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Chemical Characteristics of Dark-Brown Humic-like Substances Formed from the Abiotic Condensation of Maillard Precursors with Different Glycine Concentrations - https://doi.org/10.3390/agronomy12092199

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Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

Reviewer #1: Partly

Reviewer #2: Yes

Reviewer #3: Yes

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2. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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3. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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4. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: No

Reviewer #2: Yes

Reviewer #3: Yes

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Reviewer #1: This study optimized the concentration of glycine to enhance the gibbsite-catalyzed Maillard reaction process. While it holds certain research significance, the exploration of the underlying mechanisms is insufficient. Several issues require detailed revisions:

1. The title is too lengthy and needs further refinement.

2. The introduction should clearly compare existing research with the innovations of this study, explaining why gibbsite was chosen as the catalyst.

3. It is recommended to include a separate paragraph in the introduction outlining the research content and objectives.

4. Why was gibbsite synthesized instead of purchased commercially? Was the synthesized product tested for purity?

5. How were the concentrations of catechol, glucose, and glycine determined? The materials and methods section lacks supporting references.

6. Section 2.2 needs further reorganization to effectively distinguish between experimental design and humic-like acid extraction methods.

7. The rationale for selecting 28°C in the experiments should be clarified.

8. The discussion should address the limitations of this study and propose future validation methods.

9. Unnecessary numerical data should be reduced in the conclusion.

10. Abbreviations are inconsistent throughout the text (e.g., HS and HSs).

Reviewer #2: This manuscript focuses on optimizing glycine concentration to enhance the abiotic humification of catechol and glucose through a gibbsite-catalyzed Maillard reaction—a topic of considerable significance in the field of humic-like substance (HLS) formation. The study employs a sterile liquid shake-flask system and utilizes multiple analytical techniques (UV-Vis, TOC, FTIR, elemental analysis) to investigate the role of glycine, yielding valuable experimental data. However, several sections suffer from unclear logical connections, insufficient methodological details, and inadequate depth in interpreting results and contextualizing contributions. Minor revisions are required prior to publication.

1. The introduction highlights the limited understanding of gibbsite's synergy with amino acids but does not sufficiently contextualize the knowledge gaps regarding Al(III) (hydr)oxides in abiotic humification. For instance, it should elaborate on why Al(III) (hydr)oxides are less studied compared to Fe(III) or Mn(IV) oxides and what distinct properties of gibbsite may influence its catalytic behavior in Maillard-type humification.

2. The electrodialysis process for gibbsite preparation is described as lasting 3 months at room temperature, but key parameters (e.g., current density, voltage, membrane type) are omitted. These details are critical for reproducibility and could influence gibbsite’s surface properties and catalytic activity.

3. The selection of specific culture timepoints (0–360 hours) lacks justification. Rationale for sampling intervals (e.g., early vs. late stages) should be provided to support the experimental design.

4. The concentration of “concentrated HCl” used for acidification in HLA/FLA extraction is not specified. Variability in HCl concentration may influence the precipitation and quantification of humic-like fractions.

5. Figure 1 includes error bars, but numerical standard deviation values are not provided in the main text or supplement. Additionally, statistical comparisons among treatments at the final time point are missing.

6. Table 1 reports FTIR relative intensities as percentages but does not specify how the total area was defined (e.g., sum of selected bands or full spectrum). This ambiguity hinders interpretation and cross-study comparison.

7. The peak in E4/E6 ratios of HLA at 124 hours is noted but not explained. Potential reasons—such as accumulation of low-molecular-weight intermediates—should be discussed.

8. While glycine’s promoting effect is acknowledged, the molecular-level mechanism(s) behind its interaction with gibbsite, catechol, and glucose remain unclear. For example, does glycine facilitate surface-mediated condensation or act as a bridging molecule?

9� Comparisons with previous studies (e.g., Zhang et al., 2019; Zou et al., 2020) are descriptive rather than critical. The discussion should address how differences in precursor types and experimental systems (sterile model vs. composting) impact the applicability of the findings.

10. The limitations of the sterile liquid system are not acknowledged. Potential influences of environmental factors (e.g., microbes, additional minerals) on extrapolation to natural systems should be discussed.

Reviewer #3: This manuscript explored the optimization of glycine concentration to enhance the abiotic humification of catechol and glucose in a gibbsite-catalyzed Maillard system. It held theoretical value (deepening mechanistic understanding of abiotic humification) and practical potential (valorizing lignin-rich waste), with methodological strengths: a sterile liquid shake flask setup, integrated analyses (UV-Vis, TOC, FTIR, elemental analysis) for evaluating glycine’s effects on humic-like acids (HLA) properties, comprehensive data, and a key finding that 0.12 mol/L glycine optimized humification by balancing aromatic polymerization and functional group enrichment—demonstrating scientific novelty and relevance. Minor refinements were needed to improve the manuscript’s clarity, coherence, and scientific rigor (without compromising core findings). The comments are provided below.

