the Creative Commons Attribution 4.0 License.
the Creative Commons Attribution 4.0 License.
| 19 Sep 2025
Role of phosphorus concentration and the nitrogen to phosphate ratio in the synergistic stimulation of alkaline phosphatase activity in Laizhou Bay, China, coastal waters
Abstract. In coastal ecosystems, microbial alkaline phosphatase (AP) production is primarily induced by low phosphate (PO4-P) availability but is additionally regulated by the dissolved inorganic nitrogen to phosphate (DIN:PO4-P) ratio and seasonal temperature variation. However, the dominant driver of APA surges and potential synergistic effects among these factors remain unclear. Through integrated seasonal field surveys and an enclosure experiments in Laizhou Bay, China, we demonstrate that PO4-P seawater concentration serves as the primary control for APA induction, with a consistent threshold of 0.05 . Below this threshold, APA exhibits a significant positive correlation with the DIN:PO4-P ratio in both the field and an enclosure experiment under P limitation (combined analysis of field and experimental data, p＜0.01; n=36). Notably, phytoplankton-dominated APA is evidenced in autumn. Genetic analysis confirms that AP-related gene expression increases only when PO4-P falls below the identified threshold. These findings refine the conceptual framework for AP regulation in coastal ecosystems, highlighting the hierarchical control of phosphorus limitation over stoichiometric effects.
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CC1: 'Comment on egusphere-2025-4047', zhenjun kang, 12 Oct 2025
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RC1: 'Reply on CC1', Dongliang Lu, 11 Nov 2025
General Comments
The authors present a comprehensive study investigating the regulation of alkaline phosphatase activity (APA) in the coastal waters of Laizhou Bay, China. By effectively integrating seasonal field surveys with a mesocosm experiment, the manuscript convincingly identifies a phosphate concentration threshold for APA induction and demonstrates a synergistic role of the DIN:PO₄-P ratio under phosphorus-limited conditions. The incorporation of genetic data strengthens the mechanistic insights. The study is well-structured, addresses a relevant topic in coastal biogeochemistry, and is suitable for publication in Water Research after minor revisions to enhance clarity and precision in several specific sections.
Specific Comments
Lines 24-27 (Abstract): The phrase "combined analysis of field and experimental data, p<0.01; n=36" is vague. Please specify the variables between which this significant correlation was observed to make the abstract more informative.
Suggestion: Rephrase to, for example, "... a significant positive correlation was observed between APAtotal and the DIN:PO₄-P ratio in the combined dataset (p < 0.01; n=36)."
Lines 114-118 (Mesocosm Experiment Methods): While the target DIN:PO₄-P ratios are stated, the absolute initial concentrations of DIN and PO₄-P for the Control (CG) and Treatment (TG) groups are not provided in the main text, relying solely on a reference to Table S1. Including these critical values in the text would improve readability.
Suggestion: Briefly state the key initial conditions, e.g., "The initial DIN and PO₄-P concentrations for the CG were [X] μmol L⁻¹ and [Y] μmol L⁻¹, respectively. For the TG, concentrations were adjusted to [A] μmol L⁻¹ and [B] μmol L⁻¹ to achieve the target ratio."
Lines 318-320 (Results 3.4): The basis for categorizing the data into "high APA" and "low APA" groups for the regression analysis is not defined. The reproducibility of this analysis requires a clear, objective criterion.
Suggestion: Please specify the statistical criterion used for this grouping (e.g., median split, a specific APA threshold value).
Lines 437-443 (Discussion on DON): The discussion on the potential mechanisms linking DON to APA, while insightful, could be further strengthened. The argument would benefit from a more direct connection to the DON dynamics observed in your own experiment (e.g., Figure 5d) or from citing more specific literature on how particular DON compounds influence microbial P metabolism.
Suggestion: Consider elaborating briefly on how your data aligns with the proposed mechanisms or referencing studies that identify specific DON components as regulators.
Line 49-50 and throughout the text (Terminology): The abbreviation "AP" for Alkaline Phosphatase is used in the abstract without the full term being presented first. Additionally, specialized terms like "pho regulon" (Line 423) may be unfamiliar to a general readership.
Suggestion: Please define "AP" upon its first appearance in the abstract as "alkaline phosphatase (AP)". Consider adding a brief explanatory phrase for "pho regulon", such as "...the pho regulon (a gene suite responsible for phosphorus scavenging)".
Typos and Minor Corrections
Line 22: "an enclosure experiments" -> "an enclosure experiment" or "enclosure experiments"
Line 25: "ssignificant" -> "significant"
I commend the authors on a robust and valuable study. I believe these suggested revisions will further enhance the clarity, impact, and overall quality of the manuscript.
Citation: https://doi.org/10.5194/egusphere-2025-4047-RC1 -
AC1: 'Reply on RC1', Yanqun Yang, 09 Jan 2026
Dear Reviewers and Editor,
Thank you for giving us the opportunity to revise our manuscript. We are deeply grateful to the reviewers for their insightful comments and constructive suggestions, which have significantly helped us to improve the quality and clarity of our work. We have carefully considered all the points raised and have made corresponding revisions to the manuscript. Our point-by-point responses to the comments are detailed below.
