Sustainable Remediation Using Hydrocarbonoclastic Bacteria for Diesel-Range Hydrocarbon Contamination in Soil: Experimental and In Silico Evaluation

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

Review on the manuscript with the title: Bioremediation of Diesel-range Hydrocarbons in Soil Using Hydrocarbonoclastic Bacteria in Microcosms and its Approximation Through in Silico Studies. I suggested a major revision with the following comments:

1. The authors should discuss the latest progress in this field in Introduction to demonstrate the innovation of this study.
2. The authors should further describe the environmental hazards of organic pollutants. The reference (Carbon Research 4, 13 (2025)) may be helpful for the section and is suggested to be cited.
3. The authors should list the commonly used repair techniques currently and demonstrate the advantages of bioremediation through comparison. The reference (Chem. Eng. J. 506 (2025) 160050) may be helpful for the section and is suggested to be cited.
4. Please provide the initial concentration of hydrocarbons in the soil sample.
5. The authors should explain the meanings of H1, F1, F2, and N2 when they first appear.
6. The authors should explain the reason for adjusting the C:N:P ratio to 100:10:1 and provide evidence to support this adjustment.
7. The authors should determine the physicochemical properties of the soil after the remediation is completed and analyze the reasons for the differences compared with those before the remediation, thereby highlighting the practical application of this study. The reference (J. Hazard. Mater. 489 (2025) 137512) may be helpful for the section and is suggested to be cited.
8. The fact that A. globiformis has been recorded as a dominant microbial community only proves its high adaptability, but its ability to degrade hydrocarbons in the soil requires further experimental verification.
9. Figure 4 showed that the variation of RMSD for the complex of C₁₀ with the AlKB from P. agglomerans was more than 2 Å after 30 ns of simulation. This result is inconsistent with the phenomenon observed by the authors.
10. The authors should summarize and generalize the conclusions of this study.

Author Response

REVIEWER #1

Review on the manuscript with the title: Bioremediation of Diesel-range Hydrocarbons in Soil Using Hydrocarbonoclastic Bacteria in Microcosms and its Approximation Through in Silico Studies. I suggested a major revision with the following comments:

1. The authors should discuss the latest progress in this field in Introduction to demonstrate the innovation of this study.

Answer: Thank you for your observation. We have included some additional references of advances in bioremediation of soil in lines 54-63 and the specific use of computational chemistry in environmental sciences in lines 77-83.

1. The authors should further describe the environmental hazards of organic pollutants. The reference (Carbon Research 4, 13 (2025)) may be helpful for the section and is suggested to be cited.

Answer: We appreciate your kind suggestion. Undoubtedly, all organic pollutants significantly affect the environment and addressing them is urgent. On the other hand, since the primary focus of this manuscript is on a specific type of organic pollutant, hydrocarbons, we have oriented specifically on them.

1. The authors should list the commonly used repair techniques currently and demonstrate the advantages of bioremediation through comparison. The reference (Chem. Eng. J. 506 (2025) 160050) may be helpful for the section and is suggested to be cited.

Answer: Thank you for your fine suggestion. We have included some examples of remediation methods and advantages of bioremediation in the Introduction section in lines 54-63.

1. Please provide the initial concentration of hydrocarbons in the soil sample.

Answer: Thank you for your kind observation. We have included this information in line 119.

1. The authors should explain the meanings of H1, F1, F2, and N2 when they first appear.

Answer: Thank you for your kind observation. We have included this information in lines 131-132.

1. The authors should explain the reason for adjusting the C:N:P ratio to 100:10:1 and provide evidence to support this adjustment.

Answer: The adjustment of the C:N:P ratio to 100:10:1 was in accordance with EPA recommendations to stimulate the growth of microorganisms for the treatment of contaminated soils (EPA, 2002). This information was added to the document on lines 149-150.

1. The authors should determine the physicochemical properties of the soil after the remediation is completed and analyze the reasons for the differences compared with those before the remediation, thereby highlighting the practical application of this study. The reference (J. Hazard. Mater. 489 (2025) 137512) may be helpful for the section and is suggested to be cited.

Answer: We recognize that the physicochemical and biological characteristics of remediated soil can change, but in this work, we focused primarily on diesel removal. We greatly appreciate your recommendation and will consider it for future research. This recommendation was included in the document on lines 258-262.

