Biochar and Bacillus sp. MN54 Assisted Phytoremediation of Diesel and Plant Growth Promotion of Maize in Hydrocarbons Contaminated Soil

Round 1

Reviewer 1 Report

The presented publication entitled "Biochar and microbes assisted phytoremediation of diesel and plant growth promotion of maize in hydrocarbons contaminated soil" is an extremely interesting research topic in the field of soil remediation. Remediation of soils from petroleum products, as the authors noted, is associated with the continuous development of civilization and the increasing demand for fuel. This is related to the ever-increasing problem of petroleum products getting into the environment as a result of a accidents, for example. For this reason, in the field of soil remediation from contamination by diesel fuel, the search for new solutions as well as improvements and modifications to the existing ones are still ongoing. The authors decided to modify the already existing methods. The article is written in a very clear and simple language that is required for scientific publications, contains the necessary references, descriptions of research methods, analyzes. However, some information that is quite important in the experiment conducted in this way has not been included.

1. The first missing information is the particle size distribution of the soil used in the pot experiment, which was not taken into account in the present form of the article.
2. There is no information about the basic chemical and physical properties of the tested soil, there is only a reference that they were tested but there is no data on this.
3. The authors should add in the text of the article information on the capacity of the vases used in the experiment and the amount of soil mass added to the vases.
4. The whole article should be checked for typos and errors, such as in line 132 "potash", or in the caption of table 2 no superscript "mg-1".
5. The chapter on materials and methods should contain information on the methodologies used, among others, for the determination of PHs.
6. Authors should also provide values for basic physical and chemical properties of soils after harvesting the maize crop.
7. In the discussion, the Authors should check typos and spaces, and the text in the discussion should not be in italics.
8. Section 6 on patents can be deleted.
9. Conclusions should be extended.
10. When referring to phytotoxicity, the authors should consider applying phytotoxicity tests to the tested soil combinations using test plants to determine the potential impact of contamination on plants on various substrates.
11. The aspect of the statistical calculations of which LSD test was used to compare the results also needs clarification.
12. When describing the efficiency of pollutant uptake by plants, the authors should refer to the BCF factor and especially to TF factor. An example of a helpful publication covering this topic: https://doi.org/10.3390/min11030268
13. The graphs in Figure 1 should have the same scale, otherwise they should not be adjacent to each other and the text should contain information about the N: P: K ratio in the pot experiment.
14. It is worth the Authors to read the publications on phytoremediation and soil remediation and add some information in introduction section. https://doi.org/10.3390/ijerph17051606 https://doi.org/10.1016/j.jes.2019.08.010

Taking into account the above remarks, the article will be suitable for publication in the journal due to the current research topic and originality of the work.

Author Response

Reviewer #1:

General remarks: The presented publication entitled "Biochar and microbes assisted phytoremediation of diesel and plant growth promotion of maize in hydrocarbons contaminated soil" is an extremely interesting research topic in the field of soil remediation. Remediation of soils from petroleum products, as the authors noted, is associated with the continuous development of civilization and the increasing demand for fuel. This is related to the ever-increasing problem of petroleum products getting into the environment as a result of a accidents, for example. For this reason, in the field of soil remediation from contamination by diesel fuel, the search for new solutions as well as improvements and modifications to the existing ones are still ongoing. The authors decided to modify the already existing methods. The article is written in a very clear and simple language that is required for scientific publications, contains the necessary references, descriptions of research methods, analyzes. However, some information that is quite important in the experiment conducted in this way has not been included:

Response: Authors are thankful to the reviewer for his/her comments on the manuscript and suggestions for increasing the quality of our work.

Comment 1: The first missing information is the particle size distribution of the soil used in the pot experiment, which was not taken into account in the present form of the article.

Response: The information on particle size distribution has already been presented in Table S2. Physicochemical properties of the soil used in the study are provided in the supplementary material file (please see the supplementary materials).

Comment 2: There is no information about the basic chemical and physical properties of the tested soil, there is only a reference that they were tested but there is no data on this.

Response: The information on the basic chemical and physical properties has already been provided in Table S2 (please see the supplementary materials).

Comment 3: The authors should add in the text of the article information on the capacity of the vases used in the experiment and the amount of soil mass added to the vases.

Response:  Each pot (with dimension 26 L× 20 W × 20 D cm) was filled with 8 kg of contaminated or uncontaminated soil. The information has been provided in the manuscript (please see line 157 page 4).

Comment 4: The whole article should be checked for typos and errors, such as in line 132 "potash", or in the caption of table 2 no superscript "mg-1".

Response:  The whole manuscript has been checked carefully and all “typos and errors” have been removed and superscripts or subscripts have also been corrected (please see Table 2).

Comment 5: The chapter on materials and methods should contain information on the methodologies used, among others, for the determination of PHs.

Response:  The information on methodologies for the determination of PHs has been provided (please see line 210-216 page 5).

Comment 6: Authors should also provide values for basic physical and chemical properties of soils after harvesting the maize crop.

Response:  Basic physical and chemical properties of soil at the launch of experiment were checked and presented in Table S2; however, we did not analyze these properties after harvesting of crop except residual PHs analysis as post-harvest soil analysis was not the scope of the study.

Comment 7: In the discussion, the Authors should check typos and spaces, and the text in the discussion should not be in italics.

