This manuscript addresses an important and timely topic: how estuarine mixing reshapes the partitioning, composition, and potential bioavailability of organic nitrogen. The distinction among PON, CON, and tDON is meaningful, and the comparison between biologically active mixing (BAM) and biologically inhibited mixing (BIM) is a potentially strong feature of the study. The analytical framework is also relatively comprehensive, combining TFF-based fractionation, fluorescence signatures, FTIR, stable isotopes, and ancillary particle and enzyme measurements. Overall, the study has clear potential and is built on a potentially publishable dataset.

Specific Comments

1. The central message should be sharpened.At present, the manuscript tries to emphasize too many things at once: ON partitioning, DON bioavailability, PON refractoriness, nitrogen sequestration, eutrophication risk, and a broadly generalized estuarine framework. This weakens the paper’s main contribution. In my view, the strongest and most defensible message is that estuarine mixing generates a dual response: humic-like ON is preferentially transferred into particulate/refractory pools through adsorption and flocculation, while biological processing simultaneously alters the residual dissolved pool toward a more potentially labile composition. I encourage the authors to reorganize the abstract, discussion, and conclusions around this narrower and clearer core advance.
2. The manuscript interprets increasing protein-like fluorescence, higher FI and BIX, lower SUVA254, and NH₄⁺ accumulation as evidence for enhanced DON bioavailability. These are useful and suggestive indicators, but they remain indirect proxies. The study does not include direct DON bioassays, uptake experiments, or response experiments demonstrating that the transformed dissolved nitrogen pool is more readily used by microbes or phytoplankton. Therefore, I suggest that the manuscript consistently distinguish between demonstrated transformation and inferred increases in potential bioavailability.
3. L84 provides coordinates for the riverine and marine endmembers and notes that the seawater site was located approximately 40 km downstream of the river mouth, but there is no figure showing the sampling region, the river mouth, and the relative positions of the two endmembers. A map is a basic but important element for a study of this type. It would help readers assess the geographic setting, the representativeness of the chosen endmembers, and the realism of the experimental river–sea mixing framework. I strongly recommend adding a location figure.
4. L92-93 states that river water and seawater were mixed at 10:0, 8:2, 6:4, 5:5, 4:6, 2:8, and 0:10, but the manuscript does not explain why these particular ratios were selected. Were they chosen to represent specific salinity zones in the estuary, to match field observations, or simply to generate a broad mixing gradient? This matters because the interpretation of non-conservative behavior may depend on how well the critical salinity range is resolved. The authors should provide a clear rationale for this design choice.
5. Figure 4 presents direct experimental observations, including FI, SUVA254, BIX, FTIR, POC, and C/N changes along the salinity gradient. They are primary results. Moving Figure 4 to the Results would also improve the structure of the paper.
6. The manuscript ends by linking its findings to coastal eutrophication risk and bloom development. This is a reasonable broader implication, but it extends beyond what is directly shown in a short-term controlled mixing experiment based on one summer sampling event. The transformed ON pool may indeed have ecological implications, but the paper does not directly demonstrate downstream biological responses in the receiving coastal system. I recommend framing this as a likely implication or testable hypothesis, not as an outcome already established by the present study.

Technical Corrections

1.  L87 “dissolved oxygen (DO) were measured in Table S1”
2. L171-172 “While in the BAM treatments” it should almost certainly refer to BIM？
3. There is an apparent inconsistency regarding Figure 4d: the figure caption says FTIR is shown for BAM only, whereas the Discussion interprets the FTIR trends as occurring in the BIM.
4. Supplement

(1) Text S1the TFF cleaning protocol appears to omit the concentrations for NaOH and HCl (“mol L^-1 NaOH” and “mol L^-1 HCl”)

(2) “Pdl” should presumably be “PdI,” and “Phl” also appears to be a typo or mislabeled parameter. Please check both the text and the figure labeling.

(3) The supplement still contains typographical errors such as “doenotes” in the Figure S8 caption.

(4) Table S1 “Sampling data” should be “Sampling date.”

(5) Table S2 in the supplement incorrectly refers to “truly dissolved organic carbon (tDON)” and should be corrected to nitrogen.

General comments

This manuscript investigates organic nitrogen transformation and bioavailability during estuarine mixing using a dual-treatment design (BAM vs. BIM) to separate biological from physicochemical processes. The topic is relevant to BG's scope, and the experimental approach — combining tangential flow filtration, EEM-PARAFAC, stable isotopes, FTIR, and enzyme assays — is comprehensive. The three-pathway conceptual model and the "enhanced nitrogen pump" framework are valuable contributions.