1. The fact that A. globiformis has been recorded as a dominant microbial community only proves its high adaptability, but its ability to degrade hydrocarbons in the soil requires further experimental verification.

Answer: We agree that the adaptability of A. globiformis to diesel does not necessarily mean that it can degrade it. Although there are few records, we consider it to be a species with a high degradation potential, and we agree that further studies will be necessary to confirm this assertion. However, we believe that the approach with computational analysis and laboratory experiments provides more data to propose it as a species that participates in the remediation process. We have added this discussion in lines 282-287.

1. Figure 4 showed that the variation of RMSD for the complex of C₁₀ with the AlKB from P. agglomerans was more than 2 Å after 30 ns of simulation. This result is inconsistent with the phenomenon observed by the authors.

Answer: Thank you for your fine observation. It this particular case of the P.agglomerans-decane commplex, it can be seen that de RMSD value after 30 ns is around 7 A. Around 70 ns there is slight change in conformation that sets RMSD values between 6 and 8 A, which are still in the range of 2 Å to consider a structure in the equilibrium. (Kufareva, I., Abagyan, R. (2011). Methods of Protein Structure Comparison. In: Orry, A., Abagyan, R. (eds) Homology Modeling. Methods in Molecular Biology, vol 857. Humana Press. https://doi.org/10.1007/978-1-61779-588-6_10)

1. The authors should summarize and generalize the conclusions of this study.

Answer: Thank you for your kind suggestion. We have included a Conclusions section to summarize our main findings.

Reviewer 2 Report

Comments and Suggestions for Authors

This study investigates the bioremediation of diesel-range hydrocarbons in soil using hydrocarbonoclastic bacteria, supported by in silico analyses. Bacterial strains isolated from hydrocarbon-exposed soils demonstrated significant diesel degradation, especially for short- and medium-chain alkanes. Experimental results were complemented by molecular docking and dynamics simulations to understand enzyme-substrate interactions, particularly involving the AlkB monooxygenase. The findings highlight the synergy between biological and computational approaches in enhancing soil remediation strategies and optimizing microbial selection for effective hydrocarbon degradation.

 

 

1. Line 15: “Soil bioremediation through bacterial bioaugmentation is an alternative for increasing the degradation.” Suggestion: “is an alternative to increase the degradation.”
2. Line 18: “close to areas with a risk of spills due to pipelines with hydrocarbons.” Suggestion: “pipelines carrying hydrocarbons.”
3. Line 27: “processes involved in the microbial degradation of hydrocarbons with different chain lengths.” Suggestion: “hydrocarbons of varying chain lengths.”
4. Line 36: “20,772 thousand barrels respectively[1].” Suggestion: “.20,772 thousand barrels, respectively [1].”
5. Line 50: “with 10 to 28 carbons.” Suggestion: “with 10 to 28 carbon atoms.”
6. Line 52: “is biodegradable, which increases its impact on ecosystems.” Suggestion: “is poorly biodegradable, which increases its impact on ecosystems.”
7. Line 58: “soil nutrient ratio (C; N; P)” Suggestion: “soil nutrient ratio (C:N:P)”
8. Line 62: “microbial cultures, autochthonous microbial communities or genetically engineered microbes.” Suggestion: “communities, or genetically engineered microbes.”
9. Line 108: “was diluted in hexane and added until it was perfectly mixed.” Suggestion: “until it was homogeneously mixed.”
10. Line 117: “Ashby Mannitol medium.” Suggestion: “Ashby’s Mannitol medium.”
11. Line 233: “decrease cellular activity, but high levels are undesirable because spaces in the soil are occupied by water.” Suggestion: “because excess water reduces soil aeration.”
12. Line 258: “indicating high adaptability and an important role in hydrocarbon biodegradation rates in soil.” Suggestion: “an important role in enhancing hydrocarbon biodegradation in soil.”
13. Line 324: “with the results of their experimental biodegradation remotion.” Suggestion: “with the results of their experimental biodegradation.”
14. Line 70: “the gene sequence to encode AlkB has been used in various studies.” Issue: It’s not the gene sequence being used, but rather the gene itself or its detection. Suggestion: “The AlkB gene has been studied to evaluate bacterial alkane degradation potential.”
15. Line 86: “to evaluate whether the affinity of the alkanes. correlates with the microorganism's degradation capacity.” Issue: Affinity alone cannot explain degradation capacity; enzyme expression, environment, etc., also matter. Suggestion: Add nuance: “correlates with one aspect of the degradation capacity.”
16. Line 130: “to identify the bacteria using the BLAST bioinformatics program.” Issue: “Identify the bacteria” is vague implies a broad capability. Suggestion: “to identify bacterial species by comparing sequences with known entries in the BLAST database.”
17. Line 195: “with an extension of 10 Å larger on each side of the complex.” Issue: This wording is vague. What does “on each side” mean in 3D space? Suggestion: Clarify that a cubic or rectangular box was extended 10 Å in all directions.
18. Line 233: “low moisture levels can decrease cellular activity, but high levels are undesirable.” Issue: It implies there is no ideal range. Suggestion: Add: “Thus, maintaining an optimal moisture range is essential.”
19. Line 282: “in all bacteria, there is a tendency for greater removal in the short carbon chains C10 and C12” Issue: The logic assumes cause-effect without acknowledging possible enzyme specificity. Suggestion: “This trend may reflect differences in enzyme-substrate compatibility.”

