the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 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.
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Status: final response (author comments only)
- RC1: 'Comment on egusphere-2025-3548', Anonymous Referee #1, 20 Nov 2025
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RC2: 'Comment on egusphere-2025-3548', Anonymous Referee #2, 02 Dec 2025
In this study, Große et al performed a lab experiment to monitor how biofilm structure and function affect nutrients uptake. The amount of work for getting the results and the number of parameters measured is remarkable. However, the authors did not manage to aggregate those results in a coherent way. I think the manuscript has an immense potential, but the authors also need to put a lot of effort to improve the manuscript. On a separate note, I would like to apologise for not providing a timely review.
The manuscript would be far easier to understand and the results far easier to appreciate if the authors invest more time in the writing. The manuscript is understandable, but the flow of ideas and structure of sentences often sometimes unclear. Sentences containing random facts are placed within the text and it is unclear why. The text is wordy in general. I am pretty sure the authors can significantly improve the quality of the writing.
The number of replicates is not mentioned in the methods section. I assume it is 5 based on the results section but it is important to clarify that in the methods section. It would be great to have the full factorial design better explain in Figure 1. The authors also need to provide further information about why they used different methods at the “startpoint” and “endpoint”, especially for Nox and NH4 which are central for the study. It is unclear how gastight the mason jars were. Have the authors tested that? Also, it is hard to believe such a jar was completely free of air bubbles. I assume the authors did not put the jars upside down to check for the presence of air bubble, because that would severely compromise the experiment. Therefore, it is unclear how the authors can guarantee there was no air bubbles, and more importantly that the same amount of air bubble was present between the treatments. Typically, we use screw cap with cone insert/liner to ensure no air bubbles are present. This could explain why the authors find that “many measured GPP values were negative, which is not biologically possible” besides the typical issue with instrument sensitivity.
In the results section, the authors need to report the results of the test when saying something was significantly affected. For instance, section 3.1 and 3.2 start by saying there are significant results, but the p-value, test statistics, and degree of freedom are not presented. Oddly enough, such information is present in section 3.4 and 3.5. Also, where are the results for the Ecoplate and the many parameters measured by the authors? It feels like a lot of information is missing.
The discussion is repeating the results section quite a lot. The discussion does not manage to bring all results together and does not help the reader understanding how this study advances our understanding of nitrate uptake in benthic biofilms. The discussion describes why some of the results might or might not be accurate but rarely brings us back to what is happening in nature.
Further comments:
In the abstract, it is really hard to follow the logic between the first and second sentence. The abstract is not really down to the point. The study is about nitrate cycling in streams exposed to different anthropogenic pressure. The abstract does not help us understand that until we reach the results sentences. Please work on this and try to refocus the introduction sentences within the abstract.
Line 19: It is unclear why this idea is brought up. It feels disconnected from the previous and following sentence.
Line 24: the term extracellular polymeric substances was coined much earlier than Battin et al., 2016. Instead of having a reference to Battin et al 2016 after EPS, maybe combine the reference to Battin et al 2016 and Freeman and Lock at the end of the sentence. Otherwise, without necessarily citing the original 1982 paper, consider citing some of the older literature besides Battin et al 2016 to avoid giving a false impression that EPS are something relatively new when they are not.
Line 25: The sentence “The most reactive zone of stream sediments are the upper 2 cm, also known as the benthic biolayer (Knapp et al., 2017).” Is disconnected from the previous and following sentence.
Line 47: The Redfield ratio is related to marine plankton so it is unclear why we would expect to get such ratio in headwater streams. Maybe I missed something.
Line 107: Maybe add “specifically” or similar at the beginning of the second sentence. Initially, I thought there was no information about how the calculations were done.
Line 244: It is unclear what is meant by Biofilm structure. This was not presented in the methods. The methods (preferred) or the results section need to clearly indicate which parameters are used for characterising. We cannot read anything in Fig. A1.
Line 393: “We used acetate as a non-natural DOC source” Acetate is a natural DOC source, and its degradation leads to important methane flux. It is unclear why the authors claim acetate is non-natural.
Figures: We cannot read the labels on any of the ordinations (i.e. Figure 2 and all the other ordinations in the supplementary) because they are stacked together or placed below the points. Also, some of the figures are in the text whereas others are not. It is confusing. From a reviewer’s perspective, having the figure in the text is far better.
Citation: https://doi.org/10.5194/egusphere-2025-3548-RC2
Data sets
Acute changes in macronutrient stoichiometry alter nitrate uptake in benthic biofilms A. Große et al. https://zenodo.org/records/15878295?token=eyJhbGciOiJIUzUxMiJ9.eyJpZCI6ImMwYzU0ZDNlLTAzNjUtNGI1ZS05M2YyLTAyOWE2ODA2NjAyNiIsImRhdGEiOnt9LCJyYW5kb20iOiJmNzY2NjhjZWE0NzdkZjJmNjI4ZWZiZTE0OWJmMWE0ZiJ9.YJCyTiB4-7AIV9VBno0mJN66O-QevH3onkswKk8HnFj-CUPt9adDn2prJaIgZbvvlipVGcPHj48Tu5XMmppvIA
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- 1
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.