Specific Comments
Lines 24-27 (Abstract): The phrase "combined analysis of field and experimental data, p<0.01; n=36" is vague. Please specify the variables between which this significant correlation was observed to make the abstract more informative. Suggestion: Rephrase to, for example, "... a significant positive correlation was observed between APAtotal and the DIN:PO₄-P ratio in the combined dataset (p < 0.01; n=36)."
Response: Result seaction 3.4 (Lines 329-333).A significant positive correlation was observed between APA and the DIN:PO₄-P ratio below this threshold (0.05 ) , as analyzed in the combined dataset of field and enclosure experiments (p < 0.01, n = 36).
Lines 114-118 (Mesocosm Experiment Methods): While the target DIN:PO₄-P ratios are stated, the absolute initial concentrations of DIN and PO₄-P for the Control (CG) and Treatment (TG) groups are not provided in the main text, relying solely on a reference to Table S1. Including these critical values in the text would improve readability. Suggestion: Briefly state the key initial conditions, e.g., "The initial DIN and PO₄-P concentrations for the CG were [X] μmol L⁻¹ and [Y] μmol L⁻¹, respectively. For the TG, concentrations were adjusted to [A] μmol L⁻¹ and [B] μmol L⁻¹ to achieve the target ratio." Response: Discussion Section 2.3. Field enclosure experiments Line 122-125 The initial DIN and PO4-P concentrations for the CG were 1.90 and 0.04 , respectively. For the TG, concentrations were adjusted to 50.01 and 1.78 to achieve the target ratio.
Lines 318-320 (Results 3.4): The basis for categorizing the data into "high APA" and "low APA" groups for the regression analysis is not defined. The reproducibility of this analysis requires a clear, objective criterion. Suggestion: Please specify the statistical criterion used for this grouping (e.g., median split, a specific APA threshold value).
Response: We appreciate the reviewer's comment regarding the transparency of our method. We have now clarified the grouping criterion in Result seaction 3.4 (Lines 329-333) . The revised caption includes the following sentence: During the former, this linear relationship could be further divided into two groups by K-means cluster analysis based on APA, such that the slope of the fitted straight line was significantly higher in the high APA group (P < 0.01; line read in Figure 6) relative to that of low APA group (P < 0.01; line black in Figure 6).
Lines 437-443 (Discussion on DON): The discussion on the potential mechanisms linking DON to APA, while insightful, could be further strengthened. The argument would benefit from a more direct connection to the DON dynamics observed in your own experiment (e.g., Figure 5d) or from citing more specific literature on how particular DON compounds influence microbial P metabolism. Suggestion: Consider elaborating briefly on how your data aligns with the proposed mechanisms or referencing studies that identify specific DON components as regulators.
Response:Discussion Section 4.2 Line 477-488 In the cultivation experiments, it is possible that the growth of phytoplankton in the culture system provided a supplement for DON and DOP, resulting in the absence of a positive correlation between DON and APA. This correlation was only obtained from the field survey data. The revised caption includes the following sentence: It is noteworthy that prior studies have found that under the same N:P ratio and active phosphate concentration, phytoplankton APA in DON-enriched cultures was significantly higher than that in cultures without DON (Fitzsimons et al., 2020). Our winter survey data revealed a significant positive correlation between DON and APAbac (Figure 4), similar to findings of APAphyto and DON concentration (Ou et al., 2024), indicating that not only PO4-P but also nutrient composition and ratios, can affect APA. Regulation of APA by DON may potentially operate through specific DON components or their degradation products influencing P metabolic pathways, or through DON serving as an alternative N source that stimulates phytoplankton growth and P demand (Ma et al., 2018; Forchhammer et al., 2022). Meanwhile, DON serving as alternative nitrogen source may enhance phytoplankton growth and phosphorus demand.
Line 49-50 and throughout the text (Terminology): The abbreviation "AP" for Alkaline Phosphatase is used in the abstract without the full term being presented first. Additionally, specialized terms like "pho regulon" (Line 423) may be unfamiliar to a general readership. Suggestion: Please define "AP" upon its first appearance in the abstract as "alkaline phosphatase (AP)". Consider adding a brief explanatory phrase for "pho regulon", such as "...the pho regulon (a gene suite responsible for phosphorus scavenging)".
Response:Discussion Section 4.2 Line 463-469 When ambient PO4-P concentrations fall below the 0.05-0.2 (Dyhrman et al., 2007), phytoplankton activate the pho regulon (a gene suite responsible for phosphorus scavenging) operon (a gene cluster containing the pstSCAB phosphate transport system and phoA/phoX alkaline phosphatase genes) through the PhoB/PhoR two-component system (a P-sensing system consisting of histidine kinase PhoR and the response regulator PhoB) (Lin et al., 2016).