Author Response

Reviewer # 2

This study investigates the bioremediation of diesel-range hydrocarbons in soil using hydrocarbonoclastic bacteria, supported by in silico analyses. Bacterial strains isolated from hydrocarbon-exposed soils demonstrated significant diesel degradation, especially for short- and medium-chain alkanes. Experimental results were complemented by molecular docking and dynamics simulations to understand enzyme-substrate interactions, particularly involving the AlkB monooxygenase. The findings highlight the synergy between biological and computational approaches in enhancing soil remediation strategies and optimizing microbial selection for effective hydrocarbon degradation.

Answer: We are thankful for your kind comments to our work. As you have mentioned, we tried to show the value of the synergy between both biological and computational approaches to provide explanations and solutions to bioremediation problems.

  Line 15: “Soil bioremediation through bacterial bioaugmentation is an alternative for increasing the degradation.” Suggestion: “is an alternative to increase the degradation.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 18: “close to areas with a risk of spills due to pipelines with hydrocarbons.” Suggestion: “pipelines carrying hydrocarbons.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 27: “processes involved in the microbial degradation of hydrocarbons with different chain lengths.” Suggestion: “hydrocarbons of varying chain lengths.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 36: “20,772 thousand barrels respectively[1].” Suggestion: “.20,772 thousand barrels, respectively [1].”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 50: “with 10 to 28 carbons.” Suggestion: “with 10 to 28 carbon atoms.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 52: “is biodegradable, which increases its impact on ecosystems.” Suggestion: “is poorly biodegradable, which increases its impact on ecosystems.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 58: “soil nutrient ratio (C; N; P)” Suggestion: “soil nutrient ratio (C:N:P)”

  Line 62: “microbial cultures, autochthonous microbial communities or genetically engineered microbes.” Suggestion: “communities, or genetically engineered microbes.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 108: “was diluted in hexane and added until it was perfectly mixed.” Suggestion: “until it was homogeneously mixed.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

 

  Line 117: “Ashby Mannitol medium.” Suggestion: “Ashby’s Mannitol medium.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 233: “decrease cellular activity, but high levels are undesirable because spaces in the soil are occupied by water.” Suggestion: “because excess water reduces soil aeration.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 258: “indicating high adaptability and an important role in hydrocarbon biodegradation rates in soil.” Suggestion: “an important role in enhancing hydrocarbon biodegradation in soil.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 324: “with the results of their experimental biodegradation remotion.” Suggestion: “with the results of their experimental biodegradation.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 70: “the gene sequence to encode AlkB has been used in various studies.” Issue: It’s not the gene sequence being used, but rather the gene itself or its detection. Suggestion: “The AlkB gene has been studied to evaluate bacterial alkane degradation potential.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 86: “to evaluate whether the affinity of the alkanes. correlates with the microorganism's degradation capacity.” Issue: Affinity alone cannot explain degradation capacity; enzyme expression, environment, etc., also matter. Suggestion: Add nuance: “correlates with one aspect of the degradation capacity.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 130: “to identify the bacteria using the BLAST bioinformatics program.” Issue: “Identify the bacteria” is vague implies a broad capability. Suggestion: “to identify bacterial species by comparing sequences with known entries in the BLAST database.”

