the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 10 Oct 2025
Fluvial biofilm phosphorus entrapment shifts from intracellular to extracellular dominance along a multifactorial longitudinal river gradient
Abstract. Phosphorus (P) entrapment by biofilms can be distinguished into the intracellular P entrapment (the P uptake by microbial cells) and the extracellular P entrapment via the extracellular polymeric substances. It is unknown how these two P entrapment pathways behave in natural ecosystems in which gradients of environmental drivers such as P, labile dissolved organic matter (DOM) and light occur. Another key aspect in P dynamics at the sediment-water interface is microbial activity. Microbially mediated mineralization of DOM and microbial activity in general is suspected to be a driver of internal P mobilization from the sediments. Here we brought such expectations into a real-world context by analysing the P entrapment patterns of benthic biofilms and the P release potential from the sediments along a longitudinal gradient in a third order river in front of the background of key microbial metabolic variables. The gradient consists of increasing P availability, DOM lability and light availability.
We found a gradual shift in the dominance of P entrapment from higher intracellular P entrapment in the upstream biofilms to higher extracellular P entrapment in the downstream biofilms. This shift towards dominance of extracellular P entrapment was accompanied by an increase in the P release potential from the sediment. The increasing P release potential was also connected to high extracellular enzyme activity of alkaline phosphatase, an enzyme involved in the mineralization of P from organic compounds. We further found that a balanced ratio between intracellular and extracellular P was connected to a higher C metabolic diversity.
All this evidence suggests an influence of the benthic biofilms on P dynamics at the sediment-water interface. This research advocates for a more integrated perspective that accounts for both intracellular and extracellular biofilm-mediated processes.
- Preprint
(2361 KB) - Metadata XML
- BibTeX
- EndNote
Status: open (until 20 Apr 2026)
- RC1: 'Comment on egusphere-2025-4844', Anonymous Referee #1, 02 Dec 2025 reply
-
RC2: 'Comment on egusphere-2025-4844', Anonymous Referee #2, 09 Apr 2026
reply
The manuscript presents the phosphorus and carbon contribution in biofilms along a longitudinal gradient across different reaches of a low-order stream. These patterns may be further influenced by climate change modifying nutrient biogeochemical processes. Results provide a comprehensive representation of both structural and functional characteristics of biofilms, particularly focusing the attention on intra and extracellular phosphorus acquisition, as well as the contribution of dissolved organic carbon. This study globally embedded the contribution of fluvial biofilm in this longitudinal gradient with extensive length covered.
The manuscript clearly introduces the key concepts related to biofilms and low-order streams, which are currently threatened by global change. However, despite the extensive research conducted by the authors, several issues and questions can arise. The introduction is relatively long, and particularly the first paragraph is too general. Focusing more directly on the key aspects would strengthen it. Additionally, some references are old (e.g., lines 39, 41, 43, 56, 74, among others). I recommend including more recent citations where possible.
The methods are well described. The field site is clearly characterised, and assessing half of the total stream length is an important aspect that should be explicitly highlighted. The authors also acknowledge challenges encountered during sampling, which is appreciated. The description of biofilm parameters evaluated is clear, and the statistical approaches are well explained with scientific rigour.
I suggest replacing Table 1 with a map showing the different stream reach categories to strengthen this section. Table 1 could be moved to the supplementary material. Additionally, incorporating land-use analysis of each reach’s surroundings with differentiating agricultural, forestry, or urban predominance could help interpret the characteristics of each reach and improve the discussion, particularly in subsection 4.1.
For clarification: how was phosphorus concentration in the biofilm extract analysed? If inductively coupled plasma mass spectrometry (ICP-MS) was used, I suggest briefly describing this method.
I think it is important to mention the six samples that were incompletely homogenised to obtain good results, but in this case, I suggest modifying the paragraph to start mentioning the relevance that a minimum of 3 observations for each sampling site were obtained, and due to this issue, these six samples were discarded.
The CLPP analysis from Perujo et al., 2020 is highly relevant and strengthens the interpretation of functional variables.
Some specific comments from the Methods section:
Lines 152-154: the explanation of the x-intercept determination is unclear.
Lines 166-167: Why was Optiprep density gradient medium used?
Line 171: why was dilution performed with Milli-Q water instead of Ringer solution (to avoid osmotic shock)?