Citation: https://doi.org/10.5194/egusphere-2025-4047-AC1
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AC1: 'Reply on RC1', Yanqun Yang, 09 Jan 2026
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CC2: 'Reply on CC1', Yanqun Yang, 16 Nov 2025
Dear Reviewers and Editor,
Thank you for giving us the opportunity to revise our manuscript. We are deeply grateful to the reviewers for their insightful comments and constructive suggestions, which have significantly helped us to improve the quality and clarity of our work. We have carefully considered all the points raised and have made corresponding revisions to the manuscript. Our point-by-point responses to the comments are detailed below.
Comment 1: Elaboration on the spatial coupling of high Chl a and APA. Figure 3 shows a spatial overlap between areas of high Chl a concentration and high APA, particularly near the Xiaoqing River (XQR) and Yellow River (YR) estuaries. It is suggested to explicitly point out this coupling relationship in Section 3.2 or 3.3 of the Results. For example, adding a sentence like: "Notably, the spatial distributions of high Chl a concentrations and high APA_total values showed remarkable consistency, especially in the adjacent areas of the XQR and YR estuaries." This would visually strengthen the conclusion that phytoplankton biomass is an important driver of APA.
Response: We thank the reviewer for this excellent suggestion. We have added the following sentence to section 3.3. Alkaline phosphatase activity and kinetics in the LZB (Line 255-257 in the revised manuscript) to explicitly highlight this important spatial pattern: “The spatial distributions of high Chl a concentrations and high APAtotal values showed consistency, especially in the adjacent areas of the XQR and YR estuaries (Figure 3). “This addition strengthens the visual connection between phytoplankton biomass and APA, as suggested.
Comment 2: Regarding the universality of the PO₄-P concentration threshold. The study identifies a distinct PO₄-P threshold (0.05 μmol·L⁻¹), which is well-supported by data from autumn and the enclosure experiment. However, the winter data show no significant correlation between APA and either PO₄-P or the DIN:PO₄-P ratio. It is recommended that the authors further discuss the potential reasons for this seasonal discrepancy in the discussion section. For instance, is it due to direct suppression of microbial activity and enzyme production by low winter temperatures, or does the phytoplankton community structure in winter (e.g., potentially diatom-dominated) inherently respond differently to P stress compared to summer/autumn communities? A deeper analysis of these factors would help clarify the boundary conditions under which the "threshold" concept applies.
Response: We agree with the reviewer that discussing the seasonal discrepancy is crucial for our findings. We have now deepened the discussion in Section 4.2 (Lines 436-443) to include an analysis of the potential reasons. “The absence of a significant APA response to low PO4-P or high DIN:PO4-P ratios in winter may be attributed to several factors. Low temperatures likely directly suppress microbial metabolic rates and enzyme production, including APA. Additionally, seasonal shifts in phytoplankton community composition—such as a predominance of diatoms in winter, which may exhibit an inherently lower responsiveness to phosphorus stress compared to the autumn assemblages—could also contribute to the observed seasonal discrepancy in APA regulation (Ivancic et al., 2016; Ou et al., 2024).”
Comment 3: Clarifying the role of dominant taxa in APA. The manuscript states that APA in autumn was primarily derived from phytoplankton. Could the authors briefly discuss the potential role of the dominant species (e.g., specific genera within diatoms) or functional groups (e.g., groups known to strongly induce APA under P stress) present in the phytoplankton community during that season? Even in the absence of species-level data, referencing existing literature on which common taxa tend to exhibit high APA under P stress would make the conclusion of "phytoplankton-dominated APA" more concrete and enriched.
Response: This is a valuable point. While our dataset does not include species-level identification in field monitoring, we have enriched our discussion by referencing literature on phytoplankton functional groups and culture experiment data. In the Discussion section 4.2 (Lines 449-458), we now state that: Different phytoplankton species responded differently to variations in active phosphate concentrations. For instance, dinoflagellates expressed APA at relatively higher active phosphate concentrations than diatoms, indicating a greater demand for active phosphate, potentially due to their higher DNA content (Hackett et al., 2005; Nicholson et al., 2006). Changes in the dominant phytoplankton species were also observed during the culture experiments (Figure 5). Significant differences in phytoplankton biodiversity between the two cultures (Figure 5) suggest that individual phytoplankton species adapt differently to phosphorus limitation. This leads to varying growth trends among species, resulting in changes to phytoplankton community structure and biodiversity (Ivancic et al., 2016).
Comment 4: Regarding the explanation of the ecological significance of kinetic parameters. The study measured the enzyme kinetic parameters Km and Vmax and observed their changes below the identified threshold. It is recommended that the discussion include an explanation of the ecological implications of these parameter changes using more intuitive ecological terminology. For instance, a decrease in Km may indicate enhanced enzyme affinity for the substrate, representing an adaptive strategy under low-phosphorus conditions. Adding such an explanation would make these biochemical parameters more accessible to a broader audience of ecologists, thereby enhancing the readability and impact of the manuscript.
