Evaluating the Effect of Bacterial Inoculation and Fertilization on the Soil Nutrient Status of Coal Mine Soil by Growing Soybean (Glycine max) and Shrub Lespedeza (Lespedeza bicolor)
Round 1
Reviewer 1 Report
In my opinion, this paper is interesting and the topic fits the overall scope of the journal. Before a possible publication, the paper needs an overall revision of English in order to further improve the paper's quality. So, I suggest the acceptance of the paper afterv minor revision.
Comments for author File: 
Comments.pdf
Author Response
Response to Reviewer 1 Comments
Manuscript ID: sustainability-388429
Dear reviewers,
We have replied to the reviewer’s comments in the red text shown below each comment.
Point 1: Use "N" for nitrogen, here and elsewhere.
Response 1: "N" was used for nitrogen in the manuscript according to the reviewer’s suggestion.
Point 2: Replace "However" by "Thus" in the abstract.
Response 2: "However" was replaced by "Thus".
Point 3: Do not use capital for soybean and shrub lespedeza, here and elsewhere.
Response 3: soybean and shrub lespedeza was written in small letter in the manuscript.
Point 4: Put "," after (T2) in abstract.
Response 4: "," was put after (T2).
Point 5: "Available" should be in small letter in the "Abstract" section.
Response 5: "Available" was replaced by "available".
Point 6: "the" should be deleted in line number 28.
Response 6: "the" was deleted.
Point 7: "the" should be deleted in line number 29.
Response 7: "the" was deleted.
Point 8: Revise the sentence, "Legume species together with symbiotic N-fixing bacteria (generally known as rhizobia), improving their potential for use in revegetation programs."
Response 8: The sentence was revised and changed to "The association between legume species and symbiotic N-fixing bacteria can improve the potentiality of legume species for use in revegetation programs" in the "Introduction" section.
Point 9: Revise the sentence, "Some of that nitrogen is taken in by plants in that area, and some of it escapes into the atmosphere."
Response 9: The sentence was revised and changed to "After nitrification, some of available NO3−-N is taken in by plants, and some of it escapes into the atmosphere."
Point 10: "Barley" should be written in small letter.
Response 10: "Barley" was written as "barley" in the "Discussion" section.
Point 11: "Application" should be written in small letter.
Response 11: "Application" was written as "application" in the "Discussion" section.
Point 12: "control" should be deleted from line 371.
Response 12: "control" was deleted from line 371 in the "Conclusion" section.
# All changes were highlighted with yellow highlighter in the manuscript.
# After deleting the reference number 36, the sequence of references was changed from reference no. 36 to 42.
Author Response File: Author Response.pdf
Reviewer 2 Report
The Introduction section is appropiated, as well as the description of M& M; exept that there is no indication of experiment the duration .
The main constraint of the article relay on data presentation and its discussion. In my opinión Tables and Figures are redundant. Thus, in the Tables can be set up the statisticall significance of the studied parameters between treatments. In general, except for T0 treatment (control, no inoculation no fertilization) there were no statistically significant differences at p(<0.05), the regular level of significance in most greenhouse experiments. I attached the document with my corrections.
Comments for author File: Comments.pdf
Author Response
Response to Reviewer 2 Comments
Manuscript ID: sustainability-388429
Dear reviewers,
We have replied to the reviewer’s comments in the red text shown below each comment.
Point 1: Number 12 reference is mostly related with proteomics leaf proteins.
Response 1: Number 12 reference was changed in the "Introduction" section.
Point 2: Delete the sentence "is a species of flowering plant in the legume family. It".
Response 2: "is a species of flowering plant in the legume family. It" was deleted according to the reviewer’s suggestion.
Point 3: "Symbiosis" must be replaced by pre-inoculated seeds or seedlings, or the rhizobia inoculants.
Response 3: "Symbiosis" was replaced by rhizobia inoculants.
Point 4: Indicate size or volume of the pots in the "Materials and methods" section.
Response 4: Volume of the pot was mentioned "Materials and methods" section.
