Nitrogen Biostimulation of Petroleum-Contaminated Sandy Podzolic Soil Under Boreal Conditions: Effects of Temperature, Nitrogen Form, and Contamination Level
Round 1
Reviewer 1 Report
Comments and Suggestions for AuthorsThe manuscript investigates nitrogen-assisted biostimulation for petroleum hydrocarbon remediation in sandy podzolic soil from Western Siberia, using a factorial experimental design to assess the combined effects of temperature regime, nitrogen source (urea vs. ammonium nitrate), contamination level (25, 50, and 100 g·kg⁻¹), and nitrogen dosage. System response was monitored via residual hydrocarbon content and hydrocarbon-oxidizing microbial abundance. The study concludes that ammonium nitrate generally outperformed urea, and that biostimulation efficacy is governed by factor interactions rather than by nitrogen addition alone.
The study aims to quantify the individual and combined effects of the aforementioned variables on hydrocarbon degradation in a cold-region boreal soil, applying a factorial design to overcome the limitations of single-factor optimization approaches, and to provide a scientific basis for future pilot-scale remediation in cold climates.
Line 121 – The term "moderate anthropogenic influence" requires further elaboration and justification.
Line 126 – Please specify the drying procedure (48 h): what method was used and what is the scientific rationale for this choice?
Line 142 – The origin of the crude oil used must be clarified: was it commercially sourced or collected directly from the contaminated site?
Line 144 – The rationale for selecting three contamination levels that are exceptionally high and of limited environmental representativeness should be explicitly justified.
Line 145 – The mixing procedure must be described in detail, including the instrument or method employed.
Lines 149–150 – Were urea and ammonium nitrate of commercial grade? Additional supplier and purity details are required. Furthermore, the selection of the tested dosages must be clearly justified.
Line 152 – Incubation conditions are insufficiently described: Where were the samples incubated? How were they collected? Were conditions aerobic or anaerobic? Full details on the incubation system must be provided.
Line 155 – Given the complexity of the experimental design and the large number of variants, a summary table would substantially aid reader comprehension.
Lines 177–178 – "A weighed portion" is vague: the exact dry weight must be specified. Regarding mechanical shaking, the speed (rpm) must be reported, as a duration of 15 minutes is meaningless without this information. Additionally, all materials of the flasks used for sampling and analysis must be specified, as hydrocarbons may adsorb onto plastic or hydrocarbon-derived materials, potentially compromising analytical accuracy.
Line 188 – The plate count method described does not appear sufficient to selectively discriminate hydrocarbon-oxidizing microorganisms from the total microbial community. As applied, it appears to reflect total microbial abundance rather than the specific functional group of interest.
Line 209 – This information is repetitive and has already been stated multiple times in the manuscript. Please consolidate.
Line 247 – The validity of the results does not appear sufficient to support the conclusions drawn. In particular, it must be clarified whether time zero represents a theoretical or a measured value — based on the current presentation, it appears to be theoretical, which substantially reduces the interpretive significance of the reported trends, as the first actual measured time point would be the subsequent one.
Regarding Figure 2: the legend could be abbreviated using "PH" as a shorthand notation, which would allow for larger font sizes on the legend, axes, and tick labels, the figure is currently difficult to read. Furthermore, the x-axis presents a non-equidistant time scale, which may lead to misleading graphical interpretation; this must be corrected or explicitly addressed. A more detailed figure caption is needed. The differences between panels A and B do not appear visually significant; statistical significance, if present, must be clearly indicated.
Regarding Figure 3: the readability of the graph must be improved. Again, time zero appears to be a theoretical value, lacks error bars, and the first measured time point is at day 30. Panel labels (A and B) are missing and must be added. The figure caption requires substantially more detail.
Line 273 – The use of "strongly" is not adequately supported by the data presented. The language should be moderated accordingly.
Regarding Figure 4: panel labels are again missing between the left and right graphs, consistent with the issues noted for other figures. Time zero appears theoretical and lacks error bars. The differences between treatments are not clearly evident. Given that only two measured time points are available, the authors should consider adopting an alternative graphical representation, for instance, a bar chart reporting percentage removal efficiency relative to t0, or hydrocarbon removal rate, which would likely be more informative and interpretable. A more thorough data analysis is strongly recommended.
Line 296 – A plate count method alone is insufficient to determine and discriminate the abundance of hydrocarbon-degrading bacteria from the broader microbial community.
Regarding Figure 5: the figure remains difficult to read; font sizes are too small throughout.
Line 313 – The authors themselves acknowledge the limitations of the microbial enumeration technique. In the reviewer's opinion, this approach is not effective even in a supporting role relative to the chemical data.
