Genomic Science Program
U.S. Department of Energy | Office of Science | Biological and Environmental Research Program

Metabo-Lipidomics Unveil Root Exudate Molecular Diversity and Functional Impacts on Soil Microbial Communities


Kirsten S. Hofmockel1,2*, Sneha P. Couvillion1 (, Robert E. Danczak1, Isabella Yang1, Dylan Hermosillo1, Josie Eder1, Sheryl Bell1


1Biological Sciences Division, Pacific Northwest National Laboratory; 2Department of Agronomy, Iowa State University



PNNL’s Phenotypic Response of Soil Microbiomes Science Focus Area (SFA) aims to achieve a systems-level understanding of the soil microbiome’s phenotypic response to changing moisture. Researchers perform multi-scale examinations of molecular and ecological interactions occurring within and between members of microbial consortia during organic carbon decomposition, using chitin as a model compound. Integrated experiments address spatial and inter-kingdom interactions among bacteria, fungi, viruses, and plants that regulate community functions throughout the soil profile. Data are used to parametrize individual- and population-based models for predicting interspecies and inter-kingdom interactions. Laboratory and field experiments test predictions to reveal individual and community microbial phenotypes. Knowledge gained provides a fundamental understanding of how soil microbes interact to decompose and sequester organic carbon and enables prediction of how biochemical reaction networks shift in response to changing moisture regimes.


The rhizosphere, where plant roots meet soil, is a hub of biogeochemical activity. The impact of the small molecule metabolites and lipids in root exudates on microbial community structure, gene expression, and processes that control cycling and long-term storage of carbon (C) are poorly understood. Here the goal was to discover the molecular chemodiversity of metabolites and lipids in root exudates and root-associated soils to advance the understanding of plant-microbial feedbacks that regulate C cycling. Researchers worked with mature, field-grown tall wheatgrass (Thinopyrum ponticum), a deep-rooted perennial plant from the Tall Wheatgrass Irrigation Field Trial, in Prosser, WA, which features marginal, low-carbon aridisols. Researchers optimized exudate collection protocols to enable the capture of non-polar lipids in addition to polar and semi-polar metabolites. Researchers found that rates of C input via hydrophobic exudates were approximately double that of aqueous exudates and C/N ratios were markedly higher in hydrophobic compared to aqueous exudates (459 ± 90 vs 14.40 ± 0.58), emphasizing the importance of lipids, due to their high carbon content. Researchers used liquid chromatography coupled tandem mass-spectrometry (LC-MS/MS) for paired untargeted metabolomics and lipidomics or metabo-lipidomics for maximizing molecular coverage. To address the challenge of metabolite annotation, a major bottleneck in metabolomics, the team employed both MS/MS spectral library searching and deep learning-based chemical class assignment. The tandem approach substantially increased the characterization of the chemodiversity of root exudates. Notably, in an unprecedented characterization of intact lipids in root exudates, the team discovered the presence of a diverse variety of lipids, including substantial levels of triacylglycerols (~19 μg/g fresh root per min), fatty acyls, sphingolipids, sterol lipids, and glycerophospholipids. To understand how the spatial gradient of rhizodeposition impacts microbial community composition and metabolism, researchers performed metabo-lipidomics, metagenomics, and metatranscriptomics on a gradient of soil fractions with varying proximity to the roots. Lipids in exudates and soils had lower nominal oxidation state of C (NOSC) compared to more polar metabolites, suggesting increased persistence and less susceptibility to microbial breakdown. The team observed that microbial expression in members of the Actinomycetota, Acidobacteriota, Bacteroidota, Methylomirabilota, Myxococcota, Thermoproteota increased close to the root. Nucleoside metabolites (structural components of RNA) were more abundant near the root, suggesting higher microbial activity. Community expression related to the biosynthesis of secondary metabolites and fatty acid degradation increased closest to the roots while nitrogen metabolism decreased. Focusing on the phyla responsible for these metabolisms, the Actinomycetota and Methylomirabilota had the most significant gradients in abundance as distance toward the root decreased. Triacyglycerols and microbial phospholipids were abundant in bare soil while most secondary metabolites and organic acids increased close to the root. Here researchers show that metabo-lipidomics enables direct measurements of the functional molecules that govern metabolism, signaling, and resource sharing among microbes and in microbe-plant interactions. This builds on recent work that demonstrated the value of intact lipids in soil ecosystems as sensitive indicators of environmental stress response and substrate availability and highlights their great potential for interrogating interkingdom interactions and soil C accrual (Couvillion et al 2023; Naasko et al 2023).


Couvillion, S. P., et al. 2023. “Rapid Remodeling of the Soil Lipidome in Response to a Drying-Rewetting Event,” Microbiome 11(1), 34.

Naasko, K. I., et al. 2023. “Influence of Soil Depth, Irrigation, and Plant Genotype on the Soil Microbiome, Metaphenome, and Carbon Chemistry,” mBio 14(5), e01758-01723.

Funding Information

PNNL is a multi-program National Laboratory operated by Battelle for the DOE under Contract DE-AC05-76RLO 1830. This program is supported by the U.S. DOE, Office of Science, through the GSP, BER Program, under FWP 70880. A portion of this work was performed in the William R. Wiley Environmental Molecular Sciences Laboratory (EMSL), a national scientific user facility sponsored by BER Program and located at PNNL. IY and DH were supported by the DOE Science Undergraduate Laboratory Internship (SULI) program.