Response: We thank the reviewer for this suggestion to improve the accessibility of our work. We have revised the relevant paragraph in the Discussion seaction 4.2 (Lines 426-432) to explicitly explain the ecological implications of the kinetic parameters. Specifically, we now state: Moreover, enzyme kinetics data from field investigations in LZB further validated the phosphate concentration threshold, showing that AP activity and substrate affinity increased significantly when phosphate concentration dropped below the threshold (Figure S7). The in autumn was lower than in winter, while was much higher in autumn than in winter. Higher AP production () and stronger substrate affinity () in autumn resulted in higher APA (Figure 3).
Comment 5: Improving language clarity and chart specifications. The MS is generally well-writton, but still need improve language clarity and fluency. Some sentences are unnecessarily long or awkwardly phrased.
Response: We have thoroughly reviewed the entire manuscript and revised it for language clarity and fluency. Several long and complex sentences have been broken down into shorter, more direct statements to improve readability. We believe these edits have significantly improved the overall clarity of the manuscript.
Comment 6: In Figure 6, the scatter plot in the lower panel demonstrates a positive correlation between APA and the DIN:PO₄-P ratio, distinguishing between "high APA" and "low APA" groups. It would be beneficial to briefly state in the figure caption or main text the criterion used to separate these two groups (e.g., based on a specific APA value, or using a statistical clustering method). This would enhance the transparency of the analytical approach.
Response: We appreciate the reviewer's comment regarding the transparency of our method. We have now clarified the grouping criterion in Result seaction 3.4 (Lines 329-333) . The revised caption includes the following sentence: During the former, this linear relationship could be further divided into two groups by K-means cluster analysis based on APA, such that the slope of the fitted straight line was significantly higher in the high APA group (P < 0.01; line read in Figure 6) relative to that of low APA group (P < 0.01; line black in Figure 6).
Citation: https://doi.org/10.5194/egusphere-2025-4047-CC2 -
CC3: 'Reply on CC2', Yanqun Yang, 16 Nov 2025
Dear Reviewers and Editor,
Thank you for the opportunity to revise our manuscript. We are deeply grateful to the reviewers for their thoughtful and constructive comments, which have been invaluable in improving our work. We have carefully addressed all the points raised, and our detailed point-by-point responses are provided below.
Comment 1: Lines 24-27 (Abstract): The phrase "combined analysis of field and experimental data, p<0.01; n=36" is vague. Please specify the variables...
Response 1: We thank the reviewer for this suggestion to improve the clarity of our abstract. We have rephrased the sentence as recommended. The relevant text in the abstract now reads:
"A significant positive correlation was observed between APA and the DIN:PO4-P ratio in the combined dataset (p < 0.01, n = 36)."
Comment 2: Lines 114-118 (Mesocosm Experiment Methods): ...Including these critical values in the text would improve readability.
Response 2: We agree with the reviewer that including the initial concentrations improves readability. We have added the following sentence to Section 2.3 (Lines 122-125):
"The initial DIN and PO4-P concentrations for the CG were 1.90 μmol L⁻¹ and 0.04 μmol L⁻¹, respectively. For the TG, concentrations were adjusted to 50.01 μmol L⁻¹ and 1.78 μmol L⁻¹ to achieve the target ratio."
Comment 3: Lines 318-320 (Results 3.4): The basis for categorizing the data into "high APA" and "low APA" groups... is not defined.
Response 3: We appreciate the reviewer's comment regarding the transparency of our method. We have now clarified the grouping criterion in Section 3.4 (Lines 329-333). The revised text states:
"During the former, this linear relationship could be further divided into two groups by K-means cluster analysis based on APA, such that the slope of the fitted straight line was significantly higher in the high APA group (P < 0.01; red line in Figure 6) relative to that of the low APA group (P < 0.01; black line in Figure 6)."
Comment 4: Lines 437-443 (Discussion on DON): The discussion on the potential mechanisms linking DON to APA... could be further strengthened.
Response 4: We thank the reviewer for this insightful suggestion. We have significantly strengthened this part of the discussion in Section 4.2 (Lines 477-488) by integrating our own observations and citing more specific literature. The added text reads: "It is noteworthy that prior studies have found that under the same N:P ratio and active phosphate concentration, phytoplankton APA in DON-enriched cultures was significantly higher than that in cultures without DON (Fitzsimons et al., 2020). Our winter survey data revealed a significant positive correlation between DON and APAbac (Figure 4), similar to findings for APAphyto and DON concentration (Ou et al., 2024), indicating that not only PO4-P but also nutrient composition and ratios can affect APA. Regulation of APA by DON may potentially operate through specific DON components or their degradation products influencing P metabolic pathways, or through DON serving as an alternative N source that stimulates phytoplankton growth and P demand (Ma et al., 2018; Forchhammer et al., 2022). Meanwhile, DON serving as an alternative nitrogen source may enhance phytoplankton growth and phosphorus demand."