Point 5: First sentence of the "Seed germination and transplantation" part in "Materials and methods" section is redundant.
Response 5: The sentence was deleted.
Point 6: "under the Department" should be replaced by "of the Department".
Response 6: "under the Department" was replaced by "of the Department"
Point 7: Last sentence of the "Greenhouse experiment setup" part in "Materials and methods" section is redundant. Duration of the experiment should be included.
Response 7: The sentence was deleted and duration of the experiment was included in the first line of the same paragraph.
Point 8: Inoculation of bacteria after 15 days of germination is too late for soybean.
Response 8: Actually shrub lespedeza was inoculated after 15 days and soybean was inoculated after 8 days of germination because shrub lespedeza seedlings were transplanted after two weeks of germination and soybean seedlings were transplanted after one week of germination which was mentioned in the "Seed germination and transplantation" part of "Materials and methods" section. We have corrected the sentence in the "Materials and methods" section.
Point 9: Experimental soil means the mixture 1:1, coal mine soil: piedmont soil.
Response 9: yes, the meaning of experimental soil was added in Table 1.
Point 10: There is no need of the equation for the measurement of NH4+ and NO3−, just cited the method used.
Response 10: The equation was deleted and the name of the method was mentioned.
Point 11: Data presentation in both ways (Tables and Figures) is redundant and did not afford more information. So, just include the significance of the values in Tables.
Response 11: We have tried to represent the data in the tables and significance of the values in the figures by using GraphPad software (GraphPad Prism version 7.00). According to reviewer it is redundant to represent the data in both ways, so table 2 and table 3 were deleted from the main manuscript. Those tables were merged in one table and added as a supplementary table in a separate file and was mentioned as table S1. Figure 1 and figure 2 were kept in the main manuscript to represent the significance of the values.
Point 12: Data of figures 3, 4, 5, 6 and 7 are already presented in Tables 6 and 7.
Response 12: Similar to table 2 and table 3, table 6 and table 7 were also deleted from the main manuscript. Those tables were added as a supplementary table in a separate file and was mentioned as table S2 and table S3, respectively. Figures 3, 4, 5, 6 and 7 were kept in the main manuscript to represent the significance of the values.
Point 13: pH values of 7.3 and 7.1 are really the same. In my opinion there was no any real change in the soil pH after growing plants in relation to the initial pH of the experimental soil. A light decrease of the initial pH may be observed, due to fertilizers (T1 and T3) and nodulated plants (T2), as BNF decrease soil pH.
Response 13: After doing the data analysis again, we also did not find any significance difference between pH value 7.3 and 7.1. Therefore, figure 3 has been corrected. Only a slight decrease of the initial pH was found due to the effect of treatments as reviewer mentioned.
Point 14: There was a light increase in the content of NH4+, in treatments: T1, T2 and T3 of soybean, with respect to the initial soil value (1.09 ppm), but these values are not significantly different between them, except with the T0 (control).
Response 14: After the revision of the data analysis, some changes were found in the significant difference between treatments. We found low significant difference in increasing concentration of NH4+ between treatment T1 and T2 for shrub lespedeza (Figure 1) and in increasing concentration of NO3- between treatment T2 and T3 for soybean (Figure 2) which were discussed in line number 290-292 and 309-310 of "Discussion" section. But there was no significant difference between other treatments. Mainly we tried to compare T1, T2 and T3 with the control and found significant difference between control and other treatments that means application of fertilizer and inoculation of bacteria has significant effect in increasing the concentration of NH4+ and NO3-.
Point 15: "Single celled" should be deleted from line number 283 in the "Discussion" section.
Response 15: "Single celled" was deleted from line number 283 in the "Discussion" section.
Point 16: The soil of T0 treatment, in both legumes (soybean and lespedeza) at the end of the assay has been decreased in both NH4+ and NO3-, with respect to the initial values: 1.09 ppm (NH4+) and 4.36 ppm (NO3-) (Table 1); as developing plants have been used both N forms to grow.
