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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Status: open (until 18 Dec 2025)
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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
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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 -
CC2: 'Reply on CC1', Yanqun Yang, 16 Nov 2025
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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
reply
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
reply
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RC1: 'Reply on CC1', Dongliang Lu, 11 Nov 2025
reply
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- 1
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.