Comment 5: Line 49-50 and throughout the text (Terminology): The abbreviation "AP" for Alkaline Phosphatase is used in the abstract without the full term being presented first.
Response 5: We apologize for this oversight. The abbreviation "AP" has now been properly defined upon its first appearance in the abstract. Furthermore, in the Discussion (Lines 463-469), we have added brief explanatory phrases for specialized terms as suggested: "When ambient PO4-P concentrations fall below the 0.05-0.2 μmol L⁻¹ threshold (Dyhrman et al., 2007), phytoplankton activate the pho regulon (a gene suite responsible for phosphorus scavenging) operon (a gene cluster containing the pstSCAB phosphate transport system and phoA/phoX alkaline phosphatase genes) through the PhoB/PhoR two-component system (a P-sensing system consisting of histidine kinase PhoR and the response regulator PhoB) (Lin et al., 2016)."
Comment 6: Typos and Minor Corrections. Line 22: "an enclosure experiments" has been corrected to "an enclosure experiment". Line 25: "ssignificant" has been corrected to "significant".
Response 6: We sincerely thank the reviewer for their meticulous reading. All typos and minor errors have been corrected throughout the manuscript.
Once again, we extend our sincere gratitude to the reviewers and the editor for their valuable comments and guidance. We believe the manuscript has been significantly improved and hope it is now suitable for publication.
Citation: https://doi.org/10.5194/egusphere-2025-4047-CC3
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CC3: 'Reply on CC2', Yanqun Yang, 16 Nov 2025
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RC1: 'Reply on CC1', Dongliang Lu, 11 Nov 2025
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RC2: 'Comment on egusphere-2025-4047', Anonymous Referee #2, 09 Jan 2026
Yang et al. investigate the relationships among phosphorus concentrations, N:P ratios, and alkaline phosphatase activity (APA) in Laizhou Bay, China. By integrating intensive field observations with enclosure experiments, the authors demonstrate that phosphate availability is a key regulator of APA induction. In addition, genetic analyses show that the expression of alkaline phosphatase–related genes increases only when phosphate concentrations fall below a threshold identified in this study. These findings support a conceptual framework in which APA-mediated dissolved organic phosphorus (DOP) utilization enhances phytoplankton biomass, accelerates phosphate depletion, and intensifies N:P imbalance, thereby favoring phytoplankton genotypes with elevated APA. Overall, the manuscript is well structured, and the conclusions are generally well supported by extensive field and experimental data. However, the quality of writing needs substantial improvement, and an overall language polish is recommended to enhance readability. Moreover, several textual descriptions are inconsistent with the figures, and the entire manuscript should be carefully checked for accuracy and consistency.
Specific comments:
Line 24: Delete “an”.
Line 27: Spelling correction needed for “significant”.
Line 37: The term “emission” is typically used for gases; consider alternatives such as “inputs” or “loading”.
Line 57: Please clarify “OP”; it likely refers to phosphate or dissolved inorganic phosphorus (DIP).
Line 59: Replace “seas” with “coastal systems”.
Line 66: This statement is repetitive; N:P limitation is equivalent to a high N:P ratio.
Line 68: Please clarify why freshwater inputs would enhance APA—does this relate to freshwater having intrinsically high N:P ratios?
Line 87: Consider explicitly stating the objectives of the study at the end of the Introduction.
Line 92: Specify whether “mean depth” is intended. Also clarify the meaning of “half-exchange”.
Line 98: The phrase “affect the ecosystems” is vague; please provide region-specific examples.
Line 110: More detail on field sampling is needed, including sampling frequency and methods for water collection, transport, and preservation.
Line 121: Please justify the choice of an N:P ratio of 28 (half of 55). Clarify the rationale for the nitrogen and phosphorus concentrations used in the treatment groups, which appear substantially higher than in the control.
Line 123: Explain the rationale for sampling 17 times at 2-day intervals.
Line 198: Given the dominant nutrient inputs from the Yellow and Xiaoqing Rivers (Fig. 2), analyses of N:P ratios for all rivers may be unnecessary, as the remaining tributaries likely contribute only a small fraction of total loading.
Line 216: Clarify how temperature and other environmental parameters were spatially interpolated.
Lines 231–233: The N:P ratio results should be presented in the main text rather than in the supplementary material.
Lines 261–265: The analysis described here does not appear consistent with the corresponding figure; please verify and revise.
Line 273: Add subtitles to the figure indicating “summer” and “winter”.
Lines 283–286: The text is not fully consistent with the figure content and should be revised.
Line 310: APAtotal is referenced but not provided; please include this information.
Lines 412–414: The proposed approach to reducing the N:P ratio by increasing phosphorus inputs may not be practical; please reconsider or further justify this recommendation.