Response 16: As developing plants use both N forms to grow, so both NH4+-N and NO3−-N would be decreased at non-fertilized non-inoculated treatment (control) but the concentration of NH4+-N and NO3−-N at the end of the current experiment was increased from the initial values at control. The initial value of NH4+ was 1.09 ppm and after finishing of the study, the value of NH4+ was 1.76 ppm for soybean and 1.21 ppm for shrub lespedeza, respectively at T0 (control). So, the increasing concentration of NH4+ at T0 (control) for soybean was 1.76-1.09= 0.67 ppm and for shrub lespedeza was 1.21-1.09= 0.12 ppm which were shown in the table S1 and figure 1. Similarly, the initial value of NO3− was 4.36 ppm and after finishing of the study, the value of NO3− was 6.22 ppm for soybean and 6.50 ppm for shrub lespedeza, respectively at T0 (control). So, the increasing concentration of NO3− at T0 (control) for soybean was 6.22-4.36= 1.86 ppm (table S1 and figure 2) and for shrub lespedeza was 6.50-4.36= 2.14 ppm (table S1 and figure 2). The reason for increasing the concentration of NH4+-N and NO3−-N at non-fertilized non-inoculated treatment (control) might be due to mineralization and nitrification of organic N and decomposed materials present in the soil and formation of few number of nodules in both plants (table 2 and table 3).
Point 17: 323-324 line of the "Discussion" section should be deleted.
Response 17: The sentence was deleted according to the reviewer’s suggestion.
Point 18: The sentence from 332-335 lines of the "Discussion" section should be deleted.
Response 18: The sentence was deleted according to the reviewer’s suggestion.
Point 19: Year of some references should be in black font and the author name "Arreseigor" in reference number 34 should be written as "Arrese-Igor".
Response 19: Year of reference number 4, 11, 13, 17, 18, 36, 39 and 40 were changed to black font and the author name "Arreseigor" in reference number 34 was written as "Arrese-Igor".
# All changes were highlighted with green highlighter in the manuscript.
# After deleting the reference number 36, the sequence of references was changed from reference no. 36 to 42.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
In my opinión still redundance in the presentation of data (Results) in both ways, Tables and Figures.
It would be enough to add the statistical significance between treatments to the values presented in the Tables.
Still, no increase in N-NO3 in the soil after growing soybeans, with respect to the original value (Table 1, N-NO3= 4.36 ppm)
Author Response
Response to Reviewer 2 Comments (Round 2)
Manuscript ID: sustainability-388429
Dear reviewers,
We have replied to the reviewer’s comments in the red text shown below each comment.
Point 1: According to the opinion of the reviewer, it is still redundant to present the data (results) in both ways, Tables and Figures. It would be enough to add the statistical significance between treatments to the values presented in the tables.
Response 1: All the figures were deleted in the "Results" section and the data were presented only in the tables with statistical significance according to the reviewer’s suggestion.
Point 2: Still, no increase in N-NO3 in the soil after growing soybeans, with respect to the original value (Table 1, N-NO3= 4.36 ppm)
Response 2: Actually, the concentration of NO3−-N at the end of the current experiment was increased from the initial values (4.36 ppm) at all treatments for soybean. The initial value of NO3−-N was 4.36 ppm and after finishing of the study, the value of NO3−-N was 6.22 ppm for soybean at T0 (control). So, the increasing concentration of NO3−-N at T0 (control) for soybean was 6.22-4.36= 1.86 ppm (table 3). Similarly, the concentration of NO3−-N for soybean at treatment T1, T2 and T3 were 7.52, 7.42 and 7.95 ppm, respectively after finishing of the study. Therefore, the increasing concentration of NO3−-N for soybean at treatment T1, T2 and T3 were 7.52-4.36= 3.16, 7.42-4.36= 3.06 and 7.95-4.36= 3.59 ppm, respectively which were shown in the table 3. So, it could be said that the concentration of NO3−-N in the soil was increased after growing soybeans.
Author Response File: Author Response.pdf