General comment on the Discussion – Throughout the Discussion section, conclusions are drawn that are not adequately supported by the experimental data, and the authors repeatedly acknowledge the limitations of their own findings. Specific examples are found at lines 361, 385, 405, 414, 423, 428, 436, 453, and 459, where the authors explicitly state, on multiple occasions, that the data are insufficient to support in-depth interpretations. The Discussion as a whole fails to substantiate the initial hypothesis; on the contrary, it explicitly exposes the limitations of the experimental approach.
General comment on the Conclusions – The Conclusions present scientific evidence that is in direct contrast with what emerges from the Discussion, where no such evidence is adequately established. This internal inconsistency is further undermined by the authors' own closing statement: "At the same time, the results should be interpreted within the limitations of controlled laboratory conditions, simplified microbiological assessment, and pairwise statistical analysis."
Overall assessment – This manuscript is not ready for publication. The authors themselves repeatedly and explicitly acknowledge its limitations throughout the Discussion, and the experimental evidence presented does not support the conclusions stated.
Author Response
Response to Reviewer 1
Comment #1: The manuscript investigates nitrogen-assisted biostimulation for petroleum hydrocarbon remediation in sandy podzolic soil from Western Siberia, using a factorial experimental design to assess the combined effects of temperature regime, nitrogen source (urea vs. ammonium nitrate), contamination level (25, 50, and 100 g·kg⁻¹), and nitrogen dosage. System response was monitored via residual hydrocarbon content and hydrocarbon-oxidizing microbial abundance. The study concludes that ammonium nitrate generally outperformed urea, and that biostimulation efficacy is governed by factor interactions rather than by nitrogen addition alone.
The study aims to quantify the individual and combined effects of the aforementioned variables on hydrocarbon degradation in a cold-region boreal soil, applying a factorial design to overcome the limitations of single-factor optimization approaches, and to provide a scientific basis for future pilot-scale remediation in cold climates.
Response #1: Dear Reviewer, we sincerely thank you for your detailed and critical evaluation of our manuscript. Your comments were highly valuable and helped us substantially improve the clarity, methodological transparency, and internal consistency of the study. We have carefully revised the manuscript accordingly. All changes are highlighted in the revised version.
Below we provide a point-by-point response.
Comment #2: Line 121 – The term "moderate anthropogenic influence" requires further elaboration and justification.
Response #2: We agree and have clarified this description (see lines 128-131).
Comment #3: Line 126 – Please specify the drying procedure (48 h): what method was used and what is the scientific rationale for this choice?
Response #3: The drying procedure has been clarified (see lines 135-138).
Comment #4: Line 142 – The origin of the crude oil used must be clarified: was it commercially sourced or collected directly from the contaminated site?
Response #4: Clarified (see lines 161-162).
Comment #5: Line 144 – The rationale for selecting three contamination levels that are exceptionally high and of limited environmental representativeness should be explicitly justified.
Response #5: We added explicit justification (see lines 164-167).
Comment #6: Line 145 – The mixing procedure must be described in detail, including the instrument or method employed.
Response #6: Now specified (see lines 167-169).
Comment #7: Lines 149–150 – Were urea and ammonium nitrate of commercial grade? Additional supplier and purity details are required. Furthermore, the selection of the tested dosages must be clearly justified.
Response #7: Both aspects clarified (see lines 173-176).
Comment #8: Line 152 – Incubation conditions are insufficiently described: Where were the samples incubated? How were they collected? Were conditions aerobic or anaerobic? Full details on the incubation system must be provided.
Response #8: The incubation system has been fully described (see lines 181-186).
Comment #9: Line 155 – Given the complexity of the experimental design and the large number of variants, a summary table would substantially aid reader comprehension.
Response #9: A summary table has been added (Table 1).
Comment #10: Lines 177–178 – "A weighed portion" is vague: the exact dry weight must be specified. Regarding mechanical shaking, the speed (rpm) must be reported, as a duration of 15 minutes is meaningless without this information. Additionally, all materials of the flasks used for sampling and analysis must be specified, as hydrocarbons may adsorb onto plastic or hydrocarbon-derived materials, potentially compromising analytical accuracy.
Response #10: Clarified (see lines 223-226).
Comment #11: Line 188 – The plate count method described does not appear sufficient to selectively discriminate hydrocarbon-oxidizing microorganisms from the total microbial community. As applied, it appears to reflect total microbial abundance rather than the specific functional group of interest.
Response #11: We agree and revised the interpretation (see lines 245-247).
