrous oxide; permafrost affected soils.

FIGURE 1

Acetate (A,C) and nitrate (E,G) consumption concomitant to CO₂ (B,D) and N₂O (F,H) production in anoxic incubations of acetate supplemented cryoturbated peat circle sediments without (A,B,E,F) and with (C,D,G,H) acetylene. pH is shown in panels (F) and (H). Arrows indicate time of acetate or nitrate supplementation. Shaded and non-shaded areas highlight different periods of nitrate consumption, and lines above the graphs indicate phases (see Table 2). (A–D) show ¹³C-acetate treatments only. For details on acetate and CO₂ in ¹²C-acetate treatments, please refer to Supplementary Figure S1. (E–H) include data of ¹³C- and ¹²C-acetate treatments as indicated in the insert. 59C-acetate, unlabeled ¹²C₂-acetate; 60C-acetate, half-labeled ¹³C₁-acetate; and 61C-acetate, fully labeled ¹³C₂-acetate. Values are means ± standard deviation of triplicate incubations except for isotope analyses where 1-3 values were obtained (see text for details).

FIGURE 2

Mean relative abundance of bacterial families (>2% of relative abundance in at least one sample, A) delineated from analysis of density resolved 16S rRNA sequences retrieved from cryoturbated peat circle sediments prior to and after 16 days of anoxic incubation (see Figure 1). Values are means of triplicate incubations. Heavy (H) and Light (L) indicate fractions representing relative abundances of ¹³C-labeled and unlabeled 16S rRNA sequences after isopycnic centrifugation (see section “Materials and Methods” for further details) where sequences have been retrieved from. ¹³C- and ¹²C-acetate refer to treatments with ¹³C- and ¹²C-acetate, respectively. t₀ indicates peat circle sediments prior to incubation. Relative abundances in amplicon libraries of archaeal family (B) and genus (C) level taxa in all samples are displayed. Please note that Log₁₀ of sequence counts per genus are given to highlight rare genera (C). For a more detailed overview of archaeal taxa see Supplementary Figure S7.

FIGURE 3

Principal coordinates analysis (PCoA; A) based onBray–Curtis dissimilarity calculated from relative abundance data of bacterial species-level OTUs from density resolved 16S rRNA sequences retrieved from cryoturbated peat circle sediments prior to (t₀) and after 16 days of anoxic incubation (see Figure 1). Hierarchical heat map clustering is presented in panel (B). Color code indicates levels of dissimilarity. Sample code: H and L indicate heavy and light fractions, respectively; t₀, ¹³C- and ¹²C- represent peat circle sediments prior to incubation, ¹³C- and ¹²C-acetate treatments, respectively. Numbers are indicative of replicate. Heavy fractions of treatemnts with labelled acetate are printed in bold.

FIGURE 4

Genus-level taxa with significantly different relative abundances (p < 0.05) in heavy and light fractions of ¹³C-acetate treatments only, i.e., such taxa were not differentially abundant in heavy than light fractions at t₀ or ¹²C-acetate treatments.

FIGURE 5

Correlation network analysis of density resolved 16S rRNA sequences retrieved from cryoturbated peat circle sediment incubations (see Figure 1) using the SparCC algorithm (A). Nodes represent genus-level taxa, and red and blue edges represent positive and negative correlations of labeled OTU 1 (Burkholderia-Caballeronia-Paraburkholderia). For more details on correlations please refer to Supplementary Table S1. Node size reflects taxon abundance and colors indicate the relative proportion per sample. The box plots (B,C) show the abundances of Burkholderia-Caballeronia-Paraburkholderia (B) and of one positively correlated taxon (Rhodanobacter spp.; C). Sample code: H and L indicate heavy and light fractions, respectively; 0, ¹³ C-, and ¹²C- represent peat circle sediments prior to incubation, ¹³C- and ¹²C-acetate treatments, respectively.