Line 175: Chl-a was determined using ethanol 90% instead of acetone 90%, could you justify this choice?
Line 178: consider using “biofilm functional variables” instead of “metabolic variables”
Line 235: the absorbance range (250-400 nm) requires further clarification.
Lines 244-247: why was the Benjamini-Yekutieli multiple test selected over other non-parametric approaches?
Lines 250-251: why was Chl-a expanded for zero inflation? Is this comparable to a log(x+1) transformation?
Lines 254-255: why was one-way ANOVA used for microbial C metabolic profiles, substrate guilds, and EEAs? Would glm models be more appropriate?
Results were clearly representative to demonstrate the differences among sampling stream reaches and respective contribution of biofilm structural and functional variables. Most of the current studies in this field were considering longitudinal gradient patterns on nutrient release or uptake from urban wastewater treatment plants. This article goes even further with specific contributions on phosphorus and carbon dynamics to widely represent the biofilm capacities on their longitudinal shifts.
In several figure captions, the colour gradient indicating distance from the source is unclear and could be redundant, as distance is already shown on the x-axis. Additionally, given the limited number of replicates, presenting mean values with standard deviation for each distance site may be enough.
Specific comments from the Results section:
Lines 283-284: does an increasing slope ratio indicate decreasing DOM molecular weight (Fig.1C)? This is unclear or can be misunderstood. High variability may also affect the interpretation. Similar concerns apply to Fig.1D.
Line 286: I suggest defining BIX as “biological index” when first mentioned.
Lines 285-290: is statistical analysis missing here? Could it be an option to interpret statistically the variability of these variables against distance in section 3.1? Similarly, as you performed at section 3.2.
Line 309: in figure 3 caption, the P release potential should be added as EPC0 acronym.
Line 322: consider using “biofilm structural variables” only.
Lines 322-331: consider including standard deviation alongside mean values.
Lines 347-349: consider including key CLPP results in the main text and moving the diversity indices figure of Shannon and Evenness to supplementary material.
Line 354: define EEA abbreviations in figure 6 caption.
Lines 373-374: results from the log EEA ratios should be briefly described in the main text, only referring to the supplementary material for figure and table.
Line 387: consider replacing “uppermost sampling site” with “upstream”. And consistently use upstream/downstream throughout the manuscript.
Finally, regarding discussion and conclusions sections. I would appreciate the work done by the authors of this research, extensively discussing their results. Nevertheless, some specifications and recommendations are also needed to consider improving the readability and strength its interpretation. Subsections 4.1 and 4.2 are particularly interesting. However, incorporating recent citations could strengthen the discussion, especially with the connection between Jarvie et al., 2002 and Weitere et al., 2021 focusing on their temporal assessment, compared to your study which was focused on longitudinal and specific temporal period evaluated. Given your focus on longitudinal gradients, I expected to observe a stronger connection with WWTP effluents discharged into these low-order streams. For instance, SRP loads (lines 435-438) may be originated from urban WWTP discharges.
Only a specific mesocosm experiment was cited (lines 441-443). Nevertheless, I suggest adding other studies that can corroborate it.
In some subsections you referenced the hypothesis mentioned in the introduction. However, there are other subsections such as 4.3 and 4.4 that can be commented in the main manuscript for enhanced readability.
Additionally, there are several conditional sentences that some of them can be improved with specific citations. In this case, adding recent citations could strengthen the discussion.
Subsection 4.5 is highly relevant and well-written, although some parts are repetitive from the other subsections (e.g., lines 500-502). A few sentences are also unclear; for instance, between lines 539 and 542, you described a lack of downstream connection with APA, followed by correlations with Chl-a and EPC0. Clarifying these relationships would improve interpretation. Structuring sentences around observed correlations could improve readability and clarity.
Some specific comments from the Discussion section:
Line 453: replace with “upstream sampling sites” and homogenise terminology throughout the manuscript.
Line 464: could you mention additional underlying factors?
Line 470: specify land-use types? Agricultural or urban shift?
Line 480: avoid speculative sentences; support statements with evidence. For instance, in the sentence “likely caused by an increase in DOM diversity”. I suggest avoiding supposition and corroborating it, as you explained in the following sentences with the examples provided.
