The Influence of Sewage Sludge and Fly Ash Fertilization on the Total Number of Bacteria (TNB) and Bradyrhizobium Species in Soybean Agroecosystem

Round 1

Reviewer 1 Report

Major revisions will be required for the publication of this manuscript entitled “The influence of selected soil parameters modified with sewage sludge and fly ash fertilization on the total number of bacteria and Bradyrhizobium species at soybean agroecosystem”. The research conducted by the author is interesting, but not clearly explained.

1.     Authors should revise the title of this manuscript to make it more understandable.

2.     Please improve the language of whole manuscript and abstract. What the authors are trying to say is unclear.

3.     Please mention how many field experiments were conducted??Line 12.

4.     Line 14 it should be like total viable count of bacteria and Bradyrhizobium sp. were determined using viable plate count method.

5.     Line 22. Please add the values or fold/percentage increase in total bacterial count and Bradyrhizobium. Which treatment showed better results. Explain in detail.

6.     Update the introduction section with latest reference from last five years (2018-2022). Improve the introduction with recent references related to the soil microbial population and soil enzyme activities responsible for maintenance of plant growth and soil health (Line 75-85). Authors can read these papers and explain accordingly. Authors may consider these works: https://doi.org/10.1007/s11104-022-05351-2, http://doi.org/10.1016/j.apsoil.2020.103836, 10.3389/fenvs.2021.769871 (Line 82).

7.     Importance of microbial fertilizers for soyabean growth, soil health explain in introduction section.

8.     Why authors studied only total bacteria and Bradyrhizobium no other microbial population?? Authors use rhizosphere soil?? Please explain clearly.

9.     Improve the language of material method section.

10.  There are many things which are not clear like whether the soil sample collected in triplicate or not. Is all the experiment related to soil conducted in triplicates individually or the soil sample of the different area collected then pooled and further the experiments are carried out?

11.  If authors have microbial enzyme activities of soil data. Please include them.

12.  Line 146 how many seeds were inoculated in soil??

13.  Please mention the references for total count and Bradyrhizobium count??

14.  Line 157. After this time?? Improve the sentence.

15.  Line 156. Authors have counted the bacteria using streaked plate?? How is it possible??

16.  Standard method for total bacteria count is pour plate method. Please write the formula for calculation of total bacterial count in manuscript.

17.  Which dilution used for bacterial count mention it.

18.  Please include the pictures of cfu count plates and field of soyabean where you have conducted the experiment.

19.  Is there any correlation of soil type, soil pH on microbial community? Explain in details in discussion section.

20.  Discussion section is not written properly. Please write it in a good manner and explain in detail with recent references. How population increased what factors responsible for this. Effect of different treatments (sewage, heavy metals, fly ash) on soil physicochemical properties. How they improve/decrease soil health parameters.

21.  Line 179. Of inorganic?? Improve the sentence.

22.  Table 3: remove the comma in mean values and SD it should be dot. Like 0.72, 1.73, 3.15.

23.  P should be Capital letters and italics in whole manuscript correct it. Line 286,287,288,289 P= 0.001 not 0,001

24.  Authors used objects in whole manuscript. You can change this term.

25.  Improve the conclusion part of this study.

Author Response

Ad.1.

The title of the manuscript was revised.

Ad.2.,Ad.9., Ad.14

Before uploading the manuscript was proofread by a native speaker. After revision another native speaker proofread it.

Ad.3., Ad.10

Field experiment was carried out with the random plot method (consisted of 24 plots: six treatments and four replications). Soil samples were collected from each plot (24) for analysis. The results in the tables are the arithmetic mean of 4 replicates.

Ad. 4

A series of ten-fold dilutions of the material was made and the number of microorganisms was determined by the spread plate method. The number of microorganisms was expressed as colony forming units (CFU) per gram of soil or root nodule.

Ad.5

The values of percentage increase/decrease of TNB and Bradyrizobium spp. were included at section 3. “Results and discussion” (Abstract can consist of max. 200 words).

