Metagenomics coupled with biogeochemical rates measurements provide evidence that nitrate addition stimulates respiration in salt marsh sediments

Abstract

High-throughput sequencing has enabled robust shotgun metagenomic sequencing that informs our understanding of the genetic basis of important biogeochemical processes. Slower to develop, however, are the application of these tools in a controlled experimental framework that pushes the field beyond exploratory analysis toward hypothesis-driven research. We performed flow-through reactor experiments to examine how salt marsh sediments from varying depths respond to nitrate addition and linked biogeochemical processes to this underlying genetic foundation. Understanding the mechanistic basis of carbon and nitrogen cycling in salt marsh sediments is critical for predicting how important ecosystem services provided by marshes, including carbon storage and nutrient removal, will respond to global change. Prior to the addition of nitrate, we used metagenomics to examine the functional potential of the sediment microbial community that occurred along a depth gradient, where organic matter reactivity changes due to decomposition. Metagenomic data indicated that genes encoding enzymes involved in respiration, including denitrification, were higher in shallow sediments, and genes indicative of resource limitation were greatest at depth. After 92 d of nitrate enrichment, we measured cumulative increases in dissolved inorganic carbon production, denitrification, and dissimilatory nitrate reduction to ammonium; these rates correlated strongly with genes that encode essential enzymes in these important pathways. Our results highlight the importance of controlled experiments in linking biogeochemical rates to underlying genetic pathways. Furthermore, they indicate the importance of nitrate as an electron acceptor in fueling microbial respiration, which has consequences for carbon and nitrogen cycling and fate in coastal marine systems.

Not All Nitrogen Is Created Equal: Differential Effects of Nitrate and Ammonium Enrichment in Coastal Wetlands

“…By contrast, subsystems involved in fermentation and other low energy producing metabolisms were more abundant in the unamended treatment (figure 6 c) as were genes responsible for N fixation (figure 6 d). The metagenomics findings from our FTR experiment are consistent with the biogeochemical rates we measured (Bulseco et al 2020 ) and provide a mechanistic link that connects microbial genetics to the NO3− loss and DIC production we observed under NO3− addition.…”

“…In light of these results, it is not surprising that we were unable to detect a strong signal of nutrient enrichment in the microbial metagenomes from our field experiment (figure 6 a). In the FTR experiments, however, where NO3− was added at higher concentrations and in a continuous manner, we saw broad shifts in the metagenomes (figure 6 b) that are consistent with the biogeochemical rate measurements (Bulseco et al 2020 ). Many genes involved in central carbon metabolism were enhanced in the NO3− addition (figure 6 c, supplemental table S1), including increases in the Entner–Doudoroff pathway for the generation of pyruvate from glucose, and decarboxylates that harness that pyruvate for cellular respiration, supporting the hypothesis that the addition of NO3− allows the microbial community to access otherwise inaccessible organic matter.…”

“…Surprisingly, we also observed an increase in genes associated with CO2 fixation as a result of nutrient enrichment, suggesting that added NO3− could also promote autotrophic denitrification pathways (Bulseco et al 2020 ). Many of the genes responsible for the cycling of nitrogen, including all the genes found in the denitrification pathway, were also significantly higher in the NO3− amended FTRs, compared with the unamended reactors (figure 6 d, table S1).…”

“…(d) Heat map indicating the 35 biggest differences between amended and unamended FTRs for genes involved in the nitrogen cycle. Source: The data used in panels (c) and (d) were adapted from Bulseco and colleagues ( 2020 ).…”

Soil Organic Carbon Development and Turnover in Natural and Disturbed Salt Marsh Environments

“…It is unlikely that measured δ 13 C‐CO 2 values are methodological artifacts, as they spanned a greater range than predicted by kinetic fractionation and enrichment did not increase monotonically with temperature (Figure 3; Hemingway et al., 2017b). This result highlights that thermal properties provide an index of reactivity that may not reflect bioavailability, which depends on environmental conditions, among other factors (Bulseco et al., 2019, 2020; Lehmann et al., 2020).…”