1. Introduction Section

The introduction provides a general background on the Maillard reaction and humic-like substances (HLSs) but lacks a clear research gap that explicitly highlights why investigating gibbsite-catalyzed abiotic humification with varying glycine concentrations is urgently needed. Existing studies on metal oxides (e.g., MnO₂, Fe₂O₃) are mentioned, but the unique advantages or research value of gibbsite (a layered Al(III) hydroxide) compared to these well-studied oxides are not sufficiently elaborated.

2. Materials and Methods Section

2.1 In Section 2.1 (Preparation of Materials), the rationale for choosing a phosphate buffer with pH 8.0 is not explained. Since pH significantly affects the Maillard reaction and the surface charge of gibbsite (with a point of zero charge (PZC) of 8.2), why this specific pH was selected (e.g., to match gibbsite’s PZC, or to simulate a specific environmental condition) should be clarified.

2.2 The experimental design (Section 2.2) mentions that all reactions were conducted in triplicate, but details on how experimental reproducibility was validated (e.g., statistical analysis of replicate data, coefficient of variation) are missing. Additionally, the duration of 360 hours (15 days) for the incubation is chosen without justification—why this timeframe was sufficient to capture the full humification process (e.g., based on preliminary experiments or literature precedents) needs to be addressed.

2.3 In Section 2.3 (Chemical Analysis), the procedure for extracting humic-like acid (HLA) and fulvic-like acid (FLA) mentions acidifying the dark-brown residue to pH 1.0 and equilibrating for 24 hours. However, the criteria for confirming that HLA precipitation was complete (e.g., no further precipitate formation after extended equilibration) are not provided, which may raise concerns about the accuracy of CHLA and CFLA measurements.

3. Results Section

Table 2 (Atomic ratios of HLA) presents significant differences among treatments using lowercase letters, but the statistical method (e.g., one-way ANOVA with LSD test) is only mentioned in Section 2.4 and not explicitly linked to the table. It is also unclear whether the "±" values represent standard deviation or standard error—this should be specified for clarity.

4. Discussion Section

4.1 The discussion links the TOC retention in the Gly0.24 treatment to Al–C complex formation but provides limited mechanistic details. How exactly gibbsite (Al(OH)₃) interacts with glycine and other precursors to form Al–C complexes (e.g., through ligand exchange, electrostatic adsorption) should be elaborated, with references to specific studies on Al-based complexes in humification.

4.2 The discussion mentions that higher glycine concentrations (0.12–0.24 mol/L) promote N-compound accumulation in HLA, but it does not address a potential contradiction: why the Gly0.24 treatment, despite high N accumulation, had a lower O/C ratio and higher E4/E6 ratio (indicating less mature HLSs) compared to the Gly0.12 treatment. This inconsistency needs to be resolved to strengthen the conclusion about optimal glycine concentration.

4.3 The practical implications of the study (e.g., for lignin-rich waste valorization) are briefly mentioned in the introduction and conclusion but are not discussed in depth. How the optimal glycine concentration (0.12 mol/L) identified in this lab-scale study can be translated to real-world composting systems (e.g., adjusting precursor ratios in agricultural waste) should be explored.

5. Conclusion Section

The conclusion summarizes the key findings but does not explicitly highlight the study’s novelty (e.g., first systematic investigation of glycine concentration effects on gibbsite-catalyzed Maillard humification) or address limitations (e.g., the sterile, lab-scale system may not fully simulate natural soil conditions with microorganisms). Acknowledging limitations and suggesting future research directions (e.g., testing mixed metal oxides, incorporating microbial communities) would improve the section’s completeness.

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Reviewer #1: No

Reviewer #2: No

Reviewer #3: No

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Optimizing glycine concentration to enhance gibbsite-catalyzed abiotic humification of catechol and glucose

PONE-D-25-40528R1

Dear Dr. Wang,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

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Kind regards,

Muammar Qadafi

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

Reviewer #1: All comments have been addressed

Reviewer #3: All comments have been addressed

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2. Is the manuscript technically sound, and do the data support the conclusions??>

Reviewer #1: Yes

Reviewer #3: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously? -->?>

Reviewer #1: Yes

Reviewer #3: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available??>

The PLOS Data policy

Reviewer #1: Yes

Reviewer #3: Yes

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5. Is the manuscript presented in an intelligible fashion and written in standard English??>

Reviewer #1: Yes

Reviewer #3: Yes

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Reviewer #1: (No Response)

Reviewer #3: The authors have addressed all of my concerns, and the manuscript is now acceptable in its current form.

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what does this mean? ). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy

Reviewer #1: No

Reviewer #3: No

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