Scientific significance: The BAM/BIM design effectively isolates biological and physicochemical contributions to ON fate, which is difficult to achieve in field studies. The global estuarine ON synthesis (Fig. 5, Table S4) adds breadth.

Scientific quality: The analytical methods are sound, but several issues in data interpretation weaken key arguments — notably an apparent inconsistency between the FTIR description and figure caption (Line 257–258), a questionable zeta potential argument (Line 189–192), and insufficient support for the claimed humic-to-protein transformation pathway (Line 260–275).

Presentation quality: Generally well-organized, but inconsistent abbreviations, ambiguous method descriptions, and a disproportionately long limitations section reduce clarity. The Conclusions section lacks focus.

Specific Comments

Lines 119–122: The description of Zetasizer measurements immediately follows FTIR analysis of freeze-dried DON, making it unclear what matrix was actually measured for particle size and zeta potential. The measurement target should be specified.

Lines 142–143: The statement that declining CON and tDON concentrations cause the DON/ON ratio to decrease is logically incomplete — the ratio also depends on concurrent PON changes.

Lines 189–192: The claim that negative zeta potentials (ζ < 0) promote DON adsorption onto SPM is inconsistent with colloidal chemistry, where strongly negative ζ indicates greater electrostatic repulsion and colloidal stability. The roles of surface adsorption and colloidal destabilization/flocculation need to be distinguished.

Lines 257–258: FTIR evidence is attributed to the BIM treatments, but the Fig. 4d caption states these spectra are from BAM only. This contradiction undermines a key argument in Section 4.2.

Lines 260–275: The strong negative correlation between C2 and (C1+C3) does not establish that humic-like components are directly transformed into protein-like components. Alternative explanations (e.g., de novo microbial production of C2) could be considered.

Text S1 describes a two-step TFF (10 kDa + 1 kDa) yielding three size fractions, but only two are reported. The rationale for merging the >10 kDa and 1–10 kDa fractions is not explained.

Section 4.5 (Limitations) is disproportionately long and risks overshadowing the study's strengths. The Conclusions section partly repeats the Discussion and could be more concise and quantitative.

Technical Corrections

Line 30: "where critically mediate" — missing subject.

Line 95–96: "to inhibited" — should be "to inhibit."

Line 127 and Fig. S2 caption: "Phl" — should be "PdI."

Line 188: "are govern by" — should be "are governed by."

Line 309: "This study highlight" — should be "highlights."

Line 351: "component effects" — unclear; perhaps "compound effects"?

Fig. 2 legend: "phasa" — should be "phase."

Fig. S2a: Treatment labels "BEM"/"APM" are inconsistent with the defined abbreviations "BIM"/"BAM."

Manuscript egusphere‑2026‑216 employs laboratory incubation experiments to investigate the transformation of dissolved organic nitrogen (DON) and particulate organic nitrogen (PON), as well as nitrogen bioavailability. The study offers valuable insights with clear relevance to future research on nitrogen biogeochemical cycling in coastal waters. Overall, the experimental design is robust and the results are interesting and potentially impactful. However, several aspects require further clarification:

1. Between line 50 and 55: is there a citation for “Humic-like (terrestrial and microbial sources) and protein-like components are considered as the dominant terrestrial DON components entering the sea”?
2. Between line 110 and 115: authors mentioned the PN was considered equivalent to PON because PIN is less than 5%. Wonder how much would it affect the isotopic ratios? Especially if the nitrogen isotopic values of PIN vary along the salinity gradient, would it affect the observed trend?
3. Between line 115 and 120: does the S in “Additional water quality parameters including S, pH, SPM, and chlorophyll-a (Chl-a) were measured” stand for sulfur? Why is sulfur collected and how would affect nitrogen? I don’t think I have seen the relevant discussion in the manuscript.
4. In results, the manuscript mentioned the DON indicated a non-conservative mixing and PON indicated a conservative mixing. Do you have data to support it? All I can see from the paragraph and figures is that DON decreases with increasing salinity, which looks like conservative for DON.
5. Between line 155 and 160: what are the theoretical fluorescence values?
6. Between line 170 and 175: numbers from −28.3‰ to −26.5‰, 1.1‰ to 8.8‰, −26.1‰ to −25.9‰, and 6.2‰ to 7.2‰, are them statistically different? If not, it would be a stretch to say they decline along the salinity gradient, especially Figure 3 didn't look like these numbers decreased when the salinity increased.
7. Between line 225 and 230: How does the Fig S5a indicate the decrease in the intensity of C1? I didn't found a decrease trend in the figure and also decrease with what? With time, treatment numbers, or salinity?
8. Between line 230 and 235: what are the fluorescence index and biological index and how are them measured? You would want to address them in the methods section first.