Ad.6. Ad.7.

Introduction was updated.

Ad.8

The rhizosphere, i.e. the space covering the root surface and the soil surrounding it, is an environment with the high microbiological activity and the relationship between the plant, soil and microorganisms, therefore, the total number of microorganisms in the ectorosphere was determined.

Ad.11.

The enzymatic activity of microorganisms was not determined.

Ad.12.

HiStick® contains at least 2 x 10⁹ live cells from the Rhizobium group (Bradyrhizobium japonicum). 400 g of HiStick® was used to inoculate 100 kg of soybeans by dry inoculation before sowing.

Ad.13.

All references were updated.

Ad.15.

The spread plate method was used.

Ad. 16

The number of colonies formed in the plates were enumerated and the average number of the colony-forming unit (CFU/g and CFU/root nodule) were calculated by using following formula:

 

CFU/g or CFU/root nodule =  x  dilution factor

Ad. 17

 

A series of ten-fold dilutions were made of the tested materials.  Dilutions from 10^-4 to 10^-6 were selected from soil samples, and from 10^-1 to 10^-3 dilutions from nodules and spread on the media. After incubation, plates containing 30 to 300 colonies were selected for counting. To determine the number of bacteria, the mean dilution number of colonies, the dilution factor and the plated volume were taken into account.

 

Ad.18

Pictures of the experimental field

 

 

 

Ad.19, Ad.20

Discussion was updated.

 

Ad.21 – 24

All suggestions were followed.

 

Author Response File: Author Response.docx

Reviewer 2 Report

This manuscript investigated the impact of selected soil parameters modified with sewage sludge and fly ash fertilization on the total number of bacteria and Bradyrhizobium species in soybean agroecosystem. Results showed that high correlation coefficients between χ and Fe, Zn, Cr and TOC indicated that the source of these elements was external organic matter. Significant relationships between the total content of bacteria and Bradyrhizobium species showed the resistance of the microbial community in the field cultivation of Lissabon soybean to heavy metal contamination from sewage sludge and fly ash. Although some interesting results were found in this study, the presentation at the present form is far below the standards of the international journal.  The English has so many errors – inconsistent and incorrect tense, subject-verb disagreement, and convoluted sentence structures. I had a hard time understanding parts of the text because of all the problems.  It is not the job of the reviewer to copy-edit bad writing, and I will not do so here.  The authors can hire a capable English technical editor to get rid of all the presentation problems.  Following are comments and suggestions for the authors to further improve this work.

- The main file needs to be completely improved as which looks like an experiment report but not an academic paper. 

- The title should be further improved and "soil parameters" should be more specific. Moreover, punctuation should be avoided in the title.

- Abstract: so many descriptions about the experiment design, more results, discussion and the key findings should be added in this section. Please avoid using abbreviations in this part, such as TOC.

- Unreadable information in figure 1 due to the missing contents of Y axis and unclear information of X axis. Please further improve it.

- Figure 2 shows the effect of heavy metal concentration on bacterial growth, but no heavy metal concentrations were shown in this figure. Unclear information of X axis, “No of objects” means what? Please make it clear.

- Please double check the standard format of the three-line-table, all tables in this manuscript should be improved.

- Please double check the standard format of the references, which should be met the journal requirements.

- How about the potential of the practical application of the findings? Please add more related information.

Author Response

Both the title and abstract were revised.

Before uploading, the manuscript was proofread by a native speaker. After revision another native speaker proofread it.

The figures were corrected.

The format of references was adapted to the journal requirements.

The potential practical application of our study was presented.

We tried to follow all the suggestions.

Reviewer 3 Report

Manuscript title: The influence of selected soil parameters modified with sewage sludge and fly ash fertilization on the total number of bacteria and Bradyrhizobium species at soybean agroecosystem.   

Manuscript id: agriculture-1990665

Authors: Pisarek and Grata

 

The manuscript is particularly strong regarding the less studied topic and the experimental setup on soybean……. The manuscript regarding the topic and results presented is of interest to plant science community and revisions based on the comments below are recommended before considering for publication.

