Stability of stream biofilm community composition to transient shifts in dissolved organic carbon characteristics

Lines, Oliviah; Silvester, Ewen; Acharya, Suman; Holland, Aleicia; Shackleton, Michael

doi:https://doi.org/10.5194/egusphere-2025-4143

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https://doi.org/10.5194/egusphere-2025-4143

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16 Sep 2025

| 16 Sep 2025

Status: this preprint is open for discussion and under review for Biogeosciences (BG).

Stability of stream biofilm community composition to transient shifts in dissolved organic carbon characteristics

Oliviah Lines, Ewen Silvester, Suman Acharya, Aleicia Holland, and Michael Shackleton

Abstract. Microbial communities within biofilms are widely recognised as important contributors to ecological food webs and elemental cycles within stream systems. Yet, little is known about how these biofilm communities respond compositionally to storm-event-driven changes in dissolved organic carbon (DOC) characteristics. Alpine headwater peatland-draining streams offer a unique opportunity to investigate this response as these systems are known to export high loads of DOC during storm events, with little further upstream input. This study investigated how sub-alpine peatland-draining stream biofilm composition changed in response to storm-event-driven pulses of DOC. It was found that during the peak of the DOC pulses, the composition of DOC changed to include increased contributions of organic acids, protein-like substances and microbially derived DOC. Despite this change in DOC composition, the composition of most biofilm microbial communities did not significantly shift following each pulse; rather, differences in biofilm community composition appeared to be more closely linked to peatland stream site. The findings of this study suggest biofilm microbial communities maintain compositional stability following short-term rapid changes in stream water chemistry, and that site-specific environmental factors may be more important in determining biofilm microbial community composition in sub-alpine headwater peatland-draining streams.

Received: 25 Aug 2025 – Discussion started: 16 Sep 2025

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Oliviah Lines, Ewen Silvester, Suman Acharya, Aleicia Holland, and Michael Shackleton

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RC1:
'Comment on egusphere-2025-4143', Anonymous Referee #1, 04 Nov 2025 reply
The manuscript by Lines et al. investigates the response of stream biofilm microbial communities to storm-event-driven pulses of dissolved organic carbon (DOC) in a sub-alpine peatland catchment. This is a relevant topic, given the projected increase in storm intensity under climate change and the critical role of biofilms in aquatic carbon cycling. The study employs a comprehensive multi-method approach that combines detailed hydrology, sophisticated DOC characterisation, and high-throughput sequencing of algal, bacterial, and fungal communities. The central finding – that biofilm community composition remained largely stable following short-term DOC pulses and was instead more strongly influenced by site-specific factors – is noteworthy and contributes a valuable perspective to the field. The manuscript is generally well-written, and the data appear robust. However, several points require clarification and further discussion to strengthen the interpretation of the results and the overall impact of the study before it can be considered for publication.
Major Comments
Temporal Resolution of Biofilm Sampling: Biofilms were sampled only “within 24 hours prior to zero storm time” and “12–16 days after each storm event.” This gap misses short-term (e.g., 1–7 days post-storm) community dynamics, which could reveal transient shifts that recover by 12–16 days. For example, if biofilms temporarily respond to DOC pulses but revert to pre-storm composition by the post-storm sampling, the current design would mischaracterise “stability” as absence of change rather than resilience. If retrospective sampling is not possible, discuss this limitation explicitly and propose future studies with intermediate time points (e.g., 3, 7 days post-storm) to capture potential short-term responses.

Interpretation of the PERMANOVA Results: The significant three-way interaction (Event × Site × Storm-time) is reported, but the subsequent analysis and discussion focus heavily on the overwhelming effect of 'Site'. The PERMANOVA model uses Type I (sequential) sums of squares, meaning the variance explained by 'Site' (entered first as a random factor?) is removed before testing the other factors. The authors should clarify the order of factors in the model and consider whether the significance of the three-way interaction is largely an artefact of the strong site effect, as they allude to. A more detailed presentation of the variance explained by each term (e.g., via a more complete table than S10) would be helpful. Furthermore, for the pairwise tests following the significant interaction, the use of Monte-Carlo p-values due to low permutation numbers should be explicitly stated in the main text methods, not just the supplement.

Linking DOC Change to Biofilm Function: The study expertly characterises DOC composition but does not link these changes to any functional response of the biofilms (e.g., metabolic activity, extracellular enzyme assays). The authors hypothesise that the biofilm matrix may buffer against change, but without concurrent data on microbial function or carbon utilisation, this remains speculative. Whilst a comprehensive functional analysis may be beyond the scope of this paper, the discussion on why the communities are stable (Section 4.2) would be strengthened by acknowledging this gap and suggesting that functional stability (or plasticity) could occur without compositional shifts. Are the observed communities functionally redundant?

Climate Change Implications: The manuscript concludes that biofilm stability “suggests that with the predicted changes in the frequency and intensity of storm events under a warming climate, these communities may be resilient” (p. 29). However, it does not address whether repeated storms (e.g., the three events between SE1 and SE2, p. 12) could exhaust resilience over time. For example, if each storm depletes EPS stores, successive pulses might eventually trigger community shifts. Discuss the limitations of extrapolating from two storms to long-term climate change, and propose future studies on the effects of repeated DOC pulses on biofilm resilience.

Minor Comments
Introduction: The introduction is comprehensive but could be more sharply focused. The link between DOC characteristics, bioavailability, and expected microbial community shifts could be articulated more directly early on to set a clearer hypothesis.

Methods (Section 2.7.1): The use of a correlation cut-off (r ≥ 0.7) to remove variables from the PCA is a valid approach to reduce multicollinearity. However, the choice of the cut-off is somewhat arbitrary. A brief justification for this threshold would be helpful. Furthermore, stating that DOC is "best represented by Electrical Conductivity" is an overinterpretation; it is correlated with, not a direct proxy for.

Discussion (Section 4.2): The argument that short water residency times limited community response is compelling. Could the authors provide a rough estimate of water travel times during peak flow vs. baseflow for their sites to quantitatively support this line of reasoning?

Language and Typography: The manuscript is well-written but would benefit from a final proofread for minor grammatical inconsistencies (e.g., consistent use of present/past tense). Ensure all acronyms are defined at first use.

I recommend major revisions.

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Citation: https://doi.org/10.5194/egusphere-2025-4143-RC1

Oliviah Lines, Ewen Silvester, Suman Acharya, Aleicia Holland, and Michael Shackleton

Supplement

https://doi.org/10.5194/egusphere-2025-4143-supplement

Data sets

Data (zOTU and water chemistry as csv files), data processing and analysis workflow (pdf) and R scripts (as html files) for Lines et al. (2025) "Peatland stream biofilm community composition response to a storm pulse of dissolved organic carbon (DOC)". O. Lines et al. https://doi.org/10.26181/29757092

PRJNA1301096 O. Lines https://www.ncbi.nlm.nih.gov/sra/?term=PRJNA1301096

Oliviah Lines, Ewen Silvester, Suman Acharya, Aleicia Holland, and Michael Shackleton

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Short summary

Stream biofilm microbial communities play key roles in elemental cycles and stream food webs, yet little is known about their response to transient changes in dissolved organic carbon (DOC) composition and characteristics. Using chemical analyses and eDNA metabarcoding, this study found that DOC composition shifted during storm events, but the composition of most biofilm communities remained stable, suggesting ecological resilience to transient shifts in DOC composition and characteristics.


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