Functional capacities of microbial communities to carry out large scale geochemical processes are maintained during ex situ anaerobic incubation

Abstract

Mechanisms controlling CO2 and CH4 production in wetlands are central to understanding carbon cycling and greenhouse gas exchange. However, the volatility of these respiration products complicates quantifying their rates of production in the field. Attempts to circumvent the challenges through closed system incubations, from which gases cannot escape, have been used to investigate bulk in situ geochemistry. Efforts towards mapping mechanistic linkages between geochemistry and microbiology have raised concern regarding sampling and incubation-induced perturbations. Microorganisms are impacted by oxygen exposure, increased temperatures and accumulation of metabolic products during handling, storage, and incubation. We probed the extent of these perturbations, and their influence on incubation results, using high-resolution geochemical and microbial gene-based community profiling of anaerobically incubated material from three wetland habitats across a permafrost peatland. We compared the original field samples to the material anaerobically incubated over 50 days. Bulk geochemistry and phylum-level microbiota in incubations largely reflected field observations, but divergence between field and incubations occurred in both geochemistry and lineage-level microbial composition when examined at closer resolution. Despite the changes in representative lineages over time, inferred metabolic function with regards to carbon cycling largely reproduced field results suggesting functional consistency. Habitat differences among the source materials remained the largest driver of variation in geochemical and microbial differences among the samples in both incubations and field results. While incubations may have limited usefulness for identifying specific mechanisms, they remain a viable tool for probing bulk-scale questions related to anaerobic C cycling, including CO2 and CH4 dynamics.

Fig 1

CO₂ (panel a) and CH₄ (panel b) production in the incubations. Linear regression analyses were used to calculate production rates in the incubations over time. The results for significant linear regressions are given in each panel.

Fig 2

Alpha compared across habitats in the field (panel a) and and in the incubations averaged over 0–5 days (panel b), 20–30 days (panel c), 45–55 days (panel d), 70–80 days (panel e), and 90–100 days (panel f) throughout the incubation for each habitat. The first panel contains significance results of anova comparing the field and timepoints for each habitat. Neither depth of the collapsed palsas or BogD field were significantly different from any time point in the respective incubations. FenD was not significantly different until the end (day 75) of the incubations. BogS and FenS were the only sites that were significantly different from the incubations at all time points.

Fig 3. Optical measurements of bulk DOM.

Individual field samples for each parameter appear in the shaded gray region on the left of each panel. Individual incubation samples are plotted against time in the white region of each plot. Both peat depths for each habitat (except fen which was not sample) are included, S indicates the 8–18 cm deep peat and D indicates the 28–38 cm deep peat. Text in each panel presents the significant results from statistical comparison of field vs. day 0 samples. Panel a) Spectral slope ratio of UV/vis results. Panel b) Specific UV absorbance at 254 (SUVA₂₅₄; Tfaily et al. 2013) from UV-Vis analysis. Panel c) through f) provides ratios of EEMS peaks based on canonical peak areas (Coble 1996). Panel c) indicates the peak C:peak M ratio which provides a measure of diagenetic alteration. Panel d) indicates the peak C:peak T ratio which provides a measure of humic-like vs. fresh organic matter. Panel e) indicates the peak C:peak A ratio which provides a measure of humic-like versus fluvic like OM. Panel f) indicates the peak A:peak T ratio which indicates fulvic-like vs. fresh organic matter.

Fig 4

van Krevelen diagrams (H/C vs O/C ratios) of FTICRMS identified compounds for each site/depth: shallow collapsed palsa (panel a), shallow bog (panel b), shallow fen (panel c), deep collapsed palsa (panel d), and deep bog (panel e). Circles indicate compounds that are unique to the field samples for each site/depth. Triangles indicate compounds that are unique to the incubation samples for each site/depth. Venn diagrams to the right of each graph indicate the total number of compounds in the field, in the incubations, and the number of compounds that are found in both for each site/depth. The percentage indicates the percent of compounds from the field that were also found in the incubations. Panel (f) contains venn diagrams illustrating the number of samples in the shallow and deep field samples, as well as the percent of shallow field compounds that were also observed in the deep samples for each site/depth.