Response:  All the typos and spaces issues have been checked and corrected, and the text (in italics) in discussion has also been corrected (please see the discussion section of the revised manuscript).

Comment 8:   Section 6 on patents can be deleted.

Response: The suggested change has been made in the revised version of manuscript.

Comment 9: Conclusions should be extended.

Response: The conclusions section has been revised and extended (please see line 147-159).

Comment 10: When referring to phytotoxicity, the authors should consider applying phytotoxicity tests to the tested soil combinations using test plants to determine the potential impact of contamination on plants on various substrates.

Response: Maize was used as a test plant in this study and potential phytotoxic effects of PHs were observed on maize growth and physiology as compared to control please see table 3. PHs were only substrate tested in this study.

Comment 11: The aspect of the statistical calculations of which LSD test was used to compare the results also needs clarification.

Response: Least Significance Difference (LSD) test was applied on the results of experiment and mean values of different treatments were compared according to this test (please see line 218-227)

Comment 12: When describing the efficiency of pollutant uptake by plants, the authors should refer to the BCF factor and especially to TF factor. An example of a helpful publication covering this topic: https://doi.org/10.3390/min11030268

Response: Unfortunately, we could not check the uptake/accumulation of PHs in plants body. We only determined the residual PHs contents in contaminated soil after the termination of the experiment. So, we could not determine BCF. In future investigations this aspect can be studied thanks to reviewer for suggestion

Comment 13: The graphs in Figure 1 should have the same scale, otherwise they should not be adjacent to each other and the text should contain information about the N: P: K ratio in the pot experiment.

Response: The graphs in Figure 1 have been corrected and the information about the N: P: K ratio in the pot experiment have also been incorporated in text (please see page 9  line 27-35).

Comment 14: It is worth the Authors to read the publications on phytoremediation and soil remediation and add some information in introduction section. https://doi.org/10.3390/ijerph17051606 https://doi.org/10.1016/j.jes.2019.08.010

Response: We have read the suggested publications and relevant information has also been incorporated in the introduction section (please see line 69-77).

Reviewer 2 Report

Comments on agronomy-1341321

The concept of the manuscript is interesting. The overall presentation is also good; however, I have the following suggestions and queries, kindly incorporate, and explain respectively:

• Firstly, as in the title (Biochar and microbes assisted phytoremediation of diesel and plant growth promotion of maize in hydrocarbons contaminated soil), microbes are mentioned but, in the whole manuscript, only Bacillus sp. MN54 is used. So, please explain about it.
• Write scientific names of bacteria and plants as the guidelines.
• Term co-addition can be replaced with co-supplementation [Line 38]
• CO2 and H2O: check for subscript [Line 66]
• Use full form of TPH at least once.
• There are some minor grammatical mistakes, please check critically.
• I suggest including a Figure to depict or propose the mechanism of how the supplementation of biochar and microbe improved the phytoremediation along with growth and developmental benefits to maize.
• Add some recent references to support the current research outcomes (authors may consider: DOI: 10.3390/plants10050841; DOI: 10.1007/s10653-019-00505-1), especially in the discussion part.

Author Response

General remarks: The concept of the manuscript is interesting. The overall presentation is also good; however, I have the following suggestions and queries, kindly incorporate, and explain respectively:

Response: Authors are thankful to the reviewer for his/her valuable comments and suggestions on the manuscript.

Comment 1: Firstly, as in the title (Biochar and microbes assisted phytoremediation of diesel and plant growth promotion of maize in hydrocarbons contaminated soil), microbes are mentioned but, in the whole manuscript, only Bacillus sp. MN54 is used. So, please explain about it.

Response: To reflect the reviewer comment, the title of the manuscript has been changed to “Biochar and Bacillus sp. MN54 assisted phytoremediation of diesel and plant growth promotion of maize in hydrocarbons contaminated soil”.

Comment 2: Write scientific names of bacteria and plants as the guidelines.

Response: Thanks to reviewer, scientific names of bacteria and plants are added (please see the line 43, 72,73)

Comment 3: Term co-addition can be replaced with co-supplementation [Line 38]

Response:  Term co-addition has been replaced with co-supplementation (please see the line 42-243).

Comment 4: CO2 and H2O: check for subscript [Line 66]

Response:  Correction has been made (please see the line 215).

Comment 5: Use full form of TPH at least once.

Response:  the term “TPH” has been changed to “Portable hydrocarbon analyzer” (please see the line 175)

Comment 6: There are some minor grammatical mistakes, please check critically.

Response:  Whole manuscript has been revised carefully to remove the grammatical mistakes.

Comment 7: I suggest including a Figure to depict or propose the mechanism of how the supplementation of biochar and microbe improved the phytoremediation along with growth and developmental benefits to maize.

Response:  Figure has been added in supplementary material (please see Figure S1 and see the line 87, 123)

Comment 8: Add some recent references to support the current research outcomes (authors may consider: DOI: 10.3390/plants10050841; DOI: 10.1007/s10653-019-00505-1), especially in the discussion part.

Response: We have read the suggested publications and relevant information has also been incorporated in the discussion section (please see line 87, 104-105).

Round 2

Reviewer 2 Report

The authors critically revised the manuscript entitled “Biochar and Bacillus sp. MN54 assisted phytoremediation of diesel and plant growth promotion of
maize in hydrocarbons contaminated soil”  according to the reviewer's directions and suggestions. The current version can be accepted for publication.