Major comments

·       Insufficient Abstract: In the abstract, the main aim and background of the manuscript are missing, the current version it only highlights the result. In addition, it would be even better to have a sentence as a future perspective.

·       The unit/abbreviation is not mentioned before, consider defining the abbreviation when mentioned for the first time…. Please check throughout the manuscript to define the abbreviations.

·       Line 100-103, the aim or hypothesis of the study is clear, however, the approach is missing ….

·       Lake of scientific literature to support the statements and findings throughout the manuscript…... I have made some suggestions for that and more need it….

·       More information is needed for ALL TABLE captions and define the abbreviation and units that are used. And adjust the significant figures for the table and manuscript.

·       Grammar and punctuation issuers need to be addressed. I have selected/mentioned some as examples.

·       I have a major concern about the results and discussion section. The authors describe the results and compare the results with previous studies, however, insight mechanisms are still insufficient.

·       Authors used ‘’soybean’’, ‘’soy’’ and ‘’legume’’ to refer to soybean. Please consider harmonizing and using only one term throughout the manuscript.

·       The term ‘’object’’ is not clear what authors referring to? Please consider elaborating more or name the parameters

 

Detailed comments:

Abstract

If the unit/abbreviation is not mentioned before, consider defining the abbreviation when mentioned for the first time.

Line 16-22: A complicated sentence, please revise and check the grammar

Line 23: Add a comma after them………...

 

Introduction:

Line 36: A reference is needed here.

Line 37-38: Term ‘’Poland used 2 times in the same sentence.

Line 8-10: A complicated sentence, please revise and check the grammar

Line 42: correct the temperature degree, I guess you mean (22 ◦C), right?

Line 37-44: A reference is needed here, for example, you can use:

https://doi.org/10.3390/app12157830

or

https://doi.org/10.5513/JCEA01/20.1.2210

 

Line 70: A reference is needed here, for example, you can use:

https://doi.org/10.1021/acs.est.1c01477

Line 71-90: How does this discussion help to build the research question? Which some of the parameters discussed here are not well related to this study. Please consider to reparse it and make the connection between the literature and the study.

 

In MM section

Literature references are missing for all sub-section. It would be better to cite the references that the procedure adopted.

Additional info is needed for the table caption, most importantly significant figures.

In MM section, what is the quality control (QC) data? There is no mention of the QC.

What is the accuracy of the instruments, recovery, LOD, and LOQ ……. These parameters are needed to report the efficiency of any analytical system.

In general, how many times you’ve recorded the data,? duplicate? Triplicate?..... what you mentioned in the text is not clear, please elaborate more on this

And abbreviations: P, K, Mg, Fe, Zn, Mn, Cu, [mg kg-1] stands for what? Consider a reader who is not a chemist

 

 

R&D section

These two paragraphs belong to the introduction section, please consider rephrasing or moving the paragraph to the introduction, Line 177-184, Line 197-200

Line:179: A complicated sentence, please revise and check the grammar

These sections are repeating information already presented and explain things in an unnecessarily complicated way. The quality of the manuscript would benefit from the whole section being condensed, Line 201-234, Line 245-277,

Figure 1 and 2: why there is no y-axis? And please consider highlighting the significant on the figure, the current version is not visualized well.

Conclusion

 Important conclusions! However, the future perspectives for the following research are highly crucial here …..

 

Author Response

Both the title and abstract were revised.

Before uploading, the manuscript was proofread by a native speaker. After revision another native speaker proofread it.

The figures were corrected.

The format of references was adapted to the journal requirements.

The potential practical application of our study was presented.

We tried to follow all the suggestions.

Round 2

Reviewer 1 Report

I gave an opportunity to the authors during first round of review to improve the article, but they did not do so to a satisfactory level.