Citation: https://doi.org/10.5194/egusphere-2025-4047-RC2 -
AC2: 'Reply on RC2', Yanqun Yang, 12 Jan 2026
We sincerely thank the reviewer for their thorough evaluation and the positive assessment of our study's scientific contribution, structure, and supporting data. We are particularly encouraged by their recognition of the integrated field-experimental approach and the conceptual framework.We fully acknowledge the reviewer's critical points regarding the need for substantial improvement in writing quality and the inconsistencies between the text and figures. We have taken these comments very seriously and have implemented comprehensive revisions to address them:
The entire manuscript has undergone extensive language polishing by a professional editing service by V. Monica Bricelj, Ph.D. from TechMar Resrch Inc. We have focused on improving sentence structure, clarity, terminology, and overall flow to enhance readability, while strictly preserving the scientific content.
We have carefully and systematically checked the entire manuscript against all figures and tables. All identified inconsistencies have been corrected. The text now accurately and precisely reflects the data presented in the figures.
We believe these revisions have significantly improved the clarity, accuracy, and professionalism of the manuscript. We are grateful for the reviewer's constructive suggestions, which have undoubtedly strengthened our paper.
All point-by-point responses to the specific comments are provided below.
Specific comments:
Line 24: Delete “an”.
Response: Thank you for pointing out this typographical error. The word “an” has been deleted from Line 24.
Line 27: Spelling correction needed for “significant”.
Response: Thank you for your careful reading. The spelling of “significant” on Line 27 has been corrected.
Line 37: The term “emission” is typically used for gases; consider alternatives such as “inputs” or “loading”.
Response: Thank you for this precise suggestion. As recommended, the term “emissions” has been changed to “inputs” in the revised manuscript (Line 37, now reads: “With the intensification of nitrogen (N) inputs and the control of phosphorus (P) emissions, P limitation has become widespread in global coastal waters (Zhang et al., 2024; Maavara et al., 2020; Liang et al., 2023) and has triggered adverse ecological consequences, including exacerbated eutrophication and shifts in phytoplankton community structure (Xin et al., 2019; Peñuelas and Sardans, 2022). ”).
Line 57: Please clarify “OP”; it likely refers to phosphate or dissolved inorganic phosphorus (DIP).
Response: Thank you for raising this point. The abbreviation “OP” did indeed refer to organophosphates. To avoid confusion, the text has been clarified as follows: “To cope with P limitation, microorganisms have evolved a range of strategies, including an increase in inorganic phosphate transporters, induction of hydrolases for scavenging organophosphates (OP, organic phosphorus compounds), and a reduction in P demand by replacing phospholipids with sulfur- or N-containing lipids (Van et al., 2006; Karl, 2014; Lin et al., 2016). ”.
Line 59: Replace “seas” with “coastal systems”.
Response: Thank you for the suggestion to improve the terminology. The word “seas” has been replaced with the more precise term “coastal systems” as recommended.
Line 66: This statement is repetitive; N:P limitation is equivalent to a high N:P ratio.
Response: We agree with the reviewer's point. The redundant phrasing has been revised for conciseness. The sentence now reads: “A high DIN:PO4-P ratio (indicating N excess and P limitation) has been confirmed to be positively correlated with APA...” . This revision merges the two concepts into a single, clearer statement.
Line 68: Please clarify why freshwater inputs would enhance APA—does this relate to freshwater having intrinsically high N:P ratios?
Response: Thank you for this suggestion, which helped clarify the mechanism. As recommended, we have explicitly linked freshwater inputs to high N:P stoichiometry. The revised text now reads: “Freshwater inputs, which often carry nutrients with intrinsically high N:P ratios, can thereby enhance APA...” .
Line 87: Consider explicitly stating the objectives of the study at the end of the Introduction.
Response: Thank you for this constructive suggestion. As recommended, we have added a dedicated “Objectives of the study” paragraph at the end of the Introduction. It explicitly states our aims to identify the PO4-P concentration threshold, evaluate the synergistic role of the DIN:PO4-P ratio, assess seasonal and community-level APA partitioning, and validate these dynamics with genetic evidence, thereby refining the conceptual framework for AP regulation.
Line 92: Specify whether “mean depth” is intended. Also clarify the meaning of “half-exchange”.
Response: Thank you for these suggestions to improve clarity. We have revised the sentence as follows: “It covers an area of approximately 7,000 km² with a coastline of 320 km, a mean depth of less than 10 m, and a water half-exchange time (i.e., the time for 50% water renewal) of 55 days (Wu et al., 2023).”
Line 98: The phrase “affect the ecosystems” is vague; please provide region-specific examples.
Response: Thank you for the suggestion to make the ecological impact more concrete. The text has been revised to include specific, regionally-relevant examples. It now reads: “The resulting increase in N availability, coupled with intensifying P limitation, has triggered significant ecological shifts in the bay, including more frequent harmful algal blooms and changes in dominant phytoplankton species from diatoms to dinoflagellates (Song et al., 2017; Xin et al., 2019).”
Line 110: More detail on field sampling is needed, including sampling frequency and methods for water collection, transport, and preservation.
