Organic Matter Composition and Water Stoichiometry Are Main Drivers of Heterotrophic Nitrate Uptake in Mediterranean Headwater Streams

Heterotrophic bacteria can contribute to improve stream water quality by taking up nitrate (NO₃⁻) from the water column, although microbial demand for this nutrient is usually lower than for other inorganic nitrogen (N) forms, such as ammonium. Heterotrophic NO₃⁻ uptake has been related to the availability of dissolved organic carbon (DOC) relative to nutrients (i.e., DOC: nutrients ratios). Yet, how dissolved organic matter (DOM) composition and specific microbial assemblages influence NO₃⁻ uptake remains poorly understood. We conducted laboratory incubations to investigate heterotrophic NO₃⁻ uptake kinetics in 9 Mediterranean freshwater ecosystems, primarily headwater streams, exhibiting wide variation in DOC:NO₃ ratios (from 1.5 to 750). Moreover, we characterized DOM composition using spectroscopic indexes and its degradation via a reactivity continuum model approach. Microbial community composition and functioning were assessed by analyzing extracellular enzymatic activities and the potential abundance of N-cycling genes. Our results revealed that NO₃⁻ uptake rates (k_NO3) were positively related with DOC:NO₃ ratios (r² = 0.4) and to NO₃:SRP ratios as well (r² = 0.6). Furthermore, k_NO3 was negatively correlated to the humification index (r² = 0.7), suggesting that a higher proportion of humic-like compounds slow down heterotrophic NO₃⁻ uptake. A partial least squares regression model (PLS) pinpointed that DOC and nutrient stoichiometry, DOM composition and reactivity, and microbial composition and activity collectively contributed to explain the variability in k_NO3 observed across treatments. Our findings suggest that heterotrophic NO₃⁻ uptake may show significant responsiveness to shifts toward more labile DOM sources and nutrient imbalances induced by global change.