Author Response

• We tried to do the best revision used to your suggestions.
• The title of the manuscript was revised.
• Before uploading the manuscript was proofread by a native speaker. After revision another native speaker proofread it.
• Field experiment was carried out with the random plot method (consisted of 24 plots: six treatments and four replications). Soil samples were collected from each plot (24) for analysis.The results in the tables are the arithmetic mean of 4 replicates.
• A series of ten-fold dilutions of the material was made and the number of microorganisms was determined by the spread plate method. The number of microorganisms was expressed as colony forming units (CFU) per gram of soil or root nodule.
• The values of percentage increase/decrease of TNB and Bradyrizobium spp. were included at section 3. “Results and discussion” (Abstract can consist of max. 200 words).
• Introduction was updated. In fact, we had left out references which were suggested, we included them in the present version:

[23] Agri, U., Chaudhary, P., Sharma, A., Kukreti, B. Physiological response of maize plants and its rhizospheric microbiome under the influence of potential bioinoculants and nanochitosan. Plant Soil 2022, 474, 451–468. https://doi.org/10.1007/s11104-022-05351-2

[24] Chaudhary, P., Sharma, A. Chaudhary, A., Khati, P.,Gangola, S., Maithani, D. Illumina based high throughput analysis of microbial diversity of maize rhizosphere treated with nanocompounds and Bacillus sp. Applied Soil Ecology 2021, 159, 103836. https://doi.org/10.1016/j.apsoil.2020.103836

[25] Chaudhary, P., Chaudhary, A., Bhatt, P., Kumar, G., Khatoon, H., Rani, A., Kumar, S., Sharma, A. Assessment of Soil Health Indicators Under the Influence of Nanocompounds and Bacillus spp. in Field Condition. 2022, Front. Environ. Sci. 9:769871. https://doi.org/10.3389/fenvs.2021.769871

 

• Introduction was updated.
• The rhizosphere, i.e. the space covering the root surface and the soil surrounding it, is an environment with the high microbiological activity and the relationship between the plant, soil and microorganisms, therefore, the total number of microorganisms in the ectorosphere was determined.
• Before uploading the manuscript was proofread by a native speaker. After revision another native speaker proofread it.
• Field experiment was carried out with the random plot method (24 plots: six treatments and four replications). Soil samples were collected from each plot (24) for analysis.The results in the tables are the arithmetic mean of 4 replicates. For each replicate we made 4 lab analysis for the confirmation of each value – the value of each replicate were calculate as the arithmetic mean.
• The enzymatic activity of microorganisms was not determined.
• HiStick® contains at least 2 x 10⁹ live cells from the Rhizobium group (Bradyrhizobium japonicum). 400 g of HiStick® was used to inoculate 100 kg of soybeans by dry inoculation before sowing.
• Standard method for total bacteria count is pour plate method. The number of colonies formed in the plates were enumerated and the average number of the colony-forming unit (CFU/g and CFU/root nodule) were calculated by using following formula:

 

CFU/g or CFU/root nodule =  x  dilution factor

• A series of ten-fold dilutions were made of the tested materials. Dilutions from 10^-4 to 10^-6 were selected from soil samples, and from 10^-1 to 10^-3 dilutions from nodules and spread on the media. After incubation, plates containing 30 to 300 colonies were selected for counting. To determine the number of bacteria, the mean dilution number of colonies, the dilution factor and the plated volume were taken into account.
• We did not take a pictures of cfu count plates. The scheme of experimental field and pictures of soyabean were included.
• Discussion was updated. All suggestions were followed.
• We defined all treatments in our study. The Conclusions section was changed and future prospects were included.

Author Response File: Author Response.docx

Reviewer 2 Report

As the authors further improved the manuscript based on all the reviewers comments and suggestions, this paper could be consider published in this journal at the present version.

Author Response

Thank you for revision of our paper.

For the request of other Reviewers we included more references and the scheme of field experiments.