Response: The revision specifies that marine surface water was collected using a 5-L Niskin bottle. For riverine water samples collected at the mouths of ten major rivers, sampling was conducted following standard protocols for surface water monitoring in China (specifically, HJ 494-2009). An organic glass water sampler or a pre-cleaned polyethylene container was used to collect subsurface water at a depth of approximately 0.5 m. All samples (riverine and marine) underwent immediate on-site processing into aliquots for different analyses (nutrients, APA, molecular biology) to ensure comparability. It also clarifies that all samples were preserved according to the specific protocols detailed in sections 2.4.1–2.4.4 and transported on ice to the laboratory within 12 hours.
Line 121: Please justify the choice of an N:P ratio of 28 (half of 55). Clarify the rationale for the nitrogen and phosphorus concentrations used in the treatment groups, which appear substantially higher than in the control.
Response: Thank you for this suggestion to clarify the experimental design. We have revised the text to explicitly justify both aspects. The choice of the DIN:PO4-P ratio of 28 for the treatment group (TG) was based on creating a clear contrast to the ambient ratio (55) and moving toward the classical Redfield stoichiometry (16:1) to test responses under mitigated imbalance. Furthermore, the higher absolute nutrient concentrations in the TG (DIN: 50.01, PO4-P: 1.78 ) compared to the control (CG) (DIN: 1.90, PO4-P: 0.04 ) were necessary to ensure that nutrients remained non-limiting throughout the 29-day incubation, allowing the target N:P ratio to be the primary variable influencing the system over time.
Line 123: Explain the rationale for sampling 17 times at 2-day intervals.
Response: Thank you for prompting us to clarify the sampling strategy. The sampling regimen (17 times over 29 days) was designed to capture the full temporal dynamics of the system. As detailed in the revised text, the frequency was actively adjusted: In parallel, culture water samples were collected 17 times over the 29-day incubation. The sampling regimen was actively adjusted based on observed system dynamics: a high frequency (approximately daily) was maintained during the initial period of rapid change. Once nutrients were largely depleted and biological activity plateaued, sampling intervals were lengthened to monitor the sustained response without unnecessary density.
Line 198: Given the dominant nutrient inputs from the Yellow and Xiaoqing Rivers (Fig. 2), analyses of N:P ratios for all rivers may be unnecessary, as the remaining tributaries likely contribute only a small fraction of total loading.
Response: Thank you for this comment. We agree that the Yellow and Xiaoqing Rivers are the dominant point sources. The comprehensive analysis of ten rivers, however, was conducted to evaluate the nutrient stoichiometry of the entire watershed. This approach confirms that elevated N:P ratios are a pervasive characteristic of the regional riverine inputs, not an artifact of the largest sources alone. This broader perspective strengthens the conclusion that anthropogenic nutrient imbalance is a basin-wide issue, which is relevant for managing non-point source pollution. The data have been retained in Fig. 2 to support this finding.
Line 216: Clarify how temperature and other environmental parameters were spatially interpolated.
Response: Thank you for this suggestion to improve methodological transparency. As requested, a sentence has been added to clarify the interpolation method (Line 216-218). The spatial distribution maps for all parameters (e.g., temperature, salinity, nutrients, APA, Chl a) were generated using ordinary kriging interpolation in Surfer (version 16, Golden Software LLC), a geostatistical method that accounts for spatial autocorrelation to produce optimal, unbiased estimates for continuous surfaces.
Lines 231–233: The N:P ratio results should be presented in the main text rather than in the supplementary material.
Response: Thank you for this suggestion. We agree that the DIN:PO4-P ratio is a key parameter for interpreting the ecological status. In response, we have moved the relevant data from the supplementary material to the main text. The spatial distribution of the DIN:PO4-P ratio is now included in Figure 3, alongside PO4-P and Chl a, providing readers with a direct visual comparison of these critically linked variables. The corresponding description and discussion have been integrated into Sections 3.2.2.
Lines 261–265: The analysis described here does not appear consistent with the corresponding figure; please verify and revise.
Response: We thank the reviewer for their careful scrutiny. We have re-examined the correlation analysis and the corresponding Figure 4. The description in the original text was indeed not fully aligned with the figure. The relevant paragraph in Section 3.3 has been thoroughly revised to accurately reflect the patterns shown in Figure 4.
Line 273: Add subtitles to the figure indicating “summer” and “winter”.
Response: Thank you for this suggestion to improve the clarity of the figure. As recommended, subtitles clearly indicating “Autumn” and “Winter” have been added to the respective panels of Figure 4.
Lines 283–286: The text is not fully consistent with the figure content and should be revised.
Response: Thank you for highlighting this inconsistency. We have carefully revised the text in Lines 283–286 to ensure it accurately describes the nutrient dynamics presented in Figure 5. The updated description now clearly and correctly reflects the observed changes in DIN:PO4-P uptake ratios, the temporal trends of the DIN:PO4-P ratio in both the CG and TG, and the dynamics of DON and DOP concentrations during the initial phase of the enclosure experiment.
Line 310: APAtotal is referenced but not provided; please include this information.