Comment #12: Line 209 – This information is repetitive and has already been stated multiple times in the manuscript. Please consolidate.
Response #12: Redundant text has been removed and consolidated.
Comment #13: Line 247 – The validity of the results does not appear sufficient to support the conclusions drawn. In particular, it must be clarified whether time zero represents a theoretical or a measured value — based on the current presentation, it appears to be theoretical, which substantially reduces the interpretive significance of the reported trends, as the first actual measured time point would be the subsequent one.
Response #13: This issue has been fully corrected. The initial reference point is now day 2 (first measured value). All figures and captions have been updated accordingly.
Comment #14: Regarding Figure 2: the legend could be abbreviated using "PH" as a shorthand notation, which would allow for larger font sizes on the legend, axes, and tick labels, the figure is currently difficult to read. Furthermore, the x-axis presents a non-equidistant time scale, which may lead to misleading graphical interpretation; this must be corrected or explicitly addressed. A more detailed figure caption is needed. The differences between panels A and B do not appear visually significant; statistical significance, if present, must be clearly indicated.
Response #14: Figure 2 has been fully revised: “PH” abbreviation introduced, equidistant time scale used, captions expanded, graphical readability improved.
Comment #15: Regarding Figure 3: the readability of the graph must be improved. Again, time zero appears to be a theoretical value, lacks error bars, and the first measured time point is at day 30. Panel labels (A and B) are missing and must be added. The figure caption requires substantially more detail.
Response #15: Figure 3 has been revised: panel labels (a, b) added, error bars included, time axis corrected (day 2, 30, 60), caption expanded.
Comment #16: Line 273 – The use of "strongly" is not adequately supported by the data presented. The language should be moderated accordingly.
Response #16: The wording has been moderated.
Comment #17: Regarding Figure 4: panel labels are again missing between the left and right graphs, consistent with the issues noted for other figures. Time zero appears theoretical and lacks error bars. The differences between treatments are not clearly evident. Given that only two measured time points are available, the authors should consider adopting an alternative graphical representation, for instance, a bar chart reporting percentage removal efficiency relative to t0, or hydrocarbon removal rate, which would likely be more informative and interpretable. A more thorough data analysis is strongly recommended.
Response #17: Figure 4 has been revised analogously to Figure 3. We retained the kinetic representation (time-dependent trends), as it is consistent with the experimental design, but improved clarity and data presentation.
Comment #18: Line 296 – A plate count method alone is insufficient to determine and discriminate the abundance of hydrocarbon-degrading bacteria from the broader microbial community.
Response #18: We agree and have explicitly acknowledged this limitation in both Methods and Results.
Comment #19: Regarding Figure 5: the figure remains difficult to read; font sizes are too small throughout.
Response #19: Figure 5 has been redesigned with improved font size and clarity.
Comment #20: Line 313 – The authors themselves acknowledge the limitations of the microbial enumeration technique. In the reviewer's opinion, this approach is not effective even in a supporting role relative to the chemical data.
Response #20: We agree that the microbiological dataset is limited. The manuscript has been revised to clearly position these results as supportive rather than mechanistic evidence.
Comment #21: General comment on the Discussion – Throughout the Discussion section, conclusions are drawn that are not adequately supported by the experimental data, and the authors repeatedly acknowledge the limitations of their own findings. Specific examples are found at lines 361, 385, 405, 414, 423, 428, 436, 453, and 459, where the authors explicitly state, on multiple occasions, that the data are insufficient to support in-depth interpretations. The Discussion as a whole fails to substantiate the initial hypothesis; on the contrary, it explicitly exposes the limitations of the experimental approach.
Response #21: The Discussion has been substantially rewritten: overly speculative interpretations removed, statements aligned strictly with experimental evidence, limitations retained but no longer undermine the study.
Comment #22: General comment on the Conclusions – The Conclusions present scientific evidence that is in direct contrast with what emerges from the Discussion, where no such evidence is adequately established. This internal inconsistency is further undermined by the authors' own closing statement: "At the same time, the results should be interpreted within the limitations of controlled laboratory conditions, simplified microbiological assessment, and pairwise statistical analysis."
Response #22: The Conclusions section has been revised to ensure full consistency with the Discussion and to avoid overinterpretation.
Comment #23: Overall assessment – This manuscript is not ready for publication. The authors themselves repeatedly and explicitly acknowledge its limitations throughout the Discussion, and the experimental evidence presented does not support the conclusions stated.
Response #23: We respectfully submit that the manuscript has been substantially improved in response to all comments: methodological transparency significantly enhanced, internal consistency between Results and Discussion ensured, figures and data presentation corrected, interpretation aligned with experimental evidence.
