| 26 Aug 2025
Acute changes in macronutrient stoichiometry alter nitrate uptake in benthic biofilms
Abstract. Benthic biofilms, located at the sediment-water interface, are hot-spots for macronutrient cycling in headwater streams. Here, the supply of dissolved organic carbon (DOC), nitrogen (N), and phosphorus (P), affects nutrient cycling processes such as nitrate uptake. Flushing events can add short-term pulses of DOC, N and P to streams, changing the macronutrient ratios in the stream water, altering stoichiometric imbalances between water and microbial macronutrient ratios. However, there is little information on whether these short-term changes in macronutrient imbalances can alter biofilm nitrate uptake. To better understand how acute changes to DOC, N, and P stoichiometric imbalances affect nitrate uptake, we sampled stream biofilms from four different sites in Florida and incubated them in the lab in mesocosms after changing their macronutrient ratios by adding DOC and/or nitrate. Here we show that biofilms from anthropogenically less impacted streams with less N excess increased their nitrate uptake after 48 h of incubation in different macronutrient stoichiometric ratios, but biofilm structure remained mainly unaffected. Furthermore, nitrate uptake was positively related to biofilm metabolism, differentiating in sites with more autotrophic- or more heterotrophic-driven nitrate uptake. Our study reveals that acute changes in macronutrient stoichiometric imbalance between stream water and biofilm microorganisms changes nitrate uptake. This needs to be considered when assessing short-term nitrate uptake capacity of stream reaches.
The paper investigates benthic biofilms as hotspots for macronutrient cycling in Florida headwater streams. It presents an incubation experiment to assess biofilm nitrate uptake under varying macronutrient ratios. The study has the potential to make a valuable contribution to the literature; however, it contains several major flaws that need to be thoroughly addressed. The manuscript can be reconsidered for publication only after these primary issues are properly resolved.
Introduction: The motivation for the study is not clearly presented. It is unclear why these specific sites were selected and what unique characteristics make them suitable for this type of investigation. The introduction does not sufficiently explain how this work contributes to the broader literature, how findings from Florida watersheds can be extrapolated to other stream systems globally, or what the key biogeochemical implications of the study are. These elements are missing despite the introduction being very lengthy. Although hypotheses are stated, they are not adequately introduced or explained.
Experimental: A map of the study sites is required. Several sites are described as being more anthropogenically impacted than others, but this distinction is not clearly illustrated. A map or other visual representation should be included to show the relative locations of the sites, along with the corresponding land-use characteristics. This would allow readers to understand the spatial context of the study and how site differences may influence the observed patterns.
Results: None of the measured parameters are clearly presented in the manuscript. I could not find results for any variables measured before and after the incubations. While the dataset is available in an online repository, the manuscript does not reference or describe any of these measurements. This makes it impossible to follow what was done during the incubation experiments or how the parameters changed over time. Results section heavily refers statistical tests, without presenting the underlying data, which undermines the clarity and interpretability of the study.
Discussion: I stopped reading the paper after noting that the empirical results were not actually presented in the results section. Once the results section is corrected and the measured data (before and after incubations) are fully reported and displayed, the discussion must be thoroughly rewritten. Such that: it should be grounded in the actual measurements including means, ranges, changes etc. rather than starting from statistical interprations. Every interpretive statement must point to the corresponding table, figure, or data. Statistical models should be presented and used as complementary to support the observed patterns.