Response: Thank you for pointing out this omission. As suggested, the average values for all APA components in the treatment group (TG), including APAtotal, have now been explicitly provided in the revised text. The sentence now reads: “Average values of APAphy, APAbac, APAfree, and APAtotal in the CG were 47.5, 22.6, 54.8, and 687.6 nmol·L-1·h-1.Average values of APAphy, APAbac, APAfree, and APAtotal in the TG were 5.1, 2.9, 42.2, and 122.8 nmol·L-1·h-1.” This addition ensures that both the control and treatment group data are fully presented.
Lines 412–414: The proposed approach to reducing the N:P ratio by increasing phosphorus inputs may not be practical; please reconsider or further justify this recommendation.
Response: We agree with the reviewer that increasing phosphorus inputs to lower the N:P ratio would be counterproductive and impractical, as it could exacerbate eutrophication. Our intended recommendation was precisely the opposite: to emphasize source control of nitrogen to mitigate the N:P imbalance and restore ecological balance as the primary management lever. We have revised the text in the Discussion to clearly state that: Management strategies must prioritize the reduction of nitrogen inputs at their source to mitigate the N:P imbalance and restore ecological balance, rather than relying on intrinsic biological compensation within the coastal zone. We thank the reviewer for highlighting this ambiguity, which allowed us to clarify this critical point.
Citation: https://doi.org/10.5194/egusphere-2025-4047-AC2
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AC2: 'Reply on RC2', Yanqun Yang, 12 Jan 2026
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This manuscript presents a comprehensive investigation by land-sea synchronous field surveys and enclosure experiments to reveal the potential regulatory mechanisms of alkaline phosphatase activity (APA) in the coastal waters of Laizhou Bay (LZB). The authors proved that the absolute phosphate (PO₄-P) concentration and the ratio nitrogen-to-phosphate ratio (DIN:PO₄-P) have synergistic effects on regulation of the APA secretion and organophosphorus utilization. The combination of geochemical measurements and molecular biological evidence provides valuable insights into phosphorus cycling driven by anthropogenic nitrogen inputs in coastal zones of LZB.
Overall, this is a well-structured and well-supported manuscript that meets the standards for publication. However, several issues should be addressed or clarified to further strengthen the manuscript.
Figure 3 shows a spatial overlap between areas of high Chl a concentration and high APA, particularly near the Xiaoqing River (XQR) and Yellow River (YR) estuaries. It is suggested to explicitly point out this coupling relationship in Section 3.2 or 3.3 of the Results. For example, adding a sentence like: "Notably, the spatial distributions of high Chl a concentrations and high APA_total values showed remarkable consistency, especially in the adjacent areas of the XQR and YR estuaries." This would visually strengthen the conclusion that phytoplankton biomass is an important driver of APA.
The study identifies a distinct PO₄-P threshold (0.05 μmol·L⁻¹), which is well-supported by data from autumn and the enclosure experiment. However, the winter data show no significant correlation between APA and either PO₄-P or the DIN:PO₄-P ratio. It is recommended that the authors further discuss the potential reasons for this seasonal discrepancy in the discussion section. For instance, is it due to direct suppression of microbial activity and enzyme production by low winter temperatures, or does the phytoplankton community structure in winter (e.g., potentially diatom-dominated) inherently respond differently to P stress compared to summer/autumn communities? A deeper analysis of these factors would help clarify the boundary conditions under which the "threshold" concept applies.
The manuscript states that APA in autumn was primarily derived from phytoplankton. Could the authors briefly discuss the potential role of the dominant species (e.g., specific genera within diatoms) or functional groups (e.g., groups known to strongly induce APA under P stress) present in the phytoplankton community during that season? Even in the absence of species-level data, referencing existing literature on which common taxa tend to exhibit high APA under P stress would make the conclusion of "phytoplankton-dominated APA" more concrete and enriched.
The study measured the enzyme kinetic parameters Km and Vmax and observed their changes below the identified threshold. It is recommended that the discussion include an explanation of the ecological implications of these parameter changes using more intuitive ecological terminology. For instance, a decrease in Km may indicate enhanced enzyme affinity for the substrate, representing an adaptive strategy under low-phosphorus conditions. Adding such an explanation would make these biochemical parameters more accessible to a broader audience of ecologists, thereby enhancing the readability and impact of the manuscript.
The MS is generally well-writton, but still need improve language clarity and fluency. Some sentences are unnecessarily long or awkwardly phrased.
In Figure 6, the scatter plot in the lower panel demonstrates a positive correlation between APA and the DIN:PO₄-P ratio, distinguishing between "high APA" and "low APA" groups. It would be beneficial to briefly state in the figure caption or main text the criterion used to separate these two groups (e.g., based on a specific APA value, or using a statistical clustering method). This would enhance the transparency of the analytical approach.
Recommendation
Minor revision. The manuscript is timely, novel, and well-supported by data, but the above points should be addressed to improve scientific rigor, clarity, and broader impact.