Bioenergy production often focuses on the aboveground feedstock production for conversion to fuel and other materials. However, the belowground component is crucial for soil carbon sequestration, greenhouse gas fluxes, and ecosystem function. Roots maximize feedstock production on marginal lands by acquiring soil resources and mediating soil ecosystem processes through interactions with the microbial community. This belowground world is challenging to observe and quantify; however, there are unprecedented opportunities using current methodologies to bring roots, microbes, and soil into focus. These opportunities allow not only breeding for increased feedstock production but breeding for increased soil health and carbon sequestration as well. A recent workshop hosted by the USDOE Bioenergy Research Centers highlighted these challenges and opportunities while creating a roadmap for increased collaboration and data interoperability through standardization of methodologies and data using F.A.I.R. principles. This article provides a background on the need for belowground research in bioenergy cropping systems, a primer on root system properties of major U.S. bioenergy crops, and an overview of the roles of root chemistry, exudation, and microbial interactions on sustainability. Crucially, we outline how to adopt standardized measures and databases to meet the most pressing methodological needs to accelerate root, soil, and microbial research to meet the pressing societal challenges of the century.

Hydroxyl radical (•OH) is produced in soils from oxidation of reduced iron (Fe(II)) by dissolved oxygen (O₂) and can oxidize dissolved organic carbon (DOC) to carbon dioxide (CO₂). Understanding the role of •OH on CO₂ production in soils requires knowing whether Fe(II) production or O₂ supply to soils limits •OH production. To test the relative importance of Fe(II) production versus O₂ supply, we measured changes in Fe(II) and O₂ and in situ •OH production during simulated precipitation events and during common, waterlogged conditions in mesocosms from two landscape ages and the two dominant vegetation types of the Arctic. The balance of Fe(II) production and consumption controlled •OH production during precipitation events that supplied O₂ to the soils. During static, waterlogged conditions, •OH production was controlled by O₂ supply because Fe(II) production was higher than its consumption (oxidation) by O₂. An average precipitation event (4 mm) resulted in 200 µmol •OH m⁻² per day produced compared to 60 µmol •OH m⁻² per day produced during waterlogged conditions. These findings suggest that the oxidation of DOC to CO₂ by •OH in arctic soils, a process potentially as important as microbial respiration of DOC in arctic surface waters, will depend on the patterns and amounts of rainfall that oxygenate the soil.

Toxic cyanobacterial harmful algal blooms (cyanoHABs) threaten freshwater resources globally and are intensifying with increasing eutrophication. Bloom toxicity is strongly influenced by intraspecific variation in the biosynthetic repertoires of toxic cyanobacteria, yet few studies examine the diversity of cyanobacterial cyanopeptides beyond hepatotoxic microcystins. To understand the dynamics and drivers of cyanopeptide diversity in cyanoHABs, we analyzed temporal patterns of cyanobacteria, metabolites, and their biosynthetic gene clusters (BGCs) in western Lake Erie using a 7-year time series (2016–2022) of metagenomic and metabolomic data. Our findings demonstrate that shifts from Microcystis to Dolichospermum occur later in the bloom season, coinciding with lower temperatures. Modules of co-varying BGCs (biosynthesis modules) from these genera were identified with hierarchical clustering, with uncharacterized BGCs among the most abundant. Biosynthesis modules rich in nonribosomal peptide synthetases (NRPS) peaked in early August, coinciding with elevated levels of inorganic nitrogen, warmer temperatures, and high Microcystis abundance. In contrast, modules rich in polyketide synthases (PKS) and ribosomally synthesized and post-translationally modified peptides (RiPPs) peaked following the Microcystis maximum in mid-August. Metabolomic analyses confirmed that metabolites followed shared seasonal patterns with their associated biosynthesis modules, forming three phases characterized by (i) microcystins, (ii) anabaenopeptins and aeruginosins, and (iii) aerucyclamides. These phases co-varied with bottom-up and top-down pressures, with later phases coinciding with increased microbially processed organic nitrogen and reduced detection of grazers. This study demonstrates consistent seasonal patterns of cyanobacterial metabolite succession and co-occurrence beyond microcystins, suggesting tradeoffs between biosynthetic resource demands and ecological controls.