Author Response File: Author Response.docx

Reviewer 3 Report

Dear Authors, 

I can't see the response file, where you show how you addressed my comments. From what I can see, you haven't addressed most of my scientific comments, including references, and editing the R&D section .....   I have attached my comment for the 1st version of the manuscript, please address the comment, I would be more than happy to read it again.    best wishes

Comments for author File: Comments.pdf

Author Response

Major comments

• The abstract was revised. We added to the Abstract sentences about future prospects (“The study confirmed that external organic matter, such as sewage sludge, can be an alternative to natural fertilizers for soybean production.”). Additionally, the potential practical application of our study was presented in the section Conclusions. The title was also changed.
• The units/abbreviations were checked in the manuscript.
• The section Introduction was revised and the approach to the research was defined as: “Therefore, the aims of this study were: (1) to analyse the changes of chemical parameters of soil and their magnetometry in analysed treatments with sewage sludge and fly ash, and (2) to investigate the effects of analysed treatments on the TNB and Bradyrhizobium community.”
• In fact, we had left out references which were suggested, we included them in the present version:

[3]Hama, J.R.; Kolpin, D.W.; LeFevre, G.H.; Hubbard, L.E.; Powers, M.M.; Strobel, B.W. Exposure and transport of alkaloids and phytoestrogens from soybeans to agricultural soils and streams in the Midwestern United States. Environ. Sci. Technol. 2021, 55, 16, 11029–11039. https://doi.org/10.1021/acs.est.1c01477

 [4]Wenda-Piesik, A.; Ambroziak, K. The choice of soybean cultivar alters the underyielding of protein and oil under drought conditions in Central Poland. Appl. Sci. 2022, 12, 7830. https://doi.org/10.3390/app12157830

[16]Żarski, J.;  Kuśmierek-Tomaszewska, R.;  Dudek, S.;  Kropkowski, M.;  Kledzik, R.  Identifying climatic risk to soybean cultivation in the transitional type of moderate climate in Central Poland.  Journal of Central European Agriculture. 2019, 20(1), 143-156.  https://doi.org/10.5513/JCEA01/20.1.2210

-  The Tables and Figures were corrected.

- Before uploading the manuscript was proofread by a native speaker. After revision another native speaker proofread it.

- In the section Discussion we compare our results to the results of other authors, including our previous study. In our opinion this practice is accepted in scientific studies.

- We used soybean in the manuscript.

       - We defined all treatments in our study.

 

Detailed comments:

• The abstract was revised. The units/abbreviations were checked in the manuscript. Before uploading the manuscript was proofread by a native speaker. After revision another native speaker proofread it.
• References in the line 36 were added. Lines 37-38 were changed: (Poland only once): “Due to climatic and geographical conditions, only cultivars of soybeans grown or tested for their usefulness in Poland may be grown there.” Grammar in the manuscript was corrected. In line 42 we wrote about the sum of daily temperatures, not diurnal (“the sum of daily temperatures up to 2200°C, and their growing season does not exceed 140 days”). The suggested references were included: [3], [4], [16].
• Sewage sludge and fly ash are a source of contamination. The magnetometry is a method used to determine slight changes in soils including the changes after unconventional fertilization. At the same time, humic substances can create different complexes with oxides, hydrox-ides and elements from the group of ferromagnetics, and we would like to show it at our study. The creation of organo-mineral complexes is a possibility of soil protection, especially its biological parts (i.e. bacterial community), which we tried to prove.
• The literature references were supplemented. The figures were corrected. The accuracy of the instruments was added.
• Field experiment was carried out with the random plot method (24 plots: six treatments and four replications). Soil samples were collected from each plot (24) for analysis.The results in the tables are the arithmetic mean of 4 replicates. For each replicate we made 4 lab analysis for the confirmation of each value – the value of each replicate were calculate as the arithmetic mean.
• The abbreviations as P, K, Mg, Fe, Zn, Mn, Cu etc. were explained.
• The grammar and in generally English was proofread; the figures were corrected.
• The Conclusions section was changed and future prospects were included.

 

Author Response File: Author Response.docx

Round 3

Reviewer 3 Report

The revised manuscript has improved compared to the original version. The authors tried to address my questions as much as possible. I recommend the manuscript to be published!