We believe the revised manuscript now provides a clear and scientifically sound contribution to the field.
Reviewer 2 Report
Comments and Suggestions for Authors- L135 -139, Typical soil parameter measurements should be mentioned and cited.
- L209-212, these sentences are repeated content and can be removed.
- L235-242, the reduction percentages should be given.
- Fig. 3 captions, cold and warm conditions should be given.
- L259, where are the figures for the urea dosage of 10g/kg and 25g/kg?
- Fig. 4, where are the figures for the Ammonium nitrate dosage of 5 and 15 g/kg?
- L319-321, the significance of the contribution should be further analyzed through principal component analysis.
- L364-365, I don’t see the significant differences between the two nitrogen supplies in the remaining hydrocarbon contents, according to your Fig. 5.
- L409-410, I don’t see the microbial biomass data for the other two contamination levels in Fig. 5.
- Section 4 discussion should be subdivided to convey a clearer structure and more coherent perspectives.
Author Response
Response to Reviewer 2
Comment #1: L135 -139, Typical soil parameter measurements should be mentioned and cited.
Response #1: Thank you for this comment. The description of the initial soil characterization has been clarified and supported with appropriate references (see lines 138-140).
Comment #2: L209-212, these sentences are repeated content and can be removed.
Response #2: We agree with the reviewer. The repeated content has been removed, and the corresponding section has been streamlined to avoid redundancy.
Comment #3: L235-242, the reduction percentages should be given.
Response #3: Thank you for this suggestion. Reduction percentages relative to the initial measured concentrations have now been explicitly included in the Results section (see lines 358-364 and 405-410).
Comment #4: Fig. 3 captions, cold and warm conditions should be given.
Response #4: The figure captions have been revised to explicitly indicate temperature regimes.
Comment #5: L259, where are the figures for the urea dosage of 10g/kg and 25g/kg?
Response #5: All tested urea dosages (10, 25, and 50 g kg⁻¹) are included in Figure 3. The figure legend and graphical representation have been improved to enhance clarity and ensure that all treatments are clearly distinguishable.
Comment #6: Fig. 4, where are the figures for the Ammonium nitrate dosage of 5 and 15 g/kg?
Response #6: All ammonium nitrate treatments (5, 15, and 30 g kg⁻¹) are included in Figure 4. The graphical presentation has been revised (line styles, markers, legend clarity) to ensure that all dosage levels are clearly visible and distinguishable.
Comment #7: L319-321, the significance of the contribution should be further analyzed through principal component analysis.
Response #7: We appreciate this suggestion. However, multivariate statistical methods such as principal component analysis (PCA) were not applied in this study due to the structure of the dataset (limited number of response variables and focus on predefined factorial contrasts). This limitation has now been explicitly clarified in the manuscript (see lines 674-678). At the same time, we have clarified that interpretation is based on factorial comparison of treatments rather than multivariate decomposition.
Comment #8: L364-365, I don’t see the significant differences between the two nitrogen supplies in the remaining hydrocarbon contents, according to your Fig. 5.
Response #8: We agree that the differences between nitrogen sources are moderate and condition-dependent rather than universally strong. The wording in the Discussion has been revised to moderate the interpretation. For example, see lines 574-581. This revision ensures that conclusions are consistent with the magnitude of the observed differences.
Comment #9: L409-410, I don’t see the microbial biomass data for the other two contamination levels in Fig. 5.
Response #9: We thank the reviewer for this observation. Microbial data were indeed obtained only for the intermediate contamination level (50 g kg⁻¹). This limitation has now been clearly stated in both the Results (see line 426-431) and Discussion sections.
Comment #10: Section 4 discussion should be subdivided to convey a clearer structure and more coherent perspectives.
Response #10: We agree with the reviewer. The Discussion section has been reorganized into clearly defined subsections to improve readability and logical structure:
4.1. Temperature-Driven Constraints on Natural Attenuation
4.2. Effects of Nitrogen Form and Dosage
4.3. Role of Contamination Level and Microbiological Evidence
4.4. Practical Implications and Study Limitations
This restructuring improves clarity and provides a more coherent interpretation of the results.
Round 2
Reviewer 1 Report
Comments and Suggestions for AuthorsThe authors have addressed each of my comments point by point and have also revised the figures, which are now much easier to read. The paper has improved significantly in terms of clarity and scientific rigor; I believe it has benefited greatly from my comments!
Reviewer 2 Report
Comments and Suggestions for AuthorsThe authors have addressed all the issues and it can be accepted now.
