Fungi present distinguishable isotopic signals when grown on glycolytic versus tricarboxylic acid cycle intermediates

Abstract. Microbial activity in soils controls both the size and turnover rates of large carbon (C) inventories stored in the subsurface, having important consequences for the partitioning of C between terrestrial and atmospheric reservoirs as well as the recycling of mineral nutrients such as nitrogen or phosphorus, often bound to the C, that support plant growth. Fungi are major decomposers of soil organic matter (SOM); however, uncertainty in the predominant C substrates that fuel respiration confound models of fungal production and SOM turnover. To further define the signals of microbial heterotrophic activity, we applied a dual hydrogen (H) and C stable isotope probing (SIP) approach on pure fungal cultures representing the phyla Ascomycetes, Basidiomycetes, and Zygomycetes growing on monomeric (glucose, succinate) or complex substrates (tannic acid, β-cyclodextrin). Our findings demonstrate that the investigated species incorporated only minor amounts of inorganic C (provided as bicarbonate) into their membrane lipids, amounting to < 3 % of lipid-C, with no consistent patterns observed between species or growth substrates. The net incorporation of water-derived H (i.e., α_W) into lipids also did not differ significantly between incubations with monomeric versus complex substrates; however, growth on succinate solicited significantly higher α_W values than glucose or β-cyclodextrin. This finding suggests that ²H-SIP assays have the potential to distinguish between microbial communities supported predominantly by substrates that are catabolized by the tricarboxylic acid cycle versus glycolytic pathway. Furthermore, the average α_W value of heterotrophic fungal incubations [0.69 ± 0.03 (SEM)] is consistent with that observed for bacterial heterotrophs, and may be applied for upscaling lipid-based estimates of fungal production in environmental assays.