  Line 195: “with an extension of 10 Å larger on each side of the complex.” Issue: This wording is vague. What does “on each side” mean in 3D space? Suggestion: Clarify that a cubic or rectangular box was extended 10 Å in all directions.

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 233: “low moisture levels can decrease cellular activity, but high levels are undesirable.” Issue: It implies there is no ideal range. Suggestion: Add: “Thus, maintaining an optimal moisture range is essential.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

  Line 282: “in all bacteria, there is a tendency for greater removal in the short carbon chains C10 and C12” Issue: The logic assumes cause-effect without acknowledging possible enzyme specificity. Suggestion: “This trend may reflect differences in enzyme-substrate compatibility.”

Answer: Thank you for your kind suggestion. We have changed appropriately.

We thank the reviewer for all her/his kind suggestions that have helped to clarify our ideas.

Reviewer 3 Report

Comments and Suggestions for Authors

A manuscript describes loss of different fraction of diesel from soil amended with 4 different bacterial strains isolated from the soils historically contaminated with petroleum products. Namely, strains were Arthrobacter globiformis, two strains of Pantoea agglomerans, and Nitratireductor soli. A detailed description of fractions from C10 to C28 presented. To explain different rate of loss of fraction, in silico study has been performed, which included molecular docking and molecular dynamics simulations. It was intended to find interrelationship between loss of different diesel fraction and peculiarities of their binding to 1-monooxygenase. The results suggested that diesel fractions’ loss related to their affinity to alkane 1-monooxygenase presented in the studied microorganisms. The issue of the manuscript seems to be relevant and topical. Paper is written rather logical and contains detailed information on state of art, performed experiment, and data obtained. Results are described in a good manner, statistically treated and explained. However, the manuscript contains one fundamental flaw concerning an overly simplified view of the behavior of a diesel in the system under study. The authors postulate that diesel is a non-volatile substance and believe that the disappearance of diesel from the soil is solely due to its biodegradation. One cannot agree with this (see, for example, 10.1016/j.chemosphere.2013.10.073 and others). Therefore, it is necessary to provide additional results demonstrating the absence of significant diesel evaporation. Below please find some particular comments.

 

Particular comments

line 52. Diesel comes in several grades differing in properties including volatility. Some diesel fuels are rather volatile. So, this statement should be corrected

line 67. May be the authors want to change “alkane monooxygenase” for “alkane 1-monooxygenase (EC 1.14.15.3)”

line 103. Please specify what kind of pH (water, KCl, CaCl2) was measured

line 108. Please add more information about studied diesel

lines 113-141. Please describe how amount of VOCs was accounted. Please specify replication of the experiment

line 134. Please specify which sieve was used for sieving (diameter of holes)

line 137. Please add information how many ml were added

line 138. Please use superscript for 8

line 139. Names of variants is unclear. Though they are explained later in the manuscript, the meaning of abbreviations should be clear at the first mention in the text

lines 249-250. Here and further along the text. Please italicize Latin names

Figure 1. Please specify what error bars relate to (standard deviation or whatever). Also, please specify replications. Please add data on control (no bacteria added)

line 325. Please decipher or describe meaning of clogP. In addition, please add a Ref where the information on clogP was obtained from

line 355. Abbreviation was introduced in the text earlier

line 341. Did the authors mean Figure 3?

Author Response

Reviewer# 3

A manuscript describes loss of different fraction of diesel from soil amended with 4 different bacterial strains isolated from the soils historically contaminated with petroleum products. Namely, strains were Arthrobacter globiformis, two strains of Pantoea agglomerans, and Nitratireductor soli. A detailed description of fractions from C10 to C28 presented. To explain different rate of loss of fraction, in silico study has been performed, which included molecular docking and molecular dynamics simulations. It was intended to find interrelationship between loss of different diesel fraction and peculiarities of their binding to 1-monooxygenase. The results suggested that diesel fractions’ loss related to their affinity to alkane 1-monooxygenase presented in the studied microorganisms. The issue of the manuscript seems to be relevant and topical. Paper is written rather logical and contains detailed information on state of art, performed experiment, and data obtained. Results are described in a good manner, statistically treated and explained. However, the manuscript contains one fundamental flaw concerning an overly simplified view of the behavior of a diesel in the system under study. The authors postulate that diesel is a non-volatile substance and believe that the disappearance of diesel from the soil is solely due to its biodegradation. One cannot agree with this (see, for example, 10.1016/j.chemosphere.2013.10.073 and others). Therefore, it is necessary to provide additional results demonstrating the absence of significant diesel evaporation. Below please find some particular comments.

