Redox-Related Metabolic Dynamics Imprinted on Short-Chain Carboxylic Acids in Soil Water Extracts: A ¹³C-Exometabolomics Analysis

Abstract

Soil microbial metabolism is critical to carbon cycling, but direct annotation of metabolic activities is challenging. Here, we present an exometabolomics approach coupled with 13C profiling for in situ probing of metabolic activities following a 13C-glucose pulse in oxic or anoxic soil incubations. In the soil water extracts, we monitored both abundance and isotopic enrichment of short-chain carboxylic acids (SCCAs) involved in different metabolic pathways: peripheral sugar oxidation, glycolysis, fermentation, and the tricarboxylic acid cycle. The water-extractable SCCAs captured redox-dependent metabolic dynamics. First, under both redox conditions, increased concentration (up to 50 μM) along with near-complete 13C-labeled fractions for both gluconate and 2-ketogluconate revealed activation of the peripheral sugar oxidation pathway. Second, greater citrate depletion during the oxic condition than during the anoxic condition (6.2 versus 2.3 μM) was consistent with an expected decrease in carbon usage in the anoxic incubations. Third, accumulation of 13C-labeled succinate and malate only in the anoxic incubations demonstrated metabolic overflow typical of anaerobic metabolism. Fourth, production of 13C-labeled lactate under the anoxic conditions highlighted glucose fermentation but, under the oxic conditions, lactate depletion implied its role as a carbon source. Such 13C-assisted exometabolomics data offer insights to interpret gene-based predictions of metabolic potentials.

Tracing carbon metabolism with stable isotope metabolomics reveals the legacy of diverse carbon sources in soil

“…All nine carbon sources were added together and only the identity of the single 13 C-labeled substrate was varied between treatments to allow for stable isotope probing of the metabolome over time. 12 C-control microcosms contained all unlabelled carbon sources. Triplicate microcosms for each of the nine carbon sources were destructively sampled over time (1,3,6,14,30, and 48 days after amendment, n = 104 samples), freezing immediately at -70˚C until use.…”

“…Atom % 13 C served as a measure of the balance of source-derived carbon in metabolite pools. An increase in atom % 13 C reflects the channeling of 13 C into a given metabolite pool, while a decrease can indicate a pool dilution (channeling of 12 C from unlabeled carbon sources into a given metabolite pool). Changes in atom % 13 C may also reflect metabolite losses from the differential consumption of a 13 C-versus 12 C-enriched metabolites among segregated cells / populations, or vice versa.…”

“…An increase in atom % 13 C reflects the channeling of 13 C into a given metabolite pool, while a decrease can indicate a pool dilution (channeling of 12 C from unlabeled carbon sources into a given metabolite pool). Changes in atom % 13 C may also reflect metabolite losses from the differential consumption of a 13 C-versus 12 C-enriched metabolites among segregated cells / populations, or vice versa. Thus, atom % 13 C represents the relative balance between sourcederived carbon entering a pool versus its dilution or loss.…”

“…We interpreted any lack of change in atom % 13 C enrichment over time as an indication that this metabolite was no longer being consumed or synthesized. The % atom 13 C of each metabolite was calculated by apportioning the peak area of each isotopologue using the following equations: equation 1 13 C area = equation 2 12 C area = equation 3 atom % 13 C = 13 C area / ( 13 C area + 12 C area ),…”

“…SIP has been used to quantify the persistence of microbial biomass carbon in soil food webs by tracking 13 C into lipids, amino acids, and carbohydrates [9,10]. The quantification of 13 C in whole metabolite pools (i.e., SIP-metabolomics) has been used to study amino acid turnover in soil [11], and contrast the metabolism of glucose in oxic and anoxic soils [12], as well as in other applications [13,14]. Here, we used SIP-metabolomics to examine community metabolism with respect to nine carbon sources that are common to soils.…”