Fig 5. DOM characteristics identified by FTICRMS.

In panels a-d: field results are outlined by dashed boxes and plotted preceding day 0 and day 50 of incubation. Text indicates results of a t-test comparing field and day 0 incubation samples. Symbols indicate average for each characteristic; inverted triangles are the 8–18 cm samples and circles are the 28–38 cm samples from each site. Panel a) denotes the nominal oxidation state of carbon (NOSC), panel b) denotes the double bond equivalence, panel c) denotes the oxygen to carbon ratio, and panel d) denotes aromaticity index.

Fig 6

Top five transforms by percentage for each habitat: collapsed palsa (panel a), bog (panel b), and fen (panel c). Squares indicate means of replicates of the shallow field, deep field, shallow incubations and deep incubations results respectively, with whiskers denoting 1 standard deviation. Counts for all transforms identified in each sample are provided in S2 Table.

Fig 7. Solid-phase peat chemical functional group composition comparison for surface samples.

FTIR results showing the baseline-corrected peak areas of carbohydrates (1030 cm^-1, panel a), lignin-like aromatics (1510 cm^-1, panel c), aromatics and deprotonated carboxylic acids (1630 cm^-1, panel b), organic acids (1720 cm^-1, panel d), and lipids (2850 cm^-1, 2920 cm^-1, panel e and f, respectively) as percentages of total spectral area. Day 0 of the incubations was not different from the field for any parameter (Anova, p > 0.05). The different depth peat are indicated by symbol shape, shallow peat are circles, and inverted triangles are for the deep peat incubations.

Fig 8. The cell concentrations (by copy-corrected 16S rRNA counts; and per ul of extract) in field and incubation samples, by quantitative polymerase chain reaction.

Two replicates were averaged at each time point and error bars represent standard deviation. ‘Setup’ denotes post-storage, at the establishment of pre-incubations. 0, 25 and 50 refer to the elapsed incubation day. Matched field samples of identical depth range were missing for PHB.

Fig 9. Alpha diversity metrics of microbial communities at field collection, at incubation setup (post-storage), and over incubation time within each habitat/depth.

An asterisk indicates a significant difference (p-value < 0.05) between the field sample and the average of the incubation (including the incubation setup) samples. The significance of these differences is reported in S4 Table.

Fig 10. Relative abundances of microbial phyla in field and incubation samples.

Relative abundances were averaged between duplicates at each time point. “Other phyla” were microbial phyla below 1% relative abundance. “Unclassified” bacteria and archaea were OTUs that did not have a BLAST-assigned taxonomy at the phylum level.

Fig 11

Principal coordinate analysis of microbial communities by weighted Unifrac distances, with samples identified by habitat (collapsed palsa as yellow/orange/brown, bog as green, fen as blue), depth (shallow as square, deep as circle), and field (red outline) versus increasing time since collection/incubation (darker shades of each habitat color). Habitat-specific principal coordinates analysis of microbial communities by weighted Unifrac distances top: collapsed palsa middle: bog; bottom: fen).

Fig 12. Ordination and metabolic pathways for PICRUSt-predicted microbial community functions.

Principal coordinate analysis of predicted microbial community functions, with samples identified by habitat (collapsed palsa as yellow/orange/brown, bog as green, fen as blue), depth (shallow as square, deep as circle), and field (red outline) versus increasing time since collection/incubation (darker shades of each habitat color).

Fig 13. Methanogen abundance and diversity in bog and fen field and incubations, via the methanogen marker gene mcrA.

Fig 14

Boxplots showing CO₂:CH₄ ratios calculated for the field (panel a) and in the incubations averaged over 0–5 days (panel b), 20–30 days (panel c), 45–55 days (panel d), 70–80 days (panel e), and 90–100 days (panel f) throughout the incubation for each habitat. Anova was used to compare the means of each date with the field for each habitat. In panel (a) the results of that comparison are given (n.s. = p > 0.05). PHBD, BogS, and BogD did not differ from the field on any date as indicated by n.s. (i.e. not significant).