Answer: Thank you for your kind comments to our manuscript. We hope that in the following text we have answered all your concerns.

Particular comments

line 52. Diesel comes in several grades differing in properties including volatility. Some diesel fuels are rather volatile. So, this statement should be corrected

Answer: Thank you for your kind suggestion. We have changed appropriately. We corrected this statement in lines 52-53.

line 67. May be the authors want to change “alkane monooxygenase” for “alkane 1-monooxygenase (EC 1.14.15.3)”

Answer: Thank you for your kind suggestion. We have changed appropriately.

line 103. Please specify what kind of pH (water, KCl, CaCl2) was measured

Answer: Thank you for your kind observation. In was in water. We included this information in line 112.

Line 108. Please add more information about studied diesel

Answer: Thank you for your kind observation. We included this information in line 117-119.

lines 113-141. Please describe how amount of VOCs was accounted. Please specify replication of the experiment

Answer: Thank you for your kind observation. In our study, we assume that all C₁₀-C₂₈ carbon chains in diesel in soil are biodegraded by the added microorganisms because they produce the enzyme alkane monooxygenase. But we are aware that a portion of the C₁₀ to C₁₄ hydrocarbons may volatilize, but they could also be enzymatically degraded by stopping the volatilization of short-chain hydrocarbons by water, fine particles, and organic matter contained in the soil. However, further research would be necessary to quantify the VOCs and determine how many of them biodegrade and how many volatilize naturally during the soil treatment period with diesel. We greatly appreciate your recommendation and will consider it for future research. We discussed this and suggested these complementary analyses for further research in the lines 345-355.

line 134. Please specify which sieve was used for sieving (diameter of holes)

Answer: Thank you for your kind observation.  The sieve used was ASTM No. 40 (425 µm). We included this information in lines 146-147.

line 137. Please add information how many ml were added

Answer: Thank you for your kind suggestion. We have clarified that we added 1 mL of the bacterial suspension.

line 138. Please use superscript for 8

Answer: Thank you for your kind suggestion. We have changed appropriately.

line 139. Names of variants is unclear. Though they are explained later in the manuscript, the meaning of abbreviations should be clear at the first mention in the text

Answer: Thank you for your kind suggestion. We clarified in lines 131-132 with the text: Isolated hydrocarbonoclastic bacterial strain was labeled as H1, two phosphorus-solubilizing strains were labeled P1 and P2 and one nitrogen-fixing strain was coded N2.

lines 249-250. Here and further along the text. Please italicize Latin names

Answer: Thank you for your kind suggestion. We have changed where it was needed.

Figure 1. Please specify what error bars relate to (standard deviation or whatever). Also, please specify replications. Please add data on control (no bacteria added)

Answer: Thank you for your kind observation. We have added the information in the description below the figure and added the data from the control (no bioaugmentation). We included this information in lines 310-314.

line 325. Please decipher or describe meaning of clogP. In addition, please add a Ref where the information on clogP was obtained from

Answer: Thank you for your kind suggestion. clogP stands for calculated partition coefficient which predicts the lipophilicity of the analyzed compound.

line 355. Abbreviation was introduced in the text earlier

Answer: Thank you for your kind suggestion. We have removed it in this part of the manuscript, as it was introduced earlier.

line 341. Did the authors mean Figure 3?

Answer: Thank you for your kind observation. You are right, the Figure 3 shows the overlap tridimensional structures predicted by computational studies. It was clarified.

 

 

Round 2

Reviewer 1 Report

Comments and Suggestions for Authors

The authors have answered my questions and responded to my comments, and the manuscript has been improved. I recommend the publication of the manuscript in its present form.

Reviewer 2 Report

Comments and Suggestions for Authors

Agreed with the revisions made by the authors.