Lines 504-507: the reference of Zhou et al. (2017) may be out of context? They performed an experiment based on Synechocystis sp., but I am not sure if they considered different microbial communities such as occurred with biofilms.
Line 508: why did you consider the upstream reach as transient P storage due to EPS? A brief explanation about the reference you found could enhance this interpretation.
Overall, I recommend a minor revision, addressing the previous comments mentioned above.
Citation: https://doi.org/10.5194/egusphere-2025-4844-RC2
Viewed
| HTML | XML | Total | BibTeX | EndNote | |
|---|---|---|---|---|---|
| 309 | 163 | 31 | 503 | 30 | 37 |
- HTML: 309
- PDF: 163
- XML: 31
- Total: 503
- BibTeX: 30
- EndNote: 37
Viewed (geographical distribution)
| Country | # | Views | % |
|---|
| Total: | 0 |
| HTML: | 0 |
| PDF: | 0 |
| XML: | 0 |
- 1
The authors present data on phosphorus and carbon turnover characterization in benthic biofilms of a river stretch. The data include results from a large number of laboratory classification approaches (including biofilm composition, enzyme activity and P release experiments) for 7 positions in a 30km stream stretch, sampled once in autumn of 2023 under baseflow. The results are imbedded in a research question that surrounds the role of the biofilms as P retainers of various form (cells and their biofilm) and how their relation is driven.
The manuscript is well structured, characterizing the field site and its boundary conditions and then successively introducing the dynamic biofilm characteristics. The discussion section describes the systems effectively without repetition from the results section. Methods and statistics seem rigorous.
Despite the technical benefits, I struggle with the overall novelty of the manuscript. I study aquatic biogeochemistry across various environments, not stream biofilms in particular, and it seems to me as if this manuscript suffers from an overly deep insight paired with too limited spatial and temporal coverage. With their dataset, the authors try to establish a novel connection between carbon metabolism and P retention mechanisms. It escaped me, unfortunately, what the larger benefit of the knowledge of such a tight-knit dependence would be. This may be due to my limited insight to biofilm mechanics, but also to the limited structure of the introduction section. Especially the “open questions” are mixed with background knowledge and presented independent from the (too many and unclear) hypotheses. I’d like to motivate the authors to review this section with great care. This assessment it seems to me is mirrored in the discussion section. Next to the (solid, but to a large extent already published) presentation of the riverine gradient, there is a lot of text that revolves around the argumentation why the analyzed system is three-fold partitioned and why some of the other patterns observed in the previous studies trigger some of the observations made here (e.g. DOM quality, 490f). Section 4.5, which would be the heartpiece of the manuscript as currently presented, is not sufficiently clear. Overall my conclusion is that the authors go beyond what the limited observational density can provide.
For a revision, I think this manuscript needs a better focused introduction and a better imbedding of the results. I understand that the many analyses were time-consuming and could not be rolled out to the full network as in Weitere et al. network description. Despite that, I think the data has the potential to couple the observations from the 7 sites with the findings across the larger network and maybe even pair it with numerical data from other sites and studies. I don’t want this review to be read as a straight-out recommendation for rejection, as I see merit in the rigorous work. However, I feel for a strong broad-topic biogeo journal as BG, the study motivation, depths and conclusions should be more accessible to a broad community of researchers, which I think is not achieved in the current form, but authors and data have the potential for.
Some more specific comments
46-51 is it possible to add quantities to these mechanistic background. It becomes more tangible if the pool sizes of intra and extra-P are better known
57 “increasing gradients” is maybe not a good terminology here. It is gradients of increasing XYZ
64 not sure if Hupfer and Lewandowski, 2008 really had dissolved OM in mind, it is more about solid-phase OM
60-80 maybe it is possible to state the 2 open research question in a more systematic fashion (e.g. in two paragraphs, each focused on one of the topics: labile DOM->APA and the P entrapment gradient)
100 write out upstream and downstream
149 do you put the sediments in the P buffer solutions?
250 I do not understand why the zero-inflation was necessary in the data structure
401 Increasing gradient.. again this seems like a wrong terminology. What is “light DOM availability”
404 As an example here but also elsewhere, the discussion section could be clearer if it more directly links to the results figure in question.
447 the biofilm as natural P buffer… this is a relatable concept that may be useful also elsewhere
459 -465 Unclear what this statement is leading to. Discussion is not developing more than just the presentation of results.